Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Article (PDF Available)inVaccine 23(50):5751-9 · January 2006with212 Reads
DOI: 10.1016/j.vaccine.2005.07.098 · Source: PubMed
Abstract
Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80-120nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.
Vaccine 23 (2005) 5751–5759
Influenza virus-like particles comprised of the HA, NA,
and M1 proteins of H9N2 influenza virus induce
protective immune responses in BALB/c mice
Peter Pushko
a,
, Terrence M. Tumpey
b
, Fang Bu
b
, John Knell
a
,
Robin Robinson
a
, Gale Smith
a
a
Novavax, Inc., Vaccine Technologies, 1 Taft Court, Rockville, MD 20850, USA
b
Influenza Branch, Centers for Disease Control and Prevention, Atlanta, 1600 Clifton Road, GA 30333, USA
Received 25 April 2005; accepted 28 July 2005
Available online 15 August 2005
Abstract
Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the
H9N2subtype,weexpressedininsectcellsvirus-likeparticles(VLPs)consistingofthreestructuralproteinsofinfluenzaA/HongKong/1073/99
(H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1)
proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120nm in diameter.
VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs
elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after
challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Avian influenza virus; Virus-like particles; Influenza vaccine
1. Introduction
Avian influenza A viruses are considered likely culprits
of future influenza pandemics [1,2]. In the last decade, the
H9 subtype of avian influenza has been isolated from several
species of domestic birds worldwide [3,4]. In 1999, the avian
H9N2 influenza virus was isolated from two children who
recovered from influenza-like illnesses in Hong Kong [5].
An additional five human cases of H9N2 influenza infection
in southern China were reported [6]. Genetic reassortment
between avian and human influenza viruses may lead to the
emergence of a pandemic influenza virus that could spread
rapidly in the human population [7,8]. The difficulty of the
conventional production of avian influenza vaccines includ-
ing necessity for BSL-3 containment facilities, have led to
Corresponding author. Tel.: +1 301 738 1106; fax: +1 301 738 1109.
E-mail address: ppushko@novavax.com (P. Pushko).
a push to develop strategies to produce recombinant avian
influenza vaccines as a priority for pandemic preparedness.
Influenza A virus is a member of the Orthomyxoviri-
dae family [9]. The most abundant structural protein of the
virion is the matrix protein, M1, which lies beneath the virus
envelope.Theenvelopeisderivedfromtheinfectedcellmem-
braneandcontainsmultiplecopiesofthehemagglutinin(HA)
and neuraminidase (NA) glycoproteins. Successful prophy-
lactic influenza vaccineselicit efficient HA-specific systemic
antibody, which can bind the virus and inhibit early events in
the influenza virus infection.
Recombinant influenza HA proteins expressed in insect
cells using a baculovirus expression system were shown to
prevent lethal influenza in chickens caused by avian H5 and
H7 influenza subtypes [10]. For many viruses, VLPs have
also been shown to be effective immunogens, because VLPs
are comprised of numerous copies of epitopes in native con-
formation [11,12]. In a recent study, co-expression of four
0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2005.07.098
5752 P. Pushko et al. / Vaccine 23 (2005) 5751–5759
genes,HA, NA,M1, andM2, ofhuman influenzaH3N2virus
ininsect cells resultedin the self-assemblyof influenzaVLPs
and protection against H3N2 virus challenge in mice [13,14].
In this report, we describe the development of an H9N2
influenza VLP vaccine comprised of only three influenza
virus structural proteins, HA, NA, and M1, which were
derived from avian influenza A/Hong Kong/1073/99 (H9N2)
virus. The H9N2 VLPs derived from insect cells exhibited
hemagglutination and neuraminidase activities and elicited
protective immune responses in BALB/c mice. These results
suggest that VLPs represent a promising vaccine candi-
date for H9N2 influenza and likely other subtypes of avian
influenza viruses with pandemic potential.
2. Materials and methods
2.1. Cloning of HA, NA, and M1 genes
Influenza A/Hong Kong/1073/99 virus was provided
kindly by Dr.K. Subbarao and Dr. N. Cox (Influenza Branch,
CDC, Atlanta, GA). Viral RNA was extracted using Tri-
zol LS (Invitrogen, Carlsbad, CA) under BSL-3 contain-
ment conditions. Reverse transcription (RT) and PCR were
performed on extracted viral RNA using the One-Step RT-
PCR system (Invitrogen) with gene-specific oligonucleotide
primers. The following primer pairs were used for the syn-
thesis of the HA, NA, and M1, respectively: 5
-AGGATCC-
ATGGAAACAATATCACTAATAAC-3
and 5
-AGGTA-
CCTTATATACAAATGTTGCATCTGC-3
;5
-AGAATTC-
ATGAATCCAAATCAAAAGATAATA-3
and 5
-AGATAT-
CTTATATAGACATGAAATTGATATTC-3
; and 5
-AGAA-
TTCATGAGTCTTCTAACCGAGGTCG-3
and 5
-AGGT-
ACCTCACTTGAATCTCTGCATTTGC-3
(ATG codons
shown in italics). Following RT-PCR, cDNA fragments con-
taining influenza HA, NA, and M1 genes were cloned
into the pCR2.1-TOPO vector (Invitrogen). The nucleotide
sequences of the HA, NA, and M1 genes were determined by
DNA sequencing and found to be identical to the previously
published sequences (GenBank AJ404626, AJ404629, and
AJ278647, respectively).
2.2. Generation of recombinant baculoviruses
The HA gene was cloned as a BamHI-KpnI DNA frag-
ment (1.7 kb) downstream of the AcMNPV polyhedrin pro-
moter within pFastBac1bacmid transfer vector (Invitrogen)
digested with BamHI and KpnI. Similarly, the NA and M1
genes were cloned as EcoRI-EcoRI DNA fragments (1.4 and
0.8kb, respectively) into EcoRI-digested pFastBac1 plasmid
DNA (Fig. 1a). The three resulting baculovirus transfer plas-
mids containing influenza virus genes were designated pHA,
pNA, and pM1.
A bacmid transfer vector expressing both HA and M1
genes was prepared by cloning a SnaBI-HpaI DNA fragment
from pM1plasmid into the HpaIsite of pHA. This resultedin
Fig. 1. (a) Constructs for individual expression of influenza proteins. (b)
Constructs for co-expression of influenza proteins. Indicated are the poly-
hedrin promoter (Polh), polyadenilation signal, Tn7 regions, gentamicin
resistance gene (Gm), and influenza A/Hong Kong/1073/99 (H9N2) genes.
HA, hemagglutinin; NA, neuraminidase; M1, matrix protein.
a plasmid encoding both the HA and M1 genes from the sep-
arate polyhedrin promoters and designated pHAM (Fig. 1b).
Finally, a bacmid transfer vector that permitted expression
of the three influenza genes was constructed by cloning a
SnaBI-AvrII DNAfragment frompHAM thatcontained both
theHAandM1expressioncassettesintoHpaI-AvrII-digested
pNA plasmid DNA. This resulted in a plasmid, pNAHAM,
that encoded the HA, NA, and M1genes, each within its own
expression cassette that included a polyhedrin promoter and
transcription termination sequences (Fig. 1b).
Recombinant bacmids were produced by site-specific
homologous recombination following transformation of
bacmid transfer plasmids containing influenza genes into
E. coli DH10Bac competent cells, which contained the
AcMNPV baculovirus genome (Invitrogen). The recombi-
nant bacmid DNA was transfected into the Sf9 insect cells
seeded in 6-well platesat 0.5× 10
6
cells/ml using CellFectin
reagent (Invitrogen). At 72h post-transfection, cells were
harvested for protein expression analysis and recovery of
recombinant baculoviruses in the culture medium.
2.3. Cell culture and baculovirus infections
Spodoptera frugiperda Sf9insectcells(ATCCCRL-1711)
were maintained as suspension cultures in HyQ-SFX insect
serum free medium (HyClone, Logan, UT) at 28
C. Plaque
isolates expressing influenza proteins were amplified by
infecting Sf9 cells seeded in shaker flasks at 2× 10
6
cells/ml
at a multiplicity of infection (MOI)=0.05. At 72h post-
infection, culture supernatants containing the recombinant
baculoviruseswereharvested,clarifiedbycentrifugation,and
stored at 4
C. Titers of recombinant baculovirus stocks were
determined by agarose plaque assay in Sf9 cells.
2.4. Protein expression
For protein expression, Sf9 cells were infected in 200 ml
volume for 72h at a cell density of 2 × 10
6
cells/ml with
P. Pushko et al. / Vaccine 23 (2005) 5751–5759 5753
recombinant baculoviruses at a MOI= 3. Expression was
determined by SDS–PAGE using 4–12% gradient polyacry-
lamide gels (Invitrogen) and Coomassie staining and by
Western blotting using antigen-specific sera (not shown for
individual proteins). Expression of recombinant HA and
M1 proteins was also confirmed by indirect immunoflu-
orescence assay (IFA) using infected cells fixed with
acetone.
H9N2 specific sera included rabbit polyclonal sera R#55
(obtained from A. Klimov, CDC) and chicken polyclonal
sera antibody C99-26 (obtained from R. Webster, St. Jude
Children Research Hospital, Memphis, TN) raised against
influenza A/Hong Kong/1073/99 (H9N2) virus. Addition-
ally, murine monoclonal antibody MCA401 to influenza M1
protein was used; this antibody was generated using both
influenza A/PR/8/34 (H1N1) and A/Bangkok/1/79 (H3N2)
viruses (Serotec, Oxford, UK). Secondary antibodies were
goat anti-IgG directed to rabbit, chicken, or mouse IgG
(H+L) and conjugated to alkaline phosphatase or fluores-
cein isothiocyanate (Kirkegaard and Perry, Gaithersburg,
MD).
2.5. Sucrose gradient ultracentrifugation
Culture supernatants (200ml) from Sf9 cells infected
with recombinant baculoviruses were harvested and clari-
fied by centrifugation for 1 h at 10,000× g at 4
C. VLPs
were pelleted for 3 h at 27,000rpm and 4
C using a Sor-
vall AH-629 swinging bucket rotor. The pellets were resus-
pended in 1ml of phosphate buffered saline (PBS) solu-
tion (pH 7.2), loaded onto a 20–60% (w/v) discontinuous
sucrose step density gradient, and sedimented by ultracen-
trifugation for 16h at 27,000rpm and 4
C. As a control,
influenza A/Sydney/5/97 (H3N2) virus was used. Fractions
(1ml) werecollected fromthe topof the gradient using a1ml
syringe. Proteins were analyzed by SDS–PAGE and West-
ern blotting, and fractions containing influenza VLPs were
pooled.
2.6. Chromatography
Gel filtration chromatography of pooled gradient frac-
tions containing influenza proteins was performed using
Sepharose CL-4B resin in PBS solution, pH 7.2, in a col-
umn 7 mm in diameter with a bed volume of 14ml. Prior
to loading, the column was equilibrated with PBS and cal-
ibrated with dyes that included dextran blue 2000, dextran
yellow, and vitamin B12 with apparent molecular weights
of 2,000,000, 20,000, and 1357Da, respectively (Amersham
Biosciences, Piscataway, NJ). Void volume of the column
was determined using dextran blue 2000. As a control,
influenza A/Sydney/5/97 (H3N2) virus was used. An aliquot
of VLPs or a control virus (100g) was loaded onto
the column at a flow rate of 0.1ml/min. Fractions (0.2ml)
were collected and analyzed by SDS–PAGE and Western
blotting.
2.7. Electron microscopy
Aliquot of the pooled sucrose gradient fractions was
treated for 24 h at 4
C with 2% glutaraldehyde in PBS,
pH7.2, adsorbed onfreshly dischargedplastic/carbon-coated
grids, and washed with deionized water (Charles River Lab-
oratories, Durham, NC). Washed samples were stained with
2%sodiumphosphotungstate,pH6.5,andstainedVLPswere
observed by transmission electron microscope at magnifica-
tions ranging from 6000× to 100,000×.
2.8. Hemagglutination and hemagglutination inhibition
A series of two-fold dilutions of influenza VLPs in PBS
were prepared and incubated at 25
C for 2h with 40 lof
0.6% guinea pig red blood cells. The extent of hemagglutina-
tion was inspected visually, and the highest dilution capable
of agglutinating guinea pig red blood cells was determined.
A hemagglutination inhibition assay (HAI) was carried out
essentially as described previously [15] using 4 hemaggluti-
nation units of -propiolactone inactivated influenza virus
A/Hong Kong/1073/99 (H9N2), which were mixed with
serial dilutions of antibodies from VLP-immunized BALB/c
mice prior to addition of guinea pig red blood cells.
2.9. Neuraminidase assay
Neuraminidase activity was determined by a neu-
raminidase enzyme assay using fetuin as a substrate for
cleavage of sialic acid. An aliquot of influenza VLPs was
incubated with fetuin for 16h at 37
C [16]. The amount of
sialicacid liberatedwas determined chemically withthe thio-
barbituric acid that produced a pink color in proportion to the
amount of free sialic acid, which was measured spectropho-
tometrically. The NA activity was expressed as the optical
density (OD) at a wavelength of 549nm.
2.10. Vaccination and challenge
Influenza VLPs pooled from sucrose gradients were
diluted with two volumes of PBS, pelleted by ultracentrifu-
gation as described above, and resuspended at a final protein
concentration of 1mg/ml in PBS, pH 7.2. In the first experi-
ment,10femaleBALB/cmicepergroupwereinoculatedsub-
cutaneously (s.c.) with 10g of VLPs in PBS on days 0 and
28.AnegativecontrolgroupofBALB/cmicereceivedPBSin
place ofVLPs. Serum samples weretaken before theprimary
and boosterinoculations (days 0 and 28),as well as at day57.
Serum antibodies were determined by HAI titration as well
as by ELISA and Western blotting using beta-Propiolactone-
inactivated influenza A/Hong Kong/1073/99 influenza virus
asantigen.Inthesecondexperiment,thirteenfemaleBALB/c
mice per group were inoculated intramuscularly (i.m.) in the
left hind leg with 10 g of influenza VLPs on days 0 and
28 whereas group of negative control animals received PBS
only. Serum was collected before virus challenge and tested
5754 P. Pushko et al. / Vaccine 23 (2005) 5751–5759
by ELISA for the presence of anti-H9 HA-specific IgG anti-
body as previously described [17]. ELISA plateswere coated
with a purified baculovirus-expressed recombinant HK/1073
HA protein (Protein Sciences Corporation, Meriden, CT;
1g/ml). The bound antibody was detected by the addition
of goat anti-mouse IgG conjugated to horseradish peroxi-
dase(Kirkegaard&Perry). Titersareexpressedas thehighest
dilution that yielded on optical density greater than the mean
plus twostandard deviationsof similarly diluted control sera.
On day 60, animals were transferred into a BSL3+ con-
tainment facility and challenged intranasally (i.n.) with 100
mouse infectious doses, MID
50
(equivalent to 10
5.5
50% egg
infectious doses, EID
50
) of influenza A/Hong Kong/1073/99
virus [17]. Five mice were sacrificed on day 3 and another
four on day 5 post-challenge for determination of influenza
virus replication in lung and nose tissues. Tissues were col-
lected, snapfrozen at 70
C andlater thawed,homogenized
in 1 ml of cold PBS and pelleted by centrifugation. Clari-
fied homogenates were titrated for virus infectivity in eggs
from initial dilutions of 1:10. The limit of virus detection
was 10
1.2
EID
50
/ml. The remaining mice were observed and
weighed daily for 14 days for morbidity.
3. Results
3.1. Expression of VLP vaccine for influenza A/Hong
Kong/1073/99 (H9N2) virus
Recombinantbaculovirusesweregeneratedforexpression
of individual HA, NA, or M1 proteins as well as for co-
expression of these proteins (Fig. 1). Baculoviruses express-
ing individual influenza proteins contained a single expres-
sion cassette comprised of the AcMNPV polyhedrin pro-
moter, influenza gene of interest (HA, NA, or M1), and tran-
scription termination and polyadenylation signals (Fig. 1a).
Baculovirus bHAM encoded both HA and M1 genes from
twoseparate polyhedra expressioncassettes; and baculovirus
bNAHAM encoded HA, NA, and M1 genes from three sep-
arate polyhedra expression cassettes (Fig. 1b).
To determine whether avian influenza VLPs were formed
by self-assembly from three structural proteins, HA, NA,and
M1, Sf9 insect cells were co-infected with the three recom-
binant baculoviruses encoding each of these influenza genes
(Fig. 1a). Sf9 cells showed a high degree of cytopathology,
possibly due to high MOI, as each of the baculovirus con-
structswasusedatMOI=3.Theattemptstorecoverinfluenza
VLPs from culture supernatants harvested from co-infected
cells or from cells co-infected with BacHAM and BacNA
were inconclusive (data not shown). Alternatively, infection
ofSf9cellswithbNAHAM,whichco-expressedallthreepro-
teins (Fig. 1b), resulted in the expression and self-assembly
of VLPs that were secreted from infected cells into the cul-
ture medium (Fig. 2). The influenza proteins co-localized at
the surface of the infected cells as determined by indirect
immunofluorescence assays (data not shown).
Influenza VLPs were isolated from 200ml cultures by
pelleting and ultracentrifugation in 20–60% sucrose den-
sity gradients. Fractions were analyzed for the presence of
influenza proteins by Western blot (Fig. 2a), hemagglutina-
tion,andneuraminidaseassays(Fig.2b).Theauthenticityand
the expected molecular weights of HA and M1 proteins were
confirmed by Western blot using polyclonal serum to H9N2
influenza and a monoclonal antibody to M1. The majority
Fig. 2. Purification of influenza VLPs from Sf9 culture supernatants by sucrose density gradient centrifugation. (a) Analysis of fractions 1through 9 by Western
blot using chicken serum C99-26 specific forH9 (upperpanel) andmurine monoclonalantibody MCA401to M1 (bottom panel). Positions of influenzaHA and
M1 proteins are indicated on the right. Influenza A/Sydney/5/97 (H3N2) virus was used as control (not shown). (b) Analysis of fractions by hemagglutination
(upper panel) and neuraminidase assays (bottom panel). For hemagglutination, guinea pig red blood cells were used. Neuraminidase assay was carried out
using fetuin as a substrate. Data points represent average of two experiments, bars indicate standard deviation values.
P. Pushko et al. / Vaccine 23 (2005) 5751–5759 5755
of the HA and M1 proteins were detected in fractions 5,
6, and 7 corresponding to sucrose densities of 32.8–41.0%.
The maximal hemagglutination activity (1:4048) was also
observed in fractions 5 through 7 (Fig. 2b). Similarly, the
majority of the NA activity was also detected in fractions
5, 6, and 7 as determined by neuraminidase assay (Fig. 2b).
Another peak of neuraminidase activity was detected in frac-
tion 3 suggesting that NA may also be present in culture
supernatants in soluble form or in association with the cel-
lular components. As expected, the recombinant HA protein
derived from insect cells wasin the uncleavedHA
0
precursor
form with a molecular weight of approximately 70 kDa. No
significant amounts of processed HA
1
and HA
2
polypeptides
weredetected.Thesedatademonstratedthatuponinfectionof
insect cells with bNAHAM, all three influenza proteins were
co-released from infected cells, and co-purified in the same
sucrose density gradient fractions suggesting that HA, NA,
and M1 proteins self-assembled into VLPs, which retained
hemagglutination and neuraminidase functions. The pres-
ence of baculoviruses in the sucrose gradient fractions was
determined by an agarose baculovirus plaque assay. In frac-
tion 5, the baculovirus titer was 3.1× 10
6
pfu/ml, whereas
the majority of baculovirus was detected in fractions 8 and 9,
with titers of>3.0 × 10
8
pfu/ml.
3.2. Characterization of influenza VLPs by gel filtration
chromatography and electron microscopy
In order to confirm that H9N2 influenza HA, NA, and M1
proteins self-assembled into high-molecular weight VLPs,
fractions5, 6, and7 from thesucrose densitygradient (Fig. 2)
were pooled and loaded onto a Sepharose CL-4B column.
BecausetheexpectedmolecularweightofinfluenzaVLPisat
least 2,000,000Da, the column was calibrated using dextran
blue 2000 (MW 2,000,000Da) and influenza A/Sydney/5/97
(H3N2) virus.As expectedfor H9N2 VLPs, theHA, NA, and
M1 proteins co-migrated in the void volume of the column
(Fig. 3a). In column fractions, HA and NA were detected in
multiple bands, which may reflect incomplete dissociation of
oligomeric protein complexes within VLPs. The M1 protein
was detected as 29kDa protein with monoclonal antibody
MCA401 but not with rabbit serum. In addition, a diffuse
band of 42–48kDa was detected, which may result from the
proteolytic processing of influenza glycoproteins in insect
cells.
Electron microscopic examination of negatively-stained
samples revealed the presence of influenza A/Hong
Kong/1073/99 (H9N2) VLPs with a diameter of approxi-
mately 80–120nm, which is consistent with the morphology
and size of influenza virus particles. VLPs were associated
frequently as groups resembling bead-like structures, and
showed surface spikes, characteristic of influenza HA pro-
tein on virions (Fig. 3b).
3.3. Influenza VLPs are immunogenic and inhibit
replication of influenza A/Hong Kong/1073/99 virus in
mice
Because veterinary avian influenza vaccines are adminis-
tered s.c., in the first experiment, BALB/c mice received two
inoculations s.c. of influenza VLPs from pooled fractions 5
through 7. Animals showed no ill effects after administra-
tion of 10g of influenza VLPs. Blood samples were taken
before the primary and booster inoculations as well as after
the booster. Sera were tested for the presence of antibodies to
influenza A/Hong Kong/1073/99 (H9N2) virus by ELISA,
Fig. 3. (a) Characterization of influenza VLPs using Sepharose CL-4B chromatography, by Western blot using rabbit H9N2 influenza-specific serum (upper
panel) and murine M1-specific monoclonal antibody MCA401 (bottom panel). Positions of elution of dextran blue 2000 (2,000,000 Da) and dextran yellow
(20,000Da) are indicated with arrows. Positions of HA, NA, and M1 proteins are indicated on the right. (b) Negative staining electron microscopy of H9N2
influenza VLPs comprised of the HA, NA, and M1 proteins. Bars represent 100 nm.
5756 P. Pushko et al. / Vaccine 23 (2005) 5751–5759
Fig. 4. Induction of serum antibody in BALB/c mice vaccinated with influenza VLPs. (a) Experiment 1, specificity of antiserum induced in VLP-vaccinated
mice, experiment 1, by Western blot. Serum from individual animals was pooled, diluted 1:25 in PBS, pH 7.2, and probed with inactivated influenza A/Hong
Kong/1073/99 virus. Lanes 1–2, pooled serum from VLP-vaccinated mice; lanes 3–4, rabbit antiserum to influenza A/Hong Kong/1073/99 virus. Lanes 1,
3, inactivated influenza A/Hong Kong/1073/99 (H9N2) virus antigen; lanes 2, 4, control H3N2 influenza virus antigen. (b) Experiment 2, end-point average
ELISA (upper panel) and HAI (bottom panel) titers of individual sera from VLP-vaccinated (filled bars) and control (empty bars) BALB/c mice. Also shown
are standard deviation values.
HAI assay, and Western blotting. Ten out of ten animals
seroconverted after primary immunization, and the antibody
responses increased after booster inoculation. In the sera of
animals, end-point ELISA titers ranged from 10
4
to 10
5
after
two inoculations, whereas HAI titers ranged from 320 to 640
(data not shown). In Western blot, sera recognized proteins
with molecular weights of 29, 50, and 70kDa, as shown by
using inactivated influenza A/Hong Kong/1073/99 virus as
antigen (Fig. 4a, lane 1). These bands corresponded well to
HA
2
,HA
1
, and uncleaved HA
0
precursor proteins expected
to be found in control H9N2 influenza antigen; these proteins
reacted also with the reference rabbit antibody to influenza
A/Hong Kong/1073/99 virus (Fig. 4a, lane 3). Rabbit anti-
body cross-reacted with 49 and 28 kDa proteins, which may
have represented HA
2
and HA
1
from the control H3N2 virus
antigen (lane 4). No such cross-reactivity was detected with
the serum from VLP-immunized mice (lane 2). Addition-
ally, multiple protein bands of higher molecular weight were
detected, which possibly represented oligomeric HA and NA
complexes. Asexpected,noantibodies toinfluenza viruspro-
teins were detected in the control animals inoculated with
PBS (data not shown).
In the second experiment, BALB/c mice were vaccinated
i.m. with two doses of influenza VLPs. An alternative route
of i.m. administration was chosen for this experiment to best
parallel the conventional route of influenza vaccination for
humans. Again, following a single inoculation, serum anti-
body was detected by ELISA and HAI in all animals except
the PBS controls and the titers increased after booster vac-
cination (Fig. 4b). Following i.n. challenge with A/Hong
Kong/1073/99 virus, virus replication and morbidity (mea-
sured by weight loss) were determined. Average weight loss
of up to 17% were observed in non-vaccinated control ani-
mals, whereas animals vaccinated with VLPs showed less
weight loss (Fig. 5a). Active replication of influenza virus
was detected in lung and upper respiratory tissues of control
animals at days 3 and 5 post-challenge (Fig. 5b). In oppo-
site, a significant (p <0.05) reduction of virus titers in the
lung and nose tissues were demonstrated among the VLP-
vaccinated animals at day 3 post-challenge. Finally, on day
5 post-challenge, three out of five vaccinated animals had no
detectable virus in the lungs, and no virus was detected in
nose tissues of all challenged animals (Fig. 5b).
4. Discussion
Avian influenza virusesof H9N2 subtype have beenfound
in domestic birds [3,4,18] and their recent transmission to
humans [5,6] raised concern of their potential for pandemic
spread in an immunologically na
¨
ıve population.
For many viruses, recombinant VLPs have been shown
to be a promising recombinant vaccine approach because of
robust immunogenicity, elicitation of protective neutralizing
antibodies due to the presence of conformational epitopes
on VLPs, and their advantageous safety profiles [11,12,19].
Assembly of influenza VLPs was demonstrated previously
in mammalian cells expressing virus-encoded polypeptides
from plasmids [20–22]. However, development of recombi-
nant VLPs for vaccine purposes was hampered by low yields
and the relatively complex structure of influenza particles,
which consist of multiple structural components. Morpho-
genesis of influenza virus particles and the critical structural
requirements for the efficient VLP formation in cell culture
are not yet well understood. One recent attempt to address
these issues was undertaken in baculovirus-infected insect
P. Pushko et al. / Vaccine 23 (2005) 5751–5759 5757
Fig. 5. Protective response in BALB/c mice vaccinated with VLPs derived from influenza A/Hong Kong/1073/99 (H9N2) virus. (a) Weight loss in VLP-
vaccinated(black diamonds)vs. non-vaccinated (open circles) mice (8mice/group).(b) Titers of replicating influenza A/Hong Kong/1073/99 virus in lungs and
nose tissues of VLP-vaccinated (filled bars) vs. non-vaccinated (open bars) mice (5mice/group). Detection limit was 1.2 log
10
EID
50
/ml of virus. Also shown
are standard deviation values. Standard deviation for day 3 in lungs of the VLP-vaccinated animals was 0.2 (not visible at this scale). An asterisk indicates the
VLP-vaccinated group was significantly (p < 0.05) different from the unimmunized-control group by using analysis of variance (ANOVA).
cells where VLPs were generated from four human H3N2
influenza virus proteins, HA, NA, M1, and M2 [13].
In this study, the requirements for the assembly of
influenza VLPs and their potential as an influenza vaccine
were elucidated using an H9N2 influenza virus. The results
demonstrated that VLPs can be generated in baculovirus-
infected Sf9 cells from only HA, NA, and M1 proteins
of influenza A/Hong Kong/1073/99 (H9N2) virus. These
“three-protein” VLPs exhibited hemagglutination and neu-
raminidase functions, elicited influenza-specific antibody,
and inhibited replication of influenza A/Hong Kong/1073/99
virus following i.n. challenge in mice. The process of VLP
assembly likely depended on the presence of M1 protein,
which was previously shown to play the critical role in virus
assembly and budding [23,24]. Although in insect cells HA
was not processed into HA
1
and HA
2
polypeptides, the anti-
body elicited with VLPs in mice efficiently recognized the
HA
1
and HA
2
proteins derived from the wild type influenza
virus. Further, the conformation of HA
0
and NA on the
outer surface of VLPs did not abrogate VLP self-assembly,
hemagglutination, and neuraminidase functions. The size,
80–120nm, and morphology of avianinfluenza VLPs resem-
bled those of influenza virionssuggesting authentic influenza
particle geometry and architecture. The VLP size variation
observed is similar to previous observations of pleomorphic
influenza virions. Size variation was also documented for
recombinant VLPs derived from other viruses. For example,
recombinant HBcAg antigen of hepatitis B virus formed par-
ticles of different sizes, which had distinct architectures and
triangulation number [25].
The results demonstrated that influenza VLPs derived
from insect cells and consisting of HA, NA, and M1 proteins
may be promising vaccine candidate for H9N2 influenza.
In mice, VLPs elicited titers of antibody that are similar
to those elicited with purified whole H9 virus vaccine and
considered adequate for protection in humans [17]. Further,
inactivated influenza A/Hong Kong/1073/99 (H9N2) virus,
which belongs to a G1 antigenic group, protected BALB/c
mice against both G1 and G9 groups of H9N2 influenza,
whereas immunization of mice with G9 lineage virus pro-
videdprotectionagainstG9butincompleteprotectionagainst
influenza A/Hong Kong/1073/99 (H9N2) virus [17]. These
data suggested that influenza A/Hong Kong/1073/99 VLPs
may provide broad coverage against H9N2 viruses.
Previous studies showed that individual influenza HA,
NA, and M1 proteins were capable of inducing protec-
tive immune responses to influenza [26–29]. However,
baculovirus-expressed recombinant HA from H5 avian
influenza virus was poorly immunogenic in people [26].It
is possible that expression in insect cells and differences
in post-translational modification such as glycosylation may
negatively affect immunogenicity. However, relatively high
doses of baculovirus-expressed HA are well tolerated in
humans and it is our hope that high doses of VLP will also
be well tolerated. Furthermore, avian VLP vaccine may be
moreimmunogenic than the HAalone, butmore experiments
are needed to compare the efficacy of influenza VLPs and
individualproteinsasvaccinesinotheranimalmodelsinclud-
ing ferrets, as well as in clinical trials in people. It appears
that the glycosylation pattern of H9N2 VLPs derived from
insect cells did not have a large impact on the murine anti-
body response. Future development of VLPs as influenza
vaccine may also include generation of “chimeric” VLPs
containing proteins from different influenza strains in order
to improvecoverage and protection. Another way to increase
the immunogenicity of the vaccine candidates may be using
5758 P. Pushko et al. / Vaccine 23 (2005) 5751–5759
advanced adjuvants [30,31]. The fact that influenza VLPs
can be assembled from only three structural proteins can
also facilitate the use of other expression methods for gen-
erating influenza VLPs including DNA and virus vectors
[32–34].
The challenging task for the vaccine development of
influenza VLPsmay be purification of VLPsfrom the recom-
binant baculoviruses. Although significant levels of purifi-
cation were afforded by centrifugation in sucrose density
gradients, residual baculoviruses were consistently detected
in VLP preparations. Recently, because high speed centrifu-
gation is difficult to scale for the manufacture of vaccines,
we have developed an efficient, scaleable, and rapid pro-
cess for the manufacture of influenza VLP vaccines, which
should be commercially viable (unpublished results) mak-
ing the further development of this approach all the more
important.
Taken together, the results suggested that influenza VLPs
with functional characteristics of influenza virions can be
generated readily in insect cells by the self-assembly of only
three structural proteins, HA, NA, and M1, and that these
influenza VLPs may be a promising vaccine candidate for
pandemic influenza.
Acknowledgements
We thank R. Compans, N. Cox, A. Klimov, K. Subbarao,
and R. Webster for support and valuable reagents; and L.
Potash, M. Massare, and the Vaccine Group staff for advice
and expert help. This work was supported in part by NIH
grant AI49509.
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    • "A recombinant baculovirus expression system was used to generate VLPs consisting of the HA, NA, and M1 proteins of A/Changchun/01/2009/ (H1N1) with or without the membrane-anchored versions of CTB or RTB using previously described methods [36]. The membrane-anchored versions of CTB and RTB were constructed by fusing the CTB and RTB coding sequences with sequences encoding the honeybee melittin signal peptide and the transmembrane and cytoplasmic regions of the A/Changchun/01/2009 (H1N1) HA protein (Figure 1A). "
    [Show abstract] [Hide abstract] ABSTRACT: Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.
    Full-text · Article · Apr 2016
    • "For preparation of triple-clade H555 VLPs, the HA proteins were derived from three clades of H5N1 HPAI avianorigin influenza viruses, strains A/chicken/Germany [16,20] and therefore were included in the expression construct. In a previous study, we have shown that influenza VLP can within the same particle [16] . "
    [Show abstract] [Hide abstract] ABSTRACT: Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.
    Full-text · Article · Feb 2016
    • "While these conserved viral epitopes individually have shown their potentials as universal influenza candidates, a new innovation is the combination of these epitopes as a vaccine. A combination of these epitopes is seen to have the potential to give a universal influenza vaccine that can give cross-protection against subtypes of influenza viruses. Pushko et al. (2005), Mahmood et al. (2008) and Tao et al.,(2009 all demonstrated protective immune responses by VLPs made of HA1, NA and M1 at different times and locations. Recently Price et al. (2014) showed that a candidate "universal" vaccine based on NP and M2 formulation protected animals against lethal infection and reduced transmission of influenz"
    [Show abstract] [Hide abstract] ABSTRACT: Africa is experiencing reoccurrence of avian influenza outbreaks with huge negative impact on the economy of the continent as a result of high mortality rate and extreme contagiousness of the disease. The epidemiology of highly pathogenic avian influenza (HPAI) in Africa during the 2006-2008 outbreaks was complex and linked to movements of poultry commodities and wild birds. The peculiar risk factors, negative economic impact and the potential of being used as a biological weapon necessitates the development of a comprehensive control programme for the prevention or eradication of the disease. It is the opinion of this paper that development of new influenza vaccine technologies will provide affordable comprehensive control programmes for avian influenza prevention in Africa. To keep pace with the variability of the viruses, there is need for frequent redesign of avian influenza (AI) vaccines to match the circulating subtypes and on this is predicated the necessity of the development of influenza vaccine technology for a country, zone or region. The new vaccine technologies have been shown to have the potentials of giving vaccines with required criteria of purity, safety, efficacy, potency, low cost and short response time. The concept of most new vaccine technologies is biased towards removal of influenza virus from the system of vaccine development and at the same time obtaining more effective, potent and safe influenza vaccines. The new influenza vaccine technologies include gene-based, genomics-based, subunit, plant-based, VLPs and universal vaccine technologies. These technologies have the potential to provide vaccines that will not just be used as intervention strategies to lessen severity of the disease but as preventative vaccination. Also routine vaccination will not just be as a tool of last option in disease endemic areas, but one to prevent the disease.
    Full-text · Article · Aug 2015 · Vaccine
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