characterization of persistent hiv-1 in a broad spectrum of cd4 + t-cells isolated from peripheral...

Characterization of persistent HIV-1 in a broad spectrum of CD4+ T-cells isolated from peripheral blood, bone marrow,

lymphoid tissue and gut associated lymphoid tissue from patients on

long-term suppressive therapyLina Josefsson

Karolinska Institutet,Solna, Sweden

“Towards an HIV Cure” Pre-Conference Symposium20 & 21 July 2012

Background

• Persistent HIV can still be detected in plasma and cellular reservoirs even after several years of suppressive antiretroviral therapy

• Identifying the source and dynamics of persistent HIV in patients on suppressive therapy is an important step towards HIV eradication

Objectives of the study • Use single-genome and single-proviral sequencing assays, to investigate the:

– Source of persistent HIV-1• What cells are HIV reservoirs during suppressive therapy?

– Dynamics of persistent HIV-1• Does low-level viral replication maintain HIV during suppressive

therapy? • 8 patients on suppressive antiretroviral therapy (4-12 years)

– 5 initiating therapy during acute/early infection (< 6months)– 3 initiating therapy during chronic infection (> 1.5year)

• Peripheral blood (PB)• Bone Marrow (BM)• Gut Associated Lymphoid Tissue (GALT)• Lymph Node Tissue (LN)

Layout of the study

20 april 2023Lina Josefsson 4

Initiation of cART

Plasma1) Chronic: 1-3years

Plasma2) Chronic:1-6 months

Acute/early:0-12 days

Cells:Progenitor (CD34+ Lin-)Late progenitor (CD34-, Lin-) (CD34+, Lin-)Lymphoid cells (Lin+ CD4+)Naïve (CD45RO-CD27+)Central Memory (CD45RO+CD27+)Effector Memory (CD45RO+CD27-)Myeloid (CD3- CD4lo)

5 patients treated during acute/early infection

3 patients treated during chronic infection

1. Investigate the source of persistent HIV-1 - Characterization of HIV-1 in infected cells after long-term suppressive cART

2. Investigate the dynamics of persistent HIV-1 - Compare pre-therapy HIV-1 populations to HIV-1 populations after long-term suppressive cART

Treatment naive HIV-1 < 75 copies/ml

4 – 12 years

LNPBGALTBM

P6, protease, RTgag-pro-pol

Higher frequency of infection in cells isolated from patients treated during chronic infection compared to patients treated during

acute/early infection

Peripheral blood

a p <0.05

1 2 3 4 5 6 7 8 9 10

Estimated Frequency of Infection (%)

95% CIEstimated

Frequency of Infection (%)

95% CIEstimated


95% CIEstimated

Frequency of Infection (%)*

95% CIEstimated


95% CI

1 N/A N/A N/A N/A 0.0094 0.0079, 0.0112 0 0.0000, 0.0006 0 0.0000, 0.00022 0.0213 0.013, 0.0349 0.0091 0.005, 0.0164 0.0163 0.0130, 0.0205 0.0021 0.0009, 0.0050 0 0.0000, 0.000073 0.0048 0.0029, 0.0078 0.0102 0.007, 0.0149 0.0055 0.0038, 0.0081 0.0003 0.0002, 0.0005 0 0.0000, 0.00024 0.0004 0.0002, 0.0006 0.001 0.0004, 0.0026 0.0006 0.0003, 0.0012 0.0001 0.0000, 0.0004 0 0.0000, 0.0002

5 0.0475 0.0317, 0.0713 0.0877 0.0668, 0.1151 0.1817 0.1373, 0.2404 0.0026 0.0016, 0.0043 0, (0.0001)**0.0000, 0.0002

(0.0000,0.0009)

0.0065 0.0096 0.0098 0.0005 0.0000

6 0.1351 0.1017, 0.1794 0.0008 0.0005, 0.0012 0.4190 0.3155, 0.5565 0.0359 0.0259, 0.0499 0, (0.0003)0.0000, 0.0001

(0.0001, 0.0006)7 0.0316 0.0218, 0.0457 0.2615 0.2086, 0.3277 0.1646 0.1311, 0.2066 0.0221 0.0152, 0.0320 0 0.00028 0.0498 0.0375, 0.0662 0.0878 0.0618, 0.1247 0.0284 0.0180, 0.0447 0.0022 0.0014, 0.0036 0 0.0003

0.0597 0.0259 0.1251 0.0121 0.0001Geometric Mean*** Frequency of Infection - Chronic infection

Initiated therapy during acute/early infection

Initiated therapy during chronic infection

Myeloid (CD3- CD4+)Patient

All Memory(CD45RO+ CD27-/+)

Effector Memory(CD45RO+ CD27-)

Central Memory(CD45RO+ CD27+)

Naive (CD45RO- CD27+)

Geometric Mean*** Frequency of infection - Early infection

* One cannot rule out the possibility of low but nonzero infection rates in cases where estimates were zero, as shown by the upper confidence bounds.** Numbers in parenthesis denotes the frequency of infection found in myeloid lysate with T-cell receptors present.*** For calculation of geometric means, estimated infection rates of zero were replaced with 1/(number of cells tested)

0.007%

0.06%

X9

0.01%

0.03%

X3

0.01%

0.13%

X13a

0.0005%

0.01%

X24a

Gut Associated Lymphoid Tissue

Higher frequency of infection in cells isolated from patients treated during chronic infection compared to patients treated during

acute/early infection

1 2 3 4 5 6 7 8

Estimated Frequency of Infection (%)*

95% CIEstimated


95% CIEstimated


95% CIEstimated


95% CI

1 0.0171 0.0014, 0.2046 0.0278 0.0023, 0.3330 N/A N/A 0 0.0000, 0.01692 0.0212 0.0046, 0.0974 0.0287 0.0056, 0.1464 0 0.0000, 0.1108 0 0.0000, 0.06403 0.0155 0.0032, 0.0761 0.0313 0.0050, 0.1967 0 0.0000, 0.3181 0 0.0000, 0.02734 0 0.0000, 0.0152 0.0135 0.0014, 0.1281 0 0.0000, 0.1209 0 0.0000, 0.0124

5 0.002 0.0003, 0.0125 0.0236 0.0103, 0.0540 0.1392 0.0147, 1.3195 0, (0.0083)**0.0000, 0.012,

(0.0009, 0.0784)

0.0089 0.0240 0.0685 0.0082

6 0.1231 0.0840, 0.1802 0.1497 0.0959, 0.2337 0.7585 0.2469, 2.3300 0, (0.0228)0.0000, 0.0084, (0.0074, 0.0703)

7 0.014 0.0035, 0.0560 0.1959 0.1346, 0.2853 0.1233 0.0059, 2.5807 0, (0.0288)0.0000, 0.0208, (0.0034, 0.2480)

8 0.02 0.0054, 0.0733 0.1191 0.0753 0.1882 0 0.0000, 0.0190 0, (0.0225)0.0000, 0.0061, (0.0058, 0.0881)

0.0325 0.1517 0.0840 0.0246Geometric Mean* Frequency of Infection - Chronic Infection

Central Memory(CD45RO+ CD27+)

Naive (CD45RO- CD27+) Myeloid (CD3- CD4+)



Patient

Effector Memory(CD45RO+ CD27-)

Geometric Mean*** Frequency of infection - Early infection

* One cannot rule out the possibility of low but nonzero infection rates in cases where estimates were zero, as shown by the upper confidence bounds.** Numbers in parenthesis denotes the frequency of infection found in myeloid lysate with T-cell receptors present.*** For calculation of geometric means, estimated infection rates of zero were replaced with 1/(number of cells tested)

0.009%

0.03%

0.02%

0.15%

0.07%

0.08%

X4 X6a X1.2

a p <0.05

No infected progenitor cells isolated from bone marrow

* One cannot rule out the possibility of low but nonzero infection rates in cases where estimates were zero, as shown by the upper confidence bounds.

Bone marrow

a p < 0.05 Josefsson et al, JID 2012


Upper 95% CI


Upper 95% CI


Upper 95% CI (patient 1-5)

95% CI (patient 6-8)

Estimated Frequency of Infection (%)

95% CI

1 0 0,0004 0 0,0001 0 0,0003 0,0045 0.0037, 0.00552 0 0,0006 0 0,0001 0 0,0002 0,0103 0.0071, 0.01493 0 0,0007 0 0,0002 0 0,0003 0,0013 0.0008, 0.00214 0 0,0030 0 0,0002 0 0,0008 0,0003 0.0001, 0.00085 0 0,0006 0 0,0001 0 0,0002 0,0090 0.0066, 0.0124

6 0 0,0003 0 0,0002 0,0003 0.0001, 0.0005 0,0980 0.0698, 0.13757 0 0,0004 0 0,0004 0,0004 0.0002, 0.0012 0,1157 0.0892, 0.14998 0 0,0009 0 0,0005 0,0008 0.0003, 0.0020 0,0165 0.0111, 0.0245



Patient

Bone Marrow Lin-/CD34+ HPCs Lin-/ CD34- HPCs Lin-/CD4+ Cells Lin+/CD4+ Lymphoid cells

X13a

Phylogenetically indistinguishable HIV-1 sequences from plasma collected before initiation of cART and cells isolated

after several years of suppressive therapy

Patient 4 Treated during early infection> 6 years on therapy

Peripheral blood – Central memoryPeripheral blood – Effector memory

On-therapy Peripheral blood – All memory

Peripheral blood – Naive

Pre-therapy

Plasma

GUT – Effector memory

Lymph node – Effector memoryLymph node– Transitional memory

Bone marrow – Lin+CD4+

0.001

Phylogenetically indistinguishable HIV-1 sequences from plasma collected before initiation of cART and cells isolated

after several years of suppressive therapy

Patient 8Treated during chronic infection>8 years of therapy

Peripheral blood – Central memoryPeripheral blood – Effector memory

On-therapy Peripheral blood- All memory

Peripheral blood – Naive

Pre-therapy

Plasma

GUT – Effector memoryGUT – Central memoryGUT– Naive

Lymph node– Effector memoryLymph node – Central memory

Lymph node– Transitional memory

Lymph node– Naive

Bone marrow – Lin+CD4+Bone marrow – Lin-CD4+

0.01

No clear detection of viral evolution between sequences isolated from pre-therapy plasma and sequences isolated from peripheral

blood or GALT collected after 4-12 years of suppressive cART

• 3 different approaches to investigate the amount of viral evolution between sequences from plasma before the initiation of therapy and sequences from cells after 4-12 years of suppressive cART

Genetic distance Correlation of genetic divergence and time Evolutionary rate analysis

Genetic distance between:

1. Sequences from pre-therapy and on-therapy

2.Sequences from pre-therapy 3.Sequneces from on-therapy

Correlation of genetic divergence and time between:

1. Pre-therapy and on-therapy2.Pre-thearpy (1) and pre-therapy(2) for patients treated during chronic infection

Nucleotide substitutions per site per year between:1. Pre-therapy and on-therapy

2. Pre-therapy (1) and pre-therapy(2) for patients treated during chronic infection

Comparison of genetic distance between sequences from the

same time point differ no more than the comparision of

sequences between PT and OT indicating no evolutionary change

during suppressive therapy

No correlation between genetic divergence and time between pre-therapy and on-

therapy sequences indicating no evolutionary change during suppressive thereapy.

Correletion between pre-therapy (1) and pre-thearpy (2).

Nucleotide substitutions per site per year:

1. Patients treated during early infection: mean = 10e-5

2. Patients treated during chronic infection: mean = 10e-4

3. Pre-therapy sequences from patients treated during chronic infection

mean = 10e-3

Genetic distance

Genetic distance between: 1. Sequences from pre-therapy and on-therapy

2.Sequences from pre-therapy 3.Sequneces from on-therapy

Comparison of genetic distance between sequences from the same time point differ no more than the comparison of sequences between PT and OT

indicating no evolutionary change during suppressive therapy

Correlation of genetic divergence and time

Correlation of genetic divergence and time between: 1. Pre-therapy and on-therapy

2.Pre-thearpy (1) and pre-therapy(2) for patients treated during chronic infection

No correlation between genetic divergence and time between pre-therapy and on-therapy sequences indicating no evolutionary change during suppressive therapy.

Correlation between pre-therapy (1) and pre-therapy (2).R2 = 0.0015 – 0.1556

Evolutionary rate analysis

Nucleotide substitutions per site per year between:1. Pre-therapy and on-therapy

2. Pre-therapy (1) and pre-therapy (2) for patients treated during chronic infection

Nucleotide substitutions per site per year: 1. Patients treated during early infection:

mean = 10e-5 2. Patients treated during chronic infection:

mean = 10e-43. Pre-therapy sequences from patients treated during chronic infection

mean = 10e-3

Nucleotide substitutions per site/year: Early infection: 10e-5

Chronic infection: 10e-4Pre-therapy: 10e-3

Conclusions• T-cells were found to be infected in all patients after long-term

suppressive therapy. No progenitor cells were found to be infected and myeloid cell samples contained T-cell receptors

• Early initiation of effective therapy results in lower reservoir size• No clear evidence for evolution could be detected in patients

treated during early and chronic infection• The lack of clear evidence for HIV-1 genetic evolution in the HIV-1

infected CD4+ T-cell populations after years of therapy argues against virus replication as a major cause of persistence in these cell populations. – The role of replication in other tissues and cell types however

remains to be defined.

AcknowledgementsWe acknowledge with gratitude the participation of all the patients in this study

University of California, San FranciscoF. HechtE. SinclairP. BacchettiP. LewisP. HuntM.SomsoukH. HantanoS. DeeksL. EplingM. Kilian T. HoA. TanJ. CusterL. LoebR. HohL. Poole

Karolinska Institutet The Swedish Institute for Communicable Disease ControlS. PalmerS. ErikssonV. DahlM. MildJ. Albert Rega Institute, BelgiumN. FariaP. Lemey

SAIC/NCIW. Shao

VRC/NIAIDD. DouekW. Wylie

Patient information

Patient Viral Load

Pre Therapy(RNA copies/ml)

Viral Load On Therapy*

(RNA copies/ml)

CD4+ T-cellCount

Pre Therapy(cells/ul)

CD4+ T-cellCount

On Therapy (cells/ul)

Lenght of infection(years)

Time on therapy(years)

Treated during acute/early infection

1 88359 <40 165 470 8.6 8.5

2 44960 <40 494 1048 7.9 7.6

3 40930 <40 792 1279 12.8 12.5

4 3583 <40 648 867 6.7 6.6

5 118888 <40 243 571 4.3 4.3

Treated during chronic infection

6 85999 <40 406 491 7.3 5.3

7 74117 <40 400 726 11.4 9.8

8 70510 <40 342 891 10.6 8.8

* Viral RNA levels at the time of the study

Results – genetic distance

PatientGenetic distance (%)

(Pre-therapy sequences and on-therapy sequences)

Genetic distance (%) (Pre-therapy sequences)

Genetic distance (%)(On-therapy sequneces)


1 1.36 1.34 1.22

2 0.18 0.19 0.13

3 0.07 0.05 0.09

4 0.12 0.12 0.12

5 0.12 0.1 0.13


6* 0.87 N/A N/A

6 0.91 0.98 0.78

7* 5.51 N/A N/A

7 1.11 0.98 0.97

8* 1.43 N/A N/A

8 1.38 1.22 1.27

* Pre-therapy samples collected 1-3 years apart

Results – regression analysis

PatientCorrelation of genetic divergence and time

R2

Initiated therapy during acute/early infection1 2.78E-022 1.87E-023 2.28E-024 1.29E-025 2.63E-02

Initiated therapy during chronic infection 6* 0.7767

6 1.54E-03 7* 0.8795

7 0.1556 8* 0.3769

8 0.1205* Pre-therapy samples collected 1-3 years apart

Results – evolutionary rate analysis

Treated during early infection Treated during chronic infection

1818

Sort by cell type using FACS

Single-proviral Sequencing

PCR Amplify each HIV genomefrom the lysate and sequence


Calculate genetic relationship to plasma sequences from sample taken before and after long-term therapy

Align sequences

Patient Cells from peripheral blood, bone marrow,

lymphoid tissue and GUTLyse

sorted cells 1 2 3 4 5 6 7 8 9 10

DNA endpoint dilutionDNA endpoint dilution

Cell lysate

Positive PCR

reactions

Josefsson et. al. JID 2012

20 april 2023Namn Efternamn 19

Multiple Alignment File

cDNA endpoint dilutioncDNA endpoint dilution

11 22 33 44 55 66 77 88 99 1010

Agarose gelAgarose gel


Sequence individual genomes

Palmer et. al. J Clin Micro 2005Kearney et. al. J Virol 2008

Single-Genome Sequencing of Plasma Samples

characterization of persistent hiv-1 in a broad spectrum of cd4 + t-cells isolated from peripheral...

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