AIDSVaccine2010 Atlanta, Georgia Willy Bogers. NIH HIVRad Grant nr 5P01AI066287

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1 HIV-1 envelope-cd4 receptor complexes elicit broad T- and B- cell immune responses as well as cross-reactive neutralizing antibodies in Rhesus macaques NIH HIVRad Grant nr 5P01AI AIDSVaccine2010 Atlanta, Georgia Willy Bogers B I O M E D I C A L P R I M A T E R E S E A R C H C E N T R E, R I J S W I J K, T H E N E T H E R L A N D S

2 Stabilizing the CD4-bound Conformation of HIV Env Use scyllatoxin scaffold Improve Optimization CD4M48 Transfer CD4 binding site Interface with gp120 by combinatorial chemistry gp120 interface CD4M9 Gp120-CD4 binding inhibition CD4M33 Exploration of Phe43 cavity CD4 % fixation Binding % CD4M9 CD4M33 CD4M48 scd4 M48-U1 Van Herrevege et al (2008) J. Antimicrob. Chemother. 61 : 818 Patent IB2006/ M48-U Conc. (M) Using the optimized mini-cd4, we produced a complex which stabilized the CD4-bound conformation of gp120 (and gp140). gp120 -Claudio Vita -Gregoire Martin -Loïc Martin CEA, France M48-U1-SH = ML106

3 NHP CD4ML106-Env Immunization Study Examining the protective role of CD4i NAbs Hypothesis: gp140-cd4mimi complex Immunization induces broader Immune responses then protein only Immunization Group N= Protein Dose (µg) Adjuvant Route Time (Weeks) 1 6 o-gp140 V2 SF MF59 IM 0, 4, 24, CD4ML MF59 IM 0, 4, 24, CD4ML106/o-gp140 V2 100 MF59 IM 0, 4, 24, MF59 IM 0, 4, 24, 36

4 Cellular Responses Following Immunization IFNγ T cell ELIspot

5 Cellular Responses Following Immunization IL2 T cell ELIspot

6 Cellular Responses Following Immunization IL4 T cell ELIspot

7 Cellular Responses Following Immunization Mean T cell ELIspots

8 Humoral Responses Following Immunization Serum binding titers A. Mean titers of binding antibodies against gp120 and minicd4 antigens. Only the protein immunized group (group1) and the complex group (group 3) developed anti-gp120 responses. B. The area under the curve (AUC) for all individual animals has been given separately.

9 Humoral Responses Following Immunization Anti-gp120 specific B-mem ELIspot 2wP2 2wP3 2wP4 t = 26: p = 0,0029 (Group 1 vs Group 3) t = 38: p = 0,037 (Group 1 vs Group 3) A. Box-whisker plots showing the ranges and medians (horizontal lines) of anti-gp120 Bmemory responses (expressed as Spot Forming Units per 10 6 PBMC of the various groups at indicated time points. B. The total amount of antigen specific Bmemory responses (expressed as the area under the curve) in the complex immunized group 3 is significantly higher as compared with the protein only immunized group 1.

10 Neutralization Titers (ID50) scd4 at 2wp3 Pseudovirus virus (BPRC)

11 Neutralization Titers (ID50) scd4 at 2wp4 Replicating infectious virus (BPRC) * * * ID50 is <20 for all control animals lowest dilution tested was 540 *

12 Neutralization Titers (IC50) ±scd4 at 2wp4 Pseudoviruses from SHIV challenge strains (Montefiori) Clade B Clade C Clade C

13 Conclusions -Both gp140-cd4mimetic Complex and gp140 Protein immunization induces strong T-cell responses (IFNγ, IL4 and to lesser extend IL2). -Complex immunization induces better T helper responses then Protein only immunization -Complex immunization induces earlier and higher antigen specific Ab secreting B- cell responses than protein only immunization - both protein only and gp140-cd4mimetic Complex immunization induces homologous, heterologous (clade B) and cross-clade neutralization. The Complex group has a broader neutralization spectrum

14 Acknowledgements Novartis, Cambridge, USA: -Antu Dey -Yide Sun -Brian Burke -Indresh Srivastava -Susan Barnett CEA, France: -Gregoire Martin -Loïc Martin Duke University, Durham, USA: -David Montefiori Cambridge University, UK: -Rachel Pei-Jen Lai -Jonathan Heeney Dana-Farber Cancer Institute, Boston, USA: -Nagadenahalli Siddappa -Ruth Ruprecht B I O M E D I C A L P R I M A T E R E S E A R C H C E N T R E, R I J S W I J K, T H E N E T H E R L A N D S

15 Department of Virology, BPRC

16 Humoral Responses Following Immunization Inhibition/competition assay: sera 2 wk post 4th Imm -Rachel Pei-Jen Lai, -Jonathan Heeney Cambridge University, UK Percentage of residual monoclonal antibody binding that has not been outcompeted by the individual rhesus macaque sera. Lower percentage is equivalent to having higher affinity/stronger competition of the animal sera. Both group 1 and 3 had similar levels of antibodies against the epitopes in the CD4bs, CD4i and gp120 V3.

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