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 Journal of Clinical Virology 46 (2009) 161–164

Contents lists available at ScienceDirect

 Journal of Clinical Virology

 j o u r n a l h o m e p a g e :   w w w . e l s e v i e r . c o m / l o c a t e / j c v

Prevalence of influenza A and B antibodies in pregnant

women and their offspring

Peter Wutzler a, Ruprecht Schmidt-Ott b, Heike Hoyer c, Andreas Sauerbreia,∗

a Institute of Virology and Antiviral Therapy, Friedrich-Schiller University of Jena, Hans-Knoell-Straße 2, 07745 Jena, Germanyb GlaxoSmithKline, Theresienhöhe 11, 80339 Munich, Germanyc Institute of Medical Statistics, Computer Sciences and Documentation, Friedrich-Schiller University, Bachstraße 18, 07743 Jena, Germany

a r t i c l e i n f o

 Article history:Received 5 December 2008

Received in revised form 24 June 2009

Accepted 24 June 2009

Keywords:

Influenza

Antibody prevalence

Placental antibody transfer

a b s t r a c t

Background: Influenza is associated with substantial morbidity and mortality in pregnant women andneonates, but few countriesoffer annualinfluenzavaccinationwith theinactivated vaccine to all women

who are, or intend to become, pregnant.

Objectives: To provide seroepidemiological information on influenza A and B antibodies in pregnant

women and their offspring in Germany.

Study design: Anti-influenza antibodies were determined using commercially available enzyme-linked

immunosorbent assays (ELISA) on serum obtained from 209 women and their newborns at delivery.

Results: Theprevalenceof antibodies against influenza A virus was 93.8% [89.6–96.6%]in themothersand

96.7% [93.2–98.6%] in the newborns. The prevalence of antibodies against influenza B virus was 42.1%

[35.3–49.1%] in themothers and78.5%[72.3–83.8%] in theirnewborns, which wasa significant difference.

The antibody concentrations against both influenza A and influenza B viruses were significantly lower in

mother than in their newborns.

Conclusions: Because of active placental transport of IgG antibodies, neonates have higher prevalence

and/or concentrations of influenza A and B virus-specific antibodies induced by natural infections than

their mothers. Considering these serological findings, especially the lower prevalence of maternal anti-

body against influenza B virus, annual influenza vaccination may improve the protection of pregnantwomen and their offspring against influenza.

© 2009 Elsevier B.V. All rights reserved.

1. Background

The most significant respiratory infection that affects pregnant

women with the risk of severe illness and death is influenza.

Women of child-bearing age have increased exposure to young

children who have a high prevalence of infection during influenza

epidemics.1 The rate of seasonal influenza infection during the

secondand thirdtrimester isreported torange from 2%to 22%.2 Epi-

demiological studies during the past 25 years document increasedrates of influenza in pregnant women compared with healthy non-

pregnant women.3,4 Influenza-associated excess deaths among

pregnant women were documented during the pandemic “Spanish

Flu” of 1918–1919 and the pandemic “Asian Flu” of 1957–1958.5,6

Providing pregnant women withannual influenza vaccination is

considered the mosteffective public health intervention to prevent

influenza during pregnancy. The American College of Obstetri-

∗ Corresponding author. Tel.: +36 41 9395700; fax: +36 41 9395702.

E-mail address:  Andreas.Sauerbrei@med.uni-jena.de (A. Sauerbrei).

cians and Gynecologists (ACOG), along with theCenters for Disease

Control and Prevention, recommend that all women who are, or

intend to become, pregnant during the influenza season should be

vaccinated with the inactivated influenza vaccine.7,8 In Germany,

influenza vaccine is not recommended for pregnant women.9 Such

a policy would not only be beneficial for pregnant women, but

their offspring might also be protected by placentally transmit-

ted maternal antibodies during the first months of life.10 Influenza

in neonates and very young children is associated with substan-tialmorbidity and occasionalmortality.11,12 R atesof hospitalization

during the influenza season are highest in very young children,

particularly those under 6 months of age, and in high-risk elderly

patients.13,14

2. Objectives

The objective of this study was to provide information on the

prevalence of influenza A and B antibodies in pregnant women and

their offspring in selected populations in Germany.

1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.jcv.2009.06.024

http://www.sciencedirect.com/science/journal/13866532http://www.elsevier.com/locate/jcvmailto:Andreas.Sauerbrei@med.uni-jena.dehttp://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.jcv.2009.06.024http://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.jcv.2009.06.024mailto:Andreas.Sauerbrei@med.uni-jena.dehttp://www.elsevier.com/locate/jcvhttp://www.sciencedirect.com/science/journal/13866532

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3. Study design

 3.1. Subjects and serum samples

Allhealthywomenwho delivered at theDistrict Hospital Rudol-

stadt, German federal state Thuringia, between January 1999 and

 January 2000, and their healthy newborn infants agreed to be con-

secutively enrolled in this study. At delivery, the women and their

offspring were clinicallyhealthyand routine laboratory parameters

were normal. Two hundred and nine women aged between 17 and

40 years (mean 26.1 years, standard deviation, SD 4.3) and their

offspring were included in this study. One hundred and eighty-

nine (89.0%) infants were delivered spontaneously; 13 (6.2%) by

caesarean section; and 10 (4.8%) by vacuum extraction, at gesta-

tional age of 34–42 weeks (mean 39.8 weeks, SD 1.3). Data on

maternal occupation and history of influenza immunization were

not available. Blood samples were taken from all women at the

time of delivery and from the umbilical cord of the neonates by the

gynaecologist shortly after birth. Contamination of newborn blood

withmaternalblood wasavoided.Maternaland newborn serawere

obtained without delay by centrifugation and stored in aliquots of 

0.5 mlat−20 ◦C forseveral yearsunderequal conditions that would

not lead to concentration of the infant samples. Informed consent

was obtained from the parents of the infants.

 3.2. Serological testing 

The Influenza Virus A IgG ELISA (no. INFG0290, lots INFAG-023

and INFAG-27) and Influenza Virus B IgG ELISA (no. INFG0300, lots

INFBG-024 and INFBG-29) manufactured by NovaTec Immundi-

agnostica (Dietzenbach, Germany) were used for qualitative and

semi-quantitative antibody testing. The micro-titration strip wells

were pre-coated with influenza virus type-specific antigens con-

sisting of matrix proteins and nucleoproteins of influenza A virus

strain Texas 1/77 (H3N2) and influenza B virus strain Hong Kong

5/72, respectively. Testing of sera was carried out according to the

manufacturer’s instructions. In short, sera diluted 1 in 100 were

incubated with antigen-coated wells, washed, and incubated with

horseradish peroxidase-labeled anti-human IgG. The wells were

then washed, after which they were incubated with tetramethyl-

benzidine substrate; sulfuric acid was added to stop the reaction.

Plateswere read photometrically at 450and 620nm. Samples were

considered positive if the absorbance value was higher than 10%

above the cut-off. Negative samples had an absorbance value lower

than 10% below the cut-off. Samples with an absorbance value less

10% above or below the cut-off were regarded as equivocal. By

using the arbitrary units “NovaTec Units” (NTU), a relative quan-

titation within the test was possible. The NTU was calculated with

the following formula:

mean absorbance× 10

cut-off   = [NovaTec Units = NTU]

The cut-off was defined by the manufacturer as 10 NTU; posi-

tive sera contained>11 NTUand negative sera correspondedto NTU

levels of

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P. Wutzler et al. / Journal of Clinical Virology 46 (2009) 161–164   163

 Table 2

Distribution of qualitativeIgG antibody againstinfluenza B virus (IBV) in newbornscompared withtheir mothers. The numbers represent the absolute numbers of newborns

tested negative, positive or equivocal for IBV antibodies. Percentages are given in parenthesis.

Antibodies against IBV of mothers Antibodies against IBV of newborns Summary

Negative Positive Equivocal

Negative 29 (13.9%) 40 (19.1%) 11 (5.3%) 80 (38.3%)

Positive 2 (1.0%) 86 (41.2%) 0 (0%) 88 (42.1%)

Equivocal 3 (1.4%) 38 (18.2%) 0 (0%) 41 (19.6%)

Summary 34 (16.3%) 164 (78.5%) 11 (5.3%) 209 (100%)

Fig. 1.  Boxplotof IgGantibodyconcentration[NovaTec-Units,NTU] againstinfluenza

A virus(IAV)and influenzaB virus(IBV) inmothers(m)andnewborns (n)at thetime

of deliveryshowingmedian, 25thand 75thpercentile, minimum, maximum and/or

outliers (values >1.5 inter-quartile range from the end of the box).

SD 5.1 NTU). Gestational age did not significantly influence IgG

concentrations against influenza B virus in the infants ( p = 0.314).However, newborns who were delivered by vacuum extraction

(n = 10) had significantly lower antibody concentrations (11.4 NTU,

SD 5.4NTU) than newborns who were delivered spontaneously

(n = 189, 14.5NTU, SD 5.0NTU) or by caesarean section (n = 13;

14.5NTU, SD 6.1 NTU) ( p = 0.046). Figs. 1 and 2 demonstrate the dis-

tribution of IgG antibody concentrations against influenza A and

influenza B virus in the mothers and newborns at the time of birth.

5. Discussion

Thehemagglutination inhibition(HI) assay andthecomplement

fixation (CF) test are used routinely for quantitation of influenza

virus-specific serumantibodies.15 However, theseassaysarelabori-

ous, difficult to incorporate into automatic procedures, and requirea readily available source of appropriate erythrocytes. ELISAs mea-

suring virus-specific serum IgG antibodies are more sensitive than

the HI or the CF assay.16–18 Thus, they are more suitable for the

determination of prevalence of antibodies against influenza A and

influenza B viruses. Furthermore, since the matrix and nucleopro-

tein antigens used in these ELISAs are highly conserved within

both the influenza A and influenza B viruses,19–21 we could detect

antibodies induced by influenza A and B viruses circulating dur-

ing the last few years without subtype-specific assay. The ELISA

assays do not require the annual adjustment of the viral anti-

gen preparations and are not affected by antibodies induced by

hemagglutinin antigens of split influenza vaccines that are used

currently in Germany. A limitation is that quantitative results

cannot be calibrated in the absence of an international stan-

Fig. 2.  Boxplot of mother–newborn pair differences of IgG antibody concentration

[NovaTec-Units, NTU] against influenza A virus (IAV) and influenza B virus (IBV) at

thetimeof deliveryshowingmedian,25thand75thpercentile,minimum, maximum

and/or outliers (values >1.5 inter-quartile range from the end of the box).

dard. Thus, the arbitrary NovaTec Units were used. Even though

antibodies to influenza virus matrix and nucleoprotein antigensmay not contribute to protection, they indicate the presence of 

subtype-independent T cell-mediated protection22 and may be

informative for the development and application of a new live

attenuated influenza vaccine inducing both humoral and cellular

immunity.

In thepresent study,the prevalenceof theanti-influenza B virus-

specific antibodies was significantly greater in neonates than in

their mothers. In addition, the antibody concentrations against the

influenza A virus as well as the influenza B virus were significantly

higher in newborns than in their mothers. The mean newborn-to-

mother ratios between cord and maternal blood were between 1.2

and 1.5 for antibodies against influenza A virus and between 1.3

and 1.7 for antibodies against influenza B virus. These data suggest

active placental transfer of maternal influenza virus-specific IgGduring the third trimester of pregnancy. Apart from the quantita-

tive differences, theprevalence of influenza B antibodyin newborns

supports this hypothesis. As shown in Table 2, 11 sera from new-

borns whose mothers were seronegative had equivocal results and

38 newbornswhosemothershad equivocal results tested antibody-

positive. Thus, a higher prevalence of antibodies in the cord blood

sera can be the consequence of actively transported antibodies if 

maternalconcentrationswere below thedetectable assay levels. No

significant association of antibody concentration with gestational

age was found for either influenza virus A or B. This is most likely

because only 10 out of 209 babies were born at a gestational age of 

≤37 weeks and only 3 after

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164   P. Wutzler et al. / Journal of Clinical Virology 46 (2009) 161–164

their offspring, but has been reported for antibodies to measles,

mumps, rubella, parainfluenza and varicella-zoster viruses, as well

astetanusantitoxin, diphtheriaantitoxinandanti-Bordetella pertus-

sis antibodies.23–28 Specific receptor-mediated binding of the Fc

portion of IgG at the maternal surface of the placenta has been

considered part of the mechanism involved in neonatal antibody

levels.29 Why newborns who were delivered by vacuum extrac-

tionhad significantly lower antibodylevels thanneonatesdelivered

spontaneously or by caesarean section cannot be explained. Since

only 10 newborns delivered by vacuum extraction were included

in this study, theanalysis of different factors that could have biased

this result is not possible.

The high prevalence of influenza A virus-specific antibodies

in pregnant women suggests high incidence of recent infection.

Depending on the persistence of ELISA antibodies, theseresults cor-

respond to the epidemiological situation in 1999 and 2000 when

the sera included in this study were taken from the mothers and

their newborns. During these seasons, there was a moderate activ-

ityof influenzaA viruses ofthe subtype H3N2 inGermany.30,31 Since

women at child-bearing agehaveincreased exposureto youngchil-

dren who have higher infection rates of influenza than adults,32,33

it is likely that these women have an increased risk of becoming

infected with influenza. However, this does not apply to women

having theirfirst child. Theconsequenceof thehighantibodypreva-lence of pregnant women is that nearly all neonates also acquired

antibodies. In the case of influenza, passive immunity is important

sincecurrentlyavailable influenza vaccines arenot indicatedbelow

6 months of age.

In contrast, there was a lower prevalence of maternal antibody

against influenza B virus, which also reflects the epidemiological

data reported in 1999 and 2000 in Germany. Whereas one third

of all isolated influenza viruses could be classified as influenza B

viruses in 1999, only a few influenza B virus isolates were obtained

in the following season.30,31,34 This would lead to a relatively low

number of booster infections caused by the influenza B virus dur-

ing these seasons. Although less common and usually associated

with lower mortalitythaninfluenza A, influenza B cancausesignifi-

cant morbidityand mortalityin neonates and other children.11,35,36Offering influenza vaccine to pregnant women, who will deliver

during influenza season, may potentially reduce the frequency and

severity of this neonatal disease.

Conflict of interest

There are no competing interests.

Ethical approval

The work has been approved by the Ethical Committee of the

University of Jena (Germany) and subjects gave informed consent

to the work.

 Acknowledgments

This work was supported by grants of the GlaxoSmithKline

GmbH & Co. KG, Munich, Germany.

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