Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus

Abstract

The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

Stable production of peptide antigens in transgenic tobacco chloroplasts by fusion to the p53 tetramerisation domain

“…The parenteral immunisation protocol was performed as previously described (Molina et al 2005). Briefly, antigen-enriched leaf extracts (29% purity; 20 µg 2L21-TD/dose and 70 µg total protein/dose) were administered with complete (first dose) or incomplete (second and third doses) Freund's adjuvant.…”

Chloroplast-Derived Therapeutic and Prophylactic Vaccines

“…CTB-2L21 fusion protein accumulated to 31% TSP in tobacco chloroplast and was subsequently analyzed for immunogenicity. Parenteral injections of enriched plant-extracts in mice and rabbits induced high titer antibody production against 2L21 (Molina et al 2005 ). Rabbit sera displayed the same capacity for neutralizing 2L21 antigen as the monoclonal antibody 3C9.…”

“…While the priming generated a humoral IgG response, oral boosting was critical to drive iso-type switching to mucosal IgA. However, the antibodies induced by oral immunization were ineffective at neutralizing 2L21 during in vitro assays suggesting the breakdown of key epitopic determinants (Molina et al 2005 ). The high yield of subunit vaccine generated through this approach vindicates the generation of similar chloroplast-derived vaccines in edible food systems.…”

“…Demonstrating effi cacy in these mouse-models is required to warrant examination in higher animal models. Advancing through trials in rabbits has been accomplished by some groups (Molina et al 2005 ) ; however, this trend must become more widespread and continue to advance through other systems such as gerbils, pigs, dogs, and eventually non-human primates before human clinical evaluation of chloroplast-derived antigens may occur. These models will aid in the development of antigen doses, and immunization strategies that will be effective in humans.…”

The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts

“…Based on these advantages, several vaccine antigens and biopharmaceuticals have been expressed in tobacco chloroplasts and their eYcacy evaluated (Bock 2007;Chebolu and Daniell 2009). For example, vaccine antigens have been expressed against numerous pathogens and shown to be immunogenic and oVer protection against the pathogen challenger (Koya et al 2005;Molina et al 2005;Arlen et al 2008). Similarly, several therapeutic human proteins including somatotropin (Staub et al 2000), interferon alpha (Arlen et al 2007), proinsulin (Ruhlman et al 2007) and cardiotrophin-1 (Farran et al 2008), were expressed in chloroplasts and revealed to be properly folded and fully functional.…”