Identification and cloning of a new western Epstein-Barr virus strain that replicates efficiently in primary B cells.

The Epstein-Barr virus (EBV) causes human cancers, and epidemiological studies have shown that lytic replication is a risk factor for some of these tumors. This fits with the observation that EBV M81, which was isolated from a Chinese patient with nasopharyngeal carcinoma, induces potent virus production and increases the risk of genetic instability in infected B cells. To find out whether this property extends to viruses found in other parts of the world, we investigated 22 viruses isolated from Western patients. While one-third of the viruses hardly replicated, the remaining viruses showed variable levels of replication, with three isolates replicating at levels close to that of M81 in B cells. We cloned one strongly replicating virus into a bacterial artificial chromosome (BAC); the resulting recombinant virus (MSHJ) retained the properties of its nonrecombinant counterpart and showed similarities to M81, undergoing lytic replication in vitro and in vivo after 3 weeks of latency. In contrast, B cells infected with the nonreplicating Western B95-8 virus showed early but abortive replication accompanied by cytoplasmic BZLF1 expression. Sequencing confirmed that rMSHJ is a Western virus, being genetically much closer to 695-8 than to M81. Spontaneous replication in rM81- and rMSHJ-infected B cells was dependent on phosphorylated Btk and was inhibited by exposure to ibrutinib, opening the way to clinical intervention in patients with abnormal EBV replication. As rMSHJ contains the complete EBV genome and induces lytic replication in infected B cells, it is ideal to perform genetic analyses of all viral functions in Western strains and their associated diseases.IMPORTANCE The Epstein-Barr virus (EBV) infects the majority of the world population but causes different diseases in different countries. Evidence that lytic replication, the process that leads to new virus progeny, is linked to cancer development is accumulating. Indeed, viruses such as M81 that were isolated from Far Eastern nasopharyngeal carcinomas replicate strongly in B cells. We show here that some viruses isolated from Western patients, including the MSHJ strain, share this property. Moreover, replication of both M81 and of MSHJ was sensitive to ibrutinib, a commonly used drug, thereby opening an opportunity for therapeutic intervention. Sequencing of MSHJ showed that this virus is quite distant from M81 and is much closer to nonreplicating Western viruses. We conclude that Western EBV strains are heterogeneous, with some viruses being able to replicate more strongly and therefore being potentially more pathogenic than others, and that the virus sequence information alone cannot predict this property

Variable morphology of human immunodeficiency virus-associated lymphomas with <em>C-MYC</em> rearrangements.

Burkitt lymphoma (BL) and immunoblastic lymphoma (IL) are the most frequent lymphoid tumors encountered in human immunodeficiency virus (HIV)-infected patients. Tumors with a morphology intermediate between BL and IL, and the existence of Burkitt&#39;s type translocations in some IL cases makes the classification of these tumors sometimes unclear. We have studied 14 cases of BL and IL in HIV-seropositive individuals with regard to clonality, Epstein-Barr virus (EBV) association, and the presence of c-myc rearrangement. Of seven tumors with morphology of BL, all were monoclonal, six showed a c-myc rearrangement and four were associated with EBV. Five tumors with morphology of IL were associated with EBV and devoid of c-myc rearrangement. Three were polyclonal representing EBV-driven lymphoproliferations similar to those observed in transplant recipients. Two tumors, one with a morphology of IL and the other intermediate between IL and BL were monoclonal, associated with EBV, and harbored a c-myc rearrangement. We propose that these last two tumors represent cases of BL that have adopted an immunoblastic morphotype in the context of acquired immunodeficiency syndrome (AIDS), reflecting the morphologic evolution of Burkitt lymphoma cells observed in culture

Mitogenicity of anti-Thy-1 monoclonal antibodies attributable to an Fc-dependent mechanism.

We analyzed the mechanism by which certain anti-Thy-1 monoclonal antibodies (mAb) activate T cells directly without additional stimuli. Using a panel of rat anti-Thy-1 antibodies which included more than 30 IgG2c mAb, we found that only the IgG2c isotype was able to induce a strong proliferative response in both resting T cells and a T cell lymphoma, suggesting that this form of T cell activation is isotype restricted and might be a consequence of a unique physico-chemical property of the IgG2c heavy chain. Results from surface distribution studies of Thy-1 molecules, following specific interactions with anti-Thy-1 antibodies of different isotypes, again showed that only IgG2c mAb formed Thy-1 aggregates of high valence on the surface of a T cell lymphoma, and such clustering always evoked a biological response. This led us to propose that IgG2c mAb have the inherent tendency to self-associate, probably through homophilic Fc-Fc contacts, and that this feature renders anti-Thy-1 mAb mitogenic. To prove this, we set up cross-inhibition studies with randomly selected mitogenic (IgG2c) and non-mitogenic (IgG2b) anti-Thy-1 mAb. The results clearly demonstrated that IgG2c antibodies enhance their own binding, analogous to the new form of antibody binding that was recently demonstrated between murine IgG3 mAb and a multivalent antigen. Confirmation of this was also provided by IgG2c-derived F(ab&prime;)2 fragments, which were unable to cause proliferation. Furthermore, masking the Fc part of cell-bound IgG2c mAb with a monomeric and thus non-aggregating IgG-binding protein A-derived fragment cancelled their mitogenic ability. Finally, induction of T cell proliferation appeared to be independent of cross-linking via Fc&gamma;R. The results support a model in which noncovalent intermolecular homophilic contacts of the Fc regions of the IgG2c isotype bring about effective aggregation of Thy-1 molecules, thereby stimulating the mitotic apparatus of the cell

Standardized and highly efficient expansion of Epstein-Barr virus-specific CD4+ T cells by using virus-like particles.

Epstein-Barr virus (EBV)-specific T-cell lines generated by repeated stimulation with EBV-immortalized lymphoblastoid B-cell lines (LCL) have been successfully used to treat EBV-associated posttransplant lymphoproliferative disease (PTLD) in hematopoietic stem cell transplant recipients. However, PTLD in solid-organ transplant recipients and other EBV-associated malignancies respond less efficiently to this adoptive T-cell therapy. LCL-stimulated T-cell preparations are polyclonal and contain CD4(+) and CD8(+) T cells, but the composition varies greatly between lines. Because T-cell lines with higher CD4(+) T-cell proportions show improved clinical efficacy, we assessed which factors might compromise the expansion of this T-cell population. Here we show that spontaneous virus production by LCL and, hence, the presentation of viral antigens varies intra- and interindividually and is further impaired by acyclovir treatment of LCL. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4(+) T cells, whereas human serum from EBV-seropositive donors diminished viral antigen presentation. To overcome these limitations, we used peripheral blood mononuclear cells pulsed with nontransforming virus-like particles as antigen-presenting cells. This strategy facilitated the specific and rapid expansion of EBV-specific CD4(+) T cells and, thus, might contribute to the development of standardized protocols for the generation of T-cell lines with improved clinical efficacy

Epstein&ndash;barr virus: From the detection of sequence polymorphisms to the recognition of viral types.

The Epstein&ndash;Barr virus is etiologically linked with the development of benign and malignant diseases, characterized by their diversity and a heterogeneous geographic distribution across the world. The virus possesses a 170- kb-large genome that encodes for multiple proteins and non-coding RNAs. Early on there have been numerous attempts to link particular diseases with particular EBV strains, or at least with viral genetic polymorphisms. This has given rise to a wealth of information whose value has been difficult to evaluate for at least four reasons. First, most studies have looked only at one particular gene and missed the global picture. Second, they usually have not studied sufficient numbers of diseased and control cases to reach robust statistical significance. Third, the functional significance of most polymorphisms has remained unclear, although there are exceptions such as the 30-bp deletion in LMP1. Fourth, different biological properties of the virus do not necessarily equate with a different pathogenicity. This was best illustrated by the type 1 and type 2 viruses that markedly differ in terms of their transformation abilities, yet do not seem to cause different diseases. Reciprocally, environmental and genetic factors in the host are likely to influence the outcome of infections with the same virus type. However, with recent developments in recombinant virus technology and in the availability of high throughput sequencing, the tide is now turning. The availability of 23 complete or nearly complete genomes has led to the recognition of viral subtypes, some of which possess nearly identical genotypes. Furthermore, there is growing evidence that some genetic polymorphisms among EBV strains markedly influence the biological and clinical behavior of the virus. Some virus strains are endowed with biological properties that explain crucial clinical features of patients with EBV-associated diseases. Although we now have a better overview of the genetic diversity within EBV genomes, it has also become clear that defining phenotypic traits evinced by cells infected by different viruses usually result from the combination of multiple polymorphisms that will be difficult to identify in their entirety. However, the steadily increasing number of sequenced EBV genomes and cloned EBV BACS from diseased and healthy patients will facilitate the identification of the key polymorphisms that condition the biological and clinical behavior of the viruses. This will allow the development of preventative and therapeutic approaches against highly pathogenic viral strains

Immunoinformatic analysis reveals antigenic heterogeneity of Epstein-Barr virus is immune-driven.

Whole genome sequencing of Epstein-Barr virus (EBV) isolates from around the world has uncovered pervasive strain heterogeneity, but the forces driving strain diversification and the impact on immune recognition remained largely unknown. Using a data mining approach, we analyzed more than 300 T-cell epitopes in 168 published EBV strains. Polymorphisms were detected in approximately 65% of all CD8+ and 80% of all CD4+ T-cell epitopes and these numbers further increased when epitope flanking regions were included. Polymorphisms in CD8+ T-cell epitopes often involved MHC anchor residues and resulted in changes of the amino acid subgroup, suggesting that only a limited number of conserved T-cell epitopes may represent generic target antigens against different viral strains. Although considered the prototypic EBV strain, the rather low degree of overlap with most other viral strains implied that B95.8 may not represent the ideal reference strain for T-cell epitopes. Instead, a combinatorial library of consensus epitopes may provide better targets for diagnostic and therapeutic purposes when the infecting strain is unknown. Polymorphisms were significantly enriched in epitope versus non-epitope protein sequences, implicating immune selection in driving strain diversification. Remarkably, CD4+ T-cell epitopes in EBNA2, EBNA-LP, and the EBNA3 family appeared to be under negative selection pressure, hinting towards a beneficial role of immune responses against these latency type III antigens in virus biology. These findings validate this immunoinformatics approach for providing novel insight into immune targets and the intricate relationship of host defense and virus evolution that may also pertain to other pathogens

The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation.

The Epstein-Barr virus (EBV) transforms B cells by expressing latent proteins and the BHRF1 microRNA cluster. MiR-BHRF1-3, its most transforming member, belongs to the recently identified group of weakly expressed microRNAs. We show here that miR-BHRF1-3 displays an unusually low propensity to form a stem-loop structure, an effect potentiated by miR-BHRF1-3&#39;s proximity to the BHRF1 polyA site. Cloning miR-BHRF1-2 or a cellular microRNA, but not a ribozyme, 5&#39; of miR-BHRF1-3 markedly enhanced its expression. However, a virus carrying mutated miR-BHRF1-2 seed regions expressed miR-BHRF1-3 at normal levels and was fully transforming. Therefore, miR-BHRF1-2&#39;s role during transformation is independent of its seed regions, revealing a new microRNA function. Increasing the distance between miR-BHRF1-2 and miR-BHRF1-3 in EBV enhanced miR-BHRF1-3&#39;s expression but decreased its transforming potential. Thus, the expression of some microRNAs must be restricted to a narrow range, as achieved by placing miR-BHRF1-3 under the control of miR-BHRF1-2

Antibodies conjugated with viral antigens elicit a cytotoxic T cell response against primary CLL ex vivo.

Chronic lymphocytic leukemia (CLL) is the most frequent B cell malignancy in Caucasian adults. The therapeutic armamentarium against this incurable disease has recently seen a tremendous expansion with the introduction of specific pathway inhibitors and innovative immunotherapy. However, none of these approaches is curative and devoid of side effects. We have used B-cell-specific antibodies conjugated with antigens (AgAbs) of the Epstein-Barr virus (EBV) to efficiently expand memory CD4(+) cytotoxic T lymphocytes (CTLs) that recognized viral epitopes in 12 treatment-naive patients with CLL. The AgAbs carried fragments from the EBNA3C EBV protein that is recognized by the large majority of the population. All CLL cells pulsed with EBNA3C-AgAbs elicited EBV-specific T cell responses, although the intensity varied across the patient collective. Interestingly, a large proportion of the EBV-specific CD4(+) T cells expressed granzyme B (GrB), perforin, and CD107a, and killed CLL cells loaded with EBV antigens with high efficiency in the large majority of patients. The encouraging results from this preclinical ex vivo study suggest that AgAbs have the potential to redirect immune responses toward CLL cells in a high percentage of patients in vivo and warrant the inception of clinical trials