The Challenge of an HIV Cure HIV provirus integration and expression - - PowerPoint PPT Presentation

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The Challenge of an HIV Cure HIV provirus integration and expression - - PowerPoint PPT Presentation

Feb 19, 2023 25 likes •450 views

HIV Drug Resistance Program National Cancer Institute at Frederick The Challenge of an HIV Cure HIV provirus integration and expression on long-term suppressive therapy John M. Coffin Tufts University Objectives 1. To understand the role of

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The Challenge of an HIV Cure

HIV provirus integration and expression on long-term suppressive therapy John M. Coffin Tufts University

National Cancer Institute at Frederick

HIV Drug Resistance Program

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  • 1. To understand the role of HIV in causing AIDS
  • 2. To understand how antiretroviral drugs control, but do not

cure HIV infection

  • 3. To understand the role of persistently infected, dividing, cells

in HIV persistence

Objectives

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  • 1. Financial:

Tocagen, Inc. SAB member and shareholder

  • 2. Off-label or unapproved drug recommendations:

None

Disclosures

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Global Burden of HIV Infection

UNAIDS,2015

Total: 36.7 million [34.0-39.8 million]

On Therapy 38,200

People undergoing antiretroviral therapy: 17 million

On Therapy: 2.1 million On Therapy: 320,000 On Therapy: 1.8 million On Therapy: 10.2 million On Therapy: 1.4 million On Therapy: 1.1 million WHO, 2015

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Challenges of HIV Infection

  • Current

antiviral therapies that can fully suppress viral replication and allow infected people to live a mostly normal life, but there are still both individual andand global challenges.

  • Only about half the affected population has access to drugs,

particularly in the worst-hit areas.

  • Effective means of blocking transmission (PREP) are known, but

not widely used. A vaccine is still a dream.

  • Development of resistance to antiviral drugs is still a major

issue, particularly in poorly-resourced areas.

  • Antiviral drugs effectively suppress HIV infection, but will never

cure it.

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Timothy Brown was cured with a bone marrow transplant. Why can’t we cure HIV Infection in everyone?

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Years on ART

  • 1

1 2 3 4 5 6

Plasma HIV-1 RNA (copies/ml)

10-1 100 101 102 103 104 105 106 107

Clinical LOD (50 copies/mL) Initiate ART Phase I t½ = 1.5 days Phase II t½ = 28 days Phase III t½ = 273 days

Courtesy Ben Hilldorfer, UPitt

HIV-1 RNA Decays in Four phases after Initiating ART

Phase IV t½ = ∞

1.0E-07 1.0E-06 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01 1.0E+00 1.0E+01 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

101 100 10-1 10-2 10-3 10-4 10-5 10-6 10-7 HIV DNA in PBMC (relative to start of ART)

HIV-1 DNA Decays Much Less Than Plasma Virus RNA after Initiating ART

Interrupt therapy 7 ≥10 Years

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Two Features of HIV Biology are Helpful in Understanding Persistence

  • Generation of genetic diversity during ongoing

virus replication.

  • Stable integration of DNA copy (provirus) of the

viral genome at one of millions of sites in the host cell DNA

  • The first important point is to distinguish

between two models:

  • ngoing

low-level replication and latent proviruses in long-lived cells.

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Inferring Virus Replication from Genetic Diversity

van Zyl et al Retrovirology 2018

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Clonal Expansion of RNA and DNA Sequences During Suppressive ART (NO Evidence for Evolution)

  • 3 weeks

6 weeks 2.7 years 6.7 years 7.2 years Reference

*G>A hypermutant

stop codon

*

99 99 99 99 90 99 99 48 99 99 64 99 26 27 53 15 88 25 21 6 3 32 15 79 65 16 91 69 26 37 63 59 98 32 85 66 51 57 3 52 41 44 31 63 3 29 26 22 25 4 22 13 3 1 23 5 26 16 5 4 21 5 21 1 15 1 5

Different clonal populations of RNA and DNA appear after years of therapy *Highly variable from patient to patient

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PT 3

pre- Rx (0.7%) 7 yr rebound (0.8%)

Pt 4

pre- Rx (1.0%) 5 yr rebound (0.6%)

0.005 0.005

divergence = 0.2% divergence = 0.01%

No Evolution from Pre-therapy in Rebound Viremia After Long-term ART

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  • The problem with the previous studies was that

the high diversity of HIV in chronically-infected individuals made it difficult to detect additional diversification on ART.

  • Therefore we studied HIV populations in a set of

infected people who were diagnosed and started on antiretroviral therapy (ART) within a few weeks

  • f

infection, when the virus population has had very little time to evolve.

  • Although

very difficult to identify, these patients provide a much stronger signal to detect HIV evolution.

Is There Ongoing HIV Replication on ART?

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No Evidence for Ongoing HIV Replication on ART?

(At least in blood)

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  • No evidence for on-going cycles of viral replication in

individuals fully suppressed on ART

  • Implies that the HIV reservoir is not maintained by on-going

cycles of viral replication and therefore developing more potent ART will not cure HIV infection

  • Others have proposed that ongoing replication during ART is

not seen in blood, but does occur in the lymphoid compartment

  • What’s going on in lymph nodes?

Summary and Implications

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Does HIV Replication Persist in Lymphoid Tissues During ART?

  • Sequenced paired lymph node and PBMC samples in

four suppressed individuals and compared to pre-ART populations

  • Sequenced longitudinal lymph node samples from

individuals on ART

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Proviral Populations Are Not Different in PB and LN After 5 Years of Suppression on ART

Diversity: Pre ART PB: 0.5% Long-term ART PB: 0.1% Long-term ART LN: 0.3%

0 days before ART (PBMC DNA) 5.4 years on ART (PBMC DNA) 5.4 years on ART (LNMC DNA)

6 nts

Probable clone Probable clone hypermutants

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SLIDE 17

hypermutants

No Difference in HIV Populations Across Lymph Nodes

6 nts

Right Inguinal LN Left Inguinal LN

Panmixia: p=0.6 Diversity: Right LN: 1.6% Left LN: 1.3%

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  • No evidence for on-going cycles of viral replication in lymph

nodes of adults fully suppressed on ART

  • No evidence for “compartmentalization” of infected cells

among lymph nodes and peripheral blood

  • The HIV reservoir is not maintained by on-going cycles of viral

replication and therefore developing more potent ART will not cure HIV infection

  • Are identical sequences expanded clones or are they identical

HIV variants (founder virus maybe?) with different integration sites?

Summary and Implications

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NO Evidence for a Role of HIV Replication in Maintaining the True Reservoir

What does maintain the reservoir?

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The Persistent Steady State

Long - lived cells Short - lived cells

ART Pre-ART Erosion Proliferation

The number of infected cells remains about the same, but clonal populations appear

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  • HIV DNA can irreversibly integrate at many millions of possible

sites in the cell gnome, and sites of integration can uniquely “tag” single infected cells and their progeny.

  • In long-lived HIV-infected cells, sites of integration are

determined both by initial preferences (i.e.., “hot spots”), by selection after integration, and by chance.

  • The assay we used (thanks to Rick Bushman and Charles

Bangham) involves shearing infected cell DNA, ligating linkers, PCR amplification with LTR and linker-specific primers, and paired-end Illumina sequencing.

  • Integration site is adjacent to LTR primer, and breakpoint is net

to linker-specific primer.

  • Multiple sequences with the Identical integration site and

multiple breakpoints imply clonal expansion of the cell after infection.

Integration Site Preferences 1.

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  • We have assessed integration site distribution in cultured cells

(PBMC, stem cells, various cell lines (>150,000 sites each) or in HIV infected patients during suppressive ART, and compared with gene expression (RNA-seq).

  • In the next slides, cumulative integration sites in each interval
  • f chromosome 16 are shown in red, and the expression level
  • f each gene or region in blue.
  • As you will see, the distribution of integration sites is highly

similar, even between freshly isolated PBMCs and highly aneuploid epithelial cell lines, like HeLa.

Multi-Scale Analysis of HIV Integration in and ex Vivo

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Jurkat Chromosome 16, 1-8882254 HeLa Chromosome 16, 1-8882254 HEK 293Chromosome 16, 1-8882254 PHA+ PBMC Chromosome 16, 1-8882254

  • A. Whole Chromosome 16 (350 kb/bin)

Patient 1 Chromosome 16, 1-8882254 All Patients Chromosome 16, 1-8882254

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Jurkat Chromosome 16, 9263001 -19263000 HeLa Chromosome 16, 9263001 -19263000 HEK 293Chromosome 16, 9263001 -19263000 PHA+ PBMC Chromosome 16, 9263001 -19263000

  • B. Chromosome 16 ca 10X from A (40 kb/bin)

Patient 1 Chromosome 16, 9263000 -19262999 All Patients Chromosome 16, 9263000 -19262999

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Jurkat Chromosome 16, 13763001-1476300 HeLa Chromosome 16, 13763001-1476300 HEK 293Chromosome 16, 13763001-1476300 PHA+ PBMC Chromosome 16, 13763001-1476300

  • C. Chromosome 16 10X from B (4kb/bin)

Patient 1 Chromosome 16, 13763001-1476300 All Patients Chromosome 16, 13763001-1476300

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Translation Start Site Transcription Intron 6 Intron 5 Intron 4

PBMC

Integration Sites in MKL2 in Patient 1

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Integration Site Preferences 2. Selection for Specific Regions

  • In long-lived HIV-infected cells, sites of integration are

determined both by initial preferences (i.e.., “hot spots”) and by selection after integration.

  • Integrations in MKL2 (and a couple of other genes) in patient 1

are clearly due to selection for preferential growth or survival due to effects

  • f

provirus

  • n

host gene expression, reminiscent of well-known models of retroviral oncogenesis.

  • Only a very small fraction of proviruses seem to be involved in

such effects. What does the overall distribution look like?

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Integration Site Analysis Identifies Highly Expanded Clones in Patient 1

Maldarelli et al. Science 2014 Expanded clones with fewer than 7 integrants

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Are all HIV Integrations in Patients on ART in Expanded Clones?

  • We think it likely that they are, but we sample only about 10-6
  • f the CD4+ T cell population.
  • We might be able to figure it out if we knew the underlying

distribution of clones, but this is a very difficult problem…

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What does the City Look Like?

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Oh

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Clonal Expansion of Infected Cells can Arise in Several Ways

  • Direct effects of the integrated provirus on cell multiplication or survival
  • Chance outgrowth of a memory cell during homeostatic replacement
  • Antigen driven expansion during an active immune response

The latter two effects have nothing to do with the provirus, but it serves to mark all descendants of an originally infected cell.

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Case Report: Patient 1:

Persistence and Rebound of HIV Viremia on cART

Rebound Treatment Interruption

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Cancer diagnosis

HIV Viremia:

Persistence and Rebound on cART

Therapy switch

1nt 63 53 53 33 45 62 36 71 100 45 56 58 99

Prior to change in cART

WT Virus

M184V K103N Resistance Mutations Present

After change in cART

WT Virus

Resistance Mutations Present

99 1nt 99 96

After change in cART

WT Virus ONLY

1nt 100

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AMBI-1 Full Provirus is Intact

Encodes Infectious virus!

Amplify in overlapping fragments and sequence

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Log copies/ml

The Predominant Virus in Plasma is Produced by the AMBI-1 Provirus

*Bootstrap values >.85 AMBI-1 provirus

P6-RT Sequences

HIV RNA 12.14.09 Plasma HIV DNA 12.09.11 CD4 DNA HIV RNA 12.09.11 Plasma HIV RNA 07.23.12 Plasma HIV RNA 08.31.12 Plasma

1 10 100 1,000 10,000 100,000 6/1/08 10/14/09 2/26/11 7/10/12

Predominant Plasma Clone (PPC)

Date

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A Highly Expanded HIV Clone Carries an Infectious Provirus

This is the first known case where we could identify and characterize a clone of latently-infected cells responsible for infectious virus in blood Only a small fraction of cells express virus RNA (not shown). What is the epigenetic state of this provirus? (We don’t know yet. It’s not DNA methylation) What is driving clonal expansion? (We know a little bit)

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Despite the potential for viral cytopathic effect and immune-mediated killing of expressing cells, cell clones can both expand and harbor intact proviruses that produce infectious virus over time. Ca 1% of cells with the AMBI-1 provirus express small amounts of RNA at any one time. (M. Kearney) Persistent Clone Persistent Clone is responsible for Residual Viremia

cART blocks new infections

Antigen

Cells infected with an intact provirus can expand and produce infectious HIV

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Summary

  • We found no evidence for either ongoing replication or

compartmentalization of HIV in blood or lymph nodes during suppressive ART, implying that infection is primarily maintained as long-lived cells infected prior to ART.

  • Integration site may influence persistence and clonal expansion and

therefore, the virus that rebounds

  • Expanded clones CAN contain infectious proviruses, and are almost

certainly the source of rebound virus when therapy is stopped.

  • Curing HIV infection will require completely new strategies for

eliminating or otherwise dealing with this expanding persisting reservoir, which must be very large, and is highly variable from one patient to the next.

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Acknowledgments

  • Translational Research Unit

– Mary Kearney

  • Jon Spindler
  • Wei Shao
  • Andrew Musick
  • Ann Weigand
  • Michael Bale
  • Retroviral Replication Laboratory

– Stephen Hughes

  • Andrea Ferris
  • Clinical Retrovirology Section

– Frank Maldarelli

  • Shawn Hill
  • Francesco Simonetti
  • Leidos Biomedical Inc

– Xiaolin Wu

  • Amber Guo
  • Daaria Wells

– HMMC

  • Jeff Lifson
  • Brandon Keele
  • Rob Gorelick
  • University of Pittsburgh

– John Mellors

  • Michelle Sobolewski
  • UCSF DARE Collaboratory

– Steve Deeks

NCI Contract No. HHSN261200800001E NIH Bench to Bedside Program

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And the Patient Participants!

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