PDF file - 44K, Comparison of the 99bp and 213bp PCR for measuring the EBV DNA load in NP brushings. Both primer pairs gave similar results.

PDF file, 21K, Primers used for quantitative LightCycler PCR.

Background Studies suggest that the most common type of nasopharyngeal carcinoma (NPC) is WHO-3, which is strongly associated with Epstein-Barr virus (EBV). Objective To assess NPC patient characteristics in a national general referral hospital in Indonesia, with regards to EBV DNA load and treatment response. Methods Twenty-three pediatric patients diagnosed with NPC were included in the study. Data collected were history, physical examination, tissue biopsy, CT scan, staging and EBV DNA load from nasopharyngeal (NP) brushing as well as blood specimens. The NP brushing, blood specimens and CT scan evaluations were done two months post-treatment. Results Pediatric patients with symptoms such as blood tinged secretion, lymph node enlargement, and nasal congestion were more likely to have higher EBV DNA loads in their NP brushings (P<0.05) (including T3 and higher). Despite significant reduction of EBV DNA load in NP brushing post-treatment, it was not associated with treatment response, as evaluated by CT scan. Conclusion Higher DNA load from NP brushings is associated with a higher tumor stage. Larger sample size and follow-up data are needed to assess the usefulness of EBV DNA load assessment in pediatric patients.

Background: Epstein-Barr virus associated gastric carcinoma (EBVaGC) is considered a distinct GC disease entity, with the virus persisting in a latent phase. Treatment with Epirubicin, Capecitabine and Cisplatin (ECC combination) showed survival benefit in patients with GC in clinical trials (MAGIC study and CRITICS study) when compared to chemotherapy with Capecitabine and Cisplatin (GCb/Cis). Current treatment protocols for GC do not consider virus involvement. Methods: In this study, we tested a CytoLytic Virus Activation (CLVA) strategy consisting of the ECC combination or GCb/Cis together with the HDAC inhibitor Valproic acid (VPA) to define whether EBV reactivation and subsequent antiviral treatment with Ganciclovir (GCV) could be used as virus-targeted therapy for EBVaGC. Drug combinations with VPA and GCV were evaluated in multiple cell lines and in an EBVaGC mouse model based on human naturally EBV-infected SNU-719 cells. Results: EBV reactivation was demonstrated by lytic mRNA transcripts and proteins in treated cells, and the virus-reactivating capacity of different CLVA drug combinations was compared in C666.1, AGS-BX1 and SNU-719 cell lines. In an EBVaGC mouse model, GCb/Cis with VPA and GCV strongly reduced tumor volume and showed the highest potential for EBV-reactivation. Upon a single round of CLVA treatment, EBV DNA levels in circulation decreased, and loss of EBV-positive cells in treated tumors was observed. In vivo EBV-reactivation was revealed by the presence of lytic gene transcripts and proteins in tumor tissues 6 days after treatment. Conclusion: In EBVaGC model systems, CLVA treatment showed a more potent virus reactivation and killing of tumor cells when compared to standard chemotherapy alone, suggesting that addition of VPA plus GCV to the ECC or GCb/Cis combination should be considered in future clinical studies.

Epstein–Barr Virus (EBV) BamHI-A rightward frame 1 (BARF1) protein is considered a viral oncogene in epithelial cells and has immune-modulating properties. During viral lytic replication BARF1 is expressed as an early gene, regulated by the immediate early EBV protein R. However, in viral latency BARF1 is exclusively expressed in epithelial tumors such as nasopharyngeal (NPC) and gastric carcinoma (GC) but not in lymphomas, indicating that activation of the BARF1 promoter is cell type specific. Undifferentiated NPC is characterized by high expression of ΔNp63 isoforms of the epithelial differentiation marker p63, a member of the p53 family of transcription factors. Transcription factor binding site analysis indicated potential p53 family binding sites within the BARF1 promoter region. This study investigated ability of various p53 family members to transactivate the BARF1 promoter. Using BARF1 promoter luciferase reporter constructs we demonstrate that only p63 isoform ΔNp63α is capable of transactivating the BARF1 promoter, but not the TAp63 isoforms, p53 or p73. Direct promoter binding of ΔNp63α was confirmed by Chromatin Immune Precipitation (ChIP) analysis. Deletion mutants of the BARF1 promoter revealed multiple ΔNp63 response elements to be responsible for BARF1 promoter transactivation. However, ΔNp63α alone was not sufficient to induce BARF1 in tumor cells harboring full EBV genomes, indicating that additional cofactors might be required for full BARF1 regulation. In conclusion, in EBV positive NPC and GC, BARF1 expression might be induced by the epithelial differentiation marker ΔNp63α, explaining BARF1 expression in the absence of lytic reactivation.

Undifferentiated nasopharyngeal carcinoma (NPC) is 100% associated with Epstein‐Barr virus (EBV). Expression of viral proteins in the tumor cells is highly restricted. EBV reactivation by CytoLytic Virus Activation (CLVA) therapy triggers de novo expression of early viral kinases (PK and TK) and uses antiviral treatment to kill activated cells. The mechanism of tumor elimination by CLVA was analyzed in NPC mouse model using C666.1 cells. Valproic acid (VPA) was combined with gemcitabine (GCb) to stimulate EBV reactivation, followed by antiviral treatment with ganciclovir (GCV). A single cycle of CLVA treatment resulted in specific tumor cell killing as indicated by reduced tumor volume, loss of EBV‐positive cells in situ, and paralleled by decreased EBV DNA levels in circulation, which was more pronounced than treatment with GCb alone. In vivo reactivation was confirmed by presence of lytic gene transcripts and proteins in tumors 6 days after GCb/VPA treatment. Virus reactivation was visualized by [124I]‐FIAU accumulation in tumors using PET‐scan. This studied showed that CLVA therapy is a potent EBV‐specific targeting approach for killing tumor cells. The [124I]‐FIAU appears valuable as PET tracer for studies on CLVA drug dosage and kinetics in vivo, and may find clinical application in treatment monitoring.

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers.

This study estimated the value of quantitative measurements of EBV markers in the clinical management of nasopharyngeal carcinoma in a non-endemic area. The aim was to predict prognosis and detect recurrent and residual disease. In 72 patients, EBV DNA load in blood and nasopharyngeal brushes, and IgA VCA-p18 and EBNA1 in plasma were measured at different time points. At diagnosis and post-treatment, a cut-off value was used for detecting disease [positive (PPV) and negative (NPV) predictive value]. The markers were correlated as a continuous variable with tumor stage, disease-free survival (DFS) and overall survival (OS). The Cox hazard ratio model assessed hazard ratios. At diagnosis, the markers were above the COV in 45, 92, 85 and 83 % of the patients, respectively. Post-treatment, DNA load test in blood and brush had the best discriminating power (blood DNA load test: PPV 39 % and NPV 97 %, brush for local disease: PPV 75 % and NPV 99 %). Post-treatment, DNA load in blood was the best predictor for OS and DFS [hazard ratio 3.2 (95 % CI 1.51–3.5) and 2.3 (95 % CI 1.72–5.8)]. Assessing the EBV DNA load in blood has significant prognostic value, although the clinical value is for discussion. The EBV DNA load in the brush might improve early detection of local failures post-treatment.

Undifferentiated nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr virus (EBV) as oncogenic driver. NPC is often diagnosed late due to initial vague complaints and obscured location. Prior studies suggest that measurement of EBV DNA load and RNA transcripts in nasopharyngeal (NP) brushings is useful for minimally invasive NPC diagnosis. However, whether these EBV markers relate to local virus replication or reflect tumor origin remains to be demonstrated. To resolve this, we analysed EBV-DNA characteristics and quantified latent and lytic viral RNA transcripts in NP brushings and matching frozen NP-biopsy specimens from patients suspected of having NPC. We observed non-fragmented and Cp-promotor methylated EBV-DNA in both NP brushings and biopsies suggestive of tumor origin. Using quantitative RT-PCR we determined expression levels of 7 critical latent (EBER1, Qp-EBNA1, EBNA2, BART, LMP1, LMP2, BARF1) and 5 lytic (Zta, Rta, TK, PK and VCA-p18) RNA transcripts. Although latent and early lytic RNA transcripts were frequently detected in conjunction with high EBV viral load, in both brushings and biopsies the latent transcripts prevailed and reflected a typical NPC-associated latency-II transcription profile without EBNA2. Late lytic RNA transcripts were rare and detected at low levels mainly in NP brushings, suggestive of abortive viral reactivation rather than complete virus replication. EBV-IgA serology (EBNA1, VCA, Zta) did not correlate to the level of viral reactivation in situ. Overall, viral RNA profiling, DNA fragmentation and methylation analysis in NP brushings and parallel biopsies indicate that NP brush sampling provides a true and robust indicator of NPC tumor presence.

Epstein-Barr virus (EBV) is causally associated with multiple cancers of epithelial and lymphoid origin. Generally, EBV establishes a lifelong quiescent homeostasis (latency) exerting its immune modulating and oncogenic properties only under certain conditions. Lytic replicative EBV infection is restricted to differentiated oropharyngeal epithelia and plasma B-cells. In EBV-associated tumor cells viral gene expression is limited to defined latency programs, without overt virus replication. Artificial triggering of EBV lytic replication with chemical agents (drugs) forms the basis of cytolytic virus activation (CLVA) therapy currently under clinical investigation for treating EBV-positive malignancies. Reactivation from latency requires expression of viral transactivator proteins BZLF1 (Zta) and BRLF1 (Rta). In tumor cells, the BZLF1 promoter (Zp) is highly methylated and generally inactive. However, Zp can be triggered into a self-enhancing activation loop in response to chemical or biological agents, including cytostatic drugs and epigenetic modifiers. This process is orchestrated by Zp-binding cellular transcription factors, such as myocyte enhancer factor 2 (MEF2), specificity protein 1 (SP1), and zinc finger E-box binding homeobox (ZEB) proteins. Understanding the mechanism of lytic induction by chemical inducers is essential for developing effective therapies. This review provide a short overview of EBV biology and will focus on branded and novel agents (drugs) used for EBV lytic induction and discuss molecular mechanisms by which the EBV lytic switch is triggered and controlled.

Despite successful primary treatment of nasopharyngeal carcinoma (NPC), the incidence of distant metastasis remains 25–34 %. Treatment options are limited, and survival is poor. Intratumoural Epstein–Barr virus (EBV) was used as treatment target. In NPC, EBV is present in a latent state, expressing only few non-immunogenic viral products. Gemcitabine and valproic acid can trigger EBV to the lytic state, wherein viral kinases are expressed, making EBV-positive tumour cells susceptible for antiviral therapy with, i.e. valganciclovir, and inducing an EBV-specific immune response.

Epstein-Barr virus (EBV) BARF1 (BamHI-A rightward frame 1) protein is a viral oncogen and immunomodulator. BARF1 mRNA is highly and selectively expressed in nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC), however, evidence for presence of BARF1 protein in situ is lacking. In vitro, the hexameric form of BARF1 is rapidly secreted (sBARF1). Secretion of BARF1 from NPC tumor cells may result in the presence of sBARF1 in sera of NPC patients. This study describes the development of new monoclonal and polyclonal antibodies that strongly and selectively bind to ‘native’ NPC-derived sBARF1 protein in its natural hexameric conformation. Using these antibodies, a capture enzyme linked immunosorbent assay (ELISA) was developed to detect sBARF1 protein in sera of NPC patients. Although a detection limit of 10 ng/ml sBARF1 in serum was reached for the ELISA, only 3 of 71 NPC patients at diagnosis showed elevated levels of sBARF1 protein. Possibly sBARF1 is complexed with serum proteins shielding antibody recognition sites. However, sample denaturation prior to ELISA testing did not affect the levels observed. A MALDI-MS/MS approach was developed to overcome this problem, yielding a specific BARF1 identifier peptide. However, the sensitivity of the current setup was less than BARF1-capture ELISA. The BARF1 identifier peptide can be used to further develop a quantitative targeted proteomics approach. Using immunohistochemical staining with various anti-BARF1 antibody reagents, BARF1 could be detected in paraffin-embedded BARF1 expressing cell lines revealing a cytoplasmic localisation. Unfortunately, in patient material, no BARF1 protein expression could be demonstrated. In conclusion, the sensitive sBARF1 capture ELISA (10 ng/mL serum) demonstrated only low levels of sBARF1 in NPC patient sera, conflicting with previously published data. A higher sensitivity of both ELISA and proteomic approach is needed for a diagnostic tool. sBARF1 remains a potential serum biomarker for NPC and GC and its diagnostic value using a targeted proteomics approach should be further evaluated. Chapter 8 EBV BARF1 detection

Background: Nasopharyngeal carcinoma (NPC) is endemic in Indonesia and 20% of the patients are diagnosed before the age of 31. This study evaluates presentation and treatment outcome of young patients in Jakarta, in a tertiary referral centre.Methods: Forty-nine patients under the age of 31, diagnosed with NPC between July 2004 and January 2007, were evaluated. Baseline data included histological type, stage of disease and presenting symptoms. We intended to follow all patients after diagnosis to reveal treatment outcome and overall survival (OS).Results: All but two patients had advanced stage disease (94%), 7 (14%) had distant metastasis. The median interval between start of complaints and diagnosis was 9 months. Forty-two patients were planned for curative intent treatment. Eleven patients (26%) never started treatment, 2 patients did not complete treatment and 3 patients did not return after finishing treatment. Four patients died before radiation could start. Three patients died within 4 months after treatment. Nine patients (21%) had a complete response. Due to the high number of patients who were lost to follow-up (LFU), OS was analyzed as follows: a best-case (patients censored at last contact) and a worst-case scenario (assuming that patients who did not finish treatment or had disease at last contact would have died). The 2-year OS for patients without distant metastases was 39-71%.Conclusion: Treatment outcome for young patients with NPC in this institute was poor. Improvement can be achieved when NPC is diagnosed at an earlier stage and when there is better treatment compliance.

Background Epstein-Barr virus (EBV) is causal in WHO-III nasopharyngeal carcinoma (NPC) and 10% of gastric cancers (GC) worldwide. Since all tumour cells carry EBV, the virus itself is a potential target for therapy. However, EBV expresses few non-immunogenic gene products essential for EBV-DNA maintenance and tumour growth. We developed a cytolytic virus activation (CLVA) therapy, reactivating latent EBV into the lytic cycle, triggering immune recognition and inducing susceptibility to antiviral therapy. Methods CLVA combines a cytostatic drug with valproic acid (VPA) for virus reactivation with (val) ganciclovir (GCV) as an antiviral drug preventing virus replication and killing reactivated tumour cells. CLVA treatment was validated in cell lines and mouse models and in a phase-1/2 clinical trial with Dutch NPC patients refractory to conventional treatment. A similar trial is about to start in Indonesia. Findings In NPC and GC cell lines, various cytostatic drug combinations induce the EBV lytic cycle, which is enhanced by VPA. Addition of GCV resulted in higher viable cell elimination. CLVA therapy eliminated NPC tumours in nude mice more efficiently and completely than single drugs alone. Using 131FIAU, CLVA responsive tumours in mice could be visualised by PET scan and DNA load in blood, mRNA profiling, and in situ staining confirmed in vivo virus reactivation. A phase 1/2 study with six cycles of CLVA treatment showed that CLVA was well-tolerated in 11 patients with end-stage NPC. Patients showed increased viral DNA in blood with lytic mRNA in nasopharyngeal brushings during treatment. Treatment showed a good response in five patients, two patients developed stable disease and in four patients no (early) effect was seen and therapy was ended. Approval for a phase 1/2 trial for NPC was obtained in Yogyakarta, Indonesia. Interpretation CLVA successfully activated EBV from latency in NPC and GC tumour cell lines in vitro and cleared NPC tumours from nude mice. In patients, CLVA was well tolerated and showed a biological effect. This new virus-specific CLVA therapy could open a generic approach for treatment of multiple EBV-associated malignancies. Epstein-Barr virus (EBV) is causal in WHO-III nasopharyngeal carcinoma (NPC) and 10% of gastric cancers (GC) worldwide. Since all tumour cells carry EBV, the virus itself is a potential target for therapy. However, EBV expresses few non-immunogenic gene products essential for EBV-DNA maintenance and tumour growth. We developed a cytolytic virus activation (CLVA) therapy, reactivating latent EBV into the lytic cycle, triggering immune recognition and inducing susceptibility to antiviral therapy. CLVA combines a cytostatic drug with valproic acid (VPA) for virus reactivation with (val) ganciclovir (GCV) as an antiviral drug preventing virus replication and killing reactivated tumour cells. CLVA treatment was validated in cell lines and mouse models and in a phase-1/2 clinical trial with Dutch NPC patients refractory to conventional treatment. A similar trial is about to start in Indonesia. In NPC and GC cell lines, various cytostatic drug combinations induce the EBV lytic cycle, which is enhanced by VPA. Addition of GCV resulted in higher viable cell elimination. CLVA therapy eliminated NPC tumours in nude mice more efficiently and completely than single drugs alone. Using 131FIAU, CLVA responsive tumours in mice could be visualised by PET scan and DNA load in blood, mRNA profiling, and in situ staining confirmed in vivo virus reactivation. A phase 1/2 study with six cycles of CLVA treatment showed that CLVA was well-tolerated in 11 patients with end-stage NPC. Patients showed increased viral DNA in blood with lytic mRNA in nasopharyngeal brushings during treatment. Treatment showed a good response in five patients, two patients developed stable disease and in four patients no (early) effect was seen and therapy was ended. Approval for a phase 1/2 trial for NPC was obtained in Yogyakarta, Indonesia. CLVA successfully activated EBV from latency in NPC and GC tumour cell lines in vitro and cleared NPC tumours from nude mice. In patients, CLVA was well tolerated and showed a biological effect. This new virus-specific CLVA therapy could open a generic approach for treatment of multiple EBV-associated malignancies.

Epstein-Barr virus (EBV) causes several benign and malignant disorders of lymphoid and epithelial origin. EBV-related tumors display distinct patterns of viral latent gene expression, of which the BamHI-A rightward frame 1 (BARF1) is selectively expressed in carcinomas, regulated by cellular differentiation factors including ΔNp63α. BARF1 functions as a viral oncogene, immortalizing and transforming epithelial cells of different origin by acting as a mitogenic growth factor, inducing cyclin-D expression, and up-regulating antiapoptotic Bcl-2, stimulating host cell growth and survival. In addition, secreted hexameric BARF1 has immune evasive properties, functionally corrupting macrophage colony stimulating factor, as supported by recent functional and structural data. Therefore, BARF1, an intracellular and secreted protein, not only has multiple pathogenic functions but also can function as a target for immune responses. Deciphering the role of BARF1 in EBV biology will contribute to novel diagnostic and treatment options for EBV-driven carcinomas. Herein, we discuss recent insights on the regulation of BARF1 expression and aspects of structure-function relating to its oncogenic and immune suppressive properties.

Abstract Purpose: Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) and highly prevalent in Indonesia. EBV-DNA load can be used for early diagnosis and may have prognostic value. In this study, EBV-DNA load was evaluated in minimal invasive nasopharyngeal (NP) brushings and whole blood for initial diagnosis and therapy assessment against the standard-of-care diagnosis by biopsy with EBV-RISH and standard EBV-IgA serology. Experimental Design: NP brushings and blood samples were collected from 289 consecutive ENT patients suspected of NPCs and 53 local healthy controls. EBV-DNA load was quantified by real-time PCR and serology by peptide-based EBV-IgA ELISA. Tissue biopsies were examined by routine histochemistry and by EBER RNA in situ hybridization. Results: Repeated NP brushing was well tolerated by patients and revealed high viral load in the 228 NPC cases at diagnosis than 61 non-NPC cancer cases and healthy controls (P < 0.001). The diagnostic value of EBV-DNA load in blood and EBV-IgA serology was inferior to the NP brush results. The level of EBV-DNA load in brushes of patients with NPC was not related to T, N, or M stage, whereas elevated EBV-DNA load in blood correlated with N and M stage. EBV-DNA levels in brushings and whole blood showed a significant reduction at 2 months after treatment (P = 0.001 and P = 0.005, respectively), which was not reflected in EBV-IgA serology. Conclusions: NP brush sampling combined with EBV-DNA load analysis is a minimal invasive and well-tolerated diagnostic procedure, suited for initial diagnosis and follow-up monitoring of NPCs. Clin Cancer Res; 19(8); 2175–86. ©2013 AACR.

Abstract Purpose: Nasopharyngeal carcinoma (NPC) is causally linked to Epstein–Barr virus (EBV) infection. Because all tumor cells carry EBV, the virus itself is a potential target for therapy. In these tumor cells, EBV hides in a latent state and expresses only a few non-immunogenic proteins for EBV maintenance and contributes to tumor growth. We developed a cytolytic virus activation (CLVA) therapy for NPC treatment, reactivating latent EBV, triggering immune recognition, and inducing susceptibility to antiviral therapy. Experimental Design: CLVA therapy combines gemcitabine (GCb) and valproic acid (VPA) for virus activation and tumor clearance with (val)ganciclovir (GCV) as the antiviral drug to block virus replication and kill proliferating virus-infected cells. CLVA treatment was optimized and validated in NPC cell lines and subsequently tested in 3 Dutch patients with NPC that was refractory to conventional treatment. Results: In NPC cell lines, both GCb and VPA can induce the lytic cycle of EBV. Their combination resulted in a strong synergistic effect. The addition of GCV resulted in higher cytotoxicity compared with chemotherapy alone, which was not observed in EBV-negative cells. CLVA therapy was analyzed in 3 patients with end-stage NPC. Patients developed increased levels of viral DNA in the circulation originating from apoptotic tumor cells, had disease stabilization, and experienced improved quality of life. Conclusions: Our results in the initial CLVA-treated patients indicate that the therapy had a biological effect and was well tolerated with only moderate transient toxicity. This new virus-specific therapy could open a generic approach for treatment of multiple EBV-associated malignancies. Clin Cancer Res; 18(18); 5061–70. ©2012 AACR.

ABSTRACT Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides −554 and −327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.