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IntroductionThe frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear.MethodsWe designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptide-keyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund’s adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay.ResultsSeroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomain-based vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro. Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30.DiscussionThe findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections.