Oral Polio Vaccine
Currently, the oral polio vaccine (OPV), is the major tool in the WHO Global Polio Eradication Initiative, consists most frequently of a trivalent mixture of three strains corresponding to each of the three serotypes of polio. Monovalent and bivalent preparations are also increasingly in use. The master strains from which master seeds and working virus stocks for production are prepared are those developed by Sabin in the 1950s by passage of polioviruses under various laboratory conditions followed by intensive evaluation of virulence and genetic stability in primate models, including old world monkeys and chimpanzees. Molecular studies of the vaccine strains suggest that for at least the Type 2 and 3 strains, only two or three mutations account for the attenuated phenotype, which allows the virus to infect recipients and induce an immune response without causing disease. In fact, in a small number of instances estimated at one in 500,000 first-time vaccine recipients, the strains do cause poliomyelitis. This is such a low level of adverse event that for many years it proved difficult to get useful figures; for instance, with a birth cohort of approximately 650,000, the UK could expect one or fewer cases per year. However, when it happens it is clearly a very serious event, and as polio moves closer to being eradicated it becomes ever more significant. Moreover, it was recognized from the very beginning that vaccine-associated poliomyelitis was a real possibility. The initial need was, therefore, to ensure that the final product was as safe as possible. This was achieved by a combination of production controls and final product testing.
The Sabin vaccine strains are all temperature sensitive in their growth, so the first step was to ensure that the temperature of production was controlled at 35°C or less, with tight limits. It was also clear that the more growth cycles the virus was subjected to the more likely it was to acquire a virulent phenotype; therefore, the number of growth cycles was tightly controlled: the multiplicity of infection in terms of numbers of infectious virus particles per production cell was relatively high and the duration of the production run was restricted to 2 or 3 days to ensure that it involved only a limited number of growth cycles. Finally, the origin of the master and working seed viruses were documented. The original pool of the viruses produced by Sabin in the laboratory has been designated 'Sabin original' or 'SO' and the number of passages from this material is recorded.
These controls on the production method were based on testing of the final individual vaccine bulks in the monkey neurovirulence test, which showed that the virulence of the Type 3 vaccine in particular was unacceptable after four passages in monkey cells, that is Sabin original + 4. The combination of production controls and final product testing gave a vaccine of acceptable attenuation and consistency, and helped to define the production conditions needed. Currently, additional tests of greater precision are possible, including assessing the proportion of virulent mutants present by molecular means (mutant analysis by PCR and restriction enzyme cleavage) and the use of transgenic mice carrying the human polio receptor as an alternative neurovirulence test to that in monkeys. This enables production parameters to be explored and set without using primates in the first instance. The need to control passage level from vaccine seeds shown to give an acceptable vaccine is a general principle that applies to yellow fever, measles, mumps and all live attenuated vaccines. It is also applied to killed vaccines including influenza as described below, the principle being that the more a virus is grown in culture the greater the chance that it will mutate from the virus whose properties are known by clinical experience.
In addition to the properties of the virus itself, extraneous viruses were often introduced into the product from the cells used to grow it. In the early days, most production was on primary cell cultures from monkey kidney, and some manufacturers continue with this method, which has been shown to give a vaccine that is satisfactory in its neurovirulence. However, virtually all monkeys are inapparently infected with viruses. The classic example was simian virus 40 (SV40), a polyoma virus that produces no symptoms in the live animal, and no sign of infection in the cell cultures produced from rhesus macaques that were the preferred substrate. Most polio vaccines, either live or killed, were contaminated with SV40 up until the 1960s. The precautions used to address the issue included using monkeys other than rhesus to provide production cells that do give a visible cytopathic effect when infected so that a contaminant, if present, is obvious, and testing the product on susceptible cells. Other precautions included the use of monkeys screened and found negative for antibodies to SV40, which were presumably not infected. There is evidence that the precautions were effective. Primary monkey cells can be infected by other agents, notably foamy virus and simian cytomegalovirus. The monkeys may also be infected with Herpes simiae (or B virus), which is extremely hazardous, or on occasion the filovirus Marburg virus first isolated from a polio vaccine production facility where it killed several workers.In vitro and in vivo tests have accumulated over the years to deal with these potential contaminants. For example, polio vaccines made in primary monkey cells are tested in rabbit kidney cells to detect B virus and the animals are kept in quarantine before being used to generate cell cultures to ensure that they are free of other pathological agents including filoviruses.
However, the best approach is to use established and characterized cell banks, such as the human diploid cell line MRC5, or the continuous cell line Vero. Frozen banked cells can be evaluated for contamination and suitability before use in a way that primary cells cannot, but there has been extensive consideration of the suitability of using transformed, potentially cancerous cells for the production of vaccines. The original concern has reduced over the years with increasing experience. Vaccines are in any case extensively tested for adventitious viruses and cell banks are exhaustively evaluated for their suitability.
In addition, care is taken over the sourcing of the media components, which may have a biological origin and carry viruses or prions from the donor; for example, bovine serum may be used in production and may be contaminated with bovine virus diarrhea or bovine polyoma or, in theory, the agent of bovine spongiform encephalopthy. Much concern has been expressed over possible bovine spongiform encephalopthy contamination, and many manufacturers choose to use cells adapted to serum-free growth media.
Once a preparation free of unwanted agents and of suitable attenuation has been manufactured, it will be formulated in stabilizers at the concentration required to immunize the recipient. This concentration, defined in terms of cell culture infectious dose, has been established by clinical trial and should be in excess of the amount required to produce good levels of seroconversion. Occasionally, clinical trials have resulted in an alteration of the formulation required so as to give the required seroconversion rates, but the principle, as for Smallpox, is to include enough to immunize a very high proportion of recipients. Moreover, while the vaccine includes materials intended to render it stable over the shelf life it will inevitably lose potency on storage and it must be certain that it has sufficient material at the end of its shelf life to immunize satisfactorily. In the past, the loss of titer at higher than normal storage temperatures such as 37°C was considered a surrogate of stability at the specified temperature; usually it is not accurately predictive of the titer at the end of shelf life and the thermal stability is now usually regarded by manufacturers as an additional indication of quality and consistency.
The specifications for the final product for OPV, therefore, include parameters related to the attenuation of the virus, its freedom from extraneous infectious agents, the amount of infectious material in the final container and the stability of the virus at elevated temperatures. However, these product specifications are supplemented by in-process specifications, which are crucial to the final quality; they include the passage level of seed viruses, the origin of production materials and other factors. The consequence is that OPV in use today is of high quality and predictable safety and efficacy when used in populations of recipients.