1. Phenotypic properties influencing HIV-1 transmission fitness IAS Conference on HIV Pathogenesis, Treatment & Prevention Vancouver July 21 st 2015 Katja Klein U I have no conflict of interest to disclose.

2. Primary HIV-1 infection Primary HIV-1 infection requires the penetration of the virus across the mucosal barrier and the establishment of infection in target cells. In the vast majority of cases, mucosal HIV infection is initiated by a single infectious virus or a small number of HIV-1 virions. To better understand the infectious process, we need an in-depth comprehension of the phenotypes of transmitted viruses and how it contributes to the genetic bottleneck selection process. U Donor Recipient

3. Study Aim To explore transmission fitness and analyse possible phenotypic differences between acute and chronic HIV-1 that may effect the genetic bottleneck selection process. Find if there is an advantage of the virus that gets through the mucosal barrier and establishes a systemic infection. U

4. Acute and chronic viruses used in this study Constructed Env-chimeric viruses from: Eleven acute subtype B envelopes received from the Center for HIV/AIDS Vaccine Immunology (CHAVI, USA). Chronic subtype B envelopes from the Institute of Tropical Medicine (Belgium). Env-chimeras were generated by yeast-based cloning of the env gene into NL4-3 backbone at Case Western Reserve University (USA). Assays performed: Replication efficiency Sensitivity to entry inhibitors Entry kinetics Entry efficiency Transmission fitness U

5. Phenotypic analysis of acute and chronic HIV A.A. B.B. King et al. 2013 JVI U Replication fitness in PBMCs by competing each of the acute virus against three chronic viruses. Test for differential sensitivity to entry inhibitors. Entry kinetics: Analyse how long it takes for the virus to get into the host cell. Entry efficiency in affinophile cells with variable CCR5 and CD4 expression. Replication kinetics in different cell types No selective advantage of acute over chronic viruses.

6. HIV-1 competitions in explant tissue; surrogate for “transmission” fitness U HIV-1 competitions: Mix 4-5 acute viruses + 3 chronic viruses Acute viruses compete in every possible combination →“round robin” B1, B2, B3, B4+ Q0, K44, I10 B4, B7, B8, B9+ Q0, K44, I10 B9, B14, B17, B19+ Q0, K44, I10 B19, B20, B2, B7+ Q0, K44, I10 B2, B7, B17+ Q0, K44, I10 B14, B20, B3, B8, B17 + Q0, K44, I10 B9, B20, B1, B7+ Q0, K44, I10 B1, B8, B4, B19 + Q0, K44, I10 …… Acute Chronic Acute: B1, B2, B3, B4, B7, B8, B9, B14, B17, B19, B20 Chronic: Q0, K44, I10

7. “transmission” fitness U

8. Acute vs chronic HIV-1 competition in cervical tissue U Percent replication in tissue Percent replication in MC + PM-1 cells chronic virus Cervix

9. Acute vs chronic HIV-1 competition in penile tissue U Penis Percent replication in tissue Percent replication in MC + PM-1 cells chronic virus

10. Summary of transmission fitness in mixed virus infections of penile tissue U

11. Accumulative analysis of mixed virus infections of penile tissue U

12. Potential influence of lectins on the infection process U Extracellular mannose binding lectins/proteins (MBL/Ps) and C-type lectins on epithelial cells bind and trap pathogens with high mannose, branched oligosaccharides. Mannose receptors on vaginal epithelial cells are thought limit pathogen infections and may even induce MBPs upon direct pathogen binding. Homozygote carriers for a variant MBL allele that decreases MBL concentrations (nearly 1000-fold in sera) were at high risk for HIV infection whereas heterozygotes showed slower disease progression. (Garred, P. et al. Lancet. 1997)

13. Role of Env glycosylation on transmission fitness U Daniel Stieh

14. Conserved N-linked glycosylation sites in HIV-1 Env U

15. Primary infection in the cervix vs blood >100 Ugandan and >180 Zimbabwean women were recruited within 3 months of infection U

16. Conclusion HIV-1 genetic diversity may be higher at the site of infection (e.g. vaginal tract) than in the plasma at acute/early infection. In our ex vivo models, chronic HIV-1 isolates appear to be “trapped and replicating” in the mucosal tissue whereas acute HIV-1 preferentially pass through the mucosal tissue via LC/DCs and are transmitted to T cells. Higher transmission fitness of acute HIV-1 correlates with reduced envelope N- linked glycosylation.  High levels of extracellular soluble lectins in tissue and C-type lectins expressed on epithelial cells are thought to prevent infection of high mannose-containing pathogens. As such, HIV-1 with reduced glycan shield may be passively selected for transmission by escaping the “lectin trap” due to reduced binding ability. U

17. Acknowledgements UCLA Samantha Johnston Sandra Nguyen Benhur Lee Imperial College Deborah King Asna Siddiqui Viviana Buffa Lucia Fishetti Robin J. Shattock CHAVI Investigators/Leaders Norman Letvin Joe Sodroski John Kappes Christina Jambor Brandon Keale Beatrice Hahn George Shaw Jeff Anderson Carolyn Williamson Ron Swanstrom and others…. University of Manchester John Archer David Robertson U R01 AI49170 University of Western Ontario/ Case Western Reserve University Eric Arts Awet Abraha Denis Tebit Yong Gao Michael Lobritz Kara Lassen Annette Ratcliff Immaculate Nankya Gabriele Nickel Kenneth Henry