Deep mutationally scanned (DMS) CHIKV E3/E2 virus library maps viral amino acid preferences and predicts viral escape mutants of neutralizing CHIKV antibodies

Abstract

As outbreaks of chikungunya virus (CHIKV), a mosquito-borne alphavirus, continue to present public health challenges, additional research is needed to generate protective and safe vaccines and effective therapeutics. Prior research has established a role for antibodies in mediating protection against CHIKV infection, and the early appearance of CHIKV-specific IgG or IgG neutralizing antibodies protects against progression to chronic CHIKV disease in humans. However, the importance of epitope specificity for these protective antibodies and how skewed responses contribute to development of acute and chronic CHIKV-associated joint disease remains poorly understood. Here, we describe the deep mutational scanning of one of the dominant targets of neutralizing antibodies during CHIKV infection, the E3/E2 (also known as p62) glycoprotein complex, to simultaneously test thousands of p62 mutants against selective pressures of interest in a high throughput manner. Characterization of the virus library revealed achievement of high diversity while also selecting out non-functional virus variants. Furthermore, this study provides evidence that this virus library system can comprehensively map sites critical for the neutralization function of antibodies of both known and unknown p62 domain specificities. IMPORTANCEChikungunya virus (CHIKV) is a mosquito-borne alphavirus and re-emerging pathogen of global health concern that causes fever and debilitating acute and chronic joint pain. Prior studies established a critical role for antibodies in protection against CHIKV infection, however, the importance of different antibody epitopes engaged on the virus for these functions is poorly understood. Here, we describe the generation of a high-throughput, functional virus library capable of simultaneously identifying critical neutralization sites for multiple antibodies. We find that, through generation of the virus library, the plasticity of the viral glycoproteins tested varied by subdomains. Additionally, our study revealed new sites in the major CHIKV surface glycoprotein important for neutralization by previously proposed therapeutic monoclonal antibodies. Overall, this study describes a new tool that can be used to better understand antibody responses associated with distinct CHIKV infection outcomes and could contribute to the development of efficacious vaccines and antibody-based therapeutics.