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Investigate the vaccine for malaria

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Following this protocol, youngsters take part in the research to develop a vaccine against malaria, which, in combination with the current measures, could contribute significantly to a better control …

Following this protocol, youngsters take part in the research to develop a vaccine against malaria, which, in combination with the current measures, could contribute significantly to a better control of this important parasite-caused disease. Students will test different vaccine candidates using a technique called ELISA and they will decide which is the most effective. The experiment protocol is an opportunity for science centres, museums and schools to replicate a real experiment done in a real lab doing research on the malaria vaccine.

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  • 1. Investigate thevaccine for malariaExperimental workshopPROTOCOL
  • 2. IntroductionMalaria is considered to be the most impor- of different measures that include the use oftant parasite-caused disease in the world, and mosquito nets impregnated with insecticide,is responsible for the death of approximately spray insecticides, preventive treatments, the800,000 people every year, particularly children implementation of educational programmesunder 5 and pregnant women. According to the and environmental intervention, among others.World Health Organization (WHO), some threebillion persons are at risk of contracting the The scientific community is working hard to de-infection, while in the year 2010 there were velop a vaccine which, in combination with the225 million cases of malaria in the world, of current measures, could contribute significantlywhich 90% were in Africa. to a better control of malaria. There is already a vaccine in the clinical study phase, which wouldCurrently, malaria is endemic in more than be effective in 50% of cases.100 countries located in sub-Saharan Africa andregions of South Asia, Latin America and Oceania.The most recent reports indicate that half theworlds population lives in areas where there isa risk of contracting the disease, and where inaddition to its consequences for the health of thepopulation, malaria contributes to further weak-ening the area’s economic situation. In this workshop you will do researchTo eradicate this disease in areas with a high with different vaccine candidates torisk of transmission, various interventions are decide which is the most effective.being conducted that require a combination Countries or areas where the transmission of malaria occurs. Countries or areas with limited risk of transmission of malariaSource: World Health Organization (WHO): 2009 data 2
  • 3. How is malaria transmitted? Why is a vaccine against malaria necessary?Malaria is an infectious disease which is caughtfrom the bite of the Anopheles mosquito, whichtransmits Plasmodium parasites, thereby act- Historically, vaccines have been one of the mosting as a vector. In the human body, the parasites efficient measures for the prevention of dis-multiply in the liver and then infect the red eases and saving lives, especially in the case ofblood cells. Prominent symptoms of malaria are infectious diseases. Obtaining a vaccine that isfever, headache and vomiting, and they appear partially effective could save hundreds of thou-from 10 to 15 days after the mosquito bite. sands of lives. Obtaining a vaccine would be a great step for- ward that could be added to the current arsenal of measures used for the prevention of malaria, such as insecticide-impregnated mosquito nets and the prompt and appropriate treatment of persons diagnosed with malaria.What is being done to Since its short-term effectiveness would becontrol malaria? partial, it would be a substitute for theseBasic interventions to control malaria are di- measures, but rather complement them. Thevided into several groups: two together would represent a comprehensive response to the prevention of malaria.1 Strategies directed against the mosquito, or vector, such as spraying enclosed spaces with insecticides.2 Strategies to avoid contact between the vector and host, such as the use of mosquito nets impregnated with insecticide.3 Strategies directed against the parasite. One of these strategies consists of treatment with combinations of medicines based on amolecule called artemisinin, which is rapidand effective. A vaccine would also be a controlstrategy directed against the parasite which,in combination with other strategies, couldcontribute significantly to the eradication ofmalaria. 3
  • 4. What are we doing at ISGLOBAL, the Barcelona Institute for Global Health?The Barcelona Institute for Global Health 1. The study of the molecular basis of the(ISGlobal) is a not-for-profit organisation whose disease as well as the variety of immuneobjective is to improve the health and devel- responses.opment of the most vulnerable populationsthrough the creation, management, transmis- 2. The development of new medicines and thesion and application of knowledge. Its vision is assessment of their safety and efficacy.of a world in which we can all enjoy good health,and it receives support from the “La Caixa” 3. The assessment of the epidemiologicalFoundation, among others. characteristics of malaria in different set- tings and of the social and cultural factorsOne of the essential pillars of ISGlobal is re- that surround it.search that concentrates on health problemsthat affect the most vulnerable populations, 4. The analysis of the effectiveness of variouswhich is conducted at its Barcelona Centre for prevention tools and the cost-effectivenessInternational Health Research (CRESIB). The ratio of these interventions.research on malaria which is done at CRESIBfocuses on: As part of this role (4) CRESIB carries out clini- cal studies of the safety and efficacy of vaccines. It is currently participating in the development of the RTS,S vaccine against malaria, which is showing itself to be effective in more than 50% of infected children. At the same time, CRESIB investigators are conducting research to identify new vaccine candidates. 4
  • 5. Workshop objectives 1In this workshop we invite you to analyse vari-ous candidates for the vaccine against malariaalong with those that CRESIB is researchingto identify which is the best candidate. Thecandidates for the CRESIB vaccine have beenobtained from parasite proteins that have beenpurified beforehand.To analyse them, we have a number of bloodsamples from people living in malaria-affectedareas who have had the disease on various oc-casions and who are now immunised.To confirm that our vaccine candidates are ef-fective, we must show that immunised peoplehave developed a response to these candidates.If activation of the immune response againstthe candidate proteins is found in these people,it will mean that they could be good vaccine 2candidates because they could also trigger theresponse necessary to protect people againstfuture infection. 3 5
  • 6. The objective of this workshop is to familiarise that they are capable of activating a good im-you with one of the techniques most often used mune response, and that they could thereforein biomedical laboratories, the ELISA (enzyme- be good candidates.linked immunosorbent assay) technique. Using this technique, we will specificallyThis is an analysis that detects whether anti- discover which of the antigens available in ourbodies are present in blood samples. The pres- laboratory is a good candidate for a vaccineence of candidate-specific antibodies indicates against malaria. Basic principles of the ELISA techniqueThe basic principle behind this technique is To carry out this identification, the antibodiesbased on the interaction of the vaccine candi- used have a molecule attached to them calleddate, or antigen (1), with the antibody (2). A spe- an enzyme (3), which has the ability to reactcific antibody will bind to a specific antigen to with a substance called a substrate (4), whichcreate an exclusive antibody-antigen complex. we will add to produce a colour.The ELISA technique allows us to identify Therefore, if the sample contains the antibodywhether antibodies were present, and whether, we wish to detect, it will bind to what we haveas a result, antibody-antigen complexes were added, which is bound to an enzyme that willformed when they came into contact with blood in turn cause the substrate to change colour,samples containing the vaccine candidates. thereby telling us that the results are positive. Substrate Enzyme Antibody Antigen1. Antigen: any foreign substance that binds specifically to the specified antibodies or lymphocytes and activates an immune response. In general antigens have a high molecular weight; normally they are proteins or polysaccharides.2. Antibody: proteins (immunoglobulins, Ig) from serum that are formed as a response to the invasion of the body by foreign molecules, whether due to natural exposure or an antigen introduced by vaccine immunization. They are in the form of a Y, and are made up of four polypeptide chains that are kept connected by interchain disulfide bonds. Antibodies have a constant region and a variable region.3. Enzyme: a protein that facilitates specific reactions of the metabolism.4. Substrate: a solution that contains a compound acted upon by an enzyme. 6
  • 7. Organisation of the workshopTo analyse which vaccine candidate or antigen against malaria have antibodies against them.is the most effective, we will test whether the To do so we will divide the experiment into threeblood samples from persons who are immunized main stages.1. BINDING OF THE CANDIDATE PROTEINS (OR ANTIGENS) TO THE SURFACE OF THE WELLSThe first step will be to fix the candidate proteins that are being studied to a solid support2. FORMATION OF ANTIBODY-ANTIGEN COMPLEXESWe then add the blood sample, specifically blood serum (blood samples with the cells and clottingfactors removed), and an antibody marked with an enzyme that we will call the secondary antibody.In the blood samples in which the antibody being studied was present, antibody-antigen complexeswill be formed, which will in turn bind to the antibody marked with the enzyme. Serum Secondary from the antibody marked patients with an enzyme3. READING THE REACTIONFinally, we will add the enzyme substrate which, if the antibody antigen complex is present, willchange colour. In this way we will learn if the blood samples contain the antibody being studied, andin what quantity. Substrate from the enzyme Results and conclusionsOnce the results are obtained, we can decide intense immune response and may therefore bewhich vaccine candidate activates the most the best candidate. 7
  • 8. Equipment and materialrequiredLaboratory instruments and utensils Magnetic stirrer (1) (for Stir bar and Micropipettes preparing PBS-Tween) “stir bar retriever” of 20 to 200 µl (2) Test tube, Glass bottle, Timer Funnel 100 ml 250 mlConsumables Strips with 12 wells Graduated plastic Pasteur Tips for the micropipettes for ELISA, and supports pipettes Absorbent paper Permanent marker Gloves, goggles and apron1. If you do not have a magnetic stirrer, the PBS liquid may be purchased2. If you do not have micropipettes, you may use small-volume Pasteur pipettes 8
  • 9. Reagents and samples PBS buffer solution (3) Tween-20 detergent (4) Distilled water Secondary antibody A B C+ C- Vaccine candidates Positive controls Secondary antibody (antigens) and negative controls (5) with enzyme (peroxidase) Substrate 1 2 3 4 Substrate, or colouring Serum samples from four people residing in areas in which solution malaria is endemic, and who are immune to the disease3. Helps to maintain the solution pH thanks to sodium and potassium phosphates4. Helps prevent binding of nonspecific antibodies5. C+: contains a mixture of serum from people residing in areas where malaria is endemic and whoare immune to the disease. C-: mixture of serum from people who have never been exposed to malaria 9
  • 10. ProceduresTo identify the presence or absence of specific blood serum) of different patients residing inantibodies to the vaccine candidate antigens areas where malaria is endemic to see if wewe have in the laboratory, we will use these find specific antibodies against our antigens inantigens to challenge the blood (actually the their serum. 1 Binding of the antigens to the surface of the wellsThe micro-well strips are covered with the vac- The binding of these antigens to the surface ofcine candidates (antigens) that we wish to test the wells is easily brought about since they areto see if they would be good candidates for a made of a treated plastic that has a great abilityvaccine against malaria. to bind proteins.PROTOCOL FOR BINDING ANTIGENS TO THE SURFACE OF THE WELLS1 Note below what you will place in each well (controls, blood samples and the names of the antigens that you will analyse).2 Permanently mark the wells where you place each sample. 10
  • 11. 3 Prepare a washing solution (PBS-Tween 0.05%).A B Measure 200 ml of distilled water using the test tube, adjusting the volume with the Place the stir bar in the bottle, and dilute Pasteur pipette. Use the funnel to add 200 ml one PBS tablet with water using the of distilled water to the bottle. magnetic stirrer.C D When the tablet has dissolved, extract the stir bar and add 100 µl of Tween-20 using a Remove it carefully inverting the bottle plastic Pasteur pipette. several times.Note: The washing solution contains PBS (phosphate buffer saline), which allows the antibodies to be kept in a stable envi-ronment that helps to preserve their structure. Tween-20 is a detergent that helps to eliminate the proteins that have beenable to bind in a nonspecific manner, and also adheres to the portions of the well that are not covered by the antigen, therebyreducing background noise. 11
  • 12. 4 5 Add the two test antigens to the respective wells using the micropipette (50 µl per well). It is important that you use a clean tip to dispense the antigens to avoid contamination. Allow it to incubate for 5 minutes at room temperature.6 7 Wash it to eliminate the excess antigen Eliminate the remainder of the antigen not bound to the strip. To do this, fill the by inverting the strip over the absorbent wells with the washing solution using a paper. plastic Pasteur pipette.8 9 Discard the washing solution by inverting the strip over absorbent paper. Repeat steps 7 and 8. 12
  • 13. 2 Formation of antibody-antigen complexesIn this step we will first condition the serum of called peroxidase. As each primary antibody canthe patients to determine whether they contain bind with more than one secondary antibody,antibodies against the vaccine candidate. The the amount of colour obtained in step three willantibodies that we wish to test will be called be enhanced. The sensitivity of the technique isprimary antibodies. We will then add a second- thereby increased.ary antibody which is marked with an enzyme Serum Secondary from the antibody marked patients with enzymePROTOCOL FOR FORMATION OF ANTIBODY-ANTIGEN COMPLEXES1 2 Add the positive and negative controls Add the different serum samples to the respective wells using the from 4 residents of the areas micropipette (50 µl per well). where malaria is endemic to the The positive control (C+) contains a corresponding wells using the mixture of serum from people residing micropipette (50 µl per well). in areas where malaria is endemic and who are immune to the disease. The negative control (C-) contains a mixture of serum from people who have never been exposed to malaria. 13
  • 14. 3 4 Allow it to incubate for 5 minutes at Eliminate the excess antigen by inverting room temperature. the strip over absorbent paper.5 6 Wash all the wells to eliminate the antibodies that have not reacted with the antigens and which are therefore not specific. Fill the wells with the washing Discard the washing solution by inverting solution using a plastic Pasteur pipette. the strip over absorbent paper.7 8 Using the micropipette, add the secondary antibody which is bound to an Repeat the steps five or six more times. enzyme to all the wells (50 µl per well). 14
  • 15. 9 10 Eliminate the excess secondary Allow it to incubate for 5 minutes at antibodies by inverting the strip over room temperature. absorbent paper.11 12 Wash the wells by filling them with the Discard the washing solution by inverting washing solution using a plastic Pasteur the strip over absorbent paper. pipette.13 Repeat the two previous steps three more times. 15
  • 16. 3 Reading the reactionAfter washing to eliminate all the markedmolecules that have not been fixed in the formof antibody-antigen complexes, the enzymesubstrate solution is added to facilitate thechange of colour. Substrate from the enzymePROTOCOL FOR READING THE REACTION1 2 Add the enzyme substrate to all the Allow it to incubate for 5 minutes. During wells using the micropipette this time the substrate will bind to the (50 µl per well). enzyme at room temperature and the colour will begin to appear. 16
  • 17. 3 Assemble the results in the form of bar graphs.ANTIGEN 1Maximum IntensityMinimum Samples C+ C- M1 M2 M3 M4ANTIGEN 2Maximum IntensityMinimum Samples C+ C- M1 M2 M3 M4 17
  • 18. Results and conclusions Interpret and record the results 1. Which of the antigens that you have tested do you believe is the best vaccine candidate? Do you believe that the antigens that you have tested are good vaccine candidates? Why? 2. When is a reaction positive and when is it negative? Why? 3. Why do you think the controls are used? 18
  • 19. 4. Which part of the primary antibody is recognised by the secondary antibody? The constantregion or the variable region? Give reasons for your answer.5. What would happen if we did not do the washing before adding the colouring substrate?6. Could we use blood from your classmates to determine whether our laboratory antigensare good candidates for a vaccine against malaria? Give reasons for your answer.7. Do you believe that this experiment has shown that the selected candidate stimulates theimmune response? Would you have to do some other type of experiment to assess whether itis also capable of activating some other type of response? 19
  • 20. Annex I OBLIGATORY USE OBLIGATORY USE OBLIGATORY USE OF GOGGLES OF APRON OF GLOVESSafety precautionsBE INFORMED or corrosive products. Do not place reagentFind out where the safety equipment of the containers near a flame. Do not heat inflam-laboratory or the place where you are experi- mable liquids. Carry bottles holding them frommenting is located (fire extinguishers, show- beneath, never by the neck.ers or baths, exits, etc.). Read the instructionscarefully before doing an experiment. Do not WASTE DISPOSALforget to read the safety labelling for reagents Deposit broken glass, reagents that are toxic,and equipment. noxious or harmful to the environment and biological waste in special and appropriately la-USE PROPER CLOTHING belled receptacles. Never dispose of solid wasteGloves, apron and goggles. using the sink.GENERAL STANDARDS In case of accident, advise the instructor im-Smoking, eating or drinking in the laboratory or mediately. Remember: If you have a question,area where you are experimenting is prohibited. ask the trainer.Wash your hands before leaving the labora-tory. Work in a neat and orderly fashion without SPECIFIC PRECAUTIONShurry. If any product should spill, clean it up FOR THIS WORKSHOPimmediately. Always leave materials clean and During this practice session you must followorderly. Never use equipment or apparatuses the usual precautions for handling of chemi-without perfectly understanding how they work. cal products. Below are listed only those that present the following degrees of hazard:  HANDLING OF GLASSProtect your hands when handling materials • PBS:made of glass. Do not use cracked glass items. toxic when ingested, inhaled or in contact with the skin.      CHEMICAL PRODUCTSDo not use unlabelled containers of reagents. • Tween 20:Do not sniff, inhale, taste or touch chemical toxic when ingested, inhaled or in contact withproducts. Never pipette by mouth. Wear gloves the skin. Irritant.and wash your hands frequently if you use toxicAnnex IIReagent referencesNAME: REFERENCES COMMERCIAL MANUFACTURERPBS P4417-50TAB SigmaTween-20 P1379-100ML SigmaCHK IGY, bagged (= ANTIGEN) 1662406EDU BioRadRB ANTI-CHK, bagged (= PLASMA) 1662407EDU BioRadGAR-HRP, bagged (= SECONDARY ANTIBODY) 1662408EDU BioRadCOLOURING SUBSTRATE 1662402EDU BioRad 20
  • 21. Learn more at Xplore Health!Researchers who have contributed content: Laura Puyol, investigator for CRESIB, ISGlobal. DEVELOPED BY This work has been included with an Attribution-NonCommercial-NoDerivs 3.0 Unported Creative Commons license. to see a copy of the license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/