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Vaccine 28 (2010) 5582–5590

Contents lists available at ScienceDirect

Vaccine

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

ublingual immunization with an HIV subunit vaccine induces antibodies andytotoxic T cells in the mouse female genital tract

atherine Hervoueta,b,c,1, Carmelo Lucia,b,c,1, Nicolas Cuburub,c,1, Magali Cremelb,c, Selma Bekria,b,ene Vimeuxd,e, Concepcion Maranond,e, Cecil Czerkinskyc,f, Anne Hosmalind,e, Fabienne Anjuèrea,b,c,∗

Inserm, U634, Faculté de Médecine Pasteur, 06107 Nice cedex 2, FranceUniversity of Nice-Sophia Antipolis, 06 107 Nice cedex 2, FranceInserm, U721, Faculté de Médecine Pasteur, 06107 Nice cedex 2, FranceInstitut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, FranceInserm, U1016, Paris, FranceInternational Vaccine Institute, Seoul 151-818, South Korea

r t i c l e i n f o

rticle history:eceived 23 November 2009

a b s t r a c t

A vaccine against heterosexual transmission by human immunodeficiency virus (HIV) should generatecytotoxic and antibody responses in the female genital tract and in extra-genital organs. We report that

eceived in revised form 6 May 2010ccepted 10 June 2010vailable online 25 June 2010

eywords:

sublingual immunization with HIV-1 gp41 and a reverse transcriptase polypeptide coupled to the choleratoxin B subunit (CTB) induced gp41-specific IgA antibodies and antibody-secreting cells, as well as reversetranscriptase-specific CD8 T cells in the genital mucosa, contrary to intradermal immunization. Conjuga-tion of the reverse transcriptase peptide to CTB favored its cross-presentation by human dendritic cellsto a T cell line from an HIV+ patient. Sublingual vaccination could represent a promising vaccine strategy

smis

ublingual vaccinationenital immunityIV

against heterosexual tran

. Introduction

The genital mucosa constitutes a major entry site for sexuallyiral transmitted infections such as those due to herpes simplexirus, human papilloma virus (HPV), and human immunodeficiencyirus (HIV). Administration of an antigen by different mucosaloutes has a broader impact on adaptative immune responseshan by systemic injection. Indeed, mucosal immunization cannduce local and loco-regional production of secretory antibodiesIgA subtypes) contrary to systemic immunization. Furthermore,

ucosal immunization can also induce systemic antibody and cel-ular immune responses as well as mucosal cytotoxic T CD8 cellesponses [1–4]. It is now widely accepted that the generationf mucosal antibody and cellular responses by vaccination at aotential mucosal site of microbial entry might greatly reduce theransmission of mucosal pathogens [5,6].

Heterosexual infection is a major cause of HIV transmission7,8]. HIV-specific antibodies, including secretory IgA and IgG, areresent in the cervicovaginal secretions of seropositive patientsnd some of them were shown to block HIV transcytosis through

∗ Corresponding author at: Inserm, U634, Faculté de Médecine Pasteur, 06107ice cedex 2, France. Tel.: +33 493 37 77 93; fax: +33 493 37 77 52.

E-mail address: anjuere@unice.fr (F. Anjuère).1 These authors equally contributed to this work.

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.06.033

sion of HIV-1.© 2010 Elsevier Ltd. All rights reserved.

an artificial epithelium [9–12]. Antiviral cytotoxic T cells weredetected in the vaginal mucosa of female macaques after intravagi-nal infection by SIV strains [13]. Furthermore, HIV-specific T CD8responses are present in cervicovaginal samples from HIV-exposedand non-infected female sex workers [14–16]. Altogether, theseobservations suggest that B and T cell immune responses locatedin the female genital tract might play a protective role against het-erosexual transmission of HIV-1. Thus, an efficient vaccine shouldbe able to induce antibodies and cytotoxic CD8 T cells (CTLs) in thegenital tract to control infection and to block genital transmissionof HIV-1.

Local intravaginal immunization was shown to induce geni-tal antibodies and CTLs in mice and humans but its effectivenessappears to be markedly influenced by the host’s hormonal sta-tus [17–19]. The nasal mucosa constitutes an alternative potentroute of immunization for inducing systemic and genital anti-body and cellular responses [17,20,21]. Nasal immunization witha non-replicative SIV antigen together with a strong mucosaladjuvant was shown to induce B and T cell responses in the gen-ital tract of female macaques [22]. Furthermore, the nasal routealso constitutes a potent site of immunization to induce antiviral

immunity against mucosal infection by influenza virus [23]. Inter-estingly, sublingual immunization was recently shown to be aseffective but comparatively safer than the nasal route in a mouseinfluenza model of infection [24]. Moreover, sublingual administra-tion of ovalbumin (OVA), used as model antigen, together with the

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ucosal adjuvant cholera toxin (CT) induces CTLs and secretoryntibodies in the genital tract, as efficiently as local intravaginalmmunization [19,25].

Mucosal immunization with non-replicative antigens requireshe use of adjuvants and delivery systems in order to break mucosalolerance and to facilitate uptake of immunogens through epithe-iae [26,27]. The AB5 bacterial toxin CT is one of the most potent

ucosal adjuvant [28]. The pentameric B subunit of cholera toxinCTB) is responsible for binding of the toxin to GM1 gangliosides,hich are expressed by all nucleated cells. CTB, which can be pro-uced independently of the CTA moiety in a stable pentameric form,onstitutes a good antigen-delivery vector for different types ofovalently linked proteins and polysaccharides [28]. Interestingly,e recently demonstrated that CTB favored the cross-presentation

f murine MHC class I-restricted epitopes by dendritic cells (DCs) toD8 T cells, increasing the priming of CD8 T cells after intravaginal

mmunization [19].We now report that sublingual administration of HIV-1 antigens,

ncluding the HXB2 gp41 ectodomain and a reverse transcrip-ase peptidic sequence covalently coupled to CTB, co-administeredith CT adjuvant, induces anti-gp41 IgG and IgA antibodies in

enital secretions as well as reverse transcriptase-specific IFN-�-roducing CD8 T cells and CTLs in the genital tract.

. Material and methods

.1. Mice

Six- to eight-week-old female BALB/c mice were purchased fromharles River Laboratories (L’Arbresle, France) and HLA-A2 trans-enic mice (HHD-2 mice) on C57BL/6 background were a kindift from Dr F. Lemonnier, Institut Pasteur, Paris) [29]. All miceere maintained in mouse pathogen-free conditions at the ani-al facilities of the Faculty of Medicine (Nice, France), according to

nstitutional guidelines and all experiments were approved by theocal committee for animal experimentation.

.2. Antigen preparation

CT was obtained from List Biologicals Laboratories Inc. (USA).MP grade recombinant CTB was obtained from SBL Vaccine (Swe-en). An HXB2-derived gp41452-682 polypeptide was obtained fromebu-Bio Laboratories (Le Perray en Yvelines, France). Reverseranscriptase 33-mer peptide (Pol463-495, called Pol: TEEAELE-AENREILKEPVHGVYYDPSKDLIAE), a protein sequence from therototypic HXB2 HIV isolate obtained from the NIH databasewww.hiv.lanl.gov), was synthesized by Polypeptid Group (Stras-ourg, France). A cystein amino acid residue was added at themino-terminal end of the Pol463-495 sequence in order to allowts covalent conjugation to CTB through a disulfide bridge. Peptidicequences were conjugated to CTB using LC-SPDP (N-succinimidyl--(2-pyridyldithio)-propionate) (Thermo Fisher Scientific Inc.,SA) as described earlier [30]. The ability of resulting CTB con-

ugates to bind GM1 was checked with a solid phase ELISA usingM1 ganglioside as capture system and antibodies to CTB as detec-

ion reagents [31]. LPS contamination of the different preparationsas below 0.12 ng/mL as determined using the Limulus Amebocyte

ysate Pyrogent® Plus (Lonza, Switzerland).

.3. Immunizations

Mice were immunized with antigen preparations [gp41 (10 �g)r CTB-Pol (25 �g of Pol) alone or mixed with CT (1 �g)] by differentoutes on days 1, 14 and 21. Mice immunized with CT alone (1 �g),p41 alone (10 �g) or Pol alone (25 �g) and non-immunized miceere used as control groups. For sublingual immunization, mice

8 (2010) 5582–5590 5583

were anaesthetized with pentobarbital and received a 5 �L dose ofantigen in saline delivered with a pipette under the tongue. Anaes-thetized mice were maintained with heads placed in ante flexionfor 30 min in order to avoid leakage of the antigen. For intravagi-nal immunization, mice were given the same doses of the differentantigen preparations in a 20 �L volume. For intradermal immuniza-tion, mice received the same doses of antigens in a 10 �L volume.

2.4. Cell preparation

Mice were sacrificed by cervical dislocation. For B ELISPOT assayand T cell intracellular staining, the genital tract tissue (vaginaand uterus) from 2, 3 or mice was pooled in different experimentsand finely cut before digestion for 1 h at 37 ◦C with 2 mg/mL col-lagenase A (Roche Diagnostics) in RPMI medium (Gibco Europe)supplemented with 0.1 mg/mL DNase 1 (Roche Diagnostics). Geni-tal tract cell suspensions were collected by filtration through a cellstrainer and then, enriched in immune cells by purification onto aPercoll gradient for B ELISPOT assay. Alternatively, for T cell intra-cellular staining, genital tract cell suspensions were enriched inhematopoietic cells by immunomagnetic selection of CD45+ cellsusing a biotinylated anti-CD45 antibody (30F11 clone, BD Bio-sciences) and streptavidin-MACS beads (Miltenyi Biotec) accordingto manufacturer’s recommendations. Cell suspensions from spleenand ileo-sacral lymph nodes (ILN), obtained by pressing the organsthrough nylon sieves were then freed from erythrocytes by treat-ment with ammonium chloride.

2.5. Measurement of specific antibodies

Vaginal washes were collected from anaesthetized mice byflushing three times 50 �L of PBS using a micropipette. Mucosalsamples were clarified by centrifugation and immediately stored at−20 ◦C in presence of protease inhibitors. Blood samples were col-lected from the tail vein and sera were separated by centrifugation,and stored at −20 ◦C. Sera and genital secretions from immunizedmice as well as pre-immune samples were assayed for antibodylevels to gp41 by ELISA using HRP-conjugated affinity-purifiedgoat antibodies to mouse total IgG or IgA (Southern Biotechnol-ogy) as detection reagents as described previously [32]. The titerof a sample was defined as the reciprocal of the highest sampledilution yielding an absorbance value at least equal to 3-fold thatof background [which is the absorbance value of the correspond-ing pre-immune sample at the lowest dilution (1/50 for sera and1/3 for vaginal washes)]. For each group of immunization, resultswere expressed as geometric mean titer (GMT) + Standard Devia-tion (S.D.) or +Standard Error of the Mean (S.E.M.) when data weregenerated from one individual experiment or several individualexperiments, respectively.

2.6. B ELISPOT assay

Organs were collected 8 days after the third immunization andprepared as described above. Spleen, ILN and genital tract cell sus-pensions from pooled organs (between 2 and 4 mice) were assayedfor numbers of specific antibody-secreting cells (ASCs) using a BELISPOT assay [33,34]. Briefly, graded numbers of cells (between105 and 8 × 105 cells per well) were incubated for 12 h at 37 ◦C innitrocellulose-bottom 96-well plates previously coated with gp41(5 �g/mL) for detection of gp41-specific ASCs or with goat antimouse kappa and lambda chains (2 �g/mL) (Southern Biotechnol-

ogy Associates) for detection of total ASCs. Gp41-specific ASCs werepreferentially measured when cell numbers in the cell suspensionswere not sufficient to run all conditions. Plates were then exten-sively washed and antibodies produced were detected by stepwiseincubations with HRP-conjugated goat antibodies to mouse IgG or

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gA (Southern Biotechnology Associates), wash buffer (PBS), andEC-H2O2 chromogenic substrate (Sigma-Aldrich) as previouslyescribed [17]. Spots were counted using an automated reader (CTL

mmunospot, BD Biosciences, Mountain View, CA). Control groupsncluding non-immunized, CT-immunized and gp41-immunized

ice gave only rare spots on gp41-coating (maximal spot numberser million cells ≤3).

.7. IFN-� intracellular staining

Organs or tissues (spleen, ILN or genital tract) were collecteddays after the third immunization and cell suspensions pre-

ared as described in Section 2.4. Specimens from non-immunizedice and mice immunized with Pol alone were used as con-

rol groups. Cell suspensions were assayed for IFN-� productionsing intracellular staining after a 12 h culture of cell suspensions2 × 105/well) with Pol476-84-pulsed syngeneic splenic antigen-resenting cells (APCs) at a T:APC ratio of 2:1 in the presencef brefeldin A according to manufacturer’s recommendations (BDiosciences). Briefly, after in vitro stimulation, cell surface Fc recep-ors were blocked by incubation with purified anti-Fc�RII/III mAbclone 2.4G2) and then, cell suspensions were stained with surfacentibodies including PE-Cy7-conjugated anti-CD8� (Clone 53-6-), PerCP-conjugated anti-CD19 (Clone 1D3) and PE-conjugatedD62L (Clone MEL14). After fixation and permeabilization using aytoFix/Perm kit (BD Biosciences), cells were directly stained withPC-conjugated anti-IFN-� antibody (clone XMG1.2) or an isotypeontrol (PE-conjugated IgG1 rat antibody, clone R3-34). All anti-odies used were from BD Biosciences. Analysis was performed onFACSCanto flow cytometer (BD Biosciences) at the Flow Cytom-

try facility of IFR50 (Faculty of Medicine Pasteur, Nice, France).bsolute numbers per organ were determined by multiplying theumber of cells by the relative percentages of IFN-�+ CD8+ T cellsetermined by flow cytometry analysis.

.8. In vivo T CD8 cytotoxic assay

In vivo cytolysis assays were conducted as described elsewhere35]. Briefly, syngeneic splenocytes were split into two popula-ions and labeled with CFSE at a concentration of 4 �M (CFSEhigh)r 0.4 �M (CFSElow). CFSEhigh cells were pulsed with 1 �M ofhe Pol476-84 epitope (ILKEPVHGV) for 1 h at 37 ◦C, while CFSElow

emained un-pulsed. Mice, anaesthetized by isoflurane inhalation,ere injected with equal numbers (2 × 106 total cells) of the two

plenocyte populations (CFSEhigh pulsed and CFSElow un-pulsed)irectly in the mucosa using an insulin syringe (Becton Dickinson)days after the third immunization. 12–15 h later, genital tractas excised and was successively treated by digestion in dispase II

Roche Diagnostics) for 45 min at 37 ◦C and in collagenase/dispasen RPMI medium supplemented with 0.1 mg/mL DNase I for 1 ht 37 ◦C in order to recover cells for cytolysis analysis by flowytometry. Specific cytolysis activity was calculated as previouslyescribed [36] using the formula: [1 − [(ratio of CFSElow/CFSEhigh

ells in control mice)/(ratio of CFSElow/CFSEhigh cells in immunizedice)] × 100].

.9. Pol476-484 epitope cross-presentation by human HLA-A2+

endritic cells

Monocyte-derived DCs were obtained from HLA-A2+ healthyonors from the French Blood Bank (EFS) within a convention with

NSERM. DCs were pulsed with different antigens used at differentoncentrations during 24 h. After loading, DCs (20 000/well) werextensively washed and used in a 12–16 h IFN-� ELISPOT assaysing a Pol476-484 specific CD8 T cell line (60 000/well) as effec-ors as previously described [37]. The Pol476-484 specific CD8 T cell

8 (2010) 5582–5590

line was generated by using PBMC of HIV+ individuals from cohortstudies with the approval of Cochin Hospital’s ethics committee asdescribed elsewhere [38]. All conditions were tested in triplicate.The stimulation of CD8 T cells by unloaded DCs gave a backgroundlevel of around 50 spots/well. This background level is the oneobserved in the different control groups presented including DCsloaded with Pol463-95, CTB + Pol463-95, CTB alone, Tax11-19.

2.10. Statistical analysis

One-way ANOVA analysis was used to compare experimentalgroups and was followed by pairwise multiple comparisons usinga Tukey’s t test or Dunn’s test. A p value <0.05 was considered sig-nificant. All statistic calculations were computed with SigmaStatsoftware (SPSS).

3. Results

3.1. Sublingual immunization with HIV-1 gp41 ectodomaininduced strong IgA and IgG responses in genital secretions

We first evaluated whether sublingual immunization with theHIV-1 gp41 ectodomain (gp41452-682) alone or mixed with CT adju-vant induced gp41-specific antibodies. We compared the intensityof IgG and IgA gp41-specific antibodies induced in blood and geni-tal secretions after immunization by different routes. As shown inFig. 1A and C, sublingual administration of gp41 and CT inducedserum and genital anti-gp41 IgG antibodies with titers compara-ble to those seen after intravaginal or intradermal immunizations.Furthermore, mice immunized sublingually with gp41 and CTgave serum gp41-specific IgA responses with titers almost 25-fold higher than those observed in intradermally immunized mice(Fig. 1B). Interestingly, sublingual immunization induced anti-gp41IgA antibodies in genital secretions with titers comparable to thoseseen after intravaginal immunization, while intradermal immu-nization induced only negligible IgA responses in genital tract[geometric mean titer (GMT) = 970 ± 450, range 81–6561, n = 19and GMT= 4 ± 1, range 0–54, n = 7, respectively] (Fig. 1D). Sublin-gual immunization with gp41 in the absence of CT adjuvant failedto induce gp41-specific IgG and IgA (gp41 SL group, Fig. 1), point-ing to the need to use a mucosal adjuvant in combination withsuch non-replicative antigens. Furthermore, control groups includ-ing CT-immunized mice and non-immunized mice did not give anydetectable antibodies directed against gp41 (Fig. 1 and data notshown).

These results indicate that sublingual immunization with gp41mixed with CT adjuvant induces HIV-1 anti-gp41 antibodies includ-ing IgG and IgA isotypes in genital secretions as efficiently aslocal intravaginal immunization whereas intradermal immuniza-tion failed to induce antigen-specific IgA antibodies in the genitaltract.

3.2. Sublingual immunization with HIV-1 gp41 ectodomaininduced IgA and IgG antibody-secreting cells in the genital tract

To further determine to which extent the genital antibodyresponses observed after sublingual immunization resulted fromlocal antibody formation rather than from blood transudation,ELISPOT analyses of genital tract specimens were carried out 8days after the third sublingual immunization with gp41 alone, gp41mixed with CT adjuvant or with CT alone used as a control. Total

and gp41-specific antibody-secreting cells (ASCs) with IgA and IgGisotypes were quantified in the spleen, ILN and genital tract (Fig. 2).Antigen-specific ASCs were detected in mice which received gp41mixed with CT (Fig. 2A and B) whereas total ASCs were presentin all mice (Fig. 2C and D). Of note, gp41-specific ASCs induced in

C. Hervouet et al. / Vaccine 28 (2010) 5582–5590 5585

Fig. 1. Sublingual immunization induces HIV-1 gp41-specific IgG and IgA antibody responses. Anti-gp41 IgG (A and C) and IgA (B and D) were measured in the serum (A andB) and genital secretions (C and D) of BALB/c mice after intravaginal (IVAG), sublingual (SL) or intradermal (ID) immunizations. Mice received 3 consecutive immunizationsof gp41 alone or co-administered with CT on days 1, 14 and 21. Sera and genital secretions were collected 10 days after the last immunization and assayed by ELISA.A s per in linguS CT + g

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ntibody titers are calculated as indicated in material and methods. Antibody titeron-immunized control mice (CTRL, n = 4), mice immunized with gp41 alone by subL (n = 19) or ID (n = 7) routes. Statistical differences (p < 0.05, Dunn’s test) between

he genital tract after sublingual immunization were dominatedy the IgA isotype (Fig. 2A and B). Indeed, sublingual administra-ion of gp41 mixed with CT induced eight times more IgA thangG gp41-specific ASCs (54 ± 14 IgA ASCs versus 7 ± 3 IgG ASCs).

similar, but less marked tendency was observed in the spleen36 ± 7 IgA ASCs versus 19 ± 5 IgG ASCs). In contrast, only rare gp41-pecific IgA and IgG ASCs were detected in ILN, draining the genitalucosa after sublingual immunization, suggesting that the gp41-

pecific ASCs observed in the genital tract were mainly recruitedrom extra-genital lymphoid organs, including sublingual lymphodes.

These results demonstrate that gp41-specific ASCs generatedfter sublingual immunization are recruited in the genital tract,hich suggests that they contribute to the production of the gp41-

pecific antibodies detected in genital secretions.

.3. Sublingual immunization with CTB-Pol conjugate inducedenital IFN-� producing T cells and CTLs

Our previous experiments in the OVA model showed thatonjugation of a peptidic antigen to the CTB improved the pre-entation of MHC class I-restricted epitopes by dendritic cells andncreased cytotoxic CD8 T cell responses after intravaginal immu-

ndividual (symbols) and geometric mean titers (horizontal lines) are indicated foral route (gp41 SL, n = 8), and mice immunized with CT + gp41 either by IVAG (n = 7),p41 immunized groups are indicated in graphs.

nization [19]. Here, we conjugated a 33-amino acid sequence fromthe HIV-1HXB2 reverse transcriptase (Pol) to CTB (Fig. 3A). Thissequence includes a prominent MHC class I HLA-A2-restricted epi-tope (hereafter called Pol476-484). We compared the frequency ofgenital Pol-specific IFN-�-producing CD8 T cells after immuniza-tion of HLA-A2+ transgenic mice by different routes, with CTB-Poland CT adjuvant. CD8 T cell analysis was performed after a stepof T cell in vitro restimulation with Pol476-484-loaded APCs for12 h. Sublingual and intravaginal immunizations induced notablepercentages of IFN-�-producing CD8 T cells in the genital tract(13.6 ± 2.4% and 9.6 ± 0.5% IFN-�-producing CD8 T cells, respec-tively). In contrast, intradermal immunization induced percentagesof IFN-�-producing CD8 T cells in the genital tract comparable tothe non-immunized control group (6.5 ± 0.5% and 6.1 ± 0.1% IFN-�-producing CD8 T cells, respectively, Fig. 3B, upper right panel).Similarly, mice immunized with Pol alone sublingually had fre-quencies and absolute numbers of IFN-�+ CD8 T cells comparableto those obtained in the control group (Fig. 3B), indicating the need

to use a mucosal adjuvant to differentiate Pol-specific CD8 T cellsafter sublingual immunization. Furthermore, sublingual and intrav-aginal immunizations induced substantial numbers of Pol-specificIFN-�+ CD8 T cells in the genital tract contrary to intradermalimmunization. In contrast, the three immunization routes tested

5586 C. Hervouet et al. / Vaccine 28 (2010) 5582–5590

Fig. 2. Sublingual immunization induces gp41-specific antibody-secreting cells in the genital mucosa. BALB/c mice were given CT alone (n = 12), gp41452-682 alone (n = 8) ortogether with CT (n = 19) in saline on days 1, 14 and 21 by sublingual route. The frequency of gp41-specific (A and B) and total (C and D) IgA (A and C) and IgG (B and D)a l tractp f ASCo group

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ntibody-secreting cells (ASCs) in spleen, ileo-sacral lymph nodes (ILN) and genitaer million cells were expressed as individual values (symbols) and as the mean organs. Statistical differences (p < 0.05, Tukey test) between CT, gp41 and CT + gp41

nduced comparable numbers of IFN-� producing CD8 T cells in thepleen (Fig. 3B). In ILN, IFN-� producing CD8 T cells were mainlyetected after local intravaginal immunization (Fig. 3, central pan-ls).

The potential of the sublingual mucosa to generate antigen-pecific cytotoxic T cell responses in the genital tract was thenvaluated using an in vivo cytolytic assay. We determined the fre-uency of cytotoxic Pol-specific CD8 T cell responses after threeublingual administrations of CTB-Pol alone or mixed with CT onays 1, 14 and 21. As a control, we immunized mice with an irrele-ant antigen, here CTB-OVA (ovalbumin) mixed with CT. Sublingualmmunization induced Pol-specific cytotoxic T cells in the gen-tal mucosa (37 ± 5% Pol-specific lysis) (Fig. 4A). No cytolysis ofol-pulsed target cells was observed in mice sublingually immu-ized with CTB-OVA + CT or in control non-immunized mice (Fig. 4And data not shown). In a set of experiments, we determinedhether the mucosal adjuvant CT was needed to induce mucosalTLs after sublingual immunization. As shown in Fig. 4B, a poorol-specific cytolytic activity was observed in the genital tract ofice immunized sublingually with CTB-Pol alone in contrast toice which received CTB-Pol mixed with CT (3 ± 1% and 32 ± 6%

ol-specific lysis, respectively), demonstrating the need to use aucosal adjuvant to elicit mucosal CTLs after sublingual immu-

ization. Furthermore, no Pol-specific cytolysis was obtained afterublingual administration of Pol mixed with CT suggesting that,t least at the antigen concentration used in our experimental set-

(GT) for the different experimental groups was assayed by ELISPOT. ASC numbersnumbers (horizontal lines) per group. Each value corresponds to 2, 3 or 4 pooleds are indicated in graphs.

tings, CTB favored the in vivo delivery of covalently coupled antigen(Fig. 4C).

In order to evaluate whether a vaginal challenge may increasethe cytolytic activity observed in the genital tract, HLA-A2+ trans-genic mice were immunized twice by sublingual route (days 1 and14) and received a vaginal challenge on day 21 with the same anti-gen. No significant increase in specific cytolysis was observed inthe genital tract after vaginal challenge in comparison with micewho had received all three doses sublingually (34 ± 7% and 37 ± 5%Pol-specific lysis, respectively) (Fig. 4A).

These results, altogether, indicate that the sublingual adminis-tration of HIV antigens with a mucosal adjuvant induces specificCTLs as well as gp41-specific IgA antibodies in the genital mucosacontrary to systemic immunization. This suggests that such anapproach is worth evaluating in non-human primates or inhumans.

3.4. Human DCs loaded in vitro with CTB-Pol conjugatecross-presented Pol476-484 to MHC class I-restricted CD8 T cells

We previously showed that CTB favors the cross-presentation

of MHC class I-restricted epitopes in mice when used as mucosaldelivery system [19]. We evaluated whether the HLA-A2-restrictedepitope (Pol476-484) could be efficiently cross-presented by humanHLA-A2+ DCs to a Pol-specific CD8 T cell line generated from periph-eral blood mononuclear cells from an HIV-infected patient. The

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Fig. 3. Sublingual administration of CTB-Pol induces IFN-�-producing CD8 T cells in the genital mucosa. (A) A 33 amino acid long HIV-1 reverse transcriptase peptide(463-495) containing the HLA-A2-restricted epitope Pol476-484 (dotted box), was covalently linked to CTB by its amino-terminal end through a disulfide bridge (CTB-Pol).HLA-A2+ transgenic mice were immunized with Pol or CTB-Pol together with the mucosal adjuvant CT by (IVAG), sublingual (SL) or intradermal (ID) routes on days 1, 14and 21. Eight days after the third immunization, the frequency of CD8+ IFN-�+ cells in spleen, ILN and genital tract (GT) was determined by flow cytometry analysis afterIFN-� intracellular staining performed on cell suspensions stimulated 12 h in vitro with syngeneic APCs loaded with the MHC class I-restricted Pol476-484. Data in B representthe mean percentages (upper panels) and absolute numbers (lower panels) of IFN-�+ CD8+ T cells per organ + S.E.M. for non-immunized mice (CTRL, white bars), for miceimmunized with Pol by sublingual route (Pol SL, grey bars) or for mice immunized with CTB-Pol and CT (black bars) by different routes. Data correspond to 6 mice perexperimental group. Horizontal dashed lines in graphs indicate the limit of non-specific background. Statistical differences between mucosal (SL, IVAG) and ID immunizedmice are indicated in graphs (p < 0.05, Tukey test).

Fig. 4. Sublingual immunization with CTB-Pol induces antigen-specific CTLs in the genital tract. (A) HLA-A2+ transgenic mice were immunized with CTB-Pol (25 �g, blackbars) or CTB-OVA (25 �g, grey bars) together with CT adjuvant (1 �g) given in saline either three times by sublingual route (SL) on days 1, 14 and 21 or twice by sublingualroute (days 1 and 14) and once by intravaginal route (IVAG) on day 21. Seven days after the third immunization, mice received an intravaginal injection of both CFSEhigh-labeled target cells pulsed with Pol476-484 and un-pulsed CFSElow target cells. Fifteen hours later, in vivo specific cytolysis was assessed in the genital tract by measuring therelative percentages of CFSEhigh and CFSElow target cells by flow cytometry analysis. Bars represent the mean percentages (+S.E.M.) of specific cytolysis calculated as indicatedin material and methods for CTB-Pol immunized group (black bars) and CTB-OVA immunized group (grey bars) (n = 8 mice from two independent experiments). (B) Micewere immunized three times by sublingual administration of CTB-Pol alone or mixed with CT and the Pol-specific cytolytic activity in the genital tract was measured. Barsrepresent the mean percentages (+S.D.) of specific cytolysis (n = 4 mice per group). (C) Mice were immunized three times by sublingual administration of Pol or CTB-Pol mixedwith CT and the Pol-specific cytolytic activity in the genital tract was measured. Bars represent the mean percentages (+S.D.) of specific cytolysis (n = 3 mice per group).

5588 C. Hervouet et al. / Vaccine 2

Fig. 5. CTB delivery vector favors the DC cross-presentation of the MHC class IHLA-A2-restricted epitope Pol476-484 from a 33-amino acid transcriptase peptidicsequence. Human HLA-A2+ dendritic cells (DCs) generated from circulating mono-cytes of healthy donors were incubated with the reverse transcriptase MHC class IHLA-A2-restricted peptide Pol476-84, or with Pol463-495 covalently linked to CTB (CTB-Pol463-495) or mixed with CTB (Pol463-495 + CTB), or with Pol463-495 alone, or with CTBalone, or with an irrelevant MHC class I HLA-A2-restricted peptide HTLV-1 Tax11-19

(Tax11-19). The antigen-loaded DCs were then tested for antigen presentation using aCD8+ T cell line specific for the MHC class I Pol476-484 epitope at a DC:effector ratio of1:3 in a 12 h IFN-� ELISPOT assay. Data represent IFN-� spots per well ± S.D. for thedcd

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jugated to CTB induces potent antibodies and CTL responses in

ifferent peptide preparations at a concentration of 4 �M (A) or at different peptideoncentrations (B). Data are representative of 3 independent experiments. Asterisksenote statistical differences between CTB-Pol463-95 (*) or Pol476-84 (**) and controls.

ross-presentation efficacy was assayed in an IFN-� ELISPOT assay.notable activation of the Pol-specific CD8 T cell line, as mea-

ured by increased IFN-� spot numbers, was observed when DCsere pre-incubated with CTB-Pol or Pol476-484 used as a positive

ontrol in comparison with different negative controls includingTB + Pol463-495, Pol463-495 alone or CTB alone. These control groupsave a basal activity independent of the antigen concentrationnd similar to DCs loaded with an irrelevant HLA-A2-restrictedpitope (HTLV-1 Tax11-19) (Fig. 5). Furthermore, the activation ofol-specific CD8 T cells was antigen dose-dependent (Fig. 5B). Theseata indicate that such an HIV antigen-CTB conjugate might be used

n humans to activate human CD8 T cells.

. Discussion

In the present study, we demonstrate that the sublingual immu-ization with a subunit vaccine comprising HIV-1 gp41 and a

everse transcriptase Pol polypeptide sequence induced gp41-pecific IgG and IgA antibody responses which disseminate in theenital tract. Furthermore, we also provide evidence of the pres-nce in the genital mucosa of cytotoxic CD8 T cell responses specific

8 (2010) 5582–5590

for an HLA-A2-restricted epitope comprised in the Pol polypep-tide.

In an attempt to develop effective mucosal vaccinationstrategies against genital infections and in particular against het-erosexual infection by HIV-1, the choice of the most appropriateroute of immunization is important due to the fact that the mucosalimmune system is known to be compartimentalized [5,39]. Theintravaginal and nasal routes of vaccination have proven to beeffective for inducing specific immunity in the genital tract, and insome cases protection against a genital infection [6]. We previouslyreported that similarly to intranasal immunization, sublingualimmunization with a non-replicating model antigen combinedwith CT adjuvant induced mucosal antibodies and CTL in the sys-temic compartment, in the upper aerodigestive tract and in thelung mucosa [21]. We demonstrate here the potential of the sub-lingual immunization to generate HIV-specific secretory antibodiesand cytotoxic T cells in the reproductive tract mucosa, suggestingthat this approach might be successful to limit the genital trans-mission of HIV-1 during heterosexual intercourse. In comparisonto the intranasal route, the sublingual immunization offers theadvantage to limit the risk of antigen passage through the brainbarrier, which led to the recent withdrawal of an influenza vaccine[40].

Even if the relative roles of mucosal IgG and IgA for providingprotective immunity through HIV neutralization in the genital tractis not completely established, it is generally considered that thepresence in genital secretions of specific neutralizing antibodiesof both isotypes might be beneficial to control genital HIV trans-mission [10,11,41]. Our data indicate that sublingual immunizationinduces IgA and IgG anti-HIV antibodies in the genital tract contraryto systemic immunization. Our results are consistent with thosefrom a recent report comparing nasal and systemic immunizationwith another HIV subunit vaccine, which showed that the mucosalroute of immunization was important to induce mucosal anti-HIVantibodies [42]. Two injectable vaccines (Cervarix® and Gardasil®),which induce protection against genital HPV infection through gen-ital transudation of IgG antibodies derived from the circulation,have been recently registered. The present study demonstrates thatsublingual immunization induced HIV-specific IgG antibodies ingenital secretions as intradermal immunization, but in additionHIV-specific IgA antibodies and CTLs, two types of responses whichmight play a critical role against genital transmission of HIV-1.Immune responsiveness in the reproductive tract is under controlof sex hormones [18,43] which are known to influence the amountof mucosal antibodies present in vaginal secretions, especially afterlocal intravaginal immunization. The observation that sublingualimmunization is less dependent on sex hormones than intravaginalimmunization (Cremel M, Anjuère F, unpublished data), might offeran advantage to the sublingual route to deliver vaccines targetinggenital pathogens.

The gp41 protein contains two B epitopes known to be the tar-gets of the broadly neutralizing antibodies 4E10 and 2F5 [44,45].Furthermore, a recent report indicates the presence of neutralizingantibodies of unknown specificity directed against gp41 in patientsinfected by HIV-1 clades B and C, suggesting that gp41 constitutesa major target antigen for cross-neutralizing antibodies [46]. Theability of sublingual immunization to produce neutralizing anti-gp41 IgG and IgA antibodies in cervicovaginal secretions might becritical to limit genital HIV transmission. This issue is now beingaddressed in cynomolgus macaques.

Sublingual immunization with HIV antigens alone and con-

the genital tract, respectively, when co-administered with smallamounts of the mucosal adjuvant CT. This observation confirmsprevious reports demonstrating that the generation of HIV-specificantibody and/or cellular immune responses in mucosal tissues after

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ucosal immunization (oral, nasal) with non-replicative HIV anti-ens was dependent on CT co-administration [22,47].

CTB was shown to be an efficient mucosal carrier-deliveryystem [5], able to promote MHC class I cross-presentation ofxogenous antigens by murine DCs [19]. We show here that theonjugation of an HIV antigen containing an HLA-A2 epitope toTB favors its cross-presentation by human DCs (Fig. 5). Impor-antly, such CTB-antigen conjugates are better than free antigens tonduce genital cytotoxic antigen-specific CD8 T cells after intravagi-al immunization [19]. We confirm here this observation showinghat CTB-Pol is efficient to differentiate Pol-specific CD8 T cells afterublingual immunization in presence of the CT mucosal adjuvantontrary to the non-conjugated Pol polypeptide (Figs. 3 and 4). Nev-rtheless, we cannot exclude that free Pol polypeptide used at aighest concentration would be able to prime CD8 T cells. Alto-ether, these data indicate that such HIV-CTB conjugates may beuitable for mucosal vaccination in humans.

CTB constitutes a good mucosal antigen-delivery vector in vivoo generate T CD8 responses when administered by sublingualFigs. 3 and 4) or by intravaginal routes, due to its ability to targeto-linked antigens to GM1 gangliosides expressed by all nucleatedells and, consequently to reduce antigen leakage [19]. Further-ore, CTB favors the presentation by dendritic cells of T CD8

pitopes in the MHC class I pathway (Fig. 5 and [19]) by a molec-lar mechanism involving retrograde transport and proteasome19]. Nevertheless, CTB is not sufficient in vivo to efficiently primeytotoxic CD8 T cells. CD8 T cell generation needs the additionf the mucosal adjuvant CT (Fig. 5B). CT is known to be able toransiently recruit potent antigen-presenting cells, namely den-ritic cells, at the site of mucosal immunization by a mechanismependent of the CCL20 chemokine, and then to induce the appro-riate maturation of dendritic cells migrating in the draining lymphodes [21,48,49]. These immunostimulatory properties are nothared by the non-toxic B subunit used in our experimental settings21,48].

The generation of B and T cell responses in the genital tract afterublingual vaccination needs the use of CT adjuvant which is toxic,recluding its use in humans. In future developments, CT shoulde substituted by adjuvants usable in humans. Certain adjuvantserived from CT might be good candidates. Among them, we canention the CTA1-DD adjuvant recently described to be efficient to

nduce mucosal anti-Env antibodies after intranasal immunization42]. Furthermore, a recently designed adjuvant derived from CTalled TCTA1 is not toxic and was successful to induce mucosal anti-odies in the lungs after intranasal and sublingual immunizationsDr M. Song, IVI, Korea, personal communication). This adjuvant isow under investigation in our laboratory.

In summary, sublingual immunization with appropriately for-ulated HIV antigens induces B and T cell immune responses

omprising secretory antibodies and cytotoxic CD8 T cells in theouse genital tract. These findings underscore the potential of

he sublingual mucosa to serve as a potent route of vaccine deliv-ry for inducing protective genital antibody and cellular immuneesponses able to limit the genital transmission of HIV-1.

cknowledgements

We thank Dr Déborah Rousseau (INSERM U895, Nice, France),hierry Juhel (INSERM U634, Nice, France) and Anne-Claire RipocheInstitut Cochin, Paris, France) for expert technical assistance. These

tudies were supported by Institut National de la Santé et de laecherche Médicale (France), the Agence Nationale de Rechercheur le SIDA (France), the SIDACTION (France), the Association Faireace au SIDA (France) and the Association de Recherche sur le Can-er (ARC).

[

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