# Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages

## TL;DR

This study explores how adeno-associated viruses (AAVs) evolved by identifying positively selected sites in their capsid genes and linking them to immune evasion and host interactions.

## Contribution

The study experimentally links specific AAV2 capsid mutations to immune evasion and provides new insights into AAV evolution in natural hosts.

## Key findings

- Site 548 of AAV2 capsid is under positive selection due to host humoral immunity and affects immune evasion from neutralizing antibodies.
- Site 410 of AAV2 capsid is not driven by selection pressure from human CD4+ T-cell responses.
- Evolutionary analysis identified multiple positively selected sites across AAV lineages.

## Abstract

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.

Adeno-associated virus (AAV) is a viral vector that is universally applied in human gene therapy. Despite their versatile applications in gene therapy, the origin and evolution of AAV serotypes have not been extensively studied. In this study, positive selection upon diversification of AAV capsid genes was carefully investigated by evolutionary analysis. Numerous diversifying selected sites were dispersed among the AAV lineages, and evolutionary pathways were inferred for two selected sites of the AAV2 capsid gene. Furthermore, the putative immunological drives underlying the selection of AAV2 capsid genes were experimentally dissected. This work provides a novel interpretation of the evolution and diversity of AAV serotypes and might be helpful for their engineering and application in gene therapy.