(1) Goals of project: - To study the activities of monoclonal antibodies (mAbs) or polyclonal antibodies raised against HIV-1 envelope (gp120), CD4, gp120/CD4 complexes, gp41-fusion intermediates, or the HIV-1 co-receptors, on HIV-1 fusion/infection of primary human cells. - To examine the effects of such mAbs on the normal biological functions of primary human cells. - To compare the HIV neutralization capacity of different HIVIG subclasses (2) Experimental approach: - We have tested a panel of rabbit polyclonal antibodies generated against the the prptides representing the fusion-intermediate transitinal structures in HIV-1 envelope (gp41). Kinetics experiments and suboptimal temperature (31.5 oC) were used to slow down the fusion process and evaluate the viral neutralization and fusion inhibition. - HIVIG was fractionated into IGG1, IGG2, and IGG3 subclasses using preparative Protein A column. The purified subclasses were tested in a variety of binding assays ans HIV fusion inhibition and neutralization assays. (3) Major Findings: - Using HIVIG preparations that were used in human trials, we demonstrated that the IGG3 subclass (which reperesents only 10% of the total IG in the original HIVIG preparation) is superior to other subclasses in its abilty to block HIV-1 mediated fusion and to neutralize multiple cell-free HIV-1 strains, including primary isolates. This superior biological activity may be explained by the unusually long """"""""Hinge"""""""" region, unique to IGG3 molecules. -Rabbit antibodies against peptides modeling the N-heptad repeat (HR) and six-helix bundle of gp41 blocked fusion (and viral infection) at 37oC, only if preincubated with Env-expressing cells and target cells at the suboptimal temperature for 1-2 hr. - Our data demonstrate that antibodies targeting gp41 fusion-intermediates are able to bind to gp41 and to arrest HIV-1 fusion. They also support future investigations of the prehairpin and six-helix bundle structures as targets for rational design of novel HIV-1 inhibitors and vaccines.
