Scientific Report 2005

Immunology

Structural Analysis of the Host-Pathogen Interface

E. Ollmann Saphire, M.L. Havert, D.M. Abelson, C.R. Kimberlin, J.E. Lee

We are crystallizing proteins that play key roles in the pathogenesis and lethality of viruses that cause hemorrhagic fever. The resulting crystal structures will provide (1) information for design of vaccines and inhibitors against the viruses as the microbes exist naturally and (2) structural templates that will enable us to anticipate and rapidly respond to newly emerging and synthetic versions of the virus and viral proteins.

Ebola and Marburg Viruses

At least 10 recognized outbreaks of infection with Ebola virus in humans have occurred; in each outbreak, 50%–90% of those infected died. Six outbreaks of infection by the closely related Marburg virus have also occurred. In outbreaks of Marburg virus, typically 25%–40% of those infected die; however, in a recent outbreak in Angola, mortality was 90%. To date no vaccines or treatments are available for infections caused by either virus.

With these 2 viruses, death usually occurs 7–12 days after infection. Events early in infection and innate immune responses are critical for survival in those infected. However, filoviruses have evolved mechanisms by which the host immune system is suppressed. For example, the viral protein VP35 is a required component of the Ebola and Marburg viral capsids and transcription complexes. VP35 also blocks activation of immunomodulatory genes by type I interferon and may play a significant role in viral suppression of the host immune system. Hence, structural analysis of the VP35 protein will provide insights into viral replication and type I interferon suppression and will provide the structural basis for the design of antiviral compounds and attenuated viral strains.An additional, unusual feature of the genome of Ebola virus is its ability to encode 2 different glycoproteins, sGP and GP, from the same gene. These 2 glycoproteins share 295 amino acids of N-terminal sequence, but a transcriptional editing event causes them to have different C-terminal sequences that result in unique patterns of disulfide bonding, structures, and roles in pathogenesis. Comparative structural analysis of sGP and GP should explain how 2 structures arise from the same sequence and should provide templates for the design of vaccines that elicit antibodies that target the virus rather than the secreted proteins.

In contrast, Marburg virus expresses only the membrane-embedded GP. Although Ebola and Marburg viruses are closely related, antibodies to Ebola GP do not cross-react with Marburg GP. Comparative structural analysis of Ebola and Marburg GP should illustrate the fusion machinery required for infection and the structural mechanisms by which the viruses escape from immune surveillance. Additional crystal structures of these proteins in complex with rare human antibodies derived from survivors of infection will assist in vaccine design.

Dengue Virus

Dengue virus is a mosquito-borne flavivirus that causes up to 100 million infections each year. Infection with dengue virus results in either dengue fever or the much more severe disease dengue hemorrhagic fever. Dengue hemorrhagic fever usually occurs upon secondary infection with a different viral subtype or in infants born to dengue virus–immune mothers. This potential antibody-mediated enhancement of infection is a major concern in the testing and use of vaccines against dengue virus because antibodies elicited by the vaccines could trigger severe disease. To aid in vaccine design, we are determining crystal structures of envelope proteins of contemporary field isolates of dengue virus, alone and in complex with antibodies, to determine structural features of epitopes associated with neutralization and enhancement.