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 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.

A monoclonal antibody that targets the conserved core/lipid A region of lipopolysaccharide affects motility and reduces intestinal colonization of both classical and El Tor Vibrio cholerae biotypes

“…The plates were incubated at 37°C and the diameter of bacterial migration away from the point of inoculation was determined at 4 and 8 h time points. When treated with PBS or an isotype antibody control, V. cholerae strain O395 had a migration diameter of ~0.8 cm at 4 h and 1.5 cm at 8 h. In the presence of ZAC-3 IgG, bacterial migration was reduced 4–10 fold (Figure 1Ai), which is consistent with our previous results [15]. The motility studies were then repeated with the El Tor Inaba strain C6706.…”

“…However, we recently reported that ZAC-3, a monoclonal antibody against the core/lipid A region of V. cholerae O1 LPS, abolishes migration of V. cholerae O1 strain O395 in a semi-solid agar motility assay, an observation that may have a number of implications for cholera vaccine development [15]. ZAC-3 was first isolated as a monoclonal IgA from a B cell hybridoma derived from mouse Peyer’s patches [16].…”

“…ZAC-3 was first isolated as a monoclonal IgA from a B cell hybridoma derived from mouse Peyer’s patches [16]. As the ZAC-3 hybridoma secretes very little antibody (K. Levinson and N. Mantis, unpublished results), we generated a recombinant ZAC-3 antibody in which the heavy (V H ) and light (V L ) chains of ZAC-3 IgA were cloned onto a human IgG 1 framework [15]. Chimeric ZAC-3 IgG was successfully expressed in a Nicotiana benthamiana -based rapid antibody-manufacturing platform (RAMP), thereby ensuring a ready supply of antibody.…”

Inhibition of invasive salmonella by orally administered IgA and IgG monoclonal antibodies

“…The addition of greater amounts of Sal4 further reduced the CI with a maximal reduction occurring at concentrations above 1.2 μg Sal4 IgA. Invasion of Peyer's patch tissues by AR05 and AR04 was unaffected by 2D6, an anti-Vibrio cholerae IgA mAb that served as the isotype control for these studies [13,21].…”

“…For the soft agar motility assays, LB medium with 0.3% Bacto agar (Becton Dickinson) was prepared with 15 μg/ml of each mAb of interest poured into 60 mm Petri dishes (n = 3 per treatment group) and allowed to set at room temperature for 30 min. Individual colonies of ATCC 14028 STm were then picked from a freshly-streaked LB agar plate and stabbed directly into the center of the plate [21]. The plates were placed in a 37˚C incubator and the diameters of the concentrically growing bacterial cultures were measured at 60-minute intervals.…”