JOURNAL OF VIROLOGY, Nov. 2011, p. 10945–10954
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 21
H5N1 Virus-Like Particle Vaccine Elicits Cross-Reactive Neutralizing
Antibodies That Preferentially Bind to the Oligomeric Form of
Influenza Virus Hemagglutinin in Humans?†
Surender Khurana,1¶ Jian Wu,1¶ Nitin Verma,1Swati Verma,1Ramadevi Raghunandan,2
Jody Manischewitz,1Lisa R. King,1Eloi Kpamegan,2Steven Pincus,2Gale Smith,2
Gregory Glenn,2and Hana Golding1*
Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, Maryland 20892,1and Novavax Inc., Rockville, Maryland 208502
Received 14 June 2011/Accepted 14 August 2011
Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans
is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple
vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade
1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach
to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans
vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs
were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins.
In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 ?g HA/dose
resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other
clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage
display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human
sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose
groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited
sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The
oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally,
the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to
a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing
the quality of the antibody responses in humans following AIV VLP immunization and support further
development of such vaccines against emerging influenza virus strains.
The recent global spread of swine-origin H1N1 influenza
viruses highlights the need for rapid development of effective
vaccines against pandemic strains. Much of our recent knowl-
edge about the potential for pandemic spread is derived from
studies with the highly pathogenic (HP) H5N1 avian influenza
A viruses (AIV) (12). H5N1 viruses cause severe human dis-
ease and may undergo adaptation permitting human-to-human
transmission. As of 21 April 2011, there have been 552 human
cases of H5N1, resulting in 322 deaths (fatality rate ? 59%)
The production of hemagglutinin by using recombinant
technology overcomes the constraints of traditional inactivated
influenza virus vaccine manufacturing, which requires months
to generate vaccine viruses using reassortant/reverse genetics
and adaptation for high growth in eggs. Technologies that can
be easily translated into a well-controlled large-scale manufac-
turing process will have a great advantage. Thus far, various
vaccine prototypes produced in a baculovirus-insect cell ex-
pression system have undergone preclinical and clinical devel-
opment (22, 49). Baculovirus-expressed recombinant hemag-
glutinin (HA) was shown to be immunogenic in humans (42,
44, 45) and protected against lethal infection in poultry chal-
lenged with avian H5 and H7 influenza virus subtypes (8).
Virus-like particles (VLPs) are multiprotein structures that
mimic the organization and conformation of authentic viruses
but lack the viral genome and can be rapidly produced in insect
vector systems by coexpression of key viral membrane compo-
nents (23, 25, 29, 32, 38). VLPs are predicted to elicit stronger
protective immunity than recombinant monomeric proteins
because they present numerous copies of oligomeric spike-like
structures that efficiently cross-link antigen-specific receptors
on B cells. Several VLP products have been evaluated in clin-
ical trials. In addition to a hepatitis B virus VLP-based licensed
vaccine, two human papillomavirus (HPV) VLP vaccines were
recently licensed in the United States (6, 9, 15, 38).
To address the need for improved influenza virus vaccines
and the limitation of egg-based manufacturing, VLPs based on
diverse human and avian influenza virus strains using baculo-
virus vectors expressing the HA, neuraminidase (NA), and
matrix 1 (M1) proteins were engineered (Novavax Inc.). In
previous studies, these VLPs were shown to be immunogenic
in mice and ferrets and to provide protection against respira-
* Corresponding author. Mailing address: Division of Viral Prod-
ucts, Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD 20892. Phone: (301) 827-0784. Fax:
(301) 496-1810. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
¶ These authors contributed equally to this study.
?Published ahead of print on 24 August 2011.
tory challenge with homologous and heterologous influenza
virus strains (4, 5, 37).
In the current study, we evaluated in depth the human an-
tibody responses in a subset of serum samples from an FDA-
approved clinical trial phase I/II study of an H5N1 (A/Indo-
nesia/05/2005) VLP vaccine containing the HA, NA, and M1
proteins, produced in Sposoptera frugipertda (Sf9) insect cells.
In addition to HA inhibition (HAI), NA inhibition (NAI), and
virus neutralization assays, the immune sera were analyzed by
use of H5N1 whole-genome fragment phage display libraries
(GFPDL) expressing HA and NA inserts and by surface plas-
mon resonance to measure kinetics of antibody binding to
oligomeric and monomeric recombinant HA (rHA) proteins.
In our previous studies, influenza virus-specific GFPDL (FLU-
GFPDL) expressing protein fragments from all the open
reading frames of the avian influenza A/Vietnam/1203/2004
(H5N1) virus HA and NA genes were used for mapping of
broadly neutralizing human monoclonal antibodies (MAbs)
derived from these H5N1 survivors (18). These MAbs were
found to recognize nonlinear conformational epitopes pre-
sented by large hemagglutinin fragments encompassing the
receptor binding domain (RBD). FLU-GFPDL (H5N1) al-
lowed the identification of large viral epitopes recognized by
antibodies in sera of individuals who recovered from HP H5N1
infection (18). In a follow-up study we deciphered the role of
oil-in-water adjuvant in augmenting the immune response follow-
ing vaccinations against H5N1 (A/Vietnam) and H1N1pdm09
viruses (16, 19).
In the current study, we provide evidence that the H5N1
A/Indonesia/05/05 VLPs can elicit antibodies with preferential
binding to oligomeric HA reminiscent of HA spikes on the
influenza virus. The oligomeric/monomeric HA1 binding ratios
correlated with neutralization titers and possibly explain the
broad cross-reactivity of antibodies induced by VLP-based in-
fluenza virus vaccines.
MATERIALS AND METHODS
Vaccine product. Influenza A/Indonesia/5/2005 (H5N1) virus VLPs were pro-
duced with recombinant baculovirus Autographa californica nuclear polyhedrosis
virus (AcMNPV) expressing the HA, NA, and M1 genes. HA, NA, and M1
protein sequences were from A/Indonesia/05/2005 (H5N1), with GenBank ac-
cession numbers ABP51969, ABW06107, and ABI36004, respectively. All three
genes were codon optimized for insect cells and biochemically synthesized by
Geneart AG (Regensburg, Germany). The HA, NA, and M1 genes were cloned
into pFastBac1 baculovirus transfer vector (Invitrogen, Carlsbad, CA) in a tan-
dem fashion with NA/HA/M1 gene order through restriction enzyme digestion
and ligation. Each gene was under the control of its own AcMNPV polyhedrin
promoter and poly(A) termination signals. Recombinant baculoviruses express-
ing the HA, NA, and M1 genes were generated using a Bac-to-Bac baculovirus
expression system (Invitrogen) as described previously (36). Briefly, the tandem
pFastBac1 plasmid was transformed into Escherichia coli DH10Bac competent
cells containing the AcMNPV baculovirus genome to produce a recombinant
bacmid through site-specific homologous recombination. Bacmid DNA was pu-
rified from transformed E. coli and transfected into Spodoptera frugiperda (Lep-
idoptera) Sf9 insect cells. Recombinant baculovirus was recovered, plaque puri-
fied, and amplified to infect Sf9 cells for VLP production. A/Indonesia/5/2005
VLPs were purified through tangential filtration, sucrose density gradient cen-
trifugation, and ion-exchange chromatography to ?95% purity as measured by
scanning densitometry of Coomassie blue-stained SDS-polyacrylamide gels. The
dose of vaccine was based on HA content as measured using a single radial
immunodiffusion (SRID) assay.
Clinical trial. A phase I/IIa blinded, randomized, placebo-controlled study was
conducted to evaluate the safety and immunogenicity of an A/Indonesia/05/2005
(H5N1) influenza virus VLP vaccine in subjects 18 to 40 years of age. The study
was conducted in 2008 under informed consent (clinical trial number NCT 005
19389, approved by IRB ENC 1-07-324). The H5N1 VLP vaccine and placebo
were packaged in 2-ml, single-dose glass vials, with each 0.5-ml dose of the
vaccine formulated to contain 15, 45, or 90 ?g of HA in sterile phosphate (25
mM HPO4)-buffered saline (0.5 M NaCl), pH 7.2, with calcium (10 ?M) and
polysorbate 80 (0.01%). The placebo injection (0.5 ml) contained the vehicle
utilized for suspension of the VLPs.
The study was conducted in two stages. In stage A, 70 subjects received 15 or
45 ?g of the H5N1 VLP vaccine or placebo (7:3 ratio) at days 0 and 28. Stage A
safety and immunogenicity results were reviewed by a Data Safety Monitoring
Board, which recommended that the study progress to stage B with a higher VLP
vaccine dose. In stage B, 160 subjects received 15, 45, or 90 ?g of H5N1 vaccine
or placebo (1:1:1:1 ratio) at days 0 and 28. Subjects were assessed for all adverse
events (AEs) through 28 days after receiving the second dose of vaccine, and
serious AEs (SAEs) were recorded through 6 months after dose 2. For the 6-day
period following each injection, subjects recorded the presence and severity of
local symptoms (pain, tenderness, redness, and swelling) and systemic symptoms
(fever, headache, fatigue, myalgia, nausea, vomiting, and diarrhea) as well as
body temperature. Serum samples for evaluation of immunogenicity were ob-
tained at days 0, 28, and 56. Day 0 and day 56 samples were used for the analyses
performed in the studies described in this report.
All samples provided to CBER were deidentified except for dose number. The
protocols were evaluated and approved by CBER Research Involving Human
Subjects Committee (RIHSC) and were conducted under RIHSC exemption
Outcomes and statistical methods. The stage A and stage B phases of the
clinical trial were analyzed separately. The primary objective was to evaluate
the safety of the H5N1 VLP vaccine. Secondary objectives were to assess the
immunogenicity of the influenza virus H5N1 VLP vaccine as determined by
hemagglutination inhibition (HAI) antibody responses as measured against a
reassortant influenza A/Indonesia/05/2005(H5N1)/PR8-IBCDC-RG2 virus and
microneutralization (MN) responses to the homologous influenza A/Indonesia/
05/2005 virus. HAI and MN titers were measured as previously described (33),
and the seroprotection rate (SPR) (the percentage of individuals with a post-
vaccination HAI or MN titer of ?1:40) and seroconversion rate (SCR) (defined
as either a prevaccination titer of ?1:10 with a postvaccination titer of ?1:40 or
a prevaccination HAI titer of ?1:10 with a minimum 4-fold rise in HAI titer in
postimmunization serum relative to preimmunization serum) were determined.
Cross-clade microneutralization assay. Cross-clade virus-neutralizing activity
was analyzed in a subset of day 0 and day 56 serum samples in a virus micro-
neutralization (MN) assay based on the method used in the pandemic influenza
virus reference laboratories of the Centers for Disease Control and Prevention
(CDC). The following low-pathogenicity and reassortant H5N1 viruses gener-
ated by reverse genetics were obtained from CDC: A/Indonesia/5/2005 (PR8-
IBCDC-RG2; clade 2.1), A/Turkey/1/05 (NIBRG-23; clade 2.2), and A/Anhui/
1/05 (IBCDC-RG5, clade 2.3.4). The experiments were conducted with three
replicates for each serum sample and performed at least twice.
NA inhibition assay for serum immunized with H5N1 VLP vaccine. NA inhi-
bition was tested using H5N1 A/Indonesia/5/2005 VLPs as the source of active
enzyme and with inhibition of NA activity assessed following incubation with
serum samples. The NA activity was measured using a modified fluorometric
assay with 2?-(4-methylumbelliferyl)-?-D-N-acetylneuraminic acid sodium salt
hydrate (MUNANA) as a substrate and fluorometric detection of the fluorescent
reaction product, 4-methylumbellipherone (MU) (35). This assay was validated
and performed at Novavax as previously described (13).
Briefly, VLP samples were diluted with assay buffer containing 32.5 mM MES
(morpholineethanesulfonic acid) sodium salt (Sigma), 4 mM CaCl2(Sigma), and
0.1% Tween 20 (Sigma) (pH 6.5) to provide final NA activity within the analyt-
ical range for MU measurement. For each pre- and postimmunization serum, six
3-fold serum dilutions in assay buffer starting from dilution at 1:2 were prepared.
Diluted VLP sample (30 ?l) was added into each well of 96-well black microplate
(Greiner Bio-One), mixed with 30 ?l of each serum dilution in duplicate or with
30 ?l of assay buffer for the VLP control, and then incubated at room temper-
ature with shaking for 40 min. Next, 30 ?l of 300 ?M MUNANA in assay buffer
with 60 ?g/ml bovine serum albumin BSA) was added to each well containing
VLP-serum mixtures and to wells containing 60 ?l of AB1 (substrate blank), and
reaction mixtures were incubated at 37°C with shaking on a Jitterbug-4 incuba-
tor-shaker (Boekel) for 60 min. The reaction was stopped by adding 150 ?M stop
solution (0.1 M glycine in 25% ethanol at pH 10.7), and fluorescence signals were
measured with a Modulus microplate multimode reader (Turner Biosystems) at
an excitation wavelength of 365 nm and an emission wavelength of 410 to 460 nm.
Readings for samples were corrected on substrate blank fluorescence and were
used to generate the inhibition curve as percent residual NA activity (NAA)
10946KHURANA ET AL. J. VIROL.
relative to the initial NAA for the VLP control with no serum added versus log2
serum dilution. The NA inhibition titer (NIT) was defined as the inverse of the
log2serum dilution that inhibited neuraminidase activity ?75% relative to the
The groups were compared using an analysis of covariance (ANCOVA) model
with fixed effects for treatment group and baseline NAI titers (day 0 titer) as a
covariate to adjust the variability seen at day 0. The data were log10transformed
prior to statistical analysis. The overall treatment difference was assessed using a
global F test. Pairwise comparisons of vaccine groups were analyzed and esti-
mated by least-squares mean (LSMEANS) at day 56.
Construction of H5N1 (A/Indonesia/5/2005) HA/NA GFPDL and panning of
H5-GFPDL with polyclonal human vaccine sera. The phage display libraries
expressing inserts spanning the HA and NA genes of the H5N1 A/Indonesia/05/
2005 strains were constructed as previously described for H5N1 A/Vietnam/
1203/04 GFPDL (16, 18). For each round of panning, equal volumes of day 56
sera obtained from five individuals in each VLP vaccine dose group were pooled.
Prior to panning of GFPDL, serum components that might nonspecifically in-
teract with phage proteins were removed by incubation with UV-killed M13K07
phage-coated petri dishes. Each subsequent GFPDL selection was carried out in
solution (with protein A/G-Sepharose), and inserts of bound phages were PCR
amplified and sequenced as previously described (16, 18).
Generation of H5N1 HA1 recombinant proteins. The DNA gene segments
corresponding to the HA1(1-320) and HA(28-320) proteins of H5N1 A/Indone-
sia/05/2005 were cloned as NotI-PacI inserts into a T7 promoter-based pSK
expression vector, in which the desired polypeptide can be expressed as a fusion
protein with a His6tag at the C terminus.
E. coli Rosetta Gami cells (Novagen) were used for expression of H5N1
A/Indonesia/05/2005 HA1 proteins. Following expression, inclusion bodies (IBs)
were isolated by cell lysis and multiple washing steps with 1% Triton X-100. The
final pellets containing IBs were resuspended in denaturation buffer containing
6 M guanidine hydrochloride and dithioerythritol (DTE) at a final protein con-
centration of 10 mg/ml and were centrifuged to remove residual debris. For
refolding, supernatant was slowly diluted 100-fold in redox folding buffer, fol-
lowed by dialysis against 20 mM Tris HCl (pH 8.0) to remove the denaturing
agents (16, 18, 20, 21). The dialysate was filtered through a 0.45-?m filter and was
subjected to purification by HisTrap fast-flow chromatography following the
Gel filtration chromatography. HA1(1-320) and HA(28-320) proteins at a
concentration of 5 mg/ml were analyzed on a Superdex S200 XK 16/60 column
(GE Healthcare) preequilibrated with phosphate-buffered saline (PBS). Protein
elution was monitored at 280 nm. Protein molecular weight marker standards
(GE Healthcare) were used for column calibration and generation of standard
curves to identify the molecular weights of the test protein sample.
Hemagglutination assay. Human erythrocytes were separated from whole
blood (Lampire Biologicals). After isolation and washing, 30 ?l of 1% (vol/vol)
human red blood cell (RBC) suspension in 1% BSA-PBS was added to 30-?l
serial dilutions of purified HA1 proteins or influenza virus in 1% BSA-PBS in a
U-bottom 96-well plate (total volume, 60 ?l). Agglutination was read after
incubation for 30 min at room temperature.
Binding to oligomeric and monomeric HA1 proteins and off-rate measure-
ments by surface plasmon resonance. Steady-state equilibrium binding of
postimmunization H5N1 human vaccine sera, selected for highest HAI titers by
group from day 56 sera (n ? 5 per group and corresponding to the samples used
in the GFPDL analysis), was monitored at 25°C using a ProteOn surface plasmon
resonance biosensor (Bio-Rad) as previously described (19). The H5N1 rHA
proteins were coupled to a GLC sensor chip with amine coupling with 500
resonance units (RU) in the test flow cells. Samples of 60 ?l freshly prepared
sera at 10-fold dilutions were injected at a flow rate of 30 ?l/min (120-s contact
time) for association, and dissociation was performed over a 600-s interval (at a
flow rate of 30 ?l/min). Responses from the protein surface were corrected for
the response from a mock surface and for responses from a separate, buffer-only
injection. MAb 2D7 (anti-CCR5) was used as a negative control in these exper-
iments. Binding kinetics for the selected human vaccine sera and data analyses
were calculated using Bio-Rad ProteOn manager software (version 2.0.1).
Antibody off-rate constants, which describe the stability of the complex, i.e.,
the fraction of complexes that decays per second, were determined directly from
serum sample interaction with HA1 protein using surface plasmon resonance (as
described above) and calculated using the Bio-Rad ProteOn manager software
for the heterogeneous sample model. For all polyclonal sera, it was important to
demonstrate that the dissociation rates were independent of total HA binding
antibody titers. To that end, parallel dissociation curves for 10-fold and 100-fold
dilutions for postvaccination human sera were established as previously de-
scribed (19). To improve the measurements, the off-rate constants were deter-
mined from two independent surface plasmon resonance runs.
Vaccination with H5N1 A/Indonesia/05/2005 VLP elicits
HAI and neutralizing antibodies against homologous and het-
erologous influenza virus strains. Participants in the phase
I/IIa clinical trial were immunized on days 0 and 28 with 15, 45,
or 90 ?g HA/dose of the A/Indonesia/5/2005 (H5N1) VLP
vaccine. In the initial safety and immunogenicity arm (stage
A), 70 subjects were given either placebo or H5N1 VLP at 15
?g and 45 ?g HA/dose. The stage A study indicated that the
vaccine was generally well tolerated, but the desired SCRs and
SPRs were not reached. This was followed by a dose escalation
study using 15, 45, and 90 ?g of H5N1 VLP vaccine compared
to placebo (stage B, n ? 160).
Overall, 91% of subjects completed at least 4 weeks of safety
follow-up after the second dose, and 87% completed 7 months of
safety observation. Only a single subject was discontinued due to
an adverse event, a clinically silent laboratory abnormality that
reversed spontaneously. There were no serious adverse events.
Transient injection site pain was approximately twice as fre-
quently reported by active vaccinees (39 to 56%) as placebo
recipients (19 to 23%) and was dose responsive in frequency in
both stage A and stage B. Injection site erythema and swelling
were less frequent, but also more common in active vaccinees
than placebo recipients in stage B, although not in stage A. Sys-
temic reactogenicity symptoms were not notably different among
active vaccines than placebo recipients, although in stage B, my-
algia was reported by 19% of vaccines versus 8% of placebo
recipients. There was no apparent association of clinical labora-
tory abnormalities with active vaccine, and no significant associ-
ation of any spontaneously offered adverse event with active vac-
cine when both stages were considered.
The sera were analyzed for HAI and MN titers as previously
described (14, 32). HAI for the stage B subjects on day 0 was
negative, and day 56 percent seroconversion and seroprotec-
tion are shown in Table 1. No seroconversion was observed
TABLE 1. Seroconversion and seroprotection rates after the
second dose of H5N1 VLP vaccinea
Assay and HA
40 (23, 59)
57 (37, 76)
61 (42, 78)
3.1 (0, 16)
40 (23, 59)
54 (34, 73)
55 (36, 73)
3.1 (0, 16)
39 (23, 58)
52 (34, 69)
76 (58, 89)
0 (0, 13)
39 (23, 58)
52 (34, 69)
76 (58, 89)
0 (0, 13)
aData are for post-second-vaccination sera collected on day 56 as tested
against A/Indonesia/05/2005 (clade 2.1) influenza virus by HAI and MN.
bSCR, fraction of subjects demonstrating a 4-fold increase in HAI titers over
the prevaccination titer. The 95% confidence interval values are shown in pa-
cSPR, fraction of subjects demonstrating HAI titers of ?1:40. The 95%
confidence interval values are shown in parentheses.
VOL. 85, 2011ANTIBODY RESPONSES TO AIV VLP IMMUNIZATION IN HUMANS 10947
after the first vaccination in any of the dose group. There was
a dose-related trend toward increased seroconversion (4-fold
increase in HAI titers) and seroprotection (HAI titer of ?40)
rates based on HAI and MN testing. There was a 61% sero-
conversion rate based on HAI and 76% seroconversion rate
based on MN in the 90-?g dose group after two injections
(Table 1). Unexpectedly, one placebo recipient had a posi-
tive titer on day 56.
A subset of samples representing each of the vaccine doses
were selected for cross-clade analysis. Ten samples from sub-
jects with low or undetectable HAI titers from the 15-?g im-
munization group and 10 samples each from the 45- and 90-?g
dosing groups with high HAI titers were evaluated in the MN
assay for cross-reactivity against other clade 2 viruses (data for
the 45- and 90-?g dosing groups are shown in Table 2). In this
subset, the neutralization titers against the vaccine H5N1
strain A/Indonesia/05/2005 (clade 2.1) ranged between 1:20
and 1:640, titers against A/Anhui/1/05 (clade 2.3.4) ranged
between 1:10 and 1:160, and titers against A/Turkey/1/05
(clade 2.2) ranged between 1:10 and 1:160. Similar serocon-
version rates were observed in the preclinical studies con-
ducted in ferrets vaccinated with A/Indonesia/05/2005 (H5N1)
GFPDL analyses of anti-HA and anti-NA antibodies follow-
ing H5N1 VLP (A/Indonesia/5/2005) vaccination. Five post-
second-immunization samples (day 56 sera) from each vaccine
dose group were used for panning of A/Indonesia/5/2005
GFPDL as previously described (16) (Fig. 1). The number of
bound phages in the placebo group was low, and all bound to
epitopes that mapped to the HA2 region, which is highly con-
served with seasonal H1N1 strains. Similar findings were ob-
served in the post-H5N1 subunit vaccination study with
A/Vietnam/1194/2004 (16). In the 15-?g group with low or
undetectable H5N1 HAI titers, a 5-fold increase in the number
of bound phage clones was observed. However, the majority of
the epitopes detected still mapped to the same HA2 region,
with only a few epitopes mapping to HA1, resulting in an
HA1/HA2 ratio of 0.07 (Fig. 1A and B; see Table S1 in the
supplemental material). In contrast, pooled sera from both the
45- and 90-?g/dose groups exhibited a 10- to 20-fold increase
in the number of bound phages. In addition to an increase in
the frequency of HA2-specific phage clones detected, there
was a significant expansion of HA1 fragments displaying phage
clones observed using the day 56 (post-second-vaccination)
immune sera (HA1/HA2 ratio ? 0.4). Importantly, the HA1
epitopes captured included large segments encompassing the
receptor binding domain (RBD) (shown in yellow in Fig. 1B)
(Fig. 1A and B; see Table S1 in the supplemental material).
Binding to neuraminidase epitopes also increased in the 45-?g
and 90-?g vaccine groups (7- and 15-fold, respectively). This
increase in NA binding was expected, since both HA and NA
proteins are present in the VLP vaccine. Furthermore, at the
two higher antigen doses (45 ?g and 90 ?g), the NA epitopes
captured by the post-second-vaccination serum antibodies
spanned the entire NA protein and included the C-terminal
epitope predicted to border the sialic acid binding site (shown
in green in Fig. 1B) (18).
H5N1 VLP (A/Indonesia/5/2005) vaccine elicits antibodies
with a high capacity for binding to the functional oligomeric
HA1 globular domain. In previous studies, we demonstrated
that the HA1 globular domains of multiple influenza virus
strains can form functional trimers and oligomers in the ab-
sence of HA2 (17, 20, 21). The oligomerization was dependent
on the first 8 amino acids in the N terminus of HA1 (20).
Furthermore, HA1 globular domain proteins containing
?80% oligomeric forms (mimicking the HA spikes) were
shown to elicit broadly neutralizing antibodies and protected
ferrets against challenge with pandemic influenza virus strains
(20, 21). In the current study, oligomeric and monomeric HA1
recombinant proteins were used in surface plasmon resonance
to capture the real-time kinetics of antibody association and
dissociation rates, which reflects the overall antibody binding
avidity (19). Both oligomeric rHA1(1-320) and monomeric
rHA1(28-320) proteins from A/Indonesia bind to conforma-
tion-dependent H5N1-specific broadly neutralizing human
monoclonal antibodies FLD21.140 and FLA3.14 (16, 18, 40)
(Fig. 2C and D). However, only the oligomeric rHA1(1-320)
agglutinated human RBC (Fig. 2A and E), while the mono-
meric rHA1(28-320) did not (Fig. 2B and E).
Binding of post-second-vaccination sera from the low-dose
VLP (15 ?g HA) to rHA1(1-320) was very low (?300 RU)
(Fig. 3A). In contrast, binding of antibodies to the oligomeric
rHA1(1-320) was much higher with sera obtained following
administration of VLP vaccine containing either 45 ?g or 90
?g of HA/dose, ranging from 500 to 1,500 RU and 1,000 to
2,000 RU, respectively (Fig. 3B and C). The total serum anti-
body binding to rHA1 correlated with the serum HAI titer
trend within each vaccine group.
To further investigate if two vaccinations with H5N1 VLP
(45 ?g and 90 ?g HA/dose) lead to an increase in antibody
avidity compared to that with the low-dose vaccine (15 ?g
HA), we determined the off-rate constants of the post-second-
vaccination human sera following binding to the functional
oligomeric rHA1 using surface plasmon resonance. As de-
picted in Fig. 4, immune sera obtained from subjects given 15
?g/dose demonstrated low binding affinity with high dissocia-
tion rate constants (?10?3) (Fig. 4, black diamonds), while
sera obtained from responders in the 45-?g and 90-?g dose
TABLE 2. Cross-clade MN serum titers in selected sera after the second dose of H5N1 VLP vaccinea
VLP vaccine (n ? 10 subjects per group)
45 ?g HA90 ?g HA
GMT ? 95% CI (range) SCR (%)b
GMT ? 95% CI (range)
A/Indonesia/05/2005 (clade 2.1)
A/Turkey/1/05 (clade 2.2)
A/Anhui/1/05 (clade 2.3.4)
166 ? 117 (20–320)
48 ? 29 (10–80)
62 ? 30 (10–80)
208 ? 180 (40–640)
75 ? 61 (10–160)
90 ? 52 (20–160)
aData are for post-second-vaccination sera collected on day 56 and selected for high HAI titers. GMT, geometric mean titer; CI, confidence interval.
bSCR, fraction of subjects demonstrating a 4-fold increase in MN titer over the prevaccination titer.
10948 KHURANA ET AL.J. VIROL.
groups showed around ?1 log lower dissociation off-rate con-
stants (?10?4) than sera from subjects from the low-dose
group (Fig. 4, blue and red circles).
H5N1 A/Indonesia/5/2005 VLPs elicit antibodies with pref-
erential binding to oligomeric rHA1. The VLP platform was
designed to express the recombinant HA proteins in a form
that mimics native virus (5, 37). Therefore, we determined if
the surface plasmon resonance assay can differentiate relative
binding of postvaccination human sera to oligomeric versus
monomeric rHA1 proteins. As shown in Fig. 5A, the same
serum samples from all the vaccine arms, which were analyzed
by GFPDL (Fig. 1), bound preferentially to the oligomeric
rHA1(1-320) compared to the monomeric rHA1(28-320) (Fig.
5A, red versus black bars). Furthermore, the ratios of maxi-
mum RU (oligomeric/monomeric) values from 10 individuals
each from the 45-?g and 90-?g HA groups after the second
vaccination correlated with the microneutralization (MN) ti-
ters against A/Indonesia/5/2005 (r ? 0.83) (Fig. 5B). These
findings suggest that HA oligomer binding antibodies are im-
portant in virus neutralization, as we previously demonstrated
in ferrets (20, 21).
High-dose H5N1 VLP (A/Indonesia/5/2005) vaccine elicits
neuraminidase-inhibiting antibodies that differentially map to
the sialic acid binding site. Antineuraminidase antibodies
were also detected in the stage B subjects (Fig. 6A). The
analysis of NAI responses indicated an overall treatment dif-
ference (global F test, P ? 0.0001). The pair wise comparison
(LSMEANS) of the vaccine groups revealed that the 45-?g
and 90-?g groups were significantly different from the placebo
group, with P values of ?0.0001 and 0.0008, respectively. In
FIG. 1. Analysis of antibody repertoires elicited in adults following vaccination with VLP-based H5N1 A/Indonesia/5/2005 vaccine. (A) Dis-
tribution of phage clones after affinity selection with sera obtained from adults before and after two doses of H5N1 VLP vaccine. (B) Schematic
alignment of the peptides recognized by post-dose 2 H5N1 sera as identified by panning with H5N1 GFPDL-A/Indonesia/5/2005. The amino acid
designation is based on the H5N1 A/Indonesia/5/2005 HA protein sequence (see Fig. S1 in the supplemental material). Bars indicate identified
inserts in HA1 (red bars), HA2 (blue bars), and NA (black bars). Phage displaying peptides from sequences within the HA1 receptor binding
domain (RBD) are depicted with yellow bars. Peptides encompassing the C terminus of NA are shown in green. The thickness of each bar represent
the frequency of repetitively isolated phage inserts (only clones with a frequency of ?2 are shown; all sequenced clones are shown in Table S1 in
the supplemental material).
VOL. 85, 2011 ANTIBODY RESPONSES TO AIV VLP IMMUNIZATION IN HUMANS10949
addition, the 15-?g group was significantly different from the
45-?g group. However, no significant difference was observed
between the 15-?g group and the placebo group.
By comparing the patterns of epitope recognition found in
the sera from individuals receiving high and low vaccine doses,
it is clear that only sera from subjects immunized with the
high-dose vaccine contained antibodies that recognize seg-
ments encompassing the neuraminidase amino acid sequence
positions 978 to 1004 (Fig. 1A and B; see Table S1 in the
supplemental material). Antigenic mapping of this C-terminal
epitope on the NA structure identified a region which juxta-
poses the sialic acid binding site of neuraminidase, as previ-
ously observed in patients who survived H5N1 infection in
Vietnam (Fig. 6B, shown in green) (18).
Together, these data suggest that the H5N1 VLPs generated
functional antibodies against targets in both HA and NA, in-
cluding epitopes dependent on the multimeric forms of these
Recombinant vaccine technology has the potential to pro-
vide a rapid response to emerging influenza virus infections
with pandemic potential, since it does not rely on the deriva-
FIG. 2. Characterization of bacterially expressed and purified H5N1 HA proteins. (A and B) Characterization of purified H5N1 HA proteins
from E. coli by Superdex S-200 gel filtration chromatography. Purified H5N1 HA1 proteins with an intact N terminus (positions 1 to 320) (A) and
HA1 with an N-terminal deletion (28 to 320) (B) were subjected to gel filtration. The panels show superimposed elution profiles of purified HA1
proteins (red lines) overlaid with calibration standards (gray lines). The elution volumes of protein species are shown in parentheses. (C and D)
Steady-state binding equilibrium analysis of conformation-dependent human H5N1 neutralizing MAbs FLD21.40 (C) and FLA3.14 (D) at 10
?g/ml to purified bacterially expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group and on a blank flow cell,
free of peptide. Binding was recorded using a ProteOn system surface plasmon resonance biosensor instrument (Bio-Rad Labs, Hercules, CA).
(E) Agglutination of human RBCs by properly folded oligomeric H5N1 rHA1(1-320) protein and its monomeric H5N1 rHA1(28-320) counterpart
along with rgH5N1 virus. Serial dilutions of purified HA1 proteins were mixed with washed RBCs, and hemagglutination was read after 30 min
at room temperature. Reassorted virus rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 (clade 1) was used as a positive control.
10950 KHURANA ET AL. J. VIROL.
tion of vaccine viruses with high growth in eggs. The VLP
platform is particularly attractive for vaccines against highly
pathogenic zoonotic influenza virus strains, since it does not
require the use of live virus in any stage of the manufacturing
process. Furthermore, VLPs present the key antigenic targets
of influenza virus, HA and NA, in a native-like conformation
similar to the intact virion. Studies in animal models also pro-
vided evidence for generation of cross-clade protective anti-
bodies following H5N1 VLP (but not rHA) immunization (4, 5,
The current study was conducted with immune sera from a
subset of participants in the first human phase I/II trial of an
A/Indonesia/05/2005 (H5N1) VLP vaccine candidate. The vac-
cine was generally well tolerated, and after two vaccinations,
HAI seroconversion rates (SCRs) of 40%, 57%, and 61% and
MN SCRs of 39%, 52%, and 76% were achieved in the 15-,
45-, and 90-?g groups, respectively. Cross-reactivity against
other H5N1 clade 2 subtypes was also demonstrated with high-
responder sera. GFPDL analyses (A/Indonesia/5/2005; HA/
NA) revealed broad epitope repertoires recognized by immune
sera from the two higher-antigen-dose groups, which included
large segments spanning the RBD in the HA1 globular head
and the NA C terminus. We identified an immunodominant
epitope in the HA2 domain in preimmune sera that was sig-
nificantly boosted following vaccination. A similar epitope was
identified in our previous studies with H5N1 A/Vietnam/1203/
2004 vaccine trials (16). This HA2 epitope sequence is highly
conserved (98%) within H5N1 and seasonal H1N1 strains and
therefore may be present due to preexisting immunity from
either vaccination or exposure to seasonal H1N1 strains. Since
reactivity to this HA2 epitope was also boosted with the 15-?g
VLP dose, which had no significant virus-neutralizing activity,
it is unlikely that antibodies against this HA2 epitope contrib-
ute to protection.
Binding of antibodies to properly folded oligomeric H5N1
rHA1(1-320) protein was significantly higher than that to mo-
nomeric rHA1(28-320) for all sera obtained following high-
dose vaccinations. The oligomeric/monomeric HA1 binding
ratio correlated with virus neutralization titers. Neuraminidase
inhibition ratios of above 2 (post-second vaccination/prevacci-
nation) were detected in sera from the high-dose groups and
correlated with binding to an NA C-terminal epitope that is
located close to the sialic acid binding site.
The HAI titers and cross-clade reactivity of sera in the hu-
man trial are in agreement with the preclinical studies involv-
ing ferrets and a similar VLP vaccine product (30). The H5N1
VLP vaccine was immunogenic, and the HAI titers were com-
parable to those reported with an inactivated egg-derived
FIG. 3. Kinetics of binding of post-H5N1 vaccination human serum
to properly folded oligomeric H5N1 HA1 proteins using surface plas-
mon resonance. Steady-state equilibrium analysis of the binding of
human postvaccine serum to properly folded functional HA1 oligo-
mers was done using surface plasmon resonance and is shown for
representative samples from each group. Ten-fold-diluted individual
post-H5N1 vaccination sera from the three VLP dose vaccine groups
(15 ?g [A], 45 ?g [B], and 90 ?g [C]) after a second dose were injected
simultaneously onto HA1 proteins immobilized on a sensor chip
through the free amine group and onto a blank flow cell, free of
peptide. Binding of the antibodies to the immobilized protein is shown
as resonance units (RU). Data are shown for three representative
samples for each dose group, and their HAI titers against H5N1
A/Indonesia/5/2005 are given in parentheses.
FIG. 4. Serum antibody affinity to hemagglutinin as measured by
off-rate constants in post-H5N1 vaccination human sera in different
VLP dose groups. Surface plasmon resonance analysis of human sera
postvaccination with H5N1 VLP vaccine from three VLP dose groups
of the vaccine trial was performed with properly folded functional
oligomeric H5N1 HA1 (A/Indonesia/5/2005). Serum antibody off-rate
constants were determined as described in Materials and Methods.
Correlation statistics of the off-rate constants of the postvaccine hu-
man sera between different vaccine groups were highly significant for
the 45-?g versus 15-?g and the 90-?g versus 15-?g dose groups (P ?
0.005 by t test).
VOL. 85, 2011 ANTIBODY RESPONSES TO AIV VLP IMMUNIZATION IN HUMANS10951
H5N1 (A/Vietnam/1203/04) vaccine previously licensed in the
United States (1, 2, 43). Furthermore, we measured heterolo-
gous neutralization of other clade 2 subtypes not found after
vaccination with the egg-based inactivated subunit A/Vietnam
vaccine unless combined with oil in water adjuvants (16, 24, 28,
43). A plant-based H5N1 VLP vaccine was also reported to
elicit cross-reactive neutralizing antibodies in animals and hu-
mans (23). This difference could also reflect a difference in the
immunogenicities of A/Indonesia/05/2005 HA and A/Vietnam/
1203/04, as previously observed in animal experiments (4).
More recently, a VLP vaccine against the pandemic H1N1
strain was evaluated in the Mexican population and was found
to be safe and immunogenic (27).
The role of oligomeric HA in eliciting protective antibodies
is an important finding. There is growing evidence that oligo-
meric vaccines present epitopes that are specific to virion
spikes and that are absent on monomeric proteins. Further-
more, antibodies that bind preferentially (with high avidity) to
oligomeric compared with monomeric HA proteins were re-
ported to have broader cross-reactivity (31, 34, 39, 48, 52).
Therefore, several recombinant influenza virus and HIV vac-
cines were designed to include trimerization domains in order
to elicit broadly neutralizing antibodies (3, 50, 51). Since the
H5N1 VLP vaccine used in the current trial naturally presents
multimeric HA and NA to the immune system, it was impor-
FIG. 5. Binding of oligomeric versus monomeric HA1 proteins by
human sera following immunization with H5N1 VLPs (A/Indonesia/
5/2005). (A) Maximum RU values for oligomeric HA1(1-320) (red
bars) versus monomeric HA1(28-320) (black bars) binding by serum
antibodies from individuals after a second dose of A/Indonesia/5/2005
H5N1 VLP (placebo, 15 ?g, 45 ?g, or 90 ?g) in surface plasmon
resonance are shown for the same serum samples that were analyzed
by GFPDL (Fig. 1). The differences in binding of post-H5N1 vaccina-
tion human sera to oligomeric HA1 between different VLP dose
groups were statistically significant for the 45-?g versus 15-?g and the
90-?g versus 15-?g dose groups (P ? 0.05 by t test). (B) Correlation
between in vitro neutralizing titers and oligomeric/monomeric HA1
binding ratio in human sera following immunization with H5N1 VLP
vaccine in different dose groups. Oligomeric/monomeric HA1 binding
ratios [HA(1-320) versus HA(28-320), from panel A] correlated with
microneutralization titers against A/Indonesia/5/2005 (r2? 0.83).
FIG. 6. Antineuraminidase antibodies and antigenic mapping of
neuraminidase-inhibiting antibodies elicited by human sera following
immunization with H5N1 VLPs (A/Indonesia/5/2005). (A) Neur-
aminidase enzymatic activity inhibition (NAI) titers for prevaccine and
post-second-vaccination human sera were measured as described in
Materials and Methods. The pairwise comparison (LSMEANS) of the
vaccine groups revealed that the 45-?g and 90-?g groups were signif-
icantly different from the placebo group, with P values of ?0.0001 and
0.0008, respectively. In addition, the 15-?g group was significantly
different from the 45-?g group. (B) The immunodominant epitope
[NA(978-1004)] in NA is shown on the tetrameric NA structure (Pro-
tein Data Bank [PDB] identifier 2HTY) (corresponding to the green
bars in Fig. 1B) and is aligned with the bound sialic acid shown in red.
Side (top) and bird’s-eye (bottom) views are shown.
10952 KHURANA ET AL.J. VIROL.
tant to determine if the human immune sera contained anti-
bodies that bind differentially to oligomeric versus monomeric
structures. In earlier studies we have expressed recombinant
HA1 proteins from multiple influenza virus strains that were
enriched for oligomeric or monomeric HA structures. We pre-
viously demonstrated that oligomeric rHA1 proteins were
highly immunogenic and induced superior protective immunity
against homologous and heterologous strains compared with
monomeric HA1 or HA0 recombinant proteins (references 20
and 21 and unpublished data). In the current study, we dem-
onstrated that the H5N1 VLPs generated antibodies that pref-
erentially bound to oligomeric A/Indonesia/5/2005 rHA1(1-
320) compared with monomeric rHA1(28-320) protein (Fig.
5). Furthermore, a positive correlation between the oligomer-
ic/monomeric HA1 binding ratios and the in vitro neutraliza-
tion titers for individual postvaccination human sera was dem-
onstrated. In concordance, we found that the oligomeric/
monomeric binding ratios of human immune sera from egg-
based H5N1 subunit vaccination (A/Vietnam/1203/04, SP) (1)
were around 1, with no clear preference for oligomeric HA1
binding (data not shown).
Neuraminidase-neutralizing antibodies elicited by vaccines
may provide added value to the protective immunity against
both seasonal and avian influenza viruses. In the case of tra-
ditional inactivated subunit vaccines, the neuraminidase con-
tent is not monitored, and the fraction of functional protein
may vary considerably with the manufacturing process (7, 10,
11, 26, 41, 46, 47). In contrast to the inactivated influenza virus
vaccines, the VLP platform presents NA in its native tetra-
meric form, thereby increasing the opportunity to elicit pro-
tective antibodies against this important viral membrane pro-
tein. The reactivity of the antibodies against the NA C
terminus, demonstrated in the current VLP vaccine trial, is
likely to interfere with the binding of NA to sialic acid, which
is required for its enzymatic activity and release of virions from
In summary, our data provide the first insight into the quality
of the epitope repertoire and avidity of the antibodies elicited
by H5N1 VLP vaccine in humans. The increased binding to
oligomeric HA and the C terminus of NA may contribute to
the broader cross-reactivity of the immune sera seen in sub-
jects administered VLP vaccines. However, no clear dose spar-
ing was provided by the VLP platform compared with the
inactivated H5N1 vaccine. Future trials should determine if a
VLP-adjuvant combination could improve immunogenicity
and dose response.
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