1. No Evidence Of Ongoing Replication In Tissue Compartments During cART
Giorgio Bozzi, MD
HIV Dynamics and Replication Program, NCI, NIH, Frederick, MD

2. Proposed Mechanisms Contributing to HIV Persistence During cART
Background: Understanding HIV Persistence is Key to Curative Strategies
Combination antiretroviral therapy (cART) controls but does not cure HIV
Mechanisms responsible for persistence may be diverse
Proposed Mechanisms Contributing to HIV Persistence During cART
PRIMARILY VIRAL
Proviral integration into long lived cells
Cycles of viral replication
PRIMARILY HOST
Immune response
Cycles of cell replication (clonal expansion)
Sanctuary

3. Background: HIV Genetic Diversity Increases Over Time
Early Diagnosis
0.005
6 nt
Overall
diversity
(APD) = 0.35%
+ 32 Months
APD = 1.1%
0.005
6 nt
Composite
0.005
6 nt

4. Hypothesis and Objective
Ongoing cycles of HIV replication are detectable in tissues by accumulation of genetic change
OBJECTIVE
Analyze HIV in tissue and blood compartments in individuals undergoing long term cART

5. Autopsy, Primary Effusion Lymphoma
Methods
Colonoscopy, healthy
[N = 2]
Plasma, PBMC (longitudinal) and Gut-Associated Lymphoid Tissue (GALT) nucleic acid extraction
Single Genome Sequencing
(SGS, pro-pol, c nt)
Phylogenetic analysis
(Neighbor Joining)
Population genetics analysis
Autopsy, Primary Effusion Lymphoma
[N = 1]
PBMC (longitudinal) and tissue
nucleic acid extraction
Quantification of HIV DNA
(RT-PCR, ddPCR)

6. Study Population cART initiated early after diagnosis
Prolonged viral suppression by cART

7. No Evidence of Molecular Evolution in GALT over 14-16 years cART
HXB2
0.005 (6 nucleotides)
Plasma RNA
PBMC DNA
Colon DNA
Ileum DNA DX 2 7
16 Y
106
105
103
102
101
104
HIV VL
APD = 0.2%
PID2
PID1
Plasma RNA
PBMC DNA
Colon DNA
Ileum DNA DX 12 15
16 Y
106
105
103
102
101
104
HIV VL
HXB2
0.005 (6 nucleotides)
APD = 0.4%
PLASMA RNA
PBMC DNA
ILEUM DNA
COLON DNA

8. Participant 3: Case Summary
2003: HIV negative
11/2007: Diagnosed with HIV (RNA log 5.2, CD4 538 cells/µl), Kaposi Sarcoma (KS)
12/2007: Started cART with TDF/FTC/EFV, consistently < 50 c/ml plasma
05/ : Chemotherapy for KS, Castleman’s, Primary Effusion Lymphoma (PEL)
Primary
Effusion
Lymphoma
CD4 (Cells/µl)
Chemo- therapy
Chemotherapy with Pomalidomide
Autopsy
cART
103
107
105 10
HIV RNA (c/ml)

9. HIV DNA is Detectable in Most Tissues 12 Hours Post Mortem
HIV Recovery
HIV DNA (c/106 cells)
Location
1.8
Spinal Cord
Cerebellum
Frontal Lobe
1.9
KS skin lesion
Genetic Analysis N
Single Genome
Sequences 30
Lymph node 38
Spleen 12
Jejunum 23
Ileum 5
Colon 3
Lung 1
Testes
Kidney 10
Effusion cells
127
Total Tissue 75
Lymph node 12
Spleen 54
Jejunum 90
Ileum 2
Colon 52
Lung 3
Testes 7
Kidney 6
Effusion cells
107
PBMC
234

10. No Evidence Of Molecular Evolution In Tissue Compartments
HXB2 88 89 81 85
0.005 (6 nucleotides)
NO EVIDENCE OF POPULATION SHIFTS OR COMPARTMENTALIZATION
APD = 0.3%
PBMC (6/2013)
PBMC (4/2015)
LYMPH NODE
SPLEEN
JEJUNUM
ILEUM
COLON
EFFUSION
KIDNEY
LUNG
TESTES

11. HIV Infected Cells Undergo Clonal Expansion In PBMC And GALT
0.005 (6 nucleotides)
HXB2
APOBEC3G
HYPERMUTANTS
(replication incompetent)
Josefsson et al., PNAS 2013
Plasma RNA
PBMC DNA
Colon DNA
Ileum DNA
106
105
103
102
101
104
HIV VL #2 DX 2 7
16 Y
One of these mechanisms of host restriction to HIV infection is mediated by the members of the apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) 3G 3F possibly others
are incorporated into nascent viral particles and act during the next infection cycle
Deamination of cytidine to uracil predominantly occurs on the minus strand intermediate (Lecossier et al., 2003), which can be evidenced as guanine-to-adenine (G→A) changes in the plus strand.
HIV genomes with APOBEC-mediated G→A changes can either be degraded or can be integrated into the host chromosome.

12. HIV Infected Cells Undergo Clonal Expansion In PBMC And Tissues
100 96
0.005
APOBEC3G
HYPERMUTANTS
(replication incompetent)
PBMC (6/2013)
PBMC (8/2013)
PBMC (8/2014)
PBMC (4/2015)
LYMPH NODE
SPLEEN
JEJUNUM
ILEUM
COLON
EFFUSION
KIDNEY
LUNG
TESTES

13. Summary
No evidence of ongoing cycles of HIV replication in tissue compartments and PBMC in study participants undergoing long term cART
8-16 years suppression
450 single genome sequences
Clonal expansion of HIV infected cells can take place across multiple tissue compartments
Strategies to eradicate HIV reservoirs will require elimination of clonally expanded populations
HIV DNA was recovered from most, but not all, tissues post mortem
HIV persistence studies will benefit from autopsy analyses

14. Acknowledgements STUDY PARTICIPANTS HIV And AIDS Malignancy Branch
K. Aleman
T. Uldrick
R.Yarchoan
HIV Molecular Monitoring Core
R. Gorelick
B. Fullmer
NIAID/CCMD Clinic
C. Rehm
S. Jones
H.C. Lane
H. Masur
NIDDK
S. Kumar
S. Wank
University of Milan
C. Balotta
M. Galli
A. Lai
HIV Dynamics and Replication Program
Clinical Retrovirology Section
F. Maldarelli
E. Anderson
J. Bell
J. Hattori
S. Hill
Z. Grossman
C. Lange
F. Simonetti
S. Watters
Translational Research Unit
M. Kearney
Tufts University
J. Coffin
Viral Mutation Section
V. Pathak
R. Burdick
LEIDOS
W. Shao
R. Dewar
STUDY
PARTICIPANTS