An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose–response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.     

An international collaborative study on the First International Standard of bovine luteinizing hormone for immunoassay

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.     

Report of a collaborative study for assessing the potency of hepatitis B vaccines

A collaborative study was carried out to examine the suitability of a hepatitis B vaccine derived from plasma as an immunogenicity reference for vaccines produced by recombinant DNA technology in yeast. The use of a plasma derived vaccine as reference appeared satisfactory, although the use of homologous reference improved agreement in potency estimates. The use of a recombinant standard did not however improve agreement for a recombinant vaccine produced by a different manufacturer. The variation in the dilution of vaccine required to induce antibodies in 50% of test animals and in potency estimates varied widely between laboratories (25-fold and 10-fold respectively). However this was similar to the variation found in a previous collaborative study.     

An international collaborative study of an anti-infectious bursal disease virus reference serum

Fourteen laboratories participated in a collaborative study of a freeze-dried preparation of anti-infectious bursal disease virus serum to assess the suitability of the serum as a standard for use in the infectious bursal disease virus neutralization test. Ten laboratories carried out micro-virus neutralization tests and six carried out plaque reduction tests, two laboratories carrying out both tests. When titres were expressed as a proportion of that obtained for a reference preparation there was a marked reduction in variation between results from different laboratories. The use of a reference preparation was therefore of value when comparing results from different laboratories. It is proposed that the reference preparation used in this study be used as a standard to facilitate the comparison of results from different laboratories. The proposed standard contains by definition 10,000 UK units.     

Biomarkers in veterinary medicine: establishing a new international forum for veterinary biomarker research

Background: Mid-regional pro-atrial natriuretic peptide (MR-proANP) increases with severity in community-acquired pneumonia (CAP). We investigated whether changes of MR-proANP correlated to bacteremia.

Methods: 392 adult patients with CAP visiting emergency department from a prospective observational multicenter study.

Results: MR-proANP levels increased in patients with positive bacteremia (92.8 pmol/L vs. 84.3 pmol/L, p?=?0.04). Performance of MR-proANP to detect bacteremia (0.60) was equivalent to CRP (0.59) but less accurate than PCT (0.69).

Conclusion: MR-ANP poorly predicts bacteremia in CAP patients.     

An international collaborative study on foot and mouth disease virus assay methods. 2. Quantification of 146S particles

Workers in 11 laboratories in Europe and one in North America participated in a collaborative study to assess the variability of a sucrose gradient procedure used for the quantification of foot and mouth disease virus (FMDV). To this end, a range of standards was distributed from one of the participating laboratories. A series of adenine preparations were used to assess the various spectrophotometers/UV monitors and it showed most to be accurate and linear in their responses. The FMDV and MS2 ribophage standards were used to assess the sucrose gradient procedure and indicated low levels of within laboratory variation whereas between laboratory variation was greater. Recalculation of results from the unprocessed data in the coordinating laboratory permitted the identification and reduction of one source of between laboratory variation. Despite the observed variations, the results indicated that meaningful comparisons could be made between the data of different laboratories provided that a procedure similar to the one described in this report is used.     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.     

SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes

This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes.     

SFTG international collaborative study on in vitro micronucleus test IV. Using CHL cells

In this report, are presented the results of an international collaborative study on the in vitro micronucleus assay, using CHL cells. Fourteen laboratories participated in this study which was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances, having different modes of action and at different levels were assessed in the in vitro micronucleus test, using a common protocol. Mitomycin C was used as a positive control. In order to help to define a standard protocol on CHL cells, short and long treatment periods followed by various recovery times, with or without cytochalasin B, were compared. After an evaluation of the acceptability of the assays, the tested chemicals were classified as negative, positive or equivocal. Mannitol and clofibrate were judged as negative in all treatment schedules. Bleomycin was positive in all the treatment schedules, with an increase in the number of micronucleated cells in both mononucleate and binucleate cells when using cytochalasin B. This was also shown for the aneugens colchicine, diethylstilboestrol and griseofulvin, as expected. Urethane was judged as equivocal only after long treatment with cytochalasin B, and negative in all other treatment schedules. In any case, no genotoxic compound would have been missed with schedules including a short and a long treatment time, whether the treatment was followed by a recovery period or not and whether cytochalasin B was used or not. Thus, these results show that CHL cells were suitable for accurately detecting clastogenic and aneugenic compounds of various types in the in vitro micronucleus test.     

Long survival in acute myelogenous leukaemia: an international collaborative study.

A group of 82 adult patients with acute myelogenous leukaemia had survived in continuous first remission for more than three years was studied. These long-surviving patients were being treated at 12 referral centres in Europe and the USA, and they were compared with other patients with acute myelogenous leukaemia from 10 of these centres. There was no clear difference in the amount of induction chemotherapy or the time taken to achieve remission. Immunotherapy was not found to improve chances of long-term survival. The 82 patients were also compared with a group of 115 patients who had no appreciable difference in the number of blood or marrow myeloblasts between these two groups at presentation, but the long survivors had significantly higher initial platelet counts and were slightly younger. The long survivors also tended to have a lower total white cell count at presentation and lower granulocyte counts; there was no obvious explanation for these differences. Eight of the 82 patients relapsed from three to four years after remission and two (of 69 patients) after four to five year. Thereafter relapse was rare, and it seems likely that some of the 40 patients who have survived for five years or more are cured.     

Testing for viral contaminants of veterinary vaccines in Hungary

The safety of veterinary vaccines is of paramount importance and it is significantly jeopardised by extraneous agents such as bacteria, mycoplasma, Chlamydia and viruses. Several critical steps of vaccine manufacture involve a potential risk of viral contamination. Viruses, as extraneous, agents can be divided into two main groups. Group 1 agents, such as Pestivirus, chicken anaemia virus (CAV), and egg drop syndrome virus (EDSV) are well-known to manufacturers and authorities. Compendial detection methods, clear guidelines and legislation have been established to minimise the risk of contamination with these agents. Contrary to group 1, group 2 agents like Torque Teno virus (TTV) or RD114, a replication-competent feline γ-retrovirus, have only recently been recognised and their role as contaminants needs further investigation.Randomly selected veterinary vaccines used between 1992 and 2009 were tested by nucleic acid amplification for CAV, EDSV, and TTV. Pestivirus contamination was examined in 33 vaccines used between 1996 and 2006 and a further 27 vaccines used between 2007 and 2009 based on random selection of these vaccines. In addition to random tests done on vaccines used from 2007 on, 12 batches of live Aujeszky's disease vaccines submitted to our laboratory for Official Control Authority Batch Release (OCABR) were also tested for Pestivirus.     

International collaborative study by in vitro bioassays of the first international standard for porcine inhibin.

A lyophilized preparation of inhibin from porcine ovarian follicular fluid, ampoule code 86/690, was made internationally available as a research standard for in vitro bioassays in 1987. A study involving ten participants in eight countries assessed the stability and suitability of this research standard to serve as an international standard. Each of the participants used in vitro assays, the majority of which depended upon the inhibition of release of follicle-stimulating hormone from dispersed rat anterior pituitary cells. The research standard 86/690 was compared with coded ampoules of 86/690 stored under conditions of accelerated thermal degradation and with inhibins from different species. Intra- and interlaboratory variation for estimates of potency of a coded duplicate ampoule of the research standard provided the basis for comparisons of non-identical inhibins, but the fourfold variability of potency estimates for identical ampoules was such that no conclusions about the differences seen for non-identical inhibins could be made. Predictions of stability from consensus estimates of potency of ampoules that have undergone accelerated thermal degradation indicated that the research standard had satisfactory stability. On the basis of this study, the research standard 86/690 was deemed sufficiently stable and suitable to serve as a standard for in vitro bioassays and was established by the World Health Organization Expert Committee on Biological Standardization as the First International Standard for Porcine Inhibin. The possible presence, in biological extracts (standard or sample), of other bioactive proteins, such as activin and follistatin, complicates the quantitative interpretation of bioassay data. A standard of highly purified human inhibin is now required as a standard for immunoassays used for clinical research purposes; sufficient quantities of recombinant human inhibins have recently been donated for ampouling and evaluation by bio- and immunoassay in the subsequent phase of the standardization of inhibins.     

An international collaborative study of ''genetic drift'' in Salmonella typhimurium strains used in the Ames test

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded phi X174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded phi X174 DNA than for its single-stranded counterpart. However, the efficiency of inducible recovery of the double-stranded DNA molecule decreased as its exposure to ultraviolet light increased until it became constant at a value 1.5 times less than that for single-stranded form of phi X174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of phi X174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded phi X174 DNA was also 1.5 times less than that for the single-stranded molecule. These results suggest that the Weigle reactivation of double-stranded phi X174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.     

The International Standard for Endotoxin: evaluation in an international collaborative study

An ampouled preparation of bacterial endotoxin, coded 84/650, was evaluated in 35 laboratories in 12 countries for its suitability to serve as the International Standard for Endotoxin. The ampouled preparation was calibrated in terms of the USA National Standard, EC5, in Limulus Amoebocyte Lysate gelation, turbidimetric and chromogenic tests and in rabbit pyrogen tests. On the basis of the results reported here, with the agreement of the participants in the study, and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 84/650 was established in 1986 as the International Standard for Endotoxin for Limulus Gelation Tests with an assigned unitage of 14,000 IU of endotoxin per ampoule.     

An international collaborative study on foot and mouth disease virus assay methods. 1. Virus infectivity and neutralizing antibody assays

In a collaborative study sponsored by the European Commission for the Control of Foot and Mouth Disease, workers in 19 laboratories participated in the early phases (1, 2 and 4). All three phases were devoted to assessments of virus infectivity and the neutralizing activity of sera. Virus preparations and antisera distributed from one laboratory were tested either by 'in house' or suggested methods. Analyses of the results clearly showed that whilst 'within' laboratory variation in the results of replicate tests was low, there were significant differences in the results from various laboratories. By attempting to standardize test conditions with distributed materials, e.g. media and cells, etc., the 'between' laboratory variation was reduced.     

Transgenic plants for the production of veterinary vaccines

The expression of antigens in transgenic plants has been increasingly used in the development of experimental vaccines, particularly oriented to the development of edible vaccines. Hence, this technology becomes highly suitable to express immunogenic proteins from pathogens. Foot and mouth disease virus, bovine rotavirus and bovine viral diarrhoea virus are considered to be the most important causative agents of economic loss of cattle production in Argentina, and they are thus optimal candidates for alternative means of immunization. Here, we present a review of our results corresponding to the expression of immunogenic proteins from these three viruses in alfalfa transgenic plants, and we discuss the possibility of using them for the development of plant-based vaccines.