Quantification of SIV RNA and evaluation of infectivity

1. Quantification of SIV RNA and evaluation of infectivity Neil Berry Division of Retrovirology NIBSC, UK.

2. Transfer of Simian Immunodeficiency viruses • Simian immunodeficiency viruses (SIV) are highly prevalent in non-human primates with 30+ species infected with a diverse range of SIVs • Potentially large reservoir of infection – examples of co-evolution over millenia and recombinants of different SIVs • Represent ‘natural infections’ - largely apathogenic/mild pathology • Contrast with HIV infection in humans which is a relatively recent phenomenon due to cross-species/zoonotic transfers • HIV-1 infection of humans (SIVcpz, chimpanzee) – Group M, N and O • HIV-2 (SIVsm, sooty mangabey) – 8 independent transmissions (mainly subtypes A and B). • Asian macaques have no SIV but SIVsm has also crossed the species barrier into captive macaques resulting in a spectrum of infection/pathology and disease course which mimics HIV infection of humans • Model for HIV pathogenesis and vaccination/therapeutic strategies

3. SIV infection in macaques • Indian rhesus – USA/Europe • Cynomolgus macaques - Europe • Indonesian • Mauritian Range of factors affecting susceptibility - host genetics, species differences - intrinsic factors : anti-retroviral resistance

4. Establishment of infection • To establish infection a number of events need to occur • During primary infection HIV/SIV targets CD4+/CCR5+ T cells are among the first cells to be productively infected after exposure • Local replication at the site of exposure is thought to amplify an initially restricted viral inoculum – expansion and replication to establish systemic infection with sufficient vigour to generate a self-propagating infection • Major site(s) of early replication – centrally located mucosal sites – Gut-associated lymphoid tissue – but inoculum administered at numerous peripheral sites is initial point of exposure • Within 10 days after infection the majority of extra-lymphoid tissue based CD4+ Tem target cells are productively infected or have interacted in a specific manner • Accompanied by a massive burst of replication detected as a viraemia in the peripheral blood • Plasma viral load (PVL) measured by vRNA > 108 RNA copies/ml

5. Titration of virus stocks: infectivity in different species • Virus stocks need to be titrated in vivo before a challenge expnt can proceed to determine the MID50 -varies between different species • Due to the small numbers involved it is important that all controls become infected but that a biologically relevant challenge dose is used • MID50 calculated for a challenge in rhesus will need to be re-assessed before a challenge in cynos can be attempted and vice-versa

6. Rhesus Dilution Number infected factor 10 4/4 100 4/4 1000 2/4 0/3 1000 MID50 RKI stock/rhesus Cynomolgus Dilution Number infected factor 3 - 2/2 10 2/2 1/2 100 0/2 0/2 1000 1/2 0/2 10 MID50 RKI stock/cyno Intra-rectal titration of SIVmac251/RKI in macaques 100 fold difference between the two species. In subsequent challenge expnts in the respective species infection was established in all naïve challenge controls.

7. 102 103 102 103 102 104 103 103 102 400 400 200 0 200 0 400 400 600 0 200 0 200 Infected Cells/106 PBMC ELISA Titre Neutralising Ab Titre 10 12 14 10 12 14 16 12 16 12 14 16 10 16 14 10 0 4 4 2 8 0 6 0 2 8 4 6 8 2 6 0 4 6 2 8 Comparison of Cell-Associated Virus Load,ELISA Titre and Neutralising Antibody Titre 104 D6P FJK AKE 2WV 103 10-1 102 101 100 E2H AA18 3AA EDF 103 10-2 102 101 100 P2T 2XJ AA2D 2XN 103 102 10-3 101 100 W6A DPW AA2K 103 102 10-4 101 100

8. 10 D6P FJK AKE 2WV 8 6 4 2 0 E2H AA18 3AA EDF 8 6 2XN 4 2 0 2XJ W6A DPW AA2K 2 4 6 8 10 12 14 16 8 P2T AA2D 6 8 4 6 2 4 0 2 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 16 0 Plasma Virus Loads (qRT-PCR) 10-1 10-2 RNA copies/well 10-3 10-4

9. Titration data SIVsmE660 in cynomolgus macaques Titration 2 Titration 1 Highly vigorous and pathogenic challenge virus in cynomolgus macaques High peak and set-point viraemia Irrespective of the amount or level of the initial challenge inoculum when challenged in 10-fold serial steps End-point at 10-5 dilution, Group F.

10. Multi-centre SIV RNA study • Reference materials to allow validation and comparison of HIV-1 developed • HIV-2 currently under development • No comparable materials available for SIV RNA – fewer centres performing assays though this is now increasing • Desirable to have the ability and confidence to compare vRNA data generated in different centres • Established a multi-centre vRNA study where the participating laboratories used varying approaches in their evaluation and validation systems • Materials prepared from a stock of high titre SIV (SIVmac251/32H) – 2 macaques exsanguinated at 14 day (peak) viraemia. • Diluted to an extinction end-point in negative macaque plasma and initially evaluated by qRT-PCR at NIBSC • Both gag and LTR targets have been used to characterise these materials in-house • Goal was to determine the ability of different assay systems to detect the wide dynamic range of SIV RNA levels encountered in experimental vaccine/challenge trials • Materials were tested ‘blind’ by participating centres and the code broken

11. Participating centres • ENVEP ‘umbrella’ and CDC/USA • Centre A: NIBSC, UK (qRT-PCR, real-time ); • Centre B: BPRC, Holland (qRT-PCR, conventional) • Centre C: ISS, Italy (qRT-PCR, real-time) • Centre D: SIIDC, Sweden (Cavidi, RT/RNA conversion) • Centre E: CDC, USA - (qRT-PCR independently validated by virion counting)

12. Multi-centre comparative analysis of SIV RNA levels in plasma Centre A: NIBSC (qRT-PCR); Centre B: BPRC, Holland (qRT-PCR) ; Centre C ISS, Italy (qRT-PCR); Centre D SIIDC, Sweden (Cavidi, RT) Centre E, CDC (qRT-PCR validated by virion counting).

13. Assay detects all members of the HIV-2/SIVmac/SIVsm phylogenetic group with comparably high efficiency Breadth of SIV/HIV-2 LTR real-time PCR assay HIV-2 ROD SIVsmE660 HIV-2 EHO SIVmac SIVmac

14. Summary • In different primate species the limiting dose of infectivity needs to be verified independently • Provides information regarding the establishment of infection and kinetics at low dose where a ‘single-hit’ is sufficient to establish infection • vRNA profiles at the limiting dose were indistinguishable from high levels of inoculum where infection was established • Assessment of vRNA levels in macaque challenge studies for both vaccine trials and studies of pathogenesis are essential • Further comparison of assay methodologies and some standardisation across study centres in Europe/US is desirable • SIV RNA reference materials have been established at NIBSC • Aim to expand these studies to include more centres (SoGAT/Europrise) to facilitate collaboration in this area

15. Acknowledgements • NIBSC : Neil Almond, Mark Page, Debbie Ferguson, Richard Stebbings Claire Ham • Collaborators (ENVEP): Steve Norley, RKI, Germany. Jonathan Heeney, Ernst Vershoor, BPRC, Holland Rigmor Thorstensson, SIID, Sweden Fausto Titti, ISS,Italy. Priya Srinivasan, CDC, Atlanta