DNA Immunization with Plasmids Encoding Fusion and Nucleocapsid Proteins of Bovine Respiratory Syncytial Virus Induces a Strong Cell-Mediated Immunity and Protects Calves against Challenge

0022-538X/07/$08.00

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doi:10.1128/JVI.00502-07

Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DNA Immunization with Plasmids Encoding Fusion and Nucleocapsid

Proteins of Bovine Respiratory Syncytial Virus Induces a Strong

Cell-Mediated Immunity and Protects Calves against Challenge

䌤

Mathieu Boxus,

* Maryle

`ne Tignon,

Stefan Roels,

Jean-Franc¸ois Toussaint,

Karl Walravens,

Marie-Ange Benoit,

Philippe Coppe,

Jean-Jacques Letesson,

Carine Letellier,

and Pierre Kerkhofs

Veterinary and Agrochemical Research Centre (VAR), 1180 Brussel, Belgium,

and Faculte´s Universitaires Notre-Dame de la Paix,

Unite´ de Recherche en Biologie Mole´culaire, 5000 Namur, Belgium

Received 9 March 2007/Accepted 13 April 2007

Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and

cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed two

codon-optimized plasmids encoding the bovine RSV fusion (F) and nucleocapsid (N) proteins and assessed

their immunogenicity in young calves. Two administrations of both plasmids elicited low antibody levels but

primed a strong cell-mediated immunity characterized by lymphoproliferative response and gamma interferon

production in vitro and in vivo. Interestingly, this strong cellular response drastically reduced viral replication,

clinical signs, and pulmonary lesions after a highly virulent challenge. Moreover, calves that were further

vaccinated with a killed-virus vaccine developed high levels of neutralizing antibody and were fully protected

following challenge. These results indicate that DNA vaccination could be a promising alternative to the

classical vaccines against RSV in cattle and could therefore open perspectives for vaccinating young infants.

Bovine respiratory syncytial virus (BRSV) and human

respi-ratory syncytial virus (HRSV) belong to the

Pneumovirus

genus

of the

Paramyxoviridae

family (52). These two negative

single-stranded RNA viruses share common genomic, antigenic,

epidemiological, and pathological characteristics (62).

BRSV and HRSV are major causative agents of severe

res-piratory tract diseases in cattle and infants worldwide,

respec-tively (20, 31, 62). Both BRSV infection and HRSV infection

can remain asymptomatic or cause severe respiratory tract

diseases leading sometimes to death (62). Seventy percent of

calves exhibit a positive serological response against BRSV at

the age of 12 months, and mortality can reach up to 20% in

some outbreaks (31, 61). From figures available in

industrial-ized countries, the number of annual HRSV infections

world-wide can be estimated around 64 million and mortality could

be as high as 160,000 (20).

For these reasons, efficient vaccines against HRSV and

BRSV are needed. However, their development has been

ham-pered since the dramatic vaccine failure in the 1960s. In fact,

vaccination with formalin-inactivated, alum-adjuvanted virus

predisposed children to a far more serious, and sometimes

fatal, form of pathology in the case of natural infection (29).

Subsequently, it was found that a similarly inactivated BRSV

vaccine could induce strikingly similar immunopathology (47).

Further studies in mice and cattle suggested that exacerbation

of disease resulted from a polarized type 2 T-helper cell

re-sponse characterized by increased production of interleukin-4

(IL-4) and IL-5 cytokines, high levels of immunoglobulin G1

(IgG1) and IgE, and a lack of BRSV-specific CD8

T cells,

resulting in enhanced pulmonary eosinophilia (10, 13, 18, 25,

27, 63, 67).

Recently, DNA vaccines have emerged as a promising

alter-native to the modified live and killed-virus (KV) vaccines.

Direct immunization with naked DNA results in the

produc-tion of immunogenic antigens in the host cell which can readily

go through processing and presentation via both class II and

class I pathways and engender long-lasting humoral and

cell-mediated immunity. Furthermore, DNA vaccines mimic live

attenuated virus in their ability to induce both humoral and

cellular responses but are considered to be safer and to offer

several technical advantages (21, 22). Finally, since the

immu-nizing protein is not present in the vaccine preparation,

plas-mid DNA is not susceptible to direct inactivation by maternal

antibodies (44).

So far, DNA vaccination against HRSV has been mainly

investigated in mice or cotton rats (6, 8, 32, 33, 58). These

studies demonstrated that plasmids encoding the HRSV fusion

(F) or attachment (G) proteins primed both humoral and

cell-mediated immunity and protected against HRSV infection

without significantly enhancing pulmonary pathology following

challenge. Despite these promising results, very few studies

confirmed the ability of DNA vaccines to protect against RSV

infection in a natural host. DNA immunization with plasmid

encoding BRSV F or G protein primed the humoral response

of young calves, reduced virus excretion, and partially

pro-tected them after experimental infection (48, 53). Similarly,

DNA immunization against BRSV F and nucleocapsid (N)

proteins was shown to be safe, immunogenic, and partially

protective in infant rhesus monkeys (64). Even if these reports

highlight the potential of DNA vaccination, it seems that the

efficacy of this strategy has to be improved in terms of the

quality and intensity of the response induced.

Codon optimization and protein boost following DNA

vac-* Corresponding author. Present address: Biologie Cellulaire et

Mole

´culaire, Faculte

´ des Sciences Agronomiques, 5030 Gembloux,

Belgium. Phone: 32 (81) 62.21.57. Fax: 32 (81) 61.38.88. E-mail:

[email protected].

䌤

Published ahead of print on 25 April 2007.

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cination are two commonly used methods that improve the

efficacy of DNA immunization (21, 66). In this report, we

designed codon-optimized plasmids encoding BRSV F and N

proteins and assessed their immunogenicity in young calves.

MATERIALS AND METHODS

Plasmids.Full-length nonoptimized F and N genes of BRSV were amplified by reverse transcription-PCR (RT-PCR) from viral mRNA extracted from cell culture supernatant infected with the BRSV strain RB94 as previously described (7). Synthetic constructs carrying BRSV F (Fsyn) and N (Nsyn) genes were manually designed by reverse translation of the amino acid sequence of the RB94 strain in order to avoid negativelycis-acting sequences and restore bovine codon usage. The prediction of splicing sites was performed at the NetGene2 server (http://www.cbs.dtu.dk/services/NetGene2/) (9). The presence of othercis-acting elements like the polyadenylation sequence (AAUAAA) (65) or the Shaw-Kamen instability motif (AUUUA) (49) was detected manually. The CUSP software available at EMBOSS (http://emboss.sourceforge.net/) was used to re-store bovine codon frequency. The codon adaptive index (CAI), which is a measurement of the relative adaptativeness of the codon usage of a gene com-pared with the codon usage of highly expressed genes, was calculated using the CAI software available at EMBOSS with a user-defined set of codon relative adaptiveness values based on codon usage in a compendium of bovine genes (Ebovsp.cut) (ideal value of 1.0). The resulting synthetic sequences for F and N genes were submitted to GenBank under accession no. AJ971803 and AJ971804, respectively, and were provided by Geneart GmbH (Germany). Native and optimized genes encoding F (pF and pFsyn, respectively) and N (pN and pNsyn, respectively) proteins were cloned into the HindIII-BamHI window of the pCDNA3.1 plasmid (Invitrogen) with an ATG initiation codon presented in an optimized Kozak sequence context (30). All plasmids were restriction verified, sequenced, and purified with Endofree plasmid Giga kits (QIAGEN) according to the manufacturer’s instructions.

Antibodies. Monoclonal antibodies specific for BRSV F (AK13A2 and AK8D1) were produced by immunizing BALB/c mice with the human Long strain and have been previously described (35). Mab19 was kindly provided by G. Taylor (Institute for Animal Health, Compton, United Kingdom) and has been described by Arbiza et al. (4). Monoclonal antibodies specific for BRSV N (AL5G1 and MoAb␣-N) were produced by immunizing BALB/c mice with the RB94 strain by a protocol similar to that described by Matheise et al. (35). The specificity of the AL5G1 and MoAb␣-N antibodies was tested by immunoblot-ting and immunoprecipitation procedures (data not shown). The K71 bovine polyclonal anti-BRSV antibody was produced in gnotobiotic calves after re-peated inoculation with the RB94 BRSV strain and was provided by G. Welle-mans (Veterinary and Agrochemical Research Centre, Brussels, Belgium).

Cell culture.Vero and COS-7 cells were grown in minimal Eagle’s medium (Gibco) supplemented with 10% fetal calf serum, 2.5 mM glutamine, and anti-biotics.

Viruses. BRSV RB94 was grown on Vero cell monolayers. The challenge inoculum consisted of lung lavage fluid collected 6 days after intratracheal inoculation of a calf with the BRSV strain VRS 3761 (kindly provided by Gilles Meyer, Ecole Nationale Ve´te´rinaire de Toulouse, France). This strain was suc-cessively passaged on three young calves by a protocol similar to that described by Tjørnehøj et al. (56). This inoculum contained 103.5 _{50% tissue culture}

infective doses (TCID50)/ml and was free of bovine viral diarrhea virus, bovine

herpesvirus 1, bovine parainfluenza virus 3, bovine coronavirus, bovine adeno-virus 5, endotoxins, bacteria, and mycoplasmas.

In vitro expression of plasmids in COS-7 cells.COS-7 cells were grown in 24-well plates at 8⫻104

cells per well and were transiently transfected with 400 ng of recombinant or empty pcDNA3.1 plasmids using Lipofectamine as

de-scribed by the manufacturer (Invitrogen). Twenty-four hours after transfection, monolayers were fixed in 4% paraformaldehyde and the expression of the F and N proteins was checked by an indirect immunostaining procedure using bovine polyclonal bovine anti-BRSV serum (K71) and monoclonal antibodies specific for the F protein (AK13A2, AK8D1, and mAb19) or for the N protein (AL5G1 and MoAb␣-N).

DNA immunization of mice.Four groups of five 6- to 8-week-old, BALB/c mice were housed in animal facilities. Mice were immunized twice by intramus-cular injections with 50␮g of plasmid construct (pCDNA3, pF, pN, pFsyn, or pNsyn). Three weeks after the second immunization, sera were collected in each group and pooled, and BRSV-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA) performed as previously described (47). Briefly, RB94 cell cultures from classic culture were sedimented by an overnight centrifugation at 90,000⫻g. The pellet was suspended in phosphate-buffered saline (PBS) containing 0.1%N-octyl␤-D-glucopyranoside (Sigma) for perme-abilization of virus envelopes. A predetermined optimal dilution of this prepa-ration was adsorbed on polyvinyl microplates (Maxisorp; Nunc) overnight at 4°C. Plates were washed with PBS–0.1% Tween 20 and blocked with 100␮l/well of hydrolyzed casein in PBS–0.1% Tween 20 for 2 h at room temperature. Serial fivefold dilutions of murine sera (50␮l/well) were added to wells, and the plates were incubated for 1 h. Goat anti-mouse IgG antibody conjugated to horseradish peroxidase (Dako) was used as secondary antibody at a dilution of 1:2,000 in PBS containing hydrolyzed casein. Antibody titer was defined as the logarithm of the inverse of the dilution giving an optical density (OD) value two times over that of the corresponding preimmune serum.

Immunization of calves.Seventeen 1-month-old calves, deprived of colostrum, were housed in separate isolation rooms. The calves were free of bovine viral diarrhea virus, bovine herpesvirus 1, BRSV, and bovine parainfluenza virus 3. As shown in Table 1, three groups of four calves were immunized intramuscularly and intradermally twice at a 3-week intervals (day 0 and day 21) with either 250

␮g of each of the pFsyn and pNsyn plasmids (F⫹N/KV and F⫹N) or 500␮g of the empty pCDNA3.1 vector (pCDNA3/KV). The other calves received saline. Three weeks after the second DNA vaccination (day 42), the calves of the F⫹N/KV and pCDNA3/KV groups were vaccinated once by intramuscular im-munization with 5 ml of a commercially available, Quil-A-adjuvanted vaccine, containing 105.5

TCID50of inactivated BRSV (Bovipast; Intervet). The other

groups received saline solution. Three weeks after the third vaccination (day 63), all of the calves were challenged with BRSV strain VRS 3761 by intratracheal injection of 15 ml of inoculum. One calf (control) was not infected.

All calves were bled weekly for examination of humoral and cellular responses. Bronchoalveolar lavage (BAL) fluids were collected from days 63 to 77 by instillation and aspiration of 50 ml of PBS directly into the lungs using a 120-cm-length PVC dual-flow tube (Vygon, Belgium). On days 68, 70, and 82, one or two calves of each group were euthanized. Immediately after euthanasia, lungs were excised and macroscopic lesions were photographed and recorded on a standard lung diagram to score the extent of pneumonic consolidation as previ-ously described (56). A score of 0 was given to a lung without macroscopic lesion. A score of between 1 and 5 was given according to the extent of the lung lesions: 1 represents 1 to 5%, 2 represents 5 to 15%, 3 represents 15 to 30%, 4 represents 30 to 50%, and 5 represents⬎50%. Finally, six pieces of tissue from the right cranial, middle, and caudal lobes (two pieces in each lobe) were excised.

Evaluation of clinical signs.Clinical signs were recorded daily after challenge. Respiratory rate was measured via a stethoscope for a full minute. Rectal tem-perature, the presence of nasal discharge, spontaneous or induced cough, lung sounds, depression, and anorexia were also noted. Examination of calves was done by qualified people who were not aware of group assignments. Clinical scores were then assigned to each calf as previously described (23).

Humoral immune response of calves.The ELISA anti-BRSV kit (CER, Bel-gium) was used to determine BRSV-specific antibody titers. IgG, IgG1, or IgG2

TABLE 1. Study design

Group (no. of calves)

Vaccination(s) _Challenge

(day 63)

No. of calves euthanized

Day 0 Day 21 Day 42 Day 67 Day 69 Day 82

Mock (4)

Saline

Saline

Saline

BRSV

1

1

2

F

⫹

N/KV (4)

pFsyn

⫹

pNsyn

pFsyn

⫹

pNsyn

KV

BRSV

1

1

2

F

⫹

N (4)

pFsyn

⫹

pNsyn

pFsyn

⫹

pNsyn

Saline

BRSV

1

1

2

pCDNA3/KV (4)

pCDNA3.1

pCDNA3.1

KV

BRSV

1

1

2

Control (1)

Saline

Saline

Saline

Saline

0

1

0

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was detected by the following horseradish peroxidase-conjugated antibodies: a rabbit anti-bovine IgG antibody (Sigma), an IgG1-specific monoclonal antibody 1C8 (kindly provided by J.-J. Letesson, University of Namur, Belgium), or a sheep anti-bovine IgG2 (Serotec), respectively. All sera were tested at a 100-fold dilution, and the corrected OD (COD) was calculated for each serum by sub-traction of the OD of the serum on control antigen from the OD of the same serum on BRSV antigen. The COD was then expressed as a percentage of the COD of a positive reference serum (K71).

Neutralizing antibody titers were determined in a virus seroneutralization assay as previously described (47). Briefly, twofold-diluted sera were incubated with 100 TCID50of RB94 virus for 1 h at 37°C and added on Vero cells grown

in 96-well culture microplates. Each dilution was tested in four wells. After 4 days of incubation, monolayers were fixed in 4% paraformaldehyde and viral expres-sion on the monolayer was revealed by an immunoperoxidase assay with a bovine anti-BRSV serum (K71). Virus neutralization titers were expressed as the recip-rocal of the highest dilution of serum that completely prevented viral expression.

Lymphocyte proliferation assay.Heparinized blood samples were 10-fold di-luted in RPMI 1640 medium (Gibco) supplemented with 2 mM glutamine, 5⫻ 10⫺5M␤-2-mercaptoethanol, 100 U/ml of gentamicin, and 2.5␮g/ml ampho-tericin B (Fungizone). Two hundred microliters of diluted blood samples was added to wells of U-bottom microplates (Nunc). After 6 days of incubation with 20␮l of BRSV antigen (sonicated and heat-inactivated culture supernatant of Vero cells infected with RB94 virus, containing 105_TCID

50/ml) or 20␮l of

control antigen (sonicated and heat-inactivated culture supernatant of mock-infected Vero cells), 0.8␮Ci of methyl [3_{H]thymidine in 25␮l RPMI 1640}

medium was added to each well. After 18 h of incubation, cells were harvested and radioactivity incorporated into the DNA was measured by liquid scintillation counting (Betaplate; Pharmacia). The results were expressed as stimulation indexes (SI) corresponding to the number of cpm obtained with the BRSV antigen divided by the number of cpm obtained with the control preparation. The lymphoproliferation was considered significant when the SI was equal or superior to 2.2. All tests were set up in sextuplicate cultures.

IFN-␥assay.Gamma interferon (IFN-␥) production was measured after a 24-h in vitro stimulation of heparinized blood with 20␮l of the above-described BRSV or control antigens. The amounts of IFN-␥in the plasma were quantified by ELISA with the bovine IFN-␥EASIA kit (Biosource Europe). The results were expressed as the SI corresponding to the OD obtained with the BRSV antigen divided by the OD obtained with the control preparation. IFN-␥ pro-duction was considered significant when the SI was equal or superior to 1.8.

Hpt assay.The haptoglobin (Hpt) concentration was determined by measuring hemoglobin binding capacity in serum (50). Briefly, 10␮l of serum, blank, or standard was incubated at 25°C with 90␮l of methemoglobin for 10 min. Guaiacol (1.5 ml; Sigma) was added immediately followed by 0.5 ml of hydrogen peroxide. After 8 min incubation at 25°C, the OD at 470 nm was read. The Hpt concentration

in the sample was calculated as follows: [(sample absorbance⫺blank absorbance)/ (standard absorbance⫺blank absorbance)]⫻standard concentration (g/liter).

IF.For immunofluorescence (IF), frozen lung samples from the right cranial, middle, and caudal lobes were sectioned at 5␮m, put on slides, and fixed in acetone for 15 min at 4°C. Slides were incubated with a biotinylated bovine anti-BRSV serum (K71) for 1 h at 37°C. After washing, fluorescein isothiocya-nate (FITC)-conjugated streptavidin (Sigma) was added, the mixture was incu-bated for 1 h at 37°C, and the slides were observed on a fluorescence microscope. Slides were then examined without any information on the group assignments.

Histopathology.Samples of the macroscopic lung lesions were taken, forma-linized, embedded in paraffin wax, sectioned at 5␮m, and stained with hema-toxylin and eosin or using May-Gru¨nwald-Giemsa stains. Slides were then ex-amined without any information on the group assignments.

RNA extraction and cDNA synthesis.RNA extraction and cDNA synthesis were performed as previously described (7). RNA was extracted from 200␮l of lung homogenate or BAL fluids with an RNeasy RNA purification kit (QIAGEN) as recommended by the manufacturer. Purified RNA was dissolved in 20␮l of RNase-free water. Five microliters of RNA solution was used as a template for cDNA synthesis in the presence of 1␮l of 200 U of Moloney murine leukemia virus reverse transcriptase (200 U) (Invitrogen), 4␮l of 5⫻First Strand buffer (Invitrogen), 2␮l of 0.1 M dithiothreitol, 2␮l of 1⫻hexamer (Roche), 1

␮l of 10 mM deoxynucleoside triphosphates (dNTPs; Roche), and 5␮l of RNase-free water. After a 50-min incubation at 37°C, the reverse transcriptase was inactivated at 70°C for 10 min.

Real-time PCR.BRSV and bovine␤-actin sequences were amplified by real-time PCR as previously described (7). Each PCR was performed on 1␮l of cDNA mixed with 12.5␮l of Master Mix PlatinumTaqpolymerase (Invitrogen), 0.5 ␮l of ROX dye (6-carboxyl-X-rhodamine; Invitrogen), 100 nM of each primer, 200 nM of probe, and RNase-free water for a final volume of 25␮l. Amplifications were performed as follows: 2 min at 50°C, 10 min at 95°C, and 45 cycles at 95°C for 15 s and 59°C for 1 min. The detection limit of the assay was 102

RNA copies. The standard curves demonstrated a linear range from 103

to 108_copies.

For IFN-␥and IL-4, real-time PCRs were performed with Taqman Master Mix kit (Applied Biosystem) in a final volume of 25␮l containing 5␮l of cDNA, 300 nM of each primer, and 900 nM of the probes described in Table 2. Ampli-fications were performed as follows: 10 min at 95°C and 40 cycles at 95°C for 15 s and 60°C for 1 min. In each assay, control RNAs were used to construct standard curves. The detection limits for IFN-␥and IL-4 were 103_{RNA copies, and}

standard curves demonstrated a linear range up to 108

copies (J.-F. Toussaint, unpublished data).

Statistics.The data collected were statistically analyzed using Graphpad Prism software. Two-way analysis of variance was used to compare the evolution of IgG titers, lymphoproliferation, and IFN-␥SI. Peak clinical signs and viral load values were compared by one-way analysis of variance and Tukey’s test with a family error rate of 0.05. Logarithmic transformation was applied to fulfill the condi-tions of variances in homogeneity and normality when necessary.

RESULTS

Codon optimization of the BRSV F and N genes.

Codon

optimization of the BRSV F and N genes was performed to

restore bovine codon usage and remove potential negatively

cis

-acting elements such as splicing sites, polyadenylation signals, and

Shaw-Kamen instability motifs. As a result of codon optimization,

the GC contents of the F and N genes were increased from 35%

and 39% to 47% and 51%, respectively, and the CAI was

im-proved from 0.551 to 0.710 for the fusion gene and from 0.559 to

0.736 for the nucleocapsid gene (Table 3).

TABLE 2. Sequences of primers and probes used to detect IFN-

␥

and IL-4 by real-time RT-PCR

Target and primer or probe Sequencea

Bovine IFN-␥

Forward primer ...ATGGATATCATCAAGCAAGACATGTT Reverse primer ...TCATCCACCGGAATTTGAATC Probe...FAM-TCCTCCAGTTTCTCAGAGCTGCC

ATTCA-TAMRA Bovine IL-4

Forward primer ...TTCTGCAGGGTTGGAATTGAG Reverse primer ...TCATTCACAGAACAGGTCTTGCTT Probe...FAM-GTTCAAGCACGTGTGGCTCCTG

TAGATAC-TAMRA

a

FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.

TABLE 3. Effects of codon optimization

Gene No. of instability sites native/optimizeda

CAI for native/ optimized

No. (%) of native nucleotides altered/total

% GC content native/optimized

Fusion

6 P, 3 SK, 4 S/0

0.551/0.710

240/1,725 (14)

35/47

Nucleocapsid

1 SK, 18 S/0

0.559/0.736

150/1,176 (13)

39/51

a_{P, polyadenylation signal; SK, Shaw-Kamen instability motif; S, splicing site.}

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In vitro protein expression of the pFsyn and pNsyn

plas-mids and immunogenicity in mice.

The impact of codon

opti-mization on protein expression was investigated by transfection

of COS-7 cells with equal amounts of native or optimized

plasmids encoding BRSV F and N proteins. Twenty-four hours

after transfection, protein expression was revealed by a

poly-clonal bovine anti-BRSV serum (K71) (Fig. 1). Expression of F

or N proteins was detected in cells that received native or

optimized plasmids, but no signal was observed in cells

trans-fected with the empty pcDNA3.1 plasmid. Importantly, the

results showed that expression levels were greater in cells

transfected with codon-optimized plasmids. Similar results

were obtained when monoclonal antibodies specific for

differ-ent epitopes of the fusion protein (AK13A2, AK8D1, and

mAb19) or the nucleocapsid protein (AL5G1 and MoAb

␣

-N)

were used instead of the polyclonal K71 serum (data not

shown).

The immunogenicity of the plasmids was then investigated in

mice. Five groups of four mice twice were given 50

␮

g of

plasmid at a 3-week interval. Three weeks after the second

immunization, sera were pooled and BRSV-specific antibodies

were measured by ELISA. No antibody response was detected

after injection of mice with either the empty pCDNA3 plasmid

or nonoptimized vectors, but pFsyn and pNsyn immunizations

elicited IgG titers of 3.01 and 1.86, respectively.

Overall, these results showed that the codon optimization of

the BRSV F and N genes increased both their in vitro

expres-sion as well as their immunogenicity in mice.

BRSV-specific immune response of calves following

vacci-nations and challenge.

The global immune response against

BRSV induced by coadministration of pFsyn and pNsyn

plas-mids was evaluated weekly by measuring levels of

BRSV-spe-cific IgG and lymphoproliferative response after antigenic

stimulation of whole blood cells. Immunization with pFysn and

pNsyn plasmids resulted in seroconversion 5 weeks after the

second vaccination (day 56), as shown by stable antibody levels

in F

⫹

N calves (Fig. 2A). KV induced the production of

BRSV-specific antibodies 2 weeks after injection in the pCDNA3/KV

groups and raised antibody levels in the F

⫹

N/KV group

com-pared to the F

⫹

N calves (

P

⬍

0.01). Ten days after challenge

(day 73), seroconversion of mock-vaccinated calves was

ob-served as well as enhanced amounts of IgG in the other groups,

especially in the pCDNA3/KV calves.

DNA priming induced a lymphoproliferative response 5

weeks after the second vaccination (day 56) in F

⫹

N and

FIG. 1. In vitro expression of pFsyn and pNsyn plasmids in COS-7

cells. COS-7 cells in 24-well plates were transfected with 400 ng of the

plasmids described. Twenty-four hours after transfection, expression of

the BRSV F and N proteins was revealed in an immunoperoxidase

assay with a polyclonal anti-BRSV antibody (K71).

FIG. 2. BRSV-specific immune response following vaccination and

challenge. Calves were vaccinated twice with DNA (days 0 and 21)

followed by protein boost (day 42) and challenge (day 63) at 3-week

intervals. (A) IgG levels were measured by ELISA, and COD values

were transformed to percentages of a standard positive serum. (B) In

vitro lymphocyte proliferation was evaluated by measuring [

H]thymi-dine incorporation after antigenic stimulation of whole blood cells with

control or BRSV antigen, and values were expressed as an SI. Bars

represent means (

⫹

standard deviations) in each group on days 0, 21,

42, 56, 63, 69, and 73.

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F

⫹

N/KV calves, as shown by higher SI compared to the

mock-vaccinated and pCDNA3/KV groups (

P

⬍

0.05) (Fig. 2B). The

KV boost was not able to stimulate lymphoproliferation in the

pCDNA3/KV group even if it induced slightly higher SI values

in F

⫹

N/KV calves compared to the F

⫹

N calves (

P

⬎

0.05).

The challenge did not enhance the SI values measured in F

⫹

N

and F

⫹

N/KV groups but it induced a lymphoproliferative

re-sponse in pCDNA3/KV and mock-vaccinated calves on day 73.

Ig isotype profile and neutralizing antibody titers.

BRSV-specific humoral immunity primed by DNA immunization and

KV boost was further investigated by analyzing the IgG isotype

profile and neutralizing antibody levels on days 63 and 69. On

these days, F

⫹

N calves had low but significant BRSV-specific

IgG1 (Fig. 3A) and IgG2 (Fig. 3B) compared to

mock-vacci-nated calves (

P

⬍

0.05). Nevertheless, these antibodies did not

possess a neutralizing activity (Fig. 3C). KV boost resulted in

significantly higher levels of IgG1 (

P

⬍

0.01), IgG2 (

P

⬍

0.01),

and neutralizing antibodies (

P

⬍

0.05) in F

⫹

N/KV and

pCDNA3/KV animals compared to F

⫹

N and mock-vaccinated

calves. Interestingly, on days 63 and 69, F

⫹

N/KV calves had

significantly lower IgG1 levels (

P

⬍

0.05) and similar amounts

of

IgG2

but

larger

neutralizing

antibody

titers

than

pCDNA3/KV calves (

P

⬍

0.05). SN/IgG

and IgG2/IgG1

ratios were then calculated for each calf in the F

⫹

N/KV and

pCDNA3/KV groups. These ratios could not be determined in

the two other groups because the titers before and after

chal-lenge were too weak. As shown in Fig. 3D, animals that

re-ceived the F

⫹

N priming had higher IgG

/IgG

and SN/IgG

ratios than pCDNA3/KV calves (

P

⬍

0.05).

Cytokine production ex vivo and in vivo.

The effect of DNA

priming on the type of immune response induced was

exam-ined by quantifying cytokine production in vitro and in vivo.

Secretion of IFN-

␥

was measured after antigenic stimulation of

whole blood cells at challenge (day 63) and 6 days after

chal-lenge (day 69). As shown in Fig. 4A, DNA-primed calves

(F

⫹

N and F

⫹

N/KV groups) displayed significant IFN-

␥

pro-duction on the day of the challenge and this response was

slightly improved on day 69. Similarly to what we observed in

lymphoproliferation assay, KV boost did not enhance SI

mea-sured in F

⫹

N/KV calves nor did it stimulate IFN-

␥

production

in the pCDNA3/KV group.

The amounts of IFN-

␥

and IL-4 mRNAs produced in the

right caudal lung lobes of calves euthanized four (day 67) and

six days (day 69) following challenge were measured by

real-time RT-PCR. Levels of IFN-

␥

mRNA measured in the lungs

of calves that received pFsyn and pNsyn priming (F

⫹

N and

F

⫹

N/KV) were up to 100-fold higher than those of the other

calves (Fig. 4B, left panel), confirming the results observed

after in vitro stimulation of whole blood cells. Analysis of IL-4

mRNA levels did not reveal such differences between groups

as there was no obvious IL-4 induction in the lung (Fig. 4B,

middle panel). Finally, ratios between IFN-

␥

and IL-4 were

calculated as a measure of Th bias for each calf (Fig. 4B, right

panel). These ratios were higher in calves that received the

FIG. 3. Immunoglobulin isotype profile and neutralizing antibody titers at challenge (day 63) and 6 days after challenge (day 69). IgG1 (A) and

IgG2 (B) levels were measured by isotype-discriminating ELISA, and COD values were expressed as percentages of a standard positive serum.

Neutralizing antibody titers (C) were determined by neutralization assay and expressed as the base 2 log of the maximum positive dilution. Bars

represent means

⫹

standard deviations measured in each group on days 63 and 69. Individual IgG2/IgG1 and SN/IgG

ratios (D) were calculated

in the F

⫹

N/KV and pCDNA/KV groups on days 63 and 69.

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pFsyn and pNsyn priming compared to mock-vaccinated and

pCDNA3/KV calves, reflecting an increase in Th1 response.

Clinical signs.

After experimental infection on day 63, a

clinical score was calculated for each calf as previously

de-scribed (23). Whereas the mock-vaccinated calves had an

im-portant increase of rectal temperature and respiratory rate two

days after infection (day 65), calves from either F

⫹

N,

F

⫹

N/KV or pCDNA3/KV groups didn’t display any clinical

signs earlier than 4 to 5 days after infection (Fig. 5). In all

groups, peak clinical scores were recorded between days 68

and 70. Mock-vaccinated calves had the most severe

respira-tory disease compared to pCDNA3/KV (

P

⬍

0.01), F

⫹

N (

P

⬍

0.001) and F

⫹

N/KV (

P

⬍

0.001) animals. DNA-primed calves

displayed reduced clinical signs compared to pCDNA3/KV

group (

P

⬍

0.05), and there was no significant difference

be-tween F

⫹

N and F

⫹

N/KV groups.

Postmortem examination.

On days 67, 69, and 82, gross

pneumonic lesions were recorded and scored as previously

described (56). On day 67, only limited lesions were observed

in cranioventral lung parts of F

⫹

N, pCDNA3/KV, and

mock-vaccinated calves (Table 4). On days 69 and 82, extended

consolidated areas were seen, covering up to 50% of the lung

surface

(score

of

5)

in

the

mock-vaccinated

group.

pCDNA3/KV and F

⫹

N calves displayed reduced lesions

com-pared to mock-vaccinated calves, but consolidation was

ob-served in cranioventral and caudal regions of the lung.

Inter-FIG. 4. Cytokine production in vitro and in vivo before and after

challenge. (A) IFN-

␥

production was measured by ELISA after

anti-genic stimulation of whole blood cells with control or BRSV antigen,

and values were expressed as SI. Bars represent means

⫹

standard

deviations measured in each group on days 63 and 69. (B) Levels of

IFN-

␥

(left panel) and IL-4 (middle panel) transcripts were quantified

by real-time RT-PCR in lung homogenates of calves euthanized on

days 67 and 69, using

␤

-actin (

␤

-act) as an internal reference, and

ratios between IFN-

␥

and IL-4 mRNAs were calculated for each calf

(right panel).

FIG. 5. Clinical signs following challenge. Rectal temperature,

respiratory rhythm, presence of nasal discharge, lung sounds, and

anorexia were recorded daily after challenge, and clinical scores were

calculated as previously described (56). Data represent means (

⫹

standard

deviations) in each group from day 63 (challenge) to day 77.

TABLE 4. Lesion score, peak Hpt concentration, and BRSV

detection following challenge

Group and day euthanized

Lesion scorea

Peak Hpt concn (mg/ml)

Viral

loadb,c IF detectionc,d

Mock

67

1

0

4.4

⫾

0.1

cr, ca, m

69

3

0

4.2

⫾

0.2

cr, ca, m

82

5–3

1.80–0.68

⫺

ND

F

⫹

N/KV

67

0

0

1.6

⫾

0.2

⫺

69

1

0.05

0.5

⫾

0.3

⫺

82

0–0

0.28–0

⫺

ND

F

⫹

N

67

1

0

2.2

⫾

0.2

cr

69

2

0.01

1.8

⫾

0.3

⫺

82

1–2

0.14–0.79

⫺

ND

pCDNA3/KV

67

1

0

3.0

⫾

0.6

cr, m

69

2

0

3.3

⫾

0.2

cr, m

82

4–2

1.81–0.9

⫺

ND

Control

69

0

0

⫺

⫺

a_{As described by Tjornehoj et al. (56).}

b_{Mean (⫾standard deviation) viral load expressed as the log of BRSV}

mRNAs per 106_{b-actin mRNAs detected in the right cranial, middle, and caudal}

lung lobes.

c_{⫺, not detected.}

d_{Lung lobes in which viral expression was detected by IF. cr, cranial; m,}

middle; ca, caudal; ND, not done.

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estingly, only one F

⫹

N/KV calf had one spot of consolidated

lung tissue in the cranial lobe. The assessment of

macroscop-ical lesions was confirmed by measuring the concentration of

an acute-phase-response protein, Hpt. Serum Hpt was

de-tected in all groups from day 68 and peaked around days 70 to

72. The highest Hpt concentrations were measured in

pCDNA3/KV and mock-vaccinated calves, and there was a

good correlation between the magnitude of the peak Hpt

con-centration and the extent of lung lesions. Overall, the observed

clinical signs were severe. Finally, viral load and expression of

the virus in cranial, middle, and caudal lung lobes were

as-sessed by real-time RT-PCR and immunofluorescence (IF),

respectively. As shown in Table 4, real time RT-PCR revealed

the presence of BRSV in all infected calves but the viral load

measured in calves that received the F

⫹

N priming was lower

than those in to mock-vaccinated and pCDNA3/KV calves.

Viral expression was detected by IF in all four lobes of

mock-vaccinated calves, in two lobes (cranial and caudal) of

pCDNA3/KV calves, and on the cranial lobe of the F

⫹

N calf

killed at day 69. Samples collected from the control calf that

was mock infected did not display gross lesions and were free

of BRSV.

Histopathological lesions.

Examination of pulmonary

histo-pathology of the control calves revealed the classical signs of

BRSV infection. Four days after infection (day 67), all sections

showed local alveolar emphysema, local lobular thickening of

the alveolar septa with syncytium formation, and localized

peribronchiolar cuffing (Fig. 6A). On day 69, pCDNA3/KV

and mock-vaccinated calves had severe bronchopneumonia

(Fig. 6B) characterized by the presence of eosinophils in the

peribronchiolar infiltrate (Fig. 6C). Eosinophils were observed

between the hyperplastic epithelium, mucosa, and submucosa.

Their presence was focal: sometimes solitary (e.g., epithelium)

and sometimes in small groups. Sections of F

⫹

N/KV calf

dis-played moderate alveolar emphysema, scattered thickening of

the alveolar septa, and syncytial formation (Fig. 6D). In the

F

⫹

N calf there was prominent inflammatory reaction with

infiltration of lymphocytes and neutrophils in the septa, near

bronchi and bronchioli, and scattered sites of consolidation

(Fig. 6E). On day 82, a prominent fibrovascular reaction,

prob-ably due to repair, was present on lung sections of

pCDNA3/KV and mock-vaccinated calves, and to a lesser

ex-tent on sections of F

⫹

N calves (Fig. 6F). Thus, the only calves

that did not display histological lesions were those from the

F

⫹

N/KV group.

Viral replication.

The real-time RT-PCR was used to

deter-mine the viral loads in BAL fluids. As shown in Fig. 7, the

presence of BRSV was not detected before infection. Virus

load peaked around day 67 in all calves and decreased around

day 70, except in mock-vaccinated calves in which the decrease

was observed at day 72. As expected, peak viral load was

significantly higher in pCDNA3/KV and mock-vaccinated

groups compared to F

⫹

N and F

⫹

N/KV groups (

P

⬍

0.001).

Similar observations were made from the analysis of viral load

in lungs (Table 4). At day 73, F

⫹

N and F

⫹

N/KV groups were

free of BRSV, whereas viral RNA was detected up to day 75

and day 77 in pCDNA3/KV and mock-vaccinated groups,

re-spectively.

DISCUSSION

DNA vaccines are promising alternatives to the classical

modified live virus or KV vaccines. In fact, DNA immunization

often primes a Th1 immune response and provides large

num-FIG. 6. Histopathological features on lung section after challenge. (A, B, and F) Lung sections of mock-vaccinated calves on days 67 (A), 69

(B), and 82 (F). (C) Eosinophil infiltration (arrow) in pneumonic lung of a pCDNA3/KV calf on day 69. (D and E) General aspects of the F

⫹

N/KV

(D) and F

⫹

N (E) calf lung sections on day 69.

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bers of memory B and T cells. Furthermore, DNA

immuniza-tion might avoid the inhibitory effect of maternal antibodies

(for reviews, see references 21, 22, and 44). Efficacy of DNA

immunization against RSV F and G proteins has well been

described in rodents (6, 8, 32, 33, 58). However, immunization

of large animals like cattle or monkeys offered only partial

protection and require more innovative approaches, like codon

optimization and protein boost following DNA vaccination

(48, 53, 64).

In this report, we designed and constructed codon-optimized

plasmids encoding BRSV F and N proteins and evaluated the

immunogenicity of these plasmids in calves. Construction of

genes was performed in order to increase the overall GC content,

avoid negatively

cis

-acting sequence motifs, and restore codon

adaptation to frequently used bovine codons. We have shown that

codon optimization of the BRSV F and N genes led to an increase

in antigen levels expressed in mammalian cells and improved

antibody response in mice. These results are consistent with

sim-ilar codon optimization studies reported for genes encoded by

different pathogenic organisms (1, 37–39, 43).

Vaccination of young calves with a DNA plasmid containing

the BRSV F gene or recombinant vaccinia virus expressing the

F or N proteins was shown to stimulate both humoral and

cell-mediated immunity (16, 53, 54). More precisely, in these

studies, the F gene construct rather primed humoral immunity

while immunization against N rather promoted cellular

immu-nity. Consistent with these reports, two immunizations with

pNsyn elicited lower IgG levels than pFsyn but resulted in

in vitro lymphoproliferation and IFN-

␥

production, whereas

pFsyn did not (M. Tignon, unpublished results). Thus,

coim-munization against F and N proteins should stimulate both

humoral and cell mediated immunity and could be an

inter-esting vaccine approach. To answer this question, young calves

were vaccinated with both codon-optimized pFsyn and pNsyn

plasmids. Moreover, in an attempt to increase the immune

response against the virus, we boosted the DNA priming with

a heterologous boost consisting of a BRSV KV vaccine.

Re-cently, a similar strategy was shown to be efficient in the

con-text of the bovine herpesvirus 1 (57). Here, two administrations

of both pFsyn and pNsyn plasmids stimulated the two arms of

the immune system, as shown by antibody production, in vitro

lymphoproliferation, and IFN-

␥

production. Furthermore, the

use of the KV vaccine considerably increased the levels of

BRSV-specific antibody. Overall, these observations confirm

the potential of coimmunization against F and N proteins as

well as the efficacy of an alternative protein boost following

DNA immunization in large animals.

DNA priming with pFsyn and pNsyn elicited lower levels of

antibody than the KV vaccine and failed to induce the

pres-ence of a detectable neutralizing humoral response.

Neverthe-less, we have shown that pFsyn/pNsyn priming influenced the

antibody response induced by KV vaccination. Indeed, pFsyn/

pNsyn-primed calves had lower levels of IgG, especially IgG1,

but higher neutralizing antibody titers after KV vaccination

than nonprimed calves. As the presence of RSV-neutralizing

antibodies is much more important than the absolute antibody

levels in the serum (59, 60), our results suggest that pFsyn/

pNsyn priming, even if it elicits low BRSV-specific IgG titers,

improves the humoral immune response induced by the KV

boost.

The role of cell-mediated immunity is more complicated and

is a critical parameter in the outcome of RSV infection (for a

review, see reference 40). In rodents and calves, T cells are

essential for virus clearance but also appear to mediate

immu-nopathology. The CD8 cells seem to play an important role in

protection against the disease, possibly through RSV-specific

cytotoxic activity, but a strong cytotoxic T lymphocyte response

has been also implicated in the pathological outcome of

vac-cination (11, 12, 17, 25, 45, 51, 55). The role of CD4 Th cells is

more uncertain and complicated, but the imbalance of Th1 and

Th2 cytokines is likely to play a role in the pathogenesis of

RSV disease.

DNA vaccination often promotes a Th1-biased immune

re-sponse that is characterized by high levels of antigen-specific

lymphoproliferative response and the production of large

amounts of IFN-

␥

(5). After two inoculations of the pFsyn and

pNsyn constructs, both IFN-

␥

production and

lymphoprolif-erative response were detected. In contrast to what we

ob-served for the humoral response, KV boost did not enhance

the IFN-

␥

production in calves that were DNA primed and

showed poor efficacy for the induction of both IFN-

␥

produc-tion and lymphoproliferative response in nonprimed calves

before and after challenge. In a previous report, we

demon-strated that this KV vaccine did not stimulate detectable or

strong cell-mediated immunity even after two inoculations

(28). To approach the problem of IL-4 production, we

per-formed real-time RT-PCR to quantify the amounts of IFN-

␥

and IL-4 mRNAs in the lungs of calves during the first week of

the infection. IFN-

␥

mRNA levels were increased in calves

primed with pFsyn and pNsyn plasmids, whereas there was no

clear difference in IL-4 transcripts between groups, confirming

what we observed after whole-blood-cell sensitization. Thus,

DNA priming using pFsyn and pNsyn plasmids seems to

mote a Th1-biased immune response characterized by the

pro-duction of IFN-

␥

both in vitro and in vivo. IFN-

␥

has been

shown to promote the production of IgG2, resulting in a higher

IgG2/IgG1 ratio (15). Here, in DNA-primed calves, we did not

measure higher levels of IgG2. However, higher IgG2/IgG1

FIG. 7. Evolution of viral load in BAL fluids. Virus load was

de-termined by real-time RT-PCR in BAL fluid samples collected on days

63, 66, 68, 70, 73, 75, and 77. Bars represent mean (

⫹

standard

devia-tion) standardized amounts (BRSV mRNAs/10

␤

-actin mRNAs) in

each group.

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ratios were observed after challenge, further confirming that

pFsyn/pNsyn priming elicits a Th1 immune response.

The protection conferred by these vaccination protocols was

evaluated after experimental infection of calves using a BRSV

field strain (VRS 3761) successively passaged on young calves.

This method has previously been shown to induce an

excep-tionally severe pathology in mock-vaccinated calves compared

to challenge procedures based on the usage of cell

culture-passaged strains (56). In the present study, the first clinical

signs of BRSV infection were recorded 3 days following

chal-lenge and they peaked 1 week after infection. Postmortem,

consolidated areas covered up to 50% of the lung in

mock-vaccinated animals. Histopathologically, syncytia, emphysema,

alveolitis, and bronchiolitis with infiltration of numerous

neu-trophils and eosinophils appeared first, rapidly evolving in

bronchopneumonia and a prominent fibrovascular reaction

in-dicative of repair. As shown by others, the serum concentration

of Hpt, an acute-phase-response protein, peaked 7 to 9 days

after infection and the magnitude of the response was well

correlated with the severity of the clinical signs and the extent

of lung lesions (24). Thus, BRSV challenge resulted in severe

pathology similar to that seen in naturally infected calves,

allowing reliable evaluation of the protection conferred by

each vaccine strategy.

Even if it was not sufficient to fully prevent histopathological

changes and gross pneumonic lesions after challenge, the

im-mune response generated by pFsyn and pNsyn plasmids clearly

reduced both clinical signs of the infection and virus

replica-tion while KV failed to do so. In cattle and mice, secrereplica-tion of

IFN-

␥

was shown to contribute to NK activation and CD8

cytotoxic activity, resulting in viral clearance and resolution of

the infection in the airways (14, 26, 41, 55, 68). Here, we have

also shown that the production of IFN-

␥

was correlated with

viral clearance as pFsyn/pNsyn-primed calves efficiently

elimi-nated the virus after challenge. In contrast, KV vaccination,

which elicited high levels of neutralizing antibody but did not

induce IFN-

␥

production, failed to reduce viral replication and

pneumonic lesions. Thus, it seems that the induction of a

strong cell-mediated immunity is a critical parameter for the

protection against BRSV infection as the pFsyn/pNsyn

vac-cine-induced cell-mediated immunity allowed viral clearance

even in absence of a neutralizing humoral response. However,

prevention of the disease was only achieved when calves were

further vaccinated with the KV boost.

In contrast to previous reports, KV vaccination did not

in-duce a strong lymphoproliferative response and did not result

in enhanced pathology following challenge (2, 3, 19, 27, 46).

However, a single inoculation of the vaccine poorly reduced

viral load, clinical signs, histological changes, and gross

pneu-monic lesions compared to mock-vaccinated calves.

Further-more, it did not prevent eosinophilia which had previously

been linked to immunopathology following challenge (25).

These results are consistent with a previous report exploring

the efficacy of the same KV vaccine (36). This supports the

interest in DNA priming before KV vaccination. Similarly, KV

vaccines formulated with CpG oligodeoxynucleotides have also

been shown to result in a better Th1-type response and lack of

immunopathology (34, 42).

In summary, we have demonstrated for the first time that

DNA priming against BRSV F and N protein primed a

Th1-biased immune response that reduces viral replication and

partially protected calves against a highly virulent challenge.

Furthermore, KV boost potentiated the immune response

primed by DNA vaccination and this heterologous

DNA-prim-ing KV vaccine boost offered complete protection against

BRSV. In a future work, immune response induced by specific

heterologous prime-boost protocols against F or N protein as

well as strategies for improving the efficacy of DNA

vaccina-tion in cattle like microparticle encapsulavaccina-tion will be evaluated.

ACKNOWLEDGMENTS

This study was supported by the Federal Public Service Health,

Food Chain Safety and Environment (Brussels, Belgium).

The authors are grateful to K. Lentz, C. Didembourg, P. Van

Muylem, S. Durand, C. Rodeghiero, and R. Geeroms for their skilled

technical assistance. They also would like to thank all the staff of the

animal facilities of the VAR in Machelen for taking care of the

animals.

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