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.

A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5′- LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 940 bp DNA fragment spanning between the LTR and Gag gene in spleen, liver, heart, lung, and kidney as well as in circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 upon delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau ("residual viremia") that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification.

The ligation of programmed death-ligand 1 (B7-H1) to T cells results in the preferential production of interleukin 10 (IL-10). We investigated if B7-H1 would be upregulated in HIV infection, a disease characterized by increased IL-10 production, by measuring B7-H1, B7-1 (CD80), and B7-2 (CD86) expression and mRNA in 36 HIV-infected patients and in 22 healthy controls (HCs). Results showed that (1) B7-H1 expression and mRNA are augmented in cells of HIV patients; (2) increased IL-10 production in these patients is largely induced by B7-H1-expressing CD14 ؉ cells; (3) an inverse correlation is detected between B7-H1 expression and CD4 counts, whereas the up-regulation of B7-H1 is directly associated with HIV plasma viremia; (4) antiviral therapy results in the parallel down modulation of IL-10 production and B7-H1 expression/ synthesis; and ( IntroductionThe activation of T lymphocytes is dependent on the presentation of processed antigenic peptides in association with major histocompatibility (MHC) molecules to lymphocytes that express a T-cell receptor specific for that binary complex. 1,2 However, optimal lymphocyte activation requires a second signal that is delivered by the interaction between costimulatory, accessory molecules. 3,4 The cross-linking of CD28 on the surface of T lymphocytes allows for the activation of these cells. CD28 binds to a family of ligands on the surface of non-T cells that are collectively known as B7 molecules. 5,6 Beside B7.1 (CD80) and B7.2 (CD86), a number of other B7-like ligands have been described. 7-9 Among these ligands, B7-H1 is particularly interesting. B7-H1, also called PD-L1, is constitutively present on monocytes and could be induced on activated T cells. 7 B7-H1 does not interact with CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), or inducible costimulator (ICOS) but was shown to bind to programmed death 1 (PD-1) 10,11 a CTLA-4-like molecule belonging to the immunoglobulin superfamily. Studies, however, suggested that receptors other than PD-1 can also ligate B7-H1. 7 Ligation of B7-H1 to T cells results in the preferential production of interleukin-10 (IL-10) 7 and in increased T-helper-dependent synthesis of trinitrophenyl (TNP)-specific immunoglobulin G2a (IgG2a) 12 in mice. These results suggest that ligation of B7-H1 may be responsible for promoting type 2 cytokine-biased responses.Interleukin-10 production by peripheral blood mononuclear cells (PBMCs) is augmented in infectious and noninfectious pathologies, including HIV disease. [13][14][15][16] In particular, cell-mediated immunity (CMI) is characteristically impaired in HIV infection. [17][18][19][20] The progression of this infection is associated with increased HIV replication, reduction of circulating CD4 ϩ T lymphocytes, functional defects of CMI, and augmented apoptosis of T lymphocytes. 21,22 An impairment in the production of type 1 cytokines accompanied by increased secretion of type 2 cytokines has also repeatedly been suggested to accompany progression of HIV disease. 14,16,23,24 Because IL-10, ...