CN107007829B

CN107007829B - Pneumonia multivalent conjugate vaccine and preparation method thereof

Info

Publication number: CN107007829B
Application number: CN201710277403.4A
Authority: CN
Inventors: 艾智武; 刘昊智; 史晋; 吴克
Original assignee: Bravovax Co ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2014-07-25
Filing date: 2014-09-23
Publication date: 2020-08-11
Anticipated expiration: 2034-09-23
Also published as: CN107007829A; CN104208671A; CN104208671B

Abstract

The invention discloses a pneumonia polyvalent combined vaccine, which is formed by connecting polyvalent pneumococcal polysaccharide with two or more than two carrier proteins through a connector, wherein the connector is a magnetic nano microsphere. The preparation method of the pneumonia multivalent conjugate vaccine is characterized in that multivalent pneumococcal polysaccharide and two or more than two carrier proteins are coupled with nano microspheres respectively. The pneumonia multivalent conjugate vaccine adopting the nano microspheres as the connecting object can enhance Th1 type immune response of mice, and the immune persistence, specificity and affinity of polysaccharide specific antibodies, and can induce the mice to generate rotavirus antibodies; has the prevention effect of two vaccines; therefore, the method has very wide application prospect.

Description

Pneumonia multivalent conjugate vaccine and preparation method thereof

Technical Field

The invention relates to a vaccine, in particular to a pneumonia multivalent conjugate vaccine and a preparation method thereof, belonging to the technical field of biology.

Background

Harm and countermeasure of microorganism to human body

The microorganisms generally refer to a biological population with the individual volume diameter of generally less than 1mm, the biological population is simple in structure, mostly single cells, even cells and structures do not exist, the microorganisms do not exist at all times, and the shapes and the structures of the microorganisms can be seen by means of a microscope or an electron microscope; the pathogenic microorganism is a microorganism which can cause diseases of human, animals and plants and has pathogenicity. The pathogenicity of a pathogen depends on its ability to invade the host and to reproduce and defend against the host's resistance without being destroyed. The pathogenicity of microorganisms is characterized by species, and the degree of the pathogenicity is called virulence. The establishment of infectious diseases is not unilaterally determined by the virulence of the microorganism, but rather depends on the health and immune function of the host. In general, virulent microorganisms infect non-immunized organisms, causing pathological lesions such as dominant infections, while normal organisms are resistant to many less virulent microorganisms (e.g., opportunistic pathogens), but are susceptible to disease when host resistance is reduced. The virulence of the pathogen and the resistance of the host are compared, so that the occurrence, development, outcome and prognosis of infectious diseases are led, and the counter results of the pathogen and the host are different due to different adaptation degrees, so that different infection spectrums, namely different manifestations of the infection process, are generated. Pathogenicity is that for a particular host, some are pathogenic only to humans, some are pathogenic only to certain animals, and some are zoonotic microorganisms. In this case, the host can usually only infect the microorganism through its own immune system, and the death rate is very high, so that researchers develop vaccines against the invasion of pathogenic microorganisms to human bodies.

Vaccines are classified into therapeutic vaccines and prophylactic vaccines, in which diseases are treated by therapeutic vaccines and the body is protected from pathogenic microorganisms by prophylactic vaccines. The prevention of disease by vaccination is a proven effective tool in human clinical practice for over a century. Over the course of many years of effort, the medical community has developed a variety of vaccines to prevent various diseases caused by infections such as bacteria, viruses and fungi, which have greatly improved the health level of humans. The continuous development of biotechnology promotes the diversification of vaccine varieties. Nowadays, vaccines developed by inactivated virus technology, such as Japanese encephalitis vaccine, polio vaccine, influenza vaccine, etc., are used for preventing infectious diseases caused by viruses; attenuated live vaccines developed by attenuated virus technology, such as rotavirus vaccine, oral poliovirus vaccine, measles virus vaccine, mumps virus vaccine, rubella virus vaccine, varicella vaccine and the like. The bacterial vaccines developed by the purification technology of biological macromolecules such as protein and polysaccharide for preventing bacterial infectious diseases, such as tetanus toxoid, diphtheria toxoid, pertussis toxoid, subcellular components thereof, epidemic meningococcal polysaccharide, 23-valent pneumococcal polysaccharide and the like. Vaccines developed by using gene recombinant protein technology, such as hepatitis B surface antigen (for preventing hepatitis B), human papilloma virus (for preventing cervical cancer), etc. Bacterial vaccines developed by the semi-chemical conjugation technique for preventing meningitis and pneumonia, such as polysaccharide-protein conjugate vaccine of Haemophilus influenzae type b, polysaccharide-protein conjugate vaccine of pneumococcus with 7 or 10 valences, and polysaccharide-protein conjugate vaccine of meningococcus with 4 valences. Therefore, the development of medical biotechnology is the prime power for the development of vaccine products, and more novel vaccine products can be developed to deal with the challenges of different infectious diseases on human health through the continuous improvement on the biotechnology.

Second, summary of conjugate vaccines

Polysaccharides are an important immunologically active ingredient in pathogenic bacteria, and are classified into the fungal polysaccharides (OPS) and Capsular Polysaccharides (CPS). When pathogens invade the body, they act as immunogens which stimulate the body to produce a protective immune response. However, polysaccharide molecules belong to T cell independent antigens (Ti-Ag), have weak immunogenicity, are not particularly ideal for immunization after being inoculated to infants, and currently, many serious diseases such as meningitis caused by Haemophilus influenzae (Hib), hemorrhagic diarrhea dehydration in infants caused by E.Coli O157: H7 and the like have high incidence and no clinically effective treatment method, and have high fatality rate (Wang Yan et al, combined with vaccine summary [ J ], microbiological immunological progress, 2000,28(1): 60-63). In order to improve the immunogenicity of polysaccharide vaccines, a polysaccharide and protein chemical conjugate vaccine, namely a conjugate vaccine, is developed in the beginning of the 20 th century abroad, and the conjugate vaccine (bacterial polysaccharide using protein as a carrier) is prepared by covalently binding polysaccharide on a protein carrier by a chemical method to prepare a polysaccharide-protein conjugate vaccine for improving the immunogenicity of a bacterial vaccine polysaccharide antigen, such as a b-type haemophilus influenzae conjugate vaccine, a meningococcus conjugate vaccine, a pneumococcal conjugate vaccine and the like.

Third, the research on the hazard of pneumococcus and the epidemiology thereof

Infections caused by pneumococci (the lung chain) are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia and meningitis are the most common manifestations of invasive pneumococcal disease, and bacterial spread in the respiratory tract can lead to middle ear infections, sinusitis or recurrent bronchitis. Non-invasive manifestations are usually less severe, but more common, than invasive diseases. The likelihood of pneumococcal disease onset during influenza is further increased due to the spread of antibiotic-resistant infections, and pneumococcal pneumonia often follows influenza infection.

The disease caused by streptococcus pneumoniae has become an important public health problem worldwide. Pneumococci have become the first killer of children worldwide. The fatality rate of pneumonia in China is 16.4%, wherein the fatality rate of middle-aged and old people over 50 years old and infants under 1 year old is respectively as high as 28.6% and 22.0%. The carriage rate of pneumococcus in healthy children in China is high, and data statistics shows that the carriage rate of pneumococcus in healthy children in northern regions is 24.2%, and the carriage rate of pneumococcus in southern regions is 31.3%. Which is a significant cause of death in children under the age of 5. The main reasons are that the development of the immune system of infants is not complete and the immunity is weak. And the younger the infant, the weaker the immunity. There are about 90 serotypes (strains) of pneumococcus, and statistics in China show that the first several serotypes of pneumococcus infection strains are: 5. types 6, 19, 23, 14, 2, 4. A study in which 860 pneumococcal isolates were collected showed that 109 (12.7%) showed serogroup 6 with 100% erythromycin resistance in pneumococcal serotype 6 in china, with types 6A, 6B and 6C of 62 (56.9%), 38 (34.9%) and 9 (8.2%) respectively.

Chemically, the above pathogens possess a cell surface Capsular Polysaccharide (CPS) or Lipopolysaccharide (LPS) shell, or both, which function to help the pathogen infect the host. Capsular polysaccharide can shield the functional components on the surface of bacterial cells from being recognized by the immune system of a host, prevent the complement system from being activated by bacterial surface proteins and phagocytosis by immune cells, and prevent bacteria from being killed if the bacteria are phagocytosed. In most pathogenic bacteria, different strains express capsular polysaccharides and lipopolysaccharides of different structures, resulting in a variety of different serotype strains. Pneumococcal pneumonia and meningitis are caused by infection with a large proportion of the 90 known serotypes of the strain.

It follows that most bacterial polysaccharide vaccines must contain a number of different bacterial polysaccharides to increase the coverage of pathogenic strains, and that optimizing and selecting which bacterial or serotype polysaccharide to include is a very complex epidemiological problem in vaccines. Once it is clear which polysaccharide antibodies have a protective effect, the polysaccharide can be used as an immunogen to produce vaccines.

23-valent pneumococcal vaccines produced by China biotechnology group Chengdu biological product research institute are prepared by selecting 23 most common pathogenic bacteria (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), fermenting, culturing, separating and purifying various polysaccharides on pneumococcal capsules respectively, and mixing the polysaccharides in equal proportion to prepare the vaccine. Bacterial polysaccharides are a thymus-independent antigen, which differs from thymus-dependent antigens primarily in that the former do not require the help of T lymphocytes to produce antibodies. Clinical use proves that the vaccine prepared from the capsular polysaccharide is definitely effective and widely used in a plurality of countries. However, such polysaccharide vaccines have the following problems: (1) only weak immune response can be generated in the bodies of young animals or infants, even no immune response can be generated, and the immune response is enhanced along with the increase of the age; (2) generating low affinity antibodies; (3) only produces transient immune response, and does not have immune memory and immune enhancement effect during repeated inoculation; (4) immune tolerance is easy to generate; (5) the common adjuvant has the function of immune enhancement to the antigen, the protection rate of the 23-valent polysaccharide vaccine to invasive lung chain infection is 50-70%, and the vaccine can only be used for the inoculation of people over 2 years old, and the peak incidence age of pneumonia is 6-12 months old; (6) the polysaccharide with the repeated structure is T cell independent type 2 immunogen without T cell participation, and the T cell independent type 2 immunogen cannot induce immunological memory effect, and the antibodies produced by the body are mainly IgM and IgG2, so the polysaccharide cannot effectively activate the complement system.

The approach to develop vaccines by semi-chemical conjugation (also known as conjugation) was the development of bacterial vaccines since the 80's of the 20 th century. John Robbins initiated a new generation of bacterial vaccine development technology by synthesizing polysaccharide PRP-tetanus toxoid conjugate vaccine (PRP-TT) of Haemophilus influenzae type b (Haemophilus fluuenzaetypeb, Hib) by covalently linking PRP to a protein carrier (tetanus toxoid). Of particular interest is that for infants younger than 2 years of age, due to the poor development of the immune system, polysaccharides are a T-cell independent antigen for infants and are not able to stimulate the body to produce long-lasting protective IgG antibodies specific to the bacteria from which they are derived, as in adults. Thus, polysaccharide is a hapten for infants younger than 2 years of age and cannot be used as a vaccine to inoculate children younger than 2 years of age. When a bacterial polysaccharide is covalently linked to a protein carrier, such as Tetanus Toxoid (TT), the protein is a T-cell dependent antigen, and the covalently linked polysaccharide can be converted to a T-cell dependent antigen, thereby stimulating the body to produce specific IgG antibodies directed against the polysaccharide and protecting the body from bacterial infection. The success of the development of the polysaccharide-tetanus toxoid (PRP-TT) conjugate vaccine of the haemophilus influenzae type b opens up a technical platform for developing bacterial vaccines, namely, the conjugate vaccine prepared by covalently bonding bacterial polysaccharides such as capsular polysaccharide, O-specific polysaccharide (O-specific polysaccharide) or Oligosaccharide (Oligosaccharide) to a protein carrier. Based on the success of this concept, the medical and biological research community has developed conjugate vaccines of different bacteria by using the same chemical synthesis method; conjugate vaccines of the same bacteria were also developed using different synthetic techniques.

Capsular polysaccharide can shield the functional components on the surface of bacterial cells from being recognized by the immune system of a host, and prevent the complement system from being activated by proteins on the surface of the bacteria and phagocytosis of immune cells. Capsular polysaccharides also prevent bacteria from being killed if they are phagocytosed by immune cells. Capsular polysaccharide is one of the main antigen components of Neisseria meningitidis, and has certain protection for larger children as a vaccine. However, capsular polysaccharides have poor immune efficacy in infants under 2 years of age, the elderly, and people with B-cell immunodeficiency, vaccinants fail to achieve antibody protection, and antibodies quickly disappear. The polysaccharide vaccine, like other polysaccharide vaccines, is a T cell independent antigen, has age-related immunogenicity, and does not induce T cell dependent booster responses. By covalently binding a polysaccharide to a protein, the polysaccharide can be converted to a T cell-dependent antigen, thereby stimulating T cell-dependent antibody synthesis in infants, producing a booster response, and increasing the antibody ratio of immunoglobulin (IgG). The polysaccharide conjugate vaccine can protect infants (children under 2 years old) and greatly enhance the resistance of patients with poor resistance to bacterial infection, so that the polysaccharide conjugate vaccine has a very wide application prospect. In 1980, John Robbins randomly activated Hib capsular polysaccharide with cyanogen bromide, then Added Dihydrazide (ADH) as a linker to the activated polysaccharide, and finally covalently linked the derivatized polysaccharide to the carrier protein tetanus toxoid by EDC method, synthesized the Haemophilus influenzae type b polysaccharide-tetanus toxoid conjugate vaccine (PRP-TT). Since there are multiple activation sites on each polysaccharide chain and multiple attachment sites on the protein carrier are the same, the conjugate formed is a cross-linked macromolecule of polysaccharide and protein with an average molecular weight of about 5X 106 Da. In 1980, Harold Jennings in U.S. Pat. No. 4356170, it was stated that a polysaccharide of group A, C of meningococcus was covalently linked to BSA by the reductive amine method using Bovine Serum Albumin (BSA) as a carrier, and an epidemic meningitis polysaccharide-BAS conjugate vaccine was synthesized. In 1987 and 1990, Porter Anderson described the synthesis of a haemophilus influenzae type b oligosaccharide-variant avirulent diphtheria toxin 197(HbOC-CRM197) conjugate vaccine using variant avirulent diphtheria toxin 197(Cross reaction material sub197, CRM197) as a carrier protein in a reduced amine process in us patents 4673574 and 4902506, respectively. The specific method is to oxidize Hib capsular polysaccharide with sodium periodate to generate oligosaccharide with aldehyde group at two ends, and to link the oligosaccharide to protein carrier via covalent bond with reducing agent sodium cyanoborohydride (sodium cyanoborohydride). Lipopolysaccharide conjugates with a molecular weight of about 90kDa were formed, and conjugate vaccines containing 30% polysaccharides and 6 saccharide molecules per protein were prepared. Subsequently, american Merck (Merck, sharp and Dohme) synthesized an epidemic haemophilus B type polysaccharide-bacterial surface protein complex (PRP-OMP) conjugate vaccine using purified Neisseria meningitidis group B bacterial surface protein complex (OMP) as a protein carrier by thiol chemistry. By applying the synthesis technology, three kinds of epidemic haemophilus type b combined vaccines which are widely used for clinical inoculation at present, namely PRP-TT, PRP-HbOC and PRP-OMP, are developed successively. The success of Hib conjugate vaccines provides a theoretical and technical basis for the development of other bacterial conjugate vaccines, and subsequent development has entered the more technically complicated multivalent conjugate vaccine stage, because some infectious diseases, such as pneumonia by pneumococci, meningitis by epidemic meningococci, etc., can be caused by infection with many different serotypes or strains, and because there is no cross-immune reaction between the serotypes or strains due to the chemical structure of the bacterial surface polysaccharides, and therefore, vaccination with a single serotype or strain does not protect the vaccinated body from infection by other serotypes or strains. For this reason, the synthesis and formulation of multivalent conjugate vaccines to expand the protective coverage of the vaccine is a major goal of development.

Through years of effort, various multivalent conjugate vaccines with broad coverage have been developed using conjugation techniques. Bacterial capsular polysaccharide-protein conjugate vaccines were first shown in the 30's of the 20 th century, and Goebel and Avery linked pneumococcal type 3 polysaccharides to equine serum globulin, producing conjugates that produced polysaccharide-specific antibodies in animals while providing corresponding immunoprotection. In 1987, the first polysaccharide protein conjugate vaccine in the world, the Haemophilus influenzae type B (HiB) polysaccharide-Tetanus Toxoid (TT) conjugate vaccine, was approved by the U.S. FDA for entry into the market. The HiB polysaccharide-tetanus toxoid conjugate vaccine and the epidemic meningitis polysaccharide-tetanus toxoid conjugate vaccine were successively developed by merck, buffy and noval and successfully marketed. In 2000, 7-valent pneumococcal polysaccharide-CRM 197 conjugate vaccine was successfully developed and marketed by the American Huishh (Wyeth) company, and is a multivalent vaccine prepared by covalently bonding 7 different pneumococcal serotype polysaccharides to CRM197 protein carriers respectively and mixing the polysaccharides to prevent infantile pneumonia, wherein the 7 serotype pneumococci cover more than 90% of the popular pneumococcal different serotype strains in North America and Europe. In 2006, Sanofi Pasteur developed a 4-valent meningococcal polysaccharide-tetanus toxoid conjugate vaccine for the prevention of meningitis caused by 4 epidemic meningococcal flora, namely A, C, Y, W135. In 2009, glaxosmithkline (gsk) also developed a 10-valent pneumococcal polysaccharide-protein conjugate vaccine to prevent pneumonia caused by 10 pneumococcal serotypes. The vaccine uses three proteins as Protein carriers, wherein the most important carrier is Protein-D (PD), and 8 serotype polysaccharides use the Protein as a carrier. protein-D is an esterificated surface protein expressed by a gene recombination method of non-separable haemophilus influenzae, can stimulate the organism to generate protective antibodies, has potential to prevent acute otitis media caused by non-separable haemophilus influenzae infection, and is used as a carrier for tetanus toxoid and diphtheria endotoxin which are used as protein carriers of conjugate vaccines of serotypes 18C and 19F respectively. From the above design of conjugate vaccine products, it can be seen that the development of conjugate vaccines has transitioned from monovalent vaccines to more technically complex multivalent vaccines, improving the coverage of bacterial vaccines.

Polysaccharide protein conjugate vaccine (conjugate vaccine) is the most advanced vaccine technology at present, and a protein carrier is added on a specific antigen to increase the immunogenicity of the vaccine. The protein carrier has T cell dependent characteristic, and the polysaccharide protein conjugate vaccine can convert polysaccharide antigen with non-T cell dependent property into antigen with T cell dependent property, and excite T helper cell of body to produce a series of immune enhancement effects. The capsular polysaccharide conjugate vaccine is prepared by adding a protein carrier on polysaccharide, so that the T cell independent antigen is changed into the T cell dependent antigen, and the immunogenicity of the vaccine is improved. The quality and quantity of the antibody generated after combined vaccination are 1000 times of those of the first-generation vaccine, so that the generated immune protection is wider and stronger, the protection time is longer, and the high-efficiency protection is achieved. In 2000, the first 7-valent pneumococcal conjugate vaccine of pfeiffe americana was marketed; pfeiri developed a more targeted 7(4, 6B, 9V, 14, 18C, 19F and 23F) valent pneumococcal protein vaccine for infants and children under 5 years of age. The capsular polysaccharide protein conjugate vaccine is prepared by adding a protein carrier to polysaccharide, and changing non-T cell dependent antigen into T cell dependent antigen, so that the immunogenicity of the vaccine can be improved, and the vaccine can be used for children over 6 weeks old. Currently, the company pfeiri is registering 13-valent conjugate vaccines in china, adding 6 serotypes (1, 3, 5, 7F, 6A, 19A). However, the data in China show that the serotypes of pneumococcal infection strains are sequentially as follows: 5. the coverage rate of 6, 1, 19, 23, 14, 2, 4, 7-valent pneumococcal conjugate vaccine of the pfeiffer is only about 50 percent for common pathogenic bacteria types in China, and the coverage rate of 13-valent conjugate vaccine is only 70 percent. The Huishi 7-valent pneumococcal conjugate vaccine needs to be inoculated with 4 injections of 860 yuan each, and is expensive and not beneficial to popularization. A 13-valent vaccine would be estimated to be more expensive. Therefore, the 7-valent and 13-valent conjugate vaccines of the American pfeiffer company are not suitable for preventing the pneumonia of children in a large range in China.

Although 13-valent pneumococcal conjugate vaccines are commercially available, several of these serotypes have a lower immune response than others, such as type 3. And with the increase of different serotypes and the repeated use of the same carrier protein, the dosage of the carrier protein is increased, and the immunogenicity of the carrier protein is reduced. GlaxoSmithKline selected protein-D as a carrier in the design of developing a 10-valent pneumococcal polysaccharide conjugate vaccine for two reasons. First, it is to avoid the repeated use of tetanus toxoid and diphtheria toxoid, which have been components of a diphtheria vaccine, as carriers. Clinical trials show that when the multivalent pneumococcal conjugate vaccine is inoculated with other univalent vaccines or multiple vaccines containing the same components with protein carriers, such as Hib-TT, diphtheria-Hib polysaccharide conjugate vaccine-IPV (inactivated polio vaccine) -hepatitis B vaccine (6-combination vaccine for short), the immunogenicity of most of serotype polysaccharides in the multivalent pneumococcal conjugate vaccine can be inhibited, and particularly, the immunogenicity of the serotype polysaccharides using tetanus toxoid as carriers is obviously influenced. The reason is that the total concentration of tetanus toxoid and diphtheria toxoid as carriers in the multivalent pneumococcal conjugate vaccine is too high, and when the conjugate vaccine containing diphtheria and tetanus toxoid is simultaneously inoculated, such as the 6-conjugate vaccine, a so-called carrier receptor competitive inhibition effect is caused, and the immunogenicity of the polysaccharide part of the conjugate vaccine is reduced. The 11-valent pneumococcal polysaccharide-TT and DT mixed carrier protein conjugate vaccine of SanofiPasteur and the 7-valent pneumococcal polysaccharide-OMP conjugate vaccine (PCV-OMP) of Merck are examples of product development failures caused by poor clinical test results, and the reason is that the two vaccines are combined. Secondly, the protein carrier is endowed with a real protective immunogenicity function. Clinical tests prove that the protein-D can stimulate the organism to generate protective antibodies and has potential prevention of acute otitis media caused by non-separable haemophilus influenzae infection. The 10-valent pneumococcal polysaccharide conjugate vaccine of GlaxoSmithKline selects protein-D as a carrier, so that an antibody generated by the protein carrier has a clinically significant protective effect, and the method is a great progress in the development process of a conjugate vaccine technology.

However, the practical clinical significance of non-typeable haemophilus epidemicus is limited by the low rate of infection by the bacteroides, which results in a low incidence of acute otitis media. However, these conjugate vaccines have a common disadvantage in that the protein carrier does not confer a protective function of immunogenicity, i.e., although the conjugate vaccine carrier can stimulate the body to produce antibodies, the vaccine designer does not utilize the protection of the antibodies to prevent infectious diseases, and at the same time, does not determine whether the antibodies produced reach a protective titer level. The conventional proteins, tetanus toxoid, diphtheria toxoid and diphtheria non-toxic variant toxin, were selected as carriers not for their protective properties of the antibodies produced, but for their safety and their ability to enhance the immunogenicity of the polysaccharides in the conjugate. Obviously, tetanus toxoid and diphtheria toxoid are two components of the diphtheria-tetanus triple vaccine and are inoculated conventionally; therefore, it is not important whether the tetanus toxoid and diphtheria toxoid carriers in the conjugate vaccine can stimulate the body to produce protective antibody titers, but rather, the antibody titers of the polysaccharide portion of the conjugate vaccine are the main issues of concern to the vaccine designer. In addition, vectors for use in some combination vaccine products under development, such as recombinant pseudomonas aeruginosa exotoxin a (repa) detoxified by e.coli expressed gene deletion mutations, recombinant cholera toxin detoxified by e.coli expressed gene deletion mutations, and the like, are also based on the same considerations.

The types and types of new polysaccharide conjugate vaccines are increasing year by year, while the types of carrier proteins selected are fewer, and the number of carrier proteins reused in different vaccines is more. Vaccination with different combinations of the same carrier protein may produce immunosuppressive effects, leading to interplay of the immune effects between different vaccines. Moreover, the main carrier protein of the polysaccharide conjugate, such as TT, is used as a vaccine to be inoculated to infants in a large range, and the high-titer specific antibody aiming at the carrier protein originally in the human body may inhibit the specific immune response of the body to the polysaccharide in the conjugate vaccine.

Chinese patent (zl02159032.x) discloses a preparation method of polysaccharide-protein conjugate vaccine, which is also one of the most commonly used techniques for preparing polysaccharide conjugate vaccines at present. In the technique, adipic Acid Dihydrazide (ADH) is used as a linker to bind the polysaccharide to the protein. The combination mode firstly needs to activate the polysaccharide by cyanogen bromide, namely cyanogen bromide is used for acting on hydroxyl groups on polysaccharide molecules under alkaline conditions to form cyanate ester, and then the cyanate ester reacts with ADH; one carbon-oxygen bond in the cyanate is broken, and the cyanate and the amino at one end of the ADH generate addition reaction, so that ester hydrazide (AH) groups are introduced into polysaccharide molecules to form polysaccharide-AH derivatives; polysaccharide-AH derivatives form stable conjugates with carrier proteins mediated by carbodiimide (EDAC). The combination mode can reduce the steric hindrance of the combination of the polysaccharide and the carrier protein, retain the epitope of the polysaccharide, avoid the solubility of the polysaccharide, and reduce the side effect of the reaction of the polysaccharide and antiserum.

However, the above-mentioned conventional polysaccharide-protein binding techniques have the following disadvantages: (1) the polysaccharide-AH derivative can continuously react with cyanogen bromide activated polysaccharide to form self-polymer of the polysaccharide, so that the polysaccharide-protein combination efficiency is reduced; (2) EDAC, while mediating the binding of ADH-derived polysaccharides to carrier proteins, tends to cause self-crosslinking of the polysaccharides with the carrier proteins, thereby reducing the polysaccharide-protein binding efficiency; (3) the polysaccharide and the carrier protein are large biomolecules, the polysaccharide and the carrier protein are connected by ADH with the length of only 6 carbon atoms, and the structures of the polysaccharide and the protein are bound to influence each other, so that some important antigen epitopes of the polysaccharide are easily shielded by the protein, and the immunogenicity of the polysaccharide is further reduced. Therefore, the immunogenicity and antibody duration of polysaccharide-protein conjugate vaccines still need to be further improved, for example, polysaccharide conjugate vaccines need to be immunized three times to generate immune effect. These deficiencies limit the further development of polysaccharide conjugate vaccines.

Application of nano-microsphere in biotechnology and prospect

The nano material is a single crystal or a polycrystal with a grain size less than 100 nm, has a unique small size effect and a surface or interface effect, has many excellent or brand new properties, and is increasingly paid attention to. For example, the constant penetration and influence of nanomaterials on the field of drug research has triggered a profound revolution in the field of drugs. Drugs are important substances for resisting and preventing diseases of human beings, and research and development of the drugs have provided a plurality of treatment means for clinic for a long time and bring a plurality of benefits to patients. However, the existing drugs still have many problems, such as the drugs can not be retained in the circulation system and reach effective concentration, can not reach specific therapeutic targets, can not pass through the blood brain barrier, can not form higher concentration in a certain local part and simultaneously can not generate toxic and side effects, etc. (Wu Xinrong. application and research progress of drug-loaded nanoparticles [ J ]. China Hospital Pharmacology, 2001,21(3): 171-. The magnetic nano microsphere medicine carrier is a product combining nanotechnology and modern medicine, and has the advantages of small size effect, good targeting property, biocompatibility, biodegradability, functional groups and the like, so that the defects brought by the traditional medicine are expected to be overcome. The magnetic nanoparticles can also be used for purification and recovery of proteins and enzymes and immobilization of the enzymes, the operation is simple, and the stability of the enzymes is improved. The magnetic nanoparticles are used for immunoassay, and have the characteristics of good specificity, quick separation and good reproducibility. The magnetic microsphere carrier is used for interventional therapy, and embolization is performed in a magnetic control blood vessel, so that the magnetic control blood vessel has the advantages of magnetic control guiding, target embolization and the like.

The application of the magnetic nano-microsphere in biomedicine is gradually widened along with the depth of research, and specifically comprises the following components:

1. immobilized enzyme

Biological macromolecules such as enzyme molecules and the like have a plurality of functional groups, and can be immobilized on the surfaces of the magnetic particles through physical adsorption, crosslinking, covalent coupling and the like. The magnetic nano microsphere immobilized enzyme has the advantages that: easy separation of the enzyme from the substrate and product; improving the biocompatibility and the immunocompetence of the enzyme; improves the stability of the enzyme, has simple operation and can reduce the cost. Bendkiene et al prepared chitosan magnetic microspheres for use as immobilized carriers. After the enzyme is immobilized on the carrier, the enzyme can be easily separated and recovered from the reaction mixture by a magnetic device. Researchers in China also searched this aspect, Dibin et al synthesized Fe3O4/(St-MPEO) (St-styrene, MPEO-polyethylene oxide macromonomer) microspheres by dispersion polymerization, which had amphiphilic structures and good swelling properties in most polar and nonpolar media, so that the microsphere-immobilized compounds had high activity in various media (Ding XB, Wei L, ZHao HZ. Synthesis and catalysis of aliphatic polycarboxylic acids [ J ]. Applied Polymer science, 2001,79(3):1847 and 1851). The magnetic nano microsphere carrier is expected to be used in the fields of purification and recovery of protein and enzyme, immobilization of enzyme, cell separation and the like.

2. Targeted drugs

The targeted drug carrier microspheres mean that the drug-loaded microspheres can be distributed in an action object with high selectivity, so that the curative effect is enhanced, and the side effect is reduced. The initial targeting drug carrier microsphere is prepared by selecting carriers with different affinities to various tissues or pathological parts of an organism to prepare drug-carrying microspheres according to clinical requirements, or combining a monoclonal antibody with the carriers so that the drug can be delivered to a specific part expected to be achieved by treatment. With the increasing requirements of people on treatment, the targeted positioning is limited by the matrix and cannot be completely satisfied, so that a magnetic nano microsphere drug-loading system appears. Under the action of an external magnetic field, the system leads a carrier to be directed to a pathological part (target position) by injecting a human to the pathological tissue in a moving (static) pulse mode, so that the contained medicine is positioned and released and concentrated to the pathological part for action (A Paul, Alivisatos. ultrasensitive macrobiosense for homogeneous immunology [ J ]. Science, 2001,12(5): 53-60). Lubbe et al, Germany, completed the first clinical trial worldwide using magnetic drug targeted therapy. In magnetic targeted therapy of 14 patients with advanced solid tumors, the patients were found to be well-tolerated by magnetic targeted drugs. Lexion et al also used magnetic microspheres as drug carriers to treat rabbit squamous cell carcinomas (Lexion C, Amold W, Klein RJ, et al, Locotegenic Cancer with magnetic drug targeting [ J ]. Cancer Res, 2000,60(23):6641-6648), domestic Dorkaton et al used doxorubicin magnetic protein microspheres to treat mouse vegetative gastric tumors (Kangndron, Sunworu, Chendada, etc.. doxorubicin magnetic protein microspheres targeted to treat mouse vegetative gastric tumors [ J ]. J. China J. Experimental surgery, 2000,17(1):63-64)), Guogu et al used pingyangmycin magnetic microspheres to treat oral-facial spongiform hemangiomas 25 cases. In addition, the strong magnetic field also has the cancer inhibition effect (Guojun, Li Cheng, Wuhanjiang Pingyangmycin magnetic microspheres target to treat cavernous hemangioma of oral maxillofacial part 25 cases of clinical report [ J ]. Nanjing railway college of medicine, 2000,19(2): 112-. Xuhui development and the like, the glucose magnetic microsphere immobilized L-asparaginase is used for treating acute lymphoblastic leukemia, and a good treatment effect is obtained. These results show that the concentration of the magnetic drug at the tumor site increases with the increase of the magnetic field strength, and the drug is fixed at the target site by the magnetic guiding effect of the external magnetic field, thereby exerting the concentrated and efficient anti-tumor effect. Due to the targeting property of the magnetic nano-microsphere and the surface-bound specific carrier, people are expected to track and kill cancer cells which are metastasizing by using the magnetic nano-microsphere, so that the magnetic nano-microsphere becomes a biological missile for killing the cancer cells in a human body. However, the tumors to be treated by the magnetic drugs are mostly on the body surface or in the body surface, so the strength of the external magnetic field can be weaker and is easy to control. However, it is difficult to increase the magnetization of the magnetic microsphere, because the coating layer on the surface of the magnetic particle can greatly reduce the magnetic property, and in addition, the controllability of the particle size is a problem to be solved.

3. Cell isolation and immunoassay

When specific antibody with bioactivity is introduced to the surface of magnetic particle, under the action of external magnetic field, the specific combination between antibody and cell can obtain Immunomagnetic microspheres (IMMS) or Immunomagnetic beads (IMBS), which can be used to separate cell or perform immunoassay. Especially, when the IMBS is adopted to separate the specific antigen substances on the surfaces of cells and organelles, the method has the characteristics of simplicity, convenience, rapidness, high separation purity, reservation of the activity of the target substances and the like. Mccole et al use IMBS to separate T lymphocytes in peripheral blood of adult cattle infected by liver fluke, and the separated cells are pure and have good effect when used in liver fluke infection mechanism. John uses IMMS connected with monoclonal antibody to detect salmonella, the whole detection process only needs 2-3h, the sensitivity is 103-104 thalli/ml, the existence of a certain amount of blood and feces has no interference to analysis, and compared with the agglutination method and the immunofluorescence method, the sensitivity is improved by 103 times. Glenn uses IMBS to separate respiratory syncytial virus, combines enzyme-linked immunoassay, reduces diffusion and nonspecific adsorption in the traditional tube and micropore analysis, the formation of the complex only needs 7min, and the traditional micropore method needs 120 min. Cell isolation techniques can also be used for cancer therapy. The Kangji super-grade monoclonal antibody for resisting human wing cancer is connected to the surface of a polystyrene magnetic microsphere carrier prepared in advance by a method of combining physical adsorption with chemical bond covalent bonding, so that the immunomagnetic microsphere which can be specifically combined with target cells and endows the target cells with magnetic responsiveness is constructed. The results show that the constructed IMMS can be effectively combined with target cells, and preliminary experiments of separating cancer cells from animal bone marrow by using IMMS show that the IMM can effectively eliminate the cancer cells, and the bone marrow cells are only slightly lost. Immunoassay is an important method in modern bioanalytical techniques, and plays a great role in the quantitative analysis of proteins, antigens, antibodies and cells. The antigen or antibody combined with the magnetic nanoparticle carrier is used for immunoassay, and has the characteristics of high specificity, quick separation, good reproducibility and the like. Jingyan and the like adopt an emulsion-free polymerization method, potassium persulfate is used as an initiator in an alcohol-water system, an initiation point is formed on the surface of Fe3O4 magnetic fluid particles, Acrylic Acid (AA) is used as a stabilizer, Styrene (ST) and acrylamide are copolymerized to prepare monodisperse amino magnetic microspheres, and the microspheres can be directly and rapidly crosslinked with antibody (antigen) protein, so that the defect that protein is required to be used as a crosslinking agent when other functional group microspheres are combined with an immunological reagent is avoided (Jingyan, Wangjun, Li Ru Min, and the like).

4. Magnetic control detection plug

In the general process of interventional treatment, ectopic embolism, infarction and other phenomena can occur, and serious complications are caused, which is a difficult problem to be solved in clinic urgently, while interventional treatment using a magnetic microsphere carrier has the advantages of magnetic control guiding, target position embolism and the like when embolism is carried out in a magnetic control blood vessel, and provides a way for solving the problems. The researchers focus on the detailed research on the particle size of the magnetic sphere, the magnetic control time, the magnetic field intensity, the wrapping material of the magnetic microsphere (rough, carrying positive charge and having hydrophobic property), and the like. Minalnimura et al used the combination of hyperthermia and arterial embolization for the study of mouse liver cancer models. They developed DM-MS arterial catheter local drug delivery with the addition of a 500kHz magnetic field. After 3 days of treatment, the tumor growth rates (embolization-hyperthermia, embolization only and control) were 28%, 124% and 385%, respectively (Minalnimura T, Sato H, Kasaoka S, et al. tumor regression by induced hyperthermia combined with fibrosis using dextran associated intraspecific microspheres in rates [ J ]. IntJ Oncol, 2000,16(6): 1153-. The research shows that the DM-MS thermotherapy and embolism combined therapy is an anti-tumor feasible therapy and has wide research and application prospects. Goodwin et al have studied doxorubicin magnetic microsphere hepatic artery embolization and toxicity of drug-targeted anti-tumor therapies (Goodwin SC, Bittner CA, Peterson CL, equivalent. Single-dose invasion study of pathological intra-arterial infusion of viral induced to a novel magnetic targeted drug carrier [ J ]. local, 2001,60(1): 117. sup. 183). The results of the pig liver cancer model established by the method show that the adriamycin magnetic microspheres have low dose and no toxic or side effect. Only when the content of the magnetic microspheres is equal to 75mg (with or without adriamycin), the target area has better effect, the necrosis degree of the liver cancer cells is in direct proportion to the embolism degree, and the adriamycin can not freely circulate in the whole body to be successfully controlled at the target area. The experiments show that the PMMA magnetic microsphere of 30-50um has the advantages of strong magnetic response capability, good magnetic control embolization effect, capability of realizing target embolization under the condition of high blood flow speed and the like, and is a better magnetic control intravascular embolization material (Huihahui, Hilida, before any ability. the experimental research on the polymethyl methacrylate magnetic microsphere intravascular embolization [ J ]. Sichuan medicine, 2001,22(10): 928-) -929)). In magnetron embolization, the size of the magnetic microsphere carrier is the most important factor in influencing the target area localization. If the particle size is smaller, the magnetic responsiveness is weak, the magnetic control degree is poorer, and the magnetic control embolism can not be used for the magnetic control embolism in the blood vessel with high blood flow rate or larger caliber. Therefore, unlike the application of magnetic microspheres in other aspects, magnetic microspheres with larger particle size are generally used in the magnetic control embolism intervention treatment.

It is also reported that the nanospheres can be used as carrier protein and adjuvant of DNA vaccine, but the application of the nanospheres in preventive polysaccharide and/or protein vaccine is not reported.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to provide a multivalent conjugate vaccine for pneumonia, which is more immunogenic.

In order to achieve the purpose, the invention adopts the following technical scheme:

a pneumonia multivalent conjugate vaccine is a pneumonia multivalent conjugate vaccine which is formed by connecting multivalent pneumococcal polysaccharide and two or more than two carrier proteins through a connector, wherein the connector is a magnetic nano microsphere.

As a preferable scheme, the magnetic particles of the magnetic nanospheres are located inside the magnetic nanospheres as cores, and the polymer material is coated outside the magnetic particles.

More preferably, the magnetic fine particles are Fe₃O₄。

In a further preferred embodiment, the particle size of the magnetic nanospheres is 0.1-10 μm, preferably 0.1-5 μm.

As another preferable scheme, the polymer material is a biological polymer material, and is selected from one or more of chitosan, polyethylene glycol, polylactic-co-glycolic acid (PLGA), and polylactic-co-polyethylene glycol (PELA); most preferred is PLGA or PELA.

As another preferred embodiment, the multivalent pneumococcal polysaccharide is a plurality of pneumococcal capsular polysaccharides, preferably capsular polysaccharides on capsules of isolated and purified serotypes of pneumococci, wherein the serotypes of the pneumococci comprise 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F.

As a further preferable scheme, the mass ratio of the multivalent pneumococcal polysaccharide to the two carrier proteins is (0.5-2): 1, preferably (0.5-1): 1, wherein the mass ratio between the carrier proteins is preferably 1: 1.

as a preferred embodiment, the carrier protein is selected from the group consisting of recombinant human rotavirus protein, diphtheria toxoid, tetanus toxoid, carrier protein CRM197, haemophilus influenzae surface protein HiD, pertussis Prn surface protein, pertussis Fha antigen and/or pneumococcal surface protein a (rppa).

In a further preferred embodiment, one of the carrier proteins is the Haemophilus surface protein HiD or the pneumococcal surface protein A (rPspA).

As a further preferred scheme, the recombinant human rotavirus protein is a partial amino acid sequence or a complete sequence of P gene type rotavirus protein, preferably one of P gene type rotavirus strains P8, P4, P6 or P11; wherein the P gene type rotavirus strain is one selected from P8G 1, P4G 2, P8G 3, P8G 4, P8G 9, P8G 5 or P6G 8.

As a further preferred variant, the rotavirus strain of genotype P [8] P is selected from one of the Wa, Ku, P, YO, MO, VA70, D, AU32, CH-32, CH-55, CHW2, CH927A, W161, F45, Ai-75, Hochi, Hosokawa, BR1054, WT78 or WI79 strains.

As a further preferred variant, the rotavirus strain of genotype P4P is selected from one of the DS-1, RV-5, S2, L26, KUN, E210, CHW17, AU64, 107E18, MW333 or TB-Chen strains.

As a further preferred variant, the rotavirus strain of genotype P6P is selected from one of the M37, 1076, RV-3, ST3, SC2, BrB, McN13, US1205, MW023, US585 or AU19 strains.

As another further preferred embodiment, the recombinant human rotavirus protein is selected from one of VP8, VP4, VP8 polypeptide chain fragments, nuclear VP8, VP4 polypeptide chain fragments, VP 8-specific antigenic cluster peptide chain or VP 4-specific antigenic cluster peptide chain.

As another further preferred embodiment, the recombinant rotavirus protein is a partial amino acid sequence or a complete sequence of VP7 protein of a G serotype rotavirus.

As a still further preferred version, the G serotype rotavirus strain is selected from one of the G1, G2, G3, G4, G9, G5, G8, G10 or G11 serotypes.

As a still further preferred embodiment, the G serotype rotavirus strain is selected from one of the P8G 1, P4G 2, P8G 3, P8G 4, P8G 9, P8G 5 or P6G 8 serotypes.

The invention also aims to provide a preparation method of the pneumonia multivalent conjugate vaccine, which is specifically formed by coupling multivalent pneumococcal polysaccharide and two or more carrier proteins with magnetic nano microspheres respectively.

As a preferable scheme, the preparation method of the pneumonia multivalent conjugate vaccine specifically comprises the following steps:

a) respectively separating and purifying capsular polysaccharide on capsules of various serotype pneumococci;

b) respectively preparing, separating and purifying the selected carrier proteins;

c) respectively coupling various capsular polysaccharides and magnetic nano microspheres to form polysaccharide-magnetic nano microsphere couplets;

d) coupling and combining the polysaccharide-magnetic nano microsphere couplet with various carrier proteins respectively;

e) separating and purifying the couplet obtained in the step d) to obtain the pneumonia multivalent conjugate vaccine stock solution.

As a further preferred scheme, the preparation method of the pneumonia multivalent conjugate vaccine specifically comprises the following steps:

b) respectively separating and purifying the selected multiple carrier proteins;

c) respectively coupling various capsular polysaccharides and magnetic nano microspheres into polysaccharide-magnetic nano microsphere couplets, and then carrying out chemical modification, so as to modify-OH, which does not react with polysaccharides, on the surfaces of the polysaccharide-magnetic nano microsphere couplets into-CHO;

As a further preferred scheme, the preparation method further comprises the step of preparing the magnetic nano-microspheres, wherein the step of preparing the nano-magnetic particles first and then preparing the magnetic nano-microspheres is included; the magnetic nano-particles can be prepared by a chemical coprecipitation method, a hydrothermal method or a sol pyrolysis method, the magnetic nano-microspheres can be prepared by an embedding method, a monomer polymerization method or an in-situ method, preferably, the magnetic nano-particles are prepared by the chemical coprecipitation method, and then the magnetic nano-particles and a high polymer material are subjected to rapid film emulsification combined with a solvent extraction method and/or the embedding method or the monomer polymerization method to prepare the magnetic nano-microspheres; most preferably, a chemical coprecipitation method is adopted to prepare nano magnetic particles, and then the nano magnetic particles and a high polymer material are subjected to rapid membrane emulsification combined with a solvent extraction method and/or an embedding method to prepare magnetic nano microspheres with uniform particle sizes; wherein the magnetic fine particles are preferably Fe₃O₄。

As a further preferable mode, the preparation method of the nano magnetic ions can refer to the Fe in the prior art₃O₄The method for preparing the magnetic nano-microsphere can be seen in the following specific embodiments, and is not taken as a key factor for realizing the invention.

As a further preferred option, the process for the isolation and purification of the polysaccharides in step a can be carried out according to the prior art, depending on the desired type of polysaccharide and protein.

As a further preferred embodiment, the process of preparing, separating and purifying the protein in step b can be performed according to the prior art according to the type of polysaccharide and protein desired.

As a further preferable scheme, the specific operation of the step c is as follows: c, in a coupling medium reaction buffer solution, reacting the polysaccharide obtained in the step a with the magnetic nano microspheres for 6-24 hours at room temperature and at the pH value of 4.0-9.0 to obtain a polysaccharide-magnetic nano microsphere couplet, and further modifying the polysaccharide-magnetic nano microsphere couplet by 25% glutaraldehyde to modify-OH, which is not reacted with the polysaccharide, on the surface of the polysaccharide-magnetic nano microsphere couplet, into-CHO; wherein the coupling medium is preferably PB, PBS or TBS, most preferably 0.1M TBS solution; the optimal pH of the reaction buffer is 6; the optimum reaction time is 12-16 hours.

As a further preferable scheme, the specific operation of the step d is as follows: c, in a coupling medium reaction buffer solution, reacting the polysaccharide-magnetic nano microsphere couplet obtained in the step c with the carrier protein for 12-24 hours at the temperature of 4 ℃ and the pH value of 4.0-9.0; wherein the coupling medium is preferably PB, PBS or TBS, most preferably 0.1M TBS solution; the optimal pH of the reaction buffer is 6; the optimum reaction time is 24 hours.

As a further preferred embodiment, the separation and purification of step e is to separate and purify the conjugate and the unreacted polysaccharide and carrier protein, and the purification method can be chromatography or ultrafiltration; the pneumonia multivalent conjugate vaccine can be separated and purified according to the molecular size, and the chromatography is preferably performed by a Superdex200 gel filtration column, Sepharose CL-4B or Sepharose CL-6B; ultrafiltration is a process that uses membranes of different molecular weight retention to separate the conjugate from the unreacted reactants.

The pneumonia multivalent conjugate vaccine preparation can adopt aqua or freeze-dried agent. In order to enhance the immunogenicity, an adjuvant can be added, and the commonly used adjuvant is an aluminum adjuvant, such as aluminum hydroxide and aluminum phosphate, and the aluminum phosphate is preferably selected in the invention. The solvent for the conjugate may be 0.2 sodium chloride solution, 1 XPBS buffer, or other buffer capable of stabilizing the polysaccharide or conjugate. The preparation method of each preparation of the pneumonia multivalent conjugate vaccine adopts conventional means in the technical field for preparation. Among them, lyophilization is preferably performed in the presence of a sugar (e.g., sucrose or lactose).

The multivalent conjugate pneumonia vaccine provided by the invention can be used for immunization by any existing route, including the forms of dermal or skin administration, muscle administration and the like. Wherein the amount administered can be determined by one skilled in the art based on common general knowledge.

Definition of terms

Nucleus VP 8: is a polypeptide chain with the function of bonding with sialic acid (sialic acid) on the cell surface in rotavirus protein VP8, and generally comprises 160 amino acid residues.

VP8 polypeptide chain fragment: is any polypeptide chain with a lower molecular weight than full length VP 8.

VP4 polypeptide chain fragment: is any polypeptide chain with a lower molecular weight than full length VP 4.

VP8 specific antigen cluster chain: the full VP8 polypeptide chain contains multiple antigenic determinants, and is spliced out by genetic recombination to contain no essential amino acids, while the polypeptide chain containing the specific antigenic cluster remains, typically of a smaller molecular weight than the full length VP8 polypeptide chain.

VP4 specific antigen cluster chain: the full VP4 polypeptide chain contains multiple antigenic determinants, and is spliced out by genetic recombination to contain no essential amino acids, while the polypeptide chain containing the specific antigenic cluster remains, typically of a smaller molecular weight than the full length VP4 polypeptide chain.

VP8 fusion protein: by using a gene recombination method, the VP8 protein and other soluble protein polypeptide chains are subjected to fusion expression so as to improve the solubility of the VP8 in an aqueous solution; or enhancing the immunogenicity of VP 8.

NSP4 protein: is a non-structural protein with enterotoxin property, has the molecular weight of 28kDa and contains 175 amino acids.

VP8-NSP4 fusion protein: by means of gene recombination, VP8 and NSP4 polypeptide chain are expressed through fusion to raise the solubility of VP8 in water solution and make the fusion protein possess the function of stimulating body to produce protective antibody to NSP 4.

Capsular polysaccharide fragment: polysaccharide fragments obtained by degrading (depolymerisation) polysaccharides (called whole or original polysaccharides) purified from bacterial cultures by physical methods (e.g. ultrasound, particle spray), chemical methods (e.g. acid, alkali, enzymatic digestion), etc., usually have a lower molecular weight than the original polysaccharides. Capsular oligosaccharide: polysaccharide fragments obtained by degrading (depolymerisation) polysaccharides (called whole or original polysaccharides) purified from bacterial cultures by physical methods (e.g. ultrasound, particle spray), chemical methods (e.g. acid, base, enzymatic digestion), etc., the monosaccharide residues in the molecular structure are usually below 10. Although the number of monosaccharide residues is defined differently, there are references to oligosaccharides with polysaccharide chains having more than 10 monosaccharide residues and less than 20 monosaccharide residues.

Compared with the prior art, the invention has the following advantages:

1. the pneumonia multivalent conjugate vaccine is an immunoconjugate containing two or more than two different carrier proteins, and compared with the existing pneumococcal conjugate vaccine, the pneumonia multivalent conjugate vaccine has stronger immunogenicity, the induced polysaccharide antibody level is higher than that of a single carrier conjugate, and immune response can be caused in wider crowds, particularly infants; carrier epitope overload can be avoided by reducing the respective carrier dose; helper T cell activity can be enhanced by both vectors;

2. because the protective protein antigen epitopes in the two carrier proteins can also induce higher immunoreaction than that of the two proteins when the two proteins are mixed and injected, the immunogenicity of the carrier proteins is further improved and the immunoreaction of organisms to polysaccharide is increased due to the mutual synergistic effect; the other is adjuvant carrier protein, which can further enhance immunogenicity and has certain immunological memory;

3. the invention adopts the nano microspheres as the connecting body of the polysaccharide and the multi-carrier protein for the first time, effectively avoids the self coupling of the capsular polysaccharide and the protein in the preparation process, can improve the yield of the combined product and is beneficial to the quality control of the product; the space distance between the capsular polysaccharide and the carrier protein can be effectively prolonged, the space shielding effect of the carrier protein on the capsular polysaccharide epitope is reduced, and the immunogenicity of the capsular polysaccharide is favorably improved;

4. the surface of the nano microsphere is firstly created in the preparation process of the pneumonia multivalent conjugate vaccineFirstly, the-OH is coupled with polysaccharide, and then the coupling body is chemically modified to change the unreacted-OH into-NH of-CHO in the carrier protein₂The coupling combination is more stable than the combination method in the prior art;

5. the preparation method is simple, is suitable for the requirement of large-scale industrial production, and does not obviously change the structural characteristics of the capsular polysaccharide and the carrier protein.

In conclusion, the pneumonia multivalent conjugate vaccine provided by the invention has a simple preparation process, and the pneumonia multivalent conjugate vaccine adopting the nano microspheres as the connecting objects can enhance the Th1 type immune response of mice, the immune persistence, the specificity and the affinity of polysaccharide specific antibodies, and can induce the mice to generate rotavirus antibodies; has the prevention effect of two vaccines; therefore, the method has very wide application prospect.

Drawings

FIG. 1 shows the multivalent conjugate vaccine for pneumonia prepared in example 1 of the present invention¹H-NMR spectrum;

FIG. 2 is a schematic diagram showing the results of an immune response experiment of polysaccharide specific antibodies of the multivalent conjugate vaccine for pneumonia provided by the present invention;

fig. 3 is a schematic diagram of the results of an experiment on the immune persistence of polysaccharide-specific antibodies of the multivalent conjugate vaccine for pneumonia provided by the present invention.

Detailed Description

The present invention will be described more fully hereinafter with reference to the following examples. The reagents or equipment used below are all commercially available varieties, and if no special description is provided, the operations are performed according to the description, which is not described herein.

The present invention will be further described with reference to specific examples, but the present invention should not be construed as being limited thereto.

Example 1

Firstly, preparing magnetic nano-microsphere

1. Taking 2.24g of FeSO₄-7H₂O and 3.24gFeCl₃-6H₂O dissolved in 10mL and 15mL dddH filtered to remove oxygen₂Dissolving in O, mixing, adding 100mL dddH for filtering and deoxidizing₂O；

2. In N₂Stirring for 5min under the protection of (1 mol/L) NaOH solution of 50mL at one time, adjusting the pH value of the solution to 9-10, accelerating the stirring speed to 200 and 250r/min, and continuously stirring for 30 min;

3. the reaction vessel was transferred to a water bath at 65-70 ℃ and continued under N₂Stirring and aging for 30min under the protection of (1);

4. after the reaction is finished, the volume is determined to be 100mL, and the synthesis condition of the magnetic particles is observed under a microscope;

5. 400mgPLGA was dissolved in 10mLEA solvent to obtain oil phase (O), and 3mL of the above magnetic particle solution was added to obtain internal water phase (W)₁) Preparing colostrum by pre-emulsifying in ice water bath with ultrasonic cell disruptor (120W, 60s), and adding water solution (external water phase, W) containing 15g/LPVA and 0.9% (omega) NaCl₂) Magnetic stirring (300r/min, 2min) to prepare a pre-emulsion (W)₁/0/W₂) Then pouring the pre-compounded emulsion into a storage tank for rapid membrane emulsification, and adding a certain amount of N₂Repeatedly pressing the nano microsphere composite emulsion through an SPG membrane under pressure to obtain nano microsphere composite emulsion droplets with uniform particle sizes. In addition, the nanometer microsphere can be made into freeze-dried agent for use.

Or by taking Fe₃O₄Adding magnetic particles, 50mL and anhydrous ethanol in equal volume, ultrasonically activating for 30min, placing in 60 deg.C water bath, and slowly dropwise adding 10mL of PELA to carry out-NH treatment on magnetic nanoparticles₂End-modified and PELA encapsulated in Fe₃O₄Stirring the magnetic particles outside the magnetic particles under the protection of nitrogen to react with the mixture I0 h to prepare magnetic nano microspheres; after the reaction is finished, washing the paint with 50mL of absolute ethyl alcohol for 3 times, then washing the paint with 0.01MPBS for three times, fixing the volume to 50mL, observing the modification condition of the magnetic beads under a microscope till the surface of the magnetic nano microsphere is-NH₂The end was changed to the-OH end.

Preparation of pneumococcal polysaccharide

1. Selecting 24 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F) for culturing pneumococcus;

2. respectively purifying the capsular polysaccharide with strong antigenicity in the various serotype pneumococci: inactivating pneumococcus, centrifuging to collect supernatant, ultrafiltering, concentrating, adding appropriate amount (volume fraction of 70%) of precooled ethanol according to serotype characteristics of pneumococcus, centrifuging, and collecting to obtain crude polysaccharide; the crude polysaccharide was dissolved in sodium acetate solution and then mixed as 1: mixing with cold phenol at a ratio of 2, centrifuging to remove protein, repeatedly extracting with phenol for 5-6 times, collecting supernatant, dialyzing with distilled water, adding 2mol/L calcium chloride solution into the dialyzed liquid, adding ethanol, stirring, centrifuging to remove nucleic acid, collecting supernatant, supplementing ethanol (final concentration of 80% stirring), centrifuging to collect precipitate, washing the precipitate with ethanol and acetone, dehydrating and drying to obtain polyvalent refined capsular polysaccharide, and storing at-20 deg.C for use.

Preparation of tri-rotavirus carrier protein

The preparation of rotavirus carrier protein can be referred to the preparation methods of various recombinant proteins in the prior art, and the preparation in this example by the method mentioned in CN 101972475 can be simplified into the following steps, and specific parameters are not described in detail:

1. establishment of a cDNA library of Rotavirus

The VP4 gene of the Wa strain VP8 protein is selected, and the amplification primers are designed as follows: sense primer HWaVP4 ρ ET 28:

5'-TTACATATGGCTTCGCTCATTTATAG-3', anti-sense primer AHWaVP4 ρ ET 28:

5’-CCGGATCCCTAGTCTTCATTAACTTGTGCT-3’。

2. construction of pET28aWaVP8 expression full-Length VP8 plasmid

3. Expression of recombinant VP8 protein

1) The resulting pET28aWaVP8 plasmid was transformed into BL21(DE3) component cells, which were seeded into 50. mu.g/mL kanamycin LB plates at 37 ℃ in CO₂The incubator was overnight.

2) One colony was picked, inoculated into 10mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH7.5) containing 50. mu.g/mL kanamycin, amplified, and cultured overnight at 37 ℃.

3) The culture medium was inoculated to 100mL of 50. mu.g/mL kanamycin LB medium, and the culture was continued. When the OD of 600nm absorbance reached 1.0, the culture was inoculated into 6 liters to 50. mu.g/mL kanamycin LB culture, and the culture was continued in a shaker at 37 ℃ and a shaking speed of 200rpm, and when the OD of 600nm absorbance reached 0.6 to 0.8, 0.3mM IPTG (isopropyl-. beta. -D-thiogalactopyranoside) was added to induce the expression of VP 8.

4) After 4 hours of induction under the same culture conditions, the cells were centrifuged at 4000g at 10 ℃ for 20 minutes, and then collected.

5) The bacteria were suspended in 20mL of 1 XPBS solution, disrupted in a French press (French press), centrifuged at 10000g at 10 ℃ for 30 minutes, the supernatant was discarded, and the precipitated inclusion bodies were collected. The inclusion bodies were stored at-40 ℃ prior to further purification.

3. Purification of VP8 protein from Inclusion bodies

1) The prepared inclusion bodies of VP8 were weighed at 0.5 g (wet weight), suspended in washing buffer (10mM Tris, 100mM phosphate buffer, 2Murea, pH8.0), incubated at room temperature for 30 minutes, centrifuged at 10,000g for 10 minutes, and the inclusion bodies were collected. The above steps are repeated three times to remove contaminating proteins.

2) The inclusion body pellet was dissolved in a dissolution buffer (10mM Tris-HCl, 100mM phosphate buffer, 8M urea, pH8.0) and incubated on ice with stirring for 1 hour. Centrifuge at 16,000g for 30 minutes, collect the supernatant and discard the insoluble precipitate.

3) His-tagged recombinant VP8 protein was purified by immobilized metal ion affinity chromatography (IMAC).

4) The eluate containing VP8 was collected, transferred into a dialysis bag and dialyzed against TBS (pH4.0) containing 20mM β -mercaptoethanol 1 and 8M urea, and the urea concentration was gradually decreased (i.e., 8, 6, 4, 2, and 1M) and dialyzed overnight at 4 ℃. Then dialyzed twice against TBSpH5.5 solution containing 2mM β -mercaptoethanol and finally against TBS solution. Final dialysis was performed depending on the strain from which the recombinant VP8 protein was derived.

Preparation of pneumococcal surface protein A (rPspA)

Cloning the PspA protein into Escherichia coli for expression, separation and purification, and specifically comprising the following steps:

1. optimization of target gene and construction of recombinant expression plasmid

Obtaining PspA gene sequence (GI: 193804931) from GenBank, optimizing, adding His label, carrying out whole gene synthesis, carrying out SacI and NdeI double digestion on the synthesized sequence, directionally cloning into expression vector pET-30a (+) of the same double digestion, transforming competent Escherichia coli BL21Star (DE3), overnight culturing at 37 ℃, selecting positive single clone colony, extracting plasmid after amplification culture, carrying out double digestion identification by NdeI and SacI, and sending to sequencing, wherein the recombinant expression plasmid with correct sequencing is named as pET-30 a-rPspA.

2. Induced expression and purification of recombinant protein

Recovering engineering bacteria in a ratio of 1: inoculating 2 × LB culture medium at a ratio of 100, performing amplification culture at 37 deg.C and 237r/min, when the thallus value is about 12, adding IPTG to the final concentration of 1mmol/L, inducing at 37 ℃ for 4h, sampling, carrying out SDS-PAGE analysis, centrifugally collecting induced thallus, adding physiological saline, resuspending and washing for 2 times, adding a buffer solution of 05mol/LNaCl5mmol/L imidazole 20mmol/LPB (pH7.4) according to the proportion of 1:10(g/mL) for resuspending the thalli, carrying out ultrasonic disruption on the thalli, centrifuging for 40min at 8000 Xg, collecting supernatant, purifying in a nickel ion chromatographic column, purifying products according to the operation of instructions and analyzing the whole thalli of escherichia coli BL21Star (DE3) transformed by a carrier pET-30a (+) (control) and the purified sample obtained in the previous step, carrying out SDS-PAGE separation, then electrically transferring to a nitrocellulose membrane, and slightly shaking and sealing for 2h by using a 5% skimmed milk shaking table; adding His mouse source monoclonal antibody (diluted 1: 800), and standing overnight at 4 deg.C; TBST washing 3 times, adding goat anti-mouse IgG labeled with HRP (diluted 1: 2000), and incubating at room temperature for 1 h; washing for 3 times, and DAB developing.

Preparation of pneumonia multivalent conjugate vaccine

1. Polysaccharide-magnetic nanosphere coupling

0.1 adding any one or more of capsular polysaccharide and magnetic nanospheres prepared in the above steps into an MTBS solution buffer solution at a pH of 6.0, wherein in the present embodiment, 13-valent capsular polysaccharide (1, 3, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) is adopted, the mass ratio of the capsular polysaccharide to the magnetic nanospheres is 1 (0.5-1), and reacting at room temperature for 6-24 hours; wherein the optimized mass ratio is 1:1, and the acetalization reaction is carried out for 16 hours at room temperature. And after the reaction is finished, fully dialyzing by using a dialysis bag to remove the unreacted magnetic nano microspheres.

2. Modification of couplets

Taking 30mL of the polysaccharide-magnetic nano microsphere couplet in the step 1, slowly dropwise adding 2mL of 25% glutaric acid under the stirring condition, and stirring at 200r/min for reaction for 6 hours; after the reaction is finished, washing the solution for three times by using 0.01MPBS, fixing the volume to 30mL, and observing the modification condition of the magnetic nano-microsphere under a microscope to modify-OH which is not reacted with the polysaccharide on the surface of the polysaccharide-magnetic nano-microsphere couplet into-CHO; n is a radical of₂And storing at 4 ℃ for later use.

3. Co-conjugation to PspA Carrier proteins and Rotavirus proteins

And (3) under the buffer of 0.1MTBS solution and at the temperature of 4 ℃ and the pH value of 4.0-9.0, respectively carrying out a co-reaction on the modified polysaccharide-magnetic nano microspheres and rotavirus protein and PspA protein for 24 hours (in order to ensure sufficient carrier protein and magnetic nano microspheres, the adding amount of excessive nano carrier protein is adopted, and the mass ratio of polysaccharide to magnetic nano microspheres to PspA to rotavirus protein is 1: 1: 2: 2).

4. Separation and purification of pneumonia multivalent conjugate vaccine

The separation and purification were carried out by a Superdex200 gel filtration column (2.6 cm. times.60 cm), and the eluate was 20mM phosphate buffer (pH7.4) at a flow rate of 3 mL/min. And collecting an elution peak corresponding to the polysaccharide-magnetic nanosphere-double carrier protein.

Composition analysis of the prepared multivalent conjugate vaccine for pneumonia

By using¹H-NMR is carried out to detect the pneumonia multivalent conjugate vaccine, and the detection result is shown in figure 1. As shown in fig. 1, the conjugate showed a characteristic peak at 0.4-1.4ppm, corresponding to the fatty chain amino acid residues of the carrier protein, compared to the capsular polysaccharide molecule. A characteristic peak corresponding to aromatic amino acid residues of the carrier protein appeared at 7.2 ppm. This indicates that the capsular polysaccharide molecule successfully couples two carrier proteins, rotavirus protein and PspA carrier protein. A characteristic peak corresponding to succinimide appears at 6.2ppm, indicating that the connecting bridge of the polysaccharide conjugate vaccine contains succinimide. In addition, the characteristic peaks of PELA are shown in lines of 5.2, 1.6 and 3.6, which proves that the magnetic nanosphere PELA is used as a connector. Thus, capsular polysaccharides combine two carriersBefore and after the protein, the structure of the protein is not obviously changed.

Detecting the molecular weight distribution of the polysaccharide-protein conjugate by a CL-4B (SEC-MALLS) method; determining protein and polysaccharide species in the polysaccharide protein conjugate by the method of immune double-expansion using different antibody sera; detecting the polysaccharide content of the polysaccharide protein conjugate by an anthrone method; detecting the total protein content of the polysaccharide-protein conjugate by the protein content of Lowry method, and calculating to obtain the polysaccharide-protein binding Ratio (Ratio) of the conjugate; the concentrations of rotavirus protein and PspA protein are detected by an enzyme-linked immunosorbent assay. The results show that: in the pneumonia multivalent conjugate vaccine stock solution, the concentration ratio of each substance is about: polysaccharide: magnetic nano-microspheres: PspA: rotavirus protein ═ 1: 1: 1: 1).

Comparative example 1

This comparative example differs from example 1 only in that: the vaccine is prepared without magnetic nanoparticles as a linker and is obtained by conjugating a dual carrier protein to a polysaccharide by the methods mentioned in the prior art.

Comparative example 2

This comparative example differs from example 1 only in that: the prepared vaccine does not contain PspA carrier protein, and only adopts polysaccharide-magnetic nanoparticles-rotavirus carrier protein as the pneumonia multivalent combined vaccine.

Example 2

The present example only differs from example 1 in that the carrier proteins of the multivalent conjugate pneumonia vaccine are tetanus toxoid carrier protein and PspA.

The analysis result of the composition of the prepared pneumonia multivalent conjugate vaccine is consistent with the result obtained in example 1 within the theoretical error range.

Example 3

This example differs from example 1 only in that: the carrier protein of the pneumonia multivalent conjugate vaccine is rotavirus carrier protein and tetanus toxoid carrier protein.

Example 4

This example differs from example 1 only in that: the carrier protein of the pneumonia multivalent conjugate vaccine is rotavirus carrier protein and haemophilus influenzae surface protein HiD.

Example 5

This example differs from example 1 only in that: the carrier proteins of the pneumonia multivalent conjugate vaccine are carrier proteins CRM197 and PspA.

Example 6

This example differs from example 1 only in that: the connector is PLGA magnetic nano-particles.

Example 7

This example differs from example 1 only in that: the linker is a PEG magnetic nanoparticle.

Vaccine evaluation

The vaccines obtained in examples 1 to 7 and comparative examples 1 to 2 were evaluated for their immune effects by the following evaluation experiments for conventional titer of vaccines such as immunogenicity:

A. multivalent conjugate vaccine immunogenicity experiments for pneumonia

100 female Blab/C mice, 5 weeks old, were selected weighing 15-22 grams. The groups were randomly divided into 10 groups, i.e., examples 1-7, comparative examples 1-2 and a positive control group (Prevnar 13), each of which was 10 mice. Intraperitoneal injection is carried out, each injection is 5 micrograms, and the injection is carried out 1 time per week and 3 times in total. Blood was collected from the orbit 21 days later. Anti-capsular polysaccharide IgG, IgG1 and IgG2a were detected in mouse plasma by ELISA. The results are shown in Table 1

TABLE 1

As shown in table 1, the multivalent pneumonia conjugate vaccines obtained in examples 1 to 7 can significantly improve the antibody titer (p < 0.0001), as shown in fig. 2, and are significantly better than those of comparative examples 1 and 2 and the positive control group; therefore, the pneumonia multivalent conjugate vaccine prepared from the double-carrier protein, the magnetic nano microspheres and the pneumonia polysaccharide can obviously enhance Th1 type immune response, and the immune effect is obviously superior to that of the pneumonia multivalent conjugate vaccine prepared from the nano-magnetic microsphere-free connector in the comparative example 1 and the single-carrier protein in the comparative example 2; however, it can be seen from the immune response results obtained in examples 1 to 7 that the immune response effect is better when PspA or HiD is one of the dual carrier proteins, and the PELA and PLGA in the magnetic nano-carrier have better immune response effect than PEG.

B. Experiment of immune persistence, specificity and affinity of polysaccharide specific antibody

The following experiment was performed with the pneumonia multivalent conjugate vaccines obtained in example 1, comparative examples 1 and 2, and the positive control, respectively.

1. Persistence of immunity

The immune persistence of polysaccharide-specific antibodies was studied by measuring the titer of polysaccharide-specific antibodies within 20 weeks after three immunizations. FIG. 3 is a schematic representation of the persistence of the immunization, as shown in FIG. 3, which shows a lower polysaccharide-specific IgG titer, which gradually decreased with increasing injection time and was not detectable after week 4. Comparative examples 1 and 2 produced polysaccharide-specific IgG titers that were lower than those of the example 1 group and higher than those of the positive control group. Polysaccharide specific IgG titers peaked at week 2 and declined gradually between weeks 4-20, with the positive control group declining most rapidly. After week 18, the polysaccharide-specific IgG titers of the example 1 group, the comparative example 1, the comparative example 2 and the positive control group were 20%, 15%, 12% and 10% of their peaks, respectively. Therefore, the pneumonia multivalent conjugate vaccine provided by the invention can enhance the immune persistence of the polysaccharide specific antibody.

2. Specificity and affinity

Different amounts of capsular polysaccharide were added to the plasma of mice in the PS-TT group, the PS-PLGA-TT group and the PS-PELA-TT group diluted by 200 times, and the antibody level against capsular polysaccharide in the plasma of the mice was measured by ELISA method, and the results are shown in Table 2.

TABLE 2

As shown in table 2, the ability of polysaccharide-specific antibodies to bind to polysaccharides in 96-well plates gradually decreased with increasing amounts of polysaccharide added. When the added polysaccharide reaches 20. mu.g, the ability of the antibody to bind to the polysaccharide is lost. This indicates that the anti-capsular polysaccharide antibodies generated by the multivalent conjugate pneumonia vaccine provided by the present invention induced mice are capable of specifically binding to capsular polysaccharide.

Antibody affinity to capsular polysaccharides was determined by ammonium thiocyanate method. The polysaccharide-specific antibody affinity index of the capsular polysaccharide group (negative control) was 1.18mol/L, while the antibody affinity index of the positive control group, the comparative example 1 group, the comparative example 2 group and the example 1 group were 2.65mol/L, 2.80mol/L, 3.02mol/L and 3.21mol/L, respectively. This shows that the pneumonia multivalent conjugate vaccine provided by the invention can obviously improve the affinity of polysaccharide specific antibody.

C. Immunogenicity comparison experiment of simplex pneumococcal polysaccharide in multivalent pneumonia combined vaccine

A13-class coating antigen type of each single pneumococcal polysaccharide conjugate vaccine is prepared by the method described in example 1 (namely only one pneumococcal polysaccharide is combined with two carrier proteins to form a conjugate vaccine through magnetic nanoparticles), the antibody titer is respectively detected by an indirect ELISA method, each type of pneumococcal polysaccharide conjugate vaccine is dissolved in 0.05mol/L carbonate buffer solution with pH of 9.6, an enzyme label plate is coated at the concentration of 20ug/ml, and 2% BSA solution is sealed. During the experiment, the serum to be detected is diluted by 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 102400, 204800 and 409600 times, then is added into an enzyme label plate, the reaction is carried out for 40min at 37 ℃, and after the routine washing, a goat-anti-mouse secondary antibody marked by horseradish peroxidase is added. Buffer without serum was also added as a negative control. The color is developed by using tetraaminobenzidine, and the A value is read at the wavelength of 450nm by using an enzyme-labeling instrument. The ratio of the A value in each well to the A value in the negative well is calculated, but the largest dilution multiple when the ratio is more than 2.1 is the antibody titer in the serum, the titers of 10 mice are geometrically averaged, and the logarithm value is calculated by taking 10 as the base, and the specific results are shown in Table 3.

TABLE 3 evaluation of immunogenicity of various pneumococcal haplotype conjugate vaccines

As can be seen from Table 3: the pneumonia polysaccharide conjugate vaccine obtained by using the double-carrier protein with the magnetic nanoparticles provided by the invention as the connector can obviously enhance the immune response of mice to the lung chain polysaccharide, wherein the types 1, 3, 5 and 6A/B are especially obvious, and the pneumonia polysaccharide conjugate vaccine has a better immune response effect; the product is more suitable for people in China.

D. Results of rotavirus neutralization experiments

Each mouse serum of each group of mouse sera prepared by the method of example 1 (one, two and three injections of example 1, comparative example 1 and comparative example 2, respectively) was mixed in 10. mu.L each for neutralizing the serum of the test sample.

Neutralization assays of anti-WaVP 8 antibodies generated by injected mice were performed on microplates (microplates) with BSC-1 cells. The heat-inactivated serum was serially diluted, mixed with a Wa rotavirus strain of 100TCID50, and then cultured at 4 ℃ for 1 hour. BSC-1 cells were then seeded onto microplates and incubated for 1 hour. DMEM (without serum) was added to each well and incubated at 37 ℃ for 1 hour. Finally, the concentration at which the diluted antiserum prevents the onset of rotavirus cytopathic effect (CPE) is the neutralizing titer. Polysaccharide control group sera were used as negative controls. The results are shown in Table 4.

Table 4: test result of neutralizing rotavirus Wa strain by pneumonia multivalent conjugate vaccine injected mouse antibody

As shown in Table 3, the pneumonia multivalent conjugate vaccine has obviously better rotavirus neutralizing effect than the comparative example 1 and the comparative example 2, and can obviously induce the generation of rotavirus antibodies.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A multivalent conjugate pneumonia vaccine, characterized by: the pneumonia multivalent conjugate vaccine is formed by connecting multivalent pneumococcal polysaccharide and two carrier proteins through a connector, wherein the connector is a magnetic nano microsphere, magnetic particles of the magnetic nano microsphere are positioned inside the magnetic nano microsphere and are cores, a high polymer material is coated outside the magnetic particles, the high polymer material is polylactic acid-polyethylene glycol copolymer (PELA), and the mass ratio of the multivalent pneumococcal polysaccharide to the two carrier proteins is (0.5-2): 1, the carrier proteins are carrier proteins CRM197 and pneumococcal surface protein A (rPspA), the multivalent pneumococcal polysaccharide is capsular polysaccharide on a capsule of an isolated and purified serotype pneumococcus, and the serotypes of the serotype pneumococcus comprise 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F.

2. The method of claim 1, wherein the multivalent conjugate vaccine of pneumonia is prepared by: the polyvalent pneumococcal polysaccharide and the two carrier proteins are coupled with the magnetic nano-microspheres respectively to form the magnetic nano-microsphere.

3. The method for preparing a multivalent conjugate vaccine for pneumonia according to claim 1, comprising the following steps:

b) respectively preparing, separating and purifying the selected multiple carrier proteins;

e) a separation and purification step;

d) the obtained couplet becomes the pneumonia multivalent conjugate vaccine stock solution.

4. The method for preparing a multivalent conjugate vaccine for pneumonia according to claim 3, comprising the following steps:

c) respectively coupling various capsular polysaccharides and magnetic nanospheres into polysaccharide-magnetic nanosphere couplets, and then carrying out chemical modification, so that-OH which does not react with the polysaccharides on the surfaces of the polysaccharide-magnetic nanosphere couplets is modified into-CHO;

e) a separation and purification step;