Antibodies play a crucial role in humoral immunity. Since physiologically, they are either expressed on cell surface or secreted into body fluid, they work exclusively extracellularly. Therefore, they play a limited role in defense against many intracellular pathogens. Recently, several anchor domains responsible for intracellular localization of a given protein have been identified. As a result, it is becoming possible to target genetically engineered molecules to specific intracellular compartments. The purpose of this project is to determine whether antibody genes can be directed and expressed in appropriate intracellular compartments and, if so, whether they will effect physiologic and pathologic processes. The model system being developed involves the expression of anti-HIV-1 antibody genes in human CD4+ T cell lines. During the past year, we have progressed in several areas of this project. 1) We have isolated and sequenced the full-length cDNA's containing heavy and light chain antibody genes specific for HIV-1 protease from a hamster B hybridoma. Single-chain Fv (scFv) and scFv/Ckappa gene constructs specific for HIV-1 protease have been made. 2) We have expressed the scFv specific for HIV-1 gp41 in bacteria and found that the scFv can specifically bind to antigen(s). 3) The anti-HIV- 1 gp41 scFv's with or without ER and TGN anchor domains have been expressed in COS and human Jurkat T cell lines. The results showed that without the ER- or TGN-anchor the scFv's are secreted from cells and with ER- or TGN-anchor the scFv's are captured inside cells. The kinetic study indicates that the half life of the scFv-ER is over 24 hours and the half life of the scFv-TGN is about 9 hours. We are now testing the effect of these scFv's on the infectivity of HIV-1. 4) We have generated transgenic mice containing scFv-ER and scFv-TGN specific for HIV-1 gp41. We will mate them to our HIV-1 transgenics to test the in vivo effect of the intracellular scFv.
