"Mapping the Specificity of Neutralizing HIV Immune Sera"

1. "Mapping the Specificity of Neutralizing HIV Immune Sera" -mAbs -HIV+ patient sera -vaccine sera

2. Aim: What specificities underlie HIV+ patient sera neutralization? Challenges:*polyclonal*distinguishing neutralizing and non-neutralizing fractions

3. Mapping methods Neutralization mechanism Native PAGE

4. HIV neutralizing antibodies b12 overlaps CD4 binding site 2G12 binds high mann. gp120 gp41 2F5, 4E10, Z13 (MPER region of gp41)

5. Behavior of mAbs in different neutralization assay formats Anti-gp120 Anti-gp41 standard post-CD4/ CCR5 Assay b12 2G12 2F5 Crooks et al. Human Antibodies 14:101 (2005) Binley et al J.Virol. 77:5678 (2003)

6. Mapping HIV+ plasma neutralization *Lack of post-CD4/CCR5 neutralization indicates mostly - gp120 neutralization (not - gp41) Crooks et al. Human Antibodies 14:101-113 (2005)

7. Neutralization mechanism Native PAGE

8. Native PAGE trimer binding assay trimer Infer: trimer binding directly correlates with neutralization P.Moore et al. J.Virol. 80:2515-2528 (2006)

9. 2G12 and soluble CD4 [ligand] µg/ml 2G12 (trimer IC50=0.3ug/ml neut IC50=0.5ug/ml) [ligand] µg/ml soluble CD4 (trimer IC50=0.3ug/ml neut IC50=0.8ug/ml) *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?)

10. Comparison of 2G12 and Z13 [ligand] µg/ml 2G12 (trimer IC50=0.3ug/ml neut IC50=0.5ug/ml) [ligand] Z13 (anti-gp41) (trimer IC50=3ug/ml neut IC50=45ug/ml) *trimer binding is more sensitive than neutralization for detecting anti-gp41 Abs Perhaps more gp41 Ab required before neut effect (3x Abs)?

11. Trimer shifts are mediated by neutralizing plasmas

12. high mannose K L K S I R 290 N T H 600 N I N A V3 T K G G I T N P F I N N S C V A G T T P I N I P C E complex D T R R V C V Q W L K S S A C2 E 610 G H N C F R N A Y W A I I Q T I S R V S T N V I G G G R D W E E L F K G I N I L E G 180 S E L A A L L S T G Q K P L K N C T W D K N 340 590 K V Q N S S N C L S K T D D Y V Q V 620 L T K V Q L D F D K T H N E V T V L R C G G Q G N L I I P P P I S A R I A L A N A F G W I Y C N V N R W Y Y E D H I P I N T N E V2 200 M V N I K V4 A T T L S K R E W L P N 170 Q E E N Q N M V F F G R E I S T A V T F W V I Q N Q E E K S S L A C N P E D T T L K R Q Y Q E R T K I I D S I T R P C3 K V C I T T L T C T D I L K I V N G P S I S W Y L L V L 420 N F N C S T N S P S H T F S V Q C L E G C I Q T S C4 D Y R G L W Y C L F I N T D M K L L N R C F Q Q P I S K K I D E I I E E I Q E I Q N I E D M V A E G S M M E G H Q V 380 S F G N R Q W C E I 370 N N E A Q R Q V1 V L F H E A T M N V C S E S Q 150 M N M R E L T N V T K N G N E N W I N K I N G A N K Q L Q E M T T G Q E Q 430 Y T R Q E S G N V V D I A V E NH2 L L Q P P I P L G L E S P L N L A T T W T V V D H D N P R L C A D K L G H L L G E W I R S Y N A T T V F L T C A E R V Q A A K 460 S W N E S L E A G 670 Y W N D 50 T G K D V A K N D R T I K M V5 T Y D A W W C1 G E L G V I F F G V R T G V D A P P C5 K V M G I Y I Y V V T T S E 490 V NH2 F V E K L W P K A L K R 40 L A R COOH A K V G W V Q R E K R G G T V M A F T P L S L G G A W A 500 Y Gp120 and gp41 mutations in Env trimers. gp120 gp41 ∆2G12 ∆b12 ∆2F5/4E10

13. Shifts of 2G12 and b12 knockout trimers (295 mutant) (368 mutant) WT D2G12 Db12

14. N308 plasma binding to 2G12 and b12 “knockout” trimers (-) b12 (-) b12 N308 titrate N308 titrate *368 (∆b12) mutant knocks out most of HIV+ plasma (N308) neutralization.

15. ....BUT: a fraction of trimer binding doesn’t overlap the b12 epitope N308 titrate ...i.e. something completely different? Gp41 Abs?

16. Summary Neutralizing HIV+ plasma: -368-sensitive -butnot all b12 overlapping b12 2G12 ? 2F5/4E10

17. Plans… *Make additional point mutants *Make clade A, B, C trimers *Investigate vaccine sera

18. NICD Thanks Emma Crooks, Phenix Jiang Devin Shrestha Doug Richman Dennis Burton Lynn Morris Penny Moore James Robinson