1. Partners AIDS Research Center Massachusetts General Hospital
HLA-B27-Associated Escape Mutations Dramatically Reduce Viral Fitness of HIV-1
Arne Schneidewind
Partners AIDS Research Center
Massachusetts General Hospital
Boston

2. Background
• HLA-B27 is a “protective” MHC class I allele in HIV infection
- Genetic effect observed late in disease (Gao et al, Nat Med., 2005)
- immunodominant CD8 response targets KK10 in p24 capsid
- CTL escape mutations in KK10 occur very late in disease
• Studies have illustrated that drug resistance mutations and CD8 escape mutations can impact viral replication
Hypothesis
The effect of CD8 escape mutations on viral fitness may contribute to immune control associated with HLA-B27

3. Goals
• Examine the impact of CD8 escape mutations in B27- KK10 (KRWIILGLNK) on HIV-1 replicative fitness
• Assess the ability of compensatory mutations to rescue the fitness defects of a CD8 escape mutation
• Explore mechanisms for the late escape from this immunodominant CD8 T cell response

4. Viral Escape in B27-KK10
• Various CD8 escape and compensatory mutations in KK10 have been previously described :
Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001;
Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005
Upstream mutation 90 aa

5. Viral Escape in B27-KK10
• Various CD8 escape and compensatory mutations in KK10 have been previously described :
Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001;
Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005
Upstream mutation 90 aa
• L268M occurs early after infection and is a weak CD8 escape mutation

6. Viral Escape in B27-KK10
• Various CD8 escape and compensatory mutations in KK10 have been previously described :
Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001;
Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005
Upstream mutation 90 aa
• Mutation R264K at the anchor position P2 diminishes binding to HLA-B27 and represents the predominant CD8 escape mutation in KK10

7. Viral Escape in B27-KK10
• Various CD8 escape and compensatory mutations in KK10 have been previously described :
Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001;
Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005
Upstream mutation 90 aa
• An upstream compensatory mutation (S173A) has been proposed
- Kelleher / Brander (manuscript in preparation)

8. Compensatory Mutation lies in Proximity to Escape Mutations
N-term
p24 capsid

9. Compensatory Mutation lies in Proximity to Escape Mutations
N-term
p24 capsid
R264K
L268M

10. Compensatory Mutation lies in Proximity to Escape Mutations
N-term
p24 capsid
S173A

11. Escape Mutation R264K Reduces Infectivity
- 12%
- 96%
Replace with powerpoint title
Xhrs post infection
Assay to measure p24

12. Escape Mutation R264K Reduces Replicative Capacity
can we split this into 2 figures?
First with WT, LM, RK, SA
2nd adding in RKLM, SARKLM
Let’s talk about the insert too...
Need to highlight SA reduction too

13. Escape Mutation R264K Reduces Replicative Capacity
can we split this into 2 figures?
First with WT, LM, RK, SA
2nd adding in RKLM, SARKLM
Let’s talk about the insert too...
Need to highlight SA reduction too

14. Escape Mutation R264K Reduces Replicative Capacity
can we split this into 2 figures?
First with WT, LM, RK, SA
2nd adding in RKLM, SARKLM
Let’s talk about the insert too...
Need to highlight SA reduction too

15. Escape Mutation R264K Reduces Replicative Capacity
can we split this into 2 figures?
First with WT, LM, RK, SA
2nd adding in RKLM, SARKLM
Let’s talk about the insert too...
Need to highlight SA reduction too

16. Reduces p24 Production on PBMCs
Escape Mutation R264K
Reduces p24 Production on PBMCs

17. Escape Mutation R264K doesn’t alter Release of p24

18. Escape Mutation R264K doesn’t alter Cleavage of GagWT RK
SARKLM WT RK
SARKLM
p24 
p55 
p24 
supernatant
intracellular

19. Conclusions
CD8 escape mutation R264K exhibits a dramatic defect in infectivity and replicative capacity
Upstream mutation S173A in helix 2 effectively compensates for the fitness defect of R264K
• The dramatic fitness cost of R264K, and the requirement for a simultaneous compensatory mutation, may explain the late escape from this CD8 response
R264K variant seems to be blocked at an early step of the replicative cycle
Add in the ability of other upstream compensatory mutations to rescue other KK10 mutations (data not shown)

20. This project was supported by NIH Grant R21 AI067078
Acknowledgment
Mark A. Brockman
Sylvie Le Gall
Rahma I. Adam
Matthew P. Lahaie
Christian Brander
Tony Kelleher
Bruce D. Walker
Todd M. Allen
This project was supported by NIH Grant R21 AI067078