Jim Brewer and Virgil Schijns describe how modern vaccination approaches have developed and how animals will continue to play a fundamental role in understanding the control of the immune response to vaccines and the development of novel vaccines.
Edward Jenner derived the term “vaccination” from the Latin word “vacca”, meaning “cow”, following his original description of protection against smallpox (variola) through prior exposure to cowpox (vaccinia). It is estimated that 300 million people died from smallpox last century.
However, through the application of vaccinia, the last case of infection was reported in Ethiopia in 1977. Animals have played a critical role in the development of vaccination as sources of vaccine material, experimental models of clinically relevant infections, and as recipients and beneficiaries of veterinary vaccines.
A brief history
In the demonstration of vaccination against smallpox, Jenner tested a clearly formed hypothesis. He employed the 18th century technique of “variolation”, whereby patients were inoculated with small amounts of “variolous material” from smallpox pustules.
Although variolation was known to induce infection, this was of reduced severity and associated with protection against subsequent smallpox exposure. Indeed, the onset of symptomatic disease was seen as evidence of successful variolation.
Jenner described a number of individuals with a clinical history of cowpox infection evidenced by appearance of sores, typically on the hands, together with a moderate fever (they could not be successfully variolated).1
This led him to test his hypothesis that vaccination with cowpox would protect against smallpox variolation. The public health challenge that smallpox posed in the 19th century was enormous, for example, one fifth of all deaths in Glasgow were a result of smallpox infection and nine out of 10 people who died of smallpox were under five years of age.2
Consequently, the practice of vaccination spread rapidly in Europe, facilitated by the ability to cultivate cowpox on calf skin.
It can be seen how prescient Jenner’s observations and hypothesis were in light of the available knowledge at the time. Further vaccine development required the rejection of the spontaneous generation theory of infection and establishment by Robert Koch of the germ theory of disease and its associated methodology.3
However, it was Louis Pasteur who applied germ theory in a series of seminal studies that paved the way for the next 50 years of vaccine development.
Pasteur developed techniques of laboratory attenuation that produced weakened strains of disease-causing bacteria (cholera, plague and anthrax) and inactivated viruses (rabies), and successfully demonstrated protection mediated by these vaccine strains in animal models of infection before testing in man.
The subsequent identification and isolation of bacterial toxins that were not infective but nonetheless pathogenic when injected into animals led Emil von Behring to develop chemically modified toxoids. Although these toxoids were no longer pathogenic, they could confer protection against infection in recipient animals. Importantly, von Behring identified in immunised animals a serum component, antitoxin, which could passively transfer disease resistance.4
Ehrlich’s subsequent work allowed quantification of antitoxin and antibodies, which made their therapeutic use feasible. This work provided some of the basis for his theories on the “lock-and-key” chemical specificity of antibodies and drugs.5
During this time, development of viral vaccines lagged behind despite the clear public health challenges of influenza pandemics, polio and measles. Importantly, the requirement of an intracellular habitat for virus growth posed a particular problem.
Ernest Goodpasture’s demonstration in 1931 of virus cultivation in embryonated hens’ eggs overcame this obstacle, allowing large-scale production of influenza and yellow fever viruses for production of inactivated vaccines, which remain essentially unchanged to this day.
Further development of embryonic cell lines for large-scale culture allowed more diverse viruses to be cultivated, such as macaque or Old World monkey renal cells for poliovirus propagation for both the killed Salk and live Sabin vaccines.6
Using these approaches, development of attenuated live and killed vaccines, which have formed the mainstay of our paediatric vaccine regimens, has continued.
However, bulk cultivation and production could not be achieved for a number of significant infectious agents, such as viruses that are difficult to cultivate (eg, hepatitis) and parasites. These infections required a revolution in the way vaccines were produced, which resulted from the introduction of recombinant DNA technology.
The first licensed recombinant protein vaccine was the hepatitis B surface antigen in 1986, which was expressed in yeast and could be grown in bulk culture. Before this development, hepatitis B surface antigen was biochemically purified from plasma recovered from individuals harbouring the virus.
However, more importantly, this development heralded the possibility — for the first time — of creating vaccines against almost any infectious disease without having to cultivate the infectious agent.
A golden age of vaccinology
Picking the right antigen
Whole attenuated or killed organisms have a diverse array of antigens on display. Which antigens can be (and are) recognised and will stimulate a protective immune response is a matter of selection by the immune system and the organism. The ability to produce recombinant antigens for vaccination offered a number of advantages over and above ease of production.
First, there were improvements in biosafety, for example, early cell lines used to produce polio vaccine were contaminated with host viruses, such as SV40. Secondly, it was possible to exclude non-useful, counter-productive or even pathogenic antigens from the vaccine. In theory, it would now be possible to make new vaccines against infectious diseases, which had been previously characterised by failure.
However, this required identification of the protective antigen. Advances in immunology, particularly by Peter Doherty and Rolf Zinkernagel,7 ultimately led to an understanding of the molecular basis of antigen recognition and the crucial role of the polymorphic MHC Class I and II molecules in presenting processed antigen for recognition by the immune system — specifically, T lymphocytes.
Although this understanding led to the rational development of the polysaccharide conjugate vaccines, these studies also explained why, as vaccines became increasingly refined and contain fewer antigens, the impact of host genetics on immune responsiveness has increased.
The challenge that remains is, therefore, to identify a suitable antigen that is preferably conserved between pathogen species and is able to offer protection within a genetically heterogeneous outbred population.8
Antigen identification has been greatly refined by structure-function studies of MHC-antigen interactions. Crystallography has revealed the structure of the MHC Class I and II molecules, including the binding clefts where peptides derived from vaccine antigens associate.
Combinatorial techniques have revealed the motifs in peptide antigens that are important for these interactions. This has allowed identification of potential antigens in silico.
More recently, the ability to apply predictive vaccine algorithms to complete pathogen genomes (reverse vaccinology)8 has turned the flow of potential vaccine antigens into a flood. However, the ability of the immune system to recognise a particular antigen is not predictive of vaccine-induced protection.
In contrast to Jenner’s era, current experimental vaccines cannot be tested directly in volunteers or patients; protection studies must still be performed in laboratory animals. These studies are facilitated by suitable animal model systems, which recapitulate the human or veterinary disease.9
However, laboratory animals are not always susceptible to human pathogens and this requires careful selection of strain or species or host-adaption of pathogens.
It is important to note that, despite considerable progress and success with these techniques, they do not always accurately mimic human or veterinary pathology.
Recent work points to the potential value in vaccine development of mice with humanised immune systems created by engraftment of human peripheral blood cells and immune-system tissues into severe combined immune deficient (SCID) mice,10 which lack an adaptive immune response.
Such “humanised” animals allow human immune responses to be analysed following vaccination. Unfortunately, most of these animal (rodent) studies cannot be replaced by in vitro studies, although alternative assays for vaccine potency and safety are actively investigated.
Thus, although protection models are definitely valuable and have often been found to be predictive, as a whole, they must be designed and interpreted with care.
Stimulating the right response
Having the ability to identify immunogenic proteins that stimulate protective immune responses is not sufficient to make a vaccine. As vaccines have undergone increasing refinement for the reasons outlined, this has had the unwanted side effect of reducing their immunogenicity (ie, the ability of an antigen to induce immune responses).
In experimental situations, this problem could be remedied by the addition of a vaccine adjuvant. Although a number of potent adjuvants are available for use in experimental systems, until recently, the only vaccine adjuvant licensed for use in human vaccines were aluminium compounds.
These adjuvants are effective in inducing the types of antibody responses required to neutralise bacterial toxins or block cell invasion by viruses.
However, these types of responses (known as humoral or Th2 responses) are not effective in controlling a number of important diseases caused by intracellular infections, such as HIV and AIDS, malaria or tuberculosis.
Importantly, in contrast to antigen identification, we know little about how the immune system senses and responds to vaccine adjuvants at a cellular or molecular level. This state of affairs prompted the eminent scientist Charles Janeway to refer to them as “the immunologist’s dirty little secret”.11
It is clear that identifying the factors that determine the induction, magnitude, phenotype and persistence of immune responses will significantly contribute to the rational design of vaccines.
For example, a relevant development in our understanding of microbial recognition by the immune system has been the emergence of the toll-like receptors12 (TLRs). Toll was initially revealed to play an essential role in immunity to fungal infections in fruit flies (Drosophila melanogaster).
Subsequent studies in a variety of species, including mice and humans, revealed TLRs to be a highly conserved set of microbial sensors that recognise evolutionarily conserved microbial motifs, including bacterial cell walls and nucleic acids. TLRs clearly play a significant role in initiation of immune responses and were found to be considerably useful as vaccine adjuvants.
For example, a hepatitis B vaccine containing a TLR4 agonist (MPL) was recently licensed in Europe (Fendrix) and clinical trials employing TLR4 agonists to adjuvant malaria vaccines have proven promising.
Almost everything we know about how the immune system works has been derived from animal models and most of these findings have subsequently been validated and substantiated through clinical studies.
The immune system is a hugely complex system of organs, compartments, cells and molecules that integrate through a series of short- and long-range interactions. Although reductionism approaches, such as gene knockouts and cell reporters, are revealing the critical components in co-ordinating the immune response, so far we have not reached a situation where immune responsiveness can be modelled or understood in vitro.
Animals have played an important role in the development of our current battery of human and veterinary vaccines. We have identified areas where this role is waning, in particular in the identification of protective pathogens, which is now dominated by genomic sequencing, in silico analysis and the production of vaccine antigens, which now relies on recombinant proteins. We have also highlighted the problems where animal models of disease have been of variable predictive value.
Importantly, in the absence of valid alternatives, of primary concern is the careful design and analysis of these studies to ensure that their full predictive value is realised. However, one of the greatest obstacles facing vaccine development is our lack of understanding of the control of vaccine-induced immune responses.
In this respect, animal models are essential because the highly complex cellular and molecular interactions involved can only be analysed within a physiological environment.
This fundamental information will help produce agents to enhance the immune response to vaccines against infections or cancers, as well as to turn off inappropriate immune responses associated with diseases, such as allergy, asthma and autoimmunity. This is a fundamental objective with profound implications in the management of animal and human health.
Jim Brewer is reader in immunology at the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde.
Virgil Schijns is professor in immune intervention at the Department of Cell Biology and Immunology, Wageningen University, The Netherlands
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