Developments in biological standardization最新文献

Safety tests in animals are an important part of the licensing procedures for human and veterinary medicines. Pharmacopoeial and other legal regulations require immunobiologicals to be approved for safety on a batch-to-batch basis. A large number of animals is needed to perform these general and specific safety tests. The search for alternatives according to the famous 3R concept proposed by Russel & Burch [1] is also relevant to safety tests. This session of the conference will highlight recent progress in the application of this concept. However, before considering potential alternatives to a test, it is advisable to re-evaluate the necessity of the safety test in question. Many animal-based safety test procedures were introduced because the production of biologicals was difficult and methods to control this production were limited. Nowadays much progress has been achieved due to a better knowledge of the prophylaxis of infectious diseases and the standardisation of methods of production and control. Therefore, some animal tests may no longer be necessary. A good example of this is the deletion of the abnormal toxicity test from the European Pharmacopoeia. Efforts to limit the use of animals in the quality control of immunobiologicals should therefore include a reassessment of the value of the safety test. Consequently, reassessment should be the first R used to evaluate whether an animal test can be removed. The 3R concept of Russel & Burch [1] to replace, reduce and refine animal tests has to be considered if that does not prove to be possible.

In the control of biologicals, animals are used largely to measure the concentration of a specific substance, rather than as a "model" of humans, so there is considerable scope for the development of replacement alternatives. When animals continue to be be used, a critical analysis of guidelines and regulations has suggested many ways in which the use of animals could be reduced [1]. However, the widespread failure to use genetically and microbiologically defined animals is scientifically questionable and almost certainly results in the use of excessive numbers. International standardisation on a small number of genetically defined F1 hybrid mice should lead to greater precision in individual tests as well as greater comparability among different laboratories.

The immunoresponse to vaccination in the Neonatal Tetanus Program (NNT) for pregnant women was studied in Vietnam using the Toxin Binding Inhibition Test (ToBI). The vaccination schedule consisted of two primary doses of adsorbed tetanus toxoid (TT) vaccine given with a one month interval. The seroconversion rate in the women was 98%. Two and a half months after birth, 63% of the children born from these women had tetanus antibody values higher than 0.01 IU/ml. Four women who had anti-tetanus titres < 0.01 IU/ml at delivery, despite two doses of primary vaccination, received a third booster with vaccine one year after the first injection. Their antibody levels were well above 0.01 IU/ml one month after this additional booster, suggesting that (when economically feasible) a third TT injection could be considered into the NNT to confer optimal anti-tetanus antibody levels in women for subsequent pregnancies. This study confirmed the effectiveness of the TT vaccines investigated and indicates their potential to replace, in immunosurveillance studies under field conditions, the in vivo mouse neutralisation test by in vitro alternative methods such as the ToBI test.

The United States Code of Federal Regulations requires that all influenza virus vaccines produced for use in the United States adhere to specific regulatory standards including the demonstration of safety and efficacy. For vaccines produced in cell lines, rigorous characterization for manufacturing is particularly important. Influenza vaccines produced by the passage of viruses in mammalian cell lines will require careful evaluation to ensure the removal or inactivation of potential adventitious agents.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bio-assay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bio-assay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin from the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bio-assay. It is envisaged that such assays will prove realistic alternatives to animal based tests.

The testing of vaccines for use in chickens requires a large number of animal trials. Especially for poultry vaccines, quality testing of each batch consists of testing for extraneous agents in chickens for all products and potency tests for inactivated products. For the licensing of a vaccine a number of safety and efficacy tests is necessary. The safety testing covers dose and overdose studies, and the influence on reproductive performances and immunological functions. For live vaccines some additional trials concerning spread of vaccine strains, dissemination in the vaccinated animals and reversion to virulence are required. Some possibilities for combining several tests are presented. The purpose is to reduce the number of animals needed in these trials. The efficacy testing mostly requires challenge tests to define onset, level and duration of immunity. Serological test systems to replace the challenges are rarely implemented. The example of efficacy testing of infectious bursal disease vaccines demonstrates the possible replacement of a challenge by a serological test system. Parameters are morbidity, mortality, histological findings, bursa/body-ratios, and humoral antibodies detected by serum neutralization and ELISA.

The intracerebral mouse protection test (Kendrick test) for the potency assay of pertussis vaccines is a complex and time consuming in vivo test which has a significant intra- and interlaboratory variation. Thus, there is a pressing need to develop a replacement for the Kendrick test. There is now convincing evidence to suggest that Bordetella pertussis can be taken up and survive within macrophages in the lungs and that cell-mediated immunity plays a role in protection. It was hypothesised that murine macrophages could be activated by immunisation with whole cell pertussis vaccines and therefore induce NO production. An alternative in vitro assay based on the determination of reactive nitrogen intermediates produced as a result of macrophage activation has been examined as a possible replacement for the current intracerebral (i.c.) mouse protection test. NO induction was studied in the peritoneal macrophages of female NIH mice immunised with normal and denatured whole cell B. pertussis vaccines respectively. Compared with controls receiving diluent only, macrophages and spleen cells from mice immunised with whole cell pertussis vaccine responded in vitro to selected pertussis antigens by NO synthesis. The production of NO in response to in vitro culture with bacterial antigen was immunisation dose dependent and was correlated with protective immunity in vivo as determined by i.c. challenge. The results suggest that NO production may serve as a marker of macrophage activation in mice immunised with whole cell vaccine, and could form the basis of a potential replacement potency assay.

Alternative tests have a role in vaccine testing, especially to confirm production consistency. Given the characteristics of these alternative tests and of the products for which they may be used, there are several factors which will influence their use. These include a good understanding of the test and the product to be tested, strong national regulatory infrastructure, a laboratory run in accordance with the principles of laboratory quality systems, and the ability to validate the alternative method. This means that national regulatory authorities will need strong expertise in epidemiology and quality assurance to complement laboratory experience.