CN107029225B - Conjugate vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses a conjugate vaccine which is prepared 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 conjugate vaccine is that the polyvalent pneumococcal polysaccharide and two or more than two carrier proteins are respectively coupled with the magnetic nano-microspheres to form the conjugate vaccine. The conjugate vaccine adopting the magnetic nano microspheres as the connecting substances 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

A kind of conjugated vaccine and preparation method thereof

technical field

The present invention relates to a vaccine, in particular to a conjugated vaccine vaccine and a preparation method thereof, belonging to the field of biotechnology.

Background technique

1. Harm of microorganisms to human body and countermeasures

Microorganisms usually refer to those biological groups whose individual volume and diameter are generally less than 1mm. They have simple structures, most of which are single cells, and some even have no cellular structure. There are microorganisms around us all the time, usually only with the help of a microscope or electron microscope. Their morphology and structure; among them, pathogenic microorganisms refer to pathogenic microorganisms that can cause diseases of humans, animals and plants. The pathogenicity of a pathogen depends on its ability to invade the host and reproduce in the body and resist host resistance without being destroyed by it. The pathogenicity of microorganisms has species characteristics, and the degree of pathogenicity is called virulence. The establishment of infectious diseases is not unilaterally determined by the virulence of microorganisms, but also depends on the health and immune function of the host. In general, highly virulent microorganisms infect unimmunized organisms, which can cause pathological damage and overt infection, while normal organisms can resist the damage of many low-virulence microorganisms (such as opportunistic pathogens), but when the host resists When the force is reduced, it can be susceptible to these microorganisms and cause disease. The contest between the virulence of pathogens and host resistance leads to the occurrence, development, outcome and prognosis of infectious diseases. Due to the different degrees of adaptation between pathogens and hosts, the outcome of the confrontation between the two parties is different, resulting in various differences. The spectrum of infection, that is, the different manifestations of the infection process. Pathogenicity is for a specific host, some are only pathogenic to humans, some are only to certain animals, and some are zoonotic microorganisms. At this time, the host usually can only fight against the infection of microorganisms through its own immune system, and the mortality rate is very high, so that various researchers have developed vaccines to combat the damage of pathogenic microorganisms to the human body.

Vaccines are divided into two types: therapeutic and preventive. Therapeutic vaccines are used to treat diseases, and preventive vaccines are used to protect the human body from pathogenic microorganisms. Preventing disease by inoculating preventive vaccines is a proven and effective means of human clinical practice for more than a century. After years of efforts, the medical community has developed various vaccines to prevent various diseases caused by infections such as bacteria, viruses and fungi, which have greatly improved human health. The continuous development of biotechnology has promoted the diversification of vaccine varieties. Today, there are vaccines developed with inactivated virus technology to prevent infectious diseases caused by viruses, such as Japanese encephalitis vaccine, polio vaccine, influenza vaccine, etc.; live attenuated vaccines developed with attenuated virus technology, such as rotavirus Virus vaccine, oral poliovirus vaccine, measles virus vaccine, mumps virus vaccine, rubella virus vaccine and chickenpox vaccine, etc. Bacterial vaccines developed by purification technology of biological macromolecules such as useful proteins and polysaccharides for the prevention of bacterial infectious diseases, such as tetanus toxoid, diphtheria toxoid, pertussis toxoid and its subcellular components, meningococcal polysaccharide and 23-valent pneumococcal polysaccharide, etc. Vaccines developed with gene recombinant protein technology, such as hepatitis B surface antigen (prevention of hepatitis B), human papilloid virus particle virus (prevention of cervical cancer) and so on. Bacterial vaccines developed by semi-chemical combination technology to prevent meningitis and pneumonia, such as Haemophilus influenzae type b polysaccharide-protein conjugate vaccine, 7-valent or 10-valent pneumococcal polysaccharide-protein conjugate vaccine, and 4-valent meningococcal polysaccharide - Protein conjugate vaccines. It can be seen that the development of medical biotechnology is the driving force for the continuous development of vaccine products. Through the continuous improvement of biotechnology, more new vaccine products can be developed to meet the challenges of different infectious diseases to human health.

2. Overview of conjugate vaccines

Polysaccharide is an important immune active component in pathogenic bacteria, and it can be divided into bacterial polysaccharide (OPS) and capsular polysaccharide (CPS). When pathogens invade the body, they act as immunogens and can stimulate the body to produce a protective immune response. However, polysaccharide molecules belong to T cell-independent antigens (Ti-Ag), and their immunogenicity is weak, and the immune effect is particularly unsatisfactory after vaccination for infants and young children. At present, many serious diseases such as Haemophilus influenzae (Hib) cause Meningitis, hemorrhagic diarrhea and dehydration in children caused by E.Coli O 157:H7 are high in infants and young children, and there is no effective clinical treatment method, and the mortality rate is high (Wang Yan et al. Overview of combined vaccines [J], Microbiology Immunology Progress, 2000, 28(1):60-63). In order to improve the immunogenicity of polysaccharide vaccines, chemical conjugate vaccines of polysaccharides and proteins emerged in the early 1920s, that is, conjugate vaccines. The conjugate (bacterial polysaccharide with protein as carrier) vaccine uses chemical methods to combine polysaccharides. The polysaccharide-protein conjugate vaccine is prepared by valence binding on a protein carrier, which is used to improve the immunogenicity of bacterial vaccine polysaccharide antigens, such as Haemophilus influenzae type b conjugate vaccine, meningococcal conjugate vaccine and pneumococcal conjugate vaccine. It has developed rapidly for decades and has achieved remarkable results.

3. The harm of pneumococcus and its epidemiological research

Infections caused by pneumococcus (lung chain) are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common forms of invasive pneumococcal disease, while 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 disease. The likelihood of pneumococcal disease flares during influenza is further increased due to the spread of antibiotic-resistant infectious diseases and the fact that pneumococcal pneumonia often follows influenza infection.

Disease caused by Streptococcus pneumoniae has become a major public health problem worldwide. Pneumococcus has become the number one killer of children worldwide. The fatality rate of pneumonia in my country is 16.4%, of which the middle-aged and elderly people over 50 years old and infants and young children under 1 year old are as high as 28.6% and 22.0% respectively. The carrier rate of pneumococcus in healthy children in my country is relatively high. Statistics show that the carrier rate among healthy children in northern regions is 24.2% and that in southern regions is 31.3%. The disease is an important cause of death in children under 5 years of age. The main reason is that the development of the immune system of infants and young children is not perfect and the immunity is weak. And the younger the baby, the weaker the immune system. There are about 90 serotypes (strains) of pneumococcus. Statistics in my country show that the first serotypes of pneumococcal infection strains are: 5, 6, 19, 23, 14, 2, and 4. According to a study that collected 860 pneumococcal isolates, 109 (12.7%) showed serogroup 6, and the erythromycin resistance of pneumococcal serotype 6 in China reached 100%, of which 6A, Types 6B and 6C were 62 strains (56.9%), 38 strains (34.9%) and 9 strains (8.2%).

In terms of chemical structure, the above pathogens possess a cell surface capsular polysaccharide (CPS) or lipopolysaccharide (LPS) shell, or both, whose function is to help the pathogens infect the host. Capsular polysaccharides can shield bacterial cell surface functional components from being recognized by the host immune system, preventing the complement system from being activated by bacterial surface proteins and phagocytosed by immune cells. If bacteria are phagocytosed, capsular polysaccharides can prevent bacteria from being killed. In most pathogenic bacteria, different strains express different structures of capsular polysaccharides and lipopolysaccharides, 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.

It can be seen that most bacterial polysaccharide vaccines must contain a variety of different types of bacterial polysaccharides to improve the coverage of pathogenic strains. Optimizing and selecting which bacteria or serotype polysaccharides to contain is a very complex epidemiology in vaccines. question. Once it is clear which polysaccharide antibodies are protective, this polysaccharide can be used as an immunogen to produce vaccines.

The 23-valent pneumococcal vaccine produced by Chengdu Institute of Biological Products of China Biotechnology Group is selected from the 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, 33F), respectively ferment and culture and separate and purify various types of polysaccharides on the pneumococcal capsule, and mix them in equal proportions to make a vaccine. Bacterial polysaccharide is a thymus-independent antigen. The main difference between this antigen and thymus-dependent antigen is that the former does not require the help of T lymphocytes to produce antibodies. Clinical use has proved that the vaccine made by capsular polysaccharide is clearly effective and widely used in many countries. However, this type of polysaccharide vaccine has the following problems: (1) in young animals or infants, it can only produce a weak immune response, or even no immune response, and the immune response increases with age; (2) produce low-affinity antibodies (3) It only produces a short-term immune response, and does not have the immune memory and immune enhancement effects during repeated vaccination; (4) It is easy to produce immune tolerance; (5) Ordinary adjuvants are not easy to play an immune enhancing effect on this antigen. The 23-valent polysaccharide vaccine has a protection rate of 50-70% against invasive lung chain infection, and can only be used for vaccination in people over 2 years old, while the peak age of onset of pneumonia is 6-12 months old; (6) with Polysaccharides with repetitive structures are T cell-independent type 2 immunogens. Without the participation of T cells, they cannot induce immune memory effects. The antibodies that stimulate the body to produce are mainly IgM and IgG2, which cannot effectively activate the complement system.

The method of developing vaccines by semi-chemical combination technology (also known as combination technology) is a technology for developing bacterial vaccines that emerged in the 1980s. John Robbins synthesized Haemophilus influenzae b by covalently linking the capsular polysaccharide (Polyribosylribitolphosphate, PRP) of Haemophilus influenzaetypeb (Hib) to a protein carrier (tetanus toxoid). Type polysaccharide PRP-tetanus toxoid conjugate vaccine (PRP-TT) can produce bactericidal protective antibodies after immunizing animals, thus creating a new generation of bacterial vaccine development technology. Of particular significance is that for infants and young children younger than 2 years old, due to the immature immune system, polysaccharide is a T-cell-independent antigen for infants and young children, and cannot stimulate the body to produce long-term effects like adults. IgG antibodies are specifically protected against bacteria from which the polysaccharide is derived. Therefore, polysaccharide is a hapten for infants and young children under the age of 2, and cannot be used as a vaccine to vaccinate children under 2 years of age. When bacterial polysaccharides are covalently linked to protein carriers, such as Tetanus Toxoid (TT), since protein is a T cell-dependent antigen, it can convert covalently linked polysaccharides into T cells. dependent antigen, thereby stimulating the body to produce specific IgG antibodies against the polysaccharide, and protecting the body from bacterial infection. The successful development of Haemophilus influenzae type b polysaccharide-tetanus toxoid (PRP-TT) conjugate vaccine has created a technological platform for the development of bacterial vaccines, namely bacterial polysaccharides, such as capsular polysaccharides, O-specific polysaccharides (O -specific polysaccharide), or oligosaccharide (Oligosaccharide), a conjugate vaccine made by covalently linking to a protein carrier. Based on the success of this concept, the medical biological research community has developed conjugate vaccines of different bacteria by using the same chemical synthesis method;

Capsular polysaccharides can shield bacterial cell surface functional components from being recognized by the host immune system, preventing the complement system from being activated by proteins on the bacterial surface and phagocytosed by immune cells. Capsular polysaccharides can also prevent bacteria from being killed if they are engulfed by immune cells. Capsular polysaccharide is one of the main antigenic components of Neisseria meningitidis, and as a vaccine, it has certain protection for older children. However, capsular polysaccharide has poor immune effect on infants and young children under 2 years old, the elderly and people with B cell immunodeficiency. The polysaccharide vaccine, like other polysaccharide vaccines, is a T-cell-independent antigen, has age-related immunogenicity, and does not induce a T-cell-dependent booster response. By covalently combining polysaccharides with a certain protein, the polysaccharides can be converted into T-cell-dependent antigens, thereby stimulating the synthesis of T-cell-dependent antibodies in infants and young children, and can produce a booster response, while also increasing the immunoglobulin (IgG) ) of the antibody ratio. This polysaccharide conjugate vaccine can not only protect infants and young children (children under 2 years old), but also can greatly enhance the resistance of patients with poor resistance to bacterial infection, so it has a very broad application prospect. In 1980, John Robbins used cyanogen bromide to randomly activate the Hib capsular polysaccharide, then, Adipic Dihydrazide (ADH) was added to the activated polysaccharide as a linker, and finally the derivatized polysaccharide was derivatized by the EDC method. The polysaccharide is covalently linked to the carrier protein tetanus toxoid to synthesize the Haemophilus influenzae type b polysaccharide-tetanus toxoid conjugate vaccine (PRP-TT). Since there are multiple activation points on each polysaccharide chain, there are also multiple connection points on the protein carrier, and the formed conjugate is a macromolecule cross-linked between polysaccharide and protein, with an average molecular weight of about 5×106Da. In 1980, Harold Jennings stated in U.S. Patent No. 4,356,170 that bovine serum albumin (BSA) was used as a carrier to covalently link the A and C groups of meningococcal polysaccharides to BSA by reducing amine method. Synthesized meningitis polysaccharide-BAS conjugate vaccine. In 1987 and 1990, Porter Anderson described in U.S. patents 4,673,574 and 4,902,506, respectively, that the mutated avirulent strain of diphtheria toxin 197 (Cross reaction material sub197, CRM197) was used as a carrier protein to synthesize Haemophilus epidemic by reducing amine method. Type b oligosaccharide-variant avirulent diphtheria toxin 197 (HbOC-CRM197) conjugate vaccine. The specific method is to oxidize the Hib capsular polysaccharide with sodium periodate to generate oligosaccharides with aldehyde groups at both ends, and the oligosaccharides are covalently linked to the protein carrier through the reducing agent sodium cyanoborohydride (sodium cyanoborohydride). superior. A lipopolysaccharide conjugate with a molecular weight of about 90kDa was formed, and a conjugate vaccine containing 30% polysaccharide and 6 sugar molecules on each protein was prepared. Subsequently, Merck, Sharpe and Dohme in the United States used thiol chemistry to use purified Neisseria meningitidis groups B bacterial surface protein complex (outer membraneprotein complex, OMP) as a protein carrier, Haemophilus influenzae type b polysaccharide-bacterial surface protein complex (PRP-OMP) conjugate vaccine was synthesized. Using these synthetic techniques, three types of Haemophilus epidemic b conjugate vaccines, which are now widely used in clinical vaccination, have been developed successively, namely PRP-TT, PRP-HbOC and PRP-OMP. The success of Hib conjugate vaccine provides a theoretical and technical basis for the development of other bacterial conjugate vaccines, and subsequent development has entered the stage of more complex multivalent conjugate vaccines. The reason is that some infectious diseases, such as pneumonia caused by pneumococcus, epidemic Meningitis caused by meningococcus, etc., can be caused by a variety of different serotypes or strains of infection, and due to the difference in the chemical structure of bacterial surface polysaccharides between serotypes or strains, their antibodies have no cross-immunity reaction. Therefore, Vaccination with a single serotype or strain conjugate vaccine does not protect the vaccinated person from infection by other serotypes or strains. For this reason, synthesis and formulation of multivalent conjugate vaccines to expand the protective coverage of vaccines has become a major goal of development.

Through years of efforts, a variety of multivalent conjugate vaccines with wide coverage have been developed by combining technology. Bacterial capsular polysaccharide-protein conjugate vaccines first appeared in the 1930s. Goebel and Avery linked type 3 pneumococcal polysaccharides to horse serum globulin, and the resulting conjugates produced polysaccharide-specific antibodies in animals. Provide corresponding immune protection. In 1987, the world's first polysaccharide protein conjugate vaccine, Haemophilus influenzae type B (HiB) polysaccharide-tetanus toxoid (TT) conjugate vaccine was approved by the US FDA to enter the market. Merck, Pfizer and Novartis have successively developed HiB polysaccharide-tetanus toxoid conjugate vaccine and meningitis polysaccharide-tetanus toxoid conjugate vaccine, which have been successfully marketed. In 2000, Wyeth Corporation of the United States successfully developed and marketed a 7-valent pneumococcal polysaccharide-CRM197 conjugate vaccine, which is composed of seven different pneumococcal serotype polysaccharides covalently linked to the CRM197 protein carrier and mixed and formulated. A multivalent vaccine for the prevention of pediatric pneumonia, the 7 serotypes of pneumococcus cover more than 90% of the different serotype strains of pneumococcus circulating 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 meningococcal groups, 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, among which the most important carrier is protein-D (Protein D, PD), and 8 serotype polysaccharides use this protein as the carrier. Protein-D is a non-esterified surface protein expressed by the genetic recombination method of non-typeable Haemophilus influenzae, which can stimulate the body to produce protective antibodies and has the potential to prevent non-typeable Haemophilus influenzae For acute otitis media caused by infection, other tetanus toxoid and diphtheria endotoxin are used as carriers, which are used as protein carriers for conjugate vaccines of serotypes 18C and 19F, respectively. From the design of the above conjugated vaccine products, it can be seen that the development of conjugated vaccines has transitioned from monovalent vaccines to technically more complex multivalent vaccines, improving the coverage of bacterial vaccines.

Polysaccharide protein conjugate vaccine (conjugated vaccine) is the most advanced vaccine technology at present. Adding protein carrier to specific antigen can increase its immunogenicity. Protein carriers have T cell-dependent properties, and polysaccharide-protein conjugate vaccines can convert T-cell-independent polysaccharide antigens into T-cell-dependent antigens, and stimulate the body's T helper cells to produce a series of immune-enhancing effects. Capsular polysaccharide conjugate vaccine, adding a protein carrier to the polysaccharide, changes from a T cell-independent antigen to a T-cell-dependent antigen, increasing its immunogenicity. The quality and quantity of antibodies produced by the combined vaccine are 400-1000 times that of the first-generation vaccine, resulting in broader and stronger immune protection, longer protection time, and high-efficiency protection. In 2000, the first 7-valent pneumococcal conjugate vaccine was launched by Pfizer in the United States; Pfizer in the United States developed a more targeted 7 (4, 6B, 9V, 14, 18C, 19F and 23F) pneumococcal protein vaccine for infants and young children. Also effective for children under 5 years old. Capsular polysaccharide protein conjugate vaccine, adding a protein carrier to the polysaccharide, changes from a T cell-independent antigen to a T-cell-dependent antigen, which can increase its immunogenicity and can be used for children over 6 weeks old. Pfizer is currently registering a 13-valent conjugate vaccine in China, adding 6 serotypes (1, 3, 5, 7F, 6A, 19A). However, my country's data show that the serotypes of pneumococcal infection strains are: 5, 6, 1, 19, 23, 14, 2, and 4. The Pfizer 7-valent pneumococcal conjugate vaccine covers only about 50% of common pathogenic bacteria in my country. , and the coverage of 13-valent conjugate vaccine is only 70%. Wyeth 7-valent pneumococcal conjugate vaccine requires 4 injections, 860 yuan per injection, which is expensive and not conducive to promotion. The 13-valent vaccine is estimated to be more expensive. Therefore, the 7- and 13-valent conjugate vaccines of Pfizer in the United States are not suitable for the prevention of large-scale childhood pneumonia in my country.

Although 13-valent pneumococcal conjugate vaccines are already on the market, several serotypes are less effective than others, such as type 3. And with the increase of different serotypes and the repeated use of the same carrier protein, the amount of carrier protein increases, but its immunogenicity decreases. GlaxoSmithKline chose protein-D as the carrier in the design of the 10-valent pneumococcal polysaccharide conjugate vaccine for two reasons. One is to avoid the repeated use of tetanus toxoid and diphtheria toxoid that have been used as DTP vaccine components as carriers. Clinical trials have shown that when the multivalent pneumonia conjugate vaccine is administered simultaneously with other monovalent vaccines or multiple vaccines containing the same components as the protein carrier, such as Hib-TT, DTP-Hib polysaccharide conjugate vaccine-IPV (inactivated polio). Inflammation vaccine)-hepatitis B vaccine (abbreviated as 6 combined vaccine), the immunogenicity of most serotype polysaccharides in the multivalent pneumococcal conjugate vaccine will be inhibited, especially for the serotype polysaccharide immunogen using tetanus toxoid as a carrier Sexual influence is particularly pronounced. The reason is that the total concentration of tetanus toxoid and diphtheria toxoid as carriers in the multivalent pneumococcal conjugate vaccine is too high. The competitive inhibitory effect reduces the immunogenicity of the polysaccharide portion of the conjugate vaccine. SanofiPasteur's 11-valent pneumococcal polysaccharide-TT and DT mixed carrier protein conjugate vaccine and Merck's 7-valent pneumococcal polysaccharide-OMP conjugate vaccine (PCV-OMP) are examples of poor clinical trial results that lead to product development failures. The reasons right here. The second is to endow the protein carrier with a real protective immunogenic function. Clinical trials have shown that protein-D can stimulate the body to produce protective antibodies and has the potential to prevent acute otitis media caused by non-typeable Haemophilus epidemic infection. GlaxoSmithKline's 10-valent pneumococcal polysaccharide conjugate vaccine selects protein-D as a carrier, which makes the antibodies produced by the protein carrier have clinically meaningful protective effects, which is a major progress in the development of conjugate vaccine technology.

However, the actual clinical significance of the non-typeable Haemophilus epidemicis is limited by the low infection rate of this class of bacteria, which causes a low incidence of acute otitis media. However, these conjugate vaccines have a common disadvantage, that is, the protein carrier does not confer the protective function of immunogenicity. That is, although the conjugate vaccine carrier can stimulate the body to produce antibodies, the vaccine designer does not take advantage of the protection provided by the antibodies. sex to prevent infectious diseases, at the same time, it has not been determined whether the antibody produced reaches protective titer levels. Traditional proteins such as tetanus toxoid, diphtheria toxoid and diphtheria avirulent variant toxin were chosen as carriers not because the antibodies they produced were protective, but because of their safety and ability to enhance the binding Consider the immunogenicity of polysaccharides. Obviously, tetanus toxoid and diphtheria toxoid are two components of DTP vaccine and are routinely vaccinated; therefore, whether the tetanus toxoid and diphtheria toxoid carriers in the conjugate vaccine can stimulate the body to produce protective effects Antibody titers are not important, on the contrary, antibody titers that bind to the polysaccharide moiety in the vaccine are the main concern for vaccine designers. In addition, the vectors used in some conjugate vaccine products under development, such as the recombinant Pseudomonas aeruginosa exotoxin A (rEPA) expressed by E. Recombinant cholera toxin, etc. are also based on the same considerations.

The types and types of new polysaccharide conjugate vaccines are increasing year by year, but there are fewer types of carrier proteins to choose from, and many carrier proteins are reused for different vaccines. Inoculation of different conjugate vaccines with the same carrier protein may produce immunosuppressive effects, resulting in the mutual influence of immune effects between different vaccines. Moreover, the main carrier proteins of polysaccharide conjugates, such as TT, have been widely used as vaccines for infants and young children. The original high titer specific antibodies against the carrier proteins in the population may inhibit the body's specificity to the polysaccharides in the conjugate vaccine. Sexual immune response.

A Chinese patent (ZL02159032.X) discloses a method for preparing a polysaccharide-protein conjugate vaccine, which is also one of the most commonly used technologies for preparing polysaccharide conjugate vaccines. In the technique, adipic acid dihydrazide (ADH) is used as a linker to bind polysaccharides and proteins. This combination method first needs to activate the polysaccharide by cyanogen bromide, that is, use cyanogen bromide to act on the hydroxyl group on the polysaccharide molecule under alkaline conditions to form cyanate ester, and then react with ADH; a carbon oxygen in the cyanate ester The bond is broken, and an addition reaction occurs with the amino group at one end of ADH, so that the ester hydrazide (AH) group is introduced into the polysaccharide molecule to form a polysaccharide-AH derivative; the polysaccharide-AH derivative is mediated by carbodiimide (EDAC) It forms stable conjugates with carrier proteins. Such a binding method can reduce the steric hindrance of the binding of the polysaccharide to the carrier protein, retain the antigenic epitope of the polysaccharide, avoid the solubility of the polysaccharide itself, and reduce the side effects of the reaction between the polysaccharide and the antiserum.

However, the above-mentioned traditional polysaccharide-protein binding technology has the following shortcomings: (1) The polysaccharide-AH derivative will continue to react with the polysaccharide activated by cyanogen bromide to form a self-polymer of the polysaccharide, which reduces the binding of the polysaccharide-protein. (2) EDAC mediates the binding of ADH-derived polysaccharides to carrier proteins, and at the same time easily causes self-crosslinking of polysaccharides and carrier proteins, thereby reducing the binding efficiency of polysaccharides and proteins; (3) Both polysaccharides and carrier proteins are large organisms. The molecules are connected by ADH with a length of only 6 carbon atoms in the middle. The structures of polysaccharides and proteins are bound to affect each other, making some important epitopes of polysaccharides easily shielded by proteins, thereby reducing the immunogenicity of polysaccharides. Therefore, the immunogenicity and antibody persistence of the polysaccharide-protein conjugate vaccine still need to be further improved. For example, the polysaccharide conjugate vaccine needs to be immunized three times to produce an immune effect. These shortcomings limit the further development of polysaccharide conjugate vaccines.

4. The harm of human rotavirus and its epidemiological study

Worldwide, rotavirus is the main pathogen causing severe diarrhea in children. In developed countries, the detection rate of rotavirus accounts for 35-52% of children hospitalized for acute diarrhea. In the United States, an estimated 3 million children suffer from rotavirus diarrhea each year, resulting in 82,000 hospitalizations and 150 deaths. Rotavirus is also the most common causative agent of severe gastroenteritis in infants and young children under 2 years of age in developing countries, with more than 125 million children under 5 years of age estimated to suffer from rotavirus diarrhoea each year, of whom 18 million suffer from Moderate diarrhea, 870,000 deaths. In China, the birth rate of children is about 17 million, and it is estimated that about 350,000 children die each year due to rotavirus diarrhea, ranking second in the world. Since infection with the virus has a significant effect on the increased morbidity and mortality of diarrhea in infants under 2 years of age, the development of an effective and safe vaccine to prevent rotavirus infection is a top priority.

Rotaviruses belong to the Reoviridae genus and are pathogens that cause diarrhea in humans and many animals. The diameter of the whole virus is 70nm, and it has a special three-shell structure. The innermost nucleocapsid structure is nucleocapsid protein, which is wrapped with virus genes. The gene of the virus consists of 11 segmented segments of double-stranded RNA, encoding 6 structural and 5 nonstructural proteins. The nucleocapsid protein is composed of three viral proteins, VP1, VP2 and VP3; the intermediate capsid protein is VP6, and the coat protein is composed of VP4 and VP7. Since the rotavirus gene is a double-stranded RNA with a multi-segment structure, the gene of this structure can undergo a certain degree of recombination between genes, that is, the reassortment of the rotavirus gene. When two or more viruses infect a host cell at the same time, in the packaging stage of new virus particles, the gene segments of each virus will be recombined in the cell, resulting in the occurrence of reassortment genes. The ability of rotavirus to reassort genes has led to the diversity of the body's immune response to it, and it has also increased the difficulty of making specific rotavirus vaccines.

Rotavirus can be divided into different types, subtypes and serotypes according to the different antigenicity of the virus. Seven serotypes (A-G) have been identified, and most human pathogens belong to A, B, and C types. Epidemiological investigations have found that type A is the most common type of rotavirus that causes disease in humans and animals, and is the main target for vaccine development. Type A rotaviruses are further classified into subtypes according to their VP6 antigenic characteristics, and most virus strains belong to one of subtypes I or II. Different rotavirus surface capsid proteins VP7 and VP4 have different neutralizing antigen clusters and can induce their own neutralizing antibodies independently. The serotype of the virus can be determined by the specificity of VP4 and VP7 antigens. Type A rotavirus can be further classified into G serotype and P serotype according to VP7 and VP4. Since the rotavirus gene is composed of 11 segments, the coding genes for VP7 and VP4 can be separated and combined independently, resulting in a binary reassortment of genes. VP7 is a glycoprotein that is classified into 15 different G serotypes due to its antigenic properties, corresponding to its specific nucleic acid sequence, the genotype; that is, each G serotype Has its specific genotype, therefore, usually G serotype and G genotype can be used in common. Of the human pathogenic rotavirus strains identified globally, more than 90% are G1, G2, G3, G4, and G9 strains, and a total of 10 serotypes have been isolated from humans (see Table 1). VP4 is a virus protein that can be cleaved by trypsin into two different viral proteins, namely VP8 and VP5 viral proteins. The serotype determined by its antigenicity is P serotype, and some P serotypes can be further divided into 2 subserotypes. Serotyping of VP4 is hindered by the lack of serum or monoclonal antibodies that distinguish different VP4 serotypes. The application of RT-PCR makes it possible to genotype VP4 and apply it to epidemiological investigation of samples. Therefore, VP4 is basically classified according to the gene sequence, and a total of 14 P serotypes and at least 26 P genotypes (marked in brackets) have been found so far. The P serotype and P genotype may not correspond and need to be marked together, for example, the Wa strain of rotavirus is marked as the P1A[8]G1 virus. In human pathogenic strains, the combination of P and G serotypes for G1, G3, G4, and G9 is usually P1A [8], while G2 is usually P1B [4]. It can be seen that the rotaviruses circulating worldwide share the same cross-neutralizing antigenic cluster (epitopes) P1 serotype, and at least seven VP4 serotypes have been found in human rotavirus. Epidemiologically significant human strains of G serotypes 1, 3, and 4 are subserotype 1A belonging to serotype P, and serotype G 2 is subserotype 1B of serotype P.

Since natural rotavirus infection induces immune protection well, at least against severe rotavirus infection, most efforts to develop vaccines have been placed on live attenuated vaccines. Initial research focused on the use of animal rotavirus strains, by what is known as the Jennerian method, since naturally attenuated animal strains are safe in humans and yield predominantly mixed immune protection.

In 1983, 10 years after the discovery of rotavirus as the causative agent of severe diarrhea in children, the first live attenuated rotavirus was produced using the porcine rotavirus strain RIT4237 (G6P[1] type). The vaccine was clinically tested, and the results of the trial in Finland showed that the vaccine was safe and effective, with 80% protection against severe rotavirus diarrhea through the action of heterotypic human rotaviruses. However, subsequent clinical trials in other countries were disappointing, showing little or no protection, and the trials failed. In 1987, the simian rotavirus RRV strain was used to develop a live attenuated vaccine, another of the first rotavirus vaccines to be developed. Clinical trials have shown that although the vaccine can induce the body to produce protective antibodies, the results are not stable. When the serotype is G3, the effect of the vaccine is significant; if it is a different type of G serotype infection, the effect is not good. Subsequently, the RRV strain introduced the VP7 gene from the human strain into the strain by gene reassortment, so that the other three G serotypes G1, G2 and G4 commonly found in human rotaviruses can also be reassorted in RRV. expression on the matched strain, which led to the successful development of the 4-valent Rotashield vaccine. The vaccine was approved for marketing by the FDA in 1998, but was removed from the market in 1999 due to the occurrence of a few but significantly increased cases of intussusception side effects after large-scale vaccination. In 1988, clinical trials with another rotavirus strain, the WC3 swine strain (G6P[5] type), were initially shown to be effective, but subsequent trials showed no significant protection, The vaccine was also discontinued from continuing trials. By 1990, in order to make the antigenic structure of the WC3 strain closer to that of human rotavirus, the genes encoding VP4 and VP7 proteins were introduced from human rotavirus into WC3 reassortment by gene reassortment. On strains, this method is called the modified Jennerian method. The strains and methods described are the development methods used by Merck to develop the pentavalent vaccine for RotaTeq. In 2006, RotaTeq, a 5-valent WC3-gene reassortment vaccine produced by Merck, was also approved for marketing. The vaccine contains two human rotavirus protein replacement genes, VP7 and VP4, that is, the corresponding genes in G1, G2, G3, and G4. The VP7 protein gene, and the corresponding VP4 of P[8]. Clinical trials show that the vaccine has no intussusception side effect, the protection rate against gastroenteritis due to G1-G4 rotavirus is 74%, the protection rate against severe gastroenteritis is 98%, and the protection rate against hospitalization and emergency The protection rate for access is 94.5%. In the same year, Rotarix, a live attenuated human rotavirus vaccine produced by GSK, was also approved. . The virus was isolated from a clinical sample of a patient suffering from rotavirus gastroenteritis, and obtained after multiple passages of histiocytes attenuated by mutation. Clinical trials have shown that following two doses of injection, protection against all rotavirus infections is over 87%, protection against severe gastroenteritis is 96%, and protection against gastroenteritis requiring hospitalization is 100% %. Further experiments showed that the scope of protection of the vaccine includes not only gastroenteritis caused by the G1P[8] strain, but also gastric gastroenteritis caused by the VP4-related G3P[8], G4[8] and G9P[8] strains enteritis. Efficacy of protection against G3, G4 and G9 rotavirus infection was the same as that of G1, exceeding 95%, while the efficacy against G2 strain was 75%, and the protection rate against all rotavirus-induced gastroenteritis hospitalization was 75% %.

Statistics show that the G1 strain is the most frequently detected strain worldwide, with G1-G4 strains accounting for 97.5% of total rotavirus infections in Asia, North America and Europe. In South America, Africa, Australia accounted for 83.5%-90.4%, meanwhile, G5, G8 and G9 strains began to become important in these regions. In terms of P serotypes, the P1A[8] strain is the most common, followed by the P1B[4] strain. This result is predictable because the VP7 serotype G1 is P1A [8], the most commonly detected VP7 serotype; the other two epidemiologically important VP7 serotypes, G3 and G4, also share the same VP4 serotype. VP4 of the other major serotype, G2, is characteristic of P1B[4]. Although there are many different combinations of G and P serotypes or genotypes, there are four P-G combinations, namely P[8]G1, P[4]G2, P[8]G3, and P[8]G4 constitutes 88.5% of the common pathogenic strains.

It can be seen that if the VP7 protein of the G serotype is used to configure the vaccine, it is necessary to include a variety of different G serotype strains to improve the coverage of the vaccine, namely G1, G2, G3, G4, G9, G8 and G5, 7 Valuable live attenuated rotavirus vaccine is also the current effort to develop a new generation of vaccines. If rotavirus vaccines are developed from another strategy, a new generation of vaccines can be formulated with differences in the P serotype-determining protein VP4, which can greatly reduce the number of strains included in the vaccine to achieve the same coverage. It can be seen from the above analysis that if the vaccine is formulated with two P serotype strains, P[8] and P[4], the immunization effect will be equivalent to that of the 7-valent G serotype. From the clinical use of Rotarix and Roateq, there is little difference, and Rotarix appears to be more effective than Roateq based on the available statistics. From the analysis of the composition of the serotype-determining proteins of the strains contained in these two vaccines, Rotarix contains only one VP7 viral protein, G1; The effect was not enhanced; and judging from the amount of P serotype-determining protein VP4 contained in both, Rotarix contains P[8] one, while Rotateq contains P[8] (one of the strains) and P[5] ( The original strains of WC-3 are G6P[5]); however, the content of the important antigenic component P[8] in Rotarix will be higher than that in Roateq. It can be seen that from the perspective of P serotype, the VP4 strain rotavirus vaccine containing P[8] is of great significance. If the new generation vaccine contains the three VP4 antigen components of P[8], P[4] and P[6] of the P serotype, it can cover the circulating strains in most parts of the world.

5. Application and prospect of nano-microspheres in biotechnology

Nanomaterials refer to single crystals or polycrystals with a grain size of less than 100 nanometers. The unique small size effect and surface or interface effects make them have many excellent or brand-new properties, and they are increasingly attracting people's attention. For example, the continuous penetration and influence of nanomaterials on the field of pharmaceutical research has triggered a profound revolution in the field of pharmaceuticals. Drugs are important substances for human beings to resist and prevent diseases. For a long time, the research and development of drugs has provided many clinical treatments and brought many benefits to patients. However, there are still many problems with the existing drugs, such as the inability of the drugs to stay in the circulatory system and reach an effective concentration, unable to reach specific therapeutic targets, unable to pass through the blood-brain barrier, unable to form a high concentration in a local area without Toxic and side effects, etc. (Wu Xinrong. Application and research progress of drug-loaded nanoparticles [J]. Chinese Journal of Hospital Pharmacy, 2001,21(3):171-173.). Magnetic nanosphere drug carrier is the product of the combination of nanotechnology and modern medicine. Because of its advantages of small size effect, good targeting, biocompatibility, biodegradability and functional groups, it is expected to overcome traditional drugs. these defects. Magnetic nanoparticles can also be used for purification and recovery of proteins and enzymes and immobilization of enzymes, which are easy to operate and improve the stability of enzymes. The use of magnetic nanoparticles for immunoassay has the characteristics of good specificity, fast separation and good reproducibility. The use of magnetic microsphere carriers for interventional therapy and embolization in magnetron blood vessels has the advantages of magnetron guidance and target embolization.

The application of magnetic nano-microspheres in biomedicine is becoming more and more extensive with the deepening of research, specifically:

1. Immobilized enzyme

Biopolymers such as enzyme molecules have many functional groups, which can be immobilized on the surface of magnetic particles by physical adsorption, cross-linking, covalent coupling, etc. The advantages of immobilizing enzymes with magnetic nano-microspheres are: easy separation of enzymes from substrates and products; improved biocompatibility and immune activity of enzymes; improved stability of enzymes, and simple operation can reduce costs. Bendkiene et al. prepared chitosan magnetic microspheres as immobilization carriers. After the enzyme is immobilized on this carrier, it can be easily separated and recovered from the reaction mixture by a magnetic device. Domestic researchers have also explored this aspect. Ding Xiaobin et al. used dispersion polymerization to synthesize Fe3O4/(St-MPEO) (St-styrene, MPEO-polyethylene oxide macromonomer) microspheres. The microspheres have an amphiphilic structure and have good swelling properties in most polar and non-polar media, making the compounds immobilized on the microspheres have high activity in a variety of media (Ding XB, Wei L, Zhao HZ. Synthesis and characterization of aliphatic polycarbonatediols [J]. Applied Polymer Science, 2001, 79(3): 1847-1851). The magnetic nano-microsphere carrier is expected to be used in the fields of purification and recovery of proteins and enzymes, immobilization of enzymes, cell separation and the like.

2. Targeted drugs

Targeted drug carrier microspheres refer to drug-loaded microspheres that can be selectively distributed to the target, thereby enhancing curative effect and reducing side effects. The initial targeted drug carrier microspheres are based on clinical needs, by selecting carriers with different affinities to various tissues or lesions of the body to make drug-loaded microspheres, or combining monoclonal antibodies with carriers, so that the drug can be delivered to the desired therapeutic target. specific parts reached. As people's requirements for treatment are getting higher and higher, the targeting positioning is also not completely satisfactory due to the limitation of the matrix, so the magnetic nano-microsphere drug-carrying system has appeared. Under the action of an external magnetic field, this system injects people into the diseased tissue through arterial (intravenous) veins, and orients the carrier to the diseased site (target site), so that the contained drugs are localized and released, and concentrated to the diseased site for action (A Paul , Alivisatos. Ultrasensitive magneticbiosensor for homogeneous immunoassay [J]. Science, 2001, 12(5):53-60). Germany's Lubbe et al. completed the world's first clinical trial using magnetic drug targeted therapy. In the magnetic targeting therapy of 14 patients with advanced solid tumors, the patients were found to be well tolerated by the magnetic targeting drugs. Lexion et al. also applied magnetic microspheres as drug carriers to treat squamous cell carcinoma in rabbits (Lexion C, Amold W, Klein RJ, et al. Locotegional cancer treatment with magnetic drug targeting[J]. Cancer Res, 2000, 60(23): 6641- 6648), domestic Tao Kaixiong et al. used doxorubicin magnetic protein microspheres to treat mouse implanted gastric tumors (Tao Kaixiong, Sun Hongwu, Chen Daoda, et al. Doxorubicin magnetic protein microspheres targeted therapy for mouse implanted gastric tumors[J]. Chinese Journal of Experimental Surgery, 2000, 17(1): 63-64)), Guo Jun et al. used Pingyangmycin magnetic microspheres to treat 25 cases of cavernous hemangioma of the oral and collar. In addition, strong magnetic field also has tumor suppressor effect (Guo Jun, Li Cheng, Wu Hanjiang. Clinical report of 25 cases of oral and maxillofacial cavernous hemangioma targeted with Pingyangmycin magnetic microspheres [J]. Journal of Nanjing Railway Medical College, 2000, 19(2):112-114)). Xu Huixian et al. used glucose magnetic microspheres to immobilize L-aspartame to treat acute lymphoblastic leukemia, and also achieved good therapeutic effects. These results all show that with the increase of the magnetic field strength, the aggregation of magnetic drugs at the tumor site increases, and the magnetic guidance effect of the external magnetic field is used to make the drug localize at the target site and exert a concentrated and efficient anti-tumor effect. It is precisely because of the targeting and surface-bound specific carrier of magnetic nanospheres that people are expected to use it to track and eliminate cancer cells that are metastasizing, thus becoming a "biological missile" for eliminating cancer cells in the human body. However, most of the tumors that are currently treated with magnetic drugs are located on the body surface or close to the body surface, so the intensity of the applied magnetic field can be weak and easy to control. If the tumors in deep organs or tissues are treated, it is necessary to further optimize the magnetic field intensity and positioning. and drug carrier particle size, etc. However, it is difficult to improve the magnetization of magnetic microspheres, because the coating layer on the surface of the magnetic particles will greatly reduce their magnetic properties. In addition, the controllability of particle size is also a problem to be solved.

3. Cell isolation and immunoassay

If a specific antibody with biological activity is introduced on the surface of the magnetic particle, and under the action of an external magnetic field, the specific binding between the antibody and the cell can be used to obtain immunomagnetic microspheres (Immunomagnetic microspheres, IMMS) or immunomagnetic beads (Immunomagnetic beads). , IMBS), which can be used to rapidly and efficiently isolate cells or perform immunoassays. Especially when IMBS is used to separate antigenic substances specific to the surface of cells and organelles, it has the characteristics of simplicity and rapidity, high separation purity, and retention of target substance activity. McCole et al. used IMBS to isolate T lymphocytes from the peripheral blood of adult bovines infected with liver flukes. John uses IMMS with monoclonal antibody to detect Salmonella. The whole detection process only takes 2-3 hours, and the sensitivity is 103-104 bacteria/ml. The presence of a certain amount of blood and feces does not interfere with the analysis, compared with agglutination method and immunofluorescence method , the sensitivity is increased by 103 times. Glenn used IMBS to separate respiratory inclusion body viruses, combined with ELISA, to reduce diffusion and non-specific adsorption in traditional tube and microwell assays. The formation of complexes only took 7 minutes, while the traditional micropore method took 120 minutes. Cell isolation techniques can also be used for cancer treatment. Kang Jichao et al. used the method of physical adsorption and chemical bond covalent bonding to connect the anti-human bladder cancer monoclonal antibody to the surface of the pre-prepared polystyrene magnetic microsphere carrier, and constructed a structure that can specifically bind to target cells and endow them with Magnetically responsive immunomagnetic microspheres. The results show that the constructed IMMS can effectively bind to the target cells. Preliminary experiments using IMMS to isolate cancer cells from animal bone marrow show that IMM can effectively remove cancer cells, while only a small amount of bone marrow cells are lost. Immunoassay is an important method in modern bioanalytical technology, which plays a huge role in the quantitative analysis of proteins, antigens, antibodies and cells. The use of antigens or antibodies bound to magnetic nanoparticle carriers for immunoassay has the characteristics of high specificity, fast separation, and good reproducibility. Jing Xiaoyan et al. adopted the emulsion-free polymerization method. In the alcohol-water system, potassium peroxodisulfate was used as an initiator to form an initiation point on the surface of Fe3O4 magnetic fluid particles, and acrylic acid (AA) was used as a stabilizer. ) and acrylamide to prepare monodisperse amine-based magnetic microspheres, which can be directly and quickly cross-linked with antibody (antigen) proteins, avoiding the need to use protein as a Disadvantages of cross-linking agents (Jing Xiaoyan, Wang Jun, Li Rumin, et al. Preparation of Magnetic Functional Polymer Microspheres [J]. Applied Science and Technology, 2000, 27(1): 16-17)).

4. Magnetic control plug

During the usual interventional therapy, phenomena such as ectopic embolism and infarction will occur and cause serious complications. This is a thorny problem that needs to be solved urgently in clinical practice. However, the interventional therapy using magnetic microsphere carriers is very important in magnetic control. Intravascular embolization has the advantages of magnetron guidance and target embolization, which provides a way to solve the above problems. Scholars have made meticulous research on the particle size of magnetic spheres, magnetron time, magnetic field strength, and magnetic microsphere encapsulation materials (rough, positively charged, and hydrophobic). Minalnimura et al. used a combination of hyperthermia and arterial embolization in a mouse model of hepatocellular carcinoma. They developed a DM-MS arterial catheter for local administration with an applied 500kHz magnetic field. After 3 days of treatment, the tumor growth rates (embolization-hyperthermia group, simple embolization group, and control group) were 28%, 124%, and 385%, respectively (Minalnimura T, Sato H, Kasaoka S, et al. Tumor regression by inductive hyperthermia combined with hepaticembolization using dextran magnetite incorporated microspheres in rats[J]. IntJ Oncol, 2000, 16(6):1153-1160). This study shows that the combination of DM-MS hyperthermia and embolization is a feasible anti-tumor therapy and has broad research and application prospects. Goodwin et al. studied the toxicity of doxorubicin magnetic microspheres hepatic artery embolization and drug-targeted antitumor therapy (Goodwin SC, Bittner CA, Peterson CL, et al. Single-dose toxicity study of hepatic intra-arterial infusion of doxorubicin coupled to a novel magnetically targeted drug carrier[J]. Toxical, 2001, 60(1):117-183). The results of the pig liver cancer model they established showed that the low dose of doxorubicin magnetic microspheres had no toxic side effects. Only when the content of magnetic microspheres >= 75mg (with or without doxorubicin) in the target area has a better effect, the degree of necrosis of liver cancer cells is proportional to the degree of embolism, and doxorubicin cannot circulate freely throughout the body and successfully controlled in the target area. Hui Xuhui et al. discussed intravascular embolism with self-made polymethyl methacrylate magnetic microspheres. Experiments showed that 30-50um PMMA magnetic microspheres have strong magnetic response ability, good magnetic control embolization effect, and high blood pressure. It can still achieve target embolization and other advantages under the condition of flow rate, and it is a better magnetically controlled intravascular embolization material (Hui Xuhui, Gao Lida, He Nengqian. Experimental study on intravascular embolization of polymethyl methacrylate magnetic microspheres [J]. Sichuan Medicine, 2001, 22(10):928-929)). In magnetron embolization, the size of the magnetic microsphere carrier is the most important factor affecting the positioning of the target area. If the particle size is small, the magnetic responsiveness is weak and the degree of magnetic control is poor, so it cannot be used for magnetron embolization in blood vessels with high blood flow or large diameter. Therefore, different from the application of magnetic microspheres in other aspects, magnetic microspheres with larger particle size are generally used in magnetron embolization interventional therapy.

There are also reports that nanospheres can be used as carrier proteins and adjuvants for DNA vaccines, but there is no report on the application of prophylactic polysaccharide and/or protein vaccines.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the object of the present invention is to provide a conjugated vaccine with stronger immunogenicity.

For realizing the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is as follows:

A conjugated vaccine is a conjugated vaccine formed by linking a polyvalent pneumococcal polysaccharide and two or more carrier proteins, wherein the linker is a magnetic nano-microsphere.

As a preferred solution, the magnetic particles of the magnetic nano-microspheres are located inside the magnetic nano-microspheres as cores, and the polymer material is wrapped around the outside of the magnetic particles.

As a further preferred solution, the magnetic particles are Fe ₃ O ₄ .

As a further preferred solution, the particle size of the magnetic nano-microspheres is 0.1-10 μm, preferably 0.1-5 μm.

As another preferred solution, the polymer material is a biopolymer material, selected from chitosan, polyethylene glycol, polylactic acid-glycolic acid copolymer (PLGA), polylactic acid-polyethylene glycol copolymer (PELA) One or more of the mixtures; the best is PLGA or PELA.

As another preferred solution, the polyvalent pneumococcal polysaccharide is a variety of pneumococcal capsular polysaccharides, preferably the capsular polysaccharide on the capsule of isolated and purified serotype pneumococcus, and the serotypes of the serotype pneumococcus include 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 preferred solution, the mass ratio of the polyvalent 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 preferred solution, the carrier protein is selected from 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 (rPspA).

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

As a further preferred solution, the recombinant human rotavirus protein is the partial amino acid sequence or the whole sequence of the P genotype rotavirus protein, preferably the P genotype rotavirus strains P[8], P[4], P[6 ] or one of P[11]; wherein, the P genotype rotavirus strain is selected from P[8]G1, P[4]G2, P[8]G3, P[8]G4, P[8 ]G9, P[8]G5, or P[6]G8.

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

As a further preferred solution, the rotavirus strain of P genotype P[4] is selected from DS-1, RV-5, S2, L26, KUN, E210, CHW17, AU64, 107E18, MW333 or TB-Chen strains one of the.

As a further preferred solution, the rotavirus strain of P genotype P[6] is selected from one of M37, 1076, RV-3, ST3, SC2, BrB, McN13, US1205, MW023, US585 or AU19 strains .

As another further preferred solution, the recombinant human rotavirus protein is selected from VP8, VP4, VP8 polypeptide chain fragment, nuclear VP8, VP4 polypeptide chain fragment, VP8-specific antigenic cluster peptide chain or VP4-specific antigenic cluster peptide chain A sort of.

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

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

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

As a further preferred solution, the G serotype rotavirus strain is selected from P[8]G1, P[4]G2, P[8]G3, P[8]G4, P[8]G9, P[8] 8] One of the G5 or P[6]G8 serotypes.

Another object of the present invention is to provide a method for preparing the conjugated vaccine, which is specifically prepared by coupling the polyvalent pneumococcal polysaccharide and two or more carrier proteins to magnetic nano-microspheres, respectively.

As a preferred version, the preparation method of the conjugated vaccine specifically includes the following steps:

a) Separate and purify the capsular polysaccharides on the capsules of various serotypes of pneumococcus respectively;

b) respectively preparing and separating and purifying the selected carrier protein;

c) Respectively coupling various capsular polysaccharides and magnetic nano-microspheres into polysaccharide-magnetic nano-microsphere conjugates;

d) Conjugate the polysaccharide-magnetic nano-microsphere conjugates with various carrier proteins respectively;

e) Separation and purification of the conjugate obtained in step d) into the conjugate vaccine stock solution.

As a further preferred version, the preparation method of the conjugated vaccine specifically includes the following steps:

a) Separate and purify the capsular polysaccharides on the capsules of various serotypes of pneumococcus respectively;

b) Separate and purify the selected multiple carrier proteins respectively;

c) Respectively coupling various capsular polysaccharides and magnetic nano-microspheres into polysaccharide-magnetic nano-microsphere conjugates, and then chemically modifying them, so that the surface of the polysaccharide-magnetic nano-microsphere conjugates is free from polysaccharide The -OH of the reaction is modified to -CHO;

d) Conjugate the polysaccharide-magnetic nano-microsphere conjugates with various carrier proteins respectively;

e) Separation and purification of the conjugate obtained in step d) into the conjugate vaccine stock solution.

As a further preferred solution, the preparation method further includes the preparation of magnetic nano-microspheres, including the process of first preparing nano-magnetic particles and then preparing magnetic nano-microspheres; wherein the nano-magnetic particles can be heated by chemical co-precipitation method, hydrothermal method or sol method. Solution preparation, magnetic nano-microspheres can be prepared by embedding method, monomer polymerization method or in-situ method, preferably by chemical co-precipitation method to prepare nano-magnetic particles, and then the nano-magnetic particles and polymer materials are emulsified and combined with solvent by rapid membrane Magnetic nano-microspheres are prepared by extraction method and/or embedding method or monomer polymerization method; most preferably, nano-magnetic particles are prepared by chemical co-precipitation method, and then nano-magnetic particles and polymer materials are subjected to rapid membrane emulsification combined with solvent extraction method and/or or embedding method to prepare magnetic nano-microspheres with uniform particle size; wherein the magnetic particles are preferably Fe ₃ O ₄ .

As a further preferred solution, the preparation method of the above-mentioned nano-magnetic ions can be prepared by referring to the method of Fe ₃ O ₄ magnetic nano-microspheres in the prior art. For details, please refer to the following specific embodiments, which is not the key to whether the present invention can be realized or not. factor.

As a further preferred solution, the separation and purification process of the polysaccharide in step a can be operated according to the prior art according to the type of polysaccharide and protein required.

As a further preferred solution, the preparation, separation and purification process of the protein in step b can be operated according to the prior art according to the type of polysaccharide and protein required.

As a further preferred solution, the specific operation of step c is: in the coupling medium reaction buffer, at room temperature and pH 4.0-9.0, co-react the polysaccharide obtained in step a with the magnetic nano-microspheres for 6-24 hours - Magnetic nano-microsphere conjugate, which is further modified by 25% glutaraldehyde, so that the -OH that is not reacted with polysaccharide on the surface of the polysaccharide-magnetic nano-microsphere conjugate is modified to -CHO; wherein the coupling The medium is preferably PB, PBS or TBS, the best is 0.1M TBS solution; the best pH of the reaction buffer is 6; the best reaction time is 12-16 hours.

As a further preferred solution, the specific operation of step d is as follows: in the coupling medium reaction buffer, at 4°C and pH 4.0-9.0, the chemically modified polysaccharide-magnetic nano-microsphere conjugate obtained in step c is combined Co-react with carrier protein for 12-24 hours; wherein the coupling medium is preferably PB, PBS or TBS, the best is 0.1M TBS solution; the best pH of the reaction buffer is 6; the best reaction time is 24 hours.

As a further preferred solution, the separation and purification of step e is to separate and purify the conjugated conjugate, unreacted polysaccharide and carrier protein, and the purification method can be chromatography or ultrafiltration; it can be carried out according to the molecular size of the obtained conjugated vaccine. For separation and purification, chromatography is preferably performed using Superdex200 gel filtration column, Sepharose CL-4B or Sepharose CL-6B; while ultrafiltration uses membranes with different retention molecular weights to separate conjugates and unreacted substances.

The conjugated vaccine formulations can be in aqueous or lyophilized formulations. In order to enhance its immunogenicity, adjuvants can be added. Commonly used adjuvants include aluminum adjuvants, such as aluminum hydroxide and aluminum phosphate. In the present invention, aluminum phosphate is preferred. The solvent of the conjugate can be 0.2 sodium chloride solution, 1×PBS buffer or other buffers capable of stabilizing the polysaccharide or the conjugate. The preparation method of each formulation of the conjugated vaccine of the present invention is prepared by conventional means in the technical field. Of these, lyophilization in the presence of sugars such as sucrose or lactose is preferred.

The conjugate vaccine provided by the present invention can be immunized by any existing route, including dermal layer or skin administration, intramuscular administration and the like. Wherein, the administered amount can be determined by those skilled in the art according to common sense.

Definition of Terms

Nuclear VP8: It is a polypeptide chain in the rotavirus protein VP8 that has the function of binding with the cell surface containing sialic acid, usually containing 160 amino acid residues.

VP8 polypeptide chain fragment: any polypeptide chain whose molecular weight is lower than the full-length VP8.

VP4 polypeptide chain fragment: any polypeptide chain whose molecular weight is lower than the full-length VP4.

VP8-specific antigenic cluster chain: The full VP8 polypeptide chain contains multiple antigenic determinants, which are cleaved by genetic recombination to remove important amino acids, while retaining the polypeptide chain containing specific antigenic clusters, the molecular weight is usually smaller than the full-length VP8 polypeptide chain.

VP4-specific antigenic cluster chain: The full VP4 polypeptide chain contains multiple antigenic determinants, which are cleaved without important amino acids by genetic recombination, while the polypeptide chain containing specific antigenic clusters is retained, and the molecular weight is usually smaller than the full-length VP4 polypeptide. chain.

VP8 fusion protein: by gene recombination, VP8 protein and other soluble protein polypeptide chains are fused and expressed to improve the solubility of VP8 in aqueous solution; or to enhance the immunogenicity of VP8.

NSP4 protein: It is a non-structural protein with enterotoxin properties, with a molecular weight of 28kDa and 175 amino acids.

VP8-NSP4 fusion protein: The VP8 and NSP4 polypeptide chains are fused and expressed by gene recombination to improve the solubility of VP8 in aqueous solution, and at the same time, the fusion protein can stimulate the body to produce protective antibodies against NSP4.

Capsular polysaccharide fragment: obtained by depolymerization of purified polysaccharide (called whole polysaccharide or original polysaccharide) in bacterial culture solution by physical (such as ultrasonic wave, microparticle spray), chemical (such as acid, alkali, enzymatic digestion) and other methods Fragments of polysaccharides, usually lower in molecular weight than the original polysaccharide. Capsular oligosaccharide: obtained by depolymerization of purified polysaccharide (called whole polysaccharide or original polysaccharide) in bacterial culture solution by physical (such as ultrasonic wave, microparticle spray), chemical (such as acid, alkali, enzymatic digestion) and other methods The polysaccharide fragment of the molecular structure is usually less than 10 monosaccharide residues. However, there are differences in the definition of the number of monosaccharide residues, and some literatures refer to polysaccharide chains with more than 10 and less than 20 monosaccharide residues as oligosaccharides.

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

1. The conjugated vaccine is an immunoconjugate containing two or more different carrier proteins. Compared with the existing pneumococcal conjugate vaccine, its immunogenicity is stronger, and the induced polysaccharide antibody level is higher than that of single Carrier conjugates, which can elicit immune responses in a wider population, especially infants and young children; can avoid carrier epitope overload by reducing the dose of each carrier; can enhance helper T cell activity by both carriers;

2. Since the protective protein epitopes in the two carrier proteins will also induce a higher immune response than when the two proteins are injected together, they synergize with each other, further enhancing the immunogenicity of the carrier protein and increasing the body's ability to respond. Immune response to polysaccharides;

3. The use of magnetic nano-microspheres as the linker between polysaccharide and multi-carrier protein, which is pioneered in the present invention, can effectively avoid the self-coupling between capsular polysaccharide and protein during the preparation process, can improve the yield of the combined product, and is beneficial to the product. It can effectively extend the spatial distance between the capsular polysaccharide and the carrier protein, reduce the space shielding effect of the carrier protein on the epitope of the capsular polysaccharide, and help improve the immunogenicity of the capsular polysaccharide;

4. In the preparation process of the conjugated vaccine, the first -OH on the surface of the magnetic nano-microspheres is firstly reacted with the polysaccharide, and then the unreacted -OH is modified into -CHO by chemical modification of the conjugated body. Coupling with -NH ₂ in the carrier protein is more stable than the binding method in the prior art;

5. The preparation method is simple, suitable for the needs of large-scale industrial production, and the structural characteristics of the capsular polysaccharide and the carrier protein are not significantly changed.

In summary, the preparation process of the conjugated vaccine provided by the present invention is simple, and the conjugated vaccine using magnetic nano-microspheres as the connector can enhance the Th1 immune response of mice, as well as the immune persistence, specificity and affinity of polysaccharide-specific antibodies. In addition, it can induce mice to produce rotavirus antibodies; it has the preventive effect of two vaccines; therefore, it has a very broad application prospect.

Description of drawings

Fig. 1 is the ¹ H-NMR spectrum of the conjugated vaccine prepared in Example 1 of the present invention;

Figure 2 is a schematic diagram of the results of an immune response experiment of the polysaccharide-specific antibody of the conjugated vaccine provided by the present invention;

FIG. 3 is a schematic diagram showing the results of the immunosustainability experiment of the polysaccharide-specific antibody of the conjugated vaccine provided by the present invention.

Detailed ways

The present invention will be further described in detail and completely below in conjunction with the examples. The reagents or equipment used in the following are all commercially available varieties. If there is no special instruction, they are operated in accordance with the instructions, and will not be repeated here.

The present invention is further described below with reference to specific embodiments, but should not be regarded as a limitation of the present invention.

Example 1

1. Preparation of magnetic nano-microspheres

1. Dissolve 2.24g FeSO ₄ -7H ₂ O and 3.24g FeCl ₃ -6H ₂ O in 10mL and 15mL of dddH ₂ O filtered and deoxygenated, respectively, and mix well after dissolving, and add 100 mL of dddH ₂ O filtered and deoxygenated;

2. Stir for 5 min under the protection of N ₂ , add 50 mL of 1mol/L NaOH solution at one time, adjust the pH of the solution to 9-10, speed up the stirring speed to 200-250 r/min, and continue to stir for 30 min;

3. Transfer the reaction vessel to a 65-70°C water bath, and continue to stir and age for 30min under the protection of N ₂ ;

4. After the reaction is completed, the volume is adjusted to 100 mL, and the synthesis of magnetic particles is observed under a microscope;

5. Dissolve 400mg PLGA in 10mL EA solvent as oil phase (O), add 3mL of the above magnetic particle solution as inner water phase (W ₁ ), use ultrasonic cell disruptor (120W, 60s) to prepare initial emulsification in ice water bath Colostrum, then poured the colostrum into a certain amount of aqueous solution (outer water phase, W ₂ ) containing 15g/LPVA and 0.9% (ω) NaCl, magnetic stirring (300r/min, 2min) to prepare pre-reconstituted emulsion (W ₁ / 0/W ₂ ), then pour the pre-recombination emulsion into the storage tank for rapid membrane emulsification, and repeatedly press it through the SPG membrane with a certain N ₂ pressure to obtain nano-microsphere re-emulsion droplets with uniform particle size. In addition, the unused nano-microspheres can be made into lyophilizer for use.

Or by taking Fe ₃ O ₄ magnetic particles and adding 50 mL and anhydrous ethanol in an equal volume, after ultrasonic activation for 30 min, put them in a 60 ℃ water bath, slowly add 10 mL PELA dropwise to the magnetic nano-microspheres to modify the -NH ₂ end and change the PELA was wrapped in Fe ₃ O ₄ magnetic particles, and the reaction was stirred for 10 h under nitrogen protection to prepare magnetic nano-microspheres; Make up to 50mL, observe the modification of the magnetic beads under a microscope, and change the -NH ₂ end to the -OH end on the surface of the magnetic nano-microsphere.

2. Preparation of pneumococcal polysaccharides

1. Select 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, 33F) pneumococcal culture;

2. Purify the capsular polysaccharides with strong antigenicity in the above various serotypes of pneumococcus: pneumococcus. After inactivation, the supernatant is collected by centrifugation, concentrated by ultrafiltration, and an appropriate amount (volume fraction) is added according to the characteristics of each pneumococcal serotype. 70%) pre-cooled ethanol, collected by centrifugation to obtain crude polysaccharide; dissolved crude polysaccharide in sodium acetate solution, then mixed with cold phenol at a ratio of 1:2, centrifuged to remove protein, and repeated phenol extraction for 5-6 Second, collect the supernatant, dialyze it with distilled water, add 2 mol/L calcium chloride solution to the liquid after dialysis, add ethanol and stir, centrifuge to remove nucleic acids, collect the supernatant, add ethanol (final concentration 80% stirring), and collect the precipitate by centrifugation. The precipitation was washed with ethanol and acetone, dehydrated and dried to obtain the polyvalent refined capsular polysaccharide, which was stored at -20°C for later use.

3. Preparation of rotavirus carrier protein

The preparation of rotavirus carrier protein can refer to the preparation methods of various recombinant proteins in the prior art. This embodiment adopts the method mentioned in CN 101972475 to prepare, which can be simplified into the following steps, and the specific parameters are not repeated:

1. Establishment of rotavirus cDNA library

Selected is the VP4 gene of the Wa strain VP8 protein, and the amplification primers are designed as follows: sense primer HWaVP4ρET28:

5'-TTACATATGGCTTCGCTCATTTATAG-3', anti-sense primer AHWaVP4ρET28:

5'-CCGGATCCCTAGTCTTCATTAACTTGTGCT-3'.

2. Construction of pET28aWaVP8 expression full-length VP8 plasmid

3. Expression of recombinant VP8 protein

1) Transform the prepared pET28aWaVP8 plasmid into BL21(DE3)competent cells, inoculate the cells into a 50 μg/mL kanamycin LB dish, and incubate overnight at 37°C in a CO ₂ incubator.

2) Pick out a colony and inoculate it into 10 ml of LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.5) containing 50 μg/mL kanamycin, and expand at 37°C Incubate overnight.

3) Inoculate the culture solution into 100 ml of 50 μg/mL kanamycin LB culture solution, and continue to culture. When the OD of the absorbance at 600nm reaches 1.0, transfer the culture solution to 6 liters of 50μg/mL kanamycin LB culture solution, and continue to cultivate in a shaker at 37°C with a shaking speed of 200rpm, until the OD of the absorbance at 600nm When reaching 0.6-0.8, 0.3 mM IPTG (isopropyl-β-D-thiogalactoside) was added to induce VP8 expression.

4) Under the same culture conditions, after 4 hours of induction, the cells were collected by centrifugation at 4000 g for 20 minutes at 10°C.

5) Suspend the bacteria in 20 mL of 1×PBS solution, disrupt the bacteria with a French press, centrifuge at 10,000 g for 30 minutes at 10° C., discard the supernatant, and collect the precipitated inclusion bodies. Inclusion bodies were stored at -40°C prior to further purification.

3. Purification of VP8 protein from inclusion bodies

1) Weigh 0.5 g (wet weight) of the prepared VP8 inclusion bodies, suspend the inclusion bodies with washing buffer (10 mM Tris, 100 mM phosphate buffer, 2 Murea, pH 8.0), incubate at room temperature for 30 minutes, and centrifuge at 10,000 g For 10 minutes, the inclusion bodies were collected. Repeat the above steps three times to remove contaminating impurities.

2) The inclusion body pellet was dissolved in lysis buffer (10 mM Tris-HCl, 100 mM phosphate buffer, 8 M urea, pH 8.0), and incubated on ice for 1 hour with stirring. Centrifuge at 16,000 g for 30 minutes, collect the supernatant, and discard the insoluble pellet.

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

4) Collect the eluate containing VP8, transfer it to a dialysis bag and dialyze it in TBS (pH 4.0) containing 20 mM β-mercaptoethanol 1 and 8 M urea, and gradually reduce the concentration of urea (ie, 8, 6, 4, 2). , and 1M), dialyzed overnight at 4°C. It was then dialyzed twice against TBS pH 5.5 solution containing 2 mM β-mercaptoethanol and finally against TBS solution. The final dialysis is performed depending on the strain from which the recombinant VP8 protein is derived.

4. Preparation of pneumococcal surface protein A (rPspA)

The PspA protein was cloned into E. coli for expression, isolation and purification, including:

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

The PspA gene sequence (GI: 193804931) was obtained from GenBank and optimized. After adding the His tag, the whole gene was synthesized. The synthesized sequence was double digested by SacⅠ and NdeⅠ, and then cloned into the expression vector pET with the same double digestion. -30a(+), transform competent Escherichia coli BL21Star (DE3), cultivate overnight at 37°C, pick positive monoclonal colonies, and extract plasmids after amplifying and culturing. The correctly sequenced recombinant expression plasmid was named pET-30a-rPspA.

2. Inducible expression and purification of recombinant protein

Resuscitate the engineered bacteria, inoculate 2×LB medium at a ratio of 1:100, and expand the culture at 37°C and 237r/min. When the bacterial cell value is about 12, add IPTG to a final concentration of 1mmol/L, and induce induction at 37°C. 4h, take samples for SDS-PAGE analysis and centrifuge to collect induced bacteria, add physiological saline to resuspend and wash twice, and add 05mol/LNaCl5mmol/L imidazole 20mmol/LPB (pH7.4) at a ratio of 1:10 (g/mL). The bacteria were resuspended in buffer solution, disrupted by ultrasonic wave, centrifuged at 8000 × g for 40 min, the supernatant was collected, and purified in a nickel ion chromatography column. Escherichia coli BL21Star (DE3) whole cells (control) and the purified samples in the previous step were separated by SDS-PAGE, and then electrotransferred to nitrocellulose membranes, and sealed with 5% nonfat milk powder shaker for 2 hours; Antibody (1:800 dilution), overnight at 4°C; washed 3 times with TBST, added HRP-labeled goat anti-mouse IgG (1:2000 dilution), incubated at room temperature for 1 h; washed 3 times, DAB developed color.

V. Preparation of Conjugate Vaccines

1. Polysaccharide-magnetic nanosphere coupling

Under 0.1MTBS solution buffer, pH6.0 adds any one or more capsular polysaccharides and magnetic nano-microspheres obtained by the above steps, and in the present embodiment, 13-valent capsular polysaccharides (1, 3, 5, 6A, 6B) are used. , 7F, 9V, 14, 18C, 19A, 19F and 23F), the mass ratio of capsular polysaccharide and magnetic nano-microspheres is 1:(0.5-1), and the reaction is carried out at room temperature for 6-24 hours; wherein the optimized mass ratio At 1:1, the acetal was reacted at room temperature for 16 hours. After the reaction, fully dialyzed with a dialysis bag to remove unreacted magnetic nanospheres.

2. Conjugate modification

Take 30 mL of the polysaccharide-magnetic nano-microsphere conjugate in step 1, slowly add 2 mL of 25% glutaric acid dropwise under stirring conditions, and stir at 200 r/min for 6 hours; The modification of the magnetic nano-microspheres was observed under a microscope, so that the -OH that was not reacted with the polysaccharide on the surface of the polysaccharide-magnetic nano-microsphere conjugate was modified to -CHO; N ₂ , 4°C was stored for later use.

3. Coconjugate with PspA carrier protein and rotavirus protein

Under 0.1MTBS solution buffer, 4°C, pH 4.0-9.0, the modified polysaccharide-magnetic nanospheres were reacted with rotavirus protein and PspA protein for 24 hours (in order to ensure a sufficient amount of carrier protein and PspA protein). For magnetic nano-microspheres, an excess amount of nanocarrier protein is used, such as the mass ratio of polysaccharide: magnetic nano-microspheres: PspA: rotavirus protein = 1:1:2:2).

4. Isolation and purification of conjugated vaccines

Use Superdex200 gel filtration column (2.6cm×60cm) for separation and purification, the eluent is 20mM phosphate buffer (pH7.4), and the flow rate is 3mL/min. The elution peaks corresponding to the polysaccharide-magnetic nanosphere-double carrier protein were collected.

Compositional analysis of the prepared conjugate vaccine

The conjugated vaccine was detected by ¹ H-NMR, and the detection results are shown in Figure 1 . As shown in Figure 1, compared with the capsular polysaccharide molecules, the conjugates exhibited characteristic peaks at 0.4-1.4 ppm, corresponding to the aliphatic chain amino acid residues of the carrier protein. A characteristic peak corresponding to the aromatic amino acid residues of the carrier protein appeared at 7.2 ppm. This indicated that the capsular polysaccharide molecule was successfully coupled to the two carrier proteins, the rotavirus protein and the PspA carrier protein. A characteristic peak corresponding to succinimide appeared at 6.2 ppm, which indicated that succinimide was contained in the linking bridge of the polysaccharide conjugate vaccine. In addition, the characteristic peaks of PELA appeared in 5.2, 1.6, and 3.6, which proved that PELA of magnetic nanospheres was used as a linker. Therefore, the structure of the capsular polysaccharide did not change significantly before and after binding to the two carrier proteins.

The molecular weight distribution of the polysaccharide-protein conjugates was detected by CL-4B (SEC-MALLS) method; the protein and polysaccharide species in the polysaccharide-protein conjugates were determined by the method of double immunostaining using different antibody sera; the polysaccharide was detected by the anthrone method The polysaccharide content of the protein conjugate; the protein content of the Lowry method detects the total protein content of the polysaccharide-protein conjugate, and then calculates the polysaccharide-protein binding ratio (Ratio) of the conjugate; Immunoassay detection. The results show that: in the conjugated vaccine stock solution, the concentration ratio of each substance is about: polysaccharide: magnetic nanosphere: PspA: rotavirus protein = 1:1:1:1).

Comparative Example 1

The only difference between this comparative example and Example 1 is that the prepared vaccine has no magnetic nanoparticles as linkers, and is obtained by conjugating dual carrier protein and polysaccharide by the method mentioned in the prior art.

Comparative Example 2

The only difference between this comparative example and Example 1 is that there is no PspA carrier protein in the prepared vaccine, and only polysaccharide-magnetic nanoparticles-rotavirus carrier protein is used as the conjugated vaccine.

Example 2

The only difference between this example and Example 1 is that the carrier proteins of the conjugated vaccine are tetanus toxoid carrier protein and PspA.

The composition analysis results of the prepared conjugated vaccines were consistent with the results obtained in Example 1 within the theoretical error range.

Example 3

The only difference between this example and Example 1 is that the carrier proteins of the conjugated vaccine are rotavirus carrier protein and tetanus toxoid carrier protein.

The composition analysis results of the prepared conjugated vaccines were consistent with the results obtained in Example 1 within the theoretical error range.

Example 4

The only difference between this example and Example 1 is that the carrier proteins of the conjugated vaccine are rotavirus carrier protein and Haemophilus influenzae surface protein HiD.

The composition analysis results of the prepared conjugated vaccines were consistent with the results obtained in Example 1 within the theoretical error range.

Example 5

The only difference between this example and Example 1 is that the carrier proteins of the conjugated vaccine are carrier proteins CRM197 and PspA. The composition analysis results of the prepared conjugated vaccines were consistent with the results obtained in Example 1 within the theoretical error range.

Example 6

The only difference between this example and Example 1 is that the connector is PLGA magnetic nanoparticles.

The composition analysis results of the prepared conjugated vaccines were consistent with the results obtained in Example 1 within the theoretical error range.

Example 7

The only difference between this example and Example 1 is that the linker is PEG magnetic nanoparticles.

The composition analysis results of the prepared conjugated vaccines were consistent with the results obtained in Example 1 within the theoretical error range.

Vaccine Evaluation

The immune effects of the vaccines obtained in Examples 1 to 7 and Comparative Examples 1 to 2 were evaluated by the evaluation experiments of the conventional titers of vaccines such as immunogenicity:

A. Conjugate vaccine immunogenicity experiments

100 5-week-old female Blab/C mice with a body weight of 15-22 g were selected. Randomly divided into 10 groups, namely Examples 1-7, Comparative Examples 1-2 and positive control group (Prevnar 13), with 10 mice in each group. Intraperitoneal injection, each injection of 5 micrograms, once a week, a total of 3 injections. Orbital blood was collected after 21 days. The anti-capsular polysaccharide IgG, IgG1 and IgG2a in mouse plasma were detected by ELISA. The experimental results are shown in Table 1

Table 1

As shown in Table 1, the conjugated vaccines obtained in Examples 1 to 7 can significantly increase the antibody titer (p<0.0001**), as shown in Figure 2, and are significantly better than Comparative Example 1 and Comparative Example 2 and positive Control group; it can be seen that the conjugated vaccine obtained by using dual carrier protein and magnetic nano-microspheres and pneumococcal polysaccharide can significantly enhance the Th1 immune response, and the immune effect is significantly better than that without nano-magnetic microspheres in Comparative Example 1. However, according to the immune response results obtained in Examples 1 to 7, it can be seen that the immune response effect of PspA or HiD as one of the dual carrier proteins is better, and the magnetic PELA and PLGA in nanocarriers had better immune response than PEG.

B. Immune persistence, specificity and affinity experiments of polysaccharide-specific antibodies

The following experiments were carried out with the conjugated vaccines obtained in Example 1, Comparative Examples 1 and 2, and the positive control, respectively.

1. Immunity persistence

The immune persistence of polysaccharide-specific antibodies was investigated by measuring the titer of polysaccharide-specific antibodies within 20 weeks after three-needle immunization. Figure 3 is a schematic diagram of the immune persistence. As shown in Figure 3, the titer of polysaccharide-specific IgG was low, gradually decreased with the increase of injection time, and could not be detected after the 4th week. The polysaccharide-specific IgG titers produced by Comparative Examples 1 and 2 were lower than those of the Example 1 group, but higher than that of the positive control group. The titer of polysaccharide-specific IgG peaked in the 2nd week and gradually decreased in the 4th to 20th week, and the decline rate of the positive control group was the fastest. After the 18th week, the polysaccharide-specific IgG titers of the Example 1 group, Comparative Example 1, Comparative Example 2 and the positive control group were 20%, 15%, 12% and 10% of their peak values, respectively. Therefore, the conjugated vaccine provided by the present invention can enhance the immune persistence of polysaccharide-specific antibodies.

2. Specificity and Affinity

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

Table 2

As shown in Table 2, with the increase of the amount of polysaccharide added, the ability of polysaccharide-specific antibodies to bind to polysaccharides in the 96-well plate gradually decreased. When the added polysaccharide reached 20 μg, the ability of the antibody to bind the polysaccharide was lost. This indicates that the anti-capsular polysaccharide antibody induced by the conjugated vaccine provided by the present invention can specifically bind to the capsular polysaccharide.

Antibody affinity to capsular polysaccharide was determined by the 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 2.65, respectively mol/L, 2.80 mol/L, 3.02 mol/L and 3.21 mol/L. This shows that the conjugated vaccine provided by the present invention can significantly improve the affinity of polysaccharide-specific antibodies.

C. Rotavirus neutralization test results

10 μL of each mouse serum from each group of mouse serum prepared by the method in Example 1 (one injection, two injections and three injections were injected in Example 1, Comparative Example 1 and Comparative Example 2, respectively) were mixed , used to neutralize test sample serum.

Neutralization assays by injection of anti-WaVP8 antibodies produced in mice were performed on microplates with BSC-1 cells. The heat-inactivated serum was serially diluted, mixed with a Wa rotavirus strain of 100 TCID50, and incubated at 4°C 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°C for 1 hour. Finally, the concentration at which the diluted antiserum prevents rotavirus cytopathic effect (CPE) is the neutralizing titer. The polysaccharide control serum was used as a negative control. The experimental results are shown in Table 3.

Table 3: Results of antibody neutralization of rotavirus Wa strain in mice injected with conjugate vaccine

As shown in Table 3, the effect of the conjugate vaccine in neutralizing rotavirus is significantly better than that of Comparative Example 1 and Comparative Example 2, and can significantly induce the production of rotavirus antibodies.

Finally, it is necessary to explain here: the above embodiments are only used to further describe the technical solutions of the present invention in detail, and should not be construed as limiting the protection scope of the present invention. Non-essential improvements and adjustments belong to the protection scope of the present invention.

Claims (25)

1. A conjugated vaccine, characterized in that: the conjugated vaccine is a conjugated vaccine formed by a polyvalent pneumococcal polysaccharide and two carrier proteins through a linker, wherein the linker is a magnetic nano-microsphere, and the The magnetic particles of the magnetic nano-microsphere are located inside the nano-microsphere as the core, and the polymer material is wrapped around the outside of the magnetic particles, the polymer material is polyethylene glycol, the magnetic particles are Fe ₃ O ₄ , and the magnetic particles are The diameter is 0.1-10 μm; the mass ratio of the polyvalent pneumococcal polysaccharide to the two carrier proteins is (0.5-1): 1, wherein the mass ratio between the carrier proteins is 1: 1, and one of the carrier proteins is the P gene Type rotavirus protein, another is Haemophilus influenzae surface protein HiD or pneumococcal surface protein A. 2 . The conjugated vaccine according to claim 1 , wherein the particle size of the magnetic nano-microspheres is 0.1-5 μm. 3 . 3. The conjugate vaccine according to claim 1, wherein the polyvalent pneumococcal polysaccharide is the capsular polysaccharide on the capsules of various isolated and purified serotypes of pneumococcus, and the serotypes of the serotypes of pneumococcus include 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. 4. The conjugated vaccine according to claim 1, wherein the polyvalent pneumococcal polysaccharide is a variety of pneumococcal capsular polysaccharides. 5. The conjugate vaccine according to claim 1, wherein the virus strain of recombinant human rotavirus protein is P genotype rotavirus strain P[8], P[4], P[6] or One of P[11]. 6. The conjugate vaccine according to claim 1, wherein the virus strain of recombinant human rotavirus protein is selected from the group consisting of P[8]G1, P[4]G2, P[8]G3, P[8 ]G4, P[8]G9, P[8]G5, or P[6]G8. 7. The conjugate vaccine according to claim 1, wherein the virus strain of recombinant human rotavirus protein is selected from Wa, Ku, P, YO, MO, VA70, D, AU32, CH-32, CH One of the -55, CHW2, CH927A, W161, F45, Ai-75, Hochi, Hosokawa, BR1054, WT78 or WI79 strains. 8. The conjugate vaccine according to claim 1, wherein the virus strain of recombinant human rotavirus protein is selected from DS-1, RV-5, S2, L26, KUN, E210, CHW17, AU64, 107E18 , MW333 or one of the TB-Chen strains. 9. The conjugate vaccine according to claim 1, wherein the virus strain of recombinant human rotavirus protein is selected from M37, 1076, RV-3, ST3, SC2, BrB, McN13, US1205, MW023, US585 or one of the AU19 strains. 10. The conjugate vaccine according to claim 1, wherein the recombinant human rotavirus protein is selected from one of VP8, VP4 or nuclear VP8. 11. The conjugate vaccine of claim 1, wherein the recombinant human rotavirus protein is the VP7 protein of serotype G rotavirus. 12. The conjugate vaccine according to claim 1, wherein the serotype G rotavirus strain of the recombinant human rotavirus is selected from G1, G2, G3, G4, G9, G5, G8, G10 or G11 one of the serotypes. 13. The conjugate vaccine according to claim 1, wherein the serotype G rotavirus strain of the recombinant human rotavirus is selected from the group consisting of P[8]G1, P[4]G2, and P[8]G3 , P[8]G4, P[8]G9, P[8]G5, or P[6]G8 serotype. 14. The method for preparing a conjugated vaccine according to any one of claims 1 to 13, characterized in that: the polyvalent pneumococcal polysaccharide and two or more carrier proteins are respectively coupled with nanospheres. 15. The method for preparing a conjugated vaccine according to claim 14, characterized in that it specifically comprises the following steps: a) Separate and purify the capsular polysaccharides on the capsules of various serotypes of pneumococcus respectively; b) respectively preparing and separating and purifying the selected rotavirus carrier protein and another carrier protein; c) Respectively coupling various capsular polysaccharides and nano-microspheres into polysaccharide-nano-microsphere conjugates; d) coupling and combining the polysaccharide-nano-microsphere conjugates with the rotavirus carrier protein and another carrier protein respectively; e) Separation and purification of the conjugate obtained in step d) into the conjugate vaccine stock solution. 16. The method for preparing a conjugated vaccine according to claim 14, characterized in that it specifically comprises the following steps: a) Separate and purify the capsular polysaccharides on the capsules of various serotypes of pneumococcus respectively; b) Separate and purify the selected multiple carrier proteins respectively; c) Respectively coupling various capsular polysaccharides and magnetic nano-microspheres into polysaccharide-magnetic nano-microsphere conjugates, and then chemically modifying them, so that the surface of the polysaccharide-magnetic nano-microsphere conjugates does not react with polysaccharides The -OH is modified to -CHO; d) Conjugate the polysaccharide-magnetic nano-microsphere conjugates with various carrier proteins respectively; e) Separation and purification of the conjugate obtained in step d) into the conjugate vaccine stock solution. 17. The method for preparing a conjugated vaccine according to claim 14, further comprising: preparing magnetic nano-microspheres, including the process of first preparing nano-magnetic particles, and then preparing magnetic nano-microspheres; wherein the nano-magnetic particles can pass through It can be prepared by chemical co-precipitation method, hydrothermal method or sol pyrolysis method, and magnetic nano-microspheres can be prepared by embedding method, monomer polymerization method or in situ method. 18. The method for preparing a conjugated vaccine according to claim 17, wherein the magnetic nanoparticle is prepared by chemical co-precipitation, and then the magnetic nanoparticle and the polymer material are subjected to rapid membrane emulsification combined with a solvent extraction method and/or Magnetic nano-microspheres were prepared by embedding method or monomer polymerization method. 19. The method for preparing a conjugated vaccine according to claim 18, characterized in that: using chemical co-precipitation method to prepare nano-magnetic particles, and then subjecting the nano-magnetic particles and polymer materials to rapid membrane emulsification combined with solvent extraction method and/or Magnetic nano-microspheres with uniform particle size were prepared by embedding method. 20 . The method for preparing a conjugated vaccine according to claim 14 , wherein the magnetic particles are Fe ₃ O ₄ . 21 . 21. The method for preparing a conjugated vaccine according to claim 16, wherein the specific operation of step c is as follows: in a coupling medium reaction buffer, at room temperature and at pH 4.0-9.0, the resulting The polysaccharide and magnetic nano-microspheres reacted together for 6-24 hours, and then the polysaccharide-magnetic nano-microsphere conjugate was further modified by 25% glutaraldehyde, so that the surface of the polysaccharide-magnetic nano-microsphere conjugate did not interact with The -OH of the polysaccharide reaction is modified to -CHO. 22. The method for preparing a conjugated vaccine according to claim 21, wherein the reaction time is 12-16 hours. 23. The method for preparing a conjugated vaccine according to claim 16, wherein the specific operation of step d is: in the coupling medium reaction buffer, at 4°C and pH 4.0-9.0, the resultant obtained in step c The chemically modified polysaccharide-magnetic nano-microsphere conjugates were co-reacted with carrier protein for 12-24 hours. 24. The method for preparing a conjugated vaccine according to claim 23, wherein the reaction time is 24 hours. 25. The method for preparing a conjugated vaccine according to claim 21 or 23, wherein the coupling medium is PB, PBS or TBS.

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