Transcript In thinking about vaccines, recall that there are two arms

In thinking about vaccines, recall that there are
two arms of the adaptive immune response
• Cellular (Cytotoxic T Lymphocyte or CTL) response -- works well
for controlling (most) viral infections because viruses replicate
intracellularly. Less adapted for controlling extracellular
pathogens such as bacteria or protozoa.
• Humoral (antibody-mediated) response -- works well for
controlling extracellular pathogens.
– Passive transfer of antibodies alone (e.g., mother to offspring) can
protect against viral infection.
– Neutralizing antibodies can prevent reinfection by some viruses
(e.g., influenza).
Vaccination
• For most viruses, you are immune to reinfection by the same
virus (e.g., chicken pox).
• Immunization or vaccination: Process by which one is
exposed to a live or inactivated virus, or to components of
the virus, in order to establish a state of immunity.
• Immunizations against smallpox introduced >1000 years ago.
Variolation: introduce dried smallpox scabs into nose of an
uninfected person, who then contracted a mild form of the
disease but was immune to smallpox.
– 1-2% died after variolation compared with 30% after smallpox.
• Vaccination against smallpox introduced by Edward Jenner in
1796. Jenner infected a boy with cowpox (a live-attenuated
virus), then exposed him to smallpox, which he failed to
contract.
– Jenner later experimented on other children, including his son.
These sorts of experiments are illegal today.
Clicker question
Most vaccines today are given in the form of wholekilled virus or live-attenuated virus.
Whole-killed virus vaccines induce _____ immunity.
Live-attenuated virus vaccines induce ____ immunity.
1)
2)
3)
4)
5)
cellular and humoral; cellular and humoral
cellular; cellular
humoral; humoral
cellular; cellular and humoral
humoral; cellular and humoral
Polio vaccine has been very successful in the US
•
Polio epidemics in 1950s affected >50,000 people in US.
•
Salk vaccine is an inactivated virus given in a series of injections. The
vaccine induces circulating antibodies, but no cellular immunity. Prevents
spread of virus from gastrointestinal tract to the central nervous system,
but doesn’t prevent infection of the gastrointestinal tract by the virus.
•
Sabin vaccine is a live-attenuated virus given orally. Produces cellular
immunity and circulating antibodies and prevents subsequent infection by
wild-type virus. Worldwide use of Sabin vaccine has eradicated polio in the
US and Americas.
•
We will discuss both of these methodologies (whole-killed virus and liveattenuated virus approaches) as potential ways to make an HIV vaccine.
Clicker question
What chemical did Jonas Salk use to inactivate
poliovirus to make the famous vaccine?
1) Formaldehyde
2) Polyethylene Glycol
3) Methanol
4) Ammonia
Clicker question
What chemical did Jonas Salk use to inactivate
poliovirus to make the famous vaccine?
1) Formaldehyde
2) Polyethylene Glycol (in many things, including in
Dr. Pepper: prevents over-foaming)
3) Methanol
4) Ammonia
Clicker question
How many American children contracted polio in 1955 because
of an incompletely inactivated batch of poliovirus vaccine
produced by Cutter Laboratories?
1) 10
2) 100
3) 20000
4) 40000
Source: Offit, PA (2005) The Cutter Incident, 50 years later. New England Journal of Medicine
Clicker question
How many American children contracted polio in 1955 because
of an incompletely inactivated batch of poliovirus vaccine
produced by Cutter Laboratories?
1) 10
2) 100
3) 20000
4) 40000
120,000 doses contained live virus, resulting in 40,000
cases of abortive polio (headache, stiff neck, fever,
muscle weakness). 51 children were permanently paralyzed,
5 died; 113 family members were paralyzed, 5 died.
Source: Offit, PA (2005) The Cutter Incident, 50 years later. New England Journal of Medicine
Cases of polio per year in US
From Morbidity and Mortality Weekly Report
(MMWR), Vol. 46, p. 79 (1997)
Flu vaccines
• WHO* specifies the contents of the vaccine each
year to contain the most likely strains of viruses
that will attack the next year.
• Annually updated trivalent flu vaccine for 20082009 season consisted of H3N2, H1N1 and B
influenza viruses. (But the vaccine H1N1 strain
doesn’t confer protection to H1N1 swine flu.)
• Viruses are grown in hens’ eggs.
• Inactivated viruses are injected or live attenuated
viruses are given as a nasal spray (not currently
approved in children younger than 5).
*World Health Organization
Why we need an HIV vaccine:
Anti-retroviral therapy hasn’t eradicated HIV
• Anti-retroviral treatment regimens are complex, expensive,
and can result in serious side effects.
• Developing safe, effective and affordable vaccines that can
prevent HIV infection in uninfected people is the best hope
for controlling and/or ending the AIDS epidemic.
• In 1984, Margaret Heckler (President Reagan’s Secretary of
the Department of Health and Human Services) announced
that the virus responsible for causing AIDS had been
identified, and that a vaccine would be ready for testing within
two years.
•We still don’t have a vaccine. Why?
“CD4” means CD4 T cells; “CTL” means CD8 cytotoxic T lymphocytes; “VLP” means virus-like particle
Pantello and Koup, 2004, Nature Medicine 10: 806-810
Pantello and Koup, 2004, Nature Medicine 10: 806-810
Traditional approach to an HIV vaccine:
whole-killed virus
Problems:
• Safety issues: are the viruses really dead?
• Production of strain-specific responses: would need to
inject a mixture of MANY different killed HIVs.
• A big problem: gp120 is shed from HIV, so what is being
injected isn’t a form to which the host will make effective
neutralizing antibodies.
• Another big problem: where do you get the HIV to kill
and then inject? Answer: grow in cells in the lab, but
laboratory strains are antigenically different than primary
HIV isolates.
http://www.niaid.nih.gov/hivvaccines/whole.htm
Future research needs
(from http://www.niaid.nih.gov/hivvaccines/whole.htm)
• Identify HIV isolate(s) that retain sufficient envelope
glycoprotein to effectively mimic wild-type HIV and generate
a strong, protective immune response.
• Develop and test inactivation procedures that will retain
antigenic integrity of the envelope glycoprotein.
• Develop cells and culture methods that will permit largescale production of primary HIV isolates.
• Develop alternative methods that can circumvent some of
the safety and technical concerns posed by manufacturing and
then inactivating infectious HIV virions.
Live-attenuated virus approach to
making an HIV vaccine
• Initial optimism because live SIV with a deletion in the Nef
gene* caused no disease in macaques and protected against initial
infection by wild-type** SIV.
• However, Nef-deletion mutants only slow disease progression,
but don’t abrogate it completely
• Adult macaques eventually showed immune dysfunction and 18%
developed AIDS.
• This vaccine shows limited or no cross protection against infection
by other strains of SIV.
• Live-attenuated HIV vaccines have not been tested in humans
due to safety concerns.
• Long-term growth of live-attenuated viruses would be done in
culture, but laboratory strains of HIV are antigenically different
than primary isolates.
*Nef down-regulates surface expression class I MHC molecules and CD4
**Wild-type: A normal, non-mutant form of a macromolecule, cell or organism
Possible vaccine approaches
• Induction of antibodies by injection with
whole-killed virus or viral proteins.
• Induction of antibodies and cellular immunity using
live-attenuated virus.
• Induction of only cellular immunity (cytotoxic T
lymphocyte (CTL) responses) by inducing expression of
viral proteins.
• DNA vaccines -- injection of purified DNA, usually into muscle, results in
transient expression of proteins encoded by introduced genes. Peptides derived
from these proteins are presented by MHC class I proteins to CD8 CTLs.
• Note that these sorts of vaccines can never prevent infection because T cells
do not recognize or kill free viruses. A strong anti-HIV CTL response, however,
has the potential to reduce or eliminate virally-infected cells and therefore
greatly reduce viral load.
DNA vaccines: http://people.ku.edu/~jbrown/dnavac.htm
Possible HIV Vaccine Strategies
• Peptide vaccines
• Recombinant subunit protein vaccines
– gp120, gp41, or gp160 produced by genetic
engineering
• Live vector vaccines
– non-HIV viruses engineered to carry HIV genes
• Virus-like particle vaccines
– non-infectious HIV containing one or more HIV
genes
• DNA vaccines
– HIV genes inserted into plasmids
• Combinations of vaccines
–Prime-boost regimens (e.g., DNA vaccine followed by
injection of recombinant protein)
• Current HIV Vaccine trials:
Merck’s HIV vaccine
• Merck V520 ad5 vaccine -- a live attenuated
virus designed to produce cellular and humoral
immunity
– Three stretches of HIV genome (from HIV gag, pol
and nef genes)
• Note HIV genes derived from only one HIV strain
– Shell from adenovirus type 5 (normally causes cold
symptoms)
• Virus was replication incompetent -- could
infect cells, but couldn’t produce more virus -Vaccine cannot cause HIV infection.
STEP trial
• 1500 volunteers (high-risk for HIV infection)
in Americas and Australia recruited in December
2004. 3000 volunteers by March 2007.
–62% male, Average age = 29
• Trial stopped in September 2007.
– Vaccine conferred no protective effects against HIV
infection and no effect on course of infection.
– More infections in vaccine recipients (49) than
placebo (33).
• Much analysis now to figure out what went
wrong.
– Is difference in infection rates significant?
– Did adenovirus immunity increase infection risk?
RV144 – the Thai Prime-Boost AIDS Vaccine Trial
• Prime with ALVAC-HIV (to induce cellular immunity)
– Recombinant canarypox vector vaccine genetically
engineered to express HIV-1 gp120 (clade E) linked to the
transmembrane anchoring portion of gp41 (clade B), and
HIV-1 gag and protease (clade B)
– Induces HIV-specific CD8 T cell responses in ~20% of
recipients
• Boost with AIDSVAX (to induce humoral immunity)
– HIV gp120 envelope glycoprotein vaccine (one clade E gp120 and
one clade B gp120)
– Results in non-neutralizing antibodies in most recipients
– Human trials in 2003 with AIDSVAX alone showed no protection
• Reported in fall 2009 – First vaccine to show
reduced risk of HIV infection
RV144 – the Thai Prime-Boost AIDS Vaccine Trial
Cost: ~$105,000,000
• Reported to lower rate of HIV infection by 1/3
• 16,402 participants (low to moderate risk for HIV
infection); 1:1 vaccinated versus placebo (HIV
testing every 6 months after vaccination)
– 74 in placebo group became infected
– 51 in vaccinated group became infected
– 31.2% efficiency; 96% confidence level (p < 0.05)
• HIV replication, CD4 T cell loss same in HIVinfected vaccine and placebo groups
Is there any hope for an antibody-based
therapeutic approach against HIV given that
people don’t normally make broadly neutralizing
antibodies against HIV?
• HIV rapidly mutates so that
antibodies are no longer effective.
• HIV spike proteins are covered
with carbohydrates, which are
poorly or non-immunogenic.
• Antibodies are too big to access
some regions of the HIV spike.
Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 14943-14948
A limited number of “broadly” neutralizing antibodies have
been isolated from HIV-infected individuals
Name
Epitope
Affinity (KD)
Structure
4E10
Conserved (98%) linear
WFXI sequence of gp41 Cterminal to the 2F5 epitope
1 – 70 nM
(n = 8)
2.2 Å
2F5
Conserved linear ELDKWA
sequence of the membrane
proximal ectodomain of
gp41
0.5 – 5 nM
(n = 8)
2.0 Å
b12
Discontinous epitope
overlapping the CD4
binding site of gp120
0.2 – 8 nM
(n = 5)
1.8 Å
2G12
a12 mannose cluster on
the “silent” face of gp120
2 nM
(n = 1)
1.8 Å
3.0 Å
17b
CD4-induced discontinuous
epitope overlapping the
coreceptor binding site
0.2 - 15 nM
(n = 11)
2.5 Å
X5
(PD)
CD4-induced discontinuous
epitope near the
coreceptor binding site
0.1 – 100 nM
(n = 3)
1.9 Å
Locations of antibody binding sites on
schematic model of trimeric viral spike
Kwong, P.D. et al., J. Virol. 2000. 74: 1961-72
Burton, D.R., R.C. Desrosiers, et al., Nat Immunol, 2004. 5(3): 233-6.
PD means “Phage display”, an in vitro method.
HIV has evolved to thwart the immune
system it has to confront
• Few, if any, HIV-infected individuals clear the virus.
• We can, however, make neutralizing reagents (antibodies
or antibody-like proteins) that prevent HIV infection.
• What we cannot yet do is design an immunogen that will
elicit broadly neutralizing reagents in a person (traditional
vaccine approach).
• Can we reprogram a human immune system so that it makes
broadly neutralizing anti-HIV reagents?
Gene therapy -- could it be used to treat or
prevent AIDS?
Adenovirus is not
a retrovirus, so
the inserted
genes are not
integrated into
the host cell
genome or
inherited by the
descendents of
infected cell.
Use recombinant DNA technology to insert a new gene into a virus, then use that to
infect cells. Infected cells will transcribe the new gene and make the encoded protein.
http://en.wikipedia.org/wiki/Gene_therapy
Gene therapy using retroviral vectors
• Advantage: Don’t have to re-administer the recombinant virus (as
required if using recombinant adenoviruses) because genetic
material is incorporated into the host cell genome, so all
descendents of infected cell will inherit it.
• Disadvantage: Retroviral integrase can insert the viral genetic
material anywhere in the host genome.
• Insertion into the middle of a host cell gene will disrupt it.
• Insertion upstream of a gene can disrupt its regulation.
• Disruption of a gene regulating cell growth could lead to
uncontrolled cell division (i.e., cancer).
• Gene therapy trials in France to treat X-linked Severe Combined
Immunodeficiency (X-SCID) were successful in curing the disease,
but resulted in leukemia in several patients.
Gene therapy may be safer using lentiviral vectors
than onco-retroviral* vectors
• French trial treating X-SCID - 3 of 11 developed leukemia-like condition. Used murine
leukemia virus (MLV)-based vector for ex vivo** infection of hematopoietic stem cells.
• Lentiviral vectors (HIV,SIV) have different integration properties than the oncoretroviral vectors (MLV).
• Infection with HIV is not known to cause integration-related cancers.
from Hematti, P.,
et al. PLoS Biol 2:
e234 (2004)
* The prefix “onco” refers to oncology, which is the study of cancer.
**ex vivo: Pertaining to a biological process or reaction taking place outside of a living cell or organism.