Measurement of plasma HIV-1 RNA below the limit of quantification (<20 copies/mL) of commercial assays with the integrase HIV RNA single-copy assay.

BACKGROUND: Plasma HIV-1 RNA (viral load, VL) is measured routinely in HIV-infected persons with FDA-approved commercially available assays such as the Cobas-TaqMan HIV-1 Assay v2.0. This assay provides quantification of viremia ≥20 copies/mL. More sensitive methods, able to quantify low-level persistent viremia below the detection limit of commercially available assays, are needed to assess the impact of current HIV cure strategies on viremia. OBJECTIVES: The novel integrase HIV-1 RNA single-copy assay (iSCA) was evaluated for measurement of low-level persistent viremia in clinical trial samples (n = 151) from subjects participating in Gilead HIV clinical research. STUDY DESIGN: Paired plasma samples from HIV-1-infected patients treated with combination ART were assessed using both HIV-1 Cobas-TaqMan and iSCA; results from the two assays were compared. RESULTS: Paired Cobas-TaqMan/iSCA data were obtained for 151 HIV-infected adults. Most samples (117/151, 77%) had non-quantifiable Cobas-TaqMan result, either <20 copies/mL ("<20") or "Target Not Detected" (TND). All 117 non-quantified samples were quantified with iSCA and showed higher HIV-1 RNA levels in samples with <20 than TND Cobas-TaqMan results (p < 0.0001). CONCLUSIONS: In this large sample collection from virologically suppressed HIV-infected adults, use of iSCA led to quantification of low-level viremia below the limit of detection of the Cobas-TaqMan assay in all 117 previously non-quantifiable plasma samples. These data confirm the value of the iSCA as a helpful addition to the classical HIV VL assays and its potential for use in HIV cure studies to assess whether experimental interventions alter viremia.

HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells.

The persistence of HIV-infected cells in individuals on suppressive combination antiretroviral therapy (cART) presents a major barrier for curing HIV infections. HIV integrates its DNA into many sites in the host genome; we identified 2410 integration sites in peripheral blood lymphocytes of five infected individuals on cART. About 40% of the integrations were in clonally expanded cells. Approximately 50% of the infected cells in one patient were from a single clone, and some clones persisted for many years. There were multiple independent integrations in several genes, including MKL2 and BACH2; many of these integrations were in clonally expanded cells. Our findings show that HIV integration sites can play a critical role in expansion and persistence of HIV-infected cells.

Prevalence of XMRV nucleic acid and antibody in HIV-1-Infected men and in men at risk for HIV-1 Infection.

Xenotropic MLV-Related Virus (XMRV) was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS). Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs) and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA). Although 9 of 332 (2.7%) samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy.

BACKGROUND: Combination antiretroviral therapy suppresses but does not eradicate human immunodeficiency virus type 1 (HIV-1) in infected persons, and low-level viremia can be detected despite years of suppressive antiretroviral therapy. Short-course (28-day) intensification of standard antiretroviral combination therapy is a useful approach to determine whether complete rounds of HIV-1 replication in rapidly cycling cells contribute to persistent viremia. We investigated whether intensification with the integrase inhibitor raltegravir decreases plasma HIV-1 RNA levels in patients receiving suppressive antiretroviral therapy. METHODS: Subjects (n = 10) with long-term HIV-1 suppression receiving combination antiretroviral regimens had their regimens intensified for 4 weeks with raltegravir. Plasma HIV-1 RNA level was determined before, during, and after the 4-week intensification period, using a sensitive assay (limit of detection, 0.2 copies of HIV-1 RNA/mL of plasma). A 4-week intensification course was chosen to investigate potential HIV-1 replication in cells with relatively short (approximately 1-14-day) half-lives. RESULTS: There was no evidence in any subject of a decline in HIV-1 RNA level during the period of raltegravir intensification or of rebound after discontinuation. Median levels of HIV-1 RNA before (0.17 log10 copies/mL), during (0.04 log10 copies/mL), and after (0.04 log10 copies/mL) raltegravir intensification were not significantly different (P > .1 for all comparisons in parametric analyses). High-performance liquid chromatography and mass spectroscopy experiments confirmed that therapeutic levels of raltegravir were achieved in plasma during intensification. CONCLUSIONS: Intensification of antiretroviral therapy with a potent HIV-1 integrase inhibitor did not decrease persistent viremia in subjects receiving suppressive regimens, indicating that rapidly cycling cells infected with HIV-1 were not present. Eradication of HIV-1 from infected persons will require new therapeutic approaches. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00618371.

Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy.

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

Human immunodeficiency virus type 1 population genetics and adaptation in newly infected individuals.

Studies on human immunodeficiency virus type 1 (HIV-1) diversity are critical for understanding viral pathogenesis and the emergence of immune escape variants and for design of vaccine strategies. To investigate HIV-1 population genetics, we used single-genome sequencing to obtain pro-pol and env sequences from longitudinal samples (n = 93) from 14 acutely or recently infected patients. The first available sample after infection for 12/14 patients revealed HIV-1 populations with low genetic diversity, consistent with transmission or outgrowth of a single variant. In contrast, two patients showed high diversity and coexistence of distinct virus populations in samples collected days after a nonreactive enzyme-linked immunosorbent assay or indeterminate Western blot, consistent with transmission or outgrowth of multiple variants. Comparison of PR and RT sequences from the first sample for all patients with the consensus subgroup B sequence revealed that nearly all nonsynonymous differences were confined to identified cytotoxic T-lymphocyte (CTL) epitopes. For HLA-typed patients, mutations compared to the consensus in transmitted variants were found in epitopes that would not be recognized by the patient's major histocompatibility complex type. Reversion of transmitted mutations was rarely seen over the study interval (up to 5 years). These data indicate that acute subtype B HIV-1 infection usually results from transmission or outgrowth of single viral variants carrying mutations in CTL epitopes that were selected prior to transmission either in the donor or in a previous donor and that reversion of these mutations can be very slow. These results have important implications for vaccine strategies because they imply that some HLA alleles could be compromised in newly acquired HIV infections.

A robust measure of HIV-1 population turnover within chronically infected individuals.

A simple nonparameteric test for population structure was applied to temporally spaced samples of HIV-1 sequences from the gag-pol region within two chronically infected individuals. The results show that temporal structure can be detected for samples separated by about 22 months or more. The performance of the method, which was originally proposed to detect geographic structure, was tested for temporally spaced samples using neutral coalescent simulations. Simulations showed that the method is robust to variation in samples sizes and mutation rates, to the presence/absence of recombination, and that the power to detect temporal structure is high. By comparing levels of temporal structure in simulations to the levels observed in real data, we estimate the effective intra-individual population size of HIV-1 to be between 10(3) and 10(4) viruses, which is in agreement with some previous estimates. Using this estimate and a simple measure of sequence diversity, we estimate an effective neutral mutation rate of about 5 x 10(-6) per site per generation in the gag-pol region. The definition and interpretation of estimates of such "effective" population parameters are discussed.

Longitudinal patterns of HIV type 1 RNA among individuals with late disease progression.

Longitudinal measurements of plasma HIV RNA were analyzed using novel segmented regression models for 62 men in the Multicenter AIDS Cohort Study who at enrollment in 1985 were HIV seropositive and who had stable CD4+ lymphocyte counts and no clinical disease progression for a 6-year period between 1985 and 1991. Through 1996, 20 of the men developed clinical AIDS or died (late progressors) and 42 remained asymptomatic (nonprogressors). Using segmented regression model methods, we estimated, for each individual, the time when a change in HIV RNA trajectory was most likely to have occurred. Prior to this time, late progressors and nonprogressors had stable plasma HIV RNA levels, although the mean level in late progressors was 0.42 log10 copies/ml higher than in nonprogressors (p = 0.018). Furthermore, late progressors showed significant increases in HIV RNA levels of 0.23 log10 copies/ml/year (1.7-fold increase/year). This increase in HIV RNA in the late progressors began approximately 1.1 years prior to the onset of their decline in CD4+ lymphocytes, and 4.8 years prior to the onset of AIDS. These results provide evidence that an increase in the slope of plasma levels of HIV RNA is a sign of incipient progression of HIV disease.

Antiretroviral activity and safety of abacavir in combination with selected HIV-1 protease inhibitors in therapy-naive HIV-1-infected adults.

OBJECTIVE: To assess antiretroviral efficacy and safety of abacavir in combination with selected HIV-1 protease inhibitors. DESIGN: A 48-week, open-label study. MATERIALS AND METHODS: Eighty-two antiretroviral naive HIV-1-infected adults (CD4 cell count > or = 100 cells/mm3, plasma HIV-1 RNA > or = 5,000 copies/ml) were randomly assigned to receive abacavir (300 mg twice daily) in combination with standard doses of one of five protease inhibitors: indinavir, saquinavir soft-gel, ritonavir, nelfinavir or amprenavir. Adults who met protocol-defined switch criteria at or after week 8 could modify their randomized therapy. Antiretroviral activity was assessed by the proportion of subjects with plasma HIV-1 RNA < or = 400 and < or = 50 copies/ml, and by changes in plasma HIV-1 RNA levels and CD4 cell counts. Safety was assessed by monitoring clinical adverse events and laboratory abnormalities. RESULTS: At week 48, the proportion of subjects in the indinavir, saquinavir, ritonavir, nelfinavir and amprenavir groups with plasma HIV-1 RNA < or = 400 copies/ml was 53, 50, 50, 41 and 56%, respectively, and the proportion with HIV-1 RNA < or = 50 copies/ml was 47, 56, 50, 47, and 44%, respectively (by intent-to-treat analysis). Median reductions from baseline in plasma HIV-1 RNA for each group ranged from 1.7 to 2.4 log10 copies/ml. The median CD4 cell count increase from baseline was 195, 131, 116, 136 and 259 cells/mm3 in the indinavir, saquinavir, ritonavir, nelfinavir, and amprenavir groups, respectively. Overall, the most common adverse events attributed to study drugs were diarrhoea, nausea, malaise/fatigue, headache and perioral paresthesia. The frequency of treatment-limiting adverse events did not differ between groups. CONCLUSIONS: Abacavir is safe and effective when used in combination with a protease inhibitor.

Alkylglycerol prodrugs of phosphonoformate are potent in vitro inhibitors of nucleoside-resistant human immunodeficiency virus type 1 and select for resistance mutations that suppress zidovudine resistance.

Phosphonoformate (foscarnet; PFA) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), but its use for the treatment of HIV-1 infection is limited by toxicity and the lack of an orally bioavailable formulation. Alkylglycerol-conjugated prodrugs of PFA (1-O-octadecyl-sn-glycero-3-PFA [B-PFA]) having sn-2 substituents of hydrogen (deoxybatyl-PFA [DB-PFA]), methyl (MB-PFA), or ethyl (EB-PFA) are more-potent inhibitors of wild-type HIV-1 in vitro than unmodified PFA and are orally bioavailable in mice. We have evaluated the activities of these compounds against a panel of nucleoside-resistant HIV-1 variants and have characterized the resistant variants that emerge following in vitro selection with the prodrugs. Except for an HIV-1 variant encoding the K65R mutation in RT that exhibited 3.3- to 8.2-fold resistance, the nucleoside-resistant viruses included in the panel were sensitive to the PFA prodrugs (<3-fold increase in 50% inhibitory concentration), including multinucleoside-resistant variants encoding the Q151M complex of mutations or the T69S[SA] insert. Viruses resistant to the PFA prodrugs (>10-fold) were selected in vitro after 15 or more serial passages of HIV-1 in MT-2 cells in escalating prodrug concentrations. Mutations detected in the resistant viruses were S117T, F160Y, and L214F (DB-PFA); M164I and L214F (MB-PFA); and W88G and L214F (EB-PFA). The S117T, F160Y, and M164I mutations have not been previously identified. Generation of recombinant viruses encoding the single and double mutations confirmed their roles in prodrug resistance, including 214F, which generally increased the level of resistance. When introduced into a zidovudine (AZT)-resistant background (67N 70R 215F 219Q), the W88G, S117T, F160Y, and M164I mutations reversed AZT resistance. This suppression of AZT resistance is consistent with the effects of other foscarnet resistance mutations that reduce ATP-dependent removal of AZT monophosphate from terminated template primers. The favorable activity and resistance profiles of these PFA prodrugs warrant their further evaluation as clinical candidates.

Immunologic and virologic response to highly active antiretroviral therapy in the Multicenter AIDS Cohort Study.

OBJECTIVES: To evaluate prior antiretroviral therapy experience and host characteristics as determinants of immunologic and virologic response to highly active antiretroviral therapy (HAART). METHODS: We studied 397 men from the Multicenter AIDS Cohort Study (MACS) who initiated HAART between October 1995 and March 1999. CD4 cell count and HIV-1 RNA responses to HAART were measured at the first visit following HAART (short-term) and extending from the first visit to approximately 33 months after HAART (long-term). Prior antiretroviral experience was classified into three groups based on antiretroviral therapy use during the 5 years prior to HAART. Age, race and host genetic characteristics also were assessed for their effects on treatment response. RESULTS: Better short- and long-term CD4 cell and HIV-1 RNA responses were observed in the treatment-naive users. Intermittently and consistently experienced users did not significantly differ in response. Whereas race did not independently affect response, among those initiating HAART with > 400 x 10(6) CD4 cells/l, younger age and the Delta32 CCR5 genotype were associated with a better short-term CD4 cell response. There was a suggestion that having the protective CCR5 genotype also was associated with a better long-term CD4 cell response. CONCLUSION: Immunologic and virologic response to HAART was stronger in individuals who had no prior experience with the antiretroviral therapy agents subsequently included in their initial HAART regimen. Age, level of immune competence and immunogenetics appeared to play a role in the subsequent immune reconstitution following use of highly effective HIV therapy.

Restoration of anti-human immunodeficiency virus type 1 (HIV-1) responses in CD8+ T cells from late-stage patients on prolonged antiretroviral therapy by stimulation in vitro with HIV-1 protein-loaded dendritic cells.

We demonstrate that dendritic cells loaded in vitro with human immunodeficiency virus type 1 (HIV-1) protein-liposome complexes activate HLA class I-restricted anti-HIV-1 cytotoxic T-lymphocyte and gamma interferon (IFN-gamma) responses in autologous CD8+ T cells from late-stage HIV-1-infected patients on prolonged combination drug therapy. Interleukin-12 enhanced this effect through an interleukin-2- and IFN-gamma-mediated pathway. This suggests that dendritic cells from HIV-1-infected persons can be engineered to evoke stronger anti-HIV-1 CD8+ T-cell reactivity as a strategy to augment antiretroviral therapy.

In vitro anti-HIV-1 activity of sn-2-substituted 1-O-octadecyl-sn-glycero-3-phosphonoformate analogues and synergy with zidovudine.

Monoalkyl ether lipid analogues of foscarnet (phosphonoformate, PFA) exhibit substantially greater in vitro antiviral activity than unmodified PFA against human immunodeficiency virus type 1 (HIV-1). Our previous studies indicate that the length of the alkyl chain must be 14-22 carbons for optimal antiviral activity. To further evaluate the structure-activity relationship, we prepared 1-O-octadecyl-sn-glycerol analogues of PFA with various substitutions at the sn-2 position of glycerol and determined the effect of structure on in vitro antiviral activity and selectivity against HIV-1 in MT-2 and CD4-expressing HeLa cells (HT4-6C). We also studied combinations of zidovudine with PFA, 1-O-octadecyl-2-O-methyl-sn-glycero-3-PFA, or 1-O-octadecyl-sn-glycero-3-PFA and calculated their combination index values against HIV-1 in HT4-6C cells. Alkyl substitutions of one to four carbons at the sn-2 position of glycerol showed optimal antiviral activity. Both alkyl ether lipid analogues were strongly synergistic with zidovudine over a wide range of drug ratios and concentrations. 1-O-octadecyl-sn-glycerol analogues of PFA have selective antiviral properties and warrant further evaluation as potential antiretroviral drugs.

3-year suppression of HIV viremia with indinavir, zidovudine, and lamivudine.

BACKGROUND: Antiretroviral regimens containing HIV protease inhibitors suppress viremia in HIV-infected patients, but the durability of this effect is not known. OBJECTIVE: To describe the 3-year follow-up of patients randomly assigned to receive indinavir, zidovudine, and lamivudine in an ongoing clinical trial. DESIGN: Open-label extension of a randomized, double-blind study. SETTING: Four clinical research units. PATIENTS: 33 HIV-infected, zidovudine-experienced patients with serum HIV RNA levels of at least 20,000 copies/mL and CD4 counts ranging from 50 to 400 cells/mm3. INTERVENTION: Indinavir, zidovudine, and lamivudine. MEASUREMENTS: Safety assessments, HIV RNA levels, CD4 cell counts, and genotypic analyses. RESULTS: After 3 years of follow-up, 21 of 31 contributing patients (68% [95% CI, 49% to 83%]) had serum viral load levels less than 500 copies/mL. Twenty of 31 (65% [CI, 45% to 80%]) had levels less than 50 copies/mL. The median increase in CD4 count from baseline was 230 cells/mm3 (interquartile range, 150 to 316 cells/mm3). Nephrolithiasis occurred in 12 of 33 patients (36%). CONCLUSION: A three-drug regimen of indinavir, zidovudine, and lamivudine suppressed viremia in two thirds of patients for at least 3 years.

In vitro selection of mutations in the human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (-)-beta-D-dioxolane-guanosine and suppress resistance to 3'-azido-3'-deoxythymidine.

Human immunodeficiency virus type 1 (HIV-1) isolates resistant to (-)-beta-D-dioxolane-guanosine (DXG), a potent and selective nucleoside analog HIV-1 reverse transcriptase (RT) inhibitor, were selected by serial passage of HIV-1(LAI) in increasing drug concentrations (maximum concentration, 30 microM). Two independent selection experiments were performed. Viral isolates for which the DXG median effective concentrations (EC(50)s) increased 7.3- and 12.2-fold were isolated after 13 and 14 passages, respectively. Cloning and DNA sequencing of the RT region from the first resistant isolate identified a K65R mutation (AAA to AGA) in 10 of 10 clones. The role of this mutation in DXG resistance was confirmed by site-specific mutagenesis of HIV-1(LAI). The K65R mutation also conferred greater than threefold cross-resistance to 2',3'-dideoxycytidine, 2', 3'-dideoxyinosine, 2',3'-dideoxy-3'-thiacytidine, 9-(2-phosphonylmethoxyethyl)adenine, 2-amino-6-chloropurine dioxolane, dioxolanyl-5-fluorocytosine, and diaminopurine dioxolane but had only marginal effects on 3'-azido-3'-deoxthymidine (AZT) susceptibility. However, when introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q), the K65R mutation reversed the AZT resistance. DNA sequencing of RT clones derived from the second resistant isolate identified the L74V mutation, previously reported to cause ddI resistance. The L74V mutation also decreased the AZT resistance when the mutation was introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q) but to a lesser degree than the K65R mutation did. These findings indicate that DXG and certain 2',3'-dideoxy compounds (e.g., ddI) can select for the same resistance mutations and thus may not be optimal for use in combination. However, the combination of AZT with DXG or its orally bioavailable prodrug (-)-beta-D-2, 6-diaminopurine-dioxolane should be explored because of the suppressive effects of the K65R and L74V mutations on AZT resistance.

Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society-USA Panel.

OBJECTIVE: Assays for drug resistance testing in human immunodeficiency virus type 1 (HIV-1) infection are now available and clinical studies suggest that viral drug resistance is correlated with poor virologic response to new therapy. The International AIDS Society-USA sought to update prior recommendations to provide guidance for clinicians regarding indications for HIV-1 resistance testing. PARTICIPANTS: An International AIDS Society-USA 13-member physician panel with expertise in basic science, clinical research, and patient care involving HIV resistance to antiretroviral drugs was reconvened to provide recommendations for the clinical use of drug resistance testing. EVIDENCE AND CONSENSUS PROCESS: The full panel met regularly between January and October 1999. Resistance and resistance testing data appearing in the last decade through April 2000 and presentations at national and international research conferences were reviewed. Recommendations and considerations were developed by 100% group consensus, acknowledging that definitive data to support final recommendations are not yet available. CONCLUSIONS: Emerging data indicate that despite limitations, resistance testing should be incorporated into patient management in some settings. Resistance testing is recommended to help guide the choice of new regimens after treatment failure and for guiding therapy for pregnant women. It should be considered in treatment-naive patients with established infection, but cannot be firmly recommended in this setting. Testing also should be considered prior to initiating therapy in patients with acute HIV infection, although therapy should not be delayed pending the results. Expert interpretation is recommended given the complexity of results and assay limitations.

CD8(+) T-cell gamma interferon production specific for human immunodeficiency virus type 1 (HIV-1) in HIV-1-infected subjects.

The CD8(+)-T-cell response to human immunodeficiency virus type 1 (HIV-1) is considered to be important in host control of infection and prevention of AIDS. We have developed a single-cell enzyme immunoassay (enzyme-linked immunospot assay) specific for gamma interferon (IFN-gamma) production stimulated by either autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia virus vectors expressing HIV-1 proteins or synthetic peptides representing known HIV-1 CD8(+) cytotoxic T-lymphocyte (CTL) epitopes. Single-cell IFN-gamma production stimulated by HIV-1 Gag-, Pol-, and Env-expressing B-LCL was a reliable measure of HIV-1-specific T-cell immunity in peripheral blood CD8(+) T cells from HIV-1 infected individuals. This method was more sensitive than stimulation of IFN-gamma by direct infection of the cultures with HIV-1-vaccinia virus vectors. Comparable results were found for IFN-gamma production in CD8(+) T cells from HIV-1-negative, cytomegalovirus (CMV)-seropositive, healthy donors stimulated with B-LCL expressing the CMV pp65 lower matrix protein. HIV-1 peptides were immunodominant for both CD8(+) single-cell IFN-gamma production and CTL precursor frequencies. The number of cells producing IFN-gamma decreased in individuals with late-stage HIV-1 infection and was temporally enhanced during combination antiretroviral therapy with two reverse transcriptase nucleoside inhibitors and a protease inhibitor.

Natural history of human immunodeficiency virus type 1 viremia after seroconversion and proximal to AIDS in a large cohort of homosexual men. Multicenter AIDS Cohort Study.

The natural history of human immunodeficiency virus type 1 (HIV-1) viremia and its association with clinical outcomes after seroconversion was characterized in a cohort of homosexual men. HIV-1 RNA was measured by reverse-transcription polymerase chain reaction (RT-PCR) in stored longitudinal plasma samples from 269 seroconverters. Subjects were generally antiretroviral drug naive for the first 3 years after seroconversion. The decline in CD4 lymphocyte counts was strongly associated with initial HIV RNA measurements. Both initial HIV RNA levels and slopes were associated with AIDS-free times. Median slopes were +0.18, +0.09, and -0.01 log10 copies/mL, respectively, for subjects developing AIDS <3, 3-7, and>7 years after seroconversion. In contrast, HIV RNA slopes in the 3 years preceding AIDS and HIV RNA levels at AIDS diagnosis showed little variation according to total AIDS-free time. HIV RNA load at the first HIV-seropositive visit ( approximately 3 months after seroconversion) was highly predictive of AIDS, and subsequent HIV RNA measurements showed even better prognostic discrimination.