Emily Adland

CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

HLA-A*7401-mediated control of HIV viremia is independent of its linkage disequilibrium with HLA-B*5703.

The potential contribution of HLA-A alleles to viremic control in chronic HIV type 1 (HIV-1) infection has been relatively understudied compared with HLA-B. In these studies, we show that HLA-A*7401 is associated with favorable viremic control in extended southern African cohorts of >2100 C-clade–infected subjects. We present evidence that HLA-A*7401 operates an effect that is independent of HLA-B*5703, with which it is in linkage disequilibrium in some populations, to mediate lowered viremia. We describe a novel statistical approach to detecting additive effects between class I alleles in control of HIV-1 disease, highlighting improved viremic control in subjects with HLA-A*7401 combined with HLA-B*57. In common with HLA-B alleles that are associated with effective control of viremia, HLA-A*7401 presents highly targeted epitopes in several proteins, including Gag, Pol, Rev, and Nef, of which the Gag epitopes appear immunodominant. We identify eight novel putative HLA-A*7401–restricted epitopes, of which three have been defined to the optimal epitope. In common with HLA-B alleles linked with slow progression, viremic control through an HLA-A*7401–restricted response appears to be associated with the selection of escape mutants within Gag epitopes that reduce viral replicative capacity. These studies highlight the potentially important contribution of an HLA-A allele to immune control of HIV infection, which may have been concealed by a stronger effect mediated by an HLA-B allele with which it is in linkage disequilibrium. In addition, these studies identify a factor contributing to different HIV disease outcomes in individuals expressing HLA-B*5703.

Impact of HLA-driven HIV adaptation on virulence in populations of high HIV seroprevalence

Significance Factors that influence the virulence of HIV are of direct relevance to ongoing efforts to contain, and ultimately eradicate, the HIV epidemic. We here investigate in Botswana and South Africa, countries severely affected by HIV, the impact on HIV virulence of adaptation of HIV to protective HLA alleles such as HLA-B*57. In Botswana, where the epidemic started earlier and reached higher adult seroprevalence than in South Africa, HIV replication capacity is lower. HIV is also better adapted to HLA-B*57, which in Botswana has no protective effect, in contrast to its impact in South Africa. Modelling studies indicate that increasing antiretroviral therapy access may also contribute to accelerated declines in HIV virulence over the coming decades. It is widely believed that epidemics in new hosts diminish in virulence over time, with natural selection favoring pathogens that cause minimal disease. However, a tradeoff frequently exists between high virulence shortening host survival on the one hand but allowing faster transmission on the other. This is the case in HIV infection, where high viral loads increase transmission risk per coital act but reduce host longevity. We here investigate the impact on HIV virulence of HIV adaptation to HLA molecules that protect against disease progression, such as HLA-B*57 and HLA-B*58:01. We analyzed cohorts in Botswana and South Africa, two countries severely affected by the HIV epidemic. In Botswana, where the epidemic started earlier and adult seroprevalence has been higher, HIV adaptation to HLA including HLA-B*57/58:01 is greater compared with South Africa (P = 7 × 10−82), the protective effect of HLA-B*57/58:01 is absent (P = 0.0002), and population viral replicative capacity is lower (P = 0.03). These data suggest that viral evolution is occurring relatively rapidly, and that adaptation of HIV to the most protective HLA alleles may contribute to a lowering of viral replication capacity at the population level, and a consequent reduction in HIV virulence over time. The potential role in this process played by increasing antiretroviral therapy (ART) access is also explored. Models developed here suggest distinct benefits of ART, in addition to reducing HIV disease and transmission, in driving declines in HIV virulence over the course of the epidemic, thereby accelerating the effects of HLA-mediated viral adaptation.

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.

Ongoing burden of disease and mortality from HIV/CMV coinfection in Africa in the antiretroviral therapy era

Human Cytomegalovirus (CMV) is a well-recognized pathogen in the context of HIV infection, but since the roll out of ART, clinical and scientific interest in the problem of HIV/CMV coinfection has diminished. However, CMV remains a significant cofactor in HIV disease, with an influence on HIV acquisition, disease progression, morbidity, and mortality. Disease manifestations may be a result of direct interplay between the two viruses, or may arise as a secondary consequence of immune dysregulation and systemic inflammation. The problem is most relevant when the rates of coinfection are high, most notably in sub-Saharan Africa, and in children at risk of acquiring both infections early in life. Understanding the interplay between these viruses and developing strategies to diagnose, treat and prevent CMV should be a priority.

Nef-Specific CD8+ T Cell Responses Contribute to HIV-1 Immune Control

Recent studies in the SIV-macaque model of HIV infection suggest that Nef-specific CD8+ T-cell responses may mediate highly effective immune control of viraemia. In HIV infection Nef recognition dominates in acute infection, but in large cohort studies of chronically infected subjects, breadth of T cell responses to Nef has not been correlated with significant viraemic control. Improved disease outcomes have instead been associated with targeting Gag and, in some cases, Pol. However analyses of the breadth of Nef-specific T cell responses have been confounded by the extreme immunogenicity and multiple epitope overlap within the central regions of Nef, making discrimination of distinct responses impossible via IFN-gamma ELISPOT assays. Thus an alternative approach to assess Nef as an immune target is needed. Here, we show in a cohort of >700 individuals with chronic C-clade infection that >50% of HLA-B-selected polymorphisms within Nef are associated with a predicted fitness cost to the virus, and that HLA-B alleles that successfully drive selection within Nef are those linked with lower viral loads. Furthermore, the specific CD8+ T cell epitopes that are restricted by protective HLA Class I alleles correspond substantially to effective SIV-specific epitopes in Nef. Distinguishing such individual HIV-specific responses within Nef requires specific peptide-MHC I tetramers. Overall, these data suggest that CD8+ T cell targeting of certain specific Nef epitopes contributes to HIV suppression. These data suggest that a re-evaluation of the potential use of Nef in HIV T-cell vaccine candidates would be justified.

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.

Evaluation of the NucliSens EasyQ v2.0 Assay in Comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in Quantification of C-Clade HIV-1 in Plasma

Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 – Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 – Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 – NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.

Impact of HLA Selection Pressure on HIV Fitness at a Population Level in Mexico and Barbados

ABSTRACT Previous studies have demonstrated that effective cytotoxic T lymphocyte (CTL) responses drive the selection of escape mutations that reduce viral replication capacity (VRC). Escape mutations, including those with reduced VRC, can be transmitted and accumulate in a population. Here we compared two antiretroviral therapy (ART)-naive HIV clade B-infected cohorts, in Mexico and Barbados, in which the most protective HLA alleles (HLA-B*27/57/58:01/81:01) are differentially expressed, at 8% and 34%, respectively. Viral loads were significantly higher in Mexico than in Barbados (median, 40,774 versus 14,200; P < 0.0001), and absolute CD4+ T-cell counts were somewhat lower (median, 380/mm3 versus 403/mm3; P = 0.007). We tested the hypothesis that the disparate frequencies of these protective HLA alleles would be associated with a higher VRC at the population level in Mexico. Analysis of VRC in subjects in each cohort, matched for CD4+ T-cell count, revealed that the VRC was indeed higher in the Mexican cohort (mean, 1.13 versus 1.03; P = 0.0025). Although CD4 counts were matched, viral loads remained significantly higher in the Mexican subjects (P = 0.04). This VRC difference was reflected by a significantly higher frequency in the Barbados cohort of HLA-B*27/57/58:01/81:01-associated Gag escape mutations previously shown to incur a fitness cost on the virus (P = 0.004), a difference between the two cohorts that remained statistically significant even in subjects not expressing these protective alleles (P = 0.01). These data suggest that viral set points and disease progression rates at the population level may be significantly influenced by the prevalence of protective HLA alleles such as HLA-B*27/57/58:01/81:01 and that CD4 count-based guidelines to initiate antiretroviral therapy may need to be modified accordingly, to optimize the effectiveness of treatment-for-prevention strategies and reduce HIV transmission rates to the absolute minimum. IMPORTANCE Immune control of HIV at an individual level is strongly influenced by the HLA class I genotype. HLA class I molecules mediating effective immune control, such as HLA-B*27 and HLA-B*57, are associated with the selection of escape mutants that reduce viral replicative capacity. The escape mutants selected in infected patients can be transmitted and affect the viral load and CD4 count in the recipient. These findings prompt the hypothesis that the frequency of protective alleles in a population may affect viral set points and rates of disease progression in that population. These studies in Mexico and Barbados, where the prevalence rates of protective HLA alleles are 8% and 34%, respectively, support this hypothesis. These data suggest that antiretroviral therapy (ART) treatment-for-prevention strategies will be less successful in populations such as those in Mexico, where viral loads are higher for a given CD4 count. Consideration may therefore usefully be given to ART initiation at higher absolute CD4 counts in such populations to optimize the impact of ART for prevention.

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.