BR102020026607A2 - IMMUNOGENIC FORMULATION AND USES OF IMMUNOGENIC FORMULATION - Google Patents

Abstract

immunogenic formulation and uses of the immunogenic formulation. the invention relates to an immunogenic formulation that confers protection against respiratory viruses that comprises an attenuated recombinant strain of bacillus calmette and guerin (bcg), in a concentration of between 104 to 109 bacteria, which expresses at least one immunogenic protein or fragment of the virus respiratory syncytial (rsv). and the use of this immunogenic formulation to prepare a vaccine to prevent, treat or attenuate respiratory virus infections, especially hrsv and/or hmpv.

Description

IMMUNOGENIC FORMULATION AND USES OF IMMUNOGENIC FORMULATION DESCRIPTION FIELD OF THE INVENTION

[001] An immunogenic formulation useful for the preparation of a vaccine against respiratory viruses, such as respiratory syncytial virus (RSV, human orthopneumovirus) and/or human metapneumovirus (hMPV), is disclosed, wherein this formulation comprises at least an attenuated strain of Mycobacterium, preferably a strain of Bacillus Calmette-Guérin (BCG), which recombinantly expresses one or more immunogenic proteins or fragments of RSV.

BACKGROUND OF THE INVENTION

[002] Among respiratory viruses, respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) stand out for their clinical importance, as they can cause very serious infections in infants and young children. The clinical manifestations of both viruses are indistinguishable from each other, causing a wide range of clinical cases, which can be mild, such as rhinitis, or much more severe, such as pneumonia or bronchiolitis, with the most serious conditions being seen in children, premature babies, children with congenital heart disease, and in immunosuppressed patients. RSV is the main causative agent of acute respiratory tract infections in infants worldwide. According to the WHO, this virus infects 64 million people annually, of which 160,000 die. RSV is the leading cause of infection and hospitalization in children under 2 years of age. (www.who.int). In turn, the human metapneumovirus is the second cause of hospitalizations for acute respiratory infections in children under 5 years of age.

[003] Infections with these viruses are extremely frequent and recurrent, as virtually 100% of children over three years of age have had at least one episode of RSV and/or hMPV infection by age 5 years. As these infections do not leave a complete immunological memory, reinfections are frequent, decreasing in severity with increasing age of the patient.

[004] The health situation generated by these respiratory infections has a high economic impact for the affected countries. Studies carried out in developed countries estimate that the individual cost of RSV infection is over 3,000 euros (approximately $2,250,000 Chilean pesos), with a ceiling of up to 8,400 euros (approximately $6,800,000 Chilean pesos).

[005] Both RSV and hMPV are classified in the Pneumoviridae family, a family that has two genera, Metapneumovirus and Orthopneumovirus. While hMPV belongs to the Metapneumovirus genus, RSV belongs to the Orthopneumovirus genus.

[006] Both viruses contain non-segmented negative single-stranded RNA with a lipid envelope and similar but not identical organization. RSV has a genome of approximately 15 kb, with 10 genes encoding a total of 11 proteins. Five of these proteins have structural functions, corresponding to the transmembrane glycoproteins F, G, and SH, the nucleocapsid N protein, and the M matrix protein. The other four proteins, M2-1, M2-2, P, and L are involved. in viral replication and transcription. The two remaining proteins, designated NS1 and NS2, are nonstructural and are involved in virulence. On the other hand, hMPV has a genome of approximately 13 kb and comprises 8 genes encoding nine proteins: N, P, M1, F, M2-1, M2-2, SH, G, and L. hMPV does not contain the proteins NS1 and NS2. The N protein is the most conserved between the two viruses, RSV and hMPV.

[001] The RSV that infects humans has different strains or subtypes, with subgroups A and B being those that predominate in the population. The main antigenic difference between the subgroups corresponds to the G protein, which conserves only 40-44% of the amino acids between the different subgroups.

[007] Due to the differences that exist between the two viruses, it is not obvious or predictable that a single vaccine can provide simultaneous protection for RSV and hMPV. As we will explain in detail later, the vaccine of the invention is a formulation containing the Bacillus Calmette-Guérin (BCG) strain, in a concentration of between 1 x 104 to 1 x 109 colony forming units per dose, which expresses at least one protein or immunogenic fragment of respiratory syncytial virus (RSV). Surprisingly, the inventors found that this immunogenic formulation confers protection against RSV and hMPV infections.

[008] In the state of the art, we found some unsuccessful attempts to obtain a vaccine against RSV. An attempt made in the 1960s consisted of formalin-inactivated whole virus (RSV-FI), which was administered intramuscularly to volunteers in the presence of adjuvant alum (1). Contrary to expectations, this immunization produced a much more severe respiratory condition in children vaccinated after natural infection with RSV, which led to the hospitalization of 80% of those vaccinated and the death of two of them (2). The respiratory-pulmonary picture presented by the vaccinees was characterized by an unusual infiltration of eosinophils and neutrophils, together with a high titer of complement-fixing antibodies (2). Analysis of lung tissues from children vaccinated with RSV-FI who died due to presentation of RSV infection is for complement deposition, immune complexes, and the presence of eosinophils in the peribronchial regions (3). Furthermore, studies in animal models show that vaccination with RSV-FI produces a Th2-type immune response, based on CD4+ T lymphocytes. These same characteristics were observed in animals that were immunized with the G protein (4) or that received CD4+ T lymphocytes specific for this glycoprotein before infection with RSV.

[009] In the same sense, the Th2 response is not able to provide protection against viral infection sustained over time, and stimulates, through the secretion of IL-5, the eosinophils responsible for allergic reactions and inflammatory hyperreactivity, which can be even more dangerous than the viral infection. In this type of Th2 response, T cells also secrete IL-4, a cytokine that induces the generation of IgG1 and IgE isotype antibodies, inhibiting the IgM, IgG3, IgG2a, and IgG2b isotypes.

[0010] For these reasons, an effective and safe vaccine against RSV and hMPV respiratory viruses must generate a Th1-type immune response, based on CD4+ helper T lymphocytes and interferon-gamma (IFN-γ)-secreting cytotoxic T cells. This response is capable of providing sustained protection over time against these viral infections, also inducing the proliferation and differentiation of B cells that secrete antibodies of the IgG2a isotype and inhibiting the production of immunoglobulins of the IgG3, IgE, IgG2b and IgG1 isotypes.

[0011] Ongoing research on RSV vaccines has focused on the development of assays and viral subunits, such as the F (5), M2 (6) proteins, and also some conserved segments of the G protein (7). On the other hand, the generation of vaccines from mutant strains of RSV, such as those sensitive to temperature (8), with deletion of some genes (9) or recombinants for cytokines such as GM-CSF (10) have also been studied. Some of these vaccines have been tested in phase I and II clinical trials, with variable results (11-13). Another type of RSV vaccine trial is the case of vaccines based on F and G proteins, administered with adjuvants such as ISCOMs. Immunization with this type of vaccine produces increased infiltration of eosinophils into lung tissue against a new viral infection (14), which increases lung tissue damage.

[0012] The infant's immune system is characterized by preferentially developing Th2-type immune responses possibly due to the immaturity of the immune system during the first six months of life (15, 16). However, if properly stimulated, it can mount a Th1-type response (17).

[0013] In order to formulate an effective and safe vaccine against RSV and/or hMPV, the inventors carried out a final study of the immune response generated against the different proteins of RSV, in order to identify those that allow the generation of an immune system of the Th1 type response, based on cytotoxic T lymphocytes. The use of bacterial vectors for heterologous expression of viral antigens has the advantage that they can be used as live attenuated vectors, since they have intact invasiveness and are recognized as non-pathogenic. An additional advantage of certain bacterial vectors used for the expression of heterologous antigens is their recognized ability to induce Th1-type immunities (18, 19), which is highly attractive in the case of vaccine development. Bacillus Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis, used as a vaccine in newborns against Mycobacterium tuberculosis. Since BCG was approved as a TB vaccine, it has been given to over 3.3 billion people worldwide. Its massive use has been facilitated by several advantageous characteristics of this bacterium, such as its great thermostability in lyophilized form. Furthermore, immunizing newborn babies with this bacteria is not risky and produces only minimal side effects. Regarding the safety and stability of BCG, reversion to virulence has never been observed throughout all the years it has been in use. BCG is highly immunogenic and with just one dose it is possible to generate an immune response that is maintained for long periods. Importantly, BCG induces a potent Th1-type immune response in adults and children (20). This phenomenon in neonates is evidenced by the cell-type immune response that is generated against M. tuberculosis antigens (PPD), a response that can be maintained for prolonged periods (20).

[0014] To date, several bacterial, parasitic, viral antigens have been successfully expressed in this bacterial system, which when evaluated in animal models have been shown to be capable of generating humoral and cellular immunity against these antigens (21, 22). In addition, BCG has the particularity of not being neutralized by antibodies present in breast milk, and can be used as an inducer of immunity in infants (22). The present invention corresponds to an immunological formulation that contains one or more attenuated recombinant strains of Mycobacterium, bacteria, preferably the BCG strain, for RSV proteins and can be used in the preparation of vaccines against respiratory viruses, especially RSV and hMPV.

[0015] The formulation of the invention makes it possible to prevent or attenuate lung damage caused by infection by the respiratory viruses RSV and hMPV, thanks to the generation of an efficient immune response favorable to the elimination of the virus. As attenuated strains of Mycobacterium such as BCG are potent inducers of Th1-type immune responses, the immune response induced by recombinant BCG strains to RSV provides protection against infections caused by both viruses.

BRIEF DESCRIPTION OF THE INVENTION

[0016] The present invention consists of an immunogenic formulation that induces protection against infection caused by respiratory viruses, especially infections caused by respiratory syncytial virus (RSV) and/or human metapneumovirus (hMPV), or at least attenuates the pathologies caused by these viruses in mammals. The immunogenic formulation of the present invention can be used to prepare vaccines and contains colony forming units (CFU) of live attenuated recombinant strains of Mycobacterium, preferably the Bacillus Calmette-Guérin (BCG) strain that recombinantly or heterologously expresses one or more more proteins or immunogenic fragments of respiratory syncytial virus.

[0017] These RSV proteins or immunogenic fragments correspond to the RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G proteins of the RSV A or RSV B subtypes and are found inserted into the bacterial genome of attenuated Mycobacterium strains or in extrachromosomal plasmids, in one or more copies, and their expression is managed by endogenous or exogenous, constitutive or inducible BCG promoters. These proteins or immunogenic fragments of RSV can be expressed by BCG or other attenuated strains of Mycobacterium, in soluble cytoplasmic form, secreted extracellularly or as proteins bound to the cell membrane.

[0018] The immunogenic formulation disclosed in the present invention can be used in conjunction with immunogenic formulations that contain other attenuated strains of Mycobacterium or BCG and that differ in the immunogenic RSV proteins they express, as well as in the location of the genes (inserted into the genome or extrachromosomal), the number of copies of the gene protein, the promoter that induces the expression of the protein, or the fate of the immunogenic protein or RSV fragments (soluble cytoplasmic proteins, secreted extracellularly or bound to the cell membrane).

[0019] The immunogenic formulation described above is useful for the preparation of a vaccine, which can be applied to the individual subcutaneously, percutaneously, or subdermally, in conjunction with a physiological or buffered saline solution.

[0020] The immunogenic formulation of the invention can be used to vaccinate individuals who have or have not had prior contact with a respiratory syncytial virus or human metapneumovirus, to confer protection against respiratory viruses such as RSV and/or hMPV.

DESCRIPTION OF THE FIGURES

[0021] Figure 1 shows the percentage of CD8+/CD69+ cells (A) and (B) IFN-γ secretion from spleen cells of BALB/c mice immunized with BCG in saline ((PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, 0.02% Tween 80 pH 7.4, at 4°C) expressing N protein. 5 x 105 spleen cells from non-immunized animals, immunized with 1 x 10 7 RSV UFP, immunized with BCG (1 x 10 8 CFU/mouse in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1 mM KH2PO4 .47 mM, 0.02% Tween 80 pH 7.4, 4°C) or immunized with recombinant BCG for RSV N protein (1 x 10 8 CFU/mouse in PBS: 137 mM NaCl; 2. 7 mM; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, 0.02% Tween 80 pH 7.4, 4 °C) stimulated for 72 hours with 0.5 μM RSV N protein. the percentage of positive cells for CD8 and CD69 markers (A) was determined by flow cytometry. Cell supernatants were submitted to ELISA to detect the presence of secret IFN-γ ted. (B) **, p value of 0.002, Student's t test. It can be concluded that the recombinant BCG strain for the N protein of RSV produces, in mice immunized with this immunogenic formulation, a favorable response of T lymphocytes.

[0022] These results show that in response to immunization with recombinant BCG for the N protein of RSV, these CD8+ T lymphocytes are activated, as they express activation markers on their surface (CD69+) and secrete IFN-γ into the extracellular environment, the which evidences that the vaccine of the invention generates a Th1-type immune response.

[0023] Figure 2 shows a curve of body weight variation of BALB/c mice immunized with a recombinant BCG strain expressing the RSV N protein (1 x 10 8 CFU/mouse, in PBS: 137 mM NaCl; KCl at 2.7; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, 0.02% Tween 80 pH 7.4, 4°C) or with a recombinant BCG strain for RSV M2 protein (1 x 10 8 CFU / mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, 0.02% Tween 80 pH 7.4, 4°C). Unvaccinated BALB/c mice (□), immunized with 1 x 107 plaque forming units (PFU) of UV-inactivated RSV (UV lamp, 312 nm, 8 watt power) for 20 minutes (RSV-UV) (□) , immunized with a strain of wild-type BCG (WT), which does not express RSV (□) (1 x 10 8 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; KH2PO4 1.47 mM, 0.02% Tween 80 pH 7.4, 4°C), immunized with a BCG strain transformed with pMV361-N (□) (1 x 10 8 CFU/mouse, in PBS: NaCl at 137 mM; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, 0.02% Tween 80 pH 7.4, 4°C) or immunized with a BCG strain transformed with pMV361- M2 (□) (1 x 10 8 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, 0.02% Tween 80 pH 7 .4, 4 °C) were infected intranasally with 1 x 10 7 PFU of RSV, strain 13018-8. As a control, a group of unvaccinated and uninfected mice (□) was included. The change in body weight from day 0 was recorded daily for 4 days. **, p-value 0.002, Student's t test.

[0024] It can be concluded that immunization of mice with a recombinant BCG strain for RSV N or M2 proteins produces a favorable response against RSV infection, since after infection, the body weight of these mice does not vary significantly when compared to unvaccinated mice, in which a decrease in body weight is observed post-infection.

[0025] Figure 3-A shows the percentage of CD11c-/ CD11b+/ Gr1+ cells in bronchoalveolar lavage of BALB/c mice from four experimental groups: non-immunized and non-infected, non-immunized and infected intranasally with 1 x 107 strain of hMPV cz0107, mice immunized with recombinant BCG for RSV N protein (1 x 10 8 CFU/mouse) and mice immunized with recombinant BCG for hMPV P protein (1 x 10 8 CFU/mouse).

[0026] It is observed that groups immunized with the recombinant BCG strain for hMPV P protein and RSV N protein show significantly less polymorphonuclear infiltration than the group infected without immunization.

[0027] Figure 3-B shows the percentage of CD11c-/CD11b+/Gr1+ cells in the lung for the four groups studied.

[0028] As in the previous case, it is observed that the groups immunized with the recombinant BCG strain for hMPV P protein and RSV N protein show significantly lower inflammatory cell infiltration than the infected group without immunization. It can be seen that the group immunized with the recombinant BCG for hMPV P even presented a similar result to the uninfected control group (1).

[0029] Figure 4 shows the viral load in lung tissue as assessed by qPCR for the four study groups, expressed as the number of hMPV N protein copies per 5,000 copies of β-actin.

[0030] It is observed that groups immunized with both the recombinant BCG for hMPV P protein strain and the recombinant BCG for N of RSV have significantly lower viral load than the unimmunized infected group.

[0031] Figure 5 shows the levels of specific antibodies against RSV (expressed at OD 450 nm) evaluated by ELISA in sera from animals immunized with the recombinant BCG-N vaccine, at three different time points: day 0 (pre-immune ), day 21 (pre-viral challenge), and day 14 post-infection.

[0032] The results show that animals immunized with BCG-N produce anti-RSV antibodies even before viral challenge. After challenge with RSV, the levels of specific IgG immunoglobulins against the virus are significantly higher in the group of animals immunized with BCG-N in relation to the control groups.

[0033] Figure 6 shows the isotypic characterization of specific antibodies against RSV induced by the recombinant BCG-N vaccine, expressed in O.D. 450 nm, in sera from immunized and non-immunized animals evaluated by ELISA. The immunoglobulin isotype was analyzed by specific secondary antibodies against IgG2a (Figure 6A) or IgG1 (Figure 6B) at three different times. Figure 6C shows the O.D. 450 nm of IgG2a in IgG1 from data obtained by ELISA on day 14 post-infection. It is shown that the ratio of anti-RSV immunoglobulins of the IgG2a/IgG1 isotype after infection is higher in animals immunized with the vaccine of the invention BCG N.

[0034] Figure 7 shows an RSV-GFP seronetralization assay in HEp-2 cells. In Figure 7A, HEp-2 cells were treated with a mixture of serum and RSV-GFP previously incubated at 37 °C for 1 hour and after 48 hours they were visualized under an epifluorescence microscope. In Figure 7B, quantification of plaque forming units (PFUs) visualized by microscopy. In Figure 7C, quantification of GFP expression in HEp-2 cells by flow cytometry. The absence of bars in B and C indicates that viral GFP was not detected in the treated cells.

[0035] It is evident that the antibodies produced by immunization with the vaccine of the invention are capable of reducing RSV infection in vitro, which is why these immunoglobulins have neutralizing functions against respiratory syncytial virus.

[0036] Figure 8 shows the levels of specific antibodies against hMPV (expressed at O.D. 450 nm), in sera from animals immunized with the recombinant vaccine BCG-N, BCG-P and unimmunized and uninfected controls (Mock), in two different times: day 21 (pre-viral challenge) and day 7 post-infection.

[0037] The results show that immunization with recombinant BCG for RSV N protein induces an increase in hMPV-specific IgG levels before and after viral challenge, in the same proportion as immunization with recombinant BCG for P of hMPV.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention consists of an immunogenic formulation that can be used for the preparation of vaccines that induce protection against infections caused by respiratory viruses, specifically respiratory syncytial virus and/or human metapneumovirus, or that attenuate the pathologies caused by these viruses in mammals. The vaccines of the invention contain live attenuated strains of Mycobacterium, preferably Bacillus Calmette-Guérin (BCG), for example the Danish or Pasteur BCG strains which recombinantly or heterologously express one or more proteins or immunogenic fragments of RSV. The vaccines of the invention comprise between 1 x 10 4 to 1 x 10 9 CFU (colony forming units) of the described strains per dose and can be preserved, prior to administration, in a lyophilized form or in cold stabilizing saline.

• - Solução diluída de Sauton SSI (125 μg de MgSO4, 125 μg de K2HPO4, 1 mg de L-asparagina, 12,5 μg de citrato de amônio férrico, 18,4 mg de glicerol a 85%, 0,5 mg de ácido cítrico, em 1 ml de H2O) em 4 °C,
• - PBS (137 mM de NaCl; 2,7 mM de KCl; 4,3 mM de Na2HPO4; 1,47 mM de KH2PO4, pH 7,4) suplementada com Tween 80 a 0,02% e Glicerol a 20% a -80 °C, ou
• - Volume da solução: volume de lactose 25% e meio de Proskauer e Beck’s suplementado com glicose e Tween 80 (PBGT: 0,5 g de asparagina; 5,0 g de fosfato monopotássico; 1,5 g de citrato de magnésio; 0,5 g de sulfato de potássio; 0,5 ml de Tween 80 e 10,0 g de glicose por litro de água destilada) liofilizado e armazenado a uma temperatura entre 4 °C e 25 °C.

[0039] Examples of suitable stabilizing solutions for the immunogenic formulations or vaccines of the invention are:

• - Diluted Sauton SSI solution (125 μg of MgSO4, 125 μg of K2HPO4, 1 mg of L-asparagine, 12.5 μg of ferric ammonium citrate, 18.4 mg of 85% glycerol, 0.5 mg of acid citrus, in 1 ml of H2O) at 4 °C,
• - PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with 0.02% Tween 80 and 20% Glycerol a - 80°C, or
• - Volume of solution: volume of 25% lactose and Proskauer and Beck's medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate; 1.5 g of magnesium citrate; 0 .5 g of potassium sulfate, 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) lyophilized and stored at a temperature between 4 °C and 25 °C.

[0040] The attenuated recombinant Mycobacterium bacteria of the immunogenic formulation of the present invention contain genes encoding at least one RSV protein, or immunogenic fragment of RSV A or RSV B subtypes or both. The respiratory syncytial virus genome was previously described in the GeneBank database, accession numbers NC-001803 and NC-001781.

[0041] Where genes encoding at least one RSV protein, or immunogenic fragment of subtypes RSV A or RSV B or both of the present invention have at least 80% identity with the genes described in said sequences disclosed from GeneBank, accession numbers NC-001803 and NC-001781.

[0042] The immunogenic RSV proteins, or fragments, correspond to the RSV proteins NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F, or G. preferred embodiment, the immunogenic proteins or fragments correspond to N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G of RSV.

[0043] To obtain the recombinant strains of the invention, the genes encoding these proteins or their immunogenic fragments are inserted into a plasmid, which is incorporated into the bacterium by any available technique. In one embodiment, plasmid pMV361 is used, is incorporated into bacteria by electrotransformation, and is integrated into the bacterial genome by the action of mycobacteriophage integrases (23). These genes can also be inserted into extrachromosomal plasmids, such as pMV261, which is incorporated into Mycobacterium by electrotransformation and remains extrachromosomal in bacteria (23). These genes can be in one or more copies, and their expression is controlled by endogenous, constitutive or inducible BCG promoters, for example the hsp60 gene promoter and the acr gene promoter respectively. These proteins, or immunogenic fragments of RSV, can be expressed by BCG or other attenuated strains of Mycobacterium, in a soluble cytoplasmic form, secreted extracellularly or as proteins linked to the cell membrane thanks to the fusion of respiratory syncytial virus genes, or immunogenic fragments, with DNA sequences that code for peptides that function as protein targeting signals for different bacterial compartments, for example, the N-terminal sequence of the gene for the alpha antigen for extracellular secretion and the N-terminal sequence of the gene for the protein of 19 kD for membrane bound proteins.

[0044] The immunogenic formulation disclosed in the present invention can be used in conjunction with immunogenic formulations that contain one or more attenuated strains of Mycobacterium or BCG and that differ in the immunogenic RSV proteins they express, as well as in the location of the genes (inserted into the genome or extrachromosomal), the number of copies of the protein gene, the promoter that induces the expression of the protein, or the target of the protein or immunogenic fragments of RSV (cytoplasmic soluble, extracellular secreted or cell membrane bound proteins).

[0045] The inventors have demonstrated that the vaccines of the invention induce a Th1-type immune response, which includes both IgG2a isotype antibody-producing B lymphocytes, and an efficient response of IFN-γ-producing T lymphocytes. This guarantees humoral protection against these respiratory viruses and an efficient cellular response that increases the efficacy and applicability of the immunogenic formulation of the invention.

[0046] The vaccine of the invention can be administered to the subject subcutaneously, percutaneously, or subdermally, in conjunction with a buffered or physiological saline solution.

[0047] As indicated, the immunogenic formulation of the invention can be used to vaccinate individuals who have or have not had prior contact with a respiratory virus, such as respiratory syncytial virus or human metapneumovirus, in order to confer protection against these respiratory viruses or to alleviate the pathology caused by these in the future.

[0048] The following examples are applicable to immunological formulations containing an attenuated recombinant strain of Mycobacterium expressing the NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G proteins of RSV, and also all combinations of these formulations. Likewise, the examples are applicable to immunological formulations containing one or more attenuated recombinant strains of Mycobacterium; wherein said recombinant bacterium contains protein genes, or immunogenic fragments of RSV that are inserted into the bacterial genome or extrachromosomal plasmids, in one or more copies, and their expression is managed by endogenous or exogenous expressed promoters, constitutive or inducible, in a soluble cytoplasmic form, secreted extracellularly or as cell membrane bound proteins

EXAMPLES

[0049] These examples are illustrative only and are not intended to limit the range of production or application of the invention. While specific terms are used in the descriptions that follow, their use is descriptive only and not limiting.

EXAMPLE I: IMMUNOGENIC FORMULATION CONSISTING OF 108 BACTERIA FROM THE RECOMBINANT DANISH BCG STRAIN FOR RSV N GENE SUBTYPE A.

[0050] The gene is inserted in one copy in the genome of the bacteria under the regulation of the endogenous constitutive hsp60 promoter of BCG and the expression of the protein is cytoplasmic. The immunogenic formulation can be found in a diluted Sauton SSI solution (125 μg of MgSO4, 125 μg of K2HPO4, 1 mg of L-asparagine, 12.5 μg of ferric ammonium citrate, 18.4 mg of 85% glycerol , 0.5 mg of citric acid in 1 ml of H2O) at -80°C. The formulation can also be found in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4), supplemented with 20% glycerol and 0.02 80% Tween at a final concentration of 108 bacteria per 100 μΙ and stored at -80 °C. Likewise, the strains can be resuspended in a solution of volume: volume of 25% lactose and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate ; 1.5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) to later be lyophilized and stored at 25 °C.

[0051] The Danish strain of BCG was transformed by electrotransformation (24) with the plasmid pMV361/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene encoding the RSV subtype A N protein, which is expressed under the endogenous promoter and constitutive of the BCG hsp60 gene. The resulting recombinant colonies were grown (at 37 °C in supplemented Middlebrock 7H9 culture medium) until OD600nm = 1, they were centrifuged at 4,000 rpm for 20 min (Eppendorf rotor model 5702/R A-4-38) and resuspended in solution. PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4), supplemented with 20% glycerol and 0.02% Tween 80 at a final concentration of 108 bacteria per 100 μΙ and stored at -80 °C. Likewise, the strains can be resuspended in a solution of volume: volume of 25% lactose and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate ; 1.5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) to later be lyophilized and stored at 25 °C. By Western blot using antibodies to RSV N protein, the inventors observed that this BCG strain recombinantly expresses RSV subtype A N protein in the cytoplasm. Immunization of BALB/c mice with the described formulation, as a vaccine, provides protection to these animals against an intranasal infection with 107 plaque forming units of RSV subtype A (Figures 1 and 2). This immunogenic formulation can confer immunity against the N protein of RSV subtypes A and B.

EXAMPLE II: IMMUNOGENIC FORMULATION CONSISTING OF 5 X 107 RECOMBINANT DANISH BCG STRAIN BACTERIA FOR RSV SUBTYPE A N GENE AND 5 X 107 RECOMBINANT DANISH BCG STRAIN BACTERIA FOR RSV SUBTYPE A M2 GENE.

[0052] In each of the bacteria that make up the immunogenic formulation, the RSV genes are inserted into one copy in the bacterial genome under the regulation of the constitutive endogenous BCG hsp60 promoter and the protein expression is cytoplasmic. The immunogenic formulation is preserved in PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na 2HPO4; 1.47 mM KH 2PO4, pH 7.4), supplemented with 20% glycerol and Tween 80 to 0.02% at a final concentration of 108 bacteria per 100 μΙ and these were stored at -20 °C. Likewise, the strains can be resuspended in a bulk solution: 25% lactose and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1 .5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) to later be lyophilized and stored at 4 °C.

[0053] The Danish strain of BCG was transformed by electrotransformation (24) with plasmid pMV361/N or pMV361/M2, derived from plasmid pMV361 (25), which are inserted only once into the genome of the bacterium. These plasmids contain the genes for RSV subtype A N and M2 proteins, respectively, under the constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were cultured at 37 °C in Middlebrock 7H9 culture medium supplemented to OD600nm = 1, centrifuged at 4000 rpm for 20 min (Eppendorf rotor model 5702/R A-4-38) and resuspended in a of PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4), supplemented with 20% glycerol and 0.02% Tween 80 at a final concentration of 107 bacteria per 100 μl and stored at -20 °C. Likewise, the strains can be resuspended in a bulk solution: 25% lactose and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1 .5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) to later be lyophilized and stored at 4 °C. By Western blot, using antibodies to RSV N and M2 proteins, the inventors observed that these Danish BCG strains recombinantly express RSV subtype A N and M2 proteins. This immunogenic formulation can confer simultaneous immunity against the M2 and N proteins of RSV subtypes A and B.

EXAMPLE III: IMMUNOGENIC FORMULATION CONSISTING OF 106 BCG BCG PASTEUR STRAIN BACTERIA FOR A RSV SUBTYPE B PROTEIN F SEGMENT.

[0054] The gene is found in bacteria extrachromosomally in multiple copies (2-4 copies per bacterium) and encodes a fragment of RSV subtype B F protein (segment ranging from amino acid 5 to 200). The expression of this gene is under the control of the endogenous promoter constitutive of the gene encoding the BCG alpha (85 kD) antigen protein. In addition, the protein encoded by this gene has, at its N-terminus, the signal peptide HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG of the 19 kDa protein of BCG, which induces its expression in the bacterial membrane. The immunogenic formulation is preserved in PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with 0.02% Tween 80 and Glycerol at 20% at -80°C. Likewise, the strains can be resuspended in a bulk solution: 25% lactose and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1 .5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) to later be lyophilized and stored at 4 °C.

[0055] The Pasteur BCG strain was transformed by electrotransformation (24) with the plasmid pMV261/F5-200, derived from the plasmid pMV261 (25), which resides extrachromosomally in multiple copies in the bacterium. This plasmid encodes a fragment of the F gene of RSV subtype B (a segment ranging from amino acid 5 to 200) fused at its N-terminus with the signal peptide:
HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG
of the 19 kD protein of BCG, which induces its expression in the bacterial membrane. The expression of this gene is under the control of the endogenous promoter constitutive of the gene encoding the BCG alpha (85 kD) antigen protein. The resulting recombinant colonies were grown to OD600nm = 1 at 37 °C in supplemented Middlebrock 7H9 culture medium and centrifuged at 4000 rpm for 20 min (Eppendorf rotor model 5702/R A-4-38) and resuspended in PBS ( 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with 0.02% Tween 80 and 20% glycerol to a final concentration of 106 bacteria per 100 μΙ. Likewise, the strains can be resuspended in a solution of volume: volume of 25% lactose and Proskauer and Beck's medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate ; 1.5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) to later be lyophilized in aliquots with 106 bacteria each and stored at 4°C. This immunogenic formulation can confer immunity against the F protein of RSV subtypes A and B.

EXAMPLE IV: IMMUNOGENIC FORMULATION CONSISTING OF 105 RECOMBINANT DANISH BCG STRAIN BACTERIA SIMULTANEOUSLY FOR RSV SUBTYPE A N AND M2 GENES.

[0056] The N gene is inserted in a copy in the genome of the bacteria under the regulation of the endogenous constitutive promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The M2 gene is found extrachromosomally in bacteria in multiple copies (2-4 copies per bacterium) under the control of the endogenous promoter constitutive of the gene encoding the BCG alpha antigen protein (85 kD). The protein encoded by the M2 gene has, at its N-terminal end, the signal peptide:
HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG
of the 19 kDa protein of BCG, which induces its expression in the bacterial membrane. The immunogenic formulation is preserved at 4 °C lyophilized from the bacteria resuspended in a volume of solution: 25% lactose and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate; 1.5 g of magnesium citrate; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of lyophilized distilled water stored at 4 °C. Likewise, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with Tween 80 at 0.02% and 20% glycerol at a final concentration of 105 bacteria per 100 μΙ.

[0057] The Danish strain of BCG was transformed by electrotransformation (24) with the plasmid pMV361/N, derived from the plasmid pMV361 (25), which is inserted only once into the bacterial genome. This plasmid contains the gene encoding the RSV subtype A N protein, which is expressed under the endogenous promoter and constitutive of the BCG hsp60 gene. After verifying that the resulting BCG strain is recombinant for RSV N protein, it was transformed by electrotransformation (24) with plasmid pMV206/M2, derived from plasmid pMV206 (25), which resides extrachromosomally in multiple copies in the bacterium. The protein encoded by the M2 gene has, at its N-terminal end, the signal peptide:
HMKKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTTAAGTTASPGG
of the 19 kD protein of BCG, which induces its expression in the bacterial membrane. The resulting recombinant colonies were grown (at 37 °C in supplemented Middlebrock 7H9 culture medium) to OD600nm = 1, centrifuged at 4,000 rpm for 20 min (Eppendorf rotor model 5702/R A-4-38) and resuspended in one volume of solution: 25% lactose and Proskauer and Beck's medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate; 1.5 g of magnesium citrate; 0. 5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) at a final concentration of 105 bacteria per 1 ml. Finally, 1 ml aliquots were lyophilized with 10 5 bacteria and the aliquots were stored at 4°C. Likewise, strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with Tween 80 at 0.02% and 20% Glycerol at a final concentration of 105 bacteria per 100 μΙ. This immunogenic formulation can confer immunity against the N and M2 proteins of RSV subtypes A and B.

EXAMPLE V: IMMUNOGENIC FORMULATION CONSISTING OF 104 BACTERIA FROM THE RECOMBINANT DANISH BCG STRAIN FOR RSV N GENE SUBTYPE A.

[0058] The gene is inserted into one copy in the bacterial genome under the regulation of the endogenous inducible acr promoter of BCG, which is active in response to nitric oxide, low oxygen concentrations and stationary phases of growth. Protein expression is cytoplasmic. The immunogenic formulation is lyophilized from one volume: 25% bulk lactose solution and Proskauer and Beck medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate; 1.5 g of citrate, magnesium; 0.5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water) stored at 25 °C in a dilute solution of Sauton SSI (125 μg of MgSO4, 125 μg of K2HPO4, 1 mg of L-asparagine, 12.5 μg of ferric ammonium citrate, 18.4 mg of 85% glycerol, 0.5 mg of citric acid in 1 mL of H2O). Likewise, strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with Tween 80 at 0.02% and 20% Glycerol at a final concentration of 104 bacteria per 100 μΙ.

[0059] The Danish strain of BCG was transformed by electrotransformation (24) with the plasmid pMV361pacr/N, derived from the plasmid pMV361 (25), which is inserted only once into the bacterial genome. This plasmid contains the gene encoding the RSV subtype A N protein, which is expressed under the endogenous and inducible promoter of the BCG acr gene (26). The resulting recombinant colonies were grown (at 37 °C in supplemented Middlebrock 7H9 culture medium) to OD600 nm = 1, centrifuged at 4000 rpm for 20 min (Eppendorf rotor model 5702/R A-4-38) and resuspended. in one volume of solution: 25% lactose and Proskauer and Beck's medium supplemented with glucose and Tween 80 (PBGT: 0.5 g of asparagine; 5.0 g of monopotassium phosphate; 1.5 g of magnesium citrate; 0 .5 g of potassium sulfate; 0.5 ml of Tween 80 and 10.0 g of glucose per liter of distilled water). Finally, 1 ml aliquots were lyophilized with 104 bacteria and stored at 25°C. Likewise, strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with Tween 80 at 0.02% and 20% Glycerol at a final concentration of 108 bacteria per 100 μΙ. This immunogenic formulation can confer immunity against the N protein of RSV subtypes A and B.

EXAMPLE VI: IMMUNOGENIC FORMULATION CONSISTING OF 109 BACTERIA FROM THE RECOMBINANT DANISH BCG STRAIN FOR RSV N GENE SUBTYPE A.

[0060] The gene is inserted into a copy in the genome of the bacterium under the regulation of the exogenous T7 phage promoter of constitutive expression in BCG strains that co-express the T7 phage polymerase. Protein expression is cytoplasmic. The immunogenic formulation is in a diluted Sauton SSI solution (125 μg of MgSO4, 125 μg of K2HPO4, 1 mg of L-asparagine, 12.5 μg of ferric ammonium citrate, 18.4 mg of 85% glycerol, 0 .5 mg of citric acid in 1 ml of H2O) and stored at -20°C, or it can be lyophilized and stored at 4°C. Likewise, strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, pH 7.4) supplemented with Tween 80 at 0.02% and 20% Glycerol at a final concentration of 109 bacteria per 100 μΙ.

[0061] The Danish strain of BCG was transformed by electrotransformation (24) with the plasmid pMV361PT7/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene encoding RSV subtype A N protein, which is expressed under the T7 promoter activated by T7 phage polymerase expression (27).

[0062] The resulting BCG strain was transformed by electrotransformation (24) with plasmid pMV261Amp/ PolT7, derived from plasmid pMV261 (25), which resides extrachromosomally in the bacterium in multiple copies. In this plasmid, resistance to the antibiotic kanamycin (27) was replaced by resistance to the antibiotic hygromycin (Higr). The T7 phage T7 polymerase is under the control of the constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were cultured at 37 °C in Middlebrock 7H9 culture medium supplemented to OD600nm = 1, centrifuged at 4000 rpm for 20 min (Eppendorf rotor model 5702/R A-4-38) and resuspended in a diluted Sauton solution. SSI (125 μg of MgSO4, 125 μg of K2HPO4, 1 mg of L-asparagine, 12.5 μg of ferric ammonium citrate, 18.4 mg of 85% glycerol, 0.5 mg of citric acid in 1 ml of H2O) and stored at -80°C. This immunogenic formulation can confer immunity against the N protein of RSV subtypes A and B.

EXAMPLE VII: PROTECTION OF RSV BCG-N AGAINST HMPV INFECTIONS.

[0063] To determine that the vaccines of the invention provide protection against human metapneumovirus (hMPV), mice previously immunized with one of the vaccines of the invention or with a recombinant BCG vaccine for hMPV P protein effective against hMPV infection. These results were compared with mice infected with hMPV without previous immunization and with mice without infection.

• 1) Controle não infectado, não imunizado;
• 2) Infectado com hMPV sem imunização;
• 3) Imunizado com hPMV BCG-P, infectado com hMPV; e
• 4) Imunizado com RSV BCG-N, infectado com hMPV.

[0064] The experimental groups, of 3 mice each, are as follows:

• 1) Non-infected, non-immunized control;
• 2) Infected with hMPV without immunization;
• 3) Immunized with hPMV BCG-P, infected with hMPV; and
• 4) Immunized with RSV BCG-N, infected with hMPV.

[0065] First, the infiltrate of polymorphonuclear cells in the lung and bronchial alveolar lavage (BAL) was analyzed by flow cytometry for the 4 groups, determining the percentage of CD11c-/CD11b+/Gr1+ polymorphonuclear cells. The presence of these cells is directly related to the inflammatory response to viral infection. Figure 3-A shows the percentage of CD11c-/CD11b+/Gr1+ cells in the BAL for the four groups studied. It is observed that the groups immunized with BCG-P and BCG-N have significantly less inflammatory reaction than the group infected without immunization.

[0066] Figure 3-B shows the percentage of CD11c-/CD11b+/Gr1+ cells in the lung for the four groups studied. As in the previous case, it is observed that the groups immunized with BCG-P and BCG-N have significantly lower inflammatory cell infiltration than the group infected without immunization. It is observed that group 3, immunized with BCG-P, presents a similar result to the uninfected group (1).

[0067] Second, the viral load in lung tissue of the 4 groups was determined by quantitative PCR (qPCR) (Figure 4), expressed as the number of hMPV N protein copies per 5,000 copies of β-actin. It is observed that the immunized groups have a significantly lower viral load than the infected non-immunized group.

[0068] As can be appreciated, the vaccine of the invention provides similar protection against an hMPV infection to the protection afforded by a recombinant BCG vaccine to a protein from the same hMPV virus, in this case the P protein.

EXAMPLE VIII: INDUCTION OF HUMORAL RESPONSE BY THE BCG RSV-N VACCINE

[0069] To determine that the vaccines of the invention induce a Th1-type response, BALB/cJ mice aged 6 to 8 weeks were given a subcutaneous injection in the back with 1 x 10 8 CFU of BCG WT or recombinant BCG expressing the RSV protein. -N, in a final volume of 100 μΙ per dose. After 14 days, they were boosted with the same starting dose. After 21 days of the first injection of BCG WT or BCG-N, the animals were challenged, for which RSV strain A2 13018-8 obtained from a clinical isolate was used. Preimmune serum samples were obtained (day 0), before viral challenge (day 21) and 14 days after infection. These samples were analyzed by ELISA at a dilution of 1/500. The results show that animals immunized with BCG-N produce anti-RSV antibodies even in phases prior to viral challenge, this production is significantly higher than non-immunized controls and animals immunized with BCG WT. After challenge with RSV, levels of virus-specific IgG immunoglobulins show an even greater increase that is maintained at day 14 post-infection (Figure 5).

[0070] To establish the isotype of the immunoglobulins produced through immunization, the levels of RSV-specific IgG1 and IgG2a isotype antibodies were analyzed by ELISA in the serum of the test animals. From day 7 post-infection, a significantly greater increase in the production of antibodies of the IgG2a isotype is observed in animals immunized with the BCG RSV-N vaccine of the invention, compared to control animals, which includes animals vaccinated with wild type. of BCG, and not immunized. On the other hand, IgG1 isotype anti-RSV immunoglobulins show an increase in animals vaccinated with BCG-N before infection, however, the RSV control group and the animals vaccinated with BCG WT are already able to equal their production on day 14 after the infection. viral challenge (Figures 6-A, 6-B). The latter is better evidenced in Figure 6-C, where it is shown that the ratio of anti-RSV immunoglobulins of the IgG2a/IgG1 isotype is higher in animals immunized with the BCG RSV-N vaccine of the invention than in the control group infected only with RSV. . As we have already pointed out, this relationship is indicative of an immune response polarized to a Th1 phenotype.

[0071] In order to determine whether the humoral response induced by vaccination was able to protect against RSV infection, after complement inactivation, the RSV-GFP virus was incubated with sera from animals from all experimental groups on day 14 post -infection and before viral challenge, and then HEp-2 cells were infected with these mixtures. In Figure 7, epifluorescence microscopy images show a decrease in RSV-GFP expression in cells previously treated with serum from animals immunized with BCG-N in relation to the other treatments. When quantifying the plaque forming units, a significant decrease in this parameter is evidenced, only in the treatment with serum from day 14 post-infection of animals immunized with BCG-N (Figure 7-B) and in the analysis of RSV expression -GFP by flow cytometry, the same trend is evidenced (compare Figure 7-C with 7-B).

EXAMPLE IX: PRODUCTION OF ANTI-HMPV IGG ANTIBODIES IN ANIMALS IMMUNIZED WITH RSV BCG-N.

[0072] In order to evaluate the production of specific immunoglobulins against hMPV, serum samples were obtained from BALB/cJ animals challenged with this virus and previously immunized with the vaccine of the invention, recombinant BCG for RSV N protein (BCG-N ) or with a recombinant BCG vaccine for hMPV P protein (BCG-P). These results were compared with animals infected with hMPV without previous immunization and with an uninfected control (Mock). Sera are compared at 2 different times: previral challenge and day 7 post-infection. Samples were analyzed by ELISA at 1/1000 dilution. The results are seen in Figure 8, where it is shown that total anti-hMPV IgG immunoglobulins significantly increased in the vaccinated animals compared to the control groups.

[0073] It is highly significant that animals immunized with RSV BCG-N and hMPV BCG-P achieve the same levels of specific antibodies against human metapneumovirus before and after viral challenge. This means that the vaccines of the invention, which include RSV proteins, provide protection against human metapneumovirus, similar to that obtained by a specific vaccine for that virus based on a recombinant BCG for the P protein of hMPV.

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Claims (13)

IMMUNOGENIC FORMULATION that provides protection against respiratory viruses, characterized by comprising an attenuated recombinant strain of Mycobacterium, preferably the Bacillus Calmette-Guerin (BCG) strain, in an amount between 104 to 109 CFU per dose, which expresses at least one immunogenic protein or fragment of respiratory syncytial virus (RSV) in a pharmaceutically acceptable saline buffer solution. IMMUNOGENIC FORMULATION according to claim 1, characterized in that at least one RSV protein corresponds to RSV A or RSV B subtypes or to both subtypes. IMMUNOGENIC FORMULATION according to claim 1, characterized in that at least one RSV protein or immunogenic fragment belongs to the group consisting of RSV proteins NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 3, characterized by the genes coding for the proteins or immunogenic fragments of RSV to be inserted into the Mycobacterium genome, preferably BCG or extrachromosomal plasmids, in one or more copies. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 4, characterized in that the expression of protein genes is controlled by endogenous or exogenous, constitutive or inducible Mycobacterium BCG promoters. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 5, characterized in that immunogenic proteins or RSV fragments can be expressed by BCG in soluble cytoplasmic form, secreted extracellularly or as proteins bound to the cell membrane. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 6, characterized in that the formulation can also be used in conjunction with other immunogenic formulations, which express RSV proteins or different immunogenic fragments of NS2, N, P, M, SH, M2 proteins (ORF1), M2 (ORF2), L, F or G. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 6, characterized in that the formulation can also be used in conjunction with other immunogenic formulations, which differ in the way in which they express soluble, soluble-cytoplasmic, extracellularly secreted or extracellularly secreted proteins or immunogenic fragments. proteins bound to the cell membrane. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 6, characterized in that the formulation can also be used together with other immunogenic formulations or formulations that differ in the number of copies of the genes of the proteins or immunogenic fragments of RSV, as well as in the way in which they the expression of RSV proteins or immunogenic, constitutive or inducible fragments are controlled and also differ in the fate of the RSV protein or immunogenic fragment. IMMUNOGENIC FORMULATION, according to any one of claims 1, 7, 8 or 9, characterized in that it is stabilized by freezing, lyophilization or buffered saline solution for conservation before use. IMMUNOGENIC FORMULATION, according to any one of claims 1 to 10, characterized in that the immunogenic formulation is stabilized by freezing, lyophilization or buffered saline solution for its preservation before use. USE OF THE IMMUNOGENIC FORMULATION as defined in any one of claims 1 to 11, characterized in that it is used to prepare a vaccine to prevent, treat or attenuate RSV and/or hMPV infections, wherein said formulation contains between 1 x 104 to 1 x 109 colony forming units of the attenuated recombinant Mycobacterium strain of claims 1 to 11 stabilized with saline. USE OF THE IMMUNOGENIC FORMULATION, as defined in any one of claims 1 to 11, characterized in that it is used to prepare a vaccine to prevent, treat or attenuate RSV and/or hMPV infections to be administered subcutaneously, percutaneously or subdermally in saline solution acceptable physiological.

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