TW201930594A

TW201930594A - Hepatitis B virus (HBV) vaccines and uses thereof

Info

Publication number: TW201930594A
Application number: TW107145775A
Authority: TW
Inventors: 丹尼爾包登; 海倫荷頓; 簡馬克艾德蒙弗內德馬利尼夫斯; 蘇米特拉羅伊
Original assignee: 愛爾蘭商健生科學愛爾蘭無限公司
Priority date: 2017-12-19
Filing date: 2018-12-18
Publication date: 2019-08-01

Abstract

Polynucleotides encoding hepatitis B virus (HBV) core antigen and polymerase antigen, and related combinations are described. Also described are vectors, such as DNA plasmids or viral vectors, expressing the HBV core and polymerase antigens, and immunogenic compositions containing the expression vectors. Methods of inducing an immune response against HBV or treating a HBV-induced disease, particularly in individuals having chronic HBV infection, using the immunogenic compositions are also described.

Description

B型肝炎病毒(HBV)疫苗及其用途Hepatitis B virus (HBV) vaccine and uses thereof

B型肝炎病毒(HBV)係編碼四個開放閱讀框架及七種蛋白質的3.2 kb親肝性小DNA病毒。有約二十億人感染HBV，且有約兩億四千萬人患有慢性B型肝炎感染(慢性HBV)，該病以病毒及亞病毒粒子持久存在於血液中超過6個月為特徵(1)。持久的HBV感染經由病毒肽及循環抗原長期刺激HBV特異性T細胞受體而導致循環及肝內HBV特異性CD4+及CD8+ T細胞的T細胞耗盡。因此，T細胞多功能性減弱(亦即，IL-2、腫瘤壞死因子(TNF)-α、IFN-γ含量降低，及增殖缺乏)。Hepatitis B virus (HBV) is a 3.2 kb hepatotropic small DNA virus that encodes four open reading frames and seven proteins. About two billion people are infected with HBV and about 240 million people have chronic hepatitis B infection (chronic HBV), which is characterized by the persistent presence of viruses and subviral particles in the blood for more than 6 months ( 1). Persistent HBV infection stimulates HBV-specific T cell receptors through viral peptides and circulating antigens for a long time, resulting in depletion of circulating and intrahepatic HBV-specific CD4 + and CD8 + T cells. As a result, T cell multifunction is reduced (i.e., reduced levels of IL-2, tumor necrosis factor (TNF) -α, IFN-γ, and lack of proliferation).

自1980年代起，已有可用的針對HBV感染之安全且有效的預防性疫苗，且成為B型肝炎預防之主要手段(3)。世界衛生組織(World Health Organization)建議所有嬰兒接受疫苗接種，且在存在較低或中等B型肝炎地方性流行的國家中，建議所有兒童及青少年(＜18歲)以及處於風險群體類別的某些人接受疫苗接種。由於接受疫苗接種，使得全世界感染率大幅下降。不過，預防性疫苗不能治癒已確定之HBV感染。Since the 1980s, safe and effective preventive vaccines against HBV infection have been available, and they have become the main means of preventing hepatitis B (3). The World Health Organization recommends that all infants be vaccinated and that in countries with a low or moderate endemic hepatitis B epidemic, all children and adolescents (<18 years) and some in the risk group category are recommended Man receives vaccination. Due to vaccination, infection rates worldwide have fallen dramatically. However, prophylactic vaccines do not cure established HBV infections.

慢性HBV當前係用IFN-α及核苷或核苷酸類似物進行治療，但由於在感染之肝細胞中存留有作為病毒RNA之模板起重要作用的稱為共價閉合環狀DNA (cccDNA)之細胞內病毒複製中間物且因此存留有新病毒粒子而最終無法治癒。普遍認為，誘發病毒特異性T細胞及B細胞反應可以有效地除去載有cccDNA之肝細胞。當前靶向HBV聚合酶之療法抑制病毒血症，但對存在於核中之cccDNA及相關之循環抗原產生的作用有限。最嚴格的治癒形式可以除去生物體中之HBV cccDNA，此既不為觀察到的自然發生之結果，亦非任何治療性干預之結果。然而，HBV表面抗原(HBsAg)之喪失係臨床上可信的治癒等效結果，因為疾病復發僅在嚴重免疫抑制情況下才會發生，而此可接著藉由預防性治療加以預防。因此，至少自臨床觀點看，HBsAg之喪失與針對HBV的最嚴格免疫重建形式相關。Chronic HBV is currently treated with IFN-α and nucleoside or nucleotide analogs, but covalently closed circular DNA (cccDNA), which plays an important role as a template for viral RNA, remains in infected liver cells The intracellular virus replicates intermediates and therefore retains new virions that ultimately cannot be cured. It is generally believed that inducing virus-specific T cell and B cell responses can effectively remove cccDNA-containing hepatocytes. Current therapies targeting HBV polymerase inhibit viremia, but have limited effect on the production of cccDNA and related circulating antigens present in the nucleus. The most stringent form of cure removes HBV cccDNA from the organism, which is neither an observed naturally occurring result nor the result of any therapeutic intervention. However, the loss of HBV surface antigen (HBsAg) is a clinically credible cure-equivalent result, as disease recurrence occurs only in cases of severe immunosuppression, which can then be prevented by preventive treatment. Therefore, at least from a clinical standpoint, the loss of HBsAg is associated with the most stringent form of immune reconstitution against HBV.

舉例而言，經證實，利用聚乙二醇化干擾素(pegIFN)-α進行之免疫調節在維持有限治療療程之治療結束後反應方面優於核苷或核苷酸療法。除直接抗病毒作用外，據報導，IFN-α在細胞培養物及人類化小鼠中對cccDNA起到表觀遺傳抑制作用，使病毒粒子生產率及轉錄本減少(4)。然而，此療法仍伴隨副作用且且部分由於IFN-α對HBV特異性T細胞僅具有較弱的調節作用，總體反應相當低。詳言之，治癒率較低(＜10%)且毒性較高。同樣，直接作用於HBV之抗病毒劑，即HBV聚合酶抑制劑恩替卡韋(entecavir)及替諾福韋(tenofovir)，作為單藥療法有效誘導病毒抑制作用且針對耐藥性突變體之出現具有較高基因屏障作用且由此預防肝病之進展。然而，利用此類HBV聚合酶抑制劑很少實現由HBsAg喪失或血清轉化定義的慢性B型肝炎之治癒。因此，該等抗病毒劑理論上需要無限期投與以預防肝病之復發，與針對人類免疫缺陷病毒(HIV)之抗反轉錄病毒療法類似。For example, immunomodulation with pegylated interferon (pegIFN) -α has proven to be superior to nucleoside or nucleotide therapy in maintaining a response after the end of treatment for a limited course of treatment. In addition to direct antiviral effects, IFN-α has been reported to exert epigenetic inhibitory effects on cccDNA in cell cultures and humanized mice, reducing virion productivity and transcripts (4). However, this therapy is still accompanied by side effects and in part because IFN-α has only a weaker regulatory effect on HBV-specific T cells, and the overall response is quite low. In detail, the cure rate is low (<10%) and the toxicity is high. Similarly, antiviral agents that directly act on HBV, namely the HBV polymerase inhibitors entecavir and tenofovir, are effective as single-agent therapies to induce viral suppression and are more resistant to the emergence of drug-resistant mutants. High genetic barrier effect and thus prevent progression of liver disease. However, the use of such HBV polymerase inhibitors has rarely achieved a cure for chronic hepatitis B as defined by HBsAg loss or seroconversion. Therefore, these antiviral agents theoretically need to be administered indefinitely to prevent recurrence of liver disease, similar to antiretroviral therapy against human immunodeficiency virus (HIV).

治療性疫苗接種有可能除去長期感染患者之HBV(5)。已經研究許多策略，但迄今為止，尚未證實治療性疫苗接種之成功性。Therapeutic vaccination has the potential to remove HBV from chronically infected patients (5). Many strategies have been studied, but to date, the success of therapeutic vaccination has not been demonstrated.

因此，由於具有較高治癒率的良好耐受之治療方法有限，對於B型肝炎病毒(HBV)，特別是慢性HBV治療之醫療需求尚未得到滿足。本發明藉由提供用於誘發針對B型肝炎病毒(HBV)感染之免疫反應的免疫原性組合物及方法滿足此需求。本發明之免疫原性組合物及方法可以用於向個體，諸如患有慢性HBV感染之個體提供治療性免疫。Therefore, due to limited well-tolerated treatments with high cure rates, the medical needs for hepatitis B virus (HBV), especially chronic HBV treatment, have not been met. The present invention meets this need by providing immunogenic compositions and methods for inducing an immune response against a hepatitis B virus (HBV) infection. The immunogenic compositions and methods of the invention can be used to provide therapeutic immunity to an individual, such as an individual with a chronic HBV infection.

在一通用態樣中，本申請案係關於一種編碼HBV抗原，諸如截短之HBV核心抗原或HBV聚合酶抗原的非天然存在之核酸分子。根據本申請案之一個實施例的HBV抗原係能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應(體液及細胞免疫反應)，較佳在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應，更佳地，在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應的共同抗原。In a general aspect, the present application relates to a non-naturally occurring nucleic acid molecule encoding a HBV antigen, such as a truncated HBV core antigen or a HBV polymerase antigen. The HBV antigen system according to an embodiment of the present application is capable of inducing an immune response (humoral and cellular immune response) against at least two HBV genotypes in a mammal, preferably in mammals against at least HBV genotype B, C and D T cell responses, more preferably, a common antigen that elicits CD8 T cell responses against at least HBV genotypes A, B, C, and D in a human individual.

在一個實施例中，本申請案之非天然存在之核酸分子編碼由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原，且該非天然存在之核酸分子包含與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致的聚核苷酸序列。較佳地，該非天然存在之核酸分子包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。In one embodiment, the non-naturally occurring nucleic acid molecule of the present application encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14, and the non-naturally occurring nucleic acid molecule Contains a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15. Preferably, the non-naturally occurring nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

在一個實施例中，本申請案之非天然存在之核酸分子編碼包含與SEQ ID NO: 4至少98%一致之胺基酸序列的HBV聚合酶抗原，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H(RNAse H)活性。較佳地，HBV聚合酶抗原包含SEQ ID NO: 4之胺基酸序列。更佳地，非天然存在之核酸分子包含與SEQ ID NO : 3或SEQ ID NO: 16至少90%一致，最佳與SEQ ID NO: 3或SEQ ID NO: 16達100%一致之聚核苷酸序列。In one embodiment, the non-naturally occurring nucleic acid molecule of the present application encodes a HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have a reverse transcriptase Activity and RNAse H activity. Preferably, the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4. More preferably, the non-naturally occurring nucleic acid molecule comprises a polynucleoside at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16 and most preferably 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16 Acid sequence.

在另一通用態樣中，本申請案係關於一種包含本申請案之非天然存在之核酸分子的載體，較佳地為DNA質體或病毒載體。In another general aspect, the present application relates to a vector, preferably a DNA plastid or a viral vector, comprising a non-naturally occurring nucleic acid molecule of the present application.

在另一通用態樣中，本申請案係關於一種包含本申請案之非天然存在之核酸分子或載體的重組宿主細胞。In another general aspect, the present application relates to a recombinant host cell comprising a non-naturally occurring nucleic acid molecule or vector of the present application.

在另一通用態樣中，本申請案係關於一種由本申請案之非天然存在之核酸分子編碼的非天然存在之多肽。In another general aspect, the present application relates to a non-naturally occurring polypeptide encoded by a non-naturally occurring nucleic acid molecule of the present application.

在又一通用態樣中，本申請案係關於一種組合物，其包含本申請案之非天然存在之核酸分子、載體、重組宿主細胞及非天然存在之多肽中的至少一種，及醫藥學上可接受之載劑。In another general aspect, the present application relates to a composition comprising at least one of a non-naturally occurring nucleic acid molecule, a vector, a recombinant host cell, and a non-naturally occurring polypeptide of the present application, and a pharmacological Acceptable vehicle.

在另一通用態樣中，本申請案係關於一種免疫原性組合，特定言之套組，其包含：
(a) 包含編碼HBV聚合酶抗原之第一聚核苷酸的第一非天然存在之核酸分子，該HBV聚合酶抗原具有與SEQ ID NO: 4至少98%一致的胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性；
(b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二非天然存在之核酸分子，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成；以及
(c) 醫藥學上可接受之載劑，
其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一非天然存在之核酸分子中或兩個不同的非天然存在之核酸分子中。In another general aspect, the present application relates to an immunogenic combination, specifically a set, comprising:
(a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity or ribonuclease H activity;
(b) a second non-naturally-occurring nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen, the truncated HBV core antigen being an amino group of SEQ ID NO: 2 or SEQ ID NO: 14 Acid sequence composition;
(c) a pharmaceutically acceptable carrier,
The first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule exist in the same non-naturally occurring nucleic acid molecule or two different non-naturally occurring nucleic acid molecules.

在一些實施例中，本申請案之免疫原性組合，特定言之套組包含存在於第一載體中之第一聚核苷酸及存在於第二載體中之第二聚核苷酸。較佳地，該第一載體不同於該第二載體。更佳地，該載體係質體載體或病毒載體。更佳地，該第一載體及該第二載體各自為質體DNA載體。In some embodiments, the immunogenic combination of the present application, specifically the set comprises a first polynucleotide present in a first vector and a second polynucleotide present in a second vector. Preferably, the first carrier is different from the second carrier. More preferably, the vector is a plastid vector or a viral vector. More preferably, the first vector and the second vector are each a plastid DNA vector.

在一個實施例中，本申請案之免疫原性組合，特定言之套組包含：
a) 包含編碼HBV聚合酶抗原之第一聚核苷酸序列的第一質體DNA載體，該HBV聚合酶抗原具有SEQ ID NO: 4之胺基酸序列； b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二質體DNA載體，該截短之HBV核心抗原由SEQ ID NO: 2或14之胺基酸序列組成；以及
c) 醫藥學上可接受之載劑，
其中該第一質體DNA載體及該第二質體DNA載體各自進一步包含抗生素抗性基因及複製起點，且
其中該第一質體DNA載體與該第二質體DNA載體係存在於同一組合物中或兩種不同組合物中。In one embodiment, the immunogenic combination of the present application, specifically the set includes:
a) a first plastid DNA vector comprising a first polynucleotide sequence encoding an HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence of SEQ ID NO: 4; b) comprising a core encoding a truncated HBV A second plastid DNA vector of a second polynucleotide of an antigen, the truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or 14; and
c) a pharmaceutically acceptable carrier,
The first plastid DNA vector and the second plastid DNA vector each further include an antibiotic resistance gene and an origin of replication, and wherein the first plastid DNA vector and the second plastid DNA vector are in the same composition. Medium or in two different compositions.

在一個特定實施例中，本申請案之免疫原性組合，特定言之套組包含：
a) 第一質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的強化子序列、含SEQ ID NO: 5之聚核苷酸序列的信號肽編碼序列、含SEQ ID NO: 3之聚核苷酸序列的第一聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的聚腺苷酸化信號序列； b) 第二質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的強化子序列、含SEQ ID NO: 5之聚核苷酸序列的信號肽編碼序列、含SEQ ID NO: 1之聚核苷酸序列的第二聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的聚腺苷酸化信號序列；以及 c) 醫藥學上可接受之載劑，其中該第一質體DNA載體及該第二質體DNA載體各自進一步包含具有SEQ ID NO: 12之聚核苷酸序列的卡那黴素抗性基因及具有SEQ ID NO:10之聚核苷酸序列的複製起點，且
其中該第一質體DNA載體與該第二質體DNA載體係存在於同一組合物中或兩種不同組合物中。In a specific embodiment, the immunogenic combination of the present application, specifically the set includes:
a) The first plastid DNA vector, from the 3 'end to the 5' end, comprising a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7 and a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 8 Enhancer sequence, signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and A polyadenylation signal sequence of a polynucleotide sequence; b) a second plastid DNA vector, from the 3 'end to the 5' end, comprising a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, An enhancer sequence containing the polynucleotide sequence of SEQ ID NO: 8, a signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, a second sequence containing the polynucleotide sequence of SEQ ID NO: 1 A polynucleotide sequence and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11; and c) a pharmaceutically acceptable carrier, wherein the first plastid DNA vector and the second Each of the plastid DNA vectors further comprises a kanamycin resistance gene having a polynucleotide sequence of SEQ ID NO: 12 and an origin of replication having a polynucleotide sequence of SEQ ID NO: 10, and wherein the first One plastid DNA vector and the second plastid DNA vector exist in the same composition or in two different compositions.

在其他實施例中，本申請案之免疫原性組合，特定言之套組包含存在於同一載體中之第一聚核苷酸及第二聚核苷酸。較佳地，該載體係質體載體或病毒載體。更佳地，該載體係腺病毒載體，諸如Ad26或Ad35載體。In other embodiments, the immunogenic combination of the present application, specifically the set comprises a first polynucleotide and a second polynucleotide present in the same vector. Preferably, the vector is a plastid vector or a viral vector. More preferably, the vector is an adenoviral vector, such as an Ad26 or Ad35 vector.

在一個實施例中，本申請案之免疫原性組合，特定言之套組包含：
a) 載體，其包含編碼具有SEQ ID NO: 4之胺基酸序列之HBV聚合酶抗原的第一聚核苷酸序列，及編碼由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原之第二聚核苷酸；以及 b) 醫藥學上可接受之載劑。In one embodiment, the immunogenic combination of the present application, specifically the set includes:
a) A vector comprising a first polynucleotide sequence encoding a HBV polymerase antigen having an amino acid sequence of SEQ ID NO: 4 and an amino acid encoding an amino acid consisting of SEQ ID NO: 2 or SEQ ID NO: 14 The sequence consists of a second polynucleotide of a truncated HBV core antigen; and b) a pharmaceutically acceptable carrier.

在一個特定實施例中，本申請案之免疫原性組合，特定言之套組包含病毒載體，較佳地腺病毒載體，自5'端至3'端，該載體包括含SEQ ID NO: 17之聚核苷酸序列的啟動子序列、含SEQ ID NO: 23之聚核苷酸序列的調控序列、含SEQ ID NO: 18之聚核苷酸序列的信號肽編碼序列、含SEQ ID NO: 15之聚核苷酸序列的第二聚核苷酸序列、含SEQ ID NO: 22之聚核苷酸序列的連接子編碼序列、含SEQ ID NO: 16之聚核苷酸序列的第一聚核苷酸序列，及含SEQ ID NO: 24之聚核苷酸序列的聚腺苷酸化信號序列，及醫藥學上可接受之載劑。In a specific embodiment, the immunogenic combination of the present application, specifically the set comprises a viral vector, preferably an adenoviral vector, from the 5 'end to the 3' end, the vector comprising a sequence comprising SEQ ID NO: 17 The promoter sequence of the polynucleotide sequence, the control sequence of the polynucleotide sequence of SEQ ID NO: 23, the signal peptide coding sequence of the polynucleotide sequence of SEQ ID NO: 18, the sequence of SEQ ID NO: The second polynucleotide sequence of the polynucleotide sequence of 15, the linker coding sequence containing the polynucleotide sequence of SEQ ID NO: 22, and the first polymer sequence of the polynucleotide sequence containing SEQ ID NO: 16. A nucleotide sequence, a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 24, and a pharmaceutically acceptable carrier.

本申請案之其他態樣係關於製造本申請案之聚核苷酸、載體、多肽及組合物以及免疫原性組合或套組的方法。Other aspects of this application relate to methods of making the polynucleotides, vectors, polypeptides, and compositions and immunogenic combinations or sets of this application.

且在又一通用態樣中，本申請案係關於一種在有需要之個體中誘發針對B型肝炎病毒(HBV)之免疫反應的方法，該方法包含向該個體投與免疫原性有效量的本申請案之組合物或免疫原性組合。較佳地，該方法在個體中誘發針對至少兩種HBV基因型之免疫反應，諸如抗體反應及/或T細胞反應。較佳地，該方法在個體中誘發針對至少HBV基因型B、C及D之T細胞反應。更佳地，該方法在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。在一實施例中，該方法進一步包含向該個體投與另一免疫原性藥劑，較佳地另一HBV抗原。And in another general aspect, the present application relates to a method for inducing an immune response against hepatitis B virus (HBV) in an individual in need, the method comprising administering to the individual an immunogenicly effective amount of The composition or immunogenic combination of this application. Preferably, the method elicits an immune response, such as an antibody response and / or a T cell response, in an individual against at least two HBV genotypes. Preferably, the method induces a T-cell response in an individual against at least HBV genotypes B, C, and D. More preferably, the method elicits a CD8 T cell response against at least HBV genotypes A, B, C, and D in a human individual. In one embodiment, the method further comprises administering to the individual another immunogenic agent, preferably another HBV antigen.

在另一態樣中，本申請案係關於一種治療有需要之個體的B型肝炎病毒(HBV)誘發之疾病的方法，該方法包含向該個體投與治療有效量的本申請案之組合物或免疫原性組合。較佳地，該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。在一實施例中，該方法進一步包含向該個體投與另一治療劑，較佳地另一抗HBV抗原。In another aspect, the present application relates to a method for treating a hepatitis B virus (HBV) -induced disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present application Or immunogenic combination. Preferably, the individual has a chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In one embodiment, the method further comprises administering to the individual another therapeutic agent, preferably another anti-HBV antigen.

本申請案亦係關於用於誘發針對B型肝炎病毒(HBV)之免疫反應的本申請案之組合物、免疫原性組合或套組；以及本申請案之組合物、免疫原性組合或套組在製造用於誘發針對B型肝炎病毒(HBV)之免疫反應的藥物中的用途。該用途可以進一步包含與另一免疫原性藥劑，較佳地另一HBV抗原之組合。較佳地，該個體患有慢性HBV感染。This application is also related to the composition, immunogenic combination or kit of the present application for inducing an immune response against hepatitis B virus (HBV); and the composition, immunogenic combination or kit of the present application Use of the group in the manufacture of a medicament for inducing an immune response against hepatitis B virus (HBV). The use may further comprise a combination with another immunogenic agent, preferably another HBV antigen. Preferably, the individual has a chronic HBV infection.

本申請案進一步係關於用於治療有需要之個體的HBV誘發之疾病的本申請案之組合物、免疫原性組合或套組；以及本申請案之組合物、免疫原性組合或套組在製造用於治療有需要之個體的HBV誘發之疾病之藥物中的用途。該用途可以進一步包含與另一治療劑，較佳地另一抗HBV抗原之組合。較佳地，該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。The present application further relates to a composition, immunogenic combination or set of the present application for treating HBV-induced diseases in an individual in need; and a composition, immunogenic combination or set of the present application Use in the manufacture of a medicament for the treatment of HBV-induced diseases in an individual in need. The use may further comprise a combination with another therapeutic agent, preferably another anti-HBV antigen. Preferably, the individual has a chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

自以下揭示內容，包括本發明之詳細說明及其較佳實施例以及所附申請專利範圍，將易於瞭解本發明之其他態樣、特徵及優勢。Other aspects, features, and advantages of the present invention will be readily understood from the following disclosure, including a detailed description of the present invention, its preferred embodiments, and the scope of the attached patent application.

相關申請案之交叉引用Cross-reference to related applications

本申請案主張2017年12月19日申請之臺灣專利申請案第106144659號之優先權，其揭示內容以全文引用之方式併入本文中。
對以電子方式提交之序列表之引用This application claims priority from Taiwan Patent Application No. 106144659, filed on December 19, 2017, the disclosure of which is incorporated herein by reference in its entirety.
Reference to a Sequence Listing Submitted Electronically

本申請案含有序列表，該序列表係以2018年12月10日創建的檔案名稱為「688097-403 Sequence Listing」且大小為46.6 KB之ASCII格式序列表經由EFS-Web以電子方式提交。此經由EFS-Web提交之序列表係說明書之一部分且以全文引用的方式併入本文中。This application contains a sequence listing, which is filed electronically via EFS-Web with an ASCII format sequence listing created on December 10, 2018 with the file name "688097-403 Sequence Listing" and a size of 46.6 KB. This sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

先前技術及本說明書通篇引用或描述各種出版物、文章及專利；該等參考文獻各自以全文引用的方式併入本文中。本說明書中所包括的文獻、操作、材料、器件、文章或類似物之論述係出於提供本發明之內容的目的。此類論述並非承認任何或所有該等內容形成關於所揭示或所要求的任何發明之現有技術的一部分。Various publications, articles, and patents are cited or described in the prior art and throughout this specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles, or the like included in this specification is for the purpose of providing the present invention. Such discussions are not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

除非另外定義，否則本文所用的所有技術及科學術語均具有與本發明所屬領域的一般技術者通常所理解相同的含義。另外，本文中使用之某些術語具有如本說明書中所述之含義。本文中引用的所有專利、公開之專利申請案及出版物均以引用的方式併入，就如同在本文中完整闡述一般。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, certain terms used herein have the meanings as described in this specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

必須注意的是，除非上下文另外明確指示，否則如本文及所附申請專利範圍中所使用，單數形式「一個(種)(a/an)」及「該(the)」包括複數個(種)指示物。It must be noted that, unless the context clearly indicates otherwise, as used herein and in the scope of the attached patent application, the singular forms "a / an" and "the" include plural Counter.

除非另外指示，否則在一系列要素之前的術語「至少」應理解為指該系列中之每一要素。熟習此項技術者將認識到或能夠僅使用常規實驗即確定本文所描述的本發明之具體實施例的許多等效物。本發明意欲涵蓋此類等效物。Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. This invention is intended to cover such equivalents.

在本說明書通篇及隨後的申請專利範圍中，除非上下文另有要求，否則詞語「包含(comprise)」及變化形式諸如「包含(comprises)」及「包含(comprising)」應理解為暗示包括一個所述整數或步驟或者一組整數或步驟，但不排除任何其他整數或步驟或者整數或步驟組。當在本文中使用時，術語「包含」可以用術語「含有」或「包括」取代，或有時當在本文中使用時，用術語「具有」取代。Throughout this specification and the scope of subsequent patent applications, unless the context requires otherwise, the words "comprise" and variations such as "comprises" and "comprising" should be understood to imply the inclusion of a The integer or step or a group of integers or steps does not exclude any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced with the terms "containing" or "including" or sometimes when used herein with the term "having."

當在本文中使用時，「由……組成」不包括所要求要素中未說明之任何要素、步驟或成分。當在本文中使用時，「基本上由……組成」不排除不會實質上影響技術方案之基本及新穎特徵之材料或步驟。每當本文中在本申請案之態樣或實施例之上下文中使用時，前述術語「包含」、「含有」、「包括」及「具有」中之任一個可以用術語「由……組成」或「基本上由……組成」替代以改變本發明之範圍。As used herein, "consisting of" does not include any elements, steps or ingredients not stated in the required elements. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel features of the technical solution. Whenever used herein in the context of aspects or examples of the present application, any of the foregoing terms "comprising," "containing," "including," and "having" may use the term "consisting of" Or "consisting essentially of" to change the scope of the invention.

如本文所使用，在多個所述要素之間的連接性術語「及/或」係理解為涵蓋個別及組合選擇兩種。舉例而言，當兩個要素藉由「及/或」連結時，第一個選擇係指第一要素之適用性，不含第二要素。第二個選擇係指第二要素之適用性，不含第一要素。第三個選擇係指第一要素與第二要素一起之適用性。此等選擇中之任一個應理解為在該含義之範圍內，且因此滿足如本文所使用之術語「及/或」之要求。該等選擇中多於一個之同時適用性亦應理解為在該含義之範圍內，且因此滿足術語「及/或」之要求。As used herein, the connectivity term "and / or" between a plurality of stated elements is understood to cover both the individual and combination options. For example, when two elements are connected by "and / or", the first choice refers to the applicability of the first element, excluding the second element. The second option refers to the applicability of the second element, excluding the first element. The third option refers to the applicability of the first and second elements together. Any of these choices should be understood to be within the meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one of these choices should also be understood to be within the meaning and therefore meet the requirements of the term "and / or".

除非另外規定，否則任何數值，諸如本文所描述之濃度或濃度範圍，應理解為在所有情況下以術語「約」修飾。因此，一個數值通常包括所述值±10%。舉例而言，1 mg/mL濃度包括0.9 mg/mL至1.1 mg/mL。同樣，1 mg/mL至10 mg/mL之濃度範圍包括0.9 mg/mL至11 mg/mL。除非上下文另外明確地指示，否則如本文所使用，使用的數字範圍明確地包括所有可能的子範圍及在該範圍內的所有個別數值，包括該等範圍內之整數及該等值之分數。Unless otherwise specified, any numerical value, such as the concentration or range of concentrations described herein, is understood to be modified in all cases by the term "about." Therefore, a value usually includes ± 10% of the stated value. For example, a 1 mg / mL concentration includes 0.9 mg / mL to 1.1 mg / mL. Similarly, the concentration range from 1 mg / mL to 10 mg / mL includes 0.9 mg / mL to 11 mg / mL. Unless the context clearly indicates otherwise, as used herein, a numerical range used explicitly includes all possible subranges and all individual numerical values within that range, including integers within those ranges and fractions of those values.

短語「序列一致性百分比(%)」或「%一致性」或「與……%一致」當參照胺基酸序列使用時描述兩個或兩個以上比對之胺基酸序列中匹配(「相配(hit))」的一致胺基酸之數量與構成該等胺基酸序列之總長度的胺基酸殘基之數量的比較。換言之，當比較兩個或兩個以上序列且對準達到最大對應性，使用此項技術中已知之序列比較演算法量測時，或當手動地比對並目視檢查時，使用比對，可以確定該等序列中相同胺基酸殘基之百分含量(例如在胺基酸序列全長內90%、91%、92%、93%、94%、95%、97%、98%、99%或100%一致性)。因此，供比較以確定序列一致性的序列可能因胺基酸之取代、添加或缺失而不同。適合用於比對蛋白質序列之程式係熟習此項技術者已知的。蛋白質序列之序列一致性百分比可以例如用諸如CLUSTALW、Clustal Omega、FASTA或BLAST之程式，例如使用NCBI BLAST演算法確定(Altschul SF等人(1997),Nucleic Acids Res . 25:3389-3402)。The phrase "percent sequence identity (%)" or "% identity" or "% identity" when used with reference to an amino acid sequence describes a match in two or more aligned amino acid sequences ( A comparison of the number of "hits" consistent amino acids with the number of amino acid residues that make up the total length of the amino acid sequences. In other words, when two or more sequences are compared and the alignment reaches the maximum correspondence, measured using a sequence comparison algorithm known in the art, or when manually aligned and visually inspected, using alignment, you can Determine the percentage content of the same amino acid residues in the sequences (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% over the full length of the amino acid sequence Or 100% consistency). Therefore, the sequences used for comparison to determine sequence identity may differ due to amino acid substitutions, additions, or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. The percent sequence identity of a protein sequence can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA, or BLAST, for example, using the NCBI BLAST algorithm (Altschul SF et al. (1997), Nucleic Acids Res . 25: 3389-3402).

如本文在向個體投與兩種或兩種以上療法或組分之情形中所使用的術語及短語「組合」、「與……組合」、「共遞送」及「與……一起投與」係指同時投與兩種或兩種以上療法或組分，諸如兩個載體，例如DNA質體，或免疫原性組合及佐劑。「同時投與」可以為至少在同一天內投與兩種組分。當兩種組分係「一起投與」或「組合投與」時，其可以在較短時間段內，諸如在24、20、16、12、8或4小時內或在1小時內以獨立組合物依序投與，或其可以單一組合物形式同時投與。使用術語「與……組合」並不限定向個體投與療法或組分之次序。舉例而言，第一療法或組分(例如編碼HBV抗原之第一DNA質體)可以在投與第二療法或組分(例如編碼HBV抗原之第二DNA質體)之前(例如5分鐘至一小時之前)、伴隨地或同時地，或之後(例如5分鐘至一小時之後)投與。在一些實施例中，第一療法或組分(例如編碼HBV抗原之第一DNA質體)及第二療法或組分(例如編碼HBV抗原之第二DNA質體)係以同一組合物投與。在其他實施例中，第一療法或組分(例如編碼HBV抗原之第一DNA質體)及第二療法或組分(例如編碼HBV抗原之第二DNA質體)係以獨立組合物投與。The terms and phrases "combination", "combination with", "co-delivery", and "administration with" as used herein in the context of administering two or more therapies or components to an individual "Means the simultaneous administration of two or more therapies or components, such as two vectors, such as DNA plastids, or immunogenic combinations and adjuvants. "Simultaneous administration" can be the administration of two components at least on the same day. When the two components are "administered together" or "combined administration", they can be separated within a short period of time, such as within 24, 20, 16, 12, 8, or 4 hours or independently within 1 hour The compositions are administered sequentially, or they can be administered simultaneously in the form of a single composition. The use of the term "in combination with" does not limit the order in which the therapy or components are administered to the individual. For example, the first therapy or component (e.g., the first DNA plastid encoding the HBV antigen) may be administered before the second therapy or component (e.g., the second DNA plastid encoding the HBV antigen) (e.g., 5 minutes to One hour ago), concomitantly or simultaneously, or after (eg, 5 minutes to one hour later). In some embodiments, the first therapy or component (e.g., the first DNA plastid encoding the HBV antigen) and the second therapy or component (e.g., the second DNA plastid encoding the HBV antigen) are administered in the same composition . In other embodiments, the first therapy or component (e.g., the first DNA plastid encoding the HBV antigen) and the second therapy or component (e.g., the second DNA plastid encoding the HBV antigen) are administered in separate compositions .

如本文所使用，「非天然存在之」核酸或多肽係指自然界中不存在的核酸或多肽。「非天然存在之」核酸或多肽可以為經合成、處理、製造及/或以其他方式在實驗室及/或製造環境中操作的。非天然存在之核酸或多肽可以包含經處理、加工或操作而展現在處理之前天然存在之核酸或多肽中不存在之特性的天然存在之核酸或多肽。如本文所使用，「非天然存在之」核酸或多肽可以為自發現其之天然來源分離或分開的核酸或多肽，且其與在天然來源中與其關聯之序列不具有共價鍵。「非天然存在之」核酸或多肽可以以重組方式或其他方法，諸如化學合成製備。As used herein, a "non-naturally occurring" nucleic acid or polypeptide refers to a nucleic acid or polypeptide that is not found in nature. A "non-naturally occurring" nucleic acid or polypeptide may be synthesized, processed, manufactured, and / or otherwise manipulated in a laboratory and / or manufacturing environment. Non-naturally occurring nucleic acids or polypeptides can include naturally occurring nucleic acids or polypeptides that have been processed, processed, or manipulated to exhibit properties that were not present in the nucleic acids or polypeptides that naturally existed prior to processing. As used herein, a "non-naturally occurring" nucleic acid or polypeptide may be a nucleic acid or polypeptide that is isolated or separated from the natural source in which it is found, and which does not have a covalent bond with a sequence to which it is associated in a natural source. A "non-naturally occurring" nucleic acid or polypeptide can be prepared recombinantly or by other methods, such as chemical synthesis.

如本文所使用，「個體」意謂將利用根據本申請案之一個實施例的方法治療或已利用該方法治療的任何動物，較佳為哺乳動物，最佳為人類。如本文所使用，術語「哺乳動物」涵蓋任何哺乳動物。哺乳動物之實例包括(但不限於)牛、馬、綿羊、豬、貓、狗、小鼠、大鼠、兔、天竺鼠、非人類靈長類動物(NHP)諸如猴或猿、人類等，更佳為人類。As used herein, "individual" means any animal, preferably a mammal, and most preferably a human, that will be treated or has been treated using the method according to one embodiment of the present application. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHP) such as monkeys or apes, humans, etc., and more Better for humans.

如本文所使用，術語「可操作地連接」係指鍵聯或毗連，其中如此描述之組分係呈允許其以其預期方式發揮作用的關係。舉例而言，可操作地連接至所關注核酸序列之調控序列能夠引導該所關注核酸序列之轉錄，或可操作地連接至所關注胺基酸序列之信號序列能夠將該所關注胺基酸序列分泌或轉位至膜上。As used herein, the term "operably linked" means linked or contiguous, where the components so described are in a relationship that allows them to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest can direct transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest can direct the amino acid sequence of interest Secreted or translocated to the membrane.

為了幫助本申請案之讀者，說明書分成各種段落或部分，或針對本申請案之各種實施例。該等分離不應認為一個段落或部分或實施例之物質與另一段或部分或實施例之物質無關聯。相反，熟習此項技術者應理解，本說明書具有廣闊應用且涵蓋可以涵蓋之各種部分、段落及語句之所有組合。任何實施例之論述僅意欲為例示性的，且並不意欲表明本發明之範疇，包括申請專利範圍，侷限於此等實例。舉例而言，儘管本文所描述的本申請案之HBV載體(例如質體DNA或病毒載體)之實施例可以含有按特定次序佈置之特定組分，包括(但不限於)某些啟動子序列、強化子或調控序列、信號肽、HBV抗原編碼序列、聚腺苷酸化信號序列等，但一般熟習此項技術者應瞭解，本文所揭示之概念可以同等地應用於按可以在本申請案之HBV載體中使用的其他次序佈置之其他組分。本申請案涵蓋使用具有可以用於本申請案之HBV載體中之任何序列的呈任何組合形式之可應用組分中之任一種，無論是否明確地描述特定組合。To assist the reader of this application, the description is divided into various paragraphs or sections, or directed to various embodiments of this application. Such separations should not be considered as unrelated to the substance of one paragraph or part or embodiment and the substance of another paragraph or part or embodiment. On the contrary, those skilled in the art should understand that this description has broad applications and covers all combinations of various parts, paragraphs, and sentences that can be covered. The discussion of any embodiment is intended to be illustrative only, and is not intended to indicate the scope of the invention, including the scope of patent applications, which is limited to such examples. For example, although embodiments of the HBV vectors (such as plastid DNA or viral vectors) of the present application described herein may contain specific components arranged in a specific order, including (but not limited to) certain promoter sequences, Enhancers or regulatory sequences, signal peptides, HBV antigen coding sequences, polyadenylation signal sequences, etc., but those skilled in the art should generally understand that the concepts disclosed herein can be equally applied to HBV that can be used in this application. Other components arranged in other sequences used in the carrier. This application covers the use of any of the applicable components in any combination with any sequence that can be used in the HBV vector of this application, whether or not a particular combination is explicitly described.

BB 型肝炎病毒Hepatitis virus (( HBVHBV ))

如本文所使用，「B型肝炎病毒」或「HBV」係指肝DNA病毒科之病毒。HBV係編碼四個開放閱讀框架及七種蛋白質的親肝性小(例如3.2 kb)DNA病毒。參見圖 1A 。HBV編碼之七種蛋白質包括小(S)、中等(M)及大(L)表面抗原(HBsAg)或包膜(Env)蛋白質、前核心蛋白、核心蛋白、病毒聚合酶(Pol)及HBx蛋白質。HBV表現三種表面抗原，或包膜蛋白，即L、M及S，其中S最小且L最大。M及L蛋白質中之額外結構域分別命名為前S2及前S1。核心蛋白係病毒核衣殼之次單元。Pol係合成病毒DNA(逆轉錄酶、核糖核酸酶H及引子)所需的，其出現在定位於受感染肝細胞之細胞質的核衣殼中。前核心蛋白係具有N末端信號肽之核心蛋白且在自受感染細胞分泌之前，在其N及C末端經歷蛋白水解加工為所謂的B型肝炎e抗原(HBeAg)。HBx蛋白質係共價閉合環狀DNA(cccDNA)高效轉錄所需的。HBx並非病毒結構蛋白。除核心及聚合酶共有mRNA外，HBV之所有病毒蛋白均具有其自身mRNA。除前核心蛋白之外，該等HBV病毒蛋白均不經歷轉譯後蛋白質水解加工。As used herein, "hepatitis B virus" or "HBV" refers to a virus of the hepatic DNA virus family. The HBV line encodes four open reading frames and seven hepatophilic (eg, 3.2 kb) DNA viruses of seven proteins. See Figure 1A . Seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-core proteins, core proteins, viral polymerases (Pol), and HBx proteins . HBV displays three surface antigens, or envelope proteins, namely L, M, and S, with S being the smallest and L being the largest. The extra domains in the M and L proteins are named pre-S2 and pre-S1, respectively. The core protein is a subunit of the viral nucleocapsid. Pol is required for the synthesis of viral DNA (reverse transcriptase, ribonuclease H, and primers) and appears in the nucleocapsid that is localized to the cytoplasm of infected liver cells. Procorein is a core protein that has an N-terminal signal peptide and undergoes proteolytic processing at its N and C-termini to the so-called hepatitis B e antigen (HBeAg) before being secreted from infected cells. HBx proteins are required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA, except for the core and polymerase shared mRNAs. With the exception of pre-core proteins, none of these HBV viral proteins undergo post-translational proteolytic processing.

HBV病毒粒子含有病毒包膜、核衣殼，及部分呈雙股之DNA基因組的單一複本。核衣殼包含120個核心蛋白二聚體且經內嵌有S、M及L病毒包膜或表面抗原蛋白質之衣殼膜覆蓋。在進入細胞之後，病毒去殼且與病毒聚合酶共價結合的含衣殼之鬆環DNA(rcDNA)遷移至核中。在該過程期間，核心蛋白磷酸化誘導結構變化，暴露出核定位信號，使得衣殼能夠與所謂的內輸蛋白(importin)相互作用。該等內輸蛋白介導核心蛋白與核孔複合物之結合，在該結合後，衣殼解離且聚合酶/rcDNA複合物釋放至核中。在核內，rcDNA變得去蛋白化(移除聚合酶)且經宿主DNA修復機構轉變成共價閉合環狀DNA(cccDNA)基因組，自該基因組，重疊之轉錄本編碼HBeAg、HBsAg、核心蛋白、病毒聚合酶及HBx蛋白質。核心蛋白、病毒聚合酶及前基因組RNA(pgRNA)在細胞質中締合並自組裝成不成熟的含pgRNA之衣殼粒子，該等衣殼粒子進一步轉化成為成熟rcDNA-衣殼且充當共同中間物，經包覆且以感染病毒粒子形式分泌，或轉運回到核中以補充及維持穩定cccDNA池。參見圖 1B 。HBV virions contain a single copy of the virus envelope, nucleocapsid, and partially double-stranded DNA genome. The nucleocapsid contains 120 core protein dimers and is covered by a capsid membrane embedded with S, M and L virus envelopes or surface antigen proteins. After entering the cell, the capsid-containing pine-loop DNA (rcDNA) that is unshelled by the virus and covalently bound to the viral polymerase migrates into the nucleus. During this process, core protein phosphorylation induces structural changes, exposing nuclear localization signals, enabling the capsid to interact with so-called importins. These introproteins mediate the binding of the core protein to the nuclear pore complex, after which the capsid dissociates and the polymerase / rcDNA complex is released into the nucleus. In the nucleus, rcDNA becomes deproteinized (removes polymerase) and is transformed into a covalently closed circular DNA (cccDNA) genome by the host DNA repair mechanism. From this genome, overlapping transcripts encode HBeAg, HBsAg, and core proteins , Viral polymerase and HBx protein. Core protein, viral polymerase and pre-genomic RNA (pgRNA) are associated and self-assembled into immature pgRNA-containing capsid particles in the cytoplasm. These capsid particles are further transformed into mature rcDNA-capsids and serve as common intermediates. Coated and secreted as infectious virions, or transported back to the nucleus to replenish and maintain a stable cccDNA pool. See Figure 1B .

迄今為止，HBV基於包膜蛋白上存在之抗原性抗原決定基而分成四種血清型(adr、adw、ayr、ayw)，且基於病毒基因組之序列而分成八種基因型(A、B、C、D、E、F、G及H)。HBV基因型分佈於不同地理區域。舉例而言，亞洲最流行之基因型係基因型B及C。基因型D主要存在於非洲、中東及印度，而基因型A在北歐、撒哈拉沙漠以南非洲(sub-Saharan Africa)及西非較為普遍。So far, HBV has been divided into four serotypes (adr, adw, ayr, ayw) based on the antigenic epitopes present on the envelope protein, and eight genotypes (A, B, C) based on the sequence of the viral genome. , D, E, F, G, and H). HBV genotypes are distributed in different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is mainly found in Africa, the Middle East, and India, while genotype A is more common in Northern Europe, sub-Saharan Africa, and West Africa.

HBVHBV 抗原antigen

如本文所使用，術語「HBV抗原」、「HBV之抗原性多肽」、「HBV抗原性多肽」、「HBV抗原蛋白質」、「HBV免疫原性多肽」及「HBV免疫原」皆指能夠在個體中誘發針對HBV之免疫反應，例如體液及/或細胞介導之反應的多肽。HBV抗原可以為HBV多肽、其片段或抗原決定基，或多個HBV多肽、其部分或衍生物之組合。HBV抗原能夠在宿主中產生保護性免疫反應，例如誘發針對病毒性疾病或感染之免疫反應，及/或在個體中針對病毒性疾病或感染產生免疫(亦即，接種疫苗)，由此保護個體免受病毒性疾病或感染影響。舉例而言，HBV抗原可以包含來自來源於任何HBV基因型，例如基因型A、B、C、D、E、F、G及/或H之任何HBV蛋白質，諸如HBeAg、前核心蛋白、HBsAg(S、M或L蛋白質)、核心蛋白、病毒聚合酶或HBx蛋白質，或其組合的多肽或其免疫原性片段。As used herein, the terms "HBV antigen", "HBV antigenic polypeptide", "HBV antigenic polypeptide", "HBV antigen protein", "HBV immunogenic polypeptide" and "HBV immunogen" all refer to the ability to Polypeptide that induces an immune response against HBV, such as a humoral and / or cell-mediated response. The HBV antigen may be a HBV polypeptide, a fragment or an epitope thereof, or a combination of a plurality of HBV polypeptides, portions or derivatives thereof. HBV antigens can protect individuals by generating a protective immune response in the host, such as by eliciting an immune response against a viral disease or infection, and / or by generating immunity (ie, vaccinating) against a viral disease or infection in an individual Free from viral diseases or infections. For example, the HBV antigen may comprise any HBV protein from any HBV genotype, such as genotypes A, B, C, D, E, F, G, and / or H, such as HBeAg, precore protein, HBsAg S, M or L protein), core protein, viral polymerase or HBx protein, or a combination of polypeptides or immunogenic fragments thereof.

(1) HBV(1) HBV 核心抗原Core antigen

如本文所使用，術語「HBV核心抗原」、「HBcAg」及「核心抗原」均係指能夠在個體中誘發針對HBV核心蛋白之免疫反應，例如體液及/或細胞介導之反應的HBV抗原。術語「核心」、「核心多肽」及「核心蛋白」均係指HBV病毒核心蛋白。全長核心抗原通常係183個胺基酸長度且包括組裝結構域(胺基酸1至149)及核酸結合結構域(胺基酸150至183)。該34個殘基之核酸結合結構域係前基因組RNA衣殼化所需的。此結構域亦用作核輸入信號。其包含17個精胺酸殘基且具有較高鹼性，與其功能相符。HBV核心蛋白在溶液中呈二聚體形式，且該等二聚體自組裝成二十面體衣殼。每個核心蛋白二聚體具有在任一側上側接一個α-螺旋結構域的四個α-螺旋束。不含該核酸結合結構域的截短之HBV核心蛋白亦能夠形成衣殼。As used herein, the terms "HBV core antigen", "HBcAg", and "core antigen" all refer to HBV antigens capable of eliciting an immune response against an HBV core protein, such as a humoral and / or cell-mediated response in an individual. The terms "core", "core polypeptide" and "core protein" all refer to the HBV virus core protein. The full-length core antigen is usually 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). This 34-residue nucleic acid binding domain is required for capsidization of pregenomic RNA. This domain is also used as a nuclear input signal. It contains 17 arginine residues and is highly basic, consistent with its function. The HBV core protein is in the form of a dimer in solution, and the dimers self-assemble into an icosahedral capsid. Each core protein dimer has four alpha-helical bundles flanked by an alpha-helical domain on either side. A truncated HBV core protein without the nucleic acid binding domain can also form a capsid.

在本申請案之一個實施例中，HBV抗原係截短之HBV核心抗原。如本文所使用，「截短之HBV核心抗原」係指不含完整長度之HBV核心蛋白但能夠在個體中誘發針對HBV核心蛋白之免疫反應的HBV抗原。舉例而言，HBV核心抗原可以經修飾成使核心抗原中通常含有十七個精胺酸(R)殘基的帶大量正電荷(富含精胺酸)之C末端核酸結合結構域的一或多個胺基酸缺失。本申請案的截短之HBV核心抗原較佳為不包含HBV核心核輸入信號的C末端截短之HBV核心蛋白及/或已缺失C末端HBV核心核輸入信號的截短之HBV核心蛋白。在一個實施例中，截短之HBV核心抗原包含在C末端核酸結合結構域中之缺失，諸如缺失該C末端核酸結合結構域之1至34個胺基酸殘基，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33或34個胺基酸殘基，較佳缺失全部34個胺基酸殘基。在一個較佳實施例中，截短之HBV核心抗原包含C末端核酸結合結構域中之缺失，較佳缺失全部34個胺基酸殘基。In one embodiment of the present application, the HBV antigen is a truncated HBV core antigen. As used herein, "truncated HBV core antigen" refers to a HBV antigen that does not contain a full-length HBV core protein but is capable of eliciting an immune response against the HBV core protein in an individual. For example, the HBV core antigen can be modified so that the core antigen typically contains seventeen spermine (R) residues, one or Multiple amino acids are missing. The truncated HBV core antigen of the present application is preferably a C-terminal truncated HBV core protein that does not contain the HBV core nuclear input signal and / or a truncated HBV core protein that has been deleted from the C-terminal HBV core nuclear input signal. In one embodiment, the truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues in the C-terminal nucleic acid binding domain, such as 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33 or 34 amino acid residues, preferably all 34 amino acid residues are deleted. In a preferred embodiment, the truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, preferably all 34 amino acid residues are deleted.

本申請案之HBV核心抗原可以為來源於多種HBV基因型(例如基因型A、B、C、D、E、F、G及H)之共同序列。如本文所使用，「共同序列」意謂基於同源蛋白質之胺基酸序列比對，例如藉由比對(例如使用Clustal Omega)同源蛋白質之胺基酸序列所測定的人工胺基酸序列。其可以為基於來自至少100個天然HBV分離株之HBV抗原(例如核心、pol等)之序列計算的在序列比對中各位置處所發現的最常見胺基酸殘基之次序。共同序列可以為非天然存在的且不同於原生病毒序列。共同序列可以藉由使用多序列比對工具比對來自不同來源之多個HBV抗原序列，且在有變化之比對位置選擇最常見的胺基酸來設計。較佳地，HBV抗原之共同序列係來源於HBV基因型B、C及D。術語「共同抗原」用以指具有共同序列之抗原。The HBV core antigen of the present application may be a common sequence derived from multiple HBV genotypes (eg, genotypes A, B, C, D, E, F, G, and H). As used herein, "common sequence" means an amino acid sequence alignment based on homologous proteins, such as an artificial amino acid sequence determined by aligning (eg, using Clustal Omega) amino acid sequences of homologous proteins. It can be the order of the most common amino acid residues found at various positions in the sequence alignment, calculated based on sequences of HBV antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates. The common sequence may be non-naturally occurring and different from the native viral sequence. The common sequence can be designed by using multiple sequence alignment tools to align multiple HBV antigen sequences from different sources, and selecting the most common amino acid at the position where the alignment is changed. Preferably, the common sequence of HBV antigens is derived from HBV genotypes B, C and D. The term "common antigen" is used to refer to antigens having a common sequence.

根據本申請案的例示性截短之HBV核心抗原不具有核酸結合功能，且能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應。較佳地，截短之HBV核心抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應。更佳地，截短之HBV核心抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。The exemplary truncated HBV core antigen according to the present application does not have a nucleic acid binding function and is capable of eliciting an immune response in mammals against at least two HBV genotypes. Preferably, the truncated HBV core antigen is capable of inducing a T-cell response in mammals against at least HBV genotypes B, C, and D. More preferably, the truncated HBV core antigen is capable of eliciting a CD8 T cell response against at least HBV genotypes A, B, C, and D in a human individual.

較佳地，本申請案之HBV核心抗原係共同抗原，較佳為來源於HBV基因型B、C及D之共同抗原，更佳為來源於HBV基因型B、C及D的截短之共同抗原。根據本申請案的例示性截短之HBV核心共同抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，諸如與SEQ ID NO: 2或SEQ ID NO: 14至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致之胺基酸序列組成。SEQ ID NO: 2及SEQ ID NO: 14係來源於HBV基因型B、C及D之核心共同抗原。SEQ ID NO: 2及SEQ ID NO: 14缺失天然核心抗原中C末端帶大量正電(富含精胺酸)之核酸結合結構域的34個胺基酸。Preferably, the HBV core antigen of the present application is a common antigen, preferably a common antigen derived from HBV genotypes B, C, and D, and more preferably a truncated common antigen derived from HBV genotypes B, C, and D. antigen. Exemplary truncated HBV core common antigens according to the present application are at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, such as at least 90%, 91 corresponding to SEQ ID NO: 2 or SEQ ID NO: 14, 91 %, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical amino acid sequence composition. SEQ ID NO: 2 and SEQ ID NO: 14 are core common antigens derived from HBV genotypes B, C, and D. SEQ ID NO: 2 and SEQ ID NO: 14 delete the 34 amino acids in the C-terminus of the natural core antigen with a large number of positively charged (sinine-rich) nucleic acid binding domains.

在本申請案之一個特定實施例中，HBV核心抗原係由SEQ ID NO: 2之胺基酸序列組成的截短之HBV抗原。在另一特定實施例中，HBV核心抗原係由SEQ ID NO: 14之胺基酸序列組成的截短之HBV抗原。In a specific embodiment of the present application, the HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 2. In another specific embodiment, the HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 14.

(2) HBV(2) HBV 聚合酶抗原Polymerase antigen

如本文所使用，術語「HBV聚合酶抗原」、「HBV Pol抗原」或「HBV pol抗原」係指能夠在個體中誘發針對HBV聚合酶的免疫反應，例如體液及/或細胞介導之反應的HBV抗原。術語「聚合酶」、「聚合酶多肽」、「Pol」及「pol」均係指HBV病毒DNA聚合酶。HBV病毒DNA聚合酶具有四個結構域，自N末端至C末端，包括充當負股DNA合成之引子的末端蛋白質(TP)結構域；對於聚合酶功能不重要的間隔子；用於轉錄之逆轉錄酶(RT)結構域；以及核糖核酸酶H結構域。As used herein, the terms "HBV polymerase antigen", "HBV Pol antigen" or "HBV pol antigen" refer to those capable of eliciting an immune response, such as a humoral and / or cell-mediated response, to HBV polymerase in an individual. HBV antigen. The terms "polymerase", "polymerase polypeptide", "Pol" and "pol" all refer to HBV virus DNA polymerase. HBV viral DNA polymerase has four domains, from the N-terminus to the C-terminus, including the terminal protein (TP) domain, which serves as a primer for negative-strand DNA synthesis; spacers that are not important for polymerase function; reverse for transcription Transcriptase (RT) domain; and ribonuclease H domain.

在本申請案之一個實施例中，HBV抗原包含HBV Pol抗原，或其任何免疫原性片段或組合。HBV Pol抗原可以含有改善該抗原之免疫原性的其他修飾，諸如藉由將突變引入聚合酶及/或核糖核酸酶結構域之活性位點中以降低或基本上除去某些酶活性。In one embodiment of the application, the HBV antigen comprises a HBV Pol antigen, or any immunogenic fragment or combination thereof. The HBV Pol antigen may contain other modifications that improve the immunogenicity of the antigen, such as by introducing mutations into the active site of the polymerase and / or ribonuclease domains to reduce or substantially remove certain enzyme activities.

較佳地，本申請案之HBV Pol抗原不具有逆轉錄酶活性及核糖核酸酶H活性，且能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應。較佳地，HBV Pol抗原能夠在哺乳動物中發針對至少HBV基因型B、C及D之T細胞反應。更佳地，HBV Pol抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。Preferably, the HBV Pol antigen of the present application does not have reverse transcriptase activity and ribonuclease H activity, and can induce an immune response in mammals against at least two HBV genotypes. Preferably, the HBV Pol antigen is capable of eliciting a T-cell response in mammals against at least HBV genotypes B, C, and D. More preferably, the HBV Pol antigen is capable of eliciting a CD8 T cell response against at least HBV genotypes A, B, C, and D in a human individual.

因此，在一些實施例中，HBV Pol抗原係失活之Pol抗原。在一個實施例中，失活之HBV Pol抗原在聚合酶結構域之活性位點中包含一或多個胺基酸突變。在另一個實施例中，失活之HBV Pol抗原在核糖核酸酶H結構域之活性位點中包含一或多個胺基酸突變。在一個較佳實施例中，失活之HBV pol抗原在聚合酶結構域及核糖核酸酶H結構域兩者之活性位點中包含一或多個胺基酸突變。舉例而言，可以例如藉由用天冬醯胺殘基(N)置換一或多個天冬胺酸殘基(D)，消除或降低金屬配位功能，由此降低或基本上消除逆轉錄酶功能來使HBV pol抗原之聚合酶結構域中核苷酸/金屬離子結合所需之「YXDD」基元突變。作為「YXDD」基元突變之替代或除該突變之外，可以例如藉由用天冬醯胺殘基(N)置換一或多個天冬胺酸殘基(D)及/或用麩醯胺酸(Q)置換麩胺酸殘基(E)，由此降低或基本上消除核糖核酸酶H功能來使HBV pol抗原之核糖核酸酶H結構域中Mg² ⁺ 配位所需之「DEDD」基元突變。在一個特定實施例中，HBV pol抗原係藉由以下方式修飾：(1)使聚合酶結構域之「YXDD」基元中的天冬胺酸殘基(D)突變成天冬醯胺殘基(N)；以及(2)使核糖核酸酶H結構域之「DEDD」基元中的第一個天冬胺酸殘基(D)突變成天冬醯胺殘基(N)及使第一個麩胺酸殘基(E)突變成麩醯胺酸殘基(N)，由此降低或基本上消除pol抗原之逆轉錄酶及核糖核酸酶H功能。Thus, in some embodiments, the HBV Pol antigen is an inactivated Pol antigen. In one embodiment, the inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the polymerase domain. In another embodiment, the inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the ribonuclease H domain. In a preferred embodiment, the inactivated HBV pol antigen contains one or more amino acid mutations in the active site of both the polymerase domain and the ribonuclease H domain. For example, reverse transcription can be reduced or substantially eliminated, for example, by replacing one or more asparagine residues (D) with asparagine residues (N) to eliminate or reduce metal coordination functions. Enzyme function to mutate the "YXDD" motif required for nucleotide / metal ion binding in the polymerase domain of the HBV pol antigen. As an alternative to or in addition to the "YXDD" motif mutation, for example, by replacing one or more aspartic acid residues (D) with asparagine residues (N) and / or with gluten leucine (Q) substitutions glutamic acid residue (E), thereby reducing or substantially eliminate the RNase H function to make the desired Mg ² ⁺ ligand RNase H domain antigen of HBV pol "DEDD Primitive mutation. In a specific embodiment, the HBV pol antigen system is modified by: (1) mutating an asparagine residue (D) in the "YXDD" motif of the polymerase domain to an asparagine residue ( N); and (2) mutating the first asparagine residue (D) in the "DEDD" motif of the ribonuclease H domain to the asparagine residue (N) and making the first bran The amino acid residue (E) is mutated to the glutamate residue (N), thereby reducing or substantially eliminating the reverse transcriptase and ribonuclease H functions of the pol antigen.

在本申請案的一個較佳實施例中，HBV pol抗原係共同抗原，較佳為來源於HBV基因型B、C及D之共同抗原，更佳為來源於HBV基因型B、C及D的失活之共同抗原。根據本申請案的例示性HBV pol共同抗原包含與SEQ ID NO: 4至少90%一致，諸如與SEQ ID NO: 4至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 4至少98%一致，諸如與SEQ ID NO: 4至少98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致的胺基酸序列。SEQ ID NO: 4係在聚合酶及核糖核酸酶H結構域之活性位點中包含四個突變的來源於HBV基因型B、C及D之pol共同抗原。詳言之，該四個突變包括聚合酶結構域之「YXDD」基元中的天冬胺酸殘基(D)突變成天冬醯胺殘基(N)；以及核糖核酸酶H結構域之「DEDD」基元中的第一個天冬胺酸殘基(D)突變成天冬醯胺殘基(N)及麩胺酸殘基(E)突變成麩醯胺酸殘基(Q)。In a preferred embodiment of the present application, the HBV pol antigen is a common antigen, preferably a common antigen derived from HBV genotypes B, C, and D, and more preferably a HBV genotype B, C, or D. Common antigen of inactivation. Exemplary HBV pol common antigens according to the present application contain at least 90% identity to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5, and SEQ ID NO: 4 %, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% consistent, preferably at least 98% consistent with SEQ ID NO: 4, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6 %, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences. SEQ ID NO: 4 is a pol common antigen derived from HBV genotypes B, C, and D that contains four mutations in the active sites of the polymerase and ribonuclease H domains. Specifically, the four mutations include mutations in the asparagine residue (D) in the "YXDD" motif of the polymerase domain to asparagine residue (N); and the " The first aspartic acid residue (D) in the "DEDD" motif is mutated to asparagine residue (N) and glutamic acid residue (E) to glutamic acid residue (Q).

在本申請案之一個特定實施例中，HBV pol抗原包含SEQ ID NO: 4之胺基酸序列。在本申請案之其他實施例中，HBV pol抗原由SEQ ID NO: 4之胺基酸序列組成。In a specific embodiment of the present application, the HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 4. In other embodiments of the present application, the HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 4.

(3) HBV(3) HBV 核心抗原與Core antigen and HBVHBV 聚合酶抗原之融合物Polymerase antigen fusion

如本文所使用，術語「融合蛋白」或「融合物」係指具有至少兩個通常不存在於單一天然多肽中之多肽結構域的單一多肽鏈。As used herein, the term "fusion protein" or "fusion" refers to a single polypeptide chain having at least two polypeptide domains that are not normally present in a single natural polypeptide.

在本申請案之一個實施例中，HBV抗原包括包含截短之HBV核心抗原可操作地連接至HBV Pol抗原或HBV Pol抗原可操作地連接至截短之HBV核心抗原的融合蛋白，該連接較佳經由連接子進行。In one embodiment of the present application, the HBV antigen includes a fusion protein comprising a truncated HBV core antigen operably linked to the HBV Pol antigen or a HBV Pol antigen operably linked to the truncated HBV core antigen, the linkage being This is preferably done via a linker.

如本文所使用，術語「連接子」係指充當分子橋以可操作地連接兩種不同分子的化合物或部分，其中該連接子之一部分可操作地連接至第一分子，且其中該連接子之另一部分可操作地連接至第二分子。舉例而言，在含有第一多肽及第二異源多肽之融合蛋白中，連接子主要用作該第一與第二多肽之間的間隔子。在一個實施例中，連接子係由經肽鍵連接在一起的胺基酸構成，較佳由經肽鍵連接的1至20個胺基酸構成，其中胺基酸係選自20種天然存在之胺基酸。在一個實施例中，該1至20個胺基酸係選自甘胺酸、丙胺酸、脯胺酸、天冬醯胺、麩醯胺酸及離胺酸。較佳地，連接子係由大量無空間位阻之胺基酸，諸如甘胺酸及丙胺酸構成。例示性連接子係聚甘胺酸，尤其是(Gly)₅ 、(Gly)₈ ；聚(Gly-Ala)及聚丙胺酸。如以下實例中所示的一種例示性適合連接子係(AlaGly)_n ，其中n係2至5之整數。As used herein, the term "linker" refers to a compound or moiety that acts as a molecular bridge to operably link two different molecules, where a portion of the linker is operably linked to a first molecule, and wherein the linker The other part is operably linked to the second molecule. For example, in a fusion protein containing a first polypeptide and a second heterologous polypeptide, a linker is mainly used as a spacer between the first and second polypeptides. In one embodiment, the linker system is composed of amino acids linked together via peptide bonds, preferably from 1 to 20 amino acids linked via peptide bonds, wherein the amino acid system is selected from 20 naturally occurring Amino acids. In one embodiment, the 1 to 20 amino acids are selected from the group consisting of glycine, alanine, proline, asparagine, glutamine and lysine. Preferably, the linker system is composed of a large number of sterically hindered amino acids such as glycine and alanine. Exemplary linkers are polyglycine, especially (Gly) ₅ , (Gly) ₈ ; poly (Gly-Ala), and polyalanine. An illustrative suitable linker line (AlaGly) _n is shown in the examples below, where n is an integer from 2 to 5.

較佳地，本申請案之融合蛋白能夠在哺乳動物中誘發針對至少兩種HBV基因型之HBV核心及HBV Pol的免疫反應。較佳地，融合蛋白能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應。更佳地，該融合蛋白能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。Preferably, the fusion protein of the present application is capable of eliciting an immune response in mammals against the HBV core and HBV Pol of at least two HBV genotypes. Preferably, the fusion protein is capable of eliciting a T-cell response against at least HBV genotypes B, C, and D in a mammal. More preferably, the fusion protein is capable of eliciting a CD8 T cell response against at least HBV genotypes A, B, C, and D in a human individual.

在本申請案之一個實施例中，融合蛋白包含具有與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，諸如至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致之胺基酸序列的截短之HBV核心抗原；連接子；及具有與SEQ ID NO: 4至少90%一致，諸如至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致之胺基酸序列的HBV Pol抗原。In one embodiment of the present application, the fusion protein comprises a protein having at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 14, such as at least 90%, 91%, 92%, 93%, 94%, 95% , 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9 Truncated HBV core antigen with a% or 100% identical amino acid sequence; a linker; and having at least 90% identity to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94% , 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8 %, 99.9%, or 100% identical HBV Pol antigens.

在本申請案的一個較佳實施例中，融合蛋白包含由SEQ ID NO: 14之胺基酸序列組成之截短之HBV核心抗原；包含(AlaGly)_n 之連接子，其中n係2至5之整數；及具有SEQ ID NO: 4之胺基酸序列的HBV Pol抗原。更佳地，根據本申請案之一個實施例的融合蛋白包含SEQ ID NO: 20之胺基酸序列。In a preferred embodiment of the present application, the fusion protein comprises a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 14; a linker comprising (AlaGly) _n , where n is 2 to 5 An integer; and a HBV Pol antigen having an amino acid sequence of SEQ ID NO: 4. More preferably, the fusion protein according to one embodiment of the present application comprises the amino acid sequence of SEQ ID NO: 20.

在本申請案之一個實施例中，融合蛋白進一步包含信號序列。較佳地，信號序列具有SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列。更佳地，融合蛋白包含SEQ ID NO: 21之胺基酸序列。In one embodiment of the present application, the fusion protein further comprises a signal sequence. Preferably, the signal sequence has an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19. More preferably, the fusion protein comprises the amino acid sequence of SEQ ID NO: 21.

聚核苷酸及載體Polynucleotides and vectors

在另一通用態樣中，本申請案提供編碼根據本申請案之HBV抗原的非天然存在之核酸分子，及包含該非天然存在之核酸的載體。非天然存在之核酸分子可以包含編碼本申請案之HBV抗原的任何聚核苷酸序列，其可以根據本發明，使用此項技術中已知之方法製備。較佳地，聚核苷酸編碼本申請案之截短之HBV核心抗原及HBV聚合酶抗原中的至少一種。聚核苷酸可以呈藉由重組技術(例如選殖)獲得或合成製造(例如化學合成)之RNA形式或DNA形式。DNA可以為單股或雙股的，或者可以含有雙股及單股序列之部分。DNA可以例如包含基因組DNA、cDNA或其組合。聚核苷酸亦可為DNA/RNA雜合體。本申請案之聚核苷酸及載體可以用於製造重組蛋白、在宿主細胞中表現蛋白質或製造病毒粒子。較佳地，聚核苷酸係DNA。In another general aspect, the present application provides a non-naturally occurring nucleic acid molecule encoding a HBV antigen according to the present application, and a vector comprising the non-naturally occurring nucleic acid. Non-naturally occurring nucleic acid molecules may comprise any polynucleotide sequence encoding the HBV antigen of the present application, which may be prepared according to the present invention using methods known in the art. Preferably, the polynucleotide encodes at least one of the truncated HBV core antigen and the HBV polymerase antigen of the present application. Polynucleotides can be in the form of RNA or DNA obtained by recombinant techniques (e.g., breeding) or synthetically produced (e.g., chemical synthesis). DNA can be single-stranded or double-stranded, or it can contain portions of double-stranded and single-stranded sequences. The DNA may, for example, comprise genomic DNA, cDNA, or a combination thereof. Polynucleotides can also be DNA / RNA hybrids. The polynucleotides and vectors of the present application can be used to make recombinant proteins, express proteins in host cells, or make virions. Preferably, the polynucleotide is DNA.

在本申請案之一個實施例中，非天然存在之核酸分子包含編碼截短之HBV核心抗原的聚核苷酸，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，諸如與SEQ ID NO: 2至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 2或SEQ ID NO: 14有98%、99%或100%一致的胺基酸序列組成。在本申請案之一個特定實施例中，非天然存在之核酸分子編碼由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原。In one embodiment of the present application, the non-naturally occurring nucleic acid molecule comprises a polynucleotide encoding a truncated HBV core antigen, and the truncated HBV core antigen is composed of SEQ ID NO: 2 or SEQ ID NO: 14 At least 90% consistent, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5, and SEQ ID NO: 2 %, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably the same as SEQ ID NO: 2 or SEQ ID NO: 14 consists of 98%, 99% or 100% identical amino acid sequences. In a specific embodiment of the present application, the non-naturally occurring nucleic acid molecule encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14.

本申請案的編碼由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原之聚核苷酸序列的實例包括(但不限於)與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致，諸如與SEQ ID NO: 1或SEQ ID NO: 15至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 1或SEQ ID NO: 15有98%、99%或100%一致之聚核苷酸序列。編碼截短之HBV核心抗原的例示性非天然存在之核酸分子具有SEQ ID NO:1或15之聚核苷酸序列。Examples of polynucleotide sequences encoding truncated HBV core antigens consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14 in this application include, but are not limited to, SEQ ID NO: 1 Or at least 90% identical to SEQ ID NO: 15, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5 %, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% are consistent, preferably A polynucleotide sequence that is 98%, 99%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15. An exemplary non-naturally occurring nucleic acid molecule encoding a truncated HBV core antigen has a polynucleotide sequence of SEQ ID NO: 1 or 15.

在本申請案之一個實施例中，非天然存在之核酸分子編碼HBV聚合酶抗原，該HBV聚合酶抗原包含與SEQ ID NO: 4至少90%一致，諸如與SEQ ID NO: 4至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 4達100%一致的胺基酸序列。在本申請案之一個特定實施例中，非天然存在之核酸分子編碼由SEQ ID NO: 4之胺基酸序列組成的HBV聚合酶抗原。In one embodiment of the present application, the non-naturally occurring nucleic acid molecule encodes an HBV polymerase antigen, which comprises at least 90% identity to SEQ ID NO: 4, such as at least 90% identity to SEQ ID NO: 4, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4% , 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% of the amino acid sequence, preferably 100% of the amino acid sequence of SEQ ID NO: 4. In a specific embodiment of the present application, the non-naturally occurring nucleic acid molecule encodes a HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 4.

本申請案的編碼包含SEQ ID NO: 4之胺基酸序列之HBV Pol抗原的聚核苷酸序列之實例包括(但不限於)與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致，諸如與SEQ ID NO: 3或SEQ ID NO: 16至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 3或SEQ ID NO: 16有98%、99%或100%一致的聚核苷酸序列。編碼HBV pol抗原的例示性非天然存在之核酸分子具有SEQ ID NO:3或16之聚核苷酸序列。Examples of polynucleotide sequences encoding the HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 4 in this application include, but are not limited to, at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16 , Such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98% with SEQ ID NO: 3 or SEQ ID NO: 16 , 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably consistent with SEQ ID NO: 3 or SEQ ID NO: 16 has 98%, 99%, or 100% identical polynucleotide sequences. An exemplary non-naturally occurring nucleic acid molecule encoding a HBV pol antigen has a polynucleotide sequence of SEQ ID NO: 3 or 16.

在本申請案之另一實施例中，非天然存在之核酸分子編碼包含截短之HBV核心抗原可操作地連接至HBV Pol抗原或HBV Pol抗原可操作地連接至截短之HBV核心抗原的融合蛋白，該連接較佳經由連接子進行。在一個特定實施例中，本申請案之非天然存在之核酸分子編碼：截短之HBV核心抗原，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，諸如與SEQ ID NO: 2或SEQ ID NO: 14至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 2或SEQ ID NO: 14達100%一致，更佳與SEQ ID NO: 14達100%一致的胺基酸序列組成；連接子；以及HBV聚合酶抗原，該HBV聚合酶抗原包含與SEQ ID NO: 4至少90%一致，諸如與SEQ ID NO: 4至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 4有98%、99%或100%一致的胺基酸序列。在本申請案之一個特定實施例中，非天然存在之核酸分子編碼融合蛋白，該融合蛋白包含由SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原；包含(AlaGly)_n 之連接子，其中n係2至5之整數；以及包含SEQ ID NO: 4之胺基酸序列的HBV Pol抗原。在本申請案之一個特定實施例中，非天然存在之核酸分子編碼包含SEQ ID NO: 20之胺基酸序列的融合蛋白。In another embodiment of the present application, a non-naturally occurring nucleic acid molecule encodes a fusion comprising a truncated HBV core antigen operably linked to a HBV Pol antigen or a HBV Pol antigen operably linked to a truncated HBV core antigen Protein, this connection is preferably made via a linker. In a specific embodiment, the non-naturally occurring nucleic acid molecule of the present application encodes: a truncated HBV core antigen, which is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14 , Such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98% with SEQ ID NO: 2 or SEQ ID NO: 14 , 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably consistent with SEQ ID NO: 2 or SEQ ID NO: 14 up to 100% identical, more preferably amino acid sequence composition identical to SEQ ID NO: 14 up to 100% identical; linker; and HBV polymerase antigen comprising at least 90% of SEQ ID NO: 4 % Consistent, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, SEQ ID NO: 4, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably 98%, 99%, or SEQ ID NO: 4 100% consistent amino acid sequence. In a specific embodiment of the present application, a non-naturally occurring nucleic acid molecule encodes a fusion protein comprising a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 14; comprising (AlaGly) _n A linker in which n is an integer from 2 to 5; and a HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 4. In a specific embodiment of the present application, the non-naturally occurring nucleic acid molecule encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

本申請案的編碼融合蛋白之聚核苷酸序列的實例包括(但不限於)與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致，諸如與SEQ ID NO: 1或SEQ ID NO: 15至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 1或SEQ ID NO: 15有98%、99%或100%一致的聚核苷酸序列可操作地連接至與SEQ ID NO: 22至少90%一致，諸如與SEQ ID NO: 22至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 22有98%、99%或100%一致的連接子編碼序列，該連接子編碼序列進一步可操作地連接至與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致，諸如與SEQ ID NO: 3或SEQ ID NO: 16至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳地與SEQ ID NO: 3或SEQ ID NO: 16有98%、99%或100%一致的聚核苷酸序列。在本申請案之特定實施例中，非天然存在之核酸分子編碼融合蛋白，該融合蛋白包含SEQ ID NO: 1或SEQ ID NO: 15可操作地連接至SEQ ID NO: 22，SEQ ID NO: 22進一步可操作地連接至SEQ ID NO: 3或SEQ ID NO: 16。Examples of a polynucleotide sequence encoding a fusion protein of the present application include, but are not limited to, being at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15 such as, for example, SEQ ID NO: 1 or SEQ ID NO: 15 At least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably 98%, 99%, or 100% consistent with SEQ ID NO: 1 or SEQ ID NO: 15 The polynucleotide sequence is operably linked to be at least 90% identical to SEQ ID NO: 22, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5% of SEQ ID NO: 22 , 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100 % Consistent, preferably 98%, 99%, or 100% identical linker coding sequence, which is further operably linked to SEQ ID NO: 3 or SEQ ID NO: 22 16 is at least 90% consistent, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, SEQ ID NO: 3 or SEQ ID NO: 16 97.5%, 98%, 9 8.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably with SEQ ID NO: 3 or SEQ ID NO : 16 has 98%, 99% or 100% identical polynucleotide sequences. In a specific embodiment of the present application, the non-naturally occurring nucleic acid molecule encodes a fusion protein comprising SEQ ID NO: 1 or SEQ ID NO: 15 operably linked to SEQ ID NO: 22, SEQ ID NO: 22 is further operably linked to SEQ ID NO: 3 or SEQ ID NO: 16.

本申請案亦係關於一種載體，其包含編碼HBV抗原的分離之聚核苷酸。如本文所使用，「載體」係用於將遺傳物質載運至另一細胞中的核酸分子，在該另一細胞中，其可以複製及/或表現。根據本發明，熟習此項技術者已知之任何載體均可以使用。載體之實例包括(但不限於)質體、病毒載體(噬菌體、動物病毒及植物病毒)、黏質體及人工染色體(例如YAC)。較佳地，載體係DNA質體。載體可以為DNA載體或RNA載體。一般熟習此項技術者可以根據本發明，經由標準重組技術構築本申請案之載體。The present application also relates to a vector comprising an isolated polynucleotide encoding an HBV antigen. As used herein, a "vector" is a nucleic acid molecule used to carry genetic material into another cell where it can be replicated and / or expressed. According to the present invention, any carrier known to those skilled in the art can be used. Examples of vectors include, but are not limited to, plastids, viral vectors (phages, animal viruses, and plant viruses), plastids, and artificial chromosomes (eg, YAC). Preferably, the vector is a DNA plastid. The vector may be a DNA vector or an RNA vector. Those skilled in the art generally can construct the carrier of this application according to the present invention through standard recombination techniques.

本申請案之載體可以為表現載體。如本文所使用，術語「表現載體」係指包含編碼能夠轉錄之RNA之核酸的任何類型之基因構築體。表現載體包括(但不限於)用於重組蛋白表現之載體，諸如DNA質體或病毒載體；及用於將核酸遞送至個體中以在該個體之組織中表現的載體，諸如DNA質體或病毒載體。熟習此項技術者應瞭解，表現載體之設計可取決於諸如待轉型宿主細胞之選擇、所需蛋白質之表現量等因素。The carrier of this application may be a performance carrier. As used herein, the term "expression vector" refers to any type of genetic construct comprising a nucleic acid encoding a RNA capable of transcription. Performance vectors include, but are not limited to, vectors for recombinant protein expression, such as DNA plastids or viral vectors; and vectors for delivery of nucleic acids to an individual for expression in the tissues of the individual, such as DNA plastids or viruses Carrier. Those skilled in the art should understand that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the expression amount of the desired protein, and the like.

本申請案之載體可以含有多種調控序列。如本文所使用，術語「調控序列」係指允許、促成或調節核酸分子之功能調控，包括宿主細胞或生物體中核酸或其一種衍生物(亦即，mRNA)之複製、重複、轉錄、剪接、轉譯、穩定性及/或轉運的任何序列。在本發明的上下文中，此術語涵蓋啟動子、強化子及其他表現控制元件(例如聚腺苷酸化信號及影響mRNA穩定性之元件)。The vector of the present application may contain various regulatory sequences. As used herein, the term "regulatory sequence" refers to allowing, facilitating, or regulating the functional regulation of a nucleic acid molecule, including the replication, duplication, transcription, splicing of a nucleic acid or one of its derivatives (ie, mRNA) in a host cell or organism. , Translation, stability, and / or translocation of any sequence. In the context of the present invention, this term encompasses promoters, enhancers, and other performance control elements (such as polyadenylation signals and elements that affect mRNA stability).

在本申請案之一些實施例中，載體係非病毒載體。非病毒載體之實例包括(但不限於)DNA質體、細菌人工染色體、酵母人工染色體、噬菌體等。非病毒載體之實例包括(但不限於) RNA複製子、mRNA複製子、經修飾之mRNA複製子或自擴增mRNA、閉合線性脫氧核糖核酸，例如線性共價閉合DNA，例如線性共價閉合雙股DNA分子。較佳地，非病毒載體係DNA質體。「DNA質體」與「DNA質體載體」、「質體DNA」或「質體DNA載體」可互換使用，意思指能夠在適合宿主細胞中自主複製的大體上呈圓形的雙股DNA序列。用於表現編碼之聚核苷酸的DNA質體通常包含複製起點、多選殖位點及可選擇標記物，該可選擇標記物例如可以為抗生素抗性基因。可以使用的適合DNA質體之實例包括(但不限於)用於熟知表現系統(包括原核及真核系統兩種)中的可商購之表現載體，諸如pSE420(Invitrogen, San Diego, Calif.)，其可以用於在大腸桿菌中產生及/或表現蛋白質；pYES2(Invitrogen, Thermo Fisher Scientific)，其可以用於在酵母菌株釀酒酵母(Saccharomyces cerevisiae )中產生及/或表現；MAXBAC^® 完全桿狀病毒表現系統(Thermo Fisher Scientific)，其可以用於在昆蟲細胞中產生及/或表現；pcDNA^TM 或pcDNA3^TM (Life Technologies, Thermo Fisher Scientific)，其可以用於在哺乳動物細胞中高程度組成性蛋白質表現；以及pVAX或pVAX-1(Life, Thermo Fisher Scientific)，其可以用於在大部分哺乳動物細胞中高程度短暫表現所關注蛋白質。任何可商購之DNA質體的主鏈均可藉由使用常規技術及容易得到的起始物質進行修飾以使宿主細胞中蛋白質之表現達到最佳，以便逆轉某些元件(例如複製起點及/或抗生素抗性卡匣)之取向，置換質體內源性啟動子(例如抗生素抗性卡匣中之啟動子)，及/或置換編碼轉錄蛋白質之聚核苷酸序列(例如抗生素抗性基因之編碼序列)。(參見例如，Sambrook等人, Molecular Cloning a Laboratory Manual, 第二版, Cold Spring Harbor Press (1989))。In some embodiments of the application, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plastids, bacterial artificial chromosomes, yeast artificial chromosomes, phages, and the like. Examples of non-viral vectors include, but are not limited to, RNA replicons, mRNA replicons, modified mRNA replicons or self-amplifying mRNAs, closed linear DNA, such as linear covalently closed DNA, such as linear covalently closed double Strand DNA molecule. Preferably, the non-viral vector is a DNA plastid. "DNA plastid" is used interchangeably with "DNA plastid vector", "plastid DNA" or "plastid DNA vector" and means a generally circular double-stranded DNA sequence capable of autonomous replication in a suitable host cell . The DNA plastids used to express the encoded polynucleotide generally include an origin of replication, a multiple selection site, and a selectable marker, which may be, for example, an antibiotic resistance gene. Examples of suitable DNA plastids that can be used include, but are not limited to, commercially available expression vectors used in well-known expression systems, including both prokaryotic and eukaryotic systems, such as pSE420 (Invitrogen, San Diego, Calif.) , Which can be used to produce and / or express proteins in E. coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used to produce and / or express in the yeast strain Saccharomyces cerevisiae ; MAXBAC ^® fully rod-shaped Viral Expression System (Thermo Fisher Scientific), which can be used for production and / or expression in insect cells; pcDNA ^TM or pcDNA3 ^TM (Life Technologies, Thermo Fisher Scientific), which can be used for a high degree of constitutive protein in mammalian cells Expression; and pVAX or pVAX-1 (Life, Thermo Fisher Scientific), which can be used to transiently express the protein of interest to a high degree in most mammalian cells. The backbone of any commercially available DNA plastid can be modified by using conventional techniques and readily available starting materials to optimize protein performance in the host cell in order to reverse certain elements (such as the origin of replication and / Or antibiotic resistance cassette), replacing endogenous promoters in plastids (such as promoters in antibiotic resistance cassettes), and / or replacing polynucleotide sequences that encode transcription proteins (such as antibiotic resistance genes). Coding sequence). (See, eg, Sambrook et al., Molecular Cloning a Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)).

較佳地，DNA質體係適於在哺乳動物宿主細胞中表現蛋白質的表現載體。適於在哺乳動物宿主細胞中表現蛋白質的表現載體包括(但不限於)pcDNA^TM 、pcDNA3^TM 、pVAX、pVAX-1、ADVAX、NTC8454等。較佳地，表現載體係基於pVAX-1，其可以進一步經修飾以使哺乳動物細胞中蛋白質之表現達到最佳。pVAX-1係DNA疫苗中之常用質體，且含有較強的人類即刻早期巨細胞病毒(CMV-IE)啟動子，隨後為源於牛生長激素(bGH)之聚腺苷酸化序列(pA)。pVAX-1還含有pUC複製起點及由允許細菌質體繁殖之小原核生物啟動子驅動的卡那黴素抗性基因。Preferably, the DNA plasmonics system is a performance vector suitable for expressing proteins in mammalian host cells. Expression vectors suitable for expressing proteins in mammalian host cells include, but are not limited to, pcDNA ^™ , pcDNA3 ^™ , pVAX, pVAX-1, ADVAX, NTC8454, and the like. Preferably, the expression vector is based on pVAX-1, which can be further modified to optimize protein performance in mammalian cells. pVAX-1 is a commonly used plastid in DNA vaccines and contains a strong human immediate early cytomegalovirus (CMV-IE) promoter followed by a polyadenylation sequence (pA) derived from bovine growth hormone (bGH) . pVAX-1 also contains a pUC origin of replication and a kanamycin resistance gene driven by a small prokaryotic promoter that allows bacterial plastids to multiply.

本申請案之載體亦可為病毒載體。一般而言，病毒載體係載運經修飾病毒DNA或RNA的經遺傳工程改造之病毒，該病毒DNA或RNA已呈現非感染性，但仍含有病毒啟動子及轉殖基因，由此允許經由病毒啟動子轉譯轉殖基因。由於病毒載體常常缺乏感染性序列，故其需要輔助病毒或包裝株來進行大規模轉染。可以使用的病毒載體之實例包括(但不限於)腺病毒載體、腺相關病毒載體、痘病毒載體、腸病毒載體、委內瑞拉馬腦炎病毒(Venezuelan Equine Encephalitis virus)載體、勝利基森林病毒(Semliki Forest Virus)載體、菸草鑲嵌病毒載體、慢病毒載體、沙粒狀病毒(arenavirus)之病毒載體、複製缺陷型沙粒狀病毒之病毒載體或有複製能力的沙粒狀病毒之病毒載體、二片段或三片段沙粒狀病毒、感染性沙粒狀病毒之病毒載體、包含沙粒狀病毒基因組區段且其中該基因組區段之一個開放閱讀框架缺失或功能失活(且經編碼如本文所描述之HBV抗原之核酸置換)的核酸、沙粒狀病毒諸如淋巴細胞性脈絡叢腦膜炎病毒(LCMV)，例如純系13病毒株或MP病毒株，以及沙粒狀病毒諸如胡寧病毒(Junin virus)，例如Candid #1病毒株等。該載體亦可為非病毒載體。The vector in this application may also be a viral vector. Generally speaking, viral vectors are genetically engineered viruses that carry modified viral DNA or RNA. The viral DNA or RNA is non-infectious but still contains viral promoters and transgenic genes, thereby allowing viral activation. Transgenic transgenes. Because viral vectors often lack infectious sequences, they require helper viruses or packaging strains for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, enterovirus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest virus Virus) vector, tobacco mosaic virus vector, lentiviral vector, virus of arenavirus, virus vector of replication-deficient sandy virus, or virus vector, two fragments of replication-capable sandy virus Three-segment sand granulovirus, a viral vector for infectious sand granulovirus, comprising a sand granulovirus genome segment in which an open reading frame of the genome segment is missing or inactivated (and is encoded as described herein Nucleic acid replacement of the HBV antigen), sandy viruses such as lymphocytic choroid plexus meningitis virus (LCMV), such as the pure 13 strain or MP virus strain, and sandy viruses such as Junin virus, For example, Candid # 1 virus strain. The vector may also be a non-viral vector.

較佳地，病毒載體係腺病毒載體，例如重組腺病毒載體。重組腺病毒載體可以例如來源於人類腺病毒(HAdV或AdHu)，或猴類腺病毒，諸如黑猩猩或大猩猩腺病毒(ChAd、AdCh或SAdV)或恆河猴腺病毒(rhAd)。較佳地，腺病毒載體係重組人類腺病毒載體，例如重組人類腺病毒血清型26，或重組人類腺病毒血清型5、4、35、7、48中之任一種等。在其他實施例中，腺病毒載體係rhAd載體，例如rhAd51、rhAd52或rhAd53。可用於本申請案中之重組病毒載體可以根據本發明，使用此項技術中已知之方法製備。舉例而言，考慮到遺傳密碼之簡併性，可以設計出編碼相同多肽之若干種核酸序列。編碼本申請案之HBV抗原的聚核苷酸可以視情況經密碼子優化，以確保在宿主細胞(例如細菌或哺乳動物細胞)中適當表現。密碼子優化係此項技術中廣泛應用之技術，且根據本發明，用於獲得經密碼子優化之聚核苷酸的方法將為熟習此項技術者所熟知。Preferably, the viral vector is an adenoviral vector, such as a recombinant adenoviral vector. The recombinant adenoviral vector may, for example, be derived from a human adenovirus (HAdV or AdHu), or a simian adenovirus, such as a chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV) or rhesus adenovirus (rhAd). Preferably, the adenoviral vector is a recombinant human adenoviral vector, for example, a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotypes 5, 4, 35, 7, 48, and the like. In other embodiments, the adenoviral vector is a rhAd vector, such as rhAd51, rhAd52, or rhAd53. Recombinant viral vectors useful in this application can be prepared according to the present invention using methods known in the art. For example, considering the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. The polynucleotide encoding the HBV antigen of the present application may be codon optimized as appropriate to ensure proper performance in a host cell, such as a bacterial or mammalian cell. Codon optimization is a technique widely used in this technology, and according to the present invention, the method for obtaining codon-optimized polynucleotides will be well known to those skilled in the art.

本申請案之載體，例如DNA質體或病毒載體(特定言之，腺病毒載體)可以包含任何調控元件以產生該載體之習知功能，包括(但不限於)由該載體之聚核苷酸序列編碼的HBV抗原之複製及表現。調控元件包括(但不限於)啟動子、強化子、聚腺苷酸化信號、轉譯終止密碼子、核糖體結合元件、轉錄終止子、選擇標記物、複製起點等。載體可以包含一或多個表現卡匣。「表現卡匣」係載體中引導細胞機構製備RNA及蛋白質的部分。表現卡匣通常包含三種組分：啟動子序列、開放閱讀框架，及視情況包含聚腺苷酸化信號之3'非轉譯區域(UTR)。開放閱讀框架(ORF)係含有所關注蛋白質(例如HBV抗原)的自起始密碼子至終止密碼子之編碼序列的閱讀框架。表現卡匣之調控元件可以可操作地連接至編碼所關注HBV抗原之聚核苷酸序列。如本文所使用，術語「可操作地連接」係以最廣泛合理的內容解釋，且指依功能關係連接聚核苷酸元件。當聚核苷酸之放置與另一聚核苷酸具有功能關係時，其係「可操作地連接」。舉例而言，若啟動子影響編碼序列之轉錄，則其係可操作地連接至該編碼序列。適用於本文所描述之表現卡匣中的任何組件可以任何組合形式且按任何次序使用以製備本申請案之載體。The vector of the present application, such as a DNA plastid or a viral vector (specifically, an adenoviral vector) may contain any regulatory elements to produce the conventional functions of the vector, including (but not limited to) the polynucleotide of the vector Replication and expression of the sequence-encoded HBV antigen. Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation stop codons, ribosome binding elements, transcription terminators, selection markers, origins of replication, and the like. The carrier may contain one or more performance cassettes. A "performance cassette" is a part of a vector that guides the cellular machinery to prepare RNA and proteins. Performance cassettes typically contain three components: a promoter sequence, an open reading frame, and optionally a 3 'untranslated region (UTR) containing a polyadenylation signal. An open reading frame (ORF) is a reading frame containing the coding sequence from the start codon to the stop codon of the protein of interest (eg, HBV antigen). The regulatory elements of the expression cassette may be operably linked to a polynucleotide sequence encoding a HBV antigen of interest. As used herein, the term "operably linked" is interpreted in the broadest reasonable sense and refers to the attachment of polynucleotide elements in a functional relationship. A polynucleotide is "operably linked" when placed in a functional relationship with another polynucleotide. For example, if a promoter affects the transcription of a coding sequence, it is operably linked to the coding sequence. Any component suitable for use in the performance cassettes described herein can be used in any combination and in any order to make the carrier of the present application.

載體可以包含啟動子序列，較佳地在表現卡匣內包含啟動子序列，用以控制所關注HBV抗原之表現。術語「啟動子」係以習知意義使用，且指啟動該可操作地連接之核苷酸序列之轉錄的核苷酸序列。啟動子係與其轉錄之核苷酸序列位於相同股上，且鄰近該核苷酸序列。啟動子可以為組成性、誘導性或阻遏性。啟動子可以為天然存在或合成性。啟動子可以來源於包括病毒、細菌、真菌、植物、昆蟲及動物之來源。啟動子可以為同源啟動子(亦即，來源於與載體相同之基因來源)或異源啟動子(亦即，來源於不同載體或基因來源)。舉例而言，若欲採用之載體係DNA質體，則啟動子可以對於質體為內源性(同源)或來源於其他來源(異源)。較佳地，該啟動子係位於表現卡匣內編碼HBV抗原之聚核苷酸的上游。The vector may contain a promoter sequence, preferably in a performance cassette, to control the expression of the HBV antigen of interest. The term "promoter" is used in a conventional sense and refers to a nucleotide sequence that initiates transcription of the operably linked nucleotide sequence. The promoter is located on the same strand as the nucleotide sequence it transcribes, and is adjacent to the nucleotide sequence. Promoters can be constitutive, inducible or repressive. Promoters can be naturally occurring or synthetic. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter may be a homologous promoter (ie, derived from the same genetic source as the vector) or a heterologous promoter (ie, derived from a different vector or genetic source). For example, if the vector to be used is a DNA plastid, the promoter may be endogenous (homologous) to the plastid or derived from another source (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding the HBV antigen in the expression cassette.

可以使用的啟動子之實例包括(但不限於)來自猴病毒40(SV40)之啟動子、小鼠乳癌病毒(MMTV)啟動子、人類免疫缺陷病毒(HIV)啟動子諸如牛免疫缺陷病毒(BIV)長末端重複序列(LTR)啟動子、莫洛尼病毒(Moloney virus)啟動子、禽類白血病病毒(ALV)啟動子、巨細胞病毒(CMV)啟動子諸如CMV即刻早期啟動子(CMV-IE)、埃-巴二氏病毒(Epstein Barr virus，EBV)啟動子，或勞斯肉瘤病毒(Rous sarcoma virus，RSV)啟動子。啟動子亦可為來自人類基因，諸如人類肌動蛋白、人類肌凝蛋白、人類血紅蛋白、人類肌肉肌酸或人類金屬硫蛋白之啟動子。啟動子亦可為天然或合成的組織特異性啟動子，諸如肌肉或皮膚特異性啟動子。Examples of promoters that can be used include, but are not limited to, promoters from monkey virus 40 (SV40), mouse breast cancer virus (MMTV) promoters, human immunodeficiency virus (HIV) promoters such as bovine immunodeficiency virus (BIV) ) Long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter (CMV-IE) , An Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic tissue-specific promoter, such as a muscle or skin specific promoter.

較佳地，啟動子係強真核啟動子，較佳地為巨細胞病毒即刻早期(CMV-IE)啟動子。例示性CMV-IE啟動子之核苷酸序列顯示於SEQ ID NO: 7及SEQ ID NO: 17中。Preferably, the promoter is a strong eukaryotic promoter, preferably a cytomegalovirus immediate early (CMV-IE) promoter. The nucleotide sequence of an exemplary CMV-IE promoter is shown in SEQ ID NO: 7 and SEQ ID NO: 17.

載體可以包含使表現之轉錄物穩定，促進RNA轉錄物之核輸出及/或改善轉錄-轉譯偶聯之另外的聚核苷酸序列。此類序列之實例包括聚腺苷酸化信號及強化子序列。聚腺苷酸化信號通常位於載體之表現卡匣內所關注蛋白質(例如HBV抗原)之編碼序列的下游。強化子序列係當經轉錄因子結合時促進相關聯之基因之轉錄的調控性DNA序列。強化子序列較佳在載體之表現卡匣內位於編碼HBV抗原之聚核苷酸序列的上游，但在啟動子序列的下游。The vector may contain additional polynucleotide sequences that stabilize the expressed transcript, promote nuclear export of the RNA transcript, and / or improve transcription-translation coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. The polyadenylation signal is usually located downstream of the coding sequence of the protein of interest (eg, HBV antigen) in the expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, promote the transcription of associated genes. The enhancer sequence is preferably located upstream of the polynucleotide sequence encoding the HBV antigen in the expression cassette of the vector, but downstream of the promoter sequence.

根據本發明，熟習此項技術者已知之任何聚腺苷酸化信號均可使用。舉例而言，聚腺苷酸化信號可以為SV40聚腺苷酸化信號(例如SEQ ID NO: 24)、LTR聚腺苷酸化信號、牛生長激素(bGH)聚腺苷酸化信號、人生長激素(hGH)聚腺苷酸化信號或人類β-血球蛋白聚腺苷酸化信號。較佳地，聚腺苷酸化信號係牛生長激素(bGH)聚腺苷酸化信號或SV40聚腺苷酸化信號。例示性bGH聚腺苷酸化信號之核苷酸序列顯示於SEQ ID NO: 11中。例示性SV40聚腺苷酸化信號之核苷酸序列顯示於SEQ ID NO: 24中。According to the present invention, any polyadenylation signal known to those skilled in the art can be used. For example, the polyadenylation signal may be an SV40 polyadenylation signal (e.g., SEQ ID NO: 24), an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) ) Polyadenylation signal or human β-hemoglobin polyadenylation signal. Preferably, the polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or an SV40 polyadenylation signal. The nucleotide sequence of an exemplary bGH polyadenylation signal is shown in SEQ ID NO: 11. The nucleotide sequence of an exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 24.

根據本發明，熟習此項技術者已知之任何強化子序列均可使用。舉例而言，強化子序列可以為人類肌動蛋白、人類肌凝蛋白、人類血紅蛋白、人類肌肉肌酸，或病毒強化子，諸如來自CMV、HA、RSV或EBV之強化子。特定強化子之實例包括(但不限於)土拔鼠(Woodchuck) HBV轉錄後調控元件(WPRE)、來源於人類載脂蛋白A1前驅體(ApoAI)之內含子/外顯子序列、1型人類T細胞白血病病毒(HTLV-1)長末端重複序列(LTR)之非轉譯R-U5結構域、剪接強化子、合成兔β-血球蛋白內含子或其任何組合。較佳地，強化子序列係HTLV-1 LTR之非轉譯R-U5結構域、兔β-血球蛋白內含子及剪接強化子三個連續元件構成的複合序列，在本文中稱作「三強化子序列」。例示性三強化子序列之核苷酸序列顯示於SEQ ID NO: 8中。另一例示性強化子序列係SEQ ID NO: 23中顯示之ApoAI基因片段。According to the present invention, any enhancer sequence known to those skilled in the art can be used. For example, the enhancer sequence may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as an enhancer from CMV, HA, RSV, or EBV. Examples of specific enhancers include, but are not limited to, Woodchuck HBV post-transcriptional regulatory elements (WPRE), intron / exon sequences derived from the human apolipoprotein A1 precursor (ApoAI), type 1 Non-translated R-U5 domain of human T-cell leukemia virus (HTLV-1) long terminal repeat (LTR), splice enhancer, synthetic rabbit β-hemoglobin intron, or any combination thereof. Preferably, the enhancer sequence is a composite sequence consisting of three consecutive elements of the non-translated R-U5 domain of HTLV-1 LTR, rabbit β-hemoglobin intron and splice enhancer, which is referred to herein as "three Enhancer sequence. " The nucleotide sequence of an exemplary triple enhancer sequence is shown in SEQ ID NO: 8. Another exemplary enhancer sequence is the ApoAI gene fragment shown in SEQ ID NO: 23.

載體可以包含編碼信號肽序列之聚核苷酸序列。較佳地，編碼信號肽序列之聚核苷酸序列係位於編碼HBV抗原之聚核苷酸序列的上游。信號肽通常引導蛋白質之定位，促進產生蛋白質之細胞分泌蛋白質，及/或改善抗原表現及交叉呈現至抗原呈現細胞。信號肽當自載體表現時可以存在於HBV抗原之N末端，但例如在自細胞分泌時，經信號肽酶裂解。信號肽已裂解的經表現蛋白質通常稱為「成熟蛋白」。根據本發明，此項技術中已知之任何信號肽均可使用。舉例而言，信號肽可以為胱抑素S信號肽；免疫球蛋白(Ig)分泌信號，諸如Ig重鏈γ信號肽SPIgG或Ig重鏈ε信號肽SPIgE。The vector may comprise a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding the HBV antigen. Signal peptides usually guide protein localization, promote protein-producing cells to secrete proteins, and / or improve antigen performance and cross-presentation to antigen-presenting cells. The signal peptide may be present at the N-terminus of the HBV antigen when expressed from a carrier, but is cleaved by a signal peptidase when secreted from a cell, for example. Expressed proteins whose signal peptides have been cleaved are often referred to as "mature proteins". According to the present invention, any signal peptide known in the art can be used. For example, the signal peptide may be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as an Ig heavy chain γ signal peptide SPIgG or an Ig heavy chain ε signal peptide SPIgE.

較佳地，信號肽序列係胱抑素S信號肽。胱抑素S信號肽之例示性核酸及胺基酸序列分別顯示於SEQ ID NO: 5及6中。免疫球蛋白分泌信號之例示性核酸及胺基酸序列分別顯示於SEQ ID NO: 18及19中。Preferably, the signal peptide sequence is a cystatin S signal peptide. Exemplary nucleic acid and amino acid sequences of the cystatin S signal peptide are shown in SEQ ID NOs: 5 and 6, respectively. Exemplary nucleic acid and amino acid sequences for immunoglobulin secretion signals are shown in SEQ ID NOs: 18 and 19, respectively.

載體，諸如DNA質體亦可包括細菌複製起點及用於在細菌細胞，例如大腸桿菌中選擇及維持質體的抗生素抗性表現卡匣。細菌複製起點及抗生素抗性卡匣可以與編碼HBV抗原之表現卡匣相同之取向或以相反(逆向)取向定位於載體中。複製起點(ORI)係這樣一種序列，在該序列處，複製起始，使得質體能夠在細胞內複製及存活。適用於本申請案中之ORI的實例包括(但不限於)ColE1、pMB1、pUC、pSC101、R6K及15A，較佳為pUC。pUC ORI之例示性核苷酸序列顯示於SEQ ID NO:10中。Vectors, such as DNA plastids, can also include origins of bacterial replication and antibiotic resistance performance cassettes for selecting and maintaining plastids in bacterial cells, such as E. coli. The bacterial origin of replication and antibiotic resistance cassette can be positioned in the vector in the same orientation as the performance cassette encoding the HBV antigen or in the opposite (reverse) orientation. The origin of replication (ORI) is a sequence at which replication is initiated so that plastids can replicate and survive in the cell. Examples of ORIs suitable for use in this application include, but are not limited to, ColE1, pMB1, pUC, pSC101, R6K, and 15A, preferably pUC. An exemplary nucleotide sequence of pUC ORI is shown in SEQ ID NO: 10.

用於在細菌細胞中選擇及維持之表現卡匣通常包括可操作地連接至抗生素抗性基因之啟動子序列。較佳地，可操作地連接至抗生素抗性基因之啟動子序列不同於可操作地連接至編碼所關注蛋白質，例如HBV抗原之聚核苷酸序列的啟動子序列。抗生素抗性基因可以經密碼子優化，且抗生素抗性基因之序列組成通常針對細菌，例如大腸桿菌之密碼子使用進行調整。根據本發明，熟習此項技術者已知之任何抗生素抗性基因均可使用，包括(但不限於)卡那黴素抗性基因(Kan^r )、安比西林(ampicillin)抗性基因(Amp^r )及四環素抗性基因(Tet^r )，以及賦予對氯黴素(chloramphenicol)、博萊黴素(bleomycin)、大觀黴素(spectinomycin)、卡本西林(carbenicillin)等之抗性的基因。Performance cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to the antibiotic resistance gene is different from the promoter sequence operably linked to the polynucleotide sequence encoding the protein of interest, such as the HBV antigen. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is usually adjusted for the codon usage of bacteria, such as E. coli. According to the present invention, any antibiotic resistance gene known to those skilled in the art can be used, including (but not limited to) the kanamycin resistance gene (Kan ^r ), the ampicillin resistance gene (Amp ^r ) And tetracycline resistance genes (Tet ^r ), and genes that confer resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, and the like.

較佳地，載體抗生素表現卡匣中之抗生素抗性基因係卡那黴素抗性基因(Kan^r )。Kan^r 基因之序列顯示於SEQ ID NO: 13中。較佳地，Kan^r 基因經密碼子優化。經密碼子優化之Kan^r 基因的例示性核酸序列顯示於SEQ ID NO: 12中。Kan^r 可以可操作地連接至其天然啟動子，或Kan^r 基因可以連接至異源啟動子。在一個特定實施例中，Kan^r 基因可操作地連接至安比西林抗性基因(Amp^r )啟動子，稱為bla啟動子。bla啟動子之例示性核苷酸序列顯示於SEQ ID NO: 9中。Preferably, the antibiotic resistance gene in the carrier antibiotic cassette is the kanamycin resistance gene (Kan ^r ). The sequence of the Kan ^r gene is shown in SEQ ID NO: 13. Preferably, the Kan ^r gene is codon optimized. An exemplary nucleic acid sequence of the codon-optimized Kan ^r gene is shown in SEQ ID NO: 12. Kan ^r may be operably linked to its natural promoter, or the Kan ^r gene may be linked to a heterologous promoter. In a particular embodiment, the Kan ^r gene is operably linked to an ampicillin resistance gene (Amp ^r ) promoter, known as the bla promoter. An exemplary nucleotide sequence of the bla promoter is shown in SEQ ID NO: 9.

在本申請案之一個特定實施例中，載體係DNA質體，其包含表現卡匣，該表現卡匣包括編碼選自由以下組成之群之HBV抗原中之至少一種的聚核苷酸：包含與SEQ ID NO: 4至少98%一致，諸如與SEQ ID NO: 4至少98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致之胺基酸序列的HBV pol抗原，及由SEQ ID NO: 2之胺基酸序列組成的截短之HBV核心抗原；可操作地連接至該編碼HBV抗原之聚核苷酸的上游序列，自5'端至3'端，其包含啟動子序列(較佳為SEQ ID NO: 7之CMV啟動子序列)、強化子序列(較佳為SEQ ID NO: 8之三強化子序列)及編碼信號肽序列(較佳為具有SEQ ID NO: 6之胺基酸序列的胱抑素S信號肽)之聚核苷酸序列；以及可操作地連接至該編碼HBV抗原之聚核苷酸的下游序列，其包含聚腺苷酸化信號，較佳為SEQ ID NO: 11之bGH聚腺苷酸化信號。此類載體進一步包含抗生素抗性表現卡匣，其包括編碼抗生素抗性基因，較佳地Kan^r 基因，更佳與SEQ ID NO: 12至少90%一致，諸如與SEQ ID NO: 12至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 12達100%一致的經密碼子優化之Kan^r 基因的聚核苷酸可操作地連接至SEQ ID NO: 9之Amp^r (bla)啟動子，該啟動子在該編碼抗生素抗性基因之聚核苷酸的上游且可操作地連接至該聚核苷酸；以及複製起點，較佳為SEQ ID NO:10之pUC ori。較佳地，抗生素抗性卡匣及複製起點相對於HBV抗原表現卡匣以逆向取向存在於質體中。包含上述特徵的例示性DNA質體顯示於圖 2A 及 2B 中。In a specific embodiment of the present application, the carrier is a DNA plastid, which comprises a performance cassette comprising a polynucleotide encoding at least one selected from the group consisting of HBV antigens consisting of: SEQ ID NO: 4 is at least 98% consistent, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% HBV pol antigen with 99.9% or 100% identical amino acid sequences, and a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2; operably linked to the polynucleus encoding the HBV antigen The upstream sequence of the nucleotide is from the 5 ′ end to the 3 ′ end, and it contains a promoter sequence (preferably the CMV promoter sequence of SEQ ID NO: 7), an enhancer sequence (preferably SEQ ID NO: 8 ter) Enhancer sequence) and a polynucleotide sequence encoding a signal peptide sequence (preferably a cystatin S signal peptide having the amino acid sequence of SEQ ID NO: 6); and an operably linked to the HBV antigen encoding The downstream sequence of the polynucleotide comprises a polyadenylation signal, preferably the bGH polyadenylation signal of SEQ ID NO: 11. Such vectors further comprise an antibiotic resistance manifestation cassette, which includes an antibiotic resistance gene, preferably the Kan ^r gene, more preferably at least 90% identical to SEQ ID NO: 12, such as at least 90% identical to SEQ ID NO: 12 , 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4 %, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, preferably codon-optimized polynucleotides of the Kan ^r gene operatively consistent with SEQ ID NO: 12 up to 100% Linked to the Amp ^r (bla) promoter of SEQ ID NO: 9 which is upstream and operably linked to the polynucleotide encoding the antibiotic resistance gene; and the origin of replication, compared to Preferably, it is pUC ori of SEQ ID NO: 10. Preferably, the antibiotic resistance cassette and the origin of replication are present in the plastid in a reverse orientation relative to the HBV antigen expression cassette. Exemplary DNA plastids containing the above features are shown in Figures 2A and 2B .

在本申請案之另一特定實施例中，載體係病毒載體，較佳為腺病毒載體，更佳為Ad26或Ad35載體，其包含表現卡匣，該表現卡匣包括編碼選自由以下組成之群之HBV抗原中之至少一種的聚核苷酸：包含與SEQ ID NO: 4至少98%一致，諸如與SEQ ID NO: 4至少98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致之胺基酸序列的HBV pol抗原，及由SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原；可操作地連接至該編碼HBV抗原之聚核苷酸的上游序列，自5'端至3'端，其包含啟動子序列(較佳為SEQ ID NO: 17之CMV啟動子序列)、強化子序列(較佳為SEQ ID NO: 23之ApoAI基因片段序列)及編碼信號肽序列(較佳為具有SEQ ID NO: 19之胺基酸序列的免疫球蛋白分泌信號)之聚核苷酸序列；以及可操作地連接至該編碼HBV抗原之聚核苷酸的下游序列，其包含聚腺苷酸化信號，較佳為SEQ ID NO: 24之SV40聚腺苷酸化信號。In another specific embodiment of the present application, the vector is a viral vector, preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector, which comprises a performance cassette comprising a code selected from the group consisting of Polynucleotide of at least one of the HBV antigens: comprising at least 98% identical to SEQ ID NO: 4, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3% identical to SEQ ID NO: 4 , 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical HBV pol antigens, and a truncated HBV consisting of the amino acid sequence of SEQ ID NO: 14 Core antigen; operably linked to the upstream sequence of the polynucleotide encoding the HBV antigen from the 5 'end to the 3' end, which contains a promoter sequence (preferably the CMV promoter sequence of SEQ ID NO: 17) Polynucleotides of an enhancer sequence (preferably the sequence of the ApoAI gene fragment of SEQ ID NO: 23) and a signal peptide sequence (preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 19) Acid sequence; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen, which comprises a polyadenylation signal, preferably Is the SV40 polyadenylation signal of SEQ ID NO: 24.

在本申請案之一個實施例中，諸如質體DNA載體或病毒載體(較佳為腺病毒載體，更佳為Ad26或Ad35載體)之載體編碼具有SEQ ID NO: 4之胺基酸序列的HBV Pol抗原。較佳地，該載體包含HBV Pol抗原之編碼序列，其與SEQ ID NO: 3之聚核苷酸序列至少90%一致，諸如與SEQ ID NO: 3有90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 3達100%一致。In one embodiment of the present application, a vector such as a plastid DNA vector or a viral vector (preferably an adenovirus vector, more preferably an Ad26 or Ad35 vector) encodes a HBV having an amino acid sequence of SEQ ID NO: 4 Pol antigen. Preferably, the vector contains the coding sequence of the HBV Pol antigen, which is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 3, such as 90%, 91%, 92%, 93% of SEQ ID NO: 3 %, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% are consistent, preferably 100% consistent with SEQ ID NO: 3.

在本申請案之一個實施例中，諸如質體DNA載體或病毒載體(較佳為腺病毒載體，更佳為Ad26或Ad35載體)之載體編碼由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原。較佳地，該載體包含截短之HBV核心抗原的編碼序列，其與SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列至少90%一致，諸如與SEQ ID NO: 1或SEQ ID NO: 15有90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 1或SEQ ID NO: 15達100%一致。In one embodiment of the present application, a vector such as a plastid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector) is encoded by SEQ ID NO: 2 or SEQ ID NO: 14 Truncated HBV core antigen consisting of amino acid sequences. Preferably, the vector comprises a truncated HBV core antigen coding sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15, such as with SEQ ID NO: 1 or SEQ ID NO: 15 has 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% are consistent, preferably 100% consistent with SEQ ID NO: 1 or SEQ ID NO: 15.

在本申請案之又一實施例中，諸如質體DNA載體或病毒載體(較佳為腺病毒載體，更佳為Ad26或Ad35載體)之載體編碼融合蛋白，其包含具有SEQ ID NO: 4之胺基酸序列的HBV Pol抗原及由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的截短之HBV核心抗原。較佳地，該載體包含該融合物之編碼序列，其含有與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致，諸如與SEQ ID NO: 1或SEQ ID NO: 15至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 1或SEQ ID NO: 15，更佳與SEQ ID NO: 15有98%、99%或100%一致的截短之HBV核心抗原之編碼序列，其係可操作地連接至與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致，諸如與SEQ ID NO: 3或SEQ ID NO: 16至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 3或SEQ ID NO: 16，更佳與SEQ ID NO: 16有98%、99%或100%一致的HBV Pol抗原之編碼序列。較佳地，該截短之HBV核心抗原之編碼序列係經由與SEQ ID NO: 22至少90%一致，諸如與SEQ ID NO: 22至少90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致，較佳與SEQ ID NO: 22有98%、99%或100%一致之連接子編碼序列，可操作地連接至HBV Pol抗原之編碼序列。在本申請案之特定實施例中，載體包含具有SEQ ID NO: 15之融合物之編碼序列，其可操作地連接至SEQ ID NO: 22，其進一步可操作地連接至SEQ ID NO: 16。In yet another embodiment of the present application, a vector such as a plastid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector) encodes a fusion protein, which comprises a protein having SEQ ID NO: 4 HBV Pol antigen of amino acid sequence and truncated HBV core antigen consisting of amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14. Preferably, the vector comprises the coding sequence of the fusion, which contains at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 15, such as at least 90% with SEQ ID NO: 1 or SEQ ID NO: 15, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4% , 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, preferably with SEQ ID NO: 1 or SEQ ID NO: 15, more preferably 98%, 99% with SEQ ID NO: 15 or 100% consistent truncated HBV core antigen coding sequence operably linked to at least 90% identity to SEQ ID NO: 3 or SEQ ID NO: 16 such as SEQ ID NO: 3 or SEQ ID NO: 16 At least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, preferably with SEQ ID NO: 3 or SEQ ID NO: 16, more preferably 98 with SEQ ID NO: 16 %, 99% or 100% identical HBV Pol antigen coding sequences. Preferably, the coding sequence of the truncated HBV core antigen is at least 90% identical to SEQ ID NO: 22, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% , 99.9% or 100% identical, preferably 98%, 99% or 100% identical linker coding sequence corresponding to SEQ ID NO: 22, operably linked to the coding sequence of HBV Pol antigen. In a specific embodiment of the present application, the vector comprises a coding sequence having the fusion of SEQ ID NO: 15 which is operably linked to SEQ ID NO: 22 and which is further operatively linked to SEQ ID NO: 16.

編碼本申請案之HBV抗原的聚核苷酸及表現載體可以根據本發明，利用此項技術中已知之任何方法製備。舉例而言，可以使用熟習此項技術者熟知的標準分子生物學技術，例如聚合酶鏈反應(PCR)等將編碼HBV抗原之聚核苷酸引入或「選殖」至表現載體中。Polynucleotides and expression vectors encoding the HBV antigen of the present application can be prepared according to the present invention using any method known in the art. For example, standard molecular biology techniques familiar to those skilled in the art, such as polymerase chain reaction (PCR), can be used to introduce or "colony" a polynucleotide encoding an HBV antigen into a performance vector.

細胞、多肽及抗體Cells, peptides and antibodies

本申請案亦提供包含本文所描述之聚核苷酸及載體中之任一種的細胞，較佳為分離之細胞。該等細胞可以例如用於產生重組蛋白或用於產生病毒粒子。The application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein. Such cells can be used, for example, to produce recombinant proteins or to produce virions.

因此，本申請案之實施例亦係關於製備本申請案之HBV抗原的方法。該方法包括用包含可操作地連接至啟動子的編碼本申請案之HBV抗原之聚核苷酸的表現載體轉染宿主細胞，使經轉染之細胞在適於表現HBV抗原之條件下生長，及視情況純化或分離該細胞中表現之HBV抗原。HBV抗原可以藉由此項技術中已知之任何方法，包括親和層析法、尺寸排阻層析法等自細胞分離或收集。根據本發明，用於表現重組蛋白之技術將為一般熟習此項技術者熟知的。亦可在不純化或分離所表現之蛋白質的情況下，例如藉由分析用編碼HBV抗原之表現載體轉染且在適於表現HBV抗原之條件下生長之細胞的上清液來研究所表現之HBV抗原。Therefore, the examples of the present application are also related to the method for preparing the HBV antigen of the present application. The method includes transfecting a host cell with a performance vector comprising a polynucleotide encoding an HBV antigen operably linked to a promoter, and allowing the transfected cell to grow under conditions suitable for expressing the HBV antigen, And optionally purify or isolate the HBV antigen expressed in the cells. HBV antigens can be isolated or collected from cells by any method known in the art, including affinity chromatography, size exclusion chromatography, and the like. According to the present invention, techniques for expressing recombinant proteins will be familiar to those skilled in the art. The expressed protein can also be studied without purification or isolation, for example, by analyzing the supernatant of cells transfected with a expression vector encoding the HBV antigen and grown under conditions suitable for the expression of the HBV antigen. HBV antigen.

因此，亦提供包含與SEQ ID NO: 2、SEQ ID NO: 14或SEQ ID NO: 4之胺基酸序列至少90%一致之胺基酸序列的非天然存在或重組之多肽。如上下文所描述，編碼該等序列的分離之核酸分子、包含該等序列可操作地連接至啟動子之載體及包含該多肽、聚核苷酸或載體之組合物亦涵蓋在本申請案內。Accordingly, a non-naturally occurring or recombinant polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 14, or SEQ ID NO: 4 is also provided. As described above and below, isolated nucleic acid molecules encoding such sequences, vectors comprising such sequences operably linked to a promoter, and compositions comprising the polypeptide, polynucleotide or vector are also encompassed by the present application.

在本申請案之一個實施例中，重組多肽包含與SEQ ID NO: 2之胺基酸序列至少90%一致，諸如與SEQ ID NO: 2有90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致的胺基酸序列。較佳地，非天然存在或重組之多肽由SEQ ID NO: 2組成。In one embodiment of the present application, the recombinant polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 2, such as 90%, 91%, 92%, 93%, 94 %, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences. Preferably, the non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 2.

在本申請案之另一實施例中，非天然存在或重組之多肽包含與SEQ ID NO: 4之胺基酸序列至少90%一致，諸如與SEQ ID NO: 4有90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致的胺基酸序列。較佳地，非天然存在或重組之多肽包含SEQ ID NO: 4。In another embodiment of the present application, the non-naturally occurring or recombinant polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 4, such as 90%, 91%, 92% to SEQ ID NO: 4 %, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences. Preferably, the non-naturally occurring or recombinant polypeptide comprises SEQ ID NO: 4.

在本申請案之另一實施例中，非天然存在或重組之多肽包含與SEQ ID NO: 14之胺基酸序列至少90%一致，諸如與SEQ ID NO: 14有90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致的胺基酸序列。較佳地，非天然存在或重組之多肽由SEQ ID NO: 14組成。In another embodiment of the present application, the non-naturally occurring or recombinant polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 14, such as 90%, 91%, 92% to SEQ ID NO: 14 %, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences. Preferably, the non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 14.

亦提供特異性結合至本申請案之非天然存在之多肽的抗體或其抗原結合片段。在本申請案之一個實施例中，對本申請案之非天然存在之HBV抗原具有特異性的抗體不特異性結合至另一HBV抗原。舉例而言，特異性結合至具有SEQ ID NO: 4之胺基酸序列之HBV Pol抗原的本申請案之抗體將不會特異性結合至不具有SEQ ID NO: 4之胺基酸序列的HBV Pol抗原。Antibodies or antigen-binding fragments thereof that specifically bind to a non-naturally occurring polypeptide of the present application are also provided. In one embodiment of the application, an antibody specific for a non-naturally occurring HBV antigen of the application does not specifically bind to another HBV antigen. For example, an antibody of the present application that specifically binds to the HBV Pol antigen with the amino acid sequence of SEQ ID NO: 4 will not specifically bind to HBV that does not have the amino acid sequence of SEQ ID NO: 4 Pol antigen.

如本文所使用，術語「抗體」包括多株抗體、單株抗體、嵌合抗體、人類化抗體、Fv抗體、Fab抗體及F(ab')2抗體；雙功能混合物(例如Lanzavecchia等人, Eur. J. Immunol. 17:105, 1987)、單鏈抗體(Huston等人, Proc. Natl. Acad. Sci. USA 85:5879, 1988；Bird等人, Science 242:423, 1988)；以及具有改變之恆定區的抗體(例如美國專利第5,624,821號)。As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, Fv antibodies, Fab antibodies, and F (ab ') 2 antibodies; bifunctional mixtures (e.g. Lanzavecchia et al., Eur J. Immunol. 17: 105, 1987), single-chain antibodies (Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879, 1988; Bird et al., Science 242: 423, 1988); and have changes Antibodies to the constant region (eg, US Patent No. 5,624,821).

如本文所使用，「特異性結合至」抗原的抗體係指以1×10⁻ ⁷ M或更小之KD結合至抗原的抗體。較佳地，「特異性結合至」抗原之抗體以1×10⁻ ⁸ M或更小，更佳5×10⁻ ⁹ M或更小、1×10⁻ ⁹ M或更小、5×10⁻ ¹⁰ M或更小，或1×10⁻ ¹⁰ M或更小之KD結合至抗原。術語「KD」係指解離常數，其係由Kd與Ka之比率(亦即，Kd/Ka)得到且以莫耳濃度(M)表示。抗體之KD值可以根據本發明，使用此項技術中已知之方法測定。舉例而言，抗體之KD可以藉由使用表面電漿子共振，諸如藉由使用生物感測器系統，例如Biacore®系統，或藉由使用生物膜層干涉測量術，諸如Octet RED96系統測定。As used herein, "specifically binds to" refers to antigens at 1 × 10 ^- the smaller the KD ⁷ M or antigen bound to the antibody. Preferably, the antibody that specifically binds to the antigen is 1 × 10 ⁻ ⁸ M or less, more preferably 5 × 10 ⁻ ⁹ M or less, 1 × 10 ⁻ ⁹ M or less, 5 × 10 ⁻ ¹⁰ M or less, or 1 × 10 ⁻ ¹⁰ M or less KD binds to the antigen. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (ie, Kd / Ka) and is expressed in Molar concentration (M). The KD value of an antibody can be determined according to the present invention using methods known in the art. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, such as the Biacore® system, or by using biofilm interferometry, such as the Octet RED96 system.

抗體KD值越小，則該抗體結合至靶抗原之親和力越高。The smaller the antibody KD value, the higher the affinity of the antibody for binding to the target antigen.

組合物、免疫原性組合及疫苗Composition, immunogenic combination and vaccine

本申請案亦係關於包含根據本申請案之一或多種HBV抗原、編碼一或多種HBV抗原之聚核苷酸及/或載體的組合物、免疫原性組合，更特定言之套組，及疫苗。本文所描述的本申請案之HBV抗原、聚核苷酸(包括RNA及DNA)及/或載體中之任一種均可用於本申請案之組合物、免疫原性組合或套組，及疫苗中The present application also relates to compositions, immunogenic combinations, and more specifically sets, comprising one or more HBV antigens, polynucleotides and / or vectors encoding one or more HBV antigens according to the present application, and vaccine. Any of the HBV antigens, polynucleotides (including RNA and DNA), and / or vectors of the application described herein can be used in the compositions, immunogenic combinations or kits of the application, and in vaccines

本申請案提供包含編碼由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致之胺基酸序列組成的截短之HBV核心抗原，或包含與SEQ ID NO: 4至少90%一致之胺基酸序列之HBV聚合酶抗原的分離或非天然存在之核酸分子(DNA或RNA)的組合物，包含該分離或非天然存在之核酸分子的載體，及/或由該分離或非天然存在之核酸分子編碼的分離或非天然存在之多肽。The application provides a truncated HBV core antigen comprising an amino acid sequence encoding at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 14 or an amino acid sequence comprising at least 90% identity to SEQ ID NO: 4 Composition of an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) of an amino acid sequence of a HBV polymerase antigen, a vector comprising the isolated or non-naturally occurring nucleic acid molecule, and / or derived from the isolated or non-naturally occurring nucleic acid molecule Isolated or non-naturally occurring polypeptide encoded by a nucleic acid molecule.

在本申請案之一個實施例中，組合物包含編碼截短之HBV核心抗原的分離或非天然存在之核酸分子(DNA或RNA)，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成。In one embodiment of the present application, the composition comprises an isolated or non-naturally-occurring nucleic acid molecule (DNA or RNA) encoding a truncated HBV core antigen, the truncated HBV core antigen is composed of SEQ ID NO: 2 or SEQ ID NO: 14 is at least 90% identical, preferably an amino acid sequence consisting of SEQ ID NO: 2 or 100% identical to SEQ ID NO: 14.

在本申請案之一個實施例中，組合物包含編碼HBV Pol抗原的分離或非天然存在之核酸分子(DNA或RNA)，該HBV Pol抗原包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列。In one embodiment of the present application, the composition comprises an isolated or non-naturally-occurring nucleic acid molecule (DNA or RNA) encoding an HBV Pol antigen, and the HBV Pol antigen comprises at least 90% identity with SEQ ID NO: 4, preferably Amino acid sequence up to 100% identical to SEQ ID NO: 4.

在本申請案之一個實施例中，組合物包括：含編碼截短之HBV核心抗原之聚核苷酸序列的分離或非天然存在之核酸分子(DNA或RNA)，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成；以及含編碼HBV Pol抗原之聚核苷酸序列的分離或非天然存在之核酸分子(DNA或RNA)，該HBV Pol抗原包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列。截短之HBV核心抗原及HBV Pol抗原之編碼序列可以存在於同一分離或非天然存在之核酸分子(DNA或RNA)中，或兩個不同的分離或非天然存在之核酸分子(DNA或RNA)中。In one embodiment of the present application, the composition includes an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) containing a polynucleotide sequence encoding a truncated HBV core antigen, the truncated HBV core antigen Consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14 and preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14; An isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) of a polynucleotide sequence, the HBV Pol antigen contains an amine that is at least 90% identical to SEQ ID NO: 4 and preferably 100% identical to SEQ ID NO: 4 Amino acid sequence. The coding sequences of the truncated HBV core antigen and HBV Pol antigen can exist in the same isolated or non-naturally occurring nucleic acid molecule (DNA or RNA), or two different isolated or non-naturally occurring nucleic acid molecules (DNA or RNA) in.

在本申請案之一個實施例中，組合物包括含編碼截短之HBV核心抗原之聚核苷酸的載體，較佳地DNA質體或病毒載體(諸如腺病毒載體)，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成。In one embodiment of the present application, the composition includes a vector containing a polynucleotide encoding a truncated HBV core antigen, preferably a DNA plastid or a viral vector (such as an adenoviral vector), the truncated HBV The core antigen consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14 and preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

在本申請案之一個實施例中，組合物包括含編碼HBV Pol抗原之聚核苷酸的載體，較佳地DNA質體或病毒載體(諸如腺病毒載體)，該HBV Pol抗原包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列。In one embodiment of the present application, the composition includes a vector, preferably a DNA plastid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding an HBV Pol antigen, the HBV Pol antigen comprising NO: 4 is at least 90% identical, preferably an amino acid sequence that is 100% identical to SEQ ID NO: 4.

在本申請案之一個實施例中，組合物包括：含編碼截短之HBV核心抗原之聚核苷酸的載體，較佳地DNA質體或病毒載體(諸如腺病毒載體)，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成；以及含編碼HBV Pol抗原之聚核苷酸的載體，較佳地DNA質體或病毒載體(諸如腺病毒載體)，該HBV Pol抗原包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列。包含截短之HBV核心抗原之編碼序列的載體及包含HBV Pol抗原之編碼序列的載體可以為相同載體，或兩種不同的載體。In one embodiment of the present application, the composition includes: a vector containing a polynucleotide encoding a truncated HBV core antigen, preferably a DNA plastid or a viral vector (such as an adenoviral vector), the truncated The HBV core antigen consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14 and preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14; and contains an encoding HBV Polynucleotide carrier of the Pol antigen, preferably a DNA plastid or a viral vector (such as an adenoviral vector), the HBV Pol antigen contains at least 90% identity to SEQ ID NO: 4 and preferably corresponds to SEQ ID NO: 4 Up to 100% consistent amino acid sequences. The vector containing the coding sequence of the truncated HBV core antigen and the vector containing the coding sequence of the HBV Pol antigen may be the same vector, or two different vectors.

在本申請案之一個實施例中，組合物包括含編碼融合蛋白之聚核苷酸的載體，較佳地DNA質體或病毒載體(諸如腺病毒載體)，該融合蛋白包含由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成的截短之HBV核心抗原可操作地連接至包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列的HBV Pol抗原，或該HBV Pol抗原可操作地連接至該截短之HBV核心抗原。較佳地，該融合蛋白進一步包含將截短之HBV核心抗原可操作地連接至HBV Pol抗原或將HBV Pol抗原可操作地連接至截短之HBV核心抗原的連接子。較佳地，該連接子具有胺基酸序列(AlaGly)_n ，其中n係2至5之整數。In one embodiment of the present application, the composition includes a vector, preferably a DNA plastid or a viral vector (such as an adenoviral vector), containing a polynucleotide encoding a fusion protein, the fusion protein comprising : 2 or SEQ ID NO: 14 is at least 90% identical, preferably a truncated HBV core antigen consisting of an amino acid sequence that is 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14 is operably linked to HBV Pol antigen with an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4, or that the HBV Pol antigen is operably linked to the truncated HBV core antigen . Preferably, the fusion protein further comprises a linker operatively linked to the truncated HBV core antigen or HBV Pol antigen to the truncated HBV core antigen. Preferably, the linker has an amino acid sequence (AlaGly) _n , where n is an integer from 2 to 5.

在本申請案之一個實施例中，組合物包含由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成的分離或非天然存在之截短之HBV核心抗原。In one embodiment of the present application, the composition comprises at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14 and preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14 Isolated or non-naturally occurring truncated HBV core antigens consisting of amino acid sequences.

在本申請案之一個實施例中，組合物包括含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列的分離或非天然存在之HBV Pol抗原。In one embodiment of the present application, the composition includes an isolated or non-naturally occurring HBV containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, and preferably 100% identical to SEQ ID NO: 4. Pol antigen.

在本申請案之一個實施例中，組合物包含由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成的分離或非天然存在之截短之HBV核心抗原；以及包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列的分離或非天然存在之HBV Pol抗原。In one embodiment of the present application, the composition comprises at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14 and preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14 An isolated or non-naturally occurring truncated HBV core antigen consisting of an amino acid sequence; and a gene comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4 and preferably 100% identical to SEQ ID NO: 4 Isolated or non-naturally occurring HBV Pol antigen.

在本申請案之一個實施例中，組合物包含分離或非天然存在之融合蛋白，該融合蛋白包含由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致之胺基酸序列組成的截短之HBV核心抗原可操作地連接至包含與SEQ ID NO: 4至少90%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列的HBV Pol抗原，或該HBV Pol抗原可操作地連接至該截短之HBV核心抗原。較佳地，該融合蛋白進一步包含將截短之HBV核心抗原可操作地連接至HBV Pol抗原或將HBV Pol抗原可操作地連接至截短之HBV核心抗原的連接子。較佳地，該連接子具有胺基酸序列(AlaGly)_n ，其中n係2至5之整數。In one embodiment of the present application, the composition comprises an isolated or non-naturally occurring fusion protein comprising at least 90% identity with SEQ ID NO: 2 or SEQ ID NO: 14, preferably with SEQ ID NO : 2 or SEQ ID NO: 14 up to 100% identical amino acid sequence consisting of a truncated HBV core antigen operably linked to contain at least 90% identity to SEQ ID NO: 4, preferably to SEQ ID NO: 4 A HBV Pol antigen with an amino acid sequence that is 100% identical, or the HBV Pol antigen is operably linked to the truncated HBV core antigen. Preferably, the fusion protein further comprises a linker operatively linked to the truncated HBV core antigen or HBV Pol antigen to the truncated HBV core antigen. Preferably, the linker has an amino acid sequence (AlaGly) _n , where n is an integer from 2 to 5.

本申請案亦係關於免疫原性組合或套組，其包含表現根據本申請案之實施例之截短之HBV核心抗原及HBV pol抗原的聚核苷酸。編碼本文所描述的本申請案之HBV核心及pol抗原之任何聚核苷酸及/或載體均可用於本申請案之免疫原性組合或套組中。This application is also related to immunogenic combinations or sets comprising polynucleotides expressing truncated HBV core antigens and HBV pol antigens according to the examples of this application. Any polynucleotide and / or vector encoding the HBV core and pol antigens of the application described herein can be used in the immunogenic combination or set of the application.

根據本申請案之實施例，疫苗組合或套組包含：
(a) 包含編碼HBV聚合酶抗原之第一聚核苷酸的第一非天然存在之核酸分子，該HBV聚合酶抗原具有與SEQ ID NO: 4至少98%一致的胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性；
(b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二非天然存在之核酸分子，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成；以及
(c) 醫藥學上可接受之載劑，
其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一非天然存在之核酸分子中或兩個不同的非天然存在之核酸分子中。According to an embodiment of the present application, a vaccine combination or kit includes:
(a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity or ribonuclease H activity;
(b) a second non-naturally-occurring nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen, the truncated HBV core antigen being an amino group of SEQ ID NO: 2 or SEQ ID NO: 14 Acid sequence composition;
(c) a pharmaceutically acceptable carrier,
The first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule exist in the same non-naturally occurring nucleic acid molecule or two different non-naturally occurring nucleic acid molecules.

根據本申請案之實施例，疫苗組合或套組中之聚核苷酸可以連接或分開，由此使自此類聚核苷酸表現之HBV抗原融合在一起或作為獨立蛋白質產生，無論自相同抑或不同聚核苷酸表現。在一個實施例中，第一聚核苷酸及第二聚核苷酸係存在於獨立載體，例如DNA質體或病毒載體中，以同一組合物或獨立組合物形式組合使用，由此使所表現之蛋白質亦為獨立蛋白質，但組合使用。在另一個實施例中，由第一聚核苷酸及第二聚核苷酸編碼的HBV抗原可以由同一載體表現，由此產生HBV核心-pol融合抗原。視情況，核心及pol抗原可以藉由短連接子接合或融合在一起。或者，由第一聚核苷酸及第二聚核苷酸編碼的HBV抗原可以使用在核心與pol抗原編碼序列之間的核糖體滑移位點(ribosomal slippage site) (又稱為順式水解酶位點)，獨立地由單一載體表現。此策略產生雙順反子表現載體，其中個別核心及pol抗原係由單一mRNA轉錄物產生。取決於mRNA轉錄物上編碼序列之次序，由此類雙順反子表現載體產生之核心及pol抗原可以具有另外的N或C末端殘基。可用於此目的的核糖體滑移位點之實例包括(但不限於)來自***病毒(FMDV)之FA2滑移位點。另一種可能係由第一聚核苷酸及第二聚核苷酸編碼之HBV抗原可以獨立地由兩個獨立載體表現，一個載體編碼HBV核心抗原且一個編碼HBV pol抗原。According to the embodiments of the present application, the polynucleotides in a vaccine combination or set can be linked or separated, thereby fused HBV antigens expressed from such polynucleotides together or produced as independent proteins, whether from the same or Different polynucleotides behave. In one embodiment, the first polynucleotide and the second polynucleotide are present in separate vectors, such as DNA plastids or viral vectors, and are used in combination in the same composition or in the form of separate compositions. The expressed proteins are also independent proteins, but used in combination. In another embodiment, the HBV antigen encoded by the first polynucleotide and the second polynucleotide can be expressed by the same vector, thereby generating a HBV core-pol fusion antigen. Optionally, the core and pol antigens can be joined or fused together by short linkers. Alternatively, the HBV antigen encoded by the first and second polynucleotides can use a ribosomal slippage site (also known as cis-hydrolysis) between the core and the pol antigen-encoding sequence. Enzyme site), independently expressed by a single vector. This strategy produces bicistronic expression vectors in which individual core and pol antigen lines are produced from a single mRNA transcript. Depending on the order of the coding sequences on the mRNA transcript, the core and pol antigens produced by such bicistronic expression vectors may have additional N- or C-terminal residues. Examples of ribosome slip sites that can be used for this purpose include, but are not limited to, FA2 slip sites from foot-and-mouth disease virus (FMDV). Another possibility is that the HBV antigen encoded by the first polynucleotide and the second polynucleotide can be independently expressed by two independent vectors, one vector encoding the HBV core antigen and one encoding the HBV pol antigen.

在一個較佳實施例中，第一聚核苷酸及第二聚核苷酸係存在於獨立載體，例如DNA質體或病毒載體中。較佳地，該等獨立載體係存在於同一組合物中。In a preferred embodiment, the first polynucleotide and the second polynucleotide are present in separate vectors, such as DNA plastids or viral vectors. Preferably, the separate carriers are present in the same composition.

根據本申請案之較佳實施例，免疫原性組合或套組包含存在於第一載體中之第一聚核苷酸及存在於第二載體中之第二聚核苷酸。第一載體與第二載體可以相同或不同。較佳地，該等載體係DNA質體。According to a preferred embodiment of the present application, the immunogenic combination or set comprises a first polynucleotide present in a first vector and a second polynucleotide present in a second vector. The first vector and the second vector may be the same or different. Preferably, the vectors are DNA plastids.

在本申請案之一個特定實施例中，免疫原性組合或套組包括：含編碼截短之HBV核心抗原之聚核苷酸的第一載體，該截短之HBV核心抗原由與SEQ ID NO: 2至少90%一致，較佳與SEQ ID NO: 2達100%一致之胺基酸序列組成；以及含編碼HBV聚合酶抗原之聚核苷酸的第二載體，該HBV聚合酶抗原包含與SEQ ID NO: 4至少98%一致，較佳與SEQ ID NO: 4達100%一致之胺基酸序列。In a specific embodiment of the present application, the immunogenic combination or kit includes: a first vector containing a polynucleotide encoding a truncated HBV core antigen, the truncated HBV core antigen is composed of SEQ ID NO : 2 at least 90% identical, preferably 100% identical amino acid sequence composition to SEQ ID NO: 2; and a second vector containing a polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen comprising and SEQ ID NO: 4 is at least 98% identical, preferably 100% identical to the amino acid sequence of SEQ ID NO: 4.

在本申請案之一個特定實施例中，第一載體係第一DNA質體且第二載體係第二DNA質體。該第一DNA質體及該第二DNA質體各自包含複製起點，較佳為SEQ ID NO: 10之pUC ORI，及抗生素抗性卡匣，其較佳包含具有與SEQ ID NO: 12至少90%一致之聚核苷酸序列的經密碼子優化之Kan^r 基因，該基因較佳處於bla啟動子，例如SEQ ID NO: 9中顯示之bla啟動子的控制下。該第一DNA質體及該第二DNA質體各自獨立地進一步包含以下至少一個：啟動子序列、強化子序列，及編碼可操作地連接至第一聚核苷酸序列或第二聚核苷酸序列之信號肽序列的聚核苷酸序列。較佳地，該第一DNA質體及該第二DNA質體各自包含可操作地連接至第一聚核苷酸或第二聚核苷酸之上游序列，其中該上游序列自5'端至3'端包含SEQ ID NO: 7之啟動子序列、SEQ ID NO: 8之強化子序列及編碼具有SEQ ID NO: 6之胺基酸序列之信號肽序列的聚核苷酸序列。該第一DNA質體及該第二DNA質體各自亦可包含位於HBV抗原編碼序列下游的聚腺苷酸化信號，諸如SEQ ID NO: 11之bGH聚腺苷酸化信號。In a specific embodiment of the present application, the first vector is a first DNA plastid and the second vector is a second DNA plastid. The first DNA plastid and the second DNA plastid each include an origin of replication, preferably pUC ORI of SEQ ID NO: 10, and an antibiotic resistance cassette, which preferably includes at least 90% of SEQ ID NO: 12 The codon-optimized Kan ^r gene of a% identical polynucleotide sequence, which is preferably under the control of a bla promoter, such as the bla promoter shown in SEQ ID NO: 9. The first DNA plastid and the second DNA plastid each independently further include at least one of the following: a promoter sequence, an enhancer sequence, and an encoding operably linked to the first polynucleotide sequence or the second polynucleoside A polynucleotide sequence of a signal peptide sequence of an acid sequence. Preferably, the first DNA plastid and the second DNA plastid each comprise an upstream sequence operably linked to the first polynucleotide or the second polynucleotide, wherein the upstream sequence is from the 5 'end to The 3 'end contains the promoter sequence of SEQ ID NO: 7, the enhancer sequence of SEQ ID NO: 8, and a polynucleotide sequence encoding a signal peptide sequence having the amino acid sequence of SEQ ID NO: 6. Each of the first DNA plastid and the second DNA plastid may also include a polyadenylation signal located downstream of the HBV antigen coding sequence, such as the bGH polyadenylation signal of SEQ ID NO: 11.

在本申請案之一個特定實施例中，第一載體係第一病毒載體且第二載體係第二病毒載體。較佳地，該第一病毒載體及該第二病毒載體各自為腺病毒載體，更佳為Ad26或Ad35載體，其包含表現卡匣，該表現卡匣包括編碼本申請案之HBV pol抗原或截短之HBV核心抗原的聚核苷酸；可操作地連接至該編碼HBV抗原之聚核苷酸的上游序列，該上游序列自5'端至3'端包含啟動子序列(較佳為SEQ ID NO: 17之CMV啟動子序列)、強化子序列(較佳為SEQ ID NO: 23之ApoAI基因片段序列)及編碼信號肽序列(較佳為具有SEQ ID NO: 19之胺基酸序列之免疫球蛋白分泌信號)的聚核苷酸序列；以及可操作地連接至該編碼HBV抗原之聚核苷酸的下游序列，該下游序列包含聚腺苷酸化信號，較佳為SEQ ID NO:24之SV40聚腺苷酸化信號。In a specific embodiment of the present application, the first vector is a first viral vector and the second vector is a second viral vector. Preferably, the first viral vector and the second viral vector are each an adenoviral vector, and more preferably an Ad26 or Ad35 vector, which includes a performance cassette including the HBV pol antigen or a truncated antibody encoding the present application. Short HBV core antigen polynucleotide; operably linked to an upstream sequence of the HBV antigen-encoding polynucleotide, the upstream sequence comprising a promoter sequence (preferably SEQ ID from 5 'to 3') CMV promoter sequence of NO: 17), enhancer sequence (preferably ApoAI gene fragment sequence of SEQ ID NO: 23) and a signal peptide sequence (preferably with amino acid sequence of SEQ ID NO: 19) Globulin secretion signal); and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen, the downstream sequence comprising a polyadenylation signal, preferably SEQ ID NO: 24 SV40 polyadenylation signal.

在另一較佳實施例中，第一聚核苷酸及第二聚核苷酸係存在於單一載體，例如DNA質體或病毒載體中。較佳地，該單一載體係腺病毒載體，更佳為Ad26載體，其包含表現卡匣，該表現卡匣包括編碼本申請案之HBV pol抗原及截短之HBV核心抗原，較佳地編碼呈融合蛋白形式的本申請案之HBV pol抗原及截短之HBV核心抗原的聚核苷酸；可操作地連接至該編碼HBV pol及截短之核心抗原之聚核苷酸的上游序列，該上游序列自5'端至3'端包含啟動子序列(較佳為SEQ ID NO: 17之CMV啟動子序列)、強化子序列(較佳為SEQ ID NO: 23之ApoAI基因片段序列)及編碼信號肽序列(較佳為具有SEQ ID NO: 19之胺基酸序列之免疫球蛋白分泌信號)的聚核苷酸序列；以及可操作地連接至該編碼HBV抗原之聚核苷酸的下游序列，該下游序列包含聚腺苷酸化信號，較佳為SEQ ID NO: 24之SV40聚腺苷酸化信號。In another preferred embodiment, the first polynucleotide and the second polynucleotide are present in a single vector, such as a DNA plastid or a viral vector. Preferably, the single vector is an adenoviral vector, more preferably an Ad26 vector, which includes a performance cassette comprising the HBV pol antigen encoding the application and the truncated HBV core antigen, preferably encoding the presentation The polynucleotide of the HBV pol antigen and truncated HBV core antigen of the present application in the form of a fusion protein; operably linked to the upstream sequence of the polynucleotide encoding the HBV pol and truncated core antigen, the upstream The sequence from the 5 'end to the 3' end includes a promoter sequence (preferably the CMV promoter sequence of SEQ ID NO: 17), an enhancer sequence (preferably the ApoAI gene fragment sequence of SEQ ID NO: 23), and a coding signal A polynucleotide sequence of a peptide sequence (preferably an immunoglobulin secretion signal having an amino acid sequence of SEQ ID NO: 19); and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen, The downstream sequence comprises a polyadenylation signal, preferably the SV40 polyadenylation signal of SEQ ID NO: 24.

當本申請案之免疫原性組合包含第一載體，諸如DNA質體或病毒載體，及第二載體，諸如DNA質體或病毒載體時，該第一載體及該第二載體各自之量不受特別限制。舉例而言，第一DNA質體及第二DNA質體可以按重量計以10:1至1:10，諸如按重量計以10:1、9:1、8:1、7:1、6:1、5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9或1:10之比率存在。較佳地，第一DNA質體及第二DNA質體按重量計係以1:1之比率存在。When the immunogenic combination of the present application includes a first vector, such as a DNA plastid or a viral vector, and a second vector, such as a DNA plastid or a viral vector, the respective amounts of the first vector and the second vector are not affected. Special restrictions. For example, the first DNA plastid and the second DNA plastid can be from 10: 1 to 1:10 by weight, such as 10: 1, 9: 1, 8: 1, 7: 1, 6 by weight : 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8 , 1: 9, or 1:10 ratios exist. Preferably, the first DNA plastids and the second DNA plastids exist at a ratio of 1: 1 by weight.

本申請案之組合物及免疫原性組合可以包含編碼另外的HBV抗原之另外的聚核苷酸或載體及/或另外的HBV抗原或其免疫原性片段。然而，在特定實施例中，本申請案之組合物及免疫原性組合不包含某些抗原。The compositions and immunogenic combinations of the present application may comprise additional polynucleotides or vectors encoding additional HBV antigens and / or additional HBV antigens or immunogenic fragments thereof. However, in certain embodiments, the compositions and immunogenic combinations of this application do not include certain antigens.

在一個特定實施例中，本申請案之組合物或免疫原性組合或套組不包含HBsAg或編碼HBsAg之聚核苷酸序列。In a particular embodiment, the composition or immunogenic combination or set of the present application does not contain HBsAg or a polynucleotide sequence encoding HBsAg.

在另一特定實施例中，本申請案之組合物或免疫原性組合或套組不包含HBV L蛋白質或編碼HBV L蛋白質之聚核苷酸序列。In another specific embodiment, the composition or immunogenic combination or set of the present application does not include a HBV L protein or a polynucleotide sequence encoding a HBV L protein.

在本申請案之又一特定實施例中，本申請案之組合物或免疫原性組合不包含HBV包膜蛋白或編碼HBV包膜蛋白之聚核苷酸序列。In another specific embodiment of the present application, the composition or immunogenic combination of the present application does not include an HBV envelope protein or a polynucleotide sequence encoding the HBV envelope protein.

本申請案之組合物及免疫原性組合亦可包含醫藥學上可接受之載劑。醫藥學上可接受之載劑係無毒的且不應干擾活性成分之功效。醫藥學上可接受之載劑可以包括一或多種賦形劑，諸如黏合劑、崩解劑、膨潤劑、懸浮劑、乳化劑、潤濕劑、潤滑劑、調味劑、甜味劑、防腐劑、染料、增溶劑及包覆劑。醫藥學上可接受之載劑可包括媒劑，諸如脂質(奈米)粒子。該載劑或其他材料之確切性質可以取決於投與途徑，例如肌肉內、皮內、皮下、經口、靜脈內、皮膚、黏膜內(例如腸)、鼻內或腹膜內途徑。對於液體可注射製劑，例如懸浮液及溶液，適合的載劑及添加劑包括水、二醇、油、醇、防腐劑、著色劑及類似物。對於固體口服製劑，例如散劑、膠囊、囊片、膠囊錠(gelcap)及錠劑，適合的載劑及添加劑包括澱粉、糖、稀釋劑、成粒劑、潤滑劑、黏合劑、崩解劑及類似物。對於鼻噴霧劑/吸入劑混合物，水溶液/懸浮液可以包含水、二醇、油、潤滑劑、穩定劑、潤濕劑、防腐劑、芳族物、調味劑及類似物作為適合的載劑及添加劑。The compositions and immunogenic combinations of this application may also include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are non-toxic and should not interfere with the efficacy of the active ingredients. Pharmaceutically acceptable carriers may include one or more excipients such as binders, disintegrating agents, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavoring agents, sweeteners, preservatives , Dyes, solubilizers and coating agents. Pharmaceutically acceptable carriers may include vehicles, such as lipid (nano) particles. The exact nature of the carrier or other material may depend on the route of administration, such as the intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., intestinal), intranasal, or intraperitoneal routes. For liquid injectable preparations, such as suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, colorants and the like. For solid oral preparations, such as powders, capsules, caplets, gelcaps, and lozenges, suitable carriers and additives include starch, sugar, diluents, granules, lubricants, binders, disintegrating agents, and analog. For nasal spray / inhalation mixtures, the aqueous solution / suspension may contain water, glycols, oils, lubricants, stabilizers, wetting agents, preservatives, aromatics, flavoring agents, and the like as suitable carriers and additive.

本申請案之組合物及免疫原性組合可以用適於向個體投與之任何物質調配以有助於投與並改善功效，包括(但不限於)經口(腸)投與及非經腸注射。非經腸注射包括靜脈內注射或輸注、皮下注射、皮內注射及肌肉內注射。本申請案之組合物亦可調配用於其他投藥途徑，包括經黏膜、眼、直腸、長效植入、舌下投與(即在舌頭下方，自口腔黏膜投與，繞過門脈循環)、吸入或鼻內投與。The compositions and immunogenic combinations of this application can be formulated with any substance suitable for administration to an individual to facilitate administration and improve efficacy, including (but not limited to) oral (intestinal) administration and parenteral injection. Parenteral injections include intravenous or infusion, subcutaneous, intradermal and intramuscular injection. The composition of this application can also be formulated for other administration routes, including transmucosal, ocular, rectal, long-acting implantation, sublingual administration (ie, under the tongue, from the oral mucosa, bypassing the portal circulation) , Inhalation or intranasal administration.

在本申請案之一個較佳實施例中，本申請案之組合物及免疫原性組合係調配用於非經腸注射，較佳經皮下、皮內注射或肌肉內注射，更佳肌肉內注射。In a preferred embodiment of the present application, the composition and immunogenic combination of the present application are formulated for parenteral injection, preferably subcutaneous, intradermal or intramuscular injection, and more preferably intramuscular injection. .

根據本申請案之實施例，供投與之組合物及免疫原性組合通常將包含於醫藥學上可接受之載劑，例如水性載劑，諸如緩衝生理食鹽水及類似物，例如磷酸鹽緩衝生理食鹽水(PBS)中之緩衝溶液。視需要，該等組合物及免疫原性組合亦可含有醫藥學上可接受之物質以接近生理條件，諸如pH調節及緩衝劑。舉例而言，本申請案的包含質體DNA之組合物或免疫原性組合可以含有磷酸鹽緩衝生理食鹽水(PBS)作為醫藥學上可接受之載劑。質體DNA之存在濃度可以為例如0.5 mg/mL至5 mg/mL，諸如為0.5 mg/mL、1 mg/mL、2 mg/mL、3 mg/mL、4 mg/mL或5 mg/mL，較佳為1 mg/mL。According to the examples of this application, the compositions for administration and immunogenic combinations will typically include a pharmaceutically acceptable carrier, such as an aqueous carrier, such as a buffered saline solution and the like, such as a phosphate buffer Buffer solution in saline (PBS). If desired, these compositions and immunogenic combinations may also contain pharmaceutically acceptable substances to approximate physiological conditions, such as pH adjustment and buffering agents. For example, the plastid DNA-containing composition or immunogenic combination of the present application may contain phosphate buffered saline (PBS) as a pharmaceutically acceptable carrier. The plastid DNA may be present at a concentration of, for example, 0.5 mg / mL to 5 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL , Preferably 1 mg / mL.

可以根據此項技術中熟知之方法，將本申請案之組合物及免疫原性組合調配為疫苗(又稱為「免疫原性組合物」)。此類組合物可以包括用以增強免疫反應之佐劑。根據本發明，調配物中各組分之最佳比率可以藉由熟習此項技術者熟知之技術測定。The composition and immunogenic combination of the present application can be formulated as a vaccine (also known as an "immunogenic composition") according to methods well known in the art. Such compositions may include adjuvants to enhance the immune response. According to the invention, the optimal ratio of the components in the formulation can be determined by techniques well known to those skilled in the art.

在本申請案之一個特定實施例中，組合物或免疫原性組合係DNA疫苗。DNA疫苗通常包括含有處於強真核啟動子控制下的編碼所關注抗原之聚核苷酸的細菌質體。在將質體遞送至宿主之細胞質中之後，即產生並內源性加工編碼之抗原。所得抗原通常誘發體液及細胞介導之免疫反應。DNA疫苗之優勢至少係由於其提供改良之安全性，具有溫度穩定性，可以容易地用於表現抗原變異體且易於製造。可以使用本申請案之DNA質體中之任一種製備此類DNA疫苗。In a particular embodiment of the application, the composition or immunogenic combination is a DNA vaccine. DNA vaccines typically include bacterial plastids containing polynucleotides encoding the antigen of interest under the control of a strong eukaryotic promoter. After the plastids are delivered to the host's cytoplasm, the encoded antigen is produced and endogenously processed. The resulting antigen usually induces humoral and cell-mediated immune responses. The advantages of DNA vaccines are at least because they provide improved safety, temperature stability, can be easily used to express antigen variants, and are easy to manufacture. Such DNA vaccines can be prepared using any of the DNA plastids of the present application.

在本申請案之其他特定實施例中，組合物或免疫原性組合係RNA疫苗。RNA疫苗通常包含至少一個編碼所關注抗原，例如HBV抗原之單股RNA分子。與DNA疫苗類似，在將RNA遞送至宿主之細胞質中之後，即產生並內源性加工編碼之抗原，誘發體液及細胞介導之免疫反應。RNA序列可以經密碼子優化以提高轉譯效率。RNA分子可以根據本發明，藉由此項技術中已知之任何方法，諸如藉由添加例如具有至少30個腺苷殘基之聚腺苷酸尾；及/或用經修飾之核糖核苷酸，例如7-甲基鳥苷帽對5端加帽進行修飾以增進穩定性及/或轉譯，該經修飾之核糖核苷酸可以在RNA合成期間併入或在RNA轉錄之後以酶進行工程改造。RNA疫苗亦可為由α病毒表現載體開發之自我複製RNA疫苗。自我複製RNA疫苗包含來源於屬於α病毒科之病毒的複製酶RNA分子，其具有控制HBV抗原RNA複製之次基因組啟動子，隨後為位於該複製酶下游的人工聚腺苷酸尾。In other specific embodiments of this application, the composition or immunogenic combination is an RNA vaccine. RNA vaccines typically contain at least one single-stranded RNA molecule encoding an antigen of interest, such as an HBV antigen. Similar to DNA vaccines, after the RNA is delivered to the host's cytoplasm, the encoded antigen is produced and endogenously processed to induce humoral and cell-mediated immune responses. The RNA sequence can be codon optimized to improve translation efficiency. The RNA molecule can be according to the present invention by any method known in the art, such as by adding, for example, a polyadenylic acid tail having at least 30 adenosine residues; and / or using modified ribonucleotides, For example, the 7-methylguanosine cap modifies the 5-terminal cap to improve stability and / or translation. The modified ribonucleotide can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. RNA vaccines can also be self-replicating RNA vaccines developed from alphavirus expression vectors. The self-replicating RNA vaccine contains a replicase RNA molecule derived from a virus belonging to the alphaviridae family, which has a subgenomic promoter that controls HBV antigen RNA replication, followed by an artificial polyadenylic acid tail downstream of the replicase.

在某些實施例中，本申請案之組合物或免疫原性組合中包括佐劑，或將佐劑與本申請案之組合物或免疫原性組合共投與。佐劑之使用係可選的，且當該組合物係用於疫苗接種目的時，其可以進一步增強免疫反應。適於共投與或包括在根據本申請案之組合物中的佐劑應較佳地為可能安全、具有良好耐受性且有效用於人類之佐劑。佐劑可以為小分子或抗體，包括(但不限於)免疫檢查點抑制劑(例如抗PD1、抗TIM-3等)、鐸樣受體促效劑(toll-like receptor agonist)(例如TLR7促效劑及/或TLR8促效劑)、RIG-1促效劑、IL-15超級促效劑(Altor Bioscience)、突變IRF3及IRF7基因佐劑、STING促效劑(Aduro)、FLT3L基因佐劑、IL-12基因佐劑及IL-7-hyFc。佐劑亦可例如選自以下抗HBV藥劑：HBV DNA聚合酶抑制劑；免疫調節劑；鐸樣受體7調節劑；鐸樣受體8調節劑；鐸樣受體3調節劑；干擾素α受體配體；玻尿酸酶抑制劑；IL-10調節劑；HBsAg抑制劑；鐸樣受體9調節劑；親環蛋白抑制劑；HBV預防性疫苗；HBV治療性疫苗；HBV病毒進入抑制劑；靶向病毒mRNA之反義寡核苷酸，更具體地說抗HBV反義寡核苷酸；短干擾RNA (siRNA)，更具體地說抗HBV siRNA；核酸內切酶調節劑；核糖核苷酸還原酶抑制劑；B型肝炎病毒E抗原抑制劑；靶向B型肝炎病毒之表面抗原的HBV抗體；HBV抗體；CCR2趨化因子拮抗劑；胸腺素促效劑；細胞介素，諸如IL12；衣殼組裝調節劑、核蛋白抑制劑(HBV核心或衣殼蛋白抑制劑)；核酸聚合物(NAP)；視黃酸誘導性基因1之刺激劑；NOD2刺激劑；重組胸腺素α-1；B型肝炎病毒複製抑制劑；PI3K抑制劑；cccDNA抑制劑；免疫檢查點抑制劑，諸如PD-L1抑制劑、PD-1抑制劑、TIM-3抑制劑、TIGIT抑制劑、Lag3抑制劑及CTLA-4抑制劑；在免疫細胞(更具體地說T細胞)上表現之共刺激受體諸如CD27、CD28等的促效劑；BTK抑制劑；用於治療HBV之其他藥物；IDO抑制劑；精胺酸酶抑制劑；以及KDM5抑制劑。In certain embodiments, an adjuvant is included in the composition or immunogenic combination of the application, or the adjuvant is co-administered with the composition or immunogenic combination of the application. The use of an adjuvant is optional, and when the composition is used for vaccination purposes, it can further enhance the immune response. An adjuvant suitable for co-administration or inclusion in a composition according to the present application should preferably be an adjuvant that may be safe, well tolerated, and effective for use in humans. Adjuvants can be small molecules or antibodies, including (but not limited to) immune checkpoint inhibitors (such as anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (such as TLR7 (Agent and / or TLR8 agonist), RIG-1 agonist, IL-15 super agonist (Altor Bioscience), mutant IRF3 and IRF7 gene adjuvants, STING agonist (Aduro), FLT3L gene adjuvant , IL-12 gene adjuvant and IL-7-hyFc. The adjuvant may also be selected, for example, from the following anti-HBV agents: HBV DNA polymerase inhibitors; immunomodulators; Dod-like receptor 7 modulators; Dod-like receptor 8 modulators; Dod-like receptor 3 modulators; interferon alpha Receptor ligands; Hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; Duo-like receptor 9 modulators; Cyclophilin inhibitors; HBV preventive vaccines; HBV therapeutic vaccines; HBV virus entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more specifically anti-HBV antisense oligonucleotides; short interfering RNA (siRNA), more specifically anti-HBV siRNA; endonuclease modulators; ribonucleosides Acid reductase inhibitors; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting surface antigens of hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines, such as IL12 Capsid assembly regulator, nucleoprotein inhibitor (HBV core or capsid protein inhibitor); Nucleic acid polymer (NAP); Retinoic acid-inducible gene 1 stimulant; NOD2 stimulant; Recombinant thymosin alpha-1 Hepatitis B virus replication inhibitor PI3K inhibitor cccDNA inhibitor immunoassay Checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; on immune cells (more specifically T cells) Co-stimulatory receptors such as CD27, CD28, etc .; BTK inhibitors; Other drugs used to treat HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.

本申請案亦提供製備本申請案之組合物及免疫原性組合的方法。製備組合物或免疫原性組合之方法包含將本申請案的編碼HBV抗原之分離之聚核苷酸、載體及/或多肽與一或多種醫藥學上可接受之載劑混合。一般熟習此項技術者將熟悉用於製備此類組合物之習知技術。This application also provides methods for preparing the compositions and immunogenic combinations of this application. A method of preparing a composition or immunogenic combination comprises mixing the isolated polynucleotide, carrier, and / or polypeptide encoding the HBV antigen of the present application with one or more pharmaceutically acceptable carriers. Those skilled in the art will be familiar with conventional techniques for preparing such compositions.

誘發免疫反應之方法Methods to induce immune response

本申請案亦提供在有需要之個體中誘發針對B型肝炎病毒(HBV)之免疫反應的方法，其包含向該個體投與免疫原性有效量的本申請案之組合物或免疫性組合物。本文所描述的本申請案之組合物及免疫原性組合中之任一種均可用於本申請案之方法中。The application also provides a method for inducing an immune response against hepatitis B virus (HBV) in an individual in need, comprising administering to the individual an immunogenicly effective amount of the composition or immunological composition of the application . Any of the compositions and immunogenic combinations of the application described herein can be used in the methods of the application.

如本文所使用，術語「感染」係指致病物對宿主之侵襲。當致病物能夠侵襲宿主，且在宿主內複製或繁殖時，認為其具有「感染性」。感染物之實例包括病毒，例如HBV及某些物種之腺病毒、朊病毒、細菌、真菌、原蟲及類似物。「HBV感染」特指HBV對宿主生物體，諸如宿主生物體之細胞及組織之侵襲。As used herein, the term "infection" refers to the invasion of a host by a pathogen. Pathogens are considered "infectious" when they can invade the host and replicate or reproduce within the host. Examples of infectious agents include viruses such as HBV and adenoviruses, prions, bacteria, fungi, protozoa and the like of certain species. "HBV infection" specifically refers to the invasion of HBV to a host organism, such as the cells and tissues of the host organism.

當關於本文所描述之方法使用時，短語「誘發免疫反應」涵蓋在有需要之個體中引起針對感染，例如HBV感染之所需免疫反應或作用。「誘發免疫反應」亦涵蓋提供針對病原體，例如HBV之治療性免疫以進行治療。如本文所使用，術語「治療性免疫」或「治療性免疫反應」意思指經疫苗接種之個體能夠控制該疫苗接種所針對之病原體感染，例如藉由用HBV疫苗進行疫苗接種引起針對HBV感染之免疫。在一個實施例中，「誘發免疫反應」意謂在有需要之個體中產生免疫，例如以提供針對疾病，諸如HBV感染之治療作用。在某些實施例中，「誘發免疫反應」係指引起或改善針對HBV感染之細胞免疫，例如T細胞反應。在某些實施例中，「誘發免疫反應」係指引起或改善針對HBV感染之體液免疫反應。在某些實施例中，「誘發免疫反應」係指引起或改善針對HBV感染之細胞及體液免疫反應。When used in connection with the methods described herein, the phrase "eliciting an immune response" encompasses eliciting a desired immune response or effect against an infection, such as a HBV infection, in an individual in need. "Induced immune response" also covers the provision of therapeutic immunity against pathogens such as HBV for treatment. As used herein, the term "therapeutic immunity" or "therapeutic immune response" means that a vaccinated individual is able to control the infection of the pathogen to which the vaccination is directed, for example, by vaccination with the HBV vaccine. Immunity. In one embodiment, "eliciting an immune response" means generating immunity in an individual in need, for example, to provide a therapeutic effect against a disease, such as an HBV infection. In certain embodiments, "evoked immune response" refers to eliciting or improving cellular immunity, such as a T cell response, against HBV infection. In certain embodiments, "evoked immune response" refers to eliciting or improving a humoral immune response against HBV infection. In certain embodiments, "evoked immune response" refers to eliciting or ameliorating cellular and humoral immune responses against HBV infection.

如本文所使用，術語「保護性免疫」或「保護性免疫反應」意思指經疫苗接種之個體能夠控制該疫苗接種所針對之病原體感染。通常，產生「保護性免疫反應」之個體僅產生輕度至中度臨床症狀或完全無症狀。通常，針對某一病原體具有「保護性免疫反應」或「保護性免疫」之個體將不會死於該病原體感染。As used herein, the term "protective immunity" or "protective immune response" means that a vaccinated individual is able to control the infection of the pathogen to which the vaccination is directed. Generally, individuals with a "protective immune response" develop only mild to moderate clinical symptoms or are completely asymptomatic. Generally, individuals with a "protective immune response" or "protective immunity" against a pathogen will not die from an infection with that pathogen.

通常，本申請案之組合物及免疫原性組合的投與將具有治療目的，用於在HBV感染或發展HBV感染特有之症狀之後產生針對HBV之免疫反應，例如用於治療性疫苗接種。Generally, the administration of the compositions and immunogenic combinations of the present application will have therapeutic purposes for generating an immune response against HBV after HBV infection or developing symptoms specific to HBV infection, such as for therapeutic vaccination.

如本文所使用，「免疫原性有效量」或「免疫有效量」意謂足以在有需要之個體中誘發所需免疫作用或免疫反應的組合物、聚核苷酸、載體或抗原之量。免疫原性有效量可以為足以在有需要之個體中誘發免疫反應的量。免疫原性有效量可以為足以在有需要之個體中產生免疫性，例如針對疾病，諸如HBV感染提供治療作用的量。免疫原性有效量可以取決於多種因素而變化，諸如個體之身體狀況，年齡、體重、健康狀況等；具體應用，例如提供保護性免疫或治療性免疫；以及免疫需要針對之具體疾病，例如病毒感染。一般熟習此項技術者根據本發明可以容易地確定免疫原性有效量。As used herein, "immunogenically effective amount" or "immunologically effective amount" means an amount of a composition, polynucleotide, carrier, or antigen sufficient to elicit a desired immune effect or immune response in an individual in need. An immunogenicly effective amount can be an amount sufficient to elicit an immune response in an individual in need. An immunogenicly effective amount can be an amount sufficient to produce immunity in an individual in need, for example, to provide a therapeutic effect against a disease, such as an HBV infection. The immunogenic effective amount can vary depending on a variety of factors, such as the physical condition, age, weight, health status, etc. of the individual; specific applications, such as providing protective or therapeutic immunity; and specific diseases such as viruses that need to be targeted infection. A person skilled in the art can easily determine an immunogenicly effective amount according to the present invention.

在本申請案之特定實施例中，免疫原性有效量係指足以達成以下作用中之一種、兩種、三種、四種或更多種的組合物或免疫原性組合之量：(i) 降低或改善HBV感染或其相關症狀之嚴重程度；(ii)縮短HBV感染或其相關症狀之持續時間；(iii)預防HBV感染或其相關症狀進展；(iv)使HBV感染或其相關症狀消退；(v)預防HBV感染或其相關症狀之發展或發作；(vi)預防HBV感染或其相關症狀之復發；(vii)減少患HBV感染之個體的住院；(viii)縮短患HBV感染之個體之住院時間；(ix)增加患HBV感染之個體之存活期；(x)消除個體之HBV感染；(xi)抑制或減少個體中之HBV複製；及/或(xii)增強或改善另一療法之預防或治療作用。In a specific embodiment of the present application, an immunogenic effective amount refers to an amount sufficient to achieve one, two, three, four or more combinations or immunogenic combinations of the following effects: (i) Reduce or improve the severity of HBV infection or its related symptoms; (ii) shorten the duration of HBV infection or its related symptoms; (iii) prevent the progression of HBV infection or its related symptoms; (iv) make HBV infection or its related symptoms subside (V) prevent the development or onset of HBV infection or its related symptoms; (vi) prevent the recurrence of HBV infection or its related symptoms; (vii) reduce the hospitalization of individuals with HBV infection; (viii) shorten the individuals with HBV infection Length of hospital stay; (ix) increase survival of individuals with HBV infection; (x) eliminate HBV infection in individuals; (xi) inhibit or reduce HBV replication in individuals; and / or (xii) enhance or improve another therapy Preventive or therapeutic effect.

免疫原性有效量亦可為足以減小HBsAg含量以符合臨床血清轉化之發展；利用個體之免疫系統達成持續清除HBsAg以及減少受感染肝細胞；誘導HBV抗原特異性活化之T細胞群；及/或在12個月內達成持續減損HBsAg。目標指標之實例包括低於500個複本之HBsAg國際單位(IU)臨限值之較低HBsAg及/或較高CD8計數。The immunogenic effective amount may also be sufficient to reduce the HBsAg content to meet the development of clinical seroconversion; use the individual's immune system to achieve continuous elimination of HBsAg and reduce infected liver cells; T cell populations that induce specific activation of HBV antigens; and / Or achieve continuous impairment of HBsAg within 12 months. Examples of target indicators include lower HBsAg and / or higher CD8 counts below the HBsAg International Unit (IU) threshold of 500 copies.

作為指導通則，免疫原性有效量當用於DNA質體時可以在約0.1 mg/mL至10 mg/mL總DNA質體範圍內，諸如為0.1 mg/mL、0.25 mg/mL、0.5 mg/mL、0.75 mg/mL、1 mg/mL、1.5 mg/mL、2 mg/mL、3 mg/mL、4 mg/mL、5 mg/mL、6 mg/mL、7 mg/mL、8 mg/mL、9 mg/mL或10 mg/mL。較佳地，DNA質體之免疫原性有效量小於8 mg/mL，更佳小於6 mg/mL，甚至更佳為3-4 mg/mL。免疫原性有效量可以來自一個載體或質體，或來自多個載體或質體。免疫原性有效量可呈單一組合物或呈多種組合物投與，諸如1、2、3、4、5、6、7、8、9或10種組合物(例如錠劑、膠囊或可注射劑，或適於皮內遞送，例如適於使用皮內遞送貼片皮內遞送的任何組合物)，其中多個膠囊或多次注射液的投與總體向個體提供免疫原性有效量。舉例而言，當使用兩個DNA質體時，免疫原性有效量可以為3-4 mg/mL，具有1.5-2 mg/mL各質體。亦可按所謂的初打-加打療程，向個體投與免疫原性有效量，且隨後向該個體投與另一劑免疫原性有效量。此初打-加打療程之一般概念係熟習疫苗領域之技術者熟知的。可以依需要，在該療程中視情況增加投與其他追加劑。As a general guideline, an effective amount of immunogenicity when applied to DNA plastids can range from about 0.1 mg / mL to 10 mg / mL total DNA plastids, such as 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL mL, 9 mg / mL or 10 mg / mL. Preferably, the immunogenic effective amount of DNA plastids is less than 8 mg / mL, more preferably less than 6 mg / mL, even more preferably 3-4 mg / mL. An immunogenicly effective amount can be from one vector or plastid, or from multiple vectors or plastids. An immunogenicly effective amount can be administered in a single composition or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., lozenges, capsules, or injectables Or, suitable for intradermal delivery, such as any composition suitable for intradermal delivery using an intradermal delivery patch), wherein administration of multiple capsules or multiple injections generally provides an immunogenicly effective amount to the individual. For example, when two DNA plastids are used, the immunogenic effective amount can be 3-4 mg / mL with 1.5-2 mg / mL of each plastid. It is also possible to administer an immunogenicly effective amount to an individual and then to administer another dose of the immunogenicly effective amount to the individual in a so-called initial-plus treatment regimen. The general concept of this initiation-plus treatment course is well known to those skilled in the vaccine field. As needed, additional supplements can be administered during the course of treatment as appropriate.

可以藉由將兩個DNA質體(例如編碼HBV核心抗原之第一DNA質體及編碼HBV pol抗原之第二DNA質體)混合並將混合物遞送至單一解剖部位來將包含該兩個質體的免疫原性組合投與個體。或者，可進行兩次獨立的免疫接種，分別遞送單一表現質體。在此類實施例中，無論兩個質體係作為混合物以單次免疫接種投與抑或分兩次獨立的免疫接種投與，第一DNA質體及第二DNA質體均可按重量計以10:1至1:10，諸如按重量計以10:1、9:1、8:1、7:1、6:1、5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9或1:10之比率投與。較佳地，第一DNA質體及第二DNA質體按重量計係以1:1之比率投與。The two plastids can be included by mixing the two plastids (e.g., the first DNA plastid encoding the HBV core antigen and the second DNA plastid encoding the HBV pol antigen) and delivering the mixture to a single anatomical site The immunogenic combination is administered to the individual. Alternatively, two separate immunizations can be performed, each delivering a single plastid. In such embodiments, the first DNA plastids and the second DNA plastids can be weighted by 10 regardless of whether the two plastid systems are administered as a mixture in a single immunization administration or in two separate immunization administrations. : 1 to 1:10, such as 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1 by weight: Dosing at a ratio of 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1:10. Preferably, the first DNA plastid and the second DNA plastid are administered at a ratio of 1: 1 by weight.

較佳地，根據本申請案之方法治療的個體係感染HBV之個體，特別是患有慢性HBV感染之個體。急性HBV感染之特徵在於先天免疫系統之高效活化加上隨後的廣泛適應性反應(例如HBV特異性T細胞、中和抗體)，由此通常造成成功抑制複製或移除受感染之肝細胞。反之，此類反應會因高病毒及抗原負荷而減弱或減小，例如大量產生HBV包膜蛋白，且可以於亞病毒粒子中釋放超過感染性病毒1,000倍之量。Preferably, the individuals treated with the system according to the method of the present application are infected with HBV, especially those with chronic HBV infection. Acute HBV infection is characterized by the efficient activation of the innate immune system coupled with subsequent extensive adaptive responses (eg, HBV-specific T cells, neutralizing antibodies), which often results in successful inhibition of replication or removal of infected liver cells. On the contrary, such reactions will be weakened or reduced due to high virus and antigen load, for example, a large amount of HBV envelope protein is produced, and it can release 1,000 times more infectious virus in subviral particles.

慢性HBV感染係以病毒負荷、肝酶含量(壞死性發炎活性)、HBeAg或HBsAg負荷，或者針對該等抗原之抗體之存在等特徵分階段說明。cccDNA含量保持相對恆定，每個細胞有約10至50個複本，即使病毒血症可能變化極大。cccDNA物種之存留導致慢性化。更具體言之，慢性HBV感染各階段包括：(i)免疫耐受期，以高病毒負荷及正常或最小升高程度之肝酶為特徵；(ii)免疫活化HBeAg陽性期，在此階段觀察到較低或下降之病毒複製程度及明顯升高之肝酶；(iii)非活動性HBsAg攜帶期，該階段係具有低病毒負荷之低複製狀態且在血清中具有可以遵循HBeAg血清轉化之正常肝酶含量；以及(iv)HBeAg陰性期，在該階段中，定期發生病毒複製(再活化)且伴隨肝酶含量之波動，前核心及/或基礎核心啟動子中常有突變，使得受感染細胞無法產生HBeAg。Chronic HBV infection is described in stages based on characteristics such as viral load, liver enzyme content (necrotizing inflammatory activity), HBeAg or HBsAg load, or the presence of antibodies against these antigens. The cccDNA content remains relatively constant, with about 10 to 50 replicas per cell, even though viremia can vary greatly. The persistence of cccDNA species causes chronicity. More specifically, each stage of chronic HBV infection includes: (i) an immune tolerance period, characterized by a high viral load and normal or minimal elevation of liver enzymes; (ii) an immune-activated HBeAg-positive period, observed at this stage To low or decreased viral replication and significantly elevated liver enzymes; (iii) inactive HBsAg carrying period, which is a low replication state with low viral load and normal in serum that can follow HBeAg seroconversion Liver enzyme content; and (iv) HBeAg-negative phase, during which virus replication (reactivation) occurs periodically and accompanied by fluctuations in liver enzyme content, mutations in the precore and / or basal core promoters often cause infected cells Unable to produce HBeAg.

如本文所使用，「慢性HBV感染」係指個體中可偵測到HBV存在超過6個月。患有慢性HBV感染之個體可以處於慢性HBV感染之任何階段。慢性HBV感染係根據其在該領域中之普通含義理解。慢性HBV感染可例如以急性HBV感染之後HBsAg存留達6個月或更長時間為特徵。舉例而言，本文所提及的慢性HBV感染遵循疾病控制與預防中心(Centers for Disease Control and Prevention，CDC)所公開之定義，根據該定義，慢性HBV感染可藉由實驗室標準表徵，諸如：(i)針對B型肝炎核心抗原之IgM抗體呈陰性(IgM抗HBc)及針對B型肝炎表面抗原(HBsAg)、B型肝炎e抗原(HBeAg)或有關B型肝炎病毒DNA之核酸測試呈陽性；或(ii)針對HBsAg或有關HBV DNA之核酸測試呈陽性，或間隔至少6個月兩次針對HBeAg呈陽性。As used herein, "chronic HBV infection" means that the presence of HBV in an individual can be detected for more than 6 months. Individuals with chronic HBV infection can be at any stage of chronic HBV infection. Chronic HBV infection is understood in terms of its ordinary meaning in the field. Chronic HBV infection can be characterized, for example, by HBsAg persistence for 6 months or more after acute HBV infection. For example, the chronic HBV infection mentioned in this article follows the definition published by the Centers for Disease Control and Prevention (CDC). According to this definition, chronic HBV infection can be characterized by laboratory standards, such as: (i) Negative IgM antibodies against hepatitis B core antigen (IgM anti-HBc) and positive nucleic acid tests against hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) or DNA related to hepatitis B virus ; Or (ii) a positive nucleic acid test against HBsAg or related HBV DNA, or a positive test against HBeAg at least twice every 6 months.

較佳地，免疫原性有效量係指足以治療慢性HBV感染的本申請案之組合物或免疫原性組合之量。Preferably, an immunogenic effective amount refers to an amount of the composition or immunogenic combination of the present application sufficient to treat chronic HBV infection.

在一些實施例中，患有慢性HBV感染之個體正在經歷核苷類似物(NUC)治療，且受NUC抑制。如本文所使用，「受NUC抑制」係指個體具有不可偵測之HBV病毒含量及穩定丙胺酸轉胺酶(ALT)含量達至少六個月。核苷/核苷酸類似物治療之實例包括HBV聚合酶抑制劑，諸如恩替卡韋及替諾福韋。較佳地，患有慢性HBV感染之個體不會患上晚期肝纖維化或肝硬化。此類個體通常會具有小於3分的針對纖維化之METAVIR分數及小於9 kPa之肝纖維化掃描(fibroscan)結果。METAVIR分數係藉由B型肝炎患者之肝切片之組織病理學評價來評估炎症及纖維化程度的一種常用評分系統。該評分系統指定兩個標準化數字：一個反映炎症之程度且一個反映纖維化之程度。In some embodiments, individuals with chronic HBV infection are undergoing nucleoside analog (NUC) treatment and are inhibited by NUC. As used herein, "inhibited by NUC" means that an individual has an undetectable HBV virus content and a stable alanine aminotransferase (ALT) content for at least six months. Examples of nucleoside / nucleotide analog treatments include HBV polymerase inhibitors such as entecavir and tenofovir. Preferably, individuals with chronic HBV infection do not develop advanced liver fibrosis or cirrhosis. Such individuals will typically have a METAVIR score for fibrosis of less than 3 points and a fibroscan result of less than 9 kPa. METAVIR score is a commonly used scoring system to evaluate the degree of inflammation and fibrosis by the histopathological evaluation of liver sections of patients with hepatitis B. The scoring system specifies two standardized numbers: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.

咸信消除或減輕慢性HBV可以允許包括病毒誘發肝硬化及肝細胞癌在內之重度肝病的早期疾病攔截。因此，本申請案之方法亦可用作治療HBV誘發之疾病的療法。HBV誘發疾病之實例包括(但不限於)肝硬化、癌症(例如肝細胞癌)及纖維化，特別是以3分或更高的針對纖維化之METAVIR分數為特徵的晚期纖維化。在此類實施例中，免疫原性有效量係足以在12個月內達成HBsAg之持久喪失且明顯減輕臨床疾病(例如肝硬化、肝細胞癌等)的量。Eliminating or alleviating chronic HBV can allow early disease interception of severe liver diseases, including virus-induced cirrhosis and hepatocellular carcinoma. Therefore, the method of the present application can also be used as a treatment for HBV-induced diseases. Examples of HBV-induced diseases include, but are not limited to, liver cirrhosis, cancer (eg, hepatocellular carcinoma), and fibrosis, particularly advanced fibrosis characterized by a METAVIR score for fibrosis of 3 or higher. In such embodiments, an immunogenicly effective amount is an amount sufficient to achieve a permanent loss of HBsAg and significantly reduce clinical disease (eg, cirrhosis, hepatocellular carcinoma, etc.) within 12 months.

根據本申請案之實施例的方法進一步包含向有需要之個體投與另一免疫原性藥劑(諸如另一HBV抗原或其他抗原)或另一抗HBV藥劑(諸如核苷類似物或其他抗HBV藥劑)與本申請案之組合物的組合。舉例而言，另一抗HBV藥劑或免疫原性藥劑可以為小分子或抗體，包括(但不限於)免疫檢查點抑制劑(例如抗PD1、抗TIM-3等)、鐸樣受體促效劑(例如TLR7促效劑及/或TLR8促效劑)、RIG-1促效劑、IL-15超級促效劑(Altor Bioscience)、突變IRF3及IRF7基因佐劑、STING促效劑(Aduro)、FLT3L基因佐劑、IL-12基因佐劑、IL-7-hyFc；結合HBV env之CAR-T(S-CAR細胞)；衣殼組裝調節劑；cccDNA抑制劑、HBV聚合酶抑制劑(例如恩替卡韋及替諾福韋)。該一或多種其他抗HBV活性劑可以例如為小分子、抗體或其抗原結合片段、多肽、蛋白質或核酸。該一或多種其他抗HBV藥劑可例如選自HBV DNA聚合酶抑制劑；免疫調節劑；鐸樣受體7調節劑；鐸樣受體8調節劑；鐸樣受體3調節劑；干擾素α受體配體；玻尿酸酶抑制劑；IL-10調節劑；HBsAg抑制劑；鐸樣受體9調節劑；親環蛋白抑制劑；HBV預防性疫苗；HBV治療性疫苗；HBV病毒進入抑制劑；靶向病毒mRNA之反義寡核苷酸，更具體地說抗HBV反義寡核苷酸；短干擾RNA (siRNA)，更具體地說抗HBV siRNA；核酸內切酶調節劑；核糖核苷酸還原酶抑制劑；B型肝炎病毒E抗原抑制劑；靶向B型肝炎病毒之表面抗原的HBV抗體；HBV抗體；CCR2趨化因子拮抗劑；胸腺素促效劑；細胞介素，諸如IL12；衣殼組裝調節劑、核蛋白抑制劑(HBV核心或衣殼蛋白抑制劑)；核酸聚合物(NAP)；視黃酸誘導性基因1之刺激劑；NOD2刺激劑；重組胸腺素α-1；B型肝炎病毒複製抑制劑；PI3K抑制劑；cccDNA抑制劑；免疫檢查點抑制劑，諸如PD-L1抑制劑、PD-1抑制劑、TIM-3抑制劑、TIGIT抑制劑、Lag3抑制劑及CTLA-4抑制劑；在免疫細胞(更具體地說T細胞)上表現之共刺激受體諸如CD27、CD28等的促效劑；BTK抑制劑；用於治療HBV之其他藥物；IDO抑制劑；精胺酸酶抑制劑；以及KDM5抑制劑。The method according to embodiments of the present application further comprises administering to the individual in need of another immunogenic agent (such as another HBV antigen or other antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) Medicament) in combination with the composition of the present application. For example, another anti-HBV agent or immunogenic agent may be a small molecule or antibody, including (but not limited to) an immune checkpoint inhibitor (e.g., anti-PD1, anti-TIM-3, etc.) Agents (such as TLR7 agonist and / or TLR8 agonist), RIG-1 agonist, IL-15 super agonist (Altor Bioscience), mutant IRF3 and IRF7 gene adjuvants, STING agonist (Aduro) , FLT3L gene adjuvant, IL-12 gene adjuvant, IL-7-hyFc; CAR-T (S-CAR cells) that bind HBV env; capsid assembly regulator; cccDNA inhibitor, HBV polymerase inhibitor (such as Entecavir and Tenofovir). The one or more other anti-HBV active agents may be, for example, a small molecule, an antibody or an antigen-binding fragment thereof, a polypeptide, a protein, or a nucleic acid. The one or more other anti-HBV agents may be selected, for example, from HBV DNA polymerase inhibitors; immunomodulators; Dod-like receptor 7 modulators; Dod-like receptor 8 modulators; Dod-like receptor 3 modulators; interferon alpha Receptor ligands; Hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; Duo-like receptor 9 modulators; Cyclophilin inhibitors; HBV preventive vaccines; HBV therapeutic vaccines; HBV virus entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more specifically anti-HBV antisense oligonucleotides; short interfering RNA (siRNA), more specifically anti-HBV siRNA; endonuclease modulators; ribonucleosides Acid reductase inhibitors; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting surface antigens of hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines, such as IL12 Capsid assembly regulator, nucleoprotein inhibitor (HBV core or capsid protein inhibitor); Nucleic acid polymer (NAP); Retinoic acid-inducible gene 1 stimulant; NOD2 stimulant; Recombinant thymosin alpha-1 Hepatitis B virus replication inhibitor PI3K inhibitor cccDNA inhibitor immunity Checkpoint inhibitors such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; on immune cells (more specifically T cells) Co-stimulatory receptors such as CD27, CD28, etc .; BTK inhibitors; Other drugs used to treat HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.

遞送方法Delivery method

本申請案之組合物及免疫原性組合可以根據本發明，藉由此項技術中已知之任何方法投與個體，包括(但不限於)非經腸投與(例如肌肉內、皮下、靜脈內或皮內注射)、經口投與、經皮投與及鼻投與。較佳地，組合物及免疫原性組合係非經腸(例如藉由肌肉內注射或皮內注射)或經皮投與。The compositions and immunogenic combinations of this application can be administered to an individual according to the present invention by any method known in the art, including (but not limited to) parenteral administration (e.g., intramuscular, subcutaneous, intravenous) Or intradermal), oral, transdermal and nasal administration. Preferably, the composition and immunogenic combination is parenterally (e.g., by intramuscular or intradermal injection) or transdermal administration.

在組合物或免疫原性組合包含一或多個DNA質體的本申請案之一些實施例中，投與可以藉由經皮膚注射，例如肌肉內或皮內注射，較佳肌肉內注射實現。肌肉內注射可以與電穿孔組合，亦即，施加電場以有助於DNA質體遞送至細胞中。如本文所使用，術語「電穿孔」係指使用跨膜電場脈衝在生物膜中誘導產生微觀路徑(孔)。在活體內電穿孔期間，向細胞施加適當幅值及持續時間之電場，誘導短暫的細胞膜滲透性增強狀態，由此實現無法獨自跨越細胞膜之分子的細胞吸收。藉由電穿孔產生此類孔有助於諸如質體、寡核苷酸、siRNA、藥物等生物分子穿過細胞膜之一側到達另一側。經顯示，利用活體內電穿孔來遞送DNA疫苗使宿主細胞對質體之吸收明顯增加，同時亦會在注射部位引起輕度至中度炎症。因此，相較於習知注射，利用皮內或肌肉內電穿孔使轉染效率及免疫反應顯著改良(例如分別增加1,000倍及100倍)。In some embodiments of the application in which the composition or immunogenic combination comprises one or more DNA plastids, administration can be achieved by transdermal injection, such as intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, that is, an electric field is applied to facilitate the delivery of DNA plastids into cells. As used herein, the term "electroporation" refers to the use of transmembrane electric field pulses to induce microscopic paths (pores) in a biofilm. During in vivo electroporation, an electric field of an appropriate amplitude and duration is applied to the cells to induce a transient state of enhanced cell membrane permeability, thereby achieving cellular absorption of molecules that cannot cross the cell membrane alone. Creating such pores by electroporation helps biomolecules such as plastids, oligonucleotides, siRNA, drugs, etc. to pass through one side of the cell membrane to the other. It has been shown that in vivo electroporation to deliver DNA vaccines significantly increases the host cell's uptake of plastids, and also causes mild to moderate inflammation at the injection site. Therefore, compared with the conventional injection, intradermal or intramuscular electroporation can significantly improve transfection efficiency and immune response (for example, 1,000-fold and 100-fold increase, respectively).

在一個典型實施例中，將電穿孔與肌肉內注射組合。然而，亦可將電穿孔與其他非經腸投與形式，例如皮內注射、皮下注射等組合。In a typical embodiment, electroporation is combined with intramuscular injection. However, electroporation can also be combined with other parenteral administration forms, such as intradermal injection, subcutaneous injection, and the like.

經由電穿孔投與本申請案之組合物、免疫原性組合或疫苗可以使用電穿孔器件實現，該等電穿孔器件可經組態以將有效引起細胞膜中形成可逆孔的能量脈衝遞送至所希望的哺乳動物組織。電穿孔器件可以包括電穿孔組件及電極總成或手柄總成。電穿孔組件可以包括以下電穿孔器件組件中之一或多個：控制器、電流波形產生器、阻抗測試儀、波形記錄器、輸入元件、狀態報告元件、通信端口、記憶體組件、電源及電源開關。電穿孔可以使用活體內電穿孔器件實現。可以有助於遞送本申請案之組合物及免疫原性組合，特別是包含DNA質體者的電穿孔器件及電穿孔方法之實例包括CELLECTRA®(Inovio Pharmaceuticals, Blue Bell, PA)、Elgen電穿孔器(Inovio Pharmaceuticals, Inc.)、Tri-Grid^TM 遞送系統(Ichor Medical Systems, Inc., San Diego, CA 92121)，及彼等說明於：美國專利第7,664,545號、美國專利第8,209,006號、美國專利第9,452,285號、美國專利第5,273,525號、美國專利第6,110,161號、美國專利第6,261,281號、美國專利第6,958,060號及美國專利第6,939,862號、美國專利第7,328,064號、美國專利第6,041,252號、美國專利第5,873,849號、美國專利第6,278,895號、美國專利第6,319,901號、美國專利第6,912,417號、美國專利第8,187,249號、美國專利第9,364,664號、美國專利第9,802,035號、美國專利第6,117,660號，及國際專利申請公開案WO2017172838中者，其等皆以全文引用的方式併入本文中。活體內電穿孔器件之其他實例描述於與本申請案同一天提交的標題名稱為「用於遞送B型肝炎病毒(HBV)疫苗之方法及裝置(Method and Apparatus for the Delivery of Hepatitis B Virus(HBV)Vaccines)」的代理人案號為688097-405WO1之國際專利申請案中，其內容以全文引用的方式併入本文中。用於遞送本申請案之組合物及免疫原性組合的申請案亦涵蓋使用脈衝電場，如例如美國專利第6,697,669號中所描述，其以全文引用的方式併入本文中。Compositions, immunogenic combinations, or vaccines administered to the present application via electroporation can be achieved using electroporation devices that can be configured to deliver the energy pulses that effectively cause the formation of reversible pores in the cell membrane to the desired Mammalian tissue. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include one or more of the following electroporation device components: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input component, a status report component, a communication port, a memory component, a power source, and a power source switch. Electroporation can be achieved using in vivo electroporation devices. Examples of electroporation devices and electroporation methods that can facilitate the delivery of the compositions and immunogenic combinations of the present application, particularly those containing DNA plastids include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Elgen electroporation Device (Inovio Pharmaceuticals, Inc.), Tri-Grid ^TM delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121), and they are described in: U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, US Patent No. 5,273,525, US Patent No. 6,110,161, US Patent No. 6,261,281, US Patent No. 6,958,060 and US Patent No. 6,939,862, US Patent No. 7,328,064, US Patent No. 6,041,252, US Patent No. 5,873,849 U.S. Patent No. 6,278,895, U.S. Patent No. 6,319,901, U.S. Patent No. 6,912,417, U.S. Patent No. 8,187,249, U.S. Patent No. 9,364,664, U.S. Patent No. 9,802,035, U.S. Patent No. 6,117,660, and International Patent Application Publication Those in WO2017172838, all of which are incorporated herein by reference in their entirety. Other examples of in vivo electroporation devices are described on the same day as this application with the title `` Method and Apparatus for the Delivery of Hepatitis B Virus (HBV The international patent application No. 688097-405WO1, the content of which is incorporated by reference in its entirety, is the agent's case number 688097-405WO1. Applications for the delivery of compositions and immunogenic combinations of this application also cover the use of pulsed electric fields, as described, for example, in US Patent No. 6,697,669, which is incorporated herein by reference in its entirety.

在組合物或免疫原性組合包含一或多個DNA質體的本申請案之其他實施例中，投與方法係經皮投與。經皮投與可以與表皮磨蝕組合，以有助於將DNA質體遞送至細胞。舉例而言，可以使用皮膚貼片進行表皮磨蝕。在移除皮膚貼片後，組合物或免疫原性組合可以沈積於經磨蝕之皮膚上。In other embodiments of the application in which the composition or immunogenic combination comprises one or more DNA plastids, the method of administration is percutaneous administration. Transdermal administration can be combined with epidermal abrasion to help deliver DNA plastids to cells. For example, a skin patch can be used for epidermal abrasion. After removing the skin patch, the composition or immunogenic combination can be deposited on the abraded skin.

遞送方法不限於上述實施例，且用於細胞內遞送之任何手段均可使用。本申請案之方法所涵蓋的其他細胞內遞送方法包括(但不限於)脂質體包封、奈米粒子等。The method of delivery is not limited to the examples described above, and any means for intracellular delivery can be used. Other intracellular delivery methods covered by the methods of this application include, but are not limited to, liposome encapsulation, nanoparticle, and the like.

佐劑Adjuvant

在本申請案之一些實施例中，誘發針對HBV之免疫反應的方法進一步包含投與佐劑。術語「佐劑」與「免疫刺激劑」在本文中可互換地使用，且定義為刺激免疫系統之一或多種物質。在此情形下，佐劑係用於增強針對本申請案之HBV抗原及抗原性HBV多肽之免疫反應。In some embodiments of the present application, the method of inducing an immune response against HBV further comprises administering an adjuvant. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that stimulate the immune system. In this case, the adjuvant is used to enhance the immune response against the HBV antigen and antigenic HBV polypeptide of the present application.

根據本申請案之實施例，佐劑可以存在於本申請案之免疫原性組合或組合物中，或以獨立組合物形式投與。佐劑可以為例如小分子或抗體。適用於本申請案中之佐劑的實例包括(但不限於)免疫檢查點抑制劑(例如抗PD1、抗TIM-3等)、鐸樣受體促效劑(例如TLR7促效劑及/或TLR8促效劑)、RIG-1促效劑、IL-15超級促效劑(Altor Bioscience)、突變IRF3及IRF7基因佐劑、STING促效劑(Aduro)、FLT3L基因佐劑、IL-12基因佐劑及IL-7-hyFc。佐劑之實例可以例如選自以下抗HBV藥劑：HBV DNA聚合酶抑制劑；免疫調節劑；鐸樣受體7調節劑；鐸樣受體8調節劑；鐸樣受體3調節劑；干擾素α受體配體；玻尿酸酶抑制劑；IL-10調節劑；HBsAg抑制劑；鐸樣受體9調節劑；親環蛋白抑制劑；HBV預防性疫苗；HBV治療性疫苗；HBV病毒進入抑制劑；靶向病毒mRNA之反義寡核苷酸，更具體地說抗HBV反義寡核苷酸；短干擾RNA (siRNA)，更具體地說抗HBV siRNA；核酸內切酶調節劑；核糖核苷酸還原酶抑制劑；B型肝炎病毒E抗原抑制劑；靶向B型肝炎病毒之表面抗原的HBV抗體；HBV抗體；CCR2趨化因子拮抗劑；胸腺素促效劑；細胞介素，諸如IL12；衣殼組裝調節劑、核蛋白抑制劑(HBV核心或衣殼蛋白抑制劑)；核酸聚合物(NAP)；視黃酸誘導性基因1之刺激劑；NOD2刺激劑；重組胸腺素α-1；B型肝炎病毒複製抑制劑；PI3K抑制劑；cccDNA抑制劑；免疫檢查點抑制劑，諸如PD-L1抑制劑、PD-1抑制劑、TIM-3抑制劑、TIGIT抑制劑、Lag3抑制劑及CTLA-4抑制劑；在免疫細胞(更具體地說T細胞)上表現之共刺激受體諸如CD27、CD28等的促效劑；BTK抑制劑；用於治療HBV之其他藥物；IDO抑制劑；精胺酸酶抑制劑；以及KDM5抑制劑。According to embodiments of the present application, the adjuvant may be present in the immunogenic combination or composition of the present application, or administered as a separate composition. Adjuvants can be, for example, small molecules or antibodies. Examples of adjuvants suitable for use in this application include, but are not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), dorsi-like receptor agonists (e.g., TLR7 agonists and / or TLR8 agonist), RIG-1 agonist, IL-15 super agonist (Altor Bioscience), mutant IRF3 and IRF7 gene adjuvants, STING agonist (Aduro), FLT3L gene adjuvant, IL-12 gene Adjuvant and IL-7-hyFc. Examples of adjuvants can be selected, for example, from the following anti-HBV agents: HBV DNA polymerase inhibitors; immunomodulators; Dod-like receptor 7 modulators; Dod-like receptor 8 modulators; Dod-like receptor 3 modulators; interferons Alpha receptor ligands; Hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; Duo-like receptor 9 modulators; Cyclophilin inhibitors; HBV preventive vaccines; HBV therapeutic vaccines; HBV virus entry inhibitors Antisense oligonucleotides targeting viral mRNA, more specifically anti-HBV antisense oligonucleotides; short interfering RNA (siRNA), more specifically anti-HBV siRNA; endonuclease regulators; ribonucleic acid Hepatitis B reductase inhibitors; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting surface antigens of hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines, such as IL12; capsid assembly regulator, nucleoprotein inhibitor (HBV core or capsid protein inhibitor); nucleic acid polymer (NAP); retinoic acid-inducible gene 1 stimulant; NOD2 stimulant; recombinant thymosin α- 1; Hepatitis B virus replication inhibitor; PI3K inhibitor; cccDNA inhibitor; Immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; in immune cells (more specifically T cells) Co-stimulatory receptors such as CD27, CD28, and other agonists; BTK inhibitors; other drugs used to treat HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors.

本申請案之組合物及免疫原性組合亦可與至少一種其他抗HBV藥劑組合投與。適合用於本申請案的抗HBV藥劑之實例包括(但不限於)小分子、抗體，及/或結合HBV env之CAR-T療法(S-CAR細胞)、衣殼組裝調節劑、TLR促效劑(例如TLR7及/或TLR8促效劑)、cccDNA抑制劑、HBV聚合酶抑制劑(例如恩替卡韋及替諾福韋)及/或免疫檢查點抑制劑等。該至少一種抗HBV藥劑可例如選自HBV DNA聚合酶抑制劑；免疫調節劑；鐸樣受體7調節劑；鐸樣受體8調節劑；鐸樣受體3調節劑；干擾素α受體配體；玻尿酸酶抑制劑；IL-10調節劑；HBsAg抑制劑；鐸樣受體9調節劑；親環蛋白抑制劑；HBV預防性疫苗；HBV治療性疫苗；HBV病毒進入抑制劑；靶向病毒mRNA之反義寡核苷酸，更具體地說抗HBV反義寡核苷酸；短干擾RNA (siRNA)，更具體地說抗HBV siRNA；核酸內切酶調節劑；核糖核苷酸還原酶抑制劑；B型肝炎病毒E抗原抑制劑；靶向B型肝炎病毒之表面抗原的HBV抗體；HBV抗體；CCR2趨化因子拮抗劑；胸腺素促效劑；細胞介素，諸如IL12；衣殼組裝調節劑、核蛋白抑制劑(HBV核心或衣殼蛋白抑制劑)；核酸聚合物(NAP)；視黃酸誘導性基因1之刺激劑；NOD2刺激劑；重組胸腺素α-1；B型肝炎病毒複製抑制劑；PI3K抑制劑；cccDNA抑制劑；免疫檢查點抑制劑，諸如PD-L1抑制劑、PD-1抑制劑、TIM-3抑制劑、TIGIT抑制劑、Lag3抑制劑及CTLA-4抑制劑；在免疫細胞(更具體地說T細胞)上表現之共刺激受體諸如CD27、CD28等的促效劑；BTK抑制劑；用於治療HBV之其他藥物；IDO抑制劑；精胺酸酶抑制劑；以及KDM5抑制劑。此類抗HBV藥劑可以與本申請案之組合物及免疫原性組合同時或依序投與。The compositions and immunogenic combinations of this application can also be administered in combination with at least one other anti-HBV agent. Examples of anti-HBV agents suitable for use in this application include, but are not limited to, small molecules, antibodies, and / or CAR-T therapies (S-CAR cells) that bind HBV env, capsid assembly regulators, TLR agonists Agents (such as TLR7 and / or TLR8 agonists), cccDNA inhibitors, HBV polymerase inhibitors (such as entecavir and tenofovir), and / or immune checkpoint inhibitors, and the like. The at least one anti-HBV agent may be selected, for example, from a HBV DNA polymerase inhibitor; an immunomodulator; a tudor-like receptor 7 modulator; a tudor-like receptor 8 modulator; a tudor-like receptor 3 modulator; an interferon alpha receptor Ligand; Hyaluronidase inhibitor; IL-10 modulator; HBsAg inhibitor; Duo-like receptor 9 modulator; Cyclophilin inhibitor; HBV preventive vaccine; HBV therapeutic vaccine; HBV virus entry inhibitor; Targeting Antisense oligonucleotides for viral mRNA, more specifically anti-HBV antisense oligonucleotides; short interfering RNA (siRNA), more specifically anti-HBV siRNA; endonuclease modulators; ribonucleotide reduction Enzyme inhibitors; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting surface antigens of hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines such as IL12; clothing Shell assembly regulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAP); retinoic acid-inducible gene 1 stimulants; NOD2 stimulants; recombinant thymosin alpha-1; B Hepatitis virus replication inhibitor; PI3K inhibitor; cccDNA inhibitor; immunoassay Point inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; manifest on immune cells (more specifically T cells) Co-stimulatory receptors such as CD27, CD28 and other agonists; BTK inhibitors; other drugs used to treat HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors. Such anti-HBV agents can be administered simultaneously or sequentially with the compositions and immunogenic combinations of the present application.

初打First hit // 加打免疫接種方法Plus immunization method

本申請案之實施例亦涵蓋在所謂的初打-加打療程中，向個體投與免疫原性有效量之組合物或免疫原性組合，且隨後向該個體投與另一劑免疫原性有效量之組合物或免疫原性組合。因此，在一個實施例中，本申請案之組合物或免疫原性組合係用於引發免疫反應之初打疫苗。在另一個實施例中，本申請案之組合物或免疫原性組合係用於增強免疫反應之加打疫苗。本申請案之初打及加打疫苗可以用於本文所描述的本申請案之方法中。此初打-加打療程之一般概念係熟習疫苗領域之技術者熟知的。本文所描述的本申請案之組合物及免疫原性組合中之任一種可以用作初打及/或加打疫苗以引發及/或增強針對HBV之免疫反應。The examples of the present application also cover the administration of an immunogenicly effective amount of a composition or immunogenic combination to an individual during a so-called start-and-treat regimen, and subsequent administration of another dose of immunogenicity to the individual An effective amount of a composition or immunogenic combination. Therefore, in one embodiment, the composition or immunogenic combination of the present application is used to initiate an initial vaccine for an immune response. In another embodiment, the composition or immunogenic combination of the present application is an additional vaccine for enhancing an immune response. The initial and additional vaccines of this application can be used in the methods of this application described herein. The general concept of this initiation-plus treatment course is well known to those skilled in the vaccine field. Any of the compositions and immunogenic combinations of the application described herein can be used as a primary and / or additional vaccine to elicit and / or enhance an immune response against HBV.

在本申請案之一些實施例中，本申請案之組合物或免疫原性組合可以投與用於初打免疫接種。該組合物或免疫原性組合可以再投與用於加打免疫接種。視需要，該組合物或疫苗組合之進一步加打投與可以視情況添加至該療程中。佐劑可以存在於用於加打免疫接種的本申請案之組合物中，存在於欲投與本申請案之組合物或免疫原性組合一起投與以用於加打免疫接種的獨立組合物中，或獨自投與以作為加打免疫接種。在該療程中包括佐劑的該等實施例中，佐劑較佳用於加打免疫接種。In some embodiments of the present application, the composition or immunogenic combination of the present application may be administered for primary immunization. The composition or immunogenic combination can be re-administered for additional immunizations. If necessary, further administration and administration of the composition or vaccine combination can be added to the course of treatment as appropriate. The adjuvant may be present in the composition of the present application for booster immunization, in a separate composition for booster immunization that is intended to be administered with the composition of the present application or an immunogenic combination Medium, or administered alone as an additional immunization. In those embodiments in which the adjuvant is included in the course of treatment, the adjuvant is preferably used for booster immunization.

初打-加打療程之說明性且非限制性實例包括向個體投與單次劑量的免疫原性有效量之本申請案之組合物或免疫原性組合以引發免疫反應；且隨後投與另一劑免疫原性有效量的本申請案之組合物或免疫原性組合以增強免疫反應，其中該加打免疫接種先在初始投與初打免疫接種之後約兩至六週，較佳四週投與。視情況，在初始投與初打免疫接種之後約10至14週，較佳12週，再投與該組合物或免疫原性組合，或其他佐劑之加打免疫接種。Illustrative and non-limiting examples of initial-plus-treatment courses include administering to a subject a single dose of an immunogenicly effective amount of a composition or immunogenic combination of this application to elicit an immune response; and subsequent administration to another One dose of an immunogenic effective amount of the composition or combination of immunogenicity of the present application to enhance the immune response, wherein the additional immunization is first administered approximately two to six weeks after the initial immunization, preferably four weeks after administration versus. Depending on the situation, about 10 to 14 weeks, preferably 12 weeks after the initial administration of the first immunization, the composition or the immunogenic combination, or the additional immunization with other adjuvants, is administered.

套組Set

本文亦提供一種包含本申請案之免疫原性組合的套組。套組可以包含呈獨立組合物形式之第一聚核苷酸及第二聚核苷酸，或套組可以包含呈單一組合物形式之第一聚核苷酸及第二聚核苷酸。套組可以進一步包含一或多種佐劑或免疫刺激劑，及/或其他抗HBV藥劑。Also provided herein is a kit comprising the immunogenic combination of the present application. The kit may include the first polynucleotide and the second polynucleotide in the form of a separate composition, or the kit may include the first polynucleotide and the second polynucleotide in the form of a single composition. The kit may further include one or more adjuvants or immunostimulants, and / or other anti-HBV agents.

在投與動物或人類生物體中時誘發或刺激抗HBV免疫反應的能力可以使用此項技術中之多種標準分析法在活體外或活體內評價。有關可用於評價免疫反應之起始及活化之技術的大體描述，參見例如Coligan等人(1992及1994年, Current Protocols in Immunology; J Wiley & Sons Inc編, National Institute of Health)。細胞免疫性之量測可藉由量測由活化之效應細胞，包括來源於CD4+及CD8+ T細胞之該等細胞所分泌之細胞介素譜(例如藉由ELISPOT定量產生IL-10或IFN γ之細胞)、藉由確定免疫效應細胞之活化狀態(例如藉由經典的[³ H]胸苷吸收或基於流式細胞測量術進行之T細胞增殖分析)、藉由分析致敏個體中之抗原特異性T淋巴細胞(例如細胞毒性分析中之肽特異性溶解等)進行。The ability to elicit or stimulate an anti-HBV immune response when administered to an animal or human organism can be evaluated in vitro or in vivo using a variety of standard analytical methods in this technology. For a general description of techniques that can be used to assess the initiation and activation of an immune response, see, for example, Coligan et al. (1992 and 1994, Current Protocols in Immunology; edited by J Wiley & Sons Inc, National Institute of Health). Cellular immunity can be measured by measuring the interleukin profile secreted by activated effector cells, including those derived from CD4 + and CD8 + T cells (e.g., quantitative production of IL-10 or IFNγ by ELISPOT Cells), by determining the activation status of immune effector cells (such as by classical [ ³ H] thymidine uptake or T cell proliferation analysis based on flow cytometry), by analyzing antigen specificity in sensitized individuals T lymphocytes (such as peptide-specific lysis in cytotoxicity analysis).

刺激細胞及/或體液反應之能力可以藉由抗體結合及/或結合競爭來測定(參見例如，Harlow, 1989, Antibodies, Cold Spring Harbor Press)。舉例而言，可以藉由酶聯結免疫吸附分析法(ELISA)量測響應於提供免疫原之組合物之投與而產生之抗體的力價。亦可藉由中和抗體分析法量測免疫反應，其中病毒之中和定義為經由特異性抗體對該病毒之反應/抑制/中和引起之感染性損失。免疫反應亦可藉由抗體依賴性細胞吞噬作用(ADCP)分析法量測。
實施例The ability to stimulate cellular and / or humoral responses can be determined by antibody binding and / or binding competition (see, eg, Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, the potency of antibodies produced in response to administration of a composition that provides an immunogen can be measured by an enzyme-linked immunosorbent assay (ELISA). The immune response can also be measured by neutralizing antibody analysis, where virus neutralization is defined as the loss of infectivity caused by the reaction / inhibition / neutralization of the virus via specific antibodies. Immune response can also be measured by antibody-dependent cellular phagocytosis (ADCP) analysis.
Examples

實施例部分Example section 11

實施例1包含一種分離或非天然存在之核酸分子，其包含編碼截短之HBV核心抗原的聚核苷酸序列，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致，較佳與SEQ ID NO: 2或SEQ ID NO: 14達100%一致的胺基酸序列組成；較佳該截短之HBV核心抗原能夠在哺乳動物中誘發針對至少兩種HBV基因型的免疫反應；更佳該截短之HBV核心抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應；又更佳地，該截短之HBV核心抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應；且最佳地，該截短之HBV核心抗原編碼序列包含與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致，諸如90%、91%、92%、93%、94%、95%、95.5%、96%、96.5%、97%、97.5%、98%、98.5%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或100%一致的聚核苷酸。Example 1 comprises an isolated or non-naturally-occurring nucleic acid molecule comprising a polynucleotide sequence encoding a truncated HBV core antigen, the truncated HBV core antigen is composed of SEQ ID NO: 2 or SEQ ID NO: 14 At least 90% identical, preferably 100% identical amino acid sequence composition to SEQ ID NO: 2 or SEQ ID NO: 14; preferably the truncated HBV core antigen is capable of inducing mammalian targeting of at least two types of HBV Genotype immune response; more preferably, the truncated HBV core antigen can elicit a T-cell response against at least HBV genotypes B, C, and D in mammals; more preferably, the truncated HBV core antigen can A human individual induces a CD8 T cell response against at least HBV genotypes A, B, C, and D; and most preferably, the truncated HBV core antigen coding sequence comprises at least 90% consistent, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% consistent polynucleotides.

實施例2包含如實施例1之非天然存在之核酸分子，其中該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成。Example 2 includes a non-naturally occurring nucleic acid molecule as in Example 1, wherein the truncated HBV core antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14.

實施例3包含如實施例1或實施例2之非天然存在之核酸分子，其進一步包含編碼可操作地連接至該HBV聚合酶抗原之信號序列的聚核苷酸序列。Example 3 includes a non-naturally occurring nucleic acid molecule as in Example 1 or Example 2, further comprising a polynucleotide sequence encoding a signal sequence operably linked to the HBV polymerase antigen.

實施例4包含如實施例3之非天然存在之核酸分子，其中該信號序列包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，較佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Example 4 includes a non-naturally occurring nucleic acid molecule as in Example 3, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is composed of SEQ ID NO: 5 Or the polynucleotide sequence of SEQ ID NO: 18.

實施例5包含如實施例1至4中任一項之非天然存在之核酸分子，其中編碼該截短之核心HBV抗原的該聚核苷酸序列與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致。Example 5 includes a non-naturally occurring nucleic acid molecule as in any one of Examples 1 to 4, wherein the polynucleotide sequence encoding the truncated core HBV antigen and SEQ ID NO: 1 or SEQ ID NO: 15 At least 90% consistent.

實施例6包含如實施例5之非天然存在之核酸分子，其中編碼該截短之核心HBV抗原的該聚核苷酸序列包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。Example 6 includes a non-naturally occurring nucleic acid molecule as in Example 5, wherein the polynucleotide sequence encoding the truncated core HBV antigen comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15 .

實施例7包括含編碼HBV聚合酶抗原之第一聚核苷酸序列的非天然存在之核酸分子，該HBV聚合酶抗原包含與SEQ ID NO: 4至少98%一致的胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性。Example 7 includes a non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an HBV polymerase antigen, the HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and ribonuclease H activity.

實施例8包含如實施例7之非天然存在之核酸分子，其中該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應，較佳該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應，且更佳地，該HBV聚合酶抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。Example 8 includes a non-naturally occurring nucleic acid molecule as in Example 7, wherein the HBV polymerase antigen is capable of eliciting an immune response against at least two HBV genotypes in a mammal, and preferably the HBV polymerase antigen is capable of Induces a T cell response against at least HBV genotypes B, C, and D, and more preferably, the HBV polymerase antigen is capable of inducing a CD8 T cell response against at least HBV genotypes A, B, C, and D in a human individual .

實施例9包含如實施例7之非天然存在之核酸分子，其中該HBV聚合酶抗原包含SEQ ID NO: 4之胺基酸序列。Example 9 includes a non-naturally occurring nucleic acid molecule as in Example 7, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

實施例10包含如實施例7至9中任一項之非天然存在之核酸分子，其進一步包含編碼可操作地連接至該HBV聚合酶抗原之信號序列的聚核苷酸序列。Example 10 includes a non-naturally occurring nucleic acid molecule as in any of Examples 7 to 9, further comprising a polynucleotide sequence encoding a signal sequence operably linked to the HBV polymerase antigen.

實施例11包含如實施例10之非天然存在之核酸分子，其中該信號序列包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，較佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Example 11 includes a non-naturally occurring nucleic acid molecule as in Example 10, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is composed of SEQ ID NO: 5 Or the polynucleotide sequence of SEQ ID NO: 18.

實施例12包含如實施例7至11中任一項之非天然存在之核酸分子，其中該第一聚核苷酸序列與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致。Example 12 includes a non-naturally occurring nucleic acid molecule as in any of Examples 7 to 11, wherein the first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16.

實施例13包含如實施例12之非天然存在之核酸分子，其中該第一聚核苷酸序列包含SEQ ID NO: 3或SEQ ID NO: 16之聚核苷酸序列。Example 13 includes a non-naturally occurring nucleic acid molecule as in Example 12, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

實施例14包含如實施例7至13中任一項之非天然存在之核酸分子，其進一步包含編碼截短之HBV核心抗原的第二聚核苷酸序列，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成。Example 14 includes a non-naturally occurring nucleic acid molecule as in any one of Examples 7 to 13, further comprising a second polynucleotide sequence encoding a truncated HBV core antigen, the truncated HBV core antigen is represented by SEQ Consisting of the amino acid sequence of ID NO: 2 or SEQ ID NO: 14.

實施例15包含如實施例14之非天然存在之核酸分子，其中該第二聚核苷酸序列與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致。Example 15 includes a non-naturally occurring nucleic acid molecule as in Example 14, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

實施例16包含如實施例15之非天然存在之核酸分子，其中該第二聚核苷酸序列包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。Example 16 includes a non-naturally occurring nucleic acid molecule as in Example 15, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

實施例17包含如實施例14至16中任一項之非天然存在之核酸分子，其編碼包含該截短之HBV核心抗原可操作地連接至該HBV聚合酶抗原的融合蛋白。Example 17 comprises a non-naturally occurring nucleic acid molecule as in any of Examples 14 to 16, which encodes a fusion protein comprising the truncated HBV core antigen operably linked to the HBV polymerase antigen.

實施例18包含如實施例17之非天然存在之核酸分子，其中該融合蛋白包含該截短之HBV核心抗原經由連接子可操作地連接至該HBV聚合酶抗原。Example 18 includes a non-naturally occurring nucleic acid molecule as in Example 17, wherein the fusion protein comprises the truncated HBV core antigen operably linked to the HBV polymerase antigen via a linker.

實施例19包含如實施例18之非天然存在之核酸分子，其中該連接子包含胺基酸序列(AlaGly)n，且n係2至5之整數，較佳該連接子係由包含SEQ ID NO: 22之聚核苷酸序列編碼。Example 19 includes a non-naturally occurring nucleic acid molecule as in Example 18, wherein the linker comprises an amino acid sequence (AlaGly) n, and n is an integer from 2 to 5, preferably the linker is composed of SEQ ID NO : 22 polynucleotide sequence code.

實施例20包含如實施例19之非天然存在之核酸分子，其中該融合蛋白包含SEQ ID NO: 20之胺基酸序列。Example 20 includes a non-naturally occurring nucleic acid molecule as in Example 19, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 20.

實施例21包含如實施例17至20中任一項之非天然存在之核酸分子，其中該融合蛋白進一步包含信號序列，較佳該信號序列包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，更佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Embodiment 21 comprises a non-naturally occurring nucleic acid molecule according to any one of embodiments 17 to 20, wherein the fusion protein further comprises a signal sequence, preferably the signal sequence comprises an amine of SEQ ID NO: 6 or SEQ ID NO: 19 Amino acid sequence, more preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 18.

實施例22包含如實施例7至21中任一項之非天然存在之核酸分子，其進一步包含啟動子序列、視情況一或多個另外的調控序列，較佳該啟動子序列包含SEQ ID NO: 7或SEQ ID NO: 17之聚核苷酸序列，且該另外的調控序列係選自由以下組成之群：SEQ ID NO: 8或SEQ ID NO: 23之強化子序列，及SEQ ID NO: 11或SEQ ID NO:24之聚腺苷酸化信號序列。Example 22 comprises a non-naturally occurring nucleic acid molecule as in any of Examples 7 to 21, further comprising a promoter sequence, and optionally one or more additional regulatory sequences, preferably the promoter sequence comprises SEQ ID NO : 7 or the polynucleotide sequence of SEQ ID NO: 17, and the additional regulatory sequence is selected from the group consisting of the enhancer sequence of SEQ ID NO: 8 or SEQ ID NO: 23, and SEQ ID NO: 11 or the polyadenylation signal sequence of SEQ ID NO: 24.

實施例23包含如實施例7至22中任一項之非天然存在之核酸分子，其中該非天然存在之核酸分子不編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群的HBV抗原。Example 23 includes a non-naturally occurring nucleic acid molecule as in any of Examples 7 to 22, wherein the non-naturally occurring nucleic acid molecule does not encode a member selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, A group of HBV antigens composed of HBV L protein antigens.

實施例24包含一種載體，其包含如實施例1至24中任一項之非天然存在之核酸分子。Example 24 includes a vector comprising a non-naturally occurring nucleic acid molecule as in any of Examples 1-24.

實施例25包含如實施例24之載體，其中該非天然存在之核酸分子自5'端至3'端包含啟動子序列、強化子序列、信號肽編碼序列、該第一聚核苷酸序列及聚腺苷酸化信號序列，視情況該非天然存在之核酸分子進一步包含該第二聚核苷酸序列。Example 25 includes the vector of Example 24, wherein the non-naturally-occurring nucleic acid molecule comprises a promoter sequence, an enhancer sequence, a signal peptide coding sequence, the first polynucleotide sequence, and a polynucleotide from the 5 'end to the 3' end. The adenylation signal sequence, and optionally the non-naturally occurring nucleic acid molecule further comprises the second polynucleotide sequence.

實施例26包含如實施例24或25之載體，其中該載體係質體DNA載體，且該質體DNA載體進一步包含複製起點及抗生素抗性基因。Example 26 includes the vector of Example 24 or 25, wherein the vector is a plastid DNA vector, and the plastid DNA vector further includes an origin of replication and an antibiotic resistance gene.

實施例27包含如實施例26之載體，其中該質體DNA載體含有包含SEQ ID NO: 10之聚核苷酸序列的複製起點、包含SEQ ID NO: 12之聚核苷酸序列的抗生素抗性基因、包含SEQ ID NO: 7之聚核苷酸序列的啟動子序列、包含SEQ ID NO: 8之聚核苷酸序列的強化子序列、包含SEQ ID NO: 5之聚核苷酸序列的信號肽編碼序列、包含SEQ ID NO: 3之聚核苷酸序列的第一聚核苷酸序列及包含SEQ ID NO: 11之聚核苷酸序列的聚腺苷酸化信號序列，及視情況，包含SEQ ID NO: 1之聚核苷酸序列的第二聚核苷酸序列。Example 27 includes the vector of Example 26, wherein the plastid DNA vector contains an origin of replication comprising the polynucleotide sequence of SEQ ID NO: 10, and antibiotic resistance comprising the polynucleotide sequence of SEQ ID NO: 12 Gene, a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 7, a enhancer sequence comprising the polynucleotide sequence of SEQ ID NO: 8, a signal comprising the polynucleotide sequence of SEQ ID NO: 5 A peptide coding sequence, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11, and optionally, including The second polynucleotide sequence of the polynucleotide sequence of SEQ ID NO: 1.

實施例28包含如實施例24或25之載體，其中該載體係腺病毒載體，較佳為Ad26或Ad35載體。Example 28 includes a vector as in Example 24 or 25, wherein the vector is an adenovirus vector, preferably an Ad26 or Ad35 vector.

實施例29包含如實施例28之載體，其中該載體係包含編碼如實施例1至6中任一項之截短之核心HBV抗原的非天然存在之核酸的Ad26載體。Example 29 comprises a vector as in Example 28, wherein the vector is an Ad26 vector comprising a non-naturally occurring nucleic acid encoding a truncated core HBV antigen as in any of Examples 1 to 6.

實施例30包含如實施例28之載體，其中該載體係包含編碼如實施例7至13中任一項之HBV聚合酶抗原的非天然存在之核酸的Ad26載體。Example 30 includes a vector as in Example 28, wherein the vector is an Ad26 vector comprising a non-naturally occurring nucleic acid encoding the HBV polymerase antigen as in any of Examples 7-13.

實施例31包含如實施例28之載體，其中該載體係包含編碼如實施例17至21中任一項之融合蛋白的非天然存在之核酸的Ad26載體。Example 31 includes a vector as in Example 28, wherein the vector is an Ad26 vector comprising a non-naturally occurring nucleic acid encoding the fusion protein of any of Examples 17-21.

實施例31a包含如實施例28至31中任一項之載體，其中該腺病毒載體含有包含SEQ ID NO: 17之聚核苷酸序列的啟動子序列、包含SEQ ID NO: 23之聚核苷酸序列的調控序列、包含SEQ ID NO: 18之聚核苷酸序列的信號肽編碼序列、包含SEQ ID NO: 15之聚核苷酸序列的第二聚核苷酸序列、包含SEQ ID NO: 22之聚核苷酸序列的連接子編碼序列、包含SEQ ID NO: 16之聚核苷酸序列的第一聚核苷酸序列及包含SEQ ID NO:24之聚核苷酸序列的聚腺苷酸化信號序列。Example 31a comprises the vector of any one of Examples 28 to 31, wherein the adenoviral vector contains a promoter sequence comprising a polynucleotide sequence of SEQ ID NO: 17 and a polynucleoside comprising SEQ ID NO: 23 Control sequence of an acid sequence, a signal peptide coding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15, a sequence comprising SEQ ID NO: A linker coding sequence of the polynucleotide sequence of 22, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16 and a polyadenosine comprising the polynucleotide sequence of SEQ ID NO: 24 Acidify the signal sequence.

實施例32包含由如實施例1至32中任一項之非天然存在之核酸分子編碼的非天然存在之多肽。Example 32 includes a non-naturally occurring polypeptide encoded by a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 32.

實施例33包含一種宿主細胞，其包含如實施例1至23中任一項之非天然存在之核酸分子或如實施例24至32中任一項之載體。Example 33 includes a host cell comprising a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 23 or a vector as in any of Examples 24 to 32.

實施例34包含一種組合物，其包含如實施例1至23中任一項之非天然存在之核酸分子、如實施例24至32中任一項之載體或如實施例33之非天然存在之多肽，及醫藥學上可接受之載劑。Example 34 comprises a composition comprising a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 23, a vector as in any of Examples 24 to 32 or a non-naturally occurring nucleic acid as in Example 33 Polypeptides, and pharmaceutically acceptable carriers.

實施例35包含如實施例34之組合物，其包含如實施例7至13中任一項之第一聚核苷酸、如實施例14至16中任一項之第二聚核苷酸及醫藥學上可接受之載劑，其中該第一聚核苷酸及該第二聚核苷酸不包含在同一核酸分子中或同一核酸載體中。Example 35 includes a composition as in Example 34, which includes a first polynucleotide as in any of Examples 7 to 13, a second polynucleotide as in any of Examples 14 to 16, and A pharmaceutically acceptable carrier, wherein the first polynucleotide and the second polynucleotide are not included in the same nucleic acid molecule or the same nucleic acid vector.

實施例36包含如實施例35之組合物，其中該第一聚核苷酸及該第二聚核苷酸係包含在兩個獨立載體，較佳腺病毒載體，更佳Ad26載體中。Example 36 includes the composition as in Example 35, wherein the first polynucleotide and the second polynucleotide are contained in two separate vectors, preferably an adenovirus vector, and more preferably an Ad26 vector.

實施例37包含一種套組，其包含：
(a) 包含編碼HBV聚合酶抗原之第一聚核苷酸的第一非天然存在之核酸分子，該HBV聚合酶抗原具有與SEQ ID NO: 4至少98%一致的胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性； (b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二非天然存在之核酸分子，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成；以及 (c) 醫藥學上可接受之載劑，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一非天然存在之核酸分子中或兩個不同的非天然存在之核酸分子中。Embodiment 37 includes a kit comprising:
(a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and ribonuclease H activity; (b) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen, the truncated HBV The core antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and (c) a pharmaceutically acceptable carrier, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule Existing nucleic acid molecules exist in the same non-naturally occurring nucleic acid molecule or in two different non-naturally occurring nucleic acid molecules.

實施例38包含如實施例37之套組，其中該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應，較佳該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應，且更佳地，該HBV聚合酶抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。Example 38 includes the kit of Example 37, wherein the HBV polymerase antigen is capable of eliciting an immune response in mammals against at least two HBV genotypes, and preferably the HBV polymerase antigen is capable of eliciting in mammals against at least two HBV genotypes. T-cell responses to HBV genotypes B, C, and D, and more preferably, the HBV polymerase antigen is capable of inducing a CD8 T-cell response against at least HBV genotypes A, B, C, and D in a human individual.

實施例39包含如實施例37之套組，其中該HBV聚合酶抗原包含SEQ ID NO: 4之胺基酸序列。Example 39 includes the kit of Example 37, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

實施例40包含如實施例37至39中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然核酸分子中的至少一個進一步包含編碼可操作地連接至該HBV聚合酶抗原及該截短之HBV核心抗原中之至少一種的信號序列之聚核苷酸序列。Embodiment 40 includes the set of any one of embodiments 37 to 39, wherein at least one of the first non-naturally occurring nucleic acid molecule and the second non-natural nucleic acid molecule further comprises an encoding operably linked to the HBV A polynucleotide sequence of a signal sequence of at least one of a polymerase antigen and the truncated HBV core antigen.

實施例41包含如實施例40之套組，其中該信號序列獨立地包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，較佳該信號序列獨立地由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Embodiment 41 includes the set of embodiment 40, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is independently composed of SEQ ID NO: 5 or The polynucleotide sequence of SEQ ID NO: 18 encodes.

實施例42包含如實施例37至41中任一項之套組，其中該第一聚核苷酸序列與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致。Embodiment 42 includes the kit of any one of embodiments 37 to 41, wherein the first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16.

實施例43包含如實施例42之套組，其中該第一聚核苷酸序列包含SEQ ID NO: 3或SEQ ID NO: 16之聚核苷酸序列。Embodiment 43 includes the set of embodiment 42, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

實施例44包含如實施例37至43中任一項之套組，其中該第二聚核苷酸序列與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致。Embodiment 44 includes the set of any one of embodiments 37 to 43, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

實施例45包含如實施例44之套組，其中該第二聚核苷酸序列包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。Embodiment 45 includes the set of embodiment 44, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

實施例46包含如實施例37至45中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然核酸分子中的至少一個進一步包含啟動子序列、視情況之強化子序列，且亦視情況包含聚腺苷酸化信號序列，較佳該啟動子序列具有SEQ ID NO: 7或SEQ ID NO: 17之聚核苷酸序列，該強化子序列獨立地具有SEQ ID NO: 8或SEQ ID NO: 23之聚核苷酸序列，且該聚腺苷酸化信號序列獨立地具有SEQ ID NO: 11或SEQ ID NO: 24之聚核苷酸序列。Example 46 includes the set of any one of Examples 37 to 45, wherein at least one of the first non-naturally-occurring nucleic acid molecule and the second non-naturally-occurring nucleic acid molecule further comprises a promoter sequence, optionally strengthening Subsequence, and optionally a polyadenylation signal sequence, preferably the promoter sequence has a polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 17, and the enhancer sequence independently has SEQ ID NO : 8 or a polynucleotide sequence of SEQ ID NO: 23, and the polyadenylation signal sequence independently has a polynucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 24.

實施例47包含如實施例37至46中任一項之套組，其中該套組不含編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原的核酸分子，亦不含選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原。Example 47 includes the set of any one of Examples 37 to 46, wherein the set does not contain an encoding member selected from the group consisting of a hepatitis B surface antigen (HBsAg), an HBV envelope (Env) antigen, and an HBV L protein antigen. The nucleic acid molecule of the HBV antigen of the group also does not contain HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen.

實施例48包含如實施例37至47中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一載體中。Example 48 includes the set of any one of Examples 37 to 47, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same vector.

實施例49包含如實施例48之套組，其中該載體編碼呈兩種獨立蛋白質形式之HBV聚合酶抗原及截短之HBV核心抗原。Example 49 includes the kit of Example 48, wherein the vector encodes the HBV polymerase antigen and the truncated HBV core antigen in the form of two independent proteins.

實施例50包含如實施例49之套組，其中該載體編碼包含截短之HBV核心抗原可操作地連接至HBV聚合酶抗原之融合蛋白。Example 50 includes the kit of Example 49, wherein the vector encodes a fusion protein comprising a truncated HBV core antigen operably linked to an HBV polymerase antigen.

實施例51包含如實施例50之套組，其中該融合蛋白包含該截短之HBV核心抗原經由連接子可操作地連接至該HBV聚合酶抗原。Example 51 includes the kit of Example 50, wherein the fusion protein comprises the truncated HBV core antigen operably linked to the HBV polymerase antigen via a linker.

實施例52包含如實施例51之套組，其中該連接子包含胺基酸序列(AlaGly)n，且n係2至5之整數，較佳該連接子係由包含SEQ ID NO: 22之聚核苷酸序列編碼。Example 52 includes the kit of Example 51, wherein the linker comprises an amino acid sequence (AlaGly) n, and n is an integer from 2 to 5, preferably, the linker is a polymer comprising SEQ ID NO: 22 Nucleotide sequence encoding.

實施例53包含如實施例52之套組，其中該融合蛋白包含SEQ ID NO: 20之胺基酸序列。Embodiment 53 comprises the kit of embodiment 52, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 20.

實施例54包含如實施例53之套組，其中該融合蛋白進一步包含信號序列，較佳該信號序列具有SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，更佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Example 54 includes the kit of Example 53, wherein the fusion protein further comprises a signal sequence, preferably the signal sequence has an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, and more preferably the signal sequence is Encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 18.

實施例55包含如實施例54之套組，其中該融合蛋白包含SEQ ID NO: 21之胺基酸序列。Example 55 includes the kit of Example 54, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 21.

實施例56包含如實施例48至55中任一項之套組，其中該載體自5'端至3'端含有啟動子序列、強化子序列、信號肽編碼序列、該第二聚核苷酸序列、連接子編碼序列、該第一聚核苷酸序列及聚腺苷酸化信號序列。Embodiment 56 includes the kit according to any one of embodiments 48 to 55, wherein the vector contains a promoter sequence, an enhancer sequence, a signal peptide coding sequence, and the second polynucleotide from the 5 'end to the 3' end. Sequence, linker coding sequence, the first polynucleotide sequence and polyadenylation signal sequence.

實施例57包含如實施例56之套組，其中該載體係腺病毒載體，較佳為Ad26或Ad35載體。Example 57 includes the kit of Example 56, wherein the vector is an adenoviral vector, preferably an Ad26 or Ad35 vector.

實施例58包含如實施例57之套組，其中該腺病毒載體含有包含SEQ ID NO: 17之聚核苷酸序列的啟動子序列、包含SEQ ID NO: 23之聚核苷酸序列的調控序列、包含SEQ ID NO: 18之聚核苷酸序列的信號肽編碼序列、包含SEQ ID NO: 15之聚核苷酸序列的第二聚核苷酸序列、包含SEQ ID NO: 22之聚核苷酸序列的連接子編碼序列、包含SEQ ID NO: 16之聚核苷酸序列的第一聚核苷酸序列及包含SEQ ID NO: 24之聚核苷酸序列的聚腺苷酸化信號序列。Example 58 includes the kit of Example 57, wherein the adenoviral vector contains a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17 and a regulatory sequence comprising the polynucleotide sequence of SEQ ID NO: 23 A signal peptide coding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15 and a polynucleoside comprising SEQ ID NO: 22 The linker coding sequence of the acid sequence, the first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16 and the polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 24.

實施例59包含如實施例58之套組，其中該套組不含編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原的核酸分子，亦不含選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原。Embodiment 59 includes the set of embodiment 58, wherein the set does not contain a nucleic acid encoding a HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen. The molecule also does not contain HBV antigens selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen.

實施例60包含如實施例37至59中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於兩個不同的載體中。Embodiment 60 includes the set of any one of embodiments 37 to 59, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in two different vectors.

實施例61包含如實施例60之套組，其中該第一非天然存在之核酸分子係存在於第一質體DNA載體中，且該第二非天然存在之核酸分子係存在於第二質體DNA載體中。Example 61 includes the kit of Example 60, wherein the first non-naturally occurring nucleic acid molecule is present in a first plastid DNA vector, and the second non-naturally occurring nucleic acid molecule is present in a second plastid DNA vector.

實施例62包含如實施例61之套組，其中該第一質體DNA載體及該第二質體DNA載體各自包含複製起點、抗生素抗性基因，且自5'端至3'端包含啟動子序列、調控序列、信號肽編碼序列、該第一聚核苷酸序列或該第二聚核苷酸序列，及聚腺苷酸化信號序列。Example 62 includes the kit of Example 61, wherein the first plastid DNA vector and the second plastid DNA vector each include an origin of replication, an antibiotic resistance gene, and a promoter from the 5 'end to the 3' end Sequence, regulatory sequence, signal peptide coding sequence, the first polynucleotide sequence or the second polynucleotide sequence, and a polyadenylation signal sequence.

實施例63包含如實施例62之套組，其中該抗生素抗性基因係具有與SEQ ID NO: 12至少90%一致，較佳與SEQ ID NO: 12達100%一致之聚核苷酸序列的卡那黴素抗性基因。Example 63 includes the set of Example 62, wherein the antibiotic resistance gene has a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 12, and preferably 100% identical to SEQ ID NO: 12 Kanamycin resistance gene.

實施例64包含如實施例63之套組，其包含
(a) 第一質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的該調控序列、含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、含SEQ ID NO: 3之聚核苷酸序列的該第一聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列； (b) 第二質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的該調控序列、含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、含SEQ ID NO: 1之聚核苷酸序列的該第二聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列；以及 (c) 醫藥學上可接受之載劑，其中該第一質體DNA載體及該第二質體DNA載體各自進一步包含具有SEQ ID NO: 12之聚核苷酸序列的卡那黴素抗性基因及具有SEQ ID NO: 10之聚核苷酸序列的複製起點，且其中該第一質體DNA載體與該第二質體DNA載體係在同一組合物或兩種不同組合物中。Embodiment 64 includes the kit of embodiment 63, which comprises
(a) a first plastid DNA vector, from the 3 'end to the 5' end, comprising the promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7 and the polynucleotide containing SEQ ID NO: 8 The control sequence of the sequence, the signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, the first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and the sequence containing SEQ ID The polyadenylation signal sequence of the polynucleotide sequence of NO: 11; (b) a second plastid DNA vector from the 3 'end to the 5' end, which includes the polynucleotide comprising SEQ ID NO: 7 The promoter sequence of the sequence, the regulatory sequence containing the polynucleotide sequence of SEQ ID NO: 8, the signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, and the The second polynucleotide sequence of the polynucleotide sequence and the polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11; and (c) a pharmaceutically acceptable carrier, wherein The first plastid DNA vector and the second plastid DNA vector each further include a kanamycin resistance gene having a polynucleotide sequence of SEQ ID NO: 12 and a polynucleotide having SEQ ID NO: 10 Sequential Origin of replication, and wherein the first vector plasmid DNA and the second DNA plasmid vector system of the same composition or in two different compositions.

實施例65包含如實施例60之套組，其中該第一非天然存在之核酸分子係存在於第一腺病毒載體，較佳Ad26載體中，且該第二非天然存在之核酸分子係存在於第二腺病毒載體，較佳Ad26載體中。Example 65 includes the kit of Example 60, wherein the first non-naturally occurring nucleic acid molecule is present in a first adenoviral vector, preferably an Ad26 vector, and the second non-naturally occurring nucleic acid molecule is present in A second adenoviral vector, preferably an Ad26 vector.

實施例66包含如實施例65之套組，其中該第一腺病毒載體及該第二腺病毒載體各自5'端至3'端包含啟動子序列、調控序列、信號肽編碼序列、該第一聚核苷酸序列或該第二聚核苷酸序列，及聚腺苷酸化信號序列。Example 66 includes the kit of Example 65, wherein the first adenoviral vector and the second adenoviral vector each include a promoter sequence, a regulatory sequence, a signal peptide coding sequence, and a first 5 'to 3' end. A polynucleotide sequence or the second polynucleotide sequence, and a polyadenylation signal sequence.

實施例67包含如實施例66之套組，其包含
(a) 第一Ad26載體，自3'端至5'端，其包括含SEQ ID NO: 17之聚核苷酸序列的啟動子序列、含SEQ ID NO: 23之聚核苷酸序列的調控序列、含SEQ ID NO: 17之聚核苷酸序列的信號肽編碼序列、含SEQ ID NO: 15之聚核苷酸序列的第一聚核苷酸序列及含SEQ ID NO: 24之聚核苷酸序列的聚腺苷酸化信號序列； (b) 第二Ad26載體，自3'端至5'端，其包括含SEQ ID NO: 17之聚核苷酸序列的啟動子序列、含SEQ ID NO: 23之聚核苷酸序列的調控序列、含SEQ ID NO: 17之聚核苷酸序列的信號肽編碼序列、含SEQ ID NO: 16之聚核苷酸序列的第二聚核苷酸序列及含SEQ ID NO: 24之聚核苷酸序列的聚腺苷酸化信號序列；以及 (c) 醫藥學上可接受之載劑，其中該第一Ad26載體與該第二Ad26載體各自係在同一組合物或兩種不同組合物中。Embodiment 67 includes the kit of embodiment 66, which comprises
(a) the first Ad26 vector, from the 3 'end to the 5' end, comprising a promoter sequence containing a polynucleotide sequence of SEQ ID NO: 17 and regulation of a polynucleotide sequence containing SEQ ID NO: 23 Sequence, a signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 17, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 15, and a polynucleus containing SEQ ID NO: 24 A polyadenylation signal sequence of a nucleotide sequence; (b) a second Ad26 vector, from the 3 'end to the 5' end, comprising a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 17, and a sequence containing SEQ ID Regulatory sequence of polynucleotide sequence of NO: 23, signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 17, second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 16 Sequence and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 24; and (c) a pharmaceutically acceptable carrier, wherein the first Ad26 vector and the second Ad26 vector are each in In the same composition or in two different compositions.

實施例68包含如實施例64至67中任一項之套組，其中該套組不含編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原的核酸分子，亦不含選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原。Example 68 includes the set of any one of Examples 64 to 67, wherein the set does not contain an encoding member selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen The nucleic acid molecule of the HBV antigen of the group also does not contain HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen.

實施例69包含如實施例34至36中任一項之組合物，或如實施例37至68中任一項之套組，其係用於在有需要之個體中誘發針對B型肝炎病毒(HBV)之免疫反應，較佳該個體患有慢性HBV感染。Example 69 comprises the composition according to any one of examples 34 to 36, or the set according to any one of examples 37 to 68, and is used to induce hepatitis B virus in an individual in need ( HBV) immune response, preferably the individual has a chronic HBV infection.

實施例70包含一種另一免疫原性藥劑、較佳另一HBV抗原與如實施例34至36中任一項之組合物或如實施例37至68中任一項之套組的組合，其係用於在有需要之個體中誘發針對B型肝炎病毒(HBV)之免疫反應，較佳該個體患有慢性HBV感染。Example 70 comprises a combination of another immunogenic agent, preferably another HBV antigen, and a composition as in any of Examples 34 to 36 or a set as in any of Examples 37 to 68, which It is used to elicit an immune response against hepatitis B virus (HBV) in an individual in need, preferably the individual has a chronic HBV infection.

實施例71包含如實施例34至36中任一項之組合物，或如實施例37至68中任一項之套組，其係用於治療有需要之個體的B型肝炎病毒(HBV)誘發之疾病，較佳該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。Example 71 comprises a composition as in any of Examples 34 to 36, or a set as in any of Examples 37 to 68, which is used to treat hepatitis B virus (HBV) in an individual in need The induced disease, preferably the individual has a chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

實施例72包含一種另一治療劑、較佳另一抗HBV藥劑與如實施例34至36中任一項之組合物或如實施例37至68中任一項之套組的組合，其係用於治療有需要之個體的B型肝炎病毒(HBV)誘發之疾病，較佳該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。Example 72 comprises a combination of another therapeutic agent, preferably another anti-HBV agent, and a composition as in any of Examples 34 to 36 or a set as in any of Examples 37 to 68, which is Hepatitis B virus (HBV) -induced disease in an individual in need, preferably the individual has a chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) ) Group.

實施例部分Example section 22

實施例1包含一種非天然存在之核酸分子，其包含編碼HBV聚合酶抗原之第一聚核苷酸序列，該HBV聚合酶抗原包含與SEQ ID NO: 4至少98%一致之胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性。Example 1 includes a non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an HBV polymerase antigen, the HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, The HBV polymerase antigen does not have reverse transcriptase activity or ribonuclease H activity.

實施例2包含如實施例1之非天然存在之核酸分子，其中該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應，較佳該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應，且更佳地，該HBV聚合酶抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。Example 2 includes a non-naturally occurring nucleic acid molecule as in Example 1, wherein the HBV polymerase antigen is capable of eliciting an immune response against at least two HBV genotypes in a mammal, preferably the HBV polymerase antigen is capable of Induces a T cell response against at least HBV genotypes B, C, and D, and more preferably, the HBV polymerase antigen is capable of inducing a CD8 T cell response against at least HBV genotypes A, B, C, and D in a human individual .

實施例3包含如實施例1之非天然存在之核酸分子，其中該HBV聚合酶抗原包含SEQ ID NO: 4之胺基酸序列。Example 3 includes a non-naturally occurring nucleic acid molecule as in Example 1, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

實施例4包含如實施例1至3中任一項之非天然存在之核酸分子，其進一步包含編碼可操作地連接至該HBV聚合酶抗原之信號序列的聚核苷酸序列。Example 4 includes a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 3, further comprising a polynucleotide sequence encoding a signal sequence operably linked to the HBV polymerase antigen.

實施例5包含如實施例4之非天然存在之核酸分子，其中該信號序列包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，較佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Example 5 includes a non-naturally occurring nucleic acid molecule as in Example 4, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is composed of SEQ ID NO: 5 Or the polynucleotide sequence of SEQ ID NO: 18.

實施例6包含如實施例1至5中任一項之非天然存在之核酸分子，其中該第一聚核苷酸序列與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致。Example 6 includes a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 5, wherein the first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16.

實施例7包含如實施例6之非天然存在之核酸分子，其中該第一聚核苷酸序列包含SEQ ID NO: 3或SEQ ID NO: 16之聚核苷酸序列。Embodiment 7 includes a non-naturally occurring nucleic acid molecule as in Embodiment 6, wherein the first polynucleotide sequence comprises a polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

實施例8包含如實施例1至7中任一項之非天然存在之核酸分子，其進一步包含編碼截短之HBV核心抗原的第二聚核苷酸序列，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成。Example 8 includes a non-naturally occurring nucleic acid molecule as in any one of Examples 1 to 7, further comprising a second polynucleotide sequence encoding a truncated HBV core antigen, the truncated HBV core antigen is represented by SEQ Consisting of the amino acid sequence of ID NO: 2 or SEQ ID NO: 14.

實施例9包含如實施例8之非天然存在之核酸分子，其中該第二聚核苷酸序列與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致。Example 9 includes a non-naturally occurring nucleic acid molecule as in Example 8, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

實施例10包含如實施例9之非天然存在之核酸分子，其中該第二聚核苷酸序列包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。Example 10 includes a non-naturally occurring nucleic acid molecule as in Example 9, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

實施例11包含如實施例8至10中任一項之非天然存在之核酸分子，其編碼包含該截短之HBV核心抗原可操作地連接至該HBV聚合酶抗原的融合蛋白。Example 11 includes a non-naturally occurring nucleic acid molecule as in any of Examples 8 to 10, which encodes a fusion protein comprising the truncated HBV core antigen operably linked to the HBV polymerase antigen.

實施例12包含如實施例11之非天然存在之核酸分子，其中該融合蛋白包含該截短之HBV核心抗原經由連接子可操作地連接至該HBV聚合酶抗原。Example 12 includes a non-naturally occurring nucleic acid molecule as in Example 11, wherein the fusion protein comprises the truncated HBV core antigen operably linked to the HBV polymerase antigen via a linker.

實施例13包含如實施例12之非天然存在之核酸分子，其中該連接子包含(AlaGly)_n 之胺基酸序列，且n係2至5之整數，較佳地該連接子係由包含SEQ ID NO: 22之聚核苷酸序列編碼。Example 13 includes a non-naturally occurring nucleic acid molecule as in Example 12, wherein the linker comprises an amino acid sequence of (AlaGly) _n , and n is an integer from 2 to 5, preferably the linker is composed of ID NO: 22 encodes a polynucleotide sequence.

實施例14包含如實施例13之非天然存在之核酸分子，其中該融合蛋白包含SEQ ID NO: 20之胺基酸序列。Example 14 includes a non-naturally occurring nucleic acid molecule as in Example 13, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 20.

實施例15包含如實施例11至14中任一項之非天然存在之核酸分子，其中該融合蛋白進一步包含信號序列，較佳該信號序列包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，更佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Example 15 includes a non-naturally occurring nucleic acid molecule as in any one of Examples 11 to 14, wherein the fusion protein further comprises a signal sequence, preferably the signal sequence comprises an amine of SEQ ID NO: 6 or SEQ ID NO: 19 Amino acid sequence, more preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 18.

實施例16包含如實施例1至15中任一項之非天然存在之核酸分子，其進一步包含啟動子序列、視情況之一或多個另外的調控序列，較佳該啟動子序列包含SEQ ID NO: 7或SEQ ID NO: 17之聚核苷酸序列，且該另外的調控序列係選自由以下組成之群：SEQ ID NO: 8或SEQ ID NO: 23，及SEQ ID NO: 11或SEQ ID NO:24之聚腺苷酸化信號序列。Example 16 comprises a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 15, further comprising a promoter sequence, optionally one or more additional regulatory sequences, preferably the promoter sequence comprises SEQ ID NO: 7 or SEQ ID NO: 17 and the additional regulatory sequence is selected from the group consisting of SEQ ID NO: 8 or SEQ ID NO: 23, and SEQ ID NO: 11 or SEQ Polyadenylation signal sequence of ID NO: 24.

實施例17包含如實施例1至16中任一項之非天然存在之核酸分子，其中該非天然存在之核酸分子不編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群的HBV抗原。Example 17 includes a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 16, wherein the non-naturally occurring nucleic acid molecule does not encode a member selected from the group consisting of a hepatitis B surface antigen (HBsAg), an HBV envelope (Env) antigen, and A group of HBV antigens composed of HBV L protein antigens.

實施例18包含一種載體，其包含如實施例1至17中任一項之非天然存在之核酸分子。Example 18 includes a vector comprising a non-naturally occurring nucleic acid molecule as in any of Examples 1-17.

實施例19包含如實施例18之載體，其中該非天然存在之核酸分子自5'端至3'端包含啟動子序列、強化子序列、信號肽編碼序列、該第一聚核苷酸序列及聚腺苷酸化信號序列，視情況，該非天然存在之核酸分子進一步包含該第二聚核苷酸序列。Example 19 includes the vector of Example 18, wherein the non-naturally occurring nucleic acid molecule comprises a promoter sequence, an enhancer sequence, a signal peptide coding sequence, the first polynucleotide sequence, and a polynucleotide from the 5 'end to the 3' end. The adenylation signal sequence, and optionally the non-naturally occurring nucleic acid molecule further comprises the second polynucleotide sequence.

實施例20包含如實施例18或19之載體，其中該載體係質體DNA載體，且該質體DNA載體進一步包含複製起點及抗生素抗性基因。Example 20 includes the vector of Example 18 or 19, wherein the vector is a plastid DNA vector, and the plastid DNA vector further includes an origin of replication and an antibiotic resistance gene.

實施例21包含如實施例20之載體，其中該質體DNA載體含有包含SEQ ID NO:10之聚核苷酸序列的該複製起點、包含SEQ ID NO: 12之聚核苷酸序列的該抗生素抗性基因、包含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、包含SEQ ID NO: 8之聚核苷酸序列的該強化子序列、包含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、包含SEQ ID NO: 3之聚核苷酸序列的該第一聚核苷酸序列及包含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列，及視情況，包含SEQ ID NO: 1之聚核苷酸序列的該第二聚核苷酸序列。Example 21 includes the vector of Example 20, wherein the plastid DNA vector contains the origin of replication comprising the polynucleotide sequence of SEQ ID NO: 10 and the antibiotic comprising the polynucleotide sequence of SEQ ID NO: 12 Resistance gene, the promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 7, the enhancer sequence comprising the polynucleotide sequence of SEQ ID NO: 8, the polynucleoside comprising SEQ ID NO: 5 The signal peptide coding sequence of the acid sequence, the first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 3, and the polyadenylation signal comprising the polynucleotide sequence of SEQ ID NO: 11 The sequence, and optionally the second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 1.

實施例22包含如實施例18或19之載體，其中該載體係腺病毒載體，較佳為Ad26或Ad35載體。Example 22 includes the vector of Example 18 or 19, wherein the vector is an adenoviral vector, preferably an Ad26 or Ad35 vector.

實施例23包含如實施例22之載體，其中該腺病毒載體含有包含SEQ ID NO: 17之聚核苷酸序列的該啟動子序列、包含SEQ ID NO: 23之聚核苷酸序列的該調控序列、包含SEQ ID NO: 18之聚核苷酸序列的該信號肽編碼序列、包含SEQ ID NO: 15之聚核苷酸序列的該第二聚核苷酸序列、包含SEQ ID NO: 22之聚核苷酸序列的該連接子編碼序列、包含SEQ ID NO: 16之聚核苷酸序列的該第一聚核苷酸序列及包含SEQ ID NO: 24之聚核苷酸序列的該聚腺苷酸化信號序列。Example 23 includes the vector of Example 22, wherein the adenoviral vector contains the promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17 and the regulation comprising the polynucleotide sequence of SEQ ID NO: 23 Sequence, the signal peptide coding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, the second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15, the The linker coding sequence of the polynucleotide sequence, the first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16 and the polygland comprising the polynucleotide sequence of SEQ ID NO: 24 Glycosylation signal sequence.

實施例24包含一種非天然存在之多肽，其係由如實施例1至17中任一項之非天然存在之核酸分子編碼。Example 24 includes a non-naturally occurring polypeptide that is encoded by a non-naturally occurring nucleic acid molecule as in any of Examples 1-17.

實施例25包含一種宿主細胞，其包含如實施例1至17中任一項之非天然存在之核酸分子或如實施例18至23中任一項之載體。Example 25 comprises a host cell comprising a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 17 or a vector as in any of Examples 18 to 23.

實施例26包含一種組合物，其包含如實施例1至17中任一項之非天然存在之核酸分子、如實施例18至23中任一項之載體或如實施例24之非天然存在之多肽，及醫藥學上可接受之載劑。Example 26 includes a composition comprising a non-naturally occurring nucleic acid molecule as in any of Examples 1 to 17, a vector as in any of Examples 18 to 23, or a non-naturally occurring nucleic acid as in Example 24 Polypeptides, and pharmaceutically acceptable carriers.

實施例27包含如實施例26之組合物，其包含如實施例1至7中任一項之第一聚核苷酸、如實施例8至10中任一項之第二聚核苷酸及醫藥學上可接受之載劑，其中該第一聚核苷酸及該第二聚核苷酸不包含在同一核酸分子中或同一核酸載體中。Example 27 includes a composition as in Example 26, which includes a first polynucleotide as in any of Examples 1 to 7, a second polynucleotide as in any of Examples 8 to 10, and A pharmaceutically acceptable carrier, wherein the first polynucleotide and the second polynucleotide are not included in the same nucleic acid molecule or the same nucleic acid vector.

實施例28包含一種套組，其包含：
(a) 包含編碼HBV聚合酶抗原之第一聚核苷酸的第一非天然存在之核酸分子，該HBV聚合酶抗原具有與SEQ ID NO: 4至少98%一致之胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性；
(b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二非天然存在之核酸分子，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成；以及
(c) 醫藥學上可接受之載劑，
其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一非天然存在之核酸分子中或兩個不同的非天然存在之核酸分子中。Embodiment 28 includes a kit comprising:
(a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity or ribonuclease H activity;
(b) a second non-naturally-occurring nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen, the truncated HBV core antigen being an amino group of SEQ ID NO: 2 or SEQ ID NO: 14 Acid sequence composition;
(c) a pharmaceutically acceptable carrier,
The first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule exist in the same non-naturally occurring nucleic acid molecule or two different non-naturally occurring nucleic acid molecules.

實施例29包含如實施例28之套組，其中該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應，較佳該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應，且更佳地，該HBV聚合酶抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應。Example 29 includes the kit of Example 28, wherein the HBV polymerase antigen is capable of eliciting an immune response in mammals against at least two HBV genotypes, and preferably the HBV polymerase antigen is capable of eliciting in mammals T-cell responses to HBV genotypes B, C, and D, and more preferably, the HBV polymerase antigen is capable of inducing a CD8 T-cell response against at least HBV genotypes A, B, C, and D in a human individual.

實施例30包含如實施例29之套組，其中該HBV聚合酶抗原包含SEQ ID NO: 4之胺基酸序列。Example 30 includes the kit of Example 29, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

實施例31包含如實施例28至30中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然核酸分子中的至少一個進一步包含編碼可操作地連接至該HBV聚合酶抗原及該截短之HBV核心抗原中之至少一種的信號序列之聚核苷酸序列。Embodiment 31 comprises the kit of any one of embodiments 28 to 30, wherein at least one of the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule further comprises an encoding operably linked to the HBV A polynucleotide sequence of a signal sequence of at least one of a polymerase antigen and the truncated HBV core antigen.

實施例32包含如實施例31之套組，其中該信號序列獨立地包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，較佳該信號序列獨立地由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Embodiment 32 includes the set of embodiment 31, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is independently composed of SEQ ID NO: 5 or The polynucleotide sequence of SEQ ID NO: 18 encodes.

實施例33包含如實施例28至32中任一項之套組，其中該第一聚核苷酸序列與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致。Embodiment 33 includes the kit of any one of embodiments 28 to 32, wherein the first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16.

實施例34包含如實施例33之套組，其中該第一聚核苷酸序列包含SEQ ID NO: 3或SEQ ID NO: 16之聚核苷酸序列。Embodiment 34 includes the set of embodiment 33, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

實施例35包含如實施例28至34中任一項之套組，其中該第二聚核苷酸序列與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致。Embodiment 35 includes the set of any one of embodiments 28 to 34, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

實施例36包含如實施例35之套組，其中該第二聚核苷酸序列包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。Embodiment 36 includes the set of embodiment 35, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

實施例37包含如實施例28至36中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然核酸分子中的至少一個進一步包含啟動子序列、視情況之強化子序列且亦視情況包含聚腺苷酸化信號序列，較佳該啟動子序列具有SEQ ID NO: 7或SEQ ID NO: 17之聚核苷酸序列，該強化子序列獨立地具有SEQ ID NO: 8或SEQ ID NO: 23之聚核苷酸序列，且該聚腺苷酸化信號序列獨立地具有SEQ ID NO: 11或SEQ ID NO: 24之聚核苷酸序列。Embodiment 37 includes the kit according to any one of embodiments 28 to 36, wherein at least one of the first non-naturally occurring nucleic acid molecule and the second non-natural nucleic acid molecule further comprises a promoter sequence, optionally strengthening The subsequence, and optionally a polyadenylation signal sequence, preferably the promoter sequence has a polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 17, and the enhancer sequence independently has SEQ ID NO: 8 or SEQ ID NO: 23, and the polyadenylation signal sequence independently has a polynucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 24.

實施例38包含如實施例28至37中任一項之套組，其中該套組不含編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原的核酸分子，亦不含選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原。Example 38 includes the set of any one of Examples 28 to 37, wherein the set does not contain an encoding member selected from the group consisting of a hepatitis B surface antigen (HBsAg), an HBV envelope (Env) antigen, and an HBV L protein antigen The nucleic acid molecule of the HBV antigen of the group also does not contain HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen.

實施例39包含如實施例28至38中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一載體中。Embodiment 39 includes the kit according to any one of embodiments 28 to 38, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same vector.

實施例40包含如實施例39之套組，其中該載體編碼呈兩種獨立蛋白質形式之該HBV聚合酶抗原及該截短之HBV核心抗原。Example 40 includes the kit of Example 39, wherein the vector encodes the HBV polymerase antigen and the truncated HBV core antigen in the form of two independent proteins.

實施例41包含如實施例39之套組，其中該載體編碼包含該截短之HBV核心抗原可操作地連接至該HBV聚合酶抗原之融合蛋白。Example 41 includes the kit of Example 39, wherein the vector encodes a fusion protein comprising the truncated HBV core antigen operably linked to the HBV polymerase antigen.

實施例42包含如實施例41之套組，其中該融合蛋白包含該截短之HBV核心抗原經由連接子可操作地連接至該HBV聚合酶抗原。Example 42 includes the kit of Example 41, wherein the fusion protein comprises the truncated HBV core antigen operably linked to the HBV polymerase antigen via a linker.

實施例43包含如實施例42之套組，其中該連接子包含(AlaGly)_n 之胺基酸序列，且n係2至5之整數，較佳該連接子係由包含SEQ ID NO: 22之聚核苷酸序列編碼。Example 43 includes the kit of Example 42, wherein the linker comprises the amino acid sequence of (AlaGly) _n , and n is an integer from 2 to 5, preferably the linker is composed of SEQ ID NO: 22 The polynucleotide sequence encodes.

實施例44包含如實施例43之套組，其中該融合蛋白包含SEQ ID NO: 20之胺基酸序列。Embodiment 44 includes the kit of embodiment 43, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 20.

實施例45包含如實施例44之套組，其中該融合蛋白進一步包含信號序列，較佳該信號序列具有SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，更佳該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。Example 45 includes the kit of Example 44, wherein the fusion protein further comprises a signal sequence, preferably the signal sequence has an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, and more preferably the signal sequence is Encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 18.

實施例46包含如實施例45之套組，其中該融合蛋白包含SEQ ID NO: 21之胺基酸序列。Example 46 includes the kit of Example 45, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 21.

實施例47包含如實施例41至46中任一項之套組，其中該載體自5'端至3'端含有啟動子序列、強化子序列、信號肽編碼序列、該第二聚核苷酸序列、連接子編碼序列、該第一聚核苷酸序列及聚腺苷酸化信號序列。Embodiment 47 includes the kit according to any one of embodiments 41 to 46, wherein the vector contains a promoter sequence, an enhancer sequence, a signal peptide coding sequence, and the second polynucleotide from the 5 'end to the 3' end. Sequence, linker coding sequence, the first polynucleotide sequence and polyadenylation signal sequence.

實施例48包含如實施例47之套組，其中該載體係腺病毒載體，較佳為Ad26或Ad35載體。Example 48 includes the kit of Example 47, wherein the vector is an adenoviral vector, preferably an Ad26 or Ad35 vector.

實施例49包含如實施例48之套組，其中該腺病毒載體含有包含SEQ ID NO: 17之聚核苷酸序列的該啟動子序列、包含SEQ ID NO: 23之聚核苷酸序列的調控序列、包含SEQ ID NO: 18之聚核苷酸序列的該信號肽編碼序列、包含SEQ ID NO: 15之聚核苷酸序列的該第二聚核苷酸序列、包含SEQ ID NO: 22之聚核苷酸序列的該連接子編碼序列、包含SEQ ID NO: 16之聚核苷酸序列的該第一聚核苷酸序列及包含SEQ ID NO: 24之聚核苷酸序列的該聚腺苷酸化信號序列。Example 49 includes the kit of Example 48, wherein the adenoviral vector contains the promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17 and the regulation of the polynucleotide sequence comprising SEQ ID NO: 23 Sequence, the signal peptide coding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, the second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15, the The linker coding sequence of the polynucleotide sequence, the first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16 and the polygland comprising the polynucleotide sequence of SEQ ID NO: 24 Glycosylation signal sequence.

實施例50包含如實施例49之套組，其中該套組不含編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原的核酸分子，亦不含選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原。Example 50 includes the set of Example 49, wherein the set does not contain a nucleic acid encoding a HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen The molecule also does not contain HBV antigens selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen.

實施例51包含如實施例28至38中任一項之套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於兩個不同的載體中。Embodiment 51 includes the set of any one of embodiments 28 to 38, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in two different vectors.

實施例52包含如實施例51之套組，其中該第一非天然存在之核酸分子係存在於第一質體DNA載體中，且該第二非天然存在之核酸分子係存在於第二質體DNA載體中。Example 52 includes the kit of Example 51, wherein the first non-naturally occurring nucleic acid molecule is present in a first plastid DNA vector, and the second non-naturally occurring nucleic acid molecule is present in a second plastid DNA vector.

實施例53包含如實施例52之套組，其中該第一質體DNA載體及該第二質體DNA載體各自包含複製起點、抗生素抗性基因，且自5'端至3'端包含啟動子序列、調控序列、信號肽編碼序列、該第一聚核苷酸序列或該第二聚核苷酸序列，及聚腺苷酸化信號序列。Example 53 includes the kit of Example 52, wherein the first plastid DNA vector and the second plastid DNA vector each include an origin of replication, an antibiotic resistance gene, and a promoter from the 5 'end to the 3' end Sequence, regulatory sequence, signal peptide coding sequence, the first polynucleotide sequence or the second polynucleotide sequence, and a polyadenylation signal sequence.

實施例54包含如實施例53之套組，其中該抗生素抗性基因係具有與SEQ ID NO: 12至少90%一致，較佳與SEQ ID NO: 12達100%一致之聚核苷酸序列的卡那黴素抗性基因。Example 54 includes the set of Example 53, wherein the antibiotic resistance gene has a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 12, and preferably 100% identical to SEQ ID NO: 12 Kanamycin resistance gene.

實施例55包含如實施例54之套組，其包含
(a) 第一質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的該調控序列、含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、含SEQ ID NO: 3之聚核苷酸序列的該第一聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列；
(b) 第二質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的該調控序列、含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、含SEQ ID NO: 1之聚核苷酸序列的該第二聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列；以及
(c) 醫藥學上可接受之載劑，
其中該第一質體DNA載體及該第二質體DNA載體各自進一步包含具有SEQ ID NO: 12之聚核苷酸序列的卡那黴素抗性基因及具有SEQ ID NO: 10之聚核苷酸序列的複製起點，且
其中該第一質體DNA載體與該第二質體DNA載體係在同一組合物或兩種不同組合物中。Example 55 includes the kit of Example 54, which includes
(a) a first plastid DNA vector, from the 3 'end to the 5' end, comprising the promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7 and the polynucleotide containing SEQ ID NO: 8 The control sequence of the sequence, the signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, the first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and the sequence containing SEQ ID The polyadenylation signal sequence of the polynucleotide sequence of NO: 11;
(b) a second plastid DNA vector, from the 3 'end to the 5' end, comprising the promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7 and the polynucleotide containing SEQ ID NO: 8 The control sequence of the sequence, the signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, the second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 1, and the sequence containing SEQ ID The polyadenylation signal sequence of the polynucleotide sequence of NO: 11; and
(c) a pharmaceutically acceptable carrier,
The first plastid DNA vector and the second plastid DNA vector each further include a kanamycin resistance gene having a polynucleotide sequence of SEQ ID NO: 12 and a polynucleoside having SEQ ID NO: 10 The origin of replication of the acid sequence, and wherein the first plastid DNA vector and the second plastid DNA vector are in the same composition or two different compositions.

實施例56包含如實施例55之套組，其中該套組不含編碼選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原的核酸分子，亦不含選自由B型肝炎表面抗原(HBsAg)、HBV包膜(Env)抗原及HBV L蛋白質抗原組成之群之HBV抗原。Example 56 includes the set of Example 55, wherein the set does not contain a nucleic acid encoding a HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen The molecule also does not contain HBV antigens selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen.

實施例57包含如實施例26之組合物，或如實施例27至55中任一項之套組，其係用於在有需要之個體中誘發針對B型肝炎病毒(HBV)之免疫反應，較佳該個體患有慢性HBV感染。Example 57 comprises a composition as in Example 26, or a set as in any of Examples 27 to 55, which is used to elicit an immune response against hepatitis B virus (HBV) in an individual in need, Preferably, the individual has a chronic HBV infection.

實施例58包含一種另一免疫原性藥劑、較佳另一HBV抗原與如實施例26之組合物或如實施例27至55中任一項之套組的組合，其係用於在有需要之個體中誘發針對B型肝炎病毒(HBV)之免疫反應，較佳該個體患有慢性HBV感染。Example 58 comprises a combination of another immunogenic agent, preferably another HBV antigen, and a composition as in Example 26 or a set as in any of Examples 27 to 55, which is used when needed An individual induces an immune response against hepatitis B virus (HBV), preferably the individual has a chronic HBV infection.

實施例59包含如實施例26或27之組合物，或如實施例28至56中任一項之套組，其係用於治療有需要之個體的B型肝炎病毒(HBV)誘發之疾病，較佳該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。Example 59 comprises the composition as in Example 26 or 27, or the set according to any one of Examples 28 to 56, which is used to treat a hepatitis B virus (HBV) -induced disease in an individual in need, Preferably, the individual has a chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

實施例60包含一種另一治療劑、較佳另一抗HBV藥劑與如實施例26或27之組合物或如實施例28至56中任一項之套組的組合，其係用於治療有需要之個體的B型肝炎病毒(HBV)誘發之疾病，較佳該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。
實例Example 60 comprises a combination of another therapeutic agent, preferably another anti-HBV agent, and a composition as in Example 26 or 27 or a set as in any one of Examples 28 to 56, which is used to treat Hepatitis B virus (HBV) -induced disease in an individual in need, preferably the individual has chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) .
Examples

本申請案之以下實例進一步說明本申請案之性質。應理解，以下實例並不限制本申請案且本申請案之範圍係由所附申請專利範圍決定。The following examples of this application further illustrate the nature of this application. It should be understood that the following examples do not limit this application and the scope of this application is determined by the scope of the attached application patent.

實例Examples 11 ：: HBVHBV 核心及Core and PolPol 抗原序列之產生及質體優化Generation of antigen sequences and plastid optimization

肝炎核心蛋白上之T細胞抗原決定基被認為對於消除B型肝炎感染及B型肝炎病毒蛋白，諸如聚合酶極為重要，可以用來改善反應之廣度。因此，選擇B型肝炎核心及聚合酶蛋白質作為設計治療性B型肝炎病毒(HBV)疫苗之抗原。The T-cell epitope on the hepatitis core protein is thought to be very important in eliminating hepatitis B infection and hepatitis B virus proteins such as polymerase, and can be used to improve the breadth of the response. Therefore, the hepatitis B core and polymerase protein were selected as antigens for the design of a therapeutic hepatitis B virus (HBV) vaccine.

HBVHBV 核心及聚合酶抗原共同序列之衍生化Derivation of core and polymerase antigen common sequences

基於HBV基因型B、C及D，產生HBV pol及核心抗原共同序列。自不同來源獲得不同HBV序列並針對核心及聚合酶蛋白質分別進行比對。隨後將所有亞型(A至H)之原始序列比對侷限於HBV基因型B、C及D。根據各蛋白質比對，確定單獨各亞型中及所有接合BCD序列中之共同序列。在變化之比對位置中，使用共同序列中最常見的胺基酸。Based on HBV genotypes B, C and D, common sequences of HBV pol and core antigens were generated. Different HBV sequences were obtained from different sources and aligned for core and polymerase proteins, respectively. The original sequence alignment of all subtypes (A to H) was then restricted to HBV genotypes B, C, and D. Based on the alignment of each protein, common sequences were determined in each individual isoform and in all joined BCD sequences. In varying alignment positions, the most common amino acids in the common sequence are used.

HBVHBV 核心抗原之優化Optimization of core antigens

HBV核心抗原共同序列係藉由使原生病毒蛋白質中產生缺失來優化。詳言之，使對應於C末端帶大量正電之區段的前基因組RNA衣殼化所需之三十四個胺基酸缺失。The HBV core antigen common sequence is optimized by generating deletions in native viral proteins. In detail, thirty-four amino acids required for capsidization of pregenomic RNA corresponding to a large positively charged segment at the C-terminus were deleted.

HBVHBV PolPol 抗原antigen 之優化Optimization

HBV pol抗原共同序列係藉由改變四個殘基以移除逆轉錄酶及核糖核酸酶H活性來優化。詳言之，將逆轉錄酶結構域之「YXDD」基元中的天冬胺酸殘基(D)變成天冬醯胺殘基(N)以除去任何配位功能，且由此消除核苷酸/金屬離子結合。另外，將核糖核酸酶H結構域之「DEDD」基元中的第一個天冬胺酸殘基(D)變成天冬醯胺殘基(N)且將第一個麩胺酸殘基(E)變成麩醯胺酸殘基(A)以除去Mg² ⁺ 配位。另外，HBV pol抗原序列經密碼子優化以擾亂包膜蛋白，包括S蛋白質及具有N末端延伸之S蛋白質形式前S1及前S2的內部開放閱讀框架。因此，包膜蛋白(前S1、前S2及S蛋白質)及X蛋白質之開放閱讀框架移除。The HBV pol antigen common sequence was optimized by changing four residues to remove reverse transcriptase and ribonuclease H activity. In detail, the asparagine residue (D) in the "YXDD" motif of the reverse transcriptase domain is changed to asparagine residue (N) to remove any coordination function and thereby eliminate the nucleoside Acid / metal ion binding. In addition, the first aspartate residue (D) in the "DEDD" motif of the ribonuclease H domain was changed to asparagine residue (N) and the first glutamic acid residue ( E) Change to glutamate residue (A) to remove Mg ² ⁺ coordination. In addition, the HBV pol antigen sequence is codon-optimized to disrupt envelope proteins, including the S protein and the internal open reading frames of pre-S1 and pre-S2 in the form of the S protein with N-terminal extension. Therefore, the open reading frames of the envelope proteins (pre-S1, pre-S2, and S protein) and X protein were removed.

HBVHBV 核心及Core and PolPol 抗原表現策略之優化Optimization of antigen expression strategies

測試三種不同策略以自質體載體獲得核心及pol抗原的最大及相等表現：(1)HBV核心及pol抗原與在編碼序列之間***之小AGAG框內融合以產生單一核心-Pol融合蛋白(圖 2A )；(2)藉助於核糖體滑移位點，特別是來自***(FMDV)之FA2滑移位點自一個質體表現核心及pol抗原以產生自單一mRNA表現個別核心及pol蛋白質之雙順反子表現載體(圖 2B )；以及(3)兩個獨立質體分別編碼HBV核心及pol抗原(圖 2C )。Three different strategies were tested to obtain the maximum and equal performance of core and pol antigens from plastid vectors: (1) Fusion of HBV core and pol antigens with small AGAG boxes inserted between coding sequences to produce a single core-Pol fusion protein ( Figure 2A ); (2) With the help of ribosome slippage points, especially the FA2 slippage point from foot-and-mouth disease (FMDV), the core and pol antigens are expressed from a plastid to generate individual core and pol proteins from a single mRNA The bicistronic expression vector ( Figure 2B ); and (3) the two independent plastids encode the HBV core and the pol antigen, respectively ( Figure 2C ).

活體外表現分析In vitro performance analysis

將根據以上三種表現策略中之每一個的共同HBV核心及pol抗原之編碼序列選殖至可商購之表現質體pcDNA3.1中。用該等載體轉染HEK-293T細胞並藉由西方墨點法，使用HBV核心特異性抗原評價蛋白質表現。The coding sequences of the common HBV core and pol antigens according to each of the three above expression strategies were cloned into the commercially available expression plastid pcDNA3.1. HEK-293T cells were transfected with these vectors and protein expression was evaluated by HBV core-specific antigen by Western blotting method.

轉錄後調控元件之優化Optimization of post-transcriptional regulatory elements

針對藉由使主要轉錄物穩定，促進其核輸出及/或改善轉錄-轉譯偶合來增進蛋白質表現，評價四個不同的轉錄後調控元件：(1)土拔鼠HBV轉錄後調控元件(WPRE)(GenBank：J04514.1)；(2)來源於人類載脂蛋白A1前驅體之內含子/外顯子序列(GenBank：X01038.1)；(3)1型人類T細胞白血病病毒(HTLV-1)長末端重複序列(LTR)之非轉譯R-U5結構域(GenBank：KM023768.1)；以及(4) HTLV-1 LTR、合成兔β-血球蛋白內含子(GenBank：V00882.1)及剪接強化子構成之複合序列(三重複合序列)。強化子序列係引入該等質體中CMV啟動子與HBV抗原編碼序列之間。利用西方墨點法未觀察到當在存在與不存在WPRE元件下自質體表現時核心抗原表現之顯著差異(圖 2D )。然而，當藉由西方墨點法評價經具有其他三個轉錄後調控序列之質體轉染之HEK293T細胞中的核心抗原表現時，該三強化子序列引起最強的核心抗原表現(圖 2E )。Four different post-transcriptional regulatory elements were evaluated for enhancing protein performance by stabilizing major transcripts, promoting their nuclear export and / or improving transcription-translation coupling: (1) Groundhog HBV post-transcriptional regulatory element (WPRE) (GenBank: J04514.1); (2) Intron / exon sequences derived from the human apolipoprotein A1 precursor (GenBank: X01038.1); (3) Type 1 human T-cell leukemia virus (HTLV- 1) Non-translated R-U5 domain of long terminal repeat (LTR) (GenBank: KM023768.1); and (4) HTLV-1 LTR, synthetic rabbit β-hemoglobin intron (GenBank: V00882.1 ) And splicing enhancer complex sequence (triple compound sequence). Enhancer sequences were introduced between the CMV promoter and the HBV antigen coding sequence in these plastids. No significant differences in core antigen expression were observed when using Western blot methods when present in the presence and absence of WPRE elements ( Fig. 2D ). However, when the core antigen performance in HEK293T cells transfected with plastids with three other post-transcriptional regulatory sequences was evaluated by Western blot method, the three enhancer sequence caused the strongest core antigen performance ( Figure 2E ).

選擇實現高效蛋白質分泌之信號肽Select signal peptides for efficient protein secretion

評價在HBV核心抗原之N末端處框內引入之三個不同信號肽：(1) Ig重鏈γ信號肽SPIgG(BAA75024.1)；(2) Ig重鏈ε信號肽SPIgE(AAB59424.1)；以及(3)胱抑素S前驅體信號肽SPCS(NP_0018901.1)。使用Signal P預測程式，藉由計算機模擬來優化核心融合物之信號肽裂解位點。藉由分析上清液中之核心蛋白含量來測定分泌效率。利用西方墨點分析使用在N末端融合之三個不同信號肽的核心抗原分泌展示，胱抑素S信號肽引起最高效之蛋白質分泌(圖 2F )。Evaluation of three different signal peptides introduced in-frame at the N-terminus of the HBV core antigen: (1) Ig heavy chain γ signal peptide SPIgG (BAA75024.1); (2) Ig heavy chain ε signal peptide SPIgE (AAB59424.1) ; And (3) Cystatin S precursor signal peptide SPCS (NP_0018901.1). The Signal P prediction program was used to optimize the signal peptide cleavage site of the core fusion by computer simulation. Secretion efficiency was determined by analyzing the core protein content in the supernatant. Using Western blot analysis, the core antigen secretion display using three different signal peptides fused at the N-terminus, the cystatin S signal peptide caused the most efficient protein secretion ( Figure 2F ).

DNADNA 疫苗載體優化Vaccine Vector Optimization

將在具有N末端胱抑素S信號肽序列之HBV抗原編碼序列的上游含有該三複合物強化子序列之經優化表現卡匣選殖至DNA疫苗載體pVax-1(Life Technologies, Thermo Fisher Scientific, Waltham, MA)中。pVax-1中之表現卡匣含有人類CMV-IE啟動子，隨後為牛生長激素(BGH)源性聚腺苷酸化序列(pA)。細菌繁殖係由pUC複製起點驅動且卡那黴素抗性基因(Kan R)係由小真核啟動子驅動。pUC複製起點、KanR表現卡匣及由CMV-IE啟動子驅動之表現卡匣在質體主鏈內均呈相同取向。不過，在pVax-1載體中觀察到的核心抗原表現量明顯低於在pcDNA3.1載體中觀察到的表現量。An optimized expression cassette containing the triple complex enhancer sequence upstream of the HBV antigen coding sequence with the N-terminal cystatin S signal peptide sequence was cloned into the DNA vaccine vector pVax-1 (Life Technologies, Thermo Fisher Scientific, Waltham, MA). The expression cassette in pVax-1 contains a human CMV-IE promoter followed by a bovine growth hormone (BGH) -derived polyadenylation sequence (pA). Bacterial reproductive lines are driven by the origin of pUC replication and kanamycin resistance genes (Kan R) are driven by small eukaryotic promoters. The origin of pUC replication, the KanR performance cassette, and the performance cassette driven by the CMV-IE promoter all showed the same orientation in the plastid backbone. However, the expression of the core antigen observed in the pVax-1 vector was significantly lower than that observed in the pcDNA3.1 vector.

採用若干策略來改善蛋白質表現：(1)將整個pUCori-KanR卡匣倒轉成逆時針取向(pVD-核心)；以及(2)改變KanR基因之密碼子用法以及用來自pcDNA3.1載體之Amp啟動子替代Kan啟動子(pDK-核心)。兩種策略均恢復核心抗原表現，但用pDK載體之核心抗原表現量最高，該載體含有密碼子調整之Kan R基因、AmpR啟動子(代替KanR啟動子)及反向取向之pUCori-KanR卡匣。Several strategies were used to improve protein performance: (1) Invert the entire pUCori-KanR cassette into a counterclockwise orientation (pVD-core); and (2) Change the codon usage of the KanR gene and use Amp from the pcDNA3.1 vector The promoter replaces the Kan promoter (pDK-core). Both strategies restored core antigen performance, but the core antigen performance was highest with the pDK vector, which contains the codon-adjusted Kan R gene, the AmpR promoter (instead of the KanR promoter), and the reverse-oriented pUCori-KanR cassette .

將如圖 2G 中所示的四個不同HBV核心/pol抗原經優化之表現卡匣引入pDK質體主鏈中以分別測試圖 2A - 2C 中示出之三種表現策略。藉由西方墨點分析，使用核心及pol特異性抗體，針對核心及pol抗原表現在活體外測試該等質體。當核心及pol抗原係由獨立載體，即個別DNA載體pDK-核心及pDK-pol編碼時，實現細胞及分泌核心及pol抗原的最一致表現圖譜(圖 2H )。pDK-pol及pDK-核心載體之示意性表示分別顯示於圖 3A 及 3B 中。The four different HBV core as shown in / pol optimized antigen expression cassette is introduced pDK FIG. 2G backbone plastids were tested to FIG. 2A - 2C policy expressed in three of the illustrated. By Western blot analysis, core and pol specific antibodies were used to test these plastids in vitro for core and pol antigen performance. When the core and pol antigens are encoded by independent vectors, that is, individual DNA vectors pDK-core and pDK-pol, the most consistent representation map of cells and secreted core and pol antigens is achieved ( Figure 2H ). Schematic representations of pDK-pol and pDK-core vectors are shown in Figures 3A and 3B , respectively.

實例Examples 22 ：: 表現截短之Truncated performance HBVHBV 核心抗原與Core antigen and HBVHBV PolPol 抗原之融合物的腺病毒載體之產生Production of adenoviral vectors of antigen fusions

產生一種經設計為自單一開放閱讀框架表現融合蛋白的腺病毒載體。亦可設想例如使用兩個獨立表現卡匣，或使用2A樣序列分開該兩個序列以表現兩種蛋白質之另外的組態。An adenoviral vector designed to express a fusion protein from a single open reading frame was generated. It is also conceivable, for example, to use two independent expression cassettes, or to use a 2A-like sequence to separate the two sequences to represent another configuration of two proteins.

有關腺病毒載體之表現卡匣的設計Design of Adenovirus Vector Performance Cassettes

表現卡匣(圖解於圖 8A 及圖 8B 中)包含CMV啟動子(SEQ ID NO: 17)、內含子(SEQ ID NO: 23)(來源於人類ApoAI基因之片段，Genbank寄存號X01038鹼基對295-523，帶有ApoAI第二內含子)，隨後為在人類免疫球蛋白分泌信號編碼序列(SEQ ID NO: 18)之後的經優化編碼序列，即單獨核心或核心與聚合酶融合蛋白，且隨後為SV40聚腺苷酸化信號(SEQ ID NO: 24)。The performance cassette (illustrated in Figures 8A and 8B ) contains a CMV promoter (SEQ ID NO: 17), an intron (SEQ ID NO: 23) (fragment derived from the human ApoAI gene, Genbank accession number X01038 base) For 295-523, with the second intron of ApoAI), followed by an optimized coding sequence after the human immunoglobulin secretion signal coding sequence (SEQ ID NO: 18), that is, the core alone or the core and polymerase fusion protein And followed by the SV40 polyadenylation signal (SEQ ID NO: 24).

包括分泌信號係歸因於過去一些含有分泌型轉殖基因的腺病毒載體顯示可製造性之改良，同時不影響所引發之T細胞反應的經驗(小鼠實驗)。Including secretion signals is due to past experience with adenoviral vectors containing secreted transgenes showing improved manufacturability without affecting the T cell response elicited (mouse experiment).

核心蛋白的最後兩個殘基(VV)及聚合酶蛋白質之前兩個殘基(MP)若融合，則產生接合序列(VVMP)，其存在於人類多巴胺受體蛋白(D3同功異型物)以及側接同源序列上。If the last two residues (VV) of the core protein and the first two residues (MP) of the polymerase protein are fused, a junction sequence (VVMP) is generated, which is present in the human dopamine receptor protein (D3 isoform) and Flanked by homologous sequences.

核心與聚合酶序列之間AGAG連接子之***消除此同源序列且恢復成在人類蛋白質組之Blast中無其他匹配(hit)。Insertion of the AGAG linker between the core and the polymerase sequence eliminates this homologous sequence and reverts to no other hits in the Blast of the human proteome.

實例Examples 33 ：: 在小鼠中進行的Performed in mice DNADNA 疫苗之活體內免疫原性研究In Vivo Immunogenicity of Vaccine

在小鼠中測試含有編碼HBV核心抗原或HBV聚合酶抗原之DNA質體的免疫治療性DNA疫苗。本研究之目的係設計用於偵測該疫苗在經由電穿孔肌肉內遞送至BALB/c小鼠中之後誘發的T細胞反應。初始免疫原性研究集中在確定由引入之HBV抗原引發的細胞免疫反應。Immunotherapy DNA vaccines containing DNA plastids encoding HBV core antigen or HBV polymerase antigen are tested in mice. The purpose of this study was to detect the T-cell response induced by the vaccine after intramuscular delivery to BALB / c mice via electroporation. Initial immunogenicity studies have focused on identifying cellular immune responses elicited by the introduced HBV antigen.

詳言之，測試質體包括pDK-Pol質體及pDK-核心質體，分別如圖 3A 及 3B 中所示且如上文在實例1中所描述。pDK-Pol質體編碼具有SEQ ID NO: 4之胺基酸序列的聚合酶抗原，且pDK-核心質體編碼具有SEQ ID NO: 2之胺基酸序列的核心抗原。首先，個別地測試由各質體誘發之T細胞反應。使用適合用於小鼠模型中之脛前肌中的可商購之TriGrid^TM 遞送系統-肌肉內(TDS-IM)，將DNA質體(pDNA)疫苗經由電穿孔肌肉內遞送至Balb/c小鼠中。有關藉由電穿孔將DNA肌肉內遞送至小鼠之方法及器件的另外的說明，參見國際專利申請公開案WO2017172838，及與本申請案同一天提交且代理人案號為688097-405U1的題為「用於遞送B型肝炎病毒(HBV)疫苗之方法及裝置(Method and Apparatus for the Delivery of Hepatitis B Virus(HBV)Vaccines)」的美國專利申請案，其揭示內容以全文引用的方式併入本文中。詳言之，將具有電極之間之間距為2.5 mm及電極直徑為0.030吋之電極陣列的TDS-IM v1.0器件之TDS-IM陣列經皮***選定之肌肉中，其中導電長度為3.2 mm及有效穿透深度為3.2 mm，且其中電極之菱形組態的長軸平行於肌纖維取向。在電極***之後，起始注射以將DNA(例如0.020 ml)分配於肌肉中。在完成IM注射之後，在約400 ms之總持續時間內，以10%工作循環(亦即，在約400 ms持續時間內，有效地施加電壓總計約40 ms)局部施加250 V/cm電場(施加之電壓為59.4-65.6 V，施加電流之限值小於4 A，0.16 A/sec)，總計6次脈衝。在完成電穿孔程序之後，移除TriGridTM陣列且使動物恢復。如表1中所概述，向BALB/c小鼠投與高劑量(20 µg)。向六隻小鼠投與編碼HBV核心抗原之質體DNA(pDK-核心；第1組)，向六隻小鼠投與編碼HBV pol抗原之質體DNA(pDK-pol；第2組)，且兩隻小鼠接受空載體作為陰性對照。動物間隔兩週接受兩次DNA免疫接種且在最後一次免疫接種之後一週收集脾細胞。In detail, the test mass comprises pDK-Pol plasmid and plasmids pDK- core, respectively, and as shown in FIG. 3A and 3B as described above in Example 1. The pDK-Pol plastid encodes a polymerase antigen having an amino acid sequence of SEQ ID NO: 4, and the pDK-core plastid encodes a core antigen having an amino acid sequence of SEQ ID NO: 2. First, T cell responses induced by each plastid were individually tested. DNA plastid (pDNA) vaccine was delivered to Balb / c cells via electroporation using a commercially available TriGrid ^TM delivery system-intramuscular (TDS-IM) suitable for use in the tibialis anterior muscle in a mouse model. In the rat. For additional instructions on the method and device for intramuscular delivery of DNA to mice by electroporation, refer to International Patent Application Publication No. WO2017172838, and the same as the application filed on the same day as the agent case number 688097-405U1 US Patent Application "Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines", the disclosure of which is incorporated herein by reference in its entirety in. In detail, a TDS-IM v1.0 device having a TDS-IM v1.0 device with an electrode array having an electrode spacing of 2.5 mm and an electrode diameter of 0.030 inches is inserted percutaneously into a selected muscle, wherein the conductive length is 3.2 mm And the effective penetration depth is 3.2 mm, and the long axis of the diamond configuration of the electrodes is parallel to the muscle fiber orientation. After electrode insertion, an injection is initiated to distribute DNA (e.g., 0.020 ml) into the muscle. After the IM injection is completed, a 250 V / cm electric field is applied locally at a total duty cycle of about 400 ms at a 10% duty cycle (ie, effectively applying a total voltage of about 40 ms for a duration of about 400 ms) ( The applied voltage is 59.4-65.6 V, and the limit of the applied current is less than 4 A (0.16 A / sec), for a total of 6 pulses. After completing the electroporation procedure, the TriGrid ™ array is removed and the animal is allowed to recover. As outlined in Table 1, BALB / c mice were administered high doses (20 µg). Six mice were administered with plastid DNA encoding the HBV core antigen (pDK-core; group 1), and six mice were administered with plastid DNA encoding the HBV pol antigen (pDK-pol; group 2), And two mice received empty vector as negative control. Animals received two DNA immunizations at two-week intervals and splenocytes were collected one week after the last immunization.

表 1 ：預備試驗之小鼠免疫接種實驗設計 .
CT，脛前肌；EP，電穿孔。 Table 1 : Experimental design of mice immunization experiments for preliminary experiments .
CT, anterior tibialis; EP, electroporation.

藉由IFN-γ酶聯免疫斑點(ELISPOT)分析及定量抗原特異性反應。在此分析法中，將自經免疫接種之動物分離的脾細胞與包含核心蛋白、Pol蛋白質或小肽前導序列及接合序列(每種肽2 µg/ml)的肽池一起培育隔夜。該等池由重疊11個殘基之15聚體肽組成，該等殘基匹配核心及Pol疫苗載體之基因型BCD共同序列。將較大的94 kDa HBV Pol蛋白質自中間分至兩個肽池中。用同源肽池刺激抗原特異性T細胞且使用ELISPOT分析法評估IFN-γ陽性T細胞。利用適當抗體且隨後顯色偵測來觀測在微量盤上呈有色斑點(稱為斑點形成細胞(SFC))形式的單一抗原特異性T細胞釋放之IFN-γ。The antigen-specific response was analyzed and quantified by IFN-γ enzyme-linked immunospot (ELISPOT). In this assay, splenocytes isolated from immunized animals are incubated overnight with peptide pools containing core proteins, Pol proteins or small peptide leader sequences and junction sequences (2 µg / ml per peptide). The pools consist of 15-mer peptides overlapping 11 residues that match the core and genotype BCD common sequence of the Pol vaccine vector. The larger 94 kDa HBV Pol protein was split into two peptide pools from the middle. Antigen-specific T cells were stimulated with a homologous peptide pool and IFN-γ positive T cells were evaluated using ELISPOT analysis. Appropriate antibodies and subsequent colorimetric detection were used to observe the release of IFN-γ by single antigen-specific T cells in the form of colored spots (called spot-forming cells (SFC)) on microplates.

在用DNA疫苗質體pDK-核心(第1組)免疫接種之小鼠中獲得針對HBV核心之顯著T細胞反應，達到每10⁶ 個細胞1,000個SFC(圖 4 )。針對Pol 1肽池的Pol T細胞反應較強(每10⁶ 個細胞約1,000個SFC)。針對Pol-2之抗Pol細胞反應較弱可能歸因於小鼠中有限之MHC多樣性，此現象稱為T細胞免疫顯性，定義為一種抗原中之不同抗原決定基的不均等識別。進行確證研究以確證本研究中獲得的結果(資料未顯示)。Immunization of mice against HBV core obtained in the reaction of the T cells with significant pDK- core plastid DNA vaccine (Group 1), to ¹⁰⁶ cells per 10, 000 SFC (FIG. 4). For Pol 1 peptide pools Pol T cell responses stronger (about ¹⁰⁶ cells every 1,000 SFC). The weaker anti-Pol cell response to Pol-2 may be due to the limited MHC diversity in mice. This phenomenon is called T cell immunodominance and is defined as the uneven recognition of different epitopes in an antigen. Confirmation studies were performed to confirm the results obtained in this study (data not shown).

以上結果展示，用編碼HBV抗原之DNA質體疫苗進行疫苗接種誘發針對投與之HBV抗原的細胞免疫反應。The above results show that vaccination with a DNA plastid vaccine encoding a HBV antigen induces a cellular immune response against the HBV antigen administered thereto.

實例Examples 44 ：: 在小鼠中進行的組合之Combinations in mice pDKpDK -- 核心core // pDKpDK -- PolPol 質體之劑量發現研究Plastid dose discovery study

此利用組合質體之劑量發現研究的目的係評價在小鼠中使用不同DNA劑量，在一個部位施用編碼HBV核心及pol抗原之DNA質體(pDNA)載體之混合物的免疫反應。在本研究中，在小鼠中測試含有兩個質體，即實例1中所述之pDK-pol及pDK-核心質體之1:1(w/v)混合物的免疫治療性DNA疫苗。如上文在實例3中所描述，經由電穿孔將DNA疫苗肌肉內遞送至Balb/c小鼠之一個解剖部位中。如表2中所概述，每個部位用含10 µg、1 µg及0.1 µg各質體DNA的組合之核心及Pol 表現質體進行疫苗接種。測試每組中之八隻小鼠，且兩隻小鼠投與空載體作為陰性對照。動物間隔三週接受兩次DNA免疫接種且在最後一次免疫接種之後一週收集脾細胞。The purpose of this combined plastid dose discovery study was to evaluate the immune response in mice using different DNA doses and administer a mixture of DNA plastid (pDNA) vectors encoding the HBV core and pol antigen at one site. In this study, mice were tested for immunotherapeutic DNA vaccines containing two plastids, a 1: 1 (w / v) mixture of pDK-pol and pDK-core plastids as described in Example 1. As described above in Example 3, the DNA vaccine was delivered intramuscularly to an anatomical site of Balb / c mice via electroporation. As outlined in Table 2, each site was vaccinated with a core containing 10 µg, 1 µg, and 0.1 µg of each plastid DNA and Pol- expressing plastids. Eight mice in each group were tested, and two mice were administered the empty vehicle as a negative control. Animals received two DNA immunizations three weeks apart and spleen cells were collected one week after the last immunization.

表 2 ：利用組合質體之劑量發現研究的小鼠免疫接種實驗設計。
pDNA，質體DNA；CT，脛前肌；EP，電穿孔。 Table 2 : Mice immunization experimental designs using combined plastid dose discovery studies.
pDNA, plastid DNA; CT, tibialis anterior muscle; EP, electroporation.

如實例1中所描述，藉由IFN-γ酶聯免疫斑點(ELISPOT)分析及定量抗原特異性反應。在用由質體pDK-核心及pDK-Pol組成之組合DNA疫苗免疫接種之BALB/c小鼠中實現針對核心及Pol的相當大的T細胞反應(圖 5 )。就免疫反應幅值而言，用10 µg各質體免疫接種之第1組與僅用1 µg各質體免疫接種之第2組之間無統計學差異。此結果表明，在約1 µg核心抗原及Pol抗原表現質體下，T細胞反應達到最大程度。然而，在低10倍之DNA暴露下，亦即在0.1 µg各質體下，觀察到SFC明顯減少。針對Pol 1肽池之Pol T細胞反應係佔主導。針對Pol-2之抗Pol細胞反應較弱可能歸因於近親配種之小鼠中有限之MHC多樣性，此現象稱為T細胞免疫顯性，定義為一種抗原中之不同抗原決定基的不等識別。As described in Example 1, antigen-specific responses were analyzed and quantified by IFN-γ enzyme-linked immunospot (ELISPOT). A considerable T-cell response against the core and Pol was achieved in BALB / c mice immunized with a combined DNA vaccine consisting of plastid pDK-core and pDK-Pol ( Figure 5 ). In terms of the magnitude of the immune response, there was no statistical difference between group 1 immunized with 10 µg of each plastid and group 2 immunized with only 1 µg of each plastid. This result indicates that the T-cell response is maximized at approximately 1 µg of core antigen and Pol antigen-expressing plastids. However, a significant reduction in SFC was observed at 10 times lower DNA exposure, that is, at 0.1 µg of each plastid. The Pol T cell response line to the Pol 1 peptide pool predominates. The weaker anti-Pol cell response to Pol-2 may be due to limited MHC diversity in inbreeding mice. This phenomenon is called T-cell immunodominance and is defined as the inequality of different epitopes in an antigen. Identify.

以上結果展示，在用表現HBV核心及pol抗原之DNA質體之組合免疫接種的小鼠中，在1 µg各質體之劑量下發現相當大的T細胞反應，且在每一質體0.1 µg劑量下仍觀察到一定免疫反應。The above results show that in mice immunized with a combination of DNA plastids expressing HBV core and pol antigens, a considerable T cell response was found at a dose of 1 µg of each plastid, and 0.1 µg per plastid A certain immune response was still observed at the dose.

實例Examples 55 ：: 在小鼠中進行之免疫干擾研究Immune interference studies in mice

歸於實際原因，將需要開發呈組合(混合)之載體調配物形式的組合HBV核心及pol抗原DNA疫苗。然而，利用多價疫苗可能發生免疫顯性且可能缺乏針對亞顯性抗原之免疫反應。因此，評估當與在不同解剖部位免疫接種任一載體相比較時由投與兩種抗原表現質體混合在一起之組合引起的免疫干擾，亦即核心及/或Pol特異性細胞反應之降低。For practical reasons, it will be necessary to develop a combination HBV core and pol antigen DNA vaccine in the form of a combined (mixed) vector formulation. However, the use of multivalent vaccines may be immunodominant and may lack an immune response against subdominant antigens. Therefore, the immune interference caused by the combination of administration of two antigen-presenting plastids when compared to the immunization of either vector at different anatomical sites, that is, the reduction of core and / or Pol-specific cellular responses was evaluated.

如實例3中所描述，經由電穿孔，用pDK-核心及/或pDK-pol DNA質體經肌肉內對Balb/c小鼠進行疫苗接種。如表3中概述，DNA質體(pDNA)係以每個部位5 µg之劑量投與，該等DNA質體係個別地施加、在一個部位組合(混合)施加或在獨立部位組合施加。動物間隔三週接受兩次DNA免疫接種且在最後一次免疫接種之後一週收集脾細胞。As described in Example 3, Balb / c mice were vaccinated intramuscularly with pDK-core and / or pDK-pol DNA plastids via electroporation. As summarized in Table 3, DNA plastids (pDNA) are administered at a dose of 5 µg per site, and these DNA plastid systems are applied individually, combined (mixed) at one site, or combined at separate sites. Animals received two DNA immunizations three weeks apart and spleen cells were collected one week after the last immunization.

表 3 ：免疫干擾研究之小鼠免疫接種實驗設計.
CT，脛前肌。 Table 3 : Design of mice immunization experiments for immune interference studies.
CT, anterior tibialis.

如實例1中所描述，藉由IFN-γ酶聯免疫斑點(ELISPOT)分析及定量抗原特異性反應。在此實驗中，確定在BALB/c小鼠中強烈的核心及Pol特異性抗原反應(圖 6 )。基於混合兩種質體且施加於相同部位之第3組與在兩個不同部位經由電穿孔個別地施加表現核心及pol抗原之pDNA的第4組之間獲得基本上相同的T細胞反應，未觀察到明顯的免疫干擾。第1組有一隻動物顯示較低的針對Pol-2池之異常反應。在C57/Bl6小鼠中重複該實驗得到相當的結果。As described in Example 1, antigen-specific responses were analyzed and quantified by IFN-γ enzyme-linked immunospot (ELISPOT). In this experiment, a strong core and Pol-specific antigen response was determined in BALB / c mice ( Figure 6 ). Based on the mixture of two plastids applied to the same site, the third group and the fourth group of pDNA expressing the core and pol antigen were separately applied by electroporation in two different sites. Obvious immune interference was observed. One animal in group 1 showed a lower abnormal response to the Pol-2 pool. Repeating this experiment in C57 / Bl6 mice gave comparable results.

以上結果展示，當組合兩種HBV抗原表現質體pDK-核心及pDK-Pol時，觀察到基本上無免疫干擾。The above results show that when combining the two HBV antigen-expressing plastid pDK-cores and pDK-Pols, substantially no immune interference was observed.

實例Examples 66 ：: DNADNA 疫苗在非人類靈長類動物中之功效的評價Evaluation of vaccine efficacy in non-human primates

本研究之目的係評價在食蟹獼猴(Cynomolgus monkey/Macaca fascicularis)中經由電穿孔肌肉內遞送之治療性HBV DNA疫苗的功效，及誘發並量測HBV特異性T細胞反應/細胞活化。The purpose of this study was to evaluate the efficacy of therapeutic HBV DNA vaccine delivered intramuscularly through electroporation in Cynomolgus monkey / Macaca fascicularis, and to induce and measure HBV-specific T cell responses / cell activation.

疫苗vaccine

本研究中使用之疫苗係分別編碼HBV核心抗原及HBV聚合酶抗原之兩個獨立DNA質體的組合。詳言之，如圖 3A 及 3B 中分別所示且如實例1中所描述，該等DNA質體係pDK-Pol質體(編碼具有SEQ ID NO: 4之胺基酸序列的HBV聚合酶抗原)及pDK-核心質體(編碼具有SEQ ID NO: 2之胺基酸序列的HBV核心抗原)。The vaccine used in this study was a combination of two independent DNA plastids encoding the HBV core antigen and the HBV polymerase antigen. In detail, as in Figures 3A and 3B and as described in Example 1 are shown, such plastome pDK-Pol DNA plasmid (encoding SEQ ID NO: 4 is the amino acid sequence of the HBV polymerase antigen) And pDK-core plastid (encoding a HBV core antigen with an amino acid sequence of SEQ ID NO: 2).

投與該等DNA質體，將其以兩種質體之1:1(w/w)混合物形式遞送於一個解剖部位。利用適合用於非人類靈長類動物(NHP)模型中之TriGrid^TM 遞送系統-肌肉內(TDS-IM)對NHP進行電穿孔。有關藉由電穿孔將DNA肌肉內遞送至NHP之方法及器件的另外的說明，參見國際專利申請公開案WO2017172838，及與本申請案同一天提交且代理人案號為688097-405U1的題為「用於遞送B型肝炎病毒(HBV)疫苗之方法及裝置」的美國專利申請案，其揭示內容以全文引用的方式併入本文中。詳言之，將具有電極之間間距為6.0 mm且電極直徑為0.021或0.023吋之電極陣列的TDS-IM v1.0或TDS-IM v2.0之TDS-IM陣列分別經皮***選定之肌肉中，且該等電極之菱形組態的長軸平行於肌纖維取向。導電長度對於TDS-IM v1.0或TDS-IM v2.0均為5.0 mm，而有效穿透深度對於TDS-IM v1.0為15.5 mm且對於TDS-IM v2.0為9.0 mm。在電極***之後，起始注射以將DNA(例如1.0 ml)分配於肌肉中。在完成IM注射之後，在約400 ms總持續時間內，以10%工作循環(亦即，在約400 ms持續時間內，有效施加電壓總計約40 ms)局部施加250 V/cm電場(施加之電壓為142.4-157.6 V，施加電流之限值為0.6-4 A，0.16 A/sec)，且總計6次脈衝。在完成電穿孔程序之後，移除TriGridTM陣列且使動物恢復。初始免疫原性研究集中在確定由引入之HBV抗原引發的細胞免疫反應。These DNA plastids are administered and delivered to a anatomical site as a 1: 1 (w / w) mixture of the two plastids. The NHP was electroporated using a TriGrid ^™ delivery system-intramuscular (TDS-IM) suitable for use in a non-human primate (NHP) model. For additional instructions on the method and device for intramuscular delivery of DNA to NHP by electroporation, see International Patent Application Publication No. WO2017172838, and the agent's case number 688097-405U1 filed on the same day as this application and entitled " Methods and devices for the delivery of a hepatitis B virus (HBV) vaccine "US patent application, the disclosure of which is incorporated herein by reference in its entirety. In detail, a TDS-IM v1.0 or TDS-IM v1.0 with an electrode array having an electrode spacing of 6.0 mm and an electrode diameter of 0.021 or 0.023 inches is inserted percutaneously into selected muscles, respectively. The long axis of the diamond configuration of these electrodes is parallel to the muscle fiber orientation. The conductive length is 5.0 mm for both TDS-IM v1.0 or TDS-IM v2.0, and the effective penetration depth is 15.5 mm for TDS-IM v1.0 and 9.0 mm for TDS-IM v2.0. After the electrode is inserted, an injection is initiated to distribute the DNA (eg, 1.0 ml) into the muscle. After the IM injection is completed, a 250 V / cm electric field is applied locally (approximately 40 ms for a total duration of about 400 ms, that is, a total effective voltage of about 40 ms is applied for a duration of about 400 ms). The voltage is 142.4-157.6 V, the limit of the applied current is 0.6-4 A, 0.16 A / sec), and a total of 6 pulses. After completing the electroporation procedure, the TriGrid ™ array is removed and the animal is allowed to recover. Initial immunogenicity studies have focused on identifying cellular immune responses elicited by the introduced HBV antigen.

非人類靈長類動物Non-human primate

食蟹獼猴(n=30)係來源於中國(Covance Research Products Inc. USA)，在研究開始時，其年齡為2.5至5歲且重3.0至5.0 kg。根據獸醫學指南及國家研究委員會，Guide for the Care and Use of Laboratory Animals , 第8版, Washington DC: National Academies Press(2011)，將其群體性圈養於豐富環境中。在開始研究之前，使動物適應環境2週時間。在經電穿孔投與各質體之前，用***使猴麻醉。在每次免疫接種之後2週，將血液收集於含有肝素鈉之小瓶中。使用菲科爾梯度(ficoll gradient)分離PBMC並將其儲存於液氮貯罐中以待分析。Crab-eating macaques (n = 30) are from China (Covance Research Products Inc. USA). At the beginning of the study, they were 2.5 to 5 years old and weighed 3.0 to 5.0 kg. According to the Veterinary Guide and National Research Council, Guide for the Care and Use of Laboratory Animals , 8th Edition, Washington DC: National Academies Press (2011), they are housed in groups in rich environments. Before starting the study, the animals were acclimatized for 2 weeks. Monkeys were anesthetized with ketamine before each plastid was administered by electroporation. Blood was collected in vials containing heparin sodium 2 weeks after each immunization. PBMCs were separated using a ficoll gradient and stored in a liquid nitrogen storage tank for analysis.

在非人類靈長類動物中之肌肉內In muscles in non-human primates // 電穿孔投藥Electroporation administration

如表4中所概述，在第0天、第36天及第62天，進行三次質體投與(第1組)。利用設定成19 mm(短)注射深度的遞送系統，經由電穿孔將pDK-核心(1.0 mg)及pDK-Pol(1.0 mg)投與四頭肌(股外側肌)中。對於每次注射，交替腿肌肉投與。含有DNA質體之注射器裝備有適於NHP四頭肌之注射深度限制器，適合用於肌肉中約10 mm之注射目標深度且菱形組態之長軸平行於肌纖維取向。在完成IM注射之後，立即啟動電穿孔裝置，在總計40 mS持續時間內，經400 mS時間間隔以250 V/cm電極間距之幅值對肌肉進行電刺激。在第0天、第14天、第50天及第76天收集樣品，並藉由ELISPOT及細胞內細胞介素染色進行分析。As outlined in Table 4, three plastid administrations were performed on Days 0, 36, and 62 (Group 1). Using a delivery system set to a 19 mm (short) injection depth, pDK-core (1.0 mg) and pDK-Pol (1.0 mg) were administered to the quadriceps (lateral femoral muscle) via electroporation. For each injection, alternate leg muscle administration. The DNA plastid-containing syringe is equipped with an injection depth limiter suitable for NHP quadriceps, suitable for an injection target depth of about 10 mm in the muscle and the long axis of the diamond configuration is parallel to the muscle fiber orientation. Immediately after the IM injection was completed, the electroporation device was activated, and the muscles were electrically stimulated at a voltage interval of 250 V / cm over a 400 mS time interval for a total duration of 40 mS. Samples were collected on days 0, 14, 50, and 76 and analyzed by ELISPOT and intracellular interleukin staining.

表 4 ： NHP疫苗接種實驗設計
Table 4 : Design of NHP vaccination experiment

ELISPOTELISPOT 分析analysis

使用靈長類動物IFN-γ ELISpot套組(R&D Systems, USA, 目錄號EL961)，藉由IFN-γ酶聯免疫斑點(ELISPOT)分析及定量抗原特異性反應。在此分析法中，將自經免疫接種之動物分離的PBMC一式三份孔地與包含核心蛋白及Pol蛋白質之肽池(2µg/ml)一起培育隔夜。該等池由重疊11個殘基之15聚體肽組成，該等殘基匹配核心及Pol疫苗載體之基因型ABCD共同序列。合成90%純度的肽(JPT, Germany)。將較大的94 kDa HBV Pol蛋白質自中間分至兩個肽池中。利用適當抗體且隨後顯色偵測來觀測在微量盤上呈有色斑點(稱為斑點形成細胞(SFC))形式的單一抗原特異性T細胞釋放之IFN-γ。結果顯示於圖 7A 中。The primate IFN-γ ELISpot kit (R & D Systems, USA, catalog number EL961) was used to analyze and quantify the antigen-specific response by IFN-γ enzyme-linked immunospot (ELISPOT). In this analysis, PBMCs isolated from immunized animals were incubated in triplicate with peptide pools (2 µg / ml) containing core and Pol proteins overnight. The pools consist of 15-mer peptides overlapping 11 residues that match the core and genotype ABCD common sequence of the Pol vaccine vector. Synthesis of 90% pure peptides (JPT, Germany). The larger 94 kDa HBV Pol protein was split into two peptide pools from the middle. Appropriate antibodies and subsequent colorimetric detection were used to observe the release of IFN-γ by single antigen-specific T cells in the form of colored spots (called spot-forming cells (SFC)) on microplates. The results are shown in Figure 7A .

細胞內細胞介素染色Intracellular interleukin staining (( ICSICS ))

使用細胞內細胞介素染色(ICS)研究疫苗誘發之T細胞反應。將冷凍之PBMC解凍並在10% FBS、RPMI培養基中靜置隔夜，隨後在10% FBS、含有Golgiplug蛋白質轉運抑制劑(Protein Transport Inhibitor)(1 μg/μl)之RPMI培養基中，用疫苗—***序列匹配之核心、Pol-1或Pol-2肽池(2 μg/μl)、DMSO或白細胞活化混合液刺激6小時。用可固定的死活細胞鑑定染料(fixable viability dye) eFluor 780(65-0865-14, eBioscience)對經刺激之細胞染色，並用固定/透性化溶液(554714, BD Biosciences)處理20分鐘，隨後如下表5中所示，用細胞內染色混合物染色30分鐘。使用Fortessa流式細胞儀，利用適當單色補償對照獲得經染色之細胞。反應量值報導為在刺激之後表現IFN-γ、TNF-α或IL-2之CD4⁺ 或CD8⁺ T細胞的百分含量。結果顯示於圖 7B 中。Intracellular interleukin staining (ICS) was used to study vaccine-induced T cell responses. Frozen PBMCs were thawed and left in 10% FBS, RPMI medium overnight, then in 10% FBS, RPMI medium containing Golgiplug Protein Transport Inhibitor (1 μg / μl), using vaccine-insertion Sequence-matched cores, Pol-1 or Pol-2 peptide pools (2 μg / μl), DMSO or WBC activation mixed solution were stimulated for 6 hours. The stimulated cells were stained with fixable viability dye eFluor 780 (65-0865-14, eBioscience), and treated with fixed / permeabilizing solution (554714, BD Biosciences) for 20 minutes, followed by the following As shown in Table 5, the intracellular staining mixture was used for 30 minutes. Stained cells were obtained using a Fortessa flow cytometer with an appropriate monochromatic compensation control. Response magnitude values are reported as the percentage of CD4 ⁺ or CD8 ⁺ T cells that exhibit IFN-γ, TNF-α, or IL-2 after stimulation. The results are shown in Figure 7B .

表 5 ：用於細胞內細胞介素染色分析之抗體組
Table 5 : Antibody groups used for intracellular interleukin staining analysis

結果result

ELISPOT資料(圖 7A )顯示在兩次免疫接種之後出現強烈的核心及Pol-2反應。第三次免疫接種使IFN-γ量值大大增加。Pol-1肽池引發中等反應，該反應亦在第三次免疫接種下改善，不過改善程度明顯不如核心及Pol-2高。因為未能成功自第五隻猴抽取到血液，故第76天資料僅包括來自四隻NHP之結果。每一組內之較大變化係由NHP來源於遠親雜交種引起，且基因多樣性可以引起不同免疫反應。The ELISPOT data ( Figure 7A ) shows a strong core and Pol-2 response after two immunizations. The third immunization significantly increased the amount of IFN-γ. The Pol-1 peptide pool elicited a moderate response, which was also improved with the third immunization, but the improvement was not as high as the core and Pol-2. Because blood was not successfully drawn from the fifth monkey, the data on day 76 included results from only four NHPs. The larger changes within each group were caused by distant relatives of NHPs, and genetic diversity could elicit different immune responses.

ICS分析資料(圖 7B )顯示，由HBV肽刺激引起之細胞介素反應係CD8驅動的且對核心及Pol-2肽池具有特異性，與先前利用ELISPOT觀察到的相同。在ICS分析中起反應之NHP與ELISPOT分析中起反應之個體相同。儘管少數個體之ICS反應不如同在ELISPOT資料中所見一般顯示陽性，但此可以歸於ELISPOT分析之較高靈敏度。The ICS analysis data ( Figure 7B ) shows that the cytokine response CD8 driven by HBV peptide stimulation is specific to the core and Pol-2 peptide pool, which is the same as previously observed with ELISPOT. The NHPs that responded in the ICS analysis were the same as those that responded in the ELISPOT analysis. Although the ICS response of a few individuals does not show a positive result as seen in the ELISPOT data, this can be attributed to the higher sensitivity of the ELISPOT analysis.

結論in conclusion

以上結果展示，在藉由經電穿孔肌肉內注射，用pDK核心及pDK-Pol疫苗之組合免疫接種之NHP中，在1.0 mg各質體之劑量下發現明顯之T細胞反應，並在兩次免疫接種之後偵測到肽特異性反應且在三次免疫接種之後反應甚至更大。在第76天，ELISPOT分析結果顯示，肽池核心、Pol-1及Pol-2在各測試NHP(4/5隻NHP)中誘發陽性IFN-γ T細胞反應。針對來自經免疫接種NHP之PBMC的ICS分析顯示，HBV肽特異性反應係CD8驅動的，且針對核心及Pol-2肽池之反應最強。The above results show that in NHP immunized with a combination of pDK core and pDK-Pol vaccine by intramuscular injection through electroporation, a significant T cell response was found at a dose of 1.0 mg of each plastid, and twice in A peptide-specific response was detected after immunization and the response was even greater after three immunizations. On day 76, ELISPOT analysis showed that the core of the peptide pool, Pol-1, and Pol-2 induced positive IFN-γ T cell responses in each of the tested NHPs (4/5 NHPs). ICS analysis of PBMCs from immunized NHPs showed that the HBV peptide-specific response was CD8-driven and had the strongest response to the core and Pol-2 peptide pools.

實例Examples 77 ：: DNADNA 疫苗在人類個體中之功效的評價Evaluation of vaccine efficacy in human individuals

評價在人類個體中經電穿孔肌肉內遞送之治療性HBV DNA疫苗的功效。The efficacy of a therapeutic HBV DNA vaccine delivered intramuscularly by electroporation in a human individual was evaluated.

人類個體Human individual

人類個體係患有呈HBsAg陽性之慢性HBV感染的成年患者。用HBV聚合酶抑制劑(恩替卡韋或替諾福韋)治療人類個體。Adult humans with chronic HBV infection who are HBsAg positive. Human individuals are treated with an HBV polymerase inhibitor (enticavir or tenofovir).

疫苗vaccine

向人類患者投與分別編碼HBV核心抗原及HBV聚合酶抗原之兩個獨立DNA質體的組合。詳言之，如圖 3A 及 3B 中分別所示且如實例1中所描述，該等DNA質體係pDK-Pol質體(編碼具有SEQ ID NO: 4之胺基酸序列的HBV聚合酶抗原)及pDK-核心質體(編碼具有SEQ ID NO: 2之胺基酸序列的HBV核心抗原)。根據隨機分組、安慰劑對照之遞增劑量試驗，該等DNA質體係以兩個質體之1:1混合物形式以不同劑量，特定言之0.25 mg、1 mg及6 mg總質體之劑量投與。A human patient is administered a combination of two independent DNA plastids encoding the HBV core antigen and the HBV polymerase antigen, respectively. In detail, as in Figures 3A and 3B and as described in Example 1 are shown, such plastome pDK-Pol DNA plasmid (encoding SEQ ID NO: 4 is the amino acid sequence of the HBV polymerase antigen) And pDK-core plastid (encoding a HBV core antigen with an amino acid sequence of SEQ ID NO: 2). Based on randomized, placebo-controlled escalating dose trials, these DNA plastid systems are administered in different doses, specifically 0.25 mg, 1 mg, and 6 mg total plastids, in a 1: 1 mixture of two plastids. .

在人類個體中之肌肉內In the muscles of human beings // 電穿孔投與Electroporation administration

使用適合用於人類之TriGrid^TM 遞送系統-肌肉內(TDS-IM)，藉由電穿孔以2至3次肌肉內免疫接種向人類個體投與DNA質體。一些患者投與安慰劑(亦即，不含HBV抗原編碼序列之質體)作為對照。使用適合用於人類之TriGrid^TM 遞送系統-肌肉內(TDS-IM)，藉由電穿孔遞送質體DNA。有關藉由電穿孔將DNA肌肉內遞送至人類之方法及器件的另外的說明，參見國際專利申請公開案WO2017172838，及與本申請案同一天提交且代理人案號為688097-405U1的題為「用於遞送B型肝炎病毒(HBV)疫苗之方法及裝置」的美國專利申請案，其揭示內容以全文引用的方式併入本文中。舉例而言，可以將分別具有電極之間間距為6.0 mm且電極直徑為0.023吋之電極陣列的TDS-IM v2.0之TDS-IM陣列經皮***選定之肌肉中，其中該等電極之菱形組態的長軸平行於肌纖維取向。導電長度可以為5.0 mm，而有效穿透深度可以為19 mm。在電極***之後，起始注射以將DNA(例如1.0 ml)分配於肌肉中。在完成IM注射之後，在約400 ms之總持續時間內，以10%工作循環(亦即，在約400 ms持續時間內，有效地施加電壓總計約40 ms)局部施加250 V/cm電場(施加之電壓為142.4-157.6 V，施加電流之限值為0.6-4 A，0.16 A/sec)，總計6次脈衝。在電穿孔程序完成之後，移除TriGridTM陣列並使人類個體恢復。DNA plastids were administered to human subjects using electroporation with two to three intramuscular immunizations using a TriGrid ^™ delivery system suitable for humans (TDS-IM). Some patients were administered a placebo (ie, a plastid without the HBV antigen coding sequence) as a control. The plastid DNA was delivered by electroporation using a TriGrid ^™ delivery system-intramuscular (TDS-IM) suitable for humans. For additional descriptions of methods and devices for intramuscular delivery of DNA to humans by electroporation, see International Patent Application Publication No. WO2017172838, and entitled "688097-405U1" filed on the same day as this application and filed by the agent Methods and devices for the delivery of a hepatitis B virus (HBV) vaccine "US patent application, the disclosure of which is incorporated herein by reference in its entirety. For example, a TDS-IM v2.0 TDS-IM array with an electrode array having an electrode spacing of 6.0 mm and an electrode diameter of 0.023 inches can be inserted percutaneously into selected muscles, where the electrodes are diamond-shaped The configured long axis is oriented parallel to the muscle fibers. The conductive length can be 5.0 mm and the effective penetration depth can be 19 mm. After the electrode is inserted, an injection is initiated to distribute the DNA (eg, 1.0 ml) into the muscle. After the IM injection is completed, a 250 V / cm electric field is applied locally at a total duty cycle of about 400 ms at a 10% duty cycle (ie, effectively applying a total voltage of about 40 ms for a duration of about 400 ms) ( The applied voltage is 142.4-157.6 V, and the limit of the applied current is 0.6-4 A, 0.16 A / sec), for a total of 6 pulses. After the electroporation procedure is complete, the TriGridTM array is removed and a human individual is restored.

在免疫接種後之各種時間點，自患者收集血液樣品。評價在免疫接種後3至6個月患者中免疫接種後HBsAg含量的發展，特別是評價符合演變成臨床血清轉化之含量。在免疫接種後6至12個月的患者中評價HBsAg之持久喪失及臨床疾病(例如肝硬化、肝細胞癌)之減輕。Blood samples were collected from patients at various time points after immunization. The development of post-immunization HBsAg content in patients 3 to 6 months after immunization was evaluated, and in particular the evaluation was consistent with the content that evolved into clinical seroconversion. Persistent loss of HBsAg and reduction in clinical disease (eg, cirrhosis, hepatocellular carcinoma) are evaluated in patients 6 to 12 months after immunization.

實例Examples 88 ：在小鼠中進行的腺病毒載體之活體內免疫原性研究: In vivo immunogenicity study of adenovirus vectors in mice

在小鼠中測試含有編碼HBV核心抗原或HBV聚合酶抗原之腺病毒載體的免疫治療性DNA疫苗。本研究之目的係偵測該疫苗在肌肉內遞送至F1小鼠(C57BL/6×Balb/C)中之後誘發的T細胞反應。初始免疫原性研究集中在確定由引入之HBV抗原引發的細胞免疫反應。確切地說，測試的腺病毒載體含有如圖8A及8B中所示之表現卡匣。Immunotherapy DNA vaccines containing adenoviral vectors encoding HBV core antigen or HBV polymerase antigen were tested in mice. The purpose of this study was to detect the T-cell response induced by the vaccine after intramuscular delivery to F1 mice (C57BL / 6 × Balb / C). Initial immunogenicity studies have focused on identifying cellular immune responses elicited by the introduced HBV antigen. Specifically, the adenoviral vector tested contained a performance cassette as shown in Figures 8A and 8B.

活體內免疫原性研究In vivo immunogenicity studies

為了評價腺病毒疫苗之活體內免疫原性，將HBV腺病毒載體經肌肉內投與F1小鼠體內。該等免疫原性研究集中在確定由HBV抗原核心及聚合酶引發的細胞免疫反應。如表6中所概述，對F1小鼠進行投與。動物接受一次HBV載體免疫接種。在九週後收集脾細胞。To evaluate the in vivo immunogenicity of adenovirus vaccines, HBV adenovirus vectors were administered intramuscularly to F1 mice. These immunogenicity studies have focused on identifying cellular immune responses triggered by the HBV antigen core and polymerase. As outlined in Table 6, F1 mice were administered. Animals received one HBV vector immunization. Spleen cells were collected after nine weeks.

表 6 ：關於用腺病毒載體免疫接種小鼠之實驗設計
IM：肌肉內；vp：病毒粒子； Table 6 : Experimental design for immunizing mice with adenovirus vectors
IM: intramuscular; vp: virion;

關於on HBVHBV 腺病毒載體免疫原性之評價Evaluation of adenoviral vector immunogenicity

藉由IFN-γ酶聯免疫斑點(ELISPOT)分析及定量抗原特異性反應。在本分析中，將自免疫接種之動物分離的脾細胞與包含核心及Pol蛋白質(2 µg/ml各肽)之肽池一起培育。該等池由重疊11個殘基之15聚體肽組成，該等殘基匹配核心及Pol腺病毒載體之基因型ABCD共同序列。將較大的94 kDa HBV Pol蛋白質自中間分至兩個肽池中。在ELISPOT中，利用適當抗體且隨後顯色偵測來觀測在微量盤上呈有色斑點(稱為斑點形成細胞(SFC))形式的單一抗原特異性T細胞釋放之IFN-γ。The antigen-specific response was analyzed and quantified by IFN-γ enzyme-linked immunospot (ELISPOT). In this analysis, spleen cells isolated from immunized animals were incubated with a peptide pool containing core and Pol proteins (2 µg / ml of each peptide). The pools consist of 15-mer peptides overlapping 11 residues that match the core and genotype ABCD common sequence of the Pol adenovirus vector. The larger 94 kDa HBV Pol protein was split into two peptide pools from the middle. In ELISPOT, IFN-γ released by single antigen-specific T cells in the form of colored spots (called spot-forming cells (SFC)) on a microplate is observed with appropriate antibodies and subsequent colorimetric detection.

結果顯示於圖 9 中。自該等結果可以看出，HBV腺病毒載體，尤其是核心聚合酶融合物及核心腺病毒載體之組合引起核心及聚合酶特異性T細胞反應。此等資料指示，編碼HBV核心及pol抗原之腺病毒載體針對F1小鼠中之核心及pol產生穩定的T細胞反應。The results are shown in FIG. 9 . From these results, it can be seen that HBV adenovirus vectors, especially the combination of core polymerase fusions and core adenovirus vectors, cause core and polymerase-specific T cell responses. These data indicate that adenoviral vectors encoding the HBV core and pol antigens produce a stable T-cell response against the core and pol in F1 mice.

應理解，本文所描述之實例及實施例僅出於說明之目的，且在不背離其廣義發明概念情況下，可以對以上描述之實施例作出改變。因此，應理解，本發明不限於所揭示之特定實施例，而是意圖涵蓋在所附申請專利範圍所限定之本發明精神及範圍內之修改。
參考文獻 1. Cohen et al. “Is chronic hepatitis B being undertreated in the United States?”J. Viral Hepat . (2011) 18(6), 377-83. 2. Obeng-Adjei et al. “DNA vaccine cocktail expressing genotype A and C HBV surface and consensus core antigens generates robust cytotoxic and antibody responses and mice and Rhesus macaques”Cancer Gene Therapy (2013) 20, 652-662. 3. World Health Organization, Hepatitis B: Fact sheet No. 204 [Internet] 2015 March. Available from http://www.who.nt/mediacentre/factsheets/fs204/en/. 4. Belloni et al. “IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome”J. Clin. Invest . (2012) 122(2), 529-537. 5. Michel et al. “Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges.”J. Hepatol . (2011) 54(6), 1286-1296.It should be understood that the examples and embodiments described herein are for illustration purposes only, and changes may be made to the embodiments described above without departing from its broad inventive concepts. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by the scope of the appended patent applications.
References 1. Cohen et al. “Is chronic hepatitis B being undertreated in the United States?” J. Viral Hepat . (2011) 18 (6), 377-83. 2. Obeng-Adjei et al. “DNA vaccine cocktail expressing genotype A and C HBV surface and consensus core antigens generates robust cytotoxic and antibody responses and mice and Rhesus macaques ” Cancer Gene Therapy (2013) 20, 652-662. 3. World Health Organization, Hepatitis B: Fact sheet No. 204 [ Internet] 2015 March. Available from http: //www.who.nt/mediacentre/factsheets/fs204/en/. 4. Belloni et al. "IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome ” J. Clin. Invest . (2012) 122 (2), 529-537. 5. Michel et al.“ Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges. " J. Hepatol . (2011) 54 (6), 1286-1296.

當結合隨附圖式閱讀時，將更好地理解本發明之前述發明內容以及以下實施方式。應理解，本發明不限於附圖中顯示之確切實施例。The foregoing summary of the present invention and the following embodiments will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the exact embodiments shown in the drawings.

在附圖中：In the drawings:

圖 1A-1B 描繪B型肝炎病毒之基因組及病毒生命週期；圖 1A 係B型肝炎病毒(HBV)之基因組的圖式；在原生病毒中，聚合酶蛋白質(Pol)含有在不同開放閱讀框架中的包膜蛋白之編碼序列；該等包膜蛋白(前S1、前S2及S)係在同一開放閱讀框架中；圖 1B 顯示HBV之病毒生命週期； Figure 1A-1B depicts the genome and viral life cycle of hepatitis B virus; Figure 1A is a diagram of the genome of hepatitis B virus (HBV); in the native virus, the polymerase protein (Pol) is contained in different open reading frames Envelope protein coding sequence; the envelope proteins (pre-S1, pre-S2 and S) are in the same open reading frame; Figure 1B shows the viral life cycle of HBV;

圖 2A-2H 顯示如實例1中所描述的編碼HBV pol及核心抗原之表現卡匣及DNA質體的設計及優化；圖 2A 係一種表現策略之示意性表示，其中HBV核心及pol抗原之編碼序列係框內融合；圖 2B 係一種表現策略之示意性表示，其中核心及pol抗原之編碼序列係藉助於核糖體FA2滑移位點自單一質體表現；圖 2C 係一種表現策略之示意性表示，其中核心及pol抗原係由兩個獨立質體表現；圖 2D 係在存在及不存在轉錄後調控元件WPRE下表現核心抗原之質體轉染的HEK293T細胞中核心抗原表現之西方墨點(Western blot)；表現係使用α-核心抗體在細胞溶解產物(左)及上清液(sup；右)中測試；圖 2E 係西方墨點分析，顯示出用核心表現質體轉染之HEK293T細胞中核心表現的比較，該核心表現質體包括來源於人類載脂蛋白A1前驅體之內含子/外顯子序列(「AI內含子」)、1型人類T細胞白血病病毒(HTLV-1)長末端重複序列(LTR)之非轉譯R-U5結構域(「HTLV R」)，或HTLV-1 LTR、合成兔β-血球蛋白內含子及剪接強化子構成之三強化子複合序列(「triple」)；未標記之泳道係作為尺寸標記物的經純化之核心蛋白；表現係在溶解產物(左)及上清液(sup；右)中測試；利用三強化子複合序列之核心抗原表現量最高；圖 2F 係使用不同信號肽與HBV核心抗原N末端之融合物的核心抗原分泌情況之西方墨點分析；利用胱抑素S信號肽觀察到最高效之蛋白質分泌；圖 2G 係圖 2A - 2C 中所示三種表現策略各自之經優化HBV核心/聚合酶抗原表現卡匣的示意性表示；CMVpr：人類CMV-IE啟動子；TRE：三強化子序列；SP：胱抑素S信號肽；FA2：FMDV核糖體滑移位點；pA：BGH聚腺苷酸化信號；圖 2H 係有關含有圖 2G 中所示表現卡匣中之每一個的pDK載體之HBV核心及pol抗原表現的西方墨點分析；泳道1及2：pDK-核心；泳道3及4：pDK-聚合酶；泳道5及6：pDK-核心FA2聚合酶；泳道7及8：pDK-核心-聚合酶融合物：當該等抗原係由獨立載體編碼時，觀察到細胞及經分泌核心及聚合酶抗原之最一致的表現圖譜； Figures 2A-2H show the design and optimization of performance cassettes and DNA plastids encoding HBV pol and core antigens as described in Example 1; Figure 2A is a schematic representation of a performance strategy in which the HBV core and pol antigens are encoded The sequence is in-frame fusion; Figure 2B is a schematic representation of a performance strategy, in which the core and pol antigen coding sequences are expressed from a single plastid by means of the ribosome FA2 slippage point; Figure 2C is a schematic representation of a performance strategy It shows that the core and pol antigens are represented by two independent plastids; FIG. 2D is the western blot of the performance of core antigens in HEK293T cells transfected with plastids expressing core antigens in the presence and absence of post-transcriptional regulatory elements WPRE ( Western blot); performance was tested using α-core antibodies in cell lysates (left) and supernatant (sup; right); Figure 2E shows western blot analysis, showing HEK293T cells transfected with core performance plastids Comparison of core performance in this core performance plastid including intron / exon sequences derived from the human apolipoprotein A1 precursor ("AI intron"), type 1 human T cell leukemia virus (HTLV-1 Long end repeat Sequence (LTR) is a non-translated R-U5 domain ("HTLV R"), or a triple enhancer complex sequence consisting of HTLV-1 LTR, synthetic rabbit β-hemoglobin introns and splice enhancers ("triple"); Unlabeled lanes are purified core proteins as size markers; performance is tested in lysates (left) and supernatant (sup; right); core antigens using the triple enhancer complex sequence have the highest expression ; Figure 2F is a Western blot analysis of core antigen secretion using fusions of different signal peptides with the N-terminus of the HBV core antigen; the most efficient protein secretion was observed using the cystatin S signal peptide; Figure 2G is Figure 2A - 2C Schematic representation of each of the three performance strategies shown in the optimized HBV core / polymerase antigen performance cassette; CMVpr: human CMV-IE promoter; TRE: triple enhancer sequence; SP: cystatin S signal peptide; FA2 : FMDV ribosomal slippage point; pA: BGH polyadenylation signal; Figure 2H is a Western blot analysis of the HBV core and pol antigen performance of a pDK vector containing each of the performance cassettes shown in Figure 2G ; Lanes 1 and 2: pDK-core; lanes 3 and 4: pDK-poly Enzymes; lanes 5 and 6: pDK-core FA2 polymerase; lanes 7 and 8: pDK-core-polymerase fusions: When these antigens are encoded by independent vectors, cells and secreted cores and polymerase antigens are observed The most consistent performance map;

圖 3A - 3B 顯示根據本申請案之實施例之DNA質體的示意性表示；圖 3A 顯示根據本申請案一個實施例的編碼HBV核心抗原之DNA質體；圖 3B 顯示根據本申請案一個實施例的編碼HBV聚合酶(pol)抗原之DNA質體；HBV核心及pol抗原係在CMV啟動子控制下表現且在N末端帶有胱抑素S信號肽，該信號肽在自細胞分泌時自所表現之抗原裂解；該質體之轉錄調控元件包括位於CMV啟動子與編碼HBV抗原之聚核苷酸序列之間的強化子序列及位於編碼HBV抗原之聚核苷酸序列下游的bGH聚腺苷酸化序列；在該質體中以反向取向包括含處於Amp^r (bla)啟動子控制下之卡那黴素(kanamycin)抗性基因的第二表現卡匣；亦包括反向取向之複製起點(pUC)； Figure 3A - 3B shows a schematic representation of DNA embodiment of the present application of the plastid; Figure 3A shows DNA encoding the HBV core antigen according to the present application is an embodiment of the plastid; FIG. 3B shows the present application is an embodiment Example DNA plastids encoding HBV polymerase (pol) antigens; HBV core and pol antigens are under the control of the CMV promoter and carry the cystatin S signal peptide at the N-terminus. Expressed antigen cleavage; the plastid's transcriptional regulatory elements include an enhancer sequence located between the CMV promoter and the polynucleotide sequence encoding the HBV antigen, and a bGH polygland located downstream of the polynucleotide sequence encoding the HBV antigen Glycosylation sequence; a second expression cassette containing a kanamycin resistance gene under the control of the Amp ^r (bla) promoter in the reverse orientation in this plastid; also includes replication in the reverse orientation Starting point (pUC);

圖 4 顯示如實例2中所描述，用表現HBV核心抗原或HBV pol抗原之不同DNA質體免疫接種的Balb/c小鼠之ELISPOT反應；用於刺激自各種經疫苗接種之動物組分離之脾細胞的肽池以灰度指示；y軸上指示的反應性T細胞之數量以每10⁶ 個脾細胞的斑點形成細胞(SFC)數表示； Figure 4 shows the ELISPOT response of Balb / c mice immunized with different DNA plastids expressing HBV core antigen or HBV pol antigen as described in Example 2; used to stimulate spleens isolated from various groups of vaccinated animals peptide pool of cells to indicate gradation; number of reactive T cells in the y-axis indicated by dots per ¹⁰⁶ spleen cells forming cells (SFC) indicates the number;

圖 5 顯示根據實例3中描述之劑量發現研究，用表現HBV核心抗原及HBV pol抗原之DNA質體的組合免疫接種之Balb/C小鼠的ELISPOT反應；用於刺激自各種經疫苗接種之動物組分離之脾細胞的肽池以灰度指示；y軸上指示的反應性T細胞之數量以每10⁶ 個脾細胞的斑點形成細胞(SFC)數表示； Figure 5 shows the ELISPOT response of Balb / C mice immunized with a combination of DNA plastids expressing HBV core antigen and HBV pol antigen according to the dose discovery study described in Example 3; used to stimulate from various vaccinated animals peptide pool of splenocytes isolated set to indicate gradation; number of reactive T cells in the y-axis indicated by dots per ¹⁰⁶ spleen cells forming cells (SFC) indicates the number;

圖 6 顯示根據如實例4中所描述之免疫干擾研究，用表現HBV核心抗原及HBV pol抗原之DNA質體(pDNA)免疫接種之Balb/c小鼠的ELISPOT反應；第1組，僅核心 pDNA；第2組，僅Pol pDNA；第3組，混合的核心與Pol pDNA；第4組，分別在不同部位施用之核心及Pol pDNA；用於刺激自各種經疫苗接種之動物組分離之脾細胞的肽池以灰度指示；y軸上指示的反應性T細胞之數量以每10⁶ 個脾細胞的斑點形成細胞(SFC)數表示； Figure 6 shows the ELISPOT response of Balb / c mice immunized with DNA plastids (pDNA) expressing HBV core antigen and HBV pol antigen according to an immune interference study as described in Example 4; group 1, core pDNA only Group 2, Pol pDNA only; Group 3, mixed core and Pol pDNA; Group 4, cores and Pol pDNA administered at different sites, respectively; used to stimulate spleen cells isolated from various groups of vaccinated animals peptide pool indicated in gray; the number of reactive T cells in the y-axis indicates spots per 10 ⁶ spleen cells forming cells (SFC) indicates the number;

圖 7A 及 7B 顯示如實例5中所描述，在NHP中根據本申請案之一個實施例之DNA疫苗的免疫原性；圖 7A 顯示在用表現HBV核心及Pol抗原之DNA質體免疫接種之後的IFN-γ細胞介素反應；用於刺激自經疫苗接種之動物組分離之PBMC的肽池以灰度指示；y軸上指示的反應性T細胞之數量以每10⁶ 個PBMC的斑點形成細胞(SFC)數表示；圖 7B 顯示如藉由流式細胞測量術量測的針對核心、Pol-1及Pol-2肽池之CD4及CD8記憶T細胞免疫反應；圖中顯示由第76天得到的結果，以各池減去僅DMSO介質背景之後的針對該3個池之CD4或CD8 T細胞反應(IFN-γ、IL-2及TNF-α)百分比表示；CD4反應顯示於左側且CD8反應顯示於右側； 7A and 7B show as described in Example 5, the immunogenicity of DNA vaccine in the embodiment of NHP in accordance with one embodiment of the present application; FIG. 7A shows the performance with and Pol HBV core antigen DNA plasmid after immunization cytokine IFN-γ response; for stimulating peptide vaccine from the vaccinated group of animals of the separated PBMC pool to indicate gradation; number of reactive T cells in the y-axis indicated spots per 10 ⁶ PBMC forming cells (SFC) number display; Figure 7B shows CD4 and CD8 memory T cell immune responses against core, Pol-1 and Pol-2 peptide pools as measured by flow cytometry; the figure shows the results obtained on day 76 Results are expressed as the percentage of CD4 or CD8 T cell responses (IFN-γ, IL-2, and TNF-α) to the three pools after subtracting only the DMSO medium background from each pool; the CD4 response is shown on the left and the CD8 response is shown on Right;

圖 8A 及 8B 顯示根據本申請案之實施例的腺病毒載體中之表現卡匣之示意性表示；圖 8A 顯示截短之HBV核心抗原的表現卡匣，其含有CMV啟動子、內含子(來源於人類ApoAI基因之片段-GenBank寄存編號X01038鹼基對295-523，帶有ApoAI第二內含子)、人類免疫球蛋白分泌信號，隨後為截短之HBV核心抗原的編碼序列及SV40聚腺苷酸化信號；圖 8B 顯示截短之HBV核心抗原可操作地連接至HBV聚合酶抗原之融合蛋白的表現卡匣，除HBV抗原外，其在其他方面與截短之HBV核心抗原之表現卡匣相同；且 8A and 8B show a schematic representation of an adenoviral vector according to the present embodiment of the application of the expression cassette; FIG. 8A shows the expression cassettes of the truncated HBV core antigen, which contains the CMV promoter, intron ( A fragment derived from the human ApoAI gene-GenBank accession number X01038 base pair 295-523, with ApoAI second intron), human immunoglobulin secretion signal, followed by the truncated HBV core antigen coding sequence and SV40 polymer Adenylation signal; FIG. 8B shows the performance cassette of a fusion protein that is operatively linked to a truncated HBV core antigen, except for the HBV antigen, which is otherwise related to the performance card of the truncated HBV core antigen Same box; and

圖 9 顯示如實例8中所描述，用HBV腺病毒載體免疫接種之F1小鼠(C57BL/6×Balb/C)的ELISPOT反應；用於刺激自各種經疫苗接種動物組分離之脾細胞的HBV核心或聚合酶肽池以黑色(核心)及灰度(pol)指示；Pol1及pol2反應相加；X軸顯示腺病毒載體劑量及實驗組。y軸上指示的反應性T細胞之數量以每10⁶ 個脾細胞的斑點形成細胞(SFC)數表示。 Figure 9 shows the ELISPOT response of F1 mice (C57BL / 6 × Balb / C) immunized with HBV adenovirus vector as described in Example 8; used to stimulate HBV of splenocytes isolated from various groups of vaccinated animals The core or polymerase peptide pool is indicated by black (core) and gray (pol); Pol1 and pol2 reactions are added; the X-axis shows the adenovirus vector dose and the experimental group. The number of reactive T cells in the y-axis indicates spots per 10 ⁶ spleen cells forming cells (SFC) represents a number.

Claims

一種組合物，其包含： (a) 包含編碼HBV聚合酶抗原之第一聚核苷酸的第一非天然存在之核酸分子，該HBV聚合酶抗原具有與SEQ ID NO: 4至少90%一致之胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H(RNAse H)活性； (b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二非天然存在之核酸分子，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少95%一致之胺基酸序列組成；以及 (c) 醫藥學上可接受之載劑，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一非天然存在之核酸分子中或兩個不同的非天然存在之核酸分子中。A composition comprising: (a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence at least 90% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity or ribonuclease H (RNAse H) activity; (b) a second non-naturally-occurring nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen, the truncated HBV core antigen is at least 95% from SEQ ID NO: 2 or SEQ ID NO: 14 % Identical amino acid sequence composition; and (c) a pharmaceutically acceptable carrier, The first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule exist in the same non-naturally occurring nucleic acid molecule or two different non-naturally occurring nucleic acid molecules.

一種套組，其包含： (a) 包含編碼HBV聚合酶抗原之第一聚核苷酸的第一非天然存在之核酸分子，該HBV聚合酶抗原具有與SEQ ID NO: 4至少90%一致之胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性； (b) 包含編碼截短之HBV核心抗原之第二聚核苷酸的第二非天然存在之核酸分子，該截短之HBV核心抗原由與SEQ ID NO: 2或SEQ ID NO: 14至少90%一致之胺基酸序列組成；以及 (c) 醫藥學上可接受之載劑，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一非天然存在之核酸分子中或兩個不同的非天然存在之核酸分子中。A kit comprising: (a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide encoding a HBV polymerase antigen, the HBV polymerase antigen having an amino acid sequence at least 90% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity or ribonuclease H activity; (b) a second non-naturally-occurring nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV core antigen, the truncated HBV core antigen is at least 90% from SEQ ID NO: 2 or SEQ ID NO: 14 % Identical amino acid sequence composition; and (c) a pharmaceutically acceptable carrier, The first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule exist in the same non-naturally occurring nucleic acid molecule or two different non-naturally occurring nucleic acid molecules.

如請求項1或請求項2之組合物或套組，其中該第一聚核苷酸編碼包含與SEQ ID NO: 4至少98%一致之胺基酸序列的HBV聚合酶抗原，且該第二聚核苷酸編碼由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成的HBV核心抗原。The composition or kit of claim 1 or claim 2, wherein the first polynucleotide encodes a HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, and the second The polynucleotide encodes a HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14.

如請求項1或請求項2之組合物或套組，其中該第一聚核苷酸編碼含SEQ ID NO: 4之胺基酸序列的HBV聚合酶抗原。The composition or kit according to claim 1 or claim 2, wherein the first polynucleotide encodes a HBV polymerase antigen containing an amino acid sequence of SEQ ID NO: 4.

如請求項1至4中任一項之組合物或套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子中的至少一個進一步包含編碼可操作地連接至該HBV聚合酶抗原及該截短之HBV核心抗原中之至少一種的信號序列的聚核苷酸序列。The composition or kit of any one of claims 1 to 4, wherein at least one of the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule further comprises an encoding operably linked to the A polynucleotide sequence of a signal sequence of at least one of an HBV polymerase antigen and the truncated HBV core antigen.

如請求項5之組合物或套組，其中該信號序列獨立地包含SEQ ID NO: 6或SEQ ID NO: 19之胺基酸序列，較佳地該信號序列係由SEQ ID NO: 5或SEQ ID NO: 18之聚核苷酸序列編碼。The composition or kit according to claim 5, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is composed of SEQ ID NO: 5 or SEQ ID NO: 18 encodes the polynucleotide sequence.

如請求項1至6中任一項之組合物或套組，其中該第一聚核苷酸序列與SEQ ID NO: 3或SEQ ID NO: 16至少90%一致。The composition or kit according to any one of claims 1 to 6, wherein the first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16.

如請求項7之組合物或套組，其中該第一聚核苷酸序列包含SEQ ID NO: 3或SEQ ID NO: 16之聚核苷酸序列。The composition or kit of claim 7, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

如請求項1至8中任一項之組合物或套組，其中該第二聚核苷酸序列與SEQ ID NO: 1或SEQ ID NO: 15至少90%一致。The composition or kit according to any one of claims 1 to 8, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

如請求項9之組合物或套組，其中該第二聚核苷酸序列包含SEQ ID NO: 1或SEQ ID NO: 15之聚核苷酸序列。The composition or kit of claim 9, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

如請求項1至10中任一項之組合物或套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於同一載體中。The composition or kit according to any one of claims 1 to 10, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule exist in the same vector.

如請求項11之組合物或套組，其中該載體編碼包含該截短之HBV核心抗原視情況經由連接子可操作地連接至該HBV聚合酶抗原的融合蛋白。The composition or kit of claim 11, wherein the vector encodes a fusion protein comprising the truncated HBV core antigen optionally linked to the HBV polymerase antigen via a linker.

如請求項12之組合物或套組，其中該融合蛋白包含SEQ ID NO: 20之胺基酸序列。The composition or kit of claim 12, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 20.

如請求項12或請求項13之組合物或套組，其中該載體自5'端至3'端含有啟動子序列、強化子序列、信號肽編碼序列、該第二聚核苷酸序列、連接子編碼序列、該第一聚核苷酸序列及聚腺苷酸化信號序列。For example, the composition or kit of claim 12 or claim 13, wherein the vector contains a promoter sequence, an enhancer sequence, a signal peptide coding sequence, the second polynucleotide sequence, and a linker from the 5 'end to the 3' end. A daughter coding sequence, the first polynucleotide sequence, and a polyadenylation signal sequence.

如請求項14之組合物或套組，其中該載體係腺病毒載體，較佳為Ad26或Ad35載體。The composition or kit according to claim 14, wherein the vector is an adenovirus vector, preferably an Ad26 or Ad35 vector.

如請求項15之組合物或套組，其中該腺病毒載體含有包含SEQ ID NO: 17之聚核苷酸序列的該啟動子序列、包含SEQ ID NO: 23之聚核苷酸序列的調控序列、包含SEQ ID NO: 18之聚核苷酸序列的該信號肽編碼序列、包含SEQ ID NO: 15之聚核苷酸序列的該第二聚核苷酸序列、包含SEQ ID NO: 22之聚核苷酸序列的該連接子編碼序列、包含SEQ ID NO: 16之聚核苷酸序列的該第一聚核苷酸序列及包含SEQ ID NO: 24之聚核苷酸序列的該聚腺苷酸化信號序列。The composition or kit according to claim 15, wherein the adenoviral vector contains the promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17 and the regulatory sequence comprising the polynucleotide sequence of SEQ ID NO: 23 2. The signal peptide coding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, the second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15 and the poly sequence comprising SEQ ID NO: 22 The linker coding sequence of the nucleotide sequence, the first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16 and the polyadenosine comprising the polynucleotide sequence of SEQ ID NO: 24 Acidify the signal sequence.

如請求項1至10中任一項之組合物或套組，其中該第一非天然存在之核酸分子及該第二非天然存在之核酸分子係存在於兩個不同載體中。The composition or kit according to any one of claims 1 to 10, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in two different vectors.

如請求項17之組合物或套組，其中該第一非天然存在之核酸分子係存在於第一質體DNA載體中，且該第二非天然存在之核酸分子係存在於第二質體DNA載體中。The composition or kit of claim 17, wherein the first non-naturally occurring nucleic acid molecule is present in the first plastid DNA vector, and the second non-naturally occurring nucleic acid molecule is present in the second plastid DNA Vector.

如請求項18之組合物或套組，其中該第一質體DNA載體及該第二質體DNA載體各自包含複製起點、抗生素抗性基因且自5'端至3'端包含啟動子序列、調控序列、信號肽編碼序列、該第一聚核苷酸序列或該第二聚核苷酸序列，及聚腺苷酸化信號序列。The composition or kit of claim 18, wherein the first plastid DNA vector and the second plastid DNA vector each include an origin of replication, an antibiotic resistance gene, and a promoter sequence from the 5 'end to the 3' end, A control sequence, a signal peptide coding sequence, the first polynucleotide sequence or the second polynucleotide sequence, and a polyadenylation signal sequence.

如請求項19之組合物或套組，其中該抗生素抗性基因係具有與SEQ ID NO: 12至少90%一致，較佳地與SEQ ID NO: 12達100%一致之聚核苷酸序列的卡那黴素抗性基因。The composition or kit of claim 19, wherein the antibiotic resistance gene has a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 12, and preferably 100% identical to SEQ ID NO: 12 Kanamycin resistance gene.

如請求項20之組合物或套組，其包含 (a) 第一質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的該調控序列、含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、含SEQ ID NO: 3之聚核苷酸序列的該第一聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列； (b) 第二質體DNA載體，自3'端至5'端，其包括含SEQ ID NO: 7之聚核苷酸序列的該啟動子序列、含SEQ ID NO: 8之聚核苷酸序列的該調控序列、含SEQ ID NO: 5之聚核苷酸序列的該信號肽編碼序列、含SEQ ID NO: 1之聚核苷酸序列的該第二聚核苷酸序列及含SEQ ID NO: 11之聚核苷酸序列的該聚腺苷酸化信號序列；以及 (c) 醫藥學上可接受之載劑，其中該第一質體DNA載體及該第二質體DNA載體各自進一步包含具有SEQ ID NO: 12之聚核苷酸序列的卡那黴素抗性基因及具有SEQ ID NO:10之聚核苷酸序列的複製起點，且其中該第一質體DNA載體與該第二質體DNA載體係在同一組合物或兩種不同組合物中。A composition or kit as claimed in claim 20, comprising (a) a first plastid DNA vector, from the 3 'end to the 5' end, comprising the promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7 and the polynucleotide containing SEQ ID NO: 8 The control sequence of the sequence, the signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, the first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and the sequence containing SEQ ID The polyadenylation signal sequence of the polynucleotide sequence of NO: 11; (b) a second plastid DNA vector, from the 3 'end to the 5' end, comprising the promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7 and the polynucleotide containing SEQ ID NO: 8 The control sequence of the sequence, the signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, the second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 1, and the sequence containing SEQ ID The polyadenylation signal sequence of the polynucleotide sequence of NO: 11; and (c) a pharmaceutically acceptable carrier, Wherein the first plastid DNA vector and the second plastid DNA vector each further comprise a kanamycin resistance gene having a polynucleotide sequence of SEQ ID NO: 12 and a polynucleoside having SEQ ID NO: 10 Origin of replication of the acid sequence, and The first plastid DNA vector and the second plastid DNA vector are in the same composition or two different compositions.

如請求項1至21中任一項之組合物或套組，其係用於在有需要之個體中誘發針對B型肝炎病毒之免疫反應，較佳該個體患有慢性HBV感染。The composition or kit according to any one of claims 1 to 21, which is used to elicit an immune response against hepatitis B virus in an individual in need, preferably the individual has a chronic HBV infection.

如請求項1至21中任一項之組合物或套組，其係用於治療有需要之個體的B型肝炎病毒(HBV)誘發之疾病，較佳該個體患有慢性HBV感染，且該HBV誘發之疾病係選自由晚期纖維化、肝硬化及肝細胞癌(HCC)組成之群。If the composition or kit according to any one of claims 1 to 21 is for treating a hepatitis B virus (HBV) -induced disease in an individual in need, preferably the individual has a chronic HBV infection, and the HBV-induced diseases are selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

一種非天然存在之核酸分子，其包含編碼HBV聚合酶抗原之第一聚核苷酸序列，該HBV聚合酶抗原包含與SEQ ID NO: 4至少98%一致之胺基酸序列，其中該HBV聚合酶抗原不具有逆轉錄酶活性及核糖核酸酶H活性，其中該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少兩種HBV基因型之免疫反應，較佳地，該HBV聚合酶抗原能夠在哺乳動物中誘發針對至少HBV基因型B、C及D之T細胞反應，且更佳地，該HBV聚合酶抗原能夠在人類個體中誘發針對至少HBV基因型A、B、C及D之CD8 T細胞反應，視情況，該非天然存在之核酸分子進一步包含編碼截短之HBV核心抗原的第二聚核苷酸序列，該截短之HBV核心抗原由SEQ ID NO: 2或SEQ ID NO: 14之胺基酸序列組成。A non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an HBV polymerase antigen, the HBV polymerase antigen comprising an amino acid sequence at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerizes The enzyme antigen does not have reverse transcriptase activity and ribonuclease H activity. The HBV polymerase antigen can induce an immune response in mammals against at least two HBV genotypes. Preferably, the HBV polymerase antigen can be lactated T cell responses to at least HBV genotypes B, C and D are induced in animals, and more preferably, the HBV polymerase antigen is capable of inducing CD8 T cells to at least HBV genotypes A, B, C and D in human The reaction, optionally, the non-naturally occurring nucleic acid molecule further comprises a second polynucleotide sequence encoding a truncated HBV core antigen, the truncated HBV core antigen being composed of the amine of SEQ ID NO: 2 or SEQ ID NO: 14 Consisting of amino acid sequences.

如請求項24之非天然存在之核酸分子，其編碼包含該截短之HBV核心抗原視情況經由連接子可操作地連接至該HBV聚合酶抗原的融合蛋白。The non-naturally-occurring nucleic acid molecule of claim 24, which encodes a fusion protein comprising the truncated HBV core antigen optionally linked to the HBV polymerase antigen via a linker.