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.

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems.

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

Kinetic and pharmacokinetic characteristics of therapeutic methinoninе γ-lyase encapsulated in polyion complex vesicles.

Therapeutic enzymes used for the treatment of a wide range of human disorders often suffer from suboptimal pharmacokinetics and stability. Engineering approaches such as encapsulation in micro- and nanocarriers, and replacements of amino acid residues of the native enzyme provide significant potential for improving the performance of enzyme therapy. Here, we develop a nanodelivery system on the base of polyion complex vesicles (PICsomes) that includes methionine γ-lyase (MGL) as a therapeutic enzyme. We have two strategies for using the enzyme: first, methionine γ-lyase is an anticancer agent removing l-methionine from plasma, second, the binary system methionine γ-lyase/S-alk(en)yl-l-cysteine sulfoxides is effective in enzyme prodrug therapy (EPT). Various lengths polymers were synthesized, and two mutant forms of the enzyme were used. The catalytic and pharmacokinetic parameters of the nanoformulations were investigated. The catalytic efficiencies of encapsulated enzymes were comparable to that of native enzymes. Pharmacokinetic analysis has shown that inclusion into PICsomes increases half-life of the enzymes, and they can be safely administered in vivo. The results suggest the further use of encapsulated MGLs for EPT and anticancer therapy, and this strategy could be leveraged to improve the efficiency of enzyme-based therapies for managing serious human diseases.

Mesenchymal Stem Cells Can Both Enhance and Inhibit the Cellular Response to DNA Immunization by Genes of Nonstructural Proteins of the Hepatitis C Virus.

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.

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.

Dual-targeted anti-CMV/anti-HIV-1 heterodimers.

Despite the development of efficient anti-human immunodeficiency virus-1 (HIV-1) therapy, HIV-1 associated pathogens remain a major clinical problem. Human cytomegalovirus (CMV) is among the most common HIV-1 copathogens and one of the main causes of persistent immune activation associated with dysregulation of the immune system, cerebrovascular and cardiovascular pathologies, and premature aging. Here, we report on the development of dual-targeted drugs with activity against both HIV-1 and CMV. We synthesized seven compounds that constitute conjugates of molecules that suppress both pathogens. We showed that all seven compounds exhibit low cytotoxicity and efficiently inhibited both viruses in cell lines. Furthermore, we chose a representative compound and demonstrated that it efficiently suppressed replication of HIV-1 and CMV in human lymphoid tissue ex vivo coinfected with both viruses. Further development of such compounds may lead to the development of dual-targeted anti-CMV/HIV-1 drugs.

Human Herpesviruses Increase the Severity of Hepatitis.

Acute and chronic liver diseases are a major global public health problem; nevertheless, the etiology of 12-30% of cases remains obscure. The purpose of this research was to study the incidence of human herpesviruses (HHVs) cytomegalovirus, Epstein-Barr virus (EBV), and HHV-6 in patients with hepatitis and to examine the effect of HHV on the disease severity. We studied the clinical materials of 259 patients with hepatitis treated in Infectious Clinic n.1 (Moscow) and the archived materials of 118 patients with hepatitis C. HHV DNA was detected in the whole blood in 13.5% of patients with hepatitis B or C and in 10.1% of patients with hepatitis of unspecified etiology. EBV demonstrated the highest incidence (58.1%). Cirrhosis was diagnosed in 50% of patients with HHV and in 15.6% of patients without HHV. In patients with hepatitis C, the frequency of HHV was higher in liver biopsy (38.7%) compared to blood. The clinical and virological indicators of hepatitis were considerably higher in patients with coinfection. Conclusion: HHV detected in patients with viral hepatitis has been associated with a significant effect on the severity of the disease, and we suggest monitoring HHV DNA in patients with severe hepatitis and/or poor response to antiviral drugs.

Thiophene-based water-soluble fullerene derivatives as highly potent antiherpetic pharmaceuticals.

Here we report the Friedel-Crafts arylation of chlorofullerenes C60Cl6 and C70Cl8 with thiophene-based methyl esters. While C60Cl6 formed expected Cs-C60R5Cl products, C70Cl8 demonstrated a tendency for both substitution of chlorine atoms and addition of an extra thiophene unit, thus forming Cs-C70R8 and C1-C70R9H compounds. The synthesized water-soluble C60 and C70 fullerene derivatives with thiophene-based addends demonstrated high activity against a broad range of viruses, including human immunodeficiency virus, influenza virus, cytomegalovirus, and herpes simplex virus. The record activity of C70 fullerene derivatives against herpes simplex virus together with low toxicity in mice makes them promising candidates for the development of novel non-nucleoside antiherpetic drugs.

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.

DNA sequence-specific ligands. XVIII. Synthesis, physico-chemical properties; genetic, virological, and biochemical studies of fluorescent dimeric bisbenzimidazoles DBPA(n).

A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.