Download Why Vaccines and Therapies for HIV are So1

Partners in Science National Conference
M.J.Murdock Charitable Trust
San Diego, California
January 12, 2007
Why Vaccines and Therapies for HIV
are So Challenging:
New Strategies to Outwit the Virus
Nancy L. Haigwood, Ph.D.
Seattle Biomedical Research Institute
University of Washington
We envision a world where people live free from the threat of infectious disease.
[email protected]
1965 Science Fair
International School of Bangkok
The epidemic, the virus, the
genome, molecular structures
•
•
•
•
Mysteries of HIV, master of escape
Progress in drug therapy
Progress in vaccine development
Role for science teaching and
inspiration at all levels
HIV and AIDS
• Human Immunodeficiency Virus (HIV) is the virus
• AIDS = Acquired Immune Deficiency Syndrome
– Consequence of HIV infection
– Immune system unable to fight other diseases
• Death due to opportunistic infections
– Those that don’t usually cause illness
• Illness and death after ~10-20 years
– Cannot eradicate the virus once infected
– 100% mortality
Million
25 years of
Acquired Immunodeficiency Syndrome (AIDS)
People
living
with HIV
50
45
40
35
30
Children
orphaned
by AIDS in
sub-Saharan
Africa
25
20
15
10
5
0
1980
1985
1990
1995
2000
2005
HIV-1 is variable worldwide
Los Alamos Database http://hiv-web.lanl.gov/
Diversity on an INDIVIDUAL level
Homogeneous new infection
Replicates ~ 24hrs
Produces 1010 new virions a day
Average 1 mutation per replicated genome
Rapidly develop a “quasispecies”
Nancy L. Haigwood
Seattle Biomedical Research Institute
Nonhuman primate models for AIDS
Macaca mulatta (rhesus)
Macaca nemestrina (pigtailed)
Macaca fascicularis (crab-eating)
• HIV-1: only replicates in chimpanzees--disease in 10 years
• SIV: simian immunodeficiency virus; transferred from African to
Asian macaques in captivity and caused disease like AIDS
• SHIV: chimera that has the HIV Envelope and the backbone of SIV;
these viruses cause disease after passage in macaques
Nancy L. Haigwood
Seattle Biomedical Research Institute
1% divergence
Same virus,
11 macaques
89.6p consensus
89.6P
107 (0.3%)
•
•
•
•
•
191 (0.3%)
098
099 (0.3%)
168 (0.4%)
098 (0.9%)
069 (1.2%)
071 (1.4%)
156 (1.8%)
246 (1.9%)
Envelope DNA sequences
Approx 10 clones per macaque
32 weeks post-infection
Maximum likelihood tree
Average % divergence in
parentheses
• Each monkey has a different
virus collection termed
“quasispecies”
• Some animals have more virus
and more divergence--why?
108 (1.9%)
152 (1.9%)
Nancy L. Haigwood
Seattle Biomedical Research Institute
(Blay, JV 2006)
Infected cells producing HIV
Images of HIV, Boehringer-Ingelheim
Human Immunodeficiency Virus (HIV)
http://mbim.web.arizona.edu/pics/hiv.gif
Human Immunodeficiency Virus (HIV)
http://mbim.web.arizona.edu/pics/hiv.gif
Three levels of control in HIVexposed people
Images of HIV, Boehringer-Ingelheim
Activated cells with key
receptors are vulnerable to HIV
Monocyte with CD4 and CCR5
Nancy L. Haigwood
Seattle Biomedical Research Institute
Three types of “natural”
resistance to infection
1. Genetic resistance to infection and activation
2. Innate immune responses
3. Acquired immunity
Monocyte or macrophage with CD4 and CCR5
Nancy L. Haigwood
Seattle Biomedical Research Institute
Immunity to viral infection-clearance is key to health
Illness
Antibody
CTL
0
Replication
7
14
28
35
Days post-infection
Nancy L. Haigwood
Seattle Biomedical Research Institute
Acquired immunity:
antibody-producing B cells
Neutralizing (Env only)
Non-neutralizing
(all HIV proteins)
Neutralizing antibodies are a subset of HIV-specific
antibodies that block infection
Nancy L. Haigwood
Seattle Biomedical Research Institute
Acquired immunity:
cytotoxic T cells (CTL)
CTL recognize viral proteins on the cell surface
and destroy them
Killer
T-cell
CTL recognize 9 specific amino acid sequences in all
viral proteins--both variable and conserved antigens
Nancy L. Haigwood
Seattle Biomedical Research Institute
Immunity in HIV infection
• Local innate responses may control infection in some
cases (e.g. sex workers in Africa)
• Robust cellular immunity associated with long-term control
and low virus loads
• Robust neutralizing antibodies can block infection if there
before exposure and also help control
• Infection is not cleared by these mechanisms and the
virus persists in the host DNA in blood and other
reservoirs
• Loss of gut lymphocytes an early event that is critical to
survival and immunity
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV binds to the cellular receptors
Goal of drug treatment--stop the spread
Nancy L. Haigwood
Seattle Biomedical Research Institute
Fusion inhibitors block
infection
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV fuses and releases its
genome and enzymes
Nancy L. Haigwood
Seattle Biomedical Research Institute
Inhibitors to RT and INT block
infection and integration
RT=reverse transcriptase
IN=integrase
Nancy L. Haigwood
Seattle Biomedical Research Institute
Cells are permanently infected
with HIV
Nancy L. Haigwood
Seattle Biomedical Research Institute
Infected cells produce more virus,
not only in blood, but in tissues
PR=protease
Nancy L. Haigwood
Seattle Biomedical Research Institute
Protease inhibitors block HIV from
becoming infectious
Immature virions (noninfectious)
Nancy L. Haigwood
Seattle Biomedical Research Institute
More cells are susceptible…
Nancy L. Haigwood
Seattle Biomedical Research Institute
…and become infected
Nancy L. Haigwood
Seattle Biomedical Research Institute
Now the reservoir is larger
Nancy L. Haigwood
Seattle Biomedical Research Institute
More virus is made, and some are
new variants
gut
blood
brain
Nancy L. Haigwood
Seattle Biomedical Research Institute
Viral reservoirs: drug- and CTLand antibody-resistant variants
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Cells continue to produce virus in the reservoirs
Without effective combination drugs, mutants arise
New drugR strains can take over and be transferred
No current drugs can eliminate the reservoir virus
Unknown if immunity can be “enhanced” by
therapeutic vaccination
Progress in drug discovery
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Better understanding of viral regulation
More activity in drug discovery (new targets)
Additional animal models for other targets
Experience in the developing world
Drugs more widely available (political pressure)
Better application of drugs-(time, dose, patient)
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–
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Monitor for drug-resistance and adjust “cocktail”
Reduce Mother-to-Child-Transmission
Lengthen lives by earlier drug application
Reduce transmission
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV Vaccine Goals
Goal is CONTROL virus load:
–Would work like anti-HIV drug
“cocktail”
–Could slow the time to onset of AIDS
–Lengthen lives
–Reduce transmission to others
Immunity to infection
Illness
Antibody
CTL
0
Replication
7
14
28
35
Days post-infection
Nancy L. Haigwood
Seattle Biomedical Research Institute
Goal of vaccination
Live-attenuated vaccines are too risky!
CTL
Antibody
Illness
CTL
Vaccine
Antibody
Infection on challenge or exposure
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV Vaccine Approaches
• Recombinant HIV proteins
• DNA plasmids encoding
HIV genes
• Live-attenuated vector
viruses expressing HIV
proteins
Ad
• Chemically inactivated
virions
• Artificial virus-like particles
HIV Vaccine Approaches
http://www.nwabr.org/education/hiv.htm
• Recombinant HIV proteins
• T helper cells
• DNA plasmids encoding
HIV genes
• Live-attenuated vector
viruses expressing HIV
proteins
• Multiple HIV antigens
• Binding Antibodies
– Antibody-Dependent
Cellular Cytotoxicity
Ad
• Chemically inactivated
virions
• Artificial virus-like particles
• Neutralizing Antibodies
– HIV Env
– Block binding of virus to cell
• Cytotoxic T Lymphocytes
(CTL):
– Target and kill virus-infected
cells
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV Vaccine Progress
Vaccines can reduce virus loads in
nonhuman primate challenge models





Work like anti-HIV drug “cocktails”
Slow the time to onset of symptoms
Eventually all succumb to AIDS
Can be superinfected (also in humans)
Vaccines that “work” best elicit innate,
cellular, and humoral immunity
(neutralizing antibodies)
Nancy L. Haigwood
Seattle Biomedical Research Institute
Variables in vaccine development
Vaccine Mode
Whole, killed
Attenuated
DNA
Recomb. proteins
Peptides
Lipopeptides
Mimetopes
Pseudovirions
Virus-like particles
Vector
Adjuvant**
Alum
Cytokines
Pulsed DC
Co-stimulatory
QS-21
CpG oligos
PROPRIETARY
Vector
Vaccinia
MVA
Fowlpox
Canarypox
Adenovirus
VEE
Semiliki Forest Virus
Adeno-associated virus
OPV
Herpes simplex virus
Rabies virus
VSV
Measles
Moloney Leukemia virus
Hepatitis B virus
Listeria monocytogenes
BCG
Salmonella
Gene
Env
Gag
Rev
Tat
Nef
Pol
Vpu
Vif
Vpr
Design
Dose
Route
Timing
Virus
SIVmac .239/251
SIV E660
SIV mne E11S
SIV pbj14
SIVDB670
SHIV 89.6P
SHIV SF 162P
SHIV DH12R
SHIV E-P4
SHIV Grt1FX
SHIV KU2
SHIV .C2.\1
Macaque
Indian rhesus
Chinese rhesus
Crab-eating
Pigtailed
Courtesy of AS Fauci
HIV Vaccine Challenges
Vaccines elicit low level immunity humans
 Similar to macaque studies in magnitude,
duration, types of immunity
 Combination vaccines better than single
modality
 No protection in first vaccine trial (Env
protein only) NO CHALLENGES
 Excitement over adenovirus vector
delivery
 Still seeking ways to generate neutralizing
Nancy L. Haigwood
antibodies against multiple HIVs
Seattle Biomedical Research Institute
HIV Envelope, master of disguise
Envelope spike = 3 functional Envelope proteins (gp160)
gp160 = gp120 surface unit, gp41 transmembrane unit
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV Envelope, master of disguise
C1
V1V2
C2
V3
C3
V4
C4 V5
C5
5 conserved regions
5 variable regions
Glycosylated (sugars on the outside of the protein)
Nancy L. Haigwood
Seattle Biomedical Research Institute
HIV Env binds to CD4 to mediate entry
http://www.prn.org/images/prn_nb_cntnt_images/vol7num1/eron_fig1a.gif
• HIV Env must bind to CD4 to gain entry into the host cell
• This is theoretically a weak point for the virus, which could also be targeted by
neutralizing antibodies (NAbs)
Epitopes of broad NAbs from patients
adapted from: Burton DR et al. PNAS 2005
gp41
4E10
2F5
gp120
IgG1b12
2G12
These NAbs “describe” specific regions of Env but they
do not inform the mechanism of their development
The blind men and the elephant:
seeing the shape of HIV Envelope
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Photo of African elephants in Tanzania by William F. Sutton
1% divergence
Same virus,
11 macaques
89.6p consensus
89.6P
107 (0.3%)
191 (0.3%)
098
099 (0.3%)
168 (0.4%)
098 (0.9%)
069 (1.2%)
Are the envelope gene
changes in each
macaque similar or
different?
071 (1.4%)
156 (1.8%)
246 (1.9%)
108 (1.9%)
152 (1.9%)
Nancy L. Haigwood
Seattle Biomedical Research Institute
(Blay, JV 2006)
PNGs altered in “hotspots”
Prevalence
of PNG
Inoculum
1.0
0.8
0.6
0.4
0.2
0
V3
V1V2
V4
V5
Prevalence
of PNG
SHIV-89.6P
1.0
0.8
0.6
0.4
0.2
0
139C
141
V1V2
188
276
386 397
V3
V4
463
V5
N-glycosite (LANL) analysis of 89.6P
• 102 SHIV-89.6P gp120 clones
• 19/23 PNGs conserved in >90% of sequences, found on
silent face of gp120 in crystal structure
• 6 PNGs altered in V1/V2, C2, V, V5
• All are under selective pressure (dN/dS >1)
Blay et al (2006) J Virol 80:999
PNG changes map proximal to CD4 binding site
CD4bs
CD4bs
139C
276
V5
V1V2
141
460
CD4bs
186
V1/V2
397
V4
V5
386
V3
V4
CD4bs view
• Core crystal structure from
Kwong and Wyatt, Nature,
1998
• Variable loops modeled by S.
Gnanakaran, LANL
• Image modified with Protein
Explorer
V3
Blay et al (2006) J Virol 80:999
PNG changes map proximal to CD4 binding site
CD4bs
CD4bs
139C
276
V5
V1V2
141
462
CD4bs
188
V1/V2
397
V4
V5
386
V3
V4
CD4bs view
• Core crystal structure from
Kwong and Wyatt, Nature,
1998
• Variable loops modeled by S.
Gnanakaran, LANL
• Image modified with Protein
Explorer
V3
Blay et al (2006) J Virol 80:999
Pseudovirus Production
pEMC*
89.6p
gp41
293T cells
Variant gp120
HIV genome
(with no env)
Nancy L. Haigwood
Seattle Biomedical Research Institute
Neutralization assay
• Tzm-bl cell line
• Expresses CD4, CCR5, CXCR4
• HIV-1 LTR expression of -gal and luciferase
• Infect with pseudovirus or patient HIV or SHIV
HIV-1 LTR
luc
luciferase
Relative Light Units
Nancy L. Haigwood
Seattle Biomedical Research Institute
Conclusions from Envelope
studies:
HIV Envelope is plastic and utilizes many
methods to evade neutralizing antibodies
(sugars, charge change, deletions)
There is a LIMIT to where and how much
change can be tolerated (Achilles’ heel)
Expose conserved regions
Nancy L. Haigwood
Seattle Biomedical Research Institute
The E2 Protein serves as a scaffold for a
multienzyme complex
PSBD
Catalytic Domain (CD)
Nancy L. Haigwood
Seattle Biomedical Research Institute
E2 antigen display system
(E2DISP)
Heterologous protein
PSBD
Catalytic Domain (CD)
HP
Adapted from Domingo et al., 2001. J Mol Biol 305:259.
E2DISP-HIV Particles:
pure and hybrid
Express E2DISP-HIV molecules in E. coli BL21 cultures
Purification of 60mers: ammonium sulfate fractionation, ion exchange
chromatography and gel filtration
“pure” particles
Mix pure particles in varying ratios
Denature in Guanidine HCl
Renature
“hybrid” particles
Nancy L. Haigwood
Seattle Biomedical Research Institute
E2DISP Antigen Display System
• Advantages
– Ability to present up to 60 different heterologous proteins on
surface of artificial Virus Like Particles
– Present epitopes in unique conformations (conserved Env!)
– Elicit antibodies, T cell help and CTL
– Obtain up to 3 mg protein per 1 L culture
– Antibodies are long-lived and high titer
• Limitations
–
–
–
–
Prokaryotic system: No glycosylation
Each construct requires optimization of purification
Difficulties characterizing exact particle composition
Will get responses to E2 also; best used in combination
Haigwood Lab, Summer 2004
Kristina
Thorsen
Sonali
Mahalanabis
Nicole
Doria-Rose
Nancy
Haigwood
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Dina
Lauman
Wendy
Blay
Pushpa
Jayaraman
Not shown: Ruth Hotchkiss
Jeanne Ting Chowning
Bill
Sutton
Haigwood Lab, Summer 2006
Madhumita
Mahalanabis
Theresa Kasprzyk
Dina Kovarik Pushpa Jayaraman, Ph.D.
Bill Sutton
Delphine
Malherbe
Ph.D.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Cherie Ng
Mike Chester
Wendy Blay, Ph.D.
HIV and AIDS solutions 2007
• Behavior
• Drug therapy
• Vaccines
• Education of the next generation
•
•
•
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