TW200825099A

TW200825099A - Novel H5 proteins, nucleic acid molecules and vectors encoding for those, and their medicinal use

Info

Publication number: TW200825099A
Application number: TW96140489A
Authority: TW
Inventors: Eric Martin Vaughn; Paulino Carlos Gonzalez-Hernandez; Juergen Daemmgen
Original assignee: Boehringer Ingelheim Vetmed
Priority date: 2006-10-27
Filing date: 2007-10-26
Publication date: 2008-06-16
Also published as: CN101553248B; ZA200902023B; CN101553248A; AR063427A1; CL2007003102A1; TWI413647B; UA99117C2

Abstract

The present invention relates to novel hemagglutinin H5 proteins, nucleic acids and vectors encoding for those as well as vaccines comprising any of such H5 proteins, nucleic acids or vectors encoding for those H5 proteins. Moreover, the present invention also relates to the medicinal use of any of such compositions in humans and animals.

Description

200825099 九、發明說明：【發明所屬之技術領域】丨州々、吁米病領域。特定言之，本發明係關於流感蛋自· ^ 曰，編碼彼等蛋白質之核酸分子及載體；及疫苗。更特定言之，太 σ之本發明係關於該等蛋白質、核酸分子、載體或疫苗中之任一者用於>口療及預防流感感染’進一步用於預防流减病卷忒媽毋之種内及種間傳染的用途。 Γ200825099 IX. Description of the invention: [Technical field to which the invention belongs] The field of Zhangzhou 々 and Yumi disease. In particular, the present invention relates to flu eggs, nucleic acid molecules and vectors encoding the same; and vaccines. More specifically, the present invention relates to any of such proteins, nucleic acid molecules, vectors or vaccines for use in >Oral Therapy and Prevention of Influenza Infections' Further for Prevention of Flow Reduction and Attenuation Use of intraspecific and interspecies infections. Γ

【先前技術】流感感染仍然為動物及人類之重要感染。流感係由㈣連續抗原性變異/修飾且佔據動物宿主的病毒引起。：此，未來τ能發錢的流行病及廣泛性流行病，且難以實現该疾病之根除。流感病毒已熟知於此項技術且詳述於 (例如）可供進一步參考之八茗加卷，第12期，第s 82至s 86頁（2004年12月）々。兔而卞之，流感A病毒之基因組係由八個單股區段組成，且病毒顆粒在其表面上具有兩種主要醣蛋白：血球凝集素（H)及神經胺糖酸苷酶（N)。在流感病毒之間因存在至少Μ種不同的血球凝集素（H1至H16)及9種不同的神經胺糖酸苷酶 (N1至N9)亞型而存在大量的抗原性變異。已證明H5N1型禽流感病毒之流感病毒可感染禽類、豬及人類。該等病毒亦可自禽類物種直接傳染給人類（c/a似等人 ’ Lancet 1998，351: 472; Suarez等人，J· Virol· 1998, ’ 6678; Subbarao 等人，Science 1998，279· 393, 124989.doc 200825099 抑州—㈣，π料w2p)。已知人類臨床病例之死亡率接近約50%。上一個世紀，豬一直為流感廣泛性流行病之重要媒介。豬、路騎及海豹（以豬為較佳）可充當禽流感病毒之，混合室，口此代表種越過禽類（流感病毒之天然宿主）至哺乳動物之物種障礙之潛在風險因素。此通常發生為易感性動物 (例如豬）為已確定哺乳動物（豬）流感病毒與禽流感病毒雙重感染。此雙重感染可形成可導致人類或豬廣泛性流行病的新重組病毒。然而，最近有跡象表明，當前禽類H5病毒株與甫乳動物流感病毒之重組不會形成強毒性重組體。另一方面，禽流感病毒可感染豬且藉由自發突變而變得順應於豬。一旦病毒在豬（或其他哺乳動物）群體内引起橫向感染，則將越過臨界障礙。然而，東南亞豬大部分已經來源於鄰近家禽飼養業之禽 (H5)流感病毒株感染。由於彼等感染迄今為止尚為亞臨床性，因此其僅可藉由實驗室方法診斷而由此常常被忽視。存在以下高風險：彼等受亞臨床性感染之豬為病毒順應於哺乳動物系統、在豬群體内傳播以及感染人類提供了機會。當前流感疫苗包括次單位疫苗事乂， 1999，17(9-10)··1223-1238; Crawford等人，Vaccine 1999, 17(18):2265-2274; Johansson 等人，Vaccine 1999 17(15-16):2073-2080)、戚毒疫苦（Horimoto等人，Vaccine 2004, 22(17-18):2244-2247)、ΏΝΑ 爽培（Watabe 等人，Vaccine 124989.doc - 7 - 200825099 2001，19(31):4434-4444)反藏活 i 氟爽笼（Cao 等人， FaccMe 70(():235-242)，其中滅活流感疫苗以商業規稹最廣泛使用（Lipatov等人，J Viro! 2004，78(17):8951- 次單位疫苗、重組性血球凝集素及神經胺糖酸苷酶 (Babai等人，Vaccine 1999，17(9-10):1223-1238; Crawford 等人，Vaccine 1999，17(18):2265-2274; Johansson等人，[Prior Art] Influenza infection remains an important infection for animals and humans. Influenza is caused by (iv) continuous antigenic variation/modification and viruses that occupy the animal host. : This is an epidemic and a pandemic that can be used to make money in the future, and it is difficult to eradicate the disease. Influenza viruses are well known in the art and are described in detail in, for example, Gossip Plus, No. 12, pp. 82-s 86 (December 2004). Rabbits, the genome of influenza A virus consists of eight single-stranded segments, and the virus particles have two major glycoproteins on their surface: hemagglutinin (H) and neuraminidase (N) . There are a large number of antigenic variations between influenza viruses due to the presence of at least different hemagglutinin (H1 to H16) and 9 different neuraminidase (N1 to N9) subtypes. Influenza viruses of the H5N1 avian influenza virus have been shown to infect poultry, pigs and humans. These viruses can also be transmitted directly from humans to avian species (c/a like et al. Lancet 1998, 351: 472; Suarez et al., J. Virol 1998, '6678; Subbarao et al., Science 1998, 279. 393 , 124989.doc 200825099 Yizhou - (four), π material w2p). The mortality rate in human clinical cases is known to be close to about 50%. In the last century, pigs have been an important medium for the pandemic of influenza. Pigs, road rides and seals (preferably pigs) can act as avian influenza viruses, and the mixing chamber, which represents a potential risk factor for species barriers that cross the birds (the natural host of the influenza virus) to mammals. This usually occurs as a susceptibility animal (e.g., a pig) to a double infection of a mammalian (porcine) influenza virus and an avian influenza virus. This dual infection can form new recombinant viruses that can cause widespread epidemics in humans or pigs. However, there have been recent indications that the current recombination of avian H5 strains with sputum animal influenza viruses does not form highly toxic recombinants. On the other hand, the avian influenza virus can infect pigs and become compliant with pigs by spontaneous mutation. Once the virus causes a lateral infection in the swine (or other mammalian) population, the critical disorder will be crossed. However, most of the pigs in Southeast Asia have been infected with avian (H5) influenza virus strains adjacent to the poultry industry. Since their infections have so far been subclinical, they can only be diagnosed by laboratory methods and are often overlooked. There is a high risk that their subclinically infected pigs provide an opportunity for the virus to adapt to the mammalian system, spread within the swine population, and infect humans. Current influenza vaccines include sub-unit vaccines, 1999, 17 (9-10) · 1223-1238; Crawford et al, Vaccine 1999, 17(18): 2265-2274; Johansson et al., Vaccine 1999 17 (15- 16): 2073-2080), scorpion venom (Horimoto et al, Vaccine 2004, 22 (17-18): 2244-2247), 爽爽 (Watabe et al, Vaccine 124989.doc - 7 - 200825099 2001, 19(31): 4443-4444) Anti-Tibetan i Fluorine Cage (Cao et al., FaccMe 70((): 235-242), in which the inactivated influenza vaccine is most widely used by commercial regulations (Lipatov et al., J Viro! 2004, 78(17): 8951 - Subunit vaccine, recombinant hemagglutinin and neuraminidase (Babai et al., Vaccine 1999, 17(9-10): 1223-1238; Crawford et al. Vaccine 1999, 17(18): 2265-2274; Johansson et al.

Vaccine 1999，17(15-16):2073-2080)可為頗受 Μ 注的滅活疫苗替代物，儘管目前尚未作為商用疫苗投入使用。該等疫苗之製備顯然比滅活疫苗安全。此外，次單位疫苗並不對内部流感病毒蛋白產生抗體反應且從而使經接種之動物與已感染動物之間有區別。（CVaw/brd等人，Facc/w 17(18):2265-2274) 〇血球凝集素蛋白為流感病毒之受體結合及膜融合醣蛋白以及用於感染性中和抗體之標靶。H5N1之完整血球凝集素蛋白（HA)係由568個胺基酸組成，分子量為56 kDa。HA 分子係由HA1及HA2次單位組成，HA1次單位介導與細胞膜之初始接觸而HA2負責膜融合 Bioelectrochemistry 2004，63 (1-2): 129-136)。桿狀病毒/昆蟲細胞系統已用於表現自禽流感亞型分離之血球凝集素基因# 乂，Faccz·⑽/70-10): 1223-1238; Crawford 等人，Vaccine 1999, 1 7(18):2265-2274; Johansson 等人，Vaccine 1999，1 7(15-16):2073-2080): New 等人，BMC Mircobiology 2006, 124989.doc 200825099 6(16):doi:10，li86/1471-2180-6-16)。m 而，彼等重組蛋白在某些情況下似乎不具有保護性，或者僅在最低程度上對某些物種具有較低有效性（Trean〇r等人，Vaccine 2001，19: 1732-1737) 〇 • 因此’需要增強經改良之疫苗及新疫苗接種方法之可用 . 性以提供控制流感感染的更佳方法及對疾病負荷產生正面影響。【發明内容】 f) 在本發明之實施例之前，應瞭解，如本文中及隨附申請專利範圍中所使用，單數形式，，一"及，，該，，包括複數提及物，除非上下文另有明確說明。因此，舉例而言，提及一種製劑’’包括複數種該等製劑；提及該，，載劑"係指熟習此項技術者已知之一或多種载劑及其均等物，諸如此類。除非另外定義，否則本文中所使用之所有技術及科學術語具有的含義與普通熟習本發明所屬技術者通常所瞭解之含〇彡相同。除非另外指明或熟習此項技術者另外得知，否則所有給定範圍及值可變化丨至5%，因此術語，，約"在本說明曰中省去。儘官可在本發明之實施或測試中使用與本文中所述之彼等方法及物質相似或相當的任何方法及物質，但較仏之方法、裳置及物質現加以描述。為描述並揭示可配合本發明使用之如公開案中所報導之物質、賦形劑、載劑及方法之目的，本文中所提及之所有公開案以引用的方式併入本文中。不應認為本文中認可本發明無權優先於先前發明之此類揭示案。 124989.doc 200825099 藉由本說明及申請專利範圍中之特徵性實施例獲得上述技術問題之解決方法。流感蛋白及編碼彼等蛋白質之核酸分子本發明係關於流感病毒之H5蛋白，其中該H5蛋白具有胺基酸223N及修飾體328K+，其中H5蛋白之胺基酸位置編號係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上***第二離胺酸（K+)。較佳地，本發明之該H5蛋白及其他任何 H5蛋白為經分離之H5蛋白。已驚人地發現，與在位置223 及328/329上不具有相應胺基酸的H5蛋白相比，具有上述修飾體之H5蛋白具有更高的抗原性。如本文中所使用之術語’’血球凝集素5(Η5)Π或”禽流感病毒之Η5”或’’Η5蛋白’’意謂（但不限於）任何天然存在之Η5蛋白及Η5蛋白之任何經修飾形式（包括Η5蛋白之任何缺失、取代及/或***突變體），其中彼等Η5蛋白具有胺基酸223Ν 及修飾體328Κ+。如本文中所使用之Η5蛋白之胺基酸位置之編號係指如 SEQ ID ΝΟ:1中所例示性指定之胺基酸位置。SEQ ID ΝΟ··1代表病毒株鴨/China/E319_2/03之血球凝集素之胺基序列（但缺少胺基末端信號肽）。換而言之，若提及位置223 上之胺基酸（胺基酸223)，則意謂與SEQ ID ΝΟ:1中之胺基酸223對應的胺基酸殘基。然而，此並非意謂本發明之H5 蛋白具有與SEQ ID ΝΟ:1—致的胺基酸序列。其僅意謂本發明之H5蛋白之對應胺基酸編碼所明確提及之胺基酸殘 124989.doc -10- 200825099 基。在此情況下，胺基酸223為絲胺酸（S)。術語’’223N”或 Π155Ν”分別例示性意謂，處於位置223及155(根據SEQ ID ΝΟ:1之胺基酸位置編號）上之胺基酸可編碼胺基酸天冬醯胺酸（N)。換而言之，若提及’’具有胺基酸223N之H5蛋白’’，則H5胺基酸分子通常在胺基酸位置223(根據SEQ ID ΝΟ:1 之胺基酸位置編號）上編碼的胺基酸絲胺酸可經天冬醯胺酸（N)取代。術語”328〖+”或”修飾體328K+f’意謂，在H5蛋白之胺基酸位置328(根據SEQ ID ΝΟ:1之胺基酸位置編號）上***第二離胺酸（K+)。在位置328及329上天然編碼離胺酸-離胺酸之胺基酸序列之情況下，未***其他離胺酸 (K)。然而，大多數已知H5序列在胺基酸位置328及329上編碼離胺酸-精胺酸。在該等任何情況下，術語328K+修飾體意謂，第二離胺酸（K)應***位置328上之離胺酸與位置 329上之精胺酸之間。於是經修飾之序列應讀成離胺酸·離胺酸-精胺酸（KKR)。因此，本發明係關於H5蛋白及H5蛋白之任何經修飾形式（包括H5蛋白之任何缺失、取代及/或***突變體），其中彼等H5蛋白具有胺基酸223N及修飾體328K+，其中H5蛋白之胺基酸位置編號係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上***第二離胺酸（K+)。不言而喻，如本文中所提供之任一種H5蛋白具有抗原性，此意謂其在對流感病毒之標準血球凝集素抑制檢定中展示抗原特性。根據另一實施例，本發明亦係關於H5蛋白之任何部分， 124989.doc -11 - 200825099 此部分意謂在標準血球凝集素抑制檢定中展示抗原特性，至少具有胺基酸223N及修飾體328K+的任何肽片段，其中 Η5蛋白之胺基酸位置之編號係指如SEQ 1]〇 ν〇:ι中所例示性指定之胺基酸位置且其中修飾體328Κ+意謂在Η5蛋白之胺基酸位置328上***第二離胺酸（κ+)。右Η5蛋白在標準血球凝集素抑制檢定（例如，如實例2中所述）中抑制血球凝集，則其展示抗原特性。出蛋白之該抗原部分通常包含編碼如上所述之經修飾或未經修飾，在如實例2中所述之標準血球凝集素抑制檢定中展示抗原特性之Η5蛋白的胺基酸序列中之2〇〇、18〇、16〇、15〇、 140、130、120、11〇或最佳105個毗鄰胺基酸。標準血球凝集素抑制檢定例如亦描述於可供進一步參考之Vaccine 1999, 17 (15-16): 2073-2080) may be a highly inactivated vaccine replacement, although it has not yet been put into commercial vaccine. The preparation of such vaccines is clearly safer than inactivated vaccines. In addition, the secondary unit vaccine does not produce an antibody response to the internal influenza virus protein and thus distinguishes between the vaccinated animal and the infected animal. (CVaw/brd et al., Facc/w 17(18): 2265-2274) 血 Hemagglutinin protein is a receptor binding and influenza fusion glycoprotein of influenza virus and a target for infectious neutralizing antibodies. H5N1's intact hemagglutinin protein (HA) consists of 568 amino acids with a molecular weight of 56 kDa. The HA molecule consists of HA1 and HA2 subunits, HA1 subunits mediate initial contact with the cell membrane and HA2 is responsible for membrane fusion Bioelectrochemistry 2004, 63 (1-2): 129-136). The baculovirus/insect cell system has been used to represent the hemagglutinin gene isolated from the avian influenza subtype #乂, Faccz·(10)/70-10): 1223-1238; Crawford et al., Vaccine 1999, 1 7(18) : 2265-2274; Johansson et al., Vaccine 1999, 1 7(15-16): 2073-2080): New et al., BMC Mircobiology 2006, 124989.doc 200825099 6(16): doi: 10, li86/1471- 2180-6-16). m, however, their recombinant proteins do not appear to be protective in some cases or are of low efficacy to certain species to a minimum (Trean〇r et al., Vaccine 2001, 19: 1732-1737). • Therefore, there is a need to enhance the availability of improved vaccines and new vaccination methods to provide better ways to control influenza infections and have a positive impact on disease burden. [Embodiment] f) Before the embodiments of the present invention, it is to be understood that the singular forms, """ The context is clearly stated otherwise. Thus, for example, reference to a formulation' includes a plurality of such formulations; reference to the "carrier" refers to one or more carriers and their equivalents known to those skilled in the art, and the like. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Unless otherwise indicated or otherwise known to those skilled in the art, all ranges and values may vary from 5% to 5%, so the terms, "about" are omitted from this description. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the methods, skirts, and materials are now described. All publications mentioned herein are incorporated herein by reference for their purpose to describe and disclose the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure. It is not to be considered as an admission that the invention is limited to the prior invention. 124989.doc 200825099 The solution to the above technical problem is obtained by the present description and the characteristic embodiments in the scope of the patent application. Influenza protein and nucleic acid molecule encoding the same according to the invention The invention relates to an H5 protein of influenza virus, wherein the H5 protein has an amino acid 223N and a modification 328K+, wherein the amino acid position number of the H5 protein means SEQ ID NO: The amino acid position exemplified in 1 and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. Preferably, the H5 protein of the present invention and any other H5 protein are isolated H5 proteins. It has been surprisingly found that the H5 protein having the above modifications has higher antigenicity than the H5 protein which does not have the corresponding amino acid at positions 223 and 328/329. The term 'hemagglutinin 5 (Η5)Π or 'avian influenza virus Η5' or ''Η5 protein'' as used herein means, but is not limited to, any of the naturally occurring Η5 protein and Η5 protein. Modified form (including any deletion, substitution and/or insertion mutant of the Η5 protein), wherein the Η5 protein has an amino acid 223Ν and a modification 328Κ+. The numbering of the amino acid position of the Η5 protein as used herein refers to the amino acid position as exemplified in SEQ ID ΝΟ:1. SEQ ID ΝΟ··1 represents the amino acid sequence of the hemagglutinin of the virus strain Duck/China/E319_2/03 (but lacks the amino terminal signal peptide). In other words, referring to the amino acid (amino acid 223) at position 223, it means an amino acid residue corresponding to the amino acid 223 of SEQ ID: 1. However, this does not mean that the H5 protein of the present invention has an amino acid sequence identical to SEQ ID NO: 1. It merely means the amino acid residue 124989.doc -10- 200825099 base explicitly mentioned in the corresponding amino acid code of the H5 protein of the present invention. In this case, the amino acid 223 is a serine acid (S). The term ''223N' or Π155Ν', respectively, exemplarily means that the amino acid at positions 223 and 155 (based on the amino acid position number of SEQ ID: 1) encodes the amino acid aspartic acid (N). ). In other words, if reference is made to ''H5 protein with amino acid 223N'', the H5 amino acid molecule is typically encoded at amino acid position 223 (numbering according to the amino acid position of SEQ ID ΝΟ: 1) The amino acid serine can be substituted with aspartic acid (N). The term "328"+" or "modification 328K+f" means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein (numbering the amino acid position according to SEQ ID: 1) In the case of the amino acid sequence encoding the lysine-deaminase naturally encoded at positions 328 and 329, no other lysine (K) was inserted. However, most of the known H5 sequences are at the amino acid position 328 and 329 encodes the lysine-arginine. In any of these cases, the term 328K+ modification means that the second lysine (K) should be inserted into the lysine at position 328 and the arginine at position 329. Therefore, the modified sequence should be read as aminic acid-lysine-arginine (KKR). Thus, the present invention relates to any modified form of H5 protein and H5 protein (including any deletion of H5 protein, Substituting and/or inserting mutants, wherein the H5 proteins have an amino acid 223N and a modification 328K+, wherein the amino acid position number of the H5 protein refers to an amino acid as exemplified in SEQ ID NO: 1. Position and wherein the modification 328K+ means inserting a second lysine (K+) at the amino acid position 328 of the H5 protein. It is stated that any of the H5 proteins as provided herein is antigenic, which means that it exhibits antigenic properties in a standard hemagglutinin inhibition assay for influenza viruses. According to another embodiment, the invention also relates to H5 proteins. Any part, 124989.doc -11 - 200825099 This section means any peptide fragment exhibiting antigenic properties in a standard hemagglutinin inhibition assay, having at least amino acid 223N and a modified 328K+, wherein the amino acid position of the Η5 protein The numbering refers to the amino acid position as exemplified in SEQ 1] 〇ν〇: ι and wherein the modification 328Κ+ means inserting the second lysine (κ+) at the amino acid position 328 of the Η5 protein. The right Η5 protein inhibits hemagglutination in a standard hemagglutinin inhibition assay (eg, as described in Example 2), which displays antigenic properties. The antigenic portion of the protein typically contains a modified or uncoded version as described above. Modification, 2 〇〇, 18 〇, 16 〇, 15 〇, 140, 130, 120, 11 in the amino acid sequence of the Η5 protein exhibiting antigenic characteristics in the standard hemagglutinin inhibition assay as described in Example 2. Best adjacent amino acids, or 105 standard hemagglutinin inhibition assay for example is also described further in reference available

Stephenson等人，Virus Research，第 103卷，第 91-95 頁 (2004)中。然而，應瞭解，如實例2中所述之HI檢定為配合本文中所述之本發明之所有態樣的相關參考檢定：簡而言之，進行HI檢定以偵測HA特異性抗體之存在。異源H5N2病毒（A/雞/Mexic0/232/94)係以四個血球凝集單位[4 HA單位]之濃度用於HI檢定中。隨後在u形底微量滴定板中，將PBS中之連續兩倍血清稀釋液與等體積（25 叫）（含有4 HA單位）之病毒混合，且在室溫（約25。(〕）下培育 30刀鐘。將PBS中之濃度為0.5%之雞紅血球添加至含有灰清-病毒之孔中且在室溫下培育40分鐘。以觀測到血球凝集抑制的最高血清稀釋度之倒數確定HI效價。值得注意的是，發現”針對 124989.doc -12- 200825099 激發病毒之HI效價與防禦激發之保護之間可能存在關聯，，。好aaMrowd及Pe似亦測定，HI效價>40的豬，，完全抵抗激發且在受激發下呼吸道中不發生病毒複製”。因此，在經疫苗接種之豬中形成>4〇之HI效價將與保護作用有 Μ。（F· Haesebrouck及 Μ·Β· Pensaert，1986)。读先玲戚活流感H1N1疫苗免疫之肥育豬之氣管内激發的效應 (FekrMar;; M/croMo/ogj；，1 1 (1986) 239-249)。須假設等值或至少差不多等值的H5 HI效價亦將對豬產生防禦禽流感病毒的完全免疫保護。較低效價至少引起經接種之動物之血清轉化且產生對彼等動物之部分免疫保護，此亦可大大降低廣泛性流行病之風險。此外’本發明之H5蛋白之抗原部分包括（但不限於）H5蛋白之缺失突變體，其包含： i·圍繞且包括胺基酸223N之胺基酸序列中之至少35、 3〇、25、20、18、15、13、10、9或最佳8個毗鄰胺基酸；及 Π·圍繞且包括胺基酸修飾體328K+之胺基酸序列中之至少 35、30、25、20、18、15、13、10、9或最佳 8個毗鄰胺基酸；且 iii.其中H5蛋白之該抗原部分中之任一者可在如實例2中所述之標準血球凝集素抑制檢定中展示血球凝集素抑較佳地，圍繞胺基酸223N及/或328K+之彼等胺基酸係由 SEQ ID ΝΟ:1 或 SEQ ID ΝΟ··4編碼。 124989.doc -13- 200825099 此外，本發明之較佳H5蛋白為： i.上述具有胺基酸223N及修飾體328K+的彼等H5蛋白中之任一者； Π.上述具有胺基酸94N/223N及修飾體328K+的彼等H5蛋白中之任一者； iii.具有胺基酸223N及修飾體328K+的任何禽源H5蛋白，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 I iv.具有胺基酸94N/223N及修飾體328K+的任何禽源H5蛋白，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 v. 具有胺基酸1 55N/223N及修飾體328K+的任何禽源H5蛋白，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 vi. 具有胺基酸120N/155N/223N及修飾體328K+的任何禽 . 源H5蛋白，其中禽源意謂H5序列來源於最初自感染上Stephenson et al., Virus Research, Vol. 103, pp. 91-95 (2004). However, it will be appreciated that the HI assay as described in Example 2 is a relevant reference assay that is compatible with all aspects of the invention described herein: Briefly, a HI assay is performed to detect the presence of HA-specific antibodies. Heterologous H5N2 virus (A/chicken/Mexic0/232/94) was used in the HI assay at a concentration of four hemagglutination units [4 HA units]. Subsequently, in a u-bottom microtiter plate, serial two-fold serum dilution in PBS was mixed with an equal volume (25 called) (containing 4 HA units) of virus and incubated at room temperature (about 25 ()). 30 knives. Chicken red blood cells at a concentration of 0.5% in PBS were added to the wells containing the ash-virus and incubated for 40 minutes at room temperature. The HI effect was determined by the reciprocal of the highest serum dilution observed for hemagglutination inhibition. It is worth noting that there may be a correlation between the HI titer of the stimulating virus and the protection of the defensive stimulus for 124989.doc -12- 200825099. Good aaMrowd and Pe are also measured, HI titer >40 Pigs, completely resistant to excitation and no viral replication in the inflamed lower respiratory tract." Therefore, the formation of HI titers in vaccinated pigs will be associated with protective effects (F· Haesebrouck and Μ·Β·Pensaert, 1986). The effect of intratracheal challenge in pigs fed with flu-infected H1N1 vaccine (FekrMar;; M/croMo/ogj;, 1 1 (1986) 239-249). Equivalent or at least approximately equivalent H5 HI titers will also be generated for pigs Protects against complete immune protection of avian influenza viruses. Lower titers cause at least seroconversion of vaccinated animals and produce partial immunoprotection to their animals, which can also greatly reduce the risk of widespread epidemics. The antigenic portion of the H5 protein includes, but is not limited to, a deletion mutant of the H5 protein comprising: i. at least 35, 3, 25, 20, 18, 15 of the amino acid sequence surrounding and comprising the amino acid 223N , 13, 10, 9 or preferably 8 adjacent amino acids; and Π· at least 35, 30, 25, 20, 18, 15, 13 of the amino acid sequence surrounding and including the amino acid modification 328K+ 10, 9 or preferably 8 adjacent amino acids; and iii. wherein any of the antigenic portions of the H5 protein can exhibit hemagglutinin inhibition in a standard hemagglutinin inhibition assay as described in Example 2. Preferably, the amino acid groups surrounding the amino acids 223N and/or 328K+ are encoded by SEQ ID ΝΟ:1 or SEQ ID ΝΟ·4. 124989.doc -13- 200825099 Furthermore, preferred H5 proteins of the invention It is: i. among the above H5 proteins having amino acid 223N and modified 328K+ One of the above H5 proteins having amino acid 94N/223N and modified 328K+; iii. any avian H5 protein having amino acid 223N and modified 328K+, wherein the avian source The H5 sequence is derived from a virus isolate originally isolated from avian infected with avian influenza virus type 5; or I iv. any avian H5 protein having amino acid 94N/223N and modified 328K+, wherein the avian source means H5 The sequence is derived from a virus isolate originally isolated from avian infected with avian influenza virus type 5; or v. any avian H5 protein having amino acid 1 55N/223N and modified 328K+, wherein the avian source is the source of the H5 sequence a virus isolate originally isolated from avian infected with avian influenza virus type 5; or vi. any avian. source H5 protein having amino acid 120N/155N/223N and modified 328K+, wherein the avian source is the source of the H5 sequence At the initial self-infection

U 5型禽流感病毒之禽類分離的病毒分離株；或 vii. 具有修飾體94N/223N及修飾體328K+的任何H5蛋白；或， viii.具有修飾體94N/15 5N/223N及修飾體328K+的任何H5 蛋白；或； ix.具有修飾體94N/120N/15 5N/223N及修飾體 328K+的任何H5蛋白；或 X.具有修飾體223N、修飾體328K+及選自由以下各者組 124989.doc -14- 200825099 成之群的以下胺基酸團中之一或多者的任何H5蛋白：a virus isolate isolated from avian influenza virus type U 5; or vii. any H5 protein having the modification 94N/223N and the modification 328K+; or, viii. having the modification 94N/15 5N/223N and the modification 328K+ Any H5 protein; or; ix. any H5 protein having the modification 94N/120N/15 5N/223N and the modification 328K+; or X. having the modification 223N, the modification 328K+ and selected from the group of 124989.doc - 14- 200825099 Any H5 protein of one or more of the following amino acid groups:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 123-125: SDHb. aa 123-125: SDH

c. aa 128-130: SSGc. aa 128-130: SSG

d. aa 138-140: GSSd. aa 138-140: GSS

e. aa 226-228: MDFe. aa 226-228: MDF

f. aa 270-272: EVE g. aa 309-31 1: NKL ;或f. aa 270-272: EVE g. aa 309-31 1: NKL; or

xi. 具有胺基酸223N及修飾體328K+及選自由以下各者組成之群的以下胺基酸團中之一或多者的任何H5蛋白：Xi. Any H5 protein having one or more of amino acid 223N and modification 328K+ and a group of the following amino acid groups selected from the group consisting of:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 128-130: SSG c. aa 138-140: GSS ;或 xii. 具有SEQIDNO:4之胺基酸序列的任何H5蛋白。此外，如本文中所提供之較佳H5蛋白包括以下文獻所述白·· Hoffmann等人，PNAS，第 106卷，第 36期，第 /297 5-72920趸(^005丰9方6 0」，其中彼等115蛋白包括如上所述之修飾體中之一或多者，至少包括胺基酸223N及修飾體328K+，其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體 328K+意謂在H5蛋白之胺基酸位置328上***第二離胺酸 (K+)。該參考文獻之揭示内容將以引用方式全文併入本文中0 此外，如本文中所提供之較佳H5蛋白包括包含含有胺基 124989.doc -15- 200825099 酸223N及修飾體328K+之肽的H5蛋白，其中H5蛋白之胺基位置之編被係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置 328上***第二離胺酸（κ+)，及：b. aa 128-130: SSG c. aa 138-140: GSS; or xii. Any H5 protein having the amino acid sequence of SEQ ID NO: 4. In addition, the preferred H5 proteins as provided herein include those described in the following literature: H. Hoffmann et al., PNAS, Vol. 106, No. 36, No. 297 5-72920 ((^005 Feng 9 Fang 6 0) And wherein the 115 proteins include one or more of the modifications as described above, and at least include the amino acid 223N and the modification 328K+, wherein the amino acid position of the H5 protein is numbered as in SEQ ID NO: 1. The exemplarily specified amino acid position and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. The disclosure of this reference is hereby incorporated by reference in its entirety. Further, a preferred H5 protein as provided herein includes an H5 protein comprising a peptide comprising an amine group 124989.doc -15-200825099 acid 223N and a modified form 328K+, wherein the amino group position of the H5 protein is referred to as The amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means the insertion of a second lysine (κ+) at the amino acid position 328 of the H5 protein, and:

1· SEQ ID NO:l、SEQ ID ΝΟ·2、SEQ ID Ν0.3、SEQ ID NO:4、SEQIDNO··5或SEQIDNO··6之胺基酸序列；或 U·具有與i)之多肽之至少85%序列同源性、更佳至少約 9〇%序列同源性、甚至更佳至少約95%序列同源性、甚至更佳至少約97%序列同源性、甚至更佳至少約 98%序列同源性且甚至更佳至少約99%序列同源性並在如上所述之標準血球凝集素抑制檢定中展示血球凝集素抑制的任何肽；或 ill. 1)或11)之多肽之任何抗原部分，其包含i)或⑴之肽中之任一者的至少 35、30、25、20、18、15、13、10、 9或最佳8個毗鄰胺基酸。 ◎ 1V· 〇、U)或lu)之任何肽，其具有胺基酸3 6T、3 6K、 83A、83T、83D、86A、86V、120N、120S、155N、 155S、156A、156T、189R、189K、212K、212R、 212E、223N、223N或 120N/155N。 - V. i)、u)、m)或iv)之任何肽，其具有選自由以下各者組成之群的以下胺基酸團中之一或多者：1· SEQ ID NO: 1, SEQ ID ΝΟ 2., SEQ ID Ν 0.3, SEQ ID NO: 4, SEQ ID NO.. 5 or SEQ ID NO.. 6 amino acid sequence; or U· has the polypeptide of i) At least 85% sequence homology, more preferably at least about 9% sequence homology, even more preferably at least about 95% sequence homology, even more preferably at least about 97% sequence homology, even more preferably at least about Any peptide exhibiting hemagglutinin inhibition in 98% sequence homology and even better at least about 99% sequence homology and in a standard hemagglutinin inhibition assay as described above; or a polypeptide of ill. 1) or 11) Any antigenic moiety comprising at least 35, 30, 25, 20, 18, 15, 13, 10, 9 or preferably 8 contiguous amino acids of any of the peptides of i) or (1). ◎ 1V·〇, U) or lu) any peptide having amino acids 3 6T, 3 6K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A, 156T, 189R, 189K , 212K, 212R, 212E, 223N, 223N or 120N/155N. - any of P. i), u), m) or iv) having one or more of the following amino acid groups selected from the group consisting of:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 123-125: SDHb. aa 123-125: SDH

c. aa 128-130: SSG 124989.doc -16 - 200825099c. aa 128-130: SSG 124989.doc -16 - 200825099

d. aa 138-140: GSSd. aa 138-140: GSS

e. aa 226-228: MDFe. aa 226-228: MDF

f. aa 270-272: EVE g. aa 309-31 1: NKL ； ^ vi. i)、ii)、iii)或iv)之任何肽，其具有選自由以下各者組成之群的以下胺基酸團中之一或多者：f. aa 270-272: EVE g. aa 309-31 1: NKL; ^ vi. any peptide of i), ii), iii) or iv) having the following amine group selected from the group consisting of: One or more of the acid groups:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 128-130: SSG c. aa 138-140: GSS。如本文中所使用之”序列同源性”係指測定兩個序列之相關性的方法。為測定序列同源性，將兩個或兩個以上的序列最佳對齊，且必要時引入空位（gap)。與序列一致性形成對比，在測定序列同源性時，保守性胺基酸取代視為匹配。換而言之，為獲得具有與參考序列之95%序列同源性的多肽或多核苷酸，參考序列中85%、較佳9〇%、甚至更佳95%之胺基酸殘基或核苷酸必須與其他胺基酸或核苷酸匹配或包含其他胺基酸或核苷酸之保守性取代，或者可將參考序列中全部胺基酸殘基或核苷酸（不包括保守性取代）之至多15%、較佳至多1()%、甚至更佳至多5%量的胺基酸或核苷酸***參考序列中。較佳地，同源序列包含至少一段50個核苷酸、甚至更佳1〇〇個核苷酸、甚至更佳25〇個核苷馱、甚至更佳5〇〇個核苷酸。此對齊後，逐位確定序列同源性，例如，若核苷酸或胺基酸殘基在特定位置處一致，則序列在彼位置上”同源”。接著將該等位置一致之總 124989.doc -17- 200825099 數除以參考序列中核苷酸或胺基酸殘基之總數以得到序列同源性％。序列同源性可容易地藉由已知方法計算，該等方法包括（但不限於）以下文獻中所述之彼等方法：b. aa 128-130: SSG c. aa 138-140: GSS. "Sequence homology" as used herein refers to a method of determining the correlation of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced as necessary. In contrast to sequence identity, conservative amino acid substitutions are considered to match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology to a reference sequence, 85%, preferably 9%, even more preferably 95% of the amino acid residues or cores in the reference sequence The nucleotide must be matched to other amino acids or nucleotides or contain conservative substitutions of other amino acids or nucleotides, or all amino acid residues or nucleotides in the reference sequence (excluding conservative substitutions) An amino acid or nucleotide in an amount of up to 15%, preferably up to 1 (%), even more preferably up to 5%, is inserted into the reference sequence. Preferably, the homologous sequence comprises at least a stretch of 50 nucleotides, even more preferably 1 nucleotide, even more preferably 25 nucleotides, even more preferably 5 nucleotides. After this alignment, sequence homology is determined bit by bit, for example, if the nucleotide or amino acid residue is at a particular position, the sequence is "homologous" at that position. The number of total positions 124989.doc -17-200825099 which are identical in position is then divided by the total number of nucleotide or amino acid residues in the reference sequence to obtain % sequence homology. Sequence homology can be readily calculated by known methods including, but not limited to, those described in the following literature:

Computational Molecular Biology, Lesk，Α· N·編，〇xfor(jComputational Molecular Biology, Lesk, Α·N·编,〇xfor(j

University Press, New York (1988)， Biocomputing： Informatics and Genome Projects，Smith，D.W.編， Academic Press, New York (1993) ； Computer Analysis ofUniversity Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., Academic Press, New York (1993); Computer Analysis of

Sequence Data，第 I部分，Griffin，A.M·，&Griffin，H.G. 編 ’ Humana Press，New Jersey (1994) ; Sequence Analysis in Molecular Biology，von Heinge，G.，Academic Press (1987) ; Sequence Analysis Primer，Gribskov，M·及 Devereux，J.編，M. Stockton Press，New York (1991);及 Carillo, H.及 Lipman，D·，SIAM J. Applied Math·，48: 1073 (1988) ，該等文獻之教示内容以引用方式併入本文中。設計序列同源性之較佳測定方法以使所測試之序列之間達成最大匹配。序列同源性測定方法可編程為可公開獲得之測定指定序列之間的序列一致性的電腦程式。該等程式之實例包括（但不限於）GCG程式套件（Devereux，J.等人，Sequence Data, Part I, Griffin, AM·, & Griffin, HG, ed., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J. ed., M. Stockton Press, New York (1991); and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), The teachings are incorporated herein by reference. A preferred assay for sequence homology is designed to achieve a maximum match between the sequences tested. The sequence homology determination method can be programmed as a publicly available computer program for determining sequence identity between specified sequences. Examples of such programs include, but are not limited to, the GCG suite (Devereux, J. et al.

Nucleic Acids Research，12 (1):387 (1984))、BLASTP、 BLASTN及 FASTA(Altschul，S. F·等人，j· Molec. Biol·， 215:403-410 (1990))。BLASTX程式可公開獲自 nCBI及其他來源（BLAST Manual，Altschul，S·等人，NCVI NLM NIH Bethesda，MD 20894, Altschul，S· F·等人，j. Molec· Bi〇i·， 215:403-410 (1990)，該等文獻之教示内容以引用方式併入 124989.doc 200825099 本文中）。該等程式利用預設空位權重使序列最佳對齊，以使指定序列與參考序列之間達成最高水準的序列同源性。此外，較佳之H5蛋白包括包含如上所述之328K+修飾體及表1中所提供之胺基酸序列的H5蛋白或其任何免疫原部分：表1 : H5抗原Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., j. Molec. Biol., 215: 403-410 (1990)). The BLASTX program is publicly available from nCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., j. Molec. Bi〇i, 215: 403 -410 (1990), the teachings of which are incorporated herein by reference. These programs use the preset gap weights to optimally align the sequences to achieve the highest level of sequence homology between the specified sequence and the reference sequence. Furthermore, preferred H5 proteins include the H5 protein comprising the 328K+ modification as described above and the amino acid sequence provided in Table 1, or any immunogenic portion thereof: Table 1: H5 antigen

序列名稱基本序列胺基酸位置# 36 83 86 120 155 156 189 212 223 263 223N/328K+ 任何HAH5 - - N - 36T/223N/328K+ 任何HA H5 T - N - 36K/223N/328k+ 任何HAH5 K - N - 83A/223N/328k+ 任何HAH5 - A N - 83T/223N/328k+ 任何HAH5 - T - - - - - - N - 83D/223N/328k+ 任何HA H5 - D N - 86A/223N/328k+ 任何HAH5 - - A - - - - - N - 86V/223N/328k+ 任何HAH5 - - V - - - - - N - 120N/223N/328k+ 任何HA H5 - - - N - - - - N - 120S/223N/328k+ 任何HAH5 - - - S - - - - N - 155N/223N/328k+ 任何HA H5 - - - - N - - - N - 155S/223N/328k+ 任何HA H5 - - - - S - - - N - 156A/223N/328k+ 任何HAH5 - - - - - A - - N 丨- 156T/223N/328k+ 任何HA H5 - - - - - T - - N - 189R7223N/328k+ 任何HA H5 R - N - 189K/223N/328k+ 任何HAH5 K - N - 212K/223N/328k+ 任何HAH5 - - - - - - - K N - 212R/223N/328k+ 任何HAH5 R N - 212E/223N/328k+ 任何HAH5 E N - 223N/263A/328k+ 任何HAH5 - - N A 223N/263T/328k+ 任何HA H5 - - N T 120N/155N/223N/328k+ 任何HA H5 - - - N N - - - N - A/鴨/China/E319-2/03/328k+ AAR99628 T A A S D A R K N A A/鴨/China/E319-2/03__223N/328k+ AAR99628 T A A s D A R K N A A/鴨/China/E319· 2/03 120N/223N/328k+ AAR99628 T A A N D A R K N A a7 鴨/China/E319-2/03 155N/223N/328k+ AAR99628 T A A S N A R K N A a7 鴨/China/E319-2/03 120N/155N/223N/ 328k+ AAR99628 T A A s N N R K N A HA/HK/213/03/328k+ AY518362 T A A N N A R K N A HA/Vietnam/1203/04 K T V S S T K R N T 124989.doc -19- 200825099Sequence Name Basic Sequence Amino Acid Location # 36 83 86 120 155 156 189 212 223 263 223N/328K+ Any HAH5 - - N - 36T/223N/328K+ Any HA H5 T - N - 36K/223N/328k+ Any HAH5 K - N - 83A/223N/328k+ any HAH5 - AN - 83T/223N/328k+ any HAH5 - T - - - - - - N - 83D/223N/328k+ any HA H5 - DN - 86A/223N/328k+ any HAH5 - - A - - - - - N - 86V/223N/328k+ Any HAH5 - - V - - - - - N - 120N/223N/328k+ Any HA H5 - - - N - - - - N - 120S/223N/328k+ Any HAH5 - - - S - - - - N - 155N/223N/328k+ Any HA H5 - - - - N - - - N - 155S/223N/328k+ Any HA H5 - - - - S - - - N - 156A/223N/328k+ Any HAH5 - - - - - A - - N 丨- 156T/223N/328k+ any HA H5 - - - - - T - - N - 189R7223N/328k+ any HA H5 R - N - 189K/223N/328k+ any HAH5 K - N - 212K/223N/328k+ any HAH5 - - - - - - - KN - 212R/223N/328k+ any HAH5 RN - 212E/223N/328k+ any HAH5 EN - 223N/263A/328k+ any HAH5 - - NA 223N/263T/328k+ any HA H5 - - NT 120N/155N/223N/328k+ Any HA H5 - - - NN - - - N - A / Duck / China / E319-2/03/328k + AAR99628 TAASDARKNAA / Duck / China / E319-2 / 03__223N / 328k+ AAR99628 TAA s DARKNAA/Duck/China/E319· 2/03 120N/223N/328k+ AAR99628 TAANDARKNA a7 Duck/China/E319-2/03 155N/223N/328k+ AAR99628 TAASNARKNA a7 Duck/China/E319-2/03 120N /155N/223N/ 328k+ AAR99628 TAA s NNRKNA HA/HK/213/03/328k+ AY518362 TAANNARKNA HA/Vietnam/1203/04 KTVSSTKRNT 124989.doc -19- 200825099

HA/Vietnam/1203/04 223N/328k+ K T V S s T K R N T HA//Vietnam/3046/04 223N/328k+ _ T A V S s T K R N T HA/Vietnam/3062/04 223N/328k+ ~ T A V S s T K R N T HA/雞 /Vietnam/39/04 223N/ 328k+ ~ T A V s s T K R N T HA/隼/HK- D0028/04—223N/328k+ T A A s s A K E N A Ηλ/鴨 /Singapore/3/97 223N/ 328k+ T D V s N A K E N A HA/HK/156/97/328k+ T A A s s A K E N T #表1中所指定之胺基酸位置係指SEQ ID ΝΟ:1中所例示 ζ) 性指定之位置。換而言之，表1中之胺基酸223係指 SEQ ID ΝΟ:1序列中之胺基酸223。 -意謂此位置上之胺基酸與參考序列相比可變。此外，本發明亦係關於至少具有胺基酸223Ν及修飾體 328Κ+的Η5蛋白，其中Η5蛋白之胺基酸位置之編號係指如 SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體 328K+意謂在H5蛋白之胺基酸位置328上***第二離胺酸 (K+)，且包含： O i.具有以下NCBI寄存編號之序列的肽：AAT65209、 CAJ32556、ABC47656、CAF21874、CAF21870、 AAC58998、AAC58997、AAC58996、AAC58994、 AAC58993、AAC58992、AAC58991、AAC58990、 AAC58995、AAS45134、AAN17270、AAN17269、 AAN17268、AAN17267、AAN17266、AAN17265、 AAN17264、AAN17263、AAN17262、AAN17261 、 AAN17260、AAN17259、AAN17257、AAN17256、 124989.doc -20- 200825099 Ο u ΑΑΑ43083 、ΑΑΑ43082 、 ΑΑΜ49555 、AAL75843 、 ΑΑΝ17255、 ΑΑΒ19079、 ΒΑΕ48695、 ΒΑΕ48690、 ΒΑΕ48686、 AAC58999、 ΑΑΡ71992、 ΑΑΡ7201 1、 ΑΑΡ72007、 ΑΑΡ72003、 ΑΑΡ71999、 ΑΑΡ71995、 AAG38534、 AAC32101、 AAR99628、 AAL75839、 AAF04719、 AAD13570、 AAD13567、 ΑΑΝ17254、 ΒΑΕ48696、 ΒΑΕ48694、 ΒΑΕ48689、 ΒΑΕ48685、 ABC72082、 ΑΑΡ71991、 ΑΑΡ72010、 ΑΑΡ72006、 ΑΑΡ72002、 ΑΑΡ71998、 ΑΑΡ71994、 AAC32102、 AAC32098、 AAC32100、 AAD13573、 AAC34263、 AAD13575 、 ΒΑΕ48693、 ΒΑΕ48692、 ΒΑΕ48688、 ΒΑΕ48684、 AAV91149、 ΑΑΡ71990、 ΑΑΡ72009、 ΑΑΡ72005、 ΑΑΡ72001、 ΑΑΡ71997、 AAF99718、 AAC32099、 AAC32088、 AAD13568、 AAR16155 > ΒΑΕ48696、 ΒΑΕ48691、 ΒΑΕ48687、 ΒΑΕ48683、 ΑΑΡ71993、 ΑΑΡ71989、 ΑΑΡ72008、 ΑΑΡ72004、 ΑΑΡ72000、 ΑΑΡ71996、 ABF58847、 AAL75847、 AAC32078、 AAF04720、 AAD13574 、 AAD13572、AAD13569 AAD13566、ΑΑΚ57506、AAG01225 AAG01215、AAG01205、AAG01195 或 ABD83813，該等序列可以上述方式修飾，此意謂彼等序列包括上述不為野生型序列之部分的修飾體223N及328K+;或 ii.具有與i)之多肽之至少85%序列同源性、更佳至少約 90%序列同源性、甚至更佳至少約95%序列同源性、 124989.doc -21 - 200825099 甚至更佳至少約97%序列同源性、甚至更佳至少約 98%序列同源性且甚至更佳至少約99%序列同源性且在如上所述之標準血球凝集素抑制檢定中展示血球凝集素抑制的任何肽； • iH·丨）或Π)之肽中之任一者，其具有胺基酸36T、36K、 83A、83T、83D、86A、86V、120N、120S、155N、 155S、156A、156T、189R、189K、212K、212R、 212E、263A、263T或 120N/155N ;或 ιν· 1)、11)或111)之該等肽中之任一者，其具有選自由以下各者組成之群的以下胺基酸團中之一或多者：HA/Vietnam/1203/04 223N/328k+ KTVS s TKRNT HA//Vietnam/3046/04 223N/328k+ _ TAVS s TKRNT HA/Vietnam/3062/04 223N/328k+ ~ TAVS s TKRNT HA/Chicken/Vietnam/39/ 04 223N/ 328k+ ~ TAV ss TKRNT HA/隼/HK- D0028/04-223N/328k+ TAA ss AKENA Ηλ/Duck/Singapore/3/97 223N/ 328k+ TDV s NAKENA HA/HK/156/97/328k+ TAA ss The position of the amino acid specified in AKENT #Table 1 refers to the position specified by ζ in the SEQ ID ΝΟ:1. In other words, the amino acid 223 in Table 1 means the amino acid 223 in the sequence of SEQ ID ΝΟ:1. - means that the amino acid at this position is variable compared to the reference sequence. Furthermore, the present invention is also directed to a Η5 protein having at least an amino acid 223 Ν and a modification 328 Κ +, wherein the amino acid position of the Η5 protein is numbered as the amino acid position as exemplified in SEQ ID NO: 1. And wherein the modification 328K+ means inserting a second lysine (K+) at the amino acid position 328 of the H5 protein, and comprises: O i. A peptide having the sequence of the following NCBI accession number: AAT65209, CAJ32556, ABC47656, CAF21874 , CAF21870, AAC58998, AAC58997, AAC58996, AAC58994, AAC58993, AAC58992, AAC58991, AAC58990, AAC58995, AAS45134, AAN17270, AAN17269, AAN17268, AAN17267, AAN17266, AAN17265, AAN17264, AAN17263, AAN17262, AAN17261, AAN17260, AAN17259, AAN17257, AAN17256 , 124989.doc -20- 200825099 Ο u ΑΑΑ43083, ΑΑΑ43082, ΑΑΜ49555, AAL75843, ΑΑΝ17255, ΑΑΒ19079, ΒΑΕ48695, ΒΑΕ48690, ΒΑΕ48686, AAC58999, ΑΑΡ71992, ΑΑΡ7201 1, ΑΑΡ72007, ΑΑΡ72003, ΑΑΡ71999, ΑΑΡ71995, AAG38534, AAC32101, AAR99628, AAL75839 , AAF047 19, AAD13570, AAD13567, ΑΑΝ17254, ΒΑΕ48696, ΒΑΕ48694, ΒΑΕ48689, ΒΑΕ48685, ABC72082, ΑΑΡ71991, ΑΑΡ72010, ΑΑΡ72006, ΑΑΡ72002, ΑΑΡ71998, ΑΑΡ71994, AAC32102, AAC32098, AAC32100, AAD13573, AAC34263, AAD13575, ΒΑΕ48693, ΒΑΕ48692, ΒΑΕ48688, ΒΑΕ48684, AAV91149, ΑΑΡ71990, ΑΑΡ72009, ΑΑΡ72005, ΑΑΡ72001, ΑΑΡ71997, AAF99718, AAC32099, AAC32088, AAD13568, AAR16155 > ΒΑΕ48696, ΒΑΕ48691, ΒΑΕ48687, ΒΑΕ48683, ΑΑΡ71993, ΑΑΡ71989, ΑΑΡ72008, ΑΑΡ72004, ΑΑΡ72000, ΑΑΡ71996, ABF58847, AAL75847, AAC32078, AAF04720 , AAD13574, AAD13572, AAD13569 AAD13566, ΑΑΚ57506, AAG01225 AAG01215, AAG01205, AAG01195 or ABD83813, such sequences may be modified as described above, which means that the sequences include the above-described modifications 223N and 328K+ which are not part of the wild type sequence; Ii. having at least 85% sequence homology with the polypeptide of i), preferably at least 90% sequence homology, even more preferably at least about 95% sequence homology, 124989.doc -21 - 200825099 even better, at least about 97% sequence homology, even better, at least about 98% sequence homology and Even better, any peptide that exhibits at least about 99% sequence homology and exhibits hemagglutinin inhibition in a standard hemagglutinin inhibition assay as described above; • iH·丨) or Π) peptide, Having an amino acid 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A, 156T, 189R, 189K, 212K, 212R, 212E, 263A, 263T or 120N/155N; Or any one of the peptides of 1), 11) or 111) having one or more of the following amino acid groups selected from the group consisting of:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 123-125: SDHb. aa 123-125: SDH

c. aa 128-130: SSGc. aa 128-130: SSG

d. aa 138-140: GSSd. aa 138-140: GSS

e. aa 226-228: MDFe. aa 226-228: MDF

I f. aa 270-272: EVE g. aa 309-31 1: NKL ;或 ν· i)、ii)、iii)或iv)之任何肽，其具有選自由以下各者組成之群的以下胺基酸團中之一或多者：I f. aa 270-272: EVE g. aa 309-31 1: NKL; or any peptide of ν·i), ii), iii) or iv) having the following amines selected from the group consisting of One or more of the acid groups:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 128-130: SSG c. aa 138-140: GSS 〇b. aa 128-130: SSG c. aa 138-140: GSS 〇

根據另一實施例，本發明亦係關於編碼上述H5蛋白中之任一者的核酸分子。較佳地，彼等核酸分子為rNA、DNA 124989.doc -22- 200825099 或複本（c)DNA分子。因此，本發明係關於核酸分子，較佳編碼H5蛋白及H5蛋白之任何修飾形式（包括H5蛋白之任何缺失、取代及/或***突變體）的cDNa分子，其中彼等H5 蛋白具有胺基酸223N及修飾體328K+，其中H5蛋白之胺基 • 酸位置編號係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328 上***第二離胺酸（K+)。根據另一實施例，本發明亦係關於核酸分子，較佳編碼 H5蛋白之任何部分的cDna分子，其意謂編碼在上述標準血球凝集素抑制檢定中展示抗原特性且至少具有胺基酸 223N及修飾體328K+之任何肽片段的cDNA分子，其中H5 蛋白之胺基酸位置之編號係指如SEQ ID N〇:丨中所例示性指定之胺基酸位置且其中修飾體328K+意謂在115蛋白之胺基&C位置328上***第二離胺酸（κ+)。通常，編碼出蛋白之抗原部分的該等核酸分子包含編碼上述經修飾或未經修〇飾且在如本文中所述之標準血球凝集素抑制檢定中展示抗原特性之Η5蛋白的核苷酸序列中之6〇〇、54〇、48〇、45q、 420、3 90、360、330或最佳3 15個η?比鄰核苷酸。 H5蛋白之抗原部分之其他實施例在上文中加以描述。建 ^ 構任何該等核酸分子（較佳編碼上述H5蛋白之抗原部分的 cDNA分子）已為熟習此項技術者所共知。此亦包括（但不限於）建構編碼上述Η 5蛋白之抗原部分（包括H 5蛋白之缺失突變體）的核酸分子（較佳cDNA分子），其包含： 1·圍繞且包括編碼胺基酸223N之編碼序列的核苷酸序列 124989.doc -23- 200825099 中之至少 105、90、75、60、48、45、39、30、27 或最佳24個毗鄰胺基核苷酸；及 ii. 圍繞且包括編碼修飾體328K+之編碼序列的核苷酸序列中之至少 105、90、75、60、48、45、39、30、27 或最佳24個毗鄰胺基核苷酸；且 iii. 其中H5蛋白之該抗原部分中之任一者在如實例2中所述之標準血球凝集素抑制檢定中展示血球凝集素抑較佳地，圍繞編碼胺基酸223N及/或328K+之核苷酸的彼等核苷酸編碼SEQ ID ΝΟ:1或SEQ ID NO:4。此外，本發明之編碼H5蛋白之較佳核酸分子為： i. 上述編碼胺基酸223N及修飾體328K+的彼等核酸分子中之任一者； ii. 上述編碼胺基酸94N/223N及修飾體328K+的彼等核酸分子中之任一者； iii. 編碼胺基酸223N及修飾體328K+的任何禽源核酸分子，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 iv. 編碼胺基酸94N/223N及修飾體328K+的任何禽源核酸分子，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 V.編碼胺基酸1 55N/223N及修飾體328K+的任何禽源核酸分子，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 124989.doc -24- 200825099 vi.編碼具有胺基酸120N/15 5N/223N及修飾體328K+之禽源H5蛋白的任何核酸分子，其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株；或 vii·編碼具有修飾體94N/223N及修飾體328K+之H5蛋白的任何核酸分子；或 viii.編碼具有修飾體94N/155N/223N及修飾體328K+之H5 蛋白的任何核酸分子；或According to another embodiment, the invention is also directed to a nucleic acid molecule encoding any of the above H5 proteins. Preferably, the nucleic acid molecules are rNA, DNA 124989.doc -22-200825099 or a duplicate (c) DNA molecule. Accordingly, the present invention relates to a nucleic acid molecule, preferably a cDNa molecule encoding any modified form of the H5 protein and the H5 protein, including any deletion, substitution and/or insertion mutant of the H5 protein, wherein the H5 protein has an amino acid. 223N and Modification 328K+, wherein the amino acid group number of the H5 protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means the amino acid position 328 at the H5 protein. A second lysine (K+) is inserted thereon. According to another embodiment, the invention is also directed to a nucleic acid molecule, preferably a cDna molecule encoding any portion of the H5 protein, which means encoding an antigenic property in the above-described standard hemagglutinin inhibition assay and having at least an amino acid 223N and A cDNA molecule of any peptide fragment of modified 328K+, wherein the numbering of the amino acid position of the H5 protein refers to the amino acid position as exemplified in SEQ ID N: 且 and wherein the modification 328K+ means at 115 protein A second lysine (kappa+) is inserted at the amino group & C position 328. Typically, the nucleic acid molecules encoding the antigenic portion of the protein comprise a nucleotide sequence encoding the above-described modified or unmodified Η5 protein which exhibits antigenic properties in a standard hemagglutinin inhibition assay as described herein. 6〇〇, 54〇, 48〇, 45q, 420, 3 90, 360, 330 or optimal 3 15 η? Other examples of antigenic portions of the H5 protein are described above. It is well known to those skilled in the art to construct any such nucleic acid molecule, preferably a cDNA molecule encoding an antigenic portion of the above H5 protein. This also includes, but is not limited to, construction of a nucleic acid molecule (preferably a cDNA molecule) encoding an antigenic portion of the above Η5 protein, including a deletion mutant of the H5 protein, comprising: 1. surrounding and including the amino acid 223N At least 105, 90, 75, 60, 48, 45, 39, 30, 27 or optimal 24 adjacent amino nucleotides of the nucleotide sequence of the coding sequence 124989.doc -23- 200825099; and ii. At least 105, 90, 75, 60, 48, 45, 39, 30, 27 or optimal 24 contiguous amino nucleotides in the nucleotide sequence surrounding and comprising the coding sequence encoding the modification 328K+; and iii. Wherein any of the antigenic portions of the H5 protein exhibits hemagglutinin in a standard hemagglutinin inhibition assay as described in Example 2, preferably surrounding a nucleotide encoding 223N and/or 328K+ of the amino acid Their nucleotides encode SEQ ID ΝΟ:1 or SEQ ID NO:4. Furthermore, preferred nucleic acid molecules encoding the H5 protein of the present invention are: i. any of the above nucleic acid molecules encoding amino acid 223N and modified 328K+; ii. encoding amino acid 94N/223N and modified above Any of the nucleic acid molecules of 328K+; iii. any avian nucleic acid molecule encoding amino acid 223N and modification 328K+, wherein the avian source means that the H5 sequence is derived from the original infection with avian influenza virus type 5 Avian isolated virus isolate; or iv. Any avian nucleic acid molecule encoding amino acid 94N/223N and modified 328K+, wherein the avian source means that the H5 sequence is derived from a bird originally isolated from avian influenza A virus. a virus isolate; or V. any avian nucleic acid molecule encoding amino acid 1 55N/223N and a modified 328K+, wherein the avian origin means that the H5 sequence is derived from a virus isolated from avian isolated from avian influenza A virus. Or a nucleic acid molecule encoding avian H5 protein having amino acid 120N/15 5N/223N and modified 328K+, wherein the avian source means that the H5 sequence is derived from the initial self-infection. Type 5 avian influenza virus An avian isolated virus isolate; or vii. any nucleic acid molecule encoding a modified form 94N/223N and a modified 328K+ H5 protein; or viii. encoding any H5 protein having the modified 94N/155N/223N and the modified 328K+ Nucleic acid molecule; or

ix·編碼具有修飾體94N/120N/15 5N/223N及修飾體 328K+ 之Η 5蛋白的任何核酸分子；或 X·編碼具有修飾體223Ν、修飾體328Κ+及選自由以下各者組成之群的以下胺基酸團中之一或多者之Η5蛋白的任何核酸分子：Ix. Any nucleic acid molecule encoding a 蛋白5 protein having a modified form of 94N/120N/15 5N/223N and a modified form 328K+; or X·encoded with a modified form 223Ν, a modified form 328Κ+ and selected from the group consisting of: Any nucleic acid molecule of one or more of the following amino acid groups:

a. aa 93-95: GNFa. aa 93-95: GNF

b. aa 123-125: SDH c· aa 128-130: SSGb. aa 123-125: SDH c· aa 128-130: SSG

d. aa 138-140: GSSd. aa 138-140: GSS

e. aa 226-228: MDFe. aa 226-228: MDF

f. aa 270-272: EVE g· aa 309-311: NKL ;或 xi·編碼具有胺基酸223N、修飾體328K+及選自由以下各者組成之群的以下胺基酸團中之一或多者之H5蛋白的任何核酸分子：f. aa 270-272: EVE g·aa 309-311: NKL; or xi· encoding one or more of the following amino acid groups having amino acid 223N, modification 328K+, and a group selected from the group consisting of Any nucleic acid molecule of the H5 protein:

GNF a. aa 93-95: 124989.doc -25- 200825099GNF a. aa 93-95: 124989.doc -25- 200825099

b. aa 128-130: SSG c. aa 138-140: GSS ；或 xii.編碼具有SEQ ID NO:4之胺基酸序列之H5蛋白的任何核酸分子。此外，如本文中所提供之較佳Η 5蛋白包括以下文獻所述之1白·· Hoffmann等人，PNAS，第 106卷，第 36期，第 7 297 5]2920夏(^仰5卒9^(5沒」，其中彼等115蛋白包括如上所述之修飾體中之一或多者，至少包括胺基酸223N及修飾體328K+，其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中修飾體 328K+意謂在H5蛋白之胺基酸位置328上***第二離胺酸 (K+)。此參考文獻之揭示内容將以引用方式全文併入本文中。因此，根據另一實施例，本發明亦係關於任何核酸分子，較佳編碼以下文獻所述之該等蛋白質中之任一者的 cONA 分 + ·，Hoffmann 等人，PNAS，第 106 卷，第 36期， #7297 5-/2920趸丰P方6J，其中彼等H5蛋白包括上述修飾體中之一或多者，至少包括胺基酸223N及修飾體 328K+，其中H5蛋白之胺基酸位置之編號係指如SEQ ID ΝΟ:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上***第二離胺酸（K+)。將上述任何修飾體引入核苷酸序列（包括流感病毒之H5 蛋白之編碼序列）内的方法已熟知於此項技術。完整流感病毒之基因組序列可根據本發明加以修飾，例如根據可供進一步參考之US 6,951，754中所述之方法加以修飾。 124989.doc -26- 200825099b. aa 128-130: SSG c. aa 138-140: GSS; or xii. Any nucleic acid molecule encoding an H5 protein having the amino acid sequence of SEQ ID NO: 4. In addition, the preferred Η5 protein as provided herein includes one of the following documents: H. Hoffmann et al., PNAS, Vol. 106, No. 36, No. 7 297 5] 2920 Summer (^仰五卒9 ^(5未”, wherein the 115 proteins include one or more of the modifications described above, including at least the amino acid 223N and the modification 328K+, wherein the amino acid position of the H5 protein is numbered as SEQ. ID NO: The amino acid position exemplarily specified in 1 and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. The disclosure of this reference will be incorporated by reference. The manner is fully incorporated herein. Thus, according to another embodiment, the invention is also directed to any nucleic acid molecule, preferably encoding a cONA score of any of the proteins described in the following literature, +, Hoffmann et al, PNAS, Vol. 106, No. 36, #7297 5-/2920 趸丰P方6J, wherein the H5 proteins include one or more of the above modifications, including at least amino acid 223N and modified 328K+, wherein The numbering of the amino acid position of the H5 protein means that it is exemplarily specified in SEQ ID NO: 1. The acid position and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. Any of the above modifications are introduced into the nucleotide sequence (including the coding sequence of the H5 protein of influenza virus). The method of the present invention is well known in the art. The genomic sequence of the complete influenza virus can be modified according to the invention, for example according to the method described in US 6,951,754, which is incorporated by reference. 124989.doc -26- 200825099

此外，可利用此項技術技能範圍内之習知分子生物學、微生物學及重組DNA技術修飾編碼本文中所述之抗原的核酸序列。該等技術已詳釋於文獻中。參見例如人 ’Molecular Cloning: A Laboratory Manual ，第二版 (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor，N .Y.; DNA Cloning: A Practical Approach，第 I 及 II 卷（D, Ν· Glover 編，1985)，· Oligonucleotide Synthesis (M. J· Gait編，1984) ; Nucleic Acid Hybridization [B, D, Hames & S, J· Higgins 編（1985)] ; Transcription And Translation [B, D, Hames & S, J, Higgins 編(1984)]; Animal Cell Culture [R· I. Freshney編（1986)]，· Immobilized Cells And Enzymes [IRL Press，（1986)] ; B, Perbal，AIn addition, nucleic acid sequences encoding the antigens described herein can be modified using conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. These techniques are well documented in the literature. See, for example, Human 'Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D, Ν·Glover Edited, 1985), Oligonucleotide Synthesis (edited by M. J. Gait, 1984); Nucleic Acid Hybridization [B, D, Hames & S, J. Higgins (1985)]; Transcription And Translation [B, D, Hames & S, J, Higgins (1984)]; Animal Cell Culture [R. I. Freshney (1986)], Immobilized Cells And Enzymes [IRL Press, (1986)]; B, Perbal, A

Practical Guide To Molecular Cloning (1984) ； F. M, Ausuhel 等人編，Current Protocols in Molecular Biology， John Wiley & Sons，Inc. 1994) 0 根據另一實施例，本發明亦係關於包含上述該等核酸分子中之任一者的載體。換而言之，本發明係關於包括上述任何該H5蛋白或其部分之編碼序列的載體。較佳地，該載體為使上述任何該H5蛋白或其部分得到表現的表現載體。本發明之載體為適於在活體外或活體内轉染或感染細菌、酵母或動物細胞的彼等載體。載體及用於製備及/或使用表現載體（或重組體）的方法可依據或類似於以下文獻中所揭示之與DNA表現載體相關之方法：美國專利第4,603，112號、第4,769,330號、第 124989.doc -27- 200825099 5，174,993 號、第 5,505,941 號、第 5,338,683 號、第 5,494,807 號、第 4,722,848 號、第 5,942,235 號、第 5,364,773 號、第 5,762,938 號、第 5,770,212 號、第 5,942,235 號、第 382,425 號；PCT 公開案 WO 94/16716、 WO 96/39491、WO 95/30018 ； Paoletti, MApplications of pox virus vectors to vaccination: An update，’’PNAS USA 93: 1 1349-1 1353， 1996 年 10 月；Moss， ’’Genetically engineered poxviruses for recombinant gene expression， 1 vaccination，and safety，丨’ PNAS USA 93: 1 1341-1 1348, 1996 年10月；Smith等人，美國專利第4,745,051號（重組性桿狀病毒）；Richardson，C.D·(編者），Methods in Molecular Biology 39， ’’Baculovirus Expression Protocolsn( 1995 Humana Press Inc.) ； Smith 等人，’丨Production of HumanPractical Guide To Molecular Cloning (1984); F. M, Ausuhel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1994) 0 According to another embodiment, the present invention is also directed to the inclusion of such A vector for any of the nucleic acid molecules. In other words, the present invention relates to vectors comprising the coding sequences of any of the above H5 proteins or portions thereof. Preferably, the vector is an expression vector for rendering any of the H5 proteins or portions thereof described above. The vectors of the present invention are those suitable for transfecting or infecting bacterial, yeast or animal cells in vitro or in vivo. The vectors and methods for making and/or using the expression vectors (or recombinants) can be based on or similar to those disclosed in the literature, in connection with DNA expression vectors: U.S. Patent Nos. 4,603,112, 4,769,330, No. 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425 PCT Publication WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, MApplications of pox virus vectors to vaccination: An update, ''PNAS USA 93: 1 1349-1 1353, October 1996; Moss, ''Genetically engineered poxviruses for recombinant gene expression, 1 vaccination, and safety, 丨' PNAS USA 93: 1 1341-1 1348, October 1996; Smith et al., US Patent No. 4,745,051 (recombinant baculovirus) ); Richardson, CD (editor), Methods in Molecular Biology 39, ''Baculovirus Expression Protocolsn (1995 Humana Press) Inc.) ; Smith et al., '丨Production of Human

Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector’’，Molecular and Cellular Biology，1983 年 12月，第3卷，第12期，第2156-2165頁；Pennock等人，Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector’, Molecular and Cellular Biology, December 1983, Vol. 3, No. 12, pp. 2156-2165; Pennock et al.

\J ’’Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector，’’ Molecular and Cellular Biology，1984年 3月，第 4卷，第3 - 期，第 399-406 頁；ΕΡΑ0 370 573 ;美國申請案第 920，197\J ''Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector,'' Molecular and Cellular Biology, March 1984, Vol. 4, No. 3 - period, pp. 399-406; 370 573; US application 920,197

號（1986年10月16日申請）；EP專利公開案第265785號；美國專利第4,769,331號（重組性疱疹病毒）；Roizman，nThe function of herpes simplex virus genes: A primer for genetic engineering of novel vectors,” PNAS USA 124989.doc •28- 200825099 93:1 1307-1 1312，1996 年 10 月；Andreansky 等人，"The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors，’’ PNAS USA 93: 1 13 1 3-113 1 8，1996 年 1 0 月；Robertson 等人，’’Epstein-Barr virus vectors for gene delivery to B lymphocytes’，，PNAS USA 93: 1 1334-1 1340，1996年 10月； Frolov 等人， ffAlphavirus-based expression vectors:No. 265785; EP Patent No. 4,769,331 (recombinant herpesvirus); Roizman, nThe function of herpes simplex virus genes: A primer for genetic engineering of novel vectors, PNAS USA 124989.doc •28- 200825099 93:1 1307-1 1312, October 1996; Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors,'' PNAS USA 93 : 1 13 1 3-113 1 8, October 1996; Robertson et al., ''Epstein-Barr virus vectors for gene delivery to B lymphocytes',, PNAS USA 93: 1 1334-1 1340, October 1996 Frolov et al., ffAlphavirus-based expression vectors:

Strategies and applications，” PNAS USA 93: 1 1371-1 1377, 1996 年 10 月；Kitson 等人，J. Virol. 65,3068-3075,1991 ；美國專利第5,591，439號、第 5,552，143號、\¥〇 98/00166; 已受理之美國申請案第08/675,5 56號、第08/675,566號（兩者均於1 996年7月3日申請）（重組性腺病毒）；Grunhaus等人，1992，’’Adenovirus as cloning vectors，’’ Seminars in Virology(第 3卷），第 237_52頁，1993 ; Ballay等人，EMBO Journal，第 4卷，第 3861-65 頁；Graham，Tibtech 8,85-87, 1990 年 4 月；Prevec 等人，J· Gen Virol. 70,42434 ; PCT WO 91/1 1525 ; Feigner 等人，（1994), J· Biol. Chem. 269,2550·2561，Science，259: 1745-49,1993 及 McClements 等人，’’Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B? alone or in combination， induces protective immunity in animal models of herpes simplex virus_2 disease”，PNAS USA 93: 1 1414-1 1420, 1996年10月；及美國專利第5,591，639號、第5,589,466號及第 5,580,859 號；以及 WO 90/1 1092、W093/19183、 124989.doc -29- 200825099 W094/21797、WO95/1 1307、W095/20660 ; Tang 等人， Nature ;及 Furth等人，Analytical Biochemistry等。亦可泉見 WO 98/33510 ，Ju 等人，Diabetologia， 41: 736. 739,1 998(豆狀病毒表現系統）；Sanford等人，美國專利第 4,945,050 號；Fischbach 等人（Intracel)，WO 90/01543 ;Strategies and applications,” PNAS USA 93: 1 1371-1 1377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; US Patent Nos. 5,591,439, 5,552,143. \¥〇98/00166; Accepted US applications Nos. 08/675, 5 56, 08/675, 566 (both applied on July 3, 1996) (recombinant adenovirus); Grunhaus et al. , 1992, ''Adenovirus as cloning vectors,'' Seminars in Virology (Vol. 3), pp. 237_52, 1993; Ballay et al., EMBO Journal, Vol. 4, pp. 3861-65; Graham, Tibtech 8, 85 -87, April 1990; Prevec et al., J. Gen Virol. 70, 42434; PCT WO 91/1 1525; Feigner et al., (1994), J. Biol. Chem. 269, 2550·2561, Science, 259: 1745-49, 1993 and McClems et al., ''Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B? alone or in combination, induces protective immunity in animal models of herpes simplex virus_2 disease”, PNAS USA 93: 1 1414- 1 1420, October 1996; and US Patent No. 5,591,639 Nos. 5,589,466 and 5,580,859; and WO 90/1 1092, W093/19183, 124989.doc -29-200825099 W094/21797, WO95/1 1307, W095/20660; Tang et al., Nature; and Furth et al. Analytical Biochemistry et al. See also WO 98/33510, Ju et al, Diabetologia, 41: 736. 739, 1 998 (the lenticular virus expression system); Sanford et al., U.S. Patent No. 4,945,050; Fischbach et al. (Intracel), WO 90 /01543 ;

Robinson等人，seminars in Immunology 第 9卷，% 271-283 頁（1997)(DNA載體系統）；Szoka等人，美國專利第號（將 DNA***活細胞内之方法）；McCormick等人，美國專利第 5,677，178號（細胞病病毒之使用）；及美國專利第5,928,913 號（用於基因傳遞之載體）以及本文中所引用之其他文件。隶好使用例如選自豬疮療病毒（諸如Aujeszky氏疾病病毒）、豬腺病毒、痘病毒（尤其牛痘病毒、禽痘病毒、金絲雀痘病毒及豬痘病毒）之病毒載體以及DNA載體（DNA質體）來實施本發明。本發明之H5蛋白之製備方法〇根據另一態樣，本發明提供如下製備及/或回收高量重組性H5蛋白之方法：〇使培養物中之易感性細胞經含有H5 DNA編碼序列之重組性病毒載體感染，其中H5蛋白係由重組性病毋載體表現；及H)爾後自細胞培養物中回收H5蛋 • 白。H5蛋白之高量意謂（但不限於）以每毫升細胞培養物計約20 pg以上、較佳約25叫以上、甚至更佳約3〇吨以上、甚至更佳約40盹以上、甚至更佳約50 pg以上、甚至更佳、、勺60 pg以上、甚至更佳約8〇叫以上、甚至更佳約1⑼吨以上、甚至更佳約15〇叫以上、最佳約19〇叫以上。 124989.doc -30- 200825099 根據一較佳實施例，藉由收穫表現H5蛋白之全（亦即完整）SF +細胞來回收H5蛋白。較佳細胞為彼等易受含有H5 DNA並表現H5蛋白的適當重組性病毒載體感染之細胞。較佳地，該等細胞為昆蟲細胞，且更佳地，其包括以商標SF+昆蟲細胞（Pr〇tein Sciences Corporation，Meriden，CT)出售的昆蟲細胞。車交佳之細胞培養物具有約0·3χ106-2·0χ106個細胞/毫升之間，更佳約0·35χ1〇Μ·9χ106個細胞/毫升、甚至更佳約（Μχΐ〇6_ 1.8xl06個細胞/毫升、甚至更佳約〇·45χ106-1·7χ106個細胞/ 毫升且最佳約〇·5χΙΟ6-1.5xlO6個細胞/毫升的細胞數。較佳病毒載體包括桿狀病毒，諸如BaculoGold(^BD Biosciences Pharmingen，San Diego，CA)，尤其是在製備細胞為昆蟲細胞的情況下。儘管桿狀病毒表現系統為較佳的’但熟習此項技術者應瞭解，其他表現系統可用於本發明之目的，亦即用於使H5表現至細胞培養物之上清液中。該等其他表現系統可能需要使用信號序列以使H5表現至培養基中。適當生長培養基亦可由熟習此項技術者確定，較佳生長培養基為無血清昆蟲細胞培養基，諸如Excell 420(JRH Biosciences，Inc·，Lenexa，KS)及類似培養基。當用於感染易感性細胞時，含有H5 DNA序列的重組性病毒載體具有較佳約〇·〇3-1.5之間、更佳約0.05-1.3、甚至更佳約0.09-1.1且最佳約〇·1-1·〇之感染複數（MOI)。較佳地，上述ΜΟΙ係關於1 mL之細胞培養流體。較佳地，本文 124989.doc -31 - 200825099 中所述方法包含··用含有H5 DNA且表現H5蛋白的重組性病毒载體感染〇·35χ106-1·9χ106個細胞/毫升、甚至更佳約 0·4χ1〇6-1·8χ106個細胞/毫升、甚至更佳約0·45χ106_ 17><1〇6個細胞/毫升且最佳約〇.5><1〇0_1.5\1〇6個細胞/毫 • 升’該病毒載體具有約0.03-1.5之間、更佳約〇·〇5-1.3、甚至更佳約〇·〇9-ΐ·ΐ且最佳約〇·;ι·ι·〇之ΜΟΙ(感染複數）。接著將受感染細胞培育至多十天、更佳約兩天至約十天、甚至更佳約四天至約九天且最佳約五天至約八天。較佳之培育條件包括介於約22-32°C之間、更佳約24-3(TC、甚至更佳約25-29°C、甚至更佳約26-28°C且最佳約27°C之溫度。較佳地，接種之後觀測到SF+細胞之桿狀病毒誘導之特徵性變異。此觀測可包括監測感染後期間細胞密度趨向及存活力之降低。已發現，感染後3-5天觀測到峰值病毒效價，且細胞中之H5蛋白表現在第5天與第8天之間達到峰值，且/或在細胞存活力降至小於丨〇%時。 (J 因此，本發明之一態樣提供一種如下製備及/或回收重組性H5蛋白（較佳為上述量）的方法：i)使培養物中之大量易感性細胞（參見上文）經具有如上定義之M0I之重組性病毒載體感染；ii)由重組性病毒載體表現H5蛋白；及Hi)爾後自感染後第5天與第8天之間獲得且/或細胞存活力降至小於10%之細胞中回收H5蛋白。較佳地，重組性病毒載體為含有H5 DNA編碼序列的重組性桿狀病毒且該等細胞為 SF +細胞。此外，較佳定期檢查培養物受污染之宏觀及微觀跡象或感染後期間細胞形態之異常變化。任何呈現任何 124989.doc -32- 200825099 污染的培養物應棄去。為達成將用於免疫原性或免疫組合物（諸如疫苗）中之Η5 蛋白之回收，較佳包括滅活步驟以便將病毒載體滅活。Robinson et al., Seminars in Immunology, Vol. 9, pp. 271-283 (1997) (DNA Vector System); Szoka et al., U.S. Patent No. (Method of Inserting DNA into Living Cells); McCormick et al., US Patent No. 5,677,178 (the use of cytopathic viruses); and U.S. Patent No. 5,928,913 (a carrier for gene delivery) and other documents cited herein. A viral vector and a DNA vector selected from, for example, a porcine sore virus (such as Aujeszky's disease virus), porcine adenovirus, poxvirus (especially vaccinia virus, fowlpox virus, canarypox virus, and porcine pox virus) are used. DNA plastids) to practice the invention. Method for Producing H5 Protein of the Present Invention According to another aspect, the present invention provides a method for preparing and/or recovering a high amount of recombinant H5 protein: 〇 susceptibility of cells in culture to recombinant cells containing H5 DNA coding sequences A viral vector infection in which the H5 protein is expressed by a recombinant disease vector; and H) recovers H5 egg white from the cell culture. The high amount of H5 protein means, but is not limited to, about 20 pg or more, preferably about 25 or more, even more preferably about 3 tons or more, even more preferably about 40 inches or more, or even more per milliliter of cell culture. Preferably, it is more than 50 pg, even better, more than 60 pg, more preferably about 8 〇, even more preferably about 1 (9) ton or more, even more preferably about 15 〇, and most preferably about 19 〇. 124989.doc -30- 200825099 According to a preferred embodiment, the H5 protein is recovered by harvesting whole (i.e., complete) SF+ cells that express the H5 protein. Preferred cells are those which are susceptible to infection by a suitable recombinant viral vector containing H5 DNA and expressing the H5 protein. Preferably, the cells are insect cells, and more preferably, they comprise insect cells sold under the trademark SF+ insect cells (Pr〇tein Sciences Corporation, Meriden, CT). The cell culture of Chejiajiao has a ratio of about 0·3χ106-2·0χ106 cells/ml, more preferably about 0·35χ1〇Μ·9χ106 cells/ml, or even better (Μχΐ〇6_1.8x10 cells/ ML, even more preferably about χ45χ106-1·7χ106 cells/ml and optimal cell number of 〇·5χΙΟ6-1.5×10 6 cells/ml. Preferred viral vectors include baculoviruses such as BaculoGold (^BD Biosciences) Pharmingen, San Diego, CA), especially where the preparation of cells is an insect cell. Although the baculovirus expression system is preferred, it will be appreciated by those skilled in the art that other performance systems can be used for the purposes of the present invention. That is, to allow H5 to be expressed in the supernatant of the cell culture. These other expression systems may require the use of a signal sequence to allow H5 to be expressed in the medium. Suitable growth medium may also be determined by those skilled in the art, preferably growing. The medium is a serum-free insect cell culture medium such as Excell 420 (JRH Biosciences, Inc., Lenexa, KS) and the like. When used to infect susceptible cells, the weight of the H5 DNA sequence is included. The viral vector preferably has a multiplicity of infection (MOI) of between about 〇·〇3-1.5, more preferably about 0.05-1.3, even more preferably about 0.09-1.1 and optimally about 1-1·1-1·〇. The above tether is related to 1 mL of cell culture fluid. Preferably, the method described in 124989.doc -31 - 200825099 comprises: infection with a recombinant viral vector containing H5 DNA and expressing H5 protein. -1·9χ106 cells/ml, even more preferably about 0·4χ1〇6-1·8χ106 cells/ml, even more preferably about 0·45χ106_17><1〇6 cells/ml and optimal about 〇 .5><1〇0_1.5\1〇6 cells/milliliter liter' The viral vector has between about 0.03-1.5, more preferably about 〇·〇5-1.3, even better about 〇·〇9 -ΐ·ΐ and the best about 〇·; ι·ι·〇之ΜΟΙ (multiplicity of infection). The infected cells are then incubated for up to ten days, more preferably from about two days to about ten days, or even better for about four days. Approximately nine days and preferably from about five days to about eight days. Preferred incubation conditions include between about 22-32 ° C, more preferably about 24-3 (TC, even more preferably about 25-29 ° C, or even more). Good about 26-28 ° C and best about 27 ° Temperature of C. Preferably, characteristic changes in baculovirus induction induced by SF+ cells are observed after inoculation. This observation may include monitoring cell density trends and viability during post-infection. It has been found that 3-5 days after infection. Peak viral titers were observed and the H5 protein expression in the cells peaked between day 5 and day 8 and/or when cell viability decreased to less than 丨〇%. (J Accordingly, one aspect of the present invention provides a method of preparing and/or recovering a recombinant H5 protein, preferably the above amount: i) allowing a large number of susceptible cells in culture (see above) to have Recombinant viral vector infection of MOI as defined above; ii) expression of H5 protein by recombinant viral vector; and Hi) followed by day 5 and day 8 after infection and/or cell viability reduced to less than 10% The H5 protein is recovered from the cells. Preferably, the recombinant viral vector is a recombinant baculovirus containing the H5 DNA coding sequence and the cells are SF + cells. In addition, it is preferred to periodically check for macroscopic and microscopic signs of contamination of the culture or abnormal changes in cell morphology during infection. Any culture presenting any contamination of 124989.doc -32- 200825099 should be discarded. To achieve recovery of the Η5 protein to be used in an immunogenic or immunological composition, such as a vaccine, it is preferred to include an inactivation step to inactivate the viral vector.

Ο ’’免疫原性或免疫組合物”係指包含至少一種抗原的物質組合，該抗原可在宿主體内引發對受關注之組合物或疫苗之細胞性免疫反應及/或抗體介導之免疫反應的免疫反應。通常，”免疫反應”包括（但不限於）以下效應中之一或夕者·特異性針對包含於受關注之組合物或疫苗中之抗原的抗體、Β細胞、辅助性τ細胞、抑制性丁細胞及/或細胞毒性Τ細胞及/或γ_δ Τ細胞之產生或活化。較佳地，宿主可呈現治療性或保護性免疫反應，以便增強對新感染之抵抗力及/或降低疾病之臨床嚴重程度。此保護作用表現為受感染宿主通常所呈現之症狀減少或消失、恢復時間加快及/ 或文感染宿主之病毒效價降低。因此，本發明亦係關於一種如下製備及/或回收重組性 Η5蛋白（較佳為上述量）的方法：^使培養物中之大量易感性細胞(參見上文)經具有如上定義之刪之重組性病毒載體感染；Π)由重組性病毒載體表現出蛋白；出)將感毕後第5天與第8天之間獲得且細胞存活力降至小於iq%之細胞中所表現的H5回收；及iv)將重組性病毒载體滅活。較佳地’此㈣係在特進行㈣步狀前進行滤步驟剛結束之後進行，過濾 '步驟之後為較佳滅活時間。任何習知滅活方法可用於本發明之目m，減活可夢由化學及/或物理處理來進行。在較佳形式巾，娜定所^ 124989.doc -33 - 200825099 穫流體之體積且使溫度介於約32_42。〇之間、更佳介於約 34-40°C之間且最佳介於約35_39〇c之間。較佳滅活方法包括添加環化二元伸乙基亞胺（BEI)，此物質濃度較佳為約i mM至約20 mM、較佳為約2 mM至約10 mM、甚至更佳為 • 約2 mM至約8 mM、甚至更佳為約3 mM至約7 mM、最佳 • 為約5 mM。舉例而言，滅活包括將較佳為約〇·4 Μ之2-溴伸乙基胺氫溴化物溶液（其已在〇·3 N Na〇H中環化為〇·2 Μ 疒二元伸乙基亞胺BEI)添加至流體中以得到最終濃度約5 mM之BEI。較佳地，接著將流體連續攪拌72_96小時，且可將經滅活之收穫流體在-40°C或-40°C以下冷凍儲存或在約1-7 C之間儲存。滅活完成後，添加硫代硫酸鈉溶液（較佳為1·0 M)以中和任何殘餘BEI。較佳地，硫代硫酸鈉的添加量與滅活之前所添加之BEI的量相當。舉例而言，在添加BEI至最終濃度為5 mM之情況下，添加1〇 m硫代硫酸鈉溶液以得到最終最小濃度5 mM以中和任何殘餘3耵。 Q 因此，本發明之另一態樣係關於一種如下製備重組性H5 蛋白（車父佳為上述量）的方法：i)使培養物中之大量易感性細胞（參見上文）經具有如上定義之M0I之重組性病毒载體感染；ii)由重組性病毒載體表現^[5蛋白；Hi)將感染後第5 • 天與第8天之間獲得且細胞存活力降至小於1〇%之細胞中所表現之H5回收；及iv)將重組性病毒載體滅活。較佳地，重組性病毒載體為含有H5 DNA編碼序列的桿狀病毒且該等細胞為SF+細胞。較佳滅活步驟為上述彼等步驟。較佳地’滅活係在約35-39。(：之間且在2 mM至8 mM BEI之 124989.doc -34- 200825099 存在下、甚至更佳在約5 mM ΒΕΙ之存在下進行。Ο ''Immunogenic or immunological composition' refers to a combination of substances comprising at least one antigen that elicits a cellular immune response and/or antibody-mediated immunity to a composition or vaccine of interest in a host. The immune response of the reaction. Usually, the "immune response" includes, but is not limited to, one of the following effects or an evening, an antibody specific to the antigen contained in the composition or vaccine of interest, sputum cells, helper τ Production or activation of cells, inhibitory butyl cells and/or cytotoxic sputum cells and/or γ-δ Τ cells. Preferably, the host may present a therapeutic or protective immune response to enhance resistance to new infections and/or Decreased the clinical severity of the disease. This protective effect is manifested by a reduction or disappearance of the symptoms usually exhibited by the infected host, an increased recovery time and/or a decrease in the viral titer of the infected host. Thus, the present invention also relates to a preparation as follows / or method for recovering recombinant Η5 protein (preferably the above amount): ^ to make a large number of susceptibility cells in the culture (see above) have the above Deleted recombinant viral vector infection; Π) expressed by recombinant viral vector; expressed) in cells obtained between day 5 and day 8 after feeling and cell viability reduced to less than iq% H5 recovery; and iv) inactivation of the recombinant viral vector. Preferably [this (4) is performed immediately after the special (4) step is performed, and the filtration step is followed by a preferred inactivation time. The conventional inactivation method can be used for the purpose of the present invention, and the deactivation can be carried out by chemical and/or physical treatment. In the preferred form, Nadine Institute, 124989.doc -33 - 200825099, the volume of the fluid is obtained and The temperature is between about 32 and about 42. More preferably between about 34 and 40 ° C and most preferably between about 35 and 39 ° C. A preferred method of inactivation comprises adding a cyclized binary exoethylenimine ( BEI), the concentration of the substance is preferably from about i mM to about 20 mM, preferably from about 2 mM to about 10 mM, even more preferably from about 2 mM to about 8 mM, even more preferably from about 3 mM to about 7 mM, optimally • about 5 mM. For example, inactivation comprises a solution of 2-bromoethylamine hydrobromide which is preferably about 〇·4 ( (which Cyclization in 〇·3 N Na〇H to 〇·2 Μ 疒 binary exoethylenimine BEI) is added to the fluid to give a final concentration of BEI of about 5 mM. Preferably, the fluid is then continuously stirred for 72-96 hours. And the inactivated harvest fluid can be stored frozen at -40 ° C or below -40 ° C or between about 1 - 7 C. After the inactivation is completed, a sodium thiosulfate solution (preferably 1) is added. • 0 M) to neutralize any residual BEI. Preferably, the amount of sodium thiosulfate added is comparable to the amount of BEI added prior to inactivation. For example, where BEI is added to a final concentration of 5 mM A 1 μm sodium thiosulfate solution was added to give a final minimum concentration of 5 mM to neutralize any residual 3 耵. Q, therefore, another aspect of the present invention relates to a method for preparing a recombinant H5 protein (the above-mentioned amount), i) making a large number of susceptible cells in culture (see above) having the above definition Recombinant viral vector infection of MOI; ii) expressed by recombinant viral vector ^5 protein; Hi obtained between day 5 and day 8 after infection and cell viability reduced to less than 1% H5 recovery as expressed in the cells; and iv) inactivation of the recombinant viral vector. Preferably, the recombinant viral vector is a baculovirus containing the H5 DNA coding sequence and the cells are SF+ cells. Preferred inactivation steps are those described above. Preferably, the inactivation is at about 35-39. (: between and in the presence of 124 mM.doc -34 - 200825099 of 2 mM to 8 mM BEI, even more preferably in the presence of about 5 mM hydrazine.

根據本發明之另一態樣，上述方法亦包括步驟iv)之後的中和步驟。此步驟v)包含添加中和溶液中之滅活劑的等量試劑。較佳地，若滅活劑為BEI，則較佳添加等量之硫代硫酸鈉。因此，根據另一態樣，當滅活劑為BEI時，步驟 V)包含添加硫代硫酸鈉溶液直至最終濃度為約i mM至約 mM、較佳約2 mM至約1〇 mM、甚至更佳約2 mM至約8 mM、甚至更佳約3 mM至約7 mM、最佳約5 。在較佳形式中且尤其在以免疫原性組合物（諸如疫苗）使用重組性H5蛋白之形式中，將每一批所收穫之H5蛋白藉由於固著依賴性、桿狀病毒易感性昆蟲細胞(諸如⑽細胞) 中繼代來測試滅活。在此測試之一較佳形式中，將丨5〇 cm2之適當細胞培養物單層用1〇 111^經滅活之115流體接種且在25-29。。下維持14天’至少繼代兩次。在維持期結束時’對細胞單層檢查H5桿狀病毒所特有之致細胞病變作用 (CPE)。較佳地，亦使用陽性病毒對照。此等對照可由以下各物組成：一份經未滅活之參考則桿狀病毒接種之以9 培育及繼代之細胞培養物及一瓶尚未接種之Sf9細胞接著在25-29°C下維持5-6天後’經BEI處理之病毒流體中不存在受病毒感染之細胞說明滅活測試令人滿意。經參考病毒接種之對照細胞應呈現 H5桿狀病毒所特有之⑽而未接種驗應不呈現任何出桿狀病毒CPE跡象。或者，在維持期結束時，可收集上清液樣本且將其接種於Sf9 96孔板±，該孔板已裝載_細胞且接著將孔板固定且用結合 124989.doc -35- 200825099 FITC之抗·H5抗體或針對桿狀病毒特異性蛋白（亦即咖句的任何標記抗㈣色。經B戦理之病毒流體中不存在 CPE、H5表現或桿狀病毒特里松i占, t特呉^生蛋白（亦即gP64)之表現說明滅活測試令人滿意。經參考病毒接種之對照細胞應呈現 . ⑽及1FA活性，而未接種燒瓶應不呈現任何出桿狀病毒 CPE跡象且不含IFA活性。因此，本文中所述之另一態樣係關於用於測定表現幵5蛋白之重組病毒載體之滅活有效性的滅活測試，其包含以下步驟：i)使含有重組性病毒載體之培養流體之至少一部分與較佳如上所述之滅活劑接觸；i〇添加較佳如上所述之中和J以中和滅活劑，及lu)藉由如上所述之檢定測，定殘餘感染性。滅活之後，可以多種方式測定樣本中重組性115蛋白之相對畺車乂佳畺化方法包括SDS-PAGE密度測定法、ELISA 及動物接種研究，其使已知疫苗量與臨床結果（血清學等） iJ 相關。當利用SDS-PAGE量化時，將含有未知量之重組性 H5蛋白的樣本物質連同含有各種已知量之重組性H5蛋白的樣本一起在凝膠上展開。接著可基於已知樣本形成標準曲線且了藉由與此標準曲線比較來測定未知樣本中重組 .性H5之量。由於ELISA通常公認為抗原量化之行業標準，因此較佳利用ELISA進行量化。包含H5蛋白或編碼彼等蛋白質之核酸分子或載體的疫苗根據另一態樣，本發明係關於通常包含以下物質的疫苗或醫藥組合物·· 124989.doc -36- 200825099 K 一或多種如本文中所述之H5蛋白； /種如本文令所述之編碼任何該等η5蛋白之核酸分子；及/或 ⑴·或多種如本文中所述之載體，其包含如本文中所述之任何該等核酸分子丁且、、兩碼如本文中所述之任何該等 Η5蛋白；及 iv.醫藥學上可接受之載劑及/或賦形劑。 Ο 如本文中所述之術語”醫藥組合物,，、”醫藥/疫苗組合物” (不限於）用於減少或預防感染之疫苗或用於治療及減輕感染之物質組合。 +編碼流感血球凝集素之基於核酸之疫苗（較佳cDNA疫苗）的製備例如描述於以下參考文獻中：仏d事乂，According to another aspect of the invention, the above method also includes a neutralization step after step iv). This step v) contains an equal amount of reagent to add the inactivating agent in the neutralizing solution. Preferably, if the inactivating agent is BEI, it is preferred to add an equivalent amount of sodium thiosulfate. Thus, according to another aspect, when the inactivating agent is BEI, step V) comprises adding a sodium thiosulfate solution until a final concentration of from about i mM to about mM, preferably from about 2 mM to about 1 mM, or even more Preferably from about 2 mM to about 8 mM, even more preferably from about 3 mM to about 7 mM, most preferably about 5. In a preferred form and especially in the form of a recombinant H5 protein in an immunogenic composition such as a vaccine, each batch of harvested H5 protein is caused by a fixation-dependent, baculovirus-susceptible insect cell (such as (10) cells) relay generation to test inactivation. In one preferred form of this test, a suitable cell culture monolayer of 丨5〇 cm2 is inoculated with 1〇111^ inactivated 115 fluid and at 25-29. . Maintain for 14 days' at least twice. At the end of the maintenance period, the cell monolayer was examined for the cytopathic effect (CPE) characteristic of H5 baculovirus. Preferably, a positive virus control is also used. These controls may consist of a non-inactivated reference to a baculovirus inoculated with 9 bred and subcultured cell cultures and a vial of uninoculated Sf9 cells which are then maintained at 25-29 °C. After 5-6 days, 'the virus-infected cells in the BEI-treated virus fluid indicated that the inactivation test was satisfactory. Control cells inoculated with reference to the virus should be unique to the H5 baculovirus (10) and the non-vaccination test should not present any signs of baculovirus CPE. Alternatively, at the end of the maintenance period, the supernatant sample can be collected and seeded on Sf9 96-well plate ±, which has been loaded with cells and then fixed with the plate and combined with 124989.doc -35 - 200825099 FITC Anti-H5 antibody or against baculovirus-specific protein (ie, any labeled anti-(four) color of the coffee. There is no CPE, H5 expression or baculovirus Trisson i in the B-treated viral fluid, t special The performance of 呉^-protein (ie gP64) indicates that the inactivation test is satisfactory. Control cells inoculated with reference virus should present (10) and 1FA activity, while uninoculated flasks should not show any signs of baculovirus CPE and no IFA activity is included. Thus, another aspect described herein relates to an inactivation assay for determining the inactivation effectiveness of a recombinant viral vector expressing a 幵5 protein, comprising the steps of: i) rendering a recombinant virus At least a portion of the culture fluid of the carrier is contacted with an inactivating agent preferably as described above; i is preferably added as described above and J is used to neutralize the inactivating agent, and lu) is determined by the assay as described above, Residual infectivity. After inactivation, the relative sputum of the recombinant 115 protein in the sample can be determined in a variety of ways, including SDS-PAGE densitometry, ELISA, and animal vaccination studies, which allow known vaccine doses and clinical outcomes (serology, etc.) ) iJ related. When quantified by SDS-PAGE, a sample material containing an unknown amount of recombinant H5 protein was spread on a gel together with a sample containing various known amounts of recombinant H5 protein. A standard curve can then be formed based on the known samples and the amount of recombinant H5 in the unknown sample can be determined by comparison to this standard curve. Since ELISA is generally recognized as an industry standard for antigen quantification, it is preferably quantified using ELISA. Vaccines comprising H5 proteins or nucleic acid molecules or vectors encoding the same according to another aspect, the present invention relates to vaccines or pharmaceutical compositions which generally comprise the following: 124989.doc-36-200825099 K one or more as herein An H5 protein as described herein; a nucleic acid molecule encoding any of the η5 proteins as described herein; and/or (1) or a plurality of vectors as described herein, comprising any of the vectors as described herein A nucleic acid molecule, such as any of the Η5 proteins as described herein; and iv. a pharmaceutically acceptable carrier and/or excipient. The term "pharmaceutical composition,", "pharmaceutical/vaccine composition" as used herein (not limited to) a vaccine for reducing or preventing infection or a combination of substances for treating and alleviating infection. The preparation of a nucleic acid-based vaccine (preferred cDNA vaccine) is described, for example, in the following references:

Vaccine 1997，15(1):71-78 ; Ulmer 等人，Science 1993; 259.1 745 1 749，Ulmer 等人，Vaccine 1994; 12(16): 1541 - 75以。任何彼等方法可用於製備編碼如本文中所述之流感 H5蛋白之基於核酸之疫苗，較佳cDNA疫苗。此外，包含如本文中所述之H5蛋白或其部分的疫苗可藉由習知方法製備，例如藉由重組表現技術或藉由生物化學純化及分離技術製備。重組表現技術（包括在昆蟲細胞中表現）已熟知於此項技術且例如描述於以下參考文獻中： Sambrook 等人，Molecular Cloning: A Laboratory Manual，第二版（1989) Cold Spring Harbor Laboratory Press， Cold Spring Harbor，Ν·Υ· ; DNA Cloning: A Practiced Approach，第I及 II卷（D· N· Glover 編，1985)，· 124989.doc -37- 200825099Vaccine 1997, 15(1): 71-78; Ulmer et al, Science 1993; 259.1 745 1 749, Ulmer et al, Vaccine 1994; 12(16): 1541-75. Any of these methods can be used to prepare a nucleic acid based vaccine, preferably a cDNA vaccine, encoding an influenza H5 protein as described herein. Furthermore, vaccines comprising an H5 protein or a portion thereof as described herein can be prepared by conventional methods, for example, by recombinant expression techniques or by biochemical purification and separation techniques. Recombinant expression techniques, including expression in insect cells, are well known in the art and are described, for example, in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν·Υ· ; DNA Cloning: A Practiced Approach, Volumes I and II (edited by D·N·Glover, 1985), · 124989.doc -37- 200825099

Oligonucleotide Synthesis (M, J. Gait編，1984) ; Nucleic Acid Hybridization [B· D, Hames & S· J. Higgins 編 (1985) ] / Transcription And Translation [B. D. Hames & S. J· Higgins 編（1984)]，· Animal Cell Culture [R，I· Freshney M (1986)] / Immobilized Cells And Enzymes [IRL Press, (1986) ] ; B· Perbal，A Practical Guide To MolecularOligonucleotide Synthesis (edited by M, J. Gait, 1984); Nucleic Acid Hybridization [B· D, Hames & S. J. Higgins (1985)] / Transcription And Translation [BD Hames & S. J. Higgins 1984)],· Animal Cell Culture [R, I· Freshney M (1986)] / Immobilized Cells And Enzymes [IRL Press, (1986) ] ; B· Perbal, A Practical Guide To Molecular

Cloning (1984) ; F. M. Ausubel 等人（編），Current Protocols in Molecular Biology，John Wiley & Sons，Inc, /PPW e習用之重組表現系統之其他實例為細菌表現系統，諸如大腸桿菌（五.或枯草芽孢桿菌（五· ；基於酵母之表現系統，諸如釀酒酵母（& cerevb/ae)或粟酒裂殖酵母（&户謂^);或哺乳動物細胞表現系統，諸如基於BHK之表現系統、基於CHO之表現系統及/或基於NS0之表現系統。該等系統已熟知於此項技術且一般可（例如）經由 Clontech Laboratories，Inc. 4030 Fabian Way，Palo Alto,Cloning (1984); FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc, /PPW e. Other examples of recombinant expression systems are bacterial expression systems, such as E. coli (v. or Bacillus subtilis (five; yeast-based expression systems, such as Saccharomyces cerevisiae (& cerevb/ae) or Schizosaccharomyces pombe (&households); or mammalian cell expression systems, such as BHK-based expression systems CHO based performance systems and/or NS0 based performance systems. These systems are well known in the art and can generally be, for example, via Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto,

California 943〇3_46〇7，USA購得。其他表現策略例如描述於 Liischow 等人，Vaccine 第 19期（2001)，第 4249-4259 頁氣 Veit 等人，PNAS 第 103 卷（2006)，第 8197-8202 頁中。也外，重組性腺相關病毒系統為習用系統且描述於例如可供進一步參考之US 5,436，146或W0200203872中。此外，基於牛痘（痘）病毒之表現系統（例如，如可供進一步參考之 US 6,265，183中所述）亦為習用系統且適於製備如根據本發明使用之重組性抗原、抗原組合物。其他適當表現系統利用重組性popova病毒，諸如SV40、禽痘病毒、假狂犬病毒 124989.doc -38- 200825099 及逆轉錄病毒。本文中所述之相關醫藥/疫苗级合物亦可包含含有如本文中所述之H5蛋白的滅活病毒（包含如本文中所述之出蛋白之活病毒的非病原形式）、病毒之製劑及/或片段，其 • 中δ亥製劑及/或片段包含如本文中所述之H5蛋白。熟習該項技術者已知可連同抗原—起包含於該等組合物/ 疫苗中的其他組分（參見例如卿⑽心/ c Wes. υ99〇) ’ 第18版，Mack Publ，East〇n、mb 項技術者可使用已知的生理學上可接受之無菌可注射溶液可易於利用等張水溶液（諸如生理食鹽水）或相應血漿蛋白溶液來製備即用溶液。醫藥組合物/疫苗可以凍乾製劑或乾燥製劑（例如作為部分套組）提供，該等製劑可在臨用剷、在無菌條件下用已知可注射溶液復水。此外，本發明之醫樂/疫苗組合物可包括一或多種獸醫學上可接文之載劑。如本文中所使用，"獸醫學上可接受 Cj 之載劑’’包括（但不限於）任何及所有溶劑、分散介質、衣料、佐劑、穩定劑、稀釋劑、防腐劑、抗菌劑及抗真菌劑、等張劑、吸收延遲劑及類似載劑。稀釋劑可包括水、生理食鹽水、右旋糖、乙醇、甘油及類似物。等張劑尤其可包括氯化鈉、右旋糖、甘露糖醇、山梨糖醇及乳糖。穩定劑尤其包括白蛋白及乙二胺四乙酸之鹼金屬鹽。如本文中所使用之防腐劑係指抗微生物活性劑，諸如慶大黴素（Gentamycin)、硫柳汞（Merthiolate)及類似物。特定 124989.doc -39- 200825099 而0製備夕^里組合物最佳添加防腐劑。彼等抗微生物活性劑係以-定濃度添加以有效防止受關注之組合物受任何微生物污染或抑制受關注之組合物内之任何微生物生長。如本文中所使用之"佐劑"可包括氫氧化鋁及磷酸鋁、皂普（例如 QuU A、QS_21 (CambHdge Bi〇tech ⑹，California 943〇3_46〇7, USA purchased. Other performance strategies are described, for example, in Liischow et al., Vaccine, No. 19 (2001), pp. 4249-4259, Veit et al., PNAS, Vol. 103 (2006), pp. 8197-8202. In addition, the recombinant adeno-associated virus system is a conventional system and is described, for example, in US 5,436,146 or WO200203872, which is incorporated by reference. In addition, a system for the expression of vaccinia (pox) virus (e.g., as described in US Pat. Other appropriate expression systems utilize recombinant popova viruses such as SV40, fowlpox virus, pseudorabies virus 124989.doc-38-200825099 and retroviruses. The related pharmaceutical/vaccine conjugates described herein may also comprise an inactivated virus comprising a H5 protein as described herein (a non-pathogenic form comprising a live virus of a protein as described herein), a preparation of the virus And/or a fragment comprising: the H5 protein and the fragment comprising the H5 protein as described herein. Those skilled in the art are aware of other components that may be included in the compositions/vaccines along with the antigen (see, for example, Qing (10) Heart / c Wes. υ99〇) '18th Edition, Mack Publ, East〇n, The mb-technologist can readily prepare a ready-to-use solution using a known physiologically acceptable sterile injectable solution that can be readily utilized with an isotonic aqueous solution (such as physiological saline) or a corresponding plasma protein solution. The pharmaceutical compositions/vaccines may be provided as lyophilized or dry formulations (e.g., as a partial kit) which may be reconstituted with a known injectable solution under sterile conditions using a spatula. In addition, the medical/vaccine compositions of the present invention may include one or more veterinary tangible carriers. As used herein, "veterinaryly acceptable Cj carrier' includes, but is not limited to, any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial agents, and Antifungal agents, isotonic agents, absorption delaying agents and similar carriers. The diluent may include water, physiological saline, dextrose, ethanol, glycerin, and the like. The isotonic agents may especially include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The stabilizer particularly includes an alkali metal salt of albumin and ethylenediaminetetraacetic acid. Preservatives as used herein refers to antimicrobial active agents such as Gentamycin, Merthiolate and the like. Specific 124989.doc -39- 200825099 and 0 preparation of the composition is best to add preservatives. These antimicrobial actives are added at a concentration to effectively prevent the composition of interest from being contaminated by any microorganism or inhibiting the growth of any microorganisms in the composition of interest. As used herein, "adjuvant" may include aluminum hydroxide and aluminum phosphate, soap (e.g., QuU A, QS_21 (CambHdge Bi〇tech (6),

Cambridge MA)、GPI_〇1〇〇 (Galenica 扑紅邮⑽士山，Cambridge MA), GPI_〇1〇〇 (Galenica (Hong Kong) (10) Shishan,

InC.，Birmingham，AL))、油包水型乳液、水包油型乳液、水包油包水型乳液。乳液尤其可基於輕質液狀石蠟油（歐洲藥典類型）·，類異戊二烯油，諸如角鯊烷或角鯊烯；烯烴（尤其異丁烯或癸烯）之寡聚反應所產生之油；含有直鏈烷基之酸或醇之酯，尤其植物油、油酸乙酯、丙二醇二_(辛酸酯/癸酸酉曰）、甘油基二-(辛酸酯/癸酸酯）或丙二醇二油酸酯；支鏈脂肪酸或醇之酯，尤其異硬脂酸酯。油可與乳化劑組合使用以开> 成乳液。乳化劑較佳為非離子型界面活性劑，尤其脫水山梨糖醇6旨、二縮甘露糖醇酯（例如脫水甘露糖醇油酸酯）、乙二醇酯、聚甘油酯、丙二醇酯及油酸酯、異硬脂酸酯、蓖麻酸酯或羥基硬脂酸酯（其視情況經乙氧基化）；及聚氧丙烯-聚氧乙烯共聚物嵌段，尤其^犯⑽卜產口口 ’尤其 L121。參見 Hunter 等人，The Theory andInC., Birmingham, AL)), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion may especially be based on light liquid liquid paraffin oil (European Pharmacopoeia type), an isoprenoid oil such as squalane or squalene; an oil produced by oligomerization of an olefin (especially isobutylene or decene); An acid or alcohol ester containing a linear alkyl group, especially vegetable oil, ethyl oleate, propylene glycol di-(octanoate/antimony citrate), glyceryl di-(octanoate/caprate) or propylene glycol Oleate; an ester of a branched fatty acid or alcohol, especially an isostearate. The oil can be combined with an emulsifier to form an emulsion. The emulsifier is preferably a nonionic surfactant, especially sorbitan 6, dimannitol (for example, dehydrated mannitol oleate), ethylene glycol ester, polyglycerol ester, propylene glycol ester and oil. Acid ester, isostearate, ricinoleate or hydroxystearate (which is optionally ethoxylated); and polyoxypropylene-polyoxyethylene copolymer block, especially ^10 (10) Mouth 'especially L121. See Hunter et al., The Theory and

Practical Application of Adjuvants (Stewart-Tull編，D· Ε· S·)· John Wiley and Sons，NY，第 51-94 頁（1995)及 Todd等人，Vaccine 15:564-5 70 (1997)。適當水包油型乳液之實 -40- 124989.doc 200825099 例為基於Emulsigen之佐劑，諸如EMULSIGEN®、 EMULSIGEN-D®、EMULSIGEN-P®、EMULSIGEN-75® (MVP Laboratories，Inc. Omaha，NE，USA)。已驚人地發現，包含H5蛋白、較佳如本文中所述之重組性H5蛋白的醫藥/疫苗組合物有效地輔以水包油型乳液佐劑，較佳為該等基於Emulsigen之佐劑，更佳EMULSIGEN⑧及 EMULSIGEN-D®。此外，可使用M. Powell及M. Newman所編之’’Vaccine Design，The Subunit and Adjuvant Approach”第 147 頁所述之SPT乳液及該書第183頁所述之乳液MF59。佐劑之另一實例為選自丙烯酸或曱基丙烯酸之聚合物及順丁稀二酸酐與稀基衍生物之共聚物的化合物。有益的佐劑化合物為尤其與糖或多元醇之聚乙稀基醚交聯之丙稀酸或甲基丙烯酸之聚合物。該等化合物稱為卡波姆 (carbomer)(Phameuropa 第 8卷，第 2期，1996年 6月）。熟習此項技術者亦可參考美國專利第2,909,462號，其描述與具有至少3個羥基、較佳不超過8個羥基之多羥基化合物交聯的該等丙烯酸聚合物，至少三個羥基中之氫原子可置換為具有至少2個碳原子的不飽合脂族基。較佳基團為含有2至 4個碳原子的彼等基團，例如乙烯基、烯丙基及其他烯系不飽合基團。不飽合基團本身可含有其他取代基，諸如曱基。在 Carbopol (BF Goodrich，Ohio，USA)名下銷售的產品尤其適用。其與烯丙基蔗糖或烯丙基異戊四醇交聯。其中可提及 Carbopol 974P、934P及 971P。最佳為使用 Carbopol 124989.doc -41 - 200825099 971P在順丁烯—酸酐與稀基衍生物之共聚物中，共聚物 EMA (Monsanto)為順丁烯二酸酐與乙烯之共聚物。該等聚合物溶解於水中可產生酸溶液，使該酸溶液中和，較佳中 #至生里p I以便得到可免疫原性、免疫或疫苗組合物 . 自身可併入的佐劑溶液。其他適當佐劑尤其包括（但不限於）RIBI佐劑系統（Ribi Inc·)甘人段共聚物（CytRx，Atl哺& gA)、SAF_M (⑶削，Practical Application of Adjuvants (edited by Stewart-Tull, D. Ε·S·) John Wiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15:564-5 70 (1997). Appropriate Oil-In-Water Emulsions -40- 124989.doc 200825099 Examples are Emulsigen-based adjuvants such as EMULSIGEN®, EMULSIGEN-D®, EMULSIGEN-P®, EMULSIGEN-75® (MVP Laboratories, Inc. Omaha, NE ,USA). Surprisingly, it has been found that a pharmaceutical/vaccine composition comprising an H5 protein, preferably a recombinant H5 protein as described herein, is effectively supplemented with an oil-in-water emulsion adjuvant, preferably such an Emulsigen-based adjuvant. Better EMULSIGEN8 and EMULSIGEN-D®. In addition, the SPT emulsion described on page 147 of the 'Vaccine Design, The Subunit and Adjuvant Approach' by M. Powell and M. Newman and the emulsion MF59 described on page 183 of the book can be used. Examples are compounds selected from the group consisting of polymers of acrylic acid or mercaptoacrylic acid and copolymers of cis-succinic anhydride and dilute-based derivatives. Advantageous adjuvant compounds are especially crosslinked with polyethylene or polyether ethers of polyhydric alcohols. A polymer of acrylic acid or methacrylic acid. These compounds are known as carbomers (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462. No., which describes the acrylic polymer crosslinked with a polyhydroxy compound having at least 3 hydroxyl groups, preferably not more than 8 hydroxyl groups, wherein the hydrogen atoms of at least three of the hydroxyl groups may be substituted with at least 2 carbon atoms. It is saturated with aliphatic groups. Preferred groups are those having 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated group itself may contain other groups. Substituents such as sulfhydryl. In Carbo The product sold under the name pol (BF Goodrich, Ohio, USA) is especially suitable for cross-linking with allyl sucrose or allyl pentaerythritol, of which Carbopol 974P, 934P and 971P may be mentioned. Best use of Carbopol 124989.doc -41 - 200825099 971P In a copolymer of a maleic anhydride and a dilute derivative, the copolymer EMA (Monsanto) is a copolymer of maleic anhydride and ethylene. The polymers are soluble in water. An acid solution is generated to neutralize the acid solution, preferably to give an immunogenic, immunological or vaccine composition. An adjuvant solution that can be incorporated by itself. Other suitable adjuvants include, inter alia (but Not limited to) RIBI adjuvant system (Ribi Inc.) Ganren segment copolymer (CytRx, Atl feeding & gA), SAF_M ((3) cutting,

EmeiTVllle CA)、單磷醯脂A、阿夫立定（Avridine)脂質胺佐劑、獲自A腸桿菌之熱不穩冑性腸毒素（重、组體或其他形式）、霍亂毒素或胞壁醯二肽。較佳地，佐劑以每劑量約100 gg至約10 mg之量添加。甚至更佳地，佐劑以每劑量約1〇〇叫至約l〇 mg之量添加。甚至更佳地，佐劑以每劑量約5〇〇叫至約5 之量添加。甚至更佳地，佐劑以每劑量約75〇吨至約2·5 之量添加。最佳地，佐劑以每劑量約丨mg之量添加。 Ο 醫藥/疫苗組合物可進一步包括一或多種其他免疫調節劑，諸如介白素、干擾素或其他細胞因子。醫藥/疫苗組合物亦可包括慶大黴素及硫柳汞。儘管適用於本發明之上下文中之佐劑及添加劑之量及濃度可易於由熟習此項技術 '者確定，但本發明涵蓋1 ml劑量之疫苗組合物包含約5〇吨至約2〇〇〇佐劑且較佳約25〇吨佐劑的組合物。在另一較佳實施例中，本發明涵蓋包含約1 Kg/ml至約60 pg/ml抗生素且更佳小於約30 pg/ml抗生素的疫苗組合物。因此，根據另一實施例，本發明亦係關於醫藥/疫苗組 124989.doc -42- 200825099 合物，其包含： i ·治療有效量之如本文中所述之任一種流感病毒H5蛋白，其中該H5蛋白具有胺基酸223N及修飾體328K+，其中H5蛋白之胺基酸位置編號係指如SEQ ID ΝΟ··1中所例示性指定之胺基酸位置且其中修飾體328Κ+意謂在Η5蛋白之胺基酸位置328上***第二離胺酸（κ+);及 ii·如上所述之醫藥學上可接受之佐劑。較佳地，佐劑係選自由以下各物組成之群： a) EMULSIGEN⑧：一種水包油型乳液（o/w); b) EMULSIGEN-D® :—種具有溴化二甲基二-十八烷基銨 (DDA)之水包油（o/w)乳液； c) Polygen: —種共聚物； d) EMULSIGEN-P⑧，一種具有專有免疫刺激劑的水包油型（〇/w)乳液； e) Carbigen為一種交聯聚合物； f) EMULSIGEN-75® : —種包含具有交聯聚合物之水包油型（o/w)乳液的雙重佐劑； g) ISA 70為一種油包水型（w/o)乳液。最佳地，該等佐劑為水包油型乳液，諸如選自由以下各物組成之群的基於emulsigen之佐劑：EMULSIGEN®、 EMULSIGEN-D⑧、EMULSIGEN-P®、EMULSIGEN-75®、 EMULSIGEN® 及 EMULSIGEN-P®。最佳 EMULSIGEN⑧及 EMULSIGEN-P⑧用於本發明之調配物中。根據另一態樣，如本文中所提供之醫藥/疫苗組合物包 124989.doc -43- 200825099 含一或多種抗原。較佳地，其他抗原為禽類或哺乳動物病原體之抗原。根據另一實施例，其他抗原為其他流感抗原’諸如流感病毒之血球凝集素H3、H7、H9或其他任何血球凝集素。該（等）其他抗原可以純化形式、作為抗原製 • 劑之部分添加，以經滅殺微生物之形式或以經修飾之活微生物之形式添加。如本文中所使用之術語”抗原，，意謂（但不限於）肽、多 () 肽、醣肽或多醣，其能夠與免疫系統之抗原識別分子（諸如免疫球蛋白（抗體）或丁細胞抗原受體）特異***互作用以便在該抗原所投與之宿主中引發、活化或刺激針對該抗原的免疫反應。術語”抗原”亦指核酸分子，較佳為〇1^八分子或RNA分子，其各自編碼且表現能夠與免疫系統之抗原識EmeiTVllle CA), monophosphorus a lipid, Avridine lipid amine adjuvant, heat labile enterotoxin (heavy, histogram or other form) obtained from A. enterobacter, cholera toxin or cell wall Dipeptide. Preferably, the adjuvant is added in an amount from about 100 gg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of from about 1 Torr to about 1 mg per dose. Even more preferably, the adjuvant is added in an amount of from about 5 Torr to about 5 per dose. Even more preferably, the adjuvant is added in an amount of from about 75 ounces to about 2.5 times per dose. Most preferably, the adjuvant is added in an amount of about 丨 mg per dose. The pharmaceutical/vaccine composition may further comprise one or more other immunomodulatory agents, such as interleukins, interferons or other cytokines. Pharmaceutical/vaccine compositions may also include gentamicin and thimerosal. Although the amounts and concentrations of adjuvants and additives suitable for use in the context of the present invention can be readily determined by those skilled in the art, the present invention encompasses a 1 ml dose of a vaccine composition comprising from about 5 to about 2 inches. A composition of an adjuvant and preferably about 25 tons of adjuvant. In another preferred embodiment, the invention encompasses a vaccine composition comprising from about 1 Kg/ml to about 60 pg/ml of antibiotic and more preferably less than about 30 pg/ml of antibiotic. Thus, according to another embodiment, the invention is also directed to a pharmaceutical/vaccine set 124989.doc-42-200825099 composition comprising: i. A therapeutically effective amount of any of the influenza virus H5 proteins as described herein, wherein The H5 protein has an amino acid 223N and a modification 328K+, wherein the amino acid position number of the H5 protein refers to an amino acid position as exemplified in SEQ ID ΝΟ·1 and wherein the modification 328Κ+ means A second lysine (κ+) is inserted into the amino acid acid position 328 of the Η5 protein; and ii. a pharmaceutically acceptable adjuvant as described above. Preferably, the adjuvant is selected from the group consisting of: a) EMULSIGEN8: an oil-in-water emulsion (o/w); b) EMULSIGEN-D®: a species having dimethyldi-bromide Oil-in-water (o/w) emulsion of octaalkylammonium (DDA); c) Polygen: a copolymer; d) EMULSIGEN-P8, an oil-in-water type with proprietary immunostimulating agent (〇/w) Emulsion; e) Carbigen is a crosslinked polymer; f) EMULSIGEN-75®: a dual adjuvant comprising an oil-in-water (o/w) emulsion with a crosslinked polymer; g) ISA 70 is an oil Water-in-water (w/o) emulsion. Most preferably, the adjuvants are oil-in-water emulsions, such as emulsigen-based adjuvants selected from the group consisting of: EMULSIGEN®, EMULSIGEN-D8, EMULSIGEN-P®, EMULSIGEN-75®, EMULSIGEN® And EMULSIGEN-P®. The best EMULSIGEN8 and EMULSIGEN-P8 are used in the formulation of the present invention. According to another aspect, the pharmaceutical/vaccine composition package 124989.doc-43-200825099 as provided herein contains one or more antigens. Preferably, the other antigen is an antigen of an avian or mammalian pathogen. According to another embodiment, the other antigen is another influenza antigen such as hemagglutinin H3, H7, H9 or any other hemagglutinin of the influenza virus. The (other) antigen may be added in purified form, as part of an antigenic preparation, in the form of a killed microorganism or in the form of a modified living microorganism. The term "antigen, as used herein, means, but is not limited to, a peptide, a poly() peptide, a glycopeptide or a polysaccharide, which is capable of interacting with an antigen recognition molecule of the immune system (such as an immunoglobulin (antibody) or a buty cell). The antigen receptor) specifically interacts to initiate, activate or stimulate an immune response against the antigen in the host to which the antigen is administered. The term "antigen" also refers to a nucleic acid molecule, preferably a 〇1 八 molecule or an RNA molecule. , their respective coding and performance can be related to the antigenic knowledge of the immune system

別分子（諸如免疫球蛋白（抗體）或T細胞抗原受體）特異性I ^作用以便引發、活化或刺激針對由該核酸分子所編碼之抗原的免疫反應的肽、多肽或醣肽。用於製備根據本發明〇制之醫藥組合物的抗原為微生物或該微生物之抗原部分及/或製劑。就此而言，如本文中所使用之術語”免疫”意謂 (仁不限於）免疫反應之任何引起或增強。術語"免疫反應” 已於上文中加以描述。机感疫田之投藥策略已熟知於此項技術。本發明預期滅活病毋疫田及減t病毒疫苗之黏膜接種策略。儘管黏膜可由局部傳遞疫苗標乾導向，但多種策略皆已用於將免疫原性蛋白傳遞至黏膜。在一特定實施例中，可將疫苗與霍亂毒素（諸如霍亂毒 124989.doc -44- 200825099 素B或霍亂毒素A/B礙合體）混合或作為結合型或嵌合型融合白投藥（Hajishengallis，J Immunol,，154:4322-32, 】995，· Jobling 及 Holmes，Infect Immun.， 60:4915-24, /992)。基於使用霍亂毒素B次單位之黏膜疫苗已加以描述 (Lebens及 Holmgren，Dev Biol Stand 82:215-27，1994)。在另一實施例中，可製備與熱不穩定性腸毒素（LT)之混合物用於黏膜接種。其他黏膜免疫策略包括將病毒囊封於微膠囊中（us 5,075，109、US 5,820,883 及 US 5,853,763)及使用免疫增強性膜質載劑（WO 98/0558)。經口投藥之免疫原之免疫原性可藉由使用紅血球（rbc)或rbc殘骸（US 5,643,577)或藉由使用藍舌病抗原（US 5,690,938)而得以增強。根據另一態樣’本發明係關於一種製備如上所述之醫藥/ 疫苗組合物的方法，較佳為一種製備包含如上所述之經桿狀病毒表現之重組性H5蛋白之疫苗的方法。通常，此方法包括以下步驟：i)將構築體轉染至病毒内，其中該構築體包含如本文中所述之重組性H5 cDNA; Η)用經轉染之病毒感染生長培養基中之細胞；ίΗ)促使病毒表現如本文中所述之重組性H5蛋白；iv)自培養物回收經表現之仍蛋白；及v)藉由將經表現2H5蛋白與適當佐劑及/或其他醫藥學上可接受之載劑摻混而製備組合物。、較佳佐劑為上述彼等佐劑。因&，根據另_態樣，用於激發防禦流感感染之免疫反應之抗原組合物（諸如疫苗）的製備方法包含i)製備及回收H5蛋白，及Η)將此蛋白與適當 124989.doc -45- 200825099 佐劑混合。此外’本發明之疫苗組合物亦可包括稀釋劑、等張劑、穩定劑及/或防腐劑。稀釋劑可包括水、生理食蜂水、右旋糖、乙醇、甘油及類似物。等張劑尤其可包括：機鹽或有機鹽(例如氣化鈉)、右旋糖、甘露糖醇、山梨糖醇及乳糖、酷類、海藻糖、甘露糖醇、嚴糖。穩定劑尤其包括白蛋白及乙二胺四乙酸之驗金屬鹽。適當佐劑為上述彼等佐劑。 ΟA molecule, such as an immunoglobulin (antibody) or a T cell antigen receptor, specifically acts to induce, activate or stimulate a peptide, polypeptide or glycopeptide against an immune response to the antigen encoded by the nucleic acid molecule. The antigen used to prepare the pharmaceutical composition prepared according to the present invention is a microorganism or an antigenic portion and/or preparation of the microorganism. In this regard, the term "immunization" as used herein means, without limitation, any cause or enhancement of an immune response. The term "immune response" has been described above. The drug-administering strategy of the Phytophthora has been well known in the art. The present invention contemplates a mucosal vaccination strategy for inactivated disease plague and virulence vaccines, although the mucosa may be localized. Delivery of vaccines is directed, but a variety of strategies have been used to deliver immunogenic proteins to the mucosa. In a particular embodiment, vaccines can be administered with cholera toxins (such as cholera poisoning 124989.doc-44-200825099 B or cholera Toxin A/B ligands are mixed or administered as a binding or chimeric fusion white drug (Hajishengallis, J Immunol, 154:4322-32, 995, Jobling and Holmes, Infect Immun., 60:4915-24, /992). A mucosal vaccine based on the use of cholera toxin B-units has been described (Lebens and Holmgren, Dev Biol Stand 82: 215-27, 1994). In another embodiment, a thermally labile enterotoxin can be prepared. A mixture of (LT) is used for mucosal vaccination.Other mucosal immunization strategies include encapsulation of the virus in microcapsules (us 5,075,109, US 5,820,883 and US 5,853,763) and the use of immunoenhancing membrane carriers (WO 98/0558). The immunogenicity of an orally administered immunogen can be enhanced by the use of red blood cells (rbc) or rbc residues (US 5,643,577) or by the use of bluetongue antigens (US 5,690,938). According to another aspect, the invention is With regard to a method of preparing a pharmaceutical/vaccine composition as described above, preferably a method of preparing a vaccine comprising the recombinant H5 protein expressed by the baculovirus as described above. Typically, the method comprises the steps of: i) The construct is transfected into a virus, wherein the construct comprises a recombinant H5 cDNA as described herein; Η) infecting cells in the growth medium with the transfected virus; 促使) promoting viral expression as described herein Recombinant H5 protein; iv) recovering the expressed protein from the culture; and v) preparing the composition by blending the expressed 2H5 protein with a suitable adjuvant and/or other pharmaceutically acceptable carrier Preferably, the adjuvant is the above-mentioned adjuvant. According to the &, according to another aspect, the preparation method of the antigen composition (such as a vaccine) for initiating an immune response against influenza infection comprises i) preparing and recovering H5 protein And Η) mixing the protein with an appropriate 124989.doc -45-200825099 adjuvant. Further, the vaccine composition of the present invention may also include a diluent, an isotonic agent, a stabilizer, and/or a preservative. The diluent may include water. Physiological bee water, dextrose, ethanol, glycerol and the like. The isotonic agents may especially include: organic or organic salts (such as sodium carbonate), dextrose, mannitol, sorbitol and lactose, Cool, trehalose, mannitol, and sugar. Stabilizers include, inter alia, the metal salts of albumin and ethylenediaminetetraacetic acid. Suitable adjuvants are those mentioned above. Ο

G 該等H5蛋白、核酸分子、載艘及疫苗中之任一者之醫藥用途本文中所提供之H5蛋白、編碼任何該等出蛋白之核酸分子、包含任何該㈣❹子（編碼如本文巾所述之任何該等H5蛋白）的载體以及包含該H5蛋白、核酸分子或載體中之任-者的任何醫藥/疫苗組合物可用作藥物，較佳用於’口療及預防由流感病毒、最佳由流感A病毒引起的感染°如本文中所提供之H5蛋白、編碼任何該等H5蛋白之核酸分子、包含任何該等核酸分子（編碼如本文中所述之任何該等H5蛋白）之載體以及包含如本文中所述之_蛋白、核酸分子或載體中之任一者之任何醫藥/疫苗組合物可用於人類之治療或預防且可用於獸醫藥物中。當用於獸 w藥物中’車父佳為治療禽類’較佳鳥、雞、鴨、火雞及類似動物’以及哺乳動物，較佳豬、牛、馬、海豹、駱騎、狗、猶、倉氣、小鼠及類似動物。因此，根據另一態樣，本發明係關於如本文中所提供之 H5蛋白、編碼任何該等H5蛋白之核酸分子、包含任何該 124989.doc -46- 200825099 等核酸分子（編碼如本文中所述之任何該等H5蛋白）之载體以及包含如本文中所述之該H5蛋白、核酸分子或載體中之任一者之任何醫藥/疫苗組合物的用途，其可用作藥物、較佳用作人類藥物及/或獸醫藥物。 ‘ 此外，如本文中所提供之H5蛋白、如本文中所述之編石馬 #何该等H5蛋白之核酸分子、包含任何該等核酸分子(編碼任何該H5蛋白）之載體可用於製備如本文中所述之醫藥 Γ 、组合物，以便預防或治療由流感病毒引起之感染。如上所述，彼等醫藥組合物/疫苗組合物可用於人類之治療及/或預防以及動物之治療及/或預防，該等動物諸如禽類，較佳鳥、雞、鴨、火雞及類似動物，以及哺乳動物，較佳豬牛、馬、海豹、路馬它、狗、！苗、倉鼠、小鼠及類似動物。 ★如本文中所提供之出蛋白、如本文中所述之編碼任何該等^5蛋白之核酸分子、包含任何該等核酸分子（編碼任何〇該等Η5蛋白）之載體可用於製備如本文中所述之醫藥組合物’该醫藥組合物適於治療及預防較佳由禽、豬或人類流感病毒或其任何組合或混合之流感病毒感染。 ⑽ 主根據另-態樣，本發明亦係關於一種治療或預防流感病 #感染之方法’其巾該方法包含將治療有效量之如本文中所述之Η5蛋白投與需要該治療之受檢者。此外，本發明亦係關於一種治療或預防流感病毒感染之方法，其中該方法包含將治療有效量的如本文中所述之編碼如本文中所述之任何Η5蛋白的任何Η5核酸分子或載體投與需要該治療之 124989.doc -47- 200825099 :檢者。此外’本發明亦係關於一種治療或預防流感病毒感染之方法’丨中該方法包含將治療有效量之包含如本文中所述之任何細蛋白、核酸分子或栽體之疫苗投與需要 n療之文檢者。有需要之受檢者可為人類以及動物，較佳為禽類’甚至更佳為鳥、_、鴨、火雞；或哺乳動物，較佳為豬、牛、馬、海豹、駱駝、狗、貓、倉鼠、小鼠及類似動物。G. The pharmaceutical use of any of the H5 proteins, nucleic acid molecules, vectors, and vaccines, the H5 protein provided herein, a nucleic acid molecule encoding any of the such proteins, comprising any of the (four) scorpions (encoded herein) A carrier of any of the H5 proteins) and any pharmaceutical/vaccine composition comprising any of the H5 proteins, nucleic acid molecules or vectors can be used as a medicament, preferably for 'oral therapy and prevention by influenza virus Preferably, the infection caused by the influenza A virus, such as the H5 protein provided herein, a nucleic acid molecule encoding any of the H5 proteins, comprising any of the nucleic acid molecules (encoding any of the H5 proteins as described herein) The vector and any pharmaceutical/vaccine composition comprising any of the proteins, nucleic acid molecules or vectors as described herein can be used in the treatment or prevention of humans and can be used in veterinary medicine. When used in the animal w drug, 'Car father is the best way to treat birds', better birds, chickens, ducks, turkeys and similar animals' and mammals, preferably pigs, cows, horses, seals, lions, dogs, and juveniles. Cangqi, mice and similar animals. Thus, according to another aspect, the invention relates to an H5 protein, a nucleic acid molecule encoding any of the H5 proteins, as described herein, comprising any of the nucleic acid molecules of 124989.doc-46-200825099 (encoded herein) Use of a carrier of any of these H5 proteins) and any pharmaceutical/vaccine composition comprising any of the H5 proteins, nucleic acid molecules or vectors as described herein, which may be used as a medicament, preferably Used as a human drug and / or veterinary drug. In addition, a nucleic acid molecule such as the H5 protein provided herein, a chimeric horse such as described herein, or a nucleic acid molecule comprising any of the nucleic acid molecules (encoding any such H5 protein) can be used in the preparation of The pharmaceutical compositions and compositions described herein to prevent or treat infections caused by influenza viruses. As mentioned above, their pharmaceutical compositions/vaccine compositions are useful in the treatment and/or prevention of humans, such as birds, preferably birds, chickens, ducks, turkeys and the like, and for the treatment and/or prevention of animals. And mammals, better pigs, horses, seals, road horses, dogs,! Seedlings, hamsters, mice and similar animals. A nucleic acid molecule as described herein, a nucleic acid molecule encoding any of the above 5 proteins, as described herein, a vector comprising any of the nucleic acid molecules (encoding any such Η5 protein) can be used for preparation as herein Said pharmaceutical composition 'the pharmaceutical composition is suitable for the treatment and prevention of influenza virus infection, preferably by avian, porcine or human influenza virus or any combination or mixture thereof. (10) The subject is also based on another aspect, the invention is also directed to a method of treating or preventing influenza infections. The method comprises administering a therapeutically effective amount of the Η5 protein as described herein to a subject in need of the treatment. By. Furthermore, the invention relates to a method of treating or preventing an influenza virus infection, wherein the method comprises administering a therapeutically effective amount of any Η5 nucleic acid molecule or vector encoding any Η5 protein as described herein as described herein. With the need for the treatment 124989.doc -47- 200825099: the examiner. Furthermore, the invention also relates to a method of treating or preventing influenza virus infection, wherein the method comprises administering a therapeutically effective amount of a vaccine comprising any of the fine proteins, nucleic acid molecules or vectors as described herein. The examiner. Subjects in need may be humans and animals, preferably poultry 'even better birds, _, ducks, turkeys; or mammals, preferably pigs, cows, horses, seals, camels, dogs, cats , hamsters, mice and similar animals.

較佳地，當對雞進行接種時，可在i日齡時幻日齡之後 (例如在ίο日齡時，或在丨日齡至1G日齡時，或㈣日齡時或10日齡之後）使用如本文中所述之H5蛋白接種。可藉由投與任何出蛋白、編碼該任何H5蛋白之核酸分子或載體或如本文中所述之任何醫藥/疫苗組合物治療的流感感染較佳係由禽、豬或人類流感病毒或其任何組混合引起。 / 根據另一態樣，本發明係關於部分套組，其包含：i)如本文中所述之該H5蛋白、編碼任何該出蛋白之核酸分子或載體或包含如本文中所述之該H5蛋白、核酸分子或载體中任一者之任何醫藥/疫苗組合物中的任一者；及丨丨）指示该H5蛋白、核酸分子、載體或疫苗於治療或預防由流感病毒引起之感染之使用的包裝插頁。當對雞進行接種時，可在1日齡時或1日齡之後使用如本文中所述iH5蛋白接種。根據另一實施例，彼部分套組包含禽類或哺乳動物病原體之至少另一種抗原及指示彼另外抗原之醫藥、人類或獸醫學用途的資訊。 124989.doc -48- 200825099 【實施方式】實例以下實例闡述本發明之較佳物質及程序。然而應瞭解，該等實例僅為說明而提供，且不應視為對本發明之整體範圍之限制。實例1 建構編碼及表現HA H5抗原的重組性桿狀病毒如下生成含有H5 HA抗原的重組性桿狀病毒：化學合成 H5 HA(SEQ ID NO:2)之編碼序列且將其次選殖入轉移載體 pVL1392(BD Biosciences Pharmingen，San Diego，CA)内。藉由使用寡核苷酸引子及QuikChange®定點誘變套組 (Stratagene，La Jolla，CA)生成 H5 HA MutK+ (SEQ ID NO:4)且將其次選殖入轉移載體pVL 1392(BD Biosciences Pharmingen，San Diego，CA)内。接著用 DiamondBac® (Sigma)桿狀病毒DNA將含有編碼H5 HA抗原（SEQ ID NO:2)及 H5 HA MutK+抗原（SEQ ID NO:4)之基因的 pVL1392質體共轉染入Sf9昆蟲細胞（BD Biosciences Pharmingen)内以生成含有編碼SEQ ID NO:2之基因H5 HA 及編碼SEQ ID NO:4之基因H5 HA mutK+的重組性桿狀病毒。將含有編碼H5 HA(SEQ ID NO:2)及 H5 HA MutK+ (SEQ ID ΝΟ··4)之基因的重組性桿狀病毒進行空斑純化，且將主種子病毒（Master Seed Virus ; MSV)於SF +細胞株上繁殖，製成等分試樣且在-70°C下儲存。如由多株血清或單株抗體以間接螢光抗體檢定或西方墨點法所偵測，如上 124989.doc -49- 200825099 所述經H5 HA桿狀病毒感染（以生成MSV或工作種子病毒 (Working Seed Virus))之昆蟲細胞表現H5 HA抗原（SEQ ID NO:2)及 H5 HA MutK+抗原（SEQ ID NO:4)。用適量重組性桿狀病毒（分別為H5 HA及H5 HA MutK+) 接種後，接著將含有SF+細胞（Protein Sciences，Inc.， Meriden，CT)的旋轉瓶在27±2°C下培育7天且在彼期間以 100 rpm攪拌。該等旋轉瓶使用通氣蓋以使空氣流動。收穫含有經桿狀病毒感染之SF +細胞的原全細胞培養物及各 ζ\ 培養物之細胞培養上清液。實例2 製備包含HA Η5抗原之醫藥組合物（疫苗）收穫在昆蟲細胞中由基於桿狀病毒之表現系統表現的原全細胞Η5 ΗΑ蛋白及Η5 HA Mutk+蛋白。將桿狀病毒在5 mM環化二元伸乙基亞胺（BEI)(最終濃度）之存在下、在約 32°C與39它之間滅活72至96小時。滅活完成後，添加〇.3 M硫代硫酸鈉溶液直至最終濃度為5 mM，以中和任何殘餘 BEI。中和後，添加各種佐劑且生成以下疫苗/醫藥組合物0 疫苗通用產物名獾 Lx. πς --------- 501 ~~-----— OR，. AJLm ~ -- ΐ ΪΓτί胞中由基於桿狀病毒之表現系統表現的原全細胞Η5 ΗΑ蛋白調配物現ί，Η5 ΗΑ之培養昆蟲細胞及上清液的實驗性疫-田。佐劑。通用產物名稱抗原 ^^ 502 "~-------- 毒之表現系統表現的原全細 124989.doc -50- 200825099Preferably, when the chicken is inoculated, it may be after the magic age of the i day (for example, at the age of ίο, or at the age of 丨 to 1G, or at the age of (4) or after 10 days) ) vaccination with H5 protein as described herein. An influenza infection which can be treated by administering any exoprotein, a nucleic acid molecule or vector encoding the H5 protein or any of the pharmaceutical/vaccine compositions as described herein is preferably avian, porcine or human influenza virus or any thereof Group mixing caused. / According to another aspect, the invention relates to a partial kit comprising: i) the H5 protein as described herein, a nucleic acid molecule or vector encoding any such protein, or comprising the H5 as described herein Any of the pharmaceutical/vaccine compositions of any of a protein, nucleic acid molecule or vector; and 丨丨) indicating that the H5 protein, nucleic acid molecule, vector or vaccine is for treating or preventing an infection caused by an influenza virus The package insert used. When the chicken is inoculated, iH5 protein inoculation as described herein can be used at 1 day of age or after 1 day of age. According to another embodiment, the partial kit comprises at least one other antigen of the avian or mammalian pathogen and information indicative of the pharmaceutical, human or veterinary use of the additional antigen. 124989.doc -48- 200825099 EXAMPLES The following examples illustrate preferred materials and procedures of the present invention. However, it is to be understood that the examples are provided for illustration only and are not to be construed as limiting the scope of the invention. Example 1 Construction of a Recombinant Baculovirus Encoding and Characterizing the HA H5 Antigen Generates a recombinant baculovirus containing the H5 HA antigen by chemically synthesizing the coding sequence of H5 HA (SEQ ID NO: 2) and subsequencing it into a transfer vector Within pVL1392 (BD Biosciences Pharmingen, San Diego, CA). H5 HA MutK+ (SEQ ID NO: 4) was generated by using an oligonucleotide primer and a QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA) and subsequently into the transfer vector pVL 1392 (BD Biosciences Pharmingen, Within San Diego, CA). The pVL1392 plastid containing the gene encoding the H5 HA antigen (SEQ ID NO: 2) and the H5 HA MutK+ antigen (SEQ ID NO: 4) was then co-transfected into Sf9 insect cells using DiamondBac® (Sigma) baculovirus DNA ( BD Biosciences Pharmingen) was used to generate a recombinant baculovirus containing the gene H5 HA encoding SEQ ID NO: 2 and the gene H5 HA mutK+ encoding SEQ ID NO: 4. The recombinant baculovirus containing the gene encoding H5 HA (SEQ ID NO: 2) and H5 HA MutK+ (SEQ ID ΝΟ·4) was subjected to plaque purification, and the main seed virus (MSV) was used. The SF + cell lines were propagated, aliquots were made and stored at -70 °C. Infection by H5 HA baculovirus as described in 124989.doc -49-200825099, as detected by indirect fluorescent antibody assay or Western blot method, by multiple sera or monoclonal antibodies (to generate MSV or working seed virus ( The insect cells of Working Seed Virus) exhibited H5 HA antigen (SEQ ID NO: 2) and H5 HA MutK+ antigen (SEQ ID NO: 4). After inoculation with appropriate amounts of recombinant baculovirus (H5 HA and H5 HA MutK+, respectively), spinner flasks containing SF+ cells (Protein Sciences, Inc., Meriden, CT) were then incubated for 7 days at 27 ± 2 °C. Stir at 100 rpm during this period. These rotating bottles use a venting cap to allow air to flow. The original whole cell culture containing the baculovirus-infected SF + cells and the cell culture supernatant of each ζ\ culture were collected. Example 2 Preparation of a pharmaceutical composition (vaccine) comprising the HA Η5 antigen The original whole cell Η5 ΗΑ protein and Η5 HA Mutk+ protein expressed by a baculovirus-based expression system were harvested in insect cells. The baculovirus was inactivated in the presence of 5 mM cyclized binary ethylenimine (BEI) (final concentration) between about 32 ° C and 39 for 72 to 96 hours. After the inactivation was completed, 〇.3 M sodium thiosulfate solution was added until a final concentration of 5 mM to neutralize any residual BEI. After neutralization, various adjuvants are added and the following vaccine/pharmaceutical composition is produced. 0 Vaccine General Product Name 獾Lx. πς --------- 501 ~~------ OR,. AJLm ~ -- The original whole-cell Η5 ΗΑ protein formulation expressed by the baculovirus-based expression system in ΐ ί ί ί ί 现实验实验。。。。。。。。。。。。。。。培养培养培养培养培养培养培养培养培养培养培养培养培养培养培养培养Adjuvant. Generic product name antigen ^^ 502 "~-------- Poison performance system performance of the original full fine 124989.doc -50- 200825099

~mn5 HA ^ --ί 調配物 .現H5 ha之培養昆蟲細胞及上清液的實驗 Emulsigen-D 佐劑〇通用產物名稱 ----- "503 ' ------- 胞Η5 HA蛋白調配物 ^二，現，組性H5 HA之培養昆蟲細胞及上清液的實驗性疫田。疫苗辅以p〇lygen佐劑。通用產物名稱 *504 "~~一 -- 抗原 έ 由基於桿狀病毒之表現系統表現的原全細 ^配物~ — ^言，現重組性H5 HA之培養昆蟲細胞及上清液的實驗性疫田。疫苗輔以Emulsigen-P佐劑。通用產物名稱 ~505 ~- ----- 抗原士 ^蟲細胞T由基於桿狀病毒之表現系統表現的原全細胞H5HA蛋白。調配物 ^言，現重組性H5 HA之培養尾蟲細胞及上清液的實驗性疫。疫苗輔以Carbigen佐劑。通用產物名稱 Ί〇6 -- 抗原 f昆蟲細胞中由基於桿狀病毒之表現系統表現的原全細胞H5HA蛋白。調配物，言表現重組性H5 HA之培養昆蟲細胞及上清液的實驗性疫苗。疫苗辅以Emulsigen-75佐劑。通用產物名稱 T〇7 -- 抗原在把蟲細胞中由基於桿狀病毒之表現系統表現的原全細胞Η5ΗΑ蛋白。調配物包含表現重組性Η5 ΗΑ之培養昆蟲細胞及上清液的實驗佐劑。通用產物名稱 "508 ^---— 抗原在昆蟲細胞中由基於桿狀病毒之表現系統表現的原全細胞 H5 HA mutK+蛋白。調配物包含表現重組性H5 HA之培養昆蟲細胞-及上清液的實驗佐劑。 124989.doc • 51 · 200825099 通用產物名稱 509 —' ---—— 抗原弋5^=把+由1於桿狀病毒之表現系統表現的原全細胞 H5HAmutK+蛋白。調配物 2 ί現H5 HA之培養昆蟲細胞及上清液的實驗 f生疫田。疫田輔以Emulsipen-Fi佐许|。通用產物名稱 510 ---- 抗原 f te中由基於桿狀病毒之表現系統表現的原全參田一胞 H5HAmutK+蛋白。調配物 St说ί H5 HA之培養昆蟲細胞及上清液的實系— 性疫苗。疫苗辅以P〇1ygerH±劑。通甩產物名稱~~"" ΤΠ -----Ί 抗原 Ϊ 胞中由基於桿狀病毒之表現系統表現的原全細胞 H5HAmutK+蛋白。調配物 S i現H5 HA之培養昆轰細胞及上清液的實驗性疫田。疫田佐以Emulsigen-P佐劑。通用產物名稱 "512 -------Ί 抗原 f $蟲j田胞中由基於桿狀病毒之表現系▲•表現的原全f 胞 H5 HA mutK+蛋白。現ί^ H5 HA之培養昆蟲細胞及上清液的實驗性疫田。疫田輔以Carbigen佐劑。 513 ϋ 通用產物名稱抗原 ~ 之絲制原全f 胞 H5 HA muUCjj> 自〇雨上清液的保 jMi^^^i^Emulsigen-75 佐劑~mn5 HA ^ --ί Formulation. Experiment of cultured insect cells and supernatant of H5 ha. Emulsigen-D adjuvant 〇 generic product name----- "503 ' ------- Cytoplasmic 5 HA protein formulation ^ two, now, group H5 HA cultured insect cells and supernatant of experimental field. The vaccine was supplemented with p〇lygen adjuvant. Generic product name *504 "~~一--Antigen έ Experiment of culturing insect cells and supernatants of recombinant H5 HA by the original full-featured compound based on the baculovirus expression system Sexually infected fields. The vaccine is supplemented with Emulsigen-P adjuvant. Generic product name ~505 ~- ----- Antigen X. Phytophthora T is a native full-cell H5HA protein expressed by a baculovirus-based expression system. Formulations, the experimental epidemic of cultured tailworm cells and supernatants of recombinant H5 HA. The vaccine is supplemented with Carbigen adjuvant. Generic product name Ί〇6-antigen f The original full-cell H5HA protein expressed by a baculovirus-based expression system in insect cells. Formulations, an experimental vaccine for the expression of recombinant H5 HA in cultured insect cells and supernatants. The vaccine was supplemented with Emulsigen-75 adjuvant. Generic product name T〇7 - antigen The original full-cell Η5ΗΑ protein expressed in a baculovirus-based expression system in insect cells. Formulations Experimental adjuvants containing cultured insect cells and supernatants expressing recombinant Η5 。. Generic product name "508 ^---- antigen The original full-cell H5 HA mutK+ protein expressed in insect cells by a baculovirus-based expression system. Formulations Experimental adjuvants containing cultured insect cells- and supernatants expressing recombinant H5 HA. 124989.doc • 51 · 200825099 Generic product name 509 — ' --- -- Antigen 弋 5 ^ = original full cell H5HAmutK+ protein expressed by + in a baculovirus expression system. Formulation 2 ί H5 HA cultivation of insect cells and supernatants. The epidemic field is supplemented by Emulsipen-Fi. Generic product name 510 ---- Antigen f te is a H. cerevisiae H5HAmutK+ protein expressed by a baculovirus-based expression system. Formulation St says ί H5 HA is a solid-sex vaccine for cultivating insect cells and supernatants. The vaccine is supplemented with P〇1ygerH±. Byproduct name ~~"" ΤΠ -----Ί Antigen 原 The original full-cell H5HAmutK+ protein expressed by the baculovirus-based expression system. Formulation S i is an experimental field in which H5 HA is cultured with Kunming cells and supernatant. The epidemic field was supplemented with Emulsigen-P adjuvant. The generic product name "512 -------Ί antigen f $ 虫 j field cell by the baculovirus-based performance of the original full-f cell H5 HA mutK+ protein. The experimental field of cultured insect cells and supernatants of ί^ H5 HA. The epidemic field is supplemented with Carbigen adjuvant. 513 通用 Generic product name Antigen ~ Silk original cell f H5 HA muUCjj> Self 〇 Rain supernatant protection jMi^^^i^Emulsigen-75 Adjuvant

124989.doc -52- 200825099 實例3 對豬進行接種以防禦禽流感 1. 引論此研究之目的係測定含有重組性H5血球凝集素（HA)抗原之粗提取物之實驗性疫苗在豬體内誘導血球凝集抑制 (HI)效價的能力。用H5 HA抗原評價各種佐劑。此研究中評價含有來自習知H5 HA或H5 HA MutK+之抗原的HA H5原型。習知H5 HA係源自A/鴨/China/E319-2/03，而H5 HA MutK+由習知H5 HA組成，其經工程化以在S120N、D150N、S223N及328mutK+上含有三個特定胺基酸變異。其亦含有胺基酸94N。H5 HA Mut K+中之特定胺基酸變異產生更接近類似於A/HK/213/03之HA的H5 HA。目前認為A/HK/213/03之H5 HA之胺基酸組成有助於 H5 HA之抗體識別。 2. 研究設計：表1 :研究概述組豬數目疫苗原型第❶曰第21曰 ^第35曰 1 5 501 放血及肌内注射接種(頭肱組)(藉由在頸左側投藥1 ml) 放血及肌内注射接種 (頭肢組)(藉由在頸右側投藥1 ml) 放血及終止研究 2 5 502 3 5 503 4 5 504 5 5 505 6 5 506 7 5 507 8 5 508 9 5 509 10 5 510 11 5 511 12 5 512 13 5 513 14 5 514 15 5 無放血放血 124989.doc -53- 200825099 °研究開*日夺，仔豬在第21日及第35日獲得血研究開始時，仔豬為3週±5日齡臨床上為健康的。在研究第〇曰、樣0 •在研九第1曰至第35曰每曰觀測全部研究動物之—般健康狀況。在每次接種後七天，每天查看注㈣位且記錄可見反應。在研究曰第35曰動物研究期結束時，對全部動物施以無痛致死術。 3.疫苗124989.doc -52- 200825099 Example 3 Inoculation of pigs against avian influenza 1. Introduction The purpose of this study was to determine an experimental vaccine containing crude extract of recombinant H5 hemagglutinin (HA) antigen in pigs. The ability to induce hemagglutination inhibition (HI) titers. Various adjuvants were evaluated using H5 HA antigen. The HA H5 prototype containing the antigen from the conventional H5 HA or H5 HA MutK+ was evaluated in this study. The conventional H5 HA line is derived from A/Duck/China/E319-2/03, while H5 HA MutK+ consists of the conventional H5 HA, which is engineered to contain three specific amine groups on S120N, D150N, S223N and 328mutK+. Acidic variation. It also contains the amino acid 94N. The specific amino acid variation in H5 HA Mut K+ produces H5 HA that is closer to HA similar to A/HK/213/03. It is currently believed that the amino acid composition of H5 HA of A/HK/213/03 contributes to the antibody recognition of H5 HA. 2. Study design: Table 1: Summary of study Group pig number Vaccine prototype No. 21曰^35曰1 5 501 Bloodletting and intramuscular injection (head group) (by administering 1 ml on the left side of the neck) Bleeding And intramuscular injection (head group) (by administering 1 ml on the right side of the neck) bleeding and termination of the study 2 5 502 3 5 503 4 5 504 5 5 505 6 5 506 7 5 507 8 5 508 9 5 509 10 5 510 11 5 511 12 5 512 13 5 513 14 5 514 15 5 No bloodletting and bloodletting 124989.doc -53- 200825099 ° Study on the day of the day, piglets on the 21st and 35th days when the blood study begins, the piglet is 3 Weeks ± 5 days of age are clinically healthy. Studying Dijon, Sample 0 • Observing the general health status of all study animals in each of the 9th to the 35th. Seven (7) days were observed every day for seven days after each inoculation and a visible response was recorded. At the end of the study period, the 35th animal study period, all animals were given painless death. 3. Vaccine

Ο 將如實例2中所述之疫苗5〇1至514用於豬接種研究。 4 ·血球凝集素抑制檢定在第〇日及第21日用含有Η5 ΗΑ之原型對豬進行接種。在第〇日、第21日、第35日收集豬血清以便藉由血球凝集抑制（HI)檢定進行評價。進行m檢定以偵測ΗΑ特異性抗體之存在。異源H5N2病毒（A/雞/Mexico/232/94)係以四個血球凝集單位[4 ΗΑ單位]之濃度用於HI檢定中。隨後在υ形底微量滴定板中，將PB S中之連續兩倍血清稀釋液與等體積（25 μΐ^)(含有4 HA單位）之病毒混合，且在室溫（約25。〇下培育30分鐘。將PBS中之濃度為0.5%之雞紅血球添加至含有血清-病毒之孔中且在室溫下培育40分鐘。以觀測到血球凝集抑制的最高血清稀釋度之倒數確定HI效價。 5.結果 HI測試使用Mexican政府法定之H5N1抗原（A/雞/Mexico/ 232/94)[4 HA單位]，接種方案為第0日及第21日lxl mL。 124989.doc -54- 200825099 HI效價第0曰第21曰第35曰 501 H5-Emulsigen 0 0 4 502 H5-Emulsigen-D 0 0 4 503 H5 -Polygen 0 0 0 504 H5-Emulsigen-P 0 0 2 505 H5-Carbigen 0 0 4 506 H5-Emulsigen-75 0 0 16 507 H5ISA70 0 0 16 508 H5 K+-Emulsigen 0 0 128 509 H5 K+-Emulsigen-D 0 0 64 510 H5 K+-Polygen 0 0 16 511 H5 K+-Emulsigen-P 0 0 0 512 H5 K+-Carbigen 0 0 0 513 H5 K+-Emulsigen-75 0 0 16 514 H5K+-ISA70 0 4 32 對照無 0 0 0 BIV H5(來源於流感 A病毒（A/鴨 /China/E3 19-2/03(Η5Ν1)) BIV H5 K+(突變之 BIV H5，包括 S120N、D155N、 S223N，且增添 328K+) 結果證明大部分疫苗組合物在接種豬中引發免疫反應。特定言之，大部分疫苗組合物引起血清轉化，其意謂大部分接種豬產生針對HI檢定中所使用之禽流感病毒的特異性抗體。總而言之，結果清楚且無疑地證明本發明創見極其奏效。藉由用禽流感病毒之相關抗原對豬進行接種可大大降低豬（第二物種之動物）經禽流感病毒（第一物種之病原體）廣泛流行性感染之風險。此已得到清楚證明。此外，依據此接種概念，禽流感病毒對哺乳動物（包括人類）之傳染性及順應性大大降低。豬為禽病原體（包括禽流感病毒）之最重要宿主之一。若病毒在豬體内之複製且因此禽流感對豬之順應性之風險大大降低且得以控制，則禽流感病毒 124989.doc -55- 200825099 對人類之任何順應性之風險亦大大降低。在投與抗原產生較低HI效價（意謂效價低於30)之情況下，需要用抗原進一步加強以進一步改良HI效價且增強接種豬體内之免疫保護。因此，效價低並不意謂無法獲得保護，其僅教示似乎 • 需要進一步加強以改良免疫反應。接種豬體内可量測到免疫反應證明作為本發明依據之本發明創見極其奏效。換而言之，本文所提供之實驗清楚且無疑地證明本發明之創見可奏效。 Γ 實例4 對鳥進行接種以防禦禽流感 1.引論此研究之目的係測定含有重組性H5nlutk+血球凝集素 (H5 HA mutk+)抗原之粗提取物之實驗性疫苗在雞體内誘導金球凝集抑制（HI)之能力。此外，習知重組性出抗原 (H5 HA)以及滅活疫苗 Volvac® AI(Boehringer Ingelheim ^ Vetmedica，Mexico)用於對照。此外，用H5 HA抗原評價多種佐劑。 2·研究設計：將SPF鳥（15-25隻）在1曰齡或1〇曰齡時，獨立地用〇5 不同實驗性疫苗在頸背部藉由皮下途徑接種；實驗期間將所有鳥供養於隔離器中。不限量提供饋食及水。用H5N2 南病原性禽流感病毒株接種後第3 1日或第3 2日進行激發。藉由在接種後第15日、第30日自鳥頸靜脈放血獲得血清樣本。為獲得抗體效價，在進行如實例3中所述之血球凝 124989.doc -56 - 200825099 集抑制（HI)測試之前，將所得血清在4°C下儲存。 3.疫苗及激發病毒：獨立評價四種不同調配物： 1. 習知油乳液H5HA Mut k+:基於Boehringer Ingelheim Vetmedica程序將H5 HA mutk+抗原調配於油乳液（弗氏不完全佐劑（Freund incomplete adjuvant))中。 2. Seppic H5HA Mut k+ ··基於供應商建議，用非習知之佐劑（ISA 206，W/0/W，獲自 Seppic)調配 H5 HA mutk+抗原。 3. H5HA 習知油乳液：基於 Boehringer Ingelheim Vetmedica程序將H5 HA抗原調配於油乳液（弗氏不完全佐劑）中。 4. Seppic H5HA :基於供應商建議，用非習知之佐劑 (ISA 206，W/0/W，獲自 Seppic)調配H5 HA抗原。將禽流感Boehringer Ingelheim Vetmedica油乳液疫苗用作對照 Volvac® AI(Boehringer Ingelheim Vetmedica， Mexico) 〇藉由鼻内途徑，每隻鳥用〇·2 ml含有1 0 6 7 CEID之H5N2 激發病毒接種來於經接種及未經接種之雞體内進行激發。激發後，記錄病徵及死亡率。接種後第十日，根據動物實驗程序將所有存活雞施以無痛致死術。 124989.doc -57- 200825099 4.結果：結果描述於下表中： f 1日齡接種配方接種後第31日激發死亡數死亡率％ Seppic H5HA Mutk+ 8/25 32% 習知油乳液 H5HAMutk+ 0/24 0% Seppic H5HA 17/25 68% H5HA習知油乳液 4/25 16% VolvacAIKV 陰性對照 10/10 100% 10日齡接種接種後第32日激發死亡數死亡率％ 0/20 0% 0/20 0% 7/19 36.8% 2/20 10% 0/14 0% 14/14 100%疫苗 Vaccines 5〇1 to 514 as described in Example 2 were used for pig vaccination studies. 4 · Hemagglutinin inhibition test Pigs were inoculated with a prototype containing Η5 〇 on the 21st and 21st. Pig serum was collected on the 21st, 21st, and 35th days for evaluation by hemagglutination inhibition (HI) assay. A m assay is performed to detect the presence of purine-specific antibodies. Heterologous H5N2 virus (A/chicken/Mexico/232/94) was used in the HI assay at a concentration of four hemagglutination units [4 units]. Subsequently, serial two-fold serum dilutions in PB S were mixed with an equal volume (25 μM) of virus containing 4 HA units in a bottom-shaped microtiter plate and incubated at room temperature (about 25. For 30 minutes, chicken red blood cells at a concentration of 0.5% in PBS were added to the wells containing serum-virus and incubated for 40 minutes at room temperature. The HI titer was determined by the reciprocal of the highest serum dilution at which hemagglutination inhibition was observed. 5. Results The HI test used the Mexican government's legal H5N1 antigen (A/chicken/Mexico/232/94) [4 HA units], and the vaccination schedule was lxl mL on days 0 and 21. 124989.doc -54- 200825099 HI Potency No. 0, No. 21, No. 35, 501 H5-Emulsigen 0 0 4 502 H5-Emulsigen-D 0 0 4 503 H5 -Polygen 0 0 0 504 H5-Emulsigen-P 0 0 2 505 H5-Carbigen 0 0 4 506 H5-Emulsigen-75 0 0 16 507 H5ISA70 0 0 16 508 H5 K+-Emulsigen 0 0 128 509 H5 K+-Emulsigen-D 0 0 64 510 H5 K+-Polygen 0 0 16 511 H5 K+-Emulsigen-P 0 0 0 512 H5 K+-Carbigen 0 0 0 513 H5 K+-Emulsigen-75 0 0 16 514 H5K+-ISA70 0 4 32 Control no 0 0 0 BIV H5 (derived from influenza A virus (A / Duck/China/E3 19-2/03 (Η5Ν1)) BIV H5 K+ (mutated BIV H5, including S120N, D155N, S223N, and 328K+ added) The results demonstrate that most vaccine compositions elicit an immune response in vaccinated pigs. In particular, most vaccine compositions cause seroconversion, which means that most vaccinated pigs produce antibodies specific for the avian influenza virus used in the HI assay. In summary, the results clearly and unambiguously demonstrate that the inventive concept is extremely effective. By vaccinating pigs with the relevant antigens of avian influenza virus, the risk of widespread epidemic infection of avian influenza viruses (pathogens of the first species) by pigs (animals of the second species) is greatly reduced. According to this concept of vaccination, the infectivity and compliance of avian influenza viruses to mammals, including humans, is greatly reduced. Pigs are one of the most important hosts for avian pathogens, including avian influenza viruses. If the virus replicates in pigs and thus the risk of avian influenza compliance to pigs is greatly reduced and controlled, the risk of any compliance with avian influenza virus 124989.doc -55- 200825099 is also greatly reduced. In cases where administration of the antigen produces a lower HI titer (meaning a titer of less than 30), further enhancement of the antigen is required to further improve the HI titer and enhance immune protection in the vaccinated pig. Therefore, low titers do not mean that protection is not available, and it only teaches that it seems to need to be further strengthened to improve the immune response. The immunological assay can be measured in the vaccinated pigs to prove that the present invention, which is the basis of the present invention, is extremely effective. In other words, the experiments provided herein clearly and unambiguously demonstrate that the inventive concept can be effective.实例 Example 4 Inoculation of birds to protect against avian influenza 1. Introduction The purpose of this study was to determine an experimental vaccine containing a crude extract of recombinant H5nlutk + hemagglutinin (H5 HA mutk+) antigen to induce agglutination in chickens. The ability to suppress (HI). In addition, conventional recombinant antigen (H5 HA) and inactivated vaccine Volvac® AI (Boehringer Ingelheim ^ Vetmedica, Mexico) were used for the control. In addition, various adjuvants were evaluated using the H5 HA antigen. 2. Study design: SPF birds (15-25) were independently inoculated with subcutaneous route in the neck and back with 实验5 different experimental vaccines at the age of 1 or 1 ;; all birds were kept during the experiment. In the isolator. Unlimited feeding and water. Excitation was performed on the 31st or 32nd day after inoculation with the H5N2 southern pathogenic avian influenza virus strain. Serum samples were obtained by exsanguination from the jugular vein on the 15th and 30th day after inoculation. To obtain antibody titers, the resulting serum was stored at 4 °C prior to performing the hemagglutination 124989.doc -56 - 200825099 set inhibition (HI) test as described in Example 3. 3. Vaccine and challenge virus: Four different formulations were independently evaluated: 1. Conventional oil emulsion H5HA Mut k+: H5 HA mutk+ antigen was formulated on oil emulsion based on Boehringer Ingelheim Vetmedica program (Freund incomplete adjuvant) ))in. 2. Seppic H5HA Mut k+ · Based on the supplier's recommendations, the H5 HA mutk+ antigen was formulated with a non-known adjuvant (ISA 206, W/0/W, obtained from Seppic). 3. H5HA conventional oil emulsion: The H5 HA antigen was formulated in an oil emulsion (Freund's incomplete adjuvant) based on the Boehringer Ingelheim Vetmedica program. 4. Seppic H5HA: H5 HA antigen was formulated with a non-known adjuvant (ISA 206, W/0/W, obtained from Seppic) based on supplier recommendations. The avian influenza Boehringer Ingelheim Vetmedica oil emulsion vaccine was used as a control Volvac® AI (Boehringer Ingelheim Vetmedica, Mexico). Each bird was inoculated with 2 ml of H5N2 challenge virus containing 1 0 7 7 CEID by intranasal route. Excited in vaccinated and unvaccinated chickens. After the challenge, the symptoms and mortality were recorded. On the tenth day after inoculation, all surviving chickens were subjected to painless death according to the animal test procedure. 124989.doc -57- 200825099 4. Results: The results are described in the following table: f The death rate of death on the 31st day after inoculation of the 1 day old inoculation formula Seppic H5HA Mutk+ 8/25 32% Conventional oil emulsion H5HAMutk+ 0/ 24 0% Seppic H5HA 17/25 68% H5HA conventional oil emulsion 4/25 16% VolvacAIKV Negative control 10/10 100% 10 days old vaccination on the 32nd day after the death rate of death% 0/20 0% 0/ 20 0% 7/19 36.8% 2/20 10% 0/14 0% 14/14 100%

1曰齡接種(0.5 ml) 疫苗配方接種後第 30 日 HI 效價 (MG Log2) 激發後保護％ Seppic H5HA Mut k+ 0.56 68 習知油乳液 H5HAMutk+ 2.59 100 Seppic H5HA 0.18 32 H5HA習知油乳液 0.7 84 Volvac AIKV 陰性對照 10曰齡接種(0.5 ml) 接種後第 30日HI效價(MG Log2) 激發後保護 % 2.5 100 4.3 100 1.3 63.2 1.6 100 8.8 100 0 01 year old vaccination (0.5 ml) 30 days after vaccine formulation HI potency (MG Log2) Protection after challenge % Seppic H5HA Mut k+ 0.56 68 Conventional oil emulsion H5HAMutk+ 2.59 100 Seppic H5HA 0.18 32 H5HA conventional oil emulsion 0.7 84 Volvac AIKV Negative Control 10 曰 Inoculation (0.5 ml) HI titer (MG Log2) 30 days after inoculation Protection % 2.5 100 4.3 100 1.3 63.2 1.6 100 8.8 100 0 0

根據OIE標準將陽性血清效價以log2 4考量。基於此標準，血清學結果為陰性，但與基線相比，觀測到某些陽性值。在1日齡或10日齡接種之鳥體内觀測到用油佐劑及 H5HA Mut k+抗原調配之疫苗具有最佳的血清學效價。經觀測，用Seppic及H5HA抗原調配之原型具有最低的血清學效價。在激發研究中觀測到，疫苗原型，尤其H5HA 124989.doc -58- 200825099Positive serum titers were considered in log 2 4 according to the OIE criteria. Based on this criteria, serological results were negative, but some positive values were observed compared to baseline. Vaccines formulated with oil adjuvants and H5HA Mut k+ antigens were observed to have optimal serological titers in birds vaccinated at 1 day or 10 days of age. The prototypes formulated with Seppic and H5HA antigens have been observed to have the lowest serological titers. Vaccine prototypes observed in the challenge study, especially H5HA 124989.doc -58- 200825099

Mut k+抗原調配之習知油乳液疫苗，賦予保護作用。研究中觀測到Seppic H5HA具有最低的保護作用，死為68°/。。相比之下，與在一日齡接種之鳥相比，在10 種之鳥體内觀測到最高的血清學效價。激發亡率曰接 124989.doc 59- 200825099 序列表 <110 > 美商百靈佳殷格輸家畜藥品公司 <120 >新穎H5蛋白質、編碼彼等之核酸分子及載體及其醫藥用途 <130 > Case 1-2150 <140 > 096140489 <141 > 2007-10-26 <150> 60/863142 <151> 2006-10-27 <160> 6 <170> Patentln version 3.3 <210> 1 <211> 551A conventional oil emulsion vaccine formulated with Mut k+ antigen confers protection. Seppic H5HA was observed to have the lowest protection in the study, with a death of 68°/. . In contrast, the highest serological titers were observed in 10 species of birds compared to birds vaccinated at one day old. Excited death rate 124124989.doc 59- 200825099 Sequence Listing <110 > American Bailingjia Ingrid Livestock Pharmaceutical Company <120 > Novel H5 protein, nucleic acid molecule encoding the same and its use <;130> Case 1-2150 <140 > 096140489 <141 > 2007-10-26 <150> 60/863142 <151> 2006-10-27 <160> 6 <170> Version 3.3 <210> 1 <211> 551

C <212> PRT <213〉禽流感病毒 <400> 1C <212> PRT <213> Avian Influenza Virus <400> 1

Asp Gin lie Cys lie Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 15 10 15Asp Gin lie Cys lie Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 15 10 15

Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 20 25 30Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 20 25 30

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35 40 45Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35 40 45

Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60

Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr lie Val 65 70 75 80Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr lie Val 65 70 75 80

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg 工le Asn His Phe Glu 100 105 110Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg l Asn His Phe Glu 100 105 110

Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 115 120 125Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 115 120 125

Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser Ser Ser Phe Phe 130 135 140Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser Ser Ser Phe Phe 130 135 140

Arg Asn Val Val Trp Leu lie Lys Lys Asn Asp Ala Tyr Pro Thr lie 145 150 155 160Arg Asn Val Val Trp Leu lie Lys Lys Asn Asp Ala Tyr Pro Thr lie 145 150 155 160

Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 165 170 175Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 165 170 175

Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin 124989.doc 200825099 180 185 190 Asn Pro Thr 195 Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 200 205 Leu Val Pro 210 Lys 工le Ala Thr Arg Ser Lys Val Asn Gly Gin Ser Gly 215 220 Arg Met Asp 225 Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 230 235 240 ^ Phe Glu Ser Asn Gly Asn Phe 工le Ala Pro Glu Tyr Ala Tyr Lys lie 245 250 255 , Val Lys Lys Gly Asp Ser Ala 工le Met Lys Ser Glu Val Glu Tyr Gly 260 265 270 Asn Cys Asn 27 5 Γ 、 Met Pro Phe 290 Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 280 285 His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 295 300 Tyr Val Lys 305 Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 310 315 320 Pro Gin Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala lie Ala 325 330 335 Gly Phe lie Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly 340 345 350 Tyr His His 355 Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys Glu 360 365 Ser Thr Gin 370 Lys Ala lie Asp Gly Val Thr Asn Lys Val Asn Ser lie 375 380 () lie Asp Lys 385 Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn 390 395 400 Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 405 410 415 Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu 420 425 430 Asn Glu Arg 435 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr 440 445 Asp Lys Val 450 Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 455 460 Gly Cys Phe 465 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser 470 475 480Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin 124989.doc 200825099 180 185 190 Asn Pro Thr 195 Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 200 205 Leu Val Pro 210 Lys Thr Arg Ser Lys Val Asn Gly Gin Ser Gly 215 220 Arg Met Asp 225 Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 230 235 240 ^ Phe Glu Ser Asn Gly Asn Phe work le Ala Pro Glu Tyr Ala Tyr Lys lie 245 250 255 , Val Lys Lys Gly Asp Ser Ala L Le Met Lys Ser Glu Val Glu Tyr Gly 260 265 270 Asn Cys Asn 27 5 Γ , Met Pro Phe 290 Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 280 285 His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 295 300 Tyr Val Lys 305 Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 310 315 320 Pro Gin Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala lie Ala 325 330 335 Gly Phe lie Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly 340 345 350 Tyr His His 355 Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys Glu 360 365 Ser Thr Gin 370 Lys Ala lie As p Gly Val Thr Asn Lys Val Asn Ser lie 375 380 () lie Asp Lys 385 Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn 390 395 400 Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 405 410 415 Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu 420 425 430 Asn Glu Arg 435 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr 440 445 Asp Lys Val 450 Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 455 460 Gly Cys Phe 465 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser 470 475 480

Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg 485 490 495 -2- 124989.doc 200825099Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg 485 490 495 -2- 124989.doc 200825099

Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly Thr 500 505 510Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly Thr 500 505 510

Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 515 520 525Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 515 520 525

Ala 工le Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser 530 535 540Ala work le Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser 530 535 540

Leu Gin Cys Arg 工le Cys lie 545 550 >>>> 0 12 3 1 1—I iI i—I 2 2 2 2 < < < < 2 567Leu Gin Cys Arg worker le Cys lie 545 550 >>>> 0 12 3 1 1—I iI i—I 2 2 2 2 <<<< 2 567

PRT 禽流感病毒 <400> 2PRT Avian Influenza Virus <400> 2

Met Glu Lys Thr Val Leu Leu Leu Ala lie Val Ser Leu Val Lys Ser 15 10 15Met Glu Lys Thr Val Leu Leu Leu Ala lie Val Ser Leu Val Lys Ser 15 10 15

Asp Gin lie Cys lie Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 20 25 30Asp Gin lie Cys lie Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 20 25 30

Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 35 40 45Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 35 40 45

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60

Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80

Pro Met Cys Asp Glu Phe 工le Asn Val Pro Glu Trp Ser Tyr 工le Val 85 90 95Pro Met Cys Asp Glu Phe work Le Asn Val Pro Glu Trp Ser Tyr work le Val 85 90 95

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100 105 110Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100 105 110

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie Asn His Phe Glu 115 120 125Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie Asn His Phe Glu 115 120 125

Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 130 135 140Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 130 135 140

Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser Ser Ser Phe Phe 145 150 155 160Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser Ser Ser Phe 145 150 155 160

Arg Asn Val Val Trp Leu lie Lys Lys Asn Asp Ala Tyr Pro Thr lie 165 170 175Arg Asn Val Val Trp Leu lie Lys Lys Asn Asp Ala Tyr Pro Thr lie 165 170 175

Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 180 185 190Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 180 185 190

Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin 124989.doc 200825099 195 200 205Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin 124989.doc 200825099 195 200 205

Asη Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 210 215 220Asη Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 210 215 220

Leu Val Pro Lys 工le Ala Thr Arg Ser Lys Val Asn Gly Gin Ser Gly 225 230 235 240Leu Val Pro Lys work le Ala Thr Arg Ser Lys Val Asn Gly Gin Ser Gly 225 230 235 240

Arg Met Asp Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255Arg Met Asp Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255

Phe Glu Ser Asn Gly Asn Phe 工le Ala Pro Glu Tyr Ala Tyr Lys lie 260 265 270Phe Glu Ser Asn Gly Asn Phe work le Ala Pro Glu Tyr Ala Tyr Lys lie 260 265 270

Val Lys Lys Gly Asp Ser Ala 工le Met Lys Ser Glu Val Glu Tyr Gly 275 280 285Val Lys Lys Gly Asp Ser Ala L Le Met Lys Ser Glu Val Glu Tyr Gly 275 280 285

Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300 ΟAsn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300 Ο

Met Pro Phe His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 305 310 315 320Met Pro Phe His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 305 310 315 320

Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335

Pro Gin Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala lie Ala 340 345 350Pro Gin Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala lie Ala 340 345 350

Gly Phe lie Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly 355 360 365Gly Phe lie Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr Gly 355 360 365

Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys Glu 370 375 380Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys Glu 370 375 380

Ser Thr Gin Lys Ala lie Asp Gly Val Thr Asn Lys Val Asn Ser lie 385 390 395 400 lie Asp Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn 405 410 415Ser Thr Gin Lys Ala lie Asp Gly Val Thr Asn Lys Val Asn Ser lie 385 390 395 400 lie Asp Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe Asn 405 410 415

Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 420 425 430Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp Gly 420 425 430

Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu 435 440 445Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu 435 440 445

Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr 450 455 460Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr 450 455 460

Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465 470 475 480Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465 470 475 480

Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser 485 490 495Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser 485 490 495

Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg 500 505 510 -4- 124989.doc 200825099Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala Arg 500 505 510 -4- 124989.doc 200825099

Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly Thr 515 520 525Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly Thr 515 520 525

Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 530 535 540Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu 530 535 540

Ala 工le Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser 545 550 555 560Ala work le Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser 545 550 555 560

Leu Gin Cys Arg lie Cys lie 565 <210> 3 <211> 568 <212> PRT <213〉禽流感病毒Leu Gin Cys Arg lie Cys lie 565 <210> 3 <211> 568 <212> PRT <213> Avian Influenza Virus

<400> 3<400> 3

Met Glu Lys 工le Val Leu Leu Phe Ala 工le Val Ser Leu Val Lys Ser 15 10 15Met Glu Lys work le Val Leu Leu Phe Ala work le Val Ser Leu Val Lys Ser 15 10 15

Asp Thr 工le Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 35 40 45Asp Thr work le Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp lie 35 40 45

Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 7 0 75 80Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 7 0 75 80

Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr lie Val 85 90 95Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr lie Val 85 90 95

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110

Lys lie Gin lie lie Pro Lys Asn Ser Trp Ser Ser His Glu Ala Ser 130 135 140Lys lie Gin lie lie Pro Lys Asn Ser Trp Ser Ser His Glu Ala Ser 130 135 140

Leu Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Lys Ser Ser Phe Phe 145 150 155 160Leu Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Lys Ser Ser Phe Phe 145 150 155 160

Arg Asn Val Val Trp Leu lie Lys Lys Asn Asn Ala Tyr Pro Thr lie 165 170 175Arg Asn Val Val Trp Leu lie Lys Lys Asn Asn Ala Tyr Pro Thr lie 165 170 175

Asn Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 210 215 220Asn Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr Leu Asn Gin Arg 210 215 220

Leu Val Pro Lys 工le Ala Thr Arg Ser Lys Val Asn Gly Gin Asn Gly 225 230 235 240Leu Val Pro Lys work le Ala Thr Arg Ser Lys Val Asn Gly Gin Asn Gly 225 230 235 240

Arg Met Glu Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255Arg Met Glu Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255

Val Lys Lys Gly Asp Ser Ala 工le Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285Val Lys Lys Gly Asp Ser Ala L Le Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285

Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300

Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335

Pro Gin Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala lie 340 345 350Pro Gin Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala lie 340 345 350

Ala Gly Phe lie Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr 355 360 365Ala Gly Phe lie Glu Gly Gly Trp Gin Gly Met Val Asp Gly Trp Tyr 355 360 365

Gly Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380Gly Tyr His His Ser Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380

Glu Ser Thr Gin Lys Ala lie Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 lie 工le Asp Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe 405 410 415Glu Ser Thr Gin Lys Ala lie Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 lie work Le Asp Lys Met Asn Thr Gin Phe Glu Ala Val Gly Arg Glu Phe 405 410 415

Asn Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430Asn Asn Leu Glu Arg Arg lie Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430

Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 435 440 445Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 435 440 445

Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu 450 455 460Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu 450 455 460

Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480Tyr Asp Lys Val Arg Leu Gin Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480

Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495

Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala 500 505 510 -6- 124989.doc 200825099Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala 500 505 510 -6- 124989.doc 200825099

Arg Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly 515 520 525 Thr Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu 545 Ala lie Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 550 555 560 Ser Leu Gin Cys Arg lie Cys lie 565 <210> 4 <211> 568 <212> PRT <213> 禽流感病毒 <400> 4 \ Met 1 Glu Lys Thr Val Leu Leu Leu Ala 工le Val Ser Leu Val Lys Ser 5 10 15 Asp Gin 工le Cys lie Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 20 25 30 Asp Thr He Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp He 35 40 45 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro 65 Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 70 75 80 Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp Ser Tyr lie Val 85 90 95 O Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie Asn His Phe Glu 115 120 125 Lys lie Gin lie lie Pro Lys Asn Ser Trp Ser Asp His Glu Ala Ser 130 135 140 ' Ser 145 Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser Ser Ser Phe Phe 150 155 160 Arg Asn Val Val Trp Leu lie Lys Lys Asn Asn Ala Tyr Pro Thr lie 165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 180 185 190Arg Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly 515 520 525 Thr Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu 545 Ala lie Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 550 555 560 Ser Leu Gin Cys Arg lie Cys lie 565 <210> 4 <211> 568 <212> PRT <213> Avian Influenza Virus <400> 4 \ Met 1 Glu Lys Thr Val Leu Leu Leu Ala Le Val Ser Leu Val Lys Ser 5 10 15 Asp Gin L. Cys lie Gly Tyr His Ala Asn Asn Ser Thr Glu Gin Val 20 25 30 Asp Thr He Met Glu Lys Asn Val Thr Val Thr His Ala Gin Asp He 35 40 45 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro 65 Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 70 75 80 Pro Met Cys Asp Glu Phe Lie Asn Val Pro Glu Trp Ser Tyr lie Val 85 90 95 O Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie Asn His Phe Glu 115 120 125 Lys lie Gin lie lie Pro Lys Asn Ser Trp Ser Asp His Glu Ala Ser 130 135 140 ' Ser 145 Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser Ser Ser Phe Phe 150 155 160 Arg Asn Val Val Trp Leu lie Lys Lys Asn Asn Ala Tyr Pro Thr lie 165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu Leu Val Leu Trp 180 185 190

Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin -7- 124989.doc 200825099 195 200 205Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr Arg Leu Tyr Gin -7- 124989.doc 200825099 195 200 205

Arg Met Asp Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255 ^ Phe Glu Ser Asn Gly Asn Phe lie Ala Pro Glu Tyr Ala Tyr Lys lie ^ 260 265 270 . Val Lys Lys Gly Asp Ser Ala lie Met Lys Ser Glu Val Glu Tyr Gly 275 280 285Arg Met Asp Phe Phe Trp Thr lie Leu Lys Pro Asn Asp Ala lie Asn 245 250 255 ^ Phe Glu Ser Asn Gly Asn Phe lie Ala Pro Glu Tyr Ala Tyr Lys lie ^ 260 265 270 . Val Lys Lys Gly Asp Ser Ala lie Met Lys Ser Glu Val Glu Tyr Gly 275 280 285

Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300 〇 i Met Pro Phe His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 305 310 315 320Asn Cys Asn Thr Lys Cys Gin Thr Pro Met Gly Ala lie Asn Ser Ser 290 295 300 〇 i Met Pro Phe His Asn lie His Pro Leu Thr lie Gly Glu Cys Pro Lys 305 310 315 320

Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala 500 505 510 124989.doc 200825099Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gin Tyr Ser Glu Glu Ala 500 505 510 124989.doc 200825099

Arg Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly 515 520 525Arg Leu Lys Arg Glu Glu lie Ser Gly Val Lys Leu Glu Ser lie Gly 515 520 525

Thr Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540Thr Tyr Gin lie Leu Ser lie Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540

Leu Ala 工le Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555 560Leu Ala work le Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555 560

Ser Leu Gin Cys Arg lie Cys lie 565 <210> 5 <211> 253 <212> PRT <213> 禽流感病毒 <400> 5Ser Leu Gin Cys Arg lie Cys lie 565 <210> 5 <211> 253 <212> PRT <213> Avian Influenza Virus <400>

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His Ala Asn Asn Trp Thr Glu Gin Val Asp Thr lie Met Glu Lys Asn 15 10 15His Ala Asn Asn Trp Thr Glu Gin Val Asp Thr lie Met Glu Lys Asn 15 10 15

Val Thr Val Thr His Ala Gin Asp lie Leu Glu Lys Thr His Asn Gly 20 25 30Val Thr Val Thr His Ala Gin Asp lie Leu Glu Lys Thr His Asn Gly 20 25 30

Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu lie Leu Arg Asp Cys 35 40 45Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu lie Leu Arg Asp Cys 35 40 45

Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe lie 50 55 60Ser Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe lie 50 55 60

Asn Val Pro Glu Trp Ser Tyr lie Val Glu Lys Ala Asn Pro Ala Asn 65 70 75 80Asn Val Pro Glu Trp Ser Tyr lie Val Glu Lys Ala Asn Pro Ala Asn 65 70 75 80

Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu Glu Leu Lys His 85 90 95Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu Glu Leu Lys His 85 90 95

Leu Leu Ser Arg 工le Asn His Phe Glu Lys lie Gin lie lie Pro Lys 100 105 110Leu Leu Ser Arg work le Asn His Phe Glu Lys lie Gin lie lie Pro Lys 100 105 110

Asn Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys 115 120 125Asn Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val Ser Ser Ala Cys 115 120 125

Pro Tyr Gin Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu lie 130 135 140Pro Tyr Gin Gly Lys Ser Ser Phe Phe Arg Asn Val Val Trp Leu lie 130 135 140

Lys Lys Asn Asn Ala Tyr Pro Thr lie Lys Arg Ser Tyr Asn Asn Thr 145 150 155 160Lys Lys Asn Asn Ala Tyr Pro Thr lie Lys Arg Ser Tyr Asn Asn Thr 145 150 155 160

Asn Gin Glu Asp Leu Leu Val Leu Trp Gly lie His His Pro Asn Asp 165 170 175Asn Gin Glu Asp Leu Leu Val Leu Trp Gly lie His His Pro Asn Asp 165 170 175

Ala Ala Glu Gin Thr Arg Leu Tyr Gin Asn Pro Thr Thr Tyr lie Ser 180 185 190Ala Ala Glu Gin Thr Arg Leu Tyr Gin Asn Pro Thr Thr Tyr lie Ser 180 185 190

Val Gly Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Lys lie Ala Thr 124989.doc 200825099 195 200 205 Arg Ser Lys 210 Val Asn Gly Gin Asn Gly Arg Met Glu Phe Phe Trp Thr 215 220 lie Leu Lys 225 Pro Asn Asp Ala lie Asn Phe Glu Ser Asn Gly Asn Phe 230 235 240 lie Ala Pro Glu Tyr Ala Tyr Lys lie Val Lys Lys Gly Asp Ser Ala 245 250 255 lie Met Lys Ser Glu Leu Glu 260 <210> 6 <211> 290 <212> PRT <213> 禽流感病毒 <400> 6Val Gly Thr Ser Thr Leu Asn Gin Arg Leu Val Pro Lys lie Ala Thr 124989.doc 200825099 195 200 205 Arg Ser Lys 210 Val Asn Gly Gin Asn Gly Arg Met Glu Phe Phe Trp Thr 215 220 lie Leu Lys 225 Pro Asn Asp Ala Lie Asn Phe Glu Ser Asn Gly Asn Phe 230 235 240 lie Ala Pro Glu Tyr Ala Tyr Lys lie Val Lys Lys Gly Asp Ser Ala 245 250 255 lie Met Lys Ser Glu Leu Glu 260 <210> 6 <211> 290 &lt ;212> PRT <213> Avian Influenza Virus <400> 6

Gly Ser Ala 1 Thr Met Glu Lys Thr Val Leu Leu Leu Ala lie Val Ser 5 10 15 Leu Val Lys Ser Asp Gin lie Cys lie Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Glu Gin 35 Val Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His 40 45 Ala Gin Asp 50 lie Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu 55 60 Asp Gly Val 65 Lys Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp 7 0 75 80 Leu Leu Gly c, Asn Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp 85 90 95 Ser Tyr lie Val Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro 100 105 110 Gly Asn Phe - 115 Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie 120 125 Asn His Phe ^ 130 Glu Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp 135 140 His Glu Ala 145 Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser 150 155 160 Ser Ser Phe Phe Arg Asn Val Val Trp Leu lie Lys Lys Asn Asp Ala 165 170 175 Tyr Pro Thr lie Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu 180 185 190 124989.doc -10- 200825099Gly Ser Ala 1 Thr Met Glu Lys Thr Val Leu Leu Leu Ala lie Val Ser 5 10 15 Leu Val Lys Ser Asp Gin lie Cys lie Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Glu Gin 35 Val Asp Thr lie Met Glu Lys Asn Val Thr Val Thr His 40 45 Ala Gin Asp 50 lie Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu 55 60 Asp Gly Val 65 Lys Pro Leu lie Leu Arg Asp Cys Ser Val Ala Gly Trp 7 0 75 80 Leu Leu Gly c, Asn Pro Met Cys Asp Glu Phe lie Asn Val Pro Glu Trp 85 90 95 Ser Tyr lie Val Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro 100 105 110 Gly Asn Phe - 115 Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg lie 120 125 Asn His Phe ^ 130 Glu Lys lie Gin lie lie Pro Lys Ser Ser Trp Ser Asp 135 140 His Glu Ala 145 Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Gin Gly Ser 150 155 160 Ser Ser Phe Phe Arg Asn Val Val Trp Leu lie Lys Lys Asn Asp Ala 165 170 175 Tyr Pro Thr lie Lys Arg Ser Tyr Asn Asn Thr Asn Gin Glu Asp Leu 180 185 190 124989.doc -10- 200825099

Leu Val Leu Trp Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr 195 200 205Leu Val Leu Trp Gly lie His His Pro Asn Asp Ala Ala Glu Gin Thr 195 200 205

Arg Leu Tyr Gin Asn Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr 210 215 220Arg Leu Tyr Gin Asn Pro Thr Thr Tyr lie Ser Val Gly Thr Ser Thr 210 215 220

Leu Asn Gin Arg Leu Val Pro Lys lie Ala Thr Arg Ser Lys Val Asn 225 230 235 240 lie Leu Lys Pro Asn 255 lie Ala Pro Glu Tyr 270 lie Met Lys Ser Glu 285Leu Asn Gin Arg Leu Val Pro Lys lie Ala Thr Arg Ser Lys Val Asn 225 230 235 240 lie Leu Lys Pro Asn 255 lie Ala Pro Glu Tyr 270 lie Met Lys Ser Glu 285

Gly Gin Ser Gly Arg Met Asp Phe Phe Trp Thr 245 250Gly Gin Ser Gly Arg Met Asp Phe Phe Trp Thr 245 250

Asp Ala lie Asn Phe Glu Ser Asn Gly Asn Phe 260 265Asp Ala lie Asn Phe Glu Ser Asn Gly Asn Phe 260 265

Ala Tyr Lys 工le Val Lys Lys Gly Asp Ser Ala 275 280Ala Tyr Lys work le Val Lys Lys Gly Asp Ser Ala 275 280

Val Glu 290 -11 - 124989.docVal Glu 290 -11 - 124989.doc

Claims

200825099 十、申請專利範圍： 1. 一種流感病毒之H5蛋白，其中該H5蛋白具有胺基酸 223N及修飾體328K+，其中該H5蛋白之胺基酸位置之編號係指如SEQ ID NO: 1中所例示性指定之胺基酸位置且其中該修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸（K+)。 2. 如請求項1之H5蛋白，其中該H5蛋白具有胺基酸94N。 3. 如請求項1或2之H5蛋白，其中該H5蛋白具有胺基酸 f 120N。 4. 如請求項1或2之H5蛋白，其中該H5蛋白具有胺基酸 155N 〇 5. 如請求項1或2之H5蛋白，其中該H5蛋白具有選自由以下各者組成之群的以下胺基酸團中之一或多者： a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG ϋ d. aa 138-140: GSS e. aa 226-228: MDF - f. aa 270-272: EVE g. aa 309-31 1: NKL。 6. 如請求項1或2之H5蛋白，其中該H5蛋白包含肽，該肽包含·· i· SEQ ID NO:4、SEQ ID NO:5 或 SEQ ID NO:6 之胺基酸序列；或 124989.doc 200825099 11·具有與1)之夕肽之至少85%序列同源性且在標準血球凝 …集素抑制檢定中包含血球凝集素抑制的任何狀；或 m. !)或ii)之多肽之任何部分，其包含丨）或⑴之任何該等狀之至少8個晚鄰胺基酸，且其中任何該狀在標準血球凝集素抑制檢定中包含金球凝集素抑制；或 IV· 〇、u)或出）之任何肽，其具有以下胺基酸中之— 者：36T、36K、83A、83T、83D、86A、86v、、 120S、155S、156A、156T、189R、職、2UK、 212R、212E、263A或 263T ;或 v. i)、ii)、iii)或iv)之任何肽，其具有選自由以下各者組成之群的以下胺基酸團中之一或多者： a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS ) -aa 226-228: MDF f· aa 270-272: EVE g. aa 309-31 1: NKL 〇 7.如請求項1或2之H5蛋白，其中該H5蛋白係來源於禽流感病毒。 8·如請求項1或2之H5蛋白，其中該H5蛋白包含SEQ ID NCh4之胺基酸序列。 9· 一種核酸分子，其中該核酸分子編碼如請求項1至8中任一項之H5蛋白。 124989.doc 200825099 i〇. 一種載體，其包含如請求項9之核酸分子 1 1 · 一種疫苗，其包含：如請求項9之核酸分 a·如請求項1至8中任一項之H5蛋白子或如請求項10之載體；及 b·醫藥學上可接受之載劑及/或賦形劑。 12 ·如請求項11之疫苗 13 ·如請求項12之疫苗劑0 ，其中該賦形劑為一或多種佐劑。，其中該等佐劑為基於Emulsigen之佐200825099 X. Patent Application Range: 1. An H5 protein of influenza virus, wherein the H5 protein has an amino acid 223N and a modification 328K+, wherein the amino acid position of the H5 protein is numbered as in SEQ ID NO: 1. The exemplarily specified amino acid position and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. 2. The H5 protein of claim 1, wherein the H5 protein has an amino acid 94N. 3. The H5 protein of claim 1 or 2, wherein the H5 protein has an amino acid f 120N. 4. The H5 protein of claim 1 or 2, wherein the H5 protein has an amino acid 155N 〇 5. The H5 protein of claim 1 or 2, wherein the H5 protein has the following amine selected from the group consisting of: One or more of the acid groups: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG ϋ d. aa 138-140: GSS e. aa 226-228: MDF - f. aa 270-272: EVE g. aa 309-31 1: NKL. 6. The H5 protein of claim 1 or 2, wherein the H5 protein comprises a peptide comprising an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; 124989.doc 200825099 11·any having at least 85% sequence homology to the peptide of 1) and containing hemagglutinin inhibition in a standard hemagglutination inhibitor; or m. !) or ii) Any portion of a polypeptide comprising at least 8 of the ternary amino acids of any of the 丨) or (1), and wherein any of the forms comprises a lectin inhibition in a standard hemagglutinin inhibition assay; or IV·〇 , u) or any of the peptides of the following amino acids having the following amino acids: 36T, 36K, 83A, 83T, 83D, 86A, 86v, 120S, 155S, 156A, 156T, 189R, occupation, 2UK, 212R, 212E, 263A or 263T; or any peptide of v. i), ii), iii) or iv) having one or more of the following amino acid groups selected from the group consisting of: a Aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS ) -aa 226-228: MDF f· aa 270-272: EVE g. aa 309-31 1: NKL 〇 7. The H5 protein of claim 1 or 2, wherein the H5 protein is derived from an avian influenza virus. 8. The H5 protein of claim 1 or 2, wherein the H5 protein comprises the amino acid sequence of SEQ ID NCh4. A nucleic acid molecule, wherein the nucleic acid molecule encodes the H5 protein of any one of claims 1 to 8. 124989.doc 200825099 i. A vector comprising the nucleic acid molecule of claim 9 1 · a vaccine comprising: the nucleic acid according to claim 9 a. The H5 protein according to any one of claims 1 to 8 Or a carrier as claimed in claim 10; and b. a pharmaceutically acceptable carrier and/or excipient. 12. Vaccine according to claim 11 13. Vaccine 0 according to claim 12, wherein the excipient is one or more adjuvants. , wherein the adjuvants are based on Emulsigen

14·如請求項^至13中任一項 1 貝t及田，其中該疫苗包含一或多種抗原。 15.如請求項14之疫苗’其中該其他抗原為禽類或哺乳動物病原體之抗原。 16·如請求項15之疫苗，纟中該其他抗原為流感病毒之出、 H7 或 H9。 17· —種製備如請求項1至8中任一項之出蛋白的方法，其中該方法包含以下步驟： a•分離或擴增編碼該H5蛋白之核酸； b.將該H5編碼核酸選殖於表現載體内； c·表現該H5蛋白。 18·如請求項17之方法，其中該表現載體為重組性桿狀病毒。 19.如請求項17或18之方法，其中該仍蛋白係表現於昆蟲細胞中。 20· —種製備包含如請求項is 8中任一項之H5蛋白之疫苗的 124989.doc 200825099 方法，其中該方法包含以下步驟： a•獲得如請求項1至8中任一項之H5蛋白； b·將步驟a)之該H5蛋白與醫藥學上可接受之載劑及/或賦形劑混合。 ^ 21· 一種製備包含如請求項9之H5核酸或如請求項1〇之載體 . 之疫苗的方法，其中該方法包含以下步驟： a·獲得如請求項9之H5核酸分子或如請求項1〇之載體； (、 b•將步驟a)之該H5核酸分子或載體與醫藥學上可接受之載劑及/或賦形劑混合。 22. —種如請求項丨至8中任一項之H5蛋白之用途，其係用作藥物。 23. —種如請求項9之核酸分子之用途，其係用作藥物。 24· —種如請求項1〇之載體之用途，其係用作藥物。 25 · —種如請求項丨丨至丨6中任一項之疫苗之用途，其係用作藥物。 26· 一種如請求項1至8中任一項之H5蛋白之用途，其係用於裝備供預防或治療由病毒性流感引起之感染之用的醫藥組合物。 27· —種如請求項9之核酸分子之用途，其係用於製備供預防或治療由病毒性流感引起之感染之用的醫藥組合物。 28· —種如請求項1〇之載體之用途，其係用於製備供預防或治療由病毒性流感引起之感染之用的醫藥組合物。 29.如明求項26至28中任一項之用途，其中該病毒流感感染係由禽、豬或人類流感病毒或其任何組合或混合引起。 124989.doc 200825099 3 0 · —種部分套組，其包含：如請求項9之核酸分 a.如睛求項1至8中任一項之H5蛋白子、如請求項ίο之載體或如請求項丨丨至“中任一項之疫苗；及 b·—包裝紙頁，其指示a)之該H5蛋白、核酸分子、載體或疫苗用於治療或預防由流感病毒引起之感染的使用。14. The method of any one of claims 1 to 13 wherein the vaccine comprises one or more antigens. 15. The vaccine of claim 14 wherein the other antigen is an antigen of avian or mammalian pathogen. 16. The vaccine of claim 15 wherein the other antigen is influenza virus, H7 or H9. The method of producing the protein of any one of claims 1 to 8, wherein the method comprises the steps of: a: isolating or amplifying a nucleic acid encoding the H5 protein; b. selecting the H5 encoding nucleic acid Within the expression vector; c. expressing the H5 protein. 18. The method of claim 17, wherein the expression vector is a recombinant baculovirus. 19. The method of claim 17 or 18, wherein the still protein line is expressed in insect cells. A method of preparing a vaccine comprising the H5 protein of any one of claims 8 to 2008, wherein the method comprises the steps of: a: obtaining the H5 protein according to any one of claims 1 to 8 b. Mixing the H5 protein of step a) with a pharmaceutically acceptable carrier and/or excipient. A method of preparing a vaccine comprising the H5 nucleic acid of claim 9 or the vector of claim 1 , wherein the method comprises the steps of: a obtaining the H5 nucleic acid molecule of claim 9 or as claimed in claim 1 (a) b. Mixing the H5 nucleic acid molecule or vector of step a) with a pharmaceutically acceptable carrier and/or excipient. 22. The use of the H5 protein of any one of claims 8 to 8 for use as a medicament. 23. Use of a nucleic acid molecule according to claim 9 for use as a medicament. 24. The use of a carrier as claimed in claim 1 is for use as a medicament. 25. The use of a vaccine according to any one of claims 丨丨6 to 6 for use as a medicament. The use of the H5 protein according to any one of claims 1 to 8, which is for use in a pharmaceutical composition for the prevention or treatment of an infection caused by viral influenza. 27. The use of a nucleic acid molecule according to claim 9 for the preparation of a pharmaceutical composition for the prevention or treatment of an infection caused by viral influenza. 28. The use of a carrier according to claim 1 for the preparation of a pharmaceutical composition for the prevention or treatment of an infection caused by viral influenza. The use of any one of the items 26 to 28, wherein the viral influenza infection is caused by avian, porcine or human influenza virus or any combination or mixture thereof. 124989.doc 200825099 3 0 - A partial kit comprising: a nucleic acid according to claim 9 a. an H5 protein according to any one of items 1 to 8, a vector such as a request ίο or as requested The use of the H5 protein, nucleic acid molecule, vector or vaccine of the vaccine of any one of the "vaccines of any of the vaccines; and b. - packaging sheets, indication a) for the treatment or prevention of infections caused by influenza viruses.

3 1 ·如請求項30之套組，其中該套組包含至少另一種禽類或哺乳動物病原體之抗原。3. A kit according to claim 30, wherein the kit comprises at least one antigen of another avian or mammalian pathogen.

124989.doc 200825099 七、指定代表圖： (一) 本案指定代表圖為：（無） (二) 本代表圖之元件符號簡單說明：八、本案若有化學式時，請揭示最能顯示發明特徵的化學式： (無）124989.doc 200825099 VII. Designated representative map: (1) The representative representative of the case is: (none) (2) The symbol of the symbol of the representative figure is simple: 8. If there is a chemical formula in this case, please reveal the best indication of the characteristics of the invention. Chemical formula: (none)

124989.doc124989.doc