S1639 5ml

The vibriocidal responses were assessed according to previously described methods [17 (link)] with some modifications. The strains were incubated with heat-inactivated serum and exogenous guinea pig complement (Sigma Aldrich S1639-5ML) at 37°C for 1 hour, shaking (50 revs/min). Vibriocidal titers were defined as the reciprocal of the highest serum dilution resulting in a 50% reduction in optical density (595 nm) compared to controls without serum. Standard monoclonal antibody (mAb) (Boston, MA, USA, CF29.1.A2) and high titer pooled sera were used to normalize the results in case of inter-assay variations. Seroconversion was defined as a 4-fold or greater increase in vibriocidal titers after vaccination in comparison to the baseline (D0) titers with a titer of 5 assigned in cases where no vibriocidal activity was observed.

To isolate ICMs, we subjected late blastocysts to immunosurgery (Solter and Knowles, 1975 (link)). In brief, in vitro cultured single and double embryos were incubated with anti-mouse whole serum (M5774-2ML, Sigma-Aldrich) and sera complement guinea pig (S1639-5ML, Sigma-Aldrich) diluted to 20% in M2 medium for 45 min each. After elimination of trophectoderm cells by pipetting with a narrow glass capillary in M2 medium, ICMs were seeded in individual hanging drops of pre-warmed IVC-1 medium (Bedzhov et al., 2014 (link)) (M11-25, Cell Guidance Systems) and cultured for 24, 48, and 72 h at 37°C in 5% CO₂ (Figure 2A).

Vibriocidal antibody assays was also run as previously described with some modifications [18 (link)]. Local Zambian V. cholerae O1 Inaba (EDVRU/ZM/091–10) and Ogawa (EDVRU/ZM/2016) were used. These strains were quality checked at Johns Hopkins University in the United States and their performance was comparable to standard strains Inaba (T19479) and Ogawa (X25049).
Briefly, colonies from overnight cultures were inoculated in Brain Heart Infusion (BHI) Broth and incubated at 37 °C for about 4 hours before harvesting the cells. Heat inactivated serum, exogenous guinea pig complement (Sigma Aldrich S1639-5ML) and V. cholerae bacterial cells were then placed in 96-well microtitre tissue culture plates (Life sciences, Durham, USA) and incubated at 37 °C. Vibriocidal titres were defined as the reciprocal of the highest serum dilution resulting in a 50% reduction in optical density read at 595 nm compared to positive control wells without serum. Seroconversion was defined as a 4-fold or greater increase from the baseline vibriocidal titres. A standard monoclonal antibody (mAb) and a high titre standard serum was used to normalize the results in case of inter-assay variations.