cast vaccine development issue paper final rev153

8/7/2019 CAST Vaccine Development Issue Paper FINAL REV153

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Number 38

May 2008

AbstrActDevelopment o vaccination as a

tool in ghting disease has resulted inthe potential to combat almost all inec-tious agents aecting people and ani-mals. The ultimate objective o vacci-nation is to induce an immune responsethat subsequently recognizes the inec-tious agent and ghts o the disease.

Current public health threats posed bythe potential spread o highly inectiousdisease agents between animals andhumans, as well as the emergence onew diseases, impact animal agriculturesignicantly. Animal vaccinations areamong the most eective, successultools or dealing with these concerns.

This Issue Paper provides a briehistorical overview o vaccine develop-ment and describes three basic catego-ries o newer, recombinant vaccines:live genetically modied organisms,

recombinant inactivated (killed) vac-cines, and genetic vaccines. Separatesections evaluate the development ovaccines or cattle, sheep, and goats;swine; poultry; sh; and companion an-imals. Within each category, the authorsdescribe the vaccines that are commer-cially available, outline the recent ad-vances in recombinant vaccines or thecontrol o specic inectious diseases,and discuss the uture o vaccines oranimal diseases.

Future research needs to ocus onvaccine delivery methodology. In ad-dition, new eorts need to target thedevelopment o vaccines or economi-cally important diseases or which nocurrently available vaccines exist, anddiseases or which poorly eective vac-cines are currently in use. Advancesin recombinant DNA technology, inknowledge o the host immune re-sponse, and in the genetic makeup odisease agents will lead to new vac-

cines against diseases that currentlyhave ew or no control measures.

IntroductIonInectious animal diseases continue

to rank oremost among the signicantactors limiting ecient production inanimal agriculture. In addition, inec-tious agents that are transmitted romanimals to humans by way o oodand water present an increasing threatto the saety and security o the worldood supply and continue to aect hu-man health signicantly. Awareness isincreasing that animal agriculture could

lose the use o several important antimicrobial agents and drug classes ortwo reasons: (1) increased resistanceamong pathogens and (2) public healthreats posed by the potential spreadantimicrobial-resistant zoonotic1 mi-

crobes. Consequently, new approachare needed to develop improved tooland strategies or prevention and control o inectious diseases in animalagriculture. Among the most eectivand successul o these tools are animvaccines.

This material is based upon work supported by the United States Department o Agriculture under Grant No. 2005-38902-02319, Grant No. 2006-38902-035

and Grant No. 2007-31100-06019/ISU Project No. 413-40-02. Any opinions, ndings, conclusions, or recommendations expressed in this publication are tho

o the author(s) and do not necessarily refect the view o the U.S. Department o Agriculture or Iowa State University.

In laboratory tests, a vaccine injection with recombinant DNA is used to im-

munize chicks against coccidia. (Photo courtesy o the USDA Agricultural

Research Service.)

Vaccine Development Using Recombinan

DNA Technology

Animal Agricultures Future through Biotechnology, Part 7

1 Italicized terms (except genus and speciesnames) are dened in the Glossary.


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COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY

CAST Issue Paper 38 Task Force Members

Authors

Reviewers CAST Liaison

Mark W. Jackwood (Chair), PoultryDiagnostic and Research Center,University o Georgia, Athens

Leslie Hickle, Synthetic Genomics,

Inc., La Jolla, CaliorniaSanjay Kapil, Oklahoma AnimalDisease Diagnostic Laboratory,Center or Veterinary Health Sci-ences, Oklahoma State University,Stillwater

HIstoryAnd overvIewofvAccIne development

The history o vaccination dates

back to the 1798 studies by EdwardJenner, an English physician who usedcowpox virus to immunize peopleagainst smallpox (Jenner 1798). Almost200 years later, the comprehensivesmallpox vaccination program estab-lished by the World Health Organizationeventually led to the worldwide eradica-tion o that disease. That success storyis proo o the tremendous potential ovaccination and has led to the devel-opment o vaccines against almost allinectious agents aecting people and

animals. The ultimate objective o vacci-nation is to induce an immune responsethat subsequently recognizes the inec-tious agent and ghts o the disease.

Vaccination usually is accom-plished with either weakened or attenu-ated live agents; with inactivated agentsthat no longer can cause disease; orwith selected, immunogenic parts o thedisease agent called subunit vaccines.Traditional methods o creating vac-cines include using a similar agent thatdoes not cause disease, such as Jennerscowpox virus, or passing a pathogenicdisease agent through a laboratory hostsystem to weaken or attenuate the agent.Inactivating the disease agent with oneor more chemicals also can be used tocreate vaccines. In addition, extracting,puriying, and using one or more partso the disease agent can be used to in-duce a protective immune response.

An immune response is stimulat-ed when a oreign substance called anantigen is encountered by the immune

system. The animals immune systemhas the ability to distinguish between aoreign substance, such as the proteinsin a virus or bacterium, and its own pro-

teins. It does not matter whether the or-eign proteins are rom a disease agent ora vaccine against the disease agent, theimmune response is similar: when theanimal encounters the virus or bacteriaagain, the immune system recognizes itand, ideally, responds to protect the ani-mal rom the disease.

Although vaccination has savedcountless lives, it can have both avor-able and unavorable consequences.Certain vaccinesspecically, live vac-cinescan revert back to pathogenic or-ganisms and produce disease or, in someinstances, even death. The developmento rDNA technologies has provided newways o attenuating disease agents bymodiying their genetic makeup, or ge-nomes, to create saer, more ecaciousvaccines.

The genome o all living beings ismade up o many genes that dene thecharacteristics o the organism. The ge-netic material consists o nucleic acids(DNA and ribonucleic acid [RNA]) thatcarry and convey genetic inormationthrough their bases (adenine, cytosine,

guanine, and thymine); in RNA, thy-mine is replaced by uracil). The basesare uniquely ordered to make up the se-quence o the particular gene. Modiyingor deleting the genes responsible orcausing disease in an organism can beaccomplished in the laboratory usingrDNA technologies.

Typically, rDNA technology reersto laboratory methods used to break andrecombine DNA molecules rom dier-

Robert Silva, Avian Disease andOncology Laboratory, USDAARS,East Lansing, Michigan

Klaus Osterrieder, College o Vet-

erinary Medicine, Cornell University,Ithaca, New York

Christopher Prideaux, CSIROLivestock Industries, St. Lucia,Queensland, Australia

Ronald D. Schultz, School oVeterinary Medicine, University oWisconsinMadison

Alan W. Bell, CSIRO Livestock

Industries, St. Lucia, Queensland,Australia

Using recombinant deoxyribonucle-ic acid (rDNA) technologies, scientistshave been able to develop three types orecombinant vaccines: (1) live geneti-

cally modied organisms, (2) recombi-nant inactivated (killed) vaccines, and(3) genetic vaccines. These vaccines nolonger cause disease, but still induce astrong immune response. Paralleling thedevelopment o new, more ecacious,stable, and sae recombinant vaccineshas been the study o vaccine deliverymethods and immunostimulating adju-vantcompounds that enhance the im-mune response.

Advances in gene discovery o ani-mal pathogens can be expected to iden-tiy new proteins and metabolic path-ways, thereby providing a oundationor improved understanding o pathogenbiology and, ultimately, aiding in the de-sign o new and eective therapies. Newtreatments, whether vaccines or newdrugs, must rely on more than empiricalmethods o discovery and must be basedon a undamental knowledge o patho-gen biology and genetics.

This Issue Paper rst provides abrie historical overview o vaccine de-velopment and describes the three basiccategories o recombinant vaccines. The

ollowing sections evaluate the develop-ment o vaccines or cattle, sheep, andgoats; swine; poultry; sh; and compan-ion animals. Within each category, theauthors describe the vaccines that arecommercially available, outline the re-cent advances in recombinant vaccinesor the control o inectious diseases,and discuss the uture o vaccines oranimal diseases.


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ent organisms. The terminology also hasgrown to include laboratory methodsused or gene isolation, sequence modi-cation, nucleic acid synthesis, and genecloning.

Using rDNA technologies, scien-tists can isolate a disease agent, reduceit to its basic components, examine itsgenetic makeup, and modiy it so that itno longer causes disease but still inducesa strong immune response. Methodso extracting and puriying genes romextremely small amounts o even thetiniest organisms have become routineor many laboratories. Once extracted,the nucleic acids can be modied andthe genes reinserted into the organism toproduce a vaccine that is attenuated and/or capable o inducing better immuno-logical protection.

Vaccine development using rDNAtechnologies requires a thorough under-

standing o the disease agent, particu-larly the antigens critical or inducingprotection. In addition, it is importantto understand the pathogenicity o thedisease agent and the immune responseo the host, to ensure that the vaccineinduces the appropriate immunologicalreaction. Increasingly, genetic inorma-tion rom both microbial genomics stud-ies and proteomic studies is being usedto gain a better understanding o the in-teractions between the disease agent andthe host. Nonetheless, vaccines devel-

oped through recombinant technologiesare designed to be saer, more eca-cious, and/or less expensive than tradi-tional vaccines.

Categories of VaccinesRecombinant vaccines all into three

basic categories: live genetically modi-ed organisms, recombinant inactivatedvaccines, and genetic vaccines (Ellis1999).

Live Genetically Modifed Vaccines

The rst categorylive geneticallymodied vaccinescan be viruses orbacteria with one or more genes deletedor inactivated, or they can be vaccinescarrying a oreign gene rom anotherdisease agent, which are reerred to asvaccine vectors. Deletion or gene-in-activated vaccines are developed toattenuate the disease agent. Generallytwo (double-knockout) or more genesare deleted or inactivated so the vaccine

remains stable and cannot revert to apathogenic agent (Uzzau et al. 2005).

Developing a vaccine o this typerequires knowledge o the gene(s) re-sponsible or pathogenicity and assumesthat those genes are not the same genesgoverning viability and the ability o themodied organism to induce an immuneresponse. Examples o gene-deleted vac-cines include a Salmonella vaccine orsheep and poultry and a pseudorabiesvirus vaccine or pigs.

Another relatively recent methodo creating a live genetically modiedvaccine is to use an inectious clone othe disease agent. An inectious clone iscreated by isolating the entire genomeo the disease agent (usually viruses) inthe laboratory. This isolated or clonedgenome can be specically and purpose-ully modied in the laboratory and thenused to re-create the live genetically

modied organism.Vector-based vaccines are bacteria,

viruses, or plants carrying a gene romanother disease agent that is expressedand then induces an immune responsewhen the host is vaccinated. For viraland bacterial vectors, the vaccine inducesa protective response against itsel (thevector) as well as the other disease agent.Foreign genes must be inserted into thegenome o the vaccine vector in such away that the vaccine remains viable.

The rst commercial vaccine vector

was VectorVax FP-N (Zeon Corporation,Japan), a vaccine primarily used in tur-keys; it consists o a owl pox vaccinevirus that carries genes rom Newcastledisease virus. Other agents used as vec-tors o oreign genes are Salmonella,herpesviruses, adenoviruses, and adeno-associated viruses. Edible plant-derivedvaccines take advantage o the abilityo some antigens to induce an immuneresponse when delivered orally. Foreigngenes rom disease agents have been in-serted into potatoes, soybeans, and cornplants and ed to animals; the expressedproteins rom those oreign genes im-munized the animals against the diseaseagent (Streateld 2005).

Recombinant Inactivated Vaccines

The second categoryrecombinantinactivated vaccinesare subunit vac-cines containing only part o the wholeorganism. Subunit vaccines can be syn-thetic peptides that are synthesized in

the laboratory and represent the mostbasic portion o a protein that induces animmune response. Subunit vaccines alsocan consist o whole proteins extract-ed rom the disease agent or expressedrom cloned genes in the laboratory.Several systems can be used to expressrecombinant proteins, including expres-sion systems that are cell ree or thatuse whole cells. Whole-cell expressionsystems include prokaryotic (bacteria-based) systems such as Escherichia coli,and eukaryotic (mammalian, avian, in-sect, or yeast-based) systems.

The baculovirus expression sys-tem is a widely used eukaryotic systembecause it is applicable to many dier-ent proteins and because relatively largeamounts o protein can be produced.Baculovirus expression systems are en-gineered specically or expression oproteins in insect cells. It is important to

express proteins o disease agents withthe greatest possible similarity to the au-thentic molecule so that the proper im-mune response is induced when the pro-teins are used as a vaccine. Baculovirusexpression systems are eective orproperly modiying recombinant pro-teins so they are antigenically, immuno-genically, and unctionally similar to thenative protein.

Another type o recombinant sub-unit vaccines, called virus-like particles(VLPs), can be created when one or

more cloned genes that represent thestructural proteins o a virus are ex-pressed simultaneously and sel-assem-ble into VLPs. These VLPs are immu-nogenic (i.e., they look like the virus onthe outside, but cannot replicate becausethey do not contain any genetic materialon the inside).

Because subunit vaccines do notreplicate in the host, they usually are ad-ministered (injected) with an adjuvant,a substance that stimulates the immunesystem o the animal to respond to thevaccine. The mechanisms o action omany adjuvants are poorly understood;consequently, they usually are selectedon a trial-and-error basis. Adjuvants canenhance the response to a vaccine byprotecting the vaccine rom rapid deg-radation in the animal; these are usu-ally oil-based adjuvants. Or, they canattract so-called antigen-presenting cellswhich process and deliver antigens tothe immune system. Adjuvants also canbe molecules that enhance the immune


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response by stimulating immune cellsdirectly, or by stimulating immune-modiying and immune-strengthening orimmunopotentiating substances calledcytokines. In addition, adjuvants can bea combination o these types.

Genetic Vaccines

The third category o recombinantvaccines is reerred to as genetic vac-cines because they are actually DNAalone. Genetic or DNA vaccines usu-ally are circular pieces o DNA, calledplasmids, which contain a oreign generom a disease agent and a promoterthat is used to initiate the expression othe protein rom that gene in the targetanimal (Rodriguez and Whitton 2000).Plasmids can be maintained in bacteria(usually E. coli) and have been designedto accept oreign genes or expressionin animals. The recombinant plasmids

containing a oreign gene are puriedrom the bacteria, and the naked DNAis injected directly into the animal, usu-ally intramuscularly or intradermally(into the skin). The animals cells takeup the DNA, and an immune responseis induced to the protein expressed romthe oreign gene.

In addition to genes coding or im-munogenic proteins, genetic vaccinesalso have been designed to include di-erent immune-stimulatory genes thattrigger dierent compartments o the

immune system, depending on the typeo immunity desired. Unique eatures oDNA vaccines are intrinsic sequencesembedded in the DNA, so-called CpGmotis. These unmethylated motis wereshown to act as an adjuvant, stimulatingthe innate immune responses and en-hancing the eectiveness o the vaccine.

The ollowing sections describecommercially available recombinantvaccines or ruminants, swine, poul-try, sh, and companion animals. Inaddition, each section addresses recentadvances in recombinant vaccines orcontrol o inectious agents in those ani-mals, as well as uture vaccine technolo-gies being explored or animal healthand protection.

recombInAnt vAccInesfor cAttle, sHeep, AndGoAts

Clinically and economically signi-cant diseases o cattle include the bovine

respiratory disease complex (shippingever) in eedlot cattle, cal-scours andrespiratory diseases in cow-cal opera-tions, and abortions. Pathogens com-monly vaccinated against in cattle areLeptospira sp., E. coli, Clostridia sp.,Mannheimia haemolytica (also sheepand goats), Haemophilus somnus, in-ectious bovine rhinotracheitis (IBR),bovine viral diarrhea (BVD), bovine re-spiratory syncytial virus (BRSV), para-infuenza-3 (PI-3), rotavirus, and bovinecoronavirus (BCoV). Two weeks be-ore weaning/arrival at the eedlot, cattletypically are vaccinated against respira-tory disease and clostridial inections, aspart o prearrival conditioning. Vaccinesused in sheep and goats include prod-ucts or overeating disease causedby a bacterium, Clostridium perrin-gens types C and D, tetanus toxoid, soremouth (contagious ecthyma) caused by

poxvirus, diarrhea caused by E. coli,inectious causes o abortion in sheep(Campylobacter etus and Chlamydiapsittaci), ootrot in sheep caused bytwo bacteria (Bacteroides nodosus andFusobacterium necrophorum), and PI-3respiratory disease in lambs. Nearly allvaccines or cattle, sheep, and goats cur-rently licensed by the U.S. Departmento Agriculture (USDA) were developedusing conventional technologies.

Commercially Available

Recombinant VaccinesRecombinant bovine vaccines have

not been licensed or use in the UnitedStates. The European Union, however,has approved a naturally occurringglycoprotein E (gE)-deleted IBR vaccineor its IBR eradication program. Inec-tious bovine rhinotracheitis is a herpes-virus responsible or respiratory diseasein eedlot cattle as well as or reproduc-tive diseases, conjunctivitis, and nervousdisorders. Deletion o the gE geneattenuates the IBR virus and prevents it

rom being shed in bovine secretions,limiting its transmission. This modied-live, naturally occurring, gene-deletedvaccine is immunogenic and sae orcattle o all ages.

An important advantage o this vac-cine is that the gE-deleted vaccine virusand wild-type IBR viruses can be dier-entiated by the polymerase chain reac-tion, a method o ampliying very smallamounts o nucleic acid to detectiblelevels (Schynts et al. 1999). Thus, the

vaccinated cattle can be distinguishedrom naturally inected animals, whichis critical or eradication o this disease.

There are no commercially availablerecombinant vaccines used or sheepand goats in the United States.

Recent Advances

Bovine viral diarrhea virus (BVDV)causes respiratory, enteric, and eye in-ections, as well as abortions, in cattle.The virus is thought to play a role inbovine respiratory disease complex be-cause it causes an immunosuppressionthat leads to bacterial pneumonia (ship-ping ever). The E2 surace glycoproteinis a major immunogen on the virus, andDNA vaccination using the E2 pro-tein gene o BVDV provides protectionagainst the disease (Nobiron et al. 2003)

A more recent study ound thatDNA vaccination with the E2 proteingene ollowed by a boost vaccinationwith recombinant E2 protein providedgood protection against the pathogenicNY-strain o BVDV (Liang et al. 2006).Another genetic vaccine tested in cattleused RNA rom a vaccine strain oBVDV in a microparticle bombardmentmethod (Vassilev, Gil, and Donis 2001).The vaccinated cattle developed protec-tive neutralizing antibodies. In addi-tion, a genetically engineered E2-deletedBVDV isolate, which can be propagatedeciently in an E2-expressing cell line

in the laboratory, will inect and inducean immune response in cattle. But be-cause it is lacking E2, it cannot produceinectious virus particles, making itextremely sae (Reimann, Meyers, andBeer 2003).

Likewise, a gene-deletedvaccine orBRSV does not produce inectious virusparticles ater vaccination. Bovine respi-ratory syncytial virus, a cause o pneu-monia, plays an important role in ship-ping ever o cattle. The G spike proteinlocated on the surace o the virus is one

o the major immunogenic viral pro-teins. Genetically engineered BRSVlacking the G spike protein gene still canbe propagated in cell culture, and vac-cinated calves produce neutralizing anti-bodies, but no inectious virus particlesare ormed (Schmidt et al. 2002).

Capripox causes diseases such assheep pox and goat pox in domesticsmall ruminants. It also can cause lumpyskin disease in cattle and possibly inbualo. Capripox vaccine is a poxvirus


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being used as a vaccine vector or twoimportant sheep and goat diseasesrin-derpest and peste-des-petits-ruminants.Those diseases are caused by a para-myxovirus that is not endemic in theUnited States.

Future Developments

The process o distinguishing in-ected rom vaccinated animals, knownas the DIVA approach, is essential ordisease eradication programs. The useo gene-deleted or marker vaccines pro-vides a method to distinguish vaccinatesrom naturally exposed animals by test-ing or antibodies directed against themarker in the vaccine or proteins uniqueto the wild-type virus. One example isa BVDV gene-deleted vaccine that wasmodied to inactivate the RNase genewithin the viral glycoprotein E gene.That deletion resulted in an attenuatedvirus, which could be an eective vac-cine against BVD (Meyer et al. 2002).Control programs targeting the removalo persistently inected animals use vac-cination as part o an overall strategy toachieve a status o BVDV-ree. Thoseprograms would benet greatly romsuch a marker vaccine.

Vaccines or Mannheimia haemo-lytica, the major pathogen involved inshipping ever in cattle and pneumoniain sheep and goats, have ocused on itsleukotoxin, which is the primary viru-

lence actor. The leukotoxin gene hasbeen cloned and expressed in cell linesas well as in plants, and the recombi-nant protein induces leukotoxin-neu-tralizing antibodies (Lee et al. 2001).Immunodominant epitopes on a majorM. haemolytica surace lipoprotein,designated PlpE, also have been identi-ed as potential vaccine candidates, andantibodies against them are eective incomplement-mediated M. haemolyticakilling. A recombinant PlpE protein wasexpressed in the laboratory, added to a

commercial M. haemolytica vaccine,and shown to enhance protection incalves (Coner et al. 2006).

Bovine coronavirus causes caldiarrhea, or scours, inecting the en-tire length o the intestinal tract.Recombinant vaccines against BCoV aredicult to develop because it is hard toreproduce the viral proteins accuratelyin the laboratory. Viral hemagglutinin-esterase (HE) and spike surace pro-teins have been used as subunit vaccines

against BCoV, but only partial protec-tion has been induced. A recombinanthuman adenovirus vector expressingthe BCoV HE immunogen was devel-oped, and in a mouse model, antibod-ies induced against HE were detected inserum and lung washes. It is not cleari this vaccine vector will be eectiveagainst BCoV in cattle (Yoo et al. 1992).

Bovine rotavirus (BRV) is respon-sible or scours in approximately 30% ocal viral enteritis cases. The BRV spikesurace protein is cleaved by trypsin intothe viral proteins VP5 and VP8. Theimmunogenic VP8 subunit induces anti-bodies that can neutralize the virus in thelaboratory, making it a good candidateor a subunit vaccine.

A unique protein expression systemhas been developed using a tobacco mo-saic virus vector in tobacco plants. Thetobacco mosaic virus vector containing

the VP8 BRV gene is used to inect thetobacco plant, and the expressed recom-binant VP8 protein is extracted rom theplant leaves. Ater intraperitoneal (IP)inoculation o the material containingthe VP8 recombinant protein, antibodyresponses were detected against BRVin mice and protected the mice againstinection with BRV (Perez Filgueira etal. 2004). Moreover, triple-layer VLPs(complete BRV particles have threelayers) have been produced using viralproteins expressed using the baculovirus

system. Those VLPs were ound to in-duce lactogenic antibodies. But like allVLPs, they are sae in that they do notreplicate in the host because they do notcontain any genetic material.

E. coli with the K99 fmbrial an-tigen commonly causes diarrhea inyoung cattle, sheep, and goats. FanCis the major subunit o this immuno-genic protein. Agrobacterium-mediatedtransormation, a method o deliveringoreign genes to plants, has been usedsuccessully to express the FanC subunito K99 in soybeans as a rst step to de-

veloping an edible vaccine (Piller et al.2005). When mice were injected withprotein extracted rom the soybeans,they developed FanC-specic antibod-ies and cytotoxic T-cell responses, whichdemonstrates that the protein expressedin plants can unction as an immunogen.It is not clear, however, whether oralvaccination that works in animals witha monogastric digestive tract, such asmice, will work in cattle or sheep, whichhave a ruminant digestive tract.

A raccoon pox virus expressing arabies virus glycoprotein has been testedin sheep. In addition, subunit vaccinesagainst parasites such as the cestode par-asite Taenia ovis in sheep and ectopara-sites like ticks have been developed orsheep and goats.

recombInAnt vAccInesfor swIne

In the United States, approximate-ly 75% o swine producers vaccinateagainst common swine pathogens in-cluding Mycoplasma sp.; porcine repro-ductive and respiratory syndrome virus;Erysipelas; E. coli scours; parvovi-rus; Leptospira sp.; H3N2 and H1N1swine infuenza; and bacterial rhinitiscaused by Pasteurella, Bordetella, orMycoplasma. Primarily, conventional

vaccines are the ones licensed and usedin commercial swine. But rDNA vac-cines are being developed to urtherdecrease or eliminate economically im-portant diseases in domestic swine andto prevent reintroduction o these patho-gens rom eral swine populations.

Commercially AvailableRecombinant Vaccines

The DIVA approach, as discussedor cattle, also has been used or theeradication o pseudorabies virus (PRV)

in commercially grown pigs in theUnited States. Pseudorabies is an acute,highly atal disease o pigs. Although itis caused by a herpesvirus (not the virusthat causes rabies), the clinical signsare similar to rabies. Pseudorabies virusglycoprotein I (gI) and glycoprotein X(gX) gene-deleted vaccines were usedor PRV eradication in the United StatesPiglets intranasally immunized withthe gene-deleted vaccine can be distin-guished rom inected animals by a di-erential enzyme-linked immunosorbant

assay (ELISA) that detects the presenceo antibodies against gI and/or gX, indi-cating that the animals were exposed topathogenic PRV (Swenson, McMillen,and Hill 1993). The USDA-licensedgene-deleted vaccine pairs well with thePRV dierential ELISA test(IDEXXLaboratories, Westbrook, Maine). As oMay 2005, the United States achievedpseudorabies-ree status (Stage V), andvaccination no longer is practiced incommercial swine.


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Recent AdvancesProgressive atrophic rhinitis in swine

is caused by a Pasteurella multocidatoxin and is characterized by nasal turbi-nate bone degeneration, distortion o thenose and ace, and nasal hemorrhage.Inactivated toxin, or toxoid, which isused as a vaccine, can lose some o its

immunogenic properties when it is inac-tivated. To circumvent that problem butstill maintain a sae vaccine, scientiststested ragments o recombinant toxinexpressed in E. coli or the ability toprotect against progressive atrophic rhi-nitis. Vaccination o piglets resulted intoxin-neutralizing antibodies. Sows im-munized with recombinant subunit toxinhad high serum antibodies, and theirprogeny had improved survival ater ex-posure to a lethal dose o authentic toxincompared with animals immunized withconventional toxoid vaccine (Liao et al.2006).

Future DevelopmentsThe coronavirus transmissible gas-

troenteritis virus (TGEV) causes severediarrhea in piglets. The major antigenicprotein o TGEV is the spike glyco-protein. Attempts to aithully re-cre-ate these antigenic regions in recom-binant vaccines have proved dicult,but several vaccines hold promise.Recombinant pseudorabies virus carry-ing the TGEV spike gene was construct-ed and may have potential as a recombi-nant vaccine against both pseudorabiesand TGEV.

In addition, Lactobacillus casei andSalmonella enterica (serovar typhimuri-um) expressing the TGEV spike, orportions o the spike protein, have beenshown to induce neutralizing antibodiesin serum and intestinal secretions o pigsand mice, respectively. A DNA vaccine,an adenovirus serotype 5 vector, andplant-derived vaccines containing thespike or nucleoprotein genes o TGEV

also have been constructed and shownto elicit humoral and cell-mediated im-mune responses in mice.

One o the big problems with induc-ing immunity in young animals is thepresence o maternally derived antibod-ies against the antigen. To overcomeantibody intererence o vaccination inswine, a human adenovirus 5 vector,delivering the hemagglutinin and nu-cleoprotein o swine infuenza virus, is

being developed. Because piglets do nothave maternal antibodies against humanadenovirus 5, the intererence observedwith conventional swine infuenza vac-cines does not occur (Wesley and Lager2006). But i sows are vaccinated withthe human adenovirus 5 vector, the ma-ternal antibodies passed to their progenywill interere with vaccination becausehuman adenovirus 5 is susceptible toneutralization by those antibodies.

Swine mycoplasmal pneumonia iscaused by the bacterium Mycoplasmahyopneumoniae, which is worldwide indistribution. The disease is a signicantburden on the swine breeding industrybecause it causes losses associated withsevere respiratory disease and predispos-es pigs to secondary invaders that cancause atal inections. Although inac-tivated whole-cell vaccines (bacterins)are sae and currently the most eective

means o controlling the disease, theyare not always ecacious against M.hyopneumoniae because it is dicult toinduce an immune response that protectsagainst the clinical signs and lesions as-sociated with this disease.

Thus, rDNA technology is beingused to produce subunit vaccines con-taining the P97 adhesin protein respon-sible or adherence o the bacterium toswine ciliated epithelial cells. Adhesionis important or virulence, and both lo-cal and systemic antibodies have been

produced in mice to a recombinant sub-unit vaccine containing a portion o P97(the R1 repeat region) coupled to highlyimmunogenic enterotoxin subunit Brom E. coli (Conceicao, Moreira, andDellagostin 2006).

recombInAnt vAccInesfor poultry

The U.S. consumption o poultrymeat exceeds consumption o eitherbee or pork. Currently, the U.S. poultry

industry is the worlds largest producerand exporter o poultry meat. The tre-mendous numbers o animals raised inrelatively conned environments pro-motes the spread o inectious diseaseagents. The act that individual animalsare o so little value makes it economi-cally uneasible to attempt to treat sickbirds. Consequently, the poultry indus-trymore than any other industryre-lies on vaccines to prevent outbreaks

o inectious disease. Nearly all thesepoultry vaccines are conventional vac-cines that have worked well in general.But avian pathogens continue to changeand develop ways to evade the immu-nity induced by the current vaccines.Thus, there is an ongoing race to nd ordevelop new vaccines.

Commercially Available Re-combinant Vaccines

Currently, there are 11 U.S. licensedrDNA poultry vaccines (Table 1). Teno these vaccines are live recombinantviral vectors designed to deliver specicgenes to stimulate the hosts immunesystem. Seven o these use an attenuatedowl pox virus (FPV) as a vector to de-liver selected pathogen genes. Three usean attenuated vaccine, Mareks diseasevirus (MDV), or a closely related non-pathogenic turkey herpesvirus as a vec-tor. Surprisingly, only one license is ora bacterial pathogen, Salmonella, whichis a double-knockout mutant resulting ina stable attenuated bacterium. Anotherinteresting observation is that the list opathogens being targeted by rDNA vac-cines is small compared with the list oactual poultry pathogens.

Not all poultry pathogens pose thesame degree o risk to the industry. Oneexcellent source o inormation on cur-rent poultry pathogens that pose thegreatest risk or the poultry industry is

the research priorities list establishedby the Cooperative State Research,Education, and Extension Service, aUSDA granting agency. Their 2006high-unding priority list includes avianClostridium perringens, Mareks dis-ease, poult enteritis complex (a multiac-torial disease aecting young turkeys),avian infuenza, and exotic Newcastledisease. The list indicates that currentconventional and recombinant vaccinesdesigned to protect against these diseas-es are not very ecacious. It seems that

the industry needs better vaccines thanthose listed in Table 1 or MDV disease,avian infuenza, and exotic Newcastledisease, and that vaccine companies andresearchers should consider developingrecombinant vaccines or C. perringensand poult enteritis complex.

Recent AdvancesEarlier reviews listed numerous

reports o recombinant vaccines under


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development (Jackwood 1999). One othe simplest recombinant vaccines toconstruct is a subunit vaccine containing

only the immunogenic part o the inec-tious agent. As an example, research-ers have identied gametocyte antigensrom coccidia that can be administeredas a subunit vaccine (Belli et al. 2004).Other researchers have shown that add-ing immune system modiers/enhanc-ers, such as interleukins or gamma inter-eron, to subunit vaccines can decreaseoocystshedding urther and induce pro-tective intestinal immunity against coc-cidiosis (Ding et al. 2004).

In general, subunit vaccines are not

as ecacious as whole organism vac-cines. One exception seems to be thesubunit vaccine developed against theadenovirus that causes egg-drop syn-drome. A subunit vaccine using just aportion o the adenovirus ber proteinwas shown to be signicantly more e-cient than the ull-length ber protein(Fingerut et al. 2003).

Despite some success with subunitvaccines, more eort is being applied tomaking knockout mutants by deletingvirulence genes and using the result-

ing attenuated pathogens as a vector todeliver immunogenic gene products. Inone example, the UL0 gene o inec-tious laryngotracheitis virus (ILTV) wasshown to be nonessential or growth incell culture. Deletion o UL0 resulted inan attenuated virus that could be used asa vaccine to protect birds against chal-lenge with pathogenic ILTV. When UL0was replaced by the hemagglutinin generom a pathogenic avian infuenza virus,the resulting recombinant virus vector

protected against both ILTV and infu-enza virus (Veits et al. 2003).

The large genome o the FPV virus,

and its ability to incorporate large rag-ments o oreign DNA, makes FPV-based vaccine vectors a popular choice.Recently, FPV vaccine vectors weremade to express antigens rom avianinfuenza (Qiao et al. 2003), inec-tious bursal disease virus (Butter et al.2003), and MDV disease (Lee, Hilt, andJackwood 2003). Future recombinantvaccine eorts will continue the processo rening vectors and inserting ever-more immunogenic gene products. Butnewer, more imaginative approaches to

vaccine development ultimately will beneeded.

Future DevelopmentsAdvancements in technology and a

growing knowledge o avian pathogensare allowing researchers to explore nov-el approaches to vaccine development.One approach that certainly will increasewith time is the use oimmunomodula-tors. In mammalian systems, unmethyl-ated CpG motis were shown to unctionas vaccine adjuvants, which stimulate

innate immune responses. In poultry,CpG oligonucleotides were shown toenhance the resistance o chickens tococcidiosis (Dalloul et al. 2004). Otheradjuvants, such as starch microparticles,also may prove to be useul (Rydell,Stertman, and Sjoholm 2005).

The DNA vaccines based on bacte-rial plasmids have been available ormany years, are produced easily, andare sae. But DNA-based vaccines otenhave not been very eective or practical

in poultry. The primary problems havebeen to deliver sucient quantities oDNA to elicit an eective immune re-sponse, and to mass-vaccinate thousandso birds in a fock. One promising ap-proach currently being studied is the useo lipids or other micro-DNA-encapsu-lating compounds to protect and deliverthe DNA directly to the host cell (Oshopet al. 2003).

Virus-like particles are sae, immu-nogenic vaccines, but ew VLP poultryvaccines have been tested. A VLP vac-cine or inectious bursal disease hasbeen described by Rogel and colleagues(2003). That vaccine was produced byexpressing viral proteins (VP2, VP4,and VP3) in E. coli, which assembledinto VLP and protected chickens againstinectious bursal disease. Although thevaccine was injected intramuscularly,which is not a practical delivery mecha-

nism or large focks o commercialpoultry, the vaccine does show promiseor the uture.

One o the most recent technologiesadapted to inhibit pathogen growth hasbeen the use o small interering RNAs(siRNA), which can be synthesizedeasily and designed to target specicgenes, resulting in the inhibition o geneexpression. The technology is still new,however, and several problems remainto be addressed adequately. One prob-lem is to protect the siRNA molecule

rom nuclease degradation; a second isto deliver the siRNA molecules to theappropriate tissues where the pathogenis replicating. A third problem is the in-ability to predict which o several pos-sible siRNA molecules will unction toinhibit gene expression. Oten, severaldierent siRNAs must be evaluatedexperimentally beore an eective con-struct can be identied. Nevertheless,RNA intererence has been used suc-cessully to inhibit infuenza virusreplication in cell lines and embryo-nated chicken eggs, and it serves asa proo o concept (Ge et al. 2003).Undoubtedly, the use o siRNA tech-nology in uture recombinant poultryvaccines will increase.

More important than addressingnew technologies, uture recombinantvaccines must address the needs o thepoultry industry. Injection o individu-al birds is too time-consuming and noteconomically easible. Vaccines shouldbe developed that can be administered to

Table 1. Currently licensed rDNA poultry vaccines1 (Source: Data provided by Michel Carr,

USDA-Animal and Plant Health Inspection Service.)

Company Pathogen Vector

Lohmann Animal Health Salmonella typhimurium Double-deletion mutant

Merial Select AIV, FPV FPV

Merial Select NDV, FPV FPV

Merial Select IBDV, MDV HVT

Intervet MDV HVT

Intervet MDV, NDV MDV

Biomune Co. AEV, FPV, LTV FPV

Biomune Co. NDV, FPV FPV

Biomune Co. FPV, MG FPV

Biomune Co. AEV, FPV, MG FPV

Biomune Co. FPV, LTV FPV

1AIV, avian infuenza virus; NDV, Newcastle disease virus; IBDV, inectious bursal disease virus;

MDV, Mareks disease virus; AEV, avian encephalomyelitis virus; FPV, owl pox virus; LTV,

laryngotracheitis virus; HVT, turkey herpesvirus; MG, Mycoplasma gallisepticum.


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multiple birds at one time.Even better would be vaccines that

can be administered to the embryo, cur-rently the industry standard or vaccinedelivery. Researchers should evaluatetheir new vaccines in maternal antibody-positive birds. In the eld, nearly everyday-old chick has maternal antibodies,and testing vaccines in maternal anti-body-negative birds does not represent atrue picture o the ecacious nature othe vaccine.

In many instances, current vaccinescan prevent disease but do not preventreplication o the pathogen and the sub-sequent transmission o the disease agentrom one animal to another, known ashorizontal spread. Future recombinantvaccines also must prevent shedding.

Finally, new vaccines should targeteconomically important diseases orwhich there are no available vaccines,

or or which the current vaccines are notvery eective.

recombInAnt vAccInesfor fIsH

Vaccines have been used or almost20 years in commercial aquaculture.The economic nsh vaccine targetsinclude bacterial and viral pathogensaecting marine and reshwater sh.The major marine species o armedsh include salmon, trout, cod, halibut,

cobia, turbot, seabass/seabream, yellow-tail, groupers, and snappers. Freshwaterspecies include catsh, tilapia, and carp.Fish generally are vaccinated as nger-lings through immersion, oral adminis-tration, or IP.

Salmonids represent the most impor-tant segment o the sh vaccine market.Farmed salmon smolts are hand-vacci-nated by IP injection during the periodwhen they are transerred rom resh-water hatcheries to marine sea cages.The major bacterial diseases include

Yersinia ruckeri (ERM), Aeromonassalmonicida (Furunculosis), Vibriospecies, Renibacterium salmonicida(BKD), Flavobacterium columnare,Nocardia species, Streptococcus iniae,Lactococcus garvieae, Psirickettsia sal-monis (SKS), and Edwardsiella ictal-uri (ESC). Virus pathogens commonlyvaccinated or are inectious haemopo-etic necrosis virus (IHNV), inectioussalmon anemia virus (ISAV), inectiouspancreatic necrosis virus (IPNV), and

viral haemorrhagic septicaemia virus(VHSV).


There currently are no recombinantvaccines registered or sh in the UnitedStates. Norway, however, has approved

a recombinant IPNV vaccine (Midtlynget al. 2003); Chile has approved subunitrecombinant vaccines or salmon rick-ettsial septicaemia (SRS) and IPNV; andCanada has approved a DNA vaccine orIHNV in arm-raised Atlantic salmon.

The IPNV vaccines are based on theVP2 protein, which is the major com-ponent o virus particles and an impor-tant antigen. The SRS vaccine con-tains a major antigenic surace protein,OspA, used to a measles T-cell stimu-lating peptide and must be injected.

Psirickettsia salmonis is reractive to an-tibiotics and historically has respondedpoorly to live/live attenuated or singleprotein vaccination.

Canadas IHNV vaccine was the rstDNA vaccine registered or use in anyanimal. A viral coat protein carried ona DNA plasmid and injected behind thesalmons dorsal n protects northern-armed salmon against this potentiallydevastating disease. The plasmids entermuscle cells where the antigenic viralprotein is produced, which eventuallytriggers antibody production and whiteblood cell (T-cell) stimulation, providingprotection against uture exposure to thevirus. Plasmid DNA is not integratedinto the chromosome, and ater a ewmonths the cells containing the plasmidsdie, leaving virtually no traces other thanthe immunological barriers. This vac-cine was a signicant step in controllingthis disease because attenuated modiedlive IHNV vaccines have been dicultto produce.

Recent AdvancesViral hemorrhagic septicemia iscaused by a rhabdovirus that aects shworldwide and causes signicant mor-tality in trout. The recombinant, inject-able subunit vaccine produced in insectcells inected with a modied baculovi-rus and containing the major antigenicglycoprotein o the virus was eca-cious, but it was never registered withthe USDA or commercial use becauseo high costs (de Kinkelin et al. 1995).

Subsequent experimentation with aDNA vaccine carrying the glycoproteingene was shown to induce a protectiveantibody response (Boudinot et al. 1998)and has been remarkably eective. Thisvaccine may nd a niche in protectingtrout rom this virus.

Inectious salmon anemia virus isa highly inectious enveloped ortho-myxo-like virus causing acute mortality,primarily in Atlantic salmon. Studies on160 isolates show that there exist up toeight dierent combinations o hemag-glutinin A and neuraminidase subtypes.As in the closely related infuenza Avirus, antigenic drit occurs requent-ly with these highly variable, con-stantly mutating surace glycoproteins,which are the most important antigens.Conventional vaccines do not provideull protection rom the disease, butresearch on both DNA-based vaccines

as well as VLPs shows some promise(Miller and Cipriano 2002).

Inectious pancreatic necrosis vi-rus is a birnavirus aecting salmonidsworldwide. Live attenuated and killedvaccines are not 100% eective and areexpensive. The bi-segmented, double-stranded viral RNA genome includestwo structural capsid proteins, VP3 andVP2. The VP2 induces neutralizingantibodies, but there is wide antigenicdiversity between eld strains and sero-types. All isolates, however, show some

cross-reaction. Recombinant proteinsubunit vaccines have been produced,but evidence is controversial as to theiroverall protective ecacy. Current re-search is ocusing on DNA-based vac-cines containing the entire viral poly-protein and the addition o CpG motisto achieve immunostimulatory activity(Mikalsen et al. 2004).

Future DevelopmentsAntiviral DNA vaccines continue

to receive considerable attention as a

new approach and as a solution to shdiseases that are reractive to traditionalvaccines. The main advantage o thevaccines is that they mimic a naturalviral inection whereby the host reliablyreproduces a single viral protein thatinduces a protective immune response.Production o viral proteins in the natu-ral host elicits both cellular (T-cells) andhumoral (antibodies) immune responsesindicating that these vaccines have ahigh degree o ecacy (Kim et al. 2000;


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Leong et al. 1995, 1997).A major obstacle in the progress o

DNA vaccines has been delivery o su-cient quantities to elicit protective im-mune responses. Subunit vaccines alsowill continue to be developed with mul-tiple antigens (proteins) expressed roma single genetic construct. Progress inadjuvants, manuacturing, and vaccinedelivery technologies will be critical orboth DNA and subunit vaccines. Oralvaccination delivered in eed would bethe preerred method o vaccination orsh. Dierent approaches to protect theantigen (liposomes, biolms, alginatebeads) rom gastric neutralization haveyielded promising results.

recombInAnt vAccInesfor compAnIon AnImAls

(doGs

, cAts

,And

Horses

)The vaccine market or companionanimals is growing more rapidly thanany other sector o animal health. Thisgrowth is caused partly by demographicchanges in the human population andpartly by the high value associated withcompanion animals. Currently, compan-ion animals are vaccinated routinely ora variety o diseases. In the uture, vac-cinations also will address nonpathogen-ic conditions such as reproduction, aswell as cancer and periodontal health.

Canine vaccinations includethose or Leptospirosis, kennel cough(Bordetella bronchiseptica), Lyme dis-ease (Borrelia burgdoreri), rabies, dis-temper, adenovirus, parainfuenza virus,and parvovirus. Feline vaccinationstarget eline herpesvirus, eline calcivi-rus, eline panleukopenia, rabies, elineleukemia (FeLV), and eline immuno-deciency virus. Horses commonly arevaccinated or tetanus, rabies, equineinfuenza, and herpesvirus inections.Depending on the geographic region,however, they also may be vaccinat-

ed or West Nile virus (WNV), equineencephalitis viruses (eastern equineencephalitis virus, Venezuelan equineencephalitis virus, western equine en-cephalitis virus), rotavirus, and equineviral arteritis.


A poxvirus rom a canary was de-veloped into a virus vector or use in

nonavian species. Canarypox does notreplicate in nonavian species, makingit absolutely sae, although it is an ex-cellent vaccine in mammals because itcompletes one round o replication innonpermissive cells and induces a robustimmune response in the vaccinee. Thedevelopment o both humoral (antibod-ies) and cell-mediated (T-cells) immuni-ty and the duration o protection inducedby canarypox vectors in mammals arewell known (Paoletti 1996). In addition,prior exposure to canarypox in nonavianspecies does not seem to interere withsubsequent vaccinations.

A number o commercially avail-able canarypox-vectored vaccines havebeen developed by Merial (Duluth,Georgia). Canine vaccines includeRECOMBITEK Lyme or vaccinationagainst Lyme disease, RECOMBITEKCanine Distemper, RECOMBITEK

Ferret Distemper (approved or use inerrets), and PURVAX Canine Rabies.

Commercially available canarypox-vector-based eline vaccines includePUREVAX Feline Rabies, EURIFELFeline Leukemia, and PUREVAX FelineLeukemia NF. Feline vaccinations areinfuenced strongly by the appearance ovaccine-associated injection site musclecell tumors called sarcomas, which areassociated epidemiologically with tradi-tional FeLV vaccines and killed rabiesvaccines that contain strong adjuvants.

The canarypox-vectored vaccine is ad-ministered subcutaneously and does notcontain an adjuvant, markedly decreas-ing the infammation at the injection site,which is believed to be the cause o theinjection site sarcomas. The PUREVAXNF canarypox virus carrying the FeLVsubgroup A env and gag genes can begiven transdermally in the skin withoutadjuvant to decrease the infammationpossibly associated with the develop-ment o injection site sarcomas.

Several canarypox vector recombi-

nant vaccines are commercially avail-able or horses. West Nile virus is amosquito-borne virus that emerged inthe United States in 2002 and causeddisease in nearly one-third o all hors-es. RECOMBITEK WNV (Merial) isa WNV vaccine or horses that inducesprotective immunity but does not rep-licate in a horse, making it completelysae. A canarypox-based recombinantvaccine or equine infuenza (ProteqFlu,Merial) contains two dierent canarypox

vectors-one carrying the immunogenichemagglutinin gene rom a U.S. strain oequine infuenza and the other carryingthe hemagglutinin gene rom a Europeanstrain o equine infuenza virus. TheProteqFlu-Te (Merial) vaccine containsthe equine infuenza canarypox vectorsin a tetanus toxoid diluting agent.

Recent AdvancesA second-generation Lyme disease

vaccine based on the C-terminal rag-ment o the OspA protein oB. burgdor-eri has been developed to avoid the sideeects associated with the whole pro-tein, and as a result, is a saer vaccine.A unique structure-based approach wasused to determine the three-dimensionalmakeup o the protective epitopes andgenerate a stable antigen or use in thevaccine (Koide et al. 2005). Structure-based methods o vaccine development

are a promising new approach that willallow scientists to target critical im-munogenic antigens specically or in-corporation into saer, more ecaciousvaccines.

Long-term survival ater vaccinationagainst malignant melanomaa skincancerin dogs with advanced diseasehas shown some promise. The so-calledxenogeneic DNA vaccine carries a hu-man tyrosinase-encoding gene that,when expressed in the vaccinated dogs,results in cytotoxic T-cell responses that

unction to reject the tumor (Bergman etal. 2006).

Zona pellucida glycoproteins canbe used as immunogens or contracep-tion. Contraceptive vaccines based onzona pellucida glycoproteins have beenstudied or more than 10 years, but re-cent advances in recombinant vaccinetechnologyspecically recombinantproteins, synthetic peptides, and DNAvaccineshave led to longer-lasting im-munocontraceptive eects and less ovar-ian pathology (Gupta, Chakravarty, and

Kadunganattil 2005). In the uture, con-traceptive vaccines likely will substituteor the castration o domestic pets, armlivestock, and wild animals.

Future DevelopmentsRecent research in companion ani-

mal vaccines has ocused on recombi-nant vaccine development or parasiticdiseases. Resistance to drugs used tocontrol nematodes has led to the study ovarious secreted larval antigens or elic-


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COUNCIL FOR AGRICULTURAL SCIENCE AND TECHNOLOGY10

iting protective immunity. Preliminarydata in mice or protection againsthookworms, which aect dogs and catsas well as people, used DNA vaccinesand recombinant hookworm proteinsexpressed in the laboratoryas well asunique route, delivery, and adjuvant or-mulationsto induce protection.

Other approaches are being used toidentiy immunogenic molecules thatalso are protective against extremelycomplex protozoan parasites. These ap-proaches include identication o genesin parasites linked to escape rom theimmune response and the use o mi-croarrays and immune selection to mapimmunoprotective antigens in parasites.These techniques are being used to iden-tiy potential vaccine candidates or thecoccidian parasites that cause entericdisease in almost all animals.

conclusIonsVaccines induce an immune re-

sponse in the animal host that subse-quently recognizes inectious agentsand helps ght o the disease; vaccinesmust do this without causing the disease.Using recombinant DNA technologies,scientists have been able to develop livegenetically modied organisms, re-combinant killed vaccines, and geneticvaccines that no longer cause diseaseyet induce a strong immune response.

Developing vaccines using rDNA tech-nologies requires a thorough understand-ing o the disease agent, particularly theantigens critical or inducing protec-tion and the actors involved in causingdisease. In addition, it is important tounderstand the immune response o thehost to ensure that the vaccine inducesthe appropriate immunological reaction.

Paralleling the development o new,more ecacious, stable, and sae recom-binant vaccines is the study o vaccinedelivery methods. In addition to using

conventional delivery routes such asoral, intranasal, intradermal, transcuta-neous, intramuscular, and IP, scientistsare experimenting with needle-ree sys-tems and vaccine strategies that allowmass vaccination o many individualssimultaneously.

Another active area o research isthe study o compounds with the po-tential to enhance the immune responseto vaccines. These approaches includeincorporating immunomodulating com-

pounds into vaccines that can aect thetype o immune response directly andimmunopotentiating compounds thatstrengthen the immune response. Theantigenic pathway can thus be modu-lated to stimulate the appropriate arm othe immune response to provide solidprotection. Also, new compounds thatindirectly stimulate the immune re-sponse (such as microparticles and adju-vants) are being studied. These com-pounds are designed to present antigensto the immune system in such a way thatoptimal stimulation is achieved.

The promise o better vaccines tobenet animal agriculture and com-panion animals through rDNA technol-ogy is becoming a reality. A numbero recombinant vaccines are availablecommercially, and many more are pro-jected to be available soon, so the u-ture o recombinant vaccines is bright.

New eorts need to ocus on deliverymethodology as well as developmento vaccines or economically importantdiseases or which no currently avail-able vaccines exist or or diseases wherepoorly eective vaccines are currently inuse. Advances in rDNA technology, inknowledge o the host immune response,and in the genetic makeup o diseaseagents will lead to new vaccines againstdiseases or which no control measurescurrently exist.

GlossAryAdjuvant. A compound mixed with

a vaccine to enhance the immuneresponse.

Antigen. A substance (usually a pro-tein) that is oreign to the animaland stimulates a specic immuneresponse.

Birnavirus. A bi-segmented RNA virus(e.g., inectious bursal disease virus).

Capsid proteins. Proteins that makeup the shell o a virus particle that

contains the genetic material.Challenge. The process o inecting an

animal with a disease agent to test orprotective immunity.

CpG motif. A genetic element con-tained in DNA vaccines that stimu-lates the immune system.

Cytokine. Substances produced by cellso the immune system o the animalthat unction to stimulate or modiythe immune response (e.g., intereron

and interleukins).

Differential ELISA test. Detection oantibodies specic to either the vac-cine or the disease agent by the en-zyme-linked immunosorbant assay.

Epitope. A molecular region on the sur-ace o an antigen capable o elicitingan immune response and o combin-

ing with the specic antibody pro-duced by such a response.

Fimbral antigen. Proteins that make upthe hairlike organelles on the suraceo bacteria responsible or motility.

Gametocyte. A cell that makes up oneo the developmental stages in the re-production o coccidiosis.

Gamma interferon. A cytokine that en-hances the antimicrobial properties othe immune response.

Gene-deleted. An organism with oneor more genes removed rom the ge-

nome to render it nonpathogenic so itcan be used as a vaccine.

Glycoprotein. A biomolecule composedo a protein and a carbohydrate.

Hemagglutinin-esterase. A cell at-tachment protein on the surace osome viruses that recognizes sialicacid residues on the cell surace andhas both the ability to agglutinate redblood cells (hemagglutinin) and re-ceptor-destroying activity (esterase)thought to release the virus rom thecell surace.

Immunodominant epitopes. Regionso a protein that stimulate the produc-tion o antibodies.

Immunogenic. Having the ability tostimulate the immune response.

Immunomodulators. Compounds ca-pable o stimulating or down-regulat-ing the immune response.

Immunopotentiating. Strengthening othe immune system.

Immunostimulating. Capable o stim-ulating an immune response.

Interleukins. Cytokines produced bycells o the immune system that unc-tion to enhance infammation and ac-tivate the immune response.

Lactogenic antibodies. Antibodies se-creted in milk that are important orproviding protection to progeny.

Leukotoxin. A virulence actor secret-ed rom some bacteria that kills hostphagocytic cells in a species-specicmanner.


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Marker vaccine. A recombinant or-ganism containing a oreign gene inwhich, when used as a vaccine, theoreign gene or antibodies to the ex-pressed protein rom the oreign genecan be detected. Marker vaccines oranimals receiving marker vaccinescan be detected with specic diag-nostic tests.

Oligonucleotides. A linear nucleic acidragment (not necessarily DNA) con-sisting o 210 nucleotides joined byphosphodiester bonds.

Oocyst. Egg stage o the coccidianparasite.

Orthomyxo-like virus. A virus similarto viruses in the amily orthomyxo-viridae (e.g., infuenza).

Pathogenicity. The ability to causedisease.

Proteomic studies. Studies on the en-

tire protein makeup o a cell, includ-ing proteinprotein interactions, pro-tein modications, protein unctions,and the genes involved in expressiono those proteins.

Recombinant deoxyribonucleic acid(rDNA) technologies. Denotes anymanipulation o DNA in the labora-tory including, but not limited to,cloning, polymerase chain reac-tion, restriction enzyme digestion,ligation, nucleic acid replication,reverse-transcription, and ribonucleic

acid synthesis.Refractive. Not aected by. (In this

context, antibiotics do not have an in-hibitory eect on the organism.)

Spike surface proteins. Petal-like pro-jections on the surace o a corona-virus particle that are involved in at-tachment o the virus to the host cell,among other things.

Synthetic peptides. Short 230 aminoacid molecules synthesized throughchemical reactions in the laboratory.

Vectors. In recombinant DNA technol-

ogy, it can be (1) a sel-replicatingmolecule o DNA that serves totranser a gene or oreign DNA rag-ment rom one organism to another(usually bacteria) or (2) a virus orbacteria containing a oreign genethat is used to vaccinate an animal.

Virulence. The severity o the diseasecaused by an inectious agent.

Xenogeneic. Derived or obtained roman organism o a dierent species.

Zoonotic. Relating to a disease that iscommunicable rom animals to hu-mans under natural conditions.

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M. Witcombe, M. Katrib, A. Finger, M. G. Wal-lach, and N. C. Smith. 2004. Characterisation

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CAST Member SocietiesAACC INTERNATIONAL n AMERICAN ACADEMY OF VETERINARY AND COMPARATIVE TOXICOLOGY n AMERICAN AGRICULTURAL ECONOMICS ASSOCIATION n AMERICAN ASSOCIATIONFOR AGRICULTURAL EDUCATION n AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS n AMERICAN ASSOCIATION OF PESTICIDE SAFETY EDUCATORS n AMERICAN BAR ASSOCIATIONSECTION OF ENVIRONMENT, ENERGY, AND RESOURCES, COMMITTEE ON AGRICULTURAL MANAGEMENT n AMERICAN BOARD OF VETERINARY TOXICOLOGY n AMERICAN DAIRY SCENCE ASSOCIATION n AMERICAN FORAGE AND GRASSLAND COUNCIL n AMERICAN MEAT SCIENCE ASSOCIATION n AMERICAN METEOROLOGICAL SOCIETY, COMMITTEE ON AGRICULTURAL FOREST METEOROLOGY n AMERICAN PEANUT RESEARCH AND EDUCATION SOCIETY n AMERICAN PHYTOPATHOLOGICAL SOCIETY n AMERICAN SOCIETY FOR HORTICULTURASCIENCE n AMERICAN SOCIETY FOR NUTRITION n AMERICAN SOCIETY OF AGRICULTURAL AND BIOLOGICAL ENGINEERS n AMERICAN SOCIETY OF AGRONOMY n AMERICAN SOCETY OF ANIMAL SCIENCE n AMERICAN SOCIETY OF PLANT BIOLOGISTS n AMERICAN VETERINARY MEDICAL ASSOCIATION n AQUATIC PLANT MANAGEMENT SOCIETY n ASSOCIATIONFOR THE ADVANCEMENT OF INDUSTRIAL CROPS n ASSOCIATION OF AMERICAN VETERINARY MEDICAL COLLEGES n COUNCIL OF ENTOMOLOGY DEPARTMENT ADMINISTRATORS nCROP SCIENCE SOCIETY OF AMERICA n INSTITUTE OF FOOD TECHNOLOGISTS n NORTH AMERICAN COLLEGES AND TEACHERS OF AGRICULTURE n NORTH CENTRAL WEED SCIENCESOCIETY n NORTHEASTERN WEED SCIENCE SOCIETY n POULTRY SCIENCE ASSOCIATION n SOCIETY FOR IN VITRO BIOLOGY n SOCIETY FOR NUTRITION EDUCATION n SOCIETY OFNEMATOLOGISTS n SOIL SCIENCE SOCIETY OF AMERICA n SOUTHERN WEED SCIENCE SOCIETY n WEED SCIENCE SOCIETY OF AMERICA n WESTERN SOCIETY OF WEED SCIENCE

The mission of the Council for Agricultural Science and Technology (CAST)is to assemble, interpret, and communicate credible science-based inormation regionally, nationally, and internationally to legislators, regulators, policymakers, the media, the private sector, and the public. CAST is a nonproft organization

composed o 38 scientifc societies and many individual, student, company, nonproft, and associate society members. CASTs Board o Directors is composed o represen

tatives o the scientifc societies and individual members, and an Executive Committee. CAST was established in 1972 as a result o a meeting sponsored in 1970 by the

National Academy o Sciences, National Research Council. ISSN 1070-002

Additional copies o this issue paper are available rom CAST. Linda M. Chimenti, Managing Scientifc Editor. World WideWeb: http://www.cast-science.org.

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

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