News and Publications

Human Antibodies and HIV-1 Infection

P.W.H.I. Parren, R.B. Bastidas, H.J. Ditzel, A.J. Hessell, E.E. Karrer, K. Koefoed, D.J. Michaels, P.R. Poignard, D.K. Slifka, M. Wang, D.R. Burton

The perception of humans as the easy victor over microbes has changed dramatically in the last decade. Vaccination has offered protection against a number of viral pathogens, but it is increasingly recognized that the strategies used in the past will not be successful against all viruses. More understanding of viral pathogenesis and of the interaction of viruses with the immune system is required. We focus on the interplay of antibodies and viruses in humans. We make particular use of combinatorial antibody technology, which gives ready access to human antibodies. The viruses we are studying are HIV type 1 (HIV-1), respiratory syncytial virus, and emerging viruses such as Ebola virus and hantavirus.

For HIV-1, we have mapped the human antibody response to the glycoproteins of the viral envelope. We concluded that the response is directed primarily to immature forms of envelope produced during lysis of infected cells ("viral debris") rather than to the mature form found on the surface of infectious virions. Consequently, most antibodies do not bind well to virions and do not neutralize virus. We have, however, generated a human antibody that effectively neutralizes a wide range of primary isolates of HIV-1 (Fig. 1). This antibody is being used to explore the activities of antibody against HIV-1 in vivo.

In an animal model, in which mice populated with human cells can be infected with HIV-1, the antibody completely protected mice against challenge with a primary virus even when the antibody was given several hours after viral challenge. This result was the first time that an antibody had prevented infection by a primary isolate of HIV-1 in an animal model. This finding greatly enhances the possible usefulness of the antibody in prophylactic therapy given after accidental exposure to HIV and in the interruption of mother-to-child transmission of the virus. Of even greater importance, it establishes the principle of antibody protection as a target for vaccine design. Currently, the effects of antibody on established infection in the mouse model are under study in collaboration with D. Mosier, Department of Immunology.

The mechanism of antibody neutralization of HIV-1 has been poorly understood. Together with researchers in Q. Sattentau's laboratory in Marseilles, France, we have carried out 2 sets of studies with a range of recombinant Fabs and other antibodies to the HIV envelope that address the question of how antibody neutralizes HIV. The first set showed that, with one exception, neutralizing antibodies to a number of different epitopes act by blocking attachment of virus to the target cell. The second set showed that neutralization was related to occupancy of binding sites on the virus irrespective of the epitope recognized. These findings suggest that the most likely mechanism of neutralization is steric obstruction by antibody molecules on the surface of the virus. Similar conclusions are being reached on the basis of studies on the mechanism of neutralization of influenza virus.

PUBLICATIONS

Burton, D.R. Immunoglobulin, functions. In: Encyclopedia of Immunology. Roit, I.M., Delves, P.J. (Eds.). Academic Press, San Diego, in press.

Burton, D.R. A vaccine for human immunodeficiency virus type 1: The antibody perspective. Proc. Natl. Acad. Sci. U.S.A. 94:10018, 1997.

Burton, D.R., Moore, J.P. Why do we not have an HIV vaccine and how can we make one? Nature Med. 4(Suppl.):495, 1998.

Cattani, P., Rossolini, G.M., Cresti, S., Santangelo, R., Burton, D.R., Williamson, R.A., Sanna, P.P., Fadda, G. Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria. J. Clin. Microbiol. 35:1504, 1997.

Ditzel, H.J., Rosenkilde, M.M., Garred, P., Wang, M., Koefoed, K., Pedersen, C., Burton, D.R., Schwartz, T.W. The CCR5 receptor acts as an alloantigen in CCR532 homozygous individuals: Identification of chemokine- and HIV-1-blocking human antibodies. Proc. Natl. Acad. Sci. U.S.A. 95:5241, 1998.

Gauduin, M.-C., Parren, P.W.H.I., Weir, R., Barbas, C.F. III, Burton, D.R., Koup, R.A. Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1. Nature Med. 3:1389, 1997.

Glamann, J., Burton, D.R., Parren, P.W.H.I., Ditzel, H.J., Kent, K.A., Montefiori, D., Hirsch, V.M. Simian immunodeficiency virus (SIV) envelope-specific Fabs with high homologous neutralizing activity: Recovery from a long-term nonprogressor SIV-infected macaque. J. Virol. 72:585, 1998.

Mo, H., Stamatatos, L., Ip, J.E., Barbas, C.F. III, Parren, P.W.H.I., Burton, D.R., Moore J.P., Ho, D.D. Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. J. Virol. 71:6869, 1997.

Parren, P.W.H.I., Burton, D.R., Sattentau, Q.J. The neutralizing antibody response to HIV-1. In: AIDS Pathogenesis. Miedema, F., Schnitemaker, J. (Eds.). Landes Bioscience, Austin, TX, in press.

Parren, P.W.H.I., Gauduin, M.-C., Koup, R.A., Poignard, P., Sattentau, Q.J., Fisicaro, P., Burton, D.R. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett. 57:105, 1997.

Parren, P.W.H.I., Mondor, I., Naniche, D., Ditzel, H.J., Klasse, P.-J., Burton, D.R., Sattentau, Q.J. Neutralization of HIV-1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity. J. Virol. 72:3512, 1998.

Ugolini, S., Mondor, I., Parren, P.W.H.I., Burton, D.R., Tilley, S., Klasse, P.J., Sattentau, Q.J. Inhibition of virus-attachment to CD4⁺ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization. J. Exp. Med. 86:1287, 1997.

Williamson, R.A., Lazzarotto, T., Sanna, P.P., Bastidas, R.B., Dalla Casa, B., Campisi, G., Burioni, R., Landini, M.P., Burton., D.R. Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia. J. Clin. Microbiol. 35:2047, 1997.

Antibodies in Respiratory Syncytial Virus Infection

H. Sakurai, R.A. Williamson, D.R. Burton

Respiratory syncytial virus (RSV) is the most important cause worldwide of severe viral infection of the lower respiratory tract in young children. Development of a sufficiently attenuated, yet highly immunogenic, vaccine suitable for infants in their first weeks of life continues to be extremely difficult. Because antibodies can mediate resistance to RSV infection and illness, any successful vaccine should elicit high levels of antibodies that neutralize the virus.

We are using phage antibody libraries to better understand both quantitatively and qualitatively the antibody response to RSV during the course of natural infection. We have isolated a potent Fab fragment from a human monoclonal antibody that neutralizes diverse RSV isolates and is highly effective therapeutically when administered intranasally to infected mice at the time of peak viral replication in the lungs. The Fab, possibly as an aerosol, is a candidate for the treatment of serious RSV disease, and a whole antibody version could be used as a prophylactic agent in children at risk for RSV infection. Our current efforts focus on improving the neutralizing antibody by using in vitro methods and by understanding the molecular basis of antibody neutralization of this virus.

Antibodies and Emerging Viruses

T. Maruyama, M. Kaneko, M.J. Buchmeier, P.W.H.I. Parren, D.R. Burton

We are interested in determining whether antibodies can protect against and/or control some of the most extremely pathogenic human viruses. We are investigating 2 viruses: Ebola virus and hantavirus. Ebola virus attracted much attention in 1995 after an outbreak of an infection caused by this virus in Kikwit, Zaire, killed more than 300 persons, with a mortality rate of 80%. We obtained bone marrow from survivors of the epidemic, constructed phage libraries, and isolated a panel of human antibody Fab fragments. A number of these Fabs bind effectively to native viral envelope on infected cells, and one has been converted to a whole antibody. We plan to investigate the ability of this antibody to protect against viral challenge in animal models.

Sin Nombre is a hantavirus that causes a pulmonary syndrome with a high mortality rate. It is transmitted via rodents and was responsible for a major outbreak in the Four Corners region of the southwestern United States in 1993. Recently, closely related viruses have caused outbreaks of pulmonary disease in South America. We are studying antibodies from donors who recovered from infections with Sin Nombre and South American hantaviruses.

PUBLICATIONS

Maruyama, T., Parren, P.W.H.I., Sanchez, A., Rensink, I., Rodriguez, L.L., Khan, A.S, Peters, C.J., Burton, D.R. Recombinant human monoclonal antibodies to Ebola virus. J. Infect. Dis., in press.

Salonen, E.M., Parren, P.W.H.I., Graus, Y.F., Lundkvist, A., Fisicaro, P., Vapalahti, O., Kallio-Kokko, H., Vaheri, A., Burton, D.R. Human recombinant Puumala virus antibodies: Cross reaction with other hantaviruses. J. Gen. Virol. 79:659, 1998.

Probing the Structure of the Prion Protein

R.A. Williamson, R.B. Bastidas, T. Middlesworth, D. Peretz,* S.B. Prusiner,* D.R. Burton

* University of California, San Francisco, CA

The prevailing view of transmissible spongiform encephalopathies or prion diseases such as scrapie, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy ("mad cow disease") is that they are diseases of protein conformation. The infectious unit, the prion, is thought to be composed largely, if not entirely, of a modified host-encoded glycoprotein denoted PrP^Sc. Through a posttranslational process, PrP^Sc is formed from the normal cellular isoform designated PrP^C. Despite extensive efforts, no evidence indicates any covalent chemical differences between the 2 isoforms of PrP. However, the 2 forms have a number of striking differences. Most notably, PrP^C is readily degraded by proteases, whereas PrP^Sc is relatively resistant. A major recent advance has been the determination of nuclear magnetic resonance (NMR) structures for several recombinant PrP^C molecules. These structures suggest that the molecule is composed of a largely disordered N-terminal half and an autonomously folding C-terminal half centered on a 3-helix bundle.

Clearly, antibody reagents that recognize distinct PrP isoforms would have many applications in prion research, for example, in immunohistochemical studies of PrP in situ, for PrP purification and structural characterization, and for early clinical diagnosis of prion diseases in both animals and humans. However, difficult problems must be overcome. Natural scrapie infection does induce either the humoral or cellular arms of the immune system, and before 1993, only 2 high-affinity monoclonal antibodies had been generated to PrP. We overcame the problems by immunizing mice in which the gene for PrP had been ablated and then "rescuing" antibodies by using phage display libraries. Large numbers of recombinant antibodies were obtained and have been reduced to 19 different specificities that recognize linear and discontinuous epitope regions.

The antibodies have been used to study PrP conformation. Most antibodies reactive with the recombinant PrP molecules used for NMR structural studies also react with cell surface PrP^C, providing the most convincing evidence to date that the NMR structures are close to the native structures of the cell surface. This finding implies that, uniquely, approximately one half of the PrP^C molecule has a disordered structure on the surface of cells. Antibodies to the ordered C-terminal half of the molecule react with both PrP^C and a PrP^Sc-derived molecule, whereas those to the disordered N-terminal half react well with PrP^C but only poorly with PrP^Sc. These observations imply that conformational changes in the N-terminal region of the protein are pivotal to the conversion of PrP^C to PrP^Sc (Fig. 1). As an approach to understanding the folding events in the N-terminal region that most likely initiate the conversion, we are collaborating with S. Prusiner, University of California, San Francisco, in determining antibody-peptide crystal structures. Initial results suggest energetically favorable ordered structures for at least 1 peptide from the N-terminal region.

The recombinant antibodies have also enabled us to successfully pursue other novel avenues of investigation. Using immunolabeling, we have produced a unique series of high-quality images of PrP in the cell membrane and have established its caveolar location.

PUBLICATIONS

Peretz, D., Williamson, R.A., Matsunaga, Y., Serban, H., Pinilla, C., Bastidas, R.B., Rozenshteyn, R., James, T.L., Houghten, R.A., Cohen, F.E., Prusiner, S.B., Burton, D.R. A conformational transition at the N-terminus of the prion protein features in formation of the scrapie isoform. J. Mol. Biol. 273:614, 1997.

Williamson, R.A., Peretz, D., Kanyo, Z., Burton, D.R. Antibodies as tools to probe prion protein (PrP) biology. In: Biology of Prions. Prusiner, S.B. (Ed.). Cold Spring Harbor Press, Cold Spring Harbor, NY, in press.