Epstein-Barr Functional Mimicry: Pathogenicity of Oncogenic Latent Membrane Protein-1 in Systemic Lupus Erythematosus and Autoimmunity.

Systemic lupus erythematosus (SLE) and other autoimmune diseases are propelled by immune dysregulation and pathogenic, disease-specific autoantibodies. Autoimmunity against the lupus autoantigen Sm is associated with cross-reactivity to Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1). Additionally, EBV latent membrane protein-1 (LMP1), initially noted for its oncogenic activity, is an aberrantly active functional mimic of the B cell co-stimulatory molecule CD40. Mice expressing a transgene (Tg) for the mCD40-LMP1 hybrid molecule (containing the cytoplasmic tail of LMP1) have mild autoantibody production and other features of immune dysregulation by 2-3 months of age, but no overt autoimmune disease. This study evaluates whether exposure to the EBV molecular mimic, EBNA-1, stimulates antigen-specific and concurrently-reactive humoral and cellular immunity, as well as lupus-like features. After immunization with EBNA-1, mCD40-LMP1 Tg mice exhibited enhanced, antigen-specific, cellular and humoral responses compared to immunized WT congenic mice. EBNA-1 specific proliferative and inflammatory cytokine responses, including IL-17 and IFN-γ, were significantly increased (p<0.0001) in mCD40-LMP1 Tg mice, as well as antibody responses to amino- and carboxy-domains of EBNA-1. Of particular interest was the ability of mCD40-LMP1 to drive EBNA-1 associated molecular mimicry with the lupus-associated autoantigen, Sm. EBNA-1 immunized mCD40-LMP1 Tg mice exhibited enhanced proliferative and cytokine cellular responses (p<0.0001) to the EBNA-1 homologous epitope PPPGRRP and the Sm B/B' cross-reactive sequence PPPGMRPP. When immunized with the SLE autoantigen Sm, mCD40-LMP1 Tg mice again exhibited enhanced cellular and humoral immune responses to both Sm and EBNA-1. Cellular immune dysregulation with EBNA-1 immunization in mCD40-LMP1 Tg mice was accompanied by enhanced splenomegaly, increased serum blood urea nitrogen (BUN) and creatinine levels, and elevated anti-dsDNA and antinuclear antibody (ANA) levels (p<0.0001 compared to mCD40 WT mice). However, no evidence of immune-complex glomerulonephritis pathology was noted, suggesting that a combination of EBV and genetic factors may be required to drive lupus-associated renal disease. These data support that the expression of LMP1 in the context of EBNA-1 may interact to increase immune dysregulation that leads to pathogenic, autoantigen-specific lupus inflammation.

[Construction and immunological responses of recombinant adenovirus containing Epstein-Barr nuclear antigen 1 in mice].

This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.

Therapeutic vaccination against the rhesus lymphocryptovirus EBNA-1 homologue, rhEBNA-1, elicits T cell responses to novel epitopes in rhesus macaques.

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC-rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination.

A monoclonal antibody that recognizes Epstein-Barr virus nuclear antigen 2 (EBNA 2) amino acids 1-58 does not react with EBNA 2 in native form, consistent with the self-association of EBNA 2 through the amino-terminus.

We have generated a mouse IgG1 monoclonal antibody (mAb) that recognizes amino acids 1-58 of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA 2) of type 1 EBV strain B95-8. mAb Y101 also reacted with EBNA 2 of EBV type 2 strains MISP and Jijoye in immunoblots, whereas Jijoye EBNA 2 was not detected by the widely used mAb PE2. mAb Y101, in contrast to mAb PE2, reacted with faster migrated, hypophosphorylated proteins of type 1 EBNA 2 as intensely as slower migrated, hyperphosphorylated ones. mAb Y101 did not react in fixed-cell immunostaining or cell extract immunoprecipitation. The results implicate that the amino-terminal epitope is not exposed in a native form, consistent with the previously reported idea of self-association of EBNA 2 through the amino-terminus. mAb Y101 is the first mAb to the EBNA 2 amino-terminus and will be useful for further analyses of the structure and function of EBNA 2.

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) in the mouse can elicit the production of anti-dsDNA and anti-Sm antibodies.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the production of anti-nuclear antibodies. The etiology of SLE is unknown, although several viruses including the Epstein-Barr virus (EBV) have been implicated. An increase in the frequency of EBV infection has been observed in SLE patients relative to normal individuals. Some patients with SLE develop antibodies that recognize a proline rich epitope in the ribonucleoprotein Sm B/B that is similar to an epitope in EBNA-1, a major nuclear antigen of EBV. In the present study we have cloned the EBNA-1 gene under the control of the CMV promoter in the vector pcDNA3. We now report for the first time that expression of the entire EBNA-1 protein in the mouse can elicit the production of IgG antibodies to Sm and to double-stranded DNA (dsDNA). Our data suggest that the anti-Sm response arises as a consequence of antigenic cross-reactivity by anti-EBNA-1 antibodies. These results support a possible association between EBV infection and SLE.

EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) maintains the viral episome in all host cells infected with EBV. Recently, EBNA1 was found to be the main EBV latency antigen for CD4+ T cells and could be recognized in cultures from all donors tested. We now identify a polarized Th1 phenotype and obtain evidence for its presence in vivo. When T cells were stimulated with dendritic cells infected with vaccinia vectors expressing EBNA1, 18 of 19 donors secreted IFN-gamma, whereas only two of 19 secreted IL-4. Magnetic selection was then used to isolate cells from fresh blood based on EBNA1-induced cytokine production. Specific IFN-gamma CD4+ cell lines were established from six of six donors and IL-4 lines from three of six. Only the Th1 lines specifically lysed targets expressing three different sources of EBNA1 protein. When the IgG isotype of EBNA1 plasma Ab's was tested, most specific Ab's were IgG1 and of a high titer, confirming a Th1 response to EBNA1 in vivo. Ab's to other microbial antigens generally were not skewed toward IgG1. Given emerging evidence that Th1 CD4+ T cells have several critical roles in host defense to viral infection and tumors, we propose that EBNA1-specific CD4+ Th1 cells contribute to resistance to EBV and EBV-associated malignancies.