Virus-like particle vaccine for herpes zoster virus

Abstract

The invention relates to the field of biological medicine, in particular to a virus-like particle vaccine aiming at herpes zoster virus. The invention provides a virus-like particle vaccine aiming at herpes zoster virus, which is characterized in that the vaccine comprises a virus-like particle, the virus-like particle is composed of truncated gE protein, and the amino acid sequence of the truncated gE protein is as shown in SEQ ID NO: 1. The vaccine provided by the invention can effectively stimulate cellular immunity and humoral immunity of an organism, and has good immunogenicity and high safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a virus-like particle vaccine against herpes zoster virus. Background Art

[0002] Shingles is a painful rash caused by a recurrence of the varicella zoster virus (VZV). During chickenpox infection, the VZV virus spreads into the blood and infects many nerve cells (ganglia) in the spinal cord and cranial nerves, where it then lies dormant. The VZV virus remains in the ganglia in a dormant (latent or inactive) state. When the VZV virus is reactivated, it returns from the ganglia to the skin, causing a viral herpes similar to chickenpox. The herpes is arranged on the skin along the infected nerve fibers and only appears on one side of the body. Shingles can occur at any age, but is most common in patients who are elderly (such as those over 50 years old), immunocompromised, or taking immunosuppressants. The chance of developing shingles increases with age. According to the WHO, one-third of the world's population will develop shingles, and about 3 million people in China are affected by shingles each year.

[0003] Shingles causes a painful rash of fluid-filled blisters that can sometimes cause chronic pain in the affected area. The symptoms of shingles and the complications of postherpetic neuralgia, cellulitis, and vision loss are very distressing to patients. Although there are many antiviral drugs that can be used to treat shingles, these drugs can only alleviate the symptoms of shingles and shorten the course of the disease, but cannot truly cure and / or prevent shingles. Summary of the invention

[0004] In a first aspect, the present invention provides a truncated gE protein, characterized in that the amino acid sequence of the truncated gE protein is shown in SEQ ID NO: 1. Compared with conventional truncated proteins (such as the gE01 protein in the present invention), the truncated gE protein provided by the present invention can self-assemble to form virus-like particles (VLPs), which have hollow particles that are identical or similar to real virus particles, can aggregate more antigens per unit volume, but have no viral nucleic acid, cannot replicate autonomously and have no infectivity, and do not have the risk of incomplete inactivation or virulence reversion.

[0005] In a second aspect, the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the above-mentioned truncated gE protein.

[0006] In a third aspect, the present invention provides a vector, characterized in that the vector comprises the above-mentioned nucleic acid molecule.

[0007] Fourth aspect, the present invention provides a virus-like particle of varicella zoster virus, characterized in that the virus-like particle is composed of a truncated gE protein, and the amino acid sequence of the truncated gE protein is as shown in SEQ ID NO:1. Unexpectedly, the present invention discovers that the truncated gE protein as shown in SEQ ID NO:1 provided by the present invention can self-assemble into virus-like particles without introducing other proteins. The virus-like particle of varicella zoster virus provided by the present invention not only optimizes antigenic epitopes, but also can aggregate more antigens per unit volume, thereby forming virus-like particles with a high density of viral antigens on the surface, reducing the production of ineffective antibodies, and having good immunogenicity.

[0008] In some embodiments, the virus-like particle is obtained by expression, purification, and excision of the tag protein of the gene sequence of GP67-His-EK-gE02 through a baculovirus expression system, wherein the gene sequence of GP67-His-EK-gE02 is as shown in SEQ ID NO:2. The virus-like particle provided by the present invention can be prepared by a baculovirus-insect cell expression system, which is stable, efficient, and quality controllable, and is suitable for industrial production.

[0009] In some embodiments, the particle size of the virus-like particle includes 10 nm.

[0010] Fifth aspect, the present invention provides a virus-like particle vaccine against varicella zoster virus, characterized in that the vaccine comprises the above-mentioned virus-like particle. Since the virus-like particle provided by the present invention removes non-immunogenic fragments and has a high density of optimized viral antigens on the surface, it has both good immunogenicity and high safety. In addition, the virus-like particle provided by the present invention can effectively induce the immune system of the body to produce an immune protection response (including humoral immunity and cellular immunity), stimulate and promote the development and differentiation of specific CD4+ T cells, and thus prevent the activation of VZV in the skin to a certain extent. The virus-like particle provided by the present invention can also effectively induce specific antibodies, and thus prevent varicella zoster symptoms to a certain extent.

[0011] In some embodiments, the unit dose of the vaccine contains 5 μg, 25 μg, or 50 μg of the above-mentioned virus-like particle.

[0012] In some embodiments, the injection method of the vaccine includes intramuscular injection.

[0013] In some embodiments, the vaccine further comprises an adjuvant. Exemplary adjuvants include complete and incomplete Freund's adjuvant, aluminum-containing adjuvants (such as aluminum hydroxide), etc. The adjuvant can be administered together with the virus-like particle provided by the present invention. It should be emphasized that the "adjuvant" in the present invention does not refer to the truncated gE protein of the present invention and the virus-like particle formed based on it.

[0014] In some embodiments, the adjuvant includes MF59. The vaccine provided by the present invention has high immunogenicity and low side effects, which helps to increase the vaccination rate and compliance of the eligible population.

[0015] In some embodiments, the unit dosage of the adjuvant includes 25 μL.

[0016] In a sixth aspect, the present invention provides the use of the above-mentioned vaccine in the preparation of a medicament for treating and / or preventing varicella-zoster virus infection. The vaccine provided by the present invention can effectively stimulate the cellular immunity and humoral immunity of the body. It can not only stimulate and promote the development and differentiation of VZV-specific memory T cells, thereby preventing the activation of VZV in the skin to a certain extent, but also induce high-titer and persistent specific antibodies, thereby preventing varicella-zoster symptoms (such as postherpetic neuralgia caused by varicella-zoster) to a certain extent.

[0017] In a seventh aspect, the present invention provides the use of a truncated gE protein in the preparation of a virus-like particle vaccine against varicella-zoster virus, wherein the amino acid sequence of the truncated gE protein is as shown in SEQ ID NO:1.

[0018] In some embodiments, the virus-like particle vaccine against varicella-zoster virus is obtained by expressing, purifying, and removing the tag protein through self-assembly from the gene sequence of GP67-His-EK-gE02 by a baculovirus expression system, wherein the gene sequence of GP67-His-EK-gE02 is as shown in SEQ ID NO:2.

[0019] In some embodiments, specifically, the virus-like particle vaccine against varicella-zoster virus is obtained by expressing the gene sequence of GP67-His-EK-gE02 through a baculovirus expression system, purifying it by nickel ion metal chelate affinity chromatography, removing the tag protein using enterokinase, and collecting it by gel filtration chromatography protein purification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1Schematic diagram of the GP67-His-EK-gE02 gene of Embodiment 1 of the present invention;

[0022] Figure 2 Molecular sieve result diagram of the gE02 protein in Embodiment 1 of the present invention;

[0023] Figure 3 Exemplary transmission electron microscopy result diagram of the gE01 protein;

[0024] Figure 4 Exemplary transmission electron microscopy result diagram of the gE02 protein;

[0025] Figure 5 IFN-γ detection result diagram of each experimental group;

[0026] Figure 6 IL-4 detection result diagram of each experimental group. Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0029] As used herein, "a plurality" means two or more, that is, it includes two, three, four, five, etc.

[0030] It should be noted that, herein, the terms "comprise", "include" or any other variant thereof are intended to cover a non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a..." does not exclude the existence of additional identical elements in the process, method, article or device comprising that element.

[0031] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0032] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is only for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1 to 6 should be regarded as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0033] Example 1

[0034] 1. Construction, Expression, and Purification of the gE02 Expression Vector

[0035] 1.1 Selection of the gE02 Protein and Gene Synthesis

[0036] The amino acids of the extracellular region of the gE protein (gE02, 31 - 508aa) in the NCBI database were selected for gene sequence optimization. Insect cell-preferred codons were selected for codon optimization and gene synthesis. At the same time, to promote the secretion expression level of the protein, the GP67 signal peptide sequence was introduced.

[0037] For expression purification and excision of redundant tags, a 6×His tag and an EK cleavage site were introduced at the 5' end of the target gene (as Figure 1 shown). Recognition sites for the restriction enzymes EcoRⅠ and HindⅢ were introduced at the 5' end and 3' end of the target gene. The spliced full-length sequence was obtained using PCR amplification technology.

[0038] Among them, the amino acid sequence of gE02 is as follows: SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERG (SEQ ID NO:1)

[0039] Constructed in a similar method as above:

[0040] i) The coding sequence of the truncated protein gE01 (31 - 546aa) of the gE protein linked with the GP67 signal peptide;

[0041] ii) The coding sequence of the truncated protein gE04 (31 - 329aa) of the gE protein linked with the GP67 signal peptide;

[0042] iii) The coding sequence of the truncated protein gE03 (337 - 508aa) of the gE protein linked with the GP67 signal peptide.

[0043] 1.2 Construction of the gE02 protein expression vector

[0044] The GP67-His-EK-gE02 gene was introduced into the plasmid vector pFastBac1 by gene recombination technology to obtain the final recombinant plasmid pFastBac1-GP67-His-EK-gE02. The correctly identified pFastBac1-GP67-His-EK-gE02 recombinant plasmid was transformed into DH10Bac competent cells, and the recombinant bacmid was screened. The obtained recombinant bacmid was used to transfect Sf9 cells to obtain the P0 generation of recombinant baculovirus, and the P1 and P2 generations of baculovirus were amplified. After passing the qualification, they were stored at -80 °C. Then, the P2 generation of baculovirus was amplified to obtain the P3 generation of baculovirus.

[0045]

[0046] 1.3 Protein expression and purification

[0047] Resuscitate and culture sf9 cells under sterile conditions. After 3 days, transfer them to a shake flask for scale-up culture. When the cells grow to 3×10 6 cells / mL, infect the P3 generation of baculovirus at an MOI of 0.2 - 1. After 72 hours, collect the cell supernatant and store it at 4°C for later use.

[0048] Filter the collected cell supernatant through a 0.22 μm filter membrane to remove cell debris, obtaining the filtered cell supernatant. Using nickel ion metal chelate affinity chromatography, bind the target protein in the filtered cell supernatant to the nickel column through the His tag. Then, elute the miscellaneous proteins on the nickel column using PBS buffer (pH = 7.4) and low-concentration imidazole buffer (PBS, 20 mM imidazole, pH = 7.4). Elute the target protein using high-concentration imidazole buffer (PBS, 50 or 500 mM imidazole, pH = 7.4) to obtain the eluate of the target protein (recombinant protein with a tag). The elution volume of the buffer is 50 mL. Concentrate the eluate to 1 mL to obtain the purified target protein. Analyzed by SDS-PAGE, the protein band of the purified target protein (recombinant gE02 protein) obtained is approximately 60 kDa, meeting the expectation.

[0049] After excising the tag protein of the recombinant protein with a tag using enterokinase (EK), separate and purify it using molecular sieve (gel filtration chromatography protein purification method), and collect the recombinant gE02 protein ( Figure 2 ), with a yield of approximately 10 mg / L.

[0050] Based on the above similar method, collect the recombinant gE01 protein (31 - 546aa), gE03 protein (31 - 329), and gE04 protein (337 - 508aa).

[0051] Example 2

[0052] Transmission electron microscopy characterization

[0053] Drop 10 μL of the above recombinant protein sample suspension onto a copper grid and precipitate for 10 min; then drop 10 μL of 1% (w / v) phosphotungstic acid solution for negative staining and stain for 5 min; blot off the floating liquid with filter paper, dry at room temperature for several minutes, let the grid air dry, and perform electron microscopy identification imaging at 80 kV.

[0054] Under the transmission electron microscope, virus-like particles with high concentration and complete structure appear in the field of view, and their diameter is approximately 10 nm ( Figure 4 ), indicating that the gE02 protein constructed in the present invention can self-assemble into virus-like particles with complete structure.

[0055] However, gE01(31-546aa), gE03(31-329), and gE04(337-508aa) are unable to form virus-like particles, and their exemplary transmission electron microscopy images are as Figure 3 shown (taking gE01 as an example).

[0056] Example 3

[0057] Immunogenicity evaluation of gE02 protein

[0058] Although gE03(31-329) and gE04(337-508aa) seem to be two relatively independent domains in structure, perhaps due to their too short antigenic epitopes, the immunogenicity levels of gE03(31-329) and gE04(337-508aa) are relatively low, so subsequent experimental verification is not carried out on them.

[0059] The experimental groups in this example are set as follows:

[0060] gE02 group: The virus-like particles based on gE02 protein prepared in Example 1 are mixed evenly with MF59 adjuvant (supplier Invivogen) at a volume ratio of 1:1, a total of 50 μL, including 5 μg (25 μL) of protein and 25 μL of MF59 adjuvant.

[0061] gE01 group: The gE01 protein prepared in Example 1 is mixed evenly with MF59 adjuvant (supplier Invivogen) at a volume ratio of 1:1, a total of 50 μL, including 5 μg (25 μL) of protein and 25 μL of MF59 adjuvant.

[0062] Shingrix group: The gE protein of Shingrix is mixed evenly with AS01B adjuvant at a volume ratio of 1:1, a total of 50 μL, including 5 μg (25 μL) of protein and 25 μL of AS01B adjuvant.

[0063] Saline group: 50 μL of saline.

[0064] C57BL / 6 mice aged 6-8 weeks are selected and randomly grouped, with 6 mice in each group. According to the settings of the above experimental groups, the 6-8-week-old C57BL / 6 mice are immunized. On day 0, the mice in each group are immunized with the first injection (primary immunization), and on day 28, the second injection (final immunization) is carried out. The immunization route is intramuscular injection.

[0065] Seven days after the primary immunization, blood is taken from the mice in each group, and the total IgG antibody titer of the specific gE protein induced is detected by ELISA method.

[0066] Coat the 96-well ELISA plate with gE01 protein (at a concentration of 2 μg / mL, 100 μL per well) and incubate overnight at 4°C. After washing the 96-well ELISA plate 3 times, add the blocking solution and block at room temperature (25°C ± 3°C) for 1 - 4 h, then wash 2 times. Subsequently, add the diluted serum sample (dilution factor: 2000 - 128000 times), incubate at room temperature for 2 h, and then wash 4 times. Add the secondary antibody Goat Anti-Mouse IgG antibody conjugated with HRP, incubate at room temperature for 1 h, and then wash 4 times. Subsequently, add 100 μL of the chromogenic solution to each well, develop color in the dark at room temperature for 10 min, and then add 100 μL of the stop solution to each well to terminate the reaction. Measure the optical density of each well at 450 nm using an ELISA reader.

[0067] As shown in Table 1, the virus-like particles formed by the gE02 protein provided by the present invention can effectively stimulate mice to produce high levels of IgG titers against the VZV gE protein and stimulate the humoral immune response.

[0068] Collect the spleens of mice 28 days after the last immunization, isolate splenocytes, and detect the expression of IFN-γ and IL-4 using the ELISPOT method.

[0069] After the mice are euthanized, isolate the spleens and then isolate splenocytes. In a 24-well plate, set the cells at 1×10 6 cells per well (the total volume per well is 1 mL, supplemented with cell culture medium), and then add 2 μg of the polypeptide stimulator per well. The negative control wells contain 1×10 6 cells per well but are not stimulated with the protein. The positive control wells add 2 μg of the polypeptide stimulator per well. Culture in a 37°C, 5% CO2 incubator for about 48 h. Sequentially add the biotin-labeled detection antibody, streptavidin-labeled enzyme solution, and chromogenic substrate. After stopping the color development, air-dry the reaction strips in the dark at room temperature, and detect and count the spots using an ELISpot reader.

[0070] As shown in Table 1 and Figure 5 、 Figure 6 shown, the virus-like particles prepared by the present invention can effectively stimulate the cellular immune response. The cytokine response of the virus-like particles prepared by the present invention is relatively high, especially the expression of IFN-γ is significantly higher than that of the gE01 group and the Shingrix group, which may be due to the aggregation of multiple antigens per unit volume of the virus-like particles prepared by the present invention.

[0071] Table 1

[0072] The above results show that the virus-like particles prepared by the present invention have good immunogenicity and can better induce high-titer and persistent specific antibodies (humoral immunity), as well as Th1 and Th2 memory CD4 T cell immunity simultaneously. The experiments of the present invention also show that the conventional truncation methods of the prior art (for example, truncating any amino acids at 508-546aa, such as the gE01 protein) may instead affect the stability of the monomer, and thus the virus-like particles prepared by the present invention cannot be obtained.

[0073] Example 4

[0074] Safety evaluation of gE02 protein

[0075] The experimental groups in this example are set as follows:

[0076] gE02 group: Mix the virus-like particles based on gE02 protein prepared in Example 1 with MF59 adjuvant (supplier Invivogen) evenly at a volume ratio of 1:1, a total of 50 μL, including 5 μg (25 μL) of protein and 25 μL of MF59 adjuvant.

[0077] Shingrix group: Mix the gE protein of Shingrix with AS01B adjuvant evenly at a volume ratio of 1:1, a total of 50 μL, including 5 μg (25 μL) of protein and 25 μL of AS01B adjuvant.

[0078] Select C57BL / 6 mice aged 6-8 weeks and randomly divide them into groups of 6. Immunize the 6-8-week-old C57BL / 6 mice according to the settings of the above experimental groups. Administer the first dose of immunization to the mice in each group on day 0. The immunization route is intramuscular injection. Observe whether gE02 and Shingrix will cause side effects.

[0079] The observation results show that the body weights of the mice in the gE02 group did not change significantly, there was no obvious swelling and redness at the injection site, and there were no other side reactions. The body weights of the mice in the Shingrix group decreased significantly 1-7 days after injection, the injection site was swollen and red, and obvious side reactions such as pain occurred.

[0080] The above results indicate that the virus-like particles based on gE02 protein of the present invention have excellent immunogenicity and safety simultaneously and are suitable for being prepared into vaccines for treating and / or preventing herpes zoster.

[0081] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative rather than restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims, and these all fall within the protection scope of the present invention.

Claims

1. A truncated gE protein, characterized in that, The amino acid sequence of the truncated gE protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the truncated gE protein according to claim 1.

3. A carrier, characterized in that, The vector contains the nucleic acid molecule according to claim 2.

4. A virus-like particle of varicella-zoster virus, characterized in that, The virus-like particle is composed of the truncated gE protein, and the amino acid sequence of the truncated gE protein is shown in SEQ ID NO:

1.

5. The virus-like particle according to claim 4, wherein The virus-like particle is obtained by self-assembly after expression, purification, and excision of the tag protein of the gene sequence of GP67-His-EK-gE02 through a baculovirus expression system, wherein the gene sequence of GP67-His-EK-gE02 is shown in SEQ ID NO:

2.

6. The virus-like particle according to claim 4, wherein The particle size of the virus-like particle includes 10 nm.

7. A virus-like particle vaccine against varicella-zoster virus, characterized in that, The vaccine includes the virus-like particle according to any one of claims 4-6.

8. The vaccine according to claim 7, characterized in that, The vaccine further contains an adjuvant.

9. The vaccine according to claim 7, characterized in that, The adjuvant includes MF59.

10. Use of the vaccine according to any one of claims 7-9 in the preparation of a medicament for treating and / or preventing varicella-zoster virus infection.

Images