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.

Qualification of working cell banks for the Vero cell line to produce licensed human vaccines.

Since 1980, Merieux Institute has prepared on microcarriers four working cell banks from Vero Cells (137th p.) received from the ATCC in May 1979 (at 124th p.). The lots have been or are used for the production of rabies and inactivated poliomyelitis vaccines. Three lots were controlled according to WHO requirements described in the technical report 673, 1982. For the fourth lot, we have followed the WHO requirements corresponding to the technical report 745, 1987. All the tests required us to demonstrate: i) Safety and purity (tests in animals and eggs, sterility tests, cocultures with human cells and other electron microscopic observations). ii) The absence of tumorigenicity (tests in newborn rats treated with antihymocyte serum at the WBC level and on the cells propagated to at least 10 population doublings beyond the maximum passage level used for production. Assays of cell transformation with DNA from the Vero line in the standard 3T3 assay system). iii) Identity (isoenzyme technique). All were satisfactory.

Experience with preexposure rabies vaccination.

Preexposure rabies vaccination, presently limited to high-risk target populations, is facilitated by cell culture vaccines. Officially recommended programs comprise either two doses administered on days 0 and 28 or three doses given on days 0, 7, and 21 or 28. A first booster 1 year later ensures a good duration of immunity; follow-up studies now cover 5-8 years. These results were obtained by the intramuscular and subcutaneous injection routes; intradermal programs have been explored with the aim of reducing costs. Preexposure immunization is well tolerated, despite some systemic allergic reactions. Efficacy has been observed universally, but certain factors may affect results. Several lines of evidence favor an extension of preexposure vaccination. In Scandinavian countries, the majority of vaccine doses are used preexposure, while about 40%, 15%, and 10% are so used in the United Kingdom, the United States, and France, respectively. However, preventive immunization is rare in developing countries, where the risk of infection is maximal and permanent. The more economical new vaccines, such as purified Vero rabies vaccine, permit a reevaluation of preventive vaccination. Vaccine combinations including rabies may prove economical.

Influenza vaccination in the elderly: improved antibody response with Imuthiol (Na diethyldithiocarbamate) adjuvant therapy.

To improve influenza vaccine efficacy in hospitalized elderly, we compared the evolution of antibody level after vaccination in three patient groups. A sample of apparently primo vaccinated elderly were randomized to receive either Imuthiol (Na diethyldithiocarbamate: group 1) or a placebo (group P). They were compared to patients who had been vaccinated annually for several years (group C). All patients were immunized in the same week. Antibody responses increase within 15 days to reach a plateau in group P and C, while they continue to increase in the Imuthiol treated group, reaching higher antibody levels 30 days after vaccination. This higher antibody rise in group I is essentially due to higher antibody responses in patients with initially low antibody levels and who exhibited at least a four-fold antibody rise. This effect of Imuthiol on influenza antibody responses was observed in spite of a lower nutritional status in this group, a condition that induces lower antibody responses. The higher antibody responses observed in the Imuthiol treated group allow longer protection against influenza.

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.

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.

Multi-site intradermal and multi-site subcutaneous rabies vaccination: improved economical regimens.

Neutralising antibody responses to six post-exposure regimens of human diploid cell strain rabies vaccine with or without human rabies immune globulin (HRIG) were studied in 98 patients. The total amount of vaccine used was 22-34% of that required by conventional regimens. Vaccine was given at multiple sites intradermally or subcutaneously with or without adjuvant. Antibody was detectable within 7 days of the first dose in all subjects only in the groups given 0.1 ml intradermally at 8 sites. From day 14 onwards all groups showed an excellent antibody response; there was little difference between the various regimens. Suppression of the response to 8-site intradermal vaccination by a large dose of HRIG could be prevented by giving the second dose of vaccine on day 7 rather than day 14.

A human monoclonal antibody specific for tetanus toxoid.

Successful hybridization has been obtained between PWM stimulated peripheral blood lymphocytes from tetanus toxoid immunized donors and the human B lymphoma line RH-L-4. Among the hybrids selected after cloning, the average in vitro activity against tetanus toxoid was 1-5 micrograms/10(6) cells/24 hours. The specific activity of one of them was further tested in vitro (RIA) and in vivo (mouse protection test) and compared favourably with a human polyclonal antitetanus globulin.

An economical regimen of human diploid cell strain anti-rabies vaccine for post-exposure prophylaxis.

Vaccine regimens using 0.1 ml human diploid cell strain vaccine (HDCSV) given intradermally (id) in single and multiple sites, or with aluminum hydroxide adjuvant given subcutaneously (sc), were compared with the regimens of HDCSV and Semple vaccine currently suggested by WHO. Some groups were also given human rabies-immune globulin (HRIG). Neutralising antibody titres were monitored for 3 months. Antibody was detected earliest in subjects given 0.1 ml HDCSV id at each of eight sites. The highest antibody titres from day 14 onwards were found after intramuscular (im) administration of HDCSV, but the multiple-site id regimen, which requires only one quarter of the volume of vaccine required for the im regimen, gave similar results, provided that a booster was given on day 91. This finding suggests that a treatment schedule based on this regimen would be suitable for post-exposure prophylaxis. Adjuvanted vaccine gave similar results to the same amount of antigen given id. Semple vaccine produced the lowest titres. HRIG, given at the high dose of 40 IU per kg, suppressed the antibody response to some of the regimens.

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.

[The passive hemagglutination test in the evaluation of antitetanus immunity]. / Le test d'hémagglutination passive dans l'evaluation de l'immunite antitetanique.

The conditioned, passive hemagglutination method, which is simple, quick, economical and very sensitive, is suited for large-scale studies in which it is not possible to carry out the in vivo seroneutralization. However, our experience has confirmed that it has certain drawbacks and restrictions: (1) the technique must be applied very carefully and good laboratory training is required. The results obtained by this method vary according to several factors, especially the degree to which the antigen has been purified and its blood carriers, therefore these results are not always consistent; (2) the antibodies found were not a good indication of the degree of protection except at high titers. A study of correlations between HA titers and neutralizing (mice) titers carried out on the basis of 509 double titrations has demonstrated that, whereas they are satisfactory in clearly immune subjects, they show only mediocre results in subjects who are receptive or who are displaying a primary response: a wide range of variation has been observed as well as an optimalization of HA titers, perhaps because of IgM's. Therefore, the HA method, despite its usefulness, cannot provide precise evaluations for: --the amount of protection provided by an anti-tetanus vaccine, --the proportion of protected subjects in a certain group of people, --an injured person's anti-tetanus immunity. We should work to develop in vitro tests which are both sensitive and reliable in terms of the anti-tetanus protection threshold.

Mouse tumour tests for quality control of C. parvum preparations.

Description of two mouse tumor tests selected to control the quality of C. parvum pilot productions: transplanted Ehrlich's ascitic tumor in inbred Swiss mice and transplanted YC8 ascitic tumor in isogenic Balb/c mice. The results obtained during three years of control are analyzed, particularly accuracy, long range adequacy and reproducibility of tumor challenges and C, parvum stimulations. The effects of some parameters of the tumor tests, e.g. dose-responses, treatment schedules, tumor-host relationships and standard preparations are discussed.