Genotypic Analysis of Viremia and Viral Reservoirs

Robert Kauffman, University of California, Davis
Advisor: Tom North
Basic Biomedical Sciences
Dissertation Award

Human immunodeficiency virus (HIV) infection is treated using combination antiretroviral drugs. The use of a combination of drugs is known as highly active antiretroviral therapy (HAART). HAART has been shown to reduce the amount of HIV in the blood below the detection limit of standard clinical labs. HAART is able to significantly reduce the emergence of drug resistance, and may allow patients to have a normal lifespan without developing acquired immune deficiency syndrome (AIDS). Therapy has not been capable of eliminating viral infection and continual drug therapy is necessary in order to control HIV. HIV persists during therapy within the blood at levels below the detection limit of standard clinical assays; furthermore, the plasma viral load rapidly rebounds upon cessation of therapy. These results indicate that there are reservoirs in the body where HIV persists for long periods of time. These locations are believed to include latent reservoirs where infected cells exist but, due to the presence of antiviral drugs, new infections do not occur; and reservoirs where low-level residual replication occurs due to insufficient drug levels which are unable to prevent virus released by infected cells from infecting new cells. Evidence suggests that both types of reservoirs contribute to both the ubiquitous presence of HIV in the blood during therapy and to HIV which emerges upon cessation of therapy. An important tool which is used to study these two reservoirs is genetic analysis. By measuring the level of evolution of viruses found in blood and tissues one can infer whether or not the virus was produced in a location where residual replication has occurred. The specific sites in the body and the extent to which these sites contribute to both viruses present in the blood during therapy and rebound viremia are not well understood.

In order to study viral persistence during HAART, Dr. North’s lab has developed a model of human HIV therapy in rhesus macaques. This model mimics the dynamics of viral suppression and rebound of human HIV therapy. The hypothesis of this proposal is that using the rhesus macaque HAART model, viruses present in the blood during therapy and viral rebound can be distinguished as having been produced from either latent reservoirs or reservoirs undergoing residual replication; furthermore, the anatomical locales of these reservoirs can be identified by genetic analysis. Viral nucleic acids will be obtained from the plasma and tissues of approximately ten infected macaques which have been either sacrificed during HAART or when viral levels have rebounded. Important viral genes for drug resistance and immune system evasion will be sequenced by a new sequencing method known as pyrosequencing. This will be done in parallel with conventional methods. Pyrosequencing is hypothesized to permit the feasible surveillance of viral sequence diversity within dozens of anatomical sites and plasma samples and provide a saturating number of sequences in order to represent a sample’s complete viral diversity. This study will provide data regarding the replication status and anatomical locales of both latent reservoirs and reservoirs where residual replication occurs. Data generated from these analyses are critical for this study’s long term objective to create novel therapy based approaches designed to improve long term viral suppression, eliminate residual viremia, and completely eradicate HIV in infected patients.