Interleukin-1 and Interleukin-1 Fragments as Vaccine Adjuvants

Interleukin-1 and Interleukin-1 Fragments as Vaccine Adjuvants

METHODS 19, 108 –113 (1999) Article ID meth.1999.0835, available online at http://www.idealibrary.com on Interleukin-1 and Interleukin-1 Fragments as...

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METHODS 19, 108 –113 (1999) Article ID meth.1999.0835, available online at http://www.idealibrary.com on

Interleukin-1 and Interleukin-1 Fragments as Vaccine Adjuvants Diana Boraschi* ,1 and Aldo Tagliabue† *Dompe´ Research Center, L’Aquila, Italy, and †Biotechnology, University of Bologna, Bologna, Italy

The human interleukin-1b (IL-1b) domain in position 163–171, comprising the amino acids VQGEESNDK, has been synthesized as a nine-amino-acid-long peptide and used in vivo as a nontoxic HCl salt. The IL-1b nonapeptide reproduces the immunostimulatory and adjuvant effects of the whole mature IL-1b, but does not possess any of the IL-1b inflammatory, vasoactive, tumorpromoting, and systemically toxic effects, nor it can synergize with tumor necrosis factor a or other molecules in inducing toxicity and shock. The IL-1b fragment is active as adjuvant either when administered together with the antigen or if inoculated separately; it can be physically linked to the antigen or used as a discrete peptide. Moreover, the DNA sequence encoding the IL-1b domain has been included in an experimental DNA vaccine with positive results. Thus, immunostimulatory sequences can be identified within a pleiotropic cytokine like IL-1 and used in the rational design of novel vaccination strategies. © 1999 Academic Press

Interleukin 1 (IL-1) is a family of cytokines of key importance in the mechanisms of host defense because of its pivotal role in the onset and development of both immune and inflammatory reactions (1). The IL-1 family includes the two agonist proteins, IL-1a and IL-1b, with similar structure and activity, and one antagonist protein, IL-1ra, which is able to occupy the activating IL-1 receptor (IL-1R I) without exerting any agonistic effect. IL-1 binds to two types of receptors on the cell surface. IL-1R I, an 80 kDa monomeric transmembrane glycoprotein of the immunoglobulin superfamily, initiates cell activation on IL-1 binding (2). Both agonists IL-1a and IL-1b and antagonist IL-1ra bind equally well to IL-1R I, but only agonist IL-1 recruits into the IL-1/IL-1R I complex the accessory protein IL-1RAcP, 1

To whom correspondence should be addressed at Research Center Dompe´ S.p.A., Via Campo di Pile, I-67100 L’Aquila, Italy. Fax: 139/0862/338.219. E-mail: [email protected]

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which is essential for initiation of signal transduction (3). Conversely, IL-1R II is a 68 kDa receptor very similar to IL-1R I in its extracellular domain, but apparently unable to initiate cell activation (4). Indeed, IL1R II acts as IL-1 inhibitor in different ways: it can capture agonist IL-1, subtracting it from interaction with IL-1R I (5), and, once bound to IL-1, it can recruit IL-1RAcP into an inactive complex, thus limiting the availability of the accessory chain (6). A soluble form of IL-1R II also exists, represented by the protease-cleaved extracellular IL-1 binding domain of IL-1R II (7). The presence in nature of these downregulatory control mechanisms, to limit IL-1 activity, highlights the potentially detrimental effects of IL-1. Indeed, the strong inflammatory effects of IL-1 are at the basis of many acute and chronic inflammatory diseases and autoimmune derangements, where the dysregulation of IL-1 production or downregulation does not allow for its shutoff (1). These potent inflammatory effects have strongly limited the exploitation of the immunostimulatory effects of IL-1 for preventive or therapeutic use. Thus, despite early reports of the potent adjuvant capacity of whole IL-1 in experimental systems (8, 9), its use as adjuvant for vaccines has never been pursued. To exploit the adjuvant capacity of IL-1, the analysis of the IL-1b structure–function relationship has led to the identification of domains selectively endowed with different biological activities (10 –14). One of these domains, located between the fourth and fifth b-strands of the IL-1b structure (amino acids VQGEESNDK, in position 163–171 in the pro-IL-1b sequence) shows excellent immunostimulatory and adjuvant effects in vitro and in vivo in the absence of inflammatory and toxic effects and it can therefore be a perfect candidate molecule to be included as adjuvant in protein or DNA vaccines. 1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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IL-1 SEQUENCES AS ADJUVANTS

DESCRIPTION OF METHODS 1. Biological Effects of Peptide 163–171 of Human IL-1b The fragment in position 163–171 in human IL-1b has been synthesized by the solid-phase method and purified by high-performance liquid chromatography (HPLC) (14). The purified peptide obtained in this way is the peptide z trifluoroacetate (TFA) salt, estimated to contain one TFA equivalent every two peptide moles. Since TFA was found to have a profound depressing effect on the adjuvant capacity of IL-1b (15), a passage of ion chromatography on Amberlite IRA 401 (Cl) resin (BDH Italia) was performed to transform the peptide into the peptide z HCl salt. The biological effects of peptide 163–171 were then examined in a series of experimental models in vivo in comparison with the well-known inflammatory, systemic, and immunological effects of the entire mature human IL-1b. As summarized in Table 1, the synthetic fragment was unable to mimic most of the IL-1b activities, namely, all the inflammation-related and systemic effects, but it could fully reproduce the adjuvant, immunostimulatory, and immunorestorative activities of the parental molecule. In addition, the fragment could mimic, although with lower potency, the radioprotective capacity of IL-1b, as well as its ability to desensititize mice to the lethal shock induced by IL-1b in sensitive mice [either adrenalectomized or treated with tumor necrosis factor a (TNFa) or actinomycin D] (21). 2. Adjuvant Activity of Peptide 163–171 of Human IL-1b The adjuvant effect of the 163–171 IL-1b domain was assessed in a series of experimental conditions with different antigens. Data in Table 2 show that the 163– 171 fragment could enhance the immune response to all the antigens examined, independently of the route and timing of administration and of the nature of the antigen. Thus, enhancement of immune response could be seen with particulate complex T-dependent antigens such as sheep red blood cells (SRBCs), to which the peptide 163–171 could amplify both the primary and secondary responses, as well as with Thindependent antigens such as the pneumococcal polysaccharide SIII. This latter observation is of particular importance for saccharidic vaccines, which usually elicit poor responses and require effective adjuvants. The adjuvant activity of peptide 163–171 has been compared with that of the parental molecule IL-1b and found to be generally comparable, with respect to both amplitude of the effect and dose of adjuvant needed to obtain the effect (this latter observation was made with the 163–171 z HCl salt). In some experimental systems, peptide 163–171 compared favorably with other adjuvants (e.g., aluminum hydroxide, the most widely used adjuvant for human use) in terms of both the dose needed and the amplification of the response obtained.

Construction of synthetic or recombinant chimeric peptides, composed of an antigenic fragment followed or preceded by the 163–171 sequence, or physical conjugation of the antigen with the adjuvant peptide resulted in excellent enhancement of the antigen-specific immune response, better than that attainable when antigen and peptide 163–171 are not physically linked (Table 2). This has been observed with a synthetic chimeric peptide composed of a fragment of hepatitis B soluble antigen (HBsAg) followed by the VQGEESNDK sequence (26), in the case of recombinant chimeric proteins encompassing the human ferritin or the SalmoTABLE 1 In Vivo IL-1-Like Effects of the IL-1 Synthetic Fragment 163–171 In vivo activities Inflammation-related effects a Fever Anorexia Circulating PMNs Platelets Glycemia Triglyceridemia Insulinemia Corticosterone SAA Serum fibrinogen Plasmatic Fe 21 Circulating PLA 2 PGE 2 in cholitis Hepatic ED Circulating IL-6 Metastatization Systemic effects Shock and death Radioprotection Desensitization (shock and death) Immunorestoration After irradiation After cyclophosphamide In aging mice In nu/nu mice Tolerance abrogation Immunostimulation Tumor rejection Resistance to Listeria infection Adjuvanticity Protein antigens Peptides Polysaccharides Bacterial antigens Viral antigens Entire cells a

IL-1b

163–171 peptide

111 11 11 2 22 11 111 111 111 11 22 111 111 2 111 11

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

(10, 15, 16) (17) (17) (17) (15) (17) (15) (15) (15) (15) (15) (17) (18) (15) (17) (19)

111 111 111

5 1 1

(17) (15, 20) (21)

111 111 111 111 111

111 111 111 111 11

(15, 22) (22) (15, 22) (15, 22) (23)

111 111

11 11

(24) C. Cheers, unpublished

111 111 111 111 111 111

11 11 11 11 11 11

(25; unpublished) (26–28) (9, 15) (9, 15) (25, 26) (9, 15, 24, 29)

Reference

PMN, polymorphonuclear leukocytes; SAA, serum amyloid A; PLA 2, phospholipase A 2; PGE 2, prostaglandin E 2; ED, ethoxy coumarin-o-deethylase. 5, no effect; 1, 11, 111, significant, high, very high increase; 2, 22, significant, high decrease.

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BORASCHI AND TAGLIABUE

nella flagellin sequences plus the 163–171 sequence (28), and eventually in a recombinant anti-idiotype vaccine for B-cell lymphoma where the single chain Fv (scFv) antigen was genetically linked to the 163–171 fragment (27). 3. DNA Vaccination Due to the encouraging adjuvant activity of peptide 163–171 of IL-1b in practically all experimental systems in the absence of the inflammatory side effects of the parental molecule, and in the light of the observation that physical linkage between antigen and adjuvant can attain better enhancement of the immune response, an antitumor DNA vaccine has been constructed that includes the 163–171 sequence (27). The DNA sequence of the idiotypic antigen (scFv) was genetically linked to the DNA sequence coding for the

VQGEESNDK IL-1b fragment and administered intramuscularly as plasmid DNA before inoculum of B lymphoma cells in mice. Whereas control mice (receiving the empty plasmid or the plasmid containing scFv DNA alone) all died of tumor by Day 32, 40% of mice receiving the scFv/peptide DNA were alive more than 70 days from tumor inoculum (27). This effect was paralleled by significant induction of a specific antiidiotype response (27). Furthermore, a DNA vaccine containing the IL-1b sequence was tested in another tumor model, i.e., mice transgenic for the HER-2/neu oncogene, which develop mammary hyperplasia followed by appearance of carcinoma at Week 17 of age and invasion of all 10 mammary glands at 33 weeks. In this model, DNA vaccination was performed four times at 6-week intervals with a construct coding for the tumor antigen fused to the

TABLE 2 Adjuvant Capacity of IL-1b Synthetic Fragment 163–171 a 163–171

Antigen SRBC (iv) 13

Route

Time

iv

0

1 2–33

0

1 23 1 1.53 1 1.53 1 2–33 1 23 1 53 1 3–43 1 2–33 1 33 1 3–43 1 2–33 20% survival 50% survival

HRBC (iv) CE-2 tumor (sc) L1/C2 tumor (id)

ip ip ip po sc ip ip iv iv ip sc sc id

DHGG (ip)/HGG (iv)

ip

12 h 0 1 daily 8 days 11–8 days after challenge 27 days (with DHGG)

HBsAg (ip) HBsAg peptide 12-32 Renin

iv ip sc

0 Chimeric peptide Conjugate

Ferritin (ip) Flagellin (ip) scFv (ip)

ip ip ip 33

Fusion protein Fusion protein Fusion protein

im 33

DNA fusion

23

SIII (ip)

a

Adjuvanticity

22 days 12 days 0 0 0 210 days 210 days 0 0

70–80% tolerance abrogation 1 10–303 1 23 1 2–33 1 5–103 1 5–103 Appearance specific Ab 60% survival to tumor challenge 1 23 specific Ab 40% survival to tumor challenge

Comparison with other adjuvants

References

Comparable to IL-1b Better than MDP Better than aluminum hydroxide Comparable to IL-1b Comparable to IL-1b Comparable to IL-1b Better than IL-1b Comparable to IL-1b Comparable to IL-1b Comparable to IL-1b Comparable to IL-1b Comparable to IL-1b Comparable to IL-1b Better than IL-1b Worse than IL-1b (33) Worse than IL-1b (100% surv.)

(15, 16, 25, 30, 31) Unpublished Unpublished (9, 30) (9, 30) (9, 30) (30) (30) (9, 30) (9, 30) (15, 25) (25, 30) (9) (22) (24) (29)

Worse than IL-1b

(23)

Better than aluminum hydroxide Better than discrete peptides Better than discrete peptides NT NT NA

(25; unpublished) (26) C. Carelli, unpublished (28) (28) (27)

NA

(27)

SRBC, Sheep red blood cells; SIII, pneumococcal polysaccharide type III; HRBC, horse red blood cells; DHGG, deaggregated human g-globulins; HGG, aggregated human g-globulins; HBsAg, hepatitis B soluble antigen; scFv, single-chain antibody fragment Fv; iv, intravenously; ip, intraperitoneally; po, per os; sc, subcutaneously; id, intradermally; im, intramuscularly; Ab, antibody; MDP, muramyl dipeptide; NT, not tested; NA, not applicable.

IL-1 SEQUENCES AS ADJUVANTS

VQGEESNDK fragment. This treatment could reduce tumor incidence by 50% (5/10 glands invaded at 33 weeks in 20 mice) as compared with vaccination with cDNA for tumor antigen alone, which could not induce protection (10/10 glands invaded at 33 weeks) (32). These results highlight the possibility of designing new DNA vaccines carrying B- and T-cell antigen epitopes and adjuvant epitopes within the same construct, to achieve optimal protection against tumor or infectious diseases. 4. Modified Peptide Sequences The possibility of optimizing the IL-1b sequence responsible for adjuvant activity has been investigated by synthesizing a series of shorter and longer peptides encompassing the 163–171 area of IL-1b (31). As shown in Table 3, shorter sequences within the stretch VQGEESNDK could exert potent adjuvant activity, up to the minimal sequence EESN, which still had a highly significant effect. In contrast, longer peptides, although still active as adjuvants, never showed better effect as compared with the 163–171 fragment. Thus, modifications of the original 163–171 sequence could allow for improvement of the adjuvant effect. Therefore, new peptidic or peptidomimetic molecules could be designed, on the basis of the parental adjuvant sequence, optimized to achieve maximal adjuvant efficacy. 5. Biological Relevance of 163–171 Sequence of IL-1b Besides the wealth of data on the biological effects of the synthetic peptide corresponding to the IL-1b sequence 163–171, the real functional importance of this TABLE 3 Adjuvant Capacity of IL-1b Synthetic Fragments a Position

Sequence

166–169 166–170 165–169 165–170 165–171 164–171 163–171 162–171 161–171 159–171 163–176 Cyclic 163–171

EESN EESND GEESN GEESND GEESNDK QGEESNDK VQGEESNDK FVQGEESNDK SFVQGEESNDK SMSFVQGEESNDK VQGEESNDKIPVAL VQGEESNDK G

a

Adjuvant effect (vs 163–171) 2.00 0.01 60.0 0.02 10.00 90.00 1.00 0.10 0.01 1.00 .0.01 0.01

Adjuvant effect was measured as enhancement of primary response to SRBCs after intravenous inoculum of antigen and adjuvant admixed. Potency of adjuvant effect was evaluated from dose– response curves (31).

111

stretch within the IL-1b structure has been investigated following different approaches. Monoclonal antibodies raised against the synthetic peptide VQGEESNDK were found to bind effectively to both the fragment and the entire IL-1b (16). In addition, the antibody Vhp20 could inhibit the adjuvant activity of IL-1b in vivo but not its inflammatory and pyrogenic effects (16). Thus, it appears that this area of IL-1b is structurally relevant for the immunostimulatory capacity of the cytokine. Mapping of the sequence recognized by the antibody Vhp20 within the 163–171 fragment with recombinant and synthetic peptides suggested that the binding site could be tracked down to the sequence EESN (33). From another study (G. Cesareni et al., unpublished) Vhp20 apparently recognized within a series of synthetic peptides the consensus sequence SND. Indeed, a deletion mutant of IL-1b lacking the sequence SND showed a profound decrease in biological activity, paralleled by a limited decrease in receptor binding capacity (34). A series of mutant IL-1b molecules have been constructed in different laboratories carrying deletions/ modifications in the hinge area correponding to the peptide VQGEESNDK. Whereas single substitutions of amino acids within the area with other residues did not usually bring about significant changes in activity (34 – 41), of particular importance for the binding capacity of IL-1b to its receptor appears to be the immediately following residue I in position 172 (38, 42). Based on this information, two types of mutants have been constructed. The first is a modified IL-1b where the sequence 164 –173 (QGEESNDKIP) was substituted with the corresponding sequence present in the IL-1 receptor antagonist IL1ra (EPHA). It should be noted that IL-1ra, which can bind to the IL-1R I without recruiting IL-1RAcP and thus without activating capacity, strongly differs from IL-1b in terms of structural features in this particular area. The mutant SMIL-3, which misses the IL-1b hinge region between the fourth and fifth b-strands, loses its ability to bind the receptor and to exert biological activities (37). On the other hand, the reverse mutant has been also constructed, i.e., an IL-1ra carrying the IL-1b sequence QGEESN inserted within the sequence EPHA (43). This mutant IL-1ra acquires agonist activity by becoming able to recruit the IL-1RAcP into an activating complex with IL-1R I. Eventually, the crystal structure of IL-1b complexed with IL-1R I revealed that several residues of the 163– 171 stretch and those immediately adjacent (Q164, E167, N169, D170, and also F162 and I172) take part in the contact area of the cytokine with its receptor and are therefore essential for its biological effectiveness (44).

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CONCLUDING REMARKS On the basis of experimental data on the structure– function relationship within the pleiotropic cytokine IL-1b, it has been possible to define a peptide sequence endowed with immunostimulatory activity but devoid of inflammatory and pyrogenic effects. A synthetic peptide corresponding to this sequence indeed has demonstrated excellent adjuvant capacity for antigens of different nature, by several means of administration, when either physically linked to the antigen or not, in the absence of any significant side effect. On this ground, new means of vaccination have been designed and experimentally tested, in which recombinant chimeric DNA or protein vaccines are generated, encompassing the sequence of both the antigen and the adjuvant within the same construct. The successful outcome of the animal experimentation leads to the hope that the concept of built-in adjuvanticity will become the leading concept for the next generation of vaccines for poorly immunogenic antigens.

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