2.1 The disease - Introduction and Immunology

Question 1

Introduction

Answer 1

In Section 1Parasites and vectors, you learned that malaria is one of the most important infectious diseases in the world, infecting an estimated 241 million and killing 627,000 people in 2020.In this section, you will discover more about malaria as a disease that presents in a variety of ways ranging from a minorfebrileillness to life-threatening conditions. Why is it that some people experience little more than a minor inconvenience while others become seriously ill and might even die?

Question 2

Introduction

Answer 2

Part of the reason is thevariable ability of the human host to mount an effectiveimmune responsethat keeps the infection undercontrol, but the immune response itself can result in thepathologyassociated with the disease. Other reasons why the nature of the disease varies so widely include the genetic make-up of the parasite and the host, both of which can determine the outcome of the infection.

Question 3

Introduction

Answer 3

In the five sessions of this section, you will learn about the complexity of malaria and, at the end of the section, you should be able to appreciate why the immune response is involved in both protection and disease, and why the genetic make-up of both the parasite and the host make malaria such a difficult disease tocontrol. You will Return to topics such as immunology and the genetics of the mosquito host in later sections, especially Section 3 (Epidemiology andcontrol) where you will considercontrolin more detail.

Question 4

Glossary Febrile

Answer 4

Feverish

Question 5

Glossary Immune response

Answer 5

The cellular and molecular events that result in immunity.

Question 6

Glossary Pathology

Answer 6

The anatomical changes produced as a result of a disease process.

Question 7

Aims

Answer 7

To explore theimmune responsein malaria.

Question 8

Objectives

Answer 8

After working through this session, you should be able to: Describe how acquired immune responses determine the outcome of malaria infection and the development of clinical immunity. Understand the relationship between malarial pathology and immune responses to the parasite. Relate key features of the biology of malaria parasites to the development of protective immune responses. Appreciate the difficulties associated with developing malaria vaccines and the potential benefits of an effective vaccine.

Question 9

Introduction

Answer 9

If you have already studied the elective module IDM213 Immunology of Infections and Vaccines, you will find that there is quite a lot of overlap with this session. However, you will find that the articles by Rénia& Goh, Cowman et al., and Riley & Stewart (all in the IDM503 online reading list) give you a slightly different perspective on the subject. From Session 1.1 Parasites and vectors: Introduction, you should also be aware that whilst fever is the primary symptom of malaria, the major complications of infection include: Severe anaemia, Cerebral malaria, Respiratory distress, and Multi-organ failure.

Question 10

Introduction

Answer 10

Malaria is a protozoal, vector-borne disease caused by five different species of Plasmodium, of which P. falciparum, P. vivax and P. knowlesi are clinically the most important. The symptoms of malaria range from a mild, febrile syndrome accompanied by nausea and headache through to severe anaemia, cerebral malaria and multi-organ disease, which may present as renal failure, pulmonary oedema or liver failure.

Question 11

Naturally acquired immunity to malaria

Answer 11

Although malaria is potentially a very serious disease with a high fatality rate in untreated cases, individuals living in malaria-endemic areas do become partially immune to malaria. This is supported by age-related changes in disease susceptibilityand parasite prevalence. Thus, in endemic areas, children are particularly at risk of developing severe malaria while adults tend to suffer only mild symptoms of infection.

Question 12

Naturally acquired immunity to malaria

Answer 12

It is clear that the development of this resistance to severe disease depends on prior infection (that is, it is acquired or adaptive immunity) because in areas with very low levels of malaria transmission and in areas that are prone to infrequent epidemics of malaria, adults as well as children remain at risk of developing severe disease throughout their lives. Similarly, severe malaria can occur in people of all ages who live in non-endemic areas and contract malaria when travelling to the tropics.

Question 13

Naturally acquired immunity to malaria

Answer 13

Naturally acquired immunity to malaria is characterised by the ability to control levels of parasitaemia and to clear a malaria infection without developing severe symptoms. However, complete resistance to malaria infection (sterilising immunity) is very rare; most clinically immune individuals will experience periodic, asymptomatic infection.

Question 14

Naturally acquired immunity to malaria

Answer 14

The mechanisms of immunity to malaria are not fully understood but are likely to include both antibody-mediated inhibition of parasite invasion of red blood cells and inhibition of parasite growth, as well as cell-mediated cytotoxic mechanisms.

Question 15

Inappropriate immune responses

Answer 15

Whilst the parasite itself may directly cause pathology during infection (for example, rupture of infected red blood cells can contribute to malarial anaemia), most of the clinical features of malaria are due to the generation of inappropriate immune responses to the parasite, characterised by the production of high levels of circulating inflammatory cytokines.

Question 16

Inappropriate immune responses

Answer 16

Parasite products, which may include glycolipids or phospholipoproteins, can directly induce macrophages to produce tumour necrosis factor (TNF-α), interleukin-1 (IL-1),IL-6 and IL-12. This innate inflammatory response is further enhanced by interferon-γ (IFN-γ), which may come from NK (natural killer) cells, γδ T cells or malaria-specific αβ T cells.

Question 17

Inappropriate immune responses

Answer 17

This strong systemic inflammatory environment has numerous deleterious consequences, leading to many of the symptoms of infection, including fever. Thus, regulatory mechanisms that dampen down pro-inflammatory immune responses are also believed to contribute towards protection against severe disease by limiting immune mediated pathology.

Question 18

Why study the immune response to malaria?

Answer 18

The desire to develop a malaria vaccine is the most obvious reason why so much effort has been made to understand the immune response to malaria. Clearly, identifying the mechanisms involved in the elimination of parasites is an essential first step in the development of vaccines and adjunctive immunotherapies for the treatment of severe malaria.

Question 19

Why study the immune response to malaria?

Answer 19

Research over the past 50 years has greatly improved our understanding of malaria immunology and resulted in the development of antibody-based malaria diagnostics. However, although a modestly effective first generation vaccine was recommended for use by WHO in 2021, a highly effective vaccine or clinically useful immunotherapeutic procedure is not yet available.

Question 20

Why study the immune response to malaria?

Answer 20

Although insecticide-based vector control, especially the use of insecticide-impregnated bed-nets (see an example in the photo below), chemoprevention and early case management can reduce mortality from malaria, it is generally accepted that for a sustainable impact on parasite prevalence and malaria morbidity a combination of control strategies will be required. A safe and effective malaria vaccine will be an important tool for malaria control.

Question 21

Vaccine development

Answer 21

Molecular biotechnology has enabled us to identify potential target antigens, and to synthesise them in large quantities for use in vaccines. Recombinant vaccine technology is now widely utilised for developing vaccines against the various stages of the parasite’s life-cycle in the human host. A multicentre Phase 3 clinical trial of a recombinant sporozoite-based vaccine (RTS,S, details in Session 3.6 Epidemiology and prevention: Vaccination) documented approximately 55% protection against clinical malaria and against hospital admissions for severe malaria in African children. However, efficacy was lower in young infants and protection waned quite quickly in the absence of additional doses.

Question 22

Vaccine development

Answer 22

Concerns regarding the impact of the vaccine when deployed in routine systems, when all intended doses might not reliably be delivered, and lingering concerns the vaccine’s safety delayed introduction of the vaccine into routine immunisation schedules until further results from (Phase 4) studies were available. If you would like to learn more about this, read the article by Kaslow & Biernaux and see the WHO Malaria Vaccine Implementation Programme website.

Question 23

Vaccine development

Answer 23

Limited success has been achieved with recombinant blood-stage vaccines, possibly due to the highly polymorphic nature of the asexual blood-stage antigens (discussed in Section 3 Epidemiology and control), but some recently identified antigens which are less polymorphic are providing new opportunities for vaccine development. Finally, increased understanding of immune responses to sexual stages of the parasite is allowing progress in the development of vaccines that prevent parasite development in the mosquito (so-called transmission blocking vaccines). Further details are given in Session 3.6 Epidemiology and prevention: Vaccination.

Question 24

Question 1 Developing a malaria vaccine has been extremely challenging. Why do you think this might be?

Answer 24

The parasite is biologically complex – it transforms itself completely at different stages of the life cycle, expressing novel antigens at each stage. Selecting antigens that are exposed on the surface of vulnerable stages of the life cycle, that are relatively conserved between parasite lineages, and that are highly immunogenic is proving to be very difficult. Formulation of the vaccines can also be problematic. Some of the new generation adjuvants are very powerful (for example, inducing high titres of antibodies) but can also be toxic, inducing pain and swelling at the injection site and even inducing systemic side effects. Even when the vaccines are highly immunogenic, they do not seem to induce strong protective immunity. This suggests that the vaccines may be targeting the wrong antigen, or inducing the wrong kind of immune effector mechanisms. We need further research to define which are the correct antigens and the best effector mechanisms required for elimination of the parasite.

Question 25

Malaria pathology is immune-driven

Answer 25

The other important reason for studying the immune response to malaria is that much of the pathology is mediated by the immune system. Currently, between 10% and 50% of patients admitted to hospital with severe anaemia or cerebral malaria will die, despite being given effective antimalarial drugs and supportive therapy. If we can understand how the disease is caused, and how the immune response contributes to this, then we can begin to devise new approaches to treatment and so reduce the number of deaths from severe malaria.

Question 26

Malaria pathology is immune-driven

Answer 26

Because Plasmodium falciparum is the only species of malaria parasite that causes significant mortality in humans, most of the research has focused on this species. Most of what follows refers specifically to P. falciparum, but there is a short section on P. vivax and P. knowlesi at the end.

Question 27

Reading 1 It is important that you appreciate the changes in susceptibility to malaria that occur with increasing age or increasing duration of exposure to infection, as this helps us to understand the different stages in the development of antimalarial immunity. How do we know that people can become immune to malaria?

Answer 27

The graph’s vertical axis ranges from 0 to 100 and is not labelled. The horizontal axis is labelled “Age in years” and ranges from 0 to over 45. All three curves start at 0 on both axes. The death rate (small dash curve), peaks to 30 at year 1 then decreases quickly to near 0 at year 3. The parasite density (large dash curve), which is measured in µl × 10−2, peaks to 75 between years 1 and 2. The parasite prevalence (solid curve), which is measuredin %), peaks to 95 at year 4 then decreases 40 at year 35, then continues to decrease more slowly to just under 40 at year 45 and over.

Question 28

Question 2 How do each of these parameters change as people get older?

Answer 28

Parasite prevalence increases in the first year of life, peaks at about 3–6 years and declines very slowly from about 6 years onwards. Parasite prevalence can remain above 40% throughout adult life. Parasite density, which, in general, correlates with development of clinical disease,  peaks in very young children but falls dramatically in older children and adults. Deaths are essentially limited to children under the age of 3 years.

Question 29

Question 3 How could acquired immunity explain these changes?

Answer 29

If infection in childhood induces immune responses that inhibit sporozoite invasion, this might decrease parasite prevalence in the blood. If the immune response inhibits replication of blood stages, then parasite density would remain low with a consequent reduction in the incidence of clinical attacks. If specific immune responses protect against severe disease and death, then older people – who had been infected before – would be at lower risk of dying.

Question 30

Question 4 Do all the parameters change at the same time?

Answer 30

Different parameters change at different ages, indicating that some of the immune mechanisms are acquired more quickly than others. For example, immunity against the severe consequences of infection (i.e. decreased risk of dying) seems to be acquired much more quickly than immunity to infection (i.e. decreased prevalence of infection). The fact that the prevalence of parasites in the blood remains high in adults suggests that immune mechanisms that block sporozoite invasion or inhibit maturation of liver stages are not very effective. However, the fact that parasite densities are very low in adults implies that immune mechanisms that inhibit merozoite replication are very effective.

Question 31

Activity The data in the graph we've just seen in the previous activity come from a population in Tanzania where malaria transmission is very intense and where people are bitten by up to 2000 malaria-infected mosquitoes every year. The data in the graph here come from a population in The Gambia where malaria transmission is lower and people may be bitten by between one and five infected mosquitoes each year. Compare the age-related changes in malaria indices in this graph with the measurements in the previous graph.

Answer 31

The graph’s vertical axis ranges from 0 to 100 and is not labelled. The horizontal axis is labelled "Age in years" and ranges from 0 to over 50. All three curves start at 0 at year 1. The malaria death rate (small dash curve) peaks to 22 at year 4, then falls to 0 at year 7. Clinical malaria episodes (large dash curve) is measured in %. It peaks to 35 at year 5 then falls rapidly to 10 at year 13, then falls more slowly to near 0 at year 30The parasiteamia (solid curve) is measured in %. It peaks to 50 at year 7, then falls rapidly to 20 at year 17 and continues to fall more slowly to 10 at year 50 and over.

Question 32

Question 5 What are the most obvious differences between the two sets of data? How do each of these parameters change as people get older?

Answer 32

Overall, the prevalence of infection is lower in The Gambia than in Tanzania and prevalence (parasitaemia) declines more slowly (i.e. in an older age group) than in Tanzania. Parasite densities (roughly equating to incidence of clinical malarial episodes) also decline more slowly. Malaria-related deaths occur in children up to the age of 8 years.

Question 33

Question 6 What do these data tell you about the development of immunity to malaria?

Answer 33

If a minimum number of malaria infections are required to induce each type of immunity, then the age at which immunity develops will increase as transmission intensity decreases. The data suggest that relatively few infections may be needed to develop resistance to severe disease and death, but many more infections may be needed to become immune to infection per se. As a result, in areas of very low transmission intensity, immunity may never be achieved. Older individuals in areas of both high and low transmission continue to harbour parasites indicating that sterilising immunity (i.e. the ability to completely prevent infection) is seldom achieved even after lifelong exposure to very high levels of infection.

Question 34

Question 7 If you look very carefully at these two graphs, you will see that in both cases infants (children under one year of age) have a lower incidence of malaria than older children. Can you think of any reasons why this may be so? Hint: The answer may be immunological, physiological or a combination of both.

Answer 34

There are at least three possible explanations: 1 Infants may be bitten less often – they have a small surface area and are often carried close to their mother's body and are well wrapped up. 2 The parasites may grow less well in infant red blood cells because of the presence of foetal haemoglobin and a relative deficiency of p-amino benzoic acid (pABA), an essential parasite nutrient. 3 Antimalarial IgG antibodies from the mother, which cross the placenta into the child's circulation, persist for up to 4-6 months after birth and may provide passive immunity.

Question 35

Basic immunology

Answer 35

It is important to know that T lymphocytes recognise only linear epitopes. These epitopes are made up of short peptides produced by proteolytic cleavage of larger proteins inside the antigen-presenting cell (APC), which may be a dendritic cell, macrophage or a B lymphocyte. These peptides bind to major histocompatibility complex (MHC) molecules and are transported to the surface of the APC where they are available for recognition by T cells. However, B lymphocytes (and the antibodies that they produce) can recognise either linear or conformational epitopes.

Question 36

What are the mechanisms of immunity to malaria?

Answer 36

Immunity to malaria is complex and development of protective immunity requires repeated exposure to infection. As discussed on the previous pages, immunity is non-sterilising but can eventually provide complete protection against severe disease. Protective immunity requires both innate and adaptive immune effector mechanisms.

Question 37

What are the mechanisms of immunity to malaria?

Answer 37

Innate (first line, early) defences occur within minutes or hours of infection and depend upon phagocytic cells (macrophages and neutrophils), NK cells, γδ T cells and invariant NK-T (iNK-T) cells. The inflammatory cytokines released by these cells in response to activation signals from Plasmodium-infected erythrocytes mediate the immediate febrile response and shape the subsequent adaptive response.

Question 38

What are the mechanisms of immunity to malaria?

Answer 38

Specific (adaptive) immune responses involving both humoral (antibody) and cell-mediated responses start to become effective over the subsequent few days and may mature over a period of several weeks or months.

Question 39

Possible targets for host-protective antibodies

Answer 39

Immunity to malaria is complex and development of protective immunity requires repeated exposure to infection. Many antibodies to malaria antigens recognise conformational epitopes. This means that, if we are to make a synthetic malaria vaccine, we have to make sure that the synthetic antigen mimics the normal (native) three-dimensional structure of the intact parasite – so that conformational epitopes are re-created in the vaccine, which then induces antibodies that can recognise the live parasite.

Question 40

Possible targets for host-protective antibodies

Answer 40

There are other constraints on which immune effector mechanisms could operate against malaria. Antibodies can only be effective if their specific target antigen is accessible on the cell surface or if it is secreted from the parasite. It is very difficult for antibodies to bind to internal antigens. So, there are three sets of possible targets for host-protective antibodies: 1 The external surface proteins of all the extracellular stages of the parasite. 2 Proteins secreted by the apical complex of the parasite during host cell invasion. 3 Any parasite-encoded molecules that are expressed on the surface of the host cell – to modify the host cell and make it a better environment for parasite growth and replication.

Question 41

Antigen-antibody binding

Answer 41

The diagram shows a Y-shaped schematic of an antibody in the centre of the figure, with the antigen binding sites at the end of the arms of the Y shape pointing upwards. The two binding sites are identical, mirror images of each other. They are formed where the arms of the Y each have another strand alongside. The tips are coloured yellow for the main Y and brown for the alongside strand. The end has a 3D shape which fits exactly to the part of the antigen called epitope. Three different antigens with differently-shaped epitopes are shown in the upper center of the figure. One fits the antigen binding site.

Question 42

Direct killing by activated T cells

Answer 42

In contrast, T cells may be activated by peptides derived from any parasite antigen so long as the antigen has been ingested by APCs, has bound to a major histocompatibility complex (MHC) antigen molecule, and has been expressed on the surface of the APC.

Question 43

Direct killing by activated T cells

Answer 43

Once the T cell has been activated, it can either directly release cytotoxic molecules or it can cause other cells to become cytotoxic, leading to the killing of the parasite.

Question 44

Direct killing by activated T cells

Answer 44

Direct killing by cytolytic CD8+ T lymphocytes (CTLs) requires physical contact between the infected cell and the CTL. CTLs kill only infected cells that express MHC class I molecules. Human erythrocytes do not express any MHC molecules and are therefore resistant to attack by CTLs. Hepatocytes do express MHC molecules so intrahepatic parasites are theoretical targets for CTL activity.

Question 45

Question 8 Look at the diagram of the malaria life-cycle in the figure below. The large arrows indicate different parasite stages that might be vulnerable to different immune mechanisms. The letters indicate these mechanisms. Line drawing of an outline of a human torso showing the liver and superimposed with a diagram of the malaria life-cycle. The stages of the life-cycle are depicted as follows: A mosquito bites the human host and injects sporozoites in the host’s blood. The sporozoites travel to the liver and enter a liver cell. They undergo a three-stage transformation and the liver cell releases merozoites. Merozoites enter a red blood cell, some multiply and are released to infect further red blood cells, other undergo gametogenesis to form male and female gametocytes. These are picked up by a mosquito biting the human host and transported to the mosquito gut. The male and female gametes combine to form a zygote which evolves into a ookinete. This leads back to the start of the cycle. The following letters are marked on the diagram: (a) is near the liver; (b) is between where the mosquito is biting and the liver; (c) is where merozoites enter red blood cells; (d) and (e) are near an infected red blood cell; (f) is outside the human body, inside the mosquito gut; (g) is near the liver and near (a).

Answer 45

The correct answer is: a) IFN-γ+ CD8+ T cells inhibit parasite development within hepatocyte b) Antibody-mediated blocking of or attachment to host cells c) Antibodies block invasion of merozoites into red blood cells d) Killing by toxic radicals e) IFN-γ+ CD4+ T cells activate macrophages to phagocytose and kill intra-erythrocytic parasites f) Antibodies prevent fertilisation of gametes and development of zygote g) Killing by cytotoxic T lymphocytes (CTLs)

Question 46

Question 9 One important feature of malaria is that the parasite alternates between being extracellular (sporozoites, merozoites and gametes) and being intracellular (in erythrocytes and hepatocytes). How does this affect the parasite's susceptibility to different immune effector mechanisms?

Answer 46

Extracellular stages of the malaria life-cycle are particularly vulnerable to antibody attack. Antibodies can act in a number of different ways. For example, anti-sporozoite antibodies may agglutinate sporozoites in the skin, inhibiting their motility and opsonizing them for removal by phagocytes. They may also inhibit sporozoite invasion into hepatocytes by blocking essential receptor-ligand interactions at the hepatocyte surface. Similarly, anti-merozoite antibodies may opsonize and agglutinate merozoites and block invasion into red blood cells. In addition, anti-merozoite antibodies may mediate parasite killing through ADCI (antibody dependent cellular inhibition of parasite growth) where cross-linking of surface immunoglobulin Fc receptors on monocytes causes the phagocyte to release toxic molecules such as nitric oxide and oxygen radicals. Anti-gamete antibodies probably act by activating the classical complement pathway, leading to lysis of the gametes inside the mosquito. Antibodies can also bind to parasite-encoded antigens that are expressed on the surface of infected erythrocytes. These antibodies may opsonise the infected red blood cell for phagocytosis and may also prevent sequestration of infected red blood cells in peripheral organs, allowing them to be cleared in the spleen. All stages of the parasite (intracellular and extracellular) are vulnerable to toxic mediators released from T cells or phagocytes. Primed CD8+ T cells can directly target and kill infected hepatocytes that express malaria antigens in combination with MHC-I molecules. (Infected red blood cells do not express MHC-1 molecules which are essential for CD8+ T cells to recognise parasite antigens). IFN-gamma produced by CD4+ and CD8+ T cells, NK cells and iNK-T cells can activate phagocytic cells to enhance parasite killing. CD4+ T cells are also required for generation of memory CD8+ T cell responses and provide help to NK cells.

Question 47

What are the major target antigens of protective immunity to malaria?

Answer 47

The list below shows you the antigens that you may come across in major review articles and in reports of malaria vaccine trials. It is not, however, an exhaustive list. The fact that the antigens are targets of immune responses does not necessarily mean that these responses are effective in killing or inhibiting the parasite.

Question 48

What are the major target antigens of protective immunity to malaria? sporozoites

Answer 48

Surface antigens: circumsporozoite protein (CSP) Secreted antigens: thrombospondin related adhesive protein (TRAP)

Question 49

What are the major target antigens of protective immunity to malaria? liver stages

Answer 49

Surface antigens: liver stage antigen 1 (LSA-1), liver stage antigen 3 (LSA-3) Secreted antigens: exported protein-1 (EXP-1)

Question 50

What are the major target antigens of protective immunity to malaria? merozoites

Answer 50

Surface antigens: merozoite surface protein 1 (MSP 1)* merozoite surface protein 2 (MSP 2)* merozoite surface protein 3 (MSP-3) merozoite surface protein 4 (MSP-4) merozoite surface protein 5 (MSP-5) Secreted antigens: various rhoptry proteins concerned with red cell invasion (Rh, RON and RAP proteins) apical membrane antigen 1 (AMA-1)* glutamate-rich protein (GLURP)* erythrocyte-binding antigen (EBA-175) serine-rich antigen (SERA) ring-infected erythrocyte surface antigen (RESA)

Question 51

What are the major target antigens of protective immunity to malaria? infected RBCs

Answer 51

Surface antigens: erythrocyte membrane protein 1 (PfEMP1)* STEVOR* RIFIN* Secreted antigens: N/A

Question 52

What are the major target antigens of protective immunity to malaria? gametocytes

Answer 52

Surface antigens: Pfs 230, Pfs 48/45 Secreted antigens: N/A

Question 53

What are the major target antigens of protective immunity to malaria? gametes

Answer 53

Surface antigens: Pfg 25/27 Pfs 25/28 Pvg 25/28 Secreted antigens: N/A

Question 54

What are the major target antigens of protective immunity to malaria?

Answer 54

One important area of malaria research is to try to differentiate between the antigenic targets of protective immunity and antigens that induce irrelevant (or even harmful) immune responses. Many malaria antigens are known to be polymorphic; immune responses raised against one parasite isolate may not cross-react with other parasites leading to strain-specific immunity.

Question 55

What are the major target antigens of protective immunity to malaria?

Answer 55

PfEMP-1 undergoes clonal antigenic variation – an individual parasite clone may change the antigen expressed on the surface of its host red blood cell during the course of an infection, leading to ‘variant-specific’ immunity.

Question 56

Question 10 Match stage-specific antigens with the immune effector mechanisms that you saw earlier in the session.

Answer 56

Sporozoite stage: agglutination by antibody (CSP), Hepatocyte stage or liver stage: inhibition of parasite development within hepatocytes by IFNy CD8 cells killing by cytotoxic T lymphocytes (CTLs) (TRAP, LSA-1, CSP), Merozoite stage: antibodies block invasion of merozoites into RBC (MSP-1, AMA-1), RBC stage: activation of macrophages by INFy CD4 T cells to phagocytose and kill intraerythrocytic parasites (PfEMP), Mosquito gut stage: antibodies prevent fertilisation of gametes and development of zygote (Pfs230, Pfs48/45)

Question 57

Question 11 Which antigens do you think will be the targets of which immune effector mechanism?

Answer 57

The only antigens in the list that have been specifically chosen as targets of cell-mediated immunity are CSP, LSA-1 and LSA-3. Peptides derived from these antigens are believed to be expressed on the surface of the infected hepatocyte in association with MHC class I. LSA-1-specific CD8+ T cells are believed to kill parasites inside hepatocytes by localised release of IFN-γ. The surface antigens are likely to be targets of antibodies that inhibit invasion, agglutinate parasites, opsonise parasites for phagocytosis, or activate complement. The secreted antigens are potential targets for antibodies because they are released from the apical complex of the parasite at the time of invasion. Antibody binding could disrupt any of these processes for which secreted antigens are required (adhesion, provision of traction for the parasite to move into the cell, deformation of the host cell membrane).

Question 58

Mechanisms of immune evasion

Answer 58

Malaria parasites have a number of strategies for evading recognition by the immune system. The sporozoites spend a very short time in the blood before hiding in the liver cells, and the blood-stage parasites spend most of their time within red cells. Antigenic polymorphism and antigenic variation provide further evasion strategies. These properties help to explain why designing an effective vaccine is not easy.

Question 59

Malaria vaccines

Answer 59

Immune effector mechanisms are specific for each of the life-cycle stages shown in the table you saw in the previous topic. Since sporozoites and merozoites are extracellular they can only be targeted by neutralising antibodies which have to be 100% effective to prevent infection of hepatocytes and erythrocytes respectively.

Question 60

Malaria vaccines

Answer 60

Once within the hepatocyte, the parasite becomes a good target for attack by CD8+ cytotoxic T cells. Infected erythrocytes can also be targeted by specific antibodies or by mechanisms such as ADCI. Antibody responses, CD8+ T cell and macrophage activation require CD4+ T cell help.

Question 61

Malaria vaccines

Answer 61

Thus, vaccine design needs to be tailored to induce the relevant effector mechanisms for stage-specific immunity and new and improved adjuvants are required to direct the development of appropriate immune responses to the candidate vaccine antigens.

Question 62

Types of vaccines

Answer 62

In recent years, you may have seen a number of press reports about malaria vaccine trials. Details of current vaccines under development are discussed in Session 3.6 Epidemiology and prevention: Vaccination. Two main types of vaccine have been the subject of these trials.

Question 63

Types of vaccines First type

Answer 63

The first type of vaccine, which is based on the circumsporozoite protein (CSP), aims to induce high levels of antibody that inhibit sporozoite invasion of liver cells; this vaccine has recently been modified to include epitopes that also stimulate CD4+ T helper cells and IFN-γ producing T cells. The latest generation of CSP vaccines, named RTS,S, includes a powerful new adjuvant which induces very high titres of antibodies. Optimisation studies highlighted the importance of the appropriate adjuvant formulation with this vaccine as only very high titres of antibodies are associated with protection.

Question 64

Types of vaccines First type

Answer 64

Field trials in Africa have shown significant levels of protection against clinical malaria in RTS,S vaccinated infants and children (Kaslow & Berniaux 2015) (discussed in Session 3.6 Epidemiology and prevention: Vaccination). Although the precise immunological effector mechanisms have not been identified, anti-CSP antibody levels are significantly higher in children who remain free of infection after vaccination than in children who are not protected. New approaches, including modified viral vectors and prime-boost schedules, are being tested with some success.

Question 65

Types of vaccines Second type

Answer 65

The second type of vaccine targets asexual blood stages. The first to be widely tested, SPf66, was a synthetic peptide vaccine (including part of the N-terminal sequence of the merozoite surface protein, MSP-1), developed by Patarroyo and co-workers. This has now been shown not to be protective and work on it has long since been abandoned.

Question 66

Types of vaccines Second type

Answer 66

Clinical trials in endemic countries have shown partial protection with vaccines based on the c-terminal part of MSP-1 and on AMA-1. In both cases, vaccinated individuals were partially protected against infection with parasites expressing the same version of MSP-1 or AMA-1 as was contained in the vaccine but they were not protected against parasites expressing carrying different versions of the antigens. These trials demonstrate the problems caused by antigenic polymorphism.

Question 67

Types of vaccines Second type

Answer 67

Research is now focussed on identifying blood stage antigens that are highly conserved. The hope is that these antigens will provide cross-strain immunity. There is a risk that vaccination with such antigens would select for novel variants of these antigens, driving antigenic diversification. But it is also possible that these antigens are functionally constrained in the extent to which they can vary – if they mutate too much they may lose their function and the parasite would thus not be viable.

Question 68

Types of vaccines Second type

Answer 68

One such antigen is the rhoptry associated antigen, PfRh5. PfRh5 is minimally variant in the wild. It binds to the erythrocyte surface receptor basigin, and is essential for merozoite invasion. Preliminary studies indicate that anti-Rh5 antibodies provide cross-strain protection.

Question 69

Malaria vaccines

Answer 69

Developing an effective vaccine against malaria is still a very high-profile area of research. The next generation of malaria vaccines may consist of multi-antigen vaccines or multigene viral vaccines comprising genes expressed in the exoerythrocytic and erythrocytic stages of the parasite life-cycle.

Question 70

Immunopathology of malaria

Answer 70

You will learn much about the pathology and pathogenesis of malaria in the next session. It is worth remembering that the signs and symptoms of malaria are caused in part by the host's own response to infection.

Question 71

Immunopathology of malaria

Answer 71

All the clinical symptoms of malaria infection are associated with the erythrocytic phase of the life-cycle. Schizont rupture triggers the periodic release of inflammatory cytokines (giving rise to the febrile illness) and destroys erythrocytes (contributing to anaemia), degradation of haemoglobin leads to production of an insoluble haem pigment which prevents reuse of iron, leading to a functional iron deficiency. Schizont infected erythrocytes also bind to endothelial cells in post capillary venules (sequestration) causing circulatory dysfunction.

Question 72

Immunopathology of malaria

Answer 72

A number of the symptoms of malaria infection are very non-specific (fever, vomiting, headache and nausea, sometimes accompanied by diarrhoea, pneumonia, muscular aches and back pain) and the clinical picture can resemble an influenza-like illness or other systemic infection. This makes malaria quite difficult to diagnose; it also tells us something about the pathogenesis of the disease.

Question 73

Immunopathology of malaria

Answer 73

The most severe complications of malaria infection, including severe anaemia, cerebral malaria and respiratory distress can develop rapidly in infected individuals and are thus often diagnosed only when at an advanced stage; treatment with anti-malarial drugs in many of these advanced cases is ineffective and cerebral malaria and severe anaemia are often fatal unless high quality supportive care is available.

Question 74

Role of inflammatory cytokines

Answer 74

Although the spectrum of symptoms of malaria are quite diverse, accumulating evidence suggests that inflammatory cytokines, such as IFN-γ, IL-1, IL-6 and TNF-α, play a major role in the development of all the symptoms of malaria infection.

Question 75

Role of inflammatory cytokines

Answer 75

Pro-inflammatory cytokines are also released during other infections, which partially explains why the symptoms of malaria infection are non-specific and are so easy to misdiagnose. Inflammatory cytokines can be triggered by recognition of parasites and parasite materials by pathogen recognition receptors on phagocytic cells, such as macrophages and dendritic cells.

Question 76

Role of inflammatory cytokines

Answer 76

Low levels of inflammatory cytokines are beneficial to the patient because they activate macrophages to phagocytose infected erythrocytes and to release toxic radicals that kill the parasite.

Question 77

Role of inflammatory cytokines

Answer 77

However, high levels of these cytokines cause pathology and disease. In addition to causing fever, these cytokines also: increase the expression of adhesion molecules on vascular endothelium (leading to higher levels of parasite and leukocyte sequestration); decrease blood glucose levels (leading to hypoglycaemia); inhibit erythropoiesis (contributing to malarial anaemia); promote tissue damage (by inducing production of toxic radicals); and promote phagocytosis and killing of uninfected red blood cells (also contributing to malarial anaemia).

Question 78

Role of inflammatory cytokines

Answer 78

From the information above, you will understand that effective resolution of malaria infection is a constantly evolving trade-off between mounting an inflammatory anti-parasitic immune response strong enough to kill the parasite, but not so strong as to cause immunopathology and the symptoms of infection.

Question 79

Pro-inflammatory and regulatory immune responses

Answer 79

Specialised regulatory networks exist in the immune system for the precise purpose of moderating the duration and strength of pro-inflammatory immune responses to prevent immunopathology.

Question 80

Pro-inflammatory and regulatory immune responses

Answer 80

The initiation of these regulatory responses, which include specialised populations of CD4+ T cells, called regulatory T cells, and production of anti-inflammatory cytokines, including IL-10 and TGF-beta, at the correct stage of malaria infection enables the killing of parasites whilst limiting the development of immunopathology. Conversely, overactivation of regulatory responses can prevent parasite control leading to severe disease. Precisely how the immune system controls the development of pro-inflammatory and regulatory responses during malaria infection is currently unknown.

Question 81

Pro-inflammatory and regulatory immune responses

Answer 81

Understanding that much of the pathology of malaria is caused by over-activation of the cellular immune system is important for two reasons: First, it allows us to develop improved treatment regimes (including anti-inflammatory drugs). Second, it alerts us to the possibility that malaria vaccines may themselves cause some pathology and warns us to be on the lookout for potentially harmful side-effects of vaccination.

Question 82

Lymphocyte activation and immunodepression

Answer 82

Historically it was thought that malaria parasites are mitogenic (which implies that they induce activation of T and B cells without triggering through the T-cell receptor or B-cell receptor). However, it is now believed that malaria parasites express lots of antigens that cross-react with antigens on other infectious organisms and that malaria is thus able to re-activate lots of T and B cells that have been primed by other infections. Whatever the mechanism, it is true that malaria leads to activation of a large proportion of T and B lymphocytes.

Question 83

Lymphocyte activation and immunodepression

Answer 83

Suppression of allergic or autoimmune responses by malaria may be directly linked to the induction of type 1 (or inflammatory) T-cell responses; allergic reactions are mediated by type 2 T cells that are down-regulated by type 1 cells.

Question 84

Lymphocyte activation and immunodepression

Answer 84

In addition, immunodepression is a feature of acute malaria infection. At present the main reasons for immunodepression during malaria infection remain unclear. There is some evidence that it is caused through impairment of macrophage and dendritic cell function by the build up of malarial pigment (indigestible breakdown products of haemoglobin generated by the parasite as it feeds) within the cell following phagocytosis, or by reactive nitrogen intermediate effects on T cells. It may also be due to exhaustion of the immune system due to wide-scale and systemic immune activation. Immunodepression can affect the outcome of malaria infection or the response to other infections such as HIV and bacterial infections.

Question 85

P vivax malaria

Answer 85

You may be wondering why we put so much emphasis on P. falciparum at the expense of P. vivax and P. knowlesi. The simple reason is that P. falciparum causes considerably greater direct mortality than these other infections.

Question 86

P vivax malaria

Answer 86

The overall mortality rate for P. vivax is very low, despite it being highly endemic in Asia and Latin America. Historically, P. vivax was considered to be nonlethal, although recent data are beginning to challenge that assumption.

Question 87

P vivax malaria

Answer 87

Infections with P. knowlesi can certainly be fatal but since this infection was discovered only recently in humans, statistics on infection rates and fatalities are based on small numbers of cases and may not be very reliable; more studies on P. knowlesiare required before we understand the true extent of its severity.

Question 88

P vivax malaria

Answer 88

P. vivax tends to replicate more slowly than P. falciparum , so parasite densities rise more slowly and over a longer period of time, giving the host's immune response a chance to control the infection. The sheer speed at which P. falciparum reaches very high densities often makes it impossible to control.

Question 89

P vivax malaria

Answer 89

In addition, P. vivax-infected erythrocytes do not appear to sequestrate and thus very rarely cause cerebral malaria. P. vivax does, however, induce very high levels of inflammatory cytokines (especially TNF-α), which manifest as very high temperatures followed by profuse sweating - and can make the patient feel extremely ill. In this way, P. vivax infections can be as debilitating as P. falciparum infections.

Question 90

P vivax malaria

Answer 90

The relatively benign nature of P. vivax infections may be quite a recent phenomenon. In the absence of antimalarial drugs and antibiotics, and in people who are less well nourished and carry other infections, P. vivax may lead to potentially fatal secondary infections. It is believed that P. vivax selected for the 'Duffy negative' red blood cell phenotype which is present in almost all West Africans (Duffy-negative red cells are resistant to invasion by P. vivax ). If this is so, P. vivax must – once – have carried with it significant mortality. This is an example of how parasite pressure has instructed human evolution.

Question 91

Question 12 What are the strategies that malaria parasites have developed to evade the host immune response?

Answer 91

The parasite has adopted an intracellular lifestyle that protects it from the direct effects of antibodies and complement. Moreover, it spends most of its time inside red blood cells which do not express MHC molecules; the parasite can therefore 'hide' from the cellular immune response. Each stage of the life-cycle expresses different antigens so that by the time an immune response has developed to one stage of the life-cycle, the parasite has already transformed itself into another stage and cannot be recognised. Furthermore, the antigens that are targets of effective immune responses vary both between and within infections, such that an immune response induced against one parasite strain will not necessarily protect against infection with a different strain. NB: It used to be thought that malaria caused immunosuppression of the host and that this was a mechanism of immune evasion. However, the evidence for immunosuppression is not as strong as we once thought - and indeed acute malaria is an example of an overactive rather than an underactive immune system. There do seem to be some immune defects in malaria infected individuals (such as poor responses to some vaccines and increased susceptibility to concomitant bacterial infection) but these seem to reflect unfortunate consequences of malaria infection rather than a deliberate attempt by the parasite to subvert our immune systems.

Question 92

Question 13 If you were asked to design an ideal malaria vaccine, which stages would you target and which immune mechanisms would you aim to induce?

Answer 92

Ideally, you would block sporozoite invasion of liver cells (with antibody), development of liver-stage parasites (with IFN-γ released from T cells), merozoite invasion of red blood cells (with antibody), development of schizonts inside red cells (with IFN-γ), sequestration of schizont-infected cells (with antibody to PfEMP-1) and transmission of malaria by mosquitoes (by complement-mediated lysis of gametes). Attacking the pre-erythrocytic stages of infection is a very attractive approach because in doing so the development of the blood stage of infection is completely prevented.

Question 93

Question 14 What are the potential hazards of malaria vaccination?

Answer 93

You might have thought of some additional answers, but these are two possible hazards of malaria vaccination: a) Immunopathology, due to excess production of inflammatory cytokines. This is specifically a potential problem with vaccines designed to induce cell-mediated immunity to blood stage vaccines. b) Loss of naturally acquired immunity due to reduced malaria transmission in a well-vaccinated population. If the vaccination programme fails (due to lack of money, political disturbances, natural disasters or war) the population may become susceptible to epidemic malaria, which can lead to high levels of mortality.

Question 94

Summary

Answer 94

Immunity to malaria is acquired following natural infection. The rate of development of immunity depends on the intensity of malaria transmission. Different immune mechanisms operate to decrease parasite prevalence, parasite density and the risk of severe disease and death. Immunity involves both antibody-mediated and cell-mediated mechanisms, but these may be directed against different stages of the life-cycle and against different antigens. Despite intensive research and many vaccine trials, there is – as yet – no highly effective vaccine against malaria. A moderately effective vaccine, RTS,S/AS01, was recommended by WHO for use in moderate and high transmission settings in 2021 – it took 30 years to develop! Many of the symptoms of malaria are caused by overproduction of inflammatory cytokines. Low levels of these cytokines are required to mediate parasite killing.

Question 95

Glossary Vaccine

Answer 95

A suspension of a killed or attenuated microorganism, or part of a microorganism, that is given, usually in the form of an injection, to prevent or treat a specific infectious disease.

Question 96

Glossary Cerebral malaria

Answer 96

A severe consequence of malaria characterised by parasite and leukocyte sequestration in the brain associated with endothelial cell activation (cytokine mediated up regulation of adhesion molecules), raised intracranial pressure, reduced blood flow, coma and fits.

Question 97

Glossary Endemic

Answer 97

Epidemiological term describing an infection in a community where it is maintained with constant incidence of cases for many years without external inputs.

Question 98

Glossary Susceptibility

Answer 98

An uninfected individual (or population) who is able to become infected.

Question 99

Glossary Prevalence

Answer 99

The proportion of individuals with the infection or disease of interest in a defined population at a specific point in time. Prevalence is determined by dividing the number of people estimated to be infected by the total population size. Prevalence is most commonly measured at a single point in time (the ‘point prevalence’). Compare with the definition of incidence.

Question 100

Glossary Acquired or adaptive immunity

Answer 100

Antigen-specific adaptive immunity that increases during infection.

Question 101

Glossary Epidemics

Answer 101

A disease outbreak in which the number of cases increases significantly over the normal, endemic, background level, in a specified, geographically defined population group.

Question 102

Glossary Cytokines

Answer 102

A small protein messenger which mediates communication between cells of the immune system.

Question 103

Glossary Tumour necrosis factor (TNF-a)

Answer 103

A pro-inflammatory cytokine. Also called tumour necrosing factor. Abbreviated to TNF.

Question 104

Glossary Morbidity

Answer 104

Any departure, subjective or objective, from a state of physiological or psychological well-being. In practice, morbidity encompasses disease, injury, and disability.

Question 105

Glossary Efficacy

Answer 105

The impact of an intervention, often measured as the percentage reduction in the incidence of the infection or disease of interest. Calculated as 100 × (1 − RR)% where RR stands for risk ratio.

Question 106

Glossary Major histocompatibility complex (MHC)

Answer 106

Cluster of genes encoding the MHC class I and MHC class II molecules. (The MHC Class III region contains some of the complement genes). In humans, MHC is also known as human leukocyte antigen.