HK1088827B - Method of obtaining cochlear structures, vaccine compositions, adjuvants and intermediates thereof - Google Patents

Description

Method for obtaining cochlear structures, vaccine compositions, adjuvants and intermediates thereof

The invention finds application in the field of immunology, in particular in the field of adjuvants and vaccines.

In the search for effective vaccines, the discovery of appropriate antigens has proven challenging in various areas outside of vaccinology. Once the appropriate antigen has been isolated, the latter often produces an immune effect that is not satisfactory for the purpose, or does not induce the desired effect, thus requiring the application of an appropriate adjuvant. To date, there is an urgent need to prepare vaccines for diseases that are currently unprotected, and to improve existing vaccines and develop powerful adjuvants for multiple and new generation vaccines. The same is true for the development of vaccines comprising different antigens and proven to be effective in the treatment of adults and children, and more importantly in the treatment of newborns, and for the discovery of adjuvants that act at the mucosal level and are able to withstand the acidic content of the stomach.

Mucosal immunity is in increasingly widespread immunological practice because many microorganisms penetrate the body through the mucosa. The mucosa exhibits many specificities, including: the presence of the common mucus system (allowing us to induce local as well as distant responses) and the fact that Ig (immunoglobulin) a is the main antibody that engages in its immune defense mechanisms.

This type of immunization also offers many advantages, including uncomplicated administration, no need for syringes, lower production costs and low levels of reactogenicity (reactogenicity), the latter rendering all of these much safer for parenteral vaccines and for inducing mucosal and systemic reactions.

However, mucosal immunity encounters a number of obstacles: the acidic content of the stomach, which reaches extremely acidic pH levels; the basicity of the duodenum and the motility of the digestive tract, which in combination with the action of M cells from sensory organs operating at the mucosal level, act exclusively on the antigen sampling (sampling), reducing the efficacy of the antigen in the vaccine. Cilia and mucus formation in the respiratory mucosal organs also interfere with antigen sampling by M cells.

Strategies to avoid contact of the antigen with acidic pH media consist in administering the vaccine in bicarbonate solution at a time away from the meal, so as to reduce the acidity of the stomach and ensure rapid passage through it (Benitez JA et al, feed and Immun 1999, 67 (2): 539-545), or in coating the antigen with an agent resistant to acidic substances, such as liposomes.

Currently, methods for preparing liposomes, and methods for encapsulation of solid fat-soluble materials, are well known (Schneider U.S. Pat. No. 4,089,801, Ash et al U.S. Pat. No. 4,448,765, and Miller et al U.S. Pat. No. 4,133,874). The main problems posed by the encapsulation of pharmaceutical raw materials by liposomes include: its slight stability shown in laboratory tests; spillage of the encapsulating material; the efficacy of the drug is reduced; susceptibility to adverse environmental conditions, digestion in the gastrointestinal tract and indirect fusion with cell membranes: (http：//www.BDSiAdvantages.html). Liposomes, on the other hand, are unstable structures that for the most part cannot be freeze-dried-a problem that has been overcome by the development of cochlear structures.

The snail shape is a multi-layered lipid structure rolled into the form of a sea shell. The preparation of cochlear structures by fusion of unilamellar liposomes and the use of divalent cations is a well-known practice (D. Papahadjopoolous et al biochem. Biophys. acta, 1975; 394: 483). This procedure has been modified to produce suspensions containing antigen and lipid multilamellar vesicles surrounded by antigen. The latter are sonicated under nitrogen to convert into small unilamellar lipoprotein vesicles, the sonication being intended to form snail structures in the presence of divalent ions (Gould-Fogerite et al, U.S. Pat. No. 5,643,574, 1/7 1997). Fig. 1 summarizes these techniques.

Cochlear, as well as other self-assembled microstructures, have been used for the administration of therapeutic agents (Yager et al, U.S. Pat. No. 5,851,536, 22.12.1998, Gould-Forgerite et al, U.S. Pat. No. 5,994,318, 30.11.1999, and Yager et al, U.S. Pat. No. 6,180,114, 30.1.2001). These include the preparation of cochlear adjuvants (Gould-Fogerite et al, U.S. Pat. No. 5,994,318, 1999, 11/30). However, both liposomes and cochlear must be derived from negative lipids (negative lipids) in the presence of cholesterol, both of which are usually extracted from animals in expensive procedures (Mannino et al, U.S. Pat. No. 4,663,161, 5.5.1987), which are becoming increasingly unacceptable according to new pharmaceutical regulations, and in the case of vaccine preparations, preferably include purified proteins, or peptides, obtained from microorganisms. Furthermore, we should point out that the cochlear form has not previously been considered as an adjuvant with its own ability, or its formulation containing other activators that induce important signals in the immune response, such as molecular structures associated with pathogens (phylogenetically conserved structures, its receptors in the host, and recognized by the host as signs of danger).

The aim of the present invention is to obtain new cochlear structures from vesicles found in the outer membranes of living organisms, which, due to their particular protein and lipid composition, and due to the molecular structure associated with pathogens found in the organism, exhibit adjuvant and vaccine properties. Once formed, the snails are homogenized in their own size range to make them more immune.

The snail-shaped structures obtained by the present invention are characterized by a proteolipid content, which has not been tried by other authors so far, being able to assemble themselves and to produce a rolled sea-shell-like structure. The composition of the proteins and lipids of the snail structure will depend on the microorganism of the vesicle feeding its outer membrane, that is, it will depend on the characteristics of the proteins found in its membrane. In the same way, the mentioned structure contains the molecular structure associated with the pathogen in a concentration of 1-7% with respect to the protein concentration, it is supplied by the membrane of said microorganism, it is inserted and in the free state it cannot be found within the mentioned structure. Furthermore, these structures can be purified from other microorganisms and can be added to the formulation. The added and existing structures should be at a concentration between 1-30% with respect to the concentration of the protein. One of the pathogen-associated molecular structures utilized in the preparation of cochlear structures is the lipopolysaccharide of Vibrio cholerae (Vibrio cholerae) or neisseria meningitidis (n. meningitis) (example 20).

The snail-shaped structures obtained by the present invention induce cellular responses that prove effective in breast-fed infants, show resistance to heat and to acids and bases, and comprehensively prove suitable for mucosal administration (examples 2, 4,6, 8 and 10). These properties are effective in designing heterogeneous adjuvants (adjuvants for boosting vaccines other than those which produce outer membrane vesicles) and homogeneous vaccines (vaccines for combating the microorganisms which supply the outer membrane vesicles) using the mentioned structures.

With respect to vaccine compositions containing such structures, it is important to note that at various stages of the experiment, the serological response is superior to that obtained when using an alumina adjuvant-containing outer membrane vesicle-based vaccine, the latter vaccine being commercially known as VAMENGOC-BCAnd when it is administered, specific immunoglobulin a is also induced through the mucosa. Furthermore, the cochlear structure stimulates a significant portion of all immune responses of CD8 lymphocyte-intracellular organisms.

The adjuvant effect of cochlear structures was evaluated by various tests, including: production of IL12 in human tissue cell line U937 (example 11) and nitric oxide in murine macrophage line J774 (example 13) in the absence of all stimuli; stimulation of human dendrites (example 15) and challenge experiments in which lesion induration was reduced, "challenge" mice immunized with cochlear structures containing antigens derived from the protozoan organism Leishmania major (Leishmania major) (example 16).

The cochlear structures prepared by the present invention allow the adjuvant or vaccine produced to induce an earlier, stronger and more durable response "in vivo", while an efficient induction of the mediators involved in cellular structure induction, and a very good stimulation of cells presenting professional antigens (dendritic cells) was observed "in vitro" (examples 11, 13 and 15).

It is another object of the invention that vesicles found in the outer membrane of microorganisms, which constitute the starting point for the formation of the snail structure, are used as vaccines or as xenoadjuvants, thus eliminating the aluminium hydroxide adsorption without limiting the immunogenic capacity of the former. It is worth mentioning that the ability of these vesicles to induce a parenteral response by themselves has not been fully studied.

The vesicles are considered to be self-assembled microspheres and consist of a lipid bilayer into which proteins and polysaccharides are inserted. These can be extracted from any pathogen and can present different molecular structures (in particular lipopolysaccharides, peptidoglycans, lipoproteins, teichoic acids, flagellins or lipo-phoglycans). The lipo-phosphoglycans and lipopolysaccharides are obtained from Leishmania major and Neisseria meningitidis or Salmonella typhi (Salmonella typhi), respectively, and remain inserted in the vesicles at a ratio of 1 to 7% by weight of the protein and never exist in a free state during the process of obtaining the vesicles from the outer membrane of an organism.

For vaccine formulations, outer membrane vesicles extracted from salmonella typhi or from neisseria meningitidis B induced a response from IgA when inoculated intranasally and induced a good immune response when inoculated parenterally (examples 3,5, 7, 12 and 14).

The adjuvant effect of outer membrane vesicles was assessed by a number of assays, including: production of IL12 in the human tissue cell line U937 (example 12) and nitric oxide in the murine macrophage cell line J774 (example 14) in the absence of additional stimulation; the cellular response (increase in IgG2 a) was boosted by the combination of the polysaccharide with vesicles found in the outer membrane of the netherlands meningitidis compared to its fusion with tetanus toxoid (example 18), and the response against reactive antibodies against polysaccharide Vi from salmonella typhi was boosted by combination with outer membrane vesicles found in the same bacteria (example 19).

The snail structure and outer membrane vesicles serve as adjuvants or vaccines causing an unexpected boost in the immune response induced by prior doses of vaccine or by exposure to bacteria. It is important to note that the latter are administered in a different manner than those derived from snail structures or vesicles (example 22).

Also disclosed are methods for obtaining cochlear structures from vesicles found in the outer membrane of living organisms. The method is implemented by adopting the following steps: first, the outer membrane of the vesicle formed by the living microorganism or cell is purified using any method widely used by experts in the field. Preferred methods are those disclosed in EP 301992, US5,597,572, EP 11243 or US 4,271,147, Zollinger et al (J.Clin.invest.1979, 63: 836-848), Frederikson et al (NIPH Annals 1991, 14: 67-80), Sauders et al (feed.Immun.1999, 67: 113-119), Drabick et al (Vaccine2000, 18: 160-172), WO 01/09350 or EP 885900077.8 and US5,597,572. The membranes are purified so that they contain 1-7% lipopolysaccharide fully inserted into the vesicles. Solutions were prepared with a total protein concentration of 3-6mg/mL and the concentration of the non-ionic detergent was increased to 8 to 12 times the protein concentration in order to fully dissolve the vesicles. This solution was subsequently sterilized by filtration through a membrane with a pore size of 0.2 μm, wherein the assembly of vesicles, which had not yet dissolved, was also removed. After this, either rotary dialysis or tangential filtration is performed. By containing appropriate concentrations of multivalent ions (especially Ca)²⁺、Zn²⁺Or Mg²⁺Concentration ranging from 2.5 to 6.5mM) was dialyzed at pH 7.4 ± 0.2 for 24 hours. Finally, the obtained volute was subjected to mechanical treatment (in particular sonication in a water bath at a temperature between 15 ℃ and 25 ℃ for 45 minutes) in order to make the particle size uniform.

This constitutes a fast and efficient method to obtain cochlear structures containing a variety of proteins and lipids from the outer membrane of the microorganism used, as well as molecular structures associated with naturally-obtained pathogens. These structures exhibit high levels of stability and immunogenicity.

On the other hand, the simple and efficient methods for obtaining them allow us to introduce new antigens into the mentioned structures. During the dialysis process, after increasing the detergent concentration and adding multivalent ionsPreviously, the neo-antigen was added to a suspension of outer membrane vesicles prepared to obtain the structure. Antigens that may be added are carbohydrates, lipoproteins, peptides, conjugates and nucleic acids. These should be at a concentration of 0.2-2.7. mu.g per 3-9. mu.g of protein. It is also possible to introduce other molecular structures associated with pathogens to stimulate innate and acquired responses-making them effective as xeno-adjuvants. Lipopolysaccharide of vibrio cholerae, amastigotes or promastigotes of leishmania major are particularly adopted structures that allow us to induce cellular responses and the activity of reactive antibodies against them. In addition to these, plasmid DNA containing green fluorescent protein was introduced and placed in macrophage lineage; the fluorescence of this molecule then allows us to determine its presence in the cell. Also introduced into Derma tophagoides siboneyAllergen (alergenic)The induced resultant cell response was determined (examples 16 and 17).

In a specific embodiment, the cochlear structures in the vaccine composition of the invention comprise pathogen-associated molecular structures added at a concentration of 1% to 30% by weight of the proteins of the cochlear structures.

In a specific embodiment, the vaccine composition of the invention additionally comprises an antigen selected from the group consisting of: a natural or recombinant protein, peptide, carbohydrate, nucleic acid, conjugate, or allergen. In a specific embodiment, wherein the added antigen is epitope T or B.

None of the authors describe the use of living organisms as a source of raw material for obtaining cochlear structures. Nor has the method of introducing one or more pathogen-associated molecular structures into a cochlear structure as in the case of the present invention been described.

The invention will be described by the following specific examples.

EXAMPLE 1 obtaining a volute

We began with the extraction of vesicles from the outer membrane of microorganisms using the methods described in EP 885900077.8 or US5,597,572. These were resuspended in Tris-EDTA buffer containing 0.5% sodium deoxycholate. Protein concentration of the suspension was determined using the Peterson modified Lowry's method (Analyt. biochem.83, 346, 1977). The phospholipid content of vesicles was determined by determining the inorganic phosphorus content (Bartlett, J biol. chem 234, 466, 1959). Both protein and phospholipid concentrations were used to determine the optimal conditions and detergent amounts required for snail formation. Vesicle-containing solutions were prepared and adjusted to a final protein concentration of 5-6mg/mL in Tris-EDTA buffer containing sodium deoxycholate at a concentration of 6 to 15 times the total protein concentration. This solution was filtered in a dialysis apparatus with a filter having a pore size of 0.2 μm. Dialysis was performed using a rotary stirring method for a period of 24 hours with continuous and slow changes in dialysis buffer. This final solution consisted of water containing 50-150mM NaCl, 1-4mM imidazole, 1-5 mM HEPES 3-and 7mM CaCl 2-prepared under sterile conditions that remained unchanged during each step of the procedure. The formation of the snail structure was confirmed by the appearance of a white precipitate and subsequent optical and electron microscopic observation. Protein and phospholipid concentrations were recalculated and adjusted for subsequent testing. The physical and chemical properties of the proteins included in the snail structure were examined and compared with the proteins of the vesicles by electrophoresis in a coomassie blue stained polyacrylamide gel. The structural integrity of the latter was determined and confirmed using Western blot methods (fig. 2-4).

Example 2 cochlear structure-induced response in mice and vaccine VA-MENGOC-BC Comparison of induced responses

Using VA-MENGOC-BCOr cochlear configuration, Balb/c mice were immunized intramuscularly with 12. mu.g protein per mouse, two doses separated for 21 days. After the second dose, blood samples were drawn from the animals at the indicated times and the IgG serum response against the outer membrane vesicles was assessed by ELISA assay. Significant differences between cochlear structures and vaccine-induced responses (p < 0.05) were observed at 17, 27 and 180 days after the second dose, allThe former (fig. 5).

Example 3 parenteral administration of outer Membrane vesicles induced responses in mice with the vaccine VA-MENGOC-BC Comparison of induced responses

Using VA-MENGOC-BCOr cochlear configuration, Balb/c mice were immunized intramuscularly with 12. mu.g protein per mouse, two doses separated for 21 days. After the second dose, blood samples were drawn from the animals at the indicated times and the anti-vesicle IgG serum response was assessed by ELISA assay. No significant difference between vesicle-induced and vaccine-induced responses was observed (p < 0.05). These results demonstrate the usefulness of vesicles and vaccines in their own right (fig. 6).

Example 4 effectiveness of Intranasal (IN) or Intragastric (IG) immunization with cochlear structures

Balb/c mice were immunized Intranasally (IN) or Intragastrically (IG) with 100 or 12 μ g protein per mouse, respectively, and the two doses were separated for 21 days. After the second dose, blood samples were drawn from the animals at the indicated times and the anti-vesicle IgG serum response was assessed by ELISA assay. Both intranasal and intragastric inoculation of cochlear structures at both concentrations induced a good response from IgG against outer membrane vesicles. This suggests that a good systemic response was obtained by mucosal vaccination (figure 7).

Example 5 effectiveness of intranasal Immunization (IN) with outer Membrane vesicles

Balb/c mice were immunized with 12. mu.g of protein Intranasal (IN) per mouse, and the two doses were separated for 21 days. After the second dose, blood samples were drawn from the animals at the indicated times and the anti-vesicle IgG serum response was assessed by ELISA assay. The good IgG response to vesicles obtained by this vaccination method suggests that a valuable systemic response can be obtained by intranasal vaccination (figure 8).

Example 6 intranasal or intragastric administration of cochlear structures induces the effectiveness of IgA in saliva

Balb/c mice were immunized Intranasally (IN) or Intragastrically (IG) with 100 or 12 μ g protein per mouse, respectively, and the two doses were separated for 21 days. Saliva samples were taken from the animals 9 days after the last dose administration and the IgA response to the outer membrane vesicles was assessed by ELISA assay. A significant response against vesicular IgA was obtained using the IN method, and a smaller but significant increase IN IgA against vesicles was obtained using the IG method (figure 9).

Example 7 effectiveness of intranasal administration of outer Membrane vesicles to induce IgA in saliva

Balb/c mice were immunized with 12. mu.g of protein Intranasal (IN) per mouse, and the two doses were separated for 21 days. Saliva samples were taken from the animals 9 days after the last dose administration and the IgA response to the outer membrane vesicles was assessed by ELISA assay. Significant response against vesicular IgA was obtained using the IN method (fig. 10).

Example 8 subclass of reactive IgG against outer membrane vesicles in sera induced by cochlear architecture immunization

Balb/c mice were immunized Intranasally (IN), Intragastrically (IG) or Intramuscularly (IM). IN the case of the IN method, 100. mu.g of protein per mouse was given as a snail concentration, while IN the remaining cases 12. mu.g was used. In all cases the agents were separated by a period of 21 days. Vaccine VA-MENGOC-BCUsed as a positive control, was administered intramuscularly at a concentration of 12 μ g. 21 days after the second dose, blood samples were taken from the animals and titers of IgG1 and IgG2a present in the serum were determined by ELISA assay. In all cases considered (except the negative control case), significant titers of IgG2a were obtained (p < 0.05). When snails are administered intranasally, these are at their highest values. These indicate that intranasal vaccination is particularly beneficial for the induction pattern of Th1 type antibody-IgG in the cells (FIG. 11).

Example 9 subclass of IgG in serum induced by immunization with Outer Membrane Vesicles (OMVs)

Balb/c mice were immunized Intranasally (IN) and Intramuscularly (IM) with 12. mu.g protein outer membrane vesicles per mouse, two doses separated by 21 days. Vaccine VA-MENGOC-BC given at the same concentrationAs a positive control. Blood samples were drawn 21 days after the second dose and the sera were analyzed by ELISA assay for the titers of IgG1 and IgG2a against OMVs. In all cases considered, the outer membrane vesicles induced significant titers of IgG2a, indicating the induction pattern of cellular Th 1-type IgG antibodies. This was not the case with negative IM or IN controls. Complete reversal of the pattern was observed IN the case of IN vaccination, IN which the IgG2a response was almost excluded (fig. 12).

Example 10 resistance to Heat and acid of the cochlear Structure obtained from the outer Membrane vesicles

The heat resistance of the snails was evaluated by exposing the samples to a temperature of 60 c for a period of 7 days. Acid resistance was assessed by exposing the samples to a medium at pH 1 for a period of 45 minutes. After this, treatment and control samples were used to inoculate Balb/c mice intranasally at a concentration of 12 μ g per mouse, two doses, 14 days apart. Blood samples were drawn from the mice 28 days after the start of the experiment and the sera were stored alone at-20 ℃ until the time they were used. As can be observed, there was no significant difference between the induced anti-vesicular IgG responses in each animal and group (p < 0.5).

Example 11 production of IL12 in the U937 cell line stimulated with cochlear structures only

U937 cells were cultured in RPMI 1640 supplemented with gentamicin, L-glutamine (2mM), sodium pyruvate (1mM), HEPES (15mM) and 10% fetal bovine serum (Sigma) at a concentration of 50. mu.g/mL. These cells were differentiated into macrophages by PMA treatment and placed in flat-bottomed 24-well plates at 5X 10 per well⁵And (4) cells. After 24 hours, the snail structure was incubated at 250ng/mLThe concentration of the nutrient is added to them. After 24 hours stimulation, surviving cells were harvested and the presence of IL12 was determined by sandwich ELISA assay. Cochlear structures were observed to stimulate IL12 production by U937 cells (fig. 14).

Example 12 production of IL12 in the U937 cell line stimulated with Outer Membrane Vesicles (OMVs) only

U937 cells were cultured in RPMI 1640 supplemented with gentamicin, L-glutamine (2mM), sodium pyruvate (1mM), HEPES (15mM) and 10% fetal bovine serum (Sigma) at a concentration of 50. mu.g/mL. These cells were differentiated into macrophages by PMA treatment and placed in flat-bottomed 24-well plates at 5X 10 per well⁵And (4) cells. After 24 hours, outer membrane vesicles were added to them at a concentration of 250ng/mL medium. After 24 hours stimulation, surviving cells were harvested and the presence of IL12 was determined by sandwich ELISA assay. It was observed that outer membrane vesicle stimulation caused IL12 production by U937 cells (fig. 15).

Example 13 nitric oxide production in J774 murine macrophage lines stimulated with cochlear structures alone

J774 cells were cultured in DMEN medium supplemented with gentamicin at a concentration of 50. mu.g/mL, L-glutamine (2mM), sodium pyruvate (1mM), HEPES (15mM) and 10% fetal bovine serum (Sigma) previously inactivated at 56 ℃ for 30 minutes. They were placed in flat-bottomed 96-well culture dishes at 1X 10 per well⁵Concentration of individual cells, they were cultured at 37 ℃ and 5% CO2 for a period of 24 hours. After this time, adherent cells were cultured with 200L of DMEN with cochlear structures at a concentration of 250 ng/mL. Other variants in culture with the nitric oxide production inhibitor L-NMMA (at 1. mu.M) were also included. Surviving cells were harvested after 24 and 48 hours and analyzed for nitrogen content using the Greiss' reaction (Rockett, KA et al, infection. Immun.1992, 60: 3725-3730). It was observed that cells cultured with the snail structure produced significant nitric oxide. This production was inhibited using L-NMMA (fig. 16).

Example 14 nitric oxide production in J774 murine macrophage lines stimulated with Outer Membrane Vesicles (OMVs) only

J774 cells were cultured in DMEN medium supplemented with gentamicin at a concentration of 50. mu.g/mL, L-glutamine (2mM), sodium pyruvate (1mM), HEPES (15mM) and 10% fetal bovine serum (Sigma) previously inactivated at 56 ℃ for 30 minutes. They were placed in flat-bottomed 96-well culture dishes at 1X 10 per well⁵Concentration of individual cells, they were cultured at 37 ℃ and 5% CO2 for a period of 24 hours. After this, adherent cells were cultured with 200L DMEN with outer membrane vesicles at a concentration of 250 ng/mL. Other variants in culture with the nitric oxide production inhibitor L-NMMA (1. mu.M) were also included. Surviving cells were harvested after 24 and 48 hours and analyzed for nitrogen content using the Greiss' reaction (Rockett, KA et al, infection. Immun.1992, 60: 3725-3730). It was observed that cells cultured with outer membrane vesicles produced significant nitric oxide, greater than LPS-induced nitric oxide used as a control. This production was inhibited using L-NMMA (fig. 17).

Example 15 stimulation of human dendritic cells by cochlear structures

Blood was drawn and peripheral mononuclear cells were purified using ficoll. In the presence of LPS or cochlear structures at 10X 10 per ml¹⁶Cells were cultured and activation of dendritic cells was determined by flow cytometry. As observed in fig. 18, dendritic cells were activated (as determined by expression of costimulatory molecules, such as CD40, CD80, and CD86), and MHC molecule expression was increased. This shows the adjuvant properties of these structures.

Example 16 reduction of induration in Balb/c immunized with cochlear structures containing amastygote of Leishmania major and challenged with the same protozoan organism

Inclusion of amastigotes derived from leishmania major is accomplished by the first step of inclusion of the semi-purified antigen into the formation of cochlear structures. The amount of detergent used and the total protein concentration were adjusted to maintain a range of 5-6mg/mL depending on the total protein content. The ratio of vesicular protein to neoantigen contained was 12: 1. The formation of the cochlear structures was confirmed by optical and electron microscopy. Also by electrophoresis in a Coomassie blue-stained polyacrylamide gelConfirming the inclusion of proteins from Leishmania major. Balb/c mice were immunized intramuscularly with 12. mu.g of cochlear structures, 2 doses separated for 21 days. The left hind extremity was inoculated with a snail. 21 days after the second dose, the same extremities as the inoculation were treated with 3X 10⁶Individual promastigotes infected mice. Promastigotes were obtained from the stationary phase of cultures grown on solid agar-blood medium in DMEN medium. The volume of lesions was stimulated weekly, starting on the fourth week after infection. A significant reduction in lesion size was observed in the group immunized with the snail containing leishmania major antigens. This demonstrates the adjuvant properties of these structures (fig. 19).

Example 17 inclusion of plasmid DNA containing Green fluorescent protein

The purified plasmid containing the green fluorescent protein gene under the CMV promoter was included in the starting solution for obtaining the cochlear structure according to the same procedure as described in example 1 for obtaining the structure. The ratio of plasmid to vesicular protein was adjusted to 1: 100. To provoke release of their internal plasmids, EDTA was added to 2mM, followed by incubation at 37 ℃ for 30 minutes, and the inclusion of the plasmids was checked by electrophoresis in agarose gels at 1% concentration of the snail structure. The gel was stained with ethidium and observed under uv light. The presence of plasmid was detected only in the plasmid-containing snail structure after EDTA treatment. After this, transfection experiments were performed in J774 cell lines using these constructs. After 2 hours of incubation, the media was cleared of snail structures. Examination of the cells under fluorescence after 24 hours showed the presence of many cells with fluorescent signals in the cytoplasm.

Example 18 enhancement of cellular response by conjugation of outer Membrane vesicles

The Neisseria meningitidis serogroup C polysaccharides (PsC) are conjugated to Tetanus Toxoid (TT) or Outer Membrane Vesicles (OMV) from Neisseria meningitidis serogroup B. Balb/c mice were inoculated intraperitoneally three times (on days 0, 14 and 28) with 10. mu.g of PsC in combination. Blood samples were drawn from the animals before and 42 days after immunization. The response of IgG and its subclass in serum was determined. The strongest IgG2a response found in the group conjugated to outer membrane vesicles indicated that better cellular responses were obtained than those induced in the group conjugated to Tetanus Toxoid (TT) (fig. 20).

Example 19 enhancement of anti-polysaccharide Vi antibody response by conjugation

The polysaccharide Vi from salmonella typhi is conjugated to the Outer Membrane Vesicles (OMVs) of salmonella typhi. Balb/c mice were immunized intraperitoneally with two doses containing 10. mu.g Vi (administered on days 0 and 28). Blood samples were drawn from the animals before inoculation and 42 days after inoculation. The response of IgG and its subclass in serum was determined. This conjugation increased and confirmed the response against the Vi polysaccharide and detected the response provided by IgG2a (fig. 21).

Example 20 possibility of including different concentrations of pathogen-associated molecular structures in cochlear structures

Experiments were conducted with different amounts of LPS derived from neisseria meningitidis B involving molecular structures associated with different concentrations of pathogens in the cochlear structures. The ratio of LPS concentration to protein concentration used for immunization of mice was 0.05: 12, 0.5: 12, 1: 12 and 2: 12. The formation of the snail structure was confirmed by optical microscopy, which determined a 1: 12 ratio as the maximum ratio for LPS introduction without affecting the formation of the snail structure. The larger number significantly affects the structure formation, causing aggregate formation. All variants obtained (variant) were administered to Balb/c mice in two doses, separated by 21 days, with 12 μ g protein per mouse. Anti-vesicular IgG titers were determined. No differences between the results produced using the different variants were observed. However, this experiment guarantees the possibility of introducing different LPS in these structures. (FIG. 22).

EXAMPLE 21 effectiveness of the proposed method for the last step of making the volute

Cochlear structures treated with photoacoustic treatment (Tto) for 45 minutes at 20 ℃ in a water bath, as well as untreated structures were used. Balb/c mice were immunized intranasally with 100. mu.g of protein per mouse, and the two doses were separated for 21 days. Saliva samples were taken 9 days after the last dose and the IgG response against Outer Membrane Vesicles (OMVs) was assessed by ELISA assay. The immune response in those animals treated with this construct occurred significantly earlier or worked for a longer period of time as shown in figure 23.

Example 22 IgA response to anti-outer Membrane vesicles in serum, saliva and vagina induced by parenteral and mucosal Immunity and assessed by ELISA assays

Intranasal (IN) immunization with 2 or 3 doses of Outer Membrane Vesicles (OMVs), respectively, 3 doses of vaccine VA-MENGOC-BCIntramuscular administration as a control, and a combination of 1 and 2 doses of vaccine were administered to Balb/c mice using the IM and IN methods. On days 0, 21 and 42, 12 μ g of protein was administered to each mouse. Serum was taken 15 days later and saliva and vaginal fluid were taken 9 days after the last dose. The results were evaluated by ELISA assay. As can be observed, intranasal immunization induced a small increase in serum IgA levels, which was not the case with vaccines using the IM method. Mucosal response is dependent on dose number: two doses did not induce a response, while 3 doses elicited an anti-vesical IgA response. Finally, 2 doses of intranasal administration proved effective in animals that received intramuscular administration of vaccine stimulation (one dose) (fig. 24).

Advantages of the proposed solution

1. Outer membrane vesicles extracted from living organisms, allowing the selection of components during the outer membrane extraction process, which outer membrane constitutes a first defense barrier for the contact between host and pathogen, making these components suitable for animal and human protection;

2. during extraction, other proteins of interest, either native or recombined, may be included;

3. outer membrane vesicles extracted from living organisms are more stable than artificially constructed liposomes and can remain intact for months, even years, without suffering significant changes that can affect future formation of volute structures.

4. The Th1 cell response induced in animals and humans makes these adjuvants effective not only in adults and children, but also during breast feeding.

5. The resulting cochlear structures have heat resistance which may prove useful in solving problems associated with many vaccine refrigeration regimes, by virtue of their formulation as adjuvants or their development from outer membrane vesicles and cochlear structures.

6. The resulting cochlear structures are resistant to bases and acids, a consideration when considering oral administration of vaccines.

7. Antigens are introduced into the structure during the manufacturing process, so that the final product is heat, acid and alkali resistant;

8. the variability of antigens that can be included, either soluble or particulate, including nucleic acids, allows for the preparation of many vaccines, including composite vaccines.

9. The cochlear structure contains molecular structures associated with pathogens, and other substances that may be introduced at will in order to increase its adjuvant and immunological effectiveness; allowing us to reduce the potential toxicity of some structures and thus their inflammatory effects;

10. the volute structure induces an earlier, stronger and longer-lasting response in vivo;

11. the cochlear structure induces a better response in vitro at the level of cytokinins that induce a cellular immune response pattern;

12. the structure retains the properties of artificial snails (efficient introduction of hydrophobic antigens, slow unfolding system, calcium content as major mineral, reduction of lipid oxidation, freeze drying, etc.). It is superior to artificial snails in immunogenicity because it contains molecular structures associated with pathogens, and its ability to induce Th1 patterns, including T cytotoxic responses, is superior to artificial snails, and avoids the use of lipids and cholesterol derived from animal serum.

Brief Description of Drawings

FIG. 1, a method for preparing snails as described in U.S. Pat. No. 5,643,574 to Gould Fogerite et al, 1997, 7/1.

Figure 2. simplified method of obtaining a volute-object of the present invention.

FIG. 3 Electron microscopy of cochlear structures.

FIG. 4. A: proteins present in outer membrane vesicles were electrophoresed in coomassie blue stained 12.5% acrylamide gels. B: western blotting of proteins present in outer membrane vesicles and cochlear structures, human serum with high titers of reactive antibodies against outer membrane vesicles.

FIG. 5 is a schematic representation of a cell using VA-MENGOC-BCOr cochlear structure parenterally immunized mice, the IgG serum response against outer membrane vesicles was assessed by ELISA assay.

FIG. 6 is a schematic representation of a cell using VA-MENGOC-BCOr outer membrane vesicles the IgG serum response against outer membrane vesicles in mice immunized intramuscularly with outer membrane vesicles was assessed using an ELISA assay.

FIG. 7 IgG serum response against outer membrane vesicles IN mice immunized with outer membrane vesicles Intragastric (IG) or Intranasally (IN), assessed by ELISA assays.

FIG. 8 IgG serum response against outer membrane vesicles IN mice immunized Intranasally (IN) with outer membrane vesicles, assessed by ELISA assay.

FIG. 9 IgA response of anti-outer membrane vesicles in saliva of mice immunized intragastrically or intranasally with snail structure, assessed by ELISA assay.

FIG. 10 IgA response to anti-outer membrane vesicles in saliva of mice immunized intranasally with outer membrane vesicles, assessed by ELISA assay.

FIG. 11 results of IgG subclasses reactive against outer membrane vesicles in animals immunized with cochlear structures, evaluated by ELISA assay.

FIG. 12 results of IgG subclasses reactive against outer membrane vesicles in animals immunized with outer membrane vesicles, evaluated by ELISA assay.

FIG. 13. the heat and acid resistance results of the snail structure were evaluated by ELISA assay.

FIG. 14 evaluation of IL12 production by U937 cells stimulated with cochlear structures.

FIG. 15 evaluation of IL12 production by U937 cells stimulated with outer membrane vesicles from Neisseria meningitidis.

FIG. 16 production of nitric oxide by J774 cells cultured with cochlear structures.

FIG. 17 production of nitric oxide by J774 cells cultured with outer membrane vesicles derived from Neisseria meningitidis.

FIG. 18 stimulation of human dendritic cells by cochlear structures.

FIG. 19. results of immunization with amastigote-containing cochlear structures and induration in animals challenged with Leishmania major.

Figure 20. adjuvant effect results of conjugation of polysaccharides to outer membrane vesicles of neisseria meningitidis.

FIG. 21 adjuvant effect results of conjugation of polysaccharides to outer membrane vesicles of Salmonella typhi.

FIG. 22 results of the introduction of molecular structures associated with pathogens.

FIG. 23. results of the effect of sonication on the snail-induced response.

Figure 24 results of mucosal response enhancement after IN vaccination after initial intramuscular stimulation.

FIG. 25 cochlear structure-affected (proteins) IgG anti-antibodyKinetics of the OMV reaction (AFCo 1). In the presence of OMV, AFCo1 or VA-MENGOC-BC^TManti-OMV IgG responses were determined in the serum of mice immunized intramuscularly with the vaccine. AFCo1 induced a significantly high and durable IgG response.

FIG. 26. DCs can additionally process OVA peptides from OVA (OMV-OVA) contained in envelope vesicles (OMV) for MHC-II presentation. IL-2 production by OT4HT hybridoma cells incubated with OMV-Ova by the IL-2 dependent CTLL cell line [ 2 ]³H]Thymidine incorporation was quantified. Data are presented as mean + SD of triplicate experiments.

Clearly different from the other groups.

FIG. 27 IgG anti-Ova response in mice immunized with OMV-Ova. C3H/HeN mice were immunized twice with 5mg/ml OMV-Ova, phosphate buffer or Titemax-Ova emulsion (positive control), and sera were collected 21 days after the first immunization and tested by ELISA. The data show the mean IgG concentration ± standard deviation of 5 mice per group, representing three different experiments.

FIG. 28 IgG anti-Ova subclass response in mice immunized with OMV-Ova. C3H/HeN mice were immunized twice with 5mg/ml OMV-Ova, phosphate buffer or Titemax-Ova emulsion (positive control), and sera were collected 21 days after the first immunization and tested by ELISA. The data show titers of IgG1 and IgG2a in pooled sera of 5 mice per group, representing three different trials.

Figure 29 IgG response induced against hepatitis c Virus (VHC) core protein in intramuscularly immunized animals assessed by ELISA. Anti-core IgG was determined in the sera of animals immunized three times (weeks 0, 3 and 7) by the intramuscular route and samples were collected 2 weeks after the last time, indicating that the binding of the two antigens (core and capsid proteins) in AFCo1 induced a significantly high response.

Claims (12)

1. Vaccine compositions comprising cochlear structures derived from outer membrane vesicles of the genus Neisseria (Neisseria), Salmonella (Salmonella), or Leishmania (Leishmania) in a viable form and suitable excipients; the cochlear structures comprise proteins, lipids, and pathogen-associated molecular structures.

2. The vaccine composition according to claim 1, which is introduced with, or co-administered with, one or more antigens selected from the group consisting of conjugated polysaccharides Vi, hepatitis c virus, cholera lipopolysaccharide, DNA plasmids and tetanus toxoid; the concentration of the introduced or co-administered antigen ranges from 0.2 to 2.7 μ g per 3 to 9 μ g of protein.

3. The vaccine composition according to claim 1, wherein the molecular structure associated with a pathogen is selected from the group consisting of: lipopolysaccharide, peptidoglycan, lipoprotein, flagellin, and lipophosphoglycan, the concentration of the pathogen-associated molecular structure being 1% to 30% by weight of the proteins of the cochlear structure.

4.A vaccine adjuvant comprising a cochlear structure obtained from a live neisseria, salmonella, or leishmania outer membrane vesicle; the cochlear structures comprise proteins, lipids, and pathogen-associated molecular structures.

5. A vaccine adjuvant according to claim 4 wherein said pathogen-associated molecular structure is selected from the group consisting of: lipopolysaccharide, peptidoglycan, lipoprotein, flagellin, and lipophosphoglycan, the concentration of the molecular structure being 1% to 30% by weight of the protein of the cochlear structure.

6. A vaccine composition comprising vesicles obtained from the outer membrane of live neisseria, salmonella, or leishmania and a suitable excipient; the vesicles comprise proteins, lipids, and molecular structures associated with pathogens.

7. The vaccine composition according to claim 6, wherein the molecular structure associated with a pathogen is selected from the group consisting of: lipopolysaccharide, peptidoglycan, lipoprotein, flagellin, and lipophosphoglycan, the concentration of the molecular structure being 1-7% by weight of the protein of the structure.

8. A vaccine adjuvant comprising vesicles extracted from the outer membrane of live neisseria, salmonella, or leishmania; the vesicles comprise proteins, lipids, and molecular structures associated with pathogens.

9. A vaccine adjuvant according to claim 8 wherein said pathogen-associated molecular structure is selected from the group consisting of: lipopolysaccharide, peptidoglycan, lipoprotein, flagellin, and lipophosphoglycan, the concentration of the molecular structure being 1-7% by weight of the protein of the structure.

10. A method of obtaining cochlear structures from live neisseria, salmonella, or leishmania outer membrane vesicles, consisting of the steps of:

a. preparing a solution containing a total protein concentration of 3-6mg/mL from the outer membrane vesicles, and adding thereto a 10-fold protein concentration of a nonionic detergent;

b. if it is desired to introduce as vaccine adjuvant a further antigen of interest or a molecular structure associated with a pathogen selected from the group consisting of lipopolysaccharide, peptidoglycan, lipoprotein, flagellin and lipophosphoglycan, said antigen and molecular structure associated with a pathogen are added to the solution prepared in a) homogeneously to a concentration of 0.2 to 2.7 μ g of antigen per 3 to 9 μ g of protein and a protein concentration of 1-30% of molecular structure associated with a pathogen;

c. then, in order to sterilize and eliminate the vesicle aggregates still found therein, the solutions of steps a) and b) are filtered through a membrane with a pore size of 0.2 μm;

d. then subjecting the product of step c) to rotary dialysis or tangential filtration against a solution containing a multivalent ion at a concentration of 2.5-6.5mM, buffered to pH 7.4 + -0.2;

e. finally, the obtained snail-shaped structure is mechanically treated in order to make the particle size uniform.

11. The method according to claim 10, wherein said multivalent ion of step d) is Ca²⁺、Zn²⁺Or Mg²⁺。

12. The process according to claim 10, wherein the mechanical treatment in step e) is a sonication in a water bath at a temperature of 15 ℃ to 25 ℃ for a period of 45 minutes.