HK1170683A - Single-time vaccines - Google Patents

Description

Single-time vaccine

[ technical field ] A method for producing a semiconductor device

The present invention relates to the field of new vaccines, in particular to the obtaining and use of a single-time (unitemporal) vaccine for the treatment or prevention of infections of different nature and for the treatment of tumours. More particularly, the present invention relates to a single-temporal vaccination strategy that solves the problem of ineffective multi-dose vaccination (when subject absence or loss occurs for subsequent doses) and makes current parenteral vaccines more effective in order to also induce mucosal responses, using an effective mucosal adjuvant in combination with a parenteral formulation with included, conjugated or co-administered vaccine antigens.

[ Prior Art ] A method for producing a semiconductor device

Fungal, viral, bacterial, protozoan and helminth infections cause frequent pathological states throughout the world. Most of them invade the body or colonize mucosal tissues (Brandtaeg P. and Pabst R. TRENDS in Immunology, 2004, 25 (11): 570-577). There is a consensus that SIgA is the main protector of mucosa and is induced only by immunization or challenge via the mucosal route. However, nasal immunization apparently seems to be better against respiratory pathogens and to some extent is able to protect the female reproductive tract. SIgA-like antibodies in saliva can reflect mucosal responses induced in the airway in nasopharyngeal-associated lymphoid tissue (NALT). However, the nasopharyngeal route is not an alternative to vaccination for enterobacteria, which is mainly oral (Brandtzaeg P et al Immunol Today, 1999, 20: 141-151). Therefore, understanding the mechanisms of mucosal immunity and developing mucosal vaccines represent one of the major challenges in vaccinology. The presence of innate mechanisms and systemic specific IgG are also involved in mucosal protection. Thus, the parenteral route (which does not generally induce SIgA) protects against infectious diseases, but does not eliminate the carrier stage. Carriers (who carry pathogens and are not usually diseased) are those who have a greater epidemiological risk because they are constant transmitters of infection.

The adjuvant participates in: stimulation of innate responses (immunopotentiators); appropriate distribution of antigen (delivery system) (Pashine A Valianti NM. Nat. Med.2005, 11: S63-68; SinghM and O' Hagan DT. pharm. Res.2002, 19 (6): 715-; and directing the immune response to the desired protective response (immunopolarisation) (O P merez et al Pharmacology Online, 2006, 3: 762-64). The development of immunopotentiators and delivery systems, which are effective via the mucosal route, is a major goal of vaccine development and other major targets of international pharmaceutical companies. Focus has been on finding adjuvant systems combining one or more immunopotentiators with a suitable delivery system. In contrast, we started with a substance with multiple immunopotentiators acting synergistically, which in turn had delivery capacity and polarized the immune response towards a preferential Th1 response, also inducing CTL responses (Perrez O et al Scand J Immunology 2007, 66: 271-77; invention certificate OCPI 23313, 2008 Perrez MartinOG et al, WO/2004/047805; WO/2003/094964; and EP 1716866).

In general, it can be said that there are various different types of vaccines that are now in use worldwide. In the case of combination vaccines, it is meant that the various vaccine antigens are applied in the same formulation. They may be incorporated during their production or mixed at the time of immunization. This means that the number of injections performed is reduced. Simultaneous vaccine means that more than one vaccine antigen is applied simultaneously, at the same time but at different locations. These are based on the compartmentalization of the immune system, which enables the induction of different responses in each area, even in the face of simultaneous attacks.

In the case of a single dose vaccine, it is meant that immunization is achieved with a single dose. This is usually achieved with vaccines of live attenuated organisms which replicate in the body, ensuring that a suitable immune response is induced with a single dose. A recent single dose strategy for nasal influenza vaccines has been reported (f. martin et al EP 1878424). The world health organization included in its 2006-2009 strategic program, obtaining a vaccine that required fewer doses to achieve the desired level of immune response.

An alternative route of immunization is based on parenteral immunization, which requires a syringe and needle, resulting in: fear in the recipient population, particularly in prophylactic vaccines; and the possibility of repeated use of needles in a defined area or population, which thereby leads to an increased risk of disease transmission. Alternative approaches include: in particular, oral, nasal, vaginal, rectal, transdermal and sublingual routes. The regionalization of the mucosal response is meant to indicate that infections occurring through the airway are better protected by nasal immunization, which also induces protection in the female reproductive tract (Brandtsaeg P., Pabst R. TRENDS in Immunology, 2004, 25 (11): 570-577). Conversely, the oral route is recommended for protecting against infections of the gastrointestinal tract. However, only oral vaccines are mature and available, although MedImmune recently licensed a nasal vaccine against influenza (FluMisT, Belche RB, Mendelman PM, Treanor J, King J, Gruber WC, Piedra P et al NEJM 1998, 338: 11405-12).

Mucosal immune responses are often the induction of tolerance. It involves a response of SIgA in the mucosal range and does not induce a full complementSystemic IgG, which makes it effective against a variety of commensal microorganisms owned by organisms, particularly humans. However, induction of systemic specific IgG, and also SIgA, is an important backbone for mucosal immunization. IgA comprises the J chain and secretory components involved in transport to the mucosal cavity, where it is called SIgA. IgA can be present in monomeric or multimeric forms and has two subclasses: IgA for use in therapy₁And IgA₂. The latter are more resistant to proteases produced by various microorganisms. SIgA is induced almost exclusively by mucosal infection or immunization and there is a consensus that this antibody is the main protector of the mucosa.

IgA detected in saliva represents the best marker for measuring induction of immune response in the nasopharynx and it is desirable to find a good marker for oral immunization (Brandtzaeg p. int.j. med. microbiol.2003, 293: 3-15).

Adjuvants are substances that enhance the specific immune response against an antigen, leading to a faster induction of the immune response and an increase in its duration (Vogel fr. dev. biol. stand.1998, 92: 241-.

Continuous alumina is the most used adjuvant in parenteral vaccines. However, it is not a powerful adjuvant capable of adjuvanting new subunit vaccines. It is believed that only the Nature of the delivery system is mastered, although a new mechanism of action independent of the Toll-like receptor (involving Nalp3) has recently been described (Eisenbarth SC et al Nature, 2008, 453: 1122-1126). Its mucosal function was not confirmed and it was speculated that a Th2 response was preferentially induced. Licensed vaccine adjuvants are rare (MF59, ASO2, virosomes, AFPL1, AFCo1, Protellin, MPL, etc.) and few are mucosal adjuvants (CTB, mutants of LT (LT63K, LTR72), CpG, chitosan, ISCOM and AFCo1) (Singh M, O' Hagan DT. pharm. Res 2002, 19 (6): 715-728; Berez O et al Immunology and Cell biology.2004, 82: 603-610).

Its use in vaccine formulations allows for a reduction in the amount of antigen necessary, a targeting of the response to a desired pattern, and a reduction in the number of doses required. We considered adjuvant platforms based on PL (AF 'AdjuvantFinlay' PL1) derived from Neisseria meningitidis serogroup B, its coiled coil (Co) derivative (AFCo1) and analogues of other microorganisms for use in parenteral or mucosal vaccines. These adjuvants induce a preferential Th1 response in mice and humans by parenteral administration, characterized by: anti-PL IgG and Th1 subclasses, positive demonstration of interferon gamma (IFN γ), cytokine (IL)12, tumor necrosis factor α (TNF α), delayed hypersensitivity, and additionally, induction of Cytotoxic T Lymphocytes (CTL) (Berez O et al Infect Immun.2001, 69 (7): 4502' -4508; Berez O et al Immunology and Cell biology.2004, 82: 603-610; Rodr I guez T et al Vaccine 2005, 26: 1312-21; Berez O et al Scand J Immunology 2007, 66: 271-77). The mucosal action of AFCo1 and AFPL1 was also demonstrated by nasal, oral, vaginal and rectal application and it produces similar derivatives that have been expanded to other microorganisms.

VA-MENGOC-based on PL of Neisseria meningitidis serogroup BHas been used in doses in excess of 6 million and has proven to be safe, non-reactogenic and effective for protection against neisseria meningitidis serogroups B and C. It is also safe and effective to use during lactation, where the T-independent conversion of polysaccharide C into T-dependent antigen (Perez O. et al Th1 response induced by the Bcompontent of VA-MENGOC-Vitamins the same family of polysaccharides C and primers for memory in toddler, biotectolog i apalica 2002; 19(1-2): 54). This vaccine induces a preferential Th1 pattern characterized by induction of lymphoproliferation in humans and animals, anti-PL IgG antibodies, IgG1 subclass in humans and IgG2a subclass in miceClass, IFN γ, IL2 and IL12, in the messenger ribonucleic acid (mRNA) and protein ranges; no anti-PL IgE was induced, nor was total IgE increased; and no IL-4 nor IL-5 production, at the messenger ribonucleic acid (mRNA) and protein levels (Perrez O et al, infection Immun.2001, 69 (72001): 4502-4508). Proteoliposomes and derivatives thereof, such as spirosomes, have also been used as adjuvants, as disclosed in the inventor's certificate OCPI 23313, 2008, pirez Mart i o g et al, and WO/2004/047805.

Chitosan is the most abundant biopolymer in nature behind cellulose and is part of the exoskeletons of crustaceans and insects, as well as the cell walls of some microorganisms (e.g. yeast and fungi). By the alkaline deacetylation of chitin, N-acetyl-D-glucosamine polymers with different molecular weights and different degrees of deacetylation, which give them special properties, are obtained. It has many applications in the pharmaceutical industry, medicine and veterinary medicine due to its lack of toxicity and allergenicity.

Due to its natural mucoadhesive properties, chitosan is widely used in research as a transport system for drugs, peptides, proteins, vaccines and DNA. As mucoadhesive polymers, chitosan is used to increase the absorption of morphine and insulin through the nasal mucosal epithelium. Chitosan increases transepithelial transport of antigen to mucosal immune tissues via tight junctions and by reducing mucosal movement. Chitosan induces stimulatory effects of the immune system, such as activation of macrophages and induction of cytokines.

Increased paracellular transport and increased luminal antigen uptake in the intestinal epithelium. Greater uptake of antigen promotes contact with the immune system, which increases local and systemic immune responses: antigens co-administered with or encapsulated with chitosan induce higher antibody levels, although the mechanisms involved have not been described. The polysaccharide has also been used as a dietary fiber to produce changes in the intestinal microflora and microenvironment of the mucosa.

The technical aim sought is to increase vaccine coverage and to induce a combined mucosal and parenteral response against heterologous antigens (fungal, viral, protozoan, helminth or carcinogenic) or autoantigens, in particular to induce a secretory iga (siga) response, which is involved in the protection of mucosal pathogens and cancers that originally were or secondarily invade the mucosa, thus further ensuring a systemic response of specific IgG antibodies, which is also involved in mucosal protection. This extends to better receiving immunisations by reducing the number of doses and applying one or more times via a more accepted and less reactogenic route, i.e. the mucosal route. These results have led to the development of a single-time vaccine formulation that is either therapeutic or prophylactic.

In this sense, it is proposed to use Proteoliposomes (PL) and derivatives thereof (spirochete [ AFCo1]) as adjuvants in formulations having inserted in their structure, conjugated thereto or co-administered therewith heterologous antigens (fungal, viral, bacterial, protozoan, helminth or oncogenic) or autoantigens present in PL and derivatives thereof. These formulations extend the preferential type 1 helper T response (Th1) induced by PL to the native or included antigen (Perrez O et al infection Immun.2001, 69 (7): 4502-4508), the preferential mucosal response of the spiroid coil (Perrez O et al immunization and cell biology.2004, 82: 603-610) (but also induced by lipoprotein bodies), and the Cytotoxic T Lymphocyte (CTL) response (Perrez O et al Scand J immunization 2007, 66: 271-77) to two or a single application to the antigen via different routes of administration.

[ summary of the invention ]

The aim of the present invention is to use native or genetically modified bacterial PL (aflp 1), in particular those derived from neisseria meningitidis or other gram-negative bacteria, as well as the spirochetes derived from these PLs as novel adjuvants or vaccines themselves to obtain a one-time vaccine.

By "Proteoliposomes (PL)" it is meant that they are obtained from bacteria by using any known method, e.g. separation without detergent, processes involving detergents such as deoxycholate, SDS, etc. or extraction from vesicles ("bubbles") of culture supernatant, in particular the method disclosed in US5,597,572. PL contains different pathogen-associated molecular patterns (PAMPs), molecular structures conserved among pathogens that are capable of strongly stimulating the innate immune system and thereby inducing a powerful inherited response. PL comprises the structure of the bacterial outer membrane, but for the purpose of this patent is also considered to be an extract from other organisms (e.g. viruses, fungi, protozoa, worms or tumor cells). The term AFPL is reserved for the use of PL as an adjuvant.

"spiral coil (Cochleate)" refers to derivatives of PL which thus also contain several PAMPs, as described in patent (WO/2004/047805), Infect Immun.2001, 69(7) by Perrez O et al: 4502-4508, and pirez O et al Immunology and Cell biology, 2004, 82: 603, 610. Moreover, for the purposes of this patent, the concept of spiral coils is extended to produce them starting from a combination of synthetic lipids including PAMPs (which act synergistically), which is fundamentally different from other spiral coils produced from synthetic lipids and is not included in the patents mentioned above.

By "one-time" vaccine is meant that one or more mucosal doses and one parenteral dose are applied simultaneously, which enables effective immunization against one or more vaccine antigens, thereby reducing the number of doses, the number of encounters with the subject or animal to be immunized, and thereby increasing the coverage of the vaccination by eliminating losses due to subject noncooperative to subsequent doses. This concept extends to any simultaneous combination of antigen with or without adjuvant applied by different routes of immunization, either mucosal or parenteral, although it is preferred to be interested in a combination of these two routes, as well as applications with multiple mucosal routes. This concept extends to other vaccines using adjuvants different from those shown (proteoliposomes (AFPLs) and their derivatives (AFCos)). This concept does not include a single dose vaccine that is obviously also applied at a single time.

Most vaccines require two or more doses and are generally applied by the same route of immunization. Thus, it was surprisingly found that the simultaneous application of one mucosal dose and one parenteral dose induced a higher systemic specific IgG response compared to two intramuscular doses or even three mucosal doses.

Our use of a coil requires at least three nasal doses to achieve a high mucosal response, although a positive response has been obtained with two doses. Thus, it has also been unexpectedly found that a local and distal positive mucosal response is also induced using a single mucosal dose when compared to a parenteral dose.

The challenge-challenge ("prime-boost") strategy that has emerged to address the low immunogenicity induced by the promised naked DNA vaccines consists in stimulating with the DNA of interest and subsequently attacking with different vectors or purified antigens expressing the antigen of interest, in order to avoid their interference. It was also surprisingly found that only structural transformation of PL to spirochete rolls (which contain the same PAMPs, proteins and phospholipids) achieved induction of enhanced responses by reducing two vaccinations at one dose per route. It is also surprising that the same structure will also function in the same single-time application through both pathways.

The mucosal and parenteral systems are organized in different forms and include more compartmentalized mucosal systems. It is considered that the response induced in the upper respiratory tract is the best one for protection against respiratory infections, and digestive infections require oral immunization. It was therefore unexpectedly found that the simultaneous application by both mucosal and parenteral routes is enhanced and that the enhanced response is obtained by applying the formulation by several mucosal routes together with the parenteral route (nasal, oral, sublingual).

One of the important properties of adjuvants in vaccines is their delivery ability, they act as reservoirs and direct antigens to the site of T cell aggregation to allow immunopotentiators to stimulate the innate response. This ensures that antigen release occurs over several days or that they are long-term picked up by antigen presenting cells and thereby dragged to the T-zone of the peripheral lymphoid organs. This means that, at least in humans, subsequent doses are usually spaced several weeks apart from each other. Thus, it was unexpectedly observed that a similar systemic response was achieved without intervals between doses. It is more unexpected that the response was also found at the mucosal level, which ensures that a mucosal and systemic response was obtained with two administrations (one mucosal and another parenteral, simultaneously). In addition, it has also been surprisingly observed that by enhancing the delivery system for parenteral applications by using alumina, chitosan or oil containing formulations, the one-time response is also enhanced.

It was also unexpected that the simultaneous application of two or more antigens via two or more mucosal routes and parenteral application in combination vaccine formulations also induced an effective one-time response.

More surprisingly, it was found that the application of these formulations alone induced a memory response, which was demonstrated by a typical immune increase of the secondary (memory) response obtained by applying a mucosal boost for several months after the initial immunization.

The parenteral and mucosal responses induced by immunization are independent. Both routes typically require several doses and induce cellular responses, which become usually different antibody responses. The parenteral route induces a systemic specific IgG response, and the mucosal route, in particular the nasal mucosal route, induces a specific response of local and distal (genitourinary tract) SIgA and also a systemic specific IgG response. Thus, it was not surprising to find systemic specific IgG responses, as they could be the sum of the responses induced by both vaccination routes; however, it is surprising and unexpected that a single nasal dose is enhanced to a level of at least two nasal doses by parenteral application.

The invention also comprises obtaining various vaccine formulations also obtained in this one-time strategy, which exploit the ability of PL and its derivatives (spirochetes) to induce Th1 responses with CTL activity.

Poorly immunogenic antigens such as ovalbumin (Ova) or well immunogenic antigens such as Tetanus Toxoid (TT) were effectively evaluated in this one-time strategy. These antigens function in incorporated or PL-conjugated form and are then converted to spirochete coils or co-administered with them.

The vaccine formulations of the present invention may be used to protect an susceptible mammal or treat a neoplastic disease by administering the formulation in a single occasion. These applications may include injection by intramuscular, intraperitoneal, intradermal, subcutaneous or transdermal routes, with mucosal administration, especially by oral, nasal, rectal, vaginal or sublingual routes.

Typically, the number of doses is two or several by similar or different mucosal routes and one by parenteral routes with single or combined antigens. In addition, the mucosal and systemic immune responses induced can be boosted by: simultaneous similar applications (single occasion) or applications with only one of the pathways were performed to amplify the initially induced memory response.

The novelty of the present invention resides firstly in the simultaneous single use of immunization applied by two or more immunization routes.

It is particularly novel that the single-time application of the antigen via the mucosal route (oral, sublingual, vaginal, rectal or nasal) and the simultaneous parenteral route (subcutaneous, transdermal, intraperitoneal, intradermal or intramuscular) induces a similar systemic immune response compared to two parenteral dosage regimens separated by at least 14 days or at least two mucosal doses separated by 7 days required for inducing a good response via both routes.

Another aspect of novelty is that the single application of the antigen by the mucosal route and simultaneously by the parenteral route also induces local and distal mucosal immune responses that can only be achieved with at least 2 and better with 3 mucosal doses. With regard to this enhancement, there is currently no explanation on the circulating levels of lymphocytes guided by cytokine and chemokine signals.

Another aspect of novelty is that the monoparental vaccination strategy enables the use of several antigens by single or multiple mucosal routes, and the corresponding antigens by parenteral routes in combination vaccines.

The proposed solution has the following advantages:

● extends the concept of stimulation-expansion ("prime-boost") of immunizations with a new single-time vaccine, with weeks or months between them to induce an expanded secondary response.

● combine mucosal administration(s) with parenteral administration at the same vaccination time to achieve enhanced specific responses of SIgA and systemic IgG in the mucosa.

● two adjuvants of similar composition or the same adjuvant are used instead of two different adjuvants or carrier systems as used in conventional prime-boost tests.

● increase vaccination coverage through losses that occur due to non-cooperation of subsequent doses for a multi-dose regimen of immunization. This correspondingly and significantly means a reduction of the resources necessary for carrying out the immunization.

● reduces the number of vaccine doses to at least half (2-dose vaccine) or one third (3-dose vaccine) without affecting the quality of the systemic immune response and adding to the mucosal response, which is essential for the vast majority of infections that occur especially in humans.

● AFPL1 (one of the recommended adjuvants) is safe and reliable in children less than 1 year old and 2-4 years old, in pupils, adolescents and adults; also, other derivatives (AFCo1) have passed preclinical stability and toxicity tests and are intended for use via the mucosal route, which is less reactogenic and does not require sterility, but ensures a controlled microbial load.

● applied by transmucosal or parenteral route, specific responses of the SIgA, Th1 IgG subclass (with CTL activity) were induced by AFCo1 and AFPL 1.

● induce a powerful response not only to self-antigens but also to incorporated or conjugated unrelated (heterologous) antigens, but also to antigens co-administered by mucosal and parenteral routes, which extends the capacity of this single-time system to multiple vaccines.

● multiple antigens can be used by single or multiple mucosal routes and by applying the corresponding antigens as a combined vaccine via parenteral route, which means that multiple vaccines can be developed at a single time.

The invention will be described by the following specific examples.

Example 1: acquisition of lipoprotein body (PL) ]

To obtain PL, culture of neisseria meningitidis, Salmonella typhi (Salmonella typhi), Vibrio cholerae (Vibrio cholerae), escherichia coli (escherichia coli), Shigella (Shigella), Salmonella (Salmonella), or Bordetella pertussis (Bordetella pertussis) of any serogroup, in particular serogroup B, is performed and the biomass collected by centrifugation is subjected to an extraction process using detergents, enzymes, and ultrasound. Cell debris was removed by centrifugation, and then the supernatant was subjected to digestion with nuclease to remove nucleic acids. The extract was recovered by ultracentrifugation, resuspended in detergent-containing solution, and purified by molecular exclusion chromatography from other components of low and medium molecular weight. The proteoliposomes thus obtained comprise: porins (PorA and PorB), traces of bacterial DNA and less than 10% (relative to the protein) of natural LPS inserted in its structure (but never free). The porin, DNA and LPS present are PAMPs that interact with pattern recognition receptors that cause signs of risk at the cellular level involved in the innate response. The final product was subjected to a series of biological and physico-chemical examinations. In case LPS is also PAMP, which is the antigen of interest, it may be increased up to at least 35% compared to the protein. Starting from viruses and protozoa, a similar process is used to obtain liposome structures rich in outer membrane proteins.

Example 2: acquisition of Spiro-coil derived from Liposome

Starting from PL obtained by the methods described in EP 885900077.8 or US5,597,572. They were resuspended in Tris-EDTA buffer containing 0.5% sodium deoxycholate. The protein concentration of the suspension was determined using the modified Lowry method according to Peterson (analyt. biochem.83, 346, 1977). The content of PL-forming phospholipids was determined by quantification of inorganic phosphorus (Bartlett, J Biol chem.234, 466, 1959). Both protein concentration and phospholipid concentration were used to establish the optimal conditions and amount of detergent necessary for the formation of the spirochete. A solution with PL adjusted to a final protein concentration of 5-6 mg/mL in Tris-EDTA buffer containing sodium deoxycholate in an amount of 6-15 times the mass of total protein was prepared. The solution was directly filtered through a filter having a pore size of 0.2 μm on a dialysis apparatus. Dialysis is performed by rotary stirring or tangential filtration for 24 hours, with the dialysis buffer being continuously and slowly changed in the first and subsequent washes. The last solution comprises 50-150mM NaCl, 1-4mM imidazole, 3-5mM HEPES and 2-7mM CaCl, in H prepared under sterile conditions₂O, during all its steps. The formation of the spiral coil was checked by the formation of a white precipitate and subsequent microscopic observation (optical and electronic). Protein and phospholipid concentrations were re-estimated and adjusted for subsequent testing. The physico-chemical properties of the proteins contained in the spiroid coil were examined by electrophoresis on a polyacrylamide gel stained with coomassie blue and compared with PL. In addition, their structural integrity was checked and verified by Western blotting method.

Example 3: AFPL1, AFCo1 or V by intramuscular routeA-MENGOC-Application of (1) to induce a specific response of serum IgG in mice

Immunization protocol: two doses of 12.5 μ g/50 μ L of AFPL1, AFCo1 or VA-MENGOC-(Va) Balb/c mice were immunized.

Method for extracting and processing blood to obtain serum: blood was extracted 21 days after the last dose by retroorbital plexus puncture using heparinized capillary. The obtained blood sample was incubated at 37 ℃ for 1 hour, followed by centrifugation at 2000g for 10 minutes to extract serum.

Detection of anti-PL IgG by ELISA:

reagents and solutions:

coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

Standard sera of antibodies: a curve was prepared with two-fold serial dilutions of hyperimmune mouse serum obtained from reference serum from the atlanta disease control center (CDC) for meningeal serogroup B.

The method comprises the following steps:

● to 96-well plates with high binding capacity (Maxisorp, Nunc, EUA) 100. mu.L/well of PL diluted in STR at a concentration of 20. mu.g/mL was added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● Standard sera of antibodies and sera to be evaluated were diluted 1: 100 in sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● mu.L/well of anti-IgG conjugate diluted 1: 2000 in sample dilution was added and incubated for 1 hour at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution. The absorbance in Optical Density (OD) was measured at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

Parallel behavior between the curves is evaluated, and a linear fit equation and a coefficient R are calculated². Standard serum CDC1992 was defined as the independent variable (x) and standard serum of Finlay as the dependent variable (y). The final concentration was determined by substituting in the formula obtained by the value of the standard CDC1992 for specific IgG. By using a standard sigmoidal curve, 5000U/mL of anti-PL IgG antibody was assigned to the highest point of the curve and 31.25U/mL was assigned to the lowest point. The results for anti-PL IgG were calculated by interpolating the OD obtained in each serum using a model curve prepared with reference sera and expressed in U/mL. The limit of detection for this assay, i.e., the minimum amount of specific IgG that can be detected under our experimental conditions, was 26.9U/mL (FIG. 1).

To summarize:

the spiral coil (AFCo1) applied by the intramuscular route induces the vaccine VA-MENGOC-Similar specific IgG responses, and these responses were significantly higher than those induced by proteoliposomes (AFPL 1).

Example 4: AFPL1, AFCo1 or VA-MENGOC via the intramuscular routeApplication of (2) in mouse saliva without inducing secretory IgA specific response

Immunization protocol: two doses of 12.5 μ g/50 μ L of AFPL1, AFCo1 or VA-MENGOC-(Va) Balb/c mice were immunized.

Method for extracting and processing saliva: saliva extraction was performed 7 days after the last dose. To obtain saliva samples, saliva secretion was stimulated in the animals by intraperitoneal application of 50 μ L/mouse of 0.5% pilocarpine (Imefa, Cuba). The samples were maintained on ice during extraction and immediately inactivated at 56 ℃ for 15 minutes. Then, at 4 ℃ in 14000g centrifugal 15 minutes, collect the supernatant, and stored at-70 ℃ until use.

Detection of anti-PL IgA by ELISA:

reagents and solutions:

coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Peroxidase-conjugated biotinylated anti-mouse IgA (R5-140) (Sigma, St. Louis, MO, EUA)

Streptavidin-peroxidase (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

Standard saliva for antibodies: as an internal standard for antibodies, for the anti-PL IgA assay, curves prepared using saliva samples from hyperimmunized mice were used.

The method comprises the following steps:

● to 96-well plates with high binding capacity (Maxisorp, Nunc, EUA) 100. mu.L/well of PL diluted in STR at a concentration of 20. mu.g/mL was added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● Standard saliva of antibody and saliva to be evaluated were diluted 1: 2 in sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● 100 μ L/well of anti-IgA conjugate at a concentration of 2 μ g/mL in the sample dilution solution was added and incubated for 2 hours at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● then, a second conjugate (streptavidin-peroxidase) was added at a 1: 2000 dilution in the sample dilution solution and incubated at 37 ℃ for 30 minutes.

● was washed five times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution.

● Absorbance (OD) was measured by reading at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

By using a standard sigmoidal curve, two-fold serial dilutions were used, and 2000 Arbitrary Units (AU) of anti-PL IgA antibody were assigned to the highest point and 62.5AU to the lowest point of the curve. The results for anti-PL IgA were calculated by interpolating the OD obtained in each sample using a model curve prepared with reference saliva and expressed in AU. The samples were considered positive when a titer value above 250AU was measured, the cutoff was calculated after evaluating samples from 100 non-vaccinated animals, and the rating was established as the mean, 2 standard deviations of these samples (figure 2).

To summarize:

as can be observed, none of the formulations applied by the intramuscular route induced a specific IgA response.

Example 5: spiro-coil by intranasal route (AFCo1) induces a specific response of IgA in saliva in mice over proteoliposomes (AFPL1) ]

Immunization protocol: balb/c mice were immunized by the intranasal route (IN) with AFPL1 or AFCo 1. For the IN route, three doses, each with a total volume of 50 μ g IN 25 μ L/total protein concentration IN the mouse (for both formulations), were administered at 7 day intervals from each other and applied directly IN the nasal fossa (12.5 μ L/nasal fossa) using an automated pipette and sterile tip.

Method for extracting and processing saliva: the procedure was as described in example 4.

Detection of anti-PL IgA by ELISA: the procedure was as described in example 4 (fig. 3).

To summarize:

both AFCo1 and AFPL1 applied by the nasal route induced specific IgA responses in saliva, although the former induced significantly higher responses.

Example 6: spiro coil (AFCo1) by the intranasal route induces a specific response of serum IgG in mice that exceeds that of the proteoliposomes (AFPL1) ]

Immunization protocol: balb/c mice were immunized by the intranasal route (IN) with AFPL1 or AFCo 1. For the IN route, three doses, each having a total protein concentration of 50 μ g IN 25 μ L total volume/mouse (IN both formulations), were administered at 7 day intervals from each other and applied directly IN the nasal fossa (12.5 μ L/nasal fossa) using an automated pipette and sterile tip.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-PL IgG by ELISA: the procedure was as described in example 3 (fig. 4).

To summarize:

both AFCo1 and AFPL1 applied by the nasal route induced specific IgG responses in saliva, although the former induced significantly higher responses.

Example 7: intramuscular route requires two doses of proteoliposomes (AFPL1) or spirochete (AFCo1) for induction of good serum-specific IgG response in mice ]

Immunization protocol: balb/c mice were immunized by intramuscular route (IM) with AFPL1 or AFCo1 by deep puncture in one of the hind limbs of the animals. Each formulation was used by using two immunization schedules with one and two doses/group, respectively, where the latter used 14 day intervals. For each formulation, the dose used had a concentration of 12.5. mu.g/50. mu.L.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-PL IgG by ELISA: the procedure was as described in example 3 (fig. 5).

To summarize:

intramuscular administration of proteoliposomes (AFPL1) and spirochete (AFCo1) required at least 2 doses for induction of a significant systemic specific IgG response.

Example 8: the intranasal route required three doses of either spirochete (AFCo1) or proteoliposomes (AFPL1) for inducing high levels of specific IgA in saliva of mice ]

Immunization protocol: balb/c mice were immunized by intranasal route with either AFPL1 or AFCo1 using three immunization schedules with one, two and three doses of each formulation/group, respectively. Each dose was administered at a concentration of 50 μ g in 25 μ L/animal at 7 day intervals, 12.5 μ L/nasal fossa, as described in example 5.

Method for extracting and processing saliva: the procedure was as described in example 4.

Detection of anti-PL IgA by ELISA: the procedure was as described in example 4 (fig. 6).

To summarize:

both AFCo1 and AFPL1 applied by nasal route require two doses for inducing a positive response of specific IgA in saliva. However, higher responses were obtained with three doses. In all cases, AFCo1 induced a response that exceeded AFPL 1.

Example 9: the intranasal route required three doses of either spirochete (AFCo1) or proteoliposomes (AFPL1) for induction of high levels of serum-specific IgG in mice

Immunization protocol: balb/c mice were immunized by intranasal route with either AFPL1 or AFCo1 using three immunization schedules with one, two and three doses of each formulation/group, respectively. Each dose was administered at a concentration of 50 μ g in 25 μ L/animal at 7 day intervals, 12.5 μ L/nasal fossa, as described in example 5.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-PL IgG by ELISA: the procedure was as described in example 3 (fig. 7).

To summarize:

both AFCo1 and AFPL1 via the nasal route induced significant serum responses to specific IgG. However, the three doses were much higher. AFCo1 was always higher than AFPL 1.

Example 10: simultaneous immunization of one dose of the spirochete (AFCo1) (intranasal) and one dose of the proteoliposomes (AFPL1) (intramuscular) (STVS) induced specific IgG responses in mouse sera higher than the three nasal AFCo1 doses and similar to the two parenteral AFPL1 doses, with similar proportions of IgG subclasses maintained ]

Immunization protocol: balb/c mice were divided into three immunization groups and one control group. The first group was immunized by intranasal route with AFCo1 at a concentration of 50 μ g in 25 μ L/animal (12.5 μ L/nasal fossa) with three doses (0, 7, 14 days). The second group was immunized by intramuscular route with two doses (0, 14 days) of aflp 1 at a concentration of 12.5 μ g in 50 μ L/animal. The third group was immunized by the intranasal route with AFCo1(100 μ g in 25 μ L, 12.5 μ L/nasal fossa) and simultaneously by the intramuscular route with AFPL1(12.5 μ g in 50 μ L) (STVS).

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-PL IgG by ELISA: the procedure was as described in example 3.

To summarize:

single immunizations induced specific IgG responses higher than 3 nasal AFCo1 doses and similar to 2 intramuscular AFPL1 doses (fig. 8).

Detection of these subclasses by ELISA of anti-PL IgG1 and IgG2 a:

reagents and solutions:

coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Biotinylated anti-mouse IgG1 and anti-mouse IgG2a sheep MAb (Amersham, LIFE SCIENCE)

Streptavidin-peroxidase (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

The method comprises the following steps:

● to 96-well plates with high binding capacity (Maxisorp, Nunc, EUA) 100. mu.L/well of PL diluted in STR at a concentration of 20. mu.g/mL was added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● serum to be evaluated was diluted 1: 100 in sample dilution solution. Dilutions of 100. mu.L/well of serum were applied in duplicate and incubated at 37 ℃ for 2 hours.

● are washed three times with the washing solution.

● mu.L/well of anti-mouse IgG1 and anti-mouse IgG2a conjugate diluted 1: 2000 in sample dilution solution was added and incubated for 2 hours at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● then, a second conjugate (streptavidin-peroxidase) was added at a 1: 2000 dilution in the sample dilution solution and incubated at 37 ℃ for 30 minutes.

● was washed five times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution.

● Absorbance (OD) was measured by reading at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

The results for anti-PL IgG1 and IgG2a are expressed as Optical Density (OD). In this test, the sample is considered positive when the OD value is above 0.25.

To summarize:

single-time application maintained the same subclass ratio as induced by 3 nasal AFCo1 doses or 2 intramuscular AFPL1 doses (fig. 9).

Example 11: simultaneous immunization with one dose of spirochete (AFCo1) (intranasal) and proteoliposomes (AFPL1) (intramuscular) (STVS) induced high levels of anti-PL IgA in saliva, feces and vaginal washes of immunized mice

Immunization protocol: female Balb/c mice were divided into three immunization groups and one control group. The first group was immunized by intranasal route with AFCo1 at a concentration of 50 μ g in 25 μ L/animal (12.5 μ L/nasal fossa) with three doses (0, 7, 14 days). The second group was immunized by intramuscular route with two doses (0, 14 days) of aflp 1 at a concentration of 12.5 μ g in 50 μ L/animal. The third group was immunized at a single time with one dose of AFCo1(100 μ g in 25 μ L) by the intranasal route and one dose of AFPL1(12.5 μ g in 50 μ L) by the intramuscular route.

Method for extracting and processing saliva: in this example, saliva extraction was performed 7 and 14 days after immunization, the rest being performed as indicated in example 4.

The method for extracting and processing the excrement comprises the following steps: feces extraction was performed 14 days after the last dose. Three fecal "pellets"/animals were collected. The feces were weighed by subtracting the weight value of the vial. These pellets were resuspended in PBS solution with protease inhibitors (1mM PMSF and 5. mu.g aprotinin (Aprotitina)/mL) at a dilution of 20. mu.L/mg feces. The resuspended sample was vigorously stirred by using a magnetic stirrer (vortex), the insoluble material was discarded by centrifugation (40000rpm, 20 minutes at 4 ℃), and the supernatant was saved for subsequent analysis.

Methods of extracting and processing vaginal eluate: samples of vaginal washings were collected 21 days after the last dose, as with serum. These samples were obtained by: 100 μ L of sterile PBS was applied to the animal vagina, the vaginal eluate was aspirated and collected in a vial. Then, at 4 ℃ in 14000rpm centrifugal 15 minutes, preservation of supernatant for subsequent analysis.

Detection of anti-PL IgA by ELISA: the procedure was as described in example 4.

To summarize: immunization at a Single Time (STVS) of one intramuscular AFPL1 dose and one intranasal AFCo1 dose induced high levels of anti-PL IgA in saliva, feces and vaginal eluates compared to that induced by administration of three intranasal AFCo1 doses (figures 10-12).

Example 12: simultaneous immunization with a dose of ovalbumin-containing spirochete (AFCo1-Ova) (intranasal) and Ova-containing proteoliposomes (AFPL1-Ova) (intramuscular) (STVS) induced high levels of anti-Ova IgG in mice

Immunization protocol: balb/c mice were divided into four immunization groups and one control group. The first group was immunized by intranasal route with three doses (0, 7, 14 days) of AFCo1-Ova at a concentration of 50 μ g/20 μ g in 25 μ L/animal (12.5 μ L/nasal fossa). The second group was immunized by intramuscular route with two doses (0, 14 days) of AFPL1-Ova at a concentration of 12.5 μ g/10 μ g in 50 μ L/animal. The third group was immunized (STVS) by intranasal route with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L, 12.5 μ L/nasal fossa) and simultaneously by intramuscular route with a dose of AFPL1-Ova (12.5 μ g/10 μ g in 50 μ L). The fourth group was immunized by intramuscular route with Ova in two doses (0, 14 days) at a concentration of 10 μ g in 50 μ L/animal.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-Ova IgG by ELISA:

reagents and solutions:

ovalbumin, purity grade V, Sigma

Coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

The method comprises the following steps:

● to 96-well plates (Maxisorp, Nunc, EUA) with high binding capacity, 100. mu.L/well of Ova diluted in STR at a concentration of 10. mu.g/mL were added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● serum to be evaluated was diluted 1: 100 in sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● mu.L/well of anti-IgG conjugate diluted 1: 2000 in sample dilution was added and incubated for 1 hour at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution.

● Absorbance (OD) was measured by reading at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

The results of the anti-Ova IgG are expressed as Optical Density (OD). In this test, the sample is considered positive when the OD value is above 0.25.

To summarize:

single-time applications of intranasal AFCo1-Ova and intramuscular AFPL1-Ova induced significant anti-Ova IgG responses in serum. These responses were higher than those induced by three intranasal AFCo1-Ova doses and two intramuscular AFPL1-Ova doses (fig. 13).

Example 13: simultaneous immunization with a dose of ovalbumin-containing spirochete (AFCo1-Ova) (intranasal) and a dose of Ova-containing proteoliposomes (AFPL1-Ova) (intramuscular) (STVS) induced high levels of anti-Ova IgA in mice

Immunization protocol: balb/c mice were divided into five immunization groups and one control group. The first and second groups were immunized by intranasal route with three doses (0, 7, 14 days) and two doses (0, 7 days) of AFCo1-Ova at a concentration of 50 μ g/20 μ g in 25 μ L/animal (12.5 μ L/nasal fossa). The third group was immunized by intramuscular route with two doses (0, 14 days) of AFPL1-Ova at a concentration of 12.5. mu.g/10. mu.g in 50. mu.L/animal. The fourth group was immunized (STVS) with AFCo1-Ova (100. mu.g/50. mu.g in 25. mu.L, 12.5. mu.L/nasal fossa) by the intranasal route and AFPL1-Ova (12.5. mu.g/10. mu.g in 50. mu.L) simultaneously by the intramuscular route. The fifth group was immunized by intramuscular route with Ova in two doses (0, 14 days) at a concentration of 10 μ g in 50 μ L/animal.

Method for extracting and processing saliva: in this example, saliva extraction was performed 7 and 14 days after immunization, the rest being performed as indicated in example 4.

Detection of anti-Ova IgA by ELISA:

reagents and solutions:

ovalbumin, purity grade V, Sigma

Coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Biotinylated anti-mouse IgA (R5-140) conjugated with peroxidase (SIGMA, St. Louis, MO, EUA)

Streptavidin-peroxidase (SIGMA, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

Standard saliva for antibodies: as an internal standard for antibodies, for the anti-PL IgA assay, curves prepared using saliva samples from hyperimmunized mice were used.

The method comprises the following steps:

● to 96-well plates (Maxisorp, Nunc, EUA) with high binding capacity, 100. mu.L/well of Ova diluted in STR at a concentration of 10. mu.g/mL were added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● saliva to be evaluated is diluted 1: 2 in the sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● 100 μ L/well of anti-IgA conjugate at a concentration of 2 μ g/mL in the sample dilution solution was added and incubated for 2 hours at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● then, a second conjugate (streptavidin-peroxidase) was added at a 1: 2000 dilution in the sample dilution solution and incubated at 37 ℃ for 30 minutes.

● was washed five times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution.

● Absorbance (OD) was measured by reading at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

The results of anti-Ova IgA are expressed as Optical Density (OD). In this test, the sample is considered positive when the OD value is above 0.25.

To summarize:

single-time applications of intranasal AFCo1-Ova and intramuscular AFPL1-Ova induced a significant anti-Ova IgA response in saliva. These responses were higher than those induced by either two intranasal AFCo1-Ova doses or three intranasal Ova doses, and were not induced by two doses of AFPL1-Ova administered by the intramuscular route (fig. 14).

Example 14: spiro-coil (AFCo1) and proteoliposomes (AFPL1) can be used effectively in a single-time strategy by the two immunization routes (intranasal and intramuscular)

Immunization protocol: balb/c mice were divided into four immunization groups and one control group. The first group was immunized with one dose of AFCo1(100 μ g in 25 μ L) by the intranasal route and one dose of AFPL1(12.5 μ g in 50 μ L) by the intramuscular route at a single time; the second group was immunized with one dose of AFCo1(100 μ g in 25 μ L) by the intranasal route and one dose of AFCo 1(12.5 μ g in 50 μ L) by the intramuscular route at a single time; and a third group was immunized at a single time with one dose of aflp 1(100 μ g in 25 μ L) by the intranasal route and one dose of aflp 1(12.5 μ g in 50 μ L) by the intramuscular route. As a positive control, one group was immunized by intranasal route with AFCo1 at a concentration of 50 μ g/animal of 25 μ L (12.5 μ L/nasal fossa) with three doses (0, 7, 14 days).

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-PL IgG by ELISA: the procedure was as described in example 3.

Conclusion of the results: an intramuscular AFPL1 dose combined with an intranasal AFPL1 dose at a single time or intramuscular AFCo1 combined with an intranasal AFCo1 dose at a single time induced high levels of anti-PL IgG in serum compared to that induced by three doses of AFCo1 administered intranasally (figure 15). These results enable confirmation that both structures can be used in a one-time vaccine with both vaccination routes.

Example 15: the ovalbumin-containing spirochete (AFCo1) and proteoliposomes (AFPL1) (AFCo1-Ova and AFPL 1-Ova)' can be used effectively in a single-time strategy by the two immunization routes (intranasal and intramuscular)

Immunization protocol: balb/c mice were divided into five immunization groups and one control group. The first group was immunized by a single time, by intranasal route with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L) and by intramuscular route with a dose of AFPL1-Ova (12.5 μ g/10 μ g in 50 μ L). The second group was immunized with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L) by the intranasal route and a dose of AFCo1-Ova (12.5 μ g/10 μ g in 50 μ L) by the intramuscular route at a single time; and the third group was immunized by intranasal route with one dose of AFPL1-Ova (100 μ g/50 μ g in 25 μ L) and by intramuscular route with one dose of AFPL1-Ova (12.5 μ g/10 μ g in 50 μ L) at a single time. As positive controls, one group immunized with three doses (0, 7, 14 days) of AFCo1-Ova at a concentration of 50 μ g/25 μ g/animal 25 μ L (12.5 μ L/nasal fossa) by intranasal route and another group immunized with three intranasal Ova doses (25 μ g/animal 25 μ L) were used.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-Ova IgG by ELISA: the procedure was as described in example 12.

Conclusion of the results: an intramuscular AFPL1 dose combined with an intranasal AFPL1 dose at a single time or intramuscular AFCo1 combined with an intranasal AFCo1 dose at a single time (both containing Ova in their structure) induced high levels of anti-Ova IgG in serum compared to that induced by three doses of AFCo1-Ova administered by the intranasal route. However, the use of AFCo1 through both pathways induced a higher anti-OvaIgG response (fig. 16).

Example 16: the single-time vaccination strategy also included the induction of high levels of specific IgA in the feces and vaginal eluate by administering at the same time a dose of AFCo1 by other mucosal route (oral or sublingual) and AFPL1(STVS) by intramuscular route, respectively

Immunization protocol: balb/c mice were divided into six immunization groups and one control group. The first three groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L), oral route (IG) (100 μ g IN 200 μ L) or sublingual route (Sl) (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The remaining three groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal), IG (100 μ g IN 200 μ L) or Sl (50 μ g IN 25 μ L/animal), respectively.

Methods for extracting and processing fecal and vaginal eluates: extraction of feces and vaginal eluate was performed as shown in example 11.

Detection of anti-PL IgA by ELISA: the procedure was as described in example 4.

Conclusion of the results: an intramuscular AFPL1 dose (STVS) combined with an oral or sublingual AFCo1 dose at a single time induced high levels of anti-PLIgA in fecal and vaginal washings compared to that induced by three doses of AFCo1 administered orally or sublingually (figures 17 and 18).

Example 17: single-time administration of one dose of AFCo1 by oral or sublingual route and AFPL1(STVS) by intramuscular route, respectively, induced high levels of specific IgG in serum in mice at the same time

Immunization protocol: balb/c mice were divided into six immunization groups and one control group. The first three groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L), oral route (IG) (100 μ g IN 200 μ L) or sublingual route (Sl) (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route, respectively. The remaining three groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal), IG (100 μ g IN 200 μ L) or Sl (50 μ g IN 25 μ L/animal), respectively.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Detection of anti-PL IgG by ELISA: the procedure is as described in example 3

Conclusion of the results: an intramuscular AFPL1 dose (STVS) combined with an oral or sublingual AFCo1 dose at a single time induced high levels of anti-PL IgG in serum as induced by three doses of AFCo1 administered orally or sublingually (figure 19).

Example 18: in a single-time vaccination strategy, AFCo1 administered simultaneously via multiple mucosal routes and AFPL1(STVS) via intramuscular route induced high levels of specific IgA in feces in mice at the same time

Immunization protocol: balb/c mice were divided into 13 immunization groups and 1 control group. The first three groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L), oral route (IG) (100 μ g IN 200 μ L) or sublingual route (Sl) (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The other three groups were immunized at a single time with AFCo1 combining two of the aforementioned mucosal pathways and with one dose of AFPL1(12.5 μ g IN 50 μ L) by the IM pathway, IN-IG-IM, IN-Sl-IM or IG-Sl-IM. The remaining three groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal), IG (100 μ g IN 200 μ L) or Sl (50 μ g IN 25 μ L/animal), respectively.

Methods for extracting and processing fecal and vaginal eluates: extraction of feces and vaginal eluate was performed as shown in example 11.

Detection of anti-PL IgA by ELISA: the procedure was as described in example 4.

Conclusion of the results: an intramuscular AFPL1 dose (STVS) combined with an AFCo1 dose by mucosal routes induced high levels of anti-PL IgA in fecal and vaginal eluate compared to that induced by three doses of AFCo1 administered intranasally, orally or sublingually. These responses were synergistic by applying both mucosal pathways simultaneously (FIGS. 20-22).

Example 19: immunization with Tetanus Toxoid (TT), diphtheria Toxoid (TD) or cellular pertussis (P) co-administered with spirochete rolls (simultaneously with DPT vaccine added by intramuscular route (absorbed on alumina)) by intranasal, sublingual or oral routes, respectively, induced significant systemic IgG responses against all these antigens ]

Immunization protocol: balb/c mice were divided into seven immunization groups and 1 control group. The first three groups were immunized with AFCo1+ TT by intranasal route (IN) (100. mu.g/10 LF (flocculent precipitate units) IN 25. mu.L), AFCo1-TD by sublingual route (Sl) (100. mu.g/25 LF IN 25. mu.L) or AFCo1-P by oral route (IG) (100. mu.g/16 UO (turbidity units) IN 25. mu.L) and simultaneously with DPT IN Muscle (IM). Groups 4, 5 and 6 were immunized with 3 doses of AFCo1+ TT, AFCo1+ DT or AFCo1+ P (by IN, SL or 1G, respectively) at similar concentrations. The other groups were immunized with two doses of dpt (im).

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Method for extracting and processing saliva: the procedure was as described in example 4.

Detection of anti-Tetanus Toxoid (TT) IgG by ELISA:

reagents and solutions:

tetanus toxoid: ATPE-Lot 8005 Finlay Institute, c (808LF/mL)

Coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

The method comprises the following steps:

● to 96-well plates with high binding capacity (Maxisorp, Nunc, EUA) 100. mu.L/well of TT diluted in STR at a concentration of 10LF/mL was added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● serum to be evaluated was diluted 1: 100 in sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● mu.L/well of anti-IgG conjugate diluted 1: 2000 in sample dilution was added and incubated for 1 hour at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution. The absorbance in Optical Density (OD) was measured at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

The results for anti-TT IgG are expressed as Optical Density (OD). In this test, the sample is considered positive when above the control value.

Detection of anti-diphtheria Toxoid (TD) IgG by ELISA:

reagents and solutions:

diphtheria toxoid: ADPE-Lot 8001 Finlay Institute, c (1200LF/mL)

Coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping solution：2M H₂SO₄

The method comprises the following steps:

● to 96-well plates with high binding capacity (Maxisorp, Nunc, EUA) 100. mu.L/well of TD diluted in STR at a concentration of 25LF/mL was added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● serum to be evaluated was diluted 1: 100 in sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● mu.L/well of anti-IgG conjugate diluted 1: 2000 in sample dilution was added and incubated for 1 hour at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution. The absorbance in Optical Density (OD) was measured at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

The results for anti-TD IgG are expressed as Optical Density (OD). In this test, the sample is considered positive when above the control value.

Detection of anti-cellular Pertussis (PC) IgG by ELISA:

reagents and solutions:

whole cell Pertussis (PC): lot 8006 Finlay Institute, c (756UO/mL)

Coating buffer Solution (STR): 11mM Na₂CO₃，35mM NaHCO₃(pH 9.6)

Phosphate buffered saline (SSTF): 0.15M (pH 7.2)

Blocking solution (SSTF/1% SAB): 0.15M SSTF (pH 7.2), 1% (p/v) bovine serum albumin

Washing solution (SSTF, 0.1% Tween 20): SSTF, 0.1% (v/v) Tween20, pH7.4

Sample dilution solution (SSTF, 1% SAB, 0.1% Tween 20)

Peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO, EUA)

Substrate buffer solution (STS): 52mM Na₂HPO₄And 25mM citric acid (pH 5.6)

Stopping the solution: 2M H₂SO₄

The method comprises the following steps:

● to 96-well plates with high binding capacity (Maxisorp, Nunc, EUA) 100. mu.L/well of PC diluted in STR at a concentration of 16UO/mL was added and incubated overnight at 4 ℃ in a humidified chamber.

● were washed three times with SSTF.

● Add 100. mu.L/well of blocking solution and incubate for 1 hour at ambient temperature in a humidified chamber.

● are washed three times with the washing solution.

● serum to be evaluated was diluted 1: 100 in sample dilution solution. Dilutions of each serum and reference serum were applied in duplicate at 100. mu.L/well and incubated for 2 hours at 37 ℃.

● are washed three times with the washing solution.

● mu.L/well of anti-IgG conjugate diluted 1: 2000 in sample dilution was added and incubated for 1 hour at 37 ℃ in a humidified chamber.

● are washed three times with the washing solution.

● mu.L/well of a solution of 0.01% (v/v) hydrogen peroxide and 0.6mg/mL chromogen o-phenylenediamine (OPD) in STS was added and incubated for 30 minutes in the dark.

● the reaction was stopped by adding 50. mu.L of a stop solution. The absorbance in Optical Density (OD) was measured at 492nm in a microplate reader (Titertek, Multiskan Plus).

As a result: expression and calculation

The results for anti-PC IgG are expressed as Optical Density (OD). In this test, the sample is considered positive when above the control value.

To summarize:

we show that the simultaneous application of different antigens via multiple mucosal routes and the combined vaccine (DPT) via the intramuscular route in a single-time strategy is effective for inducing immune responses.

Example 20: simultaneous single dose immunization with intranasal spirochete (AFCo1) and intramuscular proteoliposomes (AFPL1) (STVS) induced a memory response that was exhibited after 4 months of immunization with one booster dose

Immunization protocol: balb/c mice were divided into two immunization groups and one control group. The first group was immunized by intramuscular route with two doses (0, 14 days) of aflp 1 at a concentration of 12.5 μ g in 50 μ L/animal. The second group was immunized by intranasal route with one dose of AFCo1(50 μ g in 25 μ L, 12.5 μ L/nasal fossa) and simultaneously by intramuscular route with AFPL1(12.5 μ g in 50 μ L) (STVS). At 120 days (4 months), challenge with AFCo1(50 μ g in 25 μ L, 12.5 μ L/fossa nasally) by intranasal route.

Method for extracting and processing blood to obtain serum: the procedure is as described in example 3, with the addition that the extractions are performed 21, 70, 90 and 120 days after the last dose administered and 21 days after challenge.

Detection of anti-PL IgG by ELISA: the procedure was as described in example 3 (fig. 23).

To summarize:

the single-time vaccination strategy induced not only a response at the effector level but also a good memory response, which was observed after administration of one booster dose at 4 months of immunization.

Example 22: simultaneous immunization with Ova-containing spirochete (AFCo1-Ova) (intranasal) and Ova-containing proteoliposomes (AFPL1-Ova) (intramuscular) (STVS) induced anti-Ova memory responses that appeared after 4 months of immunization with a booster dose of Ova

Immunization protocol: balb/c mice were divided into three immunization groups and one control group. The first group was immunized by intramuscular route with two doses (0, 14 days) of AFPL1-Ova at a concentration of 12.5 μ g/10 μ g in 50 μ L/animal. The second group was immunized (STVS) by intranasal route with one dose of AFCo1-Ova (50. mu.g/25. mu.g in 25. mu.L, 12.5. mu.L/nasal fossa) and simultaneously by intramuscular route with one dose of AFPL1 (12.5. mu.g/10. mu.g in 50. mu.L). The third group was immunized with one intramuscular Ova dose (10 μ g in 50 μ L). At 120 days (4 months), groups 1 and 2 were challenged with either 25 μ g Ova (25 μ L, 12.5 μ L/nasal fossa) contained in AFCo1 by the intranasal route or intramuscular Ova alone in group 3.

Method for extracting and processing blood to obtain serum: the procedure is as described in example 3, with the addition that the extractions are performed 21, 70, 90 and 120 days after the last dose administered and 21 days after challenge.

Detection of anti-Ova IgG by ELISA: proceed as described in example 12 (24).

To summarize:

the single-time vaccination strategy with Ova as incorporated antigen induced not only a response at the effector level, but also an anti-Ova memory response, which was observed after a booster dose of intranasal Ova administered at 4 months of immunization.

Example 23: single-time immunization strategies also work when other mucosal adjuvants are used, such as Cholera Toxin (CT) ]

Immunization protocol: balb/c mice were divided into two immunization groups and one control group. The first group was immunized by intranasal route with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L) and simultaneously by intramuscular route with a dose of AFPL1-Ova (25 μ g/10 μ g in 50 μ L). The second group was immunized with one dose of CT-Ova (5. mu.g/50. mu.g in 25. mu.L) by the intranasal route and one dose of CT-Ova (5. mu.g/10. mu.g in 50. mu.L) by the intramuscular route at the same time.

Method for extracting and processing blood to obtain serum: the procedure was as described in example 3.

Methods of extracting and processing vaginal eluate: the procedure is as described in example 11.

The method for extracting and processing the excrement comprises the following steps: the procedure is as described in example 11.

Detection of anti-Ova IgG by ELISA: the procedure was as described in example 14.

Detection of anti-Ova IgA by ELISA: the procedure is as described in example 15.

To summarize:

CT-Ova applied according to the single-time vaccine strategy induced a significant anti-OvaIgG response in serum and a significant anti-Ova IgA response in vaginal washings and faeces. Thus, with other mucosal adjuvants, the one-time immunization strategy also worked (fig. 25, 26, 27).

[ brief description of the drawings ]

FIG. 1. the intramuscular administration of AFCo1, AFPL1 or VA-MENGOC-Induced anti-PL IgG response in serum. 2 doses (0, 14 days) of AFCo1, AFPL1 or VA-MENGOC-Balb/c mice were immunized (12.5. mu.g/50. mu.L). To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 2. the intramuscular administration of AFCo1, AFPL1 or VA-MENGOC-Induced anti-PL IgA response in saliva. Two doses (0, 14 days) of AFCo1, AFPL1 or VA-MENGOC-Balb/c mice were immunized (12.5. mu.g/50. mu.L). To evaluate the level of anti-PL IgA, saliva samples extracted 7 days after the last dose of immunization were used. The determination was carried out by ELISA for anti-PLIgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicatesSignificant differences according to Tukey test (p < 0.05).

Figure 3 anti-PL IgA responses in saliva induced by AFCo1 or AFPL1 administered by the intranasal route. Balb/c mice were immunized by the intranasal route with three doses (0, 7, 14 days) of AFCo1 or AFPL 1(50 μ g in 25 μ L/animal, 12.5 μ L/nasal fossa). To evaluate the level of anti-PL IgA, saliva samples extracted 7 days after the last dose were used. Measurement was performed by ELISA for anti-PL IgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 4 anti-PL IgG responses in serum induced by AFCo1 or AFPL1 administered by intranasal route. Balb/c mice were immunized by the intranasal route with three doses (0, 7, 14 days) of AFCo1 or AFPL 1(50 μ g in 25 μ L/animal, 12.5 μ L/nasal fossa). To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 5 anti-PL IgG responses in serum induced by one and two doses of AFCo1 or AFPL1 administered by intramuscular route. Balb/c mice were immunized with 1 or 2 intramuscular doses of AFCo1 or AFPL1 (12.5. mu.g/50. mu.L). To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 6 anti-PL IgA responses in saliva induced by 1, 2 or 3 doses of AFCo1 or AFPL1 administered by the intranasal route. Balb/c mice were immunized by intranasal route with 1, 2 or 3 doses of AFCo1 or AFPL1 (50. mu.g in 25. mu.L/animal, 12.5. mu.L/nasal fossa). To evaluate the level of anti-PL IgA, saliva samples extracted 7 days after the last dose were used. Measurement was performed by ELISA for anti-PL IgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 7 anti-PL IgG responses in serum induced by 1, 2 or 3 doses of AFCo1 or AFPL1 administered by intranasal route. Balb/c mice were immunized by intranasal route with 1, 2 or 3 doses of AFCo1 or AFPL1 (50. mu.g in 25. mu.L/animal, 12.5. mu.L/nasal fossa). To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 8 anti-PL IgG responses in serum induced by single-time immunization (STVS) of intranasal AFCo1 and intramuscular AFPL 1. Balb/c mice were divided into three immunization groups and one control group. The first group was immunized by intranasal route (IN) with three doses (0, 7, 14 days) of AFCo1(50 μ g IN 25 μ L/animal, 12.5 μ L/nasal fossa). The second group was immunized by intramuscular route (IM) with two doses (0, 14 days) of AFPL1 (12.5. mu.g/50. mu.L per animal). The third group was immunized by the IM route with a dose of AFPL1 (12.5. mu.g/50. mu.L) and simultaneously by the IN route with a dose of AFCo1 (100. mu.g/25. mu.L) (STVS). To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 9 anti-PL IgG1 and IgG2a responses in sera induced by single time immunization (STVS) of intranasal AFCo1 and intramuscular AFPL 1. Balb/c mice were divided into three immunization groups and one control group. The first group was immunized by intranasal route (IN) with three doses (0, 7, 14 days) of AFCo1(50 μ g IN 25 μ L/animal, 12.5 μ L/nasal fossa). The second group was immunized by intramuscular route (IM) with two doses (0, 14 days) of AFPL1 (12.5. mu.g/50. mu.L per animal). The third group was immunized by the IM route with one dose of AFPL1 (12.5. mu.g/50. mu.L) and simultaneously by the IN route with AFCo1 (100. mu.g/25. mu.L) (STVS). To assess the level of the anti-PL IgG subclass, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA of anti-PL IgG subclass. The mean and standard deviation of the mathematical relationship of the optical density values (OD) of 2 determinations in 3 independent experiments for each formulation are shown in the figure.

FIG. 10 anti-PL IgA responses in saliva induced by single time immunization (STVS) of intranasal AFCo1 and intramuscular AFPL 1. Balb/c mice were divided into four immunization groups and one control group. The first group was immunized by intranasal route (IN) with three doses (0, 7, 14 days) of AFCo1(50 μ g/25 μ L per animal). The second group was immunized by intranasal route (IN) with one dose of AFCo1(50 μ g/25 μ L per animal); the third group was immunized by the IM route with one dose of AFPL1 (12.5. mu.g/50. mu.L) and simultaneously by the IN route with AFCo1 (100. mu.g/25. mu.L, 12.5. mu.L/nasal fossa) (STVS); and the fourth group was immunized by intramuscular route (IM) with two doses (0, 14 days) of aflp 1(12.5 μ g/50 μ L per animal). To assess the level of anti-PL IgA, saliva samples taken 7 and 14 days after the last dose were used. Measurement was performed by ELISA for anti-PL IgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) of the saliva extracted at 7 days for the AFCo1, AFPL1 group and at 14 days for the STVS group are shown in the figure, among the 3 independent experiments in which the response was maximal. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 11 anti-PL IgA response in feces induced by simultaneous immunization (STVS) of intramuscular AFPL1 and intranasal AFCo 1. Balb/c mice were divided into three immunization groups and one control group. Immunisation of the first group with one dose (12.5. mu.g/50. mu.L) of Intramuscular (IM) AFPL1 and Intranasal (IN) AFCo1 (100. mu.g/25. mu.L) at a single time; and a second group was immunized by the IN route with three doses (0, 7, 14 days) of AFCo1(50 μ g/25 μ L per animal). To evaluate the level of anti-PLIgA, fecal samples taken 14 days after the last dose of immunization were used. The assay was performed by ELISA for IgA to PL. The mean and standard deviation of the mathematical relationship of the values (UA/mL) of the feces extracted at 14 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 12 anti-PL IgA responses in vaginal eluates induced by simultaneous immunization (STVS) of intramuscular AFPL1 and intranasal AFCo 1. Balb/c mice were divided into three immunization groups and one control group. Immunisation of the first group with one dose (12.5. mu.g/50. mu.L) of Intramuscular (IM) AFPL1 and Intranasal (IN) AFCo1 (100. mu.g/25. mu.L) at a single time; and a second group was immunized by the IN route with three doses (0, 7, 14 days) of AFCo1(50 μ g/25 μ L per animal). To assess IgA levels against PL, vaginal eluate samples taken 21 days after the last dose of immunization were used. Measurement was performed by ELISA for anti-PL IgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) of the extracted vaginal eluate at 21 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 13 anti-Ova IgG responses in serum induced by single-time immunization (STVS) of intranasal AFCo1-Ova and intramuscular AFPL 1-Ova. Balb/c mice were divided into four immunization groups and one control group. The first group was immunized by the intranasal route (IN) with three doses (0, 7, 14 days) of AFCo1-Ova (50 μ g/25 μ L/animal, 12.5 μ L/nasal fossa). The second group was immunized by intramuscular route (IM) with two doses (0, 14 days) of AFPL1-Ova (12.5. mu.g/10. mu.g/50. mu.L per animal). The third group was immunized by the IM route with a dose of AFPL1-Ova (12.5. mu.g/10. mu.g/50. mu.L) and simultaneously by the IN route with a dose of AFCo1-Ova (100. mu.g/50. mu.g/25. mu.L) (STVS). The fourth group was immunized by intramuscular route with 2 doses of Ova (10. mu.g/50. mu.L per animal). To evaluate the level of anti-Ova IgG, serum samples taken 21 days after the last dose were used. The determination was carried out by ELISA for anti-Ova IgG. The mean and standard deviation of the mathematical relationship of the values (OD) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 14 anti-Ova IgA response in saliva induced by single time immunization (STVS) of intranasal AFCo1 and intramuscular AFPL 1. Balb/c mice were divided into four immunization groups and one control group. The first group was immunized by the intranasal route (IN) with three doses (0, 7, 14 days) of AFCo1-Ova (50 μ g/25 μ L/animal, 12.5 μ L/nasal fossa). The second group was immunized by intramuscular route (IM) with two doses (0, 14 days) of AFPL1-Ova (12.5. mu.g/10. mu.g/50. mu.L per animal). The third group was immunized by the IM route with a dose of AFPL1-Ova (12.5. mu.g/10. mu.g/50. mu.L) and simultaneously by the IN route with a dose of AFCo1-Ova (100. mu.g/50. mu.g/25. mu.L) (STVS). The fourth group was immunized by intranasal route with 3 doses of Ova (25 μ g/25 μ L per animal). To evaluate the level of anti-OvaIgA, saliva samples extracted 7 days after the last dose were used. The determination was carried out by ELISA for anti-OvaIgA. The mean and standard deviation of the mathematical relationship of the values (OD) of the extracted saliva at 7 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 15 anti-PL IgG responses induced by simultaneous immunization (homologous STVS) of intramuscular AFCo1 and intranasal AFCo1 at single time or intramuscular AFPL1 and intranasal AFPL1 at single time. Balb/c mice were divided into four immunization groups and one control group. The first group was immunized at a single time with one dose of AFCo1(100 μ g IN 25 μ L) by the intranasal route (IN) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM); the second group was immunized with one dose of AFCo1(100 μ g IN 25 μ L) by the IN route and one dose of AFCo 1(12.5 μ g IN 50 μ L) by the IM route at a single time; and a third group was immunized at a single time with one dose of AFPL 1(100 μ g IN 25 μ L) by the IN route and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route. As a positive control, one group was immunized by the IN route with three doses (0, 7, 14 days) of AFCo1 at a concentration of 50 μ g/animal 25 μ L (12.5 μ L/nasal fossa). To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 16 anti-Ova IgG responses induced by simultaneous immunization with intramuscular AFCo1-Ova and intranasal AFCo1-Ova at single time or with intramuscular AFPL1-Ova and intranasal AFPL1-Ova at single time (homologous STVS). Balb/c mice were divided into six immunization groups and one control group. The first group was immunized at a single time with a dose of AFCo1-Ova (100 μ g/50 μ g IN 25 μ L) by the intranasal route (IN) and a dose of AFPL1-Ova (12.5 μ g/10 μ g IN 50 μ L) by the intramuscular route (IM). The second and third groups were immunized with AFCo1-Ova by the IN and IM routes and AFPL1-Ova by the IN and IM routes, respectively, at the same concentrations as the first group. The fourth group was immunized by the IN route with three doses of AFCo1-Ova (50 μ g/25 μ g IN 25 μ L); and the remaining two groups were immunized with three and two doses of Ova by IN and IM routes, respectively. To evaluate the level of anti-Ova IgG, serum samples taken 21 days after the last dose were used. The determination was carried out by ELISA for anti-Ova IgG. The mean and standard deviation of the mathematical relationship of the values (OD) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 17 IgA responses in stool induced by single-time administration (STVS) of one dose of AFCo1 by oral or sublingual route and AFPL1 by intramuscular route, respectively, at the same time. Balb/c mice were divided into six immunization groups and one control group. The first three groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L), oral route (IG) (100 μ g IN 200 μ L) or sublingual route (Sl) (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The remaining three groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal), IG (100 μ g IN 200 μ L) or Sl (50 μ g IN 25 μ L/animal), respectively. To assess the level of anti-PL IgA, fecal samples taken 14 days after the last dose were used. Measurement was performed by ELISA for anti-PL IgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 18 IgA responses in vaginal eluates induced by single-time administration (STVS) of one dose of AFCo1 by oral or sublingual route and AFPL1 by intramuscular route, respectively, at the same time. Balb/c mice were divided into six immunization groups and one control group. The first three groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L), oral route (IG) (100 μ g IN 200 μ L) or sublingual route (Sl) (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The remaining three groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal), IG (100 μ g IN 200 μ L) or Sl (50 μ g IN 25 μ L/animal), respectively. To assess the level of anti-PL IgA, samples of vaginal eluate taken 21 days after the last dose were used. Measurement was performed by ELISA for anti-PL IgA. The mean and standard deviation of the mathematical relationship of the values (UA/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 19 IgG responses in serum induced by single-time administration (STVS) of one dose of AFCo1 by oral or sublingual route and AFPL1 by intramuscular route at the same time. Balb/c mice were divided into six immunization groups and one control group. The first three groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L), oral route (IG) (100 μ g IN 200 μ L) or sublingual route (Sl) (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The remaining three groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal), IG (100 μ g IN 200 μ L) or Sl (50 μ g IN 25 μ L/animal), respectively. To assess the level of anti-PL IgG, serum samples taken 21 days after the last dose were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (U/mL) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 20 anti-PL IgA responses in feces induced by simultaneous immunization (STVS) with intramuscular AFPL1 and intranasal-oral combination AFCo1 at a single time. Balb/c mice were divided into five immunization groups and one control group. The first two groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L) or the oral route (IG) (100 μ g IN 200 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The other groups were immunized IN combination at a single time with one dose of AFCo1 by the IN route (100 μ g IN 25 μ L) and IG route (100 μ g IN 200 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the IM route. The remaining two groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal) or IG (100 μ g IN 200 μ L), respectively. To assess IgA levels against PL, fecal samples taken 14 days after the last dose of immunization were used. The assay was performed by ELISA for IgA to PL. The mean and standard deviation of the mathematical relationship of the values of IgA (UA/mL) in feces extracted at 21 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 21 anti-PL IgA responses in feces induced by simultaneous immunization (STVS) with intramuscular AFPL1 and intranasal-sublingual combined AFCo1 at a single time. Balb/c mice were divided into five immunization groups and one control group. The first two groups were immunized at a single time with one dose of AFCo1 by the intranasal route (IN) (100 μ g IN 25 μ L) or the sublingual route (Sl) (100 μ g IN 200 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the intramuscular route (IM), respectively. The other groups were immunized IN combination at a single time with one dose of AFCo1 by the IN route (100 μ g IN 25 μ L) and the Sl route (100 μ g IN 25 μ L) and one dose of AFPL1(12.5 μ g IN 50 μ L) by the IM route. The remaining two groups were immunized with three doses (0, 7, 14 days) of AFCo1 by IN (50 μ g IN 25 μ L/animal) or sublingually (50 μ g IN 25 μ L), respectively. To assess IgA levels against PL, fecal samples taken 14 days after the last dose of immunization were used. The assay was performed by ELISA for IgA to PL. The mean and standard deviation of the mathematical relationship of the values of IgA (UA/mL) in feces extracted at 21 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 22 anti-PL IgA responses in feces induced by simultaneous immunization (STVS) with intramuscular AFPL1 and oral-sublingual combined AFCo1 at a single time. Balb/c mice were divided into five immunization groups and one control group. The first two groups were immunized at a single time with one dose of AFCo1 by the oral route (IG) (100 μ g in 200 μ L) or the sublingual route (Sl) (100 μ g in 25 μ L) and one dose of AFPL1(12.5 μ g in 50 μ L) by the intramuscular route (IM), respectively. The other groups were immunized in combination at a single time with one dose of AFCo1 by the oral route (IG) (100 μ g in 200 μ L) and the sublingual route (Sl) (100 μ g in 25 μ L) and one dose of AFPL1(12.5 μ g in 50 μ L) by the IM route. The remaining two groups were immunized with three doses (0, 7, 14 days) of AFCo1 by Sl (50 μ g in 25 μ L/animal) or IG (100 μ g in 200 μ L). To evaluate the level of anti-PL IgA, fecal samples taken 14 days after the last dose of immunization were used. The assay was performed by ELISA for IgA to PL. The mean and standard deviation of the mathematical relationship of the values of IgA (UA/mL) in feces extracted at 21 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 23 anti-PL memory responses induced by a single dose of simultaneous immunization with intranasal AFCo1 and intramuscular AFPL1 (STVS). Balb/c mice were divided into two immunization groups and one control group. The first group was immunized by intramuscular route (IM) with two doses (0, 14 days) of AFPL1 at a concentration of 12.5 μ g in 50 μ L/animal. The second group was immunized by intranasal route (IN) with one dose of AFCo1(50 μ g IN 25 μ L, 12.5 μ L/nasal fossa) and simultaneously by intramuscular route (IM) with one dose of AFPL1(12.5 μ g IN 50 μ L) (STVS). At 120 days (4 months), challenge was performed by intranasal route with 50 μ g of each antigen in 25 μ L (12.5 μ L/fossa). To assess the level of anti-PL IgG, serum samples taken 21, 70, 90 and 120 days after the last immunization dose and 14 and 21 days after challenge were used. The assay was performed by ELISA for anti-PL IgG. The mean and standard deviation of the mathematical relationship of the values (OD) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 24 anti-Ova memory responses induced by simultaneous immunization (STVS) of intranasal AFCo1-Ova and intramuscular AFPL 1-Ova. Balb/c mice were divided into three immunization groups and one control group. The first group was immunized by intramuscular route (IM) with two doses (0, 14 days) of AFPL1-Ova at a concentration of 12.5 μ g/10 μ g in 50 μ L/animal. The second group was immunized by the intranasal route (IN) with one dose of AFCo1-Ova (50. mu.g/25. mu.g IN 25. mu.L, 12.5. mu.L/nasal fossa) and simultaneously by the IM route with one dose of AFPL1 (12.5. mu.g/10. mu.g IN 50. mu.L) (STVS). The third group was immunized by the IM route with two doses of Ova (10 μ g in 50 μ L). At 120 days (4 months), challenge with AFCo1-Ova in 25 μ L (50 μ g/25 μ g) (12.5 μ L/nasal fossa) by intranasal route. To assess the level of anti-PL IgG, serum samples taken 21, 70, 90 and 120 days after the last immunization dose and 14 and 21 days after challenge were used. The determination was carried out by ELISA for anti-Ova IgG. The mean and standard deviation of the mathematical relationship of the values (OD) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 25 anti-Ova IgG response in serum induced by single-time administration (STVS) of one dose of CT-Ova via the intranasal route and CT-Ova via the intramuscular route, respectively, at the same time. Balb/c mice were divided into two immunization groups and one control group. The first group was immunized by intranasal route with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L) and simultaneously by intramuscular route with a dose of AFPL1-Ova (25 μ g/10 μ g in 50 μ L). The second group was immunized with one dose of CT-Ova (5. mu.g/50. mu.g in 25. mu.L) by the intranasal route and one dose of CT-Ova (5. mu.g/10. mu.g in 50. mu.L) by the intramuscular route at the same time. To evaluate the level of anti-Ova IgG, serum samples taken 21 days after the last dose were used. The determination was carried out by ELISA for anti-Ova IgG. The mean and standard deviation of the mathematical relationship of the values (OD) determined 2 times in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Figure 26 anti-Ova IgA response in vaginal eluates induced by single time administration (STVS) of one dose each of CT-Ova by intranasal route and CT-Ova by intramuscular route at the same time. Balb/c mice were divided into two immunization groups and one control group. The first group was immunized by intranasal route with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L) and simultaneously by intramuscular route with a dose of AFPL1-Ova (25 μ g/10 μ g in 50 μ L). The second group was immunized with one dose of CT-Ova (5. mu.g/50. mu.g in 25. mu.L) by the intranasal route and one dose of CT-Ova (5. mu.g/10. mu.g in 50. mu.L) by the intramuscular route at the same time. To assess the level of anti-Ova IgA, samples of vaginal eluate taken 21 days after the last dose were used. The determination was carried out by ELISA for anti-Ova IgA. The mean and standard deviation of the mathematical relationship of the values (OD) of the extracted vaginal eluate at 21 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

FIG. 27 anti-Ova IgA response in stool induced by single-time administration (STVS) of one dose of CT-Ova by the intranasal route and CT-Ova by the intramuscular route, respectively, at the same time. Balb/c mice were divided into two immunization groups and one control group. The first group was immunized by intranasal route with a dose of AFCo1-Ova (100 μ g/50 μ g in 25 μ L) and simultaneously by intramuscular route with a dose of AFPL1-Ova (25 μ g/10 μ g in 50 μ L). The second group was immunized with one dose of CT-Ova (5. mu.g/50. mu.g in 25. mu.L) by the intranasal route and one dose of CT-Ova (5. mu.g/10. mu.g in 50. mu.L) by the intramuscular route at the same time. To evaluate the level of anti-Ova IgA, stool samples taken 14 days after the last dose were used. The determination was carried out by ELISA for anti-Ova IgA. The mean and standard deviation of the mathematical relationship of the values (OD) of the feces extracted at 14 days in 3 independent experiments are shown in the figure. Different p indicates significant differences according to Tukey's test (p < 0.05).

Claims (28)

1. Vaccine formulation, characterized in that it comprises proteoliposomes or derivatives thereof applied simultaneously by one or more routes of administration.

2. Vaccine formulation according to claim 1, characterized in that the proteoliposomes and derivatives thereof are administered simultaneously by mucosal and parenteral routes.

3. Vaccine formulation according to claim 2, characterized in that mucosal administration can be achieved by various means.

4. Vaccine formulation according to claims 1 and 2, characterized in that the administration can be carried out simultaneously in one of the following combinations: a + B, B + C, A + D or C + D:

a: proteoliposomes, via the mucosal route;

b: proteoliposomes, by parenteral route;

c: proteoliposome derivatives, by mucosal route;

d: proteoliposome derivatives, by parenteral route.

5. Vaccine formulation according to claims 2 to 4, characterized in that mucosal administration can be by nasal, oral, sublingual, vaginal and/or rectal route.

6. Vaccine formulation according to claims 2 and 4, characterized in that parenteral administration can be by intramuscular, intradermal, subcutaneous or transdermal route.

7. Vaccine formulation according to claim 1, characterized in that the proteoliposomes and derivatives thereof act as adjuvants for self or heterologous antigens.

8. Vaccine formulation according to claim 1, characterized in that the proteoliposome derivative may be in the form of a spiral coil.

9. Vaccine formulations for single use, wherein other mucosal adjuvants are used.

10. A vaccine formulation for single use, wherein parenteral use can be extended by the addition of other delivery systems.

11. Use of a vaccine formulation in a single-time form by two or more routes of administration.

12. Use of a vaccine formulation according to claim 11 comprising the simultaneous application of proteoliposomes and derivatives thereof in a single time form by two or more routes of administration.

13. Use of a vaccine formulation according to claims 11 and 12 comprising the simultaneous administration of proteoliposomes and derivatives thereof by mucosal and parenteral routes.

14. Use of a vaccine formulation according to claim 13, characterized in that mucosal administration can be achieved by various means.

15. Use of a vaccine formulation according to claim 12, characterized in that the administration can be carried out simultaneously in one of the following combinations: a + B, B + C, A + D or C + D:

a: proteoliposomes, via the mucosal route;

b: proteoliposomes, by parenteral route;

c: proteoliposome derivatives, by mucosal route;

d: proteoliposome derivatives, by parenteral route.

16. Use of a vaccine formulation according to claims 13 to 15, characterized in that mucosal administration can be by nasal, oral, sublingual, vaginal and/or rectal route.

17. Use of a vaccine formulation according to claims 13 and 15, characterized in that the parenteral administration can be by intramuscular, intradermal, subcutaneous or transdermal route.

18. Use of a vaccine formulation according to claims 11-16 for the prevention and treatment of infections or neoplastic diseases.

19. Use of a vaccine formulation according to claim 11, wherein the different types of antigens are applied simultaneously via one or more mucosal routes and the corresponding combination vaccine via parenteral route.

20. A method of treatment characterised in that the vaccine formulation is applied in a single occasion by two or more routes of administration.

21. A method of treatment according to claim 20 which comprises the simultaneous application of proteoliposomes and derivatives thereof by two or more routes of administration in a single time form.

22. A method of treatment according to claims 20 and 21 comprising the simultaneous application of proteoliposomes and derivatives thereof by mucosal and parenteral routes.

23. The method of treatment according to claim 22, characterized in that mucosal administration can be achieved by various means.

24. Treatment according to claims 21 and 22, characterized in that the administration can be carried out simultaneously in one of the following combinations: a + B, B + C, A + D or C + D:

a: proteoliposomes, via the mucosal route;

b: proteoliposomes, by parenteral route;

c: proteoliposome derivatives, by mucosal route;

d: proteoliposome derivatives, by parenteral route.

25. A method of treatment according to claims 22 to 24, wherein mucosal administration is by nasal, oral, sublingual, vaginal and/or rectal route.

26. The method of treatment according to claims 22 and 24, characterized in that the parenteral administration can be by intramuscular, intradermal, subcutaneous or transdermal route.

27. A method of treatment according to claims 20 to 26 for the prevention and treatment of infections and neoplastic diseases.

28. The method of treatment according to claim 20, wherein the different types of antigens are applied by one or more mucosal routes simultaneously with the corresponding combination vaccine by parenteral route.