Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.

Potent antiretroviral activities and a barrier to viral resistance characterize the human immunodeficiency virus type one (HIV-1) integrase strand transfer inhibitor dolutegravir (DTG). Herein, a long-acting parenteral DTG was created through chemical modification to improve treatment outcomes. A hydrophobic and lipophilic modified DTG prodrug is encapsulated into poloxamer nanoformulations (NMDTG) and characterized by size, shape, polydispersity, and stability. Retained intracytoplasmic NMDTG particles release drug from macrophages and attenuate viral replication and spread of virus to CD4+ T cells. Pharmacokinetic tests in Balb/cJ mice show blood DTG levels at, or above, its inhibitory concentration90 of 64 ng/mL for 56 days, and tissue DTG levels for 28 days. NMDTG protects humanized mice from parenteral challenge of the HIV-1ADA strain for two weeks. These results are a first step towards producing a long-acting DTG for human use by affecting drug apparent half-life, cell and tissue drug penetration, and antiretroviral potency.

Long-acting parenteral (LAP) antiretroviral drugs have generated considerable interest for treatment and prevention of HIV-1 infection. One new LAP is cabotegravir (CAB), a highly potent integrase inhibitor, with a half-life of up to 54 days, allowing for every other month parenteral administrations. Despite this excellent profile, high volume dosing, injection site reactions and low body fluid drug concentrations affect broad use for virus infected and susceptible people. To improve the drug delivery profile, we created a myristoylated CAB prodrug (MCAB). MCAB formed crystals that were formulated into nano-particles (NMCAB) of stable size and shape facilitating avid monocyte-macrophage entry, retention and reticuloendothelial system depot formulation. Drug release kinetics paralleled sustained protection against HIV-1 challenge. After a single 45 mg/kg intramuscular injection to BALB/cJ mice, the NMCAB pharmacokinetic profiles was 4-times greater than that recorded for CAB LAP. These observations paralleled replicate measurements in rhesus macaques. The results coupled with improved viral restriction in human adult lymphocyte reconstituted NOD/SCID/IL2Rγc−/− mice led us to conclude that NMCAB can improve biodistribution and viral clearance profiles upon current CAB LAP formulations.

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human.

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8+ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.