Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors.

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels.

Evaluation of SARS-CoV-2 neutralization assays for antibody monitoring in natural infection and vaccine trials.

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays to be utilized in concert with clinical trials to establish correlates of risk and protection. This need has led to the appearance of a variety of neutralization assays used by different laboratories and companies. Using plasma samples from COVID-19 convalescent individuals with mild-to-moderate disease from a localized outbreak in a single region of the western US, we compared three platforms for SARS-CoV-2 neutralization: assay with live SARS-CoV-2, pseudovirus assay utilizing lentiviral (LV) and vesicular stomatitis virus (VSV) packaging, and a surrogate ELISA test.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs.