Acceleration of age-associated methylation patterns in HIV-1-infected adults.

Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x 10(-200) and 0.47, p<1 x 10(-200). Weighted gene correlation network analysis (WGCNA) identified 17 co-methylation modules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: ßage=0.007088, p=2.08 x 10(-9); ßHIV=0.099574, p=0.0011; Data set 2: ßage=0.008762, p=1.27 x 10(-5); ßHIV=0.128649, p=0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10(-6), odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are similar to age-associated patterns and suggest that general aging and HIV-1 related aging work through some common cellular and molecular mechanisms. These results are an important first step for finding potential therapeutic targets and novel clinical approaches to mitigate the detrimental effects of both HIV-1-infection and aging.

Differential blood and mucosal immune responses against an HIV-1 vaccine administered via inguinal or deltoid injection.

UNLABELLED: Mucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24-180 versus 180-365 days). HIV-1-specific CD8(+) T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17-24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24-180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time. TRIAL REGISTRATION: ClinicalTrials.gov NCT00076817.

The dual impact of HIV-1 infection and aging on naïve CD4 T-cells: additive and distinct patterns of impairment.

HIV-1-infected adults over the age of 50 years progress to AIDS more rapidly than adults in their twenties or thirties. In addition, HIV-1-infected individuals receiving antiretroviral therapy (ART) present with clinical diseases, such as various cancers and liver disease, more commonly seen in older uninfected adults. These observations suggest that HIV-1 infection in older persons can have detrimental immunological effects that are not completely reversed by ART. As naïve T-cells are critically important in responses to neoantigens, we first analyzed two subsets (CD45RA(+)CD31(+) and CD45RA(+)CD31(-)) within the naïve CD4(+) T-cell compartment in young (20-32 years old) and older (39-58 years old), ART-naïve, HIV-1 seropositive individuals within 1-3 years of infection and in age-matched seronegative controls. HIV-1 infection in the young cohort was associated with lower absolute numbers of, and shorter telomere lengths within, both CD45RA(+)CD31(+)CD4(+) and CD45RA(+)CD31(-)CD4(+) T-cell subsets in comparison to age-matched seronegative controls, changes that resembled seronegative individuals who were decades older. Longitudinal analysis provided evidence of thymic emigration and reconstitution of CD45RA(+)CD31(+)CD4(+) T-cells two years post-ART, but minimal reconstitution of the CD45RA(+)CD31(-)CD4(+) subset, which could impair de novo immune responses. For both ART-naïve and ART-treated HIV-1-infected adults, a renewable pool of thymic emigrants is necessary to maintain CD4(+) T-cell homeostasis. Overall, these results offer a partial explanation both for the faster disease progression of older adults and the observation that viral responders to ART present with clinical diseases associated with older adults.

Regulatory T cell expansion and immune activation during untreated HIV type 1 infection are associated with disease progression.

Regulatory T cells (Tregs) may play an important role in the immunopathology of chronic HIV-1 infection due to their potent suppressive activity of both T cell activation and effector function. To investigate the correlation between Tregs and immune activation during untreated chronic HIV-1 infection, we conducted a nested case-control study within the Multicenter AIDS Cohort Study (MACS). Twenty HIV-1-infected fast progressors (FP) and 40 slow progressors (SP) were included in our study using risk-set sampling. Nine age-matched HIV-1-uninfected men (UI) were also included. Cryopreserved peripheral blood mononuclear cells (PMBCs) were tested using flow cytometry analyses. We identified Tregs as Foxp3+CD25+CD4+ T cells and assessed the activation of CD4+ and CD8+ T cells by the expression of CD38, HLADR, or both markers simultaneously. There is a relative expansion of Tregs during HIV-1 infection, which is associated with disease progression. The increased CD38 expression on both CD4+ and CD8+ T cells expressed as either percentage or median fluorescence intensity (MFI) and the elevated proportion of CD8+ T cells that is HLADR+CD38+ were all associated with rapid HIV-1 progression. Counter to the assumed role of Tregs as the suppressors of activation, the expansion of Tregs was positively correlated with CD4+ T cell activation among HIV-1-infected fast progressors. The high level of Tregs associated with rapid HIV progression may suggest a detrimental role of these cells in the immune control of HIV-1 infection.

Premature aging of T cells is associated with faster HIV-1 disease progression.

OBJECTIVE: To determine if untreated HIV-1 infection and progression is associated with premature aging of memory CD8 and CD4 T cells and naive CD4 T cells. METHODS: Twenty HIV-1-infected fast progressors and 40 slow progressors were included in our study, using risk set sampling. The expression of cell surface markers reflecting the differentiation stages of lymphocytes was measured using flow cytometry analyses performed on cryopreserved peripheral blood mononuclear cells. RESULTS: We found that HIV-1 disease progression is associated with a decreased CD28 median florescence intensity on CD4 and CD8 T cells; an increased proportion of intermediate- and late-differentiated CD8 T cells and a decreased CD31 median florescence intensity on naive CD4 T cells of recent thymic origin. A selective depletion of peripherally expanded naive CD4 T cells was found to be associated with HIV-1 infection but not with HIV-1 disease progression. CONCLUSIONS: The overall change during HIV-1 infection and progression is associated with a shift in the T-cell population toward an aged conformation, which may be further compromised by impaired renewal of the less-differentiated CD4 T-cell population. Our results suggest that HIV-1 infection induces an accelerated aging of T lymphocytes, which is associated with the clinical progression to AIDS and death.

Differential immunogenicity of vaccinia and HIV-1 components of a human recombinant vaccine in mucosal and blood compartments.

Mucosal immune responses induced by HIV-1 vaccines are likely critical for prevention. We report a Phase 1 safety and immunogenicity trial in eight participants using the vaccinia-based TBC-3B vaccine given subcutaneously to determine the relationship between HIV-1 specific systemic and gastrointestinal mucosal responses. Across all subjects, detectable levels of blood vaccinia- and HIV-1-specific antibodies were elicited but none were seen mucosally. While the vaccinia component was immunogenic for CD8(+) T lymphocyte (CTL) responses in both blood and mucosa, it was greater in blood. The HIV-1 component of the vaccine was poorly immunogenic in both blood and mucosa. Although only eight volunteers were studied intensively, the discordance between mucosal and blood responses may highlight mechanisms contributing to recent vaccine failures.

Delayed reconstitution of CD4+ iNKT cells after effective HIV type 1 therapy.

CD1d-restricted natural killer T (iNKT) cells are increasingly recognized as key immunoregulatory cells linking innate and adaptive immunity. These fall into functionally distinct CD4+ versus CD4- subsets that are believed to steer cellular immunity toward tolerigenic/atopic versus proinflammatory phenotypes, respectively. Preferential depletion of the CD4+ subset has been observed in HIV-1 infection, but the repletion of these cells after antiretroviral therapy has not been examined in detail. T lymphocytes, CD8+ lymphocyte activation, viremia, and iNKT cell subsets in peripheral blood were compared between 18 HIV-1-uninfected (Control) and 18 seropositive (SP) men initially not on suppressive antiretroviral therapy. Compared to the Control group, the SP group demonstrated reduction of CD4+ and lesser reduction of CD4- iNKT cells at baseline. After initiation of suppressive antiretroviral treatment, the SP CD4+ iNKT cell levels remained unchanged after a year and increased by 2 years, while CD4+ iNKT cells showed a gradual increase notable after the first year. Over the first year of treatment, there was a significant correlation between changes in total CD4+ T lymphocyte and changes in CD4+ iNKT cell levels, and a significant inverse correlation between changes in CD8+ T lymphocyte activation and changes in CD4- iNKT cell levels. These results confirm preferential depletion of tolerigenic/atopic CD4+ iNKT cells by HIV-1, and suggest that disproportionate persistence of proinflammatory CD4- iNKT cells could contribute to the inappropriate immune activation believed to cause immunodeficiency in HIV-1 infection.

Assessing immunophenotyping performance: proficiency-validation for adopting improved flow cytometry methods.

BACKGROUND: The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform "switch" studies to validate performance before methods for T-cell subset analysis can be changed. METHODS: Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program. RESULTS: Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced. CONCLUSIONS: Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories' transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.

Transience of vaccine-induced HIV-1-specific CTL and definition of vaccine "response".

Many vaccine approaches emphasize producing HIV-1-specific CD8+ T-lymphocyte (CTL) responses. Towards this goal, many studies simply classify vaccinees as "responders" or "nonresponders," based on arbitrary cutoff criteria. HIV-1-uninfected participants receiving the TBC-3B vaccine were assessed for HIV-1-specific CTL by interferon-gamma ELISpot, and compared to HIV-1-infected control subjects not on antiretroviral therapy. Vaccinees also were tested for HIV-1-specific antibody responses and generalized CD8+ T-lymphocyte activation. Different criteria for vaccine "responder" status were applied to the measured CTL values. The vaccinees showed evidence of vaccine exposure by CD8+ T-lymphocyte activation and HIV-1-specific antibodies. Considering any single positive HIV-1-specific CTL measurement a vaccine "response," all vaccinees could be classified as "responders," but even slight increases in the stringency of response criteria resulted in a steep decline of the "response" rate. In contrast, HIV-1-infected persons were clearly "responders" against the same proteins by the same criteria. Quantitative assessment of CTL demonstrated low and transient HIV-1-specific CTL compared to natural infection. These analyses emphasize the pitfalls of summarizing vaccine study results using simple cutoff criteria to define response rates, and suggest the utility of more comprehensive descriptions to describe vaccine immunogenicity and persistence of responses.

Effects of HIV-1 infection on lymphocyte phenotypes in blood versus lymph nodes.

Most immunopathogenesis studies of HIV-1 use peripheral blood. Most lymphocytes reside in lymphoid tissues, however, and the extent to which blood mirrors tissues is unclear. Here, we analyze lymphocytes in blood and lymph nodes of HIV-1-uninfected and -infected persons. Baseline comparison of node and blood lymphocytes in seronegative persons demonstrates a lower ratio of CD8+ versus CD4+ T lymphocytes, a lower number of effector cells (CD28-) within the CD8+ compartment, and greater activation (D-receptor [DR+]) within the CD4+ compartment. In infected versus uninfected persons, nodes exhibit elevated CD8+ T lymphocytes with an increased memory-effector phenotype (CD62L-/CD45RA-) and activation (CD38+ and DR+) but minimal differences in the CD4+ compartment. Changes attributable to HIV-1 infection are markedly greater in node lymphocytes than in blood. Comparisons of CD8+ T-lymphocyte parameters and viremia in infected persons reveal positive correlations of CD38+ expression on cells in blood and nodes and a negative correlation of terminal effector cells (CD62L-/CD45RA+) in the nodes to viremia. Multiple linear regression analysis indicates that CD38 expression on node (not blood) CD8+ T lymphocytes is the sole independent predictor for viremia. Thus, blood indirectly reflects processes in lymphoid tissues, and caution should be applied when interpreting immunopathogenesis studies of blood.

Parallel human immunodeficiency virus type 1-specific CD8+ T-lymphocyte responses in blood and mucosa during chronic infection.

Gut-associated lymphoid tissue is the major reservoir of lymphocytes and human immunodeficiency virus type 1 (HIV-1) replication in vivo, yet little is known about HIV-1-specific CD8+ T-lymphocyte (CTL) responses in this compartment. Here we assessed the breadth and magnitude of HIV-1-specific CTL in the peripheral blood and sigmoid colon mucosa of infected subjects not on antiretroviral therapy by enzyme-linked immunospot analysis with 53 peptide pools spanning all viral proteins. Comparisons of blood and mucosal CTL revealed that the magnitude of pool-specific responses is correlated within each individual (mean r2 = 0.82 +/- 0.04) and across all individuals (r2 = 0.75; P < 0.001). Overall, 85.1% of screened peptide pools yielded concordant negative or positive results between compartments. CTL targeting was also closely related between blood and mucosa, with Nef being the most highly targeted (mean of 2.4 spot-forming cells [SFC[/10(6) CD8+ T lymphocytes/amino acid [SFC/CD8/aa]), followed by Gag (1.5 SFC/CD8/aa). Finally, comparisons of peptide pool responses seen in both blood and mucosa (concordant positives) versus those seen only in one but not the other (discordant positives) showed that most discordant results were likely an artifact of responses being near the limit of detection. Overall, these results indicate that HIV-1-specific CTL responses in the blood mirror those seen in the mucosal compartment in natural chronic infection. For protective or immunotherapeutic vaccination, it will be important to determine whether immunity is elicited in the mucosa, which is a key site of initial infection and subsequent HIV-1 replication in vivo.

Immunologic profile of highly exposed yet HIV type 1-seronegative men.

The host immune factors that determine susceptibility to HIV-1 infection are poorly understood. We compared multiple immunologic parameters in three groups of HIV-1-seronegative men: 14 highly exposed (HR10), 7 previously reported possibly to have sustained transient infection (PTI), and a control group of 14 low risk blood bank donors (BB). Virus-specific cellular immune assays were performed for CD4(+) T helper cell responses, CD8(+) cytotoxic T lymphocyte activity, CD8(+) cell chemokine release, and CD8(+) cell-derived antiviral soluble factor activity. General immune parameters evaluated included CCR5 genotype and phenotype, interferon alpha production by PBMCs, leukocyte subset analysis, and detailed T lymphocyte phenotyping. Comparisons revealed no detectable group-specific differences in measures of virus-specific immunity. However, the HR10 group differed from the BB group in several general immune parameters, having higher absolute monocyte counts, higher absolute CD8(+) T cell counts and percentages, lower naive and higher terminal effector CD8(+) cells, and lower levels of CD28(+)CD8(+) cells. These changes were not associated with seropositivity for other chronic viral infections. The PTI men appeared to have normal levels of monocytes and slightly elevated levels of CD8(+) T cells (also with increased effector and decreased naive cells). Although we cannot entirely exclude the contribution of other chronic viral infections, these findings suggest that long-lived systemic cellular antiviral immunity as detected by our assays is not a common mechanism for resistance to infection, and that resistance may be multifactorial. General immune parameters reflected by CD8(+) T cell levels and activation, and monocyte concentrations may affect the risk of infection with HIV-1, and/or serve as markers of exposure.