A Single-Component Multilayered Self-Assembling Protein Nanoparticle Vaccine Based on Extracellular Domains of Matrix Protein 2 against Both Influenza A and B

Abstract

The development of an effective and broadly protective influenza vaccine against circulating and emerging strains remains elusive. In this study, we evaluated a potentially universal influenza vaccine based on single-component self-assembling protein nanoparticles (1c-SApNPs) presenting the conserved matrix protein 2 ectodomain (M2e) from influenza A and B viruses (IAV and IBV, respectively). We previously designed a tandem antigen comprising three IAV M2e domains of human, avian/swine, and human/swine origins (termed M2ex3). The M2ex3-presenting 1c-SApNPs conferred complete protection in mice against sequential lethal challenges with H1N1 and H3N2. To broaden this protection to cover IBVs, we designed a series of antigens incorporating different arrangements of three IAV M2e domains and three copies of IBV M2e. Tandem repeats of IAV and IBV (termed influenza A-B) M2e arrayed on the I3-01v9a 60-mer 1c-SApNP, when formulated with an oil-in-water emulsion adjuvant, generated greater M2e-specific immunogenicity and protective efficacy than the soluble influenza A-B M2e trimer, indicated by higher survival rates and reduced weight loss post-challenge. Importantly, one of the influenza A-B M2e SApNP constructs elicited 100% protection against a lethal influenza A/Puerto Rico/8/1934 (H1N1) challenge in mice and 70% protection against a lethal influenza B/Florida/4/2006 (Yamagata lineage) challenge, the latter of which has not been reported in the literature to date. Our study thus provides a promising M2e-based single-component universal vaccine candidate against the two major types of influenza virus circulating in humans.

Keywords: M2e; antibody; influenza A; influenza B; protein nanoparticle; universal influenza vaccine.

Figure 1

Design and in vitro characterization of influenza A-B M2e-based immunogens. (A) Top left: Amino acid sequences for influenza A M2e from human (UniProt: P06821, residue 2–24), avian/swine (residue 2–24) [16], and human/swine (residue 2–24) [16] and influenza B M2e from human (UniProt: A4D452, residue 2–9). Top right: Series of designed antigen sequences for influenza A-B M2exA3B3, M2exB3A3, M2e(AB)x3, and M2e(BA)x3 constructs. A glycine (G4) linker was added between any two adjacent M2e peptides, regardless of IAV and IBV M2e. Bottom: Schematics of influenza A-B M2e antigens consisting of various arrangements of human, avian, and swine IAV M2e plus three copies of IBV M2e displaying on I3-01v9a-LD7-PADRE (or simply I3-01v9a-L7P) SApNPs. (B) SEC profiles of the M2exA3B3-5GS-1TD0 trimer and M2exA3B3-, M2exB3A3-, M2e(AB)x3-, and M2e(BA)x3-presenting I3-01v9a SApNPs. The trimer was analyzed on a Superdex 75 Increase 10/300 GL column and four SApNPs were analyzed using a Superose 6 Increase 10/300 GL column. (C) BN-PAGE for mAb148-purified influenza A-B M2e I3-01v9a SApNPs. (D) DLS profiles of mAb148-purified influenza A-B M2e I3-01v9a SApNPs. Average particle sizes derived from DLS are labeled. (E) nsEM micrographs of mAb148-purified influenza A-B M2e I3-01v9a-L7P SApNPs. (F) ELISA analysis of the M2eA3B3-5GS-1TD0 trimer and M2exA3B3-, M2exB3A3-, M2e(AB)x3-, and M2e(BA)x3-presenting I3-01v9a SApNPs binding to mAb148 (left) and mAb65 (right) before and after heating to 50 °C, 60 °C, and 70 °C for 30 min.

Figure 2

Protective efficacy of influenza A-B M2e-based trimer and SApNP vaccines in mice. (A) Determination of lethal dose 50 (LD₅₀) for mouse-adapted A/Puerto Rico/8/1934 (H1N1) and mouse-adapted B/Florida/4/2006 (Yamagata lineage) through benchmark challenge studies (n = 10 mice/virus dilution). Mice were challenged with various dilutions of virus stock through nasal injection, with survival, weight loss, and morbidity monitored for 14 days post-challenge. (B) Schematic representation of mouse immunization, blood collection, and sequential challenges. Mice were immunized with the influenza A-B M2e 1TD0 trimer or SApNPs adjuvanted with AddaVax (AV) and then challenged using the LD₅₀ × 10 of mouse-adapted A/Puerto Rico/8/1934 (H1N1) or LD₅₀ × 5 of mouse-adapted B/Florida/4/2006 (Yamagata lineage) at weeks 6 or 10. (C) Survival rates and weight loss of vaccinated mice after the first challenge with mouse-adapted A/Puerto Rico/8/1934 (H1N1) followed by the second challenge with mouse-adapted B/Florida/4/2006 (Yamagata lineage). Mice were monitored for survival, weight loss, and morbidity for 14 days. Tested groups (n = 10 mice/group) included mice immunized with influenza A-B M2exA3B3 1TD0 trimer and four SApNPs (10 μg/mouse), naïve mice as a negative control, and strain-matched inactivated A/Puerto Rico/8/1934 virus (10 μg/mouse) as a positive control for the first mouse-adapted A/Puerto Rico/8/1934 (H1N1) challenge. (D) Survival rates and weight loss of vaccinated mice after the first challenge with mouse-adapted B/Florida/4/2006 followed by the second challenge with mouse-adapted A/Puerto Rico/8/1934 (H1N1). A group in which mice were immunized with inactivated B/Florida/4/2006 (Yamagata) virus was included as a positive control for the first challenge with strain-matched mouse-adapted B/Florida/4/2006. Images of mouse immunization, virus challenge, and blood collection were created with BioRender.com (accessed on 21 May 2024).

Figure 3

Immunogenicity of influenza A-B M2e-based trimer and SApNP vaccines assessed by serum binding to M2e trimer probes and homotetrameric M2e on influenza-infected cells. (A) ELISA curves showing the influenza A-B M2e-immune sera of individual mice (n = 10 mice) binding to sequence-matched M2e coating antigen (M2exA3B3-5GS-foldon, M2exB3A3-5GS-foldon, M2e[AB]x3-5GS-foldon, and M2e[BA]x3-5GS-foldon). (B) Influenza A-B M2e vaccine-induced serum binding antibody responses at weeks 2 and 5 were measured by half-maximal effective concentration (EC₅₀) titers. The assay was performed in duplicate with a starting serum dilution of 40× followed by seven 10-fold titrations. (C) Serum binding to M2e on the surface of MDCK cells infected with two IAVs, A/Puerto Rico/8/1934 (H1N1) and A/Hong Kong/1/1968 (H3N2), and two IBVs, B/Florida/4/2006 (Yamagata lineage) and B/Brisbane/60/2008 (Victoria lineage). Antibody mAb148 was used as a positive control for IAVs and a negative control for IBVs. The assay was performed in duplicate with a starting serum dilution of 40 times followed by five 5-fold titrations. Antibody mAb148 was diluted to 10 μg/mL followed by five 5-fold titrations. The statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison post hoc test for each time point (B) and influenza virus strain (C). For significance, ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.