Development of a Monoclonal Antibody to a Vibriophage as a Proxy for Vibrio cholerae Detection

Abstract

Cholera is an acute watery, diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage is present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by Enzyme linked immunosorbent assay (ELISA) and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 × 10⁵ PFU by Western blotting analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.

Keywords: Bangladesh; ICP1; Vibrio cholerae; bacteriophage; cholera; diarrhea; phage; rapid diagnostic tests (RDT); vibriophage.

FIG 1

A model showing how an RDT for V. cholerae may fail when virulent phage (e.g., ICP1) are present in a stool sample (A) and how this limitation can be addressed by incorporating a mAb to phage as a proxy for V. cholerae when phage are present (B).

FIG 2

Multi-sequence alignment of ICP1 phage baseplate ORF75 (A) and capsular head ORF122 (B) amino acid sequences. Sequences from both Bangladesh (BD) and Democratic Republic of Congo (DRC). Blue boxes at the bottom of ‘A’ and ‘B’ represent the percentage of the isolates that have the same amino acid for that particular site. Temporal and divergence analysis of baseplate ORF75 (C) and capsular head ORF122 (D) nucleotide sequences from ICP1 phage isolated from Bangladesh.

FIG 3

Immunoreactivity by ELISA of ORF75 mAbs (n = 20) (A) and ORF122 mAbs (n = 20) (B) to phage particles (ICP1, ICP2, ICP3), formalin-killed V. cholerae whole-cell (VCWC), bovine serum albumin (BSA) and ORF75 and ORF122 recombinant protein. Statistically significant differences (P < 0.05) in the mean immune response from all clones are denoted with an asterisk. Symbols represent the average of three technical replicates for one mAb from one experiment; data are representative of two independent experiments. The y axis is set to the maximal absorbance in the assay.

FIG 4

Western blot analysis of candidate ICP1ORF122_mAbCL6 against ICP1 from Bangladesh (A) and Goma, DRC (B). Negative controls are ICP2 and ICP3. BD = Bangladesh, VCWC = formalin-killed V. cholerae whole-cell, bovine serum albumin = BSA, NC = negative control (only PBS), L = ladder (protein marker), ORF75 and ORF122 = ICP1 recombinant proteins.

FIG 5

ICP1 phage neutralization by ICP1ORF122_mAb CL5, CL6 and CL14. Here, PBS = phosphate-buffered saline, ICP1ORF122_mAb CL5, CL6 and CL14 represent culture supernatants from ORF122 hybridoma clones 5, 6 and 14, respectively. An asterisk denotes the statistically significant difference (P < 0.05) in plaque counts when ORF122 mAb mediated neutralization responses are compared with the control (PBS). Each symbol represents the average of three technical replicates for one mAb from one experiment.

FIG 6

Determination of the limit of detection (LOD) of ICP1ORF122_mAbCL6 against ICP1 in cholera stool supernatant. ICP1 was serially diluted in cholera stool known to be vibriophage negative (ICP1, ICP2, and ICP3 negative) in 3-fold dilution series. The concentration of the neat ICP1 stock was 2 × 10¹⁰ PFU/mL. The lane with 1:243 dilution represents 8 × 10⁵ PFU of ICP1 phage. Here, bovine serum albumin = BSA, ORF122 = ICP1 recombinant protein and L = ladder (protein marker).