ES2424831A1 - Immunological adjuvant for the formulation of vaccines and leishmaniasis vaccine comprising same - Google Patents

Abstract

The invention relates to the use of compounds comprising a molecule of lipid origin and a vinyl sulfone group as an immunological adjuvant. The lipid molecule binds to an antigen in order to form a lipopeptide that acts as a vaccine. The invention also relates to an immunnoadjuvant comprising said compounds, to a vaccine comprising said lipopeptides, i.e. the compound of the invention used as an immunoadjuvant and an antigen, and specifically to the abovementioned Leishmaniasis vaccine.

Description

IMMNULOLOGICAL ASSISTANT FOR THE FORMULATION OF VACCINES AND VACCINATION AGAINST LEISHMANIASIS THAT UNDERSTANDS

FIELD OF THE INVENTION

The present invention relates to immunoadjuvants or immunological adjuvants and to vaccines comprising them. More particularly, it refers to vaccines against Leishmaniasis comprising said immunoadjuvants.

10 STATE OF THE TECHNIQUE Vaccinations

Vaccines constitute, in the diseases for which they exist, the best method of prevention in the face of personal and public health, having as an indisputable success the

15 induction of an immunoprophylaxis created after vaccination. This has led to the elimination of some diseases that have been real blunders for world health, smallpox, and the near disappearance of many of the infectious diseases that have hit humanity, in those populations where this type of immunoprophylaxis is practiced .

20 Vaccines usually have two substantial advantages, are economical and do not induce drug resistance in the pathogens against which they are directed.

In the case of viral diseases, or for those in which chemotherapy is non-existent, expensive or very toxic, they constitute the best form of struggle.

Vaccines induce effective responses in the immune system against the infectious agent causing the disease or against the toxins released by said agent, in order to eliminate the causative effects of the disease.

30 Ultimately, they induce an immune response against pathogens or their toxins that can neutralize the adverse effects of pathogen antigens, considering antigens as substances capable of inducing an immune response. These antigens, whole organisms, may be a portion of the

The same, which are part of the infectious agent, or substances secreted by them or even any natural, synthetic or artificially obtained substance that resembles the natural ones that the pathogen possesses or excretes.

Once the immune system has been sensitized to the pathogenic organism, the subsequent exposure of the immune system to the pathogen or its substances results in a rapid immune response that cancels said pathogen before they can induce signs of disease in the vaccinated organism.

The goal of any vaccination is to induce a potent immune response capable of protecting an organism from infection as long as possible.

Adjuvants

Unlike what happens with vaccines that use living organisms, vaccines that use dead or attenuated infectious agents, antigens

15 purified, or toxins require the use of immunity enhancers called adjuvants.

The first adjuvant described was published in 1924 by Ramón [Sur la toxine et sur l’anatoxine diphthériques. Ann. Inst. Pasteur, 38, 1-10]. Since then they have

20 used aluminum salts in most vaccines. A review of adjuvants used in vaccines can be found in [Vogel FR, Powell MF. A summary compendium of vaccine adjuvants and excipients. In: Powell MF, Newman MJ, editors. Vaccine design: the subunit and adjuvant approach. New

25 York: Plenum Publishing. Corp .; 1995. p. 234-250].

The efficacy of an immunization and adjuvants will depend on the type of route of administration of the vaccine, since the same response is not obtained with immunization through mucous membranes as with parenteral administration, as well.

Depending on this, the use of new vectors, antigen release systems, and / or adjuvants will depend on the route chosen.

The adjuvants can be classified according to their mode of action or their chemical nature or origin, so we will have:

-

Antigen release systems: among which are many of the traditional adjuvants such as alumina, liposomes, microparticles; "like-virus" particles; nano antigen release particles; liposomes; polymer microspheres; ISCOMs (nanoparticles formed by phosphatidyl choline, cholesterol and Quil A).

5 - Immunity potentiators:

•

Inorganic adjuvants;

•

Adjuvants of microbial or plant origin: LPS (lipopolysaccharides); subunits of toxins of bacterial origin (cholera toxin B); Muramil dipeptide; Bacterial DNA; CpGs; or derivatives of Lipid A,

10 as monophosphorylipid A ("MPL") or chemical derivatives. All of them are being tested as adjuvants in clinical experiments.

• Other adjuvants are oily emulsions in vegetable or animal oils, or interleukins or the DNA that encodes them.

One of the most promising adjuvants currently is what are called lipopeptides, compounds based on the chemical modification of the antigens by way of immunity enhancers of bacterial origin bound to lipids equal to or similar to lipid A.

20 The first related work dates from 1984 (Hopp TP. Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier. Mol Immunol 1984;

21: 13–16). This paper describes an increase in the response to synthetic influenza virus peptides administered together with dipalmityl-lysine, thus constituting the strategy of binding proteins to lipophilic chains. Other subsequent works 25 (Schild H, Deres K, Wiesmüller KH, Falk K, Jung G, Rammensee HG. Efficiency of peptide and lipopeptide for in vivo priming of virus-specific cytotoxic T cells. Eur J Immunol 1991; 2: 2649. Deres K, Schild H, Wiesmüller KH, Jung G, Rammensee HG. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopepide vaccine. Nature 1989; 342: 561–64., Schild H, Norda M, Deres K, et al Fine specificity 30 of cytotoxic T lymphocytes primed in vivo either with virus or synthetic lipopeptide vaccine or primed in vitro with peptide. J Exp Med 1991; 174: 1665–68S) describe how an influenza virus lipopeptide triggered a cytotoxic response, This fact greatly increased the potential use of protein-bound lipids in vaccines. To date there are numerous studies in which they synthesize

35 lipopeptides or protein conjugates with lipids inducing a CTL response similar to when live vaccines have been used, thus avoiding the risk they entail.

(Brynestad K, Babbit B, Huang L, Rouse BT. Influence of peptide acylation, liposome incorporation, synthetic immunomodulators on the immunogenicity of a 1-23 peptide of glycoprotein D of herpes simplex virus: implications for subunit vaccines. J Virol 1990;

64: 680–85). The cellular mechanisms by which lipopeptides cause this

The response is also not known exactly, being a complex mechanism in which the antigen bound to the lipid interacts with the antigen presenting cells (APC). (BenMohamed L, Thomas A, Bossus M, et al. High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules. Vaccine 2000; 18: 2843–55.).

10 Lipopeptides activate macrophages by triggering a response involving IL1, IL6 and TNF-a. (Rouaix F, Gras-Masse H, Mazingue C, et al. Effect of a lipopeptidic formulation on macrophage activation and peptide presentation to T cells. Vaccine 1994; 12: 1209–14). One of the advantages they have is that they are used

15 synthetic or recombinant peptides that form micelles with fatty acids protect epitopes from proteolytic enzymes (BenMohamed L, Thomas A, Bossus M, et al. High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules Vaccine 2000; 18: 2843-55 .; BenMohamed L, Gras-Masse H, Tartar A, et al. Lipopeptide

20 immunization without adjuvant induces potent and long-lasting B, T helper; and cytotoxic T lymphocytes responses against a malaria liver stage antigen in mice and chimpanzees. Eur J Immunol 1997; 27: 1242–53) of tissues and serum thus improving the immunogenicity of the antigen by being better absorbed by APC cells (Deprez B, Sauzet JP, Boutillon C, et al. Comparative efficiency of simple lipopeptide

25 constructs for in vivo induction of virus-specific CTL. Vaccine 1996; 14: 375–82; Tsunoda I, Sette A, Fujinami R, et al. Lipopeptide particles as the immunologically active component of CTL inducing vaccines. Vaccine 1999; 17: 675–85.). And especially to be able to use said adjuvants in vaccination systems using as antigen with synthetic peptides.

Vaccines in Leishmaniasis

Leishmaniasis, in general, constitutes a disease that groups three types of lesions that vary according to the etiological species and, sometimes, the individual's immune status. These forms are divided into cutaneous, mucocutaneous and visceral. After the publication of the Leishmania genome the

possibility of the more rational use of antigens capable of conferring a certain immunity.

While there are a number of drugs for leishmaniasis that have been useful both

5 for visceral leishmaniasis as for cutaneous leishmaniasis since approximately the 20s of the last century, the high toxicity that some of these molecules possess, and the emergence of resistance to traditional and newly developed drugs forces a continuous study of research into new molecules; this leads to perhaps the most effective way of

10 control leishmaniasis, in general, be the search for a vaccination system that moves away from the risk of contracting the disease to inhabitants of endemic areas.

The first generation of Leishmania vaccines known as "leishmanization" was developed in the early 1940s and has been used for more

15 of 60 years in many countries among others in Iran. It is based on the intradermal inoculation of live promastigotes of L. major to protect from the effects of this species, the problem is that in some individuals it leads to the appearance of serious skin lesions. The experience is positive for some vaccinated, but very negative for others.

20 This first generation of vaccines uses attenuated promastigotes, dead, or extracts, with several attempts to attenuate them but the results have been negative or inconclusive. The best known are Brazil's Mayrink *, subsequently produced in Venezuela only for L. mexicana administered with BGC and the

25 vaccine against L. major produced in Iran. The prophylactic results were not convincing, but they are still used in immunotherapy.

The second generation consists of vaccines that use Leishmania sp. genetically modified, native or recombinant proteins. Many surface proteins have been identified. These include glycoprotein 63 (gp63), membrane glycoprotein 46 (gp46 or M-2), fucose-mannose ligand (FML), homologous receptor activated by kinase C (p36 / LACK), chimeric protein NH36, cysteine proteinase B and A (CP), GRP78, LD1, acylated hydrophilic surface protein (HASPB1), LCR1, saliva protein of vectors 15 (SP15), surface antigen in promastigotes 2 (PSA-2), A-2, histone H-1, MML, elongation factor (LeIF), inducible stress homologous protein 1 (LmSTI1), TSA and Leish-111f, etc.

Many defined antigens can protect experimental animals but to date only one is being clinically tested, Leish-111f together with the MPL-SE adjuvant.

5 Of all of them, the only one that has passed the experimentation phases and that is marketed in Brazil is the FML glycoprotein complex with a saponin adjuvant against canine leishmaniasis, under the name of Leishmune® (EP 1893640).

The third generation of vaccines is based on DNA vaccines. The discovery of

10 direct injection of plasmids with DNA encoding foreign proteins could lead to the biosynthesis of endogenous proteins and a specific immune response by opening new fields in vaccine development. They have advantages over the others, since they are easy to produce, stable and easy to administer. To date, vaccines have been tested with genes encoding gp63, LACK, cpa and cpb, NH36, KMP

15 11, TSA and LmSTI1 but none of them has obtained results against more than one species.

Several vaccines candidate for mass use have been tested to date, but the reports are contradictory, since the efficacy in the protection and immune response generated by these antigens is confusing. Most are capable of inducing the Th1 type response in animal models, while other authors believe that the protection of the disease involves both responses, Th1 and Th2. In addition, factors such as the number of doses, immunomodulators used, experimental protocols and the animals used complicate the question of the study. Definitely,

25 to this day, there is only one vaccine registered against canine and human leishmaniasis (using the FML fucose-mannose receptor). It is the Leish-111f + MPL-SE vaccine, which is being clinically tested [Skeiky et al., "Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL® adjuvant," Vaccine 20 : 3292-3303, 2002].

30

DESCRIPTION OF THE INVENTION

The present invention relates to the use of compounds comprising a molecule of a lipid nature and a vinyl sulfone group as an immunological adjuvant. The lipid molecule binds to an antigen to form a lipopeptide that acts as a vaccine. In addition, the present invention also relates to an immunoadjuvant that

It comprises said compounds, to a vaccine comprising these lipopeptides, that is, the compound of the invention used as an immunoadjuvant and an antigen and in particular, to the vaccine described above for Leishmaniasis.

Among the advantages of this adjuvant are:

•

Easy preparation and binding to the antigenic molecule giving stability and binding to lipophilic chains in a covalent manner, which converts them both to native, recombinant or synthetic peptides or proteins into amphipathic molecules with an apolar group consisting of the lipid chain.

•

They must surround the antigens with the hydrophobic groups protecting them from enzymatic degradation and constituting as "spherules" that favor the entry through transmembrane mechanisms, into the antigen presenting cells (APC) and their subsequent antigen presentation.

•

They can favor their action by stimulating certain TLRs (Toll type receptors) that favor an inflammatory immune response given the interleukins capable of inducing.

•

They constitute a "lipopeptide" and acts as an adjuvant giving a mostly TH17 response.

Therefore, a first aspect of the present invention relates to the use of a compound comprising a lipid as an immunological adjuvant, wherein said lipid is attached to the vinyl sulfone directly or by a linker.

The term "linker" (linker) refers to an organic molecule that covalently binds a lipid and a vinyl sulfone group.

Here, the term "adjuvant", "immune adjuvant" or "immunoadjuvant" refers to an agent, which does not have an antigenic effect by itself, but which can stimulate the immune system by increasing its response to the vaccine.

In a preferred embodiment, the compound comprising a lipid and a vinyl sulfone group is the compound of general formula (I)

(I) . where:

5 Y is a group -CH2-SO2R1- or -CH2-; preferably Y is -CH2-R1 is a radical selected from the group comprising a (C1-C10) alkyl, a (C1-C10) alkenyl, a (C1-C10) alkynyl, a (C1-C10) dialkylaryl Ar (C1- C10) or a group (CH2CH2O) nCH2CH2; preferably R1 is a group (CH2CH2O) nCH2CH2; n takes values from 1 to 20; preferably n takes values between 2 to 10, plus

10 preferably n is 2 to 5 and even more preferably n is 2.

X is a group –CO-NH-R2-Z-CH2-, -Z-CH2- or –CH2-; Z is S or O; R2 is a radical selected from the group comprising a (C1-C10) alkyl, a

(C1-C10) alkenyl, a (C1-C10) alkynyl, a (C1-C10) dialkylaryl (C1-C10) or a group (CH2CH2O) mCH2CH2; m takes values from 1 to 20; preferably m takes values from 2 to 10, more preferably m is from 2 to 5 and even more preferably m is 2; Y

It represents a lipid.

By "lipid" is meant in the present invention a molecule of a non-polar nature, such as saturated or unsaturated hydrocarbons, for example but not limited to alkyl groups (C1-C30), alkenes (C2-C30), alkynes ( C2-C30) or sterols. The majority of these types of molecules are biomolecules, composed mainly of carbon and hydrogen and to a lesser extent oxygen, although they can also contain phosphorus, sulfur and nitrogen, whose main characteristic is being hydrophobic

or insoluble in water and yes in organic solvents. Preferably, the lipid is a sterol or a saturated or unsaturated aliphatic hydrocarbon.

By "aliphatic hydrocarbon" refers, in the present invention, to organic molecules consisting of carbon and hydrogen, in which the carbon atoms form linear or branched, saturated or unsaturated chains. That is, they can be both alkyl (saturated) and alkenyl or alkynyl (unsaturated) groups. Preferably the lipid can be selected from a hydrocarbon (C4-C30), saturated or unsaturated. And more preferably the carbon number is between 10 and

twenty.

By "sterol" refers in the present invention to steroids with 27 to 29 carbon atoms. Its chemical structure derives from cyclopentaneperhydrophenanthrene or esterano, a 17-carbon molecule consisting of three hexagonal and one pentagonal rings. You can select from the list that includes, but not limited to cholesterol,

10 epicolesterol, stigmasterol, lanosterol, ergosterol and coprostenol. More preferably the sterol is cholesterol.

By "alkyl" refers in the present invention to aliphatic, linear or branched chains, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc. When we refer to lipids, they would be aliphatic chains having 1 to 30 carbon atoms, more preferably 4 to 30 carbon atoms and even more preferably 10 to 20 carbon atoms. When we refer to the R2 group, independently, they would preferably be aliphatic chains having 1 to 10 carbon atoms, more preferably 2 to 5 carbon atoms and even more

20 is preferably an ethyl.

By "alkenyl" in the present invention refers to an alkyl radical, described above, and having one or more unsaturated bonds, specifically has at least one double bond, although it can also have at least one triple bond. The

Alkenyl radicals may be optionally substituted by one or more substituents such as an aryl, halogen, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, nitro, etc.

By "alkynyl" refers in the present invention to an alkyl radical, described

30 above, and having one or more unsaturated links, specifically has at least one triple link, although it can also have at least one double link. Alkenyl radicals may be optionally substituted by one or more substituents such as an aryl, halogen, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, nitro, etc.

By "dialkylaryl" is meant in the present invention an aryl group that is substituted with two alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably having 1 to 5 carbon atoms. The alkyl, alkenyl or alkynyl groups may be the same or different, preferably they are the same. And "aryl" means in the present invention an aromatic or heteroaromatic system having 6 to 12 carbon atoms or some other atom, such as O, N, S, etc ..., can be single or multiple ring , separated and / or condensed. Typical aryl groups contain 1 to 3 separate or condensed rings and from about 6 to 10 about 18 ring carbon atoms, such as phenyl, naphthyl, indenyl, phenanthryl or anthracil radicals

In another preferred embodiment, the compound of formula (I) is the compound N- (2- (2 (vinylsulfonyl) ethoxy) ethyl) oleamide, of formula (II)

(II)

A more preferred embodiment of the present invention relates to the use of the compound of formula (II) as an adjuvant for the manufacture of vaccines. More preferably, as an adjuvant in vaccines against parasitic diseases and more preferably against Leishmaniasis.

Another aspect of the present invention relates to a vaccine comprising the immunological adjuvant described in the present invention together with an antigen or an antigenic composition.

In the context of the present invention the term "vaccine" refers to an antigen or an antigenic preparation or composition used to establish the immune system response to a disease. By "antigen" or "antigenic preparation or composition" is meant a substance foreign to an organism that, once introduced into it, causes the immune response through the production of antibodies, and an activation of the humoral and / or cellular immune response and generate immunological memory producing permanent or transient immunity. This may be a native, recombinant protein or synthetic peptide. Depending on the antigen or the antigenic composition, the vaccines of the invention may be vaccines against viruses, protozoa, parasites or tumors.

By "parasitic disease", the present invention is understood as an infectious disease caused by protozoa, vermes (cestodes, trematodes, nematodes) or arthropods. In particular the parasites are protozoa or vermes, preferably

5 trematodes or nematodes. In a more preferred embodiment the parasites belong to the Trypanosomatidae family, more preferably the parasites are of the genus Leishimania. Species thereof, may be, but not limited to, Leishmania infantum, Leishmania brazilensis, Leishmania donovani, Leishmania tropic or Leishmania chagasi, among others, known to a person skilled in the art.

10 Parasitic diseases to be treated could be leishmaniasis (or leishmaniasis). "Leishmaniosis" is a disease caused by a protozoan of the genus Leishmania and transmitted, mainly by phlebotome or jejenes mosquitoes. This disease occurs in humans and vertebrate animals, such as sermarsupials, canids,

15 rodents and primates.

In another preferred embodiment, the present invention relates to a Leishmaniasis vaccine comprising the immunological adjuvant described in the present invention and the recombinant antigens. Recombinant antigens could be

20 use, among others, glycoprotein 63 (gp63), membrane glycoprotein 46 (gp46 or M-2), fucose-mannose ligand (FML), homologous receptor activated by Kinase C (p36 / LACK), chimeric protein NH36, cysteine proteinase B and A (CP), GRP78, LD1, acylated hydrophilic surface protein (HASPB1), LCR1, saliva protein of vectors 15 (SP15), surface antigen in promastigotes 2 (PSA-2), A- 2, histone

25 H-1, MML, elongation factor (LeIF), homologous inducible stress protein 1 (LmSTI1), TSA, Leish-111f, or Prohibitins 1 and 2, among others. Preferably, the Leishmaniasis vaccine comprises the immunological adjuvant described in the present invention and the recombinant antigens consisting in this case of two types of proteins belonging to the recombinant Prohibitins, Phb 1r and Phb 2r.

Another aspect of the present invention relates to a method of preparing the vaccine of the invention comprising the following steps:

to. solubilization of the immunological adjuvant of the invention in alcohol at a pH

basic, in an incubation time that ranges from 10 minutes to 24 hours;

b.

add to the solution of step (a) the antigen or the antigenic composition;

C.

incubate the mixture from step (b) at a temperature between 0 and 37 ° C for 10 min to 24h .;

d.

add a basic buffer with an amino acid or substance capable of blocking vinyl sulfone functions that would not have reacted to the incubated mixture of the

5 step (c) and incubate for 2 to 3 hours at a temperature between 4 ° C and 40 ° C, preferably between 22 ° C and 37 ° C.

The immunological adjuvants of step (a) are the lipid-vinylsulfone compounds (LVS) described in the present invention, more preferably the compounds of

10 general formula (I) and even more preferably the compound of formula (II).

In a more preferred embodiment, the solubilization described in step (a) is carried out in methanol and carbonate buffer, preferably at 0.125 M (1: 1), pH 8 until a final concentration of 20mg / ml is reached.

In another preferred embodiment, in step (b) the mixing of the solution resulting in step (a) with the antigen or antigenic composition described above is carried out in a 5: 1 ratio.

In another preferred embodiment, in step (c) the incubation is carried out at a laboratory temperature, preferably between 4 ° C and 37 ° C, more preferably at 4 ° C, and between 10 min and 24h, preferably 12 hours.

In another preferred embodiment, in step (d) the basic buffer is a carbonate and more

Preferably the amino acid or substance capable of blocking the vinyl sulfone functions that have not reacted previously is glycine, even more preferably carbonate buffer with 0.1 M glycine.

Throughout the description and claims the word "comprises" and its

30 variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1.- Representation of the weight in grams of the spleens measured in the different test groups. The graph reflects the variation of the weight presented by the spleens of the test groups. Where Contr No Infec: corresponds to the control group no

5 infected; Cont Infect: is the weight of the spleens of the Infected groups. Ph1 + Ph2 corresponds to the weight of the spleens of animals immunized with the two recombinant proteins without adjuvant. ph1 + ph2 II corresponds to the weight of the spleens of animals immunized with the recombinant proteins together with the adjuvant of formula (II).

10 Fig. 2.- Representation of the size of the skin lesion in the focus of infection measured in the different test groups and the percentage of lesion reduction considering the maximum inflammation size of infected and non-immunized animals with any of The two preparations. Where Control: corresponds to

15 control group not infected; Infection Control: group of infected animals. p1p2 control: Group of animals immunized with the two recombinant proteins without adjuvant. p1p2 + II group of animals immunized with recombinant proteins next to the adjuvant of formula (II).

20 Fig. 3.- Representation of the Stimulation Index comparing the control group with the vaccinated group with the adjuvant of formula (II). Lympoproliferation of splenocytic cells from BALB / C mice inoculated and subsequently infected with L. major. The cells were incubated at 37 oC for three days in RPMI medium. p1p2Control: corresponds to the splenocyte stimulation index of the spleens

25 of the animals immunized with the two recombinant proteins without adjuvant; p1p2 + II corresponds to the splenocyte stimulation index of the spleens of animals immunized with the recombinant proteins together with the adjuvant of formula (II).

30 Fig. 4.- Represents the expression levels of interleukin IL2 measured in the different test groups. Interleukin corresponding to a type of TH1 response. Where; Control: unimmunized and uninfected mice; Infec Control: non-immunized and yes infected mice; p1p2 + II: Phb1r and Phb2r bound to the adjuvant of formula (II); p1p2ontrol: Phb1r and Phb 2r without the adjuvant of formula (II).

Fig. 5.- Representation of the levels of expression of interleukin IL12 measured in the different test groups. Interleukin corresponding to a type of TH1 response. Where Control: corresponds to the uninfected control group; Infec Control: group of infected animals. p1p2 control: Group of animals immunized with the two recombinant proteins without adjuvant. p1p2 + II group of animals immunized with

5 the recombinant proteins together with the adjuvant of formula (II).

Fig. 6.- Interferon γ expression levels (IFN γ). Interleukin corresponding to a type of TH1 response. Abbreviations: Control: unimmunized and uninfected mice; Infec Control: non-immunized and yes infected mice; p1p2 + II: Phb1R and Phb2R linked

10 to the adjuvant of formula (II); p1p2Control: Phb1R and Phb2R without the adjuvant of formula (II).

Fig. 7.- Expression levels of TNF-a. Where, Control: corresponds to the level of TNF-a expression of the uninfected control group; Infec Control: corresponds to

15 level of TNF-a expression of the Infected groups. p1p2Control: corresponds to the level of expression of TNF-a of the group of animals immunized with the two recombinant proteins without adjuvant. p1p2 + II corresponds to the level of TNF-a expression of animals immunized with the recombinant proteins together with the adjuvant of formula (II).

20 Fig. 8.- Expression levels of IL4. Where, Control: corresponds to the level of IL4 expression of the uninfected control group; Infec Control: corresponds to the level of IL4 expression of the Infected groups. p1p2Control: corresponds to the level of IL4 expression of the group of animals immunized with the two proteins

25 recombinants without adjuvant. p1p2 + II corresponds to the level of IL4 expression of animals immunized with recombinant proteins together with the adjuvant of formula (II).

Fig 9.- IL10 expression levels. Where, Control: corresponds to the level of

IL10 expression of the uninfected control group; Infec Control: corresponds to the level of IL10 expression of the Infected groups. p1p2Control: corresponds to the level of expression of IL10 of the group of animals immunized with the two recombinant proteins without adjuvant. p1p2 + II corresponds to the level of IL10 expression of animals immunized with recombinant proteins together with the adjuvant of

35 formula (II).

Fig. 10.-Expression levels of IL17. Where, Control: corresponds to the expression level of IL17 of the uninfected control group; Infec Control: corresponds to the level of IL17 expression of the Infected groups. p1p2Control: corresponds to the level of expression of IL17 of the group of animals immunized with the two proteins

5 recombinants without adjuvant. p1p2 + II corresponds to the level of IL17 expression of animals immunized with recombinant proteins together with the adjuvant of formula (II).

Fig. 11.- Expression levels of the TGB-�. Where, Control: corresponds to the level of

10 expression of TGB-� from the uninfected control group; Infec Control: corresponds to the level of TGB-� expression of the Infected groups. p1p2Control: corresponds to the level of TGB-� expression of the group of animals immunized with the two recombinant proteins without adjuvant. p1p2 + II corresponds to the level of TGB-� expression of animals immunized with recombinant proteins next to the

15 adjuvant of formula (II).

EXAMPLES

Tests have been conducted to demonstrate the effectiveness of this adjuvant using

20 as an infectious agent Leishmania major strain Friedlin (MHOM / IL / 81 / Friedlin, (clone VI). The response that it induces regarding immunization control without said adjuvant is compared.

For the immunization test and subsequent challenge, 20 Balb / c mice, 25 divided in batches, were used.

Two groups were inoculated intradermally as indicated:

1) Test group inoculated with 10 µg of recombinant antigens

Called as Phb 1r and Phb 2r, from Leishmania major bound to the compound of formula (II) as described below and subsequently challenged as described below.

2) Control group of the adjuvant, inoculated with 10 µg of the recombinant proteins called Phb 1r and Phb 2r, without binding said compound and subsequently challenged. 3) White Control Group with only inoculation of the parasite forms

infected at the same time and form that the challenge of the previous groups was realized. 4) Control group not vaccinated not challenged.

5 The groups were vaccinated, inoculating 3 times with the different forms and formulations of the vaccine antigens with an interval of 2 weeks the first two inoculations and after 30 days of the second inoculation were vaccinated for the last time.

10 6 months after inoculations, the groups were infected with 1000 purified metacyclic forms of the infecting metacyclic forms (Kimblin, N., et al., Quantification of the infectious dose of Leishmania major transmitted to the skin by single sand flies. Proc Natl Acad Sci USA, 2008. 105 (29): p. 10125-30) of L. major in the plantar pad of the hind limbs.

15 Three months after infection all mice were sacrificed, collecting blood and spleens. After slaughter, spleens and lesions originating in the limbs were measured, in order to determine the protection that said vaccine formulations exercised over infection.

20 Determining by means of qPCR the levels of different interleukins, as well as the levels of expression by intracellular parasites as a way to determine the level of parasitization.

25 With the sera of the mice, the absorbance rates were measured by immunoenzymatic techniques, ELISA, against the inoculated antigens when facing said sera to them.

UNION OF THE PROTEINS RECOMBINANT TO THE COMPOUND (II)

The compound (II) was solubilized in methanol and 0.125 M carbonate buffer (1: 1), pH 8 until reaching a final concentration of 20mg / ml. It was mixed in with the proteins in a 5: 1 ratio, incubating 12 hours at 4 ° C.

35 After that time, 20 µl of 0.125M1 1M glycine carbonate buffer was added to block the sulfone groups not bound to the proteins by incubating for 2 to 3 hours at 4 ° C.

ASSESSMENT OF THE ASSISTANT EFFECT

5 Measurement of injury and spleen and titration of sera

At 6 months of immunization, blood samples were taken from each mouse to subsequently titrate the serum against the recombinant proteins by the ELISA technique. After the trial period, the batches of mice were

10 slaughtered At the time of sacrifice the measurements of the injury produced in the leg and the size of the spleen were taken. In turn, the serum of each animal was collected for titling and see its evolution compared to the first titration performed.

Lymphoproliferation

The lymphoproliferation, measured as a result of DNA synthesis by splenocytes, was carried out based on the incorporation of Thymidine3H into the precipitable material of the splenic cells. To obtain the cells used in lymphoproliferation studies, splenocytes obtained from

20 the spleens of the immunized animals that after eliminating the red blood cells and determining the viability of the splenocytes were cultured in RPMI 1640 supplemented medium (5% SBF, 2 mM glutamine, 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U / ml of Penicillin and 100 µg / ml of Penicillin).

25 Splenocytes were stimulated with 25 g / ml of the two proteins used as antigens and as a positive control they were stimulated with 5 g / ml of Concanavalin A (Sigma). Splenocytes as a negative control, were maintained in the medium without the addition of any compound, and 18 hours before the stimulation measure, 1 Ci of 6-3H-Thimidine (with a specific activity of

30 26-30 Ci / mmol), diluted in RPMI 1640 added with 10% fetal bovine serum.

Duplicates of each plate were made, one for the measurement of lymphoproliferation and another for collecting cells and measuring cytokine levels.

35 The culture plate where the experiments were carried out was incubated for 72 h at 37 ° C in a humid atmosphere with 5% CO2. The cells were collected at 72 h and were

washed with PBS, centrifuging at 400g for 5 min at 4 oC. The button, containing the cells, which was to be treated to measure lymphoproliferation, was resuspended in 3 volumes of 10% cold trichloroacetic acid, incubated at 4 ° C for 2 h, and then centrifuged 10 minutes at 200g. Subsequently, they were washed with 3 ml of a 5% trichloroacetic acid solution and finally washed by centrifugation, with absolute ethanol and 80% ethanol. Once the vials were washed, they were filled with scintillation liquid (PPO 4g; POPOP 0.1g; toluene 1000 ml) and the radioactivity was measured in a spectrometer (BECKMAN LS 6000TA). The lifoproliferation index was determined by the DPM culture / Problem DPM ratio of the

10 cultures from unstimulated control cells. The maximum stimulation level was performed with splenocytes stimulated with ConA.

All experiments were repeated three times.

15 Determination of cytokines

The cytokine determination was performed by their level of expression by RTqPCR with the mRNAs extracted from the stimulated splenocytes.

20 Quantitative determination of cytokines by RTQPCR

To obtain mRNA and, subsequently, its retrotranscription to cDNA, the methodology described by (Nicolas, L., et al., Leishmania major Reaches Distant Cutaneous Sites Where It Persists Transiently while Persisting Durably in the Primary was followed)

25 Dermal Site and Its Draining Lymph Node: a Study with Laboratory Mice. Infection and Immunity, 2000. 68 (12): p. 6561-6566.)

Primer Sequence Genes

Reference gene HPTR F: 5´-TGCTGACCTGCTGGATTACA-3´ (SEQ ID (hypoxanthine-guanine NO: 1) phosphoribosyltransferase) R: 5´-TATGTCCCCCGTTGACTGAT-3´ (SEQ ID

NO: 2) TNF-a F necrosis factor: 5´-AGCCCCCAGTCTGTATCCTT-3´ (Tumor SEQ ID-a NO: 3)

R: 5´-GGTCACTGTCCCAGCATCTT-3´ (SEQ ID

NO: 4)

Interferon gamma INF-y F: 5´-CACCCTGAAGTCGTTGTGAA-3´ (SEQ ID NO: 5)

R: 5´- CGTCAAAAGACAGCCACTCA-3´ (SEQ ID

NO: 6) Growth factor TGF-F: 5´-GTGTGGAGCAACATGTGGAA-3´ (SEQ ID transformant-NO: 7)

R: 5´-CGTCAAAAGACAGCCACTCA-3´ (SEQ ID NO: 8)

Interleukin 2 IL-2 F: 5´- AAGCTCTACAGCGGAAGCAC-3´ (SEQ ID NO: 9) R: 5´-ATCCTGGGGAGTTTCAGGTT-3´ (SEQ ID NO: 10)

Interleukin 4 F: 5´-TTGAACGAGGTCACAGGAGA-3´ (SEQ ID Il-4 NO: 11)

R: 5´-CACCTTGGAAGCCCTACAGA-3´ (SEQ ID NO: 12)

Interleukin 10 F: 5´-CTGTTTCCATTGGGGACACT-3´ (SEQ ID IL-10 NO: 13)

R: 5´-AAGTGTGGCCAGCCTTAGAA-3´ (SEQ ID NO: 14)

Interleukin 12 F: 5´-CCTGAAGTGTGAAGCACCAA-3´ (SEQ ID IL-12 NO: 15)

R: 5´-TCAGGGGAACTGCTACTGCT-3´ (SEQ ID NO: 16)

Interleukin 17 IL-17 F: 5´-TTCAGGGTCGAGAAGATGCT-3´ (SEQ ID NO: 17)

R: 5´-AAACGTGGGGGTTTCTTAGG-3´ (SEQ ID

NO: 18) Table 1.- Primers used for the quantification of the expression of the genes of the different cytokines by means of RTPCR.

Expression levels were performed on a C-1000 * thermocycler attached to the CFX96 * module for Biorad Real-Time (Bio-Rad), using the SsofastEvagreenSupermix kit (Bio-Rad).

The efficiency of both amplifications was checked by quantifying serial dilutions of PCR amplified products. The efficiency percentages were determined, according to the .CT method, where the reference / test ratio = 2CT reference

5 CT test, where the reference is the HPTR gene and the test are the study genes. All these tests were carried out in triplicate.

Statistic analysis

10 Statistical analyzes of the results obtained were carried out in the Prism 5.04 version 2010 program and the Graphpat Instar version 3.06 program.

RESULTS

15 In all mice, the spleens and the skin lesion caused by the infection were weighed and measured. As can be seen in figures 1 and 2.

The lowest weight of the spleens occurred in the groups where it had been immunized with the recombinant proteins linked to vinyl sulfone fatty acid (rPhb1-rPhb2-LiVSulf), 20 where a spleen weight similar to the uninfected group (Cni) was obtained.

Regarding the size of the lesion, there was a 61% reduction in the group that was treated with the proteins in combination with (rPhb1-rPhb2 LiVsulf) with respect to the untreated group.

Study of cell stimulation levels and determination of cytokine expression

The study of the type of immune response induced in different groups of mice

30 based on the determination of interleukins, it was carried out by determining RT-PCR measurement of the expression levels of the genes of said proteins: IL2, IL4, IL10, IL12, IL17, TNF-a, TGF- and INF-y.

On the other hand, lymphoproliferation levels (stimulation indices) were analyzed, 35 as a response level to treatments from splenocytes not stimulated or stimulated with the antigens used in immunizations, incubation with said

antigens were carried out for 72 h. As a positive stimulation control the cells were incubated with the Con-A lectin. Stimulation rates were determined by the levels of incorporation of Thymidine3H, (1 µCi / well) which was added 18 hours before the sacrifice of the cells. The levels of incorporation of

The radioactive analogue to the DNA of the cells (as a marker of cell division) was determined with a scintillation counter expressing the results in counts per minute (cpm).

Stimulation levels are reflected in Figure 3, from which it follows

10 that maximum levels of stimulation occurred in the rPhb1-rPhb2-LiVSulf (p1p2 + II) groups.

DETERMINATION OF EXPRESSION LEVELS OF INTERLEUKIN ANSWER MARKERS Th1.

The expression levels of th1 type interleukins, ie IL 2, IL 12, IFN γ and the tumor necrosis factor TNF a, are reflected in the corresponding graph where the levels of expression of said interleukins are represented in unstimulated splenocytes. and stimulated with the antigens (Fig. 4, 5 and 6,

20 respectively).

IL2 levels show a significant increase in the rPhb1-rPhb2-LiVSulf group (p <0.001) (Fig. 4).

25 Figure 5 shows that IL12 levels are increased in the rPhb1-rPhb2-LiVSulf group (p <0.001) and the rest of the groups maintain levels similar to the infection control group.

INF-y levels are shown in Figure 6. The rPhb1-rPhb2-LiVSulf group

30 (p1p2 + II) shows a highly significant increase (p <0.001) with respect to the other groups.

Tumor Necrosis Factor (TNF-a) expression levels show (Fig. 7), again, an increase in the rPhb1-rPhb2-LiVSulf group (p <0.001).

DETERMINATION OF EXPRESSION LEVELS OF INTERLEUKIN ANSWER MARKERS Th2.

In this case the expression levels studied were IL4 and IIL10.

5 As can be seen in Figure 8, the groups that show a significant increase in IL4 with respect to the infection group were: p1p2 + (II) (p <0.001).

If we analyze the levels of IL 10 (Fig. 9), the only group that presents an increase is the rPhb1-rPhb2-LiVSulf group.

10 DETERMINATION OF EXPRESSION LEVELS OF INTERLEUKIN RESPONSE MARKERS Th17.

IL 17 expression levels showed that in the Phb1-rPhb2-LiVSulf group,

15 a significant increase in this type of response was induced, compared to the rest of the groups (Fig. 10).

DETERMINATION OF EXPRESSION LEVELS OF INTERLEUKIN ANSWER MARKERS Th3.

20 The expression of the tumor growth factor (TGF-) was analyzed. Fig. 11. The p1p2 + (II) group showed, again, a significant increase with respect to the rest of the groups.

From these results it follows that the compound of formula (II), bound to the antigens through the vinyl sulfone group leaving the lipid chains free, acts as an antigenic adjuvant stimulating an inflammatory response. It can be used in vaccination, intercutaneously, intraperitoneally, intramuscularly or through mucous membranes, in immunization processes in which it is required to activate this type of

30 answer

Claims (22)

one.

Use of a compound comprising a lipid and a vinyl sulfone group as an immunological adjuvant.

2.

Use according to the preceding claim, wherein the compound is a compound of general formula (I):

(I) where: Y is a group -CH2 – SO2R1- or -CH2-; R1 is a radical selected from the group comprising a (C1-C10) alkyl, a (C1-C10) alkenyl, a (C1-C10) alkynyl, a (C1-C10) dialkylaryl Ar (C1-C10) or a group ( CH2CH2O) nCH2CH2; n takes values from 1 to 20; X is a group –CO-NH-R2-Z-CH2-, -Z-CH2- or –CH2-; Z is S or O; R2 is a radical selected from the group comprising a (C1-C10) alkyl, a (C1-C10) alkenyl, a (C1-C10) alkynyl, a (C1-C10) dialkylaryl Ar (C1-C10) or a group ( CH2CH2O) mCH2CH2; m takes values from 1 to 20; Y It represents a lipid.

3.

Use according to any of the preceding claims wherein the lipid is a sterol.

Four.

Use according to the preceding claim, wherein the sterol is selected from the list comprising cholesterol, epicolesterol, stigmasterol, lanosterol, ergosterol and coprostenol.

5.

Use according to any of the preceding claims, wherein the lipid is a saturated aliphatic (C4-C30) hydrocarbon.

6.

Use according to any one of claims 1 to 4, wherein the lipid is an unsaturated aliphatic (C4-C30) hydrocarbon.

Use according to any of claims 5 or 6, wherein the lipid is an aliphatic hydrocarbon (C10-C20).

8.

Use according to any of claims 2 to 7, wherein X is a group -CO-NH-R2-Z-CH2- and Z is defined in claim 2.

9.

Use according to claim 8 wherein R2 is a (C2-C5) alkyl group.

10.

Use according to claim 9 wherein R2 is an ethyl group.

11. Use according to any of claims 2 to 7 wherein X is the group -Z-CH2- and Z is defined in claim 2. 12. Use according to any of claims 8 to 11, wherein Z is O. 20 13. Use according to any of claims 8 to 11, where Z is S.

14.

Use according to any of claims 2 to 7, wherein X is -CH2-.

fifteen.

Use according to any of claims 2 to 14, wherein the group Y is -CH2-.

16.

Use according to any of claims 2 to 14, wherein R1 is a group (CH2CH2O) nCH2CH2 and n takes values between 1 to 10.

17.

Use according to claim 16, wherein n takes values between 2 and 5.

18. Use according to claim 17, wherein n is 2. 19. Use according to claim 1, wherein the compound is of formula (II): Use of a compound described according to any one of claims 1 to 19, for the manufacture of a vaccine. 21. Use of a compound described according to any of claims 1 to 19, for the manufacture of a vaccine against a parasitic disease, preferably Leishmaniasis. 22. Use according to the preceding claim, wherein the vaccine comprises Leishmania Prohibitin 1 and 2 proteins as an antigenic component. 23. Use according to the preceding claim, wherein the vaccine is against Leishmaniasis. 24. A vaccine comprising the compound described according to any one of claims 1 to 19 and an antigen or antigen component. 25. Vaccine according to the preceding claim, wherein said vaccine is in a form suitable for administration parenterally, orally, to the mucosa, sublingual, transdermal, topical, by inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal , vaginal, by gene gun, dermal patch or in the form of eye drops or mouthwash. 26. Vaccine according to any of claims 25 or 26, wherein the antigenic component comprises Leishmania Prohibitin 1 and 2 proteins. 27. Method of preparing a vaccine as described in 30 claims 24 to 26, comprising the following steps:

to.

solubilization of the immune adjuvant described in any one of claims 1 to 19 in alcohol at a basic pH, in an incubation time ranging from 10 minutes to 24 hours;

b.

add to the solution of step (a) the antigen or the antigenic composition;

C.

incubate the mixture from step (b) at a temperature between 0 and 37 ° C for 10 min to 24 h;

d.

add a basic buffer with an amino acid or substance capable of blocking the sulfone residues that had not reacted, to the incubated mixture of step (c) and incubate for 2 to 3 hours at a temperature between 4 ° C and 40 ° C.

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11

SEQUENCE LIST

<110> <120>

University of Granada IMMNULOGICAL ADJUSTER FOR THE FORMULATION OF VACCINES AND VACCINES AGAINST LEISHMANIASIS THAT UNDERSTANDS

<130>

IPR-474

<160>

18  

<170>

PatentIn version 3.5

<210> <211> <212> <213>

1 20 DNA Artificial Sequence

<220> <223>

HPTR direct primer

<400> 1 tgctgacctg ctggattaca

     twenty

<210> <211> <212> <213>

2 20 DNA Artificial Sequence

<220> <223>

HPTR reverse primer

<400> 2 tatgtccccc gttgactgat

     twenty

<210> <211> <212> <213>

3 20 DNA Artificial Sequence

<220> <223>

TNF-alpha direct primer

<400> 3 agcccccagt ctgtatcctt

     twenty

<210> <211> <212> <213>

4 20 DNA Artificial Sequence

<220> <223>

TNF-alpha reverse primer

<400> 4 ggtcactgtc ccagcatctt

     twenty

<210> <211> <212> <213>

5 20 DNA Artificial Sequence

<220> <223>

INF-gamma direct primer

<400> 5 caccctgaag tcgttgtgaa <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> INF-gamma reverse primer <400> 6 cgtcaaaaga cagccactca <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Direct TGF-beta primer <400> 7 gtgtggagca acatgtggaa <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse TGF-beta primer <400> 8 cgtcaaaaga cagccactca <210> � <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Direct IL-2 primer <400> � aagctctaca gcggaagcac <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-2 reverse primer <400> 10 atcctgggga gtttcaggtt <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Direct IL-4 primer

<400>

11 ttgaacgagg tcacaggaga

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse IL-4 primer

<400> 12

caccttggaa gccctacaga

twenty

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Direct IL-10 primer

<400> 13

ctgtttccat tggggacact

twenty

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse IL-10 primer

<400> 14

aagtgtggcc agccttagaa

twenty

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Direct IL-12 primer

<400> 15

cctgaagtgt gaagcaccaa

twenty

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse IL-12 primer

<400> 16

tcaggggaac tgctactgct

twenty

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Direct IL-17 primer

<400> 17

ttcagggtcg agaagatgct

twenty

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse IL-17 primer

<400> 18

aaacgtgggg gtttcttagg

twenty

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201331159 SPAIN Date of submission of the application: 07.27.2013 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C07C317 / 28 (2006.01) A61K39 / 39 (2006.01) RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

P MOYLE et al., Current Medicinal Chemistry 2008, vol 15, No. 5, pp. 506-516 (Bentham Science Publishers). "Self-adjuvanting lipopeptide vaccines", the whole document. 1-27

TO

I TSUNODA et al., Vaccine 1999, vol 17, pp. 675-685. "Lipopeptide particles as the immunologically active component of CTL inducing vaccines". 1-27

TO

WO 0147553 A1 (UNIVERSITY COLLEGE LONDON) 05.07.2001, claims. 1-27

TO

WO 2006122382 A2 (UNIVERSIDADE FEDERAL DO RIO DE JANEIRO) 23.11.2006 21-27

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 25.09.2013

Examiner M. Fernández Fernández Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201331159 Minimum documentation sought (classification system followed by classification symbols) A61K Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, CAS, EMBASE, MEDLINE, BIOSIS State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201331159 Date of Completion of Written Opinion: 05.09.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-27 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-27 Claims IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201331159 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

P MOYLE et al., Current Medicinal Chemistry 2008, vol 15, No. 5, pp. 506516 (Bentham Science Publishers). "Self-adjuvanting lipopeptide vaccines", the whole document. 2008

D02

I TSUNODA et al., Vaccine 1999, vol 17, pp. 675-685. "Lipopeptide particles as the immunologically active component of CTL inducing vaccines" 1999

D03

WO 0147553 A1 (UNIVERSITY COLLEGE LONDON) 05.07.2001

D04

WO 2006122382 A2 (UNIVERSIDADE FEDERAL DO RIO DE JANEIRO) 11.23.2006

2. Statement motivated according to articles 29.6 and 29.7 of the Regulation of execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The application refers (claims 1-18) to the use of a compound of formula (I) (see claim 2), which is a lipid with a vinyl sulfone group, as an immunological adjuvant. A compound of formula (II) is specified in claim 19 and in claims 20-23 its use for the manufacture of a vaccine against leishmaniasis, wherein the antigen is Leishmania Phb 1 and 2 proteins. In claims 24-27 the vaccine and its preparation are claimed. Document D1 discloses the use of lipopeptides as adjuvants for the preparation of a vaccine and collects Examples used by these adjuvants. Document D2 discloses the use of a lipopeptide with a palmitoyl moiety as a component of the adjuvant in a influenza virus vaccine (see especially page 684 conclusion). Document D3 discloses (see claims) a lipopeptide as an adjuvant of an antigen, comprises a peptide and a lipophilic moiety, which is a fatty acid. Document D4 discloses a vaccine for leishmaniasis comprising an FML glycoprotein complex (fucose mannose ligand) and a saponin adjuvant, then is not considered related to the vaccine described in the application. None of documents D1 to D4 disclose the use of a lipid with a terminal vinyl sulfone group as an adjuvant to an antigen in a vaccine nor the leishmaniosis vaccine comprising said lipopeptide and Phb proteins as antigen. Documents D1 to D4 reflect the state of the art closest to the invention and, for a technician in the matter, it would not be possible to deduce the use of a lipid such as a fatty acid with a vinyl sulfone group, as part Lipid of a lipopeptide suitable as an adjuvant of an antigen to prepare a vaccine useful for leishmaniasis. It follows from the state of the art that the invention of claims 1-27 of the application meets the conditions of novelty and inventive activity as established in Art. 6.1 and 8.1 of Patent Law 11/1986. State of the Art Report Page 4/4