Sean Llewellyn and Dr. Gregory F. Burton, Department of Chemistry and Biochemistry
Importance of Project
HIV is known to survive on follicular dendritic cells (FDCs) for many months and is believed to contribute to persisting HIV infection. FDCs protect HIV from degradation, thereby prolonging the infectivity of the virus. Moreover, when virus is trapped on FDCs, it escapes the ability of neutralizing antibodies (NtAb) to block infection of CD4+ T cells and subsequent removal of virus from the body. We postulate that FDC CD32 and/or CD21 contribute to maintaining virus infectivity and permitting infection in the presence of NtAb. Furthermore, when HIV forms an immune complex with antibodies and binds to CD32 and/or CD21, the FDC becomes activated. FDC activation increases its expression of FDC CD32 and CD21, thereby permitting more viruses to be trapped and we reason to better escape the blocking effects from NtAb. If our hypothesis that FDC CD32 and CD21 play a role in increasing the infectivity of HIV on FDCs is correct, then we may be able to block the actions of these two receptors, thereby inhibiting the spread of infection.
Main Proposal Body
First, FDCs will be isolated from HIV non-infected human tonsils that are provided weekly by local physicians after elective surgery. In the first experiment, some of these cells will be activated with varying concentrations of lipopolysaccharide (LPS) or a similar activating substance. These activated FDCs will then be incubated for different time periods. From each of the time periods, mRNA will be extracted from the cells and reverse transcription will be performed to create cDNA. The cDNA will provide evidence for what mRNAs were transcribed and up-regulated as a result of FDC activation. The determination of upregulation will be performed through southern blotting or real-time PCR of the cDNA. I expect CD32, CD21, and, TNF-α to be expressed at higher levels when FDCs are activated. I might also see IL–6, IL–15, and BAFF up-regulated because mice have similar FDCs to humans and these genes are known to be up-regulated in mice FDCs. The expression of the proteins will be measured through ELISA and Flow Cytometry to confirm which proteins were up-regulated.
After the determination of the up-regulation effects on proteins during FDC activation, CD32 and CD21 will be the focus of the project. Using HIV-immune complexes (ICs), in vitro, measurements of activation and the level of activation of FDCs will be determined using the same methods and measures as above to detect the level of activation. If it is determined that HIV-ICs activate FDCs, I will next determine if more viruses are trapped as a consequence of the activation, which I postulate is likely. To quantify the amount of virus present on FDCs, measurements of the amount of p24 and viral RNA via ELISA and real-time PCR, respectively, will be taken. Then blocking antibodies for CD32, then for CD21, and then for both CD32 and CD21 will be used to observe if the activation is increased, decreased, or completely absent. If it is decreased or absent, I will compare the levels of the activated proteins compared to the control group (the original results from the HIV-ICs) again using ELISA and Flow Cytometry. This will give me an idea of the role of CD32 and CD21 in the infectivity of the virus on FDCs.
Finally, I will determine if CD32 and CD21 play a role in the escape of HIV from neutralizing antibodies. FDCs will be activated and the amount of virus trapped on the cell will be quantified. The viral quantification present on FDCs will be performed through the same methods as above. Then the use of blocking antibodies will be used to determine if blocking the CD32 and CD21 receptors prevent the escape of HIV from neutralizing antibodies. This will be done by blocking CD32 and CD21 with the same antibodies used in the experiment above, adding NtAb-HIV complexes, and detecting how much virus is present on the FDC. After all of these experiments, I can ascertain what role CD32 and CD21 receptors play in both the activation of human FDCs and the effect of this activation on HIV infectivity.
Anticipated Academic Outcome
Dr. Burton’s laboratory has many projects underway that all focus on the role of FDCs in HIV/AIDS. My project is an integral component of this work and the findings will be submitted for publications in a societal level, peer-reviewed journal. I also plan on attending medical school next year. Working on this project will better prepare me for medical school by teaching me more about immunology and giving me experience with complicated, problem-solving situations.
Dr. Burton received his PhD and post-doctoral work in Immunology, studying the role of FDCs in immune regulation. He is also a visiting scientist in the Department of Neurology at Johns Hopkins Medical School. Dr. Burton is an author of fifty-eight papers, and he has been a mentor to over sixty undergraduate and fifteen graduate students in the study of HIV and FDCs. I have researched with Dr. Burton for over a year now, and I have completed all of the necessary training to work with HIV. Also, I have taken or I am currently taking the following classes that will help me with this project: infection and immunity, immunology, biochemistry (both lectures and labs), molecular biology, and cell biology. Project Timetable The first experiment, using LPS to activate FDCs, has already started. Near the end of November 2009, I will start working on the second experiment, using HIV-ICs to activate FDCs. Once this phase is done, which will be during in the winter semester, the final experiment with use of NtAb-HIV complexes will start. This whole project will hopefully be done before I leave for medical school in August 2010 with a paper being published next winter.
Fit With BYU’s Mission
Two aims of a BYU education are having an intellectually enlarging environment and performing lifelong service. In working with Dr. Burton and on this project, I will constantly be in intellectually stimulating situations because new questions will always be formulated and need to be solved, allowing me to gain valuable problem solving skills and knowledge. I will also be learning a lot about immunology from Dr. Burton and the graduate students. These two aspects alone will allow my intellect to grow. This project will also help me perform lifelong service to others. This project has the ultimate goal of discovering the unknowns of HIV, which will lead to a cure of AIDS. By working on this project, I will be of service to many people by discovering one of the unknowns of HIV. Working with Dr. Burton and on this project will also help me get into medical school. Then when I become a doctor, I will be constantly serving others and helping them to have better lives. By discovering a new aspect of HIV and becoming a doctor, I can provide lifelong service to others.
- Burton, G. F., B. F. Keele, J. D. Estes, T. C. Thacker, and S. Gartner. 2002. Follicular dendritic cell contributions to HIV pathogenesis, p. 275-284, Seminars in Immunology, vol. 14.
- El Shikh, M. E., R. M. El Sayed, Y. Wu, A. K. Szakal, and J. G. Tew. 2007. TLR4 on follicular dendritic cells: an activation pathway that promotes accessory activity. J Immunol 179:4444-50.
- Estes, J. D., B. F. Keele, K. Tenner-Racz, P. Racz, M. A. Redd, T. C. Thacker, Y. Jiang, M. J. Lloyd, S. Gartner, and G. F. Burton. 2002. Follicular Dendritic Cell-Mediated Up-Regulation of CXCR4 Expression on CD4 T Cells and HIV Pathogenesis. J. Immunol. 169:2313-2322.
- Heath, S. L., J. G. Tew, J. G. Tew, A. K. Szakal, and G. F. Burton. 1995. Follicular dendritic cells and human immunodeficiency virus infectivity. Nature 377:740-744.
- Smith, B. A., S. Gartner, Y. Liu, A. S. Perelson, N. I. Stilianakis, B. F. Keele, T. M. Kerkering, A. Ferreira-Gonzalez, A. K. Szakal, J. G. Tew, and G. F. Burton. 2001. Persistence of infectious HIV on follicular dendritic cells. J Immunol 166:690-696.
- Thacker, T. C., X. Zhou, J. D. Estes, Y. Jiang, B. F. Keele, T. S. Elton, and G. F. Burton. 2009. Follicular dendritic cells and human immunodeficiency virus type 1 transcription in CD4+ T cells. J Virol 83:150-8.