An enhanced three-stage design with trend analysis for allergen immunotherapy trials.

We previously introduced a three-stage design and associated end-of-stage analyses for allergen immunotherapy (AIT) trials. End-of-stage differences alone may not provide a fuller picture of Stages 2 and 3 effects because they may depend upon stage-specific durations. Therefore, we introduce an additional trend analysis to evaluate the difference in progression curves of two groups over the entire stage. Results from such analysis are used to inform persistence of end-of-stage benefit and thus provide evidence for stagewise effects beyond the study periods. We jointly apply end-of-stage and trend analyses to support the enhanced three-stage design to determine treatment response over time and sustained response to AIT. A simulation study was performed to illustrate the statistical properties (bias and power) of trend analyses under varying statistical missing mechanisms and effect sizes. The extent of bias depended on the missing mechanism and magnitude. Powers were largely driven by effect and sample sizes as well as pre-specified success margins, particularly of relative trend. As an illustration, assuming relative treatment differences of 25-30%, stagewise dropout rate of 15%, and parallel outcome progressions, a sample size of 200 per group may achieve 97% power to demonstrate a treatment effect and 53% power to demonstrate a sustained effect post-treatment. Trend analysis supplements the end-of-stage analysis to enhance the statistical claims of stagewise effects. Inferential statistics support our proposed trend analysis for evaluating benefits of AIT over time and inform clinical understanding and decisions.

Conditional power in vaccine trials with seasonal variations.

Conditional power (CP) is widely used in clinical trial monitoring to quantify the evidence for futility stopping or sample size adaptation during the trial. When planning an interim analysis in vaccine trials for seasonal infectious diseases, CPs calculated under the hypothesized or currently estimated effect sizes may not truly reflect future data due to seasonal variations in disease incidence and/or vaccine efficacy (VE). Relying on these estimates alone could lead to erroneous decisions. Therefore, we carried out simulation studies to investigate the use of seven different choices for the drift parameter in computing CP or predictive power (PP) in end-of-season interim analysis. Our simulations showed that, when used to inform futility stopping, CP under the hypothesized effect and a weighted PP under a normal prior distribution appear to outperform others in terms of the overall type II error rate. All CPs and PPs considered in this study resulted in comparable powers and expected sample sizes when used to inform sample size adaptation. The performance of either CP or PP largely depends on the extent to which the chosen drift parameter or the prior distribution of the drift parameter matches the remainder of the trial. Weighted CP/PP tends to be less sensitive to settings where observed data and emerging data in future seasons differ substantially as they incorporate both current estimate and future variations. Therefore, weighted strategies deserve further exploration and perhaps increased usage in guiding trial operations because they are more robust to inaccuracies in prediction. In summary, for vaccine trials with seasonal variations, a decision on trial operations should be guided by a careful consideration of plausible CPs and PPs calculated under reasonable assumptions leveraging the data, prior hypotheses, and new evidence on clinical relevance.

A three-stage design for allergen immunotherapy trials.

BACKGROUND: Clinical trials of allergen immunotherapy (AIT) may require up to 5 years to complete. These lengthy trials may be complicated by high and potentially differential dropouts, especially among participants who perceive that they are receiving placebo. We propose a three-stage design in which the placebo group in Stage 1 crosses over to receive active treatment in Stage 2. In Stage 3, AIT is discontinued to determine whether benefit is maintained post-treatment. We apply inferential statistics to support the three-stage design for clinical trials to determine clinical efficacy, treatment response over time, and sustained response to AIT. METHODS: The proposed framework constitutes a series of hypothesis tests for comparing treatment responses at the end of each stage. A simulation study was performed to illustrate the statistical properties under varying statistical missing mechanisms and effect sizes. RESULTS: The statistical properties in terms of bias and statistical power were consistent with what are expected from conventional analyses. Specifically, the extent of bias depended on the missing mechanism and magnitude. The statistical powers were largely driven by effect and sample sizes as well as prespecified success margins. As an illustration, assuming relative treatment differences of 25% and stagewise dropout rate of 15%, a sample size of 200 per group may achieve 93% power to demonstrate a treatment effect and 60% power to demonstrate a maintained response post-treatment. CONCLUSIONS: Inferential statistics support our proposed study design for evaluating benefits of AIT over time and inform clinical understanding and decisions.

On the use of nonparametric tests for comparing immunological Reverse Cumulative distribution curves (RCDCs).

Reverse Cumulative Distribution Curves (RCDCs) have proven to be a useful tool in summarizing immune response profiles in vaccine studies since their introduction by Reed, Meade, and Steinhoff (RMS) (1995). They are able to display virtually all of the treatment data and characterize summary statistics such as means or even their confidence intervals (CIs) that might be obscure. RMS mentioned their similarity to survival curves often used to summarize time-to-event data which are usually not normally distributed. The RCDCs, while intuitively pleasing and useful, contain important properties which allow for more powerful statistical applications. In this paper, we will suggest several widely used rank-based tests to compare the curves in the context of vaccine studies. These rank-based tests allow for comparisons between treatments, for stratified analyses, weighted analyses, and other modifications that make them the alternative of parametric analyses without the normality assumptions. Clinical trial identification: NCT01712984 and NCT01230957.

Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control.

Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-ß-, IFN-λ-, and NF-κB-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells.IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.

Respiratory syncytial virus infection induces a subset of types I and III interferons in human dendritic cells.

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs. At 24h, A2 elicited higher ISG expression than Line 19. Both strains induced MDDCs to express genes for IFN-ß, IFN-α1, IFN-α8, and IFN-λ1-3, but only A2 induced IFN-α2, -α14 and -α21. We then show that IFN-α8 and IFN-α14 most potently induced MDDCs and bronchial epithelial cells (BECs) to express ISGs. Our findings demonstrate that RSV strain may impact patterns of types I and III IFN expression and the magnitude of the ISG response by DCs and BECs.

Revealing Facts and Avoiding Biases: A Review of Several Common Problems in Statistical Analyses of Epidemiological Data.

This paper reviews several common challenges encountered in statistical analyses of epidemiological data for epidemiologists. We focus on the application of linear regression, multivariate logistic regression, and log-linear modeling to epidemiological data. Specific topics include: (a) deletion of outliers, (b) heteroscedasticity in linear regression, (c) limitations of principal component analysis in dimension reduction, (d) hazard ratio vs. odds ratio in a rate comparison analysis, (e) log-linear models with multiple response data, and (f) ordinal logistic vs. multinomial logistic models. As a general rule, a thorough examination of a model's assumptions against both current data and prior research should precede its use in estimating effects.

Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

Statistical characteristics of moxifloxacin-induced QTc effect.

Moxifloxacin has been the most commonly used positive control in "thorough" QTc (TQT) studies. In a TQT study, the assay sensitivity is often considered to be established if the baseline corrected mean difference in QTc between moxifloxacin and placebo is greater than 5 ms in common practice at one or more prespecified time points and the observed moxifloxacin induced QTc effect over time follows the proper pharmacokinetics profile. To better understand the statistical characteristics of moxifloxacin-induced QTc prolongation and to provide guidance for future studies, 20 TQT studies that involved moxifloxacin have been evaluated. We study the QTc profile of the baseline adjusted mean difference in QTc between moxifloxacin and placebo over time. Zhang (2008) proposed that the moxifloxacin induced QTc effect can be evaluated between 1 and 4 h after a single dose (400 mg) administration near the time (T(max)) of peak concentration instead of all time points (typically 9-12 time points) at which QT was measured for the study drug evaluation. After evaluating 20 TQT studies, we confirm that the maximum moxifloxacin effect occurs in the time window between 1 and 4 h post dose. We also investigate the variability of the data as well as correlations between time points and between regimens. These findings and results can be used as a reference for future studies.

Multiple comparisons of repeatedly measured response: issues of validation testing in thorough QT/QTc clinical trials.

In order to validate the results of a thorough QT/QTc clinical trial, ICH E14 recommended that a concurrent positive control treatment be included in the trial. Zhang (2008) recommended that the study results are validated if the positive control establishes assay sensitivity, i.e., has an effect on the mean QT/QTc interval of 5 ms or more. Zhang (2008) and Tsong et al. (2008) discussed the intersection-union test approach and an alternative global average test approach for testing assay sensitivity during the validation process. In this article, we further discuss the multiple comparison issues of the repeatedly measured QT difference between positive control treatment and placebo in the validation test. We describe and discuss several approaches for type I error rate adjustment that are applicable to the situation.

Mercury levels in premature and low birth weight newborn infants after receipt of thimerosal-containing vaccines.

OBJECTIVE: We conducted a population-based pharmacokinetic study to assess blood levels and elimination of mercury after vaccination of premature infants born at > or =32 and <37 weeks of gestation and with birth weight > or =2000 but <3000 g. STUDY DESIGN: Blood, stool, and urine samples were obtained before vaccination and 12 hours to 30 days after vaccination from 72 premature newborn infants. Total mercury levels were measured by atomic absorption. RESULTS: The mean +/- standard deviation (SD) birth weight was 2.4 +/- 0.3 kg for the study population. Maximal mean +/- SD blood mercury level was 3.6 +/- 2.1 ng/mL, occurring at 1 day after vaccination; maximal mean +/- SD stool mercury level was 35.4 +/- 38.0 ng/g, occurring on day 5 after vaccination; and urine mercury levels were mostly nondetectable. The blood mercury half-life was calculated to be 6.3 (95% CI, 3.85 to 8.77) days, and mercury levels returned to prevaccination levels by day 30. CONCLUSIONS: The blood half-life of intramuscular ethyl mercury from thimerosal in vaccines given to premature infants is substantially shorter than that of oral methyl mercury in adults. Because of the differing pharmacokinetics, exposure guidelines based on oral methyl mercury in adults may not be accurate for children who receive thimerosal-containing vaccines.

Evaluation of smallpox vaccines using variola neutralization.

The search for a 'third'-generation smallpox vaccine has resulted in the development and characterization of several vaccine candidates. A significant barrier to acceptance is the absence of challenge models showing induction of correlates of protective immunity against variola virus. In this light, virus neutralization provides one of few experimental methods to show specific 'in vitro' activity of vaccines against variola virus. Here, we provide characterization of the ability of a modified vaccinia virus Ankara vaccine to induce variola virus-neutralizing antibodies, and we provide comparison with the neutralization elicited by standard Dryvax vaccination.

Two-stage group sequential robust tests in family-based association studies: controlling type I error.

In family-based association studies, an optimal test statistic with asymptotic normal distribution is available when the underlying genetic model is known (e.g., recessive, additive, multiplicative, or dominant). In practice, however, genetic models for many complex diseases are usually unknown. Using a single test statistic optimal for one genetic model may lose substantial power when the model is mis-specified. When a family of genetic models is scientifically plausible, the maximum of several tests, each optimal for a specific genetic model, is robust against the model mis-specification. This robust test is preferred over a single optimal test. Recently, cost-effective group sequential approaches have been introduced to genetic studies. The group sequential approach allows interim analyses and has been applied to many test statistics, but not to the maximum statistic. When the group sequential method is applied, type I error should be controlled. We propose and compare several approaches of controlling type I error rates when group sequential analysis is conducted with the maximum test for family-based candidate-gene association studies. For a two-stage group sequential robust procedure with a single interim analysis, two critical values for the maximum tests are provided based on a given alpha spending function to control the desired overall type I error.

Mercury levels in newborns and infants after receipt of thimerosal-containing vaccines.

OBJECTIVES: Thimerosal is a mercurial preservative that was widely used in multidose vaccine vials in the United States and Europe until 2001 and continues to be used in many countries throughout the world. We conducted a pharmacokinetic study to assess blood levels and elimination of ethyl mercury after vaccination of infants with thimerosal-containing vaccines. METHODS: Blood, stool, and urine samples were obtained before vaccination and 12 hours to 30 days after vaccination from 216 healthy children: 72 newborns (group 1), 72 infants aged 2 months (group 2), and 72 infants aged 6 months (group 3). Total mercury levels were measured by atomic absorption. Blood mercury pharmacokinetics were calculated by pooling the data on the group and were based on a 1-compartment first-order pharmacokinetics model. RESULTS: For groups 1, 2, and 3, respectively, (1) mean +/- SD weights were 3.4 +/- 0.4, 5.1 +/- 0.6, and 7.7 +/- 1.1 kg; (2) maximal mean +/- SD blood mercury levels were 5.0 +/- 1.3, 3.6 +/- 1.5, and 2.8 +/- 0.9 ng/mL occurring at 0.5 to 1 day after vaccination; (3) maximal mean +/- SD stool mercury levels were 19.1 +/- 11.8, 37.0 +/- 27.4, and 44.3 +/- 23.9 ng/g occurring on day 5 after vaccination for all groups; and (4) urine mercury levels were mostly nondetectable. The blood mercury half-life was calculated to be 3.7 days and returned to prevaccination levels by day 30. CONCLUSIONS: The blood half-life of intramuscular ethyl mercury from thimerosal in vaccines in infants is substantially shorter than that of oral methyl mercury in adults. Increased mercury levels were detected in stools after vaccination, suggesting that the gastrointestinal tract is involved in ethyl mercury elimination. Because of the differing pharmacokinetics of ethyl and methyl mercury, exposure guidelines based on oral methyl mercury in adults may not be accurate for risk assessments in children who receive thimerosal-containing vaccines.

Clinical and immunologic responses to multiple doses of IMVAMUNE (Modified Vaccinia Ankara) followed by Dryvax challenge.

Smallpox vaccination with replication deficient vaccinia strains such as Modified Vaccinia Ankara (MVA) may induce protective immunity with improved safety and tolerability profiles compared with currently available smallpox vaccines. Ninety subjects were randomized equally to six groups in a partially blinded, randomized, controlled clinical trial. IMVAMUNE (MVA-BN, Bavarian Nordic A/S, Kvistgård, Denmark) vaccine or placebo was administered at Study Days 0 and 28 by subcutaneous or intramuscular injection and five groups were challenged with Dryvax at study Day 112. Vaccination with two doses of IMVAMUNE was safe and well tolerated compared to Dryvax. IMVAMUNE produced comparable cellular and humoral immune responses to one dose of Dryvax and the immunity induced appears robust 90 days post-vaccination by evidence of attenuated primary cutaneous reaction responses following Dryvax. IMVAMUNE vaccination prior to Dryvax reduced virus replication at the Dryvax site, decreased the size of the primary cutaneous lesion, and decreased the time to healing but did not completely ameliorate the immune response.

Reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses.

BACKGROUND: When the decision was made to prepare for a deliberate release of smallpox, the United States had approximately 15 million doses of Wyeth Dryvax vaccine, which was known to induce significant morbidity when used undiluted; Sanofi Pasteur, Inc., later identified approximately 85 million additional doses in storage. METHODS: Eleven vaccine-dose groups, each with 30 vaccinia-naive subjects, were given diluted Dryvax vaccine or 1 of 2 lots of Sanofi Pasteur smallpox vaccine and were evaluated for vaccination success rates, morbidity, and immune responses. RESULTS: Estimated doses of 10(6.6)-10(8.2) pfu of virus/mL induced major reactions (or "takes") in 93%-100% of subjects in each dose group. No differences in vaccination take rates, lesion size, erythema, and induration or in serum neutralizing-antibody response were detected between the groups. However, systemic reactogenicity and missed activities were significantly lower for the vaccine groups given doses of 10(6.6)-10(7.2) pfu/mL than for those given doses of 10(7.6)-10(8.2) pfu/mL. CONCLUSIONS: These findings support the use of a higher dilution of Wyeth Dryvax vaccine and Sanofi Pasteur smallpox vaccine, given that the resulting morbidity should be significantly lower without loss of vaccine effectiveness. A plan for use of higher dilutions would create an enormous stockpile of vaccine.

Safety, immunogenicity and efficacy of modified vaccinia Ankara (MVA) against Dryvax challenge in vaccinia-naïve and vaccinia-immune individuals.

Modified vaccinia Ankara (MVA) was evaluated as an alternative to Dryvax in vaccinia-naïve and vaccinia-immune adult volunteers. Subjects received intramuscular MVA or placebo followed by Dryvax challenge at 3 months. Two or more doses of MVA prior to Dryvax reduced severity of lesion formation, decreased magnitude and duration of viral shedding, and augmented post-Dryvax vaccinia-specific CD8(+) T cell responses and extracellular enveloped virus protein-specific antibody responses. MVA vaccination is safe and immunogenic and improves the safety and immunogenicity of subsequent Dryvax vaccination supporting the potential for using MVA as a vaccine in the general population to improve immunity to orthopoxviruses.

Optimal bandaging of smallpox vaccination sites to decrease the potential for secondary vaccinia transmission without impairing lesion healing.

OBJECTIVE: To assess the optimal method for covering smallpox vaccination sites to prevent transmission of vaccinia. DESIGN: Randomized, nonblinded clinical trial. SETTING: Tertiary care medical center. PARTICIPANTS: Vaccinia-naive and vaccinia-experienced volunteers. INTERVENTIONS: After vaccination, study participants were randomized to receive 1 of 3 types of bandage: gauze, occlusive with gauze lining, or foam. Vaccination sites were assessed every 3 to 5 days until the lesion healed. During each visit, specimens were obtained from the vaccination site, the bandage surface before removal, and the index finger contralateral to the vaccination site and were cultured for vaccinia. Time to lesion healing was assessed. RESULTS: All 48 vaccinia-naive and 47 (87%) of 54 vaccinia-experienced participants developed a vesicle or pustule at the injection site 6-11 days after vaccination. Fourteen (14%) of 102 participants had bandage cultures positive for vaccinia. All but 1 of these vaccinia-positive cultures were of a bandage from participants randomized to the gauze bandage group, and all but 3 were of bandages from vaccinia-naive participants. No finger-specimen cultures were positive for vaccinia. One episode of neck autoinoculation occurred in a vaccinia-naive individual who had vaccinia recovered from his gauze bandage on multiple visits. The foam bandage was associated with more local adverse effects (skin irritation and induration). The time to healing did not differ among the bandage groups. CONCLUSIONS: The potential for transmission of vaccinia from a vaccination site is greater if the site is covered by gauze than if it is covered by occlusive or foam bandages. Use of an occlusive bandage with a gauze lining is the best choice for coverage of smallpox vaccination sites because of a reduced potential for vaccinia transmission and a lower reactogenicity rate. Bandage choice did not affect vaccination lesion healing.

A phase 1 study of 4 live, recombinant human cytomegalovirus Towne/Toledo chimeric vaccines.

BACKGROUND: Human cytomegalovirus (HCMV) infection acquired in utero often results in severe consequences, including mental retardation and deafness. Although not evaluated for this indication, live attenuated HCMV vaccines based on the Towne strain are well-tolerated and have demonstrated moderate efficacy in other clinical settings. METHODS: To produce live HCMV vaccine candidates that retain the excellent safety profile of the Towne strain but are more immunogenic, the genomes of the Towne strain and the unattenuated HCMV Toledo strain were recombined to yield 4 independent chimeric vaccine candidates. These vaccine candidates were evaluated in 20 HCMV-seropositive persons, in a phase 1, double-blinded, placebo-controlled trial. Participants received a single dose of vaccine or placebo, and the safety and tolerability of the vaccine candidates were evaluated. RESULTS: There was no difference in systemic symptoms between the vaccine and placebo recipients. As a group, vaccine recipients experienced more injection-site reactions than did placebo recipients; however, these were generally minor and short-lived. Vaccine virus could not be detected in blood, urine, or saliva samples obtained from any vaccine recipient. CONCLUSIONS: The Towne/Toledo chimeric vaccine candidates were well tolerated and did not cause systemic infection. Additional human trials are warranted to further evaluate the potential of these vaccine candidates as live virus vaccines.