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Modification of the avian coronavirus infectiousbronchitis virus for vaccine developmentPaul Brittona, Maria Armestoa, David Cavanagha & Sarah Keepa

a Avian Viral Diseases; Institute for Animal Health; Compton Laboratory; Compton, Newbury,Berkshire UKPublished online: 01 Mar 2012.

To cite this article: Paul Britton, Maria Armesto, David Cavanagh & Sarah Keep (2012) Modification of the avian coronavirusinfectious bronchitis virus for vaccine development, Bioengineered, 3:2, 114-119, DOI: 10.4161/bbug.18983

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Modification of the avian coronavirus infectious bronchitis virusfor vaccine development

Paul Britton,* Maria Armesto, David Cavanagh and Sarah KeepAvian Viral Diseases; Institute for Animal Health; Compton Laboratory; Compton, Newbury, Berkshire UK

Keywords: vaccine, poultry, coronavirus,infectious bronchitis virus, spikeglycoprotein, vaccinia virus, reversegenetics, infectious clone, homologousrecombination, avian

Submitted: 11/21/11

Accepted: 12/07/11

http://dx.doi.org/10.4161/bbug.18983*Correspondence to: Paul Britton;Email: paul.britton@iah.ac.uk

Research Note to: Armesto M, Evans S,Cavanagh D, Abu-Median A-B, Keep S, Britton P.A recombinant avian infectious bronchitis virusexpressing a heterologous spike gene belongingto the 4/91 serotype. PLoS ONE 2011; 6:e24352;PMID:21912629; http://dx.doi.org/10.1371/journal.pone.0024352

Infectious bronchitis virus (IBV) causesan infectious respiratory disease of

domestic fowl that affects poultry of allages causing economic problems for thepoultry industry worldwide. AlthoughIBV is controlled using live attenuatedand inactivated vaccines it continues tobe a major problem due to the existenceof many serotypes, determined by thesurface spike protein resulting in poorcross-protection, and loss of immuno-genicity associated with vaccine produc-tion. Live attenuated IBV vaccines areproduced by the repeated passage inembryonated eggs resulting in spontan-eous mutations. As a consequence atte-nuated viruses have only a few mutationsresponsible for the loss of virulence,which will differ between vaccines affect-ing virulence and/or immunogenicityand can revert to virulence. A newgeneration of vaccines is called for andone means of controlling IBV involvesthe development of new and safer vac-cines by precisely modifying the IBVgenome using reverse genetics for theproduction of rationally attenuated IBVsin order to obtain an optimum balancebetween loss of virulence and capacity toinduce immunity.

Modification of IBV Genome

Infectious bronchitis (IB) is an acutehighly contagious respiratory disease ofpoultry that is prevalent throughout theworld causing animal welfare issues andsevere economic losses to the poultryindustry worldwide.1-4 The etiologicalagent of IB is an avian coronavirus,infectious bronchitis virus (IBV), which

belongs to the Gammacoronavirus genus,subfamily Coronavirinae, family Coro-naviridae, order Nidovirales. IBV repli-cates primarily in epithelial cells of therespiratory tract causing IB characterizedby nasal discharge, snicking, trachealciliostasis and rales in chickens,5 but it isalso able to replicate in the epithelial cellsof other organs such as the enteric tract,oviducts and kidneys.1-3,6 The main effectsof an IBV infection are poor weight gain,renal disease, decreased egg productionand poor egg quality resulting in majoreconomic losses to poultry industriesworldwide. The IBV genome consists ofa 28 kb single-stranded RNA moleculeof positive-sense polarity. The virion con-tains the four structural proteins; spikeglycoprotein (S), small membrane protein(E), integral membrane protein (M) andnucleocapsid protein (N) which interactswith the genomic RNA. The S glycopro-tein is a type I glycoprotein composed ofthree homopolymers that is responsiblefor binding to the target cell receptor andfusion of the viral and cellular membranes.The IBV S glycoprotein (1,162 aminoacids) is cleaved into two subunits, S1(535 amino acids, 90 kDa) comprising theN-terminal subunit of the S protein andS2 (627 amino acids, 84 kDa) comprisingthe C-terminal subunit. The S1 subunitincorporates the receptor-binding activityof the S protein and is responsible forinducing neutralizing and sero-specificantibodies.7,8 The ectodomain region ofthe S2 subunit contains a fusion peptide-like region9 and two heptad repeat regionsinvolved in oligomerisation of the Sprotein10 and is required for entry intosusceptible cells.11-13 The S2 subunit asso-ciates non-covalently with the S1 subunit

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and in addition to the ectodomain con-tains the transmembrane and C-terminalcytoplasmic tail domains.

IBV is currently controlled by the useof both live attenuated and inactivatedboost vaccines. Neutralising antibodies,induced by the IBV S1 subunit, presentin the respiratory tract are responsiblefor protecting against subsequent IBVinfection and concomitant IB disease.However, amino acid variations in the S1subunit have resulted in many differentIBV serotypes requiring different vaccinesdue to lack of cross-protection. Commer-cial live attenuated vaccines are producedby multiple passages of virulent fieldisolates in embryonated domestic fowleggs as a result of spontaneous mutationsthat cause attenuation of the virus. As aconsequence, viruses that are attenuatedby this approach have only a few muta-tions responsible for loss of virulence, anddue to the nature of the procedure theattenuated viruses have different muta-tions that may affect their virulenceand/or immunogenicity. Such a processrequires a fine balance between loss ofpathogenicity and retention of immuno-genicity. Which mutations result inattenuation of pathogenicity is not known.A major drawback of this method is thatonce the virus is used to inoculate chickensthe mutations that resulted in the attenu-ation of the vaccine virus may back-mutateresulting in a virulent virus; an undesirableconsequence.1,14,15

Although the use of both live andattenuated IBV vaccines have played animportant role in the successful expansionof the poultry industry, the existence andcontinual introduction of new IBV sero-types requires alternative strategies inorder to circumvent the problem of poorcross-protection and for the production

of safer vaccines. A new generation of IBvaccines is called for. One means ofcontrolling IBV involves the developmentof new and safer vaccines by preciselymodifying the IBV genome for the pro-duction of rationally attenuated IBVs toobtain an optimum balance between lossof virulence and capacity to induceimmunity. Such vaccines would ideallybe: (1) genetically stable, have a definedand uniform stable attenuated phenotypethat is unable to back mutate to virulence;(2) have the potential for uniform immu-nogenicity, the loss of virulence shouldnot affect immunogenicity; (3) flexibility,the modified genome can be manipulatedto express different S genes or S1 subunits,allowing the same genetically definedvaccine to protect against differing sero-types and (4) allow administration in ovo.IBV vaccines generated by passage inembryonated eggs are highly virulent forembryos so in ovo application cannot beused. The IBV genome can be preciselymodified through the use of a suitablereverse genetics system. We have deve-loped such a reverse genetics system or“infectious clone” for the manipulation ofthe IBV genome and have modified anIBV genome by exchanging the S glyco-protein gene. The S genes were derivedfrom virulent IBV strains and introducedinto the genome of an attenuated IBV.The resultant recombinant IBVs werestill avirulent but were able to act asvaccines for the protection of chickensagainst challenge with the parental viru-lent viruses from which the S genes werederived. These results have demonstratedthat through the use of a reversegenetics system swapping the IBV Sprotein is a precise and effective way ofgenerating genetically defined candidateIBV vaccines.

In our recent paper in PLoS ONE,16 wehave shown that replacement of an IBVS glycoprotein from a pathogenic fieldisolate, IBV 4/91(UK), belonging to adifferent serotype as the receiver IBV(Beaudette) did not confer pathogenicitybut did induce homologous protection.This confirmed our previous work in theJournal of Virology in which the S glyco-protein we used was derived from IBVM41 that belongs to the same serotype asthe receiver virus IBV Beaudette.17,18

Our results involving replacement ofthe IBV S glycoprotein genes has demon-strated that the availability of our IBVreverse genetics system has opened upways of modifying the IBV genome forthe development of new geneticallydefined and intrinsically safer IBV vac-cines. The reverse genetics system we usefor modifying the IBV genome comprisesof two processes. The first is outlined inFigure 1 and involves the direct modifica-tion of the IBV genome and the secondprocess, outlined in Figure 2 centers onthe recovery of infectious IBV. A full-length IBV cDNA, derived from thegenomic RNA of the avirulent Beaudettestrain, was sequentially assembled invitro and ligated into a NotI site in thethymidine kinase (TK) gene of vacciniavirus19 (Fig. 1A) and is used as a templatefor modifying the IBV Beaudette genome.The IBV cDNA is under the control of abacteriopahge T7 DNA dependent RNApolymerase promoter with a hepatitisdelta virus ribozyme (HdR) sequenceplaced downstream of the coronaviruspoly(A) tail followed by a T7 DNAdependent RNA polymerase terminationsequence (Fig. 1A). The work described inour Journal of Virology17 and PLoS ONE16

papers described the replacement of theBeaudette S gene with the corresponding

Figure 1 (See opposite page). Modification of the IBV Beaudette genome. (A) Outlines the overall modification of the S gene in the full-length IBVBeaudette cDNA within the vaccinia virus genome. The positions of the T7 promoter and termination sequences are shown; the IBV cDNA is insertedwithin the vaccinia virus thymidine kinase gene. (B) Schematic diagram of the TDS process for inserting a heterologous S gene into a modified version ofthe full-length IBV cDNA that lacks the Beaudette S gene sequence. The new S gene sequence is inserted into the GPT selection plasmid flanked byBeaudette-derived sequence corresponding to sequences 5’ and 3’ to the deleted S gene sequence. A potential single-step homologous recombinationevent between the end of the replicase gene in the receiver IBV cDNA and the flanking sequence in the donor sequence in the GPT plasmid is shown.This results in a series of recombinant vaccinia viruses that are selected due to their GPT+ phenotype in the presence of MPA. Growth of a GPT+

recombinant vaccinia virus in the absence of MPA gives rise to two types of spontaneous intramolecular recombination events due to the presencetandem repeat sequences of the IBV cDNA in the recombinant vaccinia virus. This results in the generation of recombinant vaccinia viruses either with anIBV cDNA without an S gene (no modification) or a complete full-length IBV cDNA containing the heterologous S gene, the desired end product. Bothrecombination events result in the loss of the GPT gene. The IBV genes representing the structural and accessory genes are shown; a potentialrecombination event is indicated between the IBV replicase gene sequence common to both constructs.

RESEARCH NOTE

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Figure 1. For figure legend, see page 115.

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S gene sequences for the pathogenic IBVstrains M41 and 4/91(UK), respectively,and is highlighted in Figure 1A.

In order to make alterations, such asexchange of the S gene, to the IBVBeaudette genome the IBV cDNA withinthe vaccinia virus genome is modified byhomologous recombination using a vac-cinia virus-based transdominant selection(TDS) method.20,21 The procedure, as des-cribed in our recent PLoS ONE paper,for inserting the S glycoprotein genefrom IBV 4/91(UK) into the Beaudettegenomic background, is summarized inFigure 1B. The first stage consisted of asingle step homologous recombination

event between the donor IBV cDNAsequence, the IBV 4/91(UK) S genesequence, inserted into the E. coli guaninephosphoribosyltransferase (GPT) contain-ing plasmid and the receiver IBV sequence,a modified version of the IBV Beaudettefull-length genomic cDNA that lacked theBeaudette S gene sequence, in the vacciniavirus genome. The homologous recombi-nation event occurred between one of thetwo Beaudette-derived sequences flankingeither end of the heterologous 4/91(UK)S gene sequence, and the correspondingBeaudette sequence present 5' and 3' of thedeleted S gene sequence in the receiverIBV cDNA. This resulted in the integration

of the complete plasmid sequence into thereceiver IBV cDNA allowing the selectionof recombinant vaccinia viruses in thepresence of mycophenolic acid (MPA).MPA is an inhibitor of purine biosynthe-sis, therefore only viruses expressing theE. coli GPT gene, which provides analternative pathway for purine biosynthe-sis, are able to replicate in the presence ofMPA and the alternative purine precursorsxanthine and hypoxanthine. Randomlyselected GPT+ viruses were then grownin the absence of MPA which resultedin a second internal recombination eventbetween the tandem repeat IBV sequencescausing the loss of the E. coli GPT gene

Figure 2. Recovery of infectious recombinant IBV. (A) Outlines the overall scheme for the recovery of infectious IBV from the recombinant vaccinia virusDNA containing the modified IBV cDNA. The vaccinia virus DNA is transfected into primary CK cells and infectious IBV RNA synthesized using T7 DNAdependant RNA polymerase expressed from a recombinant fowlpox virus. The T7-derived RNA is recognized as eukaryotic RNA and translated by thecellular machinery to produce the IBV replicase proteins that subsequently generate IBV genomic RNA and subgenomic mRNA leading to the assemblyand release of infectious IBV virions. (B) Outlines the subsequent passage and recovery of the recombinant IBV in primary CK cells, in the case of BeauR-4/91(S) this was performed in 10-d-old embryonated eggs.16 Virus generated from P3 CK cells is analyzed for the presence of any modification and used forsubsequent experiments.

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(Fig. 1B). This second recombination stepled to two possible outcomes; one eventresulted in the original (unmodified) IBVsequence and the other, in the desiredmodification, the generation of an IBVcDNA containing the 4/91(UK) S genesequence in the Beaudette cDNA thatlacked the Beaudette S gene sequence(Fig. 1B). Recombinant vaccinia virusesthat no longer expressed the GPT genewere isolated and sequence analysis usedto identify those that contained the 4/91(UK) S gene sequence. Infectious IBVRNA was generated in situ by transfectionof the vaccinia virus DNA containing themodified Beaudette cDNA into primarychick kidney (CK) cells previously infectedwith a recombinant fowlpox virus, rFPV-T7, expressing T7 RNA polymerase22 asoutlined in Figure 2A. In this systeminfectious IBV RNA is produced from theT7 promoter immediately adjacent to the5' end of the IBV cDNA by the rFPV-T7-derived T7 RNA polymerase and termi-nates at the T7 termination sequencedownstream of the HdR sequence, whichautocleaves itself and the T7-terminationsequence from the end of the poly(A)sequence, resulting in an authentic copy ofthe IBV genomic RNA. Cell supernatantsfrom the transfected CK cells are filteredto remove any rFPV-T723 and potentialrecombinant IBVs are passaged three timesin CK cells (Fig. 2B) to produce stocks ofvirus for sequence analysis to confirm thepresence of the modified IBV sequence.We have also found that using the Sglycoprotein from field isolates of IBV,such as 4/91(UK), that have not beenadapted for growth on primary CK cellsthat we were unable to recover infectiousvirus using CK cells due to the fact thatany potential virus was refractory forgrowth on CK cells.16 To circumvent thiswe have performed the initial rescue of therecombinant IBV in CK cells and insteadof passaging any potential IBV the filteredsupernatants on CK cells this was per-formed in 10-d-old embryonated eggs.16

The resultant recombinant IBVs, apartfrom any modification, are isogenic asthey are derived from the same cDNAsequence. As indicated above we havesuccessfully introduced two differentheterologous S gene sequences into anIBV Beaudette genomic background and

recovered infectious recombinant IBVsusing our reverse genetics system. Therecombinant IBVs were found to have thecell tropism associated with the heterologousS gene sequence16,17 and have been assessedas potential IBV vaccine candidates.

Assessment of RecombinantIBVs for Pathogenicity andHomologous Protection

Infection of 8-d-old specific pathogen free(SPF) chickens with either of the recom-binant IBVs BeauR-M41(S) or BeauR-4/91(S) did not result in IBV-associatedclinical signs, snicking, tracheal rales,wheezing and nasal discharge, by 10 dpost-inoculation. In contrast, the patho-genic IBV strains M41 and 4/91(UK)resulted in clinical signs from three dayspost-infection. These results show thatthe replacement of the Beaudette S genewith a heterologous S gene from a virulentIBV strain, M41 or 4/91(UK), did notconfer pathogenicity to the resultingrecombinant viruses. This is an importantfinding for potential vaccine developmentbecause our results showed that althoughthe S glycoprotein is a known virulencefactor, with respect to receptor bindingand responsible for tissue tropism, neitherS glycoprotein derived from the twopathogenic IBVs conferred pathogenicityin vivo to the avirulent (Beaudette)receiver isolate of IBV.16,18

Three weeks after the primary inocula-tion with either BeauR-M41(S) or BeauR-4/91(S) the chickens were challenged withpathogenic IBV M41-CK or 4/91(UK),respectively. Clinical signs associated witha pathogenic IBV infection were notobserved in the vaccinated chickens indi-cating that they had been protected againstclinical disease when challenged with thehomologous pathogenic virus;16,18 demon-strating that homologous protection hadbeen induced by the appropriate recom-binant IBV. Interestingly, results fromprior inoculation of chickens with therecombinant IBV BeauR-4/91(S) andsubsequent challenge with IBV M41, adifferent serotype of IBV to 4/91(UK),indicated that under experimental condi-tions BeauR-4/91(S) had induced somelevel of cross protection against M41according to analysis of clinical signs.16

We were unable to isolate viable IBV ordetect IBV-derived RNA from the trachealcells of the vaccinated chickens follow-ing challenge with either pathogenic virus.This indicated that the recombinantviruses used to vaccinate the chickensprior to challenge had induced a protec-tive response preventing the pathogenicviruses from successfully infecting thetracheal cells. Both virus and IBV-derivedRNA was isolated from the tracheas ofchickens that had not been vaccinated.

In conclusion, we have shown, usingour IBV reverse genetics system,19-21 thatreplacement of the IBV Beaudette S glyco-protein with S gene sequences from thepathogenic IBVs M41 and 4/91(UK) didnot confer virulence to the recombinantIBVs but in the resulting viruses, BeauR-M41(S) and BeauR-4/91(S), had tissuetropisms associated with the parentalM41 and 4/91(UK) viruses. Chickensthat were vaccinated with BeauR-M41(S)or BeauR-4/91(S) were found to be pro-tected against clinical disease followingchallenge with M41 or 4/91(UK), whereaschickens vaccinated with Beaudette werenot protected against challenge. TheBeaudette isolate of IBV was attenuatedafter several hundred passages in embryo-nated hen’s eggs,24 which not only resultedin loss of virulence, but has also beenimplicated in loss of immunogenicity. Ourresults have shown that replacement ofthe Beaudette S gene with a heterologousgene from two different IBV serotypesresulted in recombinant IBVs, based onthe Beaudette genome, that were able toact as potential vaccines for the protec-tion of chickens following subsequentchallenge with the parental pathogenicviruses. The swapping of the IBV S pro-tein is a precise and effective way ofgenerating genetically defined candidateIBV vaccines.

Acknowledgments

The authors would like to thank thefollowing organizations for their financialsupport the Department of Environ-ment, Food and Rural Affairs (DEFRA;www.defra.gov.uk/) project codeOD0717, the Biotechnology and Biologi-cal Sciences Research Council (BBSRC;www.bbsrc.ac.uk/) and Intervet Schering-Plough UK.

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