NL2032309A - Varicella-zoster virus vaccine and application thereof - Google Patents
Varicella-zoster virus vaccine and application thereof Download PDFInfo
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- NL2032309A NL2032309A NL2032309A NL2032309A NL2032309A NL 2032309 A NL2032309 A NL 2032309A NL 2032309 A NL2032309 A NL 2032309A NL 2032309 A NL2032309 A NL 2032309A NL 2032309 A NL2032309 A NL 2032309A
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- zoster virus
- varicella
- virus vaccine
- vaccine
- adjuvant
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Abstract
Provided are a varicella—zoster virus vaccine and an application thereof. The varicella—zoster virus vaccine includes liposome nanoparticles, as well as a herpes zoster virus glycoprotein E and an adjuvant entrapped therein; whrein the adjuvant includes triterpenoid saponins. In the present invention, the herpes zoster virus glycoprotein E (gE) entrapped in liposome nanoparticles can effectively promote antigen presenting cells to phagocytose and efficiently deliver the antigen, and realize slow release of the vaccine, so that an organism is continuously stimulated to produce the VZV—gE—specific cellular immune response. The triterpenoid saponins used, in the present invention can effectively realize cross—presentation of the herpes zoster virus glycoprotein E, and induce an antigen—specific cellular immune response. Moreover, rich cholesterol in lipid nanoparticles can effectively neutralize the cytotoxicity of triterpenoid saponins, ensuring the safety of the vaccine.
Description
VARICELLA-ZOSTER VIRUS VACCINE AND APPLICATION THEREOF
The present invention belongs to the technical field of vac- cines, and particularly relates to a varicella-zoster virus vac- cine and an application thereof.
The varicella-zoster virus (VZV) spreading all over the world is highly infectious. Only one serotype has been found so far, and
VZV merely infects humans in nature. VZV can cause both varicella and herpes zoster (HZ). Varicella commonly appears to childhood, while herpes zoster does not occur until adulthood. After primary infection with varicella, the virus may remain dormant in the host’s ganglia. With age increase, immune function impairment or immunosuppression, VZV can be reactivated to cause herpes zoster.
The vast majority of adults are at risk of suffering from herpes zoster and its associated complications worldwide.
The Oka-strain live attenuated vaccine developed by Michiaki
Takahashi from Japan was approved by the FDA to vaccinate children and adults against varicella (with an inoculum size of 1,000- 10,000 PFU (plaque forming unit)) in 1995, and has been widely used around the world since then. Subsequent studies found that like a wild-type virus, the Oka strain would establish a latent infection, which may also lead to the occurrence of herpes zoster.
A one-time subcutaneous prime-boost high-dose live attenuated vaccine (with an inoculum size of about 19,400 PFU) for people over 50 years old who has been infected with VZV can effectively prevent herpes zoster. Zostavax, a relevant product of Merck, was launched in 2005 with protection rates for people aged 50-59, 60- 69 and over 70 being about 70%, 64% and 38% respectively. This re- duction in protection rates with age is mainly attributed to a weakened cellular immune response with aging of the immune system.
Shingrix, a genetically engineered subunit herpes zoster vac- cine launched by GSK at the end of 2017, uses a conserved virus glycoprotein E (gE) expressed in Chinese hamster ovary (CHO) cells as an antigen, and uses an adjuvant AS01B to effectively enhance a
VZV-gE-specific cellular immune response, which makes its protec- tion rate in healthy people over 50 years old become as high as 97.2% (protection rates for people aged 50-59, 60-69 and over 70 are 96.6%, 97.3% and 91.3 respectively), showing favorable safety and effectiveness in people with immunodeficiency, including HIV carriers. There is synergy between the triterpenoid polysaccharide ©S21 and the monophosphoryl lipid (MPL) A in the AS01B adjuvant system based on a liposome vector to induce gE-specific CD4- positive T cells, playing a key role in vaccine effects.
Entrapping CpG motif-containing oligodeoxynucleotides (CpG
ODN) into ionizable lipid nanoparticles (LNP) can enhance antigen- specific humoral and cellular immune responses (PMID: 33805880).
Due to the strong cytotoxicity of QS21, many studies have not been clinically applied. Therefore, how to use suitable adjuvant components to enhance the VZV-gE-specific cellular immune response and achieve immune effects similar to the Shingrix herpes zoster vaccine on the premise of ensuring the safety of vaccine compo- nents is an urgent problem to be solved in vaccine development.
In view of this, the present invention is intended to provide a varicella-zoster virus vaccine and an application thereof. The varicella-zoster virus vaccine of the present invention can effec- tively enhance the VZV-gE-specific cellular immune response, and can be used as a herpes zoster vaccine with high safety.
The present invention provides a varicella-zoster virus vac- cine, including liposome nanoparticles, as well as a herpes zoster virus glycoprotein E and an adjuvant entrapped therein; wherein the adjuvant includes triterpenoid saponins.
Preferably, a content of the herpes zoster virus glycoprotein
E in the varicella-zoster virus vaccine is 5-100 pg/dose.
Preferably, a content of the triterpenoid saponins in the varicella-zoster virus vaccine is 1-100 ug/dose.
Preferably, the triterpenoid saponins include QS21.
Preferably, the adjuvant further includes GC-rich single-
stranded oligodeoxynucleotide fragments.
Preferably, a content of the GC-containing single-stranded oligodeoxynucleotide fragments in the varicella-zoster virus vac- cine is 5 ug-2 mg/dose.
Preferably, the liposome nanoparticles include cationic lipo- somes and polyethylene glycol derivatives; wherein a molar ratio of the cationic liposomes to the polyethylene glycol derivatives is {(46-50):{(1.5-1.6).
Preferably, a particle size of the varicella-zoster virus vaccine is 20-400 nm.
Preferably, dosage forms of the varicella-zoster virus vac- cine include an injection.
The present invention further provides an application of the varicella-zoster virus vaccine of the aforesaid solution in prepa- ration of a drug for preventing or relieving herpes zoster and/or its sequelae.
The present invention provides a varicella-zoster virus vac- cine, including liposome nanoparticles, as well as a herpes zoster virus glycoprotein E and an adjuvant entrapped therein; wherein the adjuvant includes triterpenoid saponins. In the present inven- tion, the herpes zoster virus glycoprotein E (gE) entrapped in liposome nanoparticles can effectively promote antigen presenting cells to phagocytose and efficiently deliver the antigen, and re- alize slow release of the vaccine, so that the organism is contin- uously stimulated to produce the VZV-gE-specific cellular immune response. The triterpenoid saponins used in the present invention can effectively realize cross-presentation of the herpes zoster virus glycoprotein E, and induce an antigen-specific cellular im- mune response. Moreover, rich cholesterol in lipid nanoparticles can effectively neutralize the cytotoxicity of triterpenoid sapo- nins, ensuring the safety of the vaccine. Animal experiments have proved that the varicella-zoster virus vaccine of the present in- vention can enhance the cellular immune response specific to the herpes zoster virus glycoprotein E, and can be used as a herpes zoster vaccine.
FIG. 1 shows an antigen entrapment efficiency as detected in experimental example 1 (A in FIG. 1), a nucleic acid entrapment efficiency as detected in experimental example 2 (B in FIG. 1}, a
QS21 entrapment efficiency as detected in experimental example 3 (C in FIG. 1), a particle size as detected in experimental example 4 (D in FIG. 1) and a polydispersity index as obtained in experi- mental example 4 (E in FIG. 1) based on the varicella-zoster virus vaccine prepared in the example.
FIG. 2 shows a cytotoxicity as detected in experimental exam- ple 5 based on the varicella-zoster virus vaccine prepared in the example.
FIG. 3 shows a gE-specific IgG antibody titer as detected in experimental examples 6, 7 and 12 based on the varicella-zoster virus vaccine prepared in the example.
FIG. 4 shows an IL-2 concentration as detected in experi- mental examples 6, 8, 9 and 12 based on the varicella-zoster virus vaccine prepared in the example.
FIG. 5 shows an IFN-y concentration as detected in experi- mental examples 6, 8, 9 and 12 based on the varicella-zoster virus vaccine prepared in the example.
FIG. 6 shows a number of spots formed by IL-2 secreted per 2x10° splenocytes as detected in experimental examples 6, 8, 10 and 12 of the varicella-zoster virus vaccine prepared in the example.
FIG. 7 shows the number of spots formed by IFN-y secreted per 2x10° splenocytes as detected in experimental examples 6, 8, 10 and 12 based on the varicella-zoster virus vaccine prepared in the ex- ample.
FIG. 8 shows a proportion of CD4* T cells secreting IL-2 as detected in experimental examples 6, 8, 11 and 12 based on the varicella-zoster virus vaccine prepared in the example.
FIG. 9 shows a proportion of CD4* T cells secreting IFN-Y as detected in experimental examples 6, 8, 11 and 12 based on the varicella-zoster virus vaccine prepared in the example.
The present invention provides a varicella-zoster virus vac-
cine, including liposome nanoparticles, as well as a herpes zoster virus glycoprotein E and an adjuvant entrapped therein; wherein the adjuvant includes triterpenoid saponins.
In the present invention, all components in the varicella- 5 zoster virus vaccine are combined through physical electro- adsorption or physical entrapment.
In the present invention, a content of the herpes zoster vi- rus glycoprotein E in the varicella-zoster virus vaccine is pref- erably 5-100 ug/dose. In the present invention, the herpes zoster virus glycoprotein E (gE) entrapped in liposome nanoparticles can effectively promote antigen presenting cells to phagocytose and efficiently deliver the antigen, and realize slow release of the vaccine, so that the organism is continuously stimulated to pro- duce the VZV-gE-specific cellular immune response.
In the present invention, a content of the triterpenoid sapo- nins in the varicella-zoster virus vaccine is preferably 1-100 ng/dose. In the present invention, the triterpenoid saponins pref- erably include QS21 extracted from bark of Quillaja saponaria. The triterpenoid saponins used in the present invention can effective- ly realize cross-presentation of the herpes zoster virus glycopro- tein E, and induce an antigen-specific cellular immune response.
Moreover, rich cholesterol in lipid nanoparticles can effectively neutralize the cytotoxicity of triterpenoid saponins, ensuring the safety of the vaccine.
In the present invention, the adjuvant further includes pref- erably a GC-rich single-stranded oligodeoxynucleotide fragment {CpG ODN), and more preferably CpG CDN 1018. In the present inven- tion, a content of the GC-containing single-stranded oligodeoxynu- cleotide fragments in the varicella-zoster virus vaccine is pref- erably 5 ug-2 mg/dose. In the present invention, CpG ODN is en- trapped in lipid nanoparticles, which effectively avoids the deg- radation of nucleases, and allows escaping CpG ODN to be rapidly degraded by nucleases in vivo before being phagocytosed by pre- senting cells, thereby effectively avoiding systemic inflammatory side effects possibly caused by non-specific diffusion of CpG ODN from the vaccination site, so that the adjuvant exhibit “local” and “transient” characteristics to satisfy safety requirements. In addition, CpG ODN used for the varicella-zoster virus vaccine of the present invention can be taken up by TLR9 distributed in endo- somes to induce secretion of interferons, and effectively activate antigen-specific T cells by promoting cross-presentation of the antigen. For CpG ODN, class A can stimulate dendritic cells to produce type-I interferons and activate natural killer cells; class B can rapidly transfer from early endosomes to late endo- somes, and stimulate proliferation of B cells, maturation of plasmacytoid dendritic cells, and production of TNF-o, IL-6 and
IL-12; class C combines features of both classes A and B, and bal- ances the promotion of humoral and cellular immune responses.
Class C CpG ODN that escapes into the cytoplasm and can form a lo- cal stem-loop structure may induce relevant acquired immune re- sponses by activating cyclic GMP-AMP synthase (cGAS) and then passing through the stimulator of IFN genes (STING) as an innate immune pathway. The present invention combines triterpenoid sapo- nins and CpG ODN with good synergy in inducing the antigen- specific cellular immune response.
In the present invention, the liposome nanoparticles include cationic liposomes and polyethylene glycol derivatives; wherein a molar ratio of the cationic liposomes to the polyethylene glycol derivatives is (46-50):{(1.5-1.6). In the present invention, the cationic liposomes preferably include ({4- hydroxybutyl)azanediyl)bis (hexane-6,1-diyl)bis (2-hexyldecancate) (ALC-0315) and/or heptadecan-9-yl1-8-{(2-hydroxyethyl) (é-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102). In the present inven- tion, the polyethylene glycol derivatives preferably include meth- oxypoly ethylene glycol ditetradecylacetamide (ALC-0159) and 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-
PEG2000).
In the present invention, a particle size of the varicella- zoster virus vaccine is preferably 20-400 nm.
In the present invention, dosage forms of the varicella- zoster virus vaccine preferably include an injection.
In the present invention, there is no special limitation to the method for preparing the varicella-zoster virus vaccine, and all conventional plastid nanoparticle entrapment methods in the art may be used. In the specific implementation process of the present invention, microfluidic equipment is preferably used to prepare the varicella-zoster virus vaccine.
In the present invention, the varicella-zoster virus vaccine is preferably administered through injection; the injection pref- erably includes subcutaneous injection or intramuscular injection.
The present invention further provides an application of the varicella-zoster virus vaccine of the aforesaid solution in prepa- ration of a drug for preventing or relieving herpes zoster and/or its sequelae. In the present invention, the sequelae of the herpes zoster preferably include post-herpetic neuralgia.
The technical solutions of the present invention will be de- scribed below clearly and completely in conjunction with the em- bodiments of the present invention.
Amounts of components required for 20 injections in vaccine groups are calculated according to the composition of a single- injection vaccine in Table 1 below.
Table 1. Amounts for single-injection vaccine
Vaccine group gE (ug) Aluminum CpG (ug) QS21 (ug) adjuvant
Comparative example 1 10 - 10 5
Comparative example 2 10 V 10 5
Example 12 - 15 6
Blank control - - - - © - Not added; V Added.
Comparative Example 1 -- 0.2 mg of CHO-expressed extracellu- lar glycoprotein gE (purchased from Kunming Diangong Science and
Technology Co., Ltd.), 0.2 mg of thiooxidized CpG 1018 (purchased from InvivoGen) and 0.1 mg of QS21 (purchased from Alpha Diagnos- tic) were weighed and dissolved in 1 mL of PBS to obtain 20 injec- tions of the vaccine of comparative example 1.
Comparative Example 2 -- 0.2 mg of CHO-expressed extracellu- lar glycoprotein gE, 0.2 mg of thicoxidized CpG 1018 and 0.1 mg of
QS21 were weighed, dissolved in 0.5 mL of PBS and evenly mixed with the same volume of aluminum adjuvant (purchased from Thermo
Fisher) to obtain 20 injections of the vaccine of comparative ex-
ample 2.
Example -- Lipids were weighed at a molar ratio of ALC-0315 (purchased from Xiamen Sinopeg Biotech Co., Ltd.) :DSPC (purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd.):cholesterol (purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd.) :ALC- 0159 (purchased from Xiamen Sinopeg Biotech Co., Ltd.) = 46.3:9.4:42.7:1.6, dissolved in anhydrous ethanol, mixed and dis- solved in a 100 mM citric acid buffer solution (pH=4.0) containing 0.24 mg of gE, 0.3 mg of CpG 1018 and 0.12 mg of QS21 at a ratio of 1:3 using a microfluidic nanomedicine manufacturing system (Precision Nanosystems) to obtain 20 injections of the vaccine of the example.
The vaccines prepared in the example and comparative examples 1-2 described above were subjected to the following experimental determinations:
Experimental Example 1: gE concentration
The vaccine of the example was cleaved overnight in 0.1 M so- dium hydroxide and a 0.1% (w/v) sodium dodecyl sulfate buffer so- lution at a room temperature. The gE concentration was detected with a BCA colorimetric protein detection kit (Shanghai Beyotime
Biotechnology Co., Ltd.), and the protein loading efficiency was calculated.
Experimental Example 2: Nucleic acid concentration
The vaccine of the example was cleaved overnight in 0.1 M so- dium hydroxide and a 0.1% (w/v) sodium dodecyl sulfate buffer so- lution at a room temperature. The nucleic acid concentration was detected with a nucleic acid detection kit, Quant-iT OliGreen ssD-
NA Regent Kit (purchased from Thermo Fisher), and the nucleic acid loading efficiency was calculated.
Experimental Example 3: QS21 concentration
The vaccine of the example was cleaved overnight in 0.1 M so- dium hydroxide and a 0.1% (w/v) sodium dodecyl sulfate buffer so- lution at a room temperature. Taking free Q321 as a standard, the content of 9821 entrapped in the example was determined using a high performance liquid chromatograph (HPLC, purchased from Wa- ters) with a 4.6x250 mm C18 column (purchased from Waters), and the 9521 loading efficiency was calculated.
Experimental Example 4: Particle size and polydispersity in- dex
For the vaccine of the example, a particle size and a poly- dispersity index of LNPs were detected with a nanoparticle size detector (Malvern).
The results of the example and experimental examples 1-4 are shown in FIG. 1. For the LNP lipid nanovaccine prepared using the microfluidic nanomedicine manufacturing system in the example, the gE entrapment efficiency is 49.57% (A in FIG. 1), resulting in about 5.95 u4g/injection; the CpG ODN nucleic acid entrapment effi- ciency is 41.85% (B in FIG. 1), resulting in about 6.28 ug/injection; the Q321 loading efficiency is 57.15% (C in FIG. 1), resulting in about 3.43 ug/injection; the nanoparticle size is 190.3-194.7 nm (D in FIG. 1), and the polydispersity index is 0.269-0.322 (E in FIG. 1).
Experimental Example 5: Cytotoxicity
Red blood cells from femurs and tibiae of specific pathogen- free C57BL/6J mice (female, 6-8 weeks old, 16-18 g, purchased from
Chengdu Dossy Experimental Animals Co., Ltd.) were cleaved with an
ACK red blood cell lysis solution to obtain bone marrow cells. Im- mature bone marrow-derived dendritic cells (BMDC) were induced with a 1640 complete medium (purchased from Thermo Fisher) con- taining 20 ng/mL GM-CSF (purchased from PeproTech Biotech (Suzhou)
Co., Ltd.). After 2x10° cells were inoculated in each well of a 96- well plate, samples were added and culture was continued for 24 h.
The cell viability was detected with a CCK-8 kit (purchased from
MedChemExpress} .
The results of the example and experimental example 5 are shown in FIG. 2. The BMDC cell viability is only about 18% in the presence of 10 pg/mL free Q321 of comparative examples. Entrapped in LNPs, QS21 with the same concentration in the example did not exhibit significant cytotoxicity.
Experimental Example 6: Animal immunization
PRS ws taken as a blank control. At an interval of 4 weeks,
C57BL/6 mice were immunized two times by intramuscularly injecting pL of vaccines prepared in the example and comparative examples l and 2 (6 mice/group, female, primary immunization age: 6-8 weeks, weight: 16-18 g). 2 weeks after final immunization, spleens were removed. Blood was collected from hearts, placed at 4°C over- night and then centrifuged at 3,500 rpm/min for 30 min to obtain serum for subsequent immunological analysis.
Experimental Example 7: Antibody titer detection
A 2 pg/mL extracellular glycoprotein gE of a capture antigen dissolved in PBS was added to a 96-well ELISA plate (purchased from Corning) as per 100 uL/well. After overnight coating at 4°C, the plate was washed one time with PBST (0.05% (v/v) Tween20 (Sig- ma) in PBS). A blocking solution of 5% (w/v) skimmed milk powder dissolved in PBS was added. After blocking at 37°C for 1 h, the blocking solution was discarded, and the plate was washed 4 times with PBST. Antisera serially diluted in a 1% blocking solution were added as per 200 puL/well. After incubation at 37°C for 1 h, the plate was washed 5 times with PBST. A secondary antibody di- luted in the 1% blocking solution (1:10,000, Goat anti-mouse
IgG:HRP, purchased from BioRad) was added as per 100 uL/well. Af- ter incubation at 37°C for 1 h, the plate was washed 5 times with
PBST. 100 pL of a color developing solution (purchased from BD) prepared at a ratio of 1:1 was added to each well. The plate was placed in the dark for 5 min at room temperature. 100 pL of 1 M sulfuric acid was added to terminate the reaction, and the absorb- ance value was detected at 450 nm. The critical serum dilution concentration of OD450>0.15 was taken as an antibody titer, and the titer with OD450 less than 0.15 at a dilution of 1:2,000 was defined as 100 for calculation.
The results of comparative examples 1-2, the example and ex- perimental examples 6-7 are shown in FIG. 3. The gE-specific IgG titer of serum from immunized mice in the example is 170,667, which is equivalent to that in comparative example 2 and 1.3 times that in comparative example 1 (with a IgG titer of 128,000).
Experimental Example 8: Splenic lymphocyte separation
A spleen was placed in a cell strainer (purchased from Wuxi
NEST Biotechnology Co., Ltd.), and an ACK red blood cell lysis so- lution was added. The sample was placed at a room temperature for 5 min. After centrifugation at 1,800 rpm, cells were counted. The sample was resuspended at 1x10" cells/mL with a 1640 medium (pur- chased from Thermo Fisher) containing 10% fetal bovine serum (pur- chased from Thermo Fisher) and dual antibodies.
Experimental Example 9: Cytokine analysis 100 pL of splenocytes at 1x10 cells/mL were added to each well of a 96-well plate (purchased from Corning). gE with a final concentration of 10 ng/mL was added to each well. 10 pL of PMA + ionomycin (stock solution concentration: 500 ng/mL + 10 pg/mL; purchased from Dakewe) was used as a positive control. After incu- bation at 37°C and 5% CO, for 24 h, a cell supernatant was collect- ed, and contents of IL-2 and IFN-y were detected by an ELISA meth- od. A 96-well plate was coated with IL-2 (3 pg/mL) and IFN-y (4 ng/mL) capture antibodies (purchased from Thermo Fisher) dissolved in PBS at 4°C for 16 h. After blocking with a 5% skimmed milk blocking solution at 37°C for 1 h, 50 uL of the cell supernatant was added to each well, and incubation was conducted at a room temperature for 3 h. Standard curves were generated with PBS- dissolved mouse IL-2 and IFN-y protein standards (purchased from
PeproTech Biotech (Suzhou) Co., Ltd.). IL-2 or IFN-y-specific bio- tin-conjugated antibodies (2 pg/mL, purchased from Thermo Fisher) and HRP-conjugated streptavidin (1 pg/mL, purchased from Bio-
Legend) were then added, and incubation was conducted for 1.5 h.
As described in antibody titer detection, the reaction was termi- nated and detection results were obtained.
The results of comparative examples 1-2, the example, and ex- perimental examples 6, 8, 9, and 12 are shown in FIGS. 4-5. Ac- cording to ELISA analysis, the IL-2 level of the supernatant in the example is 2509 pg/mL (FIG. 4), which is 1.25 times that in comparative example 1 (2011 pg/mL, p=0.72) and 2.69 times that in comparative example 2 (934.3 pg/mL, p=0.02). The IFN-y level of the supernatant in the example is 6722 pg/mL (FIG. 5), which is 1.21 times that in comparative example 1 (5572 pg/mL, p=0.42) and 1.6 times that in comparative example 2 (4207 pg/mL, p=0.02).
Experimental Example 10: Enzyme linked immunospot assay (ELISPOT)
Both IL-2 and IFN-y detection kits were purchased from BD,
and operated according to instructions. The specific steps were as follows: A capture antibody diluted in a coating solution was add- ed to an ELISPOT plate as per 100 uL/well. After coating overnight at 4°C, the coating solution was discarded and the plate was washed one time with a blocking solution (200 uL/well). 200 uL of the blocking solution was added to each well. After blocking at a room temperature for 2 h, the blocking solution was discarded. 100 pL of a 1640 complete medium containing gE with a final concentration of 20 pg/mL was added, and spleen cells obtained through the fore- going splenic lymphocyte separation were added to a final concen- tration of 2x10° cells/well. The sample was placed in a 37°C cell incubator overnight. 800 g of the sample was subjected to centrif- ugation at for 5 min, and the supernatant was discarded. The plate was washed two times with deionized water (200 uL/well) (soaked for 5 min each time), and washed 3 times with a washing solution 1 (200 uL/well). A detection antibody diluted in a diluent was added as per 100 uL/well, and incubation was conducted at a room temper- ature for 2 h. The plate was washed 3 times with the washing solu- tion 1 (200 uL/well} (soaked for 2 min each time). An enzyme con- jugate, Streptavidin-HRP, diluted in the diluent was added as per 100 upL/well, and incubation was conducted at a room temperature for 1 h. The plate was washed 4 times with the washing solution 1 (200 uL/well) (soaked for 2 min each time). The plate was washed two times with a washing solution 2 (200 pL/well). 100 uL of a substrate solution was added to react for an appropriate period of time. The plate was washed with deionized water, and the reaction was terminated. After drying, spots were counted with an ELISPOT plate reader (AID Diagnostika GmbH).
The results of comparative examples 1-2, the example, and ex- perimental examples 6, 8, 10, and 12 are shown in FIGS. 6-7. Ac- cording to ELISPOT analysis, the number of cells secreting IL-2 after gE stimulation in the example is 224.3 per 2x10° splenocytes (FIG. 6), which is 2.1 times that in comparative example 1 (106.8 per 2x10° splenocytes, p<0.001) and 1.87 times that in comparative example 2 (119.8 per 2x10° splenocytes, p=0.002). The number of cells secreting IFN-y after gE stimulation in the example is 293.7 per 2x10 5 splenocytes (FIG. 7). which is 1.67 times that in com-
parative example 1 (175.5 per 2x10° splenocytes, p=0.008) and 1.87 times that in comparative example 2 (157 per 2x10° splenocytes, p=0.002).
Experimental Example 11: Flow analysis
All reagents for flow analysis were purchased from BioLegend.
A total of 2x10° splenocytes were incubated with 10 pg/mL protein gE for 2 h at 37°C and 5% CO;. 5 pg/mL brefeldin A was added. The splenocytes were incubated overnight under the same conditions to block cytokine release. After washing with a staining buffer solu- tion, 100 pL of Zombie NIR™ was added to each sample, and the splenocytes were incubated for 30 min. A 5 pg/mL anti-CD16/CD32 antibody was added, and the splenocytes were incubated at 4°C for 10 min to block non-specific binding to Fc receptors. PercP-Cy5.5- conjugated anti-mouse CD4 was added, and the splenocytes were in- cubated at 4°C for 30 min. PE-conjugated anti-mouse IFN-y and APC- conjugated anti-mouse IL-2 antibodies were used for intracellular staining. After staining, cells were gated (forward scatter and side scatter, FSC/SSC), and samples with over 20,000 CD4+ cell events were analyzed with a CytoFLEX flow cytometer (Beckman) and
FlowJo V10 software.
The results of comparative examples 1-2, the example, and ex- perimental examples 6, 8, 11, and 12 are shown in FIGS. 8-9. Ac- cording to analysis with the flow cytometer, the proportion of CD4*
T cells expressing IL-2 after gE stimulation in the example is 0.6633% (FIG. 8), which is 2.54 times that in comparative example 1 (0.2612%, p=0.008) and 3.04 times that in comparative example 2 (0.2183%, p=0.004). The proportion of CD4’ T cells expressing IFN-y after gE stimulation in the example is 0.7598% (FIG. 9), which is 2.11 times that in comparative example 1 (0.3598%, p=0.04) and 3.53 times that in comparative example 2 (0.2152%, p=0.004).
Experimental Example 12: Statistical analysis
Data were analyzed with GraphPad Prism 9.2 software and ex- pressed as mean + SD. Significant differences among experimental groups were analyzed through the ordinary one-way analysis of var- iance (BNOVA) and Dunnett’s multiple comparison test based on the example. Asterisks represent p-value categories: *ps0.05, **ps0.01 and ***p<0.001.
The results of experimental example 12 are shown in FIGS. 4- 9.
These embodiments are intended for a detailed description of the present invention, but they are merely a part of, rather than all of, embodiments of the present invention. Other embodiments may also be obtained based on these embodiments without making in- ventive efforts, and should all fall within the protection scope of the present invention.
Claims (10)
Applications Claiming Priority (1)
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| WO2024052882A1 (en) * | 2022-09-09 | 2024-03-14 | Access To Advanced Health Institute | Immunogenic vaccine composition incorporating a saponin |
| CN116983403B (en) * | 2022-09-30 | 2023-12-19 | 烟台派诺生物技术有限公司 | Immune composition product for preventing or treating varicella-zoster virus related diseases and preparation method thereof |
| CN116023510B (en) * | 2022-12-01 | 2023-09-22 | 北京吉诺卫生物科技有限公司 | Double-adjuvant recombinant herpes zoster vaccine |
| CN116077638B (en) * | 2022-12-20 | 2024-03-26 | 国药中生生物技术研究院有限公司 | Composite adjuvant |
| CN116747298B (en) * | 2023-08-09 | 2024-01-02 | 成都新诺明生物科技有限公司 | Varicella-zoster virus vaccine and preparation method and application thereof |
| CN117003894B (en) * | 2023-08-09 | 2024-04-16 | 成都新诺明生物科技有限公司 | gE fusion protein containing IL-2 and Fc, and preparation method and application thereof |
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| EP1541170A1 (en) * | 1999-04-19 | 2005-06-15 | GlaxoSmithKline Biologicals S.A. | Adjuvant composition comprising saponin and an immunostimulatory oligonucleotide |
| WO2023116374A1 (en) * | 2021-12-23 | 2023-06-29 | 上海泽润生物科技有限公司 | Herpes zoster vaccine composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1541170A1 (en) * | 1999-04-19 | 2005-06-15 | GlaxoSmithKline Biologicals S.A. | Adjuvant composition comprising saponin and an immunostimulatory oligonucleotide |
| WO2023116374A1 (en) * | 2021-12-23 | 2023-06-29 | 上海泽润生物科技有限公司 | Herpes zoster vaccine composition |
Non-Patent Citations (3)
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| CAO HAN ET AL: "Immunogenicity of Varicella-Zoster Virus Glycoprotein E Formulated with Lipid Nanoparticles and Nucleic Immunostimulators in Mice", VACCINES, vol. 9, no. 4, 1 January 2021 (2021-01-01), pages 310, XP093055876, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064366/pdf/vaccines-09-00310.pdf> DOI: 10.3390/vaccines9040310 * |
| GLAXOSMTIHKLINE BIOLOGICALS S.A.: "Shingrix - Annex 1 - Summary of Product Characteristics", 21 March 2018 (2018-03-21), XP093128284, Retrieved from the Internet <URL:https://www.ema.europa.eu/en/documents/product-information/shingrix-epar-product-information_en.pdf> [retrieved on 20240206] * |
| LUAN NING ET AL: "Ionizable Lipid Nanoparticles Enhanced the Synergistic Adjuvant Effect of CpG ODNs and QS21 in a Varicella Zoster Virus Glycoprotein E Subunit Vaccine", PHARMACEUTICS, vol. 14, no. 5, 30 April 2022 (2022-04-30), CH, pages 973, XP093128285, ISSN: 1999-4923, DOI: 10.3390/pharmaceutics14050973 * |
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| NL2032309B1 (en) | 2024-05-08 |
| CN114767844A (en) | 2022-07-22 |
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