Transcutaneous Immunization with Toxin-Coregulated Pilin A Induces Protective Immunity against Vibrio cholerae O1 El Tor Challenge in Mice

Abstract

Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 g of TcpA or TcpA with an immunoadjuvant (cholera toxin Cholera, a severe, dehydrating diarrhea in humans, is caused by the gram-negative bacterium Vibrio cholerae. Strains of V. cholerae that produce cholera belong to serogroup O1 or O139. V. cholerae O1 is comprised of two biotypes, classical and El Tor. Globally, O1-associated cholera is caused by the El Tor biotype. Cholera toxin (CT), the cause of the severe secretory diarrhea seen in cholera, is the major virulence factor for all toxigenic strains of V. cholerae (4). Toxin-coregulated pilus (TCP) is a second major virulence factor of V. cholerae. The major structural protein of this pilus, toxin-coregulated pilin A (TcpA), is encoded by tcpA, and its expression is regulated in V. cholerae in parallel to cholera toxin (39). TCP is essential for colonization and virulence in both animal models and human volunteers (18, 39), and recent data support its role in biofilm formation and binding to chitinous surfaces in aquatic environments (30). Although TcpA from El Tor and classical strains are approximately 80% homologous at the amino acid level, monoclonal antibodies have shown epitope differences between these proteins (19,22,31,36). TcpA proteins from El Tor and O139 strains are identical (31).[A number of observations suggest that immune responses toTcpA may contribute to protection against V. cholerae infection. TcpA has been shown to be essential for V. cholerae colonization in both mice and humans (18, 39), tcpA mRNA is up-regulated during early human infection (27), and systemic and mucosal anti-TcpA immune responses occur in over 90% of individuals infected with V. cholerae O1 El Tor in Bangladesh (1, 16). Furthermore, passive administration of both polyclonal and monoclonal antibodies against TcpA in mice is fully protective against V. cholerae challenge (36, 37), and active parenteral immunization of adult female mice with a TcpA peptide along with an immunoadjuvant induces protection against V. cholerae challenge of mice born to immunized mothers (42). For safety reasons, cholera vaccines that are available or under development all lack CT. However, CT is a potent immunoadjuvant, and immune responses induced by cholera vaccines are often less prominent than those induced by wild-type infection (32). Immunization strategies that augment immune responses to critical virulence factors may thus contribute to the development of an optimal cholera vacci...

Transcutaneous Immunization withClostridium difficileToxoid A Induces Systemic and Mucosal Immune Responses and Toxin A-Neutralizing Antibodies in Mice

“…We transcutaneously immunized mice on days 0, 14, 28, and 42, as previously described (42). Briefly, we shaved a 3-by 5-cm 2 area on the dorsa of mice by using a clipper with a no.…”

“…During TCI, cutaneously applied antigens are taken up by Langerhans cells in the epidermis, and these cells then migrate to regional lymph nodes. Interestingly, TCI induces both systemic and mucosal immune responses (6,22,23,28,41,42,48). TCI has been shown to be safe and effective in animals and humans (9,21,23,42,47,58).…”

“…TCI usually requires the presence of an immunoadjuvant, and ADP-ribosylating proteins such as cholera toxin (CT) and heat-labile enterotoxin or their derivatives have most commonly been used as immunoadjuvants during TCI (19,23,42,45,46). During TCI, cutaneously applied antigens are taken up by Langerhans cells in the epidermis, and these cells then migrate to regional lymph nodes.…”

“…Interestingly, TCI induces both systemic and mucosal immune responses (6,22,23,28,41,42,48). TCI has been shown to be safe and effective in animals and humans (9,21,23,42,47,58). In order to assess whether TCI would induce immune responses against C. difficile toxin A, we therefore transcutaneously immunized mice with a toxoid derivative of C. difficile toxin A (CDA), with or without the immunoadjuvant CT, and measured systemic and mucosal anti-CDA immune responses, including induction of toxin Aneutralizing antibodies in immunized mice.…”

“…Transcutaneous immunization (TCI) involves the needlefree application of antigens directly to hydrated skin from which the stratum corneum has been gently removed (17,18,23,42). TCI usually requires the presence of an immunoadjuvant, and ADP-ribosylating proteins such as cholera toxin (CT) and heat-labile enterotoxin or their derivatives have most commonly been used as immunoadjuvants during TCI (19,23,42,45,46).…”