Cristian Apetrei Center for Vaccine Research , University of Pittsburgh , Pittsburgh PA, USA

1. “Towards an HIV Cure” Pre-Conference Symposium 20 & 21 July 2012 SIVagm infection of rhesus macaques: a model of functional cure with persistent reservoirs of replication-competent virus Cristian Apetrei Center for Vaccine Research, University of Pittsburgh, Pittsburgh PA, USA

2. Background-Definitions (1) • HIV Eradication/Cure: complete eradication of HIV and infected cells from the body • Functional HIV cure: control of HIV infection without complete HIV eradication • undetectable viremia without ART • no disease progression • no CD4 T cell loss • lack of HIV transmission

3. Background (2) • Obstacles for an HIV cure include: • Rapid establishment of latently infected cells • Residual viral replication (hamper a proper characterization of the virus from the reservoirs) • Existence of anatomic reservoirs (privileged sites of latency insufficiently penetrated by drugs) • Obstacles for cure research: • Ethical (cannot stop therapy-knowing the risks of emergence of drug-resistant strains) • Technical: no marker for latently infected cells • Lack of an animal model (SIVmac/RM is difficult to control with ARVs, infection with molecular clones does not permit tracking the virus etc) An animal model for HIV cure/functional cure is badly needed

4. Background-Definitions (3) An animal model is a living, non-human animal used during the research and investigation of human disease, for the purpose of better understanding the disease without the added risk of causing harm to an actual human being during the process. The animal chosen will usually meet a determined taxonomic equivalency to humans, so as to react to disease or its treatment in a way that resembles human physiology as needed. (Wikipedia)

5. SIVagm infection of RMs Model of functionally-cured SIV infection Plasma Viral Loads Mucosal CD4+ T cells High levels of viral replication during acute infection, followed by complete control during the chronic stage (6 years p.i.) Massive mucosal CD4+ T cell depletion during acute infection, followed by COMPLETE mucosal CD4+ T cell restoration

6. Control of SIVagm in RMs is a functional cure and not an elite control Seroreversion of anti-SIVagm binding and neutralizing antibodies. To date, the only other case of seroreversionwas recorded in the Berlin patient

7. Functional cure of SIVagm infection in RMs • Control of SIVagm replication • Complete restoration of CD4+ T cells, including at mucosal sites (demonstrating that if virus replication is completely controlled, restoration is possible) • Control of apoptosis at mucosal sites resulting in • Control of microbial translocation resulting in • Normalization of immune activation • Seroreversion • Normalization of innate immune effectors (mDCs, pDCs, Macrophages, NKs) • Normal ratio of Treg/Th17

8. SIVagm replication in RM is not restricted to particular anatomical sites

9. Controlof SIVagm infection does not apper to be exclusively due to intrinsic immunity to the virus In vitro replication of SIVagm and SIVmac on AGM and RM PBMCs

10. Significant purifying selection is observed in SIVagm-infected RMs by deep sequencing

11. SIVagm infection of RMs Single copy VL quantification of SIVagm demonstrate the complete control of viral replication in this in vivo model of viral latency D250 pi D1440 pi Animal VL (copies/ml) RM1 19 RM2 <0.5 RM3 181 RM4 10 RM5 30 Animal VL (copies/ml) BA38 <0.8 V492 <1 P373 <1

12. Is SIVagm infection cured or functionally cured? In vivo depletion of CD8+ cells (with the human-mouse cM-T807 monoclonal antibody)

13. SIVagm rebounded during CD8+ cell depletion in RMs and was controlled with the CD8+ cell restoration

14. Is the rebounding virus replication competent or just a reactivated virus from the reservoirs unable to complete full cycles of replication?

15. Caveats • Virus levels are very low in controllers (less than 1 in 106 CD4+ T cells may contain virus) and the volume of serial samples collected from SIVagm-infected RMs is relatively small-virus isolation from the reservoirs may prove difficult • SIV is very difficult to isolate from plasma, especially from chronically-infected animals - rebounding virus isolation from plasma will likely fail • Reservoir activation protocols involve SIV passage on human T cell lines, which may alter the phenotype of the virus through truncations in the transmembrane envelope glycoprotein (gp41) or nonsense mutations in accessory genes

16. Rebounding virus after CD8+ cell depletion is replication competent Pooled plasmas at the peak of rebound and inoculated RMs

17. Rebounding virus after CD8+ cell depletion is replication competent RMs infected with the original SIVagmSab stock RMs infected with the pooled rebounding virus

18. Rebounding virus after CD8+ cell depletion is clonal A limited number of viral variants were reactivated following CD8+ cell depletion These variants were close to the original Viral stock, suggesting that the reservoirs Were seeded very early upon infection Diversity of the original SIVagmSab stock used in RMs Pooled plasmas at the peak of rebound and inoculated RMs

19. Conclusions • Our results further validate SIVagm-infected RMs as a model of functional cure of replication-competent retrovirus infection • Deciphering the mechanisms of control may identify new strategies to achieve functional cure of HIV • This model is well suited to assess new therapeutic strategies to deplete viral reservoirs without the complexity of multidrug antiretroviral therapy

20. Acknowledgements • Apetrei lab TNPRC ThaidraGaufin Rajeev Gautam Daniel Mandell • Pandrea lab TNPRC Jeanne Macfarland Melissa Pattison • Apetrei/Pandrea lab CVR PITT Jan Kristoff Fang Jeri Zhong Cui Ling Xu Dongzhu Ma ViskamVijewardana George S. Haret-Richter Kevin Raehtz • TNPRC Vet Med Jason Dufour Marion Ratterree Rudolf Bohm • Univ. of Pittsburgh Vet Services Anita Trichel • TNPRC • Ronald Veazey • Preston Marx • Andrew Lackner • Univ. of Wisconsin David O’Connor Shelby O’Connor • NIH • Vanessa Hirsch • Brandon Keele • Univ. of Pittsburgh • John Mellors Funded by NIH R01 RR025781 (IP, CA); RO1 AI065325 (CA) RO1 AI064066 (IP); AMFAR (CA)