anti-M2e Antibody Research Articles

Engineering of the PapMV vaccine platform with a shortened M2e peptide leads to an effective one dose influenza vaccine

The emergence of highly virulent influenza strains and the risks of pandemics as well as the limited efficiency of the current seasonal vaccines are important public health concerns. There is a major need for new influenza vaccines that would be broadly cross-protective. The ectodomain of matrix protein 2 (M2e) is highly conserved amongst different influenza strains and could be used as a broad spectrum antigen. To overcome its low immunogenicity we have fused a short peptide epitope derived from the human consensus sequence of M2e (amino acids 6–14, EVETPIRNE) to the N-terminus of papaya mosaic virus coat protein. The fusion harboring coat proteins were assembled around a single stranded RNA into virus-like particles (PapMV-sM2e). The resulting PapMV-sM2e rod-shaped particle was stable and indistinguishable from regular PapMV particles. A single intramuscular immunization with PapMV-sM2e was sufficient to mount appreciable levels of CD4 dependent M2e specific total IgG and IgG2a antibody in mice sera. PapMV-sM2e proved to be self-adjuvanting since the addition of PapMV as an exogenous adjuvant did not result in significantly improved antibody titers. In addition, we confirmed the adjuvant property of PapMV-sM2e using the trivalent inactivated flu vaccine as antigen and demonstrated that the newly engineered nanoparticles areas efficacious as an adjuvant than the original PapMV nanoparticles. Upon infection with a sub-lethal dose of influenza, PapMV-sM2e vaccinated animals were completely protected from virus induced morbidity and mortality. Mice immunized with decreasing amounts of PapMV-sM2e and challenged with a more stringent dose of influenza virus displayed dose-dependent levels of protection. Seventy percent of the mice immunized once with the highest dose of PapMV-sM2e survived the challenged. The survival of the mice correlated mainly with the levels of anti-M2e IgG2a antibodies obtained before the infection. These results demonstrate that PapMV-sM2e can be an important component of a broadly cross-reactive influenza vaccine.

Characterization of the M2e antibody response following highly pathogenic H5N1 avian influenza virus infection and reliability of M2e ELISA for identifying infected among vaccinated chickens

A surveillance method able to differentiate between vaccinated and infected poultry is required for those countries that practice vaccination against highly pathogenic avian influenza H5N1. The external domain of the M2 protein (M2e) of influenza virus is a potentially useful differentiating-infected from vaccinated animals (DIVA) antigen but little is known about the M2e antibody response and factors influencing its detection. In this study, the M2e antibody response was characterized in layer birds vaccinated and challenged with an Indonesian H5N1 virus isolate, using a single M2e peptide or four-branched multiple antigenic peptide form of M2e (MAP-M2e) as antigens in two separate ELISAs. Anti-M2e antibodies were absent in chicks with high level of maternal haemagglutination inhibition antibodies and also in all layers vaccinated once, twice or three times with an inactivated commercial H5N1 vaccine. In contrast, anti-M2e antibodies were detected in vaccinated layers challenged with H5N1 virus and their presence was associated with virus isolation and an increase in haemagglutination inhibition titres. The number of birds that developed M2e antibodies, as well as the strength and duration of the M2e antibody response were strongly influenced by the length of the interval between vaccination and challenge. Birds challenged at six weeks after vaccination all developed M2e antibodies by 14 days that lasted until at least 56 days after infection. In birds challenged at two weeks after vaccination, only a proportion of birds developed M2e antibodies by 14 days that lasted only until 28 days post-infection. Both single M2e peptide and MAP-M2e ELISAs had high diagnostic specificity but the diagnostic sensitivity of MAP-M2e ELISA was significantly higher and more effective in detecting M2e antibody in immune and infected birds. The results show that MAP-M2e ELISA would be useful for surveillance in countries using vaccination to control highly pathogenic avian influenza H5N1.

A lipidated form of the extracellular domain of influenza M2 protein as a self-adjuvanting vaccine candidate

The highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza A virus has been previously investigated as a potential target for an universal influenza vaccine. In this study we prepared four lipopeptide influenza vaccine candidates in which the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine, (Pam2Cys) was attached to either the N- or C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP and its analogue sequence with the two cysteine residues replaced with serine residues. The results of animal study show that each of these lipopeptides induced strong M2e-specific antibody responses in the absence of extraneous T helper cell epitope(s) which are normally incorporated in the previous studies or addition of extraneous adjuvant and that these antibodies are protective against lethal challenge with influenza virus. Comparison of different routes of inoculation demonstrated that intranasal administration of M2e lipopeptide induced higher titers of IgA and IgG2b antibodies in the bronchoalveolar lavage than did subcutaneous vaccination and was better at mitigating the severity of viral challenge. Finally, we show that anti-M2e antibody specificities absent from the antibody repertoire elicited by a commercially available influenza vaccine and by virus infection can be introduced by immunization with M2e-lipopeptide and boosted by viral challenge. Immunization with this lipidated form of the M2e epitope therefore offers a means of using a widely conserved epitope to generate protective antibodies which are not otherwise induced.

A truncated C-terminal fragment of Mycobacterium tuberculosis HSP70 enhances cell-mediated immune response and longevity of the total IgG to influenza A virus M2e protein in mice

As the importance of virus-specific IgG2a and strong induction of Th1 type immune response for virus clearance was reported, conventional influenza vaccines induce a highly humoral immune response and fail to induce cytotoxic T-lymphocyte (CTL) immunity. Hence, in agreement with heat shock protein 70 (HSP70) acting as Th1 cytokine-like adjuvant, an Escherichia coli-expressed r4M2e.HSP70c fusion protein comprising C-terminus of Mycobacterium tuberculosis HSP70 genetically fused to four tandem repeats of influenza A virus M2e was constructed. Then, the case-control study was carried out to evaluate the humoral and cellular responses elicited against M2e in Balb/C mice by intramuscular immunization with r4M2e.HSP70c alone.Our results showed that r4M2e.HSP70c rather than control groups, r4M2e, r4M2e+Alum, or rHSP70c, significantly elevated both longevity and serum level of the total M2e-specific IgG antibody, induced a Th1 skewed humoral and cellular immune responses, increased the level of IFN-γ in BALF, and promoted the proliferation of peripheral blood lymphocytes. Furthermore, a virus challenge experiment revealed that mice vaccinated with r4M2e.HSP70c limited the severity of influenza A disease by 100% survival rate, less sever body weight loss and delaying the onset of morbidity in mice for 2days rather than other control groups. Here, we used r4M2e.HSP70c to stimulate M2e-specific antibody and cellular immune responses in Balb/C mice. The mHSP70c in the fusion form induced a long lasting Th1 skewed humoral and cellular immune responses against its associated protein. It seems anti-M2e antibodies limit viral replication and ameliorate influenza infection that allows the immune system to induce sterilizing HA-antibody against whole virion that leads to full protection against virulent influenza infection.

A GFP expressing influenza A virus to report in vivo tropism and protection by a matrix protein 2 ectodomain-specific monoclonal antibody.

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1–73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1–73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1–73)GFP virus, indicate that this virus is genetically and phenotypically stable.

High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

An H5N1-based matrix protein 2 ectodomain tetrameric peptide vaccine provides cross-protection against lethal infection with H7N9 influenza virus

In March 2013, a patient infected with a novel avian influenza A H7N9 virus was reported in China. Since then, there have been 458 confirmed infection cases and 177 deaths. The virus contains several human-adapted markers, indicating that H7N9 has pandemic potential. The outbreak of this new influenza virus highlighted the need for the development of universal influenza vaccines. Previously, we demonstrated that a tetrameric peptide vaccine based on the matrix protein 2 ectodomain (M2e) of the H5N1 virus (H5N1-M2e) could protect mice from lethal infection with different clades of H5N1 and 2009 pandemic H1N1 influenza viruses. In this study, we investigated the cross-protection of H5N1-M2e against lethal infection with the new H7N9 virus. Although five amino acid differences existed at positions 13, 14, 18, 20, and 21 between M2e of H5N1 and H7N9, H5N1-M2e vaccination with either Freund's adjuvant or the Sigma adjuvant system (SAS) induced a high level of anti-M2e antibody, which cross-reacted with H7N9-M2e peptide. A mouse-adapted H7N9 strain, A/Anhui/01/2013m, was used for lethal challenge in animal experiments. H5N1-M2e vaccination provided potent cross-protection against lethal challenge of the H7N9 virus. Reduced viral replication and histopathological damage of mouse lungs were also observed in the vaccinated mice. Our results suggest that the tetrameric H5N1-M2e peptide vaccine could protect against different subtypes of influenza virus infections. Therefore, this vaccine may be an ideal candidate for developing a universal vaccine to prevent the reemergence of avian influenza A H7N9 virus and the emergence of potential novel reassortants of influenza virus.Emerging Microbes & Infections (2015) 4, e22; doi:10.1038/emi.2015.22

A Novel Subnucleocapsid Nanoplatform for Mucosal Vaccination against Influenza Virus That Targets the Ectodomain of Matrix Protein 2

In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.

Multiple heterologous M2 extracellular domains presented on virus-like particles confer broader and stronger M2 immunity than live influenza A virus infection

The influenza M2 ectodomain (M2e) is poorly immunogenic and has some amino acid changes among isolates from different host species. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced protective antibodies cross-reactive to antigenically different influenza A viruses and conferred cross protection. Anti-M2e antibodies induced by heterologous M2e5x VLPs showed a wider range of cross reactivity to influenza A viruses at higher levels than those by live virus infection, homologous M2e VLPs, or M2e monoclonal antibody 14C2. Fc receptors were found to be important for mediating protection by immune sera from M2e5x VLP vaccination. The present study provides evidence that heterologous recombinant M2e5x VLPs can be more effective in inducing protective M2e immunity than natural virus infection and further supports an approach for developing an effective universal influenza vaccine.

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F 85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F(85-93), that carries the RSV CD8(+) T cell epitope F(85-93) in its neuraminidase stalk. F(85-93)-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F(85-93) virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F(85-93) virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F(85-93) infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8(+) T cell-mediated immunity by an influenza A virus vector expressing the RSV F(85-93) epitope.

Development and ELISA-based detection of anti-M2e IgY antibodies using an encoding plasmid for M2e-Hsp70 C-terminal gene

Background: The use of IgYs in a variety of methods in different areas of research, diagnostics, medical application and biotechnology should be considered widely. Objectives: Development of antibodies against extra cellular domain of influenza M2 (M2e) protein in egg yolk of laying hens. Methods: A Fusion construct harboring C-terminal of bovine heat shock protein 70 (Hsp70) and influenza M2e coding genes was injected to laying hens. Serum and egg yolk antibodies were screened for the presence of anti-M2e antibodies by indirect enzyme-linked immunosorbent assay (ELISA). Results: Anti-M2e antibodies were detected in egg yolks and sera of injected hens from 13 and 7 days post injection (PI), with the peak titer detected on 41 and 35 days PI, respectively. Conclusions: Anti-M2e IgY titers could be an index for expression potential of pcDNA3.1-M2e-HspC-terminal construct in laying hens. This construct could be considered as a promising tool in production of anti-M2e polyclonal, monospecific IgY antibodies. Such anti-M2e antibodies could be exploited for influenza diagnostic and therapeutic measures.

Development of live attenuated Bordetella pertussis strains expressing the universal influenza vaccine candidate M2e

The attenuated Bordetella pertussis BPZE1 vaccine strain represents an attractive platform for the delivery of heterologous vaccine candidates via the nasal route. The filamentous hemagglutinin (FHA) has been used to secrete or expose the foreign antigens at the bacterial surface. In this study, one, two and three copies of the Cys-containing ectodomain of matrix protein 2 (M2e) from influenza A virus were genetically fused to full length FHA and expressed in BPZE1. The secretion efficacy of the FHA-(M2e)1,2,3 chimera in the extracellular milieu and the ability of the recombinant bacteria to colonize the mouse lungs inversely correlated with the number of M2e copies fused to FHA. Nevertheless FHA-(M2e)3-producing bacteria (BPLR3) triggered the highest systemic anti-M2e antibody response upon nasal administration to BALB/c mice. Nasal immunization with BPLR3 bacteria resulted in a significant reduction in the viral loads upon challenge with H1N1/PR8 influenza A virus, but did not improve the survival rate compared to BPZE1-immunized mice. Furthermore, since previous work reported that disulfide bond formation in Cys-containing passenger antigens affects the secretion efficacy of the FHA chimera, the dsbA gene encoding a periplasmic disulfide isomerase was deleted in the FHA-(M2e)3-producing strain. Despite improving significantly the secretion efficacy of the FHA-(M2e)3 chimera, the dsbA deletion did not result in higher anti-M2e antibody titers in mice, due to impaired bacterial fitness and colonization ability.

Safety and immunogenicity of a recombinant M2e–flagellin influenza vaccine (STF2.4xM2e) in healthy adults

BackgroundThe ectodomain of matrix protein 2 (M2e) is a promising candidate for a broadly protective influenza A vaccine because it is highly conserved and antibodies to M2e are protective in animal models. STF2.4xM2e (VAX102) is a recombinant fusion protein that links four tandem copies of the M2e antigen to Salmonella typhimurium flagellin, a TLR5 ligand used as an adjuvant. The objectives of this first-in-human study were to assess the safety and immunogenicity of VAX102 given as a prime-boost regimen to healthy adults. MethodsSixty subjects 18–49 years old were enrolled in a multicenter, double-blind, randomized, placebo-controlled trial (Study 1). Based on pre-clincial data, initial design included doses starting at 10μg, with an escalation plan. After reactogenicity was noted at the 10μg dose, the trial was redesigned to evaluate 0.3, 1.0, and 3μg doses. Following this study, 16 subjects were enrolled in Study 2, an open label, low dose study, to evaluate doses of 0.03 and 0.1μg. In both trials, vaccine or placebo was given intramuscularly (i.m.) at 0 and 28 days. Clinical and laboratory safety assessments took place 1 and 7 days after immunization. Immune responses to M2e and flagellin were assessed by ELISA at 7, 14 and 28 days after each dose. Seroconversion was defined as a serum IgG anti-M2e antibody value ≥0.174μg/ml and a fourfold rise in concentration. ResultsDoses of 0.03–1μg were safe and well tolerated in all subjects. Doses of 0.03 and 0.1μg produced limited immunogenicity (38% and 75% respectively), after the second dose of vaccine. Doses of 0.3 and 1.0μg were immunogenic in 18 (75%) of 24 vaccinees after the first dose and 23 (96%) after the second dose. In the 1.0μg group, the geometric mean M2e antibody concentration was 0.4μg/ml after the first dose and 1.7μg/ml after the booster dose. M2e antibody concentrations and seroconversion rates were not significantly different at higher doses (p>0.05). Immune response to flagellin was robust but did not appear to interfere with M2e antibody responses after the booster dose. Following the first injection of VAX102 at higher doses (3 and 10μg), self-limited but severe symptoms were noted in some subjects and were associated with elevated levels of C-reactive protein. Although not directly measured, this reaction was believed to be mediated by cytokine release. ConclusionsVAX102 was safe and induced high antibody levels to M2e at 0.3 and 1.0μg doses. The TLR5 ligand, S. typhimurium flagellin, is a novel approach to adjuvant-like activity through activation of innate immunity, and when fused to multiple copies of the M2e protein, the vaccine was able to induce a fourfold rise in antibody in humans, to a previously non-immunogenic, highly-conserved portion of the influenza virus. Clinical correlates of protection that may be afforded by M2e antibody in humans are a future focus of investigation.

Characterization of the murine Th2 response to immunization with liposomal M2e influenza vaccine

While the current influenza vaccine strategy is dependent on eliciting neutralizing antibodies to the hemagglutinin (H or HA) surface glycoprotein, antigenic drifts and occasional antigenic shifts necessitate constant surveillance and annual updates to the vaccine components. The ectodomain of the matrix 2 (M2e) channel protein has been proposed as a universal vaccine candidate, although it has not yet been shown to elicit neutralizing antibodies. Utilizing a liposome-based vaccine technology, an M2e vaccine (L-M2e-HD/MPL) was tested and shown to stimulate the production of anti-M2e antibodies which precipitated with whole virus and inhibited viral cell lysis by multiple type A strains of influenza virus using a novel in vitro assay. The anti-M2e antibodies also conferred complete protection following passive transfer from L-M2e-HD/MPL vaccinated mice to naïve mice challenged with H1N1 virus. Significantly higher levels of IL-4 compared to IFN-γ were secreted by the splenocytes of L-M2e-HD/MPL vaccinated mice incubated with M2e. In addition, depletion of CD4 cells or CD4 cells plus CD8 cells from L-M2e-HD/MPL vaccinated mice using monoclonal antibodies markedly decreased the level of protection of the vaccine when compared to just CD8 depletion of L-M2e-HD/MPL vaccinated mice. These results suggest that the protective immune response elicited by this vaccine is mediated primarily by a Th2 mechanism.

Field Application of the H9M2e Enzyme-Linked Immunosorbent Assay for Differentiation of H9N2 Avian Influenza Virus-Infected Chickens from Vaccinated Chickens

Vaccination for control of H9N2 low-pathogenicity avian influenza (LPAI) in chickens began in 2007 in South Korea where the H9N2 virus is prevalent. Recently, an enzyme-linked immunosorbent assay (ELISA) using the extracellular domain of the M2 protein (M2e ELISA) was developed as another strategy to differentiate between vaccinated and infected chickens. Here, an ELISA using the extracellular domain of the M2 protein of H9N2 LPAI virus (H9M2e ELISA) was applied to differentiate infected from vaccinated chickens using the H9N2 LPAI virus M2 peptide. The specificity and sensitivity of the optimized H9M2e ELISA were 96.1% and 83.8% (the absorbance of the sample to the absorbance for the positive control [S/P ratio] ≥ 0.6), respectively, with the cutoff value (S/P ratio = 0.6), and the criterion of avian influenza (AI) infection in a chicken house was established as >20% reactivity of anti-M2e antibody per house with this cutoff value. After infection in naïve chickens and once-vaccinated chickens with a hemagglutination inhibition (HI) assay titer of 9.25 ± 0.75 log(2) units, the sera from infected chickens were confirmed as AI infected when the chickens were 1 week old in both groups, and AI infection lasted for 24 weeks and 9 weeks in naïve and once-vaccinated chickens, respectively, although in twice-vaccinated chickens with a higher HI titer of 11.17 ± 0.37 log(2) units, anti-M2e antibody in infected sera did not reach a level indicating AI infection. In field application, anti-M2e antibody produced in infected chickens after vaccination or in reinfected chickens could be identified as AI infection, although HI test could not distinguish infected from vaccinated sera. These results indicate the utility of H9M2e ELISA as a surveillance tool in control of H9N2 LPAI infections.

Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

M2e-based universal influenza A vaccine

Human influenza causes substantial morbidity and mortality. Currently, licensed influenza vaccines offer satisfactory protection if they match the infecting strain, but they come with significant drawbacks. These vaccines are derived from prototype viruses, containing the hemagglutinin of influenza viruses that are likely to cause the next epidemic. Their usefulness against a future pandemic, however, remains problematic. A vaccine based on the ectodomain of influenza matrix protein 2 (M2e) could overcome these drawbacks. M2e is highly conserved in both human and avian influenza A viruses. The low immunogenicity against natural M2e can be overcome by fusing M2e to an appropriate carrier such as Hepatitis B virus-derived virus-like particles. Such chimeric particles can be produced in a simple and safe bacterial expression system, requiring minimal biocontainment, and can be obtained in a pure form. Experiments in animal models have demonstrated that M2e-based vaccines induce protection against a lethal challenge with various influenza A virus subtypes. Furthermore, the production and use of an effective M2e-vaccine could be implemented at any time regardless of seasonality, both in an epidemic as well as in a pandemic preparedness program. In animal models, M2e-vaccines administered parenterally or intranasally protect against disease and mortality following challenge with various influenza A strains. Adjuvants suitable for human use improve protection, which correlates with higher anti-M2e antibody responses of defined subtypes. Recently, Phase I clinical studies with M2e-vaccines have been completed, indicating their safety and immunogenicity. Further clinical development of this universal influenza A vaccine candidate is being pursued in order to validate its protective efficacy in humans.

Heterosubtypic protection conferred by combined vaccination with M2e peptide and split influenza vaccine

There is urgent need to develop influenza vaccines with broad-spectrum protection against the potential influenza pandemic. The extracellular domain of influenza M2 protein (M2e) is considered as an appropriate target to induce heterosubtypic protection. We investigated the immunity and protection induced by combined vaccination with synthetic M2e peptide and traditional split influenza vaccine. The combined vaccination was able to induce similar strain-specific hemagglutinin inhibition (HI) antibodies as vaccination of split virus alone. However, aluminum-adjuvant but not oil-in-water-emulsion adjuvant combined vaccination was able to induce high titers of anti-M2e antibodies and provoke M2e-specific T lymphocyte response. Furthermore, we found that the addition of M2e peptide greatly enhanced the cross-protective efficacy of split virus in aluminum adjuvant but slightly weakened the efficacy of vaccination in oil-in-water-emulsion adjuvant. Moreover, aluminum-adjuvant combined vaccination conferred complete cross-protection against heterosubtypic influenza virus. According to the results, we suggest that the M2e peptide should be added into split influenza vaccine in the preparation for the potential influenza pandemic.

Universal influenza A M2e-HBc vaccine protects against disease even in the presence of pre-existing anti-HBc antibodies

The extracellular domain of influenza A virus matrix protein 2 (M2e) is strongly conserved. Therefore, vaccines based on M2e can induce broad-spectrum immunity against influenza. We have mainly used recombinant virus-like particles derived from Hepatitis B virus core (HBc) as carrier for efficacious presentation of the M2e antigen. Here, we address whether pre-existing HBc-specific immunity interferes with the protective immune response obtained by M2e-HBc vaccination. Anti-HBc antibodies were induced by immunizing mice with unsubstituted HBc virus-like particles in the presence of two different adjuvants. We demonstrate that pre-existing HBc-specific antibodies affect neither the induction of M2e-specific antibody responses to vaccination with M2e-HBc particles, nor the protective efficacy of the resulting response. These results suggest that vaccination with M2e-HBc can induce protective anti-M2e antibodies even in anti-HBc positive individuals. The implications of these findings are discussed in the context of the clinical development of an M2e-based universal influenza vaccine, which recently successfully completed a Phase I trial.

Development of a universal influenza A vaccine based on the M2e peptide fused to the papaya mosaic virus (PapMV) vaccine platform

With the emergence of highly virulent influenza viruses and the consequent risk of pandemics, new approaches to designing universal influenza vaccines are urgently needed. In this report, we demonstrate the potential of using a papaya mosaic virus (PapMV) platform carrying the universal M2e influenza epitope (PapMV-CP-M2e) as a candidate flu vaccine. We show that PapMV-CP-M2e virus-like particles (VLPs) can induce production in mice of anti-M2e antibodies that can recognize influenza-infected cells. PapMV-CP-M2e discs made of 20 coat protein (CP) subunits were shown to be poorly immunogenic compared to PapMV-CP-M2e VLPs composed of several hundred CP subunits. We also show that addition of either alum or PapMV-CP VLPs as adjuvant dramatically increased the immunogenicity of PapMV-CP-M2e-containing vaccine, and led to 100% protection against a challenge of 4LD 50 with the WSN/33 strain. These results show, for the first time, the potential of a recombinant plant virus protein to serve as both peptide delivery system and adjuvant in the crucial field of influenza vaccine development.