Immunogenic particles with a broad antigenic spectrum stimulate cytolytic T cells and offer increased protection against EBV infection ex vivo and in mice.

The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection

Fbxo28 promotes mitotic progression and regulates topoisomerase IIα-dependent DNA decatenation

<p>Topoisomerase IIα is an essential enzyme that resolves topological constraints in genomic DNA. It functions in disentangling intertwined chromosomes during anaphase leading to chromosome segregation thus preserving genomic stability. Here we describe a previously unrecognized mechanism regulating topoisomerase IIα activity that is dependent on the F-box protein Fbxo28. We find that Fbxo28, an evolutionarily conserved protein, is required for proper mitotic progression. Interfering with Fbxo28 function leads to a delay in metaphase-to-anaphase progression resulting in mitotic defects as lagging chromosomes, multipolar spindles and multinucleation. Furthermore, we find that Fbxo28 interacts and colocalizes with topoisomerase IIα throughout the cell cycle. Depletion of Fbxo28 results in an increase in topoisomerase IIα−dependent DNA decatenation activity. Interestingly, blocking the interaction between Fbxo28 and topoisomerase IIα also results in multinucleated cells. Our findings suggest that Fbxo28 regulates topoisomerase IIα decatenation activity and plays an important role in maintaining genomic stability.</p

Adoptive transfer of EBV specific CD8+ T cell clones can transiently control EBV infection in humanized mice

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development

Deletion of the BART miRNAs enhances spontaneous lytic replication and tumor progression in the humanized mice model.

<p>Viral titers in peripheral blood of infected mice were determined by quantitative PCR at (A) 5 weeks post-infection. (B) The pictures show tumors that developed in the spleen. Continuous tissue sections were stained with hematoxylin and eosin (H&E), immunostained with antibodies specific for BZLF1, gp350, LMP1, EBNA2, or subjected to an <i>in situ</i> hybridization with an EBER-specific probe. Among five M81-Wt-infected mice, 3 mice (referred to as group I) had very few whilst the other 2 mice (referred to as group II) exhibited a higher percentage of BZLF1-positive cells. (C) The number of EBER positive cells per 0.04μm² (surface of the field at high magnification) is given in this boxplot. (D-G) The boxplots display the ratio between (D) BZLF1-, (E) gp350-, (F) LMP1-, or (G) EBNA2-positive cells versus EBER-positive cells. The data collected from the mice euthanized at week 5 are shown as open squares. (H) This graph shows the tumor incidence for humanized mice investigated in this study. We used a one-tailed Chi-square analysis in figure H and two-tailed unpaired student t test for all other results.</p

The miRNA subcluster1 is mainly but not exclusively responsible for the control of BZLF1 expression.

<p>The figure shows Western blot analyses of LCLs generated with M81, M81/ΔAll, M81/ΔC1, M81/ΔC2, M81/ΔC1C2, M81/Δb2, M81/ΔZR with a BZLF1-specific antibody. The LCLs were stained at 42 (A) and 101 (B) days post-infection. The relative intensity of the signals were quantified using the ImageJ software and are also displayed as a graph of bars. One more sample is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005344#ppat.1005344.s005" target="_blank">S5 Fig</a>. (C) and (D) We determined BALF5 expression level in multiple LCLs transformed with M81 and M81/ΔAll (C) and M81/Δb2 by western blot (D), and depict the results as a graph of bars after Image J quantification. All LCLs shown in (C) and (D) were investigated between 40–43 days post infection.</p

B cells infected by viruses that lack the BART miRNAs express higher levels of caspase 3 and LMP1 and are more resistant to drugs that induce mitochondria-mediated apoptosis.

<p>(A) LCLs transformed by M81 or M81/ΔAll from 4 independent donors were subjected to immunoblotting with antibodies specific to viral latent proteins (EBNA3A, 3B, 3C, EBNA2, LMP1 and LMP2A), viral lytic proteins (BZLF1), caspase 3, and actin. The levels of expression of these proteins are also represented in a bar chart. (B and C) Apoptosis was induced in five pairs of LCLs transformed by M81 or M81/ΔAll. Scatter plots represent the percentage of apoptotic cells as determined by TUNEL assays (B) or by immunostaining with antibodies directed against cleaved caspase 3 (C). DMSO or ethanol-treated samples were used as controls. P values lower than 0.05 obtained after paired t-student tests are indicated. Please also refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005344#ppat.1005344.s007" target="_blank">S7</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005344#ppat.1005344.s008" target="_blank">S8</a> Figs.</p

Replicating cells produce lower levels of EBV viral miRNAs than non-replicating counterparts.

<p>(A) CD2-positive cells were isolated from LCLs generated with a M81 mutant that expresses a truncated form of rat CD2 behind an EA-D-responsive promoter. CD2-positive or CD2-negative cell populations were submitted to RT-qPCR to assess BZLF1 mRNA expression (top graph) and to a western blot analysis with a BZLF1-specific antibody (Bottom picture). (B) The scatter plot shows expression of 6 viral microRNAs extracted from CD2-positive or CD2-negative cell populations obtained from 6 different LCLs generated with the CD2-expressing virus. (C) The experiment described in (B) was repeated with 3 cellular miRNAs expressed in EBV-transformed B cells. P values lower than 0.05, 0.01, 0.001, and 0.0001 obtained after paired t-student tests are indicated as *, **, ***, and **** in the figure.</p

The deletion of BART miRNAs enhances virus production in infected B cells.

<p>(A) This figure shows a western blot analysis performed a different time points on B cells from the same donor transformed with M81, M81/ΔAll or ΔZR with antibodies specific for gp350 and actin. The upper picture shows expression of gp350 and of its alternative spliced form gp220 in these LCLs. The relative intensity of the signals was quantified using the ImageJ software and is depicted in a graph of bars. (B) We generated LCLs by exposing B cells from 6 different donors to M81 or M81/ΔAll. These cells were immunostained with antibodies specific for gp350 as exemplified in the top pictures. The adjacent scatter plot shows the percentage of gp350-positive cells, including cells producing gp350 and B cells covered by virions, in these LCLs at different days post infection. The figure also shows the p values obtained from paired t tests performed with the two types of LCLs. (C) We quantified the EBV DNA load in supernatants from three couple of LCLs obtained by infection with M81 or M81/ΔAll by qPCR and show the results in this scatterplot. The p values of paired t tests performed with the different types of supernatants are indicated. (D) This graph gives the result of B-cell transformation assays that were performed by exposing primary B cells to supernatants from three different LCLs obtained with M81 or M81/ΔAll virions.</p