Bin Wang

Background: HIV preferentially infects CD4+ T cells, and the functional impairment and numerical decline of CD4+ and CD8+ T cells characterize HIV disease. The numerical decline of CD4+ and CD8+ T cells affects the optimal ratio between the two cell types necessary for immune regulation. Therefore, this work aimed to define the genomic basis of HIV interactions with the cellular transcriptome of both CD4+ and CD8+ T cells.Results: Genome-wide transcriptomes of primary CD4+ and CD8+ T cells from HIV+ patients were analyzed at different stages of HIV disease using Illumina microarray. For each cell subset, pairwise comparisons were performed and differentially expressed (DE) genes were identified (fold change >2 and B-statistic >0) followed by quantitative PCR validation. Gene ontology (GO) analysis of DE genes revealed enriched categories of complement activation, actin filament, proteasome core and proton-transporting ATPase complex. By gene set enrichment analysis (GSEA), a network of enriched pathways functionally connected by mitochondria was identified in both T cell subsets as a transcriptional signature of HIV disease progression. These pathways ranged from metabolism and energy production (TCA cycle and OXPHOS) to mitochondria meditated cell apoptosis and cell cycle dysregulation. The most unique and significant feature of our work was that the non-progressing status in HIV+ long-term non-progressors was associated with MAPK, WNT, and AKT pathways contributing to cell survival and anti-viral responses.Conclusions: These data offer new comparative insights into HIV disease progression from the aspect of HIV-host interactions at the transcriptomic level, which will facilitate the understanding of the genetic basis of transcriptomic interaction of HIVin vivoand how HIV subverts the human gene machinery at the individual cell type level.

Background: The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed.Result: Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings.Conclusion: These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.

Background: Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results: The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion: The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.

In a recent summary of integrase sequences, primary integrase inhibitor mutations were rare. In a review of integrase inhibitor-naïve Australian HIV-1 sequences, primary mutations were not identified, although the accessory mutation G140S was detected. A link with previous antiretroviral therapy, intra-subtype B divergence across the integrase gene and transmission of integrase polymorphisms were also noted. Based on these findings, we would recommend ongoing surveillance of integrase mutations, and integrase region sequencing for patients prior to commencement of integrase inhibitors.

Background: The efficacy of highly active antiretroviral therapy (HAART) determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6) achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6) responded intermittently. Blood samples were collected over an average of 3 years and 5–8 time points were selected for microarray assay and statistical analysis.Results: Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28) for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33) were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively.Conclusion: Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.

Elite non-progressors (plasma viral load <50 copies/ml while antiretroviral naive) constitute a tiny fraction of HIV-infected individuals. After 12 years follow-up of a cohort of 13 long-term non-progressors (LTNP) identified from 135 individuals with transfusion-acquired HIV infection, 5 remained LTNP after 23 to 26 years infection, but only 3 retained elite LTNP status. We examined the mechanisms that differentiated delayed progressors from LTNP in this cohort.

CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.

Background: Expression levels of cell surface antigens such as CD38 and HLA-DR are related to HIV disease stages. To date, the immunophenotyping of cell surface antigens relies on flow cytometry, allowing estimation of 3–6 markers at a time. The recently described DotScan antibody microarray technology enables the simultaneous analysis of a large number of cell surface antigens. This new technology provides new opportunities to identify novel differential markers expressed or co-expressed on CD4+ and CD8+ T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of the patient.Results: Using this new technology, we compared cell surface antigen expression on purified CD4+ and CD8+ T cells between 3 HIV disease groups (long-term non-progressors controlling viremia naturally; HIV+ patients on highly active antiretroviral therapy (HAART) with HIV plasma viral loads <50 copies/ml; and HIV+ patients with viremia during HAART) and uninfected controls. Pairwise comparisons identified 17 statistically differential cell surface antigens including 5 novel ones (CD212b1, CD218a, CD183, CD3 epsilon and CD9), not previously reported. Notably, changes in activation marker expression were more pronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokines and chemokines were more pronounced in CD4+ T cells.Conclusion: Our study not only confirmed cell surface antigens previously reported to be related to HIV disease stages, but also identified 5 novel ones. Of these five, three markers point to major changes in responsiveness to certain cytokines, which are involved in Th1 responses. For the first time our study shows how density of cell surface antigens could be efficiently exploited in an array manner in relation to HIV disease stages. This new platform of identifying disease markers can be further extended to study other diseases.

Here we investigated the nature and functional consequences of mutations in the HIV-1 tat gene within an epidemiologically-linked AIDS transmission cohort consisting of a non-progressing donor (A) and two normal progressing recipients (B and C). Multiple nonsynonymous mutations in the tat first exon were observed across time in all individuals. Some mutations demonstrated striking host specificity despite the cohort being infected with a common virus. Phylogenetic segregation of the tat clones at the time of progression to AIDS was also observed especially in recipient C. Tat clones supporting high levels of transactivation were present at all time points in all individuals, although a number of clones defective for transactivation were observed for recipient C in later time points. Here we show that the tat quasispecies in a linked transmission cohort diversify and evolve independently between hosts following transmission. It supports the belief that quasispecies variation in HIV-1 is a mechanism for selection towards defining a fitter gene variant that is capable of resisting the human immune system.