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Aims:  To identify proteins regulating antimicrobial peptide (AMP) resistance in Vibrio parahaemolyticus using membrane subproteome analysis. Methods and Results:  Three synthetic AMPs (Q4, Q6 and H1) and a natural one from fish (pleurocidin) were used for selection of AMP‐resistant strains. Differential expression patterns of the outer and inner membrane proteins (OMPs and IMPs) among wild‐type and the resistant strains were obtained using two‐dimensional gel electrophoresis. Two OMPs (TolC and flagellin) and five IMPs [transcription termination factor (NusA), long‐chain fatty acid transport protein (FadL), elongation factor Tu (EF‐Tu), ATP synthase F1, alpha subunit (F1‐ATPa) and dihydrolipoamide dehydrogenase (DLD)] were identified using LC‐ESI‐Q‐TOF MS/MS and Mascot program. Real‐time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. All seven membrane proteins except FadL were upregulated in the AMP‐resistant clones, both in the translational and transcriptional levels. Conclusions:  Our results suggested that V. parahaemolyticus may obtain their resistance against AMPs through upregulation of the multidrug efflux transporter, effective repair of damaged membranes and prevention of cellular penetration of AMPs. Significance and Impact of the Study:  To the best of our knowledge, this is the first report describing bacterial AMP resistance mechanism using proteomic methodologies. Elucidating the mechanism could help in the development of more sustainable antimicrobial agents.

The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 µg or 5 µg of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 μg or 5 μg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.