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 > 10 8 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 MID 50 -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 MID 50 calculated for a challenge in rhesus will need to be re- assessed before a challenge in cynos can be attempted and vice- versa

6. Intra-rectal titration of SIVmac251/RKI in macaques Rhesus Dilution Number infected factor 10 4/4 100 4/4 1000 2/4 10000 0/3 1000 MID 50 RKI stock/rhesus Cynomolgus Dilution Number infected factor 3 - 2/2 10 2/2 1/2 100 0/2 0/2 1000 1/2 10000 0/2 10 MID 50 RKI stock/cyno 100 fold difference between the two species. In subsequent challenge expnts in the respective species infection was established in all naïve challenge controls.

7. 0246810121416 0 200 400 600 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 4 024681012141602468101214160246810121416 10 2 10 3 10 4 10 2 10 3 10 2 10 3 10 2 10 3 0 200 400 0 200 400 0 200 400 10 -1 10 -2 10 -3 10 -4 Comparison of Cell-Associated Virus Load, ELISA Titre and Neutralising Antibody Titre D6PFJKAKE2WV E2HAA183AAEDF P2T2XJAA2D2XN W6ADPWAA2K Infected Cells/10 6 PBMC ELISA Titre Neutralising Ab Titre

8. W6A 0 2 4 6 8 02468101214 DPW 02468101214 AA2K 0246810121416 P2T 0 2 4 6 8 2XJAA2D2XN 246810121416 E2H 0 2 4 6 8 AA183AAEDF D6P 0 2 4 6 8 10 FJKAKE2WV 10 -1 10 -2 10 -3 10 -4 Plasma Virus Loads (qRT-PCR) RNA copies/well

9. Titration data SIVsmE660 in cynomolgus macaques Titration 1 Titration 2 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. 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). Multi-centre comparative analysis of SIV RNA levels in plasma

13. Breadth of SIV/HIV-2 LTR real-time PCR assay Assay detects all members of the HIV-2/SIVmac/SIVsm phylogenetic group with comparably high efficiency HIV-2 ROD SIVmac HIV-2 EHO SIVsm E660 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