Recombinant Polyvalent Enterotoxin Vaccines Against Enterotoxigenic Escherichia Coli

Enterotoxigenic Escherichia coli (ETEC) strains remain important pathogens causing diarrhea in children and a wide variety of newborn animals. Vaccination is an effective approach to control ETEC-induced infections which avoids the problem of antibiotics residue and antibiotics resistance. Currently available ETEC vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). Heat-stable enterotoxin a (STa) and b (STb) are important virulence fators of ETEC, but they are pooly immunogenic due to their small size. Preparation of ST-carrier toxoids which are immunogenic and devoid of toxicity continues to be a challege for ETEC vaccines development. However, the induction of antitoxic responses against STa and STb has merit, as these two poorly immunogenic toxins are frequently associated with ETEC strains.In order to obtain more effective ETEC vaccines, the aim of this study was to fuse the three coding genes of enterotoxins LTB, STa, and STb, and construct recombinant trivalent enterotoxin DNA vaccines and protein vaccines, in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. The main work of this thesis was as follows:(1) The study of polyvalent enterotoxin DNA vaccines:chicken insulin signal peptide gene ins and chicken urokinase type plasminogen activator signal peptide gene upa were amplified by splicing by overlap extension (SOE) PCR. Coding genes for LTB, STa, and STb and signal peptide gene (ins or upa) were fused to yield the polyvalent enterotoxin fusion gene with or without signal peptide gene. Two carefully selected proline-rich natural oligopeptides, PPASP and SASTTPP, were included in the fusion to link the adjacent toxins. Fusion genes were then cloned into eukaryotic expression vector pCI to obtain 3 polyvalent enterotoxin DNA vaccines pCI-ins-SLS、pCI-upa-SLS、pCI-SLS. The DNA sequences of the recombinant plasmids were verified by DNA sequencing. DNA vaccines were purified by column chromatography and used to transfect Hela cells using liposome, then indirect immunofluorescence experiments confirmed the expression of polyvalent enterotoxin in Hela cells by the recombinant plasmids. Immunization of chickens with the 3 DNA vaccines elicited low antibody level to LTB and very weak antibody responses to STa or STb, but the DNA vaccine pCI-SLS elicited a significant elevation in serum antibodies to LTB, STa, and STb in mice and the mice anti-sera were able to neutralize the toxicity of STb but not STa. Mice receiving this DNA vaccine had a survival rate of 50% in the vaccination-challenge study which is significantly higher (P<0.05) than that in the control group.(2) The study of polyvalent enterotoxin protein vaccines:Coding genes for LTB, STa, and STb were obtained by PCR and fused to yield the polyvalent enterotoxin fusion gene LTB-STa-STb by traditional "restriction enzyme digestion-ligation" method. Two similar 7-amino-acid oligopeptides were included in the fusion to link the adjacent toxins. The other fusion enterotoxin gene of STa-LTB-STb was amplified from plasmid pCI-SLS. These two fusion genes were then cloned into vector pET-30a to produce 2 polyvalent enterotoxin prokaryotic expression plasmids. Fusion enterotoxin proteins, LSS and SLS, with the molecular weight of 21 kDa, were expressed in E. coli BL21 which was confirmed by SDS-PAGE and Western blot using anti-His tag antibodies. Fusion proteins were then purified by nickel affinity chromatography. Immunization of chickens or mice with 2 protein vaccines both elicited a high level of antibody responses to LTB, STa, and STb. The IgY or the mice anti-sera were able to neutralize the toxicity of STa and STb. Mice receiving the protein vaccines had a survival rate of 50-70% in the vaccination-challenge study, which was significantly higher (P<0.05) than that in the control group.In conclusion, the two recombinant trivalent enterotoxin proteins, LSS and SLS, were immunogenic for all the three enterotoxin components (i.e., LTB, STa, and STb) and were able to induce protective antitoxins in animals. Therefore, they could be included in a multivalent vaccine as a potent component to provide broad-spectrum protection against diarrhea caused by ETEC expressing various fimbriae. Though the immunogenicity of STa and STb was enhanced in the trivalent enterotoxin DNA vaccines constructed in this work, it was still weaker than that in the corresponding recombinant protein.