In HIV-1 infection, endogenous antibodies fail to cause complete complement-mediated neutralization of all of the released HIV-1 particles, not only because of the continual mutation of immunogenic envelope epitopes, but also due to the spatial distribution of those epitopes. These spatial distributions limit the ability of adjacent lgG1 antibodies to deposit in a manner that allows the Fab regions to maximally expose the Fc region and obtain a high affinity interaction with C1q globular heads. The result is a C1 function that is quickly restricted by C1-inhibitor and only leads to very limited deposition of C4b, C4b2a and C3b on the HIV-1 envelope glycoprotein. Furthermore the HIV-1 envelope binds Factor H, promoting a rapid breakdown of C3b to iC3b then to C3d. The C3d-coated HIV-1 binds to C3d receptors on B cells (e.g., in lymph nodes). These HIV-1 reservoirs persist in HAART treated patients and remain as a serious source of infection. The first objective of this R21 application is to genetically engineer pairs of truncated bi-specific antibodies, tBiAbs that will unrelentingly fasten C1 (and the classical complement pathway) onto HIV-1 in such a way as to overcome any restrictive mechanism or any endogenous complement inhibitory factors. Our hypothesis is that if the classical pathway and the C3b-amplification loop were allowed to proceed without interruption (by C1-inhibitor or by Factor H) on the HIV-1 envelope glycoproteins, the rapidly spreading covalent C4b, C4b2a, CSbBb and C3b depositions will overrun the protection provided by the membrane complement inhibitors on B Cells, completely neutralize (completely cover and coat) the HIV-1 envelope of all infectious HIV-1 particles and substitute for the requirement to specifically target any one particular (exposed or cryptic) site on the envelope that is needed to interact with CD4. After engineering sets of the tBiAbs, our second objective is to use transfected human cell lines expressing gp160 as well as immobilized native soluble oligomeric (trimeric) gp140 to determine the degree of enhancement of the Classical Complement Pathway and of the C3b-amplification loop by the tBiAbs, via the achieved over- deposition of complement on the surface of native soluble oligomeric (trimeric) gp140 and transfected HeLa cells expressing gp160. Our third objective is to evaluate the complement-mediated neutralization of infectious HIV-1 particles by paired combinations of the truncated bi-specific antibodies. ? ? ?
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