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https://doi.org/10.1038/s41467-018-08165-y

OPEN

Cross-lineage protection by human antibodies
binding the inﬂuenza B hemagglutinin

1234567890():,;

Yi Liu1, Hyon-Xhi Tan1, Marios Koutsakos1, Sinthujan Jegaskanda1, Robyn Esterbauer1, Danielle Tilmanis2,
Malet Aban2, Katherine Kedzierska1, Aeron C. Hurt1,2, Stephen J. Kent1,3,4 & Adam K. Wheatley 1

Inﬂuenza B viruses (IBV) drive a signiﬁcant proportion of inﬂuenza-related hospitalisations
yet are understudied compared to inﬂuenza A. Current vaccines target the head of the viralhemagglutinin (HA) which undergoes rapid mutation, signiﬁcantly reducing vaccine effectiveness. Improved vaccines to control IBV are needed. Here we developed novel IBV HAprobes to interrogate humoral responses to IBV in humans. A signiﬁcant proportion of IBV
HA-speciﬁc B cells recognise both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages in a distinct pattern of cross-reactivity. Monoclonal antibodies (mAbs) were reconstituted from IBV HA-speciﬁc B cells, including mAbs providing broad protection in murine
models of lethal IBV infection. Protection was mediated by neutralising antibodies targeting
the receptor binding domain, or via Fc-mediated functions of non-neutralising antibodies
binding alternative epitopes including the IBV HA stem. This work deﬁnes antigenic crossrecognition between IBV lineages and provides guidance for the rational design of improved
IBV vaccines for broad and durable protection.

1 Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000,

Australia. 2 World Health Organization (WHO) Collaborating Centre for Reference and Research on Inﬂuenza, The Peter Doherty Institute for Infection and
Immunity, Melbourne, VIC 3000, Australia. 3 Melbourne Sexual Health Centre and Department of Infectious Diseases, Alfred Hospital and Central Clinical
School, Monash University, Melbourne, VIC 3004, Australia. 4 ARC Centre for Excellence in Convergent Bio-Nano Science and Technology, University of
Melbourne, Parkville, VIC 3010, Australia. Correspondence and requests for materials should be addressed to S.J.K. (email: skent@unimelb.edu.au)or to A.K.W. (email: a.wheatley@unimelb.edu.au)
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I

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-08165-ynﬂuenza viruses are highly infectious respiratory pathogens
that inﬂict annual epidemics and periodic global pandemics, asigniﬁcant cause of mortality and morbidity. Two types ofinﬂuenza viruses co-circulate in human populations, inﬂuenza A
viruses (IAV) and inﬂuenza B viruses (IBV). Both IAV and IBV
are formulated into current seasonal inﬂuenza vaccines. IBV
historically accounts for ~20% of inﬂuenza-related hospitalisations in any given year1, and can dominate inﬂuenza seasons as
occurred in 2017/2018 in Europe2. The clinical severity of IBV isequivalent to that of IAV3, with children and young adults mostsusceptible to IBV. Unlike IAV, which naturally resides withinaquatic bird populations, IBV infection is almost exclusively
restricted to humans, with only rare reports of infection in seals4.
Mice and ferrets can however be experimentally infected with
IBV, providing useful small animal models of human IBV
infection and disease5. Owing to the limited host range and amarkedly slower mutation rate than IAV6, circulating IBV strains
exhibit more limited antigenic diversity than H1N1 and H3N2
IAV7. Nevertheless, since the ﬁrst reports in 1940s IBV has gradually diverged into two distinct lineages—B/Victoria/2/87-likeand B/Yamagata/16/88-like8 (referred to as B/Victoria and B/
Yamagata lineages from here on), which are further divided into
antigenic clades9.
An increased awareness of the burden of IBV, coupled withgrowing antigenic complexity, prompted the recent deploymentof quadrivalent inﬂuenza vaccines, incorporating components
derived from both circulating IBV lineages in order to broaden
coverage. Quadrivalent vaccines limit the likelihood of reduced
vaccine effectiveness due to IBV lineage mismatch compared tosuperseded trivalent vaccines, which is thought to have signiﬁcantly contributed to preventable deaths10. Nonetheless, vaccine effectiveness of seasonal inﬂuenza vaccines against IBV canoften remain low even when antigenic match with circulatingstrains is good11,12. Furthermore, protective serological titres
elicited by current seasonal vaccines wane in immunised adults13.
There remains a need for improved next-generation IBV vaccines
to provide resilient and durable protection against a broad arrayof IBV strains.
While the extensive characterisation of human immune
recognition of IAV spans many decades, the principal antigenic
targets of IBV, including the degree of cross-recognition of thedifferent lineages by B cells and antibodies, remain poorly
deﬁned. Cross-reactive humoral responses between IBV lineages
were described as early as 199114, with more recent analysis of
antibody repertoires after inﬂuenza vaccination suggesting a large
fraction of antibodies elicited by primary infection15 or seasonal
immunisation16 are cross-reactive against both lineages of IBV.
Selected human monoclonal antibodies (mAbs) have been isolated and can bind and neutralise diverse IBV isolates from bothlineages17–20, highlighting that cross-protective humoral immunity is feasible. These mAbs bind conserved epitopes localised
within either the globular head, or the stem domain of hemagglutinin (HA), and can protect mice from challenge with diverse
strains of IBV. Protection likely depends, at least in part, upon theelicitation of antibody-dependent cellular cytotoxicity (ADCC)17.
In addition to HA-speciﬁc antibodies, a recent study suggests
antibody binding the viral neuraminidase might similarly allow
cross-protection against both IBV lineages21. Unlike the large
number of mAbs characterised for IAV22, human immune
recognition of IBV remains understudied. A greater understanding where and how human B cells and antibodies bind to
IBV, the capacity of each site to mediate protection, and theextent of antigenic cross-recognition between IBV lineages iscritical to guide efforts to develop improved inﬂuenza vaccines.
Using a ﬂow cytometry-based approach previously describedfor IAV23,24, we interrogated the IBV-speciﬁc memory B cell2

response following seasonal tri- and quadrivalent vaccination of
adult human volunteers. Both classical antibodies inhibiting
hemagglutination, as well as novel broadly cross-reactive antibodies binding both IBV lineages were isolated from vaccinatedsubjects and demonstrated antiviral activity in vitro and in vivo.
Furthermore, we demonstrate that protection mediated by nonneutralising antibodies was dependent upon interaction withcellular Fc receptors. The accurate deﬁnition of cross-reactive
human anti-IBV antibody speciﬁcities will guide the design ofnovel IBV vaccines for broader protection.
Results
Development of B cell probes for inﬂuenza B hemagglutinin.
Recombinant HA analogues derived from IAV24 have provenutility for characterising B cells responses to inﬂuenza A vaccination using ﬂow cytometry23,25–27. We designed analogous
constructs to enable expression of soluble trimeric IBV HA
ectodomains from B/Phuket/3073/2013 (B-PH13). We generated a wild-type (WT) and a T139G variant, selected topotentially disrupt hydrogen bonds within the receptor binding
pocket28. With the HA proteins expressed and using ﬂow
cytometry (gating in Supplementary Fig. 1), co-staining humanmemory B cells from inﬂuenza immune adults with both WT
and T139G HA probes from B-PH13 revealed largely overlapping patterns of staining (Supplementary Fig. 2), whichsuggested limited antigenic changes between WT and theT139G mutant. However, while signiﬁcant non-speciﬁc association with human B cells was previously described for IAVprobes24, this did not appear the case for B-PH13, and nonmutated HA was deemed suitable for use as B cell probes. The
antigenic conservation of the B-PH13 HA probe as compared to
the WT virus was further conﬁrmed by staining B cells in miceexperimentally infected mice with B/Phuket/3073/2013 (Supplementary Fig. 3). Analogous WT probes for two additional B/
Yamagata-lineage HAs (B/Brisbane/60/2008 (B-BR08), B/
Yamagata/16/1988), three B/Victoria-lineage HAs (B/Victoria/
2/1987, B/Malaysia/2506/2004, B/Florida/4/2006) and the historical B/Lee/1940 strain were also developed.
The utility of IBV probes for tracking IBV-speciﬁc B cell
responsiveness was ﬁrst assessed in a cohort of healthy adults(N = 30) receiving 2015 trivalent Southern Hemisphere inactivated inﬂuenza vaccine (IIV3), including a B-PH13
component23,29. Vaccine-elicited expansion of B-PH13+ classswitched memory B cells (CD19+, IgD−, IgG+) was clearly
evident between baseline (d0) and d28 post-immunisation(representative staining in Fig. 1a; gating in Supplementary
Fig. 1), with a signiﬁcant twofold rise in observed frequencies
from a median 0.18% (IQR: 0.14–0.22) to 0.35% (IQR: 0.22–0.44)(Fig. 1b). The rise in memory B cell expansion was of a similar
magnitude to levels we previously reported23 for vaccine
components H3N2 A/Switzerland/9715293/2013 (H3-SW13)
and H1N1 A/California/07/2009 (H1-CA09) (Fig. 1c). We also
observed increased proportions of B-PH13+ B cells adopting acharacteristic activated phenotype (CD27+CD21−) following
IIV3 immunisation (Fig. 1d). Moreover, a weak but signiﬁcantcorrelation (r = 0.40, p = 0.034, Spearman’s rank-order test) was
observed between the magnitude of IIV3-induced expansion in BPH13+ memory B cell frequencies and the increase in strainmatched serum hemagglutination inhibition assay (HIA) titres(Fig. 1e), conﬁrming our previous observations in IAV studies
linking B cell and serologic responsiveness to IIV323. Together,
these data highlight memory B cell dynamics following IIV3 arebroadly similar to IAV and conﬁrm the utility of IBV HA-derived
ﬂow cytometric probes for interrogating IBV-speciﬁc B cell
immunity in response to vaccination or infection.

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pre-IIV3bd28 post-IIV3

0.853%

SSC-A

0.192%% of B-PH13+ /IgD– B cells

a

1.6
1.2
0.8
0.6
0.4
0.2
0

d0

B-PH13 HA-PEd% of B-PHU13+ B cells with
CD27+ CD21– phenotype

Change in frequency
d0 vs d28 (%)

10,000

1000

100

0

p < 0.0001

30
20
10
0

B-PH13 H1-CA09 H3-SW13

d0

d28

d28

e

40
Change in frequency of
B-PH13+ IgD– B cells (%)

c

p < 0.0001

5000
r = 0.40, p = 0.034
1000

100
100
1000
5000
Change in HIA GMT (%)

Fig. 1 Memory B cell responses to B/Phuket/3073/2013 (B-PH13) HA following IIV3 immunisation. a Cryopreserved PBMC samples were stained with apanel of monoclonal antibodies and recombinant HA probes allowing frequencies of class-switched memory B cells (CD19+CD20+IgD−) B cells speciﬁc
for B-PH13 to be assessed. b Frequencies of B-PH13 + memory B cells prior to and following IIV3 vaccination (N = 30). c Degree of B-PH13+ memory B cell
expansion (%) compared with responses to inﬂuenza A vaccine components A/Switzerland/9715293/2013 (H3-SW13) and A/California/07/2009 (H1CA09). d The proportion of B-PH13+ memory B cells with an activated memory (CD27+CD21−) phenotype before and after IIV3 immunisation.
e Correlation between vaccine-elicited expansion of B-PH13+ memory B cell frequencies and changes (%) in HIA geometric mean titre (GMT). Data areshown as median ± interquartile range. Populations were compared using Mann–Whitney U tests and p values are denoted. Correlations were assessedusing Spearman’s rank-order test and r and p values are denoted

Memory B cell responses to IIV3 and IIV4. To better combat
IBV viral diversity, many health authorities recommend thequadrivalent (IIV4) over trivalent (IIV3) for seasonal immunisation programmes. The ability of IIV4 to simultaneously promote serological responses to both B/Victoria/2/87-like and B/
Yamagata/16/88-like components has been conﬁrmed30. However, the dynamics of B/Yamagata and B/Victoria-lineage crossreactivity at the level of the B cell remains unclear. We therefore
compared lineage-speciﬁc and cross-reactive B cell responses by
ﬂow cytometry following either IIV3 or IIV4 vaccines containing
B-PH13 (B/Yamagata component only) or B-PH13 and B-BR08(combined B/Yamagata and B/Victoria components), respectively. Class-switched memory B cells from subjects receiving
IIV3 or IIV4 were co-stained with B-PH13 and B-BR08 probes as
before. Four populations of HA-binding memory B cells could bedelineated (representative plot in Fig. 2a): B-PH13-speciﬁc, BBR08-speciﬁc and two cross-reactive populations, one whichbound both probes with roughly equivalent intensity (“crossreactive - equivalent” or (CR-E)) and one which showed
increased relative binding to the B/Yamagata probe (B-PH13)(“cross-reactive – B/Yamagata preference” or (CR-Y)). CR-Y
staining was difﬁcult to clearly differentiate from the B-PH13+
population of cells. However, the CR-Y was identiﬁable in many
subjects examined both at baseline and following IIV (Supplementary Fig. 4). Moreover, the speciﬁcity of the CR-Y staining
pattern was not artefactual as conﬁrmed by swapping the probe
SA conjugates on matched samples (Supplementary Fig. 5). Thus,
the CR-Y population appears to represent sub-population of BPH13 HA-speciﬁc B cells with weak cross-reactive recognition of
B-BR08. No comparable populations of B cells with preferential
recognition of B/Victoria were observed. The extent to which
preferential binding of cross-reactive memory B cells to the B/

Yamagata probes is generalisable to all IBV strains is not clear.
However, this observation was recapitulated in representative
donors using probes derived from historical IBV strains B/Florida/4/2006 (B/Yamagata-lineage) and B/Malaysia/2506/2004 (B/Victoria-lineage) (Supplementary Fig. 6).
The responsiveness of each memory B cell population
following IIV3 and IIV4 immunisation was compared. B-PH13+ memory B cells were present at a median level of 0.17% (IQR:
0.13–0.22) at baseline in both cohorts and underwent signiﬁcant
expansion (Fig. 2b) in response to both IIV3 and IIV4. In
contrast, B-BR08+ memory B cell responses were present at alower level of 0.04% (IQR: 0.03–0.06) in both cohorts andexpanded only in response to IIV4 (Fig. 2c). Interestingly, both
CR-Y and CR-E populations expanded in response to IIV3 andIIV4 (Fig. 2d, e). Therefore, seasonal vaccines elicited expansionof both memory B cells speciﬁc for the respective IBV component
antigens and cross-reactive B cell populations, however themagnitude of CR-Y expansion was signiﬁcantly greater following
IIV4 (Fig. 2f). The serological implications of cross-reactive B cell
expansion are currently unclear. However, we observed consistently greater expansion in serum endpoint titres against diverse
IBV strains in subjects receiving IIV4 compared to subjects
receiving IIV3 (Supplementary Fig. 7), suggesting antibody
binding both lineages might be elicited as part of a broad antiIBV polyclonal response.
Isolation of IBV-speciﬁc human monoclonal antibodies. We
next examined the speciﬁcity and breadth of monoclonal antibodies recovered from each of the four identiﬁed IBV HA-speciﬁc
B cell populations. As with our previous IAV work, PBMCs
from three IIV4 vaccine recipients with marked expansion of

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B-Phu13-specific (Yamagata)
Cross-reactive, Yamagata preference (CR-Y)

B-PH13 HA-PE

Cross-reactive, equivalent (CR-E)
B-Bri08-specific (Victoria)

B-BR08 HA -APC

% of B-PH13+/IgD–B cells

10

p < 0.001

p < 0.001c1% of B-BR08+/IgD–B cells

b

1

0.1

0.01
d28

d0

d28

IIV3

IIV4

p < 0.001

p = 0.002

0.1

0.01

e

1

d28

d0

d28

IIV3

IIV4

p < 0.001

p < 0.001

d0

d0

0.1

0.01

0.001

0.001
d0

d28
IIV3

f

d0

% of CR-Y/IgD–B cells% of CR-E/IgD–B cells

1

0.1

0.01
d0

d

p < 0.001

ns

d0

d28
IIV4

d28
IIV3

d28
IIV4

100
ns

p < 0.001

nsp = 0.003

Fold change infrequency d0 vs d28

IIV3
IIV4

10

1

B-PH13

B-BR08

CR-E

CR-Y

Fig. 2 Cross-reactive memory B cell responses to IBV HA following IIV3 and IIV4 immunisation. a Co-staining class-switched memory B cells with B/
Phuket/3073/2013 (B-PH13) and (B/Brisbane/60/2008) B-BR08 probes allowed the delineation of four distinct populations of B cells: B-PH13 (purple)
and B-BR08 speciﬁc (red), and two cross-reactive populations—one binding each B lineage equivalently (CR-E; green) and one with increased staining
intensity for the Yamagata antigen B-PH13 (CR-Y; blue). Frequencies of memory B cells at baseline (d0) and d28 post-immunisation for b B-PH13 speciﬁc,c B-BR08 speciﬁc, d CR-E and e CR-Y populations. f Fold change in B cell frequency of each population between d0 and d28. Data are shown as median ±interquartile range. Populations were compared using Mann–Whitney U tests and p values are denoted. ns not signiﬁcant
4

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IBV-speciﬁc B cells post-immunisation were stained as before,
followed by sorting of single B cells and BCR-sequencing as
described24,31. Out of 672 sorted B cells, 519 productive heavy
sequences were recovered, 303 of which also had productive lightchain sequences. On the basis of similarities in variable geneusage, complementarity-determining region 3 (CDR-H3)
sequence length and CDR-H3 amino acid sequence, we identiﬁed
284 unique clonal lineages with a mix of larger clonal expansions
and singletons in each population (Supplementary Fig. 8; Supplementary Data 1). A selection of clonally expanded strainspeciﬁc or cross-reactive B cell lineages (Supplementary Fig. 9)
were chosen for recombinant expression and testing by ELISA forbinding to IBV HA. Antibodies isolated from B-PH13-speciﬁc (N= 10) or B-BR08-speciﬁc (N = 10) B cell populations displayed
HA recognition largely restricted to the sample of three IBV
lineage-speciﬁc HAs, with the exception of mAb W85–3F06,
which was broadly cross-reactive (Fig. 3). The majority of theselineage-speciﬁc antibodies demonstrated both HIA and neutralisation activity in vitro, suggesting binding to canonical epitopes that surround the receptor binding site32. However, unlike
IAV where HIA+ antibodies are usually highly strain speciﬁc (i.e.
with minimal cross-reactivity even within the same HA subtype),
we found anti-IBV mAbs elicited by seasonal vaccines generally
recognised all strains tested within a respective IBV antigenic
lineage spanning over 20 years of antigenic drift.
Consistent with dual binding of both IBV HA probes by ﬂow
cytometry, mAbs isolated from the two cross-reactive B cell
populations (CR-Y and CR-E) generally displayed much broader
IBV reactivity across both lineages, with greatest breadthobserved within the CR-E-derived mAbs. A subset of crossreactive mAbs could mediate HIA and neutralisation in vitro,suggesting binding of epitopes proximal from the receptor
binding domain. Eleven broadly cross-reactive antibodies wereidentiﬁed which bound all 7 IBV HAs tested across the twolineages and the historical B/Lee/1940 strain. As many broadly
reactive IAV-speciﬁc mAbs bind the HA stem, we generated
recombinant IBV stem constructs (Supplementary Figure 10)
based upon the designs employed to generate the IAV stemdomain33. These preliminary recombinant IBV stem proteinsappeared misfolded and failed to bind the prototypic stem mAb
CR9114, indicating antigenic changes compared to full-length
HA (Supplementary Figure 10). However, the IBV stem proteins
were bound by three putative broadly cross-reactive IBV stemspeciﬁc mAbs: W85–3F06, R95–1E03 and R95–2A08. Stem
speciﬁcity was further supported based upon binding to fulllength HA but not to puriﬁed HA1 proteins by ELISA (Fig. 3).
Therefore, based on breadth of HA recognition and antiviral
activity, we deﬁned three groupings of mAbs with IBV crossreactivity for in vivo efﬁcacy testing: broad HIA+ lineages (e.g.
R95–1E07, R95–1D05, K77–2D09), broad HIA− lineages (e.g.
R95–1F04, R95–1C01, R95–1E05) and broad HIA− stem-binding
lineages (e.g. W85–3F06, R95–1E03, R95–2A08).
Protective capacity of human anti-IBV mAbs against IBV. We
next assessed the protective capacity of selected human mAbs inmice experimentally challenged with human IBV strains. Passive
infusion of classical HIA+ B/Yamagata-lineage-speciﬁc mAbs
(K77–1G12, W85–1B01) protected mice from mortality or weightloss following infection with the B/Yamagata-lineage B/Florida/4/
2006 virus (Fig. 4a). In contrast, control mice infused with a
Victoria-lineage-speciﬁc mAb R95–1E12 exhibited no protection.
All broadly cross-reactive lineages isolated from CR-E B cells
provided some measure of protection against the B/Florida/4/
2006 challenge. On the basis of area under the curve analysis ofweight maintenance, there was a consistent trend for mAbs which

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displayed HIA activity in vitro to be more protective against
weight loss than lineages lacking HIA, with the IBV stem-binding
mAb R95–1E03 showing the weakest protection.
In mice challenged with B/Victoria-lineage B/Malaysia/2506/
2004 virus, we observed three distinct patterns of protection by
the panel of mAbs studied. Passive infusion of B/Victoria-lineagespeciﬁc HIA+ mAbs provided near complete protection fromweight loss (Fig. 4b). Again, we observed superior protection
mediated by cross-reactive mAbs with HIA activity, compared tothose without HIA activity. The intermediate protection elicitedby the three cross-reactive HIA− mAbs was highly consistent. In
contrast to the weak protection observed by the stem-speciﬁc
mAb against the B/Florida/4/2006 challenge, IBV stem-speciﬁc
mAbs were not protective at all against B/Malaysia/2506/2004,
clustering closely to negative controls.
Studies using IAV have established antibody-based protectionin the murine challenge model can be mediated via direct
neutralisation of virus and/or engagement with host effector cells
via Fc receptors (FcR)34,35, with the ability to engage FcR being
highly dependent on epitope location on the viral HA13,36. We
therefore cloned and re-expressed a selection of IBV-speciﬁc
mAbs using an Fc backbone with LALA mutations (L234A/
L235A) shown to prevent engagement with murine FcRs37.
Passive infusion of either B/Yamagata-lineage-speciﬁc mAbs or
the cross-reactive mAb R95–1E07, all of which mediated HIA
activity in vitro could still fully protect animals from weight loss
in the context of a LALA Fc (Fig. 5). In contrast, cross-reactive
mAbs without HIA activity (R95–1C01, R95–1F04) and the stemspeciﬁc mAb R95–1E03, lost any protective efﬁcacy in theabsence of FcR engagement, suggesting cell-mediated antibody
effector functions are critical for protection in vivo, consistent
with observations for IAV-speciﬁc mAbs34.
Mapping cross-protective epitopes on IBV HA. Pre-dominantantigenic sites of IBV cluster around the receptor binding domain(RBD), while previously resolved epitopes from broadly reactive
mAbs bind epitopes located across both the globular head andstem domains of HA (Fig. 6a). When looking at amino acidconservation across the lineages, most divergence is concentrated
within RBD proximal regions (Fig. 6b). However, signiﬁcant areasof pan-IBV conservation exist in the stem, residual esterase
domain and in selected areas of the RBD.
In order to better understand the molecular basis of broad IBV
recognition by antibody, we mapped the epitope speciﬁcity of a
selection of IBV neutralising mAbs through generating escapeviruses by serial passaging of IBV in the presence of mAbs. Viral
supernatants were recovered and the sequence of the full-length
HA gene determined38. To conﬁrm the selection of viral escapemutants, monoclonal isolates were recovered by plaque puriﬁcation and similarly sequenced. We ﬁrst determined the sensitivityof WT and mutant viruses to mAb-directed neutralisation(Supplementary Figure 11). Any loss of recognition of the viral
HA from mutant viruses was assessed by ﬂow cytometry using
infected cells (Supplementary Figure 12) and by ELISA using
recombinant HA proteins (Supplementary Figure 13). Aminoacid substitutions conferring antibody resistance were mapped
onto resolved IBV HA structures. B/Victoria-lineage-speciﬁc
mAbs were mapped using B/Brisbane/60/2008 virus. Escapemutants displayed a T214P mutation (R95–1D08, K77–2E02),
which potentially abrogated the N-linked glycan motif at N212,
and was generally coupled with a three-residue deletion from
K177 to D179 (Fig. 6c). While the T214P mutation alone was
sufﬁcient to drive escape from antibody-mediated neutralisation,
the three-residue deletion conferred additional loss of HA
binding by mAbs CR8033 and K77–2E02. B/Yamagata-lineage-

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LEE/40

BR/08

PH/13

FRA

LEE/40

PH/13

PH-SS

BR-SS

Stem

BR-HA1HA1PH-HA1

LEE/40

VIC/87

MA/04

Victoria
BR/08

YAM/88mAb
K77-1C06

FL/06

Sort population

PH/13

Yamagata

BR/08

HIA

ELISA

ELISA

W85-1E03

EC50 <0.01 g/ml

K77-2D11

EC50 0.01–0.1 μg/ml

W85-2C03

EC50 0.1–1 μg/ml

R95-1D08
B-Bri08+

EC50 >1 μg/ml

K77-2E02

Weak binding

W85-2D09
R95-1D10
R95-1E06
R95-1E12

HIA>10,240

W85-1A06
R95-1E05

1280–5120

K77-1G12

160–640

W85-1B01

10–80

W85-1C08<10

B-Phu13+
K77-1H02
W85-1C09
W85-1D01

FRA

W85-2B04>1280

W85-3F06

1280

K77-1C02
160–640

R95-1H09
K77-1G03
R95-1B11

10–80
<10

K77-1G12
R95-1C01
R95-1D05
Cross-reactive
equivalent (CR-E)

K77-2B06
K77-2D11
R95-1E03
R95-1E05
K77-2H12
R95-1E07
R95-1F04
W85-1A07
R95-1H08
K77-1H05
W85-3E10
R95-1H09

Cross-reactive
Yamagata (CR-Y)

K77-2C08
W85-3G03
K77-2D09
K77-2E07
R95-2A08
R95-2C02
R95-2G10

Fig. 3 Speciﬁcity and antiviral activity of recovered human monoclonal antibodies. mAbs were assessed for binding to diverse IBV HA, HA1 and HA stem
proteins by ELISA, with the strength of binding calculated as the concentration yielding half-maximal binding (EC50) and denoted by the indicated shading.
HIA was assessed as the ability of mAb to inhibit hemagglutination of red blood cells. The dilution of a 1 mg/mL stock with positive HIA signal is denotedby the indicated shading. The neutralisation of IBV viruses was assessed by focus reduction assay (FRA). The dilution of a 1 mg/mL stock, which
neutralised 50% of the virus control is denoted by the indicated shading

6

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a
100

PBS
VRC01
R95-1E12 (Vic, HIA+)% Survival

80

K77-1G12 (Yam, HIA+)
W85-1B01 (Yam, HIA+)

60

R95-1D05 (CR, HIA+)
R95-1E07 (CR, HIA+)

40

K77-1G03 (CR, HIA–)
R95-1C01 (CR, HIA–)

20

R95-1F04 (CR, HIA–)
B/Florida

R95-1E03 (CR, stem)

0
0

2

4

6
Day

8

10

110

300

100

200
AUC

% Initial weight

B/Florida

90

100
80

R95-1C01

R95-1F04

R95-1E03

R95-1F04

W85-3F06

R95-1E07

K77-1G03

R95-1E03b

R95-1D05

10

W85-1B01

8

R95-1E12

6
Day

K77-1G12

4

PBS

2

VRC01

0
0

PBS

100

VRC01
W85-1B01 (Yam, HIA+)% Survival

80

R95-1E12 (Vic, HIA+)
R95-1D08 (Vic, HIA+)

60

R95-1D05 (CR, HIA+)
R95-1E07 (CR, HIA+)

40

K77-1G03 (CR, HIA–)
R95-1C01 (CR, HIA–)
R95-1F04 (CR, HIA–)

20

R95-1E03 (CR, stem)

B/Malaysia

W85-3F06 (CR, stem)

0
0

2

4

6
Day

8

10

110

300

100

200
AUC

% Initial weight

B/Malaysia

90

100
80

K77-1G03

R95-1C01

R95-1E07

R95-1D05

10

W85-1B01

8

R95-1E12

6
Day

K77-1G12

4

PBS

2

VRC01

0
0

Fig. 4 Passive protection of mAbs in mice experimentally challenged with inﬂuenza mice (N = 5 per group) were infused with 5 mg/kg of anti-IBV mAbs orcontrols (PBS, VRC01) 24 h prior to intranasal challenge with a 50 TCID50 B/Florida/4/2006 or b 2000 TCID50 B/Malaysia/2506/2004. Survival curves(NB: lines are non-overlapping for clarity) and animal weights were monitored over an 11 day period, with animals killed if weight loss exceeded 20% ofstarting weight. Data is shown as mean ± SEM. Controls (grey), B/Yamagata-speciﬁc (red), B/Victoria-speciﬁc (green), cross-reactive HIA-positive(magenta), cross-reactive HIA-negative (blue) and cross-reactive stem mAbs (yellow) are indicated. The relative protection of each mAb assessed usingarea under the curve (AUC) analysis and plotted as mean ± SEM. Yam: B/Yamagata-speciﬁc lineage, Vic: B/Victoria-speciﬁc lineage, CR: cross-reactive,
HIA+: showed HIA activity in vitro, HIA−: no HIA activity in vitro, stem: bound epitopes within the HA stem
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100
VRC01

80

% Survival

K77-1G12-LALA (Yam)
W85-1B01-LALA (Yam)60

R95-1E07-LALA (CR, HIA+)
R95-1C01-LALA (CR, HIA–)

40

R95-1F04-LALA (CR, HIA–)
R95-1E03-LALA (CR, stem)

20
B/Florida

0
0

2

4

6
Day

8

10

110

300
B/Florida

200
AUC% Initial weight

100

90

100

80

R95-1E03

R95-1F04

10

R95-1C01

8

R95-1E07

6Day

W85-1B01

4

K77-1G12

2

VRC01

0
0

Fig. 5 Protection by IBV stem or HIA− mAbs requires engagement with cellular Fc receptors mice (N = 5 per group) were infused with 5 mg/kg of anti-IBV
mAbs expressed on a human IgG1 Fc with incorporated L234A/L235A (LALA) mutations. Twenty-four hours later, mice were challenged with 50 TCID50
B/Florida/4/2006. Animal weights were monitored over an 11 day period, with animals killed if weight loss exceeded 20% of starting weight. Survival
curves and weight loss are plotted as mean ± SEM, and the relative protection of each mAb assessed using area under the curve (AUC) analysis andplotted as mean ± SEM. Controls (grey), B/Yamagata-speciﬁc (red), B/Victoria-speciﬁc (green), cross-reactive HIA-positive (magenta), cross-reactive
HIA-negative (blue) and cross-reactive stem mAbs (yellow) are indicated. Yam: Yamagata-speciﬁc lineage, CR: cross-reactive, HIA+: showed HIA activity
in vitro, HIA−: no HIA activity in vitro, stem: bound epitopes within the HA stem

speciﬁc mAbs were mapped using B/Phuket/3073/2013, andconvergent pathways of viral escape were observed with
substitutions clustered at residues G156 (W85–1B01,
K77–1G12), T214I (R95–1E05, K77–1G12) and D212N(K77–1G12, R95–1E05) (Fig. 6d). These mutations are proximal
to the receptor binding pocket and localised within the 150-loop
and 190-helix structures, respectively. A G156R substitution alone
or in combination with D212N mediated neutralisation escapeand a partial to complete loss of HA binding by W85–1B01 andK77–1G12. G156E/T214I mutations drove neutralisation escapeand a complete loss of HA recognition by K77–1G12, as well as
W85–1B01 and CR8033. In terms of cross-reactive mAbs, escapemutations were identiﬁed within the viral supernatants upon
culturing with R95–1D05, with G156R substitutions in B/Phuket/
3073/2013 and N212S glycan loss in B/Brisbane/60/2008 (Fig. 6e).
However, plaques were not recoverable from the viral supernatantpreventing further HA-binding analysis. Escape mutations weresimilarly generated for a control antibody CR8033, which elicited
a T214P substitution and K177 to D179 deletion in B/Brisbane/
60/2008. Overall, our data indicate the glycine at position 156,
and the presence or absence of glycan at position 212, constitutekey pathways of escape against both neutralising strain-speciﬁc
and cross-reactive mAbs. Escape variants could not be generated
for IBV stem-speciﬁc mAbs, nor for those lacking HIA activity
8

in vitro, with further epitope deﬁnition likely requiring X-ray
crystallographic approaches or similar.
Discussion
IBV infections make up a signiﬁcant proportion of the global
inﬂuenza burden1, with health authorities recommending
simultaneous immunisation with both IBV lineages to combat
increasing IBV diversity and minimise the chances of seasonal
vaccine mismatch. Using a novel ﬂow cytometric approach, we
provide a detailed characterisation of the memory B cell
responses to IBV HA following immunisation with seasonal triand quadrivalent vaccines. We found that each vaccine efﬁciently
drove the expansion of memory B cells binding vaccine component HAs, either B-BR08 or B-PH13. In contrast to these humandata, ferret antisera raised by IBV infection displays typicallynarrow, near strain-speciﬁc serological responses to HA withlimited inter-lineage cross-reactivity7, consistent with antigenic
changes brought about by drift. In humans, repeated lifetime
exposures to both co-circulating lineages renders antibody andB cell recognition of IBV HA decidedly more complex. In linewith this, we found a subset of human mAbs initially thought
likely to be strain-speciﬁc based on single HA probe binding often
demonstrated relatively broad cross-lineage HA recognitionin vitro and protected from heterologous challenge in vivo,

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160 loop
190 helix

Receptorbinding
pocketCR8033150 loop

120 loop
CR8071

5A7
Variable
Partially conserved
Conserved
CR9114Glycan – both lineages
Glycan – B/Victoria only

c

d

e

D212N (W85-1B01)
D212N (R95-1E05)
T214I (K77-1G12)
T214I (R95-1E05)

T214P (K77-2E02)
T214P (R95-1D08)

G156R (W85-1B01)
G156E (K77-1G12)

ΔK177-D179 (K77-2E02)
ΔK177-D179 (R95-1D08)

N212S - B-BRIS (R95-1D05)

G156R - B-PHU (R95-1D05)

ΔK177-D179 - B-BRIS (CR8033)

T214P - B-BRIS (CR8033)

B/Victoria mAb

B/Yamagata mAb

Cross-reactive mAb

Fig. 6 Areas of the IBV HA allowing cross-reactive recognition and/or escape from human mAbs. a Surface representation illustration of canonical epitopes
localised within the receptor binding domain (190-helix, 160-loop, 150-loop, 120-loop) shown on the HA trimer of B/Brisbane/60/2008 (PDB:4FQM). The
resolved epitopes of broadly cross-reactive mAbs CR8033, CR8071, 5A7 and CR9114 are illustrated on the adjacent protomer. b Amino acid conservation
across IBV strains (N = 1000 per lineage) is illustrated on the B/Brisbane/60/2008 HA trimer with shading indicating areas of low (<90%), partial(>90%) and near complete conservation. Sites of N-linked glycosylation are indicated for both lineages (yellow) and for B/Victoria only (purple). c Escapemutations elicited by B/Victoria-speciﬁc mAbs are mapped onto the HA of B/Brisbane/60/2008 (PDB:4FQM). F95 within the receptor binding pocket
indicated in red. d Escape mutations elicited by B/Yamagata-speciﬁc mAbs are mapped onto the HA of B/Yamanashi/166/1998 (PDB:4M40). e Escapemutations elicited by cross-reactive mAbs are indicated on B/Brisbane

despite binding to canonical epitopes such as the 150-loop and
190-helix, which are subject to antigenic drift in humanpopulations7.
The elicitation of IBV cross-reactive serological responses toseasonal vaccines has been previously reported14,39–41. Whenassessed directly at the level of the B cell, two interesting andreproducible populations of cross-reactive B cells (CR-Y and CRE) were identiﬁed, with recovered mAbs conﬁrming broad IBV
recognition by ELISA. Pan-IBV recognition was concentrated
within mAbs derived from the CR-E population, with 64% (9/14)

of mAbs binding all 7 IBV HA tested compared to 16% (2/12) of
the CR-Y-derived mAbs. Nevertheless, it remains possible that
the CR-E and CR-Y populations might derive from continuous
spectrum of cross-reactivity and additional mAb isolation andcharacterisation is warranted. While administration of IIV3 was
sufﬁcient to drive expansion of both CR-Y and CR-E memory
B cell populations, expansion of CR-Y was signiﬁcantly enhanced
following IIV4 immunisation. It remains to be seen if a differential ability to drive expansion of CR-Y B cells contributes to any
potential increased serological protection from IIV4 vaccines. A

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-08165-ylimitation of our study is the restricted number of mAbs and theheterogenous patterns of IBV HA binding and neutralisation.
However, in general we ﬁnd seasonal inﬂuenza vaccines drive
expansion of two pre-dominant populations of memory B cells;
one binding neutralising epitopes proximal to the receptor
binding site shared within each respective IBV lineage, and asecond population of highly cross-reactive B cells binding bothlineages and expressing antibodies that were generally nonneutralising. Although many human mAbs displayed a degree ofprotection in mice, the protective beneﬁt of cross-reactive nonneutralising antibodies in human populations remains an open
question. Nevertheless, IIV4 drove expanded serological reactivity
against a broad range of IBV strains and simultaneously expands
B cells expressing HIA+ antibody with pan-B/Yamagata or panB/Victoria reactivity, suggesting IIV4 has an improved capacity tocombat antigenic drift in either lineage while removing the risk of
lineage mismatch relative to IIV3.
Interestingly, reports from immunised children39 and in murine
models42 found that prior priming with B/Yamagata antigens followed by immunisation with B/Victoria induces strong serologicalrecall of B/Yamagata, but only low B/Victoria antibody responses.
The converse is not the case, suggesting a degree of immunologic
dominance of B/Yamagata HA. Elements of our study are consistent with these observations, such as (a) lower baseline frequencies of B/Victoria-speciﬁc memory B cells in adults comparedwith B/Yamagata-speciﬁc memory B cells, (b) a cross-reactive B cell
population (CR-Y) that shows preferential binding to B/Yamagata
HA by ﬂow cytometry and (c) the absence of cross-reactive B cells
that show the converse preferential binding to B/Victoria HA.
Further resolution of the precise epitopes recognised by IBV crossreactive B cells and antibodies may help elucidate mechanisms that
underpin any differential capacity of antigens from the two IBV
lineages to recall cross-reactive immunity.
Several human antibodies with broad recognition of both IBV
lineages have been isolated and are of interest as potential diagnostic, therapeutic and prophylactic agents for IBV infection.
Broad IBV recognition and HIA activity was ﬁrst demonstrated
by the isolation of CR8033, which binds and neutralises both IBV
lineages and has HIA activity against B/Yamagata strains20.
Subsequently, the humanised murine antibody 12G6 was shown
to potently neutralise and elicit HIA activity against both IBV
lineages19, and binds a similar epitope to CR8033 overlapping thereceptor binding domain. We identiﬁed at least four mAbs
(R95–1D05, K77–2D11, R95–1E07, K77–2D09) with analogous
breadth and HIA activity in vitro. Viral escape mutants generated
with R95–1D05 and CR8033 both entailed glycan removal at
N212, suggesting binding to highly similar epitopes. Interestingly,
conserved pathways of escape were observed for both lineagespeciﬁc and cross-reactive mAbs that mediated HIA. For B/
Yamagata, this was focused at two regions: substitutions in the
150-loop at the glycine residue 156 (G156K, G156E, G156R), andsubstitutions at residues 212 and 214 within the 190 helix. Analogous G156R and G156E mutations were reported for crossreactive mAb C12G619 and double substitutions at D212 andT214 previously reported for B/Yamagata-speciﬁc human mAbs
3A2 and 10C418. For B/Victoria, we commonly observed removalof glycan at N212 via N212S (R95–1D05) or T214P (R95–1D08,
K77–2E02, CR8033) mutations, generally coupled with deletionof K177 to D179 (R95–1D08, K77–2E02, CR8033) in the 160loop. These HA modiﬁcations lie immediately proximal to residues identiﬁed in B/Brisbane/60/2008 essential for binding bycross-reactive mAb C12G619. Common pathways of escape forboth lineage-speciﬁc and cross-reactive mAbs may complicate thedesign of IBV HA immunogens for preferential induction of panIBV immunity. In addition, HIA+ cross-reactive antibody speciﬁcities like CR8033 might be susceptible to a loss of efﬁcacy in10

the face of antigenic drift, potentially driven by strain-speciﬁc orlineage-speciﬁc humoral responses at the population level.
Broad IBV recognition and broad prophylactic and/or therapeutic protection against IBV infection in vivo can be mediatedat alternative epitopes. In particular, within the vestigial esterase
domain at the base of the HA, as seen with CR807120 and 46B817,
or localised in the IBV stem domain, as seen with CR911420.
While we have identiﬁed antibodies with properties suggestive ofboth CR8071-like (W85–1A07, W85–3E10) and HA stemreactive speciﬁcities (W85–3F06, R95–1E03, R95–2A08), theabsence of neutralisation activity in vitro means accurate epitope
localisation via crystallographic or electron microscopic approaches is required.
The ready detection of broadly cross-reactive B cells in humans
based on our study presented here is supportive of efforts tobroaden vaccine protection. Moreover, a recent study using phagedisplay to derive mAbs from a seasonal vaccine recipient also
identiﬁed multiple cross-reactive antibody lineages including
those with intra-Yamagata, inter-lineage and even CR9114-likespeciﬁcities43. However, the degree to which B cells targeting thedifferent epitope clusters are recruited into protective serologicalresponses remains unclear, although some initial studies suggest
cross-reactive serum antibody may be signiﬁcant16. In terms ofprotective capacity, we observed a clear hierarchy following
passive infusion studies whereby cross-reactive antibodies capableof mediating HIA showed the greatest protective effect in vivo.
This is consistent with the reported activity of mAb 12G6, which
demonstrated greater prophylactic and therapeutic protection
against experimental IBV challenge than antibodies binding distal
to the receptor-binding domain44. Next in that order were mAbs
that conferred intermediate protection, characterised by broad
IBV recognition but no HIA activity, in a manner akin toCR8071. Consistent with reports for IAV34,35, protection mediated by such mAbs was highly dependent upon engagement withcellular Fc receptors. Finally, we found mAbs binding the IBV
stem domain failed to neutralise in vitro and provided theweakest protection against experimental challenge. The fact thatmAb CR9114 can neutralise IAV but not IBV strains20 suggests
the IBV stem may exist in a conformation comparatively resistantto antibody-mediated neutralisation. This is supported by thepoor epitope accessibility and low relative neutralising potency of
the IBV stem-proximal mAb 5A718. In contrast, a recent reportdetailed the isolation of multiple human IBV stem-binding
antibodies that can both neutralise in vitro and provide potent
cross-lineage protection in mice45. In human populations, serum
antibodies binding the IBV stem are widely prevalent, with titres
increasing with age46 or following IBV infection15. However,while the IBV HA stem is highly conserved and can be targeted inmice to protective effect47, the utility of the IBV stem as a humanvaccine target remains to be clariﬁed.
In summary, we demonstrate broadly cross-reactive memory
B cells are common in humans, express both neutralising andnon-neutralising immunoglobulins and are efﬁciently recruited
into the humoral response elicited by seasonal immunisation.
Further, IBV cross-reactive mAbs demonstrate protective
potential in vivo and may constitute a substrate amenable toselective targeting by improved vaccine designs. Accurate
deﬁnition of conserved sites of vulnerability on the IBV HA, in
particular, clarifying the molecular basis of narrow versus broad
IBV HA recognition, will greatly inform rational vaccine design
efforts for eliciting broad and durable protective immunity
against IBV.
Methods
Ethics statement. The study protocols were approved by both the Alfred Hospital
Ethics Committee (# 432/14), and the University of Melbourne Human Research

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Ethics Committee (# 1443420) and all associated procedures were carried out inaccordance with the approved guidelines. All participants provided written
informed consent in accordance with the Declaration of Helsinki. Animal studies
and related experimental procedures were approved by the University of Melbourne Animal Ethics Committee (#1714193).
IIV3 and IIV4 clinical samples. The 2015 IIV3 immunisation trial is fully
described elsewhere29 and registered as NCT02632578. Brieﬂy, 30 healthy Australian adults (mean age 40.4 years) were vaccinated with the 2015 IIV3 (bioCSL
Fluvax®) containing 15 μg of hemagglutinin from A/California/7/2009-like(pdmH1N1), A/Switzerland/9715293/2013 (H3N2)-like and B/Phuket/3073/2013like strains. Blood samples were taken at baseline (d0) and d28 post-immunisationand sera, plasma and PBMCs were cryopreserved prior to use. For the 2016 IIV4
trial, 20 healthy adults were administered Sanoﬁ FluQuadri® vaccine containing A/
California/07/2009-like virus (pdmH1N1), A/Hong Kong/4801/2014-like virus(H3N2), B/Phuket/3073/2013-like virus and B/Brisbane/60/2008-like virus components. Sera, plasma and PBMCs were collected and cryopreserved from d0 andday 28. Participant information is summarised in Supplementary Figure 14.
HA proteins and probes. Recombinant HA proteins for use as ﬂow cytometryprobes derived for IAV A/California/7/2009 and A/Switzerland/9715293/
2013 strains were previously described23,24. Analogous probes were prepared forinﬂuenza B encompassing the HA ectodomain C-terminally fused to the trimericfoldon of T4 ﬁbritin, a biotinylatable AviTag sequence GLNDIFEAQKIEWHE, anda hexa-histidine afﬁnity tag (sequences in Supplementary Figure 15). Brieﬂy,
synthetic genes encompassing the HA ectodomain from B/Phuket//3073/2013, B/
Brisbane/60/2008, B/Yamagata/16/1988, B/Victoria/2/1987, B/Malaysia/2506/2004,
B/Florida/4/2006 and B/Lee/1940 and variants containing the T129G mutation tolimit sialic acid binding were synthesised (GeneArt) and cloned into mammalian
expression vectors. Stabilised IBV stem protein expression plasmids were constructed based upon designs established to work for IAV33. Recombinant HA andHA stem proteins were expressed by transient transfection of Expi293F (LifeTechnologies, A14527) suspension cultures and puriﬁed by polyhistidine-tag afﬁnity chromatography and gel ﬁltration. For use as ﬂow cytometric probes, HA
proteins were biotinylated using BirA (Avidity) and stored at −80 °C. Prior to use,biotinylated HA proteins were labelled by the sequential addition of streptavidin(SA) conjugated to PE, APC, Alexa488 (ThermoFisher) or BV421 (BD) and stored
at 4 °C.
Flow cytometry. HA-speciﬁc B cells were identiﬁed within cryopreserved human
PBMC by co-staining with HA probes conjugated to SA-PE, SA-APC, SA-BV421
or SA-Ax488. Monoclonal antibodies for surface staining included: CD19-ECD(Beckman Coulter IM2708U, J3–119, 1:150), CD20 Alexa700 (BD 560631 2H7,
1:300), IgM-BUV395 (BD 563903, G20–127, 1:75), CD21-BUV737 (BD 564437, Bly4, 1:150), IgD-Cy7PE (BD 561314 IA6–2, 1:500), IgG-BV786 (BD 564230,
G18–145, 1:150), CD14-BV510 (Biolegend 301841, M5E2, 1:300), CD3-BV510(Biolegend 344828, OKT3, 1:600), CD8a-BV510 (Biolegend RPA-T8, 1:1500),
CD16-BV510 (Biolegend 302047, 3G8, 1:500), CD10-BV510 (Biolegend 312220,
HI10a, 1:750) and CD27-BV605 (Biolegend 302829, O323, 1:100). Background B
cells interacting with SA were excluded by staining with SA-BV510 (BD 563261).
Cell viability was assessed using Aqua Live/Dead amine-reactive dye (ThermoFisher). For mouse samples, splenocytes or lymph node suspensions were stainedwith the following panel: B220-BUV737 (BD 564449, RA3–6B2; 1:300), IgDBUV395 (BD 564274, 11–26 c.2a, 1:300), CD45-Cy7APC (BD 557659, 30-F11,
1:300), GL7-Alexa488 (Biolegend 144612,GL7, 1:300), CD38-Cy7PE (Biolegend 90,
1:750), SA-BV786 (BD 563858, 1:300), CD3-BV785 (Biolegend 100355, 145–2C11,
1:750) and F4/80-BV785 (Biolegend 123141, BM, 1:150). Samples were collectedusing a BD Fortessa conﬁgured to detect 18 ﬂuorochromes and analysis was performed using FlowJo software version 9.5.2 (TreeStar).
Sequencing, cloning and expression of B cell immunoglobulins. The sequencing
and cloning of BCRs from single B cells and the expression of recombinant mAbs
was performed as previously described24,31,48. Productive, recombined heavy chain(V-D-J) and light chain (V-J) immunoglobulin sequences were synthesised(Genscript), cloned into expression plasmids and transfected into Expi293F cells
using ExpiFectamine (Invitrogen). Recombinant monoclonal antibodies were
puriﬁed from culture supernatants using sepharose Protein-A or G (Pierce). BCR
sequences have been deposited in Genbank accession MK311355 - MK312159.
ELISA. Antibody binding to IBV HA, HA1 and stabilised stem proteins was tested
by ELISA. 96-well Immunosorp plates (ThermoFisher) were coated overnight at
4 °C with 2 μg/mL recombinant IBV HA expressed in Expi293 cells or sourced
commercially (Sino Biological). After blocking with 1% fetal calf serum (FCS) in
PBS, duplicate wells of IBV-speciﬁc monoclonal antibodies at different dilutions(starting at 10 μg/mL, four times serial dilutions) or human sera (1:100, four times
serial dilutions) were added and incubated for one hour at room temperature.
Plates were washed prior to incubation with 1:30,000 dilution of HRP-conjugatedanti-human IgG (KPL) for 1 h at room temperature. Plates were washed anddeveloped using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma) and read

ARTICLEat 450 nm. HA-binding intensity was calculated as the antibody concentration
giving half-maximal signal (EC50) using a ﬁtted curve (4 parameter log regression).
For serum samples, endpoint titres were using a ﬁtted curve (4 parameter log
regression) and a cutoff of two times background.
Passive infusion of IBV-speciﬁc monoclonal antibodies. B/Florida/4/2006 and
B/Malaysia/2506/2004 viruses were grown in embryonated eggs and infectious
titres were determined by 50% tissue culture infective dose (TCID50) viral assays.
Challenge stocks were titrated in mice and assessed for pathogenicity (Supplementary Figure 16). Groups of ﬁve C57BL/6 female mice at 8–12 weeks of age wereused to examine prophylactic potential of mAbs generated. Mice were administeredmAbs intraperitoneally at a dose of 5 mg/kg body weight. Twenty-four hours after
passive infusion of mAbs, mice were anaesthetised by isoﬂurane inhalation andintranasally challenged with 50 μL of B/Florida/4/2006 and B/Malaysia/2506/2004
viruses at doses of 50 and 2000 TCID50, respectively. Animals were monitored forweight loss and signs of infection for 14 days, and killed if a loss of more than 20%of their pre-infection weight occurs.
Hemagglutination inhibition assay (HIA). HIA activity of recombinant mAbs
was assessed using 1% turkey erythrocytes in a standardised assay as previously
described49. Brieﬂy, mAbs were diluted to 100 μg/mL in PBS prior to incubation
with ether treated50 inﬂuenza viruses from strains B/Phuket/3073/2013, B/Brisbane/60/2008 and B/Lee/1940. HIA titres are reported as the reciprocal of thehighest dilution where hemagglutination was completely inhibited.
Focus reduction assays (FRA). Neutralisation activity of recombinant mAbs
against B/Phuket/3073/2013, B/Brisbane/60/2008 and B/Lee/1940 was examinedusing focus reduction assays as previously described51. The neutralisation titre isexpressed as the reciprocal of the highest dilution of a 1 mg/mL mAb stock at
which virus infection is inhibited by ≥50%.
Viral escape assay. The generation of IBV escape mutants was based uponpreviously described protocols38. Brieﬂy, 24-well plates were seeded with 2.5 × 105
Madin Darby Canine Kidney (MDCK) cells (ATCC CCL-34) per well to form
conﬂuent monolayers. The next day, serial dilutions of recombinant mAbs wereincubated with B/Phuket/3073/2013 and B/Brisbane/60/2008 for one hour at 37 °C
in Flu-media (Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) with 0.8% bovine
serum albumin (BSA), 1% penicillin/streptomycin and 0.1% L-1-Tosylamide-2phenylethyl chloromethyl ketone (TPCK)-treated trypsin), before adding the virusantibody mixture to MDCK cells. After 2 to 3 days culture media supernatantsfrom wells with visible cytopathic effect were collected and used to infect a freshmonolayer of MDCK cells in the presence of increasing concentrations of mAb.
After serial passaging, culture supernatants were harvested, viral RNA wasextracted and the IBV HA gene sequenced. Putative mutant viruses were identiﬁed
based upon sequence comparison to similarly passaged media-only or irrelevantmAb (anti-HIV VRC01) controls. Where mutants were identiﬁed, single virus
isolates were recovered by plaque puriﬁcation using standard techniques. Single
plaques were rescued, expanded in MDCK cells, and mutant viruses within culture
supernatants sequenced and TCID50 determined as before.
mAb neutralisation and HA-binding assays. The neutralisation activity ofrecombinant mAbs was examined using a modiﬁed microneutralisation assay52.
MDCK cells were seeded in 96-well plates at 1.5 × 105 per well. The next day, serial
dilutions of recombinant mAbs were incubated in Flu-media with 100TCID50 of
WT or mutant B/Phuket/3073/2013 and B/Brisbane/60/2008 viruses for one hour
at 37oC, before addition to MDCK cells. After 18–24 h, supernatants were removed,
cells were ﬁxed and cellular cytopathicity was visualised by ELISA using mouseanti-inﬂuenza B nucleoprotein (1:1000;Abcam) primary and goat anti-mouse HRPconjugated secondary antibodies. Plates were developed using TMB substrate andread at 450 nm. The concentration of mAb preventing 50% infectivity (IC50) was
calculated.
The ability of recombinant mAbs to bind cell-surface HA on infected cells was
examined by ﬂow cytometry. MDCK cells were seeded into 6-well plates andinfected with ~10,000 TCID50 WT or mutant B/Phuket/3073/2013 and B/Brisbane/
60/2008 viruses and incubated at 37 °C for 18–24 h. Cells were resuspended bymanual scraping and infectivity conﬁrmed by staining with mouse anti-inﬂuenza B
nucleoprotein (1:1000; Abcam) and goat anti-mouse Alexa647 (1:5000;
ThermoFisher). The binding of human anti-IBV mAbs (5 μg/mL) or an anti-HIV
negative control (VRC01) to surface expressed HA was detected using goat antihuman Alexa647 (1:5000; ThermoFisher).
Sequence analysis of viral isolates. To characterise conservation of the IBV HA,
a cross-section of 2000 B/Yamagata and B/Victoria-lineage viral sequences spanning 1988–2018 were exported from the EpiFlu database [gisaid.org], HA protein
sequences aligned using Geneious 11.1.3 (Biomatters) and weighted conservation
scores accounting for year of isolation determined at each residue position. Aminoacid conservation was visualised using Pymol.

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ARTICLE

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-08165-y

Statistical analyses. Data is generally presented as median ± interquartile range.
Statistical signiﬁcance was assessed by Mann–Whitney U tests. Correlations wereanalysed using Spearman’s rank-order tests. Curve ﬁtting was performed using 4
parameter logistic regression. All statistical analyses were performed using Prism(GraphPad).
Reporting summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this article.

Data availability
All data generated or analysed during this study are included in the publishedarticle and its supplementary information ﬁles.

Received: 30 May 2018 Accepted: 20 December 2018

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Acknowledgements
We thank all study participants and the Melbourne Cytometry Platform (MBC node) for
expert ﬂow cytometry assistance. This work was supported by NHMRC programme grant
ID1052979 (SJK), NHMRC project grant ID1129099 (AKW) and Proof-of-Concept Initiative funding co-provided by the University of Melbourne and Sanoﬁ Pasteur. YL and MK aresupported by a Melbourne International Research Scholarship and Melbourne International
Fee Remission Scholarship. KK was supported by a NHMRC Senior Research Fellowship
Level B. World Health Organization (WHO) Collaborating Centre for Reference Researchon Inﬂuenza is supported by the Australian Government Department of Health.

Author contributions
Y.L., H.-X.T., S.J.K. and A.K.W. designed the study. Y.L., H.-X.T., M.K., R.E. and S.J.
performed experiments. M.K. and K.K. provided viruses and samples. D.T., M.A. and A.
C.H. performed serological analyses and plaque puriﬁcations. Y.L., H.-X.T., S.J.K. and A.
K.W. wrote the manuscript. All authors read and revised the manuscript.

Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467018-08165-y.
Competing interests: This study was funded in part via a Proof-of-Concept Initiative
awarded to A.K.W. and co-funded by Sanoﬁ Pasteur, which is the manufacturer of

ARTICLEinﬂuenza vaccines (FluQuadri) received by some, but not all, of the participants ofthis study. The remaining authors declare no other competing interests.
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