Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new influenza A strains. Efficient control of infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A combination of several such antigens, including the conserved region of the second subunit of the hemagglutinin (HA2), the extracellular domain of the M2 protein (M2e), and epitopes of nucleoprotein (NP), which together can elicit an antibody- and cell-mediated immune response, would be preferred for vaccine development. In this study, we obtained recombinant virus-like particles formed by an artificial self-assembling peptide (SAP) carrying two epitopes from NP, tandem copies of M2e and HA2 peptides, along with a T helper Pan DR-binding epitope (PADRE). Fusion proteins expressed in Escherichia coli self-assembled in vitro into spherical particles with a size of 15–35 nm. Immunization of mice with these particles induced strong humoral immune response against M2e and the entire virus, and lead to the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with the influenza A virus. Our results show that SAP-based nanoparticles carrying conserved peptides from M2, HA, and NP proteins of the influenza A virus, as well as T helper epitope PADRE, can be used for the development of universal flu vaccines.

Background: Influenza viruses with truncated NS1 proteins show promise as viral vectors and candidates for mucosal universal influenza vaccines. These mutant NS1 viruses, which lack the N-terminal half of the NS1 protein (124 a.a.), are unable to antagonise the innate immune response. This creates a self-adjuvant effect enhancing heterologous protection by inducing a robust CD8+ T-cell response together with immunoregulatory mechanisms. However, the effects of NS1 modifications on T-follicular helper (Tfh) and B-cell responses remain less understood. Methods: C57bl/6 mice were immunised intranasally with 10 μL of either an influenza virus containing a truncated NS1 protein (PR8/NS124), a cold-adapted influenza virus with a full-length NS1 (caPR8/NSfull), or a wild-type virus (PR8/NSfull). Immune responses were assessed on days 8 and 28 post-immunisation by flow cytometry, ELISA, and HAI assay. Results: In this study, we demonstrate that intranasal immunisation with PR8/NS124 significantly increases tissue-resident CD4+ and CD8+ T cells in the lungs and activates Tfh cells in regional lymph nodes as early as day 8 post-immunisation. These effects are not observed in mice immunised with caPR8/NSfull or PR8/NSfull. Notably, PR8/NS124 immunisation also leads to the development of inducible bronchus-associated lymphoid tissue (iBALT) in the lungs by day 28, characterised by the presence of antigen-specific Tfh cells and GL7+Fas+ germinal centre B cells. Conclusions: Our findings further underscore the potential of NS1-truncated influenza viruses to drive robust mucosal immune responses and enhance vaccine efficacy.

Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum recombinant vaccines based on conserved antigens. The extracellular domain of the transmembrane protein M2 of influenza A virus (M2e) is highly conserved but poorly immunogenic and needs to be fused to an adjuvant protein or carrier virus-like particles (VLPs) to increase immunogenicity and provide protection against infection. In this study, we obtained VLPs based on capsid proteins (CPs) of single-stranded RNA phages Beihai32 and PQ465 bearing the M2e peptides. Four copies of the M2e peptide were linked to the C-terminus of the CP of phage Beihai32 and to the N and C termini of the CP of phage PQ465. The hybrid proteins, being expressed in Escherichia coli, formed spherical VLPs of about 30 nm in size. Immunogold transmission electron microscopy showed that VLPs formed by the phage PQ465 CP with a C-terminal M2e fusion present the M2e peptide on the surface. Subcutaneous immunization of mice with VLPs formed by both CPs containing four copies of the M2e peptide at the C termini induced high levels of M2e-specific IgG antibodies in serum and provided mice with protection against lethal influenza A virus challenge. In the case of an N-terminal fusion of M2e with the phage PQ465 CP, the immune response against M2e was significantly lower. CPs of phages Beihai32 and PQ465, containing four copies of the M2e peptide at their C termini, can be used to develop recombinant influenza A vaccine.

Background: The extracellular domain of the M2 protein (M2e) and the conserved region of the second subunit of the hemagglutinin (HA2, 76–130 а.а.) of the influenza A virus, could be used to develop broad-spectrum influenza vaccines. However, these antigens have low immunogenicity and require the use of special carriers to enhance it. Virus-like particles (VLPs) formed from viral capsid proteins are among the most effective carriers. Methods: In this work, we obtained and characterized VLPs based on capsid proteins (CPs) of single-stranded RNA bacteriophages Beihai32 and PQ465, simultaneously displaying M2e and HA2 peptides. Results: Fusion proteins expressed in Escherichia coli formed spherical VLPs of about 30 nm in size. Subcutaneous immunization of mice with chimeric VLPs elicited a robust humoral immune response against M2e and the whole influenza A virus, and promoted the formation of cytokine-secreting antigen-specific CD4⁺ and CD8⁺ effector memory T cells. Conclusions: VLPs based on CPs of phages Beihai32 and PQ465 carrying conserved peptides M2e and HA2 of the influenza A virus can be used for the development of universal influenza vaccines.

Conserved influenza virus proteins, such as the hemagglutinin stem domain (HA2), nucleoprotein (NP), and matrix protein (M), are the main targets in the development of universal influenza vaccines. Previously, we constructed a recombinant vaccine protein Flg-HA2-2-4M2ehs containing the extracellular domain of the M2 protein (M2e) and the aa76–130 sequence of the second HA subunit as target antigens. It demonstrated immunogenicity and broad protection against influenza A viruses after intranasal and parenteral administration. This study shows that CD8+ epitopes of NP, inserted into a flagellin-fused protein carrying M2e and HA2, affect the post-vaccination immune humoral response to virus antigens without reducing protection. No differences were found between the two proteins in their ability to stimulate the formation of follicular Th in the spleen, which may contribute to a long-lasting antigen-specific humoral response. The data obtained on Balb/c mice suggest that the insertion of CTL NP epitopes into the flagellin-fused protein carrying M2e and HA2 reduces the antibody response to M2e and A/H3N2. In C57Bl6 mice, this stimulates the formation of NP-specific CD8+ Tem and virus-specific mono- and multifunctional CD4+ and CD8+ Tem in the spleen and completely protects mice from influenza virus subtypes A/H1N1pdm09 and A/H3N2.

Background/Objectives: Although influenza A viruses with partially truncated NS1 proteins are substantially attenuated and immunogenic due to enhanced innate immune activation; residual NS1-mediated antagonism of antiviral innate responses may support viral replication in the lower respiratory tract and constrain optimal immune responses. Strategies to further improve their immunogenicity and protective efficacy by incorporating immunomodulatory cytokines, such as IL-2, have been successfully explored. Methods: Here, we extended this approach to chemokine expression by engineering an NS1-truncated PR8-based virus (PR8/NS124) to express the immunomodulatory chemokine CXCL10 from the NS segment and compared it with the parental vector. Results: The recombinant NS124_SS_CXCL10 virus replicated to high titers in embryonated chicken eggs and MDCK cells. In vivo, however, CXCL10 expression reduced viral replication in mouse lungs by ~10⁴-fold, resulting in a near-non-replicating phenotype. In contrast to the parental virus, the vector did not induce weight loss and exhibited a strongly attenuated phenotype. This effect was associated with altered innate immune signaling, including increased IRF7 expression and early induction of IFN-α responses in the lungs, together with modulation of TLR-dependent sensing pathways in the upper respiratory tract. Despite severely impaired replication, intranasal immunization induced antigen-specific T-cell responses comparable to those elicited by the parental vector. Following intraperitoneal immunization, when replication of both vectors was minimal, the CXCL10-expressing vector induced significantly higher frequencies of antigen-specific CD8⁺ and CD4⁺ effector-memory T cells. This was accompanied by enhanced antigen-specific T-cell recall responses in the lungs following intranasal challenge. Importantly, the CXCL10-expressing vector demonstrated protective efficacy comparable to that of the parental NS124 vector against heterologous H3N2 challenge while exhibiting an improved safety profile. Conclusions: These findings support the incorporation of CXCL10 as a strategy to improve the safety and T-cell immunogenicity of NS1-truncated influenza vectors.