Cultivation of E. coli carrying a plasmid-based Measles vaccine construct (4.2 kbp pcDNA3F) employing medium optimisation and pH-temperature induction techniques.

BACKGROUND: Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time. RESULTS: In this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in E. coli DH5α. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production. CONCLUSION: Production of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.

Characterization of endogenous avian leukosis viruses in chicken embryonic fibroblast substrates used in production of measles and mumps vaccines.

Previous findings of low levels of reverse transcriptase (RT) activity in chick cell-derived measles and mumps vaccines showed this activity to be associated with virus particles containing RNA of both subgroup E endogenous avian leukosis viruses (ALV-E) and endogenous avian viruses (EAV). These particles originate from chicken embryonic fibroblast (CEF) substrates used for propagating vaccine strains. To better characterize vaccine-associated ALV-E, we examined the endogenous ALV proviruses (ev loci) present in a White Leghorn CEF substrate pool by restriction fragment length polymorphism. Five ev loci were detected, ev-1, ev-3, ev-6, ev-18, andev-19. Both ev-18 and ev-19 can express infectious ALV-E, while ev-1, ev-3, and ev-6 are defective. We analyzed the full-length sequence of ev-1 and identified an adenosine insertion within the pol RT-beta region at position 5026, which results in a truncated RT-beta and integrase. We defined the 1,692-bp deletion in the gag-pol region of ev-3, and we found that in ev-6, sequences from the 5' long terminal repeat to the 5' pol region were absent. Based on the sequences of the ev loci, RT-PCR assays were developed to examine expression of ALV-E particles (EV) in CEF supernatants. Both ev-1- and ev-3-like RNA sequences were identified, as well as two other RNA sequences with intact pol regions, presumably of ev-18 and ev-19 origin. Inoculation of susceptible quail fibroblasts with CEF culture supernatants from both 5-azacytidine-induced and noninduced CEF led to ALV infection, confirming the presence of infectious ALV-E. Our data demonstrate that both defective and nondefective ev loci can be present in CEF vaccine substrates and suggest that both ev classes may contribute to the ALV present in vaccines.

[Elaboration of live measles vaccine technology on human embryo lung diploid cell culture L-68]. / Razrabotka tekhnologii proizvodstva zhivoi korevoi vaktsiny na osnove kul'tury kletok émbriona cheloveka L-68.

Measles predominates among childhood droplet infections in many countries. Immunization of all human beings sensitive to this infection is the only radical measure in controlling measles. The quality of a vaccine is primarily determined by the properties of the virus strains and cell cultures and technology of production. Now live measles vaccine is produced in or country on the basis of fibroblasts from Japanese quail embryo. The production of live measles vaccine in the primary cell cultures has a number of drawbacks caused by the nonstandard pattern of the substrate and the probability of contamination. The use the certified human diploid cells deposited in liquid nitrogen in sufficient quantities is promising. The authors have elaborated a new technology of live measles vaccine production by using the Leningrad-16 virus strain on the basis of attested L-68 diploid cell culture from the human fetal lung. Experimental batches of vaccine were obtained and attested in accordance with the present requirements for immunobiological products.

Experimental-scale measles and mumps vaccine production on microcarrier-grown cells.

Small-scale measles and mumps virus propagation in microcarrier-grown cells was studied to assess putative advantages over conventional roller-type virus propagation. Significantly higher virus yields could not be attained with microcarrier cultures in cell stirrers, therefore making the advantages purely technological. The pattern of measles virus production was slightly different for the three types of microcarriers used. Experimental measles and mumps vaccine lots obtained met vaccine quality control requirements.