Vaccine xxx (xxxx) xxx

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Antibodies to measles, mumps, and rubella virus in Thai children after

two-dose vaccination at 9 months and 2.5 years: A longitudinal study
Nasamon Wanlapakorn a,b, Jiratchaya Puenpa a, Thanunrat Thongmee a, Donchida Srimuan a,
Thaksaporn Thatsanathorn a, Sompong Vongpunsawad a, Yong Poovorawan a,c,⇑
a
Center of Excellence in Clinical Virology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
b
Division of Academic Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
c
Academy of Science, Royal Society of Thailand, Bangkok, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: Thailand changed the schedule of childhood measles–mumps–rubella (MMR) vaccination
Received 13 January 2020 in 2014, moving the second dose from the age of 6 years to 2.5 years. There are currently no data on anti-
Received in revised form 2 April 2020 body responses to the MMR vaccine since this recommendation.
Accepted 4 April 2020
Material and methods: We investigated antibody responses in a cohort of children who received two doses
Available online xxxx
of MMR vaccine at the ages of 9 months and 2.5 years that was originally established to evaluate antibody
levels to Bordetella pertussis antigens (ClinicalTrials.gov no. NCT02408926). Infants were born to mothers
Keywords:
who previously received tetanus–diphtheria–acellular pertussis vaccine at 27–36 weeks of gestation.
Seroprotection
Two-dose vaccine
Anti-measles, -mumps, and -rubella virus IgG levels were measured at birth (cord blood) and the ages
Childhood vaccination of 2 and 7 months (before the first MMR vaccination); 18 and 24 months (9 and 15 months, respectively,
Measles–mumps–rubella (MMR) after the first dose); and 36 months (6 months after the second dose) using commercially available
enzyme-linked immunosorbent assay kits.
Results: At 7 months of age, 96.2%, 99.6%, and 98.8% of infants had no protection against measles, mumps,
and rubella, respectively. Levels of antibody against all three antigens increased significantly after the
first but not the second dose. At 6 months after two-dose vaccination, 97.4%, 84.8%, and 78.7% of children
remained seroprotected against measles, mumps, and rubella, respectively.
Conclusions: Maternally derived antibodies to measles, mumps, and rubella virus disappeared by the age
of 7 months in Thai children. Two-dose MMR vaccination at 9 months and 2.5 years of age induced robust
immune responses against these viruses.
Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction
Sporadic measles outbreaks still occur in some parts of Thailand
despite universal measles vaccination in children for >30 years.
Between 2005 and 2019, over 50,000 cases of measles were
reported in people of all ages [1]. A sharp increase in measles inci-
dence was observed between 2017 and 2018, with >5000 cases
reported to the Ministry of Public Health per year (8.4 per
100,000 individuals) [2]. Severe measles occurs in children younger
than 5 years [3]. In 2018, there were 814 measles cases in Thailand
in children under the age of 1 year, who are too young to be immu-
nised with the measles vaccine. Of the 11 fatal cases of measles in
⇑ Corresponding author at: Center of Excellence in Clinical Virology, Department
of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330,
Thailand.
E-mail address: yong.p@chula.ac.th (Y. Poovorawan).
2018, 10 were children <5 years old [2]. The incidence is dispropor-
tionately high in the south of Thailand including in Narathiwat
(85.36 per 100,000 individuals) and Pattani (64.06 per 100,000
individuals) provinces [2]. This is likely due to the rejection of
measles–mumps–rubella (MMR) vaccine based on religious beliefs
or lack of access to healthcare.
Molecular epidemiology studies of measles cases in Thailand
between 1998 and 2008 identified three genotypes (G2, D5, and
D9). D9 was the predominant genotype in 2008 [4]. However,
our group has investigated measles virus genotypes in recent out-
breaks (between 2018 and 2019) in the south of Thailand and
found that B3 was the most prevalent genotype. In contrast, D8
is the genotype that is primarily responsible for outbreaks in
Bangkok.
Measles vaccination was implemented in Thailand in 1984,
starting with one dose at the age of 9 months in areas of the
outbreak and at 12 months in non-outbreak areas [5]. In 1996,

https://doi.org/10.1016/j.vaccine.2020.04.013
0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
2 N. Wanlapakorn et al. / Vaccine xxx (xxxx) xxx
the second dose of the measles vaccine, which was replaced by the
trivalent MMR vaccine in 1997, was introduced in 6-year-old chil-
dren. Due to sporadic measles outbreaks, especially among chil-
dren under the age of 5 years, the second dose of MMR vaccine
was moved from 6 to 2.5 years of age in 2014. Between 2014
and 2019, the overall coverage for the first dose of MMR vaccine
was 83%–92%, whereas that of the second dose at 2.5 years
increased from 3.3% in 2014 to 90% in 2019 [6]. Despite this high
coverage, the incidence of measles in Thailand continues to
increase. Possible reasons for this trend include failure of the vac-
cine due to increased antigen diversity among circulating strains of
measles virus; variable measles immunity in the Thai population;
primary vaccine failure due to maternal immunity; rapid antibody
waning; and low vaccine coverage.
Mumps is a disease caused by the mumps RNA virus from the
Paramyxoviridae family. Countries with a national MMR vaccina-
tion program have a low incidence of mumps, with a > 90% decline
recorded in the number of cases compared to the pre-vaccination
era [7]. In Thailand, one and two doses of mumps-containing vac-
cines were introduced in 1997 and 2010, respectively [8]; the inci-
dence of mumps decreased dramatically after implementation of
the two-dose vaccination schedule from 24.94 cases per 100,000
individuals in 2010 to between 3 and 6 cases during 2014–2019
[9], with no large outbreaks reported. Rubella is a self-limiting viral
disease but can lead to a severe illness known as congenital rubella
syndrome (CRS) in infants born of infected pregnant women.
Rubella vaccine was first introduced in female 6th-grade students
between 1986 and 1993 to prevent CRS, and later in 6-year-old
children of both sexes during 1993–1997 [5]. The two-dose
rubella-containing vaccine was implemented in Thailand in 2010
in infants 9–12 months of age and 6-year-old children. In 2014,
the second dose was changed from 6 to 2.5 years old. Since then,
the incidence of rubella in Thailand has remained low, with less
than one case per 100,000 individuals since 2013, although the
number may be substantially underreported [10].
In 2017, the World Health Organization recommended measles
vaccination at the ages of 9 months (first dose) and 15–18 months
(second dose) in countries with ongoing measles transmission [11].
There are no data on the immunogenicity of MMR vaccine admin-
istered according to the 2014 revised schedule of a two-dose vac-
cination at 9 months and 2.5 years. Here we present antibody
responses to MMR vaccine in a cohort of Thai children that was
originally established to evaluate the interference of maternal anti-
bodies against Bordetella pertussis antigens [12]. Our data provide a
basis for the development of a national vaccination policy for chil-
dren in Thailand.
2. Material and methods
2.1. Study design
This study (ClinicalTrials.gov no. NCT02408926) was conducted
according to the Declaration of Helsinki and Good Clinical Practice
Guidelines. The Institutional Review Board of the Faculty of
Medicine at Chulalongkorn University (IRB no. 604/57 [for the
enrolment of infants from birth to 19 months of age] and 315/61
[for the enrolment of children aged 24 and 36 months]) approved
this study. Written consent was obtained from the participants’
parents prior to enrolment.
A total of 370 pregnant women who visited the antenatal care
clinic at King Chulalongkorn Memorial Hospital and consented to
tetanus-diphtheria-acellular pertussis (Tdap) vaccination (Boost-
rix; GlaxoSmithKline Biologicals, Rixensart, Belgium) during the
third trimester of pregnancy were enrolled. The inclusion and
exclusion criteria for these women have been previously reported
Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
[13]. Healthy full-term and late preterm infants born at the gesta-
tional age of 36 weeks with a birth weight > 2500 g were enrolled
at birth and followed up at King Chulalongkorn Memorial Hospital
and vaccinated according to the current recommendations of the
National Vaccine Advisory Board (Table 1).
2.2. Study vaccines
The infants received intradermal bacilli Calmette-Guerin (BCG)
vaccine (Queen Saovabha Memorial Institute, Bangkok, Thailand)
and intramuscular monovalent hepatitis B (HB) vaccine at birth.
Pentavalent (Quinvaxem) or hexavalent (Infanrix hexa) vaccine
was administered at 2, 4, or 6 months (primary vaccination) and
at 18 months (first booster vaccination). Infants born to HB surface
antigen-positive mothers also received HB immunoglobulin at
birth and a monovalent HB vaccine at 1 month. Infants who
received pentavalent vaccine were also given oral poliovirus vac-
cine at 2, 4, 6, and 18 months and inactivated poliovirus (IPV) vac-
cine (IMOVAX Polio; Sanofi Pasteur, Lyon, France) containing 40, 8,
and 32 D-antigen units of IPV type 1, 2, and 3, respectively, at the
4-month vaccination visit, with the exception of two children who
reached 4 months of age before the national polio vaccination pol-
icy changed. This vaccine was separately injected into the antero-
lateral thigh.
MMR vaccine (Priorix; GlaxoSmithKline Biologicals, Rixensart,
Belgium) or M M RII (Merck & Co., Kenilworth, NJ) were admin-
istered at 9 months and 2.5 years (30 months) of age according to
the 2014 policy in Thailand. Priorix contains live attenuated
measles virus (Schwarz strain) not less than 103.0 50% tissue
culture infective dose (TCID50); live attenuated mumps virus
(RIT 4385 strain, derived from the Jeryl Lynn strain) not less than
103.7 TCID50; and live attenuated rubella virus (Wistar RA 27/3
strain) not less than 103.0 TCID50. M M RII contains live attenu-
ated measles virus (Edmonston-Enders strain) not less than 103.0
TCID50; live attenuated mumps virus (Jeryl Lynn strain) not less
than 104.1 TCID50; and live attenuated rubella virus (Wistar RA
27/3 strain) not less than 103.0 TCID50.
Children received live-attenuated Japanese Encephalitis (JE)
vaccine (CD.JEVAX; Chengdu Institute of Biological Products,
Chengdu, China) at 12 and 19 months of age; trivalent influenza
vaccine (Influvac; Abbott Biologicals, Olst, The Netherlands) at 7,
9, 24 and 36 months; and hepatitis A vaccine (VAQTA; Merck &
Co., Kenilworth, NJ) at 24 and 36 months. Some infants also
received the optional rotavirus (given orally), pneumococcal,
varicella-zoster, or rabies vaccine (administered at a separate
injection site). Parents purchased the rotavirus, pneumococcal,
varicella-zoster, or rabies vaccine for their children as they are
Table 1
Vaccination schedule for children enrolled in this study.
Age (months) Vaccine
0 (birth) BCG, monovalent HB
1 Monovalent HB*
2 Infanrix hexa or Quinvaxem + OPV
4 Infanrix hexa or Quinvaxem + OPV and IPV
6 Infanrix hexa or Quinvaxem + OPV
7 Influenza vaccine (I)
9 MMR (I), influenza vaccine (II)
12 Live-attenuated JE vaccine (I)
18 Infanrix hexa or Quinvaxem + OPV
19 Live-attenuated JE vaccine (II)
24 Hepatitis A vaccine, influenza vaccine
30 MMR (II)
36 Hepatitis A vaccine, influenza vaccine
*Only infants born to hepatitis B virus carrier mothers.
BCG, bacilli Calmette-Guerin; HB, hepatitis B; IPV, inactivated poliovirus; JE, Japa-
nese encephalitis; MMR, measles–mumps–rubella; OPV, oral poliovirus vaccine.
 N. Wanlapakorn et al. / Vaccine xxx (xxxx) xxx 3

not part of the Expanded Program on Immunization (EPI) in

Thailand.

2.3. Sample collection

The timing of blood collection was decided for the study aiming
to evaluate the antibody response to B. pertussis antigens [12]. All
residual sera from the abovementioned project were then tested
for the presence of anti-measles, -mumps, and -rubella IgG at the
following time points: at birth (umbilical cord samples), at 2 and
7 months (before the first dose of MMR vaccine), 18 months
(9 months after the first dose), 24 months (15 months after the first
dose), and 36 months (6 months after the second dose). The ethics
committee approved the use of residual sera samples collected
from participants for these analyses.

2.4. Laboratory testing

Commercial enzyme-linked immunosorbent assay kits were

used according to the manufacturer’s instructions to measure the
concentrations of IgG against measles, mumps, and rubella viruses
(EUROIMMUN, Lübeck, Germany). Serum samples were initially
diluted 1:100, and then further diluted to obtain values within
the detection range. Anti-measles, -mumps, and -rubella IgG con-
centrations are expressed in international units (IU)/l, relative
units (RU)/ml, and IU/ml, respectively. Internationally assigned
levels of protective immunity for discriminating between suscepti-
ble and protected individuals are  200 IU/l,  45 RU/ml,
and  10 IU/ml for measles, mumps, and rubella, respectively
[14]. Antibodies against measles were divided into the following Fig. 1. CONSORT flow diagram for this study. The number of serum samples tested
five categories according to immunity level: <200 IU/l, unpro- (in parentheses) and MMR vaccine administration program at 9 and 30 months of
tected; 200 to <275 IU/l, equivocal; 275 to <1000 IU/l, seropositive; age are shown. Tdap, Tetanus, diphtheria, and acellular pertussis.
1000 to <5000 IU/l, high titre; and 5000 IU/l, very high titre. Anti-
bodies against mumps were divided into the following three cate-
gories: < 45 RU/ml, unprotected; 45 to <100 RU/ml, seropositive; enrolment and one received a monovalent rubella vaccine at
and 100 RU/ml, high titre. Antibodies against rubella were 2 years prior to enrolment. From these women, 311 healthy full-
divided into the following four categories: <10 IU/ml, unprotected; term and four late preterm infants who were born at the gesta-
10 to <50 IU/ml, seropositive; 50 to <200 IU/ml, high titre; and tional age of 36 weeks with a birth weight >2500 g, were enrolled
200 IU/ml, very high titre. Antibody levels below the lower limit in the study. The baseline characteristics of the children at differ-
of quantification (LLOQ) observed in some samples were assumed ent time points are shown in Table 2. We excluded infants with
to be equivalent to half the LLOQ. conditions that potentially affected their levels of antibody against
measles, mumps, and rubella. Two children were contraindicated
for the MMR vaccine at 9 months of age and were excluded from
2.5. Statistical analysis
the analysis: one was diagnosed with Kawasaki disease at
8.5 months and received intravenous Ig and the other naturally
The geometric mean titre (GMT) was calculated. Antibody level
contracted measles at the age of 9 months, a few days before the
measurements were initially log-transformed and the geometric
scheduled MMR vaccine visit. The mean time interval between
mean of these values was calculated and exponentiated. GMT val-
birth and the first MMR vaccination was 276.2 days (min: 249;
ues are presented graphically using a log10 scale with 95% confi-
max: 392). The brand of MMR vaccine (Priorix or M M RII) was
dence intervals. Pairwise comparisons of GMT between groups of
selected according to availability at that time. Children who
children receiving penta- and hexavalent vaccines were carried
received an MMR vaccine elsewhere without a record of the brand
out using a regression model. P < 0.05 was considered statistically
were designated as uncertain. The mean time interval between
significant.
birth and the second MMR vaccination was 29.8 months (min:
28; max: 32). One child who received the second dose of the
3. Results MMR vaccine at 37 months was excluded from the analysis.

3.1. Demographic characteristics of study participants

The CONSORT flow diagram for this study is shown in Fig. 1. In
total, 370 pregnant women (age range: 18–40 years) recruited
between April 2015 and September 2016 were vaccinated with
Tdap. Since childhood vaccination records were not available for
these women, they were assumed to have received childhood vac-
cines according to the EPI at that time. Additionally, recent vacci-
nations (within 5 years of enrolment) were recorded; three
women received the MMR vaccine at 1, 2, and 3 years prior to
3.2. GMT and seroprotection rate of anti-measles IgG
Approximately 85% of newborns had an anti-measles IgG con-
centration of >200 IU/l at birth, with a GMT of 848.8 IU/l (Fig. 2
and Table S1). Two cord serum samples collected from three
women who received the MMR vaccine before pregnancy showed
high anti-measles IgG levels of 1147.7 and 3843.1 IU/l. The GMT of
anti-measles IgG decreased to 260.3 IU/l at 2 months and 68.5 IU/l
at 7 months, respectively (Table 3). At 18 months (9 months after

Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
4 N. Wanlapakorn et al. / Vaccine xxx (xxxx) xxx

Table 2 cord serum samples from two women who received the MMR vac-
Baseline characteristics of the study population. cine before pregnancy showed high anti-mumps IgG levels (120.8
All children and 59 RU/ml). The anti-mumps IgG level at birth of 24.2 RU/ml
(n = 315) decreased to 7.0 RU/ml at 2 months and 2.6 RU/ml at 7 months
Mean GA at delivery (SD) 38.7 (1.1) (Table 3). Nearly all children (99.6%) were below the seroprotective
Mode of delivery limit for anti-mumps IgG at 7 months. Antibody levels increased
Vaginal, n (%) 176 (55.8%) after the first dose of vaccine at 9 months, as evidenced by the
Caesarean section, n (%) 139 (44.2%)
Sex
GMT of 35.6 RU/ml at 18 months (9 months post first dose) and
Male, n (%) 154 (48.9%) 35.1 RU/ml at 24 months (15 months post first dose). GMT
Female, n (%) 161 (51.1%) increased significantly at 36 months after the second MMR vacci-
Weight and length according to age nation. The highest GMT was measured in samples from 36-
Birth (0 months)
month children (86.9 RU/ml).
Mean weight, g (SD) 3128.2 (358.4)
Mean length, cm (SD) 49.7 (1.9) Approximately 85% of children on the two-dose mumps vacci-
2 Months nation schedule remained seroprotected at 3 years of age. This is
Mean weight, kg (SD) 5.4 (0.6) consistent with the rate that was previously reported as adequate
Mean length, cm (SD) 57.3 (2.5) for inducing herd immunity against mumps (84%–86%) [16]. Chil-
4 Months
dren who received the hexavalent vaccine had significantly higher
Mean weight, kg (SD) 6.7 (0.8)
Mean length, cm (SD) 63.1 (2.5) GMT of anti-mumps IgG at 36 months (post-second dose) com-
6 Months pared to those who received the pentavalent vaccine (96.8 vs
Mean weight, kg (SD) 7.6 (0.9) 77.4 RU/ml, P = 0.010). There was no difference in the GMT of
Mean length, cm (SD) 67.3 (2.7)
anti-mumps IgG between children who received Priorix vs
7 Months
Mean weight, kg (SD) 7.9 (0.9) M M RII vaccine at 9 months post first dose (18 months old).
Mean length, cm (SD) 69.2 (2.8)
18 Months
Mean weight, kg (SD) 10.9 (1.5)
3.4. GMT and seroprotection rate of anti-rubella IgG
Mean length, cm (SD) 81.7 (3.4)
24 Months Approximately 84% of newborns had an anti-rubella IgG level of
Mean weight, kg (SD) 12.4 (1.9) > 10 IU/ml at birth, with a GMT of 28.6 IU/ml (Fig. 2 and Table S3).
Mean length, cm (SD) 87.0 (3.7)
The anti-rubella IgG level decreased to 8.7 and 1.2 IU/ml at 2 and
36 Months
Mean weight, kg (SD) 14.6 (2.8) 7 months, respectively (Table 3). At 18 months (9 months after
Mean length, cm (SD) 94.5 (3.8) the first MMR vaccination), the IgG level increased to 119.6 IU/
Mean time interval from birth to the first dose of MMR 276.2 days ml. As in the case of measles, the IgG level at 24 months
vaccine (SD) (±11.9 days) (119.7 IU/ml) was similar to that at 18 months. However, at
Mean time interval from birth to the second dose of 29.8 months
MMR vaccine (SD) (±0.6 months)
36 months (after the second MMR vaccination), the anti-rubella
IgG level was lower than the level at 24 months (before the second
GA, gestational age; MMR; measles–mumps–rubella; SD, standard deviation; dose).
g, grams; kg, kilograms; cm, centimeters.
The threshold for herd immunity against rubella is estimated as
85%–88% [17]. In this study, we found that after the first dose of the
the first MMR vaccination), the anti-measles IgG level increased to MMR vaccine, >99% of subjects were seroprotected. At 36 months
786.9 IU/l. The level at 24 months (858.4 IU/l) was similar to that at of age, SPR decreased to 80%.
18 months but slightly less than that at 36 months (after the sec-
ond MMR vaccination). The highest GMT was in samples from 36- 4. Discussion
month-old children (927.7 IU/l).
Approximately 96% of children were susceptible to measles at We report antibody responses to MMR vaccine in children who
the age of 7 months. After the first and second vaccinations, sero- were immunised at 9 months and 2.5 years of age according to the
protection rate (SPR) increased to 91.7% at 18 months, 91% at recently implemented childhood vaccination schedule in Thailand.
24 months, and 97.4% at 36 months, indicating that this two- Both Priorix and M M RII vaccines administered according to this
dose vaccination schedule induced herd immunity against measles protocol showed good immunogenicity profiles, although signifi-
which is achieved when the population immune is 92%–94% [15]. cant increases in anti-measles and -rubella antibody titres were
As this infant cohort was originally enrolled and randomized for observed only after the first and not the second dose. SPRs against
the study of antibody responses to B. pertussis antigens induced all antigens reached the herd immunity threshold after one- and/or
by acellular- versus whole-cell pertussis-containing vaccines, it two-dose vaccination.
would be worthy to compare the immunogenicity induced by A previous study conducted between 1994 and 1995 in Thai-
MMR vaccine among these two infant groups to see whether differ- land demonstrated that 93.5% of infants were seronegative for neu-
ent forms of pertussis vaccines administered during early infancy tralising antibody against measles at 9 months of age [18], whereas
had an impact on the antibody responses to MMR vaccine antigens. another study found that most Thai children had anti-measles IgG
There were no significant differences in GMT of anti-measles IgG concentrations below protective levels at 6 months [19]. A study in
between children receiving hexavalent vs pentavalent vaccine at Belgium showed that while newborns of naturally immune moth-
any time point (Table 3), nor between children receiving Priorix ers had higher anti-measles IgG titres than those of vaccinated
and M M RII vaccine at 9 months post first dose (18 months of mothers, over 95% had lost the antibody by 6 months of age [20].
age) (data not shown). This is in agreement with our finding that maternally acquired
antibody was undetectable by 7 months of age. The loss of mater-
3.3. GMT and seroprotection rate of anti-mumps IgG nal immunity leads to a period between 7 and 9-12 months of age
during which infants are more susceptible to measles and associ-
Our results showed that 54.2% of newborns were susceptible to ated complications, as they are too young to receive the first dose
mumps (Fig. 2 and Table S2). Similar to anti-measles IgG levels, of MMR vaccine. This is evidenced by the reported incidence of

Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
 N. Wanlapakorn et al. / Vaccine xxx (xxxx) xxx 5

Table 3
Antibody titres at different ages in children who received hexa- and pentavalent vaccines.

Antibody according to age Overall GMT GMT in children who received GMT of children who received P value
(95% CI) hexavalent vaccine (95% CI) pentavalent vaccine (95% CI) (penta- vs hexavalent)
Birth (cord)
Anti-measles IgG, IU/l 848.8 (723.6–995.7) 917.7 (739.3–1139.2) 780.3 (614.8–990.2) 0.318
Anti-mumps IgG, RU/ml 24.2 (20.0–29.3) 25.1 (19.1–33.0) 23.1 (17.6–30.4) 0.671
Anti-rubella IgG, IU/ml 28.6 (24.1–33.9) 29.7 (23.5–37.4) 27.5 (21.4–35.4) 0.664
n 264 137 127
2 Months
Anti-measles IgG, IU/l 260.3 (229.7–294.9) 277.4 (234.8–327.9) 243.5 (201.8–293.8) 0.304
Anti-mumps IgG, RU/ml 7.0 (6.0–8.2) 7.4 (5.9–9.4) 6.5 (5.2–8.1) 0.402
Anti-rubella IgG, IU/ml 8.7 (7.5–9.9) 9.5 (7.9–11.4) 7.9 (6.4–9.7) 0.183
n 280 143 137
7 Months
Anti-measles IgG, IU/l 68.5 (60.5–77.4) 67.6 (60.1–76.0) 68.5 (60.5–77.4) 0.877
Anti-mumps IgG, RU/ml 2.6 (2.3–2.9) 2.7 (2.3–3.2) 2.4 (2.1–2.9) 0.405
Anti-rubella IgG, IU/ml 1.2 (1.1–1.4) 1.3 (1.1–1.5) 1.2 (1.0–1.4) 0.581
n 262 134 128
18 Months (9 months after the first dose)
Anti-measles IgG, IU/l 786.9 (700.5–883.9) 805.6 (683.7–949.2) 768.5 (650.3–908.1) 0.690
Anti-mumps IgG, RU/ml 35.6 (31.5–40.3) 37.3 (31.0–44.9) 34.0 (28.8–40.2) 0.467
Anti-rubella IgG, IU/ml 119.6 (109.5–130.6) 128.9 (114.4–145.2) 111.0 (97.4–126.4) 0.095
n 265 133 132
24 Months (15 months after the first dose)
Anti-measles IgG, IU/l 858.4 (754.3–977.0) 889.4 (744.5–1062.5) 826.7 (683.0–1000.6) 0.579
Anti-mumps IgG, RU/ml 35.1 (30.8–40.0) 37.5 (31.3–44.9) 32.8 (27.1–39.7) 0.312
Anti-rubella IgG, IU/ml 119.7 (109.2–131.2) 131.5 (116.8–147.9) 108.3 (94.0–124.8) 0.038*
n 233 120 113
36 Months (6 months after the second dose)
Anti-measles IgG, IU/l 927.7 (824.3–1043.9) 964.5 (815.6–1140.6) 890.4 (752.3–1053.8) 0.506
Anti-mumps IgG, RU/ml 86.9 (80.0–94.6) 96.8 (86.8–107.8) 77.4 (68.4–88.2) 0.010*
Anti-rubella IgG, IU/ml 87.4 (80.5–94.9) 92.3 (82.3–103.5) 82.5 (73.2–93.0) 0.183
n 230 118 112

*P < 0.05.
CI, confidence interval; GMT, geometric mean titre; IgG, immunoglobulin G.

measles (175–814 cases per year) in infants < 1 year old in Thai-
land during 2008–2018 [1]. The optimal age for first-dose measles
vaccination has been raised especially among countries where the
disease still exists. Moving the first dose of measles vaccine to an
earlier age such as 6 months for Thai children warrants further
risk-benefit justification since previous studies demonstrated infe-
rior antibody response to measles vaccine at 6 months of age due
to the immature immune cells [21–24]. Nevertheless, early vacci-
nation in selected regions where measles outbreaks have occurred
should be considered in order to induce protective immunity in
infants and prevent severe complications. Meanwhile, infant pro-
tection against measles could be increased by increasing herd
immunity through improving vaccine coverage and by ensuring
the timely vaccination of the first dose at 9 months.
A previous study evaluating antibody responses after the first
dose of measles vaccine in Thai infants at 9 months of age found
that although the vaccine was administered at the time of disap-
pearance of maternal antibodies, the seroconversion rate was only
68.75% at 12 months and had further decreased to 53.3% at
18 months [18]. In India, >25% of infants did not respond to the
first dose of the measles vaccine [25]. In contrast, we observed that
after the first dose of the MMR vaccine, >90% of children remained
seroprotected at 18 and 24 months. This could be due to differ-
ences in vaccine immunogenicity or administration as well as
genetic background. SPR was higher at 4–6 years of age in Thai
children who received two doses of measles vaccine (at 9 and
18 months) compared to the single-dose vaccine (87% vs 76%)
[26]. We found that 97.3% of children who received the MMR vac-
cine at 9 and 30 months remained seroprotected at 3 years. This
finding can inform policy discussions regarding the appropriate
time of the second vaccine dose, as it shows that a booster at
30 months did not compromise SPR in children and should not
be moved to an earlier age.
A previous study in Portugal suggested that anti-mumps IgG is
passively transferred from mother to infant, with a reported GMT
of anti-mumps IgG in cord serum of 31 RU/ml [27]. Additionally,
67.1% of pregnant women in Spain showed seropositivity against
mumps, but only 11% of their offspring remained positive at the
age of 3 months and by 9 months, all were seronegative [28]. In
the present study, only 8% of children remained seroprotected at
the age of 2 months and by 7 months, >99% were below the protec-
tive threshold.
After two-dose MMR vaccination at 9 months and 2.5 years, the
SPR against mumps at 3 years of age was 85%. One study in Bel-
gium showed that after one-dose MMR vaccination at 12 months,
only 32% of children were seropositive for anti-mumps IgG at
5 years of age. However, 88% were positive for neutralising
antibody [29]. Another study in Belgium found that neutralising
activity against Jeryl Lynn strain vaccine was 84% in children aged
2–3 years, but only half of these children had neutralising
antibodies against circulating genotype G mumps virus, suggesting
that the vaccine-induced antibodies were ineffective in neutralis-
ing a wild-type mumps virus strain [30]. A previous serological
survey of anti-mumps antibody in the Thai population after
17 years of universal mumps vaccination showed that less than
half of 15- to 19-year-olds were seroprotected (defined as
levels  20 RU/ml) [8]. Despite this apparent waning of immunity
against mumps in Thai adolescents, there have been no mumps
outbreaks in Thailand. Additional studies investigating the
functional activity and neutralisation as well as the molecular
epidemiology of mumps virus in Thailand, are needed to clarify
the mechanism of vaccine-induced seroprotection.

Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
6 N. Wanlapakorn et al. / Vaccine xxx (xxxx) xxx

Fig. 2. Serologic status and GMT of antibodies at different ages. Antibody titres against (A) measles, (B) mumps, and (C) rubella virus at birth and at 2, 7, 18, 24, and 36 months
of age are shown. The left y-axis represents the percentage of the population with a given antibody concentration; the right y-axis represents the GMT in each age group, with
means indicated as red dots and red vertical lines denoting 95% confidence intervals. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

Anti-rubella IgG is also actively transferred from pregnant 84.1% among newborns. Although previous studies in Spain, China
women to their newborns through the placenta. In the present and Protugal reported similar GMT and SPR, the levels cannot be
work, the GMT of anti-rubella IgG was 29.7 IU/ml, with an SPR of directly compared between different studies due to the difference

Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
 N. Wanlapakorn et al. / Vaccine xxx (xxxx) xxx
in antibody assays used [14,28,31]. An anti-rubella IgG level of
>10 IU/ml was not detected in Spanish children at 9 and 12 months,
reflecting the waning of maternal immunity before the infant’s first
vaccination. Accordingly, we found that 98.8% of infants were neg-
ative for anti-rubella IgG at 7 months, but after one dose of MMR
vaccine at 9 months, >99% achieved seropositivity that lasted up
to the age of 2 years. Although our cohort received a second dose
of MMR vaccine at 2.5 years of age, the SPR at 3 years were not
as high as those observed in 5-year-old Belgian children who
received one dose of MMR vaccine at 12 months (79.7% vs 94.5%)
[29]. Possible reasons for this discrepancy are the type of vaccine
that was used, differences in the epidemiology of rubella between
the two countries, and genetic background. Notably, the GMT of
anti-rubella IgG in Thai children after the second dose of vaccine
was lower than that recorded in Indian children who received
two doses of rubella-containing vaccine at 9 and 15 months [32].
In the present study, blood samples were collected from the Thai
cohort at 6 months after the second vaccine dose; this is not the
ideal time point for detecting the peak antibody response given
that anti-rubella IgG levels decline over time. Although the sero-
protection rate against rubella following the two-dose schedule
was 79.7%, it did not imply that these vaccinated children were
susceptible. In 2017, the World Health Organization expert com-
mittee on Biological Standardization pointed out that the cut-off
level (>10 IU/ml) for seroprotection against rubella was not appro-
priate as it has been demonstrated that vaccinated individuals
often had titres < 10 IU/ml and yet are protected due to the absence
of reported rubella cases amongst vaccinated individuals [33].
Our study had some limitations. Firstly, the optimal time to
detect an antibody response is typically 1 month post-
vaccination. Since this cohort was originally established to evalu-
ate the response to pertussis vaccine, it was not possible to collect
blood samples at other time points. Nonetheless, this cohort of
healthy children offers an opportunity to assess immunity against
measles, mumps, and rubella virus at different ages. This cohort
enrolled a large group of healthy term Thai infants with >200 par-
ticipants remained at the end of the study. Results of this study can
be generalizable to the healthy Thai infants in the community,
though excluding preterm or infants with underlying chronic dis-
eases. Errors in measurement of IgG levels were minimized by
using assays that have been optimized. However, due to the lack
of standardization for IgG testing, the quantitative results from this
study cannot be directly compared with others [34–39]. Standard-
ized assays and assessment on functionality of antibody are crucial
for an accurate determination of vaccine-induced protection. The
neutralising activity of antibodies against different strains of the
measles virus was also evaluated and will be presented and dis-
cussed separately. The children in our cohort were born to mothers
who received Tdap during pregnancy, which does not affect the
development of infant immune responses to MMR vaccine anti-
gens [40]. Long-term annual follow-ups of children in this cohort
are ongoing in order to monitor the degree of immunity induced
by the two-dose MMR vaccination schedule at 9 and 30 months
based on the kinetics of antibody responses, which could help to
determine the appropriate timing for a booster.
In conclusion, our results demonstrate that maternally acquired
antibody against measles, mumps and rubella was undetectable by
7 months of age. Infant protection against measles could be
increased by increasing herd immunity through improving vaccine
coverage and by ensuring the timely vaccination of the first dose at
9 months. The second vaccine dose at 30 months did not increase
the GMT of anti-measles and -rubella IgG, although an increase in
SPR against mumps was observed. Long-term follow-up can
determine whether a third vaccine dose in this population is
needed in order to achieve the target of eradicating measles and
rubella in Thailand.
Please cite this article as: N. Wanlapakorn, J. Puenpa, T. Thongmee et al., Antibodies to measles, mumps, and rubella virus in Thai children after two-dose
vaccination at 9 months and 2.5 years: A longitudinal study, Vaccine, https://doi.org/10.1016/j.vaccine.2020.04.013
7
5. Contributors
NW and YP conceived and designed the study. NW, DS, and
Thaksaporn T acquired the data, including in the follow-up. Tha-
nunrat T performed the experiments. NW analysed the data and
drafted the manuscript. All authors read and contributed to the
writing of the manuscript.
Funding
This work was funded by Health Systems Research Institute
(HSRI) (contract no. HSRI 61-63); Ratchadaphiseksomphot Endow-
ment Fund, Chulalongkorn University 2561 (CU-GR[S] 61-36-30-
01); Center for Excellence in Clinical Virology, Faculty of Medicine,
Chulalongkorn University and Hospital; a Research Chair Grant
from the National Science and Technology Development Agency
(no. P-15-5004); Charoen Pokphand Group, MK Restaurants Group
and the Thrasher Fund (no. EWAT 12348). Grants for development
of new faculty staff, Ratchadaphiseksomphot Endownment Fund,
Chulalongkorn Univeristy.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
We thank our collaborators Dr. Elke Leuridan, Dr. Kirsten Maer-
tens, and Dr. Pierre Van Damme at the Center for the Evaluation of
Vaccination, Vaccine & Infectious Disease Institute, Faculty of Med-
icine and Health Sciences, University of Antwerp, Belgium for their
contribution in setting up the cohort of children.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.vaccine.2020.04.013.
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