Human immunodeficiency virus (HIV)

HIV-1 and HIV-2 HIV-1  Binds to CCR5 co-receptor  Most common strain of the Human Immunodeficiency Virus  What most professionals refer to when they reference HIV  HIV-2  Binds to

Human Immunodeficiency Virus (HIV)

Andrew Borst

Isabel VanDerslice

Image: http://www.npr.org/blogs/pictureshow/2011/02/22/133868260/sciviz

A World Pandemic

 HIV has enormous social, economic

and humanitarian implications

 HIV and AIDS is decimating

populations, taxing health

infrastructures, and crippling

economies, and creating

unsustainable population age

distributions

 Some parts of Africa have a

prevalence rate of 1 in 3 For many,

infection is inevitable, and

treatment beyond reach.

 From its discovery in 1981 to 2006,

AIDS killed more than 25 million

people.

The Spread of HIV

 Spread through sexual contact, intravenous

drug use (infected blood), and

mother-to-child during birthing and nursing.

 Birthing transmittance rate is 15-30% for

mothers infected with HIV.

 To minimize risk, antiretroviral therapy is

recommend prior to (for the mother) and after

birth (for the baby)

 Does not spread through mucous membranes,

only blood contact

 HIV is a fragile virus

Tip of the Day: Use condoms and clean

needles!

HIV-1 and HIV-2

 HIV-1

 Binds to CCR5 co-receptor

 Most common strain of the Human Immunodeficiency Virus

 What most professionals refer to when they reference HIV

 HIV-2

 Binds to CXCR4 co-receptor

 Uncommon; mostly found in Western Africa or individuals of whom are in

the later phases of disease progression

Higher HIV-2 prevalence-rates here

Vaccine and Treatment

 It is difficult to make a vaccine for HIV due to several

characteristics of the virus

 HAART: Highly active

anti-retroviral therapy is the only

effective treatment, but

resistance occurs, especially after many years of treatment

 Gene therapy appears to be a

good candidate for future HIV treatment options

Vaccine and

Treatment

Anti-retroviral therapy has significantly reduced

the death toll associated with AIDS.

 It is difficult to make a vaccine

for HIV due to several

characteristics of the virus

 HAART: Highly active

anti-retroviral therapy is the only

effective treatment, but

resistance occurs, especially after

many years of treatment

 Gene therapy appears to be a

good candidate for future HIV

treatment options

Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to

Nathalia Holt, Jianbin Wang, Kenneth Kim, Geoffrey Friedman, Xingchao Wang, Vanessa Taupin, Gay M Crooks, Donald B Kohn, Philip D Gregory, Michael C Holmes & Paula M Cannon

HIV-1 Infection

 Transmembrane proteins on the surface of the virus interact with receptors on the host cell

 Two interactions are needed for entry— CD4-gp120 and a chemokine coreceptor, usually CCR5.

 The main reservoirs for HIV in the body are wherever immune cells reside, such as the spleen and intestine HIV is also found

in the follicular dendritic cell (FDC)

network.

 In tonsils and adenoids of HIV-infected patients, infected macrophages fuse into multinucleated giant cells that produce huge amounts of virus.

HIV Infection Mechanism

Co-receptor CCR5 Permits HIV-1 Entry

•CCR5 is the major co-receptor

used by HIV-1 and is expressed

on key T-cell subsets and

monocytes.

•CCR5Δ32 is a relatively

common allele in Western Europe

•Confers an innate resistance to

HIV-1 infection

•CCR5 antagonists have proved to be an effective salvage therapy in patients infected with drug-resistant HIV-1 Kuby Immunology says CCR5 is only expressed on Monocytes, but this is an extremely misleading

oversimplification.

ZFN-mediated disruption of CCR5 in CD34+ HSPCs.

 ZFN: Zinc Finger Nuclease

 Artificial restriction enzymes generated

by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain

 NOD SCID IFγ-null Mice

 Non-obese diabetic severe combined immunodeficiency interferon-gamma null mice.

 Lack T, B, and NK cells

 Deficient in multiple cytokine signaling pathways

 Defects in innate immunity.

Image courtesy of Wikipedia: “The only website on the internet guaranteed to be 90% accurate!”

Fun Fact: “Cel” in Cel 1 nuclease stands for celery!

ZFN-mediated disruption of CCR5 in CD34+ HSPCs.

Figure 1 Analysis

 a) Representative gel showing extent

of CCR5 disruption in CD34+ HSPCs

24 hours after nucleofection

 Neg: No gene digestion

 Mock : No gene digestion

 ZFN: Gene digestion

 b) Mean percentage of human CD45+

cells in peripheral blood of mice 8

weeks after transplantation.

 No statistical difference between Neg,

Mock, and ZFN groups.

 c) Fluorescence-activated cell sorting

(FACS, or flow cytometry) profiles of

human cells of various organs from

one ZFN-treated mouse.

 Results indistinguishable from that of

mice transplanted with unmodified

cells.

 Holds true for both the location of cells and their frequency in that particular tissue.

Protection of human CD4+ T cells in peripheral blood of HIV-infected mice previously engrafted with ZFN-modified CD34+ HSPCs.

Figure 2 Analysis

 a) FASC readouts showing human

CD4+ and CD8+ T-cells in peripheral

blood of representative animals from

each of three cohorts.

 Uninfected: Normal CD4+ T-cell levels

 HIV-1 Infected (Negative): Complete

T-cell depletion

 HIV-1 Infected (ZFN-treated): Normal

CD4+ T-cells

 b) Ratio of human CD4+ to CD8+

lymphocytes in peripheral blood of

individual mice to which were infected

Effects of HIV-1 infection on human cells in

HSPC-engrafted NSG mice.

Figure 3 Analysis

 a) FACS analysis of human cells in

tissues of representative NSG mice

from three cohorts.

 SSC means “side scatter” of

T-helper cells post HIV-1

infection w/o ZFN treatment.

 Thymus: Complete loss of

CD4+ and CD8+ T-cells.

 Small intestine: Complete loss

of all human lymphocytes w/o

ZFN treatment.

 b) Immunohistochemical analysis of

human CD3 expression in small

intestine, and CD4 expression in

spleen of representative mice.

Reasons for CD4+ and CD8+ T-cell Depletion in the

Thymus

 Proposed to occur as a consequence of the upregulation

of CCR5 on these cells during HIV-1 infection.

 Explains observed reduction of both of these cell types in the

thymus and other tissues

Image: M Germana Paterlini (2002) Structural modeling of the Chemokine Receptor CCR5: Implications for

Ligand Binding and Selectivity Biophysical Journal 3012-3031

HIV-1 infection selects for disrupted CCR5 alleles.

Figure 4 Analysis

 a) Mean +- s.d levels of CCR5 disruption

in sequential peripheral blood samples

taken from mice.

 b) Mean +- s.d levels of CCR5 disruption

in necropsied mice 12-weeks

post-infection or with uninfected ZFN-treated

cohorts.

 Gel shows increased levels of digestion

products in infected mice indicating

increased CCR5-/- cell selection.

 c) Contour FACS analysis of human

CD4+ T-cells in small intestine and

spleen of one representative animal from

each cohort

 Expected results were expected.

 d) Mean +- s.d numbers of human CD4+

cells and CD4+CCR5+ per 5,000 cells

analyzed from different sections of the

ZFN activity produces heterogeneous mutations in CCR5.

Control of HIV1 replication in mice receiving ZFN

-treated CD34+ HSPCs

Figure 6 Analysis

 a) Mean +- s.d levels of HIV-1 RNA

and percent CD4+ T-cells in peripheral

blood.

cohorts increase post-infection, but

drop off after 6 weeks.

 Reduction in CD4+ cells limits viral reproducibility.

 T-cell levels remain mostly constant in

ZFN-treated mice barring initial

deletion of residual CD4+CCR5+

T-cells.

 b) Mean +- s.d levels of HIV-1 RNA in

small and large intestine lamina

propria from 8 and 12 week

necropsied mice.

 Significantly less HIV-1 in ZFN-treated

mice.

Control of HIV1 replication in mice receiving ZFN treated CD34+ HSPCs

 Findings suggest that transplantation of HSPCs modified by CCR5-specific ZFNs may provide permanent supply of HIV-resistant progeny

 ZFN also modified CCR2; nonspecific cleavage must be evaluated in

larger future studies

 Scientific rational for CCR5 modification stems from HIV+ Leukemia

patient being effectively cured of his infection

 Immune hyperactivation suppressed with CCR5-/- mutants

 This approach could be considered a “one-shot” treatment option

 Targeting CCR5 may promote CXCR4-tropic HIV development

 Could be used as a backup therapy, or done in addition to HAART

My science is perfect.

A quick word about using proper grammar

Now…any questions?