Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Na?ve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

Background

Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread.

Methodology/Principal Findings

DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV gag and env and one sensitive, published nested PCR assay targeting env. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for gag by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV⁺) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1⁺ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate.

Conclusions/Significance

The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific.     

In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection

Background

The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies.

Methodology/Principal Findings

We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA.

Conclusion

This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.     

HLA-Associated Immune Pressure on Gag Protein in CRF01_AE-Infected Individuals and Its Association with Plasma Viral Load

Background

The human leukocyte antigen (HLA)-restricted cytotoxic T-lymphocyte (CTL) immune response is one of the major factors determining the genetic diversity of human immunodeficiency virus (HIV). There are few population-based analyses of the amino acid variations associated with the host HLA type and their clinical relevance for the Asian population. Here, we identified HLA-associated polymorphisms in the HIV-1 CRF01_AE Gag protein in infected married couples, and examined the consequences of these HLA-selected mutations after transmission to HLA-unmatched recipients.

Methodology/Principal Findings

One hundred sixteen HIV-1-infected couples were recruited at a government hospital in northern Thailand. The 1.7-kb gag gene was amplified and directly sequenced. We identified 56 associations between amino acid variations in Gag and HLA alleles. Of those amino acid variations, 35 (62.5%) were located within or adjacent to regions reported to be HIV-specific CTL epitopes restricted by the relevant HLA. Interestingly, a significant number of HLA-associated amino acid variations appear to be unique to the CRF01_AE-infected Thai population. Variations in the capsid protein (p24) had the strongest associations with the viral load and CD4 cell count. The mutation and reversion rates after transmission to a host with a different HLA environment varied considerably. The p24 T242N variant escape from B57/58 CTL had a significant impact on the HIV-1 viral load of CRF01_AE-infected patients.

Conclusions/Significance

HLA-associated amino acid mutations and the CTL selection pressures on the p24 antigen appear to have the most significant impact on HIV replication in a CRF01_AE-infected Asian population. HLA-associated mutations with a low reversion rate accumulated as a footprint in this Thai population. The novel HLA-associated mutations identified in this study encourage us to acquire more extensive information about the viral dynamics of HLA-associated amino acid polymorphisms in a given population as effective CTL vaccine targets.     

Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes

Background

Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage.

Methodology/Principal Findings

To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling.

Conclusions/Significance

These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.     

Somatic Mutations,Allele Loss,and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

Background

The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.

Methods

We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.

Results

Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.

Conclusions

Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.     

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy,Viral Evolution and Compartmentalization

Background

Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.

Methods & Results

We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.

Conclusions

The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.     

PCR-Induced Transitions Are the Major Source of Error in Cleaned Ultra-Deep Pyrosequencing Data

Background

Ultra-deep pyrosequencing (UDPS) is used to identify rare sequence variants. The sequence depth is influenced by several factors including the error frequency of PCR and UDPS. This study investigated the characteristics and source of errors in raw and cleaned UDPS data.

Results

UDPS of a 167-nucleotide fragment of the HIV-1 SG3Δenv plasmid was performed on the Roche/454 platform. The plasmid was diluted to one copy, PCR amplified and subjected to bidirectional UDPS on three occasions. The dataset consisted of 47,693 UDPS reads. Raw UDPS data had an average error frequency of 0.30% per nucleotide site. Most errors were insertions and deletions in homopolymeric regions. We used a cleaning strategy that removed almost all indel errors, but had little effect on substitution errors, which reduced the error frequency to 0.056% per nucleotide. In cleaned data the error frequency was similar in homopolymeric and non-homopolymeric regions, but varied considerably across sites. These site-specific error frequencies were moderately, but still significantly, correlated between runs (r = 0.15–0.65) and between forward and reverse sequencing directions within runs (r = 0.33–0.65). Furthermore, transition errors were 48-times more common than transversion errors (0.052% vs. 0.001%; p<0.0001). Collectively the results indicate that a considerable proportion of the sequencing errors that remained after data cleaning were generated during the PCR that preceded UDPS.

Conclusions

A majority of the sequencing errors that remained after data cleaning were introduced by PCR prior to sequencing, which means that they will be independent of platform used for next-generation sequencing. The transition vs. transversion error bias in cleaned UDPS data will influence the detection limits of rare mutations and sequence variants.     

Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform

Background

16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform

Methodology/Principal Findings

The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.

Conclusions

Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.     

Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background

Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages.

Principal Findings

After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained.

Conclusions

Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.     

Next-generation sequencing analyses of the emergence and maintenance of mutations in CTL epitopes in HIV controllers with differential viremia control

Background

Despite the low level of viral replication in HIV controllers (HICs), studies have reported viral mutations related to escape from cytotoxic T-lymphocyte (CTL) response in HIV-1 plasma sequences. Thus, evaluating the dynamics of the emergence of CTL-escape mutants in HICs reservoirs is important for understanding viremia control. To analyze the HIV-1 mutational profile and dynamics of CTL-escape mutants in HICs, we selected 11 long-term non-progressor individuals and divided them into the following groups: (1) viremic controllers (VCs; n?=?5) and (2) elite controllers (ECs; n?=?6). For each individual, we used HIV-1 proviral DNA from PBMCs related to earliest (V_E) and latest (V_L) visits to obtain gag and nef sequences using the Illumina HiSeq system. The consensus of each mapped gene was used to assess viral divergence, and next-generation sequencing data were employed to identify SNPs and variations within and flanking CTL epitopes.

Results

Divergence analysis showed higher values for nef compared to gag among the HICs. EC and VC groups showed similar divergence rates for both genes. Analysis of the number of SNPs showed that VCs present more variability in both genes. Synonymous/non-synonymous mutation ratios were?<?1 for gag among ECs and for nef among ECs and VCs, exhibiting a predominance of non-synonymous mutations. Such mutations were observed in regions encoding CTL-restricted epitopes in all individuals. All ECs presented non-synonymous mutations in CTL epitopes but generally at low frequency (<?1%); all VCs showed a high number of mutations, with significant frequency changes between V_E and V_L visits. A higher frequency of internal mutations was observed for gag epitopes, with significant changes across visits compared to Nef epitopes, indicating a pattern associated with differential genetic pressure.

Conclusions

The high genetic conservation of HIV-1 gag and nef among ECs indicates that the higher level of viremia control restricts the evolution of both genes. Although viral replication levels in HICs are low or undetectable, all individuals exhibited CTL epitope mutations in proviral gag and nef variants, indicating that potential CTL escape mutants are present in HIC reservoirs and that situations leading to a disequilibrium of the host-virus relationship can result in the spread of CTL-escape variants.

     

Genetic analysis of HIV-1 subtypes in Nairobi, Kenya

Background

Genetic analysis of a viral infection helps in following its spread in a given population, in tracking the routes of infection and, where applicable, in vaccine design. Additionally, sequence analysis of the viral genome provides information about patterns of genetic divergence that may have occurred during viral evolution.

Objective

In this study we have analyzed the subtypes of Human Immunodeficiency Virus -1 (HIV-1) circulating in a diverse sample population of Nairobi, Kenya.

Methodology

69 blood samples were collected from a diverse subject population attending the Aga Khan University Hospital in Nairobi, Kenya. Total DNA was extracted from peripheral blood mononuclear cells (PBMCs), and used in a Polymerase Chain Reaction (PCR) to amplify the HIV gag gene. The PCR amplimers were partially sequenced, and alignment and phylogenetic analysis of these sequences was performed using the Los Alamos HIV Database.

Results

Blood samples from 69 HIV-1 infected subjects from varying ethnic backgrounds were analyzed. Sequence alignment and phylogenetic analysis showed 39 isolates to be subtype A, 13 subtype D, 7 subtype C, 3 subtype AD and CRF01_AE, 2 subtype G and 1 subtype AC and 1 AG. Deeper phylogenetic analysis revealed HIV subtype A sequences to be highly divergent as compared to subtypes D and C.

Conclusion

Our analysis indicates that HIV-1 subtypes in the Nairobi province of Kenya are dominated by a genetically diverse clade A. Additionally, the prevalence of highly divergent, complex subtypes, intersubtypes, and the recombinant forms indicates viral mixing in Kenyan population, possibly as a result of dual infections.     

Characteristics and outcomes of initial virologic suppressors during analytic treatment interruption in a therapeutic HIV-1 gag vaccine trial

Background

In the placebo-controlled trial ACTG A5197, a trend favoring viral suppression was seen in the HIV-1-infected subjects who received a recombinant Ad5 HIV-1 gag vaccine.

Objective

To identify individuals with initial viral suppression (plasma HIV-1 RNA set point <3.0 log₁₀ copies/ml) during the analytic treatment interruption (ATI) and evaluate the durability and correlates of virologic control and characteristics of HIV sequence evolution.

Methods

HIV-1 gag and pol RNA were amplified and sequenced from plasma obtained during the ATI. Immune responses were measured by flow cytometric analysis and intracellular cytokine expression assays. Characteristics of those with and without initial viral suppression were compared using the Wilcoxon rank sum and Fisher''s exact tests.

Results

Eleven out of 104 participants (10.6%) were classified as initial virologic suppressors, nine of whom had received the vaccine. Initial virologic suppressors had significantly less CD4+ cell decline by ATI week 16 as compared to non-suppressors (median 7 CD4+ cell gain vs. 247 CD4+ cell loss, P = 0.04). However, of the ten initial virologic suppressors with a pVL at ATI week 49, only three maintained pVL <3.0 log₁₀ copies/ml. HIV-1 Gag-specific CD4+ interferon-γ responses were not associated with initial virologic suppression and no evidence of vaccine-driven HIV sequence evolution was detected. Participants with initial virologic suppression were found to have a lower percentage of CD4+ CTLA-4+ cells prior to treatment interruption, but a greater proportion of HIV-1 Gag-reactive CD4+ TNF-α+ cells expressing either CTLA-4 or PD-1.

Conclusions

Among individuals participating in a rAd5 therapeutic HIV-1 gag vaccine trial, initial viral suppression was found in a subset of patients, but this response was not sustained. The association between CTLA-4 and PD-1 expression on CD4+ T cells and virologic outcome warrants further study in trials of other therapeutic vaccines in development.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00080106","term_id":"NCT00080106"}}NCT00080106