Understanding HIV. Transmitted/Founder Viruses. Brandon Keele SAIC-Frederick National Cancer Institute

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1 Understanding HIV Transmission Utilizing Transmitted/Founder Viruses Brandon Keele SAIC-Frederick National Cancer Institute AIDS Vaccine September 2011

2 Overview Several years ago, the CHAVI sought to more accurately estimate the genetic bottleneck that occurs during mucosal transmission with the goal of ultimately exploiting this potential vulnerability. Previously the restriction i in genetic diversity i from donor to recipient i was determined dto be either heterogeneous or homogeneous. Acute infection cohorts were identified/initiated (ramp-up through peak viremia). Single Genome Amplification (SGA) was used to more precisely measure viral diversity. Proportionate representation of viral diversity eliminates resampling bias. Excludes Taq induced PCR error excludeexclude sequences that have ambiguous sites. Eliminates any in vitro recombination exclude PCR reactions with >1 template. Mathematical modeling of early diversification validated sequence analysis Key feature are: The number of variants can be accurately enumerated The exact nucleotide sequence of this virus can be obtained

3 Single variant HIV-1 transmission CONSENSUS 59% id ti l t 59% identical to consensus 30% differ by one nt from consensus 11% differ by two nt from consensus

4 and a Poisson distribution of mutations

5 Multiple virus infections are not star like and do not fit a Poisson distribution of accumulated changes Lee et al. J Theor Biol

6 A model of a single virus diversifying overtime Assumptions: Exponential growth (Ro=6) Generation time: 2 days RT error rate: 2 x Identity (%) Sequence The predicted sequence identity gradually diminishes overtime due to the random accumulation of random mutations.

7 Enumerating the number of transmitted/founder variants in HIV-1 natural history studies The number of detectable variants correlates to risk behavior and exposure: Heterosexual 90% single variant infections Keele 2008, Salazar 2008, Haaland 2009, Abrahams 2009, Kearney 2009, Salazar 2009 MSM 60% single variant Keele 2008, Li 2010 IVDU 40-70% single variant Bar 2010, Masharsky 2010 Variability in the number of variants most likely due to donor s VL and quantity of contaminant. STD increases risk of multiple variant transmission Haaland 2009 ~300 subjects examined to date with consistent result Do NHP models accurately reflect this very low multiplicity of infection? Generally No. Until recently, most challenges were dosed too high

8 NHP mucosal infections can recapitulate single variant infections found in HIV-1 infections CP1W 77% identical to consensus 17% differ by one nt 6% differ by two nt i.r. Repeated low-dose challenge Keele et al. JEM 2009

9 Single-dose i.r. challenge SIVmac251 revealed one or few variant infections 1:100 dilution 1:1000 dilution Liu et al. J Virol 2010

10 Intravaginal challenge with low or intermediate dose revealed one or multiple transmitted variants 10 3 TCID TCID Stone et al. J Virol 2010

11 Cell free virus exposure to foreskin and glans penis revealed only single variant infections 1 3 exposures at 1x10 5 TCID 50 Ma et al. AIDS Hum Retro 2011

12 Mucosal SIV infection of Rhesus macaques can recapitulate HIV-1 transmissions Rectal Keele et al JEM 2008, Liu et al JVI 2010, Wilson JVI 2009 SIV and SHIV stocks Letvin; Barouch; Desrosiers; Watkins; Veazey. Vaginal Stone et al JVI 2009 SIVmac251 C. Miller; SHIV162P3; P4 Veazey/Moore Penile Ma et al. AIDS and Hum Retro 2011 Yeh et al. JVI 2011 SIVmac251 and SIVsmE660 We now have NHP models with genetically characterized stocks and a dose of viral swarms that t initiates iti t infection with one or few variants. Titered stock available from the Preclinical Research and Titered stock available from the Preclinical Research and Development Branch; NIAID

13 Does early systemic virus enumeration accurately identify the number of variants a that established s ed infection? Are viruses demonstrable at latter time points that were not identifiable at original systemic sampling? (i.e. did we miss variants looking very early that later could become important?) t?) We sought to address this concern in NHP by longitudinal analysis a s of putatively single variant a infections

14 Evidence of only a single variant infection from wk1 thru 38 following penile challenge

15 Evidence of only a single variant infection from peak viremia thru 22 months following intrarectal challenge Yeh et al. J Virol 2010

16 Is all this work worth the effort? Can this approach help in preclinical vaccine evaluations? > variants Number of transmitted/founder M891 P007 P012 P002 M899 M896 M887 M904 P136 M881 P084 P134 P015 P061 P066 P074 > P006 P018 P010 P013 P016 P014 M890 P019 M898 P008 P009 P001 P113 P017 P059 P121 P095 P071 P088 P144 M998 P130 P089 P143 G. Franchini

17 Single variant HIV-1 transmission CONSENSUS Knowing the exact nucleotide sequence of the transmitted virus allows for phenotypic analyses 59% id ti l t 59% identical to consensus 30% differ by one nt from consensus 11% differ by two nt from consensus

18 Phenotype of the transmitted Env vs. chronic

19 Transmission signature in signal peptide impacts Env processing and infectivity H G G H 69/79 (87%) transmitted viruses contain H at site 12 compared to 57/111 (51%) for chronic viruses Increased Env expression with transmission i signature H Asmal et al. PLoS One 2011

20 Generating full length infectious molecular l clones