Nompumelelo Mkhwanazi

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8+ T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein–specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8+ T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.

Detection of Polyfunctional Mycobacterium tuberculosis-Specific T Cells and Association with Viral Load in HIV-1-Infected Persons

BACKGROUND The human immunodeficiency virus type 1 (HIV-1) epidemic is associated with a significant increase in the incidence of tuberculosis (TB); however, little is known about the quality of Mycobacterium tuberculosis (MTB)-specific cellular immune responses in coinfected individuals. METHODS A total of 137 HIV-1-positive individuals in Durban, South Africa, were screened with the use of overlapping peptides spanning Ag85A, culture filtrate protein 10 (CFP-10), early secretory antigen target 6 (ESAT-6), and TB10.4, in an interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay. Intracellular cytokine staining for MTB-specific production of IFN-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2 was performed, as was ex vivo phenotyping of memory markers on MTB-specific T cells. RESULTS A total of 41% of subjects responded to ESAT-6 and/or CFP-10, indicating the presence of latent MTB infection. The proportion of MTB-specific IFN-gamma(+)/TNF-alpha(+) CD4(+) cells was significantly higher than the proportion of IFN-gamma(+)/IL-2(+) CD4(+) cells (P = .0220), and the proportion of MTB-specific IL-2-secreting CD4 cells was inversely correlated with the HIV-1 load (P = .0098). MTB-specific CD8 T cells were predominately IFN-gamma(+)/TNF-alpha(+)/IL-2(-). Ex vivo memory phenotyping of MTB-specific CD4 and CD8 T cells indicated an early to intermediate differentiated phenotype for the population of effector memory cells. CONCLUSIONS Polyfunctional MTB-specific CD4 and CD8 T cell responses are maintained in the peripheral blood of HIV-1-positive individuals, in the absence of active disease, and the functional capacity of these responses is affected by HIV-1 disease status.

High frequency of rapid immunological progression in African infants infected in the era of perinatal Hiv prophylaxis

Objectives:To determine the natural history of HIV infection following peripartum single-dose nevirapine (sd-NVP) prophylaxis in a resource-limited country, and to assess implications for antiretroviral therapy (ART) roll-out programmes. Methods:Infants of HIV-infected mothers in KwaZulu-Natal, South Africa, were tested on days 1 and 28 to detect intrauterine (IU) and intrapartum (IP) infection. Infant follow-up included monthly viral load and CD4 cell measurement. ART was initiated at infant CD4 cell% ≤ 20%. Results:In 740 infants born to 719 HIV-infected women, mother-to-child transmission (MTCT) was 10.3% (69% IU, 31% IP). Median viral load was higher in mothers of infants infected IP than IU (279 000 versus 86 600 copies/ml; P = 0.039) and lower in mothers of uninfected infants (median 26 750 copies/ml; P < 0.001). Peak viraemia was higher in infants infected IP than IU (5 160 000 versus 984 000 copies/ml; P < 0.001). Median viral load at birth in IU-infected infants (155 000 copies/ml) fell 1.4 log to 6510 copies/ml by day 5 and was beneath the detection limit using dried blood spot analysis in 38% of infants. CD4 cell% declined rapidly, to ≤ 20% in 70% and ≤ 25% in 85% [current World Health Organization (WHO) criteria for initiating ART] of infants by 6 months. Conclusions:MTCT was reduced by sd-NVP through an effect on IP transmission. Where MTCT occurred despite NVP, two-thirds of transmissions arose IU; IP-infected babies were born to mothers with very high viral load. Disease progression was particularly rapid, 85% infants meeting WHO criteria for ART within 6 months. These findings argue for more effective MTCT-prevention programmes in resource-limited countries.

Targeting of a CD8 T cell env epitope presented by HLA-B*5802 is associated with markers of HIV disease progression and lack of selection pressure

In HIV-infected persons, certain HLA class I alleles are associated with effective control of viremia, while others are associated with rapid disease progression. Among the most divergent clinical outcomes are the relatively good prognosis in HLA-B*5801 expressing persons and poor prognosis with HLA-B*5802. These two alleles differ by only three amino acids in regions involved in HLA-peptide recognition. This study evaluated a cohort of over 1000 persons with chronic HIV clade C virus infection to determine whether clinical outcome differences associated with B*5801 (n = 93) and B*5802 ( n = 259) expression are associated with differences in HIV-1-specific CD8 (+) T cell responses. The overall breadth and magnitude of HIV-1-specific CD8(+) T cell responses were lower in persons expressing B*5802, and epitope presentation by B*5802 contributed significantly less to the overall response as compared to B*5801-restricted CD8 (+) T cells. Moreover, viral load in B*5802-positive persons was higher and CD4 cell counts lower when this allele contributed to the overall CD8 (+) T cell response, which was detected exclusively through a single epitope in Env. In addition, persons heterozygous for B*5802 compared to persons homozygous for other HLA-B alleles had significantly higher viral loads. Viral sequencing revealed strong selection pressure mediated through B*5801-restricted responses but not through B*5802. These data indicate that minor differences in HLA sequence can have a major impact on epitope recognition, and that selective targeting of Env through HLA-B*5802 is at least ineffectual if not actively adverse in the containment of viremia. These results provide experimental evidence that not all epitope-specific responses contribute to immune containment, a better understanding of which is essential to shed light on mechanisms involved in HIV disease progression.

Human Immunodeficiency Virus-Specific CD8+ T-Cell Activity Is Detectable from Birth in the Majority of In Utero-Infected Infants

ABSTRACT Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8+ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8+ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4+ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8+ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.

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 in Mother and Child on Disease Progression of Pediatric Human Immunodeficiency Virus Type 1 Infection

ABSTRACT A broad Gag-specific CD8+ T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8+ T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8+ T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fishers exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8+ T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.

Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C

ABSTRACT HLA class I-mediated selection of immune escape mutations in functionally important Gag epitopes may partly explain slower disease progression in HIV-1-infected individuals with protective HLA alleles. To investigate the impact of Gag function on disease progression, the replication capacities of viruses encoding Gag-protease from 60 individuals in early HIV-1 subtype C infection were assayed in an HIV-1-inducible green fluorescent protein reporter cell line and were correlated with subsequent disease progression. Replication capacities did not correlate with viral load set points (P = 0.37) but were significantly lower in individuals with below-median viral load set points (P = 0.03), and there was a trend of correlation between lower replication capacities and lower rates of CD4 decline (P = 0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (P = 0.04), but host HLA-B-specific polymorphisms were associated with higher viral load set points (P = 0.01). Further, polymorphisms associated with host-specific protective HLA alleles were linked with higher viral load set points (P = 0.03). These data suggest that transmission or early HLA-driven selection of Gag polymorphisms results in reduced early cytotoxic T-lymphocyte (CTL) responses and higher viral load set points. In support of the former, 46% of individuals with nonprotective alleles harbored a Gag polymorphism exclusively associated with a protective HLA allele, indicating a high rate of their transmission in sub-Saharan Africa. Overall, HIV disease progression is likely to be affected by the ability to mount effective Gag CTL responses as well as the replication capacity of the transmitted virus.

Immunodominant HIV-1 Cd4+ T Cell Epitopes in Chronic Untreated Clade C HIV-1 Infection

Background A dominance of Gag-specific CD8+ T cell responses is significantly associated with a lower viral load in individuals with chronic, untreated clade C human immunodeficiency virus type 1 (HIV-1) infection. This association has not been investigated in terms of Gag-specific CD4+ T cell responses, nor have clade C HIV-1–specific CD4+ T cell epitopes, likely a vital component of an effective global HIV-1 vaccine, been identified. Methodology/Principal Findings Intracellular cytokine staining was conducted on 373 subjects with chronic, untreated clade C infection to assess interferon-gamma (IFN-γ) responses by CD4+ T cells to pooled Gag peptides and to determine their association with viral load and CD4 count. Gag-specific IFN-γ–producing CD4+ T cell responses were detected in 261/373 (70%) subjects, with the Gag responders having a significantly lower viral load and higher CD4 count than those with no detectable Gag response (p<0.0001 for both parameters). To identify individual peptides targeted by HIV-1–specific CD4+ T cells, separate ELISPOT screening was conducted on CD8-depleted PBMCs from 32 chronically infected untreated subjects, using pools of overlapping peptides that spanned the entire HIV-1 clade C consensus sequence, and reconfirmed by flow cytometry to be CD4+ mediated. The ELISPOT screening identified 33 CD4+ peptides targeted by 18/32 patients (56%), with 27 of the 33 peptides located in the Gag region. Although the breadth of the CD4+ responses correlated inversely with viral load (p = 0.015), the magnitude of the response was not significantly associated with viral load. Conclusions/Significance These data indicate that in chronic untreated clade C HIV-1 infection, IFN-γ–secreting Gag-specific CD4+ T cell responses are immunodominant, directed at multiple distinct epitopes, and associated with viral control.

A Molecular Assay for Sensitive Detection of Pathogen- Specific T-Cells

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.