Online Issues

ILAR Journal Vol 46(3)

Introduction

Laboratory Animals and Immunization Procedures: Challenges and Opportunities

Coenraad F. M. Hendriksen

Coenraad F. M. Hendriksen, D.V.M., Ph.D., is an Animal Welfare Officer and Senior Scientist at the Netherlands Vaccine Institute, Bilthoven, The Netherlands.

Key Words: adjuvants; animal welfare; antibodies; Freund's adjuvants; IACUC issues; immunization protocols; production innovation

This issue of ILAR Journal presents an overview of new developments and insights in the study of immunization procedures and adjuvant products. Substantial progress has been made, both with regard to new technologies and to understanding the immune response since publication of an earlier ILAR Journal issue devoted to Adjuvants and Antibody Production (Landi 1995). It is timely and important once again to address these developments. The objective of the current ILAR Journal issue is to discuss critical aspects of immunization in the context of what is known of the immune response and to provide an overview of new developments in the production of polyclonal antibodies (PAbs¹) and monoclonal antibodies (MAbs¹).

Immunizations in laboratory animals are performed for a wide variety of reasons. Primary purposes include (1) induction of specific B cells for the generation of hybridomas, (2) production of PAbs and MAbs, (3) development and quality control of immunobiological products, (4) fundamental immunological studies, and (5) induction of specific disease models. Clearly, these studies have been integral to scientific breakthroughs that have occurred in many areas of biomedical research. One such critical breakthrough has been the development of highly effective vaccines. In other cases, products such as PAbs and MAbs, which are obtained from immunization procedures, have become essential reagents in the laboratory and are being applied in diagnostic testing, in cancer therapy, and in numerous other ways.

Nevertheless, despite the significant medical advances that have resulted, the use of laboratory animals for immunization procedures continues to generate controversy. Investigators and members of animal care and use committees continue to have frequent and fundamental questions related to immunization and adjuvant products. Although the answers to such questions are not always clear and unequivocal, data and information in the field of immunization have increased. Indeed, tradition and myths are gradually being replaced by science-based approaches.

For example, descriptions of the adverse reactions of Freund's complete adjuvant (FCA¹), which is used for its immunostimulatory capabilities, were first published in 1950 (Leskowitz and Waksman 1950) and then in 1960 (Steiner et al. 1960). Since then, discussions regarding the potential of FCA to induce pain and distress have continued for several decades and have resulted in the preparation of numerous guidelines (e.g., institutional guidelines in the United States, and regulatory guidelines in Canada and several European countries). Currently, the use of FCA is limited in experimental animals, and the process of developing alternative adjuvant products has begun.

Concerning the production of MAbs, the Institute for Laboratory Animal Research (ILAR¹) Committee on Methods of Producing Monoclonal Antibodies concluded in 1999 that the intraperitoneal accumulation of ascites is likely to be associated with pain and distress (NRC 1999). Committee members recommended that the tissue-culture method for the production of MAbs should be adopted as the routine method unless there is clear reason why tissue culture methods cannot be used or why their use would represent an unreasonable barrier to obtaining the product at a cost consistent with the realities of funding biomedical research programs. In several European countries, the use of the ascites method is strongly discouraged, although it has not been banned entirely.

As new technologies and new experimental data become available, we are gradually unraveling the complex processes underlying immunization procedures. This information will be helpful in understanding the critical aspects involved in immunization protocols and will allow us to prepare rationally based guidelines on how to design optimal immunization protocols and how to continue reducing, refining, and replacing the use of animals.

In their article, McCullough and Summerfield (2005) introduce the basic concepts of the immune responses. The authors describe the cascade of responses that take place after the interaction between the antigen--a structure the body recognizes as a "non-self"--and the immune system. The authors elaborate on various aspects of the immune response, including the innate and specific immune responses and their interaction, the critical role dendritic cells play in "professional antigen presentation," T and B lymphocytes and memory, and cytokine profiles.

Immunization protocols are applied for a wide variety of purposes. Schunk and Macallum (2005) provide an overview of these applications. Laboratory animal models have played an integral role toward understanding the important area of infectious disease prevention. In this issue, the authors discuss vaccine development and quality control, and highlight other applications such as cancer treatment, immunotoxicology, and fertility control. They also describe routes of immunization as they relate to the animal immunization protocol, focusing on aspects such as selection of animal species, the use of inbred or outbred animals, sex, and nutrition.

To investigators, it is not always clear when the use of MAbs affords an advantage over the use of PAbs. Authors Lipman and colleagues (2005) provide information on the characteristics of both types of antibodies. They focus on structure and function in detail, and then relate these characteristics to specific applications such as their use in techniques for analysis, in purification procedures, and in immunotherapy. A particular application of antibodies involves their use in mediating the catalysis of specific synthetic organic reactions. For potential users of antibodies, an overview of information resources is provided for currently available MAbs and PAbs.

Many guidelines on PAb and MAb production exist; however, these documents generally focus on only a few aspects of the immunization protocol. Leenaars and Hendriksen (2005) analyze the critical steps in the entire production process and provide recommendations on how to optimize and refine the protocol. Aspects of their discussion include selection of animal species, adjuvant selection and preparation, injection protocol and booster immunization, and postinjection observation of laboratory animals. Also included in the discussion are recommendations regarding priming of the animals and ascites collection.

Frequently, immunization protocols require the use of adjuvant products to enhance or modify the immune response. Most adjuvants, however, also induce adverse reactions such as local inflammation and tissue destruction. These sequelae are therefore often met with concerns about the animals' welfare, particularly from the perspective of institutional animal care and use committees (IACUCs¹). This concern is particularly characteristic of FCA, the product probably most frequently used in experimental immunization protocols. Stills (2005) presents an in-depth review of the major adjuvant products being applied in the animal laboratory and focuses on mode of action, immunology, pathology, and pain and suffering. The author relies on science-based facts, in contrast to the myths that surround the use of adjuvants.

Two alternatives to the traditional protocol for PAb production are discussed in the Hau and Hendriksen (2005) article. Both alternatives have the potential to refine the production technique. The first approach is the immunization of chickens, followed by harvesting of the antibodies from the egg yolk instead of the serum. The advantages and disadvantages of this procedure are summarized. The second approach is oral immunization by voluntary intake of the antigen, or by gavage as well as oral-nasal admin-istration. In conjunction with this approach, oral adjuvant products are described. The authors conclude that the combination of both approaches will ultimately result in reduction and refinement in the use of animals.

In the next article that appears in the issue, Clough and Hauer (2005) provide practical examples of the use of PAbs and MAbs for the regulated quality control of biological products. They focus on vaccine batch release testing, and highlight the requirements these antibodies must fulfill with regard to specificity and affinity/avidity. The authors provide several case examples to illustrate these concepts. They also address the need for and availability of standardized antibodies, particularly in view of international efforts to harmonize test methods.

Dewar and colleagues (2005) provide a case study related to MAb production. These authors describe the transfer of in vivo MAb production to in vitro production in a large, multinational vaccine manufacturer. The authors address the issue of infrastructural requirements and describe the use of various in vitro systems that may be selected for the production needs. Descriptions relate both to small- and large-scale production, such as stationary and rotation suspension culture systems and hollow fiber bioreactor systems. Also included are data from comparison studies with mouse ascites production. The authors conclude that various tested in vitro culture systems are qualitatively and quantitatively equal to or even better than the ascites production method.

The use of recombinant technologies has initiated a new era in monoclonal antibody production. In the final article in the issue, Peterson (2005) describes several of these highly innovative approaches, which now allow for engineering antibodies (e.g., reducing the antibody to its functional size, or making humanized antibodies). Advances in improving MAb production efficiency and antibody repertoire are being explored by using spontaneous mutant or genetically modified mice (e.g., the XenoMouse®) and genetically modified cell lines. The author concludes that despite numerous reports on the advantages of these approaches, the commonly accepted resources for MAb production have not changed. For this reason, the author recommends generally performing standardized comparative studies to judge the validity of new technologies.

Although it is unrealistic to expect that the information provided in this overview will resolve all of the controversies that exist with regard to immunization protocols, it is important to recognize that substantial progress has been made, both with regard to new technologies and to understanding the immune response. The information in these articles is presented in an effort to help investigators, animal care committees (e.g., IACUCs), as well as all others who have concern for animal well-being to combine the best scientific output in terms of immune responses or antibody production with maximum animal welfare.

¹Abbreviations used in this Introduction: FCA, Freund's complete adjuvant; IACUC, institutional animal care and use committee; ILAR, Institute for Laboratory Animal Research; MAb, monoclonal antibody; PAb, polyclonal antibody.

References

Clough NE, Hauer PJ. 2005. Using polyclonal and monoclonal antibodies in regulatory testing of biological products. ILAR J 46:300-306.

Dewar V, Voet P, Denamur F, Smal J. 2005. Industrial implementation of in vitro production of monoclonal antibodies. ILAR J 46:307-313.

Hau J, Hendriksen CFM. 2005. Refinement of polyclonal antibody production by combining oral immunization of chickens with harvest of antibodies from the egg yolk. ILAR J 46:294-299.

Landi M, ed. 1995. Adjuvants and Antibody Production. ILAR J 37:92-152.

Leenaars M, Hendriksen CFM. 2005. Critical steps in the production of polyclonal and monoclonal antibodies: Evaluation and recommendations. ILAR J 46:269-279.

Leskowitz S, Waksman BH. 1950. Studies on immunization. 1. The effect of route of injection of bovine serum albumin in Freund's adjuvant on production of circulating antibody and delayed hypersensitivity. J Immunol 84:58-72.

Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. 2005. Monoclonal versus polyclonal antibodies: Distinguishing characteristics, applications, and information. ILAR J 46:258-268.

McCullough KC, Summerfield A. 2005. Basic concepts of immune response and defense development. ILAR J 46:230-240.

NRC [National Research Council]. 1999. Monoclonal Antibody Production. Washington DC: National Academy Press.

Peterson NC. 2005. Advances in monoclonal antibody technology: Genetic engineering of mice, cells, and immunoglobulins. ILAR J 46:314-319.

Schunk MK, Macallum GE. 2005. Applications and optimization of immunization procedures. ILAR J 46:241-257.

Steiner JW, Langer B, Schatz DL. 1960. The local and systemic effects of Freund'adjuvant and its fractions. Arch Pathol 70:424-434.

Stills JF Jr. 2005. Adjuvants and antibody production: Dispelling the myths associated with Freund's complete and other adjuvants. ILAR J 46:280-293.