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In the last 10 years, scientists' interest in the horsepox pathogen has increased sharply due to the obtaining of its chimeric copy and the discussion of whether it was used to create early smallpox vaccines and the dangers of technologies that allow the restoration of extinct pathogens of dangerous infections.The aim of the work is to summarize the materials on modern studies of the horsepox virus.The source base of the study is English-language scientific literature available via the Internet.The research method is an analysis of scientific sources on horsepox from the general to the specific. We considered the area of distribution of the virus, its epidemiological danger, phylogenetic relationship, data on the sequencing of the horsepox virus genome and the likelihood of its use in the creation of the first vaccines, as well as obtaining its chimeric copy, on the basis of which a new smallpox vaccine was created – TNX-801.Results and discussion. The horsepox virus belongs to the poxvirus family, the orthopoxvirus genus. Classical horsepox has previously been reported only in Europe (France), Mongolia, and Kenya. The complete nucleotide sequence of the horsepox virus genome MNR-76 isolated in Mongolia has been determined. In addition to genes common to all orthopoxviruses, it includes intact genes specific only to this virus, the homologues of which are fragmented in the genome of other orthopoxviruses. Phylogenetic analysis of a number of orthopoxviruses was performed and a phylogenetic tree was constructed based on the conserved central region of the genome and some of the more variable terminal regions. The data obtained indicate that horsepox virus is most closely related to vaccinia virus and rabbitpox virus strains. Although horsepox is currently considered extinct, its pathogen may persist in unknown reservoirs. The data on the sequencing of the horsepox virus genome, strain MNR-76, suggest that horsepox virus could have served as the basis for the first smallpox vaccines. A chimeric copy of the horsepox virus was obtained using synthetic biology, which was used to create a new smallpox vaccine, TNX-801. On its basis, a recombinant vaccine against SARS-CoV-2 was constructed. The restoration of "extinct viruses" using synthetic biology methods has led to intense debates about the benefits and risks of such research.Conclusion. It cannot be ruled out that the use of modern genetic engineering technologies may lead not only to the development of effective vaccines, but also to the production of new orthopoxviruses pathogenic for humans and animals, or to the reintroduction of smallpox, which is especially dangerous in the context of the virtual absence of smallpox immunity in the population and international control over experiments in the synthetic biology of dangerous pathogens.

The aim of the study was to obtain and characterize a recombinant vaccine for the immunoprophylaxis of infections caused by Pseudomonas aeruginosa. A set of two highly immunogenic P. aeruginosa recombinant proteins was used to create related vaccine. The first vaccine component was presented by recombinant outer membrane protein F (OprF), whereas the second one — by recombinant atoxic exotoxin A (toxoid). These antigens allowed to develop vaccine inducing immune response against the surface bacterial cues and promote production of neutralizing antibodies against exotoxin A, one of the most dangerous P. aeruginosa pathogenicity factors. Recombinant proteins were synthesized in Escherichia coli cells and isolated by two-step purification. In case of recombinant OprF protein, it was initially (stage 1) isolated as a precipitate containing hydrophobic fraction of producer cell-derived proteins, whereas for recombinant toxoid we purified inclusion bodies. At stage 2, a Ni Sepharose column affinity chromatography was performed. Next, purified recombinant proteins were dialyzed against buffer solution containing 50 mМ Tris-HCl (pH 9.0) and 0.01% Tween 20 followed by filtration sterilization. Three lots of the Pseudomonas Recombinant Vaccine (PRV) were obtained for the study, wherein the recombinant antigens were absorbed with aluminum hydroxide. Recombinant OprF and toxoid protein in vaccine formula were used at a dose of 25 μg and 50 μg, respectively. Absorption completeness of the recombinant antigens within the vaccine was evaluated by polyacrylamide gel electrophoresis with PRV preparations concentrated by ultrafiltration in spin columns. Authenticity of recombinant vaccine was assessed by using customized method by desorbing antigens followed by ultrafiltration concentration in spin columns. Final concentrated desorbed vaccine preparations were analyzed by polyacrylamide gel electrophoresis and immunoblotting, which allowed to confirm presence of specific recombinant antigens in the vaccine. The experimental PRV series demonstrated specific activity (protective properties) after inoculation in animals (mice) by using a dual vaccination protocol, wherein mice were immunized intraperitonially twice with a two-week interval. Next, two weeks later mice were infected by toxigenic P. aeruginosa strain PA-103 culture cells. The index of protective efficiency (IE) for experimental vaccine series was at least a value of three (IE: 3.0–3.3) that was by 1.5-fold higher than that for using single vaccine components (IE: 2.0–2.3). Thus, we confirmed an additive effect of using a set preparation to protect against infection caused by toxigenic Pseudomonas aeruginosa strains.

Development of vaccines with a broad-spectrum of protection is one of the priorities in the programs of influenza prevention. Recently, the conserved fragments of influenza virus proteins (M1, M2, NP, the second subunit of the hemagglutinin HA2) provoke interest of investigators as the object of the development a broad-spectrum vaccines. Low immunogenicity present a problem when developing vaccines based on such conserved fragments. However, fusion of low immunogenic antigens into the high immunogenic carrier protein may significantly enhance their immunogenicity. The candidate vaccine protein Flg-HA2-2-4M2e was developed which containins two highly conserved viral antigens (the ectodomain of the M2 protein (M2e), 76130 region of the second subunit of HA2), fused with flagellin as a carrier protein. Flagellin (bacterial flagella protein) is a natural ligand of TLR-5, and has a strong adjuvant activity at different ways of its administration. The purpose of this study was to assess development of humoral and T cell immune response, along with broad-spectrum protection after mice immunization with the candidate Flg-HA2-2-4M2e vaccine protein. Mice were immunized intranasally three times with two-week intervals. Two weeks after the final immunization, the mice were challenged at the 5 LD50 dose with influenza viruses A/California/07/09 (H1N1) pdm09 (phylogenetic group I), or A/Shanghai/2/2013 (H7N9) (phylogenetic group II). The results obtained in this study showed induction of strong M2e-specific humoral response (serum IgG and A) in the immunized mice. Immunization with recombinant protein stimulated formation of M2e-specific and virus-specific CD4+ and CD8+T cells in lung which produced TNFα or IFNγ. Production of antigen-specific effector and central memory T cells was also detected in lungs of immunized mice. The formation of cross-protective immunity in immunized mice was demonstrated in a model of lethal influenza infection. The experimental animals were almost completely protected from the high dose of the pandemic virus A/H1N1pdm09, and highly pathogenic avian influenza A/H7N9 (90-100% survival). We also evaluated the changes of antigen-specific immune response in immunized mice after sublethal infection with A/H3N2 influenza virus. Mice of control and experimental groups were infected with MID100 of influenza virus A/Aichi/2/68 (H3N2). It was shown that the M2e-specific response (IgG, IgA) was significantly increased in immunized mice after sublethal infection with influenza virus A/H3N2, and we detected the changes in profile of M2e-specific IgG subclasses. Following sublethal infection in immunized mice, the proportion of M2e-specific IgG2a was increased 10-fold. The results showed that the recombinant protein Flg-HA2-2-4M2e is a promising candidate for development of universal vaccines, which induces a protective humoral and T-cell response to conserved viral epitopes and protects against influenza A viruses of both phylogenetic groups.

Generating cross-reactive vaccines aimed at targeting all human influenza A virus subtypes is among high priority tasks in contemporary vaccinology. Such vaccines will be primarily demanded during pre-pandemic period as well as used to prime some population cohorts prior to vaccination with standard vaccines containing area-relevant epidemic virus. Unlike routine approach universal vaccines do not induce a sterilizing immunity, but significantly ameliorate overt infection and probable complications. Our study was aimed at evaluating characteristics of immune response in experimental animals primed with a candidate universal vaccine challenged with sublethal influenza A virus infection. Mice were immunized intranasally with the recombinant protein FlgH2-2-4M2e containing conservative peptides derived from two influenza A virus proteins: M2 protein ectodomain and 76–130 amino acid sequence from the second hemagglutinin (HA2) subunit genetically linked to bacterial flagellin protein, which is a ligand for Toll-like receptor 5 (TLR5). Control mice received saline. Two weeks after immunization, mice from both groups were infected with a sublethal dose of A/Aichi/2/68 AN3N2 influenza virus strain. Level of immunoglobulins G and A in the blood sera and bronchoalveolar lavages (BAL) were determined two weeks after immunization and 1 month post infection. Percentage of lung CD4+ T and CD4+ Tem (CD44+CD62L–) cells secreting cytokines TNFα, IFNγ, IL-2 was determined. Immunized vs. control mice responded to sublethal infection with the influenza virus by insignificant weight loss and more pronounced production of vaccine peptide-specific (M2e and aa76–130 HA2) and pan-influenza A/Aichi/2/68 virus IgG and A in the blood sera and BAL. After challenge the number of CD4+ T cells secreting cytokines TNFα and/or IL-2 in immunized mice significantly exceeded counterpart T cells in unimmunized animals that was true for both CD4+T and CD4+ Tem cells. Memory CD4+ T cells were previously shown to play a key role in the prime-boost event and heterosubtypic immune response. Thus, we were able to demonstrate a priming effect for recombinant cross-protective vaccine used in our experiment.

Current areal of anthrax among live stock and wild animals covers almost all the continents. The cause of human infection is conventionally considered to be the contact with diseased animals in the process of carrying for an animal, forced slaughter or further trimming of carcasses, as well as contact with infected raw materials of animal origin. Licensed vaccines has made an invaluable contribution to the improvement of epidemiological situation on anthrax, however, development of the vaccines complying with current scientific progress remains relevant. We have constructed a vaccine prototype containing recombinant protective antigen and S-layer EA1 protein and added state-of-the-art CpG 2006 adjuvant to the formulation. We have demonstrated the advantage of the lyophilized form of the preparation. Objective — obtainment of anthrax vaccine prototype in lyophilized state and assessment of the prototype compliance with the requirements to vaccine preparations. Materials and methods. Isolation of rPA and EA1 protein was carried out using producer-strain B. anthracis 55ΔTПА-1Spo– on an integrated end-to-end manufacturing line, including concentration, diafiltration and two-phase chromatography. CpG 2006 adjuvant was synthesized according to known sequences. Components were mixed and lyophilized in sublimation unit. Combination of 1% sucrose and 3% glycine was used as cryoprotector. Effectiveness and safety of the preparation were evaluated on the model of BALB/c mice and guinea pigs applying immunological, morphometric, and histological assays. Antibody titers in sera of immunized animals were evaluated using standard ELISA procedures. Protective properties were investigated through LD50 values for the test-strain used on immunized and control animals and immunity index. Results. We have performed complex investigation of the anthrax vaccine prototype, containing B. anthracis 55ΔTПА-1Spo– proteins as main and supplementary antigens, as well as CpG 2006 adjuvant, stabilizers and preservative agent. The prototype meets the requirements to immunobiological medicinal drugs by all physical-chemical properties. Our preparation does not have a toxic effect on the organism of laboratory animals in case of subcutaneous and intraperitoneal administration of single human dose. Pathomorphological study of guinea pigs’ organs immunized with double dose of the vaccine prototype has not revealed any evidence of damaging effect on the cells and tissues of macroorganism. The prototype protects BALB/c mice from the infection with B. anthracis 71/12 test-strain.

Virus-like HBc particles formed as a result of the self-assembly of the nuclear antigen of the hepatitis B virus can be used as a highly immunogenic carrier for the presentation of foreign epitopes when creating recombinant vaccines. We use this vehicle to create influenza vaccines based on the conservative antigens of the influenza virus, the extracellular domain of the transmembrane protein M2 (M2e) and the fragment of the second subunit of hemagglutinin (HA2). Presentation on the surface of HBc particles should improve the immunogenicity of these peptides. Using genetic engineering techniques, we obtained a fusion protein in which the HA2 sequence is attached to the N-terminus of the HBc antigen, and the M2e peptide is included in the immunodominant loop region exposed on the surface of HBc particle. The hybrid protein expressed in Escherichia coli and purified under denaturing conditions formed virus-like HBc particles after refolding in vitro. Refolding of this protein in the presence of a previously denatured HBc antigen carrying no inserts resulted in formation of mosaic virus-like particles. The developed method will allow construction of mosaic HBc particles carrying different target epitopes of the influenza virus by combining the corresponding modified HBc proteins, which opens the possibility of creating vaccines with a wider spectrum of protection.

One of the main problems in the area of influenza prophylaxis and pandemic prevention is the development of cross-reactive vaccines, i.e. vaccines directed against all subtypes of human influenza viruses. Such vaccines are being developed in many countries for more than 10 years. A number of vaccines are presently undergoing clinical trials. We created Uniflu candidate vaccine based on recombinant HBc4M2e protein consisting of 4 tandem-connected copies of the highly conserved ectodomain of M2 protein of the influenza A virus. These 4 copies were genetically fused to the carrier protein, namely hepatitis B core antigen. Commercially available Derinat was used as adjuvant in the candidate vaccine. Preclinical studies on laboratory animals (mice, ferrets) demonstrated that immunization with Uniflu leads to significantly higher level of specific immunoglobulins in the blood and bronchoalveolar lavages. Moreover, it produces immunoglobulins belonging to subtype IgG2a that is the most important mediator of antibody-dependent cytotoxicity. The vaccine under review stimulates the proliferation of T-lymphocytes, as well as the formation of CD4+ and CD8+ T-cells synthesizing ɣ-IFN. When infected with the lethal doses (5 LD50) of influenza A viruses of the subtypes H1N1, H2N2, H3N2, and H1N1pdm09, immunized animals typically developed mild form of illness. This kept them alive in 90-100% of cases, which demonstrated almost complete protection from death. Replication of the virus in the lungs of immunized mice was reduced by 1.8-4.8 log10. High immunogenicity of the vaccine, and reduced clinical symptoms following experimental infection, were demonstrated in ferrets as well. The developed recombinant vaccine Uniflu has high specific activity and cross-protection. Uniflu can be proposed as pre-pandemic vaccine, provided that it passes clinical trials.

The influenza virus is the most unique in the level of variability of antigenic and biological properties. Because of constant mutations into genes coding surface viral proteins, in modern vaccines it is necessary to replace 1–2 virus components annually. Traditional influenza vaccines are the strain – specific and have limited efficiency in prevention of new strains of influenza viruses. In this regard, creation of influenza vaccines based on conserved determinants of viral proteins with broad spectrum protection and the short period of production is one of priority tasks which decision will lead to real control of an influenza infection. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based the combination of conserved viral proteins or peptides. The goals of this study: to develop the candidate recombinant flu vaccine based on the two conserved influenza proteins (М2 and НА); to investigate immune response; and to measure the protection activity in an animal model. Results: In this study we investigated the humoral and T-cell response in mice after intranasal immunization with recombinant proteins (Flg-4M2ehs and Flg-HA2-2-4M2ehs). Both proteins induce a robust M2e-specific humoral and CD4+ T-cell response in mice lung. The recombinant protein with two target antigens (M2e and HA2) induces virusspecific CD4+ and CD8+ T-cell response and full protection (100% survival) of mice from lethal challenge human and avian influenza viruses A (A/H3N2, A/H2N2, A/H5N1). In mice immunized with Flg-4M2ehs, the survival after lethal challenge was 60–75%. Conclusion: Our results show an essential role of a conserved fragment of the HA2 in the formation of protective T-cell response and protection of mice from lethal challenge with influenza viruses A of various subtypes. The prospects of the development of vaccine formulation based on two conserved antigenic determinants of influenza virus A are shown.

Abstract. Design of an effective and safe vaccine against pathogenic streptococci is still on the agenda, in spite of numerous attempts in this area undertaken by different laboratories. In order to improve immunogenicity of recombinant vaccine preparations, a selection of effective adjuvants is necessary. Previously, two recombinant GBS polypeptides P6 and ScaAB were found to be immunogenic, and their injection in separate preparations or mixed manner boosted production of specific and protective antibodies with high affinity. Four different adjuvants (Freund adjuvant, aluminum hydroxide, Bestim and Interleukine-1β) have been tested for immunization of mice with single polypeptides, or with their mixtures. As a result of vaccination, it was demonstrated that aluminum hydroxide was providing the most desirable immunological parameters of immune response among the adjuvants tested. A mixture of polypeptides containing aluminum hydroxide was found to produce specific antibodies with better opsonizing activity against group B streptococci. (Med. Immunol., 2008, vol. 10, N 2-3, pp 215-222).

Presented are the results of experiments on the construction of Escherichia coli Top10 (pTrcSuB) strain, perspective for the development of recombinant cholera vaccine for peroral use. The obtained strain possesses inducible expression of cholera toxin B subunit. Peculiarities of cloned gene expression in strain-producer and the conditions of its maintaining were studied. The proposed approach will allow to obtain effective and safe cholera vaccines.