WO2015044909A1 - Vacunas de uso nasal contra streptococcus pyogenes en base a un vector bacteriano inocuo y antígenos específicos - Google Patents
Vacunas de uso nasal contra streptococcus pyogenes en base a un vector bacteriano inocuo y antígenos específicos Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
Definitions
- the present invention relates to the area of Immunology, in the clinical application of immunogenic formulations based on live bacteria; particularly used to prepare efficient vaccines against Streptococcus pyogenes, the causative agent of pharyngitis and necrotlzant fasceitis, among other pathologies.
- the vaccine is based on a recombinant Innocuous bacterial vector that expresses the antigens of the types of streptococci that allow greater protection against suppurative diseases of the respiratory tract and which are also the most prevalent strains in the geographical area where the vaccine will be used .
- pathogen S. pyogenes are pharyngitis, scarlet fever, localized skin infections and also some severe invasive infections such as streptococcal septic shock and necrotlzante fasce ⁇ tls, among other pathologies.
- S. pyogenes is also responsible for sequential diseases that appear several weeks after a poorly cured streptococcal infection, such as glomerulonefrltls and rheumatic fever.
- the problems caused by this pathogen and that would be preventable with the development of a vaccine are basically the costs incurred (doctors and non-doctors) and the fact of spending several days with symptoms.
- a study based on interviews with parents and their school-age children showed that children lost an average of 1, 9 days of school / nursery (range 0-7 years) per episode of pharyngitis, in 42% of The families one of the parents lost an average of 1, 8 days of work per episode, and in 14% of the families the other father lost 1, 5 days.
- the total social cost per episode of pharyngitis caused by S. pyogenes was US $ 205 (US $ 1,18 of medical costs and US $ 87 of non-medical costs). It is estimated that in the United States the costs associated with pharyngitis in children is 224-539 million dollars a year (Pfoh, et al., 2008).
- the M protein is a membrane protein of great variability, highly immunogenic and is also one of the main virulence factors of S. pyogenes, since it allows adhesion to mucous membranes and the evasion of phagocytosis.
- the M protein is encoded by the emm gene.
- US Patent Application No. US201 131 1617A1 describes the formulation of an oral vaccine against a disease caused by bacterial infections.
- This oral vaccine corresponds to a capsule formulation that contains a microorganism that expresses or secretes the antigenic protein, called transformed microorganism.
- transformed microorganism a microorganism that expresses or secretes the antigenic protein
- these microorganisms are large enteropathogenic bacteria belonging to the genus Bifidobacterium, Lactobacillus, Lactococcus and the like, with an acid-resistant bacterial cell wall.
- This vaccine is against the pathogens that cause typhoid fever, cholera and bacillary dysentery
- the Immunogenic antigen is the flagellum protein (flagellna), with only one type of fliC protein for each pathogen, which is inserted into a construct derived from E. coli pBR322.
- the vaccine is administered orally in the form of a capsule to protect microorganisms from acid as it passes through the stomach and the capsule opens in the intestine, which is the place where immunity is to be induced.
- nucleic acid molecule which comprises a nucleotide sequence encoding a hybrid recombinant of a fragment of the M protein, corresponding to a carrier attached to at least one fragment. amino-terminal peptide of said protein having an epitope to obtain opsonic antibodies for at least one S. pyogenes serotype for the prevention of rheumatic fever and not for other suppurative complications.
- the invention also provides an expression construct of the sequence described above contained in a host cell and a method for oral administration in patients.
- the structure of the construct is a hybrid protein formed by a 15 amino acid fragment of the M protein attached to a carrier by means of a linker because, since the fragment is too small to be immunogenic, the carrier is added to confer immunogenic capacity .
- the host bacteria used is Streptococcus mutans and protects the antigen until it dies in the intestinal passage where the antigen is absorbed.
- the technical problem to be addressed corresponds to the development of a vaccine against S. pyogenes, a bacterium that causes numerous diseases in humans, which vary mainly from superficial skin infections to serious systemic infections associated with high mortality.
- the present invention comprises immunogenic formulations based on innocuous recombinant live bacteria that express the types of S. pyogenes M proteins that allow greater protection against suppurative diseases.
- the present invention consists of a vaccine developed based on the types of S. pyogenes that allow greater protection against suppurative diseases of the respiratory tract and which are also the most prevalent strains and that allow generating an efficient immune response in patients with whichever is administered.
- the vaccine is also administered intranasally, so that it is as accessible as possible to the nasopharyngeal mucosa to be processed by macrophages and other cells of the immune system for the generation of IgA antibodies that constitute the main barrier against S colonization.
- pyogenes Being an intranasal administration, by means of an applicator for nasal drops, nasal spray or the like, it allows a simpler inoculation, of low administration cost and with minor complications regarding the injectable administration that facilitates the use in all types of patients, especially In the case of young children. Thus, it is a vaccine highly efficient in protection since it uses the natural route of infection of the bacteria (colonization of the mucosa of the upper respiratory tract).
- bacterial vaccines offer an economically more accessible and less complex option than protein vaccines, especially in less developed countries, since protein purification is not required, which is the most expensive part in the production of protein vaccines.
- the pathogenesis of extracellular bacteria such as S. pyogenes is not characterized by intracellular invasion of host cells. Therefore the bacteria used as a vector in the vaccine does not require invasion of host cells. For this reason the use of lactic acid bacteria as a vaccine vector is a very good option.
- the expression of heterologous proteins is efficient in these bacteria, and the use of adjuvants is not required nor is it necessary to conjugate the protein to a carrier.
- Figure 1 NICE® pNZ8149 plasmid diagram (Mobitec GmbH, Germany). Which has a Pnis promoter: nisin-inducible; a terminator T: terminator; repC and repA genes: replication C and A; and the lacF gene of the lactose metabolism of L. lactis.
- Figure 2 Colony PCR performed with the splitters that line up in the construct outside the insert (ins_chk_F: SEQ I D No: 10 and ins_chk_R: SEQ ID No: 20). Between 6 and 10 colonies of each transformation were analyzed. The expected sizes correspond to 959 bp for M1, 91 1 bp for M2, 977 bp for M9, 989 bp for M4, 965 bp for M6, 980 bp for M12, 998 bp for M28 and 980 bp for M22. 100 bp molecular weight marker.
- Figure 3A Protein analysis by SDS-PAGE for the vaccine strain M2.
- the crude extracts were loaded on a 12.5% polyacrylamide gel that was stained with Coomasie Blue.
- the concentration of nisin used for the induction of each culture is shown: 0, 1, 5 and 10 ng / ml.
- STD Standard of molecular weight of proteins.
- the M2 protein has approximately 22.7 kDa and its position in the gel is indicated by a red square.
- Figure 3B Relative quantification of M2 protein expression in the different optimization conditions. The quantification was performed by densitometry analyzing the gel photography with ImageJ software. To normalize the amount of protein loaded in each lane, a band whose intensity is maintained in all lanes (marked with black square on the gel) was quantified and the ratio between them was calculated. The graph shows the average of 2 or 3 independent experiments. A representative experiment is shown.
- Figure 4A Protein analysis by SDS-PAGE for vaccinate strains M1, M9 and M28. The crude extracts were loaded on a 12.5% polyacrylamide gel that was stained with Coomasie Blue. The induction lasted 3 h and the concentrations of nisin used were: 0, 5, 10, 20 and 40 ng / ml.
- FIG. 4B Relative quantification of the expression of M1, M9 and M28 proteins in the different concentrations of nisin. The quantification was performed by densitometry analyzing the gel photography with ImageJ software. To normalize the amount of protein loaded in each lane, a band whose intensity is maintained in all lanes was quantified and the ratio between them was calculated. The graph shows the average of 2 or 3 independent experiments. A representative experiment is shown for each strain.
- FIG. 5 Protein analysis by SDS-PAGE present in the supernatant of L. lactis cultures expressing the M2, M1, and M9 protein at 3 h induction. The culture supernatants were filtered with 0.45 ⁇ filters and the supernatant proteins were precipitated with TCA
- Figure 6A Protein analysis by SDS-PAGE for vaccinate strains M4 and M12.
- the crude extracts were loaded on a 12.5% polyacrylamide gel that was stained with Coomasie Blue. The induction lasted 3 h and the concentrations of nisin used were: 0, 5, 10 and 20 ng / ml.
- STD Standard of molecular weight of proteins. The expected sizes of the M4 and M12 proteins are 26.12 and 26.4 kDa respectively.
- Figure 6B Relative quantification of the expression of the M4 and M12 proteins in the different concentrations of nisin. The quantification was performed by densitometry analyzing the gel photography with ImageJ software. To normalize the amount of protein loaded in each lane, a band whose intensity is maintained in all lanes was quantified and the ratio between them was calculated.
- Figure 8 Weight curve of the mice used in the evaluation of the efficacy of the M9 vaccine. The differences in the initial weight decrease between the group of mice immunized with said vaccine (L. Iactis-M9) and infected mice, by Student Test where P ⁇ 0.05 ( * ).
- FIG 10 ELISA performed with sera from mice immunized with L. Iactis-M9 (M9 vaccine), L. lactis wt (empty vector) and non-immunized mice.
- the ELISA is of the "sandwich" type where the plates were sensitized with the synthetic peptide M9 (Table 2; SEQ ID NO: 29 and SEQ ID NO: 30). Differences in absorbance between the group of mice immunized with L. Iactis-M9 and mice not immunized were analyzed by Student Test where P ⁇ 0.05 ( * ).
- the vaccine according to the present invention expresses the hypervariable segments of the M protein of the most frequent types of S. pyogenes infections in a non-pathogenic bacterium for use as an intranasal vaccine against the pathologies caused by said pathogen.
- each of the selected M proteins corresponds to a specific plasmid for the bacterium to be used, without antibiotic resistance and which allows to express the protein in the membrane.
- a specific plasmid of Lactococcus lactis has been chosen.
- the S. pyogenes gene for each type of construct based on each of the 8 types of M protein selected is inserted into the multiple cloning site of the plasmid.
- the plasmid corresponds to a plasmid pNZ8149 NICE® (Mobitec GmbH, Germany), as shown in Figure 1 and having a nisin-inducible Pnis promoter; a terminator T; repC and repA genes of replication C and A; and the lacF gene of the lactose metabolism of L. lactis.
- the parent strain of the bacterium used as the host for the vaccine is strain NZ3900 of L. lactis, a lactic acid bacterium of feed quality and which also does not carry antibiotic resistance, and which is part of the NICE® system (Mobitec GmbH, Germany).
- This strain is derived from L. lactis subsp. Cremoris MG1363. a plasmid-free strain that is progeny of strain NCD0712 used in the dairy industry as a yogurt fermentation initiator.
- the hypervariable segments of the M protein have been selected from among the most frequent types detected in both localized and invasive suppurative infections, to be carried by the non-pathogenic intranasal administration bacteria.
- the vaccine is a mixture of 8 well-defined bacterial strains, each of which expresses a different type of M protein.
- Vaccines against S. pyogenes may be based on different elements of the bacteria capable of inducing antibody production. Many of them are virulence factors, such as C5a-peptidase, surface carbohydrate, exotoxins A, B or C, and protein M.
- protein M is selected which is a membrane protein that participates in the phagocytosis evasion, where the amino terminal domain exposed on the surface is responsible for inducing the production of antibodies with high protective capacity, which have greater bactericidal activity than those induced by the other virulence factors. This domain has hypervariable sequences and based on them there are more than 150 different types of M proteins.
- the main defense against pathogens such as S. pyogenes is secretory immunoglobulin A (IgA), which is poorly induced by the systemic and oral administration of protein antigens, while lactic acid bacteria offer the advantage of being potent IgA inducers. secretory and systemic immunity, and are not degraded in the gastro-intestinal tract.
- IgA secretory immunoglobulin A
- lactic acid bacteria carry several elements that enhance the immune response such as peptidoglycan, lipoteic acid, etc.
- the ideal is that the vehicle of the heterologous antigen used to develop the vaccine is also gram-positive, such as Lactococcus lactis, which is a carrier of teicoic acid, and other molecules of its own. of gram-positives as well as the real pathogen against which protection is sought.
- the use of additional adjuvants is not required nor is it necessary to conjugate the protein to a carrier, as in the case of protein vaccines.
- M protein of S. pyogenes The importance of obtaining protection against the most frequent types of M protein of S. pyogenes is that they cause the most prevalent and most serious diseases, as in the case of invasive infections.
- the types of M protein selected are the most frequent types regionally, particularly in Chile, Argentina, Brazil, Mexico, USA and other countries of America, which have high infection rates and scarce resources, without ruling out that the effectiveness of The vaccine is also extensible worldwide.
- the vaccine obtained corresponds to a mixture of recombinant strains of L. lactis that express the hypervariable region of the S. pyogenes M protein in the membrane.
- the composition is for intranasal application in a biologically acceptable and harmless diluent such as the phosphate buffered saline (PBS) and any excipient that can be incorporated into a nebulization buffer and that facilitates the administration form.
- PBS phosphate buffered saline
- the novelty of this invention lies mainly in the design of the vaccine, which is not protein. It is known that protein vaccines are not highly immunogenic for IgA, which is the really protective immune response against S pyogenes. This is a colonizing bacterium of mucous membranes and IgA is the one that prevents initial colonization, being the key starting point for infection. Since the vaccine consists of a mixture of 8 different populations of L. lactis, in which each expresses a different type of M protein, it is highly likely that interference between the different types of M protein will not occur, which could happen when Different peptides combine to form a single protein.
- each of the hypervariable segments of the M protein of types 1, 2, 4, 6, 9, 12, 22 and 28 are cloned separately in a specific expression construct of the bacteria to be used.
- the segments are cloned into plasmid pNZ8149 and introduced into L. lactis, generating each of the vaccinate strains.
- M17 medium Sigma-Aldrich, Buchs, Switzerland with lactose as the only carbon source, at a temperature of 28 ° C ⁇ 1 ° C, for 16 to 18 hours (primary culture).
- M17 medium is a specific medium for L. lactis to which the desired carbon source is added, in our case, lactose is added at a concentration of 5 g / l.
- a subculture is made by adding 1 ml of the primary culture for every 9 ml of M17 medium, freshly prepared and kept growing until it reaches an optical density of 1 ⁇ 0.1 to 600 nm
- the induction is performed by adding to the nisin culture in final concentrations of 10 and 20 ng / ml and incubating 3 h at 28 ⁇ 1.
- the culture is aliquoted in fractions that are centrifuged 5 min at 12,000 rpm and the supernatant is removed.
- the bacteria are resuspended in 30 ul of phosphate buffered saline (PBS) obtaining thus a suitable suspension for be used intranasally.
- PBS phosphate buffered saline
- the bacteria can be resuspended in culture medium with 40% glycerol and frozen at -80 ° C until used. In this case and prior to its use, its contents are thawed, centrifuged 5 min at 12,000 rpm and washed with PBS to remove glycerol. This pellet is resuspended in PBS and stored cold (4 ° C) until the moment of use.
- Each vaccine consists of doses of the immunological formulation for intranasal administration in PBS with 10 8 bacteria of each of the types M mentioned above; in total 8 x 10 8 bacteria are administered.
- each of the hypervariable segments of the M protein of types 1, 2, 4, 6, 9, 12, 22 and 28 were cloned separately in a specific expression construct of the bacteria to be used, in this case pNZ8149 and introduced in Lactococcus lactis.
- Hypervariable fragments of the gene (emm) of the protein M1, M2, M4, M6, M9, M12, M22 and M28 (emm1, emm2, emm4, emm6, emm9, emm12, emm22 and emm28) were amplified by PCR.
- the forward splitters have a queue with the cutting site for Ncol and the reverse splitters have a queue with the cutting site for Clal (Table 1).
- the membrane anchor region (MAD) was also amplified using the emm6 gene as a template.
- the forward splitter for the MAD region has a queue with the Clal cutting site and the reverse splitter has a queue with the Xbal cutting site (Table 1).
- the amino acid sequence of the hypervariable fragment introduced into each strain is detailed in Table 2.
- MAD MAD-F SEQ ID NO: 9 MAD-R SEQ ID NO: 19 plasmid ins_chk_F * SEQ ID NO: 10 ins_chk_R * SEQ ID NO: 20 * Partitioners that align in the construct and are used to verify that the insert was cloned correctly.
- Table 2 Amino acid sequence of the fragments cloned in the vaccines, each consisting of a signal peptide bound to a hypervariable fragment.
- Each of the amplified hypervariable fragments was digested with Clal and bound to the MAD fragment digested with the same enzyme, obtaining 8 inserts: M1 -MAD, M2-MAD, M4-MAD, M6-MAD, M9-MAD, M12-MAD , M22-MAD and M28-MAD.
- the verification of this ligation was carried out by PCR and showed that the 8 inserts had been correctly ligated. All the inserts thus obtained were digested with Ncol and Xbal as well as the lactococcal expression construct pNZ8149. Each insert was individually introduced into the construct by means of ligation with T4-DNA ligase.
- the ligation check was performed by PCR with splitters that line the construct (ins_chk_F and ins_chk_R) and showed that the ligation occurred correctly.
- Each plasmid contains a single copy of the insert.
- the resulting plasmids were introduced into L. lactis NZ3900 by electroporation and the transforming colonies were selected in M17-lactose medium with bromocresol purple. Yellow colonies were obtained for all transformations.
- colony PCR was performed with the splitters that line up in the construct and the fragments of the expected size were obtained (Figure 2).
- nlslna which is consistent with the use of a promoter nduclble by nlslna, as shown in Figure 1
- nlslna which is consistent with the use of a promoter nduclble by nlslna, as shown in Figure 1
- the vaccinate strain M2 was induced during different times: 2, 3 and 5 h.
- each Induction time was tested with different concentrations of nlslna: 0, 1, 5, 10, 20, 40, 100 and 200 ng / ml.
- Figure 4A shows the protein analysis by SDS-PAGE for vaccinal strains M1, M9 and M28. Induction lasted 3 h and the concentrations of nlslna used were: 0, 5, 10, 20 and 40 ng / ml. The expected sizes of the M1, M9 and M28 proteins are 24.6; 25.6; and 26, 1 kDa respectively.
- Figure 4B shows the relative quantification of the expression of the M1, M9 and M28 proteins in the different concentrations of nlslna. The graph shows the average of 2 or 3 independent experiments.
- FIG. 5 shows the protein analysis by SDS-PAGE present in the supernatant of the L. lactis cultures expressing the M2, M1, and M9 protein at 3 h induction. The culture supernatants were filtered with 0.45 ⁇ filters and the supernatant proteins were precipitated with TCA.
- Protein analysis was performed by SDS-PAGE for vaccinate strains M4 and M12.
- Figure 6A shows that the expected sizes of the M4 and M12 proteins are 26.12 and 26.4 kDa respectively.
- Figure 6B shows the relative quantification of the expression of the M4 and M12 proteins in the different concentrations of nisin.
- Vaccine evaluation is performed by testing Balb / c mice, in order to measure the protection conferred.
- the vaccination protocol consists of 3 immunizations performed at intervals of 14 days. Each immunization consists of the intranasal administration of 30 ⁇ of PBS containing 10 8 bacteria of each of the types M mentioned above; in total 8 x 10 8 bacteria are administered. Immunization with the wt (wild type) vector was also tested, which does not express any heterologous protein to verify that immunity is not due to the bacterial vector. At 14 days of 3 or immunization of mice challenged with the pathogen it was performed M9. To obtain the vaccine, each vaccine strain is grown in 3 ml of culture medium
- M17 (Sigma) with lactose as the only carbon source at 28 ° C overnight.
- a subculture is made by adding 100 ⁇ of the previous culture to 9.9 ml of medium M17-lactose fresh and grown to an optical density, OD, 600nm of about 1.
- nisin is added to the final concentration of 10 or 20 ng / ml and incubated 3 hours at 28 ° C.
- the culture is aliquoted in fractions of 1 ml which are centrifuged 5 min at 12,000 rpm. The supernatant is removed and the bacteria are resuspended in M17-lactose medium with 40% glycerol and frozen at -80 ° C until use.
- the tube On the day of its use, the tube is thawed, centrifuged 5 min at 12,000 rpm and washed with 1 ml of PBS. Finally, the pellet is resuspended in 30 ⁇ of PBS and stored cold (4 ° C) until the moment of use. No preservative is added to the preparation.
- mice Blood and saliva samples were taken from each mouse before each immunization.
- the indirect bactericidal activity assay was performed by mixing 50 ⁇ of mouse serum + 175 ⁇ of human blood and 25 ul of a suspension containing 25-100 CFU of S. pyogenes. Said mixture is incubated in 75 mm tubes for 3 h with end-to-end agitation. After 3 h the 10 ⁇ 1 and 10 ⁇ 2 dilutions are plated - The greater the bactericidal activity of the mouse serum (due to the presence of antibodies), a lower CFU number will be obtained. It is observed that the bactericidal activity of the sera of mice after the second immunization is greater than after the first and third immunization (Figure 7). After the challenge, mice are monitored to assess disease parameters. The following parameters were evaluated:
- mice vaccinated mice had a significantly greater weight decrease than vaccinated mice (L. Iactis-M9) ( Figure 8).
- mice vaccinated with L. lactis wt had a significant weight reduction similar to that suffered by infected mice ( Figure 8).
- the temperature curve shows that at 18 h after infection the temperature drop in sick mice is greater than in vaccinated mice and in healthy mice, however it is not statistically significant (Figure 9).
- mice vaccinated with L. Iactis-M9 The CFU count of S. pyogenes at 7 days post-infection showed that bacteria were recovered only in diseased mice.
- an ELISA test was performed with sera from immunized and non-immunized mice, where the plates were sensitized with the peptide. synthetic M9. The results of this assay demonstrate that the immunized mice produced anti-M9 antibodies in a significantly greater amount than the mice that were not immunized or those that were immunized with L. lactis vA ( Figure 10).
- mice show minimal disease symptoms compared to diseased mice, which indicates that the M9 vaccine is effective in disease control, and that this efficacy can be attributed to the production of anti-M9 antibodies.
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Application Number | Priority Date | Filing Date | Title |
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CL2013002782A CL2013002782A1 (es) | 2013-09-27 | 2013-09-27 | Formulación inmunogénica para inducir la respuesta inmune en un sujeto que comprende una mezcla de bacterias no patógenas que expresan cada una un segmento hipervariable de la proteína m de diferentes streptococcus pyogenes; uso de la formulación para producir una vacuna; método para producir vacuna |
CL2782-2013 | 2013-09-27 |
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WO2015044909A1 true WO2015044909A1 (es) | 2015-04-02 |
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PCT/IB2014/064870 WO2015044909A1 (es) | 2013-09-27 | 2014-09-26 | Vacunas de uso nasal contra streptococcus pyogenes en base a un vector bacteriano inocuo y antígenos específicos |
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CN112760336A (zh) * | 2020-12-30 | 2021-05-07 | 广州辉园苑医药科技有限公司 | 一种抗原表位肽的表达系统和表面展示系统及它们的构建方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MXPA04003852A (es) * | 2001-10-26 | 2005-02-17 | Univ Tennessee Res Foundation | Composiciones de vacuna estreptococica multivalente, y metodos para sus uso. |
US20070053937A1 (en) * | 1992-09-16 | 2007-03-08 | University Of Tennessee Research Foundation | Antigen of hybrid M protein and carrier for group a streptococcal vaccine |
ES2280101T3 (es) * | 1997-09-12 | 2007-09-01 | University Of Tennessee Research Foundation | Vacunas de estreptococos del grupo a. |
US20090010929A1 (en) * | 2006-12-04 | 2009-01-08 | Good Michael F | Therapeutic Antibodies, Antibody Fragments and Antibody Conjugates |
-
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070053937A1 (en) * | 1992-09-16 | 2007-03-08 | University Of Tennessee Research Foundation | Antigen of hybrid M protein and carrier for group a streptococcal vaccine |
ES2280101T3 (es) * | 1997-09-12 | 2007-09-01 | University Of Tennessee Research Foundation | Vacunas de estreptococos del grupo a. |
MXPA04003852A (es) * | 2001-10-26 | 2005-02-17 | Univ Tennessee Res Foundation | Composiciones de vacuna estreptococica multivalente, y metodos para sus uso. |
US20090010929A1 (en) * | 2006-12-04 | 2009-01-08 | Good Michael F | Therapeutic Antibodies, Antibody Fragments and Antibody Conjugates |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112760336A (zh) * | 2020-12-30 | 2021-05-07 | 广州辉园苑医药科技有限公司 | 一种抗原表位肽的表达系统和表面展示系统及它们的构建方法 |
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