Development of a cholera rapid diagnostic test that targets both Vibrio cholerae and vibriophage

Cholera is an acute watery diarrhoeal disease that can lead to severe dehydration and death in less than 16 hours. It is an important public health problem in Asia, Africa and Latin America. Globally 1.3-4 million cases and 21,000-143,000 deaths occur annually due to cholera1. Accurate diagnosis of cholera early in an epidemic is critical to reduce morbidity and mortality. Rapid diagnostic tests (RDTs) have the potential to provide immediate objective findings early in outbreaks in settings that lack conventional microbiology laboratories. Several lateral flow immunoassay-based RDTs are commercially available and target V. cholerae O1 and/or O139 specific antigens. However, the RDT performance metrics are unpredictable for unknown reasons which has resulted in limited adoption. We are specifically interested in the widely used RDT sold under the name of Crystal VC (Span Diagnostics, India) that was developed by the Institute Pasteur.

While limitations with current RDTs may include production and operator problems, there are multiple biologic reasons why the RDT may fail when deployed in field settings. One explanation is that the concentration of Vibrio cholerae may fall below the limit of detection when the bacteria are preyed upon by viruses called lytic vibriophages. This predation is dynamic to the ratio of predator / prey. We have shown that vibriophage (ICP1) negatively impacts the RDT (see preliminary data section).

Our primary research question is if the incorporation of antibodies that detect both V. cholerae and lytic vibriophage into a Rapid Diagnostic Test (RDT) will address limitations in the current RDTs used when cholera patients harbor lytic vibriophage. This novel RDT may represent a model for diagnostic tool development for enteric and nonenteric infectious diseases. To develop this RDT and answer this question we propose the following specific aims:

Aim 1

Identify lytic vibriophage structural and non-structural proteins that are antigenically unique in diarrheal stool. We hypothesize that lytic vibriophage express structural and non-structural proteins that are antigenically unique. These proteins can be used to generate a monoclonal antibodies (mAb) that can then be incorporated into an improved RDT. We will take two approaches to identify unique immunogenic vibriophage proteins with specific focus on ICP1 because it has been shown to be most common in multiple locations.
1.1 Reverse vaccinology by genomic analysis, cloning, expression, and testing immunogenicity
1.2 Proteomic analysis of immunogenic proteins separated by 2-D gel electrophoresis and MALDI-TOF

Aim 2

Produce monoclonal antibodies (mAbs) against unique lytic vibriophage protein. The vibriophage proteins identified in Aim 1 will be used to raise mAbs using both hybridoma and in vivo ascites techniques.
2.1 Immunize animal model with vibriophage proteins and screen for antibody producing hybridoma clones
2.2 Propagate selected hybridoma cells in vivo using ascites method for mAb production and affinity purification

Aim 3

Produce, test and select candidate RDTs by highest performance metrics. The monoclonal antibodies produced by hybridoma technology will be used to develop an immune-assay based lateral flow test; this test will include the existing (or equivalent) commercial mAb for V. cholerae O1 LPS. Several RDTs with different mAb candidates will be evaluated.
3.1 Conjugate colloidal gold to mAb and assemble the lateral flow strip
3.2 Evaluate the performance of the RDTs against commercially available RDTs with a clinical sample library

It is likely that rapid diagnostic tests (RDTs) for cholera intermittently fail because lytic vibriophage destroy the Vibrio cholerae target. In this proposal, we are adding an antibody to the lytic vibriophage (ICP1) to the RDT. The goal is that detection of the vibriophage can serve as a proxy for Vibrio cholerae, and therefore, increase sensitivity of the RDT when vibriophage are present.

This revised RDT will address sensitivity concerns and intermittent performance of current RDTs when lytic vibriophage are present. This new RDT will not address limitations when antibiotics are present, and therefore patient reports on antibiotic consumption need to be consider when evaluating RDT results, even with the new RDT.