Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

CD4 ligation on human blood monocytes triggers macrophage differentiation and enhances HIV infection.

UNLABELLED: A unique aspect of human monocytes, compared to monocytes from many other species, is that they express the CD4 molecule. However, the role of the CD4 molecule in human monocyte development and function is not known. We determined that the activation of CD4 via interaction with major histocompatibility complex class II (MHC-II) triggers cytokine expression and the differentiation of human monocytes into functional mature macrophages. Importantly, we determined that CD4 activation induces intracellular signaling in monocytes and that inhibition of the MAPK and Src family kinase pathways blocked the ability of CD4 ligation to trigger macrophage differentiation. We observed that ligation of CD4 by MHC-II on activated endothelial cells induced CD4-mediated macrophage differentiation of blood monocytes. Finally, CD4 ligation by MHC-II increases the susceptibility of blood-derived monocytes to HIV binding and subsequent infection. Altogether, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. Further, these events can have effects on HIV infection and persistence in the macrophage compartment. IMPORTANCE: The CD4 molecule, as the primary receptor for HIV, plays an important role in HIV pathogenesis. There are many cell types that express CD4 other than the primary HIV target, the CD4(+) T cell. Other than allowing HIV infection, the role of the CD4 molecule on human monocytes or macrophages is not known. We were interested in determining the role of CD4 in human monocyte/macrophage development and function and the potential effects of this on HIV infection. We identified a role for the CD4 molecule in triggering the activation and development of a monocyte into a macrophage following its ligation. Activation of the monocyte through the CD4 molecule in this manner increases the ability of monocytes to bind to and become infected with HIV. Our studies have identified a novel function for the CD4 molecule on peripheral monocytes in triggering macrophage development that has direct consequences for HIV infection.

In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells.

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.