Investigation of the morphology of cell clones, derived from the mammalian EBTr cell line and their susceptibility to vaccine avian poxvirus strains FK and Dessau

Development of Methods for Safe Application of Viral Vectors for Production of Gene-Engineering Vaccines

“…In this connection, the lack of CPE in mammalian cells, inoculated with initial dilutions of 10 3 CCID50/ml (with viral suspensions dilutions of 10 -3 CCID50/ml, respectively), but the presence of immature viral particles, could suggest a possibility about application of the strains in this form as material in vaccine production, including for the goals of the immunotherapy of malignancies. The higher amount of immature virions in cells, inoculated with the vaccine viral strain FK in comparison with this in the cells, infected with strain Dessau, was in support of our previous results about the proved stronger in vitro-CPE, induced by the fowl vaccine strain than the induced by the pigeon vaccine strain, as well as with other literature data [10,17,18]. In this way, a possibility for application of the vaccine fowl pox viral strain FK as a usable source for preparation of vaccines for immune-prophylaxis and immune-therapy was supposed, also in agreement with the literature finding about the high immunogenic potential of this strain [21].…”

“…The cells were incubated in combination of Modified Eagle's Medium (MEM) (Sigma) and Dulbecco's Modification of Eagle's Medium (DMEM) (Sigma), in ration 1:1, supplemented with 5% mixture of Fetal Bovine Serum (FBS) (Sigma) and Normal Bovine Serum (NMS) (Sigma), as well as of antibiotics mixture of (100 UI/ml Penicillin and 100 μg/ml Streptomycin) (Sigma), at 37°C in incubator with 5% СО2 and 95% air humidification. Three main steps for development of strategy about safe application of both viral strains were evaluated: application of heterologous for mammals and cells from them avian viral strains; of attenuated vaccine forms of both strains by many passages in cell cultures and chicken embryos, as well as of comparatively low initial infections titers of 10 3 CCID50/ml viral suspension (high initial dilutions of viral suspensions of 10 -3 CCID50/ml, respectively) [16][17][18]. Separated sub-populations of mammalian cells, inoculated with the same low dilutions of the suspensions of both viral vaccine viral strains, were freezed at -80°C or at -196°C (liquid nitrogen) after previous addition of respective volume from the cryoprotector DMSO (Sigma), added directly to the inoculated cell cultures, for 1-2 weeks.…”

Avipoxviruses: infection biology and their use as vaccine vectors

“…Nelson (1941) [52] reported mild pathology in mice following intranasal inoculation with FWPV, with no virus replication. Recent studies have also shown replication of APV in mammalian cell cultures, such as embryonic bovine tracheal cells [53] and baby hamster kidney cells [54] that are defined by the presence of infectious viral particles and CPE. These studies raise questions about the species specificity and mechanisms that restrict these viruses to certain hosts, and challenge the hypothesis that APV cannot undergo a full replication cycle in mammalian cells.…”

“…Hence, identification of new mediators that are up or down-regulated in response to APV infected mammalian and avian cells could help advance our knowledge of immune responses against APV and the related immune-mediated pathology and cell tropism. It would be of vital importance to investigate these characteristics further, especially for the cell types that were recently shown to support APV replication [53,54]. …”

“…To our knowledge, such reversions have not been identified in clinical trials of APV-vectored vaccines. In addition to concerns regarding reversion or recombination, another safety signal was recently identified in an in vitro experiment that showed APV replication in cell clones derived from embryonic bovine trachea [53] and Syrian baby hamster kidney (BHK) cells. In this experiment, infectious IMV was observed; indicating complete virus replication had taken place [54].…”