Title of Invention

IMMUNOGENIC COMPLEX FORMED BY VACCINAL ANTIGENS ENCAPSULATED BY NANOSTRUCTURED MESOPOROUS SILICA

Abstract The present invention relates to a product named "immunogenic complex", which comprises an adjuvant characterized by solid particles of highly ordered nanostructured mesoporous silica, preferably, the silica SBA-15, and vaccinal antigens from distinct nature, encapsulated in this kind of adjuvant. The immunogenic complex of the present invention allows the presentation of the antigens that compose it to lymphocytes in a safe, gradual and sustained way, which leads to a more efficient immunological memory, increases the immunogenicity of the antigen and improves the production of antibodies. It ensures an efficient immunological protection with lower amounts of antigens and/or less repetition of vaccinal doses. In addition, the characteristics of the immunogenic complex of the present invention promote effective immunity induction, homogeneous in "high and low responder" individuals.
Full Text FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. IMMUNOGENIC COMPLEX FORMED BY VACCINAL ANTIGENS ENCAPSULATED BY NANOSTRUCTURED MESOPOROUS SILICA
2.
(A) CRIST ALIA PRODUTOS QUIMICOS FARMACEUTICOS LTDA.
(B) Brazilian
(C) Rodovia Itapira/lindoia, Km 14, 13970-000 Itapira
Brazil
(a) FUNDACAO DE AMPARO A PESQUISA DO ESTADO DE SAO PAULO-FAPESP
(b) Brazilian
(C) Rua Pio Xi, 1500- Alto Da Lapa, 05468-901 Sao Paulo Brazil
The following specification particularly describes the invention and the manner in which it is to be performed.


The present invention relates to the immunology field.
The present invention relates to a product designated "immunogenical complex", effective in increasing immunogenicity, constituted by vaccinal antigens encapsulated with solid particles of highly ordinated nanostructured mesoporous silica acting as adjuvant, as shown in the present invention. The encapsulation with mesoporous silicas protects the antigens from degradation by macrophages and extends its exposure to lymphocytes, promoting improved immune response for being effective for induction of antibodies production, either in good or in bad respondents individuals. The immunogenical complex to which the present invention relates to, may bring benefit to the immunogenical activity of antigens of the types: biologically active peptides, toxins, virotic and bacterial vaccines.
The immune response of human beings to vaccinal antigens varies due to particular factors. Several individuals vaccinated with the same antigen, under the same conditions, produce responses that vary in intensity and duration. Such variation is a determinant factor of the intensity and duration of vaccines protecting effect.
After standardized antigenic stimulation, the individuals that reply producing protective titers of antibodies are named good respondents e those that do not produce protective titers are named bad respondents.
The development of safe and effective strategies for improvement of the immune response, either from good or from bad respondents is of utmost interest. In the first case, producing protective responses with lower amount of antigen or long-lasting response without re-exposure to the antigen. In the second case, producing a protective response with stimuli that, otherwise, would be insufficient.
Currently, this problem is only partially solved by the use of adjuvants that are defined as materials that extend the immune response particular of the organism to be determined as antigen [Edelman, R.; Tacket, CO.; Adjuvants Intern. Ver. Immunol, 7 (1990) 51], modifying the form through which the epitopes (antigenic determinants) are presented to the cells of immune system or raising the immunogenicity thereof. Other characteristics


desirable for an adjuvant are: to extend the stimulus period, increase the presentation time of the antigen and delay the catabolism thereof.
Apparently, many adjuvants exercise their activity by toxic actions against the macrophages. There are also adjuvants that modulate the immune response to certain antigen as, for example, inducing the predominant expression of an immunoglobulin isotype, for example an IgG. [Hadjipetrou-Kourounakis, L; Mbller, E.; Scand. J. Immunol., 19 (1984) 219].
The adjuvants licensed and largely used in human vaccines are the derivatives of aluminum salts as aluminum hydroxide or phosphate. However, these do not induce an immunological response substantially high and long lasting or qualitatively selective in relation to the subclass of IgG antibodies and to the cytokines involved.
There are other adjuvants used in veterinary such as Freund Incomplete Adjuvant [FIA] and the Freund Complete Adjuvant [FCA] that promote the undesirable formation of nodules, abscess or granulomes in the local administration. Other also known adjuvants are: A Lipid, Microspheres and Liposomes, none of which are destined for use in humans.
Thus, the interest in the development of safe and effective strategies for improvement of the immune response remains evident, either from good or bad respondents. In this way, the advancement in the sciences of materials area is enabling the preparation of new materials with improved properties and potential for application in several areas.
The inorganic porous solids present important industrial applications in catalytic and separation processes. These materials, due to the structural and surface properties thereof, allow the access of molecules to its nanostructures, thus increasing the catalytic and absorption activity thereof.
The porous materials currently used may be classified in three classes based on particulars of its microstructure: paracrystalline amorphous supports, materials with modified layers and crystalline molecular sieves. The differences in micro and mesostructure of these materials are important, either for its sortive and catalytic behavior, as well as in the properties used for characterizing it, such as: superficial area, pores size and variability of such sizes, the presence or absence of X-ray diffraction standards (DRX) and the details in


such standards, and the aspect of the materials when its microstructure is studied by transmission electronic microscopy (TEM) and electrons diffraction methods.
Amorphous and paracrystalline materials represent an important class of porous inorganic solids that have been used for many years in industrial uses. Typical examples of these materials are the amorphous silica, regularly used in formulation of catalysts and the transitive paracrystalline alumina, used as supports for acid solid catalysts and petroleum modified catalysts. The term amorphous is used in this context for indicating a material that does not present a long-range order, although nearly all materials are ordinated at a certain extent, at least in local scale. An alternative term that is being used to describe these materials is: "Indifferent X-Ray". The microstructure of silica consists of particles of 10-25 nm of condensed amorphous silica, with porosity resulting from empty spaces between particles. Since there is no long-range order in these materials, the pore size tends to be distributed within a wide range. This lack of order is also manifested in the diffraction X-ray standard (DRX), which usually appears without the characteristic peaks.
Paracrystalline materials, such as transitive alumina, have been presenting a wide distribution of the pores size, but well defined from the X-ray diffraction standard, that usually consists of some wide bands. The microstructure of these materials consists of small crystalline regions of condensed alumina phases and the porosity of the materials is the result of irregular empty spaces between these regions. Considering that in the case of one material or another, there is no long-range order controlling the pores size in the material, the variability in such sizes is typically very high. The sizes of pore in these materials comprise a band named mesopores that ranges between 1,3 to 20 nm.
In contrast with these solids, structurally little defined, are the materials which distribution of sizes of pores is very narrow, since it is controlled from the crystalline nature of the materials, accurately repeated, designated as microstructures. These materials are designated as "molecular sieves", and the most important examples are the zeolytes.
Such molecular sieves, natural or synthetic, include a wide variety of crystalline silicates containing positive ions.


In general, porous substances are divided by the pore size, for example, substances with pores size of less than 2 nm are classified as microporous, between 2 to 50 nm as mesoporous substances and over 50 nm are classified as macroporous substances.
A series of mesoporous molecular sieves, including MCM-41 and MCM-48, were described in U.S. Pat. Nos. 5,057,296 and 5,102,643. These molecular sieves show a structure in which the mesopores, uniform in size, are regularly arranged. MCM-41 has a uniform structure showing a hexagonal arrangement of direct mesopores, such as honeycomb, and has a specific surface area of 1000 m2/g obtained by BET method.
Molecular sieves have been produced using inorganic or organic cations as mold. These mesoporous molecular sieves are synthesized through a liquid crystal mechanism using surfactants as molds and have the advantage that the size of the pores may be adjusted at the rate of 1,6 to 10 nm, through the control of surfactant type or synthetic conditions employed during the production process.
Molecular sieves designated as SBA-1, SBA-2 and SBA-3 were described in Science (1995) 268:1324. Its channels are regularly arranged, while the constitutive atoms show an arrangement similar to that of amorphous silica. Mesoporous molecular sieves have regularly organized channels, larger than those existing in zeolytes, in this way capacitating its application in adsorption, isolation or reactions of catalytic conversion of relatively large molecules.
U.S. Patent No. 6,592,764 found a family of high quality mesoporous silicas, hydrothermal stability and of ultra-extensive pores size, through the synthesis with the use of a copolymer in amphophilic block in acid medium. A member of the family, SBA-15, has highly ordinated mesostructure, hexagonal in two dimensions (p6mm) similar to a honeycomb. Other structures as cubic in cage form, or three-dimensional hexagons are also formed. A calcination procedure at 500 C yields porous structures with high BET surface area of 690 to 1040 m2/g, and pores volume above 2,5 cm3/g, large interplanary distances d(100) of 7,45 to 45 nm, pores size of 4,6 to 50 nm and the thickness of silica wall of 3,1 to 6,4 nm. SBA-15 may be prepared with an extensive band of pores size and thickness of pore wall at low temperature (35 to 80 C), using a variety commercially available of


copolymer in amphiphilic block biodegrading and non-toxic, including three-blocks polyoxyalkaline.
The unique properties of SBA-15 make it an attractive material for several applications, including bio-application, for example, fixing of biologically active species. However, no document on the influence of these materials in the immunogenical response was identified, to the contrary, the literature listed would suggest its non- exploration for this purpose.
Experiments having studied the influence of amorphous silica in the immunogenical response, specifically on macrophages, were already carried out, however, silica role at that time did not involve it as adjuvant [Allison, A.C.; Harington, J.S.; Birbeck, M.; J. Exp. Med., 124 (1966) 141; Kampschmidt, R.F.; Worthington, M.L.; Mesecher, M.I.; J. Leukocyte Biol., 39 (1986) 123; Lotzova, E.; Cudkowicz, G.; J. Immunol., 113 (1974) 798; Lotzova, E; Gallagher, M.T.; Trentin, J.J. Biomedicine, 22(5) 387 1975; Vogel, S.N.; English, K.E.; O'brien, A.D.; Infect. Immun., 38 (1982) 681].
In another experiment [Gennari, M.; Bolthillier, Y.; Ibanez, O.M.; Ferreira, V.C.A.; Mevel, J.C.; Reis, M.A.; Piatti, R.M.; Ribeiro, O.G.; Biozzi, G.; Ann. Inst. Pasteur Immunol., 138 (1987) 359.], genetically modified mice were used, according to the low or high production of antibodies, in which the suspensions of colloidal silica administered during 4 consecutive days, prior to immunization with particulated antigen, namely, etherologous erythrocytes. These studies showed that there is a significant increase in the production of antibodies of bad respondent animals, and this improvement would be directly related with silica action on the macrophages, affecting some of its functions, causing non-availability of these cells and leading to the reduction of the antigen catabolism, thus favoring the presentation of the antigen to lymphocytes.
These effects were additionally analyzed particularly comparing the responses of mice strains that have distinct characteristics in relation to the functionality of its macrophages. This was achieved using an experimental model that selects the mice strains with the phenotypes of maximum or minimum response of antibodies. Such strains were obtained after crossbreeding between individuals with extreme phenotypes during


consecutive generations. After about 15 generations, the animals showing extreme phenotypes for the level of antibodies achieved homozygozis of the relevant alleles for the response against certain antigen. With this method it was possible to obtain the high strains [H] and low [L] production of antibodies of IVA Selection [Cabrera, W.H.; Ibanez, O.M.; Oliveira, S.L.; Sant'Anna, O.A.; Siqueira, M.; Mouton, D.; Biozzi, G.; Immunogenetics, 16 (1982) 583]. The differences of responses in these animals are related with the greater (L,VA mice strain) or smaller (H|VA mice strain) macrophages catabolic activity, prejudicing or favoring, respectively, the effective presentation of antigens.
The above-mentioned studies showed that when L,VA mice are previously and extensively treated with amorphous silica suspensions, and then immunized with an antigen, had its antibodies production increased, approaching to the responses of good respondents. On the other hand, [Biozzi, G.; Mouton, D.; Sant'Anna, O.A.; Passos, H.C.; Gennari, M.; Reis, M.H.; Ferreira, V.C.A.; Heumann, A.M.; Bouthillier, Y.; Ibanez, O.M.; Stiffel, C; Siqueira, M.; Current Topics In Microbiology Immunology, 85 (1979) 31.], in another experimental model, in which strains of an independent genetic selection Hm and l_w are used, which also show, respectively, high or low levels of antibodies production, which, however, owe its difference not to the functionality of its macrophages, but to the potentiality of its lymphocytes; when submitted to the same experiment, the modulation of antibodies production of bad respondent strains is not observed, when treated with the same suspension of amorphous silica.
These studies were fundamental to give support to the understanding in vivo on the role of macrophages in the immunization process, in addition to showing that for an adjuvant used in the induction of immunity is suitably efficient, the same should protect at maximum the antigen administered against the catabolism originating from macrophages and suitably present it to lymphocytes.
In large vaccine campaigns, uniform immunization products and processes are generally adopted for a large and heterogeneous group of individuals. Under these conditions, the production of variable titers of antibodies can be observed, some non-protective. This hinders the efficient immunization of part of the individuals.


Such fact is explained by the mechanisms shown in the above-mentioned experiments and originates from the phenotype variability of the individuals of the same specie, which may be interpreted by the efficient form or not of presentation of the epitope to the lymphocytes.
For example, individuals with lymphocytes effector activity that could be classified from normal to very high, or macrophages activity from reduced to normal, have a tendency to react more promptly, in relation to the production of antibodies, since the probability of the antigen to be identified more efficiently by the lymphocytes is great. These would be the "good respondent" individuals in a natural population.
To the contrary, individuals that present from normal to reduced lymphocytes activity, and very high macrophages activity have a tendency to more rapid catabolism of the antigen administered. This leads to a lower exposure of the antigen to lymphocytes and to an unstable immune response. These would be the "bad respondent" individuals in a natural population. This situation favors a natural selection of more resistant pathogens.
It is necessary to develop more efficient vaccines that would favor and promote the production of protective titers of antibodies, even in individuals that are bad respondents to the current vaccine formulations. Therefore, it is important that this differentiated cellular behavior is taken into consideration in the selection of the immunization adjuvant, seeking to minimize the influence of the differentiating factors.
The application of this concept does not exist yet, and we miss products and/or vaccines produced in accordance thereof.
One objective of the present invention is to show that antigens incorporated or encapsulated in nanostructured mesoporous silica form an immunogenical complex that is efficient in the induction of an immune response and in showing that such nanostructured mesoporous silicas do not affect the availability and phagocytic capacity of macrophages in culture.
The present invention relates to a new immunogenical complex constituted by antigens of several natures, encapsulated by highly ordinated nanostructured mesoporous


silicas that act as adjuvants, improving the induction of immunity and the production of antibodies to antigens, distinct in relation to the nature, structure and complexity.
The immunogenical complex according to the present invention relates to the product resulting from the combination of an antigen and particles of nanostructured mesoporous silicas in specific proportions.
The immunogenical complex of the present invention allows the effective immunization of the individuals that are bad respondents to products and processes currently used. This originates from more safe and effective presentation of the antigen to the lymphocytes.
The immunogenical complex of the present invention is constituted by at least one antigen, which is incorporated or encapsulated by the particles of nanostructured mesoporous silicas. In addition to effectively acting as immunization adjuvant, silica particles also serve as support or matrix for bioactive species, in this case, antigens.
The antigens that may be used in the formation of the immunogenical complex of the present invention include biologically active peptides, toxins, and virotic and bacterial vaccines.
Although a wide range of nanostructured mesoporous silica that may be used as adjuvants in the preparation of the immunogenical complex of the present invention, preferably, the silica designated as SBA-15 is used.
The highly ordinated nanostructured mesoporous silica, SBA-15, is composed of silicon oxide particles with regular cavities and uniform in size between 2 to 50 nanometers. The antigen is set in these nanocavities for the encapsulation thereof. At the same time, this protects the degradation by macrophages and carries it to gradual and more efficient presentation of lymphocytes, increasing the efficacy of the immune process.
Methods of preparation of SBA-15 silica, and similar mesoporous materials are described in scientific articles (Zhao ef a/., Science (1998) 279:548; J. Am. Chem. Soc. (1998) 120:6024; Matos etal., Chem. Mater. (2001) 13:1726) and in patent US 6,592,764.


The objective of the present invention is also to present an incorporation or encapsulation process of the antigen in nanostructured mesoporous silica, for preparation of the immunogenical complex.
The encapsulation of the antigens in silica occurs, in general, by means of a process that comprises a mixture of a solution previously prepared containing the antigen with a silica suspension, both diluted in physiological solution with pH of 7,4. The weight proportion of the antigen in relation to silica may range from 1:5 to 1:50, preferably of 1:25. This preferred proportion might be read as 1//g of antigen to 25//g of silica. The preparations are preferably carried out at room temperature and maintained under occasional stirring, about two hours, prior to inoculation time.
Another objective of the present invention is to present the use of immunogenical complex in preparation of vaccines pharmaceutical compositions for prophylactic use.
Pharmaceutical compositions, containing the immunogenical complex of the present invention and a pharmaceutically acceptable carrier, diluent or excipient, are appropriate for medical and veterinary use.
One advantage of the present invention consists of the use of immunogenical complex to promote the induction of one identical immune response with fewer amounts of antigens. This occurs either in god or bad respondent individuals. This aspect has a relevant economic and social importance to public health.
The antigen is the raw material with higher cost for production of vaccines. The reduction of the necessary amount for induction of efficient immune response may lead to a substantial reduction in production cost of many vaccines.
On the other hand, the production of larger amounts of doses with the same antigen amount has decompositions that surpass its simple economic aspects. There are antigens which production speed is limited even in the absence of economic limitative. During epidemics, the optimization and maximization of the immunization potential of smaller quantities of antigens may be essential for saving million of lives.
Another very important aspect of the present invention consists of the extension of the stimuli period, through the increase of the time for presentation of the antigen. This


results in the induction of more efficient immunological memory, guaranteeing protection with less number of doses. Several vaccines need the administration of 3 or more doses and periodic strengthen to induce efficient protection. The extended presentation of the antigen may cause the reduction in the number of revaccinations in some cases.
This brings to a forecast of great impact in public health since there exists a low adhesion of many parents to regular vaccination programs and to vaccinate their children, mainly during large campaigns published by the media. The possibility of inducing protective immunity with less number of doses would minimize the problem of lack of adhesion, taking more advantage of the campaigns and to efficiently immunize million of children, without need for returning. DESCRIPTION OF FIGURES
Figure 1. Small angle X-Ray diffraction of SBA-15 (CN) silica (natural calcinated) and SBA-15 (CT) (ground calcinated).
Figure 2. Isothermal of nitrogen adsorption at 77K and the corresponding pore distribution (PSD) of silica SBA-15/calcinated.
Figure 3. Images of Transmission Electronic Microscopy (TEM) of SBA-15/calcinated silica. Figure 4. Determination of IgG anti-lntip titers of Escherichia coli, when comparing SBA-15 with other adjuvants by oral, intraperitoneal and subcutaneous via.
The following examples are described as an illustration and there is no intention to use it for limiting the scope of the present invention.
EXAMPLE 1 - Preparation and characterization of SBA-15 silica - a component of the immunogenical complex as immunization adjuvant.
In a reactor, 4 g of three-block copolymer Pluronic P123 was dispersed, with magnetic stirring at 40°C, in 28 g of deionized water and 122 g of HCI 2 M solution. Then, 8,6 g of TEOS are added for obtaining a homogeneous solution under mechanical and magnetic stirring at 40°C. About 15 minutes, after the addition of TEOS, the growth of the precipitate with jelly aspect may be observed. The gel is maintained under stirring at 40°C for 24 hours and, then, transferred to a Teflon-lined autoclave and placed in a sterilizer at a controlled temperature of 100°C for 2 days. Then the solid product is filtered off, washed with


deionized water and air dried at room temperature. Finally, the synthesized sample is calcinated under dry N2 flow at a flow rate of 100 mLmin"1 at 540°C, using a heating ratio of 1°Cmin"1. After heating for 5 hours at 540°C, the flow of nitrogen gas is changed to air, without interruption of the process, and calcination continue for 3 hours more.
The ordinated bidimensional structure of SBA-15, in the form of channels in hexagonal symmetry, was evaluated by small angle X-Ray diffraction (SAXRD) and N2 measures of adsorption (to define the structural and surface properties, before the content of polymer present in the preparation of the material) and by transmission electronic microscopy (TEM). The results of the material characterization are resumed in Table 1 and illustrated by Figures 1, 2 and 3 of the present invention. Such characteristics are appropriate for considering the material as an excellent matrix for several molecular hosts. Table 1 - Results of SBA-15 characterization

Parameter

Result



Small angle X-Ray diffraction (SAXRD) Specific surface area (a)

12,7 nm (127 A) 900 m2/g

Total pore volume ; 1,39 cm /g

Maximum pore size (w)* Thickness of silica wall (b)

11,6 nm (116 A) 1,1 nm(11 A)

* Obtained by the pore size distribution (PSD); ** b = a-w
Figure 1 shows the results of small angle X-Ray diffraction (SAXRD) obtained for the SBA-15 sample of calcinated hexagonal type, in natural state (CN) and ground (CT). The results evidence that the structure of the ordinated mesoporous materials (diffraction peaks) does not change after grinding the powders in agate mortar. The analysis and indexation of peaks are made after removal of the non-structured spreading background. Figure 2 shows the isothermal of nitrogen adsorption for calcinated SBA-15 silica, which presented a high grade of ordination, as can be deduced from the declivity in isothermal adsorption in the step of capillary condensation.


Figure 3 shows the transmission electronic microscopy (TEM), which was used to characterize the structural order of silica SBA-15/calcinated, where the ordination of parallel channels particular of such type of material can be observed.
EXAMPLE 2 - Determination of the adsorption percentage of antigen model by SBA-15
Using bovine serum albumin [BSA - bovine serum albumin] as antigen, mixtures were made with SBA-15 at different proportions and, then, determination of adsorption percentage of antigen to silica for each proportion was made. According to the results presented in Table 2, the proportion of 1/ivg of BSA to 25/jg of SBA-15 showed high adsorption percentage of BSA by SBA-15.
Table 2 - Determination of the best proportion for adsorption of Bovine Serum Albumin [66kDa] in SBA-15 Silica.
BSA: SBA-15 Adsorption %
1:5 1:10 27,5 65,5
i
1j£5 j 91
However, it is worth to mention that due to the diversity of antigens that can compose the immunogenical complex of the present invention, the optimization of the proportion between the antigen and SBA-15 should be reconsidered due to the complexity of the antigen. EXAMPLE 3 - Demonstration of SBA-15 effects on macrophages
Experiments in vitro showed that nanostructured silica SBA-15 does not affect the availability, neither interferes in the macrophages phagocytic capacity originating from the medulla, maintained in culture for up to 30 hours. To the contrary, indicates to potentialize the phagocytosis through these cells. Table 3 shows that the treatment or not with SBA-15 does not substantially interferes in the phagocytosis process of yeast cells in Strains: genetically selected for a bad response [L|VA], genetically heterogeneous [SWISS], or isogenic [BALB/c].
Table 3 - In vitro experiment with macrophages of different mice strains


STRAIN INFECTED No. OF YEASTS
LIVA Presence of yeasts
20|jg SBA-15 + yeast 2h 68,2% 496
20|jg SBA-15 + yeast 17h 61,8% 350
10|jg SBA-15 + yeast 2h 78,9% 474
10ngSBA-15 + yeast 17h 65,2% 326
2,5pg SBA-15 + yeast 2h 79,5% 503
2,5|jg SBA-15 + yeast 17h 67,8% 379
Yeast 2h 53,9% 217
Yeast 6h 59,2% 230
Yeast 17h 82,8% 472
Yeast 21 h 59,9% 224
Yeast 30h 50,2% 164
STRAIN INFECTED No. OF YEASTS
SWISS Presence of yeasts
20pgSBA-15 +yeast 2h 84,9% 591
10|jg SBA-15 + yeast 2h 81,7% 528
10M9 SBA-15 + yeast 17h 70,8% 325
2,5|jg SBA-15 + yeast 2h 81,9% 468
2,5|jg SBA-15 + yeast 17h 74,7% 448
Yeast 2h 78,2% 479
Yeast 6h 73,3% 437
Yeast 17h 54,1% 284
Yeast 21 h 56,2% 218
Yeast 30h 53,9% 195
STRAIN INFECTED
No. OF YEASTS
BALB/c Presence of yeasts

Yeast 2h 82% 622
Yeast 6h 76,8% 438
Yeast 21 h 68,5% 424
Yeast 30h 51,5% 209
EXAMPLE 4 - Adjuvant effect of the immunogenical complex (antigen:SBA-15) on anti-/nt/b antibodies and anti-poison Micrurus ibiboca when compared with the adjuvants regularly used in mice strains.
Groups of 4-5 mice genetically selected according to high production of antibodies [Hin strain], or to the bad response [LIVA strain], and mice of isogenic strain [animals genetically identical] BALB/c were tested in distinct experiments. The potential effect of SBA-15 was tested with the measurement and the comparison of response to the recombinant protein intimine [Int1 ] of 16,5kDa of the bacteria Escherichia coli, adsorbed in SBA-15 [1:10 Int1 SBA-15] or admixed to Freund Incomplete Adjuvant (FIA). The response to antibodies formation was also evaluated for the total poison of the snake of genus Elapidae, Micrurus ibiboboca kind, composed of at least 20 proteins with molecular weight ranging from 84 to 7kDa, adsorbed in SBA-15 [1:10 M/'cnv:SBA-15], comparing the response to this poison admixed in AIF. All these experiments were carried out with following immunizations by subcutaneous via. The data obtained that are presented in Tables 4 and 5 confirm that SBA-15 is so efficient as AIF, promoting high response to antibodies and promoting efficient immunological memory.
Table 4 - Ant\-lnt1{i Titer [log2] 15 days after immunization

SBA-15 AIF
Mice strain
N X ±0 n x ± a
L|VA 4 11,3 ±0,5 4 4,5 ± 0,5
Hm 4 11,3 ±0,4 3 13,3 ±0,5
BALB/c 4 6,2 ± 3,2 5 9,8 ± 2,3


Table 5 -AnW-Micrurus Titer [log2] 14 days after immunization
^SBA-15_ AIF
Mice strain :

n x ± a n x ± a
L|VA 4 8,1 ±0,5 3 5,2 ±0,3
BALB/c 4 9,2 ±1,3 4 6,4 ±0,8
In addition, SBA-15, contrary to what occurs upon administration of AIF, does not lead to the formation of an apparent granuloma and, the local inflammatory response is insipid and, when measured at 24-48 hours after the inoculation of the immunogen in SBA-15 by subcutaneous via, presents very reduced levels of monocytes and nuclear polymorphous.
There is no apparent change in the behavior and vitality of mice that received SBA-15 relatively to the control animals and, followed for 11 months, no morphological change is observed in treated animals.
EXAMPLE 5 - The adjuvant effect of immunogenical complex (antigen:SBA-15) on anii-lntp antibodies due to time when compared with the adjuvants normally used.
In another series of assays, groups of BALB/c mice were immunized with Intip (from Escherichia coli) in SBA-15, AI(OH)3 by oral via, or Intip in SBA-15, AI(OH)3 and AIF by subcutaneous and intraperitoneal via. The anti-Intip responses were followed during a long time. Figure 4 presents the responses to the protein Intimine 1/?of Escherichia coli according to distinct immunization via. Averages and standard deviations of isogenic Strain BALB/c mice, followed up to 199 days [d] during the primary responses [PR], immunized with the known adjuvants AI(OH)3, Freund Incomplete Adjuvant (FIA) and the original SBA-15 nanostructured silica. It can be noted that the levels of antibodies remained high during throughout the analyzed period, especially in the group that received the antigen in SBA-15.


The joint results clearly show that SBA-15 is a non-immunogenical, non-toxic and efficient carrier promoting high response to antibodies and promoting efficient immunological memory.
Highly ordinated nanostructured mesoporous silicas, illustrated in the present invention by SBA-15 Silica, provide promising systems for vaccinal preparations or compositions.


We Claim:-
1- Immunogenic complex for immunity induction,
.characterized by comprising particles of highly ordered
nanostructured raesoporous silica, having size pores of 2 .to 50 nm and antigens of several natures, wherein the antigen is encapsulated by the particles of mescporous silica, which act as immunization adjuvants.
2- Immunogenic complex, according to claim 1, characterized
by the fact than the antigen is selected from the group consisting oi proteins, biologically active peptides, toxins and viral or bacterial vaccines.
3- Immunogenic complex, according to claim 1, characterized
by the fact that the highly ordered nanostructured
mesoporous silica is a mescporous silica SBA-15.
4- Immunogenic complex, according zo c'aim 1, characterized by the iact that tne antigen and the adjuvant are employed in a proportion cf 1:5 to 1:50.
5- Immunogenic complex, according to claim 4, characterized by the fact that the antigen and the adjuvant are employed in a proportion of 1:25.
6- Use of immanogenic coraplex, according to claim 1, for production of vaccinal pharmaceutical compositions for enabling the presentation of the antigen that composes it to the lymphocytes in a safe, gradual and sustained way leading to a more efficient immunological memory.
7- Use of immunogenic complex, according to claim. 1, for production
of vaccinal pharmaceutical compositions for increasing the. immunogenic:
r.y si the antigen that composes it.
8- Use of immunogenic complex, according to claim 1, for
production of vaccinal pharmaceutical compositions for


assuring immunological protection with lower amounts of antigens and/or less repetitions of vaccine doses.
9- Use of immunogenic complex, according to claim 1, for production of vaccinal pharmaceutical compositions for effective immunity induction in high- and low responder individuals, in a homogeneous way.
10- Use of immunogenic complex, according to claim 1, for

compositions
tor
:ai
production of vaccinal
effective immunizations and/or vaccinations in medicine, and veterinary.
11- Vaccinal pharmaceutical composition, characterized for containing an immunogenic complex according to claim 1 and a pharmaceuticaily acceptable carrier, diluent or excipient.
12. An iiranunogenical complex formed by vaccinal antigens encapsulated with nanostructured mesoporous silica is claimed substantially as herein described with forgoing description and figures.







ABSTRACT
The present invention relates to a product named "immunogenical complex", which comprises an adjuvant characterized by solid particles of highly ordinated nanostructured mesoporous silica, preferably, SBA-15 Silica, and vaccinal antigens of several natures, encapsulated in the referred to adjuvants. The immunogenical complex of the present invention allows the presentation of the antigens that compose it to lymphocytes, in a safe, gradual and extended way, which leads to a more efficient immunological memory, increases the immunogenicity of the antigen and improves the production of antibodies. This ensures an efficient immunological protection with fewer amounts of antigens and/or less repetitions of vaccinal doses. In addition, the characteristics of the immunogenical complex of the present invention promotes effective immunity induction, homogeneous in "god and bad respondent" individuals.

Documents:

437-MUMNP-2008-ABSTRACT(GRANTED)-(1-8-2011).pdf

437-mumnp-2008-abstract.doc

437-mumnp-2008-abstract.pdf

437-MUMNP-2008-CANCELLED PAGES(19-1-2011).pdf

437-MUMNP-2008-CLAIMS(AMENDED)-(19-1-2011).pdf

437-MUMNP-2008-CLAIMS(AMENDED)-(21-6-2011).pdf

437-MUMNP-2008-CLAIMS(GRANTED)-(1-8-2011).pdf

437-MUMNP-2008-CLAIMS(MARKED COPY)-(19-1-2011).pdf

437-MUMNP-2008-CLAIMS(MARKED COPY)-(21-6-2011).pdf

437-mumnp-2008-claims.doc

437-mumnp-2008-claims.pdf

437-mumnp-2008-correspondence(12-5-2008).pdf

437-MUMNP-2008-CORRESPONDENCE(20-3-2008).pdf

437-mumnp-2008-correspondence(24-3-2008).pdf

437-MUMNP-2008-CORRESPONDENCE(IPO)-(1-8-2011).pdf

437-mumnp-2008-correspondence(ipo)-(19-1-2010).pdf

437-mumnp-2008-correspondence-others.pdf

437-mumnp-2008-correspondence-received.pdf

437-mumnp-2008-description (complete).pdf

437-MUMNP-2008-DESCRIPTION(GRANTED)-(1-8-2011).pdf

437-MUMNP-2008-DRAWING(GRANTED)-(1-8-2011).pdf

437-mumnp-2008-drawings.pdf

437-MUMNP-2008-EPO,JPO,USPTO,SIPO PATENT DOCUMENT(21-6-2011).pdf

437-MUMNP-2008-FORM 18(24-3-2008).pdf

437-MUMNP-2008-FORM 2(GRANTED)-(1-8-2011).pdf

437-MUMNP-2008-FORM 2(TITLE PAGE)-(7-3-2008).pdf

437-MUMNP-2008-FORM 2(TITLE PAGE)-(GRANTED)-(1-8-2011).pdf

437-MUMNP-2008-FORM 26(19-1-2011).pdf

437-mumnp-2008-form 26(2-5-2007).pdf

437-mumnp-2008-form-1.pdf

437-mumnp-2008-form-2.doc

437-mumnp-2008-form-2.pdf

437-mumnp-2008-form-3.pdf

437-mumnp-2008-form-5.pdf

437-mumnp-2008-form-pct-ipea-409.pdf

437-mumnp-2008-form-pct-ipea-416.pdf

437-mumnp-2008-other document(24-3-2007).pdf

437-mumnp-2008-pct other document(6-3-2008).pdf

437-mumnp-2008-pct-search report.pdf

437-MUMNP-2008-POWER OF ATTORNEY(12-5-2008).pdf

437-MUMNP-2008-REPLY TO EXAMINATION REPORT(19-1-2011).pdf

437-MUMNP-2008-REPLY TO EXAMINATION REPORT(21-6-2011).pdf

437-mumnp-2008-table.doc

437-mumnp-2008-wo international publication report(6-3-2008).pdf

437-MUMNP-2008-WO INTERNATIONAL PUBLICATION REPORT(7-3-2008).pdf

abstract1.jpg


Patent Number 248654
Indian Patent Application Number 437/MUMNP/2008
PG Journal Number 31/2011
Publication Date 05-Aug-2011
Grant Date 01-Aug-2011
Date of Filing 07-Mar-2008
Name of Patentee CRISTÁLIA PRODUTOS QUÍMICOS FARMACÊUTICOS LTDA.
Applicant Address RODOVIA ITAPIRA/LINDOIA, KM 14, 13970-000 ITAPIRA,
Inventors:
# Inventor's Name Inventor's Address
1 PACHECO, OGARI RUA DOM PEDRO I, 109-NOVA ITAPIRA, 13974-250 ITAPIRA
2 MATOS, JIVALDO LABORATORIO DE ANALISE TERMICA PROF. DR. IVO GIOLITO(LATIG)-INSTITUTO DE QUIMICA DA USP - AV. PROF. DR. LINEU PRESTES, 748, 05508-900 SAO PAULO
3 SANT'ANNA, OSVALDO AUGUSTO INSTITUTO BUTANTAN CAT/CEPID-AV. VITAL BRAZIL, 1500-BUTANTAN, 05503-900 SAO PAULO
4 MERCURI, LUCILDES CETEC-UNIVERSIDADE CRUZEIRO DO SUL - CAMPUS ANALIA FRANCO-AV. REGENTE FEIJO, 1295,03342-000 SAO PAULO
5 FANTINI, MARCIA INSTITUTO DE FISICA-USP- DEPARTMENTO DE FISICA APLICADA- RUA DO MATAO, TRAVESSA R, 187, 05508-970 SAO PAULO
PCT International Classification Number A61K39/39,A61K39/00
PCT International Application Number PCT/BR2006/000182
PCT International Filing date 2006-09-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PI0503817-0 2005-09-12 Brazil