In vivo and in vitro models for determination of antiviral activity and resistance

Sammanfattning: The AIDS epidemic has been the driving force for the discovery of substances thatinhibit the replication of human immunodeficiency virus (HIV). Much interest hasearlier focused on a single gene product central to the life cycle of HIV and toall other retroviruses: the reverse transcriptase (RT), the enzyme responsible forsynthesis of DNA from viral RNA. Analogues to the five naturally occurring nucleosidescan inhibit the replication of retroviruses in cell culture to various degrees. Anotherenzyme critical for HIV replication is the viral protease. Inhibition of this enzymeleads to production of immature noninfectious viral progeny. The aim of this thesiswas to develop and use methods required for the preclinical development and laboratorytesting of antiviral drugs. In order to monitor in vivo challenge models for retrovirus infection, we developedassays to identify viral antigens and immune responses to them. The antibody assaywas based on a synthetic peptide, the sequence of which corresponds to a segmentof the env protein of HIV-2 which crossreacts with SIV. The peptide was successfullyused to detect IgM antibodies from days 13-16 after SIV infection and anti-SIV IgGantibody levels around days 16-20 which attained a plateau after two months. In the development of new drugs it is imperative to use animal models for thestudy of therapies against AIDS and HIV infection. In this study monkeys were infectedwith 10-100 monkey infectious doses of SIV SM and treated for ten days with foscametor one of four different nucleoside analogues. Foscamet, zidovudine, fluorothymidine,didanosine, and stavudine significantly delayed the appearance of SIV SM p24/26 antigen.Foscarnet also decreased IgM and delayed IgG antibodies. In our studies, infectionwas not prevented by any of the drugs investigated, but later studies of fluorothymidinehave prevented infection when smaller infectious doses have been used. A major clinical problem in the management of AIDS is the presence of neuropsychiatricsymptoms. It is therefore valuable if a drug against HIV penetrates the blood-brainbarrier. Microdialysis is a method of sampling extracellular fluid containing free(not protein-bound) drugs in the brain and other tissues. Using microdialysis, theconcentration of the nucleoside analogues fluorothymidine and zidovudine in the brainwere found to be approximately one-third those in the muscle and plasma in monkeysand one-fifth in rats. The in vivo unbound concentrations of both drugs in the brain,muscle and venous blood exceeded those reported to inhibit HIV replication in vitro.The method allows comparisons of the efficacy of antiviral drugs in penetrating theblood-brain barrier. Herpesvirus infections are common among AIDS patients; more than half of themhad evidence of disseminated cytomegalovirus infection at autopsy. A method for measurementof antiviral sensitivity of primary isolates of CMV was developed. The assay includessimultaneous virus titration and determination of the 50 % inhibitory concentration(IC5O). The absorbance obtained in the enzyme-linked immunosorbent assay correlatedlinearly to the number of plaques in a plaque reduction assay. The IC5O of antiviralcompounds was strongly dependent on the virus dose, except for resistant isolates,where the IC50 did not change with the viral dose. Clinical isolates exhibited differentIC50 values for ganciclovir in different cell types indicating that intracellularfactors are important for drug metabolism. The effect of drug combinations may also be primarily evaluated in vitro. Combinationsof RT and protease inhibitors not only target two different proteins but also affecttwo different stages of the viral life cycle of HIV. Combinations of foscarnet andlamivudine, both inhibitors of RT, showed clear synergy and this effect was furtherpronounced using two more potent foscarnet analogues. A weak or no synergy was foundwhen foscarnet was combined with the protease inhibitors indinavir, saquinavir orritonavir. ISBN 91-628-2716-2