High-throughput bactericidal assays for monoclonal antibody screening against antimicrobial resistant Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae (gonococcus) is an obligate human pathogen and the etiological agent of the sexually transmitted disease gonorrhea. The rapid rise in gonococcal resistance to all currently available antimicrobials has become a significant public health burden and the need to develop novel therapeutic and prophylactic tools is now a global priority. While high-throughput screening methods allowed rapid discovery of extremely potent monoclonal antibodies (mAbs) against viral pathogens, the field of bacteriology suffers from the lack of assays that allow efficient screening of large panels of samples. To address this point, we developed luminescence-based (L-ABA) and resazurin-based (R-ABA) antibody bactericidal assays that measure N. gonorrhoeae metabolic activity as a proxy of bacterial viability. Both L-ABA and R-ABA are applicable on the large scale for the rapid identification of bactericidal antibodies and were validated by conventional methods. Implementation of these approaches will be instrumental to the development of new medications and vaccines against N. gonorrhoeae and other bacterial pathogens to support the fight against antimicrobial resistance.

Keywords: Neisseria gonorrhoeae; antimicrobial resistance; bacteria; high-throughput screening; monoclonal antibodies; therapeutics.

Figure 1

Workflow for monoclonal antibody based high-throughput serum bactericidal assays. (A–C) Schematic representation of the workflows implemented for the L-ABA (A), R-ABA (B), and C-SBA (C).

Figure 2

Evaluation of different conditions for L-ABA implementation. (A–C) Graphs show the viability and non-specific killing (NSK) of bacteria tested at OD₆₀₀ 0.1 (A), 0.01 (B), and 0.001 (C) incubated with different amounts of BRC or hi-BRC (40, 20, 10, and 5%) for 1 h. (D–F) Graphs show the viability and non-specific killing of bacteria tested at OD₆₀₀ 0.1 (D), 0.01 (E), and 0.001 (F) incubated with different amounts of BRC or hi-BRC (40, 20, 10, and 5%) for 2 h.

Figure 3

2C7 bactericidal activity evaluated by L-SBA. (A,B) Graphs show the bactericidal curves of 2C7 after 1 h incubation with 10% BRC or hiBRC at OD₆₀₀ 0.1 (A) and 0.01 (B). (C,D) Graphs show the bactericidal curves of 2C7 after 2 h incubation with 10% BRC or hiBRC at OD₆₀₀ 0.1 (C) and 0.01 (D). The IC₅₀ values obtained with each condition are reported on the graphs.

Figure 4

Comparison between L-ABA, R-ABA, and C-SBA assays. (A–C) Bactericidal curves of 2C7 mAb starting from 10 μg/mL to 4 ng/mL (three-step serial dilutions) against Neisseria gonorrhoeae FA1090 tested by L-SBA (A), R-SBA (B), and C-SBA (C). The IC₅₀ values are reported on each graph. (D–F) Two-tailed Pearson coefficient correlation was performed between L-ABA and R-SBA (D), L-ABA and C-SBA (E), and R-ABA and C-SBA (F). The r squared values (r) are indicated on each graph. A nonparametric Mann–Whitney t test was used to evaluate statistical significances between groups. Two-tailed value of p significances are shown as ^**p < 0.01 and ^****p < 0.0001.

Figure 5

High-throughput assays for mAb screening. (A) Schematic representation of the two high-throughput assays (HT-L-ABA and HT-R-ABA) used to evaluate 2C7 bactericidal activity in a 96-well plate. (B) The graph shows the HT-L-ABA screening. The table describes number of replicates, positive hits, and percentage of positive hits for 2C7 supernatants and mock-transfected supernatants. (C) The graph shows the HT-R-ABA screening. The table describes number of replicates, positive hits, and percentage of positive hits for 2C7 supernatants and mock-transfected supernatants. Black lines represent the mean values, while black dotted lines display the maximum and minimum RLU values for both positive (2C7) and negative controls. The Z factor was calculated for both assays and is reported on the graphs.