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Vaccine 26 (2008) 5814–5821

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

himeric hepatitis B virus core particles carrying an epitopef anthrax protective antigen induce protectivemmunity against Bacillus anthracis

ing Yin, Jun Zhang, Dayong Dong, Shuling Liu, Qiang Guo,iaohong Song, Guanlin Li, Ling Fu, Junjie Xu ∗, Wei Chen ∗

tate Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology,0 Dongdajie, Fengtai, Beijing 100071, China

r t i c l e i n f o

rticle history:eceived 26 June 2008eceived in revised form 12 August 2008ccepted 19 August 2008vailable online 9 September 2008

a b s t r a c t

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) display-ing a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. Thetruncated hepatitis B virus core (HBc) protein (aa 1–144) was used as a carrier, and the 2�2–2�3 loopof the PA domain 2 (aa 302–325) which has been shown contains a dominant neutralizing epitope wasinserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-

eywords:nthrax protective antigenBc particleseutralizing epitopenthrax vaccine

PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electronmicroscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. Inmice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was ableto induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whetheralum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against viru-lent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope

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. Introduction

Anthrax is an epizootic disease affecting wild and domesticatederbivores, which can occasionally be transmitted to humans whoome in contact with infected animals or animal products [1,2].acillus anthracis is the etiologic agent of anthrax. Two plasmid-ncoded virulence factors, a poly-d-glutamic acid capsule and anxotoxin, have been described for the bacterium. Anthrax toxin con-ists of three large proteins that assemble at the surface of receptor-earing mammalian cells to form toxic, noncovalent complexes3,4]. Two of the proteins, lethal factor (LF) and edema factor (EF),re enzymes that covalently modify substrates within the cytosolic

ompartment; the third protein, protective antigen (PA), serves toind LF and EF to the cell and mediate their entry into the cytosol.

Antibiotics can eradicate the bacteria after the infection, but theuge load of anthrax toxin may still cause death of the victims.

∗ Corresponding authors at: Department of Applied Molecular Biology, Beijingnstitute of Microbiology and Epidemiology, 20 Dongdajie, Fengtai, Beijing 100071,hina. Tel.: +86 10 63815273; fax: +86 10 63815273.

E-mail addresses: xujunjie1@gmail.com, xujj02@yahoo.com.cn (J. Xu),henwei0226@yahoo.com.cn (W. Chen).

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.08.031

unity against Bacillus anthracis.© 2008 Elsevier Ltd. All rights reserved.

accination is an effective prophylactic measure to eliminate theisk of infection. Currently, there are two kinds of anthrax vaccinespproved for use in humans, one is live spore vaccine, which isade from the attenuated B. anthracis strain, and is mainly used in

hina and Russia; and the other is prepared by adsorbing filteredulture supernatant of an attenuated strain to aluminum hydroxide,hich is mainly used in western countries [5,6]. However, there are

ome defects about these two vaccines: the active ingredients areifficult to characterize; the schedule of vaccination is not ideal;hey are recommended to be used only in some special population;nd there are still some safety concerns about them [6–11].

The PA of anthrax toxin was found to play an essential role inmmunity and prophylaxis against anthrax. Much of the work focus-ng on PA to generate a next generation anthrax vaccine has beenone in the past decades. These include a DNA vaccine encoding PA12], as well as approaches using various micro-organism vectors toxpress PA, such as rabies virus [13], influenza virus [14], vacciniairus [15], adenovirus [16], Venezuelan equine encephalitis virus-

ased replicons [17] and a salmonella live vaccine encoding domainof PA [18], etc. Currently, the best developed vaccine candidate isrecombinant PA (rPA) adsorbed to aluminum hydroxide [19].

The immunogenicity of a protein antigen bases on differentpitopes. Only some of the epitopes in a vaccine candidate protein

Y. Yin et al. / Vaccine 26 (2008) 5814–5821 5815

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ig. 1. Schematic presentation of the chimeric HBc protein construct. The amino acidFFPS IRDLLDTASA LYREALESPE HCSPHHTALR QAILCWGELM NLATWVGSNL EDEVHVWIRTPPAY RPPNAPILST LPKLAAALEH HHHHH”. The bold represent the insertion o

ay be necessary for the protective immunity, others may beedundant epitopes, dominant no-neutralization epitopes or self-ntigen cross-reactive epitopes, etc. It was found the monomer PAs organized mainly into antiparallel �-sheets and has four domains20,4]: domain 1 contains a furin cleavage site for activating pro-eases, and is the site of interaction of PA with LF and EF; domain 2ossesses a chymotrypsin-sensitive region critical in the transloca-ion process; domain 3 plays an important role in oligomerizationf cleaved PA (PA63); and domain 4 is the receptor-binding domain.n the early studies, several neutralizing epitopes in PA domain 4ere found and antibodies against those epitopes showed protec-

ive capacities against anthrax [21–23]. Recently, the neutralizingpitopes in domain 2 and domain 1 have been revealed [24–27].reviously, we and others found 2�2–2�3 loop of PA domain 2hich is involved in the transition of PA oligomers from prepore toore contains a dominant neutralizing epitope [24,27]. So we tryo design a kind of anthrax vaccine based on this special epitope.

The design of subunit vaccines on the basis of neutralizingpitopes of infection agents is a promising tread in vaccinology.nfortunately, epitope peptides exhibit rather low immunogenic-

ty and therefore require coapplication of adjuvant to induce strongmmune response. Their immunogenicity can be increased via pre-enting target sequences on the surfaces of recombinant virus orirus-like particles (VLPs). Hepatitis B virus core (HBc) protein isne of the most promising delivery vehicles of foreign epitopes suit-ble for designing highly immunogenic vaccines [28,29]. First, HBcrotein is highly immunogenic. It induces strong B cell, T cell andTL responses in human and immunized animals. It may act as a Tell-independent immunogen, which directly activates B cells [30].econd, chimeric HBc particles can enhance the immune responseo the inserted foreign epitope, which is presumably because thepitope is presented on the surface of core particles each containing80 or 240 HBc subunits [31].

HBc particles are built from multiple subunits of a single 183mino acids (aa) core protein, with the N-terminal 144 aa con-istituting the actual assembly domain [32,33]. Foreign epitopesere inserted into HBc protein in various protein regions, includ-

ng the N- or C-termini and the immunodominant e1 loop [34].t has been demonstrated that the e1 loop in the main determi-ant (MIR, major immunodorminant region) of the core antigen ishe most promising insertion site from the immunological point ofiew [35]. Epitopes inserted there possess higher antigenicity andmmunogenicity than anywhere else [28].

In the present study, we generated a fusion protein with 144 aa

f HBc protein and 24 aa of 2�2–2�3 loop of anthrax PA domain, and confirmed this protein was able to spontaneously assem-le into chimeric HBc particles by electron microscopy. We furtherroved that these particles were able to induce PA-epitope specificntibodies and anthrax toxin-neutralizing antibodies in mice and

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nces of HBc-N144-PA-loop2 are “MASMTGGQQM GRGSMDIDPY KEFGASVELL SFLPS-V HASFFDIGGS VSAGFSPASR ELVVSYVNVN MGLKIRQLLW FHISCLTFGR ETVLEYLVSFequence and the italics are the sequences on vector pET21a (+).

uinea pigs, and partially protect the guinea pigs against anthraxpores challenge. Our results suggest that chimeric HBc particlesarrying a neutralizing epitope of PA could be a promising vaccineandidate against B. anthracis.

. Materials and methods

.1. Plasmids, bacterial strains and cells

A group of DNA fragments encoding different parts of the-terminally truncated HBc (HBc-N144) were fused by overlapxtension PCR (Table S1). The synthesized HBc-N144 gene fragmentas then amplified by primers P1u (5′-CGCGGATCCATGGACATTGA-CCGTAT-3′) and P1d (5′-CCCAAGCTTCG GAAGTGTTGATAAGAT-3′),nd cloned into the BamH I and Hind III digested vector pET21a (+)Novagen) to create the plasmid pET21a-HBc-N144. The DNA frag-

ent encoding 2�2–2�3 loop of PA domain 2 was inserted into theET21a-HBc-N144 by fusion PCR to generated the plasmid pET21a-Bc-N144-PA-loop2, with primers P1u and P2d (5′-ATATCAAAGACGACGCATGCACTTCTGCATTTCCATGTACTTCGTCTTCCAAATTACT-′); P2u (5′-GTCGTTCTTTGATATTGGTGGGAGTGTATCTGCAGGATTT-GTCCA GCATCCAGGGAAT-3′) and P1d, which inserted 24 aa302–325) of PA between aa 78 and 79 of HBc protein (Fig. 1). Forloning and expression studies, the Escherichia coli strain DH5�nd BL21 (DE3) (Novagen) were used. J774A.1 cells (ATCC) weresed for in vitro toxin neutralization assay.

.2. Expression and purification of recombinant proteins

.2.1. HBc-N144/HBc-N144-PA-loop2The recombinant plasmids were transformed into E. coli BL21

DE3) for expression. Overnight cultures of BL21 (DE3) cells har-oring the recombinant plasmids were diluted 1:400 in 1 L of LBroth containing 100 �g/ml ampicillin, and were grown until OD600eached 0.4–0.6. Expression of the proteins was then induced by.1 mM of isopropyl-�-d-thiogalactopyranoside (IPTG). After shak-

ng for 5 h at 37 ◦C, the bacteria were collected by centrifugingt 4 ◦C 6000 × g for 15 min and the pellets were resuspended in00 ml of balance buffer (pH 8.0, 50 mM Tris, 100 mM NaCl, 10 mMmidazole). For purification, the bacterial cells were lysed by ultra-onic, followed by centrifugation at 4 ◦C 17,418 × g for 20 min toemove bacterial debrits. The clear supernatant was applied to ai2+ NTA column (Qiagen) according to manufacturer’s recommen-ations. The column was washed with 50 mM Tris, 100 mM NaCl,

0 mM imidazole buffer (pH 8.0), and bound protein was elutedith 50 mM Tris, 100 mM NaCl, 500 mM imidazole buffer (pH 8.0).

he eluted was monitored at A280 and peak fractions were col-ected and analyzed by SDS-PAGE and densitometric scanning toetermine the purity. The protein was dialyzed to PBS for Electron

5816 Y. Yin et al. / Vaccine 26 (2008) 5814–5821

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ig. 2. Analysis of the recombinant proteins HBc-N144 and HBc-N144-PA-loop2 bacteria expressing HBc-N144; Lane 2: induced bacteria expressing HBc-N144; Lanane2: induced bacteria expressing HBc-N144-PA-loop2; Lane 3: purified HBc-N144

icroscopy and immunology studies. The contamination of LPS wasetermined by tachyplens amebocyte lysate (TAL) assay (Chineseorseshoe Crab Reagent Manufactory).

.2.2. Recombinant PARecombinant PA (rPA) was prepared as described previously

36]. Briefly, plasmid pAS20 carrying PA gene with an OmpAignal sequence attached to the 5′ end of PA gene was trans-ormed into E. coli DH5� and the bacteria were induced to expressPA by IPTG. The recombinant protein which was secreted tohe periplasmic space of the bacteria cell was purified with arocedure including ion-exchange, hydrophobic interaction chro-atography and gel filtration. The final protein was about 95% pure

nd the contamination of LPS was <0.2 ng/ml determined by TALssay.

.3. SDS-PAGE and Western blotting

Electrophoresis was performed in 15% SDS polyacrylamide gelsnd the recombinant proteins were detected by Western blottingsing a monoclonal antibody (mAb) against the polyhistidine tag

n the C-terminal region of the fusion protein or a mAb against�2–2�3 loop of PA domain 2 (5E12, [24]). Briefly, the transferreditrocellulose membrane was blocked with 2% (w/v) BSA in TBS forh at 37 ◦C, and washed thrice with TBS – 0.05% (v/v) Tween 20,

hen the membrane was incubated with either a 1:5000 dilutionf anti-His tag (mouse mAb, Sigma) or a 1:2000 dilution of 5E12n a 0.2% BSA-TBS – 0.05% Tween 20 solution for 1 h at 37 ◦C, andashed four times with TBS – 0.05% Tween 20. Protein bands wererobed with 1:2000 dilution of HRP-conjugated rabbit anti-mouse

gG (Sigma) and washed four times as described above. Chemilumi-escence was applied as instructed by the manufacturer (Applygenechnologies).

.4. Electron microscopy

The purified proteins, HBc protein without insert (HBc-N144)

nd the fusion protein (HBc-N144-PA-loop2) containing the 24-aansert were analyzed by negative staining electron microscopy fol-owing established techniques. Briefly, proteins were adsorbed to30 mesh carbon-coated copper grids and incubated for 3–5 min.nd then the grids were washed twice with water and finally

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-PAGE. Lane M: protein molecular weight marker in kDa; (A) Lane 1: uninducedurified HBc-N144; (B) Lane 1: uninduced bacteria expressing HBc-N144-PA-loop2;oop2.

tained for 40 s with 1% phosphotungstic acid. Specimens werevaluated using a Philips Tecnai 10 (Philips Inc.).

.5. Immunization of animals

Pathogen-free female BALB/c mice or Hartley guinea pigs wereurchased from Beijing Laboratory Animal Center. For mice exper-

ment, five female BALB/c mice (4–6 weeks) per group weremmunized subcutaneously (s.c.) with recombinant proteins HBc-144 or HBc-N144-PA-loop2 (50 �g/mouse) at 2 weeks intervals forrst three times, the forth injection was performed at the eightheek. The adjuvant of the first immunization is Freund’s adju-

ant complete; the others are Freund’s adjuvant incomplete. Themmunized animals were bled at 0, 2, 4, 6, 10 weeks for the serolog-cal tests. For guinea pigs experiment, four groups of eight femaleuinea pigs (4–6 weeks, 250 g) were vaccinated intramuscularlyi.m.) with HBc-N144, HBc-N144-PA-loop2 and rPA, respectively50 �g/animal) on weeks 0, 2, 4, 8, 12 for five immunizations. Theluminum hydroxide (final concentration was 0.5 mg/ml) was useds adjuvant unless a group of HBc-N144-PA loop2 without adjuvant.he immunized animals were bled at 0, 2, 4, 7, 10, 16 weeks for theerological tests.

.6. Serological tests

.6.1. ELISADirect ELISA was used for detection of antibodies in the sera

f immunized animals. ELISA plates (96-well) were coated with00 ng/well of HBc-N144, HBc-N144-PA-loop2 or rPA in coatinguffer (50 mM Na2CO3–NaHCO3, pH 9.6) overnight at 4 ◦C. Afterhrice washed with PBS – 0.05% (v/v) Tween 20, the plates werelocked with 2% (w/v) BSA in PBS for 1 h at 37 ◦C. Binding of theera of immunized animals with the coated antigen was monitoredsing 2-fold dilution series, and the first dilution was 100-fold. Thelates were incubated at 37 ◦C for 1 h and washed four times withBS – 0.05% Tween 20. Then, HRP-conjugated anti-mouse or anti-uinea pig antibody (Sigma) was added into each well in a 1:2000

ilution, and incubated at 37 ◦C for 1 h. The plates were washedour times and developed with TMB solution in the dark place formin, and the enzyme reaction was stopped by adding 2 M H2SO4nd the OD450 of the plates was read using a microplate readerBio-Rad).

Y. Yin et al. / Vaccine 26 (2008) 5814–5821 5817

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ig. 3. Analysis of the recombinant proteins HBc-N144 and HBc-N144-PA-loop2 byouse monoclonal anti-PA 2�2–2�3 loop antibody 5E12.

.6.2. In vitro toxin neutralization assayAnthrax toxin-neutralizing antibody titer was determined by

ssessment of the ability of immunized serum to prevent the deathf murine macrophage J774A.1 cells induced by the PA/LF toxinomplex (LeTx). J774A.1 cells were plated in 96-well flat bottomissue culture plates at a density of 3 × 104 cell/well in 100 �l MEMGIBCO) supplemented with 10% (v/v) fetal calf serum (Hyclone), 1%v/v) penicillin–streptomycin solution (Hyclone), and 2.93% (w/v)lutamine and incubated overnight at 37 ◦C. The second day, serialilutions (from 1:20, triplicate wells for each dilutions) immuneera (IS) incubated for 1 h with the LeTx (consisting of the 40 ng/�lPA and 20 ng/�l rLF [37]) in the same medium at 37 ◦C, and 100 �lf the sera–toxin mixtures (IS-LeTx) were added to J774A.1 cells (theriginal medium in each cell wells were removed) and incubated forh at 37 ◦C. Three wells in each plate with the LeTx alone were useds no protection control, and another three wells without IS-LeTxere regarded as blank control. Cell viability was assessed withTT assay. Briefly, the MTT dissolved in the medium was added

o each well at a final concentration of 0.5 mg/ml. Cells were incu-ated for another 30 min at 37 ◦C. The medium was replaced by.5% (w/v) SDS, 25 mM HCl in 90% iso-propyl alcohol, the plate wasortexed, and the absorbance reading at 570 nm were measuredith a microplate reader (Bio-Rad). Cell viability was analyzed bysing the average A570 of pairs of wells receiving IS-LeTx or LeTxlong comparing to the blank control. The IS dilution that resultedn an optical density signal of 0.1 was used as a measure of the toxineutralization antibody titer.

.7. Live Bacillus anthracis spores challenge

Challenge experiment was performed in the biosafety levelfacility at Beijing Institute of Microbiology and Epidemiology.

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ig. 4. Electron microphotographs of HBc-N144 and HBc-N144-PA-loop2 particles. (A) HBoop2. Magnification: ×135 K.

rn blot. (A) Proved with mouse monoclonal anti-His tag antibody; (B) proved with

nly some of the immunized guinea pigs above were used in thexperiment due to facility availability and they were boosted withifferent antigens 2 weeks before the challenge. Those animals andgroup of unvaccinated control were challenged subcutaneouslyith approximately 30,000 (40LD50) virulent B. anthracis spores

IVDC40048, from China institute of veterinary drug control). Theyere observed twice daily for 14 days following the challenge for

igns of clinical disease or death. Death by anthrax was confirmedn animals by plating blood on LB agar and incubating overnight at7 ◦C.

. Results

.1. Insertion of 2ˇ2–2ˇ3 loop of anthrax protective antigen intoBc protein allowed the formation of chimeric HBc particles

A carboxyl-terminally truncated HBc protein (144 aa, HBc-N144)nd a fusion protein (HBc-N144-PA-loop2) were generated in E.oli, respectively. The plasmid vector pET21a (+) (Novagen) added aix-histidine tag at the C-terminal region of recombinant proteins,hereby facilitated the proteins being detected by anit-His tag anti-ody and their purification. The HBc-N144-PA-loop2 was obtainedy inserting 2�2–2�3 loop 24 amino acids of PA (aa 302–325) intoajor immunodorminant region (MIR) of HBc-N144 (between aa

8 and 79, Fig. 1). The recombinant plasmids were transformednto E. coil BL21 (DE3), respectively, and proteins expression wasnduced by IPTG. As showed in Fig. 2, HBc-N144 (171 aa including

xtra sequence from plasmid) was about 19 kDa in SDS-PAGE imagehile the fusion protein HBc-N144-PA-loop2 (195 aa including

xtra sequence from plasmid) was about 22 kDa, both of which wereonsistent with their theoretical molecular mass. We then usedestern blot to prove the recombinant proteins. As expected, both

c particles made by HBc-N144; (B) chimeric HBc particles made by HBc-N144-PA-

5818 Y. Yin et al. / Vaccine 26 (

Fig. 5. Kinetics of antibody titer development in mice following immunizations.Bocs

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ALB/c mice were immunized with four injections of 50 �g of different proteinsn weeks 0, 2, 4, 8 and were bled at 0, 2, 4, 6, 10 weeks for the EILSA with rPA asoated protein. Each point represents the mean reciprocal log10 endpoint titers andtandard error.

Bc-N144 and HBc-N144-PA-loop2 protein bands were detectablehen anti-His tag antibody were used as primary antibody, while

nly HBc-N144-PA-loop2 was detectable when 5E12, a mAb bindingo 2�2–2�3 loop of PA domain 2 was used (Fig. 3). The recombinantroteins were purified by Ni-NTA column. According to densitomet-ic scanning of the coomassie blue-stained gel lanes (Fig. 2, lane 3),he purified proteins were more than 90% pure which was thenialyzed to PBS for further studies. The contamination of LPS was1 ng/ml determined by TAL assay.

Since HBc protein can form particles both in vivo and in vitro38–41], we then observed if our recombinant proteins can formarticles. By negative staining electron microscopy (EM), we sawoth the HBc-N144 and HBc-N144-PA-loop2 were able to formarticles with the size around 25–30 nm (Fig. 4). The results con-rmed that the chimeric HBc protein harboring 24 amino acids ofA 2�2–2�3 loop still keeps the same physical characteristic as theriginal HBc particles.

.2. Chimeric HBc particles were able to induce PA-epitopepecific antibodies in mice

To determine the immunogenicity of chimeric HBc particles, wemmunized mice with HBc-N144 and HBc-N144-PA-loop2, respec-ively, using Freund’s adjuvant. The mice were immunized at 2eeks intervals for first three times. After 4 weeks from the first

mmunization, anti-PA antibody was detectable in the serum of

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ig. 6. Kinetics of antibody titer development in guinea pigs following immunizations. Gu0 �g of different proteins on weeks 0, 2, 4, 8, 12 and were bled at 0, 2, 4, 7, 10, 16 weeks foBc-N144-PA-loop2 as coated protein to detect anti-HBc-N144 antibodies and PA 2�2–2�s a control. Each point represents the mean reciprocal log10 endpoint titers and standard

2008) 5814–5821

ice immunized with HBc-N144-PA-loop2, and at sixth week theiter was around 104, while in the serum of mice immunized withBc-N144 almost no anti-PA antibody was detectable (Fig. 5). Weoosted the immunization at the eighth week, and detected thenti-PA antibody at the tenth week. The anti-PA antibody titer ofBc-N144-PA-loop2 group was even higher, while that of the HBc-144 group was still at the background level (Fig. 5). Since wesed rPA as coated protein of the ELISA, the anti-PA titer actu-lly was the representative of PA 2�2–2�3 loop epitope specificntibodies in the sera of HBc-N144-PA-loop2 immunized mice.o our result clearly suggests that the chimeric HBc protein car-ying a PA epitope can induce PA-epitope specific antibodies inice.

.3. Chimeric HBc particles were able to induce PA-epitopepecific antibodies regardless of whether alum adjuvant was usedr not in guinea pigs

Some researches showed that the guinea pig is a better ani-al model to evaluate the efficiency of anthrax vaccine compared

o mouse [42–44]. So we used guinea pigs to comprehensivelyvaluate the immunogenicity of HBc-N144-PA-loop2 with rPA andBc-N144 as controls. For more applicable vaccine study, we usedluminum hydroxide as adjuvant, and also kept an HBc-N144-PA-oop2 group without adjuvant as it has been suggested the HBcarticles themselves were a kind of adjuvant [40]. Our resultshowed that like in mice, HBc-N144-PA-loop2 induced high anti-A (anti-epitope) antibody in guinea pigs (Fig. 6A), although thentibody titer was much lower than rPA group considering PA is anntact protein antigen while HBc-N144-PA-loop2 only contains onepitope of PA. More inspiring result was the group without adjuvantas able to induce almost as high titer as the group with adju-

ant, which means the chimeric particles themselves can inducehe PA-epitope specific antibodies without help of adjuvant, whichs promising in the future vaccine development.

We also compared the anti-HBc-N144-PA-loop2 antibodies inifferent groups (Fig. 6B). Obviously both HBc-N144 and HBc-144-PA-loop2 induced high titer of anti-HBc-N144-PA-loop2ntibodies (actually most of which should be anti-HBc-N144 anti-odies as shown in Fig. 6C), and still there was no differenceetween HBc-N144-PA-loop2 groups with or without adjuvant.nti-HBc-N144-PA-loop2 antibodies were also detectable in the

roup immunized with rPA, which was easily identified that thosentibodies were 2�2–2�3 loop epitope specific antibodies sincenly base level antibodies were detectable when HBc-N144 wassed as coating antigen in ELISA as shown in Fig. 6C. Comparingig. 6A–C, it seems that PA and HBc-N144-PA-loop2 could induce

inea pigs were immunized with five injections of 0.5 ml of preparations containingr the serological tests. (A) rPA as coated protein to detect PA specific antibodies; (B)3 loop epitope specific antibodies (for rPA group); (C) HBc-N144 as coated proteinerror.

Y. Yin et al. / Vaccine 26 (2008) 5814–5821 5819

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ig. 7. Kinetics of neutralizing antibodies to anthrax toxin following immuniza-ions. Neutralizing antibodies in the sera of immunized guinea pigs to anthrax lethaloxin were tested by in vitro neutralization assay. Each point represents the meaneciprocal log10 endpoint titers and standard error.

lmost the same titers of 2�2-2�3 loop epitope specific antibodiesaround 104) in our experiment.

.4. Chimeric HBc particles were able to induce neutralizingntibodies against anthrax lethal toxin in guinea pigs

From our previously result, the mAbs binding to the epitope inA 2�2–2�3 loop have high neutralizing activity against anthraxethal toxin (LeTx) [24]. To evaluate whether the chimeric HBcarticles containing this neutralizing epitope could induce toxin-eutralizing antibodies, sera from immunized guinea pigs wereested for the ability to neutralize LeTx with a J774A.1 cell basedssay. As shown in Fig. 7, after the second immunization, theera from group immunized with rPA were able to neutralizeeTx and prevented J774A.1 cell from death, while the neutral-zing antibodies from sera immunized with HBc-N144-PA-loop2with or without adjuvant) were detectable after the third or theorth immunization. After five times of immunization, the toxin-eutralizing antibodies titer of HBc-N144-PA-loop2 groups (withr without adjuvant) was around 2–5 × 102, which were around0–100 times less than that of rPA group (2 × 104). In the HBc-N144roup, no toxin-neutralizing activity was detectable. Since PA is an3 K protein presumably containing many toxin-neutralizing epi-opes, while HBc-N144-PA-loop2 only contains one of them, theesults is still promising for the vaccine development especially forhe no adjuvant approach.

.5. Chimeric HBc particles were able to protect guinea pigsgainst subcutaneously live Bacillus anthracis spores challenge

For more practical test of the protective immunity induced byBc-N144-PA-loop2, some immunized guinea pigs and a group ofnvaccinated animals (blank) were challenged with 40LD50 of viru-

ent B. anthracis spores. As shown in Fig. 8, the HBc-N144-PA-loop2ithout adjuvant, HBc-N144-PA-loop2 with adjuvant, and rPA with

djuvant groups had 57% (4/7), 37.5% (3/8), 66.6% (2/3) protection ofuinea pigs, respectively. HBc-N144 with adjuvant group resultedn 12.5% (1/8) protection and no animals in blank group (0/8) sur-ived after challenge. The result suggests that HBc-N144-PA-loop2as able to protect immunized guinea pigs, at least partially, against

irulent anthrax spores challenge. Since rPA provided also only aartial protection in the experiment, the result is still promisingnd other animal models (rabbits for example) may be used to eval-ate the protective immunity of HBc-N144-PA-loop2 in the futurexperiments.

etti

pores challenge in guinea pigs. Two weeks after the final additional booster withntigens, the immunized animals received a subcutaneously challenge of approxi-ately 30,000 B. anthrax spores (40LD50). The animals that died within 14 days after

hallenge were recorded.

. Discussion

In the present study, we generated a fusion HBc protein har-oring 24 aa of PA 2�2–2�3 loop which containing a dominanteutralizing epitope [24,28]. We found that the fusion proteintill kept the same physical characteristic of original HBc proteinnd was able to form chimeric HBc particles. We have shown thehimeric particles were able to induce PA-epitope specific antibodyn mice and guinea pigs. More inspiring result was the group with-ut adjuvant had as high titer of anti-PA-epitope antibody as theroup with adjuvant in guinea pigs, which is promising for the vac-ine development. However, we may have to take into account thenfluence of LPS in our preparations when evaluating the adjuvantffect of HBc particles. Although the LPS was beyond our consider-tion at the beginning of this study, we measured its concentrationn our antigen preparations after animal experiments. Data fromAL assay showed that the concentration of LPS in HBc-N144 andBc-N144-PA-loop2 preparations was below 1 ng/ml. Although theontaminated LPS may not be enough to act as an adjuvant, it isery necessary for us to pay more attention to avoid or reduce thisontamination in our future research.

In our experiment, we have also shown the chimeric HBc par-icles were able to induce neutralizing antibodies against anthraxethal toxin detected by in vitro cell based toxin neutralization assay.

direct correlation between animal survival and neutralizing-ntibody titer in the evaluation of anthrax vaccine has beeneported [45]. The ability of HBc-N144-PA-loop2 to induce anthraxoxin-neutralizing antibody was strong evidence suggests it maylso protect animal from real anthrax spore challenge. And it wasroved by our challenge experiment, in which the HBc-N144-PA-

oop2 group without adjuvant showed more than 50% of protectionfter 40LD50 of virulent anthrax spores challenge. We must indicatehat toxin-neutralizing antibody and animal protection induced byBc-N144-PA-loop2 still less than those induced by rPA, whicheans other neutralizing epitopes of PA may also contribute to the

rotective immunity. So to identify more neutralizing epitopes ofA and put them into the chimeric HBc particles may improve therotective immunity. On the other hand, to insert multi-copies ofhe same epitope may also increase the response to the insert.

HBc particles have been extensively exploited as a carrier for for-ign epitopes, and have been demonstrated to drastically improvehe immunogenicity of foreign protein segments presented onheir surface suggesting HBc particles possess an outstanding abil-ty to induce B cell, T helper cell and cytotoxic T cell response

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29,31,41,46,47]. Pumpens and Grens [48] reviewed that in HBcrotein the major RNA-recognizing sites are the aa 150–157, andNA-recognizing sites are three repeated SPRRR motifs within

he aa 157–177; and four arginines following the first 149 aminocids residues also provided RNA binding sites [49]. Accordingly,n further study, we may need to induce some modifications intohe HBc-N144-PA-loop2 to increase the immunogenicity of thehimeric particles. For example, an additional insertion of SPRRReptide at the C terminal of the protein may lead to the inclu-ion of cell RNA into chimeric particles during their formation, thusncrease immunogenic potential of these VLPs due to an adjuvantffect of incorporated RNA molecules; or introduction of an addi-ional RGD sequence at the N terminal of the construct may increasehe binding of chimeric VLPs to cells [40].

Alternative approaches towards a new anthrax vaccine haveeen reported recently. Manayani et al. [50] generated the chimericLPs displaying 180 copies of PA-binding domain of anthrax recep-

or 2 and found they had dual function as an anthrax antitoxin andaccine. Bielinska et al. [51] designed a kind of mucosal adjuvanthich was able to effectively adjuvant rPA for intranasal immuniza-

ion. All those approaches including the present study may lead tonovel and highly effective anthrax vaccine with fewer side effects

han the currently available human vaccines.

cknowledgments

We thank Drs Xiaopeng Zhang, Jianming Li, Shaoqiong Yi,ian Zhao for technical supports and helpful discussions. We arelso grateful to Wanchuan Li in the Department of pathologyor assistance with electron microscopy. This work was sup-orted by National Natural Science Foundation of China (30300016,0571745).

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.vaccine.2008.08.031.

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