Enterotoxin B Subunit Lectins as Adjuvants for Improvement of Mucosal Vaccine

<p>In comparison with whole organism vaccines, subunit vaccines may be safer for immunization but may lack sufficient immunogenicity to provide complete immunity to the pathogen. To resolve this problem, bacterial and plant enterotoxin B subunit adjuvants containing a variety of receptor-binding properties were used to enhance the immunogenicity of rotavirus subunit vaccines. Enterotoxin B subunit adjuvants were employed to enhance protection against virus infection. Pentameric cholera toxin B subunit (CTB), shiga toxin-1 B subunit (STB) and monomeric ricin toxin B subunit (RTB) molecules were genetically linked to a 90 amino acid peptide from the simian rotavirus (SA11) nonstructural protein NSP4 or to the rotavirus outer capsid protein VP7. Lectin binding assays were used to demonstrate receptor binding activity of the ligand-antigen fusion proteins synthesized in <em>E. coli</em>. Mice co-fed RTB and NSP4₉₀ protein or heat denatured NSP4₉₀::RTB fusion protein generated higher serum anti-NSP4₉₀ IgG titers than mice inoculated with the NSPT₉₀ antigen alone. Serum anti-NSP4₉₀ IgG and intestinal anti-NSP4₉₀ IgA titers were the highest in mice immunized with native NSP4₉₀::RTB or STB::NSP4₉₀ fusion proteins. Serum anti-NSPT₉₀ IgG isotype analysis and secreted cytokine assays from splenocytes isolated from mice immunized with NSP4₉₀::RTB and STB::NSP4₉₀fusion proteins generated elevated IFN-γ and IL-4 levels and increased IgG2a antibody titers confirming fusion protein stimulation of a dominant Th1 cell mediated immune response. Diarrhea in SA11 rotavirus challenged suckling neonates from dams immunized with NSP4₉₀::RTB and STB::NSP4₉₀ fusion proteins was dramatically reduced both in severity and duration in comparison with rotavirus challenged neonates from unimmunized mice, demonstrating the protective efficacy of ligand-antigen fusion proteins synthesized in bacteria.</p> <p>The feasibility of using transformed plants for production of correct posttranslational modification of adjuvanted subunit rotavirus antigens was assessed. Immunoblot analyses and lectin-receptor binding enzyme-linked immunosorbent assays showed that CTB::NSP4₉₀, CTB::VP7 and VP7::RTB fusion proteins of expected molecular mass were synthesized in transformed potato leaf and tuber tissues. The production of adjuvant ligand-rotavirus antigen fusion proteins in both bacteria and plant tissues demonstrates the feasibility of using prokaryotic and eukaryotic systems for production of adjuvanted viral antigens for generating enhanced immunity against infectious rotavirus disease.</p>

Name thesis:degree_name

Doctor of Philosophy (Medical Science)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Year

2004

Author dc:creator

• Choi, Nak-Won

Contributors dc:contributor

• William H. R. Langridge
• Penelope J. Duerksen-Hughes
• E. Clifford Herrmann
• James D. Kettering
• Thomas A. Linkhart

• Amino Acids, Peptides, and Proteins
• Animal Experimentation and Research
• Biochemistry
• Biological Phenomena, Cell Phenomena, and Immunity
• Laboratory and Basic Science Research
• Microbiology
• Vaccines, Subunit -- immunology; Enterotoxins -- therapeutic use; Rotavirus vaccines; Rotavirus infections; Lectins -- therapeutic use; Adjuvants, Immunologic.

Statement dc:rights

• This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/1455

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-2227