Background: Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Methods: Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or “deep” sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (−6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Results: Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. “Deep” sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Conclusions: Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and “deep” V3 sequencing may also be useful tools for assessing tropism.
A solid-phase enzyme immunoassay (EIA) with the capture antibody principle was developed and evaluated for detection of immunoglobulin M (IgM) specific for group B coxsackieviruses (CBV) in human serum. Of 19 patients who were culture positive for CBV type 4 infections, the EIA for CBV type 4 IgM was more sensitive (84%; 16/19 positive) than a standard microneutralization test (16%; 3/19 positive) and a microneutralization assay on the IgM fractions obtained by sucrose density gradient fractionation of human serum (68%; 13/19 positive). The attempt to produce a serotype-specific EIA was not successful, as IgM antibodies were detected to heterotypic CBV. The pooled CBV types 2 to 5 IgM EIA demonstrated an overall sensitivity of 88% (21/24 positive) in comparison to virus isolation for CBV types 4 and 5. The EIA was specific for IgM to CBV, with the exception of some cross-reactions with hepatitis A IgM-positive sera (15%; 3/20). In comparison to the microneutralization test, the EIA for pooled CBV IgM was more rapid, less cumbersome, and more sensitive. Commercially available reagents used in this study enable the CBV IgM EIA to be developed in diagnostic virology laboratories.
Objective: Selection of specific human leukocyte antigen (HLA)-restricted cytotoxic T-lymphocyte (CTL) escape mutations in key Gag epitopes has been associated with loss of HIV immune control on an individual basis. Here we undertake a population-based identification of HLA-associated polymorphisms in Gag and investigate their relationship with plasma viral load. Design: Cross-sectional analysis of 567 chronically HIV subtype B-infected, treatment-naive individuals. Methods: HLA class I-associated Gag substitutions were identified using phylogenetically corrected analysis methods featuring a multivariate adjustment for HLA linkage disequilibrium and a q-value correction for multiple tests. Presence of HLA-associated substitutions and markers of HIV disease status were correlated using Spearman’s rank test. Results: We have created a gene-wide map of HLA class I-associated substitutions in HIV-1 subtype B Gag. This features 111 HLA-associated substitutions occurring at 51 of 500 Gag codons, more than 50% of which occur within published and/or putative HLA-restricted CTL epitopes. A modest inverse correlation was observed between the total number of HLA-associated Gag polymorphic sites within each individual and plasma viral load in chronic untreated infection (R = −0.17, P in vivo contributes to viral control. A modest positive correlation was observed between the proportion of these sites exhibiting HLA-associated substitutions and plasma viral load (R = 0.09, P = 0.03), consistent with a loss of viremia control with the accumulation of CTL escape mutations. Conclusion: Results contribute to our understanding of immune-driven viral adaptation and suggest that the accumulation of CTL escape mutations in Gag results in clinically detectable consequences at the population level. These data have implications for HIV vaccines.
Next generation, "deep", sequencing has increasing applications both clinically and in disparate fields of research. This study investigates the accuracy and reproducibility of "deep" sequencing as applied to co-receptor prediction using the V3 loop of Human Immunodeficiency Virus-1. Despite increasing use in HIV co-receptor prediction, the accuracy and reproducibility of deep sequencing technology, and the factors which can affect it, have received only a limited level of investigation. To accomplish this, repeated deep sequencing results were generated using the Roche GS-FLX (454) from a number of sources including a non-homogeneous clinical sample (N = 47 replicates over 18 deep sequencing runs), and a large clinical cohort from the MOTIVATE and A400129 studies (N = 1521). For repeated measurements of a non-homogeneous clinical sample, increasing input copy number both decreased variance in the measured proportion of non-R5 using virus (p<<0.001 and 0.02 for single replicates and triplicates respectively) and increased measured viral diversity (p<0.001; multiple measures). Detection of sequences with a mean abundance less than 1% abundance showed a 2 fold increase in median coefficient of variation (CV) in repeated measurements of a non-homogeneous clinical sample, and a 2.7 fold increase in CV in the MOTIVATE/A400129 dataset compared to sequences with ≥1% abundance. An unexpected source of error included read position, with low accuracy reads occurring more frequently towards the edge of sequencing regions (p<<0.001). Overall, the primary source of variability was sampling error caused by low input copy number/minority species prevalence, though other sources of error including sequence intrinsic, temporal, and read-position related errors were detected.
Background: Acute-phase CTL responses, and the virus' ability to escape from them, shape the clinical course of HIV-1 disease. Elucidating early within-host viral evolution is key to our understanding of HIV pathogenesis. Using population-level data, the locations and kinetics of HLA-associated escape mutations in Gag, protease/RT and Nef have been identified. Here we extend these observations by characterizing sites and kinetics of escape and reversion in integrase.
Methods: HLA-associated integrase polymorphisms were defined in a cross-sectional analysis of >1200 antiretroviral naive, chronically HIV-1 subtype B-infected individuals. Using Kaplan-Meier methods, rates of HLA-associated escape and reversion at these sites were calculated in a seroconverter cohort (N=98) for whom longitudinal plasma RNA genotyping of integrase was performed in the first year of infection. CTL recognition frequencies of optimally-described epitopes and integrase-wide overlapping peptides (OLP) were assessed by IFN-γ ELISpot in independent cohorts of acute/early (N=289) and chronically-infected (N=372) individuals, respectively.
Results: We identified >100 HLA-associated polymorphisms occurring at 62 integrase codons. Of these, >45% fell within published or putative epitopes (identified by OLP responses and bioinformatic predictions). Over 400 pairwise amino acid associations were identified, illuminating potential compensatory pathways. Longitudinal data from seroconverters revealed evidence of escape within ~50% of published and putative epitopes within the first year of infection. Epitope B*57-SW10 escaped most rapidly, at a rate comparable to B*57-TW10-Gag, despite a lower recognition frequency in acute/early infection. B*44-QW11, B*58-SW10, B*51-LI9 and putative B*27-KY10 also escaped relatively rapidly. Overall, a minimum of ~20-30% of integrase substitutions in the first year post-infection were attributable to escape/reversion.
Conclusions: Immune escape occurs in integrase in the first year of infection, confirming this protein as an early target by CTL. These results extend our understanding of the earliest immune-driven viral adaptation events occurring in vivo.
Objective: Selection of specific human leukocyte antigen (HLA)-restricted cytotoxic T-lymphocyte (CTL) escape mutations in key Gag epitopes has been associated with loss of HIV immune control on an individual basis. Here we undertake a population-based identification of HLA-associated polymorphisms in Gag and investigate their relationship with plasma viral load. Design: Cross-sectional analysis of 567 chronically HIV subtype B-infected, treatment-naive individuals. Methods: HLA class I-associated Gag substitutions were identified using phylogenetically corrected analysis methods featuring a multivariate adjustment for HLA linkage disequilibrium and a q-value correction for multiple tests. Presence of HLA-associated substitutions and markers of HIV disease status were correlated using Spearman's rank test. Results: We have created a gene-wide map of HLA class I-associated substitutions in HIV-1 subtype B Gag. This features 111 HLA-associated substitutions occurring at 51 of 500 Gag codons, more than 50% of which occur within published and/or putative HLA-restricted CTL epitopes. A modest inverse correlation was observed between the total number of HLA-associated Gag polymorphic sites within each individual and plasma viral load in chronic untreated infection (R = −0.17, P < 0.0001), supporting the hypothesis that a broad ability to target Gag in vivo contributes to viral control. A modest positive correlation was observed between the proportion of these sites exhibiting HLA-associated substitutions and plasma viral load (R = 0.09, P = 0.03), consistent with a loss of viremia control with the accumulation of CTL escape mutations. Conclusion: Results contribute to our understanding of immune-driven viral adaptation and suggest that the accumulation of CTL escape mutations in Gag results in clinically detectable consequences at the population level. These data have implications for HIV vaccines.
ABSTRACT Changes in HIV tropism from R5 to non-R5 or development of drug resistance is often associated with virologic failure in patients treated with maraviroc, a CCR5 antagonist. We sought to examine changes in HIV envelope sequences and inferred tropism in patients who did not respond to maraviroc-based regimens. We selected 181 patients who experienced early virologic failure on maraviroc-containing therapy in the MOTIVATE trials. All patients had R5 HIV by the original Trofile assay before entry. We used population-based sequencing methods and the geno2pheno algorithm to examine changes in tropism and V3 sequences at the time of failure. Using deep sequencing, we assessed whether V3 sequences observed at failure emerged from preexisting subpopulations. From population genotyping data at failure, 90 patients had R5 results, and 91 had non-R5 results. Of the latter group, the geno2pheno false-positive rate (FPR) value fell from a median of 20 at screening to 1.1 at failure. By deep sequencing, the median percentage of non-R5 variants in these patients rose from 1.4% to 99.5% after a median of 4 weeks on maraviroc. In 70% of cases, deep sequencing could detect a pretreatment CXCR4-using subpopulation, which emerged at failure. Overall, there were two distinct patterns of failure of maraviroc. Patients failing with R5 generally had few V3 substitutions and low non-R5 prevalence by deep sequencing. Patients with non-R5 HIV who were failing developed very-high-prevalence non-R5 HIV (median, 99%) and had very low geno2pheno values.
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