Vaccine xxx (xxxx) xxx

Contents lists available at ScienceDirect

Vaccinejournal homepage: www.elsevier.com/locate/vaccine

Comparison of immunogenicity between candidate influenza A(H3N2)
virus vaccine strains in Japan: A randomized controlled trial using amonovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/HongKong/4801/2014 (X-263)
Tetsuo Kase a,b, Megumi Inoue c, Saeko Morikawa d, Hiroko Kumashiro c, Satoshi Hiroi d, Keiko Nakata d,Kazuya Ito a,b,f,g, Motoki Ishibashi c, Tomomi Tsuru c, Shin Irie e, Akiko Maeda a, Satoko Ohfuji a,b,
Wakaba Fukushima a,b,⇑, Yoshio Hirota g,ha

Department of Public Health, Osaka City University Graduate School of Medicine, Osaka, Japan
Research Center for Infectious Disease Sciences, Osaka City University Graduate School of Medicine, Osaka, Japan
SOUSEIKAI PS Clinic, Fukuoka, Japand
Department of Virology, Osaka Institute of Public Health, Osaka, Japane
SOUSEIKAI, Fukuoka, Japanf
Healthcare Management, College of Healthcare Management, Miyama, Japang
Clinical Epidemiology Research Center, SOUSEIKAI, Fukuoka, JapanhCollege of Healthcare Management, Miyama, Japanbca r t i c l e

i n f oArticle history:
Received 24 March 2020
Received in revised form 13 July 2020
Accepted 11 August 2020
Available online xxxx
Keywords:
Influenza A(H3N2) vaccine strains
Immunogenicity
Antigenic matching
Randomized controlled triala b s t r a c t
Background: For the 2017–18 influenza season, A/Saitama/103/2014 (CEXP-002) (Saitama strain) was
antigenically more similar to prior circulating strains than A/Hong Kong/4801/2014 (X-263) (HongKong strain) in a ferret model and was selected as the A(H3N2) vaccine virus strain in Japan. However,the Saitama strain grew poorly, and the Japanese government switched to the Hong Kong strain, raisingpublic concerns of poor effectiveness. To enhance understanding of the correlation between antigenicity
in experimental models and immunogenicity, as a surrogate measure of vaccine effectiveness, in thehuman population, we compared the immunogenicity of specially-prepared single dose monovalentinfluenza A(H3N2) vaccines containing the Saitama or the Hong Kong strain.
Methods: A randomized controlled trial of 100 healthy adults aged 20–64 years (n = 50/group) was conducted. Virus neutralization assay was performed on sera from days 0 (pre-vaccination) and 21 (postvaccination). Geometric mean titer (GMT), mean fold rise (MFR), seroconversion proportion (SCP), andseroprotection proportion (SPP) were calculated for vaccine strains and a representative circulating A(H3N2) virus strain (A/Osaka/188/2017).
Results: For the Hong Kong strain, post-vaccination GMT was significantly higher in the Hong Kong vaccine recipients (1:546 vs 1:260, p < 0.01), but MFR, SCP, and SPP were similar for both vaccine groups. For
the Saitama strain, post-vaccination GMT (1:116 vs 1:61, p = 0.01) and SPP (86% vs 68%, p = 0.03) weresignificantly higher in the Hong Kong vaccine recipients, but MFR and SCP were similar for both vaccine
groups. Against A/Osaka/188/2017, post-vaccination GMT and MFR were similar in both vaccine groups,
but SCP (32% vs 4%, p < 0.01) and SPP (28% vs. 6%, p < 0.01) were significantly higher in the Hong Kong
vaccine recipients.
Conclusion: The Hong Kong vaccine induced better or equivalent immunogenicity in comparison to the
Saitama vaccine. Our trial showed that antigenic similarity in experimental models does not necessarily
correlate with immunogenicity in the human population.
Clinical trial registration: UMIN000029293.
Ó 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

Abbreviations: WHO, World Health Organization; NIID, National Institute of Infectious Diseases; MHLW, Ministry of Health, Labour and Welfare; HA, hemagglutinin; HI,hemagglutination inhibition; TCID50, 50% tissue culture infectious doses; CPE, cytopathic effect; GMT, geometric mean titer; MFR, mean fold rise; SCP, seroconversion
proportion; SPP, seroprotection proportion; SD, standard deviation.
⇑ Corresponding author at: Department of Public Health, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
E-mail address: wakaba@med.osaka-cu.ac.jp (W. Fukushima).
https://doi.org/10.1016/j.vaccine.2020.08.025
0264-410X/Ó 2020 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

2

T. Kase et al. / Vaccine xxx (xxxx) xxx

1. Introduction

2. Materials and methods

Appropriate selection of influenza vaccine strains is critical for
providing an optimal strategy for the prevention of influenza.
Egg-adapted changes of influenza vaccine virus, particularly for
the A(H3N2) strain, can affect virus receptor-binding and altervirus antigenicity [1], and therefore has attracted increasing attention in terms of vaccine effectiveness [2–4]. This was the case of
the A/Hong Kong/4801/2014 (H3N2)-like virus, which was recommended by the World Health Organization (WHO) for the 2017–18
Northern Hemisphere influenza vaccine in March 2017 [5]. However, A/Saitama/103/2014 (CEXP-002), which was isolated in Japanand also designated a recommended A(H3N2) strain by the WHO
for the 2017–18 Northern Hemisphere influenza vaccine [6], had
fewer antigenic changes during egg-adaptation [7]. A/Saitama/103/2014 (CEXP-002) had no amino acid substitutions at
the antigenic sites of its hemagglutinin (HA) protein but multiple
substitutions in its neuraminidase protein. The National Instituteof Infectious Diseases (NIID) in Japan reported that A/Saitama/103/2014 (CEXP-002) was antigenically matched to 60% of
circulating A(H3N2) viruses in the 2016–17 season using postinfected ferret sera, while it was 3% for A/Hong Kong /4801/2014(X-263) [7,8].
In Japan, only quadrivalent, inactivated, egg-cultured, splitvirus influenza vaccines are available and these are only suppliedby domestic vaccine manufacturers. The Ministry of Health,Labour and Welfare (MHLW) in Japan annually selects influenza
vaccine strains according to the WHO’s recommendation withadditional consideration of the characteristics of circulating
viruses around Japan, proliferation and yield of candidate vaccine
strains, and the extent of antigenic matching between circulating
viruses and candidate vaccine strains. For the A(H3N2) component of the 2017–18 influenza vaccine strain in Japan, the MHLW
selected A/Saitama/103/2014 (CEXP-002) on May 2, 2017, withprioritization to antigenic matching. However, at an early stageof the manufacturing process for A/Saitama /103/2014 (CEXP002), the protein recovery was too low to meet the vaccine
demand in Japan: only 33% compared with production in the previous season. On July 12, 2017, the A(H3N2) vaccine strain was
switched to A/Hong Kong/4801/2014 (X-263) [9], which was
included in the previous season’s influenza vaccine in Japan andwas selected as one of the recommended strains for the 2017–18 season by the WHO, resulting in a vaccine shortage at the
beginning of the 2017–18 influenza season. There was also a criticism that A/Saitama/103/2014 (CEXP-002) should had been used
to provide ‘‘effective” vaccination of the public, together with thepreventive use of antiviral medication to cope with potential vaccine shortage. These arguments raised a clinical question ofwhether the vaccine containing A/Saitama/103/2014 (CEXP-002)
strain was ‘‘better” than that containing the A/HongKong/4801/2014 (X-263) strain.
Although vaccinations are expected to provide maximal protection when antigenic matching is high [10–12], antigenic similarity
between a vaccine and circulating strains confirmed by postinfected ferret sera may not necessarily correlate with clinical vaccine effectiveness as indicated in some studies [12–14]. Furthermore, no reports have directly examined the vaccine
effectiveness of multiple strains of the same subtype but with different antigenicity in single season. In this trial, we specially prepared A/Hong Kong/4801/2014 (X-263) and A/Saitama/103/2014(CEXP-002) monovalent vaccines and compared their immunogenicity as a surrogate measure of vaccine effectiveness, toenhance our understanding of the correlation between antigenicity
in experimental models and immunogenicity in the humanpopulation.

2.1. Monovalent vaccines
Two types of A(H3N2) monovalent vaccine, the Hong Kong vaccine (lot number: HAR01) containing A/Hong Kong/4801/2014 (X263) virus strain and the Saitama vaccine (lot number: HAT01)
containing the A/Saitama/103/2014 (CEXP-002) virus strain, were
produced and supplied by the Biken Foundation (Suita, Japan),which is licensed to manufacture commercial influenza vaccine
products in Japan. They were produced using the same procedure
as commercially available quadrivalent, inactivated, unadjuvanted,
egg-cultured, split-virus influenza vaccines in Japan. These preparations contained the active ingredient equivalent to 30 lg of HA
protein in 1 ml (i.e., 15 lg of HA per 0.5 ml).
2.2. Study design and subjects
This single-center, open-labelled, randomized controlled trial
was conducted from September to November 2017. A total of
100 healthy Japanese adults were recruited between midSeptember and early October through a volunteer panel for clinical
trials managed by SOUSEIKAI, Fukuoka, Japan. Subjects were eligible if they met the following criteria: (1) aged 20–64 years, (2) not
vaccinated for the 2017–18 seasonal influenza vaccine, and (3) notplanning to receive a seasonal influenza vaccine until the final
serum sample collection in this trial that was scheduled 3 weeks
after vaccination. Exclusion criteria were: fever, severe acute illness, allergy to any component of the vaccines used for this trial,
eggs, chicken, or any derivative of chicken. Subjects were also
excluded if they had received any live vaccine within 27 days or
any inactivated vaccine within 6 days. For recruitment, a total of
10 strata by sex and age were created a priori and a fixed numberof subjects was recruited for each stratum (25 subjects for 20–29/30–39 year groups, 20 subjects for 40–49/50–59 year groups,
and 10 subjects for the 60–64 year group, in both sexes).
Subjects were randomly allocated to receive the Hong Kong
vaccine (n = 50) or the Saitama vaccine (n = 50) by blocked randomization with a block size of 4. We did not employ stratified
randomization for allocation. Sample size calculations were notconducted in advance, because this trial involved monovalentinfluenza vaccines that were specially prepared under the requestof the MHLW Japan and thus financial resources for the trial werelimited.
Written consent was obtained from all subjects. This study was
performed in accordance with the Declaration of Helsinki, and thestudy protocol was approved by the ethics committee at SOUSEIKAI PS Clinic (No. K-144), Osaka City University Graduate Schoolof Medicine (No. 3276) and Osaka Institute of Public Health (No.
1711-01).
2.3. Information collection, vaccination and serum sample collection
At recruitment, a self-administrated questionnaire was used tocollect the subjects’ baseline information including sex, age, height,
body weight, smoking status, history of influenza vaccination andinfluenza diagnosis during the previous 3 seasons. Subjects
received either the Hong Kong vaccine or the Saitama vaccine via
a 0.5 ml subcutaneous injection into the extensor side of the upperarm opposite the dominant arm using a 27G needle. The subjects
were observed for 30 min following vaccination to monitor allergic
reactions and to respond and treat them quickly if they occurred.
To assess the safety of each vaccine, information on adverse
reactions (local and systemic) occurring within one week following
vaccination was collected using a self-administered questionnaire.

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

T. Kase et al. / Vaccine xxx (xxxx) xxx

Solicited adverse reactions included 6 localized reactions (redness,swelling, induration, pain, itching, and heat sensation) and 7 systemic reactions (fever, fatigue, myalgia/arthralgia, headache, nausea, diarrhea, and rash). Information on fever was collected using
8 categories (no fever, 37.0–39.9 °C by 0.5 °C, and 40.0 °C). Other
local/systemic reactions were reported with 3 categories of severity (mild, moderate and severe). Definitions of ‘‘mild/moderate/severe” reactions were as follows: ‘‘within a few centimeters within
the elbow or shoulder/spreading over the elbow or shoulder” forredness, swelling, and induration; ‘‘no requirement for medication/requirement for medication/requirement for medication withother symptoms” for rash; and ‘‘no influence on daily life/influence
on daily life/substantial influence on daily life” for other reactions.
Subjects were also asked to provide any unsolicited adverse reactions or severe adverse events following immunization in an
open-ended form.
On day 0 (i.e., pre-vaccination) and day 21 after vaccination,
blood samples were collected and centrifuged to separate supernatant and precipitation. Sample collection was completed in earlyNovember, before the start of the 2017–18 influenza season in
Japan (20 November 2017), as declared by the NIID Japan. The
serum samples were then stored at 20 °C until the neutralization
test was performed.
2.4. Measurement of antibody titers
Antigenic characterization of recent circulating A(H3N2) viruses
using an hemagglutination inhibition (HI) assay is technically difficult because many viruses do not agglutinate red blood cells.
Indeed, virus neutralization assays have supplemented HI assays
for the antigenic characterization of viruses [5]. In this trial, neutralization antibody titers were measured for each of the virus
strains contained in the vaccines and for a currently circulatingstrain [15–17]. In brief, serum samples were treated with a receptor destroying enzyme (Denka Seiken, Tokyo, Japan) and inactivated for 60 min at 56 °C to remove non-specific inhibitors. A
1:10 dilution of treated sera was prepared with phosphatebuffered saline. Two-fold serial dilutions of sera were mixed with
one hundred 50% tissue culture infectious doses (TCID50) of theinfluenza viruses and were preincubated for 60 min at 37 °C. The
mixtures were inoculated onto MDCK-SIAT1 cells (KAC, Kyoto,
Japan) in duplicate in 96-well plates and then incubated for
60 min at 37 °C. The supernatant was removed and Dulbecco’s
modified Eagle’s medium–high glucose (Sigma-Aldrich D6429)
with 3 lg/ml of acetylated trypsin (Sigma-Aldrich T6763) was
added into the wells for influenza viral growth. Then, the plates
were incubated for 4 days at 35 °C in a CO2 incubator. To determine
the neutralizing activity of the test sera, the cytopathic effect (CPE)
was observed using an inverted microscope after the cells had been
stained with 1% amid black solution to obtain the best image. The
neutralizing titer was defined as the reciprocal of the highest
serum dilution at which the infectivity of 100 TCID50 of the challenged virus was neutralized in 100% of the wells. The neutralizing
titers were calculated as the average of duplicates. Negative samples were assigned a titer of 1:5.
A/Hong Kong/4801/2014 and A/Saitama /103/1014 virus strains
were provided by the Influenza Virus Research Center, NIID, Tokyo,
Japan. A/Osaka/188/2017, belonging to the 2C1a sub-clade ofA(H3N2) strains, was isolated with MDCK-SIAT1 cells in Osaka
prefecture Japan in December 2017.
2.5. Statistical analyses
Four types of measurements were used to compare theimmunogenicity between the groups: geometric mean titer(GMT), mean fold rise (MFR), seroconversion proportion (SCP,3

4-fold rise), and seroprotection proportion (SPP, postvaccination titer 1:40). For data processing, titers <1:10 and titers
1:1280 were regarded as 1:5 and 1:1280, respectively. Reciprocal
antibody titers were analyzed after logarithmic transformation,
followed by antilogarithm to present the values in the original
scale. We also compared the occurrence of adverse reactions. Differences between the groups were assessed using the Wilcoxon
rank sum test, chi-squared test, or Fisher’s exact test as
appropriate.
Immunogenicity was also evaluated using stratification by age.
Considering the potential effect of HA imprinting, we categorized
the subjects according to the following three age groups: 20–
39 years (born after 1977, with first exposure to A[H1N1] or A
[H3N2]), 40–48 years (born between 1968 and 1977, with first
exposure to A[H3N2]), and 49–64 years (born before 1968, with
first exposure to A[H2N2] or A[H1N1]) [18].
All hypothesis testing was conducted assuming a 0.05 significance level and a two-sided alternative hypothesis. SAS software
version 9.3 (SAS Institute, Inc., Cary, NC) was used for all analyses.

3. Results
The distributions of subject background characteristics including sex, age, body weight, body mass index, smoking status, antibody titers prior to vaccination, history of influenza vaccination,and history of influenza diagnosis were similar between the Hong
Kong vaccine group and the Saitama vaccine group (Table 1). No
significant differences were observed.
Within 48 h after vaccination, many mild local adverse reactions were observed in both groups, while redness, pain or itchingwere observed only in the Saitama vaccine group from 48 h to
1 week (Fig. 1). No subjects reported moderate or severe local reactions. Three subjects (6%) in the Saitama vaccine group reported afever of 37.0–37.4 °C within 48 h after vaccination. With regard toother systemic reactions, a very small number of subjects experienced mild reactions including fatigue, headache or diarrhea(Fig. 2). No subjects reported moderate or severe systemic reactions, except for one subject (2%) in the Saitama vaccine group
who reported moderate fatigue from 48 h to 1 week following vaccination. There were no significant differences between the groups.
Neutralization antibody titers in pre- and post-vaccinationgroups are shown in Fig. 3, where all data points are plotted. Table 2
shows four types of measurements of immunogenicity against
three kinds of strains in the two vaccine groups. For A/HongKong/4801/2014, the post-vaccination GMT was significantly
higher for the Hong Kong vaccine recipients (1:546 vs 1:260,
p < 0.01), but the MFR (5.5 vs 4.5, p = 0.34), SCP (50% vs 40%,p = 0.31), and SPP (96% vs 92%, P = 0.68) were similar for both vaccine groups. For A/Saitama/103/2014, the post-vaccination GMT(1:116 vs 1:61, p = 0.01) and SPP (86% vs 68%, p = 0.03) were significantly higher in the Hong Kong vaccine recipients, but the MFR(5.3 vs 4.2, p = 0.60) and SCP (50% vs 46%, p = 0.69) were similar forboth vaccine groups. Against the A/Osaka/188/ 2017 virus, thepost-vaccination GMT (1:17 vs 1:9, p = 0.20) and MFR (2.7 vs 1.3,
p = 0.16) were similar in both vaccine groups, but the SCP (32%
vs 4%, p < 0.01) and SPP (28% vs 6%, p < 0.01) were significantly
higher for the Hong Kong vaccine recipients. The Hong Kong vaccine recipients also showed a higher SPP with a cut-off value of
1:20 (36% vs 10%, p < 0.01).
Stratified analyses by age groups are shown in Table 3. The
overall findings were similar to those in all subjects, although thestatistical significance was lost because of the small number ofsubjects in each group. Post-vaccination GMT against A/HongKong/4801/2014 or A/Saitama/103/2014 and SPP against A/Saitama/103/2014 was higher in the Hong Kong vaccine recipients

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

4

T. Kase et al. / Vaccine xxx (xxxx) xxx

Table 1
Characteristics of the subjects.
Hong Kong vaccine groupn (%) or mean [SD]Saitama vaccine groupn (%) or mean [SD]Male sex

25

(50)

25

(50)

Age (years)
20–29
30–39
40–49
50–59
60–64

13
14
9
8
6

(26)
(28)
(18)
(16)
(12)

12
11
11
12
4

(24)
(22)
(22)
(24)
(8)

Age (years)
20–39 [born after 1977]
40–48 [born between 1968 and 1977]
49–64 [born before 1968]

27
8
15

(54)
(16)
(30)

23
9
18

(46)
(18)
(36)

Body weight (kg)Body mass index (kg/m2)

61.9
22.5

[11.7]
[3.4]

61.9
22.7

[13.8][3.4]

Smoking status
Never
FormerCurrent

35
4
11

(70)
(8)
(22)

37
5
8

(74)
(10)
(16)

5
3
2
40

(10)
(6)
(4)
(80)

8
5
7
30

(16)
(10)
(14)
(60)

15
5
11
19

(30)
(10)
(22)
(38)

22
4
11
13

(44)
(8)
(22)
(26)

40
7
2
1

(80)
(14)
(4)
(2)

41
5
2
2

(82)
(10)
(4)
(4)

37
9
4

(74)
(18)
(8)

33
9
8

(66)
(18)
(16)

40
8
2

(80)
(16)
(4)

35
10
5

(70)
(20)
(10)

41
7
2

(82)
(14)
(4)

40
8
2

(80)
(16)
(4)

38
1
0
7
4

(76)
(2)
(0)
(14)
(8)

44
0
0
5
1

(88)
(0)
(0)
(10)
(2)

44
2
0
2
2

(88)
(4)
(0)
(4)
(4)

46
2
0
1
1

(92)
(4)
(0)
(2)
(2)

45
2
0
2
1

(90)
(4)
(0)
(4)
(2)

44
2
1
3
0

(48)
(4)
(2)
(6)
(0)

Pre-titer
A/Hong Kong/4801/2014
<10
10
20
40
A/Saitama/103/2014
<10
10
20
40
A/Osaka/188/2017
<10
10
20
40
Previous influenza vaccination
2014–15 season
No
Yes
Unknown
2015–16 season
No
Yes
Unknown
2016–17 season
No
Yes
Unknown
Previous influenza diagnosis
2014–15 season
No
Type A
Type B
Types unknown
Unknown
2015–16 season
No
Type A
Type B
Types unknown
Unknown
2016–17 season
No
Type A
Type B
Types unknown
Unknown

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

T. Kase et al. / Vaccine xxx (xxxx) xxx

5

Fig. 1. Proportion (%) of mild local adverse reactions following vaccination within 48 h (A) and from 48 h to 1 week (B) after vaccination. Mild local adverse reactions were
defined as ‘‘within a few centimeters” for redness, swelling and induration and ‘‘no influence on daily life” for pain, itching and heat sensation.

Fig. 2. Proportion (%) of mild systemic adverse reactions following vaccination within 48 h (A) and from 48 h to 1 week (B) after vaccination. Mild systemic adverse reactions
were defined as ‘‘no influence on daily life” for fatigue, myalgia/arthralgia, headache, nausea, and diarrhea, and ‘‘no requirement for medication” for rash.

regardless of age. Against the A/Osaka/188/2017 virus, the Hong
Kong vaccine recipients showed a higher SCP and SPP in each age
group. Focusing on immunogenicity in the Hong Kong vaccine
recipients, higher responses were observed in subjects aged 40–
48 years (born between 1968 and 1977, with first exposure to A
[H3N2]) and 49–64 years (born before 1968, with first exposure
to A[H2N2] or A[H1N1]) than those aged 20–39 years (born after
1977, with first exposure to A[H1N1] or A[H3N2]).

4. Discussion
Our trial showed that the Hong Kong vaccine induced good
immune responses not only against its homosubtypic, A/HongKong/4801/2014, but also against the heterosubtypic, A/Saitama/103/2014. Antibody responses against A/Saitama/103/2014
induced by the Hong Kong vaccine were similar to or better thanthose induced by the Saitama vaccine. Furthermore, the Hong Kong
vaccine exhibited a superior ability to induce antibody against aseasonal wild-type strain (i.e., A/Osaka/188/2017) compared with
the Saitama vaccine. Overall, the Hong Kong vaccine showed better
immunogenicity than the Saitama vaccine against a broader range
of influenza virus strains. Similar findings were also observed after
stratification by age. Our trial clearly showed that antigenic similarity between vaccine strains and circulating strains in the previous season (i.e., predicted epidemic strains in the upcoming
season) in experimental models does not necessarily correlate withimmunogenicity in the human population, probably because
humans have previous experience of virus exposure or vaccination[19–22].
Although comparing the clinical effectiveness, not immunogenicity, of the Saitama vaccine and the Hong Kong vaccine in ahuman population would be desirable to obtain direct evidence,
this is impossible under real-world settings because A/Saitama/103/2014 (CEXP-002) was not included in the commerciallydistributed influenza vaccine during the 2017–18 season in Japan.
The serum antibody titer is not a direct measurement of vaccine
effectiveness, rather it is a surrogate variable. Nevertheless,because it is expected that strong immunogenicity induced by avaccine strain induces a moderate level of antibodies against circulating strains in the influenza season [23], excellent immunogenicity may predict high influenza vaccine effectiveness [24].
Significantly higher GMT or SPP were observed in Hong Kong
vaccine recipients compared with Saitama vaccine recipients
against their homosubtypic or heterosubtypic vaccine strain. However, both vaccines induced a sufficient antibody response inaccord with the international licensing criteria. The European
Medicines Agency, Committee for Medicinal Products for Human
Use (CHMP) has established criteria as follows: MFR > 2.5,
SCP > 40%, and SPP > 70% for adults aged 18–60 years when HI
titers are assayed against the prototype strain of the vaccine [25].
Our study vaccines almost met these three criteria, although neutralization antibody titers, not HI titers, were measured. To explore
the possible reason for the different immunogenicity between the

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

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T. Kase et al. / Vaccine xxx (xxxx) xxx

Fig. 3. Antibody titers in two vaccine groups. All data points for neutralization antibody titers in pre- and post-vaccination groups are shown as scatter dot plots. Against A/
Hong Kong/4801/2014 in the Saitama vaccine group (A) and Hong Kong vaccine group (B), against A/Saitama/103/2014 in the Saitama vaccine group (C) and the Hong Kong
vaccine group (D), and against A/Osaka/188/2017 in the Saitama vaccine group (E) and the Hong Kong vaccine group (F) are shown. S0 on X axes and S1 on Y axes indicate day
0 (pre) and day 21 (post) vaccination, respectively.

vaccines, HA sequences of the two vaccine strains and the circulating strain were evaluated (Supplementary Fig. 1). Amino acidchanges (T160K or L194P substitution at an antigenic site in the
HA of recent H3N2 viruses) introduced through egg passaging

affected antigenicity. A/Hong Kong/4801/2014 (X-263) has both
T160K and L194P substitutions, while A/Saitama/103/2014(CEXP-002) only has the T160K substitution. No changes were
found in the circulating strain of A/Osaka/188/2017 at both posi-

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

7

T. Kase et al. / Vaccine xxx (xxxx) xxx
Table 2
Neutralizing antibody titers against three influenza virus strains.
GMT

SCP (%)SPP (%)S0

S1

MFR (S1/S0)(S1/S0  4)

(S1  1:40)(S1  1:20)

Against A/Hong Kong/4801/2014
Hong Kong vaccine group
Saitama vaccine group
P value*

99
57
—

546
260
<0.01

5.5
4.5
0.34

50
40
0.31

96
92
0.68

—
—
—

Against A/Saitama/103/2014
Hong Kong vaccine group
Saitama vaccine group
P value*

22
15
—

116
61
0.01

5.3
4.2
0.60

50
46
0.69

86
68
0.03

—
—
—

Against A/Osaka/188/2017
Hong Kong vaccine group
Saitama vaccine group
P value*

6
6
—

17
9
0.20

2.7
1.3
0.16

32
4
<0.01

28
6
<0.01

36
10
<0.01

GMT: geometric mean titer; MFR: mean fold rise; SCP: seroconversion proportion; SPP: seroprotection proportion; S0: sera at pre-vaccination; S1: sera at post-vaccination.
*
Wilcoxon rank sum test, chi-squared test or Fisher’s exact test, as appropriate.

Table 3Age-stratified neutralizing antibody titers against three influenza virus strains.
GMT

SCP (%)SPP (%)S0

S1

MFR (S1/S0)(S1/S0  4)

(S1  1:40)(S1  1:20)

146
113
—

435
315
0.21

3.0
2.8
0.43

33
30
>0.99

96
100
>0.99

—
—
—

40–48 years old [born between 1968 and 1977]
Hong Kong vaccine group (n = 8)
77
Saitama vaccine group (n = 9)34
P value*
—

905
132
0.02

11.8
3.8
0.33

75
33
0.15

100
78
0.47

—
—
—

49–64 years old [born before 1968]
Hong Kong vaccine group (n = 15)
Saitama vaccine group (n = 18)
P value*

55
31
—

625
285
0.07

11.3
9.2
0.77

67
56
0.72

93
89
>0.99

—
—
—

Against A/Saitama/103/2014
20–39 years old [born after 1977]
Hong Kong vaccine group (n = 27)
Saitama vaccine group (n = 23)
P value*

29
22
—

81
60
0.23

2.8
2.7
0.70

33
35
>0.99

85
70
0.30

—
—
—

40–48 years old [born between 1968 and 1977]
Hong Kong vaccine group (n = 8)
15
Saitama vaccine group (n = 9)11
P value*
—

190
38
0.04

12.3
3.6
0.18

75
33
0.15

88
44
0.13

—
—
—

49–64 years old [born before 1968]
Hong Kong vaccine group (n = 15)
Saitama vaccine group (n = 18)
P value*

16
10
—

168
78
0.08

10.8
8.0
0.58

67
67
>0.99

87
78
0.66

—
—
—

Against A/Osaka/188/2017
20–39 years old [born after 1977]
Hong Kong vaccine group (n = 27)
Saitama vaccine group (n = 23)
P value*

7
8
—

11
10
0.96

1.7
1.3
0.76

22
4
0.11

15
13
>0.99

30
13
0.19

40–48 years old [born between 1968 and 1977]
Hong Kong vaccine group (n = 8)
6
Saitama vaccine group (n = 9)7
P value*
—

38
7
0.15

5.9
1.1
0.25

50
0
0.03

50
0
0.03

50
11
0.13

49–64 years old [born before 1968]
Hong Kong vaccine group (n = 15)
Saitama vaccine group (n = 18)
P value*

22
8
0.28

4.0
1.5
0.24

40
6
0.03

40
0
<0.01

40
6
0.03

Against A/Hong Kong/4801/2014
20–39 years old [born after 1977]
Hong Kong vaccine group (n = 27)
Saitama vaccine group (n = 23)
P value*

6
5—

GMT: geometric mean titer; MFR: mean fold rise; SCP: seroconversion proportion; SPP: seroprotection proportion; S0: sera at pre-vaccination; S1: sera at post-vaccination.
*
Wilcoxon rank sum test for GMT and MFR, Fisher’s exact test for SCP and SPP.

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

8

T. Kase et al. / Vaccine xxx (xxxx) xxx

tions because it was isolated by cell-culture. Because a T160K substitution in the glycosylation site was more important in terms ofantigenicity [4], it is reasonable to suggest that the two vaccine
strains in this trial were genetically similar. Our findings indicatethat some influenza vaccine strains can maintain antigenicity eventhough substantial egg-adapted changes occur. This interpretation
might be in line with a report where the WHO recommended A/
Hong Kong/4801/2014-like viruses for the A(H3N2) vaccine strain
in the 2017–18 season because most recent isolated A(H3N2)
viruses were inhibited by ferret antisera raised against cell
culture-propagated reference viruses [5].
Our trial with unique monovalent vaccines provided a supplemental opportunity to evaluate the phenomenon of imprinting,or ‘‘original antigenic sin”, indicating that the development of
immunity against pathogens is shaped by the first exposure to a
related pathogen [26]. Regarding the possible imprinting of influenza virus using human sera, measurable differences in antibody
response according to the potential first exposure to influenza A
virus subtypes (A[H1N1], A[H2N2] and A[H3N2]) was reported[27], whereas another study reported a greater response by monovalent A(H1N1)pdm09 vaccine against the A(H1N1) pdm09 virus
rather than against the A(H1N1) virus that circulated during thechildhood of each subject [28]. Similar to the latter finding, oursubgroup analysis did not show a clear effect of imprinting. The
monovalent Hong Kong vaccine-induced antibody response
against three A(H3N2) viruses was generally high in subjects aged
40–48 years (first exposure: A[H3N2]) and aged 49–64 years (first
exposure: A[H1N1] or A[H2N2]). Overall, the lowest immunogenicity was in the youngest age group (i.e., 20–39 years, with first
exposure to A[H1N1] or A[H3N2]), although they almost met the
CHMP criteria. This indicates the importance of factors other than
the first viral exposure.
Almost all adverse reactions were mild and their frequency was
similar between the groups. Our safety profiles cannot be directlycompared with that of conventional seasonal inactivated vaccines
because the vaccines in this trial were monovalent. However, ourfrequency was similar to or lower than those in previous clinical
trials in which a monovalent, inactivated, unadjuvanted, splitvirus A(H1N1) pdm09 vaccine with 15 lg of HA per 0.5 ml wasadministered intramuscularly [29–32], except for the frequencyof redness or swelling (16%) which may attribute to the subcutaneous injection route of administration in our trial. Overall, ourstudy vaccines were well tolerated.
Strengths of our study included the unique use of speciallyprepared monovalent influenza vaccines, which enabled us toevaluate a real-world-like reaction of human antibody responses
by a single vaccine strain. Despite the constraints of time andmanufacturing processes, the trial was completed before theinfluenza epidemic, leading to a straightforward interpretationof the results without the effect of the natural infection of
influenza.
Several limitations should be considered. Because study participants were only healthy adults aged 20–64 years, the representativeness of the findings is limited. Children, adolescents aged
19 years or younger, and the elderly aged 65 or more, who may
have different immunological status from the study population,
were not evaluated in this trial. The size of the clinical trial was
small because of the feasibility of the study and budget issues.
Antibody responses against circulating strains did not meet the
CHMP criteria. Because the international licensing criteria does
not require evaluation against circulating strains and few studies
have examined such immunogenicity, it cannot be determined
whether the induced antibodies against circulating strains in thistrial were sufficient. We used only one isolated strain belonging
to the 2C1a sub-clade to determine immunogenicity against circu-

lating viruses consisting of many sub-clades. Of note, vaccinatedhuman sera may react differently to viruses belonging to othersub-clades. Finally, information on the total protein or neuraminidase content of the study vaccine was not available. This
may affect the immunogenicity of the vaccines despite standardization by HA content.
5. Conclusions
The Hong Kong vaccine showed good immunogenicity, not only
against its homosubtypic, A/Hong Kong/4801/2014, but also
against the heterosubtypic, A/Saitama/103/2014. Moreover, theHong Kong vaccine showed better immunogenicity against a seasonal wild-type strain compared with the Saitama vaccine. Ourfindings demonstrate that antigenic similarity between a predictedinfluenza epidemic strain and the influenza vaccine strain in experimental animal models does not necessarily correlate withimmunogenicity in a human population. The same explanation
might be used for vaccine effectiveness, which should be investigated in further studies.
CRediT authorship contribution statement
Tetsuo Kase: Conceptualization, Methodology, Investigation,
Data curation, Validation, Writing - original draft, Writing - review& editing. Megumi Inoue: Project administration, Methodology,
Investigation. Saeko Morikawa: Methodology, Investigation. Hiroko Kumashiro: Project administration, Resources. Satoshi Hiroi:
Methodology, Investigation. Keiko Nakata: Methodology, Investigation. Kazuya Ito: Formal analysis. Motoki Ishibashi: Resources.
Tomomi Tsuru: Resources. Shin Irie: Resources, Supervision.
Akiko Maeda: Supervision. Satoko Ohfuji: Formal analysis, Supervision. Wakaba Fukushima: Conceptualization, Project administration, Formal analysis, Data curation, Validation, Writing original draft, Writing - review & editing. Yoshio Hirota: Conceptualization, Funding acquisition, Data curation, Validation,
Supervision.
Declaration of Competing Interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competinginterests: ‘TK has received a reward for advisor tasks from The Biken
Foundation from April 2016 to March 2018. The other authors havenothing to declare’.
Acknowledgements
We thank Dr. Shinji Watanabe and Dr. Takato Odagiri in the
Influenza Virus Research Center, NIID, Tokyo, Japan, for providing
vaccine strains. We also thank Dr. Kazushi Motomura and Dr.
Yoshinobu Okuno in the Osaka Institute of Public Health, Osaka,
Japan, for allowing us to use laboratory equipment for the neutralization assays. We thank all the staff at the SOUSEIKAI PS Clinic,
Fukuoka, Japan, for helping with information collection, vaccination and serum sample collection.
Funding
This study was funded by a research grant for Research on
Emerging/Re-emerging Infectious Diseases and Promoting Vaccination Policy from the Ministry of Health, Labour and Welfare,Japan (H29-SHINKOGYOSEI-SHITEI-003).

Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025

T. Kase et al. / Vaccine xxx (xxxx) xxx

Appendix A. Supplementary material
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.vaccine.2020.08.025.
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Please cite this article as: T. Kase, M. Inoue, S. Morikawa et al., Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains
in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263), Vaccine,
https://doi.org/10.1016/j.vaccine.2020.08.025