WO2004106365A2 - Modulatory peptide motifs for inducing th1 or th2 immune response - Google Patents

Abstract

The present invention relates to techniques for inducing either a TH1 or TH2-type immune response. Specific motifs may be added or deleted from antigens, thus inducing either a TH1- or TH2-type immune response against the antigen.

Description

MODULATORY MOTIFS FOR INDUCING TH1 OR TH2 IMMUNE RESPONSE RELATED APPLICATIONS

This application claims priority to Ser. No. 60/473,642 filed May 28, 2003.

FIELD OF THE INVENTION

The present invention relates to techniques for inducing either a TH1 or TH2-type immune response. Specific motifs may be added or deleted from antigens, thus inducing either a TH1- or TH2-type immune response against the antigen.

BACKGROUND

A large variety of factors have been linked to the orientation of the T helper response towards Th1 , Th2, Th3, or ThO type (Kourilsky 2001 , Jankovic 2001). Among them, the role of endogenous or exogenous adjuvants has been shown to be critical, and the existence of a "third signal" pointed out, in addition to TCR stimulation and T cell co-stimulation such as induced by CD-CD40L interactions for instance (Kalinski 1999, Schijers 2000). In parallel, several reports have shown that some antigens have the propensity to trigger a Th1 or Th2 biased immune response on their own (Wheelan 2000, Zhang 2001 , de Jong 2002, Ausiello 2002, Wang 2002, Skeiky 1998). Such a qualitative response may be addressed for instance by measuring IFNγ (TM) and IL5 (Th2) responses, and in most mice by quantifying lgG2a(Th1) and lgG1 (Th2) antibody responses. Based on such parameters, these antigens have been shown to induce a predominant Th1 or Th2 responses in vivo (Zhang 2001), and also in vitro by exerting their effect through dendritic cells (DCs) (de Jong 2002, Whelan 2000), although the precise mechanisms involved have not been elucidated.

As the first cells to encounter antigens or pathogens are epithelial cells and antigen presenting cells (APCs) such as DCs, one can propose among other possibilities that some motifs may exist on antigens/pathogens, which would trigger one or the other type of immune responses. These motifs could interact with pattern recognition receptors (PRR) or, by molecular mimicry with some more specific receptors already know to be involved in the induction of innate and adaptive immunity (cytokine/chemokine receptors or Toll like receptors for instance). There is a need in the art for methods of manipulating the immune response toward a TH1 or TH2 phenotype. The present invention provides specific amino acid sequence motifs that may be utilized to induce either a TH1- or a TH2-biased immune response. Utilization of such motifs may result in modulation of the immune response toward a particular antigen by adding or deleting particular sequences to / from that antigen. As shown herein, allowing the definition of Th1 and Th2 consensus sequences. These motifs are termed modulotopes, and are capable of orientating the immune response in the desired direction when, for example, such sequences are fused or mixed as peptides with the antigen of interest or fused or mixed as peptides with the antigen of interest.

SUMMARY OF THE INVENTION

The present invention provides methods for producing a TH1- or TH2-biased immune response in a host. In one embodiment, a pre-selected epitope construct comprising at least two amino acid sequences of an antigen is provided. To bias the immune response towards a TH1 response, the first of these amino acid sequences has the general formula X - Z - X - Z - Z - X - Z - X where X is a hydrophobic amino acid and Z is a hydrophilic amino acid. The second of these amino acid sequences may be any sequence derived from the antigen, so long as upon expression of the first and second amino acid sequence together an α-helical-coiled structure is maintained through the first amino acid sequence.

To bias the immune response towards a TH2 response, the first of these amino acid sequences has the general formula X - Z - Z - X - X - Z - Z - X where X is a hydrophobic amino acid and Z is a hydrophilic amino acid. The second of these amino acid sequences may be any sequence derived from the antigen, so long as upon expression of the first and second amino acid sequence together an α-helical-coiled structure is maintained through the first amino acid sequence.

In certain embodiments, the first sequence is fused to the second sequence resulting in a single sequence providing for co-expression of the first and second sequences. In other embodiments, a peptide comprising the first sequence is mixed with a peptide comprising the second sequence at an immunologically effective ratio, such as 1:1 to 10:1. The present invention further provides compositions containing such constructs and / or peptides as well as methods for immunizing hosts using such constructs, peptides, and/or compositions.

BRIEF DESCRIPTION OF THE DRAWINGS Table 1. Immune responses induced by full-length and truncated H. pylori cataiase.

Outbred OF1 mice were immunized twice with 5μg of antigen in presence of 200μg of DC Choi adjuvant. Cytokine responses were measured by Elispots and antibody responses by Elisa. For cytokines, spleen cells were stimulated with whole H pylori cataiase or human cataiase; median cytokine values obtained in individual mice (5/group) are presented. Antibody responses were measured on pooled sera from the same mice against the same 2 antigens.

Figure 1. Comparison between human (hum) and H pylori cataiase (aad 0792), and identification of specific motifs. A. Alignment of human and H pylori cataiase. Identical residues are in red. The arrow indicates the beginning of recombinant C terminus. N1 to N4 Hp-specific motifs are boxed in yellow, defined as being at least 14 amino acids-long and having at least 60% differences with human cataiase, with not more than 3 consecutives residues conserved between the two species. C1 to C4 particular motifs are boxed in green, as described in the text and in Figure 1b. B. Motifs identified in Hp cataiase C terminus. These motifs have an hydrophobic core flanked by a symmetric sequence of charged/hydrophiiic and hydrophobic residues. Such motifs are present in "Th2" proteins such as ES 62 (NCBI access AAC28365), ovalbumin (230201), Hp AlpA (CAB 69511), HSP96 (NPO 35761), HIV p24 (12084543). A potential consensus motif is boxed in yellow. Φo: hydrophobic residue (PALMIVFWY); Φi: hydrophilic/charged residue (GTSQNDEKR). *: motifs which structure has been solved and identified as a short α-helix flanked by loops (ovalbumin NCBI 230 201 , p24 NCBI 12084543), or which may correspond for Hp cataiase to α-helical domains in known structure of Proteus mirabilis cataiase (NCBI 1942536). C. Motifs identified in Hp cataiase N terminus. These motifs have a charged core flanked by a symmetric sequence of charged/hydrophiiic and hydrophobic residues. Such motifs are present in "pro-inflammatory" proteins such as rat HSP 70 (NCBI 4930026), RSV-F protein (NP 056683) and human HSP60 (AAA36022). A ptential consensus motif is boxed in yellow. Φo: hydrophobic residue (PALMIVFWY); Φi: hydrophilic/charged residue (GTSQNDEKR) *^■ motifs which structure has been solved and identified as a short α-helix flanked by loops in HSP 70 (NCBI. 4930026), or which may correspond for H pylori cataiase to α-helical/loop domains in known structure of Proteus mirabilis cataiase (NCBI 1942536). D. Comparison of putative Th1 and Th2 consensus domains. Inversion of the 2 central residues with their neighbouring ones induces a shift from one motif to the other.

Table 2. Circular dichroism analysis of synthetic peptides. Peptides were analysed by circular dichroism in PBS in presence of 30% TFE or 5mM MDPC which reinforce their a- helical potential structure. The obtained % of helicity is presented, as well as the increase in helicity compared to PBS (ratio).

Figure 2. Schematic representation of potential helical structures of the identified domains. A. Comparison of putative Th2 motifs represented as a-helixes. Positively charged residues are in dark blue and negatively charged/hydrophiiic residues in light blue. Hydrophobic residues are in red. B. Comparison between potential Th2 and Th1 motifs Figure 3. Recombinant antigens and peptides used in animal and in vitro experiments. A. Constructions and associations used in animal experiments. From the top to the bottom: C terminus, N terminus, fused N1234-Cter, fused N3-Cter, fused N4-Cter, Cter mixed with N4 or N4* peptides, N ter mixed with C2/3, C2/3 T1, p24 2-3 and N3* peptides, and HIV p24 mixed with N4 or N4* peptide. Identified Nter domains are boxed in orange and Cter domains in dark green. B. Sequences of synthetic peptides used in animals and in vitro experiments. Charged/hydrophiiic residues are in blue, and hydrophobic in red. Potential Th2 motifs are boxed in green and potential Th1 motifs in grey. Crosses show the amino-acids that have been changed to shift from a potential Th1 domain to a potential Th2 one (N3 to N3*, N4 to N4*), and reciprocally (C2/3 to C2/3 T1). Figure 4. Cytokine responses induced by the different cataiase-derived antigens in outbred OF1 mice. A. Cytokine responses induced by full-length cataiase, N terminus and C terminus. OF1 mice were immunized with 5μg of the corresponding antigen as described in Methods. Two weeks after the boost, spleen cells were stimulated by terminus or C terminus, and IFNγ or IL5 measured by Elispot. The horizontal line corresponds to 5 spots/10⁶ cells, which is the maximal value reproducibly obtained in non-stimulated cells. Bars correspond to median values. B. Cytokine responses induced by fused N1234-Cter compared to Cter. Left graph: cytokine responses; right graph, individual cytokine ratios. Spleen cells were stimulated with Cter (or Nter, giving no significant response over background, not shown). C. Cytokine responses induced by fused N3 and N4-Cter compared to Cter. Spleen cells were stimulated with Cter or Nter (giving no significant response over background, not shown) Graphs are representative of 2 independent experiments.

Figure 5. Cytokine responses induced in outbred OF1 mice by Cter in presence of different synthetic peptides. A. Cytokine responses. Mice were immunized with 5 μg of Cter in presence of 5μg of N3, N4 or N4* peptides. Two weeks after the boost, spleen cells were stimulated by C terminus (or Nter, giving no significant response over background, not shown), and IFNγ or IL5 measured by Elispot. The horizontal line corresponds to 5 spots/10⁶ cells, which is the maximal value reproducibly obtained in non-stimulated cells. Bars correspond to median values. B. Individual cytokine ratios. Graphs are representative of 2 independent experiments

Figure 6. Cytokine and antibody responses induced in outbred OF1 mice by Nter in presence of different synthetic peptides. A. Individual cytokine ratios. Mice were immunized with 5 μg of Nter in presence of 5μg of C2/3, C2/3 T1 , N3* or p24 2-3 peptides. Two weeks after the boost, spleen cells were stimulated by Nter (or Cter, giving no significant response over background, not shown), and IFNγ or IL5 measured by Elispot. Bars correspond to median values. B. Individual antibody isotypes ratios. Two weeks after the boost, lgG1 and lgG2a specific responses measured by Elisa against Nter or Cter (giving no significant response over background, not shown). Bars correspond to median values. Graphs are representative of 2 independent experiments Table 3. Dose effect of peptides in OF1 mice. Mice were immunized with the corresponding antigens in presence of 5 or 25μg of the indicated peptides. Table presents the median value of individuallL5/IFNγ and lgG1/lgG2a ratios. Antibody isotype ratios were not calculated in mice immunized with Cter due to the too low number of responding mice. Figure 7. Cytokine responses induced in C57bl/6 and BALB/c mice by Cter in presence of different synthetic peptides. A. Cytokine responses induced in C57bl/6 by Cter in presence of 1 and 5μg of N4 and N4* peptides. Two weeks after the boost, spleen cells were stimulated by C terminus and IFNγ or IL5 measured by Elispot. The horizontal line corresponds to 5 spots/10⁶ cells, which is the maximal value reproducibly obtained in non- stimulated cells. Bars correspond to median values. B. Cytokine responses induced by in BALB/c mice by p24 in presence of 5μg of N4 or N4* peptides. Upper graph: cytokine responses; lower graph, individual cytokine ratios. Spleen cells were stimulated with p24. Figure 8. Alopecia induced in C57bl/6 mice by N4 and N4* peptides, in relation with IL5/IFNγ ratios. Mice were immunized and analysed as in Figure 7A. Lower. graph: % of mice presenting alopecia. Upper graph: individual cytokine ratio performed on 8/16 mice per group. Alopecia ranged from discrete depilation to large areas of naked skin. No erosion of skin has ever been observed. Peptide-dependant alopecia was observed in two independent experiments carried out on 16 mice per group.

Table 4. Alopecia observed in C57bl/6 mice. 16 mice/group were immunized SC in the lower part of the back with Cter in presence of DC Choi and different doses of petides as indicated. The results of 2 independent experiments are presented. Lesions were observed 15 days after the boost.

Figure 9. IL12 and IL10 secretion induced par type 1 (C2/T1) and type 2 (C2/3) peptides in human monocyte-derived dendritic cells in presence of LPS. Immature DCs were treated with 10 ng/ml of LPS in presence or absence of increasing doses of C2/3 or C2/3 T1 peptides. After 48 hours, supernatants were collected and cytokine levels analysed by ELISA. Figure 10. Peptide-induced modulation of LPS activity in a Limulus assay. LPS activity as a function of peptide concentration is shown. Peptide solutions in absence of LPS were shown to be free of endotoxin activity (below detection level, <0.5 Ul/ml). Grey line: LPS activity in absence of peptides. Dotted line: threshold detection level. Analyses were made in duplicates; the variation between the two measurements was less than 10%. A. Peptide- induced modulation of the activity of small amounts of LPS. All analysis were carried out in the presence of 1 ng/ml LPS (Sigma). B. Modulation of LPS activity in the absence of added LPS, but in presence of the antigen Cter. Solutions analysed contained 40 μg/ml Cter (final) and the indicated peptide concentration, but no additional LPS. C. Repetition of the experiment shown in 10a using a fixed LPS concentration of 10 ng/ml. Figure 11. General model describing the induction of Th1 or Th2 responses with respect to the nature of antigen and the presence of putative Th1 or Th2 motifs. A.

Representation of the nature and magnitude of the Th1 or Th2 responses below or above a medium ThO response (horizontal line). The induced response can be shifted to the left or to the right on the curve according to parameters such as the number and nature of Th1/Th2 putative domains, mouse strain, presence of co-stimulation by adjuvants, sex, housing conditions or other unidentified factors. B. Model applied to Cter in presence of different doses of N4 and N4* peptides in OF1 (left graph) or C57bl/6 mice (right graph), all other parameters being considered fixed. Points were placed on the curve according to the responses observed in immunized mice (see figures 5 (OF1), 7 and 8 (C57bl/6), and Table 3), initial Cter position being on the "Th2 side". In OF1 mice, fused N4-Cter would be on the "ThO" axis (see fig 4c). C. Model applied to Nter ("Th0/1" antigen) in presence of different doses of C2/3 and C2/3 T1 peptides in OF1 mice, all other parameters being considered fixed. Points were placed on the curve (left graph) according to the responses observed in immunized mice (see figure 6 and table 3), initial position of Nter being on the "Th1 side". For instance, a dose of 5μg or C 2/3 T1 peptide would correspond to a position around the ThO axis. In agreement (see right graph), both high and low/null responders were observed in such conditions while while C2/3 peptide induces more homogeneous high lgG1 responses (ThO or Th2 bias respectively, confirmed by IL5/IFNγ ratio, figure 6).

DETAILED DESCRIPTION

The present invention provides methods for producing a TH1- or TH2-biased immune response in a host. As shown herein, specific amino acid sequence motifs drive the immune response towards a TH1 or TH2-biased response, and that these motifs and may be utilized to induce the desired response. Preferably, the motifs are composed of 6 to 8 amino-acids, the charge and arrangement of which define a motif with "Th1" or "Th2" properties according to parameters such as dose and genetic background. In general, it appears that following tyrosine (Y) by positively charged (arginine or lysine R/K) residues is critical. In addition, the major difference between Th1 and Th2 motifs generally depends upon the residue preceding Y, which is R/K in the Th1 motif initially defined in OF1 mice, and a hydrophobic residue in the case of the Th2 motif.

To bias the immune response towards a TH1 response, the first immunogenic amino acid sequence has the general formula X - Z - X - Z - Z - X - Z - X where X is a hydrophobic amino acid and Z is a hydrophilic amino acid. In one embodiment, the first aminό acid sequence has the general formula X - Z1 - X - Z2 - Z3 - X1 - Z4 - X where X is a hydrophobic amino acid; Z1 is E or D; Z2 is selected from the group consisting of D, E, K and R; Z3 is K; X1 is Y; and, Z4 is K or R.

To bias the immune response towards a TH2 response, the first immunogenic amino acid sequence has the general formula X - Z - Z - X - X - Z - Z - X where X is a hydrophobic amino acid and Z is a hydrophilic amino acid. In a preferred embodiment, the first amino acid sequences has the general formula X - Z - Z1 - X - X1 - Z2 - Z - X where X is a hydrophobic amino acid; Z is a hydrophilic amino acid; Z1 is selected from the group consisting of D, E, K and R; X1 is Y; and, Z2 is K or R. The second immunogenic amino acid sequence may be any sequence derived from the same antigen the first amino acid sequence was derived. In one embodiment, a peptide comprising the first sequence may be mixed with a peptide comprising the second sequence at an immunologically effective ratio. An immunologically effective ratio is any ratio of first peptide to second peptide that is sufficient to induce an immune response against the antigen. In preferred embodiments, the immune response would be either a TH1 or TH2- biased response depending on the nature of the first peptide. A suitable ratio of first peptide to second peptide would be, for example, 1 :1 to 10:1. In more preferred embodiments, the ratio of first to second peptide is 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , or 10:1.

Suitable antigens include those related to infectious diseases (i.e., bacteria, viruses, parasites) as well as non-infectious diseases such as cancer. Exemplary infectious agents include viral agents, bacterial agents, fungal agents, parasitic agents, and prion-related agents, many antigens for which are known by those of skill in the art (see, for example, the Centers for Disease Control website, www.cdc.gov/ncidod/diseases). Exemplary infectious agents include Corynebacterium (i.e., diphtheria), Clostridium (i.e., tetanus), polio virus (i.e., IPV, OPV), hepatitis virus, Neisseria (i.e., meningitidis), Streptococcus, and Hemophiius, among others as is known in the art. Many suitable antigens known to be related to non- infectious disease targets are also known to those of skill in the art and would be useful in practicing the present invention.

An immunogenic target may also be administered in combination with one or more adjuvants to boost the immune response. Exemplary adjuvants are shown in Table I.

In other embodiments, the first amino acid sequence may be fused, either directly or indirectly, to the second amino acid sequence to form a single construct for expressing the first and second amino acid sequences. Direct fusion of the first and second sequences may be accomplished by incorporating nucleic acid sequences encoding the first and second amino acid sequences into a single nucleic acid expression construct. Indirect fusion may include the use of multiple expression cassettes within a single nucleic acid expression construct or completely separate nucleic acid constructs, each encoding either the first or the second amino acid sequences. It is important that, upon expression of the first and second amino acid sequence as a single construct, an α-helical-coiled structure is maintained through the first amino acid sequence. It may be beneficial to engineer such constructs so that the first amino acid sequence is expressed at a higher level than the second amino acid sequence (i.e., a first to second amino acid sequence expression ratio of 1:1 to 1:10 as described above). To accomplish such variations in expression level, the first amino acid sequence may be operably linked to a strong transcriptional control element (such as a promoter) and the second amino acid sequence may be operably linked to a weak transcriptional control element (such as a promoter).

A "construct" is an isolated nucleic acid molecule encoding a sequence such as the first and second amino acid sequences described herein. The nucleic acid molecule may comprise or consist of a nucleotide sequence encoding one or more immunogenic amino acid sequences, or fragments or derivatives thereof, such as that contained in a DNA insert in an ATCC Deposit. The term "nucleic acid sequence" or "nucleic acid molecule" refers to a DNA or RNA sequence.

In certain embodiments of the present invention, constructs are vectors are used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide.

A flanking sequence is preferably capable of effecting the replication, transcription and / or translation of the coding sequence and is operably linked to a coding sequence. As used herein, the term operably linked refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. However, a flanking sequence need not necessarily be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence may still be considered operably linked to the coding sequence. Similarly, an enhancer sequence may be located upstream or downstream from the coding sequence and affect transcription of the sequence. Many suitable promoters are known in the art.

While preparing reagents of the present invention, cells may need to be transfected or transformed. Transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been transfected when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art (i.e., Graham ef al., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis ef al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu ef al., 1981, Gene 13:197). Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

The present invention further provides compositions containing such constructs and / or peptides. Administration of a composition of the present invention to a host may be accomplished using any of a variety of techniques known to those of skill in the art. The composition(s) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals (i.e., a "pharmaceutical composition").

A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

EXAMPLES

EXAMPLE 1

Material and Methods

A. Mice and immunization Outbred OF1 , Balb/c or C57bl/6 female mice 6-8-weeks-old were purchased from

IFFA Credo-Charles River (France). They were housed according to European guidelines. Each experiment has been approved by Aventis Pasteur internal Ethic Committee. Mice were immunised on days 0 and 21 by subcutaneous (SC) route (200 μl under the skin of the left part of the lumbar region). Five μg of antigen (alone or in combination with various doses of peptides) were administered in presence of 200μg of DC Choi adjuvant.

B. Measurement of cytokines-secreting spleen cells by ELISPOT

Nitrocellulose plates (Millipore) were coated with 5 μg/ml of anti mouse IL5 or IFNγ

(Pharmingen). The spleens were teased through a 70 μm filter (Falcon). After treatment with Gey's solution to eliminate red cells and three further washes, the cells were counted and loaded into the wells of the plates at a final concentration of 2.10^ cells in 100 μl in each well. 5μg/ml of stimulating antigen was added into the wells to stimulate the cells for 44 hours at 37°C with 5% C02. Each assay was done in triplicate in RPMI 1640 (Gibco) supplemented with 5% decomplemented FCS, sodium pyruvate, βME, glutamine and antibiotics. A positive control (ConA, Sigma , at a 5 μg/ml final concentration) and a negative control (medium alone) were performed for each mouse. Secondary biotinylated anti mouse IL5 or IFNγ antibodies (Pharmingen) were used at 1 μg/ml. Spots were revealed with AEC substrate (Sigma) and once the plates dried, counted with an automated spot counter (Microvision, France) and manually under a stereo-microscope to check the true identity of spots. The number of spots for 10⁶ cells induced by the respective antigens was determined and the background (spots induced by medium alone, negative control) was substracted.

C. Measurement of serum antibodies by ELISA ELISAs were performed according to standard protocols (biotinylated conjugates, streptavidine peroxidase complex were from Amersham and OPD substrate from Sigma). Plates (Maxisorb, Nunc) were coated overnight at 4°C with H. pylori extracts (5 μg/ml) in carbonate buffer. After saturation with bovine serum albumin (Sigma), plates were incubated with the sera (1.5 hrs), biotinylated conjugate (1.5 hrs), streptavidin peroxidase complex (1h) and substrate (10'). A polycional mouse serum directed against H. pylori extract was used as a control in each experiment. The titres were expressed as the inverse of the dilution giving 50% of the maximal absorbance value at 492 nm.

D. Recombinant antigens 1. Cloning

Full-length cataiase had previously been cloned in and expressed from E. coli (Sodoyer R), from strain X43-2AN. For other constructs, pET28c vector (Novagen) was used for cloning synthetic genes or DNA fragments obtained by PCR. It allowed expression of recombinant proteins possessing a N-terminal Histidine tag (His-Tag) able to be purified through affinity columns. Selection of clones was done in E. coli XLIbiue (Stratagene) and protein expression was induced after transformation in £ coli BL21λDE3 (Novagen). All constructs were checked by complete sequencing of both strands. a. Cataiase C terminus (Cter) : DNA fragment corresponding to the C terminus of H. pylori cataiase was amplified by PCR from a plasmid expressing full-length cataiase and cloned between BamHI and Xho1 sites in pET28c. DNA codes for a protein which extremities downstream to His-Tag are [NH2— COOH]: [PFHS— KKKK.STOP] b. N4-Cter fusion: DNA fragment corresponding to the N4 domain (see Fig 1) was cloned between Nhe1 and BamHI sites, upstream to the fragment coding for the Cter. DNA codes for a protein which extremities downstream to His-Tag are [NH2— COOH]: [PEED— WYLQ].Cter.STOP c. N3-Cter fusion: DNA fragment corresponding to the N3 domain (see Fig 1) was cloned between Nhe1 and BamHI sites, upstream to the fragment coding for the Cter. DNA codes for a protein which extremities downstream to His-Tag are [NH2— COOH]: [HTMQ— DSNQ]_;Cter.STOP d. N1234-Cter fusion: Synthetic DNA fragment corresponding to the fused N1-

N2-N3-N4 domains (see Fig 1) was constructed with synthetic oligonucleotides and cloned between Nhe1 and BamHI sites, upstream to the fragment coding for the Cter. DNA codes for a protein which extremities downstream to His-Tag are [NH2— COOH]: N1[VNK— DW].N2 [LLQ— LAA].N3 [RFW— SNQJ.N4 [PEE— YLQJ.Cter.STOP e. Cataiase N terminus (Nter): DNA fragment corresponding to the N terminus of H. pylori cataiase was amplified by PCR from a plasmid expressing full-length cataiase and cloned between EcoRI and Xho1 sites in pET28c. DNA codes for a protein which extremities downstream to His-Tag are [NH2— COOH]: [VNK— DTHATA.STOP]. Sequence showed that a frameshift induced a premature Stop codon generating a Nter protein lacking the last 17 amino acids.

2. Expression

After cloning, all constructs have been transformed in £ coli BL21λDE3. After induction with IPTG for 4 hours, the cells were pelleted, sonicated and the protein of interest purified from the pellet after solubilization with urea.

3. Purification and characterization

The extract was passed on a sepharose column (Hi-trap chelating sepharose column, Amersham-Pharmacia) and elution done with imidazole. Purification was done with BIOCAD 700E chain. After elution and obtention of the peak at 280nm, protein was dialyzed, filtrated and stored in 0.5M arginine/PBS at -70°C. Identity of the product was checked by SDS- PAGE and western-blot using anti His-Tag and anti-catalase antibodies. Purity was evaluated between 85 and 90% by Coomassie Blue stain. LPS content was in the same range for all recombinant cataiase antigens used in this study, between 0.0125 to 0.05 ng/μg of protein. Protein concentration was measured with MicroBCA kit (Pierce). P24 of HIV1 was purchased from Fitzgerald (ref 30-AH79, batch A02012401)

E. Synthetic peptides

Synthetic peptides which sequences are presented in Figure 5 were purchased from Neosystem (Strasbourg), Immunograde quality.

F. Obtention and stimulation of Dendritic ceils

Dendritic cells (DCs) were prepared from monocytes according to standard protocols. Briefly, PBMCs were obtained from healthy donors, purified on Ficoll gradients (Lymphoprep, Nycomed) in Leucosep tubes (Deutscher). After purification of PBMCs, CD14+ cells were purified with immunobeads on Variomacs (Miltenyi Biotech). Purified cells were then differentiated in dendritic cells after 6 days of culture with 10μg/ml of IL4 (TEBU) and 1000U/ml GMCSF (Novartis). Every two days, 5 ml of fresh RPMI-GSP-10%FCS were added, as well as IL4 and GMCSF. Phenotype of cells was assessed by microscopy and FACS analysis.. Once obtained, DCs were stimulated with various concentrations of peptides in 48 wells plates in presence of LPS (Sigma, 0.01 μg/ml). Two positive controls were used in combination with LPS: cholera toxin (CT) as a Th2 inducer at 1 μg/ml and poly l/C as a Th1/Th2 inducer at 20μg/ml (de Jong 2002). After 48 hours of stimulation, supernatants were collected and immediately analysed by EIA or stored at -70°C for further analysis. IL12 p70 and IL10 were quantified using OptEAI kit (Pharmingen).

G. Determination of endotoxin activity by the Limulus test

Endotoxin activity of LPS- or Cter- peptide mixtures was determined by a quantitative assay using a test kit from Cambrex in the Quality Control Department of Aventis Pasteur. Detection threshold was 0.5 endotoxin units (EU)/ml of solution. Assays were done in duplicates, and differences between duplicates did not exceed 10%. LPS was from £ coli 026:B6 (ref L2654 Sigma, St Louis, USA).

H. Circular Dichroϊsm Measurements were made at 20 °C using a Jasco J-810 spectropolarimeter (Tokyo, Japan) using cuvettes with a pathlength of 0.1 mm. Peptides analysed were at concentrations between 1.4-2 mg/ml. Reference spectra, obtained with samples containing all components except the peptide, were recorded and subtracted from the peptide spectra. Estimates of the helical fraction of the peptide were calculated using the value of [θ]_{o s} at 222 nm and the formula: percent helix = ([θ]_{o s} ²²² - [θ]_Coii²²²) - ([θ]_heiiχ²²² - [θ]₀oi²²²) x 100 %.as described in Taylor and Kaiser

I Statistical analysis Differences between groups were estimated by Cochran test (variance check) and

ANOVA. Multiple comparisons were done using Bonferroni test.

Example 2 Identification of Th1 and Th2 Motifs A. Cataiase

It has been postulated that a balanced Th1/Th2 response is associated with protection against H. pylori (Guy 1998, Sanchez 2001). H. pylori Cataiase is able to induce such a balanced response, as shown in a mouse model using the DC Choi adjuvant (Sanchez 2001). DC Choi is known to enhance the immune response without inducing a bias towards Th1 or Th2 (Brunei 1999). Cataiase and DC Choi were therefore selected for use in these experiments.

As the N terminal domain of H. pylori cataiase is highly homologous to mammalian cataiase in its N terminal domain (Fig. 1a), only the C terminus was utilized. As presented in Table 2, elimination of N terminus effectively suppressed the antibody response against human cataiase in OF1 outbred mice (human cataiase as a coating antigen, immunization with Cter versus Cataiase). A dramatic shift towards a Th2 response against H. pylori cataiase was observed when Cter was used for immunization, as judged by IL5/IFNγ and lgG1/lgG2a ratios (stimulation or coating with H. pylori Cataiase, immunization with Cter versus Cataiase). Protection was not observed in a challenge model when Cter was compared to full-length Cataiase. B. Identification of potential Th1 or Th2 motifs

It was hypothesized that Th2 motifs would exist in the C terminus while Th1/Th2 motifs would in contrast be located in the N terminus, triggering a resulting ThO type response against the whole cataiase. Four (4) short motifs with similar structure/charge (denominated C1 to C4, fig 1b) were identified in the C terminus, all having an hydrophobic core (YY in 3 of them) flanked by charged residues. It was also noted that antigens previously considered as "pro-Th2" by other authors (ES62, Whelan 2002; gp96, Banerjee 2002) under similar conditions (AlpA, Sanchez 2001; HIV p24 and ovalbumin, unpublished) comprise one or several similar motifs (Fig. 1b). In most motifs, Y and R/K residues are conserved, and a potential consensus sequence can be identified. For instance, p24 has 3 potential "Th2" domains, two of these (1 and 3) being exactly matched to the consensus sequence (Fig. 1b and 3b).

It was also hypothesized that, as human cataiase was unable to induce any cytokine response in mice immunized with whole Hp cataiase (Table II) while Hp cataiase was able to do so, Hp specific N terminal motifs must induce the observed IFNγ response. In comparing the sequences, four (4) short non-homologous domains between Hp and human cataiase were identified as N1 , N2, N3 and N4. Domains N3 and N4 also presented a particular structure/charge organization, with an hydrophilic charged core (Fig. 1c). Equivalent motifs were also observed in some antigens known to trigger inflammatory responses (Kurt Jones 2000, Wallin 2002, Dobbin 2002, Beg 2002; Fig. 1c). Y and R/K residues were conserved, allowing identification of a potential Th1 consensus sequence.

It was then hypothesized that Cter motifs may represent Th2 motifs and N3-N4 may be Th1 motifs. As shown in Fig. 1d, th Cter and N3-N4 motifs differ by the order of their residues, but one motif can be shifted to the other by inverting the two central residues with their neighbouring residues.

These motifs were identified in p24, Ovalbumin, and HSP 70. It was determined that the potential Th2 motifs correspond to a short surface-exposed α-helix flanked by two loops, while Th1 motifs are located at the junction of an helix and a loop. The presence of proline residues flanking many of the identified domains corresponds with such a structure. Schematic representation (Fig. 2) of these helices shows the Th2 motifs as amphipatic helixes with a highly charged side and a hydrophobic/aromatic opposite side, defining a pattern capable of interacting with other structures based on charge characteristics. These motifs are also observed for instance a variety of active peptides including cathelicidin LPS- binding peptides (Nagaoka 2002) and VIP/PACAP neuropeptides (Nicole 2000, Delgado 2001). These peptides have been shown to modulating Th responses. "Th1" motifs in these amphipatic structures is disrupted, but nevertheless an alternate charged pattern is also defined.

C. Construction of cataiase variants and definition and characterization of synthetic peptides

Cataiase constructs were constructed in order to test the respective influence of identified potential Th1 or Th2 motifs (Fig. 3a). N-terminus domain was generated, and C- terminus was fused to N3, N4, or N1 to N4 domains in order to see the ability of these domains to act in cis.

In parallel, 15-16 mer peptides comprising the Th motifs were generated and combined with C or N terminus domains and address their ability to act in trans. As a control, each "Th2" peptide was mutated in a "TM" peptide and reciprocally by exchanging the central residues (Fig. 3b). Circular dichroism experiments indicated that such peptides had conserved their potential to form α-helixes in presence of agents such as TFE or MDPC, and that Th2 motifs formed more stable helixes, as shown by % helicity in these conditions as compared to TM motifs (N4 versus its mutated N4* "Th2" counterpart or versus the C2-3 Th2 domains, see Table III and Fig. 3b).

Recombinant Cter, Nter and fused Cter variants were expressed as His-tagged proteins and purified by affinity chromatography. Although the preparations contained some LPS, this was in a similar range (0.0125 to 0.05 ng/ml) for all preparations, allowing comparisons between antigens. Moreover, in most experiments, comparisons were made for the same antigen in presence of different peptides. Another "Th2" antigen, p24 of HIV1, in presence of cataiase-derived peptides, and a "Th2" peptide derived from p24 were also tested. Experiments were carried out in the presence of DC Choi adjuvant. The properties of the antigens and peptides were tested at different doses in outbred OF1 mice, inbred Balb/c and inbred C57bl/6 mice. In a second step, peptides were tested on human monocyte-derived dendritic cells.

D. Cytokine responses in OF1 mice

1. Cfer versus Nter domains. Analysis of the responses induced by the Nter and Cter domains confirmed and extended the results obtained in preliminary experiments (Fig. 4a): Cter is a Th2 domain and induced IL5 secretion. IFNγ secretion was predominant after Nter immunization. Full-length cataiase, which possesses both domains, induces very low and balanced IL5/IFNγ responses. As expected, Cter stimulation did not stimulate mice immunized with Nter and reciprocally.

2. Cfer fused with Nter domains N3, N4 and N1234. Further experiments carried out with the C-terminus fused to Nter domains 1234 showed that such domains suppressed the IL-5 response (Fig. 4b), confirming the hypothesized TM potential. Fusion with single N3 or N4 motifs indicated that the N4 motif alone induced similar suppression while N3 domain did not induce significant changes (Fig. 4c). Thus, the N4 domain by itself may be responsible of the bias induced by fused 1234 domains. Interestingly, the major difference between the sequence of N4 domain (active) and N3 domain (inactive) lies in the presence of an acidic versus basic residue following the core tyrosine (Fig. 1c).

3. Cfer mixed with "Th1" peptides. The ability of selected domains to act in trans when mixed as synthetic peptides was also tested. Figs. 5a and 5b show that 5μg of N4 peptide mixed with 5 μg of Cter reversed the Th balance induced in OF1 mice, while the mutant peptide N4* (potential Th2 domain) abolished this effect, illustrating the critical importance of the 6 amino-acid core. This confirms the TM potential of N4 domain as seen with N4-Cter fusion. On the other hand, N3 peptide mixed with Cter did not change the immune response compared to Cter alone, also in agreement with previous results obtained with N3-Cter fusion. It was also observed that the effect of the peptide was mostly a T helper one, as no significant response was induced against the N terminus containing these motifs (not shown). 4. Nter mixed with "Th2" peptides. The ability of "Th2" Cter motifs (5μg) to modulate responses induced by Nter domain (5μg) was also tested. As shown in Fig. 6a, C2-3 motif mixed with Nter increased the IL5/IFNγ ratio, while its "TM" mutant C2-3T1 decreased it, in agreement with their respective expected TM or Th2 potential. In addition, other Th2 potential domains such as N3 mutant (N3*) or a p24 motif (p24 2-3) also increased the IL5/IFNγ ratio, confirming the ability of the Th2 consensus pattern to modulate TM responses. Similar.to what was seen with TM motif, the effect of the peptide was mostly a T helper one, as no significant response was induced against the C terminus containing the C2-3 motif (not shown).

E. Antibody responses

Antibody responses were also determined in the same animals. Individual lgG1/lgG2a responses and ratios observed in Nter-immunized mice confirmed the tendencies observed with cytokines. "Th2" motif induced lgG1 responses while the C23-T1 mutant on the opposite decreased the ratios (Fig. 6b). It was observed that both IFNγ and IL5 responses were required to induce significant antibody responses in Nter-immunized mice. In (Nter + C23-T1) mice in which IL-5 responses were abolished, antibody responses were not observed in half of the mice (lgG1 or lgG2a, see Fig. 11c). Cter is poorly immunogenic and only a few OF1 mice per group presented significant antibody responses. However, among seven different experiments including a total of 300 mice (OF1, C57bl/6 or BALB/c strains), high lgG1 responders (titers >100000) were mostly seen in "Th2" conditions, i.e. when Cter or p24 were administered alone or in presence of active doses of peptides such as N4* compared to co-administration with equivalent doses of N4 peptide (44/190 mice versus 8/111 , p<0.05).

F. Dose effect in OF1 mice

The importance of the dose of added peptide was also observed in OF1 mice.

Unexpectedly, with all peptides considered, immunizing mice with presence a 5-fold higher peptide dose (25μg) resulted in the abolition or reversion of the effect seen with 5 μg of peptide (Table IV). For instance, while 5μg of N4 peptide in addition to Cter abolished IL-5 secretion, 25μg of peptide induced high levels of both cytokines, resulting in a balanced IL- 5/1 FNγ ratio. This condition was the only one in which high lgG2a levels were observed in some mice, showing the importance of both cytokines, as stated above (not shown). These results show that, as observed for instance with MHC restricted-peptides (Rogers 1999, 2000, Murray 1998, Hosken 1995, Ruedl 2000), whole antigens (Mononaka 2000) or pathogens such as Leishmania or Mycobacterium (Power 1998, Taylor Robinson 1998, Compton 2002, Menon 1998, Mencacci 1996), dose is of critical importance in the modulation of Th balance.

G. Immunization of inbred Balb/c and C57bl/6 mice.

The results obtained in OF1 mice showed the broad potential of the immunoregulatory peptides, active even in outbred mice. It was then important to study these effects in other mouse strains, in particular inbred mice such Balb/c or C57bl/6, which have been found in different studies to have a Th2 or TM bias respectively (Compton 2002). The results presented in Figs. 7a and 7b with Cter in C57bl/6 mice and p24 (or Cter, riot shown) in Balb/C mice indicate that Th2 peptides or their TM counterparts have opposite regulatory effects, and that the dose corresponding to TM or Th2 effects was different than that used in OF1 mice, which shifted towards lower doses. In these experiments, 1μg of peptide in C57 BI/6 rather corresponds to 5 μg in OF1 , and 5μg to 25μg. For instance, 1 μg of N4 peptide with Cter in C57 BI/6 induces a TM bias, while 5μg abolishes this effect, as does 1μg of N4*. In OF1 mice, such effects were observed at 5 and 25 μg respectively (Fig. 5 and Table III). N4 peptide at 1μg decreased as expected the number of high lgG1 respo ders (0/16 mice versus 6/16 mice), in agreement with its TM effect at this dose. A dose-dependent effect was observed in OF1 mice, which was "shifted " in Balb/c or C57bl/6 mice. This indicated that the dose of peptide, the nature of antigen, and the background of the mouse strain influenced the observed effect.

Interestingly, it was observed that some C57bl/6 mice presented some moderate to severe alopecia after immunization. This mouse strain may present this condition spontaneously (Festing 1998), but the number of affected mice observed was dependent upon what each received (the IL-5/IFNγ ratio was inversely proportional to the severity of alopecia; Fig. 8). Mice having an absent or very low IFNγ response, such as with 1μg of N4*, presented absolutely no lesions. A second experiment, carried out on 16 mice per group with

1 and 5μg of respective peptides, showed a similar dose-effect. This second experiment showed that as little as 0.2 μg and up to of N4* peptide was sufficient for protecting C57bl/6 mice against alopecia (Table V) while 5μg of N4 was also protective. The lesions were present after one immunization and were amplified after the boost, while the number of affected mice did not change significantly. In some mice from the groups receiving 1 μg of N4 or 5 μg of N4* peptides, alopecia was very severe, and almost the whole back was hairless. In 25% of affected mice, alopecia was the most pronounced around the site of injection. The skin itself was not damaged.

H. Stimulation of human dendritic cells

To elucidate whether the above observations can be extrapolated to humans and whether peptides interfere with "danger signals" such as LPS, experiments were carried out with monocyte-derived dendritic cells. The pro-Th1 or pro-Th2 potential of immunomodulatory peptides was determined by measuring IL12p70 and IL10 levels. Stimulation of immature DCs was performed in presence of a fixed sub-optimal dose of £ coli LPS.(Ausiello et al 2002) (10ng/ml) and increasing doses of C2/3 and C2/3 T1 peptides (0.2-25 μg/ml). In 3 independent experiments, IL12p70 levels after 48 hours were usually low (0-100 pg/ml), but detectable only in the simultaneous presence of peptides and LPS, and not in presence of LPS or peptides alone. In parallel, in agreement with its involvement in early responses to "danger" signals (1, 15), IL12p40 could be detected after 6 hours, and its level was increased

2 to 5 fold by peptides compared to LPS alone (not shown), as were IL10 levels. Importantly, these effects were dependent on peptide dose and sequence, leading to an up to 100-fold difference in ratios between peptides C2/3 and C2/3 T1 at 0.2 and 25 μg/ml doses (Fig.9). Peptides N4 and N4*pep induced similar effects as C2/3T1 and C2/3 respectively, in agreement with mice experiments.

I. Modulation of LPS activity in a Limulus assay We have thus evaluated the ability of the peptides to specifically interfere with the action of LPS in a Limulus assay. Firstly, increasing doses of N4 and N4* were shown to modulate the activity of a fixed very small amount of £ coli LPS (Fig. 10a). Low to medium peptide doses decreased LPS activity to undetectable levels whereas higher concentrations resulted in an increase. High doses of N4 motifs had a slightly higher capacity to increase LPS activity than N4*, but the observed effects were modest. Assays were then carried out without addition of LPS but in presence of Cter previously shown to contain minor LPS contaminations, and which presented no detectable activity under the conditions of the assay. Figure 10b demonstrates that peptides N4 and N4* at high doses dramatically enhance LPS activity, which was especially pronounced for N4. In contrast to N4, low doses of N4* also enhanced LPS activity. Finally, the experiments were repeated using a 10 times higher LPS concentration (Fig. 10c) and a similar V-shaped dose-response curve was obtained. Low doses of both peptides gradually decreased LPS activity, but in contrast to experiments using low LPS doses, high doses of N4* were activatory while N4 remained inhibitory. Our experiments demonstrate that, similar to their Th modulatory activity in vivo, N4pep or N4*pep can act as activators or inhibitors of LPS activity, depending partially on the LPS/peptide ratio. Jurgens ef al. observed a very similar dose-dependent effect with haemoglobin, which decreased or increased LPS activity as function of LPS concentration. However, ITC experiments (data not shown) demonstrated that N4pep and N4*pep bind to LPS with a moderate K_D of around 300 μM. A similar affinity has been measured using control peptides indicating that binding was non-specific, which was confirmed by the non-saturable nature of interaction as seen by ITC, rather suggesting an indirect interaction between peptides and LPS. Some Limulus LPS binding proteins have binding motifs similar to those found in higher eukaryotes, which are potential targets for such interactions (Schumann et al 1997), although we are aware that many proteins are involved in the Limulus assay cascade. Motifs could then act through enhancement of the biological effects of minute (sometimes undetectable) amounts of LPS or other lipidic bio-active molecules, as proposed by Walling ef al. This would explain the controversial activity of HSPs (bearing potential Th1 or Th2 motifs, Fig. 1) through TLR4, which was attributed by some authors to LPS contamination (Bausinger et al 2002, Walling et al 2002). RSV-F protein contains a Th1 motif (Fig1), and its immunostimulatory activity has also been linked to TLR4 activation (Kurt-Jones 2000), which could be explained similarly by interaction with LPS-induced pathways. One can then propose that motifs on their own are not able of triggering or enhancing immune responses, and that concomitant endogenous or exogenous "danger" signals like LPS are required . Actually, human proteins do contain these motifs, as shown by scanning databases using PROSITE (not presented), and the requirement of danger co-signals for immunomodulation might avoid deleterious undesired activation under "normal" conditions.

J. Model proposed to explain for peptide dose-effect Finally, we tried to explain the opposite immune responses induced by different doses of the same peptide, as observed for instance for TLR4-dependant LPS-induced effects, being dose and LPS-type dependant (Dabbagh 2002, Eisenbarth 2002, Yoshimura 2002). It has been demonstrated in other models that low or high doses of one given peptide triggered Th2 responses whereas medium doses induced TM responses, generating a "bell-shaped" response curve (Hosken 1995). Our experiments can be explained using similar curves. In our "oscillation" model (Fig. 11a) the initial position of an antigen, defined as TM or Th2 based on its cytokine profile, depends on factors such as its nature and dose, presence of immunomodulatory motifs and co-stimulatory (danger) signals, mouse/human genotype, hormonal/sexual influences or other undefined environmental factors. This initial position can be displaced by co-administration of modulotopes, and the magnitude and direction of displacement depend on the dose and nature of modulotopes. Experiments using preparations of the same antigen differing in some unidentified factor might result in different initial positions (Fig. 11a), which might however result in εlrr.ϋar Th responses. As a consequence co-administration of the same modulotope induces opposite effects. Our in vivo data fit well to this model as illustrated in Figure 11b and c. It also offers an explanation why under certain conditions a TM antigen behaves as Th2 antigen and vice-versa. Such apparently contradictory data have been reported for VIP peptide, shown to induce TM (Delneste 1999) or Th2 (Delgado 2001) responses. Our model may also explain "block effects" due to unanticipated changes in the conditions of the experiment. For instance, different amounts of LPS in preparations used in 2 separate experiments would place the same antigen in 2 different initial positions, resulting in opposing effects of the same modulotopes, as stated above. This oscillation model can be used to fit the data obtained from in vivo studies and is equally valid to explain the in vitro modulation of LPS activity in the Limulus assay.

In conclusion, motifs that induce either a TM or Th2 response have been identified. These motifs are capable of acting in cis or trans to modulate the induced immune response, and are termed Type 1 (i.e, TH1) or Type 2 (i.e„ TH2) modulotopes. We have reported on particular TM and Th2 consensus motifs, which does not exclude that other motifs with similar structures and activities exist. Based on these observations, one can postulate that, depending on the presence or absence of TM or Th2 motifs and on their respective numbers, each antigen has the ability to contribute to the final orientation of the Th balance, in addition to other co-stimulatory signals. Thus, adding or deleting such motifs in antigens may modify their immunogenicity, the final Th1/Th2 profile depending on their respective number and accessibility. The fact that such motifs act also in trans as peptides may favour their use as immuno-modulators mixed with the target protein.

While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.

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Claims

CLAIMSWhat is claimed is:

1. A pre-selected epitope construct or mixture for producing a TH_! immune response, the construct or mixture comprising: a) a first amino acid sequence derived from an antigen, the first amino acid sequence having the general formula:

X- Z - X-Z - Z - X- Z - X where X is a hydrophobic amino acid and Z represents a hydrophilic amino acid ; b) a second amino acid sequence to which the first amino acid sequence is fused to produce the construct, or with which a peptide containing the fist amino acid sequence is mixed at appropriate concentrations (equimolar to 10 fold higher concentration); where the construct or peptide maintain an α-helical-coiled structure through the first amino acid sequence.

2. A pre-selected epitope construct or mixture for producing a TH-i immune response, the construct comprising: a) a first amino acid sequence derived from an antigen, the first amino acid sequence having the general formula: X - Z1 - X - Z2 - Z3 - X1 - Z4 - X where X is a hydrophobic amino acid;

Z1 is E or D;

Z2 is selected from the group consisting of D, E, K and R;

Z3 is K; X1 is Y; and,

Z4 is K or R; b) a second amino acid sequence to which the first amino acid sequence is fused to produce the construct, or with which a peptide containing the fist amino acid sequence is mixed at appropriate concentrations (equimolar to 10 fold higher concentration) where the construct or peptide maintain an α-helical-coiled structure through the first amino acid sequence.

3. A pre-selected epitope construct or mixture for producing a TH₂ immune response, the construct or mixture comprising: a) a first amino acid sequence derived from an antigen, the first amino acid sequence having the general formula:

X - Z - Z -X - X - Z - Z - X where X is a hydrophobic amino acid and Z represents a hydrophilic amino acid ; b) a second amino acid sequence to which the first amino acid sequence is fused to produce the construct, or with which a peptide containing the fist amino acid sequence is mixed at appropriate concentrations (equimolar to 10 fold higher concentration) where the construct or peptide maintain an α-helical structure through the first amino acid sequence.

4. A pre-selected epitope construct or mixture for producing a TH₂ immune response, the construct comprising: a) a first amino acid sequence derived from an antigen, the first amino acid sequence having the general formula:

X - Z - Z1 - X - X1 - Z2 - Z - X where X is a hydrophobic amino acid; Z is a hydrophilic amino acid;

Z1 is selected from the group consisting of D, E, K and R; X1 is Y; and,

Z2 is K or R; and, b) a second amino acid sequence to which the first amino acid sequence is fused to produce the construct, or with which a peptide containing the fist amino acid sequence is mixed at appropriate concentrations (equimolar to 10 fold higher concentration) where the construct or peptide maintain an α-helical structure through the first amino acid sequence .

5. A composition comprising a pre-selected epitope construct of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. A composition of claim 5 further comprising an adjuvant.

7. A method for immunizing a host against an antigen comprising administering to a host a composition of claim 5 in an amount sufficient to induce an immune response against the second amino acid sequence.

8. The method of claim 7 further comprising administering an adjuvant to the host, either together with or separately from the composition of claim 5.

9. A method for immunizing a host against an antigen comprising administering to a host a composition of claim 6 in an amount sufficient to induce an immune response against the second amino acid sequence.