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Antibodies VRC01 and 10E8 Neutralize
HIV-1 with High Breadth and Potency Even
with Ig-Framework Regions Substantially
Reverted to Germline
Ivelin S. Georgiev, Rebecca S. Rudicell, Kevin O. Saunders,
Wei Shi, Tatsiana Kirys, Krisha McKee, Sijy O'Dell,
Gwo-Yu Chuang, Zhi-Yong Yang, Gilad Ofek, Mark
Connors, John R. Mascola, Gary J. Nabel and Peter D.
Kwong
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J Immunol published online 3 January 2014
http://www.jimmunol.org/content/early/2014/01/03/jimmun
ol.1302515
Published January 3, 2014, doi:10.4049/jimmunol.1302515
The Journal of Immunology
Antibodies VRC01 and 10E8 Neutralize HIV-1 with High
Breadth and Potency Even with Ig-Framework Regions
Substantially Reverted to Germline
Ivelin S. Georgiev,1 Rebecca S. Rudicell,1 Kevin O. Saunders,1 Wei Shi,1 Tatsiana Kirys,
Krisha McKee, Sijy O’Dell, Gwo-Yu Chuang, Zhi-Yong Yang, Gilad Ofek,
Mark Connors, John R. Mascola, Gary J. Nabel, and Peter D. Kwong
R
ecent years have seen an explosion in the number of
broadly neutralizing Abs (bNAbs) against HIV-1 (1–10).
Many of these bNAbs have been shown to protect from or
to provide control of infection (11–13) and, therefore, are of interest
for passive-immunization approaches (14). An underlying characteristic of anti–HIV-1 Abs is the substantially increased levels of
somatic hypermutation (15). Somatic hypermutation is part of the
diversification of Abs that occurs during affinity maturation: this
process occurs in activated B cells exposed to Ag within germinal
centers where high-affinity Abs are selected over their low-affinity
counterparts (16). Generally, chronic viral infections are associated
with the generation of Abs with increased numbers of mutations
compared with acute viral infections, suggesting that persistent Ag
exposure plays a role in stimulating repeated rounds of somatic
hypermutation and selection (17, 18). In the case of HIV-1, bNAbs
mostly show higher mutation levels compared with weakly neutralizing Abs (18). Moreover, V-gene reversion to germline of several
anti-HIV bNAbs results in lack of neutralization activity (18–20),
Vaccine Research Center, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892
1
I.S.G., R.S.R., K.O.S., and W.S. contributed equally to this work.
Received for publication September 18, 2013. Accepted for publication November
26, 2013.
This work was supported by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and the Office
of AIDS Research, National Institutes of Health.
Address correspondence and reprint requests to Dr. Peter D. Kwong, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Building 40, Room 4508, Bethesda, MD 20892.
E-mail address: [email protected]
Abbreviations used in this article: bNAb, broadly neutralizing Ab; MPER, membraneproximal external region.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302515
indicating that somatic hypermutation is important for neutralizing breadth and potency (18).
Although somatic mutations occur preferentially within the
CDR regions of Abs (21), large numbers of mutations in anti–HIV1 bNAbs are also found within the Ab-framework regions (18, 19).
Klein et al. (18) analyzed a set of anti–HIV-1 bNAbs targeting diverse epitopes on the HIV-1 envelope glycoprotein and found that
full framework reversion to germline substantially reduces or completely abrogates neutralization activity for many of these Abs;
function is partly restored in some Abs by allowing framework mature mutations in positions that are in direct contact with the Ag (18).
The results of that study underline the importance of framework
maturation for broad and potent neutralization by anti–HIV-1 Abs.
However, it has been unclear whether most or all of the framework
mutations would be necessary for retention of Ab function or
whether some of these mutations could be reverted to germline
with minimal effects on function.
To investigate this question, we selected two bNAbs that target
different sites of vulnerability on the HIV-1 Env glycoprotein: the
CD4 binding site (CD4bs) Ab VRC01 and the membrane-proximal
external region (MPER) Ab 10E8. These Abs neutralize ∼90% and
98% of HIV-1 strains at an average potency of 0.33 and 0.22 mg/
ml, respectively (4, 10). The variable regions of both of these Abs
exhibit high degrees of amino acid mutation: VRC01 V-gene,
42% heavy/28% light and 10E8 V-gene, 22% heavy/17% light.
The putative germline-reverted versions of these Abs are incapable
of neutralizing HIV-1 viral strains (19; M. Louder and J. Mascola,
unpublished observations). For VRC01, the mature CDRs, alone or
in combination with the Ag-contacting framework residues, are not
sufficient for potent neutralization: i.e., they were able to only weakly
neutralize 0 and 3 of 10 tested strains, respectively (18). These
results confirm the importance of framework mutations in VRC01.
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Abs capable of effectively neutralizing HIV-1 generally exhibit very high levels of somatic hypermutation, both in their CDR and
framework-variable regions. In many cases, full reversion of the Ab-framework mutations back to germline results in substantial to
complete loss of HIV-1–neutralizing activity. However, it has been unclear whether all or most of the observed framework
mutations would be necessary or whether a small subset of these mutations might be sufficient for broad and potent neutralization.
To address this issue and to explore the dependence of neutralization activity on the level of somatic hypermutation in the Ab
framework, we applied a computationally guided framework-reversion procedure to two broadly neutralizing anti–HIV-1 Abs,
VRC01 and 10E8, which target two different HIV-1 sites of vulnerability. Ab variants in which up to 78% (38 of 49 for VRC01)
and 89% (31 of 35 for 10E8) of framework mutations were reverted to germline retained breadth and potency within 3-fold of the
mature Abs when evaluated on a panel of 21 diverse viral strains. Further, a VRC01 variant with an ∼50% framework-reverted L
chain showed a 2-fold improvement in potency over the mature Ab. Our results indicate that only a small number of Abframework mutations may be sufficient for high breadth and potency of HIV-1 neutralization by Abs VRC01 and 10E8. Partial
framework revertants of HIV-1 broadly neutralizing Abs may present advantages over their highly mutated counterparts as Ab
therapeutics and as targets for immunogen design. The Journal of Immunology, 2014, 192: 000–000.
2
However, we conjectured that not all mutations from germline
would be necessary for retention of Ab-neutralization activity. To
test this conjecture, we created a series of VRC01 and 10E8 variants
with partial framework reversions to germline in both H and L
chains and compared their neutralization activity to that of the
mature Abs. This approach allowed us to explore the relationship
between neutralization activity and the number of framework
mutations in anti–HIV-1 bNAbs. Our results challenge the notion
that extensive framework mutations are necessary for broad and
potent neutralization by anti–HIV-1 bNAbs and suggest strategies
for Ab redesign in the context of Ab-product optimization or
immunogen design.
Ig-FRAMEWORK REVERSION OF HIV-1 Abs
proteins were expressed by cotransfection of H and L chain plasmids into
293F cells, and Abs were purified with affinity chromatography using either
Protein A Fast Flow Resin or HiTrap Protein A HP Columns (both from GE
Healthcare).
Neutralization assays
Neutralization was measured using single-round-of-infection HIV-1 Env
pseudoviruses and TZM-bl target cells, as described previously (30–32).
Neutralization curves were fit by nonlinear regression using a five-parameter
hill slope equation, as previously described (31). IC50 values were used for
the computation of neutralization breadth and potency for the different Ab
variants. Average neutralization potency for a given Ab was computed as the
geometric mean of the IC50 values for a set of 21 diverse viral strains (7),
where values . 50 mg/ml were set to 50. Neutralization breadth for a given
Ab was computed as the percentage of strains with IC50 values , 50 mg/ml.
Materials and Methods
Design and analysis of partial framework revertants to
germline
Results
Different approaches for various degrees of Ab germline reversion, ranging
from full V-gene reversion to selective point mutations, were described
previously, with applications to a number of viruses (18, 19, 22–24). In our
study, a CDR-grafting procedure typically used for humanization of nonhuman Abs (25) was used as the basis for our designs, in combination with
structural modeling and mutation analysis. Specifically, the CDR regions
(Kabat definition) of the mature Abs (VRC01 and 10E8) were grafted onto
homologous human germline genes (in effect, this replaces the germline
CDRs with their mature counterparts). Additionally, series of germline
framework residues were selected for mutation to their mature counterparts
(Fig. 1). Structural modeling, analysis, and visualization were used to determine which germline framework resides to mutate to mature. Specifically, mutations were selected based on a combination of several factors:
whether the position is implicated in supporting CDR regions (25); the
differences between germline and mature amino acid types (e.g., change in
amino acid charge, hydrophobicity, or structural properties); and proximity
to Ag or CDR regions, as determined by the Ag-bound crystal structure.
The Ag-bound crystal structure of VRC01 was obtained from Protein Data
Bank id: 3NGB (19). The Ag-bound crystal structure of 10E8 [Protein
Data Bank id: 4G6F (10)] was not available at the time of the design and
was therefore only used for retrospective analysis. Structural modeling,
analysis, and visualization were performed using OSPREY (26, 27), MolProbity (28), and PyMOL (29). Throughout this article, the mAb-nfH/mfL
nomenclature is used for designed partial framework revertants, where n is
the number of framework mutations in the H chain and m is the number of
framework mutations in the L chain: for example, VRC01-5fH/6fL refers to
the VRC01 variant with grafted mature CDRs and 5 H chain and 6 L chain
mature framework mutations. Designed H and L chains were paired in
a sparse matrix that was deemed to sufficiently represent the diversity of the
possible H–L chain combinations.
A computational design procedure was used to create partial framework reversion variants of Ab VRC01 (see Materials and Methods
and Fig. 1). The H chain of the mature VRC01 Ab contains 30
mutations within framework regions 1–4 (Fig. 2A). To test whether
a subset of these mutations could be reverted back to germline
without substantially affecting Ab activity, we created two partial
framework revertants of the H chain, with five and eight mature
framework mutations each (in the context of mature CDR regions),
as well as the CDR-grafted version of the H chain (with mature
CDR regions and no framework mutations) (Fig. 2). The selected
mutations in the VRC01-5fH variant were in proximity to the Ag
(distances of 3.6–7.7 Å) (Fig. 2A, 2B). The additional three
mutations in the VRC01-8fH variant were deemed to represent
substantial changes in amino acid type that were still in relative
proximity to the Ag (distances of 6.7–8.8 Å). The remaining 22
residue positions of maturation were more distal to the Ag (mean
distance of 16.3 6 4.7 Å; range 8.3–24.9 Å) and/or represented
more conserved amino acid changes and, thus, were not selected for
study. Similarly, the mature VRC01 L chain contains 19 framework
mutations, so we created partial framework revertants with mature
CDR regions and 0, 6, and 10 mature framework mutations (Fig. 2).
None of the framework mutations in the VRC01 L chain was in
substantial proximity to the Ag (mean distance of 21.7 6 6.9 Å;
range 9.8–32.3 Å), so the reversion analysis was focused on framework interactions with the CDR regions and/or within the overall Ab
structure. The VRC01-6fL L chain variant included mutations in
proximity to the CDR regions (distances of 2.9–5.5 Å), as well as a
two-residue FWR4 insertion that is not found in most other HIV-1
Site-directed mutagenesis or gene synthesis was used to generate the CMV/
R plasmids encoding the H or L chain framework revertants. Full-length IgG
FIGURE 1. Procedure for partial reversion of Ab
framework to germline. The CDR regions of the mature
target Ab (yellow) were grafted onto the backbone of
the putative germline precursor prior to somatic hypermutation (green) to obtain a chimeric CDR-grafted Ab.
Additionally, based on sequence and structural considerations, residues were selected for mutation to their
mature counterparts to obtain a partial framework reversion to germline of the target Ab with generally
retained function.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Protein expression and purification
Partial framework reversion of VRC01
The Journal of Immunology
3
bNAbs, including other Abs from the VRC01 class (9). Structural
modeling identified an additional four mutations that could be of
importance for the overall structure of VRC01, and the addition of
these mutations formed the VRC01-10fL variant (Fig. 2).
of framework mutations appeared to be sufficient for retention of Ab
function: for example, the VRC01-5fH/6fL construct had a ,2-fold
decrease in potency compared with the mature VRC01 (Fig. 4A, 4B).
HIV-1 neutralization by VRC01 partial framework revertants
The crystal structure of the 10E8–MPER complex was not available
at the time of the designs and, thus, was used only for retrospective
analysis. The H and L chains of mature 10E8 contain 19 and 16
mutations within framework regions 1–4, respectively (Fig. 5A).
We created 3 H chain variants with mature CDR regions and 0, 2,
and 10 framework mutations and 3 L chain variants with mature
CDR regions and 0, 4, and 10 framework mutations (Fig. 5). The
selected mutations in the 10E8-2fH and 10E8-10fH H chain variants and 10E8-4fL and 10E8-10fL L chain variants were deemed
to represent more substantial changes in amino acid type and/or
were found at positions that have been implicated in supporting
CDR regions (Fig. 5C). Post hoc structural analysis revealed that
the mutations in the 10E8-2fH H chain variant were in proximity
to the Ag (distances of 3.6 and 6.9 Å), but the distances to the Ag
or the CDR regions for the additional eight mutations in the 10E810fH H chain variant versus the remaining nine H chain mutations
were not substantially different (18.1 6 6.6 Å versus 20.6 6 4.9 Å
for Ag, 7.5 6 3.4 Å versus 6.6 6 2.1 Å for CDR regions) (Fig. 5A,
5B). None of the framework mutations in the 10E8-L chain was in
A total of 10 H–L chain combinations using the 4 H chain and 4 L
chain VRC01 variants was tested for neutralization activity. The
mature VRC01 (VRC01-30fH/19fL, consisting of 30 H chain/19 L
chain framework mutations) was among the best in terms of neutralization function (Fig. 3); however, the VRC01-30fH/10fL variant (with mature H chain and partially reverted L chain) consistently
outperformed all other constructs when using neutralization potencybreadth curves as a metric (the percentage of neutralized viral strains
at different IC50 potency cutoffs) (Fig. 4A). Six of the nine partial
framework revertants maintained good neutralization activity. In
fact, with the exception of VRC01-0fH/6fL (which did not express),
VRC01-5fH/0fL, and VRC01-0fH/0fL, the revertants generally had
an average potency within an ∼10-fold range from mature (Fig.
4B). The VRC01-0fH/0fL CDR-grafted construct showed complete
lack of neutralization activity, in concordance with previous findings (18). Generally, although a significant correlation (p = 0.0433)
was found between the number of framework mutations and the
neutralization potency for VRC01 variants (Fig. 4C), a small number
Partial framework reversion of Ab 10E8
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FIGURE 2. Framework revertants of Ab VRC01. (A) Sequence alignment (upper panel, H chain [HC]; bottom panel, L chain [LC]) of a putative
germline version, designed partial framework revertants, and mature VRC01. Residues that are identical to germline are shown as dots. CDR mutations are
shown in yellow on gray background; framework mutations are colored according to the variant in which they are first introduced (orange, closest to
germline; purple to blue, mature). For each residue position, the respective distance to Ag and CDRs is shown. (B) Mapping of framework mutations from
germline backbone onto a VRC01 complex structure [colors same as in (A)]. (C) Selection criteria for framework mutations.
4
Ig-FRAMEWORK REVERSION OF HIV-1 Abs
FIGURE 3. HIV-1 neutralization by VRC01 and 10E8 Ab variants. For each Ab, shown are IC50 values (in mg/ml) for the wild-type mature Ab, a set of partial
framework revertants, and a CDR-grafted variant in which the entire framework regions have been reverted to germline (columns; Ab names are based on the
number of framework mutations in the H and L chains). Neutralization data were obtained for 21 diverse HIV-1 strains (rows) from clades A, B, and C. Values are
colored according to potency, on a scale from green (least potent) to red (most potent), with white corresponding to a neutralization potency . 50 mg/ml.
HIV-1 neutralization by 10E8 partial framework revertants
A total of 10 H–L chain combinations using the 4 H chain and 4 L
chain 10E8 variants were tested for neutralization activity. The
mature 10E8 (10E8-19fH/16fL), along with the 10E8-19fH/10fL
and 10E8-10fH/16fL variants, displayed the best neutralization potency and breadth (Figs. 3, 6A). Unlike VRC01, the 10E8 variant
with no framework mutations (10E8-0fH/0fL) retained significant,
but reduced, neutralization activity (Fig. 6B). As with VRC01, a
significant correlation was found between the number of framework mutations and the neutralization potency for 10E8 variants
(Fig. 6C; p = 0.0022). Interestingly, however, the 10E8-0fH/4fL
variant that contained no H chain and only four L chain framework
mutations had improved neutralization potency compared with
10E8-0fH/0fL, with only an ∼3-fold decrease in potency relative
to mature 10E8 (Figs. 6A, 6B).
Discussion
The high rate of mutation and the numerous other immune-evasion
mechanisms of HIV-1 serve as a constant stimulus for HIV-1–reactive
Abs to continuously evolve and mature as they target the everchanging viral envelope. Perhaps as a consequence, broadly neutralizing anti–HIV-1 Abs typically have extreme numbers of variable
domain (CDR and framework region) mutations. In the case of
VRC01, the importance of framework mutations is underscored by
the fact that, although VRC01-5fH/0fL showed virtually no neutralization activity, the addition of six framework mutations in the L
chain resulted in VRC01-5fH/6fL being within 2-fold potency
of the mature Ab. The precise role for each of these six mutations
was not clear; however, three of these mutations (G66R, T69P,
and F71Y) clustered closely to CDR L1 (Fig. 2B, 2C), suggesting that these mutations may help to support the CDR L1 loop
in a conformation appropriate for interaction with Ag. We note that
these mutations are generally not conserved in VRC01-class Abs
from different donors, suggesting that Abs in this class may utilize
diverse pathways to achieve broad and potent neutralization (1, 9).
In the case of Ab 10E8, a variant with no mature framework mutations showed reasonable, albeit ∼10-fold lower, neutralization activity. Thus, framework maturation in anti–HIV-1 Abs does not appear
to be universally required for potent and broad neutralization activity.
Although the overall number of framework mutations can affect
neutralization activity, some mutations appeared to have a more
substantial effect on Ab activity, so the specific selection of residues for germline reversion is also important. For example, the
10E8-2fH/4fL and 10E8-2fH/0fL variants had an ∼4-fold and ∼2fold decrease in potency, respectively, compared with the analogous
FIGURE 4. HIV-1 neutralization by framework revertants of Ab VRC01. (A) Neutralization breadth at different IC50 neutralization cutoff values. (B)
Average (geometric mean) neutralization potency for the different combinations of H chain (HC) and L chain (LC) variants; gray indicates combination not
tested. DNE indicates that the variant did not express. (C) Spearman correlation of neutralization potency versus number of framework mutations (heavy +
light); colors same as in (A).
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substantial proximity to the Ag (mean distance, 22.4 6 6.8 Å;
range, 14.2–33.0 Å; Fig. 5A). The 10E8-4fL L chain variant included mutations in proximity to the CDR regions (distances of
3.1–5.9 Å), but the distances to the CDR regions for the additional
six mutations in the 10E8-10fL L chain variant versus the six
remaining residue positions of maturation were not substantially
different (10.3 6 3.2 Å versus 8.3 6 4.0 Å) (Fig. 5A, 5B).
The Journal of Immunology
5
variants without the two H chain framework mutations (10E8-0fH/
4fL and 10E8-0fH/0fL). Similarly, the VRC01-5fH/6fL variant
had an ∼2-fold better potency than the substantially more mutated
VRC01-8fH/19fL. In the case of 10E8-0fH/4fL, post hoc analysis
of the 10E8-MPER peptide structure indicated that additional reversions of mutations that do not directly interact with the Ag or
CDR regions may be possible (e.g., S2Y; Fig. 5). Further, the
reversion analysis was focused solely on framework residues;
however, it should also be possible to selectively revert CDR mutations, because not all mutations within the CDR regions are important for Ag recognition or overall Ab structure (33, 34). This
process could allow for the generation of partially reverted Ab
variants with even lower degrees of mutation and with similar, if
not better, neutralization activity as their mature bNAb counterparts.
FIGURE 6. HIV-1 neutralization by framework revertants of Ab 10E8. (A) Neutralization breadth at different IC50 neutralization cutoff values. (B)
Average (geometric mean) neutralization potency for the different combinations of H and L chain variants; gray indicates combination not tested. (C)
Spearman correlation of neutralization potency versus number of framework mutations (heavy + light); colors same as in (A).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Framework revertants of Ab 10E8. (A) Sequence alignment (upper panel, H chain [HC]; lower panel, L chain [LC]) of a putative germline
version, designed partial framework revertants, and mature 10E8. Residues that are identical to germline are shown as dots. CDR mutations are shown in
yellow on gray background; framework mutations are colored according to the variant in which they are first introduced (orange, closest to germline; purple
to blue, mature). For each residue position, the respective distance to Ag and CDRs is shown. (B) Mapping of framework mutations from germline
backbone onto a 10E8 complex structure [colors same as in (A)]. (C) Selection criteria for framework mutations.
6
Acknowledgments
We thank the Structural Biology Section, Structural Bioinformatics Core
Section, Virology Laboratory, Vector Core Section, Humoral Immunology
Section, and Humoral Immunology Core at the National Institutes of Health
Vaccine Research Center for helpful discussions and comments on the manuscript. We are grateful to J. Stuckey for assistance with graphics and thank
J. Baalwa, D. Ellenberger, D. Gabuzda, F. Gao, B. Hahn, K. Hong, J. Kim,
F. McCutchan, D. Montefiori, L. Morris, J. Overbaugh, E. Sanders-Buell,
G. Shaw, R. Swanstrom, M. Thomson, S. Tovanabutra, C. Williamson, and
L. Zhang for contributing the HIV-1 envelope plasmids used in the neutralization panels.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Disclosures
The National Institutes of Health has filed patents relating to neutralizing
Abs in this article: WO2013086533 A1, titled “Neutralizing antibodies to
HIV-1 and their use,” and WO2013070776 A1, titled “Neutralizing gp41
antibodies and their use.” The authors have no financial conflicts of interest.
22.
23.
24.
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Interestingly, we also found that reversion of selected mutations
resulted in improved Ab variants compared with the mature Abs.
In the case of VRC01, a combination of the mature H chain and an
∼50% reverted L chain (VRC01-30fH/10fL) resulted in 2-fold
improved potency over the mature Ab. For 10E8, two variants,
10E8-19fH/10fL (mature H chain/∼40% reverted L chain) and
10E8-10fH/16fL (∼50% reverted H chain/mature L chain),
exhibited similar neutralization breadth and potency to the mature
Ab. These results show the promise of partial framework reversion
for Ab optimization.
Anti–HIV-1 bNAbs with lowered mutation levels may be of use
both in vaccine design and as therapeutic agents. An effective
prophylactic HIV-1 vaccine most likely will have to elicit bNAbs.
Thus, the high mutation levels of most anti–HIV-1 bNAbs present a
challenge for vaccine development and immunogen design, because
the vaccine immunogen (or perhaps a series of different immunogens) would need to guide the Ab affinity–maturation process
from germline activation through the appropriate intermediate
stages of Ab development to the mature bNAb (35–37). However,
bNAbs with lower levels of mutations, such as those presented in
this article or from Ab ontogeny data from next-generation sequencing (1, 9), may be more easily inducible and can be suitable
targets for immunogen design, alleviating the requirement for
guiding the affinity-maturation process to extreme levels of hypermutation (alternatively, it may be that to achieve necessary CDR
mutations, high overall levels of somatic hypermutation are required,
resulting in a substantial number of “bystander” framework mutations). Abs with high mutation rates might also be problematic as
clinical products because the likelihood of immunogenicity may
be increased. Although, to our knowledge, no highly somatically
mutated Abs have been evaluated for immunogenicity in humans,
Abs that are closer to their germline ancestors (the genes that are
commonly found in most individuals) may be more easily tolerated in the general population. Thus, partial germline reversion may
be a useful addition to the armamentarium of technologies for
translating anti–HIV-1 bNAbs into clinical products. Overall, our
results suggest that at least some of the anti–HIV-1 Abs can retain
broad and potent neutralization activity even when their framework regions are substantially reverted back to germline. These
results should aid in efforts for immunogen design targeting the
elicitation of bNAbs and for Ab-based therapeutics for the treatment or prevention of HIV-1.
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