Background
HIV-1 tropism, i.e., the use of CCR5 (R5), CXCR4 (X4), or both co-receptors for cell entry may shape disease progression, immune recovery, and reservoir dynamics. We profiled tropism-specific clinical, virological, and immunological features in 1895 virally suppressed people living with HIV from the Dutch 2000HIV study, divided into a discovery and validation cohort.
Methods
We performed nested PCR to amplify env (~1.1 kb) followed by Sanger sequencing in triplicate (n=1,887). Viral tropism was inferred from V3 loops using Geno2pheno (10% FDR). Cell-associated DNA and RNA were quantified via Rainbow dPCR and multiplexed dPCR, respectively. Multi-omics profiling of PBMCs included flow cytometry (analyzed by linear model) and bulk RNA-seq (analyzed with DESeq2), and plasma proteomics was performed using Olink® Explore 3072 (analyzed with limma). Analyses were stratified by discovery (FDR <0.05) and validation (p <0.05) cohorts, adjusting for demographic and relevant covariates. Gene Set Enrichment Analysis was performed for plasma proteome, with proteins ranked based on t_stat.
Results
R5-tropic viruses were found in 1,222 participants; 336 had X4-tropic and 166 had mixed-tropic viruses. Independent of time between infection and ART, X4 group had more severe disease before ART initiation (higher odds of CDC3). Regardless of CD4 counts before ART initiation, X4 group were more commonly immunological non-responders, and CD4 recovery after 1 and 2 years of ART was lower. Viral reservoir (DNA and RNA) was larger in the X4 group. Multivariate analysis showed more diabetes II and psychiatric diagnoses along with antipsychotic and benzodiazepine use (Figure 1). Flow cytometry showed increased frequencies of HLA-DR⁺CD38⁺ CD4⁺ T cells and PD-1⁺ regulatory T cells in the X4 group, aligning with plasma proteomics revealing heightened systemic immune activation. Transcriptomics analysis showed downregulation of TSHZ2 and FAAH2 in X4 group.
Conclusions
X4-tropic HIV establishes larger reservoirs, faster disease progression before ART, poorer CD4 cell response after ART and more psychiatric disorders and diabetes II. The latter may relate to persistent immune activation, as observed through immunophenotyping and proteome analysis. The link of X4 with psychiatric disorders should be further studied, especially as impairment of FAAH2 is reported to contribute to brain dysfunction.
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