CURRENT ADVANCEMENT IN PARASITE TREATMENT AND CONTROL1
INTRODUCTION Often said that we are living in ‘post-genomic era’.1) Vaccination2) DNA/ RNA technology - Antisense DNA and RNA3) Nanotechnology4) Quantum dots5) Remoting Sensing (RS) and GIS technology 2
VACCINES AGAINST PARASITIC DISEASES Drugs may provide a complete cure for an infection but reinfection is often an almost certainty. Wherever parasite exposure is regular occurrence, long lasting protection can only come from the development of protective immune response. The purpose of vaccination is to stimulate a protective immune response without the risks associated with a natural infection. 3
For parasite vaccination effectiveness, we need to understand the biology and life cycle of parasite and also how the immune response is mounted against it. For example: anti-sporozoite vaccine. - would block new infections with malaria - very useful for people who have never been exposed to the disease and are visiting an endemic region - reduce the chances of those already infected from acquiring more serious infection through repeated challenges. - however, it will not help to cure an existing infection 4
The nature of the host immune response to parasite challenge is a crucial factor in the development of an effective vaccine More complex organism, and much more difficult to determine suitable target for vaccine preparations 5
TYPES OF VACCINES1) Attenuated – live non-virulent organism2) Killed – Dead pathogen3) Sub-unit – Antigenic pathogen4) Toxoid – Inactivated toxin5) DNA – specific gene (s) 6
ATTENUATED VACCINES Utilise live organisms that are biologically the same as the ‘wild type’ pathogen but do not induce disease or only cause mild symptoms. Can be obtained 1) through the selection of non-pathogenic strains 2) through treatment of the wild type with mutagens or passage through laboratory animals or cell cultures. The injected organism will grow and multiply within the host thus exposing it to a variety of different life cycle stages. 7
Elicit an immune response similar to that of the pathogen but without its associated pathology. More likely to induce a T cell-mediated immune response than a killed vaccine and this is important for combating intracellular parasites. 8
Problem 1) the real concerns over whether attenuated pathogen might revert back to ‘wild type’ and revert disease. 2) Many organisms cannot be grown in culture 3) Those that can be cultured change their phenotype over successive generations so that they become less like the ‘wild type’ and genetically more dangerous. Example – a vaccine against Ancylostoma caninum was developed in the 1960s and entered commercial production in 1970s. Gave up 90% protection but was withdrawn because sometimes give rise to patent infections Also due to expensive production costs and short shelf life 9
Example of vaccines:1. An anti-Leishmania attenuated vaccine2. An anti-Theileria annulata vaccine to treat cattle 10
KILLED VACCINES Involve growing the pathogen in culture and then killing it before using it as a vaccine. Obviously overcomes some of the safety worries associated with live vaccines. If the pathogen produces toxin, this must be removed during vaccine preparation. This vaccine only limited to those parasites that can be grown under culture conditions 11
Depend on the biochemically characterizing the pathogen Then testing different component for their ability to induce an immune response Once candidate has been identified, it can purified from cultured parasites. Example – Vaccine against Plasmodium, Leishmania, Neospora caninum, Toxoplasma gondii, Cryptosporidium parvum. 12
SUB UNIT/ RECOMBINANT VACCINES Enable large amount of specific antigens to be produced without the problems of parasite culture. Particularly useful for protozoan life cycle stages that can normally only obtained in very small numbers and for helminth parasites. The antigen are isolated from the rest of the pathogen and therefore they are potentially much safer than ‘live’ or ‘killed’ vaccines. 13
For cestode, recombinant sub-unit vaccines have been developed but for various reasons they not yet enter commercial production. For example, there is an effective recombinant antigen vaccine that prevents the development of the cysticerci of Taenia ovis in sheep. This vaccines was develop to reduce the prevalence of cycticercosis in older lambs before there were sent to slaughter. 14
TOXOID VACCINES Toxoid or anti-toxin vaccines are used where the toxins produced a pathogen are the main virulence factor. The vaccine is prepared by isolating the toxin and then inactivating it, for example using treatment formaldehyde. Because the chemical mimics the toxin biochemically, but it is not actually active, it is called a ‘toxoid’. Example the diphtheria and tetanus toxoids in DPT vaccine. 15
Parasite normally release antigenic excretory/secretory products that have been explored as potential vaccine candidates. These are the complex mixtures that often contain cysteine proteases which play an important part in the nutrition and the pathology they cause. Example: Vaccines using cycteine proteases have been designed against protozoa Trypanosoma cruzi, trematode Fasciola hepatica, nematode Haemonchus contortus and 16 Ostertagia ostertagia.
DNA VACCINES Prepare by cloning a gene that codes for a specific antigen into bacterial plasmid or recombinant viral factor that is then injected into subject. Promoter sequences are also incorporated into the plasmid to boost the production of antigen. The immune response to DNA vaccination has been investigated using mice but not clear whether similar response are generated in other animals. 17
The DNA vaccine stimulating both cellular and humoral immune response. Cheaper to develop and than ‘live’ attenuated and subunit vaccines. Relatively more stable and can be stored at room temperature. Vaccines are formulated with a variety of substances that help to preserve the active ingredient and have immunostimulatory properties. 18
Quite difficult to transfer candidate vaccine from laboratory situation to the field. This is because DNA vaccines not generating as strong response in humans and domestic animals as they do in mice. However progress was being made in the development of DNA vaccines against protozoa Plasmodium and Leishmania, trematode Schistosoma japonicum, nematode Haemonchus contortus and arthropod 19 Boophilus microplus.
VACCINE ADMINISTRATION Normally are given as an injection that may be:- intravenous- intramuscular- intradermal Injections are seldom popular because they cause painful or systemic flu-like reactions Some can be swallowed e.g oral polio vaccine increasing on the possibility of delivering vaccines as nasal spray Oral vaccines and nasal spray are much more ‘patient friendly’ 20
New technique – using accelerated liquids or powder grains. The injection takes as little as 40 msec using a high pressure jet This cause a little damage to underlying tissues, reduces the risk of needle borne-contamination and, virtually pain- free. Needle-free injections often provide a greater antibody response than conventional injections 21
Example: anti-malaria vaccine. Gene-guns are needle free delivery systems used to deliver DNA or RNA attach to gold nanoparticles. The gold nanoparticles are accelerated to supersonic speed in a stream of helium gas and forced into subcutaneous skin. Using gene gun gave an equivalent response to intramuscular injections. 22
DNA/ RNA TECHNOLOGY One of the fundamental discoveries in recent years is that epigenetic mechanisms are responsible for many aspects of cell regulation. Epigenetic factors are the those mechanisms that regulate genetic expression without changing the DNA sequence . Epigenetic regulation is important in all organisms and has particular relevance for host parasite relationships because its governs:1) The host’s immune response2) The parasite’s life cycle3) Virulence4) Ability to overcome the host’s immune system5) Adapt to drug Potential target because may prove possible to selectively target unique epigenetic pathways in parasites without harming the host. 23
Epigenetic factors include1. DNA methylation2. histone modification3. regulatory RNA molecules. DNA methylation occurs through the addition of methyl groups to cytosine to produce 5-methylcytosine. Normally takes place at CpG sites (cytosine-phosphate- guanine). Extensive methylation of CpG sites within a gene sequence results in the gene being silenced. 24
Chromatin consists of DNA wrapped around the large structural protein histone. If the sequence of amino acids that comprise histone is modified, it alters the three dimension shape of the molecule This affects the expression of gene activity of the associated DNA. Histone modification can occur in several different ways e.g acethylation or methylation These have different effects on gene expression. 25
There are variety of single and double stranded RNA molecules and small non-coding micro RNA molecules that are involved in the regulation of gene expression at the level of translation such as RNA interference (RNAi). RNAi regulates gene activity and is also part of cell’s natural means of protection against virus. 26
Specific double-/ stranded RNA is cleaved by ribonuclease enzyme called ‘dicer’ to form small (short) interfering RNA (siRNA) consisting 20-25 nucleotides The siRNA is then assembled to form an ‘RNA-induced silencing complex’ (RISC) that includeds the antisense strand of the target mRNA and endonuclease enzyme. The silencing complex binds to the mRNA and then the endonuclease enzyme (‘Argonaute’) brings about its degradation. mRNA not translated and the protein is codes for it not formed. 27
ANTISENSE DNA AND RNA Within a cell, the first step in the production of a protein is when the gene coding for it in the cell’s DNA is transcribed into a sequence of messenger RNA (mRNA) oligonucleotides. The single-stranded mRNA molecule then moves to ribosomes where it is translated into a sequence of amino acids. The ‘mRNA’ is referred to as a ‘sense’ strand while its non-coding complementary strand is the ‘antisense strand’. For example if the ‘sense’ strand had the sequence 5’- AACGAAUUAC-3’, its antisense strand would be 3’- UUGCUUAAUG-5’ 29
If sense and antisense strands came into contact, bind together to form a non-functional duplex molecule. Consequently the sense strand would not be translated and the protein molecule is coded for would not be formed. 30
NANOTECHNOLOGY Nano materials are solid colloidal particles 1-100 nm in diameter. Can be manufactured from elements such as gold, silver and carbon, from compounds such as iron oxide as well as from organic polymers such as chitosan. For example, gold nanoparticles have been used as a carrier of hydrophobic drugs and by conjugating an antibody to the surface of the particles, they can used to target specific cells. 31
Raman reporters or ‘Raham tags’ are molecules that are excited when stimulated by specific wavelenghts. When Raman reporters are attached to gold nanoparticles, they can be visualised after administration using a technique called Surface Enhanced Raman Spectroscopy. Consequently, the location of parasite can be determined using gold nanoparticles bearing the appropriate antibodies and Raman reporters. Certain type of gold nanoparticles convert absorbed light into near infra-red radiation and have potential for laser photoablation The basic of this approach is that the nanoparticles are targeted to specific cell types, then a laser beam is directed onto them. 32
This approach was used to kill tachyzoites of Toxoplasma gondii. Alternatively, a laser can deliver a specific wavelenghts that stimulates gold nanoparticles that have reached their target to release bound molecules, such as drugs. This ensures that the target cells experience a sudden therapeutic dose of the drug. However most potential applications are still at the experimental stage. 33
Diagram of the silica-encapsulated surface- Diagram of the SERS-based sandwich immunoassay.enhanced Raman spectroscopy (SERS) tag. Antibody-conjugated SERS tags serve as labels for the biological analyte and are captured by superparamagnetic beads which are also functionalized with antibodies specific to the analyte. A Raman laser 34 strikes the SERS tags, generating a unique spectrum that easily identifies analytes.
Some issues:1. Difficult to handle in both liquid and dry formation2. Substances are safe in particular size range may become poisonous or carcinogenic at another size3. Silver is toxic metal, and silver nanoparticles could potentially affect microbial and invertebrate communities4. Gold nanoparticles could accumulate through food chains 35
QUANTUM DOTS Quantum dots are nanocrystal semiconductor that are of great interest for their electronic and optical characteristics In biology, they have many potential uses for bioimaging because they can be attached to molecules or cells Thereby their movements to be tracked in real time. For example, quantum dots have been used to monitor the invasion of erythrocytes by Plasmodium falciparum and as tools to identify Plasmodium-infected erythrocytes using flow cytometry. Can be delivered gene silencing RNA (riRNA). 36
Also prove useful in the treatment of parasitic diseases. However, more information is required on their toxicological properties. 37
Quantum dot (QD) labelling on P. falciparum-infected erythrocytes showing that only late-stage iRBCs arelabelled.Early-stage (ring) iRBCs (A) are not labelled by the QD, while the late-stage trophozoite (B) and segmentedschizont (C) iRBCs are both labelled.The parasites were stained with Hoechst 33324 (in red, first column from the left) and PCQD (in green, 38second column).Phase contrast images (third column) and merged images (fourth column) are also shown. Bars, 5 mm.
REMOTING SENSING (RS) AND GIS TECHNOLOGY Remote sensing (RS) satellite data and Geographic Information Systems (GIS) technology useful for monitoring the epidemiology of parasites forecasting the risk of disease outbreaks 39
REMOTE SENSING (RS) RS is a means of monitoring the environment without actually making physical contact with it Commonly achieve through satellite technology using combination of passive and active monitoring devices. Passive detectors emit particular wavelengths that are emitted or reflected from land beneath. Active detectors emit particularly wavelengths and measure the time taken for them to return RS can be useful to monitor: temperature ground cover forestation etc A variety of RS satellite datasets are available including 40 LANDSAT, MODIS, NDVI, and SRTM DEM.
GEOGRAPHIC INFORMATION SYSTEMS (GIS) GIS are means of capturing, storing, updating, retrieving, analyzing, and displaying any form of geographically- referenced digital information. It is not a single entity but a collection of computer hardware, software, and geographical data. Very useful for parasite surveillance and simulating the consequences of particular intervention strategies or changes in the environment. 41
To map simultaneously one or more of the following on either a regional, national, or global scale: the occurrence of the parasite the disease it causes its host vector/ intermediate host co-infections environmental variables 42
For example, disease maps are quick and simple means of visualising spatial and temporal ‘hot spots’ of - where disease is clustering - the linkages between parasite distribution and environmental variables - the effectiveness of the control measures Can identify those environmental variables that promote the breeding of vectors and the intermediate hosts and therefore where problems are likely to arise. GIS software e.g DIVAGIS already used to identify areas suitable for colonisation by the snail intermediate 43 hosts of Fasciola hepatica
Fasciola gigantica potential distribution and abundance in the IGADD sub-region based on a GIS constructed from FAO CVIEW agroecologic zone mapfiles, 30-year-average monthly climate databases, a modification of the LSU 44climate based parasite forecast system, a base life cycle developmenttemperature of 16°C and known irrigation zones.