Systemic And Enteric Mucosal Antibody Responses Induced By Combination Of Cholera DNA Vaccine And Inactivated Vaccine

Part?. Immunogenicity of and protective immunity induced by codon optimized DNA vaccines expressing cholera toxin B subunit Objective: To construct codon optimized DNA vaccine expressing cholera toxin B (CTB), examine its immunogenicity in rabbits and mice and test the protective immunity induced by such vaccine.Methods: Based on CTB amino acid sequence, we designed the codon-optimized ctxB gene sequence using codons preferred for better expression in both mammalian cells and E. coli. The codon optimized gene was chemically synthesized and cloned into the DNA vaccine vector pJW4303. After making the CTB DNA vaccine construct, the expression of CTB protein was verified by transient transfection of 293T cells and Western blot analysis. Then, New Zealand white rabbits and Balb/c mice were immunized with CTB DNA vaccine by intramuscular injection followed by electroporation. Rabbits were immunized at Weeks 0, 2, 4 and 8, and mice were immunized at Weeks 0 and 2. Serum and fecal samples were collected prior to the first immunization and 2 weeks after each immunization. CTB-specific IgG antibodies in serum and fecal samples were evaluated by ELISA. The protective immunity induced by CTB DNA vaccine was examined against toxigenic Vibrio cholera O1 ElTor strain by a passive suckling mouse protection model using immune rabbit sera and by rabbit ileal loop test with immune rabbits.Results: The codon optimized CTB DNA vaccine was successfully constructed and could properly express CTB protein as expected. The rabbits and mice immunized with CTB DNA vaccines produced CTB-specific IgG antibody responses in immune sera. CTB-specific IgG antibody responses were also detected in rabbit fecal samples. The protection studies demonstrated that not only the immune rabbit sera produced by CTB DNA immunization could protect suckling mice against toxigenic Vibrio cholerae O1 challenge but also immune rabbit produced active protection in a rabbit ileal loop test model.Conclusions:The codon optimized DNA vaccines expressing cholera toxin B subunit were immunogenic and elicited protective immune responses against Vibrio cholerae challenge in animal models.[Key words] Vibrio cholera; cholera vaccine; DNA vaccine; CTB; codon optimizationPart?. Systemic and mucosal antibody responses induced by CTB DNA vaccine prime followed by KWC-B vaccine parenteral boostObjective: To investigate the systemic and mucosal antibody responses induced by combination of cholera CTB DNA vaccine prime and killed whole cell/recombinant cholera-toxin B subunit( KWC-B) vaccine parenteral boost. KWC-B vaccine parenteral boost.Methods: Balb/c mice were immunized twice at Weeks 0 and 2 with either the homologous DNA prime+DNA boost (DNA+DNA), KWC-B prime+KWC-B boost (KWC-B+KWC-B), or the heterologous DNA prime+KWC-B boost (DNA+KWC-B). DNA vaccine was always delivered intramuscularly followed by electroporation (EP) while the KWC-B vaccine was delivered by intramuscular injection (IM). Serum and fecal samples were collected prior to the first immunization and 2 weeks after each immunization. ELISA was conducted to measure the CTB-specific IgG, IgA and IgM in mice sera and fecal samples as well as IgG isotypes in immune sera. Mouse splenocytes were isolated at 5 month after the last immunization and CTB-specific IgG antibody secreting cells were detected by ELISPOT.Results: A single immunization with KWC-B by intramuscular injection was unable to induce high level of CTB-specific IgG, IgA and IgM antibody responses in sera nor detectable antibody in fecal samples. After two immunizations, the mice receiving CTB DNA vaccine alone, KWC-B alone or DNA prime followed by KWC-B boost all produced high level of antibody responses in sera and had detectable IgG in fecal samples. KWC-B alone induced predominant IgG1, Th2 type antibody responses while DNA vaccine alone induced better IgG2a, Th1 type antibody responses. Interestingly, DNA prime followed more balanced IgG1 and IgG2a, more balanced Th1/Th2 responses. CTB-specific antibody secreting cells were detected in all groups receiving 2 times of immunations. Conclusions: Both systemic and mucosal antibody responses against CTB were elicited in mice receiving DNA alone, KWC-B alone (IM) or DNA prime followed by KWC-B boost (IM). However, CTB DNA prime + KWC-B boost (IM) produced more balanced Th1/Th2 type antibody responses.Part?. Systemic and mucosal antibody responses induced by CTB DNA vaccine prime followed by KWC-B vaccine oral boostObjective: To investigate the systemic and mucosal antibody responses induced by combination of cholera CTB DNA vaccine prime and KWC-B vaccine oral boost. Methods: Balb/c mice were immunized twice at Weeks 0 and 2 with either the homologous DNA prime+DNA boost (DNA+DNA), KWC-B prime+KWC-B boost (KWC-B+KWC-B(O)), or the heterologous DNA prime+KWC-B boost (DNA+KWC-B (O)). DNA vaccine was always delivered intramuscularly followed by electroporation (EP) while the KWC-B vaccine was delivered orally. Serum and fecal samples were collected prior to the first immunization and 2 weeks after each immunization. ELISA was conducted to measure the CTB-specific IgG, IgA and IgM in mice sera and fecal samples as well as IgG isotypes in immune sera Mouse splenocytes were isolated at 5 month after the last immunization and CTB-specific IgG antibody secreting cells were detected by ELISPOT.Results: A single immunization with KWC-B by oral administration was unable to induce detectable antibody responses in either serum and fecal samples. After two immunizations, the mice receiving CTB DNA vaccine alone or DNA prime followed by KWC-B oral boost produced high level of antibody responses (IgG, IgA and IgM) in sera. KWC-B alone oral immunization only induced detectable IgA while DNA alone group had only detectable IgG against CTB in fecal samples. Interestingly, the group receiving CTB DNA prime followed KWC-B oral boost had detectable both IgA and IgG responses in fecal samples. CTB-specific antibody secreting cells were detected in CTB DNA alone and DNA prime + KWC-B oral boost group but not the KWC-B alone oral immunization group at 5 month after the second immunization.Conclusions: Although KWC-B oral vaccination induced mucosal antibody responses but was very inefficient in eliciting systemic antibody responses. CTB DNA prime + KWC-B oral boost induced improved both systemic and mucosal antibody responses when compared to DNA alone or KWC-B alone (oral) vaccination.