Jacob Hurst

HIV-1 DNA predicts disease progression and post-treatment virological control

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001

Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain’

Highlights • The requirement for protocol adaptation from qPCR to ddPCR is characterised.• A potential loss of sensitivity for ddPCR at low target numbers is reported.• A new bioinformatic tool ‘definetherain’ to improve droplet calling at low input target numbers is devised and applied.• ‘Definetherain’ is made available free of charge and open access at www.definetherain.org.uk.

Additive effects of HLA alleles and innate immune genes determine viral outcome in HCV infection

Background Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. Objective This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. Design We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. Results Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 ‘T’ allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome. Conclusions This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response.

Nonprogressing HIV-infected children share fundamental immunological features of nonpathogenic SIV infection

Pediatric HIV patients who do not rapidly develop AIDS are shown to have immunological features similar to nonhuman primates that experience nonpathogenic SIV infection. HIV progression at a standstill Although most people that get infected with HIV develop AIDS, rare individuals maintain immune function in the presence of virus, a phenomenon also seen in natural hosts of the closely related SIV. Muenchhoff et al. describe a cohort of pediatric HIV patients who have normal CD4 T cell counts, despite high viremia and lack of antiviral treatment. These children have low immune activation, including less chemokine receptor CCR5 expression on central memory CD4 T cells, similar to sooty mangabeys infected with SIV. The immune mechanisms described in these patients shed light on HIV pathogenesis, which may help develop future treatments. Disease-free infection in HIV-infected adults is associated with human leukocyte antigen–mediated suppression of viremia, whereas in the sooty mangabey and other healthy natural hosts of simian immunodeficiency virus (SIV), viral replication continues unabated. To better understand factors preventing HIV disease, we investigated pediatric infection, where AIDS typically develops more rapidly than in adults. Among 170 nonprogressing antiretroviral therapy–naïve children aged >5 years maintaining normal-for-age CD4 T cell counts, immune activation levels were low despite high viremia (median, 26,000 copies/ml). Potent, broadly neutralizing antibody responses in most of the subjects and strong virus-specific T cell activity were present but did not drive pediatric nonprogression. However, reduced CCR5 expression and low HIV infection in long-lived central memory CD4 T cells were observed in pediatric nonprogressors. These children therefore express two cardinal immunological features of nonpathogenic SIV infection in sooty mangabeys—low immune activation despite high viremia and low CCR5 expression on long-lived central memory CD4 T cells—suggesting closer similarities with nonpathogenetic mechanisms evolved over thousands of years in natural SIV hosts than those operating in HIV-infected adults.

Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection.

The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches.

Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression.

HIV-1-specific CD4(+) responses in primary HIV-1 infection predict disease progression.

Objectives:Immune factors determining clinical progression following HIV-1 infection remain unclear. The SPARTAC trial randomized 366 participants in primary HIV infection (PHI) to different short-course therapies. The aim of this study was to investigate how early immune responses in PHI impacted clinical progression in SPARTAC. Design and methods:Participants with PHI recruited to the SPARTAC trial were sampled at enrolment, prior to commencing any therapy. HIV-1-specific CD4+ and CD8+ ELISpot responses were measured by gamma interferon ELISPOT. Immunological data were associated with baseline covariates and times to clinical progression using logistic regression, Kaplan–Meier plots, and Cox models. Results:Making a CD4+ T-cell ELISpot response (n = 119) at enrolment was associated with higher CD4+ cell counts (P = 0.02) and to some extent lower plasma HIV RNA (P = 0.07). There was no correlation between the number of overlapping Gag CD8+ T-cell ELISpot responses (n = 138) and plasma HIV-1 RNA viral load. Over a median follow-up of 2.9 years, baseline CD4+ cell ELISpot responses (n = 119) were associated with slower clinical progression (P = 0.01; log-rank). Over a median of 3.1 years, there was no evidence for a survival advantage imposed by CD8+ T-cell immunity (P = 0.82). Conclusion:These data support a dominant protective role for CD4+ T-cell immunity in PHI compared with CD8+ T-cell responses, and are highly pertinent to HIV pathogenesis and vaccines, indicating that vaccine-induced CD4+ responses may confer sustained benefit.

Structured observations reveal slow HIV-1 CTL escape.

The existence of viral variants that escape from the selection pressures imposed by cytotoxic T-lymphocytes (CTLs) in HIV-1 infection is well documented, but it is unclear when they arise, with reported measures of the time to escape in individuals ranging from days to years. A study of participants enrolled in the SPARTAC (Short Pulse Anti-Retroviral Therapy at HIV Seroconversion) clinical trial allowed direct observation of the evolution of CTL escape variants in 125 adults with primary HIV-1 infection observed for up to three years. Patient HLA-type, longitudinal CD8+ T-cell responses measured by IFN-γ ELISpot and longitudinal HIV-1 gag, pol, and nef sequence data were used to study the timing and prevalence of CTL escape in the participants whilst untreated. Results showed that sequence variation within CTL epitopes at the first time point (within six months of the estimated date of seroconversion) was consistent with most mutations being transmitted in the infecting viral strain rather than with escape arising within the first few weeks of infection. Escape arose throughout the first three years of infection, but slowly and steadily. Approximately one third of patients did not drive any new escape in an HLA-restricted epitope in just under two years. Patients driving several escape mutations during these two years were rare and the median and modal numbers of new escape events in each patient were one and zero respectively. Survival analysis of time to escape found that possession of a protective HLA type significantly reduced time to first escape in a patient (p = 0.01), and epitopes escaped faster in the face of a measurable CD8+ ELISpot response (p = 0.001). However, even in an HLA matched host who mounted a measurable, specific, CD8+ response the average time before the targeted epitope evolved an escape mutation was longer than two years.

Sex Differences in Antiretroviral Therapy Initiation in Pediatric HIV Infection

The incidence and severity of infections in childhood is typically greater in males. The basis for these observed sex differences is not well understood, and potentially may facilitate novel approaches to reducing disease from a range of conditions. We here investigated sex differences in HIV-infected children in relation to antiretroviral therapy (ART) initiation and post-treatment outcome. In a South African cohort of 2,101 HIV-infected children, we observed that absolute CD4+ count and CD4% were significantly higher in ART-naïve female, compared to age-matched male, HIV-infected children. Absolute CD4 count and CD4% were also significantly higher in HIV-uninfected female versus male neonates. We next showed that significantly more male than female children were initiated on ART (47% female); and children not meeting criteria to start ART by >5yrs were more frequently female (59%; p<0.001). Among ART-treated children, immune reconstitution of CD4 T-cells was more rapid and more complete in female children, even after adjustment for pre-ART absolute CD4 count or CD4% (p=0.011, p=0.030, respectively). However, while ART was initiated as a result of meeting CD4 criteria less often in females (45%), ART initiation as a result of clinical disease in children whose CD4 counts were above treatment thresholds occurred more often in females (57%, p<0.001). The main sex difference in morbidity observed in children initiating ART above CD4 thresholds, above that of TB disease, was as a result of wasting and stunting observed in females with above-threshold CD4 counts (p=0.002). These findings suggest the possibility that optimal treatment of HIV-infected children might incorporate differential CD4 treatment thresholds for ART initiation according to sex.

Lower Viral Loads and Slower CD4+ T-Cell Count Decline in MRKAd5 HIV-1 Vaccinees Expressing Disease-Susceptible HLA-B*58:02

Background. HLA strongly influences human immunodeficiency virus type 1 (HIV-1) disease progression. A major contributory mechanism is via the particular HLA-presented HIV-1 epitopes that are recognized by CD8+ T-cells. Different populations vary considerably in the HLA alleles expressed. We investigated the HLA-specific impact of the MRKAd5 HIV-1 Gag/Pol/Nef vaccine in a subset of the infected Phambili cohort in whom the disease-susceptible HLA-B*58:02 is highly prevalent. Methods. Viral loads, CD4+ T-cell counts, and enzyme-linked immunospot assay–determined anti-HIV-1 CD8+ T-cell responses for a subset of infected antiretroviral-naive Phambili participants, selected according to sample availability, were analyzed. Results. Among those expressing disease-susceptible HLA-B*58:02, vaccinees had a lower chronic viral set point than placebo recipients (median, 7240 vs 122 500 copies/mL; P = .01), a 0.76 log10 lower longitudinal viremia level (P = .01), and slower progression to a CD4+ T-cell count of <350 cells/mm3 (P = .02). These differences were accompanied by a higher Gag-specific breadth (4.5 vs 1 responses; P = .04) and magnitude (2300 vs 70 spot-forming cells/106 peripheral blood mononuclear cells; P = .06) in vaccinees versus placebo recipients. Conclusions. In addition to the known enhancement of HIV-1 acquisition resulting from the MRKAd5 HIV-1 vaccine, these findings in a nonrandomized subset of enrollees show an HLA-specific vaccine effect on the time to CD4+ T-cell count decline and viremia level after infection and the potential for vaccines to differentially alter disease outcome according to population HLA composition. Clinical Trials Registration. NCT00413725, DOH-27-0207-1539.