Vaccine 27 (2009) 136–145

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Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery system

Demian Bellidoa, Patricio O. Craigb, Marina V. Mozgovoja, Diego D. Gonzaleza,Andrés Wigdorovitza, Fernando A. Goldbaumb, María José Dus Santosa,∗

a Instituto de Virología “S. Rivenson”, Centro de Investigaciones en Ciencias Veterinarias, INTA Castelar, Buenos Aires, Argentinab Fundación Instituto Leloir e Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), Buenos Aires, Argentina

a r t i c l e i n f o

Article history:Received 4 August 2008Received in revised form30 September 2008Accepted 2 October 2008Available online 28 October 2008

Keywords:BLSRotavirusSubunit vaccine

a b s t r a c t

Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. It has been previouslyevaluated as a carrier to increase the immunogenicity of peptides fused to its N-termini. VP8 is a sialic acidbinding domain of rotavirus external capsid protein VP4, which is involved in virus adhesion to host cells.In this work, the C486 bovine rotavirus (BRV) VP8 core protein (VP8d) was fused to the structure of BLSwith the aim to produce an enhancement of the immune response against BRV VP8 and to evaluate thepossible use of this antigen for vaccine development. The feasibility of using BLS as an antigen deliverysystem of polypeptides larger in size than those previously tested was also evaluated.

Groups of female mice were immunized with BLS-VP8d fusion protein, VP8d or an equimolar mixtureof purified VP8d and BLS (BLS + VP8d). Dams immunized with BLS-VP8 induced 97.5–100% protectionagainst homologous challenge with C486 BRV; while pups born to dams immunized either with VP8d orBLS + VP8d presented a significant lower level of protection. The neutralizing antibody pattern was also

significantly different among these experimental groups, and in concordance with challenge experiment.

Overall, these results demonstrate that the BLS-VP8d chimeric protein is properly folded and stable,s a poomo

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. Introduction

New vaccine strategies have been recently developed basedn important advances in biotechnology. Subunit vaccines con-aining protective epitopes against infectious organisms haveeen evaluated [9,24,30,32,34,44,52]. However, frequently, sub-nit vaccines are weak immunogens, thus, effective vaccinationequires the use of adjuvants and boosting. Different approachesre being tested to improve vaccine efficacy avoiding side effectsnd reducing the cost of immunization [49]. In this regard, these of carrier proteins constitutes a feasible strategy for thaturpose.

The bacterial enzyme lumazine synthase of Brucella spp. (BLS)as been reported as a carrier for antigen delivery based on its

hysicochemical and immunogenic properties [60]. BLS assembless a remarkably stable dimer of pentamers, with 10 N-terminusites of linkage. Structural analysis of this molecule showed thatt is possible to insert peptides and protein domains without

∗ Corresponding author at: Instituto de Virología, CICVyA, Las Cabanas y de Loseseros s/n, INTA Castelar (1712), Buenos Aires, Argentina. Tel.: +54 11 4621 1743;

ax: +54 11 4621 1743.E-mail address: mdussantos@cnia.inta.gov.ar (M.J. Dus Santos).

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.10.018

tent antigen delivery system that enhances the antibody response againsttypic passive protection in a suckling mouse model.

© 2008 Elsevier Ltd. All rights reserved.

isturbing its conformation. It has been reported that BLS pos-esses immunostimulatory properties. It can activate the cellularesponses by delivering CD8+ T-cell epitopes into the MHC classpathway [4], eliciting an antigen-specific cellular response andstrong and long-lasting humoral response in the absence of

djuvant [53]. Interestingly, BLS presents immune-enhancing prop-rties only when the foreign antigen is covalently attached to it9].

Considering its multivalence and immunogenic properties, BLSas been evaluated as a carrier for systemic immunization. Severaleptides and a small protein domain of 9 kDa were successfullyoupled by recombinant fusion to the structure of BLS. In thisense, it has been shown that BLS tolerated the insertion of the2 amino acid peptide KETc1, which is one of the peptides of a syn-hetic vaccine against Taenia solium cysticercosis. KETc1 becamentigenic in the chimera, increased its immunogenicity and itsrotective capacity [60,49]. BLS has also shown to be an efficientarrier and adjuvant for oral immunization, eliciting a systemicmmune response due to its homodecameric arrangement and

emarkable thermodynamic stability [45]. In addition, when BLSas fused to a 27-mer peptide containing the exposed loop epitopef Omp31 from Brucella spp., the chimera elicited similar protec-ive activity against Brucella ovis infection to the live Brucella Rev.1accine [7].

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Rotavirus is the principal etiological agent of severe neonatalastro-enteritis in mammalian species, including infants and younghildren worldwide. It is also responsible for extensive economicroblems in animal species. A survey conducted in Argentina during10-year period, showed that BRV represent the main pathogen

ssociated to neonatal calf diarrhea in both, beef and dairy herds,ith 64.5% overall incidence [16].

Rotavirus possesses a genome composed of 11 segments ofouble-stranded RNA contained in an inner core and surroundedy two protein layers that form the viral capsid [13]. Virus parti-les present spikes formed by homo-dimers of a nonglycosylatedrotein VP4, which pass through and extend from the virion sur-

ace layer [12]. Rotavirus infectivity requires proteolytic cleavagef VP4, giving an N-terminal sialic acid-recognizing domain (VP8)nd a C-terminal fragment (VP5) that remains associated with theirion [10].

Based on the genetic and antigenic variation of the two outerapsidic proteins VP7 (glycoprotein) and VP4 (protease-sensitive),roup A RV is further classified into G- and P-types, respectively.o far 15 G-types and at least 27 P-types have been recognized inuman and animals [2,12,17,22,25,31,36,37,42,43].

Calves are susceptible to rotavirus infection within the first 2eeks of life and for this reason, active immunization prior to expo-

ure to virulent virus is almost impossible to accomplish. However,alves can be passively immunized by vaccination of the dam inrder to increase rotavirus-specific antibody titers in colostrum,hich are then transferred to the calves, by colostrum intake [47].

imilarly, in the suckling mouse model, passive transfer of anti-odies from immunized dams to suckling mice can also preventotavirus infection [40].

Previous reports have identified several advantages of the VP8ubunit as an immunogen. The VP8 subunit has been found to playn important role in viral infectivity and neutralization of the virus33,46,58]. Studies with human and animal rotavirus have identi-ed eight neutralizing epitopes on VP4, with five of them locatedn VP8 [6,18,26,28,41,50,59]. Antibodies directed to the VP8 frag-ent, were capable of neutralizing infection in vitro by preventing

he binding of virus to cells. Furthermore, monoclonal antibodiespecific against VP8 were reported to neutralize rotavirus in vitrond passively protect mice against viral challenge in vivo [38]. Inddition, parenteral vaccination with a recombinant VP8 producedn Escherichia coli was able to boost BRV-specific neutralizing anti-odies in mice and cattle pre-immunized with BRV inactivatedarticles [29], elicited colostral antibody titers equivalent to thosebtained with a commercial vaccine in pregnant cattle [56], andnduced homotypic protection against disease in mice born to damsmmunized with this antigen [19].

Results obtained by Dormitzer et al. [10] demonstrated that thehesus rotavirus VP8 fragment (rVP8) has a compact and homo-eneus protease resistant core encompassing residues 46–231rVP846–231) that is properly folded, monomeric, and highly soluble.he nuclear magnetic resonance (NMR) analysis of this fragment11] revealed an 11-stranded antiparallel beta sandwich structure.eletion of 17 N-terminal and 5 C-terminal disordered residuesroduced the rVP862–224 fragment which has the same structure asVP846–231 as evidenced by X-ray crystallography [11], and preservets sialic acid binding capacity and all the neutralizing epitopes [26].his core contains the hypervariable and hemagglutination regions15], and is the major determinant of P serotype of rotavirus strains,hich correlates reasonably well with P genotype [21].

In this work, we fused the bovine rotavirus VP8 core protein tohe structure of BLS with the aim to: (i) produce an enhancement ofhe immune response against BRV VP8, (ii) analyze the capability ofhe chimeric protein to induce passive protection against BRV, (iii)valuate the possible use of this antigen for vaccine development,

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7 (2009) 136–145 137

nd (iv) test the feasibility of using BLS as an antigen delivery systemf polypeptides larger in size compared to those previously tested.iven the lack of a three-dimensional structure of BRV VP8, and

he structural description of the homologous protein from Rhesusotavirus previously reported (Dormitzer et al. [10]), we decided tose the 62–224 fragment of BRV (VP8d) to be fused to the structuref BLS.

Our results demonstrate that the BLS-VP8d chimeric protein isroperly folded and stable, and that the BLS scaffold is a potent anti-en delivery system that enhances the antibody response againstRV and elicits a complete homotypic passive protection in a suck-

ing mice model.

. Materials and methods

.1. Molecular modeling

The theoretical structure of the BLS-VP8d chimera wasodeled with the Swiss-PdbViewer 3.7 program (http://www.

xpasy.org/spdbv/ [20]) fusing the C-terminal end of the structuref the 62–224 region of the VP8 protein of BRV (VP8d) with the-terminal end of the crystallographic structure of BLS (PDB code:DIO [5]) through a pentapeptide linker of sequence GSGSG. Thetructure of VP8d was obtained by homology modeling with Swiss-odel [48] using the crystallographic structure of the VP862–224 of

hesus rotavirus (PDB: 1KQR [11]) as template.

.2. Virus, cells and bacteria

The C486 BRV (P[1]G6) strain was propagated in monkey kid-ey (MA104) cells. Tissue culture supernatants were used in the

mmunological assays and concentrated as described previously57], for mouse challenge.

MA-104 cells were provided by the Culture Cell Section of thenstitute of Virology, INTA-Castelar. Cells were cultured in com-lete medium (MEM-D) supplemented with 100 U/ml penicillin,00 �g/ml streptomycin and 10% heat inactivated FBS.

E. coli DH5� and BL21 (DE3) strains competent cells were usedor molecular cloning and protein expression, respectively. Bac-erial strains were routinely grown at 37 ◦C in LB broth or agarupplemented, when required, with 100 �g/ml of ampicillin.

.3. Expression of recombinant proteins

A 489-bp fragment codifying VP8d was obtained by RT-CR from C486 BRV strain using the following primers: for-ard 5′-TACGATGCATGAACCAGTGCTTGAT-3′; reverse 5′-CGTACTT-AGACCAGAACCAGAACCTAATCCATTATTTAT-3′. The sequence cor-esponding to the linker is underlined.

VP8d sequence was cloned downstream BLS gene, in a pET11ector which contains the BLS sequence [60], pET-BLS-VP8d. Addi-ionally, a vector containing only VP8d sequence (pET-VP8d) wasenerated. The plasmids were transformed into E. coli BL21(DE3)ompetent cells for expression of the recombinant proteins. Ampi-illin resistant colonies were grown until OD600 = 1 in LB mediumontaining 100 �g of ampicillin, at 37 ◦C with agitation (300 rpm).ive millilitres of this culture were diluted to 500 ml and grown toeach an OD600 = 1. At this point the culture was induced by addingmM IPTG and incubated for 4 h at 37 ◦C with agitation (300 rpm).he bacteria were centrifuged at 15,000 × g during 20 min at 4 ◦C.

.4. Protein purification and refolding

BLS-VP8d and VP8d were expressed as inclusion bodies. Thenclusion bodies were solubilized by overnight incubation in a 8 M

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rea, 50 mM Tris/HCl, 5 mM EDTA, pH 8 buffer at room tempera-ure with agitation. The solubilized proteins were purified by anionxchange chromatography in a Q-Sepharose (Pharmacia) columnnder denaturing conditions, using a HPLC apparatus (Gilson model20) connected to a UV/vis detector (Gilson, model 152). Elutionas performed using a linear gradient between 0 and 1 M NaCl in8 M urea, 50 mM Tris/HCl, 5 mM EDTA, 1 mM PMSF, pH 8 buffer.

The purified proteins were refolded by dialysis against 50 mMris/HCl, 150 mM NaCl, 5 mM EDTA, 1 mM DTT and 1 mM PMSF, pH.4 buffer at 4 ◦C.

The peaks enriched with BLS-VP8d and VP8d were further puri-ed by gel filtration on a Superose 6 (Pharmacia) and Superdex5 (Pharmacia) columns, respectively. Elution was performed with0 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, pH 7.4uffer. For BLS-VP8d a third purification step on a Superdex 200olumn was performed.

The purity of the preparations was determined by SDS-PAGE12%, w/v) and quantified by measuring at 280 nm wavelength. Theurified proteins were dialyzed against PBS, frozen in liquid N2 andtored at −80 ◦C.

.5. Western blot

After electrophoretic separation of the proteins, the gelsere blotted to an Immobilon P (Millipore) membrane, blocked

vernight with PBS–0.05% Tween 20 (PBS-T) containing 3% skimilk (all subsequent steps were performed using this buffer) and

ncubated with mouse polyclonal anti-BRV C486, for 2 h at 37 ◦C.he membrane was then washed with PBS-T, and incubated withn affinity purified alkaline phosphatase labeled goat anti-mouseg G (H + L) (KPL) for 1 h at 37 ◦C. After washing three times theeaction was developed by the addition of the substrate NBT/BCIPBIORAD).

.6. Circular dichroism (CD)

The CD spectra of BLS-VP8d, BLS, and VP8d in the far UV region250–200 nm) were measured on a JASCO J-810 spectropolarimeter,n a 50 mM sodium phosphate, 1 mM DTT, pH 7.0 buffer at 25 ◦C,sing quartz cuvettes of either 1 or 5 mm path length. Data wereonverted to molar ellipticity [�](in units of ◦ cm2 �molprot

−1) usingmolecular weight value of 35,497, 17,443, and 19,119 g/mol for

LS-VP8d, BLS, and VP8d, respectively.

.7. Thermal denaturation monitored by CD

The heat-induced denaturation of BLS-VP8d, BLS, and VP8d sam-les in 50 mM sodium phosphate, 1 mM DTT, pH 7.0 buffer wasollowed by measuring the CD signal at 222 nm of these proteins onJASCO J-810 spectropolarimeter as a function of temperature. The

amples were slowly heated by increasing the temperature withPeltier system (Jasco). The range of temperature scanning was

0–95 ◦C at a speed of 4 ◦C/min. The molar ellipticity at 222 nm waseasured every 0.5 ◦C. Fast or slow cooling back to 20 ◦C (from 95

o 20 ◦C at a speed of 1 ◦C/min) did not show a recovery of elliptic-ty of the samples demonstrating the irreversibility of their thermalnfolding. Thus the temperature midpoint of the thermal transitionas considered as an apparent melting temperature (Tm).

.8. Static light scattering (SLS)

The weight average molecular weight (Mw) of BLS-VP8d andP8d was determined on a Precision Detectors PD2010 light-cattering instrument tandemly connected to a high-performance

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7 (2009) 136–145

iquid chromatography system and an LKB 2142 differential refrac-ometer. In general, 200–400 �l of BLS-VP8d (0.5–1 mg/ml) or VP8d0.5–1 mg/ml) was loaded on a Superose 6 HR-10/30 or a Superdex5 HR-10/30 column, respectively, and eluted with 50 mM Tris/HCl,.15 M NaCl, pH 8 buffer. The 90◦ light scattering and refractive

ndex signals of the eluting material were recorded on a PC com-uter and analyzed with the Discovery32 software supplied byrecision Detectors. The 90◦ light scattering detector was calibratedsing bovine serum albumin (Mw: 66.5 kDa) as a standard.

.9. Fluorescence

Fluorescence spectra were measured on an LS 50B PerkinElmeruminescence spectrometer. The concentration of VP8d and BLS-P8d solutions were approximately 1.0 �M. A 4 nm band-width forxcitation and emission slits were used in all experiments. The exci-ation wavelength was set at 295 nm and spectra were recordedrom 300 to 500 nm. All spectra were recorded at 25 ◦C in PBS bufferpH 7.4).

.10. Mice

Female BALB/c mice, 6–8 weeks-old, were obtained from theiotery of the CICVyA, INTA. Animals were tested for the absencef rotavirus-specific antibodies by ELISA.

.11. Experimental design

Groups of female mice (n = 8) were immunized with (i) BLS-VP8dfusion protein), (ii) VP8d, (iii) mixture of BLS and VP8 (BLS + VP8d)nd (iv) BLS.

Each group was tested using four different doses (equivalent to0, 2, 0.75 and 0.25 �g of VP8d). The doses of BLS and BLS-VP8dere corrected by their molecular weights to inoculate equimolar

mounts of each protein compared to that of VP8d. Doses describedbove correspond to 0.525, 0.105, 0.040 and 0.013 nmol of VP8drotein, respectively.

A positive control group was immunized with a vaccine contain-ng 107.5 FFU/ml of C486 BRV. As negative control, a group of dams

ere inoculated with placebo (PBS).

.12. Immunization of mice

Rotavirus seronegative female mice were immunized withwo doses of 0.3 ml. Vaccines were prepared with Marcol/ArlacelBiogenesis-Bago S.A.) adjuvant containing the appropriate anti-en in a proportion adjuvant:antigen of 60:40. Preparations weredministered by the intraperitoneal route (ip) every 30 days.

Sera were obtained at 0, 15, 25 and 45 days post-first immu-ization. Milk and suckling mice sera were obtained 10 days afterhallenge.

.13. ELISA

Purified VP8d was directly adsorbed to the plate at a concen-ration of 5 �g/ml in carbonate-bicarbonate buffer, pH 9.6. Platesere then incubated with PBS-T, 5% normal horse serum (block-

ng buffer) for 1 h at 37 ◦C. Blocking buffer was discarded andouse serum samples were added to the plates, diluted 1/4 in

locking buffer and starting at a 1/40 dilution. Samples were incu-ated for 1 h at 37 ◦C. Milk samples were added to the plate at1/10 dilution. Plates were washed with PBS-T and were incu-

ated with horseradish peroxidase-conjugated goat anti-mousegG (KPL). After extensive washing, the reaction was developed with

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2O2/0.05% ABTS. Color development was stopped by the addi-ion of 5% SDS. Absorbance was measured at 405 nm (A405) in a

ultiskan Ex, Labsystems Inc.To normalize values and eliminate the variability associated to

he absorbance reading, results were expressed as a percentage ofositive control value (C+) included in each plate according to theollowing formula:

P = (Xi/C + 1/40) × 100

i corresponds to the absorbance of the sample. C+ correspondso the absorbance of positive control used as 1/40 dilution. Theositive control was a serum from a mouse immunized with BRV486.

Titers are expressed as the log10 of the reciprocal of the highesterum dilution giving a PP value above 35% which represent theverage plus two standard deviations of negative samples.

.14. Viral neutralization (VN) assay

Virus neutralizing Ab titers to C486 BRV in mice sera were deter-ined by fluorescent focus neutralization (FFN) test as described

51]. The VN titer was expressed as log10 of the reciprocal of theighest sample dilution that resulted in a >80% reduction in theumber of fluorescent foci.

.15. Passive protection assay

Two days before the second immunization, female mice wereated with rotavirus seronegative BALB/c males at a 1:3 male-to-

emale ratio. Newborn mice were left to freely suckle their dams anddays after birth were orally challenged with 105 fluorescent focusnits (FFUs) (corresponding to 30 diarrhea doses 50—DD50) of vir-lent C486 rotavirus per mouse. This infective virus dose producediarrhea in 100% of control naïve animals in mock-treated controlroups in our work, similar to that reported by Offit et al. [40]. Aroup of mock-treated and virulent BRV challenged was repeatedor each individual challenge experiment. Virus preparations weredministered by intragastric gauge directly to the stomach with aoft flexible plastic feeding tube and pups were inspected for diar-hea at 24, 48 and 72 h after challenge by gentle palpation of the

bdomen [23,39].

Mice management, inoculation, and sample collection wereonducted by trained personnel under the supervision of a vet-rinarian and in accordance with protocols approved by the INTA’sthical committee of animal welfare.

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7 (2009) 136–145 139

.16. Statistical analysis

The experimental design was analyzed using a 3×3 factorialodel (three vaccines, each evaluated in three doses) plus a control

roup. A one-way ANOVA for the proposed model plus a con-rol group was performed to compare the 10 experimental groups.ossible differences with the control were assessed by Bonferroniost-ANOVA test with a 5% level of significance.

Analysis inside the factorial model was performed by an orthog-nal partition from the ANOVA described, discriminating betweenactorial (to evaluate vaccine and concentration main effects andhe possible interaction between them) and control group, accord-ng to [55,14].

In the cases where a significant interaction was found, orthog-nal contrasts were applied to evaluate vaccine effect insideach concentration. Moreover, polynomial contrasts (lineal anduadratic) were utilized to evaluate concentration effect insideach vaccine.

The variable %protection was analyzed alter the following trans-ormation: arcsinus root of % of protection, according to [3].

Correlation between dams titer and %protection was evaluatedsing Spearman’s Rho [8,35].

. Results

.1. Molecular modeling, recombinant protein expression, andurification of VP8d and BLS-VP8d

BLS has a decameric structure formed by a 17.2 kDa subunitrranged as a dimer of pentamers [60]. In this work the 62–224ragment of BVR VP8 protein (VP8d, Mw 19.1 kDa) was fused to thetructure of BLS (BLS-VP8d chimera, MW monomer 35.5 kDa), withhe aim to increase both the immunogenicity of this protein andhe capability to induce passive protection against BRV in the suck-ing mice model. The fusion of these proteins was carried out byhe same strategy described in Laplagne et al. [27] for the construc-ion of chimeras of BLS and small peptides. In this regard, the codingequence of the first eight residues of the N-terminal end of BLS waseplaced with the coding sequence of VP8d. A flexible pentapeptideinker GSGSG was used to connect both proteins (Fig. 1). To test the

easibility of producing this fusion protein its theoretical structureas analyzed by molecular modeling (for details see Section 2).

his analysis indicated that it is theoretically possible to fuse VP8do BLS without any steric hindrance of the fused domains over theecameric structure of BLS. Thus, the BLS-VP8d chimera and the iso-

structure of these proteins was modeled as described in Section 2. The frontal andolored in the blue range, whereas the VP8d monomers fused to the structure of BLSolored in green. The figure was constructed with the program Swiss-PdbViewer.

140 D. Bellido et al. / Vaccine 27 (2009) 136–145

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iTsAihirreversibly unfolds with an apparent Tm of 65 ◦C. Accordingly,BLS-VP8d remains stable up to 58 ◦C and above this temperaturethe protein unfolds in a complex process that prevented a reli-able estimate of the apparent Tm. The CD signal of the chimera

Fig. 3. Size exclusion chromatography coupled to SLS analysis of VP8d and BLS-VP8d. VP8d (upper panel) was run on a Superdex-75 column and BLS-VP8d (bottom

ig. 2. (A) SDS-PAGE in 15% (w/v) polyacrylamide analysis of purified BLS-VP8d andntigenic characterization of the VP8d and BLS-VP8d. Western blots was probed w: BLS, lane 5: MWM.

ated VP8d domain were expressed in bacteria for immunologicalnd structural comparison of both proteins.

The proteins were expressed at 37 ◦C in E. coli with high yield.P8d was expressed as a soluble protein, even though a signifi-ant amount was obtained as inclusion bodies. On the other hand,LS-VP8d was totally expressed as inclusion bodies even at 28 ◦C.herefore, both proteins were purified from inclusion bodies (seeection 2). Refolding yields of 90% and 20% were obtained for VP8dnd BLS-VP8d, respectively. At the end of the purification approx-mately 80 mg of VP8d and 15 mg of BLS-VP8d were obtained periter of culture. The purified proteins were more than 95% pure asvidenced by SDS-PAGE analysis (Fig. 2), and positively reacted toouse polyclonal anti-BRV C486 sera.Both VP8d and BLS-VP8d demonstrated to be stable in the con-

itions normally used in the laboratory and resisted freeze–thawycles without any significant loss of material (data not shown).

.2. Structural characterization of VP8d and BLS-VP8d

The structures of VP8d and BLS-VP8d were analyzed using aariety of biophysical techniques. The molecular weight of VP8dnalyzed by SLS was 19 kDa demonstrating the monomeric naturef this domain. On the other hand, the molecular weight of BLS-P8d was 350 kDa indicating a decameric structure of the chimeraFig. 3). This result is in agreement with the quaternary structuref native BLS, suggesting that the BLS module is properly folded inhe structure of the chimera.

In order to evaluate the secondary structure of these proteins,heir CD signal in the far UV (200–250 nm wavelength range) was

easured. Fig. 4 shows the spectra of VP8d, BLS and BLS-VP8d. TheLS spectrum corresponds to a protein with mixed alpha and betatructure in agreement with the crystallographic structure of thisrotein [60]. On the other hand, the spectrum of isolated VP8d hasnegative signal with a peak at 216 nm characteristic of an antipar-llel beta sheet, in agreement with the theoretical model of thisomain. The positive peak at 232 nm probably corresponds to theontribution of aromatic residues to the CD spectrum of this pro-ein. Remarkably, the far UV CD spectrum of BLS-VP8d is practicallydentical to that theoretically calculated for this protein from theombination of the CD signals of isolated BLS and VP8d (Fig. 4), indi-ating the preservation of the secondary structure of BLS and VP8dn the structure of these modules in the structure of the chimera.

The fluorescence properties of BLS-VP8d and its modules were

nalyzed to probe the tertiary structure of the chimera (Fig. 5).he fluorescence emission spectrum of BLS is red shifted (centerf mass 360 nm) due to the solvent exposure of its single trypto-han residue, whereas the spectrum of VP8d is blue shifted (centerf mass 346 nm) in agreement with the occlusion of its three tryp-

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. Lane 1: VP8d, lane 2: BLS-VP8d, and lane 3: molecular weight marker (MWM). (B)use anti-BRV C486 serum. Lane 1: BLS-VP8d, lane 2: VP8d, lane 3: BRV C486, lane

ophan residues in the protein core as estimated by analysis of theheoretical model of this protein. The spectrum of BLS-VP8d is alsolue shifted (center of mass 348 nm) and practically identical tohat of VP8d. This result indicates that this module dominates theuorescence spectrum of the chimera and supports the idea thatot only the secondary structure, but also the tertiary structure ofP8d is preserved in the structure of the chimera.

This fact was also supported by analysis of the thermal stabil-ty of BLS-VP8d compared to the isolated VP8d and BLS modules.he thermal denaturation of these proteins was analyzed by CDpectroscopy (Fig. 6). The structure of BLS is stable up to 85 ◦C.bove this temperature the protein cooperatively unfolds in an

rreversible process with an apparent Tm of 89 ◦C. On the otherand, VP8d remains stable up to 58 ◦C and then cooperatively and

anel) was run on a Superose-6 column. Both proteins were eluted with 50 mMris/HCl, 0.15 M NaCl, pH 8 buffer. All separations were performed at a flow ratef 0.5 mL/min. The elution was monitored by light scattering at 90◦ (continuousine) and UV280 nm (dotted line) signals. The molecular weight of each sample wasalculated relating its 90◦ and RI signals, and comparing of this value with thatbtained for BSA as a standard.

D. Bellido et al. / Vaccine 27 (2009) 136–145 141

Fig. 4. Comparison of the far UV-CD spectra of BLS (dark gray dashed line), VP8d(light gray continuous line) and BLS-VP8d (black continuous line). The theoreticalspectrum of the BLS-VP8d chimera, calculated from the combination of the VP8dand BLS spectra, is represented by a black dotted line.

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Table 1Antibody titers detected by ELISA in immunized dams, pups and milk.

Treatment Dose (�g) Antibody titers (log10) (median ± S.D.)

Dams serum Pups serum Dams milk

BLS-VP8d 2 3.33 ± 0.21a 3.38 ± 0.04a 1.60 ± 0.11a

BLS + VP8d 2 2.65 ± 0.42ab 2.50 ± 0.81a 1.36 ± 0.12a

VP8d 2 2.43 ± 0.72b 2.12 ± 0.98b 0.79 ± 0.12b

BLS-VP8d 0.75 2.88 ± 0.45a 2.52 ± 0.45a 0.83 ± 0.41a

BLS + VP8d 0.75 1.22 ± 0.45b 1.15 ± 0.42b 0.10 ± 0.13b

VP8d 0.75 1.22 ± 0.64b 1.22 ± 0.43b 0.10 ± 0.12b

BLS-VP8d 0.25 2.65 ± 0.62a 1.99 ± 0.57a 0.81 ± 0.60a

BLS + VP8d 0.25 1.00 ± 0.21b 1.00 ± 0.19b 0.10 ± 0.13b

VP8d 0.25 1.00 ± 0.19b 1.00 ± 0.16b 0.10 ± 0.12b

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ig. 5. Fluorescence emission spectra of BLS (dark gray dashed line), VP8d (lightray continuous line) and BLS-VP8d (black continuous line).

onotonously decreases presumably due to an aggregation phe-omena of the denatured VP8d domains in the decameric structuref the chimera that perturb the structure of the BLS modules pro-

oting their unfolding. However, the agreement in the thermal

tability of BLS-VP8d and VP8d, indicates that this domain is prop-rly folded in the structure of the chimera.

ig. 6. Thermal denaturation of BLS (dark gray dashed line), VP8d (light gray contin-ous line) and BLS-VP8d (black continuous line) followed by the 222 nm CD signalf these proteins as a function of temperature.

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ean values within each dose, with different superscript letters (a, b) differ signifi-antly, 3×3 factorial ANOVA, p < 0.05.

.3. Immune response to VP8d and BLS-VP8d

In order to test the potential increase in the immunogenicity ofP8d produced by its recombinant fusion to the structure of BLS,roups of female mice were immunized with four different doses0.25, 0.75, 2 and 10 �g) of VP8d fused to BLS (BLS-VP8d), VP8ds a monomeric protein, and a mixture of isolated BLS and VP8dBLS + VP8d). In addition, the same schedule was used for immu-izing with BLS as control. Sera and milk from immunized damsnd their pups were tested by VP8-ELISA (Fig. 7).

IgG antibodies were detected at high titers in the groups immu-ized with doses equivalent to 10 �g of VP8d. In this regard, sincehe large amount of antigen used for immunization could maskhe immune-enhancing property of BLS, we decided to excludehis group in the subsequent experiments. When a 2 �g dose wassed to immunize mice, a strong specific antibody response againstP8d was found in all groups (BLS-VP8d, VP8d and BLS + VP8d).

n this condition, BLS-VP8d evoked a significantly higher antibodyesponse than VP8d, whereas the BLS + VP8d mixture produced anntermediate response that did not differ significantly from none ofhe above (Table 1). On the other hand, when lower doses were inoc-lated (0.75 and 0.25 �g), only mice immunized with the BLS-VP8dhimera were able to induce a strong antibody response againstP8d. In these conditions, BLS-VP8d elicited a significantly higherntibody response than VP8d and BLS + VP8d.

Differences in the antibody titers observed in the immunizedams were also found in their offspring, indicating that antibod-

es were passively transferred to pups. It is important to note thatpecific IgG detection in milk was clearly associated with antibodyevels in the sera of dams and pups. According to this, antibody titersnduced by BLS-VP8d were significantly higher than those gener-ted by BLS + VP8d and VP8d in the groups immunized with dosesquivalent to 0.75 and 0.25 �g of VP8d. No significant differencesere found between the titers elicited by VP8d and VP8d + BLS in

ll the doses evaluated (Table 1).The ability of anti-VP8d antibodies to prevent viral infection in

itro was examined by a virus neutralization assay. Neutralizingntibodies were detected at significantly higher titers in BLS-VP8dmmunized mice when compared to mice immunized with VP8dnd BLS + VP8d (Table 2).

.4. Protection against challenge

To test the immunogenicity of the BLS-VP8d chimera, using ann vivo model, a challenge experiment was performed in passivelymmunized suckling mice. Immunized female were allowed to pro-uce litters which averaged about five new born mice each. Pups

142 D. Bellido et al. / Vaccine 27 (2009) 136–145

Fig. 7. Detection of specific antibodies in mice immunized with the recombinant proteins and their offspring. Antibody response developed by mice immunized with BLS-VP8d, VP8d and BLS + VP8d was evaluated by ELISA. As control, a group of female mice was also immunized with BLS. Three doses of VP8 were used: 2 �g (A), 0.75 �g (B) and0.25 �g (C). White bars represent titer values from dams, while black bars represent those from pups. Titers are expressed as the log10 of the reciprocal of the highest serumdilution which gives a PP value above 35%. Squares on the top indicate milk anti-VP8 IgG detection. Black squares represent positive detection and white squares representnon-detection.

D. Bellido et al. / Vaccine 2

Table 2Neutralizing antibodies induced in immunized dams.

Treatment

BLS-VP8d VP8d BLS + VP8d

Dose2 �g 3.2 ± 0.03a 2.1 ± 0.05b 1.9 ± 0.05b

0.75 �g 3.1 ± 0.06a 1.4 ± 0.05b 1.4 ± 0.04b

0.25 �g 2.9 ± 0.05a 1.6 ± 0.04b 1.7 ± 0.06b

Values are expressed as SN titer mean (log10) ± S.E.M. Different superscript letters (a,b) in each row indicate significantly differences between treatments (3×3 factorialANOVA, p < 0.05). Possible differences with the control were assessed by Bonferronipost-ANOVA test with a 5% level of significance.

Table 3Challenge of suckling mice born to immunized dams.

Doses (�g)a Treatmentb

BLS-VP8d VP8d VP8d + BLS

2 100%a 67.44%b 80%ab0.75 97.5%a 36.07%b 44.2%b0.25 100%a 37.3%b 57.14%b

Controls

BRV C486 100%BLSc 12.5%Mock 0%

Different letters (a, b) in each row indicate significantly differences between treat-ments (3×3 factorial ANOVA, p < 0.05). Possible differences with the control wereassessed by Bonferroni post-ANOVA test with a 5% level of significance.

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ere born between 1 and 3 weeks after their dams received the lastooster. Litters were challenged with the homologous BRV. All theemale mice immunized with BLS-VP8d chimera developed a com-lete protective immune response, since they were able to passivelyrotect 98–100% the offspring. Passive protection levels inducedere significantly lower when the same amount of VP8d was

dministrated alone or in a mixture with isolated BLS (p < 0.001)Table 3). Additionally, a positive and significant correlation wasound between antibody titers elicited by dams and passive pro-ection levels conferred to pups (Spearman’s Rho = 0.773, N = 81,< 0.01). This result indicated that the higher level of protectionenerated by the immunization with BLS-VP8 is proportional tohe increased antibody response observed in the dams.

. Discussion

The development of effective, safe and stable delivery systemsemains an important objective in vaccine research. We have pre-iously established a modular system to produce chimeric proteinsy fusing BLS to target antigens and have shown that the folding andtability of the modules of chimeric constructs are similar to thatound in their isolated native state [9,27]. Previous data obtainedsing BLS as a carrier for antigen delivery support the usefulness ofhis strategy to develop new vaccines. BLS is highly immunogenic54] presumably due to its decameric arrangement and remark-ble stability as shown by biophysical studies [60]. BLS has beenhown to confer partial protection against Brucella abortus as a

NA vaccine or as a recombinant protein [53,54] and it is also aotent activator of bone marrow dendritic cells [4]. Moreover, BLSas been recently described to be an antigen delivery system toffectively induce mucosal immunity [45]. Small peptide epitopesrom Brucella spp. (Omp3148–74) [7] and T. solium (KETc1) [45] have

mwdps

7 (2009) 136–145 143

een successfully fused to BLS, and increased their immunogenic-ty. But short peptides usually fail to induce neutralizing antibodiesecause of the complex three-dimensional structure of the neutral-

zing epitopes. Thus, for many pathogens, whole protein domainseed to be expressed as effective immunogens.

VP8 region of VP4 constitutes an interesting target for vac-ine development since it is involved in rotavirus infectivity andeutralization. Studies have identified eight neutralizing epitopesn VP4, with five of them located on VP8. Furthermore, mono-lonal antibodies against VP8 were reported to neutralize rotavirusn vitro [6,41,50] and passively protect mice against challenge inivo [34]. Antibody competition experiment and escape mutantnalyses indicate that VP8 contains several interrelated epitopesecognized by neutralizing antibodies [6,18,33,50,59].

VP8 contains a compact and homogeneous protease-resistantore from residues A46–R231 [10]. This core contains all mappedntigenic sites on VP8, the hypervariable region (residues72–C203) and the hemagglutination region (residues V93–I208)nd since the VP8 core is monomeric, does not agglutinate red bloodells [11]. In this study, we demonstrated that it is possible to pro-uce a stable chimera by fusing to BLS a large domain as it is theore of BRV VP8 protein.

Both, BLS-VP8d and VP8d were efficiently expressed in bac-eria. Nevertheless, refolding efficiency for BLS-VP8d (20%) wasower than for plain VP8d (90%). It may be, therefore, importanto optimize BLS-VP8d production in bacteria as well as to evaluatelternative expression systems. The structural characterization ofLS-VP8d demonstrated that the different modules of the chimeraere properly folded and preserved their stability. These results

ndicate that the general structure of the chimera is approximatelyhat of the theoretical model shown in Fig. 1. The capability ofLS to tolerate the insertion of large proteins without disturbing

ts oligomeric structure is an important feature compared to someirus-like particles (VLPs) postulated as carrier for vaccine develop-ent. In this regard, there are several examples about the structural

onstrains that the fusion of whole proteins produce over virusike-particles subunits. The decameric order of BLS may have andvantage over VLPs for the display of proteins, given its smaller andompact structure and its thermodynamic stability, which allowsor an increased capacity to accommodate protein domains.

An important feature of BLS-VP8d is that it was able to induceigorous IgG responses against the fused protein. The vaccinatedams developed high titers of VP8-specific antibodies in serums well as neutralizing antibodies to the homologous strain. Alear association between IgG antibodies in dams and pups wasound indicating that specific antibodies were passively trans-erred. Detection of IgG in milk was in agreement to those detectedn serum.

BLS-VP8d chimera induced higher antibodies titers and protec-ion levels than those induced by the several VP8 subunit vaccinesreviously described. Moreover, the protective immune responsebserved with BLS-VP8d was obtained by immunizing mice with–50 times lower antigen doses [1,19,29,39]. We have previouslyeported that immunization with three doses containing 4 �g ofP8 produced in tobacco plants generated 85% protection againstirulent BRV. The results presented here showed that immuniza-ion with two doses of only 0.25 �g of the chimera BLS-VP8droduced 97.5–100% protection in suckling mice.

As it was reported by Gil et al. [19], VP8 itself is a highly immuno-enic polypeptide that induces an effective homotypic protection

ediated by neutralizing antibodies. Our results are in agreementith those found by Gil et al. [19] and suggested that the polymericisplay of VP8d in the structure of BLS enhanced its immunogenicroperties. The repetitive and spatially ordered presence of theame epitopes would produce a strong signal transduction medi-

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ted by B cell receptors, as has been described using haptens asodel antigens. In this sense, the three-dimensional structure of

LS shows that any given epitope would be presented at 10 differentoints being each other at a distance of about 40 Å [5]. This anal-sis is in agreement with the previous proposal that the immuneystem has evolved to respond strongly to antigens with an epi-ope spacing of 50–100 Å, typically found on microbial surfaces [7].emarkably in accordance with previous results [4,9] the immuno-enicity of VP8d is enhanced only when it is covalently attached tohe structure of BLS, suggesting that the adjuvant and carrier prop-rties are tightly coupled. The fact that BLS is able to activate DCshrough TLR4 would explain to some extent this phenomenom [4].

Expression of VP8d as a fusion protein with BLS may consti-ute an effective strategy to induce a protective immune responsegainst rotavirus disease. The most important finding is that VP8dxpressed in the chimera not only preserved its immunogenicityt a large extent, but also exhibit total protective capacity in suck-ing mice. It is interesting that VP8d alone induce a very low levelf protection which could not be increased by addition of isolatedLS.

Overall, results obtained herein support the feasibility of usingLS as a novel and effective delivery system to induce a protective

mmune response against rotavirus disease. In particular, previousesults showing the plasticity of the BLS scaffold for the productionf polyvalent chimeras suggest that VP8 from different strains cane coupled to BLS in order to elicit wide-protecting neutralizingntibodies against different field strains of rotavirus.

cknowledgments

We want to thank to Laura Marangunich for the statistical assis-ant. We also want to thank Dr Viviana Parreno for her helpfuleading of the manuscript.

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