Antiretroviral Therapy Concentrations Differ in Gut vs. Lymph Node Tissues and Are Associated With HIV Viral Transcription by a Novel RT-ddPCR Assay.

BACKGROUND: Most HIV-infected cells during antiretroviral therapy (ART) persist in lymphoid tissues. Studies disagree on whether suboptimal tissue ART concentrations contribute to ongoing HIV replication during viral suppression. METHODS: We performed a cross-sectional study in virally-suppressed HIV+ participants measuring lymphoid tissue ART [darunavir (DRV), atazanavir (ATV), and raltegravir (RAL)] concentrations by LC-MS/MS assay. Tissue and plasma ART concentrations were used to estimate TPRs and drug-specific tissue:inhibitory concentration ratios (TICs). HIV DNA and sequentially produced HIV RNA transcripts were quantified from rectal biopsies using droplet digital PCR (ddPCR) assays. RESULTS: Tissue samples were collected in duplicate from 19 participants: 38 rectal, 8 ileal (4 RAL, 2 DRV, 2 ATV), and 6 lymph node (4 RAL, 2 DRV) samples. Overall, median TICs were higher for RAL than DRV or ATV (both P = 0.006). Median TICs were lower in lymph nodes vs. ileum (0.49 vs. 143, P = 0.028) or rectum (33, P = 0.019), and all ART levels were below target concentrations. Higher rectal TICs were associated with lower HIV RNA transcripts (read-through, long LTR, and Nef, P all < 0.026) and a lower long LTR RNA/long LTR DNA ratio (P = 0.021). CONCLUSIONS: We observed higher tissue ART concentrations in ileum and rectum compared with lymph nodes. We observed higher HIV transcription in participants with lower rectal ART concentrations. These findings add to the limited data supporting the idea that viral transcription may be influenced by ART concentrations in lymphoid tissues. Further exploration of tissue pharmacokinetics is needed in future HIV eradication strategies.

Correction: Rapid Progressing Allele HLA-B35 Px Restricted Anti-HIV-1 CD8+ T Cells Recognize Vestigial CTL Epitopes.

[This corrects the article DOI: 10.1371/journal.pone.0010249.].

Rapid progressing allele HLA-B35 Px restricted anti-HIV-1 CD8+ T cells recognize vestigial CTL epitopes.

BACKGROUND: The HLA-B*35-Px allele has been associated with rapid disease progression in HIV-1 infection, in contrast to the HLA-B*35-Py allele. METHODOLOGY/PRINCIPAL FINDINGS: Immune responses to two HLA-B*35 restricted HIV-1 specific CTL epitopes and their variants were followed longitudinally during early HIV-1 infection in 16 HLA-B*35+ individuals. Subjects expressing HLA-B*35-Px alleles showed no difference in response to the consensus epitopes compared to individuals with HLA-B*35-Py alleles. Surprisingly, all the HLA-B*35-Px+ individuals responded to epitope-variants even in the absence of a consensus response. Sequencing of the viral population revealed no evidence of variant virus in any of the individuals. CONCLUSIONS/SIGNIFICANCE: This demonstrates a novel phenomenon that distinguishes individuals with the HLA-B*35-Px rapid progressing allele and those with the HLA-B*35-Py slower progressing allele.

Immunity to HIV-1 is influenced by continued natural exposure to exogenous virus.

Unprotected sexual intercourse between individuals who are both infected with HIV-1 can lead to exposure to their partner's virus, and potentially to super-infection. However, the immunological consequences of continued exposure to HIV-1 by individuals already infected, has to our knowledge never been reported. We measured T cell responses in 49 HIV-1 infected individuals who were on antiretroviral therapy with suppressed viral loads. All the individuals were in a long-term sexual partnership with another HIV-1 infected individual, who was either also on HAART and suppressing their viral loads, or viremic (>9000 copies/ml). T cell responses to HIV-1 epitopes were measured directly ex-vivo by the IFN-gamma enzyme linked immuno-spot assay and by cytokine flow cytometry. Sexual exposure data was generated from questionnaires given to both individuals within each partnership. Individuals who continued to have regular sexual contact with a HIV-1 infected viremic partner had significantly higher frequencies of HIV-1-specific T cell responses, compared to individuals with aviremic partners. Strikingly, the magnitude of the HIV-1-specific T cell response correlated strongly with the level and route of exposure. Responses consisted of both CD4(+) and CD8(+) T cell subsets. Longitudinally, decreases in exposure were mirrored by a lower T cell response. However, no evidence for systemic super-infection was found in any of the individuals. Continued sexual exposure to exogenous HIV-1 was associated with increased HIV-1-specific T cell responses, in the absence of systemic super-infection, and correlated with the level and type of exposure.

Target cell APOBEC3C can induce limited G-to-A mutation in HIV-1.

The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.

Cerebrospinal fluid HIV infection and pleocytosis: relation to systemic infection and antiretroviral treatment.

BACKGROUND: Central nervous system (CNS) exposure to HIV is a universal facet of systemic infection. Because of its proximity to and shared barriers with the brain, cerebrospinal fluid (CSF) provides a useful window into and model of human CNS HIV infection. METHODS: Prospective study of the relationships of CSF to plasma HIV RNA, and the effects of: 1) progression of systemic infection, 2) CSF white blood cell (WBC) count, 3) antiretroviral therapy (ART), and 4) neurological performance. One hundred HIV-infected subjects were cross-sectionally studied, and 28 were followed longitudinally after initiating or changing ART. RESULTS: In cross-sectional analysis, HIV RNA levels were lower in CSF than plasma (median difference 1.30 log10 copies/mL). CSF HIV viral loads (VLs) correlated strongly with plasma VLs and CSF WBC counts. Higher CSF WBC counts associated with smaller differences between plasma and CSF HIV VL. CSF VL did not correlate with blood CD4 count, but CD4 counts <50 cells/microL associated with a low prevalence of CSF pleocytosis and large differences between plasma and CSF VL. CSF HIV RNA correlated neither with the severity of the AIDS dementia complex (ADC) nor abnormal quantitative neurological performance, although these measures were associated with depression of CD4 counts. In subjects starting ART, those with lower CD4 counts had slower initial viral decay in CSF than in plasma. In all subjects, including five with persistent plasma viremia and four with new-onset ADC, CSF HIV eventually approached or reached the limit of viral detection and CSF pleocytosis resolved. CONCLUSION: CSF HIV infection is common across the spectrum of infection and is directly related to CSF pleocytosis, though whether the latter is a response to or a contributing cause of CSF infection remains uncertain. Slowing in the rate of CSF response to ART compared to plasma as CD4 counts decline indicates a changing character of CSF infection with systemic immunological progression. Longer-term responses indicate that CSF infection generally responds well to ART, even in the face of systemic virological failure due to drug resistance. We present simple models to explain the differing relationships of CSF to plasma HIV in these settings.

HIV-1 chemokine coreceptor utilization in paired cerebrospinal fluid and plasma samples: a survey of subjects with viremia.

BACKGROUND: Chemokine receptors serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, influence cell tropism, and may critically determine central nervous system infection pathogenesis. Using an in vitro functional entry assay, we examined utilization of 2 principal coreceptors in cerebrospinal fluid (CSF) and plasma in 46 subjects. METHODS: Paired CSF and plasma samples were selected from subjects with a range of CD4 T cell counts. Amplified populations of env sequences were characterized as using CCR5 (R5), CXCR4 (X4), or both receptors (R5+X4). Individual clones derived from 3 subjects were analyzed for viral tropism and phylogeny. RESULTS: CSF and plasma pairs were mainly concordant for R5 (36/46) or R5+X4 (5/46) viruses. However, 5 pairs were discordant, 2 of which had the R5+X4 phenotype in CSF despite having the R5 phenotype in plasma. Although R5+X4 tropism was associated with advanced immunodeficiency, all 4 subjects with acquired immunodeficiency syndrome dementia complex had R5 tropism in CSF. Clones derived from R5+X4-tropic populations revealed mixtures of R5 and X4 viruses and viruses able to utilize either coreceptor, suggesting both virus exchange between compartments and autonomous CSF virus evolution. CONCLUSIONS: Although R5 viruses predominate in the CSF, HIV-1 populations able to utilize CXCR4 are also present. Discordant tropism in CSF and plasma may have implications for R5 inhibitor therapy.

Persistence of primary drug resistance among recently HIV-1 infected adults.

OBJECTIVES: Primary, or transmitted, drug resistance is common among treatment naive patients recently infected with HIV-1, and impairs response to anti-retroviral therapy. We previously observed that patients with secondary resistance (developed in response to anti-retroviral treatment) who chose to stop an anti-retroviral regimen experience rapid overgrowth of drug resistant viruses by wild-type virus of higher pol replication capacity. We sought to determine if primary drug resistance would be lost at a rapid rate, and viral pol replication capacity would increase, in the absence of treatment. METHODS: We tracked drug resistance phenotype, genotype, viral pol replication capacity (single cycle recombinant assay incorporating a segment of the patient pol gene [pol RC]), plasma HIV-1 RNA, and CD4 T cell counts in the absence of treatment among patients in early HIV-1 infection. RESULTS: Six of 22 patients had evidence of primary drug resistance to at least one class of drug; three resistant to protease inhibitors, three resistant to non-nucleoside reverse transcriptase inhibitors, and four resistant to nucleoside reverse transcriptase inhibitors. All six patients maintained evidence of drug resistance for the period of observation. Among patients with baseline primary drug resistance pol RC did not increase over time. CONCLUSION: The selection environment of early infection is determined by immune pressure, and stochastic events, not viral pol replication capacity. In contrast to secondary resistant infections that are rapidly overgrown when therapy is stopped, primary drug resistance persists over time. Surveillance and clinical detection of primary resistance is feasible in the first year of infection.

Higher CD4+ T cell counts associated with low viral pol replication capacity among treatment-naive adults in early HIV-1 infection.

BACKGROUND: Infection with primary drug-resistant human immunodeficiency virus type 1 (HIV-1) has been associated with higher CD4(+) T cell counts in drug-naive patients, suggesting that altered viral pol replication capacity (RC) associated with drug resistance diminishes immune injury in vivo, independent of exposure to drugs. METHODS: Virus replication over a single cycle was measured by use of a viral test vector containing patient-derived HIV-1 protease and reverse transcriptase gene segments. RESULTS: Among 191 recently infected patients, pol RC ranged widely, with only 6% of the variance explained by drug-resistance mutations. Patients infected with a virus with a low pol RC (