A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.

BACKGROUND: Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. METHODS: A multicenter study was conducted to validate an updated assay design for 454 Life Sciences' GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940-447,400 copies/mL, two dilution series (52,129-1,340 and 25,130-734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10-99, RT codons 1-251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. RESULTS: The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592-3,488) and 2,410 for V3 (IQR 786-3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2-5.2 ± 0.04-0.65). In the two dilutions series, no variants >20% were missed, variants 2-10% were detected at most sites (even at low VT), and variants 1-2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. CONCLUSIONS: This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.

Deep sequencing of HIV-1 variants from paired plasma and cerebrospinal fluid during primary HIV infection.

BACKGROUND: Limited data exist comparing viral quasispecies between cerebrospinal fluid (CSF) and plasma compartments during primary HIV infection. Deep sequencing is a new method to examine the HIV plasma and CSF quasispecies. METHODS: In this pilot study, deep sequencing of protease (PR) and reverse transcriptase (RT) was performed in plasma and CSF from participants during primary HIV infection. Estimated mutational load was calculated by mutant variant frequency multiplied by HIV-RNA level. RESULTS: Paired plasma and CSF samples were studied from five antiretroviral therapy-naïve male participants with median 109 days post estimated transmission, age 32 years, CD4 cell count 580 cells/µL, HIV-RNA 5.18 log10 copies/mL in plasma and 3.67 log10 copies/mL in CSF. Plasma samples averaged 7,124 reads of PR and 2,448 reads of RT, whereas CSF samples averaged 7,082 and 2,792 reads, respectively. A distinct drug-resistance pattern with linked mutations present at significant levels (5-10%) was detected in one participant in CSF. Other low abundance variants (>0.2%) were detected in plasma and CSF of four out of five participants. CONCLUSIONS: Deep sequencing of CSF HIV is technically possible with sufficient HIV-RNA levels. Differences between the quasispecies in the two compartments detected in one participant, which were present with a high mutational load in CSF at an estimated 3.6 months after HIV infection, suggest that early CNS compartmentalisation may be revealed by sensitive deep-sequencing methods. The presence of distinct low abundance (<1%) resistance variants in plasma and CSF of three other subjects may be significant, but further investigation is needed.

Low-frequency NNRTI-resistant HIV-1 variants and relationship to mutational load in antiretroviral-naïve subjects.

Low-frequency HIV variants possessing resistance mutations against non­nucleoside reverse transcriptase inhibitors (NNRTI), especially at HIV reverse transcriptase (RT) amino acid (aa) positions K103 and Y181, have been shown to adversely affect treatment response. Therapeutic failure correlates with both the mutant viral variant frequency and the mutational load. We determined the prevalence of NNRTI resistance mutations at several RT aa positions in viruses from 204 antiretroviral (ARV)-naïve HIV-infected individuals using deep sequencing, and examined the relationship between mutant variant frequency and mutational load for those variants. Deep sequencing to ≥0.4% levels found variants with major NNRTI-resistance mutations having a Stanford-HIVdb algorithm value ≥30 for efavirenz and/or nevirapine in 52/204 (25.5%) ARV-naïve HIV-infected persons. Eighteen different major NNRTI mutations were identified at 11 different positions, with the majority of variants being at frequency >1%. The frequency of these variants correlated strongly with the mutational load, but this correlation weakened at low frequencies. Deep sequencing detected additional major NNRTI-resistant viral variants in treatment-naïve HIV-infected individuals. Our study suggests the significance of screening for mutations at all RT aa positions (in addition to K103 and Y181) to estimate the true burden of pre-treatment NNRTI-resistance. An important finding was that variants at low frequency had a wide range of mutational loads (>100-fold) suggesting that frequency alone may underestimate the impact of specific NNRTI-resistant variants. We recommend further evaluation of all low-frequency NNRTI-drug resistant variants with special attention given to the impact of mutational loads of these variants on treatment outcomes.

An international multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing.

The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600-573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10-99 and reverse transcriptase (RT) codons 1-251 were generated using molecular barcoded primers. 454-UDS was performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1364 reads per position (IQR=809-2065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1-25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1 and 10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the method. HIV-1 drug resistance testing using 454 ultra-deep pyrosequencing results in an accurate and highly reproducible, albeit complex, approach to the analysis of HIV-1 mutant spectra, even at frequencies well below those detected by routine population sequencing.

The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.

Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.

Prevalence of low-level HIV-1 variants with reverse transcriptase mutation K65R and the effect of antiretroviral drug exposure on variant levels.

BACKGROUND: It has been reported that treatment-naive individuals infected with HIV-1 subtype C may be more likely to harbour viral variants possessing a K65R reverse transcriptase gene mutation. The objectives of this study were to determine the prevalence of low-level K65R variants within different HIV-1 subtypes and to assess the effects of antiretroviral exposure on K65R variant levels. METHODS: Treatment-naive individuals infected with different HIV-1 subtypes were genotyped by ultra-deep sequencing. Samples were evaluated for low-level variants to 0.4% or 1% levels depending upon viral load. Estimated mutational load was calculated by multiplying the percentage of the variant by the plasma viral load. RESULTS: A total of 411 treatment-naive individuals were evaluated by ultra-deep sequencing to 1% levels; 4 subjects (0.97%) had K65R variants at ≥1% or had a very high mutation load. All four subjects had variants with linked drug resistance mutations suggesting transmitted resistant variants. 147 ARV-naive subjects were sequenced to 0.4% levels; 8.8% (13/147) had K65R low-level variants identified: 2.2% (2/92) in subtype B, 35.7% (10/28) in subtype C (P<0.001 for B versus C) and 3.7% (1/27) in non-B/C subtypes. The 13 ARV-naive subjects with K65R variants at <1% received tenofovir plus emtricitabine plus a ritonavir-boosted protease inhibitor (TDF+FTC+PI/r) and 5 subsequently experienced virological failure. There was no enhancement in K65R levels by percentage or mutational load compared to pre-therapy levels. CONCLUSIONS: Low-level K65R variants were more frequently identified in subtype C. K65R variants at >1% levels likely represent transmitted resistant variants. The clinical implication of low-level K65R variants below 1% in treatment-naive subjects who receive TDF+FTC+PI/r remains to be determined as the majority are very low-level and did not increase after antiretroviral exposure.

Effect of mucosal fluid from women with bacterial vaginosis on HIV trans-infection mediated by dendritic cells.

Women with bacterial vaginosis (BV) have a higher risk of HIV transmission but the cause of risk is unknown. Dendritic cells (DC) are implicated in transmission of HIV and we previously observed that DC mature when exposed to mucosal fluid from women with BV. We hypothesized that maturation of DC by BV mucosal fluid would enhance DC-mediated trans-infection of HIV. Monocyte-derived DC (MDDC) were treated with mucosal fluid, incubated with HIV(Bal), and HIV trans-infection was evaluated. While LPS-treated MDDC increased HIV(Bal)trans-infection, BV fluid reduced trans-infection. HIV(Bal) DNA levels in MDDC were not affected by BV fluid or LPS but productive infection of MDDC was decreased by LPS and BV fluid. Mucosal fluid from women with BV does not increase MDDC-mediated trans-infection suggesting that BV does not increase HIV susceptibility by increasing DC-mediated trans-infection. However, indirect effects of DC maturation on HIV transmission cannot be ruled out.

Dendritic cell activation and maturation induced by mucosal fluid from women with bacterial vaginosis.

Dendritic cells (DC) at mucosal surfaces mature when exposed to "danger" signals such as LPS. Bacterial vaginosis (BV) is a prevalent alteration of the vaginal bacterial flora associated with preterm childbirth and increased risk for HIV acquisition. We examined the effect of mucosal fluid from women with BV or healthy flora on DC function. IL-12, IL-23 and p40 production by monocyte-derived dendritic cells (MDDC) were all induced by BV samples. Activation/maturation markers HLA-DR, CD40 and CD83 on MDDC incubated with BV CVL were also induced. BV CVL also decreased the endocytic ability of MDDC and increased proliferation of T cells in allogeneic MLR. Plasmacytoid dendritic cell (pDC) CD86 expression was induced by BV CVL. Healthy flora CVL had little effect in any of the tests. This study suggests that BV, but not healthy flora, affects local dendritic cell function in vivo suggesting a mechanism through which BV affects mucosal immunity.