Vaccine 22 (2004) 3274–3284

The artificial antimicrobial peptide KLKLLLLLKLK inducespredominantly a TH2-type immune response to co-injected antigens

Jörg H. Fritza,∗, Sylvia Brunnera,1, Max L. Birnstiela,2, Michael Buschlea,3,Alexander v. Gabaina,4, Frank Mattnerb,1, Wolfgang Zaunera,5

a Intercell AG, Campus Vienna Biocenter 6, 1030 Vienna, Austriab Institute for Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9/4, 1030 Vienna, Austria

Received 16 October 2003; received in revised form 26 February 2004; accepted 5 March 2004

Available online 9 April 2004

Abstract

Cationic antimicrobial peptides (CAMPs) are active defence components of the innate immune system. Several artificial CAMPs havebeen designed as antibiotic peptide therapeutics, but none have been reported to exert adjuvant activity in animal models. Here we show forthe first time that an artificial CAMP, KLKLLLLLKLK (KLKL5KLK), is a potent inducer of adaptive immunity to co-injected antigens invivo. High levels of antigen-specific antibodies were obtained after co-injection of KLKL5KLK with the model antigen ovalbumin (OVA) ora commercially available influenza vaccine. We show that KLKL5KLK induces a sustained immune response with a prevalent TH2 profilewhen co-injected with proteinaceous and peptide-based antigens. Furthermore, the immuno-enhancing activity of peptide KLKL5KLK wasretained when C-terminally amidated or synthesised as retro-all-d-peptide. We provide evidence that KLKL5KLK enhances the associationof antigen to antigen-presenting cells and forms a depot of antigen at the site of injection, making it an interesting adjuvant for novel vaccinedesign.© 2004 Elsevier Ltd. All rights reserved.

Keywords:Innate immunity; Vaccination; Depot of antigen

1. Introduction

Antimicrobial peptides as part of the innate immunesystem are ancient responses evolved to fend off a widerange of microbes. Numerous naturally occurring peptideantibiotics have been characterised[1–4], including a largegroup of cationic antimicrobial peptides (CAMPs,[5]).CAMPs are mainly found at anatomical sites exposed to

Abbreviations:7-AAD, 7-amino-actinomycin D; Ag, antigen; Alum, aluminiumhydroxide; CAMPs, cationic antimicrobial peptides; CRT, calreticulin;DMF, dimethylformamide; ELISA, enzyme-linked immunosorbent assay; ELIspot, enzyme-linked immunospot; FACS, fluorescence-activated cell sort-ing; HI, haemagglutination inhibition; HNP, human neutrophil-derived�-defensin; IFN, interferon; IL, interleukin; MALDI-TOF, matrix-assisted laserdesorption/ionisation-time of flight mass spectrometry; MBL, mannose binding lectin; MFI, mean fluorescence intensity; MHC, major histocompatibilitycomplex; OD, optical density; ODN, oligodeoxynucleotide; OVA, ovalbumin; PMA, phorbol 12-myristate 13-acetate; RP-HPLC, reversed phase highperformance liquid chromatography; SFX, fluorescein-succinimidylester; TAMRA, tetramethylrhodamine

∗ Corresponding author. Tel.:+43-1-20620-311; fax:+43-1-20620-800.E-mail addresses:joerghfritz@hotmail.com (J.H. Fritz), sylvia.brunner@affiris.com (S. Brunner), max.birnstiel@bluewin.ch (M.L. Birnstiel),

mbuschle@intercell.com (M. Buschle), agabain@intercell.com (A.v. Gabain), frank.mattner@univie.ac.at, frank.mattner@affiris.com (F. Mattner),wzauner@intercell.com (W. Zauner).

1 Present address: AFFIRIS GmbH, Campus Vienna Biocenter 2, Viehmarktgasse 2a, 1030 Vienna, Austria. Tel.:+43-1-4277-54632;fax: +43-1-4277-9546.

2 Tel.: +41-438446591; fax:+41-438446592.3 Tel.: +43-1-20620-105; fax:+43-1-20620-801.4 Tel.: +43-1-20620-101; fax:+ 43-1-20620-801.5 Tel: +43-1-20620-250; fax:+43-1-20620-800.

microbial invasion. They are secreted into internal bodyfluids or stored in cytoplasmic granules of professionalphagocytes and are produced in large quantities at sites ofinfection and inflammation. In mammals, four groups havebeen described:�-defensins,�-defensins,�-defensins andcathelicidins[6–8], all of which are known for their abil-ity to kill or inhibit the growth of a variety of infectiousagents, including bacteria, fungi, viruses and parasites[9].

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2004.03.007

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Beside their microbicidal activity, CAMPs have severalother activities which have an impact on the quality, theeffectiveness, and direction of immune responses under-lining their role in linking innate and adaptive immunity[10]. It became apparent, for example, that certain CAMPsare able to recruit distinct subsets of immune cells[11].Interestingly, chemokines and chemokine-derived peptideshave been shown to have antimicrobial properties, indi-cating a functional overlap of chemokines and CAMPs[12–14].

Recently, immuno-enhancing activities of certain CAMPswere reported. Human neutrophil-derived�-defensins(HNPs) enhance the production of Ag-specific serum im-munoglobulin G (IgG) and the release of interferon-�(IFN-�), interleukin 5 (IL-5), interleukin 6 (IL-6) and in-terleukin 10 (IL-10) by CD4+ T-cells [15,16]. Because oftheir immunopotentiating properties defensins are interest-ing candidates for vaccine design. However, due to threeintramolecular disulfide bonds, the synthesis and purifica-tion of defensins is laborious and expensive.

Beside the work on naturally occurring CAMPs, a largenumber of artificial CAMPs have been designed to de-velop novel microbicidal drugs. Several of these are al-ready in clinical trials [17,18]. An interesting examplefor an artificial CAMP is an undecapeptide originallyderived from sapecin B, but modified to increase its ac-tivity against Staphylococcus aureus[19]. This artificialpeptide, H-KLKLLLLLKLK-NH 2 (KLKL 5KLK-NH 2),exhibits significant chemotherapeutic activity in Staphy-lococcus aureus-infected mice[20]. Furthermore, it hasbeen shown that KLKL5KLK-NH 2 activates human neu-trophils and U937 monocytes to produce superoxide anionsthrough binding to cell surface calreticulin[21,22]. It hasbeen assumed[21,22] that the chemotherapeutic activityof KLKL 5KLK-NH 2 is related to its ability to change theactivation status of neutrophils and monocytes. Thus, weinvestigated if KLKL5KLK-NH 2 can generally influencethe onset of adaptive immunity.

We report here that the artificial antimicrobial pep-tide, KLKLLLLLKLK (KLKL 5KLK), is a potent in-ducer of adaptive immunity to co-injected antigens. Whenco-injected with the model antigen ovalbumin (OVA), allthe three peptides, KLKL5KLK-NH 2 and its derivativesd-KLKL 5KLK-NH 2 and KLKL5KLK-OH, enhanced theproduction of OVA-specific IgG1 antibodies. Ex vivo stimu-lation of spleen cells with OVA revealed increased numbersof IL-4 and IL-5 producing cells indicating a predominantTH2-type polarization profile elicited by KLKL5KLK. Weprovide evidence that KLKL5KLK enhances the associ-ation of antigen to antigen presenting cells and forms adepot of antigen at the site of injection. Furthermore, by ad-ministration of KLKL5KLK together with a commerciallyavailable non-adjuvanted influenza vaccine, we obtainedmarkedly elevated influenza-specific antibody levels andhaemagglutination inhibition titers, as compared to thosewith aluminiumhydroxide (Alum). Thus, the artificial an-

timicrobial peptide KLKL5KLK is a novel adjuvant suitablefor vaccine design.

2. Materials and methods

2.1. Animals

C57BL/6 mice (Harlan Winkelmann, Borchen, Germany)were used at the age of 6–8 weeks. All animal experimentswere performed according to local guidelines.

2.2. Reagents

Peptides were synthesised by solid phase synthesis usingstandard Fmoc chemistry. Crude peptides were purified byRP-HPLC on a C18 column. Identity and purity were con-firmed by mass spectroscopy using a MALDI-TOF massspectrometer Reflex III (Bruker, Leipzig, Germany) andanalytical RP-HPLC. The following peptides were used:OVA257–264 (H-SIINFEKL-OH, H2-Kb [23]), OVA265–280(H-TEWTSSNVMEERKIKV-OH, I-Ab [24]) both derivedfrom chicken OVA, H-KLKLLLLLKLK-OH (KLKL 5-KLK-OH), H-KLKLLLLLKLK-NH 2 (KLKL 5KLK-NH 2)and H-KLKLLLLLKLK-NH 2 synthesised withd-aminoacids (d-KLKL 5KLK-NH 2).

Cy-5 labelled KLKLLLLLKLK-OH (Cy5-KLKL 5KLK-OH): after assembly of the peptide on the resin and removalof the Fmoc-protecting group, the resin was washed withDMF. Reactive dye (15 mg, Cy-5 succinimidyl ester; Amer-sham, Uppsala, Sweden) was dissolved in 0.5 ml DMF andadded to 200 mg of resin (∼50–75�mol peptide). The re-action was allowed to proceed over night at room tempera-ture. Unreacted dye was washed away with DMF. Labeledpeptide was then cleaved from the resin and purified as de-scribed above.

Labelling of OVA with fluorescein (SFX) or tetramethyl-rhodamine (TAMRA) was carried out as described[25].

Cy5-labelled CpG1668 oligodeoxynucleotide (CpG1668-Cy5; 5′-(Cy5)TCCATGACGTTCCTGATGCT-3′, phospho-rothioate backbone) was purchased from Purimex (Göttin-gen, Germany).

For vaccination experiments OVA (Sigma, St. Louis, MO)or the non-adjuvanted trivalent influenza split-vaccine FLU-VIRIN (Evans Vaccines, Liverpool, UK; season 2001/2002)containing inactivated virus surface antigens (15�g haemag-glutinin per strain) of strains: A/New Caledonia/20/99IVR-116 (H1N1), A/Panama/2007/99 RESVIR-17 (H3N2),B/Guangdong/120/00 were used. Aluminiumhydroxide(Alhydrogel 2%) was obtained from Accurate Chemical,Westbury, NY.

2.3. Vaccination of mice

Mice (four per group) were vaccinated subcutaneouslywith a mixture of protein or peptide and adjuvant in a totalvolume of 100�l sorbitol buffer (0.27 M d-sorbitol and

3276 J.H. Fritz et al. / Vaccine 22 (2004) 3274–3284

5 mM Tris pH 7.4) at intervals of 28 days. All componentsof the vaccine tested negative in Limulus Amebocyte LysateAssays (BioWhittaker, Apen, Germany). Fourteen daysafter vaccination, pools of spleen cells were analysed byELIspot assays as described below. Blood was drawn fromtail veins 26 days after each injection and sera of individ-ual mice per group were pooled. The long-term responseof vaccinations with OVA was analysed 109 days after theboost immunisation.

2.4. Analysis of the immune response by ELIspot assay

ELIspot plates (MAHA S4510 for IFN-� and MAIPS4510 for IL-5, Millipore, Molsheim, France) were coatedwith capture antibodies (IFN-�: R4-6A2 at 1�g/ml; IL-5:TRFK5 at 2�g/ml; Becton Dickinson, San Diego, CA) andthe assay was carried out as described[25]. In brief, freshlyisolated splenocytes were plated at 1× 106 or 3× 105 cellsper well. Plates were incubated (IFN-� for ∼18 h, IL-5for ∼40 h) at 37◦C/5% CO2 in the presence of the fol-lowing stimuli (triplicates): OVA (50�g/ml), bovine serumalbumin (50�g/ml), OVA257–264 (10�g/ml), OVA265–280(10�g/ml), irrelevant H2-Kb- and I-Ab-restricted peptides(10�g/ml) and the peptide-adjuvant (10�g/ml) used forvaccination. As positive controls Concanavalin A (IFN-�;10�g/ml) or PMA/Ionomycin (IL-5; 2×10−8/7.5×10−7 M)were applied. Complete medium was used as negativecontrol. Results are expressed as the number of IFN-� orIL-5 producing cells per 1× 106 splenocytes± standarddeviation of triplicates.

2.5. Analysis of the immune response by ELISA

Ninety-six well plates (MaxiSorp, Nalgene Nunc,Roskilde, Denmark) were coated with antigen (10�g/mlOVA or 0.1�g/ml total protein of the FLUVIRIN vaccine)and incubated with serial dilutions of mouse sera. Boundantibodies were detected by sequential incubation with bi-otinylated anti mouse-Ig antibodies (IgG, IgG1 or IgG2,respectively, Southern Biotechnology Associates, Birming-ham, AL) and horseradish peroxidase-conjugated strepta-vidin (Roche Diagnostics, Mannheim, Germany). Colourreaction was done as described[15]. Absorbance at 405 nmwas measured using a microplate reader (Sunrise, Tecan,Salzburg, Austria). Titers at half maximal OD were deter-mined by linear interpolation between the measured pointsneighbouring the half maximal OD. Linear interpolationwas calculated using the logarithm of the titer values. Eachserum titration was assayed in triplicates.

2.6. Analysis of the in vivo distribution of fluorescentlylabelled vaccine compounds

At indicated time points after vaccination photos of in-jection sites were taken. Three mice per group were inves-tigated.

2.7. Haemagglutination inhibition assay

After injections with FLUVIRIN, serum antibody levelswere analysed by haemagglutination inhibition (HI) assayfor each of the three strains by standard microtiter methods.Sera were diluted two-fold and eight HA units of each anti-gen were added. Plates were incubated at room temperaturefor 30 min. Human erythrocytes were added and incubatedfor 75 min before plates were visually analysed for haemag-glutination. The HI titer given is the inverse of the highestdilution of serum exhibiting complete haemagglutination in-hibition.

2.8. Flow cytometric quantification of antigen delivery(TRANSloading assay)

Flow cytometric quantification of antigen delivery wascarried out by incubating 1× 106 cells of the murinemonocyte-macrophage line P388D1 (ATCC, Manassas,VA, TIB-63) with OVA-SFX alone or with a combina-tion of OVA-SFX and “carrier-peptide” (KLKL5KLK-OH,KLKL 5KLK-NH 2 or d-KLKL 5KLK-NH 2). The appropriateamount of “carrier-peptide” was added to Ovalbumin-SFX(diluted in 0.27 M sorbitol, 5 mM Tris pH 7.4 (both pur-chased from Sigma, St. Louis, MO)) in a final volume of200�l and vortexed gently. After incubating the mixturefor 30 min at room temperature, 1× 106 cells in 800�lDMEM containing 5% heat-inactivated FCS were addedand incubated for 60 min at 37◦C. Cells were washed threetimes with ice-cold wash buffer (1× PBS, 0.2% BSA and0.2% NaN3) and analysed by flow cytometry (FACScan;Becton Dickinson, Franklin Lakes, NJ). Dead cells wereexcluded from analysis by 7-AAD counter-stain. Meanfluorescence intensities (MFI) were taken to calculate theenhancement (fold increase) of antigen delivery due to theeffect of “carrier-peptide”.

Fold increase

=[(MFI of OVA-SFX mixed with carrier peptide)

−MFI of cells alone]

[(MFI of OVA − SFX) − MFI of cells alone]

3. Results

3.1. KLKL5KLK enhances the delivery of OVA to antigenpresenting cells

We tested if KLKL5KLK is capable to enhancethe delivery of antigens to antigen-presenting cells(APCs). Cells of the murine monocyte-macrophage lineP388D1 were incubated with fluorescein-labelled pro-tein, OVA-SFX (OVA-SFX) alone or in combination withKLKL 5KLK-NH 2, its d-form d-KLKL 5KLK-NH 2, or withthe peptide synthesised without an amidated C-terminus,KLKL 5KLK-OH. The mean fluorescence intensity of cells

J.H. Fritz et al. / Vaccine 22 (2004) 3274–3284 3277

Fig. 1. Flow cytometric quantification of antigen delivery (TRANSload-ing assay). The mouse APC cell line P388D1 was pulsed with aconstant amount (10�g/ml) of the fluorescein-tagged model proteinovalbumin (OVA-SFX) alone or in combination with KLKL5KLK-OH,KLKL 5KLK-NH 2 or d-KLKL 5KLK-NH 2 at the indicated concentrations.The mean fluorescence intensity of cells was determined by FACS anal-ysis. The enhanced delivery of OVA-SFX to cells due to the use ofKLKL 5KLK is given as the fold increase over cells pulsed with OVA-SFXonly. Data shown are representative of two separate experiments.

was measured by FACS analysis and the association ofantigen to APCs was determined. All the three peptidesmarkedly enhanced the association of OVA-SFX to APCsin a concentration-dependent manner (Fig. 1). Cytotoxicitywas only observed when more than 50 nmol of KLKL5KLKwere used. Comparable results were obtained by usingfluorescein-labelled peptides in the same experimental setup(data not shown).

3.2. KLKL5KLK elicits a sustained adaptive immuneresponse to co-injected antigens with a predominantTH2-type polarisation profile

The induction of an immune response critically dependson antigen being available in lymphoid organs[26]. In viewof this consideration, increased immune responsiveness maypartly be the result of enhanced translocation of vaccineantigen from the peripheral site of injection towards thedraining lymph node. Antigen presenting cells residing inthe periphery, such as dendritic cells, play a central role inpriming naive T- and B-cells[27].

Since KLKL5KLK enhances the antigen associationto APCs in vitro, we tested whether co-injection ofKLKL 5KLK leads to an increased immune response. We

chose OVA as a model antigen (Ag) to address the questionof whether adaptive immunity could be induced by vac-cination of mice with a mixture of OVA and the artificialCAMP, KLKL 5KLK-OH. We measured the systemic anti-body response to OVA after prime-boost vaccinations withincreasing amounts of KLKL5KLK-OH. Immunizationwith OVA alone set the baseline for antibody production,adsorption of OVA to Alum served as positive control.

On the basis of antibody isotypes, several immunopoten-tiators have been shown to preferentially elicit a TH1- ora TH2-type response[28]. Using ELISA we analysed theOVA-specific profile of IgG subclasses. We monitored theonset and the development of the response by analysing serataken after each (prime-boost) immunisation of vaccinatedC57BL/6 mice. To assess the duration of the immune re-sponse we analysed sera 3–4 months after booster immuni-sation (long-term).

As expected with Alum, we observed a strong induc-tion of total IgG and IgG1 specific for OVA (Fig. 2).KLKL 5KLK-OH also markedly elevated OVA-specific totalIgG and IgG1 production. Although the response peakedafter the second immunisation, we still found sustainedlevels of OVA-specific titers 3–4 months after booster im-munisation (Fig. 2). We did not detect OVA-specific IgG2levels with any of the stimuli tested (data not shown). Intests with various amounts of KLKL5KLK-OH mixed with

Fig. 2. Analysis of OVA-specific IgG subclasses. Prime-boost vaccinations(day 0 and day 28) were carried out. On day 26 (prime), day 54 (boost)and day 137 (long-term) sera were taken. OVA-specific immunglobulinisotype production was analysed by ELISA. Total IgG and IgG1 titers± standard deviation assayed in triplicates are given. Data shown arerepresentative of two separate experiments.

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Fig. 3. Analysis of OVA-specific IL-5 producing T-lymphocyte frequen-cies. Prime-boost vaccinations (day 0 and day 28) were carried out.On day 26 (prime), day 54 (boost) and day 137 (long-term) spleenswere taken. The frequencies of IL-5 producing spleen cells specific forOVA-protein, an OVA-derived MHC class I H2-Kb peptide (OVA257–264)and an OVA-derived MHC class II I-Ab peptide (OVA265–280) were de-termined by ELIspot assay. Data shown are representative of two separateexperiments.

100�g OVA we observed the highest OVA-specific serumIgG response when 100 nmol of KLKL5KLK-OH wereused (data not shown).

We further determined the frequencies of IFN-� andIL-5 producing T-lymphocytes specific for OVA-derivedMHC class I-(OVA257–264, H2-Kb) [23] and MHC classII-(OVA 265–280, I-Ab) [24] derived peptides by ELIspot.Compared to the effect of vaccination with OVA alone,mice given a mixture of OVA and KLKL5KLK-OHelicited markedly higher numbers of IL-5 producingspleen cells (Fig. 3). After a single injection of OVA plusKLKL 5KLK-OH we observed∼30 Ag-specific IL-5 pro-ducing cells among a million spleen cells whereas wedetected no OVA-specific IL-5 producing spleen cells inanimals injected with OVA alone. Although the boosterimmunisation led to a slight increase in IL-5 produc-ing cells in animals given OVA alone, co-injection withKLKL 5KLK-OH induced a striking increase of spot num-bers. In the latter case, even after 3–4 months, without fur-ther boost, the number of Ag-specific IL-5 producing spleencells remained high (Fig. 3). A single injection of OVA ad-sorbed to Alum resulted in higher numbers of OVA-specificIL-5 producing spleen cells compared to vaccination withOVA alone. However, a further boost with OVA/Alum didnot markedly raise the level of OVA-specific IL-5 produc-

ing cells compared to animals injected with OVA alone.On the contrary, we observed that over time the number ofOVA-specific spleen cells producing IL-5 dropped belowcontrols injected with OVA alone (Fig. 3).

To investigate whether also a TH1-type response was in-duced, we performed IFN-� ELIspot assays. Vaccinationwith a mixture of OVA plus KLKL5KLK-OH or Alum re-sulted in an insignificant increase of IFN-� producing spleencells compared to the OVA controls (data not shown).

3.3. KLKL5KLK-NH2 and d-KLKL5KLK-NH2 are aseffective as KLKL5KLK-OH

We investigated the immunopotentiating activities oftwo derivatives of KLKL5KLK-OH: we examined the in-fluence of C-terminal amidation (KLKL5KLK-NH 2) aswell as the activity of KLKL5KLK-NH 2 synthesised en-tirely with d-amino acids (d-KLKL 5KLK-NH 2). As in thecase of KLKL5KLK-OH, total IgG and IgG1 levels spe-cific for OVA were markedly elevated by co-injection ofKLKL 5KLK-NH 2 andd-KLKL 5KLK-NH 2 (Fig. 2) whereasno OVA-specific IgG2 was detected (data not shown). Fur-thermore, the kinetics of the response and the sustained lev-els of OVA-specific titers 3–4 months after booster immu-nisations with KLKL5KLK-NH 2 and d-KLKL 5KLK-NH 2were similar to KLKL5KLK-OH (Fig. 2).

After the initial immunisation as well as after booster im-munisations mice vaccinated with OVA plus KLKL5KLK-NH2 or d-KLKL 5KLK-NH 2 exhibited levels of Ag-specificIL-5 producing spleen cells comparable to those exhibitedby KLKL 5KLK-OH. However, at a later time point (day 109after boost) we observed sustained levels of OVA-specificIL-5 producing spleen cells in animals injected with OVAplusd-KLKL 5KLK-NH 2, while in animals given OVA plusKLKL 5KLK-NH 2 the number of OVA-specific spleen cellsproducing IL-5 dropped to the level of controls injected withOVA alone (Fig. 3).

In all IL-5 ELIspot assays we only observed significantspot numbers by ex-vivo restimulation of spleen cells withOVA protein and with OVA-derived MHC class II-restrictedpeptide OVA265–280. No spots above medium control weredetected upon restimulation with OVA-derived MHC classI-restricted peptide OVA257–264. IL-4 ELIspot assays gavecomparable patterns of OVA- and OVA265–280-specificcell numbers confirming the results obtained with IL-5ELIspot (data not shown). We did not detect spot num-bers above medium control in any of the ELIspot assayswhen spleen cells were restimulated with irrelevant H2-Kb-and I-Ab-restricted peptides nor with KLKL5KLK-OH,KLKL 5KLK-NH 2 and d-KLKL 5KLK-NH 2. The same ex-perimental settings in Balb/c mice yielded comparableresults (data not shown). Only an insignificant increase ofIFN-� producing spleen cells was observed in animals vac-cinated with a mixture of OVA and KLKL5KLK-NH 2 ord-KLKL 5KLK-NH 2, comparable to KLKL5KLK-OH (datanot shown).

J.H. Fritz et al. / Vaccine 22 (2004) 3274–3284 3279

3.4. KLKL5KLK enhances a TH2 type-like response whenco-injected with MHC class II epitopes

Most traditional vaccines contain rather undefined prepa-rations of pathogens as antigens. Peptides are very at-tractive antigens in that they can contain T- and B-cellepitopes, are chemically defined and stable, overcomingmost of the restrictions of traditional vaccines. Since weobserved that co-injection of OVA and KLKL5KLK yieldeda predominant TH2-type polarization profile, we aimed attesting if KLKL5KLK also elicits a comparable responsewhen mixed and co-injected with a MHC class II peptide.Thus, we vaccinated C57BL/6 mice once with a mixtureof an OVA-derived MHC class II peptide (OVA265–280,I-Ab [24]) and KLKL5KLK-OH, KLKL 5KLK-NH 2 ord-KLKL 5KLK-NH 2 and determined the frequencies ofIFN-� and IL-5 producing T-lymphocytes specific forOVA265–280 by ELIspot.

Compared to the effect of vaccination with OVA265–280alone, mice given a mixture of OVA265–280and KLKL5KLK-OH, KLKL 5KLK-NH 2 or d-KLKL 5KLK-NH 2 elicitedhigher numbers of OVA265–280-specific IL-5 producingspleen cells (Fig. 4). A single injection of OVA265–280adsorbed to Alum also resulted in higher numbers ofOVA265–280-specific IL-5 producing spleen cells com-pared to vaccination with OVA265–280 alone. IL-4 ELIspotassays gave comparable patterns of OVA265–280-specificcell numbers confirming the results obtained with IL-5ELIspot (data not shown). Similar to vaccinations with the

Fig. 4. Analysis of OVA265–280-specific IL-5 producing T-lymphocytefrequencies. One-shot vaccinations were carried out and on day 14 spleenswere taken. The frequencies of IL-5 producing spleen cells specific for theOVA-derived MHC class II I-Ab peptide (OVA265–280) were determined byELIspot assay. Data shown are representative of two separate experiments.

whole protein OVA, only an insignificant increase of IFN-�producing spleen cells was observed in animals vacci-nated with a mixture of OVA265–280 and KLKL5KLK-OH,KLKL 5KLK-NH 2, d-KLKL 5KLK-NH 2 or Alum (data notshown).

Taken together, these results indicate that KLKL5KLK-OH,KLKL 5KLK-NH 2 andd-KLKL 5KLK-NH 2 induce a strongTH2 type-like response with a very low concomitant type 1like response when co-injected with proteinaceous antigenssuch as OVA or peptide-based antigens such as OVA265–280.

3.5. KLKL5KLK traps the antigen at the site of injection

Antigen maintenance at the site of injection by depotformation is achieved by aluminium salts and oil-basedadjuvants and this is thought to be a prerequisite for elic-iting an enhanced immune reaction[29]. To investigatewhether KLKL5KLK-OH was retaining the vaccine-antigenat the injection site, we injected mice subcutaneously withfluorophor-labelled compounds and followed their subse-quent distribution (Fig. 5). Since it was shown that short im-munostimulatory oligodeoxynucleotides (ODNs) containingunmethylated CpG motifs distribute rapidly upon injec-tion [25], we used Cy5-labelled CpG1668 (CpG1668-Cy5,blue) as positive control. As anticipated, injection ofCpG1668-Cy5, regardless of whether injected alone or to-gether with antigen, was rapidly distributed all over theskin (Fig. 5F and G). Tetramethylrhodamine-labelled OVA(OVA-TAMRA, red) was administered together with or with-out Alum or KLKL5KLK-OH. Injection of OVA-TAMRAalone resulted in its rapid distribution within 30 min after in-jection. After 3 h we could hardly detect the molecule whilstafter 24 h it had completely disappeared (Fig. 5A). Whenco-injected with Alum (white), we saw a sustained depot ofOVA-TAMRA at the site of injection. Alum/OVA-TAMRA(pink) was visible within this depot even 30 days afteradministration (the latest time point analysed,Fig. 5B).

When co-injected with KLKL5KLK-OH, we observeda transient retention of OVA-TAMRA. We detected a de-pot of OVA-TAMRA (red) at day 5 after administration.Over time this depot shrank (day 14) and only traces ofOVA-TAMRA were visible after 30 days (Fig. 5C). We ob-tained the same results from co-injection of OVA-TAMRAand KLKL5KLK-NH 2 or d-KLKL 5KLK-NH 2 (data notshown).

We further examined if retention of antigen by KLKL5KLK-OH is independent of the fluorescent label at-tached to the antigen by injection of fluorescein-labelledovalbumin (OVA-SFX, yellow). We also investigatedwhether KLKL5KLK-OH itself is retained in the de-pot by applying Cy5-KLKL5KLK-OH (blue). WhileOVA-SFX administered alone rapidly distributed all overthe skin (data not shown), we noticed a transient reten-tion of OVA-SFX/Cy5-KLKL5KLK-OH (black–dark blue;Fig. 5D). Although we found a reduction in the size ofthe depot of OVA-SFX/Cy5-KLKL5KLK-OH over time,

3280 J.H. Fritz et al. / Vaccine 22 (2004) 3274–3284

Fig. 5. Examination of the maintenance of vaccine compounds at the site of injection. Images of injection sites were taken at indicated timepoints after injection of: OVA-TAMRA only (A), OVA-TAMRA/Alum (B), OVA-TAMRA/KLKL5KLK-OH (C), OVA-SFX/Cy5-KLKL5KLK-OH (D),Cy5-KLKL5KLK-OH only (E), OVA-SFX/CpG1668-Cy5 (F) and CpG1668-Cy5 only (G). Representative images from five mice (a–c) and three mice(d–g) per group of two independent experiments are shown.

we were still able to detect it 14 days after administra-tion (the latest time point analysed,Fig. 5D). Furthermore,Cy5-KLKL5KLK-OH (blue) on its own forms a depot atthe injection site (Fig. 5E). However, this depot disap-pears more rapidly than OVA-SFX/Cy5-KLKL5KLK-OH(compare day 14 ofFig. 5D versus 5E).

3.6. KLKL5KLK enhances adaptive immunity toco-injected clinically relevant antigens

We chose the trivalent influenza split-vaccine FLUVIRIN(season 2001/2002) containing inactivated virus surfaceantigens of three different strains (H1N1, H3N2 and B) to

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Table 1Characterisation of haemagglutination inhibition titers

Number of injections Tested influenza strain

H1N1 H3N2 B

First Second Third First Second Third First Second Third

Fluvirin (1�g) n.d. 80 40 40 40 40 n.d. n.d. 20Fluvirin (1�g) + KLKL 5KLK-OH (100 nmol) 40 160 640 80 320 320 80 320 640Fluvirin (1�g) + KLKL 5KLK-OH (300 nmol) 80 640 640 80 640 640 160 1280 1280Fluvirin (1�g) + KLKL 5KLK-OH (900 nmol) 80 640 640 160 640 2560 80 1280 320Fluvirin (1�g) + KLKL 5KLK-NH 2 (100 nmol) n.d. 160 640 80 640 1280 40 640 640Fluvirin (1�g) + d-KLKL 5KLK-NH 2 (100 nmol) 40 320 1280 80 320 1280 80 320 320Fluvirin (1�g) + Alum 20 160 160 160 320 160 160 160 640

Prime-boost–boost vaccinations (day 0, day 28 and day 56) with FLUVIRIN were carried out. On day 26 (prime), day 54 (first boost) and day 82(second boost) sera were taken and analysed for haemagglutination inhibition for each of the three strains included in the FLUVIRIN vaccine (H1N1,H3N2 and B). The inverse of the highest serum dilution exhibiting complete haemagglutination inhibition is given. n.d. is not detected.

test the immunopotentiating activity of KLKL5KLK-OHand its derivatives. One dose of FLUVIRIN applied tohumans contains 45�g of total protein. Thus, to adaptthe amount of antigen for rodents we chose to inject

Fig. 6. Analysis of FLUVIRIN-specific IgG isotypes. Prime-boost-boostvaccinations (day 0, day 28 and day 56) were carried out. On day 26(prime), day 54 (first boost) and day 82 (second boost) sera were taken andFLUVIRIN-specific immunglobulin isotypes were analysed by ELISA.Total IgG, IgG1 and IgG2 titers± standard deviation assayed in triplicatesare given. Representative data of two independent experiments are shown.

1�g of FLUVIRIN per mouse. Since we only used 1�gof total protein for vaccination, we again titrated theamount of KLKL5KLK-OH for maximal immune re-sponse. After prime-boost-boost vaccinations of C57BL/6mice, we analysed the FLUVIRIN-specific profile ofIgG subclasses. In addition we measured the functionalactivity of the antibody response to single antigensby haemagglutination-inhibition (HI). Mice vaccinatedwith a mixture of FLUVIRIN and KLKL5KLK-OH,KLKL 5KLK-NH 2 or d-KLKL 5KLK-NH 2 exhibitedmarkedly higher levels of FLUVIRIN-specific IgG, IgG1,and IgG2 than animals vaccinated with FLUVIRIN alone.The magnitude of the response was higher than lev-els elicited by Alum. The use of 300 and 900 nmolKLKL 5KLK-OH were superior to 100 nmol (Fig. 6).Haemagglutination-inhibition assays revealed that all threepeptides elicited markedly higher HI levels than FLUVIRINalone (Table 1).

4. Discussion

Besides their antimicrobial properties, naturally occurringantimicrobial peptides, such as�-defensins, also possessimmuno-enhancing activities[15,16] making them interest-ing novel adjuvant candidates for vaccine design. Becauseof these potent features a lot of effort has been put into de-signing non-naturally occurring (artificial) CAMPs for ther-apeutic use[18], but none so far has been reported to exertadjuvant-like activities in vivo.

Here we show that the artificial CAMP KLKL5KLK [19]is a potent inducer of adaptive immunity to co-injected anti-gens. We employed the model antigen OVA to study the na-ture of the response induced. When administered with KLKthe production of OVA-specific serum IgG1 was clearly en-hanced but not that of IgG2. We observed increased num-bers of IL-5 and IL-4 producing spleen cells when restim-ulated with OVA protein or with an OVA-derived MHCclass II-restricted peptide, but not when restimulated withan OVA-derived MHC class I-restricted peptide. In contrast,

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we detected only a slight increase of OVA-specific IFN-�producing cells by KLKL5KLK. Even 3–4 months after thebooster immunisation we found sustained OVA-specific IgGtiters and markedly elevated numbers of IL-4 and IL-5 pro-ducing OVA-specific spleen cells, providing evidence thatKLKL 5KLK is able to induce immunological memory.

While co-injections with 100�g OVA per mouse en-hanced Ag-specific serum IgG1 but not IgG2, co-administ-ration of 1�g FLUVIRIN with KLKL 5KLK augmentedboth, the production of FLUVIRIN-specific serum IgG1and IgG2. The different IgG profiles observed for OVA andFLUVIRIN might result from injecting different amountsof antigen. However, the IgG profile did not change byapplying less OVA (data not shown). Hence, the differentIgG profiles observed for OVA and FLUVIRIN could be aconsequence of the nature of the antigens.

We furthermore provide evidence that the immunoenhanc-ing property of KLKL5KLK is not restricted to proteina-ceous antigens, but can also be of use for peptide-based anti-gens. When co-injected with an OVA-derived MHC classII peptide, OVA265–280, elevated numbers of IL-4 and IL-5producing spleen cells were detected. Taken together, thisindicates that KLKL5KLK induces a strong TH2 type-likeresponse with a very low concomitant type 1 like response.

It has been reported that human neutrophils and U937monocytes are activated by KLKL5KLK-NH 2 to producethe superoxide anion via binding to cell surface calretic-ulin (CRT) [21,22]. Calreticulin, a multifunctional protein[30] expressed at the cell surface of macrophages[31] anddendritic cells[32], is part of a receptor complex for de-fence collagens like mannose binding lectin (MBL), surfac-tant proteins A and D and C1q. MBL and C1q engagementof CRT initiates macropinocytosis in human macrophages[31], a process of antigen capture by rapid engulfment ofsubstantial volumes of the extracellular milieu[33].

Although a receptor for KLKL5KLK has been described,the question whether CRT triggering by KLKL5KLK playsa pivotal role in the observed immune-enhancement hasto be raised. Since CRT-deficient mice die in utero, andCRT-deficient embryonic stem cells were not available tous, we aimed at studying the structure-function relationshipof KLKL 5KLK by comparing KLKL5KLK-NH 2 with itsd-form, d-KLKL 5KLK-NH 2, and with the peptide synthe-sised without an amidated C-terminus, KLKL5KLK-OH.

Although the kinetics of the response induced by thethree peptides, KLKL5KLK-OH, KLKL 5KLK-NH 2, andd-KLKL 5KLK-NH 2, slightly differ, we observed that allthree peptides are able to induce sustained adaptive im-munity to co-injected antigens. Due to the palindromicsequence of KLKL5KLK, d-KLKL 5KLK-NH 2 can also beviewed as the retro-all-d-form of KLKL 5KLK-OH. The ori-entation of the amino acid side chains in retro-all-d-peptidesis identical to that of thel-form, whereas the orientationof the backbone amide bonds is reversed[34]. Thus, theimmunoenhancing activity of KLKL5KLK is neither in-fluenced by C-terminal amidation nor relying on direct

stereo-specific interactions of its peptide backbone. Thisindicates that binding of KLKL5KLK to CRT either fullyrelies on its specific amino acid side chains sequence oris not required for its immuno-enhancing activity. Furtherinvestigations are needed to find out whether binding ofKLKL 5KLK to CRT correlates with the ability to induceadaptive immunity to co-injected antigens. At present weare investigating the sequence requirements of KLKL5KLKneeded for its immunoenhancing activity.

The facilitation of antigen transport, uptake and presenta-tion by APCs draining the vaccine injection site is of majorimportance for the effectiveness of vaccines[26,35]. Thisaim can be reached e.g. by repeated or prolonged accessibil-ity of antigen at the site of injection and/or increased load-ing of APCs with antigen[29]. Prolonged antigen mainte-nance at the injection site is effectively established by Alumand oil in water-based emulsions like incomplete Freund‘sadjuvant[29]. We report here that KLKL5KLK markedlyenhances the association of antigen to APCs in vitro. Fur-thermore, our results show that KLKL5KLK-OH as well asKLKL 5KLK-NH 2 andd-KLKL 5KLK-NH 2 are able to forma transient depot of antigen at the site of injection. Surpris-ingly, also KLKL5KLK-OH administered without antigen,although to a lesser extent, is kept at the injection site.

Development of TH1 and TH2 effector lymphocytes isdriven primarily by IL-12 or IL-4[36]. Pathogens and ad-juvants differ in their capacity to prime APCs for IL-12production[28]. Those that induce IL-12 production willdirectly prime TH1 responses. In contrast, when APCs areprimed by stimuli that do not elicit IL-12 production, TH2responses may be generated[37]. Preliminary results showthat IL-12 production of murine as well as human APCs isnot primed when stimulated with KLKL5KLK. A furtherparameter influencing the acquisition of T-cell effector func-tions is the duration of TCR stimulation. In the presenceof IL-12 a short TCR stimulation can lead to efficient TH1polarisation. IL-12 exerts its effect when present during aswell as after TCR signalling. In contrast, TH2 polarizationbesides IL-4 requires a prolonged TCR stimulation[38]. Weshow here that KLKL5KLK is able to form a depot of vac-cine adjuvant at the site of injection. This prolonged antigenavailability might increase the duration of TCR stimulation.Together with the inability to prime APCs such as DCs forIL-12 production, this might explain the predominant induc-tion of TH2 responses by KLKL5KLK.

In conclusion, we have shown that an artificial, chemicallydefined CAMP that can easily be synthesised is an interest-ing novel vaccine adjuvant. The peptide KLKL5KLK is apotent inducer of sustained adaptive immune responses toco-injected antigens of clinical relevance. Testing the func-tional activity of the antibody response revealed markedlyhigher haemagglutination inhibition titers by KLKL5KLKcompared to vaccine alone. Depending on the individual in-fluenza strain, the HI titers with KLKL5KLK range fromsimilar to strikingly higher levels compared to Alum. Pre-liminary pre-clinical toxicology studies indicate a favourable

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toxicological profile of KLKL5KLK. Taken together, the re-sults obtained with a commercially available influenza vac-cine and OVA protein indicate that a prevalent TH2-type re-sponse is induced by KLKL5KLK. Furthermore, our resultsprovide strong evidence that a main action of KLKL5KLKis the formation of a depot of antigen at the site of injec-tion, but other roles for KLKL5KLK in linking innate andadaptive immunity cannot be excluded. Depot formation ofantigen might also be a contributing factor for the induc-tion of adaptive immunity in the case of naturally occurringCAMPs and this clearly remains to be investigated.

Our results imply that the artificial CAMP KLKL5KLKis a novel adjuvant suitable for vaccine design. A large num-ber of other artificial CAMPs have already been designed,primarily for the use as microbicidal drugs. It might be ofutmost interest to investigate, similar to KLKL5KLK, alsotheir adjuvant activity.

Acknowledgements

We thank M. Chipchase for editing the manuscript, A.Habel for help with conducting FLUVIRIN experimentsand constant support, M. Berger, H. Kirlappos, J. Lipp-mann, A. Otava and M. Schunn for excellent technical help,Kuei-Tai Lai for inspiring discussions, B. Kruspel and M.Kindl for co-ordinating the animal care facility, I. Baum-gartner, C. Cseh and C. Walter for peptide synthesis, W.Schmidt for constant supportive advice, P. Steinlein for helpwith the FACS analysis, and Hanns Hofmann for carryingout haemagglutination inhibition assays.

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