Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®.

HIV-1 typically infects cells via the CD4 receptor and CCR5 or CXCR4 co-receptors. Maraviroc is a CCR5-specific viral entry inhibitor; knowledge of viral co-receptor specificity is important prior to usage. We developed and validated an economical V3-env Illumina-based assay to detect and quantify the frequency of viruses utilizing each co-receptor. Plasma from 54 HIV+ participants (subtype B) was tested. The viral template cDNA was generated from plasma RNA with unique molecular identifiers (UMIs). The sequences were aligned and collapsed by the UMIs with a custom bioinformatics pipeline. Co-receptor usage, determined by codon analysis and online phenotype predictors PSSM and Geno2pheno, were compared to existing Trofile® data. The cost of V3-UMI was tallied. The sequences interpreted by Geno2pheno using the most conservative cut-off, a 2% false-positive-rate (FPR), predicted CXCR4 usage with the greatest sensitivity (76%) and specificity (100%); PSSM and codon analysis had similar sensitivity and lower specificity. Discordant Trofile® and genotypic results were more common when participants had specimens from different dates analyzed by either assay. V3-UMI reagents cost USD$62/specimen. A batch of ≤20 specimens required 5 h of technical time across 1.5 days. V3-UMI predicts HIV tropism at a sensitivity and specificity similar to those of Trofile®, is relatively inexpensive, and could be performed by most central laboratories. The adoption of V3-UMI could expand HIV drug therapeutic options in lower-resource settings that currently do not have access to phenotypic HIV tropism testing.

Longitudinal cervicovaginal microbiome and virome alterations during ART and discordant shedding in women living with HIV.

Despite successful suppression of plasma HIV replication by antiretroviral therapy (ART), some women living with HIV (WLHIV) can still experience genital HIV shedding (discordant shedding). Female genital tract (FGT) microbiome and virome dynamics during long-term ART in WLHIV are poorly understood but might contribute to discordant HIV shedding, as the microbiome and virome are known to influence FGT health. To understand FGT microbial communities over time during ART usage and discordant shedding, we characterized the microbiome and virome in 125 cervicovaginal specimens collected over two years in 31 WLHIV in Lima, Peru. Intrapersonal bacterial microbiome variation was higher in HIV shedders compared to non-shedders. Cervicovaginal virome composition changed over time, particularly in non-shedders. Specifically, anellovirus relative abundance was inversely associated with ART duration and CD4 counts. Our results suggest that discordant HIV shedding is associated with FGT microbiome instability, and immune recovery during ART influences FGT virome composition.

Development and Optimization of Oligonucleotide Ligation Assay (OLA) Probes for Detection of HIV-1 Resistance to Dolutegravir.

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART. Probes to detect G118R, Q148H/K/R, N155H and R263K in HIV-1 subtypes A, B, C, D and CRF01_AE were designed using sequence alignments from the Los Alamos database and validated using 61 clinical samples of HIV-1 subtypes A, B, C, D, CRF01_AE genotyped by PacBio (n = 15) or Sanger (n = 46). Initial OLA probes failed to ligate for 16/244 (6.5%) codons (9 at G118R and 7 at Q148H/K/R). Probes revised to accommodate polymorphisms interfering with ligation at codons G118R and Q148R reduced indeterminates to 3.7% (5 at G118R and 4 at Q148H/K/R) and detected DTG-mutations with a sensitivity of 96.5% and 100% specificity. These OLA DTG resistance probes appear highly sensitive and specific across HIV-1 subtypes common in RLS with high burden of HIV infection.

Observational study of effects of HIV acquisition and antiretroviral treatment on biomarkers of systemic immune activation.

To assess whether biomarkers of systemic inflammation are associated with HIV acquisition or with the timing of ART initiation ("immediate", at diagnosis, versus "deferred", at 24 weeks post-diagnosis) in men-who-have-sex-with-men (MSM) and transgender women, we conducted a retrospective study comparing inflammatory biomarkers in participants' specimens collected before infection and after ≥2 years of effective ART. We measured biomarkers in four longitudinally collected plasma, including two specimens collected from each participant before and two after HIV acquisition and confirmed ART-suppression. Biomarkers were quantified by enzyme-linked immuno-assay or Meso Scale Discovery. When evaluating systematic variation in these markers over time, we found that multiple biomarkers consistently varied across participants' two pre-infection or two post-ART-suppression specimens. Additionally, we compared changes in biomarkers after vs before HIV acquisition. Across 47 participants, the levels of C-reactive protein (CRP), monocyte chemo-attractant protein-1, tumor necrosis factor-α and interferon gamma-induced protein-10 significantly increased while leptin and lipopolysaccharide binding protein (LBP) significantly decreased following HIV infection. Randomization to deferred-ART initiation was associated with greater increases in CRP and no decrease in LBP. Acquisition of HIV appeared to induce systemic inflammation, with elevation of biomarkers previously associated with infections and cardiovascular disease. Initiation of ART during the early weeks of infection tempered the increase in pro-inflammatory biomarkers compared to delaying ART for ~24 weeks after HIV diagnosis. These findings provide insight into potential mediators by which immediate-ART initiation improves health outcomes, perhaps because immediate-ART limits the size of the HIV reservoir or limits immune dysregulation that in turn trigger systemic inflammation.

Antigen specificities and proviral integration sites differ in HIV-infected cells by timing of antiretroviral treatment initiation.

Despite effective antiretroviral therapy (ART), persons living with HIV harbor reservoirs of persistently infected CD4+ cells, which constitute a barrier to cure. Initiation of ART during acute infection reduces the size of the HIV reservoir, and we hypothesized that in addition, it would favor integration of proviruses in HIV-specific CD4+ T cells, while initiation of ART during chronic HIV infection would favor relatively more proviruses in herpesvirus-specific cells. We further hypothesized that proviruses in acute ART initiators would be integrated into antiviral genes, whereas integration sites (ISs) in chronic ART initiators would favor genes associated with cell proliferation and exhaustion. We found that the HIV DNA distribution across HIV-specific versus herpesvirus-specific CD4+ T cells was as hypothesized. HIV ISs in acute ART initiators were significantly enriched in gene sets controlling lipid metabolism and HIF-1α-mediated hypoxia, both metabolic pathways active in early HIV infection. Persistence of these infected cells during prolonged ART suggests a survival advantage. ISs in chronic ART initiators were enriched in a gene set controlling EZH2 histone methylation, and methylation has been associated with diminished long terminal repeat transcription. These differences that we found in antigen specificities and IS distributions within HIV-infected cells might be leveraged in designing cure strategies tailored to the timing of ART initiation.

Genome sequences of human anelloviruses in the Lamedtorquevirus, Memtorquevirus, and Samektorquevirus genera identified from the female genital tract.

We identified and characterized seven anellovirus genome sequences in the female genital tract through virome metagenomic sequencing of cervicovaginal lavage specimens from women living with HIV in Peru. Phylogenetic and genomic analyses indicate that they belong to three newly proposed Lamedtorquevirus, Memtorquevirus, and Samektorquevirus genera in the Anelloviridae family.

Impact of Human Immunodeficiency Virus Drug Resistance Mutations Detected in Women Prior to Antiretroviral Therapy With Efavirenz + Tenofovir Disoproxil Fumarate + Lamivudine (or Emtricitabine).

Background: Two large studies suggest that resistance mutations to only nonnucleoside reverse transcriptase inhibitors (NNRTI) did not increase the risk of virologic failure during antiretroviral therapy (ART) with efavirenz/tenofovir disoproxil fumarate/lamivudine (or emtricitabine). We retrospectively evaluated a third cohort to determine the impact of NNRTI resistance on the efficacy of efavirenz-based ART. Methods: Postpartum women living with human immunodeficiency virus (HIV) were studied if they initiated efavirenz-based ART because of the World Health Organization's recommendation for universal ART. Resistance was detected by Sanger genotyping plasma prior to efavirenz-based ART and at virologic failure (HIV RNA >400 copies/mL). Logistic regression examined relationships between pre-efavirenz genotypes and virologic failure. Results: Pre-efavirenz resistance was detected in 169 of 1223 (13.8%) participants. By month 12 of efavirenz-based ART, 189 of 1233 (15.3%) participants had virologic failure. Rates of virologic failure did not differ by pre-efavirenz NNRTI resistance. However, while pre-efavirenz nucleos(t)ide reverse transcriptase inhibitors (NRTI) and NNRTI resistance was rare (8/1223 [0.7%]) this genotype increased the odds (adjusted odds ratio, 11.2 [95% confidence interval, 2.21-72.2]) of virologic failure during efavirenz-based ART. Age, time interval between last viremic visit and efavirenz initiation, clinical site, viremia at delivery, hepatitis B virus coinfection, and antepartum regimen were also associated with virologic failure. Conclusions: Resistance to NNRTI alone was prevalent and dual-class (NRTI and NNRTI) resistance was rare in this cohort, with only the latter associated with virologic failure. This confirms others' findings that, if needed, efavirenz-based ART offers most people an effective alternative to dolutegravir-based ART.

Characterizing HIV drug resistance in cases of vertical transmission in the VESTED randomized antiretroviral treatment trial.

Introduction: VESTED (NCT03048422) compared the safety and efficacy of three antiretroviral treatment (ART) regimens in pregnant and postpartum women: dolutegravir+emtricitabine/tenofovir alafenamide fumarate; dolutegravir+emtricitabine/tenofovir disoproxil fumarate (TDF); efavirenz/emtricitabine/TDF. Vertical HIV transmission (VT) occurred to 4/617 (0.60%) live-born infants, who were evaluated for HIV drug resistance (HIVDR) and other risk factors. Setting: In 2018-2020, pregnant (weeks-14-28) women living with HIV and ≤14 days of ART were enrolled at 22 international sites and followed with their infants through 50 weeks postpartum. Methods: HIV sequences derived by single genome amplification (SGA) from longitudinally collected specimens were assessed from VT Cases for HIVDR in protease, reverse transcriptase, integrase, and the nef 3'polypurine tract (3'PPT). Results: The four Case mothers were prescribed efavirenz-based-ART for 1-7 days prior to randomization to study ART. Their infants received postnatal nevirapine+/-zidovudine prophylaxis and were breastfed. A total of 833 SGA sequences were derived. The "major" (Stanford HIVDR Score ≥60) non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation (K103N) was detected persistently in one viremic mother, and likely contributed to VT of HIVDR. Major NNRTI HIVDR mutations were detected in all three surviving infants. No integrase, nor high frequencies of 3'PPT mutations conferring dolutegravir HIVDR were detected. The timing of HIV infant diagnosis, plasma HIV RNA levels and HIVDR suggests one in utero, one peripartum, one early, and one late breastfeeding transmission. Conclusions: VT was rare. New-onset NNRTI HIVDR in Case mothers was likely from efavirenz-ART prescribed prior to study dolutegravir-ART, and in one case appeared transmitted to the infant despite nevirapine prophylaxis.