Analysis of temporal virus evolution and intra-host diversity in long-term non-progressors by bulk next-generation sequencing

Long-term non-progressors (LTNPs) provide key insights into HIV-1 pathogenesis and potential functional cure strategies. Whether viral evolution persists in LTNPs remains debated. Previous studies have mainly focused on partial HIV-1 genome fragments using single-gene amplification. In contrast, our study employs near full-length bulk next-generation sequencing (NGS) to investigate viral quasispecies evolution, diversity, and CTL-associated mutations at the nucleotide level, offering new perspectives on intra-host viral dynamics. Blood samples of two LTNPs were collected at four time points over a approximately 2 years. The HIV-1 near full-length genome of plasma RNA and proviral DNA were amplified, and bulk NGS was performed. Consensus sequences were used for phylogenetic analysis and divergence calculation. Intra-host single-nucleotide variants (iSNVs), amino acid variations, and mutations in CTL epitopes were also analyzed. Both LTNPs displayed limited evolution over time, as identified from the phylogenetic tree and genetic distance, with higher divergence observed in DNA sequences than in RNA sequences. Evolutionary divergence was observed between HIV-1 plasma RNA and proviral DNA sequences. Intra-host diversity indices, including the number of iSNVs and Shannon entropy, also indicated greater diversity in DNA sequences than in RNA sequences. A considerable number and high frequency of mutations in CTL were identified consistently both in DNA and in RNA sequences, which showed slight frequency changes over time. These findings support the limit HIV-1 evolution in LTNPs and greater intra-host diversity of proviral DNA than plasma RNA. Our findings also suggest substantial, high-frequency, and sustained mutations in CTL epitopes in LTNPs with a relatively high level of viral replication despite optimal immune control.IMPORTANCELong-term non-progressors (LTNPs) are a rare group of HIV-1-infected individuals who maintain stable CD4⁺ T cell counts and low viral loads in the absence of antiretroviral therapy. Understanding the virological and immunological mechanisms underpinning this phenotype could inform strategies for functional cure. Using bulk next-generation sequencing of near full-length genomes of both HIV plasma RNA and proviral DNA over multiple time points, our study provides high-resolution insights into intra-host viral evolution in LTNPs. We reveal limited viral evolution and divergence between plasma RNA and proviral DNA. Higher diversity was found in proviral DNA than in plasma RNA, contributed by a substantial number of low-frequency mutations. HLA-restricted CTL escape mutations were stable and maintained at high frequency across all time points. These results challenge the notion of viral stasis in LTNPs and underscore the complex interplay between viral persistence and immune control, offering critical clues for vaccine design and reservoir-targeting therapeutic approaches. These findings support the hypothesis that LTNPs maintain durable viral control and non-progressive disease by achieving a balance between viral evolution and immune containment.