title: Enhanced immunogenicity of a functional enzyme by T cell epitope modification.
abstract: BACKGROUND
  T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines.
  
  
  RESULTS
  Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone.
  
  
  CONCLUSIONS
  With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics.
authors.affiliation: Department of Immunology, Genencor International, Palo Alto, California 94303, USA. jmucha@genencor.com
authors.forename: Jeanette M
authors.initials: JM
authors.lastname: Mucha
authors.forename: Marcia M
authors.initials: MM
authors.lastname: Stickler
authors.forename: A J
authors.initials: AJ
authors.lastname: Poulose
authors.forename: Grant
authors.initials: G
authors.lastname: Ganshaw
authors.forename: Mae
authors.initials: M
authors.lastname: Saldajeno
authors.forename: Kathy
authors.initials: K
authors.lastname: Collier
authors.forename: Manley T
authors.initials: MT
authors.lastname: Huang
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Harding
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D004795:Enzyme Stability; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006168:Guinea Pigs; D007114:Immunization; D007074:Immunoglobulin G / Q000097:blood; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D007074:Immunoglobulin G; D013381:Subtilisins