Insertion of a HIV-1-neutralizing epitope in a surface-exposed internal region of the cholera toxin B-subunit.
The non-toxic B-subunit of cholera toxin (CTB) is a powerful immunogen and has been investigated as a carrier for foreign peptide epitopes, with peptides genetically fused to either the N- or C terminus of CTB. In the present study, we have constructed a plasmid encoding a novel intrachain CTB fusion protein with a peptide epitope inserted into an internal region of CTB: eight amino acids (aa) in CTB (56-63) were substituted with a 10-aa peptide from the third variable (V3) loop of the HIV-1 envelope protein gp120. The resulting chimeric protein retained important functional characteristics of the native CTB including pentamerization and GM1 ganglioside receptor binding. The internal hybrid protein was also shown to be resistant to proteolytic degradation during production in Vibrio cholerae, whereas a terminal hybrid protein, where the same gp120-epitope was fused to the N terminus of CTB, was rapidly cleaved during culture. The inserted epitope, which is known to give rise to HIV-1 neutralizing Ab, could be detected with a V3 loop-specific monoclonal Ab when the chimeric protein was analyzed in ELISA and immunoblot, indicating that the epitope inserted at this site is presented on the surface of the protein. Consistent with these observations, immunization of mice with the CTB::HIV hybrid protein elicited a high titered serum Ab response to the CTB moiety and also, in some but not all animals, a detectable response to the inserted gp120 epitope.
Bäckström
M
M
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
Lebens
M
M
Schödel
F
F
Holmgren
J
J
D016915:AIDS Vaccines / Q000096:biosynthesis* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010957:Plasmids; D055503:Protein Multimerization; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D015183:Restriction Mapping; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000276:immunology
D016915:AIDS Vaccines; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D002772:Cholera Toxin