Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.     

Phytochemical Compounds from the Crop Byproducts of Tunisian Globe Artichoke Cultivars

The phytochemical composition in two Tunisian globe artichoke cultivars (bracts, leaves, and floral stems) was evaluated in the plant byproducts. The results indicated that the bracts contain the highest levels of total phenols, o‐diphenols, and flavonoids, whereas tannins seem to be more abundant in the leaves. Bracts from the ‘Violet d'Hyères’ cultivar possessed more total phenols (160.8 mg/g DW), flavonoids (64.9 mg/g DW), and anthocyanins (15.3 μg/g DW) than the ‘Blanc d'Oran’ bracts (134.5 mg/g DW, 51.2 mg/g DW, and 8.3 μg/g DW, resp.). Sixty‐four volatile compounds were identified in the headspace of globe artichoke material, particularly in the bracts. The volatile profile showed that sesquiterpene hydrocarbons and non‐terpene derivatives were the main volatiles emitted by the bracts in both cultivars. These results suggest that globe artichoke byproducts might represent a potential source of natural compounds, which could be used as nutraceuticals or as ingredients in the design of functional foods.     

Hand,Foot, and Mouth Disease in China: Modeling Epidemic Dynamics of Enterovirus Serotypes and Implications for Vaccination

Background

Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by serotypes of the Enterovirus A species in the genus Enterovirus of the Picornaviridae family. The disease has had a substantial burden throughout East and Southeast Asia over the past 15 y. China reported 9 million cases of HFMD between 2008 and 2013, with the two serotypes Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) being responsible for the majority of these cases. Three recent phase 3 clinical trials showed that inactivated monovalent EV-A71 vaccines manufactured in China were highly efficacious against HFMD associated with EV-A71, but offered no protection against HFMD caused by CV-A16. To better inform vaccination policy, we used mathematical models to evaluate the effect of prospective vaccination against EV-A71-associated HFMD and the potential risk of serotype replacement by CV-A16. We also extended the model to address the co-circulation, and implications for vaccination, of additional non-EV-A71, non-CV-A16 serotypes of enterovirus.

Methods and Findings

Weekly reports of HFMD incidence from 31 provinces in Mainland China from 1 January 2009 to 31 December 2013 were used to fit multi-serotype time series susceptible–infected–recovered (TSIR) epidemic models. We obtained good model fit for the two-serotype TSIR with cross-protection, capturing the seasonality and geographic heterogeneity of province-level transmission, with strong correlation between the observed and simulated epidemic series. The national estimate of the basic reproduction number, R ₀, weighted by provincial population size, was 26.63 for EV-A71 (interquartile range [IQR]: 23.14, 30.40) and 27.13 for CV-A16 (IQR: 23.15, 31.34), with considerable variation between provinces (however, predictions about the overall impact of vaccination were robust to this variation). EV-A71 incidence was projected to decrease monotonically with higher coverage rates of EV-A71 vaccination. Across provinces, CV-A16 incidence in the post-EV-A71-vaccination period remained either comparable to or only slightly increased from levels prior to vaccination. The duration and strength of cross-protection following infection with EV-A71 or CV-A16 was estimated to be 9.95 wk (95% confidence interval [CI]: 3.31, 23.40) in 68% of the population (95% CI: 37%, 96%). Our predictions are limited by the necessarily short and under-sampled time series and the possible circulation of unidentified serotypes, but, nonetheless, sensitivity analyses indicate that our results are robust in predicting that the vaccine should drastically reduce incidence of EV-A71 without a substantial competitive release of CV-A16.

Conclusions

The ability of our models to capture the observed epidemic cycles suggests that herd immunity is driving the epidemic dynamics caused by the multiple serotypes of enterovirus. Our results predict that the EV-A71 and CV-A16 serotypes provide a temporary immunizing effect against each other. Achieving high coverage rates of EV-A71 vaccination would be necessary to eliminate the ongoing transmission of EV-A71, but serotype replacement by CV-A16 following EV-A71 vaccination is likely to be transient and minor compared to the corresponding reduction in the burden of EV-A71-associated HFMD. Therefore, a mass EV-A71 vaccination program of infants and young children should provide significant benefits in terms of a reduction in overall HFMD burden.     

Assessment of a Standardized Pre-Operative Telephone Checklist Designed to Avoid Late Cancellation of Ambulatory Surgery: The AMBUPROG Multicenter Randomized Controlled Trial

Objectives

To assess the impact of a standardized pre-operative telephone checklist on the rate of late cancellations of ambulatory surgery (AMBUPROG trial).

Design

Multicenter, two-arm, parallel-group, open-label randomized controlled trial.

Setting

11 university hospital ambulatory surgery units in Paris, France.

Participants

Patients scheduled for ambulatory surgery and able to be reached by telephone.

Intervention

A 7-item checklist designed to prevent late cancellation, available in five languages and two versions (for children and adults), was administered between 7 and 3 days before the planned date of surgery, by an automated phone system or a research assistant. The control group received standard management alone.

Main Outcome Measures

Rate of cancellation on the day of surgery or the day before.

Results

The study population comprised 3900 patients enrolled between November 2012 and September 2013: 1950 patients were randomized to the checklist arm and 1950 patients to the control arm. The checklist was administered to 68.8% of patients in the intervention arm, 1002 by the automated phone system and 340 by a research assistant. The rate of late cancellation did not differ significantly between the checklist and control arms (109 (5.6%) vs. 113 (5.8%), adjusted odds ratio [95% confidence interval] = 0.91 [0.65–1.29], (p = 0.57)). Checklist administration revealed that 355 patients (28.0%) had not undergone tests ordered by the surgeon or anesthetist, and that 254 patients (20.0%) still had questions concerning the fasting state.

Conclusions

A standardized pre-operative telephone checklist did not avoid late cancellations of ambulatory surgery but enabled us to identify several frequent causes.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01732159","term_id":"NCT01732159"}}NCT01732159     

Social Patterning in Adiposity in Adolescence: Prospective Observations from the Chinese Birth Cohort ‘‘Children of 1997’’

Introduction

Low early life socio-economic position is more strongly associated with adiposity among women than men. We examined whether the sex difference of social patterning in general and central adiposity exists before adulthood.

Methods

In Hong Kong’s “Children of 1997” birth cohort, we used multivariable regression to examine the association of parental education, a marker of early life socio-economic position, with body mass index (BMI) (n = 7252, 88% follow-up) and waist-height ratio (n = 5636, 68% follow-up), at 14 years.

Results

Parental education of Grade 9 or below, compared to Grade 12 or above, was associated with higher waist-height ratio z-score particularly in girls (0.30, 95% confidence interval (CI) 0.19, 0.41) compared to boys (0.12, 95% CI 0.02, 0.22) (p for sex interaction = 0.02). Lower parental education was associated with greater BMI z-score in adolescents of locally born mothers, but not adolescents of migrant mothers, with no difference by sex.

Conclusions

Different social patterning in different markers of adiposity may imply different sociological and biological mediating pathways. A stronger association between low early life socio-economic position and waist-height ratio in adolescent girls may indicate sex-specific influences of SEP related early life exposures on central adiposity.     

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOB_F, MOB_Q and MOB_P families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.