Published Ahead of Print 31 October 2012.
2013, 87(2):1261. DOI: 10.1128/JVI.02625-12.
Katherine V. Houser, Jacqueline M. Katz and Terrence M.
Virus in Ferrets
Emerging Variants of Influenza A (H3N2v)
Vaccine Does Not Protect against Newly
Seasonal Trivalent Inactivated Influenza
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Seasonal Trivalent Inactivated Influenza Vaccine Does Not Protect
against Newly Emerging Variants of Influenza A (H3N2v) Virus in
Katherine V. Houser,a,bJacqueline M. Katz,aTerrence M. Tumpeya
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USAa; Graduate Program in
Immunology and Molecular Pathogenesis of the Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, Georgia, USAb
species since the 1918 pandemic (2). The classical H1N1 virus
late 1990s, when human H3N2 viruses infected this species and
subsequently spread widely in North American pigs (3). Since
that time, multiple reassortment events that have presumably
occurred in swine have resulted in the emergence of an H3N2
The TRIG cassette, which shares host gene lineage origins with
the A(H1N1)pdm09 virus, highlights the public health threat
posed by swine-origin influenza subtypes (5).
Until recently, transmission of novel variants of H3N2
[A(H3N2)v] from swine to humans was rare, with only 7 con-
firmed cases documented in 2009-2010 (6–8). In 2011, public
health laboratories in the United States detected an additional 12
cases of human infection (9, 10), caused by a novel A(H3N2)v
virus that had acquired the M gene from A(H1N1)pdm09 virus
2012, there have been 307 additional confirmed cases (including
hospitalizations) among 11 U.S. states (12). Clinical characteris-
tics of the A(H3N2)v cases have been generally consistent with
signs and symptoms of seasonal influenza, and there is no evi-
dence at this time that sustained human-to-human transmission
is occurring. However, rare instances of probable human-to-hu-
and findings from an experimental study suggest that A(H3N2)v
in mammals (13).
The seasonal H3N2 vaccine component present in the 2010-2011
and 2011-2012 trivalent inactivated influenza vaccine (TIV) is
A/Perth/16/2009 (Perth/16; H3N2)-like viruses (14). Although
serological studies indicate that Perth/16 (H3N2) and A(H3N2)v
viruses are antigenically distinct from each other (7, 8), the effi-
cacy of seasonal influenza vaccination against A(H3N2)v has not
been adequately evaluated in vivo.
In this study, we evaluated whether the 2011-2012 TIV pro-
nfluenza A viruses have been isolated from swine since 1930
(1) and have been known to spread and cause disease in this
rus challenge. Male Fitch ferrets (Triple F Farms, Sayre, PA), 8
to 12 months of age and seronegative against currently circu-
lating human influenza H1, H3, and type B viruses, were vac-
cinated and twice boosted (3 to 4 weeks between injections)
intramuscularly with an adult human dose (0.5 ml) of the
2011-2012 seasonal inactivated split-product TIV or phos-
phate-buffered saline (PBS) (controls) (15). Prior to vaccine
boost and viral challenge, ferret sera were collected to assess
hemagglutination inhibition (HI) antibody responses against
IN/11 virus and the three homologous viruses in the 2011-2012
TIV. As shown in Table 1, all TIV-vaccinated ferrets displayed
HI antibody titers of ?80 against all three homologous viruses
present in the TIV; however, cross-reactive HI antibodies to
A(H3N2)v IN/11 virus were not observed.
We first determined the level of protection, induced by sea-
sonal TIV against seasonal homologous Perth/16 (H3N2) virus
challenge. The Perth/16 virus stock was grown in the allantoic
cavities of 10-day-old embryonated hens’ eggs at 34°C for 48 h
and titrated in a standard plaque assay expressed as PFU. Fer-
rets were challenged intranasally with 106PFU of virus, and
vaccine protection was measured by reduction in fever, weight
loss, and upper respiratory tract virus replication (15). Viral
challenge with the seasonal Perth/16 virus resulted in minimal
morbidity among vaccinated and control ferrets, causing 3.4%
and 3.9% maximum weight loss, respectively (Table 2). No
significant differences in body temperatures were detected be-
tween TIV-immune and unimmunized control ferrets ob-
served for 14 days postchallenge (p.c.), although there was a
trend toward reduced fevers among TIV-immune animals. The
extent of virus replication in the upper respiratory tract was
determined by titrating nasal wash samples collected on alter-
nating days p.c. The TIV did not provide sterilizing immunity
Received 24 September 2012 Accepted 25 October 2012
Published ahead of print 31 October 2012
Address correspondence to Terrence M. Tumpey, firstname.lastname@example.org.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
January 2013 Volume 87 Number 2Journal of Virologyp. 1261–1263jvi.asm.org
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against homologous viral challenge, as seen previously (15),
and viral titers were observed in all TIV-immunized ferrets and
control ferrets (Fig. 1). However, in comparison to control
ferrets, TIV-immunized ferrets displayed a significant reduc-
tion in viral titers on every day analyzed (day 2, P ? 0.007; day
4, P ? 0.03; and day 6, P ? 0.04), until viral clearance was
observed in both groups on day 8 p.c.
Next, we assessed the degree of cross-protection against the
A(H3N2)v IN/11 virus conferred by seasonal TIV. Ferrets were
challenged intranasally with 106PFU of the IN/11 stock virus,
which was grown in Madin-Darby canine kidney (MDCK)
cells. Overall, ferrets challenged with IN/11 virus displayed
higher temperatures and greater weight loss than ferrets chal-
lenged with Perth/16 virus (Table 2). On day 2 p.c., all unim-
munized control ferrets exhibited an early spike in body tem-
perature, ranging from 0.5 to 1.8°C over baseline (mean
maximum ? 1.2°C) (Table 2). Similarly, TIV-immune animals
also displayed an early spike in body temperature, ranging
from 0.75 to 1.8°C over baseline (mean maximum ? 1.5°C).
Moreover, in comparison to control animals, TIV-immune
ferrets did not display significant differences in weight loss and
virus titers on peak days (2 to 6 days p.c.) of replication (Table
2 and Fig. 1). However, TIV-immune ferrets showed a modest
reduction in viral titers on day 8 (P ? 0.02), perhaps due to a
low level of anti-N2 neuraminidase cross-reactive antibodies
induced by the TIV (16).
The results of this study suggest that previous immunization
with the commercially available seasonal TIV may provide mini-
mal to no cross-protection against A(H3N2)v virus. These data
are consistent with human serologic studies demonstrating that
of cross-reactive A(H3N2)v antibodies in immunologically naive
children (age, ?3 years) and failed to substantially improve the
level of cross-reactive antibodies in adults (17, 18). Because the
majority of the population lacks specific immunity against this
new virus variant, an A(H3N2)v-specific vaccine is needed for
optimal protection for all ages.
TABLE 1 Serum hemagglutination inhibition antibody responses to TIV immunization in ferrets
HI antibody titera
A/Perth/16/2009 B/Brisbane/60/2008 A/Indiana/08/11
Pre-first booster vaccination
Pre-second booster vaccination
aTiters generated by HI with 0.5% turkey RBCs, against A/Indiana/08/11 A(H3N2)v and viruses in the TIV 2011-2012 formulation: A/California/07/2009 (H1N1), Perth/16
(H3N2), and B/Brisbane/60/2008. Assays were performed using the WHO influenza reagent kit (according to instructions) obtained through the Influenza Reagent Resource,
Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention (Atlanta, GA). Range of
antibody titers shown for each ferret group, with geometric mean in parentheses. Eleven ferrets per group. All sera were initially diluted in receptor-destroying enzyme (RDE) from
Vibrio cholerae (Denka Seiken, Tokyo, Japan) for a final dilution of 1/10.
b?10, below the limit of detection in this assay.
TABLE 2 Clinical symptoms observed in TIV-immune ferrets
challenged with homologous or A(H3N2)v virus
5.5 ? 0.65
6.7 ? 0.42
5.6 ? 0.79
6.8 ? 0.42
aMean maximum weight loss percentage values shown (day 7 postchallenge) for 5 or 6
ferrets per group.
bTemperature increases over ferret baseline of 38.3 ? 0.5°C, all maximum
temperatures from day 2 p.c.
cMean peak titer shown as log10PFU/ml including standard deviation. All peak titers
from day 2 p.c.
FIG 1 TIV vaccine efficacy following challenge with A/Perth/16/2009 (Perth/
PBS on even-numbered days postchallenge. Titers were determined by stan-
A/Indiana/08/11 (B) virus. Bars display average values, with standard devia-
were analyzed by the Student t test (*, P ? 0.05; **, P ? 0.01).
Houser et al.
jvi.asm.orgJournal of Virology
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tute for Science and Education, Oak Ridge, TN.
The findings and conclusions in this report are those of the authors
and do not necessarily reflect the views of the funding agency.
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