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International Journal for Computational Biology (IJCB)
Vol.5, No.1, August 2016, pp. 88~94
ISSN: 2278-8115 88
Exploring The Pre-erythrocytic Stage Of The Malaria Parasite For
Possible Target Proteins To Develop An Effective Vaccine And
Looking Into Available Preventive Measures Against Malaria: Can
SPECT And SPECT2 Act As Potential Targets For An Effective
Vaccine?
Manas Joshi1, Shama Mujawar2
1 Department of Microbiology, Abasaheb Garware College, Pune
2 RASA Life Science Informatics, Pune
Article Info
ABSTRACT
Article history:
Received Sep 30th, 2015
Revised Nov 2nd, 2015
Accepted Dec 26th, 2015
Malaria is most commonly found in tropical and sub-tropical countries of
Africa. In a year, 3.2 billion people are at risk of getting malaria of which
infection occurs in over 200 million of which one million results in death;
hence it is one of the most infectious parasite diseases. 90% of the deaths occur
in south of Saharan desert and most of the cases are of children under the age
of five. In the year 2012, malaria caused estimated total deaths of 627,000. In
the year 2013, malaria caused estimated total deaths of 500,000. 40% of total
malaria cases in the world are from Nigeria and the Democratic Republic of
Congo (Statistics provided by WHO). These stats strongly suggest the need of
a preventive malaria vaccine. Here we analyze the malaria parasite cycle,
specifically pre-erythrocytic stage, and present some possible target proteins.
We also look at the current options for prevention of malaria.
Keyword:
Pre-erythrocytic life stage of
Malaria parasite
Preventive vaccine for Malaria
Malaria
SPECT
SPECT2
Copyright © 2015 International Journal for Computational Biology,
http:// www.ijcb.in, All rights reserved.
Corresponding Author:
Manas Joshi,
Department of Microbiology,
Abasaheb Garware College
Pune,India
Email: manaswwm@gmail.com
DOI:
How to Cite: Manas Joshi et. al. Exploring The
Pre-erythrocytic Stage Of The Malaria Parasite
For Possible Target Proteins To Develop An
Effective Vaccine And Looking Into Available
Preventive Measures Against Malaria: Can
SPECT And SPECT2 Act As Potential Targets
For An Effective Vaccine?. IJCB. 2016; Volume
5 (Issue 1): Page 88-94.
Journal homepage: http://www.ijcb.in
89 ISSN: 2278-8115
Fig1: Schematic representation of the areas where malarial transmission occurs (Source:
http://www.cdc.gov/malaria/malaria_worldwide/impact.html)
Fig2: A typical malaria parasite life cycle (Source: http://encarta.msn.com/media_461541582/Life_Cycle _of_the_Malaria_Parasite.htm)
Parasite Life-cycle: Pre-erythrocytic stage:
The malaria parasite exhibits a complex life cycle consisting of an insect vector (Anopheles mosquito) and
vertebrate host (humans). The species of Plasmodium which infect humans are: Plasmodium falciparum,
Plasmodium ovale, Plasmodium vivax, Plasmodium malariae; of which Plasmodium falciparum is considered to be
the most morbid due to high levels of parasitemia. These species are inoculated in the avascular portion of skin in
the form of sporozoites by an Anopheles mosquito when it takes a blood meal, these sporozoites then reach the liver
of human host by invading host cells.
IJCB Vol. 5, No. 1, August 2016, 88 – 94 http://www.ijcb.in
90 ISSN: 2278-8115
The number of sporozoites delivered by an individual mosquito is naturally variable [1]. The sporozoites invasion
can be brought about by either cell-traversal (disrupting host cell’s plasma-membrane and migrating through them)
or by cell-infection (forming a parasite-cell junction and settling inside an intracellular vacuole) [2]. Sporozoites can
actively cross the walls of both blood and lymphatic vessels in the dermis [2]. The elongated sporozoite cell displays
an active gliding locomotion on solid substrate reaching upto speed of 4µm/sec, energy for this locomotion is
provided by actin-myosin motor[3]. This cell traversal ability of sporozoites is considered important for many
reasons: for moving freely until endothelial barriers are reached, for resisting attacks by phagocytic cells and for
crossing the liver sinusoid barrier. This cell traversal ability is brought about by two proteins SPECT and SPECT2
(SPECT – Sporozoite microneme protein Essential for Cell-Traversal) which are individually critical for membrane-
damaging capacity of sporozoite, of these SPECT2 has homology with pore-forming proteins [2] . For complete
development of sporozoites, invasion of hepatocytes is a mandatory step. For this, sporozoites on inoculation enter
blood capillaries and are arrested in liver. This arrest is most likely mediated Extracellular matrix (ECM)
proteoglycans of liver [1]. In vitro studies show that spect-disrupted and spect2-dsirupted sporozoites completely
lose cell passage activity, but preserve normal infectivity to hepatocytes. Hence, lack of previous cell passage has no
influence on infectivity to hepatocytes [4]. Once the sporozoites reach hepatocytes they undergo an asexual
replication known as exoerythrocytic schizogony. This results in formation of merozoites which are now released in
bloodstream. These merozoites infect erythrocytes and reproduce either sexually or asexually within these
erythrocytes. A portion of liver-stage parasites go through a dormant period instead of asexual reproduction, these
are termed are hypnozoites. Hyponozoites reactivate themselves after several weeks-months of primary infection
and are responsible for relapses. From the Plasmodium species, Plasmodium vivax and Plasmodium ovale can gives
rise to such hypnozoites. To reach hepatocytes, their first site of multiplication in the mammalian host, Plasmodium
sporozoites must cross the continuous cell layer lining the sinusoids, a layer composed of specialized fenestrated
endothelia interspersed with Kupffer cells, the resident stationary macrophages of liver[6]. Biochemical,
physiological, microscopic as well as various genetic approaches have suggested that sporozoites reach hepatocytes
by passing through Kupffer cells [4][5]. These Kupffer cells can be recognized by their peroxidase activity [9]. As
stated above, SPECT and SPECT2 are responsible for cell-traversal activity of sprozoites, hence they are responsible
for traversal of sporozoites through Kupffer cells into the liver.
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Fig3: (A): Nucelotide sequence of spect cDNA(top lane) and the deduced amino acid sequence(bottom lane). The predicted N-terminal sequence
is underlined while the termination codon is shown by asteriks.
(B): A comparison of deduced amino acid sequences from Plasmodium berghei(top) and Plasmodium falciparum(bottom). Asteriks indicate
conserved region and dots indicate similar region. (Source: Ishino et al., 2004)
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Fig4: Comparison of Membrane pore forming proteins: SPECT2, Human complement component 9, Human Perforin (Source: Ishino et al., 2004)
Current available Preventive measures:
Incubation period for a malaria parasite varies between 8-40 days depending upon the species of parasite. This
incubation period then gives rise to various typical symptoms of malaria; these symptoms are a result of
synchronous lysis of infected erythrocytes. These symptoms include shaking chills, fever, body-ache, head-ache,
general malaise, hyperparasitemia, metabolic acidosis etc. Severe cases of malaria include symptoms like severe
anemia, cerebral malaria, acute kidney failure etc. By the time typical malarial symptoms appear, it is very much
possible that an Anopheles mosquito has picked up macro or micro-gametocytes, which is a result of sexual
replicative cycle, from the infected individual. This complicated life cycle of parasite calls for an effective vaccine
development program. Careful study of cycle also indicates that the vaccine should target the pre-erythrocytic stage
of the parasite. An ideal malaria vaccine should elicit both Cell Mediated Immune (CMI) and Humoral Mediated
Immune (HMI) Response. Point worth noting here is, individual infected with malaria for the first time acquires a
degree of natural immunity i.e. the same individual will be less prone to a second time infection [9].
Various preventive measures are used for controlling transmission of malaria, these measures include: using
mosquito nets, dicholorodiphenyltrichloroethane (DDT) insecticide sprays, pyrethroid insecticide sprays, genetically
modified Metarhizum anisopliae sprays. Use of DDT is restricted as it has various human side-effects such as
reduced fertility, genital birth defects. Cases of mosquitoes being resistant to pyrethroid insecticide have been
reported. Genetically modified Metarhizum anisopliae sprays have given positive results; this modified fungus
inhibits the attachment of sporozoites to the salivary glands of vector mosquito. These sprays are effective for 3-6
months. Mosquito nets are effective for 3-5 years depending upon their model and condition of use. Various
preventive drugs are also currently available against malaria such as Mefloquine (initiates swelling of parasite’s food
vacuole), Doxycycline (inhibits protein synthesis in the parasite), Proguanil (inhibits synthesis of purines and
pyrimidines in the parasite). RTS,S (Main partner: GlaxoSmithKline) is a pre-erythrocytic malaria vaccine which
uses Circumsporozoite protein and Hepatitis B surface antigen as antigenic targets [8], mixed with an adjuvant. This
candidate vaccine went under Clinical Trials Phase III in African countries.
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Results showed that the vaccine administration reduced the number of clinical malaria cases in children (aged 5-17
months at first vaccination) by 36% and in infants (aged 6-12 weeks at first vaccination) by 26%. Both groups were
administered with ‘booster-dose’ of 18 months (Press release from Malaria Vaccine Initiative PATH, MVI PATH is
one of the partners in the development of the vaccine RTS,S). In July 2015, European Medicines Agency
recommended that the vaccine be licensed for use of young children in Africa. Similar recommendation from WHO
is still awaited. In addition to RTS,S, PfSPZ (Pf- Plasmodium falciparum, SPZ- Sporozoite) is a candidate malaria
vaccine. PfSPZ (developed by
Sanaria Inc.) consists of non-replication irradiated whole sporozoites. Higher dosage of this vaccine has shown some
promising results as 3 out of 15 candidates injected with PfSPZ were prone to infection by malaria parasite [7]
.WHO has set a target for developing malaria vaccine with 80% efficacy rate by 2025. At present there is no
preventive vaccine recommended by WHO.
SPECT and SPECT2 as target proteins:
The cell-traversal ability of sporozoites seems to play a major role in its survival. This ability prevents the
sporozoites from being phagocytosed by cells in blood stream, also this ability makes it possible for them to traverse
through Kupffer cells and infect hepatocytes. As stated above, SPECT and SPECT2 proteins are responsible for the
cell-traversal ability of sporozoites. Hence, these proteins can be used as potential preventive-drug targets in near
future. Blocking the cell-traversal ability of sporozoites will make them prone to phagocytosis and also block their
entry into liver through Kupffer cells. The fact that sporozoites traverse through Kupffer cells might not hold true in
some cases as liver sinusoidal barrier is not perfect and it contains few openings through which the sporozoites can
pass directly and infect hepatocytes [4]. SPECT2, as stated above, shows homology with pore-forming
proteins.Perforin-1 is a pore-forming protein found in granules of Cytotoxic T Lymphocytes and Natural Killer
Cells. Also, Complement Component 9, known to be the effecter molecule in cytolysis mediated by T-cells and
Natural Killer (NK) cells, is also a pore-forming protein. Hence, if SPECT2 is considered as a target for preventive
vaccine, immunological complexity may arise where in the vaccine can act on such self-molecules.
Fig5: (a): Normal route for the introduction of sporozoites into the liver by traversing Kuffer cells. (b): Openings in the liver sinusoidal barrier
through which some sporozoites pass for infecting hepatocytes (Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC314478/)
Discussion:
RTS,S can be seen as a potential vaccine, but the results obtained after the trails are not very satisfactory. There are
possibilities of developing a more effective vaccine in the near future. PfSPZ adopts the method of injecting
irradiated and metabolically active sporozoites for developing immunity against malaria; this approach has shown
some promising results. The limitation of PfSPZ vaccine is its feasibility i.e. large scale production and delivery to
areas which are in need of the vaccine. SPECT and SPECT2 can be used as target proteins for vaccine development,
but the immunological complexity that may arise due to which the vaccine can act on self-molecules should also be
taken into consideration.
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In addition to that, development of resistance to the vaccine by the parasite is a factor worth considering.
Development of resistance to vaccine by parasite can significantly reduce the efficacy of the vaccine. Plasmodium
falciparum has shown a high degree of evolutionary change, hence prone to drug-resistance. The process of
evolutionary change by the parasite is one of the key factors to consider for developing a potential preventive
vaccine. There are various drugs available for curing malaria, the most famous curing drug is Artemisinin-based
Combination Therapy (ACT), but recent findings have shown the parasite being resistant to this therapy. This
resistance is observed in countries like Cambodia, Loas, Myanmar, Thailand and Vietnam. ACT remains a popular
therapy for treating uncomplicated cases of malaria and is recommended by WHO. ACT consists of
Artemether/Lumefrantine fixed dosage .For fast action, various companion drugs belonging to different category can
be used, these include mefloquine, amodiaquine, sulfodoxine/pyrimethamine, piperaquine and
choloroproguanil/dapson .Majority of drugs are active against the erythrocytic stages of the parasite and these
include Chloroquine, Mefloquine etc. In addition to these several other curing drugs are available such as:
Qualaquin(Interferes with parasite’s ability to digest hemoglobin), Hydroxychloroquine(Alters DNA of the parasite,
increases pH of the parasite’s food vacuole, inhibits phospholipid metabolism), Atovaquone(acts of cytochrome
BC1 complex, mechanism of this action is not fully understood), Pyrimethamine(used in chloroquine resistant
parasites, acts on nucleic acid precursos), Atovaquone(Attacks cytochrome BC1 complex) etc (Drugbank.ca) . The
seat for a more effective preventive malaria vaccine is still vacant and can be filled up in near future. But this
candidate vaccine will need to be developed taking into consideration various immunological complexities and it
should target the pre-erythrocytic stage of the parasite and also should be feasible to produce and transport to areas
in need. It should be considered that the parasite undergoes evolutionary changes hence is prone to resistance.
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