HK1124791B - Immunogenic complex formed by vaccinal antigens encapsulated by nanostructured mesoporous silica - Google Patents

Description

Immunogenic complexes formed from vaccine antigens encapsulated using nanostructured mesoporous silica

The present invention relates to the field of immunology.

The present invention relates to a product named "immunogenic complex" effective in increasing immunogenicity, consisting of a vaccine antigen encapsulated with highly ordered nanostructured solid particles of mesoporous silica acting as adjuvant, as demonstrated by the present invention. The use of mesoporous silica encapsulation protects the antigen from degradation by macrophages and prolongs its contact with lymphocytes, thereby promoting an improved immune response that effectively induces antibody production in high or low response individuals. The immunogenic complexes of the invention may confer the benefit of overall immunological activity to different types of antigens as follows: biologically active peptides, toxins, viral and bacterial vaccines.

The immune response of a human to a vaccine antigen varies depending on specific factors. Several individuals vaccinated with the same antigen produced responses of varying intensity and duration under the same conditions. This variation is a decisive factor in the strength and duration of the protective effect of the vaccine.

Individuals who rely on the production of protective antibody titers after standardized antigen stimulation are referred to as high responders, while those who do not produce protective titers are low or even non-responders.

The development of safe and effective strategies for improving immune responses from high or low responders is of most interest. In the first case, protective responses are elicited by using lower amounts of antigen, or long-lasting responses are generated without further exposure to antigen. In the second case, the protective response is triggered by the use of a stimulus, which otherwise would be insufficient.

Currently, this problem is only partially solved by the use of adjuvants, defined as substances that prolong the specific immune response of an organism to certain antigens [ Edelman, r.; pocket, c.o.; adjuvants lntern. ver. immunol, 7(1990)51], altered the form by which epitopes (antigenic determinants) are presented to cells of the immune system or increased their immunogenicity. Other properties required for adjuvants are: maintaining the stimulation cycle, increasing the time of antigen presentation and delaying its catabolism.

Obviously, many adjuvants exert their activity through toxic effects on macrophages. Adjuvants are also present that modulate the immune response to certain antigens, for example, adjuvants that induce the predominant expression of immunoglobulin isotypes, such as IgG [ hadjipietrou-korurounakis, l.;E.；Scand.J.Immunol.，19(1984)219]。

adjuvants approved and widely used in human vaccines are derivatives of aluminium salts, such as aluminium hydroxide or aluminium phosphate. However, they do not induce an immune response that is substantially higher and longer lasting or qualitatively selective than the ideal subclass of IgG antibodies and than the cytokines involved.

Other adjuvants used in veterinary medicine, such as Freund's incomplete adjuvant [ IFA ] and Freund's complete adjuvant [ CFA ], promote the formation of undesirable nodules, abscesses or granulomas in the local area of administration. Other adjuvants are: lipid a, microspheres and liposomes, none of which are intended for use in humans.

Thus, the focus on developing safe and effective strategies for improving immune responses from high or low responders remains evident. In this way, advances in the field of material science have made it possible to prepare new compounds with improved properties and potential for application in several fields.

Inorganic porous solids provide important industrial applications in catalytic and separation processes. These materials, due to their structure and surface properties, enable molecules to readily enter their nanostructures, thereby increasing their catalytic and adsorptive activities.

The porous materials currently used can be divided into three classes based on their specific microstructure: a paracrystalline amorphous support; materials with modified layers and crystalline molecular sieves. The difference in the microstructure and the mesostructure of these materials is important in terms of their adsorption and catalytic properties and the characteristics used to characterize it, such as: a surface area; pore size and variability of such pore size; the presence or absence of the X-ray diffraction standard (DRX) and detailed descriptions in such standards; and the appearance of the material when its microstructure is studied by Transmission Electron Microscopy (TEM) and electron diffraction methods.

Amorphous and paracrystalline materials represent an important class of porous inorganic solids that have been used for many years in industrial applications. Typical examples of these materials are amorphous silica, which is commonly used in formulating catalysts, and transmissive sub-crystalline alumina, which is used as a support for acidic solid catalysts and petroleum-modified catalysts. The term amorphous, when used in this context, means a material that does not exhibit long range order, however, nearly all materials are ordered to some degree, at least locally. Alternative terms used to describe these materials are: "X-ray without difference". The microstructure of the silica consists of 10-25nm aggregated amorphous silica, wherein the porosity is created by the spatial zones between the particles. Since there is no long range order in these materials, the pore size tends to be distributed over a wide range. This lack of ordering is also manifested in the diffraction X-ray standard (DRX), which usually occurs without characteristic peaks.

Paracrystalline materials, such as transmissive alumina, have exhibited a broad distribution of pore sizes and are well defined according to X-ray diffraction standards that typically consist of a certain broad band. The microstructure of these materials consists of small crystalline regions of aggregated alumina phase and the porosity of the material is a result of irregular spatial regions between these regions. The variability of such pore sizes is generally extremely high, considering that there is no long range order between materials that controls the pore size in the material. The pore size in these materials comprises bands called mesopores in the range of 1,3-20 nm.

In contrast to these solids, the structure is almost uncertain in materials with extremely narrow pore size distribution, since it can be controlled, precisely repeated, according to the crystalline properties of the material, named microstructure. These materials are referred to as "molecular sieves" and the most important example is zeolites (zeolytes).

Such natural or synthetic molecular sieves include various crystalline silicates containing cations.

In general, porous substances are separated according to pore size, for example, substances having a pore size of less than 2nm are classified as microporous, 2-50nm are classified as mesoporous substances, and substances exceeding 50nm are classified as macroporous substances.

A series of mesoporous molecular sieves, including MCM-41 and MCM-48, are described in US patents US5,057,296 and US5,102,643. These molecular sieves exhibit a structure in which mesopores having a uniform size are regularly arranged. MCM-41 has a uniform structure exhibiting a hexagonal arrangement of oriented mesopores, such as honeycombs, and has a 1000m obtained by the BET method²Specific surface area per gram.

Molecular sieves have been produced using inorganic or organic cations as templates (molds). These mesoporous molecular sieves are synthesized by a liquid crystal mechanism using a surfactant as a template (mold) and they have an advantage in that the pore diameter is adjusted at a ratio of 1.6 to 10nm by controlling the type of surfactant or the synthesis conditions used in the production process.

Molecular sieves designated SBA-1, SBA-2 and SBA-3 are described in Science (1995) 268: 1324. The channels are regularly arranged and the constituent atoms exhibit an arrangement similar to that of amorphous silica. The mesoporous molecular sieve has channels with a regular structure, larger than those present in the zeolite, in such a way that it can be used in adsorption, separation or catalytic conversion reactions of relatively large molecules.

A family of high quality hydrothermally stable and ultra-broad pore size mesoporous silicas was found in U.S. patent No. 6,592,764, synthesized by using amphiphilic block copolymers in acidic media. The member SBA-15 in this family has a highly ordered mesoporous structure, i.e., a two-dimensional hexagonal shape similar to a honeycomb (p6 mm). Other structures such as a cage-shaped cube or a three-dimensional hexagon are also formed. Calcination operation at 500 ℃ results in a porous structure having 690-1040m²In terms of/gHigh BET surface area and greater than 2.5cm³Pore volume/g, interplanar spacing d (100) of 7.45-45nm, pore diameter of 4.6-50nm and silica wall thickness of 3.1-6.4 nm. SBA-15 can be prepared with a wide range of pore sizes and pore wall thicknesses at low temperatures (35-80 deg.C) using a variety of commercially available amphiphilic block biodegradable and non-toxic copolymers, including triblock polyoxazolines (polyxykaline).

The unique properties of SBA-15 make it an attractive material for several applications, including bio-applications, such as immobilization of bioactive species. However, no comparison document has been established reporting the effect of these materials on immune responses, rather the listed literature may suggest that no study was made for this purpose.

Experiments have been conducted on the effect of amorphous silica in immune responses, particularly on macrophages, however, at that time they did not involve the effect of silica as an adjuvant [ Allison, a.c.; harington, J.S.; birbeck, m.; mep.med., 124(1966) 141; kampschmidt, r.f.; worthington, m.l.; mescher, m.i.; 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) 3871975; 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.; measure, j.c.; reis, m.a.; piatti, r.m.; ribeiro, o.g.; biozzi, g.; ann. inst. pasteur immunol., 138(1987)359.], a genetically modified mouse with low or high antibody production was used and in which a colloidal silica suspension was administered during the consecutive 4 days prior to immunization with the particulated antigen, i.e. the heterologous red blood cells. These studies demonstrated that there was significant antibody production in hypo-responder animals, and that this improvement was directly linked to the effect of silica on macrophages, thereby affecting certain of their functions, altering the viability of these cells and leading to a reduction in antigen catabolism, thereby facilitating antigen presentation to lymphocytes.

Thus, these effects were analyzed by comparing the response of mouse strains expressing characteristics that differ with respect to their macrophage functionality. This is achieved using an experimental model that selects mouse strains with the phenotype of highest or lowest response to antibodies. Such lines are obtained after crossing between individuals with extreme phenotypes during serial passages. After about 15 passages, animals exhibiting extreme phenotypes at antibody level achieved homozygosity for the relevant alleles of certain antigen control responses. Using this model, one can obtain a high [ H ] in select IVAs]And low [ L ]]Antibody-responsive strain [ Cabrera, w.h.; ibanez, o.m.; oliveira, s.l.; sant' Anna, o.a.; siqueira, m.; mouton, d.; biozzi, g.; immunogenetics, 16(1982)583]. The difference in response among these animals was higher (H)_IVAMouse strain) or lower (L)_IVAMouse strain) macrophage catabolic activity, respectively, is detrimental or beneficial to the efficient presentation of antigen.

The above studies demonstrate that when L is treated beforehand and extensively with amorphous silica suspensions_IVAWhen a mouse is then immunized with antigen, it has increased antibody production, approaching H_IVAResponse in mice. Another aspect [ 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 toppcs In Microbiology Immunology, 85(1979)31.]In another similar animal model, H obtained by independent genetic selection of III was used_IIIAnd L_IIIMice, after treatment with the same silica suspension, no modulation of antibody production was observed in low responder mice. It must be emphasized that in these H_IIIAnd L_IIIIn animals, high or low levels of antibody production are not associated with the functionality of their macrophages, but rather with the potential of lymphocytes.

These studies are the basis for developing support for understanding the role in vivo on macrophages in vaccination protocols, and also show that for an effective adjuvant for inducing immunity, the administered antigen should be protected from the high catabolic activity of macrophages and the appropriate presentation of antigenic determinants to macrophages.

In a larger vaccine campaign, the homogeneous immunization products and methods are generally suitable for large and heterogeneous groups of individuals. Under these conditions, variable titers of antibody production can be observed, some non-protective. It prevents effective immunization of a subset of individuals.

This fact is explained by the mechanisms shown in the above experiments and which derive from the phenotypic variability of individuals of the same species, which can be explained by the effective form of presentation or non-presentation of the epitope to the lymphocytes.

For example, lymphocytes with effector activity that can be classified from normal to very high or individuals with reduced to normal macrophage activity have a tendency to respond more rapidly than antibodies because of the greater likelihood of more efficient identification of antigens by lymphocytes. They are "high-response" individuals in the natural population.

In contrast, individuals exhibiting decreased lymphocyte activity from normal to extremely high macrophage activity have a tendency to more rapidly catabolize the administered antigen. This results in a lower degree of antigen exposure to lymphocytes and an ineffective immune response. These individuals are "low-response" individuals in the natural population. This situation facilitates the natural selection of more resistant pathogens.

More effective vaccines must be developed that may facilitate and promote the production of protective antibody titers even in individuals who are low responders to current vaccine preparations. It is therefore important to take this differentiated cell behaviour into account in selecting a vaccination, seeking to minimise the effect of differentiation factors.

However, the application of this concept does not exist and we do not get a product and/or a vaccine produced according to the product.

It is an object of the present invention to show that antigens incorporated or encapsulated into nanostructured mesoporous silica form immunogenic complexes that are effective in inducing immune responses and that such nanostructured mesoporous silica does not affect the survival and phagocytic capacity of macrophages in culture.

The present invention relates to a novel immunogenic complex consisting of antigens of several nature, encapsulated by highly ordered nanostructured mesoporous silica acting as an adjuvant, which can improve the induction of immunity and produce antibodies directed against antigens that differ in nature, structure and complexity.

The immunogenic complex of the invention relates to the product resulting from the combination of an antigen with nanostructured mesoporous silica particles in a specific ratio.

The immunogenic compositions of the invention are effective in immunizing individuals who are low responders to currently used products and methods. The complex is derived from a safer and more effective antigen presentation to lymphocytes.

The immunogenic complex of the invention is composed of at least one antigen, which is incorporated into or encapsulated by the nanostructured mesoporous silica particles. In addition to acting effectively as an adjuvant for immunization, the silica particles also serve as a support or matrix for the biologically active species, in this case the antigen.

Antigens that may be used to form the immunogenic complexes of the invention include biologically active peptides, toxins and viral and bacterial vaccines.

Although a wide range of nanostructured mesoporous silicas may be used as adjuvants for the preparation of the immunogenic complexes of the invention, it is preferred to use the silica known as SBA-15.

Highly ordered nanostructured mesoporous silica SBA-15 is composed of silica particles with regular cavities and uniform sizes of 2-50 nanometers. Antigens are inserted into these nanocavities so that they are encapsulated. At the same time, this will prevent degradation by macrophages and carry it with it to progressively and more efficiently present lymphocytes, thereby increasing the efficacy of the immune response.

Methods for preparing SBA-15 silica and similar mesoporous materials are described in scientific literature (Zhao et al, Science (1998) 279: 548; J.Am.chem.Soc. (1998) 120: 6024; Matos et al, chem.Mater. (2001) 13: 1726) and in U.S. Pat. No. 6,592,764.

It is also an object of the present invention to provide a method for incorporating and encapsulating antigens into nanostructured mesoporous silica for the preparation of immunogenic complexes.

The encapsulation of the antigen on the silica is generally carried out by a method comprising the use of a previously prepared solution mixture comprising the antigen and a suspension of silica, both diluted with a physiological solution of pH 7.4. The weight ratio of antigen to silica may be in the range 1: 5 to 1: 50, preferably 1: 25. This preferred ratio can be read as the ratio of 1. mu.g antigen to 25. mu.g silica. Preferably, the preparation is carried out at room temperature and maintained under temporary stirring until about 2 hours before inoculation.

It is another object of the invention to provide the use of the immunogenic complex in the preparation of a vaccine pharmaceutical composition for prophylactic use.

Pharmaceutical compositions comprising the immunogenic complex of the invention and a pharmaceutically acceptable carrier, diluent or excipient are suitable for medical and veterinary applications.

An advantage of the present invention is the use of an immunogenic complex to facilitate the induction of the same immune response in high and low responding individuals using lower amounts of antigen. This aspect has relevant economic and social importance to public health.

Antigens are raw materials with the high cost of producing vaccines. The reduction in the amount necessary to induce an effective immune response may result in a significant reduction in the production costs of many vaccines.

On the other hand, the use of the same amount of antigen to produce large doses has implications over its purely economic aspects. There are antigens that limit the production rate even in the absence of economic limiting factors. Optimizing and maximizing the vaccination potential of smaller amounts of antigen may be necessary to save millions of lives during an epidemic.

Another very important aspect of the invention is to extend the stimulation period by increasing the time of antigen presentation. This results in the induction of more effective immunological memory, thereby ensuring protection with lower number of doses. Several vaccines need to be given 3 or more doses and periodically boosted to induce effective protection. Prolonged antigen presentation may lead to a reduction in the number of replicates in some cases.

This possibility has a great impact on well-known health, since there are many parents who are on a regular basis performing vaccination programs or vaccinate their children to a lesser extent, mainly during the huge battle period of media publishing. The possibility of inducing protective immunity using lower numbers of doses can minimize the lack of adherence problems, thereby making more use of the campaign and effectively immunizing millions of children without the need for multiple races.

Description of the drawings

FIG. 1 is a small angle X-ray diffraction of SBA-15(CN) silica (natural calcined) and SBA-15(CT) (ground calcined).

FIG. 2 nitrogen adsorption isotherms at 77K and pore distribution (PSD) of the corresponding calcined silica SBA-15.

FIG. 3 is a Transmission Electron Microscopy (TEM) image of calcined silica SBA-15.

FIG. 4. determination of mouse antibodies of the IgG isotype anti-Intimin 1. beta. of Escherichia coli (Escherichia coli) when SBA-15 is compared with other adjuvants after administration by oral, intraperitoneal and subcutaneous routes.

The following examples are described by way of illustration and are not intended to limit the scope of the invention.

Example 1 preparation and characterization of silica SBA-15-Components of immunogenic complexes as Immunity-vaccinating adjuvants

In a reactor, 4g of the tri-block copolymer pluronic P123 was dispersed in 28g of deionized water and 122g of 2M HCl solution by magnetic stirring at 40 ℃. Then 8.6g TEOS was added to obtain a homogeneous solution at 40 deg.C with mechanical and magnetic stirring. A jelly-like precipitate was observed to form 15 minutes after the addition of TEOS. The gel was kept under stirring at 40 ℃ for 24 hours and then transferred to a teflon-jacketed autoclave and placed in a 100 ℃ controlled temperature sterilizer for 2 days. The solid product was then filtered off, washed with deionized water and air dried at room temperature. Final at 540 ℃ and 100mLmin^-1Dry N at a flow rate of₂In air flow, use for 1 deg.C min^-1The synthesized sample was calcined at the heating rate of (1). After heating at 540 ℃ for 5 hours, the nitrogen flow was changed to air and the calcination was continued for another 3 hours without interrupting the process.

By small angle X-ray diffraction (SAXRD) and N₂The ordered two-dimensional structure of SBA-15 in the form of hexagonal symmetric channels was evaluated by measurement of adsorption (in order to determine the structure and surface properties, relative to the amount of polymer present in the preparation of the material) and Transmission Electron Microscopy (TEM). The characterization results of the materials are summarized in table 1 and exemplified by fig. 1, 2 and 3 of the present invention. Such characteristics are suitable for considering the material as an excellent matrix for several molecular hosts.

TABLE 1-results of SBA-15 characterization

Obtained by Pore Size Distribution (PSD); a-w

FIG. 1 shows the small angle X-ray diffraction (SAXRD) results obtained for calcined hexagonal-like SBA-15 samples in the natural state (CN) and in the Ground (GC). The results demonstrate that the structure (diffraction peaks) of the ordered mesoporous material is unchanged after grinding the powder in an agate mortar. The peaks after removal of the unstructured extended background were analyzed and indexed.

FIG. 2 shows the nitrogen adsorption isotherms used to characterize the fumed silica SBA-15 providing high order, as can be inferred from the slope of isothermal adsorption in the capillary condensation step.

FIG. 3 shows a Transmission Electron Microscope (TEM) examination, which is used to characterize the structural ordering of the fumed silica SBA-15, in which in particular the order of parallel channels of this type of material can be observed.

Example 2 determination of the adsorption percentage of SBA-15 model antigen

Mixtures were prepared using bovine serum albumin [ BSA ] as the antigen, using SBA-15 at different ratios, and then the percent adsorption of antigen by silica was determined for each ratio. According to the results presented in Table 2, the ratio of 1. mu.g BSA to 25. mu.g SBA-15 shows a high percentage of adsorption of SBA-15 to BSA.

TABLE 2 determination of the optimal ratio for adsorption of bovine serum albumin [66kDa ] in silica SBA-15

However, it is worth mentioning that due to the diversity of the antigens that may constitute the immunogenic complex of the invention, the ratio between antigen and SBA-15 should be reconsidered, due to the complexity of the antigen.

Example 3 demonstration of the Effect of SBA-15 on macrophages

In vitro experiments demonstrated that nanostructured silica SBA-15 did not affect viability nor interfere with macrophage phagocytic ability derived from medulla maintained in culture for up to 30 hours. Instead, it is shown that phagocytosis can be enhanced by these cells. Table 3 shows that treatment with or without SBA-15 does not substantially interfere with the phagocytosis process of yeast cells in the following strains: genetically selected for low response [ L_IVA]Genetically heterologous [ SWISS]Or isogenic [ BALB/c]。

TABLE 3 in vitro experiments with macrophages of different mouse strains

EXAMPLE 4 adjuvant Effect of the immunogenic Complex (antigen: SBA-15) on anti-Int β antibody and the antidote Micrurus ibboca when compared to the adjuvant regularly used in mouse strains

Testing of antibodies in high yield [ H ] in different experiments_IIIIs a system]Or low response [ L_IVAIs a system]Groups of 4-5 genetically selected mice and isogenic lines [ genetically identical animals]BALB/c mice. By adsorbing to SBA-15[ 1: 10 Int1 beta: SBA-15]Recombinant protein beta-intimine [ Int1 beta ] of the 16.5kDa bacterium Escherichia coli in medium or in admixture with Freund's incomplete adjuvant (IFA)]The potential effects of SBA-15 were evaluated by determination and comparison of responses. Adsorption on SBA-15[ 1: 10M ] was also evaluatedicru∶SBA-15]Of the genus Elapidae (Elapidae), Micrurus iboboca, consisting of at least 20 proteins having a molecular weight of 84-7kDa, compared the response to this venom mixed in IFA. All these experiments were performed after immunization by the subcutaneous route. Data provided in tables 4 and 5[ mean ± standard deviation (log)₂)]SBA-15 was shown to be as effective as IFA, promoting high antibody titers and to be effective in immunological memory induction.

TABLE 4 anti-Int 1 beta titers 15 days post-immunization [ log ]₂]

TABLE 5 anti-Elapidae (Micrurus) titers at 14 days post-immunization [ log₂]

Furthermore, in contrast to what happens when IFA is administered, SBA-15 does not lead to significant granuloma formation and the local inflammatory response is negligible and presents very reduced levels of monocytes and polymorphonuclear when measured 24-48 hours after vaccination with immunogen by subcutaneous route.

The mice receiving SBA-15 had no significant changes in behavior and viability compared to the control animals, and no morphological changes were observed in the treated animals at the 11 month follow-up.

EXAMPLE 5 adjuvantation of the immunogenic Complex (antigen: SBA-15) anti-Int β antibody over time when compared to the normally used adjuvant

In addition toIn a series of assays, groups of BALB/c mice were immunized with Int1 β (from Escherichia coli) in SBA-15 by oral route, or SBA-15, Al (OH) by subcutaneous and intraperitoneal routes₃And Int1 β in IFA. anti-Int 1 β responses were followed over a long period of time. FIG. 4 presents the response to the protein Intimin 1. beta. of Escherichia coli according to different immunization routes. Mean and standard deviation of isogenic strain BALB/c mice, primary response [ PR]The process lasts for 199 days [ d ]]Follow-up of (1), immunization with the known adjuvant Al (OH)₃Freund's incomplete adjuvant (IFA) and original SBA-15 nanostructured silica. It can be noted that antibody levels remained high throughout the assay, especially in the group that received the antigen in SBA-15.

Together, these results clearly demonstrate that SBA-15 is a non-immunogenic, non-toxic and effective carrier that promotes a high response to antibodies and an effective immunological memory.

The highly ordered nanostructured mesoporous silica exemplified by the SBA-15 silica in the present invention provides a promising system for vaccine preparations or compositions.

Claims (9)

1. Immunogenic complex for immune induction, characterized by comprising highly ordered nanostructured mesoporous silica particles having a pore size of 2-50nm and an antigen of proteinaceous nature, wherein the antigen is encapsulated by mesoporous silica particles acting as an immunization adjuvant.

2. The immunogenic complex of claim 1, wherein said antigen is selected from the group consisting of proteins, biologically active peptides, toxins, viral vaccines and bacterial vaccines.

3. Immunogenic complex according to claim 1, characterized in that the highly ordered nanostructured mesoporous silica is SBA-15 mesoporous silica.

4. Immunogenic complex according to claim 1, characterized in that the antigen and the adjuvant are used in a weight ratio of 1: 5 to 1: 50.

5. Immunogenic complex according to claim 4, characterized in that the antigen and the adjuvant are used in a weight ratio of 1: 25.

6. Use of the immunogenic complex of claim 1 in the manufacture of a vaccine pharmaceutical composition capable of presenting the antigens that make up it to lymphocytes in a safe, stepwise and continuous manner, thereby resulting in a more effective immunological memory.

7. Use of the immunogenic complex of claim 1 in the manufacture of a vaccine pharmaceutical composition that increases the immunogenicity of the antigen of which it is composed.

8. Use of an immunogenic complex according to claim 1 in the manufacture of a vaccine pharmaceutical composition for effective vaccination and/or vaccination in medicine and veterinary medicine.

9. A vaccine pharmaceutical composition characterized by comprising the immunogenic complex of claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.