Intranasal Vaccination with 1918 Influenza Virus-Like Particles Protects Mice and Ferrets from Lethal 1918 and H5N1 Influenza Virus Challenge

Abstract

Influenza vaccines capable of inducing cross-reactive or heterotypic immunity could be an important first line of prevention against a novel subtype virus. Influenza virus-like particles (VLPs) displaying functional viral proteins are effective vaccines against replication-competent homologous virus, but their ability to induce heterotypic immunity has not been adequately tested. To measure VLP vaccine efficacy against a known influenza pandemic virus, recombinant VLPs were generated from structural proteins of the 1918 H1N1 virus. Mucosal and traditional parenteral administrations of H1N1 VLPs were compared for the ability to protect against the reconstructed 1918 virus and a highly pathogenic avian H5N1 virus isolated from a fatal human case. Mice that received two intranasal immunizations of H1N1 VLPs were largely protected against a lethal challenge with both the 1918 virus and the H5N1 virus. In contrast, mice that received two intramuscular immunizations of 1918 VLPs were only protected against a homologous virus challenge. Mucosal vaccination of mice with 1918 VLPs induced higher levels of cross-reactive immunoglobulin G (IgG) and IgA antibodies than did parenteral vaccination. Similarly, ferrets mucosally vaccinated with 1918 VLPs completely survived a lethal challenge with the H5N1 virus, while only a 50% survival rate was observed in parenterally vaccinated animals. These results suggest a strategy of VLP vaccination against a pandemic virus and one that stimulates heterotypic immunity against an influenza virus strain with threatening pandemic potential.

Full text

JOURNAL OF VIROLOGY, June 2009, p. 5726–5734 Vol. 83, No. 11
0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00207-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Intranasal Vaccination with 1918 Influenza Virus-Like Particles
Protects Mice and Ferrets from Lethal 1918 and H5N1
Influenza Virus Challenge
䌤
Lucy A. Perrone,
1
Attiya Ahmad,
2
Vic Veguilla,
1
Xiuhua Lu,
1
Gale Smith,
2
Jacqueline M. Katz,
1
Peter Pushko,
2
and Terrence M. Tumpey
1
*
Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases,
Collaborating Centers for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,
1
and
Novavax, Inc., Rockville, Maryland
2
Received 28 January 2009/Accepted 17 March 2009
Influenza vaccines capable of inducing cross-reactive or heterotypic immunity could be an important first
line of prevention against a novel subtype virus. Influenza virus-like particles (VLPs) displaying functional
viral proteins are effective vaccines against replication-competent homologous virus, but their ability to induce
heterotypic immunity has not been adequately tested. To measure VLP vaccine efficacy against a known
influenza pandemic virus, recombinant VLPs were generated from structural proteins of the 1918 H1N1 virus.
Mucosal and traditional parenteral administrations of H1N1 VLPs were compared for the ability to protect
against the reconstructed 1918 virus and a highly pathogenic avian H5N1 virus isolated from a fatal human
case. Mice that received two intranasal immunizations of H1N1 VLPs were largely protected against a lethal
challenge with both the 1918 virus and the H5N1 virus. In contrast, mice that received two intramuscular
immunizations of 1918 VLPs were only protected against a homologous virus challenge. Mucosal vaccination
of mice with 1918 VLPs induced higher levels of cross-reactive immunoglobulin G (IgG) and IgA antibodies
than did parenteral vaccination. Similarly, ferrets mucosally vaccinated with 1918 VLPs completely survived
a lethal challenge with the H5N1 virus, while only a 50% survival rate was observed in parenterally vaccinated
animals. These results suggest a strategy of VLP vaccination against a pandemic virus and one that stimulates
heterotypic immunity against an influenza virus strain with threatening pandemic potential.
Influenza A viruses represent a substantial public health
burden, with a yearly average of more than 220,000 hospitaliza-
tions and approximately 36,000 deaths in the United States alone
(http://www.cdc.gov/flu/keyfacts.htm). In addition to seasonal out-
breaks caused by antigenic variants of circulating influenza A and
B viruses, another pandemic influenza virus strain may emerge at
any time. The threat of a pandemic is greater than it has been in
decades. Confirmed cases of human infection with several sub-
types of avian influenza viruses have been reported since 1997 (2,
3, 38; http://www.who.int/csr/disease/avian_influenza/country/en
/index.html). Of the avian subtypes that have recently been intro-
duced into humans, the highly pathogenic avian influenza virus
H5N1 subtype is the most immediate public health problem.
More than 400 human H5N1 virus infections have occurred,
and approximately 60% have been fatal (http://www.who.int/csr
/disease/avian_influenza/country/en/index.html). Should these vi-
ruses acquire the ability to spread efficiently among humans lack-
ing immunity to the H5 hemagglutinin (HA), a pandemic could
occur. If the case fatality rate of the virus remained high, an H5
pandemic could recapitulate the devastating consequences of the
“Spanish” influenza pandemic of 1918, which resulted in an esti-
mated 50 million deaths and a 10-year reduction in the average
life expectancy in the United States (13). Although recent studies
suggest that most 1918 (H1N1) pandemic deaths were attributed
to secondary bacterial pneumonia (5), the inherent ability of the
virus to replicate efficiently and cause severe acute infection of the
respiratory tract was a critical underlying cause of this historic
public health disaster (24, 26, 28, 50, 72, 74, 78, 79). Thus, the 1918
influenza virus represents an ideal candidate for the study of
protective immunity to a pandemic influenza virus strain.
Traditional influenza vaccines provide optimal protection
against viruses that are antigenically closely matched with
those contained in the vaccine but have been less effective
against antigenic variants within a subtype and historically pro-
vide only minimal protection against viruses of novel HA sub-
types (1). Thus, there has been interest in developing a vaccine
or vaccine strategy that can induce broader cross-reactive im-
munity against multiple subtypes of influenza viruses contain-
ing multiple combinations of surface proteins, also known as
heterosubtypic immunity. In addition to a decrease in overall
morbidity following infection, heterosubtypically immune ani-
mals show decreased viral titers and duration of viral shedding
within the respiratory tract (23, 27, 36, 54, 65, 70, 75).
Pandemic influenza vaccines must meet a number of criteria,
which include low production cost, ease of manufacture, and
rapid production and delivery. In recent years, several dif-
ferent approaches have been tested. A promising technology
uses recombinant noninfectious virus-like particles (VLPs)
that present structurally native, immunologically relevant viral
antigens. VLP vaccines have proven effective in preventing
diseases in humans, as exemplified by the recently approved
human papillomavirus VLP vaccine for the prevention of cer-
vical cancer (21; http://www.fda.gov/cber/vaccines.htm). Non-
* Corresponding author. Mailing address: 1600 Clifton Rd. NE, MS
G-16, Atlanta, GA 30333. Phone: (404) 639-5444. Fax: (404) 639-2350.
E-mail: tft9@cdc.gov.
䌤
Published ahead of print on 25 March 2009.
5726

infectious VLPs morphologically resemble their live-virus
counterparts and are recognized and processed readily by an-
tigen-presenting cells of the immune system (4, 30, 60, 69, 76,
81). Recombinant VLPs do not involve the use of infectious
influenza virus and thus require no exceptional biosafety con-
tainment to produce and can be manufactured quickly for an
emergency response. Influenza VLPs have been generated in
insect cells by using three influenza virus proteins, i.e., HA,
neuraminidase (NA), and matrix (M1). To date, these VLPs
have been produced from the H1N1, H3N2, H5N1, H5N3, and
H9N2 subtypes (6, 7, 19, 41, 51–53). Unlike conventional split
and inactivated vaccines based only on HA content, VLPs
contain known quantities of the NA and M1 proteins. There-
fore, VLPs may have a greater potential for eliciting cross-
reactive antibody and T-cell immunity against newly emerging
antigenic drift (61) and shift variants (71). Influenza VLPs
have been shown to induce high neutralizing antibody titers
against homologous and heterologous strains in mice and fer-
rets (6, 7, 41, 53, 54), and a phase I clinical trial with an
investigational H5N1 VLP vaccine is ongoing (6).
In this study, we evaluated a 1918 VLP vaccine for the ability
to protect mice against the 1918 pandemic virus in the hope
that such a preclinical evaluation may pave the way for a 1918
vaccine which could be offered to laboratory workers working
with the virus to mitigate biosafety concerns. In addition, the
ability of the 1918 (H1N1) VLP vaccine to elicit protective
heterotypic immunity against a lethal challenge with a contem-
porary highly pathogenic avian influenza H5N1 virus was as-
sessed in mice and ferrets. The ferret model of influenza dis-
ease has been used to evaluate H5N1 virus virulence previously
(17, 40, 82), as well as the safety and efficacy of other H5N1
vaccine candidates (6, 18, 20, 35, 41). Mice immunized intra-
nasally (i.n.) with the 1918 VLP vaccine were protected not
only from death caused by the homologous 1918 virus but also
from a heterotypic H5N1 virus, whereas mice immunized par-
enterally were only protected against a homotypic 1918 virus
challenge. A study with ferrets further demonstrates that mu-
cosal VLP vaccination was clearly superior to parenteral vac-
cination for the induction of heterotypic immunity against an
H5N1 virus. Although subtype-cross-reactive neutralizing an-
tibodies were not associated with mouse and ferret survival,
high circulating and respiratory mucosal immunoglobulin G
(IgG) and IgA antibody levels correlated with heterotypic im-
munity protection in mice.
MATERIALS AND METHODS
Viruses and cells. The replication-competent influenza viruses used in these
experiments included (i) the reconstructed 1918 H1N1 (abbreviated 1918) virus
(72) possessing the A/South Carolina/1/18 HA and (ii) the A/Vietnam/1203/2004
H5N1 (abbreviated VN/1203) virus previously shown to be highly virulent for
both mice and ferrets (42, 74). The 1918 virus was generated with the 12-plasmid
reverse genetics system in a mixture of Madin-Darby canine kidney (MDCK;
ATCC, Manassas, VA) and 293T cells (ATCC) as previously described (72). The
VN/1203 virus was grown in embryonating hen’s eggs. All virus stock titers were
determined by plaque assay on MDCK cells, and virus stocks were maintained in
Dulbecco’s modified Eagle’s medium culture (Gibco, Grand Island, NY) sup-
plemented with 10% fetal calf serum (HyClone, Logan, UT) and 1% penicillin/
streptomycin (Gibco). All virus challenge experiments were performed under the
guidance of the U.S. National Select Agent Program in negative-pressure
HEPA-filtered biosafety level 3⫹(BSL-3⫹) enhanced laboratories with the use
of a battery-powered Racal HEPA filter respirator and according to Biomedical
Microbiological and Biomedical Laboratory procedures (58).
VLP generation. 1918 VLPs were made with recombinant baculovirus express-
ing the genes for HA, NA, and M1. The genes for HA, NA, and M1 used for the
generation of the VLPs were synthesized by reverse transcription-PCR with viral
RNA extracted from reconstructed 1918 influenza virus (72). Following reverse
transcription-PCR, the genes for the NA, M1, and HA cDNAs were combined in
this order within the pFastBac1 transfer vector (Invitrogen) essentially as de-
scribed previously (52). This resulted in a plasmid, pNVAX1250, that encoded
the genes for HA, NA, and M1, each within its own expression cassette that
included a polyhedrin promoter and transcription termination sequences. The
DNA sequences of the genes and flanking regulatory sequences were confirmed.
The HA gene was identical to that of influenza A virus A/South Carolina/1/18
(H1N1) (GenBank AAD17229), whereas the NA and M1 genes were identical to
the influenza A/Brevig Mission/1/18 virus genes (accession no. AAF77036 and
AAN06597, respectively). The DNA fragment from pNVAX1250 containing the
NA, M1, and HA expression cassettes was then transferred into bacmids and
transfected into Spodoptera frugiperda (Sf9) insect cells (ATCC CRL-1711) to
generate recombinant baculovirus (52). Recombinant baculoviruses were used to
infect Sf9 cells. VLPs were harvested at 72 h posttransfection. Recombinant
VLPs were purified to approximately 90% by sucrose density gradient ultra-
centrifugation, followed by ion-exchange chromatography. The influenza viral
proteins in the VLP preparations were confirmed by sodium dodecyl sulfate-
polyacrylamide electrophoresis and Western blot analysis. Semiquantitative
densitometry of the Coomassie-stained gel containing the purified VLP prepa-
ration showed that the HA, NA, and M1 proteins were present at a ratio of
approximately 61:5:22. The remaining ⬃12% of the Coomassie-stained material
contained impurities derived from Sf9 cells and baculovirus. A single-radial-
immunodiffusion assay was used to quantitate the HA content of the VLPs as
described previously (80). Reference reagents for the single-radial-immunodif-
fusion standard curve included A/New Caledonia/20/99 (H1N1) IVR-116 (79
␮g/ml of HA) antigen and anti-HA reference sheep antiserum S-6706 (obtained
from the Center for Biologics Evaluation and Research, Food and Drug Admin-
istration, Rockville, MD). Control VLPs derived from human immunodeficiency
virus (HIV)-type Con-S gp145 (77) were prepared and quantitated similarly.
Mouse vaccination and virus challenge. All animal research was conducted
according to the guidance of the CDC Institutional Animal Care and Use
Committee in an Association for Assessment and Accreditation of Laboratory
Animal Care International-accredited facility. Eight- to 10-week-old female
BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized by
intraperitoneal injection of 0.2 ml of 2,2,2-tribromoethanol in tert-amyl alcohol
(Avertin; Sigma-Aldrich, Milwaukee, WI) (73) and vaccinated either i.n. or
intramuscularly (i.m.) (right hind leg) with 5 ␮g (based on HA content) of 1918
VLPs in 50 ␮l of phosphate-buffered saline (PBS). Fourteen days post primary
vaccination, mice were boosted with a second dose of 5 ␮g of 1918 VLPs by
either the i.m. or the i.n. route. Mice were bled for collection of serum prior to
and following the first and second vaccinations. Lung and nasal washes were
collected as previously described in 1 ml of sterile PBS for antibody titration (75).
Two weeks post boost vaccination, mice were challenged i.n. with 50 50% lethal
doses (LD
50
) of either the 1918 or the VN/1203 virus in a volume of 50 ␮l. Mice
were weighed daily and observed for illness. On day 5 post virus challenge (p.c.),
four mice per group were exsanguinated and euthanatized and their lungs were
removed for virus titration. Lungs were homogenized in 1 ml of sterile PBS, and
clarified homogenate virus titers were determined by a standard plaque assay for
virus infectivity. Briefly, 10-fold serial dilutions of lung homogenate were placed
onto confluent monolayers of MDCK cells in the presence of tosylsulfonyl
phenylalanyl chloromethyl ketone (TPCK) trypsin (1 ␮g/ml). Following1hof
adsorption, cells were washed with PBS and a 2x L-15 medium (Cambrex/Lonza
Inc., Walkersville, MD) agar overlay was placed into each well. The statistical
significance of virus titer data was determined by using the analysis-of-variance
(ANOVA) test. Mouse survival data and mean time to death (MTD) were
subjected to Kaplan-Meier survival analysis with SPSS software.
Ferret vaccination and virus challenge. Eighteen male Fitch ferrets, 10 months
of age (Triple F Farms, Sayre, PA), that were serologically negative by hemag-
glutination inhibition (HI) assay for currently circulating influenza viruses were
used in these studies. 1918 VLP vaccinations were conducted under BSL-2
conditions, and H5N1 virus challenges were performed under BSL-3⫹condi-
tions within a Duo-Flo Bioclean unit (Lab Products, Seaford, DE) with a max-
imum capacity of 18 ferrets. Serum samples, nasal washes, and temperature and
weight measurements were taken throughout the duration of the study. Tem-
peratures were monitored following the virus challenge by a subcutaneously
implanted transponder (BioMedic Data Systems, Seaford, DE). Prior to the
study, baseline serum was collected via the anterior vena cava from all 18 ferrets.
Six ferrets were vaccinated with 15 ␮g/500 ␮l 1918 VLPs by the i.n. route (250 ␮l
in each nostril), and six ferrets were vaccinated by the i.m. route. The remaining
VOL. 83, 2009 HETEROTYPIC PROTECTION BY MUCOSAL VLP VACCINATION 5727

six ferrets received PBS in place of vaccine by the i.n. and i.m. routes. Fourteen
days following the initial VLP or PBS inoculation, serum was collected from all
of the ferrets and they were then boosted with 15 ␮g/500 ␮l by the same route
used previously. Fourteen days following the VLP boost, all 18 ferrets were
challenged i.n. with 50 50% ferret infectious doses in 1 ml of A/VN/1203/04
(H5N1) virus. Fifty percent ferret infectious doses were determined by inocu-
lating groups of ferrets each at 10
4
,10
3
,10
2
, and 10
1
50% egg infective doses and
calculating the outcome of infection by the method of Reed and Muench (57).
Ferrets were weighed daily and monitored for signs of illness. Nasal cavities of
infected ferrets were washed with 1 ml of PBS every other day, beginning on day
2 p.i., and measured for virus replication by 10-fold serial titration in hen’s eggs.
Serology. Sera collected from mice and ferrets prior to a virus challenge were
treated with receptor-destroying enzyme (RDE; Denka Seiken, Tokyo, Japan)
and tested for reactivity to the ␤-propiolactone-inactivated VN/1203 and 1918
viruses by the HI assay with 1.0% horse or 0.5% turkey red blood cells, respec-
tively, starting with a serum dilution of 1:10. Briefly, virus stocks were treated
with 0.1% ␤-propiolactone (Sigma-Aldrich) for 24 h at 4°C. Stocks were safety
tested by two serial passages in embryonating eggs before use in the HI assays.
Geometric mean titers (GMTs) and their standard deviations were calculated for
each treatment group. For the microneutralization assays, 100 50% tissue culture
infective doses of infectious VN/1203 or 1918 virus was incubated with twofold
serial dilutions of sera, starting at a 1:10 dilution, and incubated overnight on
MDCK cells (59). Titers represent the reciprocal dilution of serum giving
50% neutralization activity. An indirect enzyme-linked immunosorbent assay
(ELISA) was performed on RDE-treated mouse sera to determine the IgA and
IgG responses following vaccination (75). Briefly, IgA and IgG titers were mea-
sured with purified, formalin-inactivated VN/1203 and recombinant 1918HA/
NA:A/Texas/36/91 (49) viruses. Immulon microtiter plates (Thermo Scientific
Inc., Waltham, MA) were coated with 100 hemagglutination units of each virus,
and mouse antibody was detected with horseradish peroxidase-conjugated anti-
body (KPL Laboratories, Gaithersburg, MD). The ELISA endpoint titers were
expressed as the highest dilutions that yielded an optical densities greater than
the mean optical density plus 3 standard deviations of similarly diluted negative
control sera. Titers are expressed in the log
2
format, and the statistical signifi-
cance of differences between treatment groups was measured by the ANOVA
test.
RESULTS
Mucosal, but not parenteral, VLP vaccination induces het-
erotypic immunity in mice. 1918 (H1N1) VLPs generated in a
baculovirus expression system (Fig. 1A) from Sf9 cells were
purified from culture medium and negatively stained for elec-
tron microscopic examination. As shown in Fig. 1B, 1918 VLPs
had a diameter of approximately 80 to 120 nm with pro-
nounced surface protein spikes and a morphology characteris-
tic of replication-competent 1918 virions (Fig. 1C). We com-
pared the mucosal (i.n.) and parenteral (i.m.) vaccination
routes for the ability to induce protection against a homolo-
gous 1918 virus challenge, as well as an H5N1 heterotypic virus
challenge. BALB/c mice were vaccinated twice with 5 ␮gof
1918 VLPs or control HIV VLPs and challenged i.n. 14 days
post VLP boost with 50 LD
50
of either VN/1203 (H5N1) or
1918 (H1N1) virus (Fig. 2). The extent of vaccine protection
was measured by (i) morbidity (weight loss), (ii) survival over
a 14-day p.c. period, and (iii) virus titers in the lower respira-
tory tract (lung) at 5 days p.c. Mice vaccinated with 1918 VLPs
by either the i.n or the i.m. vaccination route uniformly sur-
vived the lethal challenge with the homologous 1918 virus,
while control animals succumbed to infection (MTD ⫽8.8
days; *, P⬍0.05, Kaplan-Meier survival analysis) (Fig. 2A, left
side). In contrast to the control animals, mice in both vaccina-
tion groups showed no signs of sickness and gained weight
every day p.c. although mucosally vaccinated mice gained, on
average, 5% more body mass over time than parenterally vac-
cinated mice (Fig. 2B, left side). The 1918 virus replicated
efficiently in the lungs of control mice, with titers of approxi-
mately 5.7 log
10
PFU/ml (Fig. 2C, left column). However, 1918
VLP-vaccinated/1918 virus-challenged mice did not have de-
tectable lung virus titers on day 5 p.c.
In contrast to the homologous subtype challenge data, all mice
that received the 1918 VLPs by the i.m route succumbed to a
VN/1203 (H5N1) heterotypic virus challenge (MTD ⫽6.4 days),
as did the HIV VLP control mice (MTD ⫽5.5 days) (Fig. 2A,
right column). Moreover, parenterally vaccinated mice exhibited
dramatic weight loss from 2 days p.c. until death, similar to those
animals vaccinated with the HIV VLPs (Fig. 2B, right column). In
contrast, five of six mice that received the same dose of 1918 VLP
vaccine administered i.n. were protected against an H5N1 het-
erotypic virus challenge (Fig. 2A, right column). The surviving
mice exhibited morbidity that reached a mean maximum weight
loss of 17% on day 5 p.c. before weight gain was observed from
day 6 p.c. onward (Fig. 2B, right column). The mean lung virus
titers of mice administered 1918 VLP vaccine by the i.n. route
were approximately 300-fold lower than those of control mice
receiving PBS or HIV VLP vaccine (Fig. 2C, right column). Mice
vaccinated i.m. and challenged with the H5N1 virus exhibited
titers of nearly 10
6
PFU/ml, only twofold lower than those of
control mice. Collectively, these results demonstrate that mucosal
H1N1 VLP immunization provides greater heterotypic immunity
against the H5N1 virus than does parenteral VLP vaccination.
Mucosal vaccination with 1918 VLPs results in higher IgG
and IgA antibody titers in mice than does parenteral vaccina-
tion. Characterization of postvaccination antibody responses was
performed in attempts to identify cross-reactive antibodies that
may be responsible for conferring protection against a lethal
H5N1 heterotypic virus challenge. First, the virus neutralization
(v.n.) antibody titers against the 1918 (H1N1) and VN/1203
(H5N1) viruses were measured in pooled sera (n⫽8or9per
group) collected from mice vaccinated twice with the 1918 VLPs
(Table 1). Mice vaccinated by either the i.n or the i.m. route
displayed v.n. antibody titers against the homologous 1918 virus
FIG. 1. 1918 VLP generation. VLPs were constructed in a baculo-
virus genetic background with the sequences of the genes for HA, NA,
and M1 of the 1918 pandemic virus and produced from Sf9 cells.
(A) Baculovirus construct for the expression of 1918 influenza VLPs.
Indicated are the polyhedrin promoter (PolH), polyadenylation signal
(pA), and influenza virus genes. 1918 VLPs had an average diameter
of 100 nm, as illustrated in this negatively stained transmission electron
micrograph. While not replication competent, the 1918 VLPs (B) mor-
phologically resemble the reconstructed 1918 virions that were col-
lected from supernatants of 1918 virus-infected MDCK cell cultures
(C). Mag, magnification.
5728 PERRONE ET AL. J. VIROL.

(1:800 to 1:1,600) but exhibited no detectable serum v.n. antibody
titer against the heterotypic H5N1 virus. As expected, mice vac-
cinated with the HIV VLPs had no detectable serum v.n. antibody
titer (⬍10) to either influenza virus. The HI antibody response
among individual mice was also measured 2 weeks after the vac-
cine boost. Similarly, vaccination of mice with 1918 VLPs elicited
a detectable HI antibody response against the homologous 1918
virus and the mucosally vaccinated mice elicited a GMT (individ-
ual sera ranged from 320 to 1,280; GMT ⫽607) that was 3.4-fold
greater than that of the parenterally vaccinated mice (range, 80 to
320; GMT ⫽180). However, sera from either 1918 VLP vacci-
nation group did not exhibit any cross-reactivity to the H5N1 virus
in the HI assay (⬍10). Mice vaccinated with HIV VLPs or given
PBS did not exhibit any HI antibody titer (⬍10) against either the
1918 or the VN/1203 virus.
We next performed ELISAs to investigate the presence of
H5N1-cross-reactive IgG and IgA antibodies in serum samples
and respiratory washes from mice administered two doses of
the 1918 H1N1 VLP vaccine (Fig. 3). Antiviral IgG and IgA
antibodies in the sera and mucosal washes of i.n. and i.m.
vaccinated mice were detected by using ELISA plates coated
with purified whole H1N1 or heterologous H5N1 virus. Over-
all, 1918 VLP immunization by either route induced IgG an-
tibody to the homologous 1918 virus in serum and respiratory
FIG. 2. 1918 VLP vaccine efficacy in mice following a lethal H1N1 or H5N1 virus challenge. Mice were vaccinated with 5 ␮g/50 ␮l 1918 VLPs or
control HIV VLPs and challenged with 50 LD
50
of either 1918 (H1N1) (left column) or VN/1203 (H5N1) (right column) virus. Mice were monitored
daily for 14 days p.c. Survival rates (A) following the challenge were calculated based percent survival within each experimental group (n⫽6 mice per
experimental group; *,P⬍0.05, Kaplan-Meier survival analysis). Mice were weighed daily (B) to measure morbidity (loss of 25% of the original body
weight necessitates euthanasia). Average weights in each treatment group were measured for the duration of the study, and percent original body weight
was calculated based on the average starting weight for each group on day 0. On day 5 p.c., four mice per experimental group were euthanized and their
lungs were removed for virus titration (C). Lungs were homogenized in 1 ml PBS and clarified before titer determination by standard plaque assay on
MDCK cells in duplicate (limit of detection, 10 PFU). Bars represent the average titer for each experimental group; error bars indicate the standard
deviation between mice in each respective group (n⫽4 mice per experimental group; *,P⬍0.05, ANOVA).
TABLE 1. Neutralizing and HI antibody titers from mouse sera
Vaccination group
(route)
c
Neutralizing
antibody titer
a
HI antibody titer
b
1918 H5N1 1918 H5N1
1918 VLP (i.n.) 1,600 ⬍10 607 ⬍10
1918 VLP (i.m.) 800 ⬍10 180 ⬍10
HIV VLP (i.n.) ⬍10 ⬍10 ⬍10 ⬍10
PBS (i.n. or i.m.) ⬍10 ⬍10 ⬍10 ⬍10
a
Mouse serum samples from each treatment group were pooled and tested for
neutralizing antibody activity against either the 1918 or the VN/1203 virus in a
microneutralization assay with MDCK cells. The titers represents the reciprocal
serum dilution with virus-neutralizing activity.
b
HI assays used either 0.5% turkey (BPL-1918 virus) or 1% horse (BPL-VN/
1203 virus) red blood cells. The titers shown are GMTs.
c
Mice were treated twice with 1918 VLPs, HIV VLPs, or PBS.
5729

mucosa washes and these samples also exhibited cross-reactive
IgG titers against the H5N1 virus (Fig. 3A). As shown in Fig.
3A (top), i.n. vaccinated mice exhibited significantly more
H5N1-cross-reactive serum IgG antibody than did mice vacci-
nated by the parenteral route (ˆ, P⬍0.05). Although homol-
ogous virus serum IgG titers were similar in both 1918 VLP
vaccination groups (GMT [log
2
]⫽20.3 [i.m.] versus 21.5
[i.n.]), i.n. vaccination resulted in lung mucosal IgG antibody
titers against the homologous H1N1 (*, P⬍0.05) and heter-
ologous H5N1 (ˆ, P⬍0.05) viruses that were significantly
higher than those measured in i.m. vaccinated mice (Fig. 3A,
middle). This trend was also observed in nasal wash samples, in
which the differences between the IgG titers of the two vacci-
nation groups were even more pronounced (* and ˆ, P⬍0.05)
(Fig. 3A, bottom). IgA antibodies to whole H1N1 and H5N1
virus were also examined. As shown in Fig. 3B, mucosal ad-
ministration of 1918 VLPs induced significantly higher homol-
ogous virus IgA titers in serum (top), and particularly lung
(middle) and nasal mucosal (bottom) washes, than did paren-
teral VLP vaccination (*, P⬍0.05). Likewise, circulating levels
of cross-reactive IgA antibody to the H5N1 virus were signif-
icantly higher (ˆ, P⬍0.05) in mice vaccinated by the i.n. route
(GMT [log
2
]⫽10.3) than in those vaccinated by the i.m. route
(GMT [log
2
]⫽2.9) (Fig. 3B, top). The differences in IgA titer
between these two vaccination groups was most pronounced in
lung wash samples (Fig. 3B, middle), and these trends were
also observed in nasal wash samples (Fig. 3B, bottom) (ˆ, P⬍
0.05). Together, these results show that vaccination with 1918
H1N1 VLPs induced a population of HI- and v.n.-negative,
cross-reactive IgG and IgA antibodies that were greater following
mucosal vaccination than following parenteral vaccination.
Ferrets mucosally vaccinated with 1918 VLPs are protected
from a lethal H5N1 virus challenge. Because of its natural
susceptibility to influenza A viruses, the ferret is currently
accepted as an excellent mammalian host for influenza vaccine
efficacy studies. We next compared mucosal versus traditional
parenteral administration of the 1918 VLP vaccine for the
ability to induce heterotypic immunity in this species. Ferrets
were vaccinated twice with 15 ␮g of the 1918 VLPs either i.n.
or i.m. and subsequently challenged 14 days post VLP boost
with 50 LD
50
of VN/1203 (H5N1) virus. Strikingly, all of the six
ferrets vaccinated with the 1918 VLPs i.n. survived a lethal
challenge with the H5N1 virus (Table 2), whereas 50% of the
ferrets vaccinated by the i.m. route and 100% of the control
animals succumbed to the lethal H5N1 virus infection. More-
over, mucosally vaccinated ferrets also exhibited less weight
loss, as a group, over time than did ferrets vaccinated by the i.m
route or with PBS alone (Fig. 4A). Ferrets vaccinated by the
i.m route did exhibit a longer survival time (MTD ⫽8.3 days)
than did PBS-inoculated control animals (MTD ⫽6.3 days).
While there were no significant temperature change differ-
ences observed between ferrets in the i.n. and i.m. vaccination
groups, overall the vaccinated animals exhibited less fever than
did the PBS-inoculated control animals (Fig. 4B). Ferrets vac-
cinated i.n. also exhibited reduced virus titers in their nasal
mucosa following infection than did the i.m. vaccinated ferrets,
and the differences between these two groups were statistically
significant on days 2, 4, 6, and 8 p.c. (Fig. 4C). All vaccinated
FIG. 3. Antibody responses in 1918 VLP-vaccinated mice. BALB/c mice were vaccinated with 1918 VLPs or HIV VLPs or given PBS. Serum,
lung wash, and nasal wash sample IgG (A) and IgA (B) antibody titers were determined following two vaccinations with 1918 VLPs. Sera was
treated with RDE, and all of the samples were individually tested in fourfold serial dilutions against the formalin-inactivated 1918 and VN/1203
viruses in an antibody capture ELISA. Plots show the geometric mean antibody titers (log
2
) of all of the mice in each treatment group, and error
bars indicate the standard deviation observed among the mice in each group. Significant differences between the antibody titers elicited by the
different vaccination routes are indicated (*[1918] and ˆ [VN/1203], P⬍0.05, ANOVA).
5730 PERRONE ET AL. J. VIROL.

ferrets exhibited lower virus titers than did the PBS-inoculated
animals, indicating that while the i.m. vaccinated ferrets were
not completely protected against death, VLP vaccination still
induced an immune response capable of suppressing some
virus replication in the upper respiratory tract. The HI anti-
body responses to the H1N1 (1918) and H5N1 (VN/1203)
viruses were measured in individual serum samples collected
from ferrets (n⫽6) vaccinated twice with 1918 VLPs. Ferrets
vaccinated by the i.n route had slightly higher circulating HI
antibody titers to the homologous virus (GMT ⫽254) than did
i.m. vaccinated ferrets (GMT ⫽160) (Table 2). HI antibody
against the H5N1 virus was not detected in either group of
VLP-vaccinated ferrets. Collectively, these results from the
ferret challenge model confirm the experimental outcomes
observed in the mouse studies, showing that mucosal influenza
immunization with 1918 VLPs can provide greater heterotypic
immunity than parenteral influenza vaccination.
DISCUSSION
The emergence of new influenza virus subtypes to which the
human population has little immunity is of great public health
concern, and vaccination remains the principal and most econom-
ically prudent public health intervention strategy against both
seasonal and pandemic influenza. The ability of the 1918 (H1N1
subtype) virus to spread rapidly and cause high rates of human
illness makes it an ideal candidate for studying vaccine efficacy
against a pandemic strain. Furthermore, scientists working with
the reconstructed 1918 virus would benefit from the development
of and access to an effective vaccine against this recognized killer.
The high mortality rate associated with human infections by
H5N1 viruses (15, 25, 34; http://www.who.int/csr/disease/avian
_influenza/country/en/index.html) highlights the need for the de-
velopment of improved vaccine technologies. To date, clinical
evaluations of many H5N1 vaccine candidates indicate the need
for improved adjuvants or alternative approaches that could en-
hance vaccine immunogenicity (31–33, 46, 47). Moreover, vaccine
strategies capable of inducing more cross-reactive immunity may
overcome limitations in vaccine efficacy imposed by antigenic
variability of influenza A viruses (11). VLPs have been generated
previously from a number of subtypes and have been shown to
promote a strong immune response to a challenge with a homol-
ogous virus (14, 41, 52, 53); however, their capacity to induce
heterotypic immunity has not been adequately addressed.
In the present study, we tested the ability of nonadjuvanted
1918 VLPs to provide protection against the reconstructed 1918
pandemic virus, as well as elicit cross-protection against a lethal
H5N1 virus challenge. Two routes of vaccination (mucosal and
parenteral) were compared to assess the potential effect of the
route of VLP administration on vaccine efficacy, and two mam-
malian models of highly pathogenic influenza disease were used
(39, 42, 72, 74). All of the mice vaccinated with the 1918 VLPs
and lethally challenged with the 1918 virus survived and were well
protected, regardless of the vaccination route, supporting results
from previous homologous virus challenge studies with baculo-
virus-expressed influenza VLPs (4, 41, 53, 55). Importantly, these
studies showed that mucosal VLP vaccination was superior to
parenteral vaccination for the induction of heterotypic immunity.
The cross-protective effect of mucosal vaccination was associated
with a reduction in weight loss and reduced H5N1 virus replica-
tion in the respiratory mucosa.
Antibodies are prime candidates for contributors to het-
erotypic immunity because they are generated to conserved
antigens, and further support comes from studies in which
FIG. 4. Ferret morbidity following 1918 VLP vaccination and an
H5N1 virus challenge. Eighteen ferrets were divided into three exper-
imental groups. Six ferrets were vaccinated by the i.n. or i.m. route with
15 ␮g/500 ml 1918 VLPs. Six control ferrets received the same volume
of PBS i.n. plus i.m. Twenty-eight days following the administration of
two doses of 1918 VLPs or PBS, ferrets were challenged i.n. with 50
LD
50
of the A/Vietnam/1203/04 (H5N1) virus and monitored daily for
signs of illness. The ferrets were weighed (A) and their temperatures
were measured individually (B) for 14 days p.c. Control ferrets all
succumbed to infection by day 6 p.c. (†). The nasal cavities of infected
ferrets were washed with 1 ml PBS every other day beginning on day
2 p.c., and virus titers were determined (C). Differences in virus titers
between vaccination groups were analyzed by ANOVA (*,P⬍0.005).
TABLE 2. Ferret mortality following 1918 VLP vaccination and
lethal H5N1 challenge
Group No. of
ferrets
No. that
survived virus
challenge
(% mortality)
MTD (days
postchallenge)
Serum HI
antibody GMT
prior to virus
challenge
1918 H5N1
VLP i.n. 6 6 (0) NA
a
254 ⬍20
VLP i.m. 6 3 (50) 8.3 180 ⬍20
PBS 6 0 (100) 6.3 ⬍20 ⬍20
a
NA, not applicable.
VOL. 83, 2009 HETEROTYPIC PROTECTION BY MUCOSAL VLP VACCINATION 5731

B-cell-deficient mice failed to survive a heterotypic chal-
lenge (45, 75). In the present study, characterization of the
postvaccination antibody responses identified differences
between the two routes of vaccination. ELISAs revealed the
presence of H5N1-cross-reactive IgG and IgA antibodies
among H1N1 VLP-vaccinated animals, suggesting the pres-
ence of common cross-reactive epitopes in the HA, NA, or
M1 protein. Mice vaccinated by the mucosal route generally
had higher levels of antiviral IgG and IgA antibodies than did
parenterally vaccinated mice. The differences in cross-reactive
antibody titers between the two vaccination groups were most
pronounced in the lung and upper respiratory tract. Polymeric
IgA antibody may contribute to heterotypic immunity by its
ability to pass through the lung epithelium and, during this
transcytosis, interfere with the production of viral proteins in
an infected cell (43, 44). However, heterotypic immunity can
be effective in IgA
⫺/⫺
mice, suggesting that this class of anti-
body is unlikely playing a role (11). Cross-reactive IgG anti-
body was found in the lungs along with IgA and has been
considered a correlate of heterotypic protection (75). In mice,
mucosal but not parenteral vaccination induced subtype-cross-
reactive lung and serum IgG anti-HA antibodies, suggesting
the presence of a common cross-reactive epitope in the HA of
the H3 and H5 subtypes (75).
Although mucosally vaccinated animals were largely pro-
tected against a lethal H5N1 virus challenge, neutralizing an-
tibody against the H5N1 virus was not detected in any group of
VLP-vaccinated animals. It is conceivable that the cross-reac-
tive anti-HA antibodies produced in VLP-vaccinated hosts act
by additional mechanisms in vivo to neutralize progeny virus
and/or enhance the clearance of virus-infected cells (11, 16, 37,
75). While the identity between the HA1 amino acid sequences
of the VN/1203 H5N1 and 1918 viruses is only 58%, antibodies
to the conserved HA2 stem region are generated and may
affect overall virus binding and immune recognition (13, 48,
64). More recent support for our findings presented here
showed that neutralizing antibody epitopes exist in the stem
region of the HA molecule and that while antibody bound to
the HA2 region does not classically “neutralize” the receptor
binding site, it prevents the molecule from conformationally
changing upon endocytosis and exposure to a low pH, thereby
preventing viral fusion with the cell membrane (9, 48, 63, 64,
67). It is reasonable to speculate that mucosal vaccination with
VLPs generates a population of v.n.-negative, cross-reactive
IgG and IgA antibodies to regions of the virus HA molecule
that promote virus clearance. A cross-reactive epitope(s) on
the NA may also be generating nonneutralizing antibodies.
Anti-NA antibodies can partially reduce the viral yield and
plaque size of influenza viruses from infected cells. The 1918
and VN/1203 viruses exhibited 85% identity in their NA amino
acid sequences, and N1-specific antibodies have been shown to
provide partial cross-protection against H5N1 virus infection
(61). Studies are under way to delineate the cross-reactive
epitope(s) on the HA and NA glycoproteins.
Mucosal vaccination with these VLPs may also be inducing
a stronger protective immune response because of the inherent
stimulation of innate immune sentinels such as Toll-like recep-
tors on the mucosal surface (22), as these VLPs contain viral
surface proteins in their native conformation and it is pre-
sumed that these particles are capable of binding to cells ex-
pressing the viral receptor ␣-2-3 or -2-6 sialic acid, as do in-
fectious virus particles (66, 74). Mucosal vaccination may be
better than parenteral administration at delivering viral anti-
gens to the pertinent anatomical compartment, specifically to
dendritic cells in the respiratory tissue which play a critical role
in communicating with T and B cells to develop immunological
memory against influenza viruses (62). Supporting this hypoth-
esis is our observation that mice vaccinated mucosally express
twofold greater amounts of interleukin-2 (IL-2) (a cytokine
released by activated CD4
⫹
T cells to allow for clonal expan-
sion of antigen-specific T cells and also a potent activator of B
cells) in the lungs 5 days following an H5N1 virus challenge
than do mice vaccinated parenterally (data not shown), which
may contribute to the overall survival associated with this vac-
cination group. Also elevated in mucosally VLP-vaccinated/
H5N1-challenged surviving mice were the cytokines IL-9 and
IL-17 (data not shown), both of which are recognized to facil-
itate B-cell proliferation. The broadly cross-neutralizing anti-
bodies reported recently were of the IgG1 subclass (10, 67),
B-cell products that help mediate the Th2 CD4
⫹
T-cell and
cytokine response, including IL-9 and L-10 production. IL-10
was recently shown to be important in regulating pulmonary
inflammation in influenza virus-infected mice, and inhibition
of this cytokine results in severe lung inflammation (68). We
found that levels of IL-10 in lungs from VLP-vaccinated mice
were three times higher in those infected with the H5N1 virus
than in 1918 virus-challenged mice (data not shown), possibly
contributing to their survival. While the protective role of
CD8
⫹
cytotoxic T lymphocytes and memory B cells following
1918 VLP vaccination warrants further investigation, previous
studies on the induction of heterotypic immunity has revealed
CD8
⫹
T cells to be generally accessory to mouse survival
following a lethal virus challenge, while B cells are critical (8,
12, 45, 56, 75).
Our studies raise important questions regarding the appli-
cation of this vaccination technology, in both seasonal and
epidemic outbreak situations. Specifically, could mucosal ad-
ministration of influenza VLP vaccine bearing seasonal or pan-
demic influenza virus proteins reduce the widespread morbid-
ity and lethality due to a newly emerging subtype prior to the
production of a strain-specific vaccine? Other research has
indicated that this may be possible (23, 29). A vaccine that
could induce or boost heterotypic immunity through the stim-
ulation of a cross-reactive antibody could be an important
preventative measure against a novel subtype, allowing time
for the development of a pandemic strain-specific vaccine.
ACKNOWLEDGMENTS
L.A.P. was supported by a fellowship from the American Society for
Microbiology and the CDC Coordinating Center for Infectious Dis-
eases.
We thank the Vietnamese Ministry of Health for use of the A/Viet-
nam/1203/04 isolate and Jessica Belser for the ferret LD
50
determina-
tion of that virus stock. We thank Debra Wadford, Neal Van Hoeven,
Joshua DeVos, and Ebonee Butler for providing reagents and assisting
with the serological assays. We also thank Ye Liu and Tom Kort for
expert assistance in VLP purification and Feng Lui for his assistance in
statistical analysis of mouse mortality data.
The findings and conclusions in this report are ours and do not
necessarily represent the views of the funding agency.
We have no competing interests to report.
5732 PERRONE ET AL. J. VIROL.

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