Determinants of HIV Transmission

Eric Hunter

Emory Vaccine Center

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?

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.

Genetic bottleneck during transmission of HIV

Transmission

Diverse virus population in chronically infected “donor”

Re-emergence of viral diversity

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).

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.

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

• 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

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

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

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.

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.

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

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.

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

Multiple variants can be confirmed in the Highlighter analysis

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

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

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.

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.

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.

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.

Donor - GF Recipient - Blood

Could the genetic bottleneck be the result of limited diversity in genital fluids?

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