Hepatitis B virus (HBV) is a blood-born-associated virus that has infected over 2 billion people worldwide. It is also one of the major causes of liver cancer. The overall goal of this study is to derive the 3-dimensional crystal structure of the HBsAg protein, the antigenic component of the currently licensed vaccine for HBV. Despite its worldwide usage and its application as a carrier for epitope presentation, the tertiary structure of this important protein is still unknown. In addition, the lack of a HBsAg structure has (1) prevented us from understanding the effects of mutations on polymerase drug-resistance and on the antigenic properties of the HBsAg major neutralizing antigenic epitope and (2) impeded the development of bivalent vaccines where HBsAg is used as a carrier for foreign antigenic epitopes. Due to the importance of the HBsAg structure the proposed research falls under a number of CDC's Health Protection Goals including (1) """"""""Healthy People in Every Stage of Life"""""""" where new and better vaccines will impact disease in infants, children, adults and the aged, (2) """"""""People Prepared for Emerging Health Threats"""""""" where again new and better vaccines will strengthen our resources to prevent, mitigate and control disease, and (3) """"""""Healthy People in a Healthy World"""""""" where better vaccines developed based on the HBsAg structure would greatly impact the health of people in Asia and Africa. We propose to: (1) Produce diffraction quality crystals of HBsAg that can be used for X-ray diffraction studies. (2) Carryout single crystal X-ray diffraction experiments on the HBsAg crystals necessary to produce a crystal structure. (3) Refine the HBsAg crystal structure to the highest resolution possible. (4) Use the refined crystal structure to understand HBsAg mutations and their effect on vaccine effectiveness and to design. We are collaborating with Dr. Yury Khudyakov, Chief Molecular Epidemiology &Bioinformatics, Division of Viral Hepatitis, CDC Atlanta, who is supplying the highly pure HBsAg samples needed for the proposed studies. This application seeks support for Mr. Quentin Florence who has been heavily involved in the initial HBsAg sample characterization (protein purity, homnogenity, etc) and in setting up the initial crystallization screens at the University of Georgia (using locally developed screens) and at the high-throughput screening laboratory at the Hauptman-Woodward Institute (using a newly developed membrane protein crystallization screen). The results from the initial trials (24576 images) are encouraging with six leads being produced. Mr. Florence is currently supported by a teaching assistantship which requires ~20 hrs/week classroom contact, grading and preparation. This award would allow him to completely focus on the structure determination of HBsAg, his dissertation project. In summary, the results of the proposed studies will provide the basis for understanding how various mutations and fusion of foreign epitopes may affect the tertiary structure of the HBsAg neutralizing epitope. In addition, the studies should provide sufficient understanding of molecular recognition between the immunogenic site ('a'-determinant region) of HBsAg and antibodies. Such knowledge is necessary for the development of improved HBV vaccines, bivalent vaccines and understanding the role of drug-resistant mutations in HBV immune escape.

Public Health Relevance

Hepatitis B virus (HBV) is a blood-born-associated virus that has infected over 2 billion people worldwide. It is also one of the major causes of liver cancer. Despite its worldwide usage as a licensed vaccine for HBV the tertiary structure of the hepatitis B surface antigen (HBsAg) is still unknown. This lack of structure prevents us from understanding mutational effects on the antigenic properties of the epitope and in the design of new and better vaccines.

National Institute of Health (NIH)
National Center for HIV, Viral Hepatitis, STDS and Tb Prevention (NCHHSTP)
Dissertation Award (R36)
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Special Emphasis Panel (ZCD1-SGI (14))
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University of Georgia
Schools of Arts and Sciences
United States
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