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Epitope-Specific Human Influenza Antibody
Repertoires Diversify by B Cell Intraclonal
Sequence Divergence and Interclonal
Convergence
Jens C. Krause, Tshidi Tsibane, Terrence M. Tumpey,
Chelsey J. Huffman, Bryan S. Briney, Scott A. Smith,
Christopher F. Basler and James E. Crowe, Jr.
Supplementary
Material
http://www.jimmunol.org/content/suppl/2011/08/29/jimmunol.110182
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J Immunol published online 31 August 2011
http://www.jimmunol.org/content/early/2011/08/29/jimmun
ol.1101823
Published August 31, 2011, doi:10.4049/jimmunol.1101823
The Journal of Immunology
Epitope-Specific Human Influenza Antibody Repertoires
Diversify by B Cell Intraclonal Sequence Divergence and
Interclonal Convergence
Jens C. Krause,* Tshidi Tsibane,† Terrence M. Tumpey,‡ Chelsey J. Huffman,*
Bryan S. Briney,* Scott A. Smith,x Christopher F. Basler,† and James E. Crowe, Jr.*,{
I
nduction and maintenance of a diversity of broadly neutralizing Abs against viruses is desirable for immunity against
reinfection, but the molecular features of human Ab repertoires specific for particular agents or epitopes are poorly understood. Isolation of limited panels of epitope-specific human mAbs
to viruses has suggested that the circulating human B cell response
often is dominated by major clonal populations. In vivo selection in
germinal centers of particular B cell clones using BCRs with highaffinity binding to virus epitopes likely leads to expansion of
dominant clonal populations. The extent to which a dominant clone
of B cells responding to a viral epitope represents a single BCR
with an optimal affinity for binding, versus a family of related
*Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN
37232; †Department of Microbiology, Mount Sinai School of Medicine, New York
City, NY 10029; ‡Influenza Division, Centers for Disease Control and Prevention,
Atlanta, GA 30333; xDepartment of Medicine, Vanderbilt University Medical Center,
Nashville, TN 37232; and {Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232
Received for publication June 21, 2011. Accepted for publication August 2, 2011.
This work was supported by National Institutes of Health Grants P01 AI058113,
R21AI085306, and a pilot project from UL1RR029887 (to C.F.B.) and by Department of Defense Grant HDTRA1-08-10-BRCWMD-BAA and National Institute of
Allergy and Infectious Diseases Contract HHSN272200900047C. The Genome Sciences Resource was supported by Cancer Center Support Grant CA068485, the
Vanderbilt Digestive Disease Research Center (DK058404), and the Vanderbilt Vision Research Center (EY008126). J.E.C. is a Burroughs Wellcome Fund Clinical
Scientist in Translational Research.
The Ab nucleotide sequences presented in this article have been submitted to GenBank under accession numbers JF806284–JF806293.
The findings and conclusions in this report are those of the authors and do not
necessarily reflect the views of the funding agency.
Address correspondence and reprint requests to Dr. James E. Crowe, Jr., Vanderbilt
Vaccine Center, Vanderbilt University Medical Center, T-2220 Medical Center North,
1161 21st Avenue South, Nashville, TN 37232-2905. E-mail address: james.crowe@
vanderbilt.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: HA, hemagglutinin; HAI, hemagglutination activation inhibition; IMGT, international ImMunoGeneTics information system; VLP,
virus-like particle.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101823
somatic variants, has not been determined in the past because of the
difficulty in generating large numbers of human Abs.
The 2009 H1N1 influenza pandemic was the first influenza pandemic in .40 y. Pediatric death rates were 10 times the rates for
seasonal influenza in previous years (1). Elderly people had preexisting cross-reactive Abs against this 2009 H1N1 virus (2–4).
Preserved epitopes within H1N1 hemagglutinin (HA) were the likely
structural correlate for this cross-reactivity, particularly the Sa antigenic site on the globular head (5–7). We had shown previously that
the Sa site-specific Ab 2D1 that was cloned from a survivor of the
1918 pandemic potently neutralized 2009 pandemic virus (5, 8). We
elucidated the crystal structure of Ab 2D1 in complex with 1918 HA
(9). Ab 2D1 uses the VH2-70 germline gene with a unique insertion
close to CDR H2 that enhances the function of the Ab (8, 10).
In this study, we describe a panel of H1N1-specific Abs that was
cloned from a 47-y-old healthy woman after the pandemic. Like Ab
2D1, Abs from this new panel bound the Sa site, but they shared
VH3-7/JH6 germline gene usage and had hemagglutination activation inhibition (HAI) activity against a broader panel of H1N1
strains than 2D1, including viruses with glycosylation sites in the
Sa site. These VH3-7/JH6 Abs belonged to four different clones
that arose independently, yet converged toward similar amino acid
sequences. Ultra-deep sequencing has been used previously to
determine the combinatorial diversity of Abs (11–14). We used
this technology to elucidate the VH3 repertoire of this donor to
find related Ab sequences using the VH3-7/JH6 H chain gene
segments to more fully define the molecular diversity of an
epitope-specific human Ab repertoire. The data revealed unexpected features of the evolution of Ab repertoires and the persistence of corresponding B cells in the peripheral blood.
Materials and Methods
Hybridoma generation and recombinant Ab expression
Acquisition of human blood samples was approved by the Vanderbilt University Institutional Review Board. The animal studies were approved by the
Institutional Review Boards of the Centers for Disease Control. PBMCs were
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We generated from a single blood sample five independent human mAbs that recognized the Sa antigenic site on the head of influenza
hemagglutinin and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the VH3-7 and JH6 gene
segments, but at least four independent clones were identified by junctional analysis. High-throughput sequence analysis of
circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple
diverging lineages of these clones, including many containing very few mutations in the Ab genes, persisted in the circulation.
Conversely, we noted frequent instances of amino acid sequence convergence in the Ag combining sites exhibited by members of
independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide
diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly
mutating virus Ags, such as influenza hemagglutinin. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human Abs. The Journal of Immunology, 2011, 187: 000–000.
2
DIVERGENCE AND CONVERGENCE IN Ab REPERTOIRES
isolated from a 47-y-old healthy female donor with Histopaque-1077 (SigmaAldrich), EBV-transformed in 384-well plates (Nunc) in the presence of
2.5 mg/ml CpG ODN 2006 (Invivogen), 10 mM Chk2 inhibitor II (C3742;
Sigma-Aldrich), and 1 mg/ml cyclosporine A (Sigma-Aldrich), essentially as
described previously (10, 15). Supernatants from wells containing EBVtransformed lymphoblastoid cell lines were screened for binding activity
by ELISA against a panel of recombinant soluble HA proteins. Positive wells
were fused with HMMA2.5 myeloma cells and cloned molecularly using
previously described primer sets (16) into pGEM-T Easy vector (Promega)
and eventually into pEE12.4/pEE6.4 mammalian expression vectors (Lonza),
from where they were expressed (9) and purified on a protein G column
using an ÅKTA chromatography instrument (GE Healthcare). All following
studies were performed using recombinant Abs. We used Kabat numbering
as determined using the Abnum server (17) for the Abs and an H3 numbering
scheme (18) for HA. Ab clonality was defined strictly by shared VH gene,
shared VDJ junction, and a sequence of shared somatic mutations.
h after inoculation, mice were each administered 200, 20, or 2 mg (∼10, 1,
or 0.1 mg/kg) of Ab 4K8 or an equal volume of polyclonal human IgG
(Sigma-Aldrich) by the i.p. route in groups of 10 mice. Mice were observed for weight loss for 14 d. Subsets of four animals treated with Abs
were euthanized on day 4 after inoculation, and whole lungs were homogenized in 1 ml sterile PBS. Virus titers in lung tissue homogenates
were determined by plaque titration in Madin-Darby canine kidney cell
monolayer cultures and expressed as log10 PFU per milliliter.
Generation and purification of recombinant soluble HA
molecules
Isolation and characterization of Ab escape mutant viruses
We selected new Ab escape mutant viruses by incubating virus with neutralizing Abs followed by inoculation in 10-d-old embryonated chicken eggs,
essentially as described (21, 22). RNA was extracted from virus-infected
allantoic fluid, and then cDNA was generated by RT-PCR, cloned molecularly, and sequenced. The K166 escape mutations found in selected virus
mutants were built back into 1918 VLPs for use in HAI assays to confirm
that these alterations mediated escape from neutralization (10).
Pyrosequencing
ELISA
The 384-well clear plates (242757; Nunc) were coated with HA at 1 mg/ml in
Dulbecco’s PBS overnight, blocked with 0.5% cow milk, 0.2% goat serum,
and 0.05% Tween 20 (Sigma-Aldrich) in Dulbecco’s PBS. Five microliters
hybridoma supernatant per well was transferred to 25 ml blocking solution
with a multichannel pipette. Secondary goat anti-human IgG Ab (W99008A;
Meridian Life Science) was diluted 1:8000 in blocking solution and added
after four automated washing steps. After another wash, phosphatase substrate (S0942; Sigma-Aldrich) was dissolved in substrate buffer per the
instructions of the manufacturer and dispensed onto the plates. The plates
were read at 405 nm on a PowerWave HT (BioTek).
Virus-like particles expression and HAI assays
Expression plasmids encoding the parenteral or mutated 1918 HA were
coexpressed with N1 neuraminidase to produce virus-like particles (VLPs)
in 293T cells (10, 19). Two days posttransfection, supernatants were collected. HAI assays were performed as described (20) using VLPs (for 1918
H1N1 or H2) or live virus (all other influenza strains). Briefly, serially
diluted Abs were preincubated with eight hemagglutinating units of virus
or VLP per well. Chicken RBCs were added to a final concentration of
0.5%, and the plate was incubated on ice for 30–60 min.
In vivo antiviral effect of Ab 4K8
Female 8-wk-old BALB/c mice were inoculated intranasally with 53 LD50
in a 50 ml volume of the virulent reconstituted 1918 influenza virus. At 24
Total RNA was extracted from 30 million PBMCs (RNeasy kit; Qiagen). A
first-round PCR was performed for 35 cycles using BIOMED-2 VH3
framework 1/JH primers (23) and the OneStep RT-PCR kit (Qiagen). The
454-specific adapters (Roche) were added by 10 cycles of a second-round
PCR. The PCR products were sequenced on a GS Junior instrument (Roche).
The primers were cartridge-purified (Invitrogen); the sequences were 59CCATCAGCTGGGGGGTCCCTGAGACTCTCCTG-39 (forward) and 59CGCTCAGCTTACCTGAGGAGACGGTGACC-39 (reverse) for the first
round and 59-CGTATCGCCTCCCTCGCGCCATCAGCTGGGGGGTCCCTGAGACTCTCCTG-39 (forward) 59-CTATGCGCCTTGCCAGCCCGCTCAGCTTACCTGAGGAGACGGTGACC-39 (reverse) for the second
round. The gene-specific elements are in bold; the key is underlined; and
Roche-specific primer A/B sequences are in italics. Sequence analysis was
performed with the international ImMunoGeneTics information system
(IMGT)/HighV-Quest (24). IMGT output was analyzed in Access 2010
(Microsoft).
Results
Hybridoma generation, Ab reactivity, and animal studies
We isolated a panel of five human mAbs named 4A10, 2O10, 4K8,
6D9, and 2K11 from a single blood sample of a human subject. All
Abs showed HAI activity against both 1918 and 2009 H1N1
pandemic viruses, the related swine influenza viruses from 1930
and 1976, and the H1N1 virus from 1977, but not against strains
from the 1940s or H1N1 strains after 1977 (Table I). Ab 6D9 tested
negative for functional activity against representative H2N2,
H3N2, or H5N1 viruses by HAI (data not shown). Generally, Ab
4K8 was the most potent Ab, with an HAI activity ,0.01 mg/ml
against pandemic H1N1 viruses. We selected this Ab for testing in
a lethal mouse model of 1918 influenza virus infection (Fig. 1,
Table II). Ab 4K8 protected six out of six animals from death at
Table I. Specific HAI activity of human Abs against influenza viruses or VLPs
HAI Activity of Indicated Ab (mg/ml)
Influenza A Strain
4A10
2O10
4K8
6D9
2K11
A/South Carolina/1/1918 wt (VLP)
A/South Carolina/1/1918 K166E (VLP)
A/South Carolina/1/1918 K166N (VLP)
A/South Carolina/1/1918 K166Q (VLP)
A/South Carolina/1/1918 K166R (VLP)
A/swine/Iowa/15/1930
A/Weiss/1943
A/L3/47
A/New Jersey/11/1976
A/USSR/92/1977
A/New Caledonia/20/1999
A/Brisbane/59/2007
A/California/04/2009
0.08
.
.
.
10
0.63
.
.
2.5
0.32
.
.
0.16
0.04
.
.
.
.
0.08
.
.
2.5
2.5
.
.
0.32
,0.01
.
.
.
.
,0.01
.
.
0.16
0.32
.
.
,0.01
0.2
.
.
.
20
0.4
.
.
1.3
2.5
.
.
0.7
0.16
.
.
.
.
0.16
.
.
1.3
0.32
.
.
0.63
., activity was not detected at the highest concentration tested, 20 mg/ml.
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The 1918 or 2009 influenza HA constructs were ordered sequenceoptimized for expression in human cells (GenScript) based on the extracellular domain of the respective HA, a thrombin recognition cleavage site,
a fibritin trimerization domain, and a 6X His-tag (18). The constructs were
cloned into pcDNA3.1(+) (Invitrogen), expressed in 293F cells (Invitrogen), purified over nickel columns using an ÅKTA chromatography
instrument (GE Healthcare), and concentrated with Amicon Ultra centrifugal filters with a 30-kDa molecular mass cutoff (Millipore).
The Journal of Immunology
both the highest and the intermediate dose levels and two out of
six animals at the lowest dose (Fig. 1A). Ab 4K8 reduced lung
virus titers as compared with the human IgG control by 3.1 log10
PFU/ml at the 200 mg dose, 2.6 log10 PFU/ml at the 20 mg dose,
and still 1.3 log10 PFU/ml at the 2 mg dose (Table II).
Generation and validation of escape mutations in epitopes
recognized by these five Abs
Ab 4K8 selected for a K166R mutation in the A/USSR/97/1977
context and a K166E mutation in the A/California/04/2009
H1N1 context. To confirm that these mutations alone were sufficient to confer escape, we produced 1918 VLPs with mutant HA.
The entire panel of Abs was unable to inhibit hemagglutination of
VLPs with K166E, K166N, or K166Q mutations (Table I). Abs
4A10 and 6D9 showed modest HAI activity against VLPs with the
K166R mutation, but not 2O10, 4K8, or 2K11 (Table I). These
data suggested that all five Abs recognized a common epitope,
possibly with minor differences in the mode of binding among
those five Abs.
Next, we determined whether shared germline genes were the
basis for the recognition of this epitope. Nucleotide sequence
determination and sequence analysis of variable gene sequences
(Supplemental Fig. 1, Table III) using IMGT (24) revealed that all
of the five Abs shared usage of the VH3-7*01 gene and the JH6*02
gene; furthermore, 4A10, 2O10, and 2K11 used the same VL140*01 gene. Also, 4K8 and 6D9 used both VK3-20*01 and JK2.
The D genes of these five Abs were predicted to be of different
origins, except for 4K8 and 6D9, which shared the D6-13*01 gene
(Table III). These data suggested that 4K8 and 6D9 might be
derived from the same B cell ancestor clone. Careful review of
the junctional sequences showed that indeed 4K8 and 6D9 were
Table II. Therapeutic efficacy of Ab 4K8 against virus replication in
mice inoculated with 1918 influenza A virus
Ab
Dose (mg/Mouse)
Ab 4K8
200
20
2
200
20
2
Human IgG control
Mean Lung Virus Titera
(log10 PFU/ml 6 SD)
3.6
4.3
5.3
6.7
6.9
6.6
6
6
6
6
6
6
0.2
0.3
0.2
0.1
0.0
0.4
Four mice were inoculated intranasally with 53 LD50 and administered 4K8 Ab
or human IgG i.p. 24 h later. Mice were euthanized on day 4 after inoculation for the
determination of lung titers.
a
At the a = 0.025 level, controlling the overall type I error at 7.5%, the lung
homogenates of the 4K8 groups had lower virus titers than the human IgG control
groups for all dose levels by the Wilcoxon rank sum test (p = 0.01429 for each
level).
clonally related, whereas 4A10, 2O10, and 2K11 were derived
independently of each other and of the 4K8/6D9 clone (Fig. 2).
Despite four different clonal origins, the CDR H3s of these Abs
were remarkably similar (Fig. 2); for instance, the CDR H3 length
of 18 aa was identical across this panel. The amino acids in
positions 93–95, 96, 100, 100A, 100B, 100D, 100F, and 100H–
103 were fully conserved. Interestingly, somatic mutations were
shared between the Abs: for example, the tyrosine to histidine
mutation in position 100E of 2K11 and 4A10 or the glycine to
alanine mutation in position 100G of 2O10 and 4K8 (Fig. 2). Also,
several common mutated amino acid residues were found despite
differing sequences in the inferred germline origin sequence: the
glycine in position 96 was encoded entirely by the N1 segment
(2K11, 2O10), by the D segment (4A10), or by both (4K8/6D9).
The S97 was encoded by the D segment alone (4A10, 4K8/6D9)
or by both N1 and D1 (2K11). An aspartic acid was found in
position 100 of 2K11 and 2O10 because of their germline; clones
4A10 and 4K8/6D9 acquired this aspartic acid through a somatic
mutation, suggesting that this panel converged toward a consensus
sequence (Fig. 2). Position 100A was predicted by IMGT to be
encoded by the N2 segment alone (2K11, 4K8/6D9), by the D
chain and the N2 segment (2O10), or by the D and the J chain
(4A10; Fig. 2). No matter the origin, a threonine was found in all
five CDR H3s in this position (Fig. 2). Residues from 100B onward were encoded by the JH6 gene in all clones; four to six
successive tyrosine residues are typically encoded by that germline gene segment (25), although somatic mutations were found in
positions 100C (2K11) and 100E (4A10, 2K11).
We reviewed the variable gene encoded N-terminal part of H
variable chains for common somatic mutations: 4A10 and 2K11
shared a threonine to serine mutation in position H28. All five Abs
shared a threonine instead of the germline serine in position H35
(Fig. 3). Abs 2O10 and 4K8 shared a lysine to asparagine mutation
in position H52. Abs 2O10 and 6D9 mutated toward a threonine
from an asparagine in position H76 (Fig. 3). Abs 2O10, 4K8, and
Table III. Genetic features of H1N1-specific human mAbs
FIGURE 1. Therapeutic efficacy of Ab 4K8 against disease caused by
the 1918 A(H1N1) virus in mice. Mice were inoculated on day 0 and
treated on day 1 with the indicated Ab and dose. In each group, six mice
were monitored for survival every day (A) and weight every other day (B).
At all dose levels, the differences in survival distribution between the 4K8
and the human IgG control groups were significant by log-rank test
(p = 0.001 for the high-dose level, p , 0.001 for the medium-dose level,
p , 0.01 for the low-dose level).
H Chain Genes
L Chain Genes
Ab
VH
D
JH
VK/VL
JK/JL
l/k
4A10
2O10
4K8
6D9
2K11
3-7*01
3-7*01
3-7*01
3-7*01
3-7*01
3-16*02
3-22*01
6-13*01
6-13*01
4-17*01
6*02
6*02
6*02
6*02
6*02
1-40*01
1-40*01
3-20*01
3-20*01
1-40*01
2*01 or 3*01
1*01
2
2
3*02
l
l
k
k
l
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Sequence analysis of the Ab variable gene sequences in the
hybridomas
3
4
DIVERGENCE AND CONVERGENCE IN Ab REPERTOIRES
2K11 were found to have a valine instead of an alanine in position
H84 (Fig. 3). An aspartic acid took the place of a glutamic acid in
position H85 of Abs 4A10 and 4K8. Finally, both Abs 4A10 and
2K11 have a histidine to tyrosine mutation in position L34 of the l
chains in common (Supplemental Fig. 1).
clones for the presence of this motif, but none of them shared this
DTY motif in CDR H3, suggesting that we likely had identified
most of the clones of the VH3-7/JH6 gene segment-encoded Abs
that recognized the influenza HA Sa site.
Deep sequencing of the VH3 gene-encoded repertoire of this
donor
Although we identified sequences highly related to those of Abs
4A10, 2O10, or 4K8/6D9, none of the sequences from the high
throughput analysis was completely identical to those in the
original clones. Remarkably, we were able to recover a sequence
that was virtually identical to the 2K11 hybridoma cell IgH sequence in the high-throughput sequence BQ2Y7. This occurrence
may have been linked to the fact that sequences recovered that were
related to Ab 2K11 represented by far the biggest clonal family
identified and thus provided a more comprehensive snapshot of the
evolution of this Ab. Within the panel related to Ab 2K11, the
AE441 and AIF9Z sequences embodied essentially a germline state
with just a single, nonsilent, somatic mutation in the variable geneencoded amino acid sequence, the valine in position 84. In contrast,
the Ab 2K11 sequence itself and related sequences were highly
mutated, particularly in the CDR H1, with up to five changes in
amino acid sequence. Although both germline states and highly
mutated states were present simultaneously in the peripheral blood,
sequences representing many of the intermediate predicted steps
were not detected. For example, it was not clear in what order the
mutations within CDR H1 of the hybridoma 2K11 occurred except
that S31N probably occurred first because it was present in A32OY
by itself. Also, the S30I mutation probably occurred last within
CDR H1 because AUOJR contained all of the other mutations
except the aforementioned one. Because the other three clones had
fewer representatives than the 2K11 clone, the order of mutations
would be more difficult to establish for those clones. Nevertheless,
sequence divergence from the germline sequence was readily apparent. Notably, insertion/deletion events seemed to play a minor
role in this Ab panel with only sequence BQVKK within the 2K11
clone bearing evidence of a three base pair deletion, leading to the
loss of a single amino acid residue within CDR H1.
The response of this donor toward 2009 H1N1 was dominated by
Abs encoded by the VH3-7/JH6 gene segments. We hypothesized
that this limited panel of five clones might represent only a small
portion of the circulating antigenic site Sa-specific repertoire in
this individual. To test this idea, we extracted mRNA from total
PBMCs from this donor 6 mo after hybridoma generation and
isolated Ab genes using an RT-PCR amplification specific for
VH3. We used pyrosequencing to delineate the entire VH3 repertoire. We identified sister clones of the above Abs based on shared
V/J usage and identical VDJ junctions to more fully define the
genetic diversity within these clones in circulating cells. We
obtained 26.2 megabases of data including 80,687 sequences that
passed filter reads. The sequences had an average length of 325 bp
after removal of primer sequences. Analysis with IMGT identified
60,484 of those sequences as productive; 60,447 belonged to the
VH3 germline. VH3-23 and VH3-11 each accounted for 17% of
productive VH3 sequences (the first IMGT assignment was used in
case of ambiguities), VH3-30 for 13%, and VH3-7 for 9% (Supplemental Fig. 2). A total of 1917 VH3-7/JH6 IgH were identified,
of which 203 shared a CDR H3 length of 18 aa with the five
neutralizing influenza Abs we had isolated. Of these 203 sequences, 138 were nonredundant on the protein level belonging
to a total of 69 clones based on review of the VDJ junctions. Eight
of those sequences belonged to the 4A10 lineage (5 to clone 2O10,
2 to clone 4K8/6D9, and 23 to clone 2K11), meaning that every
single clone was still found in the peripheral blood of the individual 6 mo after the initial blood draw for hybridoma generation. Because residues DTY at positions 100-100B were completely conserved across all Abs, we screened the remaining
Intraclonal sequence divergence of the 2K11 clone
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FIGURE 2. Comparison of Ab gene junctional sequences reveals four independent clones. The IgH gene segment junctions of the five VH3-7/JH6 Abs
4A10, 2O10, 4K8, 6D9, and 2K11 are shown in amino acid and DNA sequence. Mutated amino acids and nucleotides are underlined. The amino acid
residues are color-coded per standard IMGT color scheme based on chemical properties (44). Briefly, aliphatic (A, I, L, V) residues are dark blue, phenylalanine light blue, sulfur (C, M) residues cyan, glycine dark green, residues with hydroxyl groups (S, T) medium green, tryptophan purple, tyrosine light
green, proline yellow, acidic (D, E) residues light orange, amide (N, Q) residues dark orange, and basic (H, K, R) residues red. Kabat numbering for amino
acids is used instead of IMGT numbering and is shown at the top level; the CDR H3 margins are denoted in red. The contributions of the VH, D, and JH
genes are shown in light gray (VH), medium gray (D), and dark gray (JH).
The Journal of Immunology
5
Interclonal sequence convergence
We defined convergence as the same altered amino acid introduced
by somatic mutation present in two or more independent clones.
Despite sequence divergence within the individual clones, sequence comparison revealed many further examples of interclonal
sequence convergence not evident in the hybridoma sequences such
as valine in H23 (members of clones 4A10/2K11) or threonine in
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FIGURE 3. Phylogram and sequence alignment to
the VH3-7*01 germline sequence of the H variable
chain genes of Abs 4A10 (cyan), 2O10 (orange), 4K8/
6D9 (medium blue), and 2K11 (green) from hybridoma technology and pyrosequencing. Five-letter alphanumeric labels denote sequences derived from pyrosequencing; hybridoma names are italicized. The
location of the CDRs (based on IMGT analysis) is
shown on top; Kabat numbering is shown at the bottom. Amino acids similar to the VH3-7*01 germline
sequence (for the VH region) or to the consensus sequence (for the D/J regions) are in light gray and
dissimilar amino acids in white. Variable gene segment
(V-GENE) encoded sequences are separated from the
diversity and joining gene segment (D/J-GENE)
encoded sequences by a dashed line. Residues within
the VH region with evidence of convergence are
identified with an asterisk at the bottom. The phylogram was generated based on the protein sequences
with MacVector software version 12 using neighbor
joining, best tree, symmetric tie breaking, uncorrected
(“p”) distance settings, and rooted to the deduced VH37*01 germline protein sequence.
the same position (2O10, 4K8/6D9, 2K11), leucine in H29 (4A10/
2K11), asparagine in H31 (4A10/2K11), glutamine in H46 (4A10/
2K11), asparagine in H58 (2O10/4K8/6D9), histidine in H59
(4A10, 2K11), and several others (Fig. 3). Overall, there were 20
positions within the VH protein sequence with evidence of convergence. Only 8 of those 20 positions were found within CDR
H1, H2, or the V-GENE–encoded portion of H3, so 12 conver-
6
DIVERGENCE AND CONVERGENCE IN Ab REPERTOIRES
gence positions were located in the framework regions. Remarkably, a subclone each within two different clones converged toward a set of similar somatic mutations within the CDR H1
(amino acids SLIN in positions H28-31 in the 2K11 clones and
amino acids SLKN in the same positions of the 4A10 lineage). An
analysis of silent versus nonsilent mutations by framework and
CDR is presented in Supplemental Table I.
in general seemed to neutralize 2009 H1N1 best among those
three candidates, we hypothesized that these Abs were created in
response to the 2009 virus and that the process of several Abs
independently hitting the same epitope with a similar genetic
makeup was entirely stochastic.
Discussion
The ultra-deep sequencing in this study revealed that the VH3-7/JH6
clones represented large circulating phylogenies. Because pyrosequencing was performed on PBMCs isolated 6 mo after the initial
hybridoma generation, the phylogenies seemed to persist for at least
several months in the peripheral blood. B cells that are not stimulated because they do not express high-affinity Ab are supposed not
to undergo further proliferation (30). Still, wide divergence of sequences from essentially unmutated germline states to extensively
mutated sequences was found within clones in the peripheral blood
of this donor. Persistent low-affinity memory populations may aid
in the immunologic response toward a related HA Ag that the individual subsequently encounters (31, 32).
B cell development has been traced by molecular analysis of
single cells picked from histological sections in human germinal
centers (33, 34) and spleen (34, 35) showing the presence of both
naive and memory B cells. An alternative method to track the
development of single memory-lineage B cells has been the use of
a specific anti-idiotypic Ab E4, which recognizes a canonical V
region (36). In the mouse study by Liu et al. (36), this E4+ pool is
derived from fewer than five canonical precursors. A lack of
shared somatic mutations across clonally related cells by day 13
indicates that the selective expansion of mutant subclones typical
of memory responses did not yet occur as it did in our panel of
Abs. The strength of our study was the combination of functional
data from human hybridoma technology with ultra-deep sequencing. A limitation of the deep sequencing was that we were
not able to document the concurrent evolution of the corresponding L chains, although the hybridoma technology allowed us
to find at least one hybridoma per clone with a matching H and L
chain, adding further evidence that these hybridomas represented
four distinct clones.
Despite different clonal origins within the VDJ junction, these
four independent clones converged toward common amino acid
residues within this junction and throughout the shared VH3-7
gene. This suggested a strong selection for optimal binding sites
across clones. Because three clones also share the VL1-40 gene,
but different J genes, CDRs H1, H2, H3, L1, and L2 would be
expected to make contacts with the HA. Given the frequency of
mutations within the CDR H1 segment and the conservation of the
typically very variable CDR H3, we hypothesize that those two
loops would be especially critical in the Ab–Ag interaction. The
majority of the convergence positions are within framework
regions; these residues can be at the surface, the core, or the H
chain–L chain interface of the Ab. Core mutations partially determine stability (37) and may change the conformation of adjacent and distant CDR loops (8).
Glycosylation within site Sa does not always confer escape
from neutralization
Redundancy of the immune response
Interestingly, this VH3-7/JH6 panel of four independent clones was
derived from a single donor; her response to pandemic 2009 H1N1
may have been VH3-7/JH6 dominant and oligoclonal because we
were only able to generate another H1N1 globular head Ab, Ab
5J8, from this donor that selected for escape mutations along the
receptor-binding pocket (26). The redundancy of this response
may ensure adequate neutralization of a given pathogen such as
influenza A virus. This redundancy may be quite common, but
may only be detected more frequently through advances in Ab
engineering and sequencing technology.
Same genes suggest a common epitope
Wrammert et al. (27) described cross-reactive human Abs in
subjects convalescing from infection with 2009 H1N1 pandemic
virus; this work did not feature neutralizing VH3-7/JH6 Abs, potentially due to a limited number of Abs. Although a similar genetic makeup (like VH3-7/JH6) suggests a common epitope, the Sa
antigenic site can likely be reached from a variety of different
germline configurations such as VH3-7/JH6 or the VH2-70 of the
2D1 Ab (5, 8, 10).
The VH3-7/JH6 Abs described in this study showed strongest
HAI activity against 1918 VLPs of a virus that circulated well
before the birth of this donor and thus could not have served as the
inciting agent. In fact, given that this donor was born between
1957 and 1977 when H1N1 did not circulate in humans, subtype
H1N1 infection was unlikely to have been the cause of her first
exposure to influenza or original antigenic sin (28). These Abs
could have been created in response to vaccination with the 1976
swine influenza virus (29) or vaccination or infection with a
USSR/77-like virus or the 2009 H1N1 virus. Because these Abs
Sequence convergence has been found in a variety of proteins
across individuals and species
Abs of the human VH1-69 germline from diverse phage display
libraries, plasmablasts, and EBV-transformed B cells neutralize
the stem of H1N1 and H5N1 influenza viruses (27, 38–41).
Likewise, the phage-derived HIV Abs Fab 8066 and D5 from
entirely different Ab libraries and panning procedures show convergence in their sequences and also in the conformation of the
CDR H2 loop (42). Indeed, pyrosequencing of zebrafish Abs
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We describe a panel of four independent clones of human Abs.
Like Ab 2D1, they were Sa site Abs based on the selection of
escape mutations in position K166 and that showed potent HAI
activity against pandemic H1N1 influenza (5, 9, 10). Unlike Ab
2D1, however, this panel had HAI activity against USSR/77 H1N1
as well. This finding is striking because the USSR virus possesses
three predicted N-linked glycosylation sites within its Sa antigenic
site (9). Glycosylation at these sites is thought to shield HA from
neutralization (7, 9). In support of this model, experimental introduction of glycosylation sites into the 1918 or 2009 HAs
conferred resistance to neutralization by antiserum elicited by the
unglycosylated HAs (7). The fact that neither the 1918 virus nor
the 2009 pandemic virus possess glycosylation sites within the
Sa antigenic site, whereas human H1N1 viruses acquired N-linked
glycosylation over the course of the 20th century, likely explains,
at least in part, why elderly people had pre-existing cross-reactive
humoral immunity against the 2009 pandemic virus (7, 9). The
fact that VH3-7/JH6 Abs inhibited USSR virus, despite the presence of the glycosylation sites, demonstrated that the presence of
glycosylation sites within site Sa did not always confer escape
from Sa-specific Abs.
Intraclonal sequence divergence and interclonal sequence
convergence
The Journal of Immunology
showed evidence of convergence, in which different individuals
made the same Ab (12), but this was not correlated with functional
data. Sequence convergence is not only seen in Abs across individuals of the same species (or, as demonstrated in this study,
within a single individual), but also even in proteins that serve
a similar purpose across distinct mammalian species (43). With
continued progress in B cell technology, there will likely be further discoveries of converging sequence-related Abs from one or
multiple individuals that share common epitopes. Once common
germline gene responses to defined epitopes of pathogens are
better understood, it might be possible to diagnose an infection of
an individual based on the immune repertoire documented by
ultra-deep sequencing.
7
15.
16.
17.
18.
19.
20.
Acknowledgments
21.
22.
23.
Disclosures
Vanderbilt University submitted a patent covering the diagnostic and
therapeutic use of the Abs described in this article. The authors have
no financial conflicts of interest.
24.
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