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

Invasive nontyphoidal Salmonella (iNTS) disease is a major cause of deaths among children and HIV-infected individuals in sub-Saharan Africa. Acquisition of IgG to iNTS lipopolysaccharide (LPS) O-antigen in Malawi in early childhood corresponds with a fall in cases of iNTS disease suggesting that vaccines able to induce such antibodies could confer protection. To better understand the acquisition of IgG to iNTS in other African settings, we performed a cross-sectional seroepidemiological study using sera from 1090 Ugandan individuals aged from infancy to old age. Sera were analysed for IgG to LPS O-antigen of S. Typhimurium and S. Enteritidis using an in-house ELISA. Below 18 months of age, most children lacked IgG to both serovars. Thereafter, specific IgG levels increased with age, peaking in adulthood, and did not wane noticeably in old age. There was no clear difference in antibody levels between the sexes and the few HIV-infected individuals in the study did not have obviously different levels from uninfected subjects. While IgG to iNTS is acquired at a younger age in Malawian compared with Ugandan children, it is not clear whether this is due to differences in the populations themselves, their exposure to iNTS, or variations between assays used. In conclusion, there is a need to develop a harmonised method and standards for measuring antibodies to iNTS across studies and to investigate acquisition of such antibodies with age across different sites in sub-Saharan Africa.


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 cases 1 . 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 strains 2 , development of a vaccine is increasingly vital 3 .
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 IgG 5 . 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 killing 6 .

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. 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 infancy 4 , 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

Centre for Infectious Disease Research in Zambia (CIDRZ), Lusaka, Zambia
The article describes an important aspect of research which needs more data. Knowing when children are exposed to salmonella infection can inform schedules for vaccination and other control interventions.
The aim of the study should be clear, given the limitation in the standardized method for immunological assessment (I suspect it was to determine the kinetics of antibody acquisition by age). The differences in assays could be discussed in the limitations and maybe how the difference can affect the date explained as well.
The assay is not described (just called in house) -reviewers could have advised how best to analyse or interpret the data. Did they try to do an EQA with another lab that used a different method to maybe determine concordance?
While the infants in this study have antibodies much later than Malawi, it is not clear if the mean concentrations increase with time or if it is just the number(%) (i.e. positivity rates) of participants with antibodies that increase over time. The authors could have compared the reported concentrations between the countries.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes 1.

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed. Competing Interests: Reviewer Expertise: Enteric disease, vaccinology, immunology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This study aims to assess the presence of serum IgG to S. Enteritidis and Typhimurium by age and sex in a broad Ugandan cohort. It is of value because the sero-epidemiology of NTS in Uganda, as opposed to other African countries, is not well understood.
More detail is needed for the in-house ELISA. Presumably, it was conducted with purified OPS, as with other studies from this group (e.g. Fiorino , 2017 ). At the moment, it is not clear whether et al. the serum IgG responses are directed against the OPS (and therefore serogroup B/D-specific) as opposed to the core oligosaccharide or lipid A (all other NTS). What is an Antibody Unit?
The authors mention that the lack of seroconversion to OAg-specific IgG in Ugandan children could be due to variation in exposure to NTS. To further support this hypothesis, it would be interesting to assess the kinetics of IgM since both anti-Typhimurium IgG and IgM can demonstrate bactericidal activity (Goh , 2016 ). If these titers are available, it would add a et al. thought-provoking element to the data set.
Please state the statistical test used to compare groups.
Serovars do not need to be italicized. Panels A and B are blurry, and the font size for the axes is small. Please add a label to the x-axis of panels C and D. Is the work clearly and accurately presented and does it cite the current literature? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Infectious disease, bacterial vaccine development, humoral immunity I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.