Vaccine 24 (2006) 2238–2247

Intranasal immunization with human papillomavirus type 16 capsomeresin the presence of non-toxic cholera toxin-based adjuvants elicits

increased vaginal immunoglobulin levels

Kerstin Della,∗, Robert Koestersb, Michael Linnebacherc,Corinna Kleina, Lutz Gissmanna

a Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Germanyb Universitatsklinik Heidelberg, Pathologisches Institut, Abteilung fur Angewandte Tumorbiologie,

Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germanyc Universitatsklinik Rostock, Abteilung fur Allgemeinchirurgie, Schillingallee 35, 18057 Rostock, Germany

Received 9 August 2005; received in revised form 26 October 2005; accepted 10 November 2005Available online 13 December 2005

Abstract

re directeda tigens. Herew sera andv CTB. Wef gnificantlyi ponses.©

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Prophylactic immunization against human papillomaviruses (HPVs) aims preferentially at the generation of antibodies, which against the virus capsid proteins. DNA-free virus-like particles or their pentameric subunits, the capsomeres represent suitable ane investigated if anti-HPV16 L1 specific antibodies and L1-specific CTL induced by intranasal immunization with capsomeres inaginal washings of C57Bl6 mice can be enhanced by co-application of the non-toxic cholera toxin adjuvants CTA1-D2D1 oround that CTA1-D2D1 elevated L1-specific serum IgG antibodies in a dose-dependent manner and both CTA1-D2D1 and CTB sincreased L1-specific IgA antibody levels in the vaginal lumen. Furthermore, CTA1-D2D1 and CTB enhanced L1-specific CTL res

2005 Elsevier Ltd. All rights reserved.

eywords: Intranasal immunization; Capsomeres; CTA1-D2D1; CTB

. Introduction

Cervical cancer is attributed to persistent infection withhigh-risk” human papillomaviruses (HPV) most commonlyPV type 16 [1]. Infection of the cervical epithelium

s thought to occur after penetration of virions throughicroabrasions and subsequent entry into epithelial stem

ells located in the basal epithelial cell layer[2]. The HPVapsid consists of two proteins, the major capsid protein L1nd the minor L2. The viral capsid possesses a highly orderedtructure, which is composed of 72 pentameric capsomeres.eutralizing antibodies recognizing conformational epitopesre directed against sites within the solvent-facing apicesf three loops formed by the L1 protein[2–4]. Antibody-

∗ Corresponding author. Tel.: +49 6221 424662; fax: +49 6221 424932.E-mail address: k.dell@dkfz.de (K. Dell).

capturing of virions inhibits the attachment and invasiothese pathogens into the apical surface of mucosal epitcells by sterical hindrance and aggregation[5].

The predominant immunoglobulin (Ig) class in secretof the human genital tract is IgG, followed by secretory(sIgA) [6]. Secretory IgA is transported across epithecells in a receptor-mediated process known as transsis [5]. It is still under debate whether IgG is transporvia transcytosis or by passive transudation[5]. IntravaginasIgA is derived locally from plasma cells that reside witthe mucosallamina propria. Activation of mucosal B celloccurs after antigen-uptake via highly specialized M cand antigen-transport to underlying antigen-presenting(APC). In turn, these APC activate T helper cells, whichduce interleukins that effect IgA class switching and affimaturation of germinal centre residing B cells[5,7]. The sensitized lymphocytes leave the site of antigen-entry, migra

264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2005.11.060

K. Dell et al. / Vaccine 24 (2006) 2238–2247 2239

local lymph nodes, where they enter the circulation and seedeither at the mucosa of origin or at specifically interconnectedtissues, where they differentiate into IgA-producing plasmacells [8]. Vaccination via the nasal route leads to specificmucosal IgA immune responses first at the site of immuniza-tion and subsequently in the genital-vaginal mucosa[8] thusmaking intranasal vaccination strategies attractive for immu-nization against HPV16.

Virus-like particles (VLPs) and capsomeres are able toinduce neutralizing antibodies[9–12]. Capsomeres wereshown to eradicate L1-positive tumors in mice after sub-cutaneous (s.c.) immunization[13] and protected againstchallenge in the canine oral papillomavirus model[11]. Inmice, intranasal VLP administration prevented the growthof L1-expressing tumors and even eradicated pre-existingtumors [14]. It is well known that mucosal immunizationis superior to parenteral immunization with regard to thegeneration of mucosal antigen-specific antibodies. Previ-ous studies proved that intranasal administration of eitherVLPs [9,15] and capsomeres[13] are able to induce anti-L1 humoral immune responses both in serum and vaginalsecretions.

Cholera toxin (CT) is composed of two subunits:the potential toxic CTA1 subunit exhibiting strong ADP-ribosyltransferase activity and the non-toxic pentameric CTBoligomer, which binds to the membrane ganglioside GM1p -t o adc fullyi -va ofC nce,f B-s ebyc ects[ viai t toe[ ll asg cificC edw n[

f theC a-t igen.CVf IgGl andC ev-e ginalI thel ease

of L1-specific CTL numbers in the spleen of immunizedmice.

2. Materials and methods

2.1. Mice

C57BL/6 female mice were obtained from Charles RiverWiga breeding laboratories (Sulzfeld, Germany). The micewere maintained under pathogen-free conditions and wereused at 6 to 8 weeks of age.

2.2. Cell lines and culture conditions

Both cell lines used were of C57BL/6 origin (H2b).RMA cells [28], a Rauscher virus-induced T lymphoma cellline, and RMA-S cells[29], a RMA-derived TAP-deficientmutant cell line were cultured in RPMI 1640 medium supple-mented with 5% fetal calf serum (FCS), 2 mMl-glutamine,100 U/ml penicillin, 100�g/ml streptomycin and 0.01 mM�-mercaptoethanol (complete medium).

2.3. Generation of HPV16 L1 capsomeres

The pGEX-4T-3 plasmid (Amersham Biosciences,F the1 rase( ebs-f -c -f ngt

2v

b -CA -7 ro-t eDa -3 ainsa iquer er-i ersw -t asd

CRuGT

resent on all nucleated cells[16]. CTA1-DD is a nonoxic 37 kDa fusion polypeptide where CTA1 is linked timer of the IgG/IgM/IgA-binding fragment ofStaphylococ-us aureus protein A. CT as adjuvant was used successn intranasal VLP immunization[17–19] resulting in eleated titers of vaginal IgA[17,18] and IgG [17] as wells serum IgG[17,18]. However, a limitation for the useT resides in its cell-type associated toxicity. For insta

ollowing intranasal immunization with CT in mice, itsubunit accumulated within olfactory nerve cells theronferring a high risk of central nervous system side eff20]. In contrast, the non-toxic CT-based CTA1-DD bindsts DD-dimer specifically to Ig expressing B cells but nopithelial and nerve cells[21]. CTA1-DD [20,22] and CTB

23] revealed enhancing effects on total serum Ig as weenital tract IgA levels and increased the extent of L1-speTL responses[24] when administered nasally combinith either ovalbumin[20,22] or hepatitis surface antige

23].In our study, we investigated the adjuvant potential o

TA1-DD derived CTA1-D2D1 and CTB in nasal vaccinion in the presence of HPV16 L1 capsomeres as antapsomeres represent a highly stable[25,26] alternative toLPs that can be produced at low cost inE. coli [27]. We

ound that CTA1-D2D1 enhanced L1-specific serumevels in a dose-dependent manner. Both CTA1-D2D1TB significantly increased L1-specific IgA antibody lls in vaginal secretions and, to a lesser extent, also va

gG. Furthermore, both adjuvants positively influencedevel of cellular immune response as shown by an incr

reiburg, Germany) containing an L1 sequence lacking0 N-terminal residues fused to glutathione-S transfeGST) was kindly provided by Dr. P. Sehr, Deutsches Krorschungszentrum, Heidelberg[30]. Expression and purifiation of the resulting plasmid pGEX16L1�N10 in transormedEscherichia coli BL21 cells was performed accordio the protocol described inOhlschlager et al.[13].

.4. Construction of the pT7-CTA1-D2D1 expressionector

Domain D (D1) of protein A fromS. aureus was amplifiedy PCR using the D1 primers 5′-TTCGGCCCCAAAGGCGATGCGCAACAAAA-3′ and 5′-AACTCGAGTTATCC-TCAGCTTTCGGTGCTTGAGA-3′ and plasmid pSPA235 [31] as template. A second domain D (D2) of p

ein A from S. aureus was amplified by PCR using th2 primers 5′-GGAGCTCAAGCTCCAAAAGCTGAT-3′nd 5′-AGGCCTTTGGGGCCTGAGATTCGTTTAATTTT′. D1 and D2 PCR products both encode identical D domt the amino acid level but differ by the presence of unestriction sites to facilitate cloning. D1 and D2 were dimzed bySfiI digestion, ligated and subsequently D2D1 dimere digested withSacI and XhoI and cloned into the vec

or pCRII (Invitrogen, Karlsruhe, Germany). This vector wesignated pCR-D2D1.

The A1 subunit of cholera toxin was amplified by Psing CTA1 primers 5′-GGGAAGCTTCATATGGGTAAT-ATGATAAGTTATATC-3′ and 5′-AAAGAGCTCCCGA-GATCTTGGAGCATTC-3′ and plasmid pRG-CT[32] as

2240 K. Dell et al. / Vaccine 24 (2006) 2238–2247

template. The PCR product with flankingHindIII and SacIsites was cloned into pCR-D2D1 atHindIII/SacI. The result-ing fusion gene CTA1-D2D1 was then cut byNdeI/XhoIand inserted into the prokaryotic expression vector pDEST17(Invitrogen) atNdeI/SalI. This construct was designated pT7-CTA1-D2D1 because it allows expression of recombinantprotein in T7-RNA Polymerase expressingE. coli cells, e.g.BL21 star cells (Invitrogen).

2.5. Expression and purification of the CTA1-D2D1fusion protein

For the production of the fusion proteinE. coli BL21StarTM cells (Invitrogen) transformed with pT7-CTA1-D2D1were grown in 1× LB medium supplemented with 100�g/mlampicillin. Induction with 1 mM IPTG was performed atOD600 nm= 0.5. Three hours post induction cultures were har-vested by centrifugation and solubilized using 6 M guanidine-HCl. After addition of distilled water to allow refolding, thefusion protein was purified by affinity-chromatography usingIgG-Sepharose (Amersham Pharmacia, Freiburg, Germany)as described[33] and stored at−80◦C.

2.6. FACS analysis

In order to investigate the ability of CTA1-D2D1 to bet edfw D1i A1-D andt del-b ec per-m BS,0 itro-g inedw teinA en)i dt FCSb seda wasd del-b ,000c

2s

klyi puna puni wasai n.

alone or in combination with 5�g of cholera toxin subunit B(CTB, diluted in PBS; Sigma, Taufkirchen, Germany), 5�gor 10�g CTA1-D2D1. Negative control mice received 5�gCTA1-D2D1 i.n. alone and the positive control mice werevaccinated with 5�g capsomeres subcutaneously (s.c.). Micewere sacrificed one week after the last boost, spleens wereremoved and blood was taken by heart puncture. Vaginallavage samples were collected on 5 consecutive days cov-ering the complete murine estrus cycle[19] just prior to thetermination of the experiment. The vaginal cavity was rinsedwith 100�l PBS by repeated up and down pipetting underisoflurane inhalation anaesthesia. Immediately after collec-tion, 1 mM PMSF was added. Samples were stored at−80◦Cuntil further testing.

2.8. Generation of cytotoxic T lymphocyte (CTL) lines

After splenectomy, single cell cultures were prepared.During the first 5 days, 2× 107 spleen cells of immunizedmice were co-cultured with 2× 106 �-irradiated RMA-Scells (200 Gy) pulsed with 10�M of the L1-derived H-2Db MHC class I restricted CTL epitope AGVDNRECI[13] (L1165–173) in 10 ml �-modified minimum essentialmedium (Sigma) supplemented with 10% FCS, 4 mM glu-tamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and0.1 mM ß-mercaptoethanol (CTL medium). For long-termc latesa mMm tantf urceo tim-u llsa y)f ell.Ci

2

ter-m enH y)w FNga el-b eteR s)w atesw with0 l orw hi nds FNgs -ga gen)w

aken up by B cells, 1× 106 activated human B cells isolatrom peripheral blood mononuclear cells (described in[34])ere incubated for 1h with varying amounts of CTA1-D2

n medium containing 5% FCS. To remove residual CT2D1, the cells were washed twice with PBS/1% FCS

hen fixed with cold 4% paraformaldehyde (Serva, Heierg, Germany) in PBS for 10 min at 4◦C. Subsequently, thells were washed twice again with PBS/1% FCS andeabilized by a short treatment with saponin buffer (P.1% saponin (Serva), 1% FCS and 0.01 M Hepes (Inven)) for 10 min at ambient temperature. B cells were staith fluorescein-labeled chicken-anti-staphylococcal proantibody (clone WL-9; Immunsystems, Uppsala, Swed

n saponin buffer for 20 min at 4◦C. Cells were washewice with saponin buffer and resuspended in PBS/1 %efore sorting. Cells treated without CTA1-D2D1 were us negative controls. Intracellular immunofluorescenceetermined on a FACSCalibur (Becton Dickinson, Heierg, Germany) using CellQuest software. Typically, 20ells were collected from each sample.

.7. Immunizations and collection of vaginal lavagepecimens

C57BL/6 mice were immunized three times in bi-weentervals intranasally (i.n.) under deep ketamin/romnaesthesia [75 mg/kg Ketamin plus 10 mg/kg Rom

ntraperitoneal]. The L1 capsomere/adjuvant mixturepplied in a maximum volume of 20�l slowly drop by drop

nto one nostril. Mice received either 5�g capsomeres i.

ulture, spleen cell cultures were expanded in 24-well pnd maintained in CTL medium supplemented with 25ethyl-�-mannopyranoside (Sigma) and 5% superna

rom rat spleen cells treated with Concanavalin A as a sof interleukin (IL)-2. Once a week, bulk cultures were reslated with 2× 105 �-irradiated peptide-loaded RMA-S ces stimulators and 5× 106 �-irradiated spleen cells (33 G

rom naıve C57BL/6 mice as syngeneic feeder cells per wultures were grown at 37◦C and 7.5% CO2 in a humified

ncubator.

.9. Interferon-γ (IFNg) Elispot assay

The number of L1-specific CTLs ex vivo was deined by IFNg Elispot assay. Sterile 96-well MultiScreA plates (MAHAS4510, Millipore, Eschborn, Germanere coated overnight with a monoclonal anti-mouse Intibody (0.6�g/well; clone R4-6A2, Pharmingen, Heiderg, Germany) at 4◦C, washed, and blocked with complPMI medium for 2 h at 37◦C. Splenocytes (in triplicateere seeded in serial two-fold dilutions. One of the triplicas left untreated, the others were restimulated either.2�g/well Pokeweed Mitogen (Sigma) as positive controith 0.025�g/well L1165–173CTL epitope peptide. After 24

ncubation at 37◦C and 7.5% CO2, plates were washed aubsequently incubated with an biotinylated anti-mouse Iecondary antibody (0.2�g/well, clone XMG1.2, Pharminen) for 24 h at 4◦C. After washing, 100�l/well streptavidinlkaline phosphatase (diluted 1:500 in PBS; Pharminas added for 2 h at RT. Plates were stained with 75�l/well

K. Dell et al. / Vaccine 24 (2006) 2238–2247 2241

5-bromo-4-chloro-3-indolylphosphate (BCIP/NBT, Sigma)and the spots were counted in an ELISPOT reader (Autoim-mundiagnostik GmbH, Straßberg, Germany). The number ofspecific spots per 106 cells was determined for each mouseby substracting the number of spots after incubation of cellswith medium alone from the number of spots after incubationwith L1-specific peptide.

2.10. 51Chromium (51Cr) release assay

The ability of CTLs to lyse antigen-presenting cells wasanalyzed by51Cr release assay after two restimulations invitro, which corresponds to 12 days of in vitro cultivation.Ten thousand target cells (RMA-S loaded with L1165–173andthe appropriate parental RMA cells) labelled with 50�CiNa2

51CrO4 (Perkin-Elmer, Rodgau, Germany) for 1 h at37◦C were co-incubated with different numbers of effec-tor cells (CTLs) in 200�l complete medium per well ona 96-well round bottom tissue culture plate (Nunc, Wies-baden, Germany). After 4 h incubation at 37◦C, 50�l ofthe supernatant from each well was transferred onto a 96-well LumaPlateTM (Packard Bioscience BV, Groningen,The Netherlands) and air-dried overnight at RT. Releasedradioactivity was measured in a Microbeta counter (Wallac,Turku, Finland). Specific lysis of target cells was calculatedaccording to the formula [(release by CTL− spontaneousr -tl totalr ma)t aluesg

2i

ighta 1V ,p 5%m aw d-i y) in2 sh-i h at3 dase-c am-b sh-i te,5 4.2a sa ini valueoa unes

2.12. Measurement of total and HPV16 L1-specific IgAand IgG antibody contents in vaginal washes by ELISA

The amount of total IgA or IgG in vaginal washes wasdetermined by coating 96-well plates overnight (4◦C) eitherwith 100 ng/well rat anti-mouse IgA monoclonal antibody (inPBS; Becton Dickinson) or with 100 ng/well goat anti-mouseIgG monoclonal antibody (in PBS; Jackson ImmunoRe-search, Hamburg, Germany). After washing and blockingwith MPBS-T, pre-diluted vaginal specimens in MPBS-T(1:10 to 1:640 for IgA- and 1:50 to 1:6400 for IgG-detection)were incubated for 1 h at 37◦C. After washing, vaginalmouse IgA or IgG was detected by incubation for 1 h at37◦C either with a peroxidase-labelled goat anti-mouse IgAsecondary antibody (1:2500 in MPBS-T; KPL, Wedel, Ger-many) or with a horseradish peroxidase-coupled donkeyanti-mouse IgG secondary antibody (1:5000 in MPBS-T;SouthernBiotech, Eching, Germany). ABTS was used as sub-strate (described above). Monoclonal mouse IgA (0.5 mg/ml)or monoclonal mouse IgG (0.5 mg/ml; both Becton Dick-inson) were used as standards. The L1 specific IgA andIgG antibodies were detected by coating the ELISA plateswith VLPs. Vaginal lavage specimens were pre-diluted 1:5in MPBS-T and detected by the appropriate IgA- or IgG-specific secondary antibodies. For comparison, the specificIgA or IgG L1-specific OD values were normalized tot ell(

2

chm d byr ithin5 limi-n paree nor-m ed ton con-t lysis.Da val-i ns aredb

3

ub-l L1c (i.n.)i 1-s art-m veda

elease)/(total release− spontaneous release)]× 100. Sponaneous chromium release was determined by using51Cr-abeled target cells in the absence of effector cells, andelease was determined by adding 2% TritonX-100 (Sigo target cells in the absence of effector cells. Sample vreater than 10% lysis were scored positive.

.11. HPV16 L1-specific serum IgG enzyme-linkedmmunosorbent assay (ELISA)

ELISA plates (Becton Dickinson) were coated overnt 4◦C with 30 ng/well of baculovirus-derived HPV16 LLPs (production described in[35]) in PBS. After washinglates were blocked with milk (PBS + 0.05% Tween +ilk powder [MPBS-T]) for 1 h at 37◦C. Meanwhile, serere pre-diluted in MPBS-T on 96-well non-protein bin

ng PS-microplates (Greiner, Frickenhausen, German-fold dilutions starting from 1:800. After blocking and wa

ng, the plates were incubated with sera dilutions for 17◦C, washed and subsequently incubated with a peroxioupled goat anti-mouse secondary antibody (Dianova, Hurg, Germany) diluted 1:2500 in MPBS-T. After wa

ng, ABTS (1 mg/ml; Sigma) in 100 mM sodium aceta0 mM sodium-dihydrogenphosphate-monohydrate, pHnd freshly added 30% H2O2 (0.4�l/ml) as substrate wadded. Optical density (OD405 nm) was measured after 30 m

ncubation at RT. Titers are presented as the reciprocalf the highest serum dilution which reached an OD405 nmbove 0.03 (= cut-off: mean OD values of pre-immera + 3× S.E.M.).

405 nmhe total amount (ng) of total IgA or IgG added per wOD405 nm/ng).

.13. Data normalization and statistical analysis

The total and L1-specific mucosal IgA or IgG of eaouse collected on 5 consecutive days was normalize

eferring them to the animal’s lowest value measured wdays in order to flatten cycle stage dependencies. To eate differences between members of a group and to comach experimental group with the control groups, medianalized values of each experimental mouse were referrormalized median values of each of the appropriate

rol mice. These data were subjected to statistical anaifferences were considered significant whenp-values <0.05pplying the Kolmogorov–Smirnov test, and were further

dated by the unpaired Student’st-test. Reciprocal mediaerum antibody levels of the different groups were compy unpaired Student’st-test.

. Results

This study represents the continuation of previously pished work evaluating the immunogenicity of HPV16apsomeres following subcutaneous (s.c.) or intranasalmmunization of mice[13]. Here we intended to improve Lpecific antibody levels especially in the mucosal compents of the body by using two cholera toxin (CT)-deridjuvants, i.e. CTA1-D2D1 and CTB.

2242 K. Dell et al. / Vaccine 24 (2006) 2238–2247

Fig. 1. Characterization and uptake of CTA1-D2D1 by B cells. (A) Structure of the CTA1-D2D1 fusion gene. (B) Expression and purification of the CTA1-D2D1fusion protein. Recombinant CTA1-D2D1 protein was assayed by SDS-Polyacrylamid gel electrophoresis and Coomassie staining. Lanes: 1, 9: protein sizestandards; 2, 3, 4: total bacterial cell lysates before (2) and after (3, 4) induction with IPTG; 5, 6, 7, 8: different fractions collected after IgG-Sepharose columnchromatography. (C) Intracellular FACS staining. FACS staining profiles of B cells are shown after incubation with 0, 10, or 100�g CTA1-D2D1 protein,permeabilization and staining using a fluorescein-labelled chicken-anti-staphylococcal protein A antibody. Positive cells are presented as percentage of gatedcells.

3.1. Characterization of CTA1-D2D1 and uptake by Bcells

CTA1-D2D1 was generated similar to the previously pub-lished CTA1-DD[22]. The difference between CTA1-DDand CTA1-D2D1 consists in the spacing of the two D-domains ofS. aureus protein A. The two amino acid insertion(PE) in CTA1-DD is lacking in both the individual Ig bindingdomains of CTA1-D2D1 and the native protein A ofS. aureus(Fig. 1A). The CTA1-D2D1 protein displays all of the pre-dicted properties with regard to molecular size (Fig. 1B) anduptake by B cells (Fig. 1C). Uptake rates in 106 B cells wereshown to increase arithmetically from 37 to 77% in the pres-ence of 5–100�g CTA1-D2D1 (Fig. 1C and data not shown).We conclude from these data that CTA1-D2D1 exhibits sim-ilar properties as described for CTA1-DD[22,36,37].

3.2. CTA1-D2D1 enhances L1-specific serum IgG levelsafter intranasal immunization in a dose-dependentmanner

Mice (3 per group) were immunized three timesintranasally (i.n.) either with 5�g capsomeres in combina-tion with 5�g CTB, 5�g or 10�g CTA1-D2D1. Controlmice received either 5�g capsomeres i.n. or s.c. alone or 5�gCTA1-D2D1 i.n. alone (n = 2). The data are summarized in

Table 1. Immunization via the intranasal route in the presenceof 10�g CTA1-D2D1 showed the highest L1-specific serumIgG-levels (reciprocal titers: 80,000/80,000/51,200 versus51,200/51,200/6400 in mice immunized i.n. with capsomeresalone). In contrast, the use of 5�g CTA1-D2D1 or CTBtogether with antigen was similar to i.n. immunization withcapsomeres alone (each group: 51,200/51,200/25,600). Ele-vated serum antibody levels were obtained in all i.n. immu-nized groups when compared to the group after s.c. immu-nization (25,600/12,800/12,800;Table 1). In the presence ofadjuvant, this difference reached statistical significance.

3.3. CTA1-D2D1 or CTB i.n. significantly increasesL1-specific IgA levels in vaginal secretions

Following immunization, vaginal specimen samples werecollected on 5 consecutive days prior to splenectomy (seeSection2). Recruitment of several vaginal lavage specimensspanning one estrous cycle is important since Ig contentsin mucosal secretions are highly variable during this period[19], which was confirmed in this study both for IgA and IgG(Fig. 2A and B andFig. 3A and B).

As shown inFigs. 2A and 3A, the vaginal secretionscontained significantly more total IgG than total IgA through-out the estrous cycle. Intranasal immunizations seemed toenhance the total vaginal IgA levels when compared to the

K. Dell et al. / Vaccine 24 (2006) 2238–2247 2243

Table 1Reciprocal median anti L1-specific serum IgG titers of mice after three times vaccination either with 5�g capsomeres intranasally in combination with adjuvantsas indicated (n = 3), with capsomeres i.n. (n = 3) or s.c (n = 3) alone or with 5�g CTA1-D2D1 i.n. alone (n = 2)

Antigen Adjuvant Mode of Application Median reciprocal titer (±S.E.M) Significance vs. capsomeresi.n. (vs. capsomeres s.c.)

Capsomeres 5�g CTB i.n. 51,200 0.36(±8533) (0.028)

Capsomeres 5�g CTA1-D2D1 i.n. 51,200 0.36(±8533) (0.028)

Capsomeres 10�g CTA1-D2D1 i.n. 80,000 0.06(±9600) (0.004)

Capsomeres – i.n. 51,200 –(±14933) (0.14)

Capsomeres – s.c. 12,800 0.14(±4267) –

– 5�g CTA1-D2D1 i.n. 0 0.07(0.03)

Levels of significance between the groups immunized with or without adjuvants were evaluated byt-test (p-values <0.05 were considered significant; in bold).All but the adjuvant only group (n = 2) encompassed 3 mice.

Fig. 2. Total IgA (A) and normalized anti L1-specific IgA (B) contents invaginal lavage specimens collected on 5 consecutive days prior to splenec-tomy determined by ELISA. Mice were immunized three times intranasallyeither with 5�g capsomeres in the presence of adjuvants as indicated, with5�g capsomeres i.n or s.c alone or with 5�g CTA1-D2D1 i.n. (A) For mea-suring the total IgA content [ng/ml], samples were pre-diluted 1:10 to 1:640and total IgA was calculated by using an IgA standard. Results of individualmice are shown. (B) The L1-specific IgA OD405 nmvalues were normalizedto the amount of total IgA [ng] added per well (OD405 nm/ng IgA). Resultsof individual mice are shown. Significantly increased immunoglobulin lev-els between the groups are indicated as follows: # vs. non-immunized mice(=CTA1-D2D1 i.n.), * vs. capsomeres i.n. and◦ vs. capsomeres s.c. Statis-tical analysis was performed by Kolmogorov–Smirnov test (see statistics).p-values <0.05 were considered significant. Caps., capsomeres.

Fig. 3. Total IgG (A) and normalized anti L1-specific IgG (B) contents invaginal lavage specimens collected on 5 consecutive days prior to splenec-tomy determined by ELISA. Mice were immunized three times intranasallyeither with 5�g capsomeres in the presence of adjuvants as indicated, with5�g capsomeres i.n or s.c alone or with 5�g CTA1-D2D1 i.n. (A) For mea-suring the total IgG content [ng/ml], samples were pre-diluted from 1:50 to1:6400 and total IgG was calculated by using an IgG standard. Results ofindividual mice are shown. (B) The L1-specific IgG OD405 nm values werenormalized to the amount of total IgG [ng] added per well (OD405 nm/ngIgG). Results of individual mice are shown. Caps., capsomeres.

2244 K. Dell et al. / Vaccine 24 (2006) 2238–2247

s.c. immunized mice, but this difference was statisticallynot significant (Fig. 2B). Any kind of application of cap-someres increased the total amount of IgG in the vaginalsecretions in a statistical significant manner (Fig. 3A). Co-application of adjuvants (i.n.) did not increase the total IgAor IgG (Fig. 2A and 3A).

In order to compare the intra- and inter-individual L1-specific IgA and IgG antibody levels, we determined thetiters in vaginal secretions and normalized the values ofeach mouse to the total amount [ng] of the respective iso-type. Only intranasal immunization induced L1-specific IgAin vaginal washings (Fig. 2B) whereas vaginal L1-specificIgG was obtained independent of the route of immuniza-tion (Fig. 3B). The addition of adjuvant in i.n. immuniza-tion significantly increased vaginal L1-specific IgA but notIgG (Fig. 2B and 3B). Compared to the mice who hadreceived capsomeres s.c., there was no elevated L1-specificIgG after i.n. immunization even in the presence of adju-

vants (Fig. 3B). Interestingly, the L1-specific fraction ofvaginal IgA was much higher than the specific amount ofIgG (Figs. 2B and 3B).

3.4. Immunization (i.n.) in the presence of CTA1-D2D1increases L1-specific cytotoxic T lymphocyte frequency

We measured the in vivo priming of HPV16 L1-specificcytotoxic T cells (CTLs) by IFNg-Elispot and51Cr releaseassay. The results of the ex vivo Elispot assay are shownin Fig. 4A. Intranasal immunization with capsomeres alonereached L1-CTL numbers slightly above the ones obtainedafter subcutaneous immunization. The highest L1-specificCTL frequency was measured when antigen was combinedwith 5�g or 10�g CTA1-D2D1. Co-application of CTB alsoincreased the number of L1-CTLs, but to a lesser extent.

Functionality of the generated L1-CTL was investigatedafter two rounds of in vitro restimulation (Fig. 4B). Com-

FwCswe

ig. 4. L1-specific CTL responses of spenocytes ex vivo (A) and after two rith 5�g capsomeres in combination with adjuvants as indicated, with capsoTL ex vivo. Splenocytes were assayed by IFNg Elispot. Cells were restimulpot counts per million cells of each mouse (median values± S.E.M. are shownere assayed by51Cr release assay. CTL were co-cultured with L1-CTL restrffector to target ratios. Results of individual mice are shown. Caps., capsom

ounds of in vitro restimulation (B). Mice were immunized three times i.n. eithermeres i.n. or s.c. alone, or with 5�g CTA1-D2D1 i.n. (A) Number of L1-specificated with the L1-CTL restricted epitope. Each grey bar represents the L1-specificas black bars). (B) Cytolytic activity after two in vitro restimulations. Cellsicted epitope-loaded RMA-S cells or with the parental RMA cells at indicatederes.

K. Dell et al. / Vaccine 24 (2006) 2238–2247 2245

pared to intranasal immunization with capsomeres alone,co-administration of CTB and 10�g CTA1-D2D1 increasedthe cytolytic activity in vitro. Only one of three mice showedpositive lytic activity values after co-immunization with 5�gCTA1-D2D1 (27% at an effector to target ratio of 20). In con-trast to the relative low numbers of L1-specific CTL revealedby Elispot ex vivo, lytic activities of the mice immunizedwith capsomeres subcutaneously ranged within the ones ofmice immunized intranasally (Fig. 4B).

We conclude from these data that CTA1-D2D1 has posi-tive immunomodulatory effects on both, the humoral and thecellular immune response.

4. Discussion

Intranasal (i.n.) immunization is the most effective routefor inducing immune responses in the genital tract[8]. Pre-vious studies have shown that virus-like particles (VLPs)[9,15,17] given by this route induce L1-specific neutraliz-ing antibodies within the serum and vaginal secretions ofmice with vaginal specific IgG exerting a higher neutralizingpotential than IgA[17].

Our data confirm earlier published results that s.c. immu-nization induces only antigen-specific IgG whereas induc-tion of vaginal antigen-specific IgA requires i.n. applica-t tionow -t cifics theSC nitop andC f thet sa tivep -l thec rated[ t oni wnt ontp ellsw u-lfi hei fore,w tudyi

ithc L1-s se-q ccine

preparation. In a previous study[13] we determined the endo-toxin content as 0.002 ng/�g L1 as measured by the Limulusassay. The endotoxin activity ofE. coli-derived CTA1-DDpreparations corresponded to <5 ng of LPS/�g of protein[37]. Thus, the total dose is at least 5 times less than theamount required to exert a significant augmenting effect onantigen-specific serum IgG levels[21].

CTA1-D2D1 also exerted an adjuvant effect on total andL1-specific vaginal Ig. Vaginal washings from the mice weretaken on 5 consecutive days in order to cover a completeestrous cycle. In rodents and humans both total and spe-cific daily IgA and IgG contents vary, since the amount ofimmunoglobulins in genital secretions are influenced by sexhormones[19]. In our study, the amount of total IgG exceededIgA by 10–30 fold, which is in contrast to published data[19]revealing a dominance of IgA in the mice. Because of the dif-ferent antibodies used for coating of the ELISA plates in ourand the other study[19] differences in sensitivity are feasible.Our data match results obtained in humans where an ELISAformat identical to ours was used[19]. Both CTA1-D2D1and CTB increased L1-specific IgA but not IgG in vaginalsecretions significantly. In contrast to CTA1-D2D1 the holo-toxin (CT) was shown to elevate both vaginal IgG and IgA[17]. However, CTA-D2D1 has two major advantages whencompared to CT: it is non-toxic and preferentially binds tothe surface of Ig-expressing B cells. Therefore CTA1-D2D1,u ccu-m ausen

ellu-l pre-v uldo tedc ationt ione TBn d tot herL resa mmu-n ands llswi hal-l

asyi ala bi-n

A

siss theH d by

ion [17,38,39]. Others have shown that the co-applicaf cholera toxin (CT) enhanced serum IgG[17,18] asell as vaginal IgA[17,18] and IgG[17]. CTB, the non

oxic binding-subunit of CT also stimulated antigen-speerum IgG and vaginal IgA when mixed with VLPs ofimian Immune Deficiency Virus[40]. Another non-toxicT-derived adjuvant is CTA1-DD where the CTA1-subuf CT is coupled to the two Ig-binding D-domains ofS. aureusrotein A. The basic difference between CTA1-DDTA1-D2D1 (used in our study) consists in the spacing o

wo D-domains ofS. aureus protein A. In CTA1-DD, there itwo amino acid (PE) insertion, which is lacking in the narotein A of S. aureus and in CTA1-D2D1. Immunostimu

ating properties of CTA1-DD on both the humoral andellular arm of the immune system have been demonst20] but there is no information about its adjuvant effecmmunization in combination with HPV particles. It is knohat the immunomodulating properties of CTA1-DD relyhe enzymatic activity of the CTA1-subunit[37]. Whereas inrevious studies binding of CTA-DD to Ig-expressing B cas shown[37] we demonstrated the intracellular accum

ation of CTA-D2D1 within B cells in vitro (Fig. 1C). Thisnding is in line with the apparent activity of CTA1 at tnner leaflet of the target cell plasma membrane. Theree speculate that the adjuvant effect observed in this s

s due to activation of antigen presentation by B cells.In this study we showed that i.n. immunization w

apsomeres in the presence of CTA1-D2D1 increasedpecific serum IgG. This effect is unlikely to be the conuence of endotoxin contamination in the capsomere va

nlike the holotoxin and the CTB subunit, does not aulate in olfactory cells and thus is not expected to ceurotoxic side effects[20,21].

Not only the humoral immune response, but also the car arm of the adaptive immune system may play a part inention of HPV-associated lesions. L1-specific CTL woffer an improved protection by early clearing of infecells as suggested by the published results of the vaccinrials [41,42]. In our study, i.n. L1-capsomere immunizatlicited CTL responses in the spleen. CTA1-D2D1 and Cot only enhanced the number of L1-CTLs but also le

he generation of functional L1-specific CTLs with hig1-specific cytotoxicity than immunization with capsomelone. Others have presented evidence that mucosal iization induces Ag-specific CTLs in both the mucosalystemic compartment[43]. Mucosal antigen-specific T ceere shown to migrate to distinct mucosal tissues[44,45]and

nduce protection against a distinctly localized mucosal cenge of a virus that expresses the relevant CTL epitope[43].

Together, robust and specific immune-stimulation, en vivo delivery and lack of toxicity promise broad clinicpplication of antigen-specific anti-HPV vaccines in comation with cholera-toxin based vaccine adjuvants.

cknowledgements

We are grateful to Dr. I.G. Bravo for statistical analyupport and to Birgit Aengeneyndt for the production ofPV16 L1 capsomeres. pSPA7235 was kindly provide

2246 K. Dell et al. / Vaccine 24 (2006) 2238–2247

Dr. Tim Foster, Trinity College, Dublin, Ireland. pRG-CTwas kindly provided by Dr. Frank H. Burton, University ofMinnesota, Minneapolis, Minnesota.

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