Persistent HIV transcription and variable antiretroviral drug penetration in lymph nodes during plasma viral suppression.

OBJECTIVE: The ability of antiretroviral drugs to penetrate and suppress viral replication in tissue reservoir sites is critical for HIV remission. We evaluated antiretroviral concentrations in lymph nodes and their impact on HIV transcription. METHODS: Participants of the RV254/SEARCH010 Acute HIV Infection Cohort in Thailand were enrolled. Group 1 (n  = 6) initiated and continued antiretrovirals with two nucleoside reverse transcriptase inhibitors (NRTIs), dolutegravir (DTG) and mar- aviroc (MVC). Group 2 (n = 12) initiated antiretrovirals with two NRTIs as well as efavirenz and were switched to two NRTIs as well as DTG. Antiretroviral concentrations were measured by mass spectroscopy. HIV RNA+ and DNA+ cells were measured by in-situ hybridization. RESULTS: All participants were MSM. At lymph node biopsy, all had plasma HIV RNA less than 20 copies/ml. Group 2 had longer durations of antiretroviral and DTG use (medians of 135 and 63 weeks, respectively) compared with Group 1 (median 44 weeks for both). TFV-DP, 3TC-TP, DTG and MVC were quantifiable in all lymph node samples from participants receiving those drugs versus carbovir-triphosphate (CBV-TP) in four out of 14. Median ratios of lymph node to peripheral blood concentrations were DTG, 0.014; MVC, 6.9; CBV-TP, 0.38; 3TC-TP, 0.32; and TFV-DP, 3.78. Median inhibitory quotients [ratios of lymph node concentrations to in-vitro inhibitory levels (IC50-or-90)] were DTG, 0.8; MVC, 38.8; CBV-TP, 0.5; 3TC- TP, 4.1; and TFV-DP, 1.8. Ongoing viral transcription was detected in lymph node of all participants. Median lymph node RNA+ cells were 71 350 versus 99 750 cells/g for Groups 1 and 2, respectively (P = 0.111). CONCLUSION: MVC has enhanced lymph node penetration and thereby may contribute to more complete viral suppression in the lymph node.

Longitudinal Analysis of Peripheral and Colonic CD161+ CD4+ T Cell Dysfunction in Acute HIV-1 Infection and Effects of Early Treatment Initiation.

CD161 expression on CD4+ T cells is associated with a Th17 functional phenotype, as well as with an innate capacity to respond to interleukin (IL)-12 and IL-18 without T cell receptor (TCR) stimulation. Chronic HIV-1 infection is associated with loss of the CD161+ CD4 T cell population, and non-human primate studies suggest that their depletion is associated with disease progression. However, the dynamics of the CD161+ CD4+ T cell population during acute HIV-1 infection remains unknown. In this study, we characterize peripheral blood CD161+ CD4+ T cells in detail, and examine how they are affected during the earliest stages of HIV-1 infection. Unbiased surface proteome screening and principal component analysis indicated that CD161+ CD4+ T cells are relatively phenotypically homogeneous between donors, and are intermediates between conventional CD4 T cells and innate-like T cells. In acute untreated HIV-1 infection, the circulating CD161+ CD4+ T cell population decreased in frequency, as did absolute cell counts starting from peak viral load, with elevated levels of activation and exhaustion markers expressed throughout acute HIV-1 infection. The capacity of these cells to respond to stimulation with IL-12 and IL-18 was also reduced. Early initiation of anti-retroviral treatment (ART) during acute HIV-1 infection restored the functionality of peripheral blood CD161+ CD4+ T cells, but not their frequency. In contrast, early ART initiation prevented the decline of colonic CD161+ CD4+ T cells that otherwise started during acute infection. Furthermore, loss of peripheral and colonic CD161+ CD4+ T cells in untreated infection was associated with levels of viral load. These results suggest that acute HIV-1 infection has profound effects on the CD161+ CD4+ T cell population that could not be completely prevented by the initiation of ART.

Feasibility and safety of research sigmoid colon biopsy in a cohort of Thai men who have sex with men with acute HIV-1.

BACKGROUND: The gut-associated lymphoid tissue (GALT) is a major reservoir of HIV-1 established early in acute HIV-1 infection (AHI). Sampling tissue from GALT can provide information about viral reservoirs and immune responses but may be complicated during AHI for reasons such as high viral replication, CD4 T cell depletion and immune activation. Risk of adverse events (AEs) associated with research sigmoid colon biopsies was assessed in participants with AHI in Bangkok, Thailand. METHODS: Between 2009 and 2016, 170 biopsies collected from the sigmoid colon were performed during AHI and at follow-up visits (median 24 weeks post AHI diagnosis). Adverse event incidence was evaluated, as well as the associations of procedure timing, repetition and clinical parameters with AE risk. Negative binomial regression models were used to calculate incidence rate ratios and 95% confidence intervals. RESULTS: Among 103 participants (median age of 27 years, 97.1% male, 96.1% men who have sex with men), 87 sigmoidoscopies were completed during AHI and 83 at a follow-up visit. Approximately 30 biopsies were obtained per procedure for assessment of colonic viral load and HIV-1 reservoir, immunohistochemistry or phenotypic assays. All 11 AEs were grade 1 (6.5%) and included abdominal discomfort (n = 5, 2.9%), mild rectal bleeding (n = 5, 2.9%) and difficulty passing stool (n = 1, 0.6%). Biopsy-related AE risk was not significantly associated with age, HIV-1 RNA, CD4 T cell count, or number and time of biopsy. CONCLUSIONS: Complications of sigmoidoscopy with biopsy in participants with AHI were infrequent and mild. Longitudinal sampling of the sigmoid colon to evaluate the gut-associated HIV-1 reservoir can be safely performed as part of research studies.

Brief Report: Safety and Tolerability of Inguinal Lymph Node Biopsy in Individuals With Acute HIV Infection in Thailand.

INTRODUCTION: Latent HIV reservoirs are rapidly established in lymphoid tissues during acute HIV infection (AHI). Sampling these tissues provides important information about HIV pathogenesis. This period is associated with viral replication and immune activation that may affect procedure-related adverse events (AEs). We examined the safety and tolerability of inguinal lymph node (LN) biopsy in research participants with AHI in Bangkok, Thailand. METHODS: Between 2013 and 2016, 67 AHI participants in the RV254/SEARCH010 study underwent at least one optional inguinal LN biopsy during AHI at the baseline visit and/or after antiretroviral therapy (median 48 weeks after antiretroviral therapy). Biopsy-related AEs were graded according to NIH Division of AIDS guidelines. Poisson regression was used to calculate incidence rate ratios and 95% confidence intervals to evaluate associations of demographic and HIV characteristics, procedure timing, and repetition with AE incidence. RESULTS: Of the 67 participants, 97% were male with a median age of 26. Among 78 LN biopsies (39 at baseline and 39 at follow-up), 10 (12.8%) AEs were reported: 6 (7.7%) grade 1 and 4 (5.1%) grade 2. The AEs were biopsy-site discomfort (n = 8, 10.2%) and hematoma (n = 2, 2.6%). No factors were significantly associated with AE incidence. All biopsy-related AEs were transient and self-limited. CONCLUSIONS: Inguinal LN biopsies were safe and well tolerated in mostly Thai men with AHI. As LN biopsies become an integral part of HIV research, this study provides information to participants, researchers, and institutional review boards that these samples can be safely obtained.

Distribution of Human Immunodeficiency Virus (HIV) Ribonucleic Acid in Cerebrospinal Fluid and Blood Is Linked to CD4/CD8 Ratio During Acute HIV.

Background: Human immunodeficiency virus (HIV) ribonucleic acid (RNA) levels in the plasma and cerebrospinal fluid (CSF) are correlated in chronic HIV infection, but their dynamics have not been characterized during acute infection. Methods: This study analyzed predictors of CSF HIV RNA and relative degree of CNS viral transmigration expressed as plasma minus CSF HIV log10 RNA (PCratio) during untreated acute HIV infection. Cerebrospinal fluid immune markers were compared between groups with different PCratio. Results: One hundred seventeen mostly male (97%) participants in the RV254 cohort in Bangkok, Thailand, had a median age of 28 years and an estimated median 18 days duration of infection; 43 (37%) were Fiebig stages I/II. Twenty-seven (23%) had CSF HIV RNA <80 copies/mL. Those with quantifiable levels (n = 90) had median CSF HIV RNA and PCratio of 3.76 and 2.36 log10 copies/mL, respectively. Human immunodeficiency virus RNA peaked at Fiebig III in plasma and Fiebig IV in CSF. In multivariable analyses, plasma HIV RNA and CD4/CD8 ratio independently correlated with CSF HIV RNA (P < .001), whereas CD4/CD8 ratio predicted PCratio (P = .018). Participants with PCratio <1 had higher CSF neopterin, soluble (s)CD163, interleukin-6, and sCD14 levels (all P < .05). Conclusions: CD4/CD8 ratio independently correlated with CSF HIV RNA and PCratio, suggesting that immune responses modulate central nervous system viral entry at early infection.

Acute Retroviral Syndrome Is Associated With High Viral Burden, CD4 Depletion, and Immune Activation in Systemic and Tissue Compartments.

Background: Many individuals with acute human immunodeficiency virus infection (AHI) experience acute retroviral syndrome (ARS), which is associated with adverse long-term clinical outcomes. Methods: Participants presenting for voluntary human immunodeficiency virus (HIV) testing were enrolled during AHI in Bangkok, Thailand. ARS was defined by ≥3 qualifying signs/symptoms. HIV burden, immunophenotypes, and biomarkers were stratified by ARS diagnosis at enrollment and after up to 96 weeks of antiretroviral therapy (ART). Results: From 212382 samples screened, 430 participants were enrolled during AHI, including 335 (78%) with ARS. Median age was 26 years and 416 (97%) were men. Sixty (14%) underwent sigmoid biopsy and 105 (24%) underwent lumbar puncture during AHI. Common symptoms included fever (93%), fatigue (79%), pharyngitis (67%), and headache (64%). Compared to those without ARS, participants with ARS were in later Fiebig stages with higher HIV RNA in blood, colon, and cerebrospinal fluid; higher total HIV DNA in blood; CD4 depletion in blood and colon; and elevated plasma tumor necrosis factor alpha (TNF-α), C-reactive protein, and D-dimer (all P < .05). Subgroup analyses of Fiebig I/II participants (95 with ARS, 69 without) demonstrated similar findings. After 96 weeks of ART, TNF-α and interleukin 6 were elevated in the ARS group (P < .05) but other biomarkers equilibrated. Conclusions: ARS was associated with high viral burden, CD4 depletion, and immune activation across multiple body compartments during AHI and prior to ART. Persistent inflammation despite suppressive ART could contribute to increased morbidity in individuals who experience ARS.

Factors associated with intention to take non-occupational HIV post-exposure prophylaxis among Thai men who have sex with men.

BACKGROUND: Men who have sex with men (MSM) are disproportionately infected with HIV in Thailand. Factors affecting their intention to take non-occupational HIV post-exposure prophylaxis (nPEP) are not well understood. This study sought to determine factors associated with an intention to take nPEP in this population. METHOD: This is a two-phase mixed-method study. Phase I was a cross-sectional survey of intention to take nPEP in 450 MSM attending for HIV testing, using a self-administered questionnaire. Phase II was a prospective descriptive study, using an in-depth interview among 40 MSM who had been exposed to HIV in the past 72 hours. Multiple logistic regression was used to evaluate factors relating to the intention to use nPEP. RESULTS: Among 450 MSM seeking HIV testing in Bangkok, 7% had ever taken nPEP. Only 40% expressed an intention to take it to prevent HIV acquisition, despite the fact that they were at high risk as evidenced by an 18.9% prevalence of HIV-positive status. Factors associated with an intention to take nPEP were awareness about nPEP, HIV knowledge, mode of sexual intercourse and circumcision. Among 40 MSM who were eligible for and offered nPEP, 39 agreed to take it, and all but one completed the 4-week course. Condom use increased and all 32 individuals who could be contacted tested HIV negative after nPEP. CONCLUSION: A high HIV prevalence was found in MSM testing for HIV in this study. However, fewer than half of the participants expressed the intention to take nPEP if they were at risk for HIV infection. Efforts to create nPEP awareness and improve HIV knowledge in MSM are crucial to the successful implementation of nPEP as part of a combination package for HIV prevention in this high-risk population.

Persistent, Albeit Reduced, Chronic Inflammation in Persons Starting Antiretroviral Therapy in Acute HIV Infection.

BACKGROUND: Serious non-AIDS events cause substantial disease and death despite human immunodeficiency virus (HIV) suppression with antiretroviral therapy (ART). Biomarkers of inflammation, coagulation cascade activation, and fibrosis predict these end-organ events. We aimed to determine whether ART initiation during acute HIV infection would attenuate changes in these biomarker levels. METHODS: Plasma samples were obtained from participants starting ART during acute or chronic HIV infection and from HIV-uninfected participants from Bangkok, Thailand. Biomarkers of inflammation (C-reactive protein [CRP], interleukin 6, soluble interleukin 6 receptor [sIL-6R], soluble gp130, tumor necrosis factor [TNF]), enterocyte turnover (intestinal fatty acid binding protein [I-FABP]), lipopolysaccharide-induced monocyte activation (soluble CD14 [sCD14]), coagulation cascade activation [D-dimer], and fibrosis (hyaluronic acid [HA]) were measured at baseline and through 96 weeks of ART. RESULTS: CRP, TNF, sIL-6R, I-FABP, sCD14, D-dimer, and HA levels were elevated in acute HIV infection. Early ART was associated with increased I-FABP levels but normalization of TNF, sIL-6R, and D-dimer levels. CRP, sCD14, and HA levels decreased during ART but remained elevated compared with HIV-uninfected participants. Higher sCD14, CRP, and D-dimer levels were associated with higher peripheral blood mononuclear cell and gut integrated HIV DNA levels. Decreases in sCD14 and CRP levels were correlated with increases in CD4 T-cell counts. CONCLUSIONS: ART initiated in early acute HIV infection was associated with normalization of the coagulation cascade and several systemic inflammatory biomarkers, but the acute-phase response, enterocyte turnover, monocyte activation, and fibrosis biomarkers remained elevated. Additional interventions to attenuate inflammation may be needed to optimize clinical outcomes in persons with HIV infection.

Initiation of antiretroviral therapy before detection of colonic infiltration by HIV reduces viral reservoirs, inflammation and immune activation.

INTRODUCTION: Colonic infiltration by HIV occurs soon after infection, establishing a persistent viral reservoir and a barrier to cure. We investigated virologic and immunologic correlates of detectable colonic HIV RNA during acute HIV infection (AHI) and their response to antiretroviral treatment (ART). METHODS: From 49,458 samples screened for HIV, 74 participants were enrolled during AHI and 41 consented to optional sigmoidoscopy, HIV RNA was categorized as detectable (≥50 copies/mg) or undetectable in homogenized colon biopsy specimens. Biomarkers and HIV burden in blood, colon and cerebrospinal fluid were compared between groups and after 24 weeks of ART. RESULTS: Colonic HIV RNA was detectable in 31 participants (76%) and was associated with longer duration since HIV exposure (median 16 vs. 11 days, p=0.02), higher median plasma levels of cytokines and inflammatory markers (CXCL10 476 vs. 148 pg/mL, p=0.02; TNF-RII 1036 vs. 649 pg/mL, p<0.01; neopterin 2405 vs. 1368 pg/mL, p=0.01) and higher levels of CD8+ T cell activation in the blood (human leukocyte antigen - antigen D related (HLA-DR)/CD38 expression 14.4% vs. 7.6%, p <0.01) and colon (8.9% vs. 4.5%, p=0.01). After 24 weeks of ART, participants with baseline detectable colonic HIV RNA demonstrated persistent elevations in total HIV DNA in colonic mucosal mononuclear cells (CMMCs) (median 61 vs. 0 copies/10(6) CMMCs, p=0.03) and a trend towards higher total HIV DNA in peripheral blood mononuclear cells (PBMC) (41 vs. 1.5 copies/10(6) PBMCs, p=0.06). There were no persistent differences in immune activation and inflammation. CONCLUSIONS: The presence of detectable colonic HIV RNA at the time of ART initiation during AHI is associated with higher levels of proviral DNA after 24 weeks of treatment. Seeding of HIV in the gut may have long-lasting effects on the size of persistent viral reservoirs and may represent an important therapeutic target in eradication strategies.

High prevalence of transmitted drug resistance in acute HIV-infected Thai men who have sex with men.

: As use of antiretroviral therapy in Thailand increases, so does the potential for transmission of drug-resistant HIV. We describe the prevalence of WHO surveillance drug resistance mutations among 120 subjects who underwent genotypic testing during acute HIV infection in Bangkok, Thailand. In this cohort of predominantly men who have sex with men, we observed an overall transmitted drug resistance prevalence of 9.2%, including nucleoside/nucleotide analog reverse transcriptase inhibitor 5.0%, nonnucleoside analog reverse transcriptase inhibitor 3.4%, and protease inhibitor 3.4%. These prevalence estimates are higher than previous reports of transmitted drug resistance in Thailand. Baseline drug resistance testing may be warranted, particularly among men who have sex with men.

A novel acute HIV infection staging system based on 4th generation immunoassay.

BACKGROUND: Fourth generation (4thG) immunoassay (IA) is becoming the standard HIV screening method but was not available when the Fiebig acute HIV infection (AHI) staging system was proposed. Here we evaluated AHI staging based on a 4thG IA (4thG staging). FINDINGS: Screening for AHI was performed in real-time by pooled nucleic acid testing (NAT, n=48,828 samples) and sequential enzyme immunoassay (EIA, n=3,939 samples) identifying 63 subjects with non-reactive 2nd generation EIA (Fiebig stages I (n=25), II (n=7), III (n=29), IV (n=2)). The majority of samples tested (n=53) were subtype CRF_01AE (77%). NAT+ subjects were re-staged into three 4thG stages: stage 1 (n=20; 4th gen EIA-, 3rd gen EIA-), stage 2 (n=12; 4th gen EIA+, 3rd gen EIA-), stage 3 (n=31; 4th gen EIA+, 3rd gen EIA+, Western blot-/indeterminate). 4thG staging distinguishes groups of AHI subjects by time since presumed HIV exposure, pattern of CD8+ T, B and natural killer cell absolute numbers, and HIV RNA and DNA levels. This staging system further stratified Fiebig I subjects: 18 subjects in 4thG stage 1 had lower HIV RNA and DNA levels than 7 subjects in 4thG stage 2. CONCLUSIONS: Using 4th generation IA as part of AHI staging distinguishes groups of patients by time since exposure to HIV, lymphocyte numbers and HIV viral burden. It identifies two groups of Fiebig stage I subjects who display different levels of HIV RNA and DNA, which may have implication for HIV cure. 4th generation IA should be incorporated into AHI staging systems.

Long-term lopinavir/ritonavir monotherapy in HIV-infected children.

BACKGROUND: Long-term data are limited on lopinavir/ritonavir monotherapy (mLPV/r) as a treatment simplification strategy in virologically suppressed children. METHODS: Children with confirmed plasma HIV viral load (VL) <50 copies/mL while receiving double protease inhibitors (dPI) were switched to mLPV/r therapy. Virologic failure (VF) was defined as 2 consecutive VL ≥ 500 or 3 consecutive VL ≥ 50 copies/mL. dPI was resumed within 4 weeks in children with VF. Primary endpoint was the proportion of children with VL < 50 copies/mL while still receiving mLPV/r at week 144. RESULTS: Forty children were enrolled; 90% were receiving LPV/r + saquinavir and 10% LPV/r + indinavir before simplifying to mLPV/r. Median age was 11.7 years; 50% were female. Median CD4% was 27%. Four (10%) had VL > 50 copies/mL at entry. At week 144, the proportion of children still receiving mLPV/r who had VL < 50 copies/mL was 22 of 40 (55%). The proportion of all children with VL < 50 copies/mL at week 144 was 33 of 40 (82.5%). Among 16 children who had VF and resumed dPI, 11 (69%) achieved VL < 50 copies/mL at week 144. No children with VF had major LPV/r mutations. Having detectable VL at entry and adherence by pill count <95% for >3 times at any visits during the study period significantly predicted VF on mLPV/r (both P = 0.025). The proportion of children with elevated total cholesterol (>200 mg/dL) decreased from 65% at baseline to 40% at week 144 (P = 0.007). CONCLUSIONS: About half of children maintained virologic suppression on mLPV/r for almost 3 years. VF was common but the majority achieved suppression after resuming dPI and none had major LPV/r mutations. mLPV/r should only be considered for simplified maintenance therapy if frequent VL monitoring to detect VF is available.

Efavirenz, in contrast to nevirapine, is associated with unfavorable progesterone and antiretroviral levels when coadministered with combined oral contraceptives.

BACKGROUND: Effective contraception has been widely promoted for HIV-positive women. However, there are limited data on the interactions between combined hormonal contraceptives and nonnucleoside reverse transcriptase inhibitors . METHODS: This study assessed the steady-state contraceptive effectiveness and safety of combined oral contraceptive (COC) containing 0.150 mg desogestrel /0.030 mg ethinyl estradiol with either nevirapine (NVP) or efavirenz (EFV) in 34 HIV-positive women. The targeted level for contraceptive effectiveness was endogenous progesterone level < 3.0 ng/mL. We measured NVP/EFV plasma concentrations 12 hours after administration (C12) with and without COC. The desired therapeutic levels were >3.1 mg/L for NVP and 1.0-4.0 mg/L for EFV, respectively. RESULTS: All 18 subjects in the NVP group had serum progesterone <1.0 ng/mL. Four of 16 subjects (25%) in the EFV group had serum progesterone >1.0 ng/mL, including 3 subjects with >3.0 ng/mL (might indicate ovulation). The difference in progesterone levels between the 2 groups was statistically significant (P = 0.04). The median C12 of NVP increased insignificantly by 17% with COC; the median C12 of EFV decreased significantly (P = 0.02) by 22%. In 3 of 16 subjects (19%) in the EFV group, C12 of EFV dropped below 1.0 mg/L. CONCLUSIONS: In contrast to NVP, coadministrating desogestrel/ethinyl estradiol containing COC with EFV was associated with unfavorable progesterone and antiretroviral levels. Our results suggest that NVP may be superior to EFV when used with COC in HIV-positive women.