**6. Consequences in terms of diagnosis and therapy**

The observation of ZEBRA transduction properties and abortive cycles has prompted us to imagine the existence of a circulating ZEBRA form, especially in the serum of patients, particularly those with lymphoid tumors. Many articles have previously mentioned the existence of the lytic cycle, especially in transplant patients with PTLD. Studies conducted on EBV lytic proteins, especially the IE proteins like ZEBRA, in patients with PTLD or HIV-associated NHL are still scarce, mostly relating to the role of EBV proteins and gene products in neoplastic tissues [18, 40, 90–95]. It must be pointed out that several authors exploring BZLF1 transcripts in the peripheral blood lymphocytes (PBL) of PTLD patients demonstrated that both a high EBV genome number and strong BZLF1 mRNA expression are sensitive

**105**

**Figure 5.**

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis…*

markers of EBV-related PTLD [96]. In a previous study, we demonstrated ZEBRA expression in the whole peripheral blood mononuclear cells (PBMCs) from a patient exhibiting a LPD using flow cytometry. In these patients who underwent nonmyeloablative allogeneic stem cell transplantation, the ZEBRA antigen was found in mostly 5% of PBMCs [97]. Moreover infected cells were detected in the peripheral

Recently we succeeded in detecting soluble ZEBRA (s-ZEBRA) protein in serum from transplant patients (measured by an antibody-based ELISA). The s-ZEBRA (>100 ng/mL) was predictive in 80% of PTLD patients within 10 weeks, prior to PTLD diagnosis (p < 0.0001) [84]. We applied this technique in both solid organ transplant patients and in hematopoietic stem cell (HSC) patients. During the HSCT patient follow-up, the availability of iterative serum samples enabled us to investigate the kinetics of s-ZEBRA appearance in comparison to that of EBV DNA qPCR and anti-ZEBRA IgG antibodies. As for patient followup, it was interesting to notice that the circulating ZEBRA protein could be detected during periods in which the viral DNA was not detectable by qPCR. This could be explained by certain inconsistencies observed between the qPCR and s-ZEBRA detection results (**Figure 5**). This discrepancy may be accounted for by the precocity of the ZEBRA signal measured over the course of EBV infection in this patient population. This precocity of s-ZEBRA detection (with respect to the qPCR) was independent of the PCR format, since we observed the same phenomenon in PTLD patients who were investigated by measuring the EBV load (expressed in copies/150,000 cells). In two PTLD patients, s-ZEBRA was detected at 2 and 6 weeks, respectively, prior to the PTLD episode and before the increase in qPCR signals [100]. It is interesting to note that the s-ZEBRA potentially correlated the symptomatology, as only one patient (#P3) (**Figure 5**) exhibited very high levels (3690 ng/mL) compared to the two others (#P4 and #P10) without

B cells in persistently infected healthy individuals) [99].

B lymphocytes [1, 98]

*DOI: http://dx.doi.org/10.5772/intechopen.88607*

versus 1 and 50 per 106

blood at higher levels (e.g., 1–10 lytic-infected cells per 104

any PTLD (162 and 300 ng/mL, respectively) (**Table 3**).

*Follow-up of the HSCT patients #P3 (see Table 3): Levels of s-ZEBRA (ng/mL), titers of anti-ZEBRA IgG, and EBV DNA load (copies/mL) are noted, respectively. This patient experienced a lymphoproliferative episode (Oct. 27) and then was treated by anti-CD20 therapy, with decrease of the markers explored. The re-increase in* 

*s-ZEBRA during the course of the GvHD is worth noticing, until the patient finally died (Nov. 10).*

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88607*

markers of EBV-related PTLD [96]. In a previous study, we demonstrated ZEBRA expression in the whole peripheral blood mononuclear cells (PBMCs) from a patient exhibiting a LPD using flow cytometry. In these patients who underwent nonmyeloablative allogeneic stem cell transplantation, the ZEBRA antigen was found in mostly 5% of PBMCs [97]. Moreover infected cells were detected in the peripheral blood at higher levels (e.g., 1–10 lytic-infected cells per 104 B lymphocytes [1, 98] versus 1 and 50 per 106 B cells in persistently infected healthy individuals) [99].

Recently we succeeded in detecting soluble ZEBRA (s-ZEBRA) protein in serum from transplant patients (measured by an antibody-based ELISA). The s-ZEBRA (>100 ng/mL) was predictive in 80% of PTLD patients within 10 weeks, prior to PTLD diagnosis (p < 0.0001) [84]. We applied this technique in both solid organ transplant patients and in hematopoietic stem cell (HSC) patients. During the HSCT patient follow-up, the availability of iterative serum samples enabled us to investigate the kinetics of s-ZEBRA appearance in comparison to that of EBV DNA qPCR and anti-ZEBRA IgG antibodies. As for patient followup, it was interesting to notice that the circulating ZEBRA protein could be detected during periods in which the viral DNA was not detectable by qPCR. This could be explained by certain inconsistencies observed between the qPCR and s-ZEBRA detection results (**Figure 5**). This discrepancy may be accounted for by the precocity of the ZEBRA signal measured over the course of EBV infection in this patient population. This precocity of s-ZEBRA detection (with respect to the qPCR) was independent of the PCR format, since we observed the same phenomenon in PTLD patients who were investigated by measuring the EBV load (expressed in copies/150,000 cells). In two PTLD patients, s-ZEBRA was detected at 2 and 6 weeks, respectively, prior to the PTLD episode and before the increase in qPCR signals [100]. It is interesting to note that the s-ZEBRA potentially correlated the symptomatology, as only one patient (#P3) (**Figure 5**) exhibited very high levels (3690 ng/mL) compared to the two others (#P4 and #P10) without any PTLD (162 and 300 ng/mL, respectively) (**Table 3**).

#### **Figure 5.**

*Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*

**5. Evidence of abortive lytic cycle and their role in tumorigenesis**

newly formed infective viral particles [13].

**6. Consequences in terms of diagnosis and therapy**

*tumorigenesis without relying on a complete lytic (from Ref. [13] with permission).*

The observation of ZEBRA transduction properties and abortive cycles has prompted us to imagine the existence of a circulating ZEBRA form, especially in the serum of patients, particularly those with lymphoid tumors. Many articles have previously mentioned the existence of the lytic cycle, especially in transplant patients with PTLD. Studies conducted on EBV lytic proteins, especially the IE proteins like ZEBRA, in patients with PTLD or HIV-associated NHL are still scarce, mostly relating to the role of EBV proteins and gene products in neoplastic tissues [18, 40, 90–95]. It must be pointed out that several authors exploring BZLF1 transcripts in the peripheral blood lymphocytes (PBL) of PTLD patients demonstrated that both a high EBV genome number and strong BZLF1 mRNA expression are sensitive

*The existence of abortive lytic cycles (presence of ZEBRA protein, with absence of other lytic genes, particularly those encoding late structural proteins) would reconcile the requirement of lytic expression in view of viral* 

In the absence of other lytic genes, particularly those encoding late structural proteins, without the formation of infective viral particles, BZLF1 expression is termed the "abortive lytic cycle" (**Figure 4**) [13]. The existence of abortive cycles was demonstrated in EBV-associated malignancies through the detection of either the ZEBRA protein (via monoclonal antibodies) or mRNA: Hodgkin disease [17], Non-Hodgkin lymphoma (NHL) [18, 88], NPC [20], or Burkitt lymphoma [21]. Decades ago, we revealed the early stages of EBV replication in lymphomas in scid/hu mice, assessed by the expression of ZEBRA expression, whereas the VCA expression late replicate protein proved to be weak [89]. In a recent review, the authors discussed evidence supporting an abortive lytic cycle with several lytic genes expressed, such as immunomodulatory (BCRF1, BARF1, BNLF2A, BGLF5, and BILF1) and anti-apoptotic (BHRF1 and BALF1) proteins. In their paper, the authors also discussed how the EBV immunomodulatory mechanisms result in paracrine signals that feed tumor cells. The existence of such abortive lytic cycles would reconcile the requirement of lytic expression in view of viral tumorigenesis without relying on a complete cycle that would induce cell lysis, thus releasing the

**104**

**Figure 4.**

*Follow-up of the HSCT patients #P3 (see Table 3): Levels of s-ZEBRA (ng/mL), titers of anti-ZEBRA IgG, and EBV DNA load (copies/mL) are noted, respectively. This patient experienced a lymphoproliferative episode (Oct. 27) and then was treated by anti-CD20 therapy, with decrease of the markers explored. The re-increase in s-ZEBRA during the course of the GvHD is worth noticing, until the patient finally died (Nov. 10).*

#### *Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*


*Only one patient (#P3) experienced a PTLD episode. For patients #P3, #P4, and #P10, the OD450 values correspond to 3690, 162, and 300 ng/mL of circulating s-ZEBRA, respectively. NT, not tested. EBV DNA load in serum was measured by qPCR (see Ref. [84]).*

#### **Table 3.**

*Summary of the s-ZEBRA ELISA, anti-ZEBRA IgG titration, and EBV PCR assay results in the control populations (immunocompetent EBV-seronegative/EBV-seropositive subjects, patients with primary infection) and in 11 hematopoietic stem cell transplant patients.*

Therefore, s-ZEBRA detection could be a potential diagnostic marker for EBV follow-up in immunocompromised patients. Given this particular setting, our findings suggest that s-ZEBRA testing could help identify patients likely to develop severe outcomes during the critical posttransplant period. Based on our findings, we assume that the circulating ZEBRA form to be a useful target for the rapid and early diagnosis of PTLD, meaning that ZEBRA antigen-capture ELISA is likely to be a good confirmatory test for assessing EBV load in this patient population. Future evaluations of the prognostic value of ZEBRA detection should focus on the sampling time and clinical phase of lymphoproliferative disease. To improve the sensitivity and specificity of PTLD diagnosis [101–103], we hypothesize that combining approaches to detect both the circulating antigen and EBV load would be likely to increase the sensitivity and reliability of tests designed to identify such malignant EBV-related diseases.

These data focused on the relevance of the lytic cycle have already attracted the attention of the EBV community due to the potential usefulness of targeting certain lytic proteins (**Figure 6**). Investigations using both in vitro and in vivo systems revealed that FDA-approved leflunomide, a teriflunomide metabolite that targets EBV replication, inhibited the earliest step of lytic EBV reactivation (BZLF1 and BMRF1 expression) and thus prevented the development of EBVinduced lymphomas in both a humanized mouse model and a xenograft model [104].

**107**

EBV malignancies [109].

**Figure 6.**

cially the IE proteins, such as ZEBRA.

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis…*

More recently duvesilib (a molecule inhibiting the PIU3K/akt signaling pathway, thereby inhibiting BCR signaling) was shown to reduce the expression of EBV lytic genes like BZLF1 and gp350/220, in EBV-positive cell lines and cell growth, suggesting that this molecule was able to suppress the lytic EBV cycle induced by BCR signaling [105]. The histone acetylase and DNA methyl transferase inhibitors are possible avenues to suppress the ZEBRA expression and entire lytic cascade [106]. Immunotherapeutic approaches such as vaccination against IE proteins or IE-specific therapeutic monoclonal antibodies (mAbs) look likewise promising. A recent study demonstrated that vaccination of hu-PBL-SCID mice against the ZEBRA protein could enhance specific cellular immunity and significantly delay the development of lethal EBV-LPD [107]. Efforts are additionally being made to improve the quality of CD4+ T-cell line infusions responding to EBV lytic antigens [108]. Recently authors demonstrated the role of BARF1 as a novel EBV-specific antigen suitable for immunotherapeutic approach. These authors provided evidence that mABs anti-BARF1 are likely to be a potent tool for managing several

*Contributions of latent and lytic EBV in tumorigenesis and tumor progression. Consequences in clinical settings: In PTLD patients, EBV DNA quantification (qPCR) in blood samples corresponds to the burden of memory B cells (not proliferating lymphoblasts) infected by EBV. Thus, the increased EBV load in PBMCs, currently measured in the routine patients' follow-up, is accounted for by an increase in the number circulating EBV-positive cells (memory B cells), similar to latently infected resting B cells. Conversely, the measurement of circulating s-ZEBRA could be a novel biomarker for PTLD prognosis. The potential usefulness of targeting* 

*certain lytic proteins is also critical for managing several EBV malignancies in the future.*

In conclusion, the relevance of the lytic cycle and, particularly, the role of ZEBRA in lymphomagenesis is a new paradigm pertaining to the prevention and treatment strategies for EBV-associated cancers. Therefore, it now appears relevant to investigate the lytic EBV infection in immunocompromised patients, such as organ transplant recipients, who are highly prone to developing EBV-associated malignancies. With respect to circulating s-ZEBRA, we have made the following assumptions: (i) it may be a marker of over-immunosuppression by triggering the expression of immunomodulating cytokines; (ii) it may thus consequently play a specific role in the oncogenic process, even tumor progression. More efforts should be invested to examine the potential of drugs that target EBV lytic proteins, espe-

*DOI: http://dx.doi.org/10.5772/intechopen.88607*

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88607*

#### **Figure 6.**

*Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*

**s-ZEBRA (OD450 value)**

Infectious mononucleosis 50 0.238 24,000 NT No

P1 3 0.461 2833 93 No

 P3 (deceased) 7 3.26 5214 1300 Yes P4 3 0.92 2000 947 No P5 4 0.274 2300 136 No P6 3 0.255 200 2417 No P7 2 0.24 2250 42 No P8 2 0.201 4000 117 No P9 2 0.248 4000 NT No P10 1 1.236 20,000 0 No P11 1 0.323 20,000 0 No

P2 3 0.321 3333 28

**Means (m) PTLD**

**EBV DNA load (copies/mL)**

**anti-ZEBRA (IgG titer)**

30 0.067 NT 0 No

25 0.092 NT NT No

**serum samples**

**Patients Nr of** 

Hematopoietic stem cell transplant patients

Immunocompetent

Immunocompetent

Seronegative

Seropositive

Therefore, s-ZEBRA detection could be a potential diagnostic marker for EBV follow-up in immunocompromised patients. Given this particular setting, our findings suggest that s-ZEBRA testing could help identify patients likely to develop severe outcomes during the critical posttransplant period. Based on our findings, we assume that the circulating ZEBRA form to be a useful target for the rapid and early diagnosis of PTLD, meaning that ZEBRA antigen-capture ELISA is likely to be a good confirmatory test for assessing EBV load in this patient population. Future evaluations of the prognostic value of ZEBRA detection should focus on the sampling time and clinical phase of lymphoproliferative disease. To improve the sensitivity and specificity of PTLD diagnosis [101–103], we hypothesize that combining approaches to detect both the circulating antigen and EBV load would be likely to increase the sensitivity and reliability of tests designed to identify such

*Only one patient (#P3) experienced a PTLD episode. For patients #P3, #P4, and #P10, the OD450 values correspond to 3690, 162, and 300 ng/mL of circulating s-ZEBRA, respectively. NT, not tested. EBV DNA load in* 

*Summary of the s-ZEBRA ELISA, anti-ZEBRA IgG titration, and EBV PCR assay results in the control populations (immunocompetent EBV-seronegative/EBV-seropositive subjects, patients with primary infection)* 

31 m = 0.727 m = 6012 m = 507.9

These data focused on the relevance of the lytic cycle have already attracted the attention of the EBV community due to the potential usefulness of targeting certain lytic proteins (**Figure 6**). Investigations using both in vitro and in vivo systems revealed that FDA-approved leflunomide, a teriflunomide metabolite that targets EBV replication, inhibited the earliest step of lytic EBV reactivation (BZLF1 and BMRF1 expression) and thus prevented the development of EBVinduced lymphomas in both a humanized mouse model and a xenograft model [104].

**106**

malignant EBV-related diseases.

*serum was measured by qPCR (see Ref. [84]).*

*and in 11 hematopoietic stem cell transplant patients.*

**Table 3.**

*Contributions of latent and lytic EBV in tumorigenesis and tumor progression. Consequences in clinical settings: In PTLD patients, EBV DNA quantification (qPCR) in blood samples corresponds to the burden of memory B cells (not proliferating lymphoblasts) infected by EBV. Thus, the increased EBV load in PBMCs, currently measured in the routine patients' follow-up, is accounted for by an increase in the number circulating EBV-positive cells (memory B cells), similar to latently infected resting B cells. Conversely, the measurement of circulating s-ZEBRA could be a novel biomarker for PTLD prognosis. The potential usefulness of targeting certain lytic proteins is also critical for managing several EBV malignancies in the future.*

More recently duvesilib (a molecule inhibiting the PIU3K/akt signaling pathway, thereby inhibiting BCR signaling) was shown to reduce the expression of EBV lytic genes like BZLF1 and gp350/220, in EBV-positive cell lines and cell growth, suggesting that this molecule was able to suppress the lytic EBV cycle induced by BCR signaling [105]. The histone acetylase and DNA methyl transferase inhibitors are possible avenues to suppress the ZEBRA expression and entire lytic cascade [106]. Immunotherapeutic approaches such as vaccination against IE proteins or IE-specific therapeutic monoclonal antibodies (mAbs) look likewise promising. A recent study demonstrated that vaccination of hu-PBL-SCID mice against the ZEBRA protein could enhance specific cellular immunity and significantly delay the development of lethal EBV-LPD [107]. Efforts are additionally being made to improve the quality of CD4+ T-cell line infusions responding to EBV lytic antigens [108]. Recently authors demonstrated the role of BARF1 as a novel EBV-specific antigen suitable for immunotherapeutic approach. These authors provided evidence that mABs anti-BARF1 are likely to be a potent tool for managing several EBV malignancies [109].

In conclusion, the relevance of the lytic cycle and, particularly, the role of ZEBRA in lymphomagenesis is a new paradigm pertaining to the prevention and treatment strategies for EBV-associated cancers. Therefore, it now appears relevant to investigate the lytic EBV infection in immunocompromised patients, such as organ transplant recipients, who are highly prone to developing EBV-associated malignancies. With respect to circulating s-ZEBRA, we have made the following assumptions: (i) it may be a marker of over-immunosuppression by triggering the expression of immunomodulating cytokines; (ii) it may thus consequently play a specific role in the oncogenic process, even tumor progression. More efforts should be invested to examine the potential of drugs that target EBV lytic proteins, especially the IE proteins, such as ZEBRA.

*Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*
