Plant-based production of two chimeric monoclonal IgG antibodies directed against immunodominant epitopes of Vibrio cholerae lipopolysaccharide

Abstract

We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest.

Keywords: Antibodies; Cholera; Plant expression; Vaccine.

Figure 1. Reactivity of chimeric 2D6 and ZAC-3 with purified LPS and whole cell V. cholerae

2D6 (left panels) and ZAC-3 (right panels) IgA and IgG reactivity with (A) purified Ogawa LPS, (B) whole cell V. cholerae strain O395, (C) strain El Tor C6706 or (D) strain 9459. ELISAs were performed as described in the Materials and Methods. Sal4 and SyH7 were used as isotype mouse IgA and human IgG1 controls.

Figure 2. Effects on 2D6 and ZAC-3 on V. cholerae agglutination and motility

(A) Bacterial macro-agglutination assays: mid-log phase V. cholerae O395 was mixed with an equal volume of LB medium containing two-fold serial dilutions of Sal4 (control), 2D6 or ZAC-3 IgA or IgG mAbs and then seeded in U-bottom 96-well plates. Microtiter wells were monitored for the appearance of aggregated bacteria over a period of 2 hr. 2D6 IgA promoted the appearance of visible clumps at concentrations ≥18 μg/ml, whereas ZAC-3 IgA promoted agglutination at ≥0.25 μg/ml (arrowheads). Neither 2D6 nor ZAC-3 IgG produced visible agglutination at any concentration tested. (B and C) Semi-solid liquid motility assays: single colonies of V. cholerae O395 were stabbed into 0.3% semi-solid LB agar containing 9 μg/ml of ZAC-3 IgG1 or ZAC-3 Fab fragments. Plates were incubated at 37°C and the diameter of the bacterial spread was measured every 60 min. These data demonstrate that both ZAC-3 IgG and Fab fragments significantly reduced V. cholerae motility (P=0.0003, Student t test), although the Fab fragments effects were only apparent at the earliest time point.