Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model.

COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

In Vitro Stimulation with Live SARS-CoV-2 Suggests Th17 Dominance In Virus-Specific CD4+ T Cell Response after COVID-19.

The SARS-CoV-2 and influenza viruses are the main causes of human respiratory tract infections with similar disease manifestation but distinct mechanisms of immunopathology and host response to the infection. In this study, we investigated the SARS-CoV-2-specific CD4+ T cell phenotype in comparison with H1N1 influenza-specific CD4+ T cells. We determined the levels of SARS-CoV-2- and H1N1-specific CD4+ T cell responses in subjects recovered from COVID-19 one to 15 months ago by stimulating PBMCs with live SARS-CoV-2 or H1N1 influenza viruses. We investigated phenotypes and frequencies of main CD4+ T cell subsets specific for SARS-CoV-2 using an activation induced cell marker assay and multicolor flow cytometry, and compared the magnitude of SARS-CoV-2- and H1N1-specific CD4+ T cells. SARS-CoV-2-specific CD4+ T cells were detected 1-15 months post infection and the frequency of SARS-CoV-2-specific central memory CD4+ T cells was increased with the time post-symptom onset. Next, SARS-CoV-2-specific CD4+ T cells predominantly expressed the Th17 phenotype, but the level of Th17 cells in this group was lower than in H1N1-specific CD4+ T cells. Finally, we found that the lower level of total Th17 subset within total SARS-CoV-2-specific CD4+ T cells was linked with the low level of CCR4+CXCR3- 'classical' Th17 cells if compared with H1N1-specific Th17 cells. Taken together, our data suggest the involvement of Th17 cells and their separate subsets in the pathogenesis of SARS-CoV-2- and influenza-induced pneumonia; and a better understanding of Th17 mediated antiviral immune responses may lead to the development of new therapeutic strategies.

Cross-Reactivity of SARS-CoV-2 Nucleocapsid-Binding Antibodies and Its Implication for COVID-19 Serology Tests.

The emergence of the new coronavirus SARS-CoV-2 in late 2019 led to the global pandemic COVID-19, causing a profound socioeconomic crisis. Adequate diagnostic tools need to be developed to control the ongoing spread of infection. Virus-specific humoral immunity in COVID-19 patients and those vaccinated with specific vaccines has been characterized in numerous studies, mainly using Spike protein-based serology tests. However, Spike protein and specifically its receptor-binding domain (RBD) are mutation-prone, suggesting the reduced sensitivity of the validated serology tests in detecting antibodies raised to variants of concern (VOC). The viral nucleocapsid (N) protein is more conserved compared to Spike, but little is known about cross-reactivity of the N-specific antibodies between the ancestral B.1 virus and different VOCs. Here, we generated recombinant N phosphoproteins from different SARS-CoV-2 strains and analyzed the magnitude of N-specific antibodies in COVID-19 convalescent sera using an in-house N-based ELISA test system. We found a strong positive correlation in the magnitude of anti-N (B.1) antibodies and antibodies specific to various VOCs in COVID-19-recovered patients, suggesting that the N-binding antibodies are highly cross-reactive, and the most immunogenic epitopes within this protein are not under selective pressure. Overall, our study suggests that the RBD-based serology tests should be timely updated to reflect the constantly evolving nature of the SARS-CoV-2 Spike protein, whereas the validated N-based test systems can be used for the analysis of sera from COVID-19 patients regardless of the strain that caused the infection.

Development of a T Cell-Based COVID-19 Vaccine Using a Live Attenuated Influenza Vaccine Viral Vector.

The COVID-19 pandemic emerged in 2020 and has caused an unprecedented burden to all countries in the world. SARS-CoV-2 continues to circulate and antigenically evolve, enabling multiple reinfections. To address the issue of the virus antigenic variability, T cell-based vaccines are being developed, which are directed to more conserved viral epitopes. We used live attenuated influenza vaccine (LAIV) virus vector to generate recombinant influenza viruses expressing various T-cell epitopes of SARS-CoV-2 from either neuraminidase (NA) or non-structural (NS1) genes, via the P2A self-cleavage site. Intranasal immunization of human leukocyte antigen-A*0201 (HLA-A2.1) transgenic mice with these recombinant viruses did not result in significant SARS-CoV-2-specific T-cell responses, due to the immunodominance of NP366 influenza T-cell epitope. However, side-by-side stimulation of peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents with recombinant viruses and LAIV vector demonstrated activation of memory T cells in samples stimulated with LAIV/SARS-CoV-2, but not LAIV alone. Hamsters immunized with a selected LAIV/SARS-CoV-2 prototype were protected against challenge with influenza virus and a high dose of SARS-CoV-2 of Wuhan and Delta lineages, which was confirmed by reduced weight loss, milder clinical symptoms and less pronounced histopathological signs of SARS-CoV-2 infection in the lungs, compared to LAIV- and mock-immunized animals. Overall, LAIV is a promising platform for the development of a bivalent vaccine against influenza and SARS-CoV-2.

Dysregulated Immune Responses in SARS-CoV-2-Infected Patients: A Comprehensive Overview.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in humans more than two years ago and caused an unprecedented socio-economic burden on all countries around the world. Since then, numerous studies have attempted to identify various mechanisms involved in the alterations of innate and adaptive immunity in COVID-19 patients, with the ultimate goal of finding ways to correct pathological changes and improve disease outcomes. State-of-the-art research methods made it possible to establish precise molecular mechanisms which the new virus uses to trigger multisystem inflammatory syndrome and evade host antiviral immune responses. In this review, we present a comprehensive analysis of published data that provide insight into pathological changes in T and B cell subsets and their phenotypes, accompanying the acute phase of the SARS-CoV-2 infection. This knowledge might help reveal new biomarkers that can be utilized to recognize case severity early as well as to provide additional objective information on the effective formation of SARS-CoV-2-specific immunity and predict long-term complications of COVID-19, including a large variety of symptoms termed the 'post-COVID-19 syndrome'.

Dysregulated Immune Responses in SARS-CoV-2-Infected Patients: A Comprehensive Overview

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in humans more than two years ago and caused an unprecedented socio-economic burden on all countries around the world. Since then, numerous studies have attempted to identify various mechanisms involved in the alterations of innate and adaptive immunity in COVID-19 patients, with the ultimate goal of finding ways to correct pathological changes and improve disease outcomes. State-of-the-art research methods made it possible to establish precise molecular mechanisms which the new virus uses to trigger multisystem inflammatory syndrome and evade host antiviral immune responses. In this review, we present a comprehensive analysis of published data that provide insight into pathological changes in T and B cell subsets and their phenotypes, accompanying the acute phase of the SARS-CoV-2 infection. This knowledge might help reveal new biomarkers that can be utilized to recognize case severity early as well as to provide additional objective information on the effective formation of SARS-CoV-2-specific immunity and predict long-term complications of COVID-19, including a large variety of symptoms termed the 'post-COVID-19 syndrome'.

Ferristatin II Efficiently Inhibits SARS-CoV-2 Replication in Vero Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a significant impact on global public health. Multiple mechanisms for SARS-CoV-2 cell entry have been described; however, the role of transferrin receptor 1 (TfR1) in SARS-CoV-2 infection has received little attention. We used ferristatin II to induce the degradation of TfR1 on the surface of Vero cells and to study the consequences of such treatment on the viability of the cells and the replication of SARS-CoV-2. We demonstrated that ferristatin II is non-toxic for Vero cells in concentrations up to 400 µM. According to confocal microscopy data, the distribution of the labeled transferrin and receptor-binding domain (RBD) of Spike protein is significantly affected by the 18h pretreatment with 100 µM ferristatin II in culture medium. The uptake of RBD protein is nearly fully inhibited by ferristatin II treatment, although this protein remains bound on the cell surface. The findings were well confirmed by the significant inhibition of the SARS-CoV-2 infection of Vero cells by ferristatin II with IC50 values of 27 µM (for Wuhan D614G virus) and 40 µM (for Delta virus). A significant reduction in the infectious titer of the Omicron SARS-CoV-2 variant was noted at a ferristatin II concentration as low as 6.25 µM. We hypothesize that ferristatin II blocks the TfR1-mediated SARS-CoV-2 host cell entry; however, further studies are needed to elucidate the full mechanisms of this virus inhibition, including the effect of ferristatin II on other SARS-CoV-2 receptors, such as ACE2, Neuropilin-1 and CD147. The inhibition of viral entry by targeting the receptor on the host cells, rather than the viral mutation-prone protein, is a promising COVID-19 therapeutic strategy.

A Phase 1 Randomized Placebo-Controlled Study to Assess the Safety, Immunogenicity and Genetic Stability of a New Potential Pandemic H7N9 Live Attenuated Influenza Vaccine in Healthy Adults.

This study describes a double-blind randomized placebo-controlled phase I clinical trial in healthy adults of a new potential pandemic H7N9 live attenuated influenza vaccine (LAIV) based on the human influenza virus of Yangtze River Delta hemagglutinin lineage (ClinicalTrials.gov Identifier: NCT03739229). Two doses of H7N9 LAIV or placebo were administered intranasally to 30 and 10 subjects, respectively. The vaccine was well-tolerated and not associated with increased rates of adverse events or with any serious adverse events. Vaccine virus was detected in nasal swabs during the 6 days after vaccination or revaccination. A lower frequency of shedding was observed after the second vaccination. Twenty-five clinical viral isolates obtained after the first and second doses of vaccine retained the temperature-sensitive and cold-adapted phenotypic characteristics of LAIV. There was no confirmed transmission of the vaccine strain from vaccinees to placebo recipients. After the two H7N9 LAIV doses, an immune response was observed in 96.6% of subjects in at least one of the assays conducted.

Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2.

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.