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

Pre-transplant immune factors may be associated with BK polyomavirus reactivation in kidney transplant recipients

BK polyomavirus (BKPyV) reactivation in kidney transplant recipients can lead to allograft damage and loss. The elements of the adaptive immune system that are permissive of reactivation and responsible for viral control remain incompletely described. We performed a prospective study evaluating BKPyV-specific T-cell response, humoral response and overall T-cell phenotype beginning pre-transplant through one year post-transplant in 28 patients at two centers. We performed an exploratory analysis of risk factors for the development of viremia and viruria as well as compared the immune response to BKPyV in these groups and those who remained BK negative. 6 patients developed viruria and 3 developed viremia. BKPyV-specific CD8+ T-cells increased post-transplant in viremic and viruric but not BK negative patients. BKPyV-specific CD4+ T-cells increased in viremic, but not viruric or BK negative patients. Anti-BKPyV IgG antibodies increased in viruric and viremic patients but remained unchanged in BK negative patients. Viremic patients had a greater proportion of CD8+ effector cells pre-transplant and at 12 months post-transplant. Viremic patients had fewer CD4+ effector memory cells at 3 months post-transplant. Exploratory analysis demonstrated lower CD4 and higher total CD8 proportions, higher anti-BKPyV antibody titers and the cause of renal failure were associated BKPyV reactivation. In conclusion, low CD4, high CD8 and increased effector CD8 cells were found pre-transplant in patients who became viremic, a phenotype associated with immune senescence. This pre-transplant T-cell senescence phenotype could potentially be used to identify patients at increased risk of BKPyV reactivation

JC Virus Latency in the Brain and Extraneural Organs of Patients with and without Progressive Multifocal Leukoencephalopathy▿

JC virus (JCV) is latent in the kidneys and lymphoid organs of healthy individuals, and its reactivation in the context of immunosuppression may lead to progressive multifocal leukoencephalopathy (PML). Whether JCV is present in the brains or other organs of healthy people and in immunosuppressed patients without PML has been a matter of debate. We detected JCV large T DNA by quantitative PCR of archival brain samples of 9/24 (38%) HIV-positive PML patients, 5/18 (28%) HIV-positive individuals, and 5/19 (26%) HIV-negative individuals. In the same samples, we detected JCV regulatory region DNA by nested PCR in 6/19 (32%) HIV-positive PML patients, 2/11 (18%) HIV-positive individuals, and 3/17 (18%) HIV-negative individuals. In addition, JCV DNA was detected in some spleen, lymph node, bone, and kidney samples from the same groups. In situ hybridization data confirmed the presence of JCV DNA in the brains of patients without PML. However, JCV proteins (VP1 or T antigen) were detected mainly in the brains of 23/24 HIV-positive PML patients, in only a few kidney samples of HIV-positive patients, with or without PML, and rarely in the bones of HIV-positive patients with PML. JCV proteins were not detected in the spleen or lymph nodes in any study group. Furthermore, analysis of the JCV regulatory region sequences showed both rearranged and archetype forms in brain and extraneural organs in all three study groups. Regulatory regions contained increased variations of rearrangements correlating with immunosuppression. These results provide evidence of JCV latency in the brain prior to severe immunosuppression and suggest new paradigms in JCV latency, compartmentalization, and reactivation

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

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 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

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

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