Lipopeptides: adjuvanticity in conventional and genetic immunization

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Lipopeptides: adjuvanticity in conventional and genetic immunization K. Mittenbu«hler a

a;


, U. v.d. Esche a , L. Heinevetter b , W.G. Bessler a , M. Huber

a

Institut fu«r Molekulare Medizin und Zellforschung, Universita«tsklinikum Freiburg, AK Tumorimmunologie/Vakzine, Stefan-Meier-Str. 8, D-79104 Freiburg, Germany b Deutsches Institut fu«r Erna«hrungsforschung, Abteilung Immunologie, Bergholz-Rehbru«cke, Germany Received 9 September 2002; received in revised form 21 November 2002 ; accepted 21 November 2002 First published online 20 March 2003

Abstract Synthetic lipopeptides derived from the bacterial cell wall component lipoprotein activate B-lymphocytes and macrophages/monocytes in vitro. In vivo they constitute potent immunoadjuvants for a broad range of different antigens and species comparable or superior to Freund’s adjuvant. Here, we demonstrate that P3 CSK4 , representing a highly active lipopentapeptide derivative in vitro, significantly enhances and accelerates the humoral immune response to tetanus toxoid. P3 CSK4 could substitute for up to 90% of the antigen without any decrease in the specific IgG level, and the presence of the lipopeptide resulted in a prolonged production of specific IgG in time. Investigations using P3 CSK4 as an adjuvant in genetic immunization confirmed earlier data demonstrating that lipopeptides constitute adjuvants for low-immunogenic DNA constructs and/or for application routes resulting in weak immune responses. We monitored a lipopeptide-dependent shift from a Th1-type to Th2-type response, when DNA immunization was followed by i.p. administration of the lipopeptide adjuvant. < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Vaccination ; Immunoadjuvant; Lipopeptide

1. Introduction Synthetic lipopeptides derived from bacterial lipoprotein constitute potent macrophage/monocyte activators in vitro, inducing the release of various interleukins, tumor

* Corresponding author. Tel. : +49 (761) 2035472; Fax : +49 (761) 2035492. E-mail address : [email protected] (K. Mittenbu«hler). Abbreviations : BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HB-s, recombinant hepatitis B surface antigen; IL, interleukin; MDP, muramyl dipeptide ; NF-UB, nuclear factor-UB; N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)P3 CSK4 , cysteinyl-seryl-(lysyl)3 -lysine; PBS, phosphate-bu¡ered saline; TLR, tolllike receptor; TNF, tumor necrosis factor; TT, tetanus toxoid

necrosis factor (TNF) K, reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor-UB (NF-UB) [1^5]; in vivo they act as e¡ective adjuvants in parenteral and in mucosal immunization [6^10]. Investigations on lipopeptide derivatives from lipopentapeptide and lipopeptide fatty acid libraries indicate that the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)cysteinyl-seryl-(lysyl)3 -lysine (P3 CSK4 ) constitutes one of the most e¡ective leukocyte activators [3,11]. This compound represents an e⁄cient immunoadjuvant in conventional peptide/protein (parenteral, oral, and nasal application) and genetic immunization either in combination with or after covalent linkage to antigen [6^11]. The adjuvanticity of P3 CSK4 has been further increased by linking Th cell epitopes to the lipopeptide backbone [12,13]. Lipopeptides are suitable for the generation of polyclonal and monoclonal antibodies in di¡erent species, and constitute adjuvants for the in vitro immunization of human mononuclear cells or mouse B-cells, resulting in a markedly increased yield of antigen-speci¢c antibody-secreting hybridomas [13,14]. The in£uence of lipopeptide adjuvants on the Th1/Th2 bias has also been investigated in peptide/

0928-8244 / 03 / $22.00 < 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-8244(03)00069-5

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protein immunizations; the response varies depending on the antigen and application route [15]. In DNA immunization studies a shift was monitored from Th1 towards a Th2-type response [16]. Bacterial and viral vaccines based on synthetic lipopeptides, e.g. Enterobacteriaceae vaccines and vaccines against foot and mouth disease, provide protection in animal models [8]. Investigations on the molecular mode of action showed that lipopeptide (P3 CSK4 )induced activation of macrophages is toll-like receptor 2(TLR2) and CD14-dependent, and TLR4-independent; the activation is mediated via the mitogen-activated protein kinase signal transduction pathway, resulting in NFUB translocation and activation/repression of at least 140 genes partly involved in signal transduction and regulation of the immune response, including the transcriptional activation of p53, c-rel, inhibitor of NF-UBK, type 2 (inducible) nitric oxide synthase, CD40-LR, intercellular adhesion molecule 1, and interleukin (IL)-1/6/15 [1,17]. Lipopeptides are well-de¢ned, non-toxic, non-in£ammatory, and can be synthesized in large amounts.

2. Materials and methods 2.1. Adjuvants, antigens, and vector DNA P3 CSK4 (Fig. 1) was obtained from EMC, Tu«bingen, Germany. The pCMV-s vector was kindly supplied by R. Whalen, Centre National de la Recherche Scienti¢que, Paris, France. Kirre protein and the corresponding pKirre vector were provided by M. Struenkelnberg, Biologie II/ III, University of Freiburg, Germany. Tetanus toxoid (TT, Clostridium tetani) was purchased from Calbiochem, Schwalbach, Germany. Recombinant hepatitis B surface antigen (HB-s) was obtained from Advanced ImmunoChemical, Long Beach, CA, USA (catalog number AIHS7).

Palmitoyl

O

CH2

Palmitoyl

O

CH CH2 S CH2

Palmitoyl

NH CH

CO

Ser

(Lys)4

Fig. 1. Structural formula of the N-terminal lipopeptide P3 CSK4 from the lipoprotein of Escherichia coli.

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2.2. Mice BALB/c mice (female), 6^8 weeks old, were obtained from the breeding facilities of the Max-Planck-Institut fu«r Immunbiologie, Freiburg, Germany. 2.3. DNA/protein immunizations DNA immunizations were performed either by gene gun or by i.m. application of the corresponding vectors. For administration of DNA by gene gun, we used the Helios Gene Gun System (Bio-Rad, Heidelberg, Germany). All gene gun experiments were performed according to the instructions of the manufacturer, using a microcarrier loading quantity (MLQ, mg gold particles per administration) and a DNA loading ratio (Wg DNA mg31 gold particles) of 0.5 and 2.0, respectively, resulting in the application of 1 Wg DNA per shot. Administration of DNA was performed at 400 psi. Intramuscular application of DNA was performed by injecting the corresponding vector in 50 Wl phosphate-bu¡ered saline (PBS) (10 Wg DNA) into both tibialis anterior muscles, using a 0.3-ml Micro-Fine U-100 insulin syringe (Becton Dickinson, Basel, Switzerland). Mice were anesthetized prior to the application of the DNA. TT was applied i.p. as indicated in the ¢gure legends. On the days indicated, blood was taken from the lateral tail veins or the plexus retroorbitalis, and sera were prepared. Sera were subsequently stored in 10 Wl aliquots at 320‡C. 2.4. Determination of antibodies by ELISA Serum IgG levels were determined by enzyme-linked immunosorbent assay (ELISA) as described earlier [6,7]. In short, 96-well microtiter plates (M 129 A Dynatech, Dynex Technologies, Denkendorf, Germany) were coated with TT (50 ng per well), Kirre protein (50 ng per well) or HB-s protein (50 ng per well), at 37‡C for 2 h or at 4‡C overnight. The remaining binding sites were saturated with 1% bovine serum albumin (BSA) in PBS. Serum samples were diluted serially 1:2, starting at 1:40 or 1:80 in PBS (with 1% BSA and 0.1% Tween 20), added in 50 Wl aliquots per well and incubated for 3 h at 37‡C. The wells were washed three times with 0.2% Tween 20/PBS and incubated for 1 h at 37‡C with horseradish peroxidaseconjugated goat anti-mouse Ig (isotype)-speci¢c antibody. After washing three times, 100 Wl of substrate solution (0.4 mg ml31 ortho-phenylenediamine (Sigma, Taufkirchen, Germany), 0.02% H2 O2 (30%) in 0.1 M citrate bu¡er pH 4.2) was added and the plates were incubated for 20 min at room temperature. The reaction was stopped by the addition of 50 Wl per well 2 N H2 SO4 . Absorbance at 490 nm (reference wavelength 690 nm) was measured by using an automated ELISA reader (MRX Dynatech, Denkendorf, Germany).

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3. Results and discussion 3.1. Lipopeptides constitute adjuvants for in vivo immunization 3.1.1. Lipopeptides as adjuvants in peptide/protein immunization P3 CSK4 enhanced/accelerated the generation of antiTT-directed antibodies in BALB/c mice when administered i.p. together with the antigen. As can be seen in Fig. 2, a signi¢cant enhancement of the speci¢c serum IgG level was detected already on day 11, when 0.1 Wg of TT was applied in the presence of P3 CSK4 . The adjuvant e¡ect of lipopeptide was more pronounced on days 39 and 129. Control mice and mice treated with lipopeptide alone showed no anti-TT-directed antibodies. Furthermore, 0.1 Wg antigen ^ coapplied with P3 CSK4 ^ resulted in speci¢c IgG levels similar to the levels obtained after i.p. administration of 1 Wg TT alone (days 11, 39, and 129). Investigations on the long-term e¡ect of lipopeptides on the immune response showed an enhanced antigen-speci¢c IgG level on days 129 and 249 (Fig. 3). Thus, we could con¢rm earlier results [11], indicating that P3 CSK4 functions as an e¡ective adjuvant not only enhancing/accelerating the immune response, but also resulting in the ex-

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tended presence of speci¢c antibodies. The antibody levels obtained using lipopeptide adjuvants are comparable or even superior to the levels detected after coapplication of antigen and Freund’s adjuvant [11]. In contrast to Freund’s adjuvant, lipopeptide derivatives exhibit no toxic side e¡ects and no lipopeptide-dependent tissue damage has been observed. Immunostimulating properties were also shown for further bacteria- and plant-derived components, including cholera toxin, muramyl dipeptide (MDP), CpG oligonucleotides, saponin, and phorbol esters. When immunizing with saponin as an adjuvant, the IgG2a as well as the IgG1 response were both enhanced [15]. Cholera toxin, a Th2-type adjuvant, promotes the elevation of IgG and IgA in antigen-speci¢c responses [18^22]. MDP, representing a puri¢ed peptidoglycan moiety of the cell wall of Gram-negative bacteria, has been shown to provide enhanced T-cell help [23,24]. Finally, CpG oligonucleotides ^ activating cells via TLR9 [25] ^ are not only e¡ective adjuvants with respect to DNA immunizations, but also represent strong immune adjuvants in conventional peptide/protein immunization [26^28]. However, synthetic lipopeptides ^ in contrast to the above-mentioned immunoadjuvants ^ can be prepared under quality assurance conditions in large quantities/high purity, are chemically stable, non-toxic, non-pyrogenic and do not cause

Fig. 2. Short-term immunostimulating e¡ect of lipopeptide : in vivo immunization of mice with TT in the presence and absence of P3 CSK4 . BALB/c mice (6^8 weeks old, female; three mice per group) were primed i.p. with 0.1 Wg TT and either with or without 50 Wg P3 CSK4 in a ¢nal volume of 300 Wl 0.9% NaCl. Two further groups of BALB/c mice (6^8 weeks old, female; three mice per group) were treated identically with 1 Wg TT or 50 Wg P3 CSK4 . Boost injections were performed on days 15 and 28, using the same doses and application routes. Control mice received no antigen or lipopeptide. Blood was taken from the lateral tail veins on days 11, 39, and 129, and the resulting sera were analyzed for anti-TT-speci¢c immunoglobulins (IgG) by ELISA. ELISA plates were coated with TT (50 ng per well). Data represent means of two determinations.

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Fig. 3. Long-term immunostimulating e¡ect of lipopeptide: in vivo immunization of mice with TT in the presence and absence of P3 CSK4 . Priming and boost injections were performed as described in Fig. 2. Blood was taken from the lateral tail veins on days 129, 249, and 362/346, and the resulting sera were analyzed for anti-TT-speci¢c IgG by ELISA. ELISA plates were coated with TT (50 ng per well). Data represent means of two determinations.

tissue damage. Therefore, these novel immunostimulatory agents, derived from the cell wall of Gram-negative bacteria, constitute a signi¢cant improvement in animal immunization. 3.1.2. Lipopeptides as immunostimulants in genetic (DNA) immunization Earlier investigations we have performed indicated that P3 CSK4 may also be an adjuvant with respect to DNA immunization [3]. These results were con¢rmed by additional experiments. Since co-administration of vector and lipopeptide adjuvant (i.m.) resulted in no enhanced immune responses, lipopeptide adjuvants were administered 1 or 2 days after the DNA application, in order to use the immunostimulating e¡ect of lipopeptides when protein expression in transfected cells occurs [16]. As can be seen in Fig. 4, gene gun immunization with pKirre vector DNA and subsequent i.p. administration of P3 CSK4 resulted ^ as early as 16 days post immunization ^ in a signi¢cant increase of anti-Kirre-speci¢c IgG (Fig. 4, left panel). Signi¢cance was con¢rmed by statistical analysis (Mann^ Whitney U-test), using the OD490 values (at a serum dilution of 1:80); the obtained signi¢cance level (a) was 0.01. Increased antibody levels were still seen on day 28 (Fig. 4, center panel), but not on day 103 (Fig. 4, right panel), indicating that P3 CSK4 only accelerates the generation of speci¢c antibodies in the early phase of the hu-

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moral immune response. In contrast, the response against highly immunogenic DNA, e.g. pCMV-s encoding the HB-s protein, could not be further increased by P3 CSK4 . No di¡erences in antigen-speci¢c antibody levels (IgG) could be detected on day 14 (Fig. 5, left panel), 28 (Fig. 5, center panel), and 84 (Fig. 5, right panel) between the groups receiving no or subsequent i.p. administration of P3 CSK4 2 days after DNA application. However, a P3 CSK4 -dependent enhancing e¡ect was observed when the pCMV-s constructs were applied i.m. in the presence of lipopeptide. This adjuvant e¡ect could be caused ^ apart from the immunostimulating properties ^ by the cationic character of P3 CSK4 , resulting in an increase of DNA uptake by the cells, as also known for lipofectin [3]. Besides P3 CSK4 , a broad range of adjuvants for DNA immunization have been described : various components were used to facilitate the uptake of DNA into cells, e.g. the snake poison cardiotoxin and the anesthetic bupivacaine, as well as cationic lipids [16]. More promising were attempts to co-inject recombinant cytokines or cytokine-encoding vectors [16,29]. Recent investigations showed the adjuvant e¡ects of vectors encoding cholera toxin or Escherichia coli heat-labile enterotoxin [30]. In addition, bacterial DNA itself exhibits adjuvant activity, in£uencing the immune response towards Th1 [16]. Taken together, our results suggest that P3 CSK4 enhances and accelerates weak immune responses due to low-immuno-

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Fig. 4. P3 CSK4 as adjuvant in genetic immunization (gene gun application/pKirre vector DNA). BALB/c mice were immunized via gene gun using pKirre vector DNA as described in Section 2, with or without a subsequent 1-day delayed i.p. administration of 50 Wg P3 CSK4 . Sera were prepared on days 16, 29, and 103 post immunization and monitored for antigen-speci¢c (anti-Kirre) IgG. The x-axis represents serum dilutions. Serum samples were diluted serially 1:2, starting at 1:40. Symbols represent immunized individual animals.

genic antigens or application routes in DNA vaccination, and therefore constitutes a valuable novel adjuvant for DNA immunization. However, further investigations have to be performed in order to determine the optimal timing and route of P3 CSK4 application in relation to DNA administration.

3.2. Lipopeptides modulate Th1/Th2 bias CD4þ T-lymphocytes, which can di¡erentiate into functionally distinct subsets, are a key component of the immune defence against pathogens [31]. Th1 cells, which generate interferon-Q, IL-2, and TNF-L, are responsible

Fig. 5. P3 CSK4 as adjuvant in genetic immunization (gene gun application/pCMV-s vector DNA). BALB/c mice were immunized via gene gun using pCMV-s vector DNA as described in Section 2, with or without a subsequent 2-day delayed i.p. administration of 50 Wg P3 CSK4 . Sera were prepared on days 14, 28, and 84 post immunization and monitored for antigen-speci¢c (HB-s) IgG. The x-axis represents serum dilutions. Serum samples were diluted serially 1:2, starting at 1:40. Symbols represent immunized individual animals.

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Fig. 6. In£uence of P3 CSK4 on the Th1/Th2 balance in genetic immunization, i.m. application. Mice were immunized i.m. (tibialis anterior, both legs) with a total of 20 Wg pCMV-s vector DNA as described in Section 2. Blood was taken on day 66 and the corresponding sera were monitored for IgG subtypes by ELISA. The x-axis represents serum dilutions. Serum samples were diluted serially 1:2, starting at 1:40. Symbols represent immunized individual animals.

for cell-mediated immunity. In contrast, Th2 cells, producing IL-4, -5, -6, -9, -10, and -13, evoke strong antibody responses and eosinophil accumulation, but inhibit several functions of phagocytic cells [32]. Therefore, active modulation of the Th1/Th2 balance by adjuvants is most important for the development of new types of vaccines resulting in e¡ective, target-tailored immune responses. We here continue our investigations on the potential of lipopeptides to in£uence the Th1/Th2 bias. Preliminary results with respect to genetic immunization suggested that the Th1-type response usually induced by i.m. application of DNA [33^35] is directed towards a Th2-type response in the presence of the lipopeptide adjuvant [16]. We performed these studies, using the vector pCMV-s (encoding the HB-s protein) for immunization and P3 CSK4 as an adjuvant. As shown in Fig. 6, the speci¢c IgG1 level was markedly increased when 20 Wg pCMV-s DNA was applied i.m. (tibialis anterior) in the presence of 50 Wg P3 CSK4 (Fig. 6, right panel), compared to vector i.m. injection without lipopeptide (Fig. 6, left panel). These results indicate that P3 CSK4 changes the Th1-type response observed in genetic immunization towards a Th2type response. With respect to conventional (peptide/protein) immunization, the lipopeptide-induced speci¢c IgG1 and IgG2a (respectively Th2- and Th1-type) responses were investigated in previous studies, for di¡erent antigens and application routes. Oral immunization with gliadin in the presence of P3 CSK4 resulted in a high gliadin-speci¢c IgG2a level; a signi¢cantly lower level of gliadin-speci¢c IgG1 was detected, indicating a shift towards a Th1-type im-

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mune response [15]. Nasal immunizations of BALB/c mice with gliadin, melittin or ovalbumin in the presence of the lipopeptide P3 CSK4 resulted in enhanced serum antibody responses compared to immunizations performed in the absence of the lipopeptide adjuvant. With respect to gliadin (35 kDa), the gliadin-speci¢c serum IgG1 level was enhanced, but no IgG2a or IgA was detected. Similar results were obtained with the model antigen melittin (3 kDa) [10]. In contrast, nasal immunization with ovalbumin (45 kDa) in the presence of the lipopeptide resulted in markedly enhanced antigen-speci¢c IgG2a and IgA levels, suggesting that a Th1-type response is favored. However, i.p. administration of ovalbumin in the presence of lipopeptide led to high levels of antigen-speci¢c and total IgE, indicating a Th2-type response (M. Ayoub et al., unpublished results). Thus, our investigations suggest that, using lipopeptides as adjuvants in peptide/protein immunization, the antigen-speci¢c IgG1 /IgG2a relation depends on the route of application and the antigen which is used for immunization. 3.3. Advantages of lipopeptide adjuvants Lipopeptides, and especially P3 CSK4 , represent most suitable adjuvants with respect to conventional peptide/ protein immunization and DNA vaccination. No, or only marginal immune responses against lipopeptide adjuvants were detected up to now, using a wide range of antigens and various application routes. However, coupling of lipopeptides to low-molecular-mass antigens plus T-helper epitopes can result in additional anti-T-helper

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epitope-directed antibodies. Either in combination with or covalently bound to antigen, lipopeptide adjuvants have the following advantages [11] : b Lipopeptides are superior to conventional adjuvants or ISCOM preparations with respect to their high inertness against heat, light, and di¡erent solvents. b No toxic side e¡ects or tissue damages have been observed in animals. b Lipopeptides are completely biodegradable into amino and fatty acids, and S-glyceryl-cysteine is a metabolite from E. coli lipoprotein found in urine and faeces. b Lipopeptides are an e¡ective substitute for Freund’s adjuvant with its well-known detrimental characteristics. b The low-molecular-mass lipopeptide antigen conjugates are recognized by antigen-speci¢c B-lymphocytes and/or by other antigen-presenting cells. They are internalized by endocytosis, and the corresponding T-cell epitopes are exposed in context with MHC class I or class II molecules. In addition, TLR2-dependent cell activation occurs. b Lipopeptides covalently linked to antigen epitopes, which can be presented on MHC class I molecules, activate cytotoxic T-lymphocytes and are therefore an ef¢cient substitute for viral or other viable vaccines. b Because of the rapid uptake of the immunogen^lipopeptide constructs into cell membranes or the formation of micellar deposits, enzymatic degradation of the antigen is decreased. b Clearly de¢ned routine methods of peptide synthesis, including scaled-up synthesis and multiple peptide synthesis, are used for the preparation of lipopeptides. These products can be analyzed by mass spectrometry and do not contain endotoxin contaminations.

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

Acknowledgements

[13]

We appreciate the funding of this investigation by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. We appreciate the excellent technical assistance of A. Haber, C. Heine, and N. Kegel.

[14]

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