Cross-sectional study of IgG antibody levels to invasive nontyphoidal Salmonella LPS O-antigen with age in Uganda

Introduction

Invasive non-typhoidal Salmonella (iNTS) disease is principally caused by serovars S. Typhimurium and S. Enteritidis and is thought to be responsible for up to 680,000 deaths annually, with Africa accounting for more than half of cases¹. Much of this burden is in children under 5 years and HIV-infected adults. In view of this major global burden of disease, and rapid emergence of multidrug resistant iNTS strains², development of a vaccine is increasingly vital³.

Studies in Malawian children indicate that anti-S. Typhimurium antibodies, notably IgG to O-antigen of LPS and flagellin, and serum bactericidal activity rises rapidly with age in the first few years of life corresponding with a fall in cases of iNTS disease^4,5. One study found a positive correlation between serum bactericidal assay (SBA) killing and acquisition of anti-LPS IgG⁵. However, there is no standardised assay for measurement of iNTS-specific IgG, and the clinical significance of the iNTS SBA is unknown. Given that incidence of iNTS disease drops in children over 2 years, it has been suggested that a rise in specific antibodies and bactericidal activity correlates with protection. This hypothesis is complicated by the observation that among HIV-infected Malawian adults, high LPS-specific IgG was associated with a lack of in vitro bacterial killing⁶.

Methods

In a cross-sectional study, we investigated NTS-specific antibody responses in the rural Ugandan General Population Cohort (GPC)⁷. Levels of IgG against serovars S. Typhimurium and S. Enteritidis LPS O-antigens were measured using a standardised in-house ELISA in stored sera from a cross-section of 1,090 Ugandans of all ages, 10 of whom were HIV-infected. Sera from adults (≥ 16 years) were collected from January 2014 to November 2015, and children (<16 years) from January 2016 to November 2017. Antibody units (AU) were calculated using Gen5 software (version 2.0) using a five-parameter logistic (5PL) curve generated with a standard serum from an iNTS-exposed individual. Sera were defined as seronegative if below the lower limit of detection (4 AU for S. Typhimurium and 5 AU for S. Enteritidis) at 1:100 serum dilution.

Written informed consent for the use of clinical records and biological samples for research purposes was obtained from all GPC participants following Uganda National Council of Science and Technology guidelines. Ethical approval for the use of samples for this study was obtained from The UVRI Research and Ethics Committee and from the Uganda Council for Science and Technology (Ref: GC/127/19/10/710).

Results and discussion

In this assay, overall O-antigen seropositivity was 82% for S. Typhimurium, and was 70% for S. Enteritidis. Levels of antibody were undetectable in at least 50% of children until 18 months for both serovars and a similar pattern of increasing IgG level was observed with increasing age (Figure 1A, B). There were no observable differences in antibody levels by sex (Figure 1C, D). HIV-infected individuals did not have notably high IgG antibody responses, although the study was not powered to demonstrate this.

Figure 1.

Plots showing antibody units (AU) for S. Typhimurium (A and C) and S. Enteritidis (B and D) by age. Orange dots indicate HIV infected individuals. (A, B). Females are indicated in red and males in blue (C, D). The box shows the interquartile range (IQR) with middle line representing the median. The whiskers represent the adjacent values, defined as 1.5 × IQR from the edge of the box, with values outside this range shown individually. LLOQ, lower limit of quantification.

Although performed using a flow cytometric assay, previously published data from Malawi suggest that NTS-specific IgG is present in the majority of children throughout infancy⁴, contrasting with our results from Uganda. This could be due to variation in exposure to iNTS in Uganda compared to Malawi, or differences in assays. However, burden of, and exposure to, iNTS disease in Uganda is not well understood. A standardised assay is key to understanding variation in exposure across geographic locations to support vaccine development.

Data availability

Open Science Framework: Invasive Non-Typhoidal Salmonella serology in Uganda. https://doi.org/10.17605/OSF.IO/68BYT⁸.

This project contains the age, sex, antibody levels, HIV status and Salmonella status of each participant.

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).