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Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site

January 2016
Cell Reports 14(1)

January 2016
14(1)

DOI:10.1016/j.celrep.2015.12.017

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CC BY-NC-ND 4.0

Authors:

Tamsin Bradley

University of Portsmouth

Daniela Fera

Swarthmore College

Jinal N Bhiman

National Institute for Communicable Diseases

Show all 28 authorsHide

Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

Glycosylation of the V5 Loop Blocks Autologous Tier-2 Neutralization (A) Highlighter plot showing mutations in the Env that occur in the longitudinal immunogens from the T/F sequence. (B) Amino acid alignment of the CAP206 V5 regions. (C) Macaque 5173 plasma neutralization of wildtype and mutant CAP206 6-month viruses over the course of immunization measured as the plasma dilution at which RLUs were reduced 50% compared to control wells in the TZM-bl neutralization assay. (D) CAP206 plasma neutralization of autologous 2-month and 12-month viruses compared with 12-month reverted mutant viruses over the course of infection.

…

DH427 and DH428 Neutralize the Tier-2 CAP206 6-Month Virus (A) Neutralization of the CAP206 6-month virus and engineered glycosylation site mutant measured as the antibody concentration at which RLUs were reduced 50% compared to control wells (IC 50 ) in the TZM-bl neutralization assay (mg/ml). (B) CAP206 T/F V5 sequence and mutated V5 sequence to match the 6-month Env. Red amino acids were deleted. (C) DH427 and DH428 neutralization of the T/F and T/F mutant that had a V5 that matched the 6-month Env in the TZM-bl neutralization assay (mg/ml; orange, IC 50 < 5.0; red, IC 50 < 1.0). See also Figure S5.

…

DH427 and DH428 Neutralize the Tier-2 CAP206 6-Month Virus

…

Reconstruction of the DH427 Clonal Lineage

…

Isolation of Antibodies that Specifically Target the CAP206 6-Month Env

…

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Article

Structural Constraints of Vaccine-Induced Tier-2

Autologous HIV Neutralizing Antibodies Targeting

the Receptor-Binding Site

Graphical Abstract

Highlights

dHIV-1 TF Env immunization induced potent Tier-2 neutralizing

antibodies (nAbs)

dVaccine-elicited nAbs target CD4bs and mimic autologous

nAbs in infected individual

dAutologous nAb-Env complex structure reveals mechanism

of strain-speciﬁc neutralization

Authors

Todd Bradley, Daniela Fera,

Jinal Bhiman, ..., Sampa Santra,

Stephen C. Harrison, Barton F. Haynes

Correspondence

todd.bradley@duke.edu (T.B.),

barton.haynes@duke.edu (B.F.H.)

In Brief

HIV-1 vaccine elicitation of antibodies

against neutralization-resistant (Tier-2)

viruses is rare. Bradley et al. demonstrate

induction of antibodies that can

neutralize a vaccine-matched Tier-2 virus

in a rhesus macaque immunized with HIV

trimers isolated from a HIV-1-infected

individual. Structural analysis revealed

the mechanism of restricted

neutralization breadth.

Accession Numbers

5F6H

5F6I

5F6J

Bradley et al., 2016, Cell Reports 14, 1–12

January 5, 2016 ª2016 The Authors

http://dx.doi.org/10.1016/j.celrep.2015.12.017

Cell Reports

Article

Structural Constraints of Vaccine-Induced Tier-2

Autologous HIV Neutralizing Antibodies Targeting

the Receptor-Binding Site

Todd Bradley,

1,11,

*Daniela Fera,

2,11

Jinal Bhiman,

4,10

Leila Eslamizar,

Xiaozhi Lu,

Kara Anasti,

Ruijung Zhang,

Laura L. Sutherland,

Richard M. Scearce,

Cindy M. Bowman,

Christina Stolarchuk,

Krissey E. Lloyd,

Robert Parks,

Amanda Eaton,

Andrew Foulger,

Xiaoyan Nie,

Salim S. Abdool Karim,

6,7

Susan Barnett,

Garnett Kelsoe,

Thomas B. Kepler,

S. Munir Alam,

David C. Monteﬁori,

M. Anthony Moody,

Hua-Xin Liao,

Lynn Morris,

4,6,10

Sampa Santra,

Stephen C. Harrison,

2,3

and Barton F. Haynes

Duke Human Vaccine Institute, Departments of Medicine, Surgery and Immunology, Duke University School of Medicine, Durham,

NC 27710, USA

Laboratory of Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA

Howard Hughes Medical Institute, Boston, MA 02115, USA

National Institute for Communicable Diseases, Johannesburg 2131, South Africa

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

Center for AIDS Program of Research in South Africa, University of KwaZulu-Natal, Durban 4013, South Africa

Columbia University, New York, NY 10032, USA

Novartis Vaccines and Diagnostics, Inc., Cambridge, MA 02139, USA

Boston University, Boston, MA 02118, USA

Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2131, South Africa

Co-ﬁrst author

*Correspondence: todd.bradley@duke.edu (T.B.), barton.haynes@duke.edu (B.F.H.)

http://dx.doi.org/10.1016/j.celrep.2015.12.017

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARY

Antibodies that neutralize autologous transmitted/

founder (TF) HIV occur in most HIV-infected individ-

uals and can evolve to neutralization breadth. Autol-

ogous neutralizing antibodies (nAbs) against neutral-

ization-resistant (Tier-2) viruses are rarely induced by

vaccination. Whereas broadly neutralizing antibody

(bnAb)-HIV-Envelope structures have been deﬁned,

the structures of autologous nAbs have not. Here,

we show that immunization with TF mutant Envs

gp140 oligomers induced high-titer, V5-dependent

plasma neutralization for a Tier-2 autologous TF

evolved mutant virus. Structural analysis of autolo-

gous nAb DH427 revealed binding to V5, demon-

strating the source of narrow nAb speciﬁcity and

explaining the failure to acquire breadth. Thus, oligo-

meric TF Envs can elicit autologous nAbs to Tier-2

HIVs, but induction of bnAbs will require targeting

of precursors of B cell lineages that can mature to

heterologous neutralization.

INTRODUCTION

The HIV-1 envelope protein (Env) is the primary target of neutral-

izing antibodies (nAbs) (Wyatt and Sodroski, 1998; Zhou et al.,

2007). One major obstacle to developing an effective HIV-1 vac-

cine is ﬁnding an immunogen that can elicit broadly nAbs (bnAbs)

with the capacity to overcome variability of the virus and to retain

neutralizing activity for most circulating HIV-1 strains (Burton

et al., 2012; Mascola and Haynes, 2013).

Between 3 and 12 months after HIV-1 transmission, most in-

fected individuals develop autologous, strain-speciﬁc nAbs to

the transmitted/founder (TF) virus and TF variants (Ariyoshi

et al., 1992; Richman et al., 2003; Wei et al., 2003). The autolo-

gous nAb response drives viral escape and stimulates additional

speciﬁcities of nAbs that neutralize escape viruses (Richman

et al., 2003; Wei et al., 2003). This antibody virus co-evolution

persists throughout infection, and in 20% of individuals, it

leads, after years of infection, to development of high levels of

bnAbs (Doria-Rose et al., 2010; Gray et al., 2011; Liao et al.,

2013a; Tomaras et al., 2011; Walker et al., 2011).

Two recent studies mapped the ontogeny of bnAbs and TF

viruses from the time of transmission to bnAb development

and showed that bnAbs arise from autologous nAb B cell

clonal lineages but that only a small number of the autologous

nAb lineages ultimately evolve to neutralization breadth (Do-

ria-Rose et al., 2014; Liao et al., 2013a). Identiﬁcation of im-

munogens that can induce nAbs against autologous, neutral-

ization-resistant (Tier-2) viruses is a major challenge for HIV

vaccine design, and examples of vaccine-matched, Tier-2

nAb responses elicited by vaccination in primates are few

(Sanders et al., 2015; Willey et al., 2003). Moreover, no vac-

cine-induced Tier-2 nAbs have yet been isolated and charac-

terized, nor have structures of their Env complexes been

determined.

CAP206 is an HIV-infected African individual who later

developed gp41-targeted bnAbs (Gray et al., 2009a; Morris

Cell Reports 14, 1–12, January 5, 2016 ª2016 The Authors 1

Please cite this article in press as: Bradley et al., Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the

Receptor-Binding Site, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.017

et al., 2011). As a critical ﬁrst step in HIV vaccine design, we

sought to map the autologous nAb response and to elicit

Tier-2 nAbs that mimicked this early autologous nAb response

by immunization with Env proteins isolated over the course of

infection. We report here that immunization of rhesus ma-

caques with HIV-1 TF variants from CAP206 induced strain-

speciﬁc nAbs to vaccine-matched Tier-2 autologous viruses

in three of six animals. After only two immunizations, one ma-

caque had a high-titer nAb response that targeted the CD4-

binding site (bs) and mimicked the autologous nAb response

observed in CAP206. We isolated a vaccine-induced nAb

clonal lineage (DH427) that potently neutralized the Tier-2

CAP206 6-month virus and recapitulated the observed plasma

neutralization response. A crystal structure of DH427 in com-

plex with HIV Env showed that DH427 bound close to the

CD4bs but also interacted with variable regions in the HIV

Env (loop E and V5-loop), explaining the restricted neutraliza-

tion breadth and the failure of DH427 to evolve to heterologous

neutralization.

RESULTS

Immunization of Rhesus Macaques Elicits Tier-2

Autologous Neutralization

We tracked the evolution of the CAP206 env gene from the TF vi-

rus until 39 months after transmission (Figure 1A). We selected

the CAP206 TF and six additional representative mutant env

genes from 2-, 6-, 12-, 21-, 24-, and 30-month time points and

produced them as recombinant gp140 oligomers that were pre-

dominately trimers (Figure S1A). The antigenic and functional

epitopes expressed on each of the recombinant CAP206 Envs

were determined by SPR assays (Figure S1B). All Envs bound

to CD4 and mAb A32, which binds well to uncleaved trimers,

and the magnitude of CD4 binding increased in Envs isolated

from later CAP206 time points (Figure S1B). The seven Envs

showed binding to a panel of nAbs and bound to 17b, an anti-

body that binds to the CD4-induced conformation of Env, in

the absence of CD4 (Figure S2). Some Envs lacked binding of

bnAbs that target the CD4bs, and others only reacted weakly,

indicating disruption of canonical CD4bs bnAb epitopes in the

CAP206 Envs.

We immunized six rhesus macaques with a cocktail of

all seven CAP206 recombinant gp140 oligomers and collected

plasma and PBMCs before the ﬁrst immunization and 2 weeks

after each subsequent immunization (Figure 1B).Wetested

plasma from Env-immunized animals for the presence of

anti-HIV neutralizing activity using the TZM-bl pseudovirus

assay. Plasmas collected before the ﬁrst immunization

(week 0) and 2 weeks after the last immunization (week 38)

were tested for neutralization of heterologous, neutralization-

sensitive (Tier-1) isolates MN (clade B) and MW965 (clade

C) and of autologous, Tier-2, vaccine-matched isolates

of the seven CAP206 Envs (Figures 1CandS3A). We

observed potent neutralization titers against the Tier-1 MN

and MW965 viruses in all animals. Low levels of neutralizing

activity against Tier-2 autologous viruses were present in

two animals (Figures 1CandS3B).Oneanimal,rhesusma-

caque 5173, had potent plasma neutralization activity against

the Tier-2 autologous 6-month CAP206 TF variant virus (Fig-

ures 1CandS3B).

Neutralization of the 6-month virus emerged in 5173 plasma

2 weeks after the second immunization and was boosted by re-

petitive immunization and persisted throughout the remainder of

the immunization regimen (Figure 1D). Plasma neutralization re-

sponses waned 20 months after the last immunization (data not

shown). Thus, immunization with CAP206 Envs effectively eli-

cited potent Tier-1 heterologous virus neutralization in all six an-

imals and low levels of Tier-2 nAbs in two of them; in one animal,

it rapidly induced an early and robust neutralization response to

an autologous Tier-2 virus.

Immunization-Induced Neutralization Mimics Early

Autologous CAP206 Plasma Neutralization Response

Autologous Tier-2 neutralization in macaque 5173 was speciﬁc

for the CAP206 6-month virus. Env sequences that were isolated

from later time points post-infection from CAP206 contained

numerous mutations that were candidate sites of immune pres-

sure. Two of the very ﬁrst observed Env sequence changes re-

tained in the 6-month Env were in the V1/V2 and V5 regions

(Figure 2A).

Closer inspection of the V5 sequences revealed extensive di-

versity in this region among longitudinal CAP206 Envs. Specif-

ically, the 6-month Env had a ﬁve-amino-acid deletion and

lacked a predicted glycosylation site at position 463 (Figure 2B).

To determine whether 5173 neutralization of the 6-month

CAP206 virus targeted V5, we engineered a mutation in the

6-month virus that introduced a glycosylation site at position

463 (S463N). We found persistent plasma neutralization of the

wild-type 6-month virus for samples across the full immunization

period but no neutralization of the glycosylation site variant

(Figure 2C).

To examine the role of mutations in the V5 loop in early autol-

ogous escape of the infecting virus in CAP206, we tested

CAP206 plasma isolated post-infection for the ability to

neutralize the autologous 2-month and 12-month viral isolate.

Early plasma samples exclusively neutralized the 2-month virus,

and only later plasma samples acquired neutralization activity for

the 12-month virus (Figure 2D). When we reverted the 12-month

V5 region back to the 2-month sequence, the chimeric virus

became sensitive to neutralization by early CAP206 plasma

time points. Reverting mutations in the V1V2 in the 12-month

back to the 2-month did not have any effect on early neutraliza-

tion. Thus, the V5 loop was a target for autologous nAbs in

CAP206 after infection and a V5-dependent autologous nAb

response was elicited in a rhesus macaque by immunization

with a CAP206 TF variant.

Immunization Elicited CD4bs Autologous nAbs

We isolated single Env-speciﬁc memory B cells from macaque

5173 PBMC using tetramers of the neutralization-sensitive

CAP206 6-month Env as well as the neutralization-resistant

CAP206 30-month Env. Memory cells (CD20

and CD27

) that

bound the 6-month, but not the 30-month, Env were sorted

into individual wells of a 96-well plate (Figure 3A). The frequency

of 6-month Env-speciﬁc memory B cells was 0.07% (Figure 3A).

Single-cell PCR ampliﬁcation and transient expression of

2Cell Reports 14, 1–12, January 5, 2016 ª2016 The Authors

Please cite this article in press as: Bradley et al., Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the

Receptor-Binding Site, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.017

immunoglobulin (Ig) genes of the sorted memory B cells identi-

ﬁed two mAbs (DH427 and DH428) that speciﬁcally bound the

CAP206 6-month Env in ELISA (Figure 3B). As expected from

their sensitivity to changes at position 463, DH427 and DH428

competed for binding of soluble CD4 to CAP206 6-month Env

(Figure 3C). Sequences of the heavy- and light-chain Ig genes

showed that DH427 and DH428 are two members of the same

B cell clonal lineage, which used the rhesus orthologs of the hu-

man V

3-23 and V

2-11 genes (Figures 3D and S4). Members of

the DH427 lineage have short HCDR3s (10 aas); the variable

heavy regions were mutated 4.5% in DH427 and 2.4% in

DH428 (Figure 3D).

DH427 CD4bs Antibody Lineage Neutralized the Tier-2

CAP206 6-Month Virus

DH427 and DH428 neutralized the 6-month virus but did not

neutralize any of the other autologous or heterologous viruses

tested (Figures 4A and S5). Moreover, DH427 and DH428 did

not neutralize the CAP206 6-month virus with an N463 mutation,

recapitulating the pattern of neutralization observed in the 5173

plasma (Figure 4A).

The CAP206 6-month Env lacked a glycosylation site in

V5; introduction of a glycosylation site in V5 of the CAP206

6-month Env eliminated neutralization by both 5173 plasma

and DH427 lineage antibodies. The CAP206 TF Env also lacked

0.003

CAP206 T/F

1 mo

2 mo

6 mo

12 mo

15 mo

21 mo

24 mo

30 mo

33 mo

39 mo

Week

Plasma sample

#1 #2 #3 #4 #5 #6 #7

Immunization

MF59 + CAP206 gp140

(T/F, 2 month, 6 month, 12 month, 21 month, 24 month, 30 month)

Immunization

Tier-1 Tier-2

100

150

200

250

300

350

400

450

02 8 14 20 26 32 38

Week

CAP206 6 month T/F

variant virus

ID50

CAP206 T/F and other

variant viruses

2 month

6 month

12 month

21 month

24 month

30 month

Animal Week B.MN C.MW965 C.CAP206.2mo C.CAP206.6mo C.CAP206.12mo

0 <20 <20 <20 <20 <20

38 990 5499 25 <20 28

0 <20 <20 <20 <20 <20

38 1036 2125 <20 <20 <20

0 <20 <20 <20 <20 <20

38 804 1682 <20 <20 <20

0 <20 <20 <20 <20 <20

38 281 2464 <20 437 <20

0 <20 <20 <20 <20 <20

38 579 1379 <20 <20 <20

0 <20 <20 <20 <20 <20

38 400 1663 38 <20 <20

5160

5165

5167

5173

5183

5184

Figure 1. Immunization with T/F Envs Elicits Tier-2 Neutralization

(A) Neighbor-joining phylogenic tree of isolated env sequences from South African HIV-1-infected individual CAP206 from the time of transmission to 39 months

post-infection. Red boxes indicate envs selected for production of recombinant immunogens.

(B) Immunization regimen; six rhesus macaques immunized seven times every 6 weeks with a swarm of seven CAP206 Envs. Plasma samples were collected

2 weeks post-immunization.

(C) Plasma neutralization of heterologous Tier-1 and autologous Tier-2 viruses before immunization (week 0) and 2 weeks after the last immunization (week 38)

measured as the plasma dilution at which relative luminescence units (RLUs) were reduced 50% compared to control wells in the TZM-bl neutralization assay

(yellow, ID

> 20; orange, ID

> 200; red, ID

> 1,000).

(D) Plasma neutralization over the course of immunization for rhesus monkey number 5173 of the CAP206 autologous Tier-2 viruses (T/F, 2-month, 6-month,

12-month, 21-month, 24-month, and 30-month) in the TZM-bl neutralization assay. Dashed line indicates immunization time points.

See also Table S2.

ing B cell responses to the CD4bs. More-

over, modifying the CAP206 Env to have a

more-closed structure by SOSIP modiﬁ-

cation may also increase immunogenicity.

Such structural considerations coupled

with understanding host control of induc-

tion of nAbs should lead to design of stra-

tegies for inducing autologous nAbs to

Tier-2 HIV with greater heterologous

neutralization breadth (Liao et al., 2013a).

EXPERIMENTAL PROCEDURES

Donor Subject

CAP206 is an HIV-1 subtype C chronically infected

individual used for this study. This participant was

part of the CAPRISA 002 Acute infection cohort

whose antibody neutralization proﬁle has been

studied since the point of seroconversion (Gray

et al., 2007, 2009b). This study was approved by

the IRBs of the Universities of KwaZulu Natal and

Witwatersrand in South Africa and Duke University. Written informed consent

was obtained from all study participants.

Design and Production of Recombinant gp140 Proteins Derived from

CAP206

The cloning and sequencing of env sequences was performed as described

previously from longitudinal samples from CAP206 (L.M., unpublished data;

Figure 5. Reconstruction of the DH427 Clonal Lineage

(A) Binding of DH427, DH428, and the inferred intermediate (I1) and unmutated common ancestor (UCA) to the CAP206 6-month Env measure by SPR. An-

tibodies were immobilized on a CM5 chip, and protein was ﬂowed over at 100 mg/ml.

(B) Binding of the DH427 I1 and I1-UCA hybrids to the CAP206 6-month Env measured by ELISA.

(C) Amino acid alignment of the germline IGVL2-F*01 with DH427 UCA and I1 antibodies compared with IGVL2-F*02 allele discovered by sequencing the IGVL2-F

genomic DNA from animal 5173.

(D) Binding of newly designed DH427 UCA (DH427 UCA2) to the CAP206 6-month Env by SPR. DH427 UCA2 was immobilized on a CM5 chip, and protein was

ﬂowed over at the indicated concentrations.

(E) Neutralization of DH427 I1, UCA2, and chimeric (UCA V

:I1V

) of the CAP206 6-month virus and glycosylation site mutant measured as the antibody con-

centration (mg/ml) at which RLUs were reduced 50% compared to control wells (IC

) in the TZM-bl neutralization assay.

(F) Reactivity of DH427UCA2 and DH427 on a panel of 9,400 human proteins tested using a ProtoArray microchip compared with a nonreactive rhesus mAb. Axis

values are relative ﬂuorescent signal intensity. Each dot represents an average of duplicate array proteins. Dashed lines indicate 500-fold signal/background ratio

as cutoff for reactivity.

See also Figures S6 and S7 and Table S1.

8Cell Reports 14, 1–12, January 5, 2016 ª2016 The Authors

Please cite this article in press as: Bradley et al., Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the

Receptor-Binding Site, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.017

Gray et al., 2007; Keele et al., 2008). The single TF and six mutant clones from

later time points (CAP206 TF, 2 months, 6 months, 12 months, 21 months,

24 months, and 30 months) were selected for production as soluble gp140

trimeric immunogens. The recombinant Envs were expressed in 293T cells

and puriﬁed using lectin and size-exclusion chromatography as described pre-

viously (Liao et al., 2013b).

Immunization of Rhesus Macaques

Six rhesus macaques each received seven intramuscular immunizations at 6-

week intervals with seven CAP206 Env immunogens (100 mg total) in MF59

adjuvant (Novartis). Blood samples were collected 2 weeks after each immu-

nization. All rhesus macaques were housed at Bioqual. All rhesus macaques

were maintained in accordance with the Association for Assessment and

Accreditation of Laboratory Animals with the approval of the Animal Care

and Use Committees of the NIH and Harvard Medical School. Research was

conducted in compliance with the Animal Welfare Act and other federal stat-

utes and regulations relating to animals and experiments involving animals

and adheres to principles stated in the Guide for the Care and Use of Labora-

tory Animals, NRC Publication, 2011 edition.

Antibody Isolation

CAP206 6-month gp140 and CAP206 30-month gp140 proteins were ﬂuores-

cently labeled with AF647 and BV421 (Invitrogen), respectively. Peripheral

blood mononuclear cells (PBMCs) from rhesus monkey 5173 at week 38

were stained with ﬂuorescently labeled antibodies for cell surface markers

and both CAP206 Envs. Memory B cells that were stained positive for the

CAP206 6-month Env and not the CAP206 30-month Env were sorted into sin-

gle wells of 96-well PCR plates containing RT-PCR buffer as previously

described (Wiehe et al., 2014). Antibody variable heavy and variable light

genes were ampliﬁed using nested PCR and puriﬁed and sequenced as

described previously (Wiehe et al., 2014). VDJ arrangements, clonal related-

ness, and identiﬁcation of the intermediate and unmutated common ancestor

were inferred using previously described computational methods (Kepler,

2013; Munshaw and Kepler, 2010).

Sequencing of Germline Variable Region

Genomic DNA was isolated from all six animals from PBMCs at 2 weeks af-

ter the ﬁrst immunization (QIAmp DN A Blood mini kit; QIAGEN). IGLV2 -F se-

quences were ampliﬁed using two independent primer sets. To ensure

ampliﬁcation of non-rearranged variable sequences, both primer sets

reverse primers aligned to sequences present in the non-coding genomic

DNA downstream the V-recombination site. The forward primer for set 1

resided in the IGV2-F leader sequence and upstream of the leader in set

2. The PCR fragments were cloned into a pcDNA2.1 (TOPO-TA kit; Life

Technologies) and transformed into bacteria for sequencing of individual

colonies.

Expression of Recombinant Antibodies

Transient small-scale expression of antibodies was achieved by assembling

VH, VK, or VL sequences with linear cassettes that contain the CMV pro-

moter, respective Ig constant region, and poly A signal sequence using over-

lapping PCR and transfection into 293T cells as described previously (Liao

et al., 2009). Supernatants were directly used to screen for binding of Env an-

tigens in ELISA.

For production of puriﬁed recombinant mAbs, the VH and VL genes from

DH427 lineage antibodies were cloned into expression vectors and expressed

and puriﬁed as described previously (Liao et al., 2011). Site-directed mutagen-

esis of antibody genes was performed using the Quikchange II lightening multi-

site-directed mutagenesis kit (Agilent).

ELISA binding of transiently transfected supernatants and puriﬁed re-

combinant mAbs was performed as described previously (Liao et al.,

2011).

Surface Plasmon Resonance

Surface plasmon resonance assays were performed on a BIAcore 4000 in-

strument, and data analysis was performed with BIAevaluation 4.1 soft-

ware (BIAcore). Anti-gp120 mAbs or sCD4 in 10 mM Na-acetate buffer

(pH 4.5) were directly immobilized to CM5 sensor chips using a standard

amine coupling protocol for protein immobilization. Puriﬁed CAP206 Env

glycoproteins were ﬂowed over CM5 sensor chips at concentrations of

2–100 mg/ml. Binding of CAP206 envelope proteins was monitored in

real time at 25C with a continuous ﬂow of PBS (150 mM NaCl, 0.005%

surfactant P20 [pH 7.4]) at 10–30 ml/min (Alam et al., 2011; Gao et al.,

2014).

Neutralization Assays

Neutralization activities of animal plasma and puriﬁed antibodies were deter-

mined by the TZM-bl cell-based neutralization assay (Sarzotti-Kelsoe et al.,

2014). Tier phenotyping of the pseudoviruses was assayed by sensitivity to

a pool of HIV-infected serum as described previously (Seaman et al., 2010).

Antibody Reactivity with Host Proteins

The polyreactivity of the DH427 lineage was assayed in the AtheNA multi-lyte

system (Zeus Scientiﬁc). The rhesus mAbs were also tested for reactivity with

human host cellular antigens using ProtoArray 5 microchip (Life Technologies)

compared to a rhesus isotype-matched control antibody as described previ-

ously (Yang et al., 2013).

Expression and Puriﬁcation of Proteins for Crystallization

The heavy- and light-chain variable and constant domains of the DH427 and

DH428 Fabs were cloned into the pVRC-8400 expression vector using Not1

and Nhe1 restriction sites and the tissue plasminogen activator signal

sequence. The C terminus of the heavy-chain constructs contained a non-

cleavable 63histidine tag. Fabs were expressed and puriﬁed as described

previously (Fera et al., 2014).

The codon-optimized synthetic construct of the ZM176.66 HIV-1 subtype C

gp120 containing aa 41–492 (HXB2 numbering) DV123 (core) was produced by

GenScript with an N-terminal 63-histidine tag and inserted into the pVRC-

8400 expression vector as described for Fabs. The V5 loop of this gp120

core was mutated to that of the CAP206 6-month envelope by site-directed

mutagenesis using manufacturer’s protocols (Stratagene) and expressed in

293S GnTi-suspension adapted cells using linear PEI. After 5 days of expres-

sion, supernatants were clariﬁed by centrifugation and passed over galanthus

nivalis lectin resin pre-equilibrated with 13PBS. Bound protein was washed

with 13PBS and eluted with 13PBS and 500 mM methyl a-D mannopyrano-

side. The glycoprotein was then deglycosylated overnight at 37C with Endo

H in a buffer of 50 mM sodium acetate (pH 6.0), 5 mM EDTA, 500 mM NaCl,

1mg/ml leupeptin, and 1 mg/ml aprotonin. Deglycosylated gp120 core was

then puriﬁed by gel ﬁltration chromatography in buffer B (2.5 mM Tris [pH

7.5], 350 mM NaCl, and 0.02% sodium azide) using a superdex 200 analytical

column (GE Healthcare). To make the complex for co-crystallization, DH427

and the ZM176.66 gp120 mutant were mixed in a 1.2:1 molar ratio and incu-

bated for at least 1 hr before passing over a superdex 200 preparatory column

(GE Healthcare) in buffer B. Fractions corresponding to the complex were

combined and concentrated to 10 mg/ml for co-crystallization trials.

Crystallization, Structure Determination, and Reﬁnement

All His-tagged Fabs were crystallized at 15 mg/ml, and the DH427/ZM176.66

mutant gp120 core complex was crystallized at 10 mg/ml. Crystals were

grown in 96-well format using hanging drop vapor diffusion and appear ed after

24–48 hr at 20C. DH427 crystals were obtained in a condition of 20% polyeth-

ylene glycol (PEG) 2K monomethyl ether (MME), 100 mM sodium citrate (pH

4.0), and 100 mM NaCl; and DH428 crystals were grown over a reservoir of

40% PEG 400 and 100 mM sodium acetate (pH 5.0). Initial crystals of

DH427/gp120 core complex were obtained in a condition of 1.5 M ammonium

sulfate and 100 mM Tris (pH 8.0) and were optimized to 24-well format to

obtain larger crystals. All crystals were harvested and cryoprotected by the

addition of 20%–25% glycerol to the reservoir solution and then ﬂash cooled

in liquid nitrogen. Crystals of complex were cryoprotected with a series of

different cryoprotectants including 15%–30% glycerol, 15%–30% ethylene

glycol, and 1 or 2 M sodium malonate; however, the best diffraction was ob-

tained with 20%–30% glycerol or ethylene glycol as the cryoprotectant.

Diffraction data were obtained at 100 K from beam lines 24-ID-C and 24-

ID-E at the Advanced Photon Source using a single wavelength. Data sets

Cell Reports 14, 1–12, January 5, 2016 ª2016 The Authors 9

Please cite this article in press as: Bradley et al., Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the

Receptor-Binding Site, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.017

from individual crystals (two crystals for DH427 and one crystal for DH428)

were processed with HKL2000 (Otwinowski and Minor, 1997) and XDS

(Kabsch, 2010) for DH427 and DH428, respectively. Molecular replacement

calculations for the free Fabs were carried out with PHASER (McCoy, 2007),

using I3.2 from the CH103 lineage (PDB ID 4QHL) as the starting model. The

I3.2 model was separated into its variable and constant domains for the

DH427 and DH428 Fab structure determinations. Crystals of the DH427 and

DH428 Fabs had four and one molecules per asymmetric unit, respectively.

For both Fabs, subsequent reﬁnement steps were carried out with PHENIX

(Adams et al., 2010), and all model modiﬁcations were carried out with Coot

(Emsley and Cowtan, 2004). During reﬁnement, maps were generated from

combinations of positional, group B-factor, and TLS (translation/ libration/

screw) reﬁnement algorithms. Secondary-structure restraints were included

at all stages for all Fabs; noncrystallographic symmetry restraints were applied

to the DH427 Fab throughout reﬁnement.

Data from multiple crystals of the DH427/gp120 core complex were pro-

cessed using HKL2000 (Otwinowski and Minor, 1997), and molecular replace-

ment calculations were carried out with PHASER (McCoy, 2007), using the

reﬁned DH427 coordinates and gp120 core from the VRC01/gp120 complex

(PDB ID 4LST) as the starting models. DH427 was separated into its variable

and constant domains for structure determinations. There were two molecules

per asymmetric unit. The resulting electron density map for the complex was

further improved by solvent ﬂattening, histogram matching, and noncrystallo-

graphic symmetry averaging using the program Parrot (Winn et al., 2011).

Phase combination was disabled in these calculations. After density modiﬁca-

tion, rigid-body reﬁnement was performed using Refmac in Coot.

Structure validations were performed periodically during reﬁnement using

the MolProbity server (Davis et al., 2007). The ﬁnal reﬁnement statistics are

summarized in Table S1.

Protein Structure Analysis and Graphical Representations

The core domain of the chimeric ZM176.66 gp120 core mutant designed in our

study was superposed on other gp120 cores from the PDB by least-squares

ﬁtting in Coot. All graphical representations with protein crystal structures

were made using PyMol.

ACCESSION NUMBERS

The accession numbers for the rhesus antibodies reported in this study are

GenBank: KU216183–KU216190. The accession numbers for the structures

of DH427, DH428, and DH427-Env complex reported in this paper are PDB:

5F6H, PDB: 5F6I, and PDB: 5F6J, respectively.

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven ﬁgures and two tables and can be

found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.12.017.

AUTHOR CONTRIBUTIONS

T.B. isolated and characterized antibodies, designed assays, analyzed and

interpreted data, and wrote and edited the manuscript. D.F. conducted

structural and sequence analyses, protein expression, data analyses and

interpretation, and edited the manuscript. J.B. performed CAP206 plasma

analysis. S.S. assisted with animal study and analysis. K.A. and S.M.A.

performed protein puriﬁcation and SPR assays. R.Z. and A.F. contributed to

rhesus PCR and antibody production. X.L. assisted with CAP206 Env protein

production. H.-X.L. led protein and antibody production. X.N. and G.K.

performed Protoarrays. K.E.L., C.S., and R.P. performed antibody-binding

assays. L.L.S., R.M.S., and C.M.B. performed rhesus immunizations. L.M. pro-

vided CAP206 Envs and sequences. S.S.A.K. assisted with clinical protocols.

S.B. provided vaccine adjuvant. M.A.M. and D.C.M. performed neutralization

assays. T.B.K. designed software and performed computational analyses of

antibody sequences and inferred unmutated common ancestors. M.A.M. pro-

duced ﬂuorophor-labeled Env proteins for memory B cell staining; S.C.H. led

structural studies and analysis and edited the manuscript; and B.F.H. de-

signed the study, oversaw all experiments, analyzed all data, and wrote and

edited the manuscript.

ACKNOWLEDGMENTS

This work was supported by the Center for HIV/AIDS Vaccine Immunology-

Immunogen Discovery (CHAVI-ID; UMI-AI100645) grant from NIH/NIAID/

DAIDS. D.F. is supported by a F32 fellowship (1F32AI116355-01) from the

NIH. S.C.H. is an investigator with the Howard Hughes Medical Institute. We

thank Dawn J. Marshall and John Whitesides for expert technical assistance

with ﬂow cytometry and Kelly Soderberg and Samantha Bowen for project

management. We also thank beam line staff at Advanced Photon Source

24-ID-C and 24-ID-E for support during data collection.

Received: August 6, 2015

Revised: November 20, 2015

Accepted: December 4, 2015

Published: December 24, 2015

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Cell Reports

Supplemental Information

Structural Constraints of Vaccine-Induced Tier-2

Autologous HIV Neutralizing Antibodies Targeting

the Receptor-Binding Site

Todd Bradley, Daniela Fera, Jinal Bhiman, Leila Eslamizar, Xiaozhi Lu, Kara Anasti,

Ruijung Zhang, Laura L. Sutherland, Richard M. Scearce, Cindy M. Bowman, Christina

Stolarchuk, Krissey E. Lloyd, Robert Parks, Amanda Eaton, Andrew Foulger, Xiaoyan

Nie, Salim S. Abdool Karim, Susan Barnett, Garnett Kelsoe, Thomas B. Kepler, S. Munir

Alam, David C. Montefiori, M. Anthony Moody, Hua-Xin Liao, Lynn Morris, Sampa

Santra, Stephen C. Harrison, and Barton F. Haynes

Trimer

CAP206 gp140

T/F

2mo

6mo

12mo

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KDa

CAP206 gp140

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Blue Native Page SDS-Page; Reducing

Dimer

Monomer

-200

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-50 0 50 100150200250

sTime

A32

sCD4

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CAP206 T/F gp140 CAP206 2month gp140 CAP206 6month gp140

CAP206 12month gp140 CAP206 21month gp140 CAP206 24month gp140

CAP206 30month gp140

CAP206

A32 T8 sCD4

Figure S1. Expression and Purification of CAP206 Env gp140 Proteins, Related to Figure 1.(A) Blue-native PAGE and SDS-

PAGE of the 7 CAP206 Envs used as immunogens. (B) Binding of sCD4, A32 and control mAb T8 to each of the CAP206 Envs by

SPR.

Figure S2. CAP206 Envs can Bind bnAbs Isolated from Infected Individuals, Related to Figure 1. Binding of bnAbs to the 7

CAP206 gp140 immunogens by ELISA (Dark blue, V1/V2; Aqua,V3-glycan; Red,CD4bs; Orange, Other; Green, gp41).

CAP206 T/F gp140

Log AUC

H58

2G1

CH31

C01

D4/

CAP206 2 month gp140

Log AUC

CH5

PG9

G12

PGT

PGT128

CH1

D4/17

7B2

CAP206 6 month gp140

Log AUC

CH5

H59

CH01

PGT125

T12

CH3

VRC01

sCD4

2F5

4E10

CAP206 12 month gp140

Log AUC

CH01

PGT12

GT1

CH3

CH1

sCD

2F5

CAP206 21 month gp140

Log AUC

H01

PGT128

H31

H106

RC01

7B2

E10

CAP206 24 month gp140

Log AUC

CH58

CH59

T125

CH3

sCD

H59

PGT12

T128

CH31

17B

2F5

4E1

Log AUC

CAP20630 month gp140

Figure S3. Plasma Neutralization of CAP206 Env-immunized Animals, Related to Figure 1. (A) Tier phenotype of CAP206 env

pseudotyped viruses. CAP206 autologous viruses were tested for neutralization by a characterized panel of sera from clade C HIV

infected individuals in the TZM-bl neutralization assay.(B)Plasma neutralization of heterologous tier-1 (B.MN, C.MW965) and

autologous tier-2 (CAP206 T/F, 2mo, 6mo, 12mo, 21mo,24mo, 30mo) before immunization (week 0) and 2 weeks after the last

immunization (week 38) measured as the plasma dilution at which relative luminescence units (RLUs) were reduced 50% compared to

control wells in the TZM-bl neutralization assay(Yellow, ID50 > 20 Orange, ID50 > 200; Red, ID50>1000).

Figure S4. Amino Acid Alignment of DH427 Antibody Lineage, Related to Figure 3.

DH427_UCA EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLEWVSY ISNGGGSTYY ADSVKGRFTI SRDNSKNTLS LQMNSLRAED TAVYYCAKEG WAYFDYWGQG VLVTVSS

DH427_UCA2 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......

DH427_I1 .......... .......... .......... .......... .......... ..IS...... .......... .......... .......... .......... .......... .......

DH427 .......... .......... .......... NS..I..... .......... ..LS.AN... .......... .....Q.... .......V.. ..M....... .S...F.... .......

DH428 .......... .......... .......... T......... .......... ..IS...... T......... .......... ..I....P.. .......... .......... .......

DH427_UCA QAALTQPPSV SKSLGQSVTI SCTGTSNDVG GYNDVSWYQQ HPGTAPRLLI YDVSKRPSGV SDRFSGSKSG NTASLTISGL QAEDEADYYC CSYRSGSTYI FGAGTRLTVL

DH427_UCA2 .S........ .......... ......S.I. ...G...... .S........ .E........ .......... .......... .......... G......... ..........

DH427_I1 .S........ .......... ......S.I. D..G...... .S........ .......... .......... .......... .......... ....T.G... ..T.......

DH427 .S........ .......... ......S.I. A.TG...... .S........ .......... .......... .......... .TD....... ....T.A... ..T...V...

DH428 .S........ .......... .....NS.I. D..G...... .S........ .......... .......... .......... .......... ....T.G... ..T.......

CDR1 CDR2 CDR3

Heavy

Light CDR1 CDR2 CDR3

Virus

DH427

DH428

CAP206 T/F

>50

CAP206 2mo

>50

CAP206 6mo

0.02

CAP206 12mo

>50

CAP206 21mo

>50

CAP206 24mo

>50

CAP206 30mo

>50

B.MN

>50

C.MW965

>50

C.1086

>50

TV1

>50

Q842

>50

Q23

>50

Du172

>50

Du422

>50

Figure S5. DH427 and DH428 Neutralization of Autologous and Heterologous Viruses in the TZM-bl Assay, Related to Figure

4. Values are IC50 of neutralization.

Figure S6. Binding of DH427, DH428 and the Intermediate (I1) and Unmutated Common Ancestor (UCA) to the CAP206

Immunogens by SPR, Related to Figure 5.

CM5 Sensor Chip

Anti-human IgG Fc Antibody

Capture mAbs:

DH427_UCA, DH427_I1, DH427, DH428

CAP206 Envs

-10

100

-50 0 50 100150200250300350400450500

DH427_UCA

DH427_I1

DH427

DH428

-10

100

-50 0 50 100150200250300350400450500

-10

100

-50 0 50 100150200250300350400450500

-10

100

-50 0 50 100150200250300350400450500

-10

100

-50 0 50 100150200250300350400450500

s-10

100

-50 0 50 100150200250300350400450500

Binding response (RU)

CAP206 Env gp140 proteins

T/F 2 month

12 month 21 month

24 month 30 month

Time

L1 L2 VL2-F V-RS

10 20 30 40 50 60 70 80 90

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|....|....| ....|....| ....|...

IGVL2-F*01QAALTQPPSVSKSLGQSVTISCTGTSNDVGGYNDVSWYQQHPGTAPRLLIYDVSKRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCCSYRSGST

5173_1.S....A............................................E............FV....................I...S.......

5173_2.S........................S.I.....................................................................

5173_1.S........................S.I....G.......S.........E......................................G.......

5173_2.S........................S.I. ...G.......S.........E......................................G.......

5173_1.S........................S.I....G.......S.........E......................................G.......

5173_1.......R...G.P............S.I....Y............K.M..A.........................................AGSY.

5173_1...........G.P............S.I....R.........K..K.M..E......................................S..AGSD.

5160_1 .S....A............................................E............FV....................I...S.......

5160_1 .S........................S.I....G.......S........ .E.........................F............G.......

5160_1 .S........................S.I....G.......S.........E..N....................I..............S..A.S..

5160_2 .S........................S.I....G.......S........................................................

5160_1 ......S....G.P............S.I....R.........K..K.M..E..N....................I..............S..A.S..

5160_1.......R...G.P............S.I....Y............K.M..A.........................................AGSY.

5165_1 ..........................S.I............A........................................................

5165_2 .S........................S.I....G.......S.........E.........................................TTS..

5165_1 .........M.G.P............S.I....R.........K..K.M..E......................................S..AGSN.

5167_2 .S....A............................................E............FV....................I...S.......

5167_1 .S....A............................................E............FV........................S..AGSD.

5167_1 ......................A...S.I....G.......S.........E......................................G.......

5167_2 .S........................S.I....G.......S.........E......................................G.......

5167_1 .T....................A...SGIAS.S.................HR..N...........F...S...............I...........

5183_1 .S........................S.I....G.......S.........E......................................G.......

5183_2 .S........................S.I....G.......S...................................... ..........S..AGSN.

5183_1 .S.P.......G.P............S.I.Y..A............K.M..G..N...........................................

5183_1 ...........G.P............S.I....Y.........K..K.M.........................................S..AGSN.

5183_1 .......R...G.P............S.I....Y............K.M..E.........................................AGSY.

5183_1 ...........G.P............S.I....Y.........K..K.M.........................................S..AGSN.

5184_2 ..........................S.I............A........................................................

5184_1 .........M.G..............S.I....G.......S.........E......................................G.......

5184_1 .S........................S.I....G.......S.........E..............................................

5184_2 .S........................S.I....G.......S.........E......................................G.......

5184_1 ......S....G.P............S.I....R.........K..K.M..E......................................S..A.S..

5184_1 .......R...G.P............S.I....Y............K.M..E.........................................AGSY.

5184_1 .........M.G.P............S.I....R.........K..K.M..E......................................S..AGSN.

Log AUC

DH427 I1 I29V D31G G34D S42PS27N

CAP206 6 month Env B

Animal

Week

SSA

SSB

RNP

Scl 70

Jo 1

dsDNA

Cent B

Histone

5160

5165

5167

5173

5183

5184

5160

5165

5167

5173

5183

5184

4E10

50µg/ml

122

190

186

Synagis

50µg/ml

Antibody

(50µg/m l)

SSA

SSB

RNP

Scl 70

Jo 1

dsDNA

Cent B

Histone

DH427

UCA2

DH427 I1

DH427

DH428

4E10

100

192

210

Synagis

Figure S7. Sequencing Genomic DNA to Identify Genomic VL Protein Sequences, Related to Figure 5 (A) Binding of DH427 I1

and DH427 I1 with light chain mutations to the CAP206 6 month Env measured by ELISA. (B) the primer design for determination of

IGVL2-F sequences are shown with leader sequences (blue), coding sequence (red), V recombination site (V-RS; green) and

noncoding genomic regions (black). 2 primer sets were used to ensure amplification of unrearranged germ-line DNA (green and red

arrows). (C) Amino acid sequence alignments of germ-line IGVL2-F alleles from all 6 CAP206 animals compared with the annotated

IGVL20F allele. 1 indicates sequences amplified with primer set 1 (green) and 2 indicated sequences amplified with primer set 2 (red).

Red box highlights residue critical for DH427 binding to the CAP206 Env. (D-E) Plasma (D) and DH427 antibody lineage (E)

screened for reactivity with auto-antigens in the AtheNA ANA assay. Values greater than 100 considered positive (orange), and 4E10

(auto-reactive HIV mAb) and Synagis (RSV-specific mAb) used as positive and negative control, respectively.

Table S1. DH427UCA2 Reactive Host Proteins, Related to Figure 5.

Description

DH427UCA2 MFI

Control MFI

family with sequence similarity 21, member C (FAM21C)

6195

-6

family with sequence similarity 21, member C (FAM21C)

6490

-6

G-protein signaling modulator 3 (AGS3-like, C. elegans)

(GPSM3)

3085

G-protein signaling modulator 3 (AGS3-like, C. elegans)

(GPSM3)

2824

SNAP25-interacting protein (SNIP)

1057

SNAP25-interacting protein (SNIP)

1645

nuclear gene encoding mitochondrial protein(ATP5H)

1230

nuclear gene encoding mitochondrial protein (ATP5H)

1203

WD repeat domain 45 (WDR45), transcript variant 1

452

WD repeat domain 45 (WDR45), transcript variant 1

584

regulator of G-protein signaling 8 (RGS8), transcript variant 1

2709

regulator of G-protein signaling 8 (RGS8), transcript variant 1

2961

lactate dehydrogenase A (LDHA)

1086

lactate dehydrogenase A (LDHA)

868

Microtubule-associated serine/threonine-protein kinase-like

323

Microtubule-associated serine/threonine-protein kinase-like

9011

126

SUPPLEMENTAL INFORMATION

Bradley et al. “Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the

Receptor Binding Site.” (2015)