WO2013128424A1

WO2013128424A1 - Vaccine for prophylaxis or treatment of an allergen-driven and/or a chronic airways disease

Info

Publication number: WO2013128424A1
Application number: PCT/IB2013/051660
Authority: WO
Inventors: Bruno Pitard
Original assignee: Institut National De La Sante Et De La Recherche Medicale
Priority date: 2012-03-02
Filing date: 2013-03-01
Publication date: 2013-09-06

Abstract

The present invention relates to a vaccine comprising at least one nucleic acid, and at least one organic or mineral salt of a tetrafunctional copolymer of formula (I) used in a cationic form, and wherein x and y represent, independently of one another, an integer of between 1 and 500 with x having a value such that said molecule comprises at least 40% by weight of ethylene oxide units, for use in prophylaxis or treatment of anallergen-driven and/or a chronic airways disease.

Description

VACCINE FOR PROPHYLAXIS OR TREATMENT OF AN ALLERGEN-DRIVEN AND/OR A CHRONIC AIRWAYS DISEASE

FIELD OF THE INVENTION

The present invention is directed, in a main capacity, toward a vaccine for prophylaxis or treatment of an allergen-driven airways disease and/or a chronic airways disease, and in particular allergic asthma.

Asthma is a chronic, inflammatory, respiratory disease caused by an abnormal reactivity against allergens. The most promising treatments for asthma are based on specific immunotherapies, but they lack efficiency and can induce deleterious side effects. Among new modalities of immunotherapy currently in development, DNA vaccination presents a promising approach, as it enables targeted immunotherapy in association with reduced allergenicity. The invention relates to an innovative, DNA-based vaccine able to induce a strong humoral and cellular response, as well as to lead to a reduction of airway hyperresponsiveness and a significant decrease in the level of inflammatory cytokines in vaccinated individuals.

BACKGROUND OF THE INVENTION

Allergic asthma is a chronic respiratory disease affecting 300 million people worldwide (Global Initiative for Asthma (GINA), 2011). The number of individuals with asthma has doubled during the last ten years and around 250,000 people die prematurely each year due to this condition. In the majority of cases, asthma is caused by an abnormal reactivity against some environmental antigens, also called allergens. In Western Europe, the prevalence of atopic diseases (including asthma and rhinitis) is more than 30%, thus allergic diseases are considered to be an important public health issue.

Considering the pathophysiological aspects, allergic asthma is a bronchial inflammatory disease resulting from the exposition of a predisposed subject to different allergens. In Europe and the USA, the most-commonly encountered species of mite are Dermatophagoides pteronyssimus and Dermatophagoides farinae. In Europe, Dermatophagoides farinae 1 (Derfl) is one of the major allergens of Dermatophagoides farinae, and 50% of the allergic population are carriers of Derfl -IgE specific antibodies Asthma patients are usually treated with corticosteroids, which, however, only suspend the disease and are associated with deleterious side effects. An alternative treatment for allergic asthma is based on a specific immunotherapy protocol: the repeated administration of increasing doses of allergen to induce hyposensitivity, and hence reduced symptoms when another subsequent exposure to this allergen occurs. Nevertheless, the efficacy of immunotherapy remains limited, and its efficacy is very variable between patients. Moreover, immunotherapy may cause an anaphylactic crisis when administered subcutaneously.

Thus, new modalities for immunotherapy are under development, notably based on DNA vaccination, which presents a promising approach. Here, DNA encoding immunodominant peptide sequences of the allergen are administered instead of allergen extracts, leading to a targeted immunotherapy associated with reduced allergenicity. Some studies have shown therapeutic potential of this strategy. Jarman et al. reported that intramuscular administration to asthmatic mice of a naked plasmid encoding an immunodominant peptide of Der p 1 , which is a one of the major allergens in allergic asthma, led to a decrease in type-2 cytokines in bronchoalveolar lavages compared with untreated mice [2]. The antigen-encoding plasmids may also be formulated with adjuvants which are able to modulate the immune response toward a Thl or Th2 bias as reported by Kim et al, who used the Calmette and Guerin bacilli, known to induce a Thl bias, in a DNA vaccination protocol against Der p 2 [3]. Nevertheless, the amounts of DNA used in these studies were too large to consider a future application in humans. Moreover, no study has demonstrated that DNA vaccination against the specific allergen, Derfl , could prevent or reduce the development of asthmatic symptoms in a relevant animal model.

Thus, the future application of DNA vaccines in humans requires the development of a new, efficient and safe adjuvant that is capable of inducing a strong humoral and cellular response with a low dose of injected DNA.

SUMMARY OF THE INVENTION

The object of the present invention is precisely to propose a novel use of specific chemical molecules for significantly increasing the efficiency of DNA-based vaccine.

More precisely, the present invention relates to a vaccine comprising at least one nucleic acid, and

at least one organic or mineral salt of a tetrafunctional copolymer of formula (I)

CH₃ CH,

I I

H-co-CHj-oy, -(o-CH-ay ₍CH,-CH-O) -₍cn-ci o₎ -H

J I y ^χ

H-(0-CH₂-CH₂) _X -(0-CH-CH₂) _Y (CH₂-CH-0) _Y -(CH₂-CH₂-0) _Χ -H CH₃ CH₃

(I) used in a cationic form, and

wherein x and y represent, independently of one another, an integer of between 1 and 500 with x having a value such that said molecule comprises at least 40% by weight of ethylene oxide units,

for use in prophylaxis or treatment of an allergen- driven airways disease and/or a chronic airways disease.

The abovementioned molecules, which are specific poloxamines, consist of hydrophobic segments (propylene oxide bearing the y indices), of hydrophihc segments (ethylene oxide bearing the x indices) and of a positively charged central ethylenediamine component (NCH₂-CH₂N)

Previous studies showed that formulations comprising plasmid DNA in association with such tetrafunctional block copolymer, were able to safely increase the transfection efficiency of reporter or therapeutic genes in lung, skeletal and cardiac muscle in healthy and animal models compared with results achieved using naked DNA [4-6]. It has also been shown that the tetrafunctional block copolymer 704 is able to promote low- dose DNA vaccination efficiency [7]. Recently, this novel class of vector has also been used to treat hepatocellular carcinoma in a highly autochonus relevant mouse model [8]. This class of vector has also been proposed for intracellular delivery of nucleic acids (WO 03/066104).

The inventors have now surprisingly shown that a formulation of the synthetic vector pVAX-Derfl with a 304, a 704 or a 904 poloxamine led to a significant reduction of inflammatory cell infiltration and cytokines in the BAL of asthmatic mice, as well as to a reduction of airway hyperresponsiveness and of inflammatory cytokine levels. The results obtained by the inventors show that vaccination with a poloxamine-based vaccine induces a strong and specific humoral and cellular immune response, characterized by a strong Thl-bias (increase of IgG2a/IgGl ratio) and Tel (increase in IFN-γ produced by CD8+ cells by stimulation of splenocytes of vaccinated mice with antigen, such as Derfl, class I restricted peptides). Advantageously, the immunization protocol using the 704 polymer may be performed with a low-dose of DNA, which offers a number of practical advantages, such as ease of production and purification in large quantities and the absence of special handling or storage conditions. The inventors have therefore validated an immunotherapy strategy for the prevention of allergic asthma and have shown that immunotherapy to prevent allergic disease represents an attractive alternative to classical, specific immunotherapy.

According to another of its objects, the invention relates to a vaccine comprising

- at least one nucleic acid encoding for a protein Derf, or an immunogenic fragment thereof, and

- at least one organic or mineral salt of a tetrafunctional copolymer of formula

(I)

CH₃ CH,

I I

H-iO-CHj-CH,) , -(0-CH-CH_;) (OL-CH-O) -(CH,-CH,-0) -H

J I y x

H-(0-CH₂-CH₂) _x -(CH₂-CH₂-0) _χ -H

(I) used in a cationic form, and

wherein x and y represent, independently of one another, an integer of between 1 and 500 with x having a value such that said molecule comprises at least 40% by weight of ethylene oxide units.

The present invention also relates to a method of treating a patient with an allergen driven and/or a chronic airway disease comprising administering to said patient an effective amount of a vaccine according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Tetrafunctional copolymer

A tetrafunctional copolymer suitable for the invention may be a compound of formula (I)

CH₃ CH₃

H-(0-CH₂-CH₂) _X -(0-CH-CH₂) _Y (CH₂-CH-0) -(CH₂-CH₂-0) -H

I I

N-CH₂-CH₂-N

HKO-CH--CH-) -(O-CH-OL) (CH₂-CH-0) -(CH₂-CH₂-0) -H

I I

CH, CH₃ CO

The invention may also be implemented with derivatives of compounds of formula (I).

For the purpose of the present invention, the term "derivative" is intended to cover compounds which have the chemical structure of general formula I but which also carry secondary chemical or biological functions or entities capable of conferring on them complementary properties. Particularly representative of these derivatives are compounds of general formula (I) in which there is also as least one intra- or extracellular targeting unit. By way of non-limiting illustration of these targeting units, mention may more particularly be made of peptides carrying a nuclear localization sequence, or peptides which recognize receptors present at the surface of certain cells.

A compound of general formula (I) has preferably no more than 85% by weight of ethylene oxide units.

A compound of general formula (I) has in particular approximately between 40 and 80% by weight of ethylene oxide units.

According to a preferred variant of the invention, the molecules of compounds of general formula (I) also have a molecular weight of at least 800 g/mol, and more preferably of between 1000 and 25 000 g/mol.

According to a preferred embodiment of the invention, the compounds of general formula have an ethylene oxide/propylene oxide (EO/PO) unit ratio of between 0.5 and 1.5, and preferably of the order of 1 ± 0.2.

As compounds of general formula (I) that are most particularly suitable for the present invention, mention may more particularly be made of molecules having, respectively, a molecular weight of 1650 g for an ethylene oxide/propylene oxide (EO/PO) ratio of 15: 16 (for example poloxamine 304), of 5500 g/mol for an ethylene oxide/propylene oxide (EO/PO) ratio of 50:56 (for example poloxamine 704) and of 6700 g/mol for an ethylene oxide/propylene oxide (EO/PO) ratio of 61 :68 (for example poloxamine 904).

According to a preferred embodiment a compound of formula (I) may be selected in the group consisting of poloxamine 304, 704, 904, and mixture thereof.

According to a preferred embodiment, the composition is free of sodium phosphate and/or of glucose.

A tetrafunctional copolymer suitable for the invention is used in a cationic form.

A compound of general formula (I) is preferably used in the form of one of its salts, and more preferably in a cationic form. To do this, the composition claimed combines with said compound a preferably mineral salt, and more preferably an alkali metal salt or an alkaline-earth metal salt. It may in particular be chosen from sodium chloride, potassium chloride or lithium chloride and sodium thiocyanate, or more preferably calcium chloride (CaCl₂) or magnesium chloride (MgCl₂).

This salt may be introduced in isotonic, hypotonic or hypertonic amount. The inventors have also established the advantage of controlling the pH and/or the ionic composition of the formulation, in order to be sure that the copolymer of formula (I) is in its cationic form.

A pH of 6.5 to 8, preferably 7 to 7.8, more preferably 7.4, proves to be particularly advantageous.

According to a preferred embodiment of the invention, the composition is formulated in a medium referred to as Tyrode's (medium containing 3 mM CaCl₂, 2 mM MgCl₂, 6 mM KC1, 140 mM NaCl, 10 mM glucose and 10 mM Hepes, pH 7.4) (Tyrode Pharmacology. Philadelphia, 1908, 2nd edition, 1912). The presence of the Tyrode's makes it possible in particular to control the ionic composition of the formulation and the pH and, consequently, the use of the compound of formula (I) in a cationic form.

The preparation of a nucleic acid molecule with a poloxamine in a salt medium, and in particular in presence of Tyrode medium may be made as described in WO 03/066104.

Nucleic acid

For the purpose of the present invention, the term "nucleic acid" covers both a deoxyribonucleic acid and. a ribonucleic acid.

A nucleic acid of the invention may be a sequence of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, small interference RNA (iRNA) hybrid sequences, or synthetic or semi-synthetic sequences of oligonucleotides which may or may not have been modified. These nucleic acids may be of human, animal, plant, bacterial, viral, etc. origin. They may be obtained by any technique known to those skilled in the art, and in particular by screening libraries, by chemical synthesis or by mixed methods including the chemical or enzymatic modification of sequences obtained by screening libraries. They may be chemically modified.

As regards more particularly the deoxyribonucleic acids, they may be single- stranded or double-stranded, just as short oligonucleotides or longer sequences. These deoxyribonucleic acids can carry therapeutic genes, regulatory sequences for transcription or for replication, modified or unmodified antisense sequences, regions for binding to other cellular components, etc. They may in particular direct the synthesis of a polypeptide specific for an infectious agent or may be capable of remedying a genetic or acquired deficiency.

Preferably, a nucleic acid useful for the invention encodes for a peptide or a protein antigen.

The nucleic acid may comprise one or more genes encoding an antigenic peptide or protein capable of generating an immune response in humans or animals. In this particular embodiment, the invention therefore makes it possible to produce either vaccines or immuno-therapeutic treatments applied to humans or to animals, in particular against microorganisms, viruses or cancers. In a more preferred embodiment, an antigen encoded by a nucleic acid of the invention may induce a strong humoral and cellular immune response, with a pro-Thl bias.

Preferably, the nucleic acid also comprises sequences which allow the expression of the therapeutic gene and/or of the gene encoding the antigenic peptide in the desired cell or organ. They may be the sequences which are naturally responsible for the expression of the gene under consideration when these sequences are capable of functioning in the infected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or even synthetic sequences). In particular, they may be promoter sequences for eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell that it is desired to infect. Similarly, they may be promoter sequences derived from the genome of a virus. In this regard, mention may be made, for example, of the promoters of the El A, MLP, CMV and RSV genes, and tissue-specific promoters such as myosin chain promoters, for example, etc. In addition, these expression sequences can be modified by the addition of activation sequences, regulatory sequences, etc. They may also involve an inducible or repressible promoter.

Moreover, the nucleic acid can also comprise, in particular upstream of the therapeutic gene, a signal sequence which directs the synthesized therapeutic product into the target cell's secretion pathways. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid can also comprise a signal sequence which directs the synthesized therapeutic product to a particular compartment of the cell. In a preferred embodiment, a nucleic acid of the invention may encode a protein Derfl , or an immunogenic fragment thereof.

The protein Derfl and its coding nucleic acid are well-known in the art. A sequence of the protein Derfl useful for the invention may be identified in the UniProtKB/Swiss-Prot databank under the Reference PI 6311 or may be SEQ ID NO 17. A nucleic acid coding for a protein Derfl useful for the invention may be identified in the EMBL-EBI databank under the reference CAA46316.1 or may be SEQ ID NO 18.

A nucleic acid useful for the invention may also encode for an immunogenic fragment of Derfl .

An immunogenic fragment useful for the invention may comprise from 8 to 20 consecutive amino acids from the amino acids sequence of Derfl, in particular from 8 to 16 amino acids, and preferably from 8 to 12 consecutive amino acids from the amino acids sequence of Derfl .

As example of immunogenic fragments useful for the invention, one may mention the following peptides SEQ ID NO 1 a SEQ ID NO 16: anfe m awite

Pepides BiMAS

Derfl L2 FVLAIASL - S

Derfl. _ 5 VYARPAS! _ 1Q

D¾m 32 FNKNYATV T

Derfl_, _82 ^■SAEAFEQL 9 8

Derfl. .131 ■SCWAFS&V _ 1

Derfl _ .147 LAYRHTSL 4 2

Derfl 190 RSYPYVAR - 4

Derfl. .206 NSQHYGiS 6 -

Derfl. 211 G mrcQi 3 -

Derfl 2S4 YQPNYHAV S 9

Derfl. 2S0 QGVDY iV 10 -

Derfl. 290 SGYGYFQA 2 6

Derfl. .302 YFQAG L 7 -

Derfl 31 Q !EQYPYV - 3

Derfl. 313 EQYPYWI 1 -

Derfl 314 QYPVWi:^ 8 -

Airways diseases

The airways diseases considered in the invention are allergen-driven airways diseases and/or chronic airways diseases. Within the invention, chronic airways diseases intend to relate to diseases with symptoms persisting over time, and even installing definitively, and caused by a pathogen agent, such as a bacteria, a virus, or a fungus, or caused by a cancer.

An airways disease considered in the invention may be selected from the group consisting of asthma, in particular allergic induced-asthma or exercise-induced asthma, hay fever, and interstitial lung diseases.

Asthma results from bronchospasm which may be triggered by many factors such as allergen or exercise. Asthma is mainly associated with coughing, wheezing, shortness of breath, chest tightness, fatigue or stomach ache.

In a particular embodiment, airways diseases considered in the invention may be asthma, in particular allergic asthma or exercise-induced asthma. Exercise-induced asthma occurs in most of individuals suffering from asthma.

Vaccine composition

Further to compound of general formula (I) and nucleic acid, a vaccine of the invention may comprise one or more adjuvant(s), and in particular a surfactant.

By way of representation of these adjuvants, mention may more particularly be made of celluloses, such as carboxymethylcellulose or hydroxypropylcellulose, hyaluronate or alginate salts, pectins, polyethylene glycols, dextrans, polyvinylpyrrolidones, chitosans, polyvinyl alcohols, propylene glycols, polyvinyl acetates, lecithins, polylactic and polyhydroxybutyric acids, and poloxamers of the pluronics.RTM. (PEO-PPO-PEO) and reverse pluronics.RTM. (PPO-PEO-PPO) series.

Formulation of the vaccines of the present invention can be accomplished using art recognized methods. The amount of vaccines of the invention to be administered to a subject and the regime of administration can be determined in accordance with standard techniques well known to those of ordinary skill in the pharmaceutical and veterinary arts taking into consideration such factors as the adjuvant (if present), the age, sex, weight, species and condition of the particular subject and the route of administration. The administration of the vaccine is usually in a single dose. Alternatively, the administration of the vaccine of the invention is made a first time (initial vaccination), followed by at least one recall (subsequent administration), with the vaccine. The vaccines of the present invention can be formulated in any suitable manner. In general, the vaccines of the present invention can be administered orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added carriers.

A vaccine of the present invention can be administered as a capsule or tablet, as a dry powder or in liquid form. Administration can for example be achieved by injection (eg, subcutaneous, or intravenous), orally such as by dosage unit form (e.g., tablet, capsule or dosed liquid form), or by inhalation.

Typically the vaccines can be administered by nasal administration or by inhalation. This type of administration is low in costs and enables the distribution of the vaccine of the invention in the respiratory tract. Nasal administration may be accomplished with a vaccine of the invention under the form of liquid solution, suspension, emulsion. Solutions and suspensions are administered as drops. Solutions can also be administered as a fine mist from a nasal spray bottle or from a nasal inhaler. Gels are dispensed in small syringes containing the required dosage for one application. Inhalation may be accomplished with a vaccine under the form of solutions, suspensions, and powders; these formulations are administered via an aerosol, droplets or a dry powder inhaler. The powders may be administered with insufflators or puffers.

According to a specific embodiment, combinatorial methods may be implemented according to the present invention. They involve the administration of at least two agents to a patient, the first of which is a vaccine according to the present invention, and the second of which is a second therapeutic agent.

The combinatorial therapy methods of the present invention can result in a greater than additive effect, providing therapeutic benefits where neither the vaccine nor second therapeutic agent administered in an amount that is alone effective for treatment of asthma.

In the present methods, the vaccine and the second therapeutic agent can be administered concurrently or successively. As used herein, the vaccine and the second therapeutic agent are said to be administered concurrently if they are administered to the patient on the same day, for example, simultaneously, or 1 , 2, 3, 4, 5, 6, 7 or 8 hours apart. In contrast, the vaccine and the second therapeutic agent are said to be administered successively if they are administered to the patient on the different days, for example, the vaccine and the second therapeutic agent can be administered at a 1-day, 2- day or 3 -day, one-week, 2-week or monthly intervals. In the methods of the present invention, administration of the vaccine can precede or follow administration of the second therapeutic agent.

As a non-limiting example, the vaccine and second therapeutic agent can be administered concurrently for a period of time, followed by a second period of time in which the administration of the vaccine and the second therapeutic agent is alternated.

Most people with persistent asthma use a combination of long-term control medications and quick-relief medications, taken with a hand- held inhaler. Asthma symptoms triggered by airborne allergens, such as pollen or pet dander, are also treated with allergy medications.

Accordingly, suitable second therapeutic agents include long-term control medications, quick-relief medications, and allergy medications.

The second therapeutic agent may be corticosteroids, short-acting beta-2 agonists (SABAs) such as albuterol or albuterol sulfate, anti-histamines, for example.

The vaccines according to the invention may also use one or more targeting elements for directing the nucleic acid complexes to receptors or ligands at the surface of the cell. By way of example, the vaccines of the present invention may comprise one or more antibodies directed against cell surface molecules, or else one or more membrane- receptor ligands such as insulin, transferrin, folic acid or any other growth factor, cytokines or vitamins. Advantageously, the vaccines can use modified or unmodified lectins in order to target specific polysaccharides at the surface of the cell or on the neighboring extracellular matrix. Proteins containing an RGD unit, peptides containing a tandem of RGD units, which may or may not be cyclic, and also polylysine peptides or ligand peptides, which may be natural or synthetic, can thus be used.

Usually, the vaccines are administrated by intramuscular route.

A compound of general formula (I) may be present in a composition of the invention in a proportion of 0.005% to 15% by weight/volume, preferably from 0.01% to 10%) by weight/volume, and more preferably from 0.05%> to 5% by weight/volume. Also a compound of general formula (I) may be present in a composition of the invention in a proportion of 0.1 to 5% by weight/volume, and preferably in a proportion of 0.15 to 0.3% by weight/volume.

More particularly, when a compound of general formula (I) is a 704 poloxamine, it may be used in a proportion of 0.15 to 0.3% by weight/volume.

More particularly, when a compound of general formula (I) is a 304 poloxamine, it may be used in a proportion of 5 to 10% by weight/volume.

More particularly, when a compound of general formula (I) is a 904 poloxamine, it may be used in a proportion of 0.01 to 0.1% by weight/volume.

The claimed vaccines are obtained by mixing the nucleic acid under consideration with at least one compound of general formula (I) in a solution compatible with in vivo administration. By way of illustration, these vaccines may be prepared using the following protocol: a solution containing the nucleic acid (in particular DNA) two- times concentrated in 300 mM NaCl or 2 times, (two-times concentrated) Tyrode's and a two-times more concentrated aqueous solution containing a compound of general formula (I) are mixed volume for volume. The entire mixture is stirred, preferably by means of a VORTEX.

It has thus been noted that the claimed compounds of general formula (I) make it possible to increase the amount of protein synthesized by the muscle by a factor ranging from 5 to 20, compared to naked DNA. Moreover, with the vaccine according to the invention, no expression of a transgene has been noted in cells in culture in vitro, using conventional cell culturing methods.

The vaccine of the invention preferably contains a vehicle that is pharmaceutically acceptable for an injectable formulation, in particular for injection directly into the desired organ or for topical administration, for example to the skin and/or mucous membranes. They may in particular be sterile isotonic solutions or dry, in particular lyophilized, compositions which, by means of the addition, according to the case, of sterilized water or of physiological saline, make it possible to constitute injectable solutes.

It is clear that the doses of nucleic acid used for the injection and also the number of administrations can be adjusted by means of various parameters, and in particular as a function of the method of administration under consideration, of the pathology involved, of the nature of the gene to be expressed or of the desired duration of treatment.

The vaccines according to the invention are particularly advantageous from a therapeutic point of view, in particular in the field of immunization. In this case, a DNA encoding a bacterial, viral or other antigen is injected into cells, preferably muscle cells. Insofar as the claimed vaccines are particularly advantageous for increasing the amount of proteins synthesized by the transfected cells, it is possible, by virtue of this, to obtain higher concentrations of antibodies and of cytotoxic T lymphocytes.

The use of a compound of general formula I as defined above, for preparing a vaccine intended for prophylaxis or treatment of an allergen-driven and/or a chronic airways disease.

The invention also relates to a method for immunization of an individual in need thereof, comprising at least a step of administering one nucleic acid together with at least one compound of general formula I as defined above or one of its organic or mineral salts.

The administration can be carried out topically, directly into the cells under consideration, or by means of one of the routes of administration discussed above.

According to a preferred variant of the invention, the target cells are muscle cells or cardiac cells.

According to a preferred embodiment of the invention, poloxamines 304, 704 or 904 are used in accordance with the invention. Advantageously, they are formulated in a composition containing Tyrode's medium.

The present invention will be described more fully by means of the examples and figures which follow, which should be considered as non-limiting illustrations.

FIGURE LEGENDS

Figure 1: Influence of the amount of pVAX-Derfl plasmid on the humoral and cellular immune response. Groups of mice (n=5) were injected intramuscularly (i.m.) with various amounts of pVAX-Derfl, ranging from 1 to 50 μg, formulated with 704, at day 0 and day 21. (a) Humoral response was measured at day 21 (□) and at day 42 (■). Results are expressed as anti-Derfl IgG antibody titers. Mean titers are shown for each group ± standard deviation, (b) For cellular response, splenocytes were stimulated overnight with a pool of Derfl immunodominant peptides and the number of IFNy SFC was determined. Mean numbers of IFNy SFC are shown for each group ± standard deviation, c) Isotypes profile of Derfl -specific antibody. IgGl and IgG2a were titered in sera at day 42 and the ratio IgG2a/IgGl is shown as a mean for each group ± standard deviation. * P< 0.05 by the Student Newman-keuls Test. Doses given are per tibialis anterior.

Figure 2: Influence of the number and time of boost injection on the amplitude and kinetics of the humoral response. Groups of mice (n=5) were injected with 10 μg pVAX-Derfl formulated with 704 and anti-Derfl antibody IgG titer was determined at different time points. Mean titers are shown for each group ± standard deviation. Doses given are per tibialis anterior. Groups of mice were injected at day 0 only ( ^A), at days 0 and 7 (♦), at days 0 and 14 (X), at days 0 and 21 (■) or at days 0 and 28 (·). Doses given are per tibialis anterior.

Figure 3: Immunization protocols against Derfl in asthmatic mice, a) Prophylactic approach. Groups of mice (n=5) exhibiting an asthmatic phenotype following sensitization and challenge with total extract of HDM, were administered i.m. with 50 μg pVAX-Derfl plasmid formulated with 704 at days 0 and 21. b) Early vaccination approach. Groups of mice (n=5) were administered i.m. with 10 μg pVAX-Derfl plasmid formulated with 704 at days 0 and 21 and then sensitized and challenged with total extract of HDM.

Figure 4: Effect of prophylactic immunization protocol in asthmatic mice on immune response. Groups of mice (n=5) were injected i.m. at day 0 and day 21 following a prophylactic protocol (Figure 7) with 50 μg plasmid formulated with 704. (a) Healthy and asthmatic mice were injected i.m. at day 0 and day 21 with 50 μg pCMV-Pgal formulated with 704. Humoral response was measured at day 21 (□) and at day 52 (■). Results are expressed as anti-Pgal IgG antibody titers. Mean titers are shown for each group ± standard deviation, (b) Asthmatic mice were injected i.m. at day 0 and day 21 with 50 μg pVAX-Derfl formulated with 704. Humoral response was measured at day 21 (□) and at day 52 (■). Results are expressed as anti-Derfl IgG antibody titers. Mean titers are shown for each group ± standard deviation, (c) For cellular response, splenocytes were stimulated overnight with a pool of Derfl immunodominant peptides and the number of IFNy SFC was determined. Mean numbers of IFNy SFC are shown for each group ± standard deviation, (d) Isotypes profile of Derfl -specific antibody. IgGl and IgG2a were titered in sera at day 42 and the ratio IgG2a/IgGl is shown as a mean for each group ± standard deviation. Doses given are per tibialis anterior.

Figure 5: Effect of immunization protocol in asthmatic mice on asthmatic phenotype. Groups of asthmatic mice (n=5) were injected i.m. at day 0 and day 21 with pVAX-Derfl plasmid formulated with 704. Doses given are per tibialis anterior, (a) Airway responsiveness was measured at day 52 using a plethysmography system following intranasal instillation of 5 to 20 mg/ml methacholine in healthy mice (^A), asthmatic mice (■) and asthmatic, vaccinated mice (♦). Results are expressed in enhanced pause (Penh), as a mean for each group ± standard deviation, (b) BAL cellular composition. Cell proportions in BAL were established after May Grunwald Giemsa coloration on about 300 cells in healthy mice (□), asthmatic mice (□) and asthmatic, vaccinated mice (■). Results are expressed as absolute number of cells in 1 ml BAL, as a mean for each group ± standard deviation. * P< 0.05 by the Student Newman-keuls Test, (c - f) IL-4, IL-5, IL-10 and IFN-γ levels in BAL were determined using Luminex technology.

Figure 6: Effect of prophylactic immunization protocol in asthmatic mice on asthmatic phenotype. Groups of asthmatic mice (n=5) were injected i.m. at day 0 and day 21 with pVAX-Derfl plasmid formulated with 704. Doses given are per tibialis anterior, (a) Airway responsiveness was measured at day 73 using a plethysmography system following intranasal instillation of 5 to 40 mg/ml methacholine in healthy mice asthmatic mice (■) and asthmatic vaccinated mice (♦). Results are expressed in enhanced pause (Penh), as a mean for each group ± standard deviation, (b - e) IL-4, IL-5, IL-10 and IFN-γ levels in BAL of control mice or asthmatic mice immunized with pVAX- Derfl or pCMV- Gal plasmids formulated with 704, were determined using Luminex technology. * p<0.05 by the T test (Mann- Wit hney). Groups included at least 17 different mice. BIBLIOGRAPHY Zock, J. P., et al. (2006). Distribution and determinants of house dust mite allergens in Europe: the European Community Respiratory Health Survey II. J Allergy Clin Immunol 118: 682-690.

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Richard, P., Bossard, F., Desigaux, L., Lanctin, C, Bello-Roufai, M., and Pitard, B. (2005). Amphiphilic block copolymers promote gene delivery in vivo to pathological skeletal muscles. Hum Gene Ther 16: 1318-1324.

Mcllroy, D., et al. (2009). DNA/amphiphilic block copolymer nanospheres promote low-dose DNA vaccination. Mol Ther 17: 1473-1481.

Cany, J., et al. AFP-specific immunotherapy impairs growth of autochthonous hepatocellular carcinoma in mice. J Hepatol 54: 115-121.

EXAMPLES

MATERIALS AND METHODS

Animal procedures

Balb/C mice (Elevage Janvier, Le Genest, France) were housed in conventional conditions according to INSERM guidelines. Mice aged 8 weeks were used for all experiments. For intramuscular (i.m.) DNA vaccination, mice were anesthetized by isofiurane inhalation, then different DNA-polymer formulations were injected into both tibialis anterior muscles using an Insumed Pic Indolore 30G syringe (Artsana, Grandate, Italy). Two sites were injected per animal, DNA doses are given per tibialis anterior. In all cases, the injection volume was 50 μΐ per injection site. After vaccination, sera and spleens were recovered for analysis of the immune response at the end of each experiment.

Plasmid preparation and formulation

The pCMV-Pgal plasmid (Clontech, St Germain en Laye, France) encoding β- galactosidase, and the pVAX-Derfl plasmid encoding Derfl, under the control of the human cytomegalovirus immediate promoter, were used as antigen. The pVAX-Derfl plasmid was obtained by cloning the Derfl gene sequence into pVAX (Invitrogen, Courtaboeuf, France) with Hindlll and Xhol restriction enzymes. pQE30 (Qiagen, Courtaboeuf, France) was used as carrier DNA. All plasmids were purified using EndoFree plasmid purification columns (Qiagen). The tetrafunctional block copolymers 304, 704 and 904 were kindly supplied by In-Cell-Art (Nantes, France). Plasmid DNA was formulated immediately prior to i.m. injection in Tyrode's medium, as previously described [7].

Derfl expression

For Derfl -expression experiments, tibialis anterior muscles were dissected, and immediately frozen in liquid nitrogen. Derfl expression was then quantified in muscle extracts using the Derfl ELISA kit (Indoor Biotechnologies, Wiltshire, UK), according to the manufacturer's protocol.

Measurement of the immune response

Humoral immune responses were measured by enzyme-linked immunoassay. For anti-Derfl IgG, IgGl and IgG2a antibody dosage, 96-well plates (Nunc Maxisorp, Roskilde, Denmark) were coated overnight at 4°C with 2.5 μg/ml purified Derfl (Indoor Biotechnologies, Wiltshire, UK) in 50 mmol/1 NaHCOs pH 9.5 and then blocked for 1 hour at room temperature with PBS 0.05% Tween-20 1% bovine serum albumin, before distributing diluted sera in duplicate. Plates were incubated at 37°C for 90 min, then Derfl - specific IgG, IgGl and IgG2a were detected using peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Newmarcket, UK) diluted 1/5000, goat anti-mouse IgGl (Abd Serotec, Oxford, UK) diluted 1/10,000 and goat anti-mouse IgG2a (Abd Serotec, Oxford, UK) diluted 1/10,000 in PBS 0.05% Tween-20, respectively. Peroxidase activity was revealed with 1 mg/ml ortho-phenylene diamine in pH5 citrate buffer. Reactions were stopped by addition of 1 mol 1 H₂S0₄, and optical density was measured at 490 nm. Anti- Derfl IgG titers were calculated from the regression curve of a standard Derfl -specific mouse serum included in each ELISA plate. The absolute titer of the standard was 204,800 and was diluted from 1/1000 to 1/64,000 to build a calibration curve. Each tested sample was diluted two times and dilutions with OD included in the linear part of the calibration curve were conserved for antibody titer determination. Other dilutions were excluded. Similarly, for anti-P-gal IgG antibody dosage, 96-well plates (Nunc Maxisorp, Roskilde, Denmark) were coated overnight at 4°C with 5 μg/ml purified β-gal (Roche, Rosny-sou- bois, France) in 50 mmol 1 NaHC0₃ pH 9.5 and then blocked for 1 hour at room temperature with PBS 0.05%> Tween-20 1% bovine serum albumin, before distributing diluted sera in duplicate. Plates were incubated at 37 °C for 90 min, then β-gal-specific IgG was detected using peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Newmarcket, UK) diluted 1/5000 in PBS 0.05%> Tween-20. Peroxidase activity was revealed with 1 mg/ml ortho-phenylene diamine in pH 5 citrate buffer. Reactions were stopped by addition of 1 mol/1 H₂S0₄, and optical density was measured at 490 nm. Anti-P-gal IgG titers were calculated from the regression curve of a standard β- gal-specific mouse serum included in each ELISA plate. The titer of the standard was arbitrarily fixed at 5000 and was diluted from 1/1000 to 1/64,000 to build a calibration curve. Each tested sample was diluted two times and dilutions with OD included in the linear part of the calibration curve were conserved for antibody titer determination. Other dilutions were excluded.

As a paradigm read out of the cellular response to 704 vaccine formulation, Derfl -specific CD8+ response was tested by class I-restricted IFNy secretion determined by ELISPOT (Abcys, Paris, France). Sixteen 8-mer Derfl -derived peptides predicted to bind with H-2Kb were selected based on the binding score, as calculated by the BIMAS and Net MHC software (Supplementary Table 1) and were used as representative Derfl epitopes. The negative control was the KRWIILGLNK peptide (HIV gag 263-272). Live splenocytes were counted on a hematocytometer slide by Trypan blue exclusion, resuspended at 10⁶/ml in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 rnmol/1 L-glutamine, penicillin and streptomycin, all from Invitrogen, Paisley, UK), then distributed in triplicate at 10⁵ cells/well. Cells were incubated overnight at 37 °C in 5% C0₂ in the presence of 4 μg/ml peptide. Spot-forming colonies (SFC) were detected according to the manufacturer's protocol, counted automatically on an AID ELISPOT Reader (Autoimmun Diagnostika, Strassberg, Germany) and results expressed as SFC/million splenocytes after subtraction of the nonspecific signal obtained with the negative control.

Allergic asthma mouse model

Balb/C mice were sensitized and challenged with total extract of Dermatophagoides farinae, kindly supplied by Stallergenes (Anthony, France), according to the protocol described in Figure 5. Mice were sensitized percutaneously in each ear with 250 μg total extract of HDM diluted in 10 μΐ dimethylsulfoxyde, and challenged by intranasal inhalation of 250 μg total extract of HDM diluted in 40 μΐ PBS.

Measurement of airway responsiveness

Airway responsiveness was measured in conscious, unrestrained mice using barometric, whole-body plethysmography by recording respiratory pressure curves (EMKA Technologies, Paris, France) in response to inhaled methacholine (Sigma Chemical Co.) at concentrations of 5-20 mg/ml for 1 min. Airway responsiveness was expressed in enhanced pause (Penh) units, a calculated value, which correlates with the measurement of airway resistance, impedance and intrapleural pressure in the same mouse: Penh = (te/tr-1) x Pef/Pif ( (te = expiration time, tr = relaxation time, Pef = peak expiratory flow, Pif = peak inspiratory flow).

Bronchoalveolar lavage analysis

Mice were tracheotomized and administered 1 ml sterile PBS intratracheally through a flexible catheter. Cells and supematants from recovered fluid were separated by centrifugation. Total cell number was determined by optical microscopy and a sample was spotted by Cytospin. Cell proportions were established after May Grunwald Giemsa coloration on about 300 cells. Cytokine analysis

The expression levels of cytokines IL-4, 11-5, IL-13 and IFN-γ were determined in BAL supernatant using Luminex® technology. A bead-based Bio-plex kit (Bio-Rad Laboratories, Munich, Germany) was used to measure IL-4, IL-5, IL-13 and IFN-γ. The assays were performed according to the manufacturer's specifications. Cytokines were quantified using an eight-point calibration curve constructed from the standard provided. Data analysis was performed using the Bio-PlexManager Software version 4.0.

Statistical analysis

Statistical analysis was conducted using Sigma Stat 2.03 software. All data are expressed as mean ± standard deviation. Statistical analysis was performed using the Studen-Newman Test. A p value < 0.05 was considered as significant.

Example 1

Optimization of the formulation containing various amounts of DNA encoding Derfl

Mice were injected intramuscularly with the plasmid encoding the Derfl antigen formulated with 704. Five days after injection, muscles were collected and evaluated for Derfl expression levels by ELISA. Injection of naked plasmid DNA resulted in no significant expression of the Derfl protein in the muscle, irrespective of the amount of injected DNA. Conversely, injection of plasmid DNA formulated with the block copolymer 704 led to a significant increase in Derfl expression in muscle when the amount of injected DNA encoding Derfl was increased.

The ability of this synthetic formulation to elicit specific humoral and cellular immune responses against Derfl was then evaluated. Mice were injected i.m. at days 0 and 21 with various amounts of pVAX-Derfl formulated with 704. Cellular and humoral responses were analyzed at days 21 and 42 after the first injection. Dose-response experiments showed a maximal humoral response for 10 μg pVAX-Derfl, which then remained at this level for higher doses (Figure la). To analyze the cellular response, we synthesized sixteen 8-mer immunodominant peptides from Derfl, predicted to bind to the H-2Kb-restricted MCH 1 molecule.

Rank to s¾:oriJ :m

-m Pepides Sequence S«AS

Derfl _2 FVUUASL - S

Derfl _15 VYARPASi 10

Derfl 32 FNKNYATY - 7

Derfl _82 SAEAFEQL 9 8

Derf1_131 SCWAFSGV - 1

Derf1_147 LAYRINTSL 4 2

Derfl 190 RSYPYVA - 4

Der!1_206 NSQHYGiS S -

Derfl _21 1 GISMYCQl 3- -

Derfl 264 YQPNYHiAV 9

Derf1_280 QGVDY iV 10 -

Derfl _2§8 SGYGYFQA 2 6

Derfl _302 YFQAGMML 7 -

Derfl 310 Mi!EQYP - 3

Derfl _313 EQYFYYV! 1 -

Derfl 314 QYPYVViM S - The tables displays the sequence of the sixteen selected 8-mer peptides susceptible to bind with H-2Kb with their relative ranks (when higher than 12^th) in BIMAS (www-bimas.cit.nih.gov/molbio) and NetMHC (http://www.cbs.dtu.dk/services/NetMHC/) algorithms.

These peptides were used in IFNy ELISPOT assays to evaluate the specific

CD8+ T-cell response induced by Derfl DNA/704 immunizations in mice. The results showed that the cellular response increased with increasing amount of injected pVAX- Derfl formulated with 704 and reached a plateau at 10 μg pVAX-Derfl (Figure lb). To measure the orientation of the immune response, we measured different, specific anti- Derfl antibody isotypes for various amounts of injected, formulated plasmid DNA. Formulation with 704 led to the induction of a Thl-bias, as indicated by the predominance of Derfl -specific IgG2a compared with Derfl -specific IgGl (Figure lc).

Example 2

Optimization of the vaccine formulation and protocol

To optimize the anti-Derfl vaccine formulation, we tested the effect of three different block copolymers which were previously shown to enhance reporter gene expression in vivo and vaccination efficiency. Mice were injected i.m. on days 0 and 21 with 10 μg pVAX-Derfl plasmid formulated at two doses of the block copolymers 304, 704 or 904. Each formulation led to a significant and specific humoral immune response at day 42, while the maximal humoral response was obtained with block copolymer 704 at the concentration of 0.15%. All formulations tested were able to elicit a cellular response. Interestingly, the block copolymer 704 at 0.15 % promoted a high cellular response, as measured by INF-γ ELISPOT assay.

Given that DNA vaccination usually requires a repeated injection scheme to elicit the development of a strong immune response characterized by high antibody titers and memory B cell activation, we investigated the influence of the prime-boost injection protocol on the immune response. Mice were injected i.m. on day 0 with 10 μg pVAX- Derfl plasmid formulated with the block copolymer 704, and then boosted one, two, three or four weeks later. Antibody titers were analyzed each week during 10 weeks (Figure 2). It was clear that the mice that received a boost one or two weeks after the primary injection, presented very low antibody titers, comparable with those obtained using only one injection. On the contrary, mice receiving a boost injection three or four weeks after the primary injection showed a very robust increase in their antibody titers, occurring two or one week after the boost injection, respectively. Seventy days after the primary injection, antibody titers were still detectable for all conditions. As boosting at 21 days after the primary injection led to the sustained presence of antibodies in the serum, we selected this injection scheme for Derfl vaccination in an allergic mouse model.

Example 3

Derfl vaccination in an allergic asthma mouse model

The efficiency of our vaccination formulation against Derfl was tested in a model of asthmatic mice. The allergic asthma phenotype was induced using a total house- dust-mite (HDM) extract in Balb/C mice. Firstly, mice were sensitized by percutaneous administration of 250 μg total HDM extract, once a week for 4 weeks (days 0, 7, 14, 21). They were then challenged three times by intranasal inhalation of 250 μg total HDM extract, at days 28, 42, and 49. Two different pVAX-Derf 1/704 vaccination schemes of asthmatic mice were used to study the impact of anti-Derfl vaccination in these mice.

In the first setting, Balb/C mice were vaccinated at the onset of the HDM sensitization (Figure 3a). First, we evaluated the influence of the mouse model on the 704- mediated vaccination efficiency. To this end, mice were immunized using the β- galactosidase antigen at the onset of HDM sensitization. Mice were immunized twice, according to the protocol described in Figure 3, using pCMV-Pgal reporter plasmid formulated with the block copolymer 704. Humoral response was assessed at day 52, after the last HDM challenge. The dosage of the specific, anti-P-galactosidase IgG antibodies indicated that no significant differences between healthy or asthmatic mice were obtained. This result shows that the induction of an asthmatic phenotype does not modify the immune response against an antigen expressed by gene transfer with 704 (Figure 4a). As a control, we also investigated whether the immunization protocol with 704 against Derfl could induce, by itself, an asthmatic phenotype. Analysis of respiratory parameters of healthy mice immunized with the synthetic vector, pVAX-Derfl/704, showed no differences compared with untreated healthy mice, indicating that the immunization protocol did not promote an asthmatic phenotype. Then, at the same time as the HDM sensitization, mice were immunized at days 0 and 21 , using pCMV-P-gal plasmid as control plasmid or using pVAX-Derfl plasmid formulated with the block copolymer 704. At day 52, after the last HDM challenge, different parameters were assessed. The results showed that asthmatic mice vaccinated against Derfl present high levels of Derfl -specific IgG antibodies (Figure 4b), associated with a strong Thl bias, as attested by the predominance of Derfl -specific IgG2a compared with Derfl -specific IgGl (Figure 4d). As a control, the dosage of the specific Derfl IgG antibodies in non- vaccinated healthy, and in β-galactosidase-vaccinated asthmatic mice, did not show the presence of Derfl -specific IgG antibodies. This indicates that the production of the asthmatic mouse model did not induce a specific immune response against Derfl . Under the same experimental conditions, healthy and asthmatic mice vaccinated against β-galactosidase did not present a significant cellular response, whereas the asthmatic mice vaccinated against Derfl presented a high level of IFN-y-secreting splenocytes stimulated by a cocktail of Derfl peptides (Figure 4c).

Asthma is usually characterized by airway hyper-responsiveness and by an influx of inflammatory cells and cytokines in the lung. Airway responsiveness was measured following inhalation of methacholine (5 to 20 mg/ml), which induces bronchoconstriction, expressed as enhanced pause (Penh). The results show that asthmatic mice presented a high Penh value compared with healthy mice (Figure 5 a). Asthmatic mice immunized against β-galactosidase or Derfl antigen presented no significant variation of Penh compared with nonvaccinated, asthmatic mice. The influx of inflammatory cells in the lung was assessed by cell counts in bronchoalveolar lavage (BAL) and by histological analysis of lung tissues. The BAL of healthy mice contained essentially macrophages, whereas the BAL of asthmatic mice contained macrophages and also numerous neutrophils, eosinophils and lymphocytes. Interestingly, the asthmatic, Derfl -vaccinated mice presented significantly reduced amounts of macrophages and eosinophils compared with the asthmatic mice vaccinated against β-galactosidase antigen (Figure 5b). The numbers of neutrophils and lymphocytes were also reduced. We also determined the expression of inflammatory cytokines in the BAL using Luminex technology. The BAL of asthmatic mice immunized against the control antigen, β-galactosidase, presented high levels of IL-4, IL-5 and IL-13, which are proinflammatory cytokines, and reduced levels of IFN-γ, which is a pro-Thl cytokine. Conversely, asthmatic mice presented reduced levels of IL-5 and IL-13 and an increased level of IFN-γ in BAL, compared with asthmatic mice vaccinated against the β-galactosidase antigen (Figure 5c,d,e,f). Altogether these data show that, in this first setting, vaccination of asthmatic mice against the major allergen, Derfl, promoted a significant reduction in inflammatory cells and cytokines observed in the BAL, as compared with asthmatic mice vaccinated against a control antigen.

In the second setting, mice were given two vaccination injections at days 0 and 21. On day 28, they were HDM-sensitized followed by HDM challenges (Figure 3b). Mouse airway hyperreactivity was measured at day 73. Asthmatic mice immunized with the control antigen, β-galactosidase, presented a high Penh value compared with healthy mice, while asthmatic mice vaccinated with pVAX-Derfl formulated with 704 presented a reduction of Penh for all amounts of methacholine used, indicating a reduction of airway hyperresponsiveness (Figure 6a). Measurements of inflammatory cytokine expression levels in the BAL, using Luminex technology, showed that the BAL of asthmatic mice immunized against the control antigen, β-galactosidase, presented high levels of IL-4, IL-5 and IL-13, which are proinflammatory cytokines, and reduced levels of IFN-γ, which is a pro-Thl cytokine. Conversely, asthmatic mice presented reduced levels of IL-4, IL-5 and IL-13 and increased levels of IFN-γ in BAL, compared with asthmatic mice vaccinated against β-galactosidase antigen (Figure 6b,c,d,e) Altogether these data show that early vaccination of asthmatic mice against the major allergen, Derfl, promotes a significant reduction of airway hyper-responsiveness and of cytokines observed in the BAL, as compared with asthmatic mice vaccinated with a control antigen. Thus, early vaccination of asthmatic mice may confer a protective effect on the development of asthmatic symptoms.

Claims

1. A vaccine comprising

at least one nucleic acid, and

CH₃ CH,

I I

Η-(ο-α¾-<¾⁾ -(o-CH-ciy ₍CH,-CH-O) -(CH,-CH,-O) -H

J I y χ

(I) used in a cationic form, and

2. The vaccine according to claim 1, wherein the airways disease is selected from the group consisting of asthma, in particular allergic induced-asthma or exercise- induced asthma, hay fever, and interstitial lung diseases.

3. The vaccine according to claim 1 or 2, wherein the compound of general formula (I) has no more than 85% by weight of ethylene oxide units.

4. The vaccine according to any one of the preceding claims, wherein the compound of general formula (I) has approximately between 40 and 80% by weight of ethylene oxide units.

5. The vaccine according to any one of the preceding claims, wherein the compound of general formula (I) has a molecular weight of at least 800 g/mol, and more preferably of between 1000 and 25 000 g/mol.

6. The vaccine according to any one of the preceding claims wherein the ethylene oxide/propylene oxide (EO/PO) ratio is between 0.5 and 1.5, and preferably of the order of 1 ± 0.2.

7. The vaccine according to any one of preceding claims, wherein the compound of formula (I) is chosen from the compounds having a molecular weight of 1650 g/mol with an ethylene oxide/propylene oxide (EO/PO) ratio of 15: 16, a molecular weight of 5500 g/mol for an ethylene oxide/propylene oxide (EO/PO) ratio of 50:56, and a molecular weight of 6700 g/mol for an ethylene oxide/propylene oxide (EO/PO) ratio of 61 :68.

8. The vaccine according to any one of the preceding claims, wherein the compound of formula (I) is selected in the group consisting of poloxamine 304, 704, 904, and mixture thereof.

9. The vaccine according to any one of the preceding claims, wherein the compound of general formula (I) is present in a proportion of 0.005% to 15% by weight/volume, preferably from 0.01% to 10%> by weight/volume, and more preferably from 0. 05%) to 5% by weight/volume.

10. The vaccine according to any one of the preceding claims, further comprising at least one salt and in particular selected in the group consisting of calcium chloride, magnesium chloride, potassium chloride, lithium chloride, sodium thiocyanate and mixtures thereof.

11. The vaccine according to any one of the preceding claims, formulated with Tyrode's medium, i.e. 140 mM NaCl, 6 mM KC1, 3 mM CaCl₂, 2 mM MgCl₂, 10 mM

Hepes, pH 7.4, and 10 mM glucose.

12. The vaccine according to any one of the preceding claims, wherein the nucleic acid is a deoxyribonucleic acid, a ribonucleic acid, an antisense, an interfering R A or therapeutic gene.

13. The vaccine according to any one of the preceding claims, wherein the nucleic acid encodes for a protein DERF1, or an immunogenic fragment thereof.

14. The vaccine according to any one of preceding claims, being administrable by nasal route, by inhalation, or by intramuscular route.

15. Vaccine comprising

- at least one nucleic acid encoding for a protein DERF1, or an immunogenic fragment thereof, and

(I)

CH, CH,

H-(0-CH₂-CH₂) _x -(0-CH-CH₂) (CH₂-CH-0) -(CH₂-CH₂-0) -H

H-(0-CH₂-CH₂) -(0-CH-CH₂) (CH₂-CH-0) -(CH₂-CH₂-0) _χ -H

CH, CH,

(I)

used in a cationic form, and

16. The vaccine according to claim 15 wherein the copolymer of formula (I) is as defined in any one of claims 3 to 9.

17. The vaccine according to claim 15 or 16 further comprising at least one salt as defined in claims 10 to 11.