Benign by Recoding - Coronavirus Vaccines Through Genetic Deoptimization

Benign by Recoding - Coronavirus Vaccines Through Genetic Deoptimization Project Summary Respiratory viruses cause billions of infections worldwide and their spread and evolution are difficult to control with parenterally administered vaccines. Coronaviruses are among the respiratory pathogens of greatest concern given their devastating pandemic potential, against which broadly applicable vaccine strategies are needed. Applying large-scale genetic recoding, we have previously constructed a live attenuated SARS-CoV-2 vaccine with excellent preclinical safety and efficacy superior to currently used mRNA vaccines. In this vaccine, genetic recoding of the distal end of the viral ORF1ab results in a complete loss of virulence, while permitting viral replication and facilitating induction of protective mucosal immune responses, effectively preventing virus transmission. Our preliminary data suggests that genetic recoding by codon-pair deoptimization results in a benign viral phenotype, maintaining viral replication in absence of considerable pro-inflammatory host response. With this project, we aim to identify the exact mechanisms of attenuation achieved by recoding of ORF1ab in coronaviruses. We hypothesize that attenuation is not directly related to a lack of protein expression from the recoded region, but rather a result of a broader dysregulation of viral protein biosynthesis. This project will uncover the effect of genetic recoding by codon-pair-deoptimization across viral transcriptome and proteome, by applying techniques from long-read direct RNA sequencing to 5' single-cell RNA sequencing and virus proteomics. We will further exemplify the general applicability of our recoding strategy to coronaviruses by extending our work to MERS-CoV, a zoonotic respiratory pathogen of highest pandemic potential. The comparison of different degrees of recoding in MERS-CoV will allow us to define the optimal level of recoding to achieve attenuation of coronaviruses and maintain protective efficacy in vaccine candidates. Furthermore, this project is aimed at defining causes and correlates of mucosal protection conferred by locally applied live attenuated vaccines. To this end, we will employ models from rodents to human tonsil organoids to advance our understanding of adaptive mucosal immunity in response to vaccination. Taken together, this project is designed to uncover mechanisms behind an innovative approach to coronavirus vaccine design and to demonstrate the broad applicability of our concept to one of the most relevant families of zoonotic, respiratory viruses.