Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44⁺CD62L⁻) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP₃₆₆); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.

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.

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.

BACKGROUND: Influenza viruses cause worldwide epidemics, and the most effective method to prevent influenza disease is regular vaccinations. The development of new generation vaccines is aimed primarily at the formation of an immune response against a wide range of influenza viruses. One of the promising approaches is sequential vaccination with chimeric influenza viruses with identical stem domains of the hemagglutinin surface protein. AIM: The development of an experimental vaccine strain of influenza B virus with chimeric hemagglutinin consisting of head and stem domains of influenza B viruses belonging to different genetic lineages. MATERIALS AND METHODS: A chimeric influenza hemagglutinin gene was obtained by genetic engineering from the genetic material of B/Victoria and B/Yamagata influenza strains. The gene was inserted into the vector for the reverse genetics of the influenza virus. The influenza B virus strain with chimeric hemagglutinin was obtained by transfection of Vero cells using an 8-plasmid system. The rest of the genes were obtained from the attenuated influenza B virus with cold-adapted and temperature-sensitive phenotypes. The biological properties of the obtained recombinant strain, its infectious titer in developing chicken embryos and MDCK cell culture were evaluated. RESULTS: A recombinant vaccine strain has been successfully rescued. The head domain of the hemagglutinin of the virus is inherited from the B/Victoria influenza virus, and the stem domain from the B/Yamagata virus. The virus actively replicated in eggs and MDCK cells, with temperature-sensitive and cold-adapted phenotypes identical to classical live attenuated influenza vaccine viruses. The thermal stability of the chimeric hemagglutinin did not differ significantly from the thermal stability of the hemagglutinins of the donor viruses. CONCLUSIONS: The results obtained indicate the possibility of creating a strain with chimeric hemagglutinin, fragments of which are inherited from different genetic lineages. The growth characteristics and biological properties of the strain make it a promising candidate for the experimental evaluation of the possibility of inducing a cross-protective immune response by sequential vaccination with vaccine strains with identical stem hemagglutinin domains.

BACKGROUND: The development of a universal influenza vaccine remains a critical goal to enhance protection against diverse influenza virus strains. While vaccines against influenza type A viruses have benefited from the use of chimeric hemagglutinin designs, strategies for influenza type B vaccines still lag. AIM: The study aimed to investigate the efficacy of chimeric vaccine strains to induce humoral immune response targeting conserved antigenic sites of influenza type B virus. METHODS: A chimeric hemagglutinin was engineered by combining the head domain from the B/Brisbane/60/2008 virus (Victoria lineage) with the stalk domain from the B/Phuket/3073/2013 virus (Yamagata lineage). The gene encoding the chimeric hemagglutinin was incorporated into a vaccine virus based on the cold-adapted B/USSR/60/69 master donor virus to produce live-attenuated influenza vaccine. Mice were sequentially vaccinated with the conventional live-attenuated influenza vaccine and then with the recombinant live-attenuated influenza vaccine expressing the chimeric hemagglutinin. Immune responses and cross-protection against both homologous and heterologous influenza type B virus strains were assessed. RESULTS: The engineered chimeric hemagglutinin did not impair the replication or assembly of the vaccine virus. Sequential vaccination induced a robust humoral immune response and provided protection against both homologous and heterologous influenza type B virus strains in the mouse model. CONCLUSION: Live-attenuated influenza type B vaccines expressing chimeric hemagglutinin show promise in broadening protection against influenza type B virus infection. These findings support the development of a universal influenza type B vaccine using a chimeric hemagglutinin design.

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections.