BackgroundEbola virus disease (EVD) is one of the most dangerous and lethal diseases affecting humans. There are several licensed vaccines against EVD, but it remains one of the priority diseases for research and development of effective vaccines.MethodsA double-blind randomized placebo-controlled trial was performed to evaluate safety and immunogenicity of rVSV- and rAd5-vectored vaccine GamEvac-Combi in healthy adults of both sexes between 18 and 60 years. Safety and immunogenicity were assessed during the observation period of 12 months. Immunogenicity was assessed with GP-specific ELISA, IFN-γ ELISA, and plaque pseudoneutralization assay.ResultsVaccinated participants showed marked GP-specific IFN-γ response at day 28 and neutralizing response at day 42 (GMT = 32.6, seroconversion rate 96.3%). GP-specific IgG antibody levels in vaccinated participants peaked at day 42 (GMT = 9345) and persisted for a year after vaccination (GMT = 650).ConclusionThe vaccine showed favorable safety profile and induced robust cell-mediated immune response and strong humoral immune response that lasts at least for a year from the start of vaccination.Clinical trial registrationClinicalTrials.gov, identifier NCT03072030; Pan African Clinical Trial Registry, identifier PACTR201702002053400.

This work highlights the importance of nucleotide sequence optimization of a target gene in the development of vaccines. The purpose of the study was to compare the immunogenicity of recombinant adenoviruses expressing the S glycoprotein gene of the SARS-CoV-2 Omicron (BA.5.3.1) variant with an optimized (Ad5-S-BA5opt) or nonoptimized (Ad5-S-BA5) nucleotide sequence. Recombinant adenoviruses were grown on HEK293 cell culture and purified by ultracentrifugation in a stepwise CsCl density gradient. The resulting adenovirus preparations were characterized by the authenticity (by real-time PCR and immunoblotting), the number of viral particles (spectrophotometrically), and the infectious titer (by endpoint dilution titration TCID50). The titer of RBD-specific IgG antibodies in the blood serum of immunized animals was assessed by enzyme-linked immunosorbent assay (ELISA). Based on the results of studies in an animal model, a vector expressing the optimized gene (Ad5-S-BA5opt) resulted in the formation of higher levels of IgG-specific antibodies compared with a similar vector containing the wild-type protein gene. It is particularly interesting that an approach aimed solely at optimizing the nucleotide sequence of the target gene, and not other vector components, can significantly contribute to changes in the immunogenicity. The obtained results are important for the development of new, more effective strategies for creating vaccines and gene therapy drugs. Optimization of the nucleotide sequence can lead to an improved mRNA stability and a significant increase in translational efficiency and, as a consequence, to enhanced protective properties of the candidate drug. It can be supposed that this method will be promising for the development of vaccines against viruses containing low-immunogenic antigens.

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

Introduction. The World Health Organization considers the values of antibody titers in the hemagglutination inhibition assay as one of the most important criteria for assessing successful vaccination. Mathematical modeling of cross-immunity allows for identification on a real-time basis of new antigenic variants, which is of paramount importance for human health. Materials and methods. This study uses statistical methods and machine learning techniques from simple to complex: logistic regression model, random forest method, and gradient boosting. The calculations used the AAindex matrices in parallel to the Hamming distance. The calculations were carried out with different types and values of antigenic escape thresholds, on four data sets. The results were compared using common binary classification metrics. Results. Significant differentiation is shown depending on the data sets used. The best results were demonstrated by all three models for the forecast autumn season of 2022, which were preliminary trained on the February season of the same year (Auroc 0.934; 0.958; 0.956, respectively). The lowest results were obtained for the entire forecast year 2023, they were set up on data from two seasons of 2022 (Aucroc 0.614; 0.658; 0.775). The dependence of the results on the types of thresholds used and their values turned out to be insignificant. The additional use of AAindex matrices did not significantly improve the results of the models without introducing significant deterioration. Conclusion. More complex models show better results. When developing cross-immunity models, testing on a variety of data sets is important to make strong claims about their prognostic robustness.

Bacillus cereus sensu lato comprises genetically, morphologically, and physiologically similar gram-positive spore-forming bacterial species with high pathogenic potential, such as B. anthracis, B. cereus, and B. thuringiensis. Toxin-producing strains of B. cereus s.I. pose a major threat to human health. The high degree of similarity between these species makes it very difficult to identify them and to take adequate measures to treat the diseases they cause. Previously, we characterized the clinical isolate CCGC 19/16 belonging to B. cereus s.l. that exhibited features of both B. cereus and B. cytotoxicus. In the present work, CCGC 19/16 was identified as B. cytotoxicus using multilocus sequence typing (MLST) and mass spectrometric analysis. It was also shown that, unlike other representatives of the B. cytotoxicus species, strain CCGC 19/16 is not thermotolerant. Unlike B. cereus, strain CCGC 19/16 is sensitive to most antibiotics and shows increased motility. Like B. cereus strain CCGC 19/16 forms β-hemolysis zones in blood agar. In addition, it has been shown that prolonged storage of samples prior to analysis can lead to misidentification of the isolate. Our results indicate that "rapid methods" of analysis using single genes have insufficient resolving power in the identification of B. cereus s.l. species. The combination of MLST analysis with MALDI-TOF MS provides sufficient resolution.

Objectives. To develop a technology for obtaining recombinant antibodies in a suspension culture of human HEK293 cells using transduction with recombinant adenovirus serotype 5 (rAd5) carrying genes expressing heavy and light chains of antibodies on the example of two broadspectrum anti-influenza antibodies 27F3 and CR9114.Methods. Ad5-27F3-H, Ad5-CR9114-H, and Ad5-27F3-L recombinant adenoviruses carrying the 27F3 antibody heavy chain gene, CR9114 antibody heavy chain gene, and 27F3 light chain gene, respectively, were generated using the AdEasy™ Adenoviral vector system. To accumulate preparative amounts of recombinant r27F3 and rCR9114 antibodies, the HEK293 suspension cell line was transduced with recombinant adenoviruses carrying genes for heavy and light chains of antibodies. The cells were cultured in a wave-type bioreactor. Chromatography was used to purify recombinant antibodies from the culture medium. After analyzing the molecular weights of purified antibodies using protein electrophoresis, their ability to interact with influenza A and B viruses was analyzed using the Western blot technique, while their ability to neutralize influenza A and B viruses was evaluated using the virus neutralization assay.Results. A method for the accumulation and purification of recombinant r27F3 and CR9114 antibodies from the culture medium of a suspension culture of human cells following transduction with its recombinant adenoviruses carrying the genes for heavy and light chains of these antibodies was developed. The ability of the r27F3 antibody to interact with and neutralize influenza A viruses of group 1 (except influenza A virus subtype H2) and group 2 was shown. The ability of the rCR9114 antibody to interact with influenza A viruses of group 1 and influenza B viruses, as well as to neutralize influenza A viruses of group 1, was demonstrated.Conclusions. A technology for obtaining recombinant antibodies in a suspension culture of HEK293 cells using transduction with recombinant adenoviruses carrying genes expressing heavy and light chains of antibodies was developed along with a confirmation of their specificity.

— Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

—As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation ( R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus.

Introduction.Recombinant adenoviral vectors become the leading technological platform in the development and production of modern vaccines.In Russia and other countries of the world, vaccines based on recombinant adenoviruses have been designed and registered against the Ebola virus and SARS-CoV-2 coronavirus infections.Clinical trials of vaccines against infl uenza, Marburg virus, human papillomaviruses are ongoing.The target antigen encoded in the DNA of the adenoviral vector is expressed in the body of the vaccine recipient, and adaptive immune responses develop against this target antigen protein.The vector particle is viral and contains dozens of viral antigens.Therefore, along with immune responses to the encoded target antigen, immune responses to the antigens of the vector itself can develop in the body of the vaccinated.The aim of this work is to investigate the intensity and quality of two immune responses that are different in their antigenic specifi city, i.e. directed against the target coronavirus antigen and adenovirus vector antigens.Material and methods.In C57BL/6 mice, the intensity of immune responses of CD4-and CD8-T-cells was studied in response to immunization with a recombinant adenoviral vector encoding the SARS-CoV-2 S-protein (Ad5-S). 2 and 3 months after immunization, the number and antigenic specifi city of CD4-and CD8-T memory cells were determined in the spleen of mice.The T-cell response was induced in vitro in co-culture with antigen-presenting dendritic cells.Purifi ed CD4-and CD8-T-cell populations were obtained by sorting on a BD FACS Aria II laser fl ow sorter machine.Antigen-presenting cells were transduced with the Ad5-S adenoviral vector encoding the SARS-CoV-2 S-protein or a control recombinant adenoviral vector without a target insert (Ad5-0).The T cells response was determined by ELISPOT according to the number of cells secreting IFN-γ.In some experiments, to reactivate CD4 T cells, antigen-presenting dendritic cells were loaded with a recombinant RBD-fragment of the SARS-CoV-2 S-protein.To enhance the T-cell response, antigen presenting dendritic cells were stimulated with a TLR4 agonist.Results.A single intranasal immunization with the Ad5-S vector at a dose of 10 8 PFU induced strong systemic T-cell immune responses in C57BL/6 mice.Two months after immunization, about 100 000-200 000 antigen-reactive memory T-cells were found in the spleen of mice, secreting IFN-γ when reactivated in vitro by dendritic cells presenting the target SARS-CoV-2 S-antigen.Most antigen-reactive CD8-T memory cells were specifi c for the SARS-CoV-2 S-antigen.The concentration of such cells after immunization with the Ad5-S vector exceeds 1 % of all CD8-T cells.The number of antigen-reactive CD8-T memory cells specifi c to the adenoviral antigens of the vector was approximately 3-fold lower than the number of antigen-reactive CD8-T memory cells specifi c to the target S-antigen.The intensity

Актуальные направления современной иммунологии

One of the primary tasks in vaccine design and development of immunotherapeutic drugs is to predict conformational B-cell epitopes corresponding to primary antibody binding sites within the antigen tertiary structure. To date, multiple approaches have been developed to address this issue. However, for a wide range of antigens their accuracy is limited. In this paper, we applied the transfer learning approach using pretrained deep learning models to develop a model that predicts conformational B-cell epitopes based on the primary antigen sequence and tertiary structure. A pretrained protein language model, ESM-1v, and an inverse folding model, ESM-IF1, were fine-tuned to quantitatively predict antibody-antigen interaction features and distinguish between epitope and non-epitope residues. The resulting model called SEMA demonstrated the best performance on an independent test set with ROC AUC of 0.76 compared to peer-reviewed tools. We show that SEMA can quantitatively rank the immunodominant regions within the SARS-CoV-2 RBD domain. SEMA is available at https://github.com/AIRI-Institute/SEMAi and the web-interface http://sema.airi.net.

Ebola fever is an acute, highly contagious viral disease with a mortality rate that can reach 90%. There are currently no licensed therapeutic agents specific to Ebola in the world. Monoclonal antibodies (MAbs) with viral-neutralizing activity and high specificity to the Ebola virus glycoprotein (EBOV GP) are considered as highly effective potential antiviral drugs. Over the past decade, nanobodies (single-domain antibodies, non-canonical camelid antibodies) have found wide use in the diagnosis and treatment of various infectious and non-infectious diseases. In this study, a panel of nanobodies specifically binding to EBOV GP was obtained using recombinant human adenovirus 5, expressing GP (Ad5-GP) for alpaca (Vicugna pacos) immunization, for the first time. Based on specific activity assay results, affinity constants, and the virus-neutralizing activity against the recombinant vesicular stomatitis virus pseudotyped with EBOV GP (rVSV-GP), the most promising clone (aEv6) was selected. The aEv6 clone was then modified with the human IgG1 Fc fragment to improve its pharmacokinetic and immunologic properties. To assess the protective activity of the chimeric molecule aEv6-Fc, a lethal model of murine rVSV-GP infection was developed by using immunosuppression. The results obtained in lethal model mice have demonstrated the protective effect of aEv6-Fc. Thus, the nanobody and its modified derivative obtained in this study have shown potential protective value against Ebola virus.

Вирусы гриппа птиц подтипов Н1 и Н5 участвовали в формировании высокопатогенных вариантов вирусов, вызвавших пандемии и панзоотии в XX–XXI веках. С целью оценки зоонозного потенциала вирусов этих подтипов, выделенных от диких уток в черте Москвы, была проведена адаптация вирусов к размножению в легких мышей, изучены их фенотипические свойства и идентифицированы генетические изменения, возникшие при адаптации. Изначально апатогенные для мышей вирусы A/duck/Moscow/4970/2013 (H1N1) и A/duck/Moscow/4182 C/2010 (H5N3) после 7–10 пассажей через легкие мышей изменили фенотип на патогенный. Полногеномное секвенирование выявило в адаптированных к мышам вирусах 2 аминокислотные замены в вирусе гриппа H1N1 (Glu627Lys в белке PB2 и Asp35Asn в гемагглютинине (HA) — нумерация по H3) и 6 мутаций в белках вируса H5N3 (Glu627Lys в PB2, Val113Ala в PB1, Ser82Pro в PB1 F2, Lys52Arg в HA2, Arg65Lys в NP и Ser59Ile в NA). Возрастание вирулентности для мышей, скорее всего, обусловлено общей для обоих вирусов заменой – Glu627Lys в белке PB2. Замена Asp35Asn в HA адаптированного к мышам вируса гриппа H1N1 ассоциирована с возрастанием значения рН конформационного перехода HA с 5.0 до 5.5 относительно HA дикого вируса. Обнаруженные в адаптированном варианте H5N3 мутации в белках НА, NA и PB1 F2 — уникальные. Мутации Glu627Lys в PB2, Arg65Lys в NP и Val113Ala в PB1, скорее всего, носят адаптационный характер.

Ebola virus disease (EVD) (former Ebola hemorrhagic fever) is one of the most dangerous infectious diseases affecting humans and primates. Since the identification of the first outbreak in 1976, there have been more than 25 outbreaks worldwide, the largest of which escalated into an epidemic in 2014-2016 and caused the death of more than 11,000 people. There are currently 2 independent outbreaks of this disease in the eastern and western parts of the Democratic Republic of the Congo (DRC) at the same time. Bats (Microchiroptera) are supposed to be the natural reservoir of EVD, but the infectious agent has not yet been isolated from them. Most animal viruses are unable to replicate in humans. They have to develop adaptive mutations (AM) to become infectious for humans. In this review based on the results of a number of studies, we hypothesize that the formation of AM occurs directly in the human and primate population and subsequently leads to the development of EVD outbreaks.

The influenza virus infection claims ~650,000 lives annually. Taking into account the evolving resistance of the pathogen to antiviral drugs and the waning effectiveness of vaccination among certain populations, new approaches to the treatment of influenza are needed. The current study is aimed at obtaining single-domain antibodies (Nanobodies®) to the highly conserved stem domain of influenza A virus hemagglutinin by phage display. Two high-affinity neutralizing clones of Nanobodies® with a particular specificity were selected; they ensured 100% neutralization of the H1N1 and H5N2 influenza viruses in vivo. The obtained data demonstrate that it is possible to develop highly effective VHH-based drugs for the treatment of influenza.

The objective of the study is to carry out preclinical research on the immunogenicity and protective properties of the trivalent influenza vaccine AdeVac-Flu in laboratory mice. The AdeVac-Flu vaccine is a mixture of recombinant human adenoviruses of the fifth serotype expressing the hemagglutinin genes of the A/California/07/2009(H1N1), A/Perth/16/2009(H3N2), and B/Brisbane/60/2008 influenza vaccine strains, as well as the molecular adjuvant Immunomax. The Ultravac live influenza vaccine (Microgen, Russia) was used as a reference. To study the protective properties of the vaccine, mice of the line Balb/c were immunized once intranasally and, 28 days after immunization, infected with lethal doses of influenza viruses A H1N1, H3N2, and B. The level of viral shedding from the lungs of mice was assessed on the third and sixth days after infection by titration of lung homogenates on MDCK cell culture. The level of specific antibodies to influenza viruses was determined by indirect ELISA, HAI, and SVN. It was shown that the trivalent influenza vaccine AdeVac-Flu is more immunogenic than the reference drug. The levels of protection against homologous strains of influenza viruses are either slightly less than or comparable to those of the reference drug. In this case, it was shown that the AdeVac-Flu, like the live influenza vaccine, effectively protects mice from a heterologous strain of the influenza virus, although the mice exhibited excretion of the virus from the lungs and a slight decrease in body weight (less than 10%). It has been shown that the AdeVac-Flu vaccine based on recombinant human adenoviruses of the fifth serotype expressing hemagglutinin genes of A/California/07/2009 (H1N1), A/Perth/16/2009 (H3N2), and B/Brisbane/60/2008 influenza viruses has immunogenicity and protective properties in relation to both homologous and heterologous (within the subtype) strains of the influenza virus.

Иммуногенные и защитные свойства рекомбинантного аденовируса человека 5-го серотипа, экспрессирующего ген гликопротеина G вируса бешенства вакцинного штамма РВ-971 Федеральное государственное бюджетное учреждение «Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф.Гамалеи» Министерства здравоохранения Российской Федерации

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

Influenza is a highly contagious disease that causes annual epidemics and occasional pandemics. Birds are believed to be the source of newly emerging pandemic strains, including highly pathogenic avian influenza viruses of the subtype H7. The aim of the study: to evaluate the ability of the recombinant human adenovirus, serotype 5, which expresses genes of influenza A highly conserved antigens (ion channel M2 and nucleoprotein NP), to provide protection to laboratory mice against infection with a lethal dose of avian influenza virus, subtype H7. To achieve this goal, it was necessary to adapt influenza A virus, subtype H7 for reproduction in the lungs of mice, to characterise it, and to use it for evaluation of the protective properties of the recombinant adenovirus. Materials and methods: avian influenza virus A/Chicken/NJ/294508-12/2004 (H7N2) was adapted for reproduction in the lungs of mice by repeated passages. The adapted strain was sequenced and assessed using hemagglutination test, EID50 and LD50 for laboratory mice. BALB/c mice were immunised once with Ad5-tet-M2NP adenovirus intranasally, and 21 days after the immunisation they were infected with a lethal dose (5 LD50) of influenza virus A/Chicken/NJ/294508-12/2004 (H7N2) in order to assess the protective properties of the recombinant adenovirus. The level of viral shedding from the lungs of the infected mice was evaluated by titration of the lung homogenates in MDCK cell culture on days 3 and 6 after infection. The level of specific antibodies to H7 avian influenza virus was determined by indirect enzyme immunoassay. Results: the use of Ad5-tet-M2NP adenovirus for immunisation of the mice ensured 100% survival of the animals that had disease symptoms (weight loss) after their infection with the lethal dose (5 LD50) of H7 avian influenza virus. The study demonstrated a high post-vaccination level of humoral immune response to H7 avian influenza virus. The virus titer decreased significantly by day 6 in the lungs of mice that had been immunised with Ad5-tet-M2NP compared to the control group. Conclusion: the Ad5-tetM2NP recombinant adenovirus can be used to create a candidate pandemic influenza vaccine that would protect against avian influenza viruses, subtype H7, in particular.