Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

The broadly-neutralizing anti-HIV antibody 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. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. 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, but is inconsistent with splicing factor 3B3. 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

Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies

Effects of HLA single chain trimer design on peptide presentation and stability

MHC class I “single-chain trimer” molecules, coupling MHC heavy chain, β2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation

Innate and humoral immune responses to HIV-1

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

Discovery of Novel DNA Gyrase Inhibitors by High-Throughput Virtual Screening▿

The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains

HLA-F and MHC-I Open Conformers Bind Natural Killer Cell Ig-Like Receptor KIR3DS1 - Fig 3

<p>Surface staining with KIR3DS1-His and HLA-F (A) PBMC from three individuals were stained with NK markers (CD3-, CD56+) and with mAb Z27 and DX9 to detect KIR3DL1 (DX9+, Z27+) and KIR3DS1 (DX9-, Z27+), and stained with HLA-F tetramer as indicated. Three individuals were chosen based on their KIR haplotypes, containing homozygous KIR3DL1, heterozygous KIR3DL1, KIR3DS1, and homozygous KIR3DS1 as indicated. (B) Cells described in A were activated and subjected to the same staining protocols 11 days post activation. (C) Isolated T cells from two individuals with the indicated KIR3DS1L1 genotypes were stained with mAb 3D11 and with KIR3DS1-His as marked beneath each pair of before (<i>left</i>) and after (<i>right</i>) activation profiles.</p

Western analysis using the indicated mAbs for detection of gel fractionated protein after pull-down with KIR3DS1-D0-D2stem-His (<i>above</i>).

<p>Four cell lines were incubated with KIR3DS1-His and pull-downs performed followed by Western blot analysis with HCA2 and 3D11. Heterodimerization of KIR-His and HLA-F without tag followed by Ni column purification (<i>below</i>). After gel fractionation, gels were stained with Coomassie blue and proteins visualized. Heterodimerization assays were carried out with KIR alone or with KIR + HLA-F as indicated above each lane. Recombinant HLA-F is included alone for comparison. MW markers are indicated for both gels.</p

Functional measurement of HLA-F and KIR3DS1 interaction.

<p>PBMC from three donors were activated and stimulated with refolded HLA-F, HLA-control tetramers, or no protein as described in <i>Materials and Methods</i>. (<b>A)</b> A representative FACS staining for the analysis of CD107a expression on cell populations gated in the top panel indicated as KIR3DS1+ and KIR3DS1-. Expression of CD107a was detected with percentages of the total cell population analyzed in bold in the lower right quadrant of each profile. Experiments were performed in the presence of HLA-F and D^b-TRP bound to streptavidin beads and no-protein as indicated above the panels. The percentages CD107a positive cells within the KIR3DS1+ populations (<b>B</b>) and in the KIR3DS1- population (<b>C</b>) for each donor are graphed for comparison. <b>(B and C)</b> Five experiments were performed for each of four KIR3DS1+ donors (Donors CW and EB: KIR3DL1/KIR3DS1, Donors TG and DG KIR3DS1/KIR3DS1). The graphs show the percentage of total cells using the formula showing the mean of 5 replicates for each individual, the error bars represent the standard error of the mean (SEM) of the replicates, and the p-values calculated by performing a paired t-test comparing all values F and D^b across replicates and individuals.</p

Steric clashes with presented peptides introduced by KIR substitutions.

<p>(<i>Left</i>) View of the KIR3DL1/HLA-B*5701 interface (PDB accession code 3VH8; <a href="http://www.rcsb.org/" target="_blank">www.rcsb.org</a>). (<i>Right</i>) Model of KIR3DS1 in complex with HLA-B*5701, shown as at left. The steric clash between Arg166 with p8 is highlighted in red. One rotomer of Arg166 is depicted; however, all accessible Arg166 rotomers sterically clash with residue p8 of the bound peptide. HLA-B*5701 is shown in cartoon representation, colored teal; the bound peptide (LSSPVTKSF) is shown as a ribbon with side-chains in licorice stick representation, colored yellow. KIR3D molecules are shown in semi-transparent surface representation, colored grey. Residue 166 from the KIR3D molecules is shown in licorice stick representation, colored grey (KIR3DL1) or grey and red (KIR3DS1).</p

Analysis of the effects of salt concentration, mutation and overall cassette charge on 4E10/liposome interactions.

<p>Corrected SPR responses are shown for Annexin V or 4E10 IgG analytes (300 nM; duplicate runs) to liposomes incorporating biotinylated lipids captured on streptavidin-coated biosensor chips; liposome compositions are indicated above each frame. (<b>A</b>) SPR responses of wild-type 4E10 IgG analytes at different salt concentrations are plotted. (<b>B</b>) SPR responses of Annexin V (300 nM), wild-type 4E10 IgG (300 nM) or the 4E10 [G(L50)E] mutant IgG (400 nM) are plotted. A higher concentration of 4E10 mutant IgG was used with the expectation that binding might be significantly reduced. Since this did not occur, mutant IgG responses appear elevated due to the concentration differential. (<b>C</b>) The net charge at neutral pH of the Fv cassettes of the anti-HIV Abs with structures currently available through the PDB <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Berman1" target="_blank">[103]</a> was calculated with the structure-based algorithm PDB2PQ <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Dolinsky1" target="_blank">[51]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Unni1" target="_blank">[54]</a>. Two Abs, PG16 and NIH45-46, were excluded because their structures included modified amino acids that could not be accommodated by PDB2PQ. Fvs are plotted with their names, with assigned PDB accession codes in parentheses. Ab labels are colored by the locale of their epitopes on Env, as indicated; 4E10 is also bolded.</p