The present invention relates to immortalized avian cell lines suitable for production of biologicals or viruses for vaccination. In particular, the cell lines are derived from primary cells which are transformed with at least two viral or cellular genes, one of which causes cell cycle progression whereas the other interferes with innate protective mechanisms of the cell induced by dysregulated replication. The invention moreover relates to the production of said immortalized cell lines and their use for producing biologicals or viruses for vaccination.

Immortalized avian cell lines for virus production

Although mycoplasmas are generally considered to be harmless commensals, some mycoplasma species are able to cause infections in pediatric, geriatric, or immunocompromised patients. Thus, accidental contamination of biologics with mycoplasmas represents a potential risk for the health of individuals who receive cell-derived biological and pharmaceutical products. To assess the efficiency of inactivation of mycoplasmas by the agents used in the manufacture of egg-derived influenza vaccines, we carried out a series of experiments aimed at monitoring the viability of mycoplasmas spiked into both chicken allantoic fluid and protein-rich microbiological media and then treated with beta-propiolactone, formalin, cetyltrimethylammonium bromide, Triton X-100, and sodium deoxycholate, which are agents that are commonly used for virus inactivation and disruption of viral particles during influenza vaccine production. Twenty-two mycoplasma species (with one to four strains of each species) were exposed to these inactivating agents at different concentrations. The most efficient inactivation of the mycoplasmas evaluated was observed with either 0.5% Triton X-100 or 0.5% sodium deoxycholate. Cetyltrimethylammonium bromide at concentrations of ≥0.08% was also able to rapidly inactivate (in less than 30 min) all mycoplasmas tested. In contrast, negligible reductions in mycoplasma titers were observed with 0.0125 to 0.025% formaldehyde. However, increasing the concentration of formaldehyde to 0.1 to 0.2% improved the mycoplasmacidal effect. Incubation of mycoplasmas with 0.1% beta-propiolactone for 1 to 24 h had a marked mycoplasmacidal effect. A comparison of the mycoplasma inactivation profiles showed that strains of selected species (Mycoplasma synoviae, Mycoplasma gallisepticum, Mycoplasma orale, Mycoplasma pneumoniae, and Acholeplasma laidlawii) represent a set of strains that can be utilized to validate the effectiveness of mycoplasma clearance obtained by inactivation and viral purification processes used for the manufacture of an inactivated egg-based vaccine.

Evaluation of Mycoplasma Inactivation during Production of Biologics: Egg-Based Viral Vaccines as a Model

Contamination of Japanese quail, strain Pharaoh, cell culture with oncogenous and infectious avian viruses was studied. The susceptibility of the embryonal cell cultures of the Japanese quail, strain Pharaoh, to measles, parotitis and fixed rabies viruses was also determined. It was found that the sera of pubertal quails had no antibody to Rous sarcoma virus (RSV), strains Brian, RSV (RAV-1), Schmidt-Ruppin, Carr-Zilber, as well as to Marek's disease and Newcastle disease viruses. No reverse transcriptase activity was detected in the embryonal alantoic fluid of this avian species. The quails were less susceptible, as compared to the chicken, to Schmidt-Ruppin and Carr-Zilber strains of RSV. Measles, parotitis and fixed rabies viruses reproduced actively in the Japanese quail, Pharaoh strain, embryonal cell cultures. It is suggested that the embryonal cell cultures of this avian species can be used as a leukemia-free substrate for experimental studies and manufacturing of viral vaccines.

["Pharaoh" line culture of Japanese quail cells as a leukosis-free system for virus reproduction].

Agents identified as adenovirus CELO were isolated from organ suspensions of 1500 Japanese quails (JQ) in 4 experiments. No contaminating viruses were found in examinations of cell cultures prepared from the kidneys of 80 JQ. Negative results were obtained in examinations of 163 antigens from JQ organs in the COFAL test. Agents identified as mycoplasma were isolated in 16 cases from 736 specimens of the virus-containing fluid used for manufacture of measles virus. According to the results of the CFT, 59 antigens prepared from Japanese quail embryo cell cultures contained no oncornavirus antigens and were negative in the COFAL test. Among 1848 JQ sera examined for the presence of antibody to leukemia-sarcoma complex viruses only 2 sera were found in 1976 to contain antibody to Rous sarcoma virus. No antibody to Marek disease virus in 414 serum samples or to Newcastle disease virus in 554 sera were detected. Among 500 sera tested 30.8% contained antibody to adenovirus CELO.

I A Brudno

Papers

["Pharaoh" line culture of Japanese quail cells as a leukosis-free system for virus reproduction].

Study of viral contamination of Japanese quail and cell cultures from their embryos