Research Articles

influenza viruses (17) has raised

hopes that a broadly protective
vaccine may indeed be feasible
(812). The majority of these
bnAbs are directed toward
Antonietta Impagliazzo,1* Fin Milder,1 Harmjan Kuipers,1
highly conserved conformational
Michelle Wagner,2|| Xueyong Zhu,3 Ryan M. B. Hoffman,3
epitopes in the HA stem (1, 2, 4,
5, 7) suggesting that this region
Ruud van Meersbergen,1 Jeroen Huizingh,1 Patrick
1
1
1
may have the potential to induce
Wanningen, Johan Verspuij, Martijn de Man, Zhaoqing
broad
protective
immunity,
2
1
1
1
Ding, || Adrian Apetri, Baak Kkrer, Eveline Sneekes-Vriese,
provided
it
is
well
exposed
and
Danuta Tomkiewicz,1 Nick S. Laursen,3 Peter S. Lee,3 Anna
properly presented. Various
Zakrzewska,1 Liesbeth Dekking,1 Jeroen Tolboom,1 Lisanne
strategies to enhance exposure of
the HA stem to the immune
Tettero,1 Sander van Meerten,1 Wenli Yu,3 Wouter Koudstaal,1
1
3
1
system are being explored,
Jaap Goudsmit, Andrew B. Ward, Wim Meijberg, Ian A.
including presentation on self3
1
Wilson, * Katarina Radoevi #
assembling nanoparticles (13),
1
Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, Archimedesweg 4-6, 2301 CA Leiden,
chimeric HAs (14, 15), epitope
Netherlands. 2Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 3210 Merryfield Row, San
transplantation on a virus-like
Diego, CA 92121, USA. 3Department of Integrative Structural and Computational Biology, The Scripps Research Institute,
particle (VLP) (16), and immune
10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
refocusing (17). Yet another
approach involves removal of the
*Corresponding author. E-mail: aimpagli@its.jnj.com (A.I.); wilson@scripps.edu (I.A.W.)
HA head while stabilizing the
Present address: Janssen Prevention Center, Janssen Pharmaceutical Companies of Johnson & Johnson,
HA stem. A prerequisite for
Archimedesweg 4-6, 2301 CA Leiden, Netherlands.
generating a broadly protective
These authors contributed equally to this work.
soluble HA stem immunogen is
that it is stable and structurally
Present address: Infectious Diseases and Vaccines Therapeutic Area, Janssen Research and Development, Janssen
resembles the stem of full-length
Pharmaceutical Companies of Johnson & Johnson, Archimedesweg 4-6, 2301 CA Leiden, Netherlands.
(FL) HA. This simple and elegant
||Present address: Janssen Prevention Center, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210
concept is complicated by
Merryfield Row, San Diego, CA 92121, USA.
serious structural constraints
Present address: Department of Molecular Biology and Genetics, Aarhus University, Aarhus 8000, Denmark.
(18). HA is a metastable trimeric
surface glycoprotein (19, 20) that
#Present address: Global Biotherapeutics, Sanofi, 94400 Vitry-sur-Seine, France.
undergoes
extensive
The identification of human broadly neutralizing antibodies (bnAbs)
conformational rearrangements
targeting the hemagglutinin (HA) stem revitalized hopes of developing a
at low pH (21). Removal of the
universal influenza vaccine. Using a rational design and library approach,
transmembrane domain and HA
we engineered stable HA stem antigens (mini-HAs) based on an H1
head
without
extensive
subtype sequence. Our most advanced candidate exhibits structural and
structural
modifications
to
bnAb binding properties comparable to full-length HA, completely protects
stabilize the remaining molecule
mice in lethal heterologous and heterosubtypic challenge models, and
inevitably leads to loss of native
reduces fever following sublethal challenge in cynomolgus monkeys.
conformation of the HA stem
Antibodies elicited by this mini-HA in mice and nonhuman primates bind a
(18) and concomitant loss of
wide range of HAs, compete with human bnAbs for HA stem binding,
conformational bnAb epitopes.
neutralize H5N1 viruses, and mediate antibody-dependent effector activity.
Previously reported soluble HA
These results provide proof-of-concept for design of HA stem mimics that
stem-derived immunogens have
elicit bnAbs against influenza A group 1 viruses.
exhibited lower affinity with
bnAbs compared to FL HA,
The ultimate goal of influenza vaccinology is the indicating suboptimal conformation (2225).
development of a universal vaccine that protects against a
wide range of strains and subtypes, thereby eliminating the Design and initial characterization
need for seasonal reformulation of vaccines and providing
Starting
with
the
HA
sequence
of
H1N1
an effective defense against viruses with pandemic A/Brisbane/59/2007, changes were introduced using a
potential. The relatively recent discovery of broadly combination of rational design and library approaches (26).
neutralizing human monoclonal antibodies (bnAbs) against In the first design phase (Stages I-III, Fig. 1A), we aimed to

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A stable trimeric influenza hemagglutinin

stem as a broadly protective immunogen

generate a soluble and stable HA stem monomer containing

bnAb epitopes in the correct conformation, whereas in the
final stages (Stage IV and V), we aimed to create a mini-HA
in its natural trimeric form. Details on the design, sequences
and screening are outlined in Supplementary Materials and
figs. S1 and S2. As a readout for a native-like stem
conformation of the mini-HA constructs, binding of bnAbs
CR9114 and CR6261 (both of which recognize epitopes in the
HA stem) was used (27). From each design stage, the best
performing candidates were selected for further
optimization. Key mutations in these lead candidates are
schematically depicted in fig. S2 and on a model structure of
mini-HA in Fig. 1B.
Selected mini-HAs #4157 (stage II), #4454 (stage III),
#4650 (stage IV) and #4900 (stage V) were expressed in
HEK293F cells and purified. Based on size, mini-HAs #4157
and #4454 appeared predominantly monomeric in solution;
#4650 was larger, suggesting at least partial formation of
dimers, whereas #4900 appeared trimeric (fig. S3A).
Formation of an intermonomer cysteine bridge in trimeric
mini-HA #4900 was confirmed using SDS-PAGE under nonreducing and reducing conditions (fig. S3B). All selected
mini-HA constructs have four putative N-linked
glycosylation sites (Fig. 1B). Under reducing conditions, the
mini-HA bands were diffuse and ran at higher molecular
weight (3545 kDa) than expected (29 kDa), indicating that
the mini-HAs were glycosylated (fig. S3B). Accordingly,
deglycosylation with PNGase F resulted in sharper bands
(~30 kDa; as for mini-HA #4900 in fig. S3B). The glycans
appear relevant for proper folding since attempts to
efficiently produce mini-HA with retention of bnAb epitopes
in bacteria were not successful (28).
Size exclusion chromatography with inline multi-angle
light scattering (SEC-MALS) analysis revealed a single peak
(fig. S4A), with average molecular weights of 40 kDa for
#4157 and #4454, 60 kDa for #4650 and 108 kDa for #4900
(Table 1), which also indicated that #4157 and #4454 are
monomeric in solution, #4650 forms dimers (at least in part)
and #4900 forms trimers. In the presence of CR9114 or
CR6261 Fabs, a peak shift to shorter retention time was
observed for all mini-HAs, indicating formation of
complexes (fig. S4A), which was confirmed by MALS (Table
1), with masses in agreement with binding of one Fab
(monomeric mini-HAs #4157 and #4454), one to two Fabs
(dimeric mini-HA #4650) or three Fabs (trimeric mini-HA
#4900) (29).
Binding of CR9114 and CR6261 to all four candidate
immunogens was in the nanomolar range with affinity
improving as the design process progressed (Table 1 and fig.
S4B). The strongest binding was exhibited by mini-HA
#4900, with KD values comparable to trimeric FL HA,
indicating that this mini-HA recapitulates the bnAbs
epitopes and thus accurately mimics the native
conformation of the HA stem.

Structural characterization
Modifications introduced throughout the design
gradually increased the conformational stability as
determined by hydrogen/deuterium exchange mass
spectrometry
(HDX-MS) (30, 31). In particular,
modifications introduced in Stage III (from #4157 to #4454)
and stage V (from #4650 to #4900) stabilized the overall
structure and the shorter A helix (Fig. 2). Furthermore, we
observed only a low level of deuterium exchange on the long
CD helix of mini-HA #4900, likely due to the shielding effect
caused by trimerization. Most importantly, the HDX pattern
of mini-HA #4900 is very similar to trimeric FL HA, further
demonstrating that the final design mimics the structure
and dynamics of native HA. In line with these findings, the
overall stability of the mini-HAs also evolved, as is apparent
from measurements of the thermal stability (fig. S5).
Complexes of mini-HAs with CR9114 Fab were first
studied by negative-stain electron microscopy (EM) (Fig.
3A). The EM class averages illustrate the progression from
monomeric #4454, to a more heterogeneous mixture of
monomers and dimers with #4650, and finally to a trimeric
form with #4900. To provide further structural insights,
crystal structures of #4454CR9114 and #4900CR9114 Fab
complexes were determined to 4.3 and 3.6 resolution,
respectively (Fig. 3, B and C; table S1; see also
Supplementary Materials). In the #4454CR9114 complex
(Fig. 3B), CR9114 binds monomeric mini-HA and recognizes
essentially the same epitope as in FL HA through contacts
only with its heavy chain (table S2) (10). The CR9114 epitope
residues in #4900 share similar solvent accessible surfaces
as in A/California/04/2009 FL H1 HA (table S3). The #4900
trimer differs only slightly from the stem of trimeric
A/California/04/2009 FL HA, and perhaps other HAs, as it
adopts a more open, splayed conformation at the trimer
base with the HA2 H helix, shifted by about 14.5 (Fig. 3, C
and D), perhaps as a consequence of insertion of the GCN4
trimerization motif at the top of the long helix in #4900 (fig.
S6), or from differences in the sequences of Brisbane 2007
and California 2009 HA. The primary interactions are
maintained between CR9114 and #4454 as compared with
native H5 HA (3) and the CR9114 complex was trimeric (Fig.
3C). The #4900 protomer is superimposable with an HA
protomer from the H1 A/California/04/2009 trimer (PDB ID
4M4Y) with a C rmsd of 1.2 , and only 1.0 for the
CR9114 epitope residues. As a consequence of the removal of
the HA1 head in #4900 and due to a more open trimer base
(table S2), some residues in the HA2 A helix, B loop, C and
D helix, and E strand become solvent accessible. These
results convincingly demonstrate that the iterative design
process led to antigens with an increasingly improved HA
stem conformation and ultimately yielded HA-like stem
antigens with a preserved CR9114 epitope.
In vivo effects
When tested in mice, all mini-HAs were highly

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immunogenic, eliciting high titers of antibodies binding to

the FL HA of the A/Brisbane/59/2007 (H1N1) strain used as
the basis for mini-HA generation (fig. S7A), as well as to FL
HAs from a number of other group 1 (H1, H5 and H9) and
group 2 (H3, H7) influenza A strains (fig. S7B). These results
demonstrate that antibodies induced with mini-HAs
efficiently recognize epitopes that are present in native
sequences of FL HA and conserved among different group 1
and even some group 2 influenza A strains (32).
Next, we evaluated the ability of mini-HA immunogens
to provide protection against stringent lethal challenges and
characterized the elicited immune response (Fig. 4 and fig.
S7). Protection against influenza challenge with H1N1
A/PR/8/34 (heterologous to the parental strain used for
design of the mini-HAs) was assessed following three, two
or one immunizations with mini-HAs #2759 (monomer, long
variant of #4157; fig. S2), #4650 (mix of monomers and
dimers), and #4900 (trimer). Mice immunized three times
with monomeric mini-HA #2759 were only partially
protected against mortality and showed severe bodyweight
loss and clinical symptoms, while #4650 and #4900 provided
complete protection, with #4900-immunized mice showing
neither bodyweight loss nor clinical symptoms (Fig. 4A and
fig. S7C). Only mini-HA #4900 showed the same full
protective ability when administered twice. Even after one
immunization, #4900 exhibited 90% protection against
mortality, although animals exhibited some (recoverable)
bodyweight loss and clinical symptoms, whereas #4650 and
#2759 were not able to protect animals. Antibodies elicited
with mini-HA #4900 after three and two immunizations
competed with bnAb CR9114 for binding to the FL HA stem
(Fig. 4A), proving that this mini-HA could readily elicit
antibodies specific for bnAb stem epitopes.
To evaluate the breadth of protection, mice immunized
three times with an expanded set of mini-HAs were
challenged with heterosubtypic H5N1 A/Hong Kong/156/97
virus. Mini-HAs #2759 and #4157 (monomers) failed to elicit
protection, #4454 (improved library monomer) and #4650
exhibited partial protection while #4900 exhibited complete
protection with neither bodyweight loss nor clinical
symptoms (Fig. 4B and fig. S7D). To further investigate
antibody-mediated effector mechanisms, pre-challenge sera
were tested in a neutralization assay using pseudoparticles
derived from H5N1 A/Vietnam/1194/04 (33). Sera from mice
immunized with mini-HA #4900, and to some extent #4650,
showed detectable neutralization, demonstrating the ability
of these mini-HAs to elicit bnAbs (Fig. 4C). Besides direct
virus neutralization, Fc-mediated effector mechanisms, such
as antibody-dependent cellular cytotoxicity (ADCC),
contribute substantially to protection against influenza,
with stem-directed bnAbs being particularly effective in
these mechanisms (34). We therefore tested pre-challenge
sera using a mouse-adapted ADCC surrogate assay (3537).
The evolving mini-HAs demonstrated an increasing ability
to elicit antibodies with potential to induce ADCC (Fig. 4D),

with ADCC potency of serum antibodies corresponding to

survival proportions observed in the H5N1 challenge model.
In accordance with the binding data (fig. S7, A and B), miniHA #4900 elicited ADCC responses to a wide range of group
1 influenza A HAs (fig. S7E). To assess whether serum
antibodies are indeed responsible for the observed in vivo
protection, we passively transferred serum from mice
immunized with #4650 and #4900 to nave mice and
subsequently challenged the recipient mice with
A/HK/156/97 (H5N1) virus (Fig. 4E and fig. S7F). In contrast
to mice that received mock-serum, mice that received serum
from mini-HA immunized mice were significantly protected,
proving that antibodies play a key role in the protection
elicited by mini-HAs.
These combined results demonstrate that the progressive
structural refinement and stabilization of mini-HA directly
translates to increasing ability to elicit stem-targeting,
neutralizing, and ADCC-mediating antibodies that can
protect mice against heterologous and heterosubtypic group
1 influenza strains, with trimeric #4900 exhibiting the most
favorable features. In a recent study (25) a bacterially
produced trimeric HA stem construct also seemed more
effective than monomeric versions in eliciting protection in
an animal influenza challenge model.
We next tested the immunogenicity and protective
efficacy of a three-dose regimen of mini-HA #4900 in six
cynomolgus monkeys (Macaca fascicularis). As controls,
groups of six monkeys received three injections with PBS, or
two human doses of a trivalent seasonal influenza vaccine
(the standard-of-care for nave children). As previously
observed in mice, #4900 elicited high titers of antibodies in
monkeys that were able to bind to a wide range of group 1
FL HAs (Fig. 5A and fig. S8A), compete with stem-binding
bnAb CR9114 for binding to homologous, heterologous and
heterosubtypic HAs (Fig. 5B), and elicit ADCC responses to
these divergent HAs (Fig. 5C). Furthermore, #4900 elicited
H5N1 neutralizing antibodies that were not only detectable
in a pseudoparticle-based neutralization assay (fig. S8B), as
for the mouse sera (Fig. 4C), but also in a standard
microneutralization assay (Fig. 5D). Animals vaccinated
with mini-HA #4900 had significantly reduced fever
following challenge with A/Mexico/InDRE4487/2009 (H1N1)
virus, compared to mock-immunized monkeys (Fig. 5E and
fig. S8E) (38), although no significant effect on the tracheal
viral load was detected (fig. S8C). These results are
comparable to those from monkeys vaccinated with the
trivalent seasonal influenza vaccine (Fig. 5E and fig. S8C).
However, the immune response elicited by mini-HA #4900
differs from the response elicited with the trivalent vaccine.
As expected, vaccination with the seasonal vaccine, but not
with headless #4900, elicited hemagglutination inhibiting
(HI) antibodies against A/California/07/09 (H1N1) virus
from which the H1 component of the trivalent vaccine is
derived and which is closely related (two AA differences) to
the A/Mexico/InDRE4487/2009 (H1N1) virus used to

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challenge the monkeys (fig. S8D). Furthermore, due to the

presence of the A/Texas/50/2012 HA antigen in the trivalent
seasonal vaccine, the vaccine elicited higher antibody titers
against this H3 strain than mini-HA #4900 (fig. S8A).
However, unlike the mini-HA immunized monkeys, no
antibodies able to compete with CR9114, induce ADCC, or
neutralize H5N1 virus were detectable in the sera of
monkeys immunized with the seasonal vaccine (Fig. 5, B to
E, fig. S8B), showing that the mini-HA elicited a
qualitatively different, and much broader immune response
than the seasonal vaccine.
In these in vivo studies, we used single antigen dose and
a potent adjuvant. Although this adjuvant has not been
registered for human use yet, it has been tested in a number
of clinical trials with favorable safety and immunogenicity
outcome (39). Future studies will need to address the
minimal doses of mini-HA and adjuvant for a protective
response. Since pre-existing immunity may have a profound
effect on the breadth of the response and no animal model
recapitulates the complex pre-existing immunity against
influenza found in humans, such studies should be
performed in humans (40).
Concluding remarks
Here we have described the design and characterization
of a series of soluble HA immunogens solely composed of
the HA stem. While all selected mini-HAs elicited
comparable levels of antibodies to FL HA, the breadth and
protective ability of the elicited antibodies progressively
increased with the structural evolution of mini-HA
configuration. The final candidatestabilized trimeric miniHA #4900 demonstrated its unique ability to elicit broad
and protective immune response in mice and nonhuman
primates. It has been reported previously (41) that
stabilization of respiratory syncytial virus F antigen
improves immune response and protection. Our results
demonstrate that the same principle holds for influenza HA
and provide further direction for design of an epitope-based,
universal influenza vaccine.
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ACKNOWLEDGMENTS
We thank R. Vogels, R. van der Vlugt, D. Zuijdgeest, N. Kroos, V. Klaren, S. SchmitTillemans, L. Kil, S. Barens and O. Diefenbach for scientific input and technical support; J. Klap, M. Koldijk and G.J. Weverling for statistical support; Algonomics for
input in early designs; Novavax for supplying Matrix-M; Promega for early access to
mouse surrogate ADCC assay; E. Montomoli, D. Perini and S. Piccirella from
VisMederi for pseudoparticle assay; Janssen Diagnostics for quantitative PCR of
tracheal swabs; P. Mooij and G. Koopman of the Biomedical Primate Research Center (BPRC), TNO Triskelion, Central Veterinary Institute and PreClinBiosystems AG
for performing the animal studies; and L. Dolfing and A. Dingemans for critical reading of the manuscript. The data presented in this manuscript are tabulated in the
main paper and in the supplementary materials. X-ray diffraction data sets were
collected at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2.
Use of the SSRL, SLAC National Accelerator Laboratory, is supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy Sciences under
Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program
is supported by the DOE Office of Biological and Environmental Research, and by the
National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of
the authors and do not necessarily represent the official views of NIGMS or NIH.
Coordinates and structure factors of the crystal structures are deposited in the
Protein Data Bank as entries 5CJQ and 5CJS. Crucell Holland B.V., a Janssen company, has the following pending patent applications in this field: WO 2013/079473,
WO 2014/191435, WO2008/028946, WO2013/007770, US 62/062,746 and US
62/062,754. Sharing of materials will be subject to standard material transfer
agreements.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/cgi/content/full/science.aac7263/DC1
Materials and Methods
Figs. S1 to S8
Tables S1 to S3
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10.1126/science.aac7263

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Table 1. Binding of bnAbs to selected mini-HA candidates. The bnAbs CR9114 and CR6261 form high-affinity
complexes with mini-HAs. The KD values were determined using biolayer interferometry and steady state analysis.
Multi Angle Light Scattering (MALS) analysis and size determination were used to confirm Fab binding and
complex formation. The numbers are mean values and standard deviations of at least two independently produced
protein batches. The values below in parentheses represent the expected molecular weights (Mw) for monomeric
(#4157, #4454), dimeric (#4650) and trimeric (#4900) mini-HA / Fab complexes, using the determined Mw of the
mini-HA and 48kDa for the Fabs.
KD values (nM)
Construct

CR9114

Mw (kDa)

CR6261

Mini-HA with Fab

Mini-HA

CR9114

CR6261

CR8020

Mini-HA
#4157

28.3 7.4

490 192

40.3 1.2

78.3 0.6 (88)

63.7 3.2 (88)

42.3 2.1

#4454

9.5 0.7

105 36

40.0 1.4

81.5 2.1 (88)

72.5 2.1 (88)

40.5 2.1

#4650

2.7 0.6

4.5 1.3

59.5 2.1

129.5 2.1 (156)

109.5 0.7 (156)

62.0 1.4

#4900

0.50

0.6 0.1

107.5 0.7

241.5 0.7 (252)

214.5 2.1 (252)

110.5 0.7

0.04
FL HA
monomer

6.7 4.0

11.7 4.2

trimer

0.8 0.3

0.6 0.2

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Fig. 1. The mini-HA design strategy and key candidates. (A) The mini-HA design strategy is described in five
stages, each characterized by specific modifications leading to selection of the best construct for each stage. Key
modifications for each stage are schematically depicted and color coded. Mini-HA #4157 was also generated in an
alternative, 11 C-terminal residues longer form as mini-HA #2759 (fig. S1F). (B) Ribbon mini-HA model (from PDB
ID 1RD8) with color-coded modifications (per stage as in Fig. 1A) and putative N-glycosylation sites (CG1-3, CG7;
taupe).

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Fig. 2. Dynamic structural properties of mini-HAs

and
trimeric
FL
HA
analyzed
by
hydrogen/deuterium
exchange
mass
spectrometry. Relative deuterium uptake over time
(0-60 min), illustrating the local dynamics of
different structural units, is depicted on model miniHA and FL HA structures using a color code from
blue (1% deuterium uptake: highly protected) to red
(100% deuterium uptake: highly exposed or flexible).

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Fig. 3. EM and crystal structures of mini-HAs in complex with CR9114 Fab. (A) Representative referencefree class averages of mini-HAs #4454, #4650 and #4900 in complex with CR9114 Fab using negative-stain
EM. The colored panels indicate how densities are attributed to either mini-HA (red) or Fab (blue.) The yellow
scale bar shown is 36.5 for the #4454 and #4650 complexes, and 73.0 for the #4900 complex. (B)
Crystal structure of monomeric mini-HA #4454 (HA1 in pink and HA2 in cyan) in complex with CR9114 Fab
(light chain in yellow and heavy chain in orange). The GCN4 motif, the B-loop and the glycine linker are shown
in dashed lines as they are disordered in the structure. (C) Crystal structure of trimeric #4900 in complex with
CR9114 Fab. One monomer of #4900-CR9114 Fab trimer is shown and color coded as in (B). The other two
monomers of #4900-CR9114 Fabs trimer are shown in gray for clarity. The GCN4 motif at the top of long helix
in #4900 is ordered and can be modeled. (D) Superimposition of mini-HA #4900 with a mini-HA model
computationally extracted from the Cali04 HA structure (HA from A/California/04/2009 (H1N1), PDB ID
4M4Y) (one monomer with HA1 green in and HA2 in yellow, and the other two monomers in dark grey). See
also tables S2 and S3.
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Fig. 4. Immunogenicity and protective efficacy of mini-HA candidates in mice. (A) KaplanMeier
survival curves (upper panels) and mean bodyweight change (middle panels) after three, two or one
immunization with mini-HA (n=10) or vehicle (mock; n=14), four weeks later followed by challenge with
25LD50 H1N1 A/PR/8/34. Serially diluted pre-challenge sera were individually tested for competition with
bnAb CR9114 (bottom panels). Error bars denote standard deviation of the mean for CR9114 competition
graphs. (B) KaplanMeier survival curves and mean bodyweight change of animals (n=10 for mini-HA
groups, n=12 for mock group) immunized three times and challenged with 12.5LD50 H5N1 A/Hong
Kong/156/97. (C) Neutralization of H5 HA A/Vietnam/1194/04 pseudovirus, with pre-challenge serum
pooled per immunization group. Data shown are the geometric mean SD of three technical replicates
per measurement. Results are representative for three independent experiments. (D) ADCC surrogate
assay with target cells expressing H5N1 A/Hong Kong/156/97 HA and pre-challenge serum pooled per
immunization group. Individual data points and the geometric mean of two technical replicates per
measurement are shown, representative for two independent experiments. (E) KaplanMeier survival
curves and mean bodyweight change for animals receiving serum from animals immunized three times
with mini-HA or mock (n=10 per group). Four weeks after the last immunization, serum was pooled and
transferred to nave recipient mice on three consecutive days before challenge, followed by challenge
with 12.5LD50 H5N1 A/Hong Kong/156/97. Symbols indicate significantly improved survival proportion
(Fishers Exact test; *), survival time (log-rank test; #) or bodyweight change (ANOVA; ^) compared to
mock-immunized animals. Three symbols (i.e., ***, ###, ^^^) indicate p<0.001, two symbols p<0.01,
and one symbol p<0.05.

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Fig. 5. Immunogenicity and protective efficacy of mini-HA #4900 in cynomolgus monkeys. Animals (n=6
per group) were immunized with mini-HA #4900, Inflexal 2013 or vehicle (mock) followed by challenge with
4x106 TCID50 H1N1 A/Mexico/InDRE4487/09. (A) Pre-challenge serum antibody titers to HA from H1N1
A/Brisbane/59/07 (left panel), H1N1 A/California/07/09 (middle panel) and H5N1 A/Vietnam/1203/04
(right panel). Open symbols indicate samples that were below limit of detection using a serum start dilution of
1/50. Statistical analysis between treatments was performed using pairwise t test with Tukey-Kramer
adjustment for multiple comparisons. Bars represent group medians. (B) Serially diluted pre-challenge sera
from individual animals tested for competition with bnAb CR9114, using FL HA from H1N1 A/Brisbane/59/07
(left panel), H1N1 A/California/07/09 (middle panel) and H5N1 A/Vietnam/1203/04 (right panel) as antigen.
Data shown are the mean of two technical replicates per measurement. (C) ADCC surrogate assay with target
cells expressing HA from H1N1 A/Brisbane/59/07 (left panel), H1N1 A/California/07/09 (middle panel) and
H5N1 A/Vietnam/1203/04 (right panel) and pre-challenge serum from individual animals. Data shown are the
geometric mean of two technical replicates per measurement. (D) Neutralization of H5N1
A/HongKong/156/97 reassortant virus using serially diluted pre-challenge serum from individual animals.
Data shown are the mean of two technical replicates per measurement. (E) Cumulative net body temperature
increase post challenge per animal. The AUC of the net temperature increase was calculated over intervals of
day 0-3 (left panel), day 0-8 (middle panel) and day 0-21 (right panel). Statistical analysis between treatments
was performed using pairwise t test with Tukey-Kramer adjustment for multiple comparisons. Bars denote
median. One animal of the #4900 group was excluded due to data logger failure, and one animal of the Inflexal
group died at the end of day 8 and was excluded from the day 0-21 interval calculation.

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