Differences in the genetic characteristics of HIV likely influence the rate of disease progression and the response to antiretroviral therapy (ART). One important unanswered question is the biological basis for the apparent higher virulence of HIV subtype D compared with subtype A.1-6 Surprisingly, knowledge in this area is limited, and no conclusive recommendations currently exist regarding subtype and treatment guidelines or therapeutic monitoring. The level of viral replication and its subsequent effect on the immune system influences CD4+ T-cell depletion. During chronic HIV infection, more CD4+ T cells die than can be accounted for by direct infection.7 This bystander loss of uninfected T cells is believed to be associated with the generalized HIV-induced immune activation.8 We have previously reported higher T-cell activation in Uganda compared with Western cohorts (HIV subtype B).9 We hypothesize that the infecting HIV subtypes exert independent influence on the outcome of immunity in response to antiretroviral treatment in our study population.
MATERIALS AND METHODS
Study Subjects and Samples
HIV-positive ART-naive volunteers were randomly recruited from a prospective observational cohort study from individuals receiving care at the Mbarara Hospital Uganda AIDS Rural Treatment Outcomes in Mbarara, Uganda.10 Demographic information, CD4+ T-cell count, and HIV viral load were obtained at the time of enrollment, and then again at 3 and 6 months after initiation of ART. All patients initiated ART within 2 weeks of being enrolled into the study. HIV-1 RNA levels were determined from plasma using the Roche Amplicor 1.5 (Roche, Branchburg, NJ), as per manufacturer's recommendations. Peripheral blood mononuclear cells (PBMCs) were obtained from enrolled volunteers. PBMC samples were isolated from whole blood by the use of density gradient centrifugation over ficoll-hypaque solution, cryopreserved immediately, and stored in liquid nitrogen. Institutional Review Board approvals were obtained from the Uganda National Council of Sciences and Technologies, the UCSF committee on Human Research, and the California Department of Public Health. All study patients participated voluntarily and gave written informed consent.
Subtyping was based on plasma sequencing for Pol as previously reported.11 Briefly, RNA was extracted from plasma using the Qiagen viral RNA kit with a BioRobot 9600/9604. HIV Pol genes were amplified using nested reverse transcriptase polymerase chain reaction and sequenced in both the 5′ and 3′ directions. The National Institutes of Health's HIV-1 Genotyping Tool Web site (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.html) in conjunction with the Los Alamos HIV sequence database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html) was used to determine the HIV-1 subtype.
HIV-1 infection-induced cleavage of procaspases results in cell apoptosis. Flow cytometry analysis approach allows for the detection of apoptotic cells at the single cell level. We measured caspase activity by using the Vybrant FAM Poly Caspases Assay Kit (Molecular Probes). The kit employs a fluorescently labeled inhibitor of caspases reagent containing a fluoromethyl ketone group, a caspase specific recognition sequence (VAD), and a carboxyfluorescein (FAM) group as a reporter. Cryopreserved PBMCs were thawed, washed, and resuspended at 1 × 106 cells per milliliter in 300 μL RPMI media with 10% fetal bovine syndrome. After adding 10 μL of 30× fluorescently labeled inhibitor of caspases working solution to each culture, the suspensions were incubated for 1 hour at 37°C and 5% CO2. The cells were washed twice with 1× wash buffer supplied by the manufacturer before proceeding with subsequent surface staining. Apoptosis was analyzed based on caspase-positive and caspase-negative populations, with gating based on a fluorescence minus one control.
Immune Activation and Functional Immunophenotype
Activated T cells were measured as previously described.9 Further immunophenotyping was performed on cryopreserved PBMCs, which were thawed and subsequently stained with antibody panels for activation and PD-1. For activation panels, cells were stained with the following antibodies: HLADR FITC, CD38 PE, CD3 PerCp Cy5.5, CD4 APC CY7, CD27 APC, CD45RA PECy7, and CD8 Pacific Blue (BD BioSciences, San Jose, CA). For the PD-1 panels, cells were stained with antibodies including CD3 PerCP Cy5.5, CD4 PeCy7, CD8 Pacific Blue, HLADR FITC, CD38 PE, and PD-1 APC (BD BioSciences). Data were collected on an 8-color flow cytometer (LSRII; BD Biosciences) and analyzed using FLOWJO software (TreeStar, San Carlos, CA). A minimum of 30,000 CD4+ cells per sample was acquired on the LSRII. Dead cells were first gated out using a violet excited viability dye (LIVE/DEAD Fixable Dead Cell Stain; Invitrogen). Immune activation was defined as the percent of CD38+ HLADR+ T cells. PD-1 level was defined as the percent expression of PD-1 APC on CD3+ CD8+ T cells. Gating was standardized and set using fluorescence controls for HLADR, CD38, and PD-1.
Groups were compared using the Mann-Whitney U test or the paired t test using PRISM software version 4.02 (GraphPad, San Diego, CA). Statistical significance was defined as P < 0.05.
We analyzed subtype distribution in our study population based on the Pol region of the genome, and valid data were obtained for 172 samples. We found that subtype distribution in our study population (Fig. 1) is composed primarily of subtypes A and D (n = 84 and 76, respectively). No correlation between viral subtypes (A and D) for CD4 count (mean values 95 and 70 count/mL; P > 0.05; subtype A and D, respectively) or viral load (mean values 203, 000 and 151,000 copies/mL; P > 0.05; subtype A and D, respectively) was observed before ART initiation (Table 1). In addition, CD4 cell gain after ART suppression did not differ significantly between the 2 infecting subtypes within 6 months (Fig. 1C). There was no significant difference in subtype distribution by sex or age (P > 0.05; data not shown).
CD4 Immune Activation Decreases Post ART Suppression in Subtypes A and D
Different HIV-1 subtypes may vary in pathogenesis, resulting in distinct profiles of immune activation. Others and we have described heightened immune activation in HIV-infected individuals from Africa compared with those from Europe and the United States.9,12 Activation staining was performed as previously described.9,13 CD8 and CD4 activation was defined as the percentage of CD3+ CD8+ CD38+ HLADR+ or CD3+ CD4+ CD38+ HLADR+ lymphocytes, respectively. Both CD8 and CD4 T-cell immune activation were examined separately at baseline (when the patients were naive) and again at 6 months after the initiation of antiretroviral treatment. Because the majority of our population was infected with subtypes A and D, we focused our immune activation analysis on these 2 subtypes. We observed no significant differences in CD4 and CD8 immune activation between these subtypes (Fig. 2; data not shown).
Differential Levels of Apoptosis in Activated Cells Between Subtypes A and D
We next examined the level of apoptosis in CD4+ T cells between the 2 subgroups. Overall levels of apoptosis were highest in the activated CD4 cells compared with the total fraction of CD4+ caspase+ T cells and significantly decreased with viral suppression (Fig. 3A; P < 0.0001). Interestingly, the apoptosis of activated CD4 cells in individuals infected with subtype D was significantly higher compared with those infected with subtype A (Fig. 3B; P = 0.028) before antiretroviral treatment. After ART-mediated viral suppression, we observed normalization of activation to similar levels between subtypes.
PD-1 Expression on CD4+ T Cells
The inhibitory programmed cell death receptor (PD-1) has been compared with activation marker CD38 and viral load as a predictor for clinical progression. PD-1 is upregulated in chronic HIV infection, and its expression correlates positively with plasma viral load and inversely with CD4+ T-cell count. We found that levels of expression of PD-1 on CD4 cells in subtype D was substantially higher compared with that in subtype A (Fig. 4A; P = 0.01). This difference was not observed in the CD8 population (Fig. 4B; P = 0.97). The level of PD-1 expression in the 2 populations was not significantly different after antiretroviral suppression (P = 0.25; data not shown).
The mechanism by which HIV causes immune destruction is linked to its ability to induce a generalized immune hyperactivation. Discordant responses to ART have been linked to the level of cellular activation.14-16 How chronic activation might be detrimental to the immune system remains unclear. Bystander activation of uninfected CD4+ T cells has been demonstrated to increase susceptibility to infection and subsequent virus production ex vivo. T-cell apoptosis and subsequent immune exhaustion are likely key factors in the eventual failure of viral control.
HIV-1 subtypes may confer distinct pathogenesis and impact HIV disease progression. Subtypes A and D account for the majority of infection in our treatment-naive study population in Uganda. CD4+ T-cell immune activation significantly decreased after ART-mediated viral suppression as we have previously reported.17 Although subtypes did not correlate with generalized levels of immune activation, we observed significant differences in CD4 dysfunction in individuals infected with subtype D, as demonstrated by higher level of apoptosis and PD-1 expression. These data support the hypothesis that subtype D is more pathogenic14,15 and is consistent with previous cohort studies demonstrating faster disease progression in subtype D that is independent of viral load levels.1
The role of HIV subtype and viral gene products may be critical in determining the level of immunopathogenesis associated with HIV disease progression to AIDS. We propose that the differential subtype-induced activation and subsequent apoptosis is one such mechanism that leads to differential clinical disease outcome in Uganda.1 The difference in the level of activation-associated apoptosis between subtypes A and D becomes insignificant after 6 months of ART. This finding may reflect on the specific subtype's ability to induce the expression of apoptosis ligands and disrupt the effective generation of T-cell immunity. However, the full explorations of all mechanisms involved in treatment failure and immune recovery cannot be assessed in this relatively short follow-up, and the small sample size of our study limits definitive long-term outcome. The true clinical impact of T-cell dysfunction in association with HIV subtypes in predicting disease outcome will require a larger longitudinal cohort study. The recognition of the different HIV-1 subtypes has led many to question whether properties of HIV-1 infection and its consequences as a whole can be generalized among different subtypes. Our findings suggest that infection with one subtype may be more aggressive than others with regard to disease progression. Such diversity could potentially affect the overall efficacy of HIV therapy or intervention strategies in the affected population.
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