Yulia Desheva

Background/Objectives: The impact of repeated annual influenza vaccination on long-term immunogenicity and protection remains debated. Although reduced fold-increases in antibody titers are often observed in repeatedly vaccinated individuals, the effect on the duration of the humoral response remains poorly understood. This study evaluated the magnitude and persistence of the humoral immune response over four consecutive seasons. Methods: An open-label observational study was conducted between 2018 and 2022 and involved 28 healthy adults (median age, 55 years) immunized with seasonal inactivated influenza vaccines. Hemagglutination inhibition (HAI) titers were assessed at five time points annually. Secondary endpoints included neutralizing (MNA) and neuraminidase-inhibiting (ELLA) antibody responses measured at baseline and three weeks after vaccination. The duration of antibody responses above the seroprotection threshold (titer ≥ 40) was compared across seasons using the Mantel–Cox test. Results: Antibody dynamics differed by vaccine component. The highest GMT and fold-increase were observed for A/H1N1pdm09 in 2018/19, followed by a significant decline after the second vaccination, that was more pronounced for neutralizing antibodies. The response to N1 neuraminidase remained weak throughout the study. For A/H3N2, baseline HAI GMTs increased significantly after each subsequent vaccination, whereas N2 neuraminidase responses peaked after the first dose and declined thereafter. Influenza B responses were weaker than those to influenza A. A significant increase in B/Yamagata titers in 2020/21 was attributed to the introduction of quadrivalent vaccines. A/H1N1pdm09 HAI responses were long-lasting, with titers remaining at or above the seroprotection threshold for one year. The duration of the A/H3N2 antibody response increased significantly over four seasons, likely because of annual strain updates. A similar trend was observed for B/Victoria after strain changes. Conclusions: Repeated annual influenza vaccination did not negatively affect post-vaccination antibody titers to the HA vaccine antigens in the studied cohort. Regular updates to vaccine strains may enhance the persistence of HA-targeted humoral immune responses.

Background/Objective: This study is a cross-sectional investigation of long-term immune responses measured at different time intervals after COVID-19 infections, vaccinations, or combined exposure. The focus is on immune reactivity against recombinant spike (S) and nucleocapsid (N) protein antigens. Materials and Methods: Serum antibody levels were assessed up to four to four and a half years after infection or immunization, including virus-specific immunoglobulin G (IgG), IgA and IgM antibodies, as well as neutralizing antibodies against the S-protein. Cellular immunity was assessed by analyzing peripheral blood mononuclear cells (PBMC; n = 43 in first cohort, n = 32 in second cohort), including T-helper memory and cytotoxic subsets, and cytokine production after in vitro stimulation with recombinant SARS-CoV-2 proteins. A multiplex cytokine assay was used to analyze effector and regulatory immune responses. Results: Virus-specific IgG antibodies persisted for years after exposure to SARS-CoV-2, with IgG against the receptor-binding domain (RBD) correlating most strongly with neutralizing activity. Vaccinated individuals demonstrated higher IgA responses, whereas antibodies to the N-protein were associated with previous infection. No IgM antibodies were detected in any subjects, suggesting an immune response based on memory rather than ongoing infection. PBMCs from individuals with a history of both COVID-19 exposure and vaccination exhibited enhanced responsiveness, characterized by increased frequencies of memory T cells compared to vaccination alone. Stimulating with the S-protein induces higher cytokine production, including IFN-gamma, TNF-alfa, and IL-12(p70), compared with stimulation by the N-protein. Cytokines such as IL-10 and TGF-beta are also elevated, suggesting immune regulation rather than persistent inflammation. Conclusions: SARS-CoV-2 infection and vaccination are associated with persistent humoral and cellular immune responses detectable several years after exposure. Individuals with hybrid immunity exhibit broader and functionally enhanced immune reactivity, indicating more robust long-term immune memory. Future studies should focus on the long-term consequences of hybrid immunity and optimize other vaccine strategies, including recombinant antigen vaccines.

Background/Objective: This study Investigates long-term immune responses (up to 4–4.5 years) to SARS-CoV-2 in individuals with: Past COVID-19 infection, Vaccination, Combined exposure, with focus on immune reactivity to recombinant spike (S) and nucleocapsid (N) proteins. Materials and methods: Serum antibody responses were assessed up to 4–4.5 years after infection or immunization, including virus-specific IgG, IgA, IgM, and neutralizing antibodies. Cellular immunity was evaluated by phenotypic and functional analysis of peripheral blood mononuclear cells (PBMCs), including memory T-helper and cytotoxic T-cell subsets, as well as cytokine production following in vitro stimulation with recombinant SARS-CoV-2 proteins. Multiplex cytokine profiling was used to characterize effector and regulatory immune responses. Results: Virus-specific IgG antibodies persisted for several years after SARS-CoV-2 exposure, with anti-RBD IgG showing the strongest correlation with virus-neutralizing activity, whereas antibodies to the N- protein primarily reflected prior infection. Vaccinated individuals exhibited a distinct immunoglobulin profile characterized by a higher prevalence of IgA. No IgM detected suggesting the detected immune responses reflect immunological memory rather than active infection. PBMCs from individuals with combined COVID-19 and vaccination history demonstrated enhanced responsiveness and more memory T cells. Hybrid immunity (infection and vaccination) provides stronger and broader immune responses.. Stimulation with S - protein induces stronger cytokine production (IFN-γ, TNF-α, IL-12p70) than N- protein. Regulatory cytokines (IL-10, TGF-β) also elevated which suggests immune regulation rather than chronic inflammation.Conclusion: SARS-CoV-2 infection and vaccination induce persistent humoral and cellular immunity. Neutralizing activity correlates only with anti-RBD and anti-S IgG. Future research should focus on long-term effects, hybrid immunity, and optimizing other vaccine types, in addition to Adenovector vaccines, such as recombinant antigen-based vaccines .

Aim: Develop A/H1N1pdm09-based live attenuated influenza vaccines (LAIVs) presenting chimeric hemagglutinin (HA) fused to fragments of Streptococcus pneumoniae (PspA, Spr1875) or S. agalactiae (ScaAB) to elicit combined anti-influenza and anti-bacterial immunity. Methods: Recombinant LAIVs were generated by reverse genetics. Replicative fitness was measured in embryonated chicken eggs (CE; log10 EID50/0.1 mL, n = 5 per delution) and MDCK cells (log10 TCID50/mL, n = 5). BALB/c mice (n = 20 per group; serology n = 6 per group; lung titers n = 5 per group) received intranasal 106 EID50. Systemic IgG and mucosal IgA to influenza and to the recombinant pneumococcal peptide were quantified by ELISA (GMT ± SD). Early cytokine responses were profiled in THP-1 cells. Results: All recombinant strains replicated in CE at 33°C but were temperature-sensitive at 39°C. H1-ScaAB retained relatively high replication and exhibited a cold-adapted phenotype despite a large N-terminal insert. In MDCK cells, H1-PspA showed significantly reduced replication compared with the parental LAIV. In mouse lungs, replication on day 3 post-immunization was significantly lower for H1-ScaAB and H1-PspA compared with the parental LAIV strain (p < 0.05). The parental LAIV induced robust systemic anti-influenza IgG and, uniquely for H1-ScaAB, significant mucosal anti-influenza IgA (p < 0.05). H1-Spr generated stronger antibody responses to the inserted pneumococcal peptide (p < 0.05). THP-1 assays revealed construct-specific cytokine patterns (unmodified H1N1: highest IFN-α; H1-Spr: elevated IL-6; H1-ScaAB: greatest MCP-1). Conclusions: Multiple A/H1N1pdm09-based recombinant LAIVs with chimeric HA can replicate in eggs and murine respiratory tract and induce dual influenza/pneumococcal antigen responses. Expanded biophysical validation, functional antibody assays and challenge studies are needed to optimize insert design without compromising viral fitness.

Influenza B viruses, divided into B/Victoria and B/Yamagata lineages, have not had B/Yamagata isolates after 2020. A study evaluated immunity to influenza B surface antigens hemagglutinin (HA) and neuraminidase (NA) in 138 patient sera from 2023 and 23 pairs of sera from 2018 to 2019 vaccine recipients. The phylogenetic tree of the influenza B virus, based on HA and NA genes, shows that the Yamagata lineage evolves gradually, while the Victoria lineage exhibits rapid mutations with short branches. In 2023, mean levels of antibodies to HA and NA of B/Yamagata virus were higher than to B/Victoria, despite no cases of B/Yamagata lineage isolation after 2020. The titers of antibodies to NA of B/Yamagata statistically significantly differed among individuals born before and after 1988. Among patients examined in 2018–2019, neuraminidase-inhibiting (NI) antibody titers before vaccination were higher to B/Yamagata than to B/Victoria, and NI antibodies to B/Victoria and B/Yamagata positively correlated with neutralizing antibodies to B/Victoria virus before and after vaccination. Immunity to B/Yamagata virus was stronger in 2023, despite no isolation since 2020, probably due to the presence of cross-reactive antibodies from B/Victoria infections or vaccinations. Antibodies to NA of B/Victoria and B/Yamagata in 2023 correlated significantly in patients born before 1988, potentially supporting the concept of ‘antigenic sin’ phenomenon for influenza B viruses. The fact that NI antibody titers to B/Victoria and B/Yamagata correlated with neutralizing antibody titers to B/Victoria may suggest broad cross-protection. Studying influenza B virus NA antigenic properties helps understand the evolution and antigenic competition of HA and NA.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underwent significant mutations, resulting in the Omicron variant. Methods: In this study, we analyzed blood samples from 98 patients with acute coronavirus disease 19 (COVID-19) hospitalized during the initial SARS-CoV-2 wave and the onset of Omicron in 2021. High-resolution melting (HRM) analysis of PCR products was used to analyze RNA extracted from clinical samples collected in July and November 2021 from patients infected with SARS-CoV-2. Results: HRM analysis revealed a characteristic deletion in the N protein RNA of the virus isolated in November 2021, associated with the Omicron variant. Elevated levels of inflammatory markers and interleukin-6 (IL-6) were observed in both waves of COVID-19. Complement levels and IgG and IgM antibodies to SARS-CoV-2 were detected more often during the second wave. An increase in hemagglutinin-inhibiting (HI) antibodies against influenza viruses was observed in paired blood specimens from moderate to severe COVID-19 patients during both outbreaks. Conclusions: Patients admitted during both waves of COVID-19 showed a significant rise in inflammatory markers, suggesting that Omicron triggers inflammatory responses. The rapid formation of IgM and IgG in Omicron may indicate a faster immune response. Seasonal flu may negatively impact the clinical course of coronavirus infections.

Introduction : Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has undergone significant mutations since its emergence, resulting in a variety of antigenic variants. One of these variants is Omicron. Methods : In this study, the blood samples from 98 patients with acute coronavirus disease-19 (COVID-19) who were hospitalized during the initial SARS-CoV2 wave and the onset of Omicron infection in 2021 were analyzed. We used high-resolution melting (HRM) of PCR products to analyze RNA extracted from two clinical samples collected in July and November of 2021 from patients infected with the SARS-COV-2 virus. Results : HRM-analysis detected a characteristic deletion in the N-protein RNA of the virus isolated from November 2021 that is associated with the Omicron variant. Elevated levels of C-reactive protein (CRP), neutrophil-lymphocyte ratio (NLR), and interleukin-6 (IL-6) were observed in patients during both initial wave of COVID-19 and 2021 hospitalization. Additionally, complement levels were more frequently detected at the start of hospitalization during the second wave. IgG and IgM antibodies to SARS-CoV-2 were detected more often at the beginning of hospitalization during the second wave of COVID-19. During both outbreaks, an increase in hemagglutinin-inhibiting (HI) antibodies against Influenza A and B was observed in paired blood specimens from moderate to severe COVID-19 patients. Conclusions : Patients admitted during both waves of COVID-19 showed a significant rise in several inflammatory markers, suggesting that Omicron triggers significant inflammatory responses. The rapid formation of IgM and IgG in Omicron patients may indicate a faster immune response due to memory B cell formation after previous infections or vaccinations. Seasonal flu may negatively impact the clinical course of coronavirus infections. Further research is needed to determine if clinical presentation and laboratory parameters change in response to variations in the SARS-CoV virus in breakthrough cases and in patients with post-COVID syndrome.

Aim: The objectives of our study were to evaluate a range of circulating biomarkers in COVID-19-related long-term neurological dysfunction. Methods: The study involved 30 patients with post-COVID syndrome (PCS) and 28 patients after COVID-19 without PCS. The third cohort consisted of 29 patients with acute COVID-19 of varying severity. The severity of COVID-19 was classified as mild and moderate to severe. The Montreal Cognitive Assessment (MOCA) and the SAGE test were used to study cognitive functions. The Hospital Anxiety and Depression Scale (HADS), the Sheehan Anxiety Scale, and the Beck Depression Inventory were used to study affective functions. The levels of serum cytokines and IgM, IgG, IgA to the SARS-CoV-2 coronavirus were determined using the Vector-Best test systems (Novosibirsk, Russia). We also studied the IgG subclasses to the spike protein of the SARS-CoV-2. Results: А mild to moderate COVID-19 infection primarily increases the risk of affective disorders and asthenia and, to a lesser extent, the development of cognitive impairment. The levels of IFN-α, IL-6, as well as serum antibodies to the SARS-CoV-2 among patients with PCS were significantly higher compared to convalescents without PCS. IgM to the SARS-CoV-2 was detected in the blood of patients with PCS during 2–7 months after the disease. After moderate and severe COVID-19, IgG2 and IgG4 were predominant in the blood of patients with PCS and neurological symptoms. The levels of IL-1, IL-4, IL-6, IL-8 in the blood serum of patients with PCS were higher after moderate and severe COVID-19 compared to patients who had mild COVID-19. Conclusions: The obtained data on an elevated level of cytokines and IFN-α in the blood of PCS patients can suggest the hypothesis about the participation of chronic inflammation in neurological disorders. The main limitation of the study is the relatively small sample size, which limits the statistical analyses.

Live influenza vaccine (LAIV) is an effective tool in the fight against influenza infection. LAIV is easy to administer, economical and fast to produce, and stimulates a systemic and local immune response. The aim of the study was to study the early protective effect of LAIV in mice against homologous and heterologous influenza infection within one week of LAIV immunization and possible pathways of immune system activation. We studied expression of early cytokines and type I interferons in response to LAIV introduction in THP-1 cell line. Mice were immunized intranasally with the vaccine virus A/17/South Africa/2013/01(H1N1)pdm09 at a dose of 6 lg EID50. The production of Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α) and type I interferons in the respiratory tract of mice was determined by ELISA. Infection was carried out on the 3rd or 5th day after immunization with the influenza viruses A/South Africa/3626/2013(H1N1)pdm09 or A/Indonesia/5/2005(H5N1) IDCDC-RG2. When both the vaccine virus and the parent virus A/South Africa/3626/2013(H1N1)pdm09 were introduced into THP-1 cell culture, an increase in the expression of type I interferon was observed. Immunization with LAIV lead to increase in the production of early cytokines and type I interferons in the respiratory tract of mice. The mice were completely protected on 5 after immunization against lethal homologous and heterologous influenza infection and partially protected on day 3. The data obtained may indicate the benefit of using LAIV during the seasonal increase in acute respiratory viral infections due to stimulation of innate immune factors. HIGHLIGHTS Live influenza vaccine (LAIV) has long been successfully used for the prevention of seasonal influenza. The LAIV provides early protection against homologous and heterologous influenza infection in the 1st week after immunization before the formation of an adaptive immune response. Introduction of LAIV into a cell culture of macrophage origin and into the nasal passages of mice causes an increase in the expression and production of early cytokines and type I interferon. The use of LAIV is useful during the period of circulation of not only influenza viruses, but also other pathogens of respiratory infections GRAPHICAL ABSTRACT