CN107137703B - A kind of rabies immunogenic conjugate and vaccine and preparation method thereof - Google Patents

Abstract

The invention relates to a novel Rabies Immunogenic Conjugate (Rabis Immunogenic Conjugate RIC) and a preparation method thereof, the Rabies Immunogenic Conjugate comprises a Rabies virus antigen (RabAg) and a Carrier Protein (Carrier Protein CP), the Rabies virus antigen and the Carrier Protein are connected through a chemical bond, the Carrier Protein and the Rabies virus antigen are connected through a chemical bond, and the chemical bond connects the Rabies virus antigen and the Carrier Protein to form the Rabies virus antigen-Carrier Protein Immunogenic Conjugate. The invention also relates to a novel Rabies combined Vaccine (Rabis Conjugate Vaccine RCV) prepared according to the Rabies immunogenic Conjugate and a preparation method thereof. The preliminary proof proves that the invention overcomes the defects of the prior art and has better effect.

Description

A kind of rabies immunogenic conjugate and vaccine and preparation method thereof

technical field

The invention relates to the medical field, in particular to vaccines and vaccine preparations, in particular to a rabies immunogenic conjugate and a preparation method thereof, and a rabies conjugate vaccine and a preparation method thereof.

Background technique

Rabies (Rabies) is an acute infectious disease caused by Rabies Virus (Rabies Virus) infection, and is a zoonotic acute infectious disease of natural foci. The rabies virus is mainly transmitted to humans through direct contact with damaged skin or mucous membranes through the saliva of infected animals. Once rabies occurs, its case fatality rate is almost 100%. Dogs are the main host of rabies virus, and up to 99% of human rabies is transmitted by dogs. Bats are a major source of rabies deaths in the Americas, and bat rabies has recently emerged in Australia and Western Europe as a public health threat. Rabies occurs on all continents except Antarctica, and WHO estimates that the number of deaths from rabies worldwide is as high as 59,000 per year, of which more than 95% of human rabies cases occur in Asia and Africa (http://www.who.int/mediacentre/factsheets /fs099/en/). The incidence of human rabies in China was at its peak in 2007, and the annual number of reported cases reached 3,300. From 2004 to 2014, the number of deaths from rabies has always been among the top three in the number of deaths from infectious diseases in my country (Chinese Center for Disease Control and Prevention. Rabies Prevention and Control Technical Guidelines .2016).

Rabies virus belongs to the genus Lyssaviruses of the family Rhabdovividae. The genus Lyssavirus includes 12 species, 4 serotypes, and 7 genotypes. Rabies virus is the most important species that causes human infection with rabies, and belongs to type I serotype I genotype. The typical shape of rabies virus particles is flat and slightly concave at one end, and semicircular at the other end, which resembles a bullet, hence the name rhabdovirus (Rhabdoyivus). The size of the virus is 75～80×170～180nm. The virus genome is a single-stranded negative-strand RNA with 11162 nucleotides, which encodes five structural proteins, namely nucleoprotein (N), phosphoprotein (P), Matrix protein (Matrixprotein, M), glycoprotein (Glycoprotein, G) and RNA-dependent RNA polymerase (RNA dependent RNA polymerase or Large protein, L). Rabies virus is relatively stable genetically, and rabies viruses isolated from all over the world can be neutralized by a single antiserum.

Currently there is no test to diagnose rabies virus infection before the onset of clinical symptoms. So far, rabies is still an incurable disease for humans, because there is no treatment drug at present, and once it occurs, its case fatality rate is almost 100%. The use of antiviral drugs, interferon and large doses of specific rabies immunoglobulin can only prolong the course of the disease, but still cannot prevent death. Unlike many other infectious diseases, rabies is a vaccine-preventable disease, and even timely vaccination after exposure to rabies virus and in combination with rabies immune globulin is effective in preventing mortality (Rabies vaccines: WHO position paper 2010 ).

The incubation period of rabies usually ranges from weeks to months, and can also be as short as a few days or as long as several years (ACIP. Use of a Reduced (4-Dose) Vaccine Schedule for Postexposure Prophylaxis to Prevent Human Rabies. 2010). Huang Shaoxin reported that the incubation period of 249 cases of rabies in Guilin was as short as 1 day and as long as 3650 days (Huang Shaoxin. Occupation and Health. 2013, 29(17): 2123-2126). Liu Fuqiang et al. reported that the incubation period of 1059 cases of rabies in Hunan Province was as short as 2 days and as long as 4636 days (Liu Fuqiang et al. Chinese Journal of Natural Medicine. 2008,10(4):263-268). Cao Minghua et al. reported that the incubation period of 416 cases of rabies in Anhui Province was as short as 2 days and as long as 5606 days (Cao Minghua et al. Anhui Journal of Preventive Medicine. 2013,19(1):1-4). Yu Chun et al. reported that the incubation period of 2065 cases of rabies in Guizhou Province was as short as 2 days and as long as 13 years (Yu Chun et al. Medical Animal Control. 2016(2):145-147).

Most of the rabies vaccines are used for post-exposure immunity (Post Exposure Prophylaxis PEP), which is blocked before the rabies virus invades the central nervous system (Central Nervous System CNS) through passive or active immunity to exert the effectiveness of PEP (Wilde H. Vaccine. 2007, 25(44):7605-9). Since there are cases in which the incubation period of rabies is only a few days, it is necessary for the vaccine to induce an immune response quickly in humans, produce early immune protection, and provide long-lasting immune protection. However, for cases with a short incubation period, rabies virus will quickly invade the nervous system, and PEP measures cannot block virus replication and cannot prevent rabies virus from infecting the brain (Terryn S, etal.PLOS Neglected Tropical Diseases.2016,8,2:1 -15).

Since Louis Pasteur invented the rabies vaccine in 1885, the rabies vaccine has experienced early animal nerve tissue vaccines, poultry embryo vaccines, and crude vaccines from cell culture, and has developed to the current primary hamster kidney cells, primary Purified vaccines cultured from chicken embryo cells, primary duck embryo cells, African green monkey kidney cells (Vero cells), rhesus monkey fetal diploid cells, and human diploid cells. Regardless of the early crude vaccines or the current purified vaccines, the antigens of rabies vaccines have been inactivated whole virus particles (Virus Particles) since 130 years ago. The inactivated whole virus particle vaccine stimulates the body to produce rabies virus-specific CD4+ T cells, B cells and antibodies (Faber M et al.Proc Natl Acad Sci USA.2009,106(27):11300-11305). These inactivated rabies whole virus particle antigens are mainly presented by MHC class Ⅱ molecules in the body to induce CD4+ T cells and produce antibodies, which belong to the immune response mechanism mainly based on humoral immunity (Li Maoguang. Japanese encephalitis vaccine and rabies vaccine Research on Cellular Immunity. Master's Degree Thesis. 2008). The inactivated rabies whole virus particle vaccine in mice induces Th2 to produce IgG1 antibody humoral immunity as the main immune response (Ertl H. Plos Neglected Tropical Diseases, 2009, 3(9): 1-9). The Rabies Working Committee of the American Advisory Committee on Immunization analyzed 12 vaccine clinical studies published between 1976 and 2008. It takes more than 14 days for the existing rabies inactivated whole virus particle vaccine to produce neutralizing antibodies in the human body (Rupprecht CE, et al .Vaccine.2009.27:7141-8). Chinese scholars reported that the neutralizing antibody GMT of Vero cell rabies vaccine was only 0.24-0.27IU/ml on the 7th day after the first dose of rabies vaccination, and the seroconversion rate was 12.2-28.9%. The positive conversion rate is 100% (Xiao Qiyou et al. Chinese Journal of Zoonoses. 2009, 25(5): 493-494; Yang Shuhong et al. Disease Surveillance. 2009, 24(6): 409-411).

In the absence of co-administration of immunoglobulins, PEP alone cannot prevent rabies deaths (Servat A, et al. Vaccine. 2003.22(2):244-9). Institut Pasteur of France, Wuhan Institute of Biological Products and Institute of Military Veterinary Medicine, Academy of Military Medical Sciences applied mice and Beagle dogs to three independent post-exposure experiments, that is, subcutaneous or muscle infection of rabies wild strain virus, and then immunization with rabies vaccine. The results all show that without the combined application of antiserum, the protection rate of commercially available human rabies vaccines at home and abroad is only 0-30% (Lin Haixiang et al. Chinese Journal of Experimental and Clinical Virology. 2016.30(1):37-40) . Humans infected with rabies virus are rarely able to induce protection by inducing their own immune system and clear the virus if they do not receive post-exposure immunization. The inactivated whole virus particle vaccine has poor immunogenicity because it cannot induce inflammatory response and thus cannot effectively activate Th1 lymphocyte and B lymphocyte response (Marciani DJ.DrugDiscovToday.2003.8(20):934-943). Studies have shown that humoral immunity induced by inactivated whole rabies virus particle vaccine or injected passive immunization preparations can only clear the peripheral rabies virus and prevent it from invading nerve endings, the effect of cellular immunity is limited, and once the virus enters the CNS, it cannot provide protection ( Hooper DC, et al. J. Virol. 1998.72(5): 3711-3719; Lafon M. Human rabies vaccines. 2nd ed. SanDiego: Academic Press. 2007. p489-504). This is because the ability of neutralizing antibodies to cross the blood-brain barrier (BBB) is very limited (Hooper DC et al. Future Virol. 2011.6(3):387-397).

It can be seen that the existing inactivated whole virus particle vaccines are based on the fatal defects of no or weak cellular immunity and slow humoral immunity, and the production of antibodies obviously lags behind the speed and speed at which rabies virus reproduces in local muscles and invades peripheral nerves. Time, especially for those exposed with severe bites, highly dangerous parts, and short incubation periods, cannot produce effective protection (Wang Shusheng. Applied Preventive Medicine. 2010.16(1):1-4).

In view of the above reasons, it is necessary to revolutionize the existing inactivated rabies whole virus particle vaccine, so that the new vaccine can produce an immune response faster in the human body, induce a higher level of immunity, and provide a longer-lasting immune protection effect. In order to meet the urgent needs of people to prevent rabies after exposure.

In the present invention, the carrier protein and the rabies virus antigen are chemically bonded to form a rabies immunogenic conjugate, specifically, the carrier protein and the rabies whole virus particle antigen are chemically bonded, or the carrier protein and the rabies virus-like particle (Virus Like Particle VLP ) antigens are linked by chemical bonds, or the carrier protein and the lysed Fragment of Virus Envelope antigen are linked by chemical bonds, or the carrier protein and rabies virus glycoprotein (G) antigen are linked by chemical bonds to form A new rabies immunogenic conjugate. The carrier protein in the rabies immunogenic conjugate induces the body to generate a cellular immune response to the rabies virus antigen, and makes the cellular immune response and humoral immune response synergistic in vivo, thereby making the new rabies immunogenic conjugate more effective in the human body. Quickly generate an early immune response, induce a higher immune level, and provide a longer-lasting immune protection effect.

In recent years, it has been reported that rabies vaccine has a certain therapeutic effect on malignant melanoma, cervical cancer, and malignant glioma, and it is further found that anti-phosphatidylinositol (phosphatidylinositol PI) antibodies exist in the blood of all malignant tumor patients, while phospholipids Acyl inositol is an important component with a high content in the rabies virus envelope. It is believed that a large dose of rabies vaccine stimulates the activity of lymphocytes to a greater extent and affects cancer cells, and it is proposed that rabies vaccine can be used as a new treatment for cancer. Methods and improving the efficacy of existing cancer immunotherapy (FaiderbeS, et al. C R Acad Sci III. 1989; 310(3): 49-52; Altinoz MA, et al. Clin TranslOncol. Published online: 16January 2017; Bongiorno E, et al. J Immunol. 2013; 190(1 Supplement): 126-5.). However, a novel rabies immunogenic conjugate of the present invention can stimulate the body to generate cellular immune response, and realize synergy with humoral immunity, thereby generating stronger immune response efficacy. Therefore, we believe that the rabies immunogenic conjugate of the present invention can also be used for the prevention or treatment or adjuvant treatment of cancers including malignant melanoma, cervical cancer and malignant glioma.

The patents on the vaccine containing tetanus toxoid and rabies virus antigen and the preparation method described in the published patent texts of the existing rabies vaccine and its preparation method include CN200910177427.8, CN201010607735.2, CN201110458972.1, CN201210137897.3, US9,296,796B2, US9,220,770B2, US9,200,042B2, US9,056,901B2, US8,956,812B2, US7,090,853B2, CN201210137897.3, however, the technical prevention of the above patents all have the above technical defects and deficiencies.

In summary, the rabies immunogenic conjugate of the present invention focuses on solving the defects that the existing rabies whole virus particle vaccine does not produce cellular immunity or weak cellular immunity and slow antibody production, and greatly improves the protective efficacy of existing vaccines. The reaction time of the early immune response of the vaccine is accelerated.

Contents of the invention

In order to overcome the deficiencies of the existing rabies whole virus particle vaccine, the object of the present invention is to provide a new rabies immunogenic conjugate and its preparation method, and a new rabies vaccine prepared according to the above immunogenic conjugate. Conjugate vaccines and methods for their preparation. Compared with the prior art, its difference and beneficial effect are:

(1) The structure of the antigen contained in the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate is different from that of the prior art and literature. In the existing literature reports and the scheme disclosed in the above-mentioned patent texts, the rabies virus antigen and the protein represented by tetanus toxoid exist in a mixed form, that is, there is no chemical bond connection between the rabies virus antigen and the tetanus toxoid, and rabies The virus antigen and the tetanus toxoid are not structurally connected, and the rabies virus antigen and the tetanus toxoid exist in a form completely independent of each other. However, in the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate, the rabies virus antigen and the carrier protein including tetanus toxoid are connected by a chemical bond, rabies Viral antigens and carrier proteins form a structural link.

(2) The function of the carrier protein contained in the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate is different from that of the prior art and literature. In the existing literature reports and the schemes disclosed in the above-mentioned patent texts, the protein represented by tetanus toxoid mostly acts as an adjuvant to achieve the purpose of enhancing the immunogenicity of the rabies virus antigen. However, in the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate, the function of the carrier protein is to stimulate the body to produce cellular immunity against the rabies antigen, producing both humoral immunity and cellular immunity. Immunization, so as to achieve the purpose of generating an immune response faster, enhancing immunogenicity, and improving immune persistence.

(3) The preparation method of the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate is different from the prior art and literature. In the existing literature reports and the schemes disclosed in the above-mentioned patent texts, the protein represented by tetanus toxoid is added to the rabies virus stock solution when the semi-finished vaccine stock solution is prepared, and there is no interaction between the tetanus toxoid and the rabies virus antigen. Combining chemical bonds, they exist independently of each other in semi-finished stock solutions and finished products. In the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate, the carrier protein and the rabies virus antigen are chemically bonded to form a rabies immunogenic conjugate, and then the rabies immunogen Sex conjugate is formulated into rabies conjugate vaccine as stoste, and described method comprises the following steps:

(a) Purified to make rabies virus antigen stock solution;

(b) The rabies virus antigen stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies virus antigen-carrier protein immunogenic conjugate.

(c) diluting, preparing and dispensing the rabies virus antigen-carrier protein immunogenic conjugate prepared in step (b) to form a finished vaccine.

(4) The preparation method of the rabies immunogenic conjugate of the present invention and the rabies virus conjugate vaccine prepared according to the rabies immunogenic conjugate is different from the prior art and literature. In the prior art, rabies vaccine needs to add human albumin as a protective agent or stabilizer. In the present invention, the rabies virus antigen is chemically bonded to the carrier protein that completely replaces the function of adding human albumin as a protective agent or stabilizer in the existing rabies vaccine, so the rabies conjugate vaccine of the present invention does not include human albumin.

The present invention provides a rabies immunogenic conjugate (Rabies Immunogenic Conjugate, RIC), comprising a rabies virus antigen (RabAg) and a carrier protein (Carrier Protein, CP), characterized in that the rabies virus antigen at least includes a carrier protein The rabies virus antigen whose protein is linked by a chemical bond, the carrier protein at least includes a carrier protein linked to the rabies virus antigen by a chemical bond, and the chemical bond connects the rabies virus antigen and the carrier protein to form a rabies virus antigen-carrier protein immunogenic conjugate. Linking the carrier protein and the rabies virus antigen through a chemical bond to form a rabies immunogenic conjugate can firstly promote Th1 cells to recognize the carrier protein and stimulate B cells to generate a stronger immune response to the rabies virus antigen-carrier protein immunogenic conjugate, And can induce Th1 cell-mediated immune recall response, and promote more B cells to produce specific antibodies; secondly, the rabies virus antigen-carrier protein immunogenic conjugate is more useful for rapid induction than the increase in the molecular weight of rabies virus antigen The immune response in the body; in addition, the rabies virus antigen and the carrier protein are directly linked by chemical bonds and compared with the preparation of the rabies virus antigen-carrier protein immunogenic conjugate by means of fusion expression, it has the following effects and technical effects and overcomes the Following defect: 1) because rabies virus antigen and carrier protein are connected by chemical bond, keep the respective protein structure and effective action site of rabies virus antigen and carrier protein, keep the immunogenicity of rabies virus antigen; 2) overcome It eliminates the defects that larger fusion proteins are difficult to express, purify and inactivate during the purification process; 3) Rabies virus antigen and carrier protein are easier to obtain, which saves production cost and time. The carrier protein and the rabies virus antigen are connected by chemical bonds to form a rabies immunogenic conjugate, so that the cellular immune response and the humoral immune response can be coordinated in vivo, so that the new rabies immunogenic conjugate can generate an early immune response faster in the human body response, induce higher immune levels, and provide longer-lasting immune protection.

Preferably, the rabies virus antigen and the carrier protein are connected by chemical bonds to the rabies virus antigen, and the carrier protein is connected to the rabies virus antigen by chemical bonds. - a carrier protein immunogenic conjugate.

Preferably, any one of the above-mentioned rabies virus antigens includes whole rabies virus particles, recombinantly expressed rabies virus-like particles, outer membrane fragments of rabies virus prepared after splitting whole rabies virus particles, rabies virus isolated and purified from rabies virus Viral glycoprotein, nucleoprotein isolated and purified from rabies virus, phosphoprotein isolated and purified from rabies virus, matrix protein isolated and purified from rabies virus, polymerase isolated and purified from rabies virus, recombinant expressed rabies virus glycoprotein, recombinant At least one of expressed rabies virus nucleoprotein, recombinantly expressed rabies virus phosphoprotein, recombinantly expressed rabies virus matrix protein and recombinantly expressed rabies virus polymerase.

In any one of the above, preferably, the whole rabies virus particle is a rabies virus antigen obtained by cell culture, inactivation and purification of rabies virus.

In any one of the above, preferably, the rabies virus-like particle is a rabies virus antigen prepared by recombinant expression.

In any one of the above, preferably, the outer membrane fragment of rabies virus is a rabies virus antigen containing spike and matrix protein prepared by lysing whole rabies virus particles.

Preferably in any one of the above, the rabies virus glycoprotein is a rabies virus antigen isolated and purified from rabies virus or recombinantly expressed.

Any one of the above preferably, the carrier protein includes tetanus toxoid (TT), diphtheria toxoid (DT), avirulent variant of diphtheria toxin (CRM197), group B meningococcal outer membrane protein (OMP), Pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), Haemophilus influenzae protein D (PD), pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), Escherichia coli LT, Escherichia coli ST, tuberculin pure protein derivative (PPD), Pseudomonas aeruginosa Bacterial exotoxin A (PEA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg), tetanus At least one of toxin fragment C (TTC).

Any one of the above preferably, the rabies virus antigen is present in the rabies immunogenic conjugate in the form of a rabies virus antigen-carrier protein conjugate, and the rabies virus antigen-carrier protein conjugate comprises a single independently existing and/or more than two rabies virus antigen-carrier protein conjugates, which contain a single independent and/or more than two rabies virus antigens, i.e. rabies virus antigens and RabAg -CP, RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg-CP-RabAg and (RabAg-CP) _n -RabAg-RabAg in at least one form present in rabies In the immunogenic conjugate, wherein "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, and "-" is the chemical bond connecting the rabies virus antigen and the carrier protein, the single independent rabies virus antigen does not pass through the The chemical bonds between the carrier proteins establish structural connections with other single independent rabies virus antigens, n≥1, and the two or more rabies virus antigens establish structural connections with each other through the chemical bonds between the carrier proteins. A single independent and/or two or more rabies virus antigen-carrier protein conjugates effectively increase the immunogenicity of the obtained vaccine, can promote Th1 cells to recognize the carrier protein, and stimulate B cells to be immune to the rabies virus antigen-carrier protein The original conjugate produces a stronger immune response, and can induce Th1 cell-mediated immune recall response, promote more B cells to produce specific antibodies, and make the cellular immune response and humoral immune response synergistic in vivo, thereby enabling new The rabies immunogenic conjugates in humans produce early immune responses faster, induce higher immune levels, and provide longer-lasting immune protection.

Any one of the above preferably, the rabies virus antigen is present in the rabies immunogenic conjugate in the form of a rabies virus antigen-carrier protein conjugate, and the rabies virus antigen-carrier protein conjugate exists as a single independent The rabies virus antigen-carrier protein conjugate (RabAg-CP), the rabies virus antigen-carrier protein conjugate only contains a single rabies virus antigen, that is, the rabies virus antigen exists in the form of RabAg-CP in the rabies immunogenic combination Among them, "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, and "-" is the chemical bond connecting the rabies virus antigen and the carrier protein. A single independent rabies virus antigen does not pass through the chemical bond between the carrier protein Structural links are established with other single independent rabies virus antigens. A single independently existing rabies virus antigen-carrier protein conjugate effectively increases the immunogenicity of the obtained vaccine, can promote Th1 cells to recognize the carrier protein, and stimulate B cells to produce stronger rabies virus antigen-carrier protein immunogenic conjugates It can induce immune memory response mediated by Th1 cells, promote more B cells to produce specific antibodies, and make cellular immune response and humoral immune response synergistic in vivo, thus making new rabies immunogenic conjugates In the human body, an early immune response is generated faster, a higher immune level is induced, and a longer-lasting immune protection effect is provided.

In any one of the above, preferably, the rabies virus antigen is present in the rabies immunogenic conjugate in the form of a rabies virus antigen-carrier protein conjugate, and there are more than two rabies virus antigen-carrier protein conjugates The rabies virus antigen-carrier protein conjugate ((RabAg-CP) _n -RabAg, n≥1), the rabies virus antigen-carrier protein conjugate contains more than 2 rabies virus antigens, that is, the rabies virus antigen is represented by RabAg at least one form of CP-RabAg, RabAg-CP-RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg-CP-RabAg, (RabAg-CP) _n- RabAg is present in the rabies immunogenic conjugate , wherein "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, "-" is the chemical bond connecting the rabies virus antigen and the carrier protein, n≥1, and the two or more rabies virus antigens pass between the carrier protein chemical bonds to establish structural connections with each other. More than two rabies virus antigen-carrier protein conjugates effectively increase the immunogenicity of the obtained vaccine, can promote Th1 cells to recognize the carrier protein, and stimulate B cells to produce stronger rabies virus antigen-carrier protein immunogenic conjugates It can induce immune memory response mediated by Th1 cells, promote more B cells to produce specific antibodies, and make cellular immune response and humoral immune response synergistic in vivo, thus making new rabies immunogenic conjugates In the human body, an early immune response is generated faster, a higher immune level is induced, and a longer-lasting immune protection effect is provided.

In any one of the above, preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein.

Any one of the above preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein and include hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO) and At least one of amino (-NH2).

In any one of the above, preferably, the carrier protein contains one or more than two functional groups chemically bonded to the rabies virus antigen.

Any one of the above preferably, the carrier protein contains one or more than two functional groups that are chemically bonded to the rabies virus antigen, including hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), At least one of amino (-NH2).

Any one of the above preferably, the rabies virus antigen is based on at least one selected from the group consisting of PAS strains, PV strains, PM strains, CVS strains, Nishigara strains, Flury strains, Vnukovo-32 strains, CTN-1V strains, and aGV strains. A rabies virus antigen prepared from a fixed strain of rabies virus, or according to at least one selected from PAS strain, PV strain, PM strain, CVS strain, Nishigara strain, Flury strain, Vnukovo-32 strain, CTN-1V strain, and aGV strain The rabies virus antigen prepared by gene recombination expression of a fixed strain of rabies virus.

Any one of the above is preferably, the chemical bond connection method of the rabies virus antigen and the carrier protein includes carbodiimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester method), N-hydroxysuccinyl At least one of the imide ester method, glutaraldehyde method, diazotization method, succinic anhydride method, carbonyldiimidazole method, maleimide method, disulfide bond method, periodic acid oxidation method, and carboxymethylhydroxylamine method kind.

Any one of the above-mentioned is preferably, the chemical bond connecting agent of the rabies virus antigen and the carrier protein comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-cyclohexyl-3-(2-N-morpholinoethyl)carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 2-ethyl-5-phenylisoxazole-3'-sulfonate (Woodward's reagent K), N,N'-carbonyldiimidazole (CDI), Schiff base formation and reductive amination Reagents such as sodium cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4) at least one.

Any one of the above-mentioned is preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is a direct connection, and the direct connection does not include at least a linker arm (Linker Arm), a spacer arm (Spacer Arm) and a bridging molecule ( Bridging Molecule), the rabies virus antigen and carrier protein are zero-length connection (Zero-LengthLinking) or zero-length cross-linking (Zero-Length Crosslinking) or zero-length bridging (Zero-LengthBridging), connecting rabies virus antigen and carrier protein The chemical bond does not contain additional atoms or molecules between the rabies virus antigen and the carrier protein.

Any one of the above is preferred, the chemical bond connection between the rabies virus antigen and the carrier protein is through at least one connection of a linking arm, a spacer or a bridge molecule, and the newly created linking arm or spacer or bridge The linkage molecule connects the rabies virus antigen and the carrier protein through a chemical bond to form a rabies virus antigen-carrier protein immunogenic conjugate, and the chemical bond connecting the rabies virus antigen and the carrier protein contains new atoms or molecules between the rabies virus antigen and the carrier protein.

Any one of the above preferably, said linking arm or spacer arm or bridging molecule comprises dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimide suberate (DSS), bis(sulfosuccinimidyl) suberate sodium salt (BS3), disuccinimide tartrate (DST), Disulfosuccinimidyl tartrate ester (sulfo-DST), bis(2-(succinimidyloxycarbonyloxy)ethyl)sulfone (BSOCOES), bis(2-(sulfosuccinimidyloxycarbonyloxy) ) ethyl) sulfone (sulfo-BSOCOES), ethylene glycol-bis(succinimidyl ester succinate) (EGS), ethylene glycol-bis(sulfosuccinimidyl ester succinate) ( sulfo-EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimide ester (DMA), pimeloyl Dimethylimidate (DMP), Dimethylsuberine Dinitrate (DMS), 3,3'- Dithiobispropioimidate (DTBP), 1,4-Di(3'- (2'-dithiopyridine)propionylamino)butane (DPDPB), bismaleimidohexane (BMH), difluorodinitrobenzene (DFDNB), difluorodinitrophenyl Sulfone (DFDNPS), bis(β-(4-azidosalicylamino)ethyl) disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol glycidyl ether, adipic diacyl Hydrazine (ADH), carbohydrazide, diaminodimethylbiphenyl, p-diaminobiphenyl, nitrogen-succinate argonamine-3(2-pyridyldithio)-ester (SPDP), long chain-nitrogen -Succinyl argon ammonia-3(2-pyridinedithio)-ester (LC-SPDP), sulfo long chain-nitrogen-succinate argon ammonia-3(2-pyridinedithio)-ester (sulfo -LC-SPDP), succinimidyloxycarbonyl-methyl-(2-pyridylthio)benzene (SMPT), sulfo long chain-succinimidyloxycarbonyl-methyl-(2 -pyridylthio)benzene (sulfo-LC-SMPT), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), 4-(N -Maleimidomethyl)cyclohexane-1-carboxylic acid 3-thio-N-succinimide ester sodium salt (sulfo-SMCC), maleimidobenzoic acid succinimide ester (MBS), M-maleimide succinimidyl benzoate (sulfo-MBS), N-succinimide (4-iodoacetyl)aminobenzoic acid (SIAB), sulfo-N - Succinimidyl (4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), succinimidyl 4-(4-maleimidophenyl)butanoic acid ester (SMPB), sulfosuccinate Imido-4-(P-maleimidophenyl)butyrate (sulfo-SMPB), 4-maleimidobutanoic acid-N-succinimide ester (GMBS), sulfomaleimidobutyric acid-N-succinimidyl ester (sulfo-GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX ), succinimidyl-6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)caproic acid (SIAXX), succinimidyl-4-(((iodoacetyl) base)amino)methyl)cyclohexane-1-carboxylic acid (SIAC), succinimidyl-6-(((((4(iodoacetyl)amino)methyl)cyclohexane-for-1 -carbonyl)amino)caproic acid (SIACX), 4-nitrophenyl iodoacetic acid (NPIA), 4-(4-N-maleimidobenzylamino ester) butyric acid hydrazide (MPBH), 4- N-maleimidomethylcyclohexane-1-carboxyhydrazide (M2C2H), 3-(2-pyridyldithio)propionohydrazide (PDPH), N-hydroxysuccinimide-4 -Azidosalicylic acid (NHS-ASA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-ASA), sulfosuccinimide-4-azide Salicylaminocaproic acid (sulfo-NHS-LC-ASA), sulfosuccinimidyl-2-(P-azido-salicylamido)ethyl-1,3'-dithiopropane ester (SASD), succinimidyl-4-azidobenzoate (HSAB), sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB), N- Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH), sulfosuccinimidyl-6-(4'-azido- 2'-Nitrophenylamino)hexanoate (sulfo-SANPAH), 5-azido-2-nitrobenzoic acid-N-succinimide ester (ANB-NOS), sulfosuccinimide -2-(M-nitroazido-benzamido)-ethyl-1,3'-dithiodipropionate (SAND), N-succinimidyl-(4-azido phenyl)-1,3'-dithiopropionate (SADP), N-sulfosuccinimidyl-(4-azidophenyl)-1,3'-dithiopropanoic acid Ester (sulfo-SADP), sulfosuccinimidyl-4-(P-azidophenyl)butanoic acid (Sulfo-SADP), sulfosuccinimidyl-2-(7-azido-4- Methylmethylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (SAED), sulfosuccinimide-7-azido-4-methylcoumarin Sulfo-3-acetate (Sulfo-SAMCA), p-nitrophenyldiazopyruvate (pNPDP), p-nitrophenyl-2-diazo3,3,3-trifluoropropionic acid (PNP-DTP ), 1-(p-azidosalicylamino)-4-(iodoacetamido)butane (ASIB), N-(4-(p-azidosalicylamido)butyl)-3' -(2'-pyridyldithio)propionamide (APDP), two Benzophenone-4-iodoacetamide, benzophenone-4-maleimide, p-azidobenzohydrazide, 4-(P-azidosalicylamido)-butylamine (ASBA) , P-azidophenylglyoxal (APG), 4-azido-2-nitrophenylbiotin-4-nitrophenyl ester (ABNP), sulfosuccinimidyl-2-( 6-(Biotinamido)-2-(p-azidobenzamido)caproylamino)ethyl-1,3-dithiopropionate (sulfo-SBED), methanethiosulfonate Nitrotetrafluoro-long chain-biotin (MTS-ATF-biotin), methanethiosulfonate azidotetrafluorobiotin (MTS-ATF-LC-biotin), tris (hydroxymethyl) phosphine ( THP), at least one of tris(hydroxymethyl)phosphopropionic acid (THPP).

In any one of the above, preferably, the rabies virus antigen is activated and then connected to the carrier protein through a chemical bond.

In any one of the above, preferably, the carrier protein is activated and then linked with the rabies virus antigen through a chemical bond.

In any one of the above, preferably, the rabies virus antigen and the carrier protein are respectively activated and then connected by chemical bonds.

In any one of the above, preferably, the rabies immunogenic conjugate is used for the diagnosis, prevention and treatment of rabies.

In any one of the above, preferably, the rabies immunogenic conjugate is used for the prevention, treatment or adjuvant treatment of cancer.

The present invention also provides a method for preparing a rabies immunogenic conjugate, said method comprising the following steps:

(a) Purified to make rabies virus antigen stock solution;

(b) The rabies virus antigen stock solution prepared in step (a) is chemically bonded to a carrier protein, and then purified to prepare a rabies virus antigen-carrier protein immunogenic conjugate.

Linking the carrier protein and the rabies virus antigen through a chemical bond to form a rabies immunogenic conjugate can firstly promote Th1 cells to recognize the carrier protein and stimulate B cells to generate a stronger immune response to the rabies virus antigen-carrier protein immunogenic conjugate, And can induce Th1 cell-mediated immune recall response, and promote more B cells to produce specific antibodies; secondly, the rabies virus antigen-carrier protein immunogenic conjugate is more useful for rapid induction than the increase in the molecular weight of rabies virus antigen The immune response in the body; in addition, the rabies virus antigen and the carrier protein are directly linked by chemical bonds and compared with the preparation of the rabies virus antigen-carrier protein immunogenic conjugate by means of fusion expression, it has the following effects and technical effects and overcomes the Following defect: 1) because rabies virus antigen and carrier protein are connected by chemical bond, keep the respective protein structure and effective action site of rabies virus antigen and carrier protein, keep the immunogenicity of rabies virus antigen; 2) overcome It eliminates the defects that larger fusion proteins are difficult to express, purify and inactivate during the purification process; 3) Rabies virus antigen and carrier protein are easier to obtain, which saves production cost and time. The carrier protein and the rabies virus antigen are connected by chemical bonds to form a rabies immunogenic conjugate, so that the cellular immune response and the humoral immune response can be coordinated in vivo, so that the new rabies immunogenic conjugate can generate an early immune response faster in the human body response, induce higher immune levels, and provide longer-lasting immune protection.

Preferably, the rabies virus antigens include whole rabies virus particles, recombinantly expressed rabies virus-like particles, outer membrane fragments of rabies virus prepared after cleavage of whole rabies virus particles, canine virus glycoproteins isolated and purified from rabies virus , nucleoprotein isolated and purified from rabies virus, phosphoprotein isolated and purified from rabies virus, matrix protein isolated and purified from rabies virus, polymerase isolated and purified from rabies virus, recombinantly expressed rabies virus glycoprotein, recombinantly expressed rabies At least one of viral nucleoprotein, recombinantly expressed rabies virus phosphoprotein, recombinantly expressed rabies virus matrix protein, and recombinantly expressed rabies virus polymerase.

Preferably, in any one of the above, in step (a), the rabies virus antigen stock solution is obtained by inoculating cells with fixed rabies virus seeds, culturing, harvesting the virus liquid, inactivating, and purifying to prepare the stock solution of whole rabies virus particles.

Preferably, in any one of the above, in step (a), the rabies virus antigen stock solution is a rabies virus-like particle stock solution prepared through recombinant expression and purification.

Any one of the above is preferably, in step (a), the rabies virus antigen stock solution is the rabies virus outer membrane fragment stock solution obtained according to the following steps, rabies virus fixed virus seed inoculated cells, cultured, harvested virus solution, inactivated, Purified whole rabies virus particle stock solution; the rabies whole virus particle stock solution prepared in the above steps is cracked and purified to make rabies virus outer membrane fragment stock solution.

Preferably, in any one of the above, in step (a), the rabies virus antigen stock solution is purified rabies virus glycoprotein stock solution.

Any one of the above preferably, the carrier protein is selected from tetanus toxoid (TT), diphtheria toxoid (DT), avirulent variant of diphtheria toxin (CRM197), group B meningococcal outer membrane protein (OMP) , pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), Haemophilus influenzae protein D (PD), pertussis toxin (PT), pertussis filamentous hemagglutination FHA, pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), Escherichia coli LT, Escherichia coli ST, tuberculin pure protein derivative (PPD), pseudomonas aeruginosa Bacterial exotoxin A (PEA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg), broken At least one of the typhoid toxin fragment C (TTC).

Any one of the above preferably, the rabies virus antigen is based on at least one selected from the group consisting of PAS strains, PV strains, PM strains, CVS strains, Nishigara strains, Flury strains, Vnukovo-32 strains, CTN-1V strains, and aGV strains. A rabies virus antigen prepared from a fixed strain of rabies virus, or according to at least one selected from PAS strain, PV strain, PM strain, CVS strain, Nishigara strain, Flury strain, Vnukovo-32 strain, CTN-1V strain, and aGV strain The rabies virus antigen prepared by gene recombination expression of a fixed strain of rabies virus.

In any one of the above, preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein.

Any one of the above preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein and include hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO) and At least one of amino (-NH2).

In any one of the above, preferably, the carrier protein contains one or more than two functional groups chemically bonded to the rabies virus antigen.

Any one of the above preferably, the carrier protein contains one or more than two functional groups that are chemically bonded to the rabies virus antigen, including hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), At least one of amino (-NH2).

Any one of the above is preferably, the chemical bond connection method of the rabies virus antigen and the carrier protein includes carbodiimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester method), N-hydroxysuccinyl At least one of the imide ester method, glutaraldehyde method, diazotization method, succinic anhydride method, carbonyldiimidazole method, maleimide method, disulfide bond method, periodic acid oxidation method, and carboxymethylhydroxylamine method A sort of.

Any one of the above-mentioned is preferably, the chemical bond connecting agent of the rabies virus antigen and the carrier protein comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-cyclohexyl-3-(2-N-morpholinoethyl)carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 2-ethyl-5-phenylisoxazole-3'-sulfonate (Woodward's reagent K), N,N'-carbonyldiimidazole (CDI), Schiff base formation and reductive amination Reagents such as at least one of sodium cyanoborohydride (NaBH3CN) or sodium borohydride (NaBH4).

Any one of the above-mentioned is preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is a direct connection, and the direct connection does not include at least a linker arm (Linker Arm), a spacer arm (Spacer Arm) and a bridging molecule ( Bridging Molecule), rabies virus antigen and carrier protein are zero-length linking (Zero-Length Linking) or zero-length cross-linking (Zero-Length Crosslinking) or zero-length bridging (Zero-Length Bridging), connecting rabies virus antigen and carrier protein The chemical bond between the rabies virus antigen and the carrier protein does not contain additional atoms or molecules.

Any one of the above preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is through the connection of the linking arm, spacer or bridging molecule, and the newly created linking arm, spacer or bridging molecule will rabies virus The antigen and the carrier protein are linked by a chemical bond to form a rabies virus antigen-carrier protein immunogenic conjugate, and the chemical bond connecting the rabies virus antigen and the carrier protein contains new atoms or molecules between the rabies virus antigen and the carrier protein.

Any one of the above preferably, said linking arm or spacer arm or bridging molecule comprises dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimide suberate (DSS), bis(sulfosuccinimidyl) suberate sodium salt (BS3), disuccinimide tartrate (DST), Disulfosuccinimidyl tartrate ester (sulfo-DST), bis(2-(succinimidyloxycarbonyloxy)ethyl)sulfone (BSOCOES), bis(2-(sulfosuccinimidyloxycarbonyloxy) ) ethyl) sulfone (sulfo-BSOCOES), ethylene glycol-bis(succinimidyl ester succinate) (EGS), ethylene glycol-bis(sulfosuccinimidyl ester succinate) ( sulfo-EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimide ester (DMA), pimeloyl Dimethylimidate (DMP), Dimethylsuberine Dinitrate (DMS), 3,3'- Dithiobispropioimidate (DTBP), 1,4-Di(3'- (2'-dithiopyridine)propionylamino)butane (DPDPB), bismaleimidohexane (BMH), difluorodinitrobenzene (DFDNB), difluorodinitrophenyl Sulfone (DFDNPS), bis(β-(4-azidosalicylamino)ethyl) disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol glycidyl ether, adipic diacyl Hydrazine (ADH), carbohydrazide, diaminodimethylbiphenyl, p-diaminobiphenyl, nitrogen-succinate argonamine-3(2-pyridyldithio)-ester (SPDP), long chain-nitrogen -Succinyl argon ammonia-3(2-pyridinedithio)-ester (LC-SPDP), sulfo long chain-nitrogen-succinate argon ammonia-3(2-pyridinedithio)-ester (sulfo -LC-SPDP), succinimidyloxycarbonyl-methyl-(2-pyridylthio)benzene (SMPT), sulfo long chain-succinimidyloxycarbonyl-methyl-(2 -pyridylthio)benzene (sulfo-LC-SMPT), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), 4-(N -Maleimidomethyl)cyclohexane-1-carboxylic acid 3-thio-N-succinimide ester sodium salt (sulfo-SMCC), maleimidobenzoic acid succinimide ester (MBS), M-maleimide succinimidyl benzoate (sulfo-MBS), N-succinimide (4-iodoacetyl)aminobenzoic acid (SIAB), sulfo-N - Succinimidyl (4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), succinimidyl 4-(4-maleimidophenyl)butanoic acid ester (SMPB), sulfosuccinate Imido-4-(P-maleimidophenyl)butyrate (sulfo-SMPB), 4-maleimidobutanoic acid-N-succinimide ester (GMBS), sulfomaleimidobutyric acid-N-succinimidyl ester (sulfo-GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX ), succinimidyl-6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)caproic acid (SIAXX), succinimidyl-4-(((iodoacetyl) base)amino)methyl)cyclohexane-1-carboxylic acid (SIAC), succinimidyl-6-(((((4(iodoacetyl)amino)methyl)cyclohexane-for-1 -carbonyl)amino)caproic acid (SIACX), 4-nitrophenyl iodoacetic acid (NPIA), 4-(4-N-maleimidobenzylamino ester) butyric acid hydrazide (MPBH), 4- N-maleimidomethylcyclohexane-1-carboxyhydrazide (M2C2H), 3-(2-pyridyldithio)propionohydrazide (PDPH), N-hydroxysuccinimide-4 -Azidosalicylic acid (NHS-ASA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-ASA), sulfosuccinimide-4-azide Salicylaminocaproic acid (sulfo-NHS-LC-ASA), sulfosuccinimidyl-2-(P-azido-salicylamido)ethyl-1,3'-dithiopropane ester (SASD), succinimidyl-4-azidobenzoate (HSAB), sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB), N- Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH), sulfosuccinimidyl-6-(4'-azido- 2'-Nitrophenylamino)hexanoate (sulfo-SANPAH), 5-azido-2-nitrobenzoic acid-N-succinimide ester (ANB-NOS), sulfosuccinimide -2-(M-nitroazido-benzamido)-ethyl-1,3'-dithiodipropionate (SAND), N-succinimidyl-(4-azido phenyl)-1,3'-dithiopropionate (SADP), N-sulfosuccinimidyl-(4-azidophenyl)-1,3'-dithiopropanoic acid Ester (sulfo-SADP), sulfosuccinimidyl-4-(P-azidophenyl)butanoic acid (Sulfo-SADP), sulfosuccinimidyl-2-(7-azido-4- Methylmethylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (SAED), sulfosuccinimide-7-azido-4-methylcoumarin Sulfo-3-acetate (Sulfo-SAMCA), p-nitrophenyldiazopyruvate (pNPDP), p-nitrophenyl-2-diazo3,3,3-trifluoropropionic acid (PNP-DTP ), 1-(p-azidosalicylamino)-4-(iodoacetamido)butane (ASIB), N-(4-(p-azidosalicylamido)butyl)-3' -(2'-pyridyldithio)propionamide (APDP), two Benzophenone-4-iodoacetamide, benzophenone-4-maleimide, p-azidobenzohydrazide, 4-(P-azidosalicylamido)-butylamine (ASBA) , P-azidophenylglyoxal (APG), 4-azido-2-nitrophenylbiotin-4-nitrophenyl ester (ABNP), sulfosuccinimidyl-2-( 6-(Biotinamido)-2-(p-azidobenzamido)caproylamino)ethyl-1,3-dithiopropionate (sulfo-SBED), methanethiosulfonate Nitrotetrafluoro-long chain-biotin (MTS-ATF-biotin), methanethiosulfonate azidotetrafluorobiotin (MTS-ATF-LC-biotin), tris (hydroxymethyl) phosphine ( THP), at least one of tris(hydroxymethyl)phosphopropionic acid (THPP).

In any one of the above, preferably, the rabies virus antigen is activated and then connected to the carrier protein through a chemical bond.

In any one of the above, preferably, the carrier protein is activated and then connected to the rabies virus antigen through a chemical bond.

In any one of the above, preferably, the rabies virus antigen and the carrier protein are respectively activated and then connected by chemical bonds.

Preferably, any one of the above is that the prepared rabies immunogenic conjugate is used for the diagnosis, prevention and treatment of rabies.

Preferably, any one of the above is that the prepared rabies immunogenic conjugate is used for the prevention, treatment or adjuvant treatment of cancer.

The present invention also provides a rabies conjugate vaccine (Rabies Conjugate Vaccine RCV), comprising a rabies immunogenic conjugate, the rabies immunogenic conjugate comprising a rabies virus antigen and a carrier protein, and the rabies virus antigen at least includes a carrier protein The rabies virus antigen whose protein is linked by a chemical bond, the carrier protein at least includes a carrier protein linked to the rabies virus antigen by a chemical bond, and the chemical bond connects the rabies virus antigen and the carrier protein to form a rabies virus antigen-carrier protein immunogenic conjugate. Linking the carrier protein and the rabies virus antigen through a chemical bond to form a rabies immunogenic conjugate can firstly promote Th1 cells to recognize the carrier protein and stimulate B cells to generate a stronger immune response to the rabies virus antigen-carrier protein immunogenic conjugate, And can induce Th1 cell-mediated immune recall response, and promote more B cells to produce specific antibodies; secondly, the rabies virus antigen-carrier protein immunogenic conjugate is more useful for rapid induction than the increase in the molecular weight of rabies virus antigen The immune response in the body; in addition, the rabies virus antigen and the carrier protein are directly linked by chemical bonds and compared with the preparation of the rabies virus antigen-carrier protein immunogenic conjugate by means of fusion expression, it has the following effects and technical effects and overcomes the Following defect: 1) because rabies virus antigen and carrier protein are connected by chemical bond, keep the respective protein structure and effective action site of rabies virus antigen and carrier protein, keep the immunogenicity of rabies virus antigen; 2) overcome It eliminates the defects that larger fusion proteins are difficult to express, purify and inactivate during the purification process; 3) Rabies virus antigen and carrier protein are easier to obtain, which saves production cost and time. The carrier protein and the rabies virus antigen are connected by chemical bonds to form a rabies immunogenic conjugate, so that the cellular immune response and the humoral immune response can be coordinated in vivo, so that the new rabies immunogenic conjugate can generate an early immune response faster in the human body response, induce higher immune levels, and provide longer-lasting immune protection.

Preferably, the rabies virus antigen and the carrier protein are connected by chemical bonds to the rabies virus antigen, and the carrier protein is connected to the rabies virus antigen by chemical bonds. - a carrier protein immunogenic conjugate.

Preferably, any one of the above-mentioned rabies virus antigens includes whole rabies virus particles, recombinantly expressed rabies virus-like particles, outer membrane fragments of rabies virus prepared after splitting whole rabies virus particles, rabies virus isolated and purified from rabies virus Viral glycoprotein, nucleoprotein isolated and purified from rabies virus, phosphoprotein isolated and purified from rabies virus, matrix protein isolated and purified from rabies virus, polymerase isolated and purified from rabies virus, recombinant expressed rabies virus glycoprotein, recombinant At least one of expressed rabies virus nucleoprotein, recombinantly expressed rabies virus phosphoprotein, recombinantly expressed rabies virus matrix protein and recombinantly expressed rabies virus polymerase.

In any one of the above, preferably, the whole rabies virus particle is a rabies virus antigen obtained by cell culture, inactivation and purification of rabies virus.

In any one of the above, preferably, the rabies virus-like particle is a rabies virus antigen prepared by recombinant expression.

In any one of the above, preferably, the outer membrane fragment of rabies virus is a rabies virus antigen containing spike and matrix protein prepared by lysing whole rabies virus particles.

Preferably in any one of the above, the rabies virus glycoprotein is a rabies virus antigen isolated and purified from rabies virus or recombinantly expressed.

Any one of the above preferably, the carrier protein includes tetanus toxoid (TT), diphtheria toxoid (DT), avirulent variant of diphtheria toxin (CRM197), group B meningococcal outer membrane protein (OMP), Pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), Haemophilus influenzae protein D (PD), pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), Escherichia coli LT, Escherichia coli ST, tuberculin pure protein derivative (PPD), Pseudomonas aeruginosa Bacterial exotoxin A (PEA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg), tetanus At least one of toxin fragment C (TTC).

Preferably in any one of the above, the rabies conjugate vaccine includes an adjuvant.

Any one of the above preferably, the adjuvant includes aluminum adjuvant, calcium phosphate adjuvant, cholera toxin (CT), cholera toxin B subunit (CTB), pertussis toxin (PT), pertussis toxin B subunit (PTB ), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), saponin QS-21, α-tocopherol, squalene, liposomes, liposomes, monophosphates Lipid A (MPL-A), MF59, Virosomes, Polyglycolide (PLA) Microspheres, Polylactic-Glycolic Acid (PLGA) Microspheres, Lipid-Cholesterol (DC-Chol) , dimethyl dioctadecyl quaternary ammonium bromide (DDA), immunostimulatory complex (ISCOM), Montanide ISA50, Montanide ISA51, Montanide ISA206, Montanide ISA720, Montanide ISA series adjuvants, AS01, AS02, AS03, AS04, AS series adjuvants, muramyl dipeptide (MDP), bacterial lipopolysaccharide (OM-174), Escherichia coli heat-labile toxin (LT), IL-1, IL-2, IL-6, IL-12, IL -15, IL-18, IFN-γ, GM-CSF, CpG oligonucleotides, trehalose dipycolate (TDM), containing polyinosine nucleotide (Poly I) and/or polycytosine core At least one of the substances of the pica adjuvant of glycoside (Poly C).

Any one of the above preferably, the rabies virus antigen is present in the rabies immunogenic conjugate in the form of a rabies virus antigen-carrier protein conjugate, and the rabies virus antigen-carrier protein conjugate comprises a single independently existing and/or more than two rabies virus antigen-carrier protein conjugates, which contain a single independent and/or more than two rabies virus antigens, i.e. rabies virus antigens and RabAg -CP, RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg-CP-RabAg and (RabAg-CP) _n -RabAg in at least one form present in the rabies immunogen Among them, "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, and "-" is the chemical bond connecting the rabies virus antigen and the carrier protein, and the single independent rabies virus antigen does not pass through the carrier protein The chemical bonds between them establish structural connections with other single independent rabies virus antigens, n≥1, and the two or more rabies virus antigens establish structural connections with each other through chemical bonds with carrier proteins. A single independent and/or two or more rabies virus antigen-carrier protein conjugates effectively increase the immunogenicity of the obtained vaccine, can promote Th1 cells to recognize the carrier protein, and stimulate B cells to be immune to the rabies virus antigen-carrier protein The original conjugate produces a stronger immune response, and can induce Th1 cell-mediated immune recall response, promote more B cells to produce specific antibodies, and make the cellular immune response and humoral immune response synergistic in vivo, thereby enabling new The rabies immunogenic conjugates in humans produce early immune responses faster, induce higher immune levels, and provide longer-lasting immune protection.

Any one of the above preferably, the rabies virus antigen is present in the rabies immunogenic conjugate in the form of a rabies virus antigen-carrier protein conjugate, and the rabies virus antigen-carrier protein conjugate exists as a single independent The rabies virus antigen-carrier protein conjugate (RabAg-CP), the rabies virus antigen-carrier protein conjugate only contains a single rabies virus antigen, that is, the rabies virus antigen exists in the form of RabAg-CP in the rabies immunogenic combination Among them, "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, and "-" is the chemical bond connecting the rabies virus antigen and the carrier protein. A single independent rabies virus antigen does not pass through the chemical bond between the carrier protein Structural links are established with other single independent rabies virus antigens. A single independently existing rabies virus antigen-carrier protein conjugate effectively increases the immunogenicity of the obtained vaccine, can promote Th1 cells to recognize the carrier protein, and stimulate B cells to produce stronger rabies virus antigen-carrier protein immunogenic conjugates It can induce immune memory response mediated by Th1 cells, promote more B cells to produce specific antibodies, and make cellular immune response and humoral immune response synergistic in vivo, thus making new rabies immunogenic conjugates In the human body, an early immune response is generated faster, a higher immune level is induced, and a longer-lasting immune protection effect is provided.

In any one of the above, preferably, the rabies virus antigen is present in the rabies immunogenic conjugate in the form of a rabies virus antigen-carrier protein conjugate, and there are more than two rabies virus antigen-carrier protein conjugates The rabies virus antigen-carrier protein conjugate ((RabAg-CP) _n -RabAg, n≥1), the rabies virus antigen-carrier protein conjugate contains more than 2 rabies virus antigens, that is, the rabies virus antigen is represented by RabAg at least one form of CP-RabAg, RabAg-CP-RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg-CP-RabAg, (RabAg-CP) _n- RabAg is present in the rabies immunogenic conjugate , wherein "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, "-" is the chemical bond connecting the rabies virus antigen and the carrier protein, n≥1, and the two or more rabies virus antigens pass between the carrier protein chemical bonds to establish structural connections with each other. More than two rabies virus antigen-carrier protein conjugates effectively increase the immunogenicity of the obtained vaccine, can promote Th1 cells to recognize the carrier protein, and stimulate B cells to produce stronger rabies virus antigen-carrier protein immunogenic conjugates It can induce immune memory response mediated by Th1 cells, promote more B cells to produce specific antibodies, and make cellular immune response and humoral immune response synergistic in vivo, thus making new rabies immunogenic conjugates In the human body, an early immune response is generated faster, a higher immune level is induced, and a longer-lasting immune protection effect is provided.

In any one of the above, preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein.

Any one of the above-mentioned is preferably, the functional group that the rabies virus antigen is chemically bonded to the carrier protein includes at least hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO) amino (-NH2) A sort of.

In any one of the above, preferably, the carrier protein contains one or more than two functional groups chemically bonded to the rabies virus antigen.

Any one of the above-mentioned is preferably, the functional groups that the carrier protein is chemically bonded to the rabies virus antigen include hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), amino (-NH2) at least one.

Any one of the above preferably, the rabies virus antigen is based on at least one selected from the group consisting of PAS strains, PV strains, PM strains, CVS strains, Nishigara strains, Flury strains, Vnukovo-32 strains, CTN-1V strains, and aGV strains. A rabies virus antigen prepared from a fixed strain of rabies virus, or according to at least one selected from PAS strain, PV strain, PM strain, CVS strain, Nishigara strain, Flury strain, Vnukovo-32 strain, CTN-1V strain, and aGV strain The rabies virus antigen prepared by gene recombination expression of a fixed strain of rabies virus.

Any one of the above is preferably, the chemical bond connection method of the rabies virus antigen and the carrier protein includes carbodiimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester method), N-hydroxysuccinyl At least one of the imide ester method, glutaraldehyde method, diazotization method, succinic anhydride method, carbonyldiimidazole method, maleimide method, disulfide bond method, periodic acid oxidation method, and carboxymethylhydroxylamine method kind.

Any one of the above-mentioned is preferably, the chemical bond connecting agent of the rabies virus antigen and the carrier protein comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-cyclohexyl-3-(2-N-morpholinoethyl)carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 2-ethyl-5-phenylisoxazole-3'-sulfonate (Woodward's reagent K), N,N'-carbonyldiimidazole (CDI), Schiff base formation and reductive amination Reagents such as sodium cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4) at least one.

Any one of the above preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is a direct connection, and the direct connection does not include at least a linker arm (Linker Arm), a spacer arm (Spacer Arm) and a bridging molecule (Bridging Molecule). ), the rabies virus antigen and carrier protein are zero-length connection (Zero-LengthLinking) or zero-length crosslinking (Zero-Length Crosslinking) or zero-length bridging (Zero-LengthBridging), the chemical bond connecting rabies virus antigen and carrier protein No additional atoms or molecules are included between the rabies virus antigen and the carrier protein.

Any one of the above preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is connected by at least one way of connecting arm, spacer or bridging molecule, and the newly created connecting arm or spacer or bridging molecule The rabies virus antigen and the carrier protein are connected through chemical bonds to form a rabies virus antigen-carrier protein immunogenic conjugate, and the chemical bond connecting the rabies virus antigen and the carrier protein contains new atoms or molecules between the rabies virus antigen and the carrier protein.

Any one of the above preferably, said linking arm or spacer arm or bridging molecule comprises dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimide suberate (DSS), bis(sulfosuccinimidyl) suberate sodium salt (BS3), disuccinimide tartrate (DST), Disulfosuccinimidyl tartrate ester (sulfo-DST), bis(2-(succinimidyloxycarbonyloxy)ethyl)sulfone (BSOCOES), bis(2-(sulfosuccinimidyloxycarbonyloxy) ) ethyl) sulfone (sulfo-BSOCOES), ethylene glycol-bis(succinimidyl ester succinate) (EGS), ethylene glycol-bis(sulfosuccinimidyl ester succinate) ( sulfo-EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimide ester (DMA), pimeloyl Dimethylimidate (DMP), Dimethylsuberine Dinitrate (DMS), 3,3'- Dithiobispropioimidate (DTBP), 1,4-Di(3'- (2'-dithiopyridine)propionylamino)butane (DPDPB), bismaleimidohexane (BMH), difluorodinitrobenzene (DFDNB), difluorodinitrophenyl Sulfone (DFDNPS), bis(β-(4-azidosalicylamino)ethyl) disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol glycidyl ether, adipic diacyl Hydrazine (ADH), carbohydrazide, diaminodimethylbiphenyl, p-diaminobiphenyl, nitrogen-succinate argonamine-3(2-pyridyldithio)-ester (SPDP), long chain-nitrogen -Succinyl argon ammonia-3(2-pyridinedithio)-ester (LC-SPDP), sulfo long chain-nitrogen-succinate argon ammonia-3(2-pyridinedithio)-ester (sulfo -LC-SPDP), succinimidyloxycarbonyl-methyl-(2-pyridylthio)benzene (SMPT), sulfo long chain-succinimidyloxycarbonyl-methyl-(2 -pyridylthio)benzene (sulfo-LC-SMPT), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), 4-(N -Maleimidomethyl)cyclohexane-1-carboxylic acid 3-thio-N-succinimide ester sodium salt (sulfo-SMCC), maleimidobenzoic acid succinimide ester (MBS), M-maleimide succinimidyl benzoate (sulfo-MBS), N-succinimide (4-iodoacetyl)aminobenzoic acid (SIAB), sulfo-N - Succinimidyl (4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), succinimidyl 4-(4-maleimidophenyl)butanoic acid ester (SMPB), sulfosuccinate Imido-4-(P-maleimidophenyl)butyrate (sulfo-SMPB), 4-maleimidobutanoic acid-N-succinimide ester (GMBS), sulfomaleimidobutyric acid-N-succinimidyl ester (sulfo-GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX ), succinimidyl-6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)caproic acid (SIAXX), succinimidyl-4-(((iodoacetyl) base)amino)methyl)cyclohexane-1-carboxylic acid (SIAC), succinimidyl-6-(((((4(iodoacetyl)amino)methyl)cyclohexane-for-1 -carbonyl)amino)caproic acid (SIACX), 4-nitrophenyl iodoacetic acid (NPIA), 4-(4-N-maleimidobenzylamino ester) butyric acid hydrazide (MPBH), 4- N-maleimidomethylcyclohexane-1-carboxyhydrazide (M2C2H), 3-(2-pyridyldithio)propionohydrazide (PDPH), N-hydroxysuccinimide-4 -Azidosalicylic acid (NHS-ASA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-ASA), sulfosuccinimide-4-azide Salicylaminocaproic acid (sulfo-NHS-LC-ASA), sulfosuccinimidyl-2-(P-azido-salicylamido)ethyl-1,3'-dithiopropane ester (SASD), succinimidyl-4-azidobenzoate (HSAB), sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB), N- Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH), sulfosuccinimidyl-6-(4'-azido- 2'-Nitrophenylamino)hexanoate (sulfo-SANPAH), 5-azido-2-nitrobenzoic acid-N-succinimide ester (ANB-NOS), sulfosuccinimide -2-(M-nitroazido-benzamido)-ethyl-1,3'-dithiodipropionate (SAND), N-succinimidyl-(4-azido phenyl)-1,3'-dithiopropionate (SADP), N-sulfosuccinimidyl-(4-azidophenyl)-1,3'-dithiopropanoic acid Ester (sulfo-SADP), sulfosuccinimidyl-4-(P-azidophenyl)butanoic acid (Sulfo-SADP), sulfosuccinimidyl-2-(7-azido-4- Methylmethylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (SAED), sulfosuccinimide-7-azido-4-methylcoumarin Sulfo-3-acetate (Sulfo-SAMCA), p-nitrophenyldiazopyruvate (pNPDP), p-nitrophenyl-2-diazo3,3,3-trifluoropropionic acid (PNP-DTP ), 1-(p-azidosalicylamino)-4-(iodoacetamido)butane (ASIB), N-(4-(p-azidosalicylamido)butyl)-3' -(2'-pyridyldithio)propionamide (APDP), two Benzophenone-4-iodoacetamide, benzophenone-4-maleimide, p-azidobenzohydrazide, 4-(P-azidosalicylamido)-butylamine (ASBA) , P-azidophenylglyoxal (APG), 4-azido-2-nitrophenylbiotin-4-nitrophenyl ester (ABNP), sulfosuccinimidyl-2-( 6-(Biotinamido)-2-(p-azidobenzamido)caproylamino)ethyl-1,3-dithiopropionate (sulfo-SBED), methanethiosulfonate Nitrotetrafluoro-long chain-biotin (MTS-ATF-biotin), methanethiosulfonate azidotetrafluorobiotin (MTS-ATF-LC-biotin), tris(hydroxymethyl)phosphine ( THP), at least one of tris(hydroxymethyl)phosphopropionic acid (THPP).

In any one of the above, preferably, the rabies virus antigen is activated and then connected to the carrier protein through a chemical bond.

In any one of the above, preferably, the carrier protein is activated and then linked with the rabies virus antigen through a chemical bond.

In any one of the above, preferably, the rabies virus antigen and the carrier protein are respectively activated and then connected by chemical bonds.

In any one of the above, it is preferred that the titer unit of the rabies conjugate vaccine is ≥ 2.5 IU/dose.

Preferably in any one of the above, the rabies conjugate vaccine includes a protective agent and/or a stabilizer.

In any one of the above, preferably, the protective agent and/or stabilizer of the rabies conjugate vaccine includes human albumin.

According to any one of the above, it is preferred that the rabies conjugate vaccine does not include protective agents and/or stabilizers.

Preferably in any of the above, the rabies conjugate vaccine does not include human albumin.

Preferably in any of the above, the rabies conjugate vaccine does not contain any preservatives.

According to any one of the above, it is preferred that the rabies conjugate vaccine contains a preservative.

Preferably in any of the above, the preservative includes at least one of thimerosal, 2-phenoxyethanol, and benzyl alcohol.

Any one of the above is preferably that the rabies conjugate vaccine is used for the diagnosis, prevention and treatment of rabies.

Any one of the above is preferably that the rabies conjugate vaccine is used for the prevention, treatment or adjuvant treatment of cancer.

The present invention also provides a method for preparing rabies conjugate vaccine, said method comprising the following steps:

(a) Purified to make rabies virus antigen stock solution;

(b) The rabies virus antigen stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies virus antigen-carrier protein immunogenic conjugate.

(c) diluting, preparing and dispensing the rabies virus antigen-carrier protein immunogenic conjugate prepared in step (b) to form a finished vaccine.

Linking the carrier protein and the rabies virus antigen through a chemical bond to form a rabies immunogenic conjugate can firstly promote Th1 cells to recognize the carrier protein and stimulate B cells to generate a stronger immune response to the rabies virus antigen-carrier protein immunogenic conjugate, And can induce Th1 cell-mediated immune recall response, and promote more B cells to produce specific antibodies; secondly, the rabies virus antigen-carrier protein immunogenic conjugate is more useful for rapid induction than the increase in the molecular weight of rabies virus antigen The immune response in the body; in addition, the rabies virus antigen and the carrier protein are directly linked by chemical bonds and compared with the preparation of the rabies virus antigen-carrier protein immunogenic conjugate by means of fusion expression, it has the following effects and technical effects and overcomes the Following defect: 1) because rabies virus antigen and carrier protein are connected by chemical bond, keep the respective protein structure and effective action site of rabies virus antigen and carrier protein, keep the immunogenicity of rabies virus antigen; 2) overcome It eliminates the defects that larger fusion proteins are difficult to express, purify and inactivate during the purification process; 3) Rabies virus antigen and carrier protein are easier to obtain, which saves production cost and time. The carrier protein and the rabies virus antigen are connected by chemical bonds to form a rabies immunogenic conjugate, so that the cellular immune response and the humoral immune response can be coordinated in vivo, so that the new rabies immunogenic conjugate can generate an early immune response faster in the human body response, induce higher immune levels, and provide longer-lasting immune protection.

Preferably, the rabies virus antigens include whole rabies virus particles, recombinantly expressed rabies virus-like particles, rabies virus outer membrane fragments prepared after splitting whole rabies virus particles, rabies virus glycoproteins isolated and purified from rabies virus, Nucleoprotein isolated and purified from rabies virus, phosphoprotein isolated and purified from rabies virus, matrix protein isolated and purified from rabies virus, polymerase isolated and purified from rabies virus, recombinantly expressed rabies virus glycoprotein, recombinantly expressed rabies virus At least one of nucleoprotein, recombinantly expressed rabies virus phosphoprotein, recombinantly expressed rabies virus matrix protein, and recombinantly expressed rabies virus polymerase.

In any one of the above, preferably, the rabies virus antigen stock solution is obtained by inoculating cells with fixed rabies virus seeds, culturing, harvesting virus liquid, inactivating, and purifying to prepare a stock solution of whole rabies virus particles.

Preferably, in any one of the above, in step (a), the rabies virus antigen stock solution is a rabies virus-like particle stock solution prepared through recombinant expression and purification.

Any one of the above is preferably, in step (a), the rabies virus antigen stock solution is the rabies virus outer membrane fragment stock solution obtained according to the following steps, rabies virus fixed virus seed inoculated cells, cultured, harvested virus solution, inactivated, Purified whole rabies virus particle stock solution; the rabies whole virus particle stock solution prepared in the above steps is cracked and purified to make rabies virus outer membrane fragment stock solution.

Preferably, in any one of the above, in step (a), the rabies virus antigen stock solution is purified rabies virus glycoprotein stock solution.

Any one of the above preferably, the carrier protein is selected from tetanus toxoid (TT), diphtheria toxoid (DT), avirulent variant of diphtheria toxin (CRM197), group B meningococcal outer membrane protein (OMP) , pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), Haemophilus influenzae protein D (PD), pertussis toxin (PT), pertussis filamentous hemagglutination FHA, pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), Escherichia coli LT, Escherichia coli ST, tuberculin pure protein derivative (PPD), pseudomonas aeruginosa Bacterial exotoxin A (PEA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg), broken At least one of the typhoid toxin fragment C (TTC).

In any one of the above, preferably, the rabies conjugate vaccine comprises an adjuvant.

Any one of the above preferably, the adjuvant comprises aluminum adjuvant, calcium phosphate adjuvant, cholera toxin (CT), cholera toxin B subunit (CTB), pertussis toxin (PT), pertussis toxin B subunit (PTB ), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), saponin QS-21, α-tocopherol, squalene, liposomes, liposomes, monophosphates Lipid A (MPL-A), MF59, Virosomes, Polyglycolide (PLA) Microspheres, Polylactic-Glycolic Acid (PLGA) Microspheres, Lipid-Cholesterol (DC-Chol) , dimethyl dioctadecyl quaternary ammonium bromide (DDA), immunostimulatory complex (ISCOM), Montanide ISA50, Montanide ISA51, Montanide ISA206, Montanide ISA720, Montanide ISA series adjuvants, AS01, AS02, AS03, AS04, AS series adjuvants, muramyl dipeptide (MDP), bacterial lipopolysaccharide (OM-174), Escherichia coli heat-labile toxin (LT), IL-1, IL-2, IL-6, IL-12, IL -15, IL-18, IFN-γ, GM-CSF, CpG oligonucleotides, trehalose dipycolate (TDM), containing polyinosine nucleotide (Poly I) and/or polycytosine core At least one of the substances of the pica adjuvant of glycoside (Poly C).

Any one of the above preferably, the rabies virus antigen is based on at least one selected from the group consisting of PAS strains, PV strains, PM strains, CVS strains, Nishigara strains, Flury strains, Vnukovo-32 strains, CTN-1V strains, and aGV strains. A rabies virus antigen prepared from a fixed strain of rabies virus, or according to at least one selected from PAS strain, PV strain, PM strain, CVS strain, Nishigara strain, Flury strain, Vnukovo-32 strain, CTN-1V strain, and aGV strain The rabies virus antigen prepared by gene recombination expression of a fixed strain of rabies virus.

In any one of the above, preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein.

Any one of the above preferably, the rabies virus antigen contains one or more than two functional groups that are chemically bonded to the carrier protein and include hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO) and At least one of amino (-NH2).

In any one of the above, preferably, the carrier protein contains one or more than two functional groups chemically bonded to the rabies virus antigen.

Any one of the above preferably, the carrier protein contains one or more than two functional groups that are chemically bonded to the rabies virus antigen, including hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), At least one of amino (-NH2).

Any one of the above is preferably, the chemical bond connection method of the rabies virus antigen and the carrier protein includes carbodiimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester method), N-hydroxysuccinyl At least one of the imide ester method, glutaraldehyde method, diazotization method, succinic anhydride method, carbonyldiimidazole method, maleimide method, disulfide bond method, periodic acid oxidation method, and carboxymethylhydroxylamine method A sort of.

Any one of the above-mentioned is preferably, the chemical bond connecting agent of the rabies virus antigen and the carrier protein comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-cyclohexyl-3-(2-N-morpholinoethyl)carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 2-ethyl-5-phenylisoxazole-3'-sulfonate (Woodward's reagent K), N,N'-carbonyldiimidazole (CDI), Schiff base formation and reductive amination Reagents such as at least one of sodium cyanoborohydride (NaBH3CN) or sodium borohydride (NaBH4).

Any one of the above-mentioned is preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is a direct connection, and the direct connection does not include at least a linker arm (Linker Arm), a spacer arm (Spacer Arm) and a bridging molecule ( Bridging Molecule), rabies virus antigen and carrier protein are zero-length linking (Zero-Length Linking) or zero-length cross-linking (Zero-Length Crosslinking) or zero-length bridging (Zero-Length Bridging), connecting rabies virus antigen and carrier protein The chemical bond between the rabies virus antigen and the carrier protein does not contain additional atoms or molecules.

Any one of the above preferably, the chemical bond connection of the rabies virus antigen and the carrier protein is through the connection of the linking arm, spacer or bridging molecule, and the newly created linking arm, spacer or bridging molecule will rabies virus The antigen and the carrier protein are linked by a chemical bond to form a rabies virus antigen-carrier protein immunogenic conjugate, and the chemical bond connecting the rabies virus antigen and the carrier protein contains new atoms or molecules between the rabies virus antigen and the carrier protein.

Any one of the above preferably, said linking arm or spacer arm or bridging molecule comprises dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimide suberate (DSS), bis(sulfosuccinimidyl) suberate sodium salt (BS3), disuccinimide tartrate (DST), Disulfosuccinimidyl tartrate ester (sulfo-DST), bis(2-(succinimidyloxycarbonyloxy)ethyl)sulfone (BSOCOES), bis(2-(sulfosuccinimidyloxycarbonyloxy) ) ethyl) sulfone (sulfo-BSOCOES), ethylene glycol-bis(succinimidyl ester succinate) (EGS), ethylene glycol-bis(sulfosuccinimidyl ester succinate) ( sulfo-EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimide ester (DMA), pimeloyl Dimethylimidate (DMP), Dimethylsuberine Dinitrate (DMS), 3,3'- Dithiobispropioimidate (DTBP), 1,4-Di(3'- (2'-dithiopyridine)propionylamino)butane (DPDPB), bismaleimidohexane (BMH), difluorodinitrobenzene (DFDNB), difluorodinitrophenyl Sulfone (DFDNPS), bis(β-(4-azidosalicylamino)ethyl) disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol glycidyl ether, adipic diacyl Hydrazine (ADH), carbohydrazide, diaminodimethylbiphenyl, p-diaminobiphenyl, nitrogen-succinate argonamine-3(2-pyridyldithio)-ester (SPDP), long chain-nitrogen -Succinyl argon ammonia-3(2-pyridinedithio)-ester (LC-SPDP), sulfo long chain-nitrogen-succinate argon ammonia-3(2-pyridinedithio)-ester (sulfo -LC-SPDP), succinimidyloxycarbonyl-methyl-(2-pyridylthio)benzene (SMPT), sulfo long chain-succinimidyloxycarbonyl-methyl-(2 -pyridylthio)benzene (sulfo-LC-SMPT), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), 4-(N -Maleimidomethyl)cyclohexane-1-carboxylic acid 3-thio-N-succinimide ester sodium salt (sulfo-SMCC), maleimidobenzoic acid succinimide ester (MBS), M-maleimide succinimidyl benzoate (sulfo-MBS), N-succinimide (4-iodoacetyl)aminobenzoic acid (SIAB), sulfo-N - Succinimidyl (4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), succinimidyl 4-(4-maleimidophenyl)butanoic acid ester (SMPB), sulfosuccinate Imido-4-(P-maleimidophenyl)butyrate (sulfo-SMPB), 4-maleimidobutanoic acid-N-succinimide ester (GMBS), sulfomaleimidobutyric acid-N-succinimidyl ester (sulfo-GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX ), succinimidyl-6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)caproic acid (SIAXX), succinimidyl-4-(((iodoacetyl) base)amino)methyl)cyclohexane-1-carboxylic acid (SIAC), succinimidyl-6-(((((4(iodoacetyl)amino)methyl)cyclohexane-for-1 -carbonyl)amino)caproic acid (SIACX), 4-nitrophenyl iodoacetic acid (NPIA), 4-(4-N-maleimidobenzylamino ester) butyric acid hydrazide (MPBH), 4- N-maleimidomethylcyclohexane-1-carboxyhydrazide (M2C2H), 3-(2-pyridyldithio)propionohydrazide (PDPH), N-hydroxysuccinimide-4 -Azidosalicylic acid (NHS-ASA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-ASA), sulfosuccinimide-4-azide Salicylaminocaproic acid (sulfo-NHS-LC-ASA), sulfosuccinimidyl-2-(P-azido-salicylamido)ethyl-1,3'-dithiopropane ester (SASD), succinimidyl-4-azidobenzoate (HSAB), sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB), N- Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH), sulfosuccinimidyl-6-(4'-azido- 2'-Nitrophenylamino)hexanoate (sulfo-SANPAH), 5-azido-2-nitrobenzoic acid-N-succinimide ester (ANB-NOS), sulfosuccinimide -2-(M-nitroazido-benzamido)-ethyl-1,3'-dithiodipropionate (SAND), N-succinimidyl-(4-azido phenyl)-1,3'-dithiopropionate (SADP), N-sulfosuccinimidyl-(4-azidophenyl)-1,3'-dithiopropanoic acid Ester (sulfo-SADP), sulfosuccinimidyl-4-(P-azidophenyl)butanoic acid (Sulfo-SADP), sulfosuccinimidyl-2-(7-azido-4- Methylmethylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (SAED), sulfosuccinimide-7-azido-4-methylcoumarin Sulfo-3-acetate (Sulfo-SAMCA), p-nitrophenyldiazopyruvate (pNPDP), p-nitrophenyl-2-diazo3,3,3-trifluoropropionic acid (PNP-DTP ), 1-(p-azidosalicylamino)-4-(iodoacetamido)butane (ASIB), N-(4-(p-azidosalicylamido)butyl)-3' -(2'-pyridyldithio)propionamide (APDP), two Benzophenone-4-iodoacetamide, benzophenone-4-maleimide, p-azidobenzohydrazide, 4-(P-azidosalicylamido)-butylamine (ASBA) , P-azidophenylglyoxal (APG), 4-azido-2-nitrophenylbiotin-4-nitrophenyl ester (ABNP), sulfosuccinimidyl-2-( 6-(Biotinamido)-2-(p-azidobenzamido)caproylamino)ethyl-1,3-dithiopropionate (sulfo-SBED), methanethiosulfonate Nitrotetrafluoro-long chain-biotin (MTS-ATF-biotin), methanethiosulfonate azidotetrafluorobiotin (MTS-ATF-LC-biotin), tris (hydroxymethyl) phosphine ( THP), at least one of tris(hydroxymethyl)phosphopropionic acid (THPP).

In any one of the above, preferably, the rabies virus antigen is activated and then connected to the carrier protein through a chemical bond.

In any one of the above, preferably, the carrier protein is activated and then connected to the rabies virus antigen through a chemical bond.

In any one of the above, preferably, the rabies virus antigen and the carrier protein are respectively activated and then connected by chemical bonds.

In any one of the above, it is preferred that the titer unit of the rabies conjugate vaccine is ≥ 2.5 IU/dose.

Preferably in any one of the above, the rabies conjugate vaccine includes a protective agent and/or a stabilizer.

In any one of the above, preferably, the protective agent and/or stabilizer of the rabies conjugate vaccine includes human albumin.

According to any one of the above, it is preferred that the rabies conjugate vaccine does not include protective agents and/or stabilizers.

Preferably in any of the above, the rabies conjugate vaccine does not include human albumin.

Preferably in any of the above, the rabies conjugate vaccine does not contain any preservatives.

According to any one of the above, it is preferred that the rabies conjugate vaccine contains a preservative.

Any one of the above preferably, the preservative contains at least one of thimerosal, 2-phenoxyethanol, and benzyl alcohol.

Preferably, any one of the above is that the prepared rabies conjugate vaccine is used for rabies diagnosis, prevention and treatment.

Preferably, any one of the above is that the prepared rabies conjugate vaccine is used for the prevention, treatment or adjuvant treatment of cancer.

One aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a rabies virus antigen and a carrier protein, the rabies virus antigen and the carrier protein are linked by a chemical bond, the carrier protein and the rabies virus antigen The chemical bond connects the rabies virus antigen and the carrier protein to form a rabies virus antigen-carrier protein immunogenic conjugate. In other words, the present invention includes the rabies virus antigen-carrier protein immunogenic conjugate formed by linking the rabies virus antigen with the carrier protein through chemical bonds. The invention also includes the rabies virus antigen-carrier protein immunogenic conjugate formed by linking the rabies virus antigen with the carrier protein through chemical bonds. The invention also includes the rabies virus antigen-carrier protein immunogenic conjugate formed by linking the carrier protein and the rabies virus antigen through chemical bonds. The rabies virus antigen-carrier protein immunogenic conjugate of the invention is used for the diagnosis, prevention and treatment of human rabies. The rabies virus antigen-carrier protein immunogenic conjugate of the present invention can also be used for prevention or treatment or adjuvant treatment of cancers including malignant melanoma, cervical cancer and malignant glioma.

Another aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a single independently existing rabies virus antigen-carrier protein, and the single independently existing rabies virus antigen is chemically bonded to the carrier protein respectively Connection, the single independently existing rabies virus antigen connected with the carrier protein through chemical bonds does not establish a structural connection with other single independently existing rabies virus antigens, and exists in the form of a single independently existing rabies virus antigen-carrier protein In the immunogenic conjugate, the rabies virus antigen-carrier protein conjugate contains only a single rabies virus antigen, that is, the rabies virus antigen exists in the form of RabAg-CP, RabAg-CP, RabAg-CP, ... in the rabies immunogenicity In the combination, "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, and "-" is the chemical bond connecting the rabies virus antigen and the carrier protein. Therefore, the present invention includes a single independently existing rabies virus antigen linked to a carrier protein via a chemical bond to form a rabies immunogenic conjugate. The rabies virus antigens are present in the rabies immunogenic conjugate in a structurally linked form.

Another aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises two or more rabies virus antigen-carrier proteins, and the two or more rabies virus antigens are chemically bonded to the carrier protein Connection, the carrier protein connects two or more rabies virus antigens through chemical bonds, and establishes a structural connection between the rabies virus antigens connected to the carrier protein through chemical bonds and other rabies virus antigens, in the form of two or more rabies virus antigens-carrier proteins It exists in the rabies immunogenic conjugate, and the rabies virus antigen-carrier protein conjugate contains more than two rabies virus antigens, that is, the rabies virus antigen is RabAg-CP-RabAg, and/or RabAg-CP-RabAg- CP-RabAg, and/or RabAg-CP-RabAg-CP-RabAg-CP-RabAg, ... exist in the form of rabies immunogenic conjugates, wherein "RabAg" is the rabies virus antigen, "CP" represents the carrier protein, " -" is the chemical bond connecting the rabies virus antigen and the carrier protein. Therefore, the present invention includes two or more rabies virus antigens linked to carrier proteins via chemical bonds to form rabies immunogenic conjugates, and the two or more rabies virus antigens are structurally connected to each other through chemical bonds between carrier proteins The form exists in the rabies immunogenic conjugate.

Another aspect of the present invention provides a rabies immunogenic conjugate, which includes a single independent and/or two or more rabies virus antigen-carrier proteins, a single independent and/or two More than two rabies virus antigens are separately and/or jointly connected to the carrier protein via chemical bonds, and the carrier protein does not and/or connects a single independent and/or two or more rabies virus antigens through chemical bonds, and the carrier protein is chemically bonded. The independently existing rabies virus antigen does not establish and/or establish a structural connection with other independently existing rabies virus antigens, and exists in the form of a single and/or two or more independently existing rabies virus antigens-carrier proteins in the rabies immune system. In the original conjugate, the rabies virus antigen-carrier protein conjugate contains a single independent and/or two or more rabies virus antigens, that is, the rabies virus antigen is RabAg-CP, and/or RabAg-CP-RabAg , and/or RabAg-CP-RabAg-CP-RabAg, and/or RabAg-CP-RabAg-CP-RabAg-CP-RabAg, ... are present in the rabies immunogenic conjugate, wherein "RabAg" is rabies virus Antigen, "CP" represents the carrier protein, "-" is the chemical bond connecting the rabies virus antigen and the carrier protein. Therefore, the present invention includes a single independently existing and/or two or more rabies virus antigens connected to a carrier protein via a chemical bond to form a rabies immunogenic conjugate, the single independently existing and/or two or more independently existing rabies virus antigens The antigen is not present in the rabies immunogenic conjugate in a form that is not structurally linked by and/or through chemical bonds with the carrier protein.

Another aspect of the present invention provides a rabies immunogenic conjugate, which includes a rabies virus antigen and a carrier protein, and the rabies virus antigen contains one or more than two compounds that can be chemically bonded to the carrier protein. Functional groups, the functional groups include hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), amino (-NH ₂ ).

Another aspect of the present invention provides a rabies immunogenic conjugate, which includes a rabies virus antigen and a carrier protein, and the carrier protein contains one or more than two rabies virus antigens that can be chemically bonded to the rabies virus antigen. Functional groups, the functional groups include hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), amino (-NH ₂ ).

Another aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises rabies whole virus particles and a carrier protein, the rabies whole virus particles are chemically bonded to the carrier protein, and the carrier protein is connected to the carrier protein The whole rabies virus particles are connected by chemical bonds, and the chemical bonds connect the whole rabies virus particles and the carrier protein to form the whole rabies virus particles-carrier protein immunogenic conjugate. Therefore, the present invention includes the whole rabies virus particle-carrier protein immunogenic conjugate formed by linking the whole rabies virus particles with the carrier protein through chemical bonds. The whole rabies virus particle is the rabies virus antigen obtained by cell culture, inactivation and purification of rabies virus.

Another aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises rabies VLP and a carrier protein, the rabies VLP is connected to the carrier protein through a chemical bond, and the carrier protein and the rabies VLP are chemically bonded The chemical bond connects the rabies VLP and the carrier protein to form a rabies VLP-carrier protein immunogenic conjugate. Therefore, the present invention includes the rabies VLP-carrier protein immunogenic conjugate formed by connecting the rabies VLP with the carrier protein through chemical bonds. The VLP is a rabies virus antigen prepared through recombinant expression.

Another aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a rabies virus outer membrane fragment and a carrier protein, the rabies virus outer membrane fragment is chemically bonded to the carrier protein, and the carrier The protein is connected with the rabies virus outer membrane fragment through a chemical bond, and the chemical bond connects the rabies virus outer membrane fragment and the carrier protein to form a rabies virus outer membrane fragment-carrier protein immunogenic conjugate. Therefore, the present invention includes the rabies virus outer membrane fragment-carrier protein immunogenic conjugate formed by connecting the rabies virus outer membrane fragments with the carrier protein through chemical bonds. The rabies virus outer membrane fragment is a rabies virus antigen containing a spike and a matrix protein prepared by lysing whole rabies virus particles.

Another aspect of the present invention provides a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises rabies virus glycoprotein (G) and a carrier protein, the rabies virus glycoprotein and the carrier protein are linked by a chemical bond, the The carrier protein is connected with the rabies virus glycoprotein through a chemical bond, and the chemical bond connects the rabies virus glycoprotein and the carrier protein to form a rabies virus glycoprotein-carrier protein immunogenic conjugate. Therefore, the present invention includes the rabies virus glycoprotein-carrier protein immunogenic conjugate formed by linking the rabies virus glycoproteins with the carrier protein through chemical bonds. The rabies virus glycoprotein is a glycoprotein-containing rabies virus antigen isolated and purified from rabies virus or recombinantly expressed.

Another aspect of the present invention provides a rabies conjugate vaccine (Rabies Conjugate Vaccine RCV), the rabies conjugate vaccine includes a rabies immunogenic conjugate, the rabies immunogenic conjugate includes a rabies virus antigen and a carrier protein, the rabies The viral antigen is connected to the carrier protein through a chemical bond, the carrier protein is connected to the rabies virus antigen through a chemical bond, and the chemical bond connects the rabies virus antigen and the carrier protein to form a rabies virus antigen-carrier protein immunogenic conjugate. In other words, the present invention includes a rabies conjugate vaccine prepared by preparing the rabies virus antigen-carrier protein immunogenic conjugate formed by linking the rabies virus antigen with the carrier protein through chemical bonds. The present invention also includes the rabies conjugate vaccine which is prepared from the rabies virus antigen-carrier protein immunogenic conjugate formed by linking the rabies virus antigen with the carrier protein through chemical bonds. The present invention also includes the rabies conjugate vaccine prepared by the rabies virus antigen-carrier protein immunogenic conjugate formed by chemically linking the carrier protein and the rabies virus antigen. The rabies conjugate vaccine of the invention is used for the prevention and treatment of human rabies.

Another aspect of the present invention provides a rabies conjugate vaccine, the rabies conjugate vaccine comprises a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a rabies whole virus particle and a carrier protein, and the rabies whole virus particle and a carrier The protein is connected through a chemical bond, and the carrier protein is connected with the whole rabies virus particle through a chemical bond, and the chemical bond connects the whole rabies virus particle and the carrier protein to form the whole rabies virus particle-carrier protein immunogenic conjugate. In other words, the present invention includes the rabies whole virus conjugate vaccine which is prepared by the whole rabies virus particle-carrier protein immunogenic conjugate formed by linking the whole rabies virus particles with the carrier protein through chemical bonds. The whole rabies virus particle is the rabies virus antigen obtained by cell culture, inactivation and purification of rabies virus.

Another aspect of the present invention provides a rabies conjugate vaccine, the rabies conjugate vaccine comprises a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a rabies VLP and a carrier protein, and the rabies VLP and the carrier protein are linked by a chemical bond The carrier protein is connected to the rabies VLP through a chemical bond, and the chemical bond connects the rabies VLP and the carrier protein to form a rabies VLP-carrier protein immunogenic conjugate. In other words, the present invention includes the rabies VLP conjugate vaccine which is prepared by formulating the rabies VLP-carrier protein immunogenic conjugate formed by connecting the rabies VLP with the carrier protein through chemical bonds. The VLP is a rabies virus antigen prepared through recombinant expression.

Another aspect of the present invention provides a rabies conjugate vaccine, the rabies conjugate vaccine comprises a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a rabies virus outer membrane fragment and a carrier protein, and the rabies virus outer membrane fragment The carrier protein is connected with the carrier protein through a chemical bond, and the carrier protein is connected with the rabies virus outer membrane fragment through a chemical bond, and the chemical bond connects the rabies virus outer membrane fragment and the carrier protein to form a rabies virus outer membrane fragment-carrier protein immunogenic conjugate. In other words, the present invention includes a split rabies conjugate vaccine prepared by preparing the rabies virus outer membrane fragment-carrier protein immunogenic conjugate formed by linking the rabies virus outer membrane fragments with the carrier protein through chemical bonds. The rabies virus outer membrane fragment is a rabies virus antigen containing a spike and a matrix protein prepared by lysing whole rabies virus particles.

Another aspect of the present invention provides a rabies conjugate vaccine, the rabies conjugate vaccine comprises a rabies immunogenic conjugate, the rabies immunogenic conjugate comprises a rabies virus glycoprotein (G) and a carrier protein, and the rabies virus saccharide The protein is connected with the carrier protein through a chemical bond, the carrier protein is connected with the rabies virus glycoprotein through a chemical bond, and the chemical bond connects the rabies virus glycoprotein and the carrier protein to form a rabies virus glycoprotein-carrier protein immunogenic conjugate. In other words, the present invention includes a rabies glycoprotein-conjugated vaccine prepared by linking rabies virus glycoproteins with carrier proteins through chemical bonds to form a rabies virus glycoprotein-carrier protein immunogenic conjugate. The rabies virus glycoprotein is a glycoprotein-containing rabies virus antigen isolated and purified from rabies virus or recombinantly expressed.

Another aspect of the present invention provides a method for preparing a rabies immunogenic conjugate, said method comprising the following steps:

(a) Purified to make rabies virus antigen stock solution;

(b) The rabies virus antigen stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies virus antigen-carrier protein immunogenic conjugate.

Another aspect of the present invention provides a method for preparing a rabies immunogenic conjugate, said method comprising the following steps:

(a) inoculating cells with fixed virus seeds of rabies virus, culturing, harvesting virus liquid, inactivating, purifying to make rabies whole virus particle stock solution;

(b) chemically linking and purifying the stock solution of whole rabies virus particles prepared in step (a) with a carrier protein to prepare an immunogenic conjugate of whole rabies virus particles-carrier protein.

Another aspect of the present invention provides a method for preparing a rabies immunogenic conjugate, said method comprising the following steps:

(a) making rabies VLP stock solution through recombinant expression and purification;

(b) The rabies VLP stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies VLP-carrier protein immunogenic conjugate.

Another aspect of the present invention provides a method for preparing a rabies immunogenic conjugate, said method comprising the following steps:

(a) inoculating cells with fixed virus seeds of rabies virus, culturing, harvesting virus liquid, inactivating, purifying to make rabies whole virus particle stock solution;

(b) cracking and purifying the stock solution of rabies whole virus particles prepared in step (a) to make a stock solution of rabies virus outer membrane fragment;

(c) chemically linking and purifying the rabies virus outer membrane fragment stock solution prepared in step (b) with a carrier protein to prepare a rabies virus outer membrane fragment-carrier protein immunogenic conjugate.

Another aspect of the present invention provides a method for preparing a rabies immunogenic conjugate, said method comprising the following steps:

(a) Purified to make rabies virus glycoprotein stock solution;

(b) The rabies virus glycoprotein stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies virus glycoprotein-carrier protein immunogenic conjugate.

Another aspect of the present invention provides a method for preparing a rabies conjugate vaccine, the method comprising the following steps:

(a) Purified to make rabies virus antigen stock solution;

(b) The rabies virus antigen stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies virus antigen-carrier protein immunogenic conjugate.

(c) diluting, preparing and dispensing the rabies virus antigen-carrier protein immunogenic conjugate prepared in step (b) to form a finished vaccine.

Another aspect of the present invention provides a method for preparing a rabies conjugate vaccine, the method comprising the following steps:

(a) inoculating cells with fixed virus seeds of rabies virus, culturing, harvesting virus liquid, inactivating, purifying to make rabies whole virus particle stock solution;

(b) chemically linking and purifying the stock solution of whole rabies virus particles prepared in step (a) with a carrier protein to prepare an immunogenic conjugate of whole rabies virus particles-carrier protein.

(c) diluting, preparing and dispensing the rabies whole virus particle-carrier protein immunogenic conjugate prepared in step (b) into a finished vaccine.

Another aspect of the present invention provides a method for preparing a rabies conjugate vaccine, the method comprising the following steps:

(a) making rabies VLP stock solution through recombinant expression and purification;

(b) The rabies VLP stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies VLP-carrier protein immunogenic conjugate.

(c) diluting, preparing and dispensing the rabies VLP-carrier protein immunogenic conjugate prepared in step (b) to form a finished vaccine.

Another aspect of the present invention provides a method for preparing a rabies conjugate vaccine, the method comprising the following steps:

(a) inoculating cells with fixed virus seeds of rabies virus, culturing, harvesting virus liquid, inactivating, purifying to make rabies whole virus particle stock solution;

(b) cracking and purifying the stock solution of rabies whole virus particles prepared in step (a) to make a stock solution of rabies virus outer membrane fragment;

(c) chemically linking and purifying the rabies virus outer membrane fragment stock solution prepared in step (b) with a carrier protein to prepare a rabies virus outer membrane fragment-carrier protein immunogenic conjugate.

(d) diluting, preparing and dispensing the rabies virus outer membrane fragment-carrier protein immunogenic conjugate prepared in step (c) into a finished vaccine.

Another aspect of the present invention provides a method for preparing a rabies conjugate vaccine, the method comprising the following steps:

(a) Purified to make rabies virus glycoprotein stock solution;

(b) The rabies virus glycoprotein stock solution prepared in step (a) is chemically bonded to the carrier protein and purified to prepare a rabies virus glycoprotein-carrier protein immunogenic conjugate.

(c) diluting, preparing and dispensing the rabies virus glycoprotein-carrier protein immunogenic conjugate prepared in step (b) into a finished vaccine.

In the terminology of the present invention, Rabies Immunogenic Conjugate RIC refers to a specific immunogenic conjugate against rabies virus. The rabies immunogenic conjugate includes a rabies virus antigen and a carrier protein, the rabies virus antigen and the carrier protein are connected by a chemical bond, the carrier protein and the rabies virus antigen are connected by a chemical bond, and the chemical bond connects the rabies virus antigen and the carrier protein to form a rabies virus antigen- Carrier protein immunogenic conjugates. In other words, the rabies immunogenic conjugate is a rabies virus antigen-carrier protein conjugate formed by linking the rabies virus antigen and the carrier protein through chemical bonds.

In the terms of the present invention, Rabies Conjugate Vaccine RCV is a vaccine that specifically prevents or treats rabies virus infection. The antigen of the rabies conjugate vaccine is a rabies immunogenic conjugate. The rabies immunogenic conjugate includes a rabies virus antigen and a carrier protein. The rabies virus antigen and the carrier protein are connected by a chemical bond. The carrier protein and the rabies virus antigen are connected by a chemical bond. The rabies virus antigen and the carrier protein are connected to form the rabies virus antigen-carrier protein immunogenic conjugate. In other words, the rabies conjugate vaccine is prepared from a rabies virus antigen-carrier protein conjugate formed by chemically bonding the rabies virus antigen and the carrier protein.

In the terms of the present invention, rabies virus antigen (RabAg) refers to a substance capable of generating specific immune responses against rabies virus, and the rabies virus antigen contains one or more than two functional groups that can be chemically bonded to the carrier protein. The functional groups for the chemical linkage between the rabies virus antigen and the carrier protein include hydroxyl (-OH) and/or carboxyl (-COOH) and/or aldehyde (-CHO) and/or amino (-NH ₂ ). Rabies virus antigens can be natural, such as from cultured whole rabies virus particles, or recombinantly expressed substances, or semi-synthetic or synthetic substances. Rabies virus antigens include rabies whole virus particles, or include recombinantly expressed rabies VLP, or include outer membrane fragments prepared after cleavage of whole rabies virus particles, or include glycoproteins isolated and purified from rabies virus, or include isolated and purified rabies virus Purified nucleoprotein, or comprising phosphoprotein isolated and purified from rabies virus, or comprising matrix protein isolated and purified from rabies virus, or comprising polymerase isolated and purified from rabies virus, or comprising recombinantly expressed rabies virus glycoprotein, or comprising The recombinantly expressed rabies virus nucleoprotein, or includes the recombinantly expressed rabies virus phosphoprotein, or includes the recombinantly expressed rabies virus matrix protein, or includes the recombinantly expressed rabies virus polymerase.

In the terms of the present invention, the carrier protein (Carrier Protein CP) is a protein substance that is chemically bonded to a hapten or an antigen and has immunogenicity, and the hapten or antigen is connected to the carrier protein to enhance the immunogenicity of the hapten or antigen and T cell-dependent immune responses. The carrier protein contains one or more than two functional groups that can be chemically bonded to the hapten or antigen. The functional groups for the chemical linkage between the carrier protein and the rabies virus antigen include hydroxyl (-OH) and/or carboxyl (-COOH) and/or aldehyde (-CHO) and/or amino (-NH ₂ ). Carrier proteins can be natural, such as proteins from bacteria or viruses, or recombinantly expressed substances, or semi-synthetic or synthetic substances. Carrier proteins include tetanus toxoid (TT), or include diphtheria toxoid (DT), or include diphtheria toxin avirulent variant (CRM197), or include group B meningococcal outer membrane protein (OMP), or include pneumococcal Surface protein A (PspA), or include pneumococcal surface adhesin A (PsaA), or include pneumolysin (Ply), or include Haemophilus influenzae protein D (PD), or include pertussis toxin (PT), Or include pertussis filamentous hemagglutinin (FHA), or include pertussis adhesin (PRN), or include cholera toxin (CT), or include muramyl dipeptide (MDP), or include Escherichia coli LT, or include Escherichia coli Bacillus ST, or include tuberculin pure protein derivative (PPD), or include Pseudomonas aeruginosa exotoxin A (PEA), or include ovalbumin, or include keyhole limpet hemocyanin (KLH), or include bovine serum Albumin (BSA), or including hepatitis B virus surface antigen (HBsAg), or including hepatitis B virus core antigen (HBcAg), or including Tetanus Toxin Fragment C (Tetanus Toxin Fragment C, TTC).

In the terms of the present invention, chemical bond connection means that the rabies virus antigen and carrier protein in the rabies virus antigen-carrier protein immunogenic conjugate are in the form of chemical bond connection (Linking) or crosslinking (Crosslinking) or bridge connection (Bridging) . In other words, the chemical bond links or cross-links or bridges the rabies antigen and the carrier protein into a rabies antigen-carrier protein immunogenic conjugate. Therefore, in the terms of the present invention, a chemical bond connection is equivalent to or identical to a chemical bond crosslink, and a chemical bond connection is also equivalent to or identical to a chemical bond bridge.

In the terms of the present invention, the connecting arm (Linker Arm) is the material that connects the rabies virus antigen and the carrier protein with a chemical bond in the rabies virus antigen-carrier protein immunogenic conjugate, and is a new atom created between the rabies virus antigen and the carrier protein or new molecules. Therefore, in the terms of the present invention, the tether is equal to or the same as the spacer arm (SpacerArm), and the tether is also equal to or the same as the bridging molecule (Bridging Molecule).

The invention provides a rabies immunogenic conjugate formed by linking a rabies virus antigen and a carrier protein through a chemical bond, and further provides a rabies conjugate vaccine prepared by formulating the rabies immunogenic conjugate. In a preferred embodiment of the present invention, the rabies virus fixed strain for preparing rabies virus antigen is selected from PAS strain, PV strain, PM strain, CVS strain, Nishigara strain, Flury strain, Vnukovo-32 strain, CTN-1V strain, aGV strain or any other rabies virus fixed strain. The fixed rabies virus strain is more preferably PV strain, PM strain, Flury strain, CTN-1V strain, aGV strain. The fixed strains of rabies virus are most preferably PM strains, Flury strains, and CTN-1V strains. Fixed strains of rabies virus are sold by American Standard Biology Collection Center (ATCC) and China National Institutes for Food and Drug Control.

The invention provides a rabies immunogenic conjugate formed by linking a rabies virus antigen and a carrier protein through a chemical bond, and further provides a rabies conjugate vaccine prepared by formulating the rabies immunogenic conjugate. In a preferred embodiment of the present invention, the rabies virus antigen is selected from the group consisting of whole rabies virus particles, recombinantly expressed rabies VLP, outer membrane fragments prepared after cleavage of whole rabies virus particles, glycoproteins isolated and purified from rabies virus, Nucleoprotein isolated and purified from rabies virus, phosphoprotein isolated and purified from rabies virus, matrix protein isolated and purified from rabies virus, polymerase isolated and purified from rabies virus, recombinantly expressed rabies virus glycoprotein, recombinantly expressed rabies virus Nucleoprotein, recombinantly expressed rabies virus phosphoprotein, recombinantly expressed rabies virus matrix protein, recombinantly expressed rabies virus polymerase. The rabies virus antigen is more preferably rabies whole virus particle, rabies VLP through recombinant expression, the outer membrane fragment prepared after cracking the whole rabies virus particle, glycoprotein isolated and purified from rabies virus, and rabies virus glycoprotein through recombinant expression. Rabies virus antigens are most preferably whole rabies virus particles. Other lyssavirus antigens may also be used if desired, and the rabiesvirus antigens given here are merely illustrative of the invention.

The invention provides a rabies immunogenic conjugate formed by linking a rabies virus antigen and a carrier protein through a chemical bond, and further provides a rabies conjugate vaccine prepared by formulating the rabies immunogenic conjugate. In a preferred embodiment of the present invention, the carrier protein is selected from tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria toxin avirulent variant (CRM197), group B meningococcal outer membrane protein (OMP ), pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), Haemophilus influenzae protein D (PD), pertussis toxin (PT), pertussis filamentous blood Lectin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), Escherichia coli LT, Escherichia coli ST, tuberculin pure protein derivative (PPD), aeruginosa Monascus exotoxin A (PEA), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg), tetanus toxin Fragment C (TTC). Carrier proteins are more preferably TT, DT, CRM197, PD and OMP. Carrier proteins are most preferably TT and CRM197. Other protein carriers may also be used if desired, and the protein carriers presented here are merely illustrative of the present invention.

In the present invention, the preparation and quality control methods and standards of the rabies virus antigen are well known to those skilled in the art. In a preferred embodiment of the present invention, technicians can according to literature (Li Zhiqiang et al. Journal of Microbiology. 2010.30 (1): 96-99; Lewei et al. Chinese Virology. 2004.19 (4): 373-375), the world Rabies virus whole virus particle stock solution was prepared and quality tested according to technical documents such as the manufacturing and testing procedures of inactivated rabies vaccines for human use (WHO Technical Report Series No 941, 2007) issued by the World Health Organization and the third part of the Pharmacopoeia of the People's Republic of China (2015 edition).

In the present invention, the preparation and quality control methods and standards of the rabies virus antigen are well known to those skilled in the art. In a preferred embodiment of the present invention, the skilled person can according to literature (Fontana D, et al.Vaccine. , preparation and immune effect evaluation. Changchun. Jilin University. 2015), the Manufacture and Verification Regulations of Inactivated Rabies Vaccine for Human Use issued by the World Health Organization (WHO Technical Report Series No 941, 2007) and the Pharmacopoeia of the People’s Republic of China (2015 Edition) ) and other technical documents to prepare rabies VLP stock solution and carry out quality inspection.

In the present invention, the preparation and quality control methods and standards of the rabies virus antigen are well known to those skilled in the art. In a preferred embodiment of the present invention, technicians can according to the inventor's another invention technical scheme (CN100413536C), the human rabies inactivated vaccine promulgated by the World Health Organization and the verification regulations (WHOTechnical Report Series No 941,2007) And "Pharmacopia of the People's Republic of China" (three 2015 editions) and other technical documents to prepare the stock solution of rabies virus outer membrane fragment and carry out quality inspection.

In the present invention, the preparation and quality control methods and standards of the rabies virus antigen are well known to those skilled in the art. In a preferred embodiment of the present invention, technical personnel can according to literature (Amann R, et al. :772-776; Kan Haiping et al. Biotechnology Communications. 2015.26(4):493-496; Zhang Jianming et al. Live vaccine manufacturing and testing procedures (WHO Technical Report Series No 941, 2007) and the third part of the Pharmacopoeia of the People's Republic of China (2015 edition) and other technical documents prepared rabies virus glycoprotein stock solution and carried out quality inspection.

In the present invention, the principle and method that the rabies virus antigen and the carrier protein are connected through chemical bonds can refer to the covalent bond coupling reaction technology between biomacromolecules and biomacromolecules (Hermanson G. (2008) BioconjugateTechnigues, 2nd Edition. Academic Press, London and New York.). In a preferred embodiment of the present invention, the chemical bond connecting method of rabies virus antigen and carrier protein is selected from carbodiimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester method), N-hydroxysuccinimide Ester method, glutaraldehyde method, diazotization method, succinic anhydride method, carbonyldiimidazole method, maleimide method, disulfide bond method, periodic acid oxidation method, carboxymethyl hydroxylamine method, etc. The chemical bonding method is more preferably a carbodiimide method, a glutaraldehyde method, a diazotization method, a disulfide bond method, and a periodic acid oxidation method. The most preferred chemical bonding method is the carbodiimide method and the periodic acid oxidation method. Other chemical bond connection methods can also be used if the requirements are met, and the chemical bond connection methods given here are only for illustrative purposes of illustrating the present invention.

In the present invention, the chemical bond connection of the rabies virus antigen and the carrier protein is a direct connection without a linker arm (LinkerArm) or a spacer arm (Spacer Arm) or a bridging molecule (Bridging Molecule), also known as a zero-length connection (Zero-Length Linking) or zero-length crosslinking (Zero-Length Crosslinking) or zero-length bridging (Zero-Length Bridging), that is, the chemical bond connecting the rabies virus antigen and the carrier protein does not contain new atoms or molecules. In a preferred embodiment of the present invention, the chemical bond connection of the rabies virus antigen and the carrier protein is a direct connection without a link arm or a spacer arm, and the linker is selected from 1-(3-dimethylaminopropyl)-3-ethyl carbon Diimine hydrochloride (EDC), 1-cyclohexyl-3-(2-N-morpholinoethyl) carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N,N '-Diisopropylcarbodiimide (DIC), 2-ethyl-5-phenylisoxazole-3'-sulfonate (Woodward's reagent K), N,N'-carbonyldiimidazole (CDI) And reagents for Schiff base formation and reductive amination reactions such as sodium cyanoborohydride (NaBH3CN) or sodium borohydride (NaBH4). The linker is more preferably EDC, CMC, DCC, NaBH3CN, NaBH4. The linking agent is most preferably EDC, NaBH3CN. Other linking agents may also be used if desired, and the linking agents given here are merely illustrative of the invention.

In the present invention, the chemical bond connection of rabies virus antigen and carrier protein is through the connection of connecting arm or spacer arm or bridging molecule, and newly created connecting arm or spacer arm or bridging molecule connects rabies virus antigen and carrier protein through chemical bond The formation of rabies virus antigen-carrier protein immunogenic conjugates means that the chemical bond connecting the rabies virus antigen and the carrier protein contains additional atoms or molecules. In a preferred embodiment of the present invention, the chemical bond connection of rabies virus antigen and carrier protein is through the connection of linking arm or spacer arm or bridging molecule, and linking arm or spacer arm or bridging molecule are selected from dithiobis(succinyl iminopropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimide suberate (DSS), bis(thiosuccinimidyl Amino) suberate sodium salt (BS ³ ), disuccinimide tartrate (DST), disulfosuccinimide tartrate (sulfo-DST), bis(2-(succinimidyloxycarbonyl Oxygen)ethyl)sulfone (BSOCOES), bis(2-(sulfosuccinimidyloxycarbonyloxy)ethyl)sulfone (sulfo-BSOCOES), ethylene glycol-bis(succinimidyl ester succinate ) (EGS), ethylene glycol-bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinyl Imino carbonate (DSC), dimethyl adipimide (DMA), dimethyl pimelimide (DMP), dimethyl octane dinitrate (DMS), 3,3'- Dimethyl dithiobispropionimide (DTBP), 1,4-bis(3'-(2'-dithiopyridine) propionamido)butane (DPDPB), dimaleimide Difluorodinitrobenzene (DFDNB), difluorodinitrophenyl sulfone (DFDNPS), bis(β-(4-azidosalicylamino)ethyl) disulfide (BASED ), formaldehyde, glutaraldehyde, 1,4-butanediol glycidyl ether, adipic acid dihydrazide (ADH), carbohydrazide, diaminodimethylbiphenyl, p-diaminobiphenyl, nitrogen-succinate Star argon ammonia-3 (2-pyridine dithio)-ester (SPDP), long chain-nitrogen-succinyl ammonium-3 (2-pyridine dithio)-ester (LC-SPDP), sulfo Long-chain-nitrogen-succinylamino-3(2-pyridyldithio)-ester (sulfo-LC-SPDP), succinimidyloxycarbonyl-methyl-(2-pyridylthio) Benzene (SMPT), sulfo long chain-succinimidyloxycarbonyl-methyl-(2-pyridylthio)benzene (sulfo-LC-SMPT), 4-(N-maleimido N-hydroxysuccinimide methyl)cyclohexanecarboxylate (SMCC), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate 3-thio-N-succinimide Sodium imide ester (sulfo-SMCC), maleimido succinimide benzoate (MBS), M-maleimide succinimide benzoate (sulfo-MBS), N -succinimide (4-iodoacetyl)aminobenzoic acid (SIAB), sulfo-N-succinimide (4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), 4-(4 -Maleimidophenyl)succinimidyl butyrate (SMPB), sulfosuccinimidyl-4-(P-maleimide Iminophenyl)butyrate (sulfo-SMPB), 4-maleimidobutyrate-N-succinimidyl ester (GMBS), sulfomaleimidobutyrate-N- Succinimidyl ester (sulfo-GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX), succinimidyl-6-(6-(((4- Iodoacetyl)amino)hexanoyl)amino)hexanoic acid (SIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid (SIAC) , Succinimidyl-6-(((((4(iodoacetyl)amino)methyl)cyclohexane-for-1-carbonyl)amino)hexanoic acid (SIACX), 4-nitrophenyl iodide Acetic acid (NPIA), 4-(4-N-maleimidobenzylamino ester) butyric acid hydrazide (MPBH), 4-N-maleimidomethylcyclohexane-1-carboxyhydrazide (M ₂ C ₂ H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimide-4-azidosalicylic acid (NHS-ASA), N -Hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-ASA), sulfosuccinimide-4-azidosalicylaminocaproic acid (sulfo-NHS-LC-ASA ), sulfosuccinimidyl-2-(P-azido-salicyloylamino)ethyl-1,3'-dithiopropionate (SASD), succinimidyl-4- Azidobenzoate (HSAB), sulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB), N-succinimidyl-6-(4'-azido Syl-2'-nitrophenylamino)hexanoate (SANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo -SANPAH), 5-azido-2-nitrobenzoic acid-N-succinimidyl ester (ANB-NOS), sulfosuccinimide-2-(M-nitroazido-benzoic acid Amino)-ethyl-1,3'-dithiodipropionate (SAND), N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropane Acid ester (SADP), N-sulfosuccinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (sulfo-SADP), sulfosuccinimidyl -4-(P-azidophenyl)butanoic acid (Sulfo-SABP), sulfosuccinimide-2-(7-azido-4-methylmethylcoumarin-3-acetamide) ethyl 1,3'-dithiopropionate (SAED), sulfosuccinimide-7-azido-4-methylcoumarin-3-acetate (Sulfo-SAMCA), p- Nitrophenyldiazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropanoic acid (PNP-DTP), 1-(p-azidosalicylamino) -4-(iodoacetamido)butane (ASIB), N-(4-(p-azidosalicylamido)butyl)-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, diphenyl Ketone-4-maleimide, p-azidobenzoyl hydrazide, 4-(P-azidosalicylamido)-butylamine (ASBA), P-azidophenylglyoxal (APG ), 4-azido-2-nitrophenyl biotin-4-nitrophenyl ester (ABNP), sulfosuccinimidyl-2-(6-(biotinamide)-2-( p-azidobenzamido)hexamido)ethyl-1,3-dithiopropionate (sulfo-SBED), methanethiosulfonate azidotetrafluoro-long chain-biotin (MTS -ATF-biotin), methanethiosulfonate azidotetrafluorobiotin (MTS-ATF-LC-biotin), tris(hydroxymethyl)phosphine (THP), tris(hydroxymethyl)phosphopropyl acid (THPP). Tether or spacer or bridging molecule is more preferably DTSSP, BS ³ , sulfo-DST, sulfo-BSOCOES, sulfo-EGS, DSG, DSC, DMA, DMP, BMH, glutaraldehyde, ADH, carbohydrazide, sulfo- LC-SPDP, sulfo-LC-SMPT, sulfo-SMCC, sulfo-MBS, Sulfo-SIAB, sulfo-SMPB, Sulfo-GMBS, MPBH, M ₂ C ₂ H, PDPH, Sulfo-NHS-LC-ASA, Sulfo- HSAB, Sulfo-SANPAH, Sulfo-SADP, ASIB, ABH, ASBA, sulfo-SBED. The linker or spacer or bridging molecule is most preferably DTSSP, DMP, ADH, carbohydrazide, sulfo-LC-SPDP, sulfo-SMCC, sulfo-MBS, MPBH, PDPH, sulfo-SBED. Other tethers or spacers or bridging molecules may also be used if desired, and the tethers or spacers or bridging molecules given here are merely illustrative of the invention.

In a preferred embodiment of the present invention, the carrier protein is first activated with a cyanating agent (1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) or cyanogen bromide (CNBr)) , and then add adipic acid dihydrazide (ADH) as a homologous bifunctional linker to the activated carrier protein to form a carrier protein-ADH derivative, then add rabies virus antigen, and undergo carbodiimide-mediated condensation Function The carrier protein-ADH derivative is combined with the rabies virus antigen through a chemical bond to form a rabies virus antigen-carrier protein immunogenic conjugate, which is further purified to obtain the antigenic component for preparing the rabies conjugate vaccine. In another preferred embodiment of the present invention, earlier use cyanide agent (1-cyano group-4-dimethylaminopyridine tetrafluoroboric acid (CDAP) or cyanogen bromide (CNBr)) to carry out activation treatment to rabies virus antigen , and then add adipic hydrazide (ADH) as a homologous bifunctional linker to the activated rabies virus antigen to form a rabies virus antigen-ADH derivative, then add carrier protein, and undergo carbodiimide-mediated condensation The rabies virus antigen-ADH derivative is combined with the carrier protein through a chemical bond to form a rabies virus antigen-carrier protein immunogenic conjugate, which is further purified to obtain an antigenic component for preparing a rabies conjugate vaccine. In another preferred embodiment of the present invention, the carrier protein and the The rabies virus antigen is activated, and then adipic acid dihydrazide (ADH) is added to the activated carrier protein and rabies virus antigen respectively to form a carrier protein-ADH derivative and a rabies virus antigen. -ADH derivatives, then the carrier protein-ADH derivatives and rabies virus antigen-ADH derivatives are mixed, and through the condensation action mediated by carbodiimide, the carrier protein-ADH derivatives and rabies virus antigen-ADH derivatives are combined by chemical bonds , form the rabies virus antigen-carrier protein immunogenic conjugate, and then further purify to obtain the antigenic component for preparing the rabies conjugate vaccine. There are many methods for the chemical bond connection of the rabies virus antigen and the carrier protein, and the connection methods described here are only used to illustrate the present invention. Therefore, various equivalent methods can be used to realize the connection described in the present invention, so any method and process or process for chemical bond connection of rabies virus antigen and carrier protein may be used to implement the present invention.

The rabies conjugate vaccine provided by the present invention is prepared by formulating the rabies immunogenic conjugate, which includes an effective dose of rabies virus antigen combined with carrier protein through chemical bonds. According to the Manufacture and Verification Regulations of Inactivated Rabies Vaccines for Human Use issued by the World Health Organization (WHO Technical Report Series No 941, 2007), the European Pharmacopoeia. Rabies Vaccine for Human Use in Prepared Cell Cultures and the European Pharmacopoeia. According to the requirements of the Pharmacopoeia of the People's Republic of China (Three Parts, 2015 Edition), a human rabies vaccine contains ≥ 2.5 IU of rabies virus antigen. WHO, European Pharmacopoeia and Chinese Pharmacopoeia have promulgated NIH methods and standards for human rabies vaccine potency determination. In a preferred embodiment of the present invention, the potency of the rabies immunogenic conjugate and the rabies conjugate vaccine is determined by the NIH potency test method, and the potency unit is marked as ≥2.5 IU/dose, and each dose is 0.5ml or 1ml.

The present invention provides a rabies conjugate vaccine prepared by formulating a rabies immunogenic conjugate. In a preferred embodiment of the present invention, the rabies conjugate vaccine includes human albumin as a protective agent or a stabilizer. In another preferred embodiment of the present invention, the carrier protein chemically bonded to the rabies virus antigen completely replaces the function of adding human albumin as a protective agent or stabilizer in existing rabies vaccines. In the prior art, one of the main functions of human albumin is to prevent the aggregation of rabies virus particles, thereby avoiding the reduction of rabies virus antigenic sites in vaccines, thereby realizing the function as a protective agent or stabilizer of rabies virus antigens. Because the rabies virus antigen in the present invention is the antigen that has been combined with the carrier protein through chemical bonds, the rabies virus antigen that has been combined with the carrier protein through chemical bonds has prevented the aggregation of the rabies virus antigen because of the space barrier of the carrier protein, thereby avoiding the rabies virus in the vaccine. The reduction of antigenic sites, therefore the rabies conjugate vaccine of the present invention does not include human albumin. In another preferred embodiment of the present invention, the rabies conjugate vaccine also does not include protective or stabilizing agents with functions similar to human albumin.

The present invention provides a rabies conjugate vaccine prepared by formulating a rabies immunogenic conjugate. In a preferred embodiment of the present invention, the rabies conjugate vaccine may also contain adjuvants commonly used in the art. Preferred adjuvants are aluminum adjuvant, calcium phosphate adjuvant, cholera toxin (CT), cholera toxin subunit B (CTB), pertussis toxin (PT), pertussis toxin subunit B (PTB), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), saponin QS-21, α-tocopherol, squalene, liposomes, liposomes (liposomes), monophosphate lipid A (MPL-A), MF59, virus-like particle liposomes (Virosomes), polyglycolide (PLA) microspheres, polylactic-glycolic acid (PLGA) microspheres, lipid-cholesterol (DC-Chol), dimethyl dioctadecyl Quaternary ammonium bromide (DDA), immunostimulatory complex (ISCOM), Montanide ISA50, MontanideISA51, Montanide ISA206, Montanide ISA720, Montanide ISA series adjuvants, AS01, AS02, AS03, AS04, AS series adjuvants, muramyl di peptide (MDP), bacterial lipopolysaccharide (OM-174), Escherichia coli heat labile toxin (LT), IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN- Gamma, GM-CSF, CpG oligonucleotides, trehalose dipycolate (TDM), picacto containing polyinosine (Poly I) and/or polycytosine (Poly C) agent. More preferred adjuvants are QS-21, MPL-A, MF59, Virosomes, Montanide ISA50, Montanide ISA51, Montanide ISA206, Montanide ISA720, AS01, AS02, AS03, AS04, CpG, Picard adjuvants. Most preferred adjuvants are MF59, Virosomes, Montanide ISA51, AS03, AS04, CpG, Picard adjuvants.

The present invention provides a rabies conjugate vaccine prepared by preparing the rabies immunogenic conjugate. In a preferred embodiment of the present invention, the rabies conjugate vaccine contains preservatives, and the preferred preservatives are thimerosal, 2-phenoxy Ethanol, benzyl alcohol. A more preferred preservative is 2-phenoxyethanol. In another preferred embodiment of the present invention, the rabies conjugate vaccine is a vaccine that does not contain any preservatives.

In the rabies conjugate vaccine provided by the present invention, the dosage form and compatibility of the rabies conjugate vaccine are determined according to the technical standards known to those skilled in the pharmaceutical field. In a preferred embodiment of the present invention, the dosage form of the rabies conjugate vaccine is preferably a liquid dosage form, a lyophilized dosage form, a pill, a tablet, a capsule dosage form, more preferably a liquid dosage form or a lyophilized dosage form, and most preferably a lyophilized dosage form.

In the rabies conjugate vaccine provided by the present invention, the rabies conjugate vaccine is prepared by formulating the rabies immunogenic conjugate, the rabies conjugate vaccine vaccination route includes intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and also includes nasal cavity, oral cavity injection, etc. , anal, vaginal, and mucosal routes of administration.

illustrate:

The newly created connecting arm or spacer or bridging molecule in the present invention refers to the connecting arm or spacer or bridging molecule formed during the reaction process of rabies virus antigen and carrier protein being connected by chemical bonds; The atom or molecule refers to the newly formed atom or molecule between the rabies virus antigen and the carrier protein during the reaction process in which the rabies virus antigen and the carrier protein are linked by chemical bonds.

Biological or chemical preparations such as fixed strains of rabies virus, adjuvants, chemical bonding agents, and protein reagents described in the present invention are all existing commercially purchased products unless the preparation method and process are specified otherwise. In the present invention, the fixed rabies virus strain can be purchased from China National Institute for Drug and Food Control, American Standard Biological Collection (ATCC) or American Center for Disease Control and Prevention (CDC).

The content disclosed above has generally described the present invention, and the core content or spirit of the present invention can be further understood through the following examples, but not limiting. The examples are only used to illustrate the concept of the present invention and several preferred embodiments, and should not be construed as limiting the scope of the present invention in any way. It should be understood that the present invention is not limited to the following specific embodiments, and various changes, adjustments, modifications, modifications, changes, improvements, improvements, reductions, simplifications, additions, additions, etc. to the following examples can be implemented by those skilled in the art, without departing from the scope or spirit of the invention. Other embodiments of the invention will envision those skilled in the art without departing from the core of the invention.

Description of drawings

Fig. 1 is the column chromatography purification pattern of the rabies immunogenic conjugate of preferred embodiment 3 of the present invention

Detailed ways

The present invention can be further discussed in conjunction with the following examples. The rabies virus antigen and carrier protein are chemically bonded to make a rabies immunogenic conjugate, and then formulated into a rabies conjugate vaccine. The rabies conjugate vaccine has been proved by animal experiments and laboratory tests. It has good safety and excellent immunogenicity and excellent protective efficacy. Several preferred implementations are selected in the examples, just to better illustrate the present invention, but not to limit the content of the invention.

Example 1

Rabies whole virus particle antigen stock solution preparation and vaccine preparation:

Take ^the cells from the Vero cell working bank for recovery and expansion culture at 37°C, and pass through 3-5 ^generations continuously. bioreactor for tank-flow culture. Microcarriers are cultured in high-density suspension for 5 to 7 days, and the cell density reaches 1.0×10 ⁶ to 1.0×10 ⁷ /ml, and the maintenance solution is replaced. The rabies virus fixed virus CTN-1V strain with a virus titer of not less than 7.5 lg LD ₅₀ /ml was inoculated at a MOI of 0.001-0.1. Replace with maintenance medium, and culture in tank flow at 33-35°C. The virus fluid can be continuously harvested on the third day, and the virus fluid can be continuously harvested for more than 20 days. The harvested virus fluid is clarified and filtered, and concentrated by 100-300KD membrane ultrafiltration for 10-30 times. Add β-propiolactone at a ratio of 1:2000～1:8000, put it at 4℃ for 18～24 hours to inactivate the virus, and then put it in a water bath at 37℃ for 2 hours to completely hydrolyze the residual β-propiolactone, and inactivate the virus verify. The inactivated virus liquid is purified by Sepharose 4FF gel column chromatography, and the V ₀ peak containing the whole virus particles is collected. The purified virus liquid is added with an antigen stabilizer of 1-10% human serum albumin, which is the rabies whole virus particle antigen stock solution.

Use ELISA to measure the rabies virus antigen content in the rabies whole virus particle antigen stock solution, add disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and water for injection, prepare, adjust pH to 7.0-8.0, and dispense to form a vaccine. According to the human rabies vaccine manufacturing and testing regulations promulgated by the World Health Organization (WHO Technical Report Series, No.941, 2007), the "People's Republic of China Pharmacopoeia" (2015 edition) promulgated the human rabies vaccine manufacturing and testing regulations The method and The standard is tested, and after the test is qualified, it is a human rabies whole virus particle vaccine (RV-Vero).

Example 2

Preparation of carrier protein-tetanus toxoid (TT)

Open the tetanus freeze-dried bacterial strain preserved at low temperature, inoculate it into a semi-solid medium containing tryptone beef infusion agar, and cultivate it at 33-37°C for 24-48 hours. Transfer to semi-solid medium, and cultivate at 33-37°C for 24-48 hours. Then transfer to the strain bottle containing tryptone beef extract liquid medium to carry out enrichment culture, and cultivate at 33-37 DEG C for 24-48 hours. The culture in the strain bottle is transferred to a fermenter of double peptone liquid medium, and cultured at 33-37° C. for 60-80 hours. After the fermentation is completed, formaldehyde with a final concentration of 0.1-0.5% is added for sterilization treatment, and the supernatant is collected by centrifugation. The supernatant was clarified and filtered through a 0.45-1.2 μm filter, and then concentrated by a 50-100KD membrane ultrafiltration. Add NaHCO ₃ to a final concentration of 0.2-0.8% and (NH ₄ ) ₂ SO ₄ to a final concentration of 15.0-20.0%, respectively, stir for 30 minutes, and stand at room temperature for 12-48 hours. The supernatant was collected by centrifugation. (NH ₄ ) ₂ SO ₄ with a final concentration of 6.0-12.0% was added to the collected supernatant, stirred for 30 minutes, and left to stand at room temperature for 12-24 hours. The precipitate was collected by centrifugation and dissolved in 0.1% NaHCO ₃ solution, and the ammonium was removed by ultrafiltration with a 50-100KD membrane. Add NaCl to a final concentration of 0.85%, then add formaldehyde to a final concentration of 0.15-0.25%, and place at 37°C for 15-31 days for detoxification. Use 50-100KD membrane ultrafiltration to remove formaldehyde, purify by Sephacryl S-300 gel column chromatography, and then sterilize and filter through a 0.22μm filter to obtain the stock solution. The stock solution after detoxification and purification was tested according to the items and methods specified in "Pharmacopoeia of the People's Republic of China" (2015 Edition) "Adsorbed Tetanus Vaccine". After passing the test, it was the stock solution of tetanus toxoid, which was stored at 2-8°C for future use.

Example 3

Rabies whole virus particle-carrier protein immunogenic conjugate preparation and vaccine preparation:

Measure the stock solution of tetanus toxoid and dilute it to 1-10 mg/ml with 0.1 M PBS with pH 7.5. Add 100mg/ml CDAP (1-cyano-4-dimethylamino-pyridine tetrafluoroboric acid) acetonitrile solution to a final concentration of 0.5-1.5mg/mgTT, react for 30 seconds, then add 0.2M TEA (triethylamine), The ratio of TEA addition to CDAP addition is 1-3:1 (v/v), adjust the pH to 9.0, and maintain the reaction for 2-5 minutes. Add ADH (adipic hydrazide) NaHCO ₃ (0.1M) solution to a final concentration of 2.0-4.0 mg/mgTT, and maintain the reaction for 120 minutes. Use 50-100KD membrane ultrafiltration to remove residual chemical reagents, which is the stock solution of tetanus toxoid-ADH derivatives. Measure the purified stock solution of whole rabies virions prepared according to the method of Example 1 and without adding human serum albumin, according to the ratio of the protein content of the whole rabies virus particles stock solution to the protein content of the tetanus toxoid-ADH derivative stock solution is 1-5 : 1 (w/w) ratio, mix rabies whole virus particle purified stock solution and tetanus toxoid-ADH derivative stock solution. Adjust the pH to 5.0-5.8, add sulfo-NHS (N-hydroxysulfosuccinimide) with a final concentration of 10-25mM, then add EDAC (carbodiimide) to a final concentration of 25-100mM, and maintain the reaction for 240 minutes . After the reaction is finished, adjust the pH to 6.8-7.5 and let stand overnight. Purify with Sepharose 4FF gel column chromatography, collect the V ₀ peak containing the whole virion-carrier protein immunogenic conjugate, and sterilize and filter through a 0.22 μm filter to obtain the stock solution of the rabies whole virion-carrier protein immunogenic conjugate , stored at 2-8°C for later use.

Figure 1 shows the 280nm profile of the rabies virus whole virion-carrier protein immunogenic conjugate purified by Sepharose 4FF gel column chromatography. After binding, the signal of the carrier protein moves forward and forms a conjugate with the rabies whole virion antigen, the carrier The protein was bound to whole rabies virus particles to form an immunogenic conjugate with little unbound free protein.

Use ELISA to measure the rabies virus antigen content in the rabies whole virus particle-carrier protein immunogenic conjugate stock solution, add polysorbate 80 (50-300μg/ml), succinate buffer (300-800μg/ml), sodium chloride (7.5-9.5mg/ml), water for injection, after preparation, adjusting the pH to 5.0-7.0, and dispensing 0.5-1.0ml/dose, it is the vaccine. According to the human rabies vaccine manufacturing and testing regulations promulgated by the World Health Organization (WHO Technical Report Series, No.941, 2007), the "People's Republic of China Pharmacopoeia" (2015 edition) promulgated the human rabies vaccine manufacturing and testing regulations The method and The standard is tested, and after the test is qualified, it is a rabies whole virus particle conjugate vaccine (RCV-VP) for human use.

Example 4

Rabies virus outer membrane fragment-carrier protein immunogenic conjugate preparation and vaccine formulation:

According to the method described in another invention of the inventor (CN200610152928.7), the purified stock solution of rabies whole virus particles prepared by the method in Example 1 and without adding human serum albumin was lysed with Triton-100 at a final concentration of 0.2-1.0%, 10 ~60% sucrose density gradient centrifugation or Sepharose 4FF gel column chromatography purification, 50 ~ 100KD membrane ultrafiltration concentration, preparation of rabies virus outer membrane fragment stock solution containing spike and matrix protein. Then the rabies virus outer membrane fragment stock solution was condensed with tetanus toxoid-ADH derivatives under EDAC condensation according to the method in Example 3 to prepare a rabies virus outer membrane fragment-carrier protein immunogenic conjugate stock solution.

Further, the rabies virus outer membrane fragment-carrier protein immunogenic conjugate stoste was measured by ELISA according to the method in Example 3 for the rabies virus antigen content in the rabies virus outer membrane fragment-carrier protein immunogenic conjugate stoste, and polysorbate 80 ( 50-300μg/ml), succinate buffer (300-800μg/ml), sodium chloride (7.5-9.5mg/ml), water for injection, after preparation, adjust pH to 5.0-7.0, dispense 0.5-1.0ml /dose is the vaccine. According to the Manufacture and Verification Regulations of Rabies Vaccines for Human Use (WHO Technical Report Series, No.941, 2007) promulgated by the World Health Organization, and the Manufacture and Verification Regulations of Rabies Vaccines for Human Use promulgated by the Pharmacopoeia of the People's Republic of China (2015 Edition), the method and The standard is tested, and after passing the test, it will be human rabies split conjugate vaccine (RCV-split).

Example 5

Rabies virus-like particle (VLP)-carrier protein immunogenic conjugate preparation and vaccine formulation:

Refer to Qi Yinglin's method (Qi Yinglin. Construction, preparation and immune effect evaluation of rabies virus-like particles. Changchun. Jilin University. 2015) to prepare the rabies virus-like particle stock solution, and then mix the rabies virus-like particle stock solution with the tetanus class according to the method in Example 3. The toxin-ADH derivative is condensed by EDAC to prepare the rabies virus-like particle stock solution-carrier protein immunogenic conjugate stock solution.

Further, the rabies virus-like particle stock solution-carrier protein immunogenic conjugate stock solution was used to measure the rabies virus antigen content in the rabies virus-like particle-carrier protein immunogenic conjugate stock solution by ELISA according to the method in Example 3, and polysorbate 80 (50 -300μg/ml), succinate buffer (300-800μg/ml), sodium chloride (7.5-9.5mg/ml), water for injection, after preparation, adjust the pH to 5.0-7.0, dispense 0.5-1.0ml/ Dosage is a vaccine. According to the Manufacture and Verification Regulations of Rabies Vaccines for Human Use (WHO Technical Report Series, No.941, 2007) promulgated by the World Health Organization, and the Manufacture and Verification Regulations of Rabies Vaccines for Human Use promulgated by the Pharmacopoeia of the People's Republic of China (2015 Edition), the method and The standard is tested, and after passing the test, it is a rabies virus-like particle conjugate vaccine (RCV-VLP) for human use.

Example 6

Rabies virus glycoprotein-carrier protein immunogenic conjugate preparation and vaccine preparation:

With reference to Zhang Jianming's method (Zhang Jianming etc. Straits Preventive Medicine Journal. 2009,15 (2): 1-4) the rabies virus glycoprotein (GP) stoste of recombinant expression is prepared, then the rabies virus glycoprotein stoste is pressed the present embodiment 3 method and Tetanus toxoid-ADH derivatives are condensed with EDAC to prepare the stock solution of rabies virus glycoprotein-carrier protein immunogenic conjugate.

Further, the rabies virus glycoprotein-carrier protein immunogenic conjugate stoste was determined by ELISA according to the method in Example 3 for the rabies virus antigen content in the rabies virus glycoprotein-carrier protein immunogenic conjugate stoste, and polysorbate 80 (50- 300μg/ml), succinate buffer (300-800μg/ml), sodium chloride (7.5-9.5mg/ml), water for injection, after preparation, adjust the pH to 5.0-7.0, dispense 0.5-1.0ml/dose , which is the vaccine. According to the Manufacture and Verification Regulations of Rabies Vaccines for Human Use (WHO Technical Report Series, No.941, 2007) promulgated by the World Health Organization, and the Manufacture and Verification Regulations of Rabies Vaccines for Human Use promulgated by the Pharmacopoeia of the People's Republic of China (2015 Edition), the method and The standard is tested, and after passing the test, it is human rabies glycoprotein conjugate vaccine (RCV-G).

Example 7

Rabies conjugate vaccine specific toxicity test, toxicity reversal test

The 4 kinds of rabies virus-carrier protein immunogenic conjugate stock solution and 4 kinds of human rabies conjugate vaccines prepared in Example 3, Example 4, Example 5, and Example 6 were sampled respectively and combined into 8 parts of test samples in equal amounts . Each test sample was injected into 4 guinea pigs with a body weight of 250-350g. The injection route was subcutaneous in the abdomen, and the injection dose was 2ml/guinea pigs. The symptoms of the guinea pigs were observed on the 7th, 14th and 21st days after the injection. During the observation period, there was no suppuration, no necrosis, no desquamation, no depilation, no late paralysis and other tetanus symptoms in the injected parts of all test samples, and the body weight of each guinea pig increased compared to before injection.

The 4 kinds of rabies virus-carrier protein immunogenic conjugate stock solution and 4 kinds of human rabies conjugate vaccines prepared in Example 3, Example 4, Example 5, and Example 6 were sampled respectively and combined into 8 parts of test samples in equal amounts , placed at 37°C for 42 days. Each test sample was injected into 4 guinea pigs with a body weight of 250-350 g. The injection route was subcutaneous in the ventral side, and the injection dose was 5 ml/piece. The symptoms of the guinea pigs were observed on the 7th day, the 14th day and the 21st day after the injection. During the observation period, there was no suppuration, no necrosis, no desquamation, no depilation, no late paralysis and other tetanus symptoms in the injected parts of all test samples, and the body weight of each guinea pig increased compared to before injection.

It shows that the tetanus toxoid carriers used in the 4 kinds of rabies virus-carrier protein immunogenic conjugate stock solutions prepared by the present invention and the 4 kinds of human rabies conjugate vaccines do not have tetanus toxin specificity after a series of special combination process steps. There was no toxic reaction, and there was no toxic reversal phenomenon.

Example 8

Rabies Conjugate Vaccine Safety Check

The 4 kinds of rabies virus-carrier protein immunogenic conjugate stock solution and 4 kinds of human rabies conjugate vaccines prepared in Example 3, Example 4, Example 5, and Example 6 were sampled respectively and combined into 8 parts of test samples in equal amounts . For each test sample, 5 mice weighing 18-22 g and 2 guinea pigs weighing 250-350 g were selected, each mouse was intraperitoneally injected with 0.5 ml of sample, and each guinea pig was injected intraperitoneally with 5.0 ml, observed for 7 days, and body weight changes were recorded. The results showed that all the mice and guinea pigs survived the observation period without any abnormal reaction, and the body weight of each mouse and guinea pig increased compared with that before the injection.

It shows that the stock solutions of the 4 kinds of rabies virus-carrier protein immunogenic conjugates prepared by the present invention and the 4 kinds of human rabies conjugate vaccines are not contaminated with exogenous toxic substances, and there are no unexpected unsafe factors.

Example 9

Immunogenicity of Rabies Conjugate Vaccine-Antibody Dynamic Determination

Sampling the whole human rabies virus particle vaccine (RV-Vero) and human rabies whole virus conjugate vaccine (RCV-VP) prepared by the traditional technology of Example 1 and Example 3 and immunizing with physiological sodium chloride solution for 6 to 8 weeks Age BALB/c mice, immunize 6 mice for each sample, immunization dose 0.1ml/mouse, immunization route subcutaneous, immunization procedure 0, 3, 7, 14 days, on 3, 7, 14, 28 days by abdominal administration Arterial blood was collected, and serum was separated by centrifugation. Serum anti-RabAg-specific IgG, IgG1, IgG2a antibodies were measured by ELISA method, and the anti-rabies virus neutralizing antibody titer (Virus Neutralization Antibody, VNA) was measured by RFFIT method. The results are shown in Table 1, Table 2, Table 3, and Table 4, respectively. The data showed that the IgG GMT level and seroconversion rate, IgG1 GMT level and seroconversion rate, IgG2a GMT level and seroconversion rate, VNA GMT level and seroconversion rate were all high in the early stage of human rabies whole virus conjugate vaccine (RCV-VP) Vero cell human rabies vaccine (RV-Vero) prepared by existing traditional technology. The dynamic test results of the rabies conjugate vaccine antibody showed that the early antibody titer and antibody positive conversion rate of the human rabies whole virus conjugate vaccine (RCV-VP) were superior to the Vero cell human rabies vaccine prepared by traditional technology. Therefore, the rabies immunogenic conjugate and vaccine provided by the present invention, and the rabies immunogenic conjugate and vaccine prepared by the method of the present invention have beneficial technical effects, and can induce an immune response in the body more rapidly in the early stage of vaccination , which is beneficial to realize the efficacy of the drug in the early incubation period or before the onset of the disease after the patient is infected with rabies virus, and save the patient's life.

Table 1. Comparison of IgG GMT (positive conversion rate) produced by rabies conjugate vaccine and Vero cell rabies vaccine

Table 2. Comparison of IgG1GMT (positive conversion rate) produced by rabies conjugate vaccine and Vero cell rabies vaccine

Table 3. Comparison of IgG2a GMT (positive conversion rate) produced by rabies conjugate vaccine and Vero cell rabies vaccine

Table 4. Comparison of VNA GMT (positive conversion rate) produced by rabies conjugate vaccine and Vero cell rabies vaccine

Example 10

Determination of Protective Efficacy of Rabies Conjugate Vaccine

The 5 kinds of human rabies conjugate vaccines prepared in Example 1, Example 3, Example 4, Example 5, and Example 6 and the human rabies reference vaccine were diluted by 1:25, 1:125, and 1:625 respectively 16 mice of 14-16 g were immunized with different dilutions of human rabies conjugate vaccine and human rabies reference vaccine, and each mouse was immunized with 0.5ml intraperitoneally, twice with an interval of 7 days. 14 days after the primary immunization, the rabies virus strain CVS containing 5-100 LD ₅₀ virus load was used for intracerebral challenge, each 0.03ml. Observe for 14 days, count the mice that died after the 5th day and showed typical rabies brain symptoms, calculate the ED ₅₀ value, and then calculate the vaccine potency. From the data in Table 5, it can be seen that compared with the RV-Vero prepared by the existing traditional method, the rabies conjugate vaccine has a higher potency.

Table 5. Results of titer determination of rabies conjugate vaccine

Claims (21)

1.A method of rabies immune originality conjugate is prepared, the described method comprises the following steps：

(a) purified that Rabies Virus Antigen stoste is made；

(b) the Rabies Virus Antigen stoste for preparing step (a) carries out chemical key connection with carrier protein, then purified system At Rabies Virus Antigen-carrier protein immunogenic conjugates, carrier protein is tetanus toxoid (TT), chemistry key connection It is to removePeptide connectsBeing connected chemically in addition, the chemical bond connection method of Rabies Virus Antigen and the carrier protein includes carbon two Imines method, mixed anhydride method, N- hydroxyl succinimides ester process, glutaraldehyde method, diazotising method, succinic anhydride method, carbonyl dimidazoles At least one of method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay.

2. according to the method described in claim 1, it is characterized in that, the Rabies Virus Antigen includes rabies totivirus Grain, the hydrophobin outer membrane prepared after cracking through the hydrophobin sample particle of recombinant expression, rabies whole virus particles Segment, the dog disease viral glycoprotein isolated and purified from hydrophobin, the nucleoprotein isolated and purified from hydrophobin, from mad dog The phosphoprotein of sick virus isolation, is isolated and purified from hydrophobin the stromatin isolated and purified from hydrophobin Polymerase, the rabies virus glucoprotein through recombinant expression, the rabies virus nucleoprotein through recombinant expression, through recombinant expression Hydrophobin phosphoprotein, the hydrophobin stromatin through recombinant expression, the hydrophobin polymerase through recombinant expression At least one of.

3. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste is will Hydrophobin fixes that seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, rabies whole virus particles are former made of purifying Liquid.

4. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste is warp Hydrophobin sample particle stoste made of recombinant expression, purifying.

5. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste be by According to the hydrophobin outer membrane segment stoste that following steps obtain, hydrophobin fixes seed culture of viruses inoculating cell, culture, harvest disease Venom, inactivation, purifying and manufactured rabies whole virus particles stoste；The rabies whole virus particles prepared in above-mentioned steps are former Hydrophobin outer membrane segment stoste is made through cracking, purifying in liquid.

6. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste is warp Rabies virus glucoprotein stoste made of purifying.

7. method according to any one of claims 1 to 6, which is characterized in that the Rabies Virus Antigen is according to choosing Extremely from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, aGV plants Rabies Virus Antigen prepared by a kind of few hydrophobin fixed virus.

8. method according to any one of claims 1 to 6, which is characterized in that the Rabies Virus Antigen is according to choosing At least from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, aGV plants Rabies Virus Antigen prepared by a kind of hydrophobin fixed virus DNA recombinant expression.

9. the method according to the description of claim 7 is characterized in that the Rabies Virus Antigen contains one or more The functional groups of chemical key connection are carried out with carrier protein.

10. according to the method described in claim 9, it is characterized in that, the Rabies Virus Antigen contain one or two with The upper functional groups that chemistry key connection is carried out with carrier protein include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO) and Amino (- NH₂) at least one.

11. the method according to the description of claim 7 is characterized in that the carrier protein contain one or more with it is mad Dog disease viral antigen carries out the functional groups of chemical key connection.

12. according to the method for claim 11, which is characterized in that the carrier protein contain one or more with it is mad The functional groups that dog disease viral antigen carries out chemical key connection include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO), ammonia Base (- NH₂) at least one.

13. the method according to the description of claim 7 is characterized in that the Rabies Virus Antigen and the carrier protein Chemical bond bridging agent includes 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), 1- cyclohexyl -3- (2-N- Morpholinyl ethyl) carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimide (DIC), 2- Ethyl -5- phenyl isoxazole -3'- sulfonate (Woodward ' s reagent K), N, N'- carbonyl dimidazoles (CDI), schiff bases Generate the reagent sodium cyanoborohydride (NaBH with reductive amination process₃) or sodium borohydride (NaBH CN₄At least one of).

14. the method according to the description of claim 7 is characterized in that the chemistry of the Rabies Virus Antigen and carrier protein Key connection is to be directly connected to, described to be directly connected to not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridge Join molecule (Bridging Molecule), Rabies Virus Antigen is that zero-length connects (Zero-Length with carrier protein Linking) or zero-length is crosslinked (Zero-Length Crosslinking) or zero-length bridging (Zero-Length Bridging), the chemical bond of connection Rabies Virus Antigen and carrier protein is between Rabies Virus Antigen and carrier protein And not comprising newly-increased atom or molecule.

15. the method according to the description of claim 7 is characterized in that the chemistry of the Rabies Virus Antigen and carrier protein Key connection is by the connection of linking arm, spacerarm or bridging molecules, and the linking arm, spacerarm or the bridging molecules that newly create will be mad Dog disease viral antigen and carrier protein form Rabies Virus Antigen-carrier protein immunogenic conjugates through chemistry key connection, The chemical bond for connecting Rabies Virus Antigen and carrier protein is former comprising increasing newly between Rabies Virus Antigen and carrier protein Son or molecule.

16. the method according to claims 14 or 15, which is characterized in that the linking arm or spacerarm or bridging molecules are Adipic acid dihydrazide (ADH).

17. according to claim 1 to 6,9 to 14 any one of them methods, which is characterized in that the Rabies Virus Antigen warp It is keyed again by chemistry with carrier protein after activation.

18. according to claim 1 to 6,9 to 14 any one of them methods, which is characterized in that after the carrier protein is activated It is keyed again by chemistry with the Rabies Virus Antigen.

19. according to claim 1 to 6,9 to 14 any one of them methods, which is characterized in that the Rabies Virus Antigen and It is keyed again by chemistry after the carrier protein difference is activated.

20. according to any one of claim 1 to 6,9 to 14 the method, which is characterized in that the rabies immune being prepared is former Property conjugate preparing the application prevented and treated on rabic drug.

21. according to any one of claim 1 to 6,9 to 14 the method, which is characterized in that the rabies immune being prepared is former Property conjugate prepare prevent, treat or auxiliary for treating cancer drug on application.