Experimental Models

Question 1

Why is malaria still a problem?

Answer 1

• environmental conditions
• poverty, poor housing, lack of infrastructure, economics, wars etc
• vector (mosquito) hard to control
• parasites have short generation time
• rapid parasite evolution in response to intervention eg drug resistance, potential for vaccine escape mutants

Question 2

How are malaria infections studied in humans?

Answer 2

controlled human malaria infection (CHMI)

used as treatment for neurosyphilis in early 1900 (p. vivax and p. falciparum)
– high fevers caused by malaria killed bacteria pre antibiotics

now used to test drugs and vaccines, and research into malaria immunobiology

infection of human volunteers via infected-mosquito bite or blood passage
– infections are cured by drugs at low parasite density (severe malaria cannot be studied)

Question 3

What are the advantages of CHMI?

Answer 3

infections are highly controlled and monitored

parasites used are well characterised

detailed evaluation of anti-malarial drug kinetics, accessibility, toxicity, and parasite-killing rate

proof of concept efficacy data for vaccines, including titre and longevity of Ab response and degree of protection conferred

detailed data on immune responses and parasite adaptations

possibility of long follow up, re-infections etc

Question 4

What are the limitations of CHMI?

Answer 4

hosts differ from main affected global population (african children)
– chmi mostly carried out in naive, healthy adult volunteers
– few trials in malaria endemic regions

little genetic diversity in parasite inoculum (<5 parasite genotypes available for CHMI)

infections can only be studied at v low parasitaemia (outcome if left untreated?)
– no severe malaria
– no chronic infections

obvs cannot study liver stage

Question 5

What is the course of untreated P. falciparum infection?

Answer 5

• high initial numbers (>10 000 parasite/microlitre)
• numbers soon decrease
• threshold for detection through good RDT/microscopy = >100 parasite/microlitre
  – numbers after every burst = threshold for microscopy
• threshold for detection through PCR/LAMP = >10 parasite/microlitre
  – <10 parasite/microlitre = asymptomatic

Question 6

What are the advantages of rodent models?

Answer 6

fewer ethical problems than using humans or primates

in vivo

defined genetic background of hosts
- genetics more easily manipulated to test particular cell types/cytokines

direct imaging of host-parasite interactions possible

potential of humanised mice

can study liver infection

Question 7

What are the limitations of rodent models?

Answer 7

course of infection different to human malaria (lack of or limited recrudescence (reactivation of malaria due to incomplete eradication))

clinical features differ: hypothermia, heart failure

major difference is pathological features: no widespread sequestration of infected RBCs in tissues/organs

differences in immune responses

differences in liver infection compared to humans
– P. berghei can potentially complete cycle in skin fibroblasts at site of inoculation without requiring hepatic cycle

Question 8

In the context of detection by the immune system, what advantage do mice have over humans?

Answer 8

mice express TLR 11 and 12, which recognises profilin, a protein expressed in protozoan parasites including plasmodium

profilin responsible for motility through skin using actin filaments

Question 9

Give some info on P. chabaudi.

Answer 9

seen as most similar to human infection; acute infection followed by a resolving chronic infection
– in susceptible mouse strains, pathology/death associated w severe malaria
– pathological features different (limited sequestration of infected RBCs)

many genetically distinct strains of P. chabaudi are available
- investigation of strain specific immunity
- effect of co-infection on development of immune responses

Question 10

Give some info on P. berghei.

Answer 10

forms acute infection that does not resolve and is lethal
– death due to severe anaemia/organ failure called ‘experimental cerebral malaria’
– pathology is completely different to human cerebral malaria (controversial?)
– sequestration does not occur in mice

easiest malaria parasite to genetically manipulate

Question 11

Why is P. berghei easy to genetically manipulate?

Answer 11

It has a high transfection efficiency
– DNA only needs to cross the erythrocyte membrane, the parasite plasma membrane, and the nuclear membrane to be transfected

(how is this different from other species?)

Question 12

How has P. berghei been used in gene identification and vaccine research?

Answer 12

– widely used for analysis of gene function throughout life cycle
– identification of essential genes including those for transmission and development in liver

– preliminary work leading to design of transmission-blocking and attenuated-sporozoite vaccines in humans

Question 13

What Old World primate is used as a NHPIM, and what strains are used as models for human malaria?

Answer 13

macaque
many basic elements of the immune system are shared

the four known human malaria species (P. falciparum,P. vivax,P. malariaeandP. ovale) do not develop in the blood of macaques

– P. coatneyimodel for P. falciparum
– P. cynomolgias a model forP. vivax
- is also v genetically manipulated
– P. knowlesias a model for both

Question 14

What Old World primate is used as a NHPIM, and what strains are used as models for human malaria?

Answer 14

Saimiri and Aotus

– P. simium,which is genetically and biologically similar toP. vivax,
– P. brasilianum, which is genetically and biologically similar toP. malariae

new world can be infected w P. falciparum

Question 15

What are the benefits to using NHPs as models for malaria infection?

Answer 15

able to study relapsing malaria caused by P. vivax hypnozoites (P. cynomolgi in NHPs)

all of the primate plasmodium species and NHP genomes have been sequenced, allowing studies of influences of infection on gene expression of both parasite and host

NHPs can be used to validate studies performed in rodents
* rodents –> NHPs –> humans

peripheral blood B cell subsets and T-cells can be phenotyped and classified similarly between humans and macaques

NHPs can develop severe malaria syndromes similar to humans, such as cerebral malaria and severe anemia

generation of many parasites for use in other experiments

study of CM via MRI brain scans

Question 16

What are the downsides to using NHPs as models for malaria infection?

Answer 16

obvious ethical concerns
– high cognitive abilities and capacity for suffering

smaller sample sizes

expensive to take care for

New World monkey systems-based research limitations include repeated tissue sampling and volume limitations imposed by their small size (~ 1 kg)

studying chronic infections difficult as it would require monitoring of a single individual over a long period of time

fewer genetic tools and transgenic approaches are available

Question 17

How was a CHMI study used to look at differences is gene expression between individuals after infection with malaria?

Answer 17

14 volunteers injected w P. falciparum-infected blood

followed for 7-11 days, blood collected every 2 days

3 sets of people found:
– inflammatory = increased gene expression
– suppressive = decreased gene expression
– unresponsive = no change in gene expression

clinical symptoms also varied and matched transcriptional responses

who would go on to develop severe malaria?

Question 18

Do host immune cells contribute to severe malaria pathology?

Answer 18

no widespread presence of inflammatory cells such as neutrophils and monocytes at sites of sequestration

limited evidence for monocytes or T cells at sites of brain sequestration in fatal human CM
– pronounced feature in experimental CM mouse model

Question 19

How does the spleen restructure in malaria infection?

Answer 19

mouse models

during infection, open circulation turns into a closed system
– forms closed fibroblast barrier associated w fibronectin and collagen

white pulp become disorganised
– causing loss of B cells in marginal zones
– T and B cells scattered due to loss of GCs

Question 20

How does haematopoiesis change during malaria infection?

Answer 20

increasing demand for myeloid cells, recuing BM capacity to produce erythrocytes and lymphocytes

loss of B cell progenitors during peak parasitaemia is associated w increased B cell development in spleen
– mouse models show the development of atypical B cells

Question 21

Can gametocytes be studied in experimental models?

Answer 21

it is common for lab isolates to lose the ability to produce gametocytes in vitro after prolonged cultivation due to the absence of selection pressure to transmit

Question 22

What are humanised mice?

Answer 22

mice that express human genes or have been engrafted with human tissues

Question 23

How are humanised mice generated?

Answer 23

transplantation with either primary human hepatocytes or red blood cells under immunodeficiency conditions to prevent xenorejection

Question 24

What are the benefits to humanised mice?

Answer 24

becoming attractive and suitable models as a potential substitute to NHPs

various organs and or tissues can be transplanted into mice, becoming a more convenient approach to modelling several aspects of malaria parasite biology as well as disease pathogenesis

for example, recent experiments have shown in vivo P. falciparum cytoadhesion

Human liver chimaeric mice are currently available to examine the biology of sporozoite invasion of hepatocytes

Question 25

What are the current downsides to humanised mice?

Answer 25

still lack sufficient tissue or organ penetration of the engrafted cells due to the hostile mouse microenvironment for foreign human cells to survive and proliferate

the use of immunocompromised mice (SCID) limits the study of certain aspects of host–pathogen interactions such as host immune responses

Question 26

What is the P. berghei ANKA strain?

Answer 26

an experimental cerebral malaria (ECM) model, in which mice develop similar neurological features to human CM
– paralysis, coma, and death

Question 27

What are the differences between ECM and human CM?

Answer 27

sequestration of iRBCs can occur in the lung but accumulation in the brain does not correlate w pathogenesis
– use of a different P. berghei strain (NK65) shows occlusion of brain micro-vessels

However, desth in human CM is caused by brain swelling depressing brain stem which regulates breathing
– not been shown in ECM

ECM also appears to be largely an inflammatory syndrome

Question 28

How has the liver stage in mice been used to come up w new treatments?

Answer 28

Recently, it has been demonstrated that immunization of mice with late-stage arresting sporozoites that lack a type II fatty acid synthesis enzyme (Pyfabb/f−) result in robust humoral as well as CD4 T-cells immune responses capable of blocking follow-up challenges with wild-type sporozoite

Question 29

What type of research are humanised mice used in?

Answer 29

used for pathogenesis rather than immune response
– however, mice transplanted with immune system components (CD4 and CD8 T cells, B cells) are being developed to combat this limitation

Question 30

How have in vitro assays been used?

Answer 30

used in exploratory evaluation of inhibitory activities of immune sera raised against sporozoites infecting cultured human hepatocytes

absence of the complex three-dimensional architecture of the ideal liver microenvironment always limit their physiological relevance

Question 31

Can humanised mice replicate human disease pathology?

Answer 31

Liver chimaeric mice which can sustain a low-level parasitaemia in the blood if transfused with human red blood cells
– parasites demonstrate tissue sequestration as hypnozoites

parasites are only maintained for short time due to rapid clearance of hRBCs