Characterization of V3 loop-Pseudomonas exotoxin chimeras. Candidate vaccines for human immunodeficiency virus-1.
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.
FitzGerald
D J
DJ
Biotherapy Section, Laboratory of Molecular Biology, Division of Basic Science, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Fryling
C M
CM
McKee
M L
ML
Vennari
J C
JC
Wrin
T
T
Cromwell
M E
ME
Daugherty
A L
AL
Mrsny
R J
RJ
D036002:ADP Ribose Transferases; D016915:AIDS Vaccines; D000163:Acquired Immunodeficiency Syndrome / Q000097:blood / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D002294:Carcinoma, Squamous Cell; D002470:Cell Survival / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005098:Exotoxins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D015483:HIV Antibodies / Q000097:blood / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D015497:HIV-1 / Q000187:drug effects* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D014407:Tumor Cells, Cultured; D014614:Vaccines, Synthetic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D016915:AIDS Vaccines; D001427:Bacterial Toxins; D005098:Exotoxins; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A