Impact of Early Initiation of Combination
Antiretroviral Therapy on Measures of
Virus in Peripheral Blood of Vertically
HIV-1-Infected Children
Jason Brophy1, Tae-Wook Chun2, Lindy Samson1, Fatima Kakkar3,
Hugo Soudeyns3, Mario Ostrowski4, S. Mujib5, John Kim6, Paul
Sandstrom6, Richard Harrigan7, Stanley E. Read8, Ari Bitnun8
1. Children’s Hospital of Eastern Ontario, University of Ottawa; 2. National Institute of Allergy &
Infectious Diseases, National Institutes of Health, U.S.A.; 3. CHU Sainte-Justine, Université de
Montréal; 4. Department of Immunology and Medicine, University of Toronto, 5. University of
Toronto, Institute of Medical Sciences, Department of Medicine; 6. National HIV & Retrovirology
Laboratories, Public Health Agency of Canada; 7. University of British Columbia, British Columbia
Centre for Excellence in HIV/AIDS; 8. Hospital for Sick Children, University of Toronto
Early Combination Antiretroviral
Therapy in Infants
 The “Mississippi baby” received early cART –
experienced a 2-year “viral remission” after
stopping treatment until a recent viral rebound
 raises the possibility of this as an intervention to limit
reservoir establishment and enable “viral remission”
 Triple cART as HIV-post exposure prophylaxis has
been routinely administered to newborns at high
risk for HIV infection in our centres for many years
 SickKids (Toronto), CHEO (Ottawa), and CHU Ste-Justine
(Montreal)
Objectives

To investigate HIV-1 reservoirs in peripheral blood
of HIV-1-infected children with SVS following
initiation of cART within 72 hours of birth

SVS: defined as absence of detectable virus in standard viral load (VL)
assay subsequent to having achieved an undetectable VL (< 50
copies/mL)
Methods
 Retrospective review at our 3 centres of all children
born to HIV-infected mothers who received triple
cART within 72 hours of birth
 Evaluation of HIV reservoir, immune responses, and
genetic characteristics in those infected children
with SVS after cART
Results –
Retrospective Review
 136 infants received triple cART
 HIV infection in 12 (8.8%)
 In utero infection probable in at least 50% (n=6; HIV PCR
positive within 48 hours of birth)
 Timing uncertain in 50% (n=6) as testing done after 48 hrs
 Four HIV-infected children achieved SVS (Cases 1-4)
 Eight HIV-infected children did not achieve SVS
 6 of 8 did not achieve virologic suppression due to poor
adherence
 2 of 8 initially suppressed for 2-3 years, then experienced
viral rebound after poor adherence (one during the course of
our study – Case 5)
Reservoir Evaluation of
4 Early-Treated, HIV-Infected Children
 Cases 1 to 4 had SVS from 2-7 years
 All 4 remained on their original cART regimen of
zidovudine, lamivudine, and nevirapine
Maternal Characteristics and Infant Diagnostic Testing
Case 1
Case 2
Case 3*
Case 4*
30 years
32 years
29 years
29 years
Viral load pre-delivery
97,701 c/mL
Unknown**
6326 c/mL
6326 c/mL
CD4 count pre-delivery
190 cells/L
10 cells/L
61 cells/µL
61 cells/µL
G (CRF 6)
Unknown
C
C
Emergency C/S
Spontaneous
Spontaneous
Spontaneous
34 weeks
27 weeks
36 weeks
36 weeks
2980 grams
1070 grams
2270 grams
1640 grams
Maternal Characteristics
Age
Clade
Infant Characteristics
Mode of delivery
Gestational age at birth
Birth weight
HIV PCR (age)
Positive (day 1) Positive (day 2) Positive (day 1) Positive (day 1)
* Non-identical twins; ** mother died soon after delivery from OI
Case 1
 Now 7.5 years old
 Remains on same ART combination
CD4 count
3940 (55%)
cART initiated (day 1)
HIV DNA PCR positive (day 1)
 Has maintained an undetectable
viral load
Case 2
 Now 7.5 years old
 Remains on same ART
combination
 Undetectable viral load
CD4 count 1447 cells/L (28.9%; day 20)
cART initiated (day 1)
HIV DNA PCR positive (day 2)
Cases 3 & 4
CD4 count 2663 cells/L
(63%, day 15)
cART initiated (day 1)
HIV DNA PCR positive (day 1)
CD4 count 1997 cells/L
(41%, day 17)
795 c/mL (day 12)
cART initiated (day 1)
HIV DNA PCR positive (day 1)
 Now 3 years old
 Remain on same ART combination and have sustained virologic suppresSion
Case 1
Case 2
Case 3
Case 4
7 years/F
7 years/F
2.5 years/F
2.5 years/M
Serology (ELISA, WB) Negative
Negative
Negative
Negative
HIV-specific T-cell
Undetectable
Undetectable
Undetectable
Undetectable
Plasma viremia ‡
< 1.5 copies/mL
< 1.5 copies/mL
< 1.5 copies/mL
< 1.5 copies/mL
Cell-associated
< 2.6 copies/g
< 2.6 copies/g
< 2.6 copies/g
< 2.6 copies/g
20.0 copies/1.5g
19.5 copies/1.5g
130 copies/1.5g
RNA
RNA
RNA
RNA
HIV RNA in
Not detected (5.4
Not detected (7.2
Not detected (8.0
Not detected (8.0
stimulated CD4 T-
million cells)
million cells)
million cells)
million cells)
Not detected
0.1 infectious
Not detected
Not done
Age/Sex
responses (Gag, Nef)
proviral DNA
Cell-associated RNA § 24.9 copies/1.5g
cells
Quantitative CD4 Tcell co-culture
units/10^6 CD4 T-cells
CCR-5 32 status
Wild type
Wild type
Wild type
Wild type
HLA typing
A*01–A*02; B*27–
A*30–A*66; B*44–
A*01–A*66; B*55–
A*01–A*66; B*55–
B*58; C*02–C*03
B*45; C*03–C*04
B*58; C*03–C*03
B*58; C*03–C*03
HLA-B variation ¥
67CM; 70K/S; 97R/T 67S; 70N; 97R
67Y/M; 70Q/S; 97R/T 67Y/M; 70Q/S; 97R/T
‡ Limit of detection: 1.5 copies/mL; each assay performed on 6 mL of whole blood; § Limit of detection: 1.5 g RNA; performed in duplicate; ¶ Limit of detection; 20 copies/ml; ¥ HLA-B
variation at positions associated with better virologic control (lower set-point) and specific protective amino acid substitutions are indicated in bold
Case 5 –
Prior to Treatment Interruption
Initial findings before TI
Case 5
Age/Sex/Clade
3 years/M/Clade B
Serology (ELISA & WB)
Negative
Plasma viremia ‡
< 1.5 copies/mL
Cell-associated proviral DNA
< 2.6 copies/g
Cell-associated RNA §
149 copies/1.5g RNA
HIV RNA in stimulated T-cells
Not detected (6.7 million cells)
CCR-5 32 status
Wild type
HLA typing
HLA-B variation ¥
A*01-A*02; B*08-B*27; C*01-C*07
67C/F; 70N/Q; 97N/S
Case 5
CD4 2568 (46.2%)
CD4 1995 CD4 2145
(38.3%) (40.0%)
HIV DNA
PCR positive
(Day 4)
cART changed (day 21) - AZT/3TC/LPV
cART started (day 1) - AZT/3TC/NVP
CD4 2299
(37.3%)
CD4 2584
(39.3%)
1000000
102172
100000
7797
Viral Load Log 10
10000
9944
11358
HIV ELISA,WB,
DNA PCR:
POSITIVE
3915
CMI TESTING:
gag/nef POSITIVE
1000
AZT/3TC/LPV
Re-started
100
<40
10
10
30
50
70
Days After Stopping Therapy
90
110
130
Discussion

Absence of detectable HIV DNA and absent/very low levels
of replication-competent virus in peripheral blood and lack
of HIV-specific immune responses demonstrated in a
subgroup of children initiated on cART <72 hours of birth

Suggests early cART initiation can greatly reduce HIV
reservoir size

Genetic factors may also play an important role - protective
HLA genotypes were found in 3 of 4 children
 HLA B*58
 Sequence variation at HLA-B positions 67, 70 and 97 (associated with
superior control of HIV replication)
 The child with replication competent virus did not have these
protective genotypic features
International HIV Controllers Study, Science 2010
Lazaryan, J Virology 2006; Lazaryan, J Virology 2010
Discussion

Our 5th case with rapid viral rebound after interruption of
therapy despite limited reservoir size demonstrates that
early cART will not be effective in all patients

Multiple factors may have influenced this patient’s outcome


Baseline NNRTI resistance, sub-optimal initial regimen

Low-level detectable VL after initial suppression
This, along with late relapse of viral replication in Mississippi
baby, underscore the need for better understanding of
contributing factors to reservoir & viral control



Host and viral genetics
Timing and completeness of initial viral suppression
Selection of components of cART regimen in infants
Conclusions

Based on our study findings and other reports, early cART
for HIV-infected infants clearly limits size of viral reservoir

Additional non-blood reservoir sites require investigation

The clinical significance and benefit of this remain to be seen

Accurate estimation of size of HIV viral reservoir in children
is significantly impacted by limitations in collection of
adequate blood volumes in children compared with adults

A prospective multi-centre observational study (EPIC4) is
underway in Canada to determine the impact of early versus
later treatment on reservoir size and HIV control in children
Acknowledgments

The children and parents who agreed to participate in this
study

The Canadian Institutes for Health Research, Canadian
Association for AIDS Research, and the International AIDS
Society for funding to carry out EPIC4 study
QUESTIONS?
Reservoir, Immunologic Responses
and Genetics
Level of cell-associated HIV-1 DNA in CD4+ T cells
real-time PCR
Level of cell-associated HIV RNA
Cobas Ampliprep/Cobas Taqman HIV‐1 assay
Residual plasma viremia
modified Cobas Ampliprep/Cobas Taqman HIV‐1 assay
Presence of replication competent virus
level of virion-associated HIV RNA in culture supernatant after
mitogenic stimulation
co-culture assay
HIV serology; HIV-specific cell-mediated immune responses;
HLA typing and CCR5 delta 32 genotyping
Case 1
Case 2
Case 3
Case 4
Age/Sex
7 years/F
7 years/F
2.5 years/F
2.5 years/M
HLA typing
A*01–A*02; B*27–
A*30–A*66; B*44–
A*01–A*66; B*55–
A*01–A*66; B*55–
B*58; C*02–C*03
B*45; C*03–C*04
B*58; C*03–C*03
B*58; C*03–C*03
HLA-B variation ¥
67CM; 70K/S; 97R/T 67S; 70N; 97R
67Y/M; 70Q/S; 97R/T 67Y/M; 70Q/S; 97R/T
CCR-5 32 status
Wild type
Wild type
Wild type
Wild type
Serology (ELISA, WB) Negative
Negative
Negative
Negative
HIV-specific T-cell
Undetectable
Undetectable
Undetectable
Undetectable
Plasma viremia ‡
< 1.5 copies/mL
< 1.5 copies/mL
< 1.5 copies/mL
< 1.5 copies/mL
Cell-associated
< 2.6 copies/g
< 2.6 copies/g
< 2.6 copies/g
< 2.6 copies/g
responses (Gag, Nef)
proviral DNA
Cell-associated RNA § 24.9 copies/1.5g
20.0 copies/1.5g RNA 19.5 copies/1.5g
RNA
130 copies/1.5g
RNA
RNA
HIV RNA in
Not detected (5.4
Not detected (7.2
Not detected (8.0
Not detected (8.0
stimulated CD4 T-
million cells)
million cells)
million cells)
million cells)
Not detected
0.1 infectious
Not detected
Not done
cells
Quantitative CD4 Tcell co-culture
units/10^6 CD4 T-cells
‡ Limit of detection: 1.5 copies/mL; each assay performed on 6 mL of whole blood; § Limit of detection: 1.5 g RNA; performed in duplicate; ¶ Limit of detection; 20 copies/ml; ¥ HLA-B
variation at positions associated with better virologic control (lower set-point) and specific protective amino acid substitutions are indicated in bold