Production, assessment, and regulation of vaccines

Production, assessment, and regulation of vaccines

Given their critical importance in controlling infectious diseases, it is essential that there be a reliable, consistent supply of safe and effective vaccines available to veterinarians and animal producers. Veterinary vaccines constitute about a quarter of the global market for animal health products. Infectious diseases are highly diverse, and the significance and benefits of vaccination obviously depend on the disease. The potential economic returns on animal vaccines are generally much less than for human vaccines with lower prices and smaller markets, so there must be a lower investment in research. On the other hand, there are less stringent regulatory requirements and the cost of preclinical studies is much less. It usually takes a shorter time to take an animal vaccine to market and thus to deliver a return on investment. Additionally, veterinarians can determine the effectiveness of a vaccine in the appropriate target species rather than extrapolate from results in rodents.

Vaccine categories

The term vaccine encompasses all products designed to induce active immunity in animals against disease. Both live and inactivated vaccines may also contain adjuvants, stabilizers, preservatives, and diluents. Products generated by recombinant DNA technology do not differ fundamentally from conventional products. The use of modern molecular and genetic techniques has generated new and improved vaccines. For administrative and regulatory purposes, these vaccines are conveniently divided into four categories.

Category I: Antigens generated by gene cloning

Gene cloning can be used to produce large quantities of pure antigen in culture. In this process, DNA coding for an antigen of interest is first isolated from the pathogen. This DNA is then inserted into a plasmid and then into a bacterium or yeast in such a way that it is functional and the recombinant antigen is expressed in large amounts. Category I vaccines are not viable and present no unusual safety concerns.

Category II: Genetically attenuated organisms

It is possible to alter the genes of an organism so that it becomes irreversibly attenuated. These are live organisms modified by adding or deleting a gene. The added genes can encode marker antigens. The deleted genes reduce virulence. It is critical that these genetic modifications do not increase the virulence, pathogenicity, or survivability of the agent.

Category III: Live recombinant organisms

Genes coding for protein antigens can be cloned directly into a variety of organisms, especially DNA viruses. These live viral vectors may carry one or more foreign genes. The recombinant organism itself may then be used as a vaccine. Safety issues need to be determined for each vaccine.

Category IV: Polynucleotide vaccines

DNA or RNA encoding protective antigens may be used as vaccines. In the case of DNA, it is inserted into a bacterial plasmid that acts as a vector. The vaccine antigen gene is placed under the control of a strong mammalian promoter sequence. When the engineered DNA-plasmid is injected intramuscularly into an animal, it is taken up by host cells. It is usual to incorporate an antibiotic resistance gene into these plasmids to serve as a marker.

Vaccine production

Vaccine manufacturing is a challenging task. The most basic steps required to produce a safe, effective, and consistent vaccine can be difficult to execute. There is a high cost to establishing complex processes. The variability in the starting materials, the infectious agent itself, environmental conditions, the manufacturer’s expertise, and also the basic production steps all add to the complexity. Many of the assays required to validate each step also have high inherent variability. Inability to manage these risks can result in vaccine failure and costly recalls.

The production of both live attenuated and inactivated vaccines requires the generation of large quantities of pathogens, both viruses and bacteria. This results in a significant time elapsing between the start of production and vaccine delivery. It requires specialized facilities that if they fail, may expose both the operators and the environment to unacceptable risks.

Regulatory authorities in the United States issue licenses for general sale and distribution of biologics manufactured domestically. Biologics manufactured outside the United States that meet all regulatory requirements are issued permits for general sale and distribution. Biologics have to meet minimum standards of potency, safety, purity, and efficacy in accordance with their label claims.

Regulatory authorities license not only the specific biological product, but also the processes by which that biologic is produced, tested, and released. Minor changes in the production process may significantly alter the final product. Clinical trials may be required to validate any new processes. These risks make vaccine manufacture more challenging than many pharmaceuticals. Additionally, as emphasized throughout this book, the commercial realities of vaccine usage, especially in the food animal industries place severe pressures on profit margins. This is one reason why the number of major animal vaccine manufacturers remains relatively small—it is not a job for amateurs.

Conditional licenses are used to meet emergency situations or other special circumstances. Products released conditionally need only a “reasonable expectation” of efficacy, but they must meet the same safety and purity standards as other vaccines. These licenses are usually issued for one or two years, but may be renewed.

Key steps

The production of approved, licensed, or permitted vaccines requires strict adherence to key steps in vaccine production that can be collectively classified as quality assurance (Fig. 11.1). In producing vaccines, the quality of the starting materials needs to be assured by rigorous testing. These are especially complex for antiviral vaccines. Thus a first step is characterization of the vaccine virus. A master seed virus (MSV) is prepared from a suitable strain of the virus. The identity of this virus must be confirmed, its history known, and its quality assessed by determining its purity and freedom from extraneous agents. (A major threat is the risk of transmission of spongiform encephalopathies). Not only must the master seed be tested, but also the cells in which it is grown. Known as the master cell stock (MCS), this must also be completely characterized. The cell line may also be tested for its ability to induce tumors. Thus it must be shown that both the MSV and MCS are free from mycoplasma, bacteria, fungi, cytopathic or hemadsorbing viruses, and other extraneous agents.

Only approved MSV and MCS can be used when making vaccines. In-process control tests must then be established to verify the consistency of the final product. It is usual to release the final product in batches, referred to as serials, which are then tested to confirm the reproducibility of the quality of the finished vaccine. Required quality control tests include ensuring that killed products, are nonviable by demonstrating their inability to grow in tissue culture. Safety must be tested by inoculating the reconstituted vaccine into animals. It is usual to use a greatly increased dose, usually tenfold, to confirm this. No abnormal or local reactions should occur. The potency of each batch must be demonstrated by comparing the results with previous potency tests. Efficacy must be assessed by challenge studies that are standardized for some pathogens or must be customized for other agents. Likewise, when producing live viral vaccines, the virus content should be measured and found to be within appropriate limits.

If there is no preservative in the vaccine, the manufacturer must demonstrate that it remains effective for a suitable period of time after opening the vial. If it contains preservatives, the vaccine stability and sterility in multidose containers must be demonstrated. It must also be shown that the vaccine will retain its stability throughout the designated shelf life. Many finished products require cold (4°C) or even ultra-cold (liquid nitrogen) storage to maintain stability and potency. Stability may be tested over several years. In general, vaccines in liquid and lyophilized forms are deemed to have shelf-lives of one and two years respectively. They are formulated to ensure that there will always be a minimal immunizing dose remaining by the expiration date.

All possible hazards must be indicated. This is especially important if the agent poses a risk to human health. The manufacturer must also indicate all the conditions for the correct use of the vaccine such as species, age, and route of administration. Safety requirements are absolutely critical, so any local and general reactions in response to the vaccine must be carefully examined. If the vaccine contains a live organism then the exact safety properties of the strain must be documented. These will include testing the vaccine by the recommended route in live animals known to be susceptible to natural infections. Reversion to virulence following serial passage has to be examined. Usually a minimum of four passages is required to show lack of reversion. Living vaccine strains should be tested for their ability to pass from vaccinated to naïve animals through secretions and excretion. This may have to be repeated several times to provide assurance of its inability to spread.

In the case of inactivated/killed vaccines, safety is also critical. In addition to inactivation assurance, animals need to be vaccinated by the recommended route and any local and systemic reactions assessed. These local reactions should be assessed by dissection at slaughter in food animals to ensure that they have not degraded carcass quality. Local tissue reactions help to dictate the withdrawal period before slaughter and are largely dependent on the reactivity of the adjuvant.

A vaccine must be able to do what the manufacturer claims. Efficacy and potency requirements include assessment of induced immunity. The precise nature of these assays will depend on both the animal species and the nature of the claims made for the vaccine. Any claims regarding duration of immunity must also be based on the results of challenge trials and also possible serologic assays. As discussed previously surrogates of protection have not been established for many infectious diseases.

Although it is important to test vaccines in the field before they can be licensed, it is almost impossible to assess vaccine efficacy in field trials because disease outbreaks cannot be predicted, and it is very difficult to have available unvaccinated control animals held under the same conditions. Field trials are much more useful for assessing safety under field conditions.

The release of genetically modified live organisms (categories II and III) for field-testing or distribution requires additional assurances that there will be no risk to animals, humans, or the environment. Thus before release a careful risk assessment needs to be performed. This should analyze such issues as the characteristics of the vaccine organism; human health risks; animal health risks for target and nontarget species; and environmental persistence including the possibility of increased virulence. If any environmental effect is anticipated then an environmental impact statement must be prepared.

Vaccine excipients

To improve the antigenicity of a vaccine it is often necessary to add adjuvants (Chapter 7). It may also be necessary to enhance the stability of the vaccine by adding excipients. These may include, binders, preservatives, buffering agents, emulsifiers, wetting agents, nonviral vectors, and transfection facilitating compounds. In general, no preservative is used in single dose vials or syringes. Preservatives are, however, required in multidose vials of vaccines. Because of perceived (but unsubstantiated) health risks the organic mercury compound thiomerosal (Merthiolate) is no longer widely used as a preservative in human vaccines, but it remains in use in some veterinary vaccines. An alternative vaccine preservative is 2-phenoxyethanol (2-PE). It is both stable and effective.

Some vaccines are unstable in aqueous solutions, especially if they cannot be held in uninterrupted cold storage. Vaccines containing live viruses are susceptible to heat inactivation, but are much more resistant when lyophilized. As a result, many vaccines are stored as a lyophilized powder. They store well but should be kept cool and away from light and should only be reconstituted with the diluent provided by the manufacturer.

However, lyophilization may also alter the product pH and solute concentration and may denature the vaccine antigens. Thus stabilizers must be added to the vaccine. These can include sugars and sugar alcohols, which encase the proteins and stabilize them. Other polymers and amino acids may also be added to improve stability. Remember, however, that intense sunlight and heat can destroy even lyophilized vaccines.

Because the living organisms or antigens found in vaccines normally die or degrade over time, it is necessary to ensure that they will be effective even after storage. Therefore it is usual to use an antigen in excess of the protective dose under laboratory conditions, and its potency is tested both before and after accelerated aging. Vaccines that contain killed organisms, although more stable than living ones, contain an excess of antigens for the same reason. Vaccines approved for licensing on the basis of challenge exposure studies must usually show evidence of protection in 80% of vaccinated animals, whereas at least 80% of the unvaccinated controls must develop evidence of disease after challenge exposure (the 80:80 efficacy guideline). The route and dose of administration indicated on the vaccine label should be scrupulously heeded because these were probably the only routes and doses tested for safety and efficacy during the licensing process. Vaccines usually have a designated shelf-life, and although properly stored vaccines may still be potent after the expiration of this shelf-life, this should never be assumed, and all expired vaccines should be discarded. Adverse reactions should always be reported to the appropriate licensing authorities in addition to the vaccine manufacturer (Chapter 10). Because modified live virus vaccines carry the risk of residual virulence and of contamination, some countries will not approve their use.

Inactivated vaccines are commonly available in liquid form and usually contain suspended or emulsified adjuvant. These should not be frozen, and they should be shaken well before use. The presence of preservatives will not control massive bacterial contamination, and multidose containers should be discarded after partial use.

Cost/benefits of vaccination

Economics is a key consideration when deciding whether to vaccinate livestock. The agricultural sector is price sensitive so that vaccines need to be produced at low cost in large quantities. When considering vaccination costs, economic benefits must also be considered. Control of disease by vaccination will result in savings on veterinary care, reduced animal husbandry labor, improved animal health, and survival reflected in faster growth and improved feed conversion, protection of unvaccinated animals through herd immunity, and reduction in uncertainty about future outcomes. Costs will include the cost of purchasing, storing, and administering vaccines.

Vaccination against zoonotic diseases has both direct and indirect benefits. Indirect benefits include increased productive time and income, and also reduced pain and suffering. Direct benefits include the reduced costs for medical services. For example, rabies is an expensive disease. If an unvaccinated animal bites a human, costs include postexposure vaccination of the victim in addition to quarantine or euthanasia of the biting animal. The brain of the animal must be examined for the presence of the virus. These costs, of course, do not account for the stress and worry associated with this disease. In Texas, aerial vaccination by dropping vaccinia-vectored rabies vaccine enclosed in food bait has been employed to vaccinate coyotes against rabies. The costs of this were the total expenditures of the program—vaccine, food, planes, fuel, and so forth. The benefits were the savings associated with human postexposure prophylaxis, and animal rabies tests within the affected area. The calculated cost of the rabies vaccination program is about US$26 million. The benefits were estimated at between US$89 million and US$346 million. Depending on the frequency of postexposure prophylaxis and animal testing, the cost/benefit ratio therefore ranged from 3.38 to 33.13.

Many other analyses have been conducted into the benefits and costs of animal vaccines. For example, a study on vaccination against paratuberculosis in dairy cattle in North America indicated that vaccination cost US$15/cow, and the returns minus costs were US$142 per cow.

The consolidation of large livestock enterprises has resulted in fewer and bigger farms. Animal health decisions may now involve thousands of animals. The cost of an incorrect decision is so large that guessing is not an option. In general, the cost of a vaccine and its administration are relatively easy to calculate. For example, poultry vaccines sell for US$0.01 to US$0.05 per dose. Cattle vaccines cost about 20 to 30 times this, and human vaccines sell for 100 to 500 times the cost of a poultry vaccine. The benefits of vaccination are more difficult to calculate. The prime benefit expected will be improved productivity, but reduced treatment costs are also important. Measures of productivity would typically include, mortality, average daily gain and feed conversion (Feed conversion is critical, given that feed is by far the largest cost in intensive livestock systems.) Once productivity is determined this needs to be translated into revenues, costs, and profits. A benefit/cost ratio of greater than 1 indicates that the benefits exceed the costs.

Combined/polyvalent vaccines

The development of effective combined (many different organisms) and polyvalent (many strains of the same organism) vaccines also presents manufacturing issues. Usually bulk lots of monovalent vaccines are produced and then combined into a single product. Alternatively, the individual components may be stored separately and combined just before administration. In each case both the monovalent lots and the final combined product have to undergo quality testing. Thus these vaccines require an increased investment in resources and facilities. Manufacturers must demonstrate that there are no differences in the physical, chemical, and immunological properties for the individual lots and the combined product. Likewise, vaccines that are polyvalent and combine different strains must be balanced to maximize infectivity and immunogenicity. Antigens, adjuvants, and preservatives must be compatible. For example, preservatives or buffers may alter vaccine potency.

Vaccine presentation

Vaccine labels

Proper labeling will allow a lay person to be able to understand the correct need, administration, revaccination, and precautions that must be taken to use the product effectively and safely.

US Department of Agriculture (USDA)–approved label claims for veterinary vaccines are now based on the statistical significance of their efficacy data and their clinical relevance. Efficacy indication statements now simply state, “This product has been shown to be effective for the vaccination of a specific species of healthy animal of a specific age against a specific disease.” This is accompanied by a standardized summary of efficacy and safety data. There is also a cautionary statement telling the public to consult a licensed veterinarian for interpretation of the data. These labels do not automatically carry a default recommendation for annual revaccination. Revaccination statements are based on available data. If this is unavailable the label will say so. There is also a required statement on labels referring the end-user to a website where basic, yet relevant, data regarding efficacy and safety is available. There is a single claim that carries the implication that if the product is licensed, it is efficacious and meets USDA requirements.

Vaccine regulation

The production of veterinary vaccines is regulated by national agencies. The Animal and Plant Health Inspection Service (APHIS), Center for Veterinary Biologics (CVB) of the USDA in the United States; by the Canadian Centre for Veterinary Biologics of the Canadian Food Inspection Agency; and by the Veterinary Medicines Directorate in the United Kingdom; in addition to appropriate government agencies in other countries. In Europe, the European Medicines Agency grants licenses for human and animal use of GMOs, in collaboration with national authorities. In general, regulatory authorities have the right to license establishments where vaccines are produced and to inspect these premises to ensure that the facilities are appropriate and that the methods employed are satisfactory. All vaccines must be checked for purity, efficacy, safety, and potency.

APHIS has the responsibility for regulating vaccine production in the United States (Box 11.1). Vaccine licensure is managed by the CVB within this agency, located in Ames, Iowa. The CVB ensures that veterinary biologics (products used to diagnose, treat, and cure disease) are free from disease producing agents, especially foreign animal diseases. It develops the appropriate procedures and standards for products to be released, and the biologics receive either a license (domestically produced) or a permit (foreign manufacture) for general sale and distribution. It monitors and inspects both products and facilities and regulates field tests and the release of veterinary biologics. In essence the CVB ensures that vaccines sold to the public are pure, safe, potent, and effective. It also plays a role in the international harmonization of product regulations.

BOX 11.1 ■

The Virus-Serum-Toxin Act

In the United States, the Virus-Serum-Toxin (VST) Act of 1913 (amended in 1985) was designed to protect farmers and ranchers by regulating vaccine production and assuring safety and efficacy. It originated as a result of issues with the production of anti-hog cholera serum. It made the Secretary of Agriculture responsible for licensing and regulating the manufacture, import, and export of veterinary biologicals. In practice, this is managed and enforced by the Animal and Plant Health Inspection Service. Border and import inspection is a function of the Department of Homeland Security.

The VST Act makes it unlawful to:

Jan 21, 2021 | Posted by in GENERAL | Comments Off on Production, assessment, and regulation of vaccines

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