FitzGerald D J, Fryling C M, McKee M L, Vennari J C, Wrin T, Cromwell M E, Daugherty A L, Mrsny R J
Biotherapy Section, Laboratory of Molecular Biology, Division of Basic Science, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
J Biol Chem. 1998 Apr 17;273(16):9951-8. doi: 10.1074/jbc.273.16.9951.
To develop a candidate vaccine for human immunodeficiency virus, type 1 (HIV-1), chimeric proteins were constructed by inserting sequences derived from the V3 loop of gp120 into a nontoxic form of Pseudomonas exotoxin (PE). Inserts of 14 or 26 amino acids, constrained by a disulfide bond, were introduced between domains II and III of PE. V3 loop-toxin proteins expressed in Escherichia coli and corresponding to either MN (subtype B) or Thai (subtype E) strains, were recognized by strain-specific monoclonal anti-gp120 antibodies. When loop sequences were introduced into an enzymatically active form of the toxin, there was no loss of toxin-mediated cell killing, suggesting that these sequences were co-transported to the cytosol. Sera from rabbits injected with nontoxic PE-V3 loop chimeras were reactive for strain-specific gp120s in Western blots, immunocapture assays, enzyme-linked immunosorbent assays, and neutralized HIV-1 infectivity. Since toxin vectors were designed to receive oligonucleotide duplexes encoding any V3 loop sequence, this approach should allow for the production of V3 loop-toxin chimeras corresponding to multiple HIV isolates.
