Comparison of Self-report to Biomarkers of Recent HIV Infection: Findings from the START Trial.

Identifying individuals with recent HIV infection is critical to research related to viral reservoirs, outbreak investigations and intervention applications. A multi-assay algorithm (MAA) for recency of infection was used in conjunction with self-reported date of infection and documented date of diagnosis to estimate the number of participants recently infected in the Strategic Timing of AntiRetroviral Treatment (START) trial. We tested samples for three groups of participants from START using a MAA: (1) 167 individuals who reported being infected ≤ 6 months before randomization; (2) 771 individuals who did not know their date of infection but were diagnosed within 6 months before randomization; and (3) as controls for the MAA, 199 individuals diagnosed with HIV ≥ 2 years before randomization. Participants with low titer and avidity and a baseline viral load > 400 copies/mL were classified as recently infected. A significantly higher percentage of participants who self-reported being infected ≤ 6 months were classified as recently infected compared to participants diagnosed ≥ 2 years (65% [109/167] vs. 2.5% [5/199], p < 0.001). Among the 771 individuals who did not know their duration of infection at randomization, 206 (26.7%) were classified as recently infected. Among those diagnosed with HIV in the 6 months prior to enrollment, the 373 participants who reported recent infection (n = 167) or who had confirmed recent infection by the MAA (n = 206) differed significantly on a number of baseline characteristics from those who had an unknown date of infection and were not confirmed by the MAA (n = 565). Participants recently infected by self-report and/or MAA were younger, more likely to be Asian, less likely to be black, less likely to be heterosexual, more likely to be enrolled at sites in the U.S., Europe or Australia, and have higher HIV RNA levels. There was good agreement between self-report of recency of infection and the MAA. We estimate that 373 participants enrolled in START were infected within 6 months of randomization. Compared to those not recently infected, these participants were younger, had higher HIV RNA levels and were more likely to come from high income countries and from populations such as MSM with more regular HIV testing.

The effects of storage temperature on PBMC gene expression.

BACKGROUND: Cryopreservation of peripheral blood mononuclear cells (PBMCs) is a common and essential practice in conducting research. There are different reports in the literature as to whether cryopreserved PBMCs need to only be stored ≤ -150 °C or can be stored for a specified time at -80 °C. Therefore, we performed gene expression analysis on cryopreserved PBMCs stored at both temperatures for 14 months and PBMCs that underwent temperature cycling 104 times between these 2 storage temperatures. Real-time RT-PCR was performed to confirm the involvement of specific genes associated with identified cellular pathways. All cryopreserved/stored samples were compared to freshly isolated PBMCs and between storage conditions. RESULTS: We identified a total of 1,367 genes whose expression after 14 months of storage was affected >3 fold in PBMCs following isolation, cryopreservation and thawing as compared to freshly isolated PBMC aliquots that did not undergo cryopreservation. Sixty-six of these genes were shared among two or more major stress-related cellular pathways (stress responses, immune activation and cell death). Thirteen genes involved in these pathways were tested by real-time RT-PCR and the results agreed with the corresponding microarray data. There was no significant change on the gene expression if the PBMCs experienced brief but repetitive temperature cycling as compared to those that were constantly kept ≤ -150 °C. However, there were 18 genes identified to be different when PBMCs were stored at -80 °C but did not change when stored < -150 °C. A correlation was also found between the expressions of 2'-5'- oligoadenylate synthetase (OAS2), a known interferon stimulated gene (IFSG), and poor PBMC recovery post-thaw. PBMC recovery and viability were better when the cells were stored ≤ -150 °C as compared to -80 °C. CONCLUSIONS: Not only is the viability and recovery of PBMCs affected during cryopreservation but also their gene expression pattern, as compared to freshly isolated PBMCs. Different storage temperature of PBMCs can activate or suppress different genes, but the cycling between -80 °C and -150 °C did not produce significant alterations in gene expression when compared to PBMCs stored ≤ -150 °C. Further analysis by gene expression of various PBMC processing and cryopreservation procedures is currently underway, as is identifying possible molecular mechanisms.

HIV populations are large and accumulate high genetic diversity in a nonlinear fashion.

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.

HIV-1 infected monozygotic twins: a tale of two outcomes.

BACKGROUND: Replicate experiments are often difficult to find in evolutionary biology, as this field is inherently an historical science. However, viruses, bacteria and phages provide opportunities to study evolution in both natural and experimental contexts, due to their accelerated rates of evolution and short generation times. Here we investigate HIV-1 evolution by using a natural model represented by monozygotic twins infected synchronically at birth with an HIV-1 population from a shared blood transfusion source. We explore the evolutionary processes and population dynamics that shape viral diversity of HIV in these monozygotic twins. RESULTS: Despite the identical host genetic backdrop of monozygotic twins and the identical source and timing of the HIV-1 inoculation, the resulting HIV populations differed in genetic diversity, growth rate, recombination rate, and selection pressure between the two infected twins. CONCLUSIONS: Our study shows that the outcome of evolution is strikingly different between these two "replicates" of viral evolution. Given the identical starting points at infection, our results support the impact of random epigenetic selection in early infection dynamics. Our data also emphasize the need for a better understanding of the impact of host-virus interactions in viral evolution.

Detection of a substantial rate of multidrug-resistant tuberculosis in an HIV-infected population in South Africa by active monitoring of sputum samples.

BACKGROUND: Tuberculosis (TB) coinfection with human immunodeficiency virus (HIV) is a substantial problem in South Africa. There has been a presumption that drug-resistant strains of TB are common in South Africa, but few studies have documented this impression. METHODS: In Phidisa, a joint observational and randomized HIV treatment study for South African National Defence Force members and dependents, an initiative was launched to test subjects (by use of microbiologic TB test) who appeared to be at high risk. We report results for HIV-infected subjects. RESULTS: TB was identified by culture in 116 (19.9%) of 584 patients selected for sputum examination on the basis of suggestive symptoms. Smear was an insensitive technique for confirming the diagnosis: only 33% of culture-positive patients were identified by smear, with a 0.2% false-positive rate. Of the 107 culture-positive individuals with susceptibility testing, 22 (20.6%) were identified to be multidrug resistant (MDR), and 4 (3.7%) were identified to be extensively drug resistant. Culture-positive cases with a history of TB treatment had more than twice the rate of MDR than those without (27.1% vs 11.9%; P = .05). CONCLUSIONS: TB is common in this cohort of HIV-infected patients. Smear was not a sensitive technique for identifying culture-positive cases in this health system. Drug susceptibility testing is essential to proper patient management because MDR was present in 20.6% of culture-positive patients. Better management strategies are needed to reduce the development of MDR TB, because so many of these patients had received prior antituberculous therapy that was presumably not curative.

Defective human immunodeficiency virus-specific CD8+ T-cell polyfunctionality, proliferation, and cytotoxicity are not restored by antiretroviral therapy.

Identifying the functions of human immunodeficiency virus (HIV)-specific CD8+ T cells that are not merely modulated by the level of virus but clearly distinguish patients with immune control from those without such control is of paramount importance. Features of the HIV-specific CD8+ T-cell response in antiretroviral-treated patients (designated Rx <50) and untreated patients (long-term nonprogressors [LTNP]) matched for very low HIV RNA levels were comprehensively examined. The proliferative capacity of HIV-specific CD8+ T cells was not restored in Rx <50 to the level observed in LTNP, even though HIV-specific CD4+ T-cell proliferation in the two patient groups was comparable. This diminished HIV-specific CD8+ T-cell proliferation in Rx <50 was primarily due to a smaller fraction of antigen-specific cells recruited to divide and not to the numbers of divisions that proliferating cells had undergone. Exogenous interleukin-2 (IL-2) induced proliferating cells to divide further but did not rescue the majority of antigen-specific cells with defective proliferation. In addition, differences in HIV-specific CD8+ T-cell proliferation could not be attributed to differences in cellular subsets bearing a memory phenotype, IL-2 production, or PD-1 expression. Although polyfunctionality of HIV-specific CD8+ T cells in Rx <50 was not restored to the levels observed in LTNP despite prolonged suppression of HIV RNA levels, per-cell cytotoxic capacity was the functional feature that most clearly distinguished the cells of LTNP from those of Rx <50. Taken together, these data suggest that there are selective qualitative abnormalities within the HIV-specific CD8+ T-cell compartment that persist under conditions of low levels of antigen.

Comparisons of CD8+ T cells specific for human immunodeficiency virus, hepatitis C virus, and cytomegalovirus reveal differences in frequency, immunodominance, phenotype, and interleukin-2 responsiveness.

To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8(+) T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8(+) T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8(+) T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8(+) T cells were predominantly CD27(+)45RO(+) for HIV and CD27(-)45RA(+) for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8(+) T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8(+) T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8(+) T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.

Idiopathic CD4+ lymphocytopenia: natural history and prognostic factors.

Idiopathic CD4(+) lymphocytopenia (ICL) is a rare non-HIV-related syndrome with unclear natural history and prognosis. This prospective natural history cohort study describes the clinical course, CD4 T lymphocyte kinetics, outcome, and prognostic factors of ICL. Thirty-nine patients (17 men, 22 women) 25 to 85 years old with ICL were evaluated between 1992 and 2006, and 36 were followed for a median of 49.5 months. Cryptococcal and nontuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In 32, CD4 T-cell counts remained less than 300/mm(3) throughout the study period and in 7 normalized after an average of 31 months. Overall, 15 (41.6%) developed an opportunistic infection in follow-up, 5 (13.8%) of which were "AIDS-defining clinical conditions," and 4 (11.1%) developed autoimmune diseases. Seven patients died, 4 from ICL-related opportunistic infections, within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4 but not CD8 T lymphocytes. CD8 T lymphocytopenia (< 180/mm(3)) and the degree of CD4 T cell activation (measured by HLA-DR expression) at presentation were associated with adverse outcome (opportunistic infection-related death; P = .003 and .02, respectively).

Human immunodeficiency virus viremia induces plasmacytoid dendritic cell activation in vivo and diminished alpha interferon production in vitro.

Human immunodeficiency virus type 1 (HIV-1) infection has been associated with perturbations of plasmacytoid dendritic cells (PDC), including diminished frequencies in the peripheral blood and reduced production of type I interferons (IFNs) in response to in vitro stimulation. However, recent data suggest a paradoxical increase in production of type 1 interferons in vivo in HIV-infected patients compared to uninfected controls. Using a flow cytometric assay to detect IFN-alpha-producing cells within unseparated peripheral blood mononuclear cells, we observed that short-term interruptions of antiretroviral therapy are sufficient to result in significantly reduced IFN-alpha production by PDC in vitro in response to CpG A ligands or inactivated HIV particles. The primary cause of diminished IFN-alpha production was reduced responsiveness of PDC to de novo stimulation, not diminished per cell IFN-alpha production or migration of cells to lymphoid organs. Real-time PCR analysis of purified PDC from patients prior to and during treatment interruptions revealed that active HIV-1 replication is associated with upregulation of type I IFN-stimulated gene expression. Treatment of hepatitis C virus-infected patients with IFN-alpha2b and ribavirin for hepatitis C virus infection resulted in a profound suppression of de novo IFN-alpha production in response to CpG A or inactivated HIV particles, similar to the response observed in HIV-infected patients. Together, these results suggest that diminished production of type I interferons in vitro by PDC from HIV-1-infected patients may not represent diminished interferon production in vivo. Rather, diminished function in vitro is likely a consequence of prior activation via type I interferons or HIV virions in vivo.

Virological outcome after structured interruption of antiretroviral therapy for human immunodeficiency virus infection is associated with the functional profile of virus-specific CD8+ T cells.

A clear understanding of the antiviral effects of CD8(+) T cells in the context of chronic human immunodeficiency virus (HIV) infection is critical for the development of prophylactic vaccines and therapeutics designed to support T-cell-mediated immunity. However, defining the potential correlates of effective CD8(+) T-cell immunity has proven difficult; notably, comprehensive analyses have demonstrated that the size and shape of the CD8(+) T-cell response are not necessarily indicative of efficacy determined by measures of plasma viral load. Here, we conducted a detailed quantitative and qualitative analysis of CD8(+) T-cell responses to autologous virus in a cohort of six HIV-infected individuals with a history of structured interruption of antiretroviral therapy (ART) (SIT). The magnitude and breadth of the HIV-specific response did not, by themselves, explain the changes observed in plasma virus levels after the cessation of ART. Furthermore, mutational escape from targeted epitopes could not account for the differential virological outcomes in this cohort. However, the functionality of HIV-specific CD8(+) T-cell populations upon antigen encounter, determined by the simultaneous and independent measurement of five CD8(+) T-cell functions (degranulation and gamma interferon, macrophage inflammatory protein 1beta, tumor necrosis factor alpha, and interleukin-2 levels) reflected the emergent level of plasma virus, with multiple functions being elicited in those individuals with lower levels of viremia after SIT. These data show that the quality of the HIV-specific CD8(+) T-cell response, rather than the quantity, is associated with the dynamics of viral replication in the absence of ART and suggest that the effects of SIT can be assessed by measuring the functional profile of HIV-specific CD8(+) T cells.

ART suppresses plasma HIV-1 RNA to a stable set point predicted by pretherapy viremia.

Current antiretroviral therapy is effective in suppressing but not eliminating HIV-1 infection. Understanding the source of viral persistence is essential for developing strategies to eradicate HIV-1 infection. We therefore investigated the level of plasma HIV-1 RNA in patients with viremia suppressed to less than 50-75 copies/ml on standard protease inhibitor- or non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy using a new, real-time PCR-based assay for HIV-1 RNA with a limit of detection of one copy of HIV-1 RNA. Single copy assay results revealed that >80% of patients on initial antiretroviral therapy for 60 wk had persistent viremia of one copy/ml or more with an overall median of 3.1 copies/ml. The level of viremia correlated with pretherapy plasma HIV-1 RNA but not with the specific treatment regimen. Longitudinal studies revealed no significant decline in the level of viremia between 60 and 110 wk of suppressive antiretroviral therapy. These data suggest that the persistent viremia on current antiretroviral therapy is derived, at least in part, from long-lived cells that are infected prior to initiation of therapy.

The benefit of immediate compared with deferred antiretroviral therapy on CD4+ cell count recovery in early HIV infection.

OBJECTIVE: To assess the impact of immediate vs. deferred antiretroviral therapy (ART) on CD4 recovery among individuals early in HIV infection. DESIGN: Using serologic markers of early infection together with self-reported dates of infection and HIV diagnosis, ART-naive participants who were randomized to immediate vs. deferred ART in the Strategic Timing of Antiretroviral Treatment trial were classified into subgroups of duration of HIV infection at baseline. CD4 cell count recovery over follow-up according to duration of HIV infection was investigated. METHODS: Three subgroups were defined: first, infected 6 months or less (n = 373); second, infected 6-24 months (n = 2634); and third, infected 24 months or longer (n = 1605). Follow-up CD4, CD8, and CD4 : CD8 ratio for the immediate and deferred ART groups were compared by subgroup using linear models. For the deferred ART group, decline to CD4 less than 350 cells/µl or AIDS according to infection duration was compared using time-to-event methods. RESULTS: Follow-up CD4 cell count differences (immediate minus deferred) were greater for those recently infected (+231 cells/µl) compared with the two other subgroups (202 and 171 cells/µl; P < 0.001). CD4 : CD8 ratio treatment differences varied significantly (P < 0.001) according to duration of infection. In the deferred ART group, decline to CD4 less than 350 cells/µl or AIDS was greater among those recently infected (16.1 vs. 13.2 and 10.5 per 100 person years for those infected 6-24 and ≥24 months; P = 0.002). CONCLUSION: In this randomized comparison of immediate vs. deferred ART, the CD4 cell count difference was greatest for those recently infected with HIV, emphasizing the importance of immediate ART initiation.

In vivo expansion of CD4CD45RO-CD25 T cells expressing foxP3 in IL-2-treated HIV-infected patients.

Administration of IL-2 to HIV-infected patients leads to expansion of a unique subset of CD4CD45ROCD25 cells. In this study, the origin, clonality, and function of these cells were investigated. Analysis of TCR excision circles revealed that the CD4CD45ROCD25 cells were the product of peripheral expansion but remained polyclonal as determined by TCR repertoire analysis. Phenotypically, these cells were distinct from naturally occurring Tregs; they exhibited intermediate features, between those of memory and naive cells, and had lower susceptibility to apoptosis than CD45ROCD25 or memory T cells. Studies of intracellular cytokine production and proliferation revealed that cytokine-expanded naive CD25 cells had low IL-2 production and required costimulation for proliferation. Despite elevated expression of forkhead transcription factor P3 (foxP3), they exerted only weak suppression compared with CD45ROCD25 cells (Tregs). In summary, in vivo IL-2 administration to HIV-infected patients leads to peripheral expansion of a population of long-lived CD4CD45ROCD25 cells that express high levels of foxP3 but exert weak suppressive function. These CD4CD25 cytokine-expanded naive cells, distinct from antigen-triggered cells and Tregs, play a role in the maintenance of a state of low turnover and sustained expansion of the CD4 T cell pool.

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients.

HIV infection leads to decreases in the number of CD4 T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells. This increased survival was time-dependent: the median half-life, as determined by semiempirical modeling, of labeled CD4 cells in 6 patients increased from 1.7 weeks following an early IL-2 cycle to 28.7 weeks following a later cycle, while CD8 cells showed no change in the median half-life. Examination of lymphocyte subsets demonstrated that phenotypically naive (CD27+CD45RO-) as well as central memory (CD27+CD45RO+) CD4 cells were preferentially expanded, suggesting that IL-2 can help maintain cells important for host defense against new antigens as well as for long-term memory to opportunistic pathogens.

Systemic Inflammation, Coagulation, and Clinical Risk in the START Trial.

BACKGROUND: The Strategic Timing of AntiRetroviral Treatment (START) trial demonstrated that immediate (at CD4+ >500 cells/µL) vs deferred (to CD4+ <350 cells/µL or AIDS) antiretroviral therapy (ART) initiation reduced risk for AIDS and serious non-AIDS (SNA). We investigated associations of inflammation, coagulation, and vascular injury biomarkers with AIDS, SNA or death, and the effect of immediate ART initiation. METHODS: Biomarkers were measured from stored plasma prior to randomization and at month 8. Associations of baseline biomarkers with event risk were estimated with Cox regression, pooled across groups, adjusted for age, gender, and treatment group, and stratified by region. Mean changes over 8 months were estimated and compared between the immediate and deferred ART arms using analysis of covariance models, adjusted for levels at entry. RESULTS: Baseline biomarker levels were available for 4299 START participants (92%). Mean follow-up was 3.2 years. Higher levels of IL-6 and D-dimer were the only biomarkers associated with risk for AIDS, SNA or death, as well as the individual components of SNA and AIDS events (HRs ranged 1.37-1.41 per 2-fold higher level), even after adjustment for baseline CD4+ count, HIV RNA level, and other biomarkers. At month 8, biomarker levels were lower in the immediate arm by 12%-21%. CONCLUSIONS: These data, combined with evidence from prior biomarker studies, demonstrate that IL-6 and D-dimer consistently predict clinical risk across a broad spectrum of CD4 counts for those both ART-naïve and treated. Research is needed to identify disease-modifying treatments that target inflammation beyond the effects of ART.

Immune plasma for the treatment of severe influenza: an open-label, multicentre, phase 2 randomised study.

BACKGROUND: Influenza causes substantial morbidity and mortality despite available treatments. Anecdotal reports suggest that plasma with high antibody titres to influenza might be of benefit in the treatment of severe influenza. METHODS: In this randomised, open-label, multicentre, phase 2 trial, 29 academic medical centres in the USA assessed the safety and efficacy of anti-influenza plasma with haemagglutination inhibition antibody titres of 1:80 or more to the infecting strain. Hospitalised children and adults (including pregnant women) with severe influenza A or B (defined as the presence of hypoxia or tachypnoea) were randomly assigned to receive either two units (or paediatric equivalent) of anti-influenza plasma plus standard care, versus standard care alone, and were followed up for 28 days. The primary endpoint was time to normalisation of patients' respiratory status (respiratory rate of ≤20 breaths per min for adults or age-defined thresholds of 20-38 breaths per min for children) and a room air oxygen saturation of 93% or more. This study is registered with ClinicalTrials.gov, number NCT01052480. FINDINGS: Between Jan 13, 2011, and March 2, 2015, 113 participants were screened for eligibility and 98 were randomly assigned from 20 out of 29 participating sites. Of the participants with confirmed influenza (by PCR), 28 (67%) of 42 in the plasma plus standard care group normalised their respiratory status by day 28 compared with 24 (53%) of 45 participants on standard care alone (p=0·069). The hazard ratio (HR) comparing plasma plus standard care with standard care alone was 1·71 (95% CI 0·96-3·06). Six participants died, one (2%) from the plasma plus standard care group and five (10%) from the standard care group (HR 0·19 [95% CI 0·02-1·65], p=0·093). Participants in the plasma plus standard care group had non-significant reductions in days in hospital (median 6 days [IQR 4-16] vs 11 days [5-25], p=0·13) and days on mechanical ventilation (median 0 days [IQR 0-6] vs 3 days [0-14], p=0·14). Fewer plasma plus standard care participants had serious adverse events compared with standard care alone recipients (nine [20%] of 46 vs 20 [38%] of 52, p=0·041), the most frequent of which were acute respiratory distress syndrome (one [2%] vs two [4%] patients) and stroke (one [2%] vs two [4%] patients). INTERPRETATION: Although there was no significant effect of plasma treatment on the primary endpoint, the treatment seemed safe and well tolerated. A phase 3 randomised trial is now underway to further assess this intervention. FUNDING: National Institute of Allergy and Infectious Diseases, US National Institutes of Health.

Selection and persistence of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 in patients starting and stopping non-nucleoside therapy.

BACKGROUND: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy. OBJECTIVE: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy. METHODS: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10 copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis. RESULTS: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy. CONCLUSIONS: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.

Interleukin-15 (IL-15) Strongly Correlates with Increasing HIV-1 Viremia and Markers of Inflammation.

OBJECTIVE: IL-15 has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. We sought to measure the level of IL-15 in a large, well characterised cohort of HIV-1 infected patients and correlate this with well known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14. DESIGN AND METHODS: IL-15 levels were measured in 501 people (460 patients with HIV-1 infection and 41 uninfected controls). The HIV-1 infected patients were divided into 4 groups based on viral load: <50 copies/ml, 51-10,000 copies/ml, 10,001-100,000 copies/ml and >100,000 copies/ml. The Mann Whitney test (non-parametric) was used to identify significant relationships between different patient groups. RESULTS: IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 ± 1.53 pg/ml) compared to both uninfected controls (1.69 ± 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 ± 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001). CONCLUSIONS: IL-15 levels are significantly elevated in HIV-1 infected patients with viral loads >100,000 copies/ml compared to uninfected controls, with a significant direct correlation noted between IL-15 and HIV-1 viremia and an inverse correlation between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation.

Preferential survival of CD4+ T lymphocytes engineered with anti-human immunodeficiency virus (HIV) genes in HIV-infected individuals.

The present study examined the safety and relative in vivo survival of genetically engineered CD4+ T lymphocytes in human immunodeficiency virus (HIV)-infected individuals. Ten pairs of identical twins discordant for HIV infection were recruited, with the uninfected twin serving as the lymphocyte donor. Ten subjects were treated with a total of 19 separate infusions of retroviral vector-transduced CD4+ enriched T cells. Control (neo gene) or anti-HIV gene (antisense trans-activation response [TAR] element and/or trans-dominant Rev)-engineered lymphocytes were monitored in peripheral blood for 3 years, using a vector-specific PCR assay. Data from 9 of the 10 patients (15 of the 19 infusions) demonstrated preferential survival of CD4+ lymphocytes containing the anti-HIV gene(s) in the immediate weeks after infusion. In six of six patients studied long term (>100 weeks), only T cells containing the anti-HIV genes were consistently detected. In addition, a marked survival advantage of anti-HIV gene-containing T cells was observed in a patient treated during a period of high viral load. Thus, these data strongly support the hypothesis that anti-HIV genes afford a survival advantage to T cells and potential benefit to HIV-1+ individuals.

Five-year follow-up of a cohort of profoundly immunosuppressed patients discontinuing therapy for cytomegalovirus retinitis.

Potent antiretroviral therapy stimulates robust and durable immune reconstitution among profoundly immunosuppressed patients that is clinically important. Fifteen patients with cytomegalovirus retinitis (CMVR) were followed after discontinuing anti-cytomegalovirus therapy. No patients had progression or relapse for a median of 51 months. Survival from the diagnosis of CMVR was universal for up to 95 months, with an observation period averaging more than 6 years. The CD4 cell count continued to rise through the fourth year of follow-up.