Browsing by Subject "HIV (Viruses)"
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Item Kinetic analysis of HIV-1 reverse transcriptase in the presence of non-nucleoside inhibitors(2004) Wang, Louise Zhiying, 1973-; Johnson, Kenneth A. (Kenneth Allen)Current treatment for human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS) delays the conversion of infected helper T cells to monoclonal malignant cells but does not eradicate HIV. A novel non-nucleoside inhibitor, CZ-1 (2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5- hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2- naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt), was designed to bind at an unconventional site on HIV type 1 reverse transcriptase (RT) (1). I examined the effect of CZ-1 on the kinetic parameters governing the single nucleotide polymerization. CZ-1 decreased the amplitude of the reaction as previously shown for other non-nucleoside inhibitors due to the slow equilibration of the inhibitor with RT. CZ-1 also weakened the apparent DNA binding affinity. Likewise, DNA weakened the apparent CZ-1 binding affinity. In contrast, CZ-1 did not affect the Kd and the maximum incorporation rate of the incoming nucleotide. We therefore conclude that CZ-1 represents a new class of NNRTIs distinct from Nevirapine and related NNRTIs. CZ-1 can bind to RT in both the absence and presence of DNA. In the ternary enzyme-DNA-CZ-1 complex, the binding of DNA is weakened and incorporation of the next nucleotide onto the primer is inhibited. A possible mode of inhibition for CZ-1 is the distortion of RT conformation and the consequent misalignment of DNA at the active site. Unexpectedly, CZ-1 also inhibited human mitochondrial DNA polymerase (pol γ), although I did not see evidence of nonspecific inhibition via self-aggregation or intercalation with DNA. The second part of my project aimed to characterize a non-nucleoside drug-resistant mutant form of HIV-1 RT, containing a single amino acid substitution at position 103 from lysine to asparagine. Three non-nucleoside RT inhibitors, CZ-1, HBY 097, and α-APA, exhibited similar inhibition kinetics on this mutant as on the wild type RT. The third part of my project determined that HIV RT exhibited similar polymerization kinetics whether utilizing an RNA template or DNA template. This validation suggests that the measured kinetic parameters were similar for RNA-directed minus-strand DNA synthesis and DNA-directed plus-strand DNA synthesis.Item A novel vaccine with beta₂-microglobulin linked to a viral epitope stimulates a CTL response and provides immunity to the virus(2003) Piper, John Daniel; Kitto, George B.Designing a vaccine that can elicit a strong cytotoxic T-lymphocytes (CTL) response will be an important component of vaccines against many viruses, but in particular against HIV. CTL stimulation occurs through the recognition of epitopes presented in the context of the class-I major histocompatibility complex (MHC-I). As a way to achieve this, the physical coupling of a viral epitope to the N-terminus of β2-microglobulin (β2m) via a flexible linker has been explored. With the stimulation of CTLs against the Sendai virus as a model, both the effect of linker size on the vaccine and the ability of the β2m-viral epitope fusion vaccine to trigger a primary immune response, as either a recombinant protein expressed in E. coli or as part of a mammalian expression vector are presented in this work. Here we show that as a DNA vaccine, β2mlinked to the immunodominant Sendai NP(324-332) epitope, administered intramuscularly (IM) with or without a plasmid encoding for the cytokine IL-12, can cause the proliferation of viral specific splenocytes. However, when the vaccine was administered IM or subcutaneously as recombinant protein, no response was observed. We also show that a construct with a 21 amino acid (AA) linker stimulates more viral specific splenocytes than constructs with either a 10 AA or a 15 AA linker. Further, DNA vaccine dosages of 100 µg and 200 µg generate more viral specific splenocytes than dosages of 400 µg or 800 µg. We suggest that the reason for this might be high-localized concentrations of β2m. Finally, we show that in response to a sub-lethal challenge with the Sendai virus, mice primed with both the β2m-Sendai epitope DNA vaccine and the IL-12 plasmid can trigger the early arrival of viral specific CD8+ memory cells in the lungs.