↵5 Senior Research Fellow of the National Health and Medical Research Council of Australia. Therefore, effective antiviral drugs are needed to control the lytic phase of such acute infections in these patients. The KSHV replication and transcription activator (K-RTA) is necessary and sufficient for induction of KSHV lytic replication. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. BZLF1-expressing cells had increased p53-specific DNA binding activity in electrophoretic mobility shift assays, increased p53 phosphorylation at multiple residues (including serines 6, 9, 15, 33, 46, 315, and 392), and increased acetylation at lysine 320 and lysine 382. When sera were fractionated, this blocking effect was observed only in the serum immunoglobulin G fractions. Immunocytochemistry and confocal microscopy revealed that BILF1 localizes predominantly to the plasma membrane, similar to the localization of KSHV ORF74.
Moreover, HHV-8 could be serially passaged from HHV-8+/EBV+ LCLs to fresh PBMC. These findings suggest that the widely expressed A3C cytidine deaminase can function as a restriction factor for some human herpesviruses. The molecular events involved in the switch between the latent and lytic replication phases of the EBV life cycle can be studied in cultured lymphoblastoid or lymphoma cell lines. The eye of the American AIDS patient was diagnosed with primary intraocular lymphoma at necropsy. Therefore, PELs exhibit a restricted latency pattern, with expression of EBNA1 in all cases, and low LMP1 and LMP2A levels. Infection is often asymptomatic. The HIV protease inhibitor nelfinavir inhibits Kaposi’s sarcoma-associated herpesvirus replication in vitro.
EBV infection has been associated with the development of Buikitts’ lymphoma (BL), Hodgkin’s disease, nasopharyngeal carcinoma and others –. During primary EBV infection many lytic cycle genes are expressed which are likely responsible for stimulating the intense cellular immune response associated with acute infectious mononucleosis. During convalescence a minor population of circulating B cells remain latently infected, harbor multiple EBV episomes, and express only EBNA-1 and possibly LMP-2a (type-I latency). Thus, latency type-I infected B cells in-vivo express a much more restricted spectrum of latent proteins and are therefore not subject to elimination by the same virus-specific CTL as are type-III EBV latently infected cells. Accordingly, many mechanisms have been proposed to explain EBV persistence including; restricted expression of EBV latent genes, reduced levels of cellular adhesion molecules, downregulation of MHC class-I molecules, absence of EBNA-1 T-cell-epitopes, and most recently, EBNA-1-mediated inhibition of antigen processing. While these mechanisms may contribute to ineffective T cell surveillance against latency type-I EBV- infected cells, B cells expressing the full spectrum of latent proteins (type-III) also exist transiently in vivo and maintain detectable humoral and CTL responses to most latent proteins. Our first goal was to identify the virus-encoded immunodominant antigens recognized by in-vivo activated MHC class-I restricted CTL isolated from college students experiencing primary EBV infection, manifested as acute IM.
Following a prodromal period of several weeks, newly EBV infected patients present with signs and symptoms of acute IM, including elevated numbers of activated CD8+ T cells in their peripheral blood, many of which, like memory CTL, are EBV-specific and HLA-restricted. In order to address the issue of EBV persistence and the immune control of EBV-induced lymphoproliferation, we also studied the long-term EBV-specific memory CTL response in these same individuals. Blood from acute IM patients and healthy EBV seropositive donors served as a source of peripheral blood lymphocytes to generate bulk CTL cultures and autologous target cells. The infecting strain of EBV was determined for each patient by DNA-PCR amplification of virus from saliva. Lymphocytes were isolated from whole blood by Ficoll-Paque density centrifugation and T- and B-cell enriched populations were obtained by AET-sheep red cell rosette selection. Autologous B cell blasts served as a source of target cells and recombinant vaccinia virus constructs were used to introduce individual EBV latent genes into target cells. Expression of individual EBV genes in target cells was confirmed by both western blot and immunofluorescence.
Primary CTL responses to EBV were evaluated in standard 5lCr release assays using freshly isolated, T-cell enriched PBL from acute IM patients as effector cells. EBV-specific memory CTL responses were evaluated with bulk CTL culture generated by in-vitro restimulation with autologous B-LCLs. FACS analyses were routinely performed on bulk cultures of effector CTL populations in order to more clearly characterize their phenotype. Lastly, monoclonal antibody blocking studies and cold target competition assays were performed in order to accurately identify the viral antigen and MHC components responsible for target cell recognition. Our results based upon evaluation of 35 acute IM patients and 32 convalescent patients demonstrate that the virus-specific primary CTL response is broadly directed against the full spectrum of latent proteins, including EBNA1 and the viral coat glycoprotein gp350, while the memory CTL response, which essentially lacks EBNA1 reactivity, is directed primarily against the EBNA 3 family of proteins (3A, 3B, 3C). Importantly, the immunodominant response by both primary and memory CTL was directed against the EBNA3 proteins. Our data suggest that tvRNAs are an integral, multifunctional part of the rich biology of EBV and probably other herpesviruses as well.
Similarly, CTL from 7 of 35 acute IM patients recognized gp350 transfected targets, while no gp350-specific memory CTL responses were observed. While the phenotype of in-vivo primed CTL effectors were CD8+/HLA-DR+/CD11b+, the major subpopulation of memory CTL were CD8+/HLA-DR+/CD11b-. The structure of EBV protease was solved by X-ray crystallography . In contrast, the CD11b marker was quickly shed from in vitro propogated CTL, over a period of 5-10 days. Target cell lysis by in-vivo activated CTL was almost completely blocked by antibody directed againt [against] class-I molecules (BBM.1), whereas the effect of blocking target cell lysis by anti-CD8 mAb varied between 40-75%. These findings are consistent with an absolute need for class-I restricted antigen presentation, and imply that CD8 was variably required, likely for the lower affinity TCR/ Ag combinations. Cell lysis mediated by in-vitro-restimulated memory CTL was also largely inhibited by anti-class-I mAb, while anti-CD8 mAb was only mild/moderately effective in blocking target cell lysis, in keeping with the concept that memory CTL bear higher avidity TCR which can recognize antigen independent of CD8.
El-Guindy AS, Heston L, Endo Y, Cho MS, Miller G. The rare ability to detect an EBNA1-specific memory CTL responses remains a relatively unexplained phenomenon and may involve a number of tolerizing mechanisms including the induction of anergy by presentation of EBNA-1 in the absence of costimulation, clonal deletion of low affinity T cells, the absence of dominant T cell epitopes within EBNA1 or a result of the recently described inhibiting properties of EBNA-1 on antigen processing and presentation. Alternatively, the absence of detectable EBNA1-specific memory CTL may be the result of insufficient or inappropriate restimulation of memory CTL in vitro. We addressed this possibility by attempting to selectively restimulate and expand EBNA1-specific CTL from acute IM patients by using EBNA1 expressing B cells blasts as a stimulus. Effector cells generated in this manner killed target cells in an MHC class-I restricted manner but were specific for an unspecified vaccinia antigen. Interestingly, the phenotype of the effector cells was predominantly CD3+/CD4-/CD8-/γδ T cells. In summary, our findings suggest that a multitude of previously unrecognized, EBV-specific CTL are present in the peripheral blood during acute IM, and include EBNA-1-specific CTL.
The importance of accurately defining the in-vivo immune response to EBV is underscored by the ever-growing list of EBV associated malignancies. In addition to providing insights into the oncogenesis and potential treatment of NPC, a newly described link between precursor lesions and EBV infection raises the possibility that heightened immunity to EBV or EBV-infected cells may prevent the development of NPC. An obvious expectation would include extension of such knowledge to other EBV associated malignancies such as B and T cell lymphomas, Hodgkin’s lymphomas, and smooth muscle tumors. First however, existing gaps in knowledge regarding the immune response to EBV and EBV-associated malignancies must be closed. We provide evidence that gp150 shields the recognition of these surface molecules in a glycan-dependent manner. Adenovirus vectors and infections.An E1- and E3-deficient adenovirus type 5 vector expressing EBV IE protein BZLF1 cDNA (under the control of a cytomegalovirus promoter) was made by using the recombinant Cre-Lox-mediated recombination system as previously described (AdBZLF1) (91). Increased understanding of viral escape mechanisms is also likely to contribute to therapeutic modalities to treat autoimmune disorders.