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Statistics in medicine  2009;28(4):716-719.
In replying to Andrew Dunning’s helpful letter, we endeavor to clarify the value of incorporating baseline participant predictors of the potential surrogate endpoint and of the clinical endpoint into the evaluation of a surrogate endpoint. Such baseline predictors are useful for the evaluation of surrogates defined within each of the statistical and principal surrogate frameworks, which are complementary.
PMCID: PMC4510047  PMID: 19140113
Clinical trial; Counterfactual; Immune correlate; Potential outcomes; Principal surrogate
2.  Temporal and spatial evolution of somatic chromosomal alterations: A case-cohort study of Barrett’s esophagus 
All cancers are believed to arise by dynamic, stochastic somatic genomic evolution with genome instability, generation of diversity and selection of genomic alterations that underlie multi-stage progression to cancer. Advanced esophageal adenocarcinomas (EAs) have high levels of somatic copy number alterations. Barrett’s esophagus (BE) is a risk factor for developing EA, and somatic chromosomal alterations (SCAs) are known to occur in BE. The vast majority (~95%) of individuals with BE do not progress to EA during their lifetimes, but a small subset develop EA, many of which arise rapidly even in carefully monitored patients without visible endoscopic abnormalities at the index endoscopy. Using a well-designed, longitudinal case-cohort study, we characterized SCA as assessed by SNP arrays over space and time in 79 “progressors” with BE as they approach the diagnosis of cancer and 169 “nonprogressors” with BE who did not progress to EA over 20,425 person-months of follow-up. The genomes of nonprogressors typically had small localized deletions involving fragile sites and 9p loss/copy neutral LOH that generate little genetic diversity and remained relatively stable over prolonged follow-up. As progressors approach the diagnosis of cancer, their genomes developed chromosome instability with initial gains and losses, genomic diversity, and selection of SCAs followed by catastrophic genome doublings. Our results support a model of differential disease dynamics in which nonprogressor genomes largely remain stable over prolonged periods whereas progressor genomes evolve significantly increased SCA and diversity within four years of EA diagnosis, suggesting a window of opportunity for early detection.
PMCID: PMC3904552  PMID: 24253313
Somatic genomic evolution; Barrett’s esophagus; overdiagnosis; genome doubling in cancer; temporal and spatial neoplastic evolution
3.  Statistical approaches to analyzing HIV-1 neutralizing antibody assay data 
Neutralizing antibody assays are widely used in research toward development of a preventive HIV-1 vaccine. Currently, the neutralization potency of an antibody is typically quantified by the inhibitory concentration (IC) values (e.g., IC50), and the neutralization breadth is estimated by the empirical method. In this paper, we propose the AUC and pAUC measures for summarizing the titration curve, which complement the commonly used IC measure. We present multiple advantages of AUC over IC50, which include no complications due to censoring, the capability to explore low-level neutralization, and improved coverage probabilities and efficiency of estimators. We also propose statistical methods for determining positive neutralization and for estimating the neutralization breadth. The simulation results suggest that the AUC measure is preferable in particular as IC50s get closer to the highest concentration of antibodies tested. For the majority of the assay data, the AUC method is more powerful than the IC50 method. However, since these methods test different hypotheses, it is not unexpected that some virus-antibody combinations are AUC positive but IC50 negative or vice versa.
PMCID: PMC3959164  PMID: 24660049
AUC; breadth; HIV-1; neutralization assay; polynomial model; titration curve
4.  Finding gene clusters for a replicated time course study 
BMC Research Notes  2014;7:60.
Finding genes that share similar expression patterns across samples is an important question that is frequently asked in high-throughput microarray studies. Traditional clustering algorithms such as K-means clustering and hierarchical clustering base gene clustering directly on the observed measurements and do not take into account the specific experimental design under which the microarray data were collected. A new model-based clustering method, the clustering of regression models method, takes into account the specific design of the microarray study and bases the clustering on how genes are related to sample covariates. It can find useful gene clusters for studies from complicated study designs such as replicated time course studies.
In this paper, we applied the clustering of regression models method to data from a time course study of yeast on two genotypes, wild type and YOX1 mutant, each with two technical replicates, and compared the clustering results with K-means clustering. We identified gene clusters that have similar expression patterns in wild type yeast, two of which were missed by K-means clustering. We further identified gene clusters whose expression patterns were changed in YOX1 mutant yeast compared to wild type yeast.
The clustering of regression models method can be a valuable tool for identifying genes that are coordinately transcribed by a common mechanism.
PMCID: PMC3906880  PMID: 24460656
Clustering; Microarray; Regression; Replications; Time course
5.  HIV-1 Vaccine-Induced T-Cell Reponses Cluster in Epitope Hotspots that Differ from Those Induced in Natural Infection with HIV-1 
PLoS Pathogens  2013;9(6):e1003404.
Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.
Author Summary
The HIV epidemic is a major global health challenge leading to more than 1.8 million deaths annually, and despite significant efforts, the search for an efficacious and safe vaccine continues. Several candidate vaccines were designed to elicit CD8+ T-cell responses and were based on using recombinant Adenovirus serotype 5 (rAd-5) vector that expresses HIV-derived antigens. While none of these vaccines had protective effects, they provide an opportunity to study vaccine-induced T-cell responses on a population level. Here, we analyze data from the three largest epitope mapping studies performed in three clinical trials testing two rAd-5 vaccines. We find that vaccine-induced responses tend to cluster in “epitope hotspots” and that these hotspots are different for each vaccine and more surprisingly in two different vaccine trials testing the same vaccine. We also compared vaccine-induced hotspots to those elicited by natural infection and found that some of the vaccine-induced hotspots are not observed in natural infection. Finally, we show that epitope prediction methods can be useful for predicting vaccine induced hotspots based on participants HLA alleles.
PMCID: PMC3688560  PMID: 23818843
6.  Focus and Breadth of Cellular Immune Responses Elicited by a Heterologous Insert Prime-Boost Vaccine Regimen in Rhesus Monkeys 
Vaccine  2011;30(3):506-509.
The global sequence diversity of HIV-1 presents a daunting challenge for vaccine development. We investigated whether a heterologous insert prime-boost regimen could expand global coverage by selectively boosting cellular immune responses to conserved epitopes. Rhesus monkeys were primed and boosted with recombinant adenovirus vectors expressing homologous or heterologous HIV-1 Gag sequences that were optimized to focus responses on highly conserved epitopes. We observed comparable responses directed to specific regions of the Gag protein in all experimental groups without evidence of improved coverage or expanded breadth in the heterologous insert group. These data suggest that antigen-independent factors contribute to the immunodominance patterns of vaccine-elicited cellular immune responses.
PMCID: PMC3246049  PMID: 22133509
7.  A Comparison of Testing Parameters and the Implementation of a Group Sequential Design for Equivalence Studies Using Paired-sample Analysis 
We address the problem of establishing two-sided equivalence using paired-sample analysis of two treatments or two laboratory tests with a binary endpoint. Through real data examples and monte carlo simulations, we compare three commonly used testing parameters, namely, the difference of response probabilities, the ratio of response probabilities, and the ratio of discordant probabilities based on score test statistics for constructing equivalence hypothesis tests of paired binary data. We provide suggestions on the choice of these three testing parameters and proper equivalence margins in hypothesis formulation of equivalence testing. In addition, we describe the implementation of a group sequential design in the context of equivalence testing with early stopping to reject, as well as to declare equivalence.
PMCID: PMC3481840  PMID: 23110243
binary data; double one-sided testing; equivalence boundary; interim analysis; sample sizes
8.  A Sequential Phase 2b Trial Design for Evaluating Vaccine Efficacy and Immune Correlates for Multiple HIV Vaccine Regimens 
Five preventative HIV vaccine efficacy trials have been conducted over the last 12 years, all of which evaluated vaccine efficacy (VE) to prevent HIV infection for a single vaccine regimen versus placebo. Now that one of these trials has supported partial VE of a prime-boost vaccine regimen, there is interest in conducting efficacy trials that simultaneously evaluate multiple prime-boost vaccine regimens against a shared placebo group in the same geographic region, for accelerating the pace of vaccine development. This article proposes such a design, which has main objectives (1) to evaluate VE of each regimen versus placebo against HIV exposures occurring near the time of the immunizations; (2) to evaluate durability of VE for each vaccine regimen showing reliable evidence for positive VE; (3) to expeditiously evaluate the immune correlates of protection if any vaccine regimen shows reliable evidence for positive VE; and (4) to compare VE among the vaccine regimens. The design uses sequential monitoring for the events of vaccine harm, non-efficacy, and high efficacy, selected to weed out poor vaccines as rapidly as possible while guarding against prematurely weeding out a vaccine that does not confer efficacy until most of the immunizations are received. The evaluation of the design shows that testing multiple vaccine regimens is important for providing a well-powered assessment of the correlation of vaccine-induced immune responses with HIV infection, and is critically important for providing a reasonably powered assessment of the value of identified correlates as surrogate endpoints for HIV infection.
PMCID: PMC3502884  PMID: 23181167
HIV vaccine efficacy clinical trial; immune correlate of protection; one-way crossover design; surrogate endpoint for HIV infection; two-phase sampling
9.  Safety and Immunogenicity of the MRKAd5 gag HIV Type 1 Vaccine in a Worldwide Phase 1 Study of Healthy Adults 
The safety and immunogenicity of the MRK adenovirus type 5 (Ad5) HIV-1 clade B gag vaccine was assessed in an international Phase I trial. Three-hundred and sixty healthy HIV-uninfected adults were enrolled on five continents. Subjects received placebo or 1 × 109 or 1 × 1010 viral particles (vp) per dose of the MRKAd5 HIV-1 gag vaccine at day 1, week 4, and week 26. Immunogenicity was evaluated using an IFN-γ ELISPOT gag 15-mer assay with positive responses defined as ≥55 SFC/106 PBMCs and ≥4-fold over mock control. The vaccine was well tolerated. The most common adverse events were injection site reactions, headache, pyrexia, diarrhea, fatigue, and myalgia. At week 30, geometric mean ELISPOT responses were 24, 114, and 226 SFC/106 PBMCs in the placebo, 1 × 109 vp/dose, and 1 × 1010 vp/dose groups, respectively. Overall, responses to 1 × 1010 vp were 85% and 68% in subjects with low (≤200) and high (>200) baseline Ad5 titers, respectively. The MRKAd5 HIV-1 gag vaccine was immunogenic in diverse geographic regions. Gag ELISPOT responses were greater in the 1 × 1010 vp/dose groups than in the 1 × 109 vp/dose groups. Data from this first international study indicate that adenovirus-vectored vaccines are well tolerated and may be immunogenic in subjects from regions with high prevalence of preexisting Ad5 immunity.
PMCID: PMC3422055  PMID: 20854108
10.  Mapping HIV-1 Vaccine Induced T-Cell Responses: Bias towards Less-Conserved Regions and Potential Impact on Vaccine Efficacy in the Step Study 
PLoS ONE  2011;6(6):e20479.
T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1.
Trial Registration NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.
PMCID: PMC3112144  PMID: 21695251
11.  Equivalence of ELISpot Assays Demonstrated between Major HIV Network Laboratories 
PLoS ONE  2010;5(12):e14330.
The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.
We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study.
Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (−1.5%, 1.5%) and CEF (−0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [−15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.
The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.
PMCID: PMC3001861  PMID: 21179404
12.  Estimation of the Initial Viral Growth Rate and Basic Reproductive Number during Acute HIV-1 Infection▿  
Journal of Virology  2010;84(12):6096-6102.
During primary infection, the number of HIV-1 particles in plasma increases rapidly, reaches a peak, and then declines until it reaches a set point level. Understanding the kinetics of primary infection, and its effect on the establishment of chronic infection, is important in defining the early pathogenesis of HIV. We studied the viral dynamics of very early HIV-1 infection in 47 subjects identified through plasma donation screening. We calculated how fast the viral load increases and how variable this parameter is among individuals. We also estimated the basic reproductive ratio, the number of new infected cells generated by an infectious cell at the start of infection when target cells are not limiting. The initial viral doubling time had a median of 0.65 days with an interquartile range of 0.56 to 0.91 days. The median basic reproductive ratio was 8.0 with an interquartile range of 4.9 to 11. In 15 patients, we also observed the postpeak decay of plasma virus and found that the virus decay occurred at a median rate of 0.60 day−1, corresponding to a half-life of 1.2 days. The median peak viral load was 5.8 log10 HIV-1 RNA copies/ml, and it was reached 14 days after the virus was quantifiable with an assay, with a lower limit of detection of 50 copies/ml. These results characterize the early plasma viral dynamics in acute HIV infection better than it has been possible thus far. They also better define the challenge that the immune response (or therapeutic intervention) has to overcome to defeat HIV at this early stage.
PMCID: PMC2876646  PMID: 20357090
13.  Ontogeny of Toll-Like Receptor Mediated Cytokine Responses of Human Blood Mononuclear Cells 
PLoS ONE  2010;5(11):e15041.
Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1β varied by stimuli. Our data contradict the notion of a linear progression from an ‘immature’ neonatal to a ‘mature’ adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation.
PMCID: PMC2994830  PMID: 21152080
14.  Power to Detect HIV Vaccine Effects in Repeated Low-Dose Challenge Experiments 
The Journal of infectious diseases  2009;200(4):609-613.
Simulation studies were conducted to estimate the statistical power of repeated low-dose challenge experiments in non-human primates to detect a candidate HIV vaccine’s effect. The effect of various design parameters on power was explored. Simulation results indicate repeated low-dose challenge studies with total sample size 50 (25 per arm) typically provide adequate power to detect a 50% reduction in the per-exposure probability of infection due to vaccination. Power generally increases with the maximum number of allowable challenges per animal, the per-exposure risk of infection in controls, and the proportion susceptible to infection.
PMCID: PMC2754821  PMID: 19591571
HIV; Macaque; Vaccine
15.  Protective Efficacy of a Single Immunization of a Chimeric Adenovirus Vector-Based Vaccine against Simian Immunodeficiency Virus Challenge in Rhesus Monkeys▿  
Journal of Virology  2009;83(18):9584-9590.
Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for human immunodeficiency virus type 1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing simian immunodeficiency virus (SIV) Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, single-shot immunization may ultimately be preferable for global vaccine delivery. We therefore evaluated the immunogenicity and protective efficacy of a single immunization of chimeric rAd5 hexon hypervariable region 48 (rAd5HVR48) vectors expressing SIV Gag, Pol, Nef, and Env against a homologous SIV challenge in rhesus monkeys. Inclusion of Env resulted in improved control of peak and set point SIV RNA levels following challenge. In contrast, DNA vaccine priming did not further improve the protective efficacy of rAd5HVR48 vectors in this system.
PMCID: PMC2738237  PMID: 19553307
16.  HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis 
Lancet  2008;372(9653):1894-1905.
In the Step Study, the MRKAd5 HIV-1 gag/pol/nef vaccine did not lower post-infection plasma viremia, and HIV-1 incidence was higher in vaccine-treated than placebo-treated males with pre-existing adenovirus serotype 5 (Ad5) immunity. We evaluated vaccine-induced immunity and its potential contributions to infection risk.
To assess immunogenicity, HIV-specific T-cells were characterized ex vivo using validated IFN-γ ELISpot and intracellular cytokine staining (ICS) assays, employing a case-cohort design. To determine effects of vaccine and pre-existing Ad5 immunity on infection risk, flow cytometric studies measured Ad5-specific T-cells and circulating activated (Ki67+/Bcl- 2lo) CD4+ T-cells expressing CCR5.
IFN-γ-secreting HIV-specific T-cells (range, 163–686/106 PBMC) were detected ex vivo by ELISpot in 77% (258/354) of vaccinees; the majority recognized 2–3 HIV proteins. HIV- specific CD4+ T-cells were identified by ICS in 41%; ~85% expressed IL-2, and two-thirds of these co-expressed IFN-γ and/or TNF-α. HIV-specific CD8+ T-cells (range, 0.4–1.0%) were observed in 73%, expressing predominantly either IFN-γ alone or with TNF-α. No major differences were found in vaccine-induced HIV-specific immunity, including response rate, magnitude, and cytokine profile comparing vaccinated male cases (pre-infection) with non-cases. Interestingly, Ad5-specific T-cells were lower in cases than non-cases in several subgroup analyses. The percent circulating Ki67+Bcl-2lo/CCR5+ CD4+ T-cells did not differ between cases and non-cases.
Consistent with previous trials, the MrkAd5/HIV-1 gag/pol/nef vaccine was highly immunogenic for inducing HIV-specific CD8+ T-cells. Comparative analyses did not reveal differences in HIV-specific immunologic responses between cases and non-cases that explain the lack of vaccine efficacy and potential infection enhancement. If T-cell immunity is critical in vaccine-induced HIV protection, our findings suggest that future candidate vaccines must elicit responses that either exceed in magnitude or differ in breadth and/or function from those observed in this trial.
National Institute of Allergy and Infectious Diseases, U.S. National Institute of Health; Merck Research Laboratories
PMCID: PMC2774110  PMID: 19012957
17.  Induction of a Striking Systemic Cytokine Cascade prior to Peak Viremia in Acute Human Immunodeficiency Virus Type 1 Infection, in Contrast to More Modest and Delayed Responses in Acute Hepatitis B and C Virus Infections ▿  
Journal of Virology  2009;83(8):3719-3733.
Characterization of the immune responses induced in the initial stages of human immunodeficiency virus type 1 (HIV-1) infection is of critical importance for an understanding of early viral pathogenesis and prophylactic vaccine design. Here, we used sequential plasma samples collected during the eclipse and exponential viral expansion phases from subjects acquiring HIV-1 (or, for comparison, hepatitis B virus [HBV]or hepatitis C virus [HCV]) to determine the nature and kinetics of the earliest systemic elevations in cytokine and chemokine levels in each infection. Plasma viremia was quantitated over time, and levels of 30 cytokines and chemokines were measured using Luminex-based multiplex assays and enzyme-linked immunosorbent assays. The increase in plasma viremia in acute HIV-1 infection was found to be associated with elevations in plasma levels of multiple cytokines and chemokines, including rapid and transient elevations in alpha interferon (IFN-α) and interleukin-15 (IL-15) levels; a large increase in inducible protein 10 (IP-10) levels; rapid and more-sustained increases in tumor necrosis factor alpha and monocyte chemotactic protein 1 levels; more slowly initiated elevations in levels of additional proinflammatory factors including IL-6, IL-8, IL-18, and IFN-γ; and a late-peaking increase in levels of the immunoregulatory cytokine IL-10. Notably, there was comparatively little perturbation in plasma cytokine levels during the same phase of HBV infection and a delayed response of more intermediate magnitude in acute HCV infection, indicating that the rapid activation of a striking systemic cytokine cascade is not a prerequisite for viral clearance (which occurs in a majority of HBV-infected individuals). The intense early cytokine storm in acute HIV-1 infection may have immunopathological consequences, promoting immune activation, viral replication, and CD4+ T-cell loss.
PMCID: PMC2663284  PMID: 19176632
18.  Development of an automated analysis system for data from flow cytometric intracellular cytokine staining assays from clinical vaccine trials 
Intracellular cytokine staining (ICS) by multiparameter flow cytometry is one of the primary methods for determining T cell immunogenicity in HIV-1 clinical vaccine trials. Data analysis requires considerable expertise and time. The amount of data is quickly increasing as more and larger trials are performed, and thus there is a critical need for high throughput methods of data analysis.
A web based flow cytometric analysis system, LabKey Flow, was developed for analyses of data from standardized ICS assays. A gating template was created manually in commercially-available flow cytometric analysis software. Using this template, the system automatically compensated and analyzed all data sets. Quality control queries were designed to identify potentially incorrect sample collections.
Comparison of the semi-automated analysis performed by LabKey Flow and the manual analysis performed using FlowJo software demonstrated excellent concordance (concordance correlation coefficient >0.990). Manual inspection of the analyses performed by LabKey Flow for 8-color ICS data files from several clinical vaccine trials indicates that template gates can appropriately be used for most data sets.
The semi-automated LabKey Flow analysis system can analyze accurately large ICS data files. Routine use of the system does not require specialized expertise. This high-throughput analysis will provide great utility for rapid evaluation of complex multiparameter flow cytometric measurements collected from large clinical trials.
PMCID: PMC2591089  PMID: 18615598
Flow Cytometry; Intracellular Cytokine Staining; HIV-1; vaccine; T cell; immunogenicity; data analysis; automation
19.  Initial B-Cell Responses to Transmitted Human Immunodeficiency Virus Type 1: Virion-Binding Immunoglobulin M (IgM) and IgG Antibodies Followed by Plasma Anti-gp41 Antibodies with Ineffective Control of Initial Viremia▿ †  
Journal of Virology  2008;82(24):12449-12463.
A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.
PMCID: PMC2593361  PMID: 18842730
20.  Evaluating a surrogate endpoint at three levels, with application to vaccine development 
Statistics in medicine  2008;27(23):4758-4778.
Identification of an immune response to vaccination that reliably predicts protection from clinically significant infection, i.e. an immunological surrogate endpoint, is a primary goal of vaccine research. Using this problem of evaluating an immunological surrogate as an illustration, we describe a hierarchy of three criteria for a valid surrogate endpoint and statistical analysis frameworks for evaluating them. Based on a placebo-controlled vaccine efficacy trial, the first level entails assessing the correlation of an immune response with a study endpoint in the study groups, and the second level entails evaluating an immune response as a surrogate for the study endpoint that can be used for predicting vaccine efficacy for a setting similar to that of the vaccine trial. We show that baseline covariates, innovative study design, and a potential outcomes formulation can be helpful for this assessment. The third level entails validation of a surrogate endpoint via meta-analysis, where the goal is to evaluate how well the immune response can be used to predict vaccine efficacy for new settings (building bridges). A simulated vaccine trial and two example vaccine trials are presented, one supporting that certain anti-influenza antibody levels are an excellent surrogate for influenza illness and another supporting that certain anti-HIV antibody levels are not useful as a surrogate for HIV infection.
PMCID: PMC2646675  PMID: 17979212
clinical trial; counterfactual; immune correlate; meta-analysis; potential outcomes; principal surrogate; statistical surrogate
21.  The Use of Nonhuman Primate Models in HIV Vaccine Development 
PLoS Medicine  2008;5(8):e173.
Cecilia Morgan and colleagues outline a two-stage nonhuman primate screening strategy for T cell-based HIV-1 vaccines.
PMCID: PMC2504486  PMID: 18700814
22.  Genital Herpes Has Played a More Important Role than Any Other Sexually Transmitted Infection in Driving HIV Prevalence in Africa 
PLoS ONE  2008;3(5):e2230.
Extensive evidence from observational studies suggests a role for genital herpes in the HIV epidemic. A number of herpes vaccines are under development and several trials of the efficacy of HSV-2 treatment with acyclovir in reducing HIV acquisition, transmission, and disease progression have just reported their results or will report their results in the next year. The potential impact of these interventions requires a quantitative assessment of the magnitude of the synergy between HIV and HSV-2 at the population level.
Methods and Findings
A deterministic compartmental model of HIV and HSV-2 dynamics and interactions was constructed. The nature of the epidemiologic synergy was explored qualitatively and quantitatively and compared to other sexually transmitted infections (STIs). The results suggest a more substantial role for HSV-2 in fueling HIV spread in sub-Saharan Africa than other STIs. We estimate that in settings of high HSV-2 prevalence, such as Kisumu, Kenya, more than a quarter of incident HIV infections may have been attributed directly to HSV-2. HSV-2 has also contributed considerably to the onward transmission of HIV by increasing the pool of HIV positive persons in the population and may explain one-third of the differential HIV prevalence among the cities of the Four City study. Conversely, we estimate that HIV had only a small net impact on HSV-2 prevalence.
HSV-2 role as a biological cofactor in HIV acquisition and transmission may have contributed substantially to HIV particularly by facilitating HIV spread among the low-risk population with stable long-term sexual partnerships. This finding suggests that prevention of HSV-2 infection through a prophylactic vaccine may be an effective intervention both in nascent epidemics with high HIV incidence in the high risk groups, and in established epidemics where a large portion of HIV transmission occurs in stable partnerships.
PMCID: PMC2377333  PMID: 18493617
23.  Direct Inference of SNP Heterozygosity Rates and Resolution of LOH Detection 
PLoS Computational Biology  2007;3(11):e244.
Single nucleotide polymorphisms (SNPs) have been increasingly utilized to investigate somatic genetic abnormalities in premalignancy and cancer. LOH is a common alteration observed during cancer development, and SNP assays have been used to identify LOH at specific chromosomal regions. The design of such studies requires consideration of the resolution for detecting LOH throughout the genome and identification of the number and location of SNPs required to detect genetic alterations in specific genomic regions. Our study evaluated SNP distribution patterns and used probability models, Monte Carlo simulation, and real human subject genotype data to investigate the relationships between the number of SNPs, SNP HET rates, and the sensitivity (resolution) for detecting LOH. We report that variances of SNP heterozygosity rate in dbSNP are high for a large proportion of SNPs. Two statistical methods proposed for directly inferring SNP heterozygosity rates require much smaller sample sizes (intermediate sizes) and are feasible for practical use in SNP selection or verification. Using HapMap data, we showed that a region of LOH greater than 200 kb can be reliably detected, with losses smaller than 50 kb having a substantially lower detection probability when using all SNPs currently in the HapMap database. Higher densities of SNPs may exist in certain local chromosomal regions that provide some opportunities for reliably detecting LOH of segment sizes smaller than 50 kb. These results suggest that the interpretation of the results from genome-wide scans for LOH using commercial arrays need to consider the relationships among inter-SNP distance, detection probability, and sample size for a specific study. New experimental designs for LOH studies would also benefit from considering the power of detection and sample sizes required to accomplish the proposed aims.
Author Summary
More than 99% of each person's genome is identical to everyone else's. Many of the differences involve single base pairs, termed single nucleotide polymorphisms (SNPs). SNPs are used as genetic markers to facilitate identification of disease-causing genes, as well as in cancer studies by aiding in determining which regions of the genome may be lost (LOH) or amplified during neoplastic progression. One drawback to SNPs is their low informativity: a SNP is only informative if it is polymorphic on the two different alleles found on each chromosome of a pair; and if there is not an informative SNP in the region of genome of interest, it is impossible to detect alterations occurring there through LOH. A common solution to this problem is to use arrays containing hundreds of thousands of SNPs to ensure adequate coverage, but for many studies this is prohibitive on a cost and sample amount basis. In addition, SNP distribution itself can constrain the size of loss that can be reliably detected at the population level. We examined the relationship between chromosome loss sizes and detection probability of LOH genome-wide. The study provides useful information for researchers designing LOH-related studies and evaluating results obtained from such studies.
PMCID: PMC2098867  PMID: 18052545
24.  On Modeling HIV and T Cells In Vivo: Assessing Causal Estimators in Vaccine Trials 
PLoS Computational Biology  2006;2(6):e64.
The first efficacy trials—named STEP—of a T cell vaccine against HIV/AIDS began in 2004. The unprecedented structure of these trials raised new modeling and statistical challenges. Is it plausible that memory T cells, as opposed to antibodies, can actually prevent infection? If they fail at prevention, to what extent can they ameliorate disease? And how do we estimate efficacy in a vaccine trial with two primary endpoints, one traditional, one entirely novel (viral load after infection), and where the latter may be influenced by selection bias due to the former? In preparation for the STEP trials, biostatisticians developed novel techniques for estimating a causal effect of a vaccine on viral load, while accounting for post-randomization selection bias. But these techniques have not been tested in biologically plausible scenarios. We introduce new stochastic models of T cell and HIV kinetics, making use of new estimates of the rate that cytotoxic T lymphocytes—CTLs; the so-called killer T cells—can kill HIV-infected cells. Based on these models, we make the surprising discovery that it is not entirely implausible that HIV-specific CTLs might prevent infection—as the designers explicitly acknowledged when they chose the endpoints of the STEP trials. By simulating thousands of trials, we demonstrate that the new statistical methods can correctly identify an efficacious vaccine, while protecting against a false conclusion that the vaccine exacerbates disease. In addition to uncovering a surprising immunological scenario, our results illustrate the utility of mechanistic modeling in biostatistics.
In traditional biostatistics, mechanistic modeling of the relevant biology usually plays no role, because regulatory agencies will not, quite understandably, license vaccines or drugs on the basis of theories. But the second wave of trials of HIV vaccines will test two conjectures simultaneously. The theoretical possibility that these new, nonclassical, T cell–directed vaccines will prevent some infections while only ameliorating disease in others required biostatisticians to invent new ways of estimating vaccine efficacy. When only the one traditional endpoint—infection—is analyzed, the randomization to vaccine or placebo groups protects against bias. But the new techniques required input from experts on the plausible range of bias introduced by post-randomization selection (by infected state) for the second analysis. Here mechanistic modeling can play a role in evaluating the statistical methodology in biologically plausible settings. By simulating thousands of trials using their models, Wick, Gilbert, and Self were able to demonstrate that the methods protected from falsely concluding a harmful effect of the vaccine on disease. They also noted that the so-called killer T cells, as opposed to antibodies raised by a traditional vaccine, may actually be able to prevent some infections—a conclusion rather surprising for most immunologists and virologists, but which had to be allowed for when designing the vaccine trials.
PMCID: PMC1479086  PMID: 16789816
25.  How Many Human Immunodeficiency Virus Type 1-Infected Target Cells Can a Cytotoxic T-Lymphocyte Kill? 
Journal of Virology  2005;79(21):13579-13586.
The antiviral role of CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.
PMCID: PMC1262579  PMID: 16227278

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