EPITOPE MAPPING OF HIV PROTEINS USING PROTEOLYTIC FOOT PRINTING AND MS

点击翻译按钮获取中文摘要

Summary 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 glyco-protein 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 have identified the core epitope on recombinant HIV p26 identified by the monoclonal antibody 13-102-100 as being residues 102-112. The cyclophilin A binding region is also contained within these residues. We have also determined the antigenic determinant of the envelope glycoprotein gp120 recognized by a polyclonal antibody raised against the C-terminus of the protein. Antibodies against the C- terminus often show protective effects in vitro. We are currently mapping an epitope on gp120 recognized by a MAb obtained from sera of an HIV infected individual characterized as a slow progressor, i.e., an individual who may be producing protective antibodies. We are also mapping a discontinuous epitope on HIV p24. This latter example utilizes the proteolytic footprinting results combined with the known molecular structure to identify amino acids involved in the epitope.