CN109602901B

CN109602901B - Herpes zoster virus vaccine and preparation method and application thereof

Info

Publication number: CN109602901B
Application number: CN201910016746.4A
Authority: CN
Inventors: 陈德祥; 董丽春
Original assignee: Chengdu Maikekang Biotechnology Co ltd
Current assignee: Chengdu Huarenkang Biotechnology Co.,Ltd.; Chengdu Maikekang Biotechnology Co ltd
Priority date: 2019-01-08
Filing date: 2019-01-08
Publication date: 2022-05-27
Anticipated expiration: 2039-01-08
Also published as: CN109602901A

Abstract

The invention relates to a herpes zoster virus vaccine and a preparation method and application thereof. The composite adjuvant contains a nano carrier and at least one molecular adjuvant inducing cellular immunity. The vaccine disclosed by the invention can induce strong antibody reaction and T cell reaction, can achieve the optimal protection effect and has small side effect.

Description

Herpes zoster virus vaccine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of vaccine preparation, and relates to a herpes zoster virus vaccine.

Background

Shingles is an acute infectious skin disease caused by varicella-zoster virus. After infection of children, chickenpox occurs. Because the virus is neurotropic, it can remain latent in the neurons of the dorsal root ganglion of the spinal nerve for a long period of time after infection. When the old people have low resistance or fatigue, infection and cold, the virus can grow and propagate again and move to the skin along nerve fibers, so that the invaded nerve and the skin generate strong inflammation. According to the statistics of the united states centers for disease control, approximately 1 in every 3 people in the united states will develop shingles in their lifetime. An estimated 100 million cases of herpes zoster occur each year. The risk of herpes zoster is greater in special people. This includes those who maintain immune hypofunction, such as cancers like leukemia and lymphoma, as well as Human Immunodeficiency Virus (HIV), and receive immunosuppressive drugs such as steroids and drugs administered after organ transplantation. Most people who develop herpes zoster have only one episode in life. However, some people suffer from recurrent morbidity.

The incidence and severity of herpes zoster increase with age and increase significantly after age 50, which is associated with a decline in the cellular immune function of the elderly. It is estimated that approximately half of the elderly over the age of 85 have had at least one herpes zoster. Existing epidemiological data on the burden of herpes zoster disease are from high-income regions. Studies conducted in canada, israel, japan, taiwan and the united states show that the age-adjusted population has a shingles incidence of 3.4-5.0% for the total population and 8-11% for the population over 65. A study conducted in 27 countries in Europe found that the incidence of herpes zoster varies from 2.0 to 4.6% in different countries, but does not show significant regional differences. Data in recent years have shown that the incidence of young adults has also increased.

The united states has long used attenuated vaccines to prevent herpes zoster (Zostervax). The vaccine and the vaccine for preventing varicella are the same strain, namely, Oka strain. The vaccine is used for people over 60 years old, and the protective effect is about 50% on average. Its protection is age-related, with the greater the age, the less protective. The protection rate of Zostavax against herpes zoster is 69.8% and 51% in the population 50-59 years and 60 years old respectively, and the protection rate decreases with the age, and is only 18% in the population 80 years old, and the other limitation of the attenuated vaccine is that the attenuated vaccine cannot be used in the population with defective immune system, which is the population most in need of protection.

The 2017 subunit vaccine (Shringrix) began to be applied in various countries. Shingrix is a recombinant subunit vaccine prepared from varicella-zoster virus surface glycoprotein gE (expressed by CHO cells) and adjuvant AS01B (liposome adjuvant containing MPL and QS21 AS main ingredients), and is injected intramuscularly and immunized twice. The protection rate of Shingrix for herpes zoster of people over 50 years old and over 70 years old is 97.2 percent and 89.8 percent respectively, the protection rate is not obviously influenced by the age, and the protection rate is still 89.1 percent in people over 80 years old. However, subunit vaccines have a strong side effect.

Since most people carry herpes zoster virus, the virus is latent within neurons. The protective mechanism of herpes zoster vaccines is by inducing a T cell immune response that inhibits the spread of the virus through the nerves to the skin upon activation within neurons. Although the primary protection mechanism of vaccines is T cells, antibodies also have some protective effect. The main evidence is that inactivated viral vaccines formulated with aluminum adjuvants also have a 50% protective effect, and vaccines formulated with aluminum adjuvants generally do not induce a T cell response.

The incidence of herpes zoster in China is very high, and no vaccine for preventing herpes zoster exists at present. The patient is mainly treated by interferon, Chinese herbal medicines and the like. The aluminum hydroxide and aluminum phosphate adjuvants commonly used for human vaccines can only enhance antibody responses and cannot induce T cell immunity necessary for preventing herpes zoster, so that the aluminum hydroxide and aluminum phosphate adjuvants are not suitable for developing herpes zoster vaccines. A barrier to the development of safe and effective herpes zoster vaccines is the lack of novel adjuvants that safely and effectively activate T cell immunity and neutralizing antibodies.

The invention discloses a preparation method of a varicella-zoster virus glycoprotein E extracellular domain protein, and relates to a recombinant vector of the varicella-zoster virus glycoprotein E extracellular domain protein, a transformant prepared by using the vector, a protein produced by using the transformant, and application of the protein in the research and development of related vaccines such as varicella vaccines, herpes zoster vaccines, multivalent vaccines containing the varicella-zoster vaccines and combined vaccines, and the detection of corresponding antigens and antibodies. The invention utilizes gene engineering technology to clone and connect VZV-gE extracellular region nucleic acid sequence to prokaryotic expression vector pET-21b to obtain recombinant vector; transforming the recombinant vector into escherichia coli BL21(DE3) to obtain a transformant; and inducing the transformant to express to obtain the target protein. The expression system can efficiently express VZV-gE extracellular region antigen protein, and the expressed VZV-gE extracellular region protein antigen has good immunogenicity and can be used in the fields of research and development of corresponding vaccines and detection of antigen and antibody. However, the disadvantage of this patent (201611119488) is that the resulting protein is not glycosylated and the epitope of the antigen may not be authentic. Whether it can be used for developing a vaccine remains to be verified. Also, the above patents do not relate to adjuvant technology necessary for the development of effective vaccines.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a herpes zoster virus vaccine, which comprises the preparation of an antigen and the screening of an adjuvant. The vaccine disclosed by the invention can induce strong antibody reaction and T cell reaction, can achieve the optimal protection effect and has small side effect.

The invention provides the following technical scheme:

a herpes zoster virus vaccine is prepared from varicella-zoster virus glucoprotein E or its fragment and compound adjuvant.

Preferably, the varicella-zoster virus glycoprotein E is in particular a herpes zoster virus glycoprotein E.

In any of the above embodiments, the varicella-zoster virus glycoprotein E is specifically a glycoprotein E of the Dumas strain of herpes zoster virus, and the amino acid sequence is shown in SEQ ID No. 1.

Preferably, in any of the above embodiments, the fragment of varicella-zoster virus glycoprotein E comprises an extracapsular fragment gE-1 of herpes zoster virus glycoprotein E and/or a fusion protein gE-2 of the extracapsular fragment and the intracapsular fragment of glycoprotein E.

Any of the above embodiments is preferably made from a fusion protein gE-2 of an extracapsular fragment and an intracapsular amino acid fragment of the glycoprotein E of herpes zoster virus.

In any of the above embodiments, preferably, the amino acid sequence of the extracapsular fragment of herpes zoster virus glycoprotein E is SEQ ID No.2 or an amino acid sequence with a similarity of more than 95%.

In any of the above embodiments, preferably, the amino acid sequence of the fusion protein gE-2 of the extracapsular fragment and the intracapsular amino acid fragment of the glycoprotein E of herpes zoster virus is SEQ ID No.3 or an amino acid sequence which has a similarity of more than 95%.

In any of the above embodiments, preferably, the fragment of varicella-zoster virus glycoprotein E is expressed in a eukaryotic expression system, and the glycosylation of the protein is of the same degree and type as the glycosylation on the surface of the virus.

Preferably, in any of the above embodiments, the composite adjuvant comprises a carrier and a molecular adjuvant for inducing cellular immunity.

In any of the above embodiments, preferably, the composite adjuvant comprises a nano-carrier and at least one molecular adjuvant inducing cellular immunity.

In any of the above embodiments, preferably, the carrier is any one of a nanoemulsion, a liposome, and an aluminum salt adjuvant. The carrier is a nano carrier. The preparation method of the nano-carrier is common knowledge in the field.

In any of the above embodiments, preferably, the aluminum salt adjuvant comprises any one of aluminum hydroxide, aluminum phosphate and calcium phosphate.

Preferably in any of the above embodiments, the nanoemulsion is an oil-in-water emulsion.

Preferably, in any of the above embodiments, the nanoemulsion comprises any one of MF59, Stable emulsion, AS04, AF03, or the like.

In any of the above embodiments, the liposome is preferably a liposome (hollow) made of cholesterol, lecithin, ceramide, etc., and has a vesicle membrane of monolayer or bilayer structure with a diameter of 25-1000 nm.

In any of the above embodiments, preferably, the molecular adjuvant inducing cellular immunity is any one of TLR3 ligand, an agonist of TOLL-7/8 receptor, or an agonist of TOLL-like receptor.

Preferably, in any of the above embodiments, the TOLL-like receptor agonist is any one of TLR4, TLR 5 and TLR 9.

In any of the above embodiments, the composite adjuvant is preferably any one of aluminum hydroxide adsorbed with TLR3 ligand, a nanoemulsion containing an agonist of TLR-7/8, and a nanoemulsion containing an agonist of TLR-7/8.

In any of the above embodiments, the amount of aluminum hydroxide is preferably 0.1-1 mg, and the amount of TLR3 ligand (TLR3L) adsorbed on aluminum hydroxide is preferably 10-1000 micrograms.

In any of the above embodiments, the amount of TLR3 ligand (TLR3L) adsorbed on aluminium hydroxide is preferably 100 and 500 micrograms.

Preferably, in any of the above embodiments, the amount of gE-1 or gE-2 is 10 to 200. mu.g.

In any of the above embodiments, the amount of gE-1 or gE-2 is preferably 10 to 100. mu.g.

Preferably, in any of the above regimens, the TLR7/8 agonist is administered in an amount of 0.1 to 30 micrograms.

Preferably in any of the above regimens, the amount of TLR7/8 agonist is 1-10 micrograms.

The preparation method of herpes zoster virus vaccine is prepared from varicella-zoster virus glycoprotein E or fragment thereof and a composite adjuvant, and comprises the following steps:

(1) preparing a composite adjuvant;

(2) mixing glycoprotein E or its fragment with compound adjuvant, and mixing to obtain vaccine.

Preferably, the composite adjuvant is prepared by adsorbing TLR3 ligand on aluminum hydroxide or by using an agonist of TOLL-7/8 receptor and a nano emulsion or liposome.

Preferably in any of the above embodiments, the fragment of glycoprotein E comprises an extracapsular fragment gE-1 of glycoprotein E of herpes zoster virus and/or a fusion protein gE-2 of the extracapsular fragment and the intracapsular fragment of glycoprotein E.

Preferably, in any of the above embodiments, the amino acid sequence of glycoprotein E is as set forth in SEQ ID No. 1.

In any of the above embodiments, the amino acid sequence of the extracapsular fragment of glycoprotein E is SEQ ID No.2 or an amino acid sequence that has a similarity of 95% or more.

In any of the above embodiments, it is preferable that the amino acid sequence of the fusion protein gE-2 of the extracapsular fragment and the intracapsular amino acid fragment of glycoprotein E is SEQ ID No.3 or an amino acid sequence that has a similarity of 95% or more.

Preferably in any of the above embodiments, the glycoprotein E is a herpes zoster virus glycoprotein E.

Preferably, in any of the above embodiments, the glycoprotein E is glycoprotein E of the Dumas strain of herpes zoster virus.

Preferably, the composite adjuvant comprises a nano carrier and at least one molecular adjuvant inducing cellular immunity.

In any of the above embodiments, preferably, the fragment of varicella-zoster virus glycoprotein E is expressed in a eukaryotic expression system, and the glycosylation of the protein is the same as the degree and type of glycosylation on the surface of the virus. Glycoprotein E is expressed in eukaryotic cells (Chinese Hamster Ovary cell, or CHO cell); primers were designed based on the existing heavy chain sequence and PCR amplification was performed with Hind III/EcoRI sites inserted into the Multiple Cloning Site (MCS) upstream of the pGenHT1.0 vector. The vector was designed with 1 MCS and CMV promoters. Meanwhile, the vector contains Glutamine Synthetase (GS), and the promoter is SV40early promoter.

CHO-K1SP cell suspension cells were transfected with plasmid vectors. And confirming that the stable cell line expressing the target gene is constructed at the beginning according to the expression evaluation result of transfection for 48 h. Transfected cells were seeded into 24-well plates and pressure-screened using cell pool screening media. After the Cell pool screening was completed, the supernatants of all Cell pools were collected for ELSIA to detect expression levels. And selecting Cell pool with high expression quantity to perform monoclonal screening, adopting a limiting dilution method for the monoclonal screening, performing monoclonal imaging shooting, confirming the monoclonality, and finally selecting 10 clones with the monoclonality to perform 70 generations of passage stability tests. Selecting a strain (Clone 6) with high expression and stability for library construction.

CHO high expressing cell clones were grown in serum free medium from 6-well plates, 25ml, and 250 ml shake flasks to a 2 liter reactor. The supernatant was collected and filtered through a 0.22 micron membrane to remove cell debris. Anion chromatography was then performed by Q-sepharose column. Collecting eluate containing gE protein, and performing hydrophobic chromatography and ultrafiltration to obtain purified glycoprotein. Both proteins were purified by molecular sieving to about 95% pure protein. Since the protein is glycosylated, it is dispersed on SDS-PAGE gel and has a molecular weight of 60-70 KD.

The molecular adjuvant inducing cellular immunity is TLR3 ligand, agonist of TOLL-7/8 receptor or agonist of TOLL-like receptor (TLR4, TLR 5, TLR9 and the like) which is a purchased finished product.

In any of the above embodiments, preferably, the composite adjuvant is any one of aluminum hydroxide adsorbed with TLR3 ligand, a nanoemulsion containing an agonist of TLR-7/8, and a nanoemulsion containing an agonist of TLR-7/8.

Preferably, in any of the above embodiments, the amount of TLR3 ligand (TLR3L) adsorbed on aluminium hydroxide is 10-1000 micrograms.

In any of the above embodiments, the amount of gE-1 or gE-2 is preferably 10 to 200. mu.g.

Preferably, in any of the above regimens, the TLR7/8 agonist is administered in an amount of 1 to 10 micrograms.

Use of a composition or vaccine comprising an antigen consisting of the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 and an adjuvant in the manufacture of a medicament for preventing or ameliorating herpes zoster and/or post herpetic neuralgia.

Preferably, it is suitable for adults over 20 years of age, especially for people over 50 years of age.

In the invention:

the virus surface glycoprotein E (gE) is an important virulence factor of herpes zoster virus and also an important vaccine component. During the spread of the virus, gE binds to receptors of the host cell, allowing the virus to invade and spread rapidly to neighboring cells. The gE protein is a transmembrane protein. The extracapsular polypeptide is an antigen component of the current foreign vaccine (Shringrix). This vaccine can block the spread of virus from neurons to the skin, etc., but does not eliminate virus that does not replicate in vivo.

We find that several important T cell sites are on the segment of polypeptide outside the cyst membrane by testing the T cell response of the person inoculated with the attenuated vaccine. To our surprise, we found that polypeptide fragments within the capsular membrane also contain important sites for T cell reflection. We expressed the extracapsular polypeptide and the intracapsular polypeptide fused thereto and demonstrated that both polypeptides were able to induce high levels of T cell responses in animals. The MHC will vary even more among people in different countries and regions. Recombinant proteins containing the polypeptide gE within the capsular membrane may be more broadly protective.

Adjuvants are important components in the development of effective vaccines. In the absence of adjuvant, subunit antigens typically induce only antibody responses and Th2 responses. The commonly used aluminum adjuvants often only enhance the Th 2-like response and are not able to induce the Th 1-like response. Therefore, the successful development of a novel subunit geriatric bleb vaccine requires the use of novel adjuvants to enhance both the antibody response and the cellular immune response. The marketed Shingrix subunit vaccine employs AS01B adjuvant. The main component is liposome adjuvant of MPL and QS 21. In animal and human experiments, very strong antibody and T cell responses can be induced. The MPL component is considered to be a core component for inducing cellular immunity.

There are many immunological adjuvant compositions in the literature that are capable of inducing cellular immune responses in vivo.

The chemical components of Toll-like Receptor 3 ligand (TLR3L) Receptor activators are double-stranded ribonucleic acids, and specific TLR3 activators include TLR3 ligand, polyinosinic cell (polymer formed by kanamycin), TLR3 ligand LC (polymer formed by polylysine) and the like. The TLR3 receptor activator is capable of binding to the intracellular receptor TLR3, stimulating antigen presenting cells and antigen presenting functions, inducing T cell responses.

In addition to the above mentioned MPL extracted from microorganisms, there are also artificially synthesized molecules including glucopyranosyl lipid A (glucopyranosyl lipid A), artificially synthesized lipoprotein A (synthetic-lipid A), which are capable of binding to TLR4 receptors on the surface of antigen presenting cells to activate the cells to stimulate the induction of T cell production.

QS21 is a saponin substance extracted from quillaja saponaria molina in south america, has surfactant activity, and induces T cell responses in humans. The specific mechanism of operation is not clear. Similar molecules include saponin molecules extracted from ginseng or its stem and leaves.

Toll-like Receptor 7 ligand (TLR7L) Receptor activator activators include Rashimod (Resiqimod), imiquimod (Imiqimod),3M-052 and the like. These molecules, which are capable of binding to TLR7 on the surface of antigen presenting cells, activate their antigen presenting function and induce a T cell response.

Toll-like receptor 9 ligand (TLR9L) is a short-chain CpG deoxyribonucleic acid. Many of these sequences, after binding to the intracellular TLR9 receptor, activate antigen presenting cells and induce a T cell response.

In our experiments we found that TLR3 ligand activator, TLR7 receptor activator, and even QS-21 alone had a strong immunostimulatory effect, and that these adjuvants induced a stronger T cell response than MPL. We further prove that the immune effect of the specific immune adjuvant and a nano-carrier compatible composite adjuvant is better than that of an immune molecular adjuvant or a nano-carrier adjuvant which is used independently.

The nano-carrier comprises nano aluminum salt, liposome, oil-in-water nano emulsion and the like. The nano-carrier can adsorb or combine with molecular adjuvant, on one hand, it can be transferred into lymphatic system, even cell, on the other hand, it can prevent the molecular adjuvant from diffusing into blood to cause side reaction resulted from cytokine release.

Aluminum salt adjuvants, including aluminum hydroxide, aluminum phosphate and calcium phosphate, are used in vaccines in a very wide range of applications.

Liposomes are vaccines and drug delivery systems formulated from biological materials of lipids such as phospholipids, cholesterol, and the like.

The nanoemulsion is prepared from various oils with immunostimulating function (such as squalene, squalane, etc.) and surfactant. Commonly used nanoemulsions include MF59, Stable emulsion, AS04, AF03, and the like.

Advantageous effects

The herpes zoster virus vaccine can induce strong antibody reaction and T cell reaction, and can achieve the optimal protection effect. The traditional vaccine prepared by the aluminum salt adjuvant can only induce antibody reaction, cannot induce cellular immunity, and has very limited protective effect on the herpes zoster of the elderly. Most adults carry varicella-zoster virus, and when resistance is reduced, the virus is activated and spreads to the nerve endings of the skin causing inflammation, blistering and neuropathic pain. Because the virus is latent in the cells, the antibody induced by the traditional vaccine prepared by the aluminum salt adjuvant cannot permeate into the cells, and the activation and diffusion of the virus cannot be inhibited. The vaccine of the invention can induce strong cellular immune response. Cytokines (gamma-interferon, IL-2, etc.) produced by T cells can permeate into virus-infected cells, and effectively inhibit activation and diffusion of viruses. Can completely prevent the herpes zoster and the skin pain which often occurs before and after the onset of the herpes zoster. The vaccine induces neutralizing antibodies capable of killing viruses exposed outside or on the surface of cells, preventing the viruses from further infecting other normal cells. Compared with the traditional attenuated vaccine or the vaccine prepared by the traditional aluminum salt adjuvant, the vaccine product of the invention can greatly improve the effectiveness of the vaccine by using glycosylated protein E expressed by eukaryotic cells and a novel adjuvant; can effectively prevent herpes zoster from happening, and the effective rate is far higher than that of the traditional vaccine. Can prevent neuralgia caused by herpes zoster, and has low side effect.

Drawings

FIG. 1 is an SDS-PAGE image of purified gE-1 and gE-2 proteins;

FIG. 2 is a graph of the production of interferon-gamma by recombinant gE proteins in vitro stimulating T cells;

FIG. 3 shows the antibody titers induced by the combination of recombinant gE protein with different adjuvants;

FIG. 4 shows the induction of interferon-gamma producing T cells by recombinant gE protein in combination with various adjuvants;

figure 5 is an antibody and cell response induced by recombinant gE protein adjuvanted with TLR3L and aluminum hydroxide;

figure 6 is the T cell response of recombinant gE protein to TLR3L and aluminum hydroxide adjuvant-adjuvanted gamma interferon;

figure 7 is an antibody response induced by different doses of recombinant gE protein with adjuvant-complex adjuvant of TLR3L and aluminum hydroxide;

figure 8 is an antibody response induced by recombinant gE protein with TLR7L and nanoemulsion composite adjuvant;

figure 9 is the cellular response induced by recombinant gE protein with TLR7L and nanoemulsion co-adjuvant;

figure 10 is an antibody response induced by recombinant gE protein with adjuvant-complexed adjuvant of TLR3L and aluminum hydroxide.

Detailed Description

In order to further understand the technical features of the present invention, the present invention is described in detail with reference to the specific embodiments below.

A herpes zoster virus vaccine is prepared from varicella-zoster virus glucoprotein E or its fragment and compound adjuvant. Wherein, the composite adjuvant contains a nano carrier and at least one molecular adjuvant inducing cellular immunity.

(1) The glycoprotein E is the glycoprotein E of the herpes zoster virus Dumas strain, the amino acid sequence is shown as SEQ ID No.1, the fragment can be the amino acid sequence of the polypeptide fragment outside the cyst membrane of the strain of the glycoprotein E of the herpes zoster virus Dumas strain, the amino acid sequence is shown as SEQ ID No.2, the fragment can also be the fusion protein of the polypeptide outside the cyst membrane and the fragment inside the cyst membrane of the glycoprotein E of the Dumas strain, and the amino acid sequence is SEQ ID No. 3.

(2) Glycoprotein E was expressed in eukaryotic cells and purified by anion chromatography and hydrophobic chromatography.

Glycoprotein E is expressed in eukaryotic cells (Chinese Hamster Ovary cell, or CHO cell); primers were designed based on the existing heavy chain sequence and PCR amplification was performed with Hind III/EcoRI sites inserted into the Multiple Cloning Site (MCS) upstream of the pGenHT1.0 vector. The vector was designed with 1 MCS and CMV promoters. Meanwhile, the vector contains Glutamine Synthetase (GS), and the promoter is SV40early promoter.

CHO-K1SP cell suspension cells were transfected with plasmid vectors. According to the expression evaluation result of 48h of transfection, the establishment of a stable cell line expressing the target gene is confirmed. Transfected cells were seeded into 24-well plates and pressure-screened using cell pool screening media. After the Cell pool screening was completed, the supernatants of all Cell pools were collected for ELSIA to detect expression levels. And selecting Cell pool with high expression quantity to perform monoclonal screening, adopting a limiting dilution method for the monoclonal screening, performing monoclonal imaging shooting, confirming the monoclonality, and finally selecting 10 clones with the monoclonality to perform 70 generations of passage stability tests. Selecting a strain (Clone 6) with high expression and stability for library construction.

CHO high expressing cell clones were grown in serum free medium from 6-well plates, 25ml, and 250 ml shake flasks, up to a 2 liter reactor. The supernatant was collected and filtered through a 0.22 micron membrane to remove cell debris. Anion chromatography was then performed by Q-sepharose column. Collecting eluate containing gE protein, and performing hydrophobic chromatography and ultrafiltration to obtain purified glycoprotein. Both proteins were purified by molecular sieving to about 95% pure protein. Since the protein is glycosylated, it is dispersed on SDS-PAGE gel and has a molecular weight of 60-70 KD.

(3) The carrier can be nano emulsion, liposome or aluminum salt adjuvant.

(4) Molecular adjuvants that induce cellular immunity are TLR3 ligand, agonists of the TOLL-7/8 receptor, or other TOLL-like receptor (TLR4, TLR 5, TLR9, etc.) agonists.

Example 1:

a herpes zoster virus vaccine is prepared from the extracapsular fragment of varicella-zoster virus glucoprotein E and aluminium hydroxide compound adjuvant adsorbed with TLR3 ligand.

(1) The extracapsular fragment of the glycoprotein E is an extracapsular polypeptide fragment g-E1 of a strain of the glycoprotein E of the Dumas strain of the herpes zoster virus, and the amino acid sequence is shown as SEQ ID No. 2.

(2) gE-1 was expressed using CHO cells and purified by anion chromatography and hydrophobic chromatography (see example 9).

(3) The amount of gE-1 is 10-200. mu.g, preferably 50. mu.g.

(4) The aluminum component is aluminum hydroxide, and its production method is described in reference 1 (the reference: Li German Juannan He Wei Rui Pailna Zhao Lili Jiangyu Jinlijie. optimization of preparation process of aluminum hydroxide. China biological products journal 2010, 10.23, 10 th, 1135, 1137. page.) its dosage is 0.1-1 mg, 0.2-0.8 mg, and most preferably 0.5 mg.

(5) Aluminium hydroxide-adsorbed TLR3 ligand (TLR3L) was supplied by metropolis haitong pharmaceutical co. The dosage of the TLR3 ligand is 100-500 micrograms, and the optimal dosage is 300 micrograms.

The preparation method of the vaccine comprises the following steps: the vaccine is prepared in sterile and A-grade purified environment, and the temperature is controlled at 20-24 ℃. The following formulation method is a method of formulating 1 liter of vaccine, and can be scaled up for large-scale production.

(1) The aluminum hydroxide adjuvant was diluted with normal saline to an aluminum concentration of 1 mg/ml. 500 ml was poured into a two-liter dispensing bottle and magnetic stirring was initiated. The stirring speed was 150 revolutions per minute.

(2) 600 mg of TLR3 ligand was diluted to 100 ml of normal saline and then added to a stirred aluminum adjuvant container at a rate of 10 ml per minute. Stirring was continued for 1 hour after completion.

(3) 100 mg of gE protein (gE-1 or gE-2) was diluted to 100 ml with physiological saline and then added to a vessel containing the aluminum adjuvant TLR3 ligand at a rate of 10 ml per minute. Stirring was continued for 1 hour after completion.

(4) 300 ml of physiological saline was added thereto, and stirring was continued for 1 hour.

(5) The mixture is divided into 1000-1300 penicillin bottles, and each bottle is 0.7 ml (0.5 ml per dose).

(6) The prepared vaccine is stored at 2-8 ℃.

Example 2 of implementation:

a herpes zoster virus vaccine: a vaccine formulated with a co-adjuvant of the extra-capsular fragment of varicella-zoster virus glycoprotein E (gE-1) with a nanoemulsion containing an agonist of TLR-7/8.

(2) gE-1 was expressed in CHO cells and purified by anion chromatography and hydrophobic chromatography (see example 9).

(3) The amount of gE-1 is 10-200. mu.g, preferably 50. mu.g.

(4) The nano-emulsion is an oil-in-water emulsion. The preparation method is described in reference 2(Gary Ott, Ramachandran Ramachakrishnan, Jia-Hwa Fang, and Maninder Hora The addition MF59: A10-Yeast Perspecific. methods in Molecular Medicine, Vol.42, Page 211:vaccineadditions 228: Preparation Methods and Research Protocols。Edited by:D.T.O’Hagan

humana Press, inc., Totowa, NJ) consisting of 4-5% squalene, 0.5-1% tween 80, and 0.5-1% Span 85, the nanoemulsion was used in an amount of 0.1-1 ml, 0.2-0.5 ml, preferably 0.25 ml.

(5) The amount of TLR7 agonist used was 0.1-30 micrograms, and TLR7 agonist was supplied by 3M company in the united states. The dosage is 1-10 micrograms, preferably 3 micrograms.

The preparation method of the vaccine comprises the following steps: the preparation is operated in an aseptic and A-grade purified environment, and the temperature is controlled to be 20-24 ℃. The following formulation method is a method of formulating 1 liter of vaccine. Can be produced in large scale with equal scale.

(1) After mixing 45 g of squalene, 4.5 g of tween 80,4.5 g of harvest 85, 6 mg of TLR7 ligand, 450 ml of 10mM citrate buffer (ph6.5) were mixed, homogenized 5 times by microfluidization with a pressure of 1000bar, and sterilized by filtration through a 0.22 μm membrane.

(2) The emulsion was poured into a sterile 2-liter container and stirring was started with a magnetic bar at a speed of 150 revolutions per minute.

(3) 100 mg of gE protein (gE-1 or gE-2) was diluted to 500 ml with physiological saline and then quickly poured into a container containing the nanoemulsion. Stirring was continued for 1 hour.

(4) The mixture is divided into 1000-1300 penicillin bottles, and each bottle is 0.7 ml (0.5 ml per dose).

(5) The prepared vaccine is stored at 2-8 ℃.

Example 3:

a herpes zoster virus vaccine: the vaccine is prepared by a fusion protein of an extracapsular fragment and an amino acid fragment in an cyst membrane of varicella-zoster virus glycoprotein E and a composite adjuvant of aluminum hydroxide adsorbed with TLR3 ligand.

(1) The fusion protein is a fusion protein gE-2 of the extracapsular polypeptide and the intracapsular fragment of the glycoprotein E of the herpes zoster virus Dumas strain, and the amino acid sequence is shown as SEQ ID No. 3.

(2) gE-2 was expressed in CHO cells and purified by anion chromatography and hydrophobic chromatography (see example 9).

(3) The amount of gE-2 is 10-200. mu.g, preferably 50. mu.g.

(4) The aluminum component is aluminum hydroxide, and the dosage is 0.1-1 mg.

(5) The amount of TLR3 ligand adsorbed on aluminum hydroxide is 10-1000 micrograms, preferably 100-300 micrograms.

The preparation method of the vaccine comprises the following steps: the vaccine is prepared in sterile and A-grade purified environment, and the temperature is controlled at 20-24 ℃. The following formulation method is a method of formulating 1 liter of vaccine. Can be produced in large scale with equal scale.

(2) 600 mg of TLR3 ligand was diluted to 100 ml of physiological saline and then added to a stirred aluminum adjuvant container at a rate of 10 ml per minute. Stirring was continued for 1 hour after completion.

(6) The prepared vaccine is stored at 2-8 ℃.

Example 4:

a herpes zoster virus vaccine: vaccine formulated with a composite adjuvant of an extracapsular fragment of varicella-zoster virus glycoprotein E, a fusion protein with an intracapsular amino acid fragment, and a nanoemulsion containing an agonist of TLR-7/8.

(2) gE-2 was expressed in CHO cells.

(3) The dosage of gE-2 is 10-200 micrograms, preferably 10-100 micrograms.

(4) The nano-emulsion is an oil-in-water emulsion.

(5) The amount of TLR7 agonist is 0.1-30 micrograms, preferably 1-10 micrograms.

The preparation method of the specific vaccine comprises the following steps: the vaccine is prepared in sterile and A-grade purified environment, and the temperature is controlled at 20-24 ℃. The following formulation method is a method of formulating 1 liter of vaccine. Can be produced in large scale with equal scale.

(3) 100 mg of gE protein (gE-1 or gE-2) was diluted to 500 ml with physiological saline, and then quickly poured into a container containing the nanoemulsion. Stirring was continued for 1 hour.

(5) The prepared vaccine is stored at 2-8 ℃.

Example 5: screening for herpes zoster virus antigens

Varicella-zoster virus is a very complex virus that co-expresses over 70 different proteins, including 9 glycoproteins across the membrane. Both these glycoproteins and internal proteins are likely to be useful as antigens. By comparing the T cell responses to 71 different proteins before and after 24 adults vaccinated with the attenuated zona vesiculosis vaccine, we found that gene 68 protein was the target protein for the major T cell response. The specific experimental methods and results are as follows:

the 71 genes of the herpes zoster virus Oka strain were PCR-amplified and cloned into a plasmid of pDONR207 or pDONR221(Invitrogen, Grand Island, New York) containing a T7 promoter, and then transferred into a pDEST vector containing a T7 promoter, and His-tagged proteins were expressed using E.coli expression kits (IVTT Expressway, Invitrogen). Expression of each protein was confirmed by dot blot experiments on His-tag antibodies.

Polyclonal CD4 cell lines cultured from isolated blood leukocytes from 24 patients prior to and 14 days after vaccination were cultured and stimulated in vitro for 24 hours with each of the proteins expressed above. Then, the supernatant of the culture was collected and the concentration of interferon-. gamma.was determined. As a result, it was found that all 24 vaccinated persons had T cell responses to the protein expressed by gene 68, and some other persons had T cell responses to genes 9,18,40,67,59,12 and 62. The experiment proves that the protein expressed by the gene 68 contains important T cell reaction sites and can be used for developing vaccines.

Example 6: antigenic site identification of proteins expressed by the herpes zoster Virus Gene 68

Partially overlapping 13-peptide or 20-peptide fragments covering the entire amino acid sequence were synthesized for gene 68. Using these fragments, we cultured and stimulated 24 in vitro polyclonal CD4 cell lines cultured from isolated blood leukocytes before and 14 days after vaccination for 24 hours, and then measured the concentration of interferon-gamma in the supernatant. The site of the T cell response resulting from each protein was thus determined.

The protein expressed by gene 68 is the gE protein. The results demonstrate that the sites of the primary CD4T cells are in the amino acid sequences 190-209 and 280-299. 75% of vaccinees' T cells reacted positive to both sites. Another at least one third of the sites for T cell response in the vaccinee included the amino acid sequences 46-65, 154-173, 199-219, 334-353 and 415-434. In addition, one third of the inoculators had a strong T cell response against the sequences in the viral envelope (577-596, 588-607). Sequences within the capsular membrane may be of significant value in developing a vaccine.

Through the above work, we found an important T cell site of the protein expressed by gene 68, as shown in table 1, confirming the potential for vaccine development.

Table 1 the zona herpesviruses contain important T cell response site proteins.

Example 7: isolation and preparation of T cell clones of the major T cell site of the gE protein

The two major T cell sites of the gE protein are the amino acid sequence 190-210(RIYGVRYTETWSFLPSLTCT) and the amino acid sequence 280-299 (EIEPGVLKVLRTEKQYLGVY). We cloned T cells from two seeded human blood leukocytes at these two sites and expanded them for culture.

First, the tetranectors of the two polypeptides were synthesized separately and labeled with allophycycanin. The labelled tetranects were used to react with isolated blood leukocytes of the vaccinees. Allophytocyanin chromophil labeled CD4 positive cells were isolated by flow cytometry. The isolated cells were placed in 96-well cell culture plates (1 cell per well) and cultured with gamma-irradiated whole blood leukocytes (20 ten thousand cells per well) and phytohemagglutinin (1.6. mu.g/well empty). And the next day human IL-2 (32U per ml) was added. The culture was continued for 14 days. Fresh medium was added every three days. After the completion of the culture, the grown T cells were cultured together with the above-mentioned 20 amino acid polypeptide and whole blood leukocytes of the same patient, and positive clones were confirmed by staining with intracellular cytokine (. gamma. -interferon). Positive T cell clones were then expanded. The number of cells of positive clones was expanded by seeding with 20 ten thousand positive CD4T cells, 500 ten thousand irradiated LCL B cell lines, 2500 ten thousand irradiated blood leukocytes from the same inoculum, and 30ng/mL of OKT3 monoclonal antibody in 25mL of culture medium for 2-3 weeks. The resulting CD 4-positive cells directed against the gE protein site were aliquoted and stored in liquid nitrogen. These preserved T cells are capable of producing interferon-gamma and IL-2 when stimulated in vitro by a gE protein or a specific polypeptide. The above work yielded 5T cell clones in total, as shown in Table 2. Five clones corresponded to two different HLA' s

TABLE 2T cell cloning of important T cell reactive sites for the gE protein

Example 8: construction of Stable cell line CHO expressing gE protein

The gE protein is a glycosylated protein and requires expression in eukaryotic cells to obtain a more structurally authentic protein. Therefore, we constructed two Chinese Hamster Ovarian (CHO) cell lines expressing different gE protein sequences. The first cell line expressed the sequence outside the membrane envelope of gE without the signal peptide (amino acid sequence 31-538). The second CHO cell line expressed a fusion protein of the sequence outside the envelope of the gE protein sequence (amino acid sequence 31-538) and the sequence inside the envelope (amino acid sequence 560-623). The protein expressed by the second CHO cell line did not contain the transmembrane sequence (amino acid sequence 539-559) and was easily precipitated due to poor solubility of the product of the transmembrane sequence.

The cell line was constructed according to a method other than the conventional method. Designing a primer according to the existing heavy chain sequence, and carrying out

PCR was performed to insert HindIII/EcoRI sites into the Multiple Cloning Site (MCS) upstream of the pGenHT1.0 vector. The vector was designed with 1 MCS, and CMV promoter. Meanwhile, the vector contains Glutamine Synthetase (GS), and the promoter is SV40early promoter.

Example 9: culture and purification of Stable CHO cell lines

CHO high expressing cell clones were grown in serum free medium from 6-well plates, 25ml, and 250 ml shake flasks, up to a 2 liter reactor. The supernatant was collected and filtered through a 0.22 micron membrane to remove cell debris. Anion chromatography was then performed by Q-sepharose column. Collecting eluate containing gE protein, and performing hydrophobic chromatography and ultrafiltration to obtain purified glycoprotein. Both proteins were purified by molecular sieving to about 95% pure protein. Since the protein is glycosylated, it is dispersed on SDS-PAGE gel and has a molecular weight of 60-70 KD. The molecular weight of the gE-1 protein is slightly smaller than that of the gE-2 protein, as shown in FIG. 1.

The purified protein of amino acid sequence 31-538 was named gE-1. The purified fusion protein of amino acid sequence 31-538 and amino acid sequence 560-623 was designated gE-2. The yield of gE-1 purified per liter of culture supernatant was 2.6 g, and the yield of gE-2 was 1.8 g.

Example 10 in vitro activation of T cell clones by gE-1 and gE-2 recombinant proteins

Using the two recombinant gE proteins, we performed in vitro stimulation experiments on 5 cloned T cells with different gE (see experiment 4) to confirm the biological activity of the recombinant proteins. 5 ten thousand cloned T cells were cultured for 18 hours on 5 ten thousand gamma-ray treated blood leukocytes of a different person of the same HLA type. The amount of interferon-gamma in the supernatant was measured by ELISA. If stimulated with the recombinant proteins gE-1 or gE-2 (50 ng/ml), each T cell line clone produced large amounts of interferon-gamma (as shown in FIG. 2). The control of the medium without the recombinant protein did not produce any interferon-gamma. Indicating that the expressed protein has biological activity and can be used as vaccine development. There was no significant difference between the two recombinant proteins.

Example 11 immunogenicity of gE recombinant proteins and screening of adjuvants

We immunized mice with recombinant gE-1 and gE-2 proteins, respectively, in combination with different adjuvants, and measured the antibody titers produced and the levels of interferon-gamma producing T cells. Adjuvants evaluated in this assay include activators of TLR3 (TLR3 ligand), activators of TLR7 (TLR7 ligand), aluminum hydroxide, and nanoemulsions. The vaccine immunized by each mouse contains 20 micrograms of gE-1 or gE-2 protein, 20 micrograms of TLR3 ligand, 1 microgram of TLR7 ligand, 100 micrograms of aluminum hydroxide (average particle size is 50-160 nm), or 0.2 milliliter of nanoemulsion (average particle size is 100-160 nm). Saline (without any adjuvant) was used as a control. Mice were immunized subcutaneously on days 1 and 28, respectively, and blood was drawn on day 42 to check for antibody levels. Spleens were also collected to examine T cell responses.

The detection of the antibody was performed by ELISA. The enzyme label plate is coated by gE-1 or gE-2 protein, then serum with different dilutions is added, and finally the second antibody is used for detection and substrate reaction. A serum dilution greater than three times that of a control well without serum is used to establish the titer of the serum. The mean titers of five animals per group are shown in figure 3. The results demonstrate that TLR3 ligand,3M-052, aluminum hydroxide, and nanoemulsion induced high titers of serum antibodies, respectively. There was no significant difference in the antibody titers induced by gE-1 and gE-2.

The T cell response induced by the recombinant gE protein was determined by the cellular proportion of intracellular interferon-gamma after in vitro spleen cell stimulation. After spleen collection, single leukocytes were prepared. In a 96-well cell culture plate, 10 ten thousand leukocytes and 1 microgram of recombinant protein gE-1 or gE-2 per well are added. After 1 hour of incubation in a 37 degree cell culture chamber, GolgiPlug (BD Pharmingen) was added to prevent intracellular cytokines from escaping the cells, and incubation was continued for 5 hours. The collected cells were stained with different fluorescein-labeled antibodies against CD3 and CD4 molecules. Then, the cell membrane-penetrating fluid was treated, and staining was performed with fluorescein-labeled anti-gamma-interferon monoclonal antibody. Finally, after the cells were fixed with 1% formaldehyde, gamma-interferon positive T cells were detected by flow cytometry, and the percentage of the total number of CD4 positive cells was calculated.

The results are shown in FIG. 4. Mice immunized with gE-1 and gE-2 with physiological saline induced fewer gamma-interferon positive T cells, less than 0.2%, and similarly, vaccines formulated with aluminum hydroxide and nanoemulsion induced fewer gamma-interferon positive T cells. The vaccines compatible with TLR3L and TRL7L respectively induce a large amount of gamma-interferon positive T cells, and the ratio is between 1.5 and 2.5 percent. The two adjuvants are proved to be effective adjuvant components for developing herpes zoster vaccines.

This experiment tested if there was a synergistic effect between TLR3L and the aluminium hydroxide adjuvant and the effect of the adsorbed TLR3L dose on the immune efficacy of the vaccine. Different doses of TLR3L are respectively adsorbed to the surface of a certain amount of aluminum hydroxide to prepare a composite adjuvant, and then gE-1 or gE-2 is adsorbed. The vaccine thus formulated, containing 200 micrograms of aluminium, 10 micrograms of antigen and 1, 10, 100, or 300 micrograms of tlr3l per group of 5 mice per 0.2 ml, was injected subcutaneously per mouse on days 1 and 28, respectively. The titers of gE-1 and gE-2 specific antibodies in the sera were determined by ELISA on day 42. As shown in figure 5, the results demonstrate a synergistic effect of TLR3L and an adjuvant of aluminum hydroxide. The effect of the compound adjuvant is 5 times higher than that of each adjuvant used alone. The optimal dose of TLR3L adsorbed onto aluminium hydroxide surface was 10-300 micrograms. The immune enhancement effect of the compound adjuvant on gE-1 and gE-2 is the same.

The spleen of the harvested mice was analyzed for interferon-gamma producing cells on day 45. The procedure is as in example 7. The results are shown in figure 6, where high levels of interferon-gamma positive cells were induced with 10 microgram of TLR3L itself or with a vaccine surface compatible adsorbed to 200 microgram of aluminium hydroxide, respectively. The vaccine formulated with aluminum hydroxide induced almost no gamma-interferon positive cells. This experiment demonstrates that TLR 3L-aluminum hydroxide adjuvanted with adjuvant can improve the efficacy of vaccines

Example 12 Effect of the dose of recombinant protein on the immunogenicity of vaccines formulated with TLR 3L-aluminum hydroxide composite adjuvant

The composite adjuvant formulated with 10 micrograms of TLR3L and 200 micrograms of aluminum hydroxide was adsorbed with varying doses of gE-1 or gE-2 protein, and then mice were immunized and serum antibody titers determined as in example 7. As shown in FIG. 7, the results showed that 10-200. mu.g of E-1 or gE-2 recombinant protein could induce strong antibody response

Example 13: antibody and cell reaction induced by recombinant gE protein, TLR7L and nano-emulsion composite adjuvant

This experiment tested whether TLR7L and nanoemulsion had a synergistic effect and the effect of adsorbed TLR7L dose on the immune efficacy of the vaccine. Different doses of TLR7L and nanoemulsion are prepared into a composite adjuvant, and then the composite adjuvant is mixed with gE-1 or gE-2. The vaccine thus formulated, containing 10 micrograms of antigen and 0.01, 0.5, 2, or 10 micrograms of tlr7l per 0.2 ml of nanoemulsion, was then used to immunize mice as in example 7, and the antibody and cellular immune responses were detected. As shown in fig. 8, the results demonstrate a synergistic effect of TLR7L and nanoemulsion. The optimal dose of TLR7L is 0.5-10 microgram. The immune enhancement effect of the compound adjuvant on gE-1 and gE-2 is the same.

Spleen from harvested mice were analyzed on day 45 for interferon-gamma producing cells. The procedure is as in example 7. The results are shown in FIG. 9. 2 microgram of TLR7L alone or vaccine with nanoemulsion induced high levels of gamma-interferon positive cells, respectively. The vaccine compatible with the nano emulsion hardly induces gamma-interferon positive cells.

Example 14 Effect of different doses of recombinant gE protein on immunogenicity of vaccines formulated with TLR 7L-nanoemulsion Complex adjuvant

We adsorbed complex adjuvants formulated with 2 μ g of TLR7L and 200 μ l of nanoemulsion with varying doses of gE-1 or gE-2 protein, then immunized mice and assayed for serum antibody titers as in example 7. As shown in FIG. 10, the results showed that 10-200. mu.g of E-1 or gE-2 recombinant protein could induce a strong antibody response.

Example 15: mouse antibody induced by gE recombinant protein vaccine can neutralize VZV virus

We formulated the vaccine and immunized mice with either normal saline, LR 3L-aluminum hydroxide complex adjuvant, or LR 7L-nanoemulsion complex adjuvant with gE-1 and gE-2, respectively, neutralizing antibody titers in serum 14 days after the second immunization were determined using a complement-dependent neutralization assay. The Oka strain of the vaccinia virus was first diluted with PBS-gelatin-sucrose (PGS) solution to 3000 Plaque Forming Units (PFU) per ml, and 1: 8 diluted guinea pig complement sera and mixed and then mixed in equal volumes with diluted mouse immune sera to be tested. After 1 hour of 37 degrees incubation, the cells were plated out on human diploid cells. Each well contained 300 PFUs. Infected cells were cultured for 8 days at 37 neutralizing antibody titers were the highest serum dilution that was able to reduce PFU 50%. The results show that the two vaccines formulated with the composite adjuvant respectively induce high titer of neutralizing antibodies as shown in table 3.

TABLE 3 neutralizing antibody Titers

The above embodiments are only illustrative and not restrictive, and any insubstantial modifications made by those skilled in the art based on the present invention shall fall within the scope of the present invention.

Claims

1. A herpes zoster virus vaccine, comprising:

prepared by varicella-zoster virus glycoprotein E segment and compound adjuvant;

the amino acid sequence of the fragment of the varicella-zoster virus glycoprotein E is shown as SEQ ID No. 2;

the composite adjuvant comprises a carrier and a molecular adjuvant for inducing cellular immunity;

the carrier is a nano emulsion;

the molecular adjuvant inducing cellular immunity is an agonist of TOLL-7 receptor.

2. The herpes zoster virus vaccine of claim 1, wherein: the nano-emulsion is an oil-in-water emulsion and comprises the following components: 4-5% squalene, 0.5-1% tween 80 and 0.5-1% Span 85.

3. A method of preparing the herpes zoster virus vaccine of claim 1 or 2, which is prepared from varicella-zoster virus glycoprotein E or a fragment thereof and a composite adjuvant, comprising the following steps:

(1) preparing a composite adjuvant; the composite adjuvant comprises a carrier and a molecular adjuvant for inducing cellular immunity, wherein the carrier is a nano emulsion; the molecular adjuvant inducing cellular immunity is an agonist of a TOLL-7 receptor;

(2) the glycoprotein E segment with the amino acid sequence shown as SEQ ID No.2 is mixed with a composite adjuvant and evenly mixed to prepare the vaccine.