Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma.

Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal plasma using quantitative real time PCR technology.

Higher HIV-1 genetic diversity is associated with AIDS and neuropsychological impairment.

Standard methods used to estimate HIV-1 population diversity are often resource intensive (e.g., single genome amplification, clonal amplification and pyrosequencing) and not well suited for large study cohorts. Additional approaches are needed to address the relationships between intraindividual HIV-1 genetic diversity and 2 disease. With a small cohort of individuals, we validated three methods for measuring diversity: Shannon entropy and average pairwise distance (APD) using single genome sequences, and counts of mixed bases (i.e. ambiguous nucleotides) from population based sequences. In a large cohort, we then used the mixed base approach to determine associations between measure HIV-1 diversity and HIV associated disease. Normalized counts of mixed bases correlated with Shannon Entropy at both the nucleotide (rho=0.72, p=0.002) and amino acid level (rho=0.59, p=0.015), and APD (rho=0.75, p=0.001). Among participants who underwent neuropsychological and clinical assessments (n=187), increased HIV-1 population diversity was associated with both a diagnosis of AIDS and neuropsychological impairment.

A combined screening platform for HIV treatment failure and resistance.

BACKGROUND: To develop a low cost method to screen for virologic failure of antiretroviral therapy (ART) and HIV-1 drug resistance, we performed a retrospective evaluation of a screening assay using serial dilutions of HIV-1 RNA-spiked blood plasma and samples from patients receiving >6 months of first-line ART. METHODS: Serial dilution testing was used to assess sensitivity of a simple PCR-based assay (targeted at ≥1,000 HIV RNA copies/mL). We created blood plasma minipools of five samples, extracted HIV RNA from the pools, PCR amplified the reverse transcriptase (RT) coding region of the HIV-1 pol gene from extracted RNA, sequenced PCR product of positive pools, and used sequences to determine drug resistance. Sensitivity, specificity, and predictive values were determined for different levels of virologic failure based on maximum viral loads of individual samples within a pool. RESULTS: Of 295 samples analyzed, 43 (15%) had virologic failure at ≥50 copies/mL (range 50-10,500 copies/mL, four at ≥1,000 copies/mL). The assay demonstrated 100% sensitivity to detect virus from these four samples, requiring only one round of PCR, and 56% and 89% sensitivity to detect samples with ≥50 and ≥500 copies/mL using two rounds. Amplified PCR products of all positive pools were successfully sequenced and 30% harbored ≥1 major resistance mutation. This method would have cost 10% of the combined costs of individual viral load and resistance testing. CONCLUSIONS: We present a novel method that can screen for both virologic failure of first-line ART and drug resistance. The method is much less expensive than current methods, which may offer sustainability in resource-limited settings.

In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

The use of pooled viral load testing to identify antiretroviral treatment failure.

BACKGROUND: To develop less costly methods to virologically monitor patients receiving antiretroviral therapy, we evaluated methods that use pooled blood samples and quantitative information available from viral load assays to monitor a cohort of patients on first-line antiretroviral therapy for virologic failure. METHODS: We evaluated 150 blood samples collected after 6 months of therapy from participants enrolled in a San Diego primary infection program between January 1998 and January 2007. Samples were screened for virologic failure with individual viral load testing, 10 x 10 matrix pools and minipools of five samples. For the pooled platforms (matrix and minipools), we used a search and retest algorithm based on the quantitative viral load data to resolve samples that remained ambiguous for virologic failure. Viral load thresholds were more than 500 and more than 1500 copies/ml for the matrix and more than 250 and more than 500 copies/ml for the minipool. Efficiency, accuracy and result turnaround times were evaluated. RESULTS: Twenty-three percent of cohort samples were detectable at more than 50 HIV RNA copies/ml. At an algorithm threshold of more than 500 HIV RNA copies/ml, both minipool and matrix methods used less than half the number of viral load assays to screen the cohort, compared with testing samples individually. Both pooling platforms had negative predictive values of 100% for viral loads of more than 500 HIV RNA copies/ml and at least 94% for viral loads of more than 250 HIV RNA copies/ml. CONCLUSION: In this cohort, both pooling methods improved the efficiency of virologic monitoring over individual testing with a minimal decrease in accuracy. These methods may allow for the induction and sustainability of the virologic monitoring of patients receiving antiretroviral therapy in resource-limited settings.

Select resistance-associated mutations in blood are associated with lower CSF viral loads and better neuropsychological performance.

BACKGROUND: When antiretroviral therapy does not fully suppress HIV replication, suboptimal levels of antiretrovirals can select for antiretroviral resistant variants of HIV. These variants may exhibit reduced replication capacity and result in lower viral loads in blood. Our study evaluated whether antiretroviral resistance was associated with viral loads in the cerebrospinal fluid (CSF) and better neuropsychological (NP) performance. METHODS: We enrolled 94 participants and each participant underwent a comprehensive neuromedical evaluation that used structured clinical assessments of medical history, ART and other medication use, comprehensive NP testing, and neurological and general physical signs of disease. Blood was collected by venipuncture, and all participants were offered lumbar puncture. Univariate and multivariate statistical methods were used to analyze the relationship between antiretroviral resistance, blood and CSF HIV RNA levels, substance use, and NP performance. RESULTS: Antiretroviral resistance, detected in blood, was associated with lower CSF viral loads (p<0.01) and better NP performance (p=0.04) in multivariate analyses, independent of past and current ARV use and blood viral loads (model: p<0.01). However, HIV RNA levels in CSF did not independently correlate with NP performance. Low viral loads in the CSF limited our ability to investigate the relationship between antiretroviral resistance detected in CSF and NP performance. CONCLUSIONS: Even in the absence of ART, antiretroviral resistance-associated mutations correlate with better NP performance possibly because these mutations reflect reduced neurovirulence compared with wild-type HIV.

Persistence of transmitted drug resistance among subjects with primary human immunodeficiency virus infection.

Following interruption of antiretroviral therapy among individuals with acquired drug resistance, preexisting drug-sensitive virus emerges relatively rapidly. In contrast, wild-type virus is not archived in individuals infected with drug-resistant human immunodeficiency virus (HIV) and thus cannot emerge rapidly in the absence of selective drug pressure. Fourteen recently HIV-infected patients with transmitted drug-resistant virus were followed for a median of 2.1 years after the estimated date of infection (EDI) without receiving antiretroviral therapy. HIV drug resistance and pol replication capacity (RC) in longitudinal plasma samples were assayed. Resistance mutations were characterized as pure populations or mixtures. The mean time to first detection of a mixture of wild-type and drug-resistant viruses was 96 weeks (1.8 years) (95% confidence interval, 48 to 192 weeks) after the EDI. The median time to loss of detectable drug resistance using population-based assays ranged from 4.1 years (conservative estimate) to longer than the lifetime of the individual (less conservative estimate). The transmission of drug-resistant virus was not associated with virus with reduced RC. Sexual transmission of HIV selects for highly fit drug-resistant variants that persist for years. The prolonged persistence of transmitted drug resistance strongly supports the routine use of HIV resistance genotyping for all newly diagnosed individuals.

Dynamics of total, linear nonintegrated, and integrated HIV-1 DNA in vivo and in vitro.

BACKGROUND: In patients infected with human immunodeficiency virus type 1 (HIV-1), HIV-1 DNA persists during highly active antiretroviral treatment, reflecting long-lived cellular reservoirs of HIV-1. Recent studies report an association between HIV-1 DNA levels, disease progression, and treatment outcome. However, HIV-1 DNA exists as distinct molecular forms that are not distinguished by conventional assays. METHODS: We analyzed HIV-1 RNA levels in plasma, CD4 cell counts, and levels of integrated and nonintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from patients with early or chronic infection before and during antiretroviral treatment. We also studied HIV-1 DNA decay in primary CD4 T cells infected in vitro. HIV-1 DNA was analyzed using an assay that is unaffected by the location of HIV-1 integration sites. RESULTS: HIV-1 RNA levels and total HIV-1 DNA levels decayed rapidly in patients during receipt of suppressive antiretroviral therapy. Ratios of total HIV-1 DNA levels to integrated HIV-1 DNA levels were high before initiation of therapy but diminished during therapy. Levels of linear nonintegrated HIV-1 DNA decayed rapidly in vitro (t (1/2) = 1- 4.8 days). CONCLUSION: Total HIV-1 DNA decays rapidly with suppression of virus replication in vivo. Clearance of HIV-1 DNA during the first 6 months of therapy reflects a disproportionate loss of nonintegrated HIV-1 DNA genomes, suggesting that levels of total HIV-1 DNA in PBMCs after prolonged virus suppression largely represent integrated HIV-1 genomes.

Long-term persistence of transmitted HIV drug resistance in male genital tract secretions: implications for secondary transmission.

BACKGROUND: Transmitted drug-resistant HIV slowly reverts in the blood to drug-sensitive virus. The environment of the male genital tract (MGT) may result in even slower rates of reversion to drug susceptibility. METHODS: We measured the decay of resistance in longitudinally collected blood and semen samples from 5 individuals newly infected with HIV containing resistance mutations to nonnucleoside reverse-transcriptase inhibitors (NNRTIs). We also investigated the sexual transmission of HIV to and from these participants. RESULTS: In 3 of the 5 individuals, NNRTI resistance persisted in blood and semen throughout follow-up (mean, 296 days after the estimated day of infection [EDI]). In the other 2 individuals, NNRTI resistance persisted in blood and semen for 871 and 1179 days after the EDI; however, even after NNRTI resistance had fully reverted in blood, it remained readily detectable in semen. Two transmission groups were identified among these participants--one as the recipient partner and the other as the source partner. CONCLUSIONS: Transmitted drug-resistant HIV, which persists in blood for years, may revert to wild type even more slowly in the MGT. This prolonged persistence in the MGT may contribute to the high prevalence rates of transmitted drug resistance.

Determinants of CD4+ T cell recovery during suppressive antiretroviral therapy: association of immune activation, T cell maturation markers, and cellular HIV-1 DNA.

BACKGROUND: Suboptimal CD4+ T cell recovery during antiretroviral therapy (ART) is a common clinical dilemma. METHODS: We analyzed viral and immunologic predictors of CD4+ T cell recovery in 116 human immunodeficiency virus type 1 (HIV-1)-infected subjects who had suppressed viremia (< or = 50 copies/mL) while receiving ART. Successive measurements of T cell immunophenotypes and cellular HIV-1 DNA levels were obtained before and during receipt of ART. On the basis of increases in the CD4+ T cell count, subjects were classified as immunologically concordant (demonstrating an increase of > or = 100 CD4+ T cells/mm3) or discordant (demonstrating an increase of <100 CD4+ T cells/mm3) after 48 weeks of ART. RESULTS: In adjusted analyses, CD4+ and CD8+ T cell activation at baseline was negatively associated with immunologic concordance at week 48 of ART (odds ratio [OR], 0.80 [P = .04] and 0.67 [P = .02], respectively). High memory (CDRA(-)CD62L-) CD8+ T cell counts at baseline (OR, 0.33 [P = .05]) predicted less CD4+ T cell recovery, whereas increased naive CD4+ T cell counts were associated with higher increases in CD4+ T cells (OR, 1.19 [P = .052]). Neither the cell-associated HIV-1 DNA level at baseline (P = .32) nor the cell-associated HIV-1 DNA level at week 48 of ART (P = .42) was associated with immunologic concordance during ART. CONCLUSIONS: These results support the potential clinical usefulness of the baseline determination of immune activation and maturation subsets in the prediction of CD4+ T cell recovery during viral suppression. Furthermore, identification of individuals with reduced potential for CD4+ T cell recovery during ART may provide a rationale for the initiation of early therapy for some patients.

HIV drug resistance acquired through superinfection.

OBJECTIVE: HIV interclade B superinfection has previously been described in individuals initially infected with drug resistant virus who then become superinfected by a drug susceptible strain. We report an individual initially infected with a drug-sensitive clade B strain of HIV who was superinfected with another clade B strain resistant to two classes of antiretroviral drugs. METHODS AND DESIGN: To differentiate superinfection from possible co-infection we applied three independent molecular techniques: dye-primer sequencing of a pol fragment, length polymorphism analysis of the V4-5 coding region of the env gene and clonal sequencing of the V3 coding region of the env gene. To assess viral fitness we performed replication capacity assays of the pol gene. RESULTS: These investigations supported the conclusion that this was a case of superinfection and not co-infection. Coincident with acquiring the new strain, the individual's viral load increased by about 10,000 copies/ml with a decrease of 150 x CD4 T cells/mul over the next 6 months. The greater in vivo fitness of the second virus was not supported by the replication capacity assay. Furthermore, superinfection negatively impacted this individual's treatment course. It was not known that he had acquired a drug resistant strain before entering a treatment study, and he had an incomplete response to therapy most likely because the superinfecting viral strain had a decreased susceptibility to two of the prescribed medications. CONCLUSION: HIV drug resistance acquired through superinfection significantly lowers the likelihood of successful antiretroviral therapy and undermines the clinical value of a patient's prior drug resistance testing and lack of prior antiretroviral use.

Effect of treatment, during primary infection, on establishment and clearance of cellular reservoirs of HIV-1.

Patients in whom virologic suppression is achieved with highly active antiretroviral therapy (HAART) retain long-lived cellular reservoirs of human immunodeficiency virus type 1 (HIV-1); this retention is an obstacle to sustained control of infection. To assess the impact that initiating treatment during primary HIV-1 infection has on this cell population, we analyzed the decay kinetics of HIV-1 DNA and of infectivity associated with cells activated ex vivo in 27 patients who initiated therapy before or <6 months after seroconversion and in whom viremia was suppressed to <50 copies/mL. The clearance rates of cellular reservoirs could not be distinguished by these techniques (median half-life, 20 weeks) during the first year of HAART. The clearance of HIV-1 DNA slowed significantly during the subsequent 3 years of treatment (median half-life, 70 weeks), consistent with heterogeneous cellular reservoirs being present. Total cell-associated infectivity (CAI) after 1 year of treatment was undetectable (<0.07 infectious units/million cells [IUPM]) in most patients initiating treatment during primary infection either before (9/9) or <6 months after (6/8) seroconversion. In contrast, all 17 control patients who initiated HAART during chronic infection retained detectable CAI after 3-6 years of treatment (median reservoir size, 1.1 IUPM; P<.0005). These results suggest that treatment <6 months after seroconversion may facilitate long-term control of cellular reservoirs that maintain HIV-1 infection during treatment.

Predictors of residual viremia in HIV-infected patients successfully treated with efavirenz and lamivudine plus either tenofovir or stavudine.

In human immunodeficiency virus (HIV)-infected patients successfully treated with highly active antiretroviral therapy (HAART), a low level of HIV RNA persists in plasma at steady state for years and varies among patients. To understand predictors of residual viremia, we measured HIV RNA levels <50 copies/mL in patients after 1 year of treatment with efavirenz and lamivudine plus either tenofovir disoproxil fumarate (n=55) or stavudine (n=45), by use of an HIV RNA assay with a limit of detection of 2.5 copies/mL. The mean posttreatment HIV RNA levels were 0.58 log(10) copies/mL (3.8 copies/mL) in the tenofovir arm and 0.61 log(10)copies/mL (4.1 copies/mL) in the stavudine arm (P=.24). Forty-seven percent of patients receiving tenofovir, compared with 29% of patients receiving stavudine, had undetectable residual viremia (P=.07). In multivariate analyses, we found that lower baseline HIV RNA levels in plasma, lower HIV DNA levels in peripheral blood mononuclear cells, and inclusion in the tenofovir arm each independently predicted undetectable residual viremia (P<.05). However, a level of residual viremia <50 copies/mL was not associated with CD4 cell count changes or risk of virologic rebound through 72 weeks of follow-up.

The prostate as a reservoir for HIV-1.

As the prostate can harbor bacterial and fungal pathogens, it was investigated as a reservoir for HIV. Nine men chronically infected with HIV participated in a crossover trial in which weekly semen samples were collected with and without previous prostate massage (PM). Six of the nine participants had undetectable seminal plasma(SP) HIV RNA in all samples without previous PM, but had detectable SP HIV RNA (> 25 copies/ml) in one to three samples collected after PM.

Clade B HIV-1 superinfection with wild-type virus after primary infection with drug-resistant clade B virus.

BACKGROUND: The immunological response to HIV-1 infection has been postulated to impede superinfection with a second virus; however, a few recent reports have documented cases of HIV-1 superinfection in humans either from different viral clades or from the same clade. OBJECTIVE: To differentiate between co-infection and superinfection in a patient harboring a distinct wild-type HIV 4 months after primary infection with drug-resistant HIV. METHODS: Detailed dye primer and clonal sequencing along with length polymorphism analysis was used to investigate the evolutionary linkage between viral populations sampled at different timepoints. RESULTS: After a set point viral load of -6000 copies HIV RNA/ml, the viral load jumped to 34 000 copies/ml at month 4 and, shortly after, to almost 200 000 copies/ml. At that time a second viral strain was first detected by dye primer sequencing of a pol fragment. These findings were confirmed by analysis of a 1300 bp gag-pol fragment and clonal sequencing and phylogenetic analysis of the V3 region. Length polymorphism analysis of the gp120 V4-V5 region showed that the second viral population was absent even as a minority population until month 4, when it was found to be the majority population, and the initial variant was present only as a minority. Both strains were subtype B. CONCLUSION: These data support intraclade HIV-1 superinfection by wild-type virus in the absence of antiretroviral therapy in a patient initially infected with drug-resistant HIV. The substantially different in-vivo viral growth characteristics observed illustrate the potential for superinfection to impact disease progression.