viernes, 17 de agosto de 2012


Animal Models in Vaccinology Coenraad F.M. Hendriksen CONTENTS Introduction ..................................................................................................................................... 1 Vaccine Development Technologies ................................................................................................ 3 First-Generation Vaccines....................................................................................................... 4 Second-Generation Vaccines .................................................................................................. 4 Third-Generation Vaccines ..................................................................................................... 4 Adjuvants ............................................................................................................................... 5 The Historical Role of Animal Models in Vaccinology.................................................................. 5 Characteristics of the Use of Laboratory Animals in Vaccinology ................................................ 7 Animal Models in Vaccinology ....................................................................................................... 8 Animal Models in Vaccine Development .............................................................................. 8 Animal Models in Vaccine Production ................................................................................ 10 Animal Models in Vaccine Quality Control ........................................................................ 11 Conclusion..................................................................................................................................... 13 References ..................................................................................................................................... 13 INTRODUCTION Vaccines belong to the category of immunobiologicals — products that are produced by or derived from a living organism. Immunobiologicals include a variety of products, such as vaccines, immunoglobulins, monoclonal antibodies, and antisera. The characteristic feature of vaccines is that these preparations are capable of inducing a specific and active immunity against an infecting agent or its toxin. 1 Vaccination is one of the most powerful and cost-effective tools in modern medicine. The worldwide immunization campaigns against a number of infectious diseases (e.g., diphtheria, tetanus, and measles) have led to substantial decreases in morbidity and mortality rates and, in the cases of smallpox and poliomyelitis, to complete and almost complete eradication, respectively. In the coming years, the importance of vaccines will continue to increase because of the emergence of antibiotic-resistant strains of bacteria such as Mycobacterium tuberculosis , the impact of new viral infections such as human immunodeficiency virus (HIV) and severe acute respiratory syndrome (SARS), increased biological warfare threats (e.g., smallpox and anthrax), 2 HANDBOOK OF LABORATORY ANIMAL SCIENCE global travel and tourism, the high incidence and economic effects of infectious diseases in large livestock industries, and various other factors. 2 Vaccination is not without controversy. Some groups oppose vaccination programs for religious reasons. Others adhere to the speculative concept that infant vaccination may stimulate allergic sensitization. It has been hypothesized that due to today’s comprehensive immunization program, children would be more prone to develop allergic diseases due to a shift of the Th1/Th2 balance in the immune system. 3,4 Although studies have been initiated to scientifically underpin this theory, no proof is yet available. The main reason that parents withhold immunizations from their children, however, is fear of vaccine-associated adverse effects. No vaccine is totally safe and totally effective, and adverse reactions have been reported with all vaccines, although products differ in the extent of their effects. 5 Because public acceptability of immunizing children is inversely related to the extent of adverse reactions, much attention has been given to improving existing vaccines. One of the best examples is the whooping cough (pertussis) whole cell vaccine. Due to campaigns in the U.K. tabloid press in the mid-1970s, which magnified the adverse reactions of whole cell pertussis immunization out of proportion, vaccine uptake dropped sharply from 80 to 30% in the U.K. and also decreased in other countries. Later studies showed that the claimed adverse reactions were highly exaggerated. 6 Table 1.1 summarizes the frequency of adverse reactions from postimmunization surveillance data as well as morbidity data from natural whooping cough infections. It can be seen from this table that whooping cough vaccination is highly cost effective. Nevertheless, public concern about adverse effects stimulated a renewed interest in basic research toward a safer product, 7 ultimately resulting in the first acellular pertussis (acP) vaccine, 8 which only includes the protective epitopes of the pertussis microorganism. The pertussis case is a good example of how public concern can affect the use of laboratory animals. Animal models played a crucial role in the development and screening of new pertussis vaccine candidates, and literally hundreds of thousands of animals were used in the development of the new product. A traditional link exists between laboratory animals and vaccines. As far back as the end of the 19th century, vaccine research provided a major impetus for the development of animal models. Some of the animal models currently used in routine vaccine quality control are in fact slight modifications of the tests developed by Emile von Behring or Paul Ehrlich in the 1890s. A close association between laboratory animals and vaccines still exists. Animals are particularly required for vaccine development and for quality control. Few animals are currently needed for vaccine production. This chapter focuses on the animal models used in vaccine research and testing. It provides information on technologies in vaccine development, on the historical context of animal models in vaccine research, and on characteristics of animal use in current vaccine development, production, and quality control. Table 1.1 Benefits of Vaccination against Natural Whooping Cough Incidence (No. per 100,000 Cases) Following Adverse Reactions Infection Vaccination Ratio of Infection/Vaccination Shock — 15 — Convulsions 4000 45 89 Encephalitis 2000 1.5 1300 Permanent brain damage 1300 0.3 4300 Death 2000 0.2 10,000 Source : Galazka, A.M., Lauer, B.A., Henderson, R.H., and Keja, J., Bull. WHO , 62, 357, 1984. ANIMAL MODELS IN VACCINOLOGY 3 VACCINE DEVELOPMENT TECHNOLOGIES The work of Edward Jenner on smallpox is generally considered to be the first scientific approach to vaccine development. After 25 years of study, Jenner published in 1798 the results of his successful experiment in which an 8-year-old boy, James Phipps, was inoculated with cowpox material and subsequently challenged with smallpox virus. Jenner’s study was entirely based on epidemiology and observation. Animal experiments did not contribute to it in any way. 10 It took almost 100 years before Louis Pasteur discovered several new vaccines, against fowl cholera (1880), anthrax (1881), and rabies (1885). All these vaccines were partly developed by trial and error without full understanding of the pathogenesis of the diseases. Nevertheless, Pasteur was the first to approach vaccine development in a systematic and coherent way. The virulent rabies virus, obtained from the saliva of infected dogs and humans, was attenuated in rabbits by multiple passages of the virus in cerebral and spinal cord tissue and finally by exposure to atmospheric oxygen. After Pasteur’s pioneering work, vaccination as a means of combating infectious diseases was taken up and extended by many researchers. In fact it was Pasteur who came up with the word “vaccination” as a tribute to Edward Jenner (the Latin word vacca means “cow”). The term “vaccinology,” introduced by Jonas Salk in 1977, can be defined as “the study and the application of the requirements for effective immunization,” meaning nothing other than the science of vaccines from A to Z, 11 thus including development, production, quality control, and research on vaccine-related issues such as adjuvants. Figure 1.1 shows the major vaccines for human use that have been developed since Jenner’s smallpox vaccine. They amount to a total of 21 products. Although the number is small, these products all have had tremendous impact on human health care. Several diseases with high morbidity and mortality rates in the 19th century, such as diphtheria and measles, are now almost unknown, * Meningococcal C * Hepatitis A * H. influenzae b * Hepatitis B * Pneumococcus * Meningococcus * Rubella * Mumps * Measles * Polio (Sabin) * Polio (Salk) * Yellow fever * Influenza * Pertussis * Cholera * Tuberculosis * Tetanus * Diphtheria * Typhoid 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 * Diphtheria and Tetanus IgG * Smallpox * Rabies 1800 1900 Figure 1.1 History of vaccine development. (Only vaccines for human use are shown.) (Adapted from van der Zeist, B.A.M. and Lenstra, J.A., LAB/ACBC , 9, 18, 1988.) 4 HANDBOOK OF LABORATORY ANIMAL SCIENCE at least in Western countries, while smallpox has been eradicated and poliomyelitis is almost eradicated. 12 Roughly, vaccine development from the early days until now can be divided in three time periods, with each era characterized by specific production technologies. First-Generation Vaccines These conventional products consist of attenuated or inactivated (whole) microorganisms. Pasteur’s technique of attenuation was subsequently used for the production of several other vaccines, such as the oral poliomyelitis vaccine developed by Sabin in 1954 and the measles vaccine in 1958. Inactivated vaccines are produced by inactivating the whole microorganism or bacterial products (toxins) using heat or chemicals such as formaldehyde. Examples of inactivated vaccines are the whole cell pertussis vaccine, tetanus toxoid, and the inactivated poliomyelitis vaccine developed by Salk in 1953. Bacterial microorganisms have been grown on culture media, and viruses have been cultured in laboratory animals (e.g., rabies virus in rabbit or suckling mouse brains), in embryonated eggs, and after the 1950s also in cell cultures. In addition, immune response to vaccine antigens is enhanced by addition of an adjuvant product, generally aluminum salts, AlPO 4 and Al(OH) 3 . Conventional vaccines are characterized by high complexity in composition and structure. As a consequence, emphasis has to be given to extensive batch-related quality control. Vaccine batches are produced in volumes of up to 1000 L. Second-Generation Vaccines A rational approach to vaccine development started in the late 1950s. The approach was based on the subunit principle — the isolation and purification of only those antigenic components (antigenic epitopes) of the microorganism that underlie the protective immune response. Examples of subunit viral vaccines are the influenza vaccines based on surface protein and a subviral hepatitis B vaccine. A breakthrough in subunit vaccine development only occurred after the introduction of hybridoma technology to produce monoclonal antibodies, 14 as these antibodies allowed selection and identification of protective epitopes. The acellular pertussis (acP) vaccine, including one or multiple antigenic components of the pertussis microorganism, 8 is an example of a bacterial subunit vaccine, now used in most Western countries. A group of products of particular interest in this context is the bacterial polysaccharide vaccines, derived from capsular polysaccharides of Grampositive or Gram-negative bacteria. Polysaccharides are poorly immunogenic — a problem that can only be overcome by chemical linkage to proteins. Examples of polysaccharide vaccines available are those against Neisseria meningitides, Haemophilus influenzae, and Streptococcus pneumoniae. Third-Generation Vaccines Advances in biotechnology and concomitant developments in the fields of molecular immunology and protein chemistry opened the door to new vaccine production strategies in the 1980s. Recombinant DNA (rDNA) technology allowed for the insertion of one or more genes from microorganisms encoding immunologically relevant proteins into host systems such as viruses (e.g., vaccinia and adenovirus), yeast, or animal cells. Thus, expression of these genes by the vectors induces immunity after administration. An example of a licensed rDNA product is the yeast- and Chinese hamster ovary (CHO)-derived recombinant hepatitis B (rHBsAg) vaccine. However, most experimentally produced rDNA (carrier) vaccines still have not passed official registration. Another approach to rDNA vaccines involves deleting those genes that encode for pathogenicity. One such product has been produced for Aujeszky disease. 15 Another example is the double mutant acP vaccine. 16 ANIMAL MODELS IN VACCINOLOGY 5 Advances in biochemistry have increased interest in polypeptide vaccines produced by chemical synthesis. The appeal of this approach is that it permits the manufacture of chemically well-defined products on an industrial scale. However, it has meanwhile become clear that, though the required antigenic peptides can be synthesized, it is extremely difficult to obtain the spatial configuration (glycosylation) of the peptide chains necessary for immunogenicity. To date, not one peptide vaccine has reached the stage of clinical trials. Finally, the use of plasmid expressing recombinant genes of pathogens encoding for immune responses has become the basis of the DNA vaccine strategy. Intramuscular injection of the plasmid results in uptake by muscle cells, expression of the recombinant gene, and the elicitation of immunity against the protein product of the pathogen. 17 DNA vaccination has been studied for a long list of pathogens such as influenza, rabies, and tuberculosis, but few products have entered clinical trials yet. 18 Adjuvants An integral part of vaccinology is research on adjuvant products. Adjuvants are chemicals or biomolecules which, when given in conjunction with a vaccine, enhance and/or modify immune responses. Currently, no adjuvant products exist that are absolutely safe. Consequently, safety and adjuvanticity must be balanced between obtaining maximum immune stimulation and minimum adverse effects. 5 Since early vaccine development, research has been performed to develop effective formulations. However, the only adjuvants approved by regulatory authorities for use in humans include aluminium phosphate and aluminum hydroxide. As side effects are somewhat less a problem in the livestock industry, the range of products allowed for use in the veterinary clinic is much larger. New vaccine production strategies have resulted in more defined but smaller antigenic structures such as subunits and peptides. These products are less immunogenic compared to whole cell vaccines. In addition, some new vaccines require modulation of immune response toward cellmediated immunity (CMI). Unfortunately, aluminum “alum” is a weak adjuvant and stimulates mostly humoral immunity. Therefore, significant efforts are now underway to develop new generations of adjuvants. Promising new products, such as muramyl-dipeptide (MDP) or immunostimulatory complexes (ISCOM), not only induce strong antibody responses but also induce more balanced Th1/Th2 responses; they therefore might be useful for vaccines that require responses of the Th1 (CMI) type. 19 Furthermore, an urgent need now exists for adjuvants that support new vaccine administration strategies, such as oral vaccines. THE HISTORICAL ROLE OF ANIMAL MODELS IN VACCINOLOGY In 1884, the German microbiologist Robert Koch published his book Die Aethologie und die Bekämpfung der Tuberkulose , which elaborated on the criteria that should be met in relating a microorganism to a given infection. These criteria, also known as Koch’s postulates, have been of historical importance regarding the role of animal models in the study of infectious diseases. Apart from the isolation of the microorganism in pure culture, the postulates stated that these pure cultures, following the introduction into a suitable animal model, should result in the typical clinical signs of the disease in the laboratory animal. Koch’s postulates gained general acceptance in microbiology and thus in vaccine development. When it was difficult to find a suitable animal model, as was the case for poliomyelitis, progress in vaccine development stagnated. By contrast, rapid results were obtained in research on diphtheria and tetanus when it was found that animal models were easily available. 20 As an example, a historical overview is given of the use of animal models in the production and quality control of diphtheria therapy and prophylaxis. In the 1800s, diphtheria was a highly contagious disease in young children with a mortality rate of up to around 40%. The 6 HANDBOOK OF LABORATORY ANIMAL SCIENCE disease was also known as the “strangling angel of children”; infected children suffocated due to a pseudomembranous inflammation of the pharynx. For a number of reasons diphtheria makes an interesting case. It nicely shows the crucial contribution animal models made to a major breakthrough in medical history (Table 1.2). Further, it also played a crucial role in the heated discussions about the moral status of animal experimentation that took place at the end of the 19 th century. Diphtheria antitoxin and vaccine completely changed the previously hopeless treatment of a child with diphtheria. Shortly after the introduction of diphtheria antiserum, morbidity and mortality rates declined significantly. The fact that the use of animal models had made the treatment of this feared disease in young children possible convinced many of the value of this type of experimentation and undermined the criticism leveled at it. As a result, the animal welfare movement lost power for several decades.20 From the end of the 19th century, the animal model played an important role in vaccinology, first in gaining insight into the etiology and course of disease, second in the development of prevention and, in the case of antisera, of treatment, and finally, in the development of quality control of these preparations. As an example, an overview is given of the animal model in the development of diphtheria antitoxin and vaccine quality control (Table 1.3). In fact, Koch’s postulates still do apply to today’s vaccine development, and the role of the animal model remains indisputable. However, new technologies have been or are about to be introduced that will modify the role of the animal model. Information will be provided in the following sections, but first statistics and some characteristics of the use of laboratory animals in vaccinology will be presented. Table 1.2 Animal Models in the Development of Diphtheria Treatment and Prevention Development Year Scientist Animal Speciesa Isolation of the causal microorganism Corynebacterium diphtheriae 1884 Loeffler Pigeon, chicken, rabbit, guinea pig Production of the exotoxin 1884 Roux and Yersin Various animal species, guinea pig Demonstration of the therapeutic value of antitoxin 1890 Behring and Kitasato Guinea pig, dog, mouse, rat, various animal species Large-scale production of antitoxin 1894 Roux and Martin Dog, sheep, goat, cow, horse Toxin-antitoxin mixtures for active immunization 1913 Behring Guinea pig Diphtheria toxoid (vaccine) 1923 Ramon Various animal species aThe animal species finally chosen is in bold type. Source: Adapted from Hendriksen, C.F.M., in Replacement, Reduction, and Refinement of Animal Experiments in the Development and Control of Biological Products, Brown, F., Cussler, K., and Hendriksen, C., Eds., Developments in Biological Standardization 86, Karger, Basel, 1996, 3. Table 1.3 Animal Models in the Quality Control of Diphtheria Antitoxin and Vaccine Development Year Scientist Animal Species Quality control of antitoxin 1892 Behring and Wernicke Guinea pig Introduction of a standard preparation in potency testing 1897 Ehrlich Guinea pig Potency test using parallel-line bioassay 1937 Prigge Guinea pig Multiple intradermal challenge test 1974 Knight Guinea pig Serological potency test 1985 Kreeftenberg Mouse Source: Adapted from Hendriksen, C.F.M., in Replacement, Reduction, and Refinement of Animal Experiments in the Development and Control of Biological Products, Brown, F., Cussler, K., and Hendriksen, C., Eds., Developments in Biological Standardization 86, Karger, Basel, 1996, 3. ANIMAL MODELS IN VACCINOLOGY 7 CHARACTERISTICS OF THE USE OF LABORATORY ANIMALS IN VACCINOLOGY Few specific data are available on the use of laboratory animals in vaccinology. In the Netherlands, yearly statistics on the use of animals in biomedical research and testing have included a specific question on biologicals. Figure 1.2, which specifies the purposes of animal use in the Netherlands in 2002, shows that the category of biologicals accounted for 22.7% of total use. Although the use of animals for biologicals has been reduced in the last 20 years, the number has increased as a relative percentage of total use for biomedical research (Figure 1.3). Figure 1.4 further specifies the category of biologicals. Since this information is not included in the official Dutch statistics, the data given are percentages and are based on personal inquiry. As can be seen, vaccine development and vaccine quality control are particularly animal demanding. Although no data are available, it is assumed that the percentage of animal use for biologicals as compared to total animal use will be about the same in other European countries as well as in the U.S. In nonindustrialized countries, the percentage might even be higher as vaccine production and quality control quite often are major areas of biomedical research in those countries. Some other characteristics of the use of animals for the category of biologicals are given in Table 1.4, which shows that many of the animals are required for regulatory purposes — the registration of a new product and routine batch release testing. Furthermore, animal use is also characterized by a high level of pain and suffering. Particular, vaccine potency testing is based on animal models of a general design dating back to Behring and Ehrlich and often includes an immunization–challenge procedure. Figure 1.2 Purposes of use of laboratory animals in the Netherlands and percentage of total use. (From Zo doende 1978–2002, Annual statistics on the use of laboratory animals in the Netherlands, Keuringsdienst van Waren (KvW), The Hague, 2002.) Figure 1.3 Use of laboratory animals in the Netherlands in the period between 1978 and 2002: total use and use for the purpose of biologicals. (From Zo doende 1978–2002, Annual statistics on the use of laboratory animals in the Netherlands, Keuringsdienst van Waren (KvW), The Hague, 2002.) Fundamental Biologicals Education Diagnostic Pharmaceuticals Toxicity testing 0 200 400 600 800 1000 1200 1400 1600 1978 1980 1984 1988 1992 1996 2000 Year No. animals × 1000 Total Biologicals 8 HANDBOOK OF LABORATORY ANIMAL SCIENCE ANIMAL MODELS IN VACCINOLOGY Laboratory animals are still essential in vaccinology for vaccine development, vaccine production, and finally for vaccine batch release. Although in vitro methods are used and their contribution to vaccinology is becoming more important, many scientific questions still require an integrated immune system and consequently, an intact animal. A broad range of animal models is used, depending on the type of vaccine, the target animal species, and whether animals are used in development activities, in production, or in quality control. A general outline of these models will be given in the following paragraphs. Animal Models in Vaccine Development The first step in vaccine development is the establishment of an infection model to study pathogenesis. Aspects that are dealt with include route of infection, target organs, incubation time, virulence, and clinical disease progress. Well-characterized and relevant infection models are decisive for the success of further studies. For veterinary vaccines, it will be clear that the target animal species is the model of choice, unless there are reasons (e.g., cost or availability) to establish a laboratory animal model. For human vaccines, there is a need for specific laboratory animal models. History shows that when it was difficult to find a suitable animal model, progress stagnated. For instance, this was the case for poliomyelitis vaccine development. Although monkeys could be infected by intracerebral inoculation, for a number of reasons this appeared not to be a particularly suitable experimental animal for this kind of research. Figure 1.4 Use of laboratory animals for the category of biologicals: specification of purposes. The 0.1% of laboratory animals used for vaccine production is too small to indicate in the diagram. Animals are no longer used for monoclonal production. Table 1.4 Characteristics of Use of Animals for the Category of Biologicals and for Total Animal Usea Characteristic Percentage for the Category of Biologicals Percentage for Total Animal Use (All Categories) Percentage of use of animals 23 100 Use of animals for regulatory purposes 69 28 Substantial pain and suffering 21 13 No pain relief 1 5 a Biologicals include antisera, immunoglobulins, hormones, vaccines, blood products, cytokines, etc. Source: Zo doende 2002. Annual statistics on the use of laboratory animals in the Netherlands. Keuringsdienst van Waren (KvW), The Hague, 2002. Vaccine dev. (10%) Vaccine q.c. (49%) Hormones (25%) Polyclonal antibodies (5%) Blood products (5%) Others (5%) ANIMAL MODELS IN VACCINOLOGY 9 Recent examples of infection models to study vaccine development are those for emerging infections such as HIV and SARS. Several papers have discussed the use of a chimpanzee model for HIV research (e.g., reference 22). However, apart from economical and ethical constraints, the model only partly reproduced clinical progress as seen in humans. Another approach focused on studying animal viruses analogous to HIV, particularly the lentiviruses that induce acquired immunodeficiency syndrome (AIDS)-like illness in animals — SIV (simian immunodeficiency virus) infections in cynomolgus monkeys23 and FIV (feline immunodeficiency virus) infection in cats.24 Thus information could be obtained about genetic diversity, infection characteristics, etc. More recent is research on SARS. To date, infection models have been described using monkeys (cynomolgus macaques),25 cats, and ferrets.26 Having an infection model also offers a way to upscale virus production or to attenuate the virus by serial passage. Vaccine development generally starts with obtaining information about immunogenicity. Depending on the type of vaccine, microorganisms will be attenuated/inactivated as is the case for conventional vaccines, or studies will start to identify and select the antigenic structures (epitopes) that are relevant for immunogenicity and could be used as (subunit) vaccine leads. Part of this work will be done in vitro, but particularly information on complex immunological processes such as type of immune response (humoral and/or cytotoxic T-cell), antibody classes produced, duration of immune response, protective activity, and antigenic stability often requires the use of laboratory animals. In the case of conjugate vaccines, the effect on immune response due to protein linkage has to be studied, and in the case of vector vaccines, the best vector has to be identified and characterized. Additionally, adjuvants have to be identified that are optimal in enhancing and/or modifying immune responses. Thus, animal studies are performed to study antigen–adjuvant interactions and to select an adjuvant that is potent and safe. Another focus of study is safety aspects. In the case of conventional vaccines, information has to be provided on loss of virulence, thermal stability, and antigenic stability of attenuated vaccines and on protocols for effective inactivation of inactivated vaccines. In the last few decades safety requirements have increased in diversity, particularly with the introduction of new production technologies. For instance, the safety requirements for registration of rDNA vaccines specify studies on local and systemic toxicity including histopathological effects, virulence of the vector, stability of the integrated sequence, transmission from vaccinated to nonvaccinated animals, reversion of virulence, hypersensitivity, and drug interactions. Apart from the development of new vaccines, continuing efforts are made to improve existing vaccines, generally to reduce adverse effects, such as is the case for the pertussis vaccine, but also to optimize vaccine efficacy, as for tuberculosis and measles, or to overcome problems of antigenic variation (influenza vaccine).19 Another area of research is aimed toward the development of combined vaccines. The number of products in pediatric immunization programs has increased significantly in the last few decades and will further increase in the near future. As the acceptance rate for immunization is inversely correlated with the number of injections, there is a need to combine vaccines and to limit the number of injections. Thus, studies focus on the interaction of the various vaccine components as well as on new administration approaches such as prefilled ampules and gene gun “injection.” Early-phase vaccine development is based on fundamental research. Study protocols, although fulfilling the conditions of Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP), are not laid down in formal procedures. However, toward the end of development, studies, in particular those on immunogenicity and safety, become part of the registration dossier for obtaining the product license. Registration dossiers for product licenses are approved in Europe by the European Agency for the Evaluation of Medicinal Products (EMEA) and in the U.S. by the Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA). Generally, registration is indicative of the animal models that will be specified for batch release testing. 10 HANDBOOK OF LABORATORY ANIMAL SCIENCE Animal Models in Vaccine Production Laboratory animal use in vaccine production is limited in numbers. Bacterial microorganisms are grown in culture media that might require blood products or beef extract. Due to the BSE (bovine spongiform encephalopathy) crisis, however, there now is a strong tendency to use synthetic media only, without any animal products. Virus production needs a cell substrate for propagation. Until the introduction of technologies that allowed for large-scale cell culture, viruses had to be grown in living animals (rodents or nonhuman primates) or in embryonated eggs. For example, the rabies vaccine was produced in brains of suckling mice until cell culture–produced vaccines became available in the seventies.27 Now, rabies vaccines are generally produced in cell culture, but in vivo production is still used, particularly in developing countries, because these vaccines are cheap and easy to produce, despite the safety disadvantages of animal-produced vaccines. Only a few viral vaccines are still based on ex vivo production. One of these is the polio vaccine. Some manufacturers still rely on virus propagation in primary or subcultured monkey kidney cells, although the virus can also be produced in continuous cell lines (CCLs) such as Vero cells or human diploid cells. Table 1.6 presents an overview of cell culture and non–cell culture virus vaccines. Apart from the greater safety of vaccines produced in CCLs, the use of CCL in the manufacture of viral vaccines has other advantages: 1. The cell culture substrate is more consistent and “clean” than primary culture. 2. The use of large-scale tissue culture procedures allows more efficient and more reproducible cell growth. 3. It provides higher yields of virus. 4. It could reduce or even preclude the use of animals.29 Despite these advantages, the acceptability of CCL as a substrate in the production of viral vaccines has been controversial due to the suspected oncogenic potential of CCLs — that is, the potential to induce cancer in humans after vaccine administration. However, it has now been well established that DNA obtained from these cells has no detectable activity in vivo.30 The use of primary cell cultures has continued since then because vaccine safety is not only a scientific issue but a political issue as well. However, there is reason to believe that virus propagation in CCLs will gradually but completely replace the use of animals. Table 1.6 Cell Culture and Non–Cell Culture Virus Vaccines Cell Culture Virus Vaccines Non–Cell Culture Virus Vaccines Polio Smallpox (vaccinia): bovine, lymph, skin Measles Rabies: various brain or spinal cords (rabbit, sheep, goat, mouse), duck embryo Mumps Japanese encephalitis: mouse brain Rubella Yellow fever: mouse brain, hen’s egg Adenovirus Influenza: chick embryo allantoic fluid Rabies Varicella Hepatitis A Rotavirus Cytomegalovirus Source: Katz, S.L., Wilfert, C.M., and Robbins, F.C., in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds, Elsevier, Paris, 1986, 213. ANIMAL MODELS IN VACCINOLOGY 11 Animal Models in Vaccine Quality Control Central to the vaccine batch release process is quality control. In particular, conventionally produced vaccines have a tight testing program because these products are complex mixtures that might differ from batch to batch. New-generation vaccines can be produced more consistently, and consequently quality control, and thus the use of laboratory animals, is less extensive. Quality control takes place within a regulatory framework, and guidelines for quality control tests are laid down by (inter)national regulatory bodies such as FDA and USDA for the U.S. and the European Pharmacopoeia for the Member States of the Council of Europe. Broadly, controls are directed almost entirely towards the safety or the lack of toxicity and the efficacy or potency of the vaccine. Control on safety ensures that the vaccine does not contain ingredients that are harmful to humans or animals after administration. The harm can be traceable to the agent (the bacterial or viral strain or toxin), to chemicals added (intentionally or unintentionally), or to the substrate used (culture media, culture cells, or embryonated hen’s eggs). Table 1.7 specifies the tests that, depending on the type of vaccine, can be part of the safety-testing scheme. Next to safety testing, by far the greatest effort in quality control goes into potency testing to demonstrate that the vaccine induces protective immunity after its administration. In the case of live vaccines (e.g., mumps, oral polio, bacille Calmette-Guérin [BCG]), the efficacy of each vaccine batch is related to the number of live particles, determined either by counting or by titration, that is, entirely in vitro. Potency testing of inactivated vaccines, however, generally requires the use of experimental animals. The tests often rely on a limited number of basic principles, quite often relating to procedures already established in the early days of vaccinology. One of the approaches (the “parallel-line” potency test) is to immunize groups of animals with serial dilutions (three or four) of the vaccine under study and a reference preparation with known potency, respectively. Generally, these ranges of dilutions include groups of animals receiving a low, an intermediate, and a high vaccine dose. After a number of weeks, the animals are challenged with the virulent microorganism or toxin, and specific clinical signs or death is recorded for the observation period given. Based on the information obtained from the various groups, a dose–response curve can be plotted, both for the vaccine under study and for the reference preparation, and the dose that protects Table 1.7 Safety Tests in Quality Control of Vaccines and Animal Models Used Safety Aspect Specific Safety Test Animal Model Used The agent Specific toxicity test (bacterial vaccines)a Mice, guinea pigs Identity test (all) (Neuro)virulence test (live vaccines)a Monkeys (intracerebral and intraspinal) Test for residual live virusa (live vaccines) Various animal species Test for endotoxina Rabbits Vaccine additives Abnormal toxicity (= innocuity) testa (all) Mice and guinea pigs Target animal safety test (veterinary vaccines)a Target animals Thimersal content Sterility test (all) Test for endotoxin levelsa Rabbits Chemical tests to characterize components pH (all) Substrate used Tests for extraneous microorganisms* (live) Various animal species, e.g., suckling mice, chickens Test for tumorigenicity Mice a Tests that usually are based on animal models. Source: Adapted from Hendriksen, C.F.M., Laboratory Animals in Vaccine Production and Control. Replacement, Reduction, and Refinement, Kluwer Academic, Dordrecht, the Netherlands, 1988. 12 HANDBOOK OF LABORATORY ANIMAL SCIENCE 50% of the animals is calculated. An alternative strategy for toxoid vaccines is to immunize one group of animals with the vaccine under study, to bleed these animals after a number of weeks, and to estimate levels of protective antibodies by titration of serum samples mixed with fixed doses of toxin in groups of animals. Both approaches require large numbers of animals (e.g., 140 for the parallel-line potency test) and induce substantial levels of suffering. Attention is now being given to the development of methods that could replace, reduce, and refine (the 3Rs) the use of laboratory animals. The reasons for this trend include: • Concern about the extensive use and the substantial levels of pain and distress inflicted on the animals • The questionable relevance of some animal models, such as the rabies vaccine and whole cell pertussis vaccine potency tests, which show very poor reproducibility • The fact that animal tests are time consuming and interfere with the limited shelf life of vaccines • New developments and strategies in vaccine production such as standardization of production processes and the introduction of GMP, Quality Assurance (QA), and in-process control that make extensive quality control less relevant and even superfluous. Extensive overviews of 3R developments have been published.31–33 A summary of some of the achievements is given in Table 1.8. Although these developments had an impact on the numbers of animals used, a breakthrough can only take place if the concept of “demonstration of consistency” is generally accepted. The key issue of consistency has emerged from the new generation of vaccines. These vaccines, which are based on new technologies, are produced in a consistent way and the stress of quality control is on in-process monitoring rather than on final batch testing. In-process testing is almost exclusively based on in vitro biochemical and physicochemical tests. The consistency concept has become state of the art for the new generations of vaccines. Also, in the field of conventional vaccines, continued advances in production technology have resulted in more defined and thus less variable products. This, together with the implementation of GMP and QA, makes people feel that for a conventionally produced vaccine, the extent of batch release testing should reflect the level of consistency obtained with the vaccine. Thus, a vaccine manufacturer should perform extensive testing (including animal testing) during the development phase and on the first few batches of the new product to characterize the vaccine thoroughly. However, if consistency in production is demonstrated, then testing could rely on a battery of easy-to-use in vitro assays to characterize (fingerprint) the vaccine and confirm consistency. If this new approach is applied, the number of animals used for quality control of conventional vaccines will be reduced to an absolute minimum. Table 1.8 Summary of Major 3R Developments in Vaccine Quality Control Vaccine Animal Test 3R Alternative Toxoids Potency test based on challenge procedure Serological-based potency test Erysipelas Potency test based on challenge procedure Serological-based potency test All Abnormal toxicity test Deleted from test specificationsa Hepatitis B Mouse potency test In vitro method (ELISA) Polio (live) Neurovirulence test in monkeys MAPREC assay and transgenic mouse test Relevant vaccines Lethal challenge procedure Use of humane endpoints Note: ELISA = enzyme-linked immunosorbent assay; MAPREC = mutant analysis by polymerase chain reaction (PCR) and restriction enzyme cleavage. a Deleted only from the European Pharmacopoeia monographs but still specified by other regulatory bodies. ANIMAL MODELS IN VACCINOLOGY 13 CONCLUSION Animal models have played and still play an essential role in vaccinology. They have made possible the worldwide immunization of children with pediatric vaccines that are both safe and efficacious. Until recently, laboratory animals were required for development, production (in case of viral vaccines), and particularly, for quality control. This resulted in the use of large numbers of animals for these purposes, often in models that induced severe pain and suffering. Changes are now taking place that will affect this situation. Newer generations of vaccines are more defined and can be produced more consistently than the conventionally produced vaccines were. As a consequence, a shift in the need for laboratory animals will take place. The burden of animal research with the newer generation of vaccines will be on development, while for the conventional vaccines it was on routine batch quality control. Therefore, it can be anticipated that the numbers of animals needed will be reduced in the near future. However, limited numbers will still be needed to evaluate the interaction of the vaccine with the complex immune system in an intact organism. REFERENCES 1. European Pharmacopoeia, Vaccines for human use, Pharmeuropa, 15, 3, 449, 2003. 2. Hendriksen, C., Spieser, J.-M., Akkermans, A., Balls, M., Bruckner, L., Cussler, K., Daas, A., Descamps, J., Dobbelaer, R., Fentem, J., Halder, M., van der Kamp, M., Lucken, R., Milstien, J., Sesardic, D., Straughan, D., and Valadares, A., Validation of alternative methods for the potency testing of vaccines, ATLA, 26, 747, 1998. 3. Holt, P.G., Rowe, J., Loh, R., and Sly, P.D., Development of factors associated with risk for atopic disease: implications for vaccine strategies in early childhood, Vaccine, 21, 3432, 2003. 4. Uter, W., Stock, C., Pfahlberg, A., Guillen-Grima, F., Aguinaga-Ontoso, I., Brun-Sandiumenge, C., and Kramer, A., Association between infections and signs and symptoms of “atopic” hypersensitivity — results of a cross-sectional survey among first-year university students in Germany and Spain, Allergy, 58, 580, 2003. 5. Bussiere, J.L., McCormick, G.C., and Green, J.D., Preclinical safety assessment considerations in vaccine development, in The Subunit and Adjuvant Approach, Powell, M.F. and Newman, M.J., Eds., Plenum Press, New York, 1995, 61. 6. Nicoll, A., Contraindications to whooping cough immunization — myths or realities? Lancet, i, 679, 1985. 7. Granström, M., The history of pertussis vaccination: from whole-cell to subunit vaccines, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Succesors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 107. 8. Sato, Y., Kimura, M., and Fukumi, H., Development of a pertussis component vaccine in Japan, Lancet, i, 122–126, 1984. 9. Galazka, A.M., Lauer, B.A., Henderson, R.H., and Keja, J., Indications and contraindications for vaccines used in the Expanded Programme on Immunization, Bull. WHO, 62, 357, 1984. 10. Parish, H.J., A History of Immunization, E & S Livingstone, London, 1965. 11. Moulin, A.-M., Philosophy of vaccinology, in Vaccinia, Vaccination and Vaccinology: Jenner, Pasteur and Their Successors, Plotkin, S. and Fantini, B., Eds., Elsevier, Paris, 1996, 17. 12. WHO, State of the World’s Vaccines and Immunization, revised ed., World Health Organization, Geneva, 2003, 111. 13. van der Zeijst, B.A.M. and Lenstra, J.A., Vaccinology, LAB/ABC, 9, 18, 1988. 14. Köhler, G. and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256, 495, 1975. 15. Quint, W., Gielkens, A., Van Oirschot, J., Berns, A., and Cuypers, H.T., Construction and characterization of deletion mutants of pseudo-rabies virus: a new generation of “live” vaccines, J. Gen. Virol., 68, 523, 1987. 14 HANDBOOK OF LABORATORY ANIMAL SCIENCE 16. Rappuoli, R., Podda, A., and Pizza, M., Progress towards the development of new vaccines against whooping cough, Vaccine, 10, 1027, 1992. 17. Ellis, R.W., The applications of rDNA technology to vaccines, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 303. 18. Chattergoon, M., Boyer, J., and Weiner, D.R., Genetic immunization: a new era in vaccines and immune therapeutics, FASEB J., 11, 753, 1997. 19. Klein, M., Future prospects for vaccination, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 295. 20. Hendriksen, C.F.M., A short history of the use of animals in vaccine development and quality control, in Replacement, Reduction, and Refinement of Animal Experiments in the Development and Control of Biological Products, Brown, F., Cussler, K., and Hendriksen, C., Eds., Developments in Biological Standardization 86, Karger, Basel, 1996, 3. 21. Zo doende 1978–2002, Annual statistics on the use of laboratory animals in the Netherlands, Keuringsdienst van Waren (KvW). 22. Prince, A.M., Moor-Jankowski, J., Eichberg, J.W., Schellekens, H., Mauler, R.F., Girard, M., and Goodall, J., Chimpanzees and AIDS research, Nature, 333, 513, 1988. 23. Daniel, M.D., Kirchoff, F., Czajak, S., Sehgal, P.K., and Desrosiers, R.C., Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene, Science, 258, 1938, 1992. 24. Siebelink, K.H., Chu, I.H., Rimmelzwaan, G.F., Weijer, K., van Herwijnen, R., Knell, P., Egberink, H.F., Bosch, M.L., and Osterhaus, A.D., Feline immunodeficiency virus (FIV) infection in the cat as a model for HIV infection in man: FIV-induced impairment of immune function, AIDS Res. Hum. Retroviruses, 6, 1373, 1990. 25. Kuiken, T., Fouchier, R.A., Schutten, M., Rimmelzwaan, G.F., van Amerongen, G., van Riel, D., Laman, J.D., de Jong, T., van Doornum, G., Lim, W., Ling, A.E., Chan, P.K., Tam, J.S., Zambon, M.C., Gopal, R., Drosten, C., van der Werf, S., Escriou, N., Manuguerra, J.C., Stohr, K., Peiris, J.S., and Osterhaus, A.D., Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome, Lancet, 362, 263, 2003. 26. Martina, B.E., Haagmans, B.L., Kuiken, T., Fouchier, R.A., Rimmelzwaan, G.F., Van Amerongen, G., Peiris, J.S., Lim, W., and Osterhaus, A.D., Virology: SARS virus infection of cats and ferrets, Nature, 425, 915, 2003. 27. Wiktor, T.J., Plotkin, S.A., and Koprowski, H., Development and clinical trials of the new human rabies vaccine of tissue culture (human diploid cell) origin, Develop. Biol. Stand., 40, 3, 1978. 28. Katz, S.L., Wilfert, C.M., and Robbins, F.C., The role of tissue culture in vaccine development, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1986, 213. 29. Horaud, F., Viral vaccines and cell substrate: a “historical” debate, in Vaccinia, Vaccination, Vaccinology. Jenner, Pasteur and Their Successors, Plotkin, S.A. and Fantini, B., Eds., Elsevier, Paris, 1996, 217. 30. WHO, Cells, Products, Safety: Background Papers from the WHO Study Group on Biologicals, Developments in Biological Standardization 68, Karger, Basel, 1987. 31. Hendriksen, C.F.M., Laboratory Animals in Vaccine Production and Control. Replacement, Reduction, and Refinement, Kluwer Academic, Dordrecht, the Netherlands, 1988. 32. Weisser, K. and Hechler, U., Animal Welfare Aspects in the Quality Control of Immunobiologicals, Paul Ehrlich Institute, Russell and Burch House, Nottingham, U.K., 1997. 33. Halder, M., Three Rs potential in the development and quality control of immunobiologicals, ALTEX, 18(Suppl. 1), 13, 2001.

No hay comentarios:

Publicar un comentario en la entrada