Protein nanoparticle vaccine based on flagellin carrier fused to influenza conserved epitopes confers full protection against influenza A virus challenge

Abstract

Currently marketed influenza vaccines only confer protection against matching influenza virus strains. The influenza A composition of these vaccines needs to be annually updated. Vaccines that target conserved epitopes of influenza viruses would in principle offer broad cross-protection against influenza A viruses. In our study, we investigated the specific immune responses and protective efficacy of protein nanoparticles based on fusion proteins of flagellin carrier linked to conserved influenza epitopes. We first designed the fusion protein by replacing the hyperimmunogenic region of flagellin (FliC) with four tandem copies of the ectodomain of matrix protein 2 (f4M2e), H1 HA2 domain (fHApr8) or H3 HA2 domain (fHAaichi). Protein nanoparticles fabricated from these fusion proteins by using DTSSP crosslinking retained Toll-like receptor 5 agonist activity of FliC. Intranasal immunization with f4M2e, f4M2e/fHApr8 or f4M2e/fHAaichi nanoparticles induced vaccine antigen-specific humoral immune responses. It was also found that the incorporation of the H1 HA2 domain into f4M2e/fHApr8 nanoparticles boosted M2e specific antibody responses. Immunized mice were fully protected against lethal doses of virus challenge.

Heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice

“…A recent study showed that increasing stirring speed and changing impeller shape could maintain proper mixing of desolvated gelatin nanoparticles in gram-scale batches (61). Furthermore, we showed that ultrafiltration was a viable, commercially relevant alternative to particle collection by centrifugation (62).…”

Plant-Produced Recombinant Influenza A Virus Candidate Vaccine Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin

“…A number of candidate vaccines based on M2e were able to induce a strong M2e-specific humoral immune response to protect animals against influenza A virus challenge [9,10]. A combination of M2e and HA2 in a single vaccine protein is a promising approach for the development of influenza vaccines with a broad spectrum of protection [11][12][13].…”

Peer Review #3 of "Application of built-in adjuvants for epitope-based vaccines (v0.1)"

“…Manuscript to be reviewed the portion between the N-and C-terminals comprises a highly variable flagellin antigen region (Murthy et al, 2004). It has been found that deletion of part of the highly variable region of flagellin disables the ability of the host to produce antibodies against bacterial flagellum but does not affect its adjuvant activity (Deng et al, 2017). Therefore, researchers typically replace this region with exogenous antigen epitopes (e.g., the HPV prophylactic peptide vaccine) (Nempont et al, 2008;Negahdaripour et al, 2017a).…”

“…Therefore, researchers typically replace this region with exogenous antigen epitopes (e.g., the HPV prophylactic peptide vaccine) (Nempont et al, 2008;Negahdaripour et al, 2017a). For example, replacing the FliC variable region with the M2e protein of influenza A does not obstruct TLR signaling pathways (Smith et al, 2003;Deng et al, 2017). A truncated flagellin (tFL) with deletion of the hypervariable regions was used as a carrier by chemical conjugation with a malaria antigen M.RCAg-1 (M312), and compared with the physical mixture of M312 and tFL, the conjugates M312-PEG-tFL elicited higher M312specific antibody titers (Guo et al, 2018).…”

“…In addition, two HPV epitopes and some universal Th epitopes have been linked to the different flagellin positions via different linkers, and the optimal construction of a multiepitope vaccine was screened using protein structure analysis, modeling, optimization, and an evaluation of immunogenicity (Negahdaripour et al, 2017c). In addition, four copies of the ectodomain of matrix protein 2 (f4M2e) of the influenza A virus, H1, HA2 domain (fHApr8), or H3 HA2 domain (fHAaichi) were used to replace the high immunogenicity region of flagellin, and the fusion proteins were crosslinked with propionate (DTSSP) to form protein nanoparticles, thereby retaining the agonist activity of FliC to TLR5, and ability to assist the epitope protein in stimulating the immune response against influenza A virus (Deng et al, 2017). Manuscript to be reviewed thus demonstrating a significant advantage of this strategy in enhancing the cross-protection of the HPV vaccine (Kalnin et al, 2014;Gambhira et al, 2007;Kalnin et al, 2017).…”