WO2015011334A1 - Adjuvant immunologique pour la formulation de vaccins et vaccin contre la leishmaniose le comprenant - Google Patents

Adjuvant immunologique pour la formulation de vaccins et vaccin contre la leishmaniose le comprenant Download PDF

Info

Publication number
WO2015011334A1
WO2015011334A1 PCT/ES2014/070608 ES2014070608W WO2015011334A1 WO 2015011334 A1 WO2015011334 A1 WO 2015011334A1 ES 2014070608 W ES2014070608 W ES 2014070608W WO 2015011334 A1 WO2015011334 A1 WO 2015011334A1
Authority
WO
WIPO (PCT)
Prior art keywords
use according
group
vaccine
lipid
adjuvant
Prior art date
Application number
PCT/ES2014/070608
Other languages
English (en)
Spanish (es)
Inventor
Antonio OSUNA CARRILLO DEL ABORNOZ
Teresa Cruz Bustos
Francisco SANTOYO GONZÁLEZ
Original Assignee
Universidad De Granada
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad De Granada filed Critical Universidad De Granada
Publication of WO2015011334A1 publication Critical patent/WO2015011334A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/26—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C317/28—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to acyclic carbon atoms of the carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/16—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C317/18—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton with sulfone or sulfoxide groups bound to acyclic carbon atoms of the carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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. STATE OF THE TECHNIQUE
  • Vaccines constitute, in the diseases for which they exist, the best method of prevention in the face of personal and public health, having as indubitable success the 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 civilization, in those populations where this type of immunoprophylaxis is practiced .
  • 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.
  • pathogens In short, 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.
  • antigens can be, whole organisms, a portion thereof, that are part of the infectious agent, or substances secreted by them or even any natural, synthetic or artificially obtained substance and that resembles the natural ones that the excreta possesses or excretes.
  • vaccines that use dead or attenuated infectious agents, purified antigens, or toxins require the use of immunity enhancers called adjuvants.
  • an immunization and adjuvants will depend on the type of route of administration of the vaccine, since the same response is not obtained with an immunization through mucous membranes than with a parenteral administration, thus depending on this, the use 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).
  • 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, such as monophosphorylipid A (“MPL”) or chemical derivatives. All of them are being tested as adjuvants in clinical experiments.
  • adjuvants are oily emulsions in vegetable oils or animals, or interleukins or the DNA that encodes them.
  • 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.
  • Lipopeptides activate macrophages by triggering a response involving IL1, IL6 and TNF- ⁇ .
  • Lipouaix 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 synthetic or recombinant peptides are used that form micelles with fatty acids protect epitopes against proteolytic enzymes (BenMohamed L, Thomas A, Bossus M, et al.
  • 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 has been opened.
  • 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 (LelF), homologous inducible stress protein 1 (LmSTM), TSA and Leish-1 1 1 f, etc.
  • Many defined antigens can protect experimental animals but to date only one is being clinically tested, Leish-1 1 1 f together with the MPL-SE adj
  • the third generation of vaccines is based on DNA vaccines.
  • the discovery of 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.
  • vaccines have been tested with genes encoding gp63, LACK, cpa and cpb, NH36, KMP-1 1, TSA and LmSTM but none of them have obtained results against more than one species.
  • Leish-1 1 1 f + MPL-SE vaccine It is the Leish-1 1 1 f + MPL-SE vaccine, which is being clinically tested [Skeiky et al., "Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-1 1 1 f) formulated in MPL ® adjuvant, "Vaccine 20: 3292-3303, 2002].
  • 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.
  • the present invention also relates to an immunoadjuvant comprising 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 .
  • TLRs Toll type receptors
  • 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.
  • linker refers to an organic molecule that covalently binds a lipid and a vinyl sulfone group.
  • adjuvant 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.
  • the compound comprising a lipid and a vinyl sulfone group is the compound of general formula (I)
  • Y is a group -CH 2 -S0 2 R 1 - or -CH 2 -; preferably Y is -CH 2 -R 1 is a radical selected from the group comprising an alkyl (CrC 10 ), an alkenyl (CrC 10 ), an alkynyl (CrC 10 ), a dialkylaryl (C 1 -C 10 ) Ar (C 1 -C 10 ) or a group (CH 2 CH 2 0) n CH 2 CH 2 ; preferably R 1 is a group (CH 2 CH 2 0) n CH 2 CH 2 ;
  • n takes values from 1 to 20; preferably n takes values between 2 to 10, more preferably n is 2 to 5 and even more preferably n is 2.
  • X is a group -CO-NH-R 2 -Z-CH 2 -, -Z-CH 2 - or -CH 2 -;
  • Z is S or O
  • R 2 is a radical selected from the group comprising an alkyl (C1 - C10), alkenyl (C1-C1 0), alkynyl (C1-C1 0) a dialkylaryl (Ci-Ci 0) Ar (Ci- Ci 0 ) or a group (CH 2 CH 2 0) m CH 2 CH 2 ;
  • 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.
  • 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 (C Cao), alkenes (C 2 -C 30 ), alkynes (C 2 -C 30 ) or sterols. Most 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, which have the main characteristic of being hydrophobic. or insoluble in water and yes in organic solvents.
  • the lipid is a sterol or a saturated or unsaturated aliphatic hydrocarbon.
  • 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.
  • the lipid can be selected from a hydrocarbon (C 4 -C 30 ), saturated or unsaturated. And more preferably the carbon number is between 10 and 20.
  • estero refer to steroids with 27 to 29 carbon atoms. Their chemical structure derives from the cyclopentaneperhydrophenanthrene or steran, a 17-carbon molecule consisting of three hexagonal and one pentagonal rings. It can be selected from the list comprising , but not limited to cholesterol, epicolesterol, stigmasterol, lanosterol, ergosterol and coprostenol, more preferably the sterol is cholesterol.
  • 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.
  • 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.
  • R 2 independently, they would preferably be aliphatic chains having 1 to 10 carbon atoms, more preferably 2 to 5 carbon atoms and even more preferably an ethyl.
  • alguenyl 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.
  • 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.
  • alguinyl in the present invention refers to an alkyl radical, described above, and having one or more unsaturated bonds, specifically has at least one triple bond, although it can also have at least one double bond.
  • 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.
  • dialguilaryl 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.
  • alkyl, alkenyl or alkynyl groups may be the same or different, preferably they are the same.
  • 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
  • the compound of formula (I) is the compound N- (2- (2- (vinylsulfonyl) ethoxy) ethyl) oleamide, of formula (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.
  • the term “vaccine” refers to an antigen or an antigenic preparation or composition used to establish the immune system response to a disease.
  • antigen or “preparation or antigenic 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.
  • the vaccines of the invention may be vaccines against viruses, protozoa, parasites or tumors.
  • parasites in the present invention an infectious disease caused by protozoa, vermes (cestodes, trematodes, nematodes) or arthropods.
  • the parasites are protozoa or vermes, preferably trematodes or nematodes.
  • 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.
  • leishmaniosis is a disease caused by a protozoan of the genus Leishmania and transmitted, mainly by sandfly mosquitoes or jejenes. This disease occurs in humans and vertebrate animals, such as sermarsupials, canids, rodents and primates.
  • the present invention relates to a Leishmaniasis vaccine comprising the immunological adjuvant described in the present invention and the recombinant antigens.
  • the recombinant antigens glycoprotein 63 (gp63), membrane glycoprotein 46 (gp46 or M-2), fucose-mannose ligand (FML), homologous receptor activated by Kinase C (p36 / LACK) could be used, among others.
  • 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 (LelF), homologous inducible stress protein 1 (LmSTM), TSA, Leish-1 1 1 f, or Prohibitins 1 and 2, among others.
  • the Leishmaniasis vaccine comprises the immune 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 1 r and Phb 2r.
  • Another aspect of the present invention relates to a method of preparing the vaccine of the invention comprising the following steps:
  • step (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 e C for 10 min to 24 h .;
  • step (c) 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 step (c) and incubate for 2 to 3 hours at a temperature between 4 e C and 40 e C, preferably between 22 e C and 37 e C.
  • the immunological adjuvants of step (a) are the lipid-vinylsulfone compounds (LVS) described in the present invention, more preferably the compounds of general formula (I) and even more preferably the compound of formula (II).
  • LVS lipid-vinylsulfone compounds
  • step (a) 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.
  • 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.
  • step (c) the incubation is carried out at a laboratory temperature, preferably between 4 e C and 37 e C, more preferably at 4 e C, and between 10 min and 24 h, preferably 12 hours.
  • 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 0.1 M glycine carbonate buffer.
  • 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.
  • Contr No Infec corresponds to the uninfected control group
  • 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).
  • Fig. 2. Representation of the size of the skin lesion at the site of infection measured in the different groups of the trial and the percentage of lesion reduction considering maximum the size of the inflammation of the infected and non-immunized animals with either of the two preparations.
  • Control corresponds to the uninfected control group
  • Infection Control group of infected animals.
  • p1 p2 control Group of animals immunized with the two recombinant proteins without adjuvant.
  • 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 ° C for three days in RPMI medium.
  • p1 p2Control corresponds to the splenocyte stimulation index of the spleens of animals immunized with the two recombinant proteins without adjuvant;
  • p1 p2 + ll corresponds to the splenocyte stimulation index of spleens of animals immunized with recombinant proteins together with the adjuvant of formula
  • Fig. 4. Represents the expression levels of interleukin IL2 measured in the different test groups. Interleukin corresponding to a type of TH1 response.
  • Control unimmunized and uninfected mice
  • Infec Control: non-immunized and yes infected mice
  • p1 p2 + ll Phbl r and Phb2r bound to the adjuvant of formula (II)
  • p1 p2ontrol Phb1 r 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.
  • Control corresponds to the uninfected control group; Infec Control: group of infected animals.
  • p1 p2 control Group of animals immunized with the two recombinant proteins without adjuvant.
  • Fig. 6. Interferon expression levels and (IFN ⁇ ). Interleukin corresponding to a type of TH1 response.
  • Control unimmunized and uninfected mice
  • Infec Control non-immunized and yes infected mice
  • p1 p2 + ll Phb1 R and Phb2R bound to the adjuvant of formula (II)
  • p1 p2Control Phb1 R and Phb2R without the adjuvant of formula (II).
  • Fig. 7. Expression levels of TNF- ⁇ .
  • Control corresponds to the level of TNF- ⁇ expression of the uninfected control group
  • Infec Control corresponds to the level of TNF- ⁇ expression of the Infected groups.
  • p1 p2Control corresponds to the level of TNF- ⁇ expression of the group of animals immunized with the two Recombinant proteins without adjuvant.
  • p1 p2 + ll corresponds to the level of TNF- ⁇ expression of animals immunized with the recombinant proteins together with the adjuvant of formula (II).
  • 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
  • p1 p2Control corresponds to the level of IL4 expression of the group of animals immunized with the two recombinant proteins without adjuvant
  • p1 p2 + ll corresponds to the level of IL4 expression of animals immunized with the recombinant proteins together with the adjuvant of formula (II).
  • 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
  • p1 p2Control corresponds to the level of IL10 expression of the group of animals immunized with the two recombinant proteins without adjuvant
  • p1 p2 + ll corresponds to the level of IL10 expression of animals immunized with recombinant proteins together with the adjuvant of formula (II).
  • 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.
  • p1 p2Control corresponds to the level of expression of IL17 of the group of animals immunized with the two recombinant proteins without adjuvant.
  • p1 p2 + ll corresponds to the level of IL17 expression of animals immunized with the recombinant proteins together with the adjuvant of formula (II).
  • Control corresponds to the level of TGB- ⁇ expression of the uninfected control group
  • Infec Control corresponds to the level of TGB- ⁇ expression of the Infected groups.
  • p1 p2Control corresponds to the level of TGB- ⁇ expression of the group of animals immunized with the two recombinant proteins without adjuvant.
  • p1 p2 + ll corresponds to the level of TGB- ⁇ expression of animals immunized with the recombinant proteins together with the adjuvant of formula (II).
  • mice For the immunization assay and subsequent challenge, 20 Balb / c mice were used, divided into batches.
  • Adjuvant control group inoculated with 10 ⁇ g of the recombinant proteins called Phb 1 r and Phb 2r, without binding to said compound and subsequently challenged.
  • 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.
  • mice 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.
  • 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 e C.
  • mice were taken from each mouse to subsequently titrate the serum against the recombinant proteins by the ELISA technique. After the test period, batches of mice were sacrificed. 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
  • Lymphoproliferation measured as a result of DNA synthesis by splenocytes, was carried out based on the incorporation of 3 H Thymidine into the precipitable material of the splenic cells.
  • splenocytes obtained from the spleens of the immunized animals were used, which 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 Penicillin and 100 ⁇ g / ml Penicillin).
  • 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 ⁇ of 6- 3 H-Thimidine was added to all cultures (with a specific activity of 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.
  • the culture plate where the experiments were performed was incubated for 72 hours at 37 in a humid atmosphere with 5% C0 2 .
  • the cells were collected at 72 h and washed with PBS, centrifuged at 400g for 5 min at 4 ° C.
  • the button, containing the cells, which was to be treated to measure lymphoproliferation, was resuspended in 3 volumes of cold trichloroacetic acid at 10%, it was incubated at 4 ° C for 2 h, and after that it was centrifuged 10 minutes at 200g. They were subsequently washed with 3 ml of a 5% trichloroacetic acid solution and finally washed always by centrifugation, with absolute ethanol and 80% ethanol.
  • the vials were washed, they were filled with scintillation liquid (PPO 4g; POPOP 0.1 g; 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 cultures from unstimulated control cells.
  • the maximum stimulation level was performed with splenocytes stimulated with ConA.
  • the cytokine determination was performed by their level of expression by RTqPCR with the mRNAs extracted from the stimulated splenocytes.
  • TNF-a F necrosis factor 5 ' -AGCCCCCAGTCTGTATCCTT-3 ' (Tumor SEQ ID-a NO: 3)
  • Interferon gamma INF- ⁇ F 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGAA-3 ' (SEQ ID NO: 5 ' -CACCCTGAAGTCGTTGTGAA-3 ' (SEQ ID NO: 5 '
  • Interleukin 2 IL-2 F 5 ' - AAGCTCTACAGCGGAAGCAC-3 ' (SEQ ID NO: 1
  • Interleukin 4 F 5 ' -TTGAACGAGGTCACAGGAGA-3 ' (SEQ ID NO: 1
  • Interleukin 10 F 5 ' -CTGTTTCCATTGGGGACACT-3 ' (SEQ ID NO: 1
  • Interleukin 12 F 5 ' -CCTGAAGTGTGAAGCACCAA-3 ' (SEQ ID NO: 1
  • Interleukin 17 IL-17 F 5 ' -TTCAGGGTCGAGAAGATGCT-3 ' (SEQ ID NO: 1
  • mice 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 study of the type of immune response induced in the different groups of mice based on the determination of interleukins was performed by determining RT-PCR measurement of the expression levels of the genes of said proteins: IL2, IL4, IL10, IL1 2 , IL1 7, TNF-a, TGF- ⁇ and INF- ⁇ .
  • lymphoproliferation levels were analyzed, as a level of response to treatments from splenocytes not stimulated or stimulated with the antigens used in immunizations, incubation with said antigens was 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 ⁇ / well) which was added 18 hours before the sacrifice of the cells.
  • the levels of incorporation of the radioactive analogue into the DNA of the cells were determined with a scintillation counter expressing the results in counts per minute (cpm).
  • th1 type interleukins that is to say IL 2, IL 12, IFN ⁇ and the tumor necrosis factor TNF a
  • IL 2 interleukins
  • IFN ⁇ tumor necrosis factor 1 a
  • IL2 levels show a significant increase in the rPhb1 -rPhb2-LiVSulf group (p ⁇ 0.001) (Fig. 4).
  • Figure 5 shows that IL12 levels are increased in the rPhbl-rPhb2-LiVSulf group (p ⁇ 0.001) and the rest of the groups maintain levels similar to the infection control group.
  • the levels of INF- ⁇ are shown in Figure 6.
  • the rPhb1 -rPhb2-LiVSulf group (p1 p2 + ll) shows a highly significant increase (p ⁇ 0.001) with respect to the other groups.
  • TNF- ⁇ Tumor Necrosis Factor
  • the groups that show a significant increase in IL4 with respect to the infection group were: p1 p2 + (ll) (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.
  • IL 17 expression levels showed that in the Phb1 -rPhb2-LiVSulf group, a significant increase in this type of response was induced, compared to the rest of the groups (Fig. 10).
  • TGF- ⁇ Tumor growth factor expression

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne l'utilisation de composés comprenant une molécule de nature lipidique et un groupe vinyl-sulfone comme adjuvant immunologique. La molécule lipidique se lie à un antigène pour former un lipopeptide qui agit comme vaccin. La présente invention concerne également un immunoadjuvant comprenant lesdits composés, un vaccin comprenant ces lipopeptides, c'est-à-dire le composé de l'invention utilisé comme immunoadjuvant et un antigène, et, plus particulièrement, le vaccin décrit antérieurement contre la leishmaniose.
PCT/ES2014/070608 2013-07-26 2014-07-25 Adjuvant immunologique pour la formulation de vaccins et vaccin contre la leishmaniose le comprenant WO2015011334A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP201331159 2013-07-26
ES201331159A ES2424831B1 (es) 2013-07-26 2013-07-26 Adyuvante inmnulógico para la formulación de vacunas y vacuna frente a Leishmaniasis que lo comprende

Publications (1)

Publication Number Publication Date
WO2015011334A1 true WO2015011334A1 (fr) 2015-01-29

Family

ID=49221667

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2014/070608 WO2015011334A1 (fr) 2013-07-26 2014-07-25 Adjuvant immunologique pour la formulation de vaccins et vaccin contre la leishmaniose le comprenant

Country Status (2)

Country Link
ES (1) ES2424831B1 (fr)
WO (1) WO2015011334A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047553A1 (fr) * 1999-12-23 2001-07-05 University College London Lipopeptides formant des micelles cibles sur des cellules presentatrices d'antigene utilises comme adjuvants de vaccins
WO2006122382A2 (fr) * 2005-05-16 2006-11-23 Universidade Federal Do Rio De Janeiro Composition contenant des fractions ou des sous-fractions de promastigotes ou d'amastigotes de leishmania designees ligand fucose-mannose (fml) et saponine, composition servant a preparer des vaccins bloquant la transmission de la leishmaniose chez l'homme et chez l'animal et contenant des fractions ou des sous-fractions de

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047553A1 (fr) * 1999-12-23 2001-07-05 University College London Lipopeptides formant des micelles cibles sur des cellules presentatrices d'antigene utilises comme adjuvants de vaccins
WO2006122382A2 (fr) * 2005-05-16 2006-11-23 Universidade Federal Do Rio De Janeiro Composition contenant des fractions ou des sous-fractions de promastigotes ou d'amastigotes de leishmania designees ligand fucose-mannose (fml) et saponine, composition servant a preparer des vaccins bloquant la transmission de la leishmaniose chez l'homme et chez l'animal et contenant des fractions ou des sous-fractions de

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
I TSUNODA ET AL.: "Lipopeptide particles as the immunologically active component of CTL inducing vaccines", VACCINE, vol. 17, 1999, pages 675 - 685. *
P MOYLE ET AL.: "Self adjuvating lipopeptide vaccines", CURRENT MEDICINAL CHEMISTRY, vol. 15, no. 5, 2008, pages 506 - 516 *

Also Published As

Publication number Publication date
ES2424831B1 (es) 2014-08-04
ES2424831A1 (es) 2013-10-08

Similar Documents

Publication Publication Date Title
Ghaffarifar Plasmid DNA vaccines: where are we now
Palatnik-de-Sousa Vaccines for leishmaniasis in the fore coming 25 years
ES2741729T3 (es) Vectores y construcciones de liberación de antígenos de virus de la gripe
ES2307496T3 (es) Composiciones de vacuna, antigenos y peptidos.
JP5165681B2 (ja) タンパク質断片又はペプチドカクテルとして送達される抗原を用いたワクチン接種によるサブドミナントエピトープを含むｔ細胞レパートリーの拡大
ES2312465T3 (es) Peptidos de vih de regiones conservadas en gag p17 y sus aplicaciones, tales como vacunas.
US20100047271A1 (en) Vaccine compositions
Kovacs-Nolan et al. The novel adjuvant combination of CpG ODN, indolicidin and polyphosphazene induces potent antibody-and cell-mediated immune responses in mice
EP2391383B1 (fr) Compositions et procédés qui améliorent une réponse immunitaire
Kovacs-Nolan et al. CpG oligonucleotide, host defense peptide and polyphosphazene act synergistically, inducing long-lasting, balanced immune responses in cattle
Even-Or et al. Immunogenicity, protective efficacy and mechanism of novel CCS adjuvanted influenza vaccine
Thomaz-Soccol et al. Recent advances in vaccines against Leishmania based on patent applications
Moisa et al. Synthetic peptide vaccines
Luo et al. Plasmid DNA containing multiple CpG motifs triggers a strong immune response to hepatitis B surface antigen when combined with incomplete Freund's adjuvant but not aluminum hydroxide
WO2023280303A1 (fr) Utilisation d'avc-29 en tant qu'adjuvant vaccinal et composition vaccinale contenant un adjuvant
WO2015011334A1 (fr) Adjuvant immunologique pour la formulation de vaccins et vaccin contre la leishmaniose le comprenant
JP5676452B2 (ja) ワクチンアジュバント
ES2242376T3 (es) Administracion de particulas inmunogenas mediante particulas de agshb.
US20110305720A1 (en) Peptide adjuvants
ES2301418B1 (es) Proteoliposomas y sus derivados como adyuvantes inductores de respuesta citotoxica y las formulaciones resultantes.
ES2595207T3 (es) Composición de vacuna para prevenir o tratar la leishmaniosis
Eze et al. Vaccine Delivery Using Nanoparticles: A Critical Look at ISCOMs 4 Decades but 2 and Still Counting.
WO2017163171A1 (fr) Protéine chimère, composition vaccinale contre les leishmanioses et utilisations
Acosta et al. TB VACCINES BASED ON PROTEOLIPOSOMES AND LIPOSOMES FROM NON PATHOGENIC MYCOBACTERIA
WO2018051291A1 (fr) Composition vaccinale pour protection contre la leishmaniose tégumentaire

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14828712

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14828712

Country of ref document: EP

Kind code of ref document: A1