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

Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12

Thesis (Ph.D.)--University of Washington, 2015Sequence diversity, ability to evade immune detection and establishment of human immunodeficiency virus type 1 (HIV-1) latent reservoirs present a formidable challenge to the development of an HIV-1 vaccine. Structure based vaccine design stenciled on infection elicited broadly neutralizing antibodies (bNAbs) is a promising approach, in some measure to circumvent existing challenges. Understanding the antibody maturation process and importance of the high frequency mutations observed in anti-HIV-1 broadly neutralizing antibodies are imperative to the success of structure based vaccine immunogen design. Here we report a biochemical and structural characterization for the affinity maturation of infection elicited neutralizing antibodies IgG1 b12 (b12). We investigated the importance of affinity maturation and mutations accumulated therein in overall antibody function and their potential implications to vaccine development. Using a panel of point reversions, we examined relevance of individual amino acid mutations acquired during the affinity maturation process to deduce the role of somatic hypermutation in antibody function. Biophysical characterizations of b12 point mutant interactions with gp120 monomers from two Clade B viruses (SF162 and QH0692) indicates importance of cooperative contributions by individual mutations accumulated due to the extensive maturation processes in attaining the observed broadly neutralizing properties of b12. However, effects of individual mutations on epitope binding do not correlate with their effect on virus neutralization potency. Establishment of viral latent reservoirs, rendering antibody-based immune responses ineffective as preventive treatments, precedes serum detections of infection elicited highly potent bNAbs. Bearing the goal of structure based vaccine immunogen design that recapitulate b12-like immune response, we also investigated the minimum mutations on germline antibody sequences required to garner b12 comparable epitope binding affinity and virus neutralization potency. Our progressive increase in mutations on germline b12 precursor antibody approach reveled the importance of mutations on both antibody complementarity determining (CDR) and framework regions (FWR) in epitope binding and virus neutralization. Here we report extensive mutations and prolonged affinity maturation are vital to the development of highly potent bNAbs. One potential mechanism in attaining increased affinity maturation following the antibody maturation process is conformational antibody rearrangement and rigidification of antibody variable domains. To determine the role of extensive antibody mutations in the overall antibody structural flexibility and conformational rearrangements, we examined the structures of germline precursor b12 light chain variable domains. Accordingly, our structural analyses reveal absence of apparent effect on global structure of antibody light chain variable domains following affinity maturation. However, local structural affects are observed where germline precursor antibody CDRs exhibited conformational samplings distinct from b12 light chain CDRs. While germline precursor antibody CDRs sample different conformations, the process of antibody maturation focused the epitope binding region to specific conformation enabling high affinity epitope binding. These data suggest that structure based vaccine development that utilize b12-like bNAbs as templet need to develop a scheme that enables extensive affinity maturation observed in bNAbs. The bodies of work presented here, representing work on multiple aspects of bNAb development, potentially highlight key basic science research on HIV vaccinology that inform HIV vaccine development

Control of Tumor Initiation by NKG2D Naturally Expressed on Ovarian Cancer Cells

Cancer cells may co-opt the NKG2D lymphocyte receptor to complement the presence of its ligands for autonomous stimulation of oncogenic signaling. Previous studies raise the possibility that cancer cell NKG2D may induce high malignancy traits, but its full oncogenic impact is unknown. Using epithelial ovarian cancer as model setting, we show here that ex vivo NKG2D+ cancer cells have stem-like capacities, and provide formal in vivo evidence linking NKG2D stimulation with the development and maintenance of these functional states. NKG2D+ ovarian cancer cell populations harbor substantially greater capacities for self-renewing in vitro sphere formation and in vivo tumor initiation in immunodeficient (NOD scid gamma) mice than NKG2D− controls. Sphere formation and tumor initiation are impaired by NKG2D silencing or ligand blockade using antibodies or a newly designed pan ligand-masking NKG2D multimer. In further support of pathophysiological significance, a prospective study of 47 high-grade serous ovarian cancer cases revealed that the odds of disease recurrence were significantly greater and median progression-free survival rates higher among patients with above and below median NKG2D+ cancer cell frequencies, respectively. Collectively, our results define cancer cell NKG2D as an important regulator of tumor initiation in ovarian cancer and presumably other malignancies and thus challenge current efforts in immunotherapy aimed at enhancing NKG2D function

Prediction of an ensemble of 4E10 GEPs.

<p>Sequences of 4E10 V_L (<i>top line</i>) and V_H (<i>bottom two lines</i>) domains are shown, with CDRs indicated by a blue overscore. Predicted somatic mutations are colored red in the 4E10 sequence, and the corresponding unmutated GEP residues are shown below in grey (unchanged positions are not shown for clarity). All GEP V_L domains are comprised of the IGKV3-20*01/IGKJ1*01 gene segment combination. Each GEP V_H domain comprises the IGHV1-69*06 V gene segment plus one of six D gene segments (listed to the left of the corresponding GEP in the blue boxed field), and either the IGHJ4*02 (resulting sequence differences shown in purple and bolded) or IGHJ1*01 (resulting sequence difference shown in blue and bolded) gene segment, yielding an ensemble of 12 GEPs <i>in toto</i>. GEP shorthand numbering is shown in grey (GEPs 1 to 6) and blue (GEPs 7 to 12) beside the corresponding D plus J gene segment combination sequence differences from 4E10, occurring in HCDR3. <i>Inset</i> (<i>lower right</i>): HCDR3 sequences from candidate 4E10 GEPs, determined through deep sequencing of naïve B cell germline IgH genes from four uninfected individuals <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004403#ppat.1004403-Larimore1" target="_blank">[16]</a>, show the degree of variability seen in potential 4E10 precursors present in naïve repertoires. Each germline rearrangement uses the IGHV1-69 and IGJH1 or IGJH4 gene segments. Amino acids in red designate sequence differences between GEP and 4E10. The number of nucleotide changes needed to achieve these somatic mutations is 16 for donor 1, 17 for donor 2 and 18 for donors 3 and 4.</p

Peptidome binding results.

<p>(<b>A</b>) PhIP-Seq results are plotted as −Log₁₀<i>P</i>-values, one replicate on the abscissa, the other on the ordinate, colored by Ab as indicated; note the discontinuity in axis scales. The top scoring 4E10 peptide derived from IP₃R is highlighted with a red arrow; one peptide, derived from the zinc finger Ran-binding domain-containing protein 3, which bound to both GEP 2 and GEP 4, is highlighted with purple arrows. Proximity to the diagonal indicates good replicate concordance; peptides with highly discordant replicate values, falling along the axes, were discarded from the analysis. Overall library scoring statistics are: 4E10, average = 0.32, σ = 0.35; GEP 2, average = 0.22, σ = 0.44; GEP 4, average = 0.25, σ = 0.52; 1C6, average = 0.27, σ = 0.50. (<b>B</b>) The molecular surfaces of the Fv domains of 1C6 (4LCI.pdb), unbound 4E10 (4LLV.pdb) and unbound GEP 7 (4OB5.pdb) are shown, oriented with V_H domains at left and the V_L domains at right. The surface is colored to show hydrophobic patches, defined by the program HotPatch <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004403#ppat.1004403-Pettit1" target="_blank">[80]</a>; patches are colored in descending order of total area (red, orange, yellow, …). The total surface area for red and orange hydrophobic patches is shown. The crystal structure of GEP 7 is partially disordered in HCDR1 and 3 and so patch area is underrepresented in the calculation.</p

PhIP-Seq results for GEP 2 Fv.

<p>PhIP-Seq results for GEP 2 Fv.</p

SPR sensorgrams of the interactions between ES T117 and the indicated GEPs are shown.

<p>Binding data are shown in black with the kinetic fits to the data shown in red. Details of the experiments are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004403#ppat-1004403-t001" target="_blank">Table 1</a>.</p

Interdomain movements within Fv cassettes are limited.

<p>(<b>A</b>) Superpositions of the V_H domains from two 4E10 ligand-bound structures (2FX7.pdb, 3LH2.pdb), unbound 4E10 (4LLV.pdb), ligand-bound GEP 1 (4M8Q.pdb), unbound GEP 1 (4LRN.pdb), ligand-bound GEP 2 (4M62.pdb), ligand-bound GEP 7 (4ODX.pdb), and unbound GEP 7 (4OB5.pdb) are shown in Cα backbone representations, colored as indicated. Residue P14H in each Fv, chosen as a reference point to illustrate interdomain movement upon binding between 4E10 and GEPs, is shown as a sphere and colored to match the corresponding backbone. When isolated V_H domains are superimposed, the P14H spheres are nearly coincident, indicating that the V_H domain structure is highly conserved among these Abs. (<b>B</b>) Fv cassettes of 4E10 and GEPs, colored as in (<b>A</b>), superimposed only on V_L domains (oriented on the right side of the panel), are shown as represented in (<b>A</b>). In this view, interdomain movements can be visualized as the relative movement of V_H domains, particularly at the P14H reference point. (<b>C</b>) Orthogonal view of the P14H spheres excerpted from (<b>B</b>) illustrating the pattern and degree of interdomain movements.</p