ES2391943T3 - Non-invasive genetic immunization, its expression products and its uses - Google Patents

Abstract

The use of an adenoviral vector for the preparation of a non-invasive vaccine for mucosal administration, in which the vector contains and expresses an exogenous nucleic acid encoding a lagripe hemagglutinin antigen or an epitope of this and / or an antigen of the nuclear flu protein or an epitope of this, and the vector has regions E1 and E3 selected, and in which the non-invasive vaccine for mucosal administration is to be administered to the nasal mucosa.

Description

Non-invasive genetic immunization, its expression products and its uses.

Field of the Invention

The present invention relates, in general, to the fields of immunology and vaccine technology. The present invention also relates to non-invasive gene delivery techniques for eliciting immune responses by administration to the nasal mucosa, and its uses.

Background of the invention

The activation of the vertebrate immune system is an important mechanism to protect animals against pathogens and malignant tumors. The immune system consists of many components that interact with each other, and includes the humoral and cellular sections. Humoral immunity involves antibodies that bind directly to antigens. Antibody molecules as effectors of humoral immunity are secreted by B lymphocytes. Cellular immunity involves specialized cytotoxic C lymphocytes (CTLs) that recognize and kill other cells that produce foreign antigens. CTLs respond to degraded peptide fragments that appear on the surface of the target cell bound to MHC (major histocompatibility complex) class I molecules. It is known that proteins produced within the cell are continuously degraded. to produce peptides as part of cellular metabolism. These fragments bind to the MHC molecules and are transported to the cell surface. Therefore, the immune system is constantly controlling the range of proteins produced in all body cells and is ready to eliminate any cell that produces foreign antigens.

Vaccination is the process of priming an animal to respond to an antigen. The antigen can be administered as a protein (the classical form) or as a gene that then expresses the antigen (genetic immunization). The process involves T and B lymphocytes, other types of lymphoid cells, as well as specialized antigen presentation (APC) cells that can process the antigen and present it in a way that can activate the immune system. Current modes of administration of genetic vaccines have focused on invasive procedures, including needle injections, scarification, and gene-mediated piston penetration. Vaccine inoculation in an invasive mode requires equipment and personnel with special medical training, and is usually associated with potential discomfort and danger (bleeding, infection).

The efficacy of a vaccine is measured by the degree of protection against subsequent exposure to a pathogen or tumor. Effective vaccines are immunogens that can induce long-lasting protective immunity and high titration for disease-directed intervention after a minimum number of inoculations. For example, genetic immunization is a strategy to elicit immune responses against specific proteins by expressing genes that encode proteins in an animal's own cells. Substantial antigen amplification and immune stimulation resulting from prolonged antigen presentation in vivo can induce strong immunity against the antigen. Genetic immunization simplifies the vaccination protocol to produce immune responses against specific proteins because the often difficult stages of protein purification and combination with adjuvants are eliminated, both of which are usually necessary for vaccine development. Since genetic immunization does not require protein isolation, it is especially valuable for proteins that can lose conformational epitopes when they are purified biochemically. Genetic vaccines can also be administered in combination without causing interference or affecting efficacy (Tang et al., 1992; Barry et al., 1995), which can simplify the vaccination program against multiple antigens.

Although vaccines based on topically applied proteins have been studied, their usefulness may be limited. Although the topical application of protein-based vaccines together with cholera toxin can also immunize animals in a non-invasive manner (Glenn et al., 1998), non-invasive genetic vaccines targeting the skin, as described herein. The invention activates the immune system through a different mechanism than protein-based vaccines. In addition, the efficacy of genetic vaccines in general is greater than that of protein vaccines, due to de novo synthesis of antigens similar to natural infections (McDonnell and Askari, 1996). Although US Patent No. 3,837,340 refers to a method for vaccinating animals by contacting the skin with dried viruses, the viruses used therein are not genetic vectors capable of expressing heterologous or exogenous nucleic acid transgenes or molecules. In addition, the immunogen can be a viral envelope protein, rather than a protein produced from the expression of viral genes within the animal's own cells, for example, any immune response induced by US Pat. No.

3,837,340 may be similar to that induced by topical application of protein-based vaccines that are not analogous to those of the present invention, and therefore US Patent No. 3,837,340 is not analogous to the present invention.

The prior art of vaccination usually requires equipment, for example, syringe needles or a gene gun, and special knowledge for the administration of vaccines. It is very necessary and desirable in the art to have staff without medical training and not need equipment for vaccine inoculation.

WO 99/08713, published on February 25, 1995, describes a method for inducing an immune response in a non-invasive manner, comprising the step of contacting the skin of an individual in need of such treatment by applying topically to said skin an immunologically effective concentration of a genetic vector encoding a gene of interest.

Shi et al. (in the article entitled "DNA-based non-invasive vaccination onto the skin", Vaccine, vol. 17, no. 17, March 23, 1999, pp. 2136-2141) indicate that a non-invasive vaccination on the skin (NIVS ) It could improve vaccination programs because the procedure does not require trained personnel and can eliminate many of the problems associated with needle injections. In addition, Shi et al. indicate that the results obtained can provide a preliminary efficacy study indicating that the NIVS may be a new method for the administration of DNA-based vaccines.

Papp et al. (in the article entitled "Induction of immunity in the respiratory tract and protection from bovine herpesvirus type I infection by different routes of immunization with recombinant adenovirus", Immunology, vol. 11, No. 2, 1998, pp. 79-91) describe a investigation of the ability of different immunization pathways with adenoviral recombinants competent in replication to induce antigen-specific antibody responses.

Objects and summary of the invention

A non-invasive vaccination on the skin (NIVS) can improve vaccination programs, because the skin is an immunocompetent tissue and this non-invasive procedure does not require personnel with specialized training. The administration of non-invasive genes directed to the skin can achieve the expression of localized transgenes in the skin and can cause immunological responses (Tang et al., 1997), and the mechanism for these responses is different from that of the topical application of vaccines based on in proteins together with cholera toxin (Glenn et al., 1998). These results indicate that vector-based NIVS is a new and effective method for vaccine administration. The simple, effective, cheap and painless immunization protocol described in the present invention should make vaccination less dependent on medical resources and, therefore, will increase the annual utilization rate of vaccinations.

The present invention provides the use of an adenoviral vector as defined in independent claim 1. The invention also provides an adenoviral vector for use as defined in independent claim 11.

Furthermore, the invention provides a composition, specifically a non-invasive vaccine for mucosal administration, for use as defined in independent claim 21.

The invention also includes a kit as defined in independent claim 29. This kit comprises the vector and a pharmaceutically acceptable or suitable carrier or diluent, and an optional delivery device, each in its own package; the container may be included in a unit container, or the containers may each be in separate containers or each may be its own separate container; The kit may optionally include instructions for mixing the ingredients and / or administering the composition.

Spill and spray formulations are described in US Patent Nos. 6,010,710 and 5,475,005. A ball cap applicator device is also described in US Patent No. 5,897,267. In addition, those skilled in the art will also know how to make a shampoo formulation, as well as devices for applying the formulation to an animal.

Preferred embodiments are mentioned in the dependent claims.

It is noted that, in this description, terms and expressions such as "comprises", "comprising" and the like may have the meaning ascribed in US patent law, for example, may mean "includes", "which includes ”and the like.

These and other embodiments are described or obvious from the following detailed description and are included therein.

Brief description of the figures

The following detailed description, which is offered as an example but is not intended to limit the invention to the specific embodiments described therein, can be understood together with the attached figures, incorporated herein by reference, in which:

Figure 1 shows the expression of trangenes from adenoviral recombinants in the skin by the topical application of the vectors;

Figures 2a and 2b show the characterization of potential target cells that can be transduced by adenoviral recombinants applied topically;

Figures 3a and 3b show the detection of specific antibodies in the serum of mice immunized by an adenovirus-mediated NIVS;

Figure 4 shows the survival percentage of control mice against immunized mice that were exposed to a lethal dose of tumor cells;

Figure 5 shows the characterization of tumor infiltration T lymphocytes;

Figure 6 shows the CTL characterization of tumor infiltration;

Figure 7 shows the analysis of Western blotting of antibodies against the human CEA protein in mice immunized by topical application of vaccine bandages;

Figure 8a shows the detection of specific antibodies in the serum of a mouse immunized by a DNA-mediated NIVS / adenovirus;

Figure 8b shows the detection of specific antibodies in the serum of a mouse immunized by a NIVS mediated by DNA / liposomes;

Figure 9 shows the coexpression of DNA-encoded and adenovirus-encoded transgenes in target cells;

Figure 10 shows the expression of relative transgenes from adenoviral recombinants applied topically, DNA / adenovirus complexes; and DNA / liposome complexes;

Figure 11 shows a device for the administration of non-invasive vaccines directed to the skin;

Figure 12 shows anti-influenza antibodies generated by non-invasive vaccines directed to the skin in mice;

Figure 13 shows the protection of mice against death after exposure to viruses;

Figure 14 shows ELISA antibodies generated in a pigtail macaque by means of a dermal patch containing an adenoviral vector encoding influenza HA;

Figure 15 shows the transfer of antigen spots on the skin after topical application of an adenoviral vector;

Figure 16 shows the amplification of foreign DNA in various tissues after localized gene administration in a non-invasive mode;

Figure 17 demonstrates that a hair removal agent, such as NAIR, is not fundamental to the NIVS;

Figure 18 shows protection against death after an exposure of Clostridium tetani by topical application or intranasal inoculation of a tetanus vaccine based on adenovirus.

Detailed description

Vaccine inoculation in a non-invasive mode may be unnecessary (Tang et al., 1997; Glenn et al., 1998). Since the skin forms an interface directly with the external environment and is in constant contact with potential pathogens, the immune system must constantly keep a biological army mobilized along the skin's border to reject potential infections. As a consequence, the outer layer of the skin is essentially an immunocompetent tissue. Immune components present in the skin to elicit cytotoxic and humoral cellular immune responses include epidermal Langerhans cells (which are MHC class II positive antigen presenting cells), keratinocytes, and CD4 + and CD8 + T lymphocytes. These components make the skin an ideal site for vaccine administration. The huge accessible area of the skin and its durability are other advantages to apply vaccines to this tissue. The expression of a small number of antigens in the outer layer of the skin without physical penetration can therefore cause a potent immune response by alerting the immune surveillance mechanism.

It is hereby demonstrated that genetic vaccines can be inoculated in a new way in the form of a non-invasive skin-directed vaccine, or immunogenic, immunological or therapeutic compositions. The combination of genetic vaccines with a non-invasive mode of administration produces a new class of "democratic" vaccines, or immunogenic, immunological or therapeutic compositions that may require little or no special knowledge or equipment for administration. Therefore, an individual can administer these compositions on their own skin (and this administration, advantageously, may be supervised by a physician, for example to ensure that the dosage is appropriate) or on the skin of animals (e.g., advantageously a shaved area of the skin if the animal is a mammal, although as demonstrated herein, it is not necessary to remove the hair, and more advantageously in a region where the animal cannot eliminate administration by rubbing, grooming or other activity); and thus, the present invention describes advantages in the administration of vaccines, or immunogenic, immunological or therapeutic compositions, which comprise a vector that expresses a gene product, especially with regard to the administration of said compounds to newborns, young animals, animals in general, boys and girls and the like, in which invasive administration, for example with a needle, can be somewhat difficult, bothersome or painful.

As used herein, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. As an example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (for example a segment of heterologous DNA, such as a segment of heterologous cDNA), to be transferred to a target cell.

As used herein, "AdCMV-tetC: IM" represents an adenoviral vector encoding the C fragment of the Clostridium tetani toxin; "PCMV-tetC" represents a plasmid expression vector encoding the C fragment of the Clostridium tetani toxin.

Reference is made to US Patent No. 5,990,091, issued November 23, 1999, Einat et al. or Quark Biotech, Inc., document WO 99/60164, published on November 25, 1999 of document PCT / US99 / 11066, filed on May 14, 1999, Fischer or Rhone Merieux, Inc., document WO 98/00166, published on January 8, 1998 of document PCT / US97 / 11486, filed on June 30, 1997 (which claims the priority of US applications serial number 08 / 675,556 and 08 / 675,566), van Ginkel et al., J. Immunol., 159 (2): 685-693 (1997) ("Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene"), Osterhaus et al., Immunobiology, 184 (2 -3): 180-192 (1992) (”Vaccination against acute respiratory virus infections and measles in man”), Briles et al. or UAB, WO 99/53940, published October 28, 1999, of PCT / US99 / 08895, filed April 23, 1999, and Briles et al. or UAB, US Patent No. 6,042,838, issued March 28, 2000, and Briles et al. or UAB, US Patent No. 6,004,802, for information about expressed gene products, antibodies and their uses, vectors for in vivo and in vitro expression of exogenous nucleic acid molecules, promoters to direct expression or to operably bind to Nucleic acid molecules to be expressed, methods and documents for producing these vectors, compositions comprising said vectors or nucleic acid molecules or antibodies, dosages, and modes and / or routes of administration (including compositions for mucosal, nasal administration , oral, to the oral, oral, perlingual cavity); and, therefore, US Patent No. 5,990,091, issued November 23, 1999, Einat et al. or Quark Biotech, Inc., document WO 99/60164, published on November 25, 1999 of document PCT / US99 / 11066, filed on May 14, 1999, Fischer or Rhone Merieux, Inc., document WO 98/00166, published on January 8, 1998 of document PCT / US97 / 11486, filed on June 30, 1997 (which claims the priority of US applications serial number 08 / 675,556 and 08 / 675,566), van Ginkel et al., J. Immunol., 159 (2): 685-693 (1997) ("Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene"), Osterhaus et al., Immunobiology, 184 (2 -3): 180-192 (1992) (”Vaccination against acute respiratory virus infections and measles in man”), Briles et al. or UAB, WO 99/53940, published October 28, 1999, of PCT / US99 / 08895, filed April 23, 1999, and Briles et al. or UAB, US Patent No. 6,042,838, issued March 28, 2000, and Briles et al. or UAB, US Patent No. 6,004,802, and all documents cited or referenced therein, and all documents cited or referenced in documents referenced or cited in each of US Patent No. 5,990,091, issued on 23 November 1999, Einat et al. or Quark Biotech, Inc., document WO 99/60164, published on November 25, 1999 of document PCT / US99 / 11066, filed on May 14, 1999, Fischer or Rhone Merieux, Inc., document WO 98/00166, published on January 8, 1998 of document PCT / US97 / 11486, filed on June 30, 1997 (which claims the priority of US applications serial number 08 / 675,556 and 08 / 675,566), van Ginkel et al., J. Immunol., 159 (2): 685-693 (1997) ("Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene"), Osterhaus et al., Immunobiology, 184 (2 -3): 180-192 (1992) (”Vaccination against acute respiratory virus infections and measles in man”), Briles et al. or UAB, WO 99/53940, published October 28, 1999, of PCT / US99 / 08895, filed April 23, 1999, and Briles et al. or UAB, US Patent No. 6,042,838, issued March 28, 2000, and Briles et al. or UAB, US Patent No. 6,004,802. The information in US Patent No. 5,990,091, issued November 23, 1999, WO 99/60164, WO 98/00166, van Ginkel et al., J. Immunol., 159 (2): 685- 693 (1997), Osterhaus et al., Immunobiology, 184 (2-3): 180-192 (1992), WO 99/53940 and US Pat. Nos.

6,042,838 and 6,004,802 are reliable for the practice of this description (eg, expressed products, antibodies and their uses, vectors for in vivo and in vitro expression of exogenous nucleic acid molecules, exogenous nucleic acid molecules encoding epitopes of interest or antigens or therapeutic products and the like, promoters, compositions comprising said vectors or molecules of nucleic acids or expressed products or antibodies, dosages, among others). It is noted that immunological products and / or antibodies and / or expressed products obtained according to this invention can be expressed in vitro and used in a manner in which said immunological and / or expressed products and / or antibodies are generally used, and that the cells which express said immunological and / or expressed products and / or antibodies can be used in vitro and / or ex vivo applications, for example, these uses and applications may include diagnostics, assays, ex vivo therapy (for example, in which cells expressing the gene product and / or an immune response is expanded in vitro and reintroduced into the host or animal), etc .; see U.S. Patent 5,990,091, WO 99/60164, WO 98/00166, WO 99/53940 and U.S. Patent Nos. 6,042,838 and 6,004,802, and the documents cited therein and the documents cited or referenced in said documents. In addition, the expressed antibodies or gene products that are isolated from the present methods, or that are isolated from expanded cells in vitro after the methods of administration herein, can be administered in compositions, similar to the administration of epitope subunits or antigens or therapeutic products or antibodies to induce immunity, stimulate a therapeutic response and / or stimulate passive immunity. The amount to be administered will vary depending on the patient (host) and the disorder being treated, and will vary from one or a few micrograms, or a few hundred or thousands of micrograms, for example, from 1 µg to 1 mg , from about 100 ng / kg body weight to 100 mg / kg body weight daily, and preferably will be about 10 bp / kg to 10 mg / kg daily. A vector can be administered non-invasively to a patient or host in an amount to achieve the amounts indicated for the compositions of gene products (eg, epitope, antigen, therapeutic product and / or antibody). Of course, the invention contemplates lower and higher dosages than those exemplified herein, and for any composition to be administered to an animal or human being, including its components, and for any particular method of administration, it is preferred to determine for them : toxicity, such as determining the lethal dose (DL) and LD50 in a suitable animal model, for example, a rodent, such as a mouse, and the dosage of the composition or compositions, the concentration of its components and the programming for administer the composition or compositions, which elicit an adequate response, such as by means of serum titers and their analysis, for example by ELISA and / or seroneutralization analysis. These determinations do not require undue experimentation by the knowledge of those skilled in the art, this description and the documents cited herein. And the invention also comprises the sequential administration of the compositions of the invention, or the sequential performance of the methods of the present, for example, the periodic administration of the compositions of the invention, such as in the development of a therapy or treatment for a disorder and / or the administration of reinforcement of immunological compositions and / or in priming-booster regimens; and the timing and manner of performing sequential administrations can be determined without undue experimentation. Furthermore, the invention describes compositions and methods for manufacturing and using vectors, including methods for producing gene products and / or immunological products and / or antibodies in vivo and / or in vitro and / or ex vivo (the last two being performed, for example, after isolation of cells from a host that has been subjected to a non-invasive administration according to the invention, for example after the optional expansion of said cells), and uses for said gene and / or immunological products and / or antibodies, including in diagnoses, assays, therapies, treatments and the like. Vector compositions are formulated by mixing the vector with a suitable carrier or diluent; and the compositions of gene products and / or immunological products and / or antibodies are similarly formulated by mixing the gene and / or immunological product and / or antibody with a suitable carrier or diluent; see, for example, U.S. Patent 5,990,091, WO 99/60164, WO 98/00166, WO 99/53940 and U.S. Patent Nos. 6,042,838 and 6,004,802, and the documents cited therein and other documents cited herein, and other indications herein (for example, with respect to vehicles, diluents and the like).

If a nasal or respiratory (mucosal) administration is desired, the compositions may be in suitable form and dispensed by a manual compression spray dispenser, a pump dispenser or an aerosol dispenser. These dispensers can also be used to administer the composition to the oral mucosa or the oral cavity (eg, buccal or perlingual). Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers may preferably dispense a measured dose or a dose having an appropriate particle size.

The compositions described in the invention may contain pharmaceutically acceptable aromas and / or dyes to make them more attractive, especially if they are to be administered orally (or buccally or perlingually) and, thus, these compositions may be in the form of tablets. or capsules that dissolve in the mouth or bite to release a liquid for oral or perlingual absorption (similar to oral, perlingual or oral medications for angina, such as nitroglycerin or nifedimeno). The viscous compositions may be in the form of gels, lotions, ointments, creams and the like (for example, for topical and / or mucosal and / or nasal and / or oral administration and / or oral and / or perlingual cavity and / or buccal), and will generally contain a sufficient amount of a thickening agent so that its viscosity is from about 2500 to 6500 cps, although more viscous compositions can be used, even up to 10,000 cps. The viscous compositions preferably have a viscosity of 2500 to 5000 cps, since above this range they are more difficult to administer. However, above this range, the compositions can approximate solid or gelatin forms that can then be easily administered in the form of a swallowed pill for oral ingestion and / or a pill or capsule or tablet to take in the mouth, for example, for oral or perlingual administration.

Liquid preparations are usually easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection or orally, orally or perlingually, to animals, children, particularly young children, and others who may have difficulty swallowing a pill, tablet, capsule or similar, or in multiple dose situations. The viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer periods of contact with the mucosa, such as the lining of the stomach or nasal mucosa, or for perlingual, oral or oral cavity absorption .

Obviously, the choice of suitable vehicles and other additives will depend on the exact route of administration and the nature of the specific dosage form, for example, a liquid dosage form (for example, if the composition is to be formulated in a solution, a suspension, a gel or other liquid form), or a solid dosage form (for example, if the composition is to be formulated in a pill, a tablet, a capsule, an oblong tablet, a release form in the time, or a liquid filled form).

Suspensions and gels usually contain a significant amount of water (preferably purified water) in addition to the antigen, lipoproteins and optional adjuvant. Small amounts of other ingredients may also be present, such as pH adjusters (for example, a base, such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, gelling agents (eg, methylcellulose), colorants and / or aromas. The compositions can be isotonic, that is, they can have the same osmotic pressure as blood and tear fluid.

The desired isotonicity of the compositions described in the invention can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is particularly preferred for buffers containing sodium ions.

The viscosity of the compositions can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methyl cellulose is preferred because it can be purchased easily and is cheap, and it is easy to work with it. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer and the like. The preferred concentration of the thickener will depend on the agent selected. The important point is to use an amount that can achieve the selected viscosity. The viscous compositions are usually prepared from solutions by the addition of said thickening agents.

A pharmaceutically acceptable preservative can be used to increase the expiration of the compositions. The benzyl alcohol may be suitable, although a variety of preservatives may also be employed including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight, although there may be appreciable variations depending on the agent selected.

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the vector, antigen or epitope of interest and the optional adjuvant or other active ingredients or immunity enhancers. This will not be a problem for those skilled in the chemical and pharmaceutical technique, or problems that arise can be easily avoided by referring to conventional texts or simple experiments (which do not involve undue experimentation), from this description and from documents cited therein.

The immunologically effective compositions described in the invention are prepared by mixing the following generally accepted methods. For example, the selected components may simply be mixed in a mixer or other conventional device to produce a concentrated mixture that can then be adjusted to the final concentration and viscosity by the addition of water or a thickening agent and perhaps a buffer to control the pH. or an additional solute to control tonicity. In general, the pH can be from about 3 to 7.5. The compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary art taking into consideration factors such as the age, sex, weight and disorder of the particular patient or animal, and the shape of the composition used. for administration (for example, solid vs. liquid). Dosages for humans or other mammals can be determined without undue experimentation by those skilled in the art, from this description, the documents cited therein, the following examples, and the applications, patents and other documents cited in hereby, and of the documents cited or referenced in the documents cited herein, all of which are incorporated herein by reference.

Suitable regimens for initial administration and booster doses or for sequential administrations are also variable, and may include an initial administration, followed by subsequent administrations; however, they can be determined by those skilled in the art from this description, of the citads documents and incorporated herein by reference, including the applications and patents cited herein and the documents referenced or cited in the cited documents herein, all of which are incorporated herein by reference, as well as the following examples. The compositions can be administered alone, or they can be co-administered or administered sequentially with other compositions of the invention or with other prophylactic or therapeutic compositions.

The vector expresses a gene that encodes influenza hemagglutinin and / or the nuclear flu protein.

In one embodiment of the invention, the immune response in the animal is induced by genetic vectors that express genes encoding antigens of interest in the animal's cells, specifically the hemagglutinin of the flu and / or the nuclear protein of the flu. In another embodiment, the genetic vector is used as a prophylactic vaccine or therapeutic vaccine. In another embodiment of the invention, the genetic vector comprises genetic vectors capable of expressing an antigen of interest in the animal's cells. In another embodiment of the method, the animal is a vertebrate.

With respect to the exogenous DNA for expression in a vector (for example, which encodes an epitope of interest and / or an antigen and / or a therapeutic product) and the documents that provide said exogenous DNA, as well as with respect to the expression of transcription and / or translation factors to enhance the expression of nucleic acid molecules, and with respect to terms and expressions such as "epitope of interest", "therapeutic", "immune response", "protective immune response", " Immunological composition, "" immunogenic composition "and" vaccine composition ", among others, refers to US Patent 5,990,091, issued November 23, 1999, and WO 98/00166 and WO 99/60164 , and the documents cited therein and the registration documents in the process of this patent and PCT applications. Thus, US Patent 5,990,091, and documents WO 98/00166 and WO 99/60164, and the documents cited therein and the registration documents in the process of this patent and PCT applications, and other documents cited in present or otherwise incorporated herein by reference, may be consulted in the practice of the invention; and all exogenous nucleic acid molecules, promoters, and vectors cited therein can be used in the practice of this invention. In this regard, US Patent Nos. 6,004,777, 5,997,878, 5,989,561, 5,976,552, 5,972,597, 5,858,368, 5,863,542, 5,833,975, 5,863,542, 5,843 are also mentioned. 456, 5,766,598, 5,766,597, 5,762,939, 5,756,102, 5,756,101, 5,494,807, 6,042,838, 6,004,802 and WO 99/53940.

In another embodiment of the invention, the animal is advantageously a vertebrate, such as a mammal, a bird, a reptile, an amphibian or a fish; more advantageously is a human being, or a companion or domesticated animal, or producer of food or feed, or livestock or game, racing or sports, such as a cow, a dog, a cat, a goat, a sheep, or a pig or a horse, and even poultry, such as turkeys, ducks or chickens. In another especially advantageous embodiment of the invention, the vertebrate is a human being. In another embodiment of the invention, the immune response is against influenza A. In another embodiment of the invention, the immune response against influenza A is induced by the genetic vector that expresses a gene encoding a hemagglutinin of influenza and / or a nuclear flu protein, or a fragment of these in the animal's cells. In the present invention, the genetic vector is a defective adenovirus in its E1 and E3 regions. In another embodiment of the invention, the DNA is in plasmid form. In another embodiment of the invention, all viral genes have been deleted from the vector.

In another embodiment of the present invention, the vector also contains a gene selected from the group consisting of costimulatory genes and cytokine genes. In this method, the gene is selected from the group consisting of a GM-CSF gene, a B7-1 gene, a B7-2 gene, an interleukin-2 gene, an interleukin-12 gene, and genes of interferon.

Embodiments of the invention employing recombinant adenovirals include adenoviral vectors defective in E1 and defective in E3, or adenoviral vector without coding sequences ("gutless") in which all viral genes have been deleted. Mutation of E1 increases the safety margin of the vector, because adenoviral mutants defective in E1 are incompetent for replication in non-permissive cells. The E2 mutation enhances the immunogenicity of the antigen by altering the mechanism by which the adenovirus under-regulates MHC class I molecules. The "gutless" adenoviral vector is the latest model in the family of adenoviral vectors. Its replication requires an auxiliary virus and a special human 293 cell line that expresses both E1a and Cre, a condition that does not exist in the natural environment; all the viral genes have been eliminated from the vector, therefore the vector, as a vaccine vehicle, is not immunogenic and can be inoculated many times for revaccination. The "gutless" adenoviral vector also contains a 36 kb space to introduce transgenes, thus allowing co-administration of a large number of antigen genes to the cells. Specific sequence motifs, such as the RGD motif, can be inserted into the H-I loop of an adenoviral vector to enhance its infectivity. An adenoviral recombinant is constructed by cloning specific transgenes or fragments of transgenes into any of the adenoviral vectors, such as those described above. The adenoviral recombinant is used to transduce epidermal cells of a vertebrate in a non-invasive way for use as an immunizing agent.

Embodiments using DNA / adenovirus complexes can present plasmid DNA complexed with adenoviral vectors using a suitable agent for this, such as PEI (polyethyleneimine) or polylysine. The adenoviral vector within the complex may be "alive" or "dead" by irradiation with UV or gamma rays. The irradiated inactivated adenoviral vector, as a receptor binding ligand and as an endosomolysis agent to facilitate DNA-mediated transfection (Cotten et al., 1992), may increase the safety margin of the vaccine vehicle. The DNA / adenovirus complex is used to transfect epidermal cells of a vertebrate in a non-invasive way for use as an immunizing agent.

Embodiments that employ DNA / liposome complexes may possess materials to form liposomes, and DNA / liposome complexes may be formed from these materials. The DNA / liposome complex is used to transfect epidermal cells of a vertebrate in a non-invasive way for use as an immunizing agent.

The genetic vectors described in the invention can also encode immunomodulatory molecules that can act as an adjuvant to elicit a humoral and / or cellular immune response. These molecules include cytokines, costimulatory molecules, or any molecule that can change the course of an immune response. Ways in which this technology can be modified to further enhance the immunogenicity of antigens can be devised.

Dosage forms for topical administration of the genetic vector and the gene of interest of this invention may include liquids, ointments, powders and powders. The active component may be mixed under sterile conditions with a physiologically acceptable carrier and any preservative, buffer, propellant or absorption enhancer that is necessary or desired. Reference is made to the documents cited herein, for example, U.S. Patent Nos. 5,990,091, 6,042,838 and 6,004,802, and WO 98/00166 and WO 99/60164, and WO 99 / 53940, and the documents cited therein for methods for constructing vectors, as well as for compositions for topical application, for example, viscous compositions that can be creams or ointments, as well as compositions for nasal administration.

In terms of the terminology used herein, an immunologically effective amount is an amount or concentration of the genetic vector that encodes the gene of interest that, when administered to an animal, produces an immunological response against the gene product of interest.

Various epitopes, antigens or therapeutic products can be administered topically by expression at different concentrations. In general, the amounts useful for adenoviral vectors are at least about 100 pfu, and for plasmid DNA at least about 1 ng of DNA. Other amounts may be determined from this description and knowledge of the art, including the documents cited and incorporated herein by reference, without undue experimentation.

The methods described in the invention can be appropriately applied to prevent diseases such as prophylactic vaccination, or to treat diseases such as therapeutic vaccination.

The vaccines of the present invention can be administered to an animal alone or as part of an immunological composition.

Beyond the described human vaccines, the method of the invention can be used to immunize animal cattle. The term animal means all animals, including humans. Examples of animals include humans, cows, dogs, cats, goats, sheep, horses, pigs, turkeys, ducks and chickens, etc. Since the immune systems of all vertebrates act in a similar way, the described applications can be applied in all vertebrate systems.

The following examples are offered to illustrate various embodiments of the invention, and are not intended to limit the present invention in any way.

Examples

Protocols

Mice and cell cultures

Mice spawned by inbreeding were kept at the University of Alabama in Birmingham. The cells were cultured in RPMI 1640 or DMEM medium containing 2% fetal bovine serum and 6% calf serum.

Topical application of genetic vectors

The mice were anesthetized and the hair and cornified epithelium that covered a restricted area of the abdominal skin or neck were removed by a brush or a hair remover (for example, NAIR). Genetic vectors were pipetted onto pre-ripped skin and kept in contact with bare skin for various amounts of time (for example, from 1 hour to 18 hours). Vectors can be pipetted directly onto bare skin, or into a cylinder that is attached to the skin.

Preparation of adenoviral vectors

High titre adenovirus strains were prepared from human 293 cells infected with specific adenoviral recombinants. The lysates were subjected to ultracentrifugation along a gradient of cesium chloride. The viral bands were extracted and dialyzed against 10 mM Tris (pH 7.5) / 135 nM NaCl / 5 mM KCl / 1 mM MgCl2. The purified viruses were sterilized by filtration with glycerol added up to 10% and stored in aliquots at -80 ° C. Titration for adenovirus strains was determined by a plaque assay.

Luciferase Assay

The amount of luciferase in the skin was determined as previously described (Tang, 1994). Briefly, a piece of the removed skin was homogenized with a Kontes glass tissue crusher in lysis buffer. After removing tissue debris by centrifugation, luciferase activity in the skin extract was determined with a luminometer by measuring the integrated light emission in the presence of an excess of ATP and luciferin.

B-galactosidase assay

A piece of removed skin quickly froze in the Tissue-Tek O.C.T. (Miles Laboratories Inc.) in liquid nitrogen and stored at -80 ° C until use. The frozen tissue was cut into cross sections at 4 µm, fixed in 4% paraformaldehyde, and stained for B-galactosidase activity by incubation in an X-gal staining solution as previously described (Tang et al. ., 1994). Sections were contracted with hematoxylin and eosin.

Preparation of DNA / adenovirus complexes

The DNA / adenovirus complexes were prepared by mixing 100 µg of plasmid DNA with 1 x 1011 adenovirus particles in the presence of the polylysine condensing agent for each application. The adenovirus titration was determined by absorbance.

Preparation of DNA / liposome complexes

The DNA / liposome complexes were prepared by mixing 100 µg of plasmid DNA with 100 µg of DOTAP / DOPE (1: 1, Avanti) for each application. Plasmids were prepared using Qiagen Plasmid Maxi kits.

Western blot analysis

Serums from tail bleeds were diluted from 1: 250 to 1: 500 and reacted with purified proteins that had been separated on an SDS-polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore). The reaction was visualized using the ECL kit (Amersham).

Reference Example 1

The present invention demonstrates that antigen genes can be administered to the skin of mice in a simplified manner by the non-invasive administration directed to the skin of a genetic vector without using sophisticated equipment. Figure 1 demonstrates that substantial amounts of the luciferase enzyme were produced after administration of limited amounts of AdCMV-luc (an adenoviral vector encoding firefly luciferase) (Tang et al., 1994) on the skin. Ad, adenovirus; pfu, plate forming units; LU, light units. The results are the average log [LU per cm2 of skin] ± EE (n is shown at the top of each column). Mice with fake application or coated with an adenoviral vector that does not encode luciferase produced no detectable luciferase activity on the skin. The level of expression of the transgene from the adenoviral vector in the skin does not appear to correlate with virus titration. It is possible that only a small number of cells can be transduced by the virus into a restricted subset of the skin, and 108 plaque-forming units (pfu) of adenoviral recombinants may have saturated the target cells. This variability may also be due, in part, to variations in individual mice. In addition, part of the variability probably arose from the procedure of removing the cornified epithelium that had not been standardized (Johnston and Tang, 1994). The amount of antigen produced can potentially be amplified by applying more vectors over a larger area.

Reference Example 2

The main target cells for non-invasive vaccination on the skin appear to be capillary matrix cells within the hair follicles (Figure 2a) and keratinocytes within the outermost layer of the epidermis (Figure 2b), as demonstrated by the staining of frozen sections with X-gal substrates after skin-directed non-invasive administration of an adenoviral vector encoding the E. coli B-galactosidase gene (AdCMV-Bgal) (Tang et al., 1994). No physical abrasions were found in the skin tissue undergoing treatment, and no inflammation was induced. The skin tissue undergoing non-invasive gene administration was removed from the animals one day after pipetting 108 pfu of AdCMV-Bgal onto the skin, cut into cross sections, fixed and stained with X-gal substrates according to has been described (Tang et al., 1994). Figure 2a shows capillary matrix cells transduced with adenovirus within the hair follicle, x150. Figure 2b shows keratinocytes transduced with adenovirus within the outermost layer of the epidermis, x150. No blue cells were found in the control animals, with feigned application or coated with AdCMV-luc.

Reference Example 3

Humoral immunological responses caused by adenovirus-mediated NIVS

The NIVS is a new method to vaccinate animals. To demonstrate that the procedure can elicit a specific immune response against the vector-encoded antigen, AdCMV-hcea (an adenoviral vector encoding the human carcinoembryonic antigen (CEA)) was pipetted onto the skin of mice of the C57BL / 6 breed. The serum of a vaccinated mouse one month after the non-invasive administration directed to the skin of 108 pfu of AdCMV-hcea was diluted 1: 500 and reacted with the purified human CEA protein (provided by T. Strong) and adenoviral proteins which had been separated on a 5% SDS-polyacrylamide gel, and transferred to Immobilon-P membranes (Millipore). Referring to Figure 3a: lane 1, 0.5 µg of human CEA; lane 2, 0.5 µg of BSA; lane 3, 107 pfu of adenovirus. Figure 3a shows that test sera from a vaccinated animal react in Western blot analysis with purified human CEA protein, but not with bovine serum albumin (BSA), which supports the conclusion that specific antibodies have been produced. against exogenous proteins encoded by adenoviral vectors as a result of the administration of non-invasive genes directed to the skin.

To test whether this technique can be applicable in general, AdCMV-hgmcsf (an adenoviral vector encoding the human granulocyte and macrophage colony stimulating factor (hGM-CSF)) was applied to the skin. To detect antibodies against the human GM-CSF protein, the animal was vaccinated by the non-invasive, skin-directed administration of 108 pfu of AdCMV-hgmcsf. The purified human GM-CSF protein (CalBiochem) separated on a 15% SDS-polyacrylamide gel was transferred to membranes and allowed to react with the diluted serum. Other treatments were performed as described in Figure 3a. Referring to Figure 3b: lane 1, 0.25 µg of human GM-CSF; lane 2, 0.25 µg of BSA; lane 3, 107 pfu of adenovirus. AdCMV-hgmcsf and AdCMV-hcea derived from human adenovirus defective in serotype 5 replication were produced in human 293 cells. A module containing the human CEA gene or the human GM-CSF gene, directed by the early cytomegalovirus (CMV) enhancer element, was inserted instead of the E1a deletion. Since the sequences in the E1a region were deleted, the ability of these viruses to replicate autonomously in non-permissive cells was altered.

The results (Tang et al., 1997) show that 96% (23/24) of the C57BL / 6 mice produce antibodies against the human CEA protein one month after the non-invasive administration directed to the skin of AdCMV-hcea , and 43% (6/14) of mice of the same strain produce antibodies against human GM-CSF protein after non-invasive administration directed to the skin of AdCMV-hgmcsf. Both preimmune sera were collected before the NIVS, and sera from unexposed animals did not react with human CEA and GM-CSF proteins. The possibility of oral vaccination by ingesting the vectors through grooming was eliminated (1) by rinsing the skin vectors before the animals recovered from anesthesia, (2) by pipetting the vectors onto the unshaven skin, and (3) mixing unexposed and vaccinated animals in the same cage. Cross vaccination was never observed among unexposed and vaccinated mice, and shaving appears to be a fundamental component for the NIVS, probably due to mechanical removal of the cornified epithelium along the shaving path. Thus, an adenovirus-mediated NIVS is capable of eliciting a humoral immune response against an antigen encoded by the vector.

Reference Example 4

In order to demonstrate that the techniques of the present invention can produce a protective anti-tumor immune response, genetically engineered tumor cells expressing the human carcinoembryonic antigen (CEA) gene (MC38-CEA-2) (Conry et al., 1995) were inoculated into mice. the unexposed C57BL / 6 race that had been vaccinated by topical application of an adenoviral vector encoding the human CEA gene (AdCMVhcea). Animals subjected to tumor exposure were observed for survival (Figure 4). In the control group, 90% (9/10) of the animals developed palpable tumor nodules and died 30 days after implantation of the tumor cells. In the vaccinated group, only 10% (1/10) of the animals died, and 70% (7/10) remained totally free of tumors. The mice underwent euthanasia when the tumor reached a diameter greater than 1 cm. The interval between the injection of tumor cells and euthanasia is used as the survival time of the individual. Referring to Figure 4, control mice (not given vaccines) and animals immunized with NIVS (108 pfu of AdCMV-hcea had been applied topically one month earlier) were exposed to tumors. The numbers in brackets represent the number of animals for each treatment. The results demonstrate that the non-invasive administration of genetic vaccines to the skin may be able to produce protective immune responses against tumor cells that express a specific antigen.

Reference Example 5

Construction of recombinant adenoviral vectors encoding cytokine and costimulatory genes

Adenoviral vectors were constructed that encode cytokine and costimulatory genes for co-administration of these immunomodulatory genes with antigen genes in skin cells to try to direct the immunological profile in vaccinated animals. The adenoviral vector AdCMV-mB7.1 encoding the murine B7-1 gene and the adenoviral vector AdCMV-mgmcsf encoding the murine GM-CSF gene were constructed by homologous recombination between two plasmids transfected into human 293 cells following a conventional procedure to generate new adenoviral vectors (Gómez-Foix et al., 1992). All transgenes in these vectors were transcriptionally directed by the CMV early enhancer-promoter element. AdCMVmB7.1 is characterized by staining of transduced human lung carcinoma SCC-5 cells, with the anti-CD80 antibody (PharMingen), followed by analysis by flow cytometry. AdCMV-mgmcsf was characterized by measuring the murine GM-CSF secreted from the SCC-5 cells transduced with an ELISA kit (Amersham).

Reference Example 6

Detection of antitumor immunity by an in vivo cytotoxicity test

An in vivo cytotoxicity assay was developed in which the target cells were implanted as monolayers on the muscle tissue of mice (Tang et al., 1996). Implantation of target cells as monolayers allowed an efficient collection of target cells to assess their situation after a few days of growth in vivo. This assay was particularly useful for detecting weak immune responses that are not potent enough to eradicate target cells. Immunological responses can be characterized by histological analysis of the implant bed. Without an immune response, the target cells will grow. With a potent immune response, the target cells would be eradicated in the presence of a large number of immune effector cells in the implant bed, probably due to migration to the growing target cells and in situ sensitization around them. With a weak immune response, the growing target cells would mix with the infiltrating immune effector cells in the implant bed. Implantation of 5 x 105 RMI-luc cells (RMI prostate tumor cells expressing the luciferase gene) as a monolayer in unexposed C57BL / 6 mice produced a tumor layer due to the proliferation of RMI-luc cells in alive, without evidence of immunological intervention. In contrast to control animals, RMI-luc cells were mixed with a large number of immunological effector cells in the implanted bed in vaccinated animals by non-invasive skin-directed administration of AdCMV-luc.

Reference Example 7

Characterization of immune effector cells recruited by tumor cells

The in vivo cytotoxic assay was able to concentrate a large number of immune effector cells in the implant bed by implanting a small number of target cells as a monolayer on the muscle. The characterization of specific immune effector cells in the implant bed can provide evidence to know if a cell-mediated immune response has been elicited to destroy the target cells. To characterize the T cells that had been recruited by luciferase-expressing tumor cells in vaccinated animals by non-invasive administration directed to the skin of AdCMV-luc, sections of implant bed tissue were stained with an anti-CD3 monoclonal antibody (mAb). RMI-luc cells were produced by lipofectating pHBA-luc DNA into RMI prostate tumor cells (provided by T. Thompson of Baylor College of Medicine), followed by selection in a medium containing G418. Clones expressing luciferase were characterized by a luciferase assay. 5 x 105 RMI-luc cells were implanted as a monolayer in a mouse that had been vaccinated by the non-invasive, skin-directed administration of 108 pfu of AdCMV-luc. Five days after implantation, the implant bed was frozen in O.C.T. and sections were cut at 4 µm, dried in 100% acetone, and stained with an anti-CD3 mAb (clone F500A2, provided by P. Ox of the UAB), through the procedure of ABC immunoperoxidase with diaminobenzidine as chromogen

As shown in Figure 5, a large number of T cells infiltrated the implant bed after 5 days of in vivo growth of RMI-luc cells in a vaccinated mouse by non-invasive administration directed to the skin of AdCMV. -luc (x150), while only a few T cells were found in unexposed animals. It seems that the same number of RMI-luc target cells can recruit more T lymphocytes into the implantation bed in vaccinated animals than in unexposed animals.

To characterize the CTLs that were recruited by the target cells, frozen sections of the implant bed were subjected to in situ hybridization using an antisense granzyme A RNA molecule as a probe. 5 x 105 RMI-luc cells were implanted as a monolayer in an unexposed C57BL / 6 mouse that had been vaccinated by a non-invasive administration directed to the skin with 108 pfu of AdCMV-luc. Five days after implantation, the implantation bed was frozen in O.C.T. and sections were cut at 4 µm. The frozen sections were fixed in 3% paraformaldehyde, incubated in 0.2 M HCl to inhibit endogenous alkaline phosphatase activity, and hybridized with a thermo-denatured antisense granzyme A RNA probe. The probes for in situ hybridization were single stranded RNA molecules produced by transcription from a plasmid containing bacteriophage promoters. During transcription, digoxigenin UTP was incorporated directly into the sequence. Sense sequence probes were used as negative controls. After hybridization with the sodas, the sections were washed and incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody, followed by incubation in the NBT / BCIP enzyme substrate solution.

CTLs expressing granzyme A are activated CTLs and have been used as predictive markers for tissue rejection during transplantation. Granzyme-positive CTLs were found within the RMI-luc implant bed only in animals that had been vaccinated by a non-invasive administration directed to the skin with AdCMV-luc (Figure 6). Its presence in the bed suggests that the NIVS has been able to induce a cell-mediated immune response against tumor cells that express a specific antigen.

Reference Example 8

Topical application of genetic vaccines by adhesive bandages

It was demonstrated, for the first time, that bandages can be used for vaccine administration. This development may allow personnel without medical training to administer a uniform dose of non-invasive vaccines on the skin. To transduce the skin with bandages, 50 µl of the AdCMV-luc vector described in example 7 was pipetted into the pad of an adhesive bandage (Johnson & Johnson). The bandage containing the vector was then adhered to the pre-ripened skin of a mouse. The vector was kept in contact with bare skin for 18 hours. To detect the expression of transgenes from the genetic vectors administered by the bandage, the skin was tested for luciferase (table 1). Although the results show substantial variation, expression of transgenes in the skin could be achieved using adhesive bandages.

To demonstrate that the animals can be vaccinated with non-invasive adhesive bandages, serum from tail bleeds for anti-CEA antibodies was tested two months after adhering the bandages containing AdCMVhcea on the skin of mice. As shown in Figure 7, anti-CEA antibodies were detected in 100% (10/10) of the mice that received non-invasive vaccines through adhesive bandages.

Reference Example 9

NIVS mediated by DNA / adenovirus

Adenovirus-based vectors can be made more versatile by binding plasmid DNA to the outside of an adenovirus. The resulting vector system mediates the administration of high efficiency genes in a wide variety of target cells. This strategy allows a very enhanced flexibility in terms of the size and design of the foreign genes. Thus, DNA / adenovirus complexes can deliver antigen genes to the skin through the same pathway of adenovirus receptor mediated endocytosis with more flexibility.

To demonstrate the viability of the NIVS mediated by DNA / adenovirus, plasmid DNA encoding human growth hormone (pCMV-GH) (Tang et al., 1992) was allowed to complex with a defective adenovirus in E4. Mice (strain C57BL / 6) were vaccinated by contacting DNA / adenovirus complexes with bare skin for one day. Immunized animals were then monitored for the production of antibodies against human growth hormone (hGH) protein by analyzing serum from tail bleeds. As shown in Figure 8a, lane 1, hGH (0.5 µg), and lane 2, BSA (0.5 µg), the test serum reacted in Western blot analysis with purified hGH, but not with irrelevant proteins. Of the ten mice vaccinated with DNA / adenovirus complexes, eight (80%) produced antibodies against hGH in three months, indicating that they can produce specific antibodies against exogenous proteins encoded by plasmid DNA that is complexed with adenovirus and that has been administered in a non-invasive way. Preimmune serum collected before treatment, serum from untreated animals, and serum from animals vaccinated with irrelevant vectors did not react with hGH. Thus, DNA / adenovirus complexes, like adenoviral recombinants, appear to be a legitimate vector system for NIVS.

Reference Example 10

NIVS mediated by DNA / liposomes

In addition to the development of genetic vectors that involve adenovirus as vehicles for non-invasive vaccines, it has also been shown that mice can be vaccinated by topically applying DNA / liposome complexes without viral elements. It is evident that many vectors can be applied in a creative way for the administration of non-invasive vaccines directed to the skin. As shown in Figure 8b, lane 1, hGH (0.5 µg), and lane 2, BSA (0.5 µg), the test serum from a mouse immunized by the topical application of complexes of DNA / liposomes encoding hGH reacts with hGH but not with BSA. Of the 10 mice vaccinated with the DNA / liposome complexes, the test sera reacted with purified hGH in 9 (90%) animals treated at 5 months. Thus, DNA / liposome complexes, as well as adenoviruses and DNA / adenovirus complexes, appear to be a legitimate vector system for the NIVS.

Reference Example 11

Coexpression of DNA-encoded and adenovirus-encoded transgenes

Strategies to increase the immune system response can potentially improve the clinical results of vaccines. The local production of immunomodulatory molecules involved in the activation and expansion of lymphocyte populations can significantly improve the effects of vaccination. Adenoviral vectors that encode murine GM-CSF and B7-1 genes have been manufactured. Therefore, the topical application of DNA / adenovirus complexes may be able to co-express immunomodulatory molecules or DNA-encoded antigens, with immunomodulatory molecules or adenoviruses encoded in individual dermal cells to enhance the immune response against the antigen.

Figure 9 demonstrates that the expression of transgenes from plasmid DNA in target cells depends on the presence of adenovirus, thus allowing transgenes encoded by plasmids and encoded by adenovirus to be coexpressed in the same cell. Plasmid pVR-1216 DNA (provided by Vical), AdCMV-Bgal particles and polylysine were mixed in specific proportions as shown in the figure. The complex was applied to 2 x 105 SCC-5 cells in a well and incubated for 2 hours. The complex was then removed and the cells were collected for luciferase and B-galactosidase assays the next day. White column: luciferase activity; striped column: B-galactosidase activity. The results demonstrate that DNA-encoded transgenes are not expressed in the target cells in the absence of adenovirus, while adenovirus-encoded transgenes can be expressed in the presence of DNA. It is also possible that DNA can condense on the surface of other viruses to target different cell types. Therefore, this protocol provides a simple but versatile gene delivery system that allows simultaneous expression of transgenes from a viral recombinant and an externally bound plasmid.

Reference Example 12

Relative transgene expression in the skin from different genetic vectors by topical application

It has been shown that adenoviral recombinants, DNA / adenovirus complexes, DNA / liposome complexes, and perhaps many other genetic vectors can be applied as vehicles for non-invasive vaccines. It can be thought that the greater the efficiency for the expression of transgenes, the more powerful the vehicle will be. To define the relative efficiencies of the vectors used, adenoviral recombinants, DNA / adenovirus complexes, or DNA / liposome complexes were allowed to contact the skin of a mouse by a topical application for 18 hr. The treated skin was then removed from the animal and tested for luciferase activity with a luminometer measuring the integrated light emission for 2 min using the Promega luciferase assay system, and the background was subtracted from the readings. As shown in Figure 10, it was discovered that adenoviral recombinants were the most effective vector system for non-invasive skin gene delivery. Mice treated simulated produced no detectable luciferase activity on the skin. LU, light units; Ad, AdCMV-luc; DNA / Ad, pVR-1216 DNA complexed with Ad dl1014; DNA / liposomes, pVR-1216 DNA complexed with DOTAP / DOPE. The results are the log average (LU per cm2 of skin) ± EE (n is shown at the top of each column). Although the efficacy of the DNA / adenovirus complex is less than that of the adenoviral recombinant, it is significantly greater than that of the DNA / liposome complex. In addition, the adenovirus can be inactivated by gamma or UV irradiation before forming a complex with the DNA to prevent viable viral particles from spreading. Therefore, it might appear that DNA / adenovirus complexes are the most promising vehicle system for the administration of non-invasive vaccines when considering efficacy and safety factors in the formulation of a new generation of vaccines.

Example 13

Construction of an expression vector encoding influenza antigens

A defective adenoviral recombinant was constructed in E1 / E3 encoding the HA A / PR / 8/34 gene (AdCMVPR8.ha) as described (Gómez-Foix et al., 1992). Briefly, a 1.8 kb BamHI fragment containing the complete HA coding sequence was cut from plasmid pDP122B [American Type Culture Collection (ATCC)] and then inserted into the BamHI site of pACCMV.PLPA in the correct orientation under the Transcriptional control of the human cytomegalovirus (CMV) early promoter. The resulting plasmid encoding HA was co-transfected with plasmid pJM17 in human 293 cells to generate defective adenoviral recombinants in E1 / E3. A defective adenoviral recombinant was constructed in E1 / E3 that encodes the nuclear protein gene (NP) A / PR / 8/34 (AdCMV-PR8.np) by cloning the NP gene (provided by Merck) into pACCMV.PLPA, followed of homologous recombination in 293 cells as described above.

A plasmid expression vector encoding HA (pCMV-PR8.ha) and another encoding NP (pCMVPR8.np) were constructed by cloning the HA and NP genes into pVR1012 (provided by Vical), respectively.

Example 14

Anti-influenza antibodies generated by topical application and intranasal inoculation of adenovirus-based vaccines in mice

As shown in Figure 12, BALB / c mice (3 weeks old) were immunized by a variety of vaccination modalities, including intramuscular injection of DNA, intranasal inoculation of adenoviral vectors, and topical application of a patch of adenovirus based vaccine. A non-invasive vaccination directed to the skin was performed by pipetting adenoviral vectors on the pre-delayed abdominal skin, followed by the extension of the vector as a thin film on the bare skin with a piece of the Tegaderm patch (3M). The non-absorbed vectors were washed in one hour. All animals were immunized 3 times every 3 weeks. Serum samples were tested for anti-influenza antibodies 1 week after the last booster. Anti-influenza IgG titers were determined by ELISA as described (12) using purified A / PR / 8/34 virus as capture antigen. Serum samples and goat anti-mouse IgG conjugated with peroxidase (Promega) were sequentially incubated on the plates for 1 hour at room temperature with a thorough wash between each incubation. The assessment criterion was calculated as the serum dilution that produces the same OD450 as a 1/100 dilution of the preimmune serum. Negative serum at the lowest dilution tested was assigned titration criteria of 100. The post-immunological serum also reacts with a control antigen (for example, BSA) at a low level. Hemagglutinin (HI) inhibition assay was performed to measure the ability of anti-HA antibodies to inhibit erythrocyte agglutination (RBC) by viruses, perhaps by blocking cell surface binding. Pre-absorbed serum samples were diluted with chicken RBC and mixed with 4 HA units of influenza virus A / PR / 8/34. The chicken RBCs were then added to a final concentration of 0.5%. Agglutination was determined by a visual exam. Titration was defined as the dilution that is the limit of inhibition. All preimmunological sera had a titration: 20. Group 1, 2.5 x 107 pfu intranasal inoculation of wild type serotype 5 adenovirus, followed by a topical application of 108 pfu of AdCMV-PR8.ha and 108 pfu of AdCMV -PR8.np two weeks later (n = 9); group 2, 2.5 x 107 pfu intranasal inoculation of wild type serotype 5 adenovirus, followed by an intramuscular injection of 100 µg of pCMV-PR8.ha DNA and 100 µg of pCMV-PR8.np DNA two weeks later (n = 10); group 3, 2.5 x 107 pfu intranasal inoculation of serotype 5 wild type adenovirus, followed by a 2.5 x 107 pfu intranasal inoculation of AdCMV-PR8.ha and 2.5 x 107 pfu of AdCMV-PR8 .np two weeks later (n = 8); group 4, topical application of 108 pfu of AdCMV-PR8.ha and 108 pfu of AdCMV-PR8.np (n = 10); group 5, topical application of 108 pfu of AdCMV-PR8.np (n = 10); group 6, topical application of 108 pfu of AdCMV-PR8.ha (n = 10); group 7, intramuscular injection of 100 µg of pCMV-PR8.ha DNA and 100 µg of pCMV-PR8.np DNA (n = 10); group 8, intranasal inoculation of 2.5 x 107 pfu of AdCMV-PR8.ha and 2.5 x 107 pfu of AdCMV-PR8.np (n = 9). The data were plotted as the geometric mean of the titration criteria. In the control group without exposure (n = 7) no anti-influenza antibodies were detected. The variance analysis strategy (ANOVA) was used to compare the differences in the titres of HI and ELISA. Multiple paired comparisons were made with the Tukey procedure by adjusting the overall alpha level to 0.05. The analyzes were performed at the logarithmic scale of the measurements to meet the assumption of constant variance required by the ANOVA strategy. The differences in HI and ELISA degrees between the 8 groups were significant (P <0.0001). The ELISA titration in group 8 was significantly higher than in the other groups (P <0.02). The average ELISA titration in group 1 was the lowest, but it was not significantly different from that in group 5 or 6. The HI titration in group 8 was the highest, and that in group 3 the second highest. The HI titration values in groups 1, 2, 4, 5 and 6 were not significantly different.

Example 15

Protection of mice against death after exposure to viruses

As shown in Figure 13, BALB / c mice (3 weeks) were immunized by a variety of vaccination modalities, including intramuscular injection of DNA, intranasal inoculation of adenoviral vectors, and topical application of a vaccine patch. based on adenovirus. A non-invasive vaccination directed to the skin was performed by pipetting adenoviral vectors on the pre-delayed abdominal skin, followed by the extension of the vector as a thin film on the bare skin with a piece of the Tegaderm patch (3M). The non-absorbed vectors were washed in one hour. All animals were immunized 3 times every 3 weeks. One week before the last booster, the mice were exposed intranasally to a lethal dose of influenza A / PR / 8/34 virus (1,000 units of HA) and monitored daily for survival. Data were plotted as the percentage of survival versus days after exposure. Control not exposed, mice not exposed to adenovirus; group 1, 2.5 x 107 pfu intranasal inoculation of wild type serotype 5 adenovirus, followed by a topical application of 108 pfu of AdCMV-PR8.ha and 108 pfu of AdCMV-PR8.np two weeks later; group 2, 2.5 x 107 pfu intranasal inoculation of wild type serotype 5 adenovirus, followed by an intramuscular injection of 100 µg of pCMV-PR8.ha DNA and 100 µg of pCMV-PR8.np DNA two weeks later; group 3, 2.5 x 107 pfu intranasal inoculation of serotype 5 wild-type adenovirus, followed by a 2.5 x 107 pfu intranasal inoculation of AdCMV-PR8.ha and 2.5 x 107 pfu of AdCMV-PR8 .np two weeks later; group 4, topical application of 108 pfu of AdCMV-PR8.ha and 108 pfu of AdCMV-PR8.np; group 5, topical application of 108 pfu of AdCMV-PR8.np; group 6, topical application of 108 pfu of AdCMV-PR8.ha; group 7, intramuscular injection of 100 µg of pCMV-PR8.ha DNA and 100 µg of pCMV-PR8.np DNA; group 8, intranasal inoculation of 2.5 x 107 pfu of AdCMV-PR8.ha and 2.5 x 107 pfu of AdCMV-PR8.np. AdCMV-PR8.ha, an adenoviral vector encoding the hemagglutinin of A / PR / 8/34; AdCMV-PR8.np, an adenoviral vector encoding the nuclear protein of A / PR / 8/34; pCMV-PR8.ha, a plasmid expression vector encoding the hemagglutinin of A / PR / 8/34; pCMV-PR8.np, a plasmid expression vector encoding the nuclear protein of A / PR / 8/34. The numbers in brackets represent the number of animals for each treatment.

Pre-exposure to wild-type adenovirus did not intervene in this mode of vaccination. As shown in the final phase I report, high levels of neutralizing antibodies against influenza A / PR / 8/34 can be caused by intranasal inoculation of these vectors even in animals with a pre-exposure to adenovirus. The results suggest that protection may be mediated primarily by a humoral immune response when animals are immunized by intranasal inoculation of adenoviral recombinants. In contrast to the intranasal route, animals immunized by topical application of AdCMVPR8.ha and AdCMV-PR8.np acquired 71% protection against exposure. However, animals with a pre-exposure to adenovirus were not protected by the NIVS (non-invasive skin vaccination). As shown in the final phase I report, animals immunized by NIVS produced a relatively low level of neutralizing antibodies against influenza A / PR / 8/34. Like the antibodies induced by intranasal vaccines, the production of anti-influenza antibodies, caused by the NIVS, was not intervened by pre-exposure to adenovirus. The results suggest that protective immunity can be mediated by a cellular immune response when animals are immunized by NIVS, and this immune mechanism can be suppressed by pre-existing vector immunity. For patients who can receive intranasal vaccines without complications due to respiratory problems, it may therefore be appropriate to co-administer adenovirus-based epicutaneous vaccines together with their intranasal counterparts to attempt to activate both sections of the immune system simultaneously. There is also an urgent need to develop a non-immunogenic NIVS vaccine vehicle to vaccinate animals with a pre-existing adenovirus immunity.

Reference Example 16

Production of anti-HA antibodies in pigtail macaques through NIVS

Although the NIVS can reproduce, in a reproducible way, systemic immune responses in mice (Figures 12 and 13), it may not be possible for the NIVS to immunize humans if transdermal diffusion of the vectors is necessary for the production to occur. vaccination, because human skin is thicker than its murine counterpart. However, non-invasive vaccine patches can immunize humans or other animals with thick skin if only a transient but productive wave of antigen expression is required in cells that are inside the outer layer of the skin. To address these issues, the inventors immunized a pigtail macaque with AdCMV-PR8.ha in a non-invasive manner. As shown in Figure 14, the immunized animal produced antibodies against HA in 4 weeks. The result provides evidence that non-invasive vaccine patches can immunize many different species in addition to mice.

In Figure 14, a pig-tailed macaque was immunized in a non-invasive manner by pipetting 1010 pfu of AdCMVPR8.ha onto pre-ripped abdominal skin, followed by spreading the vector as a thin film on bare skin with a Tegaderm patch (3M) . The non-absorbed vectors were washed in 5 hours. Serum samples were tested for anti-HA antibodies at 4 weeks after inoculation. Anti-HA IgG titers were determined by ELISA using purified A / PR / 8/34 virus as capture antigen. Serum samples and peroxidase-conjugated goat anti-monkey IgG (Bethyl Laboratories, Inc.) were sequentially incubated on the plates for 1 hour at room temperature with a thorough wash between each incubation. The assessment criterion was calculated as the serum dilution that produces the same OD450 as a 1/100 dilution of preimmune serum. The titers of 1 criteria were assigned to the negative serum at the lowest dilution tested.

Reference Example 17

Transfer of luciferase spots on the skin after localized gene administration in a non-invasive way

To try to determine if the antigen genes administered on the skin surface can diffuse into deeper tissues and express the antigens in the cells below the epidermis, the inventors incubated neck skin with AdCMV-luc (an adenoviral vector that encodes Luciferase) (Tang et al., 1997). As shown in Figure 15, luciferase activity can be detected in the ears (or as discrete luciferase spots in other areas within the skin) in any of the animals treated one day after the non-invasive administration of AdCMV-luc on the skin of the neck. Luciferase was undetectable in any of the internal organs, including lymph nodes, liver, spleen, heart, lung and kidney.

In Figure 15, 1 x 108 pfu of AdCMV-luc was incubated with neck skin for one hour. The skin of the neck, like the ears, was collected for the lucifera test as described (Tang et al., 1994) one day after inoculation. The numbers represent units of light having subtracted the background of the readings.

In another attempt to identify and characterize the target cells that are capable of expressing the transgene from an adenoviral vector applied topically, and putative mobile cells containing the protein expressed from the transgene, the inventors stained skin sections with X-gal after topical application of AdCMV-Bgal (an adenoviral vector encoding B-galactosidase) (Tang et al., 1994). By studying histological sections in search of dark blue cells, the inventors identified cells of the capillary matrix marked within the hair follicles and keratinocytes marked in the outermost layer of the epidermis as major target cells for adenovirus-mediated transduction when the vector it is inoculated in a non-invasive way (photographs are available upon request). None of the dermal fibroblasts were transduced by this procedure, although these cells are highly transducible when AdCMV-Bgal is injected intradermally using a needle (photographs are available upon request). The results suggest that few or no adenoviral particles applied topically could penetrate the dermis beyond the outer layer of the epidermis. Microscopic examination of histological sections revealed no physical abrasions of transduced skin. At the macroscopic level, no inflammation associated with the treated skin was observed. However, transduced cells could only be visualized within the area of inoculation (for example, the skin of the neck). The inventors were not able to identify dark blue cells in the ears or other areas within the skin when luciferase activity could be detected in these areas (Figure 4), probably because the luciferase assay is more sensitive than the assay of X-gal-mediated B-galactosidase. The inventors have hypothesized that some cells that have antigens (APC) can respond to antigens expressed on the surface of the skin

5 through the acquisition of the antigen. Protein can break down quickly, and so it is undetectable in internal organs, including lymph nodes. The biological importance of this transfer of antigens into the skin is unknown.

Reference Example 18

Amplification of foreign DNA in various tissues after localized gene administration in a non-invasive manner

To try to determine if the topical application of an adenoviral vector can also administer exogenous DNA beyond the inoculation area, DNA was extracted from various tissues, followed by an amplification of the transgene, as well as the adenovirus fiber type 5 gene by PCR after of non-invasive administration of AdCMVPR8.ha on the skin. As shown in Figure 16, the full-length fiber and HA genes can be amplified from the skin 3 hours after inoculation. Normally the full length gene is undetectable in the skin's DNA after 1 day, or in DNA extracted from other tissues. However, subfragments of the HA and fiber genes from the liver, whole blood, ear, abdominal skin, or lymph nodes assembled using different sets of primers could be amplified. No foreign DNA was detected in any of the tissues 4 weeks after inoculation. The results suggest that the topical application of

20 An adenoviral vector could administer exogenous DNA in a localized area of the skin, although foreign DNA can be rapidly acquired by some putative antigen presenting cells, degrading and moving to deep tissues. The removal of foreign DNA in 4 weeks emphasizes the safety of the NIVS. In Figure 16, AdCMV-PR8.ha and AdCMV-luc were inoculated into the pre-cleared skin in a non-invasive manner. The DNA was extracted using DNAZOL (GIBCO BRL) and amplified with the following set of primers:

Ha5.1 and Ha3.1 amplified the HA gene of 1.7 kg of almost full length; Ha5.2 and Ha3.2 amplified a 0.6 kb subfragment that includes 33% of the HA gene; Luc5.1 and Luc3.1 amplified the luciferase gene of 1.7 kb of almost full length; Luc5.2 and Luc3.2 amplified a 0.52 kb subfragment that includes 30% of the luciferase gene; Fb5.1 and Fb3.1 amplified the almost full length 1.7 kg adenovirus fiber gene; Fb5.2 and Fb3.2 amplified a 0.55 kb subfragment that includes 32% of the fiber gene. Lane M, molecular weight marker (lamdba DNA broken with HindIII); lane 1, the almost full length luciferase gene amplified by Luc5.1 and Luc3.1 from skin DNA 3 hours after the NIVS; lane 2, the almost full length luciferase gene amplified by Luc5.1 and Luc3.1 from skin DNA 1 day after the NIVS; lane 3, a subfragment of luciferase DNA amplified by Luc5.2 and Luc3.2 from mouse ear DNA 1 day after the NIVS; lane 4, a subfragment of luciferase DNA amplified by Luc5.2 and Luc3.2 from lymph node DNA 1 day after the NIVS; lane 5, a subfragment of luciferase DNA amplified by Luc5.2 and Luc3.2 from liver DNA 1 day after the NIVS; lane 6, a subfragment of luciferase DNA amplified by Luc5.2 and Luc3.2 from DNA extracted from whole blood 1 day after the NIVS; lane 7, the almost full-length HA gene amplified by Ha5.1 and Ha3.1 from skin DNA 3 hours after the NIVS; lane 8, a subfragment of the HA gene amplified by Ha5.2 and Luc3.2 from skin DNA 1 day after the NIVS; lane 9, a subfragment of the HA gene amplified by Ha5.2 and Ha3.2 from lymph node DNA 1 day after the NIVS; lane 10, a subfragment of the HA gene amplified by Ha5.2 and Ha3.2 from liver DNA 1 day after the NIVS; lane 11, a subfragment of the HA gene amplified by Ha5.2 and Ha3.2 from kidney DNA 1 day after the NIVS; lane 12, a subfragment of the HA gene amplified by Ha5.2 and Ha3.2 from DNA extracted from whole blood 1 day after the NIVS; lane 13, the almost full length fiber gene amplified by Fb5.1 and Fb3.1 from skin DNA 3 hours after the NIVS; lane 14, the almost full length fiber gene amplified by Fb5.1 and Fb3.1 from skin DNA 1 day after the NIVS; lane 15, a subfragment of the fiber gene amplified by Fb5.2 and Fb3.2 from skin DNA 1 day after the NIVS; lane 16, a subfragment of the fiber gene amplified by Fb5.2 and Fb3.2 from DNA of the ear 1 day after the NIVS; lane 17, a subfragment of the fiber gene amplified by Fb5.2 and Fb3.2 from lymph node DNA 1 day after the NIVS; lane 18, a subfragment of the fiber gene amplified by Fb5.2 and Fb3.2 from liver DNA 1 day after the NIVS; lane 19, a subfragment of the fiber gene amplified by Fb5.2 and Fb3.2 from DNA extracted from whole blood 1 day after the NIVS. The DNA of the lymph nodes was extracted by gathering inguinal, cervical and brachial lymph nodes in DNAZOL solution. The DNA was amplified for 35 cycles at optimized hybridization temperatures in a 40-degree Stratagene Robocycler thermocycler. The amplified DNA fragments were fractionated with 1% agarose gel and stained with ethidium bromide.

Reference Example 19

A hair removal agent is not necessary for the NIVS

To determine if a depilatory agent, such as NAIR (Tang et al., 1997) is necessary for the NIVS, antibody titres produced by vaccine patches were compared with or without a pretreatment using NAIR. Figure 17 demonstrates that antibody titers in mice without NAIR pretreatment are as high as their NAIR pretreatment counterparts. The elimination of NAIR simplifies the NIVS procedure.

In Figure 17, mice were injected intradermally (ID) with a dose of 108 pfu, or immunized in a non-invasive way (NIVS) by pipetting 108 pfu of AdCMV-hcea (Tang et al., 1997) on abdominal skin, followed by the extension of the vector as a thin film on the bare skin with a piece of the Tegaderm patch (3M). The non-absorbed vectors were washed. Serum samples were tested for anti-CEA antibodies 4 weeks after inoculation. Titles of anti-CEA IgG were determined by ELISA using purified human CEA (CalBiochem) as capture antigen. Serum samples and goat anti-mouse IgG conjugated with peroxidase (Promega) were sequentially incubated on the plates for 1 hour at room temperature with a thorough washing between each incubation. The assessment criterion was calculated as the serum dilution that produces the same OD450 as a 1/100 dilution of preimmune serum. The negative serum at the lowest dilution tested were assigned titration criteria of 1. The data was plotted as the geometric mean of the titration titration titres of ELISA, in which n = 4 for ID, n = 14 for 1 hr, n = 10 for NAIR (-), and n = 15 for NAIR / shaving (-). ID, intradermal injection; 1 hr, the vectors are

put in contact with the outer layer of the skin for an hour with shaving and pretreatment with NAIR; NAIR (-), the vectors were contacted with the outer layer of the skin overnight with shaving but without pretreatment with NAIR; NAIR / shaving (-), vectors were contacted with the outer layer of the skin overnight but without shaving or pretreatment with NAIR.

Reference Example 20

As shown in Figure 18, BALB / c mice (3 months old) were immunized by a variety of vaccination modalities, including intramuscular injection of DNA, topical application or intranasal inoculation of a tetanus-based vaccine based on adenovirus Non-invasive skin-directed vaccination was performed by pipetting approximately 108 pfu of AdCMV-tetC onto the pre-ripped abdominal skin, followed by the extension of the vector as a thin film on the bare skin with a piece of the Tegaderm patch (3M). The non-absorbed vectors were washed in one hour. Nasal vaccines were administered by pipetting approximately 107 pfu of AdCMV-tetC into the nasal cavity. All animals were immunized 3 times every 3 weeks. One week after the last booster, the mice were exposed to a lethal dose of Clostridium tetani by injection into the plantar pad and monitored daily for survival. Data are plotted as the percentage of survival versus days after exposure. Control not exposed, mice not exposed without vaccination before exposure. Ad-tetC: NIVS, mice immunized by the topical application of AdCMV-tetC; Ad-tetC: IN, mice immunized by intranasal inoculation of AdCMV-tetC; pCMV-tetC: IM, mice immunized by intramuscular injection of 100 g of pCMV-tetC DNA. AdCMV-tetC, an adenoviral vector encoding the C fragment of the Clostridium tetani toxin; pCMV-tetC, a plasmid expression vector that encodes the C fragment of Clostridium tetani toxin. The numbers in brackets represent the number of animals for each treatment.

Examples here involving nasal administration also illustrate that an adequate response can be achieved through mucosal administration, and that mucous membranes, such as those of the oral cavity, can be employed as routes for administration, for example. , oral and perlingual administration are provided by the invention and are demonstrated and analyzed by the examples herein that involve nasal administration, as well as by the general teachings herein.

Therefore, the invention includes the application of a recombinant vector vaccine containing one or more genetic insertions encoding an antigen or an epitope of interest or an immune stimulus, or a gene product from a recombinant vaccine, to the oral surface of the oral cavity, so that the product or products encoded by the inserted gene or genes produce an immune response that can be protective

or therapeutic against an infectious disease. The invention also includes said recombinant vector vaccine

or gene product of a recombinant vaccine incorporated on, within or adhered to a matrix, which forms a vehicle mechanism from which products for immunization can be released on the oral surface or into the oral cavity. The invention also includes embodiments in which the matrix in which the product is incorporated for immunization can be bioactive or inactive, and is composed of materials that maintain the integrity of the products for immunization; for example, the matrix material may be composed of polymeric substances, such as glucose or other sugars that are biodegradable, or other biodegradable substances, or materials that are disposable but may not be biodegradable.

Table 1: Detection of the expression of transgenes from genetic vectors administered by a bandage, skin testing for luciferase

Incubation time (hours)

LU per cm2 of skin

one

0

one

2,100

2

0

2

0

2

6,200

2

7,300

2

13,000

2

48,000

2

1,800

2

13,000

Incubation time (hours)

LU per cm2 of skin

18

 830

18

 2,400

18

 260

18

 630

18

 1,300,000

18

 24,000

18

 2,700

18

 280

Table 2: Summary of AdCMV-PR8.ha DNA transfer after topical application

Moment

 Pinna of the ear Abdominal skin Lymph nodesb Spleen Liver  Kidney  Blood Muscle

I. HA gene almost full length

Three hours

0/2 2/2 0/2 0/2  0/2  0/2  0/2  0/2

1 day

0/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3

1 month

0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2

II. HA gene subfragment

Three hours

0/2 2/2 0/2 0/2  0/2  0/2  0/2  0/2

1 day

1/3 3/3 3/3 1/3 2/3 2/3 2/3 2/3

1 month

0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2

III. Nearly full length fiber gene

Three hours

0/2 2/2 0/2 0/2  0/2  0/2  0/2  0/2

1 day

1/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3

1 month

0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2

IV. Fiber sub-fragment

Three hours

0/2 2/2 0/2 0/2  0/2  0/2  0/2  0/2

1 day

1/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3

1 month

0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2

The mice were immunized by topical application of AdCMV-PR8.ha as described in the previous examples and figures, for example, the description referred to in Figure 1. At the indicated times, the total DNA was extracted from the tissues and PCR amplified using sets of specific primers as described in the previous examples and figures, for example, the description referred to in Figure 3. The data is presented as number of animals containing detectable signals for a particular tissue by total number of animals. analyzed. a Administration site; b assembled lymph nodes; c quadriceps of the hind leg.

Table 3: Summary of pCMV-PR8.ha DNA transfer after intramuscular injection

Moment

 Pinna of the ear Abdominal skin Lymph nodes Spleen Liver  Kidney  Blood Muscleb

I. HA gene almost full length

Three hours

2/3  0/3  3/3 1/3 0/3  0/3  1/3  3/3

1 day

0/3  0/3  0/3  0/3 0/3 1/3 0/3 0/3

1 month

0/2  0/2  0/2  0/2 0/2 0/2 0/2 0/2

II. HA gene subfragment

Three hours

3/3  1/3  3/3 2/3 3/3  2/3  3/3  3/3

1 day

2/3  1/3  2/3  1/3 3/3 2/3 2/3 3/3

1 month

1/2  1/2  2/2  1/2 1/2 0/2 0/2 1/2

The mice were immunized by an intramuscular injection of pCMV-PR8.ha DNA as described in the previous examples and figures, for example, the description referred to in Figure 1. At the indicated times, the total DNA was extracted from the tissues. and was amplified by PCR using sets of specific primers as described in the previous examples and figures, for example, the description referred to in figure 3. The data is presented as number of animals containing detectable signals for a particular tissue by total number of analyzed animals.a assembled lymph nodes; b quadriceps of the hind leg (administration site).

Table 4: Summary of DNA transfer from AdCMV-PR8.ha after application of thermo-activated adenoviral vectors

Moment

 Pinna of the ear Abdominal skin Lymph nodesb Spleen Liver  Kidney  Blood Muscle

I. HA gene almost full length

1 day

0/3 (3/7) 1/3 (7/7) 0/3 (1/7) 0/3 (0/7)  0/3 (0/7)  0/3 (0/7)  0/3 (0/7)  0/3 (0/7)

II. HA gene subfragment

1 day

0/3 (4/7) 3/3 (7/7) 0/3 (2/7) 0/3 (1/7)  0/3 (1/7)  0/3 (0/7)  0/3 (0/7)  0/3 (0/7)

III. Nearly full length fiber gene

1 day

0/3 (2/7) 2/3 (6/7) 0/3 (1/7) 0/3 (0/7)  0/3 (1/7)  0/3 (0/7)  0/3 (0/7)  0/3 (0/7)

IV. Fiber sub-fragment

1 day

0/3 (2/7) 3/3 (7/7) 0/3 (2/7) 0/3 (0/7)  0/3 (2/7)  0/3 (1/7)  0/3 (1/7)  0/3 (0/7)

Summary of AdCMV-PR8.ha DNA transfer after topical application: AdCMVPR8.ha particles were inactivated by heating at 95 ° C for 10 min. The vectors were administered to mice by topical application, as described in the previous examples and figures, for example, the description referred to in Figure 1, or by intradermal injection of an equivalent amount of vectors using a needle. 15 days after the localized gene administration, the total DNA was extracted from various tissues. The almost full-length HA and fiber genes and their subfragments were amplified by PCR using sets of specific primers, as described in the legend of Figure 3. The data is presented as the number of animals containing detectable signals for a tissue. specific by total number of animals analyzed. Numbers without parentheses represent topical application; the numbers in brackets represent intradermal injection. a Administration site; b assembled lymph nodes; c quadriceps of the hind leg. Importance: It is possible that vector DNA can be transferred to distant tissues after topical application by three different mechanisms: (1) translocation through the skin by diffusion, followed by transfer through circulation; (2) translocation through the skin, followed by the subsequent pinocytotic uptake into antigen presenting cells (APC); (3) keratinocyte transduction, followed by an intracellular transfer of exogenous biomolecules to APCs. Although thermo-activated adenoviral vectors were unable to

transducing cells, as demonstrated by their inability to produce cytopathic effects (CPE) in human 293 cells probably due to the denaturation of fundamental ligands (eg, CAR motif and GDPR), are still able to spread to the skin as do the live vectors if diffusion occurs. The results show that it is not likely that the main mechanism that mediates translocation of vector DNA after NIVS is due to translocation through the skin by diffusion. Therefore, the topical application of adenoviral vectors may represent a non-invasive, rather than transdermal, vaccination modality.

Bibliographic references

Barry, M.A. et al., Protection against mycoplasma infection using expression-library immunization, Nature, 377, 632635 (1995).

Conry, R.M. et al., A carcinoembryonic antigen polynucleotide vaccine for human clinical use, Cancer Gene Ther., 2, 33-38 (1995).

Cotten, M. et al., High-efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles, Proc. Natl Acad. Sci. USA, 89, 6094-6098 (1992).

Glenn, G.M. et al., Skin immunization made possible by cholera toxin, Nature, 391, 851 (1998).

Gómez-Foix et al., Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism, J. Biol. Chem., 267, 25129-25134 (1992).

Johnston, S.A. and Tang, D.-c., Gene gun transfection of animal cells and genetic immunization, Meth. Cell Biol., 43, 353-365 (1994).

McDonnell, W.M. and Askari, F.K., DNA vaccines, New Engl. J. Med., 334, 42-45 (1996).

Tang, D.-c. et al., Genetic immunization is a simple method for eliciting an immune response, Nature, 356, 152-154 (1992).

Tang, D.-c. et al., Butyrate-inducible and tumor-restricted gene expression by adenovirus vectors, Cancer Gene Ther., 1, 15-20 (1994).

Tang, D.-c. et al., In vivo cytotoxicity assay for assessing immunity, J. Immunol. Methods, 189, 173-182 (1996).

Tang, D.-c. et al., Vaccination onto bare skin, Nature, 388, 729-730 (1997).

Claims (27)

1.- The use of an adenoviral vector for the preparation of a non-invasive vaccine for mucosal administration, in which the vector contains and expresses an exogenous nucleic acid encoding an influenza hemagglutinin antigen or an epitope thereof and / or a nuclear nuclear protein antigen or an epitope thereof, and the vector has deleted regions E1 and E3, and in which the non-invasive mucosal administration vaccine is to be administered to the nasal mucosa. 2. The use according to claim 1, wherein the expression in an animal of an influenza hemagglutinin antigen or an epitope thereof and / or a nuclear nuclear protein antigen or an epitope thereof, stimulates or modulates an immune response against influenza. 3. The use according to claim 2, wherein the response is induced by the vector that expresses the nucleic acid in the cells of an animal. 4. The use according to claim 2 or 3, wherein the animal is a vertebrate. 5. The use according to claim 4, wherein the vertebrate is a bird or a mammal. 6. The use according to claim 4, wherein the vertebrate is a human being, or a companion animal, domesticated, producer of food or feed, livestock, hunting, racing, or sports. 7. The use according to claim 5, wherein the animal is a cow, a horse, a dog, a cat, a goat, a sheep, a pig, or a chicken, a duck or a turkey. 8. The use according to any one of claims 1 to 7, wherein the adenoviral vector comprises a human adenovirus. 9. The use according to any one of claims 1 to 7, wherein the adenoviral vector comprises a canine adenovirus. 10. The use according to any one of claims 1 to 9, wherein the adenoviral vector comprises a viral vector to which all viral genes have been deleted. 11. An adenoviral vector that contains and expresses an exogenous nucleic acid that encodes an influenza hemagglutinin antigen or an epitope of this and / or an antigen of the nuclear flu protein or an epitope thereof, and which has Deleted regions E1 and E3, for use as a non-invasive vaccine for mucosal administration, in which the non-invasive vaccine for mucosal administration is to be administered to the nasal mucosa. 12. An adenoviral vector for use according to claim 11, wherein the expression in an animal of an influenza hemagglutinin antigen or an epitope thereof and / or an influenza nuclear protein antigen or a This epitope stimulates or modulates an immune response against influenza. 13. An adenoviral vector for use according to claim 12, wherein the response is induced by the vector that expresses the nucleic acid in the animal's cells. 14. An adenoviral vector for use according to claim 12 or 13, wherein the animal is a vertebrate. 15. An adenoviral vector for use according to claim 14, wherein the vertebrate is a bird or a mammal. 16. An adenoviral vector for use according to claim 14, wherein the vertebrate is a human being, or a companion animal, domesticated, producer of food or feed, livestock, hunting, racing, or sports. 17. An adenoviral vector for use according to claim 15, wherein the animal is a cow, a horse, a dog, a cat, a goat, a sheep, a pig, or a chicken, a duck or a turkey . 18. The adenoviral vector for use according to any one of claims 11 to 17, wherein the adenoviral vector comprises a human adenovirus. 19. The adenoviral vector for use according to any one of claims 11 to 17, wherein the adenoviral vector comprises a canine adenovirus. 20. The adenoviral vector for use according to any one of claims 11 to 19, wherein the adenoviral vector comprises a viral vector to which all viral genes have been deleted. 21.- A non-invasive mucosal administration vaccine comprising an adenoviral vector, in which the vector contains and expresses an exogenous nucleic acid encoding an influenza hemagglutinin antigen or an epitope thereof and / or an antigen of the nuclear protein of influenza or an epitope thereof, and in which the vector has deleted regions E1 and E3, for use to stimulate an immune response against influenza in an animal, in which the non-invasive vaccine for Mucosal administration is to be administered to the nasal mucosa. 22. A non-invasive vaccine for use according to claim 21, wherein the animal is a vertebrate. 23. A non-invasive vaccine for use according to claim 22, wherein the vertebrate is a bird or a mammal. 24. A non-invasive vaccine for use according to claim 22, wherein the vertebrate is a human being, or a companion animal, domesticated, producer of food or feed, livestock, game, racing, or sports. 25. A non-invasive vaccine for use according to claim 23, wherein the animal is a cow, a horse, a dog, a cat, a goat, a sheep, a pig, or a chicken, a duck or a Turkey. 26.- A non-invasive vaccine for use according to claim 21, wherein the adenoviral vector comprises a human adenovirus. 27. A non-invasive vaccine for use according to claim 21, wherein the adenoviral vector comprises a canine adenovirus. 28. A non-invasive mucosal administration vaccine for use according to any one of claims 21 to 27, wherein the adenoviral vector comprises a viral vector to which all viral genes have been deleted. 29. A kit comprising a non-invasive vaccine for use according to any one of claims 21 to 28 comprising, in a first container, the vector and, in a second container, a pharmaceutically acceptable carrier or diluent for administering the intranasal vector, in which the containers are optionally present in the same container or in separate packages; and the kit optionally contains instructions for mixing the vector and the vehicle or diluents and / or administration. The kit according to claim 29, further comprising an administration device in a third container, wherein the third container is optionally present in the same container as one or both of the first and second container, or is in a separate container of the first and second container; and wherein the kit optionally contains instructions for installing the vector or vector and the vehicle or diluent in or on the administration device and / or for administration or application of the administration device.