Daniel Willis and Dr. Gregory Burton, Microbiology
The development of HighlyActive Antiretroviral Therapy (HAART) against the HIV virus created a temporary excitement regarding a possible cure for HIV. This optimism resulted due to the fact that HAART blocked active HIV replication and reduced the level of HIV in the bloodstream to below standard limits of detection. However, it was soon discovered that HIV blood levels were restored shortly after cessation of HAART. This led to the discovery of HIV reservoirs, or sites where latent virus is harbored. It is this harbored virus that actively replicates and replenishes HIV viral loads when HAART is discontinued.
Several HIV reservoirs have been proposed. Among these is the Follicular Dendritic Cell (FDC). Experiments performed previously in Dr. Burton’s laboratory suggest that the FDC is indeed a valid reservoir for HIV. FDCs are specialized cells of the immune system located in secondary lymphoid tissues. They play an important role in memory immune responses by trapping and maintaining foreign antigens bound by antibody. These antibody bound antigens are known as immune complexes. Typical immune complexes are often trapped by FDCs through an interaction between receptors found on the FDC and antibodies bound to foreign antigens. Collectively, these receptors are known as Fc receptors because they recognize and bind to the Fc portion of antibodies.
Recently, HIV has been found on the FDC. However, the process by which HIV is retained on the FDC is still being elucidated. Previous experiments in Dr. Burton’s laboratory have shown that HIV specific antibody is necessary for the FDC to trap HIV. This suggests that the Fc receptors play an important role in binding HIV. Under the instruction of Dr. Burton, I studied the relationship between two specific Fc receptors and HIV trapping and retention. These Fc receptors are known as FcãRI and FcãRII.
I used a murine system to study this effect. The use of mice in studying the HIV/FDC interaction is advantageous for several reasons:
- Human and murine FDCs have identical function and are even interchangeable in most in vivo assays.
- Mice injected with HIV show significant FDC trapping and retention.
- The murine system is easily manipulated. For example, known quantities of HIV may be injected into each mouse. In addition, mice deficient in Fc receptors are readily and currently available.
- Mice are non-permissive to HIV. In other words, no HIV replication can take place within the mouse. This allows a retention study of only injected virus.
- The murine model is an in vivo study, which more closely represents actual human conditions during HIV infection than in vitro studies.
I used two types of genetically altered mice in my study. In the first group, the gene for the FcãRII receptor was defective, or knocked out (-/-). The second group had the FcãRI receptor knocked out. By comparing the HIV trapped in these genetically altered mice to the HIV trapped in normal mice, I hoped to determine if Fc receptors played an important role in HIV trapping and retention. Since I hypothesized that the Fc receptors are important in trapping infectious HIV, I expected to see less infectious HIV trapped in both Fc knockout groups. The results of my experiments were inconclusive.
The procedure I used to test this hypothesis was very long and detailed. From start to finish, it took approximately one month. The experiment involved injecting all groups of mice with anti- HIV antibody and HIV. This allowed the murine FDCs to trap HIV in the form of immune complexes. After a period of time, I isolated the murine FDCs and cocultured varying numbers of them with human T Cells. Any infectious HIV present on the murine FDCs would actively replicate and integrate within the T Cells upon coculture. At this point, I performed a detection assay involving DNA PCR and Southern Blotting to qualitatively determine the level of HIV replication from virus trapped by the three groups of mice.
Even after performing this experiment many times, my results were inconclusive. This is mostly because I was never able to perfectly optimize the assay. Because this extensive experimental procedure had so many different elements, a countless number of problems were possible. In fact, from September 2001 to June 2002, I continuously struggled to optimize and correct various problems within the assay. While this prevented me from obtaining definitive results in my time at BYU, I made significant progress that will be beneficial to those who will continue the project. I certainly gained an appreciation for the time and effort required to complete research studies from beginning to end.
Below are the results of two different experiments. The darker blots indicate higher levels of HIV. Note that the range of numbers indicate how many FDCs (in increments of 1:10 dilutions) were used in coculture. ACH-2 is a positive control.