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More than 60 million people worldwide have been infected with human immunodeficiency virus (HIV), and nearly half of these individuals have died. The development of a safe and effective HIV vaccine is a global health priority, but will require improved understanding of the immunopathogenesis of HIV infection. An ideal animal model in which to study the pathogenesis of HIV infection has been elusive, however. Non-human primates susceptible to HIV infection typically do not develop immunodeficiency, and although simian immunodeficiency virus (SIV) infection of rhesus macaques has provided many critically important insights into retroviral pathogenesis, biological and financial considerations have created some limitations to how widely this system can be disseminated. The great need for an improved animal model of HIV itself has been recently underscored by the disappointing results of human trials of an adenovirus-based HIV vaccine, which was not effective and may actually have increased some subjects' risk of acquiring HIV. In the wake of these disappointing results, there has been increased interest in humanized mouse models of HIV infection, although the absence of robust anti-HIV human immune responses in these models to date has suggested that they are not yet ready to test immune responses to vaccine candidates. Recently, we and others have developed an improved humanized mouse model of HIV by transplanting human CD34+ stem cells and autologous human thymic grafts into immunodeficient mice. In this model, we have achieved robust repopulation of mouse lymphoid tissues with human immune cells, and have generated robust anti-HIV cellular and humoral immune responses in these humanized mice. We believe this improved humanized mouse model will allow us to study questions regarding the biology of HIV not readily approachable through human studies.
Currently, we are using our humanized mouse model of HIV infection to investigate mechanisms of viral dissemination, of HIV-induced immune activation, and of HIV-induced immune dysfunction. We are studying viral dissemination in vivo in collaboration with CIID investigator Thorsten Mempel, by visualizing the migratory behavior of both HIV itself and of human T cells and dendritic cells in the lymph nodes of our humanized mice using multiphoton intravital microscopy (MV-IVM). Using fluorescently labeled virus, we are investigating how HIV is delivered to the lymph node either as a free virus or a virus associated with dendritic cells or T cells. Using an HIV mutant inducing the expression of GFP in infected cells, we are investigating the anatomy of HIV spread within the lymph node over time. Chronic systemic immune activation is an almost pathognomonic feature of progressive HIV infection, and is one of the strongest predictors of disease progression. We are investigating the contributions of several proposed mechanisms of HIV-induced immune activation, including impairment of gut mucosal barrier function leading to increased bacterial translocation, depletion of regulatory T cells by HIV, and viral production of Toll-like receptor ligands. We are also using our humanized mouse model to investigate the role of the PD-1 pathway, a member of the B7-CD28 family of cell surface receptors that negatively regulates T cell activity, in immune dysfunction induced by HIV infection in vivo. We are testing the hypothesis that inhibiting the PD-1 pathway will reinvigorate “exhausted” CD8+ T cells in vivo and lead to better control of HIV replication.
We are also working to expand the versatility of our humanized mouse models by developing the ability to genetically modify the human CD34+ hematopoietic stem cells (HSCs) used to reconstitute these mice. HSC genetic modifications will be conferred to their human immune cell progeny in the reconstituted mice, which will provide us with a novel system to molecularly dissect the human immune system and its interaction with HIV and other pathogens in vivo. We are transducing human CD34+ cells using lentiviral vectors in which the gene of interest and green fluorescent protein (GFP) are under control of a phosphoglycerate kinase promoter (PGK) and of a human elongation factor promoter (hEF1a) respectively. These promoters allow for the promiscuous expression of both genes in every cell type derived from CD34+ cells, and the expression of GFP expression will allow us to tract the transduced cells. We have begun this project by cloning rhesus monkey or mutant human versions of the anti-HIV restriction factors tripartite motif protein 5a (TRIM5a), or the apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G (A3G), into lentiviral vectors. Both TRIM5a and A3G are interesting candidates for a gene therapy approach to control of HIV infection. We will use our humanized mice to evaluate the ability of rhesus and mutant human TRIM5a, and of mutant human A3G to inhibit HIV-1 infection in human cells in vivo.
In addition to our own investigations, we have recently initiated a humanized mouse core program for the Harvard University Center for AIDS Research (HU CFAR) and the Ragon Institute of MGH, MIT and Harvard, through which we are making humanized mice available to HU CFAR and Ragon collaborators. Collaborative projects already initiated include: 1) in vivo assessment of the fitness costs to HIV of CTL escape mutations; 2) assessment of the impact of immune activation by HIV-encoded TLR ligands on HIV immunopathogenesis; 3) assessment of the role of Th17 cells in protection against HIV infection; 4) use of HIV mosaic proteins to elicit T cell responses against target and escape epitopes; and 5) assessment of the efficacy of siRNA-based microbicides to protect against vaginal transmission of HIV.
Andrew Tager, MD
Vladimir Vrbanac, DVM
AAALAC-certified animal care facilities for the mice used in this project are on the 8th and 9th floor of the MGH research building at the Charlestown Navy Yard, and are supervised by a full-time veterinarian. These animal care facilities provide viral antibody free (VAF) areas for rodents with well-established containment procedures, and contain approximately 20,700 square feet of state-of-the-art animal housing space. The facilities have a 250 square foot biocontainment level 2 room in which mice are infected with HIV and maintained while HIV-infected. Group members of the Humanized Mouse Program have laboratory bench space, as well as associated tissue culture and support space, within the Center for Immunology and Inflammatory Diseases (CIID), in the Charlestown Navy Yard Research Facility of MGH’s East campus. The Center has 20,000 square feet of laboratory and support space, including five 300 square feet tissue culture rooms, with ten6’ biosafety hoods and twentyincubators. The CIID has the equipment necessary to perform a wide range of molecular and cellular techniques, all of which are available to Program group members. This includes a FACScan flow cytometer, a Stratagene MX4000 Real Time Quantitator PCR machine and a Nikon fluorescent microscope and Spot RT color digital camera and image video equipment, multiple thermal cyclers for PCR genotyping of DNA, electrophoresis power supplies and boxes, centrifuges and microcentrifuges, water baths, hybridizers, and 4°, -20°, -80°, and liquid nitrogen storage. A darkroom, glass washing facilities, cold and warm room, seminar room and library are also available.
Please contact Dr. Andrew Tager by e-mail.
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