Vaccines and Their Production

• An animal can be made immune to infection in two general ways—passive immunization and active immunization.

• Passive immunization by administering preformed antibodies made in a normal animal provides immediate protection, but the resulting immunity is short lived.

• Active immunization using vaccines containing live or dead organisms produces slowly developing but long-lasting immunity.

• Live vaccines tend, in general, to stimulate a more effective immune response than vaccines containing killed organisms. However, vaccines containing killed organisms have tended to be somewhat safer.

• Innovative molecular techniques, such as the use of DNA vaccines or reverse vaccinology, may permit the development of vaccines against diseases for which current vaccines are ineffective or not available.

• Adjuvants are substances added to vaccines to enhance their effectiveness.

Vaccination is by far the most efficient and cost-effective method of controlling infectious diseases in humans and animals. The eradication of smallpox and rinderpest from the globe, the elimination of hog cholera and brucellosis from many countries, and the control of diseases such as foot-and-mouth disease, canine distemper, rabies, influenza, and pseudorabies would not have been possible without the use of effective vaccines. Vaccine technology continues to advance rapidly, especially through the use of modern molecular techniques and through our increased understanding of immune mechanisms and ways to optimize immune responses to achieve maximal protection.

Types of Immunization Procedures

There are two basic methods by which any animal may be made immune to an infectious disease (Figure 23-1): passive and active immunization. Passive immunization produces temporary immunity by transferring antibodies from a resistant to a susceptible animal. These passively transferred antibodies give immediate protection, but since they are gradually catabolized, this protection wanes, and the recipient eventually becomes susceptible again.

FIGURE 23-1 A classification of the different types of adaptive immunity and of the methods employed to induce protection.

Active immunization, in contrast, involves administering antigen to an animal so that it responds by mounting an immune response. Reimmunization or exposure to infection in the same animal will result in a secondary immune response and greatly enhanced immunity. The disadvantage of active immunization is that, as with all adaptive immune responses, protection is not conferred immediately. However, once established, immunity is long-lasting and capable of restimulation (Figure 23-2).

FIGURE 23-2 The levels of serum antibody (and hence the degree of protection) conferred by active and passive methods of immunization.

Passive Immunization

Passive immunization requires that antibodies be produced in donor animals by active immunization and that these antibodies be given to susceptible animals to confer immediate protection. Serum containing these antibodies (antisera) may be produced against a wide variety of pathogens. For instance, they can be produced in cattle against anthrax, in dogs against distemper, in cats against panleukopenia, and in humans against measles. They are most effective protecting animals against toxigenic organisms such as Clostridium tetani or Clostridium perfringens, using antisera raised in horses. Antisera made in this way are called immune globulins and are commonly produced in young horses by a series of immunizing injections. The clostridial toxins are proteins that can be denatured and made nontoxic by treatment with formaldehyde. Formaldehyde-treated toxins are called toxoids. Donor horses are initially injected with toxoids, but once antibodies are produced, subsequent injections may contain purified toxin. The responses of the horses are monitored, and once their antibody levels are sufficiently high, they are bled. Bleeding is undertaken at intervals until the antibody level drops, when the animals are again boosted with antigen. Plasma is separated from the horse blood, and the globulin fraction that contains the antibodies is concentrated, titrated, and dispensed.

To standardize the potency of different immune globulins, comparison must be made with an international biological standard. In the case of tetanus immune globulin, this is done by comparing the dose necessary to protect guinea pigs against a fixed amount of tetanus toxin with the dose of the standard preparation of immune globulin required to do the same. The international standard immune globulin for tetanus toxin is a quantity held at the State Serum Institute in Copenhagen. An international unit (IU) of tetanus immune globulin is the specific neutralizing activity contained in 0.03384 mg of the international standard. The U.S. standard unit (AU) is twice the international unit.

Tetanus immune globulin is given to animals to confer immediate protection against tetanus. At least 1500 IU of immune globulin should be given to horses and cattle; at least 500 IU to calves, sheep, goats, and swine; and at least 250 IU to dogs. The exact amount should vary with the amount of tissue damage, the degree of wound contamination, and the time elapsed since injury. Tetanus immune globulin is of little use once the toxin has bound to its target receptor and clinical signs appear.

Although immune globulins give immediate protection, some problems are associated with their use. For instance, when horse tetanus immune globulin is given to a cow or dog, the horse proteins will be perceived as foreign, elicit an immune response, and be rapidly eliminated (Figure 23-3). To reduce antigenicity, immune globulins are usually treated with pepsin to destroy their Fc region and leave intact only the portion of the immunoglobulin molecule required for toxin neutralization—the F(ab)′₂ fragment.

FIGURE 23-3 The fate of passively administered immune globulin when given to a homologous species (horse) or to a heterologous species (dog).

If circulating horse antibody is still present by the time the recipient animal mounts an immune response, the immune complexes formed may cause a type III hypersensitivity reaction called serum sickness (Chapter 30). If repeated doses of horse immune globulin are given to an animal of another species, this may provoke immunoglobulin E (IgE) production and anaphylaxis (Chapter 28). Finally, the presence of high levels of circulating horse antibody may interfere with active immunization against the same antigen. This is a phenomenon similar to that seen in newborn animals passively protected by maternal antibodies. Sometimes passive immunization may have unexpected side effects (Box 23-1).

Box 23-1 Serum Hepatitis of Horses

On rare occasions, horses may develop acute hepatic necrosis 30 to 70 days after vaccination. It has followed administration of horse plasma, equine immune globulin against tetanus, anthrax, strangles, influenza, and equine encephalitis. It has also occurred after active immunization against equine encephalitis and rhinopneumonitis when the vaccines were prepared using fetal equine cells. Certain serum mixtures or a single vaccine batch may be associated with a high incidence of the disease. Its etiology, mechanism of transmission, and pathogenesis are unknown. Occasional cases have been described in untreated horses living with affected animals, suggesting that a virus may transmit the disease. Nevertheless, experimental transmission and serological testing have failed to reveal a causal agent. The disease is severe, with 53% to 88% mortality. Clinical signs include anorexia, icterus, excessive sweating, and neurological abnormalities. Clinical chemistry confirms severe liver damage with high liver enzyme levels, ammonia, and bilirubin.

Monoclonal antibodies are another source of passive protection for animals. These are, however, mainly made by mouse-mouse hybridomas and thus are mouse immunoglobulins. They will therefore be antigenic when given to other species. Nevertheless, mouse monoclonal antibodies against the K99 pilus antigens of Escherichia coli may be given orally to calves to protect them against diarrhea caused by this organism. A mouse monoclonal antibody to lymphoma cells has been successfully used in the treatment of dogs with this tumor.

Active Immunization

Active immunization has several major advantages over passive immunization. These include the prolonged period of protection and the recall and boosting of this protective response by repeated injections of antigen or by exposure to infection. An ideal vaccine for active immunization should therefore give prolonged strong immunity. This immunity should be conferred on both the animal immunized and any fetus carried by it. In obtaining this strong immunity, the vaccine should be free of adverse side effects. (In effect it should stimulate adaptive immunity without triggering the inflammation associated with innate immunity.) The ideal vaccine should be cheap, stable, and adaptable to mass vaccination; ideally, it should stimulate an immune response distinguishable from that due to natural infection so that immunization and eradication may proceed simultaneously.

In addition to the requirements listed previously, effective vaccines must have other critical properties. First, antigen must be delivered efficiently so that antigen-presenting cells can process antigen and release appropriate cytokines. Second, both T and B cells must be stimulated so that they generate large numbers of memory cells. Third, helper and effector T cells must be generated to several epitopes in the vaccine so that individual variations in MHC class II polymorphism and epitope properties are minimized. Finally, the antigen must be able to stimulate memory cells in such a way that protection will last as long as possible.

Living and Killed Vaccines

Unfortunately, two of the prerequisites of an ideal vaccine—high antigenicity and absence of adverse side effects—are often incompatible. Modified live vaccines infect host cells and undergo viral replication. The infected cells then process endogenous antigen. In this way live viruses trigger a response dominated by CD8+ cytotoxic T cells, a Th1 response. This may be hazardous because the vaccine viruses may themselves cause disease or persistent infection (called residual virulence). Killed organisms, in contrast, act as exogenous antigens. They commonly stimulate responses dominated by CD4+ Th2 cells. This may not be the most appropriate response to some organisms, but it may be safer. It also appears that dendritic cells respond in a different fashion to live and killed bacteria. For example, live organisms such as salmonella upregulate more CD40, CD86, IL-6, IL-12, and GM-CSF than do killed organisms.

The practical advantages and disadvantages of vaccines containing living or killed organisms are well demonstrated in the vaccines available against Brucella abortus in cattle. B. abortus is a cause of abortion in cattle, and vaccination has been used historically to control the disease. Brucella infections are best controlled by a T cell–mediated immune response, and a vaccine containing a living avirulent strain of B. abortus is required for the control of this infection. Older live Brucella vaccines, especially strain 19, caused a lifelong immunity in cows and successfully prevented abortion. Unfortunately, strain 19 vaccine also caused systemic reactions: swelling at the injection site, high fever, anorexia, listlessness, and a drop in milk yield. Strain 19 could cause abortion in pregnant cows, orchitis in bulls, and undulant fever in humans. To eradicate brucellosis, serological tests are used to identify infected animals, and strain 19 causes an antibody response that is difficult to distinguish from a natural infection.

Because of the disadvantages associated with the use of strain 19, considerable efforts have been made to find a better alternative. Unfortunately, killed vaccines (strain 45/20) protected cattle for less than 1 year. A live attenuated strain of B. abortus called RB-51 has been used in cattle in the United States. This is a rough mutant that fails to produce the lipopolysaccharide O antigen. As a result, it produces a strong Th1 response, but unlike strain 19, it does not induce false-positive results in the standard diagnostic tests such as card agglutination, complement fixation, or tube agglutination. It is therefore possible to distinguish between vaccinated and infected cattle. RB-51 is less pathogenic for cattle than strain 19, and it is not shed in nasal secretions, saliva, or urine. RB-51 will not cause abortion in pregnant cattle. It will, however, cause disease in accidentally exposed humans, and because of its failure to stimulate antibody production, this may be difficult to diagnose.

The advantages of vaccines such as brucella strain 45/20 that contain killed organisms are that they are safe with respect to residual virulence and are relatively easy to store since the organisms are already dead (Box 23-2). These advantages of killed vaccines correspond to the disadvantages of live vaccines, such as strain 19 or RB-51. That is, some live vaccines may possess residual virulence, not only for the animal for which the vaccine is made but also for other animals. They may revert to a fully virulent type or spread to unvaccinated animals. Thus some vaccine strains of porcine reproductive and respiratory syndrome virus (PRRSV) vaccine may be transmitted to unvaccinated pigs, causing persistent infection and disease. Live vaccines always run the risk of contamination with unwanted organisms; for instance, outbreaks of reticuloendotheliosis in chickens in Japan and Australia have been traced to contaminated Marek’s disease vaccines. A major outbreak of bovine leukosis in Australia resulted from contamination of a batch of babesiosis vaccine containing whole calf blood. Abortion and death have occurred in pregnant bitches that received a parvovirus vaccine contaminated with bluetongue virus. Contaminating mycoplasma may also be present in some vaccines. Scrapie has been spread in mycoplasma vaccines. Finally, vaccines containing living attenuated organisms require care in their preparation, storage, and handling to avoid killing the organisms. Maintaining the cold chain can account for 20% to 80% of the cost of a vaccine in the tropics.

Box 23-2 Relative Merits of Living and Inactivated Vaccines

Living Vaccines	Inactivated Vaccines
Fewer doses required	Stable on storage
Adjuvants unnecessary Less chance of hypersensitivity Induction of interferon Relatively cheapSmaller dose needed Can be given by natural route Stimulate both humoral and cell-mediated response Longer-lasting protection	Unlikely to cause disease through residual virulence
	Do not replicate in recipient
	Unlikely to contain live contaminating organisms
	Will not spread to other animals
	Safe in immunodeficient patients
	Easier to store
	Lower development costs
	No risk of reversion