Wrammert, J. et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J. Exp. Med. 208, 181-193

Emory Vaccine Center, School of Medicine, Emory University, Atlanta, GA 30322, USA.
Journal of Experimental Medicine (Impact Factor: 12.52). 02/2011; 208(1):181-93. DOI: 10.1084/jem.20101352
Source: PubMed
The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.


Available from: Aneesh K Mehta
Ar ticle
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 208 No. 1 181-193
Inuenza is the seventh leading cause of death
in the United States (Beigel, 2008), with the el-
derly, the very young, pregnant women, and
otherwise immune-compromised populations
accounting for >90% of inuenza-related deaths.
The pandemic H1N1 inuenza virus strain is
immunologically distinct from other inuenza
Ra Ahmed:
Patrick C. Wilson:
Abbreviations used: GC, germi-
nal center; HA, hemagglutinin;
HAI, hemagglutinin inhibition;
MN, microneutralization;
TCID50, 50% tissue culture
infectious dose.
Broadly cross-reactive antibodies dominate
the human B cell response against 2009
pandemic H1N1 inuenza virus infection
Jens Wrammert,
Dimitrios Koutsonanos,
Gui-Mei Li,
Srilatha Edupuganti,
Jianhua Sui,
Michael Morrissey,
Megan McCausland,
Ioanna Skountzou,
Mady Hornig,
W. Ian Lipkin,
Aneesh Mehta,
Behzad Razavi,
Carlos Del Rio,
Nai-Ying Zheng,
Jane-Hwei Lee,
Min Huang,
Zahida Ali,
Kaval Kaur,
Sarah Andrews,
Rama Rao Amara,
Youliang Wang,
Suman Ranjan Das,
Christopher David O’Donnell,
Jon W. Yewdell,
Kanta Subbarao,
Wayne A. Marasco,
Mark J. Mulligan,
Richard Compans,
Ra Ahmed,
and Patrick C. Wilson
Emory Vaccine Center,
Department of Microbiology and Immunology,
Division of Infectious Diseases, Department
of Medicine, School of Medicine, Emory University, Atlanta, GA 30322
Hope Clinic of the Emory Vaccine Center, School of Medicine, Division of Infectious Disease, Decatur, Georgia, 30030
Department of Internal Medicine, Emory University, Atlanta, GA 30322
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute
Department of Medicine, Harvard Medical School, Boston, MA 02115
Department of Medicine, Section of Rheumatology, The Committee on Immunology, The Knapp Center for Lupus
and Immunology Research, The University of Chicago, Chicago, IL 60637
Center for Infection and Immunity, Columbia University Mailman School of Public Health, New York, NY 10032
Hubert Department of Global Health, Rollins School of Public Health and Department of Medicine, Emory University School
of Medicine, Atlanta, GA 30322
Laboratory of Viral Diseases,
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases,
Bethesda, MD 20892
The 2009 pandemic H1N1 inuenza pandemic demonstrated the global health threat of
reassortant inuenza strains. Herein, we report a detailed analysis of plasmablast and
monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike
antibodies elicited by annual inuenza vaccinations, most neutralizing antibodies induced
by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglu-
tinin (HA) stalk and head domain of multiple inuenza strains. The antibodies were from
cells that had undergone extensive afnity maturation. Based on these observations, we
postulate that the plasmablasts producing these broadly neutralizing antibodies were
predominantly derived from activated memory B cells specic for epitopes conserved in
several inuenza strains. Consequently, most neutralizing antibodies were broadly reactive
against divergent H1N1 and H5N1 inuenza strains. This suggests that a pan-inuenza
vaccine may be possible, given the right immunogen. Antibodies generated potently pro-
tected and rescued mice from lethal challenge with pandemic H1N1 or antigenically dis-
tinct inuenza strains, making them excellent therapeutic candidates.
© 2011 Wrammert et al. This article is distributed under the terms of an
Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first
six months after the publication date (see http://www.rupress.org/terms).
After six months it is available under a Creative Commons License (Attribution–
Noncommercial–Share Alike 3.0 Unported license, as described at http://creative-
viruses, leaving large population groups sus-
ceptible to infection (Brockwell-Staats et al.,
2009; Dawood et al., 2009; Garten et al., 2009;
The Journal of Experimental Medicine
Page 1
182 Human B cell responses to pandemic H1N1 inuenza | Wrammert et al.
most other H1N1 and H5N1 inuenza strains, especially in
high-risk populations such as immunosuppressed patients and
the elderly.
Inuenza-specic plasmablasts are persistently induced
throughout infection, providing a rich source of antiviral mAbs
B cell responses were examined in nine patients infected with
the pandemic 2009 H1N1 inuenza virus. These patients had
varying courses and severity of disease. The cases ranged from
mild disease with rapid viral clearance within a few days after
onset of symptoms to severe cases that shed virus for several
weeks and required hospitalization with ventilator support.
A majority of the patients were treated with antiviral drugs.
The diagnoses were conrmed by pandemic H1N1-specic
RT-PCR and serology. All patients had neutralizing titers of
serum antibodies at the time of blood collection. A summary
of the clinical patient data are shown in Table I. The majority
of samples were obtained around 10 d after the onset of symp-
toms, with the exception of a particularly severe case where
sampling was done 31 d after symptom onset.
Antigen-specic plasmablasts appear transiently in periph-
eral blood after vaccination with inuenza or other vaccines
(Brokstad et al., 1995; Bernasconi et al., 2002; Sasaki et al., 2007;
Wrammert et al., 2008), but the kinetics of their appearance and
persistence during an ongoing infection remain unclear. Here,
we have analyzed the magnitude and specicity of the plasma-
blast response in blood samples taken within weeks after onset of
clinical symptoms of pandemic H1N1 inuenza virus infection.
Using a virus-specic ELISPOT assay, it was possible to show a
signicant number of pandemic H1N1-reactive plasmablasts in
the blood of the infected patients, whereas none were detectable
in a cohort of healthy volunteers (Fig. 1, A and B). These cells
were also readily detectable several weeks after symptom onset in
the more severe cases. Fig. 1 (A and C) illustrates that, of the total
IgG-secreting cells, over half of the cells were producing anti-
bodies that bound pandemic H1N1 inuenza virus. Moreover,
plasmablasts specic for HA occurred at 30–50% the frequency
of virus-specic cells (Fig. 1, C and D), the specicity most likely
to be critical for protection. Most patients also had a relatively
high frequency of plasmablasts, forming antibodies that bound
to past, seasonal inuenza strains (Fig. 1 C) or recombinant HA
from the previous annual H1N1 strain, A/Brisbane/59/2007.
Based on the overall frequency of pandemic H1N1-specic
cells, it is likely that the cells binding other strains were overlap-
ping populations and cross-reactive. None of the induced plas-
mablast cells bound to recombinant HA from the H3N2 strain
from the same vaccine (A/Brisbane/10/2007). These ndings
demonstrate that inuenza-specic human plasmablasts are con-
tinuously generated throughout an ongoing infection and that a
fairly high proportion of these cells make antibodies that also
cross-react with previous annual H1N1 inuenza strains.
To analyze the specicity, breadth, and neutralizing capacity
of these plasmablasts, we used single-cell PCR to amplify the
heavy and light chain variable region genes from individually
Hancock et al., 2009). The Centers for Disease Control
reports that there were an estimated 60 million cases of
the 2009 H1N1 pandemic strain, which caused 256,000
hospitalizations. An unusually high frequency of severe dis-
ease occurred in younger and otherwise healthy patients
(Hancock et al., 2009). In addition, rare infections with avian
H5N1 inuenza strains in humans had close to a 50% mor-
tality rate (Subbarao and Joseph, 2007). Emergence of a
zoonotic or antigenically distinct strain that combined even
a fraction of the morbidity and mortality of the pandemic
H1N1 and H5N1 viruses would have dire consequences.
Antibodies play a key role in protection against inuenza in-
fection in vivo (Puck et al., 1980; Gerhard et al., 1997; Luke
et al., 2006; Simmons et al., 2007). The fact that there were
little or no preexisting antibody titers present before the
emergence of this pandemic virus, and that the virus atypi-
cally caused such severe disease in young adult, illustrates the
importance of comprehensively understanding the B cell
responses and antibody specicities induced by infection
with this inuenza virus.
Here, we have analyzed the plasmablast responses induced
by pandemic H1N1 infection and generated a panel of mono-
clonal antibodies (mAbs) from these cells to analyze their char-
acteristics in detail. In contrast to seasonal vaccination, we show
that a majority of the neutralizing antibodies induced by infec-
tion were broadly cross-reactive with all recent annual H1N1
strains, as well as the highly pathogenic 1918 H1N1 and avian
H5N1 strains. These neutralizing antibodies bound predomi-
nantly to conserved epitopes in the hemagglutinin (HA) stalk
region (Ekiert et al., 2009; Sui et al., 2009), with some binding
to novel epitopes in the HA globular head. The high frequency
of these HA-stalk binding antibodies is of particular interest, as
this epitope is a promising target for a broadly protective inu-
enza vaccine (Steel et al., 2010). Furthermore, the cross-reactive
antibodies carried highly mutated immunoglobulin genes, in-
dicative of extensive anity maturation. Together, these nd-
ings support a model in which infection predominantly activated
broadly cross-reactive memory B cells that then underwent fur-
ther anity maturation. We propose that the expansion of these
rare types of memory B cells may explain why most people did
not become severely ill, even in the absence of preexisting pro-
tective antibody titers. Recent studies in mice strongly support
the idea that consecutive immunizations with antigens from
divergent inuenza stains can indeed hone the antibody re-
sponse to preferentially target broadly protective conserved
epitopes (Wang et al., 2010; Wei et al., 2010). Our ndings
demonstrate that cross-reactive antibodies can be preferen-
tially induced in humans given the right immunogen, pro-
viding further support for the feasibility of generating a
pan-inuenza vaccine. Finally, in vivo challenge experiments
showed that the neutralizing antibodies isolated protected mice
challenged with a lethal dose of pandemic H1N1 inuenza
virus, even when administered therapeutically 72 h after
infection, and also provided protection against antigenically
distinct H1N1 influenza strains. These antibodies are thus
promising as therapeutics against pandemic H1N1, as well as
Page 2
JEM VOL. 208, January 17, 2011
Ar ticle
activated by pandemic H1N1 infection. Consistent with the
frequency of plasmablasts secreting antibodies binding annual
inuenza strains by ELISPOT analyses (Fig. 1 C), a majority
(29/46, or 63%) of the pandemic H1N1-specic antibodies
also cross-reacted with seasonal inuenza viruses (Fig. 2,
A and B). In fact, by ELISA, one third of these antibodies bind
to the prepandemic strains at lower concentrations than they
did to the pandemic H1N1 strain, suggesting higher avidity
binding. By comparison, only 22% (11/50) of plasmablasts
induced by annual H1N1 strains before the pandemic could
bind the pandemic H1N1 inuenza (Fig. S1 B). We propose
that the cross-reactivity of pandemic H1N1-induced cells
derives from the activation of memory cells originally specic
for past inuenza immunizations in an original antigenic
sin fashion.
Evidence of extensive afnity maturation suggests a high
frequency of memory cell activation against the pandemic
H1N1 strain
Based on the 10–15-fold induction of plasmablasts and ex-
pression of intracellular Ki67 during ongoing immune re-
sponses (Brokstad et al., 1995; Bernasconi et al., 2002; Sasaki
et al., 2007; Smith et al., 2009; Wrammert et al., 2008), we can
assume that most plasmablasts result from the ongoing infection
sorted cells (dened as CD19
, CD20
, CD3
, CD38
cells; Fig. 1 E; Wrammert et al., 2008; Smith et al.,
2009). These genes were cloned and expressed as mAbs in 293
cells, and the antibodies were screened for reactivity by ELISA.
Thresholds for scoring antibodies as specic to the inuenza an-
tigens were empirically determined based on being two standard
deviations greater than the background level of binding evident
from 48 naive B cell antibodies (Fig. S1 A). Of 86 antibodies
generated in this fashion, 46 (53%) bound pandemic H1N1
(Fig. 1 F) and one third (15 antibodies) were reactive to HA
(Fig. 1 G and Fig. S2 A), most of them at sub-nanomolar avidities
(based on surface plasmon resonance analyses; Fig. S2 B). On a
per donor basis, 55% of the mAbs bound to puried pandemic
H1N1 virions (range: 33 to 77%). Of the virus-specic anti-
bodies, 31% bound to recombinant HA (range: 14 to 55%). We
conclude that virus-specic plasmablasts are readily detected after
pandemic H1N1 inuenza virus infection and that virus-specic
human mAbs can be eciently generated from these cells.
Plasmablasts from patients infected with pandemic H1N1
inuenza were highly cross-reactive to prepandemic
inuenza strains
As the plasmablasts are specically induced by the ongoing
immune response, we can learn about the origin of the B cells
Table I. Summary of clinical data for patients with acute pandemic H1N1 virus infections
Patient Age Gender HAI
Initial symptoms Hospital course Sample
EM 30 F 640 1280 none Fever, cough, dyspnea Acute respiratory distress syndrome,
bacterial pneumonia, pulmonary
embolism, prolonged oscillatory
ventilator support, tracheostomy,
discharged after 2 mo
Day 31 Oseltamivir Yes
1000 37 M 80 40 Hypertension,
lung disease
of unknown
Fever, cough, shortness
of breadth, nausea,
Pneumonia, acute sinusitis, acute renal
failure, discharged after 8 d
Day 18 Oseltamivir,
70 38 F 80 160 none Fever, cough, body aches N/A Day 15 None Yes
1009 21 M 20 20 Congenital
heart disease,
repair for
Tetralogy of
Fever, cough, sore throat,
nausea, diarrhea
N/A Day 9 Oseltamivir Yes
1010 24 M 10 10 none Fever, cough, nausea,
vomiting diarrhea
N/A Day 11 Oseltamivir No
1011 25 M 20 10 none Fever, cough, sore throat,
vomiting, headache,
N/A Day 9 Oseltamivir No
1013 26 M 80 160 none Fever, cough, sore throat,
body aches, nausea,
vomiting, diarrhea
N/A Day 9 None No
1014 45 F 80 20 none Fever, chills, cough,
sore throat, body aches,
headache, nausea,
N/A Day 9 None No
The mAb column indicates whether mAbs were made from the plasmablasts of these patients.
Page 3
184 Human B cell responses to pandemic H1N1 inuenza | Wrammert et al.
Figure 1. Generation of human mAbs against pandemic H1N1 inuenza virus from infected patients. (A and B) Magnitude of the plasmablast
response observed in peripheral blood of six pandemic H1N1-infected patients and 22 healthy (noninfected/nonvaccinated) donors by ELISPOT analysis.
(A) Representative ELISPOT. Numbers of plasmablasts secreting antibody reactive to pandemic H1N1 is compared with the total number of IgG-secreting
cells from each PBMC sample (numerals). All ELISPOT assays were performed in duplicate. (B) Summary of all the donors analyzed; each dot represents
one patient or control. (C and D) Specicity of the sorted plasmablasts measured by ELISPOT analysis. Representative ELISPOT showing plasmablasts pro-
ducing antibodies reactive with total IgG or pandemic H1N1 whole virus, annual inuenza vaccine (2009/2010 TIV vaccine), or recombinant HA from pan-
demic H1N1, the previous year’s annual vaccine H1N1 strain (A/Brisbane/59/2007), or the previous years H3N2 strain (A/Brisbane/10/2007). (D) Summary
of the frequency of whole IgG secreting cells specic to pandemic H1N1 whole virus, recombinant HA from pandemic H1N1, and recombinant HA from the
previous years vaccine. Donors EM1 and SF1000 were not analyzed in this fashion, as the antigens were not available for live-cell analyses at that time point
in the pandemic. (E) Sorting of plasmablast cells from pandemic H1N1 inuenza–infected patients to generate mAbs. Flow cytometry plots show the per-
centage of CD27
cells (dot plots are gated on CD3
lymphocytes). The plasmablasts are dened herein as CD3
(right) An example of post-sort purity of ungated cells (veried for each sample). Single plasmablasts were isolated from the sorted fraction by cell sorting,
and variable antibody genes were cloned from individual cells (see Materials and methods). (F and G) Scatchard plots of binding of the isolated mAbs to
pandemic H1N1 whole-puried virus (F) and pandemic H1N1 recombinant HA (G) as measured by ELISA. Antibodies were scored positive (frequency above
plots) if they bound at least two standard deviations greater than the mean absorbance of naive B cell antibodies at 10 µg/ml (detailed in Fig. S1 A).
Page 4
JEM VOL. 208, January 17, 2011
Ar ticle
(averaging >19 per patient; Fig. 2 D and Fig. S3 A). For these
5 patients, mutations had accumulated signicantly more than
from primary IgG plasmablast responses to anthrax or vac-
cinia (small pox) vaccines, and more so than for IgG-positive
memory B cells from our historical data that averaged 14/VH
gene (Students t test P < 0.05; Zheng et al., 2004, 2005; Koelsch
et al., 2007; Wrammert et al., 2008; Fig. 2 C and Fig. S3 B) or
from 347 IgG memory cell sequences previously published
by another group (averaging 15/VH gene; de Wildt et al.,
2000). Interestingly, for patient EM (outlier in Fig. 2 D) who
had the most severe infection (Table I), mutations had accu-
mulated at a signicantly lower frequency than the IgG con-
trols (Fig. S3 A; P < 0.0001), suggesting a unique circumstance
such as a low-level or lacking primary response. Detailed se-
quence characteristics for pandemic H1N1-induced plasma-
blasts are provided in Tables S1–S3. Though based on a limited
or vaccine response. The ready detection of clonal expansions
at a mean frequency of 16.5% of the cells for the six patients
supports this view (based on CDR3 sequence similarity;
Fig. 2 C). Since the discovery of somatic mutation, it has been
appreciated that mutations progressively accumulate on vari-
able genes after repeated immunizations (McKean et al.,
1984). Thus, we can gain insight into the origin of the pan-
demic H1N1 response by comparing the somatic mutation
frequency of the plasmablasts present during H1N1 infection
to that of other plasmablast responses. The PCR strategy al-
lowed isolation of either IgG or IgA transcripts and identied
68% IgG and 32% IgA plasmablasts from the patients. Similar
to plasmablasts induced by annual vaccination (Wrammert
et al., 2008), or after a fourth booster vaccine to anthrax, the
variable genes of novel H1N1-induced cells from ve of the
six patients harbored high numbers of somatic mutations
Antibodies were tested at 10 µg/ml and threefold serial dilutions until a nonbinding concentration was determined. Each antibody was tested in at least
two (and typically more) replicates for specicity and afnity estimations. Note that only 14 of 15 HA-binding antibodies have curves in G because one of
the HA-reactive antibodies only binds HA on whole virions, not on the recombinant protein.
Figure 2. Plasmablasts induced by
pandemic H1N1 infection are highly
cross-reactive and have accumulated
particularly high levels of variable gene
somatic hypermutation. (A and B) Pandemic
H1N1 reactive mAbs isolated from infected
patients (1000, EM, 70, 1009) were assayed for
binding to annual H1N1 inuenza strain
whole virus. The minimum detectable concen-
tration is dened as two standard deviations
above the mean binding of 48 randomly cho-
sen naive B cell antibodies (Fig. S1 A). Bars are
color coded to approximate levels of cross-
reactivity to the annual vaccine (circulating)
strains of recent years. Panels A and B use the
same color scheme. Each value is representa-
tive of at least two replicate ELISAs repeated
until a single consistent minimum concentra-
tion was established. The center numeral
equals total antibodies. (C) Analysis of the
variable gene sequences from plasmablasts of
the four pandemic H1N1-infected patients
indicated that 16.5% of the pandemic
H1N1-induced plasmablasts were clonally
related (shared identical VH and JH genes and
CDR3 junctions). (D) The average number of
somatic hypermutations in the pandemic
H1N1 patient plasmablast variable region
genes compared with primary IgG plasmablast responses to vaccinia (small pox) or the anthrax vaccine, or after at least 4 boosters with the anthrax vac-
cine. To account for the obvious outlier in the pandemic H1N1 group (patient-EM), median values are indicated by the bar. Student’s t tests excluding the
outlier indicated a p-value of <0.04 for the remaining ve pandemic H1N1 samples compared with the IgG memory and germinal center (GC) cells or the
primary IgG plasmablast responses (0.2 with EM included) and a p-value of <0.0001 against the IgM populations. Notably, besides patient EM, each indi-
vidual set of VH genes averaged signicantly more mutations than the IgG memory and GC or the primary responses (Fig. S3 A). Each point represents
one individual donor and is averaged from 25–75 sequences, except for the primary response to anthrax from which only 10 VH genes could be cloned
from single cells because of the highly limited response. Mutations accumulated per individual sequence are depicted in Fig. S3. Detailed sequence char-
acteristics are provided in Tables S1–S3. The naive, IgG and IgM GC and memory populations are derived from historical data (Zheng et al., 2004, 2005;
Koelsch et al., 2007; Wrammert et al., 2008).
Page 5
186 Human B cell responses to pandemic H1N1 inuenza | Wrammert et al.
indicating that they bound to sites other than the HA active
site. Interestingly, antibodies of the latter type were predomi-
nant in the response (Fig. 3 A). This specicity is reminiscent
of antibodies against the recently discovered broadly neutral-
izing epitopes found on the HA stalk, rather than those lo-
cated on the HA globular head that is more typical for
neutralizing antibodies (Ekiert et al., 2009; Sui et al., 2009).
Importantly, ve of these antibodies are indeed of similar
specicity (including antibodies 70-5B03, 70-1F02, 1000-
3D04, and a clonal pair from donor 1009: 3B05 and 3E06).
These ve antibodies bind with high anity to most H1
strains including all from the vaccines of the past 10 yr, the
1918 pandemic strain, and to the H5 of a highly pathogenic
number of patients, the frequent cross-reactivity and high
number of somatic mutations support a model in which many
of the plasmablasts induced by pandemic H1N1 infection
arose from cross-reacting memory B cells.
A majority of the neutralizing antibodies bound to highly
conserved epitopes in both the HA stalk and head regions
A high frequency of the HA-specic antibodies was able to
neutralize the virus in vitro (totaling 73% or 11/15; Fig. 3 A).
These neutralizing antibodies could be further categorized
into two distinct groups: (a) neutralizing antibodies that
displayed hemagglutination inhibition (HAI) activity (HAI
and (b) neutralizing antibodies that had no HAI activity,
Figure 3. HA-specic antibodies induced by pandemic H1N1 infection bind cross-reactive neutralizing epitopes. (A) In vitro functional analysis
of 15 antibodies from indicated patients that bound pandemic H1N1 inuenza recombinant HA protein. The left panel shows HAI minimum effective
antibody concentration, the middle panel shows PRNT50 plaque reduction neutralization minimum effective antibody concentration, and the right panel
shows ELISA binding summarized as minimum positive concentration (as dened for Fig. 2) against recombinant HA (original curves are in Fig. 1 F and
Fig. S2 A). The antibodies are grouped based on whether they show HAI and/or neutralizing (neut) function. Antibody 1009-3B06 was only tested for
binding to whole virus, as this antibody did not bind to rHA due to binding of a quaternary or conformationally sensitive epitope that is not present in the
recombinant protein. HAI and neutralization assays were performed in duplicate and repeated at least three times. ELISA curves are provided in Fig. S2 A.
(B) ELISA binding as shown by minimum positive concentration (dened for Fig. 2) of neutralizing mAbs to rHA or whole virions from pandemic H1N1 or
other inuenza strains (ELISA binding curves are provided in Fig. S2 A). Three binding patterns (epitopes 1 and 2, and 3) were observed that coincided
with specicity comparisons by competitive ELISA, as illustrated in Fig. 4 A. (C) Three representative neutralizing antibodies (EM-4C04, 70-1F02, and
1009-3B06) were used for HAI and microneutralization (MN) activity against pandemic H1N1 and several other annual or laboratory H1N1 inuenza
strains. Experiments were performed in duplicates and repeated at least three times. Minimum effective concentration is shown for both assays.
Page 6
JEM VOL. 208, January 17, 2011
Ar ticle
FACS analysis showed that the ve antibodies bound to all 13
H5 variants tested at levels quite similar to F10, for which a crys-
tal structure had been generated to dene this epitope. Thus,
half of the neutralizing and a surprising 10% of all antibodies
induced by pandemic H1N1 infection bound to a conserved,
critical epitope on the HA stalk. By comparison, none of
50 H1N1 strain-specic antibodies that we had previously
isolated after annual vaccination before the 2009 pandemic
had this reactivity (unpublished data). The frequency of
pandemic-induced, stem-reactive antibodies (5/46) versus those
from annual vaccine (0/50) is signicantly greater (Chi-square
test, P = 0.02). Further, this specicity is only rarely seen in
human memory B cells (Corti et al., 2010) or from phage-
display libraries (Sui et al., 2009). These observations support
avian inuenza strain (Fig. 3 B and Fig. S2 A). In addition,
these ve antibodies cross-compete for a similar epitope that
was not over-lapping with the HAI
antibodies (Fig. 4 A,
epitope-1). These antibodies are competitively inhibited by a
commercial antibody referred to as C179 that binds this HA
stalk region (Okuno et al., 1993), and four of ve of these
antibodies are encoded by the hallmark VH1-69 gene (Ekiert
et al., 2009; Sui et al., 2009). To verify HA stalk reactivity,
these ve antibodies were tested for binding to H5 variants
predicted to aect the stalk epitope by the crystal structure,
and their binding patterns were compared with that of the
prototypical stalk antibody (mAb F10; Sui et al., 2009;
Fig. 4 B). Each H5 variant has a single residue mutation in
the stalk region and was transiently expressed on 293T cells.
Figure 4. The neutralizing antibodies bind to three nonoverlapping epitopes in either the stalk or the globular head of the HA molecule.
(A) Competition ELISA assays were used to determine the similarity in specicity between the various neutralizing antibodies. Shown is the percentage of
competition of each antibody in an ELISA binding assay against all other neutralizing antibodies. A 10-fold molar excess of unlabeled antibody was used
to inhibit a biotinylated antibody. Percent competition is calculated as the reduction in absorbance relative to the level of inhibition of any particular anti-
body against itself. Colors indicate degree of inhibition of antibody binding, as indicated. Antibody C179 is a commercial antibody that binds to the stalk
region of the HA molecule identifying epitope-1. Epitope-2 and -3 are each on the HA-head active site. 1000-2G06 and the nonneutralizing, but HA-
binding, antibodies had no competition with any of the other HA-reactive antibodies and are therefore not shown. VH gene usage of the individual anti-
bodies is listed on the right. All assays were performed in duplicate. (B) Plasmids encoding full-length WT H5-TH04 (A/Thailand/2-SP-33/2004 [H5N1]) and
its mutants were transiently transfected into 293T cells. 24 h after transfection, cells were harvested for FACS analysis, and binding of indicated antibodies
was tested at 10 µg/ml. The cell surface HA expression of each of the mutants were veried with a ferret anti-H5N1 serum (not depicted). Antibody F10
was one of the antibodies used to characterize the HA stalk epitope by x-ray crystallography (Sui et al., 2009) and served as a positive control for the
binding pattern expected of HA stalk–reactive antibodies to these HA mutants.
Page 7
188 Human B cell responses to pandemic H1N1 inuenza | Wrammert et al.
to all recent H1 vaccine strains and reacted strongly to the
1918 pandemic strain (antibodies 1009-3E04 and 1000-3E01;
Fig. 3 B and Fig. 4 A, epitope-3). These mAbs bind to past
vaccine strains with higher avidity than to the pandemic H1N1.
Further studies are underway to precisely identify the epitopes
of all neutralizing antibodies in this study.
Only two of 11 neutralizing antibodies were highly specic
for the pandemic H1N1 strain alone (Fig. 3 B and Fig. S2 A), in-
cluding a low avidity antibody, 1000-2G06, which only showed
slight neutralization capacity in vitro, and EM-4C04, which
was very eective at neutralizing the pandemic H1N1 inu-
enza. We conclude from these experiments that a surprising
82% (9/11) of the neutralizing plasmablasts that we isolated
during pandemic H1N1 inuenza infections were broadly
cross-reactive to multiple inuenza strains.
Potent in vivo protection and rescue of mice challenged
with a lethal dose of pandemic H1N1 or antigenically
distinct inuenza virus strains
There is a distinct interest in developing monoclonal anti-
bodies for use in a therapeutic setting. We selected three rep-
resentative antibodies of the set we have identied for detailed
functional analysis both in vitro (Fig. 3 C) and in vivo (Fig. 5
and Fig. 6), including: EM-4C04, 1009-3B06, and 70-1F02.
The antibodies EM-4C04 and 1009-3B06 are specic for the
active site of the HA molecule, whereas 70-1F02 binds to the
stalk region. Furthermore, EM-4C04 is highly specic for
pandemic H1N1, whereas 1009-3B06 and 70-F02 display
the idea that a vaccine might be developed that preferentially
targets the HA stalk, thus providing broad protection against
many inuenza strains.
The remaining neutralizing antibodies were HAI
therefore bound to the HA globular head. Based on cross-
competition analyses, these antibodies fell into two groups
binding nonoverlapping regions of the HA head, including
epitope-2 and epitope-3 (Fig. 3 B and Fig. 4 A). Indeed, using
spontaneous escape mutant selection, we found that the
EM4C04 mAb binds to the Sa region of the HA globular
head (unpublished data). Thus, by proximity based on the
competition assay (Fig. 4 A), we can predict that all of the
epitope-2 antibodies bind near the Sa/Sb region (including
EM-4C04, 1009-3B06, and 1009-3F01).
Broadly reactive antibodies binding both pandemic H1N1
strains and common annual H1N1 strains have been identi-
ed both in humans (Krause et al. 2010; Xu et al., 2010) and
in mice (Manicassamy et al., 2010). It is notable that three of
ve of the HA globular-head–binding antibodies induced by
pandemic H1N1 infection were also broadly reactive to vari-
ous H1N1 strains (Fig. 3 B). One such novel antibody was the
SF1009-3B06 antibody that reacts strongly with the pan-
demic H1N1 strain, as well as all recent H1N1 vaccine strains
(Fig. 3 B and Fig. S2 A). The precise epitope to which the
1009-3B06 antibody binds appears to be quite unique; it is
only accessible on whole virions, not on recombinant HA,
suggesting that the epitope is quaternary in nature. Finally,
two antibodies cross-reacted and inhibited hemagglutination
Figure 5. In vivo prophylactic and thera-
peutic efcacy of human mAbs against
pandemic H1N1 inuenza virus. 6–8-wk-
old BALB/c mice were infected with a 3xLD50
dose of highly pathogenic, mouse-adapted
2009 pandemic H1N1 inuenza (A/California/
04/09). 24, 48, and 60 h after infection,
200 µg (10 mg/kg of body weight) of EM-
4C04, 70-F02, or 1009-3B06 human mAb
were injected intraperitoneally. All mice were
monitored daily for body weight changes and
any signs of morbidity and mortality. Percent-
age of initial body weight is plotted, and the
number of surviving mice is shown in the
lower right of each plot. Infected, untreated
mice showed clear signs of sickness around
day 4–5 after infection and perished by day
8–9. Prophylactic treatment is shown on the
left for comparison. Antibody treatment con-
ferred signicant protection as determined by
comparison of weights in untreated versus
prophylaxis and at the time of treatment
versus 12 d after infection (unpaired, two-
tailed Student’s t test, P < 0.05). The log-rank
test indicated signicant survival as well
(P < 0.001). Figure shows one representative
experiments of at least three independent
repeat experiments.
Page 8
JEM VOL. 208, January 17, 2011
Ar ticle
for the pandemic H1N1, had no protective eect on infection
with PR/8/34 or FM/1/47. In conclusion, the antibodies
characterized herein show promise for development as broadly
reactive therapeutic agents against the pandemic H1N1 inu-
enza virus, as well as against the majority of H1N1 and H5N1
inuenza strains.
Our ndings provide insight into the human B cell responses
to a pandemic inuenza virus strain. The unique genetic
composition of the pandemic H1N1 inuenza virus meant
that our relatively young cohort probably had little or no pre-
existing specic antibody-mediated immunity to this virus
before infection (Brockwell-Staats et al., 2009; Dawood et al.,
2009; Garten et al., 2009; Hancock et al., 2009). Thus, two
sources of B cells could have contributed to this response:
newly recruited naive B cells and preexisting memory B cells
that bound to epitopes conserved between past seasonal
strains and the pandemic H1N1 strain. We theorize that pre-
dominant activation of the latter, preexisting memory cells
can account for the observed high frequency of neutralizing
antibodies (11/15 HA-binding antibodies), the majority
(9/11) of which are cross-reactive with seasonal H1N1 strains
(Fig. 3 C) and other group 1 inuenza strains, including H5 HA.
Several observations support this conjecture.
Most convincingly, there was a particularly high frequency
of cross-reactive antibodies overall, with a high level of so-
matic mutations found particularly among the variable genes
of cross-reacting cells (Fig. 2 and Fig. S3). In fact, by ELISA
most antibodies were cross-reactive and one third of the
broadly cross-reactive binding (Fig. 3 B) and have functional
activity against multiple recent and older H1N1 strains (Fig. 3 C).
These antibodies were all highly eective at providing pro-
phylactic protection against infection with a lethal dose of
mouse-adapted pandemic H1N1 in 6–8-wk-old BALB/c mice
(Fig. 5). Moreover, all three antibodies were eective thera-
peutically, even when they were administered as late as 60 h
after the lethal challenge infection, well after the mice were
symptomatic. For EM-4C04, we have successfully treated mice
as far out as 72 h post-infection (unpublished data). Infected
mice were already showing measurable weight loss that was
reversed by administration of the antibody, demonstrating
therapeutic potential even after the onset of disease. Viral
clearance was analyzed in mice treated at 48 h after infection
with EM4C04 (Fig. S4). As early as day 4, the antibody-treated
mice exhibited more than a log reduction in viral titers; titers
continued to decline, such that by day 6, virus was undetect-
able or present at very low levels. The untreated mice per-
ished by day 7 or 8, whereas the treated mice cleared the
infection with no detectable virus on day 12. Finally, 1009-
3B06 and 70-1F02, which showed activity against several
current and older H1N1 seasonal inuenza strains in vitro
(Fig. 3 C), were also tested in vivo against antigenically dis-
tinct influenza strains. For these experiments, mice were
treated with 200 µg of mAb intraperitoneally 12 h before in-
fection with a lethal dose of either pandemic H1N1 inuenza
or either of the two common influenza laboratory strains
PR/8/34 or FM/1/47. 1009-3B06 and 70-1F02 showed
protection against these antigenically distinct H1N1 inuenza
strains, as illustrated in Fig. 5. EM-4C04, which is highly specic
Figure 6. Breadth of in vivo prophylactic
efcacy in mice. 6–8-wk-old BALB/c mice
were treated with 200 µg (10 mg/kg of body
weight) EM-4C04, 70-1F02, or 1009-3B06
human mAb intraperitoneally. Control mice
were treated with PBS only, a control mAb or
polyclonal human IgG. 12 h later, they were
challenged with a 3xLD50 dose of mouse
adapted pandemic H1N1, PR/8/34, or FM/1/47
inuenza virus. All mice were monitored daily
for body weight changes and any signs of
morbidity and mortality. Percentage of initial
body weight (left) and survival curves (right)
are plotted. Infected, untreated mice showed
clear signs of sickness 4–5 d after infection
and perished by day 8–9. Figure shows one
representative experiments of at least three
independent repeat experiments. Antibody
treatment conferred signicant protection as
determined by comparison of weights in un-
treated versus prophylaxis, and at the time of
treatment versus 12 d after infection (un-
paired, two-tailed Student’s t test, P < 0.05).
The log-rank test indicated signicant survival
as well (P < 0.003).
Page 9
190 Human B cell responses to pandemic H1N1 inuenza | Wrammert et al.
seroconversion, or by the presence of highly potent antibodies,
such as EM-4C04, whose activities were less likely to titer
out. The highly specic nature of the response from this pa-
tient may have contributed to this advantage, ultimately better
targeting the epitopes of the pandemic H1N1 strain. In con-
trast, patient 1009 had relatively low HAI and MN serum titers
but the highest frequency of broadly neutralizing antibodies
and a less severe disease course. One possibility is that our
sampling from this patient was done before peak serological
responses. Another possibility is that the high frequency of
these potent antibodies in the memory B cell compartment
may have resulted in rapid resolution of infection, precluding
the development of a high serological response. A third possi-
bility is that despite broader protection, the stalk-reactive anti-
bodies are on the whole less potent and more rapidly titrated
out than the highly specic antibodies to the HA globular
head. These various possibilities will be of signicant interest
to study in the future.
Finally, we report the development of a large panel of
human mAbs induced by pandemic H1N1 infection. Pro-
phylactic therapy with polyclonal or mAbs has successfully
been used for RSV, rabies, Hepatitis A and B, and varicella.
In the case of inuenza, mAbs have been shown to provide
prophylactic or therapeutic protection in mice and other ani-
mal models (Reuman et al., 1983; Sweet et al., 1987; Palladino
et al., 1995; Renegar et al., 2004). Passive transfer of maternal
antibodies in humans has also been shown to confer protec-
tion (Puck et al., 1980). Several of the antibodies we isolated
have broad neutralization capacity in vitro against divergent
inuenza strains and show potent prophylactic and therapeu-
tic activity when used to treat mice that were lethally infected
with inuenza. These antibodies could provide much needed
pandemic therapeutics to treat severe cases of inuenza and to
protect high-risk populations.
In conclusion, analyses of the 46 mAbs induced by pan-
demic H1N1 infection indicated frequent activation of broadly
reactive B cells. We propose that these cells had a memory cell
origin caused by cross-reactivity to conserved and function-
ally important epitopes. If true, it will be important to char-
acterize the ecacy of the pandemic H1N1 vaccine to induce
a similarly cross-protective response.
Patients. All studies were approved by the Emory University, University of
Chicago, and Columbia University institutional review boards (Emory
IRB#22371 and 555–2000, U of C IRB# 16851E, CU IRB#AAAE1819).
Patient clinical information is detailed in Table I.
PBMC and plasma isolation. All work with samples from infected
patients was performed in a designated BSL2
facility at Emory University.
Peripheral blood mononuclear cells (PBMCs) were isolated using Vacutainer
tubes (BD), washed, and resuspended in PBS with 2% FCS for immediate use
or frozen for subsequent analysis. Plasma samples were saved at 80°C or
frozen in medium with 10% dimethyl sulfoxide for subsequent analysis.
Viruses and antigens. The pandemic H1N1 inuenza virus (A/California/
04/2009) was provided by R.J. Webby (St. Jude Childrens Hospital, Memphis,
TN). Inuenza virus stocks used for the assays were freshly grown in eggs,
antibodies bound to past annual viral antigens at lower con-
centrations, suggesting higher avidity to past inuenza strains
than to the current pandemic H1N1 virus. Further, cross-
reacting cells that bind with higher anity to the pandemic
H1N1 strain also have the highest frequency of variable-gene
mutations (Fig. S3 B). Antibodies that were broadly cross-
reactive were among the more highly mutated clones (Fig. S3 B).
We propose that many of these cells were specic for cross-
reactive epitopes present in annual inuenza strains that then
underwent further anity maturation and adaptation to the
infecting pandemic H1N1 virus. Supporting this conjecture,
Corti et al. (2010) rst demonstrated that naturally occurring
HA stalk–reactive memory B cells could be isolated from the
blood of people recently immunized with the annual vaccine,
before the outbreak of pandemic H1N1. The nature of that
study was to screen EBV-transformed memory cell lines, thus
precluding the determination of precise frequencies of these
stalk-reactive B cells. However, these antibodies were esti-
mated to be quite rare; occurring at one in thousands to one
in hundreds of inuenza-binding B cells, varying by individ-
ual. In contrast, we show that plasmablasts activated by infec-
tion with the highly novel pandemic H1N1 inuenza strain
have substantially increased targeting to the HA stalk region
epitopes, totaling 10% of all inuenza-specic antibodies and
half of the neutralizing antibodies (Fig. 4). In fact, most spe-
cic antibodies isolated in this study were cross-reactive to
past inuenza strains. Collectively, the data described supports
a model in which divergent viruses that are conserved only at
the most critical regions for function will elicit a higher pro-
portion of cross-reactive and neutralizing antibodies. Thus,
although the activated plasmablasts of relatively few patients
could be analyzed in detail at the monoclonal antibody level,
we proer that with the proper immunogen, the long-sought
development of a pan-inuenza vaccine might be possible.
Interestingly, the highly specic antibody EM-4C04 was
derived from a patient that had a very severe disease course,
with persistent viral shedding over several weeks. In addition,
the variable genes from the plasmablasts of this patient had the
lowest average number of somatic mutations (Fig. 2 B, outlier,
and Fig. S3 B). Collectively, the unique specicity against
pandemic H1N1, the low levels of somatic mutation, and the
unusually severe disease in the absence of predisposing condi-
tions suggest that this patient may have mounted a primary
immune response to the pandemic H1N1 inuenza infection.
The complete lack of preexisting immunity may have con-
tributed to the more severe disease observed in this patient.
In contrast, the activation of broadly cross-neutralizing mem-
ory B cells in those with immune experience to annual strains
might have contributed to the less severe disease of most in-
fected patients during the pandemic.
It is notable that there is a discrepancy between patients
for serum MN titers, the severity of disease, and the frequency
of plasmablasts expressing neutralizing antibodies (Table I and
Fig. 3). For example, patient EM, despite having the worst
disease course, had the greatest HAI and MN serum titers. This
may be caused by the time from infection (day 31), allowing full
Page 10
JEM VOL. 208, January 17, 2011
Ar ticle
conditions that were previously published (Wrammert et al., 2008; Smith
et al., 2009). Variable genes were determined using in-house analysis software
compared with the Immunogentics V gene dataset and the IMGT search
engine (Ehrenmann et al., 2010; Lefranc et al., 2009). Background mutation
rates by this method is 1 base-exchange per 1,000 bases sequenced (based
on sequences of constant region gene segments). Comparisons were made to
historical data, some of which was previously published (Zheng et al., 2005;
Wrammert et al., 2008; Duty et al., 2009).
Plaque assay and PRNT
assay. MDCK cells were grown in 6-well
plates at a density of 8 × 10
/well. On the next day, cells were washed with
PBS. 10-fold dilutions of virus were added in 500 µl DME and incubated at
37°C for 1 h, with mixing every 10 min. Cells were washed with PBS and
overlayed with 199 media containing 0.5% agarose (Seakem), 1x antibiotics
(100 U/ml penicillin and 100 mg/ml streptomycin), 0.2% BSA (Sigma-
Aldrich), and 0.5 µg/ml TPCK-Trypsin (Sigma-Aldrich). Cells were incu-
bated for 36–40 h and xed with 2% PFA for 10 min. Agarose plugs were
removed and cells were stained with 0.1% crystal violet in 25% EtOH for
1 min. After removal from the crystal violet solution, plates were dried and
used to count plaques in each well. For PRNT
assay, MDCK cells were pre-
pared as above. On the next day, mAbs were threefold-diluted (60–0.74 µg/ml).
100 PFU of virus in 250 µl DME were incubated with equal volume of di-
luted mAbs at 37°C for 1 h before the plaque assay. Plaques were counted and
the nal concentration of antibodies that reduced plaques to <50 PFU were
scored as PRNT
Determination of 50% tissue culture infectious dose (TCID50)
and MN. To determine the TCID
, MDCK cells were grown in 96-well
plate at a density of 1.5 × 10
/well. On the next day, cells were washed with
PBS and 10-fold diluted viruses in 100 µl DME were added into each well
and incubated at 37°C for 1 h. After the incubation, cells were washed
with PBS and 100 µl of DME containing 1x antibiotics (100 U/ml peni-
cillin and 100 mg/ml streptomycin), 0.5% BSA (Sigma-Aldrich), and
0.5 µg/ml TPCK-Trypsin (Sigma-Aldrich) was added. Cells were further
incubated for 60 h, and 50 µl of the supernatant was incubated with equal
volume of 0.5% of PBS-washed Turkey red blood cells (Rockland Immu-
nochemicals) for 30 min. Four replicates were performed for each dilu-
tion, and complete agglutination was scored as HA
. Virus titers were
calculated by the Reed-Muench method. For MN assay, 100 TCID
virus in 50 µl DME were incubated with 50 µl of threefold-diluted anti-
bodies (60–0.082 µg/ml) at 37°C for 1 h. Cells were washed and incu-
bated in the media as described for the HAI assay for 60 h. The MN titer
was determined to be the nal concentration of mAbs that completely in-
hibited infection.
HAI and ELISA assays. Whole virus, recombinant HA, or vaccine-specic
ELISA was performed on starting concentrations of 10 µg/ml of virus or re-
combinant HA and on 1:20 dilution of the vaccine, as previously described
(Wrammert et al., 2008). In brief, microtiter plates were coated with live
virus strains totaling 8 HAU of total virus per well or with 1 µg/ml of recom-
binant HA protein. To standardize the various ELISA assays, common high-
anity antibodies with similar anities and binding characteristics against
each virus strain were included on each plate, and the plate developed when
the absorbance of these controls reached 3.0 ± 0.1 OD units. Goat anti–human
IgG (goat anti–human I-peroxidase-conjugate; Jackson ImmunoResearch
Laboratories) was used to detect binding of the recombinant antibodies, fol-
lowed by development with horseradish peroxidase substrate (Bio-Rad labo-
ratories). Absorbencies were measured at OD415 on a microplate reader
(Invitrogen). Anity estimates were calculated by nonlinear regression analy-
sis of curves from eight dilutions of antibody (10 to 0.125 ug/ml) using
GraphPad Prism. The HAI titers were determined as previously described
(Wrammert et al., 2008). In brief, the samples were then serially diluted with
PBS in 96- well v-bottom plates and 8 HAU (as determined by incubation
with 0.5% turkey RBCs in the absence of serum) of live, egg-grown virus was
added to the well. After 30 min at room temperature, 50 µl of 0.5% turkey RBCs
prepared, and puried as previously described (Wrammert et al., 2008).
The hemagglutination inhibition activity was determined using turkey
red blood cells (Lampire Biological Laboratories) as previously described
(Wrammert et al., 2008) or purchased as inactivated preparations (ProSpec-
Tany TechnoGene Ltd.) which included the following: A/California/04/09
(H1N1), A/FM/1/47 (H1N1), A/PR8/34 (H1N1), A/New Caledonia/20/99
(H1N1), A/Solomon Island/3/06, A/Brisbane/59/07 (H1N1), and
A/Brisbane/10/07 (H3N2). Vaccines tested included the 2006/7 vaccine
from Chiron Vaccines Limited and the 2008/9 formulation from Sano Pas-
teur Inc. Recombinant HA proteins were provided by the inuenza reagent
resource (www.inuenzareagentresource.org) of the CDC (recombinant HA
from A/California/04/2009 [H1N1; #FR-180], A/Brisbane/10/2007
[H1N1; #FR-61], A/Brisbane/59/2007 [H3N2; #FR-65]) or by Biodefense
and Emerging Infections research repository (www.beiresources.org; recom-
binant HA from A/Indonesia/05/2005 [H5N1]). A/Brevig Mission/1/1918
(H1N1) was purchased from Sino Biological.
ELISPOT assay. Direct ELISPOT to enumerate the number of either total
IgG-secreting, pandemic H1N1 inuenza–specic, or vaccine-specic plasma-
blasts present in the PBMC samples were essentially done as previously
described (Crotty et al., 2003). In brief, 96-well ELISPOT lter plates (Milli-
pore) were coated overnight with either the optimized amounts of puried
pandemic H1N1 virions, recombinant HA from the pandemic H1N1 (as
above), the 08/09 inuenza vaccine at a dilution of 1/20 in PBS, or goat
anti–human Ig (Invitrogen). Plates were washed and blocked by incubation
with RPMI containing 10% FCS at 37°C for 2 h. Puried and extensively
washed PBMCs or sorted plasmablasts were added to the plates in dilution
series and incubated for 6 h. Plates were washed with PBS, followed by PBS
containing 0.05% Tween, and then incubated with a biotinylated anti-huIgG ()
antibody (Invitrogen) and incubated for 1.5 h at room temperature. After
washing, the plates were incubated with an avidin-D-HRP conjugate (Vector
Laboratories) and, nally, developed using AEC substrate (3 amino-9 ethyl-
carbazole; Sigma-Aldrich). Developed plates were scanned and analyzed
using an automated ELISPOT counter (Cellular Technologies, Ltd.).
Flow cytometry analysis and cell sorting. Analytical ow cytometry
analysis was performed on whole blood after lysis of erythrocytes and xing
in 2% PFA. All live cell sorting and single cell sorting was performed on puri-
ed PBMCs using either a FACSVantage or ARIAII cell sorter system. All of
the following antibodies for both analytical and cell sorting cytometry were
purchased from BD, except anti-CD27, which was purchased from eBioscience:
anti–CD3-PECy7 or PerCP, anti–CD20-PECy7 or PerCP, anti–CD38-PE,
anti–CD27-APC, and anti–CD19-FITC. ASCs were gated and isolated as
cells. Flow cytometry data were
analyzed using FlowJo software.
Generation of mAbs. Identication of antibody variable region genes
were done essentially as previously described (Smith et al., 2009; Wardemann
et al., 2003; Wrammert et al., 2008). In brief, single ASCs were sorted into
96-well PCR plates containing RNase inhibitor (Promega). VH and V
genes from each cell were amplied by RT-PCR and nested PCR reactions
using cocktails of primers specic for both IgG and IgA using primer sets
detailed in (Smith et al., 2009) and then sequenced. To generate recombinant
antibodies, restriction sites were incorporated by PCR with primers to the par-
ticular variable and junctional genes. VH or V genes amplied from each single
cell were cloned into IgG1 or Ig expression vectors, as previously described
(Wardemann et al., 2003; Wrammert et al., 2008; Smith et al., 2009). Antibody
sequences are deposited on GenBank (accession nos. HQ689701-HQ689792
available from GenBank/EMBL/DDBJ). Heavy/light chain plasmids were
cotransfected into the 293A cell line for expression and antibodies puried
with protein a sepharose. Antibody proteins generated in this study can be
provided in limited quantities upon request.
Mutational analysis. Antibody anti-H1N1 induced plasmablast variable
genes were amplied by single-cell RT-PCR using primer sets and PCR
Page 11
192 Human B cell responses to pandemic H1N1 inuenza | Wrammert et al.
ecacy of the mAb, mice were treated intraperitoneally with 200 µg (10 mg/kg
of body weight) of the specic mAbs. 12 h later, mice were challenged
with 3xLD50 of one of the mouse adapted inuenza viruses used in the
study. All mice were monitored daily for any signs of morbidity and mor-
tality. Body weight changes were registered daily for a period of 14 d. All
mice that lost >25% of their initial body weight were sacriced according
to the institutional animal care and use committee guidelines. To determine
the therapeutic ecacy of the mAbs, mice were challenged with 3xLD50
of the mouse-adapted pandemic H1N1 virus. At various times after infec-
tion (12, 24, 36, 48, 60, and 72 h) mice were treated intraperitoneally with
200 µg (10 mg/kg of body weight) of the specic mAbs. All mice were
monitored daily and the body weight changes were registered daily as de-
scribed above.
Statistical analysis. Data were collected and graphed using MS Excel and
GraphPad Prism software. Ecacy of the therapeutic and challenge experi-
ments was evaluated by analysis of variance using GraphPad Prism software.
Online supplemental material. Fig. S1 shows the binding characteris-
tics of control mAbs. Fig. S2 shows further binding characteristics of the
neutralizing mAbs. Fig. S3 shows further analysis of pandemic H1N1-induced
plasmablast somatic mutations. Fig. S4 shows experiments demonstrating
the therapeutic control of pandemic H1N1 viral titers in lungs after mAb
treatment. Tables S1–S3 provide detailed characteristics concerning the
variable gene sequences cloned from pandemic H1N1 induced plasmablasts.
Online supplemental material is available at http://www.jem.org/cgi/
We thank Drs. Richard J. Webby and Gillian Air for providing us with viral isolates.
We also thank Robert Karaffa and Sommer Durham for providing essential
support with cell sorting and Dr. Ruben Donis (CDC) for his generous gift of ferret
anti-H5 serum.
This work was funded in parts by National Institutes of Health (NIH)/National
Institute of Allergy and Infectious Diseases (NIAID) U19-AI057266 with ARRA
supplement funding U19 AI057266-06S2 (R. Ahmed and P.C. Wilson), by NIH/NIAID
HHSN266200700006C Center of Excellence for Inuenza Research and Surveillance
(R. Ahmed, R. Compans, and P.C. Wilson), by the Northeast Biodefense Center U54-
AI057158-Lipkin (R. Ahmed, W.I. Lipkin, and P.C. Wilson), by NIH/NIAID
HHSN266200500026C (P.C. Wilson), by NIH/NIAID 5U19AI062629-05 (P.C. Wilson),
and by NIH/NIAID U01 AI074518 (W.A. Marasco) and the National Foundation for
Cancer Research (W.A.M.). J. Wrammert was supported by a training fellowship
through the Center of Excellence for Inuenza Research and Surveillance and
D. Koutsonanos by U01-AI074579 (R. Compans). This research was supported in part
by the Intramural Research Program of the NIAID.
The authors declare no nancial or commercial conicts of interest.
Submitted: 6 July 2010
Accepted: 13 December 2010
Beigel, J.H. 2008. Inuenza. Crit. Care Med. 36:2660–2666. doi:10.1097/
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(Rockland Immunochemicals) suspended in PBS with 0.5% BSA was added to
each well and the plates were shaken manually. After an additional 30 min at
room temperature, the serum titers or minimum eective concentrations were
read based on the nal dilution for which a button was observed.
Competition ELISA. Competition ELISA was performed by inhibiting
binding of each biotinylated antibody (NHS-coupled; Thermo Fisher Scien-
tic) at the half-maximal binding concentration with a 10-fold molar excess
of puried antibody. All comparisons of dierent antibodies were based on
percentage of absorbance values for each antibody against itself (which was
scored as 100% inhibition). Detection was done using streptavidin-HRP as
described for the ELISA assay.
FACS analysis of binding of anti-HA antibodies with H5 and its
mutants. The full-length HA gene (H5-TH04) of A/Thailand/2(SP-33)/2004
(H5N1) were codon-optimized for eukaryotic cell expression and cloned
into pcDNA3.1 vector to obtain the pcDNA3.1-H5-TH04 construct (Sui
et al., 2009). All mutants of H5-TH04 were derived from pcDNA3.
1-H5-TH04 and constructed by the QuikChange method (Stratagene). The
full-length wild type H5-TH04 and mutants expressing plasmids were trans-
fected transiently into 293T cells with Lipofectamine 2000 (Invitrogen). 24 h
after transfection, cells were harvested for immunostaining. Anti-HA anti-
bodies, a control human mAb 80R (Sui et al., 2004) at 10 µg/ml, or ferret
anti-H5N1 serum at 1:300 dilution were incubated with transfected
293T cells at 4°C for 1 h. Cells were then washed three times with PBS con-
taining 0.5% BSA and 0.02% NaN
. FITC-labeled goat anti–human IgG
(Thermo Fisher Scientic) or FITC-labeled goat anti–ferret IgG (Bethyl)
were then added to cells and incubated for 30 min at 4°C. Cells were washed
as above, and binding of antibodies to cells was analyzed using a BD FACS-
Calibur with CellQuest software.
BIACORE analysis. The kinetic interactions of the mAbs with recombi-
nant A/Cal/04/09 (H1N1) HA protein were determined by surface plasmon
resonance (SPR) using a BIAcore3000 instrument. EM4CO4 and SF1009-
3FO1 antibodies were immobilized at 10 µl/min
on a CM5 sensor chip by
amine coupling and recombinant HA at concentrations ranging from 0.5 to
15 nM in HBS-EP buer were injected at 20 µl/min
over the immobilized
antibodies or reference cell surface. Running buer (HBS-EP) was then ap-
plied for 600 s, after which the sensor surface was regenerated by a single
injection of 25 mM NaOH at 100 µl/min
. For the other experiments,
recombinant HA (His-tagged) was immobilized at 5 µl/min
on NTA sen-
sor chips with a target density of 350 response units, and the antibodies at
concentrations ranging from 1 to 30 nM in HBS-P buer were injected at
20 µl/min
over the immobilized recombinant HA or reference cell surface,
followed by a 600s dissociation phase. All experiments were performed in
triplicates. For kinetic analysis, injections over reference cell surface and in-
jections with buer were subtracted from the data. Association rates (k
), dis-
sociation rates (k
) and equilibrium dissociation constants (K
) were calculated
by aligning the curves to t a 1:1 binding model using BIAevaluation 4.1
software. Antibodies 1009-3B06, 1000-3E01, and 1000-2G06 could not be
determined because these mAbs did not bind to the recombinant HA pro-
tein from baculovirus suciently well for SPR. Avidities for these mAbs and
for the antibodies that did not neutralize infection in vitro were estimated by
Scatchard plot analyses of ELISA data (shown in parentheses).
In vivo protection experiments. 6–8-wk-old female BALB/c mice were
used for the challenge studies. Mice were inoculated intranasally with
3xLD50 of a highly pathogenic, mouse-adapted pandemic H1N1 inuenza
virus (A/California/04/09), or PR/8/34 or FM/1/47 influenza virus.
The mouse adapted pandemic H1N1 virus had been serially passaged in
mice for ve generations before use herein. The LD50 for all the viruses was
determined by in vivo infection at various virus concentrations, according to
the method of Reed and Muench. The experiments were conducted in accor-
dance with ethical procedures and policies approved by the Emory University’s
Institutional Animal Care and Use Committee. To determine the prophylactic
Page 12
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Page 13
  • Source
    • "Recent studies aiming to characterize conserved epitopes of influenza virus revealed that specific regions in the HA stalk domain are highly preserved both in the structure and the sequence among various subtypes of viruses. It has been discovered that neutralizing antibodies to the HA stalk domain can be found after influenza infection (Wrammert et al., 2011) and vaccination (Corti et al., 2010 ). These anti-stalk antibodies demonstrate cross-reactivity between HAs of many strains from group 1 as well as from group 2 and it has been shown * e-mail: g.beata@biotech.ug.gda.pl "
    [Show abstract] [Hide abstract] ABSTRACT: Influenza A virus infections are the major public health concern and cause significant morbidity and mortality each year worldwide. Vaccination is the main strategy of influenza epidemic prevention. However, seasonal vaccines induce strain-specific immunity and must be reformulated annually based on prediction of the strains that will circulate in the next season. Thus, it is essential to develop vaccines that would induce broad and persistent immunity to influenza viruses. Hemagglutinin is the major surface antigen of the influenza virus. Recent studies revealed the importance of HA stalk-specific antibodies in neutralization of different influenza virus strains. Therefore, it is important to design an immunogen that would focus the immune response on the HA stalk domain in order to elicit neutralizing antibodies. In the present study, we report characterization of a conserved truncated protein, potentially a universal influenza virus antigen from the H5N1 Highly Pathogenic Avian Influenza A virus strain. Our results indicate that exposure of the HA stalk domain containing conserved epitopes results in cross reactivity with different antibodies (against group 1 and 2 HAs). Additionally, we conclude that HA stalk domain contains not only conformational epitopes recognized by universal FI6 antibody, but also linear epitopes recognized by other antibodies.
    Full-text · Article · Sep 2014 · Acta biochimica Polonica
  • Source
    • "Most adults will have been infected or vaccinated with influenza multiple times, and secondary responses are therefore the most commonly examined. During natural infection with influenza A(H1N1)pdm09, virus-specific plasmablasts are readily detected at a frequency of approximately 1,000 IgG-secreting cells per million PBMCs using B cell ELISpot assay at 7–10 days after symptom onset (Wrammert et al. 2011; Lee et al. 2011). The kinetics of these responses have been studied in more detail in the experimental human challenge model, where infection with seasonal influenza H1N1 A/Brisbane/59/07 led to an expansion of ASCs peaking at an average of 166 IgG+ ASCs per million PBMCs around 7 days postinoculation , correlating with viral shedding and symptoms (Huang et al. 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: Although vaccinesVaccines against influenza are widely available, control of the disease remains elusive. In part, this is due to the inability of current vaccines to induce durable, broadly protective immune responses. Prevention of influenza depends primarily on effective antibodyAntibody responses that block virus entry. Following infection, high-affinity IgAAntibody IgA antibodies are generated in the respiratory tract that lead to immune exclusion, while IgGAntibody IgG prevents systemic spread. These are effective and long-lasting but also exert immune pressure. Mutation of the antigenic determinants of influenza therefore rapidly leads to emergence of novel variants that evade previously generated protective responses. Not only do vaccines suffer from this strain-specific limitation, but also they are suboptimal in their ability to induce durable immunity. However, recent evidence has demonstrated the possibility of inducing broadly cross-reactive antibodyAntibody broadly cross-reactive antibody responses. Further understanding of the ways in which high-titer, long-lived antibody responses directed against such cross-reactive epitopes can be induced would lead to the development of novel vaccines that may remove the requirement for recurrent vaccination.
    Full-text · Article · Sep 2014 · Current topics in microbiology and immunology
  • Source
    • "As their epitopes are highly conserved among various influenza virus subtypes, these stem-binding antibodies have heterosubtypic neutralizing activity. Studies conducted after the initial identification of these broadly neutralizing antibodies (bnAbs) have shown that they are less rare than initially believed [13] and that infection with the 2009 pandemic H1N1 virus frequently elicited B cells producing stem-reactive antibodies [14]–[16]. Importantly, various reports indicate that B cells producing bnAbs were also induced in humans following vaccination with the 2009 pandemic H1N1 influenza vaccine [15], [17]–[20], which supports the possibility of designing vaccines that elicit bnAbs and provide broad protection against influenza. Various approaches to the design of such ‘universal’ vaccines are being explored, such as the creation of ‘headless’ HA immunogens [21], [22] and novel immunization strategies [23], [24]. "
    [Show abstract] [Hide abstract] ABSTRACT: Current influenza vaccines are believed to confer protection against a narrow range of virus strains. The identification of broadly influenza neutralizing antibodies (bnAbs) has triggered efforts to develop vaccines providing 'universal' protection against influenza. Several bnAbs were isolated from humans recently vaccinated with conventional influenza vaccines, suggesting that such vaccines could, in principle, be broadly protective. Assessing the breadth-of-protection conferred to humans by influenza vaccines is hampered by the lack of in vitro correlates for broad protection. We designed and employed a novel human-to-mouse serum transfer and challenge model to analyze protective responses in serum samples from clinical trial subjects. One dose of seasonal vaccine induces humoral protection not only against vaccine-homologous H1N1 challenge, but also against H5N1 challenge. This heterosubtypic protection is neither detected, nor accurately predicted by in vitro immunogenicity assays. Moreover, heterosubtypic protection is transient and not boosted by repeated inoculations. Strategies to increase the breadth and duration of the protective response against influenza are required to obtain 'universal' protection against influenza by vaccination. In the absence of known correlates of protection for broadly protective vaccines, the human-to-mouse serum transfer and challenge model described here may aid the development of such vaccines.
    Full-text · Article · Jul 2014 · PLoS ONE
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