Andrew Jay Thorne and Dr. Greg Burton, Chemistry and Biochemistry

Human immunodeficiency virus (HIV) infection and resultant development of autoimmune deficiency syndrome (AIDS) killed 1.8 million people and infected 2.2 million more just in 2009. Not only are those whose personal lifestyle choices put them at risk for HIV infection affected. As of 2009, there are 16.6 million orphaned children living whose parent(s) died from AIDS. These numbers are difficult to ignore, and show the need for further improvement of treatment and prevention.

HIV infects T cells, which are extremely important to the immune system. T cells perform many and varied functions such as destroying virally infected cells, regulating the immune system, and performing a crucial assisting role in many processes. T cells also serve as the body’s memory bank for previous infections, which is crucial for the development of immunity. If HIV infects and kills enough T cells, the immune system can be severely handicapped, bringing the onset of AIDS. Then even the smallest infections or sicknesses can be fatal. Because HIV is a retrovirus—meaning that the virus inserts its own genome into the genome of the host—it is unlikely that HIV can be entirely cured once infection has taken place. However, it is possible to suppress the virus so that the infected patient can live a nearly normal life. My research aims toward improving the existing suppression methods.

There are two versions of HIV: R5 virus and X4 virus. The versions are so named because of their preference for co-receptor used in cell entry. Co-receptor preference is also referred to as tropism. R5 virus uses the CCR5 co-receptor and X4 virus uses the CXCR4 co-receptor. The X4 virus is significantly more pathogenic. Over time, patients who are mostly infected by R5 virus can show an increasing amount of X4 virus, resulting in a more rapid progression to AIDS. Germinal centers are locations in the lymph system where a particular cell called the Follicular Dendritic Cell (FDC) is located. The FDC is important to the immune system because it traps foreign contaminants and presents them for recognition, which causes an immune response against that foreign contaminant. It has been shown that T cells when incubated with FDCs show a two to three fold increase in CXCR4 co-receptors, increasing the likelihood of X4 infection. With a higher likelihood of X4 infection, it is plausible that the observed increase in CXCR4 receptors from incubation with FDCs could be involved in causing the tropism change from R5 to X4 in HIV infected patients. I will be using T cells from both within and without germinal centers from an infected, deceased patient as the source of virus to be genetically examined to determine if the higher expression of X4 co-receptors will lead to preferential infection by X4 virus. Proving that there is significantly more X4 virus in the germinal center would help to elucidate the mechanisms of tropism change and contribute greatly to the development of treatment approaches to keep the X4 virus from becoming dominant.

To carry out the experiment, the genomes of numerous HIV strains are isolated from both within and without germinal centers and then sequenced to determine if they are R5 or X4 viruses. To begin, the section of the viral envelope DNA which is to be studied is amplified by polymerase chain reaction (PCR). This process involves using DNA primers that bind to specific sites on the DNA chain and signal to a special polymerase enzyme where to begin copying the DNA. The specific section of DNA is copied millions of times, which brings the concentration of the DNA high enough for further manipulation.

Then the amplified DNA is ligated, or inserted, into a specially designed plasmid. A plasmid is a circular ring of DNA found in bacteria. The specially designed plasmid has a gene that will give an E. coli bacterial cell resistance to ampicillin. The plasmid that now contains the inserted viral DNA is then placed into E. coli cells. Then the cells will be grown in an environment that contains ampicillin. Cells that have not accepted the plasmid are killed, but colonies of bacteria that have taken up the plasmid survive. This process ensures that previous steps have succeeded and it separates unique strains of viral DNA. Then numerous colonies are selected for further analysis. Next, the plasmid is harvested and purified from the E. coli cells. The viral DNA is removed from the plasmid by the restriction enzyme Eco RI. The DNA is then ready to be prepped for sequencing, which will also be done through PCR. Once sequenced, the viral genome can be analyzed by computer to predict co-receptor tropism.

To date, the project is not finished. To prepare, I began the project by using dummy samples to learn all of the procedures. In October 2010, I successfully completed a full practice run and felt comfortable enough to begin working with the patient sample. When my mentor and I began to look for it, however, we found that it was not where we expected. We looked for the records made by the graduate student who sorted the sample back in 2007. The records are lacking and we have still not located the sample. There is still some searching to be done and both my mentor and I are optimistic that the sample will be found. In the interim, I have found and am analyzing some viral DNA from a similarly sorted sample that were sequenced in 2007 by the same graduate student. Unfortunately, the record of their processing is incomplete and the data are therefore not fit for publication, but they will serve excellently as preliminary data.

I believe that we will find a significant amount more X4 virus in the germinal center than outside the germinal center. I am hopeful that I will find the sample I am searching for, but even if it has gone missing, other samples could be requested and prepared. This would take more time, but I will see the research through to completion. I am excited about the implications that my findings could have on the development of suppression methods to prevent tropism change. The findings we expect could also guide further research into the influence that the FDC has on T cell function.

References

1. Estes, Jacob D.; Keele, Brandon F.; Tenner-Racz, Klara; Racz, Paul; Redd; Michael A.; Thacker, Tyler C.; Jiang, Yongjun; Lloyd, Michael J.; Gartner, Suzanne; Burton, Gregory F. Follicular Dendritic Cell Mediated Up-Regulation of CXCR4 Expression on CD4 T Cells and HIV Pathogenesis, The Journal of Immunology, 2002, 2313–2322
2. Estes, Jacob D.; Keele, Brandon F.; Thacker, Tyler C.; Palenske, Emily A.; Burton, Gregory F.; Follicular Dendritic Cells and Regulation of Germinal Center CD4 T cell Migration, The Journal of Immunology, 2004, 6169–6178
3. UNAIDS homepage. http://www.unaids.org/en/default.asp (accessed Dec 10, 2010)