e2 expressed in tobacco plants

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Manuscript Draft

Manuscript Number: JVAC-D-11-01422R1

Title: Immunocompetent truncated E2 glycoprotein of Bovine Viral Diarrhea Virus (BVDV) expressedin Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines.

Article Type: Regular Research Articles

Keywords: Bovine Viral Diarrhoea Virus; E2; Nicotiana tabacum; Agrobacterium; plant transient

transformation; recombinant vaccine.

Corresponding Author: Dr. Maria Alejandra Alvarez, Ph.D

Corresponding Author's Institution: Instituto de Ciencia y Tecnología Dr. César Milstein (CONICET)

First Author: Guillermo Nelson, BSc

Order of Authors: Guillermo Nelson, BSc; Patricia L Marconi, PhD; Osvaldo Periolo, PhD; José L La

Torre, PhD; Maria A Alvarez, PhD

Abstract: The Bovine Viral Diarrhoea Virus (BVDV) is the etiological agent responsible for a wide

spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus

and the strongest inductor of the immune response. There are several available commercial vaccines

against Bovine Viral Diarrhoea (BVD), which show irregular performances. Here, we report the use of

tobacco plants as an alternative productive platform for the expression of the truncated version of E2

glycoprotein (tE2) from the BVDV. We have cloned the sequence of a tE2, lacking the transmembrane

domain, into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis

thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, the

KDEL endoplasmic reticulum retention signal and a His-tag to facilitate protein purification. Theresulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain

EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of

Nicotiana tabacum plants. The presence of the recombinant truncated E2 protein (rtE2) in plant

extracts was confirmed by Western Blot and ELISA assays using specific monoclonal antibodies. An

estimated amount of 20 μg of rtE2 per gram of fresh leaves was regularly obtained with this plant

system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of

BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines.

This is the first report of the production of an immunocompetent rE2 in Nicotiana tabacum plants,

having the advantage to be free of any eventual animal contaminant.



Buenos Aires, 15th August 2011

Dear EditorVaccine

I am sending you our manuscript " Immunocompetent truncated E2

glycoprotein of Bovine Viral Diarrhea Virus (BVDV) expressed in

Nicotiana tabacum plants: a candidate antigen for new generation of

veterinary vaccines " in order to be considered for being published inVaccine. We have expressed the glycoprotein E2, the main inductorof the immune response in cattle, for the first time in tobacco. Also,the recombinant E2 we have expressed was able to induce animmune response in Guinea pigs; we have had positive results inthe seroneutralisation assays and also significant antibody titres inthe ELISA test performed in sera of the immunised animals. Weconsider our work as a significant step in order to develop anefficient and competitive vaccine in a plant platform. The productionof a recombinant E2 was already performed in bacteria andbaculovirus but the yields or immunogenicity of the obtained proteinwere not competitive to the current production systems. On the otherhand, the production in the plant system has advantages from thepoint of view of biosecurity (plants do not harbour animal pathogens,

prions or bacterial endotoxins), the development and maintenanceof cultures (they are not expensive) and the scaling up, which issimple. We think that we are in the good footpath for achieving ourgoal of developing a plant- made vaccine against DVB. We will bevery honoured if we could communicate our results to therespectable scientific community of your readers and receive theirfeedback.The suggestions and comments of the reviewers have been taken inaccount as we inform in the response to them. We think that doing

so the manuscript was substantially improved, we thank them fortheir kindness.Looking forward to your decision,Yours sincerely,

Dr. María Alejandra Alvarez

ver Letter



Dear Editor,

We are sending you our revised manuscript (Ref. Ms. Ref. No.: JVAC-D-11-0142)

Ccording to the reviewer suggestions, the modifications we have made are:

Reviewer 1:

It’s true, we have made a mistake when transcribing the construct,the correct one is 2S2-tE2-His-KDEL

The reducing and non-reducing Western blots are shown in Figure 2.

Yes, the viral signal ss was removed, as we explain now in page 11,

line 7.

The titers attained in the virus neutralization assay are in page 13,

lines 1 and 12.

We have added the references suggested by de reviewer, page 13,

line 23.

We have corrected the misspelling of Kozac.

As suggested, we refer the pK7WG2 using Karimi cite, page 6. The ELISA procedure is explained in Materials and Methods section,

page 8.

The Guinea Pig Immunization procedure has been explained in the

original manuscript, maybe the reviewer did not find it. In the

present manuscript is in page 9. We have added some explanation

and reference regarding “the suitability of using guinea pigs as a

model for testing BVDV vaccines. An optimal correlation between

bovine-guinea pig models was already demonstrated indicating that

the guinea pigs could replace bovines when testing the

immunogenicity of BVDV commercial or candidate vaccines.”, page

12.

The amount of crude leaf protein extract used to immunize is in page

9.

The percentages of the total protein represented by 20 g antigen

figures in page 12, line 7.

A better description of figure 1 was introduced.

We have removed the figure 2 from the original manuscript in this

new version.

As the molecular weights in reducing and non-reducing conditions

correspond to those expected we think that the post-translational

modifications were performed.Rev. 2:

The title was modified according to the reviewers suggestions.

The abstract was improved.

More references were included regarding the current vaccines.

More references regarding to other attempt to express E2 in plants

were introduced.

A more detailed description of the results was done.

Dr. Marzocca has left research and is now working in the Argentine

Ministry of Science and Technology. However, the information about

the construct are in the references.

sponse to Reviewers



We have made the modification suggested by the reviewer, including

a better description of the samples, transformation procedure and,

immunization protocol, no purified protein was used. The formula

used were modified according was suggested.

Al the tests were done by duplicate according is specified in MM.

Rev. 3:

More information about the anti-E2 mAb figures in the text, page 8.

We have replaced, as was suggested, “concentration of 1:100” with

“1:100 dilution”, page 9.

The antibody is the same described before.

A better explanation about “ positive sera obtained from immunized

cows” is introduced in page 9.

We have introduced a comment regarding the results of the ELISAmade on plant extracts, page 11.

A paragraph about tE2 expression, tE2 amount, is added, page 12.

We have made the Western blot under reducing and non-reducing

conditions as was pointed before, the marker is in lane 4 , Figure 2A

and lane 5, Figure 2B.

Numerical data are described in page 13.

We have modified the phrase “vaccine without E2” for “Control

animals were primed and boosted with 0.5 ml of wild type tobacco

leaves extract plus each one of the two adjuvants (sham vaccine). We

also included a whole virus vaccine formulated with 106,5 titer/dose

containing the Singer strain, replicated in MDBK (Madin-Darby

bovine kidney) cells inoculated with the same schedule than the

plant vaccine (C. Seki, personnel communication). “, page 9.

We have described how the serum is inactivated, page 9, line 25.

We have explained what “the recombinant tE2” means, page

We have added the suggested references.

The jump between references was eliminated.

We have not checked the maintenance of the His-tag. We will do it in

the following steps where we will optimize the down stream process

steps.

Spelling corrections were made.

Reviewer 4:

We have modified our claim, see page 5.

Agree, we have changed the sub-title Plant transformation to Plant

transient transformation, however, our goal, in this paper, is to

express tE2 transiently and not to obtain a stable transformed

tobacco plant. The reviewer comment have made us change the title

of the manuscript to “Immunocompetent truncated E2

glycoprotein of Bovine Viral Diarrhea Virus (BVDV) expressed

inNicotiana tabacum

plants: a candidate antigen for newgeneration of veterinary vaccines. “



In Merriam Webster dictionary the definition of grind is:

http://www.webster-dictionary.net/definition/Grind We are using

the first definition.

Maybe the reviewer has not read the paragraph in the original

manuscript referred to sequence and restriction analysis. In the

present version that references are in page 6 and 7. The western blot was improved, as was explained before.

We have not tested the glycosylation pattern of our protein, we

consider that as we have the expected MW in both reducing and non-

reducing Western blot the translational modifications were correct.

The Serum Neutralization Assay is more deeply explained now, se

Materials and methods.

Figure 5 is now Figure 3 and was improved.

The purification step using His-tag will be done in future

experiments.

We agree, we have modified the statement “an effective subunitvaccine”.

Reviewer 5:

We agree and we have changed the former statement.

We have made a short description of the results obtained with other

production platforms, pages 13-14.

References regarding commercial vaccines are in the introduction,

page 4.

We have change the misspelling, s2s to 2S2

We have changed the statement “Plants are gaining widespread

acceptance as a general platform for vaccine production” assuggested for the reviewer.

True, in the former manuscript we have wrongly transcribed the

construct.

Regarding the clinical signs, if the vaccine is not well tolerated by the

animals they die.

We have introduced a discussion about other production platforms,

page 13-14.

We have checked the references and modified when necessary.

We have corrected the misspellings.

Yes both antibodies are neutralizing ones.

Yes, the primers are the same as figures in page 6.

http://www.webster-dictionary.net/definition/Grind





HIGHLIGHTS

We engineered a construct for expressing in plants a truncated version of the

glycoprotein E2 (tE2).

We have transiently expressed the recombinant tE2 en tobacco leaves by

agroinfiltration.

When subcutanously administrated our recombinant tE2 induced an immuneresponse in guinea pigs.

We could produce a vaccine against DVB in tobacco by transient transformation.

Our plant platform is competitive, efficient, safe, fast and non-expensive.

ghlights



Abstract

The Bovine Viral Diarrhoea Virus (BVDV) is the etiological agent

responsible for a wide spectrum of clinical diseases in cattle. The

glycoprotein E2 is the major envelope protein of this virus and the

strongest inductor of the immune response. There are several available

commercial vaccines against Bovine Viral Diarrhoea (BVD), which show

irregular performances. Here, we report the use of tobacco plants as an

alternative productive platform for the expression of the truncated version

of E2 glycoprotein (tE2) from the BVDV. We have cloned the sequence of

a tE2, lacking the transmembrane domain, into the pK7WG2

Agrobacterium binary vector. The construct also carried the 2S2

Arabidopsis thaliana signal for directing the protein into the plant secretory

pathway, the Kozak sequence, the KDEL endoplasmic reticulum retention

signal and a His-tag to facilitate protein purification. The resulting plasmid

(pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens

strain EHA101 by electroporation. The transformed A. tumefaciens wasthen used to express tE2 in leaves of Nicotiana tabacum plants. The

presence of the recombinant truncated E2 protein (rtE2) in plant extracts

was confirmed by Western Blot and ELISA assays using specific

monoclonal antibodies. An estimated amount of 20 g of rtE2 per gram of

fresh leaves was regularly obtained with this plant system. Injection of

guinea pigs with plant extracts containing 20 g of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than

those induced by whole virus vaccines.

This is the first report of the production of an immunocompetent rE2 in

Nicotiana tabacum plants, having the advantage to be free of any eventual

animal contaminant.

stract



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Immunocompetent truncated E2 glycoprotein of Bovine Viral Diarrhea Virus

(BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new

generation of veterinary vaccines.

Guillermo Nelson, Patricia Marconi, Osvaldo Periolo, José La Torre, María

Alejandra Alvarez *

Instituto de Ciencia y Tecnología Dr. César Milstein, CONICET- Fundación

Pablo

Cassará, Saladillo 2468, Ciudad de Buenos Aires, C11440FFX, Argentina.

*Corresponding author: [email protected]

Running title:

Expression of the glycoprotein E2 of Bovine Viral Diarrhea Virus in tobacco

plants.

nuscript

k here to view linked References

mailto:[email protected]



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http://ees.elsevier.com/jvac/viewRCResults.aspx?pdf=1&docID=14352&rev=1&fileID=402440&msid=78D8B389-C565-4AF7-8A84-3CC1EA846E1F




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Abstract

The Bovine Viral Diarrhoea Virus (BVDV) is the etiological agent responsible for

a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major

envelope protein of this virus and the strongest inductor of the immune response.

There are several available commercial vaccines against Bovine Viral Diarrhoea

(BVD), which show irregular performances. Here, we report the use of tobacco

plants as an alternative productive platform for the expression of the truncated

version of E2 glycoprotein (tE2) from the BVDV. We have cloned the sequence of

a tE2, lacking the transmembrane domain, into the pK7WG2 Agrobacterium binary

vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing

the protein into the plant secretory pathway, the Kozak sequence, the KDEL

endoplasmic reticulum retention signal and a His-tag to facilitate protein

purification. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into

Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A.

tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants.

The presence of the recombinant truncated E2 protein (rtE2) in plant extracts was

confirmed by Western Blot and ELISA assays using specific monoclonal

antibodies. An estimated amount of 20 g of rtE2 per gram of fresh leaves was

regularly obtained with this plant system. Injection of guinea pigs with plant

extracts containing 20 g of rtE2 induced the production of BVDV specific

antibodies at equal or higher levels than those induced by whole virus vaccines.

This is the first report of the production of an immunocompetent rE2 in Nicotiana

tabacum plants, having the advantage to be free of any eventual animal

contaminant.

Key words: Bovine Viral Diarrhoea Virus, E2, Nicotiana tabacum,



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Agrobacterium, plant transient expression, recombinant vaccine.

Abbreviations:

BVDV: Bovine Viral Diarrhoea Virus

KIN: Kinetin

GMP: Good Manufacture Practices

MDBK: Madin-Darby Bovine Kidney Cells

MS: Murashige and Skoog media

NAA: 1-naphtalene acetic acid

OD: optical density

PMSF: phenylmethanesulfonyl fluoride

TSP: total soluble protein

SDS: sodium dodecyl sulfate

WT: wild type

VNT: virus – neutralization test

dpt: day post infection

tE2: truncated E2

rtE2: recombinant truncated E2



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Introduction

Bovine Viral Diarrhoea (BVD) is a widespread cattle disease that causes serious

mucosal lesions and other clinical manifestations including reproductive disorders,

congenital defects, persistent infections, enteritis, and mucosal alterations. It is

estimated that BVD generates a negative economic impact in dairy operations

(between $20 and $160 per adult cow per year), which includes loss of milk

production, reproductive wastage, medication and labour to treat acute infections,

and increased mortality (1). The causative agent is the Bovine Viral Diarrhoea

Virus (BVDV), a member of the genus Pestivirus (Flaviviridae) (2) whose genome

consists of a positive-stranded RNA of about 12,300 nucleotides encoding a huge

polyprotein that is cleaved into structural and non-structural proteins. The

glycoprotein E2, the main component of the virion (3), consists of about 370 amino

acids with a theoretical molecular mass of 41 kDa. The C terminus of E2 includes a

transmembrane domain of approximately 30 amino acids, which anchors it to the

membrane of the virus-infected cells (4). E2 is the major target of the protective

immune response elicited against BVDV infection.

Commercially available inactivated and modified-live BVDV vaccines (MLV)

have been extensively used for fighting the disease. Even though both types of

vaccines are in use, there is still a controversy regarding their efficiency (5). Thus,

the E2 subunit vaccines have been considered as an alternative to induce protection

in immunized animals (1, 6, 7, 8). Nevertheless, the recombinant E2 (rE2)

expressed in bacteria did not induce neutralizing antibodies in rabbits, which was

attributed to a misfolding of E2 when expressed in prokaryotes (9) or it is produced

as an aggregated insoluble protein (10). In this last case the costs of solubilisation

and endotoxin removal have to be considered when analyzing the production costs.

On the other hand, the rE2 expressed in the baculovirus system produced



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neutralizing antibodies but at low titers (6).

Plants are gaining attention for the large-scale production of recombinant proteins

and particularly, as a promising platform for vaccine production (11, 12, 13, 14)

although a higher number of clinical trials have to be performed in order to gain

general acceptance. To date, there is more than a dozen of plant-made

biopharmaceuticals in clinical developments (15). Some of the advantages of the

plant-based systems are that they can rapidly be bulked to large biomass, its

maintenance is relatively inexpensive, and they do not harbour mammalian proteins

or pathogens. Protein production could be achieved through stable or transient

expression. The stable transformation methods, which are generally in use, modify

the genetic background of the productive plant species generating biosafety

concerns (11, 12). The transient expression strategy, where the foreign DNA is not

integrated into the plant genome, has a much shorter timeframe and the ability to

meet the stringent demands for high quality biologics and high biosafety standards

at a competitive scale and cost (16, 17). In this work we report the expression of the

BVDV truncated glycoprotein E2 in tobacco plants by transient expression with

Agrobacterium tumefaciens. Also, we tested the ability of the rtE2 to induce

specific antibodies in guinea pigs, which is an accepted animal model for testing

BVDV vaccines (18, 19). The results obtained indicate the feasibility to develop

and produce a new generation of BVDV vaccines.



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Materials and Methods

Plasmid construction:

The plasmid pss-E2 harbouring the VSV E2 glycoprotein cDNA (20) was kindly

provided by Dr. Marzocca. The E2 coding region (1029 nt) without its

transmembrane domain was amplified by polymerase chain reaction (PCR) using

5´CACCATGGCAAACAAGCTCTTCCTCGTCTGCGCAACTTTCGCCCTCTG

CTTCC

TCCTCACCAACGCTGACTTGGATTGCAAACCTG 3´as a forward primer,

carrying the NcoI restriction site, the Arabidopsis 2S2 signal (replacing the ss viral

secretory signal peptide of Dr. Marzocca construct), the Kozak sequence, and

5´TCTCGATCAATGATGATGATGATGATGTAGCTCGTCCTTGGACTC 3´ as

the reverse primer with the KDEL ER- retention signal and a His-tag. Also, the

amplified product was digested with NcoI / XhoI, and then purified and cloned into

the corresponding sites of the Gateway® pENTR-4 vector previously digested with

the same restriction enzymes (Invitrogen) rendering the pENTR-2S2-tE2-His-

KDEL plasmid. A following recombination step between the plasmid pENTR-2S2-

tE2-His-KDEL and the binary vector pK7WG2 was performed using the

Gateway® technology following the manufacturer’s instructions resulting in the

plasmid pK-2S2-tE2-His-KDEL (21) (Figure 1).

The constructs were checked by PCR, restriction endonuclease mapping and

sequencing. Sequencing was done using a DNA ABI 373A automated sequencer,

according to the Sanger method (22). We have followed standard procedures for

the plasmid manipulations (23).

Agrobacterium transformation:

The pK-2S2-tE2-His-KDEL vector was introduced in A. tumefaciens strain

EHA101 by electroporation (24). A volume of 80 μl of competent cells was mixed

with 200 ng plasmid DNA and suspended in an electroporation cuvette with an



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electrode distance of 0.1 cm (Electroporation Cuvettes Plus). A single electric pulse

of 2.5kV was applied using the 25 F capacitor (Gene pulser, Bio-Rad). After the

pulse, cells were immediately transferred into 200 μl of YEB and incubated at 28ºC

during three hours, before spreading on the YEB plates. Screening of different

transformants was performed on YEB medium containing 50 μg ml-1 kanamycine,

20 μg ml-1 rifampicin, and 100 μg ml

-1 spectinomycin. We have checked the

colonies by PCR analysis, and the purified plasmid by restriction endonuclease

map, and sequencing.

Plant transient expression:

An overnight Agrobacterium culture was inoculated in YEB medium (1:5 v/v)

supplemented with antibiotics (100 μg ml-1 kanamycine, 20 μg ml

-1 rifampicin, 100

μg ml-1 spectinomycin) and 20 μM acetosyringone. Cultures were grown overnight

at 28°C and 250 rpm. A. tumefaciens cells were then harvested by centrifugation

(4000 g at 4°C for 15 min) and re-suspended in MMA medium (MS salts, 10 mM

MES, 20 g l-1 sucrose, pH 5.6, and 200 μM acetosyringone) (25, 26, 27). The A.

tumefaciens (pK-2S2-tE2-His-KDEL) suspension was kept at room temperature for

2 h after being used for plant transient expression. N. tabacum in vitro-grown

plantlets were transferred to 330 cc pots filled with soil and kept in greenhouse

under controlled conditions. Leaves of 2-week-old- plants were infiltrated with the

A. tumefaciens (pK-2S2-tE2-His-KDEL) suspension by inoculation with a syringe

without a needle by the abaxial side of the lamina (28). We have also infiltrated

tobacco leaves with wild type A. tumefaciens strain EHA101 or with culture

medium, as negative controls. Plants were then placed in a culture chamber at 24 ±

1°C under a 16 h photoperiod given by cool white fluorescent light (57 μE.m-2.s-1,

TLT 110W/54 RS Philips day-light tubes). Samples were taken at the 4th post-

infiltration day.

Protein extraction:



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An amount of 0.5 g of sample was grinded in 1 ml of PBS extraction buffer (0.24 g

l-1 KH2PO4, 1.44 g l-1 Na2HPO4, 0.2 g l-1 KCl, 8 g l-1 NaCl, 10 μg ml-1 leupeptin, 5

mM PMSF, 50 mM EDTA, pH: 7.0-7.2), using a cold mortar and pestle and then,

centrifuged at 14,000g for 20 min at 4°C. Total protein content was determined

according to Bradford (29).

ELISA:

The sandwich ELISA test for determining the presence of tE2 in plant extracts was

performed in a polystyrene microtiter plate (Maxisorp, NUNC). Plates were coated

with anti-E2 monoclonal antibody 2.9H (18) at 4°C overnight. The plates were then

incubated with PBS- Tween 0.05% and 1% skim milk (blocking buffer) for 1 h at

37°C. The blocking buffer was discarded, and the plates were washed three times

with PBS- Tween 0.05%. Protein samples were added to the plates and incubated

for 1 h at 37º C. The plates were washed three times with PBS-Tween and

incubated with positive sera obtained from seropositive experimental infected cows

for 30 min at 37°C. Horseradish peroxidase (HRP) conjugated goat anti- bovine

IgG was used as the secondary antibody. Those plates were then incubated for 30

min at 37°C. After washing three times, the reaction was developed with H 2O2 and

0.05% 2,2´-azinobis (3- ethylbenzthiazoline-6-sulfonic acid) (ABTS) for 30 min.

Colour development was stopped by the addition of 5% SDS. The absorbance was

measured at 405 nm (30).

Western blot Semi quantification:

Protein samples were re-suspended in loading buffer, denatured at 95°C for 10 min

and subsequently analyzed on 10% SDS- PAGE under non-reducing conditions

(20). Loading buffer for reducing conditions also contained 5% (v/v) -

mercaptoethanol (Sigma) to reduce disulfide bridges. The separated protein was

blotted onto a nitrocellulose membrane (Immobilon-P, MILLIPORE).

Western blot was performed using the 2.9H mouse monoclonal antibody anti-E2,



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1:100 dilution (C. Seki, Personnel communication), and a 1:500 goat anti-gamma

mouse chain-conjugated peroxidase antibody (Sigma). As a positive control we

have used recombinant tE2 expressed in baculovirus (20). The immune complexes

were detected after incubation with Supersignal West Pico Chemiluminiscent

Substrate (Pierce Chem). Band intensity was analyzed using Gel-Pro Analyzer.

Guinea pig Immunization Schedule

Guinea pigs (60 day-old female, average weight, 413 g) were subcutaneously

immunized administering 0,5 ml of a vaccine formulated combining an oil adjuvant

(Montanide ISA 70 SEPPIC®) and 20 µg of antigen (tE2 glycoprotein accumulated

in the plant extract), in an adjuvant to antigen ratio of 60:40. In the case of the

aqueous vaccine (Al(OH)3), animals were subcutaneously inoculated administering

0,5 ml of vaccine in an adjuvant Al(OH)3 (Hydrogel®) to antigen ratio of 10:90.

Control animals were primed and boosted with 0.5 ml of wild type tobacco leaves

extract plus each one of the two adjuvants (sham vaccine). We also included a

whole virus vaccine formulated with 106,5 titer/dose containing the Singer strain,

replicated in MDBK (Madin-Darby bovine kidney) cells inoculated with the same

schedule than the plant vaccine (C. Seki, personnel communication). Blood

samples were collected at the time of immunization (0 day), and at the 15th and

30th days after vaccination (31). The bleed sera of each animal were used for

performing the Virus Neutralization Test and the ELISA. During the whole

experiment the animals were clinically evaluated.

Virus Neutralization Test:

Sera neutralising antibodies were detected by a virus neutralization test, as

recommended by OIE (31). Briefly, 100 TCID50 of BVDV (Singer strain) were co-

incubated with 75 μl of 1/4 serial dilutions of the inactivated (20 min at 56ºC)

serum samples for 1 h at 37°C. The mixture was then transferred to microtiter

plates with 3x104 MDBK cells/well. The plates were incubated for 72 h at 37°C



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with 5% CO2. The respective titers were measured using the Reed and Muench

method (32). Titers were expressed as the log of the highest dilution able to inhibit

100 TCID50. Neutralization testing for all dilutions was run in duplicate (27).

Evaluation of anti- E2 specific antibodies by ELISA:

Polystyrene microtiter plates (Maxisorp, NUNC) were coated with anti-E2

monoclonal 2.9H antibody at 4°C overnight. The plates were then incubated with

PBS -Tween 0.05% and 1% skim milk (blocking buffer) for 1 h at 37°C. The

blocking buffer was discarded, and the plates were washed three times with PBS-

Tween 0.05%. The rE2 glycoprotein expressed in baculovirus (20) was added to

the plates and incubated for 1 h at 37º C. The plates were washed three times with

PBS-Tween 0.05% and incubated with the sera obtained from the immunized

guinea pigs, in serial dilutions ranging from 1/4 to 1/16192, for 30 min at 37°C.

Horseradish peroxidase (HRP) conjugated goat anti-guinea pig IgG 1:1500

(Jackson ImmunoResearch Inc.) was used as the secondary antibody. The plates

were incubated for 30 min at 37°C. After washing three times, the reaction was

developed with H2O2 and 0.05% 2,2´-azino-bis (3-ethylbenzthiazoline-6-sulfonic

acid) (ABTS) for 30 min. Colour development was stopped by the addition of 5%

SDS. The absorbance was measured at 405 nm. (30)



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Results and Discussion

Gene construct and Agrobacter ium tr ansformation :

It is well known that when BVDV infects the cell, the viral glycoprotein E2

remains associated to the cells by an anchor domain of approximately 30 amino

acids (33). In order to facilitate downstream processing (DSP), we have cloned tE2

version deprived from its transmembrane domain as was described in Materials and

Methods (4). The construct also carried, instead of the ss viral secretory signal

peptide, the 2S2 sequence from Arabidopsis thaliana to target the protein into the

secretory pathway, and the KDEL-ER retention signal, to reduce protease

degradation (34). Finally, we included in the construct, a His-tag in order to

facilitate future purifications steps. The resulting construct (2S2-tE2-His-KDEL)

(Figure 1) was cloned into the Agrobacterium binary vector pK7WG2 rendering the

plasmid pK-2S2-tE2-His-KDEL. The plasmid was then introduced into A.

tumefaciens by electroporation. The presence of the insert (1020bp) was confirmed

by PCR (Figure 1b) and also by restriction maps and sequencing (data non-shown).

The A. tumefaciens bearing the pK-2S2-tE2-His-KDEL was used to perform

transient expressions in tobacco leaves.

Expression of the recombinant tE2 glycoprotein in tobacco leaves:

N. tabacum leaves were infected and harvested (as described in Materials and

Methods) circa 4th days post-infection (dpi) when the maximum level of tE2

expression is expected. The Western blot analysis made under non-reducing

conditions clearly shows the expression of tE2 as a dimer of approximately 80 kDa,

a pattern similar to that positive control (Figure 2) (20). The Western blot made

under reducing conditions showed a band of 35kDa, which corresponds to the

expected molecular weight of the tE2 monomer (20). Our results suggests that post-

transductional modifications of the recombinant tE2 were similar to those of the



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native protein and that the tE2 expressed was able to form dimers, which are

considered to be the most immunogenic form of tE2 (20, 35, 36, 37). No

immunoreactive bands were detected in mock-infected plants. The specific

recognition of tE2 by the 2.9H monoclonal antibody (20) confirmed that the native

E2 antigenic distinctiveness was preserved upon expression into tobacco plants.

Semi quantitative Western blot analysis indicated an expression level of

approximately 20 µg g-1 of infected leaf, corresponding to 1,3 % of total soluble

protein. This expression level suggests that our plant system is suitable for tE2

production purposes (38, 39).

Seroconversion of guinea pigs inoculated with plant recombinant tE2 :

A growing body of evidence indicates the suitability of using guinea pigs as a

model for testing BVDV vaccines. An optimal correlation between bovine-guinea

pig models was already demonstrated (18, 19), indicating that the guinea pigs could

replace bovines when testing the immunogenicity of BVDV commercial or

candidate vaccines.

Two different formulations of plant recombinant tE2 vaccines were tested. One

formulated with aqueous adjuvant (Al(OH)3) and the other one with oily adjuvant

(Montanide ISA 70). In order to establish the production of an anti-BVDV response

induced in the animals vaccinated with the recombinant tE2, sera was tested both

by an indirect ELISA (20) and by a virus neutralization test (27).

In both cases, it was established that values higher than 0.6 corresponding units

(OD405 or VNT titer) were considered positive. As it is shown in figure 3, in the

indirect ELISA, high titers of antibodies were obtained from animals inoculated

with our plant vaccine. In both cases the response was positive besides the typical

individual heterogeneity of any animal model (18). In the case of the plant-made

vaccine with an oily adjuvant (fig 3a), six out of seven animals showed titers higher



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than 0.6 indicating a clear seroconversion of vaccinated animals. Although the

number of animals used is low, the 86% of sero-conversion obtained suggests a

good potential performance for the recombinant antigen. On the contrary, titers of

sham-vaccinated (without antigen) animals remained below 0.6. In the case of the

Al(OH)3 adjuvant vaccine, the number of sera-converted animals, six out of eight

guinea pigs (75%), showed a clear sero-conversion both in the ELISA and

VNT(data not shown).

After ELISA analysis sera were controlled by VNT as described in Material and

Methods. This technique demonstrated the induction of neutralising antibodies after

inoculating guinea pigs with our plant recombinant tE2 vaccine. In the case of the

oily adjuvant vaccine only three out of seven animals produced neutralization

antibodies with titers ranging from 0,6 to 1,7. Whereas, in the case of the aqueous

vaccine adjuvant, all the animals showed neutralizing antibodies. Most important,

titers were equal or higher when inoculated with our plant vaccine rather than with

conventional vaccines consisting in whole virus formulations. In addition, sham

vaccines did not induce neutralizing antibodies. Regarding the difference between

oily and aqueous vaccine adjuvants and taking into account that the emulsion was

correctly prepared and stabilized, it can be speculated that the conformation or

presentation of the plant-made-tE2 to the immune system could be different for

each adjuvant. However, more work should be done in order to confirm this

hypothesis.

This is the first report on the expression of a BVDV tE2 glycoprotein in

tobacco plants. However, an E2 version was already expressed in alfalfa (40) by

stable transformation with a viral vector with a yield oscillating between 0.05 and

0.50 mg g-1 TSP. Other authors have reported the expression of E2 in insect cell

lysates infected with recombinant baculovirus (10 g E2 ml-1) (41), Drosophila

melanogaster cells (20), Escherichia coli (1 to 2 g E2 ml-1) (10), and human cell



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cultures (HEK293T cells) (33). As for the commercial vaccines, none of the

available veterinary vaccines have demonstrated to be optimal for controlling the

disease (41). They have several secondary effects such as immunosupression and

congenital defects. Also, vaccinated animals can generate persistent infections by

non-cytopathic BVDV biotypes. Furthermore, modified live vaccines are a

potential source of in utero infections and /or immunosupression. Finally,

modified-live, attenuated or inactivated BVDV that are produced in bovine cell

cultures can introduce contaminant pathogens adding the risk of intact virus release

(42).

Conclusions

The tE2 transiently expressed in tobacco leaves is recognized by a specific

monoclonal antibody as its native counterpart confirming that the recombinant

protein has the expected reactivity. Moreover, tE2 produced by plant transient

expression has the ability to generate a response in the immune system of guinea

pigs, with the production of specific antibodies that were revealed by the ELISA

and the Virus Neutralization Test.

Further experiments are being carried out in cattle in order to demonstrate that this

plant transient expression system would be useful for developing a vaccine for

cattle against BVD and the efficiency of our formulation. Also, future work will be

oriented to check the His-tag maintenance and to optimize the down stream process

for purification of the tE2 produced in tobacco plants.

Acknowledgements

This work was supported by the Agencia Nacional de Producción Científica y

Tecnológica (ANPCyT) (PICT 2005 Start up nº 35644) wich together with

CONICET funded the PhD grants of G. Nelson. Dr P Marconi, MA Alvarez and J



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La Torre are members of CONICET. We wish to thank Dr. Cristina Seki (ICT-

Milstein, CONICET) for her technical assistance and Ms Carlota Thompson for her

careful revision of English.

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Figure 1: Construct analysis.

a) Schematic diagram of the transformation vector pK-2S2-tE2-His-KDEL.

CaMV35S, cauliflower mosaic virus 35S promoter; npt II, neomycin

phosphotransferase gene; 35S terminator, cauliflower mosaic virus 35S

transcription terminator; 2S2, Arabidopsis secretory signal; KDEL, KDEL-ER

retention signal; tE2, truncated E2 version lacking its transmembrane domain; RB,

T-DNA right border; LB, T-DNA left border.

b) PCR of Agrobacterium carrying pK-tE2-His-KDEL. Amplification was

performed using primers E2 Fw and E2 Rv. Lane 1: ladder 100pb (Invitrogen), lane

2: transformed Agrobacterium, lane 3: vector pss-E2, (plasmid DNA as positive

control), lane 4: wild type Agrobacterium.

Figure 2: Protein analysis in plant extract.

a) Western blot analysis showing the expression of E2 glycoprotein in transient

transformed N. tabacum leaves. In non-reducing conditions (Lane 1 to 3) the E2

is revealed as an 80 kDa protein whereas in reducing conditions (Lane 5 to 7)

the protein is revealed as a 35 kDa protein. The crude protein extracted from the

leaves (30 μl) was separated on SDS- 10% PAGE and Western blot analysis was

performed using a 2.9H specific mouse monoclonal antibody against E2. Lane

1: agroinfiltrated tissue with wild type Agrobacterium, lane 2: agroinfiltrated

tissue with Agrobacterium carrying pK-tE2-His-KDEL, lane 3: positive control

(rE2 expressed in baculovirus), lane 4: Rainbow ladder (GE) lane 5:

agroinfiltrated tissue with wild type Agrobacterium, lane 6: agroinfiltrated tissue

with Agrobacterium carrying pK-tE2-His-KDEL, lane 7: positive control (rE2

expressed in baculovirus).

a) Semi-quantitative Western Blot. Lane 1 to lane 4: different amounts of

protein extract (20µl, 15µl, 10µl, 5µl). Lane 6 to lane 8: different amounts of



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positive control (200ng, 50ng, 25ng, 12,5ng). Lane 5: Rainbow ladder (GE).

b) ELISA test to determine the presence of the antigen E2 in plants extract.

We have performed an ELISA test as described in Materials and Methods, using

our plant extract as antigen. Both the positive control (rE2 expressed in

baculovirus) and the leaves agroinfiltrated with A. tumefaciens carrying pK-tE2-

His-KDEL, showed a similar absorbance at 600 nm, whereas negative control

(water) and mock agroinfiltrated tissue showed lower absorbance indicating the

presence of rE2 in the agroinfiltrated leaves. 1: Positive control (rE2 expressed in

baculovirus), 2: leaves agroinfiltrated with A. tumefaciens carrying pK-tE2-His-

KDEL, 3: Mock infected Nicotiana tabacum plants , 4: water.

Figure 3: Immune response of guinea pigs inoculated with plant extract.

ELISA and Virus Neutralization Test to determine specific anti-E2 antibodies in

guinea pigs. BVDV experimental vaccines were prepared in oily (Montanide ISA

70 SEPPIC®) and aqueous (Al(OH)3 Hydrogel®) formulations.

Control animals were immunized with sham vaccine or with whole virus vaccine.

Specific antibodies were analyzed as described in Materials and Methods. Each

point represents the mean ± SD.



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1

Figure 1: Construct analysis.

a) Schematic diagram of the transformation vector pK-2S2-tE2-His-KDEL.

CaMV35S, cauliflower mosaic virus 35S promoter; npt II, neomycin

phosphotransferase gene; 35S terminator, cauliflower mosaic virus 35S

transcription terminator; 2S2, Arabidopsis secretory signal; KDEL, KDEL-ER

retention signal; tE2, truncated E2 version lacking its transmembrane domain; RB,

T-DNA right border; LB, T-DNA left border.

b) PCR of Agrobacterium carrying pK-tE2-His-KDEL. Amplification was

performed using primers E2 Fw and E2 Rv. Lane 1: ladder 100pb (Invitrogen), lane

2: transformed Agrobacterium, lane 3: vector pss-E2, (plasmid DNA as positive

control), lane 4: wild type Agrobacterium.

Figure 2: Protein analysis in plant extract.

a) Western blot analysis showing the expression of E2 glycoprotein in transient

transformed N. tabacum leaves. In non-reducing conditions (Lane 1 to 3) the E2

is revealed as an 80 kDa protein whereas in reducing conditions (Lane 5 to 7)

the protein is revealed as a 35 kDa protein. The crude protein extracted from the

leaves (30 μl) was separated on SDS- 10% PAGE and Western blot analysis was

performed using a 2.9H specific mouse monoclonal antibody against E2. Lane

1: agroinfiltrated tissue with wild type Agrobacterium, lane 2: agroinfiltrated

tissue with Agrobacterium carrying pK-tE2-His-KDEL, lane 3: positive control

(rE2 expressed in baculovirus), lane 4: Rainbow ladder (GE) lane 5:

agroinfiltrated tissue with wild type Agrobacterium, lane 6: agroinfiltrated tissue

with Agrobacterium carrying pK-tE2-His-KDEL, lane 7: positive control (rE2

expressed in baculovirus).

a) Semi-quantitative Western Blot. Lane 1 to lane 4: different amounts of

protein extract (20µl, 15µl, 10µl, 5µl). Lane 6 to lane 8: different amounts of

ure(s)



2

positive control (200ng, 50ng, 25ng, 12,5ng). Lane 5: Rainbow ladder (GE).

b) ELISA test to determine the presence of the antigen E2 in plants extract.

We have performed an ELISA test as described in Materials and Methods, using

our plant extract as antigen. Both the positive control (rE2 expressed in

baculovirus) and the leaves agroinfiltrated with A. tumefaciens carrying pK-tE2-

His-KDEL, showed a similar absorbance at 600 nm, whereas negative control

(water) and mock agroinfiltrated tissue showed lower absorbance indicating the

presence of rE2 in the agroinfiltrated leaves. 1: Positive control (rE2 expressed in

baculovirus), 2: leaves agroinfiltrated with A. tumefaciens carrying pK-tE2-His-

KDEL, 3: Mock infected Nicotiana tabacum plants , 4: water.

Figure 3: Immune response of guinea pigs inoculated with plant extract.

ELISA and Virus Neutralization Test to determine specific anti-E2 antibodies in

guinea pigs. BVDV experimental vaccines were prepared in oily (Montanide ISA

70 SEPPIC®) and aqueous (Al(OH)3 Hydrogel®) formulations.

Control animals were immunized with sham vaccine or with whole virus vaccine.

Specific antibodies were analyzed as described in Materials and Methods. Each

point represents the mean ± SD.



3



4

e2 expressed in tobacco plants

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