Chimeric antigen of varicella-zoster virus vaccine and its application

Abstract

The present invention belongs to the field of medicine, and specifically relates to varicella-zoster virus vaccines. More specifically, it relates to a chimeric antigen of a varicella-zoster vaccine and its application. By connecting the extracellular 539 segment or the extracellular 134 segment of the gE protein in different ways in series with the Fc fragment, a chimeric antigen can be obtained, which can well induce an immune response in mice and achieve the purpose of protection.

Description

[0001] This application is a divisional application of the invention patent application with the original application number 2023104058706, the invention title of "A Chimeric Antigen of Varicella Zoster Virus Vaccine and Its Application", and the application date of April 17, 2023. Technical Field

[0002] The present invention belongs to the field of medicine, and specifically relates to varicella zoster virus vaccines. Background Art

[0003] Herpes zoster is an acute infectious skin disease caused by the reactivation of varicella-zoster virus (VZV) latent in the body. Primary infection with VZV in childhood causes chickenpox. After infection, the virus enters the sensory nerve endings of the skin and moves centripetally along the nerve fibers of the posterior roots of the spinal cord or the trigeminal ganglion, and exists in the spinal nerves or cranial nerve sensory ganglia in a persistent latent form for a long time. When the body's immunity is low or stimulated non-specifically, the virus becomes active again, grows and multiplies, causing inflammation or necrosis of the invaded ganglia, resulting in neuralgia. At the same time, the reactivated virus spreads from one or several adjacent ganglia along the corresponding sensory nerve fibers to the skin, causing clustered blisters on the basis of unilateral erythema. The incidence of herpes zoster in the Asia-Pacific region is about 1%, and the incidence increases with age. After the age of 40-50, the incidence of herpes zoster reaches more than 5%. Among patients with herpes zoster, about 9% - 34% of patients will develop postherpetic neuralgia, and the pain rating can reach above level 7, which belongs to severe pain and seriously affects the quality of life of patients. There is no specific medicine for herpes zoster and postherpetic neuralgia. Therefore, vaccination against herpes zoster is an effective means of prevention. Currently, two herpes zoster vaccines have been marketed globally, namely Zostavax (attenuated live vaccine technology) and Shingrix (recombinant protein vaccine technology).

[0004] Although from a technical level, the recombinant technology is superior to the attenuated live vaccine technology. Zostavax is an attenuated live vaccine, which was first approved by the FDA for marketing in 2006 for the prevention of the elderly aged 60 and above, and the applicable range was approved to be expanded to the population aged 50 and above in 2011. Shingrix is an adjuvanted recombinant protein vaccine containing varicella-zoster virus glycoprotein E and the AS01B adjuvant system. Shingrix was approved by the FDA for marketing in 2017 for the prevention of the elderly aged 50 and above. Shingrix is administered in 2 doses, with an interval of 2 - 6 months, intramuscular injection into the deltoid muscle of the upper arm, 1600 yuan per dose, and the total cost for the full course of vaccination is 3200 yuan. There is an urgent need for a domestic vaccine to replace foreign vaccines, reduce costs and increase efficiency, and achieve better patient compliance.

[0005] VZV is the smallest herpesvirus. Among its numbered glycoproteins, glycoprotein gE (glycoprotein E) is a late protein encoded by ORF68. It is a type I transmembrane protein with a full length of 623 amino acids (aa), including 544 aa in the extracellular region, 17 aa in the transmembrane region, and 62 aa in the intracellular region, and is rich in N- and O-glycosylation sites. The VZV structural protein gE can be highly expressed in virus-infected cells, plays an important role in virus transmission, and is also one of the main antigens inducing host humoral and cellular immunity. Some studies have shown that through Western blotting and ELISA detection methods, the extracellular 1-135 segment of the gE protein is the main antigenic activity region, and the 56-75, 86-105, and 116-135 segments among them are recognized by the sera of varicella and herpes patients. This result suggests that these three segments are the most important antigenic determinant epitopes (W J Fowler, M Garcia-Valcarcel, M S Hill-Perkins, G Murphy, D R Harper, D J Jeffries, N R Burns, S E Adams, A J Kingsman, G T Layton. Identification of immunodominant regions and linear B cell epitopes of the gE envelope protein of varicella-zoster virus. Virology. 1995 Dec 20;214(2):531-40). Summary of the Invention

[0006] Through research, the present invention has found that combining the extracellular 134 segment and the extracellular 539 segment of the gE protein has good effects. Therefore, chimeras with different combinations are designed. The antigen technical route adopts the Chinese hamster ovary (CHO) cell expression system. After being formulated with adjuvants, a vaccine with better immunogenicity is obtained, providing guarantee for independently exploring and developing subunit recombinant vaccines with better immunogenicity, and helping to reduce the dependence on international herpes vaccines.

[0007] The present invention first provides a chimeric antigen for preparing a varicella-zoster virus vaccine, which is composed of a tandem of the extracellular 539 segment of the gE protein and an Fc fragment;

[0008] or is composed of a tandem of two extracellular 539 segments of the gE protein and an Fc fragment;

[0009] or is composed of a tandem of the extracellular 539 segment, the extracellular 134 segment of the gE protein, and an Fc fragment;

[0010] or composed of the extracellular 134 segment and extracellular 539 segment of the gE protein and the Fc fragment in series;

[0011] Among them, the amino acid sequence of the extracellular 134 segment of the gE protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the extracellular 539 segment of the gE protein is as shown in SEQ ID NO: 15. Preferably, the Fc fragment is derived from the human IgG Fc fragment; more specifically, its amino acid sequence is as shown in SEQ ID NO: 16.

[0012] Preferably, the chimeric antigen for preparing the varicella zoster virus vaccine has an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

[0013] The present invention provides the coding nucleic acid of the chimeric antigen for preparing the varicella zoster virus vaccine. Preferably, the nucleotide sequences are respectively as shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

[0014] The present invention provides a recombinant expression vector and a recombinant host cell containing the nucleic acid.

[0015] The present invention provides the application of the chimeric antigen for preparing the varicella zoster virus vaccine in the preparation of the varicella zoster virus vaccine.

[0016] The present invention further provides a varicella zoster virus vaccine, including a vaccine obtained by immunizing an animal with a composition of the chimeric antigen and an adjuvant to obtain immunogenicity.

[0017] More specifically, the adjuvant is AS01B. The dosage ratio of the adjuvant to the antigen is 1:1.

[0018] In the specific embodiment, the animal is a mouse.

[0019] In the preferred embodiment, the immunization dose is: 1 μg, 2 μg, 5 μg per mouse, and the volume is 100 μl / mouse. The immunization program is that after a single immunization, a booster immunization is carried out with the corresponding dose 2 weeks later.

[0020] The chimeric antigen constructed by the present invention can obtain a higher quality protein, and the immune effect is better, which is superior to the GSK vaccine. Brief Description of the Drawings

[0021] Figure 1 N134-Fc HIC chromatography. That is, the gel electrophoresis diagram of the construction method of FA02 in the table.

[0022] Figure 2, N134-N134-Fc HIC chromatography. That is, the gel run diagram of the construction method of FA03 in the table.

[0023] Figure 3 、N134_Q540_Fc HIC chromatography. That is, the gel run diagram of the construction method of FA04 in the table.

[0024] Figure 4 , Q540-N134-Fc HIC chromatography. That is, the gel run diagram of the construction method of FA05 in the table.

[0025] Figure 5 、Q540_Q540_Fc HIC chromatography. That is, the gel run diagram of the construction method of FA06 in the table.

[0026] Figure 6 , Q540_Fc HIC chromatography. That is, the gel run diagram of the construction method of FA07 in the table. DETAILED DESCRIPTION

[0027] Example 1. Construction of 7 plasmids

[0028] Plasmids, cells and experimental animals:

[0029] pcDNA3.1-VZV-gEx / y (x is the length of extracellular amino acids of different lengths, y is the Fc mammalian system codon optimization sequence). HEK293 cells were purchased from Shanghai Duoning Biotechnology Co., Ltd. BALB / c female mice (6 weeks old) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and were raised in the company's SPF animal room in accordance with the "Regulations on the Protection of Experimental Animals". The construction methods of the 7 plasmids are as follows: Example 1, in which the sequence of 1-134 amino acids at the N terminal is shown in Sequence 1. The optimized base sequence is shown in Sequence 2. The optimized nucleotide sequence of 1-134 amino acids was concatenated with the FC optimized sequence and cloned into the pcDNA3.1 vector. The above sequence synthesis was completed by Beijing Liuhe BGI Gene Technology Co., Ltd. The Fc fragment can be selected from the immunoglobulin Fc fragment of human, mouse, rabbit, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig; the immunoglobulin Fc fragment is preferably the Fc fragment of IgG, IgA, IgD, IgE or IgM; more preferably, it is selected from IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment, or IgG4 Fc fragment; in the present invention, the immunoglobulin Fc fragment is a human IgG Fc fragment. Other schemes are similar to scheme 01, and the relevant sequences are shown in the sequence table. The construction modes of different schemes are as follows:

[0030]

[0031] Example 2. Cell transfection

[0032] One day before transfection, inoculate at a density of 1.0×10 6 cells / mL using Transpro CD 01 medium. When the cell density reaches 1.5 - 2.0×10 6 cells / mL and the cell viability is higher than 95% on the day of transfection, transfection can be carried out. In an 80 ml culture system, first add 80 μg of plasmid to 2.4 ml of PBS solution and mix well, marked as solution A. Then add 320 μl of PEI to 2.4 ml of PBS and mix well, marked as solution B. After standing for 5 minutes, add solution A and solution B together, mix well, and let it stand for 15 minutes. Subsequently, add the mixed solution dropwise to the shake flask of the 80 ml culture system and place it in a carbon dioxide incubator for cultivation. After 24 hours of transfection, add supplements to the 80 mL system: 160 μl of VPA, 200 μl of dextran sulfate, 2.4 ml of DN feed 2, and 240 μl of DN feed B2. On the second day after transfection, add 0.8 ml of 300 g / l glucose and 1.6 mL of 200 mM / L glutamine. On the third day after transfection, add 2.4 ml of DN feed 2 and 240 μl of DN feed B2. On the fourth day after transfection, add 0.8 ml of 300 g / l glucose and 1.6 mL of 200 mM / L glutamine. On the fifth day after transfection, sample for counting, centrifuge to harvest the supernatant for purification.

[0033] Example 3. Protein purification: VZVgE PA affinity chromatography

[0034] The culture supernatant of VZVgE plasmid transiently transfected cells is filtered successively with 0.8 μm and 0.45 μm filter membranes, and 80 μl of the sample is taken for electrophoresis detection.

[0035] (1) Pretreatment: First, rinse with 1M NaOH for at least 3 CVs, and then rinse with purified water for at least 10 CVs, with a flow rate of 5 - 10 ml / min;

[0036] (2) Equilibration: Wash with binding buffer for 10 CVs until the baseline is stable, with a flow rate of 5 - 10 ml / min;

[0037] (3) Loading: Load approximately 76 ml of the filtered cell supernatant sample, with a flow rate of 2.5 ml / min and a pressure limit of ≤0.3 MPa; Collect the flow-through sample at the end of loading, and take 80 μl of the sample for inspection;

[0038] (4) Washing: After loading, increase the flow rate to 5 ml / min and wash with binding buffer for at least 6 CVs until the UV baseline is stable;

[0039] (5) Elution: Elute with 100% B Buffer at an elution flow rate of 2.5 ml / min, and collect fractions according to the UV absorption peak at UV280. Collect 1 tube for each elution gradient;

[0040] (6) Regeneration: First, rinse with 1M NaOH for at least 3 column volumes (CV), and then rinse with purified water for at least 10 CV.

[0041] Example 4. Protein purification by VZVgEHIC chromatography

[0042] Prepare HIC Buffer A: 0.02M PB, 1.2 / 1.8M (NH4) 2 SO4, pH 7.8 / 8.0, HIC Buffer B: 0.02M PB, pH 7.8, HIC Buffer C: 0.02M PB, 1.8 / 2.0M (NH4) 2 SO4, pH 7.8 / 8.0

[0043] Take 10 ml of the elution sample from the previous step of VZVgE QFF anion exchange chromatography, add 20 ml of HIC Buffer C to make the ammonium sulfate concentration close to 1.2 / 1.8M, and the final volume is 30 ml.

[0044] Chromatography column: ToyoScreen Butyl-650M, column volume CV: 5 ml, flow rate: 1 - 5 ml / min, pressure limit: ≤0.3 MPa

[0045] (1) Pretreatment: Rinse the column with 2 - 5 column volumes of salt-free elution buffer (HIC Buffer B);

[0046] (2) Equilibration: Equilibrate the column with 5 - 10 column volumes of starting buffer (HIC Buffer A), or until the UV absorption returns to the baseline and the conductivity is stable;

[0047] (3) Loading: Dilute the VZVgE QFF sample to about 30 ml, flow rate: 2 ml / min; collect the flow-through during loading and take 80 μl for inspection;

[0048] (4) Washing: After loading, equilibrate the column with 5 - 10 column volumes of starting buffer, or until the UV absorption returns to the baseline and the conductivity is stable, and collect the washing sample;

[0049] (5) Elution: Gradient elution, elute with 60%, 75%, 90% and 100% B Buffer respectively, elution flow rate 2.5 ml / min, according to the UV absorption peak at UV 280 Collect fractions separately. Collect the target protein during the 90% and 100% elutions.

[0050] (6) Washing: Wash away any substances hydrophobically bound to the column with 2 - 5 column volumes of salt-free elution buffer (HIC Buffer B).

[0051] (7) Equilibrate the column with 5 - 10 column volumes of starting buffer (HIC Buffer A), or until the UV absorption returns to the baseline and the conductivity stabilizes.

[0052] Example 5. SDS-PAGE Electrophoresis and Coomassie Brilliant Blue Staining

[0053] Prepare the samples of the HIC chromatography samples collected. Take 80 μl of each sample and add 20 μl of 5× protein loading buffer. After mixing, boil in water bath for 5 min and then immediately place on ice bath, and store at -20 °C for later use. Load the samples for SDS-PAGE electrophoresis detection.

[0054] The chromatography results are shown in Figures 1 to 6 , and it can be seen that effective expression is obtained in all cases. It can be known from the figure that the molecular weights of the expressed proteins are all greater than the theoretical molecular weights of their unglycosylated monomers, that is, the proteins are effectively glycosylated and can effectively perform their functions. And after fusing with FC, the purification steps are reduced.

[0055] Example 6. Primary Immunization of Mice

[0056] Select 5 female BALB / c strain mice at 6 - 8 weeks old. Combine with AS01B adjuvant and use intramuscular injection. The antigen dose is 5 μg / 2 μg / 1 μg for immunization, and the volume is 100 μl / mouse. Boost the immunization with the corresponding dose every 2 weeks. Before each immunization, collect mouse serum, place it in a 37 °C constant temperature incubator for 30 min, centrifuge at 13000 g for 10 min, collect the serum and measure the antibody titer of serum gE. The results are shown in the following table.

[0057] Table 1. Results of Neutralizing Antibody Titer Determination

[0058]

[0059]

[0060] It can be known from the table that the effects (immunization effect and neutralizing antibody titer) of N134-N540-FC or N540-N134-FC or N540-FC are all better than those of the GSK vaccine. The immunization effect of Scheme 03 - 06 is in the order of 100,000, and the immunization effect of the GSK vaccine is in the order of 10,000.

Claims

1. A chimeric antigen for preparing a varicella-zoster virus vaccine, characterized in that, it is a fusion fragment with an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO:

9.

2. The coding nucleic acid of the chimeric antigen for preparing a varicella-zoster virus vaccine according to claim 1.

3. The coding nucleic acid according to claim 2, characterized in that, its nucleotide sequence is respectively as shown in SEQ ID NO: 8 or SEQ ID NO:

10.

4. A recombinant expression vector containing the nucleic acid according to claim 2 or 3.

5. A recombinant host cell containing the nucleic acid according to claim 2 or 3.

6. The application of the chimeric antigen for preparing a varicella-zoster virus vaccine according to claim 1 in the preparation of a varicella-zoster virus vaccine.

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