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Active immunization involves administration of a vaccine containing antigenic molecules (or genes for these molecules) derived from an infectious agent. As a result, the vaccinated animal will mount an acquired immune response and develop prolonged, strong immunity to that agent. When properly used, vaccines are highly effective in controlling infectious diseases. Several criteria determine whether a vaccine can or should be used. First, the actual cause of the disease must be determined. Although this appears self-evident, it has not always been followed in practice. For example, although Mannheimia haemolytica can be isolated consistently from the lungs of cattle with respiratory disease, these bacteria are not the sole cause of this syndrome and vaccines against the primary viral pathogens are required for full protection. In some important viral diseases (eg, equine infectious anemia, feline infectious peritonitis, and Aleutian disease in mink), antibodies may contribute to the disease process, and vaccination may therefore increase disease severity.
An ideal vaccine for active immunization should confer prolonged, strong immunity in the vaccinated animal, as well as a relatively quick onset of immunity. It should not cause adverse effects and should be inexpensive, thermo and genetically stable, and, for production animals, be adaptable to mass vaccination. It should preferably stimulate an immune response distinguishable from that due to natural infection so that vaccination and eradication may proceed simultaneously. Vaccination is not always an innocuous procedure; adverse effects can and do occur. Therefore, all vaccination must be governed by the principle of informed consent. The risks of vaccination must not exceed those caused by the disease itself.
Vaccines may contain either living or killed organisms or purified components from these organisms. Vaccines containing live organisms tend to trigger the best protective responses. Killed organisms or purified antigens may be less immunogenic than living ones. Because they are unable to grow and spread in the host, they are less likely to stimulate the immune system in optimal fashion. Living viruses from vaccines, for example, infect host cells and grow. The infected cells then process this antigen and trigger a response dominated by cytotoxic T cells, a TH1 response (see The Biology of the Immune System: Acquired Immunity). Killed organisms and purified antigens, in contrast, commonly stimulate responses dominated by antibodies, a TH2 response. This type of response may not generate an optimal protective response to some organisms. As a result, vaccines that contain killed organisms or purified antigens usually require the use of adjuvants to ensure optimal effectiveness. These adjuvants may, however, cause local inflammation, and multiple doses or high individual doses of antigen increase the risks of producing hypersensitivity reactions (see Vaccines and Immunotherapy: Adjuvants).
Inactivated vaccines should resemble the living organisms as closely as possible. Chemical inactivation should cause minimal change to the antigens. Compounds used in this way include formaldehyde, ethylene oxide, ethyleneimine, acetylethyleneimine, and β-propiolactone.
Adjuvants
To maximize the effectiveness of vaccines, especially those containing poorly antigenic components or highly purified antigens, it is usual to add adjuvants to the vaccine. Adjuvants enhance response to vaccines and/or balance/shift the TH1/TH2 immune response. They can reduce the amount of antigen injected or the numbers of doses administered, and they may promote longterm immunologic memory. It is believed that adjuvants work through 3 mechanisms.
Depot adjuvants protect antigens from degradation and prolong immune responses via the sustained release of small quantities of antigen over a period of time. Examples of depot-forming adjuvants include aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (alum) as well as calcium phosphate. Theses alum-based adjuvants also promote the local synthesis of uric acid, a very potent stimulator of toll-like receptors.
Other adjuvants consist of particles that effectively deliver antigen to antigen-presenting cells and so enhance antigen presentation. The immune system can trap and process particles such as bacteria or other microorganisms much more efficiently than soluble antigens. As a result, antigens incorporated into phagocytosable particles are much more effective than soluble ones. These adjuvants include emulsions, microparticles, ISCOMs, and liposomes. All are designed to deliver antigen efficiently to antigen-presenting cells.
Immunostimulatory adjuvants consist of molecules that cytokine production and selectivity stimulate helper cell responses. Many of them are complex microbial products that often represent pathogen-associated molecular patterns. As a result, they activate dendritic cells and macrophages through toll-like receptors and stimulate the secretion of keys cytokines such as IL-1 and IL-12. These cytokines in turn promote helper T cell responses and drive and focus the acquired immune responses. Depending on the specific microbial product, they may enhance either TH1 or TH2 responses. Commonly employed microbial immunostimulants include lipopolysaccharides (or their derivatives); killed anaerobic corynebacteria, especially Propionibacterium acnes and Bordetella pertussis; and saponins (triterpene glycosides) derived from the bark of the soapbark tree (Quillaja saponaria). Saponin-based adjuvants may selectively stimulate TH1 activity.
Very effective adjuvants can be constructed by combining particulate or depot adjuvants with an immunostimulatory agent.
Types of Vaccines
Subunit Vaccines
While vaccines containing whole killed organisms are economical to produce, they contain many antigens that do not contribute to protective immunity. They may also contain toxic components. Thus, it is often advantageous to identify, isolate, and purify the critical protective antigens. These may then be used in a vaccine by themselves. Thus, purified tetanus toxin, inactivated by treatment with formalin (tetanus toxoid), is used for active immunization against tetanus. Likewise, the attachment pili of enteropathogenic Escherichia coli can be purified and incorporated into vaccines. The antipilus antibodies protect animals by preventing bacterial attachment to the intestinal wall.
Antigens Generated by Gene Cloning
The cost of physically purifying a specific antigen may be prohibitive. In such cases it may be appropriate to clone the key antigens and isolate their DNA. This DNA may be inserted into a bacterium or yeast, which then expresses these protective antigens. The recombinant organism is propagated, and the antigens encoded by the inserted genes are harvested, purified, and administered as a vaccine. An example of such a vaccine is one directed against the cloned subunit of E coli enterotoxin. These subunits are antigenic and function as effective toxoids. A purified subunit protein vaccine, called OspA, encoded by a gene from Borrelia burgdorferi is effective in protecting dogs against Lyme disease.
It is also possible to clone antigen genes in plants. This has been successfully achieved for viruses such as transmissible gastroenteritis and Newcastle disease. The plants employed include tobacco, potato, and corn. In some cases these plants contain very high concentrations of antigen and vaccination can be achieved by feeding the plants to animals.
Attenuated Vaccines
Vaccines that contain live organisms may have a limited ability to cause disease (residual virulence) in vaccinated animals. There is also a risk of contamination with unwanted organisms in live vaccines. Considerable care must be exercised in their preparation, storage, and handling to avoid temperature extremes that can affect the viability of the organisms.
For safety reasons, the virulence of an organism must be attenuated so that it is able to replicate but is no longer pathogenic. The level of attenuation is critical to vaccine success and may be difficult to achieve. Underattenuation will result in residual virulence and disease (reversion to virulence); overattenuation will result in an ineffective vaccine. Rigorous reversion to virulence studies must be performed to demonstrate stability of the attenuation.
Attenuation has traditionally involved adapting organisms to growth in unusual conditions. Bacteria can be attenuated by culture under abnormal conditions, and viruses can be attenuated by growth in species to which they are not naturally adapted. For example, rinderpest vaccine virus has been adapted to tissue culture to produce a safe vaccine. Vaccine viruses may also be attenuated by growth in alternative media, such as tissue culture or eggs. This has been done for canine distemper, bluetongue, and rabies vaccines. Prolonged tissue culture was, for many years, the most usual method of attentuation.
For some diseases, related organisms normally adapted to another species can impart limited immunity. Examples include measles virus, which can protect dogs against distemper, and bovine viral diarrhea virus, which can protect pigs against classical swine fever.
Under rare circumstances, virulent organisms may be used for vaccination, eg, vaccination against contagious ecthyma (orf) of sheep. Lambs are vaccinated by rubbing dried, infected scab material into scratches made on the inner thigh, which produces local infection with only limited effects on the lambs; they become solidly immune. Because vaccinated animals may spread the disease, however, they must be separated from unvaccinated stock for a few weeks.
Gene-deleted Vaccines
Attenuation by prolonged tissue culture can be considered a primitive form of genetic engineering. The desired result is the development of a strain of organism that cannot cause disease. This may be difficult to achieve, and reversion to virulence is always a risk. Molecular genetic techniques make it possible to modify the genes of an organism so that it becomes irreversibly attenuated. Deliberate deletion of the genes that code for proteins associated with virulence is an increasingly attractive procedure. Gene-deleted vaccines were first used against the pseudorabies herpes-virus in swine. In this case, the thymidine kinase gene was removed from the virus. Herpesvirus requires thymidine kinase to return from latency. Viruses from which this gene has been removed can infect neurons but cannot replicate and cause disease.
Similar genetic manipulation can also be used to restrict the ability of bacteria to grow in vivo. For example, a modified live vaccine is available that contains streptomycin-dependent Mannheimia haemolytica and Pasteurella multocida. These mutants depend on the presence of streptomycin for growth. When used in a vaccine, the absence of streptomycin will eventually result in the death of the bacteria, but not before they have stimulated a protective immune response.
Additionally, it is possible to alter antigen expression so that a virus induces an antibody response distinguishable from that caused by wild strains. This allows for a way of distinguishing infected from vaccinated animals (referred to as DIVA).
Virus-vectored Vaccines
Yet another method of producing a highly effective living vaccine is insertion of the genes that code for protection antigens into an avirulent “vector” organism. These vaccines are created by recombinant technology, wherein genes are deleted from the vector and replaced by genes coding for antigens from the pathogen. The vector is then administered as the vaccine, and the inserted antigens are produced by the vaccinate's own body cells when infected by the vector virus. The vector may be attenuated so that it will not be shed from the vaccinate, or it may be host-restricted so that it will not replicate itself within the tissues of the vaccinate. Virus-vectored vaccines are well suited for preparation of vaccines against organisms that are difficult or dangerous to grow in the laboratory.
The most widely used viral vectors are poxviruses such as fowlpox, canarypox, vaccinia, and herpesvirus. These viruses have a large genome that facilitates insertion of new genes. They also express relatively high levels of the new antigen. In at least some cases, vectored vaccines appear able to induce immunity even when high levels of maternal antibody are present. Effective virus-vectored vaccines have been developed. Canarypox vector-containing genes obtained from canine distemper virus are now used to immunize dogs, and a similar vector containing the gene encoding rabies glycoprotein is effective in protecting dogs and cats against rabies.
An innovative example of a vectored vaccine involves the use of a yellow fever viral chimera to protect against West Nile virus. This technology uses the capsid and nonstructural genes of the attenuated yellow fever vaccine strain 17D to deliver the envelope genes of other flaviviruses such as West Nile virus. The resulting virus is a yellow fever/West Nile virus chimera that is much safer than either of the parent viruses. The margin of safety can be further increased by introducing targeted point mutations into the envelope genes.
Another example is a vaccine directed against Newcastle disease. The vector is fowlpox virus, into which Newcastle disease HA and F genes are incorporated. It has the benefit of conferring immunity against fowlpox as well.
Vectored vaccines are also commercially available for avian influenza, West Nile virus and influenza infection in horses, feline leukemia, and for vaccinating wildlife against rabies. These vaccines are safe, stable, can work in the absence of an adjuvant, and like the gene-deleted vaccines, allow for DIVA. Some are adaptable to mass vaccination. Field data collected on these vaccines indicate strong immunity and limited side effects.
Polynucleotide (DNA) Vaccines
Animals may also be immunized by injection of the DNA that encodes foreign antigens. For example, the DNA coding for a virus antigen can be inserted into a bacterial plasmid, a piece of circular DNA that acts as a vector. When the genetically engineered plasmid is injected, it can be taken up by host cells. The DNA is then transcribed into mRNA and translated into vaccine protein. Transfected host cells thus express the vaccine protein in association with major histocompatibility complex class I molecules. This can lead to the development of not only neutralizing antibodies but also cytotoxic T cells.
This type of DNA vaccine is used successfully to protect horses against West Nile virus infection. This approach has been applied experimentally to produce vaccines against the viruses that cause avian influenza, lymphocytic choriomeningitis, canine and feline rabies, canine parvo-virus, bovine viral diarrhea, feline immunodeficiency virus-related disorders, feline leukemia, pseudorabies, influenza, foot-and-mouth disease, bovine herpesvirus-1 related disease, and Newcastle disease, among others. Although theoretically producing a response similar to that induced by attenuated live vaccines, these nucleic acid vaccines are ideally suited for preparation of vaccines against organisms that are difficult or dangerous to grow in the laboratory. Some DNA vaccines appear to be able to induce immunity even in the presence of very high titers of maternal antibody. Immunization with purified DNA in this way allows presentation of viral antigens in their native form, which are synthesized in the same way as antigens during a viral infection. This is an improvement over the use of recombinant protein vaccines, in which it has proved difficult to create the proteins in the correct conformation.
Notwithstanding their advantages, DNA vaccines have been slow to reach the market as a result of their high development costs.
Administration of Vaccines
Route of Administration
The most common method of administration is SC or IM injection. This approach is excellent for relatively small numbers of animals and for diseases in which systemic immunity is important. In addition, the veterinarian can be sure that the animal received the appropriate dose of vaccine. However, local immunity is sometimes more important than systemic immunity, and in these cases, it is more appropriate to administer the vaccine at the site of microbial invasion. For example, intranasal vaccines are effective in protecting cattle against infectious bovine rhinotracheitis, cats against feline rhinotracheitis and calicivirus infections, and poultry against infectious bronchitis and Newcastle disease. Unfortunately, these techniques require handling each individual animal.
Aerosolization of vaccines enables them to be inhaled by all the animals in a herd, group, or flock—an obvious advantage when the unit is large. This method is commonly used in the poultry industry. Alternatively, a vaccine may be administered in feed or drinking water, eg, vaccination of poultry for Newcastle disease and avian encephalomyelitis. Fish and shrimp may be vaccinated by immersion in a solution of antigen, which is absorbed through their gills. Advances in transdermal, needle-free injections have made additional routes available and have the added benefit of providing a stronger immunity.
Combination Vaccines
Because of the complexity of many disease syndromes or to avoid giving animals multiple injections, it is common to use mixtures of organisms in single vaccines. For example, for bovine respiratory disease complex, combined vaccines are available for bovine respiratory syncytial virus, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, parainfluenza 3 virus, and Mannheimia haemolytica. Combination vaccines that save considerable time and effort are also commonly used in dogs and cats.
When a mixture of different antigens is inoculated simultaneously, they may compete with one another. However, manufacturers have recognized this and modified vaccines accordingly. Vaccines should never be mixed indiscriminately because one component may dominate and interfere with responses to the other components.
Vaccination Schedules
Although it is not possible to devise precise schedules for each vaccine, certain principles are common to all methods of active immunization. Newborn animals are passively protected by maternal antibodies and, in general, cannot be vaccinated until maternal immunity has waned. If stimulation of immunity is deemed necessary at this stage, the mother may be vaccinated during late pregnancy, timing the doses so that peak antibody levels are reached at the time of colostrum formation. Neonatal animals with antibodies are protected against disease caused by that specific pathogen while maternal antibodies are present. However, passive antibody titers decrease exponentially. These maternal antibodies may drop below protective levels while, at the same time, preventing successful immunization. Inactivated vaccines are not very effective in conferring protective immunity in the face of maternal antibodies. Modified live vaccines, however, may induce a protective primary immune response and some immunologic memory. Because the precise time of loss of maternal immunity cannot be predicted, young animals must usually be vaccinated multiple times to ensure successful immunization.
The interval between vaccine doses depends on an animal's immunologic memory. The duration of this memory depends on multiple factors, such as the nature of the antigen, the use of live or dead organisms, adjuvants used, and the route of administration. Some vaccines may induce immunity that persists for an animal's lifetime. Other vaccines may require boosting only once every 2–3 yr. Even killed viral vaccines may protect some animals against disease for many years. Unfortunately, the minimal duration of immunity has rarely been reliably measured. Annual revaccination has been the rule because this approach is administratively simple and has the advantage of ensuring that an animal is regularly seen by a veterinarian. It is likely that this is more than sufficient for most vaccines.
Individual animal and vaccine variability make it difficult to estimate the duration of protective immunity. Within a group of animals, there may be a great difference between the shortest and longest duration of protection. Vaccines may differ significantly in their composition, and although all may induce immunity in the short term, it cannot be assumed that they confer equal longterm immunity. A significant difference likely exists between the minimal level of immunity required to protect most animals and the level of immunity required to ensure protection of all animals.
A veterinarian should always assess the relative risks and benefits to an animal when determining the frequency of revaccination. Owners should be made aware that protection can be maintained reliably only when vaccines are used in accordance with the protocol approved by vaccine licensing authorities. The duration of immunity claimed by a vaccine manufacturer is the minimal duration that is supported by the data available at the time of approval.
It is now common practice to rate vaccines according to their importance. Essential (or core) vaccines should be given to all animals of a species, and veterinarians should ensure that immunity is maintained throughout an animal's life by appropriate revaccination. Optional (or noncore) vaccines protect animals against sporadic, mild, or uncommon diseases and should only be used when circumstances warrant and when the benefits clearly outweigh the risks involved. For example, essential vaccines in dogs in the USA would normally include canine distemper, parvovirus, adenovirus, and rabies. Optional vaccines may include canine coronavirus, parainfluenza, Bordetella, leptospirosis, and Lyme disease.
Prime-boost Strategies
It has long been normal practice to use exactly the same vaccine for boosting an immune response as was employed when first priming an animal. However, there is no reason why different forms of a vaccine should not be used for priming and for boosting. This approach is known as a prime-boost strategy. Under some circumstances this may result in significantly improved vaccine effectiveness. Prime-boosting has been most widely investigated in attempts to improve the effectiveness of DNA vaccines. Combinations usually involve priming with a DNA vaccine and boosting with either a recombinant vaccine or with recombinant protein antigens.
Vaccine Failure
There are many reasons why vaccination may fail. In some cases, the vaccine may not be effective because it contains strains of organisms or antigens that are different from the disease-producing agent. In other cases, the method of manufacture may have destroyed the protective epitopes, or there may simply be insufficient antigen. Such problems are relatively uncommon and can be avoided by using vaccines from reputable manufacturers. An effective vaccine may fail due to unsatisfactory administration or storage. For example, a live vaccine may be inactivated as a result of use of antibiotics in conjunction with a live bacterial vaccine or residual levels of inactivant in conjunction with a killed bacterin, chemical sterilization of syringes, or excessive use of alcohol on the skin. Route of administration may also affect efficacy. When vaccine is administered to poultry or mink by aerosol or in drinking water, the aerosol may not be evenly distributed throughout a building, or some animals may not drink adequate amounts. Also, chlorinated water may inactivate vaccines. If an animal is incubating the disease before vaccination, the vaccine may not be protective; vaccination against an already contracted disease is usually impossible.
The immune response, being a biologic process, never confers absolute protection nor is equal in all individuals of a vaccinated population. Because the response is influenced by many factors, the range in a random population tends to follow a normal distribution: the response will be average in most animals, excellent in a few, and poor in a few. An effective vaccine may not protect those with a poor response; it is difficult to protect 100% of a random population by vaccination. The size of this unresponsive population varies among vaccines, and its significance depends on the nature of the disease. For highly infectious diseases in which herd immunity is poor and infection is rapidly and efficiently transmitted (eg, foot-and-mouth disease), the presence of unprotected animals can permit the spread of disease and disrupt control programs. Problems also can arise if the unprotected animals are individually important, as in the case of companion animals or breeding stock. In contrast, for diseases that are inefficiently spread (eg, rabies), 60–70% protection in a population may be sufficient to effectively block disease transmission within that population and therefore may be satisfactory from a public health perspective.
The most important cause of vaccination failure in young animals is the inability of a vaccine to immunize in the presence of maternal antibodies. Vaccines also can fail when the immune response is suppressed, eg, in heavily parasitized or malnourished animals. (Such animals should not be vaccinated.) Stress, including pregnancy, extremes of cold and heat, and fatigue or malnourishment, may reduce a normal immune response, probably due to increased glucocorticoid production.
Adverse Reactions
Modern, commercially produced, licensed vaccines are very safe. Nevertheless, they are not always innocuous. The more common risks associated with vaccines include residual virulence and toxicity, which may cause injection-site reactions, depression, allergic responses, disease in immunodeficient hosts (modified live vaccines), neurologic complications, and rarely, contamination with other live agents. For example, lesions of mucosal disease may be seen in calves vaccinated against bovine viral diarrhea. Vaccines that contain killed gram-negative organisms may also contain bacterial cell-wall components that stimulate release of interleukin-1 and can cause fever and leukopenia and occasionally abortion. In general, it is prudent to avoid vaccinating pregnant animals unless the risks of not vaccinating are greater. Certain modified live virus bluetongue vaccines have been reported to cause congenital anomalies when given to pregnant ewes. The stress from a vaccination reaction may be sufficient to activate latent infections. For example, activation of equine herpesvirus has been demonstrated after vaccination against African horse sickness. Another adverse reaction is the “sting” that occurs when some vaccines are administered. This can cause problems for the vaccinator if the vaccinated animal objects strenuously. Some vaccines and vaccine mixtures cause mild, transient immunosuppression.
In addition to potential toxicity, vaccines, like any antigen, may provoke hypersensitivity. For example, rapid allergic reactions (type I hypersensitivity) may occur in response to any of the antigens found in vaccines, including those from eggs or tissue-culture cells. All forms of hypersensitivity are more commonly associated with multiple injections of antigen; therefore, they tend to be associated with use of inactivated products. Immune complex (type III) reactions are also potential hazards of vaccination. These may cause an intense local inflammatory reaction or a generalized vascular disturbance such as purpura. An example of a type III reaction is clouding of the cornea in dogs vaccinated against canine adenovirus 1. Delayed (type IV) hypersensitivity reactions, expressed as granuloma formation, may develop at the site of inoculation in response to the use of depot adjuvants. Some chronic inflammatory reactions to adjuvanted feline vaccines may eventually lead to development of a fibrosarcoma at the injection site.
Production of Vaccines
In most countries, government authorities regulate the production of biologics. In general, regulatory authorities license establishments that produce vaccines and inspect those premises to ensure that the facilities and the methods used are satisfactory. All vaccines are checked for safety, purity, potency, and efficacy. Safety tests include confirmation of the identity of the organism used, freedom of the vaccine from contamination with extraneous organisms, and host and non-host safety toxicity tests. Because the living organisms found in vaccines normally die over time, it is necessary to ensure that they will be effective even after storage (stability). Although properly stored vaccines may still be efficacious after the expiration of their designated shelf life, this should never be assumed; expired vaccines should not be used.
Last full review/revision March 2012 by Ian Tizard, BVMS, PhD, DACVM
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