Applications of Biotechnology in the Vaccine Industry
Huw P. A. Hughes
Director, Poultry and Aquaculture Research and Development
Intervet, Inc.
405 State Street
Millsboro, DE 19966
huw.hughes@intervet.com
Abstract
Significant strides have been made in our understanding molecular mechanisms of disease, and in some cases this knowledge has resulted in the successful application of molecular genetics for vaccine design. However, in the aquaculture industry, and particularly the catfish industry of North America, relatively little is known of either the host immunity against disease and the pathogenesis of disease. Until these are elucidated, the successful application of biotechnology to the catfish vaccine industry will be largely a matter of chance. From the perspective of the vaccine manufacturing industry, there must be some guarantee of safety and efficacy prior to embarking on a genetic approach. Vaccines that are derived from genetically modified organisms are costly and time consuming to license and launch. From the producers viewpoint it is of little consequence that the vaccines are derived from biotechnology they simply have to be effective.
Introduction
In North America, catfish remains the most significant aquatic species that is being farmed for human consumption. Successful control of disease in this industry has only recently been achieved, with vaccination only being made available for the last one or two seasons. Prior to that, disease control relied on husbandry practices, such as reducing feed given to production fish and fingerlings. These husbandry practices basically left the producer in a no-win situation if he did not reduce feed, then mortality (especially due to enteric septicemia of catfish ESC) increased. Alternatively, if he reduced feed, then the market weight would be less, eroding profits.
As with any other disease control effort, we must have an understanding not only of the aquatic host, but also of the disease itself. The pathogenesis of the disease and how the host may respond to infection and disease are both extremely important for successful vaccine design, as well as for producing the next generation of biopharmaceuticals. We are just beginning to elucidate the mechanisms of fish immunology. It is known that in some respects, fish share many attributes of higher animal immunology. They have specific and non-specific immune mechanisms, they have a fundamental immunoglobulin system, and they appear to have a T cell repertoire that may be similar to that described in other higher species. However, the ontogeny of the immune response still remains a mystery, and it may be naïve to assume that switching between different types of immunity (e.g., Th 1, Th 2 and Th 0) occurs as it does in mammals. Although fish do have cytokines, their function has yet to be described, both in immunologic and pharmacologic terms.
Just as relatively little is known about fish immunity, its activation and effector function, so little is known about many of the diseases of fish, particularly catfish. For example, Enteric Septicemia of Catfish (ESC), caused by Edwardsiella ictaluri can decimate catfish ponds at almost any stage of catfish production. Exactly where the bacterium comes from, how it becomes pathogenic and what triggers it becoming pathogenic is essentially unknown. Furthermore, very little is known regarding the nature of the protective antigens. Immunity against ESC is generally recognized as being cellular rather than humoral. Therefore conventional methods of antigen detection relying on specific antibody (e.g., ELISA or western blot), while detecting immunodominant antigens, may not elucidate the critical protective epitopes that result in generation of relevant, protective responses.
Clearly many vaccines have been produced for both human and animal health wherein the nature of the protective mechanisms and antigens is unknown. These products have relied on conventional methods for successful vaccination. These fall into two main categories, killed and modified live vaccines. Killed vaccines generally have been manufactured by growing the pathogen to high yields and treating with an inactivating agent (e.g., formalin, beta propriolactone, etc). Modified live vaccines may be generated by treating the pathogen with either a mutagenic agent, growing at different temperatures (to produce temperature sensitive mutants), or by attenuation through continuous growth in a different environment (e.g., continuous cell culture).
Below, the process that biological companies must follow in order to release a product on the market is outlined.
The Development Process Leading to Product Licensure
Following the definition of a potential product, or the discovery of a new vaccine, feasibility studies may be carried out in order to prove the concept that the vaccine is protective. In its simplest form, proof of concept may comprise killing a pathogen and testing its efficacy. Alternatively, the pathogen may be attenuated so it is no longer harmful to the host species. Recombinant vaccines may comprise both killed (e.g., expressed proteins), live (e.g., AroA- deletion mutants) or live, vectored vaccines.
Regardless of the type of vaccine that is under consideration, proof of concept must be considered before the lengthy development process is started. This is especially the case in any recombinant vaccine, as both the development time and resource allocation is far greater than those are for conventional vaccines.
Licensing Procedure
The FDA regulates pharmaceuticals and feed additives, and the USDA-APHIS has defined the procedure for licensing biological products. These regulations are documented in the Code of Federal Regulations, Chapter 9 (9 CFR). However, in the consideration of biotechnology derived products, the USDA-APHIS requires a number of studies that assess environmental impact. This Risk Assessment documentation is pivotal to the successful licensure of any biological agent (live or killed) that is derived from a genetically modified organism (GMO).
Further, if a novel adjuvant is to be used in formulation of a product for use in food animals, then prior approval must be sought by the USDA-FSIS. In many instances, the FSIS will defer to the FDA for approval of any active to be used in formulation, or require documentation that either there is no residue or that the additive poses no threat to individuals that consume the products. The USDA-APHIS, FSIS and FDA may work together in the consideration of products for licensure (Figure 1).
Licensing of a Biological (Figure 2) starts with the production of a seed material (i.e., a seed virus, cell line to grow the virus or bacterium). The seed undergoes rigorous testing to ensure purity and safety. In the case of a recombinant, the seed is also subjected to additional testing that includes sequencing information, genetic stability, and ability to infect different host species and environmental impact. Efficacy testing may be carried out on material that is made according to a filed outline of production, and in the case of polyvalent vaccines, this will also include interference with other vaccine components. Once these data are available, they are sent to the USDA-APHIS for consideration. Once the efficacy test data has been reviewed and accepted, the Company then makes prelicensing serials. In doing this, the Company must make three consecutive serials and test them according to 9CFR and the filed outline of production. The CVB/L may then carry out confirmatory testing on any or all of these serials. At this time the Company may also requests permission to use some or all of the prelicensing serials for field safety trials. Figure 2.
Field safety trials must be carried out in at least 3 different geographic locations. In all cases the data on the serials used must be reviewed, together with the protocol and list of investigators. Once the Field trial protocol has been accepted, then the field trial commences. Once completed and analyzed, the data is sent for review. At this time the Company also submits final outlines of production, which will include any potency tests for each serial, the label, insert and carton. Once this package is accepted, the USDA may grant the Company a License. However, the Company still needs to get approval from each state in which it plans to sell the vaccine. Once this final step is complete, the Company may launch the product. The length of time taken from Master Seed to launch may be as long as 5 years, but typically the development stages of a product take approximately 3 to 4 years. Recombinant products may take one to three years longer, depending on the complexity of the genetic modification, the methods used to test same, and the clarity and completeness of the material submitted in the risk analysis.
Biotechnology and Production Animal Medicine
Prior to starting the development of GMOs into products, careful consideration of regulatory agencies and market needs must be made. These may be very different in certain parts of the world, and so for any global product, these considerations must be taken into account. Regulatory restrictions may prohibit the use of live vaccines in production fish (e.g., Norway), so vectored vaccines would not be worth considering unless there is some indication that the regulatory climate will change with 5 to 7 years. However, given the resources that are required to complete a GMO registration, these indications may have to be clear and non-partisan in order for a Company to take the risk.
Killed, recombinant antigens are being used in some niche markets in animal health. Their attraction is that they may, under certain circumstances, be cheaper to produce, and also they may be safer. For instance, the lktA gene product from Pasteurella haemolytica is licensed for use in cattle in Canada, and is undergoing registration in the US. This antigen is both extremely cheap to produce and also is safer than the holotoxin.
Similarly, recombinant live organisms such as the cya crp deletion mutants of Salmonella typhimurium can provide added safety and maybe provide reliable protection where there is no conventional vaccine available. This may especially be the case with zoonotic pathogens, or for the control of diseases for which there is an eradication program, and a marker vaccine is a pre-requisite.
Vectored organisms are, by design meant to provide the farmer with a multivalent vaccine in one, simple, safe and effective product. Because of this, the choice of inserts into the vector cannot be made frivolously. The immune response to both the vector and the insert must be considered and must be compatible, or must produce the desired effect. For instance, if the vector induces a systemic, cellular response and the insert comes from an enteric pathogen that requires a strong local protective antibody response, then the vector may produce an inappropriate immune response against the insert. This has been shown for certain inserts into a Salmonella vector that made the vector more virulent, resulting in severe disease. In both human and animal health there are relatively few safe vectors that are available for foreign gene insertion. The reason for the paucity of choices is that the vectors must have certain qualities their genome must be well understood (if not entirely sequenced) and there must be an in depth knowledge of the infective process. This knowledge must be reduced to the genetic level so that certain regions of the genome may be deleted, or used as insertion sites for making the vector construct. Many vectors are based on herpesvirus, poxvirus or Salmonella based systems all of which induce primarily cellular immune responses.
In production animal medicine the market has taught that the primary concern is for efficacy. Safety cannot be trivialized, but a farmer may give a vaccine that results in side effects (e.g., transient fever, temporary weight loss) rather than risk exposing the whole herd, flock or pond to a potentially devastating disease. The use of heavily attenuated or vectored vaccines must therefore be weighed against any current alternatives. Usually this decision cannot be made in the laboratory, as it requires data that will convince the farmer that the vaccine positively affects productivity.
This underlying philosophy has direct implication in the development of GMOs and vectored vaccines. If there exists safe and efficacious products, will there be the economic return on a vectored product? The Company selling any vectored product may be commanding a premium price due to high R&D costs, alteration of production processes, etc., so will it be able to compete effectively? Further, will the general population accept food treated with a recombinant organism?
Because of these considerations, rather than reinventing the wheel for modified live and killed vaccines, the true value of GMOs and vectored vaccines may be in the emerging disease markets, those markets for which marker vaccines are require d and where environmental considerations may be paramount. Further, GMOs may be able to replace vaccines that have unwanted side effects.
Biotechnology in the North American Catfish Industry
For the North American Catfish Market, a modified live bacterial vaccine has been launched that aids in the control of ESC. The vaccine strain was licensed from the USDA-ARS, and it is safe for administration to 7 day-old fry. Thus, there now exists a conventional vaccine that will, if field data continue to support some laboratory trials, become established in the industry. Whether a second generation (GMO-based) vaccine may provide any benefit is unknown, though previous attempts to license an Edwardsiella ictaluri deletion mutant failed.
From an industrial perspective, the success or failure of biotechnology in the North American Catfish market will depend on the ability of the Companies involved in aquaculture to provide effective solutions for the catfish farmers of the Mississippi Delta and surrounding regions. It is of no consequence that these products use the latest technology they must work, and be safe.
