CN104096223B

CN104096223B - Method for enhancing immunogenicity of 13-valent pneumococcal polysaccharide protein conjugate

Info

Publication number: CN104096223B
Application number: CN201410198752.3A
Authority: CN
Inventors: 李建平
Original assignee: Kanvax Biopharmaceuticals Ltd
Current assignee: Kanvax Biopharmaceuticals Ltd
Priority date: 2014-05-11
Filing date: 2014-05-11
Publication date: 2020-06-19
Anticipated expiration: 2034-05-11
Also published as: CN104096223A

Abstract

The invention discloses a method for enhancing the immunogenicity of a 13-valent pneumococcal polysaccharide protein conjugate, which comprises the steps of adding a universal epitope peptide P30 into CRM197A, and producing P30CRM197A containing the universal epitope peptide by adopting gene recombinant escherichia coli; 13 different serotypes (including 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of polysaccharide were then covalently linked to P30CRM197A protein carriers to form 13 valent pneumococcal polysaccharide-P30 CRM197A conjugates; compared with the 13-valent pneumococcal polysaccharide-P30 CRM197A conjugate obtained by the method and the 13-valent pneumococcal polysaccharide-CRM 197A conjugate obtained by the corresponding protein carrier CRM197A which does not contain the universal epitope peptide, the immunogen of the conjugate is improved by 3-5 times compared with that of a control.

Description

Method for enhancing immunogenicity of 13-valent pneumococcal polysaccharide protein conjugate

Technical Field

The invention relates to a method for enhancing the immunogenicity of a 13-valent pneumococcal polysaccharide protein conjugate.

Background

When the polysaccharide is covalently linked to a protein carrier, the polysaccharide of the hapten can be converted to a whole antigen, resulting in enhanced immunogenicity of the polysaccharide. The polysaccharide-protein combination vaccine synthesized by the method is widely applied to children, and successfully prevents the infection of bacteria such as pneumococcus, epidemic meningococcus, and b-type haemophilus epidemicus.

The protein carriers used for synthesizing polysaccharide protein conjugates are various, such as tetanus toxoid, diphtheria toxin variant CRM197, and hemophilus epidemic surface protein D produced by gene recombination technology; however, due to the immune characteristics of different proteins, the immunogenicity of polysaccharide protein conjugates synthesized with different protein carriers is different, and after the animal is immunized, the immunogenicity of polysaccharides shown by polysaccharide protein conjugates synthesized by different protein carriers and the same polysaccharide is also different. Therefore, the immunogenicity of the polysaccharide protein conjugate synthesized by adopting protein carriers produced by different technologies is different.

After entering the animal body, the Antigen is treated by Antigen Processing Cell (APC) to generate epitope peptide (1), which is then presented on the surface of APC after being combined with Major Histocompatibility Complex (MHC) molecules, and can be recognized by T lymphocyte, which is the conclusion that immunology has been made through experiments. It is now known that the immunogenicity of a known epitope peptide depends on three factors:

generation of a first, appropriate epitope peptide;

second, presentation of major histocompatibility complex molecules bound to epitope peptides;

third, presentation of T cells recognizing conjugates of epitope peptides with major histocompatibility complexes.

Wherein the absence of either link results in a loss of immune response.

Experiments with mice have shown that the lack of a suitable epitope peptide in combination with a major histocompatibility complex forms a conjugate molecule that is most often responsible for the lack of an immune response from the animal. Major histocompatibility complex is highly polymorphic, and known epitope peptides bind to major histocompatibility complex only through one or a few Alleles (Alleles), rather than all epitope peptide fragments. In addition, experimental results have shown that inadequate antigen processing, or lack of T cell tolerance, can also lead to a loss of immune response.

It has been shown experimentally that tetanus toxin epitope peptide FNNFTVSFWLRVPKVSASHLE (designated P30) [2], which binds to a large number of different major histocompatibility complex Class II, has been shown to be recognized by T cells and possesses universal immunogenicity properties, and is called universal epitope peptide.

The universal immunogenic epitope peptide has the advantages of combining with homotype and allotype of a plurality of human major histocompatibility antigen complex Class II molecules. This random combination of universal epitope peptides and human major histocompatibility antigen complex Class II molecules is useful for the development of synthetic vaccines because it can activate the response of the adaptive immune system of a large proportion of individuals in the human population.

The P30 epitope peptide was shown to consist of the 947-967 amino acid sequence of tetanus toxin (FNNFTVSFWLRVPKVSASHLE). After the protein containing the epitope peptide enters the body, the protein is taken into APC cells and digested and degraded. However, these epitope peptides will remain intact, be presented on the APC cell surface after binding to the major histocompatibility complex, and be recognized by T cells, suggesting that the universal epitope peptides act in the same way with a variety of DR molecules.

MHC molecules are heterogeneous receptors for processed antigens and function to present epitope peptides in their antigens during induction of immune tolerance and peripheral immune response to foreign antigens in the thymus. Thus, MHC molecules must strike a balance between presenting a large amount of epitope peptide (allowing better antigen recognition, but more depleting T cell reserves), and a few epitope peptides (large T cell reserves, but little effective in presenting foreign antigens). That is, if the antigen contains a small amount of epitope peptide that is capable of preserving the integrity and is representative of the antigen after APC cell treatment, it can stimulate a certain amount of T cells to establish an immunological memory effect after MHC binding, and this process does not consume excessive T cells to avoid consuming a large amount of T cells and causing immune tolerance. Antigens containing such epitope peptides are more immunogenic, which explains why tetanus toxoid is very immunogenic.

Most of the T cell stimulatory epitope peptides found have limited effect on different MHC Class II monomers (haplotype), and different animals store and present different regions of antigenic polypeptides (epitopes) to stimulate their T cells. This genetic limitation of T cell stimulatory activity has prevented the development of vaccines by synthetic methods, which should be very effective for use by genetically diverse populations. Those found to have T cell stimulatory epitope peptides that stimulate multiple mouse individuals and/or are associated with most human MHCClass II molecules provide an efficient way to design universal activated T cells. The addition of a universal epitope peptide (also known as universal T cell antigen cluster) P30 to a common protein antigen can be recognized by MHC Class II molecules of most animals. Such T cell antigen clusters can be used to directly induce T cells, or to provide the effect of assisting B cells in producing antibodies against weak immunogens, enhancing the body's acquired immune response.

Pathogenic bacteria are usually capable of expressing high molecular weight and coated on the surface of the bacteria with capsular polysaccharide, polysaccharide for short. For adults, capsular polysaccharide is an antigen with good immunogenicity, and can be used for preparing vaccines; however, for children, i.e. infants under the age of 2 years, capsular polysaccharides are considered to be independent of T cell antigens. Experiments show that when used as an antigen, capsular polysaccharide can induce response of wild strain or T cell-deficient mice to produce polysaccharide-specific IgM antibodies; however, the conversion of IgM antibodies into IgG antibodies was not induced. Human experiments also show that, as a vaccine, polysaccharides can induce the production of protective antibodies by adults, but cannot induce immune responses in infants; that is, after repeated immunizations of the capsular polysaccharide antigen in children, there was no secondary antibody-enhanced response and no induction of sustained T cell memory.

Modern immunological experiments prove that compared with polysaccharide protein conjugate vaccines and pure polysaccharide vaccines, the polysaccharide protein conjugate vaccines have the advantage of inducing immune response. When a T cell independent capsular polysaccharide is covalently linked to a carrier protein to form a polysaccharide protein conjugate, upon immunization of a mammal, T cells can be induced to help B cells produce IgG antibodies to the polysaccharide moiety in the conjugate. Thus, the polysaccharide protein conjugate induces the conversion of polysaccharide specific antibodies IgM to IgG, differentiation of memory B cells, and long-term T cell memory.

Pneumococcus, also known as streptococcus pneumoniae, is one of the main pathogens causing human morbidity and mortality worldwide, and is especially easy to cause diseases such as bacterial pneumonia, meningitis, bacteremia and acute otitis media in infants, chronic cardiopulmonary disease patients, old people and immunocompromised people. It is estimated by the World Health Organization (WHO) epidemiological survey report in 1999 that at least one million infants and children under the age of 5 years die each year from various diseases caused by pneumococci. According to the report of industrially developed countries, the incidence of invasive pneumococcal pneumonia in infants under 2 years old is up to 160 cases per hundred thousand; in the European and American countries, 25% -40% of bacterial meningitis is caused by pneumococci. About 15 to 57 million cases of pneumococcal pneumonia and 2600 to 6200 cases of pneumococcal meningitis occur in the United states each year, and the two cases cause 4 million deaths each year; the incidence rate of pneumococcal bacteremia is about 15/10-19/10 thousands, and the fatality rate is about 25-30%. In addition, 50-67% of the bacterial otitis media are caused by pneumococcus, so that the bacterial otitis media are difficult to cure radically, have high recurrence rate and influence the healthy growth and development of children. According to statistics, the number of infants in the United states is up to seven million people for hospital visits due to otitis media, and a heavy burden is brought to a medical system. Thus, when the american hui pharmaceutical development succeeded in the heptavalent pneumococcal polysaccharide conjugate vaccine and received the us FDA production and sales license, the infectious disease council of the american pediatric society and the american immune test council immediately suggested vaccination in infants under 2 years of age and immunocompromised children 2-4 years of age in the early 2000.

Epidemiological investigations of various pneumococci have shown that the circulating strains, i.e. serotypes, of pneumococci vary from country to country and from region to region. Taking the 7-valent pneumococcal polysaccharide conjugate vaccine of Huishi company in America as an example, the coverage rate of the vaccine is 80-90% in North America, about 70-80% in Europe, and only 40-50% in Asia. The reason for this is that the distribution of pneumococcal serotypes is relatively narrow in developed countries, while the distribution of pneumococcal serotypes is relatively broad in underdeveloped countries. This is the fundamental reason why the 7-valent pneumococcal polysaccharide conjugate vaccine of hui corporation of usa cannot be popularized and applied in asia regions. According to epidemiological survey reports of pneumococcus in children aged 0-5 years, especially infants aged 2 years and younger in 5 years in China and southeast Asia, 13 serotypes were found to cover substantially 80-90% of epidemic strains, including 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Accordingly, 13-valent pneumococcal polysaccharide conjugate vaccine was developed by fevery in the united states, and 10-valent pneumococcal polysaccharide conjugate vaccine was developed by GSK in europe.

The pneumococcal polysaccharide conjugate vaccine plays a great role in preventing serious infectious diseases such as pneumonia, meningitis, otitis media and the like caused by pneumococcal infection in children. However, the existing pneumococcal polysaccharide protein conjugate vaccines in the market have large immunogenic variability, and the variability is caused by the structural difference of different serotype polysaccharides, and besides, the use of different immunogenic protein carriers is also the main reason for the immunogenic difference of 13-valent pneumococcal polysaccharide protein conjugates. In some high risk groups, such as children, the elderly or people with low immune function, the polysaccharide-protein conjugate vaccine with low immunogenicity has poor immune effect and limited protection. Therefore, the development of more immunogenic pneumococcal polysaccharide protein conjugate vaccines remains the subject of much effort in this field.

Disclosure of Invention

The invention aims to provide a method for enhancing the immunogenicity of a 13-valent pneumococcal polysaccharide protein conjugate, which adopts a protein carrier P30CRM197A containing a universal epitope peptide produced by a gene recombination technology to introduce the universal epitope peptide P30 into the polysaccharide protein conjugate. After the polysaccharide protein conjugate enters an animal body and is degraded by phagocytic digestion of Antigen Presenting Cells (APC), the combination of the universal epitope peptide P30, a part of pneumococcal polysaccharide repeating units and a main histocompatibility complex ClassII can effectively stimulate T cells, so that the immunogenicity of pneumococcal polysaccharide in the conjugate is enhanced, and the concentration of antibodies aiming at pneumococcal capsular polysaccharide is increased.

The technical scheme adopted by the invention is as follows:

a method of enhancing the immunogenicity of a 13-valent pneumococcal polysaccharide protein conjugate, comprising:

the method comprises the following steps: adding universal epitope peptide FNNFTVSFWLRVPKVSASHLE (P30) into the A chain (CRM 197A) of diphtheria toxin variant CRM197, and producing CRM197A protein carrier (P30 CRM197A) containing P30 by means of gene recombination engineering bacteria;

step two: 13 different serotypes of pneumococcal capsular polysaccharides, including 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, are respectively connected with a P30CRM197A protein carrier through covalent bonds to form 13 monovalent pneumococcal polysaccharide-P30 CRM197A conjugates;

step three: and (3) mixing the 13 monovalent pneumococcal polysaccharide-P30 CRM197A conjugates obtained in the step two to obtain 13 monovalent pneumococcal polysaccharide protein conjugates with enhanced immunogenicity.

Furthermore, the P30CRM197A protein carrier contains X P30, and X is more than or equal to 1 and less than or equal to 3.

Further, in the P30CRM197A protein carrier, P30 is linked at the N-terminus or C-terminus of CRM197A protein, or at both N-terminus and C-terminus.

Further, the P30 and the CRM197A protein are connected through the GSGSG amino acid sequence.

Furthermore, the gene recombination engineering bacteria are escherichia coli constructed by a gene recombination method.

Further, the pneumonia capsular polysaccharide is capsular polysaccharide obtained by culturing 13 different serotypes of pneumococcus respectively.

Detailed Description

The following examples illustrate specific embodiments of the present invention, but are not limited to the following examples.

First, preparation of Carrier protein and pneumococcal polysaccharide

To illustrate the effectiveness of the present invention, two carrier proteins were prepared, namely the protein carrier P30CRM197A containing P30 and the protein carrier CRM197A without P30. Wherein, the CRM197A protein carrier is used for synthesizing a 13-valent pneumococcal polysaccharide-CRM 197A conjugate sample for control.

Design of amino acid sequences of CRM197A protein carrier and P30CRM197A protein carrier

1. Design of CRM197A protein carrier amino acid sequence

Diphtheria toxin is expressed in diphtheria bacillus by β bacteriophage with diphtheria toxin gene, exists in bacterial cytoplasm in the form of polypeptide, consists of 560 amino acids, and has molecular weight of 62,000 dalton, and the amino acid sequence is as follows:

MSRKLFASILIGALLGIGAPPSAHAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQG NYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTE EFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

before secretion into the bacterial body, 25 leading amino acid sequences at the N-terminal end of the polypeptide are cut off, and the polypeptide is changed into a single-chain polypeptide which consists of 535 amino acids and has a molecular weight of 58kD and is secreted into the bacterial body, and the amino acid sequences are as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDN ENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSS VEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

diphtheria toxin secreted outside the bacterial body is cleaved into the a and B chains, which are linked by a disulfide bond to form a protein molecule. The two polypeptide chains have different functions, and the A chain is an N-terminal fragment of the diphtheria toxin protein molecule, has a molecular weight of 21kD, and consists of 193 amino acids. It is a toxic functional part of diphtheria toxin, produced by the isocitrate dehydrogenase (NAD) of adenosine triphosphate ribose (ADP-Ribosyl) in eukaryotic cytoplasm⁺) Partial transfer to Elongation Factor 2 (EF-2) to inhibitProtein synthesis in the cell, which in turn inhibits cell growth, causes cell death. The B chain is a C-terminal fragment of the diphtheria toxin protein molecule, has a molecular weight of about 37kD, and consists of 342 amino acids. The function of the B chain is to recognize specific receptors on the surface of sensitive cells, adsorbing diphtheria toxin to sensitive cells, and facilitating the entry of the a chain into the cell.

The A chain of diphtheria toxin has good water solubility, and the amino acid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDN ENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSS VEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

the research finds [3] that the toxin synthesized by β phage has no influence on the replication of phage due to the mutation of toxin gene tox, but the toxicity of the synthesized toxin may disappear or be greatly reduced to form diphtheria toxin mutant (CRM), and the serological immunogenicity of diphtheria toxin mutant is still associated with toxin.

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDN ENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSS VEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

Experimental studies show that compared with other protein carriers currently used in the market for synthesizing pneumococcal conjugate vaccines, the protein A chain polypeptide of CRM197 of diphtheria toxin variant has the advantages of immunogenicity, correlation with diphtheria toxin and diphtheria endotoxin, small molecular weight, high water solubility, easy production and macromolecular synthesis reaction. The long-term clinical use of diphtheria toxoid vaccines has proven to be safe and effective.

2. Design of P30CRM197A protein carrier amino acid sequence containing P30 universal epitope peptide

The universal epitope peptide P30 is connected to CRM197A protein carrier to constitute one new kind of protein carrier for synthesizing polysaccharide protein conjugate. The universal epitope peptide P30 can be connected to the N-terminal or C-terminal of the CRM197A protein carrier; two different universal epitope peptides can also be respectively connected to the N-terminal or the C-terminal of the CRM197A protein carrier; the other way is to connect two universal epitope peptides with each other and then connect the universal epitope peptides with the N-terminal or the C-terminal of the CRM197A protein carrier; yet another way is to attach one universal epitope peptide to the C-terminus or N-terminus and two self-attaching universal epitope peptides to the other end.

2-1, design of P30CRM197A protein carrier amino acid sequence containing universal epitope peptide P30

Design of 2-1-1, P30-N-terminal CRM197A protein vector (named P30-CRM 197A) amino acid sequence

A new protein was formed by adding the P30 amino acid sequence FNNFTVSFWLRVPKVSASHLE to the N-terminus of the CRM197A protein vector, whose amino acid sequence was as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

the protein constructed by the method is called P30-CRM 197A protein carrier by inserting GSGSG fragment between the P30 amino acid sequence and the N-terminal of the CRM197A protein carrier for connection.

2-1-2, CRM197 AC-terminal-P30 protein vector (named CRM 197A-P30) amino acid sequence design Another novel protein was formed by adding P30 amino acid sequence FNNFTVSFWLRVPKVSASHLE to the C-terminal of CRM197A protein vector, whose amino acid sequence was as follows:

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSGFNNFTVSFWLRVPKVSASHLE

the protein constructed by the method is called CRM 197A-P30 protein carrier by inserting GSGSG fragment between the P30 amino acid sequence and the C-terminal of the CRM197A protein carrier for connection.

2-1-3, P30-N-terminal CRM197 AC-terminal-P30 protein vector (named P30-CRM 197A-P30) design of amino acid sequence A new protein was formed by adding two P30 amino acid sequences FNNFTVSFWLRVPKVSASHLE to the N-terminal and C-terminal of CRM197A protein vector, respectively, and the amino acid sequence was as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSGFNNFTVSFWLRVPKVSASHLE

the protein constructed by the method is called P30-CRM 197A-P30 protein carrier by inserting GSGSG fragment between the N-terminal and C-terminal of P30 amino acid sequence and CRM197A protein carrier for connection.

2-1-4, P30-P30-N-terminal CRM197A protein carrier (named P30-P30-CRM 197A) amino acid sequence design by connecting two P30 amino acid sequences FNNFTVSFWLRVPKVSASHLE themselves and then adding to the N-terminal of CRM197A protein carrier, a new protein is formed, and the amino acid sequence is as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGFNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

the protein constructed by the method is called P30-P30-CRM 197A protein carrier by inserting GSGSG segment between two self-linked P30 amino acid sequences and between N-terminal of CRM197A protein carrier for connection.

2-1-5, CRM197 AC-terminal-P30-P30 protein carrier (called CRM 197A-P30-P30) amino acid sequence design by connecting two P30 amino acid sequences FNNFTVSFWLRVPKVSASHLE with themselves and then adding to the C-terminal of CRM197A protein carrier, a new protein is formed, the amino acid sequence of which is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSGFNNFTVSFWLRVPKVSASHLEGSGSGFNNFTVSFWLRVPKVSASHLE

the protein constructed by the method is called CRM 197A-P30-P30 protein carrier by inserting GSGSG segment between two self-connected P30 amino acid sequences and between the GSG segment and the C-terminal end of the CRM197A protein carrier for connection.

2-1-6, P30-P30-N-terminal CRM197 AC-terminal-P30 protein carrier (named as P30-P30-CRM 197A-P30) amino acid sequence is designed by connecting two P30 amino acid sequences FNNFTVSFWLRVPKVSASHLE with the protein carrier, and then adding the two P30 amino acid sequences to the N-terminal of the CRM197A protein carrier; in addition, one P30 was added to the C-terminus of CRM197A protein carrier to form another new protein whose amino acid sequence was as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGFNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSGFNNFTVSFWLRVPKVSASHLE

the protein constructed by the method is called a P30-P30-CRM 197A-P30 protein carrier by inserting a GSGSG fragment between two self-connected P30 amino acid sequences and between the two self-connected P30 amino acid sequences and the C-terminal of the CRM197A protein for connection.

Design of 2-1-7, P30-N-terminal CRM197 AC-terminal-P30-P30 protein carrier (named P30-CRM 197A-P30-P30) amino acid sequence

By attaching a P30 amino acid sequence FNNFTVSFWLRVPKVSASHLE to the N-terminus of the CRM197A protein vector; in addition, two P30 were self-ligated and then added to the C-terminus of the CRM197A protein vector to form another new protein with the following amino acid sequence:

FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSGFNNFTVSFWLRVPKVSASHLEGSGSGFNNFTVSFWLRVPKVSASHLE

the protein constructed by the method is called a P30-CRM 197A-P30-P30 protein carrier by inserting a GSGSG fragment between two self-connected P30 amino acid sequences and between the two self-connected P30 amino acid sequences and the C-terminal of the CRM197A protein carrier for connection.

Secondly, construction of CRM197A protein vector and P30CRM197A protein vector expression plasmid containing universal epitope peptide

1. Construction of CRM197A protein vector expression plasmid

The complete amino acid sequence PRF:224021 of the CRM197 protein is obtained from GenBank, the A chain fragment of the CRM197 protein is determined, and the amino acids 1-193 of CRM197 are the A chain fragment of CRM 197. Based on this, the nucleic acid sequence of the amino acids of the fragment was optimized for efficient expression in an E.coli expression system. The self-made expression plasmid is adopted, and Nde I enzyme is used for recognizing plasmid site CATATG and Bam HI enzyme recognition site GGATCC. Through analysis of the gene sequence of CRM197A, Nde I and Bam HI enzyme cutting sites are not existed in the sequence. The synthetic sequence of the CRM197A protein gene is as follows:

CATATG

GGTGCGGACGACGTTGTGGACTCCTCAAAATCGTTTGTCATGGAAAACTTCAGCTCTTAT

CATGGCACCAAACCGGGTTACGTGGACTCCATTCAGAAGGGCATCCAAAAACCGAAGTCA

GGCACCCAGGGTAACTACGATGACGATTGGAAGGAATTCTACAGCACGGACAATAAGTAT

GATGCGGCCGGCTACTCTGTTGACAACGAAAATCCGCTGAGTGGTAAAGCAGGCGGTGTG

GTTAAGGTCACCTATCCGGGTCTGACGAAAGTTCTGGCGCTGAAGGTCGATAACGCCGAA

ACCATTAAAAAGGAACTGGGCCTGTCTCTGACCGAACCGCTGATGGAACAAGTGGGTACG

GAAGAATTTATCAAACGTTTCGGCGATGGTGCATCGCGTGTCGTGCTGAGCCTGCCGTTT

GCTGAAGGCAGTTCCTCAGTGGAATACATTAACAATTGGGAACAAGCAAAAGCTCTGTCA

GTTGAACTGGAAATCAATTTCGAAACGCGTGGCAAACGCGGTCAAGATGCTATGTATGAA

TATATGGCTCAGGCGTGTGCGGGCAATCGCGTCCGTCGCTAA

GGATCC

adding Nde I enzyme and Bam HI enzyme into the blank plasmid and the synthesized PCR product of CRM197A protein gene respectively to perform double enzyme digestion reaction; and after purification, adding T4 ligase into the connecting system for connection, purifying the expression plasmid after the connection, and identifying by using a PCR identification system and an enzyme digestion map. The expression plasmid was transformed into cells using BL21(DE3) competent cells, and clones were selected. After obtaining positive expression engineering bacteria, establishing a seed bank comprising main seeds and working seeds, and storing the seed bank in a refrigerator below 20 ℃ below zero.

2. Construction of P30CRM197A protein vector expression plasmid containing universal epitope peptide

Construction of 2-1, P30-CRM 197A protein vector expression plasmid

Adopting a self-made blank expression plasmid, and using Nde I enzyme to recognize plasmid site CATATG and Bam HI enzyme recognition site GGATCC. Through the analysis of the gene sequence of the P30CRM197A protein carrier, Nde I and Bam HI enzyme cutting sites are not existed in the sequence. The P30-CRM 197A gene was synthesized in the complete sequence as follows:

CATATG

TTCAATAATTTTACGGTGTCGTTTTGGCTGCGTGTCCCGAAAGTCTCTGCGAGTCATCTG

GAAGGTTCTGGTAGCGGTGGTGCGGATGACGTGGTTGATAGCTCTAAATCTTTCGTTATG

GAAAACTTCAGTTCCTATCATGGCACCAAACCGGGTTACGTCGATTCGATTCAGAAAGGC

ATCCAAAAACCGAAAAGCGGCACCCAGGGTAACTACGATGACGATTGGAAAGAATTCTAC

TCAACGGACAACAAATACGATGCGGCCGGCTACTCCGTGGACAACGAAAATCCGCTGAGC

GGTAAAGCGGGCGGTGTCGTGAAAGTTACCTATCCGGGTCTGACGAAAGTGCTGGCTCTG

AAAGTTGATAATGCGGAAACCATCAAAAAAGAACTGGGCCTGTCCCTGACCGAACCGCTG

ATGGAACAAGTGGGTACGGAAGAATTTATCAAACGTTTCGGCGACGGTGCCTCTCGCGTT

GTCCTGAGTCTGCCGTTTGCAGAAGGCTCATCGAGCGTCGAATACATTAACAATTGGGAA

CAAGCAAAAGCTCTGAGCGTGGAACTGGAAATCAACTTCGAAACGCGTGGCAAACGCGGT

CAGGATGCGATGTATGAATACATGGCGCAAGCCTGCGCAGGTAATCGTGTTCGTCGCGGATCC

adding Nde I enzyme and BamHI enzyme into PCR product of blank plasmid and synthesized P30-CRM 197A protein gene to carry out double enzyme digestion reaction; and after purification, adding T4 ligase into the connecting system for connection, purifying the expression plasmid after the connection, and identifying by using a PCR identification system and an enzyme digestion map. BL21(DE3) competent cells were used to transform the expression plasmid into cells, clones were selected and characterized by PCR identification system and restriction mapping. After obtaining positive expression engineering bacteria, establishing a seed bank comprising main seeds and working seeds, and storing the seed bank in a refrigerator below 20 ℃ below zero.

2-2, CRM 197A-P30, P30-CRM 197A-P30, P30-P30-CRM 197A, CRM 197A-P30-P30, P30-P30-CRM 197A-P30 and P30-CRM 197A-P30-P30 protein expression plasmids:

the procedure was the same as in section 2-1, construction of P30-CRM 197A protein expression plasmid, supra.

Preparation of protein vector containing universal epitope peptide P30CRM197A and protein vector CRM197A

The experimental result of the invention shows that the characteristics of the CRM197A protein carrier and the CRM197A protein carrier containing the universal epitope peptide are similar; therefore, the purification method of these protein carriers is similar, and the method is described by taking CRM197A protein carrier containing universal epitope peptide as an example.

1. Preparation of engineering bacteria for expressing protein carrier containing universal epitope peptide P30CRM197A

The plasmid of each expression protein vector is transferred into a competent cell by using a molecular biology standard method, and expression verification is carried out. Screening out clones with high protein expression amount and establishing main seed library and working seed library via antiserum to test qualified clones.

2. Fermentation of engineering bacteria expressing protein carrier containing universal epitope peptide P30CRM197A

Taking out a working seed tube containing the universal epitope peptide P30CRM197A protein carrier from a low-temperature refrigerator of an escherichia coli engineering bacteria working seed bank, and thawing at room temperature; the bacterial solution in the seed tube was aseptically transferred to 50 ml of medium and cultured to OD on a shaker at 37 ℃ and shaking speed of 180rpm₆₀₀About 1.0; inoculating the bacterial liquid into 1L culture medium, culturing at 37 deg.C in shaker at shaking speed of 180rpm to OD₆₀₀About 1.0; inoculating the seed solution into 20L culture medium in 50L fermenter, fermenting at 37 deg.C and 240rpm, and adjusting OD₆₀₀When the protein reaches 7 to 8, IPTG is added to induce the recombinant protein to be synthesized in bacteria; after fermenting for 14 hours, stopping fermenting, centrifuging, and collecting thalli for later use.

3. Purification of P30CRM197A protein carrier containing universal epitope peptide

As the construction of the protein carriers containing different universal epitope peptides takes CRM197A as a main body, experiments show that the universal epitope peptides are added, but the influence on the parameters of protein purification is little, and other purification methods of the CRM197A protein carriers containing the universal epitope peptides can be established only by carrying out some modifications on the process parameters for purifying the CRM197A protein carriers.

Weighing 50g of wet thallus in a 2-liter centrifuge cup, adding 300ml of 1xPBpH7.0 buffer solution to suspend the thallus, and uniformly stirring on a magnetic stirrer for 30 min; centrifuging at 4000rpm at 4 deg.C for 20min, removing supernatant, and collecting thallus; this step was repeated twice; adding 300ml of 1xPBS (x PbS) with a pH value of 7.0 into a bacteria centrifugal tube, and crushing on a homogenizer; centrifuging at 10000rpm at 4 deg.C for 20 min; collecting the precipitate, and discarding the supernatant; adding 300ml of 1xPBS pH7.0 buffer solution, and stirring for 30min on a magnetic stirrer; centrifuging at 4000rpm at 4 deg.C for 20 min; discarding the supernatant, collecting the inclusion body, adding 900ml of modified buffer solution to the washed inclusion body, centrifuging at 10000rpm for 30min at 25 ℃, collecting the supernatant, and discarding the precipitate; transferring the centrifuged supernatant into a 6-8 KD dialysis bag, and sealing the dialysis bag; placing a dialysis bag in 10 liters of renaturation buffer solution 1, and stirring and dialyzing overnight on a magnetic stirrer at room temperature; the next day, the dialysis bag is transferred into 10 liters of renaturation buffer solution 2, and the mixture is stirred and dialyzed for 8 to 10 hours at room temperature; transferring the dialysis bag into 10 liters of renaturation buffer solution 3, stirring and dialyzing at room temperature overnight; the next day, the dialysis bag is transferred into 10 liters of renaturation buffer solution 4, and the mixture is stirred and dialyzed for 8 to 10 hours at room temperature; transferring the dialysis bag into 10 liters of renaturation buffer solution 5, stirring and dialyzing at room temperature overnight; the next day, the dialysis bag is transferred into 2 liters of reserve buffer solution, and the solution is stirred and dialyzed for 8 to 10 hours at room temperature; replacing the storage buffer solution twice, and dialyzing at room temperature overnight; centrifuging 1ml of dialysate at 12000rpm at room temperature for 10min, collecting supernatant, and measuring protein concentration; loading the protein solution to a pre-balanced DEAE gel column, eluting by equal gradient, and collecting a target protein peak; then loading the sample to a Phenyl hydrophobic column for further purification, and collecting an elution peak; finally, loading the SP gel column, and collecting an elution peak; the purified target protein obtained by collection was transferred to a dialysis bag, dialyzed against 0.15M sodium chloride, and after completion, transferred to 4 ℃ for storage.

Preparation of bacterial capsular polysaccharide

The invention purifies 13 serotype pneumococcal capsular polysaccharides of pneumococcus, including 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, so as to be used for synthesizing polysaccharide conjugates, and the quality of the polysaccharide conjugates reaches the polysaccharide quality standard of WHO (white WHO) synthetic polysaccharide protein conjugate vaccine.

1. Establishment of pneumococcal seed bank

13 serotypes of pneumococci purchased from ATCC, including 1 (cat # 9163), 3 (cat # 10813), 4 (cat # BAA-334), 5 (cat # BAA-341), 6A (cat # BAA-659), 6B (cat # 700675), 7F (cat # 10351), 9V (cat # 700671), 14 (cat # 6314), 18C (cat # 10356), 19A (cat # 700673), 19F (cat # 700905), and 23F (cat # 700669). Taking out the strain (original seed batch) purchased by ATCC, adding 0.5ml of pneumococcus AHC liquid culture medium to mix the strain uniformly, and taking 0.25ml of bacterial liquid to 5% sheep blood AHC culture solution. And (3) placing the inoculated 5% goat blood AHC culture solution tube on a culture shaker at 36 +/-1 ℃ and at a shaking speed of 120rpm for culture for 12-20 hours. To be OD₆₀₀When the concentration is up to 1.0, inoculating 5% sheep blood AHC culture solution onto AHC agar culture plate with inoculating loop, and placing onCulturing at 36 +/-1 deg.C for 12-20 hr. 1 to several colonies on AHC agar plates were inoculated with an inoculating loop into 10ml of AHC medium, incubated at 36 ℃. + -. 1 ℃ for 12 hours on a shaker at a shaking speed of 150-. Bacteria OD in culture₆₀₀When the growth reaches 1.0, 5ml of bacterial AHC culture solution is taken out and inoculated into 200ml of fresh AHC culture solution, and the solution is placed on a culture shaker at 36 +/-1 ℃ for culture for about 12 hours at a shaking speed of 150-200 rpm. OD₆₀₀When the concentration reaches 1.0, the bacterial culture solution is divided into 200 small test tubes by 1ml, centrifuged (4000rpm, 10min), the supernatant culture solution is removed, then 0.5ml of fresh AHC culture solution and 0.5ml of sterile skimmed milk are added, mixed evenly and frozen on ethanol dry ice. Vacuum freeze-drying, numbering, and storing in a refrigerator at 4 deg.C as the main seed batch. Taking out the main seeds for batch establishment, inoculating the bacterial culture solution into another 200ml fresh AHC culture solution according to the main seed establishment method, and culturing on a culture shaker at 36 +/-1 ℃ for 12 hours at the shaking speed of 150-200 rpm. To be OD₆₀₀When the concentration reaches 1.0, 1ml of the bacterial culture solution is dispensed into 200 small test tubes, centrifuged (4000rpm, 10min), the supernatant culture solution is removed, and then 0.6ml of fresh AHC culture solution and 0.4ml of 40% glycerol solution are added and mixed uniformly. Quick freezing on dry ice, and storing in a low temperature refrigerator at-70 deg.C as working seed.

2. Fermentation of pneumococcus

And taking out the freeze-dried working seed tube from the seed bank, and adding 1ml of AHC enrichment culture solution to dissolve the freeze-dried bacteria. Inoculating the dissolved bacterial liquid into a 5ml AHC enrichment culture solution test tube in CO₂And (5) standing and culturing in an incubator overnight. When the growth of bacteria was observed, the bacterial solution was inoculated into a 100ml AHC-enriched medium flask. Placing the culture flask in a shaker, and culturing at 36 deg.C and 200rpm/min to OD₆₀₀Is 1.0. 100ml of the bacterial culture was inoculated into 2 bottles containing 1-liter of AHC-enriched medium, respectively. Placing the culture flask in a shaker, and culturing at 36 deg.C and 200rpm/min to OD₆₀₀Is 1.0. 35 liters of sterile filtered AHC enriched broth was injected into a 50 liter fermentor. 2 l of a bacterial solution with an OD of 1 were inoculated into the fermenter. When the bacteria growth reached plateau, sterilization was performed and culture supernatant was harvested.

3. Purification of capsular polysaccharides

Filtering with deep layer filter membrane to further remove residual bacteria and debris. The sterile supernatant broth was concentrated ten-fold (about 600ml) by ultrafiltration using a 100Kd ultrafiltration membrane. The ultrafiltration wash was performed with 6 liters of 25mM sodium acetate solution. The HB stock solution was added to give a final concentration of 1% (w/v), mixed well, and the solution was placed in a freezer overnight. Centrifuging at 4000rpm for 1 hour, collecting polysaccharide/HB precipitate, and discarding centrifugate. Adding 25mM sodium acetate + 1% HB into a polysaccharide/HB precipitation container, stirring, suspending and precipitating, centrifuging at 4000rpm for 1 hour, collecting polysaccharide/HB precipitate, and discarding centrifugate. This step was repeated 3 times. The precipitate was dissolved with 600ml of 0.25M sodium chloride solution. Centrifugation at 4000rpm for 1 hour discarded insoluble nucleic acid contaminants. Adding 10% potassium iodide solution into the mixed solution of polysaccharide and HB, mixing, and placing the solution in a refrigerator overnight, wherein the final concentration of potassium iodide is 0.5%. Filtering the solution with a depth filter to remove HB/I precipitate in the solution, washing the precipitate on the depth filter with 0.25M sodium chloride/0.5% potassium iodide solution, collecting the filtrate, and discarding the precipitate. The crude polysaccharide solution was passed through a charcoal depth filter for 30 minutes of filtration by circulation (4% charcoal/0.5 mg/ml crude polysaccharide solution). Sodium phosphate buffer, pH6.8, was added to the polysaccharide solution at a final concentration of 25 mM. The above solution was passed through an HA column (50-100 ml) and circulated for 30 minutes. The column was washed with the same phosphate solution for 4-5 column volumes. The polysaccharide solution was concentrated 5-fold by ultrafiltration using a 30Kd membrane, and the polysaccharide solution was washed by ultrafiltration using pyrogen-free water. Filtering with 0.22 μm membrane, and lyophilizing.

The second step is that: preparation of 13 serotype monovalent pneumococcal polysaccharide-P30 CRM197A conjugates

The chemical structure of the capsular polysaccharide of different pneumococcal serotypes contains different groups, different synthetic methods are adopted to covalently bond the polysaccharide to a protein carrier to form a conjugate, and the yield and the immunogenicity of the conjugate synthesized by different methods are different. According to the experimental result, three synthetic methods, namely, a reduced amine method, a CDAP method (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride method) and an ADH method (adipic dihydrazide method) are adopted to synthesize the specific polysaccharide protein conjugate.

Synthesis of 13 pneumococcal serotype capsular polysaccharide-P30-CRM 197A conjugates

1. Pneumococcal serotype 1 capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5mg of Pn1 degraded polysaccharide in a reaction bottle, and weighing 0.5ml of 1mol/L NaCl in the reaction bottle; the polysaccharide was completely dissolved by magnetic stirring. Recording the initial pH of the polysaccharide solution, respectively measuring a proper amount of CDAP solution, and adding the CDAP solution into a reaction bottle. The reaction was stirred at room temperature for 1.5min and the pH of the solution was measured at 30 s. After 1.5min, the pH of the solution was adjusted to 9.5 with 0.2mol/L NaOH and the reaction was stirred at room temperature for 3min (pH was maintained at 9.5 with 0.2mol/L NaOH). Immediately after 3min, 5mg of P30-CRM 197A protein was added to the flask and the reaction was stirred at room temperature (25 ℃) for 1 h. 37.5. mu.l of 2mol/L lysine were measured and added to the reaction flask and the pH of the solution was adjusted to 9.0 with 0.1N HCl. The reaction was stirred at room temperature for 30min and the flask was transferred to 4 ℃ for overnight reaction. The reaction mixture was transferred to a dialysis bag (MWCO 6-8000) and dialyzed against 0.85% NaCl solution 3 times, 6L/time at 4 ℃. And after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

2. Pneumococcal serotype 3 capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 20mg of Pn3 degraded polysaccharide in a reaction bottle, and weighing 2ml of 0.15M NaCl in the reaction bottle; the polysaccharide was completely dissolved by magnetic stirring. An appropriate amount of CDAP solution was measured and added to the reaction flask. The reaction was stirred at room temperature for 1.5min and the pH of the solution was measured at 30 s. After 1.5min, the pH of the solution was adjusted to 9.5 with 0.2mol/L NaOH and the reaction was stirred at room temperature for 3min (pH was maintained at 9.5 with 0.2mol/L NaOH). Add final concentration of 0.8M ADH into the reaction flask, stir and mix well, 2 hours reaction at room temperature. The derivatized polysaccharide was transferred to a 10kD dialysis bag and dialyzed against 0.15M NaCl solution for three changes. The G-50 column was loaded, eluted with 0.15M NaCl and the peak of the external water volume was collected. Then transferred to a dialysis bag and dialyzed against water with three changes of fluid. 5mg of the derivatized Pn3 polysaccharide was weighed out and dissolved in 0.5ml of 0.15M NaCl solution, 5mg of P30-CRM 197A protein was added thereto, and after stirring and mixing, 30mM EDC was added thereto, and the mixture was reacted at room temperature for 4 hours and then transferred to 4 ℃ for overnight reaction. The reaction mixture was transferred to a dialysis bag (MWCO 6-8000) and dialyzed against 0.85% NaCl solution 3 times, 6L/time at 4 ℃. And after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

3. Pneumococcal serotype 4 capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5mg of activated polysaccharide into a reaction bottle, weighing 100 mul of 0.5M sodium phosphate buffer solution, adding into the reaction bottle, weighing 5mg of P30-CRM 197A protein into the reaction bottle, and magnetically stirring to completely dissolve the polysaccharide; measuring 0.5ml of pure water, adding the pure water into a reaction bottle, and uniformly stirring by magnetic force; 5.0mg of sodium cyanoborohydride was weighed and added to the reaction flask. The reaction system is placed in a dry bath at 30 ℃ for reaction for 12 h. After the reaction was completed, 1.5ml of 0.15M sodium chloride solution was measured and added to the reaction flask. 2.5mg of sodium borohydrate was weighed and added to the reaction flask. The reaction system is placed at 22 ℃ for reaction for 5 hours; the reaction mixture was transferred to dialysis bags (MWCO 12-14 Kd) and dialyzed against 0.15M sodium chloride solution at 4 ℃ for 3 times, 6L of dialysate per time; and after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

4. Pneumococcal serotype 5 capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5.0mg of activated polysaccharide, adding into a reaction bottle, and adding 100 μ l of 0.5M sodium phosphate buffer solution into the reaction bottle; weighing 4.0mg of P30-CRM 197A protein, adding into a reaction bottle, weighing 0.5ml of pure water, adding into the reaction bottle, magnetically stirring to dissolve reactants, and measuring the pH value of the reaction system; weighing 5.0mg of sodium cyanoborohydride, and adding into a reaction bottle; the reaction system is placed at room temperature for reaction for 48 hours; weighing 2.5mg of sodium borohydrate, dissolving in 10 μ l of pure water, mixing and dissolving completely with a pipette, and adding into a reaction bottle; the reaction system is placed at 23 ℃ and stirred for reaction for 5 hours; the reaction mixture was transferred to dialysis bags (MWCO 6-8 KD) and dialyzed against 0.15M sodium chloride solution at 4 ℃ for 3 times, changing the solution every 5 hours; and after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

5. Pneumococcal serotype 6A capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 6.0mg of activated Pn6A polysaccharide, dissolving in 1mL of purified water, stirring until the polysaccharide is completely dissolved, and measuring the initial pH value; adjusting the pH value of the reaction solution to 7.0 by using 0.1M NaOH; adding 4mg of P30-CRM 197A protein into the reaction system, and stirring and uniformly mixing; weighing 5.0mg of sodium cyanoborohydride, adding into the reaction flask, and reacting at room temperature for 18 hours; sampling and inspecting after the reaction is finished; 2.7mg of Sodium borohydrate is weighed and added into the reaction bottle to react for 5 hours at room temperature; sampling and inspecting after the reaction is finished; the reaction mixture was transferred to a dialysis bag and dialyzed against 0.15M sodium chloride solution 5 times, 6L/time at 4 ℃. And after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

6. Pneumococcal serotype 6B capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5.0mg of Pn6B, dissolving in 1mL of purified water, stirring until the Pn6B is completely dissolved, and measuring the initial pH value; adjusting the pH value of the reaction solution to 7.0 by using 0.1M NaOH; adding 2.5mg of P30-CRM 197A protein into the reaction system, and uniformly stirring; weighing 5.0mg of sodium cyanoborohydride, adding into the reaction flask, and reacting at room temperature for 20 hours; 2.5mg of Sodium borohydrate is weighed and added into the reaction bottle to react for 6 hours at room temperature; transferring the reaction mixture to a dialysis bag, and dialyzing against 0.15M NaCl solution at 4 ℃ for 5 times and 6L/time; and after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

7. Pneumococcal serotype 7F capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 10.0mg of Pn7F polysaccharide, dissolving in 1mL of purified water, and stirring until the polysaccharide is completely dissolved; respectively dripping 0.1M NaOH solution into the polysaccharide solution, and adjusting the pH to 7.0; adding 3.5mg of P30-CRM 197A protein into the reaction system, and uniformly stirring; weighing 5.0mg of sodium cyanoborohydride, adding into the reaction flask, and reacting at room temperature for 20 hours; adding 990 μ l of pure water into the reaction bottle, and stirring and mixing uniformly; 2.5mg of Sodium borohydrate is weighed and added into the reaction bottle to react for 6 hours at room temperature; transferring the reaction mixture to a dialysis bag (MWCO 6000-8000), and dialyzing 5 mMsuccinate/0.9% sodium chloride buffer solution at 4 ℃ for 5 times and 6L times; and after the dialysis is finished, centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting a Sepharose CL-4B gel column, collecting a conjugate peak, and sampling for inspection.

8. Pneumococcal serotype 9V capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 10mg of Pn9V activated polysaccharide, weighing 125 mul of sodium phosphate buffer solution and 125 mul of pure water, adding into a reaction bottle, and magnetically stirring to completely dissolve the polysaccharide; weighing 15mg of P30-CRM 197A protein, adding the protein into a reaction bottle, and stirring to dissolve completely; weighing 10mg of NaBH₃(CN), adding into a reaction bottle; the reaction system is placed at 22 ℃ for reaction for 48 hours; weighing 2.5mg of NaBH₄Adding the mixture into a reaction bottle, and reacting for 5 hours at the temperature of 22 ℃; and centrifuging the reaction mixed solution at 10000rpm for 10min, taking supernatant, purifying the dialyzed polysaccharide conjugate by adopting an AKTA system and a Sepharose CL-4B gel column, and collecting a binding peak.

9. Pneumococcal serotype 14 capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5mg of Pn14 activated polysaccharide, weighing 1ml of 3.9mg of P30-CRM 197A protein, adding into a reaction bottle, and magnetically stirring to completely dissolve the polysaccharide; adding weak reducing agent sodium cyanobohydride 5mg, and reacting for 48 hours at 22 ℃; adding 2.5mg of sodium borohdride serving as a strong reducing agent, and reacting for 4 hours at room temperature; transferring the reaction mixture to a dialysis bag (MWCO 12-14 KD), and rinsing the reaction bottle with 2ml of dialysate; dialyze against 0.15M sodium chloride solution at 4 ℃ for 3 times, 6L/time, and change every 5 hours. Collecting dialysate after dialysis, centrifuging at 10000rpm for 10min, collecting supernatant, purifying dialyzed polysaccharide conjugate with Sepharose CL-4B gel column, and collecting conjugate peak.

10. Pneumococcal serotype 18C capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5mg of Pn18C degraded polysaccharide, and dissolving with 1mL of 1M sodium chloride solution; measuring the initial pH value after complete dissolution; adding appropriate amount of CDAP solution, stirring at room temperature for 1.5min, adding 0.2M NaOH solution to adjust pH of the solution to 9.0. Then reacting for 3min at room temperature; adding 10mg of P30-CRM 197A protein, and reacting at 25 deg.c for 45 min; after the reaction, 37.5. mu.l of 2M lysine solution was added; reacting at 25 deg.C for 30min, and reacting at 4 deg.C overnight; transferring the reaction mixture to dialysis bag (MWCO 6000-8000), dialyzing 0.85% sodium chloride solution at 4 deg.C, and changing the solution for 3 times. The liquid is changed every 5 hours at 6L/time. Collecting dialysate after dialysis, centrifuging at 10000rpm for 10min, collecting supernatant, purifying the conjugated polysaccharide with CL-4B gel, and collecting conjugate peak; and detecting the contents of protein and polysaccharide in the conjugate solution.

11. Pneumococcal serotype 19A capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 10.0mg of Pn19A oxide polysaccharide, dissolving in 0.5mL of buffer solution, placing in a reaction bottle with a magnetic bar, and stirring until sugar is completely dissolved; adding 10mg of P30-CRM 197A protein; stirring and mixing evenly, weighing 5.0mg of sodiumcyanoborohydride, adding into a reaction bottle, and reacting for 20 hours at room temperature; weighing 2.5mg of sodiumbrohydride, adding into a reaction flask, and reacting at room temperature for 5 hours; transferring the reaction mixture to dialysis bag (MWCO 6000-8000), dialyzing 0.85% sodium chloride solution at 4 deg.C, and changing the solution for 3 times. Changing the liquid every 5 hours for 6L/time; collecting dialysate after dialysis, centrifuging at 10000rpm for 10min, collecting supernatant, purifying the conjugated polysaccharide with CL-4B gel, and collecting conjugate peak; and detecting the contents of protein and polysaccharide in the conjugate solution.

12. Pneumococcal serotype 19F capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 5.2mg of Pn19F oxide polysaccharide, dissolving in 1ml of pure water, placing a magnetic bar in a reaction bottle, and stirring on a magnetic stirrer at room temperature until the polysaccharide is completely dissolved; adding 3.0mg of P30-CRM 197A protein; after uniformly mixing, weighing 4.9mg of sodium cyanoborohydride, adding into a reaction bottle, and keeping stirring on a magnetic stirrer all the time; reacting at room temperature of 18 ℃ for 24 hours; weighing 2.5mg of sodium borohydrate, and adding into a reaction bottle; reacting for 5 hours at 18 ℃ in a constant temperature box; the reaction mixture was transferred to dialysis bags (MWCO 12-14000) and dialyzed against the buffer 5 times at 4 ℃ with 6L dialysate volume each time and with a change every 5 hours; collecting dialysate after dialysis, centrifuging at 10000rpm for 10min, collecting supernatant, purifying the conjugated polysaccharide with CL-4B gel, and collecting conjugate peak; and detecting the contents of protein and polysaccharide in the conjugate solution.

13. Pneumococcal serotype 23F capsular polysaccharide-P30-CRM 197A protein conjugate synthesis

Weighing 4.9mg of Pn23F oxide polysaccharide, dissolving in 1ml of pure water, placing a magnetic bar in a reaction bottle, and stirring on a magnetic stirrer at room temperature until the polysaccharide is completely dissolved; 5.0mg of P30-CRM 197A protein was added; weighing 5.1mg of sodium cyanohydrin, adding into a reaction flask, and stirring on a magnetic stirrer; reacting for 17 hours at 18 ℃ in a constant temperature box; weighing 2.5mg of sodium borohydrate, and adding into a reaction bottle; reacting for 5 hours at 18 ℃ in a constant temperature box; the reaction mixture was transferred to a dialysis bag (MWCO 12-14000) and dialyzed against 0.15M sodium chloride solution at 4 ℃ for 6L of dialysate at a time. Changing the solution once every 5 hours for 5 times; collecting dialysate after dialysis, centrifuging at 10000rpm for 10min, collecting supernatant, purifying the conjugated polysaccharide with CL-4B gel, and collecting conjugate peak; and detecting the contents of protein and polysaccharide in the conjugate solution.

Secondly, synthesis of 13 pneumococcal capsular polysaccharide P30CRM197A conjugates of other P30CRM197A protein carriers containing universal epitope peptides

The invention designs and produces a polysaccharide protein conjugate synthesized by 7P 30-CRM 197A protein carriers, and because the structures of the proteins are similar, the method adopted when the polysaccharide conjugate is used for synthesizing specific pneumococcal serotype polysaccharides is one of a reducing amine method, an ADH method or a CDAP method. The synthesis of the 13 pneumococcal polysaccharide-P30-CRM 197A compositions in the previous examples is similar and not necessarily a specific example.

The third step: preparation of 13-valent pneumococcal polysaccharide P30CRM197A conjugate and evaluation of immunogenicity

Preparing corresponding vaccine by the prepared pneumococcal polysaccharide protein conjugate, performing immunization injection and blood collection on mice, and detecting the concentration of polysaccharide antibody in serum and opsonophagocytosis test by an ELISA method to evaluate the immunogenicity of different conjugates.

Preparation of 13-valent pneumococcal polysaccharide-P30 CRM197A conjugate and evaluation of immunogenicity

In order to evaluate that the immunogenicity of the pneumococcal capsular polysaccharide P30CRM197A conjugate synthesized by the P30CRM197A protein carrier is better than that of the pneumococcal capsular polysaccharide CRM197A conjugate synthesized by the CRM197A protein carrier without the universal epitope peptide, 13-valent pneumococcal polysaccharide-CRM 197A synthesized by the invention is used as a control to evaluate the immunogenicity enhancing effect.

1. Preparation of 13-valent pneumococcal polysaccharide-P30-CRM 197A conjugate vaccine and 13-valent pneumococcal polysaccharide-CRM 197A conjugate vaccine

Concentrating the 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F pneumococcal serotype capsular polysaccharide protein conjugate solutions to a polysaccharide concentration of 40 μ g/ml using Millipore's Labscale ultrafiltration system, respectively; concentration of the 6B serotype conjugate solution was concentrated to a polysaccharide concentration of approximately 80 μ g/ml; the corresponding volume of monovalent serotype conjugate solution was added to the formulation vial as calculated in the following table.

Sterilizing and filtering the mixture of the conjugates by using a 0.22 mu m membrane; adding sterile aluminum phosphate gel to obtain a final aluminum ion concentration of 125 mg/ml; fixing the volume to the final volume by using a buffer solution; and (4) filling, and 0.5 ml/bottle.

2. Preparation of 13-valent pneumococcal polysaccharide P30CRM197A conjugate vaccine containing other P30CRM197A protein carriers

Referring to the method of the section "preparation of 13-valent pneumococcal polysaccharide-P30-CRM 197A conjugate and evaluation of immunogenicity", the present invention prepares the following 6 13-valent pneumococcal polysaccharide P30CRM197A conjugate with corresponding vaccine and uses it in immunogenicity evaluation experiment, including 13 Pn-CRM 197A-P30, 13 Pn-P30-CRM 197A-P30, 13 Pn-P30-P30-CRM 197A, 13 Pn-CRM 197A-P30-P30, 13 Pn-P30-P30-CRM 197-P30 and 13 Pn-P30-CRM 197A-P30-P30.

3. Injecting mice for immunization injection and blood sampling

70 CM57 mice of 5-6 weeks are selected, and each mouse is injected with the prepared 13-valent pneumococcal polysaccharide-P30-CRM 197A protein conjugate vaccine with the injection volume of 0.1 ml/mouse/time. The immune injection mice are divided into three groups, one group is injected with 13-valent pneumococcal polysaccharide-P30-CRM 197A protein vaccine prepared by the invention, the other group is injected with 13-valent pneumococcal polysaccharide-CRM 197A conjugate vaccine as a control, the third group is polysaccharide control, and the program tables of the specific injection vaccine and immune serum collection are as follows:

collecting mouse blood to a centrifuge tube, standing for 2 hours at room temperature, centrifuging for 10 minutes at 10000rpm, carefully absorbing the serum of the centrifuged supernatant by a pipette, storing in a refrigerator at 4 ℃ and detecting.

4. ELISA method for detecting polysaccharide antibody concentration in mouse serum

Stock solutions of 13 different serotypes of pneumococcal polysaccharide were prepared at 1mg/ml (in 1XPBS solution) and stored in a refrigerator at 4 ℃. The stock solution of pneumococcal polysaccharide to be tested was diluted to 2-4. mu.g/ml coating buffer, 100. mu.l of coating solution was added to each well to coat the ELISA plates, and incubated overnight at room temperature. Washing with plate washing buffer for 4 times, adding 100 μ l blocking buffer, incubating at room temperature for 2 hr, washing with plate washing buffer for 4 times, and storing at 4 deg.C for one week.

Diluting the serum to be tested obtained by injecting the conjugate vaccine and the control sample into the mouse by 1:10 to obtain the serum of a working sample, diluting by a proper multiple, adding the diluted serum into the first row of the ELISA plate with a total volume of 200 mu l, performing two-fold serial dilution from the first row downwards, and incubating for 2 hours at room temperature. The plate was washed 4 times with the washing buffer, 100. mu.l of alkaline phosphatase-labeled goat anti-mouse antibody (1:2000 dilution) was added, and the mixture was incubated at room temperature for 4 hours. The plate was washed 4 times with washing buffer, 100. mu.l of 4-nitrophenyl phosphate disodium salt substrate solution was added, and the plate was read at 405 nm.

The results of the concentration of specific pneumococcal serotype polysaccharide antibodies in mouse serum are given in the following table:

the result shows that the immunogenicity of the pneumococcal polysaccharide conjugate vaccine, namely the 13-valent pneumococcal capsular polysaccharide-P30-CRM 197A conjugate synthesized by taking P30-CRM 197A as a protein carrier is better than that of the 13-valent pneumococcal capsular polysaccharide CRM197A conjugate. The concentration of IgG antibody against specific pneumococcal polysaccharide in the serum of the mouse injected with the three needles is obviously higher than that in the serum injected with one needle and the second needle; the concentration of each serotype antibody after three injections is more than 4 times higher than that of one injection, and the WHO standard for improving the concentration of the polysaccharide protein combined vaccine is met. The concentration of polysaccharide-specific antibodies in mouse serum was higher for each serotype of pneumococcal 13 valency capsular polysaccharide-P30-CRM 197A conjugate compared to pneumococcal 13 valency capsular polysaccharide-CRM 197A conjugate after three injections.

5. Opsonophagocytic Assay (OPA)

The opsonophagocytosis test is a method for evaluating the bactericidal efficacy of 13-valent pneumococcal polysaccharide-P30-CRM 197A conjugate vaccine, and the obtained immune serum of mice is tested according to the UAB-MOPA's "Streptococcus pneumoniae capsular polysaccharide specific antibody polytype opsonophagocytosis bactericidal test method". OPA concentration results are given in the table below:

the test result shows that the OPA concentration of the antibody of the combined mouse immune serum of the 13-valent pneumococcal polysaccharide-P30-CRM 197A conjugate group and the combined mouse immune serum of the 13-valent pneumococcal polysaccharide-CRM 197A conjugate group after three injections is obviously improved.

Preparation of second and other 13-valent pneumococcal polysaccharide-P30 CRM197A conjugates and evaluation of immunogenicity

Similar to the procedure described in section "preparation of 13 valent pneumococcal polysaccharide-P30-CRM 197A conjugate and assessment of immunogenicity", the results for the anti-polysaccharide antibody IgG concentration of the 13 valent pneumococcal polysaccharide-P30 CRM197A conjugate synthesized with the other protein carriers containing P30 are given in the following table, which is a list of polysaccharide IgG antibody concentrations following three immunizations.

From the results in the table, the immunogenicity of the 13-valent pneumococcal polysaccharide conjugate synthesized by the protein carrier CRM197A containing the P30 universal epitope peptide is obviously enhanced compared with the immunogenicity of the 13-valent pneumococcal polysaccharide conjugate synthesized by the protein carrier CRM197A not containing the P30 universal epitope peptide. The immunogenicity of the 13-valent pneumococcal polysaccharide P30CRM197 conjugate synthesized by the CRM197A protein carrier containing two P30 universal epitope peptides is higher than that of the 13-valent pneumococcal polysaccharide CRM197A conjugate synthesized by the CRM197A protein carrier containing only one P30 universal epitope peptide. When the number of the P30 universal epitope peptides in the CRM197A protein carrier is increased to three, the immunogenicity of the 13-valent pneumococcal polysaccharide P30CRM197A conjugate synthesized by the protein carrier containing two P30 universal epitope peptides is not obviously changed. There was no difference in immunogenicity between the P30 universal epitope peptide in the CRM197A protein vector added to the N-terminus and the 13 valent pneumococcal polysaccharide P30CRM197A conjugate added to the C-terminus.

Reference to the literature

1、Panina‐Bordignono P,Tano A,Termijtelen A,Universally immunogenic Tcell epitopes promiscuous binding to human MHC class II and promiscuousrecognition by T cells,Eur.J.Immunol.19:2237‐2242,1989.

2、Su Y,RossiR,De Groot AS,Regulatory T cell(Tregitopes)in IgG inducetolerance in vivo and lack immunogenicity per se.J Leukoc Biol.94(2):377‐383,2013.

3、Giannini G,Rappuoli R,Ratti G,The amino‐acid sequence of two non‐toxic mutants of diphtheria toxin:CRM45and CRM197.Nucleic acids research,12:4063‐4069,1984.

Claims

1. A method of enhancing the immunogenicity of a 13-valent pneumococcal polysaccharide protein conjugate, comprising:

step three: mixing the 13 monovalent pneumococcal polysaccharide-P30 CRM197A conjugates obtained in the step two to obtain 13 valent pneumococcal polysaccharide protein conjugates with enhanced immunogenicity;

1P 30 in the P30CRM 197A; in the P30CRM197A protein carrier, P30 is linked at the N-terminus or C-terminus of CRM197A protein;

in the P30CRM197A protein carrier, the P30 and the CRM197A protein are connected through a GSGSG amino acid sequence; the gene recombination engineering bacteria are escherichia coli constructed by a gene recombination method.

2. The method of enhancing the immunogenicity of a 13 valent pneumococcal polysaccharide protein conjugate according to claim 1, wherein: the pneumonia capsular polysaccharide is capsular saccharide obtained by culturing 13 different serotypes of pneumococcus respectively.