Adjuvanticity of pGPL-Mc and LRS in the immune responses of monkeys to oral immunization with diphtheria and tetanus toxoids.

Experiments were carried out to examine the adjuvanticity of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) or the London rocket seed (LRS) when combined with diphtheria and tetanus toxoids in an oral immunization of the African green monkey. The results showed that none of the monkeys receiving diphtheria and tetanus toxoids combined with 25 mg/kg of pGPL-Mc showed an increase in the the level of diphtheria antitoxin (DA) on the third and sixth weeks following the first and the second immunizations. One monkey from this group responded with increased seroneutralizing antibodies 3 weeks after the third feeding. On the other hand, one monkey, 3 weeks after the first immunization, and three monkeys, 3 weeks after the second and third oral vaccinations, showed an increase in specific anti-diphtheria antibody responses when the toxoids were combined with 25 mg/kg of LRS. The anti-diphtheria antitoxin responses of monkeys receiving diphtheria and tetanus toxoids combined with 50 mg/kg of pGPL-Mc or 50 mg/kg of LRS were significantly enhanced compared to the groups administered 25 mg/kg of the two adjuvants. The increase was observed in four out of five pGPL-Mc administered and in three out of five LRS-receiving monkeys. The results show that pGPL-Mc induced the highest titres of anti-diphtheria antitoxin compared to LRS, whereas the level of anti-diphtheria antitoxin titre of the two monkeys receiving the toxoids alone was less than 0.1 i.u./ml of serum throughout the experiment. According to the statistical analyses, no significant differences were recorded between the diphtheria antitoxin responses of monkeys following the first, second or third administration of LRS-adjuvated diphtheria and tetanus toxoids. However, a significant difference (P < or = 0.05) was observed in the diphtheria antitoxin response between the first and the second immunization of monkeys administered with toxoids adjuvated with 50 mg/kg of pGPL-Mc. The tetanus antitoxin responses of all monkeys were less than 0.1 i.u. of antitoxin per millilitre of serum throughout the study, which is considered not to be protective. However, we have recorded an anti-tetanus antitoxin titre of more than 0.2 i.u./ml of serum in one monkey that received diphtheria and tetanus toxoids combined with 50 mg/kg of pGPL-Mc.

Stimulating role of toxoids-laden liposomes in oral immunization against diphtheria and tetanus infections.

Liposomes have been produced by injecting an ether solution of a mixture of lecithin and cholesterol into a diluted solution of prewarmed diphtheria and tetanus toxoids followed by elimination of the stream of ether vapour by vacuum. In a preliminary study, adjuvant effects of liposomes on the systemic and mucosal immune response have been studied. When a mixture of diphtheria toxoid (DT) and tetanus toxoid (TT) entrapped in liposomes were administered parenterally or orally in rabbit, a significant rise of specific antibodies against both toxoids was noticed. In monkeys receiving a mixture of DT and TT entrapped in liposomes orally, the antibody response after two and three ingestions of this product was mild but when liposomes containing toxoids were adsorbed with aluminium hydroxide in a similar experiment, a significant rise in the specific antibody response in monkey against both toxoids was recorded. Adult volunteers, similarly receiving a mixture of DT and TT, entrapped in liposomes and adsorbed with aluminium hydroxide have shown a significant rise in specific circulating antitoxins. In order to compare the efficacy of this technique of human oral immunization with the previous method, whereby a plant medicinal seed (LRS) was used as adjuvant in oral immunization of man, a second group of volunteers were simultaneously and similarly treated as suggested previously. The comparative results are discussed in the present report.

Oral immunization against diphtheria and tetanus infections by fluid diphtheria and tetanus toxoids.

Purified diphtheria toxoid incorporated in egg yolk and mixed with a medicinal plant seed was used to orally immunize rabbits against diphtheria infection. Animals were partially immunized against a lethal diphtheria toxin challenge. The immunity was complete when gastric enzyme juices were inhibited before oral vaccination by aprotinin, a natural protease inhibitor. Rabbits and monkeys were orally immunized against both diphtheria and tetanus in the same way by pre-treatment with aprotinin. Adult volunteers receiving protease inhibitor before administration of oral toxoids have shown a significant rise in specific circulating antitoxins.

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.

Measles immunization in Iran.

Mass immunization with live measles vaccine has been carried out in rural Iran since 1968. Two strains have been used: primary baby calf kidney cell-adapted Sugiyama strain and human diploid cell-adapted AIK strain. More than 94% of the susceptible children experimentally vaccinated with either of the two vaccines have shown seroconversion. Mass immunization in rural regions has covered about 80% of susceptible children. It is now recommended that the live vaccine be administered twice: the first dose at six to nine months and the second at 12-15 months of age. In all of 100 cases of subacute sclerosing panencephalitis (SSPE) observed in the Tehran region between 1977 and 1982, the patient had a history of measles infection in childhood; there was no indication that SSPE developed after vaccination. Most of the patients with SSPE have had a high titer of antibody to measles virus in serum and cerebrospinal fluid, and antibody has commonly been demonstrated in saliva as well.

Subacute sclerosing panencephalitis. Immunological findings in saliva and salivary glands.

Antimeasles antibody was detected in the saliva of 14 patients with subacute sclerosing panencephalitis. Immunofluorescent staining showed antibody in salvary gland biopsy of 7 patients.

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.

Improvement in the yield of oral poliovirus vaccine (Sabin strains) produced in human diploid cells.

When stationary cultures of human diploid cells in conventional Roux bottles were replaced by the development of cells in roller bottles, the yield of poliovirus, Sabin strains type 1 and 2, showed an increase of 0.5 to 1 log10. No significant difference was noticed in the yield of these viruses cultured in WI-38 and MRC-5 human diploid cells.

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.

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.

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.

A model for developing countries of mass serological survey of children vaccinated against diphtheria and tetanus.

PIP: To assess the efficacy of Iran's mass immunization program, 60,000 blood samples were collected from all susceptible age groups and regions of the country. Diphtheria and tetanus antitoxin levels were determined by hemagglutination test. Official reports claim that over 70% of children in Iran receive vaccine during annual mass campaigns. At least 74% of children 3 months-7 years had diphtheria antitoxin titers sufficiently high to prevent infection, while 81-91% of those 7-13 years old were protected. Similarly, 74-82% of children 3 months-7 years had tetanus antitoxin levels high enough to avoid infection, as did 80-90% of older children. Following mass vaccination in Iran, diphtheria morbidity decreased among children but increased among adults. The incidence of tetanus has been significantly decreased in recent years due to an emphasis on immunizing girls and young mothers with adsorbed tetanus toxoid. These findings confirm both the effectiveness of Iran's vaccination campaign and the boosting effect of vaccine in older children. The hemagglutination test can be performed easily in developing countries where laboratory facilities are limited and is recommended for mass serological surveys.^ieng