Experience with vero cells at Pasteur Mérieux Connaught.

The Vero cell line has been used by Pasteur-Merieux-Connaught (PMC) since 1982 with the Cell Bank's system to produce, at the 142nd passage, inactivated polio vaccine (IPV), oral polio vaccine (OPV) and rabies vaccines. The safety of the cell line has been regularly validated at the WCB level according to the WHO and European Pharmacopeia requirements for absence of bacteria, fungi, mycoplasma and viruses. Special emphasis was devoted to establishing the absence of simian viruses (SV40, SIV, Retro-D virus, simian CMV). Reverse Transcriptase (RT) activity was also negative. At low level of passage, the Vero cells are not tumorigenic. Vaccines have been prepared in low passage level Vero cells and, together with the excellent downstream purification, has resulted in excellent safety as attested by pharmacovigilance of more than 100 million doses of IPV during 12 years, more than 20 million doses of rabies vaccine during 10 years and more than one billion of OPV during six years.

Experience with the Vero cell line.

The Vero cell line has been managed with the Cell Bank system to produce at the 142nd passage IPV, OPV and rabies vaccines since 1982 by Pasteur Mérieux Serums & Vaccins (PMsv). The safety of the cell line was regularly validated at the Working Cell Bank (WCB) level according to the WHO and European Pharmacopoeia requirements for absence of bacteria, fungi, mycoplasma and viruses. A special emphasis was devoted to research on the absence of simian viruses (SV40, SIV, Retro-D virus and simian CMV). All these specific researches were negative. At a low level of passage, the Vero cells are not tumorigenic. Vaccines have been prepared in low passage level Vero cells and together with the excellent downstream purification have resulted in excellent safety as attested by pharmacovigilance of more than 100 million doses of IPV during 12 years, more than 20 million doses of rabies vaccine during 10 years and more than 1 billion of OPV during eight years.

Comparative study of poliovirus excretion after vaccination of infants with two oral polio vaccines prepared on Vero cells or on primary monkey kidney cells.

A comparative study was designed to assess the bioequivalence of 2 oral poliovaccines (OPV) produced on 2 different cell systems: primary monkey kidney (PMK) cells and the Vero cell line. The Vero cell line has been used to overcome the problem of obtaining a regular supply of high quality monkeys that are devoid of latent viruses. For this study, 9 children were vaccinated with PMK-OPV and 12 children with Vero-OPV. The comparison covered poliovirus excretion, reversion of polioviruses in the 5'-noncoding region, and immunogenicity. Major molecular markers in the 5'-noncoding region related to neurovirulence already had been identified at position 480 for type 1, position 481 for type 2, and position 472 for type 3 poliovirus. Two nucleic-acid based methods were designed for studying these positions: a RT-PCR followed by sequencing, which required preliminary culture and cloning; and a type-specific nested PCR followed by sequencing, which enabled direct detection and genotyping of polioviruses. Twenty-eight stool specimens were analyzed by this second method with no PCR inhibition problem. The use of Vero cell line did not modify the global pattern of poliovirus excretion, reversion frequency, or seroconversion. These results provide additional support for the use of the well-characterized Vero cell line in OPV manufacturing.

Computerized analysis of food records: role of coding and food composition database.

Reported dietary intake records of 30 subjects (26 men and 4 women) were analysed by three different centres using their own computerized nutrient database systems. The agreement between systems was evaluated by different statistical criteria (the correlation coefficient, the mean difference and the proportion of individuals placed in the same thirds of distribution). Significant differences between the three systems were found in the calculation of alcohol, polyunsaturated fatty acids, saturated fatty acids, linoleic acid, linolenic acid, cholesterol, magnesium, sodium and water. To ascertain the extent of mean differences that could be attributed to the coding process or to the database used, coding forms of each centre were forwarded to the other two centres. Analysis of variance showed that differences in the data obtained by the three systems were mainly due to the food composition database used.

Laser immunonephelometry reference intervals for eight serum proteins in healthy children.

Eight serum proteins were analyzed with the Behring nephelometer in samples from 479 healthy French children, ages three to 16 years. Girls had higher concentrations of IgM and albumin than boys had. Age appeared to be a main factor of variation for the proteins tested. Reference intervals are presented for IgG, IgA, IgM, albumin, transthyretin (prealbumin), retinol-binding protein, alpha 1-acid glycoprotein, and C-reactive protein. The significance of increased concentrations of C-reactive protein within a community is discussed.

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.

Retinol, beta-carotene and alpha-tocopherol status in a French population of healthy children.

The serum of 392 French healthy children aged 1-16 years was analysed for vitamin A, vitamin E, beta-carotene and cholesterol contents. The study group consisted in 185 females and 207 males, living in the area of Tours, France. The mean serum values are 42.05 +/- 12.0 micrograms/dl for vitamin A, 572 +/- 381 micrograms/l for beta-carotene and 9.5 +/- 2.5 mg/l for vitamin E. According to sex distribution, mean values of studied micronutrients levels are higher in boys than in girls, but not significantly. Vitamin A and vitamin E serum levels increased with age. Vitamin E peripheral level is strongly correlated with total cholesterol serum value. The results are compared to those of underdeveloped countries children groups studies. At least, decreased vitamins levels are observed among five per cent of children and are examined as being at risk to develop a deficiency.

Polio and rabies vaccines produced in continuous cell lines: a reality for Vero cell line.

The Vero cell line was applied to the production of Enhanced-Inactivated Polio Vaccine (E-IPV), Oral Polio Vaccine (OPV) and Rabies Vaccine, also called Purified Vero Rabies Vaccine (PVRV) for humans. The cell line was expanded through the cell bank systems. Using a microcarrier culture technique, a large-scale production combined with an efficient purification process was developed. An extensive study of purity and biological safety was applied to the cell line to validate several Manufacturer's Working Cell Banks. Special emphasis was laid on the absence of any virus in the cells or any trace of viral sequence included in the Vero cell genome. Sensitive tests were applied to check that residual cellular DNA was as low as possible per dose of vaccine. E-IPV was licensed in July 1982 and since 1983 more than 20 million doses have been inoculated into children, without any side-effects. The production capacity of this E-IPV is presently more than 60 million doses per year. For PVRV, the license was delivered in June 1985 and over 350,000 doses have been injected without any problem. For the OPV, 27 monovalent lots were produced, 9 lots for each serotype. The studies of RNA sequences in variable regions and viral proteins gave results similar to the OPV produced from primary monkey kidney cells. All the other tests, and especially the neurovirulence tests in live monkeys showed that the OPV produced in Vero cell lines was acceptable. Since the completion of clinical studies, the license has been pending. The quality of the Vero cell line, as produced and tested by the "Institut Mérieux", combined with the safety afforded by the virus purification, has helped to prevent potential problems.

Lack of adjuvant effect of the pertussis component on IPV DTP-polio vaccine in children.

On day 0, four groups of children (3 to 6 months old) randomly received IPV alone or IPV + pertussis, or IPV + DP, or IPV + DTP. At days 28 and 56, all the children received the same IPV + DTP vaccine. Polio neutralizing and diphtheria antibodies were determined at days 0, 28 and 56. No adjuvant and even some inhibitory effect of pertussis was observed at days 28 and 56 on mean polio antibody titers. These results are compared to those observed with diphtheria.

Inactivated polio vaccine: industrial production from micro-carrier Vero cells culture.

In 1980, we presented our preliminary results of large-scale production of Inactivated Polio-Vaccine (IPV). The virus was produced by Vero cells culture grown on micro-carrier in 150 litre tanks; now we produce the poliovirus in 1000 litre tanks. All the tests certifying the 'Cell Bank' have been passed; the quality of purification procedure was demonstrated; additional clinical studies were done; this new reassessed IPV was licensed in France in July 1982.

Industrial-scale production of inactivated poliovirus vaccine prepared by culture of Vero cells on microcarrier.

In 1980, the authors reported preliminary results of large-scale production of inactivated poliovirus vaccine in which virus was produced in Vero cell culture on a microcarrier. For this first stage of development, 150-liter tanks were used. The virus is now produced in 1,000-liter tanks. The main point concerning the quality of Vero cells, namely the absence of tumorigenicity, has been demonstrated, qualifying them for use in the Institut M erieux cell bank. The purity of the cell line has also been determined by checking for the absence of bacteria, fungi, mycoplasmas, and viruses. The search for oncornavirus and for reverse transcriptase activity was carried out, and the results were negative but are not described in this paper. The quality of the purification process was checked by a search for residual cellular DNA in concentrated, purified, and inactivated vaccine. With use of a molecular hybridization procedure, a specific probe was prepared to detect approximately 50 pg of DNA per filter. The preliminary results show that the purification procedure fulfills the World Health Organization's requirements. T1 oligonucleotide mapping has also shown the identity of poliovirus RNA extracted from virus grown on Vero cells and that from primary monkey kidney cells. These data have led to the awarding of a license by the French government to the Institut M erieux for production of this new, reassessed, inactivated poliovirus vaccine.

Thousand litre scale microcarrier culture of Vero cells for killed polio virus vaccine. Promising results.

Through the progress of scientific knowledge the Vero cell line was considered to be a suitable alternative cell substrate for the industrial production of Polio Virus. Using microcarrier culture, more than 10(12) cells could be obtained weekly for virus inoculation. The virus yield is around 60 D units/ml for type I; 20 D units/ml for type II, and 50 D units/ml for type III.

Angiotensin converting enzyme in bronchoalveolar lavage fluid in pulmonary sarcoidosis.

Alveolar angiotensin converting enzyme (ACE) and serum ACE were measured simultaneously in 16 patients with histologically confirmed sarcoidosis and in 16 control subjects. Although alveolar ACE was abnormally elevated in all 15 cases of active sarcoidosis, serum ACE was not. No correlations were found between radiographic staging of pulmonary sarcoidosis and the levels of these enzymes. There was a clear correlation, however, between the levels of alveolar ACE and counts made on bronchoalveolar lavage fluid. This correlation was closer than that existing between serum ACE and bronchoalveolar lavaged lymphocytes. It is suggested that alveolar ACE is an additional biological marker of pulmonary sarcoidosis which is possibly more sensitive than serum ACE.

The large-scale cultivation of VERO cells in micro-carrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine.

As the increasing shortage of monkeys is a reality, the application of an alternative cell substrate for large-scale production of Killed Poliomyelitis Vaccine (KPV) was studied. Through progress of scientific knowledge the non-tumorigenic VERO cell line was considered to be a suitable alternative cell substrate for this purpose. The Master-Cell-Bank and Working-Cell-Banks prepared by us are giving a practically inexhaustible cell source. Using micro-carrier culture, weekly more than 400 billions of cells at a concentration of 10(6) cells per ml could be obtained for virus inoculation. The virus yield per cell was at least as high as for primary monkey kidney cells. Processing of virus harvests could be performed according to the methods used at the production on primary monkey kidney cells. From a technological view-point large-scale production of KPV on VERO cells appears to be possible economically. More research on the safety control might be necessary.

Comparison of sensitivity of VERO cell line versus primary monkey kidney cells in the detection of residual live polio virus during and after inactivation.

Substitution of Primary Monkey Kidney (PMK) Cells by Monkey Cell Lines (MCL) for detection of residual live virus in poliovaccine control has been considered for various reasons. The sensitivity of VERO Cells versus PMK cells has been investigated. This study was done with poliovirus during and just after formalin inactivation. The amount of residual live virus was tested by TCID50 end-point titrations in roller-tubes and small bottles at different stages in the inactivation process. Just before or at the end of inactivation, a 1 000 doses vaccine equivalent volume was tested on Roux bottles. Thus far the results show that VERO cells seem to be less sensitive than PMK cells, especially for type 3 virus. Additional studies will be needed to investigate whether the sensitivity of the VERO cells can be improved to detect residual live poliovirus during and after inactivation with the same sensitivity as PMK cells.

[Acute myeloblastic leukaemia in the course of non-Hodgkin's lymphoma (author's transl)]. / Leucémies aiguës myéloblastiques survenant au cours de l'évolution de lymphomes malins non hodgkiniens.

Five cases of acute myeloblastic leukaemia (AML) arising in the course of non-Hodgkin's lymphoma (NHL) are described. The initial lymphoma was nodular lymphoid poorly differentiated in 4 cases. All patients were under chemotherapy for at least 28 months, and 3 of them were irradiated. AML was diagnosed 44 to 78 months after NHL and was heralded in 3 cases by a preleukaemia phase of pancytopenia. The leukaemia was typically myeloblastic in 2 cases and difficult to classify in 3 cases. Death occurred rapidly in 4 patients. The actuarial risk was 4.5% at 5 years. Since AML's occur mainly in NHL's, the management of this type of lymphoma with favourable prognosis ought to be reevaluated.

Chemotherapy--radiotherapy association in Hodgkin's disease, clinical stages IA, II2A: results of a prospective clinical trial with 166 patients.

One hundred sixty-six patients with clinical stages IA, II2A Hodgkin's disease were treated between April 1972 and December 1976 with three courses of multiagent chemotherapy (methylchlorethamine, vincristine, procarbazine, prednisone) followed by mantle irradiation--excluding mediastinum for those with initial upper cervical presentation and absence of mediastinal involvement--or inverted Y radiotherapy. None had staging laparotomy. With a follow-up of 12--84 months, median 40 months, the overall survival is 93.5% and the overall relapse-free survival 89.9%. Eight patients died, three of them with evident disease. Ten patients relapsed; four are now free of disease after retreatment. With chemotherapy-radiotherapy sequence, staging laparotomy is not indicated. Results and side effects of this treatment strategy are compared with those of other treatment policies.