1

Supporting Information SI Materials and Methods Plasma IgG adsorptions on SF162 gp120- and SF162 gp120D368R-coated beads. Plasma adsorptions were performed as previously described (1, 3), with some modifications, and the flow of the adsorptions is outlined in Figure S1. Briefly, total IgG was isolated by protein A chromatography (Pierce, Rockford Il, USA) from VC10042 plasma drawn 22 years post-infection. The purified total IgG was serially adsorbed onto SF162 gp120-coated beads (MyOne Tosylactivated Dynabeads, Invitrogen, Carlsbad, CA, USA), and the flow through collected. The antibodies bound to the gp120 coated beads were eluted by vortexing in increasingly acidic 0.1M glycine solutions, followed by buffer exchange into PBS. The anti-gp120 Ab fraction was then serially adsorbed onto SF162 gp120D368R-coupled beads to remove Abs that do not bind the CD4-BS. Each fraction described above was tested for residual neutralizing activity against 4 clade B, 3 clade C, and 2 clade A isolates in the TZM-bl neutralization assay. The depleted total IgG and the gp120D368R depleted anti-gp120 fractions were tested for the presence of anti-CD4-BS antibodies, and the absence of non-CD4-BS gp120 Abs by Luminex assay (Luminex Corporation, Austin, TX, USA) against both wild type SF162 gp120 and SF162 gp120D368R.

Figu

Figusubjeadsobeadisolaafterand tbindNAbgp12showSF16

ure S1

ure S1. Conect VC10042

orbed onto SFds, the flow tates. The var depletion wthen serially

d to the CD4-bs) were test20 antibody wn in the sec62 gp120D36

ntribution of 2. IgG was F162 gp120-through was lues in the fi

with gp120. y adsorbed on-BS. The aned against thfraction afte

cond column8R.

anti-CD4-Bfirst isolated-coated magtested for re

irst column rAnti-gp120 nto magnetic

nti-gp120 anthe 9 isolates er depletion wn. All deplet

S NAbs to thd from heat-tgnetic beads.esidual neutrrepresent theantibodies wc beads coattibodies anddescribed ab

with gp120Dtions were co

2

he overall netreated plasm After 6 rouralizing active percent reswere eluted fted with SF1d the gp120Dbove. The p

D368R (the neuonfirmed by

eutralizing pma, and thenunds of seriavity against 4sidual neutrafrom the SF162 gp120D36

D368R flow thrpercent residutralizing ac Luminex as

potential of pn antibodies tal adsorption4 clade B, 3

alizing activi162 gp120-c68R to removrough (conta

dual neutraliztivity due tossay against

plasma isolatthat bind to

n with gp120clade C, and

ity against eacoated magnve antibodiesaining the anzing activity

o anti-CD4-BSF162 gp12

ted from gp120 were

0-coated d 2 clade A ach isolate

netic beads, s that do not nti-CD4-BS y of the anti-BS NAbs) is 20 and

Figu

FiguCD4

ure S2

ure S2. A) L4+ and CD8+

Log10 viral R+ T cells per

RNA copies r microliter o

per ml of blof blood duri

3

lood during ing observat

the time of otion.

observation.

B) The nuumber of

Figu

Figucollecladevariaacid circl

ure S3

ure S3. Full ected approxe B sequencable loops Vresidues tha

les.

length gp16ximately 19 ye using Clus

V1-V5. Sitesat are known

60 amino aciyears post instal W. The s that are varn to contact C

id alignmentnfection. Repgp120, gp41

riable amongCD4 are mar

4

t of the ‘earlypresentative1 and signal g the VC100rked with red

y’ clones isoe Env clones

peptide port042 clones ard circles and

olated from p were alignetions of Envre highlighted VRC01 are

plasma that wed with the cv are labeled,ed in yellow.e marked wit

was consensus , as are the . Amino th blue

Figu

Figuapprsequloopresid

ure S4

ure S4. Full roximately 2uence using Cs V1-V5. S

dues that are

length gp162 years postClustal W. Tites that are known to co

60 amino acit infection. RThe gp120, gvariable amontact CD4 a

id alignmentRepresentatigp41 and sigong the clonare marked w

5

t of the ‘late’ve Env clon

gnal peptide nes from VCwith red circ

’ clones isolnes were alig

portions of EC10042 are hcles and VRC

ated from plgned with theEnv are labe

highlighted inC01 are mar

lasma that we consensus eled, as are tn yellow. Arked with blu

was collectedclade B

the variable Amino acid ue circles.

d

Figu

FigusubjewereMEG(JTTis locmapp100-19 y

ure S5

ure S5. Phyect VC10042e tested for thGA5.1(4) usT) substitutiocated in the ped, with no-250 coloredears post-inf

ylogenetic tr2 at 19 yearsheir neutraliing Maximu

on model andbottom left.

on-neutralized in orange, afection. (B) N

ree showing s (A) and 22zation susce

um Likelihood utilizing un The potenc

ed isolates leand IC50 titeNeutralizatio

the relations2 years (B) peptibility of Vod with 100 niform ratescy with whiceft uncoloreders above 25on with plas

6

ship betweenost infectionVC10042 plbootstrap rep. Bootstrap

ch the plasmad, IC50 titers50 colored inma isolated

n the autologn and env clolasma. Phyloplications, uvalues are lia is able to ns below 100 n red. (A) Ne

at 22 years p

gous env cloones from hogenies were

utilizing the Jisted at eachneutralize a gcolored blue

eutralization post-infectio

ones circulatiheterologous e reconstrucJones-Taylo

h node and thgiven isolatee, IC50 titerwith plasma

on.

ing in isolates that

cted using or-Thornton he scale bar e is heat s between a isolated at

t

Figu

Figuin Vmapprelev

ure S6

ure S6. The C10042. Reped on to thevant features

regions on gesidues R373e HXB2 cors.

gp120 that m3 and N386 re molecule i

mediate escapreside outsidin the b12-bo

7

pe from the de the CD4 bound state (2

neutralizingbinding pock2). Inset is s

g activity of tket (inset). Aslightly rotat

the anti-CD4All residues ted to better

4-BS NAbs were view

Figu

Figumonmuta(♦).

ure S7

ure S7. Neunoclonal broaations were iA double m

utralization seadly neutraliintroduced in

mutant, H364

ensitivity of zing antibodnto the wild

4s.R373M (o

f the clone Vdies b12 (A)type VC100

o) was also g

8

VC10042.y22

, VRC01 (B042.y22.05 (generated.

2.05 bearing ), and NIH4

(■) clone: H3

reversion m45-46G54W (C364S(▲) , R

mutations to tC). Three revR373M (●), a

the version and N386D

9

References: 1. Li, Y., S. A. Migueles, B. Welcher, K. Svehla, A. Phogat, M. K. Louder, X. Wu, G. M. Shaw, M.

Connors, R. T. Wyatt, and J. R. Mascola. 2007. Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat Med 13:1032-4.

2. Liu, J., A. Bartesaghi, M. J. Borgnia, G. Sapiro, and S. Subramaniam. 2008. Molecular architecture of native HIV-1 gp120 trimers. Nature 455:109-13.

3. Sather, D. N., J. Armann, L. K. Ching, A. Mavrantoni, G. Sellhorn, Z. Caldwell, X. Yu, B. Wood, S. Self, S. Kalams, and L. Stamatatos. 2009. Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection. J Virol 83:757-69.

4. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731-9.