NEW USE OF BCG IMMUNOGENIC FORMULATION EXPRESSING A RESPIRATORY SYNCITIAL VIRUS PROTEIN AGAINST hMPV

Abstract

The invention relates to the novel use of an immunogenic formulation containing the bacillus Calmette-Guérin (BCG) strain at a concentration between 104-109 bacteria, expressing at least one protein or immunogenic fragment of respiratory syncytial virus (RSV, Human orthopneumovirus), in a pharmaceutically acceptable saline buffer solution because it serves to prepare a vaccine useful to prevent, treat, or attenuate human metapneumovirus (hMPV) infections.

Claims

1-11. (canceled)

12. Use of an immunogenic formulation containing the Bacillus strain of Calmette and Guerin (BCG), at a concentration between 10.sup.4-10.sup.9 bacteria, expressing at least one protein or immunogenic fragment of respiratory syncytial virus (RSV), in a pharmaceutically acceptable saline buffer solution wherein the RSV immunogenic protein or fragment corresponds to the RSV nucleocapsid protein (N protein) and serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

13. Use according to claim 12 where the RSV immunogenic protein or fragment corresponds to the N protein of RSV A or RSV B subtypes or both subtypes wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

14. Use according to claim 13 where the genes encoding the proteins or immunogenic fragments of the RSV N protein are inserted into the genome of Mycobacterium, preferably BCG or in extrachromosomal plasmids, in one or more copies wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

15. Use according to claim 14 where the expression of protein genes are commanded by endogenous or exogenous promoters of Mycobacterium BCG, constitutive or inducible wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

16. Use according to claim 12 where the immunogenic RSV proteins or fragments corresponding to the RSV N protein can be expressed by BCG in a soluble-cytoplasmic form, secreted extracellularly or as cell membrane-bound proteins wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

17. Use according to claim 12 where the formulation can be used in conjunction with other immunogenic formulations, expressing proteins or immunogenic fragments different from RSV N wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

18. Use according to claim 12 where the formulation can be used in conjunction with other immunogenic formulations, which differ in the form expressed by immunogenic proteins or fragments, soluble, soluble-cytoplasmic, extracellularly secreted or proteins bound to cell membrane wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

19. Use according to claim 12 where the formulation can be used in conjunction with other immunogenic formulations or differing in the number of copies of RSV protein genes or immunogenic fragments, as well as in the way in which the expression of RSV proteins or immunogenic fragments is controlled, constitutive or inducible and also differing in the destination of the RSV protein or immunogenic fragment wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

20. Use according to claim 12 where the formulation can be stabilized by freezing, freeze-drying or saline buffer for preservation prior to use wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human methamneumovirus infections.

21. Use according to claim 12 where the formulation can be administered subcutaneously, percutaneously or subdermally in acceptable physiological saline solution wherein it serves to prepare a vaccine useful for preventing, treating, or attenuating human metapneumovirus infections.

Description

DESCRIPTION OF THE FIGURES

[0022] FIG. 1 shows the percentage of CD8+/CD69+ cells (A) and (B) IFN-7 secretion of cells from the spleen of BALB/c mice immunized with BCG in saline solution ((PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4, Tween 80 0.02% pH 7.4, a 4° C.) that expresses the N protein. 5×10.sup.5 cells from the spleen of unimmunized animals, immunized with 1×10.sup.7 PFU of RSV, immunized with BCG (1×10.sup.8 CFU/mouse in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.) or immunized with recombinant BCG for the RSV N protein (1×10.sup.8 CFU/mouse in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.) stimulated for 72 hrs. with 0.5 μM of RSV N protein. Subsequently, the percentage of positive cells for the CD8 and CD69 (A) markers was determined by flow cytometry. Cell supernatants were subjected to ELISA to detect the presence of secreted IFN-γ. (B) **, p value 0.002, Student's t-test. It can be concluded that the recombinant BCG strain for the RSV N protein produces, in mice immunized with this immunogenic formulation, a favorable response by T lymphocytes.

[0023] These results show that in response to immunization with recombinant BCG for RSV N protein, these CD8+ T lymphocytes are activated, since they express activation markers on their surface (CD69+) and secrete IFN-γ to the extracellular medium, which evidence that the vaccine of the invention generates a Th1-type immune response.

[0024] FIG. 2 shows a curve of body weight variation of BALB/c mice immunized with a recombinant BCG strain that express RSV N protein (1×10.sup.8 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.) or with a recombinant BCG strain for RSV M2 protein (1×10.sup.8 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.). Unvaccinated BALB/c mice (.square-solid.), immunized with 1×10.sup.7 plaque-forming units (PFU) of UV-inactivated RSV (UV Bulb, 312 nm, power 8 watts) for 20 minutes (RSV-UV) (.box-tangle-solidup.), immunized with a strain of wild BCG (WT), which does not express RSV (.Math.) (1×10 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.), immunized with a BCG strain transformed with pMV361-N (□) (1×10.sup.8 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.) or immunized with a BCG strain transformed with pMV361-M2 (Δ) (1×10.sup.8 CFU/mouse, in PBS: 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, Tween 80 0.02% pH 7.4, 4° C.) were infected intranasally with 1×10.sup.7 PFU of RSV, strain 13018-8. As a control, a group of unvaccinated and uninfected mice (.circle-solid.) was included. The variation in body weight with respect to day 0 was recorded daily for 4 days. **, p-value 0.002, Student's t-test.

[0025] It can be concluded that the immunization of mice with a recombinant BCG strain for RSV N or M2 proteins produces a favorable response against RSV infection since post-infection the body weight of these mice does not vary significantly when compared to that of unvaccinated mice, in which a decrease in body weight is observed post-infection.

[0026] FIG. 3-A shows the percentage of CD11c.sup.−/CD11b.sup.+/Gr1.sup.+ cells in bronchoalveolar lavage of BALB/c mice from four experimental groups: unimmunized and uninfected, unimmunized and intranasally infected with 1×10.sup.7 of hMPV strain cz0107, mice immunized with BCG recombinant for the RSV N protein (1×10.sup.8 CFU/mouse) and mice immunized with recombinant BCG for hMPV P protein (1×10.sup.8 CFU/mouse).

[0027] It is observed that the groups immunized with the recombinant BCG strain for the P protein of hMPV and the N protein of RSV show significantly less polymorphonuclear infiltration than the infected group without immunization.

[0028] FIG. 3-B shows the percentage of CD11c.sup.−/CD11b.sup.+/Gr1.sup.+ cells in the lung for the four groups studied.

[0029] As in the previous case, it is observed that the groups immunized with the recombinant BCG strain for the P protein of hMPV and the N protein of RSV show significantly less infiltration of inflammatory cells than that of the infected group without immunization. It can be seen that the group immunized with the recombinant BCG for hMPV P even shows a similar result to that of the uninfected control group (1).

[0030] FIG. 4 shows the viral load in lung tissue evaluated by qPCR for the four study groups, expressed as the number of copies of the hMPV N protein per 5,000 copies of β-actin. It is observed that the groups immunized both with the recombinant BCG strain for hMPV P protein and with the recombinant BCG for RSV N have a significantly lower viral load than the unimmunized infected group.

[0031] FIG. 5 shows the levels of specific antibodies against RSV (expressed in O.D. 450 nm) evaluated by ELISA in sera of 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 the viral challenge. After the 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] FIG. 6 shows the isotype characterization of the 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 isotype of the immunoglobulins was analyzed by specific secondary antibodies against IgG2a (FIG. 6A) or IgG1 (FIG. 6B) at three different times. FIG. 6C shows the ratio of O.D. 450 nm of IgG2a on IgG1 from data obtained by ELISA at day 14 post-infection. It is shown that the ratio of anti-RSV immunoglobulins of isotype IgG2a/IgG1 after the infection is higher in animals immunized with the vaccine of the invention BCG N.

[0034] FIG. 7 shows an RSV-GFP seroneutralization assay in HEp-2 cells. FIG. 7 A 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 in an epifluorescence microscope. FIG. 7 B Quantification of plaque-forming units (PFUs) visualized by microscopy. FIG. 7 C 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, for which reason these immunoglobulins possess neutralizing functions against the respiratory syncytial virus.

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

[0037] The results show that immunization with recombinant BCG for the RSV N protein induces an increase in the levels of specific IgG against hMPV 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 recombinant strains of Mycobacterium, preferably Bacillus Calmette-Guérin (BCG), for example, the BCG Danish or Pasteur strains that recombinantly or heterologously express one or more proteins or immunogenic fragments of RSV. The vaccines of the invention comprise between 1×10.sup.4-1×10.sup.9 CFU (colony forming units) of the strains described per dose and can be preserved, before administration, in a lyophilized form or a cold stabilizing saline solution.

[0039] Examples of appropriate stabilizer solutions for the immunogenic formulations or vaccines of the invention are: [0040] Sauton SSI diluted solution (125 μg MgSO.sub.4, 125 μg K.sub.2HPO.sub.4, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid, in 1 ml of H.sub.2O) at 4° C., [0041] PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at −80° C. or [0042] Volume solution: volume of lactose 25% 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 magnesium citrate; 0.5 g potassium sulphate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) lyophilized and stored at a temperature range of between 4° C. and 25° C.

[0043] The attenuated recombinant Mycobacterium bacteria of the immunogenic formulation of the present invention contain genes encoding for at least one RSV protein, or immunogenic fragment of the RSV A or RSV B subtypes or both. The respiratory syncytial virus genome has been previously described in the GeneBank database, access numbers NC-001803 and NC-001781.

[0044] Where the genes encoding at least one RSV protein, or immunogenic fragment of the RSV A or RSV B subtypes or both of the present invention have at least 80% identity with the genes described in said GeneBank disclosed sequences, access numbers NC-001803 and NC-001781.

[0045] The RSV immunogenic proteins, or fragments, corresponding to the RSV NS1, NS2, N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F, or G proteins. In a preferred embodiment, the immunogenic proteins or fragments correspond to N, P, M, SH, M2 (ORF1), M2 (ORF2), L, F or G of RSV.

[0046] To obtain the recombinant strains of the invention, the genes that code for these proteins or their immunogenic fragments are inserted into a plasmid, which is incorporated into the bacterium by any available technique. In one embodiment, the plasmid pMV361 is used, it is incorporated into the bacterium by electro transformation, and it 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, promoters of BCG, for example, the promoter of the hsp60 gene and the promoter of the acr gene respectively. These proteins, or immunogenic fragments of RSV, can be expressed by BCG or other attenuated strains of Mycobacterium, in a soluble-cytoplasmic way, secreted extracellularly or as proteins bound to the cell membrane thanks to the fusion of the genes of the respiratory syncytial virus, or immunogenic fragments, with DNA sequences that code for peptides that function as targeting signals of proteins to the 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 19 kD protein for membrane-bound proteins.

[0047] 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 that 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 RSV immunogenic protein or fragments (soluble-cytoplasmic, extracellularly secreted or proteins bound to the cellular membrane).

[0048] The inventors have shown that the vaccines of the invention induce a Th1-type immune response, which includes both IgG2a isotype antibody-producing B lymphocytes, and an efficient IFN-γ-producing T lymphocyte response. This guarantees humoral protection against these respiratory viruses and an efficient cellular response that enhances both the effectiveness and the applicability of the immunogenic formulation of the invention.

[0049] The vaccine of the invention can be administered to the individual subcutaneously, percutaneously, or subdermally, in conjunction with a buffered or physiological saline solution.

[0050] As indicated, the immunogenic formulation of the invention can be used to vaccinate individuals who have or have not had previous contact with a respiratory virus such as respiratory syncytial virus or human metapneumovirus, in order to confer protection against these respiratory viruses or to attenuate the pathology caused by these in the future.

[0051] 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; where said recombinant bacteria contain protein genes, or immunogenic fragments of RSV that are inserted in the bacterial genome or in extrachromosomal plasmids, in one or more copies, and their expression is managed by endogenous or exogenous, constitutive or inducible, expressed promoters, in a soluble-cytoplasmic form, secreted extracellularly or as proteins bound to the cell membrane.

EXAMPLES

[0052] These examples are illustrative only and are not intended to limit the range of production or application of the invention. Although specific terms are used in the following descriptions, their use is descriptive only and not limiting.

Example I: Immunogenic Formulation Consisting of 10.SUP.8 .Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A

[0053] The gene is inserted in a copy in the genome of the bacterium under the regulation of the constitutive endogenous promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The immunogenic formulation can be found in a diluted Sauton SSI solution (125 μg MgSO.sub.4, 125 μg K2HPO4, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg 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 Na.sub.2HPO.sub.4; 1.47 mM KH2PO4, pH 7.4), supplemented with 20% Glycerol and 0.02 Tween 80% at a final concentration of 10.sup.8 bacteria per 100 μl and stored at −80° C. In the same way, the strains can be resuspended in a volume solution: volume of lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 25° C.

[0054] The Danish BCG strain 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 coding for the RSV N protein subtype A, which is expressed under the endogenous and constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were grown (at 37° C. in supplemented Middlebrock 7H9 culture medium) until OD.sub.600nm=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 Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4), supplemented with 20% Glycerol and 0.02% Tween 80 at a final concentration of 10.sup.8 bacteria per 100 μl and stored at −80° C. In the same way, the strains can be resuspended in a volume solution: volume of lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g 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 the RSV N protein subtype A in the cytoplasm. Immunization of BALB/c mice with the described formulation, as a vaccine, confers protection of these animals against an intranasal infection with 10.sup.7 plaque-forming units of RSV subtype A (FIGS. 1 y 2). This immunogenic formulation can confer immunity against RSV N protein subtype A and B.

Example II: Immunogenic Formulation Consisting of 5×10.SUP.7 .Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A and 5×10.SUP.7 .Bacteria of the Recombinant Danish BCG Strain for the M2 Gene of RSV Subtype A

[0055] In each of the bacteria that make up the immunogenic formulation, the RSV genes are inserted in a copy in the genome of the bacterium under the regulation of the constitutive endogenous promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The immunogenic formulation is preserved in PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4), supplemented with Glycerol 20% and Tween 80 0.02% a final concentration of 10.sup.8 bacteria per 100 μl and they were stored at −20° C. In the same way, the strains can be resuspended in a volume solution: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 4° C.

[0056] The Danish BCG strain was transformed, by electrotransformation (24) with the plasmid pMV361/N or pMV361/M2, derived from the plasmid pMV361 (25), which are inserted only once into the genome of the bacterium. These plasmids contain the genes for the RSV subtype A proteins N and M2 respectively, under the constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were grown at 37° C. in supplemented Middlebrock 7H9 culture medium until OD.sub.600 nm=1, were centrifuged at 4000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and were resuspended in a solution PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4), supplemented with 20% Glycerol and 0.02% Tween 80 at a final concentration of 10.sup.7 bacteria per 100 μl and stored at −20° C. In the same way, the strains can be resuspended in a volume solution: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 4° C. By Western blot, with the use of antibodies to RSV proteins N and M2, the inventors observed that these BCG Danish strains recombinantly express RSV subtype A proteins N and M2. This immunogenic formulation can confer simultaneous immunity against the M2 and N proteins of RSV subtype A and B.

Example III: Immunogenic Formulation Consisting of 10.SUP.6 .Bacteria of the Recombinant Pasteur BCG Strain for a Segment of the F Protein of RSV Subtype B

[0057] The gene is found in bacteria extrachromosomally in multiple copies (2-4 copies per bacterium) and codes for a fragment of the RSV subtype B protein F (segment ranging from amino acid 5 to 200). The expression of this gene is under the control of the constitutive endogenous promoter of the gene encoding the alpha-antigen protein (85 kD) of BCG. Furthermore, the protein encoded by this gene has, at its N-terminal end, the peptide signal

TABLE-US-00001 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 Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at −80° C. In the same way, the strains can be resuspended in a volume solution: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized and stored at 4° C.

[0058] The Pasteur BCG strain was transformed, by electrotransformation (24) with the plasmid pMV261/F.sub.5-200, 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 that goes from amino acid 5 to 200) fused at its N-terminal end with the peptide signal:

TABLE-US-00002 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 constitutive endogenous promoter of the gene encoding the alpha antigen protein (85 kD) of BCG. The resulting recombinant colonies were grown up to OD.sub.600 nm=1, at 37° C. in supplemented Middlebrock 7H9 culture medium and were centrifuged at 4,000 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 Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10.sup.6 bacteria per 100 μl. In the same way, the strains can be resuspended in a volume solution: volume of lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g citrate magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) to later be lyophilized in aliquots with 10.sup.6 bacteria each and stored at 4° C. This immunogenic formulation can confer immunity against the F protein of RSV subtype A and B.

Example IV: Immunogenic Formulation Consisting of 10.SUP.5 .Bacteria of the Recombinant Danish BCG Strain Simultaneously for the N and M2 Genes of RSV Subtype A

[0059] The N gene is inserted in a copy in the genome of the bacterium under the regulation of the constitutive endogenous promoter hsp60 of BCG and the expression of the protein is cytoplasmic. The M2 gene is found extrachromosomally in the bacterium in multiple copies (2-4 copies per bacterium) under the control of the constitutive endogenous promoter of the gene encoding the alpha antigen protein (85 kD) of BCG. The protein encoded by the M2 gene has, at its N-terminal end, the peptide signal

TABLE-US-00003 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 solution volume: lactose 25% and Proskauer and Beck Medium supplemented with glucose and Tween 80 (PBGT: 0.5 g asparagine; 5.0 g monopotassium phosphate; 1.5 g magnesium citrate; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of lyophilized distilled water stored at 4° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10′ bacteria per 100 μl.

[0060] The Danish BCG strain 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 coding for the RSV N protein subtype A, which is expressed under the endogenous and constitutive promoter of the BCG hsp60 gene. After verifying that the resulting BCG strain is recombinant for the RSV N protein, it was transformed by electrotransformation (24) with the plasmid pMV206/M2, derived from the plasmid pMV206 (25), which resides extrachromosomally in multiple copies in the bacteria. The protein encoded by the M2 gene has, at its N-terminal end, the peptide signal

TABLE-US-00004 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) until OD.sub.600 nm=1, were centrifuged at 4,000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and were resuspended in a solution volume: lactose 25% 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 magnesium citrate; 0.5 g sulfate of potassium; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) at a final concentration of 10′ bacteria per 1 ml. Finally, 1 ml aliquots were lyophilized with 10.sup.5 bacteria and the aliquots were stored at 4° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10.sup.5 bacteria per 100 μl. This immunogenic formulation can confer immunity against the N and M2 proteins of RSV subtype A and B.

Example V: Immunogenic Formulation Consisting of 10.SUP.4 .Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A

[0061] The gene is inserted in 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. The expression of the protein is cytoplasmic. The immunogenic formulation is lyophilized from a volume: volume solution of 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 citrate. magnesium; 0.5 g potassium sulfate; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water) stored at 25° C. in dilute Sauton SSI solution (125 μg MgSO.sub.4, 125 μg K.sub.2HPO.sub.4, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H.sub.2O). Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10.sup.4 bacteria per 100 μl.

[0062] The Danish BCG strain was transformed by electrotransformation (24) with the plasmid pMV361.sub.Pacr/N, derived from the plasmid pMV361 (25), which is inserted only once into the genome of the bacterium. This plasmid contains the gene that codes for the RSV N protein subtype A, 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) until OD.sub.600 nm=1, were centrifuged at 4,000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and were resuspended in a solution volume: lactose 25% 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 magnesium citrate; 0.5 g sulfate of potassium; 0.5 ml Tween 80 and 10.0 g glucose per liter of distilled water). Finally, 1 ml aliquots were lyophilized with 10.sup.4 bacteria and stored at 25° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10.sup.8 bacteria per 100 μl. This immunogenic formulation can confer immunity against RSV N protein subtype A and B.

Example VI: Immunogenic Formulation Consisting of 10.SUP.9 .Bacteria of the Recombinant Danish BCG Strain for the N Gene of RSV Subtype A

[0063] The gene is inserted in a copy in the genome of the bacterium under the regulation of the exogenous promoter of phage T7 of constitutive expression in strains of BCG that co-express the polymerase of phage T7. The expression of the protein is cytoplasmic. The immunogenic formulation is in a diluted Sauton SSI solution (125 μg MgSO.sub.4, 125 μg K.sub.2HPO.sub.4, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H.sub.2O) and was stored at −20° C., or it can be lyophilized and stored at 4° C. Similarly, the strains can be preserved in a PBS solution (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na.sub.2HPO.sub.4; 1.47 mM KH.sub.2PO.sub.4, pH 7.4) supplemented with Tween 80 0.02% and Glycerol 20% at a final concentration of 10.sup.9 bacteria per 100 μl.

[0064] The Danish BCG strain was transformed by electrotransformation (24) with the plasmid pMV361.sub.PT7/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 N protein subtype A, which is expressed under the T7 promoter activated by the expression of phage T7 polymerase (27).

[0065] The resulting BCG strain was transformed by electrotransformation (24) with the plasmid pMV261.sub.Amp/PolT7, derived from the plasmid pMV261 (25), which resides extrachromosomally in the bacterium in multiple copies. In this plasmid, resistance to the antibiotic kanamycin (27) has been replaced by resistance to the antibiotic hygromycin (Higr). The T7 polymerase of phage T7 is under the control of the constitutive promoter of the BCG hsp60 gene. The resulting recombinant colonies were grown at 37° C. in supplemented Middlebrock 7H9 culture medium until OD.sub.600 nm=1, centrifuged at 4000 rpm for 20 min (eppendorf rotor model 5702/R A-4-38) and resuspended in a solution diluted Sauton SSI (125 μg MgSO.sub.4, 125 μg K.sub.2HPO.sub.4, 1 mg L-asparagine, 12.5 μg ferric ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid in 1 ml of H.sub.2O) and was stored at −80° C. This immunogenic formulation can confer immunity against RSV N protein subtype A and B.

Example VII: Protection from RSV BCG-N, Against hMPV Infections

[0066] 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 the P protein of hMPV effective against infection by hMPV. These results were compared with mice infected with hMPV without prior immunization and with mice without infection.

[0067] The experimental groups, of 3 mice each, are the following: [0068] 1) Control Uninfected, unimmunized [0069] 2) Infected with hMPV without immunization [0070] 3) Immunized with hPMV BCG-P, infected with hMPV [0071] 4) Immunized with RSV BCG-N, infected with hMPV

[0072] First, the infiltrate of polymorphonuclear cells in the lung and bronchoalveolar lavage (BAL) was analyzed by flow cytometry for the 4 groups, determining the percentage of CD11c.sup.−/CD11b.sup.+/Gr1.sup.+ polymorphonuclear cells. The presence of these cells is directly correlated with the inflammatory response to viral infection. FIG. 3-A shows the percentage of CD11c.sup.−/CD11b.sup.+/Gr1.sup.+ cells in BAL for the four groups studied. It is observed that the groups immunized with BCG-P and BCG-N show significantly less inflammatory reaction than the infected group without immunization.

[0073] FIG. 3-B shows the percentage of CD11c.sup.+/CD11b.sup.+/Gr1.sup.+ 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 show infiltration of inflammatory cells significantly less than that of the infected group without immunization. It can be seen that group 3, immunized with BCG-P, shows a similar result to that of the uninfected group (1).

[0074] Second, the viral load in lung tissue of the 4 groups was determined by quantitative PCR (qPCR) (FIG. 4), expressed as the number of copies of the hMPV N protein per 5,000 copies of β-actin. It is observed that the immunized groups have a significantly lower viral load than the unimmunized infected group.

[0075] As can be appreciated, the vaccine of the invention provides similar protection against an hMPV infection, to the protection granted by a recombinant BCG vaccine for a protein of the same hMPV virus, in this case, the P protein.

Example VIII: Induction of Humoral Response by BCG RSV-N Vaccine

[0076] To determine that the vaccines of the invention induce a Th1-type response, BALB/cJ mice aged 6 to 8 weeks received a subcutaneous injection in the back with 1×10.sup.8 CFU of BCG WT or recombinant BCG that expresses the RSV-N protein, in a final volume 100 μL per dose. After 14 days, they were given a booster with the same starting dose. After 21 days from the first injection of BCG WT or BCG-N, the animals were challenged, for which RSV A2 strain 13018-8 obtained from a clinical isolate was used. Pre-immune serum samples were obtained (day 0), before viral challenge (day 21), and 14 days after infection. These samples were analyzed by ELISA at a 1/500 dilution. The results show that animals immunized with BCG-N produce anti-RSV antibodies even in stages prior to viral challenge, this production is significantly higher than unimmunized controls and animals immunized with BCG WT. After the challenge with RSV, the levels of specific IgG immunoglobulins against the virus show an even greater increase that is maintained at day 14 post-infection (FIG. 5).

[0077] To establish the isotype of the immunoglobulins produced by 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 IgG2a isotype antibodies 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 BCG, and not immunized. On the other hand, IgG1 isotype anti-RSV immunoglobulins show an increase in animals vaccinated with BCG-N prior to infection, however, the RSV control group and animals vaccinated with BCG WT already manage to match their production towards day 14 after viral challenge (FIG. 6-A, 6-B). The latter is better evidenced in FIG. 6-C, where it is shown that the ratio of anti-RSV immunoglobulins of isotype IgG2a/IgG1 is higher in animals immunized with the BCG RSV-N vaccine of the invention than in the control group only infected with RSV. As we have already pointed out, this relationship is indicative of an immune response polarized towards a Th1 phenotype.

[0078] In order to determine if the humoral response induced by vaccination was capable of protecting against RSV infection, after inactivating complement, the RSV-GFP virus was incubated with sera from animals from all experimental groups on day 14 post-infection and prior to viral challenge, and then HEp-2 cells were infected with these mixtures. In FIG. 7, in the epifluorescence microscopy images, a decrease in the expression of RSV-GFP in the cells previously treated with serum from animals immunized with BCG-N in relation to the other treatments is observed. When quantifying 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 (FIG. 7-B) and when analyzing the expression of RSV-GFP by flow cytometry, the same trend is evidenced (compare FIG. 7-C with 7-B).

Example IX: Production of Anti-hMPV IgG Antibodies in Animals Immunized with RSV BCG-N

[0079] 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 the RSV N protein (BCG-N) or with a recombinant BCG vaccine for the hMPV P protein (BCG-P). These results were compared to animals infected with hMPV without prior immunization and with an uninfected control (Mock). The serum are compared at 2 different times: previral challenge and day 7 post-infection. The samples were analyzed by ELISA at a 1/1,000 dilution. The results are seen in FIG. 8, where it is shown that the total anti-hMPV IgG immunoglobulins significantly increased in the vaccinated animals compared to the control groups.

[0080] It is highly significant that animals immunized with both 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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