Background: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. Methods: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4⁺ and CD8⁺ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. Results: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4⁺CD45RA⁻CCR7⁻ phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. Conclusion: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8⁺, but not CD4⁺, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.

Current seasonal influenza vaccines have suboptimal effectiveness, especially in seasons dominated by viruses that do not match the vaccine. Therefore, finding new approaches to improve the immunogenicity and efficacy of traditional influenza vaccines is of high priority for public health. Licensed live attenuated influenza vaccine (LAIV) is a promising platform for designing broadly protective vaccines due to its ability to induce cross-reactive T-cell immunity. In this study, we tested the hypothesis that truncation of the nonstructural protein 1 (NS1) and the replacement of the nucleoprotein (NP) of the A/Leningrad/17 master donor virus with a recent NP, i.e., switching to 5:3 genome composition, could improve the cross-protective potential of the LAIV virus. We generated a panel of LAIV candidates differing from the classical vaccine by the source of NP gene and/or by the length of NS1 protein. We showed that NS1-modified LAIV viruses had reduced viral replication in the respiratory tract of mice, indicating a more attenuated phenotype compared to the LAIVs with full-length NS1. Most importantly, the LAIV candidate with both NP and NS genes modified induced a robust systemic and lung-localized memory CD8 T-cell response targeting more recent viruses, and better protected immunized mice against lethal challenge with a heterosubtypic influenza virus than the control LAIV variant. Overall, these data indicate that the 5:3 LAIVs with truncated NS1 may be beneficial for protection against heterologous influenza viruses and warrant further preclinical and clinical development.

Background/Objectives: Influenza viruses and SARS-CoV-2 are currently cocirculating with similar seasonality, and both pathogens are characterized by a high mutational rate which results in reduced vaccine effectiveness and thus requires regular updating of vaccine compositions. Vaccine formulations combining seasonal influenza and SARS-CoV-2 strains can be considered promising and cost-effective tools for protection against both infections. Methods: We used a licensed seasonal trivalent live attenuated influenza vaccine (3×LAIV) as a basis for the development of a modified 3×LAIV/CoV-2 vaccine, where H1N1 and H3N2 LAIV strains encoded an immunogenic cassette enriched with conserved T-cell epitopes of SARS-CoV-2, whereas a B/Victoria lineage LAIV strain was unmodified. The trivalent LAIV/CoV-2 composition was compared to the classical 3×LAIV in the golden Syrian hamster model. Animals were intranasally immunized with the mixtures of the vaccine viruses, twice, with a 3-week interval. Immunogenicity was assessed on day 42 of the study, and the protective effect was established by infecting vaccinated hamsters with either influenza H1N1, H3N2 or B viruses or with SARS-CoV-2 strains of the Wuhan, Delta and Omicron lineages. Results: Both the classical 3×LAIV and 3×LAIV/CoV-2 vaccine compositions induced similar levels of serum antibodies specific to all three influenza strains, which resulted in comparable levels of protection against challenge from either influenza strain. Protection against SARS-CoV-2 challenge was more pronounced in the 3×LAIV/CoV-2-immunized hamsters compared to the classical 3×LAIV group. These data were accompanied by the higher magnitude of virus-specific cellular responses detected by ELISPOT in the modified trivalent LAIV group. Conclusions: The modified trivalent live attenuated influenza vaccine encoding the T-cell epitopes of SARS-CoV-2 can be considered a promising tool for combined protection against seasonal influenza and COVID-19.

Background. Influenza and SARS-CoV-2 viruses are two highly variable pathogens. We have developed a candidate bivalent live vaccine based on the strain of licensed A/Leningrad/17-based cold-adapted live attenuated influenza vaccine (LAIV) of H3N2 subtype, which expressed SARS-CoV-2 immunogenic T-cell epitopes. A cassette encoding fragments of S and N proteins of SARS-CoV-2 was inserted into the influenza NA gene using the P2A autocleavage site. In this study, we present the results of preclinical evaluation of the developed bivalent vaccine in a non-human primate model. Methods. Rhesus macaques (Macaca mulatta) (n = 3 per group) were immunized intranasally with 7.5 lg EID₅₀ of the LAIV/CoV-2 bivalent vaccine, a control non-modified H3N2 LAIV or a placebo (chorioallantoic fluid) using a sprayer device, twice, with a 28-day interval. The blood samples were collected at days 0, 3, 28 and 35 for hematological and biochemical assessment. Safety was also assessed by monitoring body weight, body temperature and clinical signs of the disease. Immune responses to influenza virus were assessed both by determining serum antibody titers in hemagglutination inhibition assay, microneutralization assay and IgG ELISA. T-cell responses were measured both to influenza and SARS-CoV-2 antigens using ELISPOT and flow cytometry. Three weeks after the second immunization, animals were challenged with 10⁵ PFU of Delta SARS-CoV-2. The body temperature, weight and challenge virus shedding were monitored for 5 days post-challenge. In addition, virus titers in various organs and histopathology were evaluated on day 6 after SARS-CoV-2 infection. Results. There was no toxic effect of the immunizations on the hematological and coagulation hemostasis of animals. No difference in the dynamics of the average weight and thermometry results were found between the groups of animals. Both LAIV and LAIV/CoV-2 variants poorly replicated in the upper respiratory tract of rhesus macaques. Nevertheless, despite this low level of virus shedding, influenza-specific serum IgG responses were detected in the group of monkeys immunized with the LAIV/CoV-2 bivalent but not in the LAIV group. Furthermore, T-cell responses to both influenza and SARS-CoV-2 viruses were detected in the LAIV/CoV-2 vaccine group only. The animals were generally resistant to SARS-CoV-2 challenge, with minimal virus shedding in the placebo and LAIV groups. Histopathological changes in vaccinated animals were decreased compared to the PBS group, suggesting a protective effect of the chimeric vaccine candidate. Conclusions. The candidate bivalent vaccine was safe and immunogenic for non-human primates and warrants its further evaluation in clinical trials.

Cases of new COVID-19 infection, which manifested in 2019 and caused a global socioeconomic crisis, still continue to be registered worldwide. The high mutational activity of SARS-CoV-2 leads to the emergence of new antigenic variants of the virus, which significantly reduces the effectiveness of COVID-19 vaccines, as well as the sensitivity of diagnostic test systems based on variable viral antigens. These problems may be solved by focusing on highly conserved coronavirus antigens, for example nucleocapsid (N) protein, which is actively expressed by coronavirus-infected cells and serves as a target for the production of virus-specific antibodies and T cell responses. It is known that anti-N antibodies are non-neutralizing, but their protective potential and functional activity are not sufficiently studied. Here, the protective effect of anti-N antibodies was studied in Syrian hamsters passively immunized with polyclonal sera raised to N(B.1) recombinant protein. The animals were infected with 10⁵ or 10⁴ TCID₅₀ of SARS-CoV-2 (B.1, Wuhan or BA.2.86.1.1.18, Omicron) 6 h after serum passive transfer, and protection was assessed by weight loss, clinical manifestation of disease, viral titers in the respiratory tract, as well as by the histopathological evaluation of lung tissues. The functional activity of anti-N(B.1) antibodies was evaluated by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) assays. The protection of anti-N antibodies was evident only against a lower dose of SARS-CoV-2 (B.1) challenge, whereas almost no protection was revealed against BA.2.86.1.1.18 variant. Anti-N(B.1) monoclonal antibodies were able to stimulate both CDC and ADCC. Thus, anti-N(B.1) antibodies possess protective activity against homologous challenge infection, which is possibly mediated by innate Fc-mediated immune reactions. These data may be informative for the development of N-based broadly protective COVID-19 vaccines.

Background/Objectives: Cases of the new COVID-19 coronavirus infection, which manifested in 2019 and caused a global socioeconomic crisis, still continue to be registered worldwide. The high mutational activity of SARS-CoV-2 leads to the emergence of new antigenic variants of the virus, which significantly reduces the effectiveness of COVID-19 vaccines, as well as the sensitivity of diagnostic test systems based on variable viral antigens. These problems may be solved by the focusing on highly conserved coronavirus antigens, for example nucleocapsid (N) protein, which is actively expressed by coronavirus-infected cells and serves as a target for the production of virus-specific antibodies and T-cell responses. It is known that anti-N antibodies are non-neutralizing, but their protective potential and functional activity are not sufficiently studied. Methods: Protective effect of anti-N antibodies was studied in Syrian hamsters passively immunized with polyclonal sera raised to N(B.1) recombinant protein. Animals were infected with 105 or 104 TCID50 of SARS-CoV-2 (B.1, Wuhan or BA.2.86.1.1.18, Omicron) 6 hours after serum passive transfer, and protection was assessed by weight loss, clinical manifestation of disease, viral titers in respiratory tract, as well as by histopathological evaluation of lung tissues. Functional activity of anti-N (B.1) antibodies was evaluated by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) assays. Results: Protection of anti-N antibodies was evident only against lower dose of SARS-CoV-2 (B.1) challenge, whereas almost no protection was revealed against BA.2.86.1.1.18 variant. Anti-N (B.1) monoclonal antibodies were able to stimulate CDC and ADCC. Conclusions: Anti-N (B.1) antibodies possess protective activity against homologous challenge infection, which is possibly mediated by innate Fc-mediated immune reactions. These data may be informative for the development of N-based broadly-protective COVID-19 vaccines.