RV305 ADCC Update 10-February-2016 G. Ferrari

cell entry or virus budding ADCC antibodies can potentially target CD4 T cells at two different steps of HIV-1 virus life cycle: cell entry or virus budding Virus budding – Infected Cells Cell Entry – gp120 coated cells Zolla Pazner S. Nature Reviews Immunology (2004)

Methods CEM.NKRCCR5 cells were coated with the A244∆11 gp120 to generate target cells used for the flow-based assay (GTL). CEM.NKRCCR5 cells were also infected with 92TH023, CM235, and 427299 IMC to generate target cells used for the luciferase based assay (Luc). We utilized a single normal donor with the Fcγ-R IIIa F/V as source of effector cells. The 70 samples collected at visit 2 (pre-immunization), 3 (2 wks post 1st boost), and 5 (2 wks post 2nd boost) were tested starting at the 1:50 dilution with a 5 fold-scheme to the 1:102,400. ADCC responses were considered positive if % Granzyme B activity was >8% and titer of Ab >1:50 for the GTL assay. ADCC responses were considered positive if % Specific killing was >15% and titer of Ab >1:200 for the Luc assay. Data were analyzed for peak and titer ADCC response.

Percentage of responders against gp120-coated target cells Recall ADCC responses were detectable in 95 and 100% of Group 1 and 2 vaccine recipients at visit 3, respectively; they declined to 85 and 79% at visit 5, respectively. ADCC responses were not detectable in Group 3.

Peak (left) and Titers of ADCC responses (right) against A244∆11 gp120-coated target cells. Conclusions I The peak and titer of responses were significantly higher at visit 3 and 5 compared to pre-boosting in Group 1 and 2. The peak and titers of ADCC responses were not significantly different in Group 1 compared to group 2 at visit 3 and 5. The peak and titers in Group 1 and 2 were always significantly higher than in Group 3 at visit 3 and 5.

Do the polyclonal ADCC-mediating Ab recruit different populations of effector cells? Can we identify which effector cells are recruited using our in vitro ADCC-GTL assay?

Cellular staining with anti-CD14 Ab after 1 hour incubation of target and effector cells in the Granzyme B assay indicates that doublets are CD14+ positive. %CD14+ Doublets = Monocytes %CD14+ Singlets = NK CD14 Orlandi C., Pollara J., Lewis G., Ferrari G., unpublished - Confidential

Comparison of recruitment of NK and monocytes by polyclonal ADCC responses in RV144 vs RV305 The polyclonal ADCC Ab responses in RV305 at visit 3 have the tendency to recruit more NK cells than the RV144 responses at visit 8.

Tier ADCC: we have defined the susceptibility of 22 HIV-1 Envs to plasma ADCC responses (n=29) and identified their hierarchy to define the breadth of ADC responses. Arrows in green identified already tested IMC and in orange ongoing testing.

Percentage of responders based on HIV-1 IMC

Peak (at 1:250 dilution; left) and Titers of ADCC responses (right) against HIV-1 IMC-infected target cells.

Conclusions II The HIV-1 Envs were recognized according to the hierarchy observed using the plasma from infected individuals, i.e. CM235>92TH023>427299. The peak and titer of responses were significantly higher at visit 3 and 5 compared to pre-boosting in Group 1 and 2. The peak and titers of ADCC responses were not significantly different in Group 1 compared to Group 2 at visit 3 and 5. The peak and titers in Group 1 and 2 were always significantly higher than in Group 3 at visit 3 and 5.

Summary The RV305 vaccine regimen was able to boost the ADCC responses induced by RV144. The ADCC Ab titers detected after the first immunization were higher than those detected after the second immunization. The ADCC polyclonal Ab responses detected after the first RV305 boost were able to recruit more NK cells that those originally elicited by the RV144 vaccine regimen. Path forward We are investigating the breadth of these responses using target cells infected with IMC representing the subtype B (SF162) and C (TV-1 and 1086.c). We are performing additional analyses of the potency of the neutralizing and non-neutralizing mAbs generated from the RV305 vaccine recipients.