Dawn Moore-Dudley Department of Molecular biology and Microbiology

1. A single dose of an HIV-specific, vaginally-applied microbicide, PSC-RANTES (PSC) can select for a drug resistant SHIV in a macaque model Dawn Moore-Dudley Department of Molecular biology and Microbiology

2. Introduction • Resistance to many antiretroviral drugs can emerge in an HIV-1 infected individual with even a single treatment. The best example being the emergence of NVP resistance in mothers or infants given a single dose of NVP to prevent perinatal transmission • NVP resistance may also be selected during transmission from mother to infants as suggested by different NVP resistance mutations (K103N and Y181C) emerging in the mother and their infant. There is still no direct evidence that drug resistance can be selected in a drug-treated recipient from the innoculating wild type virus delivered by the donor. • Use of anti-HIV microbicide would be an example of this scenario.

3. Introduction (2)Use of macaque models for testing anti-HIV-1 microbicides • Progesterone treated macaques are successfully infected through a vaginal route with CCR5-tropic SHIVP3 • Several CCR5 inhibitors and entry inhibitors, when applied vaginally, can block SHIVP3 infection of macaques in a post-coital model • Potential drawbacks to this model are, however, • An almost exclusive use of SHIVP3 for these tests • The relative low diversity of SHIVP3 for macaque exposures as compared to the genetic diversity of the HIV-1 innoculum delivered from donor to recipient • Too many to discuss but not as relevant to this presentation

4. PSC-RANTES blocks gp120 from binding to CCR5 as well as down-regulates and sequesters CCR5 in the cell. RANTES AOP Wilken J. et al. Chemistry and Biology. 1999. PSC-RANTES as potential anti-HIV-1 microbicide • A derivative of the natural chemokine RANTES • ~8 kDa protein • May be the most potent anti-HIV compound with an ability to inhibit at sub-nanomolar concentrations • Inhibition is mediated by both CCR5 downregulation on the cell surface and competitive binding with virus • Has a higher binding affinity for CCR5 than RANTES • Does not induce CCR5-mediated inflammation like its natural counterpart.

5. PSC-RANTES did not induce vaginal irrational or inflammation in macaques PSC-RANTES effectively down-regulates CCR5 on the surface of macaque PBMCs 30 female progesterone-treated rhesus macaques were vaginally pre-treated with various concentrations of PSC-RANTES or PBS and then challenged with SHIVSF162. Testing PSC-RANTES as a microbicide Lederman et al. Science 2004

6. Viral load of macaques pre-treated with PSC-RANTES and then vaginally infected with SHIVSF162 Lederman et al. Science 2004

7. Amino acid sequence alignment surrounding the mutation in V3

8. Amino acid sequence alignment of gp41 region

9. Detecting the low frequency mutations in the inoculating SHIVSF162 versus virus Lower level of sensitivity Lower level of sensitivity

10. Detecting the low frequency mutations in the inoculating SHIVSF162 versus virus Lower level of sensitivity Lower level of sensitivity N640D <0.4% of inoculating SHIVSF162 >250-fold enrichment K315R 3% of inoculating SHIVSF162 >25-fold enrichment

11. Generating an HIV-1 chimeric virus containing the P3 and M584 SHIV env gene for phenotypic studies

12. Development of a stable U87 cell expressing huCD4 and rhCCR5 0 -RANTES and PSC-RANTES induce a calcium flux response in both U87.CD4.Rh-R5 and U87.CD4.Hu-R5 cell lines. -PSC-RANTES causes CCR5 down-regulation in U87.CD4.Rh-R5 cells.

13. Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitors P<0.001 P<0.01 The macaque treated with 100 uM (animal M584) was infected with SHIV clone resistant to PSC-RANTES. This resistance was more pronounced in cells expressing the Rh CCR5 than the Hu CCR5.

14. Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitors In the Rh CCR5 cells, PSC-RANTES could not completely inhibit the M584 SHIV whereas the innoculating virus was completely inhibited.

15. Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitors P<0.001 P=0.15 Cross resistance to TAK-779 was only observed in the Rh CCR5 cells

16. Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitors P=0.004 P=0.001 The M584 virus is hypersensitive to T20 possibly due to the mutation in the C terminal heptad repeat of gp41

17. Impact of the selection of these mutations in M584 on viral fitness The mutations in M584 have not emerged at a fitness cost to the SHIVP3. Is fitness of M584 greater in the Rh CCR5 cells?

18. Take home question….. If we can select for a PSC-RANTES resistant clone from a SHIVP3 inoculum in even one out of 10 macaques vaginally exposed and treated PSC-RANTES, That will happen if we treat humans with vaginal microbicides where the inoculating virus could be more diverse by orders of magnitude?

19. Denis-Manga Tebit Mike Lobritz Matt Lalonde Eric Arts Angel Reyes-Rodriguez Lora Angelova Ken Henry Kendall Krebs Collaborators -Ronald S. Veazey-Tulane National Primate Research Center, Covington, LA. -Oliver Hartley-Department of Structural Biology and Bioinformatics Centre Medical Universitaire, Geneva, Switzerland Dawn Moore-Dudley Yong Gao Rick Gibson Ken Nelson Immaculate Nankya

20. Measuring the frequency of K315R and N640D in the SHIVSF162P.3 inoculum. Oligonucleotide Ligation Assay (OLA) • Anneal a radiolabeled probe with identical sequence to the mutant template to both templates. • Anneal a 5’phosphorylated probe to both templates. • Allow thermostabile cycling ligase to ligate together the two probes. • Ligation will only occur when there is a perfect match between the probe and the template. • Mismatch stalls the enzyme and perhaps kicks it off the template. • Detects a mutant present at 0.4% of the population.