Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza.

Influenza and SARS-CoV-2 are two major respiratory pathogens that cocirculate in humans and cause serious illness with the potential to exacerbate disease in the event of co-infection. To develop a bivalent vaccine, capable of protecting against both infections, we inserted the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein into hemagglutinin (HA) molecule or into the open reading frame of the truncated nonstructural protein 1 (NS1) of live attenuated influenza vaccine (LAIV) virus and assessed phenotypic characteristics of the rescued LAIV-RBD viruses, as well as their immunogenicity in mouse and Syrian hamster animal models. A panel of 9 recombinant LAIV-RBD viruses was rescued using the A/Leningrad/17 backbone. Notably, only two variants with RBD insertions into the HA molecule could express sufficient quantities of RBD protein in infected MDCK cells. Intranasal immunization of mice induced high levels of anti-influenza antibody responses in all chimeric LAIV-RBD viruses, which was comparable to the LAIV virus vector. The RBD-specific antibody responses were most pronounced in the variant expressing RBD194 fragment as a chimeric HA protein. This candidate was further tested in Syrian hamsters and was shown to be immunogenic and capable of protecting animals against both infections.

Development of Cross-Reactive Live Attenuated Influenza Vaccine Candidates against Both Lineages of Influenza B Virus.

BACKGROUND: Influenza viruses continue to cause a significant social and economic burden globally. Vaccination is recognized as the most effective measure to control influenza. Live attenuated influenza vaccines (LAIVs) are an effective means of preventing influenza, especially among children. A reverse genetics (RG) system is required to rapidly update the antigenic composition of vaccines, as well as to design LAIVs with a broader spectrum of protection. Such a system has been developed for the Russian LAIVs only for type A strains, but not for influenza B viruses (IBV). METHODS: All genes of the B/USSR/60/69 master donor virus (B60) were cloned into RG plasmids, and the engineered B60, as well as a panel of IBV LAIV reassortants were rescued from plasmid DNAs encoding all viral genes. The engineered viruses were evaluated in vitro and in a mouse model. RESULTS: The B60 RG system was successfully developed, which made it possible to rescue LAIV reassortants with the desired antigenic composition, including hybrid strains with hemagglutinin and neuraminidase genes belonging to the viruses from different IBV lineages. The LAIV candidate carrying the HA of the B/Victoria-lineage virus and NA from the B/Yamagata-lineage virus demonstrated optimal characteristics in terms of safety, immunogenicity and cross-protection, prompting its further assessment as a broadly protective component of trivalent LAIV. CONCLUSIONS: The new RG system for B60 MDV allowed the rapid generation of type B LAIV reassortants with desired genome compositions. The generation of hybrid LAIV reassortants with HA and NA genes belonging to the opposite IBV lineages is a promising approach for the development of IBV vaccines with broad cross-protection.

Nucleoprotein as a Promising Antigen for Broadly Protective Influenza Vaccines.

Annual vaccination is considered as the main preventive strategy against seasonal influenza. Due to the highly variable nature of major viral antigens, such as hemagglutinin (HA) and neuraminidase (NA), influenza vaccine strains should be regularly updated to antigenically match the circulating viruses. The influenza virus nucleoprotein (NP) is much more conserved than HA and NA, and thus seems to be a promising target for the design of improved influenza vaccines with broad cross-reactivity against antigenically diverse influenza viruses. Traditional subunit or recombinant protein influenza vaccines do not contain the NP antigen, whereas live-attenuated influenza vaccines (LAIVs) express the viral NP within infected cells, thus inducing strong NP-specific antibodies and T-cell responses. Many strategies have been explored to design broadly protective NP-based vaccines, mostly targeted at the T-cell mode of immunity. Although the NP is highly conserved, it still undergoes slow evolutionary changes due to selective immune pressure, meaning that the particular NP antigen selected for vaccine design may have a significant impact on the overall immunogenicity and efficacy of the vaccine candidate. In this review, we summarize existing data on the conservation of the influenza A viral nucleoprotein and review the results of preclinical and clinical trials of NP-targeting influenza vaccine prototypes, focusing on the ability of NP-specific immune responses to protect against diverse influenza viruses.

Overview of Nucleocapsid-Targeting Vaccines against COVID-19.

The new SARS-CoV-2 coronavirus, which emerged in late 2019, is a highly variable causative agent of COVID-19, a contagious respiratory disease with potentially severe complications. Vaccination is considered the most effective measure to prevent the spread and complications of this infection. Spike (S) protein-based vaccines were very successful in preventing COVID-19 caused by the ancestral SARS-CoV-2 strain; however, their efficacy was significantly reduced when coronavirus variants antigenically different from the original strain emerged in circulation. This is due to the high variability of this major viral antigen caused by escape from the immunity caused by the infection or vaccination with spike-targeting vaccines. The nucleocapsid protein (N) is a much more conserved SARS-CoV-2 antigen than the spike protein and has therefore attracted the attention of scientists as a promising target for broad-spectrum vaccine development. Here, we summarized the current data on various N-based COVID-19 vaccines that have been tested in animal challenge models or clinical trials. Despite the high conservatism of the N protein, escape mutations gradually occurring in the N sequence can affect its protective properties. During the three years of the pandemic, at least 12 mutations have arisen in the N sequence, affecting more than 40 known immunogenic T-cell epitopes, so the antigenicity of the N protein of recent SARS-CoV-2 variants may be altered. This fact should be taken into account as a limitation in the development of cross-reactive vaccines based on N-protein.

RDE Treatment Prevents Non-Specific Detection of SARS-CoV-2- and Influenza-Specific IgG Antibodies in Heat-Inactivated Serum Samples.

Assessing the levels of serum IgG antibodies is widely used to measure immunity to influenza and the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after natural infection or vaccination with specific vaccines, as well as to study immune responses to these viruses in animal models. For safety reasons, sometimes serum specimens collected from infected individuals are subjected to heat inactivation at 56 °C to reduce the risk of infecting personnel during serological studies. However, this procedure may affect the level of virus-specific antibodies, making the results of antibody immunoassays uninterpretable. Here, we evaluated the effect of the heat inactivation of human, ferret and hamster serum samples on the binding of IgG antibodies to the influenza and SARS-CoV-2 antigens. For this, serum samples of naive and immune hosts were analyzed in three variants: (i) untreated sera, (ii) heated at 56 °C for 1 h, and (iii) treated with receptor-destroying enzyme (RDE). The samples were studied through an in-house enzyme-linked immunosorbent assay (ELISA) using whole influenza virus or recombinant proteins corresponding to nucleocapsid (N) protein and the receptor-binding domain of SARS-CoV-2 Spike (RBD) as antigens. We demonstrated that the heat inactivation of the naive serum samples of various hosts can lead to false-positive results, while RDE treatment abolished the effect of the non-specific binding of IgG antibodies to the viral antigens. Furthermore, RDE also significantly decreased the level of virus-specific IgG antibodies in SARS-CoV-2 and influenza-immune sera of humans and animals, although it is unknown whether it actually removes true virus-specific IgG antibodies or only non-specifically binding artifacts. Nevertheless, we suggest that the RDE treatment of human and animal sera may be useful in preventing false-positive results in various immunoassays, while also neutralizing infectious virus, since the standard protocol for the use of RDE also includes heating the sample at 56 °C.

Truncation of NS1 Protein Enhances T Cell-Mediated Cross-Protection of a Live Attenuated Influenza Vaccine Virus Expressing Wild-Type Nucleoprotein.

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.

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.

Sarcoidosis-related autoimmune inflammation in COVID-19 convalescent patients.

Currently, there are a large number of reports about the development of autoimmune conditions after COVID-19. Also, there have been cases of sarcoid-like granulomas in convalescents as a part of the post-COVID-19 syndrome. Since one of the etiological theories of sarcoidosis considers it to be an autoimmune disease, we decided to study changes in the adaptive humoral immune response in sarcoidosis and SARS-CoV-2 infection and to find out whether COVID-19 can provoke the development of sarcoidosis. This review discusses histological changes in lymphoid organs in sarcoidosis and COVID-19, changes in B cell subpopulations, T-follicular helper cells (Tfh), and T-follicular regulatory cells (Tfr), and analyzes various autoantibodies detected in these pathologies. Based on the data studied, we concluded that SARS-CoV-2 infection may cause the development of autoimmune pathologies, in particular contributing to the onset of sarcoidosis in convalescents.

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'.

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 Live Probiotic Vaccine Prototype Based on Conserved Influenza a Virus Antigens Protect Mice against Lethal Influenza Virus Infection.

BACKGROUND: Due to the highly variable nature of the antigenic properties of the influenza virus, many efforts have been made to develop broadly reactive influenza vaccines. Various vaccine platforms have been explored to deliver conserved viral antigens to the target cells to induce cross-reactive immune responses. Here, we assessed the feasibility of using Enterococcus faecium L3 as a bacterial vector for oral immunization against influenza virus. METHODS: we generated two vaccine prototypes by inserting full-length HA2 (L3-HA2) protein or its long alpha helix (LAH) domain in combination with four M2e tandem repeats (L3-LAH+M2e) into genome of E.faecium L3 probiotic strain. The immunogenicity and protective potential of these oral vaccines were assessed in a lethal challenge model in BALB/c mice. RESULTS: as expected, both vaccine prototypes induced HA stem-targeting antibodies, whereas only L3-LAH+4M2e vaccine induced M2e-specific antibody. The L3-HA2 vaccine partially protected mice against lethal challenge with two H1N1 heterologous viruses, while 100% of animals in the L3-LAH+4M2e vaccine group survived in both challenge experiments, and there was significant protection against weight loss in this group, compared to the L3 vector-immunized control mice. CONCLUSIONS: the recombinant enterococcal strain L3-LAH+4M2e can be considered as a promising live probiotic vaccine candidate for influenza prevention and warrants further evaluation in relevant pre-clinical models.

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.

Influenza vaccine: progress in a vaccine that elicits a broad immune response.

INTRODUCTION: The licensed seasonal influenza vaccines predominantly induce neutralizing antibodies against immunodominant hypervariable epitopes of viral surface proteins, with limited protection against antigenically distant influenza viruses. Strategies have been developed to improve vaccines' performance in terms of broadly reactive and long-lasting immune response induction. AREAS COVERED: We have summarized the advancements in the development of cross-protective influenza vaccines and discussed the challenges in evaluating them in preclinical and clinical trials. Here, the literature regarding the current stage of development of universal influenza vaccine candidates was reviewed. EXPERT OPINION: Although various strategies aim to redirect adaptive immune responses from variable immunodominant to immunosubdominant antigens, more conserved epitopes are being investigated. Approaches that improve antibody responses to conserved B cell epitopes have increased the protective efficacy of vaccines within a subtype or phylogenetic group of influenza viruses. Vaccines that elicit significant levels of T cells recognizing highly conserved viral epitopes possess a high cross-protective potential and may cover most circulating influenza viruses. However, the development of T cell-based universal influenza vaccines is challenging owing to the diversity of MHCs in the population, unpredictable degree of immunodominance, lack of adequate animal models, and difficulty in establishing T cell immunity in humans. ABBREVIATIONS: cHA: chimeric HA; HBc: hepatitis B virus core protein; HA: hemagglutinin; HLA: human leucocyte antigen; IIV: inactivated influenza vaccine; KLH: keyhole limpet hemocyanin; LAH: long alpha helix; LAIV: live attenuated influenza vaccine; M2e: extracellular domain of matrix 2 protein; MHC: major histocompatibility complex; mRNA: messenger ribonucleic acid; NA: neuraminidase; NS1: non-structural protein 1; qNIV: quadrivalent nanoparticle influenza vaccine; TRM: tissue-resident memory T cells; VE: vaccine effectiveness; VLP: virus-like particles; VSV: vesicular stomatitis virus.