Production, selection and characterization of monoclonal antibodies against outer membrane vesicles derived from Bordetella pertussis.

DANIELA F HOZBOR; MAIA L ELIZAGARAY; GRISELDA N MORENO; MARTÍN RUMBO

Cancún

Congreso; XII Congress of the Latin American Association of Immunology - ALAI; 2018

ALAI-Asociación Latinoamericana de Inmunología

Outer-membrane vesicles (OMVs) are naturally non-replicating spherical nanoparticles derived from Gram-negative bacteria. OMVs are highly immunogenic and consist of phospholipids, LPS, outer membrane proteins and entrapped periplasmic components (Ellis et al., 2010; Hozbor et al., 1999; Ormazábal et al., 2014). These vesicles participate in different biological functions such as cell to cell communication, surface modifications and host-pathogen interaction by shuttling an array of virulence factors from bacteria to host during infection (Ellis and Kuehn, 2010). Because of their immunogenic properties, selfadjuvanticity and ability to be taken up by mammalian cells, OMVs are attractive candidates for vaccine delivery platforms. We developed a new vaccine against pertussis based on OMVs, which have been shown to be safe and to induce protection in mice, with a mixed Th1/Th17/Th2 profile, a robust antibody response and adequate protection capacity against different B. pertussis genetic background including those not expressing the virulence factor pertactin (PRN) that is also a vaccine antigen (Asensio et al., 2011; Bottero et al., 2016; Gaillard et al., 2014; Roberts et al., 2008). We have characterized the composition of the pertussis nanoparticles, finding the presence of a high number of immunogens in the vaccine formulation, being an important feature since this may avoid the excessive selective pressure conferred by a single or a few protective vaccine antigens. It is estimated that the final cost per dose is less than that of existing acellular formulations based on several purified protein immunogens, which impacts on the final cost of the vaccine, which is important for its application in developing countries. In the present work we present the results obtained during the generation and characterization of anti-OMVBp monoclonal antibodies (MAbs). At the moment the production of monoclonal antibody (Mab) for the OMVBp has not been described. For the generation of MAbs, BALB/c mice were immunized three times by intraperitoneal injections (separated every 25 days) with 30 μg of OMV from B. pertussis with adjuvant. Monoclonal antibodies were derived by somatic cell hybridization as described by Galfré and Milstein (Galfrè and Milstein, 1981) using polyethylene glycol (MW 3350) as fusogenic agent. Briefly, NS0 myeloma cells were fused with spleen cells from immunized mice and the resulting hybridomas were cloned by limiting dilution. Specific reactivity of supernatant was tested by ELISA with protein lysate of B pertussis as antigen. As first screening, 30 positive clones were selected. Upon expansion and analysis of reactivity pattern by immunoblots, most of them showed reactivity mainly directed to chaperone Gro-EL, a conserved protein, found in many species of bacteria as well as in eukaryotic cells. Interestingly, anti-Gro-EL antibodies were detected after human pertussis vaccination however when the purified non-native protein was used as vaccine little protective capacity was observed (Burns et al., 1991). In the present work we assessed whether the antibodies induced by the native Gro-EL protein contained in the OMVs were able to induce protection against pertussis infection in the mice model. To this end and to obtain high levels of monoclonal antibody, hybridoma cells selected were injected intraperitoneally into mice to induce formation of ascites tumours and production of ascites fluid containing higher levels of monoclonal antibodies. Passive transfer assay was performed with MAb24G12H8 (IgG2a). Sera (100 μl) from OMVBp immunized mice were used as positive control. Challenge with sublethal dose (1.106 CFU) of BpTohama strain was performed 24 h after transfer and protection was assessed by determining CFU counts 7 days after challenge. The results expressed as log CFU/lungs (5.8±0.2 for PBS group, 6.1±0.2 for MAb group and 3.3±0.3 for positive control) showed that there was no protection for the case of MAb24G12H8 and it remains to be determined for the case of MAb52F2B9. In conclusion we were able to obtain OMV-specific mAbs with high yield that though does not present protective capacity they constitute a valuable immunochemical tool that could be used in future different assays including quality control test for the process of obtaining the OMVs.