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INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.9.5786–5793.2001

Sept. 2001, p. 5786–5793 Vol. 69, No. 9

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Towards Development of an Edible Vaccine against BovinePneumonic Pasteurellosis Using Transgenic White Clover

Expressing a Mannheimia haemolytica A1Leukotoxin 50 Fusion Protein

RAYMOND W. H. LEE,1 JUDITH STROMMER,2 DOUG HODGINS,3 PATRICIA E. SHEWEN,3

YONGQING NIU,2 AND REGGIE Y. C. LO1*

Departments of Microbiology,1 Plant Agriculture,2 and Pathobiology,3

University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 20 February 2001/Returned for modification 1 May 2001/Accepted 25 May 2001

Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly onMannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plantfor use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobictransmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of elic-iting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorterLkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant trans-formation. Constructs were screened by Western immunoblot analysis for their ability to express the fusionprotein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflowermosaic virus 35S promoter for transcription, was selected and introduced into white clover by Agrobacterium tu-mefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP wasfound to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extractcontaining Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), andrabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression ofan M. haemolytica antigen in plants and paves the way for the development of transgenic plants expressingM. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

Mannheimia haemolytica A1 is the principal microorganismresponsible for bovine pneumonia pasteurellosis, or shippingfever, a major cause of sickness, death, and economic loss inthe feedlot cattle industry (12, 33). Traditional immunizationapproaches using needle injection of various vaccine prepara-tions have provided some degree of protection. However, nee-dle injection requires the herding and restraint of the animals,inducing additional stress as well as incurring a substantiallabor cost. As an alternative, we propose to develop a nonin-vasive means of delivery of the vaccine via the oral route byusing transgenic plants expressing recombinant immunogens.Recent advances in the understanding of transgene expressionand recombinant protein accumulation, stability, and process-ing in plants have allowed the development of novel strategiessuch as using edible plants for delivery of antigens for activeimmunization (for reviews, see references 24, 28, and 30).

The leukotoxin (Lkt) of M. haemolytica A1 is one of itsmajor virulence factors (26). Lkt is secreted by M. haemolyticaA1 and acts as a pore-forming cytolysin that inserts into themembrane of target cells (3), resulting in osmotic imbalanceand cell lysis. This initiates a cascading effect that leads totissue damage, pneumonia, and death of the animals (1, 4). Lktis a member of the RTX family of cytolysins (31, 32). Several

functional domains have been identified in the typical RTXcytolysin, one of which is a transmembrane hydrophobic regionthat is involved in insertion of the toxin into the target cells (31,32). The genetic determinant that codes for Lkt has beencharacterized extensively in our laboratories. We have carriedout genetic manipulation of the lktA gene for high-level ex-pression in Escherichia coli and used this recombinant Lkt(rLkt) in a vaccine for conventional intramuscular injection(5). This rLkt was unable to cause damage to the target cellsbecause it is unstable and loses biological activity rapidly. How-ever, to completely ensure that the rLkt to be used for vaccinesis devoid of any biological activities, we constructed derivativesof Lkt by removing the section of the lktA gene that codes forthe putative hydrophobic transmembrane domains of the toxin.These derivatives, Lkt66 and the smaller Lkt50, would be in-capable of inserting into the membrane and are therefore nolonger cytotoxic. However, neutralizing antigenic epitopes ofLkt, mapped to a 227-amino-acid region at the C terminus ofthe protein (11, 17), were retained in these derivatives.

In this paper, we describe (i) the construction of Lkt66 anddemonstrate that Lkt66 is capable of eliciting anti-Lkt neutral-izing antibodies, (ii) the creation of transgenic clover plantsthat express Lkt50 fused with the green fluorescent protein(GFP), and (iii) the characterization of the Lkt50-GFP fromclover as a candidate for development of an edible vaccine.GFP was used as a marker to provide a simple and rapidmethod to screen for expression of the fusion protein in trans-genic plants.

* Corresponding author. Mailing address: Department of Micro-biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.Phone: (519) 824-4120, ext. 3363. Fax: (519) 837-1802. E-mail: [email protected].

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MATERIALS AND METHODS

Bacterial strains and culture conditions. E. coli DH5a (Table 1) was used asthe host for cloning and propagation of plasmids and was cultured in Luria-Bertani broth supplemented with thymine (50 mg/ml) and ampicillin (100 mg/ml),chloramphenicol (25 mg/ml), or kanamycin (50 mg/ml) as necessary. M. haemo-lytica A1 (ATCC 43270) was used for production of total proteins and was grownin brain heart infusion broth (Difco, Detroit, Mich.). Agrobacterium tumefaciensstrain C58C1Rifr containing the helper plasmid pMP90 (obtained from L. Erick-son, University of Guelph, Guelph, Ontario, Canada) was routinely grown inYEP (yeast extract, 10 g/liter; peptone, 10 g/liter; and NaCl, 5 g/liter) supple-mented with kanamycin (50 mg/ml) and gentamicin (25 mg/ml) when required.

Recombinant DNA methods, nucleotide sequencing, and PCR. All DNA clon-ing and ligation were carried out using standard recombinant DNA techniques(2, 25). E. coli competent cells were transformed either by the CaCl2 method orby electroporation according to our standard laboratory procedure. A. tumefa-ciens was transformed by electroporation (10). Plasmid DNA was isolated fromE. coli using kits from Qiagen (Mississauga, Ontario, Canada) or Gibco BRL(Burlington, Ontario, Canada). The constructs were confirmed by DNA sequenc-ing at the Laboratory Services Division (University of Guelph) on double-stranded plasmid DNA templates using an ABI 377 Prism automated sequencer(Applied Biosystems International, Foster City, Calif.) based on cycle sequencingwith dye-terminator dideoxynucleotides for fluorescence detection of terminatedDNA strands.

PCR was carried out in thin-walled Microfuge tubes (Gordon Technologies,Toronto, Ontario, Canada) in a Cetus DNA 480 or a Perkin-Elmer GeneAmpPCR System 2400 Thermocycler (Perkin-Elmer, Foster City, Calif.). PCR prim-ers were synthesized at the Laboratory Services Division. A typical 50-ml reactionmixture contained 10 to 100 ng of template, 50 to 100 pmol of each primer, a 0.2mM concentration of each deoxynucleoside triphosphate, and 2 to 4 mM MgSO4

in the PCR buffer supplied by the manufacturer. The reaction included a hot start of2 to 5 min at 95°C, followed by the addition of 1 U of Taq polymerase (RocheDiagnostics, Laval, Quebec, Canada) or Pwo polymerase (Roche Diagnostics)and then 30 cycles of 95°C for 1 min, 45 to 65°C for 1 min, and 72°C for 2 min.

Construction of Lkt66. The plasmid pLKT60 (Table 1) contains the lktCAgenes cloned behind the tac promoter (27) and was used as the starting materialfor construction of the Lkt derivatives. Plasmid pLKT60 DNA was digested andreligated at two NaeI sites located within the lktA sequence (Fig. 1). The ligatedDNA was transformed into competent E. coli cells. Plasmid DNA from ampi-cillin-resistant colonies was isolated and mapped by restriction endonucleases toconfirm removal of the NaeI fragment. The DNA was also sequenced using aprimer based on the lktA sequence to confirm that the nucleotides across theNaeI site had not been altered. This plasmid was designated pLKTDN andshould express a 66-kDa Lkt derivative.

Production of anti-Lkt66 antibodies in rabbits. Plasmid pLKTDN was intro-duced into E. coli DH5a also harboring the plasmid pWAM716, which carriesthe hlyBD secretion genes (7). Lkt66 was recovered from the culture supernatantof the E. coli cells using the HlyBD secretion system according to our laboratory

procedure (22). Briefly, a log-phase culture of the E. coli grown in Luria-Bertanibroth supplemented with ampicillin and chloramphenicol was induced with iso-propyl-b-D-thiogalactopyranoside (0.5 mM) for 1 h. The culture supernatant wasrecovered after two centrifugation steps at 10,200 3 g and concentrated 10-fold

FIG. 1. Gene maps for Lkt and Lkt derivatives. (A) Map of lktA,which encodes the full-length Lkt102, is shown. Shaded regions (A andB) indicate sequences encoding hydrophobic domains. Deletion of theNaeI fragment results in lktDN (B), which produces Lkt66. A naturallyoccurring ApoI site (1349) in lktA was used to construct the fusion geneshown in panel C. PCR was used to add an EcoRI site at position 2701,and the resulting ApoI-EcoRI fragment which encodes Lkt50 was in-serted between the signal peptide and GFP coding sequences. (C)Diagram showing part of the T-DNA region of pBlkt50-mgfp5 con-taining the lkt50-mgfp5 fusion gene and flanking regions from theHindIII (H) restriction site to the left border repeat (LB). Expressionof the fusion gene is driven by the constitutive cauliflower mosaic virus35S promoter (35S). The fusion gene consists of a signal peptide-encoding sequence (er), lkt50, and mgfp5. The polyadenylation signalfrom the nopaline synthase gene (nos ter) is found immediately down-stream of the fusion gene. Restriction sites and their positions onpBlkt50-mgfp5 are as follows: H, HindIII (4950); X, XbaI (5815); B,BamHI (5821); E, EcoRI (5901, 7254, and 8257); and S, SacI (7990).Numbering of pBlkt50-mgfp5 starts at the pRK2 origin of replicationas in pBin19 (8) but goes in the direction of right border to left border.

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Genotype or description Reference or source

Bacterial strainsE. coli DH5a Host strain for cloning Laboratory stockM. haemolytica A1 Wild-type strain ATCC 43270A. tumefaciens C58c1Rifr Host strain for plant transformation L. Erickson

Plant strainsTrifolium repens L. cv.Osceola

White clover for transgenic plant production Speare Seeds

N. tabacum cv. PetH4 Tobacco for transient gene expression Laboratory stock

PlasmidspLKT60 Vector containing lktA 27pLKTDN Vector containing lkt66 This studyp35S-GFP Cloning vector containing wild-type gfp Clontechp35S-mgfp5-ER Cloning vector containing mgfp5 This studyp35S-lkt50-mgfp5 Cloning vector containing lkt50-mgfp5 fusion This studyp35S-mgfp5NS Cloning vector containing mgfp5 lacking a stop codon This studypBINmgfp5-ER Binary vector containing mgfp5 14pBlkt50-mgfp5 Binary vector containing lkt50-mgfp5 fusion This studypBmgfp5-lkt50 Binary vector containing mgfp5-lkt50 fusion This studypBp mgfp5-ER Binary vector containing a promoterless mgfp5 This study

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using an Amicon (Oakville, Ontario, Canada) ultrafiltration apparatus with amembrane cutoff of 50 kDa. The concentrated fluid was dialyzed extensivelyagainst distilled water at 4°C and lyophilized. A small aliquot of the powder (10mg) was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) to confirm the presence of a 66-kDa protein corresponding to thetruncated Lkt. The lyophilized powder was dissolved in phosphate-buffered sa-line (PBS) to a concentration of 200 mg/ml, and 0.4 ml was injected intramus-cularly into rabbits for the production of antibodies as described below.

Construction of the lkt50 and gfp fusion gene. The binary vector pBINmgfp5-ER, obtained from J. Haseloff (University of Cambridge, Cambridge, UnitedKingdom), contains the mgfp5-ER gene that encodes the GFP variant mGFP5which has enhanced expression in plants and is targeted and retained in theendoplasmic reticulum (ER) (14). For simplicity, the mGFP5 variant is referredto as GFP in all subsequent descriptions.

The HindIII-SacI fragment from pBINmgfp5-ER containing the mgfp5-ERsequence was used to replace the HindIII-SacI fragment (containing wild-typeGFP) of plasmid p35S-GFP (Clontech, Palo Alto, Calif.) to produce p35S-mgfp5, a smaller vector, for the following manipulations.

To construct an lkt derivative for cloning into plants, the sense primer 59-CAAGATAATATGAAATTCTTACTGAACTTA, which annealed to positions1855 to 1884 of lktA (20) containing an ApoI site (underlined), and antisenseprimer 59-GCTATGTTTGAGGAATTCATAGTTCTCAAC, which annealed topositions 3237 to 3208 and added an EcoRI site (underlined), were used toamplify a 1.35-kbp fragment that codes for amino acids 451 to 901 of Lkt. ThePCR product was digested with ApoI and EcoRI and was cloned into p35S-mgfp5partially digested with EcoRI. Plasmid DNA from E. coli transformants wasisolated and mapped to select for the insertion of the PCR fragment in thecorrect orientation between the sequence encoding the signal peptide and GFPin p35S-mgfp5, creating p35S-lkt50-mgfp5. Subsequently, the HindIII-SacI frag-ment that contained the lkt50-mgfp5 sequence was subcloned back intopBINmgfp5-ER between the unique HindIII and SacI site to create pBlkt50-mgfp5 (Fig. 1).

To construct the binary vector containing the mgfp5-lkt50 fusion (pBmgfp5-lkt50), a vector containing mgfp5 lacking a stop codon was first made. A 933-bpPCR product was amplified from p35S-mgfp5 using the sense primer 59-GATGACGCACAATCCCACTATC, which annealed to the 35S promoter 83 nucleo-tides upstream of the XbaI site, and the antisense primer 59-GGAAATTCGAGCTCGTAAAGCTC, which removed the stop codon and added a SacI restric-tion site (underlined). The PCR product was digested with XbaI and SacI andused to replace mgfp5 in p35S-mgfp5, resulting in p35S-mgfp5NS. The lkt se-quence in pBlkt50-mgfp5 was amplified using the sense primer 59-GCCGAGCTCTTACTGAACTTAAAC, which changed the upstream EcoRI site to a SacIsite (underlined), and the antisense primer 59-TTTACTGAGCTCTTAGTTATCAACAAC which changed the downstream EcoRI site to a SacI site (under-lined) and introduced a new stop codon (boldface type). The 1.37-kbp PCRproduct was digested with SacI and inserted into SacI-digested p35S-mgfp5NS,resulting in p35S-mgfp5-lkt50. The fusion was then subcloned into the binaryvector by replacing the EcoRI fragment containing mgfp5-ER in pBINmgfp5-ERwith the EcoRI fragment from p35S-mgfp5-lkt50 containing the mgfp5-lkt50 fusion.

Transient expression in tobacco by agroinfiltration. To assess the expressionof transgenes in plants, they were transiently expressed by infiltrating tobaccoleaves (Nicotiania tabacum cv. PetH4) with A. tumefaciens cultures containingthe various constructs as previously described (6) with modifications. Briefly,A. tumefaciens (carrying the plasmid constructs) was grown in Luria-Bertanibroth with 10 mM MES (morpholineethanesulfonic acid) (pH 5.6); 20 mMacetosyringone; and antibiotics at 28°C for 16 h. After centrifugation, the culturewas resuspended to an optical density at 600 nm of 1 in Murashige and Skoog(MS) (23) salts with 2% sucrose; 0.5 mM MES (pH 5.6); and 100 mM aceto-syringone. Infiltrated plants were kept humid by covering with clear plastic bags.After 3 to 4 days, GFP fluorescence could be observed in some cases by fluo-rescence microscopy (see below). To investigate the transient production offusion protein, the infiltrated leaf areas were excised and extracted proteinsexamined by Western immunoblot analysis as described below.

A. tumefaciens-mediated plant transformation. White clover (Trifolium repensL. cv. Osceola) transformation was performed essentially as described (18) withmodifications. White clover seeds obtained from Speare Seeds (Harriston, On-tario, Canada) were surface sterilized and imbibed on 0.53 MS basal mediumsupplemented with Gamborg’s vitamins (9) (Sigma, St. Louis, Mo.) and 2%sucrose for 1 to 3 days at room temperature. Hypocotyls were cut from thegerminated seeds, leaving a 1- to 2-mm segment of the stalk attached to thecotyledons. Where possible, the apical shoot tip was removed. The two cotyle-dons were either completely or partially separated but still attached to thebisected hypocotyl. A. tumefaciens for cocultivation was grown in selective YEP

to an optical density at 600 nm of 0.5 to 0.8. Cotyledons were immersed in theA. tumefaciens culture and gently agitated for 40 min. Excess bacterial culturewas removed by blotting, and the cotyledons were cocultivated on MS basalmedium with Gamborg’s vitamins, 3% sucrose, N6-benzyladenine (1 mg/liter),a-naphthaleneacetic acid (0.1 mg/liter; Sigma), 100 mM acetosyringone (Aldrich,Oakville, Ontario, Canada) and 0.3% phytagel (Sigma), pH 5.5, at room tem-perature in the dark for 4 to 5 days. The cotyledons were then transferred toselective regeneration medium consisting of MS basal medium with Gamborg’svitamins, N6-benzyladenine (1 mg/liter), a-naphthaleneacetic acid (0.1 mg/liter),kanamycin (100 mg/liter), ticarcillin (250 mg/liter) and clavulanic acid (8.3 mg/liter) (Timentin; Smith Kline Beecham Pharma, Oakville, Ontario, Canada), and0.3% phytagel, pH 5.8. After 6 weeks, green shoots were isolated and placed inMagenta boxes (Sigma) containing a rooting medium of 0.53 MS basal mediumwith Gamborg’s vitamins, 3% sucrose, kanamycin (100 mg/liter), ticarcillin (250mg/liter), and clavulanic acid (8.3 mg/liter).

Presence of the transgene in the plantlets was confirmed by PCR using thesense primer 59-CCACTATCCTTCGCAAGAC, which anneals to the 35S pro-moter region, and the antisense primer 59-TGTTGCATCACCTTCACCCTCTC, which anneals to the mgfp5 coding region. Genomic DNA was isolated usinga commercial kit (DNeasy Plant Mini Kit; Qiagen). Selected transgenic plantletswere potted in soil and maintained in the greenhouse.

Fluorescence microscopy. Conventional epifluorescence microscopy was car-ried out using a Leica (Richmond Hill, Ontario, Canada) MZIII fluorescencestereomicroscope with a GFP3 filter set (excitation at 470 nm with a band widthof 40 nm; emission at 525 nm with a band width of 50 nm). Laser scanningconfocal microscopy (model MRC-600 microscope; Bio-Rad, Mississauga, On-tario, Canada) was used to visualize GFP fluorescence in transgenic plants. Forconfocal microscopy, observations were made on plant tissue sections mountedin water.

SDS-PAGE and Western immunoblot analysis. To prepare plant proteinextracts for SDS-PAGE, plant tissue samples were frozen in liquid nitrogen,ground, and homogenized with an extraction buffer consisting of PBS containing1 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and0.5% (wt/vol) Tween 20. One- and two-milliliter aliquots of buffer were used perg (fresh weight) of tobacco and clover tissue, respectively. Plant proteins wereseparated by SDS-PAGE as described by Lee and Huttner (19). PrestainedSDS-PAGE standards (Bio-Rad) were used for molecular mass determinations.For Western immunoblot analysis, proteins were transferred onto nitrocellulosemembranes (Schleicher & Schuell, Keene, N.H.) after SDS-PAGE (29). Themembranes were probed with either a rabbit anti-Lkt66 antiserum, a mouseanti-Lkt monoclonal antibody (MAb 601, obtained from S. Srikumaran, Univer-sity of Nebraska, Lincoln) or a rabbit anti-GFP antiserum (Clontech).

Western immunoblots were also used to detect the production of antibodiesagainst Lkt in rabbits immunized with transgenic plant extracts. In these exper-iments, an Lkt-containing M. haemolytica A1 cell suspension prepared as previ-ously described (21) was separated by SDS-PAGE, blotted, and probed with thevarious rabbit immune sera.

Lkt50-mGFP5 stability. Clover was harvested and allowed to dry at roomtemperature and ambient humidity for 1 to 4 days. Proteins were extracted bygrinding the tissue in 2 ml of PBS per g (fresh weight) with a Kontes ground-glasstissue grinder followed by centrifugation at 9,300 3 g for 10 min. The extract wasanalyzed by Western immunoblotting using the monoclonal antibody to Lkt.

Preparation of Lkt50-mGFP5 extracts for immunization. Two different cloverextracts were used for immunization. A crude extract was prepared by firstgrinding clover tissue to a fine powder in liquid nitrogen in a prechilled mortarand pestle. Proteins were extracted using 2 ml of extraction buffer (PBS con-taining 0.5% [wt/vol] Tween 20) per g (fresh weight). Insoluble material wasremoved by two rounds of centrifugation at 4°C (30,000 3 g for 30 min followedby 130,000 3 g for 1 h). The resulting supernatant was filtered through a 0.2-mm-pore-size syringe filter (Nalgene, Rochester, N.Y.) and stored at 220°C.

A second extract, enriched with recombinant fusion protein, was prepared bychromatofocusing (Pharmacia, Baie d’Urfe, Quebec, Canada). After grinding ofthe clover leaf tissue, the proteins were extracted in a buffer consisting of 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM phenylmethyl-sulfonyl fluoride, and 1% (wt/vol) Tween 20 and centrifuged at 130,000 3 g asdescribed above. The supernatant was applied to a PBE 94 column equilibratedwith 25 mM imidazole-HCl (pH 7.4). Proteins were eluted with Polybuffer74-HCl (pH 4.0), and 3-ml fractions were collected. Fractions containing Lkt50-GFP fusion protein were identified by Western immunoblotting with the anti-Lkt66 antiserum.

Rabbit immunization. New Zealand White rabbits (Charles River Laborato-ries, Wilmington, Mass.) were injected intramuscularly with 1 ml of filtered plantextract twice at a 2-week interval. The vaccine preparations contained a combi-

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nation of saponin (1.5% Quil A; Cederlane Laboratories, Hornby, Ontario,Canada) and aluminum hydroxide (23%) as adjuvant, in a ratio of 3 parts antigento 1 part adjuvant. A final dose was administered 4 weeks after the secondinjection. Blood was collected 4 weeks after the final injection of antigen. Tworabbits were immunized with each extract. Serum was analyzed by Westernimmunoblotting and for Lkt neutralization activity using a modified neutral redcytotoxicity assay (16).

RESULTS

Construction of lktDN and lkt50. An examination of the nu-cleotide sequence of lktA revealed the presence of two NaeIsites at positions 616 and 1651 within the gene (Fig. 1). Upondigestion with NaeI and religation of plasmid pLKT60, a1,035-bp fragment that coded for 345 amino acids containinghydrophobic domains was removed. The resulting Lkt deriva-tive (Lkt66) expressed from lktDN is expected to lack toxicityand thus is an ideal candidate for further vaccine developmentstudies.

Another derivative was constructed to take advantage ofsuitable nucleotide sequences for primer design to incorporaterestriction sites to amplify the lktA gene for cloning into plants.PCR was used to amplify a 1.35-kbp section of the lktA gene.This fragment coded for amino acids 451 to 901 of Lkt and didnot include the N-terminal and the C-terminal regions of Lkt,thus removing potential targeting signals that may interferewith its expression and/or localization in plants. This deriva-tive, lkt50, was used in subsequent cloning of fusion genes intoa binary vector for introduction into plants.

Immunogenicity of Lkt66. To ensure that the Lkt derivativesthat lacked the hydrophobic domains were still effective asvaccine candidates, Lkt66 was expressed in E. coli, recoveredfrom the culture supernatant, and used to immunize rabbits.The resulting rabbit anti-Lkt66 antiserum was tested in West-ern immunoblots as well as for toxin neutralization against theauthentic Lkt from M. haemolytica A1. In addition to recog-nizing Lkt66 as expected (data not shown), anti-Lkt66 anti-serum immunostained the full-length Lkt (102 kDa) fromM. haemolytica A1 (see Fig. 6A, lane 7). Moreover, anti-Lkt66antiserum exhibited a neutralizing titer (2log2) of up to 5 (1/32dilution) against the authentic Lkt. This is similar to the neu-tralizing titer obtained when the rabbits were immunized withfull-length rLkt. These results demonstrated that the hydro-phobic regions of the Lkt which were removed are not criticalfor immunogenicity. The rabbit anti-Lkt66 antiserum was usedin subsequent immunoblots in this study.

Transient expression of plasmid constructs in tobacco. Twochimeric constructs, lkt50-mgfp5 and mgfp5-lkt50, were insertedinto binary vectors and used to transform A. tumefaciens. Forrapid assessment of their ability to direct the production offusion proteins in plants, these genes were first expressed tran-siently in tobacco by infiltration. Constructs containing pro-moterless mgfp5-ER (F. Garabagi, unpublished data) and 35S-driven mgfp5-ER were used as controls for transient expression(Fig. 2A). Three to four days after infiltration, fluorescence wasobserved by microscopy only in plants injected with A. tume-faciens transformed with plasmid containing 35S-mgfp5-ER.Plants infiltrated with A. tumefaciens containing the promoter-less construct exhibited no fluorescence. Little or no fluores-cence was observed in the infiltrated regions of plants injectedwith A. tumefaciens containing either of the lkt50 fusion con-

structs (data not shown). The infiltrated areas were excised andexamined for the presence of fusion protein by Western im-munoblotting with rabbit anti-Lkt66. An immunoreactive bandof approximately 79 kDa was present only in extracts ofplants infiltrated with A. tumefaciens that carried the constructpBlkt50-mgfp5 (Fig. 2B). The size of this protein correspondedto that predicted from the nucleotide sequence of the con-struct. Thus, it appeared that only in the case in which GFP wasfused to the C-terminal side of Lkt50 was there accumulationof a significant amount of the fusion protein. This constructwas selected for the production of transgenic white clover lines.

Transgenic white clover expressing Lkt50-GFP. Transgenicclover lines expressing GFP and Lkt50-GFP were produced byA. tumefaciens-mediated transformation. PCR was used toconfirm that the transgenes were present in transformed plants(data not shown). By conventional fluorescence microscopy,green fluorescence was easily detected in GFP-expressingplants. Consistent with the results obtained with transient ex-pression, little to no fluorescence was observed in pBlkt-mgfp5-transformed plants. However, when these plants werefurther examined using laser scanning confocal microscopy,green fluorescence was detected in clover transformed withboth the pBINmgfp5-ER and pBlkt50-mgfp5 constructs (Fig.3B and C). As expected, GFP fluorescence was more intensethan that observed for Lkt50-GFP. Leaves from untrans-formed plants did not exhibit green fluorescence (Fig. 3A).Red chlorophyll fluorescence from chloroplasts was seen intissues from all plants. The pattern of green fluorescence ob-served in the clover leaves was consistent with localization ofthe recombinant protein in the ER (15). Cells contained largevacuoles, resulting in distribution of fluorescence around the

FIG. 2. Transient expression of chimeric genes in tobacco. (A)Proteins extracted from tobacco leaves infiltrated with A. tumefacienstransformed with constructs containing promoterless mgfp5-ER (lane1), 35S-mgfp5-ER (lane 2), or 35S-lkt50-mgfp5 (lane 3) were blotted andprobed with rabbit anti-Lkt66 antiserum. The resulting Western im-munoblot showed that a protein of 79 kDa was present only in lane 3,where the presence of the Lkt-containing fusion protein was expected.(B) The expression of Lkt50-GFP (lane 1) and GFP-Lkt50 after agro-infiltration (lane 2) was analyzed by Western immunoblotting with arabbit anti-Lkt66 antiserum as described above. A. tumefaciens, trans-formed with vectors containing either lkt50-mgfp5 (lane 1) or mgfp5-lkt50 (lane 2), was used for infiltration. Only in the case where GFP wasfused to the C terminus of Lkt50 (lane 1) was fusion protein expressiondetected. Migrations of the molecular mass markers are as indicated tothe left of each panel.


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cell periphery. The fusion protein exhibited a perinuclear lo-calization and was clearly excluded from the nucleus. A char-acteristic reticulate network was seen in some cells when theappropriate plane of focus was used.

Expression of a recombinant fusion protein containing bothLkt50 and GFP epitopes was confirmed by Western immuno-blot analysis (Fig. 4A and B). Both rabbit anti-Lkt66 and rabbitanti-GFP recognized a protein of 79 kDa. Preliminary scanningdensitometric analysis of gels and Western immunoblots ofextracts from one of the Lkt50-GFP-expressing clover linessuggested that the recombinant fusion protein constituted ap-proximately 1% of the soluble proteins extracted from trans-genic clover. This corresponds to approximately 18 mg ofLkt50-GFP per g (fresh weight) of plant tissue.

To assess the stability of the fusion protein in harvestedplants, transgenic clover expressing Lkt50-GFP was harvestedand allowed to dry at ambient temperatures. After 3 days, theclover tissue retained approximately 20% of its initial freshweight. Protein extracts were prepared from plant material atdifferent stages of drying and analyzed by Western immuno-blotting. After 4 days of drying, there did not appear to be sig-nificant degradation of the fusion protein (Fig. 4C). No lower-molecular-weight immunoreactive species were observed at

FIG. 3. Laser confocal microscopy of transgenic white clover ex-pressing Lkt50-GFP. A section of clover leaf was mounted in waterand observed by confocal microscopy. Images from two channels (redfor chlorophyll fluorescence and green for GFP fluorescence) weremerged to produce the micrographs shown. Leaves from untrans-formed clover (A) do not exhibit the green fluorescence that is presentin transgenic clover expressing Lkt50-GFP (B) or GFP (C). The pat-terns of green fluorescence in panels B and C were similar and areconsistent with an ER localization. Fluorescence intensity in GFP-expressing plants was higher than that observed in Lkt50-GFP-express-ing plants but was normalized in this figure to facilitate comparison.The bar in each panel indicates 100 mm. Vacuoles (V), nuclei (N), andchloroplasts (Ch) are indicated in panel B.

FIG. 4. Expression of Lkt50-GFP in transgenic white clover. Ex-pression of Lkt50-GFP in transgenic white clover was analyzed byWestern immunoblotting. Duplicate blots of proteins extracted fromone transgenic line were immunostained with either rabbit anti-Lkt66antiserum (panel A, lane 1) or an anti-GFP monoclonal antibody(Clontech) (B). Molecular masses of the prestained SDS-PAGE stan-dards (panels A and B, lanes M) are indicated at the left. Both anti-bodies detected a protein of similar size, providing evidence that afusion protein containing both Lkt and GFP sequences was indeedproduced by the transgenic clover. In addition, the size of the fusionprotein observed was close to 79 kDa, as predicted from the nucleotidesequence. Stability of the Lkt50-GFP fusion protein was also examined(C). Protein extracts prepared from fresh transgenic clover (lane 2) orfrom transgenic clover dried for 1 day, 2 days, 3 days, or 4 days atambient temperatures (lanes 3 to 6, respectively) were analyzed byWestern immunoblotting. The blot was probed with the monoclonalantibody 601 against Lkt. As controls, M. haemolytica A1 supernatant(20 times concentrated) containing full-length Lkt and an extract fromwild-type clover were loaded in lanes 1 and 7, respectively. Migrationsof molecular mass markers (lane M) are shown on the left. After 4 daysof drying at ambient temperatures, there does not appear to be sig-nificant loss of the Lkt50-GFP fusion protein.


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any stage. Dried wild-type clover did not give rise to any im-munoreactive bands.

Immunogenicity of Lkt50-GFP produced by transgenicwhite clover. To determine if Lkt50-GFP synthesized by cloverwas able to elicit an immune response, rabbits were immunizedwith either a saline extract or an Lkt50-GFP-enriched chro-matographic fraction prepared from transgenic clover.

The Lkt50-GFP-enriched fractions were produced by chro-matofocusing. A soluble protein extract prepared from trans-genic clover was applied to a PBE 94 column, and resultingfractions were analyzed by Western immunoblotting (Fig. 5).Most of the fusion protein eluted in fractions 6 to 8 (Fig. 5B);these fractions were used for rabbit immunization. The fusionprotein could be partially separated from ribulose-1,5-bisphos-phate carboxylase-oxygenase (rubisco, the most abundant pro-tein in plant tissue), most of which eluted in fractions 5 and 6(Fig. 5A). Under the conditions used in the fractionation,Lkt50-GFP was stable to degradation as indicated by the ab-sence of any major lower-molecular-weight immunoreactivebands in the fractions.

Pre- and postimmunization sera were obtained from rabbitsand tested for the presence of antibodies by Western immuno-blotting (Fig. 6). All rabbits receiving extracts containing Lkt50-GFP as antigen produced antibodies directed against theauthentic Lkt from M. haemolytica A1 (Fig. 6A, lanes 3 to 6).Preimmune sera (data not shown) and sera from mock-immu-nized rabbits (Fig. 6A, lanes 1 to 2) did not immunostain Lkt.

In addition to stimulating production of antibodies againstLkt, the fusion protein was also able to induce anti-GFP anti-body production. Rabbit immune serum contained antibodiesthat weakly recognized wild-type GFP (Clontech) on Westernimmunoblots (Fig. 6C).

To determine if neutralizing antibodies were present in therabbit sera, a toxin neutralization assay was performed (16).All rabbits immunized with Lkt50-GFP extracts as antigenexhibited neutralizing titers (2log2) up to 4 (1/16 dilution)(Table 2). Sera from rabbits immunized with wild-type whiteclover extract (data not shown), sera from mock-immunizedrabbits, or preimmune sera from all the rabbits failed to neu-tralize Lkt (Table 2).

FIG. 5. Preparation of Lkt50-GFP-enriched fractions for immuni-zation. A protein extract was prepared from transgenic white cloverand fractionated by chromatofocusing. Column fractions were ana-lyzed by Coomassie blue staining of gels after SDS-PAGE (A) andWestern immunoblotting (B). The fraction number is indicated on thetop, and molecular mass markers (lanes M) are indicated at the left.These results show that Lkt50-GFP (panel B, fractions 6, 7, and 8)could be separated from rubisco (strongly staining band migrating ataround 56 kDa) and other high-molecular-weight material (panel A,fractions 5 and 6).

FIG. 6. Immunogenicity of Lkt50-GFP produced by white clover.(A) Rabbits (duplicate rabbits used for each treatment) were mockimmunized with saline and adjuvant (lanes 1 and 2) or immunized withchromatographic fractions enriched in Lkt50-GFP (lanes 3 and 4) or asaline extract from transgenic clover (lanes 5 and 6). Immune serawere used to probe a total M. haemolytica A1 protein preparationblotted onto nitrocellulose. The rabbit anti-Lkt66 antiserum (lane 7)was used as a positive control. Immune serum from all four rabbitsimmunized with Lkt50-GFP-containing fractions recognized a 102-kDa band (arrow) migrating identically with full-length Lkt that wasimmunostained with anti-Lkt66 (lanes 3 to 7). Immune serum fromrabbit 41 (panel A, lane 6) was analyzed to see if it cross-reacted withwild-type GFP. Duplicate blots (B and C) were prepared containingM. haemolytica A1 protein extract (lanes 1) and purified recombinantwild-type GFP (Clontech) (lanes 2). Anti-GFP antibodies (B) andrabbit 41 immune serum (C) were used to probe the membranes.Rabbit 41 immune serum was able to detect GFP (panel C, lane 2).These results suggested that the immune serum contained antibodiesdirected to both Lkt (panel A, lane 6, and panel C, lane 1) and GFP(panel C, lane 2). Migrations of molecular mass markers for bothpanels B and C (lanes M) are indicated to the left of panel B. Arrows(B and C) show the position of GFP migration.


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DISCUSSION

As a member of the RTX family of toxins, the cytotoxicactivity of Lkt is mediated by its ability to insert and formtransmembrane pores in the plasma membrane of susceptiblehost cells, eventually leading to cell lysis. The hydrophobicregions in the Lkt protein have been implicated in mediatingpore formation (31, 32). The lktADN derivative which ex-pressed the Lkt66 molecule is an excellent candidate for use ina vaccine. With the removal of most of the hydrophobic do-mains, Lkt66 should be rendered incapable of inserting intothe target cells to cause cytotoxicity. On the other hand, it stillretains immunogenicity, as shown by its ability to elicit rabbitantibodies which neutralized authentic Lkt. Thus, we antici-pate that if Lkt66 were used as a vaccine in calves, it woulddrive an immune response against the Lkt of M. haemolyticaA1. A smaller derivative completely lacking all hydrophobicregions (Lkt50) was made during the subcloning of lktADNinto the binary vector for A. tumefaciens-mediated plant trans-formation. This Lkt50 contained all of the antigenic regions ofLkt66 and produced an Lkt neutralizing response in rabbits,suggesting that it would also be useful as a vaccine candidate.Indeed, when proteins containing either Lkt66 or Lkt50 se-quences were injected into rabbits, both antigens were able toelicit the production of toxin neutralizing antibodies.

GFP was used in this present study as a marker to enablerapid screening, to allow the monitoring of transgene expres-sion, and to facilitate simple Mendelian analysis of inheritance.In addition, with the current concerns about transgene move-ment in the environment, GFP could be used as a reporter fortracking transgenic plants in the field (13).

Using stable transformed transgenic plants to study trans-gene expression usually requires several months’ work. To rap-idly assess whether the transgene construct will be expressed atsignificant levels in plants, we used a more convenient tran-sient-expression assay by infiltrating tobacco leaves with A.tumefaciens carrying plasmid constructs. These studies do notrequire regeneration of plants after transformation and thuspermit assessment of gene expression within a few days byexamining transgenic protein expression directly by both fluo-rescence and Western immunoblotting. In our studies, agroin-filtration allowed us to identify the Lkt50-GFP construct as thechoice for continued studies and transformation into whiteclover. This was crucial for subsequent plant transformationexperiments since there was no previous documentation of theexpression of M. haemolytica genes in plants. With a different

codon usage bias, it is entirely possible that lkt is not expressedat significant levels in plants and would require extensivecodon replacements before it could be used for expression inthe transgenes. The failure of the GFP-Lkt50 construct toproduce significant amounts of the fusion protein could be dueto unknown factors that may cause inefficient or unstable tran-scription or translation, improper folding of the polypeptide,or rapid degradation of the fusion protein. While GFP is re-ported to be generally functional with both N- and C-terminaladditions, our results underscore the limitations of this gener-alization.

Using conventional epifluorescence microscopy, little if anyfluorescence was observed in Lkt50-GFP-expressing plants,even though the presence of fusion protein was detected byWestern immunoblot analysis. By confocal microscopy, a moresensitive technique for visualizing fluorescence, we were ableto observe fluorescence in Lkt50-GFP-expressing plants but ata lower intensity that that for plants expressing GFP alone.Even though both constructs were expressed from the same35S promoter, it may be possible that less fusion protein wasproduced in the transgenic plants than in plants expressingGFP alone. An alternative explanation may be that the recom-binant GFP was not able to properly fold when fused to Lkt50,resulting in reduced levels of fluorescence.

The Lkt50-GFP5 fusion protein contains an N-terminal sig-nal peptide derived from Arabidopsis vacuolar basic chitinaseand the C-terminal ER-retention sequence HDEL. Previousobservations have indicated that higher levels of fluorescencein transgene products could be achieved if GFP entered thesecretory pathway and was sequestered in the lumen of the ER(14, 15). Targeting proteins to the ER may improve maturationand accumulation and may protect plant cells from the photo-toxic effects of GFP (14, 15). A significantly higher level offluorescence has been observed in transgenic plants expressingmGPF5 than in plants expressing the wild-type GFP that lo-calizes to the cytoplasm (F. Garabagi, personal communica-tion).

Our results demonstrate that using plants to produce bacte-rial antigen as a vaccine component is a viable strategy. Weshowed that an Lkt fragment synthesized by plants was able toinduce an immune response in rabbits that led to the produc-tion of antibodies that neutralized the authentic Lkt. Proteinstability is important for vaccine harvest, production, and stor-age. Our results also indicated that the fusion protein wasrelatively stable in harvested material in the absence of refrig-eration. Plants are economical to grow and can yield a highlevel of recombinant proteins. While alfalfa may be the optimalsupplement for animal feed, white clover is a reasonable alter-native, and the present study paves the way for continuingresearch into the development of an edible vaccine using avariety of transgenic plants expressing antigens of M. haemo-lytica A1. Experiments are in progress to assess the immuno-genicity of plant-derived antigen in cattle and the effectivenessof feeding this transgenic material in stimulation of a mucosalimmune response against the Lkt of M. haemolytica A1.

ACKNOWLEDGMENTS

This research was supported by the Ontario Cattlemen’s Associa-tion, the Natural Sciences and Engineering Research Council of Can-

TABLE 2. Neutralizing titers of sera from rabbits immunizedwith Lkt50-GFP fusion protein

Immunogen Rabbitno.

Neutralizing antibody titera

of serum

Pre-immune Bleed 2 Bleed 3 Bleed 4

Lkt50-GFP (saline extract) 41 — — 4 3.5Lkt50-GFP (saline extract) 42 — — 1 1Lkt50-GFP (column fraction) 43 — — 1 1Lkt50-GFP (column fraction) 44 — — 1.5 1Mock 45 — — — —Mock 46 — — — —

a Values are mean reciprocal log2 serum dilutions giving at least 50% neutral-ization of toxicity; sera were assayed in duplicate. —, no neutralization activity.


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ada Strategic Grants Program, and the Ontario Ministry of Agricul-ture, Food, and Rural Affairs.

We thank Betty-Ann McBey for assistance with the rabbit immuni-zation.

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Editor: R. N. Moore


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