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.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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.





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

Thrush: Oral Candidiasis

The Discovery of the AIDS Virus - HIV in 1983



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



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



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


are needed to see this picture.Nef

• 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



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

QuickTime™ and a decompressor






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



HDAC inhibitors





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

QuickTime™ and aTIFF (LZW) decompressor


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: [email protected]: 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



- 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 ?



New York to DC



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

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

Documents

hiv slide

ccr5 slide

va slide

summers slide

hiv total

lav hiv

hiv changes

hiv genome