WO2019059817A1 - Vaccine based on fusion protein and plasmid dna - Google Patents

Abstract

The invention relates to molecular biology, biotechnology, medicine and can be used for the prevention and treatment of tuberculosis. A vaccine is proposed for prevention and treatment of tuberculosis, containing the fusion protein protected by the patent for the invention of Russia №2615440 including M. tuberculosis Ag85B, Tb10.4 proteins, fragments of S. Typhimurium FliC flagellin, connected via flexible bridges, and a plasmid DNA, encoding M. tuberculosis Ag85A protein and/or a plasmid DNA coding for M. tuberculosis Ag85B protein as active agents in an effective amount and a physiologically acceptable carrier or buffer solution, the user of the vaccine is a human or an animal. The proposed vaccine has an efficiency more than such of BCG.

Description

[Title established by the ISA under Rule 37.2] VACCINE BASED ON FUSION PROTEIN AND PLASMID DNA

The invention relates to molecular biology, biotechnology, medicine and can be used for the prevention and treatment of tuberculosis.

Tuberculosis is an infectious disease causing the greatest number of deaths. According to the world health organization (WHO), 10.4 million people worldwide fell ill with tuberculosis in 2015, and 1.8 million died from the disease [WHO | Global tuberculosis report 2016 [Electronic resource] // WHO. URL: http://www.who.int/tb/publications/global_report/en/ (accessed: 03.01.2017)]. Treatment of tuberculosis is complicated by the fact that Mycobacterium strains resistant to anti-tuberculosis drugs are widespread, causing multidrug-resistant tuberculosis (MDR) and extensively drug-resistant one (XDR). In this regard, prevention is a promising approach in the fight against this disease. According to a 2014 WHO TB report, a new vaccine that will help prevent transmission of TB among adolescents and adults in developing countries will be the most cost-effective tool in mitigating the epidemic [07_Evans_GVIRF_TB_Vaccine_Progress.pdf].

To date, the only registered and used vaccine for the prevention of tuberculosis in Russia and abroad is the BCG vaccine obtained from the attenuated Mycobacterium bovis strain and first introduced to humans in 1921. The limited efficiency of BCG vaccination and side effects cause a strong need to develop effective and safe agents for the prevention of tuberculosis.

Currently, this problem is being solved in different ways: alternative BCG vaccines such as live vaccines with improved properties, inactivated vaccines, subunit vaccines, vaccines obtained using recombinant DNA technology, combined variants are being developed and studied. However, vaccines, safe, and simple in production and use, which would have an efficiency at least comparable to BCG, does not exist at the moment. In this regard, the object of the present invention is to create a vaccine against tuberculosis having the above properties, which will also have a low cost.

Of all the areas of development of new anti-tuberculosis vaccines, the authors of this group of inventions consider the use of vaccines obtained using recombinant DNA technology the most promising. However, their effectiveness varies and is caused by a mass of factors, one of the most important of which is the selection of components and their form (structure).

For creating the present invention immunodominant secreted antigens of M. tuberculosis were selected - Ag85A and Аg85В proteins of 85 protein complex and TB10.4 protein (culture-filtrate protein-7). These proteins are highly conservative among various strains of mycobacteria, and can also cause an immune response, which suggests that it is possible to obtain a cross-reactive immune response [WO 96/15241]. It is important that these proteins do not have homology with known proteins of other organisms, which ensures the specificity of their action. It is also shown that the protein TB10.4 has an adjuvant effect when administered in combination with Mycobacterium proteins [EA 012037 B1].

The following solutions are known to be the closest to the solutions we offer on the basis of the above-mentioned M.tuberculosis proteins.

A vaccine containing a fusion protein of Ag85A, Ag85B, TB10.4 proteins is known, an adjuvant may be used additionally; or a nucleic acid fragment encoding such a fusion protein [WO 2005/061534 A2]. However, according to the authors of the present invention, the use of DNA encoding a Mycobacterium antigen, together with a fusion protein will induce the formation of an immune response, more shifted towards cytotoxic.

A composition is known containing in one embodiment a fusion protein of mycobacterial antigens Tb10.4, Ag85B, ESAT6 and no less than one vector containing DNA coding for 5 antigens of mycobacteria, including Tb10.4, Ag85A, Ag85B (WO2014009438 (A2)). The use of a suitable adjuvant would be desirable.

A pharmaceutical composition is known containing in one embodiment a fusion protein containing TB10.4 and Ag85B, and a nucleic acid encoding in one variant TB10.4 [US 6991797 B2]. The use of an adjuvant will enhance the immune response to the antigen. In this case when it is included in the fusion protein, vaccine manufacturing process is optimized due to production of lesser number of components.

A vaccine or an immunogenic composition is proposed administered to a person infected with latent TB, to prevent reactivation of tuberculosis, in one embodiment containing TB10.4 protein fused to antigen expressed by Mycobacterium tuberculosis, amplified for the adjuvant action with lipidation, as well as plasmids, from which genes are expressed encoding ESX-1 complex proteins (WO2010121618 (A1)). Protein Tb10.4 refers to the proteins of the ESX-3 complex.

The selection of the optimal adjuvant for inducing the required profile of the immune response is not an easy task in relation to mycobacteria. It is important to choose an adjuvant that will induce the most pathogen-specific immune response, in this case capable of stimulating T-cell immune response, along with the B-cell one, and which will contribute to the formation of local immunity.

With regard to the choice of proteins that dominate in the vaccine content, according to the authors of the present invention, a choice should be made in the direction of proteins Ag85A and Ag85B. These proteins provide high affinity of mycobacteria to the fibronectin mediating the attachment of M. tuberculosis to alveolar macrophages . They also help to maintain the cell wall, catalyzing the transfer of mycolic acids to the cell wall arabinogalactan and the synthesis of the flagellation factor. They have mycolyl-transferase activity and catalyze the biosynthesis of the most common glycolipids of the cell wall, including virulent cord factor [Dover L.G. et al. Regulation of cell wall synthesis and growth // Curr. Mol. Med. 2007. Vol. 7, № 3. P. 247–276]. Antigens 85 are also involved in the formation of T-cell response in individuals infected with M. tuberculosis [Tang X., Deng W., Xie J. Novel insights into Mycobacterium antigen Ag85 biology and implications in countermeasures for M. tuberculosis // Crit. Rev. Eukaryot. Gene Expr. 2012. Vol. 22, № 3. P. 179–187, Huygen K. The Immunodominant T-Cell Epitopes of the Mycolyl-Transferases of the Antigen 85 Complex of M. tuberculosis // Front. Immunol. 2014. Vol. 5. P. 321].

There is a known a composition containing in one embodiment a nucleic acid of the given structure (in one of the embodiments - for the treatment of tuberculosis), an antigen and flagellin, for the treatment of an infectious disease (RU 2010135630 A). In our case the proposed means is also a preventive one - a vaccine, antigen and flagellin are fused, an additional antigen is contained as a part of the fusion construct, a vector structure in the given application is not implied, in contrast to the variant proposed by us.

Flagellin is a protein of bacterial flagella present in many bacteria, including pathogens. Flagellin, depending on the origin of a microorganism, varies and consists of 259-1250 amino acid residues, which certainly affects its properties. Thus, the indication of the microorganism whose flagellin is used is essential. Moreover, the known flagellin adjuvants exhibit significant side effects, and, in particular, their own antigenic activity and systemic proinflammatory properties when administered in vivo.

The binding of flagellin to TLR-5 occurs only in the monomer form [of Abaturov A. E. the role of TOLL-like receptors in the recognition of pathogen-associated molecular structures of infectious pathogenic agents and the development of inflammation. Part 3. Recognition of TLR ligands / A. E. Abaturov, A. P. Volosovets, I. E. Yulish // Child Health. - Donetsk, 2012, N N 7.- P. 157-164], however, when obtaining full-size flagellins, they can polymerize, and their preservation in solution in a monomeric form, while preserving the correct conformation, requires special conditions. In this case, the joint use of such a solution and other agents, for example, antigen(s), may adversely affect the conformation of the latter and, accordingly, the properties. It turns out that the combined use of a full-size flagellin and antigen(s) may not lead to the desired result, due to inactivation of one of the agents in case of mismatch of the conditions of conservation of activity.

A composition for the vaccination is known containing a polypeptide comprising in one of the embodiments antigenic parts of Ag85B and TB10.4 proteins, also containing a nucleic acid coding for the CD40 ligand, or a peptide that possesses ability to bind with CD40 ligand and activate CD40 receptor, expressed on antibody-producing B-lymphocytes, which in one embodiment of the invention also contains flagellin [WO 2010/034974 A2, priority date 24.09.2008]. The disadvantage is that the fusion protein is not characterized structurally, only its components are indicated. It is also proposed to use a full-size flagellin, and its belonging to a particular organism is not indicated, the disadvantages of such approach are described above. Also, the flagellin type is not specified, for example, for Salmonella flagellin - FliC or FljB.

Differences in the structure of flagellin are observed among also one bacterial species. Thus, most of the Salmonella enterica serovars are two-phase, i.e. there is an alternate expression of different flagellate antigens (phases). This is due to the possibility of being in the active state of only 1 of the 2 flagellin genes (fliC and fljB), located in different loci of the chromosome [Baker S., Hardy J., Sanderson et al. A novel linear plasmid mediates flagellar variation in Salmonella Typhi. PLoS Pathog, 2007, 3 (5): e59. doi: 10.1371/journal.ppat.0030059], FliC-phase 1 flagellin (STF), fljB – phase 2 flagellin (STF2). These flagellins have the same N-terminus, homologous but differing on a few amino acids C-Terminus, as well as different D3 domain, respectively, they have different structures and can have different effects.

Anti-TB DNA vaccine is known delivered by a chitosanase delivery system containing chitosan and plasmids containing a full-sized hsp65 gene and DNA fragments encoding the T-cell epitopes of ESAT-6[189-228], Ag85A[369-405], CFP10[162-207] and Ag85B[420-459] proteins, in the form of nasal drops [CN101455846].

A polyepitopic anti-tuberculosis DNA vaccine is known, containing one type of carrier, pcDNA3 plasmid DNA, and one insert-a segment of the target gene, at the 5' end of which there is a gene encoding the HSP65 protein, at the 3' end there are successively nucleotide sequences encoding the epitopes of proteins ESAT-6, from 1 to 20 nucleotide and from 61 to 81 nucleotide, Ag85A, from 62 to 84 nucleotide, Ag85b, from 121 to 155 nucleotide, Ag85a again, from 143 to 166 nucleotide, Ag85b, from 234 to 256 nucleotide, MPT64C, from 177 to 228 nucleotide, the sequences are connected by a nucleotide sequence encoding AAY, moreover, hsp65 and the polyepitope fragment are synthesized separately, hsp65 - by PCR using Mycobacterium DNA as a matrix, the polyepitope fragment is synthesized chemically, then the fragments are crosslinked [CN101088559 A]. Thus, the creation of the structure is complicated by the introduction of stages of fragments cross-linking. It is also not clear what material is used for the synthesis of the hsp65 gene: when using Mycobacterium lysate, there is a risk of infection.

The hsp65 protein of the Mycobacterium tuberculosis complex is known to have homology with molecules present in the joints [van Eden W. et al. The mycobacterial 65 kD heat-shock protein and autoimmune arthritis // Rheumatol. Int. 1989. Vol. 9, № 3-5. 187-191], and causes the formation of an adjuvant arthritis [Kim et al. Modulation of Adjuvant Arthritis by Cellular and Humoral Immunity to Hsp65 // Front. Immunol. 2016. Vol. 7. P. 203]. So tt is not possible to use genetically engineered vaccines containing this protein.

A plasmid DNA pcDNA3.1 is known carrying a chimeric gene which contains a gene encoding the protein Ag85a of Mycobacterium tuberculosis and a gene encoding 125-282 amino acids of the protein Ag85b of Mycobacterium tuberculosis, the fragment of the gene Ag85b being in the Ag85a gene, cloned in the site of the restriction enzyme Kpn I and/or Acc I, these are fragments corresponding to aa 245-250 or 430-435, respectively [Z. Li et al. Mycobacterium Tuberculosis Ag85ab Chemical Gene Vaccine, Its Preparation Method and Application: pat. WO2011150745 (A1) USA. 2011. No. CN20101191243 20100603]. However, an adequate immune response to each of the antigens is more likely to be obtained in the absence of embedding one protein into another.

The prototype of the invention proposed by the authors, of a composition based on a fusion protein and a plasmid DNA, is invention described in US 6991797 B2 publication.

The technical result of the use of the invention is in increase of the effectiveness of the active agent for the vaccine, and, accordingly, of the vaccine against tuberculosis, by using one component that combines the functions of a specific mycobacterial antigen, and an adjuvant, that mediates the optimal immunity to suppress the development of tuberculosis infection, the correct conformation of which, mediating the full functioning of all its components, is easy to ensure also due to the absence of other protein molecules in the composition, requiring other conditions to maintain activity. This technical result is also achieved by the fact that instead of TB10.4 protein the Ag85 complex (A or B) protein is used, see the above argumentation.

The prototype of the invention proposed by the authors, a composition based on the fusion protein and the plasmid DNA, is the one described in the publication WO2010121618 (A1).

The technical result of the use of the invention is to increase the effectiveness of the active agent for the vaccine, and, accordingly, of the vaccine against tuberculosis. It is achieved using an adjuvant that mediates the optimal immunity to suppress the development of tuberculosis infection, see the argumentation above. This technical result is also achieved by the fact that instead of ESX-1 complex proteins, Ag85 complex (A and B) proteins are used, see the above argumentation.

The technical result of the use of both inventions is in the expansion of the spectrum of vaccines against tuberculosis. If there are contraindications to the use of analogues, or reluctance to use analogues due to their above described deficiencies, this vaccine will allow to carry out protection against tuberculosis. Since the problem of tuberculosis is very acute, and the introduction to the market of a drug is not possible to many, this invention will increase the chances in the fight against this infection.

The introduction of the immunogen, respectively, of a vaccine, is preferably intramuscular. It is also possible to use mucosal surface for immunization, as the immunity induced in one area of the mucosa may induce the development of immunity on the distal mucosa [McGhee, J. R. et al. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 1992, 10: 75-88]. The formation of local immunity is important to prevent infection with mycobacteria. The content of flagellin fragments in the composition of the fusion protein also contributes to this process, thus, it is known that in the respiratory tract, flagellin induces the synthesis of IgA and induces a Th2-associated response [Soloff AC, Barratt-Boyes SM. Enemy at the gates: dendritic cells and immunity to mucosal pathogens. Cell Res. 2010 Aug; 20 (8): 872-85. doi: 10.1038 / cr.2010.94. Epub 2010 Jul 6]. Immunization of the mucosal surface also greatly facilitates the delivery of the immunogen.

A vaccine has been proposed for prevention and treatment of tuberculosis comprising the fusion protein according to the Russian patent No. 2615440 and a plasmid DNA encoding the M. tuberculosis Ag85A protein and / or a plasmid DNA encoding the M. tuberculosis Ag85B protein, as active agents in an effective quantity, as well as a physiologically acceptable carrier or buffer solution, the consumer of a vaccine is a human or an animal. The proposed vaccine has an efficacy greater than that of BCG.

The proposed vaccine for the prevention and treatment of tuberculosis contains:

(1) the fusion protein protected by the Russian patent for the invention №2615440, including Ag85B, Tb10.4 Mycobacterium tuberculosis proteins, immunogenic fragments of Salmonella enterica serovar Typhimurium flagellin FliC, connected by flexible bridges, and

(2) a plasmid DNA for transient expression in mammalian cells, represented by a skeleton containing prokaryotic elements, origin of replication and a marker gene, and eukaryotic elements, a strong promoter, a leader sequence of mRNA, and regulatory sequences for said elements, no less than a single site for cloning of gene of interest, and no less than a single site for annealing of at least a single primer for analysis of plasmid DNA composition, and a polynucleotide represented by a fragment encoding M.tuberculosis Ag85A protein, in one of the embodiments also by a secretory sequence, codon-optimized for expression in mammalian cells, and a terminating sequence, and / or

(3) plasmid DNA as described in the paragraph above (2), in which M.tuberculosis Ag85B protein is used instead of M.tuberculosis Ag85A protein.

The plasmid DNA should contain elements essential for organisms of its maintaining and use, together with the corresponding regulatory sequences. Regulatory sequences are nucleotide sequences that can affect gene expression at the transcription and/or translation level, as well as affect mechanisms that ensure the existence of the plasmid DNA and maintenance of its functioning.

Essential for the prokaryotic system are an origin of replication, to maintain in a cell with an average, preferably high, copyability, and a marker gene for the possibility of selection of the producing strain. Bacterial elements of the plasmid DNA are not to negatively influence expression in mammalian cells and cause side effects from the use of the plasmid DNA. Eligible origin of replication is pM1 (der.), ColE1 (der.) and F1, F1 and pUC, but is not limited to them. Eligible marker gene is a reporter gene or a gene of resistance to an antibiotic, e.g., ampicillin, kanamycin mainly, but not limited to them. In the literature, there is evidence that the use of a gene of resistance to ampicillin as a marker gene may be undesirable in connection with the development of the reaction in patients on ampicillin, but the authors consider such effects associated with a low quality of purification of a plasmid DNA, but not by the element itself.

Essential elements of plasmids for use in mammals are a promoter, an mRNA leader sequence, a termination sequence.

A promoter is an important component of the plasmid, which triggers the expression of a gene of interest. Classic promoters for plasmid DNA - components of drugs - is a human CMV / immediate-early or CMV-chicken-β actin (CAGG) promoter. CMV promoters are used for the most DNA vaccines, as they mediate high levels of constitutive expression in a wide range of mammalian tissues [Manthorpe M, Cornefert-Jensen F, Hartikka J, et al. Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum. Gene Ther. 1993;4(4):419-431] and do not inhibit the downstream reading. The increase in expression level is observed when changing the CMV promoter, for example, by the inclusion of HTLV-1R-U5 downstream from the cytomegalovirus promoter or using a chimeric SV40-CMV promoter [Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnol. Adv. 2009;27(4):353-370]. Tissue-specific host promoters serve as an alternative to the CMV promoter, they allow to avoid constitutive expression of antigens in inappropriate tissues [Cazeaux N, Bennasser Y, Vidal PL, Li Z, Paulin D, Bahraoui E. Comparative study of immune responses induced after immunization with plasmids encoding the HIV-1 Nef protein under the control of the CMV-IE or the muscle-specific desmin promoter. Vaccine. 2002;20(27-28):3322-3331].

Promoter can be with the appropriate regulatory sequences of the natural promoters with their regulatory elements (CaM kinase II，CMV, nestin, L7, BDNF, NF, MBP, NSE, p-globin, GFAP, GAP43, tyrosine hydroxylase, subunit 1 of the kainate receptor and the glutamate receptor B subunit, and others) or synthetic promoters with regulatory sequences to obtain the desired expression pattern (ratio of duration and level of expression) of the target gene at the transcription level.

Possible regulatory sequences relative to the promoter:

- enhancer, to increase the expression level via improvement of interaction between RNA polymerase and DNA,

- insulator, for modulating the function of the enhancer,

- silencer, or fragments thereof, to decrease the level of transcription, for example, for tissue-specific expression,

- 5` noncoding region upstream of the promoter, including intron.

Plasmid DNA according to the present invention contains at least one of the above-mentioned regulatory sequences, depending on the variant of plasmid DNA, based on the choice of promoter and the desired parameters of the expression of the target gene. Based on the existing level of technology, and the known and obvious variants of such elements and their use, plasmid DNA according to the present invention may contain any combinations meeting the mentioned above conditions, in which Ag85A or Ag85B protein synthesis is performed from the plasmid DNA in mammalian cells. When a silencer is used, or insulator, as part of the construct, it is possible to regulate the expression of a target gene.

Other regulatory sequences:

- noncoding region downstream from the promoter, including intron, to increase the stability of mRNA and the expression of the target gene.

Plasmid DNA according to the present invention in one embodiment further comprises such a regulatory element.

Plasmid DNA according to the present invention contains also such an important element as a leader mRNA sequence containing Kozak sequence directly before the start codon ATG which allows to increase expression [Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J. 1997;16(9):2482–2492].

Plasmid DNA also contains a site, preferably sites, different, for cloning of a target gene, for the correct orientation of the target gene in the plasmid DNA, and a site, preferably sites, for primers annealing for its sequencing.

Plasmid DNA also contains a fragment encoding M. tuberculosis Ag85A or Ag85B protein, optimized for codon composition for expression in mammalian cells. The use of species-specific codons makes it possible to increase gene expression [Frelin L, Ahlen G, Alheim M, et al. Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene. Gene Ther. 2004;11(6):522–533]. Optimization by codon composition can be carried out manually, or using a specialized software, for example, on molbiol.ru site, based on the amino acid sequence of the protein. The amino acid sequence of Ag85A or Ag85B proteins is available on the tuberculist site, as well as in the ncbi base.

Plasmid DNA also contains a termination sequence containing sequentially a stop codon, the 3` noncoding region with signal and polyadenylation site, stop codon, with the help of which mRNA remains stable, and the proper termination of transcription and export of mRNA from the nucleus are performed. Gene expression can be affected by changing the termination sequence that is necessary to maintain mRNA stability, for proper termination of transcription and for the export of mRNA from the nucleus, including its shortening. In many modern DNA vaccines a bovine growth hormone transcription terminator sequence is used [Montgomery DL, Shiver JW, Leander KR, et al. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors. DNA Cell Biol. 1993;12(9):777–783]. Polyadenylation (polyA) is necessary to stabilize the transcript. Change in the sequence of polyA can lead to an increase in the level of gene expression [Norman JA, Hobart P, Manthorpe M, Felgner P, Wheeler C. Development of improved vectors for DNA-based immunization and other gene therapy applications. Vaccine. 1997;15(8):801–803]. In pVAX1 plasmid (Invitrogen, Carlsbad, CA), the bovine growth hormone terminator region contains a homopurine region that is sensitive to nuclease. It is shown that an alternative polyA sequence can significantly improve the plasmid stability to nuclease [Azzoni AR, Ribeiro SC, Monteiro GA, Prazeres DMF. The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression. J Gene Med. 2007;9:392–402]. The introduction of two stop codons in front of the 3` untranslated region allows to increase the efficiency of the transcription terminator. Based on the state of the art, on known and obvious variations of such an element, the plasmid DNA of the present invention may comprise any terminating sequence satisfying the abovementioned conditions, with which the synthesis of the target protein in mammalian cells is performed from the plasmid DNA.

Plasmid DNA may also contain a heterologous secretory sequence, codon-optimized for mammals. In one embodiment of the invention it contains, for example, TPA (tissue-type plasminogen activator isoform 1 preproprotein [Homo sapiens], NCBI Reference Sequence: NP_000921.1) secretory sequence, but is not limited to such. The advantage of using TPA secretory sequence is a vast previous clinical experience, and that its high performance is demonstrated in relation to the expression of the secreted protein from a variety of target genes.

The optimal plasmid design for the implementation of the function combines "bacterial" and "eukaryotic" elements, with appropriate regulatory sequences, to ensure high copyability in the production process and high expression in mammals [Saade F, Petrovsky N. Technologies for enhanced efficacy of DNA vaccines. Expert Rev Vaccines. 2012 Feb;11(2):189-209. doi: 10.1586/erv.11.188].

To create plasmid DNA according to the present invention, it is possible to use plasmid DNA, at least standardly used for the delivery of genes and their expression in the body of a mammal, including humans [Hartikka J, Sawdey M, Cornefert-Jensen F, Margalith M, Barnhart K, Nolasco M, Vahlsing HL, Meek J, Marquet M, Hobart P, Norman J, Manthorpe M. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther. 1996 Jun 20;7(10):1205-17 and others], as well as a plasmid, which can be created by an expert in this field using recommendations on the elements of vectors [“Cloning Vectors”, ed. Pouwls et al., Elsevier, Amsterdam- New York-Oxford, 1985, ISBN 0 444 904018], or optimized for the abovementioned parameters, including those detailed in the article Williams et al., 2009 [Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnol. Adv. 2009;27(4):353–370].

The preferred plasmid DNA for use in humans are vectors tested on humans containing the above-described elements with corresponding regulatory sequences, feasibly modified to match the stated criteria, which allows to reduce the number of required studies for the registration of a drug. However, the use of other plasmid DNAs containing the required described elements is possible.

In the FDA guideline (2007) it is stated that studies of the biodistribution of a substance after its introduction in the body can be waived for DNA vaccines produced by cloning of a new gene into a plasmid vector, for which acceptable biodistribution and integration profile are documented previously. In the WHO guideline (2007) it is stated that research on the biological distribution and conservation is required if there is no considerable experience of work with almost identical or similar product. In the EMEA guidance (2006) it is stated that the experience of work with the vector system will allow to optimize and focus on preclinical studies. Safety studies with the use of DNA vectors with different cloned genes showed similar biodistribution [Sheets RL, Stein J, Manetz TS, Duffy C, Nason M, Andrews C, Kong WP, Nabel GJ Gomez PL. Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts. Sheets RL, Stein J, Manetz TS, Duffy C, Nason M, Andrews C, Kong WP, Nabel GJ Gomez PL.Toxicol Sci. 2006 Jun;91(2):610-9. Epub 2006 Mar 28.] and toxicology [Sheets RL, Stein J, Manetz TS, Andrews C, Bailer R, Rathmann J, Gomez PL. Toxicological safety evaluation of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar despite differing plasmid backbones or gene-inserts. Toxicol Sci. 2006 Jun;91(2):620-30. Epub 2006 Mar 28]. For a plasmid DNA for use in mammals other than humans the requirements are less stringent, therefore it is possible to use a wider range of plasmids.

The sequence of the elements described in the plasmid DNA is understandable to an expert in this field.

Pharmaceutically acceptable carriers or buffer solutions are known in the art and include those described in various texts, such as, for example, Remington's Pharmaceutical Sciences.

The authors of this invention conducted laboratory tests confirming the possibility of implementing the described invention. The study results obtained are illustrated by examples (1-3) and Fig.1-3.

Example 1. Production of a vaccine (variants)

1.1. Production of a fusion protein

A preparation of a fusion protein (variants) with a purity of 98% to create a vaccine was obtained as described in the patent for the invention of the Russian Federation №2615440, in an amount sufficient for the study.

1.2. Obtaining of plasmid DNA encoding Ag85A or Ag85B protein of M. tuberculosis

1.2.1. The amino acid sequences of proteins Аg85а and Ag85b of Mycobacterium tuberculosis was found by using a specialized website tuberculist. The nucleotide sequence of each gene, collinear to the given amino acid sequence, was calculated with simultaneous optimization by codon composition for expression in mammalian cells and addition of flanking gene restriction sites, NheI and HindIII for cloning in pcDNA3.1 (+) plasmid, as well as the Kozak sequence before the start codon to initiate the translation, and in one of the variants of also the TPA signal sequence for the secretion of the synthesized protein from the eukaryotic cell, and of the stop codon, in one of the variants also of an additional stop codon. Calculated sequence of target genes for cloning in pcDNA3.1 (+) plasmid with TPA and one stop codon is characterized by SEQ ID NO:1 and SEQ ID NO:2. Chemical synthesis of all calculated DNA was performed.

1.2.2. Cloning of the synthesized gene into pVAX1 plasmid (Invitrogen) and pcDNA3.1(+) (Invitrogen) was performed. Also a vector pcDNA3.1(+) incapable of the expression of neomycin was received, due to the restriction of this vector with NsiI restrictase, in the region of the SV40 promoter (-71 bp). The calculated nucleotide sequences were also cloned into the obtained vector.

1.3. For the ligation reaction 3 µl of the synthesized DNA solution, 1 µl of the prepared vector solution, 5 µl of buffer for ligation ´2 and 1 µl of T4-ligase were taken. The reaction was performed at +20°C for 2 hours.

Thereafter, the mixture was warmed up at +95°C for 10 min and purified from salts by dialysis using nitrocellulose filters with pore diameter of 0.025 µm (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol, for 10 min.

1.2.4. Then the cells of E. coli strain DH10B/R (F-mcrA, ∆(mrr-hsdRMS-mcrBC), φ80dlacZΔM 15, ΔlacX74, deoR, recA1, endA1, araD139, ∆(ara,leu)769, galU, galKλ-, rpsL, nupG) were transformed with the obtained plasmid DNAs by the method of electroporation using electroporator MicroPulser (BioRad). This strain does not contain methylase, which helps minimize the possibility of mutations occurrence in DNA, including in gene cloned into the plasmid, sustained in this strain. 1 µl of dialyzed ligase mixture was added to 12 µl of the competent cells and placed between the electrodes of the poration unit and treated with a current pulse.

After transformation, cells were placed in 1 ml of SOC-medium (2% bacto-tripton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) and incubated for 40 min at +37°C.

1.2.5. Identification of clones of E. coli cells containing the plasmid DNA was performed on the selective medium containing LB-agar, 50 µg/ml of kanamycin (for plasmid DNAs based on pVAX1), or ampicillin (for plasmid DNAs based on pcDNA3.1(+)).

Plasmid DNA was isolated from the grown clones. Isolation of the plasmid DNA was performed using Wizard Minipreps DNA Purification System (Promega, USA). Purified recombinant plasmid DNA was verified by sequencing.

1.2.6. Sequencing of the cloned fragments was performed by the method of Sanger using a set of Applied Biosystems BigDye® Terminator (BDT) v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the attached instruction. For the labeling of the reaction products fluorescent dye-labeled ddNTP were used, each ddNTP corresponding to the dye. For sequencing unlabeled plasmid-specific primers were used. A PCR reaction was conducted, then the reaction mixture was purified from free labeled ddNTP using instructions to the kit BigDye X-Terminator Purification Kit (Applied Biosystems, USA), and the sequencing reaction products were separated using capillary sequencer Applied Biosystems 3500/3500xL Genetic Analyzer (Applied Biosystems, USA) and reagent 3500/3500xL Genetic Analyzer Polymer “POP-6™” (Applied Biosystems, USA).

The results of the separation of the reaction products of sequencing were recorded by scanning with laser and the detection of four fluorescent dyes contained in all types of ddNTP.

1.2.7. Computer analysis of DNA sequences was performed using a personal computer using programs Chromas and BioEdit. The nucleotide sequences of the investigated DNA fragments were aligned with the designed, the identity of the synthesized fragments to the calculated ones was demonstrated. As a result, clones of E. coli cells were selected containing the full sequences of the target genes in the plasmids - the DNA sequences encoding Ag85A and Ag85B proteins of mycobacteria.

1.2.8. A separate colony of E. coli grown on LB-agar in a Petri dish with the addition of kanamycin (or ampicillin), was placed in 10 ml of a selective media. Cells grew over night at +37°C under constant stirring (250 rpm). The resulting cells were collected by centrifugation at 4000g. Further isolation and purification of the plasmid DNA was performed using EndoFree Plasmid Mega Kit (Qiagen), which allows to obtain non-pyrogenic DNA. Isolated plasmid DNA was analyzed with electrophoresis in a 0.8% agarose gel, its concentration was measured using fluorometry.

As a control solution water without addition of test preparation was used. 1,950 ml of water and 0.05 ml of test solution were added into a unit for measuring optical density with a volume of 2 ml, mixed, and the optical density was measured at a wavelength of 260 nm. Determination of the DNA concentration was performed according to the formula:

C(µg/ml)=40А₂₆₀К,

where А₂₆₀-optical density of the preparation measured at a wavelength of 260 nm; K(µg/ml) - for DNA 50 µg/ml (50 µg/ml of double-stranded DNA in water); 40 - dilution of the test preparation.

In the end it was determined that plasmid DNAs were obtained with a concentration of (3,5 – 4,4) mg/ml. The yield of plasmid DNA ranged from 3.4 mg to 4.8 mg from 1 l of culture medium.

The purity of the obtained plasmid DNA preparation was judged by the ratio of optical density of the preparation measured at a wavelength of 260 nm to the optical density of the preparation measured at a wavelength of 280 nm (А₂₆₀/А₂₈₀) and ratio of optical density of the preparation measured at a wavelength of 260 nm to the optical density of the preparation measured at a wavelength of 230 nm (А₂₆₀/А₂₃₀). The measurements were performed in aqueous solution, as a reference solution water without addition of the test preparation was used.

For pure preparations of DNA А₂₆₀/А₂₈₀>1.80 and А₂₆₀/А₂₃₀>1.80 are characteristic. Defined in the experiment values corresponded to the values of the relations А₂₆₀/А₂₈₀ and А₂₆₀/А₂₃₀ for pure preparations, for all received preparations of plasmid DNA.

A quantitative determination of protein impurities was also conducted in the obtained preparations of plasmid DNA using the microBCA assay [Smith, P. K., et all, Measurement of protein using bicinchoninic acid. Analyt. Biochem. 150, 76-85 (1985)], measuring the optical density of the obtained colored protein complexes with copper and bicinchoninic acid at a wavelength of 562 nm. The sensitivity of the method microBCA assay is 0.5-20 µg/ml protein. The concentration of total protein in any of the studied preparations of the plasmid DNA did not exceed the norm (from 0.5 to 10 µg/mg of plasmid DNA).

The content of bacterial lipopolysaccharides was also determined in preparations of plasmid DNAs, using a gel-thrombus version of the LAL test, with a sensitivity of >0,25 EU/ml (ToxinSensor, GenScript, USA). A lysate of horseshoe crab Limulus Polyphemus amebocytes served as a LAL-reagent. LAL-reagent specifically reacts with bacterial endotoxins, as the result of the enzymatic reaction there is a change in the reaction mixture proportional to the concentration of endotoxin. The results were evaluated according to the presence or absence of a dense thrombus at the bottom of the tube by turning the tube. The gel-thrombus was not formed during the study of a sample diluted 10 times, for the half of preparations of plasmid DNAs, and 5 times for the rest half of preparations of plasmid DNAs, i.e. at the sensitivity of the method 2.5 EU/ml and 1.25 EU/ml, respectively, which, given the concentration of the plasmid DNA in the sample, confirms a valid indicator of purification from endotoxins.

1.3. The production of vaccines based on a protein and DNA

Aluminum hydroxide, in powder form, in an amount of 37 mg was added to 5 ml of a solution with a concentration of a fusion protein characterized in the patent for the invention of the Russian Federation No. 2615440, 400 µg/ml, binding was being performed to the protein for 10 min., after which further dilution was carried out. A saline solution or PBS was used as a liquid medium. Plasmid DNA(s) was(were) added to the obtained protein solution in the required amount.

Example 2. Demonstration of the preventive effect of the vaccine (variants)

The experiment was performed on white mice of noninbred lines (laboratory animal Nursery "Rappolovo"). Animals, weighing 19-22 g, were kept in standard conditions, at ambient temperature +27±2°C with constant humidity 55%, at 12-hour light day, received dry standardized food and water without restriction.

Animal groups (number of mice per group):

Intact (10) - K-

Infection control (15) - K+

BCG vaccination (15) - 1

Immunization with a fusion protein described in SEQ ID NO.:1 of Russian patent for invention №2615440, and plasmid DNAs pcDNA3.1(+)ag85a and pcDNA3.1(+)ag85b, containing TPA secretory sequence (13), intramuscularly (i/m) - 2

Immunization with a fusion protein described in SEQ ID NO.:1 of Russian patent for invention №2615440, and plasmid DNAs pVAX1ag85a and pVAX1ag85b (17), i/m – 3

Immunization with a fusion protein described in SEQ ID NO.:1 of Russian patent for invention №2615440, and plasmid DNA pcDNA3.1(+)ag85a, with deleted gene of resistance to neomycin (15), i/m – 4

Immunization with a fusion protein described in SEQ ID NO.:1 of Russian patent for invention №2615440, and plasmid DNA pcDNA3.1(+)of ag85b lacking the gene of resistance to neomycin and with an introduced second stop codon (15), i/m – 5.

One-time BCG immunization and two-time vaccine immunization based on a fusion protein and DNA were carried out with an interval of two weeks. A dose of 20 µg of protein and 50 µg of DNA (25 µg of each plasmid when using two plasmids) per mouse was used in groups of 2,4,5 and of 10 µg of protein and 25 µg of DNA (12.5 µg of each plasmid) per mouse in group 3, was administered in a volume of 100 µl intramuscularly. To achieve the required volume, saline solution was used in groups 2,4,5 and PBS in group 3. Infection was carried out two weeks after the last vaccination.

Infection was carried out 10 days after the last vaccination. M. tuberculosis Erdman standard test-strain was used to model tuberculosis. Mycobacterial suspension to infect mice was prepared ex tempore from a three-week strain, cultured on Lowenstein-Jensen medium. Infecting dose is 10⁶ colony-forming units (CFU)/mouse in 0.2 ml of saline, the route of administration - in the lateral caudal vein.

Mice were withdrawn from the experiment six weeks after infection by decapitation in accordance with the Methodological Recommendations of the USSR Ministry of Health (1985).

The indicators of severity of tuberculosis infection were determined:

1. The coefficients of the mass of the lungs (CML) and spleen (CMS), were calculated according to the formula in arbitrary units:

body weight (g) ×100

body weight (g)

2. Lung injury index (LII) was determined by the combination of exudative and productive changes in conventional units - points [12].

Exudative changes:

- lungs are aired-0

- single airless foci - 0.25

- lungs are airless at 1/2 - 0.5

- lungs are airless at 2/3 - 0.75

- whole lungs are airless - 1.0

Productive foci:

- single submilliary foci - 0.5

- numerous (no more than 20) - 1.0

- numerous submilliary (more than 20) - 1.5

- single miliary - 1,75

- numerous merging submilliarity and single miliary - 2,0

- numerous miliary (not more than 10) - 2.25

- numerous miliary, merging - 2.75

- appearance of small caseous necrotic foci - 3.0

- extensive caseous - 4.0

- continuous lung damage - 5.0

3. Bacteriological parameters. The dosed inoculation of lung tissue homogenate on a dense egg medium of Levenshtein-Jensen was carried out by the method of serial dilutions (3 and 4 dilutions). The massiveness of mycobacteria growth was determined and the organ protection index was calculated. The massiveness of mycobacteria growth was expressed in decimal lоg (lg) of the number of CFU per lung mass. The calculation of the organ index of protection was performed by the difference between the immunized mice CFU lg and infection control group mice CFU lg. In assessing the results, the protection index ≥0.5 lg is considered to be a positive effect on mycobacteria growth retardation.

The results of some of the experiments are shown on Fig. 1-3. The conducted studies have demonstrated the effectiveness of vaccination by developed vaccine candidates, being more than that of BCG, both when using two plasmid DNA, and when using a single vector, coupled with a fusion protein, characterized in the patent for the invention of the Russian Federation №2615440. It was also found that the differences between the groups were significant.

Thus, the high efficiency of the developed recombinant anti-tuberculosis vaccine (variants) was demonstrated, which turned out to be more than that of BCG in the prophylactic model, in laboratory animals, with intramuscular administration. There may be other parenteral or other mucosal methods of administration of the vaccine. Differences between groups of animals are significant.

Similar studies using other combinations of fusion protein variants from the patent for the invention of the Russian Federation №2615440 and plasmid (s) encoding Ag85a or/and Ag85b protein(s) showed similar results.

Example 3. Demonstration of the therapeutic effect of the vaccine (variants)

The experiment was performed on white mice of noninbred lines (laboratory animal Nursery "Rappolovo"). Animals, weighing 18-22 g, were kept in standard conditions, at ambient temperature +27±2°C with constant humidity 55%, at 12-hour light day, received dry standardized food and water without restriction.

M. tuberculosis Erdman standard test-strain was used to model tuberculosis. Mycobacterial suspension to infect mice was prepared ex tempore from a three-week strain, cultured on Lowenstein-Jensen medium. Infecting dose is 10⁶ colony-forming units (CFU)/mouse in 0.2 ml of saline, the route of administration - in the lateral caudal vein.

Immunogen was administered 6 days after infection. A dose of 20 μg of fusion protein and 50 μg of DNA (25 μg each plasmid when using two plasmids) per mouse was administered in a volume of 100 μl intramuscularly. Physiological saline was used to adjust the required volume.

Mice were withdrawn from the experiment six weeks after infection by decapitation in accordance with the Methodological Recommendations of the USSR Ministry of Health (1985).

Groups of animals (n-number of mice in the group):

Intact (10) - K-

Infection control (10) - K+

Isoniazid 10 mg / kg (15) - 1

Therapeutic vaccine (fusion protein described in SEQ ID NO.:1 of Russian patent for invention №2615440, and plasmid DNAs pcDNA3.1(+)ag85a and pcDNA3.1(+)ag85b (13), i/m - 5 times per week, i/m (15) – 2

Therapeutic vaccine (fusion protein described in SEQ ID NO.:2, from Russian patent for invention No. 2615440, and pVAX1ag85a and pVAX1ag85b plasmid DNAs, with TPA (15), i/m-3

Therapeutic vaccine (fusion protein described in SEQ ID NO.:3 of Russian patent for invention No. 2615440, and pcDNA3.1(+)ag85a plasmid DNA, with TPA (15), i/m-4

Therapeutic vaccine (fusion protein described in SEQ ID NO.:4 of Russian patent for invention №2615440, and plasmid DNA pcDNA3.1(+)ag85b lacking the gene of resistance to neomycin and with an introduced second stop codon (15), i/m – 5.

Etiotropic treatment was started from the 6 day after infection of animals. The duration of treatment was 36 days.

In animals receiving developed vaccine based on fusion protein along with etiotropic therapy, there was a significant decrease in the lung mass coefficient and lung injury index, as well as a decrease in the seeding rate of mycobacteria from the spleen. Similar studies using other combinations of fusion protein variants of Russian patent for invention №2615440 and plasmid (s) encoding Ag85a or/and Ag85b protein(s) demonstrated similar results.

Thus, the use of the developed vaccine (variants) together with etiotropic therapy of experimental tuberculosis significantly increases its effectiveness.

The safety of the proposed vaccine (variants) was also demonstrated: the animals of the respective groups survived and were forced out of the experiment, side effects were not observed during the trial.

<110> Dukhovlinov I.V., Fedorova E.A.

<120> Vac. based on fus. prot. and plas. DNA for the prev. and treat. of tuberc-s(var.)

<130>

<150> EA 201791906

<151> 2017-09-24

<160> 2

<210> 1

<211> 1101

<212> DNA

<213> Artificial sequence

<220>

<223> nucl. seq. enc. M.tub. Ag85a prot., cod-opt. for expr. in mam.cells

<400> 1

gctagcgcca ccatggacgc tatgaaacgc ggcctgtgct gcgtgctgct gctgtgcgga 60

gctgtgttcg tgagccccag ccagctggtg gaccgcgtgc gcggcgccgt gaccggcatg 120

agccgccgcc tggtcgttgg cgctgtggga gccgccctgg tgagcggcct ggtgggcgcc 180

gtgggcggca ccgccaccgc cggcgccttc agccgccccg gcctgcccgt ggagtacctg 240

caggtgccca gccccagcat gggccgcgac atcaaggtgc agttccaaag tggcggcgcc 300

aacagccccg ccctgtacct gctggacggc ctgcgcgccc aggacgactt cagcggctgg 360

gacatcaaca cccccgcctt cgagtggtac gaccagagcg gcctgagcgt ggtgatgccc 420

gtgggcggcc agagcagctt ctacagcgac tggtaccagc ccgcctgcgg caaggccggc 480

tgccagacct acaagtggga gaccttcctg accagcgagc tgcccggctg gctgcaggcc 540

aaccgccacg tgaagcccac cggcagcgcc gtggtgggcc tgagcatggc cgccagcagc 600

gccctgaccc tggccatcta ccacccccag cagttcgtgt acgccggcgc catgagcggc 660

ctgctggacc ccagccaggc aatgggaccc accctgatcg gcctggccat gggcgacgcc 720

ggcggctaca aggccagcga catgtggggc cccaaggagg accccgcctg gcagcgcaac 780

gaccccctgc tgaacgtggg caagctgatc gccaacaaca cccgcgtgtg ggtgtactgc 840

ggcaacggca agcccagcga cctgggcggc aacaacctgc ccgccaagtt cctggagggc 900

ttcgtgcgca ccagcaacat caagttccag gacgcctaca acgccggcgg cggccacaac 960

ggcgtgttcg acttccccga cagcggcacc cacagctggg agtactgggg cgcccagctg 1020

aacgccatga agcccgacct gcagcgcgca ctcggcgcca cccccaacac cggccccgcc 1080

ccccagggcg cctaaaagct t 1101

<210> 2

<211> 1062

<212> DNA

<213> Artificial sequence

<220>

<223> nucl. seq. enc. M.tub. Ag85b prot., cod-opt. for expr. in mam.cells

<400> 2

gctagcgcca ccatggacgc tatgaaacgc ggcctgtgct gcgtgctgct gctgtgcgga 60

gctgtgttcg tgagccccag caccgacgtg agccgcaaga tccgcgcctg gggccgccgc 120

ctgatgatcg gcaccgccgc cgccgtggtg ctgcccggcc tggtgggcct ggccggcggc 180

gccgccaccg ccggcgcctt cagccgcccc ggcctgcccg tggagtacct gcaggtgccc 240

agccccagca tgggccgcga catcaaggtg cagttccaaa gtggcggcaa caacagcccc 300

gccgtgtacc tgctggacgg cctgcgcgcc caggacgact acaacggctg ggacatcaac 360

acccccgcct tcgagtggta ctaccagagc ggcctgagca tcgtgatgcc cgtgggcggc 420

cagagcagct tctacagcga ctggtacagc cccgcctgcg gcaaggccgg ctgccagacc 480

tacaagtggg agaccttcct gaccagcgag ctgccccagt ggctgagcgc caaccgcgcc 540

gtgaagccca ccggcagcgc cgccatcggc ctgagcatgg ccggcagcag cgccatgatc 600

ctggccgcct accaccccca gcagttcatc tacgccggca gcctgagcgc cctgctggac 660

cccagccagg gcatgggacc tagcctgatc ggcctggcca tgggcgacgc cggcggctac 720

aaggccgccg acatgtgggg ccccagcagc gaccccgcct gggagcgcaa cgaccccacc 780

cagcagatcc ccaagctggt ggccaacaac acccgcctgt gggtgtactg cggtaacgga 840

acccccaacg agctgggcgg cgccaacatc cccgccgagt tcctggagaa cttcgtgcgc 900

agcagcaacc tgaagttcca ggacgcctac aacgccgccg gcggccacaa cgccgtgttc 960

aacttccccc ccaacggcac ccacagctgg gagtactggg gcgcccagct gaacgccatg 1020

aagggcgacc tgcagagcag cctgggcgcc ggctaaaagc tt 1062

Claims (2)

1. Vaccine for the prevention and treatment of tuberculosis, containing the fusion protein according to the patent for invention of Russia №2615440 and a plasmid DNA, encoding M. tuberculosis Ag85A protein and/or a plasmid DNA coding for M. tuberculosis Ag85B protein, as active agents in an effective amount and a physiologically acceptable carrier or buffer solution.
2. Vaccine according to claim 1, characterized by the fact that the consumer is a human or an animal.

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