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During HIV-1 infection, antibodies are generated against
the region of the viral gp120 envelope glycoprotein that
binds CD4, the primary receptor for HIV-1. Among these
antibodies, VRC01 achieves broad neutralization of
diverse viral strains. Here we determine the crystal
structure of VRC01 in complex with an HIV-1 gp120 core.
VRC01 partially mimics CD4 interaction with gp120. A
shift from the CD4-defined orientation, however, focuses
VRC01 onto the vulnerable site of initial CD4 attachment,
allowing it to overcome the glycan and conformational
masking that diminishes the neutralization potency of
most CD4-binding-site antibodies. To achieve this
recognition, VRC01 contacts gp120 mainly through V-
gene-derived regions substantially altered from their
genomic precursors. Partial receptor mimicry and
extensive affinity maturation thus facilitate neutralization
of HIV-1 by natural human antibodies.
Successful vaccine development often takes advantage of
clues from humoral responses elicited by natural infection.
For HIV-1, neutralizing antibody responses elicited within the
first year or two of infection are generally strain-specific (1),
and thus provide poor leads for vaccine development
[reviewed in (2)]. A few monoclonal antibodies from HIV-1
infected individuals, however, are broadly neutralizing, and
an effort has been made to facilitate vaccine design by
defining their structures (3-4).
The well-studied broadly neutralizing anti-HIV-1
antibodies, 2G12, 2F5, 4E10, and b12, have unusual
characteristics that have posed barriers to eliciting similar
antibodies in humans (5). Thus, in addition to having broad
capacity for neutralization, an appropriate antibody should be
present in high enough titers in humans to suggest that such
antibodies can be elicited in useful concentrations. We and
others have screened cohorts of sera from infected individuals
to find broadly neutralizing responses that are detectable in a
substantial percentage of subjects (6-10). One serum response
that satisfies these criteria has been mapped to the site on
HIV-1 gp120 envelope (Env) glycoprotein that binds to the
CD4 receptor (8).
While potentially accessible, the CD4-binding site is
protected from humoral recognition by glycan and
conformational masking (11). The identification of
monoclonal antibodies against this site is described in a
companion manuscript (12). In brief, we created resurfaced,
conformationally stabilized probes, with antigenic specificity
for the initial site of CD4 attachment on gp120 (22). This site,
a conformationally invariant subset of the CD4-binding
surface, is vulnerable to antibody-mediated neutralization
(22), and we used probes specific for this site to identify
antibodies that neutralize most viruses (12). Here, we analyze
the crystal structure for one of these antibodies, VRC01, in
complex with an HIV-1 gp120 core. We decipher the basis of
VRC01 neutralization, identify mechanisms of natural
resistance, show how VRC01 minimizes such resistance,
examine potential barriers to elicitation, and define the role of
affinity maturation in gp120 recognition.
Similarities of Env recognition by CD4 and VRC01
antibody. To gain a structural understanding of VRC01
neutralization, we crystallized the antigen-binding fragment
(Fab) of VRC01 in complex with an HIV-1 gp120 from the
clade A/E recombinant 93TH057 (13). The crystallized gp120
consisted of its inner domain-outer domain core, with
truncations in the variable loops V1/V2 and V3 as well as the
N- and C-termini, regions known to extend away from the
main body of the gp120 envelope glycoprotein (14).
Diffraction to 2.9 Å resolution was obtained from
Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01
Tongqing Zhou,1 Ivelin Georgiev,1* Xueling Wu,1* Zhi-Yong Yang,1* Kaifan Dai,1 Andrés Finzi,2 Young
Do Kwon,1 Johannes Scheid,3 Wei Shi,1 Ling Xu,1 Yongping Yang,1 Jiang Zhu,1 Michel C. Nussenzweig,3
Joseph Sodroski,2,4 Lawrence Shapiro,1,5 Gary J. Nabel,1 John R. Mascola,1 Peter D. Kwong1†
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
20892, USA. 2Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Pathology, Division
of AIDS, Harvard Medical School, Boston, MA 02115, USA. 3Laboratory of Molecular Immunology and Howard Hughes
Medical Institute, The Rockefeller University, New York, New York 10065 USA. 4Department of Immunology and Infectious
Diseases, Harvard School of Public Health, Boston, MA 02115, USA. 5Department of Biochemistry and Molecular Biophysics,
Columbia University, New York, NY 10032, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
orthorhombic crystals, which contained four copies of the
VRC01-gp120 complex per asymmetric unit, and the
structure was solved by molecular replacement and refined to
an R-value of 19.7% (Rfree of 25.6%) (Fig. 1 and table S1)
The interaction surface between VRC01 and gp120
encompasses almost 2500 Å2, 1244 Å2 contributed by VRC01
and 1249 Å2 by gp120 (16). On VRC01, both heavy chain
(894 Å2) and light chain (351 Å2) contribute to the contact
surface (table S2), with the central focus of binding on the
heavy chain-second complementarity-determining region
(CDR H2). Over half of the interaction surface of VRC01
(644 Å2) involves CDR H2, a mode of binding reminiscent of
the interaction between gp120 and the CD4 receptor; CD4 is
a member of the V-domain class of the immunoglobulin
superfamily (17), and the CDR2-like region of CD4 is a
central focus of gp120 binding (Figs. 2A and table S3) (18).
For CD4, the CDR2-like region forms antiparallel,
intermolecular hydrogen-bonds with residues 365-368gp120 of
the CD4-binding loop of gp120 (18) (Fig. 2B); with VRC01,
one hydrogen-bond is observed between the carbonyl of
Gly54VRC01 and the backbone nitrogen of Asp368gp120. This
hydrogen-bond occurs at the loop tip, an extra residue relative
to CD4 is inserted in the strand, and the rest of the potential
hydrogen bonds are of poor geometry or distance (Fig. 2C
and table S4). Other similarities and differences with CD4 are
found: of the two dominant CD4 residues (Phe43CD4 and
Arg59CD4) involved in interaction with gp120, VRC01
mimics the arginine interaction, but not the phenylalanine one
(Fig. 2B, C). Finally, significant correlation is observed
between gp120 residues involved in binding VRC01 and CD4
Superposition of the gp120 core in its VRC01-bound form
with gp120s in other crystalline lattices and bound by other
ligands indicates a CD4-bound conformation (PDB ID
3JWD) (14) to be most closely related in structure, with a Cα-
root-mean-square deviation of 1.03 Å (table S5). Such
superposition of gp120s from CD4-bound and VRC01-bound
conformations brings the N-terminal domain of CD4 and the
heavy chain-variable domain of VRC01 into close alignment
(Fig. 2), with 73% of the CD4 N-terminal domain volume
overlapping with VRC01 (19). This domain overlap is much
higher than observed with the heavy chains of other CD4-
binding site antibodies, such as b12, b13 or F105 (table S6).
However, when the VRC01 heavy chain is superimposed -
based on conserved framework and cysteine residues - on
CD4 in the CD4-gp120 complex, clashes are found between
gp120 and the entire top third of the VRC01 variable light
chain (Fig. 2D) (20). In its complex with gp120, VRC01
rotates 43° relative to the CD4-defined orientation, and
translates 6-Å away from the bridging sheet, to a clash-free
orientation that mimics many of the interactions of CD4 with
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gp120, though with considerable variation. Analysis of
electrostatics shows that the interaction surfaces of VRC01
and CD4 are both quite basic, though the residues types of
contacting amino acids are distinct (fig. S2). Thus, while
VRC01 mimics CD4 binding to some extent, considerable
differences are observed.
Structural basis of VRC01 breadth and potency. When
CD4 is placed into an immunoglobulin context by fusing its
two N-terminal domains to a dimeric immunoglobulin
constant region, it achieves reasonable neutralization.
VRC01, however, neutralizes better (Fig. 3A) (12). To
understand the structural basis for the exceptional breadth and
potency of VRC01, we analyzed its interactive surface with
gp120. VRC01 focuses its binding onto the conformationally
invariant outer domain, which accounts for 87% of the
contact-surface area of VRC01 (table S7). The 13% of the
contacts made with flexible inner domain and bridging sheet
are non-contiguous and are not critical for binding. In
contrast, CD4 makes 33% of its contacts with the bridging
sheet, and many of these interactions are essential (18). The
reduction in inner domain and bridging sheet interactions by
VRC01 is accomplished primarily by a 6-Å translation
relative to CD4, away from these regions; critical contacts
such as made by Phe43CD4 to the nexus of the bridging sheet-
outer domain are not found in VRC01, while those to the
outer domain (e.g. Arg59CD4) are mimicked by VRC01.
To determine the affinity of VRC01 for gp120 in CD4-
bound and non-CD4-bound conformations, we used surface-
plasmon resonance spectroscopy to measure the affinity of
VRC01 and other gp120-reactive antibodies and ligands to
two gp120s: a β4-deletion developed by Harrison and
colleagues that is restrained from assuming the CD4-bound
conformation (21) or a disulfide-stabilized gp120 core,
largely fixed in the CD4-bound conformation in the absence
of CD4 itself (18) (Fig. 3B and fig. S3). VRC01 showed high
affinity to both CD4-bound and non-CD4-bound
conformations, a property shared by the broadly neutralizing
b12 antibody (21-22). By contrast, antibodies F105 and 17b
as well as soluble CD4 showed strong preference for either
one, but not both, of the conformations.
To assess the binding of VRC01 in the context of the
functional viral spike, we examined its ability to neutralize
variants of HIV-1 with gp120 changes that affect the ability
to assume the CD4-bound state. Two of these mutations,
His66Ala gp120 and Trp69Leugp120, are less sensitive (23),
while a third, Ser375Trpgp120, is more sensitive to
neutralization by CD4 (23-24). VRC01 neutralized all three
of these variant HIV-1 viruses with similar potency (Fig. 3C),
suggesting that VRC01 recognizes both CD4-bound and non-
CD4-bound conformations of the viral spike. This recognition
diversity allows VRC01 to avoid the conformational masking
that hinders most CD4-binding-site ligands (25) and to
neutralize HIV-1 potently (26).
Precise targeting by VRC01. Prior analysis of effective
and ineffective CD4-binding-site antibodies suggested that
precise targeting to the vulnerable site of initial CD4
attachment is required to block viral entry (11, 27). This site
represents the outer domain-contact site for CD4 (22).
Analysis of the VRC01 interaction with gp120 shows that it
covers 98% of this site (Figs. 4A, 4B and fig. S4), comprising
1089 Å2 on the gp120 outer domain, about 50% larger than
the 730 Å2 surface covered by CD4. The VRC01 contact
surface outside the target site is largely limited to the
conformationally invariant outer domain and avoids regions
of conformational flexibility. This concordance of binding is
much greater than for ineffective CD4-binding-site
antibodies, as well as for those that are partially effective,
such as antibody b12 (11, 22) (fig. S4).
The outer domain-contact site for CD4 is shielded by
glycan (22). Contacts by the VRC01 light chain (Tyr28VRC01
and Ser30VRC01) are made with the protein-proximal N-acetyl-
glucosamine from the N-linked glycan at residue 276gp120
(28). Thus, instead of being occluded by glycan, VRC01
makes use of a glycan for binding. Other potential glycan
interactions may occur with different strains of HIV-1 gp120,
as the VRC01 recognition surface on the gp120-outer domain
extends further than that of the functionally constrained CD4
interaction surface, especially into the loop D and the often-
glycosylated V5 region (fig. S5).
Natural resistance to antibody VRC01. In addition to
conformational masking and glycan shielding, HIV-1 resists
neutralization by antigenic variation. In a companion
manuscript, we show that of the 190 circulating HIV-1
isolates tested for sensitivity to VRC01, 173 were neutralized
and 17 were resistant (12). To understand the basis of this
natural resistance to VRC01, we analyzed all 17 resistant
isolates by threading their sequences onto the gp120 structure
(fig. S5). Variation was observed in the V5 region in resistant
isolates, and this variation – along with alterations in gp120
loop D - appeared to be the source of most natural resistance
to VRC01 (Fig. 4C, figs. S5, S6).
Because substantial variation exists in V5, structural
differences in this region might be expected to result in
greater than 10% resistance. The lower observed frequency of
resistance suggests that VRC01 employs a recognition
mechanism that allows for binding despite V5 variation.
Examination of VRC01 interaction with V5 shows that
VRC01 recognition of V5 is considerably different from that
of CD4 (fig. S7), with Arg61VRC01 in the CDR H2 penetrating
into the cavity formed by the V5 and β24-strands of gp120
(fig. S8). Most importantly, the V5 loop fits into the gap
between heavy and light chains; thus by contacting only the
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more conserved residues at the loop base, VRC01 can tolerate
variation in the tip of the V5 loop (Fig. 4D).
Unusual VRC01 features and contribution to
recognition. We examined the structure of VRC01 for
special features that might be required for its function. A
number of unusual features were apparent, including a high
degree of affinity maturation, an extra disulfide bond, a site
for N-linked glycosylation, a 2-amino acid deletion in the
light chain, and an extensively matured binding interface
between VRC01 and gp120 (Fig. 5 and fig. S9). We assessed
the frequency with which these features were found in HIV-1
Env-reactive antibodies (SOM Appendix) or in human
antibody-antigen complexes (fig. S10 and tables S8, S9), and
measured the effect of genomic reversion of these features on
affinity for gp120 and neutralization of virus (Fig. 5A-D,
Higher levels of affinity maturation have been reported for
HIV-1 reactive antibodies in general (29), and markedly
higher levels for broadly neutralizing ones (30). These
maturation levels could be a by-product of the persistent
nature of HIV-1 infection, and may not represent a functional
requirement. Removal of the N-linked glycosylation or the
extra disulfide bond, which connects CDR H1 and H3 regions
of the heavy chain, had little effect on binding or
neutralization (Figs. 5A, 5B and table S10). Insertion of 2-
amino acids to revert the light chain deletion had moderate
effects, which were larger for an Ala-Ala insertion (50-fold
decrease in KD) versus a Ser-Tyr insertion (5-fold decrease in
KD), which mimics the genomic sequence (Fig. 5C and table
S10). Finally reversion of the interface was examined with
either single-, 4-, 7- or 12-mutant reversions. For the single-
mutant reversions of the interface to the genomic antibody
sequence, all 12 mutations had minor effects (most with less
than 2-fold effect on KD, with the largest effect for a
Gly54Ser change with a KD of 20.2 nM) (table S10). Larger
effects were observed with multiple (4, 7 or 12) changes, and
these reduced the measured KD by 5-30 fold and EC50s or
neutralization percentages by 10-100 fold (Fig. 5D and table
S10). Thus, while VRC01 has a number of unusual features,
no single alteration to genomic sequence reduced binding or
neutralization by more than 10-fold.
Elicitation of VRC01-like antibodies. The probability for
elicitation of a particular antibody is a function of each of the
three major steps in B cell maturation: 1. Recombination to
produce nascent antibody heavy and light chains from
genomic VH-D-J and Vκ/λ-J precursors; 2. Deletion of auto-
reactive antibodies; and 3. Maturation through hypermutation
of the variable domains to enhance antigen affinity. For the
recombination step, a lack of substantial CDR L3 and H3
contribution to the VRC01-gp120 interface (table S2)
indicates that specific Vκ/λ -J or VH(D)J- recombination is not
required (31) (fig. S11). The majority of recognition occurs
with elements encoded in single genomic elements or
cassettes, suggesting that specific joints between them are not
required. Within the VH cassette, a number of residues
associated with the IGHV1-02*02 precursor of VRC01
interact with gp120; many of these are conserved in related
genomic VHs, some of which are of similar genetic distance
from VRC01 (fig. S12). These results suggest that appropriate
genomic precursors for VRC01 are likely to occur at a
reasonable frequency in the human antibody repertoire.
Recombination produces nascent B cell-presented
antibodies that have reactivities against both self and nonself
antigens. Those with auto-reactivity are removed through
clonal deletion. With many of the broadly neutralizing anti-
HIV-1 antibodies, such as 2G12 (glycan reactive) (32-33),
2F5 and 4E10 (membrane reactive) (34-35), this appears to be
a major barrier to elicitation. While this remains to be
characterized for genomic revertants and intermediates, no
auto-reactivity has so far been observed with VRC01 (12).
The third step influencing the elicitation of VRC01-like
antibodies is affinity maturation, a process involving
hypermutation of variable domains combined with affinity-
based selection that occurs during B cell maturation in
germinal centers (36). In the case of VRC01, 41 residue
alterations were observed from the genomic VH-gene and 25
alterations from the VΚ-gene (including a deletion of two
residues) (fig. S13) (37). To investigate the effect of affinity
maturation on HIV-1 gp120 recognition, we reverted the VH-
and VΚ-regions of VRC01, either individually or together, to
the sequences of their genomic precursors. We tested the
affinity and neutralization of these reverted antibodies (Fig.
6A), and combined these data with the genomic reversion
data obtained while querying the unusual molecular features
of VRC01 (previous section) (Fig. 6B).
No antibodies containing genomic VH and Vκ regions
bound gp120 or neutralized virus (38). Binding affinity and
neutralization showed significant correlations with the
number of affinity-matured residues (p<0.0001).
Interestingly, binding to stabilized gp120 did not correlate
well with other types of gp120 or to neutralization (table
S11), related in part to greater retention of binding to VRC01
variants with genomically reverted Vκ regions. Extrapolation
of the correlation to the putative genomic V-gene sequences
predicted binding affinities of 0.6 μM KD for gp120 stabilized
in the CD4-bound conformation and substantially weaker
affinites for non-stabilized gp120s (Fig. 6B and fig. S14).
Notably, no single affinity maturation alteration appeared
to affect affinity by more than 10-fold, suggesting that
affinity maturation occurs in multiple small steps, which
collectively enable tight binding to HIV-1 gp120. When the
effects of VRC01 affinity maturation reversions are mapped
to the structure of the VRC01-gp120 complex, they are
broadly distributed throughout the VRC01 variable domains,
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rather than focused on the VRC01-gp120 interface. Non-
contact residues therefore appear to influence the interface
with gp120 through indirect protein-folding effects. Thus, for
VRC01, the process of affinity maturation entails incremental
changes of the nascent genomic precursors to obtain high
affinity interaction with HIV-1 Env surface.
Receptor mimicry and affinity maturation. The
possibility of antibodies using conserved sites of receptor
recognition to neutralize viruses effectively has been pursued
for several decades. The recessed canyon on rhinovirus that
recognizes the unpaired terminal immunoglobulin domains of
ICAM-1 highlights the steric role that a narrow canyon
entrance may play in occluding bivalent antibody-combining
regions (39), although framework recognition can in some
instances permit entry (40). Partial solutions such as those
presented by antibody b12 (neutralization of 40% of
circulating isolates) (22) or by antibody HJ16 (neutralization
of 30% of circulating isolates) (41), a recently identified
CD4-binding-site antibody, may allow recognition of some
With VRC01, the potency and breadth of neutralization
(over 90%) suggests a more general solution. It remains to be
seen how difficult it will be to guide the elicitation of
VRC01-like antibodies from genomic rearrangement, through
affinity maturation, to broad and potent neutralization of
HIV-1. Accumulating evidence suggests that the VRC01-
defined mode of recognition is used by other antibodies (12).
These findings suggest that VRC01 is not an isolated
example, and likely provides a template for a general mode of
recognition. The structure-function insights of VRC01
described here thus provide a foundation for rational vaccine
design based not only on the particular mode of antibody-
antigen interaction, but also on defined relationships between
genomic antibody precursors, somatic hypermutation, and
required recognition elements.
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/ www.sciencexpress.org / 8 July 2010 / Page 6 / 10.1126/science.1192819
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A.F., W.S., L.X., Y.Y. and J.Z. performed research; X.W.,
Y.K., J.Scheid, M.C.N. and J.R.M. contributed new
reagents or reference data; T.Z., I.G., X.W., J.S., L.S.,
G.J.N., J.R.M. and P.D.K analyzed the data; T.Z., I.G., J.
Sodroski, L.S., G.J.N., J.R.M. and P.D.K. wrote the paper,
on which all authors commented. We thank I.A. Wilson
and members of the Structural Biology Section and
Structural Bioinformatics Core, Vaccine Research Center,
for discussions and comments on the manuscript, J.
Gonczy for assistance with data collection, and J. Stuckey
for assistance with figures. Support for this work was
provided by the Intramural Research Program of the NIH
and by grants from the NIH and from the International
AIDS Vaccine Initiative’s Neutralizing Antibody
Consortium. Use of sector 22 (Southeast Region
Collaborative Access team) at the Advanced Photon
Source was supported by the US Department of Energy,
Basic Energy Sciences, Office of Science, under contract
number W-31-109-Eng-38. Coordinates and structure
factors for the VRC01-gp120 complex have been
deposited with the Protein Data Bank under accession
Supporting Online Material
Materials and Methods
Figures S1 to S16
Tables S1 to S14
25 May 2010; accepted 1 July 2010
Published online 8 July 2010; 10.1126/science.1192819
Include this information when citing this paper.
Fig. 1. Structure of antibody VRC01 in complex with HIV-1
gp120. Atomic-level details for broad and potent recognition
of HIV-1 by a natural human antibody are depicted with
polypeptide chains in ribbon representations. The gp120 inner
domain is shown in gray, the bridging sheet in blue, and the
outer domain in red, except for the CD4-binding loop
(purple), the D loop (brown), and the V5 loop (orange). The
light chain of the antigen-binding fragment (Fab) of VRC01
is shown in light blue with complementarity-determining
regions (CDRs) highlighted in dark blue (CDR L1) and
marine blue (CDR L3). The heavy chain of Fab VRC01 is
shown in light green with CDRs highlighted in cyan (CDR
H1), green (CDR H2), and pale yellow (CDR H3). Both light
and heavy chains of VRC01 interact with gp120: the primary
interaction surface is provided by the CDR H2, with the CDR
L1 and L3 and the CDR H1 and H3 providing additional
Fig. 2. Structural mimicry of CD4 interaction by antibody
VRC01. VRC01 shows how a double-headed antibody can
mimic the interactions with HIV-1 gp120 of a single-headed
member of the immunoglobulin superfamily such as CD4.
(A) Comparison of HIV-1 gp120 binding to CD4 (N-terminal
domain) and VRC01 (heavy chain-variable domain).
Polypeptide chains are depicted in ribbon representation for
the VRC01 complex (right) and the CD4 complex with the
lowest gp120 RMSD (left) (table S5). The CD4 complex
(3JWD) (14) is colored yellow for CD4 and red for gp120,
except for the CDR-binding loop (purple). The VRC01
complex is colored as in Fig. 1. Immunoglobulin domains are
composed of two β-sheets, and the top sheet of both ligands is
labeled with the standard immunoglobulin-strand topology
(strands G, F, C, C’, C”). (B,C) Interface details for CD4 (B)
and VRC01 (C). Close-ups are shown of critical interactions
between the CD4-binding loop (purple) and the C” strand as
well as between Asp368gp120 and either Arg59CD4 or
Arg71VRC01. Hydrogen bonds with good geometry are
depicted by blue dotted lines, and those with poor geometry
in gray. Atoms from which hydrogen bonds extend are
depicted in stick representation and colored blue for nitrogen
and red for oxygen. In the left panel of C, the β15-strand of
gp120 is depicted to aid comparison with B, though because
of the poor hydrogen-bond geometry, it is only a loop. (D)
Comparison of VRC01- and CD4-binding orientations.
Polypeptides are shown in ribbon representation, with gp120
colored the same as in A and VRC01 depicted with heavy
chain in dark yellow and light chain in dark gray. When the
heavy chain of VRC01 is superimposed onto CD4 in the
CD4-gp120 complex, the position assumed by the light chain
evinces numerous clashes with gp120 (left). The VRC01-
binding orientation (right) avoids clashes by adopting an
orientation rotated by 43° and translated by 6-Å.
/ www.sciencexpress.org / 8 July 2010 / Page 7 / 10.1126/science.1192819
Fig. 3. Structural basis of antibody VRC01 neutralization
breadth and potency. VRC01 displays remarkable
neutralization breadth and potency, a consequence in part of
its ability to bind well to different conformations of HIV-1
gp120. (A) Neutralization dendrograms. The genetic diversity
of current circulating HIV-1 strains is displayed as a
dendrogram, with locations of prominent clades (e.g. A, B
and C) and recombinants (e.g. CDR02_AG) labeled. The
strains are colored by their neutralization sensitivity to
VRC01 (left) or CD4 (right). VRC01 neutralizes 72% of the
tested HIV-1 isolates with an IC80 of less than 1 ug/ml; by
contrast, CD4 neutralizes 30% of the tested HIV-1 isolates
with an IC80 of less than 1 ug/ml (table S14). (B) Comparison
of binding affinities. Binding affinities (KDs) for VRC01 and
various other gp120-reactive ligands as determined by
surface-plasmon resonance are shown on a bar graph. White
bars represent affinities for gp120 restrained from assuming
the CD4-bound state (21) and black bars represent affinities
for gp120 fixed in the CD4-bound state (24). Binding too
weak to be measured accurately is shown as with an asterisk
and bar at 10-5 M KD. (C) Neutralization of viruses with
altered sampling of the CD4-bound state. Mutant S375Wgp120
favors the CD4-bound state, whereas mutants H66Agp120 and
W69Lgp120 disfavor this state. Neutralization by VRC01 (left)
is similar for wild-type (WT) and all three mutant viruses,
whereas neutralization by CD4 (right) correlates with the
degree to which gp120 in the mutant viruses favors the CD4-
Fig. 4. Natural resistance to antibody VRC01. VRC01
precisely targets the CD4-defined site of vulnerability on
HIV-1 gp120. Its binding surface, however, extends outside
of the target site, and this allows for natural resistance to
VRC01 neutralization. (A) Target site of vulnerability. The
CD4-defined site of vulnerability is the initial contact surface
of the outer domain of gp120 for CD4 and comprises only 2/3
of the contact surface of gp120 for CD4 (22). The molecular
surface of HIV-1 gp120 has been colored according to its
underlying domain substructure: red for the conformationally
invariant outer domain, grey for the inner domain and blue
for the highly mobile bridging sheet. Regions of the gp120
surface that interact with VRC01 have been colored green,
with the CD4-defined site of vulnerability outlined in yellow.
The view shown here is rotated 90° about the horizontal from
the view in Figs. 1 and 2. (B) VRC01 recognition. The
molecular surface of gp120 in the VRC01 bound
conformation is colored as in A. The variable domains of
VRC01 are shown in ribbon representation with the heavy
and light chains colored as in Fig. 1 and extension to constant
regions indicated. (C) Antigenic variation. The polypeptide
backbone of gp120 is colored according to sequence
conservation, blue if conservation is high and red if
conservation is low. (D) Molecular surface of VRC01 and
select interactive loops of gp120. Variation at the tip of the
V5 loop is accommodated by a gap between heavy and light
chains of VRC01.
Fig. 5. Unusual VRC01 features. The structure of VRC01
displays a number of unusual features, which if essential for
recognition might inhibit the elicitation of VRC01-like
antibodies. In A-D, unusual features of VRC01 are shown
structurally (far left panel), in terms of frequency as a
histogram with other antibodies (second panel from left), and
in the context of affinity and neutralization measurements
after mutational alteration (right two panels). Affinity
measurements were made by ELISA to the gp120 construct
used in crystallization (93TH057), and neutralization
measurements were made with a clade A HIV-1 strain
Q842.d12. Additional binding and neutralization experiments
are reported in Table S10. (A) N-linked glycosylation. The
conserved tri-mannose core is shown with observed electron
density, along with frequency and effect of removal on
affinity. (B) Extra disulfide. Variable heavy domains
naturally have two Cys, linked by a disulfide; VRC01 has an
extra disulfide linking CDR H1 and H3 regions. This occurs
rarely in antibodies, but its removal by mutation to Ser/Ala
has little effect on affinity. (C) CDR L1 deletion. A two
amino acid deletion in the CDR L1, prevents potential clashes
with loop D of gp120. Such deletions are rarely observed;
reversion to the longer loop may have a 10-100-fold effect on
gp120 affinity. (D) Somatically altered contact surface. The
far left panel shows the VRC01 light chain in violet and
heavy chain in green. Residues altered by affinity maturation
are depicted with “balls” and contacts with HIV-1 gp120 are
colored red. About half the contacts are altered during the
maturation process. Analysis of human antibody-protein
complexes in the protein-data bank shows this degree of
contact surface alteration is rare; reversion of each of the
contact site to genome has little effect (table S10), though in
aggregate the effect on affinity is larger.
Fig. 6. Somatic maturation and VRC01 affinity.
Hypermutation of the variable domain during B cell
maturation allows for the evolution of high affinity
antibodies. With VRC01 this enhancement to affinity occurs
principally through the alteration of non-contact residues,
which appear to reform the genomic contact surface from
affinity too low to measure to a tight (nM) interaction. (A)
Effect of genomic reversions. The VH- and VΚ-derived
regions of VRC01 were reverted to the sequences of their
closest genomic precursors, expressed as immunoglobulins
and tested for binding as VH- and VΚ-revertants (gHgL), as a
VH-only revertant (gH), or as a VΚ-only revertant (gL) to the
gp120 construct used in crystallization (93TH057) or to a
stabilized HXBc2 core (22). These constructs were also tested
for neutralization of a clade A HIV-1 strain Q842.d12.
Additional neutralization experiments with clade B and C
viruses are reported in the supplemental materials. (B)
Maturation of VRC01 and correlation with binding and
neutralization. Affinity and neutralization measurements for
the 19 VRC01 mutants created during the structure-function
analysis of VRC were analyzed in the context of their degree
of affinity maturation. Significant correlations were observed,
with extrapolation to VH- and VΚ-genomic revertants
suggesting greatly reduced affinity for gp120.
/ www.sciencexpress.org / 8 July 2010 / Page 8 / 10.1126/science.1192819
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