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CHAPTER 1: VACCINE TECHNOLOGY

The presence of antigens in the system of a host animal triggers the production of antibodies as part of the host's natural immune response. Recognition of a 'foreign body' – which might range from a whole organism to single proteins or polysaccharides – stimulates a primary immune response, involving the proliferation of lymphocytes that develop into short-lived antibody-producing cells or long-lived 'memory' cells. Exposure to the same antigen at a later date will provoke a secondary immune response, activating memory cells to produce high levels of the specific neutralising antibodies required to fight an infection.

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Vaccination is based on the principle of artificial exposure to pathogenic organisms in a form that provokes a primary immune response without causing the disease itself. Primary vaccine doses are followed by regular booster doses designed to maintain levels of circulating antibodies, providing continued immunity to target pathogens.

Active immunity is developed following direct contact with an infectious agent, resulting in the initiation of a primary immune response. This can be induced either naturally, through exposure in the field, or artificially, through vaccination. Passive immunity may be developed through the provision of exogenous antibodies, either naturally (through colostrum, for example), or artificially through the administration of sera.

Vaccines are designed to provide protection against a disease by administering pathogens or toxins in such a way as to trigger an immune response without inducing clinical disease symptoms. Immunisation has traditionally been achieved using three basic product types:

Live, or attenuated vaccines: derived from alternative or mutant strains of a pathogenic organism that display reduced virulence in the host while maintaining immunogenicity;

Killed or inactivated vaccines: utilising chemical or physical methods of killing highly virulent pathogenic strains;

Toxoids: based on toxins secreted from an organism, which are altered in such a way as to reduce inherent levels of toxicity – for example through formaldehyde treatment of tetanus toxin in tetanus toxoids.

Live vaccines are manufactured using micro-organisms that have little or no virulence in target animals. The vaccines and micro-organisms may be related, but non-pathogenic strains are able to confer immunity on a non-target host – for example, the use of the measles virus against distemper in dogs, or bovine viral diarrhoea virus to vaccinate pigs against classical swine fever.

Live vaccines can also be formulated using naturally occurring or artificially created mutant strains of an organism. Mutations can be introduced into wild virus strains through exposure to chemicals (phenol is widely used), or by the application of ultra-violet or X-ray radiation. More commonly, micro-organisms are passaged extensively through a non-target cell culture line. This results in the pathogen losing its specificity for – and thus virulence in – a particular animal species or target tissue.

Alternatively, chemical or physical methods can be used to kill or inactivate virulent organisms. Since production processes often reduce their potency by denaturation or chemical modification of epitopes, however, vaccines containing inactivated material require the use of strains associated with high levels of antigenicity.

Methods used to inactivate target organisms are dependent on knowledge of the molecular structures involved and the level of infectivity of an individual pathogen. A range of chemicals may be used, though alkylating agents are among the most common. Substances such as ethylene oxide or beta-propiolactone are popular, since they do not interfere with surface proteins and act directly on the DNA. Formaldehyde and alcohols, which mildly denature proteins, are also used widely.

Unlike live or inactivated vaccines, toxoids do not contain whole pathogens. Instead, they utilise specific toxins secreted by pathogens that are responsible for provoking clinical symptoms of a disease. Toxins are altered in such a way that, while they are no longer toxic to target animals, they are still antigenically related. Toxoid vaccines do not prevent infection, but protect animals against the effects of target pathogens.

Live vaccines are more potent than inactivated products, and as a result can be administered at lower doses. Although they are attenuated, they are also usually able to replicate and induce the secretion by host cells of immunoregulatory substances such as interferon, lymphokines and cytokines. Consequently, live vaccines do not generally require the use of adjuvants.

Ease of preparation and the absence of any need for immunomodulation mean that live vaccines are usually cheaper to produce. They are unstable, however, and must be refrigerated constantly in order to prevent degradation of living material. This imposes strict requirements on methods of shipment, and can compromise the viability of live vaccines in regions that are subject to climatic extremes, and where cold chain resources are either limited or unreliable.

Another potential problem with traditional live vaccines is the fact that vaccinated animals may shed live bacteria or viral particles from mucosal surfaces or in their urine and excrement. Residual virulence and the risk of mutant strains reverting back to fully infective wild types can put surrounding animals at risk of infection, and can also contaminate the immediate environment in which vaccinated animals are kept.

Residual virulence is very rare in killed or inactivated vaccines, which are inherently safer as a result. The reduced levels of antigenicity associated with such products means that adjuvants must often be added to vaccine formulations before administration, however. Adjuvants potentiate the immune response by acting at the injection site as a slow-release deposit for antigens, or through the stimulation of cell activity. The need for the inclusion of adjuvants in inactivated vaccines increases production costs, but results in greater levels of stability, making them easier to store and handle than live vaccines.

Table 1.1: Vaccine types

Source: Animal Pharm Reports.

1.2 New vaccine technologies 

Biotechnology, and recombinant molecular technology in particular, has enabled new approaches to the production and use of immunological products. By manipulating the genetic material of target pathogens, virulence-related genes from an organism can be deleted, resulting in modified live vaccines with much improved levels of safety. Alternatively, genes coding for specific immunising antigens from a disease-causing organism can be inserted into a non-virulent vector.

Several novel approaches to immunology have already been used to develop successful commercial products with improved levels of efficacy or safety. Other avenues of research are being pursued, and promise further improvements.

1.2.1 Subunit vaccines

Molecular technology has enabled the identification of specific proteins in pathogenic organisms that are responsible for triggering immune responses in host animals. Subunit vaccines, containing fractionated or purified protein or glycoprotein components that have been identified as triggers of the protective immune response, were developed and commercialised towards the end of the 20^th Century.

Improvements in production technology have since enabled the manufacture of subunit vaccines on a larger scale and at reduced cost, increasing their commercial viability. Genes encoding the required fractions, or subunits, of a pathogen are propagated in so-called expression systems (usually bacterial or yeast cultures) before being extracted and purified for use as the active ingredient in subunit vaccines.

Table 1.2: Selected subunit vaccines commercialised for use in the veterinary sector

Species	Brand name	Company	Disease target(s)
Cats	Leucogen	Virbac	Feline leukaemia
	Leukocell 2	Pfizer	Feline leukaemia
	Nobivac FeLV	Intervet	Feline leukaemia
	Quilvax-FeLV	Schering-Plough	Feline leukaemia
Dogs	Eurican Herpes	Merial	Canine herpesvirus
Horses	Pneumequine	Merial	Equine herpesvirus
Cattle	Imocolibov	Merial	E coli
	Pneumo-Star	Novartis	Pasteurella pneumonia
Pigs	Geskypur	Merial	Aujeszky's disease
	Porcilis ART DF	Intervet	Atrophic rhinitis
	Porcilis Porcoli	Intervet	E coli

Source: Animal Pharm Reports.

 

The cattle pneumonia vaccine, Pneumo-Star, developed at the Canadian Veterinary Infectious Disease Organisation (VIDO) and now part of Novartis's biologicals range, was the first subunit vaccine to reach the market. Subunit products for use in a variety of species and against a growing range of pathogens have been commercialised since then. Merial's canine Lyme disease vaccine, RM Canine Lyme, and the same company's livestock vaccines, Geskypur (Aujeszky's disease in pigs) and Ibepur (infectious bovine rhinotracheitis in cattle) are subunit products, while the leukaemia preventatives marketed by Virbac (Leucogen), Intervet (Nobivac FeLV) and Fort Dodge (Fel-O-Vax FeLV) are all based on subunit technology.

Expression system technology for the culture of viral or bacterial fractions used in subunit vaccines has been patented by several specialists in the field. The baculovirus expression vector system (BEVS) patented by Protein Sciences (US) is a leading example. Baculoviruses replicate naturally in caterpillars. The BEVS system involves splicing genes into baculoviruses and fermenting modified virus in caterpillar cell material. The virus and caterpillar cells die after about three days, leaving the desired protein material available for harvesting and purification. Protein Sciences has established alliances with a broad range of partners in both the human and animal health sectors, and has been involved in the development of vaccines for use in a range of both livestock and companion animal species.

1.2.2 Gene-deleted vaccines

Live vaccines are generally both cheaper to produce and more efficacious than inactivated products. They do possess downside risks, however, including the possibility that viral material will revert to its virulent state, or that vaccines may be contaminated by unwanted organisms.
Recombinant DNA technology has addressed the risk of reversion to virulence by enabling the deletion of genes responsible for triggering clinical disease symptoms.

Vaccine strains with deleted genes can also be used as 'markers' that enable the serological distinction between vaccinated animals and those exposed to wild virus.

Gene-deleted marker vaccines have been developed against a number of livestock diseases, and have been used successfully in eradication programmes. The most notable examples are vaccines against Aujeszky's disease (pseudorabies) in pigs. The virulence of viral vaccine strains was initially reduced through an engineered mutation of the thymidine kinase gene, which halts the enzyme's activity. More recently, products featuring glycoprotein gene deletions have been developed. The resulting attenuated virus isolates possess reduced levels of pathogenicity, and can be used as markers.

Early gene-deleted vaccines were developed for use against viral targets (usually herpesviruses), but the technique also has potential for the development of improved bacterial vaccines, in which gene deletions can be used to reduce the pathogenicity of target organisms. These may also offer greater long-term commercial potential than viral marker vaccines, since vaccination programmes against conditions such as Aujeszky's disease and infectious bovine rhinotracheitis will be halted once eradication has been achieved.

Salmonella is one of the most significant bacterial targets for gene-deleted vaccines. The first gene-deleted salmonella vaccines reached the market in the second half of the 1990s. Bioproperties Australia launched Salvax, a poultry vaccine containing a Salmonella typhimurium mutant with a deletion for the aro gene, on its home market during 1996. Two years later, Bayer handled the US-launched Argus SC, a gene-deleted Salmonella choleraesuis vaccine for use in pigs (now part of Intervet's vaccine range).

Global genes that are involved in a micro-organism's replicative cycle are favoured deletion targets. Their removal prevents vaccine strains from replicating in the host animal, and results in a vaccine that is capable of inducing an immune response in the same way as traditional live vaccines, but which possesses an improved safety profile since it will not persist in the animal or contaminate the environment.

Several novel approaches to gene deletion are being researched. The UK company, Cantab Pharmaceuticals, has developed a technique based on the removal of a single replication gene from a target virus which, as a result, can infect a cell, but can only undergo one replication cycle. So while sufficient virus is produced to trigger a full immune response in the host animal, the clinical disease state cannot develop. Cantab's disabled infectious single cycle (DISC) virus technology platform has attracted a number of commercial partners, including Pfizer Animal Health, which is investigating its application in a range of veterinary vaccines. Potential targets include Aujeszky's disease, IBR, Marek's disease and unidentified viral targets in companion animal species.

1.2.3 Marker vaccines

The development of 'marker' vaccines and companion diagnostic tests that allow differentiation between vaccinated animals and those infected with natural strains of a pathogen has already played a key role in efforts to eradicate certain livestock diseases, including infectious bovine rhinotracheitis and Aujeszky's disease (see 1.2.2 above). More recently, marker vaccines against classical swine fever have been commercialised, and products for use in the eradication of several other diseases are being developed. Many marker vaccines are gene-deleted products, but the insertion of a microbial protein code into the genome of the relevant pathogen is another technique that has been used in some products.

1.2.4 Vector vaccines

Genetic material responsible for the stimulation of an immune response can be spliced into modified viral or bacterial carriers, known as vectors, which act as a vaccine delivery vehicle. The use of vectors enables accurate and refined delivery of antigenic material, and vectors with the potential to carry more than one protective antigen can be constructed. Administration of vector material triggers a natural immune response, including the production of antibodies to the pathogen from which genetic material has been taken. As such, vector vaccines combine the benefits of modified live vaccines with those of subunit technology.

Most early work with vectored vaccines made use of the vaccinia virus as a delivery vehicle. Derived from the naturally occurring cowpox strain, the vaccinia genome is both large and relatively basic, making it an ideal subject for genetic manipulation. Vaccinia-vectored rabies vaccines, in which the G protein of the rabies virus is expressed in a vaccinia vector, have been used widely to prevent the spread of rabies in wild animal populations across parts of Europe and the US.

While vaccinia virus possesses a number of properties that make it an ideal vector vehicle, it is not species-specific, and concerns were raised by regulators – notably in Europe – about the potential danger of subsequent expression in non-target animals. Regulators elsewhere took a less conservative line, however, and most early vaccinia-vectored vaccines were commercialised in the US and other international markets.

Researchers soon began to use alternative viral vectors in order to overcome regulatory misgivings surrounding the vaccinia virus. A growing number of vectored vaccines utilising species-specific poxviruses have reached the veterinary market as a result, and more are in development. The canarypox virus, which cannot replicate in mammalian hosts, was Merial's vector of choice, and the company has registered and launched a range of vectored vaccines using canarypox as a delivery vehicle.

Production of recombinant canarypox vector vaccines involves mixing donor plasmids with wild-type canarypox virus. The donor plasmid contains genes coding for the protective viral antigens, flanked on either side by sequences that facilitate plasmid insertion into the wild-type canarypox virus. After mixing, a small amount of the resulting organisms will be a recombinant virus capable of expressing protective antigens. This material is then purified for vaccine production.

1.2.5 DNA vaccines

DNA vaccines involve the direct inoculation of host animals with 'naked' DNA. After inoculation, antigenic material is expressed within the host, stimulating its immune system. DNA vaccines offer several potential advantages. They are relatively cheap to produce, provoke strong humoral and cellular immunity, can be used to deliver multiple antigens, and present little risk of contamination with foreign pathogens. They also remain viable without the need for refrigeration during transport, since simple DNA-based material is stable over a wide range of temperatures. DNA vaccines also pose potential problems, however. Most notably, they induce a relatively limited immune response, meaning that they must be administered at high doses.

Early trials with development-stage DNA vaccines were disappointing, eliciting little or no response in target animals. Since then, however, the development of adjuvant systems designed to increase levels of immunogenicity and lower required dose levels have rekindled enthusiasm in the approach. Researchers have also developed mechanisms that ensure plasmid DNA vaccines are delivered to and expressed more effectively in prime target cells within the skin and muscle tissue of target animals. And it has become clear that inoculating host animals with plasmid DNA effectively 'primes' the immune system to recognise the same antigens more effectively when they are delivered at a later date in viral vector vaccines.

The first two DNA vaccines to receive regulatory approval for use in either animals or humans were authorised within the space of a week in July 2005. In the US, a DNA vaccine against equine West Nile virus, developed by Fort Dodge in collaboration with the US Department of Agriculture’s Centers for Disease Control, was cleared for use, and will be made available to veterinary surgeons by the company early in 2006. In Canada, regulators approved a DNA vaccine for the prevention of infectious haematopoietic necrosis (IHN) in farm-raised Atlantic salmon, which was developed by the Novartis Animal Health subsidiary, Aqua Health.

DNA vaccine candidates are being developed for use against a range of other pathogens in both the human and animal health sectors. In the veterinary field, targets include bovine respiratory syncytial virus (BRSV), infectious bronchitis in poultry, canine parvovirus and feline immunodeficiency virus. All of the leading players in the veterinary vaccines market are involved in this field, and most have filed patents in respect of specific development-stage products. Merial is believed to be involved in work on DNA vaccines for use in cattle, while Intervet has been granted patents covering a DNA-based canine parvovirus vaccine. Pfizer and Fort Dodge both possess intellectual property covering feline immunodeficiency virus vaccines, while Fort Dodge has investigated a product against feline infectious peritonitis.

1.2.6 Synthetic vaccines

Efforts to develop vaccines based on synthetic peptides have been underway for more than two decades, but while this approach offers major potential advantages it has also proved complex, and some problems have still to be overcome.

Production and quality control of synthetic peptides is relatively simple, while the absence of nucleic acid or other viral material means that they are less toxic than products using all or part of pathogenic organisms. Synthetic peptides can also be adapted relatively simply to allow for viral mutations, making them excellent candidates for protection against viruses such as influenza. They appear to be less immunogenic than conventional inactivated whole-virus vaccines, however, and primary vaccination must be followed by the administration of booster doses, even allowing for the application of adjuvants. Moreover, synthetic peptide vaccines developed to date do not appear to induce full immunogenicity. The main problem encountered by researchers has been the development of synthetic material with steric configuration that mimics exactly the configuration that occurs naturally in viral pathogens.

Researchers in the Netherlands have pursued the development of synthetic peptide vaccines against the foot and mouth disease virus for more than two decades, but have so far been unable to produce a synthetic product that matches existing, conventional vaccines against the virus in terms of efficacy. In the early 1990s, they did finally succeed in producing such a product for canine parvovirus, however, and now believe that more than one antigenic site will have to be used in the development of a successful FMD vaccine. The relevant sites of FMD virus are highly discontinuous, making them difficult to reconstruct in synthetic form. Recent technological advances now allow recombination of these sites into synthetic molecules, however, and this may eventually prove to be a crucial breakthrough.

1.2.7 Edible vaccines

Another potential avenue opened up by recombinant DNA technology is the development of edible, plant-based vaccines. Several companies are involved in this area of research, which involves the insertion of exogenous genes from a target pathogen into plant genomes. Plants subsequently produce subunit antigens that may be purified and collected from harvested crops or, in an ideal scenario, fed directly to animals.

High levels of antigen expression are required to reach required vaccine dose levels if plant-derived vaccines are to be administered orally. Reasonable levels of expression have been achieved in development-stage trials, however, and edible vaccines produced directly in fodder crops offer a number of potential advantages. Basic production costs are relatively low, and cutting out the purification process will limit costs further. Oral delivery will also rule out damage to meat and animal hides, as well as removing the risk of operator injury.

The Texas-based company, Prodigene, is a long-standing player in the plant-based vaccine sector, and has several animal vaccine projects in development. One of its most established programmes involves a maize-based vaccine for the protection of pigs against transmissible gastroenteritis virus (TGEV), which has shown promise in clinical trials.

Dow AgroSciences has entered alliances with a number of specialist research organisations in the plant-derived vaccines field. Its partners include the Boyce Thompson Institute for Plant Research in the US, the Canadian biotech company, Agrisoma BioSciences, and Guelph University, also in Canada. Among the projects being pursued by researchers at Guelph is the production of transgenic clover containing proteins from the Manheimia haemolytica pathogen that could eventually result in an edible vaccine for the protection of cattle against pneumonia caused by this organism.

Most market-end partners in the field are either crop protection or animal nutrition specialists, but Schering-Plough Animal Health entered the fray in 2003 when it concluded a deal with California-based Large Scale Biology Corporation, which uses a proprietary biomanufacturing platform technology to produce proteins as antigens. The technology involves the creation of viral mRNA vectors that infect non-food plant crops such as tobacco, causing the plant to manufacture significant quantities of target proteins. If LSBC's efforts to develop veterinary vaccines using this method are successful, the company will supply them to Schering on a toll-manufacturing basis.

1.2.8 Adjuvant technology

Adjuvants have traditionally been used to potentiate the immune response of inactivated vaccines, either by acting as a slow-release deposit for antigens at the injection site, or by stimulating an immune response. Adjuvant technology has begun to play a more significant role in veterinary vaccine development over the past decade – partly because some emerging vaccine types (notably subunit products) require adjuvants to enhance immune response levels, but also because concerns have been expressed about the impact of some traditional adjuvant types on the health of vaccinated animals.

Most classical inactivated vaccines and the subunit products that have begun to feature increasingly in the portfolios of leading players in the market contain proprietary adjuvant systems. The role of these adjuvants in maximising the immune response to vaccines and levels of product efficacy is highlighted increasingly in product literature.

Established adjuvants such as aluminium salts or mineral oil emulsions often cause modest reactions at the injection site, and may result in persistent tissue lesions. Pressure from food processors and retailers to reduce the incidence of lesions, and the issue of residues where chemical adjuvants are concerned, has been a contributing factor to renewed levels of research into adjuvant technology. The issue of injection site sarcomas in cats has also been discussed at length, and has prompted regulatory investigations into adverse reactions reported following the vaccination of companion animals in some countries. A recent review of this issue by regulators in the UK noted that injection site sarcomas in cats may be associated particularly with aluminium-based adjuvants.

New-generation adjuvant technologies containing novel ingredients have been developed by a range of specialists in the field, and have been licensed to partners – sometimes on a non-exclusive basis for application in commercial vaccines. Most adjuvant platforms being developed for application in human vaccines have potential for use in the veterinary sector.

Virbac's Leucogen feline leukaemia vaccine was an early product of novel adjuvant technology developed by the US company, Aquila Biopharmaceuticals, which was acquired by Antigenics in 2000. Leucogen contains QA-21, a version of Aquila's QS-21 adjuvant, which contains saponin – a bio-derived product of the Quillaja saponaria tree, which increases total vaccine-specific antibody response and T-cell response to antigens, increasing the ability of weak antigens to activate the immune system and to enhance the speed and duration of the immune response.

An increasing number of proprietary adjuvants now feature saponin in some form. For example, Pfizer's PreZent-A adjuvant system, which has been applied to its CattleMaster Gold range of bovine vaccines, contains a refined form of saponin that stimulates both cell-mediated and humoral immune responses. The PreZent-A adjuvant also features Amphigen, which contains micelles coated with a natural soy product, lecithin. This allows more antigen to attach to droplets than is the case with traditional oil-based droplets, and also minimises the risk of local reactions at the injection site.

Biotechnology specialists and vaccine manufacturers will continue to research new adjuvant technologies for use in both classical inactivated vaccines and novel vaccine types that require the use of adjuvants to boost levels of antigen production. These will help to maximise levels of vaccine efficacy, and will enable the commercialisation of some novel products that would otherwise not have been viable.

1.2.9 Vaccine delivery

Vaccines for use in livestock and companion animals have traditionally been administered via injection (either intramuscularly or subcutaneously). Some poultry vaccines are also administered via the injectable route, but many are now delivered orally via water rations. Alternative routes of administration have been under investigation in other species for some time, and some non-injectable products are already available commercially. The concept of in ovo vaccination pioneered by the US company, Embrex, is also well established.

Aside from the time and expense associated with mass vaccination of livestock using traditional injectable products, injections also produce lesions that can affect the quality of both carcasses and hides. Injectable products also produce systemic immune responses in internal organs rather than at mucosal surfaces. Since over 90% of pathogens enter the body at these surfaces, the ability to provide mucosal immunity offers enhanced disease protection. Alternatives to injectable routes also promise to increase safety for those administering vaccines.

Plant-derived vaccines administered in livestock feed could eventually replace injectable administration routes for a range of large animal immunologicals. Oral and intra-nasal vaccines are already being made available more widely, however, with products for use in both livestock and companion animal species launched in recent years.

Intra-nasal vaccines are generally delivered using a needle-free plastic syringe or similar applicator, which is supplied with the product. Applicators should not be used to vaccinate more than one animal in order to avoid potential spread of disease. Examples of intra-nasal vaccines brought to market recently include Fort Dodge's equine strangles preventative, Pinnacle IN, and Boehringer Ingelheim's canine vaccine, Naramune-2.

Needle-free injection technology is another field in which some leading veterinary vaccine manufacturers have taken an interest, with Merial at the forefront of developments in the sector. Merial has been working for some time with Bioject Medical Technologies (US) on the application of Bioject's needle-free injection system to a range of companion animal vaccines. A modified version of Bioject's delivery platform was developed under the alliance, and has been applied to Merial's PureVax feline leukaemia vaccine.

Launched in the US at the beginning of 2005, the product combines several of the new-generation technologies that are being applied to veterinary vaccines in a bid to increase levels of efficacy and safety. Its non-adjuvanted composition reduces the risk of chronic inflammation and other localised reactions sometimes associated with traditional adjuvanted vaccines. It also employs canarypox vector technology, allowing the vaccine to mimic natural infection, prompting a broad humoral and cell-mediated response. And finally, by utilising the Vet Jet system, PureVax is administered transdermally, allowing broad dispersion of the vaccine in the dermis, subcutaneous layer and muscle tissues, encouraging rapid and comprehensive immunity. Partly because immunity is activated in tissue layers that would be bypassed by needle injections, a relatively low dose of the vaccine is required.

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