CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

561

In-Depth Evaluation of the Intact Proviral DNA Assay for the Quantification of HIV-1 Reservoir Thuy Nguyen 1 , Mary Zipparo 2 , Lindsey Adams 2 , Annie Glassey 1 , Afra Rahman 1 , Ulisses Santamaria 3 , Catherine Rehm 2 , Jessica Earhart 2 , Chuen-Yen Lau 2 , Frank Maldarelli 2 1 National Cancer Institute at Frederick, Frederick, MD, USA, 2 National Institutes of Health, Bethesda, MD, USA, 3 Leidos Biomedical Research, Inc, Frederick, MD, USA Background: Accurate quantification of the HIV-1 reservoir is critical in understanding HIV-1 persistence and evaluating cure strategies. The Intact Proviral DNA Assay (IPDA), a droplet digital PCR assay that simultaneously targets the HIV-1 packaging signal (psi) and env , is widely used to quantify the intact or rebound-competent reservoir but has not been thoroughly evaluated. We assessed its precision in distinguishing intact from defective proviruses using genetically characterized single genome sequences (SGS). Methods: gDNA was extracted from the peripheral blood of 16 persons with HIV (PWH) at pretherapy (n=10) and multiple time points on ART (n=29). Single genome proviruses were obtained via limiting dilution and amplified using primers targeting the 5’ and 3’ LTR. Only near-full-length proviruses (NFL,>7kb) were sequenced and classified as genetically intact or defective. Corresponding >7kb single genomes were submitted to real-time amplification (qPCR) using IPDA primers/probes. A subset of proviruses < 7kb was not sequenced but submitted for IPDA qPCR. Results: For 13/16 PWH with successful IPDA, we obtained a total of 5782 proviruses by amplification and 1091 NFL SGS >7kb (479 intact and 612 defective) by sequencing. Per total amplified proviruses, a median (range) of 4.2% (0-14.8) of NFL defective proviruses at pretherapy was misclassified as intact while it was significantly lower during ART at 1.8% (0-9.4) (p<0.001). Medians (range) of 0% (0-23.3) intact proviruses at pretherapy and 0% (0-7.7) during ART were not detected by IPDA. Analysis of 158 defective misclassified as intact by IPDA showed mutations inside the psi (15.8%) or deletion/stop codons outside IPDA binding sites (19.6% in gag , 24.7% in pol , 29.7% in env ). In one PWH, 50% of the intact populations harbored mutants in the psi leading to misclassification of these intact as defective. In 350 defective proviruses <7kb obtained on ART that were not sequenced, 31 (8.8%) were misclassified as intact with a median (range) of 5.5% (2.0-27.3) per sample. Conclusions: IPDA is generally accurate in distinguishing intact from defective, but the error rate significantly varies from pretherapy to ART and across PWH. Of note, intact proviruses may heterogeneously harbor either matches or mismatches in the assay binding sites and still lead to a successful assay but erroneous quantification. Careful interpretation of IPDA results is essential, especially in evaluating interventions with minor changes in the intact proviral level. Deciphering the HIV-2 Proviral Reservoir in Antiretroviral-Naive Individuals Fella Mazouz, Romane Guilbaud, Quentin Le Hingrat, Florence Damond, Thibault Saint-Joannis, Eddy Biesaga, Romain Coppee, Diane Descamps, Jade Ghosn, Charlotte Charpentier Hôpital Bichat-Claude-Bernard, Paris, France Background: HIV-2 infection is considered as an attenuated viral infection. PBMC HIV-2 reservoir has been shown to be smaller than that of HIV-1 with a higher proportion of APOBEC3-induced defective proviruses. The aim of this study was to characterise the composition of the HIV-2 cellular reservoir by identifying the different forms of defective proviruses. Methods: ARV-naive participants were recruited from the ANRS national HIV-2 cohort during 2020. Sequencing of protease, RT and integrase was performed using GridION (Oxford Nanopore Technologies, detection threshold: 5%). We considered all hypermutated pol sequences and those containing a stop codon as defective. We set up a specific HIV-2 Intact Proviral DNA Assay (IPDA), for both HIV-2 A and B groups, targeting the Psi and env to quantify 5’- and 3’-defective (due to large internal deletions), env APOBEC3-hypermutated and intact proviral DNA. Results: Fifty-seven participants were recruited (median age: 56 years, 61% female), all with spontaneously undetectable viral load (<40 c/mL) except two (<200 c/mL). Among the 32 participants with successful prot-RT amplification, the median ratio of APOBEC3-induced mutations to the number of potential APOBEC3 mutation sites was significantly higher in RT than in protease (18% [IQR=17%-21%] vs. 11% [IQR=6%-16%], respectively; p=0.0001). Overall, five (16%) participants had APOBEC3-induced defective proviruses due to the

presence of either hypermutations or stop codons in prot-RT. Of the 20 integrase sequences generated, only one participant was found to have APOBEC3 induced defective sequences. Of the 30 participants with IPDA results, 19 (63%) harboured env-hypermutated proviruses, 15 (50%) harboured proviruses defective at either the 5' or 3' end. Intact proviruses were detected in only one participant. Median HIV-2 total DNA level was 303 c/10 6 PBMC (IQR=189 764) consisting on average of 12% 3’-defective proviruses, 19% 5’-defective proviruses and 67% env-hypermutated proviruses. Conclusions: In this cross-sectional analysis, we were able to describe the composition of part of the HIV-2 viral reservoir. This study describes for the first time the proportion of defective and intact HIV-2 proviruses within the viral reservoir and shows that a very small proportion of ARV-naïve individuals harbours intact proviruses and that most of the proviruses constituting the reservoir are hypermutated or carry large internal deletions. These findings are consistent with the attenuated profile of HIV-2 infection. CCR5 Expression Is Critical for the Maintenance of HIV Control and Reservoir Size Jéssica dos Santos 1 , Zhenhua Zhang 2 , Adriana Navas 1 , Louise E. van Eekeren 1 , Mareva Delporte 3 , Hanneke Maas 1 , Vasiliki Matzaraki 1 , Albert L. Groenendijk 4 , Wilhelm A. Vos 5 , Hans Koenen 1 , Jan van Lunzen 1 , Mihai G. Netea 1 , Linos Vandekerckhove 3 , Yang Li 2 , Andre van der Ven 1 , for the 2000HIV Human Functional Genomics Partnership Program 1 Radboud University Medical Center, Nijmegen, Netherlands, 2 Hannover Medical School, Hannover, Germany, 3 HIV Cure Research Center, Ghent University, Ghent, Belgium, 4 Erasmus University Medical Center, Rotterdam, Netherlands, 5 OLVG, Amsterdam, Netherlands Background: Natural protection against CCR5-tropic HIV-1 variants is attributed to the presence of CCR5Δ32. We aimed to identify genetic and non-genetics and factors influencing CCR5 expression on CD4 + and CD8 + T cells and to assess the role of CCR5 expression in maintaining HIV control status and reservoir size in people living with HIV (PLHIV). Methods: CCR5 expression on CD4 + and CD8 + T cell subsets was assessed using flow cytometry in PLHIV from the 2000HIV study (NTC03994835), divided into a discovery (n=1147) and validation (n=225) cohort. Part of inclusion were 114 HIV controllers (Elite controller (EC), viremic, (VC) and transient). The relationship of CCR5 expression with HIV- and non-HIV factors were assessed using Spearman’s rank correlation analysis. Genome-wide SNPs associated with CCR5 expression on CD4 + and CD8 + T cell subsets were analyzed using PLINK, with adjustments for age and gender. Significant associations were defined by a p-value < 5.32e-10 after multiple testing in the discovery cohort and a nominal P < 0.05 in the validation cohort. CCR5Δ32 was evaluated using RT-PCR. Viral reservoir size, measured as DNA copies/million CD4 + T cells, were also assessed. Results: Percentages of CD4 + T cells subsets expressing CCR5 was positively correlated with age and CMV antibody titers and negatively with female sex and CD4 nadir. Fewer factors influenced CCR5 expression on CD8 + T cells subsets, with positive associations observed for age, CMV titers and ART duration (FDR < 0.05, discovery; p < 0.05, validation). In the 2000HIV study, 14.7% of the participants were heterozygous for the CCR5Δ32, with a higher prevalence in EC (25%) and VC (35.1%) than in transient (16.6%) and non-controllers (14.03%). The percentages of naïve and T regulatory cells expressing CCR5 was significantly lower in EC and VC than non-controllers. Genome-wide association analysis identified that rs113341849-A, located upstream CCR5 and CCR2 , which co-occurs with CCR5Δ32, is linked to reduced CCR5 expression, both in terms of percentages and MFI, in a cell-type specific manner (Figure 1A). Finally, the presence of rs113341849-A and CCR5Δ32 was linked to a lower HIV reservoir size (Figure 1B) (p < 0.05 in both discovery and validation cohorts). Conclusions: Our study shows that genetic and non-genetic factors contribute to CCR5 expression and subsequently to the maintenance of viral control and reservoir size. CCR5 blocking may be especially effective in older PLHIV, males and those with higher CMV reactivity.

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Poster Abstracts

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CROI 2025 148