A robust bayesian random effects model for nonlinear calibration problems.

In the context of a bioassay or an immunoassay, calibration means fitting a curve, usually nonlinear, through the observations collected on a set of samples containing known concentrations of a target substance, and then using the fitted curve and observations collected on samples of interest to predict the concentrations of the target substance in these samples. Recent technological advances have greatly improved our ability to quantify minute amounts of substance from a tiny volume of biological sample. This has in turn led to a need to improve statistical methods for calibration. In this article, we focus on developing calibration methods robust to dependent outliers. We introduce a novel normal mixture model with dependent error terms to model the experimental noise. In addition, we propose a reparameterization of the five parameter logistic nonlinear regression model that allows us to better incorporate prior information. We examine the performance of our methods with simulation studies and show that they lead to a substantial increase in performance measured in terms of mean squared error of estimation and a measure of the average prediction accuracy. A real data example from the HIV Vaccine Trials Network Laboratory is used to illustrate the methods.

A study of vaccine-induced immune pressure on breakthrough infections in the Phambili phase 2b HIV-1 vaccine efficacy trial.

INTRODUCTION: The Merck Adenovirus-5 Gag/Pol/Nef HIV-1 subtype-B vaccine evaluated in predominately subtype B epidemic regions (Step Study), while not preventing infection, exerted vaccine-induced immune pressure on HIV-1 breakthrough infections. Here we investigated if the same vaccine exerted immune pressure when tested in the Phambili Phase 2b study in a subtype C epidemic. MATERIALS AND METHODS: A sieve analysis, which compares breakthrough viruses from placebo and vaccine arms, was performed on 277 near full-length genomes generated from 23 vaccine and 20 placebo recipients. Vaccine coverage was estimated by computing the percentage of 9-mers that were exact matches to the vaccine insert. RESULTS: There was significantly greater protein distances from the vaccine immunogen sequence in Gag (p=0.045) and Nef (p=0.021) in viruses infecting vaccine recipients compared to placebo recipients. Twenty-seven putative sites of vaccine-induced pressure were identified (p<0.05) in Gag (n=10), Pol (n=7) and Nef (n=10), although they did not remain significant after adjustment for multiple comparisons. We found the epitope sieve effect in Step was driven by HLA A∗02:01; an allele which was found in low frequency in Phambili participants compared to Step participants. Furthermore, the coverage of the vaccine against subtype C Phambili viruses was 31%, 46% and 14% for Gag, Pol and Nef, respectively, compared to subtype B Step virus coverage of 56%, 61% and 26%, respectively. DISCUSSION: This study presents evidence of sieve effects in Gag and Nef; however could not confirm effects on specific amino acid sites. We propose that this weaker signal of vaccine immune pressure detected in the Phambili study compared to the Step study may have been influenced by differences in host genetics (HLA allele frequency) and reduced impact of vaccine-induced immune responses due to mismatch between the viral subtype in the vaccine and infecting subtypes.

Comprehensive assessment of HIV target cells in the distal human gut suggests increasing HIV susceptibility toward the anus.

BACKGROUND: Prevention of rectal HIV transmission is a high-priority goal for vaccines and topical microbicides because a large fraction of HIV transmissions occurs rectally. Yet, little is known about the specific target-cell milieu in the human rectum other than inferences made from the colon. METHODS: We conducted a comprehensive comparative in situ fluorescence study of HIV target cells (CCR5-expressing T cells, macrophages, and putative dendritic cells) at 4 and 30 cm proximal of the anal canal in 29 healthy individuals, using computerized analysis of digitized combination stains. RESULTS: Most strikingly, we find that more than 3 times as many CD68 macrophages express the HIV coreceptor CCR5 in the rectum than in the colon (P = 0.0001), and as such rectal macrophages seem biologically closer to the HIV-susceptible CCR5 phenotype in the vagina than the mostly HIV-resistant CCR5 phenotype in the colon. Putative CD209 dendritic cells are generally enriched in the colon compared with the rectum (P = 0.0004), though their CCR5 expression levels are similar in both compartments. CD3 T-cell densities and CCR5 expression levels are comparable in the colon and rectum. CONCLUSIONS: Our study establishes the target-cell environment for HIV infection in the human distal gut and demonstrates in general terms that the colon and rectum are immunologically distinct anatomical compartments. Greater expression of CCR5 on rectal macrophages suggests that the most distal sections of the gut may be especially vulnerable to HIV infection. Our findings also emphasize that caution should be exercised when extrapolating data obtained from colon tissues to the rectum.

The defined attenuated Listeria monocytogenes delta mp12 mutant is an effective oral vaccine carrier to trigger a long-lasting immune response against a mouse fibrosarcoma.

Listeria monocytogenes has been proposed as a carrier to elicit major histocompatibility complex class-I restricted immune responses able to protect against tumor challenge. In this study the properties of the attenuated L. monocytogenes delta mp12 mutant has been evaluated in vivo against a highly aggressive mouse fibrosarcoma which expresses beta-galactosidase (beta-gal) as a tumor-associated antigen (TAA). Immunization with the vaccine prototypes resulted in both elicitation of specific antibodies and generation of cytotoxic lymphocytes (CTL). Oral vaccination protected 55-64% of the immunized animals from tumor take (p < 0.01) and strongly reduced the average size of the tumor in the other 34-45% (p < 0.01). Vaccinated mice developed a long-lasting response, which resulted in 100% protection from a subsequent tumor challenge. Substitution of the whole TAA by its CTL-defined immunodominant epitope resulted in 43% protection, suggesting a contribution of the humoral response to the observed antitumor effect. No statistically significant differences were observed in the antitumor response when mice were immunized with strains expressing the immunodominant TAA epitope in the context of carrier proteins which were either exported or restricted to the bacterial cytoplasm. This suggests that the topology of the recombinant antigen does not play a major role in the outcome of the protective response.

Listeriolysin O: cholesterol inhibits cytolysis but not binding to cellular membranes.

Listeriolysin O (LLO) binds to cholesterol-containing membranes in which it oligomerizes to form pores. Preincubation of the toxin with cholesterol is known to inhibit haemolysis, whereas the oxidized form of cholesterol has no inhibitory effect. Using immunoblot analyses and flow cytometry we demonstrate that preincubation with cholesterol does not influence binding of the listeriolysin-cholesterol complex to red blood cells, eukaryotic cells or artificial membranes. Lytic activity of membrane-bound LLO inactivated by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. To determine the step at which cholesterol inhibits lytic activity, we looked for pore formation using electron microscopy. Pores formed by purified listeriolysin could be directly visualized using erythrocyte ghosts. This property was lost upon incubation of the toxin with cholesterol. Quantitative analysis strongly suggest that inhibition of lysis by cholesterol is not due to decreased binding of listeriolysin to target membranes, but rather to an interference with a subsequent step leading to polymerization of the toxin.