Animal Models in Modern Vaccine Development

Animal Models The recognition that animals could be used as potential models for human infectious diseases dates back to Jenner, who in 1798 observed that milkmaids were resistant to smallpox because they were exposed to cattle infected with a related virus Pasteur investigated anthrax and rabies pathogenesis in animal models

Animal Models Pasteur's studies with the rabies virus clearly demonstrated the ability to transmit infectious agents from one species to another Pasteur further enhanced the concept of vaccination of dogs against rabies virus and finally tested his theory of vaccination on Joseph Meister, who had been bitten by a rabid dog As is now well-known, the experiment was a success and Pasteur is recognized as the individual who introduced the concept of inactivated vaccines,which is still used today

Animal Models A more recent example of an animal viral disease being used to develop a platform technology for use in licensing a human vaccine are virus-like particles (VLPs) for immunization against Papillomavirus The concept of using recombinant Papillomavirus VLPs was first established for the control of disease caused by bovine, canine and rabbit Papillomavirus, and eventually provided the basis for subsequent licensure of a bivalent and quadravalent HPV vaccine to control cervical cancer. The development of this vaccine confirms that studies in animals remain relevant to the control of infectious diseases in humans

Animal Models With availability of inbred, genetically-defined strains, mice have become the species of choice for most investigators Unfortunately, as these studies exploded in number and with different pathogens, it became clear that the mouse was not as useful a model species as was anticipated

Why Mouse is a Model for Us Humans Humans and mice have a reasonable number of genes in common– those involved in the development of major organs and major sections of our physiology such as the nervous, muscular, skeletal, cardiovascular, immune and endocrine systems. In addition to having many structures and functions similar to humans, mice are relatively short-lived and explosive reproducers (potentially producing a new litter every nine weeks). So studying these animals is one of the best ways to find out how specific genes common to mammals function over generations.

Use of Animal Models in Vaccine Production Animal models can be used in a variety of ways to study – Disease pathogenesis – Host–pathogen interactions – Mechanisms of protection following vaccination, infection or treatment of disease

Use of Animal Models in Vaccine Production

Natural or surrogate animal models Natural animal models Natural disease models make use of a specific pathogen and its natural host and have the advantage of modeling the interaction between host and pathogen, within the appropriate biological context

Natural animal models This is important since it permits analysis of virulence factors and their role in invasion, penetration and toxicity, as well as the host's immune response to the pathogen This also allows identification of specific molecules that are often required by the pathogen for infection or subsequent induction of disease and which often represent targets for the host's immune response

Natural animal models Using these molecules as vaccine antigens has proven to be a very successful strategy for developing effective vaccines against both human and animal diseases

Surrogate models Surrogate models refer to the use of species that can only be infected with the pathogen of interest under experimental conditions As a consequence, compromises such as higher infection doses or artificial routes of infection often have to be made

Surrogate models Most often, mice are the animal species of choice They offer the advantage of working in a consistent genetic background, are easy to handle, and are very cost-effective Recent advances in creating constitutive or conditional gene knockout mice have opened the door to understanding: – Role of specific host genes in pathogen recognition – Induction of acquired immunity – Cellular interactions within various immune compartments

Surrogate models Prominent examples include the successful knockout of: – β-microglobulin – Toll-like receptor – Cytokine – Chemokine genes

Surrogate models These gene deletions have created important animal models such as B- and T-cell-deficient mice: – Can help in vaccine development against many extra- and intracellular pathogens. Transfer of normal or genetically modified cells from one mouse to another is being used to characterize the function of specific immune cells in the context of infection and vaccine-induced immune responses

Surrogate models Animal models can also serve to analyze specific aspects of the immune response, such as: – The development of immune organs – The role of specific immune compartments or individual cell populations – The trafficking of immune cells following infection or vaccination – Various aspects of vaccine delivery including mucosal or topical application – Transmission amongst infected and non-infected animals – Studying transfer of passive immunity via the placenta, colostrum and milk

Surrogate models Animal models have been used to explore: – Vaccine formulation and delivery – Route of administration – Targeting to specific receptors – Induction of mucosal versus systemic immunity Surgical models allow access to intestine, lymph nodes or skin tissues for vaccine testing Large animal models provide the opportunity to evaluate vaccine efficacy, for example West Nile virus (WNV) or influenza infections in horses.

Surrogate models Since many disease models in mice utilize artificial routes of challenge, in large animals however, it is often possible to use the natural route of challenge and therefore obtain more relevant correlates of immune-mediated protection In vaccinating such population, it has frequently been observed that there are ‘low' and ‘high' responders

Surrogate models This reflects a genetic component that determines the magnitude of immune responses within individual animals Inbred lines of pigs have been established that have been defined as low and high responders following vaccination The advent of genomics has made it possible to begin defining the genetic basis of the immune response to vaccination

Surrogate models Vaccine efficacy also varies dramatically when immunizing the very young or the elderly Natural disease models including E. coli and rotavirus infections in pigs and calves have been used to establish the concept of maternal vaccination as an effective strategy to: – Reduce the risk of infection in the neonate – Optimize the passive transfer of maternal immunity to the newborn – Determine the duration of protection following passive transfer of maternal antibody

Limitations of Surrogate & Natural Animal Models The dose of pathogen used for experimental challenge frequently exceeds the dose known to cause natural infection Differences in the infectious dose may be due to passage in culture, and a consequently hypoinfectious state or the attenuation of the pathogen

Limitations of Surrogate & Natural Animal Models The use of animal models for evaluating vaccines remains critical prior to vaccine testing in humans Rarely is there an animal model that precisely replicates human infections Natural routes of infection are often unavailable owing to the lack of relevant receptors in model species Consequently, the route of entry and disease pathogenesis are different, which makes vaccine testing less relevant in this model

Overcoming these Limitations of Surrogate & Natural Animal Models Utilizing an animal model that expresses the required receptors for a specific pathogen, or using natural disease models with pathology comparable to the human disease, can assist in overcoming these difficulties For example pneumovirus (PVM) infection of mice, which has a similar pathology to respiratory syncytial virus (RSV), one of the most serious causes of respiratory illness in infants

Overcoming these Limitations of Surrogate & Natural Animal Models Likewise, bovine RSV (BRSV) is also an appropriate model for RSV owing to the genetic similarity of the viruses and similar clinical disease Transgenic mice that carry receptors for human pathogens may provide an alternative model for evaluating host responses when using the natural route of infection

Criteria for Appropriate Animal Models The quality of an animal model and its appropriateness for vaccine development can be defined by its ability to reproduce relevant human physiology, which ultimately is the target population for the vaccine Thus, good models share the same physiological characteristics, or at least reflect them as closely as possible

Criteria for Appropriate Animal Models The physiology of the skin is very similar between humans and pigs, which renders the pig a good model for studying intracutaneous or topical delivery of the vaccine The development of the immune system, in particular the maturation of the mucosa-associated lymphoid tissues, is similar in humans, sheep, cattle and pigs, which again makes these species good models for studying mucosal delivery of vaccines

Criteria for Appropriate Animal Models In these species the mucosal immune system develops well before birth, which stands in clear contrast with mice, in which the mucosal immune system only develops after birth Furthermore, the neonatal period in mice is much shorter than in man, which makes the use of mice for developing neonatal vaccines highly problematic

Criteria for Appropriate Animal Models The ethical use of animals in human vaccine research requires that we only choose animals that match the human disease as closely as possible Such a criteria will help to reduce the overall number of animals used for biomedical research

Criteria for Appropriate Animal Models DNA vaccines and recombinant viral vectors, have been developed and demonstrated to provide disease protection in horses The horse has been a very useful model for analyzing several aspects of the immune response to vaccination such as: – Maternal immmunization – Passive transfer of immunity – Response to adjuvants such as bacterial DNA – Immunization of the neonate and the elderly

Criteria for Appropriate Animal Models Large animal models, such as the horse, could provide geriatric populations for the screening of a variety of possible adjuvants Very little is known, however, regarding the functional or phenotypic changes in the immune system of most domestic species and this information will be critical to determine if these animal models are appropriate surrogates for the translation of vaccine technologies to clinical application

Conclusion When choosing an appropriate animal model for human disease, it is critical to ensure that the model simulates as closely as possible the events occurring in humans First, it is more likely that higher similarity of pattern of pathogenesis to human disease in an animal model will correlate better to immune-mediated protection resulting from that model Second, if the pathogen enters via the respiratory tract, then the model should utilize aerosol challenge to expose the pathogen to the defenses of the upper respiratory tract

Conclusion Third, the pathogen dose should be similar to that which would occur naturally, since it is always possible to overcome an adaptive immune response by excessive pathogen challenge or by using an unnatural route of infection In choosing a model for respiratory infections, the structure, function and development of the respiratory tract in the animal model should resemble that of humans