title: Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice.
abstract: We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.
authors.affiliation: Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. anthony.choi@cchmc.org
authors.forename: Anthony H-C
authors.initials: AH
authors.lastname: Choi
authors.forename: Mitali
authors.initials: M
authors.lastname: Basu
authors.forename: Monica M
authors.initials: MM
authors.lastname: McNeal
authors.forename: Judy A
authors.initials: JA
authors.lastname: Bean
authors.forename: John D
authors.initials: JD
authors.lastname: Clements
authors.forename: Richard L
authors.initials: RL
authors.lastname: Ward
mesh_terms: D000281:Administration, Intranasal; D000284:Administration, Oral; D000818:Animals; D000956:Antigens, Viral / Q000008:administration & dosage* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D036022:Capsid Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000302:isolation & purification; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D005815:Genetic Code; D006801:Humans; D007114:Immunization; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000517:prevention & control* / Q000821:virology; D022243:Rotavirus Vaccines / Q000008:administration & dosage* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; D022243:Rotavirus Vaccines; C061332:VP6 protein, Rotavirus