As noted in Chapter 4, empirical microbial attenuation by traditional processes is an unpredictable and somewhat hazardous way of making a live vaccine. Targeted attenuation by gene deletion or deliberate mutation of an organism generates a significantly safer product. But an even better solution is available—the use of recombinant vectored viruses in vaccines. Avirulent viruses may be genetically modified to express antigens from the organism of interest. When used in vaccines they can safely combine optimal antigenicity against the selected pathogen with the minimal virulence of the vector virus. These recombinant vectored vaccines have many advantages. They allow the simultaneous expression of multiple antigenic determinants and also avoid the hazards of whole pathogenic viruses, maximizing their safety. The recombinant organisms are much easier to grow than the original pathogen, and this results in a more consistent product along with a much shorter production and development time. More importantly, the ability of vectored vaccines to infect cells and so express endogenous antigens ensures that they are very efficient at inducing both antibody- and strong T cell–mediated responses against intracellular pathogens. Agents that carry selected genes encoding foreign antigens are known as vectors. Genetically engineered vectors can either be used as vaccines themselves or used to produce large amounts of antigens in vitro that can then be incorporated into vaccines. Vectors include bacteria, DNA viruses, yeasts, plasmids, and even plants (Fig. 5.1). Some microbial vectors may also replicate within an animal and as a result stimulate protective immune responses, provided they only cause abortive infections. Genes encoding protein antigens can be cloned directly into viruses. Instead of isolating and purifying the antigens, the recombinant virus vector itself may simply be used as a vaccine. Experimental recombinant vaccines used as vaccine platforms have usually used large DNA viruses such as poxviruses, adenoviruses, and herpesviruses, or bacteria such as Mycobacterium bovis, BCG, lactobacillus, or salmonella as vectors. The organisms that have been most widely employed for this purpose in veterinary medicine are poxviruses such as vaccinia, fowlpox, and canarypox. Viral vectored vaccines have the advantage of being able to induce both antibody- and cell-mediated immune responses without the need for an adjuvant. They do not require complex purification. They can generate antigens in the correct conformation and they can deliver more than one antigen at a time. As a result, these vaccines are safe, they cannot be transmitted by arthropods, and they are not excreted in body fluids. Their major advantage is that the antigens are synthesized within infected cells and thus act as endogenous antigens. They produce a “balanced” immune response compared to inactivated viral vaccines. In selecting viral vectors for vaccine use, safety is of paramount importance, whereas other considerations include vector stability and the ability to scale-up production. Poxviruses are the most widely used vectors in vaccines because they have a very large genome that can accommodate large inserts. For example, mammalian poxviruses such as vaccinia have a 190 kb genome, whereas fowlpox and canarypox have genomes of more than 300 kb. As a result, a 10 kb base-pair segment of the vaccinia genome can be removed and up to 30,000 base-pairs of foreign DNA can be inserted without affecting virus infectivity. Multiple foreign genes can therefore be inserted into a single vector. By splicing genes from selected pathogens into a poxvirus vector and using this as a vaccine, we can immunize recipients against all these pathogens. Unlike other DNA viruses, poxviruses have their own transcription machinery, RNA polymerase, and posttranscriptional modifying enzymes so this permits self-sufficient replication in the cytoplasm of infected cells. They can express high levels of the new antigen. Moreover, these recombinant proteins undergo appropriate processing steps, including glycosylation and membrane transport within the poxvirus. The poxviruses are also easy to administer by dermal scratching or by ingestion. The first widely employed vaccine vector was vaccinia virus, the “cowpox” vaccine used to protect humans against smallpox. There were some safety concerns regarding the use of vaccinia because it is not completely innocuous, especially in immunosuppressed recipients. This problem was solved by using highly attenuated, replication-deficient strains of the virus. For example, modified vaccinia virus ankara (MVA) was grown for 570 serial passages in chick embryo fibroblasts and lost about 10% of its genome. It can no longer replicate in mammalian cells. As a vector it is highly efficient and it stimulates powerful cell-mediated immune responses in children and the elderly. The earliest of such vaccines used the avirulent Copenhagen strain of vaccinia virus to express rabies virus glycoprotein. This virus strain has had 19 genes encoding virulence genes deleted, resulting in a highly attenuated organism. The cDNA for the rabies virus glycoprotein G was then inserted into the thymidine kinase (TK) gene of vaccinia. As a result, the vaccinia expressed the rabies glycoprotein and at the same time attenuated the vaccinia virus (Fig. 5.2). This glycoprotein can induce virus-neutralizing antibodies and so confer protection against rabies. Vaccination with this rabies-vaccinia recombinant (RABORAL V-RG) results in the production of antibodies to the glycoprotein and the development of immunity. This vaccine has been successfully used as an oral bait vaccine administered to raccoons, coyotes, arctic and gray foxes. This form of the vaccine can be distributed by dropping from aircraft (Chapter 20). The Copenhagen strain of vaccinia has been further attenuated by elimination of unwanted genes to produce the NYVAC strain. This strain has also been used in several animal vaccines including those directed against wildlife rabies, pseudorabies in pigs, equine influenza, Japanese encephalitis in pigs, and rabies in cats and dogs. Other mammalian poxviruses that have been investigated for their functionality as vaccine vectors include capripox, swinepox, parapox, and the myxoma virus. Highly effective recombinant vaccines were also developed for rinderpest; these consisted of a vaccinia or capripox vector containing the hemagglutinin (HA) or fusion (F) genes of the rinderpest virus. This recombinant capripox vaccine can also protect cattle against lumpy skin disease (Chapter 16). The most widely employed and successful poxvirus vector is the host-range restricted strain of canarypox virus (ALVAC). This virus grows only in birds but it is capable of entering mammalian cells, generating antigens, and so immunizing mammals. It does not replicate after entering a mammalian cell, and expression of the inserted antigens only lasts about six hours. Immunity to canarypox does not interfere with a recipient’s response to revaccination. Nor does it appear to induce the formation of canarypox neutralizing antibodies. The canarypox vector system has been used as a platform for many different vaccines including canine distemper, equine influenza, West Nile virus, feline leukemia, and rabies viruses (Table 5.1). The currently used vector was originally isolated from an infected canary, but was then passaged 200 times in chick embryo fibroblasts before being plaque purified four times. TABLE 5.1 ■
Recombinant vectored vaccines
Vectors
Poxvirus vectors
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Recombinant vectored vaccines
Some of the Recombinant Viral Vectored Vaccines That Are Commercially Available or Under Development. A Recombinant: Vaccine Vector Database, Vaxvec, is Available at http://violinet.org.vaxvec.
