Replicates
Data presented are from two independent ex-
periments. The results have been reproduced
in at least two additional experiments in this
same mouse model and in a different but re-
lated mouse model.
BG18/BG505 SOSIP structural model
The HCDR3 loop of unliganded BG18 (PDB:
5UD9) was aligned to PGT122 (PDB: 4TVP)
and several features suggested this as a plau-
sible binding mode. First, ArgL54 in LCDR2
would be positioned in a similar space as
LCDR3 ArgL94 in PGT122, a known critical
contact residue for PGT122. Second, ArgH 29
was positioned close to the N332 glycan, and
we confirmed that ArgH29 was important for
neutralization, by mutagenesis (fig. S2). Finally,
analysis of computationally predicted V1 con-
formational ensembles including protein and
glycan conformational diversity suggested that
the LC could plausibly avoid clashing with the
N137 glycan.
Design of minimally mutated versions of BG18
Design of minimally mutated versions of BG18
shown in fig. S4 was guided by analysis of
the structural model of BG18 bound to BG505
SOSIP (fig. S2B), as this work was carried out
before crystal structures were published of
BG18 bound to BG505 and B41 SOSIP trimers
( 18 ) and before we obtained a cryo-EM struc-
ture of BG18 bound to the MD39 trimer (Fig.
1A). Framework and CDR mutations were
reverted to germline if structural inspection
indicated they were not contributing to the
binding interaction. Several HC and LC var-
iants were tested. BG18.11, which we refer to
as“minBG18,”was the least mutated variant
that retained at least 50% of the breadth of
BG18 while retaining similar potency as BG18.
BG18.6 was the least mutated variant that
showed any neutralization.
Design of BG18 bnAb variants using alternate
VHor VLgenes
The VLvariant engineering was carried out
early in the study and was therefore guided
only by our model for the BG18-Env interaction
(fig. S2B). To engineer the VLvariants shown in
fig. S6, the indicated VLgene was substituted
for the BG18 VLgene and BG18 mutations were
incorporated. The engineering of VHvariants
was more complicated and was informed by
our structural and mutagenesis studies (Fig. 1A
and figs. S2, E and F, and S4). These studies
indicated that the most important feature of
the BG18 VHgene (VH4-4) was HCDR1 with a
length of nine amino acids that is rare among
human VHgenes. We therefore tested whether
BG18-like bnAbs could utilize alternate VH
genes (4-59 and 4-61) that are closely related
to VH4-4 but use the more common HCDR1
lengths of 8 and 10 amino acids found among
~74 and ~19% of human VHgenes, respec-
tively. We used mammalian display to screen
scFv libraries containing ~10^4 to 10^5 HCDR1
sequences for binding to gp120 and native-like
trimers based on several isolates (B41, 191084,
ZM197, 6811) using the directed evolution de-
sign process described previously ( 22 ). One
VH4-59 library included an NNK codon in the
HCDR2 at position 53 in addition to contain-
ing HCDR1 sequence diversity. Briefly, libraries
were integrated into 293T cells using a dox-
inducible lentivirus based system; the scFv
was anchored to the cell surface by linking
the C-terminus to a PDGFR transmembrane
domain; and the cells were incubated with
HIS-tagged Env proteins and then stained
with anti-HIS PE (miltenyi biotech). With this
process, we identified two VH4-59 clones and
one VH4-61 clone that when expressed as sol-
uble IgG showed neutralization breadth on a
BG18-sensitive virus panel (fig. S6). Because the
theoretical number of HCDR1 sequences for
lengths 8 and 10 are ~10^10 and ~10^13 , respec-
tively,therearelikelytobemanyHCDR1
sequences that can support neutralization
beyond what we identified here. We conclude
that BG18-like antibodies with diverse VHand
VLgenes can achieve broad and potent neu-
tralization. It follows that BG18-like precur-
sors containing alternate VHand VLgenes
should be targeted by vaccine design.Immunogen design by mammalian cell surface
display directed evolution
BG18 iGL 2 had detectable affinity to the 11mutB
(PGT121 germline targeting) trimer ( 22 )but
no detectable affinity to BG505 MD39 contain-
ing a native N332 epitope; therefore, we used
11mutBas a base construct to begin the BG18
germline-targeting design process. The follow-
ing libraries were screened using a previously
described mammalian cell surface display
method ( 22 ). Briefly, the following libraries
were cloned into the pLenti CMVTRE3G Puro
Dest plasmid and then stably integrated into
rtTA3G-expressing HEK 293T cells using lenti-
viral transduction. Library 1 was a screen of
all 20 aa at a subset of positions in the BG18/
PGT121 epitope. It was an NNK codon scan of
positions 294, 297, 298, 299, 300, 302, 304,
305, 326, 329, 330, 333, 386, 413, 414, 415,
416, 417, 419, and 420. NNK codons were
introduced into BG505-11mutB-gp120 using
the QuikChange Site-Directed Mutagensis
Kit (Agilent). Library 1 was screened for
binding to BG18 iGL 2 and PGT121-GLCDR3rev4.
Library 2 was intended to sample hydropho-
bic amino acids underneath the BG18 epitope.
It was a combinatorial library with amino
acids F/I/L/V introduced at positions 154,
322, 323, 326, 333, 414, 415, and 416. The li-
brary insert was assembled with overlapping
ultramers (IDT DNA) followed by Gibson clon-
ing (NEB) into BG505-11mutB-gp120. Library 2was screened for binding to BG18 iGL 2 and
PGT121-GLCDR3rev4.Library 3 was intended to
test all 20 aa at key epitope contact positions
predicted by the structural model. It was a
combinatorial library with NNK codons intro-
duced at positions 137, 325, and (F/I/L/V) at
position 326. The library insert was assembled
with overlapping ultramers (IDT DNA) followed
by Gibson cloning (NEB) into BG505-MD39-
17mutE. Library 3 was screened for binding
to the following 24 Abs: BG18 iGL 0 , BG18 iGL 1 ,
pre1 - pre6, pre8, pre10 - pre15, VL2-8, VL2-14,
VL3-21, VH1-69, VH3-33, VH4-59, VH5-51, PGT121-
GLCDR3mat, and PGT121-GLCDR3rev1. Library 4
was designed to test all 20 aa at key epitope
contact positions predicted by the structural
model and mutations at position 325 that
were isolated in the library 3 screen. It was a
combinatorial library with NNK codons intro-
duced at positions 138 and 141 and (P/H/A/D)
at position 325. The library insert was assem-
bled with overlapping ultramers (IDT DNA)
followed by Gibson cloning (NEB) into BG505-
MD39-17mutE-N137K. Library 4 was screened
for binding to BG18 iGL 0 , pre3, pre14, VL3-21,
VL2-8, VL2-14, VH3-33. Library 5 was designed
to test all amino acids at key epitope contact
positions predicted by the structural model
and mutations at position 325 that were iso-
lated in the library 3 screen. It was a combi-
natorial library with NNK codons introduced
at positions 138 and 139 and (P/H/A/D) at po-
sition 325. The library insert was assembled
with overlapping ultramers (IDT DNA) followed
by Gibson cloning (NEB) into BG505-MD39-
17mutE-N137K. Library 5 was screened for bind-
ing to BG18 iGL 0 ,VL3-21, VL2-8, VL2-14, VH1-69,
and VH5-51. Library 6 tested all 20 aa at po-
sitions not directly in the BG18 epitope to
identify mutations that may indirectly effect
binding to BG18 precursors. It was an NNK
scan of positions 167 to 308. The insert was
synthesized at SGI-DNA and Gibson cloned
(NEB) into BG505-MD39-N332-GT3. Library 6
was screened for binding to BG18 iGL 0 ,VL2-8.
Library 7 screened all 20 aa at positions in
and around the BG18 epitope, excluding the
V1 loop. It was an NNK scan of positions 309 to- The insert was synthesized at SGI-DNA
and Gibson cloned (NEB) into BG505-MD39-
N332-GT3. Library 7 was screened for binding
to BG18 iGL 0 , pre1, pre2, pre4, pre10, pre15, and
VL2-8. All constructs contained a C-terminal
myc tag and were anchored to the cell mem-
brane via a C-terminal PDGFR transmembrane
domain. Staining of the cell populations was
typically done with IgG until saturated binding
was obtained at low nanomolar IgG concen-
trations, and then Fabs were used for stain-
ing to maintain selection pressure. IgGs were
labeled with Anti-Human IgG-R-PE (Sigma)
and Fabs were labeled with Human IgG Fab
PE (LSBio). Cell surface protein expression
was detected using FITC conjugated chicken
Steichenet al.,Science 366 , eaax4380 (2019) 6 December 2019 9of13
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