Antibody Dynamics of 2009 Influenza A (H1N1) Virus in
Infected Patients and Vaccinated People in China
Ming Wang*., Jun Yuan., Tiegang Li., Yang Liu, Jibin Wu, Biao Di, Xi Chen, Xinhong Xu, Enjie Lu,
Kuibiao Li, Yanhui Liu, Yejian Wu, Xiongfei Chen, Peng He, Yulin Wang, Jianhua Liu
Guangzhou Center for Disease Control and Prevention, Guangzhou, People’s Republic of China
Background: To evaluate the risk of the recurrence and the efficiency of the vaccination, we followed-up antibody
responses in patients with the 2009 pandemic H1N1 influenza and persons who received the pandemic H1N1 vaccine in
Methods: We collected serum samples from 129 patients and 86 vaccinated persons at day 0, 15, 30, 180 after the disease
onset or the vaccination, respectively. Antibody titers in these serum samples were determined by haemagglutination
inhibition (HI) assay using a local isolated virus strain A/Guangdong Liwan/SWL1538/2009(H1N1).
Results: HI antibody positive rate of the patients increased significantly from 0% to 60% at day 15 (x2=78, P,0.001) and
100% at day 30 (x2=23, P,0.001), but decreased significantly to 52% at day 180 (x2=38, P,0.001), while that of vaccinated
subjects increased from 0% to 78% at day 15 (x2=110, P,0.001) and 81% at day 30 (x2=0.32, P=0.57), but decreased
significantly to 34% at day 180 (x2=39, P,0.001). Geometric mean titers (GMT) of HI antibodies in positive samples from the
patients did not change significantly between day 15 and day 30 (T=0.92, P=0.36), but it decreased significantly from 80 at
day 30 to 52 at day 180 (T=4.5, P,0.001). GMT of vaccinated persons increased significantly from 100 at day 15 to 193 at
day 30 (T=4.5, P,0.001), but deceased significantly to 74 at day 180 (T=5.1, P,0.001). Compared to the patients, the
vaccinated subjects showed lower seroconversion rate (x2=11, P,0.001; x2=5.9, P=0.015), but higher GMT (T=6.0,
P,0.001; T=3.6, P=0.001) at day 30 and day 180, respectively.
Conclusion: Vaccination of 2009 influenza A (H1N1) was effective. However, about half or more recovered patients and
vaccinated persons might have lost sufficient immunity against the recurrence of the viral infection after half a year.
Vaccination or re-vaccination may be necessary for prevention of the recurrence.
Citation: Wang M, Yuan J, Li T, Liu Y, Wu J, et al. (2011) Antibody Dynamics of 2009 Influenza A (H1N1) Virus in Infected Patients and Vaccinated People in
China. PLoS ONE 6(2): e16809. doi:10.1371/journal.pone.0016809
Editor: Clive Gray, University of Cape Town, South Africa
Received September 16, 2010; Accepted January 11, 2011; Published February 9, 2011
Copyright: ? 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the Ministry of Science and Technology of the People’s Republic of China (grant 2009ZX10004-306). The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
A pandemic influenza A (H1N1) virus spread worldwide since
April 2009, resulting in more than 16,000 deaths until March
2010. On 10 August 2010, WHO Director-General Dr Margaret
Chan announced that the H1N1 influenza virus has moved into
the post-pandemic period . Although 2009 pandemic influenza
A (H1N1) has been controlled, its recurrence cannot be excluded
yet . Guangzhou, the capital city of Guangdong province in
south China, is one of the earliest attacked areas by 2009
pandemic influenza A (H1N1) virus. An inactivated vaccine
against 2009 pandemic influenza A (H1N1) virus had been
urgently manufactured to be used as an economical and effective
weapon for the prophylaxis. To evaluate the risk of the recurrence
and the efficiency of the vaccination, we conducted a follow-up
study by detecting serum specimens collected from virus infected
cases in an outbreak of a boarding school and vaccinated people in
Guangzhou. The antibody dynamics characteristics would provide
useful information for evaluating risk of the potential recurrence
and efficacy of the vaccination.
We investigated antibody responses in 129 patients with the
pandemic influenza HIN1 and 86 persons who received the
pandemic H1N1 vaccine in Guangzhou China. Patients who
showed influenza symptoms, temperature $37.5u and viral RNA
and/or antibody seroconversion for the pandemic virus were
recruited in an outbreak of 2009 pandemic influenza H1N1 in a
boarding school from August 21stto October 15th, while
vaccinated study subjects were recruited from healthy persons
who received the vaccine provided by Ministry of Health of China
on October 30th2009. These patients or vaccinated persons
PLoS ONE | www.plosone.org1February 2011 | Volume 6 | Issue 2 | e16809
showed antibody negative to the pandemic virus (HI titer ,1:20)
at the onset day of the disease or when they received the
vaccination (day 0). Serum samples were collected from these
patients and vaccinated persons at day 0, 15, 30, 180 after the
onset of the disease or the vaccination, respectively. The ages of
the study subjects in patient group were from 14 to 20 years, and
that in vaccinated people were from 19 to 57 years. There are 86
males and 43 females in the patient group and 46 males and 40
females in vaccinated group.
The influenza A/H1N1 monovalent, split-virus, non-adju-
vanted vaccines were manufactured by Tianyuan Bio-Pharma-
ceutical Co., Ltd. (batch number 20090902) through the
nationwide vaccination program. Each dose of 0.5 ml product
contained 15 mg hemagglutinin as prescribed by national guide-
lines. The vaccine was administered through intramuscular
injection in the deltoid muscle.
This study was approved by the ethics committee of the
Guangzhou Center for Disease Control and Prevention and
written informed consent was obtained from the study subjects.
H1N1/2009 virus was detected by real-time RT-PCR as described
previously . Briefly, viral RNA was extracted from 140 mL
nasopharyngeal specimen using the QIAampH Viral RNA Mini
instructions. Viral RNA copies were determined by real-time
one-step RT-PCR assay using Invitrogen SuperScriptTMIII
PlatinumH One-Step Quantitative Kit (Invitrogen, Cat# 11732-
088) and primers/probe which sequences provided by WHO, i.e.
forward primer 59-GTG CTA TAA ACA CCA GCC TYC CA-
39, reverse primer 59-CGG GAT ATT CCT TAA TCC TGT
RGC-39, and probe 59-(FAM)CA GAA TAT ACA TCC RGT
CAC AAT TGG ARA A (TAMRA)-39. Reactions were first
incubated at 50uC for 30 min, denatured at 95uC for 2 min, and
then were thermal-cycled for 40 cycles (95uC for 15 sec, 55uC for
30 sec). Serially diluted positive viral RNA controls were used as
calibrators in each run.
Hemagglutination Inhibition (HI) Assay.
in these serum samples were determined by haemagglutination
inhibition (HI) assay as described previously [3,4]. The serum
samples were treated with receptor destroying enzyme (RDE) from
Vibrio cholerae (Denka Seiken, Cat#370013) for 18 h at 37uC
and then were heat-inactivated at 56uC for 30 min according to
WHO’s standard procedure. Serum samples were diluted in serial
two-fold dilutions from 1:20 to 1:640 and then mixed with 1%
suspension of chicken red blood cells and 4 hemagglutinating units
of a local isolated virus strain A/Guangdong Liwan/SWL1538/
2009(H1N1). Specific positive antiserum and negative serum
controls were included in the assay.
All the experiments were manipulated in the same laboratory of
Guangzhou center for disease control and prevention.
The existence of pandemic influenza
Data were analyzed using EpiInfo version 3.3.2 (CDC, USA)
and SPSS version 13.0 (SPSS Inc., USA). The statistically
significant criterion was P-value ,0.05. We compared the
seroconversion rate (HI $40) by x2-test between day 15 and day
30 or day 30 and day 180 or between natural infection and
vaccinated group. We compared geometric mean titers (GMT) of
Figure 1. HI antibody dynamics in infected patients and
vaccinated people. Note: Detection limitation (HI titer ,20) is
indicated by the dotted line. Error bar indicates 6 standard deviation
(SD) from different individual study subjects. * indicates significant
differences (P,0.01) between results of day 30 and day 180.
Table 1. Positive rate and GMT of 2009 H1N1 virus antibody in a follow-up study of natural infection patients and vaccinated
people, Guangzhou, 2009.
day15 day30 day180
day15day30 day180T value
natural infection 12960100 5223** 38**63 80 52 0.924.5**
vaccination 867881 34 0.3239** 100 19374 4.5** 5.1**
Antibody of 2009 Influenza A (H1N1) Virus
PLoS ONE | www.plosone.org2February 2011 | Volume 6 | Issue 2 | e16809
those with positive results by Independent-Samples T test between
day 15 and day 30 or day 30 and day 180 or between natural
infection and vaccinated group. For GMT calculations, antibody
levels below the detection limit (,1:20) were assigned the value of
Antibody seroconversion rate
HI antibody positive rate of the patients increased significantly
from 0% to 60% (95%CI: 26–88%) at day 15 (x2=78, P,0.001)
and 100% (95%CI: 93–100%) at day 30 (x2=23, P,0.001), but
decreased significantly to 52% (95%CI: 42–62%) at day 180
(x2=38, P,0.001), while that of vaccinated subjects increased
from 0% to 78% (95%CI: 68–86%) at day 15 (x2=110, P,0.001)
and 81% (95%CI: 72–89%) at day 30 (x2=0.32, P=0.57), but
decreased significantly to 34% (95%CI: 24–45%) at day 180
(x2=39, P,0.001) (Table 1). Compared to the patients, the
vaccinated subjects showed lower seroconversion rate (x2=11,
P,0.001; x2=5.9, P=0.015) at day 30 and day 180, respectively
(Fig. 1a). In the vaccinated group, we did not find a significant
difference in antibody seroconversion rate in different age groups
Geometric mean titers (GMT)
No significant difference was found in GMT of HI antibodies in
positive samples collected from the patients between 63 (95%CI:
30–135) at day 15 and 80 (95%CI: 68–93) at day 30 (T=0.92,
P=0.36), but it decreased significantly to 52 (95%CI: 47–58) at
day 180 (T=4.5, P,0.001). GMT of vaccinated persons increased
significantly from 100 (95%CI: 84–120) at day 15 to 193 (95%CI:
154–242) at day 30 (T=4.5, P,0.001), but deceased significantly
to 74 (95%CI: 63–89) at day 180 (T=5.1, P,0.001) (Table 1).
Compared to the patients, the vaccinated subjects showed higher
GMT at day 30 (T=6.0, P,0.001) and day 180 (T=3.6,
P=0.001), respectively (Fig. 1b). In the vaccinated group, we did
not find a significant difference in GMT between different age
groups (Table 2).
The 2009 pandemic influenza A (H1N1) virus is a completely
new infectious agent to human being. To investigate antibody
dynamics which induced by natural infection or vaccination of the
virus, we conducted this prospective study by following-up the
infected patients and vaccinated people in the same city for six
Consistent with previous reports [5–7], our results demonstrated
that the vaccination was effective. The antibody seroconversion
rate in vaccinated people achieved to a stable level more rapidly
than naturally infected patients (15 vs 30 days), which might be
due to the virus needs time to replicate after the infection.
However, only about 80% in vaccinated people acquired
protective antibody, whereas all subjects (100%) in the group of
infected patients yield protective antibody at 30 days after the
natural infection. It has been reported that seroconversion rates of
vaccinations with live virus vaccines reached 86–97% but that of
vaccination with inactivated vaccines achieved only 50–80% [8–
10]. Our results supported that live virus vaccines may be more
effective than inactivated vaccines. Furthermore, we found that
higher GMT of antibody was achieved in the vaccinated subjects
than in the infected patients. Previous studies also reported that
GMT in recipients of live attenuated influenza vaccines was lower
than that in recipients of inactivated vaccines [11,12]. In this
Table 2. Positive antibody and GMT in different age group of vaccinated people.
Antibody of 2009 Influenza A (H1N1) Virus
PLoS ONE | www.plosone.org3 February 2011 | Volume 6 | Issue 2 | e16809
regard, a boost dose may be help to improve protective antibody Download full-text
response for live virus vaccination.
In contrast with previous studies which reported that the
positive rate and GMT in children or elder persons were usually
lower than the other age groups , we did not find a significant
difference in antibody seroconversion rate and GMT levels in
different age groups of vaccinated people. This might be due to
that the age of study subjects recruited in this study were ranged
from 19 to 60 years, so that children and elder persons did not
In this study, we first reported that both positive rate and GMT
of antibodies to 2009 pandemic influenza A (H1N1) virus were
decreased quickly in the patients and vaccinated persons.
Antibody positive rates had been dropped down from 100% and
81% at day 30 to 52% and 34% at day 180, while GMTs were
decreased from 80 and 193 at day 30 to 52 and 74 at day 180 in
patients and vaccinated people, respective. The observation of low
level antibody responses to the pandemic influenza viral infection
and quick decrease of protective antibody levels may be able to
explain why some pandemic influenza patients acquired a re-
infection shortly as reported by Perez et al . Considering the
antibody levels tend to further decrease subsequently, these
recovered patients and vaccinated persons may probably have
no sufficient immunity against the recurrence of the viral infection.
Thus, vaccination or re-vaccination may be necessary for
prevention of the recurrence.
We thank Dr Robert E. Fontaine, Expert Advisors to CFETP, for his
valuable advice and guidance.
Conceived and designed the experiments: MW JY TL. Performed the
experiments: MW JY TL Yang Liu Xi Chen XX Yanhui Liu Y. Wang JW
BD EL KL Y. Wu PH. Analyzed the data: MW JY TL Xiongfei Chen JL.
Contributed reagents/materials/analysis tools: JW BD EL KL Y. Wu PH.
Wrote the paper: MW JY TL.
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