RU2799141C2 - Method of treatment of patients with herpes virus infection using in vitro activated autological lymphocytes - Google Patents

Abstract

FIELD: medicine; immunology.

SUBSTANCE: invention can be used for the treatment of herpes virus infection using autologous lymphocytes activated in vitro. The method includes the following: taking venous blood from a patient with a herpes virus infection, isolating lymphocytes, followed by cultivation in a CO₂ incubator in vials with a nutrient medium containing recombinant human interleukin-2, interleukin-15, sodium pyruvate, L-glutamine. Inactivated autologous serum is added to the nutrient medium, and before being added to the nutrient medium, it is inactivated at 60°C for 60 min, lymphocytes are cultivated for 14 days in a CO₂ incubator, on the 3rd, 5th, 7th, 10th day of cultivation, part of the cells is collected, half of the volume of the nutrient medium is changed, on the 14th day the cells are collected completely, living cells in the amount from 5 to 20 million are resuspended in the supernatant with a volume of 1.0 to 2.0 ml and injected into the patient intradermally paravertebral at 2 points, as in figure 1. The remaining cell product is distributed in 1.5–2 ml ampoules and stored at -30°C for subsequent use, then after 1–2 days, after the fifth injection of live cells, the cell product, previously thawed and warmed to body temperature, is also injected intradermally paravertebral at two points, in a volume of 1.5–2 ml, daily.

EFFECT: invention provides treatment of patients with herpes virus infections, including those with genital herpes, and allows to induce a high immune response, characterized by an increase in the duration of the relapse-free period.

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Description

SUBSTANCE: invention relates to medicine, namely to immunology, and can be used for the treatment of herpes virus infection using autologous lymphocytes activated in vitro.

According to the World Health Organization (WHO), herpes virus infection ranks second among human viral diseases, second only to influenza. Due to the ability of the herpes simplex virus to persist for a long time, leading to a decrease in antiviral immune defense, the treatment of patients with herpes simplex is a difficult task. Progress in the field of treatment of herpes virus infection was achieved in the 80s, and is associated with the development of the first drug from the group of acyclic nucleosides - Acyclovir. According to international standards for the treatment of herpes virus infections, the most commonly prescribed antiherpetic drugs are Acyclovir, Valtrex, Famvir. There are two main ways of using antiviral chemotherapy drugs: episodic appointment (as needed for exacerbations of herpes virus infection in a short course for 5-10 days) and prolonged therapy (daily for several months to stop this relapse). The goal of therapy in this case is to reduce the severity of symptoms of relapse and reduce the period of virus shedding [Shulzhenko A.E., Zuykova I.N. Approaches to immunotherapy of recurrent herpes simplex // Effective Pharmacology, No. 3, 2010].

The genital form of herpes is characterized by the appearance of erosive and ulcerative defects in the genital area, both in men and women. Symptoms develop with the introduction of the virus through the mucous membranes of the genital organs. According to statistics, the average prevalence of genital herpes is 20 cases per 10,000 population. In 70% of carriers, the infection proceeds in a latent form (without clinical manifestations). Symptoms of the disease occur only during relapses. Sources of infection are persons with an acute form of the disease, i.e. active isolation of the virus occurs in the phase of the formation of a herpetic rash, respectively, at this time the risk of infection of sexual partners is maximum, even if barrier methods of contraception are used. In a satisfactory state of health, the immune system suppresses the pathogen and does not allow infection to develop. However, the virus is not eliminated from the body, but hides in the nerve ganglia. The reason for the occurrence of recurrences of genital herpes is a decrease in immunity, for example, against the background of hormonal disruptions, stress, medication, or physical exertion, therefore, in the event of a weakening of the body, the pathogen is activated and again affects the mucous membranes [Ashakov M.S., Chebotarev V.V. A modern view on the course, diagnosis and treatment of herpes virus infection // Bulletin of a young scientist, No. 1, 2018, pp. 25-31].

Usually, the treatment of herpes virus infections includes antiviral agents, analgesics, antipyretics, topical preparations with antiviral components and anesthetics. However, taking antiviral drugs does not ensure complete elimination of the virus from the body, and often, after discontinuation of drugs, relapses of the disease occur with the same frequency. The patient is forced to continuously take expensive drugs that have side effects, which significantly reduces the quality of life. Great hopes are placed on immunotherapy, including immunomodulators and vaccines, because their main target is secondary immunodeficiencies, which are manifested by frequent recurrent and difficult-to-treat herpes virus infections of various localizations [Shulzhenko A.E., Zuykova I.N. Approaches to immunotherapy of recurrent herpes simplex // Effective Pharmacology, No. 3, 2010].

There are many Russian and foreign patented developments of vaccines and components against the herpes simplex virus (coding proteins, antigens) (RU No. 2575067, WO2021217206, US2021299248, WO2014164699, US5858376, EP2865386).

The disadvantages of these developments is the difficulty of obtaining such components and vaccines that require special equipment and personnel. Most developments have not been evaluated tolerability and efficacy in humans.

In Russia, the Vitagerpavak vaccine is known, containing inactivated antigens of the herpes virus (RU 2731073). The group of inventions relates to the field of biotechnology and immunology. A recombinant fusion protein from immunogenic epitopes of human herpes viruses types 1-8 for immunization against herpes is characterized by the amino acid sequence of SEQ ID NO: 1. DNA encoding the protein with the nucleotide sequence of SEQ ID NO: 2. Plasmid DNA for immunization against herpes is a plasmid pcDNA3.1 vector containing the indicated DNA. The authors declare that this vaccine is the only vaccine in the world for the treatment and prevention of relapses of herpes simplex types 1 and 2, produced according to modern international standards, which has shown safety and high efficiency. The therapeutic effect is to stimulate cellular immunity aimed at suppressing the herpes virus, reduces the degree of immune disorders from the 3rd and 2nd to the 1st degree.

The disadvantages of this invention is the complexity of obtaining the drug, you need special laboratory equipment and specially trained personnel. This invention does not guarantee 100% effectiveness against the occurrence of disease recurrence.

There are many Russian and foreign patented developments of antibodies (RU No. 2703903, RU No. 2500422, CN108064239, AU2012202218).

The disadvantages of these developments are the difficulty of obtaining, the need to use special expensive equipment and consumables, for most developments, the assessment of tolerability and effectiveness in humans has not been studied.

A known method (RU No. 2577299) for the treatment and prevention of viral infectious diseases (viral hepatitis, hepatitis C, herpes virus infection, diseases caused by the influenza virus, acute respiratory viral infection and bacterial infectious disease), characterized in that the body is administered simultaneously and a combination of a complex drug made in the form of a pharmaceutical composition containing an activated-potentiated form of affinity-purified antibodies to one or more cytokines selected from interferon gamma, interferon alpha, tumor necrosis factor alpha, and an activated-potentiated form of antibodies to one or more receptors selected from CD4, CD8. The inventors note the good tolerability of the drug and the absence of adverse events.

However, there is a difficulty in the use of the drug, which lies in the complexity of obtaining affinity-purified antibodies and the need to use special expensive equipment, as well as the use of antibodies of animal origin (rabbits).

There are many Russian patented drug developments, including Acyclovir (RU No. 2179851), a lactic acid preparation (RU No. 2459620), vaginal suppositories (RU No. 2729647), a method for producing IFN-α (RU No. 2108804), a drug based on inactivated herpes simplex virus serotypes 1 or 2 (RU No. 2264819), as well as foreign drug developments (CA2679067, WO2008118765, WO2008118763, WO2008118762, US5182104, UA80502).

There are many Russian patented regimens for the treatment of herpes virus infections with drugs (RU 2180569, RU 2492861).

The disadvantages of the methods is the need to take these antiviral drugs on an ongoing basis, because, as you know, after drug withdrawal, relapses of the disease occur with the same frequency. The constant need to take drugs is a direct deterioration in the quality of life of patients.

An invention is known (RU 2147889) for the treatment of urethrogenital infections and herpes, consisting of hydrogen peroxide at a dosage of 0.15 - 0.18% for 120 - 300 ml and a decoction of licorice roots 0.5 - 1.0 ml per 100 ml of peroxide. Intravenous infusion of the drug is carried out in courses of 10 - 15 infusions at a rate of 50 - 60 drops per minute, after 1 - 3 days, while a mixture of hydrogen peroxide is used as a preparation at a dosage of 0.15 - 0.18%, 120 - 300 ml and decoction of licorice roots 0.5 - 1.0 ml per 100 ml of peroxide.

The disadvantage of the development is the intravenous route of administration of the drug and the lack of information on the tolerability of this invention by patients.

A known method for the treatment of recurrent urogenital herpes with symptoms of chronic fatigue syndrome (RU 2492861), in which therapy is carried out by administering an antiviral drug in combination with an interferon inducer, characterized in that the oral form of Acyclovir is used as an antiviral drug, and an injection is used as an interferon inductor. or a liposomal oral form of high-polymer RNA, moreover, intramuscularly high-polymer RNA is administered at 8 mg every 72 hours for 10 days and orally Acyclovir 200 mg five times a day for 10 days or orally liposomal high-polymer RNA (Akavia) is administered daily for 6, 5 mg twice a day for 5 days followed by maintenance therapy at 6.5 mg every other day for 20 days and oral Acyclovir 200 mg five times a day for 10 days.

However, the method is laborious in obtaining liposomal high-polymeric RNA (Acavia), and special expensive equipment and consumables are required. The main disadvantage of this method is the need to use acyclovir in treatment regimens, which have a number of contraindications and side effects, such as allergic manifestations: itching, rash, urticaria, erythema multiforme, Stevens-Johnson or Lyell syndrome, angioedema, anaphylaxis.

Known developments in the field of immunology based on dendritic cells (RU 2309753). The invention relates to the field of biopharmacology and medicine. It proposes a method for obtaining blood mononuclear cells: activated lymphocytes (LAC) and DC, which includes: a) the isolation of mononuclear leukocytes (MNC) from the blood or bone marrow of a person in need of treatment or prevention of a malignant neoplasm, or an infectious disease, or an immunodeficiency state; b) incubation of isolated mononuclear lymphocytes in a culture medium; c) separation of adherent and non-adherent MNC culture flasks; e) treatment of adherent MNCs with growth factors to obtain dendritic cells from them; f) treating the DC with the patient's tumor lysate; g) cultivation of non-adherent lymphocytes with interleukin-2 to generate LAK cells. A method is proposed for the treatment and prevention of malignant and infectious diseases and immunodeficiency conditions in patients in need of this, which consists in the combined use of dendritic cells and LAK cells.

However, the method has a major drawback. It is not clear from the description of the invention whether this approach allows to induce a specific immune response against viral antigens (HCV) and what is the severity and duration of the immune response, how does this affect the clinical course of a chronic viral infection. Thus, it is not known whether this method will be effective in the treatment of herpes virus infection, which is different from other chronic viral infections. Also, the method is technologically complex and expensive.

A known method for immunotherapy of a chronic viral infection (RU 2485962), including the introduction to the patient of autologous dendritic cells generated from peripheral blood monocytes and loaded with a viral antigen in in vitro culture, characterized in that dendritic cells are cultivated in the presence of interferon-alpha, and as an antigen is used recombinant herpes virus antigen and use the obtained dendritic cells 2 courses through subcutaneous vaccinations in combination with the preparation of recombinant human interleukin-2 as an adjuvant for the treatment of frequently recurrent herpes virus infection.

The disadvantage of this method is the need for hospitalization of the patient and only during remission (at least 2 weeks after the last relapse). The method is complicated, in our opinion, by the need to generate dendritic cells (DCs) from monocytes isolated from the fraction of mononuclear cells (MNCs) of the peripheral blood of patients, a large amount of blood (200 ml) is required, the use of expensive growth factors and media for DC activation, as well as the presence of antibiotics in a cultural environment.

A known method of vaccine therapy for herpes infection (RU 2514034), which consists in using a standard anti-herpes vaccine, which is a suspension of herpes simplex viruses I and II types killed by formalin, characterized in that dendritic cells (DC) obtained from the patient's blood in vitro , "load" with viral antigens, the source of which is this vaccine; the resulting vaccine is injected intradermally at 4-6 points along the lymphatic collectors, the course of vaccine therapy consists of 3-4 injections, the intervals between vaccine injections are from 2 to 4 weeks, the initial dose of the vaccine is 1 × 106-2 × 106 DC, with the second injection the dose is increased by 2 times, with the third injection, the dose is increased by 4 times compared with the first injection.

The disadvantage of this method is the need to generate dendritic cells (DC) from monocytes isolated from the fraction of mononuclear cells (MNC) of the peripheral blood of patients. Using a source of viral antigens - an antiherpetic vaccine, which is a suspension of herpes simplex viruses I and II types killed by formalin. It should be noted that the invention only reduces the frequency of relapses, and does not eliminate them for a long time.

The closest (prototype) is a method of treating cancer patients with cytotoxic lymphocytes (EN 2596505 C1). Venous blood is taken from a cancer patient or a donor, lymphocytes are isolated, activated in a nutrient medium containing recombinant human interleukin-2, interleukin-15, albumin, sodium pyruvate, gentamicin, L-glutamine, for 9 days in a CO2 incubator , then activated cytotoxic lymphocytes in an amount of 2 to 10 million are resuspended in the cell product and injected intradermally paravertebral at 2-4 points to an oncological patient on the 3rd, 5th, 7th and 9th day of activation. On the same days, the cell product is distributed in 1.5-2 ml ampoules and stored in a freezer for subsequent use in outpatient treatment. The invention is intended for the treatment of patients with malignant tumors such as cancer of the stomach, colon, pancreas, breast and melanoma, including those with common and inoperable forms of stage 3-4 of the disease, as well as for the prevention of metastasis after surgical treatment.

The disadvantage of this invention is that it has not been studied in terms of the treatment of herpes virus infections. Indeed, the main role in antitumor and antiviral immunity is played by T-lymphocytes that activate cytotoxic T-lymphocytes, macrophages and NK cells. Immune surveillance of the normal composition of tissues is implemented due to non-specific factors that damage tumor cells: 1) NK cells, a system of mononuclear cells, the antitumor activity of which is enhanced by interleukin-2 (IL-2) and alpha- and gamma-interferons; 2) LAK cells (mononuclear cells and NK cells activated by IL-2); 3) cytokines (alpha and gamma interferons, TNF-alpha and IL-2). Target cells infected with the virus are destroyed by cytotoxic lymphocytes, NK cells and phagocytes. Interferons enhance antiviral resistance by inducing the synthesis of enzymes in cells that inhibit the formation of nucleic acids and proteins of viruses. In addition, interferons have an immunomodulatory effect, enhance the expression of major histocompatibility complex (MHC) antigens in cells. Therefore, there is reason to refine the method for obtaining activated in vitro lymphocytes and more detailed study of the use of these cells for the treatment of patients with herpes virus infection.

The technical result is the treatment of a herpes virus infection, including genital herpes, both in primary patients and often recurrent with the help of in vitro activated autologous lymphocytes, which allow to induce a high immune response.

The specified technical result in the implementation of the invention is achieved due to the fact that, just as in the known method, venous blood is taken from a patient, lymphocytes are isolated, activated in a nutrient medium containing recombinant human interleukin-2, interleukin-15, sodium pyruvate, L-glutamine , for several days in a CO2 incubator, then activated cytotoxic lymphocytes are resuspended in the cell product and injected intradermally paravertebral, and the cell product is distributed in 1.5-2 ml ampoules and stored in a freezer for subsequent use in outpatient treatment.

The peculiarity of the proposed method lies in the fact that inactivated autologous serum is added to the nutrient medium, and before being added to the nutrient medium it is inactivated at 60°C for 60 minutes, lymphocytes are cultivated for 14 days in a CO2 incubator, for 3, 5, 7 , on the 10th day of cultivation, part of the cells is collected, half of the volume of the nutrient medium is changed, on the 14th day the cells are collected completely, living cells in an amount of 5 to 20 million are resuspended in a supernatant with a volume of 1.0 to 2.0 ml and injected to the patient intradermally paravertebral in 2 point, as in figure 1, the remaining cell product is distributed in ampoules with a volume of 1.5-2 ml and stored at -30°C for later use, then after 1-2 days, after the fifth injection of living cells, it is also injected intradermally paravertebral into two points cellular product, previously thawed and warmed to body temperature, in a volume of 1.5-2 ml, daily. Moreover, the concentration of recombinant human interleukin-2 is from 200 to 500 ng/ml, and interleukin-15 is from 4 to 6 ng/ml. The concentration of autologous serum in the nutrient medium ranges from 2.5 to 5%. On the 3rd, 5th, 7th, 9th and 14th day of activation, half of the volume of the nutrient medium is renewed or the cell suspension is collected completely, viability, proliferative activity is assessed, centrifuged at 1200-2000 rpm, and the cell product is collected. If a relapse occurs, the course is repeated.

The method is illustrated by a detailed description, clinical examples and an illustration, which shows:

Fig. 1. - Photo illustration after the procedure for introducing cells - intradermally paravertebral at two points.

The method is carried out as follows.

For the treatment of herpes virus infection, including genital herpes, using in vitro activated autologous lymphocytes, venous blood is taken from a patient with herpes virus infection and lymphocytes are isolated under sterile conditions. The isolated lymphocytes are activated in a nutrient medium containing recombinant human interleukin-2, interleukin-15, sodium pyruvate, L-glutamine and autologous serum inactivated at 60°C for 60 min for 14 days in a CO2 incubator. The culture medium does not contain antibiotics. Then, on the 3rd, 5th, 7th, 10th and 14th day of activation, cytotoxic lymphocytes in an amount of 5 to 20 million under sterile conditions are resuspended in the cell product and injected into the patient intradermally paravertebral at two points (Figure 1). On the same days, the remaining cell product is distributed in 1.5-2 ml ampoules and stored at -30°C for subsequent use in outpatient treatment. Further, after 1-2 days, daily No. 5 is also injected intradermally paravertebral into 2 points of the cell product in a volume of 1.5-2 ml, pre-warmed to body temperature.

Thus, the course consists of 10 injections. After the end of the first course, it is possible to repeat the course of immunotherapy in the "on demand" mode, i.e. in the event of another exacerbation or for prevention. EFFECT: invention makes it possible to induce a high immune response characterized by an increase in the duration of the relapse-free period.

The order of implementation of the method.

To isolate lymphocytes from a patient using a vacuum system and test tubes containing heparin, venous blood is taken and diluted 2 times with saline NaCl 0.9% (Russia). The diluted blood is layered on a Ficoll solution with a density of 1.077 g/cm ³ (PanEco, Russia) and centrifuged at 2000 rpm for 25 min, which leads to sedimentation of erythrocytes and granulocytes to the bottom of the tube. As a result of centrifugation, lymphocytes form an interphase ring, which is collected with a pipette and washed twice by centrifugation in a 10-fold volume of phosphate-buffered saline (Russia) at 1800 rpm for 10 minutes.

The isolated lymphocytes are counted, resuspended at a concentration of 1.5-2×10 ⁶ cells/ml in a nutrient medium (containing sodium pyruvate, L-glutamine, with the addition of 2.5 to 5% previously obtained from the patient and inactivated at 60°C in for 60 min of autologous serum, and with the addition of 200 to 500 IU / ml of human recombinant IL-2 (Roncoleukin, Biotech, Russia), and from 4 to 6 ng / ml of human recombinant IL-15 (SciStore, Russia)) and activate for 14 days in plastic bottles with a vented cap in a CO2 incubator at a temperature of 36.9 to 37.1°C. RPMI-1640 (PanEco, Russia) can be used as a nutrient medium. On the 3rd day of cultivation receive activated cytotoxic lymphocytes.

A morphological assessment of the lymphocyte culture and a visual assessment of the nutrient medium for the absence of pathogenic microflora are carried out daily. To maintain the viability of activated cytotoxic lymphocytes, on the 3rd, 5th, 7th and 10th day of activation, half of the volume of the nutrient medium is changed, and on the 14th day they are completely collected. At the same time, the required amount of activated cytotoxic lymphocytes is collected for administration to the patient (5-20 million), and the remaining cell product is distributed in 1.5-2 ml ampoules and stored at -30°C for further use. When replacing the nutrient medium, the viability of cells and their proliferative activity are assessed.

For immunotherapy with activated lymphocytes on days 3, 5, 7, 10 and 14, the resulting cells are collected in an amount of 5 to 20 million, resuspended in a cell product with a volume of 1.0 to 2.0 ml and injected into the patient intradermally paravertebral at 2 points. Then daily No. 5 also injected intradermally paravertebral into 2 points of the cell product in a volume of 1.5-2 ml, pre-warmed to body temperature.

Thus, the course consists of 10 injections. After the end of the first course, it is possible to repeat the course of immunotherapy in the "on demand" mode, i.e. in the event of another exacerbation or for prevention.

The increase in cultivation time to 14 days was achieved by adding autologous inactivated blood serum to the culture medium, which made it possible to obtain a greater number of activated lymphocytes, therefore, to increase the number of injections of activated lymphocytes to the patient without additional blood sampling. All components and media used to implement the proposed method, Russian production

Clinical examples.

Example 1

Patient N., born in 1958 has been observed at the Center on an outpatient basis since May 2015. Diagnosis: A60.0 Herpetic infections of the genital organs and urinary tract. From the anamnesis - for about 20 years she has been suffering from chronic recurrent herpes infection of the genital organs and genitourinary tract, for the last 10 years, almost monthly exacerbations, that is, at least 12 times a year. Exacerbations were accompanied by general weakness, chills, subfebrile condition, pain during urination. She was treated with local and systemic antiviral drugs, with little or no effect. According to the words, exacerbations took place both against the background of complete well-being, and against the background of stress, hypothermia, and always before menstruation. Not examined.

A course of 10 intradermal injections of autologous activated in vitro lymphocytes was carried out. The patient was injected intradermally paravertebral at two points - on days 3, 5, 7, 10 and 14 in an amount of 10 to 12 million. warmed to body temperature.

Lymphocytes were injected daily on weekdays. Local reaction - orange peel papule, hyperemia +1.0 cm, itching. The general reaction in the form of a flu-like syndrome developed on the 3rd injection and continued until the 10th injection. Slight chills and aches in the muscles began at 18.00 and lasted about 2 hours. Body temperature did not rise.

The first exacerbation from the moment of the 1st course occurred after 4 months and was characterized by much less pronounced manifestations, literally, twice as short in time. Local treatment with Viferon gel was required 6 times a day. The second exacerbation occurred in May 2016 at the time of the second visit for a course of immunotherapy. A PCR test was carried out - DNA of herpes simplex was found. On the third day from the onset of the exacerbation, which was manifested by vaginal itching, painful urination, herpetic eruptions on the mucous membrane of the vestibule of the vagina and chills, the first injection of autologous lymphocytes was carried out on the 3rd day of incubation. The next day, the patient noted a sharp decrease in itching, no pain during urination, and no development of blisters. All existing manifestations were of a smoothed nature against the background of a course of immunotherapy and disappeared by the 7th day without the use of standard drugs. The course was carried out as planned, and the patient left for her place of residence. During the intercourse period, the patient was not observed, because she lives abroad. Arriving for the third course in May 2017, the patient reported that there was a relapse once and the rashes did not develop into vesicles, but were only in the form of slight hyperemia and mild itching. In 2018, the patient did not come, but reported that there were no exacerbations. Considering the further situation with covid restrictions, she has not come to Russia so far, however, it is known that exacerbations occur 1-2 times a year, are much less pronounced and are stopped by the local application of antiviral ointments. The patient never had Covid. PCR monitoring tests for herpes viruses were negative. The follow-up period is 7 years.

Example 2

Patient K., born in 1992 I have been outpatient since June 2018. The observation period is 4 years. Diagnosis: A60.0 Herpetic infections of the genital organs and urinary tract. From the anamnesis - from the age of 15 he suffers from chronic recurrent herpesvirus infection of the genitals and genitourinary tract. From the words, the disease continuously recurred. She received Isoprinosine continuously 2 times a day when the process subsided and 7 times a day in combination with Valtrex when the rashes resumed. PCR test - DNA of herpes simplex was detected.

Since July 2018, the first course has been held.

For this, blood was taken from the patient in the amount of 30 ml, followed by isolation and cultivation of lymphocytes. On the 3rd, 5th, 7th, 10th day of cultivation, half of the volume of the nutrient medium was changed, on the 14th day the cells were completely collected, the supernatant (cell product) was frozen at -30°C in an amount of 1.5 ml, and the cells in an amount of 5 to 8 million resuspended in the supernatant and injected into the patient intradermally paravertebral at 2 points. Then, after 2 days, daily No. 5 was also injected intradermally paravertebral at two points with a cell product in a volume of 2 ml, pre-warmed to body temperature.

Thus, the first course consisted of 10 injections. Then, intradermal injections were performed with the supernatant in the “on demand” mode, which meant immunotherapy during the exacerbation period.

In the period from June 2018 to June 2019, 20 intradermal injections were performed on an individual basis. Against the background of “on demand” immunotherapy, the relapse-free intervals lengthened, and the manifestations of a blistering rash, ailments, weakness, and chills became almost imperceptible.

In the period from June 2019 to June 2022, the patient applied three times for short courses of 3 prophylactic injections before the holiday, since genital herpes did not bother her. From the beginning of the Covid 19 epidemic until April 2022, the patient worked in the "red zone", she fell ill with Covid 19 only in March 2022 in a mild form. From May 2019 to July 2022 (38 months) there were no relapses. PCR monitoring tests for herpes viruses were negative.

Example 3

Patient G. born in 1996 has been observed on an outpatient basis since December 2020. Diagnosis: A60.0 Herpetic infections of the genital organs and urinary tract. From the anamnesis - for about two years she suffers from monthly exacerbations, was treated with Valtrex during periods of exacerbations. PCR test - DNA of herpes simplex was detected. Exacerbations were accompanied by an increase in body temperature up to 38°C, blisters of painful rashes on the labia, urethritis, loss of appetite. In the interrecurrent period, she felt weakness, headaches, loss of appetite, refused to play sports, and then could not work. Was examined by an endocrinologist. Diagnosis: autoimmune thyroiditis. Weight loss of about 3 kg in six months.

4 courses of 10 intradermal injections of autologous activated in vitro lymphocytes were carried out. The patient was injected intradermally paravertebral at two points - on days 3, 5, 7, 10 and 14 in an amount of 5 to 20 million. warmed to body temperature.

Courses were held in spring and autumn in the stated mode.

Local reaction - orange peel papule, hyperemia +1.0 cm, itching. The general reaction in the form of a flu-like syndrome developed on the 2nd injection and continued until the 10th injection. Slight chills and aches in the muscles began at 18.00 and lasted about 2 hours. Body temperature did not rise. After the first course, the exacerbations decreased to 2 per year, but in the third course they stopped, the patient began to recover and returned to work. There are currently no outbreaks. PCR monitoring tests for herpes viruses were negative. The observation period is 2 years.

Example 4

Patient M., born in 1993 Diagnosis: A60.0 Herpetic infections of the genital organs and urinary tract. From the anamnesis, since November 2020, he has been suffering from chronic herpesvirus infection, he constantly takes Acyclovir 400 mg, exacerbations occur when the dose is reduced by 200 mg. PCR test - DNA of herpes simplex was detected.

On May 17, 2022, a course of treatment with in vitro activated autologous lymphocytes was started. The patient was injected intradermally paravertebral at two points - on days 3, 5, 7, 10 and 14 in an amount of 7 to 10 million. body temperature.

She endured well, notes an improvement in mood, general tone. Against the background of treatment, an exacerbation episode occurred on the fourth week, ended in 6 days without taking systemic drugs, locally applied: Viferon gel on the vesicles, Viferon ointment on the crusts 4 times a day. Manifestations were weak - less itching, less discomfort, less rashes. Currently, he receives autolymphocytotherapy on an “on demand” basis. There is no exacerbation. Monitoring studies of PCR for herpes viruses are in the plan. The observation period is 2 months.

The proposed method allows:

- induce a high immune response and increase the duration of the relapse-free period,

- increase the time of cell cultivation up to 14 days by adding autologous inactivated blood serum to the culture medium,

- get more activated lymphocytes, thereby increasing the number of injections of activated lymphocytes to the patient without additional blood sampling.

Claims (6)

1. A method for treating patients with herpes virus infection using in vitro activated autologous lymphocytes, including taking venous blood from a patient with herpes virus infection, isolating lymphocytes, followed by cultivation in a CO ₂ incubator in vials with a nutrient medium containing recombinant human interleukins -2, interleukin-15, sodium pyruvate, L-glutamine, characterized in that inactivated autologous serum is added to the nutrient medium, and before being added to the nutrient medium it is inactivated at 60°C for 60 minutes, lymphocytes are cultivated for 14 days in under conditions of CO ₂ incubator, on the 3rd, 5th, 7th, 10th day of cultivation, some of the cells are collected, half of the volume of the nutrient medium is changed, on the 14th day the cells are collected completely, living cells in an amount of 5 to 20 million are resuspended in a supernatant with a volume of 1.0 up to 2.0 ml and injected to the patient intradermally paravertebral at 2 points, as in figure 1, the remaining cell product is distributed in 1.5-2 ml ampoules and stored at -30°C for subsequent use, then after 1-2 days, after the fifth injection of living cells, the cell product, previously thawed and warmed to body temperature, is also injected intradermally paravertebral at two points in a volume of 1.5-2 ml daily. 2. The method according to p. 1, characterized in that the concentration of recombinant human interleukin-2 is from 200 to 500 ng/ml. 3. The method according to p. 1, characterized in that the concentration of recombinant human interleukin-15 is from 4 to 6 ng/ml. 4. The method according to claim 1, characterized in that the concentration of autologous serum in the nutrient medium is from 2.5 to 5%. 5. The method according to p. 1, characterized in that on the 3rd, 5th, 7th, 9th and 14th day of activation during the renewal of the nutrient medium or the complete collection of lymphocytes to assess the viability and proliferative activity of the cells and to collect the cell product, the cell suspension is centrifuged at 1600 rpm for 5 min. 6. The method according to p. 1, characterized in that in the event of a relapse, the course is repeated.

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