Determinants of HIV Transmission
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Determinants of HIV Transmission
Eric Hunter
Emory Vaccine Center
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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?
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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.
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Genetic bottleneck during transmission of HIV
Transmission
Diverse virus population in chronically infected “donor”
Re-emergence of viral diversity
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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).
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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.
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ZM190F
ZM190M
ZM243M
ZM243F
ZM242M
ZM242F
ZM238F
ZM238M
ZM205M
ZM205F
ZM248M
ZM248F
ZM198M
ZM198F
ZM216M
ZM216F
ZM229F
ZM229M
ZM221F
ZM221M
ZM201M
ZM201F
An Extreme Genetic Bottleneck occurs during
MTF AND FTM Transmission
• In 18/19 Zambian subtype C transmission pairs this extreme genetic bottleneck was observed
RecipientDonor
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• 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
RW67M
RW67F
RW56M
RW56F
RW35F
RW35M
ZM292F
ZM292M
RW36F
RW36M
RW19M
RW19F
RW53M
RW53F
RW57M
RW57F
RW41M
RW41F
0.02
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The virus population in the acutely
infected recipient is remarkably
homogeneous
27/40 = 67% identical11/40 - one base change2/40 - two base changes
Modeling of virus replication and evolution in the absence of selection would predict:76% identical at 13 days58% identical at 26 days 45% identical at 40 days to transmitted variant
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Remarkable levels of homogeneity are observed even when full length genomes are amplified
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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.
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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.
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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
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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.
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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
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Multiple variants can be confirmed in the Highlighter analysis
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Heterogeneity in the unlinked recipient is linked to evidence of inflammatory genital
infections
Single (N=4) %
Multiple (N=3) % p
Genital inflammation (medical or lab)No 3 75.0% 0 0.0% 0.1429Yes 1 25.0% 3 100.0%
Genital ulceration (medical or lab)No 3 75.0% 2 66.7% 1.0000Yes 1 25.0% 1 33.3%
Genital inflammation or ulcerationNo 2 50.0% 0 0.0% 0.4286Yes 2 50.0% 3 100.0%
DischargeNo 4 100.0% 0 0.0% 0.0286Yes 0 0.0% 3 100.0%
Cystitis a
No 4 100.0% 2 66.7% 0.4286Yes 0 0.0% 1 33.3%
Lower abdominal pain (self report) a
No 4 100.0% 0 0.0% 0.0286Yes 0 0.0% 3 100.0%
Vaginal itching (self report) b
No 0 0.0% 1 33.3% 1.0000Yes 1 100.0% 2 66.7%
Presence of clinical and laboratory signs/symptoms of STIs on the day of and 136
days prior to seroconversion
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Heterogeneity in 5/42 newly infected recipients is linked to inflammatory or ulcerative infections
Single (N=37) %
Multiple (N=5) % p-value*
Genital inflammation (medical or lab)No 25 67.6% 2 40.0% 0.3295Yes 12 32.4% 3 60.0%
Genital ulceration (medical or lab)No 29 78.4% 2 40.0% 0.1028Yes 8 21.6% 3 60.0%
Genital inflammation or ulcerationNo 20 54.1% 0 0.0% 0.0492Yes 17 45.9% 5 100.0%
DischargeNo 28 75.7% 2 40.0% 0.3249Yes 9 24.3% 3 60.0%
Cystitis a
No 31 88.6% 4 80.0% 0.5066Yes 4 11.4% 1 20.0%
Lower abdominal pain (self report) a
No 31 88.6% 2 40.0% 0.0299Yes 4 11.4% 3 60.0%
Zambia + Rwanda
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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.
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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.
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Acknowledgements
Emory UniversityHunter LabDebi Boeras, Ph.D. Paulina HawkinsRich Haaland, Ph.D. Colleen Kraft, M.D.Ling Yue, M.D.Paul Farmer, Ph.D.Derdeyn LabCynthia Derdeyn, Ph.D.
Los Alamos National LaboratoryBette Korber, Ph.D.Karina YusimLANL Team
Emory/RZHRG cohortsThe Staff and ParticipantsSusan Allen, M.D., MPHJoseph Mulenga, M.D.LawrenceElwyn 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 BirminghamShaw/Hahn LabJesus Salazar, Ph.D.Beatrice Hahn, M.D.George Shaw, M.D., Ph.D.
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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.
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Donor - GF Recipient - Blood
Could the genetic bottleneck be the result of limited diversity in genital fluids?
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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