**4. Discussion**

Currently, there is no single approach to the treatment of CEBVI, despite the fact that there are a number of specific antiviral drugs. In particular, acyclic nucleosides are widely used, such as acyclovir, valaciclovir (Valtrex), famciclovir (Famvir), and synthetic nucleoside analogs of guanosine: ganciclovir (cymevene), valganciclovir (Valcyte). In most cases, antiviral therapy is ineffective, which has been confirmed in numerous studies. In 2016, the results of efficacy analysis of infectious mononucleosis treatment were published according to the WHO World Register of Clinical Trials, both completed and ongoing. It was shown that the effectiveness of antiviral drugs (acyclovir, valaciclovir) in acute infectious mononucleosis is doubtful. Acyclovir and valaciclovir reduce EBV replication by inhibiting viral DNA polymerase and decreasing the oral secretion of EBV in patients with infectious mononucleosis. Balfour HH, et al. showed that taking the drug in a dose of 1 g every 8 h for 14 days leads to a decrease in clinical complaints in infectious mononucleosis [46]. In the case of viral shedding, shedding was observed to suppress the shedding against the background of antiviral therapy, but this effect ceased after the end of antiviral therapy [47]. The authors did not obtain a statistically significant difference between the groups of patients receiving antiviral drugs and the control groups. Most of the studies processed were unclear or at high risk of bias. Experimental studies in vitro have shown that EBV thymidine kinase has a variable affinity for antiherpetic antiviral drugs, that is, acyclovir and dihydroxypropylmethylguanine are relatively weak substrates for EBV thymidine kinase [48]. In our study, in the group of patients receiving therapy with Valtrex (valaciclovir) at a dose of 1 g per day for 2 months, suppression of EBV DNA replication in saliva samples was obtained in 28.57% of patients. This is confirmed by literature. The effectiveness of the use of valganciclovir in suppressing EBV replication and reducing the severity of clinical complaints in patients has been shown [49]. Taking valganciclovir leads to a decrease in the amount of EBV DNA from an average of 4.3 log10 copies/ml to 1.2 log10 copies/ml by 0.77 logs (95% CI, .62–.91 logs; P < .001) [50]. Valaciclovir and famciclovir suppress EBV DNA replication by 18% and 30%, respectively [51], while valganciclovir reduces EBV DNA secretion by 46% [52]. That is, valganciclovir can be used in the treatment of CEBVI.

The research by Kure S. et al., devoted to the study of the inhibitory effect of pure recombinant human (rh) IFN-α and IFN-γ on EBV infection in B-cell lines, BJAB lines, and in normal mature B-lymphocytes, showed that the pretreatment of cells within 24 h with rhIFN-α and rhIFN-γ suppressed the production of EBV specific nuclear antigen (EBNA-1) in BJAB cells 24 h after EBV infection. Both rhIFN types also effectively inhibited EBV infection in normal mature B lymphocytes, as evidenced by a decrease in [3H] thymidine incorporation 6 days after EBV infection and the total number of proliferating cells 21 days after infection. The authors showed that rhIFN-α exhibited a more pronounced inhibitory effect than rhIFN-γ. None of the rhIFNs showed a pronounced inhibitory effect on EBNA expression in covert EBV-infected Raji and Daudi cell lines. These results indicate that rhIFNs act predominantly at the early stage of EBV infection [53]. In our work, it was shown that in group 1 (N = 51) one month after rhIFN-γ therapy, 15 (29.41%) patients had negative PCR results in saliva samples, and 36 (70.59%) patients had copies of EBV DNA decreased. That is, rhIFN-γ can completely inhibit viral replication in 29.41% of patients. However, in this group of patients, a pronounced reliable dynamics of clinical complaints were obtained after the end of therapy. In 2002, it was shown that treatment of Vero cells with IFN-β or IFN-γ inhibits HSV-1 replication by less than 20-fold, while co-treatment with IFN-β and IFN-γ inhibits

HSV-1 replication by 1000 times [41]. The authors suggested that the high level of inhibition achieved by the administration of exogenous IFN-γ is the result of a synergistic interaction with endogenous IFN-α/β, which are locally produced in response to HSV-1 infection. Our results confirm these in vitro data. If we compare the results we obtained in the groups of patients who received rhIFN-γ and monotherapy with valganciclovir in terms of the dynamics of the number of DNA copies in saliva samples, then no difference was obtained between these groups, that is, the effectiveness of monotherapy with rhIFN-γ or valaciclovir has similar efficacy (29.41% and 28.57% respectively). Our results are consistent with previously published data, in particular, the Russian literature describes the results of the study of rhIFN-γ (Ingaron) and presents evidence of the high efficiency of its use in the treatment of herpesvirus infections [54]. The authors showed that the drug has a direct antiviral effect, and the clinical effect is manifested through the activation of cellular immunity, which controls the viral antigen. In group 3, a month after taking the combination therapy valganciclovir+rhIFN-γ, a negative PCR result was obtained in 19 (71.74%) patients. The effectiveness of the therapy did not depend on the combination of drugs but on the duration of the course of rhIFN-γ administration. The best result from therapy was in patients who received 20 injections of rhIFN-γ in combination with valganciclovir. It was in this group that the number of copies of EBV DNA in saliva samples was negative in 87.50% of patients. Thus, a positive result on the number of EBV DNA copies during this treatment regimen is due not so much to the total combination course, but to the amount and duration of rhIFN-γ administration.

In 2003, an open, randomized, controlled, multicenter clinical study was conducted to study the anti-fibrotic effect of rhIFN-γ in 153 patients with chronic viral hepatitis B. RhIFN-γ was introduced i/m daily at a dose of 1 MU for three months and 1 MU every other day for the following six months. As a result, it was shown that rhIFN-γ has a pronounced anti-fibrotic effect in patients with chronic hepatitis B [55]. The effectiveness of treatment was 66% in the group of patients versus 16.2% in the control group. Later in 2011, the results of the study of rhIFN-γ monotherapy in 25 HBsAg-positive patients with stage 2-4 fibrosis who received long-term rhIFN-γ therapy were published [56]. The authors also showed that longterm therapy for nine months leads to pronounced positive dynamics of inflammation and fibrosis of the liver tissue. Our results with long-term administration of rhIFN-γ confirm these data.

With herpes viral infection, the secretion of cytokines is altered, modulating a strong and effective antiviral immune response against infected host cells. After primary infection, herpes viruses persist in the host organism for a long time [57]. One of the factors contributing to the persistence of herpes viruses is their ability to adopt two different modes of the life cycle: latent and lytic. After primary infection, herpes viruses pass into a latent, transcriptional-translational suppressed state, which can often be interrupted by lytic episodes. During the latency phase, transcripts were identified, in particular, such as microRNAs (miRs), which play a role in the mechanism of evasion of the virus from the host's immune response, including impaired interferon signaling [58].

It has been shown that the early EBV protein BZLF1 can block IFN-γ production by inhibiting the downstream IFN-γ signaling pathway. Essentially, BZLF1 stops the transcription of all expressed HLA class II molecules and, therefore, the activation of T-helper cells required for the induction of an immune response, inhibits IFN-γinduced tyrosine STAT1 phosphorylation and nuclear translocation of BZLF1, reduces the expression of the IFN-γ receptor, stimulating the mechanism, with the help of which EBV can avoid the antiviral immune response during primary infection [59]. In addition, the EBV lytic transactivator Zta suppresses the production of IFN-β,

#### *Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325*

the EBV protein LMP1 inhibits TNF-α and induces the production and secretion of IL-10, and the miR-BHRF1-2-5p EBV blocks the proinflammatory signaling of IL-1 [60]. Cytokine signaling is a very early response to viral infection and explains the presence of corresponding inhibitory viral factors in the tegument. Thus, the dysregulation of the production of proinflammatory cytokines is based on the fact that virions already contain molecules that directly target the proper cytokine signaling. After infection of host cells and transcription of viral DNA leading to translation of viral miRs into viral peptides, other mechanisms of proper immune surveillance are targeted, including, in particular, presentation of HLA class I antigen, as well as decreased expression of NKG2D ligands [61].

INF-γ plays not only an important role in modulating T-cell immunity but also, having a direct antiviral activity is used as an effective therapeutic agent in the treatment of viral infection [62]. Okano et al. conducted a study of the efficacy of therapy with recombinant IFN-γ in a patient with infectious mononucleosis and Xlinked lymphoproliferative syndrome (XLP). EBV-determined nuclear antigen and EBV DNA have been found in various tissues of the patient. After therapy with recombinant IFN-γ, there was positive dynamics in the reduction of virus-infected cells and a linear increase in the content of IFN-γ in the blood serum. NK cell activity remained within normal limits throughout the course of therapy. The authors suggested that cytotoxic cells can produce endogenous IFN-γ [63]. A. Linde et al. also revealed an increase in serum IFN-γ levels 24 h (p = 0.05) and 48 h (p = 0.01) after EBV infection, subsequently, the level of IFN-γ returned to baseline values [64]. In another study, in patients with acute infectious mononucleosis, an increase in the level of serum IFN-γ was shown only during the first week from the moment of infection, later the level of IFN-γ returned to normal [65]. Interesting data were obtained when studying the dynamics of IFN-γ level production in patients with tuberculosis, who showed a decrease in the average IFN-γ level over time (p = 0.001), but this decrease occurred during the first 8 weeks from the start of specific therapy (p = 0.019). When comparing baseline susceptible (N = 55) and drug-resistant patients (N = 18), there was no difference in the change in IFN-γ levels over time. Since the production of IFN-γ and secretion from T cells increase in response to an increase in antigenic load and then stabilize over 24 weeks, a decrease in the concentration of IFN-γ may indicate a positive response to the therapy and play the role of monitoring the response to therapy [66].

Our data indicate the absence of a significant increase in the production of the induced, serum, and spontaneous level of INF-γ three months after the end of therapy with INF-γ in the general group of patients, which is fully consistent with the previously published results of other authors. But when analyzed separately in each group of patients, it was shown that in the group with an initially low level, the administration of INF-γ led to a significant increase in the level of induced INF-γ three months after the end of therapy (p = 0.027). This is probably due to the initial low level of induced INF-γ and a more pronounced response to INF-γ therapy, which manifested itself in a significant positive dynamics of the main clinical complaints. Thus, we demonstrated that the dynamics of the production of the initially low level of induced INF-γ can be a marker of the positive effect of the therapy with INF-γ.

The absence of positive dynamics of the increase in the production of induced INF-γ in the general group of patients one and three months after the end of therapy with INF-γ indicates the absence of the effect of the drug on the level of production of endogenous INF-γ, which was previously demonstrated in studies by other authors. At the same time, INF-γ has a pronounced antiviral effect, which was shown earlier, and does not cause the increase of INF-γ production to the levels that would exceed the reference values.

When analyzing the clinical picture, we revealed that in the group of patients with a higher level of induced IFN-γ production at the time of initiation of therapy, complaints were more pronounced and more frequent. This is probably due to a more intensive inflammatory response in this group of patients. This conclusion is supported by previously published data that these inflammatory reactions are enhanced by the presence of IFN-γ, which dramatically increases the production of inflammatory mediators by macrophages [67].
