1416 AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 23, Number 11, 2007, pp. 1416–1427 © Mary Ann Liebert, Inc. DOI: 10.1089/aid.2007.0027 Peptides Selected from a Phage Display Library with an HIV-Neutralizing Antibody Elicit Antibodies to HIV gp120 in Rabbits, But Not to the Same Epitope ROYCE A. WILKINSON, 1 JODY R. EVANS, 1 JON M. JACOBS, 1, * DUSTIN SLUNAKER, 1 SETH H. PINCUS, 2 ABRAHAM PINTER, 3 CHARLES A. PARKOS, 4 JAMES B. BURRITT, 5 and MARTIN TEINTZE 1 ABSTRACT Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide–protein fusions were ex- pressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal an- tibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically in- hibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide–protein fusions produced an- tisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120. INTRODUCTION T HE HIGH MUTATION RATE OF HIV, which results in the pres- ence of hypervariable loops in the envelope protein gp120, is an obstacle to the development of an effective HIV vaccine. A successful vaccine needs to elicit a neutralizing antibody re- sponse to some of the more conserved regions of gp120, such as the CD4 binding site. Recombinant gp120 has been ineffec- tive in HIV vaccines, and has not elicited broadly neutralizing antibodies targeting conserved epitopes, because other sites are immunodominant and/or epitopes like the CD4 binding site are masked. 1–4 We attempted to find conformational mimics of the CD4 binding site region of gp120 by selecting phage that bind to antibodies, such as 5145A, that are directed at the CD4 bind- ing site. This could lead to peptide or protein constructs capa- ble of inducing the production of such broadly neutralizing an- tibodies as part of a vaccine. 5145A is a human monoclonal antibody (MAb) directed against the CD4 binding site of HIV-1 gp120. It was originally isolated from an asymptomatic seropositive hemophiliac by Ep- stein–Barr virus transformation. 5 5145A binds to gp120 from a variety of isolates including MN, IIIB, SF-2 (North America), RF, and AL (Haiti) and 9 of 10 isolates from the Central African Republic representing African subtypes A and D and the North- ern Thailand subtype E. It has potent neutralizing activity against the North American and Haitian isolates tested (MN, IIIB, SF-2, RF), but is much less potent against the African iso- lates. 5 Mutations in gp120 that abrogate 5145A binding 5 over- lap those for other CD4 binding site antibodies, such as b12, 6 F105, 7,8 and 1125H. 9 Phage display of random peptides has become an important method for the study of molecular interactions in many areas of protein science, including identification of epitopes or mimotopes of antibodies, identifying peptide agonists or antagonists of en- zymes or cellular receptors, and finding peptides capable of tar- geting to specific cellular or tissue types. 10–13 In some cases, phage display has been used to map discontinuous epitopes, in- cluding that of the CD4 binding site antibody b12. 14–17 1 Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717. 2 The Research Institute for Children, Children’s Hospital, LSU Health Sciences Center, New Orleans, Louisiana 70118. 3 Laboratory of Retroviral Biology, Public Health Research Institute, Newark, New Jersey 071031. 4 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30022. 5 Department of Microbiology, Montana State University, Bozeman, Montana 59717. *Current address: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington.