Background
Varicella-zoster virus (VZV) is one of the most common pathogens that affects humans [
1,
2]. The virus causes chickenpox with its initial infection and herpes zoster (shingles, or simply zoster) after later reactivation in the body, induced by waning VZV-specific T cell response [
1,
2]. Live-attenuated VZV vaccines have been developed and have been used for decades to prevent chickenpox [
3‐
6]. A zoster vaccine, a highly concentrated form of VZV vaccine [
7‐
9], has recently become available [
10].
T cell-mediated immunity is involved in protection of chickenpox as well as zoster [
11]. In immune compromising conditions such as aging, the reduction of VZV-specific immune memory CD4+ T cells has been observed. The impaired immunity to VZV can lead to the reactivation of the initial infectious virus, which can be followed by a zoster outbreak [
12‐
14]. In addition, T cell immunity is also crucial in primary VZV infection [
15]. Children suffering from immune deficiencies with cellular immunity can be more severe complications by varicella infection, not likely with humoral immunity such as agammaglobulinemia [
16‐
19].
There are 5 major clades and two provisional clades (VI and VII) of VZV that have been identified [
20,
21]. Several studies have demonstrated a distinctive geographic distribution of the 5 major VZV genotypes [
22,
23]: Clades 1 and 3 are common in Europe and North America; clade 2 has been found in Asia; clade 5 is common in India and Africa; and clade 4 is present in Europe and other areas. The Oka strain, the vaccine strain used in live-attenuated VZV vaccine and zoster vaccine, was isolated in Japan and belong to clade 2 along with most other virus isolates from Japanese and Korean [
24‐
26].
Another VZV vaccine strain, designated as MAV/06, was developed by attenuation of a wild-type isolate obtained from a Korean patient suffered with chickenpox in Seoul [
3]. MAV/06 vaccine (Suduvax® as its trade name) has been commercialized in Korea since 1994 and globally since 1998. MAV/06 strain is genetically similar to Oka strain and is also clustered as clade 2 [
25]. Although the MAV/06 strain has been used to produce VZV vaccines for more than 20 years, few studies have compared the characteristics of the immunological responses among different VZV strains.
A new MAV/06-based vaccine, BARYCELA®, has been developed and was approved in early 2020 by the Ministry of Food and Drug Safety in Korea. We evaluated the cross-reactivity of antibodies induced by the MAV/06 virus with VZV isolates of various genotypes. In addition, we compared both the humoral and cellular immunogenicity generated by MAV/06 vaccine to those of other VZV vaccines, including those derived from the Oka and MAV/06 viral strains.
Methods
Viruses and cells
MRC-5 cells were purchased from ECACC (European Collection of Authenticated Cell Cultures) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco) and sodium pyruvate. VZV YC strains and Jena strains were kindly provided from Dr. Hosun Park (Youngnam University, Korea) and Dr. Andreas Sauerbrei (Jena University, Germany), respectively. The 8 VZV YC isolates were clade 2 genotypes [
27] and the 6 Jena VZV isolates were clustered into the major VZV clades 1, 3 and 5 [
21] (Table
1). VZV viruses were propagated in MRC-5 cells, and titer for infectious viruses were determined with plaque assay on MRC-5 cells.
Table 1
Virus isolates used in this study
YC01 | Zoster | 2 | 19 June 2012 | M, 40 | KJ767491.1 | |
YC02 | Zoster | 2 | 18 July 2012 | M, 3 | KJ767492.1 | |
YC03 | Zoster | 2 | 07 Aug. 2012 | F, 8 | KJ808816.1 | |
YC04 | Zoster | 2 | 02 May 2013 | F, 76 | – | |
YC05 | Zoster | 2 | 01 July 2013 | F, 52 | – | |
YC06 | Zoster | 2 | 09 July 2013 | M, 73 | – | |
YC07 | Zoster | 2 | 22 July 2013 | M, 80 | – | |
YC08 | Zoster | 2 | 24 July 2013 | M, 66 | – | |
Jena 4 (432/2008) | Zoster | 1 | 07 Feb.2008 | M, 57 | JN704695.1 | |
Jena 6 (1883/2007) | Varicella | 1 | 07 Nov.2007 | F, 4 | JN704694.1 | |
Jena 12 (2308/2003) | Varicella | 3 | 22 Nov.2003 | F, 5 | JN704699.1 | |
Jena 16 (52/2007) | Zoster | 3 | 08 Jan.2007 | M, 18 | JN704701.1 | |
Jena 26 (446/2007) | Varicella | 5 | 15 Mar.2007 | M, 1 | JN704707.1 | |
MAV/06 | Varicella | 2 | 1989 | M, 3 | JF306641.2 | Lee et al. (2011) |
Animal experiment
Male Hartley guinea pigs weighing 200–250 g were purchased from Japan SLC (Japan). Thirty guinea pigs were injected subcutaneously in the scruff of the neck with the MAV/06 vaccine containing approximately 45,000 plaque forming units (PFU) 2 times with a 3-week interval between injections. Blood was collected by cardiac puncture three weeks after second immunization. Sera were pooled and stored at − 70 °C until tested for cross-reactivity.
Five to 6-week-old female C57BL/6 mice were purchased from Orient Bio (Korea). 4 heads of C57BL/6 female mice were used for the mouse experiment. MAV/06 vaccines were prepared with low (~ 600 PFU/0.1 mL), medium (~ 2000 PFU/0.1 mL), and high (~ 4000 PFU/0.1 mL) viral titers. Commercialized vaccines were used as positive control: Suduvax® (Vx1) at a minimum of 1400 PFU/0.5 mL and Zostavax® (Vx2) at a minimum of 19,400 PFU/0.5 mL. Animals were immunized intramuscularly with 0.1 mL of vaccine formulations in thigh muscle of the hind limb 2 times with a 3-week interval between injections after random allocation. Animals were sacrificed two weeks after the second immunization. The sera and spleen were collected from the sacrificed animals.
The sample size was determined by previous experiments, and in previous experiments it was determined that this sample would be appropriate. All animals were anesthetized with isoflurane using closed chambers. Animals were monitored every day and 20% of weight loss was considered for humane endpoints. According to the internal guideline, 20% of weight loss was considered for the criteria. During this study, there were no cases of animals that died.
All experiments including the procedures used and the care of animals were approved by the Institutional Animal Care and Use Committee in GC Biopharma Corp (approval No. 2018002). In addition, we confirm that all methods were carried out in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent Experiments provided by the IACUC (Institutional Animal Care and Use Committee), and all methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.
Plaque reduction neutralization test
Plaque reduction neutralization tests (PRNT
50) were performed as previously described [
28]. Briefly, twofold dilutions of heat-inactivated guinea pig sera, from fourfold through 128-fold, were each mixed with an equal volume of diluted VZV isolates at 100 PFU/0.1 mL. The mixtures were incubated for 1 h at 37 °C. Two hundred microliters of the mixture were added to 6 × 10
5 MRC-5 cells seeded in wells of 6-well culture plates. The plates were incubated for 60 min at 37 °C with agitation. DMEM with 2% FBS were overlaid and incubated for 5 days. After removing the overlays, a 0.5% crystal violet solution in 25% methanol was added to the cells. The number of stained plaques were counted. PRNT
50 titers were determined as the reciprocal of the serum dilution that demonstrated a 50% reduction in plaque counts.
Fluorescent antibody to membrane antigen (FAMA) test
FAMA assay was performed with slight modification from previously described methods [
29]. Briefly, MRC-5 cells were grown to confluency in 175 T flasks and infected with VZV isolates at 0.003 m.o.i. When cytopathic effects were observed in ~ 50- 60% of the cells, the cells were washed three times with PBS. Infected cells were detached from the flasks and incubated with serially-diluted sera at room temperature for 30 min. After washing with PBS, cell preparations were incubated with Alexa Fluor
®488 goat anti-guinea pig IgG (Invitrogen) and mounted on slides. After overlaying with mounting media containing DAPI, the slide was covered with a cover-glass and observed under a fluorescence microscope (Nikon). Titers were defined as the reciprocal of the highest dilution causing bright fluorescent ring around the surface of cells. Titers of ≥ 1:4 were considered positive. Pooled sera from MAV/06 immunized guinea pigs were pre-treated with MRC-5 cells before incubation with virus-infected cells in order to remove immune responses to cellular debris within vaccine ingredients. Non-specific reactions to mock-infected cells were confirmed using both vaccine groups.
Glycoprotein enzyme-linked immunosorbent assay (gpELISA)
Microplate (Corning) were coated with 1 μg/mL of VZV glycoprotein (QED BIO) in PBS at 4 °C overnight. Plates were washed with wash buffer (0.05% Tween in PBS) and the wells blocked with ELISA assay buffer (1% BSA, 0.1% Tween in PBS) for 2 h. A thousand-fold dilution of sera from immunized mice were added to the wells and incubated for 2 h. Plates were washed and incubated with HRP-conjugated secondary antibody mouse IgG (Southern Biotech) for 1 h. After the final wash, TMB substrate (KPL) was added to each well and incubated for 15 min. The reactions were stopped with TMB stop solution (KPL) and the 450 nm absorbance determined using a spectrophotometer (Molecular Devices, USA) and data were analyzed using SoftMaxPro (Molecular Devices, USA).
Interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assay
Spleen cells were isolated from the immunized mice and suspended at a final concentration of 5 × 106 per mL in RPMI 1640 medium (Gibco) supplemented with 10% (vol/vol) heat-inactivated FBS (Gibco), antibiotic–antimycotic solution (anti-anti; Gibco), 2 mM Glutamax (Gibco), 1 mM sodium pyruvate (Gibco), and 55 μM 2-mercaptoethanol (Gibco). Multiscreen-HA filter plates were coated with anti-mouse IFN- γ capture antibody (R&D systems) at 4 °C overnight and blocked with RPMI 1640 medium with 10% FBS. Cells were added with intact virions (VZV lysate; Mycrobix), recombinant glycoprotein E (gE), glycoprotein I (gI) (gE and gI from Peptron), IE63 (Genscript), and overlapped peptide (IE63 OLP from JPT), and incubated overnight at 37 °C. The plates were washed with PBS (3x) and biotinylated anti-mouse IFN- γ detection antibody (R&D systems) was added. After washing, streptavidin was conjugated and 3-amino-9-ethylcarbazole (AEC; BD) were added to the plates. The reaction was stopped by rinsing with tap water. Spot-forming units (SFU) were read using ELISPOT reader (Autoimmune Diagnnostika). Adjusted SFU were obtained by subtraction of mock-stimulated counts (mock lysate for VZV lysates, medium for recombinant protein, and DMSO for OLP).
Cytokine bead array (CBA)
Splenocytes were stimulated with intact virions at 37 °C for three days. Supernatants were harvested and subjected to cytometric bead array (CBA, BD Biosciences) analysis to detect levels of Th1/Th2/Th17 cytokines, tumor necrosis factor alpha (TNF-α), IFN-γ, interleukin 2 (IL-2), IL-4, IL-6 IL-10, IL-17A.
Statistical analysis
All results are expressed as the means ± standard errors of the means and compared by one-way ANOVA. Statistical analysis was performed using GraphPad Prism™ software (GraphPad, San Diego, CA, USA), and statistical significance was defined as a p value (*p < 0.05, **p < 0.01, ***p < 0.001).
Discussion
In this study, we confirmed that the newly developed MAV/06 vaccine triggers humoral immunity by production of antibodies exhibiting cross-reactivity to various VZV virus clade antigens from clade 1, 2, 3, 5. Furthermore, new MAV/06 vaccine induces cell-mediated immunity through Th1 cell response.
In general, vaccines are not administered in different titer as per body weight. The injection volume is the same not only for the adults but also for the infants. Also, for the animal models bigger than rodents, generally the same volume (human dose, 0.5 mL) is administered. However, in the mice model, the antibody titer could be saturated in human dose. For this reason, the injection volume was determined as 5 times lower than the human dose in all mice studies. In addition, the viral titer(PFU/0.5 mL) could be decreased in a time-dependent manner, since VZV vaccines are live-attenuated vaccine. Therefore, the immune responses in various viral titers were evaluated to address the broad range of human dose that could be administered to the subjects.
Currently, there are 7 different clades of VZV worldwide and most of the varicella vaccine viruses come from 5 types of clades [
20,
21]. Commercially available VZV vaccines are manufactured from Oka strain and MAV/06 strain from clade 2. Although it is common that confirming reactivity of antibodies induced by vaccination and antigens which is same as the vaccine when conducting PRNT
50 or FAMA tests, few studies have shown cross-reactivity of vaccine-induced antibodies among different virus clades. Cross-reactivity of antibodies induced by vaccination against various clades of VZV is important to estimate the prophylactic effectiveness against wild-type VZV in the field.
From the PRNT
50 and FAMA studies, we demonstrated that the newly developed MAV/06 vaccine triggers humoral immunity by the production of antibodies exhibiting cross-reactivity to viruses from VZV clades 1, 2, 3, and 5. Even though the FAMA assay was conducted once, but the reproducibility was identified by the PRNT
50 results, which was repeated in triplicate. A study conducted with Oka strain illustrated that Oka vaccine induced antibody response to a wild-type VZV that prevailed in Germany [
36]. The extent of antibody reactivity to clade 1, 2, 3, and 5 viruses was similar to that of the MAV/06 virus. These results are similar to the results from the study conducted in Germany (Fig.
1) [
36]. This study was conducted with the MAV/06 strain that used both in Suduvax and a newly developed Varicella vaccine (Trade name: BARYCELA inj. Reference;
https://nedrug.mfds.go.kr/pbp/CCBBB01/getItemDetail?itemSeq=202001448) with upgrade formulation by GC Biopharma Corp. It is the first investigation demonstrating that MAV/06 vaccination induced antibody responses against various VZV clades worldwide as well as clade 2 viruses that prevail in Asian countries, including the Republic of Korea and Japan.
Live-attenuated vaccines exhibit immunological strength that enables antibody induction and other immune reaction such as T cell immune response [
32]. Previous studies of VZV vaccines implied the importance of T cell immunity after vaccination [
1,
2,
9,
11‐
14,
33]. The occurrence of herpes zoster and the observed concomitant reduction of cell-mediated immunity (CMI) in individuals with high titers of VZV antibody indicate that the reduction in CMI is the primary cause of herpes zoster. CMI evaluation were performed using the VZV vaccine developed from the Oka strain and reported CMI induction by the VZV vaccine [
33,
37]. We investigated MAV/06 vaccine-induced CMI and humoral responses in mice.
Test results of CBA assay showed high amount of secreted IL-10 in the high viral titers compared to commercialized high-dose vaccine, Vx2. IL-10 is known as a cytokine with multiple, pleiotropic, effects in immune-regulation and inflammation. In addition, it also enhances B cell survival, proliferation, and antibody production. For this reason, highly secreted IL-10 level could induce B cell immune responses. However, the appropriate vaccine titers have to be determined by considering various factors such as antibody response, cell-mediated immunity and safety via clinical trials.
We demonstrated the vaccine evoked VZV-specific B cell and T cell immune responses that were comparable to that of the commercial vaccines in mice model. In addition, the new MAV/06 vaccine-induced T cell response was found to be mediated by Th1 rather than Th2 or Th17 cell responses, implying the MAV/06 vaccine functions via intracellular virus clearance as have been shown for other previous studies [
38]. These results indicate that the live-attenuated MAV/06 vaccine induces both humoral and cellular immunity in live organism.
Our study provided the explanation to the previously reported vaccine efficacy via immunological characterization of MAV/06 strain and will contribute to future studies for vaccine efficacy or effectiveness after new MAV/06 vaccine.
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