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Boosted Hope: AIDS Vaccine Research
by
Marjan Hezareh, Ph.D., ARA's Director of Scientific Communications
The human immunodeficiency virus (HIV) continues to spread
around the world, insinuating itself into communities previously
little troubled by the epidemic. Already almost 20 million
people have died of AIDS, over 30 million are currently living
with HIV and 16,000 new infections occur daily. HIV chemotherapy
has continued to allow substantial improvements in quality
of life and life expectancy for HIV infected individuals.
However, these therapies are out of reach for most HIV-infected
patients around the world. Even when drugs are available they
are limited by their toxicity, complex regimens and elevated
cost. Therefore, the biomedical community is redirecting efforts
towards development of an effective AIDS vaccine. In this
issue of Searchlight, we review some of the challenges facing
the scientific community in designing and developing a safe
and effective vaccine against HIV.
The
term immunity is derived from the Latin word immunitas referring
to the exemption from variety of civil duties offered to Roman
senators during their tenures in office. Historically, immunity
meant protection from disease—specifically infectious
disease.The
modern definition of immunity is a reaction to foreign substances,
including microbes, viruses, and macromolecules such as proteins
and polysaccharides. The concept of immunity existed long
before, as suggested by the ancient Chinese custom of making
children inhale powders made from the crusts of skin lesions
of patients recovering from smallpox. Edward Jenner's successful
vaccination against smallpox demonstrated for the first time
that the immune system can be manipulated under controlled
conditions. He noticed that milkmaids who had recovered from
cowpox never contracted smallpox. He established that cowpox
virus cross-reacts immunologically with smallpox virus and
could be used to protect against smallpox. This led to the
widespread acceptance of vaccination for inducing immunity
to infectious disease.
Unfortunately,
there is no population that has been cured from AIDS. Therefore,
no immune parameters have been identified that correlate with
protection from disease. Recent studies on exposed seronegative
sex workers in Nairobi provided important insights into the
mechanisms the body uses to maintain immunity, but it failed
to identify definitive markers to guide vaccine development.
Although immune correlates for protection against HIV remain
unknown, there is evidence that both cell-mediated immunity
and long term B-cell memory are crucial in immune protection.
Furthermore, transmission of HIV occurs through multiple routes.
Therefore, an effective vaccine may require stimulation of
all major immune response mechanisms including humoral (antibody
response), cellular (cytotoxic T cells and helper T cell),
and local (mucosal response at the site of the infection).
Other vaccines such as the childhood vaccine for the prevention
of pertussis have been successfully developed without precise
knowledge of correlates of immunity. Development of a vaccine
without a definitive marker of protection is possible, but
remains one of the more challenging aspects in AIDS vaccine
development.
The
genetic diversity of HIV poses another difficulty. HIV has
multiple sub-types or clades, around the world, and even in
an individual mutations occurs very quickly and with a high
frequency. Efforts to develop a universally-effective vaccine
against all strains and clades of virus have been impeded
by the unusual diversity of the HIV virus. Other vaccines
have been designed that successfully protect humans against
pathogens where strain variation occurs, such as pneumococcus,
influenza, and polio. But the strain variation of these viruses
is relatively limited compared to HIV.
Further
barriers to vaccine development include the fact that there
is no ideal animal model for HIV/AIDS. Vaccine studies for
SIV on monkeys have provided important insights into HIV immunopathogenesis,
but there are differences between HIV and SIV in their genomic
organization that could affect vaccine efficacy. Although
the animal models are not perfect they help determine the
mechanisms of protection and may help us to make decisions
about proceeding with human trials. Furthermore, we should
keep in mind that other vaccines, including measles, mumps,
and rubella, have been successfully developed where animal
models were not available or less than ideal. The true test
of a vaccine candidate will be determined in large human clinical
trials, not in animal models.
Recent
advances in immunology have led to the design and development
of new and promising vaccine strategies. As summarized below,
many of these strategies are being tried to develop an effective
HIV vaccine. Some scientists are calling for a return to traditional
methods of vaccine development such as live attenuated vaccine,
which have greater risk but may have a greater chance of success.
Others are pushing for the use of high-tech methods because
of the unique features of HIV. Either way, human clinical
trials are the crucial link between laboratory research and
an effective vaccine. It takes lots of determination and a
series of vaccines and human clinical trials to develop a
product capable of affecting the epidemic. Although the hurdles
to development of an effective HIV vaccine are high, we should
be heartened by the renewed interest in vaccine development.
Current
AIDS Vaccine Candidates
Several
types of vaccines are currently in development, either in
human clinical trials or in experiments with primates.The
following list summarizes different concepts currently in
uses for development of an AIDS vaccine.
Recombinant
Subunit Vaccines: In this strategy, individual viral components
are used to generate a protective immune response. This is
the basis of AIDSVAX™ B/B, the first vaccine currently
in Phase III clinical trials. This vaccine contains synthetic
copies of gp120 (HIV envelope) protein.
DNA
Vaccines (or "naked DNA" or "nucleic acid"): contains
an HIV immunogen under the control of an eukaryotic enhancer/promoter
signal that confers an appropriate expression of the viral
protein. Once introduced into muscle, cell surrounding the
site of injection, internalize the plasmid and transport the
DNA into nucleus, where appropriate expression of immunogene
will occur. This is the basis of Merck's "naked DNA" vaccine
currently being tested in Phase I clinical trial.
Viral
or Bacterial Vector-Based Vaccines: Recombinant vector-based
vaccines contain replication-defective forms of non-pathogenic
virus (e.g. Poxvirus, Modified Vaccinia Ankara) or bacteria
(e.g. Salmonella thyphosa) to carry one or more HIV genes.
This is the basis of Aventis's vaccine candidate, in which
a harmless canary poxvirus has been used as a vector.
Live-Attenuated Vaccines: This concept is used to protect
humans against broad range of infectious disease such as polio
and measle. Monkeys infected with live-attenuated SIV (nef-deleted
Simian Immunodeficiency Virus) were not infected upon subsequent
challenge with SIV or nef-deleted SIV. However, it was found
that significant pathology occurred in monkeys after exposure
to live-attenuated SIV. Furthermore, nef-mutated virus has
been isolated in some patients infected with HIV. This approach
may prove to be effective, but many safety concerns should
be addressed before testing such a vaccine in human clinical
trials.
Whole-Inactivated
Virus: This concept is used to protect humans against
broad range of infectious disease such as polio, influenza,
and rabies. In this approach virus is inactivated using different
methods including chemicals, irradiation or heat. Inactivation
of virus insures that every virus particle is killed while
they maintain their primary structure necessary to stimulate
HIV-specific immune response. A vaccine candidate is being
developed at UCLA by Dr. Katie Grovit-Ferbas and at NCI by
Dr. Larry Arthur based on killed, whole-inactivated HIV virus.
Virus-Like Particle Vaccines: These vaccines are made
of pseudovirion that contain non-infectious portions of HIV
viral particle but lack most or all of HIV genetic material.
The advantage of this approach lies on the fact that HIV proteins
are presented in their natural forms to the immune system.
However, it is very difficult to prove that no infectious
particles exist in the vaccine preparation.
Peptide
Vaccines: Consist of chemically-synthesized pieces of
HIV protein (peptide) known to stimulate HIV-specific immunity.
AIDS
Vaccines in the New Millennium: Meetings Updates
By
Marjan Hezareh, Ph.D.
Researchers
at both the "AIDS Vaccines in the new Millennium" conference
in Keystone (28 March-3 April) and at the "AIDS Vaccine 2000"
in Philadelphia (September 5-8) presented a broad range of
studies on HIV vaccines candidates. The need for a vaccine
has never been greater and important progress toward this
goal was presented during these meetings. NIH has increased
HIV vaccine research funding more than six-fold since 1990,
to an estimated $356.6 million for fiscal year 2002. At NIAID
(NIH institute for HIV vaccine research), an estimated $450.7
million will be devoted to all vaccine research in year 2002,
with 61 percent of that total ($276.5 million) dedicated to
HIV vaccine development.
"The
field of vaccine research owes great thanks to the largesse
of the American people, the Administration and Congress for
making these resource available," says Dr. Fauci in his keynote
lecture in Philadelphia. The data being discussed demonstrated
an increasingly complex collection of vaccine candidates and
approaches in development, which offer a new basis for new
optimism. A better knowledge of envelope structure allowed
researchers: 1. to define new HIV antigen structures to induce
useful immune response and 2. to better understand how the
immune system first recognizes and responds to HIV.
All
together, these advances helped to design new strategies that
may provoke efficient antigen uptake and presentation leading
to better T-cell activation. A growing number of studies were
directed toward the role of mucosal immunity in HIV protection
and the probability of including mucosal immunity as a component
of vaccine-induced protection. Like the skin, the mucosal
epithelia are barriers between the internal and external environments,
and are therefore an important first line of defense, specifically
in HIV infection that is transmitted sexually. Furthermore,
the ongoing phase I and II trials, and diverse vaccine approaches
including gp120 and canarypox vectored HIV-1 candidate vaccines
(ALVAC) provided increasing clarity about the safety and immunogenecity
thresholds required to take a vaccine from animal studies
into human trials and Phase III evaluation of efficacy.
Here
we summarize some important data on HIV vaccine development
presented at these conferences.
Protection
with Adeno-Associated Virus Vectors
Philip
Johnson from Children's Research Institute, Columbus, Ohio,
showed data from ongoing studies of an SIV vaccine based on
vectors of recombinant Adeno-Associated Virus (rAAV). Wild
type AAV is a replication defective parvovirus, which is non-pathogenic
in humans and animals. In this study, they generated rAAV
vectors (which lack any AAV gene) expressing SIV env, rev,
gag and protease gene and used them to immunize intramuscularly
8 monkeys (single dose). They detected a strong CTL response
and persistent neutralizing antibody titers over 14 months.
After immunization, animals were challenged intravenously
with a high dose of SIV-E660. In vaccinated animals, viral
peak was reduced by about 1.3 log and viral load by 3 logs.
After 6 months, vaccinated animals were still healthy, while
3 out of 8 control animals died. Based on these data, rAAv
vectors stimulate robust, durable and effective immune responses
against SIV. In addition, they demonstrated that immunization
with DNA prime/rAAV boost stimulate better immune responses
that rAAV alone. Dr. Johnson suggested that these data provide
evidence necessary to move forward the clinical trial of cognate
rAAV/HIV vaccine in humans.
Protective
immunity induced by whole recombinant Yeast-Based HIV Vaccine
Dr.
Franzusoff from GlobeImmune, Inc., Denver, presented data
on the development of a novel therapeutic vaccine based on
using, whole, recombinant, non-pathogenic yeast (Saccharomyces
Cerevisiae) as a vector. The optimal stimulation of CTLs requires
presentation of antigens by Dentritic Cells (DCs). These cells
are unique in their ability to process antigens into the MHC
class I pathway for presentation to CTLs. In this study they
engineered recombinant yeast expressing HIV-1HXB2 p55 gag
protein (HIVAX-2). mice (C57B1/6) were immunized subcutaneously
with 20 million intact live or heat-killed yeast.
The
vaccination stimulated strong immune response in mice, measured
by intracellular IFNg staining, by CTL-mediated cell lysis
and by T cell proliferation assays. To show protective immunity,
they used HIV-gag dependent tumor model. Mice were protected
against subsequent challenge with B16 melanoma cells stably
expressing HIV-1HXB2 p55 gag. Therefore, the recombinant yeast-based
HIV vaccine is a potent activator of cellular-immune responses
and is currently being prepared by GlobeImmune, Inc. for use
as a therapeutic HIV vaccine in a phase I clinical trial.
Merck's
DNA and Adenovirus-Based Vaccine
Merck
researchers have developed two types of vaccine; one based
on "naked DNA" and the other on an adenovirus vector. At the
Keystone meeting they presented pre-clinical studies that
led them to move these 2 vaccines candidate into phase I trials.
In his presentation, John Shiver, Director of Vaccine Research
showed data from comparative studies of several HIV vaccine
approaches. All vaccines contained SIVgag gene but no env
gene. In this study rhesus macaques were immunized with one
of the following vaccines: plasmid DNA in saline, alum or
adjuvant (CRL10050 adenovirus type 5 (Ad5)-based viral vectors
Modified Vaccinia Ankara (MVA)-based viral vector. Each vaccines
were administrated intramuscularly to 3 animals 3 or 4 times
over 32 weeks. Immunization with the Ad5 based vector resulted
in the highest immune response. Response to plasmid DNA in
saline or alum was much lower than DNA in adjuvant (CRL1005),
showing a clear advantage of adjuvanting DNA. CD4 responses
were induced by all vaccines and were higher in the DNA group.
The Ad5-based vaccine tends to induce higher CD8 responses.
Three months after immunization animals were challenged with
a high dose of SHIV89.6P, a very pathogenic lethal strain.
At day 180 after challenge all vaccinated animals were alive
(although infected), while 4 out of 6-control animals had
died. In Ad5 groups, CD4 count remained high and viral load
slowly brought under control and peak viremia was reduced
by 1.5 log. This group showed the best clinical course followed
by adjuvanted DNA with CRL1005 and MVA-based vaccine. Vaccination
with DNA in saline or alum resulted in a strong drop in CD4
cells count and the poor control of viremia.
Next,
Emilio Emini, reported on pre-clinical evaluation of Merck's
HIV vaccine candidates currently in clinical trials. In his
presentation he addressed the issue of pre-existing immunity
to adenovirus which could make this a less immunogenic vector.
About 10% of the US population have significant neutralizing
antibodies to adenovirus and 40% of them shows lower level
of antibody. Therefore they preexposed 6 monkeys to adenovirus
and then immunized them with a high dose (1011 particles)
of Ad5-gag (HIVgag). The presence of neutralizing antibodies
reduced by several folds the T-cell responses but did not
eliminate it. He also presented data on a DNA prime/Ad5 boost
study. In this study animals were given first DNA/CRL1005
adjuvant and then boosted with a high dose (107 particles)
of Ad5-gag (HIV gag). The vaccinated animals showed a 5-10
fold increase in the number of HIV-specific-T cells response
after the boost. This is generally reflected by an increase
in CD4 and CD8 T cell response, again CD8 response predominating
after Ad5 boost. Thus the preexisting immunity could be overcome,
especially if using DNA prime/Ad5 boost strategy. Vaccination
with DNA in saline or alum was a less effective prime showing
little T cell response.
Emini
suggested that a 109 particle dose should still induce a good
T cell response even if neutralized by 99%. He also said that
nef and pol would be added to Merck's vaccines in addition
to gag. This is based on data showing that HIV infected people
with good immune responses to these proteins maintain lower
viral load. A DNA gag trial began last year in uninfected
people and a trial in HIV-infected people started in March
2001.
Importance
of mucosal CTL to prevent mucosally-transmitted SIV/HIV
In
his study, Jay Berzofsky from the National Cancer Institute,
Bethesda, compared mucosal versus systemic immunization in
macaques. They demonstrated that mucosal, but not systemic
immunization, protected macaques against an SIV challenge.
In this study animals were immunized intrarectally (ir), or
subcutaneously (s.c.) with peptide HIV vaccine including SIV
gag and pol epitopes. After immunization, animals were challenged
with SHIV-ku and monitored for 200 days. All animals showed
similar viral peaks after challenge. However, following this
peak, the viral loads dropped to undetectable levels in ir-immunized
animals and they maintained a high CD4 count up to 200 days
post-infection. In contrast the sc-immunized animals had high
viral loads and a low CD4 cell-counts. All immunized animals
showed some degree of protection compared with control animals.
Two hundred days post-infection, animals were sacrificed and
HIV level in colon and jejunum was monitored. Little or no
virus was seen in the gut tissue of ir-immunized macaques,
while 10-100 times more virus has been found in control and
sc-immunized animals. The ir-immunized animals displayed a
higher level of HIV-specific CTLs in their colon than sc-immunized
animals.
These
data is in agreement with Berzofsky's study in mice. In this
study they showed a clear relationship between mucosal vaccination,
high level of mucosal CTLs and protection against subsequent
mucosal challenge. Berzofsky's study is the first showing
a correlation between mucosal immunization and improved protection
in primates. Future Directions Why there are relatively few
studies on mucosal immunity? This is mainly because analysis
of mucosal immune activity requires an invasive procedure,
while systemic immune activities can be easily measured from
blood samples. Several groups are developing techniques to
use homing markers as a way of measuring mucosal response
in blood. Homing markers are cell surface molecules that indicates
destinations of cells in blood stream. Therefore, it is possible
to monitor cells bearing these markers from blood samples
rather than from mucosa.
One
example of such a marker is alpha4beta7, the latter is an
integrin that appears on all cells trafficking to the gut.
More research has been focused on understanding the early
events in HIV infection, such as the role of dentritic cells
in the mucosa. It appear that these cells carry HIV to lymph
nodes and from there virus spread to other sites. More effort
has been focused on understanding the mechanism of action
of DC-SIGN, the receptor that carries HIV to lymph nodes,
and to find a way to inhibit its action. Although a vaccine
that prevents or delays HIV infection remains an elusive goal,
more good news is coming our way.
"Perseverance
is a great element of success. If you only knock long enough
and loud enough at the gate, you are sure to wake up somebody."
—Henry
Longfellow
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