Immunostimulatory compositions

Abstract

The invention relates to the use of melanin, complexed with an antigen, as a immunostimulatory composition.

Claims

1. A method for obtaining an immunostimulatory composition comprising: a) providing a composition containing melanin precursor and an antigen, wherein the melanin precursor is selected from the group consisting of L-dopa, L-tyrosine, D-dopa, 6-hydroxy-Dopa, dopaquinone, cyclodopa, dopachrome, DHICA, DHI, dopamine-o-quinone, dopamine, leukodopaminochrome, and dopaminochrome; and b) inducing oxidative polymerization of the melanin precursor in the presence of an oxidizing agent selected from the group consisting of oxygen, hydrogen peroxide, ammonium persulfate, ferric ions, a combination of sodium iodide and hydrogen peroxide, a salt of a transition metal cation, and a combination thereof; thereby obtaining an immunostimulatory composition able to elicit a immune response against the antigen when administered to a subject, or when incubated with cells in vitro.

2. The method of claim 1, wherein the antigen and the melanin precursor in the composition of step a) are covalenty bound.

3. The method of claim 1, wherein the antigen and the melanin precursor in the composition of step a) are not covalenty bound.

4. The method of claim 1, wherein the composition of a) also contains an adjuvant.

5. The method of claim 1, wherein an adjuvant is added to the composition obtained after step b).

6. A method for generating an immune response against an antigen in a subject, comprising administering to the subject an immunostimulatory composition comprising an antigen complexed to a macromolecule, wherein the macromolecule is obtained by oxidative polymerization of a melanin precursor, wherein the melanin precursor is selected from the group consisting of L-dopa, L-tyrosine, D-dopa, 6-hydroxy-Dopa, dopaquinone, cyclodopa, dopachrome, DHICA, DHI, dopamine-o-quinone, dopamine, leukodopaminochrome, and dopaminochrome; and wherein the oxidative polymerization of the melanin precursor is induced in the presence of an oxidizing agent selected from the group consisting of oxygen, hydrogen peroxide, ammonium persulfate, ferric ions, a combination of sodium iodide and hydrogen peroxide, a salt of a transition metal cation, and a combination thereof.

7. The method of claim 6, wherein the melanin macromolecule is in the form of particles of less than 500 nm.

8. The method of claim 6, wherein the antigen and the melanin precursor were covalently linked prior to polymerization.

9. The method of claim 6, wherein the antigen and the melanin precursor were not covalently linked prior to polymerization.

10. The method of claim 6, wherein the composition also comprises an adjuvant.

11. The method of claim 10, wherein the adjuvant is complexed with the immunostimulatory composition.

12. The method of claim 10, wherein the adjuvant is not complexed with the immunostimulatory composition.

13. The method of claim 10, wherein the adjuvant is selected from the group consisting of alum, emulsions PRR ligands, TLR3 and RLR ligands, TLR4 ligands, TLR5 ligands, TLR7/8 ligands, and TLR9 ligands.

14. The method of claim 6, wherein the antigen comprises at least one epitope selected from the group consisting of MHC epitopes and B-cell epitopes.

15. The method of claim 6, wherein the immune response includes a CD8 T lymphocytes mediated response.

16. The method of claim 6, wherein the immune response includes a CD4 T lymphocytes mediated response.

17. The method of claim 6, wherein the subject is a human being.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: schematic description of the synthesis of eumelanin starting from phenylalanine.

(2) FIG. 2: CTL response after immunization with 1 g of the Ovalbumine epitope SIINFEKL (pOVA, SEQ ID NO: 2) or 10 g or the human gp100 epitope KVPRNQDWL (hgp100, SEQ ID NO: 3). C57-B16 mice were injected twice (day 0 & day 7) with the epitope+CpG-28 (SEQ ID NO: 1), the epitope+DOPA (D)+CpG-28; or [the epitope+DOPA, co incubated for 18 hours (D incub)]+10 g CpG-28 (10 g Dopa for pOVA; 100 g Dopa for hgp100). The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the corresponding MHC class I-restricted peptide and the numbers of IFNg-SFCs (Spot forming cells) were measured. (Representative experiment with n=4 mice/group).

(3) FIG. 3: CTL response after 2 immunizations with 10 g of peptide KVPRNQDWL (hgp100, SEQ ID NO: 3) or 1 g SIINFEKL (pOVA, SEQ ID NO: 2), combined with DOPA (100 g for hgp100, 1 g for pOVA), and incubated with various molar ratio (oxidant/Dopa) of H.sub.2O.sub.2 for 4 hours, or ammonium persulfate (APS) for 2 hours, respectively. In each formulation, 10 g CpG-28 (SEQ ID NO: 1)/mouse were added before immunizations. The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the corresponding MHC class I-restricted peptide and the numbers of IFNg-SFCs (Spot forming cells) were measured. (n=4 mice/group).

(4) FIG. 4: CTL response after immunization with 1 g of pOVA (SIINFEKL, SEQ ID NO: 2). C57-B16 mice were injected twice (day 0 & day 7) with peptide+CpG-28 (SEQ ID NO: 1), [peptide+1 g DOPA+APS] incubated for 4 hours+CpG-28; or [peptide+1 g Dopamine (Dn)+APS]+CpG-28. The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the corresponding MHC class I-restricted epitope SIINFEKL and the numbers of IFNg-SFCs (Spot forming cells) were measured. (Representative experiment with n=4 mice/group).

(5) FIG. 5: CTL response after immunization with 3.6 g or the long OVA peptide (pOVAI, SEQ ID NO: 4). C57-B16 mice were injected twice (day 0 & day 7) with peptide+CpG-28 (SEQ ID NO: 1), or [peptide+100 g Dopamine (Dn)+APS]+CpG-28. The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the corresponding MHC class I-restricted epitope SIINFEKL and the numbers of IFNg-SFCs (Spot forming cells) were measured. (Representative experiment with n=4 mice/group).

(6) FIG. 6: CTL response after immunization with 10 g of the human gp100 epitope (hgp100, SEQ ID NO: 3). C57-B16 mice were injected twice (day 0 & day 7) with the epitope co-incubated for 18 hours with 100 g DOPA at pH 8.5 (Ag then D); or with 100 g DOPA previously incubated in the presence of oxygen before the epitope was added (D then Ag). In each formulation, 10 g CpG-28 (SEQ ID NO: 1)/mouse were added before immunizations. The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the corresponding MHC class I-restricted peptide and the numbers of IFNg-SFCs (Spot forming cells) were measured. (Representative experiment with n=4 mice/group).

(7) FIG. 7: CTL response after immunization with 1 g of the Ovalbumine epitope (SIINFEKL=pOVA, SEQ ID NO: 2), or with 1 g of the epitope synthesized with a DOPA at the beginning (D-pOVA) or at the end (pOVA-D) of the peptide. C57-B16 mice were injected twice (day 0 & day 7) with the peptides+10 g CpG-28 (SEQ ID NO: 1). The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the SIINFEKL (SEQ ID NO: 2) peptide and the numbers of IFNg-SFCs (Spot forming cells) were measured. (n=4 mice/group).

(8) FIG. 8: Thin-layer chromatography (TLC) after a 72-hour incubation at pH 8.5 (Tris buffer). Left: hgp100 (SEQ ID NO: 3). only; middle: hgp100 (1 mg/ml) and Dopa (10 mg/ml), co-incubated with O2; right: hgp100 (1 mg/ml) added after Dopa (10 mg/ml) had been incubated with O2. The peptide, trapped into oxidized DOPA, is not seen any more when both Dopa and the peptide are co-incubated. (TLC was performed on aluminium foils, coated with a thin layer of silica gel as the stationary phase. After the samples have been loaded, a mixture of 1-Butanol/acetic acid/H2O (2/1/1) was used as the mobile phase. The TLC plates were then sprayed with ninhydrin reagent)

(9) FIG. 9: Thin-layer chromatography (TLC) after a 72-hour incubation at pH 8.5 (Tris buffer). Left: hgp100 (, SEQ ID NO: 3) only; other lanes: hgp100 (1 mg/ml) incubated with various doses of Dopa (Dopa/hgp100 weight ratios: from 1/5 to 10/1). The peptide, complexed with melanin (oxidized DOPA), is not seen any more when the ratio is over 2. (TLC: similar conditions as FIG. 8).

(10) FIG. 10: CTL response after immunization with 3.6 g of different OVA peptides: SEQ ID NO: 5 (SMLVLLPKSVSGLSQLESIINFEKLTSWTS, neutral), or SEQ ID NO: 6 (SMLVLLPKKVSGLKQLESIINFEKLTKWTS, positively charged). C57-B16 mice were injected twice (day 0 & day 7) with the epitopes co-incubated for 18 hours with 100 g DOPA at pH 8.5. In each formulation, 10 g CpG-28 (SEQ ID NO: 1) per mouse were added before immunizations. The immune response was assessed on day 14. Splenocytes were re-stimulated in vitro with the corresponding MHC class 1-restricted epitope SIINFEKL and the numbers of IFNg-SFCs (Spot forming cells) were measured. (n=4 mice/group).

(11) FIG. 11: SDS-PAGE (16% polyacrylamide gel) of various preparations: lane MW: molecular weight marker; lane Ctrl EphA2: control peptide EphA2 alone (SEQ ID NO: 9); lane Filtrate: filtrate of complex melanin-EphA2 after filtration on a 10 kD filter; lane Retentate: retentate of complex melanin-EphA2 after filtration on a 10 kD filter, resuspension, dissolution and heating in loading buffer containing SDS. (Melanin-EphA2 was obtained by co-incubating EphA2 peptide with L-Dopa (weight ration 1:10) in arerated conditions)

(12) FIG. 12: Physico-chemical characteristics of gp100-bound melanin:UV-visible spectrum (Evolution over time during synthesis). UV visible spectra were obtained using a JASCO V630 spectrophotometer (JASCO, Lisses, France). The solution of polymerizing L-Dopa was diluted 1/20, and spectra were recorded using 1 cm path length quartz cuvette after different incubation times.

(13) FIG. 13: Numbers of IFN-secreting lymphocytes per 10.sup.5 splenocytes after immunization with various formulations. A. impact of TLR9-agonist in a specific composition with gp100 (SEQ ID NO: 3) and melanin. B. Effect of the ratio of antigen/L-Dopa in the incubation medium to prepare gp100-melanin on the immune response. C. comparison of different gp100-melanin compositions (various antigen dosage) with a composition associating gp100, the classical adjuvant (incomplete Freund's adjuvant, IFA) and a TLR9 agonist.

(14) FIG. 14: Number of IFN-secreting cells per 10.sup.5 cells (splenocytes) upon stimulation with a CD4 or CD8 epitope, after immunization of mice with a synthetic peptide (pOVALs) (SEQ ID NO: 12).) containing both a CD4 and a CD8 ovalbumine epitopes, either alone or within a composition with melanin. Results shown for mice were immunized on day 0 &14 and sacrificed on day 21 (A), or on day 0 & 21 and sacrificed on day 42 (B).

(15) FIG. 15: Mean of tumor volume over time for different vaccine formulations.

EXAMPLES

(16) Methods

(17) In all examples, epitopes presented by H-2 Kb (mouse MHC I) were used (except in example 18 which is also using a mouse MHC-II epitope).

(18) C57-B16, 5-week old, mice were immunized twice (on day 1 and day 7) with the different formulations combined to a Toll-like Receptor 9 (TLR-9) agonist (10 g of the CpG-28 oligonucleotide, TAAACGTTATAACGTTACGACGTCAT (SEQ ID NO: 1)). After 2 immunizations, the CD8+immune response was evaluated on day 14 using a gamma interferon (IFNg)-secretion Elispot assay, after restimulation of total splenocytes with the relevant MHC class-1 epitope (SIINFEKL (SEQ ID NO: 2) for Ovalbumine or KVPRNQDWL (SEQ ID NO: 3) for human gp100)

Example 1Incubation of Antigen and a Melanin Precursor in Presence of an Oxidant Induces an Immune Response

(19) The Ovalbumine epitope (SIINFEKL, SEQ ID NO: 2) alone, or mixed with 10 g of DOPA, or mixed with DOPA and further incubated for 18 hours in the presence of oxygen to promote oxidation, were used as vaccine preparations. While the epitope alone did not trigger any significant CD8 immune response, the association with DOPA, especially after incubation, was able to induce IFNg spots.

(20) Similar data were obtained with the poorly immunogenic human gp100 epitope (KVPRNQDWL, SEQ ID NO: 3). Very small amounts of epitope can be used. These results are shown on FIG. 2.

(21) In particular, in this model, as little as 10 ng of SIINEFKL (SEQ ID NO: 2) was sufficient to detect an immune response (data not shown).

Example 2The Immune Response is not Dependent on the Oxidizing Agent Used (Either Chemical or Enzymatic Oxidation)

(22) Other oxidizing agents such as hydrogen peroxide (H.sub.2O.sub.2) or ammonium persulfate (APS) can be used (FIG. 3).

(23) In each case, depending on the antigen and the oxidant used, the optimal concentration is easily defined.

(24) As previously described, a CTL response was seen without addition of any oxidant, but the efficacy of the vaccine was generally enhanced by oxidation, the optimal molar ratio of oxidant/Dopa being around 1/4.

(25) Other oxidizing agents such as ferric chlorure can also be used and lead to the same results.

(26) As shown in Table 1, enzymatic oxidation with mushroom tyrosinase also led to the obtention of a strong immune response.

(27) TABLE-US-00001 TABLE 1 number of negative controls immu- nbr IFN-secreting (Irrelevant Oxidation nization of lymphocytes epitope) procedure protocol mice mean +/ SD mean +/ SD L-Dopa + Cu.sup.2+ day 0 & 8, 4 47 +/ 47 1 +/ 1 5 mM (+O.sub.2), sacrif incubation time: day 14 18 h L-Dopa + Fe.sup.3+ day 0 & 8, 4 160 +/ 52 0 +/ 1 5 mM (+O.sub.2), sacrif incubation time: day 14 18 h L-Dopa + Fe.sup.3+ day 0 & 8, 4 162 +/ 118 1 +/ 2 20 nM (+O.sub.2), sacrif incubation time: day 14 18 h L-Dopa + day 0 & 8, 8 246 +/ 164 1 +/ 1 Mushroom sacrif Tyrosinase (+O.sub.2), day 14 incubation time: 18 h

Example 3An Immune Response is Observed with Other Melanin Precursor

(28) This example demonstrates that Dopamine can be used instead of DOPA (FIG. 4). In this experiment, 1 g DOPA or 1 g Dopamine were mixed to 1 g SIINFEKL (pOVA, SEQ ID NO: 2), incubated 4 hours with the ammonium persulfate (APS) oxidant and then used as a vaccine preparations in association with 10 g CpG-28.

Example 4Use of a Long Antigen Containing an Epitope Still Makes it Possible to Have the Epitope Processed and Adequately Presented

(29) Experiments were conducted to show that other antigens, such as a long peptide, can induce a CTL response with the same procedures. The long OVA peptide (SMLVLLPDEVSGLEQLESIINFEKLTEWTS (SEQ ID NO: 4)), containing the SIINFEKL epitope (SEQ ID NO: 2), was tested, either alone, or combined to Dopamine and incubated 4 hours with APS and then used as a vaccine preparation in association with 10 g CpG-28. Only the preparation containing Dopamine triggered an immune response (FIG. 5).

Example 5Various Adjuvants can be Used in the Composition

(30) In this experiment, it was shown that various adjuvants can be advantageously added to the formulation [Antigen+Dopa].

(31) In this experiment, the used adjuvants were substituted to the CpG-ODN, according to the same protocol than the one disclosed in the Methods above.

(32) The SIINFEKL epitope (SEQ ID NO: 2), was combined (or not) to 100 g Dopa, further incubated for 18 hours in the presence of oxygen to promote oxidation, and then used as vaccines in association with 10 g CpG-28 (TLR9 agonist), 10 g Polyinosinic:polycytidylic acid (poly I:C) (TLR3 agonist), or mixed to Freud adjuvant or aluminium salts (Alum) (vol/vol 1/1).

(33) The CD8 immune response at day 14 was significantly higher for the [DOPA-antigen] composition than the one observed for the antigen without DOPA, especially when the TLR3 or TLR9 ligands were used.

Example 6The Immune Response Comprises a Humoral Response

(34) Immunization with a composition melanin/antigen induces a humoral response (circulating antibodies).

(35) Ovalbumin protein (1 g) was mixed to Dopa (1 g) at pH 7.4, incubated for 4 hours with APS (0.3 g), and injected subcutaneously with 10 g in C57/Bml6 mice on day 0 and day 7.

(36) At day 14, significant titers of anti-ovalbumine antibodies of both IgG1 (1/8000) and IgG2b (1/5250) subtypes were seen, showing a mixed Th1 and Th2 immune response.

Example 7Formation of the Complex Through Co-Incubation is Favourable

(37) It was shown that the co-incubation of the antigen and the catechol moiety during its oxidation makes it possible to potentiate the triggered immunity.

(38) 10 g of the hgp100 epitope (KVPRNQDWL, SEQ ID NO: 3), was either mixed to 100 g DOPA at pH 8.5, then incubated for 18 hours in the presence of oxygen to promote oxidation, or mixed to a solution of DOPA that has been previously incubated in the presence of oxygen to promote oxidation before the epitope was added.

(39) A significant CD8 immune response was seen when the epitope was co-incubated with DOPA (FIG. 6).

Example 8: An Immune Response can be Obtained with Various Melanin Precursor of Derivatives

(40) L-Dopa is a convenient catechol to use, leading to soluble melanin. However other melanin precursors can be used. In this experiment, 10 g of the hgp100 epitope (KVPRNQDWL, SEQ ID NO: 3), were mixed with either 100 g DOPA,

(41) ##STR00033## 100 g L-Tyrosine,

(42) ##STR00034## 100 g 3-(4-Hydroxyphenyl)propionic acid,

(43) ##STR00035## 100 g 6-Hydroxy-DOPA,

(44) ##STR00036## 100 g Dihydro caffeic,

(45) ##STR00037## 100 g DHI,

(46) ##STR00038## 100 g DHICA,

(47) ##STR00039## 50 g DHI+50 g DHICA, or 100 g HICA,

(48) ##STR00040##
at pH 8.5 in Tris buffer.

(49) The solution was then incubated for 18 hours in the presence of oxygen to promote oxidation.

(50) In each formulations, 10 g CpG-28/mouse were added before subcutaneous immunizations with various melanin precursors. Mice were either immunized on day 0 & 8 and sacrificed on day 14, or immunized once on day 0 and sacrificed on day 8.

(51) A significant CD8 immune response was seen, especially when the epitope was co-incubated with Dopa (L-Dopa ou D-Dopa), or 6-hydroxydopa, or di-hydroxynaphtalene. Immune responses were also seen, with a reduced magnitude, with Dopamine and Boc-Dopa (N-(tert-Butyloxycarbonyl)-L-dopa).

(52) The presence of an amino-group on the side chain, and of 2 hydroxyl-moieties on the phenolic ring of the melanin precursor appeared very favorable for biological activity and is thus preferred.

Example 9: A Complex is Formed Between the Polymerized Melanin and the Antigen

(53) In vitro experiments with Thin-layer chromatography (TLC) support the hypothesis that the incubation of the peptide while the catechol is being oxidized modifies the characteristic of the final formulation and that a complex is formed between the two entities (FIG. 8).

(54) Free peptide was seen on TLC only when hgp100 is added after Dopa had been oxidized, but not when Dopa and hgp100 are co-incubated with the oxidizing agent.

(55) It is to be noted that the stability of the peptide in presence of the oxidizing agent was checked and that disparition of the peptide is thus not due to the oxidizing agent.

(56) Using Thin-layer chromatography (TLC), it is possible to easily and routinely define the minimal dosage of catechol required for the loading of a certain amount of peptide. For example, with hgp100, a Dopa/peptide weight ration above 2 is favourable (FIG. 9).

Example 10An Immune Response is Obtained After Polymerization of DOPA Covalently Linked to the Antigen

(57) Mice were immunized either with 1 g of the Ovalbumine epitope (pOVA, SIINFEKL, SEQ ID NO: 2), or with 1 g of the epitope synthesized with a DOPA at the beginning (D-SIINFEKL, D-pOVA) or at the end (SIINFEKL-D, pOVA-D) of the peptide. While the epitope alone did not trigger any significant CD8 immune response, the modified peptides, especially the one with a C-terminal DOPA induced strong CTL immune responses (FIG. 7).

Example 11the Other Adjuvant May be Added Before or after Oxidation

(58) L-DOPA (100 g) was incubated with the hgp100 epitope (KVPRNQDWL, SEQ ID NO: 3), in presence of the CpG-28 oligonucleotide, then incubated for 18 hours in the presence of oxygen to promote oxidation.

(59) Alternatively, the CpG-28 oligonucleotide was added after oxidation of L-DOPA and the hgp100 epitope.

(60) The immune responses obtained after immunization of the mice with the resulting compositions were similar, thus indicating that the other adjuvant may be added before or after the oxidation (polymerization) reaction.

Example 12the Complexed Melanin-Antigen Composition May be Obtained as a Soluble Melanin Solution

(61) Dopa (2 mg/ml) or Dopamine (2 mg/ml) solutions were incubated with or without the pOVA (0.1 mg/ml) at pH 7.4 or 8.5, and with different oxidizing agents (O2, ammonium persulfate) for 20 hours. All solutions darkened within a few hours. With Dopamine, large aggregates were seen under light microscopy. Under centrifugation, the dopamine solutions precipitated, and some dark material remained adherent to the tubes.

(62) On the contrary, in some conditions (for example when incubated with oxygen at pH8.5), the Dopa solutions did not precipitate, even after centrifugation (16 000 g for 30 minutes) and was not adherent to the tubes. These Dopa solutions remained stable for several weeks, and no aggregates can be seen with light microscopy. This solution can be filtered through a 0.2 m filter, but not through a 100 kDalton-cut off filter (approx. 0.01 m).

(63) In conclusion, although both compounds can be successfully used in vaccines formulations, their characteristics differed. Without being bound to this theory, this may be due to a different percentage of DHI and DHICA in each formulation.

(64) Using various percentages of DOPA and Dopamine in the starting composition, (especially according to the antigen characteristics and especially the charge of the antigen) can advantageously lead to various galenic formulations.

Example 13The Complexed Melanin-Antigen Composition is a Colored Solution

(65) To prepare the formulations, it is preferred when a solution of L-Dopa is mixed with a solution of peptide (weight ratio L-Dopa:epitope between 1:100 and 1:1, preferably between 1:10 and 1:2, depending upon the epitopes), and the mixture is then oxidized at pH 8.5 in aerated conditions.

(66) Under these conditions, the colorless L-Dopa solution turned black to generate synthetic melanin, a process that can be monitored using UV spectroscopy (FIG. 12). The kinetics of L-Dopa oxidation, can be assessed by the 350/280 nm ratio.

(67) Under these conditions, when filtered through a 10-kDa filter, a black material is retained in the upper chamber, and could be easily resuspended in a saline solution. The resulting product (containing both melanin and peptides) can be further characterized using Fourier transform infrared spectra (FITR), Nuclear Magnetic Resonance (NMR) or Transmission electronic microscopy.

Example 14Modifying the Antigen Charge can Improve the Immune Response and Immunogenicity of the Composition

(68) The charge of the peptide pOVAI (SEQ ID NO: 4) is negative and its sequence was thus modified to make it neutral (SMLVLLPKSVSGLSQLESIINFEKLTSWTS, SEQ ID NO: 5) or positive (SMLVLLPKKVSGLKQLESIINFEKLTKWTS, SEQ ID NO: 6). When these peptides were used with Dopa (which displays a COO-moiety that is negatively charged) as the melanin precursor, the immune response obtained after 2 immunizations was strong with the positively-charged peptide, and intermediate with the neutral peptide (FIG. 10).

(69) The results obtained after immunization with these peptides and Dopamine (which is positively charged at physiological pH) as the melanin precursor were opposite (i.e. the highest immune response was observed with SEQ ID NO:4 (negatively charged peptide) and the lowest one with SEQ ID NO: 6 (positively charged) whereas the response with SEQ ID NO: 5 was intermediate).

(70) Altogether, these results indicate that it is favourable to modify the antigen in order for its charge not to be the same than the one of the resulting melanin.

(71) Similar data were obtained with the MHC I epitope of Trp2 (VYDFFVWL, SEQ ID NO: 7), which does not contain any positively charged amino-acids.

(72) Adding arginines (R) or lysines (K) to the NH2-terminal end of the peptides enhanced the immune response when compared to unmodified Trp2 (animals were immunized once with formulations consisting of the peptide (10 g/mouse) co-incubated with L-Dopa (100 g/mouse), then mixed to CpG-28 (10 g/mouse). The CD8 immune responses were assessed on day 8).

(73) Similar data were obtained with a viral epitope in Balb/c mice (gPr73: SFAVATTAL, SEQ ID NO: 8). Adding a Lysine at the NH2-terminal end significantly enhanced the immune response. In both cases, if a glutamate (E) was added next to the lysine, the efficacy was reduced (Table 2).

(74) TABLE-US-00002 TABLE 2 number of nbr IFNy-secreting student-T-test epitope of lymphocytes (compared to structure mice mean +/ SD the native epitope) Trp2 7 30 +/ 34 / K-Trp2 7 226 +/ 79 p < 0.001 R-Trp2 7 220 +/ 56 p < 0.001 KAR-Trp2 8 210 +/ 78 p < 0.001 KE-Trp2 7 30 +/ 9 p = n.s..sup. gPr73 8 5 +/ 4 / K-gPr73 8 21 +/ 16 p = 0.02 KE-gPr73 8 14 +/ 22 p = n.s..sup.

Example 15Distribution of Melanin in Draining Lymph Nodes

(75) Ten (10) g of the hgp100 epitope (KVPRNQDWL, SEQ ID NO: 3) were mixed to 100 g DOPA at pH 8.5, then incubated for 18 hours in the presence of oxygen to promote oxidation and generate gp100-bound melanin (gp100-melanin).

(76) To assess the distribution of this formulation in vivo, mice were injected subcutaneously with [gp100-melanin+CpG-28] or saline, and sacrificed on days 2 or 7 (n=3/group). To avoid any bias caused by natural melanin, these experiments were carried out in Balb/c mice, which are naturally devoid of melanin. Black pigmentation of the draining inguinal lymph nodes was macroscopically visible on day 2 post-injection in gp100-melanin-injected animals. Fontana-Masson staining confirmed numerous melanin-laden macrophages in the sinuses and, to a lesser extent, in the paracortical areas, which is a T-cell zone. The pattern of melanin distribution was similar at days 2 and 7 post-injection. No melanin was observed in mice that received saline only. These results show that the vaccine formulation effectively reached the draining lymph nodes in vivo.

(77) This finding is consistent with the fact that induction of antigen-specific immunity relies on the direct interaction of DCs with naive T cells that occur in the T-cell zone of lymph nodes.

Example 16Improvement of the Vaccine Composition

(78) Free gp100 peptide and gp100-melanin were used as vaccine preparations alone or mixed with the TLR9 agonist CpG-28 (CpG) (n=8 mice/group).

(79) When combined with CpG, gp100-melanin, but not gp100, induced a significant number of IFN-secreting lymphocytes (p<0.001) (FIG. 13A).

(80) If the gp100 epitope was added in the vaccine formulation after L-Dopa had been oxidized instead of before, a reduction in the CTL response was observed (p<0.01).

(81) Significant immunity was obtained with a weight ratio of L-Dopa:epitope starting at 1:1, with the best response observed at a ratio of 4:1 (FIG. 13B). The minimal dose of the gp100 epitope required to induce CTLs was 0.5 g (p<0.01, when compared to the lowest concentration tested) (FIG. 3c). At both 10 and 50 g of the gp100 epitope, our vaccine formulation compared favorably (p<0.01) with the combination of incomplete Freund's adjuvant (IFA) and a TLR9 agonist, a combination that is commonly used to trigger CTL responses (FIG. 13C).

Example 17Complex Melanin-Antigen

(82) Evidence for inclusion of the peptide within melanin was obtained.

(83) A solution of L-Dopa was mixed with an EPHA2 peptide (FSHHNIIRL, SEQ ID NO: 9) at a weight ratio L-Dopa:epitope of 1:4. The mixture was then oxidized at pH 8.5 in aerated conditions.

(84) When this preparation was filtered on a 10 kD filter, the melanin was retained in the filter and no peptide could detected in the filtrate (FIG. 11, lane Filtrate), suggesting that the EPHA2 peptide was bound to melanin. Indeed, when this retentate was resuspended, dissolved and heated in a loading buffer containing a strong detergent (SDS), the EPHA2 peptide got dissociated from melanin, and can be individualized on SDS-page (16% polyacrylamide gel) (FIG. 11, lane Retentate).

Example 18Analysis of the Immune Response

(85) The ability of our formulation to trigger a CD4 immune response (MHC class II epitopes) was assessed.

(86) For this purpose, a synthetic peptide (pOVALs) containing both a CD4 (ISQAVHAAHAEINEA, SEQ ID NO: 10) and a CD8 (SIINFEKL, SEQ ID NO: 11) ovalbumine epitopes was synthetized. This peptide bears the sequence SLKISQAVHAAHAEINEAGRLRGSIINFEKLTKWR, SEQ ID NO: 12).

(87) This peptide (10 g/mouse) was mixed to a solution of L-Dopa (40 g/mouse), incubated for 18 hours in aerated conditions, to generate pOVAs-melanin, and used as vaccine (without any TLR9 agonist or any further adjuvant).

(88) Mice were immunized either on day 0 & 14 and sacrificed on day 21, or on day 0 & 21 and sacrificed on day 42.

(89) Epitope-specific IFN production by splenocytes was determined after in vitro stimulation with the CD4 or the CD8 epitopes.

(90) Formulation with melanin triggered a significant CD4 immune response, in both cases. Moreover, a CD8 immune response was also seen, showing that adjunction of a TLR9 agonist to the formulation is not needed (FIG. 14).

Example 19Use of Various Adjuvants

(91) The ability of well-described TLR9 agonists, other than CpG-28, to trigger and improve the immune response in mice with the melanin/antigen complex was assessed.

(92) The gp100 peptide (10 g/mouse) was mixed with L-Dopa (40 g/mouse), incubated for 18 hours in aerated conditions, mixed with the various TLR9 agonists CpG-28, ODN-1826 (TCCATGACGTTCCTGACGTT, SEQ ID NO: 13, or ISS (TGACTGTGAACGTTCGAGATGA, SEQ ID NO: 14), then used as vaccine preparations.

(93) The magnitude of the immune response was similar for all TLR9 agonists.

(94) TABLE-US-00003 TABLE 3 number of nbr IFNy-secreting student-T test of lymphocytes (compared to TLR9 agonist mice mean +/ SD CpG-28) CpG-28 (TLR9 agonist) 4 211 +/ 83 / 1826 (TLR9 agonist) 4 134 +/ 54 p = n.s. ISS (TLR9 agonist) 4 147 +/ 63 p = n.s.

(95) The efficacy of poly I:C (poly I:C=Polyinosinic:polycytidylic acid), a TLR3 agonist, was also assessed.

(96) C57BL/6 mice were immunized with gp100 (10 g/mouse)+poly I:C (10 g/mouse) or gp100 peptide (10 g/mouse) mixed with L-Dopa (40 g/mouse), incubated for 18 hours in aerated conditions, and mixed with poly I:C (10 g/mouse).

(97) Similarly to what was seen with TLR9 agonists, when combined to poly I:C, the melanin formation was superior the free peptide (mean number of IFN-secreting lymphocytes +/SD: 20+/18 vs 1+/1, respectively, p=0.02).

Example 20Subcutaneous Injections of pOVA30-Melanin Protect Against Established Syngenic Tumors

(98) It was next investigated whether these CD8+ T-cells were functional in vivo.

(99) Ovalbumin-transfected cells (E.G7-OVA) were injected subcutaneously into C57BL/6 mice, and the mice (n=10/group) were immunized on days 4 and 18 with [melanin+CpG-28], [pOVA30-melanin+CpG-28], [pOVA30+CpG-28]. All of the mice developed measurable tumors.

(100) The pOVA30 peptide (SMLVLLPKKVSGLKQLESIINFEKLTKWTS, SEQ ID NO: 15), contains a CD8 epitope (SIINFEKL, SEQ ID NO: 11) of the ovalbumin protein. pOVA30-melanin was generated by co-incubation of the peptide with L-Dopa in aerated conditions, as described previously.

(101) A significant decrease in the tumor growth compared with that in the control groups was observed only after immunization with [pOVA30-melanin+CpG-28] (p<0.001) (FIG. 15). Complete tumor regression occurred in 2/10 mice.

Discussion and Comments

(102) The examples above demonstrate that immunization with a complex of a melanin and an antigen makes it possible to obtain an immune response in a host. This immune response is higher than when the antigen is used alone. The immune response may be potentiated when the charge of the antigen and the charge of the melanin precursor, polymerized in the complex, are adapted to each others (preferably when they are not of the same sign).

(103) It is also to be noted that most peptides used in these experiments are containing single MHC epitopes. Consequently, an adjuvant was usually preferred for an optimal immune response to be seen.

(104) However, it was also shown that combining T-helper and MHC class-I epitopes allows the immunogenic compositions according to the invention to induce significant immune responses without the need for an adjuvant, the T-helper epitopes likely allowing recruitment of the T-helper cells and potentiation of the cytotoxic response linked to the MHC class-I epitopes.