Thesis (Ph.D.)--University of Washington, 2013Understanding cellular and humoral immune responses to HIV-1 infection and designed HIV immunogens are imperative to the development of an effective anti-HIV vaccine. In this thesis we investigate aspects of both the cellular innate and adaptive humoral arms of the immune system in response to HIV-1 infection and vaccination with designed epitope-scaffold immunogens. First, we review the anti-viral contributions from natural killer (NK) cells, focusing on NK cell recognition of MHC class I proteins through a diverse array of activating and inhibitory receptors. We also seek to answer the question of the ligand specificity of an activating NK cell receptor that is correlated with improved clinical outcomes in HIV-1 infection, KIR3DS1, through structural modeling and functional studies. We find that KIR3DS1 binds HLA -A, -B and -C alleles, but only in the absence of peptide, unusual for this class of receptor which shows fine specificity for allele type and preferences for bound peptide. Second, we explore the humoral arm of the anti-HIV immune response through characterization of the broadly neutralizing anti-HIV antibody 4E10. 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. The previously proposed candidate 4E10 autoantigen was the mitochondrial lipid cardiolipin. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies. Lastly, we characterized 4E10 ontogeny through functional and structural studies of an ensemble of 4E10 germline encoded precursors (GEPs). GEPs showed detectable, but extremely weak, binding to soluble Env gp140s and extremely limited neutralization potency, though some reverse engineered epitope-scaffolds showed robust GEP affinities, well above the B cell activation threshold. 4E10 and GEP paratopes displayed a remarkable degree of structural conservation in the antigen-bound state, with little improvement in overall shape complementarity. Frame work region mutations had little discernable affect on global or local structure. Surprisingly, 4E10 thermostability was significantly worse than its GEPs; while 4E10 and GEPs displayed similarly constrained VH/VL interdomain movements upon binding, 4E10 maturation involved negligible combining site rigidification, with both 4E10 and GEPs sampling extensive HCDR conformer ensembles. The narrowing of polyspecificity assumed to concur with maturation was not observed with 4E10, as both 4E10 and its GEPs showed similar patterns of limited polyspecificity to phage-displayed human peptidomes (PhIP-Seq). While 4E10 is demonstrably autoreactive, GEPs exhibited a distinct profile of autoantigen recognition by PhIP-Seq, suggesting that autoreactivity was acquired during ontogeny. In many respects, 4E10 provides a divergent example of Ab ontogeny, broadening the known range of affinity maturation pathways and challenging the generality of the existing paradigm. Retained combining site flexibility, and discrepancies in GEP binding of engineered versus Env-derived antigens, suggest that higher order mechanisms of neutralization are in play and that conventional vaccination protocols are unlikely to generate 4E10-equivalent Abs
