A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies
Targeting Distinct Epitopes Effectively Neutralizes Variants Found in
Leslie Goo,a,bZahra Jalalian-Lechak,aBarbra A. Richardson,c,dand Julie Overbaugha
Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAa; Graduate Program in Pathobiology, Department of Global Health,
University of Washington, Seattle, Washington, USAb; Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAc; and
Department of Biostatistics, University of Washington, Seattle, Washington, USAd
15, 18, 19, 21). However, an enormous challenge to preventing
infection in naturally exposed populations is the requirement for
NAb responses to recognize diverse circulating variants. Recently
identified HIV-1 monoclonal antibodies (MAbs) capable of po-
tently neutralizing diverse variants have spurred optimism for a
tective NAb responses and may also be candidates for gene deliv-
modify the course of infection (23). However, it is unclear how
effective these MAbs are specifically against transmitted variants,
which may comprise a unique subset of HIV variants (24) that
have distinct characteristics compared to variants in chronic in-
fection, such as shorter variable loop lengths and fewer potential
N-linked glycosylation sites (PNGS) (7, 8, 25, 29) and, in some
cases, different neutralization profiles compared to nontransmit-
ted variants (8, 9, 29, 31).
We analyzed the neutralization profiles of 45 HIV-1 envelope
variants of diverse subtypes (A, C, D), which were obtained soon
after heterosexually acquired infection (median, 59 days postin-
fection) (4, 5, 17), against 7 recently identified broadly neutraliz-
ing MAbs targeting several distinct epitopes. These included the
following: VRC01, which targets the CD4 binding site (CD4bs)
(30); NIH45-46W (10), which also targets the CD4bs but is an
engineered mutant that improves the neutralization breadth and
potency of MAb NIH45-46, a clonal variant of VRC01 (26); PG9,
PG16, and PGT145, which recognize a glycan-dependent quater-
nary epitope in V1/V2 and V3 (27, 28); and MAbs PGT121 and
PGT128 (27), which form another class of antibodies targeted to
glycan-dependent epitopes in V3. Serial dilutions of all MAbs
pseudoviruses in the TZM-bl assay as described previously (29).
This starting MAb concentration was chosen due to the limited
low concentrations (10, 27, 28, 30).
The MAbs had differing neutralizing activities against the
panel viruses, with 50% inhibitory concentration (IC50) values
ranging by more than 3 orders of magnitude from 0.0003 to ?1
tudies in nonhuman primate models have demonstrated that
passively infused neutralizing antibodies (NAbs) can protect
?g/ml (Fig. 1). The CD4bs MAb NIH45-46W neutralized 91% of
variants with a geometric mean IC50of 0.09 ?g/ml, while VRC01,
another CD4bs MAb, neutralized 71% of variants with a geomet-
ric mean IC50of 0.36 ?g/ml (Fig. 2). The glycan-dependent PG
neutralizing only 16% to 49% of variants with a geometric mean
IC50of 0.24 to 0.78 ?g/ml.
Because the PG and PGT MAbs failed to neutralize a majority
of variants, we investigated whether these variants lacked the
cases, resistance to these MAbs could be explained by the absence
of a key PNGS. For example, variants isolated from a number of
patients, including Q769, QG984, QH209, and QH359, which
were resistant to PGT121 and PGT128, lacked the N332 residue
required for neutralization (22, 27). Two of the four PG9/16-re-
glycosylation sequon that is a target for these MAbs, despite hav-
recognition by PG9 (20). However, for all other variants resistant
neutralization (N156, N160) (18, 26). Moreover, the presence of
positively charged residues at positions 168, 169, and 171, which
have been reported to be important for recognition by PG9/16
(11), did not always predict sensitivity to these MAbs (data not
Some viruses, such as those from QF495, QH343, and QA465,
had key PNGS for PGT121 and PGT128 recognition (N301 and
N332) (20) yet were resistant to one or both of these MAbs. For
other viruses, such as those isolated from Q259, Q168, and
Received 6 June 2012 Accepted 16 July 2012
Published ahead of print 25 July 2012
Address correspondence to Julie Overbaugh, firstname.lastname@example.org.
Supplemental material for this article may be found at http://jvi.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
October 2012 Volume 86 Number 19Journal of Virology p. 10857–10861jvi.asm.org
QD435, the shift of PNGS at position 332 to position 334 may
in PNGS did not always predict resistance to PGT121 and
PGT128, as exemplified by Q842 variants, which had a shift of
PNGS to position 334 yet were sensitive to these MAbs. Thus, the
presence of known residues important for neutralization by the
PG and PGT MAbs did not fully explain differing neutralization
profiles among these early variants, suggesting that there may be
other determinants of sensitivity to these MAbs. Indeed, the fact
that some viruses, such as those from QF495 and QA465, had the
expected epitope targets yet were resistant to most MAbs suggests
that these viruses may have altered conformations that result in
global neutralization resistance, as was observed for another early
subtype A virus from heterosexual transmission (6) and for sub-
types A and A/D vertically transmitted variants (14). Of note,
variants that possessed the canonical epitopes for PG and/or PGT
starting concentration of 10 ?g/ml (data not shown). An align-
ment of V1-V3 sequences of all variants did not readily reveal
signature sequences that would predict sensitivity to these MAbs
(see Fig. S2 in the supplemental material), although QF495 vari-
PNGS, that could explain their resistance to most MAbs tested
NIH45-46W neutralized all but 5 viruses in the panel, includ-
ing some viruses that were not neutralized by any other MAb
(QF495, QH359, and QA013) (Fig. 1). Interestingly, although
FIG 1 Summary of neutralization profiles of panel viruses against MAbs. Subject ID, virus subtype based on V1-V5 envelope sequence, and calendar year of
infection are shown in the first 3 columns. Each row shows the virus name, IC50for MAbs tested, and known core residues based on HXB2 numbering required
point to represent the diversity of the virus population at that time point as determined by phylogenetic analysis (5). Symbols: asterisk, estimated days
postinfection at which envelope clone was obtained; dot, amino acid is present; x, amino acid is present but not in a glycosylation sequon; shift, amino acid is
present but in position 334; D, T, S, amino acid substitutions. Darker shading indicates increasing MAb potency, as indicated by the key at the bottom, grouped
tested (1 ?g/ml). The combination of NIH45-46W and PGT128 was tested at a starting concentration of 1 ?g/ml of each MAb. IC50values shown are averages
from at least 2 independent experiments performed in duplicate.
Goo et al.
jvi.asm.org Journal of Virology
MAbs PGT121, PGT128, and PGT145 displayed limited breadth
at the highest concentration tested in these experiments (1 ?g/
ml), they potently neutralized variants that were resistant to all
supplemental material). Specifically, QA465 and QH343 variants
were only neutralized by PGT121, PGT128, and/or PGT145 (IC50
? 0.11 ?g/ml). Hierarchical clustering analyses suggested that
combining NIH45-46W and PGT128 would neutralize all but 2
variants, which were recognized by PGT121 (Fig. 3).
To test the hypothesis that NIH45-46W and PGT128 would
complement rather than interfere with each other’s neutralizing
ability, we investigated the neutralization profiles of a subset of
viruses against NIH45-46W and PGT128 alone or in a 1:1 combi-
nation. We chose viruses that were either (i) neutralized by one
but not the other MAb (Q168.A2 and QH343.21 M.ENV.A10) or
(ii) neutralized by both MAbs (QB099.391 M.ENV.B1 and
with the activity of the other MAb regardless of whether the virus
tested was sensitive to one (see Fig. S3A in the supplemental ma-
for MAbs in a 1:1 combination compared to MAbs tested alone.
Because NIH45-46W is an engineered antibody, we also con-
firmed that a naturally occurring broad and potent CD4bs MAb,
VRC01, would not interfere with PGT128 neutralization (see Fig.
S3). These results demonstrate that broad and potent MAbs tar-
geting the CD4bs and V3 do not compete for neutralization.
ized 96% of variants with a geometric mean IC50of 0.07 ?g/ml
(Fig. 1 and 2). The remaining 2 variants not neutralized by
NIH45-46W and PGT128 alone or in combination were potently
neutralized by PGT121 (Fig. 3). Because PGT121 and PGT128
have previously been shown to compete for binding (27), we in-
vestigated whether these MAbs would interfere with each other’s
neutralizing capacity against these 2 viruses. The combination of
to PGT128, to a similar extent as PGT121 alone (see Fig. S4 in the
supplemental material). These results demonstrate that the pres-
ence of PGT128 does not interfere with PGT121 neutralization
against these variants. It is possible that these observations reflect
an absence of binding of PGT128 to the viruses tested rather than
a lack of competition between the MAbs.
We observed generally similar neutralization breadth for the
breadth for PG9 and PG16 compared to previous reports (10, 28,
30). However, the neutralization breadth of PGT121, PGT128,
previously (27). In the prior study by Walker et al. (27), these
ants from chronic infection, potentially suggesting differences in
tion. However, differences in assays used and the subtypes of vi-
ruses examined could also be relevant and studies that directly
compare these variables will be needed to understand differences
in efficacy against different virus panels.
Prior studies of a subset of MAbs tested here, including
CD4bs and V1/V2 MAbs, suggested that the combination of
PG9 and VRC01 provided almost universal coverage of the
viruses tested, which included subtype B viruses (13) and vi-
ruses from diverse subtypes (12). When PG9 and VRC01 were
tested at 1 ?g/ml, 26% of viruses in our panel were resistant to
both MAbs (Fig. 1), which is more than what was reported in
the prior cross-clade study (?10%) (12). This difference could
again reflect differences in the virus panel, which included vi-
ruses from both acute and chronic infection from a study by
Doria-Rose et al. (12). Additionally, differences in the calendar
period from which viruses were isolated could also influence
sensitivity to these MAbs (13), but our sample size and distri-
bution over the sampling period (Fig. 1) were not adequate to
rigorously address this issue. Finally, the study by Doria-Rose
et al. used higher MAb concentrations (50 ?g/ml) and a larger
dilution range, and at this higher concentration, we have ob-
served a tendency to obtain lower IC50values than those ob-
tained with the lower starting concentration (1 ?g/ml) and
tighter dilution range used here for viruses that were potently
neutralized (IC50? 0.1 ?g/ml). However, this would not have
of the graph. Geometric mean IC50value for each MAb is indicated below the MAb name. IC50values greater than the highest MAb concentration tested (1
?g/ml) were assigned a value of 1 in the geometric mean IC50calculations.
HIV-1 Neutralizing MAbs against Early Variants
October 2012 Volume 86 Number 19jvi.asm.org 10859
altered our overall results, which focused on whether MAbs
could neutralize variants at1 ?g/ml, although it could lead to
small differences in the geometric mean IC50. Notably, at the
lower MAb concentration used here, only 4% of viruses in our
panel were resistant to NIH45-46W and PGT128, two MAbs
that were not included in previous studies, implying that these
MAbs may be among the most effective against variants found
early in infection. Although 7/45 viruses were obtained later in
infection (Q23, QA790, and QB099), and thus may not be rep-
resentative of recently transmitted variants, removing these vi-
ruses from the analysis did not significantly affect the results of
our study. For example, even by excluding these viruses,
NIH45-46W was still the most broad and potent MAb, while
MAbs against our panel.
In summary, we demonstrated that recently identified broadly
neutralizing HIV-1 MAbs have variable activity against variants
found early in infection. NIH45-46W, an engineered mutant of
NIH45-46 that targets the hydrophobic CD4 binding cavity in
gp120 (10), displayed remarkable breadth and potency against
these viruses. However, this MAb was unable to neutralize ?10%
of the viruses in the panel, which were potently neutralized by
glycan-dependent MAbs PGT121, PGT128, and/or PGT145.
PGT128 and NIH45-46W displayed no competition for neutral-
ization, and a combination of these MAbs neutralized 96% of
ized by this combination. Our results suggest that optimal neu-
tralization coverage of transmitted variants may be achieved by
combining a potent CD4bs NAb with one or more NAbs directed
to glycan-dependent epitopes in V3. It is currently unclear
whether this particular combination of broad and potent NAbs
can develop within a patient during natural infection, and it is
likely that eliciting such responses will be challenging. However,
the results presented here provide motivation to focus on these
epitopes, given that the antibody combination against them can
neutralize viruses representing recently transmitted variants.
FIG 3 Hierarchical clustering of MAbs NIH45-46W, PGT128, and PGT121 (bottom) and panel viruses (right). A heatmap of IC50values for each virus-MAb
combination is shown, with darker shading indicating increasing potency, as indicated by the key. Gray shading indicates that 50% neutralization was not
achieved at the highest concentration of MAb tested (1 ?g/ml).
Goo et al.
jvi.asm.orgJournal of Virology
ACKNOWLEDGMENTS Download full-text
We thank Michelle Long, Catherine Blish, Ozge Dogan, Minh-An
Nguyen, and Stephanie Rainwater for generating envelope plasmids used
in our panel, the IAVI Neutralizing Antibody Consortium for providing
Pamela Bjorkman for providing NIH45-46W.
This work was supported by NIH grant HD058304/AI103981. L.G.
was supported in part by a Fred Hutchinson Cancer Research Center
Interdisciplinary Research Fellowship.
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