Detection of JC Virus-Specific Immune Responses in a Novel Humanized Mouse Model

Progressive Multifocal Leukoencephalopathy (PML) is an often fatal disease caused by the reactivation of the JC virus (JCV). Better understanding of viral-host interactions has been hampered by the lack of an animal model. Engrafting NOD/SCID/IL-2-Rg (null) mice with human lymphocytes and thymus, we generated a novel animal model for JCV infection. Mice were inoculated with either a PML isolate, JCV Mad-4, or with JCV CY, found in the kidney and urine of healthy individuals. While mice remained asymptomatic following inoculation, JCV DNA was occasionally detected in both the blood and the urine compartments. Mice generated both humoral and cellular immune responses against JCV. Expressions of immune exhaustion marker, PD-1, on lymphocytes were consistent with response to infection. Using this model we present the first in vivo demonstration of virological and immunological differences between JCV Mad-4 and CY. This model may prove valuable for studying JCV host immune responses

Detection of JCV DNA in the urine and blood of JCV inoculated humanized BLT mice.

<p>Only positive data are shown. (A) Urine samples were collected day 7, 22, 35, 49, 77, 91, and 104 post inoculation. JCV CY inoculation resulted in an early detection of JCV DNA in the urine on day 7 compared to first detection of JCV Mad-4 in the urine on day 77. (B) Blood samples were collected 24, 44, 65, 86, and 106 days post inoculation. Inoculation with JCV Mad-4 resulted in more frequent detection of JCV DNA in the blood compared to JCV CY. Unique symbols are used for individual mice; CY: JCV CY; Mad-4: JCV Mad-4.</p

Detection of anti-JCV immune response in JCV-inoculated humanized mouse.

A<p>Mouse reinoculated with virus then sacrificed 40 hours after reinoculation.</p>B<p>Mouse reinoculated with virus then sacrificed 6 days after reinoculation.</p>C<p>IgM detected on day 67, but reverted to negative at sacrifice 96 days after inoculation.</p

JCV-inoculated humanized BLT mice showed rare detection of JCV VP1 protein in the kidney.

<p>Immunohistochemistry staining of JCV VP1 protein was not detected in the kidney of PBS-inoculated mice (A), but was detected in rare kidney cells in JCV Mad-4 inoculated mice (B). The images are magnified 40-fold, and the inset is magnified 100-fold. Scale bar = 100 µm.</p

Humanized BLT mice inoculated with JCV displayed cellular immune responses against JCV VP1 antigens.

<p>(A) Intracellular staining (ICS) of splenocytes detected an increased IFN-γ expression on both CD4⁺ and CD8⁺ T cells, after stimulation with JCV peptides. (B) Culturing splenocytes with JCV capsid protein VP1 peptide pools for 12 days and then stimulated with the peptide pool a second time for ICS increased IFN-γ expression on both CD4⁺ and CD8⁺ T cells in a mouse inoculated with Mad-4, but not in a PBS-injected mouse. (C) Tetramer staining detected JCV VP1 epitope-specific CD8⁺ T cells after stimulation with A*0201-restricted JCV VP1 p100 peptide in mice inoculated with either JCV Mad-4 or CY, but not in a PBS-injected mouse. Percentages of positive cells are indicated.</p

JCV-inoculated humanized BLT mice showed increased expression of the cell exhaustion marker, PD-1, on splenocytes.

<p>PD-1 expression was measured on splenocytes after stimulation with JCV VP1 peptide pools. A significantly higher percentage of CD4⁺ and CD8⁺ T cells expressed PD-1 in either the JCV Mad-4 or CY mice as compared to the PBS mice. Bars illustrate the means and standard deviation above the means in each group.</p

Humoral immune responses in JCV-inoculated humanized BLT mice.

<p>Anti-JCV IgM were detected 42–103 days post inoculation. JCV Mad-4 inoculation elicited a stronger humoral immune response than JCV CY inoculation. Quantitative IgM values are expressed on a logarithmic scale. Dashed line: cut-off for positive values (OD450 nm = 0.039).</p

Antigen recognition-triggered drug delivery mediated by nanocapsule-functionalized cytotoxic T-cells

Cytotoxic T-Lymphocytes (CTLs) kill pathogen-infected or transformed cells following interaction of their T-cell receptors (TCRs) with foreign (e.g. virus-derived) peptides bound to MHC-I molecules on the target cell. TCR binding triggers CTLs to secrete perforin, which forms pores in the target cell membrane, promoting target death. Here, we show that by conjugating drug-loaded lipid nanoparticles to the surface of CTLs, their lytic machinery can be co-opted to lyse the cell-bound drug carrier, providing triggered release of drug cargo upon target cell recognition. Protein encapsulated in T-cell-bound nanoparticles was released following culture of CTLs with target cells in an antigen dose- and perforin-dependent manner and coincided with target cell lysis. Using this approach, we demonstrate the capacity of HIV-specific CTLs to deliver an immunotherapeutic agent to an anatomical site of viral replication. This strategy provides a novel means to couple drug delivery to the action of therapeutic cells in vivo.Ragon Institute of MGH, MIT and HarvardNational Institutes of Health (U.S.) (IH (AI111860