James A. Roth, DVM, PhD, DACVM
Recent advances in molecular biology, immunology, microbiology, genetics and in understanding microbial pathogenesis have lead to the development of a wide variety of new approaches for developing safer and more effective vaccines. These new approaches hold tremendous promise for developing improved vaccines both for diseases we currently vaccinate against and for diseases that have defied the conventional approach of using killed or attenuated microorganisms as vaccine antigens. This presentation will briefly describe these new technologies and their potential advantages and disadvantages as compared to conventional killed and modified live vaccines. The biotechnological approaches to developing vaccines also allow the development of marker vaccines to differentiate vaccinated from infected animals and for improving vaccine thermostability.
Subunit vaccines contain only a portion (subunit) of the infectious agent which causes a disease. The first step in developing a subunit vaccine is to identify protective antigens of the infectious agent. Then those protective antigens may either be purified from a culture of the microorganism causing the disease or the genes from the protective antigens can be genetically engineered into an expression vector for production and subsequent purification of the antigen. For many diseases a single antigen or small number of antigens have not been identified that are capable of inducing broad protection when administered in a purified non-replicating form. For others, antigenic variation and/or drift in protective immunogens impacts the effectiveness of the vaccine against field strains of the pathogen. Subunit vaccines also typically require the use of potent adjuvants to induce a strong immune response, increasing the likelihood of an adverse reaction.
One method for enhancing the immunogenicity of purified subunit vaccines is to assemble the subunit antigens into a "virus-like particle" (VLP). Virus-like particles are particles that have highly repetitive subunit antigens built into their structure, much like a virus surface. These particles with highly repetitive antigens on their surface have been shown to induce potent B cell responses and efficient cytotoxic T cell responses.1 Therefore, not only is the amount of antigen important in inducing an immune response, the physical arrangement of the antigen is important as well. The assembly of rotavirus VP6 subunit proteins into VLP5 markedly enhanced its immunogenicity.2
Subunit vaccines have been investigated for use with Mycobacterium bovis3, Moraxella bovis4, Theileria parva,5,6.7 Rinderpest8, Boophilus spp.,9,10,11 Babesia spp.,12,13 and Mannheimia haemolytica.14
Gene Deleted Vaccines
Gene deleted vaccines are made from organisms which have had a specific gene or genes deleted or inactivated. In order to develop a gene deleted vaccine it is essential to first identify and remove or inactivate a gene (or genes) which are not essential for replication or induction of immunity. If the gene deleted vaccine is to be used as a marker vaccine, then it is important to delete a gene that encodes a protein which induces a long-lived antibody response when any wild strain of the organism infects a vaccinated animal. These gene deleted organisms can then be used to develop either a modified live or killed vaccine.
Gene deleted vaccines have been exploited very effectively for the production of marker vaccines for use in disease eradication programs. Animals vaccinated with the gene deleted vaccine do not produce antibodies against the protein encoded by the deleted gene. Therefore it is possible to detect animals which have been vaccinated and subsequently infected with field virus because they will in most cases produce antibodies against the protein missing from the gene deleted vaccine organism. This requires the development and use of a companion diagnostic test which detects antibody specific for the missing protein only.
Live Vectored Vaccines
Live vectored vaccines are produced by identifying a protective antigen or antigens for a particular pathogen and then engineering the genes coding for those antigens into another organism which may safely replicate and express the antigen in the target species. Both viral and bacterial vectored vaccine strains have been developed.
Live vectored vaccines can be a very useful approach for producing vaccines against organisms that replicate very poorly or not at all in vitro and for foreign animal diseases. Live vectored vaccines can make excellent marker vaccines for disease eradication programs because they only contain a portion of the antigens present on field strains of the organism. Companion diagnostic test kits can be developed which detect antibodies against antigens not found in the live vectored vaccine or against specific markers inserted in the vectored strain in order to detect vaccinated animals which have become infected with virulent organisms. A major advantage of live vectored vaccines is that they may be effective in overcoming the interference with the immune response due to the presence of maternal antibody.
Live vectored vaccines have been investigated for induction of immunity against foot and mouth disease virus,15 Bovine viral diarrhea disease virus,16 Mycobacterium bovis,17 and rinderpest.18
DNA Vaccines (nucleic acid-mediated vaccines) are produced by engineering genes for protective antigens into bacterial plasmids which are circular pieces of DNA. This plasmid DNA is then purified from that of the bacterial expression host and directly administered to animals. Many routes of administration have been used (intramuscular, subcutaneous, intradermal, and oral). To be effective, the plasmid DNA must be transported into cells, and transcribed by the mammalian host cell machinery into messenger RNA which is translated into protein. The protein then may induce an antibody and/or T cell mediated immune response. DNA vaccines are quite inexpensive to produce and are much more stable then modified live vaccines under a wide range of storage and handling conditions. It is also possible to engineer genes for selected cytokines into the plasmids. When these cytokines are co-expressed with the vaccine antigens then they may stimulate a stronger or more targeted immune response and help to induce the appropriate type of immune response for the particular organism (i.e., mucosal immunity, TH1 response, TH2 response, or a cytotoxic T cell response).
DNA vaccines have been investigated for induction of immunity to Bovine herpesvirus,19, 20 bovine viral diarrhea virus,21 Anaplasma marginale,22 and Cowdria ruminatium (the causative agent of heartwater),23 Mycobacterium bovis,24,25,26,27,3 bovine respiratory syncytial virus,28,29, 30 Staphylococcus aureus,31,32 rinderpest33 and Shistosoma spp.34
Transgenic Plants or Plant Viruses
Genes from animal pathogens can be genetically engineered into plants so that the transgenic plant produces large amounts of antigen which can be used as a vaccine. Pathogen genes can also be cloned into plant viruses which will cause the plant infected with the plant virus to produce large amounts of vaccine antigen. It is also possible to clone genes coding for monoclonal antibody production into transgenic plants. The transgenic plant will then synthesize and assemble large concentrations of monoclonal antibodies which can be used therapeutically in animals. Plants as diverse as corn, potatoes, alfalfa, spinach, tomatoes, and tobacco have been used. Transgenic plants are even capable of assembling the heavy and light chains to form IgA along with the J chain and secretory component to form secretory IgA. This is especially useful because animals can then be fed plants containing high concentrations of secretory IgA specific for enteric pathogens. Plants expressing vaccine antigens incorporated into the feed as edible vaccines are appealing because of the ease of production, storage, and administration. However incorporation of the plant based antigen into feed presents problems in standardizing the antigen dose that animals receive.
Transgenic plants or plant viruses have been investigated for production of vaccine antigens against foot and mouth disease virus,35 bovine rotavirus,35 rinderpest virus,36 and BVDV.35
Slow or Pulse Release of Antigen
Vaccine antigens can be incorporated into polymers that can be implanted subcutaneously and which will release antigen slowly or in pulse doses. This allows for single administration of antigen to deliver two or more doses of antigen over a specified time period. This prevents the need to give booster doses of vaccine and may be a particular advantage in helping to overcome maternal antibody interference in young animals. Polymers can also be used to protect antigens for oral delivery.
Adjuvants are compounds that are added to vaccines to enhance the immune response to vaccine antigens. They may increase the magnitude and duration of responses and/or they may alter the type of immune responses induced. Aluminum salts (alum) are the oldest and most commonly used vaccine adjuvants. Proteins precipitated to aluminum salts induce a stronger antibody response. Alum adjuvants are typically not very effective at inducing T cell-mediated immune responses or at inducing protection at mucosal surfaces. Considerable research has been conducted aimed at developing adjuvants capable of inducing the appropriate type of T cell response to protect against intracellular pathogens and of inducing protection at mucosal surfaces. ISCOMS have been especially promising adjuvants for this purpose and have been investigated for use with vaccines against Moraxella bovis,4 bovine respiratory syncytial virus,37 and rinderpest virus.8
Adjuvants can be classified into five main categories depending on their physical properties and mechanisms of action: the potential for depot formation, the capacity to preserve conformational epitopes, the ability to target to antigen presenting cells or to mucosal tissue, the ability to induce CD8+ cytotoxic T cells, and the capacity for immunomodulation. Rapid strides are being made in the development of safe, more effective adjuvants. Further discussion of new adjuvant technologies is beyond the scope of this manuscript, but can be found in recent review articles.
Limitations to Improving Animal Vaccines Through New Technology
The new technologies described above hold tremendous promise for the development of safer and more effective vaccines, particularly for diseases for which conventional killed and modified live vaccine approaches have not been satisfactory. There are, however, a number of factors which limit the usefulness of these new technologies. An important factor is the expense of these technologies. Research and development costs can be substantial. The effective use of these new technologies often require a thorough understanding of the molecular basis of pathogenesis and the mechanisms of protective immunity. After these are understood it is necessary to conduct extensive research to develop a vaccine that uses the protective antigens to induce the appropriate type of immune response. Some of the new technologies result in products which are more expensive to manufacture. Conversely, some of the new technologies may reduce the manufacturing costs.
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