Ellen S. Chan

Sex differences in the Toll-like receptor–mediated response of plasmacytoid dendritic cells to HIV-1

Manifestations of viral infections can differ between women and men, and marked sex differences have been described in the course of HIV-1 disease. HIV-1–infected women tend to have lower viral loads early in HIV-1 infection but progress faster to AIDS for a given viral load than men. Here we show substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce markedly more interferon-α (IFN-α) in response to HIV-1–encoded Toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in stronger secondary activation of CD8+ T cells. In line with these in vitro studies, treatment-naive women chronically infected with HIV-1 had considerably higher levels of CD8+ T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs may account for higher immune activation in women compared to men at a given HIV-1 viral load and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a new approach to reduce HIV-1–associated pathology.

The Effect of Raltegravir Intensification on Low-level Residual Viremia in HIV-Infected Patients on Antiretroviral Therapy: A Randomized Controlled Trial

In a double-blind trial, Rajesh Gandhi and colleagues detect no significant reduction in viral load after people with low-level HIV viremia added an integrase inhibitor to their treatment regimen.

Incomplete Reconstitution of T Cell Subsets on Combination Antiretroviral Therapy in the AIDS Clinical Trials Group Protocol 384

BACKGROUND Initiation of combination antiretroviral therapy (ART) results in higher total CD4 cell counts, a surrogate for immune reconstitution. Whether the baseline CD4 cell count affects reconstitution of immune cell subsets has not been well characterized. METHODS Using data from 978 patients (621 with comprehensive immunological assessments) from the AIDS [Acquired Immunodeficiency Syndrome] Clinical Trials Group protocol 384, a randomized trial of initial ART, we compared reconstitution of CD4(+), CD4(+) naive and memory, CD4(+) activation, CD8(+), CD8(+) activation, B, and natural killer cells among patients in different baseline CD4(+) strata. Reference ranges for T cell populations in control patients negative for human immunodeficiency virus (HIV) infection were calculated using data from AIDS Clinical Trials Group protocol A5113. RESULTS Patients in the lower baseline CD4(+) strata did not achieve total CD4(+) cell counts similar to those of patients in the higher strata during 144 weeks of ART, although CD4(+) cell count increases were similar. Ratios of CD4(+) naive-memory cell counts and CD4(+):CD8(+) cell counts remained significantly reduced in patients with lower baseline CD4(+) cell counts (<or=350 cells/mm(3)). These immune imbalances were most notable for those initiating ART with a baseline CD4(+) cell count <or=200 cells/mm(3), even after adjustment for baseline plasma HIV RNA levels. CONCLUSIONS After nearly 3 years of ART, T cell subsets in patients with baseline CD4(+) cell counts >350 cells/mm(3) achieved or approached the reference range those of control individuals without HIV infection. In contrast, patients who began ART with <or=350 CD4(+) cells/mm(3) generally did not regain normal CD4(+) naive-memory cell ratios. These results support current guidelines to start ART at a threshold of 350 cells/mm(3) and suggest that there may be immunological benefits associated with initiating therapy at even higher CD4(+) cell counts.

No effect of raltegravir intensification on viral replication markers in the blood of HIV-1-infected patients receiving antiretroviral therapy

Background: Controversy continues regarding the extent of ongoing viral replication in HIV-1–infected patients on effective antiretroviral therapy (ART). Adding an additional potent agent, such as raltegravir, to effective ART in patients with low-level residual viremia may reveal whether there is ongoing HIV-1 replication. Methods: We previously reported the outcome of a randomized placebo-controlled study of raltegravir intensification in patients on ART with HIV-1 RNA <50 copies per milliliter that showed no effect on residual viremia measured by single copy assay. We now report the effects of raltegravir intensification in that trial on other potential measures of ongoing HIV-1 replication as follows: 2-LTR HIV-1 circles, total cellular HIV-1 DNA, and T-cell activation. Results: Of 50 patients tested, 12 (24%) had 2-LTR circles detected at baseline. Patients who were 2-LTR–positive had higher plasma HIV-1 RNA and HIV-1 DNA levels than 2-LTR–negative individuals. At week 12 of raltegravir intensification, there was no change from baseline in 2-LTR circles, in total HIV-1 DNA or in the ratio of 2-LTR circles to total HIV-1 DNA. There was also no change in markers of T-cell activation. Conclusions: In HIV-1–infected individuals on effective ART, we find no evidence of ongoing viral replication in the blood that is suppressible by raltegravir intensification. The results imply that raltegravir intensification alone will not eradicate HIV-1 infection.

A randomized therapeutic vaccine trial of canarypox-HIV-pulsed dendritic cells vs. canarypox-HIV alone in HIV-1-infected patients on antiretroviral therapy

Targeting canarypox (CP)-HIV vaccine to dendritic cells (DCs) elicits anti-HIV-1 immune responses in vitro. We conducted a phase I/II clinical trial to evaluate whether adding DC to a CP-HIV vaccine improved virologic control during analytic treatment interruption (ATI) in HIV-1-infected subjects. Twenty-nine subjects on suppressive antiretroviral therapy were randomized to vaccination with autologous DCs infected with CP-HIV+keyhole limpet hemocyanin (KLH) (arm A, n=14) or CP-HIV+KLH alone (arm B, n=15). The mean viral load (VL) setpoint during ATI did not differ between subjects in arms A and B. A higher percentage of subjects in the DC group had a VL setpoint < 5,000 c/mL during ATI (4/13 or 31% in arm A compared with 0/13 in arm B, p=0.096), but virologic control was transient. Subjects in arm A had a greater increase in KLH lymphoproliferative response than subjects in arm B; however, summed ELISPOT responses to HIV-1 antigens did not differ by treatment arm. We conclude that a DC-CP-HIV vaccine is well-tolerated in HIV-1-infected patients, but does not lower VL setpoint during ATI compared with CP-HIV alone. New methods to enhance the immunogenicity and antiviral efficacy of DC-based vaccines for HIV-1 infection are needed.

Interleukin-2 Increases CD4+ lymphocyte numbers but does not enhance responses to immunization: results of A5046s.

To ascertain whether CD4(+) lymphocyte increases induced by interleukin (IL)-2 enhanced in vivo immune responses, 38 human immunodeficiency virus (HIV)-infected patients who had received highly active antiretroviral therapy (HAART) or HAART and IL-2 for at least 60 weeks were immunized with tetanus toxoid, inactivated glycoprotein 120-depleted HIV-1, and hepatitis A and B vaccines. Despite dramatic increases in CD4(+) lymphocyte counts, IL-2 did not enhance immunization responses.

Reanalysis of Coreceptor Tropism in HIV-1–Infected Adults Using a Phenotypic Assay with Enhanced Sensitivity

The enhanced-sensitivity Trofile assay (TF-ES; Monogram Biosciences) was used to retest coreceptor tropism samples from 4 different cohorts of HIV-1-infected patients. Nine percent to 26% of patients with CCR5-tropic virus by the original Trofile assay had CXCR4-using virus by TF-ES. Lower CD4 cell counts were associated with CXCR4-using virus in all cohorts.

Vitamin D and Calcium Attenuate Bone Loss With Antiretroviral Therapy Initiation: A Randomized Trial

Background Antiretroviral therapy (ART) initiation for HIV-1 infection is associated with 2-6% loss in bone mineral density (BMD).Context Patients with HIV who initiate antiretroviral therapy (ART) lose bone mineral density (BMD). The role of supplementation with vitamin D and calcium in preventing such loss is unknown. Contribution In a randomized clinical trial of patients with HIV who were initiating ART, BMD, 25-hydroxyvitamin D, parathyroid hormone, phosphate metabolism, and markers of bone turnover were measured at baseline. Patients who then received vitamin D3 plus calcium along with ART had smaller declines in BMD of the hip and spine than those who received placebo along with ART. Caution Nearly all of the patients were men. Implication Supplementation with vitamin D and calcium may prevent bone loss in patients with HIV initiating ART. Antiretroviral therapy (ART) has transformed HIV infection from a terminal disease to a manageable chronic illness. Although incidence of AIDS-defining conditions has decreased, it has increased for other comorbid conditions (1), including osteoporosis and fragility fractures (27). Both viral and host factorsHIV infection mediated by certain viral proteins, HIV-associated inflammation, lifestyle and behavioral factors, underlying genetic predisposition, comorbid conditions, and ARTprobably contribute to bone loss and fracture risk (814). Studies of ART initiation have confirmed that 2% to 6% of hip and spine BMD is lost over the first 24 to 48 weeks after initiation, with subsequent stabilization (1518). The magnitude of bone loss is similar to that seen with glucocorticoids or during the first year of menopausal transition (19, 20). Initial loss is marked by an increase in serum bone resorption markers followed by a delayed compensatory increase in bone formation markers (21); therefore, this catabolic window, a high bone turnover state with excess resorption, may be a central mechanism of bone loss with ART initiation. Tenofovir disoproxil fumarate (TDF), a nucleotide analogue reverse transcriptase inhibitor, has been associated with greater bone loss than other reverse transcriptase inhibitors (15, 16). Use of TDF is associated with increased parathyroid hormone (PTH), elevated vitamin D binding protein, and reduced free 1,25-dihydroxyvitamin D [1,25-(OH)2D3] levels (22, 23), suggesting that functional vitamin D deficiency with TDF use potentially contributes to excess bone loss. Initiation of efavirenz (EFV), a nonnucleoside reverse transcriptase inhibitor, is associated with a 6.2- to 12.5-nmol/L decrease in 25-hydroxyvitamin D [25-(OH)D] levels (24, 25). Efavirenz induces cytochrome P450 enzymes involved in vitamin D metabolism and may accelerate the catabolism of 25-(OH)D and 1,25-(OH)2D3, the latter being the active vitamin D metabolite (26). These ART agents are combined with emtricitabine (FTC), a nucleoside analogue reverse transcriptase inhibitor, into a fixed-dose combination, once-a-day pill (EFV/FTC/TDF) that is highly effective for treating HIV (27). Beyond effects on bone metabolism, vitamin D has immunomodulatory effects mediated through the vitamin D receptor present on cells in both the innate and the adaptive immune system (28, 29). Vitamin D increases monocyte expression of CD14 and cathelicidin, molecules involved in innate immune responses (30, 31); down-regulates cytokine expression in activated T cells; and suppresses T-cell proliferation and production of interferon- and interleukin (IL)-2, thus reducing the net state of inflammation (32, 33). These pathways are particularly relevant in HIV infection, in which excess monocyte and T-cell activation are important drivers of morbidity and mortality (3437). We hypothesized that high-dose vitamin D and calcium supplementation would attenuate bone loss associated with initiation of EFV/FTC/TDF. In addition, we evaluated immunomodulatory effects of vitamin D in the setting of HIV treatment. We report the results of the AIDS Clinical Trials Group A5280 trial, a multicenter, randomized, double-blind, placebo-controlled study assessing the effect of daily oral doses of 4000 IU of vitamin D3 and 1000 mg of calcium carbonate in adults with HIV initiating their first ART regimen with EFV/FTC/TDF. Methods Patients with ART-naive HIV without evidence of resistance to the antiretrovirals in the regimen and with HIV-1 RNA levels greater than 1000 copies/mL were eligible if they met the following criteria: screening 25-(OH)D level between 25 and 188 nmol/L; creatinine clearance of 60 mL/min/1.73 m2 or greater, estimated by the CockcroftGault formula; and serum calcium level less than 2.6 mmol/L. We excluded patients with daily calcium supplementation greater than 500 mg, daily vitamin D supplementation greater than 800 IU, any bisphosphonate use, recent steroid or chemotherapy treatments, clinically active thyroid disease, active substance or alcohol abuse, history of fragility fracture, documented osteoporosis, nephrolithiasis, or weight greater than 136 kg (limit of the dual-energy x-ray absorptiometry [DXA] scanner). Pregnant and breastfeeding women were also excluded. We did 3-day dietary recalls at entry to estimate vitamin D and calcium intake. Patients were randomly assigned to 4000 IU of cholecalciferol (vitamin D3) daily plus 500 mg of calcium carbonate twice daily or identically matching placebos (Tishcon) and were counseled to take with food to facilitate absorption. Some experts consider the current upper U.S. Dietary Reference Intake of 2000 IU to be below actual physiologic requirements; therefore, we tested the highest supplementation without risk for toxicity (3840). A dose of 4000 IU of vitamin D3 is the highest daily dose considered safe for adults by the Institute of Medicine (41) and has previously been evaluated in persons with HIV with excellent tolerability and safety data (42). Patients were randomly assigned in a 1:1 ratio using permuted blocks stratified by screening serum 25-(OH)D levels (50 and >50 nmol/L). Randomization was done using Web-based access to a central computer system maintained by the Frontier Science & Technology Research Foundation (Buffalo, New York). The institutional review boards of all participating sites approved the study, and all patients provided written informed consent. The primary end point was percentage of change in total hip BMD from baseline to 48 weeks. Secondary end points included percentage of change in lumbar spine BMD at 48 weeks, change in plasma 25-(OH)D and PTH levels, markers of bone turnover, soluble inflammatory biomarkers, and CD4 cell counts at 24 and 48 weeks. Incidence of hypercalcemia and nephrolithiasis was monitored. All DXA scans were read in a blinded fashion at the Body Composition Analysis Center at Tufts Medical Center (Boston, Massachusetts) using a standardized protocol. Biomarker Assays Screening was done at local laboratories certified by the Clinical Laboratory Improvement Amendments. Serum samples were stored at 70C at the Irving Institute Biomarkers Core Laboratory at Columbia University Medical Center (New York, New York) and underwent batched analysis. We measured 25-(OH)D2 and 25-(OH)D3 by liquid chromatographymass spectrometry, intact PTH by a radioimmunoassay (Scantibodies), procollagen-1 N-terminal peptide (P1NP) by a radioimmunoassay (Immunodiagnostic Systems), C-terminal telopeptide of type 1 collagen (CTX) by an enzyme-linked immunosorbent assay [ELISA] (Immunodiagnostic Systems), IL-6 by an ELISA (R&D Systems), soluble tumor necrosis factor receptor (sTNFR) by an ELISA (R&D Systems), and soluble CD14 by an ELISA (R&D Systems). Inflammatory biomarkers were chosen based on association with relevant end points (4346). All biomarkers, except for 25-(OH)D, were measured in duplicate, and values were averaged for analysis. Statistical Analysis To provide an intention-to-treat analysis, evaluations were done regardless of treatment change or discontinuation. Two patients who did not have the correct vitamin D test done at baseline were excluded from the efficacy analyses. Stratified Wilcoxon rank-sum tests were used to evaluate for distribution shifts between the treatment groups, which were stratified by screening vitamin D levels. Fisher exact tests and Wilcoxon rank-sum tests were used to evaluate for differences between groups for categorical and continuous secondary outcomes, respectively. Wilcoxon signed-rank tests were used to evaluate change within a treatment group. The 95% CIs for median changes within a group were estimated using a distribution-free method via percentiles. For the BMD outcomes, modification of the treatment effect by screening vitamin D stratum was evaluated by means of linear regression. Analyses of the primary outcomes of change in hip and spine BMD from baseline to 48 weeks used a multiple-imputation approach to fill in missing data with a Markov-chain Monte Carlo method. A prespecified complete-case approach was used for analyses by stratum and to assess interactions. Five imputations were used, based on screening 25-(OH)D strata, age, sex, and race/ethnicity. All statistical tests were 2-sided and interpreted at the 5% nominal level of significance without adjustment for multiple comparisons. Analyses were done using the FREQ, UNIVARIATE, REG, MI, and MIANALYZE procedures in SAS, version 9.2 (SAS Institute), and STRATIFY and PAIRED procedures in Proc-StatXact, version 9 (Cytel). Role of the Funding Source The National Institute of Allergy and Infectious Diseases funded the study. Industry sponsors (Bristol-Myers Squibb, Gilead Science, and Tishcon) provided antiretrovirals, vitamin D, calcium, and matching placebos, and additional funding was provided for completion of DXA scans and laboratory assays. The National Institute of Allergy and Infectious Diseases had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication. The manuscript was reviewed by ind

Safety and Immunogenicity of Therapeutic DNA Vaccination in Individuals Treated with Antiretroviral Therapy during Acute/Early HIV-1 Infection

Background An effective therapeutic vaccine that could augment immune control of HIV-1 replication may abrogate or delay the need for antiretroviral therapy. AIDS Clinical Trials Group (ACTG) A5187 was a phase I/II, randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of an HIV-1 DNA vaccine (VRC-HVDNA 009-00-VP) in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. (clinicaltrials.gov NCT00125099) Methods Twenty healthy HIV-1 infected subjects who were treated with antiretroviral therapy during acute/early HIV-1 infection and had HIV-1 RNA<50 copies/mL were randomized to receive either vaccine or placebo. The objectives of this study were to evaluate the safety and immunogenicity of the vaccine. Following vaccination, subjects interrupted antiretroviral treatment, and set-point HIV-1 viral loads and CD4 T cell counts were determined 17–23 weeks after treatment discontinuation. Results Twenty subjects received all scheduled vaccinations and discontinued antiretroviral therapy at week 30. No subject met a primary safety endpoint. No evidence of differences in immunogenicity were detected in subjects receiving vaccine versus placebo. There were also no significant differences in set-point HIV-1 viral loads or CD4 T cell counts following treatment discontinuation. Median set-point HIV-1 viral loads after treatment discontinuation in vaccine and placebo recipients were 3.5 and 3.7 log10 HIV-1 RNA copies/mL, respectively. Conclusions The HIV-1 DNA vaccine (VRC-HIVDNA 009-00-VP) was safe but poorly immunogenic in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. Viral set-points were similar between vaccine and placebo recipients following treatment interruption. However, median viral load set-points in both groups were lower than in historical controls, suggesting a possible role for antiretroviral therapy in persons with acute or early HIV-1 infection and supporting the safety of discontinuing treatment in this group. Trial Registration Clinicaltrials.gov NCT00125099

Higher Expression of Several Interferon-Stimulated Genes in HIV-1-Infected Females After Adjusting for the Level of Viral Replication

BACKGROUND Clinical studies have shown faster disease progression and stronger immune activation in human immunodeficiency virus (HIV)-1-infected females when compared with males for the same level of HIV-1 replication. Here we determine whether the elevated levels of HIV-1-induced interferon-alpha (IFN-α) production observed in females are associated with higher interferon-stimulated gene (ISG) expression levels in T cells, hence suggesting type-I IFN as a mechanism for the higher HIV-1-associated immune activation observed. METHODS T-cell and dendritic cell populations were isolated from treatment-naive chronically HIV-1-infected individuals enrolled in the Adult Clinical Trials Group 384 by fluorescence-activated cell sorting. The expression of 98 genes involved in Toll-like receptor and type I IFN signaling pathways were quantified using Nanostring technology. RESULTS Several ISGs were significantly correlated with HIV-1 viral load and/or CD4(+) T-cell count. Higher expression levels of a subset of these ISGs were observed in cells derived from females as compared to males after adjusting for viral load and were correlated to higher levels of T-cell activation. CONCLUSION These data show that higher IFN-α production is associated with higher ex vivo expression of several ISGs in females. This might contribute to higher levels of immune activation and the observed faster HIV-1 disease progression in females for a given level of viral replication.