A single-shot vaccine approach for the universal influenza A vaccine candidate M2e.

SignificanceAlthough the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24-amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.

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.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: This 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.

VLPs containing stalk domain and ectodomain of matrix protein 2 of influenza induce protection in mice.

BACKGROUND: As a result of antigenic drift, current influenza vaccines provide limited protection against circulating influenza viruses, and vaccines with broad cross protection are urgently needed. Hemagglutinin stalk domain and ectodomain of matrix protein 2 are highly conserved among influenza viruses and have great potential for use as a universal vaccine. METHODS: In this study, we co-expressed the stalk domain and M2e on the surface of cell membranes and generated chimeric and standard virus-like particles of influenza to improve antigen immunogenicity. We subsequently immunized BALB/c mice through intranasal and intramuscular routes. RESULTS: Data obtained demonstrated that vaccination with VLPs elicited high levels of serum-specific IgG (approximately 30-fold higher than that obtained with soluble protein), induced increased ADCC activity to the influenza virus, and enhanced T cell as well as mucosal immune responses. Furthermore, mice immunized by VLP had elevated level of mucosal HA and 4M2e specific IgA titers and cytokine production as compared to mice immunized with soluble protein. Additionally, the VLP-immunized group exhibited long-lasting humoral antibody responses and effectively reduced lung viral titers after the challenge. Compared to the 4M2e-VLP and mHA-VLP groups, the chimeric VLP group experienced cross-protection against the lethal challenge with homologous and heterologous viruses. The stalk domain specific antibody conferred better protection than the 4M2e specific antibody. CONCLUSION: Our findings demonstrated that the chimeric VLPs anchored with the stalk domain and M2e showed efficacy in reducing viral loads after the influenza virus challenge in the mice model. This antibody can be used in humans to broadly protect against a variety of influenza virus subtypes. The chimeric VLPs represent a novel approach to increase antigen immunogenicity and are promising candidates for a universal influenza vaccine.

Nanoparticles Carrying Conserved Regions of Influenza A Hemagglutinin, Nucleoprotein, and M2 Protein Elicit a Strong Humoral and T Cell Immune Response and Protect Animals from Infection.

Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new influenza A strains. Efficient control of infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A combination of several such antigens, including the conserved region of the second subunit of the hemagglutinin (HA2), the extracellular domain of the M2 protein (M2e), and epitopes of nucleoprotein (NP), which together can elicit an antibody- and cell-mediated immune response, would be preferred for vaccine development. In this study, we obtained recombinant virus-like particles formed by an artificial self-assembling peptide (SAP) carrying two epitopes from NP, tandem copies of M2e and HA2 peptides, along with a T helper Pan DR-binding epitope (PADRE). Fusion proteins expressed in Escherichia coli self-assembled in vitro into spherical particles with a size of 15-35 nm. Immunization of mice with these particles induced strong humoral immune response against M2e and the entire virus, and lead to the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with the influenza A virus. Our results show that SAP-based nanoparticles carrying conserved peptides from M2, HA, and NP proteins of the influenza A virus, as well as T helper epitope PADRE, can be used for the development of universal flu vaccines.

Is a Universal Influenza Virus Vaccine Possible?

Influenza viruses remain a severe burden to human health because of their contribution to overall morbidity and mortality. Current seasonal influenza virus vaccines do not provide sufficient protection to alleviate the annual impact of influenza and cannot confer protection against potentially pandemic influenza viruses. The lack of protection is due to rapid changes of the viral epitopes targeted by the vaccine and the often suboptimal immunogenicity of current immunization strategies. Major efforts to improve vaccination approaches are under way. The development of a universal influenza virus vaccine may be possible by combining the lessons learned from redirecting the immune response toward conserved viral epitopes, as well as the use of adjuvants and novel vaccination platforms.

Nanoparticles based on artificial self-assembling peptide and displaying M2e peptide and stalk HA epitopes of influenza A virus induce potent humoral and T-cell responses and protect against the viral infection.

The extracellular domain of the M2 protein (M2e) and conserved region of the second subunit of the hemagglutinin (HA2) could be used for the development of broad-spectrum vaccines against influenza A. Here we obtained and characterized recombinant mosaic proteins containing tandem copies of M2e and HA2 fused to an artificial self-assembling peptide (SAP). The inclusion of SAP peptides in the fusion proteins enabled their self-assembly in vitro into spherical particles with a size of 30-50 nm. Intranasal immunization of mice with these particles without additional adjuvants induced strong humoral immune response against M2e and the whole virus. Particles carrying both M2e and HA2 induced antigen-specific multifunctional CD4+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with different subtypes of influenza A virus. The obtained self-assembling nanoparticles can be used to develop a universal influenza vaccine.

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors.

Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes' presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300-400 µg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 µg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80-100 µg per gram of fresh leaf tissue; the yield after purification was up to 20 µg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.

Tetramerizing tGCN4 domain facilitates production of Influenza A H1N1 M2e higher order soluble oligomers that show enhanced immunogenicity in vivo.

One strategy for the development of a next generation influenza vaccine centers upon using conserved domains of the virus to induce broader and long-lasting immune responses. The production of artificial proteins by mimicking native-like structures has shown to be a promising approach for vaccine design against diverse enveloped viruses. The amino terminus of influenza A virus matrix 2 ectodomain (M2e) is highly conserved among influenza subtypes, and previous studies have shown M2e-based vaccines are strongly immunogenic, making it an attractive target for further exploration. We hypothesized that stabilizing M2e protein in the mammalian system might influence the immunogenicity of M2e with the added advantage to robustly produce the large scale of proteins with native-like fold and hence can act as an efficient vaccine candidate. In this study, we created an engineered construct in which the amino terminus of M2e is linked to the tetramerizing domain tGCN4, expressed the construct in a mammalian system, and tested for immunogenicity in BALB/c mice. We have also constructed a stand-alone M2e construct (without tGCN4) and compared the protein expressed in mammalian cells and in Escherichia coli using in vitro and in vivo methods. The mammalian-expressed protein was found to be more stable, more antigenic than the E. coli protein, and form higher-order oligomers. In an intramuscular protein priming and boosting regimen in mice, these proteins induced high titers of antibodies and elicited a mixed Th1/Th2 response. These results highlight the mammalian-expressed M2e soluble proteins as a promising vaccine development platform.

Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge.

PURPOSE: Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually due to constant antigenic changes in various strains. Therefore, the present study was conducted to evaluate protective immunity of the conserved influenza proteins. METHODS: For this purpose, three tandem repeats of M2e (3M2e) and NP were separately expressed in E. coli and were purified using column chromatography. Female Balb/c mice were injected intradermally with a combination of the purified 3M2e and NP alone or formulated with Alum (AlOH3) adjuvant in three doses. The mice were challenged by intranasal administration of H1N1 (A/PR/8/34) 2 weeks after the last vaccination. RESULTS: The results demonstrated that recombinant NP and M2e proteins are immunogenic and could efficiently elicit immune responses in mice compared to non-immunized mice. The combination of 3M2e and NP supplemented with Alum stimulated both NP and M2e-specific antibodies, which were higher than those stimulated by each single antigen plus Alum. In addition, the secretion of IFN-γ and IL-4 as well as the induction of lymphocyte proliferation in mice received the mixture of these proteins with Alum was considerably higher than other groups. Moreover, the highest survival rate (86%) with the least body weight change was observed in the mice immunized with 3M2e and NP supplemented with Alum followed by the mice received NP supplemented with Alum (71%). CONCLUSION: Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.

An approach to the influenza chimeric subunit vaccine (3M2e-HA2-NP) provides efficient protection against lethal virus challenge.

OBJECTIVES: Vaccination is the most effective preventive strategy for influenza disease. As the virus undergoes high antigenic drift, it requires a constant reformulation to obtain high protection. RESULTS: Immunogenicity of a purified chimeric protein containing conserved regions of influenza A/H1N1 viruses including the Hemagglutinin stalk domain, Nucleoprotein, and Matrix protein produced in a prokaryotic system was assessed in vitro and in vivo, alone or in combination with adjuvants by evaluating antibody responses, cytokine production, lymphocyte proliferative assay, and mortality rate after challenge. The animals that received the chimeric protein had specific antibody responses, elicited memory CD4 cells, cytokines of Th1 and Th2 cells and showed 75% protection against influenza virus lethal challenge. The animals injected with the chimeric protein supplemented with Alum showed improved immune responses, but they had 67% protection. In other words, although Alum adjuvant enriched the chimera specific immune responses potently, it could not enhance its protectivity. CONCLUSION: Regarding the immunogenicity and protectivity of the chimeric protein construct against influenza, findings of the study suggested that the chimeric protein could be considered as a promising influenza vaccine candidate.

The Role of Matrix Protein 2 Ectodomain in the Development of Universal Influenza Vaccines.

The influenza A virus matrix protein 2 ectodomain (M2e) is a universal influenza A vaccine candidate. Numerous studies in laboratory mice, but very few in natural influenza A virus hosts, have demonstrated that M2e-based vaccines can provide protection against any influenza A virus challenge. M2e-based immunity is largely accomplished by IgG and early stage clinical studies have demonstrated that the vaccine is safe. Yet M2e is considered a difficult target to develop as a vaccine: it does not offer sterilizing immunity and its mode of action relies on Fcγ receptor-mediated effector mechanisms, most likely in concert with alveolar macrophages. In a human challenge study with an H3N2 virus, treatment with a monoclonal M2e-specific human IgG was associated with a faster recovery compared to placebo treatment. If the universal influenza vaccine field incorporates this antigen into next generation vaccines, M2e could prove its merit when the next influenza pandemic strikes.

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.

Chimeric protein consisting of 3M2e and HSP as a universal influenza vaccine candidate: from in silico analysis to preliminary evaluation.

The 23-amino acid ectodomain of influenza virus M2 protein (M2e) is highly conserved among human influenza virus variants and represents an attractive target for developing a universal vaccine. Although this peptide has limited potency and low immunogenicity, the degree of M2e density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. The aim of this study was to design a chimer protein consisting of three tandem repeats of M2e peptide sequence fused to the Leishmania major HSP70 gene and evaluate its characteristics and immunogenicity. The structure of the deduced protein and its stability, aliphatic index, biocomputed half-life and the anticipated immunogenicity were analyzed by bioinformatics software. The oligonucleotides encoding 3M2e and chimer 3M2e-HSP70 were expressed in Escherichia coli and affinity purified. The immunogenicity of the purified recombinant proteins was preliminary examined in mouse model. It was predicted that fusion of HSP70 to the C-terminal of 3M2e peptide led to increased stability, hydropathicity, continuous B cell epitopes and antigenic propensity score of chimer protein. Also, the predominant 3M2e epitopes were not hidden in the chimer protein. The initial in vivo experiment showed that 3M2e-HSP chimer protein stimulates specific immune responses. In conclusion, the results of the current study suggest that 3M2e-HSP chimer protein would be an effective universal subunit vaccine candidate against influenza infection.

AuNP-M2e + sCpG vaccination of juvenile mice generates lifelong protective immunity to influenza A virus infection.

BACKGROUND: Influenza virus infection causes significant morbidity and mortality worldwide. Humans fail to make a universally protective memory response to influenza A because of high mutation rates in the immune-dominant influenza epitopes. We seek the development of a universal influenza A vaccine. The extracellular domain of the M2-ion channel (M2e) is an ideal antigenic target, as it is highly conserved, has a low mutation rate, and is essential for viral entry and replication. Considering the potential of a universal influenza vaccine for lifelong protection, we aimed to examine this potential using a recently published gold nanoparticle M2e vaccine with CpG as an adjuvant (AuNP-M2e + sCpG). Intranasal vaccination induces an M2e-specific memory response, which is protective against lethal infection with H1N1, H3N2, and H5N1 serotypes, in young BALB/c mice. Protection with AuNP-M2e + sCpG has been published up to 8 months after vaccination. However, the highest risk population during most influenza seasons is adults over 65 years old. Additionally, the efficacy of many vaccines decrease after aging and requiring booster vaccinations to remain effective. RESULTS: To determine if the AuNP-M2e + sCpG vaccine is a viable option as a universal vaccination capable of protection through geriatric age, we tested if the AuNP-M2e + sCpG vaccination loses efficacy after aging mice to geriatric age (over 18 months). Our data shows that mice aged 15 months after vaccination (~ 18-21 months old) retain significant M2e-specific antibody titers in total IgG, IgG1, IgG2a, and IgG2b. These mice are significantly protected from lethal influenza challenge (H1N1, 8.3 PFU). Further, these antibody titers increase upon infection with influenza A and remain elevated for 3 months, suggesting the elderly mice retain effective M2e-specific memory B cells. CONCLUSIONS: Our results demonstrate that protective M2e-specific memory in mice developed at a young age can persist until geriatric age. Additionally, this memory is protective and M2e-specific B cells produced by vaccination with AuNP-M2e + sCpG are maintained and functional. If the results of this study persist in humans, they suggest that a universal influenza A vaccine could be administered early in life and maintain lifelong protection into geriatric age.

Intranasal Nanovaccine Confers Homo- and Hetero-Subtypic Influenza Protection.

Cross-protective and non-invasively administered vaccines are attractive and highly desired for the control of influenza. Self-assembling nanotechnology provides an opportunity for the development of vaccines with superior performance. In this study, an intranasal nanovaccine is developed targeting the conserved ectodomain of influenza matrix protein 2(M2e). 3-sequential repeats of M2e (3M2e) is presented on the self-assembling recombinant human heavy chain ferritin (rHF) cage to form the 3M2e-rHF nanoparticle. Intranasal vaccination with 3M2e-rHF nanoparticles in the absence of an adjuvant induces robust immune responses, including high titers of sera M2e-specific IgG antibodies, T-cell immune responses, and mucosal secretory-IgA antibodies in mice. The 3M2e-rHF nanoparticles also confer complete protection against a lethal infection of homo-subtypic H1N1 and hetero-subtypic H9N2 virus. An analysis of the mechanism of protection underlying the intranasal immunization with the 3M2e-rHF nanoparticle indicates that M2e-specific mucosal secretory-IgA and T-cell immune responses may play critical roles in the prevention of infection. The results suggest that the 3M2e-rHF nanoparticle is a promising, needle-free, intranasally administered, cross-protective influenza vaccine. The use of self-assembling nanovaccines could be an ideal strategy for developing vaccines with characteristics such as high immunogenicity, cross-protection, and convenient administration, as well as being economical and suitable for large-scale production.

Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines.

Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Reemerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform using extracellular domain IV of the matrix 2 protein (M2e), AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous influenza virus (IV) challenges. We found that the use of AdV with tandem M2e epitopes in fiber is a potential strategy for influenza prevention.IMPORTANCE Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly and demonstrated that this 225-bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza virus infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

Flagellin-fused protein targeting M2e and HA2 induces potent humoral and T-cell responses and protects mice against various influenza viruses a subtypes.

BACKGROUND: Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new, potentially pandemic, influenza strains. Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based on combinations of various conserved epitopes of viral proteins (M1, M2, HA2, NP). In this study, we compared the immunogenicity and protective action of two recombinant proteins which feature different designs and which target different antigens. RESULTS: Balb/c mice were immunized subcutaneously with Flg-HA2-2-4M2ehs or FlgSh-HA2-2-4M2ehs; these constructs differ in the location of hemagglutinin's HA2-2(76-130) insertion into flagellin (FliC). The humoral and T-cell immune responses to these constructs were evaluated. The simultaneous expression of different M2e and HA2-2(76-130) in recombinant protein form induces a strong M2e-specific IgG response and CD4+/ CD8+ T-cell response. The insertion of HA2-2(76-130) into the hypervariable domain of flagellin greatly increases antigen-specific T-cell response, as evidenced by the formation of multi-cytokine-secreting CD4+, CD8+ T-cells, Tem, and Tcm. Both proteins provide full protection from lethal challenge with A/H3N2 and A/H7N9. CONCLUSION: Our results show that highly conserved M2e and HA2-2(76-130) can be used as important targets for the development of universal flu vaccines. The location of the HA2-2(76-130) peptide's insertion into the hypervariable domain of flagellin had a significant effect on the T-cell response to influenza antigens, as seen by forming of multi-cytokine-secreting CD4+ and CD8+ T-cells.

Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection.

Recombinant, Escherichia coli-derived outer membrane vesicles (rOMVs), which display heterologous protein subunits, have potential as a vaccine adjuvant platform. One drawback to rOMVs is their lipopolysaccharide (LPS) content, limiting their translatability to the clinic due to potential adverse effects. Here, we explore a unique rOMV construct with structurally remodeled lipids containing only the lipid IVa portion of LPS, which does not stimulate human TLR4. The rOMVs are derived from a genetically engineered B strain of E. coli, ClearColi, which produces lipid IVa, and which was further engineered in our laboratory to hypervesiculate and make rOMVs. We report that rOMVs derived from this lipid IVa strain have substantially attenuated pyrogenicity yet retain high levels of immunogenicity, promote dendritic cell maturation, and generate a balanced Th1/Th2 humoral response. Additionally, an influenza A virus matrix 2 protein-based antigen displayed on these rOMVs resulted in 100% survival against a lethal challenge with two influenza A virus strains (H1N1 and H3N2) in mice with different genetic backgrounds (BALB/c, C57BL/6, and DBA/2J). Additionally, a two-log reduction of lung viral titer was achieved in a ferret model of influenza infection with human pandemic H1N1. The rOMVs reported herein represent a potentially safe and simple subunit vaccine delivery platform.