Nanoparticles Carrying Conserved Regions of Influenza A Hemagglutinin, Nucleoprotein, and M2 Protein Elicit a Strong Humoral and T Cell Immune Response and Protect Animals from Infection.

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.

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope.

Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15-30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant "universal" vaccine against influenza A produced in plants.

Nanoparticles based on artificial self-assembling peptide and displaying M2e peptide and stalk HA epitopes of influenza A virus induce potent humoral and T-cell responses and protect against the viral infection.

The extracellular domain of the M2 protein (M2e) and conserved region of the second subunit of the hemagglutinin (HA2) could be used for the development of broad-spectrum vaccines against influenza A. Here we obtained and characterized recombinant mosaic proteins containing tandem copies of M2e and HA2 fused to an artificial self-assembling peptide (SAP). The inclusion of SAP peptides in the fusion proteins enabled their self-assembly in vitro into spherical particles with a size of 30-50 nm. Intranasal immunization of mice with these particles without additional adjuvants induced strong humoral immune response against M2e and the whole virus. Particles carrying both M2e and HA2 induced antigen-specific multifunctional CD4+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with different subtypes of influenza A virus. The obtained self-assembling nanoparticles can be used to develop a universal influenza vaccine.

Highly Immunogenic Nanoparticles Based on a Fusion Protein Comprising the M2e of Influenza A Virus and a Lipopeptide.

The highly conserved extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is a promising target for the development of broad-spectrum vaccines. However, M2e is a poor immunogen by itself and must be linked to an appropriate carrier to induce an efficient immune response. In this study, we obtained recombinant mosaic proteins containing tandem copies of M2e fused to a lipopeptide from Neisseria meningitidis surface lipoprotein Ag473 and alpha-helical linkers and analyzed their immunogenicity. Six fusion proteins, comprising four or eight tandem copies of M2e flanked by alpha-helical linkers, lipopeptides, or a combination of both of these elements, were produced in Escherichia coli. The proteins, containing both alpha-helical linkers and lipopeptides at each side of M2e repeats, formed nanosized particles, but no particulate structures were observed in the absence of lipopeptides. Animal study results showed that proteins with lipopeptides induced strong M2e-specific antibody responses in the absence of external adjuvants compared to similar proteins without lipopeptides. Thus, the recombinant M2e-based proteins containing alpha-helical linkers and N. meningitidis lipopeptide sequences at the N- and C-termini of four or eight tandem copies of M2e peptide are promising vaccine candidates.

Combination of three adjuvants enhances the immunogenicity of a recombinant protein containing the CTL epitopes of non-structural proteins of hepatitis C virus.

Hepatitis C virus (HCV) can cause chronic infection and evade the immune response. The generation and maintenance of an effective T-cell response is important for immune-mediated control of HCV infection. The purpose of this study was to obtain recombinant mosaic proteins containing the cytotoxic T lymphocyte (CTL) epitopes of HCV fused with different adjuvants and analyse their immunogenicity. A recombinant polyepitope protein comprising HLA-A2-restricted CTL epitopes of the NS3, NS4ab and NS5a proteins of HCV was designed. Adjuvant compounds, the T-helper (Th) epitope PADRE, lipopeptide from Neisseria meningiditis and interleukin 2 (IL-2) were included in the fusion proteins. Three proteins differing in their adjuvant content were expressed in Escherichia coli and purified. The purified proteins formed nanosized particles. The proteins were characterized by their ability to cause proliferation of spleen cells, induce expression of cytokine genes and production of interferon gamma by T lymphocytes of immunized mice. The obtained recombinant vaccine proteins effectively stimulate dendritic cells, which in turn specifically activate Th1 and Th2 lymphocytes. Adjuvant components act additively to enhance the stimulation of dendritic cells and polarize them in the direction of Th1 lymphocyte activation. Analysis of spleen cell proliferation, expression of Th1 and Th2 cytokines and production of interferon gamma by lymphocytes of immunized mice after specific stimulation in vitro revealed that recombinant protein comprising CTL epitopes of HCV, Th epitope PADRE, lipoprotein and IL-2 induced the highest response of T-lymphocytes.