Impact of hemagglutination activity and M2e immunity on conferring protection against influenza viruses.

To improve cross-protection of influenza vaccination, we tested conjugation of conserved M2e epitopes to the surface of inactivated influenza virus (iPR8-M2e*). Treatment of virus with chemical cross-linker led to diminished hemagglutination activity and failure to induce hemagglutination inhibiting antibodies. Conjugated iPR8-M2e* vaccine was less protective against homologous and heterosubtypic viruses, despite the induction of virus-specific binding IgG antibodies. In alternative approaches to enhance cross-protection, we developed a genetically linked chimeric protein (M2e-B stalk) vaccine with M2e of influenza A and hemagglutinin (HA) stalk of influenza B virus. Vaccination of mice with inactivated influenza A virus supplemented with M2e-B stalk effectively induced hemagglutination inhibiting antibodies, humoral and cellular M2e immune responses, and enhanced heterosubtypic protection. This study demonstrates the importance of HA functional integrity in influenza vaccine efficacy and that supplementation of influenza vaccines with M2e-B stalk protein could be a feasible strategy of improving cross-protection against influenza viruses.

A chimeric thermostable M2e and H3 stalk-based universal influenza A virus vaccine.

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.

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Thermostable H1 hemagglutinin stem with M2e epitopes provides broad cross-protection against group1 and 2 influenza A viruses.

Hemagglutinin (HA) stem-based vaccines have limitations in providing broad and effective protection against cross-group influenza viruses, despite being a promising universal vaccine target. To overcome the limited cross-protection and low efficacy by HA stem vaccination, we genetically engineered a chimeric conjugate of thermostable H1 HA stem and highly conserved M2e repeat (M2e-H1stem), which was expressed at high yields in Escherichia coli. M2e-H1stem protein presented native-like epitopes reactive to antisera of live virus infection. M2e-H1stem protein vaccination of mice induced strong M2e- and HA stem-specific immune responses, conferring broadly effective cross-protection against both antigenically distinct group 1 (H1N1, H5N1, and H9N2 subtypes) and group 2 (H3N2 and H7N9 subtypes) seasonal and pandemic potential influenza viruses. M2e-H1stem vaccination generated CD4+ and CD8+ T cell responses and antibody-dependent cytotoxic cellular and humoral immunity, which contributed to enhancing cross-protection. Furthermore, comparable broad cross-group protection was observed in older aged mice after M2e-H1stem vaccination. This study provides evidence warranting further development of chimeric M2e-stem proteins as a promising universal influenza vaccine candidate in adult and aged populations.

Enhanced cross protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes.

Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.

Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be expanding the pandemic disease across the globe. Although SARS-CoV-2 vaccines were rapidly developed and approved for emergency use of vaccination in humans, supply and production difficulties are slowing down the global vaccination program. The efficacy of many different versions of vaccine candidates and adjuvant effects remain unknown, particularly in the elderly. In this study, we compared the immunogenic properties of SARS-CoV-2 full-length spike (S) ectodomain in young adult and aged mice, S1 with receptor binding domain, and S2 with fusion domain. Full-length S was more immunogenic and effective in inducing IgG antibodies after low dose vaccination, compared to the S1 subunit. Old-aged mice induced SARS-CoV-2 spike-specific IgG antibodies with neutralizing activity after high dose S vaccination. With an increased vaccine dose, S1 was highly effective in inducing neutralizing and receptor-binding inhibiting antibodies, although both S1 and S2 subunit domain vaccines were similarly immunogenic. Adjuvant effects were significant for effective induction of IgG1 and IgG2a isotypes, neutralizing and receptor-binding inhibiting antibodies, and antibody-secreting B cell and interferon-γ secreting T cell immune responses. Results of this study provide information in designing SARS-CoV-2 spike vaccine antigens and effective vaccination in the elderly.

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin.

Recombinant baculoviruses (rBVs) have been extensively used to generate virus-like particles, and baculoviruses expressing antigenic proteins have become efficient tools for inducing protective immunity. However, current methods for generating baculoviruses are costly and inefficient. Thus, the development of a simple, rapid, and accurate method of baculovirus titration is critically important. We established a method of plaque assay using an immunostaining method by which plaques can be easily visualized in Sf9 cells under a light microscope. Sf9 cells were infected with recombinant baculoviruses expressing influenza hemagglutinin surface proteins from H1N1 (A/California/04/09) or rH5N1 (A/Vietnam/1203/04). The infected cells were incubated with anti-HA antibody and the plaques were visualized using the chromogen 3'3-diaminobenzidine (DAB). Plaques were observed from days 1 to 6 post-infection, and differences in Sf9 cell seeding densities resulted in variations in the final plaque quantification. Sf9 cells seeded at a concentration of 5.5 × 104 cells/well or 7.5 × 104 cells/well showed the higher plaque titers at days 3, 4, and 5 post-infection than those found at days 1, 2, and 6 post-infection. With 5.5 × 104 cells/well or 7.5 × 104 cells/well of cell concentrations, recombinant baculovirus for rBV-HA (H1N1) showed 6 × 107 pfu/ml of titer and rBVs for rBV-HA (rH5N1) showed 5.4 × 107 pfu/ml of titer. Three days of baculovirus incubation with a certain concentration of Sf9 cells seeded are required for a rapid, simple, and accurate plaque assay, which could significantly contribute to all baculovirus-related studies.

Cross protection by inactivated recombinant influenza viruses containing chimeric hemagglutinin conjugates with a conserved neuraminidase or M2 ectodomain epitope.

Influenza virus neuraminidase (NA) contains a universally conserved epitope (NAe, NA222-230). However, no studies have reported vaccines targeting this NA conserved epitope and inducing antibodies recognizing NAe. The extracellular domain of M2 (M2e) is considered as an attractive target for a universal influenza vaccine. We generated recombinant influenza H1N1 viruses expressing conserved epitopes in hemagglutinin (HA) molecules: NAe (NAe-HA) or M2e (M2e-HA) within the HA head domain. Inactivated recombinant NAe-HA and M2e-HA viruses were more effective in inducing IgG antibodies specific for an inserted conserved epitope than live recombinant virus. Recombinant inactivated M2e-HA virus vaccination induced cross protection against H3N2 virus with less weight loss compared to NAe-HA and was more effective in inducing humoral and cellular M2e immune responses. This study provides insight into developing recombinant influenza virus vaccines compatible with current platforms to induce antibody responses to conserved poorly immunogenic epitopes.