WO2005063287A1 - Proteoliposomas y sus derivados como adyuvantes inductores de respuesta citotóxica y las formulaciones resultantes - Google Patents
Proteoliposomas y sus derivados como adyuvantes inductores de respuesta citotóxica y las formulaciones resultantes Download PDFInfo
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- WO2005063287A1 WO2005063287A1 PCT/CU2004/000017 CU2004000017W WO2005063287A1 WO 2005063287 A1 WO2005063287 A1 WO 2005063287A1 CU 2004000017 W CU2004000017 W CU 2004000017W WO 2005063287 A1 WO2005063287 A1 WO 2005063287A1
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- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
- A61K2039/55516—Proteins; Peptides
-
- 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
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
-
- 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
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
Definitions
- the present invention relates to the field of vaccines and their use in medicine. More particularly, this relates to the use of adjuvants and the resulting vaccine formulations.
- the technical objective pursued is to favor the increase in the immune response against fungal, viral, protozoal, bacterial or carcinogenic antigens, especially to increase the induction of essential cytotoxic T response against these antigens. This work will lead to the development of therapeutic or prophylactic vaccine formulations.
- Proteoliposomes and their derivatives as adjuvants in formulations with fungal, viral, bacterial, protozoan or carcinogenic antigens, inserted in their structure, conjugated or mixed with them.
- These formulations would extend the preferential T response type 1 (Thl) induced by Proteoliposomes and their derivatives to the included antigens (Pérez O et al. Infec ⁇ Immun. 2001, 69 (7): 4502-4508), inducing a response of Cytotoxic T lymphocytes (CTL) against these antigens when applied by mucosal, parenteral route or combination thereof.
- CTL Cytotoxic T lymphocytes
- MHC Major Histocompatibility System
- Thl The immune response was phenotypically subdivided into cellular (Thl) and humoral (Th2).
- the Thl / Th2 patterns are distinguished primarily by the cytokine pattern secreted by CD4 + T cells: primarily, IFN ⁇ and IL12 by Thl and IL4 and IL5 by Th2 (Mossman, TR, Cherwinski, H., Bond, MW, Giedlin, M. A, and RL Coffman. 1986. Two types of murine helper T cells clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136: 2348-2357).
- Thl pattern preferentially induces antibodies of the IgG2a subclass (IFN ⁇ dependent), while Th2 induces IgE and the IgGl subclass (IL4 dependent).
- IFN ⁇ dependent antibodies of the IgG2a subclass
- Th2 induces IgE and the IgGl subclass (IL4 dependent).
- the Thl pattern leads to the induction of IgG1 and IgG3 subclasses and Th2 induces the IgE class.
- cytokines produced by non-lymphoid cells have also been associated with these patterns, as is the case with IL12 and IL18 associated with a Thl pattern.
- IFN ⁇ and IL10 act as inhibitors of the Th2 and Thl response, respectively.
- CD8 + T lymphocytes which, by similarity in cytokine production, have been classified as Tcl and Tc2.
- CD8 + T lymphocytes are the fundamental mediators and inducers of CTL activity. These act then, not only as effector cells, but also as regulators producing cytokines that expand one response more than the other. Therefore, the determination of the subset of CD4 + T or CD8 + T cells present in peripheral mononuclear cell lymphoproliferations of immunized subjects or secondary lymphoid organs of immunized animals, re-stimulated by an in vi / ro antigen, is a important measure that these two populations are being stimulated.
- CTL CD8 + T lymphocytes
- Successful CTL activity can be determined by any known method, both indirect and direct.
- indirect and direct methods are combined.
- the capacity of the Proteoliposome or its derivatives is determined, with antigens inserted in its structure, conjugated or co-administered to be phagocytosed, degraded and presented on the cell surface associated with the MHC-I molecules of cell lines. These then face cytotoxic cell lines against the inserted antigen and cell death is determined by radioactive or non-radioactive methods.
- Another indirect index is the stimulation, by the Proteoliposome or its derivatives with inserted, conjugated or co-administered antigens, of CD8 + T lymphocytes in peripheral mononuclear cells of immunized humans or from secondary lymphoid organs of immunized animals, re-stimulated in vitro with the antigen of interest.
- the detection of CD8 + T lymphocytes producing IFN ⁇ and IL2 in mononuclear cells re-stimulated in vitro with the antigen of interest is another indirect index that can be determined by Flow Cytometry or ELISPOT. In this case, the production of IFN ⁇ by CD8 + T cells is fundamentally determined.
- IL2 The production of IL2 by hybridomas of CD8 + T cells after recognition of the specific antigen in the context of MHC-I molecules on the surface of antigen presenting cells previously incubated with the Proteoliposome or its derivatives containing the specific antigen inserted into its structures, conjugated or co-administered with them, is another indirect way of evidencing CTL activity.
- Direct mammals are immunized with the Proteoliposome or its derivatives and the antigens of interest, inserted in its structure, conjugated or co-administered and then the secondary lymphoid organs are extracted and the presence of CTL activity against the antigen of interest associated with MHC is determined.
- the oldest method to detect antigen-specific CTL activity is based on the radioactive chromium release cytotoxicity assay, which has been designed the same for fresh cells (measuring CTL effector activity) as for CTL lines (evaluating CTL reactivity of memory). In both cases the target cells express the specific antigens of interest, which can be achieved by various methods (infection by recombinant viruses, "peptide loading” or transfection genetics). More recently, specific previously determined CTL epitopes tend to be used.
- TBA tetrameric-binding assay
- CTLs were considered the main component of the cellular response.
- adoptive adjuvants are substances that enhance the specific immune response against an antigen causing a faster induction of the antigen and increasing its duration (Vogel FR. Dev. Biol. Stand. 1998,92: 241-248). Its use in vaccine formulations allows reducing the amount of antigen needed, directing the response towards a desired pattern and reducing the number of doses needed.
- MPL ® Monophosphoryl lipid A
- LPS non-toxic lipopolysaccharide
- Adjuvants that currently induce CTL are scarce among which are MF59, CpG Oligonucleotides, QS21, MPL ® , ISCOM ® and lipid derived cochleates (O 'Hagan D., Mackicham M., and Singh M. Recent advance in adyuvants for infection disease. Biomolecular Engineering 2001 and Edelman Robert. The development and use of vaccine adyuvant. Molecular Biothecnology 2002. 21: 2, 129-148.).
- Proteoliposomes have been described for the preparation of prophylactic vaccines against infectious diseases by Ruegg CL et al. (Preparation of proteosome-based vaccines. J Immunological Methods 1990; 135: 101-109); Lowell et al. (Proteosome-Lipopeptide Vaccines: Enhancement of Immunity for Malaria CS Peptides. Science 1988; 240: 800-2) and also as disclosed in US No. 5,597,572.
- the fundamental nucleus is the Proteoliposome derived from the outer membrane of Neisseria meningitidis serogroup B, whose particulate structure, its content of native incorporated and non-free LPS, polysaccharide C, its characteristic lipid composition and its adsorption in alumina, are related to its high immunogenicity and proven protectogenicity in humans.
- This vaccine induces a preferential Thl pattern characterized by induction in human and animal lymphoproliferation; IgG anti-Proteoliposome antibodies; IgG subclasses in humans and IgG2a in mice; IFN ⁇ , IL2 and IL12 in the field of messenger ribonucleic acid (mRNA) and proteins, non-induction of anti-Propteoliposome IgE or increases in total IgE and non-production of IL4, IL5 both in the field of mRNA and protein (Perez) O et al. Infected Immun. 2001, 69 (72001): 4502-4508).
- mRNA messenger ribonucleic acid
- Proteoliposomes and derivatives thereof such as VSSPs and Cochlear Structures have also been used as adjuvants as disclosed in (US 6,149,921 of 2000) and in (Method of obtaining cochlear structures.
- the present invention has as its object the use of natural or genetically modified bacterial Proteoliposomes, especially those derived from N. meningitidis, as well as the cochlear structures derived therefrom, as new CTL inducing adjuvants. These new adjuvants are of particular importance against fungal, viral, bacterial and protozoal infections (particularly intracellular), as well as tumor diseases.
- Proteoliposome means that these are obtained from bacterial strains using any method known as: isolation without detergent; a process that includes detergent (such as deoxycholate) or the extraction from "bleb” vesicles of crop supernatants and particularly those disclosed in US 5,597,572.
- Proteoliposomes contain different Pathogen-Associated Molecular Patterns (PAMP), molecular structures conserved between pathogens capable of strongly stimulating the immune system.
- PAMP Pathogen-Associated Molecular Patterns
- Cochlear structures means an adjuvant derived from Proteoliposomes that therefore also contain PAMP as referred to in the patent (Method of obtaining cochlear structures. Vaccine compositions and adjuvants based on cochlear structures and their intermediaries. OCPI. 2002- 0292 of 11/27/02).
- the mechanisms of protection against Neisseria, Gram negative extracellular bacteria, are related to the induction of antibodies that fundamentally mediate opsonophagocytic and bactericidal functions. Therefore, it is not expected that the Proteoliposomes derived from them and the natural infection caused by this bacterium, induce CTL responses.
- N. meningitidis serogroup B infection by the natural microorganism, N. meningitidis serogroup B, induces both T CD4 + and T CD8 + anti- Proteoliposome response, which was detected in lymphocytes of individuals cured of meningococcal disease caused by N. meningitidis B.
- mice inoculated with N. meningitidis B also induced CD8 + T cell response.
- CD4 + T lymphocytes both in cells immunized with VA-ME ⁇ GOC-BC ® and in the case of individuals cured of meningococcal disease, CD4 + T lymphocytes, and surprisingly, CD8 + T secretes IF ⁇ and IL2 and not IL4 or IL5 when evaluated by Flow Cytometry. This result includes CD8 + T lymphocytes within the important cells in the production of the cytokines characteristic of the Thl response pattern.
- CTL epitopes from Ova were incorporated into the Proteoliposomes or their derivatives and thus the response induced against these epitopes was increased.
- Proteoliposomes that can express high concentrations of antigens of interest are also part of the present invention, by genetic engineering manipulation of the Proteoliposome producing strain in order to express "more or less” amount of the desired heterologous antigen or antigens.
- Antigens of interest means those from fungi, viruses, bacteria, protozoa and cancer that have been shown to require a CTL response for efficient elimination by the immune system, without restricting those already identified.
- the strain expresses more than 20, 15, 10, 5, 4, 3, 2, 1, 0.5 or 0.1% of the amount of the heterologous antigens or interest.
- the engineering strain expresses between 0.5% and 10% of the antigen of interest.
- the antigen coding gene of interest of the invention can be engineered as expressed above by known techniques.
- Associated tumor antigens, gangliosides or proteins from Leishmania were included in the Proteoliposomes or their derivatives and the induction of CD8 + response and IFN ⁇ production was demonstrated when Balb / c mice were immunized and challenged in vitro with the respective antigens.
- Vaccine formulations are also shown to be effective, where the antigens are conjugated to part of the Proteoliposomes of the vaccine composition or once conjugated to them they are transformed into cochlear structures.
- the vaccine formulations of the present invention can be used to protect a mammal susceptible to infection or to treat mineral diseases by administering said vaccine formulation systemically or mucosally. These applications may include injections by intramuscular, intraperitoneal, intradermal or subcutaneous or mucosal administration by oral / food, nasal / respiratory or genitourinary tractus.
- the amount of Proteoliposome or Cochlear Structures in each formulation is selected as the amount that shows adjuvant function, which is always lower than that used in anti-meningococcal vaccines based on these structures, so the adverse side effects are lower and are not significant. This amount will vary depending on the antigen of interest and how it is incorporated. Generally, the amount of Proteoliposome or Cochlear Structures in each dose will be between 1 and 50 ⁇ g and more typically between 5 and 25 ⁇ g. The amount of the antigen of interest will be in the range of 0.1 to 20% of the Proteoliposome or Cochlear Structures mass and preferably 0.5 to 10%.
- the number of doses will depend primarily on whether the vaccine formulation is prophylactic or therapeutic. In the first, a maximum of three doses will be applied and in the second, up to 5 doses can be reached. Both can be applied in children under one year, 2 to 5 years, school children, adolescents, adults and the elderly.
- the novelty of the invention lies primarily in the use of Proteoliposomes or Cochlear Structures derived from proteins of the outer membrane of Gram-negative bacteria and more particularly of N. meningitidis B, as adjuvants inducing CTL activity.
- Another novel aspect is its prophylactic application in fungal, viral, bacterial or protozoal vaccine formulations and its therapeutic application in subjects affected with malignant tumors in which it is desired to induce a cytotoxic response.
- the conjugation or insertion of cytotoxic epitopes to Proteoliposomes or Cochlear Structures, which increase the CTL response is particularly novel.
- Proteoliposomes and cochlear structures are obtained on an industrial scale under conditions of Good Production Practices (GMP) at the Finlay Institute and we have methodologies for the inclusion of heterologous antigens in them.
- GMP Good Production Practices
- the immunological effect achieved by this preparation allows inducing CTL responses by the Proteoliposome and Cochlear Structures against the heterologous antigens incorporated therein, which is applicable; but not limited to intracellular microorganisms and tumor diseases.
- Proteoliposomes as a vaccine and transitively as an adjuvant, where smaller amounts are used, is safe in children under one year of age, 2 to 4 years old, in schoolchildren, adolescents and adults.
- Proteoliposomes and Cochlear Structures by the preferential induced cellular response also convert to T-independent antigens (conjugated or non-covalently incorporated), such as carbohydrates into T-dependent antigens. This property is also advantageously applied to the antigens of interest of saccharide character.
- Example 1 Obtaining Proteoliposome.
- a culture of N. meningitidis B or Salmonella typhi is carried out and the biomass collected by centrifugation is subjected to an extraction process with detergent, enzymes and ultrasound.
- the cell debris is removed by centrifugation and the supernatant is then digested with nucleases to remove nucleic acids.
- the extract is recovered by ultracentrifugation, resuspended in solution with detergent and purified from the rest of the low and medium molecular weight components by molecular exclusion chromatography.
- the Proteoliposome thus obtained contains less than 10% nucleic acids and less than 10% LPS inserted in its structure; But never free.
- the LPS present is essential in triggering the danger signals to initiate the innate response.
- the final product is subjected to a set of biological and physical-chemical controls.
- Example 2 Response of CD4 + and CD8 + T lymphocytes by immunization of mice.
- Balb / c mice were immunized with two doses of 25 ⁇ g each of Proteoliposome or Cochlear Structures, intramuscularly or nasally and spaced 14 days.
- Spleen cells were taken 7 days after the second dose and challenged for 4 days with Proteoliposome
- the expanded cells were treated with anti-CD4 or anti-CD8 monoclonal antibodies and subsequently with a fluorescent anti-IgG and were evaluated by Flow Cytometry.
- Immunization stimulated both CD4 + and CD8 + T anti-Proteoliposome lymphocytes. 45% of the lymphocytes were CD4 + T and 40% were CD8 + T.
- Example 3 Example 3
- CD4 + and CD8 + T lymphocytes by human immunization. Young adults were immunized with two doses of VA-ME ⁇ GOC-BC 'of 50 ⁇ g each intramuscularly, spaced 6 weeks. Seven days after the second dose, blood was taken and peripheral mononuclear cells were purified on a Ficoll-Hipaque gradient. They faced each other for 4 days with Proteoliposome. The expanded cells were treated with anti-CD4 or anti-CD8 monoclonal antibodies and subsequently with a fluorescent anti-IgG and were evaluated by Flow Cytometry. Immunization stimulated both CD4 + T lymphocytes and anti-Proteoliposome CD8 + T lymphocytes. 50% of the lymphocytes were CD4 + T and 42% were CD8 + T.
- Example 4 Response of CD4 + and CD8 + T lymphocytes in convalescents of meningococcal disease.
- the expanded cells were treated with anti-CD4 or anti-CD8 monoclonal antibodies and subsequently with an anti fluorescent IgG and were evaluated by Flow Cytometry. Proliferation of CD4 + and T CD8 + lymphocytes was evident. 47% of the lymphocytes were CD4 + T and 39% were CD8 + T.
- Example 5 Signs of activation and production of intracellular cytokines by lymphocytes from immunized humans. Young adults were immunized with two doses of VA-MENGOC-BC ® of 50 ⁇ g each intramuscularly, spaced 6 weeks. Several months after the second dose, blood was taken. These were confronted for 27 hours with Proteoliposome at a concentration of 10 or 20 ⁇ g / ml. Subsequently, the red blood cells were used and the rest of the cell was permeabilized and faced with monoclonal antibodies for the detection of intracellular cytokines (IFN ⁇ , IL2 or IL5) and membrane markers (CD4, CD8 and CD69).
- IFN ⁇ , IL2 or IL5 monoclonal antibodies
- CD4 + T lymphocytes 3.28% were activated with 10 ⁇ g / ml Proteoliposome, while 3.86% of CD4 + T and 14.11% of CD8 + T were activated with 20 ⁇ g / ml Proteoliposome.
- CD8 + T lymphocytes produced IFN ⁇ and IL2, although some production of IL2 was also detected in activated CD4 + T lymphocytes.
- the percentages of intracellular IFN ⁇ in CD4 + and CD8 + T lymphocytes with 10 ⁇ g / ml were 0.35 and 0.89 and with 20 ⁇ g / ml were 1.54 and 1.29, respectively; but only of the IFN ⁇ producing cells were 0.57% activated with 10 ⁇ g / ml and 0.87% with 20 ⁇ g / ml of the CD8 + T.
- the percentages of intracellular IL2 in CD4 + and CD8 + T lymphocytes with 10 ⁇ g / ml were 1.19 and 1.07 and with 20 ⁇ g / ml were 1.18 and 1.6, respectively; but only IL2-producing cells were activated 0.68 and 0.7% with 10 and 20 ⁇ g / ml, respectively of CD8 + T cells.
- CD4 + T lymphocytes only low percentages of IL2 production in activated cells were detected. No production of IL5 was observed by any of the lymphocyte subpopulations.
- Example 6 Signs of activation and production of intracellular cytokines by convalescent lymphocytes of meningococcal disease. Two months after discharge from patients who had suffered meningococcal disease caused by Neisseria meningitidis B blood was taken. These clashed for 27 hours with Proteo lipo soma 10 ⁇ g / ml. Subsequently the red blood cells were used and the rest of the cell was permeabilized and faced with monoclonal antibodies for the detection of intracellular cytokines (IFN ⁇ , IL2 or IL5) and markers of membrane (CD4, CD8 and CD69). The percentages of T CD4 + and T CD8 + activated were 9.29 and 26.81, respectively.
- IFN ⁇ , IL2 or IL5 markers of membrane
- Example 7 Incorporation of exogenous antigens in the Proteoliposome.
- Ovalbumin (Ova) was incorporated into the Proteoliposome through the use of detergents, particularly deoxycholate, which allowed disassembly of its Proteoliposomal structure. The elimination of the detergent and the reassembly of the structure in the presence of Ova allowed its incorporation into the Proteoliposome.
- the vesicular structure was checked by molecular exclusion chromatography yielding similar Proteoliposome profiles only and that of the Ova. The presence of Ova was verified by the SDS-PAGE technique and its subsequent Western Blot revealed with a polyclonal anti-Ova antibody which demonstrated the presence of a band corresponding to Ova within the Proteoliposome protein profile.
- Example 8 Incorporation of exogenous antigens in cochlear structures.
- the incorporation of promishigote and amastigote proteins from Leishmania was efficiently carried out by including them in the solution during the process of formation of the cochlear structures.
- the immunization of animals with the Cochlear Structures containing these antigens demonstrated the increase in the axiou-Leishmania response, as well as the reduction of indurations caused by infection with this protozoan.
- the presence of this protein was also evidenced by the SDS-PAGE technique followed by a Western Blot revealed with anti Leishmania antibodies.
- Example 9 Conjugation of antigens to Proteoliposomes. This is described in example 1.2 of the patent "Method of obtaining conjugate vaccines. Vaccine compositions" OCPI Patent 2002-0257 of 11/14/02.
- Example 10 Incorporation of Molecular Patterns Associated with Pathogens. Lipopolysaccharides (LPS) from Neisseria meningitidis B have been incorporated into different amounts, increasing its concentrations normally present in Proteoliposomes up to 12% with an increase in the immune response obtained. Vibrio cholerae LPS have also been incorporated, which has allowed to induce Vibrio anti-LPS response.
- LPS Lipopolysaccharides
- Example 12 Incorporation of plasmid DNA in cochlear structures and release of it in the cell cytoplasm.
- Plasmid pGFP which contains the green fluorescent protein gene under an upper cell expression promoter (CMVp) was efficiently incorporated into cochlear structures.
- Cochlear structures containing said plasmid were added to a cell culture of the L929 line. The expression of the green fluorescent protein in the cell cytoplasm was checked by fluorescence optical microscopy.
- Example 13 Dedolitic cells exposed to Proteoliposome (PL) containing Ovalbumin (Ova) may present Ova peptides to T cells.
- Dedritic cells (CD) were incubated for 2 h with soluble PL-Ova or Ova and then co-cultured with T-cell hybridomas. Supernatants were collected after 24 h and tested to detect IL2 production through the incorporation of [ 3 H] thymidine by the IL2-dependent CTLL cell line.
- IL2 provides a measure of the activation of T cell hybridomas specific for Ova peptides.
- Figure 1 shows that CDs incubated with PL-Ova can present Ova peptides to the hybridoma of CD4 + T cells and that of CD8 + T.
- T-cell hybridomas produced significantly higher levels of IL2 when stimulated with CDs that had previously been incubated with PL-Ova compared to CDs incubated only with Ova.
- Significant differences between presentation levels of specific peptides of Ova in MHC-I and MHC-II by CD incubated with PL (Ova) or Ova were determined using the Duncan Multiple Comparison Test. In this way, the Proteoliposome can release CD antigens and promote efficient antigen presentation to T cells.
- FIG. 1 shows the response of the OT4H T cell hybridoma to CD of C57BL / 6 mice co-incubated with Proteoliposome (PL), Ova or PL-Ova;
- Fig. IB shows the response of the OvaCD8 + T cell hybridoma.
- the production of IL-2 by hybridomas was quantified by incorporation of [H] thymidine by the IL-2-dependent CTLL cell line. Data are presented as the mean + SD of three different experiments. CPM, scintillation per minute. * significantly different from other groups.
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Abstract
Description
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AU2004308553A AU2004308553A1 (en) | 2003-12-30 | 2004-12-28 | Proteoliposomes and derivatives thereof as cytotoxic response-inducing adjuvants and resulting formulations |
CA002552271A CA2552271A1 (en) | 2003-12-30 | 2004-12-28 | Proteoliposomes and its derivatives as adjuvants inducers of citotoxic response and the resultant formulations |
EP04802609A EP1716866A1 (en) | 2003-12-30 | 2004-12-28 | Proteoliposomes and derivatives thereof as cytotoxic response-inducing adjuvants and resulting formulations |
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CU20100083A7 (es) | 2010-05-05 | 2012-06-21 | Inst Finlay | Tolerogenos adyuvados como vacuna de malaria |
EP2689775A1 (en) | 2012-07-25 | 2014-01-29 | Instituto Finlay, Centro de Investigacion-Produccion de vacunas y sueros | Cochleate with only one mamp |
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- 2004-12-28 WO PCT/CU2004/000017 patent/WO2005063287A1/es active IP Right Grant
- 2004-12-28 BR BRPI0418332-0A patent/BRPI0418332A/pt not_active Application Discontinuation
- 2004-12-28 ES ES200650046A patent/ES2301418B1/es not_active Expired - Fee Related
- 2004-12-28 EP EP04802609A patent/EP1716866A1/en not_active Ceased
- 2004-12-28 AU AU2004308553A patent/AU2004308553A1/en not_active Abandoned
- 2004-12-28 CA CA002552271A patent/CA2552271A1/en not_active Abandoned
- 2004-12-28 KR KR1020067014392A patent/KR20070036020A/ko not_active Application Discontinuation
- 2004-12-29 AR ARP040104938A patent/AR051059A1/es unknown
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US5597572A (en) * | 1987-07-30 | 1997-01-28 | Centro Nacional De Biopreparados | Method of producing Neisseria meningitidis B vaccine, and vaccine produced by method |
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WO2013064129A1 (es) | 2011-11-02 | 2013-05-10 | Instituto Finlay. Centro De Investigación- Producción De Sueros Y Vacunas. | Adjuvantes para vacunas polisacaridicas y las formulaciones resultantes |
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Publication number | Publication date |
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ES2301418B1 (es) | 2009-05-01 |
AR051059A1 (es) | 2006-12-20 |
KR20070036020A (ko) | 2007-04-02 |
UY28706A1 (es) | 2005-08-31 |
CU23420A1 (es) | 2009-09-08 |
CA2552271A1 (en) | 2005-07-14 |
ES2301418A1 (es) | 2008-06-16 |
BRPI0418332A (pt) | 2008-02-06 |
AU2004308553A1 (en) | 2005-07-14 |
EP1716866A1 (en) | 2006-11-02 |
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