Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

7-2-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Daved H Fremont

Abstract

The four dengue viruses: DENV) are mosquito-borne flaviviruses and are considered the world's most significant arboviruses in terms of worldwide disease burden. Symptoms of dengue disease are classified into dengue fever, a mild, febrile illness, and the potentially fatal severe dengue, which can include hemorrhaging and shock. Antibody protection against DENV correlates with the production of neutralizing antibodies against the envelope: E) glycoprotein.

To understand the role of antibodies in DENV infection, we sought to dissect the relationship between epitope and function. Virologic studies had identified that the most potently neutralizing antibodies are against domain III: DIII) of the E protein. We have identified five epitopes within DENV DIII. Our data suggests that the most potently neutralizing antibodies are specific for a single serotype, while cross-reactive antibodies are relatively poorly neutralizing. Additionally, we were surprised to define neutralizing epitopes that were shown to be inaccessible on the surface of the virion in cryo-electron microscopy studies.

Fine epitope mapping was used to define the epitopes of a panel of existing DENV-2 antibodies. Antibodies against the lateral ridge were the most potently neutralizing antibodies and reacted only with the DENV-2 serotype. The second epitope was centered on the DIII A-strand, and antibodies against this epitope reacted with several serotypes of DENV. Several poorly neutralizing antibodies reacted to all four DENV serotypes, as well as West Nile virus, a related flavivirus, mapped to the highly conserved AB loop of DIII.

We expanded our studies of DIII-specific antibodies to the DENV-1 serotype. One antibody, E106, potently neutralized the five DENV-1 strains representing the five genotypes, and bound a composite epitope of the lateral ridge and A-strand epitopes. Despite the potency of E106-mediated neutralization, a combination of structural, biophysical, virologic data suggest that potent DENV-1 neutralization by E106 is coincident with bivalent engagement of the virus.

Additionally, we determined the crystal structures of E111 bound to a novel fifth CC' loop epitope on domain III: DIII) of the E protein from two different DENV-1 genotypes. The available atomic models of DENV virions revealed that the E111 epitope was inaccessible, suggesting that it recognizes an uncharacterized virus conformation. While the affinity of binding between E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC' loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.

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Permanent URL: http://dx.doi.org/10.7936/K78G8HPR

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