Structural Basis for Evasion of New SARS-CoV-2 Variants from the Potent Virus-Neutralizing Nanobody Targeting the S-Protein Receptor-Binding Domain.

COVID-19 has caused millions of deaths and many times more infections worldwide, emphasizing the unpreparedness of the global health system in the face of new infections and the key role for vaccines and therapeutics, including virus-neutralizing antibodies, in prevention and containment of the disease. Continuous evolution of the SARS-CoV-2 coronavirus has been causing its new variants to evade the action of the immune system, which highlighted the importance of detailed knowledge of the epitopes of already selected potent virus-neutralizing antibodies. A single-chain antibody ("nanobody") targeting the SARS-CoV-2 receptor-binding domain (RBD), clone P2C5, had exhibited robust virus-neutralizing activity against all SARS-CoV-2 variants and, being a major component of the anti-COVID-19 formulation "GamCoviMab", had successfully passed Phase I of clinical trials. However, after the emergence of the Delta and XBB variants, a decrease in the neutralizing activity of this nanobody was observed. Here we report on the successful crystal structure determination of the RBD:P2C5 complex at 3.1 Å, which revealed the intricate protein-protein interface, sterically occluding full ACE2 receptor binding by the P2C5-neutralized RBD. Moreover, the structure revealed the developed RBD:P2C5 interface centered around residues Leu452 and Phe490, thereby explaining the evasion of the Delta or Omicron XBB, but not Omicron B.1.1.529 variant, as a result of the single L452R or F490S mutations, respectively, from the action of P2C5. The structure obtained is expected to foster nanobody engineering in order to rescue neutralization activity and will facilitate epitope mapping for other neutralizing nanobodies by competition assays.

Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in Russia.

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 [<0·1%] of 16 427 participants in the vaccine group and one [<0·1%] of 5435 participants in the placebo group), none of which were considered related to the vaccine. INTERPRETATION: This interim analysis of the phase 3 trial of Gam-COVID-Vac showed 91·6% efficacy against COVID-19 and was well tolerated in a large cohort. FUNDING: Moscow City Health Department, Russian Direct Investment Fund, and Sberbank.

Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia.

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.

Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A.

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 LD50 of 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.

rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19.

Introduction: Numerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis. Methods: In current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing. Results and discussions: A P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants.

Safety and immunogenicity of rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine against SARS-CoV-2 in healthy adolescents: an open-label, non-randomized, multicenter, phase 1/2, dose-escalation study.

To protect young individuals against SARS-CoV-2 infection, we conducted an open-label, prospective, non-randomised dose-escalation Phase 1/2 clinical trial to evaluate the immunogenicity and safety of the prime-boost "Sputnik V" vaccine administered at 1/10 and 1/5 doses to adolescents aged 12-17 years. The study began with the vaccination of the older cohort (15-to-17-year-old participants) with the lower (1/10) dose of vaccine and then expanded to the whole group (12-to-17-year-old participants). Next, 1/5 dose was used according to the same scheme. Both doses were well tolerated by all age groups. No serious or severe adverse events were detected. Most of the solicited adverse reactions were mild. No significant differences in total frequencies of adverse events were registered between low and high doses in age-pooled groups (69.6% versus 66.7%). In contrast, the 1/5 dose induced significantly higher humoral and T cell-mediated immune responses than the 1/10 dose. The 1/5 vaccine dose elicited higher antigen-binding (both S and RBD-specific) as well as virus-neutralising antibody titres at the maximum of response (day 42), also resulting in a statistically significant difference at a distanced timepoint (day 180) compared to the 1/10 vaccine dose. Higher dose resulted in increased cross-neutralization of Delta and Omicron variants. Clinical Trial Registration: ClinicalTrials.gov, NCT04954092, LP-007632.

rAAV expressing recombinant neutralizing antibody for the botulinum neurotoxin type A prophylaxis.

Botulinum neurotoxin (BoNT) is one of the most dangerous bacterial toxins and a potential biological weapon component. BoNT mechanism of pathological action is based on inhibiting the release of neurotransmitters from nerve endings. To date, anti-BoNT therapy is reduced to the use of horse hyperimmune serum, which causes many side effects, as well as FDA-approved drug BabyBig which consists of human-derived anti-BoNT antibodies (IgG) for infant botulinum treatment. Therapeutics for botulism treatment based on safer monoclonal antibodies are undergoing clinical trials. In addition, agents have been developed for the specific prevention of botulism, but their effectiveness has not been proved. In this work, we have obtained a recombinant adeno-associated virus (rAAV-B11-Fc) expressing a single-domain antibody fused to the human IgG Fc-fragment (B11-Fc) and specific to botulinum toxin type A (BoNT/A). We have demonstrated that B11-Fc antibody, expressed via rAAV-B11-Fc treatment, can protect animals from lethal doses of botulinum toxin type A, starting from day 3 and at least 120 days after administration. Thus, our results showed that rAAV-B11-Fc can provide long-term expression of B11-Fc-neutralizing antibody in vivo and provide long-term protection against BoNT/A intoxication. Consequently, our study demonstrates the applicability of rAAV expressing protective antibodies for the prevention of intoxication caused by botulinum toxins.

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection.

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 bind to the stem domain of hemagglutinin and 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 80-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 antibodies 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.

Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern.

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.

Immunogenicity and protectivity of intranasally delivered vector-based heterologous prime-boost COVID-19 vaccine Sputnik V in mice and non-human primates.

Although unprecedented efforts aiming to stop the COVID-19 pandemic have been made over the past two years, SARSCoV-2 virus still continues to cause intolerable health and economical losses. Vaccines are considered the most effective way to prevent infectious diseases, which has been reaffirmed for COVID-19. However, in the context of the continuing virus spread because of insufficient vaccination coverage and emergence of new variants of concern, there is a high demand for vaccination strategy amendment. The ability to elicit protective immunity at the entry gates of infection provided by mucosal vaccination is key to block virus infection and transmission. Therefore, these mucosal vaccines are believed to be a "silver bullet" that could bring the pandemic to an end. Here, we demonstrate that the intranasally delivered Gam-COVID-Vac (Sputnik V) vaccine induced a robust (no less than 180 days) systemic and local immune response in mice. High immunogenic properties of the vaccine were verified in non-human primates (common marmosets) by marked IgG and neutralizing antibody (NtAb) production in blood serum, antigen-specific Tcell proliferation and cytokine release of peripheral blood mononuclear cells accompanied by formation of IgA antibodies in the nasal mucosa. We also demonstrate that Sputnik V vaccine can provide sterilizing immunity in K18-hACE2 transgenic mice exposed to experimental lethal SARS-CoV-2 infection protecting them against severe lung immunopathology and mortality. We believe that intranasal Sputnik V vaccine is a promising novel needle-free mucosal vaccine candidate for primary immunization as well as for revaccination and is worth further clinical investigation.

An open, non-randomised, phase 1/2 trial on the safety, tolerability, and immunogenicity of single-dose vaccine "Sputnik Light" for prevention of coronavirus infection in healthy adults.

BACKGROUND: While the world is experiencing another wave of COVID-19 pandemic, global vaccination program is hampered by an evident shortage in the supply of licensed vaccines. In an 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 (S) glycoprotein - "Sputnik Light". METHODS: We conducted an open label, prospective, non-randomised phase 1/2 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-CoV-2 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). FINDINGS: Most of the 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-CoV-2 antibodies revealed a group with pre-existing immunity to SARS-CoV-2. Upon this finding we separated all safety and immunogenicity data based on pre-existing immunity to SARS-CoV-2. 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 the 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. Antigen-specific T cell proliferation, formation of IFNy-producing cells, and IFNy secretion were observed in 96·7% (26/27), 96% (24/25), and 96% (24/25) of the seronegative group respectively and in 100% (3/3), 100% (5/5), and 100% (5/5) of the seropositive vaccinees, respectively. INTERPRETATION: The single-dose rAd26 vector-based COVID-19 vaccine "Sputnik Light" has a good safety profile and induces a strong humoral and cellular immune responses both in seronegative and seropositive participants. FUNDING: Russian Direct Investment Fund.

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice.

The bacterium Clostridium botulinum is the causative agent of botulism-a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A.

Development and characterization of two GP-specific monoclonal antibodies, which synergistically protect non-human primates against Ebola lethal infection.

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.

Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts.

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).