Analysis of sero-epidemiological characteristics of varicella in healthy children in Wuxi, China

ABSTRACT

The aim of this study was to evaluate seroepidemiology of varicella in Wuxi, China, after two doses of immunization of varicella vaccine (VarV) for better control of varicella transmission. Participants were selected randomly by multistage stratified sampling. The positive rate of antibody and geometric antibody titer (GMC) was determined with enzyme-linked immunosorbent assays. Logistic and linear regressions were used to analyze the correlations between antibody seropositivity, GMCs, and corresponding factors. Serum samples (n = 697) were obtained from healthy children <7 years-of-age. Seropositivity and GMC were 67.84% and 190.97 mIU/mL, respectively, in subjects receiving two doses of VarV. Multivariate analysis revealed that both the rate of varicella-zoster virus immunoglobulin G seropositivity and GMC in vaccinated subjects were higher compared to those who had not been vaccinated. The strongest overall response was observed in subjects with two doses of VarV (4.055, 95% CI: 2.774 ~ 5.928). In 470 subjects with a history of VarV application, the antibody GMC value was reduced 4 years after vaccination. Two-dose vaccination with VarV was an effective prevention strategy varicella and should be included in a national immunization program.

Introduction

Varicella is a highly contagious disease that is transmitted through the respiratory tract. It is caused by the varicella-zoster virus (VZV).¹ Children infected with VZV can experience serious complications, such as soft tissue infections, pneumonia, and central nervous system diseases. Varicella live attenuated vaccine (VarV) is efficient and reliable in reducing varicella incidence and outbreak. In 2014, the World Health Organization recommended that countries incorporate VarV into their routine vaccination programs to prevent VZV transmission and varicella outbreak.² Internationally, the recommended routine two-dose schedule has been implemented in many countries, including the United States, Germany, and Greece.^3,4 Although VarV has not been included in the national immunization program (NIP) in China, multiple areas from the northern to southern regions of the country have introduced a two-dose VarV inoculation schedule; the protective immunity of this schedule has been verified.⁵ The city of Wuxi incorporated a free two-dose VarV immunization in the local routine vaccination programs beginning in December 2018. The first dose is delivered at 12–18 months-of-age, with the second injection at 4 years-of-age. However, varicella outbreaks have continued to occur in schools. Surveillance data has revealed relatively high rates of VarV vaccination in Wuxi.⁶ Yet, rapid improvements in the number of reported cases and incidence of varicella have not been realized, and breakthrough infections have occurred occasionally.

To improve the outcomes of VarV vaccination, thorough knowledge of the immunity level of the population that is susceptible to varicella is needed. The Jiangsu Provincial Center for Disease Control and Prevention performed a legally authorized serum immunological local surveillance survey by stratified cluster random sampling in the southern, central, and northern regions of the province. Based on this surveillance data, the present study sought to discover the immunoglobulin G (IgG) levels of varicella antibodies in children <7 years of age in Wuxi and determine the levels of immune responses and protective immunity induced by administration of the two doses of VarV. The data provided scientific evidence that will be valuable in developing and optimizing a national VZV immunization strategy.

Materials and methods

Varicella serological surveillance

Since 2018, the Jiangsu Provincial Center for Disease Control and Prevention has performed serology surveillance of vaccine-preventable diseases by multistage stratified random sampling, with the goal of strengthening vaccine programs. As a research site located in the southern region of Jiangsu province, Wuxi was required to recruit eligible healthy people and collected blood samples to explore the immune against VZV. Based on the surveillance scheme, we first included districts randomly. Subjects were recruited by random selection in each district considering the distribution of demographic characteristics, such as sex and transient or permanent residence.

Study population and data collection

The inclusion criteria were: 1) age <7 years with informed consent from parents/legal guardians for blood collection, 2) residence in the region for at least 3 months, 3) lack of acute or chronic diseases, and 4) complete socio-demographic information and vaccination history. Participants whose parents/guardians refused the collection of venous blood or who were not in good physical health were excluded. Children were recruited and sampled in 2018, 2019, and 2021. Finally, 697 serum samples were obtained. Basic information and immunization history obtained were allowed from parents or legal guardians.

Vaccine

A live attenuated varicella vaccine (Oka strain, not less than 3.3 lg PFU/dose) was recommended in routine vaccination programs in Wuxi and provided by 3 domestic pharmaceutical companies (Shanghai Institute of Biological Products Co., Ltd., Shanghai, China, Sinovac (Dalian) Vaccine Technology Co., Ltd., Dalian, China, and Changchun BCHT Biotechnology Co., Ltd., Changchun, China). The vaccines were derived from the original Japanese vOka strain in the 1970s,⁷ and made into lyophilized vaccines.

The transportation and storage of vaccine were well controlled, as cold chain management policy was mandatory in Wuxi, and vaccination records were maintained in the Jiangsu Province Vaccination Integrated Service Management Information System, a traceability system where each varicella was assigned a unique identifier, in order to achieve the network management of vaccination.

Ethical approval

This investigation was performed by public health agencies as part of their legally authorized mandate and was therefore considered minimal risk research. No confidential information was involved, and the study was exempt from ethical approval by the Medical Ethics Committee of the Jiangsu Provincial Center for Disease Control and Prevention. We obtained permission for data usage from the Jiangsu Provincial Center for Disease Control and Prevention. Written informed consent was obtained from the parents/guardians of the children before enrollment.

Laboratory assays

Each collected serum sample was centrifuged, stored at −70°C, and transferred to the Jiangsu Provincial Center for Disease Control and Prevention. This facility was responsible for the laboratory assay of all surveillance sites. Antibody determination was by glycoprotein enzyme-linked immunosorbent assay (gp ELISA) using the Serion ELISA classic Varicella-Zoster Virus IgG kit (Institut Virion\Serion GmbH, Würzburg, Germany). According to the manufacturer’s protocol, quantitation of antibodies was determined by optical density measurements using software from Serion, and expressed as million international units (mIU)/mL. An antibody concentration exceeding the high cutoff value was considered positive, and a concentration below the low cutoff value was negative. Equivocal samples with a concentration between low-high cutoff value were retested; those with a result still in the gray zone were finally categorized as negative. In addition, ELISA kits distributed by Serion were used by the same staff members at the laboratory of the Department of the Expanded Program on Immunization at the Jiangsu Provincial Center for Disease Control and Prevention annually. The researchers ensured that specimen collection, storage, transfer, and complete laboratory detection complied with relevant biosafety requirements, with strict quality control.

Statistical analyses

Pearson’s chi-square test was applied to compare the differences of antibody seropositivity within groups. Statistical significance for antibody titers (geometric mean concentrations, GMCs) between groups was examined by a two independent variable t-test or one-way analysis of variance. Multiple comparisons were performed using the SNK-q test. Logistic regression was used to analyze the associations between the antibody seropositivity and related factors. Linear regression was performed to explore the relationship between GMCs and vaccination history. All statistical analyses were performed using SPSS, version 18.0 (SPSS Inc., Chicago, IL, USA). Two-tailed p < .05 was considered statistically significant.

Results

Seroepidemiology of VZV IgG

Of the 697 serum samples from healthy children <7 years of age (373 males, 324 females), 470 subjects had a history of VarV vaccination. The VZV IgG seropositivity was 54.09%, and the GMC for VZV IgG was 117.03 mIU/mL. Univariate analysis revealed a significantly higher positive rate of varicella antibodies in females (49.87% vs. 58.95%), ≥4 years of age (42.76% vs. 62.16%), and urban residence (44.38% vs. 62.33%). The antibody positive rate of antibody was highest in subjects who had been immunized with two doses of VarV (67.84%, 95% CI: 62.37 − 73.32%) compared to one dose or no inoculation. The differences of GMC for VZV IgG between sex, district, and immunization history showed similar results. The GMC for VZV IgG was 190.97 mIU/mL for subjects receiving two doses of VarV, which was higher than the GMC for subjects who had never been immunized. Significantly higher GMCs were evident in females and urban residents. Serum antibody titer was much higher in children aged ≥4 years (158.49 mIU/mL) compared with those 1 − 2 years of age (Table 1).

Table 1.

Analysis of IgG antibody levels of varicella in children aged<7 years in Wuxi via univariate analysis (n = 697).

Characteristics		Total (n)	N	Seropositivity (%, 95%CI)	GMC(mIU/ml, 95%CI)
District
	urban	377	235	62.33(57.42–67.25)	150.08(128.43–175.38)
	suburb	320	142	44.38(38.90–49.85)	87.32(74.27–102.65)
$x^2$/t value				22.481	4.725
P value				<0.001	<0.001
Sex
	male	373	186	49.87(44.77–54.96)	100.82(86.08–118.08)
	female	324	191	58.95(53.57–64.34)	138.97(118.08–163.55)
$x^2$/t value				5.763	2.773
P value				0.016	0.006
Age(years)
	<4			42.76(37.03–48.49)	77.62(64.57–93.33)
	≥4			62.16(57.43–66.89)	158.49(138.04–177.83)
$x^2$/t value				25.674	6.113
P value				<0.001	<0.001
Immunization history
	no	227	78	34.36(28.14–40.59)	51.37(41.93–62.93)
	1 dose of vaccination	187	107	57.22(50.06–64.38)	151.58(120.65–190.46)
	2 dose of vaccination	283	192	67.84(62.37–73.32)	190.97(166.53–219.01)
$x^2$/t value				57.877	58.196
P value				<0.001	<0.001
Total		697	377	54.09(50.38–57.80)	117.03(104.45–131.16)

Multivariate analysis associations of seroepidemiology of VZV IgG and related variables

Multivariate analysis findings were the same as the univariate test findings. Rates of VZV IgG seropositivity and GMCs were higher in females and urban subjects (p < .05). Compared with subjects not inoculated with VarV, a stronger overall response (OR) was observed in subjects who received one dose (OR = 3.022, 95% CI: 1.994 ~ 4.578) or two doses (OR = 4.055, 95% CI: 2.774 ~ 5.928) (Table 2).

Table 2.

Associations of seroepidemiology of VZV IgG and related variables via multivariate analysis (n = 697).

The independent variables	Seropositivity of VZV IgG		GMCs for VZV IgG
	OR(95% CI)	P-values	β	P-values
District			−0.222	<0.001
urban	1
suburb	0.435(0.314 ~ 0.602)	<0.001
Sex			0.118	0.012
male	1
female	1.453(1.055 ~ 2.003)	0.022
Immunization history			0.270	<0.001
no	1
1 dose of vaccination	3.022(1.994 ~ 4.579)	<0.001
2 dose of vaccination	4.055(2.774 ~ 5.928)	<0.001

Immunological response in children previously inoculated with VarV

We further studied the relationship between GMCs, seropositivity, and time since vaccination prior to the investigation of children vaccinated with VarV. The analysis included 470 subjects with a history of VarV vaccination. The rate (percentage) of VZV IgG seropositivity was 63.62%, and the GMC for VZV IgG was 174.18 mIU/mL. The antibody GMC value was reduced to 87.38 mIU/mL after 4 years since vaccination, suggesting that GMC had a tendency to wane with time (Table 3).

Table 3.

Relationship between GMCs, seropositivity, and time since vaccination before the sero-survey in children who had an immunization history of VarV (n = 470).

Years after vaccination	Sample size	N	Seropositivity(%, 95%CI)	P-value	GMC(mIU/ml, 95%CI)	P-value
<1	120	71	59.17(50.24–68.09)	0.088	151.54(116.15–197.72)	0.017
1-	191	134	70.16(63.61–76.70)		213.64(177.40–257.29)
2-	96	59	61.46(51.55–71.37)		169.03(130.42–219.07)
3-	39	24	61.54(45.56-77.52)		162.14(108.00–243.40)
4-	24	11	45.83(24.34–67.33)		87.38(49.76–153.46)
Total	470	299	63.62(59.25–67.98)		174.18(154.17–196.79)

Discussion

This cross-sectional serosurveillance study assessed the concentration of varicella antibody in children <7 years of age in Wuxi. We previously found that higher two-dose varicella vaccination rate could help reduce the risk of varicella, but that the immunization effectiveness of VarV via the two-dose regimen was limited.⁶ Serologic evidence is needed to improve the immune strategy of VarV. The results will provide comprehensive insights into immune states of varicella in children <7 years of age receiving the two-dose regimen.

The rate of VZV IgG seropositivity was 67.84%, 57.22%, and 34.36% for the two-dose, single-dose, and zero-dose applications of VarV. These findings were similar to previous reports.^1,5 In contrast, an observational study in Suzhou, China, involving children aged 3 − 6 years reported seropositivity percentages of 95.31% and 86.89% for children with and without the strategy, respectively.⁸ Wu et al. reported an overall seropositivity rate of 70.3% in those vaccinated for varicella in Changzhou, with positivity rates after one and two doses of varicella vaccine of 57.7% and 84.2%, respectively.⁷ Moreover, 80.3% of the VZV seropositivity rate was found in Germany⁹ and 93.0% in France.¹⁰ The relatively lower levels of seropositivity of two-dose vaccination may be related to several key factors, including variations in vaccine types, vaccination rates, time since immunization, serologic assays, and different demographic and regional distributions. Firstly, the type of vaccine used in the study was a monovalent live attenuated vaccine, but European countries utilized a tetravalent combination vaccine and immune responses varied. Secondly, the extent of vaccination coverage and the time on blood collection were critical to seropositivity,¹¹ it has been only 3 years since the addition of the two-dose regimen, and the VarV coverage in Wuxi might not yet meet the 80% threshold recommended by the WHO to achieve herd immunity,^12,13 which might obscure the vaccine’s effectiveness. In addition, the report that VZV genotypes demonstrate a specific geographical distribution¹⁴ highlights the need for data concerning the virus genotype to explain the lower conversion rate in Wuxi. Finally, the current sample size was small, and ELISA kits had lower sensitivity compared to the fluorescent-antibody-to-membrane-antigen (FAMA) test in determining protective immunity against VZV.¹⁵ Serological evidence for the assessment of VarV may bias the results. Despite the low seropositivity, the cellular immunity assessment is also essential, the evaluation of humoral immunity also provides valuable insights into the vaccine’s efficacy, and long-term efforts of vaccination has become a key public health concern in Wuxi.

In the present study, GMC was highest in children vaccinated with two doses (190.97 mU/mL). Children who were not immunized displayed the lowest antibody concentration (51.37 mU/mL). The latter level was not above the protective threshold, indicating that seropositivity rates and the level of VZV antibody titer gradually increased with multiple vaccinations. Notably, multivariate analysis revealed statistically significant differences in both seropositivity and GMC among the three groups (non-vaccinated, one-dose, and two-dose). Seropositivity in children vaccinated twice was approximately four times higher than that without varicella vaccination. A seroepidemiology study of VarV in Hangzhou, China, demonstrated a positive conversion percentage of 98.89%, with an antibody level that was four times greater after the second vaccination.⁵ Similar results have been reported in other related studies.^16,17 A strategy involving two-dose vaccination in multiple geographic areas, with voluntary vaccination of VarV being free for children, increased the vaccination rate of VarV and decreased the rate of varicella. Two doses of VarV are preferred to provide the target population and immune stimulation toward varicella that is sufficient to prevent outbreaks.^5,18 Based on the international general immunization strategy, prior population-based surveillance studies combined with laboratory-confirmed data have verified that the administration of a second dose of VarV appears to have significantly higher immunogenicity, induces stronger T-cell-mediated immune responses, and brings long-term persistence of varicella vaccine efficacy.^19,20 The collective evidence indicates that children can achieve a higher protective effect after full-course vaccination, compared with natural infection and vaccination involving only one dose of VarV.

Analyses of the overall seropositivity rates and GMC for VZV IgG considering socio-demographic factors revealed the significant influences of age, regional distribution, and sex, consistent with other studies.^21,22 Maternal antibodies against varicella declined quickly to below the protective threshold after 6 months-of-age; subjects <4 years-of-age had lower seropositivity rate and GMC values; due to later natural infections and improved immunization coverage, the rates increased. Significant regional differences were also found in our study, most likely, rural areas usually featured low population density, higher immunization coverage, and lower incidence rate of varicella. Vaccinated individuals seldom acquired sufficient immunity after being exposed to the virus. Thus, antibody levels were lower in suburb populations. These aspects deserve additional consideration in the coming years to enhance supplementary immunization activities strategy among susceptible people in rural areas to decrease breakthrough cases and maintain an adequate and persistent antibody response. Our study also found that females had significantly higher rates of VZV IgG seropositivity and GMC for VZV IgG than males. The basis of the sex difference in VZV seropositivity remains unclear and may involve the levels of virus exposure, incidence, or natural infection.⁵ Females tend to have stronger innate and adaptive immune responses than males against both infections and vaccines.²³ It is therefore understandable that males are more prone to suffer from varicella.⁵ In addition, we found that a single dose of VarV was likely to confer limited protection, and antibody GMC values declined appreciably after 4 years in vaccine recipients. Another study also revealed lower varicella antibody titers 4–5 years following vaccination.¹ A study from Shanghai also reported lower antibody levels 3–5 years after vaccination.²⁴ These study findings highlight the benefit of a second VarV vaccination beginning at 4 years of age, rather than at older ages. In contrast, another study found that the 6-year cumulative varicella antibody persistence rate was 99.5% in children 1 to 12 years of age vaccinated with the Merck Varivax vaccine in the United States.²⁵ Discrepant results are potentially due to varying immune persistence elicited by the different vaccine compositions. Our findings provide scientific evidence for determining the optimal timing for administering the second dose of the vaccine after 4 years of age.

One limitation of this study was that the subjects were <7 years of age in Wuxi. VZV antibody titers in adults were not determined, thus, the results should be extrapolated strictly; vaccination and infection of adults need to be separately investigated. Besides, although the gp ELISA (Virion\Serion) test was performed in this study, which is a widely used commercial varicella ELISA antibody kit and had relatively reliable sensitivity and specificity.²⁶ However, previous studies reported varies in sensitivity across different ELISA kits,²⁶ and we did not directly compare the results with those using the FAMA test, further seroepidemiological studies using the FAMA test or other alternative tests should be conducted in order to determine the accurate VZV seropositivity in Wuxi. Finally, this was a cross-sectional study, population-based prospective cohort studies on the persistence of VarV immunity should also be pursued.

Conclusions

This observational population-based study assessed VZV seroepidemiology of children <7 years of age. Conclusively, the results of this study support the current recommendation for the use of the two-dose varicella vaccine regimen in children. The findings will inform further efforts by the Chinese government to improve the vaccination strategy for varicella.

Biography

Xuwen Wang is a public-health physician, with 20 years of experience in Expanded Program on Immunization. Her research interests focus on vaccine effectiveness and safety.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author’s contribution

The authors’ responsibilities were as follows: XWW designed the study, instructed the data analysis and critically reviewed the manuscript, and approved the final manuscript as submitted. LLW did the data cleaning and analysis, drafted and finished the manuscript. XQ collected and analyzed the biological samples, MY reviewed and revised the manuscript. All authors read and approved the final manuscript as submitted.

Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.