Targeting HIV-1 entry and reverse transcription by vaccination

Detta är en avhandling från Stockholm : Karolinska Institutet, Microbiology and Tumor Biology Center (MTC)

Sammanfattning: Human immunodeficiency virus type I (HIV-1) is a complex retrovirus, which uses the CD4 receptor and chemokine receptors to infect its target cells. The chemokine receptor CCR5 is essential for primary HIV-1 infection. The hallmark of retroviruses is the enzyme reverse transcriptase (RT), which transcribes the virus genome from RNA to DNA. RT is a major target for HIV drugs but antiviral treatment often selects for drug resistant virus variants. RT lacks proofreading, which accounts for a great deal of the high variability of HIV-1 genomes. The aim of this thesis was to evaluate the RT gene and the CCR5 gene as immunogens and to develop an animal model for HIV- I drug resistance. The human CCR5 gene was used together with the adjuvant granulocyte macrophage colonystimulating factor (GM-CSF) for immunization of mice and monkeys (cynomolgus macaques). Immunization with the CCR5 gene induced antibody responses in both mice and monkeys. The macaques were boosted with CCR5 peptides in the rectal mucosa. This potentiated local IgA responses as well as systemic IgA and IgG antibody responses to human and macaque CCR5 peptides. The detected CCR5 antibodies demonstrate that we were able to break tolerance and immunize against the endogenous CCR5 receptor. Interestingly, the CCR5 peptide boost abolished or decreased CCR5 gene-induced antibody responses towards native CCR5. Sera from vaccinated animals blocked infection of peripheral blood mononuclear cells (PBMC) with HIV-1 and with simian immunodeficiency virus sooty mangabey (SIVsm) in vitro. Regrettably none of the CCR5-immunized monkeys were protected from SIVsm challenge. In one vaccinated animal the virus surprisingly evolved and started to use the CCR5 receptor more efficiently than before. Thus CCR5 immunization may lead to the evolution of viruses with new properties which may be negative for the patient. This has to be investigated further. Different immunization strategies were tested in order to induce strong RT specific immune responses. The adjuvant effect of bacterial DNA (CpG-oligodeoxynucleotides, ODN) was evaluated in this context. A prime with the RT gene followed by protein with CpG-ODN boost induced the most potent cellular immune responses in both mice and macaques. After challenge of mice with HIVI/murine leukemia virus (HIV-1/MuLV), cellular immune responses in splenocytes were equal in the groups: receiving a) DNA followed by protein with CpG-ODN or b) protein with CpG-ODN. In both these groups protection from challenge was observed in some of the vaccinated mice. Consequently the evaluated three-component vaccination strategy induced potent humoral and cellular immune responses to RT and provided partial protection from HIV-1. RT may therefore be an important component of an HIV- I vaccine. HIV-1 drug resistance mutations found in vitro do not always correlate to the mutations found in vivo in treated patients. An in vivo model for the study of resistance development in macaques was constructed. The occurrence, type and duration of RT mutations during treatment with the nonnucloside inhibitor nevirapine were analyzed over time. We found a good correspondence of mutation pattern and kinetics between the monkey model and known data from nevirapine treated patients. Mutation K103N found in the drug treated monkeys was infrequently selected by nevirapine in cell culture. This indicates that the monkey model might be used to detect mutations that are difficult or impossible to predict in vitro. This model can also be used to study new vaccines targeted to RT with drug-induced mutations. In conclusion we were able to induce immune responses to both the CCR5 and the RT gene products. CCR5 specific antibodies induced unpredicted virus evolution to a higher receptor affinity. RT specific immune responses partially protected mice from HIV-1/MuLV infection. A primate model for HIV-1 drug resistance was successfully developed. This model may be used in future studies of vaccines directed to RT with drug-induced mutations.

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