CN108524926B - Preparation combination of multivalent pneumococcal conjugate vaccine and application thereof - Google Patents
Preparation combination of multivalent pneumococcal conjugate vaccine and application thereof Download PDFInfo
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Abstract
The invention provides a preparation combination of a novel multivalent pneumococcal conjugate vaccine, a preparation method thereof and application of the preparation combination in preparing a medicament for preventing or treating diseases caused by pneumococcus. The invention also provides a method for controlling the molecular weight of the conjugate in the preparation combination of the multivalent pneumococcal conjugate vaccine. The pneumococcal conjugate vaccine provided by the invention solves the serum inhibition phenomenon in the multivalent pneumococcal conjugate vaccine, and effectively improves the immunogenicity of the multivalent pneumococcal conjugate vaccine in the preparation combination. The preparation method of the preparation combination of the multivalent pneumococcal conjugate vaccine can effectively control the molecular weight of the conjugate and is easier to remove free protein and free polysaccharide which are not involved in the conjugation.
Description
Technical Field
The invention relates to the technical field of vaccine product development, in particular to a preparation combination of a multivalent pneumococcal conjugate vaccine, a preparation method thereof and application of the preparation combination in preparing a medicament for preventing or treating diseases caused by pneumococci.
Background
The streptococcus pneumoniae is called pneumococcus for short, belongs to gram-positive bacteria, and has a main pathogenic factor of capsular polysaccharide. Is provided withThe virulence of the capsular strain is 10 for the non-capsular strain5And (4) doubling. Pneumococci are classified into more than 90 serotypes according to the antigenic structure of capsular polysaccharides, with strains of about 30 serotypes causing a variety of invasive diseases including bacteremia, meningitis, pneumococcal pneumonia, Invasive Pneumococcal Disease (IPD), and the like.
Pneumococcus is mainly responsible for lobal pneumonia, has high incidence rate in children and old people, and can secondarily cause pleuritis, otitis media, meningitis and septicemia. The mortality rate of IPD patients is 10% -25%, the mortality rate of IPD increases with the age of the patients, and the mortality rate of IPD of people aged more than or equal to 65 years in the United states is up to 50%.
Pneumonia patients are usually treated by antibiotics, and sulfonamides, penicillin and the like are effective on related diseases caused by pneumococcus, but with the occurrence of a large amount of antibiotic-resistant bacteria, the treatment period and the treatment cost of patients are increased sharply. Vaccine immunization is a prevention and control means, is proved to be the most effective mode at present, and is widely popularized by governments of various countries.
The research shows that the capsular polysaccharide on the surface of the pneumococcus can be used as a core antigen of the pneumonia vaccine, but the capsular polysaccharide is thymus independent antigen, so the capsular polysaccharide does not play a role in infants and the elderly with low immunity. The combined vaccine prepared by linking the capsular polysaccharide and the carrier protein can effectively solve the problem. At present, two pneumonia vaccines exist in the market, wherein one is a 23-valent pneumonia polysaccharide vaccine, and the other is a 7-valent/10-valent/13-valent pneumonia polysaccharide protein conjugate vaccine.
Pneumonia conjugate vaccines on the market at home and abroad are analyzed, wherein carrier proteins of wyeth 7-valent (serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and 13-valent pneumonia conjugate vaccines are CMR 197. However, compared with the 7-valent pneumonia conjugate vaccine, the antigen content of the 13-valent pneumonia conjugate vaccine in the finished preparation is increased by 10 percent. The carrier proteins of the 10-valent pneumonia conjugate vaccine of the GSK are TT, DT and PD, and after the 10-valent conjugate vaccine is marketed, no report related to the development of more-valent pneumonia conjugate vaccines by the GSK is found. There are many obstacles to the development of multivalent conjugate vaccines, one of which is the inhibition of the vector between the different serotypes. The production process of multivalent conjugate vaccines and the solution of vector suppression between different serotypes are technical difficulties that must be overcome in developing multivalent conjugate vaccines.
CN101378778A discloses a multivalent streptococcus pneumoniae immunogenic composition, the capsular saccharides being derived from different streptococcus pneumoniae serotypes and being conjugated to two or more different carrier proteins, wherein the composition comprises a serotype 19F capsular saccharide conjugated to DT or CRM197, optionally wherein 19F is the only saccharide in the composition conjugated to DT or CRM 197. CN103623401A discloses a multivalent pneumococcal capsular polysaccharide-protein conjugate composition and a preparation method thereof, the conjugate composition is formed by covalently connecting 14 different serotypes of pneumococcal capsular polysaccharide and carrier protein, and the conjugate composition has good adsorption effect and stability. The invention provides a preparation combination of a multivalent pneumococcal conjugate vaccine and a control method of the molecular weight of a conjugate thereof.
Disclosure of Invention
Part of serotypes in the multivalent pneumococcal conjugate vaccine show weaker immunogenicity in preparation due to carrier interference; in order to solve the serum inhibition phenomenon in the multivalent pneumococcal conjugate vaccine, TT, DT or HID is selected as a carrier protein for serotypes with weaker immunogenicity, and the immunogenicity of the serotypes in the preparation can be effectively improved.
In order to solve the technical problem that the molecular weight requirements of conjugates of different serotypes are different, the invention provides a method for increasing the molecular weight of the conjugates in the preparation combination of the multivalent pneumococcal conjugate vaccine, TT is used as a carrier protein, the molecular weight of the conjugates is larger, CRM197 is used as the carrier protein, and the molecular weight of the conjugates is smaller. The conjugates have a relatively large molecular weight, and free proteins and free polysaccharides not involved in conjugation can be more easily removed during the purification process of the conjugates, which may be more immunogenic. In the invention, TT, DT or HID is selected as a protein carrier with stricter molecular weight requirements, CRM197 is selected as a protein carrier with less stricter molecular weight requirements, and the molecular weight control of different serotype conjugates is realized.
In the 13/15 price pneumonia conjugate vaccine, the molecular weight requirements of each serotype conjugate are different, some conjugates have larger molecular weight, free polysaccharide and free protein can be easily removed by a process, and the immunogenicity of the conjugates is better. Some serotypes have smaller molecular weight and are easy to separate and purify the conjugate, and in the invention, different carrier proteins are linked through specificity of different serotypes, so that the molecular weight control of the conjugate can be easily realized.
The conjugate is purified by various production processes, such as molecular sieve, membrane-packed ultrafiltration and ion exchange column, and in the invention, the conjugate with TT as a carrier is preferentially purified by the molecular sieve, so that the conjugate has larger molecular weight, more uniform molecular weight and lower free protein and free polysaccharide. The result is a lower content of free polysaccharide for conjugates with CRM197 as carrier, preferentially ion exchange or molecular sieve purification processes.
In a first aspect of the invention, there is provided a polyvalent pneumococcal conjugate vaccine formulation combination comprising at least one first carrier protein conjugated to capsular polysaccharides from different pneumococcal serotypes, and at least one second carrier protein conjugated to capsular polysaccharides from different pneumococcal serotypes, the capsular polysaccharides of serotypes 7F and 19F being conjugated to the second carrier protein, the first carrier protein being CRM197 and the second carrier protein being TT, DT or HID, wherein capsular polysaccharides of the same serotype are conjugated to only one carrier protein, and the formulation combination comprises at least 7 capsular polysaccharides from different pneumococcal serotypes.
Preferably, the formulation combination includes at least 10 capsular polysaccharides from different pneumococcal serotypes.
It is further preferred that the formulation combination comprises at least 13 or at least 15 capsular polysaccharides from different pneumococcal serotypes.
The capsular polysaccharide of the invention is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F.
Preferably, the capsular polysaccharide is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F or 33F, or the capsular polysaccharide is selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
Preferably, according to the immunogenicity of pneumonia capsular polysaccharides of different serotypes and the adaptability of pneumonia capsular polysaccharides to carriers, the pneumonia capsular polysaccharides are divided into two types, one type uses CRM197 as a carrier protein, the other type uses TT, DT or HID as a carrier protein, and the rest capsular polysaccharides can select either CRM197 as a carrier protein or TT, DT or HID as a carrier protein. Wherein, the capsular polysaccharide with TT, DT or HID as carrier protein is selected to show weaker immunogenicity in the preparation due to carrier interference, and the capsular polysaccharide with TT, DT or HID as carrier protein can effectively improve the immunogenicity in the preparation.
Preferably, the capsular polysaccharides conjugated to TT, DT or HID carrier proteins are of serotypes 5, 7F and 19F. Further preferably, the serotypes of capsular polysaccharide conjugated to TT, DT or HID carrier proteins also include 1 and/or 4.
In a specific embodiment of the invention, the capsular polysaccharides conjugated to TT, DT or HID carrier proteins are of serotypes 5, 7F, 19F, 1 and 4.
Preferably, the serotype of capsular polysaccharide conjugated to CRM197 carrier protein is selected from one or a combination of two or more of 6B, 19A or 23F. Further preferably, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein also include combinations of one or more of 3, 6A, 9V, 14 or 18C.
In a specific embodiment of the invention, the capsular polysaccharides conjugated to CRM197 carrier protein are of serotypes 3, 6A, 6B, 9V, 14, 18C, 19A and 23F.
Preferably, the multivalent pneumococcal conjugate vaccine is selected from 7-valent pneumococcal conjugate vaccine, 10-valent pneumococcal conjugate vaccine, 13-valent pneumococcal conjugate vaccine or 15-valent pneumococcal conjugate vaccine.
Further preferably, the 7-valent pneumococcal conjugate vaccine comprises serotypes 4, 6B, 9V, 14, 18C, 19F or 23F, the 10-valent pneumococcal conjugate vaccine comprises serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F or 23F, the 13-valent pneumococcal conjugate vaccine comprises serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F, and the 15-valent pneumococcal conjugate vaccine comprises serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F or 33F.
In a specific embodiment of the invention, the multivalent pneumococcal conjugate vaccine is a 13-valent pneumococcal conjugate vaccine, wherein the serotypes of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 capsular polysaccharides in the 13-valent pneumococcal conjugate vaccine use CRM197 as a protein carrier, and the serotypes of the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 capsular polysaccharides use TT, DT or HID as a protein carrier.
In a specific embodiment of the invention, the multivalent pneumococcal conjugate vaccine is a 15-valent pneumococcal conjugate vaccine, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 serotypes of capsular polysaccharide in the 15-valent pneumococcal conjugate vaccine use CRM197 as a protein carrier, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 serotypes of capsular polysaccharide use TT, DT or HID as a protein carrier.
In one embodiment of the invention, the 13-valent pneumococcal conjugate vaccine formulation is a combination of serotypes including 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A and 23F capsular polysaccharide conjugated to CRM197 carrier protein and serotypes including 7F and 19F capsular polysaccharide conjugated to TT carrier protein.
In another embodiment of the invention, the combination of formulations of the 13 valent pneumococcal conjugate vaccine, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein include 1, 3, 4, 6A, 6B, 9V, 14, 18C, 19A and 23F, and the serotypes of capsular polysaccharide conjugated to TT carrier protein are 5, 7F, 19F.
In another embodiment of the invention, the combination of formulations of the 13 valent pneumococcal conjugate vaccine, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein include 3, 6A, 6B, 9V, 14, 18C, 19A and 23F, and the serotypes of capsular polysaccharide conjugated to TT carrier protein are 1, 4, 5, 7F, 19F.
In one embodiment of the invention, the 15-valent pneumococcal conjugate vaccine formulation is provided, wherein the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein include 1, 2, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 23F and 33F, and the serotypes of capsular polysaccharide conjugated to TT carrier protein are 7F and 19F.
In one embodiment of the invention, the 15-valent pneumococcal conjugate vaccine formulation is provided, wherein the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein include 1, 2, 3, 4, 6A, 6B, 9V, 14, 18C, 19A, 23F and 33F, and the serotypes of capsular polysaccharide conjugated to TT carrier protein are 5, 7F and 19F.
In one embodiment of the invention, the 15-valent pneumococcal conjugate vaccine formulation is a combination of serotypes including 2, 3, 6A, 6B, 9V, 14, 18C, 19A, 23F and 33F capsular polysaccharide conjugated to CRM197 carrier protein and serotypes including 1, 4, 5, 7F and 19F capsular polysaccharide conjugated to TT carrier protein. Preferably, the capsular polysaccharide is conjugated to the carrier protein directly or via a linker.
In one embodiment of the invention, the capsular polysaccharide is conjugated to the carrier protein by a CDAP or reduced amine conjugation chemistry.
The preparation combination of the pneumococcal conjugate vaccine comprises the following antigen capsular polysaccharide content of each dose: the antigen content of SP6B is higher than other serotypes, and is generally more than 2 times higher. Preferably, the antigen content of the remaining serotypes, with the exception of SP6B, fluctuates within a certain range, but the antigen dose is lower than SP 6B. Further preferably, the same dosage can be selected for the remaining serotype antigen content, except for SP 6B.
Preferably, the antigen dose of the antigen capsular polysaccharide SP6B is 2-10 mu g/dose. Further preferably, the antigen dose of the antigen capsular polysaccharide SP6B is 4.4 mu g/dose.
Preferably, the antigen dose of the antigen capsular polysaccharide other than SP6B is 1-6 μ g/dose. Preferably, the antigen dose of the antigen capsular polysaccharide other than SP6B is 2-3 μ g/dose. In one embodiment of the invention, the antigenic dose of the antigenic capsular polysaccharide other than SP6B is 2.2 or 3 μ g/dose.
Preferably, the antigen dose of the antigen capsular polysaccharide except SP6B with CRM197 as carrier is 1-6 μ g/dose. Further preferably, the antigen dose of the antigen capsular polysaccharide except SP6B with CRM197 as carrier is 1-5 μ g/dose. In one embodiment of the invention, the antigenic dose of the antigenic capsular polysaccharide with CRM197 as a carrier, other than SP6B, is 3 μ g per dose.
Preferably, the antigen dose of the antigen capsular polysaccharide except SP6B using TT, DT or HID as carrier is 1-5 μ g/dose. Further preferably, the antigen dose of the antigen capsular polysaccharide with TT, DT or HID as carrier except SP6B is 2.2 μ g/dose.
Preferably, the preparation combination of the multivalent pneumococcal conjugate vaccine further comprises an aluminum-based adjuvant, an excipient and/or a preservative.
Preferably, the preparation combination of the multivalent pneumococcal conjugate vaccine can be in the form of spray, injection, freeze-dried preparation, capsule, tablet or pill.
In a second aspect of the present invention, there is provided a method for preparing the preparation combination of the multivalent pneumococcal conjugate vaccine, comprising the following steps:
(1) preparing at least one first carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes, and obtaining a first binding stock solution, wherein the first carrier protein is CRM 197;
(2) preparing at least one second carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes, and obtaining a second binding stock solution, wherein the capsular polysaccharides of serotypes 7F and 19F are conjugated with the second carrier protein, and the second carrier protein is TT, DT or HID;
(3) combining the first combined stock solution obtained in the step (1) and the second combined stock solution obtained in the step (2) to obtain a preparation combination of the multivalent pneumococcal conjugate vaccine;
wherein capsular polysaccharides of the same serotype are conjugated to only one carrier protein, and wherein the combination of formulations comprises capsular polysaccharides from at least 7 different pneumococcal serotypes.
Preferably, the capsular polysaccharide is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F; further preferably, the capsular polysaccharide is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F or 33F, or the capsular polysaccharide is selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
Preferably, the capsular polysaccharides conjugated to TT, DT or HID carrier proteins are of serotypes 5, 7F and 19F. Further preferably, the serotypes of capsular polysaccharide conjugated to TT, DT or HID carrier proteins also include 1 and/or 4.
Preferably, the serotype of capsular polysaccharide conjugated to CRM197 carrier protein is selected from one or a combination of two or more of 6B, 19A or 23F. Further preferably, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein also include combinations of one or more of 3, 6A, 9V, 14 or 18C.
Preferably, the multivalent pneumococcal conjugate vaccine is selected from 7-valent pneumococcal conjugate vaccine, 10-valent pneumococcal conjugate vaccine, 13-valent pneumococcal conjugate vaccine or 15-valent pneumococcal conjugate vaccine.
Further preferably, the 7-valent pneumococcal conjugate vaccine comprises serotypes 4, 6B, 9V, 14, 18C, 19F or 23F, the 10-valent pneumococcal conjugate vaccine comprises serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F or 23F, the 13-valent pneumococcal conjugate vaccine comprises serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F, and the 15-valent pneumococcal conjugate vaccine comprises serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F or 33F.
In one embodiment of the present invention, the multivalent pneumococcal conjugate vaccine is 13-valent pneumococcal conjugate vaccine or 15-valent pneumococcal conjugate vaccine.
Preferably, the capsular polysaccharide is conjugated to the carrier protein directly or via a linker.
In one embodiment of the invention, the capsular polysaccharide is conjugated to the carrier protein by a CDAP or reduced amine conjugation chemistry.
In a third aspect of the invention, a method for controlling the molecular weight of the conjugate in the above polyvalent pneumococcal conjugate vaccine preparation combination is provided, the preparation combination comprises at least one first carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes and at least one second carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes, the capsular polysaccharides of serotypes 7F and 19F are conjugated with the second carrier protein, the first carrier protein is CRM197, and the second carrier protein is TT, DT or HID, wherein the capsular polysaccharide of the same serotype is conjugated with only one carrier protein, and the preparation combination comprises at least 7 capsular polysaccharides from different pneumococcal serotypes.
Preferably, the conjugate obtained by conjugating the first carrier protein is purified by column purification by membrane ultrafiltration or ion exchange, and the conjugate obtained by conjugating the second carrier protein is purified by molecular sieve. The molecular weight of the conjugate prepared based on the method meets the requirement.
Preferably, the capsular polysaccharide is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F; preferably, the capsular polysaccharide is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F or 33F, or the capsular polysaccharide is selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
Preferably, TT, DT or HID is used as a protein carrier, and CRM197 is used as a carrier protein for not strictly controlling the molecular weight of the compound. Further preferably, TT or HID is used as a protein carrier, and CRM197 is used as a carrier protein for not strictly controlling the molecular weight of the composition.
Preferably, the serotype of capsular polysaccharide conjugated to CRM197 carrier protein is selected from one or a combination of two or more of 6B, 19A or 23F.
Preferably, the capsular polysaccharides conjugated to TT, DT or HID carrier proteins are of serotypes 5, 7F and 19F. Further preferred serotypes of capsular polysaccharide conjugated to TT, DT or HID carrier proteins also include 6A and/or 14.
Preferably, the capsular polysaccharides conjugated to TT or HID carrier proteins are of serotypes 5, 7F and 19F. Further preferred serotypes of capsular polysaccharide conjugated to TT or HID carrier protein also include 6A and/or 14.
In a particular embodiment of the invention, the serotype of the capsular polysaccharide conjugated to the TT carrier protein is selected from 5, 6A, 7F, 14 and 19F.
In the multivalent pneumococcal conjugate vaccine, different serotype conjugates have different molecular weights, the effective serotype conjugate has larger molecular weight, and is easy for process production, and TT can be selected as a protein carrier for the serotypes; some serotypes have smaller molecular weights and are easy to produce in a process, and CRM197 can be selected as a protein carrier for the serotypes.
In one embodiment of the invention, the conjugate molecular weight control method in the preparation combination of the multivalent pneumococcal conjugate vaccine comprises serotypes 5, 6A, 7F, 14 and 19F of capsular polysaccharide with TT as a carrier protein and serotypes 6B, 19A and 23F of capsular polysaccharide with CRM197 as a carrier protein; capsular polysaccharides of the remaining serotypes optionally CRM197, TT, DT or HID as carrier proteins.
In a fourth aspect of the invention, the application of the preparation combination of the multivalent pneumococcal conjugate vaccine in preparing a medicament for preventing or treating diseases caused by pneumococcus is provided.
Preferably, the pneumococcal disease is selected from pneumococcal pneumonia, invasive pneumococcal disease, otitis media, chronic obstructive pulmonary disease, conjunctivitis, meningitis or bacteremia.
The pneumococcal conjugate vaccine provided by the invention solves the serum inhibition phenomenon in the multivalent pneumococcal conjugate vaccine, and the immunogenicity of the serotype with weaker immunogenicity is effectively improved in a preparation combination by taking TT, DT or HID as carrier proteins. The preparation method of the preparation combination of the multivalent pneumococcal conjugate vaccine can effectively control the molecular weight of the conjugate, TT, DT or HID is selected as a protein carrier with more strict molecular weight control, the molecular weight is easy to control, the molecular weight control is not strict, and CRM197 is selected as a protein carrier.
The TT is tetanus toxoid.
"DT" as described herein is diphtheria toxoid.
The HID is Haemophilus influenzae D.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: immunogenicity of conjugates obtained by conjugating type 5 capsular polysaccharides with different carrier proteins;
FIG. 2: immunogenicity of conjugates obtained by conjugating capsular polysaccharide type 7F with different carrier proteins;
FIG. 3: immunogenicity of conjugates obtained by conjugating type 6B capsular polysaccharides with different carrier proteins;
FIG. 4: immunogenicity of conjugates obtained by conjugation of type 9V capsular polysaccharides with different carrier proteins;
FIG. 5: comparison of immunogenicity of different carrier protein 13-valent conjugate vaccines, wherein each serotype in the figure is, in order from left to right, sample 1, sample 2, sample 3, sample 4, sample 5;
FIG. 6: the immunogenicity of the 15-valent pneumococcal conjugate vaccine is researched, wherein each serotype in the figure sequentially comprises a sample 1, a sample 2, a sample 3, a sample 4 and a sample 5 from left to right;
FIG. 7: 13-valent pneumonia conjugate vaccine immunogenicity results, wherein each serotype in the figure, from left to right, is in the order of sample 1, sample 2, sample 3, sample 4, sample 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Preparation of conjugates with different Carrier proteins
Pneumonia capsular polysaccharide of different serotypes is selected and respectively coupled with CRM197 and TT, the coupling method of polysaccharide and protein adopts a common experimental method in the industry, namely CDAP or reduced amine method coupling chemical reaction (see patent WO95/08348 in particular), and the molecular weight of the conjugate is respectively detected after purification, and the result is shown in Table 1.
TABLE 1 molecular weight size of different Carrier conjugates
Remarking: molecular weight size of the conjugates is expressed as KD < 0.35 recovery
The results show that the molecular weight of the conjugates of different carrier proteins of the same serotype is different, and generally, the molecular weight of TT as the carrier protein is higher than that of CRM197 as the carrier protein. The molecular weight of the SP6B, 19A and 23F conjugates with TT as carrier was higher than CRM197, but the difference was not significant. 5. The molecular weight of 6A, 7F, 14 and 19F with TT as carrier is obviously higher than that of CRM 197.
Formula of combined vaccine preparation for pneumonia with valence of two and 13/15
The 13-valent or 15-valent pneumonia conjugate vaccine disclosed by the invention has the following antigen content characteristics in each dose, and firstly, the antigen content of SP6B is higher than that of other serotypes, and is generally higher than 2 times. Secondly, the antigen content of the remaining serotypes, with the exception of SP6B, fluctuates within a certain range, but the antigen doses are all lower than SP6B, and thirdly, the same doses can be selected for the antigen content of the remaining serotypes, with the exception of SP 6B. The dosage of pneumococcal conjugate vaccine antigens 1, 2 and 3 are shown in tables 2-4.
TABLE 213 VALENT/15 VALENT pneumococcal conjugate vaccine antigen dose 1
| Serotype | Antigen content | Preferential dosage |
| SP6B | 2-10 mu g/dose | 4.4 μ g/dose |
| Remaining serotypes other than SP6B | 1-5 mu g/dose | 2.2. mu.g/dose |
TABLE 313 VALENT/15 VALENT pneumococcal conjugate vaccine antigen dose 2
| Serotype | Antigen content | Preferential dosage |
| SP6B | 2-10 mu g/dose | 4.4 μ g/dose |
| Serotype with CRM197 as vector in addition to SP6B | 1-6 mu g/ |
3 mu g/dose |
| Serotypes with TT/HID as carrier in addition to SP6B | 1-5 mu g/ |
2 mu g/dose |
TABLE 413 VALENT/15 VALENT pneumococcal conjugate vaccine antigen dose 3
Three, difference in immunogenicity of different carrier protein conjugates
The pneumonia capsular polysaccharide of different serotypes is selected and respectively coupled with CRM197/TT/DT/HID, the coupling method of polysaccharide and protein adopts the common experimental method in the industry, namely CDAP or reduced amine method coupling chemical reaction (see patent WO95/08348 in particular), the conjugate is purified, and then mice are immunized to determine the immunogenicity.
Based on the results of the research on the immunogenicity of the pneumonia capsular polysaccharide, pneumonia capsular polysaccharides of different serotypes are classified into 2 types, one of which is suitable for CRM197 as a carrier protein. And TT/DT/HID is suitable as a protein carrier. 2 serotypes were selected for each class, conjugated to different carrier proteins, and immunogenicity studies were performed, and the information on the test articles is shown in Table 5.
TABLE 5 summary of the test article information
| Test article | Serotype + carrier protein | |
| Group | ||
| 1 | SP5+CRM197/TT/DT/HID | Each preparation contains polysaccharide 2.2 μ |
| Group | ||
| 2 | SP7F+CRM197/TT/DT/HID | Each preparation contains polysaccharide 2.2 μ |
| Group | ||
| 3 | SP6B+CRM197/TT/DT/HID | Each preparation contains polysaccharide 4.4 μ |
| Group | ||
| 4 | SP9V+CRM197/TT/DT/HID | Each preparation contains polysaccharide 2.2 μ g |
In Table 5, the antibody titer in the serum was measured after 4 groups of test articles had been immunized into mice, and the results are shown in FIGS. 1 to 4. The immunogenicity results of different carrier protein conjugates show that the immunogenicity of TT as the carrier protein conjugate is the strongest, the immunogenicity of DT as the carrier protein conjugate is the weakest, and the immunogenicity of CRM197 and HID as the carrier protein conjugate is not greatly different.
Example 2
Immunogenicity of 13-valent pneumonia conjugate vaccine with one and different carriers
Experimental procedure for conjugation of capsular polysaccharide and carrier protein As in example 1, 13-valent pneumococcal conjugates of different carriers were prepared (Table 6) and immunogenicity was determined in a mouse model (see FIG. 5).
Summary of Carrier protein information for conjugates with valency Table 613
Remarking: all samples had the same amount of polysaccharide antigen of the same serotype
As can be seen in figure 5, some serotypes are not associated well with immunogenicity and carrier proteins, such as SP1, 3, 18C, but some serotypes are associated well with immunogenicity and carrier proteins. Overall, samples 4 and 5 are overall more immunogenic than samples 1-3.
In conclusion, the combined vaccine adopting the double vectors reduces the immune interference among different serotypes, and the immunogenicity of the combined vaccine is better than that of the combined vaccine adopting a single vector on the whole.
Immunogenicity of 15-valent pneumonia conjugate vaccine with two and different carriers
Experimental methods for conjugation of capsular polysaccharide and carrier protein as in example 1, 15-valent conjugates of pneumonia with different carriers were prepared (table 7) and immunogenicity was determined in a mouse model (fig. 6).
TABLE 715 valency conjugate Carrier proteins
As can be seen in figure 6, the immunogenicity of the SP1, 3, 18C serotypes are not strongly correlated with the carrier protein, and the immunogenicity of the other serotypes are strongly correlated with the carrier protein. Overall, samples 4 and 5 are overall more immunogenic than samples 1-3.
The results show that the combined vaccine adopting the double carriers reduces the immune interference among different serotypes, and the immunogenicity of the combined vaccine is superior to that of the combined vaccine adopting a single carrier on the whole.
Example 3
Immunogenicity comparison of novel 13-valent pneumonia conjugate vaccine
Experimental procedure for conjugation of capsular polysaccharide and carrier protein As in example 1, conjugate vaccines containing different amounts of dual carrier were prepared. All pneumonia serotypes are classified into 2 types, one type uses CRM197 as a carrier protein, and the other type uses TT/HID as a carrier protein, one (SP7F or SP19F), two (SP7F and SP19F) and all (SP1, SP4, SP5, SP19F and SP7F) are coupled with TT or HID, the sample information is shown in a table 8, and the immunized mice are subjected to immunogenicity research (see figure 7).
TABLE 8 novel 13-valent conjugate vaccines
Remarking: all samples, with the same serotype polysaccharide antigen content, were classified as TT for HID carrier protein conjugated samples
As can be seen from fig. 7, the immunogenicity of samples 5(SP1, SP4, SP5, SP19F, SP7F coupled to TT or HID carrier) and 4(SP19F, SP7F coupled to TT or HID carrier) was overall better than the pneumonia binding vaccine with CRM197 alone (sample 1), or CRM197 as the carrier of 12 serotypes (samples 2 and 3), and TT/HID protein as the carrier of the other serotype.
The results of this study indicate that at least two serotypes of SP1, SP4, SP5, SP19F, SP7F (SP19F, SP7F) are conjugated to TT or HID carriers to maintain potent immunogenicity of all serotypes of 13 valent pneumonia conjugate vaccine.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (4)
1. A preparation combination of a multivalent pneumococcal conjugate vaccine, which is characterized by comprising at least one first carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes and at least one second carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes, wherein the capsular polysaccharides are selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F, the capsular polysaccharides of 7F and 19F are conjugated with the second carrier protein, the first carrier protein is CRM197, and the second carrier protein is TT or HID, wherein the capsular polysaccharide of the same serotype is conjugated with only one carrier protein, and the multivalent pneumococcal conjugate vaccine is a 10-valent pneumococcal conjugate vaccine, A 13-valent pneumococcal conjugate vaccine or a 15-valent pneumococcal conjugate vaccine, wherein the serotype of capsular polysaccharide conjugated to CRM197 carrier protein in the 10-valent pneumococcal conjugate vaccine is 1, 4, 5, 6B, 9V, 14, 18C or 23F, and the serotype of capsular polysaccharide conjugated to TT or HID carrier protein is 7F and 19F, or the serotype of capsular polysaccharide conjugated to CRM197 carrier protein in the 10-valent pneumococcal conjugate vaccine is 6B, 9V, 14, 18C or 23F, and the serotype of capsular polysaccharide conjugated to TT or HID carrier protein is 1, 4, 5, 7F and 19F, or the serotype of capsular polysaccharide conjugated to CRM197 carrier protein in the 13-valent pneumococcal conjugate vaccine is 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A and 23F, and the serotype of capsular polysaccharide conjugated to TT or HID carrier protein is 7F and 19F, alternatively, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein in the 13-valent pneumococcal conjugate vaccine include 3, 6A, 6B, 9V, 14, 18C, 19A and 23F, the serotypes of capsular polysaccharide conjugated to TT or HID carrier protein are 1, 4, 5, 7F and 19F, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein in the 15-valent pneumococcal conjugate vaccine include 2, 3, 6A, 6B, 9V, 14, 18C, 19A, 23F and 33F, and the serotypes of capsular polysaccharide conjugated to TT or HID carrier protein are 1, 4, 5, 7F and 19F.
2. A method of preparing the combination of multivalent pneumococcal conjugate vaccine formulations of claim 1, comprising the steps of:
(1) preparing at least one first carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes, and obtaining a first binding stock solution, wherein the first carrier protein is CRM 197;
(2) preparing at least one second carrier protein conjugated with capsular polysaccharides from different pneumococcal serotypes, and obtaining a second binding stock solution, wherein the capsular polysaccharides of serotypes 7F and 19F are conjugated with the second carrier protein, and the second carrier protein is TT or HID;
(3) combining the first combined stock solution obtained in the step (1) and the second combined stock solution obtained in the step (2) to obtain a preparation combination of the multivalent pneumococcal conjugate vaccine;
wherein, the capsular polysaccharide of the same serotype is conjugated with only one carrier protein, the multivalent pneumococcal conjugate vaccine is a 10-valent pneumococcal conjugate vaccine, a 13-valent pneumococcal conjugate vaccine or a 15-valent pneumococcal conjugate vaccine, the serotype of the capsular polysaccharide conjugated with CRM197 carrier protein in the 10-valent pneumococcal conjugate vaccine is 1, 4, 5, 6B, 9V, 14, 18C or 23F, the serotype of the capsular polysaccharide conjugated with TT or HID carrier protein is 7F and 19F, or the serotype of the capsular polysaccharide conjugated with CRM197 carrier protein in the 10-valent pneumococcal conjugate vaccine is 6B, 9V, 14, 18C or 23F, the serotype of the capsular polysaccharide conjugated with TT or HID carrier protein is 1, 4, 5, 7F and 19F, or the serotype of the capsular polysaccharide conjugated with CRM197 carrier protein in the 13-valent pneumococcal conjugate vaccine is 1, 5, 7F and 19F, or the capsular polysaccharide conjugated with CRM197 carrier protein in the 13-valent pneumococcal conjugate, 3. 4, 5, 6A, 6B, 9V, 14, 18C, 19A and 23F, the serotypes of capsular polysaccharide conjugated to TT or HID carrier protein being 7F and 19F, or the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein in the 13-valent pneumococcal conjugate vaccine being 3, 6A, 6B, 9V, 14, 18C, 19A and 23F, the serotypes of capsular polysaccharide conjugated to TT or HID carrier protein being 1, 4, 5, 7F and 19F, the serotypes of capsular polysaccharide conjugated to CRM197 carrier protein in the 15-valent pneumococcal conjugate vaccine being 2, 3, 6A, 6B, 9V, 14, 18C, 19A, 23F and 33F, and the serotypes of capsular polysaccharide conjugated to TT or HID carrier protein being 1, 4, 5, 7F and 19F.
3. The method for preparing according to claim 2, wherein the conjugate obtained by conjugating the first carrier protein is purified by column purification using membrane ultrafiltration or ion exchange, and the conjugate obtained by conjugating the second carrier protein is purified by molecular sieve.
4. Use of a combination of preparations of a multivalent pneumococcal conjugate vaccine according to claim 1 in the manufacture of a medicament for the prevention of pneumococcal disease.
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