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Sequence variation in the gag p6 region in subtype B HIV-1 has been associated with changes in viral replication capacity and antiretroviral drug susceptibility. We examined sequence variation in the HIV-1 gag p6 region using plasma samples from 22 individuals with non-subtype B HIV-1 infection [subtypes A, C, D, F, and G, and circulating recombinant forms (CRFs) CRF01-AE and CRF02_AG]. An additional 105 gag sequences from the Los Alamos National Laboratory database were also analyzed. Extensive length variation was observed in the p6 gag region. Specific patterns of insertions and deletions were observed in different subtypes and CRFs, and no two subtypes or CRFs had the same general pattern. PTAP duplications were more common in subtype C than other strains (3 of 14 in subtype C vs. 2 of 113 in other strains, p = 0.004), and KQE duplications were seen only in subtype B. Further studies are needed to determine whether such genotypic differences influence viral replication capacity, antiretroviral drug susceptibility, or other phenotypic properties of these strains.

A prototype assay was used to genotype integrase (IN) from 120 HIV-1- infected IN inhibitor-naive adults from Argentina, Brazil, Cameroon, South Africa, Thailand, and Uganda. Subtype designations based on analysis of pol IN sequences were A (14), B (15), C (12), D (11), F (12), G (7), H (1), CRF01_AE (9), CRF02_AG (34), CRF22_01A1 (4), and CRF37_cpx (1). Ten (8.3%) of 120 samples had mutations associated with reduced susceptibility to the IN inhibitors, raltegravir and elvitegravir. Two samples had E92Q (both subtype B) and eight had E157Q (2A, 1C, 1D, 1F, 3 CRF02_AG). Some samples had other mutations selected by these drugs including T97A, and some had amino acid polymorphisms at positions associated with raltegravir and elvitegravir resistance. Mutations associated with other investigational HIV IN inhibitors were also identified. This suggests that HIV strains may vary in their natural susceptibility to HIV IN inhibitors.

Objectives: To compare the kinetics and magnitude of HIV-1 RNA responses to antiretroviral therapy (ART) in the cerebrospinal fluid (CSF) and plasma. Design: Repeated lumbar punctures (LPs) were performed after the initiation or change in ART in 15 HIV-1-infected subjects, with the focus on two phases of response: an acute phase within the first 11 days, for which crude estimates of viral RNA half-lives and decay rates were derived and CSF:plasma relative decay ratios quantitatively analysed; and a longer-term phase beyond 4 weeks that was descriptively assessed. Results: In 13 subjects studied during the acute phase, the crude HIV-1 RNA half-life was longer (median 2.0 compared with 1.9 days), the decay rate slower (median 0.13 compared with 0.16 log10 copies/day) and, most notably, the variability greater (intraquartile range of half-life 1.8-4.3 compared with 1.7-2.1 days) in the CSF than in the plasma. A slower decay in the CSF correlated with lower initial blood CD4 T lymphocyte counts (P=0.001). Seven of 11 subjects studied at 4 weeks or later, including some with slower acute-phase CSF responses, showed greater or more durable viral suppression in the CSF. Conclusion: Divergent acute-phase viral kinetics in the CSF and plasma, and proportionally greater long-term decrements in CSF HIV-1 RNA in slow early-responders or poor overall plasma responders indicate variable compartmentalization of CSF infection, consistent with a model of two prototypes of CSF infection: short-lived, transitory infection that predominates in early HIV-1 infection and longer-lived, more autonomous CSF infection predominating in late HIV-1 infection. Additional studies will be needed to define more precisely the acute and longer-term CSF kinetics in different clinical settings and to assess this model.

Detailed comparisons of HIV drug resistance assays are needed to identify the most useful assays for research studies, and to facilitate comparison of results from studies that use different methods. We analyzed nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in 40 HIV-infected Ugandan infants who had received nevirapine (NVP)-based prophylaxis using the following assays: an FDA-cleared HIV genotyping assay (the ViroSeq HIV-1 Genotyping System v2.0), a commercially available HIV genotyping assay (GeneSeq HIV), a commercially available HIV phenotyping assay (PhenoSense HIV), and a sensitive point mutation assay (LigAmp). ViroSeq and GeneSeq HIV results (NVP resistance yes/no) were similar for 38 (95%) of 40 samples. In 6 (15%) of 40 samples, GeneSeq HIV detected mutations in minor subpopulations that were not detected by ViroSeq, which identified two additional infants with NVP resistance. LigAmp detected low-level mutations in 12 samples that were not detected by ViroSeq; however, LigAmp testing identified only one additional infant with NVP resistance. GeneSeq HIV and PhenoSense HIV determinations of susceptibility differed for specific NNRTIs in 12 (31%) of the 39 samples containing mixtures at relevant mutation positions. PhenoSense HIV did not detect any infants with NVP resistance who were not identified with GeneSeq HIV testing. In this setting, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility.

We analyzed HIV gp41 from 195 men in the United States who were HIV-1 infected between 1999 and 2002, before enfuvirtide (ENF) was approved for clinical use in the United States. gp41 genotyping results were obtained for 175 samples. None of the samples had major ENF resistance mutations. Six (3.4%) samples had minor ENF resistance mutations in the HR1 region (V38G, N43K, L44M, L45M). Twenty-eight (16%) samples had the N42S polymorphism, which is associated with ENF hypersusceptibility. Accessory mutations in the HR2 region were identified in some samples (E137K, S138A). Five of the six samples with HR1 resistance mutations were analyzed with a phenotypic assay; one sample had reduced ENF susceptibility (a sample with N42S + L44M + E137K). Prior to the availability of ENF, some men in the United States were infected with HIV that contained mutations associated with ENF resistance or hypersusceptibility. However, most of the mutations were not associated with phenotypic ENF resistance.

The HIV fusion inhibitor enfuvirtide (ENF/Fuzeon) targets the env gp41 transmembrane domain. Mutations in gp41 are associated with ENF resistance. We developed a prototype assay to genotype a 676-bp region spanning the heptad repeat domains (HR1 and HR2) of HIV-1 gp41. Plasma samples were collected from 126 HIV-1-infected blood donors in Cameroon, Brazil, Uganda, South Africa, Thailand, and Argentina. Based on analysis of gag p24, pol integrase, and env gp41 genes, the panel was composed of subtypes A/A2 (18), B (11), C (14), D (10), F/F2 (9), G (7), CRF01_AE (9), CRF02_AG (33), and recombinant strains (15). Genotyping was successful for 119 of the 126 samples (94.4%). Although numerous amino acid polymorphisms were detected in some samples, none had primary mutations associated with ENF resistance. The gp41 HIV-1 research reagents developed by Celera are useful tools for genotyping analysis of the gp41 region in diverse HIV-1 strains.

Aim The aim of this study was to evaluate the workflow and performance of a NGS-based high resolution HLA typing prototype assay developed by GenDx. Methods 48 UCLA reference panel samples were amplified at 11 loci (HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB, DPA) using NGS-go® primers with Long-Range (LR) PCR and sequenced on the Illumina MiSeq platform. A full gene (5’ UTR to 3’ UTR) amplification strategy was applied with the exception of DRB1 which amplified from exon 2 to exon 4. Individual amplicons were generated for each sample using 100 ng of gDNA per LR-PCR reaction. Intrapatient amplicons were pooled, enzymatically fragmented, and purified. Purified fragmented dsDNA was brought through library preparation using NEBNext (New England Biolabs) for MiSeq sequencing. Due to the limited number of indices (24) in NEBNext, two 2 × 150 paired-end sequencing runs were completed with each run containing 24 samples. Demultiplexed reads were aligned and typed using GenDx NGSengine® v1.4.0 (IMGT v 3.15.0) and Omixon Target v1.8.0 (IMGT v 3.10.0) software. Results 47/48 UCLA samples successfully amplified; a single sample failed potentially due to gDNA integrity. For HLA-A, B, C, DRB1, DQB1, DQA1, DPB and DPA an average of 5 μ g of dsDNA was produced per amplicon, and 99.7% agreed with the reference SBT allele assignments. In one sample DRB1 04:05/15:02 were mistyped by both software packages. SBT reference typing was not available for DRB3/4/5, however when both software packages called a loci, there was 100% agreement at 4 digit allele typing level. Average sequencing output was 3.65 Gb of sequence (12 million paired sequence reads) per run and a mean of 459,573 (median: 433,292) paired-end reads per sample. Conclusion We have demonstrated that GenDx’s NGS HLA typing assay has high accuracy, throughput, and provided unambiguous high resolution results for 47/48 UCLA reference panel samples. This assay delivered full genomic sequences for all targeted HLA loci except DRB1 exon 1 and had the capacity to analyze 11 loci for 24 samples per MiSeq run.