       Document 0151
 DOCN  M9630151
 TI    Characterization of an internal permissive site in the cholera toxin
       B-subunit and insertion of epitopes from human immunodeficiency virus-1,
       hepatitis B virus and enterotoxigenic Escherichia coli.
 DT    9603
 AU    Bckstrom M; Holmgren J; Schodel F; Lebens M; Department of Medical
       Microbiology and Immunology, Goteborg; University, Sweden.
 SO    Gene. 1995 Nov 20;165(2):163-71. Unique Identifier : AIDSLINE
       MED/96096516
 AB    We previously described the construction of novel hybrid proteins based
       on the B-subunit of cholera toxin (CTB) [Backstrom et al., Gene 149
       (1994) 211-217], in which a neutralizing B-cell epitope from the third
       variable (V3) loop in the envelope glycoprotein gp120 from human
       immunodeficiency virus type 1 (HIV-1) was inserted within a
       surface-exposed region between amino acids (aa) 55 and 64. The resulting
       protein retained properties of native CTB and could induce strong
       anti-CTB antibody (Ab) responses, but the inserted gp120 epitope was
       only modestly immunogenic. In this study, the potential use of this
       internal permissive site in CTB for the insertion of heterologous
       epitopes has been further investigated. Six additional plasmids were
       constructed encoding HIV::CTB hybrid proteins with ten to fourteen aa
       from the V3 loop of gp120 genetically inserted at different positions
       between aa 52 and 65, with deletions of different CTB aa. Plasmids
       encoding proteins with peptides inserted between aa 53 and 64 in CTB
       gave rise to stable proteins which reacted with CTB-specific monoclonal
       antibodies (mAb) and bound to GM1 gangliosides (GM1), indicating that
       insertions between these positions do not drastically alter the
       conformation or the receptor-binding properties of native CTB. Plasmids
       were also constructed encoding CTB hybrid proteins which had either an
       11-aa peptide from hepatitis B virus (HBV) pre-S(2) or one of two
       peptides related to the heat-stable toxin (STa) of enterotoxigenic
       Escherichia coli inserted between aa 55 and 64 of CTB. This resulted in
       the production of HBV::CTB or ST::CTB hybrid proteins and illustrated
       that the internal permissive site can be used for insertion of peptides
       of varying aa composition. The reactivity of the inserted epitopes with
       epitope-specific mAb in GM1-ELISA and immunoblots varied greatly between
       hybrid proteins and depended on the position in CTB and the aa
       composition of the inserted peptides. Despite these differences, all the
       HIV::CTB, ST::CTB and HBV::CTB hybrid proteins could induce low, but
       significant, levels of serum Ab in mice against gp120, STa or pre-S(2),
       in addition to strong serum Ab responses against CTB. The Ab response
       against the internally inserted gp120 peptide was similar to that
       against the same peptide fused to the N-terminus of CTB, indicating that
       internally placed peptides had similar immunogenicity to the same
       peptides added terminally.
 DE    Amino Acid Sequence  Animal  Antibodies, Bacterial/BIOSYNTHESIS
       Antibodies, Monoclonal  Antibodies, Viral/BIOSYNTHESIS  Bacterial
       Toxins/GENETICS/*IMMUNOLOGY  Cholera
       Toxin/GENETICS/*IMMUNOLOGY/METABOLISM  Enterotoxins/GENETICS/*IMMUNOLOGY
       Epitopes/ANALYSIS/GENETICS/*IMMUNOLOGY  Epitopes,
       B-Lymphocyte/IMMUNOLOGY  G(M1) Ganglioside/METABOLISM  Hepatitis B
       Surface Antigens/GENETICS/*IMMUNOLOGY  Human  HIV Envelope Protein
       gp120/*IMMUNOLOGY  *HIV-1  Mice  Mice, Inbred BALB C  Mice, Inbred C57BL
       Molecular Sequence Data  Oligopeptides  Peptide Fragments/*IMMUNOLOGY
       Protein Precursors/GENETICS/*IMMUNOLOGY  Recombinant Fusion
       Proteins/BIOSYNTHESIS/IMMUNOLOGY  Sequence Deletion  Support, Non-U.S.
       Gov't  Vibrio cholerae/GENETICS  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

