M2e Epitope Research Articles

Intranasal delivery of a self-adjuvanted nanovaccine composed of the curli filaments and the highly conserved M2e epitope confers protection against influenza a virus in mice

Intranasal administration of vaccines is an attractive delivery route to fight viral respiratory infections. However, there are only a few intranasal vaccines used in human, emphasizing the critical need to identify novel safe mucosal adjuvants and antigen delivery systems to expand their usage. We recently revealed an immunostimulating nanoparticle based on a fragment (R4R5) of the Curli-specific gene A (CsgA) protein that confers protection against influenza A virus (IAV) when conjugated to three repeats of the highly conserved M2e epitope and administrated intramuscularly. Herein, the efficacy of this 3M2e-R4R5 nanovaccine was investigated upon administration by intranasal instillation. By triggering robust M2e-specific humoral and cellular responses, both systemic and locally in the respiratory tract, and by priming alveolar macrophages, the intranasal vaccine protected mice against a lethal IAV challenge without the use of additional adjuvant. Thus, CsgA-based nanostructures could serve as a safe and self-adjuvanted antigen delivery system for mucosal immunization.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein

BackgroundThe influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation.MethodsThe phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging.ResultsThe four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells.ConclusionThis structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

M2e-specific antibodies protect against influenza PR8 virus in an isotype and route dependent manner.

The ectodomain of influenza matrix protein 2 (M2e) is a promising target for the development of universal prophylactic and therapeutic agents against influenza viruses of different subtypes. We constructed three M2e-specific monoclonal antibody variants, M2A1-1 (IgG1), M2A1-2a (IgG2a), M2A1-2b (IgG2b), which have the same Fab region targeting the M2e epitope but different isotypes, and compared their protective efficacy in influenza PR8-infected mice. We found that anti-M2e antibodies provided protection against influenza virus in a subtype-dependent manner, with the IgG2a variant providing significantly better protection with lower virus titers and milder lung injury than IgG1 and IgG2b isotypes. Additionally, we observed that the protective efficacy was dependent on the administration routes, with intranasal administration of antibody providing better protection than intraperitoneal administration. The timing of administration was also critical in determining the protective efficacy; while all the antibody isotypes provided protection when administered before influenza challenge, only IgG2a provided minimal protection when the antibodies were administered after virus challenge. These results provide valuable information for optimizing the therapeutics usage of M2e-based antibodies and furthering the development of M2e-based universal influenza vaccines.

2318. A Composite Peptide Vaccine Comprised of Conserved SARS-CoV-2 and Influenza Epitopes Generated Antisera Reponses to Both Coronavirus and Influenza

Abstract Background While influenza and SARS-CoV-2 virus can be severe, co-infection with these viruses has the potential for worsening the clinical outcome. The combined infection of influenza A virus (IAV) and SARS-CoV-2 in animal models has also shown elevated SARS-CoV-2 viral load and pneumonia. Co-vaccination may provide a useful option in prevention of combined infection. In this study, a composite peptide vaccine comprised of highly conserved influenza neuraminidase (NA) and matrix protein (M2e) epitopes, in combination with either conserved spike protein (SP) or RNA polymerase (POL) epitopes of SARS-CoV-2 generated robust immune responses to both coronavirus and influenza virus. Methods Mice were immunized with 20µg of Coronavirus Pep02 (CorPep02, comprised of POL) or Coronavirus Pep05 (CorPep05, comprised of POL and NA+M2e) or Coronavirus Pep11 (CorPep11, comprised of SP and NA+M2e), formulated with AddaVax™. Serum antibody titers to both coronavirus and influenza peptides, and whole viruses were analyzed using ELISA. Antisera neutralizing activity against IAV was determined using microneutralization assay (MNA). Results CorPep05 immunization generated enhanced IgG1 and IgG2b antisera responses to the immunogens compared to CorPep02, starting at Day-21. Immunization with CorPep05 or CorPep11 induced strong IgG1 and IgG2b immune responses to the composite coronavirus peptides, influenza NA and M2e peptides, coronavirus POL and SP peptides. Antisera from CorPep05 and CorPep11 groups bound to both viruses (IAV and human coronavirus NL-63 (HCoV NL-63)) and demonstrated neutralizing activity against IAV, with titers >5000. Conclusion A composite peptide vaccine comprised of both influenza (NA+M2e) and coronavirus (POL) epitopes generated enhanced immune responses in comparison to peptide containing only POL. Th1 and Th2 immune responses to coronavirus POL and SP were observed. Serum neutralizing activity was demonstrated against IAV. Studies are underway to examine neutralizing antibodies against HCoV NL-63 and multiple variants of SARS-CoV-2. Highly conserved epitopes in these composite peptide vaccines may provide an important strategy to prevent infections with IAV or SARS-CoV-2 and mitigate the threat of co-infection. Disclosures All Authors: No reported disclosures.

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.

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.

Optimization of M2e cassette amino acid composition for the development of universal influenza vaccine

The development of a universal influenza vaccine with a wide spectrum and duration of action is one of the serious public health problems. This study is dedicated to optimization of an immunogen covering the M2e epitopes of influenza A viruses circulating in the natural reservoir, as well as the creation of a prototype of a universal influenza vaccine with subsequent quantitative and qualitative assessment of the induced anti-M2e responses in ferrets.

Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge.

The development of an influenza vaccine with broad protection and durability remains an attractive idea due to the high mutation rate of the influenza virus. An extracellular domain of Matrix 2 protein (M2e) is among the most attractive target for the universal influenza vaccine owing to its high conservancy rate. Here, we generated two recombinant live attenuated influenza vaccine (LAIV) candidates encoding four M2e epitopes representing consensus sequences of human, avian and swine influenza viruses, and studied them in a preclinical ferret model. Both LAIV+4M2e viruses induced higher levels of M2e-specific antibodies compared to the control LAIV strain, with the LAIV/HA+4M2e candidate being significantly more immunogenic than the LAIV/NS+4M2e counterpart. A high-dose heterosubtypic influenza virus challenge revealed the highest degree of protection after immunization with LAIV/HA+4M2e strain, followed by the NS-modified LAIV and the classical LAIV virus. Furthermore, only the immune sera from the LAIV/HA+4M2e-immunized ferrets protected mice from a panel of lethal influenza viruses encoding M genes of various origins. These data suggest that the improved cross-protection of the LAIV/HA+4M2e universal influenza vaccine candidate was mediated by the M2e-targeted antibodies. Taking into account the safety profile and improved cross-protective potential, the LAIV/HA+4M2e vaccine warrants its further evaluation in a phase I clinical trial.

Self-assembled peptide nanorod vaccine confers protection against influenza A virus

Proteinaceous nanostructures have emerged as a promising strategy to develop safe and efficient subunit vaccines. The ability of synthetic β-sheet self-assembling peptides to stabilize antigenic determinants and to potentiate the epitope-specific immune responses have highlighted their potential as an immunostimulating platform for antigen delivery. Nonetheless, the intrinsic polymorphism of the resulting cross-β fibrils, their length in the microscale and their close structural similarity with pathological amyloids could limit their usage in vaccinology. In this study, we harnessed electrostatic capping motifs to control the self-assembly of a chimeric peptide comprising a 10-mer β-sheet sequence and a highly conserved epitope derived from the influenza A virus (M2e). Self-assembly led to the formation of 100–200 nm long uniform nanorods (NRs) displaying the M2e epitope on their surface. These cross-β assemblies differed from prototypical amyloid fibrils owing to low polydispersity, short length, non-binding to thioflavin T and Congo Red dyes, and incapacity to seed homologous amyloid assembly. M2e-NRs were efficiently uptaken by antigen presenting cells and the cross-β quaternary architecture activated the Toll-like receptor 2 and stimulated dendritic cells. Mice subcutaneous immunization revealed a robust M2e-specific IgG response, which was dependent on self-assembly into NRs. Upon intranasal immunization in combination with the polymeric adjuvant montanide gel, M2e-NRs conferred complete protection with absence of clinical signs against a lethal experimental infection with the H1N1 influenza A virus. These findings indicate that by acting as an immunostimulator and delivery system, synthetic peptide-based NRs constitute a versatile self-adjuvanted nanoplatform for the delivery of subunit vaccines.

DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Self-Assembly M2e-Based Peptide Nanovaccine Confers Broad Protection Against Influenza Viruses.

The extracellular domain of influenza M2 protein (M2e) is highly conserved and is a promising target for development of universal influenza vaccines. Here, we synthesized a peptide vaccine consisting of M2e epitope linked to a fibrillizing peptide, which could self-assemble into nanoparticle in physiological salt solutions. When administrated into mice without additional adjuvant, the influenza A M2e epitope-bearing nanoparticles induced antibodies against M2e of different influenza subtypes. Comparing with other M2e-based vaccine, these M2e nanoparticles did not induce immune response against the fibrillizing peptide, demonstrating minimal immunogenicity of vaccine carrier. Furthermore, vaccination with M2e-based nanoparticles did not only protect mice against homologous challenge of influenza PR8 H1N1 virus, but also provide protection against heterologous challenge of highly pathogenic avian influenza H7N9 virus. These results indicated that M2e-based self-assembled nanoparticle vaccine is safe and can elicit cross-protection, therefore is a promising candidate of universal influenza vaccines.

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae.

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.

Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization.

Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense "grains" on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzyme-linked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

An Inactivated Influenza Virus Vaccine Approach to Targeting the Conserved Hemagglutinin Stalk and M2e Domains.

Universal influenza virus vaccine candidates that focus on the conserved hemagglutinin (HA) stalk domain and the extracellular domain of the matrix protein 2 (M2e) have been developed to increase the breadth of protection against multiple strains. In this study, we report a novel inactivated influenza virus vaccine approach that combines these two strategies. We inserted a human consensus M2e epitope into the immunodominant antigenic site (Ca2 site) of three different chimeric HAs (cHAs). Sequential immunization with inactivated viruses containing these modified cHAs substantially enhanced M2e antibody responses while simultaneously boosting stalk antibody responses. The combination of additional M2e antibodies with HA stalk antibodies resulted in superior antibody-mediated protection in mice against challenge viruses expressing homologous or heterosubtypic hemagglutinin and neuraminidase compared to vaccination strategies that targeted the HA stalk or M2e epitopes in isolation.

Design of a Universal Influenza A Vaccine Candidate Based on M2e.FliC; Immunoinformatics Analysis, Protein Modeling, and Its Expression in Escherichia coli

Background: Due to the rapid accumulation of mutations in influenza virus and the unpredictability of new influenza, the current influenza vaccines require an almost yearly reformulation. The extracellular domain of matrix protein 2 (M2e) of influenza A viruses is conserved and is an attractive alternative approach to be used as a vaccine with a broad cross- protection. Objectives: In this study, a vector containing three repeats of M2e gene of influenza A virus fused with molecular adjuvant of FliC was constructed. Methods: In silico analysis of 3M2e.FliC chimeric polypeptide was performed based on 3M2e.FliC sequence, virtual fusion construction translation, linear epitope prediction of 3M2e.FliC, 3M2e.FliC modeling, and validation score consideration through immunoinformatics approaches. Expression of 3M2e.FliC was carried out in two strains of Escherichia coli (BL21 [DE3] and ER2566). The fidelity of expression in both hosts was analyzed through a time course of sampling by SDS-PAGE and confirmed by western blotting. Results: The immunoinformatics results indicated that M2e and FliC epitopes were at the surface of protein, which would be accessible for the immune system. The expression results demonstrated that the 3M2e.FliC construct was expressed well in both strains of E. coli, although the efficiency of expression in ER2566 strain was higher than that of BL21 (DE3) strain. Conclusions: The 3M2e.Flic protein as a recombinant antigen may be considered as a universal influenza vaccine candidate after its evaluation and assessment in animal models.

Complement C3 Plays a Key Role in Inducing Humoral and Cellular Immune Responses to Influenza Virus Strain-Specific Hemagglutinin-Based or Cross-Protective M2 Extracellular Domain-Based Vaccination.

The complement pathway is involved in eliminating antigen immune complexes. However, the role of the C3 complement system remains largely unknown in influenza virus M2 extracellular (M2e) domain or hemagglutinin (HA) vaccine-mediated protection after vaccination. Using a C3 knockout (C3 KO) mouse model, we found that complement protein C3 was required for effective induction of immune responses to vaccination with M2e-based or HA-based vaccines, which include isotype class-switched antibodies and effector CD4 and CD8 T cell responses. C3 KO mice after active immunization with cross-protective nonneutralizing M2e-based vaccine were not protected against influenza virus, although low levels of M2e-specific antibodies were protective after passive coadministration with virus in wild-type mice. In contrast, C3 KO mice that were immunized with strain-specific neutralizing HA-based vaccine were protected against homologous virus challenge despite lower levels of HA antibody responses. C3 KO mice showed impaired maintenance of innate immune cells and a defect in innate immune responses upon exposure to antigens. The findings in this study suggest that C3 is required for effective induction of humoral and cellular adaptive immune responses as well as protective immunity after nonneutralizing influenza M2e vaccination.IMPORTANCE Complement is the well-known innate immune defense system involved in the opsonization and lysis of pathogens but is less studied in establishing adaptive immunity after vaccination. Influenza virus HA-based vaccination confers protection via strain-specific neutralizing antibodies, whereas M2e-based vaccination induces a broad spectrum of protection by immunity against the conserved M2e epitopes. This study revealed the critical roles of C3 complement in inducing humoral and cellular immune responses after immunization with M2e or HA vaccines. C3 was found to be required for protection by M2e-based but not by HA-based active vaccination as well as for maintaining innate antigen-presenting cells. Findings in this study have insight into better understanding the roles of C3 complement in inducing effective innate and adaptive immunity as well as in conferring protection by cross-protective conserved M2e vaccination.

Virus-Like Particles Are a Superior Platform for Presenting M2e Epitopes to Prime Humoral and Cellular Immunity against Influenza Virus.

Influenza virus M2 protein has a highly conserved ectodomain (M2e) as a cross-protective antigenic target. We investigated the antigenic and immunogenic properties of tandem repeat M2e (5xM2e) proteins and virus-like particles (5xM2e VLP) to better understand how VLP and protein platform vaccines induce innate and protective adaptive immune responses. Despite the high antigenic properties of 5xM2e proteins, the 5xM2e VLP was superior to 5xM2e proteins in inducing IgG2a isotype antibodies, T cell responses, plasma cells and germinal center B cells as well as in conferring cross protection. Mice primed with 5xM2e VLP were found to be highly responsive to 5xM2e protein boost, overcoming the low immunogenicity and protective efficacy of 5xM2e proteins. Immunogenic differences between VLPs and proteins in priming immune responses might be due to an intrinsic ability of 5xM2e VLP to stimulate dendritic cells secreting T helper type 1 (Th1) cytokines. We also found that 5xM2e VLP was effective in inducing inflammatory cytokines and chemokines, and in recruiting macrophages, monocytes, neutrophils, and CD11b+ dendritic cells at the injection site. Therefore, this study provides evidence that 5xM2e VLP is an effective vaccine platform, inducing cross-protection by stimulating innate and adaptive immune responses.

Immunogenicity and efficacy of replication-competent recombinant influenza virus carrying multimeric M2 extracellular domains in a chimeric hemagglutinin conjugate

Current influenza vaccines provide hemagglutinin (HA) strain-specific protection. To improve cross protection, we engineered replication-competent influenza A virus to express tandem repeats of heterologous M2 extracellular (M2e) domains in a chimeric HA. M2e epitopes conjugated to HA glycoproteins (M2e4x-HA) were found to be expressed on the surfaces of a replicable influenza virus as examined by electron microscopy. The recombinant influenza virus containing M2e4x-HA was moderately attenuated but superior to the parental virus in inducing M2e specific antibodies without compromising HA immunogenicity. Recombinant influenza virus immune mice showed better cross protection than parental virus immune mice. Immune sera from the mice with inoculation of live recombinant influenza virus expressing M2e4x-HA were effective in conferring protection against H1, H3, and H5 subtype influenza viruses. This study indicates that recombinant influenza virus expressing conserved protective epitopes in an HA chimeric form can provide a new approach for improving the efficacy of influenza vaccines.