search for a cure of hiv/aids dr. yuntao wu depatment of molecular and microbiology george mason...

Search for a cure of HIV/AIDS

Dr. Yuntao Wu

Depatment of Molecular and MicrobiologyGeorge Mason University, Manassas, VA

Early History of AIDS

• In March 1981, at least 8 cases of Kaposi’s Sarcoma (KS) have occurred amongst young gay man in New York.

• By April, CDC noticed an increase in PCP (pneumocystis carinii pneumonia).

• In June, CDC published a report about PCP in 5 men in Los Angeles. Then, CDC formed a Task Force on KS and Opportunistic Infections.

• In July, CDC reported KS in 26 gay men in New York City.

• Between June 1st - May 28, CDC received 355 KS/PCP reports, 79% were homosexual

• The disease were initially called GRID:

Gay-Related Immune Deficiency

- In July 1982, first non-homosexual case reports appeared that the disease was occurring in Haitians and Haemophiliacs.

- In the same month, AIDS was suggested in a meeting in Washington DC.

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From http://aidshistory.nih.gov/first_encounters/back.html

The purplish lesions of Kaposi's sarcoma, a cancer not usually seen in young men, were common among the patients with the new immune deficiency disease.

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http://www.visualdxhealth.com/child/oralCandidiasisThrush-signsAndSymptoms.htm

Thrush: Oral Candidiasis

The Discovery of the AIDS Virus - HIV in 1983

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Luc MontagnierHIV-1LAV(HIV-1Lai/LAV)

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Robert GalloHTLV-IIIB(HIV-1Lai/IIIB)

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Jay A LevyUCSF

Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS)

• F. Barre-Sinoussi; J. C. Chermann; F. Rey; M. T. Nugeyre; S. Chamaret; J. Gruest; C. Dauguet;C. Axler-Blin; F. Vezinet-Brun; C. Rouzioux; W. Rozenbaum; L. Montagnier

• Science, New Series, Vol. 220, No. 4599. (May 20, 1983), pp. 868-871.

HIV-1

gp120gp41

p17

p24

RT

IN

RNA

PR

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• What is AIDS ?

(Acquired Immunodeficiency Syndrome)

1993 CDC revised definition:

HIV-1 infected persons who have less than 200 CD4 T cells per ul or a CD4 percentage of total lymphocytes of less than 14%. 26 clinical conditions

Candidiasis of bronchi, trachea, or lungsCandidiasis, esophagealCervical cancer, invasive *Coccidioidomycosis, disseminated or extrapulmonaryCryptococcosis, extrapulmonaryCryptosporidiosis, chronic intestinal (greater than 1 month's duration)Cytomegalovirus disease (other than liver, spleen, or nodes)Cytomegalovirus retinitis (with loss of vision)Encephalopathy, HIV-relatedHerpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitisHistoplasmosis, disseminated or extrapulmonaryIsosporiasis, chronic intestinal (greater than 1 month's duration)Kaposi's sarcomaLymphoma, Burkitt's (or equivalent term)Lymphoma, immunoblastic (or equivalent term)Lymphoma, primary, of brainMycobacterium avium complex or M. kansasii, disseminated or extrapulmonaryMycobacterium tuberculosis, any site (pulmonary * or extrapulmonary)Mycobacterium, other species or unidentified species, disseminated or extrapulmonaryPneumocystis carinii pneumoniaPneumonia, recurrent *Progressive multifocal leukoencephalopathySalmonella septicemia, recurrentToxoplasmosis of brainWasting syndrome due to HIV

The Possible Origins of HIV-1

- likely transmitted to man from chimpanzees infected with SIVcpz in West Africa. However, Chimpanzees may not be the original reservoir Pan troglodytes

The Possible Origin of HIV-2

- likely transmitted to man from SIVsmm-infected sooty mangabeys in west Africa

Adults and children estimated to be living Adults and children estimated to be living with HIVwith HIV

Total: 37.8 (34.6 – 42.3) million

Western Europe580 000580 000

[460 000 – 730 000][460 000 – 730 000]

North Africa & Middle East

480 000480 000[200 000 – 1.4 million][200 000 – 1.4 million]

Sub-Saharan Africa25.0 million25.0 million

[23.1 – 27.9 million][23.1 – 27.9 million]

Eastern Europe & Central Asia1.3 million 1.3 million [860 000 – [860 000 – 1.9 million]1.9 million]

South & South-East Asia

6.5 million6.5 million[4.1 – 9.6 million][4.1 – 9.6 million]

Oceania32 00032 000

[21 000 – 46 000][21 000 – 46 000]

North America1.0 million1.0 million

[520 000 – 1.6 million][520 000 – 1.6 million]

Caribbean430 000430 000

[270 000 – 760 000][270 000 – 760 000]

Latin America1.6 million1.6 million

[1.2 – 2.1 million][1.2 – 2.1 million]

East Asia900 000900 000

[450 000 – 1.5 million][450 000 – 1.5 million]

Clinic Course of HIV Infection

G. Pantaleo, C. Graziosi, and A.S. Fauci 1993a.. N. Engl. J. Med. 328: 327-335.

HIV Replication Cycle

RNA

DNA

RT

tat,rev,nef

Env,vif,vpr,vpu

gag,pol

Transcription

Integrationrev

tatnef

CD4CXCR4

or CCR5

Reverse Transcriptase Inhibitor

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by D

an S

t ow

el l

©2

00

2- 2

00

5) .

Zidovudine (AZT), Tenofovin, ddI, ddC, d4T

nucleoside analogues

Non-nucleoside analogues

Nevirapine, Delavirdine, TMC-120, TMC-125

from Goff, Fields Virology, 4th edition

1. 3’-end processing (cytoplasm)

2. Cleavage of cell DNA (nucleus)

3. Strand-transfer4. Gap filling

-5-bp direct repeat of cellular DNA

- dinucleotides: 5’-TG; 3’-CA

Model of HIV genomic packaging

From M. F. Summers

HAART

Highly Active Anti-Retroviral Therapy (HAART) consists of 3 or more highly potent anti-HIV drugs, commonly reverse transcriptase inhibitors and protease inhibitors.

a single drug therapy may be successful for a while, but because HIV changes to avoid detection, drug-resistant strains will often arise in the patient.

The chances of a HIV genome mutating such that it can resist three separate drug treatments at once, however, is so small that the pressure of this therapy prevents the emergence of resistant strains

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HIV reservoirs

CD4 T cells- post-integration latency mostly in memory CD4 T cells- no active or low level active viral replication- rapidly generate viruses upon stimulation

Macrophages - tissue and brain macrophages, monocytes- resist to HIV mediated cell killing- actively generating virus in the body for a long period of time

Current Strategy to activate latent viral reservoirs

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HDAC inhibitors

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Histon Deacetylase Inhibitors

Limitations of re-activation strategy

1) Viral reservoirs in the body is not currently well-identified and understood

2) Not every latent virus can be re-activated by a single method

3) Reactivation does not lead to the elimination of latent cells

A “trojan horse” strategy to identify and eliminate HIV positive cells

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The making of the “trojan horse”

5’ LTR

rev

tat

gag pol env nefvpu

vif

3’ LTRD1 A5D4 A7

X X X

X

X

XXX

vprX

HIV-1 Rev-dependent gene expression

gp160, Pr55

tat, rev, nef

env, vif, vpu, vpr

gag-pol

Rev

Tat, Rev, Nef

gp160, Pr55

mRNA

The Rev-dependent lentiviral vector: pNL-GFP-RRE

Rev

Procedure for generating lentiviral particles

pMD.G(VSV)

pCMV∆R8.2

pNL-GFP-RRE293T cells

1. HIV-1(AD8) infection of macrophages

2. Super-infection with vNL-GFP-RRE +

vNL-GFP-RRE

HIV-1(NL43.HSA) + vNL-GFP-RRE

GF

PmCD24

High specificity of the Rev-dependent vector

NIH AIDS Research & Reference Reagent Program: Cat # 11466Email: ywu8@gmu.eduPhone: 703-993-4299

Rev-dependent vectors carrying toxins andpro-apoptotic genes for targeting HIV-positive cells

• Anthrolysin O• Diphtheria toxin A chain• Human TRAF-6

- secreted by Bacillus anthracis- pore-forming, cholesterol-dependent cytolysin- lethal to to human primary monocytes, macrophages- cytotoxic activity is sensitive to the inhibition by cholesterol or serum

Anthrolysin O (AnlO)

Diphtheria toxin

EF2 + NAD+ ADPR-EF2 + nicotinamide + H+DT

- DT is an ADP ribosylating enzyme- the most studied bacterial toxin- ADP ribosylation occurs at one modified histidine in EF2- highly toxic, one molecule can kill a cell- multiple previous animal and human clinical trials using DT-A for cancer therapy and immuno-therapy to kill T cellsP

rote

in S

ynth

esi

s

Time

Diphtheria Toxin

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- endogenous human gene- low level expression is tolerated and my help viral gene expression- over-expression trigger’s apoptosis- may not be as effective as bacterial toxins- safer to use than bacterial toxins

Tumor necrosis factor-associated factor 6 (TRAF6)

Killing efficiency:DT = 100%, TRAF6 = 86%, AlnO = 55%

DT-A mutants with reduced cytotoxicity

DT-A = 100%E18S = 96%E148D = 93%G128D = 65%DT∆N = 52%

Difficulties in assembly of lentiviral particles carrying toxins

pMD.G(VSV)

pCMV∆R8.2

pNL-GFP-RRE-(SA) 293T cells

Cloning and Mutagenesis of human EF-2 gene

*GGA CGAGly Arg

5H7, 46%

CB2, 28%

AB1, 24%

4H10, 14%

5E12, 9%

293T, 0%

From about 100 clones5 were DT-resistant

Resistance of 293T cells to TRAF-6

HeLa

GFP TRAF-6

0.0% 53%

24% 0.5%

7.6%

293T

0.0% 39% 25%

Cell control

Production of lentiviral particles carrying AnlO, DT-A and TRAF6

DNA construct Producer cell

p24 level at 48 hours post cotransfection

p24 level at 72 hours post cotransfection

pNL-TRAF6-RRE pCMV∆R8.2 pHCMV-G

HEK293T

1095 ng/ml

1659 ng/ml

pNL-AlnO-RRE pCMV∆R8.2 pHCMV-G

HEK293T + 6-BOCD

800 ng/ml

1500 ng/ml

pNL-DT∆N-RRE pCMV∆R8.2 pHCMV-G

5H7

109 ng/ml

102 ng/ml

Proof-of-concept: killing of HIV-positive macrophages in vitro

Proof-of-concept: killing of HIV-positive T cells in vitro

Limitations of the vector

1. Lack of selective entry

4. Toxin contamination of particles

2. Lack of ability to target resting CD4 T cells

5. Are they really safe ?

3. Integration-mediated mutagenesis

- need safety test in animals

Are these toxin particles really safe to inject ?

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New York to DC

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Acknowledgements NIAID: AI069981, AI081568 NINDS: NS051130NYCDC AIDS ride

GMU:Jessica YoungZhong-wei TangYangfan ZhengDongyang YuJeremy KellyMark SpearQuan YuBarney BishopSub IyerVladimir KarginovSerguei PopovTaissia Popova

NIH:Jon MarshDavid Neville

Harvard Medical School:Zhirui Wang

Zhong wei