Vaccine Development Using Recombinant DNA Technology Animal Agriculture’S Future Through Biotechnology, Part 7

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Number 38 May 2008

Vaccine Development Using Recombinant DNA Technology Animal Agriculture’s Future through Biotechnology, Part 7

Abstract Development of vaccination as a tool in fighting disease has resulted in the potential to combat almost all infectious agents affecting people and animals. The ultimate objective of vaccination is to induce an immune response that subsequently recognizes the infectious agent and fights off the disease. Current public health threats posed by the potential spread of highly infectious disease agents between animals and humans, as well as the emergence of new diseases, impact animal agriculture significantly. Animal vaccinations are among the most effective, successful tools for dealing with these concerns. This Issue Paper provides a brief historical overview of vaccine development and describes three basic categories of newer, recombinant vaccines: live genetically modified organisms, recombinant inactivated (“killed”) vaccines, and genetic vaccines. Separate sections evaluate the development of vaccines for cattle, sheep, and goats; In laboratory tests, a vaccine injection with recombinant DNA is used to im- swine; poultry; fish; and companion an- munize chicks against coccidia. (Photo courtesy of the USDA Agricultural imals. Within each category, the authors Research Service.) describe the vaccines that are commercially available, outline the recent advances in recombinant vaccines for the cines against diseases that currently lose the use of several important anti- control of specific infectious diseases, have few or no control measures. microbial agents and drug classes for and discuss the future of vaccines for two reasons: (1) increased resistance animal diseases. among pathogens and (2) public health Future research needs to focus on Introduction threats posed by the potential spread of 1 vaccine delivery methodology. In ad- Infectious animal diseases continue antimicrobial-resistant zoonotic mi- dition, new efforts need to target the to rank foremost among the significant crobes. Consequently, new approaches development of vaccines for economi- factors limiting efficient production in are needed to develop improved tools cally important diseases for which no animal agriculture. In addition, infec- and strategies for prevention and con- currently available vaccines exist, and tious agents that are transmitted from trol of infectious diseases in animal diseases for which poorly effective vac- animals to humans by way of food agriculture. Among the most effective cines are currently in use. Advances and water present an increasing threat and successful of these tools are animal in recombinant DNA technology, in to the safety and security of the world vaccines. knowledge of the host immune re- food supply and continue to affect hu- sponse, and in the genetic makeup of man health significantly. Awareness is 1 Italicized terms (except genus and species disease agents will lead to new vac- increasing that animal agriculture could names) are defined in the Glossary.

This material is based upon work supported by the United States Department of Agriculture under Grant No. 2005-38902-02319, Grant No. 2006-38902-03539, and Grant No. 2007-31100-06019/ISU Project No. 413-40-02. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or Iowa State University. CAST Issue Paper 38 Task Force Members

Authors Robert Silva, Avian Disease and Ronald D. Schultz, School of Oncology Laboratory, USDA–ARS, Veterinary Medicine, University of Mark W. Jackwood (Chair), Poultry East Lansing, Michigan Wisconsin–Madison Diagnostic and Research Center, University of Georgia, Athens Reviewers CAST Liaison Leslie Hickle, Synthetic Genomics, Klaus Osterrieder, College of Vet- Alan W. Bell, CSIRO Livestock Inc., La Jolla, California erinary Medicine, Cornell University, Industries, St. Lucia, Queensland, Sanjay Kapil, Oklahoma Animal Ithaca, New York Australia Disease Diagnostic Laboratory, Christopher Prideaux, CSIRO Center for Veterinary Health Sci- Livestock Industries, St. Lucia, ences, Oklahoma State University, Queensland, Australia Stillwater

Using recombinant deoxyribonucle- History and Overview of system. The animal’s immune system ic acid (rDNA) technologies, scientists has the ability to distinguish between a have been able to develop three types of Vaccine Development foreign substance, such as the proteins recombinant vaccines: (1) live geneti- The history of vaccination dates in a virus or bacterium, and its own pro- cally modified organisms, (2) recombi- back to the 1798 studies by Edward teins. It does not matter whether the for- nant inactivated (“killed”) vaccines, and Jenner, an English physician who used eign proteins are from a disease agent or (3) genetic vaccines. These vaccines no cowpox virus to immunize people a vaccine against the disease agent, the longer cause disease, but still induce a against smallpox (Jenner 1798). Almost immune response is similar: when the strong immune response. Paralleling the 200 years later, the comprehensive animal encounters the virus or bacteria development of new, more efficacious, smallpox vaccination program estab- again, the immune system recognizes it stable, and safe recombinant vaccines lished by the World Health Organization and, ideally, responds to protect the ani- has been the study of vaccine delivery eventually led to the worldwide eradica- mal from the disease. methods and immunostimulating adju- tion of that disease. That success story Although vaccination has saved vant compounds that enhance the im- is proof of the tremendous potential of countless lives, it can have both favor- mune response. vaccination and has led to the devel- able and unfavorable consequences. Advances in gene discovery of ani- opment of vaccines against almost all Certain vaccines—specifically, live vac- mal pathogens can be expected to iden- infectious agents affecting people and cines—can revert back to pathogenic or- tify new proteins and metabolic path- animals. The ultimate objective of vacci- ganisms and produce disease or, in some ways, thereby providing a foundation nation is to induce an immune response instances, even death. The development for improved understanding of pathogen that subsequently recognizes the infec- of rDNA technologies has provided new biology and, ultimately, aiding in the de- tious agent and fights off the disease. ways of attenuating disease agents by sign of new and effective therapies. New Vaccination usually is accom- modifying their genetic makeup, or ge- treatments, whether vaccines or new plished with either weakened or attenu- nomes, to create safer, more efficacious drugs, must rely on more than empirical ated live agents; with inactivated agents vaccines. methods of discovery and must be based that no longer can cause disease; or The genome of all living beings is on a fundamental knowledge of patho- with selected, immunogenic parts of the made up of many genes that define the gen biology and genetics. disease agent called subunit vaccines. characteristics of the organism. The ge- This Issue Paper first provides a Traditional methods of creating vac- netic material consists of nucleic acids brief historical overview of vaccine de- cines include using a similar agent that (DNA and ribonucleic acid [RNA]) that velopment and describes the three basic does not cause disease, such as Jenner’s carry and convey genetic information categories of recombinant vaccines. The cowpox virus, or passing a pathogenic through their bases (adenine, cytosine, following sections evaluate the develop- disease agent through a laboratory host guanine, and thymine); in RNA, thy- ment of vaccines for cattle, sheep, and system to weaken or attenuate the agent. mine is replaced by uracil). The bases goats; swine; poultry; fish; and compan- Inactivating the disease agent with one are uniquely ordered to make up the se- ion animals. Within each category, the or more chemicals also can be used to quence of the particular gene. Modifying authors describe the vaccines that are create vaccines. In addition, extracting, or deleting the genes responsible for commercially available, outline the re- purifying, and using one or more parts causing disease in an organism can be cent advances in recombinant vaccines of the disease agent can be used to in- accomplished in the laboratory using for the control of infectious diseases, duce a protective immune response. rDNA technologies. and discuss the future of vaccines for An immune response is stimulat- Typically, rDNA technology refers animal diseases. ed when a foreign substance called an to laboratory methods used to break and antigen is encountered by the immune recombine DNA molecules from differ-

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY ent organisms. The terminology also has remains stable and cannot revert to a the laboratory and represent the most grown to include laboratory methods pathogenic agent (Uzzau et al. 2005). basic portion of a protein that induces an used for gene isolation, sequence modi- Developing a vaccine of this type immune response. Subunit vaccines also fication, nucleic acid synthesis, and gene requires knowledge of the gene(s) re- can consist of whole proteins extract- cloning. sponsible for pathogenicity and assumes ed from the disease agent or expressed Using rDNA technologies, scien- that those genes are not the same genes from cloned genes in the laboratory. tists can isolate a disease agent, reduce governing viability and the ability of the Several systems can be used to express it to its basic components, examine its modified organism to induce an immune recombinant proteins, including expres- genetic makeup, and modify it so that it response. Examples of gene-deleted vac- sion systems that are cell free or that no longer causes disease but still induces cines include a Salmonella vaccine for use whole cells. Whole-cell expression a strong immune response. Methods sheep and poultry and a pseudorabies systems include prokaryotic (bacteria- of extracting and purifying genes from virus vaccine for pigs. based) systems such as Escherichia coli, extremely small amounts of even the Another relatively recent method and eukaryotic (mammalian, avian, in- tiniest organisms have become routine of creating a live genetically modified sect, or yeast-based) systems. for many laboratories. Once extracted, vaccine is to use an infectious clone of The baculovirus expression sys- the nucleic acids can be modified and the disease agent. An infectious clone is tem is a widely used eukaryotic system the genes reinserted into the organism to created by isolating the entire genome because it is applicable to many differ- produce a vaccine that is attenuated and/ of the disease agent (usually viruses) in ent proteins and because relatively large or capable of inducing better immuno- the laboratory. This isolated or cloned amounts of protein can be produced. logical protection. genome can be specifically and purpose- Baculovirus expression systems are en- Vaccine development using rDNA fully modified in the laboratory and then gineered specifically for expression of technologies requires a thorough under- used to re-create the live genetically proteins in insect cells. It is important to standing of the disease agent, particu- modified organism. express proteins of disease agents with larly the antigens critical for inducing Vector-based vaccines are bacteria, the greatest possible similarity to the au- protection. In addition, it is important viruses, or plants carrying a gene from thentic molecule so that the proper im- to understand the pathogenicity of the another disease agent that is expressed mune response is induced when the pro- disease agent and the immune response and then induces an immune response teins are used as a vaccine. Baculovirus of the host, to ensure that the vaccine when the host is vaccinated. For viral expression systems are effective for induces the appropriate immunological and bacterial vectors, the vaccine induces properly modifying recombinant pro- reaction. Increasingly, genetic informa- a protective response against itself (the teins so they are antigenically, immuno- tion from both microbial genomics stud- vector) as well as the other disease agent. genically, and functionally similar to the ies and proteomic studies is being used Foreign genes must be inserted into the native protein. to gain a better understanding of the in- genome of the vaccine vector in such a Another type of recombinant sub- teractions between the disease agent and way that the vaccine remains viable. unit vaccines, called virus-like particles the host. Nonetheless, vaccines devel- The first commercial vaccine vector (VLPs), can be created when one or oped through recombinant technologies was VectorVax FP-N (Zeon Corporation, more cloned genes that represent the are designed to be safer, more effica- Japan), a vaccine primarily used in tur- structural proteins of a virus are ex- cious, and/or less expensive than tradi- keys; it consists of a fowl pox vaccine pressed simultaneously and self-assem- tional vaccines. virus that carries genes from Newcastle ble into VLPs. These VLPs are immu- disease virus. Other agents used as vec- nogenic (i.e., they look like the virus on Categories of Vaccines tors of foreign genes are Salmonella, the outside, but cannot replicate because Recombinant vaccines fall into three herpesviruses, adenoviruses, and adeno- they do not contain any genetic material basic categories: live genetically modi- associated viruses. Edible plant-derived on the inside). fied organisms, recombinant inactivated vaccines take advantage of the ability Because subunit vaccines do not vaccines, and genetic vaccines (Ellis of some antigens to induce an immune replicate in the host, they usually are ad- 1999). response when delivered orally. Foreign ministered (injected) with an adjuvant, genes from disease agents have been in- a substance that stimulates the immune serted into potatoes, soybeans, and corn system of the animal to respond to the Live Genetically Modified Vaccines plants and fed to animals; the expressed vaccine. The mechanisms of action of The first category—live genetically proteins from those foreign genes im- many adjuvants are poorly understood; modified vaccines—can be viruses or munized the animals against the disease consequently, they usually are selected bacteria with one or more genes deleted agent (Streatfield 2005). on a trial-and-error basis. Adjuvants can or inactivated, or they can be vaccines enhance the response to a vaccine by carrying a foreign gene from another Recombinant Inactivated Vaccines protecting the vaccine from rapid deg- disease agent, which are referred to as radation in the animal; these are usu- vaccine vectors. Deletion or gene-in- The second category—recombinant ally oil-based adjuvants. Or, they can activated vaccines are developed to inactivated vaccines—are subunit vac- attract so-called antigen-presenting cells, attenuate the disease agent. Generally cines containing only part of the whole which process and deliver antigens to two (double-knockout) or more genes organism. Subunit vaccines can be syn- the immune system. Adjuvants also can are deleted or inactivated so the vaccine thetic peptides that are synthesized in be molecules that enhance the immune

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY response by stimulating immune cells respiratory disease complex (shipping vaccinated cattle can be distinguished directly, or by stimulating immune- fever) in feedlot cattle, calf-scours and from naturally infected animals, which modifying and immune-strengthening or respiratory diseases in cow-calf opera- is critical for eradication of this disease. immunopotentiating substances called tions, and abortions. Pathogens com- There are no commercially available cytokines. In addition, adjuvants can be monly vaccinated against in cattle are recombinant vaccines used for sheep a combination of these types. Leptospira sp., E. coli, Clostridia sp., and goats in the United States. Mannheimia haemolytica (also sheep Genetic Vaccines and goats), Haemophilus somnus, in- Recent Advances The third category of recombinant fectious bovine rhinotracheitis (IBR), Bovine viral diarrhea virus (BVDV) vaccines is referred to as genetic vac- bovine viral diarrhea (BVD), bovine re- causes respiratory, enteric, and eye in- cines because they are actually DNA spiratory syncytial virus (BRSV), para- fections, as well as abortions, in cattle. alone. Genetic or DNA vaccines usu- influenza-3 (PI-3), rotavirus, and bovine The virus is thought to play a role in ally are circular pieces of DNA, called coronavirus (BCoV). Two weeks be- bovine respiratory disease complex be- plasmids, which contain a foreign gene fore weaning/arrival at the feedlot, cattle cause it causes an immunosuppression from a disease agent and a promoter typically are vaccinated against respira- that leads to bacterial pneumonia (ship- that is used to initiate the expression of tory disease and clostridial infections, as ping fever). The E2 surface glycoprotein the protein from that gene in the target part of prearrival conditioning. Vaccines is a major immunogen on the virus, and animal (Rodriguez and Whitton 2000). used in sheep and goats include prod- DNA vaccination using the E2 pro- Plasmids can be maintained in bacteria ucts for “overeating disease” caused tein gene of BVDV provides protection (usually E. coli) and have been designed by a bacterium, Clostridium perfrin- against the disease (Nobiron et al. 2003). to accept foreign genes for expression gens types C and D, tetanus toxoid, sore A more recent study found that in animals. The recombinant plasmids mouth (contagious ecthyma) caused by DNA vaccination with the E2 protein containing a foreign gene are purified poxvirus, diarrhea caused by E. coli, gene followed by a boost vaccination from the bacteria, and the “naked” DNA infectious causes of abortion in sheep with recombinant E2 protein provided is injected directly into the animal, usu- (Campylobacter fetus and Chlamydia good protection against the pathogenic ally intramuscularly or intradermally psittaci), footrot in sheep caused by NY-strain of BVDV (Liang et al. 2006). (into the skin). The animal’s cells take two bacteria (Bacteroides nodosus and Another genetic vaccine tested in cattle up the DNA, and an immune response Fusobacterium necrophorum), and PI-3 used RNA from a vaccine strain of is induced to the protein expressed from respiratory disease in lambs. Nearly all BVDV in a microparticle bombardment the foreign gene. vaccines for cattle, sheep, and goats cur- method (Vassilev, Gil, and Donis 2001). In addition to genes coding for im- rently licensed by the U.S. Department The vaccinated cattle developed protec- munogenic proteins, genetic vaccines of Agriculture (USDA) were developed tive neutralizing antibodies. In addi- also have been designed to include dif- using conventional technologies. tion, a genetically engineered E2-deleted ferent immune-stimulatory genes that BVDV isolate, which can be propagated trigger different compartments of the Commercially Available efficiently in an E2-expressing cell line immune system, depending on the type Recombinant Vaccines in the laboratory, will infect and induce of immunity desired. Unique features of Recombinant bovine vaccines have an immune response in cattle. But be- DNA vaccines are intrinsic sequences cause it is lacking E2, it cannot produce embedded in the DNA, so-called CpG not been licensed for use in the United States. The European Union, however, infectious virus particles, making it motifs. These unmethylated motifs were extremely safe (Reimann, Meyers, and shown to act as an adjuvant, stimulating has approved a naturally occurring glycoprotein E (gE)-deleted IBR vaccine Beer 2003). the innate immune responses and en- Likewise, a gene-deleted vaccine for hancing the effectiveness of the vaccine. for its IBR eradication program. Infec- tious bovine rhinotracheitis is a herpes- BRSV does not produce infectious virus The following sections describe particles after vaccination. Bovine respi- commercially available recombinant virus responsible for respiratory disease in feedlot cattle as well as for reproduc- ratory syncytial virus, a cause of pneu- vaccines for ruminants, swine, poul- monia, plays an important role in ship- try, fish, and companion animals. In tive diseases, conjunctivitis, and nervous disorders. Deletion of the gE gene ping fever of cattle. The G spike protein addition, each section addresses recent located on the surface of the virus is one advances in recombinant vaccines for attenuates the IBR virus and prevents it from being shed in bovine secretions, of the major immunogenic viral pro- control of infectious agents in those ani- teins. Genetically engineered BRSV mals, as well as future vaccine technolo- limiting its transmission. This modified- live, naturally occurring, gene-deleted lacking the G spike protein gene still can gies being explored for animal health be propagated in cell culture, and vac- and protection. vaccine is immunogenic and safe for cattle of all ages. cinated calves produce neutralizing anti- An important advantage of this vac- bodies, but no infectious virus particles Recombinant Vaccines cine is that the gE-deleted vaccine virus are formed (Schmidt et al. 2002). Capripox causes diseases such as for Cattle, Sheep, and and wild-type IBR viruses can be differ- entiated by the polymerase chain reac- sheep pox and goat pox in domestic Goats tion, a method of amplifying very small small ruminants. It also can cause lumpy Clinically and economically signifi- amounts of nucleic acid to detectible skin disease in cattle and possibly in cant diseases of cattle include the bovine levels (Schynts et al. 1999). Thus, the buffalo. Capripox vaccine is a poxvirus

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY being used as a vaccine vector for two against BCoV, but only partial protec- A raccoon pox virus expressing a important sheep and goat diseases—rin- tion has been induced. A recombinant rabies virus glycoprotein has been tested derpest and peste-des-petits-ruminants. human adenovirus vector expressing in sheep. In addition, subunit vaccines Those diseases are caused by a para- the BCoV HE immunogen was devel- against parasites such as the cestode par- myxovirus that is not endemic in the oped, and in a mouse model, antibod- asite Taenia ovis in sheep and ectopara- United States. ies induced against HE were detected in sites like ticks have been developed for serum and lung washes. It is not clear sheep and goats. Future Developments if this vaccine vector will be effective The process of distinguishing in- against BCoV in cattle (Yoo et al. 1992). Recombinant Vaccines fected from vaccinated animals, known Bovine rotavirus (BRV) is respon- as the DIVA approach, is essential for sible for scours in approximately 30% of for Swine calf viral enteritis cases. The BRV spike disease eradication programs. The use In the United States, approximate- of gene-deleted or marker vaccines pro- surface protein is cleaved by trypsin into the viral proteins VP5 and VP8. The ly 75% of swine producers vaccinate vides a method to distinguish vaccinates against common swine pathogens in- from naturally exposed animals by test- immunogenic VP8 subunit induces antibodies that can neutralize the virus in the cluding Mycoplasma sp.; porcine repro- ing for antibodies directed against the ductive and respiratory syndrome virus; marker in the vaccine or proteins unique laboratory, making it a good candidate for a subunit vaccine. Erysipelas; E. coli scours; parvovi- to the wild-type virus. One example is rus; Leptospira sp.; H3N2 and H1N1 a BVDV gene-deleted vaccine that was A unique protein expression system has been developed using a tobacco mo- swine influenza; and bacterial rhinitis modified to inactivate the RNase gene caused by Pasteurella, Bordetella, or within the viral glycoprotein E gene. saic virus vector in tobacco plants. The tobacco mosaic virus vector containing Mycoplasma. Primarily, conventional That deletion resulted in an attenuated vaccines are the ones licensed and used virus, which could be an effective vac- the VP8 BRV gene is used to infect the tobacco plant, and the expressed recom- in commercial swine. But rDNA vaccine against BVD (Meyer et al. 2002). cines are being developed to further Control programs targeting the removal binant VP8 protein is extracted from the plant leaves. After intraperitoneal (IP) decrease or eliminate economically im- of persistently infected animals use vac- portant diseases in domestic swine and cination as part of an overall strategy to inoculation of the material containing the VP8 recombinant protein, antibody to prevent reintroduction of these patho- achieve a status of BVDV-free. Those gens from feral swine populations. programs would benefit greatly from responses were detected against BRV in mice and protected the mice against such a marker vaccine. Commercially Available Vaccines for Mannheimia haemo- infection with BRV (Perez Filgueira et lytica, the major pathogen involved in al. 2004). Moreover, triple-layer VLPs Recombinant Vaccines shipping fever in cattle and pneumonia (complete BRV particles have three The DIVA approach, as discussed in sheep and goats, have focused on its layers) have been produced using viral for cattle, also has been used for the leukotoxin, which is the primary viru- proteins expressed using the baculovirus eradication of pseudorabies virus (PRV) lence factor. The leukotoxin gene has system. Those VLPs were found to in- in commercially grown pigs in the been cloned and expressed in cell lines duce lactogenic antibodies. But like all United States. Pseudorabies is an acute, as well as in plants, and the recombi- VLPs, they are safe in that they do not highly fatal disease of pigs. Although it nant protein induces leukotoxin-neu- replicate in the host because they do not is caused by a herpesvirus (not the virus tralizing antibodies (Lee et al. 2001). contain any genetic material. that causes rabies), the clinical signs E. coli fimbrial an- Immunodominant epitopes on a major with the K99 are similar to rabies. Pseudorabies virus tigen commonly causes diarrhea in M. haemolytica surface lipoprotein, glycoprotein I (gI) and glycoprotein X young cattle, sheep, and goats. FanC designated PlpE, also have been identi- (gX) gene-deleted vaccines were used is the major subunit of this immuno- fied as potential vaccine candidates, and for PRV eradication in the United States. genic protein. Agrobacterium-mediated Piglets intranasally immunized with antibodies against them are effective in transformation, a method of delivering complement-mediated M. haemolytica the gene-deleted vaccine can be distin- foreign genes to plants, has been used guished from infected animals by a dif- killing. A recombinant PlpE protein was successfully to express the FanC subunit ferential enzyme-linked immunosorbant expressed in the laboratory, added to a of K99 in soybeans as a first step to de- assay (ELISA) that detects the presence commercial M. haemolytica vaccine, veloping an edible vaccine (Piller et al. and shown to enhance protection in 2005). When mice were injected with of antibodies against gI and/or gX, indi- calves (Confer et al. 2006). protein extracted from the soybeans, cating that the animals were exposed to Bovine coronavirus causes calf they developed FanC-specific antibod- pathogenic PRV (Swenson, McMillen, diarrhea, or scours, infecting the en- ies and cytotoxic T-cell responses, which and Hill 1993). The USDA-licensed tire length of the intestinal tract. demonstrates that the protein expressed gene-deleted vaccine pairs well with the Recombinant vaccines against BCoV are in plants can function as an immunogen. PRV differential ELISA test (IDEXX difficult to develop because it is hard to It is not clear, however, whether oral Laboratories, Westbrook, Maine). As of reproduce the viral proteins accurately vaccination that works in animals with May 2005, the United States achieved in the laboratory. Viral hemagglutinin- a monogastric digestive tract, such as pseudorabies-free status (Stage V), and esterase (HE) and spike surface pro- mice, will work in cattle or sheep, which vaccination no longer is practiced in teins have been used as subunit vaccines have a ruminant digestive tract. commercial swine.

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY Recent Advances being developed. Because piglets do not of infectious disease. Nearly all these Progressive atrophic rhinitis in swine have maternal antibodies against human poultry vaccines are conventional vac- is caused by a Pasteurella multocida adenovirus 5, the interference observed cines that have worked well in general. toxin and is characterized by nasal turbi- with conventional swine influenza vac- But avian pathogens continue to change nate bone degeneration, distortion of the cines does not occur (Wesley and Lager and develop ways to evade the immu- nose and face, and nasal hemorrhage. 2006). But if sows are vaccinated with nity induced by the current vaccines. Inactivated toxin, or toxoid, which is the human adenovirus 5 vector, the ma- Thus, there is an ongoing race to find or used as a vaccine, can lose some of its ternal antibodies passed to their progeny develop new vaccines. immunogenic properties when it is inac- will interfere with vaccination because tivated. To circumvent that problem but human adenovirus 5 is susceptible to Commercially Available Re- still maintain a safe vaccine, scientists neutralization by those antibodies. combinant Vaccines tested fragments of recombinant toxin Swine mycoplasmal pneumonia is Currently, there are 11 U.S. licensed expressed in E. coli for the ability to caused by the bacterium Mycoplasma rDNA poultry vaccines (Table 1). Ten protect against progressive atrophic rhi- hyopneumoniae, which is worldwide in of these vaccines are live recombinant nitis. Vaccination of piglets resulted in distribution. The disease is a significant viral vectors designed to deliver specific toxin-neutralizing antibodies. Sows im- burden on the swine breeding industry genes to stimulate the host’s immune munized with recombinant subunit toxin because it causes losses associated with system. Seven of these use an attenuated had high serum antibodies, and their severe respiratory disease and predispos- fowl pox virus (FPV) as a vector to de- progeny had improved survival after ex- es pigs to secondary invaders that can liver selected pathogen genes. Three use posure to a lethal dose of authentic toxin cause fatal infections. Although inac- an attenuated vaccine, Marek’s disease compared with animals immunized with tivated whole-cell vaccines (bacterins) virus (MDV), or a closely related non- conventional toxoid vaccine (Liao et al. are safe and currently the most effective pathogenic turkey herpesvirus as a vec- 2006). means of controlling the disease, they tor. Surprisingly, only one license is for are not always efficacious againstM. a bacterial pathogen, Salmonella, which Future Developments hyopneumoniae because it is difficult to is a double-knockout mutant resulting in induce an immune response that protects a stable attenuated bacterium. Another The coronavirus transmissible gas- against the clinical signs and lesions as- troenteritis virus (TGEV) causes severe interesting observation is that the list of sociated with this disease. pathogens being targeted by rDNA vac- diarrhea in piglets. The major antigenic Thus, rDNA technology is being protein of TGEV is the spike glyco- cines is small compared with the list of used to produce subunit vaccines con- actual poultry pathogens. protein. Attempts to faithfully re-cre- taining the P97 adhesin protein respon- ate these antigenic regions in recom- Not all poultry pathogens pose the sible for adherence of the bacterium to same degree of risk to the industry. One binant vaccines have proved difficult, swine ciliated epithelial cells. Adhesion but several vaccines hold promise. excellent source of information on cur- is important for virulence, and both lo- rent poultry pathogens that pose the Recombinant pseudorabies virus carry- cal and systemic antibodies have been ing the TGEV spike gene was construct- greatest risk for the poultry industry is produced in mice to a recombinant sub- the research priorities list established ed and may have potential as a recombi- unit vaccine containing a portion of P97 nant vaccine against both pseudorabies by the Cooperative State Research, (the R1 repeat region) coupled to highly Education, and Extension Service, a and TGEV. immunogenic enterotoxin subunit B In addition, Lactobacillus casei and USDA granting agency. Their 2006 from E. coli (Conceicao, Moreira, and high-funding priority list includes avian Salmonella enterica (serovar typhimuri- Dellagostin 2006). um) expressing the TGEV spike, or Clostridium perfringens, Marek’s dis- portions of the spike protein, have been ease, poult enteritis complex (a multifac- shown to induce neutralizing antibodies Recombinant Vaccines torial disease affecting young turkeys), avian influenza, and exotic Newcastle in serum and intestinal secretions of pigs for oultry and mice, respectively. A DNA vaccine, P disease. The list indicates that current an adenovirus serotype 5 vector, and The U.S. consumption of poultry conventional and recombinant vaccines plant-derived vaccines containing the meat exceeds consumption of either designed to protect against these diseas- spike or nucleoprotein genes of TGEV beef or pork. Currently, the U.S. poultry es are not very efficacious. It seems that also have been constructed and shown industry is the world’s largest producer the industry needs better vaccines than to elicit humoral and cell-mediated im- and exporter of poultry meat. The tre- those listed in Table 1 for MDV disease, mune responses in mice. mendous numbers of animals raised in avian influenza, and exotic Newcastle One of the big problems with induc- relatively confined environments pro- disease, and that vaccine companies and ing immunity in young animals is the motes the spread of infectious disease researchers should consider developing presence of maternally derived antibod- agents. The fact that individual animals recombinant vaccines for C. perfringens ies against the antigen. To overcome are of so little value makes it economi- and poult enteritis complex. antibody interference of vaccination in cally unfeasible to attempt to treat sick swine, a human adenovirus 5 vector, birds. Consequently, the poultry indus- Recent Advances delivering the hemagglutinin and nu- try—more than any other industry—re- Earlier reviews listed numerous cleoprotein of swine influenza virus, is lies on vaccines to prevent outbreaks reports of recombinant vaccines under

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY Table 1. Currently licensed rDNA poultry vaccines1 (Source: Data provided by Michel Carr, in poultry. The primary problems have USDA-Animal and Plant Health Inspection Service.) been to deliver sufficient quantities of DNA to elicit an effective immune re- Company Pathogen Vector sponse, and to mass-vaccinate thousands of birds in a flock. One promising ap- Lohmann Animal Health Salmonella typhimurium Double-deletion mutant proach currently being studied is the use Merial Select AIV, FPV FPV Merial Select NDV, FPV FPV of lipids or other micro-DNA-encapsu- Merial Select IBDV, MDV HVT lating compounds to protect and deliver Intervet MDV HVT the DNA directly to the host cell (Oshop Intervet MDV, NDV MDV et al. 2003). Biomune Co. AEV, FPV, LTV FPV Virus-like particles are safe, immu- Biomune Co. NDV, FPV FPV nogenic vaccines, but few VLP poultry Biomune Co. FPV, MG FPV vaccines have been tested. A VLP vac- Biomune Co. AEV, FPV, MG FPV Biomune Co. FPV, LTV FPV cine for infectious bursal disease has been described by Rogel and colleagues 1AIV, avian influenza virus; NDV, Newcastle disease virus; IBDV, infectious bursal disease virus; (2003). That vaccine was produced by MDV, Marek’s disease virus; AEV, avian encephalomyelitis virus; FPV, fowl pox virus; LTV, expressing viral proteins (VP2, VP4, laryngotracheitis virus; HVT, turkey herpesvirus; MG, Mycoplasma gallisepticum. and VP3) in E. coli, which assembled into VLP and protected chickens against development (Jackwood 1999). One of protected against both ILTV and influ- infectious bursal disease. Although the the simplest recombinant vaccines to enza virus (Veits et al. 2003). vaccine was injected intramuscularly, construct is a subunit vaccine containing The large genome of the FPV virus, which is not a practical delivery mecha- only the immunogenic part of the infec- and its ability to incorporate large frag- nism for large flocks of commercial tious agent. As an example, research- ments of foreign DNA, makes FPV- poultry, the vaccine does show promise ers have identifiedgametocyte antigens based vaccine vectors a popular choice. for the future. from coccidia that can be administered Recently, FPV vaccine vectors were One of the most recent technologies as a subunit vaccine (Belli et al. 2004). made to express antigens from avian adapted to inhibit pathogen growth has Other researchers have shown that add- influenza (Qiao et al. 2003), infec- been the use of small interfering RNAs ing immune system modifiers/enhanc- tious bursal disease virus (Butter et al. (siRNA), which can be synthesized ers, such as interleukins or gamma inter- 2003), and MDV disease (Lee, Hilt, and easily and designed to target specific feron, to subunit vaccines can decrease Jackwood 2003). Future recombinant genes, resulting in the inhibition of gene oocyst shedding further and induce pro- vaccine efforts will continue the process expression. The technology is still new, tective intestinal immunity against coc- of refining vectors and inserting ever- however, and several problems remain cidiosis (Ding et al. 2004). more immunogenic gene products. But to be addressed adequately. One prob- In general, subunit vaccines are not newer, more imaginative approaches to lem is to protect the siRNA molecule as efficacious as whole organism vac- vaccine development ultimately will be from nuclease degradation; a second is cines. One exception seems to be the needed. to deliver the siRNA molecules to the subunit vaccine developed against the appropriate tissues where the pathogen adenovirus that causes egg-drop syn- Future Developments is replicating. A third problem is the in- drome. A subunit vaccine using just a Advancements in technology and a ability to predict which of several pos- portion of the adenovirus fiber protein growing knowledge of avian pathogens sible siRNA molecules will function to was shown to be significantly more ef- are allowing researchers to explore nov- inhibit gene expression. Often, several ficient than the full-length fiber protein el approaches to vaccine development. different siRNAs must be evaluated (Fingerut et al. 2003). One approach that certainly will increase experimentally before an effective con- Despite some success with subunit with time is the use of immunomodula- struct can be identified. Nevertheless, vaccines, more effort is being applied to tors. In mammalian systems, unmethyl- RNA interference has been used suc- making knockout mutants by deleting ated CpG motifs were shown to function cessfully to inhibit influenza virus virulence genes and using the result- as vaccine adjuvants, which stimulate replication in cell lines and embryo- ing attenuated pathogens as a vector to innate immune responses. In poultry, nated chicken eggs, and it serves as deliver immunogenic gene products. In CpG oligonucleotides were shown to a proof of concept (Ge et al. 2003). one example, the UL0 gene of infec- enhance the resistance of chickens to Undoubtedly, the use of siRNA tech- tious laryngotracheitis virus (ILTV) was coccidiosis (Dalloul et al. 2004). Other nology in future recombinant poultry shown to be nonessential for growth in adjuvants, such as starch microparticles, vaccines will increase. cell culture. Deletion of UL0 resulted in also may prove to be useful (Rydell, More important than addressing an attenuated virus that could be used as Stertman, and Sjoholm 2005). new technologies, future recombinant a vaccine to protect birds against chal- The DNA vaccines based on bacte- vaccines must address the needs of the lenge with pathogenic ILTV. When UL0 rial plasmids have been available for poultry industry. Injection of individu- was replaced by the hemagglutinin gene many years, are produced easily, and al birds is too time-consuming and not from a pathogenic avian influenza virus, are safe. But DNA-based vaccines often economically feasible. Vaccines should the resulting recombinant virus vector have not been very effective or practical be developed that can be administered to

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY multiple birds at one time. viral haemorrhagic septicaemia virus Subsequent experimentation with a Even better would be vaccines that (VHSV). DNA vaccine carrying the glycoprotein can be administered to the embryo, cur- gene was shown to induce a protective rently the industry standard for vaccine Commercially Available antibody response (Boudinot et al. 1998) delivery. Researchers should evaluate Recombinant Vaccines and has been remarkably effective. This their new vaccines in maternal antibody- vaccine may find a niche in protecting positive birds. In the field, nearly every There currently are no recombinant trout from this virus. day-old chick has maternal antibodies, vaccines registered for fish in the United Infectious salmon anemia virus is and testing vaccines in maternal anti- States. Norway, however, has approved a highly infectious enveloped ortho- body-negative birds does not represent a a recombinant IPNV vaccine (Midtlyng myxo-like virus causing acute mortality, true picture of the efficacious nature of et al. 2003); Chile has approved subunit primarily in Atlantic salmon. Studies on the vaccine. recombinant vaccines for salmon rick- 160 isolates show that there exist up to In many instances, current vaccines ettsial septicaemia (SRS) and IPNV; and eight different combinations of hemag- can prevent disease but do not prevent Canada has approved a DNA vaccine for glutinin A and neuraminidase subtypes. replication of the pathogen and the sub- IHNV in farm-raised Atlantic salmon. As in the closely related influenza A sequent transmission of the disease agent The IPNV vaccines are based on the virus, antigenic drift occurs frequent- from one animal to another, known as VP2 protein, which is the major com- ly with these highly variable, con- horizontal spread. Future recombinant ponent of virus particles and an impor- stantly mutating surface glycoproteins, vaccines also must prevent shedding. tant antigen. The SRS vaccine con- which are the most important antigens. Finally, new vaccines should target tains a major antigenic surface protein, Conventional vaccines do not provide economically important diseases for OspA, fused to a measles T-cell stimu- full protection from the disease, but which there are no available vaccines, lating peptide and must be injected. research on both DNA-based vaccines or for which the current vaccines are not Psirickettsia salmonis is refractive to an- as well as VLPs shows some promise very effective. tibiotics and historically has responded (Miller and Cipriano 2002). poorly to live/live attenuated or single Infectious pancreatic necrosis vi- protein vaccination. rus is a birnavirus affecting salmonids Recombinant Vaccines Canada’s IHNV vaccine was the first worldwide. Live attenuated and killed DNA vaccine registered for use in any for Fish vaccines are not 100% effective and are animal. A viral coat protein carried on expensive. The bi-segmented, double- Vaccines have been used for almost a DNA plasmid and injected behind the 20 years in commercial aquaculture. stranded viral RNA genome includes salmon’s dorsal fin protects northern- two structural capsid proteins, VP3 and The economic finfish vaccine targets farmed salmon against this potentially include bacterial and viral pathogens VP2. The VP2 induces neutralizing devastating disease. The plasmids enter antibodies, but there is wide antigenic affecting marine and freshwater fish. muscle cells where the antigenic viral The major marine species of farmed diversity between field strains and sero- protein is produced, which eventually types. All isolates, however, show some fish include salmon, trout, cod, halibut, triggers antibody production and white cobia, turbot, seabass/seabream, yellow- cross-reaction. Recombinant protein blood cell (T-cell) stimulation, providing subunit vaccines have been produced, tail, groupers, and snappers. Freshwater protection against future exposure to the species include catfish, tilapia, and carp. but evidence is controversial as to their virus. Plasmid DNA is not integrated overall protective efficacy. Current re- Fish generally are vaccinated as finger- into the chromosome, and after a few lings through immersion, oral adminis- search is focusing on DNA-based vac- months the cells containing the plasmids cines containing the entire viral poly- tration, or IP. die, leaving virtually no traces other than Salmonids represent the most impor- protein and the addition of CpG motifs the immunological barriers. This vac- to achieve immunostimulatory activity tant segment of the fish vaccine market. cine was a significant step in controlling Farmed salmon smolts are hand-vacci- (Mikalsen et al. 2004). this disease because attenuated modified nated by IP injection during the period live IHNV vaccines have been difficult when they are transferred from fresh- to produce. Future Developments water hatcheries to marine sea cages. Antiviral DNA vaccines continue The major bacterial diseases include to receive considerable attention as a Yersinia ruckeri (ERM), Aeromonas Recent Advances new approach and as a solution to fish salmonicida (Furunculosis), Vibrio Viral hemorrhagic septicemia is diseases that are refractive to traditional species, Renibacterium salmonicida caused by a rhabdovirus that affects fish vaccines. The main advantage of the (BKD), Flavobacterium columnare, worldwide and causes significant mor- vaccines is that they mimic a natural Nocardia species, Streptococcus iniae, tality in trout. The recombinant, inject- viral infection whereby the host reliably Lactococcus garvieae, Psirickettsia sal- able subunit vaccine produced in insect reproduces a single viral protein that monis (SKS), and Edwardsiella ictal- cells infected with a modified baculovi- induces a protective immune response. uri (ESC). Virus pathogens commonly rus and containing the major antigenic Production of viral proteins in the natu- vaccinated for are infectious haemopo- glycoprotein of the virus was effica- ral host elicits both cellular (T-cells) and etic necrosis virus (IHNV), infectious cious, but it was never registered with humoral (antibodies) immune responses, salmon anemia virus (ISAV), infectious the USDA for commercial use because indicating that these vaccines have a pancreatic necrosis virus (IPNV), and of high costs (de Kinkelin et al. 1995). high degree of efficacy (Kim et al. 2000;

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY Leong et al. 1995, 1997). nonavian species. Canarypox does not vectors-one carrying the immunogenic A major obstacle in the progress of replicate in nonavian species, making hemagglutinin gene from a U.S. strain of DNA vaccines has been delivery of suf- it absolutely safe, although it is an ex- equine influenza and the other carrying ficient quantities to elicit protective im- cellent vaccine in mammals because it the hemagglutinin gene from a European mune responses. Subunit vaccines also completes one round of replication in strain of equine influenza virus. The will continue to be developed with mul- nonpermissive cells and induces a robust ProteqFlu-Te (Merial) vaccine contains tiple antigens (proteins) expressed from immune response in the vaccinee. The the equine influenza canarypox vectors a single genetic construct. Progress in development of both humoral (antibod- in a tetanus toxoid diluting agent. adjuvants, manufacturing, and vaccine ies) and cell-mediated (T-cells) immuni- delivery technologies will be critical for ty and the duration of protection induced Recent Advances both DNA and subunit vaccines. Oral by canarypox vectors in mammals are A second-generation Lyme disease vaccination delivered in feed would be well known (Paoletti 1996). In addition, vaccine based on the C-terminal frag- the preferred method of vaccination for prior exposure to canarypox in nonavian ment of the OspA protein of B. burgdor- fish. Different approaches to protect the species does not seem to interfere with feri has been developed to avoid the side antigen (liposomes, biofilms, alginate subsequent vaccinations. effects associated with the whole pro- beads) from gastric neutralization have A number of commercially avail- tein, and as a result, is a safer vaccine. yielded promising results. able canarypox-vectored vaccines have A unique structure-based approach was been developed by Merial (Duluth, used to determine the three-dimensional Recombinant Vaccines Georgia). Canine vaccines include makeup of the protective epitopes and RECOMBITEK Lyme for vaccination generate a stable antigen for use in the for Companion Animals against Lyme disease, RECOMBITEK vaccine (Koide et al. 2005). Structure- (Dogs, Cats, and Horses) Canine Distemper, RECOMBITEK based methods of vaccine development Ferret Distemper (approved for use in are a promising new approach that will The vaccine market for companion ferrets), and PURVAX Canine Rabies. animals is growing more rapidly than allow scientists to target critical im- Commercially available canarypox- any other sector of animal health. This munogenic antigens specifically for in- vector-based feline vaccines include growth is caused partly by demographic corporation into safer, more efficacious PUREVAX Feline Rabies, EURIFEL changes in the human population and vaccines. Feline Leukemia, and PUREVAX Feline partly by the high value associated with Long-term survival after vaccination Leukemia NF. Feline vaccinations are companion animals. Currently, compan- against malignant melanoma—a skin influenced strongly by the appearance of ion animals are vaccinated routinely for cancer—in dogs with advanced disease vaccine-associated injection site muscle a variety of diseases. In the future, vac- has shown some promise. The so-called cell tumors called sarcomas, which are cinations also will address nonpathogen- xenogeneic DNA vaccine carries a hu- associated epidemiologically with tradi- ic conditions such as reproduction, as man tyrosinase-encoding gene that, tional FeLV vaccines and killed rabies well as cancer and periodontal health. when expressed in the vaccinated dogs, vaccines that contain strong adjuvants. Canine vaccinations include results in cytotoxic T-cell responses that The canarypox-vectored vaccine is ad- those for Leptospirosis, kennel cough function to reject the tumor (Bergman et ministered subcutaneously and does not (Bordetella bronchiseptica), Lyme dis- al. 2006). contain an adjuvant, markedly decreas- ease (Borrelia burgdorferi), rabies, dis- Zona pellucida glycoproteins can ing the inflammation at the injection site, temper, adenovirus, parainfluenza virus, be used as immunogens for contracep- which is believed to be the cause of the and parvovirus. Feline vaccinations tion. Contraceptive vaccines based on injection site sarcomas. The PUREVAX target feline herpesvirus, feline calcivi- zona pellucida glycoproteins have been NF canarypox virus carrying the FeLV rus, feline panleukopenia, rabies, feline studied for more than 10 years, but re- subgroup A env and gag genes can be leukemia (FeLV), and feline immuno- cent advances in recombinant vaccine given transdermally in the skin without deficiency virus. Horses commonly are technology—specifically recombinant adjuvant to decrease the inflammation vaccinated for tetanus, rabies, equine proteins, synthetic peptides, and DNA possibly associated with the develop- influenza, and herpesvirus infections. vaccines—have led to longer-lasting im- ment of injection site sarcomas. Depending on the geographic region, munocontraceptive effects and less ovar- however, they also may be vaccinat- Several canarypox vector recombi- ian pathology (Gupta, Chakravarty, and ed for West Nile virus (WNV), equine nant vaccines are commercially avail- Kadunganattil 2005). In the future, con- encephalitis viruses (eastern equine able for horses. West Nile virus is a traceptive vaccines likely will substitute encephalitis virus, Venezuelan equine mosquito-borne virus that emerged in for the castration of domestic pets, farm the United States in 2002 and caused livestock, and wild animals. encephalitis virus, western equine encephalitis virus), rotavirus, and equine disease in nearly one-third of all hors- viral arteritis. es. RECOMBITEK WNV (Merial) is Future Developments a WNV vaccine for horses that induces Recent research in companion ani- protective immunity but does not rep- Commercially Available mal vaccines has focused on recombi- licate in a horse, making it completely nant vaccine development for parasitic Recombinant Vaccines safe. A canarypox-based recombinant diseases. Resistance to drugs used to A poxvirus from a canary was de- vaccine for equine influenza (ProteqFlu, control nematodes has led to the study of veloped into a virus vector for use in Merial) contains two different canarypox various secreted larval antigens for elic-

COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY iting protective immunity. Preliminary pounds into vaccines that can affect the and interleukins). data in mice for protection against type of immune response directly and Differential ELISA test. Detection of hookworms, which affect dogs and cats immunopotentiating compounds that antibodies specific to either the vac- as well as people, used DNA vaccines strengthen the immune response. The cine or the disease agent by the en- and recombinant hookworm proteins antigenic pathway can thus be modu- zyme-linked immunosorbant assay. expressed in the laboratory—as well as lated to stimulate the appropriate arm of Epitope. A molecular region on the sur- unique route, delivery, and adjuvant for- the immune response to provide solid face of an antigen capable of eliciting mulations—to induce protection. protection. Also, new compounds that an immune response and of combin- Other approaches are being used to indirectly stimulate the immune re- ing with the specific antibody pro- identify immunogenic molecules that sponse (such as microparticles and adju- duced by such a response. also are protective against extremely vants) are being studied. These com- Fimbral antigen. Proteins that make up complex protozoan parasites. These ap- pounds are designed to present antigens the hairlike organelles on the surface proaches include identification of genes to the immune system in such a way that of bacteria responsible for motility. in parasites linked to escape from the optimal stimulation is achieved. immune response and the use of mi- The promise of better vaccines to Gametocyte. A cell that makes up one croarrays and immune selection to map benefit animal agriculture and com- of the developmental stages in the re- immunoprotective antigens in parasites. panion animals through rDNA technol- production of coccidiosis. These techniques are being used to iden- ogy is becoming a reality. A number Gamma interferon. A cytokine that en- tify potential vaccine candidates for the of recombinant vaccines are available hances the antimicrobial properties of coccidian parasites that cause enteric commercially, and many more are pro- the immune response. disease in almost all animals. jected to be available soon, so the fu- Gene-deleted. An organism with one ture of recombinant vaccines is bright. or more genes removed from the ge- New efforts need to focus on delivery nome to render it nonpathogenic so it Conclusions methodology as well as development can be used as a vaccine. Vaccines induce an immune re- of vaccines for economically important Glycoprotein. A biomolecule composed sponse in the animal host that subse- diseases for which no currently avail- of a protein and a carbohydrate. quently recognizes infectious agents able vaccines exist or for diseases where Hemagglutinin-esterase. A cell at- and helps fight off the disease; vaccines poorly effective vaccines are currently in tachment protein on the surface of must do this without causing the disease. use. Advances in rDNA technology, in some viruses that recognizes sialic Using recombinant DNA technologies, knowledge of the host immune response, acid residues on the cell surface and scientists have been able to develop live and in the genetic makeup of disease has both the ability to agglutinate red genetically modified organisms, re- agents will lead to new vaccines against blood cells (hemagglutinin) and recombinant killed vaccines, and genetic diseases for which no control measures ceptor-destroying activity (esterase) vaccines that no longer cause disease currently exist. thought to release the virus from the yet induce a strong immune response. cell surface. Developing vaccines using rDNA tech- Glossary Immunodominant epitopes. Regions nologies requires a thorough understand- of a protein that stimulate the produc- ing of the disease agent, particularly the Adjuvant. A compound mixed with tion of antibodies. antigens critical for inducing protec- a vaccine to enhance the immune Immunogenic. Having the ability to tion and the factors involved in causing response. stimulate the immune response. disease. In addition, it is important to Antigen. A substance (usually a pro- Immunomodulators. Compounds ca- understand the immune response of the tein) that is foreign to the animal pable of stimulating or down-regulat- host to ensure that the vaccine induces and stimulates a specific immune ing the immune response. the appropriate immunological reaction. response. Immunopotentiating. Strengthening of Paralleling the development of new, Birnavirus. A bi-segmented RNA virus the immune system. more efficacious, stable, and safe recom- (e.g., infectious bursal disease virus). binant vaccines is the study of vaccine Immunostimulating. Capable of stim- Capsid proteins. Proteins that make delivery methods. In addition to using ulating an immune response. conventional delivery routes such as up the shell of a virus particle that contains the genetic material. Interleukins. Cytokines produced by oral, intranasal, intradermal, transcuta- cells of the immune system that func- Challenge. The process of infecting an neous, intramuscular, and IP, scientists tion to enhance inflammation and ac- animal with a disease agent to test for are experimenting with needle-free sys- tivate the immune response. tems and vaccine strategies that allow protective immunity. Lactogenic antibodies. Antibodies se- mass vaccination of many individuals CpG motif. A genetic element con- creted in milk that are important for simultaneously. tained in DNA vaccines that stimu- providing protection to progeny. Another active area of research is lates the immune system. Leukotoxin. A virulence factor secret- the study of compounds with the po- Cytokine. Substances produced by cells ed from some bacteria that kills host tential to enhance the immune response of the immune system of the animal phagocytic cells in a species-specific to vaccines. These approaches include that function to stimulate or modify manner. incorporating immunomodulating com- the immune response (e.g., interferon

10 COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY Marker vaccine. A recombinant or- Zoonotic. Relating to a disease that is Gupta, S. K., S. Chakravarty, and S. Kadunganattil. 2005. Immunocontraceptive approaches in ganism containing a foreign gene in communicable from animals to hu- females. Chem Immunol Allergy 88:98–108. which, when used as a vaccine, the mans under natural conditions. Jackwood, M. W. 1999. Current and future recom- foreign gene or antibodies to the ex- binant viral vaccines for poultry. Adv Vet Med pressed protein from the foreign gene 41:517–522. iterature ited Jenner, E. 1798. An Inquiry into the Causes and can be detected. Marker vaccines or L C Effects of the Variolae Vaccinae, a Disease animals receiving marker vaccines Discovered in Some of the Western Counties Belli, S. I., K. Mai, C. D. Skene, M. T. Gleeson, D. of England, Particularly Gloucestershire, and can be detected with specific diag- M. Witcombe, M. Katrib, A. Finger, M. G. Wal- Known by the Name of the Cow-Pox. Printed nostic tests. lach, and N. C. Smith. 2004. Characterisation for the author by S. Low, London. of the antigenic and immunogenic properties of Oligonucleotides. A linear nucleic acid Kim, C. H., M. C. Johnson, J. D. Drennan, B. E. Si- bacterially expressed, sexual stage antigens of mon, E. Thomann, and J. A. Leong. 2000. DNA fragment (not necessarily DNA) con- the coccidian parasite, Eimeria maxima. Vac- vaccines encoding viral glycoproteins induce sisting of 2–10 nucleotides joined by cine 22:4316–4325. nonspecific immunity and Mx protein synthesis Bergman, P. J., M. A. Camps-Palau, J. A. McKnight, phosphodiester bonds. in fish. J Virol 74:7048–7054. N. F. Leibman, D. M. Craft, C. Leung, J. Liao, Koide, S., X. Yang, X. Huang, J. J. Dunn, and B. J. Oocyst. Egg stage of the coccidian I. Riviere, M. Sadelain, A. E. Hohenhaus, Luft. 2005. Structure-based design of a second- P. Gregor, A. N. Houghton, M. A. Perales, parasite. generation Lyme disease vaccine based on a and J. D. Wolchok. 2006. Development of a C-terminal fragment of Borrelia burgdorferi Orthomyxo-like virus. A virus similar xenogeneic DNA vaccine program for canine OspA. J Mol Biol 350:290–299. to viruses in the family orthomyxo- malignant melanoma at the Animal Medical Lee, C. W., D. A. Hilt, and M. W. Jackwood. 2003. Center. Vaccine 24:4582–4585. viridae (e.g., influenza). Typing of field isolates of infectious bronchitis Boudinot, P., M. Blanco, P. de Kinkelin, and A. Ben- virus based on the sequence of the hypervari- Pathogenicity. The ability to cause mansour. 1998. Combined DNA immunization able region in the S1 gene. J Vet Diagn Invest disease. with the glycoprotein gene of viral hemorrhagic 15:344–348. septicemia virus and infectious hematopoietic Proteomic studies. Studies on the en- Lee, R. W., J. Strommer, D. Hodgins, P. E. Shewen, necrosis virus induces double-specific protec- Y. Niu, and R. Y. Lo. 2001. Towards develop- tire protein makeup of a cell, includ- tive immunity and nonspecific response in ment of an edible vaccine against bovine pneu- ing protein–protein interactions, pro- rainbow trout. Virology 249:297–306. monic pasteurellosis using transgenic white Butter, C., T. D. Sturman, B. J. Baaten, and T. F. tein modifications, protein functions, clover expressing a Mannheimia haemolytica Davison. 2003. Protection from infectious A1 leukotoxin 50 fusion protein. Infect Immun and the genes involved in expression bursal disease virus (IBDV)-induced immuno- 69:5786–5793. of those proteins. suppression by immunization with a fowlpox Leong, J. C., L. M. Bootland, E. Anderson, P. W. recombinant containing IBDV-VP2. Avian Recombinant deoxyribonucleic acid Chiou, B. Drolet, C. Kim, H. Lorz, D. Mourich, Pathol 32:597–604. P. Ormonde, L. Perez, and G. Trobridge. 1995. (rDNA) technologies. Denotes any Conceicao, F. R., A. N. Moreira, and O. A. Dellagos- Viral vaccines in aquaculture. J Mar Biotechnol manipulation of DNA in the labora- tin. 2006. A recombinant chimera composed 3:16–21. of R1 repeat region of Mycoplasma hyopneu- tory including, but not limited to, Leong, J. C., E. Anderson, L. M. Bootland, P. W. moniae P97 adhesin with Escherichia coli heat- cloning, polymerase chain reac- Chiou, M. Johnson, C. Kim, D. Mourich, and labile enterotoxin B subunit elicits immune G. Trobridge. 1997. Fish vaccine antigens tion, restriction enzyme digestion, response in mice. Vaccine 24:5734–5743. produced or delivered by recombinant DNA ligation, nucleic acid replication, Confer, A. W., S. Ayalew, R. J. Panciera, M. technologies. Dev Biol Stand 90:267–277. Montelongo, and J. H. Wray. 2006. Recom- reverse-transcription, and ribonucleic Liang, R., J. V. van den Hurk, L. A. Babiuk, and S. binant Mannheimia haemolytica serotype van Drunen Littel-van den Hurk. 2006. Priming acid synthesis. 1 outer membrane protein PlpE enhances with DNA encoding E2 and boosting with commercial M. haemolytica vaccine-induced Refractive. Not affected by. (In this E2 protein formulated with CpG oligodeoxy- resistance against serotype 6 challenge. Vaccine context, antibiotics do not have an in- nucleotides induces strong immune responses 24:2248–2255. and protection from bovine viral diarrhea virus hibitory effect on the organism.) Dalloul, R. A., H. S. Lillehoj, M. Okamura, H. Xie, in cattle. J Gen Virol 87:2971–2982. W. Min, X. Ding, and R. A. Heckert. 2004. Spike surface proteins. Petal-like pro- Liao, C. M., C. Huang, S. L. Hsuan, Z. W. Chen, In vivo effects of CpG oligodeoxynucleotide jections on the surface of a corona- W. C. Lee, C. I. Liu, J. R. Winton, and M. S. on Eimeria infection in chickens. Avian Dis Chien. 2006. Immunogenicity and efficacy of virus particle that are involved in at- 48:783–790. three recombinant subunit Pasteurella multo- tachment of the virus to the host cell, de Kinkelin, P., M. Bearzotti, J. Castric, P. Nou- cida toxin vaccines against progressive atrophic gayrede, F. Lecocq-Xhonneux, and M. Thiry. among other things. rhinitis in pigs. Vaccine 24:27–35. 1995. Eighteen years of vaccination against Meyer, C., M. Von Freyburg, K. Elbers, and G. Mey- Synthetic peptides. Short 2–30 amino viral haemorrhagic septicaemia in France. Vet ers. 2002. Recovery of virulent and RNase- acid molecules synthesized through Res 26:379–387. negative attenuated type 2 bovine viral diarrhea Ding, X., H. S. Lillehoj, M. A. Quiroz, E. Bevensee, chemical reactions in the laboratory. viruses from infectious cDNA clones. J Virol and E. P. Lillehoj. 2004. Protective immunity 76:8494–8503. Vectors. In recombinant DNA technol- against Eimeria acervulina following in ovo Midtlyng, P., O. Evensen, E. Rimstad, R. Stagg, E. immunization with a recombinant subunit ogy, it can be (1) a self-replicating Brun, B. Skjelstad, L. Johansen, and I. Jensen. vaccine and cytokine genes. Infect Immun molecule of DNA that serves to 2003. IPN in Salmonids, A Review. VESO and 72:6939–6944. FHL. A report supported by the Fisheries and transfer a gene or foreign DNA frag- Ellis, R. W. 1999. New technologies for making vac- Aquaculture Industries Research Fund and the ment from one organism to another cines. Vaccine 17:1596–1604. Norwegian Research Council, 118 pp. Fingerut, E., B. Gutter, G. Gallili, A. Michael, and J. (usually bacteria) or (2) a virus or Mikalsen, A. B., J. Torgersen, P. Alestrom, A. L. Hel- Pitcovski. 2003. A subunit vaccine against the bacteria containing a foreign gene lemann, E. O. Koppang, and E. 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The mission of the Council for Agricultural Science and Technology (CAST) is to assemble, interpret, and communicate credible science-based information regionally, nationally, and internationally to legislators, regulators, policymakers, the media, the private sector, and the public. CAST is a nonprofit organization composed of 38 scientific societies and many individual, student, company, nonprofit, and associate society members. CAST’s Board of Directors is composed of represen- tatives of the scientific societies and individual members, and an Executive Committee. CAST was established in 1972 as a result of a meeting sponsored in 1970 by the National Academy of Sciences, National Research Council. ISSN 1070-0021 Additional copies of this issue paper are available from CAST. Linda M. Chimenti, Managing Scientific Editor. World WideWeb: http://www.cast-science.org.

Citation: Council for Agricultural Science and Technology (CAST). 2008. Vaccine Development Using Recombinant DNA Technology Issue Paper 38. CAST, Ames, Iowa.