Introduction
Malaria is an endemic infectious disease that is caused by one of four parasitic
protozoa of the genus Plasmodium, which infect human red blood cells, namely
P. falciparum, P. vivax, P. malariae, and P. ovale. These parasites can only
be transported from host to host via the Anopheles genus of mosquito, and only
60 of the 380 species can do so (MFI 2004). Because of the widespread occurrence
of malaria many steps have been taken in order to make a vaccine. There are multiple
anti-malarial drugs that are in use, but those require daily dosage before, during,
and after time spent in endemic areas in order to be effective. The greatest
difficulty is that the parasites have begun to develop resistance to these drugs
(Warhurst 2003).As of yet there is no complete vaccination against malaria.
The Plasmodium bacteria require both a human and a mosquito host in order to
complete its life cycle, though P. malariae may also infect the higher primates.
The mosquito becomes infected with the bacteria while feeding on a human. The
bacterium then reproduces itself in the gut of the mosquito, after which it transfers
to a second human host during another mosquito feeding (CDC 2000).
Ecology:
Malaria is most common in tropical and subtropical climates, ranging between
the latitudes of 23.5o North (Tropic of Cancer) and 23.5o South but some, often
seasonal, cases also occur outside of these latitudes in areas such as South
Africa (Kruger National Park and surrounding area - 25o South) and New Delhi,
India (28.5o North). The P. falciparum is the type found in sub-Saharan Africa,
where 90% of malaria cases come from, and is the most deadly, killing approximately
1-2% of those infected; an estimated 2.7 million deaths per year. The vivax species
is common to Asia, South America, Mexico, the Middle East, and the Caribbean.
In the United States there are an estimated 1,200 cases each year, though most
are travelers returning from other countries (CDC 2000). It is also found in
North Africa, but seems to have fewer cases as many people have a recessive phenotype
for the Duffy protein which prevents the parasite from entering the red blood
cells (Schmidt 2000).
Transmission/ Clinical Features:
Plasmodium vivax is transmitted via the Anopheles spp. mosquito that is found
worldwide. The transmission cycle of malaria is started when the vivax parasite
is passed to a human during the mosquito’s blood meal. The parasite enters
the body in a sporozoite form, which is the parasite resulting from sexual reproduction.
Once the parasite is in the body it only takes minutes for it to travel to the
liver tissue where it invades the hepatocytes. The parasite undergoes asexual
reproduction once inside the cell and multiplies until the cell lysis. It takes
about 10 days for the parasite to complete a cycle in the liver. About 10,000
merozoites (offspring resulting from asexual reproduction) are released into
the circulation for every infected liver cell. Some of merozoites can remain
in the liver and become hypnozoites (dormant parasite). In the circulation the
merozoites invade young red blood cells via the Duffy binding protein found the
parasite’s surface. Using the hemoglobin in the red blood cells as a food
source, the merzoites undergo more asexual reproduction producing trophozoites
(feeding stage), microgametocytes and macrogametocytes. The trophozoites feed
on the hemoglobin and the waste product resulting is hemozoin, which when released
into the circulation causes an inflammatory response. At the height of the infection
the trophozoites occupy about 66% of all the red blood cells. The mosquito then
bites an infected person and takes up the gametocytes. The gametocyte undergoes
sexual reproduction in the gut of the mosquito and becomes an oocyst which can
produce 10,000 sporozites. The sporozoites travel to the salivary gland of the
mosquito and then are ready to be injected into the mosquito’s next victim
(Schmidt 2000).
The symptoms of the P. vivax infection take 11 to 13 days to develop. The one
symptom that malaria is known for is the cycle of high fever every 48 hours.
This fever cycle is caused by the release of waste toxins (hemozin) into the
blood, which causes an elevated level of TNFalpha. Other symptoms are heptosplenomegaly
(swollen spleen and liver), and severe anemia (Low Red Blood cell count), and
jaundice (yellowing of skin and eyes due to breakdown of hemoglobin released
from lysed red blood cells) (Schmidt, 2000). The flu like symptoms also include:
nausea, vomiting, and diarrhea ("Malaria: General…" 2000). People
visiting endemic areas are more susceptible to infection because that have not
been exposed to the parasite. Natives that live in endemic areas only have short
term immunity (~6 months) to the infection. This is because of the nature of
the parasite and its ability to induce a strong enough immune response. P. vivax
is one of the malaria parasites that can actually become dormant and the patient
can have a relapse of malaria up to 8 years later after the initial infection
(Schmidt, 2000). Malaria has such an enormous negative impact on the entire world;
it is because of this that vaccines to this bacterium are necessary.
Human Vaccine Proposal:
Malaria is an infectious disease that threatens the lives of many each year throughout
the world. Human malaria is caused by four major species that infect the general
population, of which, Plasmodium vivax is the most common. P. Falciparum is the
more life-threatening and drug resistant compared to P. Viva., However, recent
studies indicate that P. Vivax is becoming more of a problem than other species.
The recent outbreaks across the world have put enormous pressure on the scientists
to develop an economically viable, yet practical and safe vaccine for the disease.
The major problem in vaccine development has been the antigenic diversity of
the vaccine candidates among the malaria parasites. Genetic variation studies
have been conducted to identify the major antigen candidates for the vaccine:
CSP (circumsporozoite protein), MSP (merozoite surface protein), and DBP (duffy
binding protein). DBP has been the major protein evaluated in vaccine development
because it facilitates binding of the asexual merozoite blood stage of the parasite
to the red blood cells in the human. For an effective vaccine to be produced
the DBP must be targeted and parasite-red blood cell binding reduced, or potentially
eliminated, in order to prevent the blood-borne stage of the parasite. Direct
evidence for naturally occurring neutralizing antibodies to the DBP from malaria-exposed
people has recently been demonstrated and has been the cynosure for the focus
of a vaccine against the most common malaria strain. For optimal efficacy, we
propose to incorporate a vaccine that will target the CSP as well as the DBP.
The CSP will be used as well for several reasons: 1). targeting just one protein
and step in the life cycle of such a complex parasite is not likely to be optimally
effective, 2). a rational second parasite target would be the one that is responsible
for initiating the first step required to start the life cycle in the human host
– CSP on the sporozoite stage, and 3). the ligand binding domain in CSP
for the host hepatocyte receptor has been identified at the molecular level in
region II by Cerami et al and could easily be added to the herpes expression
system (discussed later), tandem to DBP, thereby targeting two key stages of
the life cycle.
Vaccines already in clinical trial are directed against three different pasrasite
stages and events in the life cycle: 1). the sporozoite (first asexual stage)
stage of the malaria parasite to prevent host hepatocyte infection, 2). the merozoite
(second asexual stage) stage to prevent host red blood cell infection and clinical
disease, or 3). the sexual stages in the mosquito vector to prevent transmission.
Proposed malaria vaccines which have been studied, most in depth, are designed
to induce immunity to the sporozoite and the infected liver cell (i.e., to stop
or reduce sporozoites invasion of liver cells and to kill any sporozoite-infected
liver cells). In early studies, sporozoite-based vaccines were shown to induce
protective immunity against experimental challenges. Under conditions of natural
exposure (for example, in semi-immune adult men in Africa) the proposed vaccine
was shown to be safe and well tolerated, with an estimated efficacy of 70% in
the first nine weeks of follow-up. However, over the 15 weeks of observation,
the efficacy (referring to incidence of infection) fell to 34%. These vaccine
approaches are thought to have sub-optimal efficiency because they do not target
all the asexual stages of the sporozoite; this becomes a major problem because
the parasite is still able to proliferate. Our vaccine proposes to target the
major asexual stages, which will prove to be more efficient because it knocks
out the proliferation mechanisms.
In developing a viable live vaccine against malaria, one must ensure that the
vaccine is attenuated, but still capable of inducing the proper immune response
in the host. An attenuated vaccine is necessary because it would induce the appropriate
protective immune responses. To achieve optimal efficacy, the DBP and CSP will
be the focus of the vaccine, and they will be isolated and tested to induce antibody
formation. Neutralizing antibodies can control infection and inducing such anti-DBP
and anti-CSP antibodies is the most rational approach for preventing and reducing
RBC infection. This is plausible because RBCs do not express class I MHC and
therefore cannot be recognized or destroyed by cell-mediated immunity, specifically
the cytotoxic T cells. Cytotoxic T cells are important because they kill infected
cells and generate immune memory. The ultimate goal of this vaccine is to generate
immune memory to protect against future infections by forming memory cytotoxic
T cells and memory B cells. Once the DBP and CSP are isolated and cloned, phase
I testing in animals can begin. The vaccine will consist of isolated DBP and
CSP genes and will be inserted into a herpesvirus simplex-1 expression vector.
The herpesvirus will be inactivated in the laboratory by targeting specific virulence
gene sequences. This will be done so it will not harm the host, but still will
be able to replicate. Herpesvirus simplex-1 is a DNA virus that has the ability
to self-replicate. This is an important property in delivering vaccine antigens,
particularly when the induction of cell-mediated immunity is critical. Moreover,
the herpesvirus is a larger sized vector, meaning it has the capability of accepting
and expressing relatively large amounts of parasite genetic material. The most
important characteristic of the herpes simplex virus-1, and the reason for its
selection, is that it establishes a persistent infection in the body. With the
persistence of the virus, it will supply constant stimulation with DBP and will
generate immune memory. The vaccine will primarily be delivered intravenously,
so it will immediately enter the bloodstream and elicit a fast immune response.
The IV route of immunization has been shown to produce the most results, meaning
the most reduced incidence of clinical disease, and has greater efficacy then
administering the vaccine orally or intranasal.
Immunology:
Innate:
After inoculation of the vaccine, the first response would be by the innate immunity
at the site of inoculation from the foreign antigen on the herpesvirus. The macrophages
are activated, which then release the cytokines IL-1, IL-6, IL-8, IL-12 and TNFalpha.
The cytokines and chemokines cause an inflammatory response, attract more leukocytes
to the site, and increase the circulation to the site through the use cell adhesion
molecules on vascular epithelium tissue. Specifically, TNFalpha increases the
expression of cellular adhesion molecules to help other leukocytes enter the
infected tissue. Also, the cytokine IL-6 will act on the liver to start producing
acute-phase proteins, which will bind to the bacterial antigen and activate complement.
The complement cascade will initiate the use of complement components to help
in opsonization and increase circulation. This allows for T cells and dendritic
cells to enter the site and bind the antigen. The T cells bind antigen-presenting
cells in the draining lymph node via the class II MHC, which activates T cells.
The dendritic cells bind the antigen and are able to migrate to the lymph node
through the use of cytokines (IL-1/IL-6/TNFa). This causes the activation of
T cells in the lymph node, thereby initiating the adaptive immune response (Parham
2000).
Adaptive:
Once the T helper cells are activated, they receive signals to generate Interleukin-2
(IL-2), which is a cytokine produced that is essential for the development and
proliferation of the adaptive immune response, mainly the cytotoxic T cells and
antibody-secreting plasma cells. Cytotoxic T cells are important in the host
defense against viruses and cytosolic pathogens. The function of the cytotoxic
T cells is to secrete perforin and granzymes to poke holes in the infected class
I MHC and host cells, which induces apoptosis. The granyzmes are serine esterase
enzymes present in the granules of the cytotoxic T cells and initiate apoptosis
resulting in DNA fragmenting and death of the target cell. The cytotoxic T cells
express VLA-4 that sends a signal which ultimately ends up with the cytoxic T
cells migrating back to the infection site. The cytotoxic T cells now express
an important TNF receptor, FasL, which binds to Fas positive cells and triggers
apoptosis in the cell. This whole process begins with cytotoxic T cell, TCR,
and CD8 binding to class I MHC. The CD8 co-receptor recognizes peptide antigen
presented by the class I MHC molecules. The cytotoxic T cell becomes fully activated,
and arranges its components so that perforins and granzymes can be secreted when
needed. Once the cytotoxic T cell is fully activated, its purpose becomes very
useful in generating immune memory. Memory CTL is important because it protects
the body from further infections by the same pathogen.
B cells are also key players in the adaptive immune response. B cells would be recruited to the infection site to make neutralizing antibodies, which would block the parasite’s binding to the host RBCs. B cells present antigen to the already activated Th2 cells and initiates the activation cascade which starts with the B cell co-receptor. The B cell co-receptor signal is needed to start the activation of the naïve B cell. The B cell co-receptor consists of three proteins: CD21 which binds to complement components deposited on a pathogen, CD19 which acts as the signaling chain of the receptor, CD81 which is a cell surface receptor for various viruses. The binding of CD21 to complement fragments on the surface of the bacteria cross-linking the B cell co-receptor with the BCR which causes them to cluster together on the B cell surface. The cytoplasmic tail of CD19 is then phosphorylated by tyrosine kinases associated with the BCR. The phosphorylated CD19 binds to extracellular signaling molecules whose signals synergize with those generated by the B cell co-receptor. The combined effects of the BCR and B cell co-receptor are still not enough to activate the naïve B cell, so final signals provided by the CD4 T helper cells are needed. TCR on CD4 T cell binds peptide on class II MHC on the B cell which facilitates the interaction between CD40L on T cell and CD40 on the B cell. The interactions between the CD40 and CD40L on B and T cells signals the B cell to activate transcription factor NF_B and increase surface expression of ICAM-1 which enables the B cell to bind to other cells. This enables the T cell to secrete important cytokines such as IL-4, which initiates the proliferation and clonal expansion of B cells. B cells differentiate into antibody secreting plasma cells. B cells begin dividing and moving into different locations. They move to the medullary cords of the lymph node and differentiate into plasma cells. The B cells then move to areas of LN and form germinal centers where B cells proliferate and undergo somatic mutation. The mutations occur in the hypervarariable regions of the heavy and light chains. B cells secrete IgM first that go to the medullary cords and isotype switch into IgA (or any other antibody specifically needed) from undergoing somatic hypermutation and somatic recombination. One complete exon is generated that encodes the variable region of the IgM and results in the production of a variant antibody, or a memory B cell. Memory B cells are important because they protect the body against future infections by the same pathogen. IgA and IgG are the most important antibodies for the purpose of malaria, because they both neutralize pathogens by blocking their binding sites. With the production of IgA/IgG, memory B cells, and memory cytotoxic T cells, the patient exposed to malaria would have immediate defense upon next encounter of the pathogen, as well as having defense against exposure for the first time.
Vaccine Tests:
After the vaccination of the proposed vaccine with the Duffy binding protein,
a primate will be used to test for immunity. By comparing the results of the
tests it will be known the effectiveness of the vaccine in cellular and humoral
immunity.
Humoral Immunity – ELISA (Enzyme-Linked-ImmunoSorbent-Assay)
Humoral immunity consists of the function of B cells, which focuses on the production
of antibodies. Immunoglobin G and A will be specifically targeted as the main
antibodies produced. Since Ig G is the one mostly produced it will be the focus
for testing. ELISA will be used to detect the production of antibody in response
to the Duffy binding protein.
An indirect ELISA is used to detect antibody in serum. The Duffy binding protein
(DBP) will be used as the antigen that will bind to the antibodies. The sample
will be serum from the mice that received the vaccination. The first step to
the assay is to bind DBP to the microtiter plate. Serum sample to be tested for
specific antibody (IgG) will be added and allow it to bind to the DBP. An anti-
Ig (from a different species) will bind to the Fc region (gamma chain) is added.
The Fc of the anti-Ig has an enzyme, which will convert a chromogenic substrate
to a colored product once there has been a reaction between the enzyme and substrate.
The more color that is detected means that more specific antibody present in
the sample. Controls must also be performed in order to realize that the assay
indeed detects the interaction of the antibody with the antigen. A negative control
is run by omitting the antigen or by adding an antibody that will not bind to
the antigen. A positive control consists of using an unknown serum instead for
a known positive serum.
Cellular Immunity-Flow Cytometry
Cellular immunity will focus to the production of CD4 T cells since Plasmodium
vivax is a parasite. Also Th cells will be needed in order to initiate the activation
of B cells to proliferate to Duffy binding protein specific and then to plasma
antibody secreting cells. Flow cytometry is used to quantify cells based on their
markers. Fluorochromes are added to antibodies that are specific for the molecules
on the T cells’ membrane. The amount of fluorescence is recorded by the
cytometer as cells pass by the detector. Blood from the vaccinated mice will
be used as the sample to be analyzed for T cells. All the erythrocytes must be
removed by centrifugation. A fluorochrome-labeled antibody specific for CD4 structure.
Negative control should also be used to control the stickiness of the antibody.
The negative control can be achieved by including an antibody of the same species
and isotype but to a different antigen.
The suspension of cells and specific antibodies is incubated then the cells
are run through the flow cytometer. As cells pass the detector the amount of
fluorescence is quantified. A fluorescence-activated cell sorter uses the fluorescence
signals to separate the cells into groups based on what fluorescence-labeled
antibody cells bound and if they bound at all. Since one cell passes the detector
and its amount of fluorescence is quantified it can be analyzed how many cells
had the CD4 molecule. Based on the number it can be identified whether the vaccine
initiated enough CD4 T cells to initiate a cellular immunity.
Primate to Human Incorporation:
The test of the vaccine will be done one primates. Primates will have the most
similar cellular functioning to humans. Ideally, it would be most beneficial
to test the vaccine on humans. However, due to the ethical problems that would
arise primates would be the next best thing. The DBP and CSP will be sought out
and isolated in the primates. With the vaccine it is hoped that by isolating
these proteins antibodies will be produced and an immune memory will be developed.
Primates have an immune response that is similar to that of humans. Inoculating
primates with the proposed vaccine would most reliably evaluate its effectiveness.
Conclusion:
The severity and prevalence of malaria necessitates an effective malarial vaccine. The current research has come close to formulating one but there still currently is no solution. It is plausible that if a vaccine to P. vivax were to target the DBP and the CSP a combatant immune responsive will be initiated. This could possibly halt attack and destruction the malarial parasite initiates on the red blood cells. Controlling P. vivax would be one step in the right direction to protecting the world from all strains of the malaria bacterium.
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