Cell - 8 September 2016

IgL-expressing B cells that could serve as potential targets for
immunization.

Induction of Neutralizing Antibodies through Stepwise
Immunization
We employed stepwise immunization to help guide affinity matu-
ration of VH1-2/LC B cells toward the end point of neutralizing
VRC01-class antibodies (Figure 4A). The overall strategy was
to prime the immune response with high-affinity germline
VRC01 binder and boost with a series of immunogens displaying

progressively decreased affinity toward germline relative to

mature VRC01 antibodies. Specifically, we primed with eOD-

GT6 60-mer, which was available and is closely related to

eOD-GT8 60-mer, but has weaker and less broad reactivity to

VRC01-class inferred precursors (Jardine et al., 2016). In subse-

quent boosts, we used a modified gp120 core protein derived

from HIV-1 strain 426c (McGuire et al., 2013). For the first boost,

we used 426c degly-3_Ferritin; in this 24-mer nanoparticle form

of gp120 core, three N-linked glycosylation sites near the CD4

binding site, which interfere with the binding of germline

E

A

B

Group 1 (G1):

Group 2 (G2):

Immunization

Week

0

Immunogens

(adj: poly I:C)

2

4

6

8

10

12

14

16

18

20

22

0

BG505 SOSIP

eOD-GT6

60mer

BG505 SOSIP

426C-degly3 core

_Ferritin

(N463D N460D N276D)

BG505 SOSIP

426c-degly2 core

monomer

(N460D N276D)

BG505 SOSIP

426c-degly1 core

monomer

(N276D)

BG505 SOSIP

426c-WT core

monomer

BG505 SOSIP

426c-WT SOSIP

Sampling

D

ΔeOD-GT6 binding

1.2%

#1531

(wk0)

1.1%

#1540

(wk2)

2.4%

#1536

(wk6)

3.8%

#1539

(wk10)

eOD-GT6 binding

Naïve G1: BG505 G2: Stepwise

#1532

(wk2)

#1533

(wk22)

1.9% 3.9%

#1534

(wk22)

3.7% 5.4% 6.4%

#1537

(wk14)

#1538

(wk22)

C

0 4 8 12 16 20 24

Week

0 4 8 12 16 20 24

101

102

103

104

105

106

107

EL

I SA En

d point Titer

-4 -2 0 2

1

2

3

4

OD 4 50

VRC01 gHvgLv

-4 -2 0 2

VRC01

0 4 8 12162024

G2

G1

eOD-GT6 60mer

426c-degly3_Ferritin

426c-degly3 core

426c-degly2 core

426c-degly1 core

426c-WT core

Iog10 Ab Conc. (μg/ml) Week

VRC01gl VRC01 eOD-GT6 vs. eOD-GT6 C13 vs. C13 426c-degly3 vs. 426c-degly3

Virus 426c.N276D.N460D.N463D 426c.N276D 426c.N460D.N463D 426c 45_01dG5 BG505.W6M.C2. T332NSIVmac251.30

N276 glycan - - + + - +

N460 glycan - + - + - -

N463 glycan - + - + + +

week 2 6 10 14 18 22 2 6 10 14 18 22 2 6 10 14 18 22 2 6 10 14 18 22 2 6 10 14 18 22 2 14 22 2 14 22

Group Mouse ID

G1

1532 <50 <50 <50 <50 <50 n.d. n.d.

1533 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50

1534 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 58 <50 <50 <50 <50 <50 55 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50

G2

1540 <50 <50 <50 <50 <50 n.d. n.d.

1536 <50 266 <50 131 <50 <50 <50 <50 <50 <50 n.d. n.d.

1535 <50 70 960 <50 101 247 <50 <50 <50 <50 <50 <50 <50 <50 <50 n.d. n.d.

1539 <50 <50 429 <50 66 198 <50 <50 <50 <50 53 <50 <50 50 212 n.d. n.d.

1537 <50 <50 1593727 <50 <50 485 156 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50

1538 <50 118 7551112 3906 3840<50 137 269 191 603 545 <50 50 <50 <50 <50 <50 <50 114 <50 <50 <50 <50 <50 81 <50 <50 58 100 <50 <50 <50 <50 <50 <50

Figure 4. Stepwise Immunization of the VH1-2/LC Mice Elicited VRC01-Class CD4bs-Specific Antibodies with Neutralizing Activity
(A) Immunization schema.
(B) Characterization of immunogens (varied symbols) for ELISA binding to VRC01 and its germline reverted variant (VRC01 gl).
(C) ELISA endpoint titers of post-immune sera to three germline-binding VRC01-class probes (solid lines) and their respective CD4bs-knock out (D) mutants
(dashed lines). Error bars indicate SDs.
(D) Splenocytes were sorted with eOD-GT6 andDeOD-GT6 probes to isolate CD4bs-specific IgG+B cells.
(E) Neutralizing activity of post-immune sera to various HIV-1 Env-pseudoviruses (ID 50 titers). n.d., not determined.
See alsoTable S4.

Cell 166 , 1471–1484, September 8, 2016 1477