CN109602899B

CN109602899B - Intradermal injection technology for enhancing virus antigen immunogenicity and application thereof in hand-foot-and-mouth disease vaccine research

Info

Publication number: CN109602899B
Application number: CN201811294631.3A
Authority: CN
Inventors: 李琦涵; 范胜涛; 张莹; 徐兴丽; 冯敏; 董承红; 杨二霞; 蒋国润; 廖芸; 姜莉; 王丽春; 廉亚茹; 崔萍芳
Original assignee: Aimei Convac Bio Pharmacy Jiangsu Co ltd; Institute of Medical Biology of CAMS and PUMC
Current assignee: Aimei Convac Bio Pharmacy Jiangsu Co ltd; Institute of Medical Biology of CAMS and PUMC
Priority date: 2018-11-01
Filing date: 2018-11-01
Publication date: 2022-12-02
Anticipated expiration: 2038-11-01
Also published as: CN109602899A

Abstract

The invention provides an intradermal injection technology for enhancing virus antigen immunogenicity and application thereof in hand-foot-and-mouth disease vaccine research, wherein the intradermal injection technology is suitable for a bivalent hand-foot-and-mouth disease vaccine for a human, and the bivalent hand-foot-and-mouth disease vaccine for the human is prepared from an enterovirus 71 (EVA 71) and a coxsackievirus A group 16 (CVA 16). The vaccine was prepared with diploid fibroblasts derived from human embryonic lung (KMB 17 cells) as the passaged cell matrix. Through comparing intracutaneous and intramuscular injections of the bivalent hand-foot-and-mouth disease inactivated vaccine for human, the result shows that the intracutaneous injection immunization technical method can more quickly stimulate related components of a natural immune system, enhance the immunogen effect of a virus antigen, induce and activate the natural immune response, start the specific immune response and effectively resist virus diseases.

Description

Intradermal injection technology for enhancing virus antigen immunogenicity and application thereof in hand-foot-and-mouth disease vaccine research

Technical Field

The invention belongs to the technical field of human preventive medicine. More specifically, the invention relates to an immunity injection technology for enhancing virus antigen immunity, which is suitable for human hand-foot-and-mouth disease inactivated vaccines.

Background

Human hand-foot-and-mouth disease (HFMD) caused by infection with enterovirus 71 (EVA 71) and coxsackievirus group a16 (CVA 16) is one of the viral infections that have been particularly emphasized in recent years by the public health regulatory layers of various countries in asia-pacific regions, and the main causes of this infection receiving widespread attention include two areas: for one, a number of large-scale outbreaks involving a large number of people and a wide spread area have been caused in various regions around the world. Secondly, in the infected population, a small number of infected individuals can be found to develop severe neurological damage and clinical features of cardiopulmonary failure, and the clinical prognosis for these patients is often quite severe. According to the statistical data of the disease prevention and control center in China, the number of HFMD cases reported in the mainland of China from 2008 to 2012 is 150 million/year, the death rate is more than 500 cases/year, and the prevalence of up to hundreds of thousands of children also appears in Taiwan during 1998 to 2000. HFMD developed in australia in 1999 was approximately 6000 people involved, with EV71 infections accounting for approximately 50% and the remainder being CoxA16 infections. In the 80 s of the 20 th century, the prevalence of hand-foot-and-mouth disease in continental countries mainly is CA16 infection, and after the 21 st century, the disease gradually turns into EVA71 infection as the main infection. Several tens of thousands of HFMD attacks and several hundreds of deaths have been reported in succession in korea, malaysia, singapore, vietnam, and the like. Thus, the public health implications of the development of a vaccine for hand-foot-and-mouth disease for the two most prominent pathogens causing HFMD, EVA71 and CVA16, are self-evident.

Both EVA71 and CVA16 belong to the enterovirus family of picornaviruses, have a positive-stranded RNA genome, a typical icosahedral structure, are non-enveloped, and are normally transmitted via the fecal tract or the respiratory tract. The clinical symptoms caused by the virus usually show that the hand, foot and mouth have characteristic herpes, and are accompanied by cold symptoms such as fever to a certain degree. Individual patients can cause lesions or complications in the nervous system, respiratory system, leading to severe illness and even death.

Since the first isolation of EVA71 in 1969 in the United states, three genotypes A, B and C, more than ten gene subtypes (B1-B5 and C1-C5), each of which includes several strains, have been developed. According to current molecular epidemiological analysis, the EVA71 virus has 7 serotypes (A, O, C, asia I, south

African terrorisis

1, 2 and 3), and the serotypes are crossed to a large extent. The predominant serotype in most areas of our country is the C4 subtype.

After CVA16 is separated and reported for the first time in the 50 th century, researchers carry out intensive research on the CVA16, the CVA is divided into three genotypes A, B and C, the two genotypes B and C are combined in a classification mode, the genotype B can be divided into subtypes B1 and B2, and the genotype B1 can be further divided into subtypes B1a, B1B and B1C. In the Pacific region, CVA16 is the dominant epidemic strain of HFMD.

Although the research on antiviral drugs and corresponding vaccines has achieved some results so far, the research on multivalent preventive vaccines against hand-foot-and-mouth disease against CVA16 and other enteroviruses has become an urgent issue with the emergence of problems such as the use of inactivated vaccines against EVA71, the change of pathogenic spectrum of hand-foot-and-mouth disease, the limited cross-protection ability between various enteroviruses, and the like. However, the vaccine development exploration is carried out under the background that the etiology is complex and the research on the infection pathogenesis is not very clear, and especially under the condition of a heavy technical bottleneck encountered in the CVA16 vaccine development, the higher requirements are put on the technical process establishment of the multivalent vaccine for the hand-foot-and-mouth disease. Therefore, the preparation of bivalent and even multivalent hand-foot-and-mouth disease vaccine and the selection of immunization method are more urgent and important by deeply knowing the existing relevant data of EVA71 and CVA16 etiology, epidemiology and infection pathogenesis and combining the research experience of EVA71 inactivated vaccine.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides an immunization technical method suitable for the human hand-foot-and-mouth disease inactivated vaccine on the basis of the experience of the EVA71 inactivated vaccine.

The purpose of the invention is realized by the following technical scheme:

an inactivated vaccine for bivalent hand-foot-and-mouth disease for human, which is prepared from enterovirus 71 (EVA 71) and coxsackievirus A group 16 (CVA 16) by taking diploid fibroblast (KMB 17 cell) derived from human embryonic lung as a passage cell matrix, and is an intradermal injected vaccine. The intradermal injection technique can more quickly and effectively activate the related components of the natural immune system, enhance the immunogen effect of the virus antigen components in the vaccine, induce and activate the natural immune response, start the specific immune response and effectively improve the immune defense capability of the organism to the virus pathogenicity.

Further, the EVA71 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 16684.

Further, the CVA16 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.6350.

Further, the vaccine comprises the following components:

EVA71 inactivated and purified antigen 100-400U/mL

CVA16 inactivated and purified antigen 100-400U/mL

0.5-0.8mg/mL of aluminum hydroxide.

Another aspect of the invention:

the use of the inactivated vaccine for the bivalent hand-foot-and-mouth disease for human in preparing the medicament for preventing or treating the diseases.

Another aspect of the invention:

a preparation method of a bivalent hand-foot-and-mouth disease inactivated vaccine for human use comprises the following steps:

(1) Cell culture: selecting KMB-17 cells growing compactly, discarding culture solution, digesting with pancreatin, then discarding pancreatin solution, and suspending cells with DMEM to obtain cell suspension;

(2) Virus inoculation and culture: adding the cell suspension into a cell factory, uniformly mixing, and placing at 37 ℃ for 3-4 days; removing the culture solution after the culture medium grows into a compact monolayer; respectively adding EVA71 or CVA16 virus seeds with the inoculation amount moi of 0.02-0.05; after mixing uniformly, placing at 37 ℃, and gently shaking timely; then adding a quantitative cell growth culture solution, and placing at 37 ℃ for 3-4 days; collecting virus harvest liquid after the cells are completely diseased;

(3) And (3) concentrating: filtering the virus harvest liquid to remove cell debris, and then performing ultrafiltration concentration to obtain a virus concentrated solution;

(4) And (3) purification: loading the virus concentrated solution into Sepharose 6FF column chromatography for purification, then carrying out iodoxanol density gradient centrifugation for purification, and collecting the purified virus particle peak; detecting the content of EVA71 or CVA16 antigen by adopting an ELISA antigen detection kit, determining the protein concentration by a Lowry method, and adjusting the antigen concentration to 2000U/mL by proportioning dilution;

(5) Inactivation: adding formaldehyde inactivated virus, dialyzing, concentrating, electrophoresing and HPLC to determine that the purity of virus particles is higher than 95%, detecting the content of EVA71 or CVA16 antigen by using an ELISA antigen detection kit, and determining the protein concentration by a Lowry method to obtain an inactivated purified antigen solution;

(6) Preparing a finished product: filtering and sterilizing the above inactivated and purified antigen solution, diluting with phosphate buffer to EVA 71U/mL and CVA 16U/mL, and adding Al (OH) ₃ And the final concentration is 0.5mg/mL, and the product is the bivalent hand-foot-and-mouth disease inactivated vaccine for human use.

Compared with the prior art, the invention has the beneficial effects that:

1. the technical scheme provided by the invention is based on that the EVA71 and the CVA16 belong to the same enterovirus member, and the research and development experience of a plurality of similar successful enterovirus vaccines can be used for reference, in particular to the research and development experience of the EVA71 inactivated vaccine which also causes hand-foot-and-mouth disease. As a vaccine with safety and emergency significance, the beneficial population is mainly the children population, and the inactivated vaccine has relative advantages. As a novel prophylactic vaccine, the quality of the vaccine can be improved by using a suitable cell matrix. The subculture cell matrix which has been widely used in the production of inactivated vaccine at present mainly includes: vero cells from african green monkey kidney and diploid fibroblasts (KMB 17 cells) from human embryonic lung. The latter, although relatively poor in the ability of the virus to support growth replication compared to Vero cells, has better safety as a human-derived cell. Meanwhile, the EVA71 inactivated vaccine production technology and quality control standard are just based on the experience of the KMB17 cell matrix, and the human bivalent hand-foot-and-mouth disease inactivated vaccine described by the invention is also suitable for preparing human diploid cells;

2. the invention provides a method for makingThe inactivated vaccine for the bivalent hand-foot-and-mouth disease for the human is prepared by using a human diploid cell (KMB 17 cell) matrix and a preparation method thereof, wherein two strains of EVA71 and CVA16 have good growth and replication capacity in the human diploid cell (KMB 17 cell) matrix, and a typical cytopathic phenomenon can occur 3-4 days after KMB17 cells are inoculated by virus adsorption; the growth kinetics curve of the virus seeds on KMB17 cells shows that the initial typical lesion appears in the inoculated cells within 8 hours, the lesion peak appears within 24-48 hours, and the titer of the virus harvest is detected at the moment, wherein the titer is usually 7.0 +/-0.5 LogCCID50; poison seed press 10 ^4.5 CCID ₅₀ Only inoculated 3-4 days old suckling mice can cause typical quadriplegia or death phenomenon of the suckling mice within 3-5 days; in addition, a bivalent vaccine and a multivalent vaccine aiming at the EVA71 and the CVA16 are not available on the market at home and abroad, and no related patent authorization is found;

3. the immunization method of the inactivated vaccine for the bivalent hand-foot-and-mouth disease for the human is intradermal injection immunization, and compared with intradermal and intramuscular routes, the intradermal injection immunization can effectively start a natural immune system so as to ensure that the vaccine can effectively induce good immune response and can effectively resist virus diseases.

Drawings

FIG. 1 is a graph of the growth kinetics of EVA71 and CVA16 on KMB17 cells;

FIG. 2 shows the detection of neutralizing antibody titer induced in one month after two intradermal and intramuscular immunization of mice with the inactivated vaccine against bivalent hand-foot-and-mouth disease for human;

FIG. 3 shows the detection of natural immune signal molecules in local tissues inoculated by intradermal and intramuscular immunized mice by the inactivated vaccine for bivalent hand-foot-and-mouth disease for human;

FIG. 4 shows the co-localization relationship between antigens and ILCs in local tissues inoculated by the bivalent hand-foot-and-mouth disease inactivated vaccine for the human through intradermal and muscle immunized mice;

FIG. 5 shows the co-localization relationship between the antigen and Dendritic Cells (DCs) in local tissues inoculated by the human bivalent hand-foot-and-mouth disease inactivated vaccine through intradermal and intramuscular immunized mice;

FIG. 6 shows the antigen presentation and T cell specific proliferation reaction of T cells by the human bivalent hand-foot-and-mouth disease inactivated vaccine for percutaneous internal immunization of mice activated Dendritic Cells (DCs) and natural Immune Lymphocytes (ILCs);

FIG. 7 shows the immunoprotection of the offspring animals of mice immunized with the inactivated vaccine for bivalent hand-foot-and-mouth disease through the skin and muscle.

Detailed Description

The invention will be illustrated in more detail by means of the following examples. It should be noted that the following examples are illustrative only, and the scope of the present invention is not limited by these examples. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The enterovirus type 71 (EVA 71): has been preserved in China general microbiological culture Collection center (CGMCC for short; address: no. 3 of Xilu No.1 of Beijing, ministry of science and technology, japan, haorizonum, beijing, china, japan, microbiological research institute; zip code: 100101) within 16 months and 10 months in 2018, and the preservation number is CGMCC No. 16684.

The Coxsackie virus type A16 (CVA 16): has been preserved in China general microbiological culture Collection center (CGMCC for short; address: no. 3 of West Lu 1 of Beijing, chaoyang, japan, institute of microbiology, china academy of sciences; zip code: 100101) at 17.07.17.2012, and the preservation number is CGMCC No.6350.

Example 1

The embodiment provides a preparation method of a bivalent hand-foot-and-mouth disease inactivated vaccine for human, which comprises the following specific steps:

(1) And (3) cell culture: selecting the compact-growing 28 th-generation KMB-17 cells according to a KMB-17 cell production verification procedure, discarding a culture solution, digesting with 0.1% of pancreatin, discarding a pancreatin solution, suspending the cells with DMEM (DMEM) with the volume of 4% of the volume of the original culture solution to obtain a cell suspension;

(2) Virus inoculation and culture: adding appropriate amount of the above cell suspension into a cell factory, mixing, and standing at 37 deg.C for 3-4 days; after the growth of a compact monolayer, removing the culture solution; respectively adding EVA71 or CVA16 virus seeds with the inoculation amount moi of 0.02-0.05; after mixing uniformly, placing at 37 ℃, and gently shaking timely; after 30min, adding a quantitative cell growth culture solution, and standing at 37 ℃ for 3-4 days; collecting virus harvesting liquid after the cells are completely diseased;

(3) Concentration: filtering the virus harvest liquid by using an SAS single-core filtering device to remove cell debris; performing ultrafiltration concentration by using a MasterFlex ultrafiltration concentration instrument to obtain virus concentrated solution;

(4) And (3) purification: loading a certain volume of virus concentrated solution into Sepharose 6FF column chromatography for purification, then carrying out iodixanol density gradient centrifugation for purification, and collecting a purified virus particle peak; detecting the content of EVA71 or CVA16 antigen by using an ELISA antigen detection kit, determining the protein concentration by using a Lowry method, and adjusting the antigen concentration to 2000U/mL by proportioning dilution;

(5) Inactivation: adding formaldehyde according to the proportion of 1;

(6) Preparing a finished product: filtering and sterilizing the above inactivated and purified antigen solution, diluting with phosphate buffer to EVA 71U/mL and CVA 16U/mL, and adding Al (OH) ₃ The final concentration is 0.5mg/mL, and the product is the finished product of the bivalent hand-foot-and-mouth disease inactivated vaccine for the needed human.

Example 2

The present example is the distribution detection of each intermediate product in the preparation process of the inactivated vaccine for the bivalent hand-foot-and-mouth disease for human described in example 1.

Wherein the virus harvest liquid, the virus concentrated liquid, the inactivated and purified antigen liquid and the semi-finished product are clear liquids without foreign matters and precipitates, and the pH value is 7.2-8.0, which is shown in Table 1, and the bacteria-free detection and the mycoplasma detection are negative.

As shown in FIG. 1, samples were taken at various time points after virus inoculation of KMB17 cells for infectious titer detection. The result shows that the primary typical lesion appears after the inoculation of the cells within 8 hours, the lesion peak appears within 24-48 hours, and the titer is usually 7.0 +/-0.5 LogCCID ₅₀ 。

Example 3

In this example, the safety of the inactivated vaccine finished product for the human bivalent hand-foot-and-mouth disease prepared in example 1 was tested.

The vaccine finished product is injected into the abdominal cavity, the skin and the muscle of a mouse with the weight of 18-20g per mouse according to 0.2mL, 20 mice in each group have no death within one month, and the weight gain of the mouse is the same as that of a normal saline control group, which is shown in a table 2.

The content of formaldehyde in the vaccine finished product is not higher than 50 mu g/dose, and the content of bacterial endotoxin is not higher than 100 EU/dose. And was negative in sterility testing, see table 3.

Example 4

In the embodiment, the finished product of the inactivated vaccine for the bivalent hand-foot-and-mouth disease for the human, which is prepared in the embodiment 1, can quickly and effectively stimulate molecules related to natural immune response through intradermal immunization, enhance the immunogenic effect of virus antigen components in the vaccine, and induce good immune response.

The vaccine finished product is injected into a mouse with the weight of 18-20g twice (0 month and 1 month) by intradermal and intramuscular routes according to 0.2 mL/mouse respectively, and can generate an anti-EVA 71 neutralizing antibody with the titer higher than 64 and an anti-CVA 16 neutralizing antibody with the titer higher than 32 in an animal body; meanwhile, the adjuvant control group can not induce the production of corresponding antibodies, and as can be seen from fig. 2, the human immunized by the bivalent hand-foot-and-mouth disease inactivated vaccine through the percutaneous internal injection method can have good immunogenicity. As can be seen from the figure 3, the detection of the natural immune signal molecules in local tissues inoculated to the mice immunized by the intradermal and intramuscular injection of the vaccine finished product proves that the immunization of the human bivalent hand-foot-and-mouth disease inactivated vaccine by the percutaneous internal injection method can activate the expression of the natural immune signal related molecules. As can be seen from FIG. 4, the co-localization of antigen and ILCs was confirmed by the co-localization of antigen and ILCs in local tissues after intradermal and intramuscular immunization of mice with the vaccine as a finished product, and showed a higher co-localization rate at 12 hours after the vaccination, and then gradually decreased toward 24 and 48 hours. As can be seen from FIG. 5, the co-localization of antigen and DCs was demonstrated by the co-localization of antigen and DCs in local tissues after intradermal, intramuscular immunization of mice with the vaccine preparations, and showed an increasing trend in the co-localization frequency of both over time. As can be seen from FIG. 6, antigen presentation and T cell specific proliferation reaction of DCS and ILCS activated by mice immunized intradermally with vaccine finished products on T cells prove that the T cell specific proliferation reaction can be activated by immunization with the bivalent hand-foot-and-mouth disease inactivated vaccine via intradermal injection method.

Example 5

This example is a protective test of the inactivated vaccine finished product for the bivalent hand-foot-and-mouth disease for human prepared in example 1.

The vaccine finished product is injected into a mouse with the weight of 18-20g twice (0 and 1 month) by an intradermal and intramuscular way according to 0.2 mL/mouse, the mouse is mated after one month to produce offspring, at least more than 90% of the offspring born by the mice immunized by the percutaneous and intramuscular way can resist the attack of EVA71 and CVA16 lethal dose viruses, and the vaccine has a good protection effect by the immunization of the bivalent hand-foot-and-mouth disease inactivated vaccine by the percutaneous internal injection method as shown in figure 7.

Claims

1. A preparation method of a bivalent hand-foot-and-mouth disease inactivated vaccine for human beings is characterized by comprising the following steps:

(1) And (3) cell culture: culturing KMB-17 cells in DMEM;

(2) Virus inoculation and culture: respectively adding EVA71 or CVA16 virus seeds into KBM-17 cells grown into compact single layers, and culturing at 37 ℃; collecting virus harvest liquid after the cells are completely diseased;

(3) Concentration: respectively filtering the virus harvest liquid to remove cell debris, and then performing ultrafiltration concentration to obtain a virus concentrated solution;

(4) And (3) purification: respectively loading the virus concentrated solution to Sepharose 6FF column chromatography for purification, then carrying out iodoxanthol density gradient centrifugation for purification, and collecting purified virus particle peaks;

(5) Inactivation: adding formaldehyde to inactivate viruses respectively, and dialyzing and concentrating to obtain inactivated and purified antigen solution;

(6) Preparing a finished product: filtering the above inactivated and purified antigen solution respectively, diluting with phosphate buffer solution to EVA 71-400U/mL and CVA 16-400U/mL, and adding Al (OH) ₃ The final concentration is 0.5-0.8mg/mL, and the product is the bivalent hand-foot-and-mouth disease inactivated vaccine for the human;

the EVA71 is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No.16684;

the CVA16 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No.6350.

2. The use of the inactivated vaccine against bivalent hand-foot-and-mouth disease for human prepared by the method of claim 1 in the preparation of a medicament for preventing diseases.