Scientific Report 2005

Immunology

Human Antibodies and Design of a Vaccine to HIV Type 1

M.B. Zwick, R.A. Pantophlet, R.O. Aguilar-Sino, R. Astronomo, D. Bowley, H. Donners, A. Gakhal, A.J. Hessell, R.C. Jensen, M. Law, J.D. Nelson, E. Scherer, S. Selvarajah, M. Wang, R.A. Dwek,* P.M. Rudd,* D. Calarese, R.L. Stanfield, I.A. Wilson, D.R. Burton

* Oxford Glycobiology Institute, Oxford, England

HIV type 1 (HIV-1) is a scourge on humanity, Nearly 42 million persons are infected with the virus, and 22 million persons have died of AIDS. It is widely recognized that a vaccine most likely is the best way to control HIV-1 infection worldwide. We wish to understand antibody responses to HIV-1 in humans and to design vaccines that will elicit protective responses to the virus.

We used phage display technology to generate panels of human monoclonal antibodies to HIV-1. We are examining human antibody responses to the virus and the antiviral activities of these antibodies. In particular, we generated a human monoclonal antibody, b12, that potently neutralizes a broad array of different viruses. The existence of this antibody indicates that some features of HIV-1 are conserved and are attractive targets for vaccines. Further, b12 and a few monoclonal antibodies with similar qualities are powerful tools for exploring antibody activity against HIV-1.

Among the first questions we tackled were the following: Can antibodies protect against HIV-1 infection, and, if so, under what conditions? On the basis of passive transfer studies in a number of animal model systems, the answer is clearly yes. Complete protection from infection is possible at serum titers of neutralizing antibody greater than about 1:100, although lower titers can provide benefit in terms of lowered or delayed viremia. Further, we showed that topically applied antibody can protect monkeys against vaginal challenge with HIV.

Another major issue is the best method for eliciting protective neutralizing antibodies. Accumulated evidence suggests that protective neutralizing antibodies are those antibodies that bind avidly to the envelope trimer on the surface of HIV-1 virions. However, such antibodies, particularly those to conserved regions of the envelope that are most important for vaccines, are difficult to elicit. Apparently the envelope trimer, which is composed of 2 glycoproteins, gp120 and gp41, has low antigenicity and immunogenicity. Several strategies to circumvent these problems are being investigated. One strategy is to study the interaction of the neutralizing antibodies with envelope glycoprotein at the molecular level and then use the knowledge gained to design antigens capable of eliciting the relevant antibodies. In these studies, we are collaborating with I.A. Wilson, Department of Molecular Biology.Currently, we are studying the epitopes recognized by 5 broadly neutralizing antibodies: b12, 2G12, 4E10, Z13, and 2F5. The antibody b12 recognizes a complex epitope that overlaps the CD4-binding site of gp120. We are using a hyperglycosylation-mutagenesis strategy to “immunofocus” responses to this epitope by blocking nonneutralizing epitopes on gp120. The strategy is based on our previous determination of the structure of b12, docking of b12 and gp120, and extensive mutagenesis studies of b12 and gp120.

The antibody 2G12 recognizes a cluster of sugar residues on gp120. Solution of the structure of 2G12 in complex with sugars enabled us, in collaboration with C.-H. Wong, Department of Chemistry, to design synthetic sugars that are being used as immunogens to elicit 2G12-like antibodies. The antibody 4E10 recognizes a linear epitope on gp41 (Fig. 1). We determined the structure of 4E10 with peptide bound, and we are working with P. Dawson, Department of Cell Biology, to design peptide immunogens that can elicit 4E10-like antibodies. The antibody Z13 recognizes an epitope on gp41 that overlaps with that of 4E10. Both 4E10 and Z13 are providing molecular insights into the structure and function of the membrane proximal region of HIV-1 gp41.

Fig 1. Model of 2F5 and 4E10, broadly neutralizing antibodies to HIV-1, bound to the membrane proximal external region of gp41.

The fifth antibody, 2F5, recognizes another linear epitope on gp41 (Fig. 1). Previous attempts to present this epitope in a wide variety of immunogens did not elicit 2F5-like neutralizing antibodies. We are investigating the molecular origins of this problem. Overall, HIV vaccine development has been distilled down to challenges in the design of protein, carbohydrate, and peptide immunogens.

Publications

Binley, J.M., Wrin, T., Korber, B., Zwick, M.B., Wang, M., Chappey, C., Stiegler, G., Kunert, R., Zolla-Pazner, S., Katinger, H., Petropoulos, C.J., Burton, D.R. A comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency type 1 monoclonal antibodies. J. Virol. 78:13232, 2004.

Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J., van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.-H., Paulson, J.C. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033, 2004.

Bryan, M.C., Fazio, F., Lee, H.-K., Huang, C.-Y, Chang, A., Best, M.D., Calarese, D.A., Blixt, O., Paulson, J.C., Burton, D., Wilson, I.A., Wong, C.-H. Covalent display of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640, 2004.

Cardoso, R.M.F., Zwick, M.B., Stanfield, R.L., Kunert, R., Binley, J.M., Katinger, H., Burton, D.R., Wilson, I.A. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22:163, 2005.

Ho, J., Uger, R.A., Zwick, M.B., Luscher, M.A., Barber, B.H., MacDonald, K.S. Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response. Vaccine 23:1559, 2005.

Koefoed, K., Farnaes, L., Wang, M., Svejgaard, A., Burton, D.R., Ditzel, H.J. Molecular characterization of the circulating anti-HIV-1 gp120-specific B cell repertoire using antibody phage display libraries generated from pre-selected HIV-1 gp120 binding PBLs. J. Immunol. Methods 297:187, 2005.

O’Connor, D.H., McDermott, A.B., Krebs, K.C., Dodds, E.J., Miller, J.E., Gonzalez, E.J., Jacoby, T.J., Yant, L., Piontkivska, H., Pantophlet, R., Burton, D.R., Rehrauer, W.M., Wilson, N., Hughes, A.L., Watkins, D.I. A dominant role for CD8⁺-T-lymphocyte selection in simian immunodeficiency virus sequence variation. J. Virol. 78:14012, 2004.

Pantophlet, R., Wilson, I.A., Burton, D.R. Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12. Protein Eng. Des. Sel. 17:749, 2004.

Zhang, M.Y., Shu, Y., Rudolph, D., Prabakaran, P., Labrijn, A.F., Zwick, M.B., Lal, R.B., Dimitrov, D.S. Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J. Mol. Biol. 335:209, 2004.

Zwick, M.B., Jensen, R., Church, S., Wang, M., Stiegler, G., Kunert, R., Katinger, H., Burton, D.R. Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1. J. Virol. 79:1252, 2005.

Antibodies and Emerging Viruses

S.K. Kurz-Camacho, W. Oswald, E.O. Saphire, P.B. Jahrling,* P.M. Rudd,** R.A. Dwek,** H. Feldman,*** D.R. Burton

* U.S. Army Medical Research Institute of Infection Diseases, Frederick, Maryland
** Oxford Glycobiology Institute, Oxford, England
*** Bureau of Microbiology, Health Canada, Winnipeg, Manitoba

We are interested in determining the immunogenicity of soluble vs surface glycoproteins of Ebola virus, a filovirus that is one of the deadliest human pathogens. Results indicate strong cross-reactivity and immunogenicity of the different glycoproteins. This finding supports the hypothesis that some of the soluble forms of the glycoproteins act as decoys.Another focus of our Ebola virus research is the early molecular events that occur during infection with the virus. We found that changes in cellular gene expression occurred as early as 1 hour after Ebola virus infection of mononuclear phagocytes, the major target cell of the virus. On the basis of our microarray data, we hypothesized that binding and entry of the Ebola virus glycoprotein cause the early changes in expression levels. To test this hypothesis, we produced soluble forms of the glycoprotein and Ebola viruslike particles that present the glycoprotein on the surface. Incubation with the viruslike particles but not with the soluble glycoproteins caused the same changes in cellular gene and protein expression in Ebola virus target cells as did infection with live Ebola virus. We detected changes in gene expression for inflammatory cytokines, chemokines, molecules involved in the development of hemorrhage, and potential receptors for the virus. Using the viruslike particles, we are testing whether the indicated receptors are involved in Ebola virus binding and signaling.

Publications

Wahl-Jensen, V., Kurz, S.K., Hazelton, P.R., Schnittler, H.-J., Ströher, U., Burton, D.R., Feldmann, H. The role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. J. Virol. 79:2413, 2005.