Bio 54

2002

Filarial Worms III: Biochemistry, Molecular Biology, and Immunology

Andrew G. Campbell, Ph.D.

Biochemistry & Molecular Biology

Methods for Study Energy Generation The Microfilarial Sheath Chitin & Chitinases Vector Resistance to Filarial Infection
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Experimental Models for Study of Filarial Worms

Lymphatic Filariae
B. malayi can be experimentally maintained in gerbils (natural reservoir hosts) and in mosquito vectors various stages of W. bancrofti and B. malayi can be transferred to mice

Nonlymphatic Filariae
Onchocercal keratitis (river blindness) can be modeled in rabbits and rodents Adult worms can be maintained in vitro
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must be obtained from human or cattle nodules

Onchocercal Keratitis

Human

Mouse

Experimental Techniques
in vitro culture
manipulation of nutrients, temperature, drugs, etc.

genetics
use of mutant Caenorhabditis elegans (nonparasitic nematode) to determine the function of parasite genes

molecular biology
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identification of important stagespecific genes via differential screening

Differential screening
cDNA probe from: worm mRNA cDNA cDNA library cDNA probe
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Day 3 L3

mosquito L3

cDNA on filter from Day 3 L3 larvae

Energy Generation in Filariae: General Considerations

Carbohydrates (CHOs) from host are a major energy source filarial worms have relatively low reserves of glycogen (CHO storage molecule) filariae have traditionally been regarded as anaerobic fermenters, producing lactate from glucose role for aerobic metabolism?
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probably varies by species environmental factors

Energy Generation in Filariae

Microfilariae
require oxygen for motility aerobic due to higher energy requirements?

Target for drug development

exploit differences in enzyme structure & regulation

Caveats
problem of extrapolating results from one species of worm to another in vitro vs. in vivo issues
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The Microfilarial Sheath

MF of W. bancrofti , Brugia spp., and Loa loa retain remnant of eggshell as an extracuticular "sheath" O. volvulus MF do not have a sheath. sheath maintained while in vertebrate blood but shed during development in insect
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Ensheathed microfilaria

The Microfilarial Sheath

Structure
at least 5 proteins, including chitinase sugars (chitin?) inorganic compounds (e.g. phosphate) host proteins about 25% unknown highly cross-linked

The sheath is the host-parasite interface

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The Microfilarial Sheath

MF form the sheath by stretching and remodeling their eggshell membranes as larval body develops within "exsheathment" is necessary for subsequent molting and development of larva in the insect blocking sheath formation or exsheathment could reduce transmission

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Chitin
polysaccharide/polymer of N-Acetyl-glucosamine (NAG) 2nd most abundant biopolymer on earth major component of insect & crustacean exoskeleton also found in the eggshells of filarial worms NOT a constituent of vertebrates Chitin metabolism could thus be exploited as a target for drug development
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Microfilarial Chitinase
two isoforms (p70 and p75) have been identified in B. malayi encoded proteins highly conserved (relative to yeast and bacteria) products of 2 different, but homologous, genes originally identified by reactivity with the monoclonal antibody MF1, which is capable of reducing microfilarial burden in infected gerbils expression weak or absent in intrauterine larvae and recently shed MF production/expression coincides with microfilarial infectivity of mosquitoes

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Possible Functions of Chitinase

Escape of parasite from digestive tract in the insect host
Plasmodium and Leishmania produce chitinase Plasmodial enzyme may allow the parasites to penetrate the peritrophic membranes surrounding the fresh bloodmeal taken by the insect vector. in Leishmania, chitinase is believed to rupture the pericardial lining and thus cause the sandfly to regurgitate its bloodmeal Microfilarial chitinase may perform similar functions

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Possible Functions of Chitinase

Support of parasite infection in insect vector
production of inhibitor oligosaccharides to prevent agglutination by lectins precedent: In gut of Tsetse fly, if trypanosome chitinase is blocked, fly becomes less susceptible to infection

MF sheath morphogenesis/ Exsheathment

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the egg contains chitin, but does the sheath?

Chitinases at Other Stages

adult female
hatching of the egg? resorption of egg shell following hatching? possible target for drug to block MF release

Infectious third stage (L3) larvae

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migration in the insect vector? escape from mouthparts?

Vector Resistance Mechanisms

species/strain specificity on part of parasites and vectors mechanical damage to larvae by blackfly teeth coagulation of bloodmeal physical barrier of the gut membranes agglutinins (lectins) in gut defense molecules in insect hemolymph

Innate

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Vector Resistance Mechanisms

expression of defense molecules in hemolymph is elevated upon infection types of inducible defense molecules
antibacterial peptides agglutinins (lectins) phenoloxidases humoral proteases

Inducible

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no memory as in the vertebrate immune system

Filarial Parasites: Immunology

General Considerations The Immune Response & Disease Evidence for Immunity Tolerance
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General Considerations
Parasites, in general, dont want to injure or kill you, rather they have an interest in keeping you alive
To satisfy their needs for nutrition and shelter To make you a long-term reservoir for dissemination

Filarial parasites tend to cause chronic infections

Most people with filarial infections dont show symptoms, but are effective parasite reservoirs There must be some mechanism to strike a balance between host and parasite

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Parasite exposure

no exposure

?
infected, no symptoms infected, disease not infected, no symptoms

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Infected, No Symptoms
microfilaria (MF) positive immunologically hyporesponsive to the parasite
impaired B and T cell proliferative responses to parasite and unrelated antigens reduced production of Th1-associated cytokines: IL-12, IL-2 and IFN- elevated IL-10 generally absent or low antibodies specific to adults or MF IgE responses and eosinophilia can be present

do not clear MF well, thus provide "reservoir" for infection of others treatment can induce symptoms in these people!
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Infected, with Disease

symptoms
early: filarial fever, lymphatic inflammation severe: TPE, elephantiasis

microfilaria (MF) negative immunologically hyperresponsive

strong B and T cell proliferative responses production of Th1 (IFN- ) and Th2 cytokines (IL-4 and IL10)

clear MF well, thus do not serve as transmission reservoir treatment generally improves symptoms, if given early
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Not Infected, No Symptoms

no overt evidence of parasites microfilaria negative
unexposed? (unlikely) undetectable infection? (unlikely)

immunity?

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Evidence for Immunity from the Mouse Model

limitation: mice are not completely permissive to lymphatic filariae, not a perfect model system antibody-mediated immunity to microfilariae
sera from mice immunized with MF extract transfers 75100% protection from MF challenge to other mice IgG2a and/or IgM probably has importance

T-cell mediated immunity to L3

transfer of primed T-cells, but not serum (antibodies) or Bcells, results in clearance of L3

T-cell mediated immunity to adults

adult worms survive longer in athymic mice
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Evidence for Immunity in Humans

onchocerciasis
putatively immune individuals make a Th1-like response to O. volvulus antigens

lymphatic filariasis
19 persons who maintained immune status for at least 17 years made much stronger parasite-specific T-cell responses than infected controls immune persons were found to make responses to the MF-1 epitope of chitinase at greater frequency than infected controls
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Factors Influencing the Immune Response to Filarial Worms

Genetics - role of HLA (MHC)
certain HLA haplotypes found to correlate with disease/infection (both positively and negatively)

neonatal exposure
MF positive mother tends to give birth to MF+ asymptomatic infant infant has elevated Th2, low Th1 response

parasite effects
parasite burden certain parasite strains may be more immunogenic than others

tolerance induction
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Tolerance Induction
evidence exists that indicates that the parasite actively induces immunological "tolerance" (unresponsiveness) in its host probably induced by soluble factors produced by the worm egg antigens? filarial cystatin seems to be directed at Th1 T-cells low IFN- and IL-2 reduced DTH responses
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Immunological Tolerance
Why induce tolerance?
prevents parasite destruction keeps host alive (good for you) and able to pass on infection (good for the worm)

is usually long-lasting breakdown causes pathology appears to be directed at certain stages (adult and MF) but not infectious L3 larvae - concomitant immunity
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Concomitant Immunity
immunity

L3

adult
insect stage

MF

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Tolerance Breakdown
immune response

L3

adult
insect stage

MF DISEASE

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Vaccination
VACCINE
Pathology reduced

L3

adult
insect stage
Transmission reduced

MF

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Take Home Points

1) An interaction of host and parasite-specific factors likely determines who gets disease 2) The pathology of filariasis is immune-mediated, but an immune response can also protect from infection 3) The worms appear to actively "tolerize" their hosts, and disease results when tolerance breaks down These points must be carefully considered if vaccination strategies are to be attempted!
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