ABSTRACT
In 2005, a single-dose varicella vaccination was incorporated into the national immunization program in Korea. Although the Oka strain is the most commonly circulating Varicella zoster virus (VZV) genotype in Korea, a domestically manufactured vaccine based on the MAV strain is widely distributed in Korea. High vaccination coverage was achieved, but breakthrough infections were frequently reported. Therefore, in this study, we aimed to analyze the maintenance of immunity after single-dose vaccination and to compare the immunity provided by the MAV and Oka vaccines. Enzyme-linked immunosorbent assays of samples from 715 vaccinated Korean children were used to assess seropositivity rates against VZV. Additionally, fluorescent-antibody-to-membrane-antigen (FAMA) tests were performed in 35 individuals in the MAV vaccine group, and seropositivity rates against the Oka strain were determined. The progressive decrease of the seropositivity rate was demonstrated from ages 1 to 4 as follows: 65% in age 1, 59% in age 2, 53% in age 3, 49% in age 4. It then increased to 62% in age 5 and 70% in age 6. Both the MAV and Oka vaccine groups showed a progressive decrease of the seropositivity rate from the age of 1 to 4 years, but the MAV vaccine group had higher seropositivity rates. In FAMA test, the MAV group demonstrated 71% seropositivity against the Oka strain. This study indicates that immunity wanes after single-dose varicella vaccination and that the MAV vaccine is not inferior to the Oka vaccine in providing immunity against VZV. The MAV vaccine also induced cross-immunity against circulating Oka strain.
Introduction
Varicella zoster virus (VZV) is a double-stranded DNA virus, and a member of the Herpesviridae family and Alphaherpesvirinae subfamily. It affects only humans and primarily manifests as chicken pox. Varicella usually affects children aged 2–8 y.Citation1-3 Based on the universal nomenclature for VZV, VZV is categorized into 5 clades (clades 1–5). While clades 1, 3, and 5 are common in Europe, clade 2 is most widely distributed in Japan and East Asia.Citation4-6 To prevent varicella, the Oka strain varicella vaccine was developed in Japan in 1974, and the MAV strain varicella vaccine was developed in Korea in 1993. Both vaccines contain live attenuated viruses that cluster in clade 2.Citation2,5,7,8
The Korea Centers for Diseases Control and Prevention (KCDC) has conducted a nationwide surveillance of varicella since 2001. The annual incidence of varicella in Korea ranged from 6,000–14,000 during 2001–2004, and children aged 5–9 y were mainly affected.Citation9-11 The high incidence of varicella among children raised public health concern; hence, KCDC incorporated varicella vaccine in the National Immunization Program in 2005, recommending that children receive a single dose between the ages of 12 and 15 months.Citation7 No documentation regarding the distribution of the vaccine types is available; however, the domestically manufactured SuduVax (MAV strain, Green Cross, Yongin, Korea) presumably has more than 50% distribution. Vari-L (Oka strain, Changchun Institute of Biological Products, Changchun, China) is the second most commonly used vaccine. Although the MAV and Oka strains both cluster in clade 2, they are molecularly different and have different genome sequences.Citation8 In sequence analysis of VZV isolated in Korea, the Oka strain was the most common strain, indicating that the Oka vaccine may be the most useful vaccine in Korea.Citation10 Although the varicella vaccination coverage was 73.2% in 2008 and 92.9% in 2012, outbreaks of varicella were constantly reported, especially among children aged 4–5 y. The incidence of varicella in children aged 4–5 y has been increasing, composed more than 50% of total incidence from 2011 to 2014, and most cases were breakthrough infections.Citation9-11
Physicians and health authorities have several concerns regarding the single-dose vaccination regime and MAV vaccine. For example, it is unclear whether immunity is maintained after administration of the single-dose varicella vaccination. Additionally whether the MAV vaccine generates a level of immunity similar to that of the Oka vaccine is not yet known, and no reports have shown whether the MAV vaccine generates cross protection against the circulating Oka strain.
Therefore, to evaluate maintenance of immunity, we performed enzyme-linked immunosorbent assay (ELISA), measured antibody titers, and determined VZV seropositivity rates in vaccinated Korean children of different ages. We then compared the seropositivity rates of children in the MAV and Oka vaccine groups and conducted fluorescent-antibody-to-membrane-antigen (FAMA) tests to investigate cross immunity provided by the MAV vaccine.
Results
Total study population
The 715 study subjects included 381 boys (53%) and 334 girls (47%) with a mean age of 5.81 ± 3.97 y. The subjects were categorized into the following age groups: 1 year, 2 year, 3 year, 4 year, 5 year, 6 year, 7–10 year, and 11–15 y. The gender distribution of each age group is described in . ELISA was performed on the residual serum samples of the 715 subjects.
Table 1. Gender distribution, seropositivity rates and GMTs of anti-VZV IgG antibodies in each age group.
FAMA test population
FAMA test was performed on the residual serum samples of only 70 out of 715 subjects due to the time and labor intensity involved. Thirty-five received the MAV vaccine (SuduVax®, Green Cross, Yongin, Korea), and 35 received the Oka vaccine (Vari-L®, Changchun Institute of Biological Products, Changchun, China). The genders and age ranges of subjects in the FAMA test population are described in .
Serosurvey according to age group
The seropositivity rates of decreased as age increased (65% at 1 year; 59% at 2 years;53% at 3 years; 49% at 4 years; P < 0.001). However, the seropositivity rates increased beginning at 5 y of age (62% at 5 years; 70% at 6 years; 73% at 7–10 years; 80% at 11–15 years; P < 0.001). Similarly, the geometric mean titer (GMT) decreased in subjects ages 1–4 y and increased in subjects ages 5 to 11–15 y (P < 0.001; ).
Serosurvey according to vaccine type and age group
The seropositivity rates of the MAV vaccine group were as follows: 66% in the age group of 1 year; 61%, 2 year; 55%, 3 year; 51%, 4 year; 59%, 5 year; 69%, 6 y. The seropositivity rates of the Oka vaccine group were as follows: 64% in the age group of 1 year; 56%, 2 year; 52%, 3 year; 46%, 4 year; 65%, 5 year; 72%, 6 y. The MAV as well as Oka vaccine group showed a decrease in the seropositivity rate from the age of 1 to 4 years, and an increase in the age group of 5 and 6 y (P < 0.001). Similar pattern was observed in the age-specific geometric mean titer; both MAV and Oka vaccine groups showed a progressive decrease from the age of 1 to 4 y and an increase at the age of 5 and 6 y (P < 0.001). The subjects who received MAV vaccine had higher seropositivity rates and geometric mean titers than those who received Oka vaccines in age groups 1 to 4 years; however, the Oka vaccine group showed higher seropositivity rates and geometric mean titers in the subjects of age groups 5 and 6 y (P < 0.001) ().
Table 3. Seropositivity rates and GMTs of anti -VZV IgG antibodies in each vaccine group.
FAMA test: MAV vaccine group's seropositivity rate against oka strain
Both the MAV and Oka vaccine groups showed seropositivity rates of 71% for the Oka strain. The seropositivity of the MAV vaccine group for the Oka strain was not lower than that of the Oka vaccine group (P < 0.001) for the Oka strain. For the MAV strain, the MAV vaccine group exhibited a seropositivity rate of 74%, while the Oka vaccine group exhibited a seropositivity rate of 63%; this difference was statistically significant (P < 0.001; ).
Table 2. Seropositivity rates of each vaccine group determined by FAMA test.
Discussion
To prevent epidemics of varicella, a universal vaccination program has been implemented in many countries including the United States, Australia, Canada, Germany, Greece, Qatar, Saudi Arabia, Taiwan, Uruguay, Korea, and in parts of Italy and Spain.Citation3,12–14 Although this decreased the morbidity and mortality due to varicella, outbreaks were frequently reported among the vaccinated population.Citation15-17 The controversial causes of breakthrough infections were the age at vaccination, vaccine type, primary vaccine failure (not mounting protective immunity), or secondary vaccine failure (waning of protective immunity).Citation17,18 In response to breakthrough infections, a necessity to administer a second dose of the vaccine has emerged.Citation16,19,20 Watson reported that a second dose would provide higher antibody levels.Citation21 The Advisory Committee on Immunization Practices had recommended a second dose of varicella vaccine at 4–6 y of age (first dose at 12–15 months and second at 4–6 years) in the United States in 2006,Citation22 and the German Standing Committee on Vaccination had recommended a second dose varicella vaccine at 15–23 months of age (first dose at 11–14 months and second at 15–23 months, with a minimum 4-week interval) in Germany, in 2009.Citation23
KCDC had implemented a single dose varicella vaccination in the National Immunization Program in 2005. The vaccine was provided free of cost, and more than 90% of the children were immunized in 2012. However, high incidence of breakthrough infection was reported, especially in children aged 4 and 5 y.Citation7,9–11 No reports are available on the causes of breakthrough infections in Korea. Hence, this study was conducted to investigate whether secondary vaccine failure was observed in the vaccinated population. The results demonstrated a gradual decrease in seropositivity rates and geometric mean titers from the age of 1 to 4 y (), which indicated waning of immunity after varicella vaccination. The increased seropositivity rates and geometric mean titers observed in the 5, 6, 7–10, and 11–15 age groups were probably due to exposure to VZV. Since most Korean children start to attend kindergarten at the age of 5 or 6, the immune response may have been enhanced by encountering children infected with varicella and the circulating wild-type virus. The increased antibody titers do not persist after a single-dose varicella vaccination, and this may have contributed to the high incidence of breakthrough infections at the ages of 4 and 5 y. To reduce breakthrough infections, a 2-dose varicella vaccination regime should be considered in Korea.
In many countries in which the varicella vaccine is incorporated into the universal vaccination program, the Oka vaccine is the most commonly used. However, in Korea, the MAV vaccine, developed by a Korean pharmaceutical company, is widely distributed.Citation1,7 The high incidence of breakthrough infections in Korea raised concerns regarding the immune response generated by the MAV vaccine; moreover, data regarding the efficacy of this vaccine are limited. In this study, seropositivity rates and GMTs of the MAV vaccine group and Oka vaccine group were compared, and the results suggested that the MAV vaccine was not inferior to the Oka vaccine. Subsequent decrease in the seropositivity rates and geometric mean titers were shown in both the MAV and Oka vaccine groups from the age of 1 to 4 years; the MAV vaccine group demonstrated higher seropositivity rates and age-specific geometric mean titers than the Oka vaccine group (). In contrast, subjects in the Oka vaccine group exhibited higher seropositivity rates and geometric mean titers than subjects in the MAV vaccine group for children ages 5 and 6 y. Antibodies in children aged 5 and 6 y could develop due to vaccination and boosted by natural exposure; thus, comparison between the vaccine groups is unnecessary in the age groups higher than 5 y.
In FAMA tests, both the MAV and Oka vaccine groups exhibited 71% seropositivity rates against the Oka strain. This result suggested that the MAV vaccine provided cross protection against the Oka strain, which is the predominatly circulating VZV serotype in Korea. Moreover, the Oka vaccine group exhibited a seropositivity rate of 63% against the MAV strain, indicating cross protection against the MAV strain (). The Oka vaccine has shown antibody responses to VZV strains that cluster to clades 1, 3, and 5 circulating in Germany and to the MAV strain.Citation5
To interpret the FAMA test results, the degree of consistency in the seropositivity rates between the ELISA and FAMA tests was analyzed. Although the FAMA test results were inherently subjective due to the interpretation of examiners, high kappa (κ) values were observed, indicating a high degree of consistency in the seropositivity rates between the 2 tests. Differences in sensitivity, specificity and correlation rates between the ELISA and FAMA tests were also analyzed, and high concordance in the results and high correlation rates were demonstrated ().
In conclusion, in this study, waning of immunity observed in vaccinated Korean children, which may have contributed to the high incidence of breakthrough infections. Our findings showed that the MAV vaccine was not inferior to the Oka vaccine in terms of providing immunity against VZV; in fact, the MAV vaccine generated higher seropositivity rates and higher antibody titers. Moreover, the MAV vaccine induced cross immunity against the Oka strain. Thus, the MAV vaccine was immunogenic and was found to be suitable for regular use in Korea. In response to the waning of immunity, a second dose of varicella vaccination should be recommended in Korea. Further studies to evaluate the antibody response after the second dose and the optimal timing of second dose should be conducted.
Materials and methods
Total study population
We collected residual serum samples from 715 subjects who underwent laboratory tests for other viral infections in 3 pediatric referral hospitals affiliated to The Catholic University of Korea in Seoul, St. Paul's Hospital, St. Vincent's Hospital, and Incheon St. Mary's Hospital. Varicella vaccination information (date of administration and trademark of the vaccine) was retrieved from the National Immunization Registry System. Subjects ages 14 months to 15 y had received one dose of varicella vaccination at 12 months of age. Subjects ages 1–6 had received SuduVax (the MAV vaccine) or Vari-L (the Oka vaccine). Children older than 6 y had their varicella vaccination date included in the registry system, but no record of the type of vaccine. Individuals who had a history of varicella, were immunocompromised, or had recently received immunosuppressive therapy were excluded from the study. The study protocol was approved by the institutional review board of each hospital (XC14TIMI0037P, XC14TIMI0037V, and XC14TIMI0037O), and written informed consents were obtained from the parents or legal representatives of all the subjects.
FAMA test population
Of the 715 patients, 15, 10, and 10 subjects ages 1, 2, and 3 years, respectively, were randomly selected from each vaccine group, and FAMA tests were performed using the extra serum samples from these 70 subjects.
ELISA
The collected serum was stored at −70°C before testing. A commercial whole cell ELISA kit, Enzygnost anti-VZV/Ig G (Siemens Healthcare Diagnostics, Marburg, Germany) was used to quantify anti-VZV IgG. As per the manufacturer's instructions, serum samples with antibody titers less than 50 mIU/mL, 50–100 mIU/mL, and more than 100 mIU/mL were considered negative, equivocal, and positive, respectively. For serum samples with antibody titers of 50–100 mIU/mL, one retest was conducted and the sample was recategorized as positive or negative in accordance with the result of the retest. Serum samples with antibody titers of 50–100 mIU/mL for both tests were categorized as equivocal. The serologic test was performed at the Vaccine Bio Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Korea.
FAMA test
The FAMA test was performed using the Williams's method with a minor modification.Citation24 A few metabotropic glutamate (MGLu) human embryonic lung fibroblast cells were infected with VZV MAV strain and other were infected with the VZV Oka strain. MGLu human embryonic lung fibroblast cells displaying 70–80% of cytopathic effects were harvested and used as antigens in the FAMA test. Two serum samples were prepared from each subject to measure the seroposivity against same and different vaccine strains. Stored serum samples were inactivated at 56°C for 30 minutes, and diluted 2-fold from 1:2 to 1:128. The VZV MAV strain- or the VZV Oka strain-infected MGLu human embryonic lung fibroblast cells were added to aliquots and incubated for 30 minutes at 37°C. After incubation, secondary antibodies, fluorescein-5-isothiocyanate (FITC) conjugated anti-human IgG antibodies (polyclonal rabbit anti-human IgG/FITC, Dako Denmark A/S, Denmark), were added to each well and incubated for 1 hour. The incubated cells were examined for ring structure around their surfaces, and graded from 1+ to 4+ depending on the intensity of the ring structure independently by 2 people using a fluorescent microscope. Serum from a child with no history of varicella and varicella vaccination was used as a negative control and anti-VZV antibodies (National Institute for Biological Standards and Control, UK) were used as positive control. Serum with grade 2 or higher fluorescence was regarded as seropositive. The subjects' seropositivity against VZV MAV or VZV Oka strain was confined as grade 2 or higher fluorescence in serum with 1:4 or greater dilution.Citation24 The FAMA test was performed at the Vaccine Bio Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Statistical analysis
The seropositivity rates against VZV were calculated with 95% confidence interval. The chi-square test was used for comparison between the age groups and vaccine groups. To assess the degree of agreement in the seropositivity rate between ELISA results and FAMA test results, chi-square tests were used, and kappa (κ) values were calculated with 95% confidence interval. The consistency was considered poor, fair, moderate, substantial, and good for κ values of ≤0.20, ≥0.21 and ≤0.40, ≥0.41 and ≤0.60, ≥0.61 and ≤0.80, and >0.80, respectively.Citation25 To analyze the correlations between the ELISA and FAMA test results, we applied the ELISA results as a reference, and to compute the FAMA test results, the number of concordant samples was divided by the total number of samples included. The differences in sensitivity, specificity and correlation rates were calculated with a 95% confidence interval using Fischer's exact test. The Statistical Package for the Social Sciences, software version 20.0 (IBM Co., Armonk, NY, USA) was used for data analysis, and P< 0.05 was regarded as statistically significant.
Disclosure of potential conflicts of interest
There are no potential conflicts of interest to disclose.
Funding
This study was supported by a research grant from the Korean Society of Pediatric Infectious Diseases. The funding institution did not have a role in the study design, data collection, data analysis, writing of the manuscript or submission of the manuscript for publication.
Related Research Data
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