title: Design of an Escherichia coli expressed HIV-1 gp120 fragment immunogen that binds to b12 and induces broad and potent neutralizing antibodies.
abstract: b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals.
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors: Sanchari
authors: S
authors: Bhattacharyya
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors: Pranveer
authors: P
authors: Singh
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors: Ujjwal
authors: U
authors: Rathore
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors: Mansi
authors: M
authors: Purwar
authors: AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative, New York, New York 11220.
authors: Denise
authors: D
authors: Wagner
authors: AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative, New York, New York 11220.
authors: Heather
authors: H
authors: Arendt
authors: AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative, New York, New York 11220.
authors: Joanne
authors: J
authors: DeStefano
authors: Department of Surgery, Duke University, Durham, North Carolina 27705.
authors: Celia C
authors: CC
authors: LaBranche
authors: Department of Surgery, Duke University, Durham, North Carolina 27705.
authors: David C
authors: DC
authors: Montefiori
authors: AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative, New York, New York 11220. Electronic address: sanjay.phogat@sanofipasteur.com.
authors: Sanjay
authors: S
authors: Phogat
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, India; AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative, New York, New York 11220. Electronic address: varadar@mbu.iisc.ernet.in.
authors: Raghavan
authors: R
authors: Varadarajan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D001703:Biophysics / Q000379:methods; D015704:CD4 Antigens / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D011817:Rabbits; D014612:Vaccines / Q000737:chemistry
chemical_list: D057134:Antibodies, Neutralizing; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D011506:Proteins; D014612:Vaccines