Soon after Louis Pasteur identified the basic principles of vaccinology, Salmon and Smith demonstrated that killed Salmonella bacteria could serve as an effective vaccine. This simple empirical approach was rapidly adopted, and many diverse killed bacterial vaccines were rapidly developed. For example, killed typhoid vaccines were administered to British soldiers by the end of the nineteenth century. They worked but were somewhat toxic and caused significant morbidity. Today whole-cell killed Bordetella pertussis vaccine is still given to children in many countries to protect against whooping cough because it is cheaper than the less toxic acellular vaccine. This approach to developing vaccines can be summarized as “isolate, inactivate, and inject.” This has worked well and is especially effective in the production of autogenous vaccines (Chapter 18). The great advantages of this type of vaccine are its safety and simplicity—and thus reduced cost. Technically speaking the term “killed vaccines” applies to the death of living organisms such as bacteria. Likewise, the term “inactivated vaccines” applies to the treatment of molecular constructs such as viruses. This type of vaccine may contain either a very complex antigenic mixture such as that found in killed whole organisms, or the vaccine components may be purified to various degrees. Unnecessary or toxic components may be removed, but the vaccine may be enriched with those antigens that are directly responsible for the protective immune response. The degree of purification demanded will depend upon the costs of the purification process and any adverse effects mediated by the unwanted/unnecessary components. The major problem with these nonliving vaccines is that they cannot invade host cells. As a result, they act as exogenous antigens and stimulate type 2 immune responses and antibody formation rather than type 1 cell mediated responses. Therefore they may provide only weak or transient immunity against viruses or intracellular bacteria. In addition, killed vaccines, depending upon their purity and toxicity, may require administration of multiple doses to generate strong prolonged immunity. Each vaccine dose increases the chances of the recipient developing allergic or other adverse reactions. In many cases these killed/inactivated/subunit vaccines also require adjuvants that carry with them the potential to induce innate immune responses resulting in other problems. Until the 1990s, most veterinary vaccines either contained inactivated/killed organisms that were adjuvanted with alum or oil, or they were modified live vaccines (Table 3.1). Increased knowledge of microbial genetics has revolutionized the way in which vaccine antigens can be generated. These vaccines may now contain cultures of organisms that have been killed by chemical or physical means; inactivated toxins (toxoids); or highly purified subunits, the antigenic components of microorganisms that have been purified from cultures by chemical or physical processes, or have been produced by recombinant DNA techniques (Fig. 3.1). TABLE 3.1 ■ CMI, Cell-mediated immunity. Organisms killed for use in vaccines must remain as structurally similar to the living organisms as possible. Therefore crude methods of killing that cause major changes in antigen structure as a result of protein denaturation are usually unsatisfactory. Chemical inactivation must not alter the structure of the antigens responsible for stimulating protective immunity. One such chemical is formaldehyde, which cross-links multiple lysine residues on the toxin to form highly stable methylene bridges. These confer structural rigidity on these proteins and neutralize their toxicity. Proteins may also be mildly denatured by acetone or alcohol treatment. Alkylating agents that cross-link nucleic acid chains are also suitable for killing organisms because by leaving the surface proteins of organisms unchanged, they do not affect their antigenicity. Examples of alkylating agents include ethylene oxide, ethyleneimine, acetyl ethyleneimine, and β-propiolactone, all of which are used in veterinary vaccines (Fig. 3.2). Vaccines containing whole, killed bacteria are called bacterins. The bacteria can be grown in large volumes in bioreactors. It is usual to then kill the bacteria with formaldehyde and add aluminum or oil-emulsion adjuvants. The formaldehyde can be neutralized with sodium thiosulfate or sodium bisulfite. The antibody responses to these vaccines are directed against many different bacterial antigens, both essential and unimportant. The immunity produced by these simple bacterins is relatively short-lived, usually lasting no longer than one year and sometimes considerably less. For instance, a formalized swine erysipelas (Erysipelothrix rhusiopathiae) bacterin protects pigs for only four to five months. Streptococcus equi bacterins protect horses for less than one year, even though recovery from a natural case of strangles may confer a lifelong immunity. One important issue, especially when using coliform and Campylobacter bacterins, is that of strain specificity. Multiple strains of each organism occur in nature, and successful vaccination requires immunization with appropriately matched strains. This is sometimes not possible if a commercial vaccine is employed. One method of overcoming this difficulty is to use autogenous vaccines. These are vaccines that contain organisms obtained either from animals on the farm where the disease problem is occurring, or from the infected animal itself. These can be very successful if carefully prepared because the vaccine will contain all the antigens required for protection in that specific location. As an alternative to the use of autogenous vaccines, some manufacturers produce polyvalent vaccines containing a mixture of antigenic types. For example, leptospirosis vaccines commonly contain up to five different serovars. This practice, although effective, is inefficient because only a few of the antigenic components employed may be required in any given location. Although simple to make and generally inexpensive, bacterins are not always effective. They also may contain unwanted toxic components such as endotoxins. For this reason, many companies purify bacterins at additional cost. Remember too that not all the antigens in a bacterium can trigger a protective immune response. Some antigens, such as those found on the bacterial surface, are of much greater importance than internal proteins. Thus to the extent afforded by economics, efficacy, and safety, inactivated vaccines may be purified. Killed bacterial vaccines are usually stable in storage so that maintenance of the cold chain is not a major issue. Once the antigens are purified, then the vaccine can be formulated by adding adjuvants to enhance its immunogenicity and stabilizers to increase its storage life. If it is to be used in multidose vials, then preservatives must also be added. Many bacterial pathogens cause disease by producing potent exotoxins. The most important are toxins from the Clostridia: Clostridium tetani, Clostridium perfringens, and Clostridium botulinum. The immunoprophylaxis of tetanus is restricted to toxin neutralization. Cl. tetani produces a toxin called tetanospasmin. This acts on presynaptic motor nerve cells to cause excessive motor neuron activity leading to extreme muscle spasms, respiratory paralysis, and death. Growing Cl. tetani in a semisynthetic medium produces large quantities of toxin. The toxin is released into the supernatant and can be treated with formaldehyde to fix the protein and block the conformational changes that make it toxic. It is then called tetanus toxoid. Next, the supernatant is ultrafiltered to get rid of unwanted proteins. Toxoids are available for most clostridial diseases and for infections caused by Mannheimia haemolytica. A toxoid is also available for vaccination against bites from Crotalus atrox, the Western diamondback rattlesnake. Tetanus toxoid with an aluminum hydroxide adjuvant is given by intramuscular injection for routine prophylaxis, and a single injection of this vaccine will induce protective immunity in 10 to 14 days. The antibodies directed against the toxin have a higher affinity for the toxin than does the toxin receptor. Thus in infected wounds the antibodies bind the toxin as it is produced. The toxin molecules cannot therefore bind their receptors on neurons and are effectively neutralized. Antitoxins to the other clostridial toxins such as Cl. botulinum or Cl. perfringens act in a similar matter. Toxoids are safe and cannot revert to virulence. They cannot spread to other animals. They are very stable and resistant to damage by temperature and light. They generally induce good humoral immunity but not cell-mediated immunity. As with other nonliving vaccines, toxoids are not always highly immunogenic. As a result, multiple doses may be needed to assure immunity. Additionally, an adjuvant, usually an aluminum salt, must be incorporated into the vaccine. Local reactions such as redness, swelling, and induration at the injection site may develop within a few hours. These usually resolve within 72 hours. Conventional immunological wisdom would suggest that the use of antibodies against tetanus toxin (tetanus immune globulin) should interfere with the immune response to toxoid and must therefore be avoided. This is not a problem in practice however, and both may be successfully administered simultaneously (at different sites) without problems. This may be because of the relatively small amount of immune globulin usually needed to protect animals. Some veterinary vaccines combine both toxoids and killed bacteria in a single dose by the simple expedient of adding formaldehyde to a whole culture. These products, sometimes called anacultures or bacterin-toxoids, are used to vaccinate against Clostridium haemolyticum and Cl. perfringens. Trypsinization of the mixture may make it more immunogenic. Other bacterin-toxoids may have improved efficacy by adding other purified immunogenic antigens. For example, Escherichia coli bacterins against enteric colibacillosis in calves and pigs may be made more effective by the addition of additional fimbrial adhesion proteins such as F4 (K88), F5 (K99), F6, F7, and F18. Antibodies to these antigens prevent Escherichia coli binding to the intestinal wall and thus contribute significantly to protection. Similarly, Mannheimia bacterins enriched with the white cell-killing leukotoxoid are more effective than conventional bacterins in preventing bovine respiratory disease. One problem with the use of whole killed organisms in vaccines is that they contain many components that are either nonantigenic or induce nonprotective responses, or, most importantly, may be toxic or allergenic. Thus in the interests of safety and efficacy it is often desirable to isolate and purify individual microbial antigens or subunits. These subunit vaccines may be isolated by classical physicochemical fractionation techniques or by gene cloning. Subunit vaccines only contain a part of the target organism and as a result vaccinated animals only respond to those subunits. By definition they are noninfectious and nonreplicating. Their great advantages include not only increased safety and reduced toxicity, but also the potential to use antibody assays to distinguish vaccinated from infected animals. An example of the importance of subunit vaccines is seen when vaccinating against either Bordetella pertussis in children or Bordetella bronchiseptica in dogs. Originally these infections were controlled by the use of whole heat-killed bacterial vaccines. Unfortunately they had significant toxicity and induced inflammation and “soreness” in vaccinated children. Because of these problems, there has been a transition to the use of “acellular” vaccines. These contain a mixture of purified bacterial subcomponents. There are several different vaccines of this type because there is no consensus on the ideal composition of an acellular vaccine. They differ in the number of subunits included such as pertussis toxin, filamentous hemagglutinin, outer membrane proteins, or fimbrial antigens. These acellular vaccines also contain much less endotoxin and are thus considerably less toxic than whole cell vaccines. Although widely accepted, they are somewhat less effective with a shorter duration of immunity besides being more expensive. Another approach to the development of subunit vaccines against gram-negative bacteria is the use of common core antigens. The outer layer of the gram-negative bacterial cell wall consists of lipopolysaccharide. This lipopolysaccharide is formed by a variable oligosaccharide (O antigen) bound to a highly conserved core polysaccharide and to lipid A. The O antigen varies greatly among gram-negative bacteria so that an immune response against one O antigen confers no immunity against bacteria expressing other O antigens. In contrast, the underlying core polysaccharide is conserved between gram-negative bacteria of different species and genera. Thus an immune response directed against this common core structure has the potential to protect against many different gram-negative bacteria. Mutant strains of E. coli (J5) and Salmonella enterica Minnesota and Typhimurium (Re) have been used as sources of core antigen. J5 is a rough mutant that is deficient in uridine diphosphate galactose 4-epimerase. As a result, the organism makes an incomplete oligosaccharide side chain, and has lost most of the outer lipopolysaccharide structure. Immunization with a J5 bacterin thus provides protection against multiple gram-negative bacteria such as E. coli, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, and Haemophilus influenzae (type B). A J5 bacterin has also been reported to protect calves against organisms such as S. enterica Typhimurium and E. coli, and pigs against A. pleuropneumoniae. The most encouraging results have been obtained when vaccinating against coliform mastitis (Chapter 16). Many important bacterial antigens consist of capsular polysaccharides or glycoproteins whose immunogenic structures are carbohydrate side-chains. Unlike protein antigens that trigger T cell-dependent B cell responses, bacterial polysaccharides cannot bind to MHC molecules and hence cannot trigger T cell help (They are “T-independent”), and they also cannot trigger the reactions needed for survival, affinity, maturation and extensive B cell proliferation. As a result, B cells can only produce low affinity immunoglobin (Ig)M antibodies against polysaccharides. They do not induce an IgM to IgG switch, they fail to induce a booster response, and they do not induce T cell memory. Thus pure bacterial polysaccharides are very poor vaccine antigens (Fig. 3.3).
Nonliving vaccines
Advantages of Killed Vaccines
Advantages of Modified Live Vaccines
No reversion to virulence or toxicity
Stimulates CMI in addition to antibodies
No mutation or recombination
Cheap
Stable on storage
No need for adjuvants
No residual virulence
Fewer boosters required
Fewer regulatory constraints
Can be used on mucosal surfaces
Bacterial vaccines
Methods of inactivation
Bacterins
Toxoids
Bacterin-toxoids
Subunit vaccines
Core antigens
Glycoconjugate vaccines
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Nonliving vaccines
The Advantages and Disadvantages of Living and Inactivated Vaccines
