Mohd Shahab

ID No. 52499
Division of Parasitology
CSIR - CDRI
Outline of the presentation


What are Neglected Tropical Diseases (NTDs) ?
Introduction to Lymphatic Filariasis (LF)
Its Immunology
Ongoing scenario on eliminating LF
Newer approaches for combating LF

Group of tropical infections which are especially endemic in
tropical & sub tropical regions and caused by diverse
pathogens, majority being parasites.

NTDs affect an estimated 100 Crore people living in:
Remote rural areas and
Urban slums of tropical countries
NTDs are endemic in 149 out of 260 (57.30%) countries
and territories
Of these,
At least 100 countries are endemic for 2 or more NTDs
And 30 countries are endemic for 6 or more NTDs


Neglected Tropical Diseases
In all 17 NTDs have been profiled in the first WHO
report on neglected tropical disease

Parasitic
Chagas disease (American trypanosomiasis)
Cysticercosis
Dracunculiasis (guinea-worm disease)
Echinococcosis
Human African trypanosomiasis (sleeping sickness)
Leishmaniasis
Lymphatic filariasis (elephantiasis)
Schistosomiasis (bilharziasis)
Onchocerciasis (river blindness)
Foodborne trematode infections
Soil-transmitted helminthiasis
Viral
Dengue
Rabies
Bacterial
Trachoma
Buruli ulcer (Mycobacterium ulcerans infection)
Leprosy (Hansen disease)
Endemic treponematoses

A large body of evidence, published in peer-reviewed
medical and scientific journals, has demonstrated that
NTDs adversely affect morbidity and mortality

This refutes the once-widespread assumptions held by
the international community

Have an important impact on morbidity and
mortality




Can be controlled, prevented and possibly eliminated
using effective and feasible solutions

WHOs Strategies:
1. Preventive chemotherapy;
2. Vector control;
3. The provision of safe water, sanitation and hygiene; and
4. Veterinary public health (that is, applying veterinary
sciences to ensure the health and well-being of humans)

These strategies make control; prevention and even
elimination of several NTDs feasible at a low cost
In 2007, WHO launched a Global Plan to Combat
Neglected Tropical Diseases
Lymphatic Filariasis
Lymphatic filariasis (LF) is a mosquito borne disease caused
by thread like lymphatic dwelling nematodes and causes
significant disability as well as loss of productivity in
developing nations.

The worms live in the human lymphatic system and can
cause:
lymphedema (swelling) and elephantiasis in limbs and
breasts
hydrocele (severe fluid accumulation) affecting mens
genitalia


Lymphatic Filariasis (LF) is one of the oldest
and most debilitating of all the NTDs.

Caused by three types of parasitic worms: Wuchereria
bancrofti, Brugia malayi, and B. timori.
Transmitted by female mosquitoes. When an infected
female mosquito bites a person, she may inject the worm
into the bloodstream.
Acknowledged as a leading cause of permanent and long
term disability.
Responsible for generating social stigma, isolation,
psychological stress.
And huge economic loss among affected individuals.
Endemic in tropical regions
Asia, Africa, Central/South America


Some key facts


Nearly 1.33 billion people in 80 countries worldwide are
threatened, commonly known as elephantiasis.
Over 129 million people are currently infected, with about
40 million disfigured and incapacitated by the disease.
In India it is endemic in 17 States and six Union Territories
with about 553 million people in peril making one-third of
the global LF burden


Cont.

LF caused by obstruction of the lymphatic system, which
results in the accumulation of a fluid called lymph in the
affected areas.
Results in an altered lymphatic system and the abnormal
enlargement of body parts, causing pain and severe
disability.
Acute episodes of local inflammation involving the skin,
lymph nodes and lymphatic vessels often accompany
chronic lymphoedema.
Diagnosis

Parasitological - Finding mf in blood
DEC provocation test - 50/100 mg DEC flushes out Mf in
blood during day
Clinical diagnosis- based on symptoms
Immunodiagnosis
Antibody detection based - IgG4 dependent using rAgs
(BmR1; BmSXP)
Antigen detection based (commercial)
- ELISA - semi-quantitative
- Simple card test (ICT immunochromatographic)


Treatments
Drugs:
DEC (Diethylcarbamazine) - Single dose recommended
(300 mg)
Ivermectin (20-200microgram, s.c. single shot)
Albendazole (400 mg tablet)-Broad spectrum anthelmintic
Antibiotics : doxycyclene, Moxidectin ( in clinical trials)



Surgery for massive leg swelling (elephantiasis) with a
fluid shunting procedure.
Pressure bandages to wrap the swollen limb and elastic
stockings to help reduce the pressure. Exercising and
elevating a bandaged limb also can help reduce its size.
Rigorous cleaning of the affected areas of the body.

Other treatments
Global Programme to Eliminate LF (GPELF)
Launched by World Health Organization (WHO) in 2000
Target elimination date of 2020
Two-pronged strategy to:
1. Interrupt the spread of infection
2. Reduce the suffering of persons already infected
To interrupt transmission WHO recommends an annual
mass drug administration of single doses of medicine to
all the eligible people in endemic areas.
At least 5 rounds on MDA are needed to interrupt
transmission

GPELF: Progress and successes
53 countries have ongoing MDA campaigns
37 countries have administered 5 or more rounds of
MDA in many target areas
2.8 billion doses of medicine delivered in first 9 years
Transmission interruption has protected 6.6 million
newborns from becoming infected with the disease

National Filaria Control Programme (NFCP) was launched
by India in 1955
The main control measures were mass DEC
administration, anti-larval measures in urban areas and
indoor residual spray in rural areas.
The programme became a part of the National Vector
Borne Disease Control Programme (NVBDCP) in 2003
and, aims to eliminate lymphatic filariasis by 2015 under
National Health Policy 2002.
India accounts for about 40% of the estimated cases
globally with either disease or infection (microfilaria
cases)

In India..
Immunology
Disease is spectral in nature
Many infected individuals showing no clinical signs,
while others suffer the consequences of chronic
stages.
Distinct antibody isotypes are associated with different
disease states
Current research in mouse models of infection is
elucidating the immunological mechanisms that can
lead to immunity against this disease


Cont.


Majorities are asymptomatic due to polarization of
immune responses to agents into either Th1 or Th2
types.
They have inability to proliferate or produce interferon
gamma (IFN-) in response to the antigen.
Initially, this regulation was ascribed to predominant IL10
response .
It is clear that a range of regulatory events are involved
including TGF-, CTLA-4.
Protective immunity in filariasis
The mechanism(s) by which different parasite stages are
killed still remain elusive, most findings result from work
comes from mouse model

It has been established that the infective larval (L
3
) and
adult stages of the parasite primarily induce type 2
responses.
Thus, during natural infection the subsequent maturation of
adult nematodes following L3 invasion, is likely to enhance
and fully establish this type 2 response.
Intriguingly, single-stage Mf injections primarily induce an
inflammatory type 1 response.
Vaccination
No vaccines are available for prevention of infection


Vaccination in experimental animals shows substantial
protective immune response.
- with attenuated infective larvae, excretory-secretory
(ES) antigens, mitochondria rich (MT) fraction, recombinant
and DNA vaccines.
A number of filarial antigens have been cloned in the past
however; many of these either await immunoprophylactic
evaluation or failed to produce adequate protection
The potential for use of live filariasis vaccines in humans is
limited because of safety issues and limited availability of
larvae.
Filarial parasite killing?
The immunological mechanism used by the host to kill large
helminth is classically thought to involve antibody-dependent
cell-mediated cytotoxicity (ADCC)



Antibody attaches to parasite surface by FcR, recruitment of
M, eosinophils and neutrophils extruding toxic mediators on
parasite surface










Despite of this vast knowledge and program
LF still remains a major problem

About 160 million people still remain infected with
further billions and more are at the risk

More and more research/studies are needed

Genomic approaches against LF, provides newer
prospects in drug discovery either in target identification
or its validation.
Large number of genomic data could possible paves the
path regarding newer methods.
Crucial for identification of vaccine antigens, their
molecular characterization, genetically modified proteins,
more effective human compatible adjuvants and vaccine
delivery systems would surely facilitate the antifilarial
vaccine discovery research.
Newer approaches for anti-filarial
drug/vaccine discovery
Screening of the target via genomic approaches opened
new opportunities.
Filtering through genomic comparison provides us to
select pathogen-specific gene products.
Functional genomics allow the selection of gene products
which are necessary for the pathogen survival.
Subsequent to this, the identified and validated targets are
put for in vitro and in vivo inhibitors studies including small
and high throughput assays, in silico drug design, lead
discovery/optimization, development and trials.
Molecular target based compound discovery
Selecting anti-filarial drug targets based on genomic data
must have two basic considerations.
1. The target genes should not possess close homologues
in the host (humans).
2. It should be possible to identify therapeutic agents
(inhibitors) with reduced or no host cross-reactivity.
Since full human and filarial genomes are available,
therefore comparative analysis along with efficient
bioinformatics can easily reveal the similarity index of
filarial parasite gene or its product to the human host

Comparative Genome Filtering

The reported B. malayi genome is

~

95 Mb
The genome is estimated to contain >11,500 genes.
Comparative assessment of the transcriptome of different
developmental stages of B. malayi helps us to determine
when and where the genes of interest are turned on or off.
Proteomic data in context to ES and somatic proteins
produced, indentified 7,103 (>60%) of the proteins
predicted in the genome in which 2,336 genes have been
assigned as hypothetical/predicted status before.
Thus transcriptomics and proteomic approach provides
valuable information on expression profile and helps in
predicting unique genes/proteins which are important for
the survival of filarial parasite
Genome analysis
Comparative genomics involves the use of computer programs that
can align multiple genomes and look for the similarity among them.

Analysis at gene level among nematodes genomes, indicate that
~
45% of the predicted protein-coding genes represent novel and
species-specific.

Comparative analysis shows that >35% of B. malayi genes have C.
elegans matches. So the functions of genes conserved among
nematodes can be investigated using C. elegans.

In silico comparison identified a set of 589 candidate drug targets in
B. malayi which are already characterized in C. elegans.

Presently, only C. elegans provides surrogate target validation and
has been endorsed by WHO as promising approach towards
discovery of new antifilarial drugs

Genome Filtering Tools

Bunch of heritable changes in gene function, that cause
different phenotypes, occurs without altering DNA sequences.
[Might be regulated by DNA methylation, histone acetylation/deacetylation,
post genomic silencing using small interfering RNA (siRNA) or micro RNA
(miRNA) via RNA interference (RNAi) etc]
miRNAs are found to be essential for the development
activities in C. elegans and has been demonstrated in various
organisms.
Genome of B. malayi contains many elements which are
required for miRNA processing. Deep sequencing and
bioinformatics approaches, identified 145 miRNA of 99 miRNA
representing families.
Of these, 60 families are conserved in other species while 11
are restricted to helminthes.

Epigenetic studies

Only 9 miRNA families have been found to be common with
other filariid species investigated further.
Since developmental processes in several parasitic nematodes
share similarities with those of C. elegans, role of miRNA in
filarial development cannot be denied.
Many novel functional miRNAs have been identified in filarial
parasites during in silico comparison studies.
It would be worth investigating the targets of B. malayi
miRNAs, in target discovery to identify and determine their
involvement in maintaining parasitism for better understanding
of epigenetic regulation.

Cont.
Identifying the function of every gene in all sequenced
organisms is one of the major objectives of the post-
genomic era.
The application of functional genomics in drug discovery
provides the opportunity to incorporate rational
approaches to the process and to prioritize and validate
the drug target.
It includes microarrays, proteomics, and RNAi.

Functional Genomic Filtering

Microarrays

Microarray analysis has been extensively applied to define
expression pattern of a gene at genomic level
In parasitic nematode, technique holds great promise as a
means of studying stage-specific or tissue-specific expression of
genes.
Genome-wide analysis of gender-associated gene transcription
in filarial nematodes identified numerous potential candidates
for rational drug design which can target worm reproductive
processes.
Though, the technique remains expensive however the data
generated from large research groups can be utilized for
screening studies, as they are freely available in the microarray
libraries.

Proteomics

There is a poor correlation between the regulation of
transcripts and actual protein quantities since genome analysis
does not account for post-translational processes such as
protein modifications and protein degradation.
Proteomics allows large-scale study of protein properties such
as expression levels, post-translational modifications and
interactions with other molecules to obtain a global view of
cellular processes at the protein level and need to be
employed in drug-discovery processes.
Functional and expression profiles from different life cycle
stages of filarial worms along with the endosymbiont
Wolbachia can be mapped down by using this approach.

RNA interference (RNAi)


Target validation - an important aspect in drug discovery as it
prioritizes the identified gene for the usefulness as a potent target
candidate.
Recently, in vivo silencing has been demonstrated in B. malayi
within the mosquito Aedes aegypti leading to suppression (83%)
in B. malayi cathepsin L-like cysteine protease gene resulting in
multiple aberrant phenotypes, motility, and partial-paralysis.
Our group as successfully silenced the Bm- iPGM and TPP gene,
which impairs the development.

Wolbachia : An anti-filarial Drug Target
A gram-negative intracellular bacteria - present in all the
stages of the life cycle of human filariids in a mutualistic
symbiotic association.
Essential for the development, fertility, and survival of the
filarial worms.
Antibiotics like Tetracycline, Doxycycline, Rifampicine and
Azithromycin kill these bacteria antifilarial drug target?

The genome sequence of the Wolbachia (wBm)
endosymbiont from B. malayi offers good opportunity for
understanding symbiosis between the bacterium and its
filarial host.
It would help in the identification of biochemical processes
and pathways of Wolbachia and that could serves as
novel anti-filarial drug targets.
It has 1.08 Mb circular genome which bears extremely low
density of predicted functional genes compared to all other
bacteria.
Lacks genes to produce lipopolysacharride but produces
inflammatory reaction via TLR4
Genome
It is predicted to encode only 806 protein-coding genes
demonstrating loss during co-evolution and co-
adaptation.
Comparing metabolic pathways, indicated that the
biosynthesis of riboflavin, flavin adenine dinucleotide
(FAD), heme and nucleotides may be important for the
symbiotic relationship with the host as genes for these
pathways are lacking in B. malayi genome.
Filarial parasite likely provides amino acids required for
Wolbachia growth, since wBm can synthesize only one
amino acid, mesodiaminopimelate, a major component of
peptidoglycan.
It has type IV secretion system.
Cont.
Wolbachia
Many of the basic biological characteristics of Wolbachia
have yet to be elucidated, but the results obtained to date
suggest that hosts can be exposed to Wolbachia:
when larvae, or adult worms, are killed by drug
when Wolbachia are expulsed- naturally
with the release of microfilariae from female
possibly through the excretory system of worms
All are involved in the pathology and adverse reactions
Wolbachia in filarial pathology
LPS is considered to be the major mediator of
inflammatory response in gram-negative bacteria.
Wolbachia Lipoprotein stimulates innate and adaptive
immune response through Toll-like Receptors which
induces disease manifestations of filariasis.

In addition, it has been speculated
Heat shock proteins
CpG motifs in DNA
Wolbachia surface protein (wsp)
Peptoglycan
May also be involved in
pathogenesis of bacterial
disease
Proteomic analysis of the various stages of B. malayi
identified 557 of the 805 wBm predicted proteins.
96 out of 166 hypothetical/predicted genes were validated
as producing a protein during one or more stages of filarial
development.
Studies have predicted some essential gene repertoire of wBm and several
essential biochemical processes that represent candidate for drug targets.
These include
heme biosymthesis
lipid II biosynthesis
lipoprotein biosynthesis
glycolytic enzymes - pyruvate phosphate dikinase (PPDK) and
cofactor-independent phosphoglycerate mutase (iPGM).
Among these several are under process for further evaluation as potent
targets or are subjected to high throughput screening for inhibitors studies
Wolbachia Cont.
The need of today
Unlocking the identity of new drug targets by
comparative and functional genomics.
Validation studies context to the role of the target
molecule in the worm and/or endosymbiont -
Wolabachia.
More and more in-vitro and in-vivo studies
Screening of the effective compounds (HTS)
Newer drug delivery approaches
Drugs with strong macrofilaricidal activity
Thank you