Viruses vaccine production

A more general example from virus vaccine production is the rigorous examination of tissue cultures to exclude contamination with infectious agents from the source animal or, in the cases of human diploid cells or cells from continuous cell lines, to detect cells with abnormal characteristics. Monkey kidney cell cultures are tested for simian herpes B virus, simian virus 40, mycoplasma and tubercle bacilli. Cultures of human diploid cells and continuous line cells are subjected to detailed kary-ological examination (examination of chromosomes by microscopy) to ensure that the cells have not undergone any changes likely to impair the quality of a vaccine or lead to undesirable side-effects. 

Optoelectronics Optosil Oraflex Oragrafin Oral care products Oral contraceptives Oral formulations Oral polio virus vaccine Oral toxicity Oramec Orange... 

Viruses replicate only in living cells so the first viral vaccines were necessarily made in animals smallpox vaccine in the dermis of calves and sheep and rabies vaccines in the spinal cords of rabbits and the brains of mice. Such methods are no longer used in advanced vaccine production and the only intact animal hosts that are used are embryonated hens eggs. Almost all of the vims that is needed for viral vaccine production is obtained from cell cultures infected with vims of the appropriate strain. 

With attenuated viral vaccines the potential hazards are those associated with reversion of the virus during production to a degree of virulence capable of causing disease in vaccinees. To a large extent this possibility is controlled by very careful selection of a stable seed but, especially with live attenuated poliomyelitis vaccine, it is usual to compare the neurovirulence of the vaccine with that of a vaccine known to be safe in field use. The technique involves the intraspinal inoculation of monkeys with a reference vaccine and with the test vaccine and a comparison ofthe neurological lesions and symptoms, if any, that are caused. If the vaccine causes abnormalities in excess of those caused by the reference it fails the test. 

A competitive assay could also be used for quantitation. In a competitive assay, unlabeled antigen competes for labeled antigen. Examples include ELISAs for vaccine product antigens, such as recombinant proteins from viruses, or nonvaccine antigens such as growth factors or cytokines. 

To conclude, an issue that is bringing great attention in recent years, the production of chimeric virus-hke particles should be briefly analysed. These chimeric VLPs are potentially valid systems for broader vaccine production, i.e. against a large number of different serotypes [34] in addition they can result in safe combination vaccines between closely related viruses [35], can be able to carry multiple foreign epitopes [36-39], or even, with the incorporation of tags (e.g. polyhistidine), allow easy single-step product recovery [40,41]. 

The basic process technology in vaccine production consists of fermentation for the production of antigen, purification of antigen, and formulation of the final vaccine, In bacterial fermentation, technology is well established. For viral vaccines, cell culture is the standard procedure. Different variations of cell line and process system are in use. For most of the live viral vaccine and other subunit vaccines, production is by direct infection of a cell substrate with the virus. Alternatively, some subunit viral vaccines can be generated by rDNA techniques and expressed in a continuous cell line or insect cells. 

Kawakita, 1963). This cell line is certified by the WHO (World Health Organization) and is usually employed for virus propagation (polio, rabies) for human vaccine production. Another important application of these cells is in cytotoxicity studies of biomaterials projected for repairing or reconstituting injured human tissues. 

The MRC-5 cell line, derived in 1966 from normal human lung tissue (Jacobs et al., 1970), is adherent and shows a fibroblast morphology. These cells are well known owing to their susceptibility to several virus types, being employed in assays related to viral transfection, in cytotoxicity evaluation, and in vaccine production. 

Transient expression Production of recombinant viruses Transient and stable expression Production of recombinant viruses Transient and stable expression Vaccine production Transient and stable expression Stable expression... 

Cell culture is the predominant and indispensable tool for virus isolation and cultivation infectivity assays and vaccine production and testing. Although some viruses are more easily isolated in animals and embryonated eggs, the modem era of virology only began when Enders et al. (1949) showed that poliovirus was able to reproduce in various kinds of human embryonic cells in culture whereas in vivo its multiplication is largely restricted to the neurons in the grey column of the spinal cord. 

In the early years primary cells were used as viral hosts. Although these have limited growth potential they support the replication of a wide variety of viruses. Continuous cell strains such as mouse L929 or human HeLa cells are now commonly used for virus growth in biochemistry laboratories, but for vaccine production untransformed, diploid cell strains are the preferred hosts. 

The major concerns with vaccine production are firstly, is the virus harmless and secondly is the vaccine free of adventitious agents. If the vaccine is produced from a virulent virus it is essential that the virus is completely inactivated before vaccine distribution. If attenuated strains are used the stability of the attenuation must be monitored. In both cases cell cultures are used in tests. 

The registration of medicinal products is governed by relative values which cannot be clearly defined or limited, for example by ethical considerations and the endeavour to ensure the best possible quality and safety of pharmaceuticals. What is possible and achievable, may soon become a standard by which subsequent products will be measured. This leads to continuous and desirable improvements, but since there is no limit, very costly and undesirable extremes may be another consequence. According to today s standards, the most successful and beneficial pharmaceutical products, such as the vaccinia virus vaccine, live poliovirus vaccine and other live vaccines, would probably not be registrable for a wide-spread use in humans. 

Figure 7.3 UV versus LIF detection of the CE separation of a 53 base pair RT-PCR product from the RNA of the polio virus vaccine, Sabin 3. An Hae Ill-digested d>X174 DNA marker was coinjected with the PCR product for size determination—note the 72 bp fragment. The same Sabin 3 concentration was used for each analysis, whereas the marker total DNA concentration varied from 200 mg/mL for UV analysis, to 20 mg/mL for LIF analysis. Note the unambiguous pattern observed with LIF for the Sabin 3 fragment compared to UV detection of the same fragment. Full scale UV detection, 0.005 absorbance unit (AU) LIF detection, 10 relative fluorescence units (RFU). [Reproduced with permission from Schwartz et al., J Capillary Electrophor 1 36 (1994). Copyright ISC Technical Publications, Inc.]...

Figure 7.9 CE separation and quantitation of the RT-PCR product (53 bp) from Sabin 3 strain of the polio virus vaccine. Discrete volumes of the template RNA were reverse transcribed and the complementary DNA PCR amplified. The resulting products were analyzed by CE-LIF using a replaceable gel matrix. With increasing amounts of RNA template, peak height and area also increased up to point (> 2.0 /nL template), whereupon the reaction plateaued.

The L-3 (Leningrad) strain was derived by combining five isolates of mumps virus from sick children. The attenuated strain is used for vaccine production, especially in Russia. 

Human diploid cell line WI-38 is derived from embryonic lung tissue with fibroblast morphology (Hayflick Moorhead, 1961). The cell line has a broad range of virus susceptibility and is used for the production of human virus vaccines rhinoviruses, measles, mumps, rubella, polio, rabies and yellow fever. The cells are anchorage dependent but will form a multilayered culture when held for long periods at 37°C with periodic pH adjustments. The cells have a hfe span of 50 10 population doublings, with a doubling time of 24h. 

Population balance modeling of influenza virus replication in MDCK cells during vaccine production... 

Keywords population balance modeling, distributed populations, virus replication, vaccine production, microcarrier cell culture. 

Despite the high social relevance of infectious diseases and widespread use of animal cell lines in vaccine production, the application of even unstructured models for quantitative analysis and parameter estimation has not been common practice in bioprocess optimization. So far, research concerning influenza vaccine production in MDCK cell cultures has focused on the characterization of metabolism, growth of different cell lines and virus yields in various production systems [1,2]. 

J. Schulze-Horsel, Y. Genzel, U. Reichl, 2007. Quantification of intracellular accumulation of Ml and NP of influenza A virus - monitoring of infection status of production cells during vaccine production by flow cjdometry. Submitted to BioMedCentral Biotechnology. 

Figure 19-5 Prostaglandin E2 production induced by the vaccines hepatitis B virus (FIBV 1 200), influenza virus vaccine (Fluzone 1 400), and Tetanus toxoid vaccine (DTaP 1 200). PTE is the peripheral tissue equivalent module containing CD33+ positively selected cells (left). HUVEC (center) is the empty PTE and also produces PGE2 when stimulated. CD33+ monocytes alone do not produce PGE2 when stimulated with vaccines (right).

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