ORCID ID

0000-0002-3726-2771

Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmacology and Experimental Neuroscience

First Advisor

Howard E. Gendelman

Abstract

Human immunodeficiency virus type one (HIV-1) exclusively infects humans and chimpanzees. Therefore, animal models are required for all investigations of viral pathogenesis, therapy and cure. Immunodeficient mice transplanted with human hematopoietic stem cells, termed humanized mice, support the development of multiple human cell lineages throughout the cell’s life span. To this end, we used humanized mice to evaluate the dynamic changes of host immunologic and viral profiles during both acute and chronic HIV-1 infection. We identified the temporal and spatial distribution of HIV-1 cell and tissue compartment infections and outlined correlations made between viral progression and host immunity by comparing two different humanized mouse models. Based on the discovery of early viral set-points, we developed a paradigm for a potential ‘HIV-1 cure’ by sequential administration of long-acting slow-effective release antiretroviral therapy (LASER ART) and CRISPR-Cas9 treatments. HIV-1 elimination was achieved in a third of dual-treated humanized mice after exhaustive works to detect HIV-1 and its gene products in peripheral blood, brain, lung, liver, spleen, kidney, gut, and bone marrow (BM). This was done by sensitive nested and digital-droplet PCR and RNAscope tests. Immune function was preserved in animals where HIV-1 was eliminated. In vivo viral outgrowth assay (VOA) was employed to confirm HIV-1 elimination by adoptive transfer of donor splenocytes and BM cells into naïve humanized mice. While virus was successfully recovered from HIV-1 control, LASER ART or CRISPR-Cas9 alone treated animals virus could not be recovered in recipients transplanted from a third of the dual-treated “cured” animals. We further characterized the impact of humanized mice-based VOA in viral isolation assay by expanding the sample sizes. We observed that replication-competent HIV-1 persisted in multiple tissues even when plasma viral load was undetectable and virus can be readily recovered from naïve humanized mice. We demonstrate that in vivo VOA using humanized mice is a sensitive assay system to interrogate replication competent HIV-1 and can be employed to determine viral eradication from donor samples. To this end, we used the humanized mouse model to study HIV-1 tissue compartmentalization, interrogate HIV-1 eradication employing innovative technologies, and assess HIV-1 elimination using humanized mice-based VOA, which will facilitate future translational studies.

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