"Mapping the Specificity of Neutralizing HIV Immune Sera"

-mAbs-HIV+ patient sera-vaccine sera

Aim:What specificities underlieHIV+ patient sera neutralization?

Challenges:*polyclonal*distinguishing neutralizing and non-neutralizing fractions

Neutralizationmechanism Native PAGE

Mapping methods

HIV neutralizing antibodiesb12 overlaps CD4 binding site

2G12binds high mann.

2F5, 4E10, Z13(MPER region of gp41)

gp120

gp41

Assay b12 2G12 2F5

0

20

40

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120

0.00010.001 0.01 0.1 1 10 50

0

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120

0.00010.001 0.01 0.1 1 10 50

concentration (µg/ml)

BBB

B

B

B

BB

B

B

0

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120

0.00010.001 0.01 0.1 1 10 50

BBBBB

B

B

BB

B

0

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0.00010.001 0.01 0.1 1 10 50

concentration (µg/ml)

E

E

E

EEE

E

EE

E

0

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0.00010.001 0.01 0.1 1 10 50

EEEEE

E

E

EE

E

0

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0.00010.001 0.01 0.1 1 10 50

concentration (µg/ml)

Crooks et al. Human Antibodies 14:101 (2005)Binley et al J.Virol. 77:5678 (2003)

Behavior of mAbs in different neutralization assay formats

Anti-gp120 Anti-gp41

J

JJJJJJ

J

0

20

40

60

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120

0.0010.01 0.1 1 10 50

B

BB

BB

B

0

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0.01 0.1 1 10 50

EEEEE

E

EE

0

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0.01 0.1 1 10 50

standard

post-CD4/CCR5

Mapping HIV+ plasma neutralization

*Lack of post-CD4/CCR5 neutralization indicates mostly - gp120 neutralization (not - gp41)

IC50 titer: human plasma gp120 titer standard post-CD4/CCR5

<50 N308 250,000 9,000 <4050-300 #11H 500,000 1,000 <40

300-1000 L92 1,000,000 400 <401000-3000 L909 1,500,000 320 <40

>3000 R2 600,000 150 <40739 80,000 100 <40A62 100,000 100 <40

TN15 603,000 64 40J864 248,000 50 <40N160 500,000 <40 <40K370 381,000 <40 <40TN11 119,000 <40 <40L503 612,000 <40 <40HIV- <300 <40 <40

spiked human plasmaJ864 248,000 50 <40

J864+b12 N.D. 950 <10J864+2G12 N.D. 120 <10J864+2F5 N.D. 110 71

format

Crooks et al. Human Antibodies 14:101-113 (2005)

Neutralizationmechanism Native PAGE

Native PAGE trimer binding assay

P.Moore et al. J.Virol. 80:2515-2528 (2006)

trimer

mAb (-) b12 b6 2G12 447D P7 X5 HIVIG 2F5 Z13 T2 T3epitope CD4bs CD4bs mann. V3 C1 CD4i gp41 gp41 gp41 gp41

Infer: trimer binding directly correlates with neutralization

2G12 and soluble CD4

[ligand]

soluble CD4(trimer IC50=0.3ug/mlneut IC50=0.8ug/ml)

(-) 0.001 0.01 0.1 1 10 µg/ml

2G12(trimer IC50=0.3ug/mlneut IC50=0.5ug/ml)

µg/ml(-) 0.3 1 3 10 30 100

*3 x 2G12's bind per trimer*direct correlation to trimer binding and neutralization IC50s*2G12 saturates over a large concentration range; sCD4 is "all or none" (co-operative?)

[ligand]

Z13 (anti-gp41)(trimer IC50=3ug/mlneut IC50=45ug/ml)

Comparison of 2G12 and Z13[ligand]

2G12(trimer IC50=0.3ug/mlneut IC50=0.5ug/ml)

µg/ml(-) 0.3 1 3 10 30 100

[ligand]

(-) 0.3 1 3 10

*trimer binding is more sensitive than neutralization for detecting anti-gp41 AbsPerhaps more gp41 Ab required before neut effect (3x Abs)?

Trimer shifts are mediated by neutralizing plasmas

-neut IC501/plasma diln - 10 100 1,000 10 100 1000

N308 L503<1:401:3,000

V E K L W V T V Y Y G V P

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SR

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RW

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WV

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D

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M

G

GGG

P

P

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F

I

I

E

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A

T

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GG

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N

N

N

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I I

ID

DL

LL

L

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NH2 490

500

COOH

complex

high mannose

V5

C4

V4

V3

V2

V1

C1

C2

C5

C3

420

370

170

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290

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E

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LLL

W

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590

600

A

620

610

NH2

670

Gp120 and gp41 mutations in Env trimers.

∆2G12

∆b12

∆2F5/4E10

gp120 gp41

Shifts of 2G12 and b12 knockout trimers

EnvmAb (-) 2G12 b12 sCD4 (-) 2G12 b12 sCD4 (-) 2G12 b12 sCD4

WT Δ2 12G Δ 12b

E EE

EE

E

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mAb conc (ug/ml)

b12

E 2G12

E E E E E E

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mAb conc (ug/ml)

b12

E 2G12

EE E

E E

E

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mAb conc (ug/ml)

b12

E 2G12

WT Δ2G12 Δb12

(295 mutant) (368 mutant)

N308 plasma binding to 2G12 and b12 “knockout” trimers

(-) b12 (-) b12N308 titrate N308 titrate

*368 (∆b12) mutant knocks out most of HIV+ plasma (N308) neutralization.

....BUT: a fraction of trimer binding doesn’t overlap the b12 epitope

N308 titrate(-) b12 Fab fixed @ 30ug/ml

...i.e. something completely different? Gp41 Abs?

Summary

b12

2G12

2F5/4E10

Neutralizing HIV+ plasma:-368-sensitive-but not all b12 overlapping

?

Plans…

*Make additional point mutants*Make clade A, B, C trimers *Investigate vaccine sera

Thanks

Dennis Burton

Emma Crooks, Phenix JiangDevin Shrestha

Doug Richman

NICDLynn MorrisPenny Moore

James Robinson