Successfully combating the COVID-19 pandemic depends on mass vaccination with suitable vaccines to achieve herd immunity. Here, we describe COVI-VAC, the only live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine currently in clinical development. COVI-VAC was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon-pair deoptimization), thereby introducing 283 silent (point) mutations. In addition, the furin cleavage site within the spike protein was deleted from the viral genome for added safety of the vaccine strain. Except for the furin cleavage site deletion, the COVI-VAC and parental SARS-CoV-2 amino acid sequences are identical, ensuring that all viral proteins can engage with the host immune system of vaccine recipients. COVI-VAC was temperature sensitive in vitro yet grew robustly (>107 plaque forming units/mL) at the permissive temperature. Tissue viral loads were consistently lower, lung pathology milder, and weight loss reduced in Syrian golden hamsters (Mesocricetus auratus) vaccinated intranasally with COVI-VAC compared to those inoculated with wild-type (WT) virus. COVI-VAC inoculation generated spike IgG antibody levels and plaque reduction neutralization titers similar to those in hamsters inoculated with WT virus. Upon challenge with WT virus, COVI-VAC vaccination reduced lung challenge viral titers, resulted in undetectable virus in the brain, and protected hamsters from almost all SARS-CoV-2–associated weight loss. Highly attenuated COVI-VAC is protective at a single intranasal dose in a relevant in vivo model. This, coupled with its large-scale manufacturing potential, supports its potential use in mass vaccination programs.

Summary The fla gene locus of Methanococcus voltae encodes the major structural components of the flagellum as well as other flagellar‐related proteins. The flaHIJ genes have been found in all flagellated archaea, suggesting a central role in flagella biogenesis. FlaI shares similarity with the type II and type IV secretion NTPases (such as PilB, VirB11 and TadA), and FlaJ exhibits similarity to putative bacterial integral membrane proteins involved in type IV pilus biogenesis such as TadB. In this study, reverse transcription polymerase chain reaction (RT‐PCR) and Northern blotting data revealed that flaHIJ are co‐transcribed with the upstream structural flagellin genes, thus demonstrating the expression of the entire fla gene cluster in vivo. Non‐polar mutants in flaI and flaJ of M. voltae were isolated using insertional inactivation via a novel mutagenic vector. These mutants were non‐motile and non‐flagellated by microscopy, demonstrating the involvement of FlaI and FlaJ in flagella biogenesis. Interestingly, all the mutants maintained the ability to produce and localize flagellins to the cytoplasmic membrane. Amino‐terminal sequencing of flagellins produced by the flaJ mutant strain revealed that the flagellins did not have their cognate leader peptides, thus indicating that preflagellin processing had occurred in vivo. This result was confirmed using an in vitro processing assay. The fla− phenotype and protein secretion characteristics of the flaI and flaJ mutants therefore implicate these respective genes in archaeal flagellin secretion and assembly. These findings further support a model describing the archaeal flagellum as a novel prokaryotic motility structure.

Rapid, yet accurate and sensitive testing has been shown to be critical in the control of spreading pandemic diseases such as COVID-19. Current methods which are highly sensitive and can differentiate different strains are slow and cannot be conveniently applied at the point of care. Rapid tests, meanwhile, require a high titer and are not sufficiently sensitive to discriminate between strains. Here, we report a rapid and facile potentiometric detection method based on nanoscale, three-dimensional molecular imprints of analytes on a self-assembled monolayer (SAM), which can deliver analyte-specific detection of both whole virions and isolated proteins in microliter amounts of bodily fluids within minutes. The detection substrate with nanoscale inverse surface patterns of analytes formed by a SAM identifies a target analyte by recognizing its surface nano- and molecular structures, which can be monitored by temporal measurement of the change in substrate open-circuit potential. The sensor unambiguously detected and differentiated H1N1 and H3N2 influenza A virions as well as the spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle-East respiratory syndrome (MERS) coronavirus in human saliva with limits of detection reaching 200 PFU/mL and 100 pg/mL for the viral particles and spike proteins, respectively. The demonstrated speed and specificity of detection, combined with a low required sample volume, high sensitivity, ease of potentiometric measurement, and simple sample collection and preparation, suggest that the technique can be used as a highly effective point-of-care diagnostic platform for a fast, accurate, and specific detection of various viral pathogens and their variants.

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that “synonymous codon deoptimization” could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.

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