Download Finding a Cure: What HIV vaccine would you choose?

Finding a Cure: Which HIV
vaccine would you choose?
Ramil Sapinoro
Life Sciences Learning Center
University of Rochester Medical Center
AIDSVax Inc.
Department of Vaccine Research
1979 Cure Drive
Rochester, NY 14620
Dear Research Technician,
We are excited to collaborate with your organization in developing a AIDS vaccine. I am
sending you 4 possible DNA vaccines that you could use in your vaccine trials. I’m not sure
which combinations of the three HIV genes are in each one. You will have to check this.
Along with the DNA, I have included some data collected from preliminary animal studies.
We injected animals with each of the three HIV genes alone: the gag gene codes for HIV
structural proteins; the env gene codes for the virus envelope proteins; and the pol gene
codes for viral enzymes. We used mice as an experimental animal.
Sincerely,
Clarke Kent, Ph.D
Director
Center for Vaccine Research
University of Rochester
How does the Immune System
fight invaders?
Antigen
MHC
APC
Virus
Antigen Presenting Cells
(ex: macrophages)
Capture invaders (pathogens), destroy
them, and display parts of them
(antigens) on proteins called MHC’s
How does the Immune System
fight invaders?
Helper T Cells
T
APC
See antigens presented by APC’s and does two things:
1) Turn on Cytotoxic T Cell – these T Cells can go on to
kill other cells that present the antigen (infected cells as
well as macrophages)
2) Activate B Cells
How does the Immune System
fight invaders?
T
Cytotoxic T Cell
B
B Cell
T
APC
How does the Immune System
fight invaders?
Helper T cell produces signaling
proteins that tell B Cells that have
antibodies against the antigen to
make more B Cells and free floating
antibodies
Cytotoxic T cell produces
signaling proteins that tell
infected cells to kill
themselves
T
B
Infected Cell
B BB
How do vaccines ‘help’ the immune
system to fight off viruses?
What would a Successful Vaccine Do?
•Stimulate the production
of antibodies
•Stimulate the production
of Cytotoxic T Cells
•Remember the pathogen
(Memory cells)
•Not cause disease
BB
Vaccine Approaches
What parts of a pathogen could we use to stimulate the
immune response and not cause disease?
Live-attenuated
Virus
Inactivated Virus
DNA
Protein subunit
Synthetic peptide
Vaccines Currently Available
Disease/Virus
Type of Vaccine
Polio
Inactivated virus
Influenza
Inactivated virus
Measles/Mumps/Rubella
Live-attenuated
Chicken Pox
Live-attenuated
Smallpox
Live vaccinia virus
Hepatitis B
Recombinant protein
Cervical Cancer
Virus-like particles
What is HIV?
HIV = Human Immunodeficiency Virus
•Small
•Replicates within cells of living
hosts
•Composed of RNA
•Pathogenic: Has the ability to
cause disease (AIDS)
T
HIV causes disease because
it kills helper T cells
APC
HIV Genome and Structure
The HIV genome has
three main genes…
gag gene
pol gene
env gene
env proteins
Viral envelope proteins
gag proteins
Capsid
Matrix
pol proteins
Viral enzymes
Four Potential Vaccines
V1
gag gene
V2
pol gene
pol gene
gag gene
V3
V4
env gene
env gene
pol gene
Which of these
possible pieces
of DNA are in
each vaccine?
How will you find
out?
gag gene
env gene
pol gene
Which genes are in the vaccines?
Cut with
Restriction Enzyme
V1 DNA Vaccine
?
V2 DNA Vaccine
?
V3 DNA Vaccine
?
V4 DNA Vaccine
?
Gel electrophoresis of digested vaccines
You have been given
vaccines that have
been pre-digested
with restriction
enzyme.
You’ll need to run the
DNA fragments out
on a gel to see what
genes are in each
vaccine.
V1
V2
V3
V4
Loading your gel
Loading your gel
Gel electrophoresis of digested vaccines
V1
V2
V3
V4
• Load the gel
• Carry the gel to a power source
• Fill the gelbox with TAE buffer (fill the box so that the top of
the gel is just covered)
• Put the lid on the gel
• Run the gel for about 10 minutes at 120 volts
Which genes are in the vaccines?
gag gene
pol gene
env gene
Restriction Enzyme
Well
gag gene
pol gene
env gene
Gel electrophoresis
Which genes are in the vaccines?
gag gene
pol gene
Restriction Enzyme
Well
gag gene
pol gene
Gel electrophoresis
Which genes are in the vaccines?
pol gene
env gene
Restriction Enzyme
Well
pol gene
env gene
Gel electrophoresis
Which genes are in the vaccines?
gag gene
Restriction Enzyme
Well
gag gene
Gel electrophoresis
Which genes are in the vaccines?
pol gene
Restriction Enzyme
Well
pol gene
Gel electrophoresis
Which genes are in the vaccines?
env gene
Restriction Enzyme
Well
env gene
Gel electrophoresis
Which one of these would you
use in a human trial?
gag gene
pol gene
env gene
pol gene
gag gene
env gene
pol gene
gag gene
env gene
pol gene
Which ones do you KNOW you would NOT want to use?
Of the others, how will you decide which ones to use in human trials?
Vaccine study in an animal model
Amount of Antibodies
BB
DNA injected into mice
Which one(s) would you use to make a vaccine?
Vaccine study in an animal model
Amount of cytotoxic T-cell Activity
BB
DNA injected into mice
Which one(s) would you use to make a vaccine?
More about HIV/AIDS…
CD4
Helper T Cell
HIV Lifecycle
Global HIV infections,
Worldwide HIV-1
Decinfections,
2006 Dec. 2002
http//www .unaids.or g/hivaidsinfo/documents.html
N. America
1.0 million
Subtypes: B
W. Europe
0.6 million
Subtypes: B
E. Eur/Cen As ia
1.2 million
Subtypes: B & ?
Africa
30.0 million
Subtypes: C, A, D, E, B
Latin America, Caribbean
1.9 million
Subtypes: B, C
Omitted
s ubtypes F-J (rare); groups N, O
Sth, SE As ia
6.0 million
Subtypes: C, E, B
E. As ia, Pacific
1.2 million
Subtypes: C & ?
Global Total (approx)
Total: 42 million
HIV Treatment: Anti-virals
Reverse Transcription
Inhibitors
Protease Blockers
X
What is in the vaccines?
Which ones would you use in human trials?
Which genes are in each of the
four potential vaccines?
Which of the potential vaccines
would you NOT use?
What other information might you
need to make a decision on which
one to use?
V1
V2
V3
V4
Clinical Trials: Testing in
Humans
Phase
Objective
Number of Volunteers
I
Safety and
dose
20–50 / trial
II
Additional
safety and
immunogenicity
100’s / trial
III
Efficacy and
protection
1000’s / trial
Phase
Objective
Number of Volunteers
I
Safety and
dose
20–50 / trial
II
Additional
safety and
immunogenicity
100’s / trial
V2 and V4
III
Efficacy and
protection
1000’s / trial
You will be
testing the
serum of a
group of
people who
have either
been injected
with V2, V4 or
a placebo to
see if they
respond to
the vaccine
by making
antibodies
against the
appropriate
proteins
V2
Each volunteer is injected
with one vaccine. After a
time, serum is collected
from the volunteer, and
analyzed for the
presence of antibodies
using an ELISA assay…
Serum
Obtain three absorbent squares and label
“G” (gag) “P” (pol) and “E” (env)…
Spot the gag, pol and env proteins on their
appropriate spots…
Gag
Pol
Env
Place serum on each of the squares – the
serum contains antibodies…
Gag
Pol
Env
If the volunteer has antibodies against the
HIV proteins, those antibodies will bind to the
proteins on the paper. Excess antibodies will
wash off…
Gag
Pol
Env
Add labeled “Secondary Antibody” This
secondary antibody binds to all human
antibodies, and has a colored tag…
Gag
Pol
Env
Upon the addition of “developing solution,”
the colored tag will become visible…
Gag
Pol
Env
You will each be given one
serum sample from one
volunteer – follow the
instructions in your handout to
determine if the volunteer
made the appropriate
antibodies…
Which vaccine worked the best in
the trial?
Vaccine
Given
Volunteer #
Gag
Pol
Env
Why do we need to test immunogenicity?
Diversity, diversity, diversity…
People have different MHC alleles
Person 1
Immune response
Person 2
No immune response
Antigen
MHC allele #1
MHC allele #2
APC
APC
MHC Polymorphism
Challenges in HIV Vaccine
Research
•
Viral Genetic Diversity: HIV is not
just one specific virus.
•
Immune Protection: Scientists don’t
know what immune responses are
needed, or how strong they need to
be.
•
Neutralizing Antibody: Difficult to
generate broadly neutralizing
antibodies.
•
Vaccine Testing: Slow process, very
expensive
…but on the Brightside…
• Precedent from other systems:
Success against other viral
infections
• Precedent from animal studies:
Long-term control of infection in
vaccinated monkeys
• Immune control of HIV-1: Infected
individuals control infection
• Vaccine Trials: In progress