**1.3 The use of interferon-γ in the treatment of herpesvirus infections**

In recent years, numerous works have been published in the world on the treatment of herpesvirus infections with recombinant IFN-γ, showing high clinical and antiviral efficacy [27, 37–39]. IFN-γ demonstrated a 7–10 times more potent antiviral effect than IFN-α or -β. When IFN-γ is added at the 3–4th days after infecting, there is a decrease in EBV-induced B cell proliferation and immunoglobulin secretion, while the addition of IFN-α and -β has an effect only within 24 h. Lotz et al. Found that EBV-infected cells can be regulated by all IFNs at an early stage. Subsequently, there comes an intermediate period when only IFN-γ is able to directly influence EBV-induced B-cell responses. In the third phase, B-lymphocytes become insensitive to the direct action of all IFNs and are exposed only to cytotoxic cells [40]. In 2002 the introduction of recombinant IFN-γ, as well as IFN-β, showed high efficiency of inhibition of replication of the herpes simplex virus type 1 (HSV-1) [41]. That is, the high level of inhibition achieved by the administration of exogenous IFN-γ was the result of a synergistic interaction with endogenous IFN-α/β, which is produced locally in response to HSV-1 infection. Other researchers revealed that IFN-β and IFN-γ interact synergistically, blocking viral DNA synthesis and nucleocapsid formation in HSV-1 infected cells, without affecting the viability of the host cells. Thus, the authors concluded that IFN-mediated suppression of HSV-1 replication plays the role of the main mechanism by which the host immune system limits the spread of infection in vivo [42]. In a doubleblind, placebo-controlled study, it was shown that the introduction of recombinant IFN-γ three times a week subcutaneously reduces the incidence of severe infections in patients with various genetic types of chronic granulomatous disease [43].

In the Russian Federation, the only IFN-γ preparation has been registered under the trade name Ingaron, developed by SPP PHARMACLON Ltd. via the microbiological synthesis in a recombinant E. coli strain and purified by column chromatography. The drug consists of 144 amino acid residues, devoid of the first three of them (Cys-Tyr-Cys), replaced by Met.

The purpose of this study is to evaluate the efficacy of IFN-γ therapy for the content of the number of EBV DNA copies in saliva samples by the Real-time PCR method, for the dynamics of INF-α and INF-γ production (spontaneous, serum, and induced levels) and the clinical picture in patients suffering from chronic Epstein-Barr virus infection (CEBVI) one and three months after the end of therapy.

## **2. Material and methods**

#### **2.1 Schemes of therapy**

All patients were divided into three groups for different therapy regimens.


(Valcyte 450 mg 2 times a day, orally) for 2 months in combination with IFN-γ (10–20 intramuscular injections of Ingaron 500,000 IU every other day). Previously, all patients in this group, as prescribed by a doctor or independently (often repeatedly), received therapy with drugs from the group of acyclic natural nucleosides, including valaciclovir for short courses (7–10 days). There was no pronounced clinical and laboratory positive effect from the previous therapy, for this reason, these patients have prescribed valganciclovir in combination with IFN-γ.

To assess the efficacy, a comparative analysis of the amount of EBV DNA in saliva samples was carried out one month after the end of the treatment course. Clinical complaints were compared for the patients of Group 1 in one and three months after the treatment course.


Patient groups and therapy are presented in **Table 1**.

#### **Table 1.**

*Characteristics of therapy in patient groups.*

The study procedures were in accordance with the Good Clinical Practice (GCP) guidelines and ethical principles of the Declaration of Helsinki. The study was approved by the Ethics Committee of Fresenius Medical Care (Dialysis Center St. Petersburg, Russia). Written informed consent was obtained from all participants before the study was initiated.

#### **2.2 Survey methods**

Clinical research methods included the collection of anamnesis, data on previous immuno- or antiviral therapy, and the presence of concomitant diseases. The clinical condition of patients was assessed according to the generally accepted method, including objective data and registration of patient complaints at the time of examination. The severity of patient complaints was recorded using a subjective assessment scale on a 3-point scale (0—no symptoms, 1—mild symptoms, 2—moderate severity of symptoms, 3—significant severity of symptoms).

Diagnosis of CEBVI was based on clinical data and positive results of EBV DNA studies in saliva samples conducted by polymerase chain reaction (PCR) with realtime hybridization-fluorescence detection. The test systems "AmpliSens EBV/ CMV/HHV6-screen-FL" (FBSI "Central Research Institute of Epidemiology", Russia) were used. The unit of measurement used to estimate the viral load during DNA extraction from saliva is the number of copies of EBV DNA per ml of sample. According to the instructions, this indicator is calculated using the formula: Number *Recombinant Human Interferon-Gamma: Prospects for the Treatment of Chronic Epstein-Barr… DOI: http://dx.doi.org/10.5772/intechopen.101325*

of DNA copies = CDNA 100, where CDNA is the number of copies of the viral DNA in the sample. The analytical sensitivity of the test system is 400 copies/ml.

We studied the dynamics of IFN-α and -γ production before the initiation of IFN-γ therapy and 1 and 3 months after the end of the course. Determined the level of IFN-α and IFN-γ in the blood serum, as well as the spontaneous and induced production of these cytokines in the culture of blood lymphocytes. Newcastle disease virus (NDV) (obtained at the L.A. Tarasevich State Institute of Culture, St. Petersburg) with an infectious titer of 8 lg EID/0.2 ml in a volume of 8 μl/hole and Phytohemagglutinin (PHA-P) (PanEko, Russia) at the dose of 10 μg/ml were used as inducers of IFN-α and -γ production respectively. The quantitative content of cytokines was determined in the serum and supernatant of a 24-h whole blood culture by solid-phase ELISA using the test systems "alpha-Interferon-IFA-BEST" and "gamma-Interferon-IFA-BEST" (JSC "VectorBest", Russia). Reference values for spontaneous, serum, and induced production of IFN-α and IFN-γ are provided by the manufacturer of the test systems.

#### **2.3 Statistical analysis**

Statistical analysis of the obtained results was carried out using the statistical software package IBM SPSS Statistics, version 26 (Armonk, NY: IBM Corp.). Group results are presented as mean standard error of the mean (SEM). Statistical processing of the results was carried out using parametric (Pearson's method) and nonparametric (Kendall's tau (τ)) criteria. To check the condition of independence of observations, linear regression analysis (with the calculation of the coefficient of determination (R Square) and the Durban-Watson criterion) and analysis of variance (ANOVA Analisis of Variance) with the calculation of the Fisher criterion (F) were carried out to test the significance of the model. The standardized coefficient ß was calculated with 95% Confidence Interval (95% CI). The critical level of significance of the difference in indicators was taken equal to 0.05.

### **3. Results**

The examination was carried out in 139 patients with СEBVI: 86 women and 53 men with average age of 35.27 1.28 years. The duration of CEBVI from the appearance of the first complaints to laboratory confirmation and the diagnosis was 2.23 0.21 years. 98 (7.720%) and 27 (24.54%) patients suffered in childhood from chronic tonsillitis with no response to antibiotic therapy and infectious mononucleosis respectively. All patients underwent differential diagnosis of CEBVI with other viral infections (HIV, viral hepatitis, cytomegalovirus infection, toxoplasmosis), helminthic invasions, autoimmune diseases associated with EBV infection.

CEBVI is characterized by a long course and frequent relapses with clinical and laboratory signs of viral activity, described in detail in the literature [44–46]. Patients worried about prolonged subfebrile condition (37.1–37.3°C), weakness, unmotivated fatigue, excessive sweating (especially at night), a constant feeling of discomfort and/or pain in the throat, lymphadenitis, swelling of the nasal mucosa with abundant drainage mucus, stomatitis. Some patients have a cough, skin rashes, arthralgia, pain in the muscles of the trunk and extremities are possible. There may be manifestations of conjunctivitis, otitis media. Neurological disorders develop headaches, memory and sleep disorders, decreased concentration, irritability, tearfulness, a tendency to depression. Perhaps an increase in internal organs (according to ultrasound, hepato- or splenomegaly) and feeling of heaviness in the right hypochondrium. Also, patients complain of frequent colds, the addition of other herpesviral infections. In the history of such patients, long-term stressful situations, psychoemotional and physical overloads often take place, against the background of which the patient's condition worsens.
