[Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines].

INTRODUCTION: Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form. OBJECTIVE: To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses. MATERIALS AND METHODS: Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay. RESULTS: 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 109 to 1 × 1010 copies/ml. CONCLUSION: Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models.

Immunogenicity and safety of the adult tbe vaccine «tick-e-vac¼.

About 3,000 cases of TBE are registered annually in the Russian Federation. Vaccination is the main way to prevent the tick-borne encephalitis disease. Comparative study of the reactogenicity and immunogenicity of a new vaccine «Tick-E-Vac¼ was held. Volunteers aged from 16 years old were twice immunized with the vaccines «Tick-E-Vac¼ or «Encevir¼ derived from strains of Far East subtype of TBE virus, according to standard and emergency schemes. The clinical study was randomized, comparative, blind, and controlled. The frequency, intensity, time of occurrence, and duration of local and general reactions had been recorded. The titers of antiviral antibodies in ELISA had been determined to assess the immunological efficacy of vaccination. According to the results of the clinical study, the severity of local and general reactions in initial seronegative recipients was weak or moderate. The symptoms were usually manifested within 1-2 days after injection and persisted for not more than 4 days, after which time the symptoms disappeared. There was no statistically significant difference in the reactogenicity of the vaccines after the first and after the second injection. The reactogenicity also did not depend on the gender of recipients. After the first immunization, the level of seroprotection was not less than 43%; the average geometric titer of antibodies (GTA), not less than 1:200. After the second injection, the level of seroprotection reached 90-100%; GTA, not less than 1:500. The data on the reactogenicity and immunogenicity to the original seropositive recipients is not significantly different from the data for the initial seronegative recipients. The data indicate weak reactogenicity of the vaccines «Tick-E-Vac¼ and «Encevir¼. Double vaccination with an interval of 14 or 30 days leads to the formation of expressed immune response. Thus, differences in the level of seroprotection and in antiviral titers in the cases of the standard and emergency vaccination schedules are not statistically significant. The correlation between the development in recipients of local and general symptoms and the immunological efficacy of the vaccines has not been identified.

Protective immunity spectrum induced by immunization with a vaccine from the TBEV strain Sofjin.

Tick-borne encephalitis (TBE) circulates widely in the territory of Eurasia with up to 10,000 cases registered annually. The TBE virus (TBEV) includes three main subtypes: European, Siberian and Far-Eastern, and two new Asiatic variants, phylogenetically distant from the others. The inactivated antigen of European or Far-Eastern strains is used in commercial TBE vaccines. A set of 14 TBEV strains, isolated in 1937-2008, with different passage histories, representing all subtypes and variants, was used in this work. The chosen set covers almost all the TBE area. Sera of mice, immunized with the TBE vaccine Moscow, prepared from the TBEV strain Sofjin, were studied in a plaque neutralization test against the set of TBEV strains. The vaccine induced antibodies at a protective titer against all TBEV strains and Omsk hemorrhagic fever virus (OHFV) with Е protein amino acid distances of 0.008-0.069, but not against Powassan virus. We showed that after a course of two immunizations, factors such as the period between vaccinations (1-4 weeks), the challenging virus dose (30-1000 LD50) and terms of challenge (1-4 weeks after the last immunization) did not significantly affect the assessment of protective efficacy of the vaccine in vivo. The protective effect of the TBE vaccine Moscow against the set of TBEV strains and the OHFV was demonstrated in in vivo experiments. TBE vaccine Moscow did not protect mice against 10 LD50 of the Powassan virus. We showed that this range of Е protein amino acid distances between the vaccine strain and challenging virus do not have a decisive impact on the TBE vaccine protective effect in vitro and in vivo. Moreover, the TBE vaccine Moscow induces an immune response protective against a wide range of TBEV variants.

[Vaccines based on the Far-Eastern and European strains induce the neutralizing antibodies against all known tick-borne encephalitis virus subtypes].

Serum of children aged 1 to 16 obtained in the course of clinical trials conducted in the sverdlovsk region in 2011 was used to study the post-vaccination immunity. Children were immunized twice with vaccines against the tick-borne encephalitis (TBE) Tick-E-Vak on the basis of the strain sofjin of the Far-Eastern subtype and FSME-IMMUN Junior based on the neudorfl strain of the european subtype. According to the plaque reduction neutralization test (PRNT), both vaccines have a high immunogenicity: after 30 days since two-time vaccination in the sera of 100% of children immunized with the vaccine Tick-E-Vak and in the 95% of children immunized with the vaccine FSME-IMMUN Junior antibodies (AT) against strain sofjin were identified in protective titers, whereas 24.5% and 21.4% of children, respectively, had antibody titers higher than 1:10000. selected sera of recipients with titers from 1:25 to 1:1000 were examined in the PRNT in a single experiment using the sofjin (Far-Eastern subtype), absettarov (European subtype) and Vasilchenko (Siberian subtype) strains. The two vaccines induced AT against the representatives of all three subtypes.

Genetic description of a tick-borne encephalitis virus strain Sofjin with the longest history as a vaccine strain.

Vaccines based on the strain Sofjin of the Far-Eastern tick-borne encephalitis virus (TBEV) subtype have been used for TBE prophylaxis for over 50 years in Russia and neighboring countries. On the wide territory, where all known TBEV subtypes are circulating, the cultural, purified, concentrated, inactivated TBE vaccine Moscow has been shown to be safe and efficacious in a massive immunization. In the present work, we describe the genome of the vaccine strain Sofjin. We have shown that it differs from TBEV strains previously published with the name "Sofjin". Moreover, we have shown the stability of the virus during the vaccine manufacturing process on the molecular level.

A synthetic peptide based on the NS1 non-structural protein of tick-borne encephalitis virus induces a protective immune response against fatal encephalitis in an experimental animal model.

Linear immunogenic peptides corresponding to amino acid sequences from the NS1 non-structural protein from tick-borne encephalitis virus (strain Sophyin) were predicted using established algorithms and synthesized. Of the 12 peptides predicted, 11 were able to induce peptide-specific antibodies in BALB/c mice but only 1 of these 11 was able to induce antibodies, which reacted with the native protein in a radio-immune precipitation assay. This peptide corresponds to amino acids 37--55, and forms one of the predicted structurally conserved alpha helices of the virus NS1 protein. It was able to protect 60% of animals against lethal challenge with the homologous highly pathogenic tick-borne encephalitis virus strain, and adoptive transfer experiments indicated the involvement of the antibodies induced by this peptide in its protective activity in mice.

Vaccinia virus recombinant expressing gene of tick-borne encephalitis virus non-structural NS1 protein elicits protective activity in mice.

A vaccinia virus recombinant containing the non-structural tick-borne encephalitis virus (TBEV) NS1 gene was developed. The recombinant expressed native dimeric form of the NS1 protein in infected cells and protected mice against lethal infection with TBEV.

A monoclonal antibody that recognizes the predicted tick-borne encephalitis virus E protein fusion sequence blocks fusion.

The fusion motif of tick-borne encephalitis virus E protein has been predicted to be located within its conserved region (98-120). Results are presented to demonstrate that non-neutralizing monoclonal antibody which recognizes a synthetic peptide corresponding to residues 98-113 of the E protein sequence can block the fusion of the virus particles with artificial membranes.

pH-dependent fusion of tick-borne encephalitis virus with artificial membranes.

pH-dependent fusion of TBE virus with artificial membranes was effective at slightly acidic pH with maximum at 6.4. The influence of various changes in E protein conformation on fusion process was studied.