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.

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.

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.

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.

Diversion of the Arbuzov reaction: alkylation of C-Cl instead of phosphonic ester formation on the fullerene cage.

We report an "inversed" Arbuzov reaction of the fullerene derivatives C60Ar5Cl with trialkyl phosphites P(OR)3 producing alkylated fullerene derivatives C60Ar5R (R = Me, Et, iPr, nBu) with almost quantitative yields. This reaction provides a convenient synthetic route for the preparation of a large variety of functionalized fullerene derivatives with tailored properties, e.g. water-soluble compounds demonstrating promising antiviral activities against HCMV, HSV1, HIV and several influenza virus strains.

DNA sequence-specific ligands. XVII. Synthesis, spectral properties, virological and biochemical studies of fluorescent dimeric bisbenzimidazoles DBA(n).

A series of DNA minor groove binding fluorescent dimeric bisbenzimidazoles DBA(n) bearing linkers of various length were synthesized and their biochemical and antiviral activities were evaluated. Their antiviral activity was assessed in model cell systems infected with human herpes simplex virus (HSV-1) and cytomegalovirus (CMV). Compounds DBA(1) and DBA(7) demonstrated in vitro inhibitory properties towards HSV-1, and DBA(7) completely blocked the viral infection. Compound DBA(11) displayed the in vitro therapeutic activity towards both HSV-1 and CMV. All of the DBA(n) could fluoresce, were well soluble in water, not cytotoxic to a concentration of 240 µM, penetrated well into cell nuclei by binding to DNA and could inhibit topo-I at low micromolecular concentrations.

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.

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.

Detection of human cytomegalovirus in motile spermatozoa and spermatogenic cells in testis organotypic culture.

BACKGROUND: The presence of human cytomegalovirus (HCMV) in male genital tract suggests its vertical transmission with spermatozoa and the development of a potentially dangerous fetal infection. The objective of the present study was to evaluate the possibility of intracellular HCMV localization in male germ cells and to examine the effect of the virus on human spermatogenesis. METHODS: Semen samples from 91 infertile and 47 fertile men were analyzed. HCMV was detected by real time PCR, rapid culture method and PCR in situ. Human testis organotypic culture and quantitative karyological analysis were used to investigate viral effects on spermatogenesis. Localization of HCMV in immature germ cells and spermatozoa was studied by immunostaining with monoclonal antibodies and ultrastructural analysis of infected organotypic culture. RESULTS: Viral DNA was detected in 12.3% samples of motile spermatozoa, while infectious activity only in 2.9% infertile and fertile men without statistically significant intergroup difference. According to PCR in situ, the mean percentage of infected cell in both groups was 1.5% (0.25%-15%), which can serve as a criterion for evaluating the risk of HCMV transmission. In HCMV-infected organotypic culture viral antigens were identified in spermatides on day 4, in spermatogonia and spermatocytes on day 8, and in spermatozoa on day 14. Empty and full capsides and virions were visualized in germ cells by electron microscopy. The number of cells before introduction in culture was taken for 100%. On day 14 infected culture contained 36.8% spermatogonia, 18.7% spermatocytes, 27.6% round spermatides and 42.5% elongated spermatides; in comparison with 82.2%, 51.5%, 70.4% and 65.7% in uninfected culture, respectively (all p < 0.05). There were no changes in the number and viability of spermatozoa. CONCLUSIONS: HCMV was detected in male germ cells, both in sperm samples and in testis organotypic culture. The virus may infect immature germ cells which develop to mature HCMV-carrying spermatozoa. A considerable decrease in the number of immature germ cells indicates that HCMV produces a direct gametotoxic effect and can contribute to male infertility.

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses.

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.

Antigenic epitopes in the hemagglutinin of Qinghai-type influenza H5N1 virus.

The highly pathogenic avian influenza H5N1 viruses have become widespread and evolved into several clades. In our previous studies, the antigenic sites of the H5 hemagglutinin (HA) were characterized by selection and sequencing of escape mutants. In the present studies we analyzed the antigenic epitopes recognized by monoclonal antibodies against avian influenza A/Duck/Novosibirsk/56/05 (H5N1) virus isolated in western Siberia and belonging to subclade 2.2 of the H5N1 viruses. The analysis revealed several antigenically relevant positions of amino acid residues in the globular head of the HA not encountered earlier in the escape mutants of the H5 subtype. The newly recognized positions (113, 117, 118, 120, and 123, mature H5 numbering) are concentrated in an area adjacent to the region described in earlier studies as corresponding to site B in H3 HA, but extending far beyond this area. The amino acid positions recognized by the monoclonal antibodies against A/Duck/Novosibirsk/56/05 (H5N1) virus differ from the positions recognized by the monoclonal antibodies against H5N2 influenza viruses. The data suggest that the evolution of the HA of H5 avian influenza viruses is associated not only with the changes of antigenic epitopes recognized by antibodies, but also with a change in the dominance of the immunogenicity of different sites in the HA.