NZ755255B2

NZ755255B2 - Herpes zoster vaccine composition

Info

Publication number: NZ755255B2
Application number: NZ755255A
Authority: NZ
Inventors: Sung Jun Hong; Eun Mi Kim; Hyo Jung Nam; Steven G Reed; Duck Hyang Shin; Kang Il Yoo
Original assignee: Infectious Disease Research Institute (Idri); Mogam Institute For Biomedical Research
Priority date: 2016-12-26
Filing date: 2017-12-20
Publication date: 2021-08-31

Abstract

The present invention relates to a herpes zoster vaccine composition which comprises glycoprotein E of Varicella zoster virus, a glucopyranosyl lipid adjuvant, and a metabolic oil and selectively increases a cell-mediated immune reaction without having disadvantages of attenuated live vaccines.

Description

Description Title of Invention HERPES ZOSTER VACCINE COMPOSITION Technical Field The present invention generally relates to a vaccine ition for preventing herpes zoster.

Background Art The primary infection of varicella-zoster virus (VZV) causes chickenpox which is characterized by highly ious skin rashes mainly on the face and trunk.

After the initial infection, the viral DNA can remain dormant for years in the asm of the host neuronal cell. The virus can be reactivated to cause herpes zoster (zoster or shingles) in adults.

Herpes zoster causes skin rashes distinct from those produced during the primary infection. The rashes are accompanied by severe pain and may lead to more severe conditions such as erpetic neuralgia (PHN).

Varicella-zoster virus (VZV), also known as human herpesvirus 3 (HHV-3), is a member of the alphaherpesvirus subfamily of the Herpesviridae family. VZV is an enveloped virus with a double-stranded DNA genome of about 125,000 nucleotides.

The genome of VZV is enclosed by an icosahedral capsid. A viral tegument ( epidermis), located in the space between the nucleocapsid and the viral envelope, is a construct consisting of virally-encoded proteins and enzymes. The viral envelope is derived from host cell nes and contains virally-encoded glycoproteins.

The VZV genome encodes y (70) or more open reading frames (ORFs), nine (9) of which encode glycoproteins (gE, gI, gB, gH, gK, gN, gL, gC, and gM) that are presumed to function at different stages in the viral replication cycle.

Glycoprotein E (gE) is essential for viral replication (Mallory et al. (1997)J.

Virol. 71: 8279-8288) and Mo et al. (2002) Virology 304: 176-186), and is the most abundant glycoprotein found in infected cells as well as in mature virions , 2002, The predominant varicella-zoster virus gE and gI glycoprotein complex, In Structure-function relationships of human pathogenic viruses, Holzenburg and Bogner (eds.), Kluwer ic/Plenum Publishers, New York, NY).

Glycoprotein I (gI) forms a complex with gE in infected cells, thereby promoting endocytosis of both glycoproteins, which are then delivered to the trans- Golgi where the final viral envelope is obtained (Olson and Grose (1998) J. Virol. 72: 1542-1551).

Glycoprotein B (gB), which is thought to play an important role in viral entry, has an e for a virus lizing antibody and is the second most glycoprotein on the virion surface (Arvin (1996) Clin. Microbiol. Rev. 9: 361-381).

Glycoprotein H (gH) is thought to have a fusion function that promotes cellto-cell spread of the virus.

Currently, live ated es, which are commonly used to prevent chickenpox or herpes zoster, have several antages. First, there is some evidence that the immunity against VZV infection decreases with time and the effect of the vaccine disappears (Chaveset al. (2007) N. Engl. J. Med. 356: 1121-1129).

Thus, the subjects vaccinated with the vaccine may remain susceptible to herpes zoster, which is a more severe condition caused by VZV. In addition, ated live es are manufactured using live viruses with weakened pathogenicity, so that vaccination subjects may become susceptible to chickenpox or herpes zoster due to vaccination. In fact, there are several cases of herpes zoster reported to have been caused by the virus strain used in the vaccine (Matsubara et al. (1995) Acta Paediatr Jpn 37: 648-50; and Hammerschlag et al. (1989) J Infect Dis. 160: 535-7). In addition, due to the live attenuated virus present in the vaccine, the use of the e may be limited for subjects whose immune function is decreased.

In order to increase the effect of preventing herpes zoster which re-emerges along the neuronal cells after the dormancy of the virus, it is important to significantly enhance the activation of cell-mediated immunity (CMI) to VZV antigen rather than the activation of humoral immunity o. For this, it is important to increase Th1/Th2 ratio by promoting the activation of Th1 among Th1 and Th2, which are T cells (helper T cells).

Therefore, there is a need to develop new herpes zoster e compositions that can selectively increase the cell mediated immune response t having the disadvantages of live attenuated vaccines.

Disclosure of Invention Technical Problem The object of the present invention is to provide a e composition having a high safety and an excellent effect of ting herpes zoster, which selectively increases the cell mediated immune response without having the disadvantages of live attenuated vaccines; and/or to at least provide the public with a useful choice.

Summary of the invention In one aspect, the invention provides a vaccine composition against chickenpox or herpes zoster comprising: glycoprotein E of varicella-zoster virus; a yranosyl lipid adjuvant of the following a 1; and squalene: [Formula 1] wherein, R1, R3, R5 and R6 are each independently C10-C12 alkyl; and R2 and R4 are each independently C8-C10 alkyl.

Other embodiments of the present description are presented in the numbered paragraphs below. Certain statements that appear below are broader than what s in the statements of the invention above. These statements are provided in the interests of providing the reader with a better understanding of the ion and its practice. The reader is ed to the accompanying claim set which defines the scope of the invention. 1. A vaccine ition against chickenpox or herpes zoster comprising: glycoprotein E of varicella-zoster virus; a glucopyranosyl lipid adjuvant of the following Formula 1; and a metabolisable oil: [Formula 1] wherein, R1, R3, R5 and R6 are each independently 2 alkyl; and R2 and R4 are each independently C8-C10 alkyl. 2. The vaccine composition of the above 1, wherein the glucopyranosyl lipid adjuvant is the one of Formula 1 wherein R1, R3, R5 and R6 are C11 alkyl. 3. The vaccine composition of the above 1, wherein the glucopyranosyl lipid adjuvant is the one of Formula 1 wherein R2 and R4 are C9 alkyl. 4. The vaccine composition of the above 1, wherein the metabolisable oil is squalene. 5. The vaccine composition of the above 4, wherein the squalene is contained in an amount of 1% (v/v) to 7% (v/v) of the total vaccine composition. 6. The vaccine composition of the above 5, wherein the squalene is contained in an amount of 1% (v/v) to 4% (v/v) of the total vaccine composition. 7. The vaccine composition of the above 1, wherein the rotein E is contained in an amount of 5 μg to 100 μg in a single dose of the vaccine composition. 8. The vaccine composition of the above 1, n the glucopyranosyl lipid adjuvant is contained in an amount of 7.5 μg to 20 μg in a single dose of the e composition. 9. The vaccine composition of the above 8, wherein the glucopyranosyl lipid nt is ned in an amount of 9 μg to 18 μg in a single dose of the vaccine composition.

. A method for preventing or treating chickenpox or herpes zoster comprising administering the composition of any one of the above 1 to 9 to a t.

Advantageous Effects of Invention The vaccine composition of the present invention is excellent in ting herpes zoster; and/or the vaccine ition of the t invention significantly increases the cell mediated immune response to the VZV antigen as compared to the humoral immune response thereto; and/or the vaccine composition of the present invention greatly increases the number of Th1 cells producing two or more Th1-specific cytokines; and/or the vaccine composition of the present invention greatly increases the production of IgG2c as compared to the production of IgG1; and/or the vaccine composition of the present invention has no possibility of infecting subject with herpes zoster due to vaccination; and/or the vaccine ition of the present invention can be administered even to ts whose immune function is decreased; and/or the e composition of the present invention has a long-lasting tive effect; and/or the vaccine composition of the present invention provides the public with a useful choice.

Brief ption of Drawings Fig. 1 illustrates the production of gE antigen-specific IgG according to the experiment of Experimental Example 3.

Fig. 2 depicts the productions of gE antigen-specific IgG2c and IgG1 according to the experiment of Experimental Example 3.

Fig. 3 indicates the number of T cells that secrete IFN-γ specifically to gE protein according to the experiment of Experimental Example 4.

Fig. 4 demonstrates the number of T cells that secrete IFN-γ specifically to the gE pping peptide according to the experiment of Experimental Example 4.

Fig. 5 displays the number of T cells that secrete IFN-γ specifically to the entire VZV according to the experiment of Experimental Example 4. presents the amount of various Th nes ed specifically to the gE antigen according to the experiment of Experimental Example 5.

Fig. 7 shows the distribution of gE antigen-specific ne-secreting cells according to the experiment of mental Example 6.

Fig. 8 illustrates the production of VZV n-specific IgG according to the experiment of Experimental Example 7.

Fig. 9 depicts the productions of VZV antigen-specific IgG2c and IgG1 according to the experiment of mental Example 7.

Fig. 10 indicates the amount of IFN-γ secreted specifically to the entire VZV according to the experiment of Experimental Example 8.

Fig. 11 demonstrates the distribution of VZV antigen-specific cytokinesecreting cells according to the experiment of Experimental Example 9.

Fig. 12 ys the production of VZV antigen-specific IgG according to the experiment of Experimental Example 10 and the number of T cells specific for gE or entire VZV according to the experiment of Experimental Example 2-2.

Fig. 13 presents the production of gE antigen-specific IgG according to the experiment of Experimental Example 11.

Fig. 14 shows the productions of gE antigen-specific IgG2c and IgG1 according to the experiment of Experimental e 11.

Fig. 15 illustrates the number of T cells that secrete IFN-γ specifically to gE protein according to the experiment of Experimental Example 12.

Fig. 16 depicts the number of T cells that secrete IFN-γ specifically to the gE overlapping peptide according to the ment of Experimental Example 12.

Fig. 17 indicates the number of T cells that secrete IFN-γ specifically to the VZV antigen according to the experiment of Experimental Example 12.

Fig. 18 demonstrates the amount of IFN-γ secreted by the gE protein stimulation according to the experiment of Experimental Example 13.

Fig. 19 displays the amount of IFN-γ secreted by the gE overlapping peptide stimulation according to the ment of Experimental Example 13.

Fig. 20 presents the number of T cells that secrete IFN-γ specifically to gE protein according to the experiment of mental Example 14.

Fig. 21 shows the number of T cells that secrete IFN-γ specifically to the entire VZV according to the experiment of Experimental Example 14.

Best Mode for ng out the Invention The present invention generally relates to a herpes zoster vaccine composition, which comprises glycoprotein E of lla-zoster virus, a yranosyl lipid adjuvant, and a metabolisable oil, and has a high safety and an excellent effect of preventing herpes zoster by selectively increasing the cell mediated immune response without having the disadvantages of live attenuated vaccines.

Described herein is a vaccine composition comprising glycoprotein E of lla-zoster virus (VZV), a yranosyl lipid adjuvant of the following Formula 1, and a metabolisable oil: [Formula 1] wherein, R1, R3, R5 and R6 are each independently C10-C12 alkyl; and R2 and R4 are each independently C8-C14 alkyl. For example, R2 and R4 may be C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, or C14 alkyl. According to a more specific example, R2 and R4 may be C9 alkyl or C13 alkyl.

Hereinafter, the present invention will be described in detail.

The vaccine composition of the present invention comprises rotein E of varicella-zoster virus (VZV), a glucopyranosyl lipid adjuvant of the following Formula 1, and a lisable oil, namely squalene: [Formula 1] wherein, R1, R3, R5 and R6 are each independently C10-C12 alkyl; and R2 and R4 are each independently C8-C10 alkyl.

The e composition of the present invention comprises glycoprotein E (gE) of VZV. Glycoprotein E (gE) in the present invention means glycoprotein E of VZV or an immunogenic derivative thereof. The immunogenic derivative in the present invention may be the one wherein a part of glycoprotein E is modified. For example, it may be the one wherein a part of glycoprotein E is cut, one or more amino acids of glycoprotein E are replaced by another amino acids, one or more amino acids of rotein E are removed, one or more amino acids are added to glycoprotein E, or one or more amino acids of glycoprotein E are chemically modified. For example, glycoprotein E useful in the present invention may be represented by the ce of SEQ ID NO: 1.

The vaccine ition of the present ion comprises a glucopyranosyl lipid adjuvant of the following Formula 1 and a lisable oil, namely squalene: [Formula 1] wherein, R1, R3, R5 and R6 are each independently C10-C12 alkyl; and R2 and R4 are each independently C8-C10 alkyl. For example, R2 and R4 may be C8 alkyl, C9 alkyl, or C10 alkyl. According to a more specific example, R2 and R4 may be C9 alkyl or C10 alkyl. For example, R2 and R4 may be C9 alkyl.

The term “metabolisable oil” as used in this description means an oil whose structure is modified by metabolism, and es vegetable oils, fish oils, animal oils, and synthetic oils, which have no biotoxicity and may undergo structural changes upon metabolic progression.

According to the present invention, the metabolisable oil useful in the present invention is squalene. Squalene is a arbon of triterpene backbone having 30 carbons. A variety of squalenes commonly known in the art to be used as metabolisable oils or emulsions may be used, for example, squalene from shark liver oil. An ary composition of squalene is described in Fox CBet al. (2013) Vaccine 31 (49): 5848-55.

In order to increase the effect of preventing herpes zoster, it is important to significantly e the tion of cell mediated immunity (CMI) to VZV antigens while minimizing the activation of humoral immunity thereto.

High levels of ecific cytokines favor induction of a humoral immune response to the provided antigen, while high levels of Th1-specific cytokines tend to prefer induction of cell mediated immune response (CMI) to the provided antigen.

Thus, the more Th1-specific cytokines are generated than the Th2-specific cytokine, the higher the degree of activation of the cell mediated immune response becomes than that of the humoral immune response.

Also, the greater the number of cells simultaneously producing two or more cytokines out of IFN- γ, TNF-α, and IL-2, the higher the degree of tion of the cell mediated immune response.

In addition, as the degree of activation of the cell mediated immune response s higher than that of the humoral immune response, the production of IgG2c antibody is y increased as compared to that of IgG1 antibody.

The vaccine composition of the present invention can greatly increase the degree of activation of the cell mediated immune se rather than that of the humoral immune response in the body of a subject.

For example, the vaccine composition of the present invention can significantly increase the production of Th1-specific cytokines (e.g., interferon- gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2)) as compared to the production of Th2-specific cytokines (e.g., interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), and the like), in the body of a subject.

Also, for example, the e composition of the present ion is capable of greatly increasing the number of cells simultaneously ing two or more cytokines out of IFN- γ, TNF-α, and IL-2 than the number of cells producing only one cytokine out of the above cytokines, among ted Th1 cells.

In addition, for example, the vaccine composition of the present invention can greatly se the production of gE-specific IgG antibodies in the body of a subject and, especially, can greatly increase the production of gE-specific IgG2c antibodies as compared to that of gE-specific IgG1 antibodies.

The e composition of the present invention may contain 5 μg to 100 μg of glycoprotein E in a single dose. For example, it may contain 5 μg to 80 μg, specifically 5 μg to 70 μg, more specifically 5 μg to 60 μg, and most specifically 5 μg to 50 μg of rotein E in a single dose.

The vaccine composition of the present invention may contain 7.5 μg to 20 μg, specifically 9 μg to 18 μg, more specifically, 9 μg to 16 μg, and most specifically 10 μg to 15 μg of the glucopyranosyl lipid adjuvant in a single dose. According to another embodiment of the present invention, the vaccine composition of the present invention may contain 13 μg to 17 μg of the glucopyranosyl lipid adjuvant in a single dose. When the glucopyranosyl lipid adjuvant is included in the above range, the cell ed immune response can be selectively maximized.

The vaccine composition of the present invention may contain 1 to 7% (v/v), more specifically 1 to 5% (v v), and most specifically 1 to 4% (v/v) of the metabolisable oil in a single dose.

In on to glycoprotein E, the glucopyranosyl lipid nt, and the metabolisable oil, the vaccine ition of the present invention may include pharmaceutically acceptable excipients, carriers, and the like. For example, the vaccine composition of the present invention may n physiological saline or PBS (phosphate buffered saline).

The vaccine composition of the present invention can be formulated and packaged in various forms. According to one embodiment, the first vial containing glycoprotein E but not comprising the glucopyranosyl lipid adjuvant and the metabolisable oil, and the second vial containing the glucopyranosyl lipid adjuvant and the metabolisable oil but not comprising rotein E may be separately packaged, and mixed prior to use de mixing). According to another ment, a vaccine composition comprising all of glycoprotein E, the glucopyranosyl lipid adjuvant and the metabolic oil may be packaged in a vial, a syringe (prefilled syringe), or the like.

Mode for the Invention Hereinafter, the present invention will be described in more detail with reference to experimental examples. These experimental examples are only intended to illustrate the present invention, and the scope of the present invention is not d to those exemplified in these mental examples. mental Example 1: Immunization Since humans have a history of chickenpox infection, in order to mimic chickenpox ion in mice, live attenuated vaccine (LAV, 3000 pfu) was subcutaneously injected once to female C57BL/6 mice to perform primary immunization (LAV priming). After 28 days from the LAV priming (Day 0), various VZV vaccine compositions with or without VZV protein gen or adjuvant were administered by intramuscular injection to perform secondary immunization.

In order to measure the humoral immune response to VZV, blood samples were taken once at the LAV priming point, and 28 days and 42 days thereafter (Day 0, Day 28 and Day 42), respectively, and leukocytes were collected from spleen samples 42 days after the LAV priming (Day 42) to measure CMI mediated immune response) against VZV.

The experimental design for primary immunization (LAV priming), secondary immunization (Immunization), and immune response measurement is ized as shown in Table 1 below. In Table 1 below, gE refers to VZV glycoprotein E of SEQ ID NO: 1, LAV refers to live attenuated virus, SLA refers to the glucopyranosyl lipid adjuvant of Formula 1 wherein R2 and R4 are C9 alkyl, and SE refers to squalene.

SLA and squalene were obtained from the Infectious Disease Research Institute (Seattle, US) and Aldrich (St. Louis, MO), respectively.

Alum ide is aluminum hydroxide; Addavax® is a squalene-based oilin-water nano-emulsion; Pam3CSK4 is a TLR 1/2 agonist, a synthetic triacylated lipoprotein of CAS number 1122081; polyIC is polyinosinic-polycytidylic acid; MPL is Monophosphoryl Lipid A; and ODN1826 is a Class B CpG oligonucleotide- Murine TLR9 ligand. In addition, the days of immunization, blood sample collection, and spleen sample collection were calculated from Day 0 as the day of LAV priming.

[Table 1] Primary ary zation Day of Day of Day of immunizat (Immunization) secondary blood spleen Group ion immunizati sample sample (LAV Antigen Adjuvant on collection collection priming*) PBS PBS-only X X LAV(1 shot) LAV PBS-only X LAV(2 shot) LAV (15,000 X pfu) gE LAV gE (5㎍) X SLA (5㎍) in aqueous gE+SLA-AF LAV gE (5㎍) formulation gE+SLA-SE LAV gE (5㎍) SLA (5㎍) + SE (2%) gE+SE LAV gE (5㎍) SE (2%) gE+liposome LAV gE (5㎍) me-only gE+Addavax LAV gE (5㎍) Addavax (50%) Day 0, Day 28 Day 28, Day 42 MPL (5㎍) + QuilA Day 42 +QuilA LAV gE (5㎍) (5㎍) gI+SLA-SE LAV gI (5㎍) SLA (5㎍) + SE (2%) IE63+SLA-SE LAV IE63 (5㎍) SLA (5㎍) + SE (2%) gB+SLA-SE LAV gB (5㎍) SLA (5㎍) + SE (2%) gC+SLA-SE LAV gC (5㎍) SLA (5㎍) + SE (2%) gL+SLA-SE LAV gL (5㎍) SLA (5㎍) + SE (2%) gE+Alum Alum hydroxide (0.5 LAV gE (5 ㎍) hydroxide mg) gE+Addavax LAV gE (5 ㎍) Addavax (50 µL) gE+Pam3CSK4 LAV gE (5 ㎍) Pam3CSK4 (11 µg) gE+polyIC LAV gE (5 ㎍) polyIC (55 µg) gE+MPL LAV gE (5 ㎍) MPL (11 µg) gE+flagellin LAV gE (5㎍) Flagellin (5.5 µg) gE+imiquimod LAV gE (5㎍) Imiquimod (55 µg) gE+ODN1826 LAV gE (5㎍) ODN1826 (35µg) ry immunization (LAV priming): Dose 100 μL/head. 3,000 pfu *Secondary immunization (Immunization): Dose 100 μL/head Experimental Example 2: Experimental methods Experimental Example 2-1: Method for measuring VZV antigen-specific IgG titer (VZV specific IgG titer) After performing the primary immunization and the secondary immunization, ELISA (enzyme-linked immunosorbent assay) was d out for measuring VZV n-specific IgG titer. A recombinant gE protein or VZV antigen (1 μg/mL) was coated onto an ELISA plate and incubated overnight at 4 ℃. The ELISA plate was washed three times and blocking was d out with PBS hate-buffered saline) solution containing 2% BSA (Bovine serum n) for 1 hour. After washing the ELISA plate, diluted serum samples were added thereto and incubated for 2 hours.

HRP (Horseradish peroxidase)-conjugated goat anti-mouse IgG, IgG1, or IgG2c antibodies were added thereto and incubated for 1 hour. After the final incubation, the ELISA plate was washed and the HRP reaction was induced by the addition of TMB (3, 3', 5, 5' - tetramethylbenzidine) substrate. The HRP reaction was d by adding ELISA stop on and optical density (OD) was measured using a spectrometer at a wavelength of 450 nm.

Experimental Example 2-2: Method for measuring VZV antigen-specific cell-mediated immune response using the enzyme-linked immunospot assay (ELISPOT assay) After performing the primary immunization and the secondary immunization, mouse IFN-γ ELISPOT (enzyme-linked immunospot) assay was carried out to confirm VZV antigen-specific cell-mediated immune response (CMI). IFN-γ capture antibody (5 μg/mL) was coated onto an ELISPOT plate and incubated overnight at 4 ℃. The ELISPOT plate was washed 3 times and blocking was carried out with a medium containing 10% FBS (fetal bovine serum) for 1 hour. After washing the ELISPOT plate, leukocytes collected from the immunized mice and gE protein, gE OLP apping e), or a VZV lysate were added thereto and incubated for 24 hours for leukocyte stimulation. Upon completion of the leukocyte ation, the ELISPOT plate was washed, and biotinylated mouse IFN-γ ion antibody (2 μg/mL) was added thereto and incubated. After washing the plate, streptavidin-HRP was added thereto and incubated again. Then, after g the ELISPOT plate, AEC substrate mixture was added thereto to induce a reaction at room temperature.

The reaction was stopped by washing the ELISPOT plate with water and the plate was dried. The number of the resulting spots were counted with a device. mental Example 2-3: Method for identifying cytokines secreted by antigen stimulation (CBA assay) After performing the primary immunization and the secondary immunization, CBA (cytometric bead array) assay was carried out to identify the types of cytokines secreted by T cells due to antigen stimulation. ytes collected from mice were ated with gE protein or a VZV lysate for 3 days and centrifuged to obtain the supernatant, which was then assayed for cytokines with mouse Th1/Th2/Th17 CBA kit. Seven (7) kinds of cytokine capture beads (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF, and IL-17A), the supernatant sample, and cytokine detection beads were reacted together for 2 hours, the beads were washed, and the amounts of nes in the supernatant were determined.

Experimental e 2-4: Method for determining the distribution of cytokine-secreting cells (ICS assay) After performing the primary immunization and the secondary immunization, the secretion of Th1-specific cytokines was measured by ICS (intracellular cytokine staining) assay to confirm the antigen-specific cell-mediated immune response.

Leukocytes collected from mice were stimulated overnight with gE protein and, at this time, GolgiStop (BFA)/GolgiPlug (monensin) was also added to prevent cytokines in the cells from being secreted to the outside. After washing the ated leukocytes, the cell surface of leukocytes was labeled with antibodies (7-AAD, CD3-FITC, CD4- V500) to identify T cells. After the tion of the reaction, the leukocytes were washed, permeabilized, and ted to a reaction with antibodies (TNF-α-PE, IFN- γ-APC, 450) which can be bound to cytokines to confirm the presence of cytokines in the cells. After the reaction, the leukocytes were washed and fixed, and the distribution of the cells ing cytokines by the antigen stimulation was analyzed. mental Example 2-5: Method for determining IFN-γ cytokine ed by gE antigen stimulation (IFN-γ ELISA) After performing the primary immunization and the secondary immunization, IFN-γ ELISA assay was carried out to determine the ion amount of IFN-γ, a typical effector cytokine secreted by T cells by antigen stimulation. Leukocytes collected from mice were stimulated with gE protein or gE overlapping peptide for 3 days and centrifuged to obtain the supernatant, which was then analyzed with a mouse IFN-γ ELISA kit. IFN-γ capture dy (4 μg/mL) was coated onto an ELISA plate and incubated overnight at room temperature. The ELISA plate was washed three times and blocking was carried out with PBS containing 1% bovine serum albumin (BSA) for 1 hour. After washing the ELISA plate, the supernatant obtained by stimulating leukocytes was added thereto and incubated at room temperature for 2 hours. After washing the ELISA plate, biotinylated mouse IFN-γ ion antibody (400 ng/mL) was added thereto and incubated at room temperature for 2 hours.

After washing, streptavidin-HRP was added thereto and incubated again for 20 minutes. After the incubation, the ELISA plate was washed and d with a substrate solution at room temperature for 20 minutes. After stopping the reaction with a stop solution, the optical y was measured using an instrument at 450 nm.

Experimental Example 3: Measurement of gE antigen-specific IgG titer (gE specific IgG titer) The gE antigen-specific IgG titer was measured according to the method of mental Example 2-1, and the results of the experiment are summarized in Figs. 1 and 2.

As shown in Fig. 1, the production of gE antigen-specific IgG was greatly increased in the gE + SLA-SE group.

As shown in Fig. 2, the production of IgG2c was y increased in the gE + SLA-SE group and, especially, the tion of IgG2c was greatly increased as compared to that of IgG1. In Fig. 2, the bar graphs on the right side based on the horizontal axis “0” represent the production of IgG2c, and the bar graphs on the left side represent the production of IgG1.

Taking the results of Figs. 1 and 2 into consideration, it was confirmed that the use of a ition comprising gE, SLA, and SE significantly increased the overall IgG production and the production of IgG2c and, especially, greatly sed the production of IgG2c as compared to IgG1. These results means that the composition comprising gE, SLA, and SE, which satisfy both high IgG2c production and high IgG2c/IgG1 ratio, has the greatest effect of preventing herpes zoster.

Experimental Example 4: Measurement of gE or VZV antigen-specific cell mediated immune responses (ELISPOT assay) The gE protein, gE OLP, or VZV lysate-specific cell-mediated immune responses were measured according to the method of Experimental Example 2-2 (IFN-γ ELISPOT assay), and the experimental results are summarized in Figs. 3, 4, and 5.

As shown in Fig. 3, when the number of T cells that specifically reacted with the gE protein after the secondary immunization was confirmed by ELISPOT assay, it can be seen that the number of T cells that secrete IFN-γ, a representative Th1 cytokine, increased when the combination of gE, SLA, and SE (gE + SLA-SE) was used.

As shown in Fig. 4, when the number of T cells specific for the gE overlapping e after the secondary immunization was confirmed by ELISPOT assay, it can be seen that, similar to the results of Fig. 3, the composition comprising gE, SLA, and SE showed significantly increased antigen-specific CMI as compared to other compositions.

As shown in Fig. 5, when the number of T cells specific for the entire VZV induced by stimulation with VZV lysate after zation with the gE antigen was confirmed by ELISPOT assay, it was confirmed that the number of T cells that secrete or cytokines specific for the entire VZV as well as the gE antigen was increased by the combination of gE, SLA, and SE.

Taking the results of Figs. 3, 4, and 5 into consideration, it was med that the composition sing gE, SLA, and SE (gE + SLA-SE) can maximally increase VZV n-specific CMI as well as gE-specific CMI. This means that the gE + SLA-SE composition has a r effect of preventing herpes zoster than other compositions.

Experimental Example 5: Confirmation of the amount of cytokines secreted by antigen stimulation (CBA assay) The confirmation of the cytokines ed by the antigen stimulation (CBA assay) was performed according to the method of Experimental Example 2-3, and the experimental results are summarized in Fig. 6. In Fig. 6, IFN-g means IFN-γ.

As shown in Fig. 6, various Th cytokines were secreted by the composition comprising gE, SLA, and SE, and a representative Th1 cytokine, IFN-γ (the fourth from the front), was greatly increased, whereas the secretion of Th2 nes, IL-4 (the second from the front) and IL-6 (the third from the front), or the Th17 cytokine, IL-17A (the sixth from the front), was minimal. This means that the composition comprising gE, SLA, and SE has a greater effect of preventing herpes zoster than other compositions.

Experimental Example 6: Confirmation of the distribution of cytokinesecreting cells The assay for ming the distribution of the cells secreting cytokines by antigen stimulation (ICS assay) was performed according to the method of Experimental e 2-4, and the s of the ment are summarized in Fig. 7.

As shown in Fig. 7, in the case of the gE + SLA-SE group, the number of T cells that simultaneously secreted two or more ecific cytokines significantly increased (69%), indicating that high quality antigen-specific T cells were induced by the immunization with gE + SLA-SE. This means that the composition comprising gE, SLA, and SE has a greater effect of preventing herpes zoster than other compositions.

Experimental Example 7: Measurement of VZV antigen-specific IgG titers (Anti-VZV glycoprotein specific IgG titer) Various VZV antigen-specific IgG titers depending on the use of SLA-SE were determined in the same manner as in Experimental Example 2-1 except that any one of gE, gI, IE63, gB, gC, and gL was used as an antigen in the ary immunization and SLA-SE was used as an adjuvant, and the results are summarized in Figs. 8 and 9.

As shown in Fig. 8, the production of VZV antigen-specific IgG was significantly increased in the gE + SLA-SE group.

As shown in Fig. 9, the production of IgG2c was greatly increased in the gE + SLA-SE group and, ally, the production of IgG2c was significantly increased as compared to that of IgG1. In Fig. 9, the bar graphs on the right side based on the horizontal axis “0” represent the production of IgG2c, and the bar graphs on the left side represent the tion of IgG1.

Taking the results of Figs. 8 and 9 into eration, it was confirmed that the use of the composition comprising gE, SLA, and SE significantly increased the overall IgG production and the production of IgG2c, and, especially, greatly increased the production of IgG2c as compared to IgG1. This means that the composition comprising gE, SLA, and SE, which satisfy both high IgG2c tion and high IgG2c/IgG1 ratio, has the greatest effect of preventing herpes zoster. mental e 8: Confirmation of the amount of cytokines secreted by VZV antigen stimulation An experiment (CBA assay) for confirming the cytokines secreted by antigen stimulation, following the immunization, was med in the same manner as in Experimental Example 2-3 except that any one of gE, gI, IE63, gB, gC, and gL was used as an antigen in the secondary immunization and SLA-SE was used as an adjuvant, and the results are summarized in Fig. 10.

As shown in Fig. 10, in the case of the gE + SLA-SE group, the secretion amount of IFN-γ, a representative Th1 cytokine, was greatly increased. This means that cell-mediated immune response (CMI) was greatly activated, and that the composition comprising gE, SLA, and SE has a greater effect of preventing herpes zoster than other compositions.

Experimental Example 9: Confirmation of the distribution of cells secreting VZV antigen-specific cytokines An experiment (ICS assay) for confirming the distribution of T cells that secrete effector cytokines by antigen stimulation, ing the immunization, was performed in the same manner as in Experimental e 2-4 except that any one of gE, gI, IE63, gB, gC, and gL was used as an antigen in the secondary immunization and SLA-SE was used as an adjuvant, and the results are summarized in Fig. 11.

As shown in Fig. 11, in the case of the gE + SLA-SE group, the proportion of CD4+ T cells that e Th1 cytokines IFN-γ, IL-2, and TNF-α was significantly sed. This means that cell-mediated immune response (CMI) was greatly activated, and that the composition sing gE, SLA, and SE has a greater effect of preventing herpes zoster than other compositions.

Experimental e 10: Identification of VZV-specific immunogenicity depending on the combination of antigens In order to identify the VZV-specific immunogenicities induced when using the gE antigen alone and when using gE antigen in combination with another VZV antigen at the time of the ary immunization, VZV antigen-specific IgG titers and VZV antigen-specific T cell immune responses were confirmed according to the methods of Experimental Examples 2-1 and 2-2, and the experimental results are summarized in Fig. 12.

As shown in Fig. 12, the VZV-specific dy response and T cell response induced when the gE antigen was used alone were not significantly different from the VZV-specific immune responses d when gE antigen was used together with gI or IE63 antigen. This means that a composition comprising gE, SLA, and SE has a greater effect of preventing herpes zoster than other compositions.

Experimental Example 11: Measurement of gE antigen-specific IgG titers (gE specific IgG titer) The gE antigen-specific IgG titers depending on the use of various adjuvant were measured in the same manner as in Experimental Example 2-1 except that gE was used as an antigen and any one of SLA-SE, alum hydroxide, Addavax, Pam3CSK4, , MPL, lin, Imiquimod, and ODN1826 was used as an adjuvant in the secondary immunization, and the results are summarized in Figs. 13 and 14.

As shown in Fig. 13, the production of gE n-specific IgG was greatly increased in the gE + SLA-SE group, compared to other adjuvants groups.

As shown in Fig. 14, the production of IgG2c was greatly increased in the gE + SLA-SE group as compared to other adjuvants groups and, especially, the production of IgG2c was significantly sed as compared to that of IgG1. In Fig. 14, the bar graphs on the right side based on the horizontal axis “0” represent the production of IgG2c, and the bar graphs on the left side represent the production of IgG1.

Taking the results of Figs. 13 and 14 into eration, it was confirmed that the use of the composition comprising gE, SLA, and SE significantly increased the overall IgG production and the production of IgG2c. This means that the composition comprising gE, SLA, and SE has the greatest effect of preventing herpes zoster.

Experimental Example 12: Measurement of gE or VZV antigen-specific cell mediated immune responses (ELISPOT assay) The gE protein, gE OLP, or VZV -specific cell-mediated immune responses were measured according to the method of Experimental Example 2-2 (IFN-γ ELISPOT assay), and the experimental results are summarized in Figs. 15, 16, and 17.

As shown in Fig. 15, when the number of T cells specifically ding to the gE protein after the secondary immunization was confirmed by ELISPOT assay, it can be seen that the number of T cells that secrete IFN-γ, a representative Th1 cytokine, icantly increased in the gE + SLA-SE group as compared to other adjuvants groups.

As shown in Fig. 16, when the number of T cells specific for the gE overlapping peptide after the secondary immunization was confirmed by ELISPOT assay, it can be seen that, similar to the results of Fig. 15, the composition comprising gE, SLA, and SE showed icantly increased antigen-specific CMI as compared to other compositions.

As shown in Fig. 17, when the number of T cells specific for the entire VZV induced by stimulation with a VZV lysate after immunization with gE antigen was confirmed by ELISPOT assay, it was confirmed that the number of T cells that secrete IFN-γ specific for the entire VZV was sed by gE + SLA-SE.

Taking the results of Figs. 15, 16, and 17 into consideration, it was confirmed that the composition comprising gE, SLA, and SE can maximally increase VZV antigen-specific CMI as well as gE-specific CMI, compared to other compositions.

This means that the gE + SLA-SE composition has a greater effect of preventing herpes zoster than other compositions.

Experimental Example 13: Confirmation of IFN-γ cytokine secreted by gE antigen or VZV antigen stimulation (IFN-γ ELISA assay) An experiment (IFN-γ ELISA assay) for confirming IFN-γ cytokine secreted by gE n or gE OLP antigen stimulation was performed according to the method of Experimental Example 2-5, and the results are summarized in Figs. 18 and 19.

As shown in Fig. 18, when the ion amount of IFN-γ cytokine secreted by gE protein stimulation after the secondary immunization was observed, it was confirmed that the secretion amount of IFN-γ, a typical Th1 or cytokine, in the gE + SLA-SE group was increased as ed to other adjuvant groups.

As shown in Fig. 19, when the secretion amount of IFN-γ cytokine secreted by gE overlapping peptide stimulation after the ary immunization was observed, it was confirmed that the secretion amount of IFN-γ was increased by the combination of gE, SLA, and SE as compared to other nt groups.

Taking the results of Figs. 18 and 19 into consideration, the composition comprising gE, SLA, and SE increased the secretion amount of a representative Th1 or cytokine, ed to other adjuvant-containing compositions, and this means that the gE + SLA-SE composition has a greater effect of preventing herpes zoster than other compositions.

Experimental Example 14: Determination of the optimum amount of SLA-SE inducing VZV-specific immunogenicity Table 2 izes the experimental design for confirming the optimum amount of SLA-SE that can most effectively induce VZV antigen-specific cellmediated immune response (CMI). Live attenuated vaccine (LAV, 3,000 pfu) was subcutaneously injected once to female C57BL/6 mice and, on day 28 thereafter, the secondary immunization (immunization) was performed. On day 56 after the LAV priming, leukocytes were collected from spleen samples to confirm cell-mediated immune se (CMI) ic for VZV.

[Table 2] Secondary immunization Primary (Immunization) Day of Day of immunization spleen Group secondary (LAV sample Antigen Adjuvant immunization priming*) collection PBS PBS-only X X gE LAV gE (5㎍) X gE + SLA 0.2 ㎍ LAV gE (5㎍) SLA (0.2㎍) + SE (2%) gE + SLA 1 ㎍ LAV gE (5㎍) SLA (1㎍) + SE (2%) Day 28 Day 56 gE + SLA 2.5 ㎍ LAV gE (5㎍) SLA (2.5㎍) + SE (2%) gE + SLA 5 ㎍ LAV gE (5㎍) SLA (5㎍) + SE (2%) gE + SLA 7.5 ㎍ LAV gE (5㎍) SLA (7.5㎍) + SE (2%) gE + SLA 10 ㎍ LAV gE (5㎍) SLA (10㎍) + SE (2%) gE + SLA 15 ㎍ LAV gE (5㎍) SLA (15㎍) + SE (2%) gE + SLA 20 ㎍ LAV gE (5㎍) SLA (20㎍) + SE (2%) gE + SLA 22.5 ㎍ LAV gE (5㎍) SLA (22.5㎍) + SE (2%) *Primary immunization (LAV priming): Dose 100 μL/head. 3,000 pfu *Secondary immunization (Immunization): Dose 100 μL/head The gE antigen-specific cell-mediated immune response (IFN-γ ELISPOT assay) was measured according to the method of Experimental Example 2-2, and the results of the experiment are summarized in Fig. 20.

The VZV antigen-specific cell-mediated immune se (IFN-γ ELISPOT assay) was measured according to the method of Experimental Example 2-2, and the s of the experiment are summarized in Fig. 21.

Taking the results of Figs. 20 and 21 into consideration, it can be seen that the optimum amount of SLA for inducing VZV antigen-specific cell-mediated immune response is in the range of 7.5 μg to 20 μg.

The term ‘comprising’ as used in this specification and claims means sting at least in part of’. When interpreting statements in this specification and claims which includes the ising’, other features besides the features prefaced by this term in each statement can also be present. d terms such as ‘comprise’ and ‘comprised’ are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such al documents is not to be construed as an admission that such documents, or such sources of ation, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

Claims

[Claim 1] A e composition t chickenpox or herpes zoster comprising: 5 glycoprotein E of varicella-zoster virus; a glucopyranosyl lipid adjuvant of the following Formula 1; and squalene: [Formula 1] 10 wherein, R1, R3, R5 and R6 are each independently C10-C12 alkyl; and R2 and R4 are each independently C8-C10 alkyl.
[Claim 2] The vaccine composition of claim 1, wherein the glucopyranosyl lipid adjuvant is the one of Formula 1 wherein R1, R3, R5 and R6 are C11 alkyl. 15 [
Claim 3] The vaccine composition of claim 1, wherein the glucopyranosyl lipid adjuvant is the one of a 1 wherein R2 and R4 are C9 alkyl.
[Claim 4] The vaccine composition of claim 1, wherein the glucopyranosyl lipid adjuvant is contained in an amount of 7.5 μg to 20 μg in a single dose of the vaccine composition.
[Claim 5] The vaccine composition of claim 4, wherein the glucopyranosyl lipid adjuvant is contained in an amount of 9 μg to 18 μg in a single dose of the vaccine composition.
[Claim 6] The e composition of claim 1, wherein the squalene is 5 contained in an amount of 1% (v/v) to 7% (v/v) of the total vaccine composition.
[Claim 7] The e composition of claim 6, wherein the squalene is contained in an amount of 1% (v/v) to 4% (v/v) of the total vaccine composition.
[Claim 8] The vaccine composition of claim 1, wherein the glycoprotein E is contained in an amount of 5 μg to 100 μg in a single dose of the vaccine composition. 10
[Claim 9] The vaccine composition of any one of claims 1 to 8 substantially as herein described with reference to any example thereof and with or without nce to the accompanying drawings.