1. Determinants of HIV Transmission Eric Hunter Emory Vaccine Center

2. Hypothesis An effective prophylactic vaccine against HIV-1 must protect against those viruses that initiate infection at the mucosal surface. These may be distinct from the bulk of the variants that have evolved to survive during their growth in the chronically infected host. What is the nature of these viruses and where do they come from?

3. Heterosexual transmission in HIV Discordant Couples HIV discordant couples represent a significant fraction of more than 50,000 couples tested in the capital cities of Zambia (20%) and Rwanda (12%). Despite counseling and condom provision, low levels of transmission still occur (7%/yr in Zambia, 3%/yr Rwanda). Approximately 80-85% of transmissions are epidemiologically linked (i.e. within the couple) allowing comparison of donor and recipient viruses.

4. Genetic bottleneck during transmission of HIV Transmission Diverse virus population in chronically infected “donor” Re-emergence of viral diversity

5. Characterization of transmitted viruses We report here today on the analysis of 21 transmission pairs from Zambia and Rwanda, where we have utilized a p24 ELISA assay to identify newly infected individuals who are antigen positive/antibody negative. To avoid resampling and in vitro recombination, we have used single genome amplification of env genes from both the transmitting partner (donor) and the newly infected partner (recipient).

6. Recipient Plasma Recipient PBMC Donor Plasma Donor PBMC An Extreme Genetic Bottleneck occurs during Transmission In this linked transmission pair the recipient virus is relatively homogeneous and originates from a single branch of the donor phylogenetic tree - thus a single genetic variant has established infection.

7. An Extreme Genetic Bottleneck occurs during MTF AND FTM Transmission In 18/19 Zambian subtype C transmission pairs this extreme genetic bottleneck was observed Recipient Donor

8. In 8/9 Rwandan and Zambian subtype-A transmission pairs examined to-date a similar severe bottle- neck has been observed following transmission. A similar genetic bottleneck is seen in Rwandan transmission pairs

9. The virus population in the acutely infected recipient is remarkably homogeneous 27/40 = 67% identical 11/40 - one base change 2/40 - two base changes Modeling of virus replication and evolution in the absence of selection would predict: 76% identical at 13 days 58% identical at 26 days 45% identical at 40 days to transmitted variant

10. Remarkable levels of homogeneity are observed even when full length genomes are amplified

11. Highlighter comparison of 0,2 and 6 month NFLG amplicons At the time of initial sample collection three genomes are identical and the bulk of the remaining genomes have a single nucleotide change However, the majority of these cluster within a potential Vpu CTL epitope, are non-synonymous changes and persist over 5 months - suggesting very early CTL escape and selection. Limited additional changes at 2 months PI, but a cluster of mutations in env at 6 months PI - suggesting antibody selection.

12. Homogeneity of recipient viruses in linked transmissions 26/28 recipient ‘quasispecies’ (17 sub-type C, 1 G, 1U and 9A) originate from a single branch of the chronically infected donor phylogenetic tree - arguing for an initiation of infection by a single virus/virus-infected cell. Recipient viruses exhibit very limited heterogeneity in both Env and Gag irrespective of whether they are amplified from p24+ve or p24-ve seroconvertors. In acutely infected recipients (virus positive, antibody negative), the homogeneous virus population is likely to represent the infecting virus genotype. Preliminary analyses of genital fluids do not support the hypothesis that the genetic bottleneck is a result of very limited viral genetic diversity in these tissues.

13. Where does the bottleneck originate In the donor? –through limited heterogeneity in the genital fluids –Unlikely - compartmentalization of genital fluid virus is observed but these enriched populations do not appear to be the source of the transmitted virus (D. Boeras - 16:45 this afternoon - Oral abstract session - Abstract # MOAA0302) In the recipient? –at the mucosal surface –selection during virus outgrowth

14. Heterogeneity in acutely infected individuals What happens if we examine heterogeneity in unlinked transmissions in the same discordant couple cohort? Studies of Overbaugh and colleagues with Kenyan sex workers (Long et al., 2000 & pers. comm.), and Swanstrom and colleagues in STD clinic participants (Ritola et al., 2004) have shown that transmission of multiple variants can occur in up to half of the cases examined. Is linked transmission in discordant couples inherently different -low frequency of STDs -single partner.

15. 4 cases of transmission of a single donor variant 3 cases of transmission of multiple donor variants (from a single source) Establishment of infection in unlinked transmissions can involve a less severe genetic bottleneck

16. Multiple variants can be confirmed in the Highlighter analysis

17. Heterogeneity in the unlinked recipient is linked to evidence of inflammatory genital infections

18. Heterogeneity in 5/42 newly infected recipients is linked to inflammatory or ulcerative infections

19. Conclusions The presence of multiple genetic variants in newly infected recipients indicates that the extreme bottleneck we have observed with linked transmissions most likely occurs at the genital mucosa rather than during virus outgrowth. The extremity of the genetic bottleneck appears to be modulated by genital infections: –These could provide an inflammatory environment with increased numbers of target cells. –They could result in breaks in the genital mucosa allowing access to underlying target cells.

20. Conclusions It seems likely that when the mucosal surface is intact that a single genetic variant establishes infection following transmission. Whereas in the presence of genital infections, or other situations that increase accessibility to target cells in the mucosa, multiple variants can be transmitted. However, because 85% of transmissions in discordant couples are linked and primarily occur in the absence of STDs (Trask et al., 2001; Fideli et al., 2001), there does not appear to be a requirement for disruption of the mucosal barrier for transmission to occur.

21. Acknowledgements Emory University Hunter Lab Debi Boeras, Ph.D. Paulina Hawkins Rich Haaland, Ph.D. Colleen Kraft, M.D. Ling Yue, M.D. Paul Farmer, Ph.D. Derdeyn Lab Cynthia Derdeyn, Ph.D. Los Alamos National Laboratory Bette Korber, Ph.D. Karina Yusim LANL Team Emory/RZHRG cohorts The Staff and Participants Susan Allen, M.D., MPH Joseph Mulenga, M.D. Lawrence Elwyn Chomba, M.D. Olivier Manigart, Ph.D. Etienne Karita, M.D. Kayitesi Kayitenkore M.D. Amanda Tichacek, MPH Funding: IAVI (Allen) NIH R01 AI-51321 (Hunter/Derdeyn) NIH R01 AI-58706 (Derdeyn) Gates Grand Challenge (Shaw) NIH P30 AI-050409 Emory Center for AIDS Research Univ. Alabama at Birmingham Shaw/Hahn Lab Jesus Salazar, Ph.D. Beatrice Hahn, M.D. George Shaw, M.D., Ph.D.

22. The genetic bottleneck is identical if instead we look at the Gag gene in linked transmissions As with our observations for env we find limited heterogeneity in gag and each recipient sequence emanates from a single branch of the donor tree.

23. Donor - GFRecipient - Blood Could the genetic bottleneck be the result of limited diversity in genital fluids?

24. Compartmentalization of viral variants in the genital fluids is not the origin of the extreme genetic bottleneck Recipient Plasma Recipient PBMC Donor Plasma Donor PBMC Donor Genital Fluids