Abstract
We developed a new chimeric M2e and H3 hemagglutinin (HA) stalk protein vaccine (M2e-H3 stalk) by genetic engineering of modified H3 stalk domain conjugated with conserved M2e epitopes to overcome the drawbacks of low efficacy by monomeric domain-based universal vaccines. M2e-H3 stalk protein expressed and purified from Escherichia coli was thermostable, displaying native-like antigenic epitopes recognized by antisera of different HA subtype proteins and influenza A virus infections. Adjuvanted M2e-H3 stalk vaccination induced M2e and stalk-specific IgG antibodies recognizing viral antigens on virus particles and on the infected cell surface, CD4+ and CD8+ T-cell responses, and antibody-dependent cytotoxic cell surrogate activity in mice. M2e-H3 stalk was found to confer protection against heterologous and heterosubtypic cross-group subtype viruses (H1N1, H5N1, H9N2, H3N2, H7N9) at similar levels in adult and aged mice. These results provide evidence that M2e-H3 stalk chimeric proteins can be developed as a universal influenza A virus vaccine candidate for young and aged populations.
Introduction
Influenza virus transmissions can be controlled by effective vaccination. However, the effectiveness of influenza vaccination inducing neutralizing antibodies to strain-specific hemagglutinin (HA) proteins is low due to constant antigenic changes in the HA1 receptor-binding globular head domain, rendering pre-existing immunity ineffective to new pandemics. For example, during the 2014–2015 season, drifting mutations in circulating H3N2 strains significantly reduced the effectiveness to 6% against H3N2 subtype virus1. Influenza A virus HA subtypes are phylogenetically divided into group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18) and group 2 (H3, H4, H7, H10, H14, H15)2. The HA on the virion is in the prefusion state and cleaved by host proteases into HA1 and HA23. In contrast to the highly variable antigenic region in the HA1 head domain, the HA2 stalk region is relatively conserved among the same HA group viruses, as recognized by broadly neutralizing antibodies among the different subtype viruses, supporting the HA2 stalk domain as a promising target for developing a universal vaccine4,5.
Previous studies reported that headless H1 stalk stabilized protein nanoparticle vaccines could provide protection against homologous H1N16,7 and heterosubtypic H5N1 virus6,8. Group 2 HA stalk proteins were reported to be more challenging in stabilizing the trimers since additional modifications had to be introduced9. Headless H3 and H7 stalk protein vaccines were constructed and shown to be immunogenic, inducing protection against homologous H3N2 and H7N9 viruses respectively9,10. The efficacy of stalk-based particularly group 2 stalk vaccines was low with homo and heterologous viruses as evidenced by substantial body weight loss in mice and the breadth of viruses tested was very limited6,9,10. In addition, challenges exist in H3N2 subtypes compared to H1N1 subtypes because of stalk mutations in circulating strains and low fitness genetic barrier for H3N2 viruses in vitro and in vivo11. These drawbacks have been difficult challenges to overcome in developing effective H3 stalk-based vaccines.
Another promising antigenic target is the highly conserved extracellular domain of matrix 2 (M2e) protein in influenza A viruses12,13. M2e-based vaccines could provide broad cross protection against different strains and subtypes in mice12,13,14,15. Recombinant M2e vaccines were safe in phase 1 trials13,16,17. Low efficacy of M2e-based vaccines inducing non-neutralizing immunity is a concern for advancing toward a stand-alone vaccine. H1 plus H3 stalk protein vaccines layered onto the M2e core nanoparticles via chemical cross linking were reported to induce enhanced cross protection, compared to stalk or M2e alone vaccines18.
In this study, we constructed a chimeric M2e and H3 stalk vaccine by genetically linking M2e repeat to the engineered H3 stalk domain with stabilizing HA1 N- and C-terminal region and point mutations (M2e-H3 stalk). E. Coli expressed M2e-H3 stalk protein displayed multi conserved M2e and stalk epitopes that are recognized by antisera of both group 1 and 2 influenza virus infections and different subtype HA proteins. Adjuvanted M2e-H3 stalk protein vaccination induced broad protection against cross-group heterologous and heterosubtypic viruses despite a wider range of antigenic differences in adult and aged mice.
Results
Rationale design and development of chimeric M2e-H3 stalk universal vaccine construct
Structural conformation of HA2 stalk domain was previously modeled to be stabilized with the N- and C-terminal HA1 parts6,8. To extend and enhance the breadth of cross protection, a genetic fusion of M2e epitopes and H3 stalk was constructed (Fig. 1A–D). The H3 shortened stalk domain contains HA1 parts [aa 37-61, aa 305-338 of H3 HA from A/Aichi], and HA2 stalk in α-helix conformation [aa 1-117, Fig. 1B, D]. Tandem 2x repeat of M2e (23 aa) epitope domains was genetically fused to the H3 stalk N-terminus (M2e-H3 stalk) from A/Aichi/H3N2 influenza A virus.
