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. Thus, they are less likely to stimulate the immune system in an optimal fashion. On the other hand, they are often less expensive and may be safer. Living viruses from vaccines, for example, infect host cells and grow briefly. The infected cells then process the viral antigens, triggering a response dominated by cytotoxic T cells, a type 1 response. Killed organisms and purified antigens, in contrast, commonly stimulate responses dominated by antibodies, a type 2 response. This type of 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. 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 beta-propiolactone.
Although vaccines containing whole killed organisms are economical to produce, they contain many components that do not contribute to protective immunity. They may also contain toxic components such as endotoxins. Thus, depending upon costs, it may be advantageous to identify, isolate, and purify the critical protective antigens. These can then be used in a vaccine by themselves. For example, 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 more appropriate to clone the genes coding for the protective antigens into a vector such as a bacterium, yeast, baculovirus, or plant. The DNA encoding the desired antigens may be inserted into its vector, which then expresses the protective antigen. The recombinant vector is grown, 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 antigen, 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 virus and Newcastle disease virus. The plants used include tobacco, potato, and corn. These plants contain very high concentrations of antigen, and protection may be achieved by simply feeding the plants to animals.
Some recombinant structural proteins may be assembled into virus-like particles (VLPs). One or more viral proteins may make up the VLP, and the particles may be either non-enveloped or enveloped. VLPs present viral antigen in a manner that more closely resembles the infectious virus. VLPs are potent immunogens and may not require adjuvants. Because VLPs contain no viral genetic material they cannot replicate in the recipient animal. A similar type of vaccine may be developed through the use of bacterial “ghosts,” bacteria that have been emptied of their contents, especially their DNA.
Animals may also be immunized by injection of DNA encoding viral antigens. This DNA is inserted into a bacterial plasmid, a piece of circular DNA that acts as a vector. When the genetically engineered plasmid is injected, it is taken up by host cells. The DNA is then transcribed, and mRNAs are translated to produce the vaccine protein. The transfected host cells thus express the vaccine protein in association with major histocompatibility complex class I molecules. This results in the development of not only neutralizing antibodies but also cytotoxic T cells.
This type of DNA plasmid vaccine is used to protect horses against West Nile virus infection. This approach has been applied experimentally to produce vaccines against:
lymphocytic choriomeningitis virus
rabies virus in dogs and cats
feline immunodeficiency virus
bovine herpesvirus-1 related disease
Because they can produce a response similar to that induced by attenuated live vaccines, these DNA plasmid vaccines are ideally suited for use against organisms that are difficult to grow in cell culture. Some DNA vaccines are able to induce immunity even in the presence of very high levels of maternal antibody. Immunization with DNA plasmids in this way allows presentation of viral endogenous antigens in their native form.
RNA vaccines also effectively induce the production of endogenous antigens. They are more stable than DNA plasmids and are more efficient because they need only enter the cell cytoplasm rather than the nucleus. RNA vaccines may also be constructed in such a way that they are self-replicating. These are usually derived from alphaviruses such as Venezuelan equine encephalitis virus. They generate large amounts of endogenous antigen when they replicate for a brief time within cells.
The use of live organisms in vaccines presents many advantages. For example, they are usually more effective than inactivated vaccines in triggering cell-mediated immune responses. Their use, however, also presents potential 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. Attenuated vaccines should not be used to vaccinate species for which they have not been tested or approved. Pathogens attenuated for one species may be over- or under-attenuated in others. 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 common method of attenuation. 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.
For some diseases, related organisms normally adapted to another species may impart limited immunity. Examples include vaccines against measles virus, which can protect dogs against distemper, and against bovine viral diarrhea virus, which can protect pigs against classical swine fever.
In rare circumstances, virulent organisms may be used for vaccination. The only current example of this is vaccination against contagious ecthyma (Orf, sore mouth) of sheep. Lambs are vaccinated by rubbing dried, infected scab material into scratches made on the inner thigh, resulting in local infection and solid immunity. 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. Likewise, vaccines such as Brucella strain RB51 and contagious ecthyma are zoonotic and present hazards to the administrator.
Traditional methods of attenuating organisms have been by prolonged tissue culture or culture in eggs. These have relied on random mutations, an unpredictable process. Although few bacterial vaccines have been attenuated in this way (the most obvious examples are Brucella strain 19 and the Sterne strain of anthrax), the bacterial genome is usually too large to generate effectively and irreversibly attenuated mutants. It has proven much easier to attenuate viruses with their relatively small genomes. Many of the currently available viral vaccine strains were attenuated in this way. 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. As an example of underattenuation, an MLV canine adenovirus 1 vaccine led to urine shedding, which sometimes caused corneal edema (blue eye) in naive dogs. This ended when the vaccine was changed to canine adenovirus 2.
Another relatively simple method is to adapt the vaccine virus to grow at a temperature approximately 10 degrees lower than normal body temperature. These cold-attenuated vaccines can be administered intranasally, where they can grow in the cool upper respiratory tract but not in the warmer lower respiratory tract or other organs.
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 Aujeszky disease 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 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 the expression of other antigens so that a vaccine will induce an antibody response distinguishable from that caused by wild strains. This creates a way to distinguish infected from vaccinated animals (referred to as DIVA).
Another way to produce a highly effective living vaccine is to insert the genes that encode protective 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 cells are infected by the vector virus. The vector may be attenuated so that it will not be shed from the vaccinated animal, 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 large DNA viruses such as poxviruses (fowlpox, canarypox), vaccinia virus, adenoviruses, and some 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 incorporating genes from canine distemper virus are now used to immunize dogs, and a similar vaccinia vector containing the gene encoding rabies glycoprotein is effective in protecting dogs and cats against rabies virus. Fowlpox virus and herpesvirus recombinant vaccines are widely used in the poultry industry. For example, one vector is fowlpox virus, into which Newcastle disease virus HA and F genes are incorporated. It has the benefit of conferring immunity against fowlpox virus as well.
An innovative example of a vectored vaccine involves the use of a yellow fever viral chimera to protect horses 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.
Vectored vaccines are commercially available for:
These vaccines are safe, stable, can work in the absence of an adjuvant, and like the gene-deleted vaccines, allow for differentiation from natural infections. Some are adaptable to mass vaccination such as in ovo vaccination of chickens.
Also see pet health content regarding vaccines and immunotherapy in animals.