Innate and Adaptive Immune Responses to Malaria

Question 1

Overall, how does the body respond to malaria?

Answer 1

body prioritises host fitness over parasite clearance

Question 2

What is so unique about the Parasite Burden and “Severity of Malaria in Tanzanian Children (2014)” paper?

Answer 2

large scale study in which nearly 900 children were examined at birth, once every two weeks during infancy, once every month after infancy, and during any illness
– blood smears collected at all visits, regardless of whether symptoms were present

this is a large scale study that was able to study the effects in v young children over a long period of time

cannot be performed again as current guidelines say that if someone in study is ill w malaria, they have to be treated

this data is only piece of its kind

Question 3

In the Tanzania study, how many children had severe malaria?

Answer 3

102 had severe malaria

87 (85.3%) had only one severe episode

70 (68.6%) had severe malaria during their first, second, or third infection of life

Question 4

In the Tanzania study, what was the trend of percentage of those who got severe malaria compared to infection number?

Answer 4

risk of severe malaria exponentially declines as the number of infections they previously has increases

engagement of tolerance, not resistance mechanisms

Question 5

What is the relationship between parasitaemia and severe malaria?

Answer 5

high parasite density is needed to progress to sever malaria
but high parasite density does not always cause severe malaria

Question 6

What is the relationship between parasite density and symptoms?

Answer 6

there is a weak correlation

Question 7

What is the difference in gene expression between the first and second exposure to malaria?

Answer 7

decrease of pro-inflammatory cytokine production in second exposure

Question 8

Do innate cells have memory?

Answer 8

experiments show when monocytes previously exposed to malaria in vivo are exposed to parasite lysate show a decrease in the amount of pro inflammatory cytokines released (Portugal 2014)

in addition, experiments on prisoner volunteers involved to exposure to endotoxin before and after they were infected with malaria
– there was a significant decrease in clinical illness
– although this study was unethical and potentially v dangerous

Question 9

Innate immune cells have a short lifespan so how would they form memory?

Answer 9

it has been shown that exposure of HSCs in the BM to the BCG vaccine (against M. tuberculosis) changes their transcriptional landscape
– leading to enhanced myelopoiesis at the expense of lymphopoiesis
– BCG-educated HSCs generate epigenetically modified macrophages that provide increased protection to M. tuberculosis compared to naive macrophages

Question 10

What functions do monocytes perform upon secondary infection to malaria, if not the pro-inflammatory response?

Answer 10

do not downregulate everything
– upregulation of blood vessel creation
– glucose uptake
– deal w stress of malaria infection
– no hypoxia –> no acidosis

in addition, there is an expansion of innate blood cells during first infection
– does not occur on second

Question 11

BCG induces epigenetic change, what about malaria?

Answer 11

experiment took place over 100 days, much longer than a monocyte lifespan, looking at epigenetic differences

no differences between unexposed and expose mice, so no innate memory?

there are functional differences however, influenced by this tissue microenvironment
– changes in liver structure due to hemozoin

Question 12

How is the NLRP3 inflammasome activated during malaria infection?

Answer 12

exposure to hemozoin activates it though Lyn and Syk kinases

Question 13

How are macrophages impaired during malaria infection?

Answer 13

impairment of macrophage function after ingestion of P. falciparum infected RBCs or hemozoin

Question 14

What is clinical immunity to malaria and how does it develop?

Answer 14

immunity that develops to the clinical symptoms of malaria, not the parasite itself

develops after years of constant exposure in hyper- or holo-endemic areas

in some areas of favourable malaria transmission, one person can be infected more than 1000 times per year

lost without re-exposure (lasts at least 5 years)

Question 15

How is it hypothesised that people eventually become immune?

Answer 15

1. immunity is due to acquisition of a repertoire of responses to many different isolates

                      and/or

2. the development of cross-protective responses to shared antigens

–> an understanding of both immune mechanisms and antigen targets is needed to understand vaccine-mediated immunity

Question 16

Is passive transfer of human malaria immunity effective?

Answer 16

12 children (4mo -2y) w malaria received IgG by injection every 8-24h for 3 days

using gambian vs UK sera - can Ab alone protect?

trophozoite counts were considerably lower using sera from ‘protected’ adults than UK adults

Question 17

How are babies protected from malaria at birth?

Answer 17

children remain v resistant to high parasitaemia, fever, SM, and death until 6 months
- susceptibility begins at 3-4 mo

protection may be associated w presence of maternal IgG –. debated, other mechanisms? eg lactoferrin

levels of these decrease over first year of life

Question 18

Why is it significant that transplacental transfer of IgG increases exponentially during final month of term pregnancy (40 weeks)?

Answer 18

any change in when baby is born could drastically change how many protective Abs they have

Question 19

How is IgG transferred during pregnancy?

Answer 19

maternal IgG Abs transferred to foetus via active transport mechanism mediated by FcRn
– neonatal Fc receptor
– selectively transports IgG

transfer efficiency highest for IgG1
– then IgG4 –> IgG3 –> IgG2

Question 20

Is there evidence to support a protective role of maternally transferred Ab?

Answer 20

not much

previous analysis based on total IgG levels

recently, however, evidence shows IgG3 should be the best IgG subclass at protecting against malaria
–> better at fixing complement + engaging FcR on phagocytes

however is transferred to foetus at low efficiency

Question 21

How can Abs block different stages of the Plasmodium lifecycle?

Answer 21

1. sporozoites
   – block trafficking to liver
2. erythrocytic stages
   – targeting merozoites and infected RBCs
3. gametocytes
   – blocking transmission

Question 22

What is the function of Abs against PfEMP1?

Answer 22

opsonic phagocytosis activity is reduced in PfEMP1 knock down
– may also block cytoadherence

no difference in function of Abs between SM and UM

therefore the amount of IgG present in an experienced host may be important for clinical presentation of disease

Question 23

Why is Ab binding to the right spot on PfEMP1 variant important?

Answer 23

PfEMP1 is large protein that contains functional binding sites

targeting these sites allows Abs to inhibit function of PfEMP1 eg binding site of EPCR and/or CD36

adults recognise PfEMP1 peptides > children

Question 24

What is the function of repetitive interspersed repeats?

Answer 24

RIFIN

like PfEMP1, highly antigenically varied
– encoded by set of 150-200 genes
– 1 expressed per parasite

function
– bind to erythrocytes (prefer group A) to from rosettes and induce cytoadherence

Question 25

How do RIFINs modulate the immune system?

Answer 25

can bind to inhibitory receptors on B, T, and NK cells (eg LILRB1 and LAIR1)

Abs to RIFINs do not correlate w protection

Question 26

What is the subtelomeric open reading frame?

Answer 26

STEVOR

family of 40 polymorphic proteins (least characterised VSA family)

biphasic expression profile
– protein expression varies at distinct life cycle stages

Question 27

What is the function of STEVORs?

Answer 27

experiments showed that STEVOR binds Glycophorin C (GPC) on the RBC surface and that its binding correlates with the level of GPC on the RBC surface

STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation)

antibodies targeting STEVOR in the merozoite can effectively inhibit invasion

Question 28

Why are merozoites the most attractive targets for antobodies?

Answer 28

they are an essential step in the extracellular asexual reproduction cycle
– problem is merozoites express large repertoire of proteins on cell surface (hard for immune system to target effective one)

Of 52 malarial antigen, MSP1 is highest exprssed
- titres of Abs to MSP family are highest detected

however it doesnt mean Abs against MSP1 would effective at neutralising it

Question 29

What are micronemes?

Answer 29

elongated secretory organelles containing proteins involved in initial stages of:
– adhesion: Microneme proteins mediate the binding of the parasite to RBC surface

– motility: enable gliding motility, which is crucial for the parasite to move through tissues and locate host cells

eg
EBA (Erythrocyte Binding Antigens)
DBL (Duffy Binding-Like)

Question 30

What are rhoptries?

Answer 30

club-shaped, secretory organelles located adjacent to micronemes
Rhoptry proteins are released after microneme proteins during the invasion process
– form parasitophorous vacuole within RBC to reside in
– modify host cell for immune evasion

eg
Reticulocyte Binding Protein Homologue 5 (RH5)
Apical Membrane Antigen 1 (AMA1)

Question 31

Why is it significant that we now know that merozoites enter RBCs via the flat end (posterior) rather than the pointed end (apical)?

Answer 31

both micronemes and rhoptries are present at the apical end on the merozoite

this changes our understanding of protein targets, and maybe these organelles arent the most effective targets
– may be why RH5 vaccine not as good as ppl thought it would be

shifts the focus to underappreciated proteins on the posterior end
– could be targeted to block initial attachment and prevent invasion

Question 32

Are microneme and rhoptry proteins good vaccine targets?

Answer 32

very important in invasion into RBCs

there are many so inhibition of one may not matter

also may only be expressed when attached to or near RBC
– small window to block them

scarce number of targets
– wouldnt be able to opsonise effectively

RH5 was promising but didnt amount to much

Question 33

How do Abs protect against merozoites?

Answer 33

1. neutralisation
2. opsonic phagocytosis
3. complement activation

Question 34

What is the function of T follicular helper cells during infection?

Answer 34

secrete IL-21 and express CD40L
–> drive B cell proliferation

function shown to be dysregulated in disease such as HIV, preventing production of effective Abs

Question 35

Why is humoral immunity inefficient in response to malaria?

Answer 35

CXCR3+ Tfh cells preferentially activated in Plasmodium infection and produce robust IFNy
– less capable of stimulating B cells
– B cell activation impaired in GCs is impaired
– IFNy actively inhibits activated B cells
– does induce phagocytosis of iRBCs

Question 36

How are Tfh cells inhibited?

Answer 36

Tregs

indirectly through IL-10 production
– however IL-10 inhibits other immune cells

or directly via cell-cell interactions w CTLA-4
– CTLA-4 also impairs GC reactions
– competitively binds CD80/86, no binding to CD28 on Tfh = no survival signals
– makes CD28 weaker = no costimulation on B cell making response even worse

Question 37

What does impairment of GC response due to Tfh and Treg activity mean for response?

Answer 37

No GC
= no long-lived plasma cells
= no class switching
= no memory B cells

experiments show circulating antibody levels drop v quickly during infection

Question 38

What are atypical B cells?

Answer 38

atMBCs

lack CD21 and CD27
– lack memory responses

no CXCR5
– not found in GC

increased expression of inhibitory receptors
– eg CD85j - inhibitory receptor also found on NK cells

heterogenous in their markers, thus may function differently

contribute to hypergammaglubulinemia
– polyclonal antibodies abundant in malaria
– IgG1

Question 39

How do atMBCs arise?

Answer 39

IFNy can stimulate expression of T-bet in human B cells = atMBC differentiation

TLR9 recognition of protozoan DNA and hemozoin triggers expression of IFNy

T-bet expression upregulates the atMBC markers–> HLA-DR, ICOS-L, CD86 –> Th1 polarisation –> expansion of Tfh cells (positive feedback loop)

Question 40

What percentage of B cells do atMBCs account for in different individuals?

Answer 40

2-4% in US adults

15-20% in Mali children
up to 40% in Mali adults

Question 41

What are current challenges w T cell responses against malaria?

Answer 41

dominant T cell epitopes targeted do not represent protective epitopes

tissue-resident T cells are present but prove difficult to access in clinical disease

Tregs abundant
- removal of Tregs resolves parasite burden

mixed characterisitcs
– TR1 and Th1
– coproduction of IL-10 and IFNy

Question 42

What are TR1 cells?

Answer 42

terminally differentiated Th1 cells that express IL-10
– arise from IL-27, IL-10, and/or Type I IFN
– FOXP3 negative, no T-bet
– also produce IFNy

Question 42

How doe TR1 cells arise?

Answer 42

CD169 macrophages (working via STING) instruct pDCs in BM to produce IFN-I
– can inhibit Th1 and drive TR1 response

via IL-10 and IFNy can vlunt Tfh cells
– knock on effect to GC-B cells
– Plasma cells polyclonal and not optimal for Ab generation

Question 43

What was the E2020 programme?

Answer 43

goal in 2016 to eliminate malaria in 21 countries by 2020

by 2021, 8 countries succeeded

Question 44

What is the E-2025 programme?

Answer 44

elimination of malaria in 25 countries by 2025

each gov has a plan

to qualify
– each needed fewer than 3000 cases in 2019
– capacity to confirm 100% of infected malaria cases

Question 45

How many merozoites are produced from a hepatocyte?

Answer 45

hepatocytes produce 10-30k merozoites per cell
– other cells such as the a skin cell only produce 100s of merozoites at most

Question 46

Why are hepatocytes crucial to Plasmodium infection?

Answer 46

sporozoites induce HO-1 (heme oxygenase) expression in hepatocytes
– protects infected cell from apoptosis, increase number of parasites that can replicate

nutrient rich
- rich in iron needed for parasite metabolism

immune privileged organ
- not as prone to immune surveillance as other cells

Question 47

How do hepatocytes respond to infection?

Answer 47

recognition of parasite through nucleic acid or hemozoin (TLR)

65 genes upregulated, all involved in innate immunity –> type I IFN

response is propagated in neighbouring cells
– Ifnar receptor detects IFN-1
– response propagated by hepatocytes, not by other cells eg macrophages

Ifnar deficient mice show 2-3 fold increase in parasite load

Question 48

How does IFN signalling combat Plasmodium infection?

Answer 48

increase in intracellular ROS and NOS

enhancement of apoptosis

upregulation of MHC

Question 49

How is the parasite detected within the cell?

Answer 49

unknown since parasite is contained within Parasitophorous vacuole, not exposed to cell receptors

Question 50

What is a merosome?

Answer 50

created when infected hepatocytes package merozoites into a single membrane-bound vesicle

These are released into the bloodstream, where they rupture and release merozoites which infect RBCs

they allow the parasite to efficiently spread to the bloodstream while minimizing immune detection

up to 1000 merozoites stored within a single merosome