Phylogenetic Analyses Comparing HIV Sequences from Plasma at Virologic Failure to Cervix Versus Blood Sequences from Antecedent Antiretroviral Therapy Suppression.

Identifying tissue sources of HIV that rebound following "failure" of antiretroviral therapy (ART) is critical to evaluating cure strategies. To assess the role of the uterine cervix and peripheral blood mononuclear cells (PBMC) as viral reservoirs, nearest-neighbor phylogenetic analyses compared genetic relatedness of tissue sequences during ART suppression to those detected in plasma at viral rebound. Blood and genital tract specimens from a natural history cohort of HIV-infected women were collected over 5 years. HIV DNA sequences extracted from PBMC and cervical biopsies during ART suppression and plasma RNA from rebound (defined as HIV RNA >3 log10 copies/mL) were derived by single-genome amplification. Phylogenetic and nearest-neighbor analyses of HIV env sequences and drug resistance in pol sequences were compared between tissues. Nine instances of plasma viral rebound (median HIV RNA 3.6 log10 c/mL; IQR: 3.1-3.8) were detected in 7 of 57 women. Nearest-neighbor analyses found rebound plasma sequences were closer to uterine cervical sequences in 4/9 (44%), closer to PBMC in 3/9 (33%), and ambiguous in 2/9 (22%) cases. Rebound plasma clades (n = 27) shared identical sequences in seven instances with the cervix versus two with PBMC. Novel drug resistance mutations were detected in 4/9 (44%) rebounds. The observed tendency for greater sharing of identical HIV variants and greater nearest-neighbor association between rebounding plasma and uterine cervical versus PBMC sequences suggests that the uterine cervix may be a relevant HIV reservoir. The cervix, a readily accessible tissue in women that can be repeatedly sampled, could help assess the HIV reservoir when evaluating cure strategies.

HIV type 1 zidovudine (ZDV) resistance in blood and uterine cervical secretions of pregnant women.

To determine if HIV-1 resistance testing of the blood predicts mutants in the genital secretions of ZDV-treated pregnant women, ZDV resistance mutations were assessed by an oligonucleotide ligation assay. ZDV resistance was detected in viral sequences from blood (16/48; 33%) and cervical secretions (6/24; 25%) collected during 57 pregnancies. The genotype of 11/69 (16%) resistance codons was discordant between blood and cervical virus of pregnant women near term. However, in only 1 (1.8%) of 57 pregnancies evaluated was resistant virus limited to the cervical secretions. ZDV-resistant virus is rarely limited to the female genital tract.

Lack of resistance to integrase inhibitors among antiretroviral-naive subjects with primary HIV-1 infection, 2007-2013.

BACKGROUND: US guidelines recommend genotyping for persons newly diagnosed with HIV infection to identify transmitted drug resistance mutations associated with decreased susceptibility to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. To date, testing for integrase strand transfer inhibitor (INSTI) mutations has not been routinely recommended. We aimed to evaluate the prevalence of transmitted INSTI mutations among persons with primary HIV-1 infection in Seattle, WA, USA. METHODS: Persons with primary HIV-1 infection have enrolled in an observational cohort at the University of Washington Primary Infection Clinic since 1992. We performed a retrospective analysis of plasma specimens collected prospectively from the 82 antiretroviral-naive subjects who were enrolled from 2007-2013, after FDA-approval of the first INSTI. Resistance testing was performed by consensus sequencing. RESULTS: Specimens for analysis had been obtained a median of 24 (IQR 18-41, range 8-108) days after the estimated date of HIV-1 infection. All subjects were infected with HIV-1 subtype B except for one subject infected with subtype C. Consensus sequencing identified no subjects with major INSTI mutations (T66I, E92Q, G140S, Y143C/H/R, S147G, Q148H/K/R, N155H). Using exact binomial CIs, the upper bound of the 95% CI was 4.4%. CONCLUSIONS: Although our sample size was small, this study does not support the need at this time to evaluate integrase mutations as part of routine consensus sequencing among persons newly diagnosed with HIV-1 infection. However, it is likely that the prevalence of transmitted INSTI mutations may increase with the recent commercial introduction of additional INSTIs and presumably greater INSTI use among persons living with HIV-1.

Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

BACKGROUND: Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. METHODOLOGY/PRINCIPAL FINDINGS: Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. CONCLUSIONS/SIGNIFICANCE: In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

Increased mutations in Env and Pol suggest greater HIV-1 replication in sputum-derived viruses compared with blood-derived viruses.

OBJECTIVE: Low-level HIV-1 replication may occur during antiretroviral therapy (ART) that suppresses plasma HIV-1 RNA to less than 50 copies/ml (suppressive ART). Antiretroviral drugs appear less effective in macrophages and monocytes compared with lymphocytes, both in vitro and as implied in vivo by greater viral evolution observed during suppressive ART. Our objective was to examine sputum, which is rich in macrophages, for evidence of increased HIV-1 replication compared with that in the blood during suppressive ART. DESIGN: A cross-sectional study during suppressive ART was performed, and HIV-1 DNA sequences derived from induced sputa and peripheral blood mononuclear cells were compared. METHODS: Multiple sequences encoding HIV-1 reverse transcriptase, protease, and envelope were generated using single-genome sequencing. Reverse transcriptase and protease sequences were analyzed for genotypic drug resistance. The evolutionary distances of env sequences from the inferred most recent common ancestor of infection were calculated, and CXCR4 usage was predicted. RESULTS: Nine hundred seventy bidirectional sequences from 11 individuals were analyzed. HIV-1 env and pol derived from sputa had greater frequency of drug-resistance mutations (P = 0.05), evolutionary divergence (P = 0.004), and tendency for CXCR4 usage (P = 0.1) compared with viruses derived from peripheral blood mononuclear cells. CONCLUSION: The greater frequency of HIV-1 drug-resistance mutations and divergence of HIV-1 env in sputa-derived viruses compared with peripheral blood mononuclear cell-derived viruses suggests greater HIV-1 replication in the respiratory tract compared with the blood. Characterization of viral evolution over time and by cell-type could identify cells that provide a sanctuary for low-level viral replication in the respiratory tract during suppressive ART.

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus.

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.

Multiple viral genetic analyses detect low-level human immunodeficiency virus type 1 replication during effective highly active antiretroviral therapy.

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by > or =0.9 log(10), resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.