investigate the vaccine for malaria

Investigate the vaccine for malaria

Experimental workshopPROTOCOL

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IntroductionMalaria is considered to be the most impor-tant parasite-caused disease in the world, and is responsible for the death of approximately 800,000 people every year, particularly children under 5 and pregnant women. According to the World Health Organization (WHO), some three billion persons are at risk of contracting the infection, while in the year 2010 there were 225 million cases of malaria in the world, of which 90% were in Africa.

Currently, malaria is endemic in more than 100 countries located in sub-Saharan Africa and regions of South Asia, Latin America and Oceania.The most recent reports indicate that half the world's population lives in areas where there is a risk of contracting the disease, and where in addition to its consequences for the health of the population, malaria contributes to further weak-ening the area’s economic situation.

To eradicate this disease in areas with a high risk of transmission, various interventions are being conducted that require a combination

In this workshop you will do research with different vaccine candidates to decide which is the most effective.

of different measures that include the use of mosquito nets impregnated with insecticide, spray insecticides, preventive treatments, the implementation of educational programmes and environmental intervention, among others.

The scientific community is working hard to de-velop a vaccine which, in combination with the current measures, could contribute significantly to a better control of malaria. There is already a vaccine in the clinical study phase, which would be effective in 50% of cases.

Countries or areas with limited risk of transmission of malariaCountries or areas where the transmission of malaria occurs.

Source: World Health Organization (WHO): 2009 data

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How is malaria transmitted?

Malaria is an infectious disease which is caught from the bite of the Anopheles mosquito, which transmits Plasmodium parasites, thereby act-ing as a vector. In the human body, the parasites multiply in the liver and then infect the red blood cells. Prominent symptoms of malaria are fever, headache and vomiting, and they appear from 10 to 15 days after the mosquito bite.

Historically, vaccines have been one of the most effi cient measures for the prevention of dis-eases and saving lives, especially in the case of infectious diseases. Obtaining a vaccine that is partially effective could save hundreds of thou-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.

Since its short-term effectiveness would be partial, it would be a substitute for these measures, but rather complement them. The two together would represent a comprehensive response to the prevention of malaria.

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Strategies directed against the mosquito, or vector, such as spraying enclosed spaces with insecticides.

What is being done to control malaria?Basic interventions to control malaria are di-vided into several groups:

Strategies to avoid contact between the vector and host, such as the use of mosquito nets impregnated with insecticide.

Strategies directed against the parasite. One of these strategies consists of treatment with combinations of medicines based on a

molecule called artemisinin, which is rapid and effective. A vaccine would also be a control strategy directed against the parasite which, in combination with other strategies, could contribute signifi cantly to the eradication of malaria.

Why is a vaccine against malaria necessary?

1 Strategies directed against the mosquito, or vector, such as spraying enclosed spaces with

vided into several groups:

2 Strategies to avoid contact between the vector and host, such as the use of mosquito netswith insecticide.

3 Strategies directed against the parasite. One of these strategies consists of treatment with combinations of medicines

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The Barcelona Institute for Global Health (ISGlobal) is a not-for-profi t organisation whose objective is to improve the health and devel-opment of the most vulnerable populations through the creation, management, transmis-sion and application of knowledge. Its vision is of a world in which we can all enjoy good health, and it receives support from the “La Caixa” Foundation, among others. One of the essential pillars of ISGlobal is re-search that concentrates on health problems that affect the most vulnerable populations, which is conducted at its Barcelona Centre for International Health Research (CRESIB). The research on malaria which is done at CRESIB focuses on:

1. The study of the molecular basis of the disease as well as the variety of immune responses.

2. The development of new medicines and the assessment of their safety and effi cacy.

3. The assessment of the epidemiological characteristics of malaria in different set-tings and of the social and cultural factors that surround it.

4. The analysis of the effectiveness of various prevention tools and the cost-effectiveness ratio of these interventions.

As part of this role (4) CRESIB carries out clini-cal studies of the safety and effi cacy 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.

What are we doing at ISGLOBAL, the Barcelona Institute for Global Health?

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Workshop objectivesIn this workshop we invite you to analyse vari-ous candidates for the vaccine against malaria along with those that CRESIB is researching to identify which is the best candidate. The candidates for the CRESIB vaccine have been obtained from parasite proteins that have been purifi ed beforehand.

To analyse them, we have a number of blood samples from people living in malaria-affected areas who have had the disease on various oc-casions and who are now immunised.

To confi rm that our vaccine candidates are ef-fective, we must show that immunised people have developed a response to these candidates. If activation of the immune response against the candidate proteins is found in these people, it will mean that they could be good vaccine candidates because they could also trigger the response necessary to protect people against future infection.

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The basic principle behind this technique is based on the interaction of the vaccine candi-date, or antigen (1), with the antibody (2). A spe-cifi c antibody will bind to a specifi c antigen to create an exclusive antibody-antigen complex.

The ELISA technique allows us to identify whether antibodies were present, and whether, as a result, antibody-antigen complexes were formed when they came into contact with blood samples containing the vaccine candidates.

To carry out this identifi cation, the antibodies used have a molecule attached to them called an enzyme (3), which has the ability to react with a substance called a substrate (4), which we will add to produce a colour.

Therefore, if the sample contains the antibody we wish to detect, it will bind to what we have added, which is bound to an enzyme that will in turn cause the substrate to change colour, thereby telling us that the results are positive.

1. Antigen: any foreign substance that binds specifi cally to the specifi ed 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 disulfi de bonds. Antibodies have a constant region and a variable region.

3. Enzyme: a protein that facilitates specifi c reactions of the metabolism.

4. Substrate: a solution that contains a compound acted upon by an enzyme.

Basic principles of the ELISA technique

The objective of this workshop is to familiarise you with one of the techniques most often used in biomedical laboratories, the ELISA (enzyme-linked immunosorbent assay) technique.

This is an analysis that detects whether anti-bodies are present in blood samples. The pres-ence of candidate-specifi c antibodies indicates

that they are capable of activating a good im-mune response, and that they could therefore be good candidates.

Using this technique, we will specifi cally discover which of the antigens available in our laboratory is a good candidate for a vaccine against malaria.

AntibodyEnzyme

Substrate

Antigen

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To analyse which vaccine candidate or antigen is the most effective, we will test whether the blood samples from persons who are immunized

1. BINDING OF THE CANDIDATE PROTEINS (OR ANTIGENS) TO THE SURFACE OF THE WELLSThe fi rst step will be to fi x the candidate proteins that are being studied to a solid support

2. FORMATION OF ANTIBODY-ANTIGEN COMPLEXESWe then add the blood sample, specifi cally blood serum (blood samples with the cells and clotting factors 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 complexes will be formed, which will in turn bind to the antibody marked with the enzyme.

3. READING THE REACTIONFinally, we will add the enzyme substrate which, if the antibody antigen complex is present, will change colour. In this way we will learn if the blood samples contain the antibody being studied, and in what quantity.

Organisation of the workshopagainst malaria have antibodies against them.To do so we will divide the experiment into three main stages.

Serum from the patients

Substrate from the enzyme

Secondary antibody marked with an enzyme

Once the results are obtained, we can decide which vaccine candidate activates the most

intense immune response and may therefore be the best candidate.

Results and conclusions

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Laboratory instruments and utensils

Consumables

1. 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

Equipment and material required

Magnetic stirrer (1) (for preparing PBS-Tween)

Timer

Graduated plastic Pasteur pipettes

Permanent marker

Micropipettes of 20 to 200 µl (2)

Strips with 12 wells for ELISA, and supports

Absorbent paper

Tips for the micropipettes

Gloves, goggles and apron

Stir bar and “stir bar retriever”

FunnelTest tube, 100 ml

Glass bottle, 250 ml

Laboratory instruments and utensils

Magnetic stirrer (1) (for “stir bar retriever”

Test tube,

of 20 to 200

Glass bottle, Funnel

Strips with 12 wells for ELISA, and supportsfor ELISA, and supports

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Reagents and samples

PBS buffer solution (3)

Substrate, or colouring solution

Tween-20 detergent (4) Distilled water

Serum samples from four people residing in areas in which malaria is endemic, and who are immune to the disease

Vaccine candidates (antigens)

Positive controls and negative controls (5)

Secondary antibody with enzyme (peroxidase)

Positive controls Positive controls

Substrate, or colouring Serum samples from four people residing in areas in which Serum samples from four people residing in areas in which Serum samples from four people residing in areas in which Serum samples from four people residing in areas in which Serum samples from four people residing in areas in which Serum samples from four people residing in areas in which Serum samples from four people residing in areas in which

A

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C+ C-

Seco

ndar

y an

tibo

dy

Subs

trat

e

B

3. Helps to maintain the solution pH thanks to sodium and potassium phosphates 4. Helps prevent binding of nonspecifi c antibodies5. C+: contains a mixture of serum from people residing in areas where malaria is endemic and who are immune to the disease. C-: mixture of serum from people who have never been exposed to malaria

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To identify the presence or absence of specifi c antibodies to the vaccine candidate antigens we have in the laboratory, we will use these antigens to challenge the blood (actually the

blood serum) of different patients residing in areas where malaria is endemic to see if we fi nd specifi c antibodies against our antigens in their serum.

The micro-well strips are covered with the vac-cine candidates (antigens) that we wish to test to see if they would be good candidates for a vaccine against malaria.

The binding of these antigens to the surface of the wells is easily brought about since they are made of a treated plastic that has a great ability to bind proteins.

Procedures

1 Binding of the antigens to the surface of the wells

PROTOCOL FOR BINDING ANTIGENS TO THE SURFACE OF THE WELLS

Note below what you will place in each well (controls, blood samples and the names of the antigens that you will analyse).

Permanently mark the wellswhere you place each sample.

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The micro-well strips are covered with the vac-cine candidates (antigens) that we wish to test to see if they would be good candidates for a vaccine against malaria.

The binding of these antigens to the surface of the wells is easily brought about since they are made of a treated plastic that has a great ability to bind proteins.

PROTOCOL FOR BINDING ANTIGENS TO THE SURFACE OF THE WELLS

Note below what you will place in each well (controls, blood samples and the names of the 1

antigens that you will analyse).

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Prepare a washing solution (PBS-Tween 0.05%).

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 been able to bind in a nonspecifi c manner, and also adheres to the portions of the well that are not covered by the antigen, thereby reducing background noise.

Measure 200 ml of distilled water using the test tube, adjusting the volume with the Pasteur pipette. Use the funnel to add 200 ml of distilled water to the bottle.

When the tablet has dissolved, extract the stir bar and add 100 µl of Tween-20 using a plastic Pasteur pipette.

Place the stir bar in the bottle, and dilute one PBS tablet with water using the magnetic stirrer.

Remove it carefully inverting the bottle several times.

A

C

B

D

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Measure 200 ml of distilled water using the

A

Measure 200 ml of distilled water using the Measure 200 ml of distilled water using the

Prepare a washing solution (PBS-Tween 0.05%).

Place the stir bar in the bottle, and dilute

B

When the tablet has dissolved, extract the When the tablet has dissolved, extract the stir bar and add 100 µl of Tween-20 using a stir bar and add 100 µl of Tween-20 using a

C

When the tablet has dissolved, extract the stir bar and add 100 µl of Tween-20 using a stir bar and add 100 µl of Tween-20 using a Remove it carefully inverting the bottle

several times.

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Wash it to eliminate the excess antigen not bound to the strip. To do this, fi ll the wells with the washing solution using a plastic Pasteur pipette.

Allow it to incubate for 5 minutes at room temperature.

Discard the washing solution by inverting the strip over absorbent paper.

Eliminate the remainder of the antigen by inverting the strip over the absorbent paper.

Repeat steps 7 and 8.

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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.

4Add 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.

Eliminate the remainder of the antigen by inverting the strip over the absorbent


Wash it to eliminate the excess antigen

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Repeat steps 7 and 8.Discard the washing solution by inverting

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In this step we will fi rst condition the serum of the patients to determine whether they contain antibodies against the vaccine candidate. The antibodies that we wish to test will be called primary antibodies. We will then add a second-ary antibody which is marked with an enzyme

called peroxidase. As each primary antibody can bind with more than one secondary antibody, the amount of colour obtained in step three will be enhanced. The sensitivity of the technique is thereby increased.

2 Formation of antibody-antigen complexes

PROTOCOL FOR FORMATION OF ANTIBODY-ANTIGEN COMPLEXES

Add the positive and negative controls to the respective wells using the micropipette (50 µl per well).The positive control (C+) contains a mixture of serum from people residing 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.

Add the different serum samples from 4 residents of the areas where malaria is endemic to the corresponding wells using the micropipette (50 µl per well).

1 2


1


2

ary antibody which is marked with an enzyme

Serum from the patients

Secondary antibody marked

with enzyme

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Eliminate the excess antigen by inverting the strip over absorbent paper.

Wash all the wells to eliminate the antibodies that have not reacted with the antigens and which are therefore not specifi c. Fill the wells with the washing solution using a plastic Pasteur pipette.


Repeat the steps fi ve or six more times.

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Allow it to incubate for 5 minutes at Eliminate the excess antigen by inverting

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Wash all the wells to eliminate the

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Using the micropipette, add the secondary antibody which is bound to an enzyme to all the wells (50 µl per well).

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Eliminate the excess secondary antibodies by inverting the strip over absorbent paper.

Wash the wells by fi lling them with the washing solution using a plastic Pasteur pipette.


Repeat the two previous steps three more times.

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Eliminate the excess secondary antibodies by inverting the strip over

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washing solution using a plastic Pasteur Discard the washing solution by inverting the strip over absorbent paper.

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After washing to eliminate all the marked molecules that have not been fi xed in the form of antibody-antigen complexes, the enzymesubstrate solution is added to facilitate the change of colour.

3 Reading the reaction

PROTOCOL FOR READING THE REACTION

Add the enzyme substrate to all the wells using the micropipette (50 µl per well).

Allow it to incubate for 5 minutes. During this time the substrate will bind to the enzyme at room temperature and the colour will begin to appear.

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Add the enzyme substrate to all the

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Allow it to incubate for 5 minutes. During

Substrate from the enzyme

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Assemble the results in the form of bar graphs.In

tens

ityIn

tens

ity

Samples

Samples

Maximum

Maximum

Minimum

Minimum

C+

C+

C-

C-

M1

M1

M2

M2

M3

M3

M4

M4

3

ANTIGEN 1

ANTIGEN 2

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Results and conclusions

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?

Interpret and record the results

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6. Could we use blood from your classmates to determine whether our laboratory antigens are good candidates for a vaccine against malaria? Give reasons for your answer.

5. What would happen if we did not do the washing before adding the colouring substrate?

4. Which part of the primary antibody is recognised by the secondary antibody? The constant region or the variable region? Give reasons for your answer.

7. Do you believe that this experiment has shown that the selected candidate stimulates the immune response? Would you have to do some other type of experiment to assess whether it is also capable of activating some other type of response?

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NAME:PBS Tween-20CHK IGY, bagged (= ANTIGEN)RB ANTI-CHK, bagged (= PLASMA)GAR-HRP, bagged (= SECONDARY ANTIBODY)COLOURING SUBSTRATE

BE INFORMEDFind out where the safety equipment of the laboratory or the place where you are experi-menting is located (fi re extinguishers, show-ers or baths, exits, etc.). Read the instructions carefully before doing an experiment. Do not forget to read the safety labelling for reagents and equipment.

USE PROPER CLOTHINGGloves, apron and goggles.

GENERAL STANDARDSSmoking, eating or drinking in the laboratory or area where you are experimenting is prohibited.Wash your hands before leaving the labora-tory. Work in a neat and orderly fashion without hurry. If any product should spill, clean it up immediately. Always leave materials clean and orderly. Never use equipment or apparatuses without perfectly understanding how they work.

HANDLING OF GLASSProtect your hands when handling materials made of glass. Do not use cracked glass items.

CHEMICAL PRODUCTSDo not use unlabelled containers of reagents. Do not sniff, inhale, taste or touch chemical products. Never pipette by mouth. Wear gloves and wash your hands frequently if you use toxic

or corrosive products. Do not place reagent containers near a fl ame. Do not heat infl am-mable liquids. Carry bottles holding them from beneath, never by the neck.

WASTE DISPOSALDeposit broken glass, reagents that are toxic, noxious or harmful to the environment and biological waste in special and appropriately la-belled receptacles. Never dispose of solid waste using the sink.

In case of accident, advise the instructor im-mediately. Remember: If you have a question, ask the trainer.

SPECIFIC PRECAUTIONS FOR THIS WORKSHOP During this practice session you must follow the usual precautions for handling of chemi-cal products. Below are listed only those that present the following degrees of hazard:

• PBS: toxic when ingested, inhaled or in contact with the skin.

• Tween 20: toxic when ingested, inhaled or in contact with the skin. Irritant.

REFERENCESP4417-50TABP1379-100ML1662406EDU1662407EDU1662408EDU1662402EDU

COMMERCIAL MANUFACTURERSigmaSigmaBioRadBioRadBioRadBioRad

Safety precautions

Reagent references

Annex I

Annex II

OBLIGATORY USE OF GOGGLES

OBLIGATORY USE OF APRON

OBLIGATORY USE OF GLOVES

Researchers who have contributed content: Laura Puyol, investigator for CRESIB, ISGlobal.

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/

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