Malaria Vaccine
Ryan Oetken and Willie Mech
Bill Gates, the third richest man in the world, is a very intelligent man. He is very knowledgeable (consider replacing the word "very", or just delete it) in running a successful business, engineering computers, and making billions of dollars. Recently, Mr. Gates has stepped out of the realm of his software giant Microsoft expertise and into the biological field. In 2007, Bill and Melinda Gates stated they wanted to eradicate malaria once and for all. Did Bill Gates do his homework before making this statement, or will his billions of dollars help fund one of the greatest vaccines known to mankind? Unfortunately for Mr. Gates and all mankind, the cure for malaria is very complex, as previous attempts to eradicate malaria have proven. ( Reference Bill Gates information and make the tone a little less matter of factly especially the very first sentence) This introduction seems very informal, the second paragraph seems like a better introduction to the content of the paper.
Malaria, one of the world’s deadliest parasites, infects 300-500 million humans and claims nearly 3 million lives annually (16). Malaria is a eukaryotic parasite (genus Plasmodium) that is predominately transferred by the bite of mosquitoes in which the parasites in their saliva will infect the erythrocytes of their vertebrate host. Plasmodium falciparum is by far the most virulent form of malaria in humans, causing major mortality and morbidity in populations where malaria is endemic. Several other species of Plasmodium infect humans, including P. vivax, P. malariae and P. ovale (11) which are much less morbid. Malaria infections can cause fever, severe anemia, coma, and renal failure in children and adults, while causing poor birth outcomes in pregnant women (2). Malaria is very endemic in third-world countries, especially in Africa where the majority of the estimated 3 million fatalities occur in children under the age of 5. Although medicines and vaccinations have slowed malaria in some points of history, this tough parasite continues to infect and reproduce successfully. I think this could be a better introduction
In the face of growing resistance to available drugs and no licensed vaccine, new approaches are urgently required to tackle its control (30). Fundamental to these is an improved understanding of the basic biology of the malaria parasite (30). Transmission of malaria is dependent on the successful completion of the Plasmodium lifecycle in the Anopheles vector (34). The Plasmodium life cycle in the Anopheles mosquitoes begins with the ingestion of a blood meal containing Plasmodium gametocytes (31). After the gametocytes (define) are fertilized in the mid-gut of the mosquito, the resulting zygotes experience many transformations. The zygotes (Define) will first transform into oocytes and undergo many mitotic divisions without cytokinesis. After a mature oocyte (define) forms, sporozoites begin to bud off and migrate into the salivary glands of the mosquito where they will remain until injected into their host. Include figures or references to figures here, the life cycle is very unusual and the physical forms of each state are unfamiliar to most people who do not study this parasite.
A malarial infection begins when an infected Anopheles mosquito injects its saliva with parasites called sporozoites into the bloodstream of humans. Sporozoites rapidly migrate to the liver, where they infect hepatocytes. Here sporozoites replicate and develop into merozoites, the blood-stage form of the parasite (32). This maturation of sporozoites is termed liver schizogony, which results in the formation of thousands of first-generation merozoites (33) through asexual reproduction. The liver cell soon ruptures as the merozoites vigorously enter the blood stream where they will go on to infect erythrocytes.
After invasion of the erythrocyte, the parasite engages in many activities of red blood cell transformation discussed later in this paper. The parasite is now currently in the ring stage that lasts about 0-20 hours after infecting the red blood cell in which little metabolic activity and little change takes place (14). The parasite then develops into the trophozoite stage, a growing and feeding stage of the parasite that will lead to DNA replication. Asexual reproduction also occurs in the trophozoite stage through schizogony. Nuclear division follows the DNA replication when the parasite enters the schizont stage after which the infected erythrocytes rupture, releasing up to 32 merozite stage parasites that infect new red blood cells (14).
Merozoites that do not undergo mitotic division undergo a type of maturation process termed gametocytogenesis. During this process male and female gametocytes are produced to later fertilize in the invertebrate host (26). Mosquitoes then take in the gametocytes to their mid-gut, where the gametocytes then breed and produce sporozoites (23), beginning the life cycle of the parasite over again. (is this suppose to be a separate paragraph?)
General knowledge of the life cycle plays a pivotal You can use the word important or essential instead of pivotal role in understanding why malaria is an astonishing yet devastating parasite. The intra-erythrocytic stage of infection is responsible for the syndrome of clinical symptoms of malaria (9). As stated before, symptoms of malaria include high fever, severe anemia, coma, and renal failure in children and adults, while causing poor birth outcomes in pregnant women (2). These disease states are in part a result of (wording here is awkward, reword to make your point clearer) the excessive binding of Plasmodium–infected erythrocytes to the vascular endothelium and to infected and uninfected erythrocytes (35). Malaria can be classified as uncomplicated or severe, depending on which Plasmodium species is contributing to the symptoms. P. falciparum contributes to severe malaria which induces organ failures and abnormalities in the blood, while the other species show uncomplicated forms with typical signs of malaria.what other parts of the life cycle contribute to the complexity of malaria?
Closer examination of severe malaria is significant because severe malaria causes many symptoms and result in the loss of human lives. One symptom of severe malaria is caused by the sequestration of mature P. falciparum-infected red blood cells to the cerebral endothelium in humans called cerebral malaria (36). Unfortunately, the process of how cerebral malaria occurs remains poorly illustrated. However, recent results strongly suggest that an antibody response to brain antigens induced by P. falciparum infection may be associated with pathogenic mechanisms in patients developing cerebral malaria (36).
In addition to cerebral malaria, anemia also accounts for severe malaria in humans. Anemia is a condition that causes the hemoglobin in red blood cells to be degraded and no longer carry oxygen to cells of the body. This can easily be noticed in the merozoite stage of the parasite in which hemolysis causes destruction of the erythrocytes and the hemoglobin cannot function properly. Hemoglobin is also used by the parasite to grow and function properly while inside the red blood cells. Anemia causes the most problems in pregnant women and children, the number one victims of malaria. These victims deserve closer examination of why they are the most vulnerable.
Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum malaria parasites binding specifically to chondroitin sulfate A in the placenta (38). By binding to the placenta, the parasites are able to evade splenic clearance and their destruction. Pregnant women are more susceptible to being infected with malaria primarily due to the sequestration in the placenta. This sequestration can cause much harm to the mother and the fetus, especially in women without pre-immunition to the malaria parasite. PAM is expressed in a range of clinical complications that include increased disease severity in pregnant women, decreased fetal viability, intra-uterine growth retardation, low birth weight, and infant mortality (37). Although these conditions are seen in many malaria pregnancies, it has been exhibited that pregnant women on subsequent pregnancies are less susceptible to malaria and are partially resistant to it (37).
Infants to five-year-old African children are most susceptible to death by malarial parasites for many reasons. Of the thousands of children dying every year from malaria, these fatalities are primarily found in endemic areas of Africa. Not only are they more susceptible to malaria, but these children are less able to defend themselves from malaria. Malnutrition, lack of funding for respectable treatment, and minimal protection from mosquito bites all play a large role in why malaria affects large amounts of children.you started to mention Africa's children in the second paragraph, so maybe move some of this information to that paragraph and possibly cite the information here unless it is common knowledge?
The battle with malaria has been tested for many years with various drugs and vaccines to give humans an advantage over the parasite. There are two main classes of antimalarial drugs: the antifolates and cinchona alkaloids that have been widely used. Examples of antifolates are proguanil and sulfadoxin that both act to inhibit nucleic acid synthesis, while atovoquone and chloroquine, cinchona alkaloids, target the electron transport chain (ETC) of the mitochondria and heme detoxification, respectively (42, 41). (How does inhibition of the ETC and heme detoxification inhibit malarial maturation or replication? Explain more on the effects of drugs on this parasite.)
Chloroquine, a synthetic 4-amminoquinoline, is a lysosomotrophic weak base compound that is the first line in antiviral drugs due to its low cost, high effectiveness, and low occurrence of side effects (8). At the peak of its use in 1994, it was priced at only $0.08 per treatment and was found to be the 3rd most widely consumed drug in the world, after aspirin and paracetamol (42). Chloroquine works by targeting a compound called heme inside the parasite. P. falciparum obtains some of its nutrients through digestion of hemoglobin and releases heme (ferriprotoporphyrin IX) into the parasites digestive vacuole (DV). Heme, toxic to the parasite, is avoided by the parasite by polymerizing it into nontoxic hemozoin. Quinine is believed to work by inhibiting heme polymerization, which will result in the buildup of toxic heme and eventually kill the parasite (8). *
Sulfadoxine is another known antimalarial drug that targets dihydropteroate synthase (DHFR) during the asexual stage of P. falciparum and is usually used in combination with pyrimethamine (42). Binding to DHFR causes a conformational change, preventing the enzyme from binding to its natural substrate. This inhibits the folate synthesis pathway and will eventually disrupt DNA synthesis, killing the parasite (41).
The quinoline-containing drug atovaquone is a very effective drug that acts on parasitic mitochondria by disrupting the pyrimidine biosynthesis pathway and ultimately crashing the membrane potential of the mitochondria (19, 10). It targets cytochrome bc1 and affects the catalytic domain and stops the protein from continuing its biosynthesis pathway collapsing the mitochondrial membrane potential (19). This will eventually cause the parasites to die. All of these drugs have worked for a period of time, but the quick resistance of malaria has proven to be a monumental force.
Chloroquine (CQ) has been the major antimalarial treatment choice during the second half of the 20th century, and resistance to this drug has made it the focus of much research (1,8). The first report of chloroquine resistance (CQR) occurred in Southeast Asia and South America in the late 1950s but can also be seen in many other global locations. Despite the multiple independent CQR antigens worldwide, five common phenotypes are common among CQR parasites. The first phenotype is increased drug inhibition concentration, at which 50% of the parasites are killed, measured by in vitro assays (IC50). Chemosensitization, reduced pH levels in the DV, and reduced CQ buildup in the DV are also shared, as well as universally shared point mutations in the gene P. falciparum choroquine resistance transporter (pfcrt), a major determinant of the parasite’s resistance (8).
The gene first linked to CQR was a 400 kb DNA segment on chromosome 7. This region was later narrowed to 36 kb region using high density microsatellite markers, while further analysis led to the discovery of the pfcrt gene, a ~3.1 kb gene containing 13 exons which encode 424 amino acids making up the PfCRT protein. Within this gene at least 2 point mutations located in isolated or laboratory clones have been found. The most common mutation is an amino acid substitution at codon 76, and it is the most prevalent of all CQR parasites worldwide (7-8). Other mutations found to occur within this gene are other amino acid substitutions at the 86th and 220th codons, which are also highly prevalent among CQR parasites worldwide (8).
The PfCRT protein belongs to a drug/metabolite transporter superfamily that contains 10 transmembrane domains and is located on the DV membrane within P. falciparum. This protein is an ion channel thought to play a role in the regulation of pH, ion exchange, and CQ concentration within the DV. Examination of this protein found that the universal mutation that substitutes an amino acid at codon 76 is a charge-loss mutation. In wild-type parasites there is a positively charged lysine residue at position 76 which is known to be near the luminal surface and acts by repelling other positively charged ions, such as the diprotic CQ2+. This charge repulsion in wild-type parasites is what helps CQ accumulate inside the vacuole, killing the parasite. There are three theories for how this mutation causes CQR: energy dependent CQ efflux mechanism, passive efflux of diprotic CQ out the DV by a “charged drug leak” mechanism, and direct CQ-PfCRT binding. The most accepted of these theories is the “charged drug leak” mechanism that ultimately leads to a decrease in CQ concentration inside the DV because the once positively charged residue inside the lumen of PfCRT that once repelled CQ2+ is now replaced by a neutral residue that allows the leak of CQ outside the DV down its concentration gradient (7).
P. falciparum has also shown resistance to sulfadoxine with its earliest onset coming under 5 years (42). Seven point mutations within the DHFR protein have been found that all cause an amino acid substitution. Some of the most conserved mutations in DHFR are mutations at codons 436, 437, 540, and 613 (40). These mutations in turn cause a change in the tertiary structure of the protein, causing a conformational change that causes (try using..that results in…instead of that causes) a diminished affinity of the binding of drugs to the enzyme target (41).
Atovaquone efficiency has also been hindered by mutations as well as many other antimalarial drugs. The target of atovaquone, cytochrome bc1, has been found to have undergone a single nucleotide mutation in the cytochrome b gene, giving the parasite high-grade resistance (10). This resistance due to an amino acid substitution is problematic for the once very potent drug. Now the drug has been used in combination with proguanil as a combination chemotherapy that is working very well but is also very expensive (39). (again, try to limit the word "very")
Along with the many drug resistant mutations of P. falciparum, a very complex lifestyle produces a wide range of proteins through each stage of life, providing scientists with hard-to-pinpoint polymorphic target antigens (9) This antigenic diversity can be a major obstacle in the development of a vaccine (42). The protozoan P. falciparum requires a rainbow of proteins that are all encoded on 23 Mb of 14 different chromosomes. This vast genome is significantly larger than any other human pathogen genome for which a successful vaccine has been created for humans. However, scientists have been advancing on vaccine candidates that have been effective in clinical trails for sporozite and blood stages of P. vivax and P. falciparum. During the sporozite stage the parasite is injected into the human host as a haploid and it then seeks out the liver where it will begin to replicate. A few key target antigens during this stage are thrombospondin-related adhesion protein (TRAP), liver-stage antigen 1 (LSA-1), and circumsporozite protein (CSP). CSP is the target of the most effective malaria vaccine RTS,S. This vaccine has been able to prevent infection in about 30% of children under the age of five when it is used in conjunction with AS02A (9).
Another major class of malaria vaccines targets the blood stage of the parasite. This is the intraerythrocytic stage that is responsible for clinical symptoms representative of malaria. Furthermore, one of the antigens presented to the human immune system is the merozite surface protein 1 (MSP-1). This 19 kDA protein contains polymorphic antigens in which many variants occur in a single infection population. Even though the MSP-119 gene is fairly conserved within the parasites, it does contain 6 polymorphic amino acid residues. MSP-119 contains two dominant haplotypes that occur in about 80% of infections. These are caused by the QKSNGL and EKSGL haplotypes; however, neither of these two are used as possible antigens. The haplotype ETRSSRL is found to be one of the leading vaccine targets, but it occurs in only about 16% of infections. Perhaps a vaccine targeting either or both of the more prevalent haplotypes would lead to a more efficient vaccine (9).
Another antigen that shows different variations on P. falciparum is the PfEMP1 protein. PfEMP1 is the major molecule that appears on the surface of an infected erythrocyte. This protein is used to mediate cytoadherence, which allows the parasite to sequester itself inside the erythrocyte. This molecule is so hard to target because it is presented on the erythrocyte for only about 30 of the 48 hours of asexual reproduction. Also, this induced surface antigen is so variable because it can stem from 50 or more var genes on multiple chromosomes (42).
The main reason these antigens are said to be polymorphic is based on the finding that the seasons affect malaria transmission. Seasonality was shown to have overwhelming effects on the prevalence of some haplotypes. In addition, to that it was also found that (To that is was also found that…this seems a little confusing) one infection population might promote two infections inside its host; an asymptomatic infection and one with clinical features could exist at the same time. This suggests some level of haplotype immunity and that at a combination of MSP-119 sequences need to be covered by a vaccine.
Combination chemotherapy has been the new technique in antimalarial treatment because it gives the therapy time to kill every parasite within its host. The theory behind combination chemotherapy is to use a rapidly acting drug along with a slower acting drug that is given over time to kill any residual parasites. Combination chemotherapy also decreases the chances that a mutant parasite will survive because resistance to both drugs is very unlikely (10). Some common antimalarial drugs used in combination are quinine-tetracycline, chloroproguanil-diapsone, artemether-lumefantrive, and the most popular being atovaquone-proguanil (42).
The atovoquone-proguanil treatment targets the pre-erythrocytic as well as erythrocytic stages of P. falciparum that act in a synergistic action that will greatly reduce the inhibition concentration of treatment (19). Proguanil acts as a fast acting drug and inhibits the parasite dihydrofolate reductase enzyme (39) inhibiting nucleic acid synthesis; while atovaquone is the slow acting drug targeting the ETC of the mitochondria. Together, the collapse of the mitochondria membrane potential as well as the inhibition of nucleic acid synthesis are very effective in killing off the parasites within the human host (19). One of the most useful things about using this combination of drugs to treat malaria is that it will take a series of mutations to inhibit proguanil’s ability to act on the parasite (39). (Have there been any studies showing the effectiveness of this type of combination therapy? If so, some stats or whatever would be sweet)
The genus Plasmodium is not only highly resistant to vaccines, but it has defense mechanisms against humans to help ensure survival. The adaptive immune system of humans requires proper dendritic cell (DC) and T-cell communication to make sure a rival pathogen is properly killed and cleared from the body. Suppression of the immune system has been exhibited in many cases of malaria infection in patients (21). These patients exhibited DC activation and function suppressed by infection or malaria pigment, causing T-cells and B-cells responses to fail in developing rapidly (21). However, different strains of the Plasmodium parasite affect the nature and function of the DC response (22). It is highly suggested that lethal parasites induce a block in DC function in migrating to the spleen to protect humans against infection (22).
The defense mechanisms of malarial parasites are used as one form of protection against humans, but its immune mimicry is another lethal weapon used against its vertebrate hosts. Plasmodium falciparum secretes a functional histamine-releasing factor homolog, translationally controlled tumor protein (TCTP), which allows the parasite to further sequester itself, making it more difficult on the immune system to attack it (2). Sequestration is also mediated by various parasite-derived molecules collectively referred to as P. falciparum erythrocyte membrane protein 1 (PfEMP-1) located on the erythrocyte membrane (6). These molecules interact with various endothelial cell (EC) surface receptors, such as CD36 and ICAM-1, stimulating them to release a tissue factor (TF) very similar to tumor necrosis factor alpha (6). The release of TF induces sequestration, a coagulation disorder, EC activation, and the production of inflammatory cytokines, all of which have been separately described as features of severe malaria pathology (6).
Not only is the ability of the parasite to mimic our immune system to evade death imperative, but the ability of the parasite to induce cell functions of the red blood cell is equally important. Plasmodium falciparum develops within the mature red blood cells of its human host in a parasitophorous vacuole (PV) that separates the host cell cytoplasm from the parasite surface (13). From here the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism (24). The establishment of an ion channel is one significant alteration that is required for parasite development (24). Among these changes are transcription and translation of many proteins that are directed to the surface of the erythrocyte. An example is the adhesion molecule PfEMP1 that is expressed on the outside of the infected red blood cell (15) to interact with many advantageous outside receptors such as CD36 and ICAM-1. Another function the malaria parasites perform after invasion of the erythrocyte is the degradation of hemoglobin as a principle source of amino acids for parasite protein synthesis (4). Plasmodium is very effective in utilizing the PV inside the erythrocytes to direct and signal cell operations. However, many portions of this process leave it vulnerable to vaccines, such as altering the anion channel, PfEMP1, and the degradation of hemoglobin.
In conclusion (delete)finding the cure to malaria is definitely not an easy venture. Malarial parasites have many advantages over mankind in continuing their existence on this planet. Complex life cycles, different hosts, high-resistance to drugs, third-world countries with poor mosquito repellent techniques, and the high costs of using these drugs are some of the reasons malaria will be difficult to eradicate completely. It is difficult to speculate whether malaria will be completely gone someday. Only time and money will tell the end to this story of the battle between man and this small protozoan parasite wreaking havoc on humans.
Oetken and Mech- You have a great topic and paper. One thing that you should consider changing is the introduction as I wrote at the beginning I think that the second paragraph explains more what your thesis statement is and what your paper is going to be about. The paper flows well and that is very important for the reader. Also you should include headings of the different topics that you mention… Good Job Guys!!!!
Ryan & William: overall this is a very well written paper. I would suggest making heading for all the different topics about malaria that are discussed. Although transitions were used effectively, I think the reader would have an easier time following if headers such as "Life cycle of Malaria Parasites", or "Symptoms of Malaria and Effects on Pregnant Women and Children".
This is a great paper. There are a ton of great facts, and the paper flows well. I would only suggest maybe adding somewhere how the immune system would recognize specific antigens on the parasite, and how it would then kill it.
