H5N1 Vaccine-Specific B Cell Responses in Ferrets Primed
with Live Attenuated Seasonal Influenza Vaccines
Xing Cheng
1.
, Michael Eisenbraun
1.
,QiXu
1
, Helen Zhou
1
, Deepali Kulkarni
1
, Kanta Subbarao
2
, George
Kemble
1
, Hong Jin
1
*
1MedImmune, Mountain View, California, United States of America, 2Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID),
National Insitutes of Health, Bethesda, Maryland, United States of America
Abstract
Background:
Live attenuated influenza H5N1 vaccines have been produced and evaluated in mice and ferrets that were
never exposed to influenza A virus infection (Suguitan et al., Plos Medicine, e360:1541, 2006). However, the preexisting
influenza heterosubtypic immunity on live attenuated H5N1 vaccine induced immune response has not been evaluated.
Methodology and Principal Findings:
Primary and recall B cell responses to live attenuated H5N1 vaccine viruses were
examined using a sensitive antigen-specific B cell ELISpot assay to investigate the effect of preexisting heterosubtypic
influenza immunity on the development of H5N1-specific B cell immune responses in ferrets. Live attenuated H5N1 A/Hong
Kong/213/03 and A/Vietnam/1203/04 vaccine viruses induced measurable H5-specific IgM and IgG secreting B cells after
intranasal vaccination. However, H5-specific IgG secreting cells were detected significantly earlier and at a greater frequency
after H5N1 inoculation in ferrets previously primed with trivalent live attenuated influenza (H1N1, H3N2 and B) vaccine.
Priming studies further revealed that the more rapid B cell responses to H5 resulted from cross-reactive B cell immunity to
the hemagglutinin H1 protein. Moreover, vaccination with the H1N1 vaccine virus was able to induce protective responses
capable of limiting replication of the H5N1 vaccine virus to a level comparable with prior vaccination with the H5N1 vaccine
virus without affecting H5N1 vaccine virus induced antibody response.
Conclusion:
The findings indicate that previous vaccination with seasonal influenza vaccine may accelerate onset of
immunity by an H5N1 ca vaccine and the heterosubtypic immunity may be beneficial for pandemic preparedness.
Citation: Cheng X, Eisenbraun M, Xu Q, Zhou H, Kulkarni D, et al. (2009) H5N1 Vaccine-Specific B Cell Responses in Ferrets Primed with Live Attenuated Seasonal
Influenza Vaccines. PLoS ONE 4(2): e4436. doi:10.1371/journal.pone.0004436
Editor: Linqi Zhang, Comprehensive AIDS Reseach Center, China
Received September 11, 2008; Accepted December 17, 2008; Published February 11, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: The funding was provided by MedImmune. This research was supported in part by the Intramural Research Program of NIAID, NIH. 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: jinh@medimmune.com
.These authors contributed equally to this work.
Introduction
Influenza pandemics can occur when new influenza subtypes
capable of both infecting and spreading easily among humans
emerge with a new hemagglutinin (HA) subtype (antigenic shift) to
which there is little or no population immunity. During the last
century, three novel influenza A hemagglutinin subtypes (H1, H2
and H3) have appeared; an H1N1 strain caused the catastrophic
‘‘Spanish flu’’ pandemic in 1918 [1] followed by milder pandemics
in 1957 and 1968 caused by H2N2 and H3N2 strains, respectively.
Importantly, the origin of the pandemic H2N2 and H3N2 viruses
has since been attributed to genetic reassortment events where
circulating human influenza viruses acquired novel HA subtypes
from avian influenza viruses [2,3]. Alarmingly, in the past decade, a
number of avian influenza viruses containing HA subtypes not
typically found in humans have crossed species barrier and infected
humans, raising concerns about a future pandemic. Highly
pathogenic avian H5N1 influenza viruses have infected only a
small number of individuals but are associated with a high mortality
rate and are perceived as a potential major global health threat.
Several strategies have been used to develop vaccines against
H5N1 viruses including inactivated whole virus vaccines, split or
subunit vaccines, live attenuated influenza vaccine (LAIV),
vectored vaccines, and DNA vaccines; many of these candidates
have shown promise in preclinical studies [4]. Seasonal LAIV has
demonstrated several attributes that would be important for an
effective pandemic vaccine including efficacy, an ability to protect
against antigenically drifted strains, an ability to elicit a rapid
immune response in an immunologically naı
¨ve population, and a
highly efficient production system for the vaccine [5,6,7,8]. Several
prototypic pandemic LAIV (pLAIV) 6:2 reassortant viruses
containing the H5N1 HA and NA gene segments have been
produced on the backbone of six internal gene segments from the
cold-adapted (ca) A/Ann Arbor/6/60 vaccine strain [9], the
master donor virus (MDV-A) used to produce influenza A vaccine
strains for the seasonal FluMistHinfluenza vaccines (MedImmune).
These candidate H5N1 vaccine strains, A/HK/491/97 (HK97
ca), A/HK/213/03 (HK03 ca), and A/VN/1203/04 (VN04 ca),
were found to provide complete protection against lethal challenge
with homologous and heterologous wild-type (wt) H5N1 viruses in
PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4436
mice and offered complete protection against pulmonary replica-
tion of wt H5N1 virus in ferrets [7].
It has been observed that individuals who have recovered from
influenza infections develop broad subtype-specific immunity that
can protect them from subsequent infection by closely related drift
variants of the same subtype [10,11,12]. Although not nearly as
common, Schulman and Kilbourne [13] reported heterosubtypic
immunity in mice, where protection was induced by an influenza
virus belonging to a different subtype. Recently, there have been
several reports describing heterosubtypic immunity against H5N1
infection induced by influenza virus infection or vaccines in mice
[8,14,15,16]. The mechanistic basis of this type of immunity
remains undetermined, however, one study demonstrated a role of
the N1 component of the vaccine [17] and other studies suggest
that structural similarity of the H5 and H1 HA may mediate this
type of protection [18,19]. Kreijtz et al. [20] reported cross-
recognition of avian H5N1 influenza virus by human cytotoxic T-
lymphocyte populations directed against human influenza viruses
and suggested that the preexisting cross-reactive T-cell immunity
in humans may dampen the impact of a next pandemic if it is
caused by an H5N1 virus. The ferret is considered to be a suitable
mammalian host for seasonal influenza vaccine research [21,22]
and for efficacy studies of HPAI H5N1 vaccines [7,23,24].
Although ferrets immunized with a H1N1 ca strain were not
protected from replication of a wild-type H5N1 virus [7], however,
because LAIV has been shown to provide protection from strains
that are antigenically different from the vaccine antigen, we
investigated whether priming with a heterologous seasonal LAIV
vaccine containing different subtypes could influence the immune
response to H5N1 viruses in the ferret model. Such studies will
also help us to understand whether live attenuated H5N1 vaccine
could induce effective immune response in individuals that have
immunity to seasonal influenza viruses.
HAI and microneutralization assays are frequently used to
measure humoral antibody responses, however, these assays may
not be sensitive enough to detect early and local antibody
responses. To assess the presence and magnitude of heterosubtypic
immunity following immunization with LAIV, a sensitive B cell
ELISpot assay was developed that could detect early induction of
immunity at a time when the HAI assay was less sensitive. Using
this assay, we show that local B cell responses induced by the
H5N1 VN04 ca and HK03 ca vaccine viruses can be detected at a
virus-specific and HA-specific level. Previous infection with an
H1N1 virus induced a faster and higher level B cell response to
H5N1 vaccination and could prevent shedding of the H5N1
vaccine virus. The data implies that priming with a non-H5
vaccine may enable a more rapid memory response to an H5
vaccine, however, whether this would be beneficial to the
effectiveness of an H5 vaccine remains to be determined.
Materials and Methods
Viruses
Influenza virus vaccine strains H1N1 A/New Caledonia/20/99
ca (NC99 ca), H3N2 A/Wyoming/03/03 ca (WY03 ca), H3N2 A/
California/7/04 ca (CA04 ca), H5N1 A/Hong Kong/213/03 ca
(HK03 ca), H5N1 A/Vietnam/1203/04 ca (VN04 ca), H2N2 A/
AA/6/60 ca, (AA60 ca or MDV-A) [22] and H1N2 reassortant ca
virus containing the H1 HA from A/New Caledonia/20/99 and
the N2 NA from A/Wyoming/03/03 were generated by reverse
genetics. All viruses were expanded at 33uC for 3 days in the
allantoic cavity of 10-day-old embryonated SPAFAS hen’s eggs
(Norwich, CT). Allantoic fluids collected from infected eggs were
examined by hemagglutination assay using 0.5% turkey (tRBC) or
horse (hRBC) erythrocytes to determine HA titer. Infectious virus
titer was determined by plaque assay (plaque forming unit, PFU)
or 50% tissue culture infectious dose (TCID
50
) using Madin-Darby
canine kidney (MDCK) cells. Viruses were inactivated by
treatment with b-propriolactone (BPL) for use as antigen in
ELISpot and ELISA assays. Trivalent LAIV (FluMistH) was
manufactured by MedImmune and contained 10
7
TCID
50
each of
6:2 reassortant vaccine strains, A/New Caledonia/20/99 ca,A/
California/7/04 ca, and B/Jilin/20/03 ca.
Animal studies
Ferrets between 7 and 10 weeks of age from Triple F Farms
(Sayre, PA) were screened prior to use in experiments for
preexisting antibodies to H1N1, H3N2 and H5N1 influenza
viruses by hemagglutination inhibition (HAI) assay. Sero-negative
ferrets were inoculated intranasally on day 0 with a predetermined
does of a monovalent vaccine virus (10
5
to 10
7
PFU or TCID
50
virus), trivalent LAIV, or medium (mock control). To examine the
B cell response and virus replication after primary infection,
animals were sacrificed 5 or 10 days post-inoculation to collect
paratracheal lymph nodes (TLN) and whole blood for B cell
ELIspot assays; serum was collected to measure serum antibody
level and nasal turbinates were harvested to examine virus
replication in the upper respiratory tract. To examine the effect
of previous influenza virus infection on the induction of H5N1-
specific immune responses, ferrets were given a second intranasal
inoculation of 10
7
or 10
8
PFU of homologous or heterologous
vaccine virus 4–6 weeks after the initial vaccination. Animals in
these latter groups were sacrificed 3–10 days later to collect TLN,
blood, serum, and nasal turbinate samples or were used to collect
serum samples for up to three weeks after the second vaccination.
All animal study protocols were approved by MedImmune’s
Institutional Animal Care and Use Committee and performed in
an AAALAC certified facility.
Measurement of virus titers in animal tissues
Nasal turbinate tissues were homogenized in MEM medium
and centrifuged at 400 g for 10 min. Serial 10-fold dilutions of
supernatants collected from each preparation were inoculated into
three 10- to 11-day old embryonated SPAFAS hen’s eggs. After
incubation at 33uC for 72 hr, allantoic fluid from each egg was
collected for HA assay using 0.5% tRBC. Virus titers in the tissues
are reported as a 50% egg infectious dose (EID
50
) per gram of
tissue processed.
B cell ELISpot assay
AcroWell
TM
96-well PVDF filter plates (Pall Life Sciences, Ann
Arbor, MI) were coated with 50 mL/well PBS containing either
2,000 HA unit/mL of BPL-treated vaccine virus or 10 mg/mL
recombinant HA protein derived from H5N1 A/VN/1203/2004
(rH5), H1N1 A/New Caledonia/20/99 (rH1), or H3N2 A/
Wyoming/03/03 (rH3) that were purified from recombinant
baculovirus infected insect cells (Protein Sciences, Meriden, CT).
After overnight incubation at 4uC, plates were washed 3 times with
PBS and blocked with RPMI-1640 medium containing 10% FBS
for 2 hr at 37uC prior to the addition of cell samples.
Whole blood samples from ferrets were collected in EDTA
tubes and processed using LympholyteH-Mammal (Cedarlane,
Ontario, Canada) to isolate peripheral blood mononuclear cells
(PBMC). PBMC were washed once with RPMI-1640/10% FBS
by centrifugation (300 g for 10 min), counted, and resuspended in
complete medium (RPMI-1640, 10% FBS, 2 mM L-glutamine,
0.5 nM ß-mercaptoethanol and penicillin/streptomycin). TLN
were harvested from each ferret and placed in cold PBS/5% FBS
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and the cells were released from TLN into the media by gently
rubbing partially minced tissue against a sterile mesh screen with a
glass pestle. The resultant cell suspension was collected, passed
through a cell strainer to remove large debris, and pelleted by
centrifugation (300 g for 10 min). Cell pellets were washed once,
counted, and resuspended in the RPMI-1640 complete medium.
PBMC and TLN cell suspensions were added to triplicate wells
(100 mL/well) at a concentration of 3610
6
/mL for PBMC or 10
5
to 10
6
/mL for TLN samples and incubated at 37uC, 5% CO2 for
5 hr. The plates were washed 5 times with PBS containing 0.05%
Tween-20 (PBS-T) to remove the cells from the plate. To measure
isotype-specific B cell responses, goat anti-ferret IgM (Rockland,
Gilbertville, PA) or goat anti-ferret IgG (Bethyl Laboratories,
Montgomery, TX) diluted 1:1000 in PBS-T/1% BSA and
incubated overnight at 4uC. After 5 washes with PBS-T, HRP-
conjugated rabbit anti-goat Ig (Dako, Carpinteria, CA) diluted
1:2000 in PBS-T/BSA was added to all wells and incubated at
37uC for 1 hr. Plates were washed 3 times with PBS-T and 3 times
with PBS before development with AEC substrate (Vector Labs,
Burlingame, CA) for 10 min at room temperature (RT). Wells
were rinsed extensively with water and allowed to dry completely
before spots in each well were counted using an ImmunoSpot plate
reader (Cellular Technologies, Ltd., Cleveland, OH).
HAI and microneutralization assays
Prior to serologic analysis, ferret sera were treated with
receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan)
that was reconstituted with 10 mL of 0.9% NaCl per vial. 0.1 mL
serum was mixed with 0.15 mL RDE and incubated at 37uC for
18 hr and adjusted to a final 1:4 dilution by adding 0.15 mL of
0.9% sodium citrate followed by incubation at 56uC for 45 min.
Strain-specific serum HAI titers were determined using 0.5%
tRBC or hRBC and the HAI titers are presented as the reciprocal
value of the highest serum dilution that did not inhibit
hemagglutination. Serum neutralizing antibody titers were
determined by microneutralization assay using MDCK cells.
RDE-treated ferret serum was 2-fold serially diluted, incubated
with 100 TCID
50
virus at 33uC for 1 hr and transferred onto
MDCK cell monolayers in 96-well culture plates (Costar, Corning,
NY). After 6 days’ incubation at 33uC, the cell monolayers were
fixed with 10% formaldehyde, incubated with chicken MDV-A
polyclonal antibody followed by incubation with an HRP-
conjugated rabbit anti-chicken IgG (Thermo, Rockford, IL), and
developed with TMB substrate (Sigma, St. Louis, MO). The
reaction was stopped with an equal volume of 0.1 N HCl and the
absorbance at 450 nm was determined using a SpectraMax plate
reader (Molecular Devices, Sunnyvale, CA). Neutralizing antibody
titers were calculated as the highest serum dilution with a value less
than that calculated by the formula of (average OD of virus-
infected wells - average OD of cell control wells)/2+average OD of
cell control wells.
ELISA analysis of HA-specific antibody responses
96-well EIA plates (Costar, Corning, NY) were coated with
0.025 mg/well of rH1, rH3 or rH5 in PBS overnight at 4uC. Plates
were washed 3 times with PBS-T and blocked with SuperBlock
Blocking Buffer (Pierce, Rockford, IL) for 1 hr at 37uC. RDE-
treated ferret sera were 2-fold serially diluted with PBS-T,
transferred to 96-well plates (50 mL/well), and incubated for
1hrat37uC. Plates were washed with PBS-T and incubated for
30 min at 37uC with 100 mL/well HRP-conjugated goat anti-
ferret IgG (Bethyl Laboratories, Montgomery, TX) diluted
1:10,000 in PBS-T/1% BSA. After washing with PBS-T, plates
were developed with TMB substrate and read as described above
in the microneutralization assay. Antibody titers are expressed as
the highest dilution with an optical density (OD) reading greater
than 2 times the mean OD+standard deviation of similarly diluted
negative control samples.
Results
Detection of H5N1-specific B cell responses after
immunization with pLAIV using a sensitive B cell ELIspot
assay
Pandemic live attenuated influenza vaccines (pLAIV) developed
for H5N1 viruses confer protection against wild-type virus
challenge in mice and ferrets [7], however, the level of the serum
HAI antibody responses induced by the VN04 vaccine was low.
We sought to implement a more sensitive ELISpot assay to
measure whole virus- and HA-specific antibody secreting cells
(ASC) in draining paratracheal lymph node (TLN) and PBMC
from vaccinated ferrets. The ELISpot assay has been shown to be
a sensitive tool for detecting cellular immunity following influenza
vaccination in humans [25,26,27]. To test the sensitivity of this
ELISpot assay, ferrets were inoculated intranasally with either the
H5N1 VN04 ca or H5N1 HK03 ca vaccine virus and were
sacrificed 5 or 10 days later to isolate TLN cells and PBMC. The
ELISpot data obtained from TLN samples (Fig. 1) showed that
H5N1 virus-specific ASC could be detected after vaccination with
either of the H5N1 ca vaccine viruses and similar data were
obtained from PBMC (data not shown). However, the magnitude
and kinetics of IgM and IgG ASC induced by the VN04 ca vaccine
appeared to be lower and slower than the HK03 vaccine virus on
both days 5 and 10 post-vaccination (Fig 1). The virus-specific IgM
ASC response was higher on day 5 than day 10 for HK03 ca virus
immunized animals, but higher on day 10 than day 5 for VN04 ca
virus immunized animals, indicating that the initial IgM response
induced by the VN04 ca virus was slower and weaker than that
induced by the HK03 ca virus. The IgG secreting ASC response
was much higher on day 10 than day 5 for both HK03 ca and
VN04 ca vaccine immunized animals. Again, HK03 ca vaccine
induced more IgG ASC than VN04 ca vaccine (p,0.05). H5 HA-
specific IgM and IgG ASC were also detected on day 10 for both
viruses. However, the level of HA-specific ASC was only about
15% of the total virus-specific ASC, indicating a majority of the
ASC induced by H5N1 ca virus vaccine were against antigens
other than HA. The level of the H5 HA-specific ASC on day 5 was
lower than on day 10 (data not shown). Thus, consistent with the
HAI data, the ELISpot data also showed that the HK03 ca vaccine
induced a better immune response than the VN04 ca vaccine.
LAIV-vaccinated animals show a more rapid immune
responses to the H5N1 ca vaccine than unvaccinated
animals
Most people have some immunity to influenza H1N1, H3N2,
and B viruses due to previous natural infections or immunization
with influenza vaccines. To mimic this sero-positive status and to
determine whether preexisting heterosubtypic immunity affects
responses to subsequent vaccination with H5N1ca vaccine viruses,
ferrets were primed intranasally with live attenuated trivalent
seasonal influenza vaccines (LAIV) or medium control prior to
vaccination with the H5N1 HK03 or VN04 ca viruses. Five days
after vaccination with H5N1 ca vaccine, TLN and PBMC samples
were collected for analysis by B cell ELISpot assay to measure
virus- and HA-specific IgG ASC. Responses detected using whole
virus reagents (Fig. 2A) revealed that the number of H2N2 (AA60,
MDV-A) and H5N1 (HK03) virus-specific IgG ASC were
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significantly higher in ferrets primed with LAIV (Fig. 2A). This
was not completely unexpected because both the H1N1 and
H3N2 viruses in LAIV share the same internal viral proteins such
as the M1 and NP that likely stimulate rapid B cell responses.
However, LAIV priming also significantly enhanced H5 HA-
specific IgG ASC responses, which were not detected in the
unprimed ferrets (Fig. 2A). This finding suggested that the
enhanced H5 HA-specific responses could occur as a result of
expansion of memory B cells that were elicited against the H1N1
and/or H3N2 influenza A virus components in the LAIV vaccine.
H1N1 vaccine primes faster B cell responses to the H5N1
vaccine than the H3N2 vaccinated animals
To determine whether the higher numbers of H5-specific B cell
responses observed after trivalent LAIV priming were due to only
one or both of the influenza A virus components, groups of ferrets
were primed with monovalent H1N1 NC99 ca, H3N2 CA04 ca,
H5N1 HK03 ca vaccine or medium six weeks prior to a second
inoculation with the H5N1 HK03 ca vaccine. Five days after
vaccination with the H5N1 HK03 ca vaccine (Fig. 2B), the level of
virus-specific IgG ASC was very low in the ferrets that initially
received medium, similar to that observed in the first study. In
contrast, ferrets that were primed with the H1N1 NC99, H3N2
CA04 or H5N1 HK03 ca vaccine viruses, had significantly higher
numbers of H5N1 HK03 ca virus-specific IgG ASC after
vaccination with the H5N1 HK03 ca vaccine. The ferrets that
were previously vaccinated with the H1N1, H3N2 and H5N1 ca
vaccine viruses also had B cell response to the H1N1 and H3N2
vaccine virus (Fig. 2B). The number of the H5 HA-specific IgG
ASC was the highest (approximately 10% of total virus-specific
ASC) in the group that received 2 doses of HK03 ca virus (Fig. 2C).
Interestingly, a significant number of H5 HA-specific IgG ASC
(approximately 180 ASC per 10
6
cells) was also observed in the
group that previously received the H1N1 NC99 ca vaccine virus. A
much lower number of ASC was found in the group that received
the H3N2 CA04 ca vaccine virus. As expected, a significant
number of H1 and H3 HA-specific IgG ASC (approximately 250
and 140 per 10
6
cells, respectively) were detected in the groups
that were primed with the H1N1 NC99 and H3N2 CA04 ca
vaccine viruses, respectively.
H1N1 ca vaccine-induced faster B cell responses to the
H5N1 vaccine virus is due to the H1 HA
The neuraminidases of the H5N1 and H1N1 viruses are of the
same (N1) subtype. Several studies have reported a role of the N1
NA-mediated immunity against H5N1 infection in the murine
model and in serology analysis of human serum samples
[14,17,28]. Because it was shown earlier that previous exposure
to H3N2 virus did not affect H5-specific ASC responses and the
H1 HA and H5 HA share some structural similarity [18] and
common epitopes [19], the effect of the H1N1-mediated priming
on the H5N1 ca vaccines was investigated. To confirm that the
enhanced B cell response to the H5N1 virus was due to a primary
immune response to the H1 HA not the N1 NA, an H1N2 virus
containing the HA from the H1N1 NC99 ca virus, the NA from
the H3N2 WY03 ca virus and the internal protein gene segments
from MDV-A was generated. Priming with this H1N2 virus in
ferrets elicited a similar level of B cell response (122 ASC per
million cells) as those (120 ASC per million cells) primed with the
H5N1 HK03 vaccine virus (Fig. 3), confirming that the enhanced
H5N1 B cell response was due to the H1 HA and not the N1 NA.
The number of the H5 HA-specific IgG ASC in the H1N2 virus
primed ferrets was higher than those primed with the H1N1 ca
vaccine virus, which was possibly due to the better replication of
the H1N2 ca virus than the H1N1 ca virus in the upper respiratory
tract of ferrets (data not shown).
H5-, H1- and H3-specific serum antibody responses
following prime-boost vaccination in ferrets
As described earlier, previous exposure to a virus containing the
H1 HA resulted in an increased H5-specific B cell response. To
examine if the increased ASC response reflected a greater
antibody response, serum antibody titers were examined by
HAI, microneutralization and ELISA assays. Groups of ferrets
were immunized with medium, the H1N1 NC99 ca, H3N2 CA04
ca, H5N1 HK03 ca or VN04 ca virus, and 6 weeks later, a second
dose of the H5N1 HK03 ca or VN04 ca vaccine was administered
intranasally. Serum samples were collected 6 weeks after the 1
st
vaccination (post dose 1) and 1 and 3 weeks after the 2
nd
vaccination (Table 1). At 6 weeks after the first dose neutralizing
antibodies against homologous vaccine virus were detected in the
ferrets that received vaccine virus but not in the control group that
Figure 1. H5N1-specific B cells were detected in ferrets infected
with live attenuated H5N1
ca
vaccines. Ferrets were intranasally
administered the H5N1 VN04 ca or HK03 ca viruses on day 0. Five and
ten days post-inoculation, ferrets were sacrificed to collect paratracheal
lymph nodes (TLN) and a B cell ELISpot assay was performed using
lymphocytes isolated from TLN and BPL-inactivated H5N1 HK03 ca virus
or rH5 HA antigens. The number of IgM ASC (A) and IgG ASC (B) are
presented as per 10
6
lymphocytes.
doi:10.1371/journal.pone.0004436.g001
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Figure 2. Previous exposure with seasonal LAIV or H1N1
ca
virus induced a faster H5N1-specific immune response. (A) Ferrets were
intranasally inoculated with either medium or trivalent LAIV on day 0. Six weeks later, ferrets were intranasally inoculated with the H5N1 HK03 ca
vaccine and five days later, lymphocytes isolated from TLN were examined for ASC against H2N2 MDV-A, H5N1 HK03 ca viruses and rH5 HA antigens
by B cell ELISpot analysis. Ferrets were intranasally inoculated with medium or the monovalent H1N1 NC99, H3N2 CA04, H5N1 HK03 ca vaccine
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received medium only. The H3N2 CA04 ca vaccine induced the
highest mean neutralizing antibody (titer of 4064) and the H1N1
NC99 ca vaccine induced a mean neutralizing antibody titer of 50.
Neither H1N1 nor H3N2 ca viruses induced antibodies that cross-
neutralized the H5N1 HK03 ca virus. The H5N1 HK03 ca
vaccine induced the H5-specific neutralizing antibody at a mean
titer of 320 (Table 1). One week after administration of the H5N1
HK03 ca vaccine, the ferrets that received two doses of the HK03
ca vaccine had levels of H5-specific neutralizing antibody that were
significantly higher than in animals that received a single dose of
the H5N1 HK03 ca virus. In contrast to the B cell ELISpots
results, animals primed with a dose of H1N1 ca and boosted with a
second dose of H5N1 ca for one week had low titers of antibodies
against the H5 antigen (Table 1). Previous vaccination with the
H3N2 CA04 ca vaccine had little effect on the antibody response
to the H5N1 ca vaccine. When measured at three weeks after
vaccination with the H5N1 HK03 ca virus, the level of the H5-
specific antibody in the animals that received a dose of medium,
the H1N1 NC99 or H3N2 CA04 ca vaccines as the first dose was
much lower (3- to 5-fold) than the animals that received two doses
of the H5N1 HK03 ca vaccine virus.
The effect of H1N1 ca vaccination on the H5N1 VN04 ca
vaccine induced antibody response was also evaluated (Table 1).
As expected, H1N1-specific antibody did not cross-react with the
H5N1 VN04 ca virus. One week after the second vaccination with
the H5N1 VN04 ca virus, H5N1-specific neutralizing (titer of 50)
antibodies were detected in the ferrets that previously received the
H1N1 NC99 ca vaccine, and this titer was significantly higher than
the ferrets that received medium only (titer of 14, p,0.005) but
much lower than the animals that received two doses of the H5N1
VN04 ca vaccine (titer of 806). However, three weeks after
vaccination with the H5N1 VN04 ca vaccine there was no
difference in antibody levels between the animals that received the
first dose of medium or the H1N1 NC99 ca vaccine. Again,
animals that received two doses of the H5N1 HK03 ca vaccine had
neutralizing antibodies more than 5-fold higher than those that
received the H1N1 or H3N2 ca vaccine as the first dose. Thus, the
H1 HA induced enhanced production of H5N1-specific neutral-
izing antibody was temporary. Similar results were also obtained
by the HAI assay (data not shown).
To further evaluate the H1 induced priming effect on the H5N1
antibody response, an ELISA assay was performed to measure
levels of HA-specific serum IgG antibodies using rHA as the
antigen (Fig. 4). Six weeks after the 1
st
vaccination, a substantial
Figure 3. The priming effect of the H1N1
ca
vaccine is induced
by the H1 HA. Ferrets were intranasally inoculated with the H1N1
NC99 ca virus, a reassortant H1N2 ca, H3N2 CA04 ca or H5N1 HK03 ca
virus on day 0 and intranasally inoculated with the H5N1 HK03 ca virus
five weeks later. The animals were sacrificed 5 days later and B cell
ELISpot analysis was performed with lymphocytes isolated from TLN
using rH5 HA as antigen. IgG antibody secreting B cells are presented as
the number of ASC per 10
6
lymphocytes.
doi:10.1371/journal.pone.0004436.g003
viruses on day 0 and intranasally inoculated with the H5N1 HK03 ca vaccine six weeks later. The B cell ELISpot analysis was performed with
lymphocytes isolated from TLN using the indicated ca vaccine virus (B) or rHA (C) as antigens. The IgG antibody secreting B cells are presented as the
number of ASC per 10
6
lymphocytes.
doi:10.1371/journal.pone.0004436.g002
Table 1. Serum antibody response to influenza viruses after one and two doses of intranasal vaccine.
1
st
dose vaccine (Day 0) 2
nd
dose vaccine (Day 42) Neutralizing antibody GMT against the indicated vaccine virus antigens
6 wk post dose-1 1 wk post dose-2 3 wk post dose-2
1st Vac H5N1 H5N1 H5N1
Medium H5N1 HK03 ca ,10 ,10 40 761
H1N1 NC99 ca 50 ,10 50 403
H3N2 CA04 ca 4064 ,10 18 419
H5N1 HK03 ca 320 320 2560 2281
Medium H5N1 VN04 ca ,10 ,10 14 71
H1N1 NC99 ca 63 ,10 50 71
H5N1 VN04 ca 45 45 806 403
Groups of three ferrets were vaccinated intranasally with the indicated 1
st
dose of vaccine and 42 days later were inoculated with a 2
nd
dose of vaccine (H5N1 HK03 ca
or VN04 ca). Serum samples were collected 6 weeks after the 1
st
dose (pre-dose 2), 1 week and 3 weeks after the 2
nd
dose, respectively, and antibody titers (geometric
mean titers from 3 animals) against the first or second vaccine viruses were determined by microneutralization assay.
doi:10.1371/journal.pone.0004436.t001
H5N1 Vaccine
PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4436
level of homologous HA-specific IgG was present (Fig. 4A). It was
noted that the H1 and H5 specific antibodies had a low level of
cross reactivity with the H5 and H1 antigens, respectively. The
level of the H5-specific ELISA antibodies was much higher in the
group that received the H1N1 NC99 ca virus as the first dose and
the H5N1 HK03 ca virus as the second dose than the ferrets that
previously received the H3N2 CA04 ca virus and those that did not
receive any virus (medium), although the titer was lower than the
ferrets that received 2 doses of the H5N1 HK03 ca vaccine
(Fig. 4B). The antibodies from the ferrets that received 2 doses of
the H5N1 HK03 ca vaccine also reacted with the rH1 HA. The
H5-specific IgG antibodies continued to increase until 3 weeks
following the 2
nd
dose and reached a level that was similar among
all the groups (data not shown). Similar data were also obtained for
Figure 4. HA-specific antibodies measured by an ELISA assay. Ferrets were intranasally inoculated with medium or the H1N1 NC99, H3N2
CA04, H5N1 HK03 ca vaccine viruses on day 0 and intranasally inoculated with the HK03 ca vaccine virus six weeks later. Serum samples were
collected 6 weeks after the 1st dose (A) and one week after the 2nd dose (B). ELISA was performed with RDE-treated serum using rH5, rH1 or rH3 HA
as antigens.
doi:10.1371/journal.pone.0004436.g004
H5N1 Vaccine
PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4436
ferrets that received the H5N1 VN04 ca vaccine as a second dose
(data not shown). Thus, these data indicated that the H1- and H5-
specific binding antibodies cross-reacted with each other and
previous exposure to H1N1 vaccine appeared to result in a more
rapid H5-specific humoral immune response to the H5 HA
protein.
H1N1 vaccine-induced immunity prevented replication
of the H5N1 ca vaccine virus
To determine whether previous vaccination with the H1N1
NC99 ca vaccine would affect subsequent replication of the H5N1
ca vaccine virus in the respiratory tract, groups of six naı
¨ve ferrets
were primed with the H1N1 NC99 ca, H3N2 WY03 ca, H5N1
HK03 ca vaccine or medium only. Four weeks later, animals were
inoculated intranasally with the H5N1 HK03 ca vaccine and
sacrificed on day 3 post-inoculation to collect nasal turbinates to
quantify replication of the H5N1 HK03 ca vaccine virus in the
upper respiratory tract of ferrets. As shown in Table 2, the mean
titer of the H5N1 HK03 ca vaccine virus detected in the upper
respiratory tract of ferrets that had previously received medium
was 10
4.5
EID
50
/g of tissue. In contrast, none of the ferrets that
were primed with the H1N1 NC99 ca or the H5N1 HK03 ca
vaccine had detectable virus in the upper respiratory tract. The
H3N2 WY03 ca vaccine priming protected 2 of 6 ferrets and the
H5N1 HK03 virus replicated to a mean titer of 10
2.2
EID
50
/g of
tissue in this group of ferrets. These results indicated that
heterosubtypic immunity from H1N1 ca virus infection or
vaccination reduced H5N1 ca vaccine virus replication.
Discussion
In this study, we demonstrate that live attenuated H5N1 vaccine
induced immunity can be detected by the B cell ELISpot assay.
The protective level of ASC is not well established yet, however,
the B cell response to the H5N1 HK03 ca vaccine is greater than to
the VN04 ca vaccine and the magnitude of the ASC response
correlates with serum antibody level as determined by micro-
neutralization assay. The H5N1 HK03 and VN04 viruses differ by
9 amino acids in the HA molecule and the amino acid at position
223 is known to contribute to receptor binding specificity [29].
The HA of the HK03 virus that preferentially binds to sialic acid
receptors with a2,6-linked oligosaccharide linkages contains serine
at residue 223 and the HA of the VN04 virus that prefers an avian-
like receptor with a2,3-linked oligosaccharide linkages contains
asparagine at this residue [30,31]. In addition, the length of the
NA of the H5N1 HK03 virus differs from the VN04 virus; the
VN04 virus, like most of the H5N1 isolates, has a deletion of 20
amino acids in the NA stalk whereas the HK03 virus does not have
this deletion. The differences in the HA and NA sequences
between the H5N1 HK03 and VN04 viruses presumably
contribute to the observed difference in vaccine immunogenicity.
Despite the lower immune response induced by VN04 ca virus,
two doses of the H5N1 VN04 ca vaccine offered complete
protection against homologous and heterologous H5N1 wt virus
lethal challenge in mice and provided protection against
replication of the H5N1 wt virus in the respiratory tracts of mice
and ferrets [7].
In addition to the local draining lymph nodes (TLN), ASC were
also detected in the PBMC of the vaccinated ferrets at a level
slightly lower than those detected in TLN, therefore, only the data
obtained with TLN are presented. Virus-specific memory B cells
in the lungs can persist for a long time along with germinal center
B cells and plasma cells and appear to be a unique feature of the
mucosal memory response [16]. In response to re-encountered
antigens, memory B cells robustly secrete antibodies against the
pathogen and this memory response is much faster than that of
primary B cells due to quantitative and qualitative changes in
antigen-specific B cells and helper T cells. In this study, we found
that previous exposure to the H1N1 ca virus could accelerate the
memory B cell response to the H5N1 virus. The heterosubtypic
antibody response as detected by ELISA in the serum could
prevent replication of the H5N1 HK03 ca vaccine virus in the
respiratory tract, suggesting that protective immunity was
enhanced by a priming dose of H1N1 ca vaccine. A recent study
also showed that the ferrets immunized with the H1N1 virus-like
particles (VLP) had a low level of neutralizing antibody against the
H5N1 virus and cleared the H5N1 challenge virus rapidly and had
reduced morbidity [32]. There was a concern that heterosubtypic
immunity might reduce vaccine efficacy by reducing vaccine virus
replication in the upper respiratory tract. However, despite
restricted replication of the H5N1 ca virus in the upper respiratory
tract of the H1N1 exposed ferrets, the level of H5N1-specific
neutralizing antibodies in the animals that previously received the
H1N1 ca vaccine was similar to the seronegative animals. Our
previous study indicated that two doses of the H1N1 A/New
Caledonia/20/99 ca virus were unable to protect ferrets from
replication of a high dose (10
7
TCID
50
) of H5N1 HK97 wt virus in
the respiratory tracts of ferrets [7]. However, it remains to be
determined whether the H1N1 ca virus could offer a protective
benefit from a lower challenge dose of H5N1 wt virus. Despite the
faster onset of immunity to the H5N1 ca vaccine, the antibodies
produced in animals that received the H1N1 ca vaccine followed
by the H5N1 ca vaccine are at least 5-fold lower that the animals
that received two doses of the H5N1 ca vaccines. Thus, it is likely
that the immunity provided by previous immunization with an
H1N1 ca virus is limited and priming with seasonal LAIV cannot
replace the use of 2 doses of an H5-specific vaccine.
Table 2. Effect of the H1N1 and H3N2 ca vaccines on replication of the H5N1 HK03 ca virus in the upper respiratory tract of ferrets.
Vaccine
#of animals
per group
GMT of homologous
HAI antibody
#of Animals with H5N1
detected in NT
Mean Virus Titer in NT
(log
10
EID
50
/g6SE)
Medium 6 ,46 4.560.5
H1N1 NC99 ca 647 0 #1.5
H3N2 WY03 ca 6 203 4 2.260.7
H5N1 HK03 ca 640 0 #1.5
Groups of ferrets were vaccinated with the indicated virus and four weeks later inoculated with the H5N1 HK03 ca vaccine. Antibody titers against homologous vaccine
virus were determined by HAI assay and expressed as geometric mean titer. The H5N1 HK03 ca virus titer in nasal turbinates (NT) on day 3 post-inoculation is expressed
as log
10
EID
50
per gram of tissues calculated from the mean of 6 animals.
doi:10.1371/journal.pone.0004436.t002
H5N1 Vaccine
PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4436
It has been observed frequently that individuals recovered from
influenza virus infection are protected against subsequent infection
by antigenic drift variant viruses within the same subtype
[10,11,12] and to a lesser extent from infection by a different
subtype due to heterosubtypic immunity [13]. Recently, several
reports have described heterosubtypic immunity from seasonal
influenza vaccines to H5N1 infection in mice and humans
[8,14,15,16]. Ichinohe et al [14] showed that intranasal inocula-
tion of an inactivated trivalent seasonal influenza vaccine provided
cross-protection against H5N1 infection in mice. Such studies are
difficult to conduct in humans. Ferrets develop symptoms upon
influenza infection that resemble those of humans including
sneezing, body temperature variation and weight loss and have
been shown to be an appropriate model for influenza virus
research. In this study, we demonstrated that the faster H5N1 B
cell response induced by the H1N1 ca vaccine in ferrets was
mediated by the H1 HA protein as demonstrated by a similar
effect caused by an H1N2 virus. Although the accelerated H5N1
response following previous exposure to the H1N1 ca virus was
barely detected by HAI and microneutralization assays, we found
a temporal rise of mincroneutralizing antibody in H5N1 VN04 ca
vaccinated ferrets that were previously exposed to the H1N1
NC04 ca virus (Table 1). We could demonstrate cross-reactivity of
H1N1 and H5N1 ca vaccine induced HA antibodies by ELISA
assay. These data confirmed that the H1 and H5 HA contain
some conserved epitopes that could elicit cross-reactive antibodies
[19,33] because of their structure similarity [18].
N1 NA-induced protection against experimental H5N1 virus
infection has been reported in mice and by the finding that human
sera are capable of inhibiting the NA enzymatic activity of the
H5N1 VN04 virus [17]. Our study was not designed to examine
the contribution of the N1 protein of the H1N1 virus to H5N1
immunity. We cannot exclude the possibility that the N1-induced
immunity might also contribute to the restricted replication of the
H5N1 ca virus in the upper respiratory tract of ferrets.
Our current study indicates that previous exposure to the H3N2
ca virus was less protective than the H1N1 ca virus in restricting
replication of the H5N1 ca vaccine virus in the upper respiratory
tract of ferrets. However, replication of the H5N1 vaccine virus in
ferrets previously primed with an H3N2 ca virus was also greatly
reduced compared to the control animals. This could be because
the H1N1, H3N2 and H5N1 ca vaccine viruses share 6 internal
protein gene segments. As shown by the ELISpot assay, the
number of ASC against the vaccine virus was much higher than
HA-specific ASC (compare Fig. 2B with Fig. 2C). In addition to
the protective immune response against the HA and NA surface
proteins, influenza viruses also induce immune responses against
conserved viral proteins such as NP and M1 that could result in
heterosubtypic protection [34] to restrict H5N1 virus replication.
An earlier report [35] showed that previous mucosal delivery of
trivalent influenza vaccine offered protection against H5N1 wt
virus lethal infection in the mouse model. We also showed
previously that H2N2 AA ca vaccinated mice were partially
protected from the lethal challenge of the H5N1 wt viruses [7].
This type of heterosubtypic response could be mediated by
secondary CTL responses involving CD8
+
or CD4
+
T cells as
reported previously [15,36].
In summary, our study supports the notion that previous
vaccination with seasonal influenza vaccine may accelerate onset
of immunity by an H5N1 ca vaccine. Since the influenza pandemic
vaccine may not be available until some time well into the first
wave or early in the second wave of a pandemic, an earlier
response may be of value in pandemic preparedness.
Acknowledgments
We are very grateful to Scott Jacobson, Kim Ngo, Brett Pickell, Ernesto
Madariaga, Stephanie Gee and Nick Nguyen for performing the ferret
studies, Anu Cherukuri for review of the manuscript, Jennifer Woo and
members of HJ’s group for technical support and discussions.
Author Contributions
Conceived and designed the experiments: ME GK HJ. Performed the
experiments: XC ME QX HZ DK. Analyzed the data: XC ME QX HJ.
Contributed reagents/materials/analysis tools: XC ME QX HZ DK HJ.
Wrote the paper: XC KS GK HJ.
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