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1
Tan, Mei Peng, Noorjahan Banu Mohamed Alitheen, Wen Siang Tan, and Wei Boon Yap. "Expression of Influenza M2e-NP Recombinant Fusion Protein in Escherichia coli BL21 (DE3) and Its Binding to Antibodies." Vaccines 10, no. 12 (2022): 2066. http://dx.doi.org/10.3390/vaccines10122066.
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The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The M2e gene was fused to the 5′-end of the NP gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the M2e-NP fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into Escherichia coli (E. coli) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate.
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Cho, Ki Joon, Bert Schepens, Jong Hyeon Seok, et al. "Structure of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus in Complex with a Protective Monoclonal Antibody." Journal of Virology 89, no. 7 (2015): 3700–3711. http://dx.doi.org/10.1128/jvi.02576-14.
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ABSTRACTThe extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e.IMPORTANCEM2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reportedin vivoescape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2.
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Reese, Kaleb A., Christopher Lupfer, Rudd C. Johnson, et al. "A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens." Veterinary Medicine International 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/316926.
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A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface ofLactococcus lactis(LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLH-M2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly (P<0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG.
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Ingrole, Rohan S., Wenqian Tao, Jatindra N. Tripathy, and Harvinder S. Gill. "Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen." Nano LIFE 04, no. 02 (2014): 1450004. http://dx.doi.org/10.1142/s1793984414500044.
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The 23 amino acid-long extracellular domain of the influenza virus transmembrane protein M2 (M2e) has remained highly conserved since the 1918 pandemic, and is thus considered a good candidate for development of a universal influenza A vaccine. However, M2e is poorly immunogenic. In this study we assessed the potential of increasing immunogenicity of M2e by constructing a nanoscale-designed protein polymer containing the M2e sequence and an elastin-like polypeptide (ELP) nanodomain consisting of alanine and tyrosine guest residues (ELP(A2YA2)24). The ELP nanodomain was included to increase antigen size, and to exploit the inherent thermal inverse phase transition behavior of ELPs to purify the protein polymer. The ELP(A2YA2)24 + M2e nanodomained molecule was recombinantly synthesized. Characterization of its inverse phase transition behavior demonstrated that attachment of M2e to ELP(A2YA2)24 increased its transition temperature compared to ELP(A2YA2)24. Using a dot blot test we determined that M2e conjugated to ELP is recognizable by M2e-specific antibodies, suggesting that the conjugation process does not adversely affect the immunogenic property of M2e. Further, upon vaccinating mice with ELP(A2YA2)24 + M2e it was found that indeed the nanodomained protein enhanced M2e-specific antibodies in mouse serum compared to free M2e peptide and ELP(A2YA2)24. The immune serum could also recognize M2 expressed on influenza virions. Overall, this data suggests the potential of using molecules containing M2e-ELP nanodomains to develop a universal influenza vaccine.
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Zhang, Yi-Nan, Keegan Braz Gomes, Yi-Zong Lee, et al. "A Single-Component Multilayered Self-Assembling Protein Nanoparticle Vaccine Based on Extracellular Domains of Matrix Protein 2 against Both Influenza A and B." Vaccines 12, no. 9 (2024): 975. http://dx.doi.org/10.3390/vaccines12090975.
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The development of an effective and broadly protective influenza vaccine against circulating and emerging strains remains elusive. In this study, we evaluated a potentially universal influenza vaccine based on single-component self-assembling protein nanoparticles (1c-SApNPs) presenting the conserved matrix protein 2 ectodomain (M2e) from influenza A and B viruses (IAV and IBV, respectively). We previously designed a tandem antigen comprising three IAV M2e domains of human, avian/swine, and human/swine origins (termed M2ex3). The M2ex3-presenting 1c-SApNPs conferred complete protection in mice against sequential lethal challenges with H1N1 and H3N2. To broaden this protection to cover IBVs, we designed a series of antigens incorporating different arrangements of three IAV M2e domains and three copies of IBV M2e. Tandem repeats of IAV and IBV (termed influenza A-B) M2e arrayed on the I3-01v9a 60-mer 1c-SApNP, when formulated with an oil-in-water emulsion adjuvant, generated greater M2e-specific immunogenicity and protective efficacy than the soluble influenza A-B M2e trimer, indicated by higher survival rates and reduced weight loss post-challenge. Importantly, one of the influenza A-B M2e SApNP constructs elicited 100% protection against a lethal influenza A/Puerto Rico/8/1934 (H1N1) challenge in mice and 70% protection against a lethal influenza B/Florida/4/2006 (Yamagata lineage) challenge, the latter of which has not been reported in the literature to date. Our study thus provides a promising M2e-based single-component universal vaccine candidate against the two major types of influenza virus circulating in humans.
6
Kim, Ki-Hye, Zhuo Li, Noopur Bhatnagar, et al. "Universal protection against influenza viruses by multi-subtype neuraminidase and M2 ectodomain virus-like particle." PLOS Pathogens 18, no. 8 (2022): e1010755. http://dx.doi.org/10.1371/journal.ppat.1010755.
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Annual influenza vaccination is recommended to update the variable hemagglutinin antigens. Here, we first designed a virus-like particle (VLP) displaying consensus multi-neuraminidase (NA) subtypes (cN1, cN2, B cNA) and M2 ectodomain (M2e) tandem repeat (m-cNA-M2e VLP). Vaccination of mice with m-cNA-M2e VLP induced broad NA inhibition (NAI), and M2e antibodies as well as interferon-gamma secreting T cell responses. Mice vaccinated with m-cNA-M2e VLP were protected against influenza A (H1N1, H5N1, H3N2, H9N2, H7N9) and influenza B (Yamagata and Victoria lineage) viruses containing substantial antigenic variations. Protective immune contributors include cellular and humoral immunity as well as antibody-dependent cellular cytotoxicity. Furthermore, comparable cross protection by m-cNA-M2e VLP vaccination was induced in aged mice. This study supports a novel strategy of developing a universal vaccine against influenza A and B viruses potentially in both young and aged populations by inducing multi-NA subtype and M2e immunity with a single VLP entity.
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Kang, Sang-Moo, Ki-Hye Kim, Jeeva Subbiah, et al. "Non-neutralizing immune-mediated cross-protection against influenza viruses by chimeric M2e-H3 stalk protein or multi-subtype neuraminidase plus M2e virus-like particle vaccine in ferrets." Journal of Immunology 212, no. 1_Supplement (2024): 0099_5226. http://dx.doi.org/10.4049/jimmunol.212.supp.0099.5226.
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Abstract Current influenza vaccine based on hemagglutinin (HA) immunity is not effective in providing cross protection against circulating variants and new pandemic viruses. In prior studies, we developed universal influenza vaccine candidates of multi-subtype neuraminidase and M2 ectodomain virus-like particles (m-cNA-M2e VLP) and chimeric M2e-H3 stalk protein vaccine (M2e-H3stalk), which were effective in inducing cross-protective immunity in mice. Here, we evaluated the immunogenicity and efficacy of these recombinant universal influenza vaccines in ferrets. Our results showed that immunization of ferrets with recombinant universal vaccines induced high levels of IgG antibody responses (M2e, H3stalk, multi-subtype NA), NA inhibition (NAI), antibody-secreting plasma cells in spleens, and IFN-γ secreting blood mononucleate cells. Ferrets with either m-cNA-M2e VLP or M2e-H3stalk vaccine were moderately protected from H1N1 and H3N2 influenza viruses as evidenced by lower viral titers in nasal washes, trachea and lung after challenge. Tests of passive immunity of vaccinated ferret antisera indicate that multi-NA plus M2e vaccine conferred more effective and broader humoral immunity in naïve mice than M2e-H3stalk vaccine. Our findings support that immunity to M2e, HA-stalk, and multi subtypes NA will induce broader cross protection in ferrets, which is likely translational to humans.
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Zharikova, Darya, Krystyna Mozdzanowska, Jingqi Feng, Manxin Zhang, and Walter Gerhard. "Influenza Type A Virus Escape Mutants Emerge In Vivo in the Presence of Antibodies to the Ectodomain of Matrix Protein 2." Journal of Virology 79, no. 11 (2005): 6644–54. http://dx.doi.org/10.1128/jvi.79.11.6644-6654.2005.
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ABSTRACT The ectodomain of matrix protein 2 (M2e) of human influenza type A virus strains has remained remarkably conserved since 1918. Because M2e-specific immunity has been shown to decrease morbidity and mortality associated with influenza virus infection in several animal models and because natural infection and current vaccines do not appear to induce a good M2e-specific antibody (Ab) response, M2e has been considered as potential vaccine for inducing cross-reactive protection against influenza type A viruses. The high degree of structural conservation of M2e could in part be the consequence of a poor M2e-specific Ab response and thus the absence of pressure for change. To assess this possibility, we studied the course of infection in SCID mice in the presence or absence of passive M2e-specific monoclonal Abs (MAbs). We found that virus mutants with antigenic changes in M2e emerged in 65% of virus-infected mice treated with M2e-specific but not control MAbs. However, the diversity of escape mutants was highly restricted since only two types were isolated from 22 mice, one with a proline-to-leucine and the other with a proline-to-histidine interchange at amino acid position 10 of M2e. The implications of these findings for the use of M2e as a broadly protective vaccine are discussed.
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Callejo, Bernadette, Tom Monath, Marla Lay, Stella Chang, and Jeff Fairman. "Highly Immunogenic JVRS-100 Adjuvanted Universal Influenza A Vaccine (52.4)." Journal of Immunology 184, no. 1_Supplement (2010): 52.4. http://dx.doi.org/10.4049/jimmunol.184.supp.52.4.
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Abstract A vaccine based on a conserved external ectodomain of M2 (M2e) offers the potential for inducing broad-immunity against divergent influenza A strains including pandemic viruses. Cohorts of mice were vaccinated 3x (IM) with M2e in the form of a multiple antigenic peptide with 4 copies of M2e (MAP4), with or without cationic lipid DNA complex adjuvant (JVRS-100). Lethal challenge using different strains of virus (H1N1, H3N2) were done to establish efficacy based on survival. The addition of JVRS-100 to M2e-MAP4 resulted in a significant decrease in morbidity and mortality following lethal challenge with H1N1 (survival 100% vs. 30% without adjuvant) or H3N2 (80% vs. 20%). Adjuvanted vaccine resulted in higher levels of IgG (p=0.0159), IgG1 (p&lt;0.02) and IgG2a (p&lt;0.005) vs. M2e-MAP4 alone. A dose titration of M2e-MAP4 inoculated with a constant amount of JVRS-100 resulted in 100% survival after challenge even at a vaccine dose of 25ng. Addition of M2e-MAP4/JVRS-100 to trivalent inactivated influenza vaccine (TIV) confers full protection with a single vaccination prior to lethal challenge. Competitive binding ELISA confirmed that the sera from vaccinated mice contained M2e conformational epitopes. Vaccination with MAP2 and MAP4 indicated the protective epitopes were dependent on dimeric and not tetrameric configuration of the MAP construct. The experiments demonstrate the potency of MAP configured M2e peptide with the JVRS-100 adjuvant in the development of universal flu vaccine.
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Heinen, Paul P., Frans A. Rijsewijk, Els A. de Boer-Luijtze, and André T. J. Bianchi. "Vaccination of pigs with a DNA construct expressing an influenza virus M2–nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus." Journal of General Virology 83, no. 8 (2002): 1851–59. http://dx.doi.org/10.1099/0022-1317-83-8-1851.
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In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broad-spectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.
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Kotomina, Tatiana, Irina Isakova-Sivak, Ki-Hye Kim, et al. "Generation and Characterization of Universal Live-Attenuated Influenza Vaccine Candidates Containing Multiple M2e Epitopes." Vaccines 8, no. 4 (2020): 648. http://dx.doi.org/10.3390/vaccines8040648.
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Influenza viruses constantly evolve, reducing the overall protective effect of routine vaccination campaigns. Many different strategies are being explored to design universal influenza vaccines capable of protecting against evolutionary diverged viruses. The ectodomain of influenza A M2e protein (M2e) is among the most promising targets for universal vaccine design. Here, we generated two recombinant live attenuated influenza vaccines (LAIVs) expressing additional four M2e tandem repeats (4M2e) from the N-terminus of the viral hemagglutinin (HA) protein, in an attempt to enhance the M2e-mediated cross-protection. The recombinant H1N1+4M2e and H3N2+4M2e viruses retained growth characteristics attributable to traditional LAIV viruses and induced robust influenza-specific antibody responses in BALB/c mice, although M2e-specific antibodies were raised only after two-dose vaccination with LAIV+4M2e viruses. Mice immunized with either LAIV or LAIV+4M2e viruses were fully protected against a panel of heterologous influenza challenge viruses suggesting that antibody and cell-mediated immunity contributed to the protection. The protective role of the M2e-specific antibody was seen in passive serum transfer experiments, where enhancement in the survival rates between classical LAIV and chimeric H3N2+4M2e LAIV was demonstrated for H3N2 and H5N1 heterologous challenge viruses. Overall, the results of our study suggest that M2e-specific antibodies induced by recombinant LAIV+4M2e in addition to cellular immunity by LAIV play an important role in conferring protection against heterologous viruses.
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Tan, Mei Peng, Wen Siang Tan, Noorjahan Banu Mohamed Alitheen, and Wei Boon Yap. "M2e-Based Influenza Vaccines with Nucleoprotein: A Review." Vaccines 9, no. 7 (2021): 739. http://dx.doi.org/10.3390/vaccines9070739.
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Discovery of conserved antigens for universal influenza vaccines warrants solutions to a number of concerns pertinent to the currently licensed influenza vaccines, such as annual reformulation and mismatching with the circulating subtypes. The latter causes low vaccine efficacies, and hence leads to severe disease complications and high hospitalization rates among susceptible and immunocompromised individuals. A universal influenza vaccine ensures cross-protection against all influenza subtypes due to the presence of conserved epitopes that are found in the majority of, if not all, influenza types and subtypes, e.g., influenza matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Despite its relatively low immunogenicity, influenza M2e has been proven to induce humoral responses in human recipients. Influenza NP, on the other hand, promotes remarkable anti-influenza T-cell responses. Additionally, NP subunits are able to assemble into particles which can be further exploited as an adjuvant carrier for M2e peptide. Practically, the T-cell immunodominance of NP can be transferred to M2e when it is fused and expressed as a chimeric protein in heterologous hosts such as Escherichia coli without compromising the antigenicity. Given the ability of NP-M2e fusion protein in inducing cross-protective anti-influenza cell-mediated and humoral immunity, its potential as a universal influenza vaccine is therefore worth further exploration.
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Vasyagin, Egor A., Anna A. Zykova, Eugenia S. Mardanova, et al. "Influenza A Vaccine Candidates Based on Virus-like Particles Formed by Coat Proteins of Single-Stranded RNA Phages Beihai32 and PQ465." Vaccines 12, no. 9 (2024): 1033. http://dx.doi.org/10.3390/vaccines12091033.
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Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum recombinant vaccines based on conserved antigens. The extracellular domain of the transmembrane protein M2 of influenza A virus (M2e) is highly conserved but poorly immunogenic and needs to be fused to an adjuvant protein or carrier virus-like particles (VLPs) to increase immunogenicity and provide protection against infection. In this study, we obtained VLPs based on capsid proteins (CPs) of single-stranded RNA phages Beihai32 and PQ465 bearing the M2e peptides. Four copies of the M2e peptide were linked to the C-terminus of the CP of phage Beihai32 and to the N and C termini of the CP of phage PQ465. The hybrid proteins, being expressed in Escherichia coli, formed spherical VLPs of about 30 nm in size. Immunogold transmission electron microscopy showed that VLPs formed by the phage PQ465 CP with a C-terminal M2e fusion present the M2e peptide on the surface. Subcutaneous immunization of mice with VLPs formed by both CPs containing four copies of the M2e peptide at the C termini induced high levels of M2e-specific IgG antibodies in serum and provided mice with protection against lethal influenza A virus challenge. In the case of an N-terminal fusion of M2e with the phage PQ465 CP, the immune response against M2e was significantly lower. CPs of phages Beihai32 and PQ465, containing four copies of the M2e peptide at their C termini, can be used to develop recombinant influenza A vaccine.
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Kim, Ki-Hye, Zhuo Li, Noopur Bhatnagar, et al. "Universal protection efficacy against influenza viruses by multi-subtype neuraminidase and M2 ectodomain virus-like particle vaccination in young and older adult mice." Journal of Immunology 208, no. 1_Supplement (2022): 64.03. http://dx.doi.org/10.4049/jimmunol.208.supp.64.03.
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Abstract Annual influenza vaccination is recommended to update the variable hemagglutinin antigens. [add more scientific justification for why NA + M2e immunity will be important?] Here, we first designed a virus-like particle (VLP) displaying consensus multi-neuraminidase (NA) subtypes (cN1, cN2, B cNA) and M2 ectodomain (M2e) tandem repeat (m-cNA-M2e VLP). Vaccination of mice with m-cNA-M2e VLP induced broad NA inhibition (NAI), M2e antibodies as well as interferon-gamma secreting T cell responses. Mice vaccinated with m-cNA-M2e VLP were protected against multi strains of different subtypes (H1N1, H5N1, H3N2, H9N2, H7N9) influenza A viruses containing substantial antigenic variations. From the T cell depletion and passive transfer experiments, we found that immunization of mice with m-cNA-M2e VLP induced cellular and humoral immune components contributing to broader cross protection. Also, surrogate effector assay indicated antibody-dependent cellular cytotoxicity as an additional protective immune contributor. Furthermore, comparable cross protection by m-cNA-M2e VLP vaccination was induced in aged mice. This study supports a novel strategy of developing a universal vaccine against influenza viruses potentially in both young and aged populations by inducing multi-NA subtype and M2e immunity with a single VLP entity. Supported by grants from NIH/NIAID (AI093772, AI154656, AI147042)
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Wang, Bao-Zhong, Harvinder S. Gill, Sang-Moo Kang, et al. "Enhanced Influenza Virus-Like Particle Vaccines Containing the Extracellular Domain of Matrix Protein 2 and a Toll-Like Receptor Ligand." Clinical and Vaccine Immunology 19, no. 8 (2012): 1119–25. http://dx.doi.org/10.1128/cvi.00153-12.
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ABSTRACTThe extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region ofSalmonella entericaserovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.
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Blokhina, Elena A., Eugenia S. Mardanova, Anna A. Zykova, et al. "Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope." Plants 12, no. 11 (2023): 2228. http://dx.doi.org/10.3390/plants12112228.
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Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15–30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant “universal” vaccine against influenza A produced in plants.
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Blokhina, Elena A., Eugenia S. Mardanova, Anna A. Zykova, et al. "Chimeric Virus-like Particles of Physalis Mottle Virus as Carriers of M2e Peptides of Influenza a Virus." Viruses 16, no. 11 (2024): 1802. http://dx.doi.org/10.3390/v16111802.
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Plant viruses and virus-like particles (VLPs) are safe for mammals and can be used as a carrier/platform for the presentation of foreign antigens in vaccine development. The aim of this study was to use the coat protein (CP) of Physalis mottle virus (PhMV) as a carrier to display the extracellular domain of the transmembrane protein M2 of influenza A virus (M2e). M2e is a highly conserved antigen, but to induce an effective immune response it must be linked to an adjuvant or carrier VLP. Four tandem copies of M2e were either fused to the N-terminus of the full-length PhMV CP or replaced the 43 N-terminal amino acids of the PhMV CP. Only the first fusion protein was successfully expressed in Escherichia coli, where it self-assembled into spherical VLPs of about 30 nm in size. The particles were efficiently recognized by anti-M2e antibodies, indicating that the M2e peptides were exposed on the surface. Subcutaneous immunization of mice with VLPs carrying four copies of M2e induced high levels of M2e-specific IgG antibodies in serum and protected animals from a lethal influenza A virus challenge. Therefore, PhMV particles carrying M2e peptides may become useful research tools for the development of recombinant influenza vaccines.
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Kannan, Nisha, Annette Choi, Mariela A. Rivera De Jesus, et al. "Intranasal Vaccination with Recombinant TLR2-Active Outer Membrane Vesicles Containing Sequential M2e Epitopes Protects against Lethal Influenza a Challenge." Vaccines 12, no. 7 (2024): 724. http://dx.doi.org/10.3390/vaccines12070724.
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Influenza is a highly contagious respiratory disease, resulting in an estimated 3 to 5 million cases of severe illness annually. While most influenza vaccines are administered parenterally via injection, one shortcoming is that they do not generate a strong immune response at the site of infection, which can become important in a pandemic. Intranasal vaccines can generate both local and systemic protective immune responses, can reduce costs, and enhance ease of administration. Previous studies showed that parenterally administered outer membrane vesicles (OMVs) that carry sequences of the M2e protein (OMV-M2e) protect against influenza A/PR8 challenge in mice and ferrets. In the current study, we measured the effectiveness of the intranasal route of the OMV-M2e vaccine against the influenza A/PR8 strain in mice. We observed high anti-M2e IgG and IgA titers post-challenge in mice vaccinated intranasally with OMV-M2e. In addition, we observed a Th1/Tc1 bias in the vaccinated mice, and an increased Th17/Tc17 response, both of which correlated with survival to A/PR8 challenge and significantly lower lung viral titers. We conclude that the intranasal-route administration of the OMV-M2e vaccine is a promising approach toward generating protection against influenza A as it leads to an increased proinflammatory immune response correlating with survival to viral challenge.
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Babapoor, Sankhiros, Tobias Neef, Christian Mittelholzer, et al. "A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection." Influenza Research and Treatment 2011 (January 12, 2011): 1–12. http://dx.doi.org/10.1155/2011/126794.
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Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P<0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.
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Samal, Sweety, Tripti Shrivastava, Praveen Sonkusre, et al. "Tetramerizing tGCN4 domain facilitates production of Influenza A H1N1 M2e higher order soluble oligomers that show enhanced immunogenicity in vivo." Journal of Biological Chemistry 295, no. 42 (2020): 14352–66. http://dx.doi.org/10.1074/jbc.ra120.013233.
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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.
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Sun, Weina, Allen Zheng, Robert Miller, Florian Krammer, and Peter Palese. "An Inactivated Influenza Virus Vaccine Approach to Targeting the Conserved Hemagglutinin Stalk and M2e Domains." Vaccines 7, no. 3 (2019): 117. http://dx.doi.org/10.3390/vaccines7030117.
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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.
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Braz Gomes, Kimberly, Sucheta D’Sa, Grace Lovia Allotey-Babington, Sang-Moo Kang, and Martin J. D’Souza. "Transdermal Vaccination with the Matrix-2 Protein Virus-like Particle (M2e VLP) Induces Immunity in Mice against Influenza A Virus." Vaccines 9, no. 11 (2021): 1324. http://dx.doi.org/10.3390/vaccines9111324.
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In this study, our goal was to utilize the extracellular domain matrix-2 protein virus-like particle (M2e VLP) that has been found to be highly conserved amongst all strains of influenza and could serve as a potential vaccine candidate against influenza. Previous studies have demonstrated that the VLP of the M2e showed increased activation of innate and adaptive immune responses. Therefore, to further explore its level of efficacy and protection, this vaccine was administered transdermally and tested in a pre-clinical mouse model. The M2e VLP was encapsulated into a polymeric matrix with the addition of Alhydrogel® and Monophosphoryl Lipid-A (MPL-A®), together referred to as AS04. The M2e VLP formulations induced IgG titers, with increased levels of IgG1 in the M2e VLP MP groups and further elevated levels of IgG2a were found specifically in the M2e VLP MP Adjuvant group. This trend in humoral immunity was also observed from a cell-mediated standpoint, where M2e VLP MP groups showed increased expression in CD4+ T cells in the spleen and the lymph node and high levels of CD8+ T cells in the lymph node. Taken together, the results illustrate the immunogenic potential of the matrix-2 protein virus-like particle (M2e VLP) vaccine.
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Subroto, Toto, Ari Hardianto, Abdul Alim Kahari, and Tika Pradnjaparamita. "Sintesis Tiga Peptida Bergugus Pelindung sebagai Prekursor Komponen Vaksin Influenza Universal." Jurnal Natur Indonesia 15, no. 2 (2015): 84. http://dx.doi.org/10.31258/jnat.15.2.84-91.
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Current highly effective conventional vaccine to halt the spread of bird flu has not been invented yet because of susceptiblemutation of influenza virus. In spite of undergoing mutation which causes the amino acid sequence change, influenzaviruses maintain conservation at ectodomain of M2 protein, especially M2e(2-16) (SLLTEVETPIRNEW). The use ofconserved epitope M2e(2-16) in epitope-based vaccine potentially produces universal influenza vaccine. In designingepitope-based vaccine, the M2e(2-16) needs to be coupled with T helper epitope, P25, which is subsequently mentioned asM2e(2-16)-K-P25 (SLLTEVETPIRNEWGKKKL IPNASLIENCTKAEL). The M2e(2-16)-K-P25 was synthesized usingconvergent solid phase peptide synthesis strategy because of the size of the sequence. In this strategy, four peptideprecursors of M2e(2-16)-K-P25; SLLTEVETP (F1), IRNEWGK (F2), KLIPNASLI (F3), and ENCTKAEL (F4); were synthesizedin advance. After the precursors ready, coupling reaction was performed to obtain M2e(2-16)-K-P25. In the previousresearch, F3 has been obtained in high purity through Fmoc/tBu solid phase peptide synthesis method. In this conductedresearch, the three remaining precursors; F1, F2, and F4; were synthesized by the same method. Each peptide was analysedby thin layer chromatography, HPLC, and mass spectroscopy methods. F1, F2 and F4 were successfully synthesized andeach of them was detected at 1490.0, 1874.8 and 1881.9 amu, respectively. However, F1 was not possible to purify becauseof its insolubility in various solvents.
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Wang, Li, Ying-Chun Wang, Hao Feng, Tamanna Ahmed, Richard W. Compans, and Bao-Zhong Wang. "Virus-Like Particles Containing the Tetrameric Ectodomain of Influenza Matrix Protein 2 and Flagellin Induce Heterosubtypic Protection in Mice." BioMed Research International 2013 (2013): 1–12. http://dx.doi.org/10.1155/2013/686549.
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The ectodomain of matrix protein 2 (M2e) is highly conserved among influenza A viruses and can be a promising candidate antigen for a broadly cross-protective vaccine. In this study, a tetrameric M2e (tM2e) and a truncated form of flagellin (tFliC) were coincorporated into virus-like particles (VLPs) to enhance its immunogenicity. Our data showed that the majority of M2e in VLPs was presented as tetramers by introducing a foreign tetramerization motif GCN4. Intranasal immunization with tM2e VLPs significantly enhanced the levels of serum IgG and IgG subclasses compared to soluble M2e (sM2e) in mice. tM2e VLPs also induced higher M2e-specific T-cell and mucosal antibody responses, conferring complete protection against homologous influenza virus infection. The immunogenicity of tM2e VLPs was further enhanced by coincorporation of the membrane-anchored tFliC (tM2e chimeric VLPs) or coadministration with tFliC VLPs as a mixture, but not the soluble flagellin, inducing strong humoral and cellular immune responses conferring cross-protection against lethal challenge with heterotypic influenza viruses. These results support the development of tM2e chimeric VLPs as universal vaccines and warrant further investigation.
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Cho, Ki Joon, Bert Schepens, Kristof Moonens, et al. "Crystal Structure of the Conserved Amino Terminus of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus Gripped by an Antibody." Journal of Virology 90, no. 1 (2015): 611–15. http://dx.doi.org/10.1128/jvi.02105-15.
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We report the crystal structure of the M2 ectodomain (M2e) in complex with a monoclonal antibody that binds the amino terminus of M2. M2e extends into the antibody binding site to form an N-terminal β-turn near the bottom of the paratope. This M2e folding differs significantly from that of M2e in complex with an antibody that binds another part of M2e. This suggests that M2e can adopt at least two conformations that can elicit protective antibodies.
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Zykova, Anna A., Elena A. Blokhina, Roman Y. Kotlyarov, et al. "Highly Immunogenic Nanoparticles Based on a Fusion Protein Comprising the M2e of Influenza A Virus and a Lipopeptide." Viruses 12, no. 10 (2020): 1133. http://dx.doi.org/10.3390/v12101133.
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The highly conserved extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is a promising target for the development of broad-spectrum vaccines. However, M2e is a poor immunogen by itself and must be linked to an appropriate carrier to induce an efficient immune response. In this study, we obtained recombinant mosaic proteins containing tandem copies of M2e fused to a lipopeptide from Neisseria meningitidis surface lipoprotein Ag473 and alpha-helical linkers and analyzed their immunogenicity. Six fusion proteins, comprising four or eight tandem copies of M2e flanked by alpha-helical linkers, lipopeptides, or a combination of both of these elements, were produced in Escherichia coli. The proteins, containing both alpha-helical linkers and lipopeptides at each side of M2e repeats, formed nanosized particles, but no particulate structures were observed in the absence of lipopeptides. Animal study results showed that proteins with lipopeptides induced strong M2e-specific antibody responses in the absence of external adjuvants compared to similar proteins without lipopeptides. Thus, the recombinant M2e-based proteins containing alpha-helical linkers and N. meningitidis lipopeptide sequences at the N- and C-termini of four or eight tandem copies of M2e peptide are promising vaccine candidates.
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Song, Manki, and Byoung-Shik Shim. "Sublingual immunization with M2-based vaccine induces broad protective immunity (P4301)." Journal of Immunology 190, no. 1_Supplement (2013): 123.14. http://dx.doi.org/10.4049/jimmunol.190.supp.123.14.
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Abstract Background: M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.
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Liu, Shenglong, Mengzhou Lin, and Xin Zhou. "T4 Phage Displaying Dual Antigen Clusters Against H3N2 Influenza Virus Infection." Vaccines 13, no. 1 (2025): 70. https://doi.org/10.3390/vaccines13010070.
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Background: The current H3N2 influenza subunit vaccine exhibits weak immunogenicity, which limits its effectiveness in preventing and controlling influenza virus infections. Methods: In this study, we aimed to develop a T4 phage-based nanovaccine designed to enhance the immunogenicity of two antigens by displaying the HA1 and M2e antigens of the H3N2 influenza virus on each phage nanoparticle. Specifically, we fused the Soc protein with the HA1 antigen and the Hoc protein with the M2e antigen, assembling them onto a T4 phage that lacks Soc and Hoc proteins (Soc−Hoc−T4), thereby constructing a nanovaccine that concurrently presents both HA1 and M2e antigens. Results: The analysis of the optical density of the target protein bands indicated that each particle could display approximately 179 HA1 and 68 M2e antigen molecules. Additionally, animal experiments demonstrated that this nanoparticle vaccine displaying dual antigen clusters induced a stronger specific immune response, higher antibody titers, a more balanced Th1/Th2 immune response, and enhanced CD4+ and CD8+ T cell effects compared to immunization with HA1 and M2e antigen molecules alone. Importantly, mice immunized with the T4 phage displaying dual antigen clusters achieved full protection (100% protection) against the H3N2 influenza virus, highlighting its robust protective efficacy. Conclusions: In summary, our findings indicate that particles based on a T4 phage displaying antigen clusters exhibit ideal immunogenicity and protective effects, providing a promising strategy for the development of subunit vaccines against various viruses beyond influenza.
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Tsybalova, L. M., L. A. Stepanova, R. Yu Kotlyarov, et al. "Strengthening the Effectiveness of the Candidate Influenza Vaccine by Combining Conserved Sequences of Hemagglutinin and M2 protein." Epidemiology and Vaccine Prevention 16, no. 3 (2017): 65–70. http://dx.doi.org/10.31631/2073-3046-2017-16-3-65-70.
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The development of universal influenza vaccine - a vaccine directed to all subtypes of human influenza A viruses - is the really actual problem task. This paper presents the comparative characteristic of the specific activity of various recombinant proteins consisting of antigenic determinants of influenza A virus - the ectodomain of the M2 protein (M2e) and a fragment of the second subunit of the hemagglutinin (the amino acid sequence 76 - 130). Flagellin - Salmonella typhimurium protein was used as carrier protein and as adjuvant. We use two forms of flagellin: full size and with deleted hypervariable region. The proteins showed high immunogenicity, and the ability to prevent lethal infection of influenza virus in mice. Full-length flagellin with HA2 (76 - 130) and M2e on the C-terminus (protein Flg-HA2-4M2e) demonstrated the most protective properties. It provides 100% survival immunized mice that were challenge with a high dose of influenza A (H3N2) - 10 LD50. Proteins containing only full sized flagellin with M2e or flagellin truncated form with M2e at the C-terminus and HA2 within the hypervariable region, protected 75% of animals from lethal infection. Protein Flg-HA2-4M2e is promising for further study as a vaccine.
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Fiers, Walter>, Sabine Neirynck, Tom Deroo, Xavier Saelens, and Willy Min Jou. "Soluble recombinant influenza vaccines." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 356, no. 1416 (2001): 1961–63. http://dx.doi.org/10.1098/rstb.2001.0980.
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Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain–specific. A third influenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human influenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain–independent, long–lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.
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Stepanova, L. A., R. Y. Kotlyarov, M. A. Shuklina, et al. "Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins." Acta Naturae 10, no. 1 (2018): 85–94. http://dx.doi.org/10.32607/20758251-2018-10-1-85-94.
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The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial -helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.
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Mezhenskaya, Daria, Irina Isakova-Sivak, Victoria Matyushenko, et al. "Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge." Viruses 13, no. 7 (2021): 1280. http://dx.doi.org/10.3390/v13071280.
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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.
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Mezhenskaya, Daria А., Irina N. Isakova-Sivak, Anastasiya E. Katelnikova, and Larisa G. Rudenko. "Optimization of M2e cassette amino acid composition for the development of universal influenza vaccine." Medical academic journal 21, no. 3 (2021): 127–30. http://dx.doi.org/10.17816/maj76326.
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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.
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Grandea, Andres, Ole Olsen, Tom Cox, et al. "Protective human MAbs to a highly conserved conformational determinant on influenza M2e (38.14)." Journal of Immunology 184, no. 1_Supplement (2010): 38.14. http://dx.doi.org/10.4049/jimmunol.184.supp.38.14.
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Abstract Specific antibody responses to the annual influenza vaccine can protect against infection if the vaccine and the infective strain are antigenically well-matched. However, due to the variability of the influenza genome, emergent strains arise for which the vaccine proves to be ineffective, and moreover, necessitate its yearly reformulation. Efforts have been made into identifying epitopes within more conserved elements of the influenza virus as an alternative attack on a wider range of strains. Through our platform of examining monoclonal antibodies (MAbs) expressed from plasmacytes derived from memory B cells from normal healthy human donors, we have found a previously unknown conformational antigenic determinant within the relatively invariable ectodomain of M2 (M2e) of influenza A. The MAbs bind to virus and cell-surface expressed M2, but not to the corresponding M2e peptide. The M2e region recognized is in almost all strains isolated to date. Furthermore, in a mouse model of infection, the MAbs can protect against lethal challenge. These results suggest that naturally expressed M2 can elicit a broadly cross-reactive and protective antibody response. And, the human recombinant anti-M2e MAbs might serve as therapeutic agents against infection.
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Deng, Lei, Ki Cho, Walter Fiers, and Xavier Saelens. "M2e-Based Universal Influenza A Vaccines." Vaccines 3, no. 1 (2015): 105–36. http://dx.doi.org/10.3390/vaccines3010105.
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Fiers, Walter, Marina De Filette, Karim El Bakkouri, et al. "M2e-based universal influenza A vaccine." Vaccine 27, no. 45 (2009): 6280–83. http://dx.doi.org/10.1016/j.vaccine.2009.07.007.
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Mytle, Nutan, Sonja Leyrer, Jon R. Inglefield, et al. "Influenza Antigens NP and M2 Confer Cross Protection to BALB/c Mice against Lethal Challenge with H1N1, Pandemic H1N1 or H5N1 Influenza A Viruses." Viruses 13, no. 9 (2021): 1708. http://dx.doi.org/10.3390/v13091708.
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Influenza hemagglutinin (HA) is considered a major protective antigen of seasonal influenza vaccine but antigenic drift of HA necessitates annual immunizations using new circulating HA versions. Low variation found within conserved non-HA influenza virus (INFV) antigens may maintain protection with less frequent immunizations. Conserved antigens of influenza A virus (INFV A) that can generate cross protection against multiple INFV strains were evaluated in BALB/c mice using modified Vaccinia virus Ankara (MVA)-vectored vaccines that expressed INFV A antigens hemagglutinin (HA), matrix protein 1 (M1), nucleoprotein (NP), matrix protein 2 (M2), repeats of the external portion of M2 (M2e) or as tandem repeats (METR), and M2e with transmembrane region and cytoplasmic loop (M2eTML). Protection by combinations of non-HA antigens was equivalent to that of subtype-matched HA. Combinations of NP and forms of M2e generated serum antibody responses and protected mice against lethal INFV A challenge using PR8, pandemic H1N1 A/Mexico/4108/2009 (pH1N1) or H5N1 A/Vietnam/1203/2004 (H5N1) viruses, as demonstrated by reduced lung viral burden and protection against weight loss. The highest levels of protection were obtained with NP and M2e antigens delivered as MVA inserts, resulting in broadly protective immunity in mice and enhancement of previous natural immunity to INFV A.
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Blokhina, Elena A., Eugenia S. Mardanova, Liudmila A. Stepanova, Liudmila M. Tsybalova, and Nikolai V. Ravin. "Plant-Produced Recombinant Influenza A Virus Candidate Vaccine Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin." Plants 9, no. 2 (2020): 162. http://dx.doi.org/10.3390/plants9020162.
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The development of recombinant influenza vaccines with broad spectrum protection is an important task. The combination of conservative viral antigens, such as M2e, the extracellular domain of the transmembrane protein M2, and conserved regions of the second subunit of hemagglutinin (HA), provides an opportunity for the development of universal influenza vaccines. Immunogenicity of the antigens could be enhanced by fusion to bacterial flagellin, the ligand for Toll-like receptor 5, acting as a powerful mucosal adjuvant. In this study, we report the transient expression in plants of a recombinant protein comprising flagellin of Salmonella typhimurium fused to the conserved region of the second subunit of HA (76–130 a.a.) of the first phylogenetic group of influenza A viruses and four tandem copies of the M2e peptide. The hybrid protein was expressed in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff up to 300 µg/g of fresh leaf tissue. The intranasal immunization of mice with purified fusion protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza A virus strain A/Aichi/2/68(H3N2). Our results show that M2e and hemagglutinin-derived peptide can be used as important targets for the development of a plant-produced vaccine against influenza.
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Song, Jin-Ha, Seung-Eun Son, Ho-Won Kim, et al. "A Model H5N2 Vaccine Strain for Dual Protection Against H5N1 and H9N2 Avian Influenza Viruses." Vaccines 13, no. 1 (2024): 22. https://doi.org/10.3390/vaccines13010022.
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Background/Objective: Highly pathogenic (HP) H5Nx and low-pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose global threats to the poultry industry and public health, highlighting the critical need for a dual-protective vaccine. Methods: In this study, we generated a model PR8-derived recombinant H5N2 vaccine strain with hemagglutinin (HA) and neuraminidase (NA) genes from clade 2.3.2.1c H5N1 and Y439-like H9N2 viruses, respectively. To enhance the immunogenicity of the recombinant H5N2 vaccine strain, N-glycans of the HA2 subunit, NA, and M2e were modified. Additionally, we replaced M2e with avian M2e to enhance the antigenic homogeneity of AIVs for better protection. We also replaced PR8 PB2 with 01310 PB2, which is the PB2 gene derived from an LP H9N2 avian influenza virus, to eliminate pathogenicity in mammals. The productivity of the model vaccine strain (rvH5N2-aM2e-vPB2) in embryonated chicken eggs (ECEs), its potential risk of mammalian infection, and the immunogenicity associated with different inactivation methods (formaldehyde (F/A) vs. binary ethyleneimine (BEI)) were evaluated. Results: The rvH5N2-aM2e-vPB2 strain demonstrated high productivity in ECEs and exhibited complete inhibition of replication in mammalian cells. Furthermore, compared with using F/A inactivation, inactivation using BEI significantly enhanced the immune response, particularly against NA. This enhancement resulted in increased virus neutralization titers, supporting its efficacy for dual protection against H5Nx and H9N2 avian influenza viruses. Furthermore, we demonstrated that M2e-specific immune responses, difficult to induce with inactivated vaccines, can be effectively elicited with live vaccines, suggesting a strategy to enhance M2e immunogenicity in whole influenza virus vaccines. Conclusions: Finally, the successful development of the model rH5N2 vaccine strain is described; this strain provides dual protection, has potential applicability in regions where avian influenza is endemic, and can be used to promote the development of versatile H5N2 recombinant vaccines for effective avian influenza control.
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Lee, Young-tae, Min-Chul Kim, Eun-Ju Ko, et al. "Novel vaccination of supplementing live attenuated influenza vaccine with M2 ectodomain tandem repeat to confer universal protection (VAC8P.1051)." Journal of Immunology 194, no. 1_Supplement (2015): 144.7. http://dx.doi.org/10.4049/jimmunol.194.supp.144.7.
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Abstract Current influenza vaccines including live attenuated influenza vaccine (LAIV) do not provide cross-protection against drift and potential pandemic strains. We hypothesized that supplementing LAIV with a highly conserved protective antigenic target M2 ectodomain (M2e) would confer broad cross-protection by inducing humoral and cellular immune responses to conserved antigenic targets (M2e, NP), overcoming strain-specific protection of current vaccines. Intranasal vaccination with LAIV (A/NL/09/H1N1) supplemented with tandem repeat M2e containing virus-like particles (M2e5x VLPs) induced M2- and H1N1 virus-specific antibodies. Upon heterosubtypic challenge with pandemic potential rgA/H5N1 virus, we found that M2e5x VLP supplemented LAIV immune mice showed significantly improved heterosubtypic protection by preventing weight loss and lowering lung viral titers compared to the LAIV alone group inducing poor heterosubtypic immunity. Further mechanistic studies on heterosubtypic immunity suggest that recall T cell responses to M2e and nucleoprotein as well as systemic and mucosal M2e antibodies were found to be major heterosubtypic immune correlates. Therefore, this study demonstrates a novel vaccination strategy to confer universal protection by supplementing current strain-specific vaccines with a highly conserved cross protective antigenic target M2e5x VLPs.
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Shuklina, Marina, Liudmila Stepanova, Olga Ozhereleva, et al. "Inserting CTL Epitopes of the Viral Nucleoprotein to Improve Immunogenicity and Protective Efficacy of Recombinant Protein against Influenza A Virus." Biology 13, no. 10 (2024): 801. http://dx.doi.org/10.3390/biology13100801.
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Conserved influenza virus proteins, such as the hemagglutinin stem domain (HA2), nucleoprotein (NP), and matrix protein (M), are the main targets in the development of universal influenza vaccines. Previously, we constructed a recombinant vaccine protein Flg-HA2-2-4M2ehs containing the extracellular domain of the M2 protein (M2e) and the aa76–130 sequence of the second HA subunit as target antigens. It demonstrated immunogenicity and broad protection against influenza A viruses after intranasal and parenteral administration. This study shows that CD8+ epitopes of NP, inserted into a flagellin-fused protein carrying M2e and HA2, affect the post-vaccination immune humoral response to virus antigens without reducing protection. No differences were found between the two proteins in their ability to stimulate the formation of follicular Th in the spleen, which may contribute to a long-lasting antigen-specific humoral response. The data obtained on Balb/c mice suggest that the insertion of CTL NP epitopes into the flagellin-fused protein carrying M2e and HA2 reduces the antibody response to M2e and A/H3N2. In C57Bl6 mice, this stimulates the formation of NP-specific CD8+ Tem and virus-specific mono- and multifunctional CD4+ and CD8+ Tem in the spleen and completely protects mice from influenza virus subtypes A/H1N1pdm09 and A/H3N2.
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Sumarningsih, Sumarningsih, Simson Tarigan, H. Farhid, and Jagoda Ignjatovic. "Characterisation of M2e Antigenicity using anti-M2 Monoclonal Antibody and anti-M2e Polyclonal Antibodies." Jurnal Ilmu Ternak dan Veteriner 24, no. 3 (2019): 122. http://dx.doi.org/10.14334/jitv.v24i3.1987.
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Matrix 2 ectodomain (M2e) protein is a potential antigen for detection of influenza A virus infection in vaccinated poultry (DIVA test). However the M2e antigenicity and immune response it induces in either humans or animals are poorly understood. Seventeen M2e peptides and sixteen recombinant M2e (rM2e) proteins with amino acid (aa) changes introduced at position 10, 11, 12, 13 14, 16, 18 and 20 were compared by western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using mouse anti-M2 monoclonal antibody (mAb) 14C2, and anti-M2e peptide chicken and rabbit polyclonal antibody (pAb). The mAb 14C had the best discriminating power and indicated that all six positions contributed to the M2e antigenicity. Position 11 was the important immunodominant and affected Mab14C binding to a greatest degree. Changes in the adjacent position 14, 16 and 18 also influenced the binding, and it detected regardless of the method (WB or ELISA), or the antigen used (M2e peptide or rM2e). For chicken pAb and rabbit pAb, the immunodominant aa was position 10 and the antibody reaction was not affected by aa change at 11. The binding of rabbit pAb was also affected by changes at 14 and 16, which confirm the contribution of these positions to the M2e antigenicity. Position 10 was the only important position for the binding of chicken pAb to M2e. Overall, the study showed that the M2e antigenic sites are located between residues 10 – 18 and that aa changes at position 10, 11, 12, 14, 16 and 18 may all affect the antibody binding within the M2e protein.
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Mardanova, Eugenia S., and Nikolai V. Ravin. "Plant-produced Recombinant Influenza A Vaccines Based on the M2e Peptide." Current Pharmaceutical Design 24, no. 12 (2018): 1317–24. http://dx.doi.org/10.2174/1381612824666180309125344.
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Background: Influenza is a widely distributed infection that almost annually causes seasonal epidemics. The current egg-based platforms for influenza vaccine production are facing a number of challenges and are failing to satisfy the global demand in the case of pandemics due to the long production time. Recombinant vaccines are an alternative that can be quickly produced in high quantities in standard expression systems. Methods: : Plants may become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Plant-based expression systems have been used to produce recombinant vaccines against influenza based on two targets; the major surface antigen hemagglutinin and the transmembrane protein M2. <P> Results: Different forms of recombinant hemagglutinin were successfully expressed in plants, and some plantproduced vaccines based on hemagglutinin were successfully tested in clinical trials. However, these vaccines remain strain specific, while the highly conserved extracellular domain of the M2 protein (M2e) could be used for the development of a universal influenza vaccine. In this review, the state of the art in developing plant-produced influenza vaccines based on M2e is presented and placed in perspective. A number of strategies to produce M2e in an immunogenic form in plants have been reported, including its presentation on the surface of plant viruses or virus-like particles formed by capsid proteins, linkage to bacterial flagellin, and targeting to protein bodies. Conclusion: Some M2e-based vaccine candidates were produced at high levels (up to 1 mg/g of fresh plant tissue) and were shown to be capable of stimulating broad-range protective immunity.
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Callejo, Bernadette J., Stella Chang, Marla Lay, Lily Liang, and Jeffery Fairman. "JVRS-100 adjuvanted universal influenza A vaccine based on a multiple antigenic peptide of M2e protects from lethal challenge (132.6)." Journal of Immunology 182, no. 1_Supplement (2009): 132.6. http://dx.doi.org/10.4049/jimmunol.182.supp.132.6.
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Abstract Background: The conventional vaccine strategy for control of influenza A is vulnerable to antigenic drift and the emergence of unmatched epidemic strains that cause primary vaccine failure. A vaccine strategy that targets an influenza antigen, which is less susceptible to antigenic variation, would be a major improvement. Methods: Balb/c mice were vaccinated three times with the M2e peptide in the context of a multiple antigenic peptide (MAP) complex and combination with the cationic lipid DNA complex adjuvant (JVRS-100). Antibody titers were monitored over the time course of vaccination and the mice received a lethal challenge with H1N1 (PR/8/34) or H3N2 (HKx31) two weeks after the final vaccination. Results: Vaccination of mice with JVRS-100-MAP4-M2e compared to MAP4-M2e resulted in increased survival following lethal challenge with 6x LD50 PR/8/34 H1N1 (100% vs. 30%, P=0.002) or 10x LD50 HKx31 H3N2 (80% vs. 20%). Recipients of 200µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (6x LD50 PR/8/34) resulted in survival of 60% (P=0.014) compared with control mice which received 200µl naive serum (0% survival). Furthermore, recipients of 300µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (2x LD50 PR/8/34) resulted in survival of 100% (P=0.0026) compared with control mice which received 200µl naive serum (0% survival). Conclusion: These studies demonstrate that a simple, synthetic M2e vaccine in a MAP configuration with a strong adjuvant may result in a viable vaccine candidate for universal flu vaccination and the protective immunity is primarily due to an antibody response.
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Shuklina, M. A., L. A. Stepanova, A. A. Kovaleva, et al. "Intranasal immunization with a recombinant protein based on the M2e peptide and second subunit of influenza A viral hemagglutinin fragment induces a cross-protective humoral and Tcell response in mice." Medical Immunology (Russia) 22, no. 2 (2020): 357–70. http://dx.doi.org/10.15789/1563-0625-iiw-1584.
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Development of vaccines with a broad-spectrum of protection is one of the priorities in the programs of influenza prevention. Recently, the conserved fragments of influenza virus proteins (M1, M2, NP, the second subunit of the hemagglutinin HA2) provoke interest of investigators as the object of the development a broad-spectrum vaccines. Low immunogenicity present a problem when developing vaccines based on such conserved fragments. However, fusion of low immunogenic antigens into the high immunogenic carrier protein may significantly enhance their immunogenicity. The candidate vaccine protein Flg-HA2-2-4M2e was developed which containins two highly conserved viral antigens (the ectodomain of the M2 protein (M2e), 76130 region of the second subunit of HA2), fused with flagellin as a carrier protein. Flagellin (bacterial flagella protein) is a natural ligand of TLR-5, and has a strong adjuvant activity at different ways of its administration. The purpose of this study was to assess development of humoral and T cell immune response, along with broad-spectrum protection after mice immunization with the candidate Flg-HA2-2-4M2e vaccine protein. Mice were immunized intranasally three times with two-week intervals. Two weeks after the final immunization, the mice were challenged at the 5 LD50 dose with influenza viruses A/California/07/09 (H1N1) pdm09 (phylogenetic group I), or A/Shanghai/2/2013 (H7N9) (phylogenetic group II). The results obtained in this study showed induction of strong M2e-specific humoral response (serum IgG and A) in the immunized mice. Immunization with recombinant protein stimulated formation of M2e-specific and virus-specific CD4+ and CD8+T cells in lung which produced TNFα or IFNγ. Production of antigen-specific effector and central memory T cells was also detected in lungs of immunized mice. The formation of cross-protective immunity in immunized mice was demonstrated in a model of lethal influenza infection. The experimental animals were almost completely protected from the high dose of the pandemic virus A/H1N1pdm09, and highly pathogenic avian influenza A/H7N9 (90-100% survival). We also evaluated the changes of antigen-specific immune response in immunized mice after sublethal infection with A/H3N2 influenza virus. Mice of control and experimental groups were infected with MID100 of influenza virus A/Aichi/2/68 (H3N2). It was shown that the M2e-specific response (IgG, IgA) was significantly increased in immunized mice after sublethal infection with influenza virus A/H3N2, and we detected the changes in profile of M2e-specific IgG subclasses. Following sublethal infection in immunized mice, the proportion of M2e-specific IgG2a was increased 10-fold. The results showed that the recombinant protein Flg-HA2-2-4M2e is a promising candidate for development of universal vaccines, which induces a protective humoral and T-cell response to conserved viral epitopes and protects against influenza A viruses of both phylogenetic groups.
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Mezhenskaya, Daria, Irina Isakova-Sivak, Tatiana Gupalova, et al. "A Live Probiotic Vaccine Prototype Based on Conserved Influenza a Virus Antigens Protect Mice against Lethal Influenza Virus Infection." Biomedicines 9, no. 11 (2021): 1515. http://dx.doi.org/10.3390/biomedicines9111515.
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Background: Due to the highly variable nature of the antigenic properties of the influenza virus, many efforts have been made to develop broadly reactive influenza vaccines. Various vaccine platforms have been explored to deliver conserved viral antigens to the target cells to induce cross-reactive immune responses. Here, we assessed the feasibility of using Enterococcus faecium L3 as a bacterial vector for oral immunization against influenza virus. Methods: we generated two vaccine prototypes by inserting full-length HA2 (L3-HA2) protein or its long alpha helix (LAH) domain in combination with four M2e tandem repeats (L3-LAH+M2e) into genome of E.faecium L3 probiotic strain. The immunogenicity and protective potential of these oral vaccines were assessed in a lethal challenge model in BALB/c mice. Results: as expected, both vaccine prototypes induced HA stem-targeting antibodies, whereas only L3-LAH+4M2e vaccine induced M2e-specific antibody. The L3-HA2 vaccine partially protected mice against lethal challenge with two H1N1 heterologous viruses, while 100% of animals in the L3-LAH+4M2e vaccine group survived in both challenge experiments, and there was significant protection against weight loss in this group, compared to the L3 vector-immunized control mice. Conclusions: the recombinant enterococcal strain L3-LAH+4M2e can be considered as a promising live probiotic vaccine candidate for influenza prevention and warrants further evaluation in relevant pre-clinical models.
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Sei, Clara J., Nimisha Rikhi, Rachmat Hidajat, et al. "2752. Peptide Vaccines Utilizing Conserved Hemagglutinin, Neuraminidase, and Matrix Ectodomain Influenza Epitopes Demonstrate Functional Activity Against Group 1 and 2 Influenza Strains." Open Forum Infectious Diseases 6, Supplement_2 (2019): S969—S970. http://dx.doi.org/10.1093/ofid/ofz360.2429.
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Abstract Background Globally, prevention and control of seasonal influenza has faced many challenges in the selection of a vaccine composition that antigenically matches circulating viruses. A universal influenza vaccine approach that targets small conserved influenza virus epitopes/peptides such as the extracellular domain of Matrix 2 (M2e) and induces broadly reactive antibodies may be helpful for both seasonal influenza outbreaks and pandemics. Here we report the ability of two composite peptide vaccines, individually and in combination, to induce broadly reactive antibodies that have binding and functional activity across several contemporary influenza strains in Group 1 and 2. Methods Mice were immunized with peptide composite vaccines against Hemagglutinin (HA), Neuraminidase (NA) and M2e, individually and in combination. Peptide composite vaccines, conjugated to CRM were administered subcutaneously with adjuvant and at least two booster doses. Serum antibody titers were analyzed using an anti-influenza ELISA for binding activity to peptides and live influenza viruses (H3N2 and H1N1) and functional activity was evaluated in vitro using Microneutralization, Hemagglutination Inhibition (HAI), and Antibody-Dependent Cellular Cytotoxicity (ADCC) assays. Results Mice given the peptide composite conjugate vaccines, individually and in combination, had strong humoral responses producing high serum anti-influenza titers post-booster immunization. Anti-influenza serum antibodies demonstrated functional activity against influenza A (H3N2 and H1N1) contemporary strains showing neutralization, HAI and ADCC activity. Conclusion Peptide conjugate vaccines were highly immunogenic in mice. Broadly reactive serum antibodies against the peptides and live influenza viruses were detected. These vaccines individually or in combination, induced antibodies that demonstrated functional activity against contemporary influenza strains in Group 1 and 2 and induced functional anti-influenza monoclonal antibodies. A vaccine that targets one or more HA, NA and M2e influenza epitopes may more closely approach the goal for a true universal influenza vaccine. In vivo protection studies are currently being designed. Disclosures All authors: No reported disclosures.
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Zhu, Wandi, Jaeyoung Park, Chunhong Dong, et al. "ISCOMs/MPLA adjuvanted SDAD protein nanoparticles induce improved immune responses and cross-protection in mice." Journal of Immunology 210, no. 1_Supplement (2023): 223.19. http://dx.doi.org/10.4049/jimmunol.210.supp.223.19.
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Abstract The development of a universal influenza vaccine to protect against influenza variants will be the priority goal in combating influenza virus infection. In this study, we exploited a novel type of protein nanoparticles consisting of influenza NP as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by using an SDAD heterobifunctional crosslinker. Intramuscular immunization of the NP/NA-M2e (core/shell) nanoparticles induced increased humoral and cellular immune responses. ISCOMs/MPLA as adjuvants further improved the nanoparticle immunogenicity and elicited stronger immune reactions conferring protection against different influenza viral challenges. The nanoparticles were delivered through intranasal route to determine the application as a mucosal vaccine. While the nanoparticles alone barely induced immune response after intranasal immunization, ISCOMs/MPLA adjuvanted nanoparticles induced strengthened antigens-specific antibodies responses, cellular responses and higher levels of mucosal IgA. Meanwhile, increased lung Trm, Brm , and alveolar macrophages, were observed from ISCOMs/MPLA adjuvanted nanoparticle group. Only the mice intranasally vaccinated with ISCOMs/MPLA adjuvanted nanoparticles fully survived during the infections, and better protection efficacy was observed when compared with other adjuvants. Our results emphasized the importance of supplementing of appropriate adjuvants to improve the immunogenicity and mucosal immune responses of vaccines in mucosal immunization. In conclusion, the ISCOMs/MPLA adjuvant combination could significantly improve the immune responses and protective efficiency of protein nanoparticles in different immunization routes.
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Mezhenskaya, Daria, Irina Isakova-Sivak, Tatiana Kotomina, et al. "A Strategy to Elicit M2e-Specific Antibodies Using a Recombinant H7N9 Live Attenuated Influenza Vaccine Expressing Multiple M2e Tandem Repeats." Biomedicines 9, no. 2 (2021): 133. http://dx.doi.org/10.3390/biomedicines9020133.
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Influenza viruses remain a serious public health problem. Vaccination is the most effective way to prevent the disease; however, seasonal influenza vaccines demonstrate low or no effectiveness against antigenically drifted and newly emerged influenza viruses. Different strategies of eliciting immune responses against conserved parts of various influenza virus proteins are being developed worldwide. We constructed a universal live attenuated influenza vaccine (LAIV) candidate with enhanced breadth of protection by modifying H7N9 LAIV by incorporating four epitopes of M2 protein extracellular part into its hemagglutinin molecule. The new recombinant H7N9+4M2e vaccine induced anti-M2e antibody responses and demonstrated increased protection against heterosubtypic challenge viruses in direct and serum passive protection studies, compared to the classical H7N9 LAIV. The results of our study suggest that the H7N9+4M2e warrants further investigation in pre-clinical and phase 1 clinical trials.
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Zykova, Anna A., Elena A. Blokhina, Liudmila A. Stepanova, et al. "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." Molecules 28, no. 18 (2023): 6441. http://dx.doi.org/10.3390/molecules28186441.
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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.