1. Natural control of HIV infection is associated with an isotype switched IgG antibody response to HIV Gag antigens in patients with 'non-protective' HLA-B alleles Martyn A French 1, Rob J Center 2, Kim M Wilson 3, Ibrahim Fleyfel 1, Sonia Fernandez 1, Anna Schorcht 2, Ivan Stratov 2, Marit Kramski 2, Stephen J Kent 2, Anthony D Kelleher 4 1. University of Western Australia, Perth, Australia 2. University of Melbourne, Melbourne, Australia 3. NRL, Melbourne, Australia 4. University of New South Wales, Sydney, Australia Your logo

2. CD8 + T cells and ‘protective’ HLA-B molecules (HLA-B*5701, -B2705, -B*14+Cw0802, -B*52) NK cells (KIR and HLA-Bw4 or HLA-C) Plasmacytoid dendritic cells Antibodies - Antibody-dependant cell-mediated cytotoxicity? - Antibodies to Gag proteins? - IgG2 antibodies? Immune control of HIV infection

3. Diversification of antibody responses through immunoglobulin isotype switching Abbas et al. 2007 IgMIgG Subclasses IgE IgA IFN-  IL-4 APRIL, BAFF, TGF-β, IL-10 IL-21 T FH IgG3 IgG1 IgG2 IgG4

4. Polysaccharide antigens induce a predominantly IgG2 antibody response Preferential binding to Fc  RIIa Deglycosylation of Fc region does not decrease binding to Fc  RIIa Covalent dimerisation Dominant IgG subclass in plasma immune complexes Functional characteristics of IgG2 antibodies

5. HIV Patients 32 HIV controllers, including 14 elite controllers 21 non-controllers with RNA >10,000 cps/mL and CD4 + T cell count <100/  L Methods IgG1 and IgG2 Abs to HIV proteins by WB assay IgG1 and IgG2 Abs to gp140 Env protein and rp55 by ELISA NK cell-mediated ADCC activity to gp140 Env protein and Env and Gag peptides Sequenced-based HLA typing on genomic DNA Patients and Methods

6. IgG1 antibodies to all HIV proteins, except gp41, are higher in HIV controllers P = 0.002P < 0.001 P = 0.006P < 0.001P = 0.440P < 0.001P = 0.001

7. Gag antigens dominate in the IgG2 antibody response against HIV P = 0.014P = 0.034P = 0.045

8. IgG2 antibodies to rp55 were only detected in HIV controllers controllersnon controllers neg 0.1 0.2 0.3 0.4 0.5 0 0.6 OD450 IgG1 0.2 0.4 0.6 0.8 1.0 1.2 controllersnon controllers OD450 neg IgG2 P=0.09P=0.14 IgG1 anti-rp55IgG2 anti-rp55

9. IgG1 and IgG2 antibodies to gp140 Env protein did not differentiate controllers and non-controllers controllers non-controllers P = 0.12 Endpoint titer (log10) controllersnon-controllers P = 0.34 Endpoint titer (log10) non-controllers IgG2 anti-gp140 Env proteinIgG1 anti-gp140 Env protein

10. HIV controllers exhibit higher ADCC activity to gp140 Env protein ADCC activity against gp140 Env protein was significantly higher in elite controllers than non-controllers (p=0.03)

11. Antibody responses to HIV proteins in controllers without or with ‘protective’ HLA-B alleles Antibody responseHIV Controllers without protective HLA-B alleles # (median, IQR) HIV Controllers with protective HLA-B # alleles (median, IQR) P value IgG1 anti-p184 (3-4)3 (2-4)0.11 IgG1 anti-p244(3-4)4 (2-4)0.65 IgG1 anti-p323.5 (2.25-4)2 (2-3)0.04 IgG1 anti-p514 (2.25-4) 0.65 IgG1 anti-p664 (4-4)4 (3.25-4)0.42 IgG1 anti-gp414 (4-4) 1.0 IgG1 anti-gp1203.5 (2-4)3 (2.25-4)0.95 IgG2 anti-p181.5 (1-3)1 (1-2)0.10 IgG2 anti-p241.5 (0-3.75)0 (0-2)0.16 IgG2 anti-gp411 (1-2) 0.91 ADCC antibodies to gp140 Env protein 0.218 (0.024 - 0.412)0.183 (0.048 - 0.634)0.51 # = HLA-B*57, -B*27, -B*52, B*14+Cw0802

12. IgG2 antibodies to p24 are higher in HIV controllers who do not carry HLA-B*57 IgG2 anti-p24 IgG1 anti-p18IgG1 anti-p32 IgG2 anti-p18

13. High-level IgG2 antibodies to one or more Gag protein (p18, p24, rp55) are most common in HIV controllers not carrying ‘protective’ HLA-B alleles IgG2 anti-GagHLA-B*57 or B*52HLA-B*27 or B*14 No ‘protective’ HLA-B allele Yes249 No1043 p=0.016 and 0.004 for trend

14. IgG2 antibodies to HIV Gag proteins may contribute to control of HIV infection Preferential binding to Fc  RIIa (major Fc  R on pDCs) Deglycosylation of Fc does not decrease binding to Fc  RIIa Phagocytosis-enhancing antibodies (bacteria, ?viruses) Such antibodies might enhance innate responses to HIV and/or adaptive immune responses to Gag Strategies for vaccination with HIV Gag proteins might include enhancement of IgG isotype switching Summary and Conclusions

16. TLR7 Gag + RNA IgG1 Fc  RIIa IFN-  and IFN- Interferon-stimulated genes pDC-dependant NK cell activation IRF-7 CD8 + T cell CD4 + T cell Pro-inflammatory cytokines IgG2 IgG3 MHC I MHC II = Endosomes B cell CD70 X NF  B Induction of innate responses to HIV and/or adaptive responses to Gag by pDC and isotype- switched IgG antibodies: a hypothesis French MA, 2012 (unpublished)

17. Control of HIV replication was better in patients possessing ‘high affinity’ Fc  RIIa genotypes who produced IgG2 anti-p24 after vaccination with FPV/Gag-Pol/IFN-  DNA P=0.002 for FC recipients ( o ) P=0.0001 for all patients French MA et al. AIDS 2010;24:1983-90