Gabriel Wagner MD of UC San Diego presents "What can we learn from HIV superinfection?" at AIDS Clinical Rounds

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AIDS CLINICAL ROUNDS

2. What%can%we%learn%%
from%HIV%superinfec6on?%
Gabriel%Wagner,%MD%
Division%of%Infec6ous%Diseases%
University%of%California%San%Diego%

3. Outline%
• Introduc6on%
• How%common%is%superinfec6on?%
• What%are%the%individual%consequences?%
• What%does%it%portend%for%ra6onal%vaccine%
design?%

4. Outline%
• Introduc6on%
• How%common%is%superinfec6on?%
• What%are%the%individual%consequences?%
• What%does%it%portend%for%ra6onal%vaccine%
design?%

5. Types%of%HIV%Dual%Infec6on%
M.Pacold%
Strain 1 + Strain 2
Coinfection
(CI)
Time
Strain 1 Strain 2
Superinfection
(SI)
Time
Intrasubtype
(same subtype)
or
Intersubtype
(different subtypes)

6. HIVI1%M%Subtypes%Global%Distribu6on%
Hemelaar%J.%Trends'Mol'Med'2012%

7. Artenstein%et%al.%JID%1995;171:805I10%%

8. Previous%Inves6ga6ve%Methods%
• Single%genome%sequencing%(SGS):%obtain%the%sequences%
of%20I30%individual%HIV%genomes%
• Lacks%sensi6vity%required%to%discern%low%minority%
popula6ons%(<5%)%
• Cannot%be%used%in%large%cohorts%

9. Next%Genera6on%Sequencing%
• Shorter%reads,%but%many%more%of%them%
• High%coverage%depth%suitable%for%detec6ng%
minority%variants%(<1%)%[Archer%et%al.,%PLOS'One'2012;%7(11)]%%
[Modified%from%Bushman%et%al.,%AIDS%2008]%
400
100
50
960 000
250 000 000
519 000 000
hbp://www.genomeweb.com/sequencing/surveyIilluminaIsolidIandI454IgainIgroundIresearchIlabsImostIusersImullIaddi6on%

10. Dual%infec6on%detec6on%
• Blood%→%%HIVI1%RNA%→%cDNA%→!PCR%3%regions%
• 454%sequencing%(Roche)%
• Bioinforma6cs%and%phylogene6c%analyses%
• DI:%nucleo6de%divergence>%2.5%%(RT,%gag)%and%>%
5%%(env),%confirmed%by%phylogene6c%bootstrap%
env: C2-V3-C3
(416 bp)
pol: RT
(534 bp)
gag: p24
(253 bp)
Pacold%et%al.,%AIDS'2012,%26:157–165%

11. Phylogenies:%Dual%vs.%Monoinfec6on%
P265%env,%3rd%year%of%infec6on%
Divergence:%16%%
D381%env,%6th%year%of%infec6on%
Divergence:%14%%
High%
bootstrap%
support%for%
dual%
infec6on%

12. Outline%
• Introduc6on%
• How%common%is%superinfec6on?%
• What%are%the%individual%consequences?%
• What%does%it%portend%for%ra6onal%vaccine%
design?%

13. Redd%et%al.%JID%2012;206:267–74%
• Heterosexual%open,%rural%cohort%
• 7%out%of%149%iden6fied%with%interI%or%intraIsubtype%superinfec6on%
• Rate%of%HIV%superinfec6on:%1.44%per%100%PYs%
• Unadjusted%primary%HIV%incidence%rate:%1.15%per%100%PYs%
• Adjusted%primary%incidence%3.28%per%100%PYs%(borderline%sta6s6cal%significance)%

14. • Seroincident%cohort%of%highIrisk%women%in%Mombasa,%Kenya%
• 21%out%of%129%iden6fied%with%interI%or%intraIsubtype%superinfec6on%
• Rate%of%HIV%superinfec6on:%2.61%per%100%Pys%
• Primary%HIV%incidence%rate:%5.75%per%100%PYs%
• Superinfec6on%incidence%significantly%lower%than%primary%HIV%incidence,%
with%a%hazard%ra6o%of%0.47%(CI%0.29–0.75,%p%=%0.0019)%
• Lower%incidence%of%superinfec6on%was%only%observed%>%6%months%
aner%ini6al%infec6on.%

15. Rates%of%HIV%dual%infec6on%in%SD%
• Of%118%cohort%par6cipants:%
– 7%coIinfec6ons%(5.9%,%95%%CI%2.4%−11.8%)%%
– 10%superinfec6ons%over%201.6%personIyears,%
resul6ng%in%overall%incidence%of%superinfec6on%of%
4.96!per!100!person.years!(95%%CI%2.67–9.22)%
• 7%superinfec6ons%occurred%in%the%first%year%(6.3%%firstI
year%incidence,%95%%CI%2.6%−12.6%),%and%3%in%the%
second%year%(2.9%%secondIyear%incidence,%95%%CI%0.6%
−8.2%)%
• Primary%HIV%incidence:%4.37!per!100!person.years!(95%%
CI%3.56–5.36)%
Wagner%et%al.,'JID%2014%Apr%1;209(7):1032I8%

16. SD%Primary%Infec6on%Cohort%
Wagner%et%al.,'JID%2014%Apr%1;209(7):1032I8%

17. Wagner%et%al.,'JID%2014%Apr%1;209(7):1032I8%

18. Cumula6ve%Prevalence%
Throughout%215%personIyears%of%followIup,%the%cumula6ve%prevalence%of%
HIVI1%dual%infec6on%(coIinfec6ons%and%superinfec6ons)%was%14.4%%(95%%CI%
8.6%−22.1%).%
Wagner%et%al.,'JID%2014%Apr%1;209(7):1032I8%

19. Timing%of%superinfec6on:%Mombasa%cohort%
Ronen%et%al.,'PLOS'Pathog'2013%9(8):%e1003593.%

20. Outline%
• Introduc6on%
• How%common%is%superinfec6on?%
• What%are%the%individual%consequences?%
• What%does%it%portend%for%ra6onal%vaccine%
design?%

24. Figure 2: Kaplan-Meier curves of time from seroconversion to AIDS or death in
dually and singly infected patients
Goblieb%et%al.,%Lancet%2004;%363:%619–22%

25. Figure 2: Kaplan-Meier curves of time from seroconversion to AIDS or death in
dually and singly infected patients
Goblieb%et%al.,%Lancet%2004;%363:%619–22%

26. Figure!3.%%Changes%in%CD4%TIcell%counts%rela6ve%to%adjusted%baseline%as%es6mated%by%
linear%mixed%modeling.%P%values%of%comparisons%of%changes%from%baseline%between%
the%groups%were%calculated%by%fipng%linear%mixed%models.%The%diagonal%lines%show%
the%longitudinal%slopes%of%CD4%TIcell%count,%as%es6mated%by%the%linear%mixed%
modeling.%
Cornelissen%et%al.,%CID%2012;54(4):539–47%%

27. Fig.!2.%(a)%Viral%load%and%(b)%CD4%progressions%of%the%three%infec6on%groups.%Mean%VL%and%
mean%square%root%CD4%per%month%of%infec6on%for%each%group%are%plobed,%and%the%linear%
mixed%model%fit%is%shown%for%each%group.%Red%arrows%indicate%the%6ming%of%SI%for%each%of%
the%seven%SI%par6cipants.%SI,%superinfec6on;%VL,%viral%load.%
Pacold%et%al.,%AIDS%2012,%26:157–165%%%

28. Table S1 – Sociodemographic and clinical characteristics of the cohort at the time of initial
infection detection* at cohort enrollment
Ronen%et%al.,%AIDS%2014,%28:2281–2286%%

29. Figure 1. Effect of superinfection on viral load and CD4+ cells.
Ronen%et%al.,%AIDS%2014,%28:2281–2286%

30. Figure 1c. Effect of superinfection on clinical events.
Ronen%et%al.,%AIDS%2014,%28:2281–2286%

31. HIV%dual%infec6on%and%
neurocogni6ve%impairment%
• HIVIassociated%neurocogni6ve%disorder%(HAND):%
– Con6nues%to%be%prevalent%among%infected%individuals%
on%an6retroviral%therapy%(ART)%
– Associated%with%lower%CD4%counts%and%AIDS%
• Neurological%sequelae%of%DI%are%unknown%
• Used%nextIgenera6on%sequencing%to:%
– Describe%prevalence%of%DI%among%ARTIsuppressed%
par6cipants%of%the%CHARTER%cohort%
– Examine%rela6onship%between%HAND%and%DI%

32. Background%
• HIV-associated neurocognitive impairment
– high prevalence despite post-ART era
– associated with decreased T-cell counts and
worsened progression to AIDS
Vivithanaporn%et%al.,%Neurology%2010%
myhivclinic.org%from%McArthur%J%et%al.%Ann'Neurol.%2010;67;%An6nori%A%et%al.%Neurology.%2007;69%

33. CHARTER%
• CNS%HIV%An6Retroviral%Therapy%Effects%Research%
– Large%mul6Isite%neuroAIDS%cohort%
– 1600%crossIsec6onal,%652%longitudinal%
– Comprehensive%neuromedical%and%neurobehavioral%
assessments%
• WellIestablished%neuropsychological%tes6ng%babery%
resul6ng%in%demographically%adjusted%TIscores%corrected%
for%age,%educa6on,%sex,%and%ethnicity%(including%norms%for%
Spanish%speakers)*%
*Heaton%RK,%et%al.%Neurology%2010;%75:2087–96.%

34. Methods%
• CHARTER participants (N=38) with longitudinal follow-up
on ART.
PBMC → HIV DNA → PCR 4 coding regions
• Next generation sequencing (454, Roche)
• Bioinformatics and phylogenetic analysis
env: C2-V3
(416 bp)
pol: PR and RT
(297 and 534 bp)
gag: p24
(253 bp)

35. Propor6on%of%HIV%dual%infec6on%and%
neurocogni6ve%impairment%%
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• High%prevalence%of%HIV%dual%infec6on%(24%)%among%chronically%infected%
individuals%on%ART.%%
• Impaired%individuals%had%higher%propor6on%of%HIV%dual%infec6on%p'=%0.058%
Wagner%et%al.,%Poster%471,%CROI%2014,%Atlanta,%GA%%

36. Neurocogni6ve%impairment%and%
HIV%dual%infec6on%
Effect!of!dual!infec?on!on!global!neurocogni?ve!impairment!
Adjusted!Analysis!
OR!(95%!CI)! P!value!
HIV!dual!infec?on! 35.913%(1.84,5978.725)% 0.014!
Current!CD4! 0.995%(0.989,0.998)% 0.001%
Es?mated!Dura?on!HIV!(months)! 0.992%(0.978,1.004)% 0.199%
Log10!CSF!Viral!Load! 5.1859497e+37%(0,NA)% 0.086%
Wagner%et%al.,%Poster%471,%CROI%2014,%Atlanta,%GA%%

37. Outline%
• Introduc6on%
• How%common%is%superinfec6on?%
• What%are%the%individual%consequences?%
• What%does%it%portend%for%ra6onal%vaccine%
design?%

38. NAb%Response%During%Primary%HIVI1%Infec6on%
Richman%et'al.,%PNAS%2003%

39. Vaccines%and%Superinfec6on%
• Neutralizing%an6bodies%(NAbs):%single%clearest%correlate%
of%protec6on%from%reIinfec6on%with%most%viruses%and%of%
protec6on%mediated%by%viral%vaccines1%
• Recent%studies%underscore%need%for%beber%
understanding%of%natural%development%of%NAbs%for%
vaccine%design2,3%
– Level%required%to%protect%from%an%infec6on%challenge%
• Superinfec6on:%unique%opportunity%to%study%correlates%
of%protec6on%
1Plotkin%SA.%C'R'Acad'Sci'III'1999,322:943I951%
2Klein%et%al.,%Nature'2012;%492:118I122%
3Walker%et%al.,%Nature%2011;%477:466I470%

40. Superinfec6on%resembles%primary%infec6on:%
similarity%to%consensus%of%early%SI%env'
Sheward%et%al.,%JID%2015%Mar%9.%pii:%jiv136%
• SI%viruses%are%not%more%similar%to%primary%virus%(colored%line)%than%would%be%
expected%for%unlinked%sequences%(grey%lines),%indica6ng%absence%of%recombina6on.%%
• HL%plot%of%early%SI%single%genome%env'sequences%indica6ve%of%a%“star”%phylogeny%of%
near%iden6cal%sequences.%Red%6cks%represent%nonIsynonymous%muta6ons.%

41. Superinfec6on%resembles%primary%infec6on:%
shorter,%less%glycosylated%variable%env%regions%
Sheward%et%al.,%JID%2015%Mar%9.%pii:%jiv136%
• Superinfec6ng%T/F%viruses%have%env'with%shorter%(A),%less%glycosylated%(B)%variable%
regions%compared%to%matched%chronic%viruses%sampled%at%3%years%post%infec6on.%

42. NAb%Response%During%HIVI1%Superinfec6on%
Smith%et'al.,%Virology%2006%

43. Comparing%NAb%ac6vity%in%HIV%
superinfec6on%vs.%monoinfec6on%
• CaseIcontrol%study%of%NAb%ac6vity%against%
autologous%and%heterologous%viruses%before%
and%aner%superinfec6on%%
– Compared%to%monoinfected%controls%matched%to%
dura6on%of%infec6on%within%3%months%
• 10%superinfec6on%cases%
• 19%monoinfec6on%controls%

44. Neutraliza6on%breadth%and%potency%
• Monogram:%neutraliza6on%assays%against%autologous%and%
heterologous%pseudoviruses.%
• Infec6vity%inhibi6on%>%IC50%6ter%>%neut%score%(breadth%and%
potency)%
%
1Richman%et%al.,%PNAS%2003;%100,7:4144I4149%
2Simek%et%al.,%J.Virol.%2009;%83(14):7337%
Tier%1%(neutraliza6onIsuscep6ble)%
viruses:%
%
SFI162%(subtype%B)%
NL4I3%(subtype%B)%
%
Tier%2%(moderately%neutraliza6onI
resistant)%viruses:%
%
94UG103%(subtype%A)%
92BR020%(subtype%B)%
JRICSF%(subtype%B)%
MGRMIC026%(subtype%C)%
93IN905%(subtype%C)%
92TH021%(CRF%AE)%

45. NAb%response%against%6er%1%subtype%B%viruses%
• Significantly%lower%NAb%against%6er%1%subtype%B%viruses%among%those%
who%will%later%acquire%intrasubtype%B%superinfec6on.%
Wagner%et%al.,%Oral%CI129,%CROI%2013%
Pre-superinfection NAb against NL43
Monoinfection Pre-Superinfection
10
100
1000
NL43logIC50
p = 0.08
Pre-superinfection NAb against SF162
Monoinfection Pre-Superinfection
10
100
1000
SF162logIC50
p = 0.01

46. NAb%response%against%autologous%viruses%
*Note:%Subject%censoring%due%to%start%of%ART.%
*%
Monoinfection After SI
100
1000
10000
100000
10
24-MonthViruslogIC50
p = 0.017
12%Months%PostIInfec6on% 24%Months%PostIInfec6on%
Monoinfection Before SI After SI
10
100
1000
10000
100000
3-MonthViruslogIC50
p = 0.055
p = 0.071
• Superinfec6on%cases%mounted%lower%NAb%against%3Imonth%autologous%
virus%and%24Imonth%contemporaneous%autologous%virus%than%
monoinfected%controls.%

47. Development!of!autologous!NAb!to!early/founder!virus!Env!is!slow!or!absent!prior!to!
superinfec?on.!Plasma%NAb%IC50%6ters%to%early/founder%virus%Env%were%determined%over%the%
course%of%the%first%year%of%infec6on%for%3%superinfected%(dashed%lines)%and%10%nonIsuperinfected%
matched%controls%(solid%lines).%Asterisks%mark%6me%at%which%superinfec6on%was%detected%in%the%
superinfected%cases.%
Low%autologous%NAb%prior%to%intrasubtype%C%superinfec6on%
Basu%et%al.,%Retrovirology%2012,%9:76%

48. Figure!5.!Cross.neutralizing!Breadth!and!Potency!against!HIV.1!Subtype!C!Env!Reference!Panel.!
Bolded%individuals%represent%superinfected%individuals%with%evidence%of%superinfec6on%from%
outsider%partnerships%(ZM282M,%ZM211F%and%ZM247F)%or%nonIsuperinfected%controls%who%selfI
reported%outside%partnerships.%Panel%included%Envs%of%both%Tier%1b%and%Tier%2%sensi6vi6es.%%
Heterologous%NAb%breadth%and%potency%%
against%subtype%C%virus%panel%
Basu%et%al.,%Retrovirology%2012,%9:76%

49. Superinfec6on%and%HLA%haplotypes%
• Frequency%analysis%of%HLA%alleles%among%
monoinfected%and%superinfected%SD%cohort%
par6cipants:%
– HLAIB*35%and%HLAIC*04%associated%with%a%
significantly%increased%risk%of%HIV%superinfec6on.%
• In%mul6variable%analysis,%HLAIA*29,%HLAIA*32,%
and%HLAIC*04%were%significantly%associated%with%
increased%superinfec6on%risk.%%
– Increased%risk%was%independent%of%sexual%risk%
behavior,%CD4+%TIcell%count%and%VL.%%

50. Overall%Conclusions%
• High%rates%of%intrasubtype%B%HIV%dual%infec6on%in%
highIrisk%cohort%
– Different%rates%depending%on%cohort%studied%
– Most%cases%occur%in%first%two%years%
• Superinfec6on%is%associated%with%higher%VL,%lower%
CD4%and%neurocogni6ve%impairment.%
• Superinfec6on%associated%with%lower%NAb%
response%in%first%2%years%aner%primary%infec6on.%
• Intrasubtype%B%superinfec6on%is%associated%with%
specific%HLA%alleles.%

51. Acknowledgments%
UCSD%
Davey%Smith%
Doug%Richman%
Caroline%Ignacio%
Melissa%Laird%(PacBio)%
Ben%Murrell%
Antoine%Chaillon%
Scob%Letendre%
Allen%McCutchan%
Donald%Franklin%
Florin%Vaida%
Siqi%Liu%
%
%
%
Funding%
U.S.%Department%of%Veterans%Affairs%
Na6onal%Ins6tutes%of%Health%
Interna6onal%AIDS%Vaccine%Ini6a6ve%
Na6onal%Science%Founda6on%
James%B.%Pendleton%Charitable%Trust%
Monogram%Biosciences%
Terri%Wrin%
Pham%Phung%
Yolanda%Lie%
All%Par6cipants%in%the%San%Diego%
Primary%Infec6on%Cohort%and%
CHARTER.%
The%Scripps%Research%Ins6tute%
Pascal%Poignard%
Elise%Landais%