HIV Vaccine Design
Broadly neutralizing monoclonal antibodies to HIV-1 are rare and are valuable tools for designing HIV vaccines. Human antibodies 4E10 and Z13 neutralize HIV-1 with potent and broad neutralizing activity, binding to conserved and overlapping epitopes on the membrane-proximal region of the viral envelope protein gp41. These gp41 epitopes are being used as templates for structure-based vaccine design. Recently, we grafted the residues of the membrane-proximal region into the V1/V2 loop region of HIV-1 gp120 to investigate the ability of the engineered antigens to elicit HIV neutralizing antibodies. To promote the correct folding and presentation of the helical 4E10 epitope, we flanked the epitope with helical domains and manipulated the helix by sequential deletion of residues preceding the epitope. Binding of the recombinant proteins to 4E10 increased gradually with the deletion of 1 and 2 residues but returned to the wild-type level when 3 residues were deleted, suggesting the possible rotation of the 4E10 epitope along the helix as anticipated.
Mice and rabbits were immunized with these constructs by using a DNA primeprotein boost approach. Although high levels of gp120-specific antibodies were elicited, no neutralizing activity was found for the membrane-proximal region or V1, V2, or V3 regions. However, we did learn that the 4E10 epitope can be manipulated by using a turn-the-helix strategy. Yet, presentation of this epitope in the immunogenic V1/V2 region did not make the epitope immunogenic in mice and rabbits. We also found that DNA vaccination with monomeric gp120-based antigens can elicit a consistent neutralizing antibody response against the homologous resistant virus by targeting epitopes outside the V1, V2, and V3 regions. These results provide support for the further exploration of a vaccination strategy in which monomeric gp120-based antigens and a DNA primeprotein boost approach are used.
Whereas the 4E10-epitope interaction has been characterized in atomic detail, less is known about Z13 and its epitope on gp41. A Z13 variant, Z13e1, with apparent affinity for gp41 that is approximately 100-fold greater than that of wild-type Z13, was identified by in vitro affinity maturation techniques with phage display of the antibody. To provide insights into the interaction between Z13e1 and its epitope, we expressed recombinant IgG Z13e1 in CHO cells, digested the IgG to Fab′ with pepsin, and extensively purified the Fab by using affinity, size exclusion, and ionic exchange chromatography. Crystallization trials of Fab and IgG Z13e1 in the free form and in complex with high-affinity peptide epitopes are in progress.
Another highly immunogenic region of the HIV-1 gp120 envelope protein is the V3 loop. Although most antibodies that target this highly variable loop are specific for only the immunizing viral strain, a few antibodies have a wider range of neutralization. We recently determined the structure for 1 of these antibodies, 2219, in complex with 3 different V3 peptides and showed that 2219 binds to a conserved hydrophobic face of V3, leaving the tip of the loop largely free from contact with Fab (Fig. 2). This unusual mode of binding allows recognition of V3 regions with unusual sequences at the tip of the loop. These HIV-1 studies are collaborations with D.R. Burton, Scripps Research; H. Katinger, University of Agriculture, Vienna, Austria; and S. Zolla-Pazner, New York University School of Medicine, New York, New York.
The Innate Immune Response Against Microbial Pathogens
CD1 molecules belong to a family of nonclassical MHC class I antigen receptors that present lipid, glycolipid, or lipopeptide ligands to specific T cells for immune recognition. Last year, we reported the structure of mouse CD1d in complex with α-galactosyl ceramide, which is the most stimulatory natural ligand known to date. To deepen our understanding of the CD1-ligand interaction, we have now determined the crystal structure of mouse CD1d in complex with α-galacturonosyl ceramide. The structure showed that the removal of a single hydroxyl group changes considerably the CD1d-ligand interaction. On the basis of this knowledge, we were able to design and synthesize new agonists with enhanced cell-based stimulatory activities compared with α-galactosyl ceramide. Work on how CD1 molecules are being recognized by T-cell receptors is also under way in collaboration with M.B. Brenner and D.B. Moody, Harvard Medical School, Boston, Massachusetts; L. Teyton and C.-H Wong, the Skaggs Institute; M. Kronenberg, La Jolla Institute of Allergy and Immunology, San Diego, California; V. Kumar, Torrey Pines Institute for Molecular Studies, San Diego, California; and W. Severn and G. Painter, Industrial Research Limited, Lower Hut, New Zealand.
Toll-like receptors (TLRs) are glycoproteins essential for immune recognition of microbial pathogens. Although some advances have been made in elucidating the TLR molecular pathways, little is known about the way TLRs recognize their ligands. The TLR extracellular domains thought to be involved in ligand recognition consist of an extensive leucine-rich repeat domain that begins with an N-terminal cap and ends with a C-terminal cap. TLR2 recognizes the widest range of ligands among TLRs and is unique because it needs other coreceptors, such as TLR6 and TLR1, to recognize ligand. We are working to determine the crystal structure of TLR2 and its coreceptors, TLR6, TLR1, and CD36, all in complex with their ligands.
Recently, cytoplasmic pattern-recognition receptors such as RIG-I, Nod1, and Nod2 were found to be involved in pathogen recognition. Nod1 and Nod2 recognize a component from the bacterial cell wall called peptidoglycan. Mutations in Nod2 have been associated with Crohns disease, and Nod1 can inhibit estrogen-dependent tumor growth. Nod proteins have 3 structural domains: a leucine-rich repeat domain, a nucleotide oligomerization domain, and a caspase recruitment domain responsible for signal transduction. As is the situation with the TLRs, the leucine-rich repeat domain is thought to be involved in ligand recognition. Different constructs of these proteins are being prepared for structural studies. These projects are being done in collaboration with R. Ulevitch and B. Beutler, Scripps Research.
Binley, J.M., Ngo-Abdalla, S., Moore, P., Bobardt, M., Chatterji, U., Gallay, P., Burton, D.R., Wilson, I.A., Elder, J.H., de Parseval, A. Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120. Retrovirology 3:39, 2006.
Brunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R., Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006.
Burton, D.R., Stanfield, R.L., Wilson, I.A. Antibody vs HIV in a clash of evolutionary titans. Proc. Natl. Acad. Sci. U. S. A. 102:14943, 2005.
Calarese, D.A., Lee, H.K., Huang, C.Y., Best, M.D., Astronomo, R.D., Stanfield, R.L., Katinger, H., Burton, D.R., Wong, C.-H., Wilson, I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. Natl. Acad. Sci. U. S. A. 102:13372, 2005.
Cheng, T.Y., Relloso, M., Van Rhijn, I., Young, D.C., Besra, G.S., Briken, V., Zajonc, D.M., Wilson, I.A., Porcelli, S., Moody, D.B. Role of lipid trimming and CD1 groove size in cellular antigen presentation. EMBO J. 25:2989, 2006.
DeMartino, J.K., Hwang, I., Xu, L., Wilson, I.A., Boger, D.L. Discovery of a potent, nonpolyglutamatable inhibitor of glycinamide ribonucleotide transformylase. J. Med. Chem. 49:2998, 2006.
Glaser, L., Stevens, J., Zamarin, D., Wilson, I.A., Garcia-Sastre, A., Tumpey, T.M., Basler, C.F., Taubenberger, J.K., Palese, P. A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity. J. Virol. 79:11533, 2005.
Huang, C.C., Tang, M., Zhang, M.Y., Majeed, S., Montabana, E., Stanfield, R.L., Dimitrov, D.S., Korber, B., Sodroski, J., Wilson, I.A., Wyatt, R., Kwong, P.D. Structure of a V3-containing HIV-1 gp120 core. Science 310:1025, 2005.
Jiang, Z., Georgel, P., Li, C., Choe, J., Crozat, K., Rutschmann, S., Du, X., Bigby, T., Mudd, S., Sovath, S., Wilson, I.A., Olson, A., Beutler, B. Details of Toll-like receptor:adapter interaction revealed by germ-line mutagenesis. Proc. Natl. Acad. Sci. U. S. A. 103:10961, 2006.
Kinjo, Y., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M.R., Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F., Khurana, A., Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A., Tsuji, M., Sellati, T.J., Wong, C.-H., Kronenberg, M. Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria. Nat. Immunol. 7:978, 2006.
Luz, J.G., Yu, M., Su, Y., Wu, Z., Zhou, Z., Sun, R., Wilson, I.A. Crystal structure of viral macrophage inflammatory protein I encoded by Kaposis sarcoma-associated herpesvirus at 1.7 Å. J. Mol. Biol. 352:1019, 2005.
Peti, W., Page, R., Moy, K., ONeil-Johnson, M., Wilson, I.A., Stevens, R.C., Wüthrich, K. Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.
Rudolph, M.G., Stanfield, R.L., Wilson, I.A. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24:419, 2006.
Shore, D.A., Teyton, L., Dwek, R.A., Rudd, P.M., Wilson, I.A. Crystal structure of the TCR co-receptor CD8αα in complex with monoclonal antibody YTS 105.18 Fab fragment at 2.88 Å resolution. J. Mol. Biol. 358:347, 2006.
Stanfield, R.L., Gorny, M.K., Zolla-Pazner, S., Wilson, I.A. Crystal structures of human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 2219 in complex with three different V3 peptides reveal a new binding mode for HIV-1 cross-reactivity. J. Virol. 80:6093, 2006.
Stanfield, R.L., Zemla, A., Wilson, I.A., Rupp, B. Antibody elbow angles are influenced by their light chain class. J. Mol. Biol. 357:1566, 2006.
Stauber, D.J., Debler, E.W., Horton, P.A., Smith, K.A., Wilson, I.A. Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor. Proc. Natl. Acad. Sci. U. S. A. 103:2788, 2006.
Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C., Wilson, I.A. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. 355:1143, 2006.
Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson, I.A. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312:404, 2006.
Wu, D., Zajonc, D.M., Fujio, M., Sullivan, B.A., Kinjo, Y., Kronenberg, M., Wilson, I.A., Wong, C.-H. Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d. Proc. Natl. Acad. Sci. U. S. A. 103:3972, 2006.
Zajonc, D.M., Maricic, I., Wu, D., Halder, R., Roy, K., Wong, C.-H., Kumar, V., Wilson, I.A. Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity. J. Exp. Med. 202:1517, 2005.
Zhu, X., Dickerson, T.J., Rogers, C.J., Kaufmann, G.F., Mee, J.M., McKenzie, K.M., Janda, K.D., Wilson, I.A. Complete reaction cycle of a cocaine catalytic antibody at atomic resolution. Structure 14:205, 2006.
Zhu, X., Wentworth, P., Jr., Kyle, R.A., Lerner, R.A., Wilson, I.A. Cofactor-containing antibodies: crystal structure of the original yellow antibody. Proc. Natl. Acad. Sci. U. S. A. 103:3581, 2006.