Identification and utilization of inter-species conserved (ISC) probesets on Affymetrix human GeneChip(® )platforms for the optimization of the assessment of expression patterns in non human primate (NHP) samples

BACKGROUND: While researchers have utilized versions of the Affymetrix human GeneChip(® )for the assessment of expression patterns in non human primate (NHP) samples, there has been no comprehensive sequence analysis study undertaken to demonstrate that the probe sequences designed to detect human transcripts are reliably hybridizing with their orthologs in NHP. By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip(® )platforms. The utility of human GeneChips(® )for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips(® )for interspecies (human vs NHP) comparisons. RESULTS: ISC probesets in each of the seven Affymetrix GeneChip(® )platforms (U133Plus2.0, U133A, U133B, U95Av2, U95B, Focus and HuGeneFL) were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip(®). Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively. CONCLUSION: It is feasible to use Affymetrix human GeneChip(® )platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies

A Double-Blind Randomized Phase I Clinical Trial Targeting ALVAC-HIV Vaccine to Human Dendritic Cells

BACKGROUND: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone. METHODOLOGY/PRINCIPAL FINDINGS: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production. CONCLUSIONS/SIGNIFICANCE: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00013572

CD4+ T-Cell Decline after the Interruption of Antiretroviral Therapy in ACTG A5170 Is Predicted by Differential Expression of Genes in the Ras Signaling Pathway*

Patterns of expressed genes examined in cryopreserved peripheral blood mononuclear cells (PBMCs) of seropositive persons electing to stop antiretroviral therapy in the AIDS Clinical Trials Group Study A5170 were scrutinized to identify markers capable of predicting the likelihood of CD4+ T-cell depletion after cessation of antiretroviral therapy (ART). A5170 was a multicenter, 96-week, prospective study of HIV-infected patients with immunological preservation on ART who elected to interrupt therapy. Study entry required that the CD4 count was greater than 350 cells/mm3 within 6 months of ART initiation. Median nadir CD4 count of enrollees was 436 cells/mm3. Two cohorts, matched for clinical characteristics, were selected from A5170. Twenty-four patients with an absolute CD4 cell decline of less that 20% at week 24 (good outcome group) and 24 with a CD4 cell decline of >20% (poor outcome group) were studied. The good outcome group had a decline in CD4+ T-cell count that was 50% less than the poor outcome group. Significance analysis of microarrays identified differential gene expression (DE) in the two groups in data obtained from Affymetrix Human FOCUS GeneChips. DE was significantly higher in the poor outcome group than in the good outcome group. Prediction analysis of microarrays (PAM-R) identified genes that classified persons as to progression with greater than 80% accuracy at therapy interruption (TI) as well as at 24 weeks after TI. Gene set enrichment analysis (GSEA) identified a set of genes in the Ras signaling pathway, associated with the downregulation of apoptosis, as significantly upregulated in the good outcome group at cessation of ART. These observations identify specific host cell processes associated with differential outcome in this cohort after TI

Sequential waves of gene expression in patients with clinically defined dengue illnesses reveal subtle disease phases and predict disease severity.

BackgroundDengue virus (DENV) infection can range in severity from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Changes in host gene expression, temporally through the progression of DENV infection, especially during the early days, remains poorly characterized. Early diagnostic markers for DHF are also lacking.Methodology/principal findingsIn this study, we investigated host gene expression in a cohort of DENV-infected subjects clinically diagnosed as DF (n = 51) and DHF (n = 13) from Maracay, Venezuela. Blood specimens were collected daily from these subjects from enrollment to early defervescence and at one convalescent time-point. Using convalescent expression levels as baseline, two distinct groups of genes were identified: the "early" group, which included genes associated with innate immunity, type I interferon, cytokine-mediated signaling, chemotaxis, and complement activity peaked at day 0-1 and declined on day 3-4; the second "late" group, comprised of genes associated with cell cycle, emerged from day 4 and peaked at day 5-6. The up-regulation of innate immune response genes coincided with the down-regulation of genes associated with viral replication during day 0-3. Furthermore, DHF patients had lower expression of genes associated with antigen processing and presentation, MHC class II receptor, NK and T cell activities, compared to that of DF patients. These results suggested that the innate and adaptive immunity during the early days of the disease are vital in suppressing DENV replication and in affecting outcome of disease severity. Gene signatures of DHF were identified as early as day 1.Conclusions/significanceOur study reveals a broad and dynamic picture of host responses in DENV infected subjects. Host response to DENV infection can now be understood as two distinct phases with unique transcriptional markers. The DHF signatures identified during day 1-3 may have applications in developing early molecular diagnostics for DHF

List of top 7 genes differentiating DF from DHF with 96% accuracy.

<p>List of top 7 genes differentiating DF from DHF with 96% accuracy.</p

Common and differential GO pathways in early acute and late acute phases.

<p>Common and differential GO pathways in early acute and late acute phases.</p

Two waves of gene expression over the disease stages.

<p>Median marker expression intensity (log 2 value) of the genes detected in the early and late acute phases from G0 to G7 was shown.</p

Daily gene expression patterns define 2 subtle phases during the disease process.

<p>a) Illness days with corresponding stages (G) and phases for samples on the HG-focus platform. The number of febrile and defervescent samples in each G was shown. b) A global overview of the gene expression pattern in G0 to G7. Using convalescent samples (G7) as a baseline, significant genes in each stage (from G0 to G6) were detected using multi-way ANOVA. PCA was performed using all of the significant genes found in G0 to G7 on the HG-focus platform. All of the samples (represented by dots) were included in the PCA. c) PCA was performed on the defervescent samples only. d) PCA was performed on the febrile samples only and are indicated according to the number of fever days. e) Samples were reassigned to the early acute, late acute and convalescent phases. Differentially expressed genes in the early acute <i>vs.</i> convalescent or late acute <i>vs.</i> convalescent phase were detected using multi-way ANOVA. Shared genes between the early and late acute phases were represented by a Venn diagram. f) PCA was performed using 313 phase-specific genes.</p

Sample information on two gene chip platforms.^*

*<p>Tables shows number of samples per category.</p>**<p>Among 97 samples tested on HG-U133plus2, 50 were also tested on HF-focus.</p