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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 aEv6Fc, 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 aEv6Fc. Thus, the nanobody and its modified derivative obtained in this study have shown potential protective value against Ebola virus.

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 20-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection.

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

Introduction Middle East respiratory syndrome (MERS) is acute inflammatory disease of respiratory system with a high mortality, caused Ьу coronavirus MERS-CoV At present moment, we still lack specific therapeutic preparations and vaccines against MERS Vaccine administration can help to limit the spread of the disease and lower the mortality Duration of vaccine-induced immune response is one of the key characteristics of a vaccine, which is connected with duration of its protective effectiveness Unfortunately, the data on duration of vaccine-induced immune response against MERS is scarce The aim of the study was to determine duration of humoral immune response in mice and primates and duration of protective immune response in mice after immunization with the heterologous virus-vectored vaccine against MERS (BVRS-GamVac-Combi), developed earlier Ьу our research group Material and methods To study duration of humoral immune response, we used mice of C57BL/6 strain and common marmosets Animals were immunized with the vaccine BVRS-GamVac-Combi, based on recomЬinant adenoviral vectors rAd26 and rAd5 Antigen-specific-antibody titers were determined with ELISA, virus-neutralizing antibody titers were measured with virus neutralization assay using MERS-CoV (EMC/2012) To study duration of protective immune response, we used a model of lethal infection on transgenic mice, carrying human DPP4 gene of viral receptor Results In present research, we showed that vaccination of animals with BVRS-GamVac-Combi induced robust humoral immune response, which persisted at least 18 months after immunization In addition, our vaccine protected 100 % of animals from lethal infection for at least 7 months after immunization Concluslon Strength of vaccine-induced immune response is generally connected with a protective effectiveness of a vaccine One of the key problems of vaccine design is to find a way to provide as long and robust immune response as possible Duration of vaccine-induced immune response is one of the key characteristics of a vaccine, which demands quality control during multiple steps of a vaccine development Введение Ближневосточный респираторный синдром (БВРС) - это острое воспалительное заболевание дыхательной системы с высокой летальностью, возбудителем которого является коронавирус БВРС-КоВ В настоящее время в мире не существует специфических профилактических и терапевтических средств против БВРС Вакцино-профилактика позволит ограничить распространение данного заболевания и снизить летальность Одной из ключевых характеристик вакцин является длительность индуцируемого иммунного ответа, от которой зависит продолжительность протективного эффекта вакцины К сожалению, данных по длительности поствакцинального иммунного ответа для вакцин против БВРС сейчас недостаточно Цель исследования - определение длительности гуморального иммунного ответа у грызунов и приматов и протективного иммунного ответа после иммунизации комбинированной векторной вакциной против БВРС (БВРС-ГамВак-Комби), разработанной нами ранее Материал и методы Длительность гуморального иммунитета исследовали на мышах линии C57BL/6 и обыкновенных игрунках Животных иммунизировали вакциной БВРС-ГамВак-Комби на основе рекомбинантных векторов rAd26 и rAd5 Титр антиген-специфических антител определяли методом иммуноферментного анализа (ИФА) Титр вирус-нейтрализующих антител определяли с помощью реакции вирус-нейтрализации против вируса БВРС-КоВ (MERS-CoV EMC/2012) Длительность протективного иммунитета исследовали на модели летальной инфекции у трансгенных мышей, несущих ген человека DPP4, кодирующий рецептор к БВРС-КоВ Результаты Исследование длительности поствакцинального гуморального иммунного ответа у грызунов и приматов показало, что вакцинация животных БВРС-ГамВак-Комби индуцирует формирование напряженного гуморального иммунного ответа к гликопротеину S БВРС-КоВ, который сохраняется на протяжении не менее 18 мес Также было показано, что вакцинация позволяет защитить 100 % животных от летальной инфекции, вызванной БВРС-КоВ (MERS-CoV EMC/2012, 100 ЛД50/мышь), через 7 мес после иммунизации Заключение Напряженность поствакцинального гуморального иммунного ответа, как правило, связана с протективностью вакцины Одной из ключевых задач при дизайне вакцин является обеспечение наиболее длительного напряженного иммунного ответа Длительность поствакцинального иммунного ответа - одна из ключевых характеристик вакцин, которая требует контроля на различных этапах их разработки

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.

Background A heterologous recombinant adenovirus (rAd)-based vaccine, Gam-COVID-Vac (Sputnik V), showed a good safety profile and induced strong humoral and cellular immune responses in participants in phase 1/2 clinical trials. Here, we report preliminary results on the efficacy and safety of Gam-COVID-Vac from the interim analysis of this phase 3 trial. Methods We did a randomised, double-blind, placebo-controlled, phase 3 trial at 25 hospitals and polyclinics in Moscow, Russia. We included participants aged at least 18 years, with negative SARS-CoV-2 PCR and IgG and IgM tests, no infectious diseases in the 14 days before enrolment, and no other vaccinations in the 30 days before enrolment. Participants were randomly assigned (3:1) to receive vaccine or placebo, with stratification by age group. Investigators, participants, and all study staff were masked to group assignment. The vaccine was administered (0·5 mL/dose) intramuscularly in a prime-boost regimen: a 21-day interval between the first dose (rAd26) and the second dose (rAd5), both vectors carrying the gene for the full-length SARS-CoV-2 glycoprotein S. The primary outcome was the proportion of participants with PCR-confirmed COVID-19 from day 21 after receiving the first dose. All analyses excluded participants with protocol violations: the primary outcome was assessed in participants who had received two doses of vaccine or placebo, serious adverse events were assessed in all participants who had received at least one dose at the time of database lock, and rare adverse events were assessed in all participants who had received two doses and for whom all available data were verified in the case report form at the time of database lock. The trial is registered at ClinicalTrials.gov (NCT04530396). Findings Between Sept 7 and Nov 24, 2020, 21 977 adults were randomly assigned to the vaccine group (n=16 501) or the placebo group (n=5476). 19 866 received two doses of vaccine or placebo and were included in the primary outcome analysis. From 21 days after the first dose of vaccine (the day of dose 2), 16 (0·1%) of 14 964 participants in the vaccine group and 62 (1·3%) of 4902 in the placebo group were confirmed to have COVID-19; vaccine efficacy was 91·6% (95% CI 85·6–95·2). Most reported adverse events were grade 1 (7485 [94·0%] of 7966 total events). 45 (0·3%) of 16 427 participants in the vaccine group and 23 (0·4%) of 5435 participants in the placebo group had serious adverse events; none were considered associated with vaccination, with confirmation from the independent data monitoring committee. Four deaths were reported during the study (three [

Background: While the world is experiencing another wave of COVID-19 pandemic, global vaccination program is hampered by obvious shortage in supply of licensed vaccines. Development of new vaccines that are easy to manufacture and administer is highly desirable to overcome hurdles in vaccine scaling up and distribution, especially in developing countries. In the effort to satisfy vaccine demands we developed a new single dose vaccine based on recombinant adenovirus type 26 (rAd26) vector carrying the gene for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein – “Sputnik Light”. Methods: We conducted an open label, two-stage, prospective, non-randomized phase I-II trial aimed to assess safety, tolerability and immunogenicity of “Sputnik Light” vaccine in a single center in Russia. Primary outcome measures were antigen-specific humoral immunity (Anti-RBD-SARS-Cov2 antibodies measured by ELISA on days 1, 10, 28, and 42) and safety (number of participants with adverse events monitored throughout the study). Secondary outcome measures were antigen-specific cellular immunity (measured by antigen-dependent CD4+ and CD8+ T-cell proliferation, number of antigen-specific interferon-γ-producing cells as well as interferon-γ concentration upon antigen restimulation) and change in neutralizing antibodies (measured in SARS-CoV-2 neutralization assay). The trial is registered at ClinicalTrials.gov (NCT04713488). Findings: Between January 14 and 27, 2021, 150 participants were screened, of whom 110 were enrolled. Most of solicited adverse reactions were mild (66.4% from all vaccinees), few were moderate (5.5%). No serious adverse events were detected. Assessment of Anti-RBD-SARS-Cov2 antibodies revealed group with pre-existing immunity to SARS-CoV-2. Upon this finding we separated all safety and immunogenicity data according to pre-existing immunity to SARS-Cov2. There were notable differences in the vaccine effects on immunogenicity by the groups. Vaccination of seropositive (N=14) volunteers rapidly boosted RBD-specific IgGs from reciprocal geometric mean titer (​GMT) 594.4 at a baseline up to 26899 comparing to 29.09 in seronegative group (N=96) by day 10. By day 42 seroconversion rate reached 100% (93/93) in seronegative group with GMT 1648. At the same time in seropositive group seroconversion rate by day 42 was 92.9% (13/14) with GMT 19986. Analysis of neutralizing antibodies to SARS-CoV-2 showed 81.7% (76/93) and 92.9% (13/14) seroconversion rates by day 42 with median reciprocal GMT 15.18 and 579.7 in the seronegative and seropositive groups, respectively. Each test to assess cell-mediated immune reactions was carried out on a separate group of 30 volunteers on day 10 upon vaccination. Antigen-specific T cell proliferation, formation of IFNy-producing cells, as well as IFNy secretion were detected in 96.7% (26/27), 96% (24/25) and 96% (24/25) seronegative and in 100% (3/3) 100% (5/5) and 100% (5/5) seropositive vaccinees, correspondingly. Interpretation: The single dose rAd26 vector-based COVID-19 vaccine “Sputnik Light” has a good safety profile and induced strong humoral and cellular immune responses both in seronegative and seropositive participants. Further investigation is needed to assess the effectiveness of this vaccine against COVID-19 used for primary and secondary vaccination. Clinical Trial Registration Details: The trial is registered at ClinicalTrials.gov (NCT04713488). Funding Information: Russian Direct Investment Fund. Declaration of Interests: OVZ, TAO, IVD, OP, DVS, DMG, ASD, AIT, DNS, IBE, EAT, AGB, ASE, FMI, NAN, NLL, ASS, SVB, BSN, DYL, ALG report patents for an immunobiological expression vector, pharmaceutical agent, and its method of use to prevent COVID-19. All other authors declare no competing interests. Ethics Approval Statement: The trial was approved by the local ethic committee and was conducted with the approval of the Ministry of Health of Russian Federation in compliance with International Conference on Harmonization and National Good Clinical Practice guidelines and Declaration of Helsinki.

The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.

Ebola fever is an acute highly contagious viral disease characterized by severe course, high mortality and development of hemorrhagic syndrome (tendency to skin hemorrhage and bleeding of mucous membranes). The mortality rate of the disease 60–90%. Nowadays, there are no licensed specific therapeutic agents for Ebola in the world. Monoclonal antibodies (MAbs) having viral neutralizing activity with high specificity to the GP protein of the Ebola virus are considered as candidate highly effective antiviral drugs. In our study, for the first time a panel of mouse monoclonal antibodies specifically binding to EBOV GP protein was obtained using recombinant human adenovirus 5 serotype, expressing GP protein (Ad5-GP). The virus-neutralizing capacities of antibodies were evaluated on the Ebola virus cell infection model, as well as recombinant vesicular stomatitis virus pseudotyped by GP Ebola virus protein (rVSV-GP) cell infection model. Based on the results of virus neutralization, two most promising clones were selected, the specific and protective capacities of which were determined. The study of the protection of selected individual antibody clones, as well as their combinations on the model of lethal infection of rhesus macaques with Ebola virus showed that intravenous administration of a mixture of antibodies in the amount of 50 mg/kg 24 h after infection leads to the survival of 100% of the animals, while individual clones of antibodies possess partial protection (0–30%). The results of the study suggest the important role of antibodies in controlling replication of the Ebola virus in vivo and show the possibility of using a mixture of antibodies specific to the GP to protect against lethal infection with the Ebola virus in the post-infected mode of administration.

To avoid outbreaks of influenza virus epidemics and pandemics among human populations, modern medicine requires the development of new universal vaccines that are able to provide protection from a wide range of influenza A virus strains. In the course of development of a universal vaccine, it is necessary to consider that immunity must be generated even against viruses from different hosts because new human epidemic virus strains have their origins in viruses of birds and other animals. We have enriched conserved viral proteins-nucleoprotein (NP) and matrix protein 2 (M2)-by B and T-cell epitopes not only human origin but also swine and avian origin. For this purpose, we analyzed M2 and NP sequences with respect to changes in the sequences of known T and B-cell epitopes and chose conserved and evolutionarily significant epitopes. Eventually, we found consensus sequences of M2 and NP that have the maximum quantity of epitopes that are 100% coincident with them. Consensus epitope-enriched amino acid sequences of M2 and NP proteins were included in a recombinant adenoviral vector. Immunization with Ad5-tet-M2NP induced strong CD8 and CD4 T cells responses, specific to each of the encoded antigens, i.e. M2 and NP. Eight months after immunization with Ad5-tet-M2NP, high numbers of M2- and NP-responding "effector memory" CD44posCD62neg T cells were found in the mouse spleens, which revealed a long-term T cell immune memory conferred by the immunization. In all, the challenge experiments showed an extraordinarily wide-ranging efficacy of protection by the Ad5-tet-M2NP vaccine, covering 5 different heterosubtypes of influenza A virus (2 human, 2 avian and 1 swine).

Influenza is a highly contagious disease and one of the most wide spreaded infection in the world. Influenza type A strains are of very great epidemiological significance. A high degree of genetic variability leads to constant modification in the antigenic structure of influenza virus. Therefore, current influenza vaccines require periodic updating of the composition of the strains. At present, it is important to develop a universal vaccine that could protect against different strains of influenza A virus and it is can be based on the conserved antigens of the virus. Recombinant adenoviral vectors expressing genes of conserved viral antigens may be used for a promising candidate vaccine against influenza A. Using a homologous recombination method, in this study we have developed a recombinant adenoviral serotype 5 vector that expresses the genes of the M2 ion channel and nucleoprotein (NP) of influenza A virus. The genes of the consensus sequences of the M2 protein and NP of human influenza A virus were inserted into the structure of the viral genome. The expression of the M2 and NP antigens using the recombinant adenovirus vector in a permissive line of eukaryotic cells was detected by Western blot assay. The high immunogenicity of the recombinant adenovirus vector obtained was demonstrated by intranasal immunization of laboratory mice.

Anthrax is a particularly dangerous infectious disease that affects humans and livestock. It is characterized by intoxication, serosanguineous skin lesions, development of lymph nodes and internal organs, and may manifest itsself in either a cutaneous or septic form. The pathogenic agent is Bacillus anthracis, a grampositive, endospore-forming, rod-shaped aerobic bacterium. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. In this study, we designed three recombinant human adenovirus serotype-5-based vectors containing various modifications of the fourth domain of the B. anthracis protective antigen (PA). Three PA modifications were constructed: a secretable form (Ad-sPA), a non-secretable form (Ad-cPA), and a form with the protective antigen fused to the Fc fragment of immunoglobulin G2a (Ad-PA-Fc). All these forms exhibited protective properties against Bacillus anthracis. The highest level of protection was induced by the Ad-PA-Fc recombinant adenovirus. Our findings indicate that the introduction of the Fc antibody fragment into the protective antigen significantly improves the protective properties of the Ad-PA-Fc adenovirus against B. anthracis.

Summary Background We developed a heterologous COVID-19 vaccine consisting of two components, a recombinant adenovirus type 26 (rAd26) vector and a recombinant adenovirus type 5 (rAd5) vector, both carrying the gene for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (rAd26-S and rAd5-S). We aimed to assess the safety and immunogenicity of two formulations (frozen and lyophilised) of this vaccine. Methods We did two open, non-randomised phase 1/2 studies at two hospitals in Russia. We enrolled healthy adult volunteers (men and women) aged 18–60 years to both studies. In phase 1 of each study, we administered intramuscularly on day 0 either one dose of rAd26-S or one dose of rAd5-S and assessed the safety of the two components for 28 days. In phase 2 of the study, which began no earlier than 5 days after phase 1 vaccination, we administered intramuscularly a prime-boost vaccination, with rAd26-S given on day 0 and rAd5-S on day 21. Primary outcome measures were antigen-specific humoral immunity (SARS-CoV-2-specific antibodies measured by ELISA on days 0, 14, 21, 28, and 42) and safety (number of participants with adverse events monitored throughout the study). Secondary outcome measures were antigen-specific cellular immunity (T-cell responses and interferon-γ concentration) and change in neutralising antibodies (detected with a SARS-CoV-2 neutralisation assay). These trials are registered with ClinicalTrials.gov, NCT04436471 and NCT04437875. Findings Between June 18 and Aug 3, 2020, we enrolled 76 participants to the two studies (38 in each study). In each study, nine volunteers received rAd26-S in phase 1, nine received rAd5-S in phase 1, and 20 received rAd26-S and rAd5-S in phase 2. Both vaccine formulations were safe and well tolerated. The most common adverse events were pain at injection site (44 [58%]), hyperthermia (38 [50%]), headache (32 [42%]), asthenia (21 [28%]), and muscle and joint pain (18 [24%]). Most adverse events were mild and no serious adverse events were detected. All participants produced antibodies to SARS-CoV-2 glycoprotein. At day 42, receptor binding domain-specific IgG titres were 14 703 with the frozen formulation and 11 143 with the lyophilised formulation, and neutralising antibodies were 49·25 with the frozen formulation and 45·95 with the lyophilised formulation, with a seroconversion rate of 100%. Cell-mediated responses were detected in all participants at day 28, with median cell proliferation of 2·5% CD4+ and 1·3% CD8+ with the frozen formulation, and a median cell proliferation of 1·3% CD4+ and 1·1% CD8+ with the lyophilised formulation. Interpretation The heterologous rAd26 and rAd5 vector-based COVID-19 vaccine has a good safety profile and induced strong humoral and cellular immune responses in participants. Further investigation is needed of the effectiveness of this vaccine for prevention of COVID-19. Funding Ministry of Health of the Russian Federation.