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Vaccine 29 (2011) 141–151

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Promoter control over foreign antigen expression in a murine cytomegalovirusvaccine vector

Paula T. Cunninghama,b,∗, Megan L. Lloyda,b, Nicole L. Harveya,b, Elizabeth Williamsa,b,Christopher M. Hardyb, Alec J. Redwooda,b, Malcolm A. Lawsona,b, Geoffrey R. Shellama,b

a Discipline of Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, Western Australia 6009, Australiab Invasive Animals Cooperative Research Centre, CSIRO Entomology, GPO 1700, Canberra, ACT 2601, Australia

a r t i c l e i n f o

Article history:Received 3 April 2007Received in revised form 3 March 2010Accepted 9 March 2010Available online 23 March 2010

Keywords:ImmunocontraceptionSpecies specificityPromoter control

a b s t r a c t

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV)containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive(VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisa-tion of the antigen expressed in order to overcome virus attenuation previously encountered. Early studiesreported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heav-ily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMVE1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant viruswas able to induce infertility in directly infected mice. However, the viruses remained attenuated.

This study demonstrated the capacity to manipulate the nature of the immune response by alteringpromoter control over foreign antigen expression and cellular localisation of the expressed antigen. Wewere able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize femaleBALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides anadded level of safety to any MCMV based VVIC approach as it only allows for transgene expression inMCMV permissive cells.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Cytomegaloviruses are large DNA viruses belonging to theBetaherpesvirinae subfamily of Herpesviridae. Several features ofcytomegaloviruses (CMVs) make them attractive candidates asvaccine vectors. These features include a large double-strandedgenome suitable for foreign gene insertion [1,2], species specificityfor their natural host [3] and their ability to provide continual stim-ulation of the immune system by establishing persistent and latentinfection [4–7]. In mice a recombinant murine cytomegalovirus(MCMV) encoding the murine zona pellucida 3 (mZP3) gene hasbeen investigated for its ability to induce infertility in female mice.This virally vectored immunocontraceptive (VVIC) is intended foruse as a population control measure in cereal growing regionsof Australia [8] where mice periodically reach plague proportions[9,10].

∗ Corresponding author at: Telethon Institute for Child Health Research,PO Box 855, West Perth, WA 6872, Australia. Tel.: +61 08 9489 7873.

E-mail addresses: paulac@ichr.uwa.edu.au, paulatc24@gmail.com(P.T. Cunningham).

Experimental studies using MCMV as a vector for VVIC havedemonstrated that a single inoculation of the MCMV express-ing mZP3 cDNA under the control of the strong constitutivelyexpressed immediate-early gene (IE1) promoter from human CMV(HCMV) induced greater than 90% infertility in female BALB/c mice[11]. The resulting infertility persisted for at least 250 days. Sev-eral subsequent studies have highlighted the value of the MCMVVVIC approach in direct injection models in female BALB/c mice[12–14] and suggest that this approach may be successful in thecontrol of mouse populations. In order to produce a freely dis-seminating virus a number of conditions must be met. The virusmust be able to transmit through the host and induce infertility inrecipient mice. The vector must also be species specific as shouldthe infertility effect. A number of factors can influence the trans-mission and species specificity of the rMCMV. Innate resistance toMCMV may limit the efficacy of any MCMV VVIC by rapid early con-trol of the infection before the adaptive immune response respondssufficiently to the expressed fertility antigen. Recent studies haveshown that strong innate resistance to MCMV in inbred mice doesin fact limit the efficacy of MCMV-mZP3 [14]. Innate resistanceto MCMV is rare in free-living mice in Australia [15]. MCMV-mZP3 is equally as effective, in fact, in pathogen free outbred

0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2010.03.012

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Australian wild mice as it is in MCMV susceptible inbred BALB/cmice [11,14]. For this reason it appears that innate resistance toMCMV may play only a limited role in preventing the dissemina-tion of an MCMV VVIC through the free-living mouse populations inAustralia.

An immediate concern for the production of a freely disseminat-ing VVIC is the consistent finding that the MCMV vectors expressingfertility antigens are heavily attenuated in vivo but not in vitro[11,13]. Recombinant MCMV vectors that express mZP3 all fail toreplicate well in the salivary gland of directly inoculated mice. Sinceviral replication in the salivary gland is believed to be responsiblefor transmission of MCMV via saliva [4], an attenuated rMCMV-mZP3 might therefore be expected to transmit poorly in a hostpopulation. The cause of this attenuation is not clear but may bedue to the strong immune response made towards the insertedtransgene. This would make the virus more “visible” to the immuneresponse and would result in immunological attenuation. Alterna-tively, the attenuation could be due to intracellular toxicity fromthe expressed antigen, or to disruption at the insertion site. Viralattenuation due to disruption at the insertion site appears unlikelyas the immediate-early 2 gene (IE2) region used in these studieshas been employed by others to express genes, such as LacZ, withminimal effect on in vivo replication [16].

The initial choice of MCMV as a vector for VVIC was based on itsstrict species specificity [8]. Studies conducted in the rat, a closelyrelated species to the mouse, have demonstrated the species spe-cific effect of MCMV-mZP3 [17]. Past experiments have used theconstitutive HCMV IE1 promoter to drive expression of mZP3 inMCMV based VVIC. The HCMV IE1 promoter produces high levelexpression in cells from a broad range of tissues [18]. This highlevel expression coupled with the capacity of MCMV to enter cellsfrom other species raises the possibility that low level transgeneexpression could occur in the cells from non-target species beforethe infection is aborted. Transgene expression could be avoided bythe design of species specific antigens [12], although the use of analternative non-constitutive promoter to drive mZP3 expressioncould also enhance the safety of a VVIC.

This study was designed to increase the safety, whilst addressingthe attenuation, of the recombinant viral vector. The first approachconsisted of substituting the HCMV IE1 promoter used to con-trol mZP3 expression in the original VVIC to an MCMV early (E)promoter. This approach allows the mZP3 to be expressed withthe same kinetics as the MCMV immune evasion genes and maytheoretically enhance the ability of the rMCMV to escape hostimmune surveillance, therefore ameliorating the attenuation seenwith transgene insertion. An added benefit of this approach is thatmZP3 expression would occur in cells capable of supporting a pro-ductive infection. The second approach employed the use of aninducible promoter. The aim was to produce a fully replicationcompetent virus that could disseminate through a target popula-tion with the transgene only being expressed when induced by abaiting approach. Lack of transgene expression should result in avirus that is not attenuated, therefore increasing dissemination ofthe virus. The bait could also be withdrawn were any non-targetspecies to become affected by the VVIC, thus increasing the safetyof the approach.

2. Materials and methods

2.1. Animals

Specific pathogen free inbred BALB/c and C57Bl/6J mice wereobtained from the Animal Resources Centre (Murdoch, Perth, West-ern Australia) and maintained under minimal disease conditions.The OT-1 transgenic mice were kindly provided by the University

Department of Medicine (University of Western Australia). Mousecare was based on the Australian Code of Practice and was approvedby the University of Western Australia Animal Experimentation andEthics Committee. Sentinel animals were found to be free of a suiteof murine pathogens including MCMV following routine testing. Allmouse inoculations used the intraperitoneal (i.p.) route.

2.2. Cells

Primary mouse embryo fibroblasts (MEFs) were prepared bytrypsin dispersion of 15- to 17-day-old embryos from inbred BALB/cmice as previously described [19]. Upon thawing, cells were grownin MEM supplemented with 10% fetal calf serum (FCS), and main-tained in MEM supplemented with 2% FCS.

2.3. Viruses

The origins of the K181 strain of MCMV have been previouslydescribed [20]. The virus RM427+ was kindly provided by Profes-sor E. Mocarski (Stanford University, Stanford, USA) [1,21]. Virusstocks were propagated in mouse embryonic fibroblasts (MEF)as previously described [11]. The previously described virus vec-tor, rMCMV-mZP3, sterilized recipient BALB/c mice. rMCMV-mZP3contains murine zona pellucida 3 (mZP3) under the control of theconstitutively expressed human cytomegalovirus (HCMV) IE1 genepromoter. In vitro replication of viruses was performed by multi-step growth curves as previously described [11]. Analysis of viraltitres in infected mice was assessed by plaque assay as previouslydescribed [11].

2.4. Plasmids

Plasmid cloning was conducted by standard methods usingrestriction endonucleases from Promega (Madison, WI, USA) orNew England Biolabs (Beverly, MA, USA) [22].

pMCMV-mOVA was constructed by excision of the TfROVAsequence from pBlueRIP-TfROVA (kindly provided by Dr F. Carbone,The University of Melbourne, Melbourne, Australia) using HindIII[23]. The TfROVA (mOVA) was blunt-ended by a fill-in reactionusing Klenow DNA polymerase (Promega) and ligated into the SmaIsite of pMV11 [11]. Next the entire expression cassette, containingthe HCMV IE1 promoter, the TfROVA and the SV40 poly-adenylationstop sequence were excised from pMCMV-mOVA by HindIII diges-tion. This fragment was end filled and inserted between the twoHpaI sites of the HindIII-L fragment of pK181-H3L [11]. The result-ing virus was named rMCMV-mOVA.

2.4.1. pMCMV-IE1-sOVAThe secreted ovalbumin gene was engineered to contain

flanking EcoRI sites. This was cloned into a shuttle plasmid,containing both the human cytomegalovirus IE1 promoter andpoly-adenylation stop sequences. A 2.6 kbp NotI fragment of thisplasmid was ligated into the pH3LN transfer plasmid [13]. ThepH3LN recombination plasmid designed to generate an insertionmutation within the MCMV HindIII-L fragment, was derived fromthe pK181-H3L plasmid received from Dr. A. Scalzo (University ofWestern Australia, Perth, Australia) with NotI linkers incorporatedinto the HpaI site. Gene cassettes of the various transfer plasmidswere ligated into the NotI site of pH3LN to construct the final trans-fer plasmids.

2.4.2. pMCMV-E1-sOVAThe SV40 poly[A] and pMCMV E1 sequences were engineered

to contain flanking HindIII and ApaI sites to allow for their liga-tion into a single plasmid. A 1878 bp EcoRI fragment containing the

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Table 1Recombinant and control viruses.

Virus Plasmid Promoter Foreign gene HpaI PCR fragment size (bp)

rMCMV-IE1-sOVA pH3LN-IE1-sOVA HCMV IE1 Secreted ovalbumin 2714rMCMV-E1-sOVA pH3LN-E1-sOVA MCMV E1 Secreted ovalbumin 3294rMCMV-E1-mZP3 pH3LN-E1-ZP3 MCMV E1 Murine zona pellucida 3 2690rMCMV-TetZP3 pH3LN-TetZP3 Tet-On (Clontech) Murine zona pellucida 3 3997K181 (Perth) – – – 160RM427+ – HCMV IE1 LacZ 4081rMCMV-IE1-mOVA – HCMV IE1 Membrane bound ovalbumin 2656rMCMV-IE1-mZP3 – HCMV IE1 Murine zona pellucida 3 2124

The shaded area of the table identifies the virus controls used in these studies: wild-type (K181(Perth), parent (RM427+), and recombinant viruses previously constructedand characterized.

sOVA gene was ligated between these two sequences. The result-ing expression cassette was excised using NotI and ligated intopH3LN.

2.4.3. pMCMV-E1-ZP3The plasmid designated pE1/ZP3/SV40p[A] was constructed

from the plasmids pCMH288 and pBSKS-/E1/SV40p[A]. pCMH288contains the murine ZP3 cDNA fragment flanked by the HCMVIE1 promoter and poly[A] sequences. pBSKS-/E1/SV40p[A] con-tains the MCMV E1 promoter and SV40p[A] sequences cloned intothe plasmid vector pBluescript KS-(Stratagene). pE1/ZP3/SV40p[A]was constructed by directionally cloning the 1.3 kb mZP3 frag-ment from pCMH288 (derived by digestion with BamHI andEcoRI) into pBSKS-/E1/SV40p[A] between the promoter and poly[A]sequences.

2.4.4. pMCMV-TetZP3Plasmids pCMH321, pCMH288 and pCMH320 were generated

in order to be able to produce the final construct pCMH324, whichcontains all the required elements of the Tet-On system and themurine ZP3. Briefly, pCMH321 was constructed by ligation ofa 337 bp PCR fragment amplified from pEGFP-1 (Clontech) intopTet-On (Clontech), this fragment contained the SV40 promoterand the rtTA sequence. pCMH288 was constructed by cloning a1302 bp BamHI and EcoRI cDNA fragment of a pGEM7Z(f-) plas-mid (Promega) containing the entire mouse ZP3 coding sequenceinto the pMV11 plasmid in between the HCMV ie1 promoter andp[A] sequences. The 1596 bp mZP3/MCMV IE poly[A] fragmentfrom this plasmid was then cloned into the HindIII/BamHI digestedpTRE-EGFP (Clontech), downstream of the PhCMV-1 promoterto generate pCMH320. To construct the gene cassette contain-ing all the elements of the Tet-On system and the mZP3 gene,a 2045 bp XhoI/HindIII fragment from pCMH320 containing thePhCMV-1/mZP3/HCMVp[A], and a 1829 bp XhoI/HindIII fragmentfrom pCMH321 containing the reverse tetracycline trans-activator(rtTA) were cloned into XhoI digested pBluescript KS(−) to producepCMH324.

2.5. RT-PCR

Heterologous gene expression was assessed by RT-PCR. RNAwas extracted from cells using TRIZOL reagent (Invitrogen), follow-ing manufacturer’s instructions. RNA was then treated with RQ1RNase-free DNase (Promega) and cDNA was generated immedi-ately afterwards using M-MLV Reverse Transcriptase (Promega).PCR for ovalbumin cDNA was performed using the primers OVAF (5′-ATC AAG CCA GAG AGC TCA TC-3′) and OVA R (5′-TGC CTGCCT CAT TGA TTT CT-3′). The OVA F and OVA R primers amplify a600 bp. PCR for ZP3 cDNA was performed using the mZP3 primersZP3/380F (5′-ACC CTC GCC CTG TGA GTG GCC-3′) and ZP3/1021R(5′-AAT TAC TAC AGT TGC CAT GGC-3′). These primers amplify a641 bp DNA fragment.

2.6. In vivo characterisation of rMCMVs

Groups of 6–8 week old BALB/c mice were inoculated intraperi-toneally (i.p.) with 2 × 104 plaque forming units (pfu) of eitherrecombinant or wild-type viruses and the spleen, liver, salivaryglands and lungs were assayed for the presence of infectious virus.Three mice were examined for each virus from each time point. DNAwas extracted from salivary glands homogenates and was tested byquantitative PCR using primers and probes designed to amplify theIE1 region of the viruses as previously described [24].

2.7. ELISA

ELISAs for anti-MCMV, anti-OVA and anti-mZP3 antibodies wereperformed as previously described [11,19], using alkaline phos-phatase anti-rat or anti-rabbit IgG (1:2000) as conjugate.

2.8. In vivo OVA CTL assay

The assay of CTL effector function in vivo was a modifica-tion of the method of Oehen and Brduscha-Rien (1998). Briefly,target cells were prepared by pulsing syngeneic spleen cells for90 min at 37 ◦C with the H2kb restricted, ovalbumin-specific pep-tide (SIINFEKL) (10 ng per 3 spleens) followed by labelling with lowintensity (5 �M) 5,6-carbofluorescein diacetate succinimidyl ester(CFSELOW) for 10 min at 37 ◦C. Antigen specificity was determinedby comparing these cells with non-pulsed syngenic spleen cellslabelled with high intensity (50 �M) CFSE (CFSEHIGH). In vivo killingwas assessed by intravenous (i.v.) injection of a 1:1 ratio of CFSElabelled cells (2 × 107 total cells) into immune C57BL/6J mice (micepreviously inoculated i.p. with 2 × 104 pfu of wild-type or recom-binant MCMVs expressing OVA) [25]. Specific killing was assessedby flow cytometric (FACScan; Benton Dickinson) analysis of sin-gle cell suspensions of splenocytes from treated mice. Cytotoxiceffector function was determined by assessing the ratio betweenthe non-pulsed CFSEHIGH cells and the peptide-pulsed CFSELOW

cells.

2.9. Proliferation assay

Lymph nodes were removed from adult OT1 mice and single cellsuspensions were prepared. Cells were labelled with CFSE (25 �M).Adult C57BL/6J mice previously inoculated with OVA expressingMCMV were inoculated i.v. with the CFSE labelled lymph node cellsuspension. To assess OVA-induced proliferation spleens were col-lected 3 days post i.v. inoculation and individual cell suspensionsprepared. The proliferation of the CFSE labelled OT1 donor cells wasdetected by flow cytometry and analysed using CellQuest software(Becton Dickinson). CD8+ T cell proliferation was determined bythe number of cell peaks of decreasing CFSE intensity that werevisualized.

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Fig. 1. Construction of recombinant MCMVs by insertion of foreign gene cassettes into the ie2 region of the virus. (A) Structure of the ie2 region of the rMCMVs withthe foreign gene cassettes inserted, the size of each inserted cassette is indicated at the right of the diagram. The dotted line above each of the rMCMV insertion cassettesrepresents the HindIII fragment the probe used to verify cassette insertion in Figs. (B) and (C) will bind to. (B) Restriction enzyme digestion of K181 and rMCMVs and Southernblot analysis for the OVA gene. HindIII restriction fragment length polymorphism (RFLP) analysis of DNA from K181 (lane 1), RM427+ (lane 2), rMCMV-IE1-mOVA (lane 3),rMCMV-IE1-sOVA (lane 4), and rMCMV-E1-sOVA (lane 5). Southern blot analysis using an OVA-specific probe. (C) Restriction enzyme digestion of MCMV-K181 (Perth) andrMCMVs and Southern blot analysis for the ZP3 gene. HindIII RFLP analysis of DNA from K181 (lane 1), RM427+ (lane 2), rMCMV-IE1-ZP3 (lane 3), rMCMV-E1-ZP3 (lane 4), andrMCMV-TetZP3 (lane 5). Southern blot analysis using a ZP3-specific probe. (D) HpaI PCR comparison of K181 with rMCMVs: 1 kb ladder (lane 1), water −ve (lane 2), K181 (lane3), RM427+ (lane 4), rMCMV-IE1-ZP3 (lane 5), rMCMV-E1-ZP3 (lane 6), rMCMV-TetZP3 (lane 7), rMCMV-IE1-mOVA (lane 8), rMCMV-IE1-sOVA (lane 9), rMCMV-E1-sOVA(lane 10), water −ve (lane 11). (E) RT-PCR analysis for mZP3 gene transcription in MEF cells infected with rMCMVs, lanes labelled (a) are reverse transcriptase positive (i.e.cDNA) and those labelled (b) are reverse transcriptase negative and are therefore a control for genomic DNA contamination.

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Fig. 2. Growth of recombinant MCMV in vitro. Multi-step growth curves comparing (A) rMCMV-IE1-sOVA, rMCMV-E1-sOVA, (B) rMCMV-E1-ZP3 and rMCMV-TetZP3 withwild-type virus shows that these recombinants do not appear to be attenuated in vitro. (C) Plaques formed by K181, rMCMV-IE1-mOVA, rMCMV-IE1-sOVA and rMCMV-E1-sOVA on MEF cells. (D) Plaques formed by K181, rMCMV-IE1-ZP3, rMCMV-E1-ZP3 and rMCMV-TetZP3 on MEF cells. All viruses form plaques in the cell monolayer. Plaquesstained with anti-Ova or anti-mZP3 show expression of foreign antigen from the recombinant viruses.

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Table 2Pathogenesis of rMCMV-IE1-sOVA, rMCMV-E1-sOVA and rMCMV-IE1-mOVA in BALB/c mice.

Days post-inoculation

3 7 21 70

K181 (Perth)Plaque assay (salivary gland)a – +++ (10665) +++ (148755) +++ 3364Serum antibody to MCMV ELISAb – +++ (40–80) +++ (80–160) +++ (40–320)Serum IgG1 antibody to OVA ELISAb – – – –Serum IgG2a antibody to OVA ELISAb – – – –qPCR (salivary gland)c – +++ (424) +++ (296626) + (3587)

rMCMV-IE1-sOVAPlaque assay (salivary gland)a – – – –Serum antibody to MCMV ELISAb – +++ (20–80) +++ (10–80) +++ (80–1280)Serum IgG1 antibody to OVA ELISAb – ++ (40) +++ (20–80) +++ (20–160)Serum IgG2a antibody to OVA ELISAb – – – –qPCR (salivary gland)c – ++ (733) + (400) +++ (103)

rMCMV-E1-sOVAPlaque assay (salivary gland)a – – – –Serum antibody to MCMV ELISAb – ++ (40) +++ (40–80) +++ (160–1280)Serum IgG1 antibody to OVA ELISAb – +++ (10–40) ++ (10) + (10)Serum IgG2a antibody to OVA ELISAb – – – –qPCR (salivary gland)c +++ (923) ++ (224) – –

rMCMV-IE1-mOVAPlaque assay (salivary gland)a – – – –Serum antibody to MCMV ELISAb – +++ (10–40) +++ (20–80) +++ (80–1280)Serum IgG1 antibody to OVA ELISAb – – ++ (10–40) ++ (80–160)Serum IgG2a antibody to OVA ELISAb – ++ (80) +++ (20–40) +++ (160–640)qPCR (salivary gland)c ++ (404) + (147) – ++ (129)

a (+) = number of animals positive (out of three). Numbers indicate the geometric mean of the number of plaque forming units per mL of organ homogenate.b (+) = number of animals positive (out of three). Numbers indicate highest positive reciprocal serum dilution per animal.c (+) = number of animals positive (out of three). Number indicates the geometric mean of the number of genome copies per mL of organ homogenate.

2.10. Immunoperoxidase staining

Immunoperoxidase staining of infected MEF monolayers todetect the expression of mZP3 and OVA was performed as previ-ously described [17], using either a rat monoclonal antibody raisedagainst murine ZP3, IE10 [26], or the commercially available rabbitanti-OVA antibody (Rockland, USA) as the primary antibody.

2.11. Histology and direct immunofluorescence

Ovaries from mice inoculated with MCMV-K181, rMCMV-ZP3,rMCMV-E1-ZP3 and rMCMV-TetZP3 were fixed in Bouin’s fixativefor 24 h and then transferred to 70% ethanol. Within 7 days of col-lection, organs were embedded in paraffin and 5 �m sections werecut and stained with haematoxylin and eosin. Direct immunofluo-

Table 3Pathogenesis of rMCMV-E1-mZP3 and rMCMV-TetZP3 in BALB/c mice.

Days post-inoculation

3 7 21 70

rMCMV-IE1-ZP3Plaque assay (salivary gland, spleen, lung) – – – –Serum antibody to MCMV ELISAa – +++ (20–160) +++ (10–80) +++ (160–320)Serum antibody to ZP3 ELISAa – – ++ (50–100) +++ (100–200)qPCR (salivary gland)b + (91) – – ++ (316)Ovarian pathologyc – ++ +++ +++

rMCMV-E1-ZP3Plaque assay (salivary gland, spleen, lung) – – – –Serum antibody to MCMV ELISAa – +++ (40–160) +++ (80–160) +++ (160–640)Serum antibody to ZP3 ELISAa – – ++ (50–100) +++ (50–100)qPCR (salivary gland)b + (91) – – ++ (316)Ovarian pathologyc – + ++ +++

rMCMV-TetZP3 + doxycyclinePlaque assay (salivary gland, spleen, lung) – – – –Serum antibody to MCMV ELISAa – +++ (10–160) +++ (40–160) +++ (40–320)Serum antibody to ZP3 ELISAa – + (0–50) ++ (0–50) +++ (50–800)qPCR (salivary gland)b – – +++ (573) +++ (954)Ovarian pathologyc – + ++ +++

rMCMV-TetZP3 − doxycyclinePlaque assay (salivary gland, spleen, lung) – – – –Serum antibody to MCMV ELISAa – ++ (10–40) +++ (10–80) +++ (10–80)Serum antibody to ZP3 ELISAa – – + (0–50) +++ (50–200)qPCR (salivary gland)b – – +++ (346) +++ (1891)Ovarian pathologyc – – + ++

a (+) = number of animals positive (out of three). Numbers indicate highest positive reciprocal serum dilution per animal.b (+) = number of animals positive (out of three). Number indicates the geometric mean of the number of genome copies per ml of organ homogenate.c (+) = degree of ovarian disruption, (+) = disrupted 3o follicles, (++) = disrupted 2o and 3o follicles, (+++) = complete disruption.

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Fig. 3. Cellular responses to OVA expressing rMCMVs. (A) In vivo proliferation of CFSE-labelled OT-1 donor cells in rMCMV infected recipient mice. Donor OVA-specificCD8+ T cells were harvested from transgenic OT-1 donor mice and CFSE labelled. Donor cells were inoculated into infected recipients at days 3, 7 and 14 p.i. After 3 daysthe percentage of cells that underwent proliferation were assayed by FACS. (B) In vivo ovalbumin-specific effector function of virally expressed OVA primed CTL. Recipientmice were generated by infecting adult female C57BL/6J mice with 2 × 104 pfu rMCMV-IE1-mOVA, rMCMV-IE1-sOVA or rMCMV-E1-sOVA TCV i.p, or with K181 to serve asa negative control. Donor target cells were prepared by pulsing syngeneic spleen cells with OVA peptide (SIINFEKL) and labeling them with a low CFSE intensity. To controlfor antigen specificity, un-pulsed syngeneic spleen cells were labelled with a high CFSE intensity. These target populations were injected i.v. into recipient mice the timespost-infection indicated. After 18 h, spleens were collected. To test for specific cytotoxic effector function the elimination of OVA peptide-pulsed CFSELOW spleen cells wasmonitored and the ratio between percentage of non-pulsed (CFSEHIGH) and percentage of pulsed indicator cells (CFSELOW) was calculated. Six mice per group were used inthis experiment and results were compared using unpaired t-tests. *P < 0.05; **P < 0.01.

rescent microscopy of frozen sections from ovaries was performedas previously described [11].

2.12. Breeding studies

For long-term breeding experiments, groups of five female 6–8week old BALB/c mice were inoculated i.p. with 2 × 104 pfu of tis-sue culture virus (TCV). One male mouse was introduced per femaleimmediately following inoculations and remained for the durationof the experiment. The introduction of males at the time of inocu-lation enabled one round of breeding before the effect of the VVICbecame evident and allowed for the initial fertility of each breed-ing pair to be confirmed. The breeding output over 100 days wasrecorded. Pups that were stillborn or eaten were not included inthe total.

2.13. Statistical analyses

Data was compared using one-way analysis of variance(ANOVA) with Tukey’s post hoc analysis or the Student’s t-test asappropriate.

3. Results

3.1. Construction and in vitro characterisation of recombinantMCMVs

Virus constructs were produced by homologous recombinationaccording to previously published methods [11] using the lacZexpressing RM427+ as the parental virus. The viruses and plasmidsused to produce them are shown in Table 1. All viruses were con-structed by insertion of the antigen cassette into the ie2 region asshown in Fig. 1A. Virus constructs were confirmed by RFLP, South-ern blot analysis (Fig. 1B and C), and by PCR across the insertion site(Fig. 1D). All rMCMVs expressed the appropriate mRNA as deter-mined by RT-PCR (data not shown). This site was chosen as earlierstudies with lacZ showed little viral attenuation. Insertion into theIE2 gene allowed for direct comparison between promoter specificeffects and earlier studies.

The in vitro growth of all recombinant viruses was comparedagainst the parental controls, K181 and RM427+. There was no sig-nificant difference in growth kinetics evident between parental or

recombinant strains (Fig. 2A and B). Heterologous protein expres-sion was confirmed by in situ immunostaining of infected cellsfor either mZP3 or OVA (Fig. 2C and D). The addition of doxycy-cline (DOX) to rMCMV-TetZP3 enhanced antigen expression overthat detected in the absence of DOX. However, there was low levelexpression of mZP3 in rMCMV-TetZP3 without the addition of DOX,consistent with the known “leakiness” of these promoter systems[27] and as confirmed by RT-PCR analysis (Fig. 1E).

3.2. Timing of ovalbumin expression

The temporal pattern of antigen exposure was investigatedto determine if delayed antigen expression could affect immuneresponses to the virus and ameliorate the attenuation previouslyencountered in other recombinant MCMVs. It was postulated thatantigen expression timed to coincide with expression of the viralimmune evasion genes would lead to less attenuation of the viralvector and could also affect host immune responses. Reagents forassessing T cell responses to the autoantigen, mZP3, in BALB/c miceare not readily available, hence along with mZP3 expressing viruses,vaccine strains of virus expressing the nominal antigen, chickenegg ovalbumin (OVA) were also constructed. In addition, becausethe mZP3 constructs in other experiments [11] contained a furincleavage site that could lead to soluble mZP3 protein expression,the effect of soluble versus membrane bound OVA expression wasassessed. These responses were compared to a previously producedvirus expressing a membrane bound OVA (mOVA) construct thatwas known to induce strong CTL responses (unpublished observa-tions).

The effects of altered temporal expression and the form of OVAwhich was expressed were assessed by infecting female BALB/cmice with 2 × 104 pfu of each virus. The presence of infectiousvirus was assessed by plaque assay of salivary gland and lunghomogenates collected at days 3, 7, 21 and 70 post-inoculation.Attenuation was seen for all rMCMVs regardless of either promoterused or the form of the antigen (Tables 2 and 3). The only exceptionwas RM427+ which expresses lacZ in the IE2 region. The attenua-tion was not due to failure of the virus to move from the originalsite of inoculation, as viral DNA could be detected in all organs byqPCR (Tables 2 and 3).

Following the inoculation of 2 × 104 pfu of virus into C57BL/6mice the rMCMVs expressing OVA were all shown to be capable of

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Fig. 4. Histological analysis of ovaries taken from mice infected with rMCMVs. (A) Detection of immunoglobulin bound to the zona of ovaries of infected mice. The ovariesof mice infected with K181, rMCMV-ZP3 and rMCMV-TetZP3 +/− doxycycline were frozen and sectioned, stained using a goat anti-mouse Ig FITC conjugate and examinedvia fluorescent microscopy. (B) Ovarian sections from infected mice stained with haematoxylin and eosin.

inducing the proliferation of OT-I CD8+ T cells specific for the dom-inant OVA peptide (SIINFEKL). At the time of peak proliferation forall OVA rMCMVs (day 7), the recombinants with OVA under thecontrol of the pIE1 exhibited the highest level of proliferation withthe rMCMV-IE1-mOVA inducing the highest levels (P < 0.01, com-pared to both sOVA rMCMVs) (Fig. 3A). Proliferation was specificto the OVA rMCMVs as no proliferation was seen to in response toK181 infection (P < 0.001) (K181 data not shown).

OVA-specific cytotoxic T cell responses in immunized mice weredetermined. Spleen cells from C57BL/6J mice were pulsed with theSIINFEKL peptide to produce target cells. Peptide-pulsed cells werelabelled using CFSE at a low intensity (CFSELOW). To control forthe elimination of target cells by noncontact-dependent or non-

specific lymphokine-mediated mechanisms, un-pulsed spleen cellswere labelled with high CFSE fluorescence (CFSEHIGH). CFSELOW

and CFSEHIGH cells were inoculated into C57BL/6 mice previouslyinfected with K181, or rMCMVs expressing sOVA or mOVA. Thetarget cell mixture was transferred into infected mice at days 3,10, 21, 42, 71 and 100 post-inoculation and CTL effector functionwas measured by monitoring the rejection of the CFSELOW targetsin the spleens by flow cytometry at 18 h after transfer. Signifi-cant differences in CTL activity were observed depending uponthe type of OVA expressed by the rMCMVs and also on the timingof foreign antigen expression (Fig. 3B). The kinetics of cytotoxic-ity mediated by CTL was similar in mice vaccinated with each ofthe OVA expressing vectors. However, significantly higher levels of

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cytotoxic activity were detected in mice infected with the rMCMVcontaining mOVA under the control of the pIE1 at days 10, 21 and100 (P < 0.05), compared with the sOVA rMCMVs. There were no sig-nificant differences seen between the two sOVA rMCMVs in eitherthe proliferation or CTL assays.

The OVA expressing viruses elicited strong antibody responsesto OVA and MCMV antigens (Table 2). Following inoculation withparental and recombinant viruses, the antibody levels elicitedagainst MCMV antigens for both IgG1 and IgG2a isotypes showedno significant differences between viral groups. Therefore, no pre-dominant Th1 or Th2 response to viral antigens was indicated.The antibody responses towards the mOVA antigen were predomi-nantly of the IgG2a isotype as previously observed (van Dommelen,unpublished observations); although at later timepoints IgG1 anti-bodies were also produced. In mice infected with sOVA expressingrMCMVs the IgG1 antibody response to OVA was dominant. NoIgG2a antibody was detected in mice receiving viruses encodingsOVA.

3.3. Timing of antigen (mZP3) expression

All recombinant viruses were heavily attenuated in vivo, asshown in Tables 2 and 3. Whilst it was possible to change theimmune responses to the foreign antigen by altering their expres-sion by different promoters this did not alter their attenuation.Ovalbumin is considered to be a strong immunogen, a fact thatmay play a role in increasing the viral attenuation. It was nextdetermined if a self-antigen (mZP3) under the control of the E1promoter would retain the ability to sterilize mice but result in avirus with less in vivo attenuation. To confirmed that the expressionof mZP3 was following IE1 and E1 expression kinetics, accordingto which promoter was present, cells infected with recombinantviruses were grown in the presence of cyclohexamide or phos-phonoacetic acid to limit expression to the IE1 or E1 stage ofvirus growth, respectively. Quantitative PCR analysis showed thatmZP3 is not transcribed from the E1-ZP3 virus when cyclohexam-ide is present but transcription is evident when phosphonoaceticacid is present, confirming early transcription kinetics of mZP3in this virus (results not shown). The immune responses elicitedto the rMCMVs expressing mZP3 were determined by examin-ing the antibody responses by ELISA at days 3, 7, 21 and 70days post-inoculation in infected mice (Table 3). Anti-MCMV IgGresponses were equivalent in mice infected with IE1-mZP3 or E1-mZP3 rMCMV. The levels of rMCMVs as detected by qPCR in thesalivary glands of infected mice were also similar for both recom-binants. Histological examination of ovaries from infected miceshowed that the vector containing mZP3 controlled by the E1 pro-moter induced a lesser degree of disruption of ovarian architecture(Fig. 4A and B). However, the infertility produced by these viruseswas equivalent (Fig. 5).

3.4. Inducible antigen expression

All of the viruses expressing either OVA or mZP3 were grosslyattenuated for in vivo growth. As K181 vectors with an ie2 deletion(identical to the deletion caused by antigen cassette insertion) orexpression of lacZ (as in RM427+) were only moderately attenu-ated in vivo, it was postulated that either the nature of the antigens(OVA or mZP3) or the level of antigen expression was responsi-ble for the attenuation. The inducible Tet-On promoter was usedin these studies to allow for antigen expression to be turned on asrequired. The aim of this was to determine if foreign antigen expres-sion or the insertion of the foreign gene sequence was the cause ofthe attenuation. The mZP3 cDNA was placed under the control ofthe externally inducible Tet-On system. Whilst it is known that thissystem is ‘leaky’ it was decided that the ability to turn low level anti-

Fig. 5. Effect of vaccination with recombinant MCMV vectors on fertility. Six femaleBALB/c mice were inoculated with 2 × 104 pfu of wild-type, parental, and ZP3rMCMVs at day 0 and mated with males immediately. The breeding output of micewas monitored for 100 days.

gen expression to high level expression would help to investigatethese questions.

Anti-MCMV IgG responses were lower in mice infected with therMCMV-TetZP3 virus but not supplied with the inducer doxycy-cline (DOX). The titres of anti-mZP3 antibodies were also muchdecreased in the absence of DOX. In contrast, levels of rMCMV DNAquantified by qPCR in the salivary glands of infected mice werehigher by day 70 in mice not supplied with DOX indicating that therecombinant virus replicated to higher levels when less mZP3 wasproduced (Table 3). The disruption of ovarian pathology was alsomuch reduced in mice infected with rMCMV-TetZP3 but not sup-plied with DOX (Fig. 4A and B). However, the fertility levels of thesemice were still significantly decreased, with levels being almostidentical to those seen in mice infected with the virus but suppliedwith DOX. These results demonstrated that whilst it was possible tosignificantly affect the immune responses to both the virus and for-eign antigen using this promoter, the low antigen levels produceddue to promoter leakiness in its off state were sufficient to induceresponses causing ovarian disruption and almost 100% infertility.

4. Discussion

In this study we demonstrated that it is possible to sterilizefemale BALB/c mice with an MCMV vector expressing mZP3 underthe control of an MCMV E gene promoter. The use of the E1 pro-moter, as opposed to the pHCMV IE1, provides an added level ofspecies specificity to any MCMV based VVIC as it only induces trans-gene expression in MCMV permissive cells. Attempts to produce aless attenuated vaccine were not successful and the investigationof alternative strategies to reduce the attenuation of the virus oralter the method of viral dissemination is required. Such alterna-tives include the use of an inducible promoter system to controlthe expression of the fertility antigen. The data presented indicatethat it is possible to construct an MCMV encoding all of the compo-nents of an inducible gene expression system and achieve increasedgene expression in vivo. It may be possible using improved sec-ond generation inducible promoters to engineer a VVIC containinga transgene that can be turned on or off by application of a baitcontaining the appropriate inducer.

Previous studies using MCMV as a VVIC vector have noted sig-nificant viral attenuation [11,13]. This study addressed issues ofviral attenuation and vaccine species specificity. Altering the tem-poral expression of the fertility antigen by changing the promoterused to control the transgene was explored. Delaying transgeneexpression until the E phase of MCMV replication was seen as apotential method of reducing attenuation due to the co-expression

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of MCMV immune evasion genes during this phase of replication.Expression of the transgene during the E phase also has the poten-tial to enhance vaccine safety as the fertility antigen would only beexpressed in cells permissive to viral replication.

Delayed gene expression was assessed using the nominal anti-gen OVA and the self-fertility antigen mZP3. In all instances theviruses expressing antigen driven by either the HCMV IE1 promoteror the MCMV E1 promoter were attenuated for replication in vivo.Attempts to reduce the in vivo attenuation and thereby potentiallyenhance viral transmission were unsuccessful. Subtle differenceswere noted in the qualitative and quantitative immune responsesto the expressed transgene with the different promoters. A reduc-tion in IgG1 antibody responses to OVA, CD8 proliferation and anearly reduction in the in vivo killing of SIINFEKL pulsed cells wasseen when responses in mice infected with rMCMV-E1-sOVA werecompared to those infected with rMCMV-IE1-sOVA. With mZP3 thiswas confirmed by a reduction in the disruption to ovarian architec-ture when rMCMV-E1-mZP3 was compared to rMCMV-IE1-mZP3.

The antibody response to soluble OVA was consistently limitedto and characterised by IgG1 responses. In the control membranebound OVA virus, the responses were comprised of both IgG1 andIgG2a antibodies. This is consistent with the studies of Boyle et al.who found that the total IgG response to mOVA was 10-fold lowerthan that of the sOVA primarily due to lower IgG1 induction, andtherefore the overall IgG response was not only lower but alsoshifted to IgG2a dominance [28]. The mZP3 typically produced anti-body responses that were composed of IgG1 and IgG2a. WhilstmZP3 contains a furin cleavage site, this study suggests that mZP3remains bound to the membrane and is in agreement with unpub-lished observations that mZP3 is mostly associated with the cellpellet and not the supernatant.

MCMV encodes a large range of immune evasion genesthat inhibit host responses [29]. These genes are predominantlyexpressed with E gene kinetics. Previous MCMV-mZP3 vaccinestudies have been carried out using a constitutive promoter[11–14]. In those studies the mZP3 gene was expressed prior tothe virus expressing its full complement of immune evasion genes.Transgene expression controlled by an IE promoter could havean effect both in the priming and effector stage of the immuneresponse and result in viral attenuation. MCMV encodes three genes(m04, m06 and m152) that inhibit antigen presentation and CD8 Tcell responses [30] as well as other mostly unidentified genes thataffect dendritic cell function [31–33]. Expression of mZP3 imme-diately upon infection prior to the production of immune evasiongene products potentially enhances priming to the encoded anti-gens. This was initially suspected to be the reason that the pp89(IE1) protein of MCMV was the immunodominant antigen in BALB/cmice [34]. In the present study there was an early reduction inthe CTL response to E1 OVA suggesting a reduction or delay inpriming of the CTL response. In the effector phase, immediate trans-gene expression prior to the down regulation of MHC class I couldenhance CTL mediated killing. In addition CTL responses inducedin the acute phase of infection are likely to adversely affect thecapacity of MCMV to enter or remain in latency as mZP3 would beconstitutively expressed in infected cells. However, delayed trans-gene expression kinetics, whilst having a moderate effect on theimmune response did not lead to reduced viral attenuation.

MCMV was originally chosen as a VVIC agent as, like other CMVs,it is considered to exhibit strict species specificity [8]. Recent stud-ies have demonstrated that rK181-mZP3 failed to sterilize femalerats [17], however infected rats did make cross-reactive antibodyresponses to mZP3. The presence of anti-mZP3 antibodies is likelyto be due to the fact that MCMV is capable of replicating in rat cells[17,35,36] although it cannot be isolated from adult rats [17]. Theterm strict species specificity cannot be interpreted as a failure toreplicate in cells from a different species. Whilst productive repli-

cation occurs only in closely related species it is known that CMVsuse promiscuous receptors that allow them to enter a wide varietyof cells. In most cells there is a post-penetration block to viral geneexpression [37–39]. In some instances this appears to be due to aninability to inhibit apoptosis in the cells of non-target species [3,40].There is a small possibility that an MCMV expressing a transgenecould enter the cells of non-target species and via a constitutivepromoter induce transient gene expression.

The data presented here suggest that an additional level ofspecies specificity can be applied to an MCMV VVIC by drivingtransgene expression by an MCMV early promoter. In this instance,mZP3 would only be expressed in cells that are permissive forinfection and are capable of supporting viral replication to the Ephase of gene expression. In studies investigating MCMV infec-tion in human macrophage and fibroblast cells, the block to virusreplication appeared to occur after the switch from IE to E pro-tein synthesis but before viral DNA replication [41]. In contrast, theHCMV IE1 promoter used in previous studies [11] is functional ina broad range of cells from several species [18]. The potential of anMCMV VVIC entering cells from another species is low and, giventhe lack of effect in directly inoculated rats, it is highly unlikelythat transient mZP3 expression could sterilize non-target species.Nevertheless this added level of safety may be an important pointwhen considering the release of a fully replicating VVIC. The use ofthe species specific promoter paired with a species specific antigenhas the potential to allay most safety concerns regarding the VVICapproach to pest management.

While the results of the E1 promoter experiments suggest ameans of increasing species specificity of the VVIC, these virusesremained attenuated. Consequently a proof of principle experimentwas initiated to determine if it was possible to drive expression ofthe self-fertility antigen with an inducible promoter. The resultspresented here demonstrate that it was possible to use all thecomponents of the Tet-On system to drive expression of a self-fertility antigen in MCMV. This is one of the few instances wherethe entire tetracycline promoter system has been incorporated intoa recombinant virus controlling the expression of a foreign anti-gen and tested in vivo [42,43]. It became evident however thatthe Tet-On promoter was leaky, and while the basal level of mZP3resulted in lower antibody responses and a much smaller degree ofovarian disruption, the expression was sufficient to result in signif-icant infertility in infected mice. This highlights the effectivenessof MCMV for antigen delivery. While these studies have confirmedthe “leakiness” of the Tet-On system it should now be possible todesign a similar VVIC with improved inducible systems [44–46].

In conclusion, these studies demonstrate that it is possible tosterilize female BALB/c mice with an MCMV vector expressingmZP3 under the control of an MCMV E gene promoter. The useof the MCMV E1 promoter has the potential to provide an addedlevel of safety to any MCMV based VVIC approach as it only allowsfor transgene expression in MCMV permissive cells. The combi-nation of a species specific virus, a promoter that functions onlyduring productive infection and a species specific antigen allowsfor the production of a safe and effective VVIC. Further examina-tion of the attenuation seen in the VVICs is required. Future studiesshould consider either alternative strategy to reduce the attenua-tion of the virus or focus on the production of an orally deliveredbait approach.

Acknowledgements

We would like to thank Professor John Papadimitriou from theDiscipline of Pathology, School of Surgery and Pathology at the Uni-versity of Western Australia for histological examination of tissuesamples. This research was supported by the Pest Animal ControlCooperative Research Centre.

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