(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) Major Findings: - HIV-1 entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Sub-optimal temperature (31.5oC) was used to prolong fusion intermediates as monitored by confocal-microscopy. After transfer to 37oC, these fusion intermediates progressed to syncytia formation with enhanced kinetics compared with effector/target (E/T) cell mixtures that were incubated only at 37oC. gp41 peptides, DP-178 , DP-107, and IQN17, blocked fusion more efficiently (5-10 fold lower ID50 values) when added to E/T cells at the suboptimal temperature prior to transfer to 37oC. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37oC only if preincubated with E/T cells at the suboptimal temperature. Similar fusion-inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site. - New immunogens mimiccking the gp41 fusion intermediates were constructed and several rabbits were immunized. The new rabbit sera inhibit HIV-1 fusion and infection of PBMC by several primary isolates when added to virus-cell mixtures at 37oC.
