of Work: Mapping epitopes of the Human Immunodeficiency Virus (HIV) is important for the diagnosis of infection and for the development of vaccines and therapeutics for Acquired Immune Deficiency Syndrome (AIDS). We have been probing epitopes on the HIV proteins gp120 and p24. The initial step in the entry of HIV into the host cell is binding of the envelope glycoprotein gp120 to the cellular receptor CD4. Also gp120 elicits the major components of the protective immune response against HIV in humans and chimpanzees. gp120 and its synthetic peptides have been investigated as potential vaccine candidates. Similarly, HIV p24 elicits the first antibodies upon HIV infection. As the HIV infection progresses to AIDS, there is a simultaneous reduction in anti-p24 antibody titer. It has been proposed that a combination vaccine eliciting antibodies to both gp120 and p24 may be useful in combating HIV infection. Thus, knowledge of the antigenic determinants on p24 and gp120, especially those eliciting the formation of protective antibodies, is extremely important in the development of a vaccine. We have combined proteolytic footprinting and MALDI/MS to map epitopes on the native proteins recognized by antibodies. In this method, proteins affinity-bound to an immobilized antibody are proteolytically cleaved and the unbound fragments are removed by washing. The bound fragments containing the epitope are characterized by directly analyzing the immobilized antibody by MALDI/MS. We are currently mapping epitopes on gp120 and gp41 recognized by human MAbs isolated from sera of HIV infected individuals. The anti-HIV gp41 MAb, 2F5, is one of a limited number (three) of antibodies that have been found to be broadly neutralizing in vivo. Using traditional methods for defining epitopes, a short 6 amino acid sequence was determined to be the antigen. All attempts, however, to use this sequence to induce neutralizing antibodies have failed. We investigated the nature of the 2F5 epitope on recombinant disulfide stabilized glycoprotein gp140. The disulfide bond stabilizes the association of gp120 and gp41ectodomains as observed during viral infection. We determined that the functional epitope recognized by the neutralizing MAb is significantly longer in length, 15-16 residues.
| Parker, Carol E; Tomer, Kenneth B (2002) MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies. Mol Biotechnol 20:49-62 |
| Peter, J F; Tomer, K B (2001) A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking. Anal Chem 73:4012-9 |
| Parker, C E; Tomer, K B (2000) Epitope mapping by a combination of epitope excision and MALDI-MS. Methods Mol Biol 146:185-201 |
| Hochleitner, E O; Borchers, C; Parker, C et al. (2000) Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis. Protein Sci 9:487-96 |
| Hochleitner, E O; Gorny, M K; Zolla-Pazner, S et al. (2000) Mass spectrometric characterization of a discontinuous epitope of the HIV envelope protein HIV-gp120 recognized by the human monoclonal antibody 1331A. J Immunol 164:4156-61 |
