(PE-Cy7,ThermoFisher, clone HIB19) and anti-
CD20 (PE-Cy7, ThermoFisher, clone 2H7), in
addition to anti-IgG (APC-Cy7, Biolegend,
clone HP6017), anti-CD3 (APC eFluor780,
ThermoFisher, clone UCHT1), anti-CD14 (APC
eFluor780, ThermoFisher, clone 61D3), anti-
CD16 (APC eFluor780, ThermoFisher, clone
eBioCB16), and Live/Dead (APC eFluor780,
ThermoFisher) for exclusion, were added for
an additional 20 min. A BD FACSAria was used
for all cell sorting. Cells were sorted at a flow
rate of 1500 events/s using an 85-mmnozzle.
Sorting stringency was set to a strict setting
to obtain one cell per well. Single B cells were
sorted directly into cold lysis buffer or N332-
specific clonal B cell lines were generated and
interrogated as in ( 9 ). cDNA synthesis, nested
BCR PCR, Sanger sequencing and sequence
analysis was carried out as in ( 15 ). Sorting was
done with FACSDiva (BD) software and post-sort
analyses were done with FlowJo (FlowJo, LLC).
In order to determine a normal HCDR3
length distribution for naïve human B cells,
we combined sequences from DeKoskyet al.
( 49 ) and Jardineet al.( 30 ). For control VL
gene frequencies, only H+L paired sequences
from DeKoskyet al.( 49 )wereused.Statistical
analysis of LC V gene usage (Fig. 3D) was done
by applying Fisher’s exact test (two-sided) to
each immunogen-probe B cell set (i.e., N332-
GT1, N332-GT2, N332-GT5) compared to the
reference normal population.
Adoptive transfer experiments and immunization
For adoptive transfer experiments, B cells
were isolated from CD45.2 C57BL/6J (“WT”)
or BG18gHKI mice of 8 to 10 weeks of age,
and cells were resuspended in 150mlofPBS
and counted by The NucleoCounter NC-200
(ChemoMetec USA Inc). The 150-mlcellsus-
pensions were injected i.v. into CD45.1 B6.
SJL-PtprcaPepcb/BoyJ recipient animals (5 ×
103 cells per mouse for BG18gHtransfers and
50 × 10^3 cells per mouse for C57BL/6 trans-
fers). One day later, recipient mice were in-
jected i.p. with 10mg GT2- or MD39-NPs with
Sigma adjuvant (Sigma, # S6322 SIGMA). After
8 days, mice were sacrificed to harvest spleen
samples. Blood samples were taken from the
submandibular vein on days 0 and 14 after
immunization. Four immunization conditions
were tested in two independent experiments,
with the following total number of mice in each
condition: (i) BG18gHB cell transfer, N332-GT2
NP immunization (N= 6 for day 8 GC analysis,
N= 5 for day 14 ELISA); (ii) BG18gHB cell
transfer, MD39 NP immunization (N= 3 for
day 8 GC analysis,N=3forday14ELISA);
(iii) WT B cell transfer, N332-GT2 NP immu-
nization (N=5forday8GCanalysis,N= 5 for
day 14 ELISA); (iv) WT B cell transfer, MD39
NP immunization (N= 3 for day 8 GC analysis,
N=3forday14ELISA).Wecompliedwith
all relevant ethical regulations. The animal
studies were approved by the Institutional Ani-
mal Care and Use Committee of Massachusetts
General Hospital.Antigen specific single-cell sorting for BCR
sequencing in mouse experiments
Antigen tetramers were prepared by conju-
gating for 1 hour (room temp.) biotinylated
N332-GT2 and N332-GT2-KO trimers with
fluorescently labeled streptavidins (Alexa Fluor
488, Alexa Fluor 647, eBioscience; Alexa Fluor
568, Thermo Fisher Scientific) in a 4:1 molar
ratio. The same streptavidins conjugated with
biotinylated Fab anti-IgM and biotinylated
BSA were used as positive and negative stain-
ing controls, respectively (data not shown).
Single-cell suspensionsgenerated from spleen
samples were depleted of red blood cells by
ACK lysis, Fc blocked (BD Biosciences), and
stained in FACS buffer (2% FCS/PBS) with
antigen tetramers for 30 min at 4°C, 50 nM
concentration. Next, a cocktail of mAbs was
added for 30 min at 4°C. For staining of
splenocytes from naïve mice (Fig. 2A), the
cocktail was B220 PerCP-Cy5.5 (Clone RA3-
6B2, Biolegend), IgD PE-Cy7 (Clone 11-26c.2a,
Biolegend), CD4 APC-eFluor780 (Clone RM4-5,
eBioscience), CD8a APC-eFluor780 (Clone 53-
6.7, eBioscience), F4/80 APC-eFluor780 (Clone
BM8, eBioscience), Ly-6G APC-eFluor780 (Clone
RB6-8C5, eBioscience). For staining of spleno-
cytes from immunized mice (fig. S13), the
cocktail was CD38 Alexa Fluor 700 (Clone 90,
Invitrogen), CD45.2 PE (Clone 104, Bioleg-
end), CD45.1 PerCP-Cy5.5 (Clone A20, Bioleg-
end), B220 PB (Clone RA3-6B2, Biolegend),
CD95 PE-Cy7 (Clone Jo2, BD Bioscience), CD4
APC-eFluor780 (Clone RM4-5, eBioscience), CD8a
APC-eFluor780 (Clone 53-6.7, eBioscience), F4/
80 APC-eFluor780 (Clone BM8, eBioscience),
Ly-6G APC-eFluor780 (Clone RB6-8C5, eBio-
science). Live-Dead staining kits (Thermo
Scientific) were used to identify dead cells
for exclusion from the analysis. Data acquisi-
tion and single-cell sorting were performed on
FACS ARIA II (BD Bioscience) and analyzed
with FlowJo v. 10 (Tree Star). Single-cell sorting
and single-cell PCR were carried out as described
previously ( 25 ).Phylogenetic analysis
We used Clustal Omega to create a multiple se-
quence alignment (input amino acid sequences
from heavy chain PCR) and iTol (EMBL) to
plot phylogeny trees:https://itol.embl.de/help/
gkw290.pdf.Thedistancebetweennodesin
the trees in Fig. 2I is a representation of dis-
similarity (or evolutionary distance); the scale
indicates the number of substitutions per site.Analysis of Ab-antigen complexes for pathogens
other than HIV
A broad but nonexhaustive set of antibody-
antigen complexes for diverse major humanpathogens were analyzed. If the HCDR3 was
judged to play an important structural role in
the interaction, then the HCDR3 sequence
was analyzed for similarity to human germline
D genes, checking all reading frames for all
D genes listed at IMGT ( 50 ). We did not have
access to nucleotide sequences for all Abs,
so amino acid sequences were used for this
analysis. If a recognizable D gene could be
identified, then the complex was subjected to
buried surface area (BSA) analysis. Antibody
BSA analysis was carried out using PDBePISA
( 51 ). Glycan interfaces were not included in
the BSA analysis. All complexes analyzed for
buried surface area were included in fig. S18,
except if the HCDR3 length was >20 amino
acids and the Ab BSA analysis indicated that
VH,VL, and HCDR3 all contributed substan-
tially to the buried area on the Ab, in which case
the complex was not considered a promising
target and was not considered further. CDRs
and FWs were specified according to IMGT
convention ( 52 ). Somatic hypermutation (SHM)
was determined by aligning the VHand VL
genes to the IMGT human germline VHand
VLgenes and calculating the % difference in
amino acid sequence from the most similar
human germline gene. Insertions or deletions
relative to the most similar human germline
VHor VLgene were identified similarly.Statistical analysis
Statistical parameters including the mean (or
geometric mean), the SEM (or geomeric stan-
dard deviation), and in some cases thePvalue,
are reported in the figures. Statistical analy-
ses were performed using Prism (GraphPad
Software) and compared with Student’sttest
(Fig.2,DtoF)orchi-squaretest(Fig.3D);
Pvalues < 0.05 were considered significant. Cor-
respondences between the number of asterisks
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