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Background/Objectives: Typhoid and paratyphoid fever together are responsible for millions of cases and thousands of deaths per year, most of which occur in children in South and Southeast Asia. While typhoid conjugate vaccines (TCVs) are licensed, no vaccines are currently available against S. Paratyphi A. Here we describe the design of a S. Paratyphi A conjugate. Methods: The serovar-specific O-antigen (O:2) was linked to the CRM197 carrier protein (O:2–CRM197) and a panel of conjugates differing for structural characteristics were compared in mice and rabbits. Results: We identified the O-antigen molecular size, polysaccharide to protein ratio, conjugate cross-linking, and O:2 O-acetylation level as critical quality attributes and identified optimal design for a more immunogenic vaccine. Conclusions: This work guides the development of the O:2–CRM197 conjugate to be combined with TCV in a bivalent formulation against enteric fever. [ABSTRACT FROM AUTHOR]

Effective vaccines against Salmonella Typhi, targeting the Vi capsular polysaccharide, have been developed and are being introduced into routine immunization in endemic countries. Vi conjugated vaccines are also being tested in new multi-component vaccine formulations. Simple, high-throughput and cost-effective assays to quantify Vi-specific IgG in clinical sera are needed. In this study we present the development and qualification of a new anti-Vi ELISA with continuous readout, which expresses results as ELISA Unit/mL (EU/mL). We have qualified the assay in terms of precision, linearity and specificity, demonstrating performance in line with a commercially available anti-Vi ELISA. We have also calibrated the assay against the 16/138 anti-Vi international standard and established conversion factor between EU/mL and international units/mL, to allow comparability of results across studies. In summary, this newassaymet all the suitability criteria and is being used to evaluate anti-Vi responses in clinical studies. [ABSTRACT FROM AUTHOR]

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies. [ABSTRACT FROM AUTHOR]

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials. [ABSTRACT FROM AUTHOR]