Viral Blips After Treatment Initiation During Acute Human Immunodeficiency Virus Infection.

Transient viral blips ≥20 copies/mL were observed in 16.9% of acutely treated adults with HIV. Blip incidence increased from 0.0 (95% CI, 0.0-2.9)/100 person-years after ART in Fiebig I to 15.9 (7.6-29.2) in Fiebig V. Increasing viral load and Fiebig stage at ART initiation were independently predictive of blips.

A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays.

Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8-11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-gamma) secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-gamma secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-gamma in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.

Concurrent cocaine withdrawal alters alcohol withdrawal symptoms.

This study compares alcohol withdrawal severity during outpatient detoxification in alcohol dependent subjects (ALC) and in subjects dependent on both alcohol and cocaine (ALC/COC). Subjects included 123 ALC and 66 ALC/COC subjects. Baseline demographic and drug use variables, alcohol withdrawal symptoms, and the total amount of oxazepam taken during alcohol detoxification were compared between the two groups. Compared to ALC subjects, ALC/COC subjects were younger, more likely to be African-American, and had less severe histories of alcohol dependence. However, alcohol withdrawal symptom severity did not differ significantly between the two groups. Nevertheless, controlling for differences in alcohol use history, ALC/COC subjects still received less oxazepam than did ALC subjects to treat alcohol withdrawal symptoms. Despite similar intensity of alcohol withdrawal symptoms, ALC/COC subjects received less oxazepam to treat alcohol withdrawal symptoms compared to ALC subjects. Both subject and clinician factors may explain the difference in oxazepam use.

Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1.

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format.

A computational framework for the analysis of peptide microarray antibody binding data with application to HIV vaccine profiling.

We present an integrated analytical method for analyzing peptide microarray antibody binding data, from normalization through subject-specific positivity calls and data integration and visualization. Current techniques for the normalization of such data sets do not account for non-specific binding activity. A novel normalization technique based on peptide sequence information quickly and effectively reduced systematic biases. We also employed a sliding mean window technique that borrows strength from peptides sharing similar sequences, resulting in reduced signal variability. A smoothed signal aided in the detection of weak antibody binding hotspots. A new principled FDR method of setting positivity thresholds struck a balance between sensitivity and specificity. In addition, we demonstrate the utility and importance of using baseline control measurements when making subject-specific positivity calls. Data sets from two human clinical trials of candidate HIV-1 vaccines were used to validate the effectiveness of our overall computational framework.

Isolation of human monoclonal antibodies from peripheral blood B cells.

Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.

Plasma IgG to linear epitopes in the V2 and V3 regions of HIV-1 gp120 correlate with a reduced risk of infection in the RV144 vaccine efficacy trial.

Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.