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Is DNA Vaccine a Viable Strategy for AIDS?
A Course Report for
MIT/CRI/AIT joint course
ED19.98 Bioengineering and Environmental Health
By
Mr. Zhang Jianjun
I.D. EVD997075
Environmental Toxicology, Technology and Management
Urban Environmental Engineering and Management Program
Asian Institute of Technology
Bangkok, Thailand
May 27-Jul 24, 2000
Mr. Zhang Jianjun/AIT
Is DNA Vaccine a Viable Strategy for AIDS?
Mr. Zhang Jianjun
EVD997075
ETTM/UEEM/AIT
1.Introduction
AIDS (Acquired Immune Deficiency Syndrome) is caused by HIV (Human
Immunodeficiency Virus). It is the final and most serious stage of HIV disease. It is
characterized by signs and symptoms of severe immune deficiency, such as
opportunistic infection and cancer (Kaposi’s sarcoma), and finally death. AIDS has
unique characteristics, such as multiple transmission routes, long latency, and
mechanisms of pathogenesis. There are no medicines to cure this insidious disease
yet. It is now prevalent around the world with a surprising growth speed, and more
than 16 million men, women and children have died from AIDS; More than 33.6
million people are living with HIV, and nearly all of them will die from AIDS-related
complications within the next two decades. An estimated 5.6 million people were
newly infected with HIV in 1999 (including almost 600,000 children). AIDS has
orphaned more than 11 million children worldwide. By some estimates their number
will reach 40 million in the next decade. By the year 2000, it is estimated that 50
million people worldwide will be infected with HIV. An estimated 16,000 people
throughout the world are infected with HIV each day.
AIDS is overwhelming health care systems and national economies. It caused a
great loss of economic development all over the world. The UN estimates that the
medical and human costs of AIDS have already reversed social and economic
development in sixteen countries. In Brazil, more than US$500 million is spent on
anti-HIV agents annually, and medications still fall short of demand. By 2005,
Kenya's GNP will be more than 14 % smaller than it would have been without AIDS.
In sub-Saharan Africa, household incomes have fallen by half and business profits
have decreased by 20 % due to AIDS deaths. More than 25 % of health expenditures
in Zimbabwe are for HIV/AIDS.
More than 95% of all new infections are in developing countries, making
HIV/AIDS among the most serious threats not only to global health, but also to global
development. Prevention programs - including education, condom and clean needle
distribution and peer counseling - have slowed the spread of HIV, but have not
stopped it. Treatment advances have yielded important new AIDS therapies, but the
cost and complexity of their use put them out of reach for most people in the countries
where they are needed the most. In industrialized nations where drugs are more
readily available, side effects and increased rates of viral resistance have raised
concerns about their long-term use. So, a safe and effective HIV preventive vaccine is
urgently needed to bring the HIV/AIDS epidemic under control. Only an AIDS
vaccine can end the HIV/AIDS pandemic.
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2.Vaccine is the cost-effective way to prevent infectious diseases
A vaccine is a preparation containing a weakened or "killed" solution of a specific
bacterium, virus, or germ believed to produce a disease. By mimicking infections by
viruses, bacteria or other pathogens, they trigger the body's immune defenses to
mount counterattacks, even though the vaccines themselves present little or no
danger. After the immune response subsides, there remains a memory of the
pathogens the vaccines pretended to be. So when the real organisms invade, the
immune system is ready. It mounts a swift and powerful response, usually enough to
eliminate the attackers well before symptoms appear.
2.1Vaccine types
There are two major components to the immune response — humoral (antibody)
and cellular. Similarly, there are two types of vaccines, those that stimulate the
immune system to produce antibodies and others that evoke the production of killer T
cells (and usually antibody as well).
2.2. Antibody inducing vaccine
Pathogens that colonize and proliferate in tissues but don't actually penetrate
individual cells evoke antibody responses. This includes most bacteria and fungi and
many parasites. Here, many of the pathogen's unique structures (antigens) are seen as
foreign by some of the immune system's B and T lymphocytes, which, stimulated by
this recognition, interact to produce high levels of antibodies. These in turn bind to
antigens on the pathogens' surfaces and mark them for destruction by the body’s
inflammatory defenses. Vaccines that fight infections this way include those against
cholera, pertussis, meningitis, plague, and Haemophilus influenza type b.
2.3. Cellular immunity vaccine
Organisms that infect and inhabit individual cells give rise to cellular immunity; a
group that includes all viruses, some bacteria, and some parasites. In this case,
antibodies can't reach the intracellular pathogens, and this arm of the immune system
is thus unable to clear them from the body. However, nearly all infected cells display
pieces of their invaders on their surfaces, which, not recognizable by antibodies, can
become the targets of killer T cells. As the infection spreads, the number of killer cells
grows until there are enough to destroy all of the infected cells. Most of the antiviral
vaccines in use today stimulate such cellular immunity, such as vaccines against
measles, rubella (German measles), mumps, polio (Sabin), and yellow fever.
3. AIDS, HIV and the immune system
3.1 The Virus
HIV is one member of the group of viruses known as retroviruses that are capable
of copying RNA into DNA. The virus has two exact copies of single-stranded RNA as
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its basic genetic material (genome) in the very center of the organism. The genome is
surrounded by a spherical core made of various proteins in tightly packed association
with one another. The core is itself surrounded by an envelope, made of fat and
various membrane-bound proteins. One of the membrane-bound proteins (gp120) can
bind to a particular protein on the surface of certain immune cells, called T-cells,
which results in the virus becoming physically attached to the cells. Upon binding, the
virus is brought inside of the T-cell, and the envelope is removed by enzymes
normally present inside the cell. The internal core is thus exposed, and it is brokendown. This last phase results in exposure of the virus's RNA genetic material. An
enzyme----reverse transcriptase, binds to the RNA, and to make a complimentary
base-pair single-strand DNA of the RNA. The single strand of DNA is also copied by
the same enzyme to form double-stranded DNA. This DNA inserts somewhere into
one of the 46 chromosomes within our cells, and there it is used as a template for
production of all of the things necessary to form new virus particles----replication of
the virus. These new virus particles can be subsequently released from the infected
cell, and can infect adjacent cells.
3.2 The Immune System
The immune system is a system within all vertebrates, which is comprised of two
important cell types: the B-cell and the T-cell. The B-cell is responsible for the
production of antibodies, and the T-cell (two types) is responsible either for helping
the B-cell to make antibodies, or for the killing of damaged or "different" cells within
the body. The two main types of T-cells are the helper-cell and the cytotoxic T-cell.
The T-helper population is further divided into those which help B-cells (Th2) and
those, which help cytotoxic T-cells (Th1). Therefore, in order for a B-cell to do its
job, it requires the biochemical help of Th2 helper T-cells; and, for a cytotoxic T-cell
to be able to eliminate a damaged cell, it requires the biochemical help of a Th1
helper T-cell. The effect of HIV on the immune system is the result of a gradual
(usually) elimination of the Th1 and Th2 helper T-cell sub-populations.
3.3 How HIV Specifically Affects the Immune System
One of the HIV surface proteins, named gp120, recognizes a protein on helper Tcells named CD4, and physically associates with it. The CD4 protein is a normal part
of a helper (both Th1 and Th2) T-cell's membrane. Thus, CD4 is a specific receptor
for HIV. This virus however, can also infect other cells, which include macrophages
and certain other kinds of cells which can engulf substances through a process known
as phagocytosis. As a consequence of the interaction with CD4 on helper T-cells, HIV
specifically infects the very cells necessary to activate both B-cell and cytotoxic Tcell immune responses. Without helper T-cells, the body cannot make antibodies
properly, nor can infected cells containing HIV be properly eliminated. Consequently,
the virus can multiply, kill the helper T-cell in which it lives, infect adjacent helper Tcells, repeat the cycle, and on and on, until eventually there is a substantial loss of
helper T-cells.
Our body responds to this onslaught through production of more T-cells, some of
which mature to become helper T-cells. The virus eventually infects these targets and
eliminates them, too. More T-cells are produced; these too become infected, and are
killed by the virus. This may continue for up to ten years before the body eventually
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succumbs, apparently because of the inability to any-longer produce T-cells. This loss
of helper T-cells finally results in the complete inability of our body to ward-off even
the weakest of organisms which are normally not ever a problem to us. This acquired
condition of immunodeficiency is called AIDS.
4. State of Current AIDS Vaccine Research
Given the scale of the AIDS pandemic and the long period required for
development and distribution of an AIDS vaccine, the number of products currently in
the development pipeline is woefully inadequate. After more than 15 years of research
and development, only one vaccine concept is undergoing wide-scale testing for
efficacy in humans (Phase III studies), and only one other type of vaccine has entered
Phase II trials. Equally serious is the shortage of vaccine candidates currently
undergoing Phase I trials.
Although there are different designs that might lead to a useful AIDS vaccine,
most seek to use specific parts of the virus (or its genes) to activate the body's immune
defenses. Once the immune system has learned to recognize these HIV components,
the hope is that it can mount a vigorous defense when it encounters the real virus.
The following list summarizes the different concepts currently under investigation
as AIDS vaccines.
4.1 Recombinant sub-unit vaccines
They introduce a harmless sub-unit or portion of an HIV protein into the body.
This is the basis of AIDSVAX, the first vaccine being tested for effectiveness in
humans and which contains a portion of HIV's outer surface (envelope) protein, called
gp120. (The hepatitis B vaccine successfully uses this approach to confer protective
immunity).
4.2 Virus-like particle and synthetic peptide vaccines
They use approaches similar to that of the sub-unit vaccine, but introduce particles
of different sizes into the body. Virus-like particles are significantly more complex
than the single proteins used in the sub-unit vaccines, consisting of several HIV
proteins engineered to mimic an HIV particle. In contrast, synthetic peptides consist
of small portions of HIV proteins chosen specifically to focus the anti-HIV immune
responses on what are thought to be the most important regions of the viral proteins.
Both strategies have shown promise in stimulating immune responses against HIV
and offer additional advantages of providing multiple HIV targets of HIV for the
immune system to recognize.
4.3 Live-attenuated vaccines
They have been used globally against many viral diseases, such as polio (Sabin
vaccine) and measles. They consist of weakened (attenuated) live virus that can not
cause disease but can still infect cells and replicate within the body. After responding
to the weakened virus, the immune system is then potentially prepared to protect
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against future infection by pathogenic strains. Live-attenuated SIV vaccines have
conferred the most consistently effective protection of any of the vaccine strategies
against SIV in monkeys; however, human trials of live-attenuated HIV vaccines have
been put on hold because of safety concerns.
4.4 Live recombinant vector vaccines
They are created by genetically engineering relatively harmless, replicating viruses
or bacteria to produce HIV proteins, theoretically providing some of the advantages of
live-attenuated vaccines without many of the safety concerns. A vector-based vaccine
using canarypox, which is harmless to humans, will probably be the next HIV vaccine
tested for efficacy in humans. Several other vector-based vaccines are currently in
development, including viral vectors such as vaccinia, Venezuelan equine encephalitis
virus (VEE) and adeno-associated virus, as well as bacterial vectors such as
salmonella and shigella.
4.5 Combination vaccines
They combine several of the approaches above, based on the premise that
protection from HIV may require a broad spectrum of immune responses. One now in
phase II trials combines the canarypox of the vector vaccine approach and the gp120
protein from HIV's outer surface. Another combination vaccine strategy uses a DNA
vaccine to prime the immune system and an MVA vaccine to boost the immune
system to produce robust anti-HIV immune responses.
5. The difficulties facing HIV vaccines
Although the creation of new vaccines is never easy, the development of a vaccine
to prevent HIV infection, or even, for that matter, to delay or temper the devastating
impact of AIDS has proven especially difficult.
5.1 The astonishing variability of the virus.
HIV mutates rapidly as the infection spreads, and the virus can literally change the
shape of the antigenic structures faster than the immune system — both the cellular
and humoral responses — can mount responses to these new elements. Thus, vaccines
based on some or all the components from a single strain or isolate of HIV is almost
surely bound to fail. Such a vaccine might protect against infection by a strain of HIV
with these identical components, but other viruses with altered antigens would be
unaffected.
5.2 There is still uncertainty about how and in what form HIV is transmitted
from one individual to another.
It is known that the virus can be passed either by direct infusion into the
bloodstream (e.g., injected drugs or contaminated blood or blood products) or
sexually by crossing the mucus membranes of the genital organs. But, it is not known
whether the viruses that actually infect the new body circulate in the bloodstream (that
is, as individual particles in blood or other fluids), or within cells. There is evidence
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that HIV can be transmitted directly from one cell to another when the cells come into
contact, and if such were the case, antibodies would be useless regardless of what
parts of the virus they were targeting.
5.3 It is unknown at this point in the AIDS pandemic what kind of immune
response or responses might actually afford protection against HIV.
Upon infection, HIV evokes both humoral and cellular immune responses.
However, which of these responses is the more important in containing the spread of
the infection, and, even more importantly, which could actually prevent or clear an
infection if an effective vaccine could be made? Could antibodies do it? Could killer
T cells? Or would it take both?
5.4 Lack of an ideal animal model.
If laboratory animals could be infected by HIV, scientist could quickly find
answers to the questions of how HIV is transmitted and what kinds of immunity are
most effective against the virus.
6. Is DNA vaccine practical to AIDS?
6.1 DNA vaccines
Also known as "naked DNA" or "nucleic acid" vaccines, it uses the actual genes of
HIV as a vaccine. Once introduced into skin or muscle, the genetic material is taken
up by cells in the body, which then produce HIV proteins; the immune system can
then mount responses against the HIV proteins. Several experimental vaccine
products using this new technology against other diseases have successfully
demonstrated protection, as have some experimental SIV vaccines in monkeys. In
early 2000 Merck began the first human trial of a DNA vaccine against HIV.
6.2 DNA vaccinations
They are a branch of gene therapy, a process through which genes are introduced
into the body's cells. It uses the genes for viral antigens, rather than the antigens
themselves, as the source of immunogen. Either through particle bombardment or
direct injection via needle, a plasmid (or loop) of DNA is injected into the organism's
tissues where it is taken in by a cell. The cell begins producing proteins that take on
the role of an antigen. If, at a later date, the organism encounters the virus for which
the antigen is required it is well prepared to protect itself
6.3 History of DNA Vaccination
1. The idea of gene therapy first sprung onto the scene in the 1950's and 1960's.
The injection of genetic material into animals produced a protein developing
response (completely independent of vaccinations).
2. In the late 1980's, Robert Zaugg of Vical, Inc., searched for a means by which
a virus could deliver DNA to cells allowing for uptake and production of
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antigens for the purpose of vaccinations (i.e.. a vehicle to get the DNA to the
cell). In 1990 Vical, Inc. and researchers at the University of Wisconsin
discovered that an injection of DNA plasmids without any vehicle produced a
protein response in mice.
3. In 1993, Merck Research Laboratories discovered that intramuscular
injections of a gene from the influenza virus into mice produced complete
immune responses.
4. In 1996, Vical, Inc. was awarded a U.S. patent for their DNA vaccination
procedure.
5. DNA was first used to elicit an immune response in humans against HIV in
1995. In 1996 trials involving T-cell lymphoma, influenza, and herpes
simplex virus were started.
Studies involving DNA vaccinations are currently at a high demand.
6.4 The mechanism of DNA vaccine---Transfection: Plasmids Into Cells
The aforementioned studies indicate that DNA plasmids can stimulate the immune
system to produce both humoral and cellular immunity in precisely the same way as
viruses. For both CTLs and antibodies to appear in response to an antigenic stimulus,
foreign genes must enter cells and make their way into the nucleus, and there begin to
make proteins. Viruses do this by carrying or injecting their cargo of genes into cells.
DNA plasmids do it by simply passing through cell membranes without any
protective shell. (Exactly how this occurs is still poorly understood).
In the recent DNA vaccine studies, the plasmids were injected intramuscularly in
fairly high amounts. DNA plasmids are degraded quickly by enzymes in blood and
extracellular fluids, and enough must be administered to assure that sufficient
quantities escape destruction to "transfect" cells. This is easily done — once created,
plasmids can be replicated in large amounts — and most if not all of the surviving
plasmids transfect into muscle cells.
Once inside, the plasmids are less prone to enzymatic attack and, when they finally
make it into the nucleus — (again, a poorly understood process) — they are stable for
weeks or even months. In these muscle cells, the plasmids do not become integrated
into the cells' chromosomes but are still able to begin making proteins. At this point,
as these newly minted foreign proteins begin to accumulate, the immune system is
alerted, and a response begins.
6.5 Positives and Negatives of DNA Vaccines
6.5.1 Positives
1. DNA is inexpensive compared to isolated proteins or organisms used for
conventional vaccines.
2. DNA vaccines can result in longer lasting production of the antigenic protein;
thereby booster shots are no longer required.
3. DNA vaccines produce stronger immune responses than conventional
vaccines.
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4. DNA vaccines are still in a "baby" stage. Therefore, the full potential has yet
to be realized. Possible cures for cancer and AIDS are now in the works!
6.5.2 Negatives
1. Although testing results have been favorable in small animals, they have been
less impressive in larger animals (including humans). DNA uptake to cells
apparently decreases with increased body size.
2. Extended immunostimulation could lead to chronic inflammation or
autoantibody production.
7. DNA vaccines in practice
7.1 DNA vaccine succeeds against tuberculosis
Bits of DNA taken from a disease organism can be used in animals as both a
Vaccine and, surprisingly, a treatment in mice against the world's most common
deadly infection, tuberculosis. The tests show that the vaccine not only blocks
infection, but also combats existing TB -- important because the body's defense, the
immune system, seems to be inadequate to erase tuberculosis infections in many
people.
7.2 DNA vaccine for malaria
A cheap vaccine for malaria which uses DNA could boost the fight against the
world's second deadliest disease, say US scientists. A study published in the journal
Science found that injecting a virus' DNA into a human would raise levels of immune
cells in the blood stream. This gave the test subjects much greater resistance to
disease.
7.3 DNA vaccine for rabies
Scientists at the Rocky Mountain Laboratories (RML), part of the National
Institute of Allergy and Infectious Diseases (NIAID), have developed a DNA vaccine
against rabies that protected eight of eight vaccinated monkeys from the disease. It is
the first DNA vaccine to show complete protection in nonhuman primates against a
virus that attacks the central nervous system (CNS). Their report describing the
successful experiment appears in the August 1998 issue of Nature Medicine.
The only drawback of the DNA vaccine is that the antibody response cannot be
detected before 30 days. Hence, as currently designed, the vaccine would not be
suitable for post-exposure prevention of disease. However, researchers will be able to
overcome this problem in the future. On the other hand, DNA vaccines typically
provide long-lasting immunity, so they could be used prophylactically to protect
people at high risk, such as veterinarians and individuals who live in developing
countries. Currently, Dr. Lodmell and his colleagues are assessing the durability of
the antibody response following just one immunization to investigate the requirement
for booster vaccinations, as well as other issues related to protection.
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8. Evidences support DNA vaccine for HIV/AIDS
Several experimental DNA vaccines for HIV/AIDS have been produced and tested
in small animals and non-human primates. In general, the results of these studies have
been quite promising. DNA vaccines delivered intramuscularly or by gene gun have
been shown to induce both neutralizing antibodies and CTL responses against HIV
and SIV antigens.
8.1 Chimpanzees got protection from HIV infection with DNA plasmids
A report in the May 1997 issue of Nature Medicine, comes from researchers at the
University of Pennsylvania and other institutions, and shows that chimpanzees can be
protected against infection by HIV following immunization with DNA plasmids
carrying four HIV genes, env, rev, and gag/pol. Chimpanzees are the only species
other than humans that can be infected by HIV, although infected chimps don't
develop AIDS or become susceptible to opportunistic infections.
The animals developed antibody and CTL responses following inoculations with
the DNA plasmids and, more importantly, were protected against infection with intact
HIV. The animals either failed to become detectably infected or became infected for a
short time until their immune systems cleared the virus.
8.2 Rhesus monkeys got protection from HIV infection with DNA plasmids
Investigators at Harvard University and the Merck Research Laboratories showed
that inoculations with DNA plasmids with a gene for an HIV protein could protect
rhesus monkeys against infection by a hybrid form of HIV. In these experiments, a
series of injections with plasmids carrying only the env gene of HIV evoked strong
levels of CTLs against HIV-infected cells.
The DNA fragments also raised high levels of antibodies capable of binding to
HIV particles, but only a fraction of the antibodies was found to be neutralizing, that
is, capable of preventing HIV from infecting cells. Accordingly, following a course of
plasmid inoculations, the animals were given a final injection of purified HIV
envelope protein (the product of the env gene, gp160). That, as the researchers had
hoped, boosted the production of neutralizing anti-HIV antibodies to high levels.
Thus primed to produce both strong CTL and neutralizing antibodies responses
against HIV, the monkeys were challenged with SHIV, a simian immunodeficiency
virus (SIV) genetically modified to express on its surface the envelope protein on
HIV. They were protected, showing no evidence of infection. Control animals, on the
other hand, were fully susceptible to the SHIV.
8.3 DNA vaccine trial will be held in China
Although China has a low number of AIDS patients until now (the official tallies
count only 670 confirmed AIDS cases and 18,143 confirmed HIV-infected people
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among the 1.2 billion population), by all indications, the epidemic is about to sweep
through the world’s most populous nation with a vengeance. The real number of
infected people probably tops 500,000, according to China’s National Center for
AIDS Prevention and Control (NCAIDS), an estimate that has risen fivefold since
1996 based on increasing intravenous drug use, changing sexual behavior, and a
burgeoning commercial sex industry. If current trends continue, NCAIDS projects
that China could have 10 million HIV-infected people by 2010.
Officials from government and NCAIDS have granted some planned DNA vaccine
trials in some provinces of China. The results should be doubtless supportive for the
future practical use in the prevention of HIV/AIDS (see table 1).
Table 1.
Province
Partners
Type of Vaccine
Planned vaccine trials
Xinjiang
Yunnan
U. Regensburg,
Rockefeller U., New
Germany/NCAIDS York/NCAIDS
European Union
IAVI
(EuroVac)
DNA vaccine
DNA vaccine
HIV clade
Start
B/C recombinant
Early 2001
Funder
C
Late 2001
Guangxi
Johns Hopkins U.,
Baltimore/CNIDA
U.S. NIAID
DNA vaccine
B/C recombinant
2001-2002
9. Conclusions
At this early stage, it is impossible to determine how successful DNA plasmids
vaccines will be. These recent experiments showing the potential of such vaccines to
raise strong immune responses against the products of retroviral genes suggest the
promise of DNA vaccines is great indeed. But experimental demonstrations are far
from proof.
Still, researchers are optimistic. With DNA vaccines, they can select and inoculate
with only those genes found to induce the strongest immunity. What's more, the
degree of safety possible with plasmids is virtually absolute; there is no possibility of
infection or complications.
With the development of molecular biology, especially genetic engineering, and
the finding of appropriate animal model as well as the detailed understanding of the
mechanism of HIV infection, the DNA vaccine is a promising, practical, effective
method for the prevention of that insidious disease around the world.
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