EPIICAL

Authors: De Rossi A, Dalzini A, Ballin G, Dominguez-Rodriuguez S, Rojo P, Foster C, Palma P, Sessa L, Nastouli E, Pahwa S, Rossi P, Giaquinto C, EPIICAL Consortium

Published in: 23rd International AIDS Conference, July 6th-10th, 2020

Background: HIV infection is linked to premature senescence, with increased risk of aging-associated illnesses. Early ART has been associated with a reduced HIV reservoir in HIV-perinatally infected children (PHIV), but its impact on the senescence process is an open question. Telomeres are critical for cellular replicative potential and their shortening is a marker of cellular senescence and aging process. We investigated the relationship between immunosenescence and HIV reservoir in PHIV enrolled in a multicenter cross-sectional study (CARMA, EPIICAL consortium).

Methods: 37 PHIV, who started ART <2 years of age and had undetectable viremia for at least 5 years, were enrolled in this study. HIV-DNA copies on CD4 cells and relative telomere length and levels of T-cell receptor rearrangement excision circle (TREC, marker of thymic output) on CD4 and CD8 cells were quantified by qPCR. Senescent and activated CD4 and CD8 cells were estimated by flow cytometry. To explore the associations between cellular parameters, HIV reservoir and age at ART initiation, data were analyzed using a multivariable Poisson regression (adjusted for baseline % CD4, plasmaviremia, age at reservoir measurement, and age at ART initiation as interaction term).

Results: HIV reservoir was significantly (p<0.001) associated with immunosenescence (1.23[1.21-1.26]) and telomere shortening (0.15[0.13-0.17]) in CD4 cells, and immune activation (3.67[3.49-3.85]) and TREC levels (1.08[1.06-1.11]) in CD8 cells. These associations decreased by 1%, 10%, 6% and 6%, respectively, for each month ART was delayed. Early treated PHIV (ART initiation ≤6 months of age) displayed significantly lower HIV-DNA level (89[56-365] vs 552[303-1001] copies/10⁶ cells)  and % CD4 senescent cells (1.0[0.5-2.7] vs 2.9[2.0-6.3]) than late treated ones.

Authors: Ruggiero A, Cotugno N, Domínguez-Rodríguez S,  Zicari S, Rinaldi S, Zangari P, Tagarro A, Foster C, De Rossi A, Nastouli E, Luzuriaga K, Giaquinto C, Rossi P, Pawha S, Palma P, on behalf of the EPIICAL consortium.

Published in: 23rd Virtual International AIDS Conference, July 6th-10th, 2020

Background The role of T-bet, an immune factor involved in adaptative and innate response, has been poorly explored in B-cells. Previous studies concentrated on HIV+ and HIV- adults and T-bet was found to be associated with ABC. This work characterizes T-bet expression in B-cell (CD19+CD10-) subsets associated with aging (activated memory AM CD21-CD27+, Tissue Like Memory TLM CD21-CD27- and Double Negative DN CD27-IgD-) and with immunological memory (Resting Memory, RM CD27+CD21+IgD-) in PHIV.

Methods We studied 40 PHIV starting ART at median 4m of age (min 0m, max 22m) and ART-suppressed for >5 years (median 14 years (y) (min 5y, max 22y). Flow Cytometry was used to define B-cell phenotype and intracellular T-bet (MFI) in PHIV and in 20 age and gender-matched controls (HC). Anti-HIV serology was measured using Western blot and ELISA. Comparisons were analysed using Mann-Whitney test (MW). Associations were explored using Spearman test (rho, p) and multivariable ridge Poisson Regression model including baseline CD4, gender, and age at ART as confounders.

Results DN were expanded in PHIV compared with HC (p=0.01 MW). T-bet levels were elevated in AM, TLM and marginally DN compared to the others.  The models demonstrated a strong association between T-bet levels and time of ART start: for each month without ART T-bet increased by 3% and 2% in DN and AM, respectively (Fig.1a). Anti-Env responses were positively associated and were identified as predictors of T-bet levels in IgG+ B-cells (rho=0.35, p=0.03, Fig 1b), IgM+ B-cells (rho=0.39, p=0.01), RM IgM+ (rho=0.33, p=0.042).

Conclusions ABC are expanded in PHIV compared with HC, despite suppressive ART. Earlier ART-start preserves from premature aging of B-cell compartment. Furthermore, elevated levels of T-bet in memory B-cells was associated with anti-Env responses. Our findings add onto the previous literature by suggesting a role of T-bet in the anti-HIV B-cell response that warrant further investigation.

Authors: Domínguez-Rodríguez S, Tagarro A, Serna Pascual M, Otwombe K, Violari A, Fernández S, Nhampossa T, Lain M,  Vaz P , Behuhuma NO, Danaviah S, Dobbels E, Barnabas S, Cotugno N, Zangari P, Palma P, Oletto A,  Nardone A, Nastouli E, Spyer M, Kuhn L, Rossi P, Giaquinto C, Rojo P on behalf of EPIICAL consortium.

Published in: 23rd international AIDS Conference, July 6th-10th 2020

Background In perinatally HIV infected children, mortality and morbidity are highest in the first months after ART initiation and is linked to advanced disease and late diagnosis. The random forest approach can deal with more predictors than classical models and has no model assumptions such as normality, linearity or hazard proportionality. The aim of this study was to predict the probability of death or clinical progression at a specific time of follow-up.

Methods EARTH (EPIICAL consortium) is an African multi-centre cohort enrolling HIV-infected infants treated within 3 months of life (n=151). A total of 134 infants with >1 follow-up visit were included in this analysis. The primary endpoint was the right-censored time to death or progression to AIDS. To predict the outcome, a log-rank random survival forest with imbalance correction was performed in a training subset (n=95, 70%). The algorithm was validated on the remaining 30% (n=39).

Results A total of 22 infants reached the primary endpoint with 13 (10%) patients dead and 9 (7%) with an AIDS defining condition. A total of 10000 trees were built with an error rate of 20%. The most important predictors of reaching the primary endpoint were baseline HIV viral load, age at diagnosis, weight-for-age, gender, age at ART initiation, and baseline CD4 count. In the validation, the model predicted a higher probability of reaching the primary endpoint among children who did indeed die or progress to AIDS, as compared to the group of children who did well (1-month: 14% vs. 0.01%, p-value=0.045; 6-months: 62% vs. 0.03%, p-value=0.019; 12-months: 76% vs. 16%, p-value=0.012). The AUC for predicting survival or progression was 0.83, 0.84, and 0.72 for 1-month, 6-months, and 1-year respectively.

Conclusions This model helps clinicians individualize the probability of death or progression to AIDS at time of diagnosis and may be useful for the early identification of high-risk patients.

Authors: Palma P, McManus M, Cotugno N, Rocca S, Rossi P, Luzuriaga K

Published in: Lancet HIV. 2020;7(5):e359-e365

Abstract Several assays have been developed to measure and characterise the replication-competent HIV-1 reservoir, which constitutes the barrier to cure. To date, the application of these assays to studies in children and in limited-resource settings has been minimal, primarily because of their expense, the large required blood volumes, and labour-intensive technologies. For children vertically infected with HIV-1 who initiated suppressive antiretroviral therapy (ART) regimens in infancy, HIV-1-specific antibody concentrations are associated with viral persistence and could be used to estimate the size of the residual latent reservoir on ART. This strategy could be particularly useful for screening children on suppressive ART for enrolment into therapeutic vaccine trials and other protocols aimed at achieving HIV-1 remission.

Authors: Dhummakupt A, Rubens JH, Andeson T, et al.

Published in: JCI Insight. 2020;5(4)

Abstract The HIV latent reservoir in resting memory CD4+ T cells precludes cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not yet in pediatric populations. We determined, ex vivo, the inducibility of the latent reservoir in perinatal infection as compared with adult infections using the Tat/rev induced limiting dilution assay (TILDA), in which a single round (12 hours) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Although rates of T cell activation with PMA/ionomycin were similar, the latent reservoir in perinatal infection was slower to reactivate and of lower magnitude compared with adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and a duration of 18 hours augmented proviral expression in perinatal but not adult infection. The baseline HLA-DR+CD4+ T cell level was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support the hypothesis that there are differences in kinetics of latency reversal and baseline immune activation in perinatal compared with adult infections, with implications for latency reversal strategies toward reservoir clearance and remission.

Authors: Dhummakupt A, Reubens JH, Anderson T, et al.

Published: Poster presented at 27th Conference on Retroviruses and Opportunistic Infections, March 8th –11th, 2020 – Boston.

Abstract The HIV latent reservoir in resting memory CD4+ T cells prevents cure. Novel therapies to reactivate and eliminate the reservoir are in clinical trials in adults, but not yet in pediatric populations.

HIV proviral reservoir size was determined in perinatal (N=11) and adult infections (N=10) by digital droplet PCR (ddPCR) and with the intact proviral DNA assay (IPDA) in perinatal samples. The inducibility of the latent reservoir was determined with the Tat/rev Induced Limiting Dilution Assay (TILDA) that uses single-round (12 hr) T cell stimulation of CD4+ T cells with PMA/ ionomycin to maximally activate cells to induce proviral expression, measured as multiply-spliced HIV RNA Units per 10⁶ CD4 cells (msRUPM). Markers of immune activation (CD69, CD25 and HLA-DR) and exhaustion (PD-1, TIM-3 and TIGIT) were also assessed. An enhanced TILDA with addition of PHA and for 18 hours was performed to enhance proviral expression in perinatal infections. Non-parametric tests were used for differences between paired and unpaired measurements; correlations were quantified by Spearman rank coefficient.

The median age was 15.8 yrs with a median duration of suppression of 6.7 yrs for perinatal infections, and 40.5 yrs with a median duration of suppression of 7.3 yrs for adult infections.  We found that despite a higher proviral reservoir size (median 132.1 vs. 66.7 c/10⁶ PBMCs) and similar rates of T cell activation with PMA/ionomycin (median %CD69 = 96.7% and 93.0%) in perinatal and adult infections, respectively, the size of the induced reservoir was significantly lower in perinatal than in adult infections (median msRUPM of 2.99 vs 11.92, p=0.020). With the enhanced TILDA, the size of the induced reservoir increased significantly in perinatal infections (1.5-fold to a median of 4.5 msRUPM; p=0.034), but not in adult infections. The proportion of induced provirus was significantly lower in perinatal infections at 1.6% compared with 4.0% in adult infections (p=0.030). At baseline, the proportion of HLA-DR+ T cells was significantly lower in perinatal compared with adult infections (median HLA-DR+ cells = 4.56% vs 10.5%, p=0.012), but not correlated with the induced reservoir size.

The inducibility of the latent reservoir is substantially lower in perinatal compared with adult infections, possibly due to differences in baseline states of immune activation, with implications for latency reversal strategies towards ART-free remission.

Authors: Schröter J, Anelone A,  Yates A, de Boer RJ;  on behalf of the EPIICAL Consortium

Published in: J Acquir Immune Defic Syndr. 2020;83(5):522-529

Background Interventions aiming for an HIV cure would benefit from rapid elimination of virus after the onset of antiretroviral therapy (ART), by keeping the latent HIV reservoir small.

Setting We investigated HIV suppression in 312 perinatally infected infants starting ART within 6 months after birth from the EPPICC (European Pregnancy and Paediatric HIV Cohort Collaboration).

Methods To better understand kinetic differences in HIV suppression among infants, we investigated their individual viral load (VL) decay dynamics. We identified VL decay patterns and determined times to viral suppression (TTS). For infants with strictly declining VLs (n = 188), we used parameter fitting methods to estimate baseline VLs, decay rates, and TTS. We subsequently identified the parameters determining TTS by linear modeling.

Results The majority of infants suppress HIV VL after the onset of ART. Some children experienced a long TTS due to an “erratic” VL decay pattern. We cannot exclude that this is partly due to treatment complications and subsequent treatment changes, but these children were characterized by significantly lower CD4 percentages (CD4%) at start of treatment compared with those with a “clean” VL decline. Focusing on this “clean” subset, the TTS could be predicted by mathematical modeling, and we identified baseline VL and CD4% as the major factors determining the TTS.

Conclusions As VL steeply increases and CD4% constantly decreases in untreated HIV-infected infants, the progression of an HIV infection is largely determined by these 2 factors. To prevent a further disease progression, treatment should be initiated early after contracting HIV, which consequently shortens TTS.

Authors: Dominguez-Rodriguez S, Rojo P, Lain MG,  Barnabas S, Lopez-Varela E, Otwombe K, Danaviah S, Nastouli E, Serna-Pasqual M, Gianuzzi V, Giaquointo C, Kuhn L, Tagarro A on behalf of the EPIICAL Consortium

Published in: 27th Conference on Retroviruses and Opportunistic Infections, March 8th –11th, 2020 – Boston.