# Viral Escape from a Candidate HIV-1 Vaccine Targeting Protease Cleavage Sites Is Associated with a Dramatic Fitness Loss in SIVmac239-Infected Cynomolgus Macaques

## TL;DR

A new HIV-1 vaccine candidate targeting protease cleavage sites reduces SIV infection and disease progression in macaques, with escape mutations causing significant fitness loss.

## Contribution

The study demonstrates that vaccine-induced immunity at protease cleavage sites limits viral escape and prevents fitness recovery in breakthrough viruses.

## Key findings

- Viral escape mutations in protease cleavage sites are associated with reduced viral load and preserved CD4+ T cell counts.
- Mutant viruses show impaired proteolytic Gag processing, reduced RNA incorporation, and poor replication in primary cells.
- Downstream mutations partially compensate for PCS2 defects but fail to restore full replicative fitness.

## Abstract

A novel HIV-1 vaccine candidate under development targeting the highly conserved protease cleavage regions reduced viral acquisition and delayed disease progression in a macaque SIV-challenge model. Breakthrough virus isolated from vaccinees and control animals were sequenced in the regions surrounding the SIV protease cleavages. We identified unique viral mutations that were associated with alterations in viral load and maintenance of CD4+ T cell counts in vaccinees. To evaluate whether the vaccine-elicited mutations were detrimental to virus fitness, we produced 11 mutant constructs and transfection-derived viral stocks harboring mutations in both PCS2 (in CA/p2) and PCS12 (in Nef) that had emerged at high frequency during breakthrough viremia. Virus preparations harboring mutations displayed impaired proteolytic Gag processing, reduced viral RNA incorporation and p27-CA content. These mutants were also compromised in their ability to replicate in primary cells and cell lines. Interestingly, we observed only partial compensation of these PCS2 defects by downstream mutation at PCS12. In sum, we demonstrate that vaccine-elicited immunity directed to viral protease cleavage regions impair viral escape, and breakthrough virus cannot easily restore replicative fitness.