Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation

<div><p>A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.</p></div

Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.

<p>Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.</p

GFP-specific T cell responses as spot forming cells (SFC)/10⁶ PBMC after rapamycin treatment.

<p>T cell responses in PBMC were measured by ELISPOT at 14, 28, and 84 days after AAV9/GFP delivery in (A) AAV9/GFP only (controls); (B) AAV9/GFP + rapamycin. Arrow indicates undetectable (< 10 SFC/10⁶ PBMC) GFP-specific responses at necropsy (84 days).</p

GFP expression in lumbar spinal cords of NHPs monitored by IHC.

<p>GFP expression in lumbar spinal cords of NHPs monitored by IHC.</p

IHC was carried out to visualize GFP expression in the lumbar spinal cord.

<p>Shown are representative 40 micron lumbar spinal cord sections of all study macaques, stained for GFP. Magnified insets show examples of GFP-positive motor neurons. Macaque ID numbers are provided in each panel, along with a qualitative scoring of ventral horn expression. The (-) indicates no GFP-positive staining above that seen in uninjected NHPs; (+) indicates some positive cells observed, above that seen in uninjected macaques; (++) indicates relatively strong expression in a large number of neurons.</p

Cytotoxic GFP-specific T cells decreased after rapamycin treatment.

<p>Multiparameter flow cytometry was used to identify CD8+ CD107+ T cells in PBMC after stimulation with GFP peptide pools. CD8+CD107+ expression in PBMC was monitored 28 and 84 days after transgene delivery in (A) AAV9/GFP (controls); (B) AAV9/GFP and rapamycin.</p

Magnitude and breadth of GFP-specific T cell responses reduced after treatment with rapamycin.

<p>Multiparameter flow cytometry to assess T cell responses was performed using peptide (GFP or AAV9 peptide pools)-stimulated PBMC. PBMC were stained with antibodies to detect the following markers: CD4, CD8, IFN-γ, IL2, Ki67, and TNFα. Panels A and B depict GFP- specific T cell responses in two treatment groups: AAV9/GFP (controls) and AAV9/GFP + rapamycin. Panels C and D depict AAV9-specific T cell responses.</p