Follicular Dentritic Cells and Retention of HIV Quasi-Species

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Candace McNaughton and Dr. Gregory Burton, Microbiology

HIV infects millions of people worldwide; in the year 2000 alone, more than 3 million people died from AIDS (Acquired Immunodeficiency Syndrome) as a result of HIV infection1. In countries, such as the United States, where effective treatment is affordable, highly active antiretroiviral therapy (HAART) can reduce viral blood loads to currently undetectable levels2. Unfortunately, the side effects of HAART can be severe, resulting in noncompliance, and upon its termination, HIV levels rapidly rebound. Several viral reservoirs within the body have been identified, including the follicular dendritic cell (FDC) network3. FDCs trap HIV antibody complexes for long periods of time in an infectious form and allow HIV infection to persist even in the presence of high concentration of neutralizing antibody4. I hypothesized that FDCs trap multiple HIV quasi-species over the course of infection (including drug-resistant strains), acting as a repository for subsequent re-ignition of HIV infection upon HAART cessation.

To begin testing this hypothesis, I used an in vitro model of HIV infection with multiple strains of HIV (IIIB, MN, LW) followed by DNA isolation and cloning techniques to determine if these HIV strains could be distinguished from each other based on based sequence differences. Nested set PCR was used to amplify HIV env, and the products were ligated and transfected into bacterial cells. Bacterial colonies were selected based on survival and ampicillin resistance. Following their isolation, the inserted plasmid was sequenced by the BYU Sequencing Core Facility. Many of my initial experiments proved unsuccessful due to problems with the experiment protocol itself; even after considerable alteration, the protocol is still undergoing revision.

In addition to my in vitro model, I used an in vivo murine model in which HIV-antibody complexes were injected into mice and the FDCs isolated from the mouse lymph nodes one week later. HIV-bearing FDCs were cultured with HIV target cells and FDCs were the only source of HIV. DNA was isolated from infected cells and analyzed as in my in vitro model.

These data from my in vitro model indicated that different strains of HIV were distinguishable by their env sequence, thus establishing the technique needed to specifically test my hypothesis in the in vivo model. Due to the technical complexity of my project, studies using the in vivo model were not successful, although this is an active area of research in the laboratory system. By the completion of this project, other members of the lab will have already met with several other researchers from other universities in an effort to reduce the complexity and increase the reproducibility of my results.

I successfully demonstrated that using PCR and cloning methods can be used to differentiate between HIV-1 strains based on sequence analysis. Work to extend the in vitro model to an in vivo murine system is still continuing; this work is needed to demonstrate that FDCs trapped different HIV quasi species. Once the murine model has been successfully completed, this assay system will then be used to isolate HIV quasi species trapped on the FDCs of HIV-positive patients over time, which will allow researchers to track HIV population dynamics over time in infected individuals. It is hoped that this will lead to development of therapies targeting HAART resistant species, allowing for the complete elimination of HIV from all reservoirs.

References

  1. Balter, M. 1999. AIDS now world’s fourth biggest killer [news]. Science 284:1101.
  2. Perelson, A.S., A.U. Neumann, M. Markowitz, J.M. Leonard, and D.D. Ho. 1996. HIVdynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271: 1582.
  3. Chun, T.W. and A.S. Fauci. 1999. Latent reservoirs of HIV: obstacles to the radication of virus. Proc.Natl.Acad.Sci.U.S.A. 96:10958.
  4. Smith, B.A., S. Gartner, Y.Liu, A.S. Perelson, N.I. Stilianakis, A.K.Szakal, J.G. Tew, and G.F. Burton. 1999. Persistence of infectious Human Immunodeficiency Virus on Follicular Dendritic Cells (FDC). (submitted)