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.

Expert opinion on the identification, risk assessment, and mitigation of microorganisms and parasites relevant to xenotransplantation products from pigs.

Xenotransplantation has the potential to address shortages of organs available for clinical transplantation, but concerns exist regarding potential risks posed by porcine microorganisms and parasites (MP) to the health of human recipients. In this study, a risk-based framework was developed, and expert opinion was elicited to evaluate porcine MP based on swine exposure and risk to human health. Experts identified 255 MP to include in the risk assessment. These were rated by experts for five criteria regarding potential swine exposure in the USA and human health risks. MP were subsequently categorized into three risk mitigation groups according to pre-defined rules: disqualifying porcine MP (due to their pathogenic potential, n = 130); non-disqualifying porcine MP (still relevant to consider for biosecurity or monitoring efforts, n = 40); and alert/watch list (not reported in the USA or MP not in swine, n = 85). Most disqualifying (n = 126) and non-disqualifying (n = 36) porcine MP can effectively be eliminated with high biosecurity programs. This approach supports surveillance and risk mitigation strategies for porcine MP in swine produced for xenotransplantation, such as documentation of freedom from porcine MP, or use of porcine MP screening, monitoring, or elimination options. To the authors' knowledge, this is the first effort to comprehensively identify all relevant porcine MP systematically and transparently evaluate the risk of infection of both donor animals and immunosuppressed human recipients, and the potential health impacts for immunosuppressed human recipients from infected xenotransplantation products from pigs.

The Effect of Mice Adaptation Process on the Pathogenicity of Influenza A/South Africa/3626/2013 (H1N1)pdm09 Model Strain.

Influenza virus strain A/South Africa/3626/2013 (H1N1)pdm09 (SA-WT) is a non-mouse-adapted model strain that has naturally high pathogenic properties in mice. It has been suggested that the high pathogenicity of this strain for mice could be due to the three strain-specific substitutions in the polymerase complex (Q687R in PB1, N102T in PB2, and E358E/K heterogeneity in PB2). To evaluate the role of these replacements, SA-WT was passaged five times in mouse lungs, and the genome of the mouse-adapted version of the SA-WT strain (SA-M5) was sequenced. SA-M5 lost E358E/K heterogeneity and retained E358, which is the prevalent amino acid at this position among H1N1pdm09 strains. In addition, in the hemagglutinin of SA-M5, two heterogeneous substitutions (G155G/E and S190S/R) were identified. Both viruses, SA-M5 and SA-WT, were compared for their toxicity, ability to replicate, pathogenicity, and immunogenicity in mice. In mice infected with SA-M5 or SA-WT strains, toxicity, virus titer in pulmonary homogenates, and mouse survival did not differ significantly. In contrast, an increase in the immunogenicity of SA-M5 compared to SA-WT was observed. This increase could be due to the substitutions G155G/E and S190S/R in the HA of SA-M5. The prospects for using SA-M5 in studying the immunogenicity mechanisms were also discussed.

The Effect of Preexisting Immunity on Virus Detection and Immune Responses in a Phase II, Randomized Trial of a Russian-Backbone, Live, Attenuated Influenza Vaccine in Bangladeshi Children.

BACKGROUND: In a 2012 Phase II clinical trial, 300 Bangladeshi children aged 24 to 59 months with no prior influenza vaccine exposure were randomized to receive a single intranasally-administered dose of either trivalent, Russian-backbone, live, attenuated influenza vaccine (LAIV) or placebo. Protocol-defined analyses, presented in the companion manuscript, demonstrate decreased viral detection and immunogenicity for A/H1N1pdm09, relative to the A/H3N2 and B strains. This post hoc analysis of the trial data aims to investigate the LAIV strain differences by testing the hypothesis that preexisting humoral and mucosal immunity may influence viral recovery and immune responses after LAIV receipt. METHODS: We used logistic regressions to evaluate the relations between markers of preexisting immunity (ie, hemagglutination inhibition [HAI], microneutralization, and immunoglobulin G and immunoglobulin A (both serum and mucosal antibodies) and LAIV viral recovery in the week post-vaccination. We then tested for potential effect modification by baseline HAI titers (ie, <10 versus ≥10) and week 1 viral recovery on the LAIV-induced serum and mucosal immune responses, measured between days 0 and 21 post-vaccination. RESULTS: Higher levels of preexisting immunity to influenza A/H3N2 and B were strongly associated with strain-specific prevention of viral shedding upon LAIV receipt. While evidence of LAIV immunogenicity was observed for all 3 strains, the magnitudes of immune responses were most pronounced in children with no evidence of preexisting HAI and in those with detectable virus. CONCLUSIONS: The results provide evidence for a bidirectional association between viral replication and immunity, and underscore the importance of accounting for preexisting immunity when evaluating virologic and immunologic responses to LAIVs. CLINICAL TRIALS REGISTRATION: NCT01625689.

Immunogenicity and Viral Shedding of Russian-Backbone, Seasonal, Trivalent, Live, Attenuated Influenza Vaccine in a Phase II, Randomized, Placebo-Controlled Trial Among Preschool-Aged Children in Urban Bangladesh.

BACKGROUND: We evaluated a Russian-backbone, live, attenuated influenza vaccine (LAIV) for immunogenicity and viral shedding in a randomized, placebo-controlled trial among Bangladeshi children. METHODS: Healthy children received a single, intranasal dose of LAIV containing the 2011-2012 recommended formulation or placebo. Nasopharyngeal wash (NPW) specimens were collected on days 0, 2, 4, and 7. Reverse transcription polymerase chain reactions and sequencing identified the influenza virus (vaccine or wild-type). On days 0 and 21, blood specimens were collected to assess immunogenicity using hemagglutination inhibition, microneutralization, and immunoglobulin A (IgA) and G enzyme-linked immunosorbent assays (ELISAs); NPW specimens were also collected to assess mucosal immunogenicity using kinetic IgA ELISA. RESULTS: We enrolled 300 children aged 24 through 59 months in the immunogenicity and viral shedding analyses. Among children receiving LAIV, 45% and 67% shed A/H3N2 and B vaccine strains, respectively. No child shed A/H1N1 vaccine strain. There were significantly higher day 21 geometric mean titers (GMTs) for the LAIV, as compared to the placebo groups, in all immunoassays for A/H3N2 and B (log10 titer P < .0001; GMT Ratio >2.0). Among immunoassays for A/H1N1, only the mucosal IgA GMT was significantly higher than placebo at day 21 (log10 titer P = .0465). CONCLUSIONS: Children vaccinated with LAIV had serum and mucosal antibody responses to A/H3N2 and B, but only a mucosal IgA response to A/H1N1. Many children shed A/H3N2 and B vaccine strains, but none shed A/H1N1. More research is needed to determine the reason for decreased LAIV A/H1N1 immunogenicity and virus shedding. CLINICAL TRIALS REGISTRATION: NCT01625689.

M2e-based universal influenza vaccines: a historical overview and new approaches to development.

The influenza A virus was isolated for the first time in 1931, and the first attempts to develop a vaccine against the virus began soon afterwards. In addition to causing seasonal epidemics, influenza viruses can cause pandemics at random intervals, which are very hard to predict. Vaccination is the most effective way of preventing the spread of influenza infection. However, seasonal vaccination is ineffective against pandemic influenza viruses because of antigenic differences, and it takes approximately six months from isolation of a new virus to develop an effective vaccine. One of the possible ways to fight the emergence of pandemics may be by using a new type of vaccine, with a long and broad spectrum of action. The extracellular domain of the M2 protein (M2e) of influenza A virus is a conservative region, and an attractive target for a universal influenza vaccine. This review gives a historical overview of the study of M2 protein, and summarizes the latest developments in the preparation of M2e-based universal influenza vaccines.

H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets.

Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines. In this study, we developed and evaluated in ferrets an intranasal live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. We demonstrate that the LAIV is attenuated and safe in ferrets and induces high hemagglutination- and neuraminidase-inhibiting and virus-neutralizing titers. The antibodies against hemagglutinin were also cross-reactive with divergent H7 strains. To assess efficacy, we used an intratracheal challenge ferret model in which an acute severe viral pneumonia is induced that closely resembles viral pneumonia observed in severe human cases. A single- and two-dose strategy provided complete protection against severe pneumonia and prevented virus replication. The protective effect of the two-dose strategy appeared better than the single dose only on the microscopic level in the lungs. We observed, however, an increased lymphocytic infiltration after challenge in single-vaccinated animals and hypothesize that this a side effect of the model.

A novel immunofluorescent test system for SARS-CoV-2 detection in infected cells.

Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt assessment of individual and herd immunity against the infection can be accomplished by using rapid tests to determine antiviral antibody levels. The microneutralization assay (MN) is one of the most widely used diagnostic methods that has been proposed to assess the qualitative and quantitative characteristics of virus-specific humoral immunity in COVID-19 convalescents or vaccine recipients. However, some aspects of the assay, such as sensitivity and time cost, need improvement. Here, we developed an express test, which may be potentially used in clinical practice for the assessment of serum-caused SARS-CoV-2 inhibition in infected cell cultures. It implies the detection and counting of coronaviral fluorescent-forming units (FFU) and includes two sequentially used developing components: biotinylated mouse monoclonal antibodies against the recombinant N protein of SARS-CoV-2 (B.1) and the recombinant EGFP-streptavidin fusion protein. Due to the universal specificity of the antibodies, our analytical tool is suitable for the detection of various strains of SARS-CoV-2 when determining both the infectious titer of viruses and the titer of serum virus-neutralizing antibodies. The developed two-component test system is characterized by high sensitivity, a reduced number of analytic stages and low assay cost, as well as by flexibility, since it may be modified for detection of other pathogens using the appropriate antibodies.

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.

Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium.

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates.

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.

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.

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.

Comparative immunogenicity and cross-clade protective efficacy of mammalian cell-grown inactivated and live attenuated H5N1 reassortant vaccines in ferrets.

Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy, LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.

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.

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.

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'.

Understanding the Variability of Certain Biological Properties of H1N1pdm09 Influenza Viruses.

The influenza virus continually evolves because of the high mutation rate, resulting in dramatic changes in its pathogenicity and other biological properties. This study aimed to evaluate the evolution of certain essential properties, understand the connections between them, and find the molecular basis for the manifestation of these properties. To that end, 21 A(H1N1)pdm09 influenza viruses were tested for their pathogenicity and toxicity in a mouse model with a ts/non-ts phenotype manifestation and HA thermal stability. The results demonstrated that, for a strain to have high pathogenicity, it must express a toxic effect, have a non-ts phenotype, and have a thermally stable HA. The ancestor A/California/07/2009 (H1N1)pdm influenza virus expressed the non-ts phenotype, after which the cycling trend of the ts/non-ts phenotype was observed in new strains of A(H1N1)pdm09 influenza viruses, indicating that the ratio of the ts phenotype will increase in the coming years. Of the 21 tested viruses, A/South Africa/3626/2013 had the high pathogenicity in the mouse model. Sequence alignment analysis showed that this virus has three unique mutations in the polymerase complex, two of which are in the PB2 gene and one that is in the PB1 gene. Further study of these mutations might explain the distinguishing pathogenicity.