Double Recombinant Vaccinia Virus: A Candidate Drug against Human Glioblastoma

Glioblastoma is one of the most aggressive brain tumors. Given the poor prognosis of this disease, novel methods for glioblastoma treatment are needed. Virotherapy is one of the most actively developed approaches for cancer therapy today. VV-GMCSF-Lact is a recombinant vaccinia virus with deletions of the viral thymidine kinase and growth factor genes and insertions of the granulocyte–macrophage colony-stimulating factor and oncotoxic protein lactaptin genes. The virus has high cytotoxic activity against human cancer cells of various histogenesis and antitumor efficacy against breast cancer. In this work, we show VV-GMCSF-Lact to be a promising therapeutic agent for glioblastoma treatment. VV-GMCSF-Lact effectively decreases the viability of glioblastoma cells of both immortalized and patient-derived cultures in vitro, crosses the blood–brain barrier, selectively replicates into orthotopically transplanted human glioblastoma when intravenously injected, and inhibits glioblastoma xenograft and metastasis growth when injected intratumorally.

EVALUATION OF IMMUNOGENICITY AND PTOTECTIVENESS OF THE VACCINIA VIRUS LIVP-GFP IN THREE LABORATORY ANIMAL SPECIES

Smallpox eradication and absence of adequate animal model of smallpox infection causes necessity of the assessment of immunogenic and protective properties of the created by genetic engineering approaches live attenuated smallpox vaccines in several animal models of orthopoxviral infections. In this research comparison of the immunogenic and protective properties of the recombinant vaccinia virus (VACV) LIVP-GFP after intradermal (i/d) injection to mice, guinea pigs and rabbits were carried out. Doses of LIVP-GFP immunization in all animal species were 2x104 or 2x106 pfu. Control animals were injected with saline. Blood sampling was done on 28 day after virus LIVP-GFP or saline injection. Blood samples were taken intravitally from the retro-orbital venous sinus of mice, from heart of guinea pigs or marginal ear vein of rabbits. Serum was isolated from blood samples by precipitating blood cells via centrifugation. The anti-VACV IgG titers in the serum samples were determined by ELISA. On 30 day of the experiment immunized by virus LIVP-GFP or control animals were intranasal infected with lethal doses of the corresponding orthopoxviruses to which every animal species was sensitive. Mice were infected by cowpox virus (CPXV) strain GRI-90 in dose 68 LD50, guinea pigs - by VACV GPA in dose 56 LD50, rabbits – by VACV HB-92 in dose 100 LD50. All control animals after that were died, but all animals immunized by attenuated recombinant virus LIVP-GFP in dose 2x106 pfu were survived. In case of the LIVP-GFP immunization dose 2x104 pfu 88% of mice were survived after CPXV infection, 67% of rabbits were survived after VACV HB-92 infection, and 50% of guinea pigs were survived after VACV GPA infection. ELISA data of the blood serums had shown correlation of the levels of VACV-specific antibodies with levels of protection in the corresponding animals. On the basis of the obtained data it could be concluded that all three studied models animal-orthopoxvirus allow give an adequate evaluation of immunogenicity and protectiveness of the created modern attenuated vaccines against smallpox and other orthopoxviral human infections. BALB/c mice are the most convenient subject of this investigation

Efficient Selection of Recombinant Fluorescent Vaccinia Virus Strains and Rapid Virus Titer Determination by Using a Multi-Well Plate Imaging System

Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte®S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.

Insights into the Organization of the Poxvirus Multi-Component Entry-Fusion Complex from Proximity Analyses in Living Infected Cells

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of eleven conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by co-immunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP β-strands. To achieve fluorescence, two additional 20-amino acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and co-transfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quanitative way do determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of eleven conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.

Preparation of Class Y Immunoglobulins that Neutralize the Marburg Virus

The aim was to study the possibility of inducing Marburg-neutralizing chicken antibodies (MARV) using various immunogens.Materials and methods. Recombinant vaccinia virus expressing the surface glycoprotein (GP) transgene MARV of Musoke strain and pseudovirus particles exhibiting GP of three strains of MARV – Popp, Musoke and DRC2000 based on lentivirus and recombinant strain of vesicular stomatitis virus (VSV) were used as immunogens. Two groups of birds were involved in the study. Chickens were immunized 9 times: first time they were injected with the recombinant vaccinia virus, and then 8 times – with pseudovirus particles (based on lentivirus and a recombinant strain of the vesicular stomatitis virus). The accumulation of specific antibodies was evaluated by enzyme-linked immunosorbent assay (ELISA). We used recombinant VSV exhibiting GP MARV, and natural MARV strain Popp for the analysis of accumulation of neutralizing antibodies.Results and discussion. We have developed an effective immunization schedule for chickens with three recombinant constructs presenting GP MARV, which results in the induction of chicken IgY antibodies against Marburg virus with a titer in ELISA from 1:100 to 1:1 million. The obtained IgY neutralize MARV pseudoviruses (Popp, DRC2000, Musoke) at a dilution of 1/256 to 1/1024 and the natural MARV virus of the Popp strain at a dilution of 1/8. More stable results were demonstrated by immunization using Freund’s incomplete adjuvant.