Domain Of Influenza M2 Protein Research Articles

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.

Self-assembly polymerization enhances the immunogenicity of influenza M2e peptide

The extracellular domain of Influenza M2 protein (M2e) was considered as a promising target for universal influenza vaccine development. Several M2e-based influenza vaccines have been developed and many of them used a mutant M2e peptide, in which the two conserved cysteine residues were substituted by serine residues. In this paper, we compared the antigenicity and immunogenicity of wild type and cysteine-mutant M2e peptides. We found that the cysteine substitution slightly affected the antigenicity of M2e epitope, but greatly reduced the immunogenicity of M2e peptide. The cysteine substitution also disabled the M2e peptide from inducing protection against influenza virus challenge in mice. Further analysis revealed that the immunogenicity of M2e peptide was enhanced by the self-assembly of the peptide through inter-peptide disulfide bonds. These results provide new information to improve the design of M2e-based vaccines against potential influenza pandemics.

Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent, cross-clade protection against H5N1 influenza virus in mice

H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8+ T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity.

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.

High copy numbers and N terminal insertion position of influenza A M2E fused with hepatitis B core antigen enhanced immunogenicity.

The extra domain of influenza M2 protein (M2e) is almost completely conserved among all influenza A virus subtypes. M2e is a promising candidate target for the development of a broad-spectrum recombinant influenza A vaccine. However, the immunogenicity of M2e needs to be improved. Copy numbers of M2e and its fusion expression with different carrier proteins may affect its immunopotency. In this study, we designed and created different constructs through genetic fusion of M2e (MSLLTEVETPTRSEWECRCSDSSD) (A/California/05/2009 (H1N1)) with the N-terminus (HBcAg1-149aa + Cys) by insertion in the N-terminus Hepatitis B Core (HBc) antigen 1-149aa and Middle 78-81aa of HBcAg1-149aa to construct a recombinant M2e-based vaccine candidate. These chimeric sequences were expressed in Escherichia coli. We constructed fusion proteins containing influenza A H1N1 influenza virus (2009), as well as one, two, and three copies of M2e and hepatitis B core antigen1-149aa amino acid-optimized codon inserted N and its intermediate. The recombinant protein was expressed and purified. Western blot analysis was employed to evaluate the expression of the M2e recombinant protein containing different copy numbers of M2e. Mice were immunized for two times with the purified fusion protein HBc/M2e BALB/c. Serum levels of M2e antibody gradually increased along with increase in immunity. The levels of different fusion protein M2e antibodies increase with increasing M2e copy number. In addition, the protein antibody level in the N terminal fusion protein is higher than that in intermediate fusion.

Immunization with high epitope density of M2e derived from 2009 pandemic H1N1 elicits protective immunity in mice

As highly conserved amongst human influenza A strains, the extracellular domain of influenza M2 protein (M2e) is considered and proved as a promising candidate for a universal influenza vaccine. However, there are four amino acid variations in the M2e sequence of the 2009 pandemic H1N1 (referred to as “swine flu M2e, SFM2e”) compared with the conventional M2e consensus sequence. Whether the sequence variation alters the immunogenicity and protection of SFM2e epitope remains unclear. In our present study, we synthesized SFM2e peptide and constructed a series of GST fusion proteins containing various copies of the SFM2e epitope and immunized mice to evaluate their immunogenicity and protective activity. We found that although the amino acid variations have weakened the immunogenicity of the SFM2e peptide, the SFM2e fusion proteins with high epitope densities induced intense and diverse antibody response as well as T cell response. Moreover, mice immunized with high epitope density of SFM2e were nearly fully protected against a lethal challenge by the mouse-adapted influenza virus A/Porto Rico/8/34. Our study could provide new available data to improve the epitope vaccine strategy against influenza pandemic.

The cytoplasmic domain of influenza M2 protein interacts with caveolin-1

The cytoplasmic domain of influenza M2 protein (M2c) consists of 54 amino acid (aa) residues from aa44 to aa97. In this paper, M2c and its deletion mutant M2c Δ47–55 were expressed using prokaryotic expression system. First, glutaraldehyde crosslinking assay showed that M2c had multimerization potential mediated by aa47–55. Then, M2c, instead of M2c Δ47–55, directed eGFP from the whole cell localization to a predominately perinuclear region in CHO cells, which indicated that aa47–55 of M2c mediated the localization. Moreover, M2c colocalized with caveolin-1 (Cav) when CHO cells were cotransfected with Cav. A caveolin-1 binding motif ΦxxxxΦxxΦ (Φ represents aromatic amino acid residues) in aa47–55 of M2c was found by sequence alignment and analysis. Further overlay ELISA result showed that M2c, but not M2c Δ47–55, bound to prokaryotically expressed cholesterol-free Cav 2–101, which illustrated the interaction could be cholesterol-independent. That was the first report of cellular protein bound to M2c.

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.

Sequence comparison between the extracellular domain of M2 protein human and avian influenza A virus provides new information for bivalent influenza vaccine design

To prevent the human and economic losses caused by human and avian influenza viruses, it is necessary to prepare safe bivalent influenza vaccines. Recent studies found that human influenza vaccines based on the extracellular domain of influenza M2 protein (M2e) induced broad-spectrum protective immunity in various antigen constructs. A prerequisite for using the M2e protein as a bivalent influenza vaccine component was to find out the sequence differences between human and non-human (avian or swine) influenza M2e proteins. Here, we completed such a comparison using 716 influenza M2e sequences available in Genbank. The results found one region on M2e protein consistent with host restriction specificities: P IRN EW GC RC N, P TRN GW EC KC S and P IRN GW EC RC N (aa10–20; the human, avian and swine specific M2e sequence, respectively). Interestingly, the comparison result was then validated by immunoblotting and enzyme-linked immunosorbent assay. The monoclonal antibody against the EVET PI RN sequence (aa6–13) of human M2e protein could weakly recognize avian M2e proteins bearing the EVET PT RN sequence (aa6–13) but failed to recognize avian M2e proteins bearing the EVET LT RN sequence (aa6–13). The data in this study provided useful information in the race to develop bivalent influenza vaccines against avian and human influenza A virus infection in human beings.