Human Mesenchymal Stem Cells Modified with the NS5A Gene of Hepatitis C Virus Induce a Cellular Immune Response Exceeding the Response to DNA Immunization with This Gene.

Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein.

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

Detection of S-HBsAg Mutations in Patients with Hematologic Malignancies.

Multiple studies of hepatitis B virus (HBV) genetic variability and its relationship with the disease pathogenesis are currently ongoing, stemming from growing evidence of the clinical significance of HBV mutations. It is becoming increasingly evident that patients with hematologic malignancies may be particularly prone to a higher frequency of such mutations. The present report is the first extensive study of the prevalence of escape mutations in S-HBsAg, performed using isolates from 59 patients from hospital hematology departments with diagnoses of leukemia (n = 32), lymphoma (n = 20), multiple myeloma (n = 3), and non-tumor blood diseases (n = 4). The isolates were serologically examined for the presence of HBV markers and sequenced using either next-generation sequencing (NGS) or Sanger sequencing. Occult hepatitis B was found in 5.1% of cases. Genetic analysis of the region corresponding to S-HBsAg demonstrated an exceptionally high mutation frequency in patients with leukemias (93.4%) and lymphomas (85.0%), along with the prominent mutation heterogeneity. Additionally, more than 15 mutations in one sample were found in patients with leukemias (6.3% of cases) and lymphomas (5.0% of cases). Most of the mutations were clinically significant. The study analyzes the mutation profile of HBV in different oncohematological diseases and the frequency of individual mutations. The data strongly suggest that the NGS method, capable of detecting minor populations of HBV mutations, provides a diagnostic advantage, lays the foundation for the development of screening methods, and allows for the study of the virological and pathogenetic aspects of hepatitis B.

New Approaches to the Prevention and Treatment of Viral Diseases.

The review discusses a new approach to the prevention and treatment of viral infections based on the use of pine needles polyprenyl phosphate (PPP) and associated with the infringement of prenylation process-the attachment of farnesol or geranyl geraniol to the viral protein. Currently, prenylation has been detected in type 1 adenovirus, hepatitis C virus, several herpes viruses, influenza virus, HIV. However, this list is far from complete, given that prenylated proteins play an extremely important role in the activity of the virus. We assume that the interferon produced in response to PPP may suppress expression of the SREBP2 transcription factor. As a result, the mevalonic acid pathway is violated and, as a result, the formation of early polyprenols precursors (geraniol, geranyl geraniol, farnesol), which are necessary for the prenylation of viral proteins, is blocked and the formation of mature, virulent virus particles is broken. As a consequence, the maturation of viral particles is inhibited, and defective particles are formed. Polyprenol was extracted from greenery (pine, fir and spruce needles, mulberry leaves, etc.), purified by chromatography, phosphorylated and identified by HPLC and NMR. Obtained PPP was used as antiviral in some experimental models in vitro and in vivo. During numerous studies, it was found that PPP manifested versatile antiviral effects, both in vitro and in vivo. The maximum effect was observed with viruses in which the presence of prenylated proteins was established, namely influenza A virus, HIV-1, tick-borne encephalitis virus, hepatitis A and C viruses, herpes simplex viruses type 1 and 2, some coronavirus. The available data obtained both in the experimental conditions and during clinical trials allow us to regard PPPs as safe and effective medicine for prevention and treatment of viral diseases.

Genetically Modified Mouse Mesenchymal Stem Cells Expressing Non-Structural Proteins of Hepatitis C Virus Induce Effective Immune Response.

Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and leads to cirrhosis and hepatocarcinoma. Despite extensive research, there is still no vaccine against HCV. In order to induce an immune response in DBA/2J mice against HCV, we obtained modified mouse mesenchymal stem cells (mMSCs) simultaneously expressing five nonstructural HCV proteins (NS3-NS5B). The innate immune response to mMSCs was higher than to DNA immunization, with plasmid encoding the same proteins, and to naïve unmodified MSCs. mMSCs triggered strong phagocytic activity, enhanced lymphocyte proliferation, and production of type I and II interferons. The adaptive immune response to mMSCs was also more pronounced than in the case of DNA immunization, as exemplified by a fourfold stronger stimulation of lymphocyte proliferation in response to HCV, a 2.6-fold higher rate of biosynthesis, and a 30-fold higher rate of secretion of IFN-γ, as well as by a 40-fold stronger production of IgG2a antibodies to viral proteins. The immunostimulatory effect of mMSCs was associated with pronounced IL-6 secretion and reduction in the population of myeloid derived suppressor cells (MDSCs). Thus, this is the first example that suggests the feasibility of using mMSCs for the development of an effective anti-HCV vaccine.

New polyprenyl phosphate based preparation Fortepren(®) as promising cytokine regulationg antiviral remedy.

Fortepren(®), a product of the phosphorylation of polyprenols from fir needles (with sodium polyprenyl phosphate being the main active ingredient), belongs to the class of antiviral drugs with immunomodulating activity. Fortepren(®) may be used as the drug of choice in the treatment of herpes diseases. It was shown that treatment with Fortepren(®) of patients with a chronic recurrent herpes infection after acute phase termination with acyclovir decreased the recurrence rate, as well as the severity of local symptoms. Fortepren(®) treatment of patients with a high incidence of recurrent herpes infection led to an increase in the interferon-producing ability of leucocytes stimulated with NDV, as well as in the production of key cytokines (IL-1ß, IL-15, MIP-1α, IFN-γ, IL-12 (p40), TNF-α, IFN-α2, IL-12 (p70), IL-6) taking part in the protection against viral infection. Data suggest that the action of the drug is directed, first of all, to the cells responsible for the natural resistance of the organism (macrophages, dendritic cells, etc.). The activation of natural immunity appears to be a leading mechanism of protection from herpesviral infection under the influence of polyprenyl phosphate.

Plant polyisoprenoids and control of cholesterol level.

The ability of plant polyisoprenoids (polyprenols and polyprenyl phosphates) to diminish the levels of serum cholesterol affecting its biosynthetic pathway are highlighted here. Possible mechanism of such process is discussed. It is also noted that polyisoprenoids can prevent toxic injuries of the liver and restore disturbed hepatic functions. The possibility of polyprenyl phosphates to reveal at the same time anti-inflammatory action suppressing lipoxygenase activity and lowering the levels of proinflammatory cytokines will be illustrated. Attention will be focused on the potential usefulness of plant polyisoprenoids in the course of prevention and treatment of hypercholesterolemia. High efficiency for combined use of polyprenyl phosphate and ß-sitosterol, which leads to substantial enhancement of the ability to overcome hypercholesterolemia versus the individual constituents will be demonstrated.

Polyprenols as possible factors that determine an instructive role of the innate immunity in the acquired immune response.

Polyprenols are an integral part of all living cells including prokaryotic and eukaryotic ones. These compounds take part in biosynthesis of glycoproteins. We have found that phosphates of polyprenols may act as effective antiviral agents with a wide spectrum of activity. One of such antiviral agents received from Pinus sativum polyprenols was named phosprenyl. Earlier we showed that phosprenyl expressed direct antiviral effect, while having mild immunomodulatory activity. In the present study we further evaluated influence of phosprenyl on the immune system. The drug was found to inhibit an early phase of IL-1 and Con A interaction in spleen cells as well as lypoxigenase activity and expression of IL-2 receptors. At the same time, phosprenyl induced NK cell activity and early TNF-alpha production. Basing on all these data we proposed that polyprenols could be considered as a "label" which grants a possibility to the innate immune system to recognize infection at the early stages and govern the acquired immunity.

Role of Cytokines in Immunomodulatory Effects of Polyprenyl Phosphate: New Generation of Antiviral Drugs.

Immunomodulatory properties of sodium polyprenyl phosphate (PP) were studied in vivo and in vitro. After injection to mice, PP was shown to increase serum levels of TNF-alpha, IL-6, and IFN-gamma. The simultaneous inoculation of tick-born encephalitis virus (TBEV) and PP to mice resulted in earlier serum appearance of IL-6, TNF-alpha and IFN-gamma (at days 1, 2 and 3, respectively) compared with mice which have received PP only. In TBEV-infected mice (not injected with PP) cytokines in serum were registered later - at day 7 after infection. Development of the disease with subsequent death was observed in 100% of infected mice. In contrast, mortality of mice infected with TBEV and simultaneously treated with PP was decreased to 40%. The study of spleen cell proliferative activity in mice injected with PP revealed a modulating effect of the latter. In vitro PP decreased spleen cell and Con A-induced blast proliferation stimulated by Con A and rIL-2 respectively. This effect was dependent upon PP inhibition of IL-2 binding to IL-2 receptors. It was concluded that PP induced early cytokine production (IL-6, TNF-alpha) by cells of monocyte/macrophage origin and, apparently, provided protection of mice against viral infection. Thus, the main properties of PP are the following: absence of the expressed direct effect on cytokine production and co-stimulating effect in combination with a bystander stimulus (in this case - TBEV).

Functional and Serological Evidences of the Non-Immune Interaction of Interleukin-2 and Immunoglobulin G.

The mechanism of non-immune inhibition of biological activity of IL-2 by IgG or its immune complexes was studied using serological analysis on the model of IL-2-dependent proliferation of Con A-induced blast cells obtained from a mouse spleen. The mechanism investigated is realized through formation of a complex between IL-2 and an effector part of IgG molecule. It is suggested that this mechanism can participate in immune regulation, formation of individual and population resistance to infections, and pathogenesis of infectious and non-infectious diseases with antigen persistence.