TECHNOLOGY AREA(S): Bio MedicalOBJECTIVE: Develop a qualified, multiplexed, functional assay that can be used to evaluate bactericidal activity of antibodies against an array of Shigella enabling high-throughput sample analysis.DESCRIPTION: Bacterial pathogens that cause diarrhea are a significant threat to the warfighter on a global scale and consistently rank at the top of the list of infectious agents for which the Army requires countermeasures. Infection with these pathogens leads to a reduction in warfighter readiness, morale, lost duty days. Shigella is a major cause of diarrhea in children and adults in low- to middle-income countries (LMICs), and among travelers and US Service Members (1). Shigella infection can lead to persistent diarrhea (≥14 days) in travelers to endemic areas, and can also have long-term health impacts including irritable bowel syndrome and reactive arthritis (2). The high incidence of infection, the rise of antibiotic resistance, the long duration of illness and the potential long-term side effects make prevention of Shigella infection a high priority for US Service Members deployed to endemic areas.One of the major technical issues facing the field when developing prophylactic or therapeutic products is the board species diversity (3) and the serotype specific immune responses that are generated after infection (4). The focus of the Shigella research has been on the development of countermeasures that are capable of protecting against multiple serotypes of Shigella. These countermeasures most often target four Shigella species, S. flexneri 2a, S. flexneri 3a, S. flexneri 6 and S. sonnei. A countermeasure that was capable of protecting against these serotypes would significantly reduce global disease burden. The need for effective prophylactic and therapeutic products to combat Shigella also requires immunological methods to evaluate these products and their efficacy. Many of the current Shigella-specific immunological assays are only quantititave, and do not offer any qualitative information about the immune response being investigated. Functional immunological assays to assess immune responses to Shigella do exist (5), but these are typically specific for only a single serotype. Evaluating responses to multiple serotypes in a single-plex assay is time consuming and also requires greater quantities of serum and mucosal samples, many of which are limited.An assay to evaluate the shigellacidal activity of antibodies is imperative for the vaccine development field, but the new push for non-vaccine countermeasures to combat Shigella will also require robust functional assays. Both vaccine and non-vaccine countermeasures will need reliable, validated assays to show product efficacy, and this will include qualitative analysis of immune responses and immunoprophylactic products. Any successful countermeasure product will need to protect against or treat infection with multiple Shigella serotypes to be highly efficacious, so the development of multiplexed immunoassays will save time, supplies, and biological sample volumes. The development and validation of a multiplexed, Shigella¬-specific, functional assay will support the rapid development and evaluation of efficacious prophylactic and therapeutic countermeasures to prevent and treat Shigella infections. The ultimate problem to be solved, and the central focus of this topic, is the development of an assay platform to measure functional antibody activity and immune responses to Shigella and other enteric bacterial pathogens. PHASE I: Phase I will focus on assay conceptualization including assay parameters, internal controls, and data analysis package. A major component of phase I will be concept design of the multiplex assay format to include discrete readouts for each bacterial serotype to be analyzed (e.g. fluorescence, antibiotic resistance cassettes). The concept design will also require that assay qualification parameters are defined including: a) bio-specimen types (e.g. blood, sera, fecal), bio-specimen volume required (e.g. finger-stick, 500 ul sera derived from venipuncture; b) generation of positive and negative controls (e.g. monoclonal antibodies, pooled sera). Specifically, the awardee will have performed the assay in a research laboratory setting and demonstrated that it can be repeated by additional users. In order to demonstrate the feasibility of multiplexing, a minimum of two Shigella serotypes will be evaluated using the proof-of-concept prototype assay and a pilot panel of control samples and monoclonal antibodies that target Shigella serotypes in the assay.PHASE II: Phase II will focus on finalizing and refining the optimal multiplex assay approach from Phase I. The workflow from Phase 1 should be refined to expand on the proof-of-concept into a product that enables high-throughput screening of serum or other clinical samples against multiple Shigella targets. The assay will be performed in a research laboratory setting to demonstrate the feasibility of multiplexing using a minimum of four Shigella serotypes. In addition to execution of the assay, a qualification of the inter- and intra-assay reproducibility in-house should be performed. This qualification will include metrics of assay precision, repeatability and reproducibility, with estimates of uncertainly around these metrics. The assay should be specific for at least four Shigella targets, but the platform should also be flexible and allow expansion to other enteric bacterial pathogens such as ETEC, Cholera, Campylobacter or Salmonella. A detailed plan for assay qualification should be developed across multiple laboratory sites. This phase should also demonstrate evidence of commercial viability of the product.PHASE III: The expected Phase II end-state is a qualified, easy to use, multiplexed, functional assay kit which can be used on a relatively low volume of biological sample of varying types and evaluates bactericidal activity to at least five Shigella serotypes simultaneously. This assay platform should also be under development for expansion to measure responses to other bacterial enteric pathogens beyond Shigella. This assay kit represents a method to evaluate functional antibody responses targeting Shigella. The development of effective anti-Shigella countermeasures relies heavily on the identification of products that are effective at functionally inhibiting bacterial infection. The assay kit described here is unique to anything currently in development, as it is multiplexed to include many cynically relevant strains of Shigella and it measures functional activity of antibodies. This end-product has the potential to be used by research laboratories to examine the potency of enteric countermeasures. These countermeasures may include hyper-immune bovine colostrum products, monoclonal antibodies, and passive vaccine strategies; all of which are aimed at preventing or treating disease caused by Shigella. A validated functional assay would help to harmonize immunological assessment of Shigella-specific countermeasures globally, which will allow for accurate comparisons between products and speed the production of efficacious prophylactics. This product would also likely be used in the immunological analysis of controlled human infection models (CHIMs) for Shigella to understand the development of serotype specific immunity, and facilitate development and evaluation of pan-Shigella countermeasures.A potential method of transition for this product will be through the Army futures command following the decision gate process. This product may also be attractive to private industry, as this multiplexed assay kit is ideally suited to evaluate immunoprophylactic products as well as commercial vaccines. Assays that evaluate functional antibody activity are essential for vaccine licensure in many current vaccines including seasonal influenza and meningococcal polysaccharide vaccines. Civilian commercialization of this product is likely to include GLP production and GMP manufacture and distribution. KEYWORDS: Shigella, Bacterial Diarrhea, Diagnostic Assay, Immunoassay, Multiplex, Validation, AntibodyReferences:1. Porter CK, Olson S, Hall A, & Riddle MS (2017) Travelers' Diarrhea: An Update on the Incidence, Etiology, and Risk in Military Deployments and Similar Travel Populations. Mil Med 182(S2):4-10.
2. Connor BA & Riddle MS (2013) Post-infectious sequelae of travelers' diarrhea. J Travel Med 20(5):303-312.
3. Anderson M, Sansonetti PJ, & Marteyn BS (2016) Shigella Diversity and Changing Landscape: Insights for the Twenty-First Century. Front Cell Infect Microbiol 6:45.
4. Formal SB, et al. (1991) Effect of prior infection with virulent Shigella flexneri 2a on the resistance of monkeys to subsequent infection with Shigella sonnei. J Infect Dis 164(3):533-537.
5. Nahm MH, et al. (2018) Development, interlaboratory evaluations, and application of a simple, high-throughput Shigella serum bactericidal assay. mSphere 3(3).
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