THE MERCK VETERINARY MANUAL
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Active Immunization

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Active immunization involves administration of vaccines containing antigenic molecules (or genes for these molecules) derived from infectious agents. As a result, vaccinated animals mount acquired immune responses and develop prolonged, strong immunity to those agents. 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, Mannheimia haemolytica can be isolated consistently from the lungs of cattle with respiratory disease; however, 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 vaccinated animals, as well as rapid onset of immunity. It should not cause adverse effects and should be inexpensive, thermo- and genetically stable, and, for production animals, adaptable to mass administration. It should preferably stimulate immune responses distinguishable from those 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 antigens from these organisms. Vaccines containing living 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 optimally stimulate the immune system. Living viruses from vaccines, for example, infect host cells and grow. The infected cells then process the viral antigens, triggering a response dominated by cytotoxic T cells, a Th1 response (see Adaptive Immunity). Killed organisms and purified antigens, in contrast, commonly stimulate responses dominated by antibodies, a Th2 response. This antibody response may not generate optimal protection against some organisms. As a result, vaccines that contain killed organisms or purified antigens usually require the use of adjuvants to maximize their effectiveness. Adjuvants may, however, cause local inflammation, and multiple doses or high doses of antigen increase the risks of producing hypersensitivity reactions (see Adjuvants).

Killed vaccines should resemble the living organisms as closely as possible. Chemical inactivation should cause minimal change to their 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, adjuvants are usually added. Adjuvants enhance response to vaccines and/or balance/shift the Th1/Th2 immune response. They can reduce the amount of antigen to be injected or the numbers of doses administered, and they may promote prolonged immunologic memory. It is believed that adjuvants work through three major mechanisms.

Depot adjuvants protect antigens from degradation and prolong immune responses as a result of the sustained release of antigen. Examples of depot-forming adjuvants include aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (alum), as well as calcium phosphate. These alum-based adjuvants also serve as very potent stimulators of toll-like receptors.

A second class of adjuvants consists of particles that effectively deliver antigen to antigen-presenting cells and so enhance antigen presentation. The immune system traps and processes particles such as bacteria or other microorganisms much more efficiently than soluble antigens. As a result, particulate antigens are much more effective than soluble ones. Examples of such adjuvants include emulsions, microparticles, ISCOMs, and liposomes.

Immunostimulatory adjuvants consist of molecules that enhance cytokine production and so selectivity stimulate helper cell responses. Many contain 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 critical cytokines such as IL-1 and IL-12. These cytokines in turn promote helper T cell responses and drive and focus the adaptive immune responses. Depending on the specific microbial product used, they may enhance either Th1 or Th2 responses. Commonly used 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.

Adjuvants can be combined. For example, very effective adjuvants can be constructed by combining particulate or depot adjuvants with an immunostimulatory agent.

Types of 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 may be advantageous to identify, isolate, and purify the critical protective antigens. These can 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.

The cost of physically purifying a specific antigen may be prohibitive. In such cases, it may be appropriate to clone the genes coding for protective antigens. The DNA encoding the desired antigens may be inserted into a bacterium or yeast, which then expresses the protective antigen. 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. The cloned subunits are antigenic and function as effective toxoids. A purified subunit protein vaccine, called OspA, encoded by a gene from Borrelia burgdorferi effectively protects dogs against Lyme disease.

It is possible to clone viral antigen genes in plants. This has been successfully achieved for viruses such as transmissible gastroenteritis and Newcastle disease. The plants used include tobacco, potato, and corn. These plants contain very high concentrations of antigen, and successful vaccination may result from feeding the plants to animals.

Some recombinant vaccines contain viral structural proteins assembled into virus-like particles (VLPs). One or more viral proteins may be present in the VLP, and the particles may be either nonenveloped or enveloped. VLPs present viral antigen in a particulate form, and these antigens more closely resemble those of the infectious virus. VLPs are potent immunogens and may not require adjuvants. Because VLPs contain no viral genetic material, they cannot replicate in the vaccinee. The effectiveness of VLPs for animal vaccination has been shown in many experimental systems, but no veterinary VLP-based vaccines are commercially available at this time.

The use of live organisms in vaccines presents many advantages. Most especially, they are usually more effective than inactivated vaccines in triggering cell-mediated immune responses. Their use, however, also presents certain hazards. Thus, the virulence of a live organism used for vaccination must be attenuated, so that it is able to replicate but is no longer pathogenic. The level of attenuation is critical to vaccine success. 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. Attenuated vaccines should not be used to vaccinate species for which they have neither been tested nor approved. Pathogens attenuated for one animal may be over- or under-attenuated in other animals. Thus, they may either cause disease or fail to provide adequate protection.

Attenuation has historically involved adapting organisms to growth in unusual conditions. Bacteria were attenuated by culture under abnormal conditions, and viruses were attenuated by growth in species to which they are not naturally adapted. 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 may 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.

Considerable care must also be exercised in the preparation, storage, and handling of modified live vaccines to avoid temperature extremes that can reduce the viability of the organisms.

Attenuation of viruses by prolonged tissue culture can be considered a primitive form of genetic engineering. Ideally, this resulted in the development of a strain of virus that was unable to cause disease. This was often difficult to achieve, and reversion to virulence was a constant hazard. Molecular genetic techniques now 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. For example, gene-deleted vaccines were first used against the pseudorabies herpesvirus 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 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 a protective immune response has been stimulated.

Additionally, it is possible to alter the expression of other antigens so that a vaccine will induce an antibody response distinguishable from that caused by wild strains. This allows for a way to distinguish infected from vaccinated animals (referred to as DIVA).

Another method to produce a highly effective living vaccine is to insert the genes that code for protection antigens into an avirulent “vector” organism. These vaccines are created by deleting genes from the vector and replacing them with genes coding for antigens from the pathogen. The recombinant vector is then administered as the vaccine, and the inserted genes express the antigens when body cells are 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 use against organisms that are difficult or dangerous to grow in the laboratory.

The most widely used vaccine 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 recombinant antigen. In at least some cases, vectored vaccines appear able to induce immunity even when high levels of maternal antibody are present. Canarypox-vectored vaccines that incorporate genes obtained from canine distemper virus are now used to immunize dogs, and a similar vector containing the gene encoding rabies glycoprotein effectively protects 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 adverse effects.

Animals may also be immunized by injection of DNA encoding viral antigens. This DNA 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, and mRNAs are translated to produce 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, bovine viral diarrhea, feline immunodeficiency virus–related disorders, feline leukemia, pseudorabies, foot-and-mouth disease, bovine herpesvirus-1 related disease, and Newcastle disease, among others. Because they can produce a response similar to that induced by attenuated live vaccines, these polynucleotide vaccines are ideally suited for use 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, because they are synthesized in the same way as antigens during a viral infection.

Administration of Vaccines

The most common method of vaccine administration is by 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 an animal has 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.

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 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.

Some veterinarians and owners have expressed concern that the use of vaccine mixtures in this way may somehow “overwhelm” the immune system. This concern is unfounded. The immune system has evolved to respond to complex organisms and multiple simultaneous challenges. The simultaneous administration of multiple vaccines to an animal does not present difficulties to the immune system of normal, healthy animals.

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 traditional 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 viral 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 supported by the data available at the time of approval.

It is 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 be used only 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.

It has long been normal practice to use exactly the same vaccine for boosting an immune response as was used 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 uncommon and can be avoided by using vaccines from reputable manufacturers. More commonly, an effective vaccine may fail because of unsatisfactory administration or storage. For example, a live bacterial vaccine may lose potency as a result of use of antibiotics. 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 may also fail when the immune response is severely 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. Recent studies have indicated that vaccines are more likely to cause adverse effects in small dogs than in large. This is because both receive the same quantity of vaccine, and the smaller animals receive a relatively larger “dose.” 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 may 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 long-acting feline vaccines may eventually lead to development of a fibrosarcoma at the injection site in cats.

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 August 2013 by Ian Tizard, BVMS, PhD, DACVM; Susan L. Payne, PhD

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