Establishment of a standard seed lot system of an Iranian Mumps virus strain; RS-12, for mass production of mumps and MMR vaccines.

BACKGROUND: At present the mumps virus strain used for production of mumps vaccine for our local use is Hoshino strain. However, according to our National public health policies, this strain should be replaced with a safer strain. Based on our previous data, the Iranian mumps strain; RS-12 has been proved to be the most suitable alternative to Hoshino strain with little or no adverse events following vaccination METHODS: The aim of the present study was to optimize propagation of RS-12 strain and prepare standard seeds for vaccine mass production. The virus was inoculated to cells using different methods and different multiplicity of infection (MOI). The viral suspensions were harvested using different methods. Quality control tests were run at different stages. RESULTS: Maximum viral yield was achieved when cell suspensions were inoculated at MOI of 1:10 and incubated at 36-37ºC for 48 hours, followed by replacement of the media and incubation at 33-34 ºC for 5-7 days. Filtration did not affect the viral titre. A standard seed lot system was successfully established and experimental batches of MMR vaccines were produced. CONCLUSION: The established seed lot system has met all requirements of WHO regulations and could be used in mass production of safe and efficacious mumps and MMR vaccines. Clinical trials are in progress for this newly produced vaccine.

Microtitration of rubella virus in monovalent vaccinal products.

BACKGROUND: Potency test for control of rubella vaccine is a significant factor to qualify production line and vaccination program. For this reason, WHO recommends to use the microtitration method by both vaccine companies and control laboratories. Then the study was done to improve this test. METHODS: Three rubella virus samples, including an in-house standard, a lot of vaccine and an in-process product, were tittered in cell culture tubes. Then micro titration steps were tested on 96-well microplate using cocultivation of standard rubella vaccine dilutions and RK-13 cell line. After 6-7 days, final reading was done and calculated the titer. Two other samples were assayed with the micromethod. RESULTS: Titer reduction less than 0.5 log was acquired for each sample during frequent tests and between two methods. CONCLUSION: The procedure was profitable and accurate for potency and identity tests of rubella virus vaccine, on the basis of WHO recommendations.

Expression of biologically active measles virus hemagglutinin glycoprotein by a recombinant baculovirus.

In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated, but it appeared to be active in hemagglutination assay.

Comparative evaluation of two combined measles-mumps-rubella vaccines based on AIK and Edmonston- Zagreb strains of measles virus.

In a previous paper, we have noticed the effectiveness of two further attenuated measles vaccines, i.e. AIK-HDC and Edmonston- Zagreb- HDC. In the present study the same strains are comparatively used for immunization of a limited number of children under 9 months of age. A seroconversion of 100% was observed. Following reimmunization, a significant increase of circulating antibodies for both strains was recorded. Two combined measles-mumps-rubella (MMR) vaccines were also produced by using the same measles strains. The seroconversion following utilisation of MMR prophylactics in susceptible children was 98.8 and 97.3 for AIK and Edmonston- Zagreb strains respectively.

Development of a new live attenuated mumps virus vaccine in human diploid cells.

A new live attenuated mumps vaccine was developed in human diploid cells. The S-12 virus was isolated from a 10-year-old girl showing typical symptoms of mumps infection, the diagnosis was confirmed by a pediatrician. The virus was isolated in green monkey kidney cells, without passage in chick embryo cavity or chick embryo fibroblasts. Attenuation of the wild virus was performed by serial passages in human diploid cells (MRC-5). The attenuated virus was characterized by identity tests, as well as by a reduction in plaque size, as marker tests. The virus was free from adventitious agents and safe for laboratory animals as well as for monkeys. The reactogenicity and immunogenicity of the S-12 virus for man was investigated by administration of a monovalent vaccine to 20 seronegative adult male volunteers and 30 children aged 1 to 5 years without history of mumps infection or vaccination. Seroconversion was obtained in 95% of the vaccinees. The new vaccine has the advantage of not requiring specific pathogen-free eggs, and being free from avian proteins and therefore can be used in sensitized patients.

Evaluation of live attenuated measles vaccines prepared in human diploid cells for reimmunization.

Two live attenuated measles vaccines developed in baby calf kidney cells, a similar vaccine produced in chick embryo chorioallantoic cells and five vaccines prepared from human diploid cells (HDC) have been studied by subcutaneous injection in groups of susceptible and immune children in three field trials. The results indicated that the vaccine developed in chick embryo cells which caused mild clinical reactions, had induced a lower seroprotection rate in susceptible children and only a low rise in hemagglutination-inhibition (HI) antibody titre in previously immunized children. The serological responses induced by vaccines developed in HDC or in calf kidney cells were satisfactory in both susceptible and immune children. The superiority of HDC grown measles vaccine for revaccination is discussed.

Isolation and characterization of a defective measles virus from brain biopsies of three patients in Iran with subacute sclerosing panencephalitis.

Three cytopathic strains of subacute sclerosing panencephalitis (SSPE) virus were isolated from brain biopsies of three patients. These strains were isolated and maintained by cocultivation of infected brain cells with fresh Vero cells. The biological characteristics of two strains were studied. It was found that these strains remain cell-associated after repeated cocultivations with Vero cells and produce plaques under fluid medium or tragacanth overlay. The correlation with measles virus was demonstrated by the plaque reduction test as well as by the immunofluorescence test. Large numbers of nucleocapsids were observed in the cytoplasm of infected cells but none in nuclei. Intracerebral inoculation of monkeys, adult guinea pigs, newborn and adult hamsters or mice was followed by acute encephalitis and death.

Age of measles immunization in tropics.

Maternal immunity to measles was studied in a group of 500 newborn children and another group of 500 children aged one to 12 months, before vaccination. The geometric mean titer of detectable hemagglutination-inhibition antibody was 16 for newborn children. This titer was absent in most children aged 3 to 5 months. Our previous studies indicate that from 1970 to 1972, children from the lower socio-economic classes aged 5 to 9 months were the main target of measles complications and deaths. Based on the present data, we suggest that children in developing countries should be vaccinated as young as 6 months and should be revaccinated 3 to 4 months later to assure full protection.

Stabilizing effect of magnesium chloride and sucrose on Sabin live polio vaccine.

A trivalent vaccine was stabilized with (a) 70% sucrose, (b) MgCl2 1M, and (c) 35% sucrose plus MgCl2 1/2M. A portion of each batch was kept at +4 degrees C and 22-25 degrees C. No change in titre for all 3 preparations was recorded after 9 weeks storage at +4 degrees C. While the potency of vaccines containing MgCl2 alone or mixed with sucrose and kept at +25-28 degrees C for 5 weeks was not altered, the vaccine containing only sucrose was less stable, and a drop of titre was noticed after 2 weeks of storage at +22-25 degrees C. Monovalent polio vaccines were also stabilized as above and kept at +4 degrees C or at -20 degrees C. It was found that regardless of the type of stabilizer used, 82 to 97 per cent of potency was retained after 9 months of storage at +4 degrees C or at -20 degrees C.

Development of a diploid cell line from fetal calf lung for virus vaccine production.

Details of isolation and replication of a fetal calf diploid cell (FCDC) is given. From the karyological point of view, the fairly large number of chromosomes existing in metaphase spreads made counting rather tedious. Lack of practical classification was another problem which made reference to individual chromosomes difficult. By increasing the population doubling of this cell, a tendency of telocentric chromosomes to undergo centric fusion was observed. Susceptibility of FCDC to different viruses is described.

Use of human diploid cell MRC-5, for production of measles and rubella virus vaccines.

A further attenuated strain of measles virus, called AIK strain, developed by Makino and his colleagues at the Kitasato Institute, Tokyo, was adapted to human diploid cells (HDC), MRC-5. In a field trial it was found that this strain initiates a low rate of clinical reactions and 100% seroconversion in vaccines. An attenuated strain of rubella virus, called Takahashi strain, isolated by Shishido at the National Institute of Health, Japan, was also adapted to HDC, MRC-5. Brief details of production of an experimental rubella vaccine are given. Up to 20 harvests of virus were made from each culture without any apparent alteration of diploid cells.

Experimental study of a further attenuated live measles vaccine of the Sugiyama strain in Iran.

After encouraging results of the mass vaccination programme in Iran, in which 5 million children in rural areas were vaccinated with the Japanese Sugiyama strain at its 82nd passage in baby calf kidney, and a progressive decrease in the incidence of measles as well as a reduction of excessive infant mortality, a further attenuated vaccine, produced with the same strain, cloned in Japan, was compared in a field trial with the parent vaccine. The new strain caused fewer reactions than the original strain. Seroconversion with a geometric mean antibody titre of 6.1 was observed in 95% of susceptible children.

Development of a camel kidney cell strain and its use in virology.

A strain of camel kidney cells was developed and carried in serial passages. The subcultures were slow-growing in the early passages and were composed of heterogeneous cell population. By the 35th passage, the growth rate increased, and more homogeneous cells, mostly of the epithelioid type, were seen. The cell strain was highly susceptible to West Nile, Sindbis, vesicular stomatitis, adeno, and vaccinia viruses, and also was susceptible to herpes simplex, rinderpest, measles, and canine distemper viruses.

Use of Vero cells for titration of rinderpest virus and its neutralizing antibody.

Primary bovine embryonic kidney cell was successfully replaced by the green monkey cell line (Vero) for the isolation and seroneutralization of rinderpest virus.