When the first vaccines were developed by Louis Pasteur and his colleagues, there was a dispute between the proponents of killed/inactivated vaccines and those that believed that living organisms were required to induce protective immune responses. It did not take long to prove that killed organisms could also induce protective immunity. However, vaccines containing living organisms that can replicate in their host do have some advantages over inactivated products. Moreover, the advantages and disadvantages of each vaccine type tend to complement each other, and each has appropriate and inappropriate uses. Unfortunately, two of the prerequisites of an ideal vaccine, high antigenicity and absence of adverse side effects, are sometimes incompatible. Vaccines containing replicating organisms may trigger innate immune responses as they infect host cells and tissues. As a result, vaccinated animals may develop a very mild, short lasting “sickness” such as fever, inappetence, or depression. Inactivated vaccines are generally less immunogenic than their modified live counterparts. Live vaccines are better able to stimulate cell-mediated immunity against the targeted pathogen because they replicate to a limited extent within the vaccinated animal. The live organisms may enter cells and be processed as endogenous antigens. As a result, they can trigger a type 1 immune response dominated by CD8+ cytotoxic T cells. This may result in a mild infection, but it generates long-lasting protection similar to that in animals that recover from the actual disease. The organisms used in these live vaccines may, however, also have the potential to gain virulence and thus cause disease. Vaccines containing killed organisms, in contrast, act as exogenous antigens. They tend to stimulate type 2 responses dominated by CD4+ T cells and antibodies. This may not generate a very strong protective response to some organisms, but they may provide adequate protection and are often safer. Note that by triggering innate immunity, live viral vaccines can retain their ability to induce interferon production. This can occur within a few hours of administration. As a result they may confer very rapid protection (Fig. 4.1). This may be important if these vaccines are used to stop an epidemic that has already started. As described in Chapter 2, cell-mediated immune responses are primarily directed against abnormal cells such as those infected with viruses. Cytotoxic T cells recognize antigen fragments bound to MHC class I molecules expressed on infected cells. To stimulate these cytotoxic responses, the foreign invader must grow inside the cells. These endogenous antigens are therefore the key to stimulating cell-mediated responses. Killed antigens or their subunits cannot do this and therefore stimulate predominantly type 2, antibody-mediated responses. Only live organisms capable of inducing intracellular protein synthesis can trigger cell-mediated responses. Thus live vaccines have an immunogenic ability lacking in inactivated vaccines. Virulent living organisms cannot normally be used in vaccines otherwise they will cause disease. As described in Chapter 1, some early vaccination attempts included administering infectious material by unusual routes. This was a hazardous procedure and only one such procedure is in use today—vaccination against contagious ecthyma of sheep. Contagious ecthyma (also called soremouth or orf ) is a viral skin disease of lambs that causes massive scab formation around the mouth, prevents feeding, and results in a failure to thrive. The disease has little systemic effect. Lambs recover completely within a few weeks and are then solidly immune. It is usual therefore to vaccinate lambs by rubbing infected scab material into scratches made in the inner aspect of the lamb’s thigh or other hairless area. The development of lesions at this site has no untoward effect on the lambs, and they become solidly immune. Because vaccinated animals may spread the disease, however, they must be separated from unvaccinated animals for a few weeks. The soremouth example notwithstanding, this is not a common procedure. Under normal circumstances microbial virulence must be reduced so that, although still living, the organisms in vaccines may replicate to a limited extent but can no longer cause disease. This process is called attenuation. The level of attenuation is critical to vaccine success. Attenuation must be a key property of the organism and not dependent on fully functional host defenses. It should not lead to the development of a persistent carrier state. However, the attenuated organisms need to be sufficiently invasive and persistent to stimulate a protective immune response. Underattenuation will result in residual virulence and disease; overattenuation may result in an ineffective vaccine. Once organisms are attenuated sufficiently to be used safely, these vaccines are classified as modified live vaccines (MLV). The traditional method of attenuating bacteria or viruses was to grow them for a long period of time in culture. It was hoped that over time, random mutations would accumulate and effectively reduce microbial virulence. This often worked, especially with agents with a small genome such as viruses. It occasionally worked with bacteria as well, but this was uncommon and unpredictable. It is difficult to attenuate bacteria. They have a large genome and, unless the genes that control virulence are specifically targeted, they will not reliably lose virulence. It is exceedingly difficult to attenuate bacteria simply by passaging them many times in vitro. The traditional methods of attenuation were empirical at a time when there was little understanding of the changes induced by the attenuation process. They usually involved adapting organisms to growth in unusual conditions so that as they mutated, they lost their adaptation to their usual host. For example, Albert Calmette and Camille Guérin passaged Mycobacterium bovis in an alkaline medium containing sterile bovine bile 230 times every 21 days from 1908 to 1921. This led to several genetic changes in the organism that resulted in its gradual attenuation for humans. This is the Bacille Calmette-Guérin (BCG) strain of M. bovis. It is widely used as a vaccine against tuberculosis, but its effectiveness varies between countries. It is widely used in Europe but not in North America. (Interestingly, Calmette also developed the first antivenom for snakebite.) The vaccine strain of Bacillus anthracis was developed by Max Sterne in 1935. He grew anthrax in 50% serum agar under an atmosphere rich in CO2 so that it lost its virulence. B. anthracis has virulence factors encoded on two plasmids, pXO1 and pXO2. The Sterne strain has lost its pXO2 plasmid. This encodes the bacterial capsule, a thick layer of polysaccharide that protects the bacteria against phagocytosis. Thus it cannot form a capsule, and as a result is susceptible to phagocytosis and is relatively avirulent. The anthrax vaccine is prepared as a spore suspension. It is administered to livestock in a dose containing up to 10 million spores with a saponin adjuvant, and it has an excellent safety record. A group of brucellosis cultures were maintained by John Buck of the US Department of Agriculture’s Bureau of Animal Industry on his desk at room temperature for “well over a year.” They were then evaluated for immunogenicity and stability. The nineteenth culture was significantly less pathogenic than the others and remained stable while passaged and transmitted. This was licensed as the Brucella vaccine strain 19 in 1941. Brucella abortus strain 19 vaccine formed the basis of Brucella eradication in many countries. Its lack of virulence is the result of a deletion of 702 base pairs that encode the erythritol catabolic genes. As a result, strain 19 is unable to use erythritol, a sugar found in abundance in the bovine placenta. Other examples of attenuated bacterial strains used in vaccines include an attenuated strain of Brucella suis (Strain 2), used in China for the prevention of brucellosis in goats, sheep, cattle, and pigs. Streptomycin was used to derive the attenuated Rev.1 strain from a virulent culture of Brucella melitensis. A rough strain of Salmonella enterica Dublin (strain 51) is used as a vaccine in Europe to protect calves at two to four weeks of age. Immunity to salmonellosis involves macrophage activation and is thus relatively nonspecific. Unfortunately, genetic stability cannot always be guaranteed in these attenuated strains. This is of special concern in bacterial vaccine strains that were generated as a result of random mutagenesis. Multiple passages of the organism in the laboratory on growth media, cell cultures, or in eggs resulted in random mutations of unknown genes. This attenuation may simply be caused by the loss of a single gene that could equally well mutate back again. Back-mutation or genome reassortment, using genes from related bacteria, may occur and thus the attenuated organisms may suddenly regain their virulence. A much more reliable method of making bacteria avirulent is by deliberate genetic manipulation. Gambling with random mutagenesis is no longer acceptable. Molecular/genetic techniques now enable us to generate gene-deleted attenuated bacteria with defined properties (Fig. 4.2). Attenuating mutations can be targeted to specific genes and designed to eliminate any signs of disease or undesirable adverse reactions. The mutations generated in this way can be made irreversible. When selecting these genes however, it is important that the mutated organism be capable of replicating for a sufficient length of time in the vaccinated animal to induce strong primary and memory responses.
Living vaccines
Antigen processing
Attenuation
Bacterial attenuation
Random mutagenesis
Gene deletion
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