DOI: 10.5061/dryad.0gb5mkm1s
title: The metabolic hormone adiponectin affects the correlation between nutritional status and pneumococcal vaccine response in vulnerable indigenous children

description: Background: Almost 200 million children worldwide are either
 undernourished or overweight. Only a few studies have addressed the effect
 of variation in nutritional status on vaccine response. We previously
 demonstrated an association between stunting and an increased
 post-vaccination 13-valent pneumococcal conjugate vaccine (PCV13)
 response. In this prospective study, we assessed to what extent metabolic
 hormones may be a modifier in the association between nutritional status
 and PCV13 response. Methods: Venezuelan children aged 6 weeks to 59 months
 were vaccinated with a primary series of PCV13. Nutritional status and
 serum levels of leptin, adiponectin and ghrelin were measured upon
 vaccination and their combined effect on serum post-vaccination antibody
 concentrations was assessed by generalized estimating equations
 multivariable regression analysis. Results: A total of 210 children were
 included, of whom 80 were stunted, 81 had a normal weight and 49 were
 overweight. Overweight children had lower post-vaccination antibody
 concentrations than  normal weight children (regression
 coefficient -1.15, 95% CI -2.22 – -0.072). Additionally, there was a
 significant adiponectin-nutritional status interaction. In stunted
 children, higher adiponectin serum concentrations were associated with
 lower post-PCV13 antibody concentrations (regression coefficient -0.19,
 95% CI -0.24 – -0.14) while the opposite was seen in overweight children
 (regression coefficient  0.14, 95% CI 0.049 – 0.22). Conclusion:
 Metabolic hormones, in particular adiponectin, may modify the effect of
 nutritional status on pneumococcal vaccine response. These findings
 emphasize the importance of further research to better understand the
 immunometabolic pathways underlying vaccine response and enable a future
 of optimal personalized vaccination schedules.
description: <strong>Study population and setting</strong> The Warao Amerindians live in Antonio Díaz, a municipality located in the Orinoco River Delta in Venezuela that can only be reached by boat. This study included Warao children aged 6 weeks to 59 months from the following nine indigenous communities in Antonio Díaz: Araguabisi, Araguaimujo, Arature, Bonoina, Guayaboroina, Ibaruma, Jobure de Curiapo, Merejina, and Winikina. Door-to-door visits were made to inform all parents of age-eligible children present in these communities during study visits. Children were included between May and November 2012. The original study was registered in a primary registry of the World Health Organization (ICTRP / RPCEC) with identifier number RPCEC00000158. This study included a subset of the original cohort as described in Verhagen et al. 2016 (14), i.e., all children who were stunted or overweight with a serum sample available and an equally large control group of normal weight children. Children who met multiple definitions, i.e. being overweight and stunted, were not included in the subset. Other exclusion criteria were known immunosuppression/deficiency, previous vaccination with any pneumococcal vaccine and major congenital malformations. <strong>Vaccination schedule</strong> Children aged 6 weeks to 6 months, 7 to 23 months and 24 to 59 months received a primary series of PCV13 including respectively 3, 2 and 1 dose(s) following CDC guidelines (30). PCV13 contains capsular polysaccharides of 13 serotypes of <em>Streptococcus pneumoniae</em> (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) conjugated to CRM-197, a non-toxic variant of diphtheria toxin, as carrier protein (Fig 1A). <strong>Data collection</strong> <strong>Patient characteristics </strong> Physical examination was performed in all included children, including anthropometric measurements. Pre-vaccination anthropometric measurements were transformed into weight-for-age Z-scores (WAZ), height-for-age Z-scores (HAZ) and Body Mass Index (BMI) Z-scores based on WHO standards (31). Stunting was defined as HAZ &lt;-2 standard deviation (SD). Overweight was defined as a BMI above +1SD. A normal nutritional status was defined as a BMI ≤ +1SD and a HAZ ≥ -2SD. <strong>Sampling and laboratory methods</strong> Blood samples were taken just before the first vaccination and again at 1.5 months (median 6.7 weeks (IQR 6.4–6.9 weeks)) after completion of the primary PCV13 vaccination series (Fig 1B). For a detailed description of the sampling and storage procedures we refer to Verhagen et al. 2016 (14). Determination of pneumococcal serotype-specific serum immunoglobulin G (IgG) concentrations was performed at the National Institute for Public Health and the Environment in Bilthoven, The Netherlands, using a fluorescent bead-based multiplex immunoassay (32). Metabolic hormones were determined in pre-vaccination serum samples. Serum ghrelin and leptin levels were analysed (in 100 µL) by radioimmunoassay (GHRT-89HK and HL-81K respectively, EMD Millipore Corp.; Missouri USA) as specified by the manufacturer’s instructions. Serum adiponectin concentration (High Molecular Weight (HMW) Adiponectin) was analysed (in 10 µL) by chemiluminescence enzyme immunoassay on a Lumipulse analyser G600II (234778, FuijRebio, Gent; Belgium) as specified by the manufacturer’s instructions. <strong>Ethical considerations</strong> Approval by the ethical committee of the Instituto de Biomedicina, Caracas, Venezuela, was granted. In addition, written permission to carry out the study was obtained from the Delta Amacuro Indigenous Health Office and from community leaders of each included community. Children were included upon written informed consent of parents or primary caregivers. <strong>Statistical analyses</strong> Categorical variables were analysed using Chi-square or Fisher’s exact test, as appropriate. For continuous variables, the unpaired Student’s t test, nonparametric Mann-Whitney U test or Kruskal Wallis test was used depending on whether the variables were normally distributed (Kolmogorov-Smirnov’s test, p &gt;0.05). We used the mean of serotype-specific log-transformed pneumococcal antibody levels as a read-out for pneumococcal vaccine response. Linear regression was performed using the log-transformed antibody concentrations. In our multivariable models we included the following ten covariates of interest and potential confounders:<em> </em>age (continuous), time from primary series completion until post-vaccination blood sampling (continuous), leptin (continuous), adiponectin (continuous) and ghrelin (continuous, per 20 pg/mL) concentration, community (categorical), mean pre-vaccination antibody response (continuous), BMI (categorical), and HAZ (categorical). Generalized estimating equations (GEE) were used to fit a multivariable linear regression model aimed at identifying possible associations between nutritional status and metabolic hormones and their interactions (independent variables) and post-vaccination log-transformed antibody concentrations (dependant variable), while adjusting for the potential confounders mentioned above. GEEs account for correlation and lack of independence of responses for individuals in clusters within communities using an independence working covariance structure and robust variance estimators. Models were run separately for both stunted vs. non-stunted (i.e. including normal weight and overweight children) and overweight vs. non-overweight children (i.e. including normal weight and stunted children). For all statistical analyses SPSS software version 25 was used. Statistical significance was set to p-value &lt;0.05.