Gates Open Res Gates Open Research 2572-4754 F1000 Research Limited London, UK 10.12688/gatesopenres.13034.1 Research Note Articles Cross-sectional study of IgG antibody levels to invasive nontyphoidal Salmonella LPS O-antigen with age in Uganda

[version 1; peer review: 2 approved, 2 approved with reservations]

 Stockdale Lisa Formal Analysis Investigation Writing – Original Draft Preparation https://orcid.org/0000-0002-2576-8783 a 1 Nalwoga Angela Methodology Writing – Review & Editing https://orcid.org/0000-0001-8259-4735 2 3 Nash Stephen Formal Analysis Visualization Writing – Review & Editing https://orcid.org/0000-0003-2086-1740 3 Elias Sean Methodology Resources Writing – Review & Editing https://orcid.org/0000-0002-6053-8238 4 Asiki Gershim Resources Writing – Review & Editing 2 Kusemererwa Sylvia Resources Writing – Review & Editing 2 Gilchrist James J. Data Curation Writing – Review & Editing 1 Newton Robert Resources Supervision Writing – Review & Editing 2 5 MacLennan Calman A. Conceptualization Writing – Review & Editing 4 Department of Paediatrics, University of Oxford, Oxford, OX3 7LE, UK MRC/UVRI and LSHTM Uganda Research Institute, Entebbe, Uganda Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7DQ, UK Clinical Epidemiology, University of York, York, UK lisa.stockdale@paediatrics.ox.ac.uk

*Equal contributors

No competing interests were disclosed.

 26 6 2019 2019 3 1501 14 6 2019 Copyright: © 2019 Stockdale L et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

 non-typhoidal salmonella NTS antibody Uganda Department for International Development, UK Government Medical Research Council MR/K012126/1 MR/J003999/1 Gates Foundation OPP1148489 This work was supported by the UK Medical Research Council (MRC): grant number MR/ J003999/1 to LS, grant number MR/K012126/1 to SN, and the Gates Foundation: grant number OPP1148489 to CM. The Ugandan General Population Cohort study is jointly funded by the UKMRC and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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.

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 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 Science Framework: Invasive Non-Typhoidal Salmonella serology in Uganda. https://doi.org/10.17605/OSF.IO/68BYT 8 .

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).

 Ao TT Feasey NA Gordon MA : Global burden of invasive nontyphoidal Salmonella disease, 2010. Emerg Infect Dis. 2015;21(6):941–949. 25860298 10.3201/eid2106.140999 4451910 Oneko M Kariuki S Muturi-Kioi V : Emergence of Community-Acquired, Multidrug-Resistant Invasive Nontyphoidal Salmonella Disease in Rural Western Kenya, 2009-2013. Clin Infect Dis. 2015;61 Suppl 4:S310–6. 26449946 10.1093/cid/civ674 Gilchrist JJ MacLennan CA : Invasive Nontyphoidal Salmonella Disease in Africa. EcoSal Plus. 2019;8(2). 30657108 10.1128/ecosalplus.ESP-0007-2018 MacLennan CA Gondwe EN Msefula CL : The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. J Clin Invest. 2008;118(4):1553–1562. 18357343 10.1172/JCI33998 2268878 Nyirenda TS Gilchrist JJ Feasey NA : Sequential acquisition of T cells and antibodies to nontyphoidal Salmonella in Malawian children. J Infect Dis. 2014;210(1):56–64. 24443544 10.1093/infdis/jiu045 4054899 Maclennan CA Gilchrist J Gordon MA : Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults. Science. 2010;328(5977):508–512. 20413503 10.1126/science.1180346 3772309 Asiki G Murphy G Nakiyingi-Miiro J : The general population cohort in rural south-western Uganda: a platform for communicable and non-communicable disease studies. Int J Epidemiol. 2013;42(1):129–141. 23364209 10.1093/ije/dys234 3600628 Stockdale L : Invasive Non-Typhoidal Salmonella serology in Uganda.2019. http://www.doi.org/10.17605/OSF.IO/68BYT 10.21956/gatesopenres.14147.r27466 Reviewer response for version 1 Simuyandi Michelo 1 Referee https://orcid.org/0000-0002-7348-2835 Enteric Disease and Vaccine Research Unit, Centre for Infectious Disease Research in Zambia (CIDRZ), Lusaka, Zambia

Competing interests: No competing interests were disclosed.

 30 7 2019 Copyright: © 2019 Simuyandi M 2019 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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.

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?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

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.

 10.21956/gatesopenres.14147.r27468 Reviewer response for version 1 Baliban Scott M. 1 Referee Center for Vaccine Development and Global Health (CVD), University of Maryland School of Medicine, Baltimore, MD, USA

Competing interests: No competing interests were disclosed.

 24 7 2019 Copyright: © 2019 Baliban SM 2019 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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 et al., 2017 1 ). At the moment, it is not clear whether 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 et al., 2016 2 ). If these titers are available, it would add a 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

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

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.

References : Immunogenicity of a Bivalent Adjuvanted Glycoconjugate Vaccine against Salmonella Typhimurium and Salmonella Enteritidis. Front Immunol .2017;8: 10.3389/fimmu.2017.00168 168 28289411 10.3389/fimmu.2017.00168 : Bactericidal Immunity to Salmonella in Africans and Mechanisms Causing Its Failure in HIV Infection. PLoS Negl Trop Dis .2016;10(4) : 10.1371/journal.pntd.0004604 e0004604 27057743 10.1371/journal.pntd.0004604 10.21956/gatesopenres.14147.r27467 Reviewer response for version 1 de Alwis Ruklanthi 1 2 Referee https://orcid.org/0000-0002-2319-3701 Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore Viral Research and Experimental Medicine Centre, SingHealth-Duke NUS, Singapore, Singapore

Competing interests: No competing interests were disclosed.

 17 7 2019 Copyright: © 2019 de Alwis R 2019 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The study investigates the sero-prevalence of iNTS, serovar Typhimurium and Enteritidis in Uganda. The study further describes the sero-epidemiology by age and sex of these pathogens in Uganda. This is a very useful study since very little is known about the basic epidemiology of iNTS in the Uganda population.

Since the authors’ main explanation for the observed differences in sero-prevalence between Malawi and Uganda are due to differences in the experimental assays used to quantify O-antigen IgG, it would be good to give more details of this in-house ELISA assay. 

Since some of the authors from the Malawi study are also authors of the present study, is it not possible to run some of the Uganda sera (probably the sera in the less than 18 months age group) on the older flow-cytometry technique described in the MacLennan et al. (2008 1 ) paper?

I agree with the authors that very little is known about the most commonly circulating and disease-causing serovars of NTS in Uganda. However, it might be good to mention one or two current literatures, such as Afema et al. (2016 2 ), where they show that neither  S. Typhimurium nor  S. Enteritidis are commonly isolated NTS serovars in Uganda (while in Malawi, Typhimurium and Enteritidis together make up over 90% of NTS cases (Feasey et al., 2015 3 )).

It wouldn’t hurt to show a graph of the O-antigen IgG data in HIV-infected and age-matched healthy individuals.

  

Minor comments:

Please add x-axis titles to Figure 1C and 1D. 

Font labelling on panels 1A and 1B is small. Increase the font size on Figure panels 1A and 1B to match the font size of panels 1C and 1D.

State the statistical test used to compare O-antigen IgG responses in females and males in Figure 1C and 1D.

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?

No source data required

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Infectious diseases, antibody responses, epidemiology, vaccines

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.

References : The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. J Clin Invest .2008;118(4) : 10.1172/JCI33998 1553-62 18357343 10.1172/JCI33998 : Potential Sources and Transmission of Salmonella and Antimicrobial Resistance in Kampala, Uganda. PLOS ONE .2016;11(3) : 10.1371/journal.pone.0152130 10.1371/journal.pone.0152130 : Three Epidemics of Invasive Multidrug-ResistantSalmonella Bloodstream Infection in Blantyre, Malawi, 1998–2014. Clinical Infectious Diseases .2015;61(suppl 4) : 10.1093/cid/civ691 S363-S371 10.1093/cid/civ691 10.21956/gatesopenres.14147.r27470 Reviewer response for version 1 Kong Qingke 1 Referee https://orcid.org/0000-0002-9185-214X University of Florida, Gainesville, FL, USA

Competing interests: No competing interests were disclosed.

 9 7 2019 Copyright: © 2019 Kong Q 2019 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

This is an ELISA assay to measure the antibodies against O-antigen from S. Typhimurium and Enteritidis from clinical sera collected from patents in Uganda. I do not have scientific questions about this assay, but hope in the future, that the authors could establish a standardized method to figure out the reason for the controversy obtained from two locations.  

Some minor grammar errors:

Please add a comma between “disease” and “suggesting” in: “…corresponds with a fall in cases of iNTS disease suggesting that vaccines able to…”.

A comma or conjunction words were missing in: “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”.

“Typhimurium” does not need to be italic.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

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

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Salmonella infection and vaccine development, polysaccharide vaccine, bacteria-derived nanoparticle vaccine.

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.

Elias Sean

Competing interests: No competing interests were disclosed.

 9 7 2019

Thank you for your comments. I can confirm that we will be publishing the optimised standardised ELISA protocol in the near future and will fully address the issues that you have highlighted.