Detection of the impairment of CD80 expression on circulating monocytes in HIV-infected Thai children

The mechanism of progressive anergic response in HIV-infected children has yet to be adequately described. One possibility is inappropriate delivery of an essential second signal for T-cell activation due to the inappropriate presentation of co-stimulatory molecules. To determine whether the ligand for the secondary signal is impaired in pediatric AIDS, we compared the level of CD80 expression by circulating monocytes in HIV-infected and-noninfected children (15 mild/asymptomatic, 13 symptomatic and 12 HIV seronegative children). By two-color flow cytometry analysis, there was no statistically significant difference in the percentage of monocytes expressing CD80 among the groups (i.e., 63.2 +/- 15.8, 60.9 +/- 12.7, 61.04 +/- 10.9 for uninfected children, mild-asymptomatic children and symptomatic children, respectively). However, both infected groups showed statistically significant lower levels of CD80 expression, with mean fluorescent intensities of 40.9 +/- 15.9 and 38.8 +/- 10.7 compared to 57.05 +/- 16.3 for the uninfected control group. Our data demonstrated a correlation between HIV infection and impairment of CD80 by circulating monocytes. Whether the impairment on CD80 expression contributes to destruction of the immunological network in HIV-infected children requires further investigation.     

Accuracy of Clinical Diagnosis of Dengue Episodes in the RV144 HIV Vaccine Efficacy Trial in Thailand

RV144 was a community-based HIV vaccine efficacy trial conducted in HIV-uninfected adults in Thailand, where dengue virus continues to cause a large number of infections every year. We attempted to document the accuracy of clinically diagnosed dengue episodes reported as serious adverse events (SAEs) and adverse events (AEs) and examine whether dengue serology would support the clinical diagnosis. Subjects without a clinical dengue diagnosis but with an infection or idiopathic fever were selected as a control population. Dengue serology was performed by hemagglutination inhibition on plasma samples. A total of 124 clinical dengue episodes were reported (103 SAEs and 21 AEs). Overall 82.6% of the clinically diagnosed dengue episodes were supported by a positive dengue serology: 71.4% of the AEs and 85.0% of the SAEs. Of the 100 subjects with both clinical dengue and positive serology, all presented with fever, 83% with leucopenia, 54% with thrombocytopenia, and 27% with hemorrhagic symptoms. All episodes resolved spontaneously without sequellae. Only two of 15 subjects with a negative serology presented with fever. The sensitivity and specificity of clinical dengue diagnosis were 90.9% and 74.4%, respectively, when compared to the control population, and with a positive predictive value of 82.6% and negative predictive value of 84.7% when compared to dengue serology. Clinical diagnosis of dengue is an accurate method of dengue diagnosis in adults in Thailand. Large-scale clinical trials offer the opportunity to systematically study infectious diseases such as dengue and other infections that may occur during the trial.     

Machine Learning Methods Enable Predictive Modeling of Antibody Feature:Function Relationships in RV144 Vaccinees

The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.     

Neutralizing monoclonal antibodies to Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are serologically related picornaviruses which cause both enteric and neurological disease in mice. The biological activities of TMEV vary between the two different TMEV subgroups (TO and GDVII) and with different passage histories of the same TMEV strain (e.g., mouse brain-passed versus tissue culture-passed DA strain of the TO subgroup). We raised neutralizing monoclonal antibodies (mAbs) against tissue culture-passed DA and GDVII strains of TMEV. We produced two mAbs against the DA strain which neutralized all members of the TO subgroup, but not the GDVII subgroup strains (GDVII and FA); these two DA mAbs reacted similarly with both mouse brain-passed DA and tissue culture-passed DA. Of six neutralizing GDVII mAbs, four reacted only to GDVII and FA, whereas two neutralized TO strains as well. These mAbs demonstrate the presence of TMEV group-specific as well as subgroup-specific neutralization and substantiate the division of TMEV into two distinct subgroups. On Western immunoblots one of the two DA mAbs reacted against isolated DA VP1, two GDVII mAbs (which were TMEV group specific) reacted against isolated GDVII VP1 and DA VP1, and the other DA mAb and four other GDVII mAbs required an intact virion conformation for reactivity. An analysis of the epitopes recognized by these mAbs may elucidate sites important in TMEV biological activities.     

The SERPINE2 gene is associated with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a complex human disease likely influenced by multiple genes, cigarette smoking, and gene-by-smoking interactions, but only severe alpha 1-antitrypsin deficiency is a proven genetic risk factor for COPD. Prior linkage analyses in the Boston Early-Onset COPD Study have demonstrated significant linkage to a key intermediate phenotype of COPD on chromosome 2q. We integrated results from murine lung development and human COPD gene-expression microarray studies with human COPD linkage results on chromosome 2q to prioritize candidate-gene selection, thus identifying SERPINE2 as a positional candidate susceptibility gene for COPD. Immunohistochemistry demonstrated expression of serpine2 protein in mouse and human adult lung tissue. In family-based association testing of 127 severe, early-onset COPD pedigrees from the Boston Early-Onset COPD Study, we observed significant association with COPD phenotypes and 18 single-nucleotide polymorphisms (SNPs) in the SERPINE2 gene. Association of five of these SNPs with COPD was replicated in a case-control analysis, with cases from the National Emphysema Treatment Trial and controls from the Normative Aging Study. Family-based and case-control haplotype analyses supported similar regions of association within the SERPINE2 gene. When significantly associated SNPs in these haplotypic regions were included as covariates in linkage models, LOD score attenuation was observed most markedly in a smokers-only linkage model (LOD 4.41, attenuated to 1.74). After the integration of murine and human microarray data to inform candidate-gene selection, we observed significant family-based association and independent replication of association in a case-control study, suggesting that SERPINE2 is a COPD-susceptibility gene and is likely influenced by gene-by-smoking interaction.     

Integration of Genomic and Genetic Approaches Implicates IREB2 as a COPD Susceptibility Gene

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and is influenced by both genetic determinants and smoking. We identified genomic regions from 56 lung-tissue gene-expression microarrays and used them to select 889 SNPs to be tested for association with COPD. We genotyped SNPs in 389 severe COPD cases from the National Emphysema Treatment Trial and 424 cigarette-smoking controls from the Normative Aging Study. A total of 71 autosomal SNPs demonstrated at least nominal significance with COPD susceptibility (p = 3.4 × 10⁻⁶ to 0.05). These 71 SNPs were evaluated in a family-based study of 127 probands with severe, early-onset COPD and 822 of their family members in the Boston Early-Onset COPD Study. We combined p values from the case-control and family-based analyses, setting p = 5.60 × 10⁻⁵ as a conservative threshold for significance. Three SNPs in the iron regulatory protein 2 (IREB2) gene met this stringent threshold for significance, and four other IREB2 SNPs demonstrated combined p < 0.02. We demonstrated replication of association for these seven IREB2 SNPs (all p values ≤ 0.02) in a family-based study of 3117 subjects from the International COPD Genetics Network; combined p values across all cohorts for the main phenotype of interest ranged from 1.6 × 10⁻⁷ to 6.4 × 10⁻⁴. IREB2 protein and mRNA were increased in lung-tissue samples from COPD subjects in comparison to controls. In summary, gene-expression and genetic-association results have implicated IREB2 as a COPD susceptibility gene.     

Identification of New Regions in HIV-1 gp120 Variable 2 and 3 Loops that Bind to α4β7 Integrin Receptor

Background

The gut mucosal homing integrin receptor α4β7 present on activated CD4⁺ T cells interacts with the HIV-1 gp120 second variable loop (V2). Case control analysis of the RV144 phase III vaccine trial demonstrated that plasma IgG binding antibodies specific to scaffolded proteins expressing the first and second variable regions (V1V2) of HIV envelope protein gp120 containing the α4β7 binding motif correlated inversely with risk of infection. Subsequently antibodies to the V3 region were also shown to correlate with protection. The integrin receptor α4β7 was shown to interact with the LDI/V motif on V2 loop but recent studies suggest that additional regions of V2 loop could interact with the α4β7. Thus, there may be several regions on the V2 and possibly V3 loops that may be involved in this binding. Using a cell line, that constitutively expressed α4β7 receptors but lacked CD4, we examined the contribution of V2 and V3 loops and the ability of V2 peptide-, V2 integrin-, V3-specific monoclonal antibodies (mAbs), and purified IgG from RV144 vaccinees to block the V2/V3-α4β7 interaction.

Results

We demonstrate that α4β7 on RPMI8866 cells bound specifically to its natural ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) as well as to cyclic-V2 and cyclic-V3 peptides. This binding was inhibited by anti-α4β7-specific monoclonal antibody (mAb) ACT-1, mAbs specific to either V2 or V3 loops, and by purified primary virions or infectious molecular clones expressing envelopes from acute or chronic subtypes A, C, and CRF01_AE viruses. Plasma from HIV-1 infected Thai individuals as well as purified IgG from uninfected RV144 vaccinees inhibited (0–50%) the binding of V2 and V3 peptides to α4β7.

Conclusion

Our results indicate that in addition to the tripeptide LDI/V motif, other regions of the V2 and V3 loops of gp120 were involved in binding to α4β7 receptors and this interaction was blocked by anti-V2 peptide, anti-V2 integrin, and anti-V3 antibodies. The ability of purified IgG from some of the uninfected RV144 vaccinees to inhibit α4β7 raises the hypothesis that anti-V2 and anti-V3 antibodies may play a role in blocking the gp120-α4β7 interaction after vaccination and thus prevent HIV-1 acquisition.     

HLA class I,KIR, and genome-wide SNP diversity in the RV144 Thai phase 3 HIV vaccine clinical trial

RV144 is the first phase 3 HIV vaccine clinical trial to demonstrate efficacy. This study consisted of more than 8,000 individuals in each arm of the trial, representing the four major regions of Thailand. Human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptor (KIR) genes, as well as 96 genome-wide ancestry informative markers (AIMs) were genotyped in 450 placebo HIV-1-uninfected individuals to identify the immunogenetic diversity and population structure of this cohort. High-resolution genotyping identified the common HLA alleles as A*02:03, A*02:07, A*11:01, A*24:02, A*24:07, A*33:03, B*13:01, B*15:02, B*18:01, B*40:01, B*44:03, B*46:01, B*58:01, C*01:02, C*03:02, C*03:04, C*07:01, C*07:02, C*07:04, and C*08:01. The most frequent three-loci haplotype was B*46:01-C*01:02-A*02:07. Framework genes KIR2DL4, 3DL2, and 3DL3 were present in all samples, and KIR2DL1, 2DL3, 3DL1, 2DS4, and 2DP1 occurred at frequencies greater than 90 %. The combined HLA and KIR profile suggests admixture with neighboring Asian populations. Principal component and correspondence analyses comparing the RV144 samples to the phase 3 International HapMap Project (HapMap3) populations using AIMs corroborated these findings. Structure analyses identified a distinct profile in the Thai population that did not match the Asian or other HapMap3 samples. This shows genetic variability unique to Thais in RV144, making it essential to take into account population stratification while performing genetic association studies. The overall analyses from all three genetic markers indicate that the RV144 samples are representative of the Thai population. This will inform subsequent host genetic analyses in the RV144 cohort and provide insight for future genetic association studies in the Thai population.