The basic objective of this proposal will be to measure the ex vivo fitness of HIV- 1 isolates from individuals infected with clade B and non-clade B HIV-1 and then compare this fitness to various predictors of disease progression. A pilot study revealed a strong correlation between ex vivo HIV-1 fitness and viral load. To extend this study, multiple HIV-1 isolates from approximately 80 HIV-infected individuals have been obtained from the Karolinska Insititute in Sweden and at the :Institute of Tropical Medicine in Antwerp, Belgium.
In SPECIFIC AIM 1, we will compete each patient isolate with our control HIV-1 strains to determine a relative ex vivo fitness value. These fitness values will then be compared to various clinical parameters such as CD4 cell counts and viral load. The objective of this aim is to extend a previous pilot study showing a strong correlation between HIV-1 fitness and disease progression. Ultimately the rate of intrapatient disease progression may play a role in distribution of HIV-1 isolates or subtypes in the epidemic.
SPECIFIC AIM 2 is to compare differences in intra- and intersubtype fitness. The fitness of multiple isolates from clades A, B, C, and D via pair-wise competitions in peripheral blood mononuclear cells. Preliminary data suggests a greater difference between isolates of different subtypes than of the same subtype. Thus, in SPECIFIC AIM 3, we will investigate if possible differences in subtype fitness may affect disease progression in non-clade B infections. As in aim 1, the ex vivo fitness of primary HIV-1 isolates from approximately 150 HIV-infected Ugandans and Zimbabweans (participating in an NICHD-funded study) will be compared to various predictors of disease progression. Since virus will be first obtained from both blood and vaginal secretions during primary infection, we will also perform intersubtype competition experiments using Langerhan cells. Viral propagation for fitness studies is a laborious technique that may be circumvented with the experiments proposed in SPECIFIC AIM 4. Using an assay to look at multiple steps in the HIV-1 replication cycle during dual virus infections, we now have preliminary data to suggest that HIV-1 entry may be controlling the competition. We will confirm this preliminary observation in SPECIFIC AIM 4 and then use a novel cloning strategy to produce HIV-1 NL4-3 clones pseudotyped with env genes directly from patient samples. Competitions between these env pseudotyped HIV-1 clones will then be compared to competitions between two propagated HIV-1 isolates from the same two patient samples.
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