Outline Of Research Work Seminar

Cloning and silencing of Subolesin, Cathepsin L and

Calreticulin genes of Hyalomma anatolicum anatolicum

and evaluation of cross-protective efficacy of

recombinant protein(s)

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Dr. Binod KumarRoll No.1374Ph.D. ScholarDivision Of ParasitologyIVRI

INTRODUCTION

Ghosh et al., 2005,2007

106 species106 species

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TICKS

Major tick species in INDIA infesting livestock

Hyalomma anatolicum anatolicumCattle, Buffalo and small ruminantsThree hosts tick

Rhipicephalus (Boophilus) microplusCattle, Buffalo, Horse, donkeys, goat, Sheep, Deer, Pig, Dog, and some wild animalsOne host tick

Direct and Indirect effects

Tick attachmentDeep bite woundAnnoyancePredispose to myasisReduced hide value

Tick secretions Tick toxicosisTick paralysisTransmission of tick borne pathogens

Blood feedingAnemiaProduction and reproduction losses

ControlHigh cost of acaricide treatmentEcological damageHuman health

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Vectorial capacity

H. a. anatolicumTheileria annulata

T. buffeli

T. lestocardi

CCHF (????)

R. (B.) microplus

Babesia bigemina

B. bovis

Anaplasma marginale

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Economic impact of tick infestation on livestock industry

De Castro, 1997;

Minjauw and Mc

Leod, 2003;

Dayton et al., 1991;

Horn, 1987;

Mukhebi et al., 1999

* * Control cost

**

In million US $

8

Methods of Control Major Control Method

Side effects of chemical control method

Chemical control (Acaricides)

Biological control

Genetic transformation

Immunological control

Herbal formulation as

acaricide

Genetically resistance breeds

Acaricides

Selection of resistant

ticks

Environmental pollution

Residues in livestock

products

High cost of repeated

application

Development of new

generation acaricide

Sustainable control ????????

Ticks control strategies

Immunological control (component of IPM)

Immunization with crude antigen

Immunization with Purified Native antigen

Immunization with recombinant antigen

1986- Bm86 identified

1994s- Bm86 based vaccine TickGARD™ and Gavac™ introduced in

market

Reduction in number of application of acaricides

Reduction in incidence of Tick borne disease

Variable efficacy of vaccine against different strain of R. (B.) microplus

(40% - 90%)Cross protective efficacy was poor except R. (B.) annulatus

de la Fuente et al., 2007

Homologue of Bm86 was identified in different ticks species and efficacy was recorded in the range of 40-60% (Liao et al., 2007; Odongo et al., 2007; Kamau et al., 2011; Nijhof et al., 2010)

In India, cross-protective efficacy of Bm86 and its homolog, Haa86 in Hyalomma anatolicum anatolicum was recorded

Antigen Ticks Efficacy Reference

Haa86 H. a. anatolicum 46-80% Azhahianambi et al., 2009; Jeyabal et al., 2010; Kumar et al., 2012

Haa86 R. (B.) microplus 36% Kumar et al., 2012

Bm86 H. a. anatolicum 25% Kumar et al., 2012

Bm86 R. (B.) microplus 44% Kumar et al., 2012

Attachment to host

Salivary gland products like

Cement protein (Kemp et al., 1982)

Anti-haemostatics (Sauer et al., 1995)

Vasodilators, anti-inflamatory , immunosuppressive factors (Champagne, 1994)

Digestive system

Molecules involve in blood meal digestion (Lara et al., 2005)

Iron metabolism (Horn et al., 2009)

Gut associated molecules (Williadsen, 2004)

Haemocoel

Transporter molecules (de la Fuente et al., 2010)

Other molecules involved in physiology of ticks

Major areas of target

Targets selected for study

CalreticulinAnti-thrombotic and complement-inhibition activities in host

Cathepsin LPart of a gut-associated multi-peptidase complex. Its endopeptidaseactivity is important in the initial phase of haemoglobinolysis.

Subolesin(Expressed in all organs and tissues)Function as transcription factors in the regulation of gene expression

Objectives

1. Cloning and sequencing of Subolesin, Calreticulin and Cathepsin L

genes of Hyalomma anatolicum anatolicum and Rhipicephalus

(Boophilus) microplus

2. Evaluation of Conservation of target genes in different isolates of H. a.

anatolicum and R. (B.) microplus

3. Characterization of target genes of H. a. anatolicum

i). Through RNA interference

ii). In-vivo immunization trial using recombinant protein(s)

.•.

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REVIEW OF LITERATURE

Native proteins as Vaccine targets

Immunogen Challenge dose Percentage protection

Larvae Nymphs Adults Immediate rejection (%)a Overall decrease in successive stageb

Reduction in egg massesc

AFF-TLE (L), 39kDa 4000 200 - 60.0 (L),44.0 (N) 34.0 (N),43.2 (A)

Aff-GHLAg (Gut), 34 kDa)

2000 140 40 pairs 24.2 (L),22.4 (N),32.2 (A) 31.2 (N), 25.2 (A), 15.0

Aff-HNAg (Nymph, 39kDa

1600 140 40 pairs 38.0 (L), 25.0 (N), 32.2 (A) 32.7 (N), 28.7 (A), 20.0

Aff-GHAAg (A), 68kDa 2000 140 - 10.3 (L) 17.6 (N) -

HGLA (L), 34 kDa 2000 - 25 pairs 39.0 (L), 28.0 (A) 16.0 (N), 15.8

HGLA (L), 34 kDa 3000 - 75 pairs 32.0 (L), 23.4 (A) 29.32 (N), 50.67

GHLgP (Gut), 37kDa - 140 - 17.0 (N) 20.7 (A) -

aa Mean differences in immediate rejection between immunized and control groups of animal. Mean differences in immediate rejection between immunized and control groups of animal.bb Mean differences in the development of successive stage of the ticks fed on immunized and control group of Mean differences in the development of successive stage of the ticks fed on immunized and control group of animals.animals.cc Mean differences in the egg masses laid by the ticks fed on immunized and control group of animals. Mean differences in the egg masses laid by the ticks fed on immunized and control group of animals.

* p < 0.01.* p < 0.01.

** p < 0.05** p < 0.05Indian J. Exp. Biol., 1998, 1999; Trop. Anim. Hlth. Prod., 2003; J. Parasitic Dis., 2003; Indian J. Exp. Biol., 1998, 1999; Trop. Anim. Hlth. Prod., 2003; J. Parasitic Dis., 2003;

Trop. Anim. Hlth. Produ., 1999; Trop. Anim. Hlth. Produ., 2001, ; Exp. Appl. Acarol., 2000; Trop. Anim. Hlth. Produ., 1999; Trop. Anim. Hlth. Produ., 2001, ; Exp. Appl. Acarol., 2000; Indian J Anim. Sci., Exp. Appl. Acarol., 2003, Parasitology Res., 2005, Trop Anim Hlth Indian J Anim. Sci., Exp. Appl. Acarol., 2003, Parasitology Res., 2005, Trop Anim Hlth Prod., 2005; J Vet Parasitol., 2007; Vaccine 2008Prod., 2005; J Vet Parasitol., 2007; Vaccine 2008

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Recombinant proteins as Vaccine -

Bm86 and its homolog

TickGARD™, Gavac™

20-30% reduction in engorge tick number

30% reduction in engorge tick weight

60-80% reduction in egg masses

50-60% reduction in number of acaricide treatment in a year (Willadsen et al., 1995, de la Fuente et al., 2007)

S.N. Tick Strain Efficacy (E%)

1 Camcord 72-91

2 Yeerongpilly 75

3 Cenapa 84

4 Tuxpan 51

5 Mora 58

6 Colombian field 60

7 Brazilian field 51

8 Argentinain strain 0

Variable efficacy of Bm86 based vaccine against different strain of R. (B.) microplus

de la Fuente et al., 1995, 2005, 2006; Garcia-Garcia et al., 2000

The use of Bm86 based vaccine on cattle tick strains located in different

geographic areas has presented variable efficacy, even the so-called vaccine

failures that seemed to be due to variations in amino acids in the protein codified

by the Bm86 locus (de La Fuente and Kocan, 2003)

de la Fuente et al. (2000) characterized at the molecular level R. (B.)

microplus strains ( 10 strains) from Latin America and Australia,

employing sequences derived from the Bm86 coding region (base

No. 1646 to 1752)

Results

1% nucleotide variation within strains

7.5% nucleotide variation between strains

These variation cause change in amino acid composition at four places

Sossai et al. (2005) collected thirty R. (B.) microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the Bm86 gene

Gene amplified and sequenced from 278–1071 base (794 base pairs)

Variations from 1.76 to 3.65% were detected in the nucleotides sequence

and 3.4–6.08% in the amino acid sequence of the Bm86 protein

Garcia-Garcia et al. (1999) suggested that variations greater than 2.8% in the amino acid sequence of the protein expressed would be sufficient to confer vaccination inefficiencies when recombinant antigens are used.

Cross-protectionVaccine molecules

Tick species Effect Reference

Bm86 Hyalomma dromedarii  DT%-27; DR%- 31; DO%- 32

Rodríguez-Valle et al., 2012

Bm86 Amblyomma cajennense No effect Rodríguez-Valle et al., 2012

Bm86 R. (B.) annulatus E%-99% Canales et al., 2009

Bm86 R. (B.) decoloratus DT%- 45; DR%- 55; DO%- 61

Odongo et al., 2007

Bm86 Rhipicephalus sanguineus Reduction in Larvae, Nymph and Adults, 38%, 29% and 31%, respectively

Perez-Perez et al., 2010

Bm86 R. appendiculatus No effect de Vos et al., 2001

Bm86 Hyalomma anatolicum anatolicum

E%- 25 Kumar et al., 2012

Bm86 Homolog

Ree86, Dr86, Hm86, Av86, Ir86, Os86 (Nijhof et al., 2010), Hl86 (Liao et

al., 2007), Ra86 (Kamau et al., 2011), Ba86 (Canales et al., 2008), Bd86

(Odongo et al., 2007), Rs86 (Fang and Xu, 2007) and Haa86

(Azhahianambi et al., 2009)

Efficacy of some of recombinant protein was evaluated which is variable

Antigens Tick species Efficacy Reference

Ba86 R. (B.) annulatus 83% Canales et al., 2009

Ba86 R. (B.) microplus 71% Canales et al., 2009

Haa86 H. a. anatolicum Larvae – 47- 60%Adults - 40-80%

Azhahianambi et al., 2009; Jeyabal et al., 2010; Kumar et al., 2012

Haa86 R. (B.) microplus 25% Kumar et al., 2012

Some other recently identified Vaccine targets-

Targeted molecules Species of Ticks Experimental animal

Vaccine Efficacy Reference

Acid phosphatase (HL-3)

41.0 kDa

Haemaphysalis longicornis Rabbit DR% = 10.6

Mortality (%) = 28.0

Zhang et al., 2011

Hc-23

43 kDa

Haemaphysalis concinna Rabbit DR% = 11

DO% = 62

Bian et al., 2011

Cathepsin L (IrCL1)

35kDa

Ixodes ricinus Not tested Franta et al., 2011

P- selectin-binding protein

(Om44), 44kDa

Ornithodorous moubata Pigs Reduction in fecundity- 44%; feeding inhibition 50%

Garcia-Varas et al., 2010

Calreticulins

55-60kDa

Haemaphysalis longicornis Mice and calves Immunogenicity tested and proposed as good target

Parizi et al., 2009

RH50; 50kDa Rhipicephalus haemaphysaloides Rabbit Mortality rate 30.5% Zhou et al., 2006b

Ferritin 2 R. microplus calves E% = 64 Hijdusek et al., 2010

64TRP R. appendiculatus Rabbit E%- 50-70 Trimnell et al., 2002; 2005

Subolesin R. microplus calves E%- 50-75 Almazan et al., 2010

Voraxin-alpha R. appendiculatus Rabbit E%- 40-50 Yamada et al., 2009

Ubiquitin R. microplus Calves E%- 30-50 Almazan et al., 2010

Subolesin

It is ortholog of akirin, an evolutionary conserved gene of insect and

vertebrate (Mangold et al., 2009)

First discovered in Ixodes scapularis by Almazan et al., 2003

The proposed function of akirins is as transcription factors required for

NF-kB-dependent gene expression (Galindo et al., 2009) and in regulation

of innate immune response in fruit fly (Goto et al., 2008)

Subolesin functions in ticks are the same as akirin in fruit fly (Goto et

al., 2008; Galindo et al., 2009; Zivkovic et al., 2010; de la Fuente et al.,

2008; 2010)

Immunization with recombinant Subolesin

Tick species Vaccine efficay (against adults)

Reference

Ixodes scapularis 71% Canales et al., 2009

Amblyomma americanum

66% de la Fuente et al., 2010

R. (B.) microplus 51% Almazan et al., 2010

R. (B.) annulatus 60% Almazan et al., 2010

Vaccination with Subolesin reduced the vactor capacity of Ixodes scapularis for

Anaplasma phagocytophilum (Almazan et al., 2010; de al Fuente et al., 2010)

Merino et al. (2011) --- 98% and 99% reduction in infection level of A.

marginale and Babesia bigemina, respectively in R. (B.) microplus fed on

Subolesin immunized animal.

Calreticulins (CRT)In general, CRT is a calcium binding protein

Found in almost every organism

In ticks, the salivary secreted CRT   involvement in evading the host's

immune system (Xu et al., 2005)

Kaewhom et al. (2008) reported, CRT is a protein found in tick

salivary glands and saliva, and CRT might facilitate tick feeding and

pathogen transmission through anti-thrombotic and complement-

inhibition activities.

Vaccination of sheep with rHqCRT conferred protective immunity

against Haemaphysalis qinghaiensis, resulting in 54.3% mortality in

adult ticks, compared to the 38.7% death rate in the control group (Gao

et al., 2008)

The possibility of using CRT to induce protective immunity against

Necator americanus and Schistosoma spp. has been suggested (El

Gengehi et al. 2000; Khalife et al. 1994; Pritchard et al. 1999).

Exhibition of necrotic lesions in the tick bite sites in Amblyomma

americanum CRT immunized rabbits indicates that immune reaction

could disrupt the feeding cycle (Jaworski et al. 1995).

Sanders et al. (1999) reported the antibody levels to A. americanum

CRT increase in humans after exposure to I. scapularis are

correlated with tick engorgement indices

Cathepsin LCathepsin family having dozen of member with protease activity.

Cathepsin L is a Cysteine protease

Sojka et al. (2008) and Horn et al. (2009) demonstrated that intestinal

haemoglobinolysis in the Ixodes scapularis relies on Clan CA papain-type

cysteine peptidases, cathepsins L (IrCL), B (IrCB) and C (IrCC), the Clan

CD asparaginyl endopeptidase, legumain (IrAE) and the Clan AA aspartic

peptidase, cathepsin D (IrCD)

Detailed analysis of the haemoglobinolytic pathway in the I. ricinus gut

demonstrated that the process is initiated by cleavage of large fragments

from haemoglobin by cathepsins D, L and Legumain

Franta et al., 2011

Silencing of IrCL by RNAi impaired weight-gain of semi-engorged

Ixodes ricinus females fed for 6 days on guinea pigs

This result suggests that IrCL has a non-redundant role in the

digestive machinery Franta et al., 2011

Targeting this enzyme using specific immunotherapeutic antibodies

provides a promising concept for the rational development of an

anti-tick vaccine (Jongejan et al., 2007)

Clara et al. (2011) through peptide phage display library shows

Cathepsin L is a potent digestive enzymes

Characterization of genes by gene silencing

RNA interference (RNAi) is a process within living cells that moderates the activity of

their genes.

Andrew Fire and Craig C. Mello, (1998) work on RNA interference in the C. elegans,

Nobel Prize in Physiology or Medicine  2006

RNAi has been shown to be valuable tools for the study of tick gene function, the

characterization of tick pathogen interface and the screening and characterization of

tick protective antigens.

de la Fuente et al., 2007

Nucleus

Cytoplasm

Exogenous dsRNA

ncRNAstRNA

mRNA

shRNA

siRNA

miRNA

Long ncRNA

Dicer

RISC

RISC

RISC

RNA gene

Tick species Target gene Phenotype References

A.americanum Histamine-binding protein (HBP)

Reduction of histamine-binding activity in salivary glands and aberrant tick feeding pattern

Aljamali et al., 2002; 2003

A.americanum Salivary Cystatin ~80% decrease in transcript level, 32% reduction in body weight, only 20% ticks was able to feed on host after injection

Karim et al., 2005

H. longicornis Leucine aminopeptidase

Delay onset of egg-laying and reduced oviposition

Hatta et al., 2007

Tick species Target gene Phenotype ReferencesR. (B.) microplus Subolesin reduction of 75%

and 99% in tick weight and egg mass, respectively and 46% mortality compare to control

Nijhof et al., 2007;

De la Fuente et al., 2005

H. longicornis Ribosomal protein P0 Low body weight, lower rate of engorgement, high mortality

Gong et al., 2008

R. (B.) microplus Ferritin 2 42% rejection, 50% reduction in weight, 53% reduction in oviposition

Hajdusek et al., 2009

Tick species Target gene Phenotype References

Amblyomma americanum

CD147 receptor homologue

~69% ticks are not able to feed properly, tick morphology was changed

Mulenga and Khumthong, 2010a

A. americanum Insulin like growth factor

Reduction in blood meal size, tick mortality, fail to lay eggs.

Mulenga and Khumthong, 2010b

R. (B.) microplus Metzincin metalloproteases

Affects average egg weight and oviposition rates

Barnard et al., 2011

R. (B.) microplus Ubiquitin-63E Knockdown of gene associated with Ubiquitin-63E

Lew-Tabor et al., 2011

Technical Program

A. Cloning and sequencing of targeted genes (Subolesin, Calreticulin and Cathepsin L) of Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus

B. Study on conservation of target genes among different isolates of H. a. anatolicum and R. (B.) microplus from India.

Ticks will be collected from different states (as much as possible).

Some isolates are already available in the Entomology laboratory,

Division of Parasitology, IVRI, Izatnagar and more will be

collected.

Total RNA will be isolated, target genes will be amplified using

suitable primers, cloned and sequenced.

Analysis of genes using bioinformatics software like Gene tool,

DNA star, Megaline, NCBI blast etc.

C. Quantification of level of transcript of targeted genes in different stages of H. a. anatolicum (IVRI line II)

Different life stages of ticks will be collected and kept in

RNAlater at -80°C

Total RNA will be isolated using standard protocol

Custom synthesis of primers

Quantification of transcriptome for each gene through

Quantitative PCR

D. Charecterization of targeted genes of H. a. anatolicum

1. Through gene silencing

2. In-vivo immunization study of recombinant

protein(s)

1. Gene silencing by RNA interference

I.Preparation of dsRNA (200-500bp) using standard protocolII.Inoculation of dsRNA in unfed adults of H. a. anatolicum

Dilution of dsRNA with injection buffer/elution buffer to make the concentration

@ 5.0 x 1010 to 5 x 1015 molecules/µl for each gene of interest.

1µl dsRNA preparations will be injected to the individual tick using specially

fabricated 34G needle fitted in micro-syringe (Hamilton, Switzerland) at posterior

to 4th coxae deep in to hemocele.

Injected ticks will be allowed to move in broad bottom tubes, incubate in BOD

incubator at 95% RH and 28°C temperature for 24 hours.

III. Assessment of biological activity of ticks

Active ticks will be selected (n = 30 for each gene inoculated and control) and

will be released on animal along with equal number of male ticks.

Feeding ticks (n= 10) will be collected at 24 hrs interval till engorgement and

will be stored in RNAlater at -80°C for RNA isolation. Engorged ticks will be

weighed and kept for oviposition at 28°C with 85% RH.

IV. Evaluation of effect of RNAi on ticks

Entomological parameters viz., percent reduction in tick number (DT%), percent

reduction in egg mass (DO%), percent reduction in tick weight (DR%) and overall

efficacy (E%) will be recorded and will be compared to control

Monitoring of inhibition of expression of gene(s) of interest in feeding ticks,

engorged ticks, eggs and larvae by q- PCR.

2. In-vivo immunization of recombinant protein(s)

I. Expression of target genes of H. a. anatolicum in prokaryotic system

Targeted genes will be expressed in suitable expression vector and

standardization will be done for good expression.

Purification and quantification of expressed protein (s).

Determination of molecular weight of recombinant proteins using

SDS-PAGE

Western blot analysis of recombinant proteins by probing with hyper

immune sera raised in rabbit against antigens prepared from H. a.

anatolicum

II. Immunization of calves with recombinant protein along with adjuvant

Cross bred caves of 3-4 month age from dairy farm (LPM), IVRI, Izatnagar

will be procured.

All the calves will be kept in tick proof shed of the Division of Parasitology.

All the animals will be dewormed after 15 days of arrivals.

Animals will be randomly divided in to different groups of four animals in

each group and immunization will be started on 6-7 month old calves.

Each animal of immunized group (s) will be inoculated with 100µg

of antigen along with adjuvant (1:1 ratio) in a three doses at one

month interval, deep intramuscularly

Control animals will be inoculated with PBS/adjuvant

For each antigen two groups will be kept, one will be challenged with

larvae (hatched from 50 mg eggs) and 50 unfed adults of H. a.

anatolicum and other with larvae (hatched from 50 mg of eggs) of R.

(B.) microplus

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III. Monitoring of immunological response against immunogen

Serum will be collected at different time (pre-immunization, post-

immunization) for estimation of

Whole serum immunoglobulins

IgG1

IgG2

by indirect ELISA

IV. Potency testing by Entomological data

For the larvae-

DT (%) = 100 (1 – NTV/NTC)

where DT(%) is the percentage reduction of challenged larvae,

MO (%) = 100 (1-MLI/MLC)

where MO (%) is the percent reduction in moulting of engorged larvae,

For the adults- DT% = 100 (1-NTV/NTC),

Where DT% is the percentage reduction in mean number of females

fed on immunized and control groups of animals.

DO (%)= 100 (1- PATV/PATC)

where DO (%) is the percentage reduction of mean weight of eggs of

ticks fed on immunized and control animals

DR (%) = 100 (1- PMTV/PMTC)

where DR (%) is the percentage reduction of mean weight of adult

females dropped from immunized and control animals

E (%) = 100 [1- (CRT X CRO)]

Where E (%) is the efficacy of immunogens. CRO is reduction in egg

laying capacity (PATV/PATC), CRT is the reduction in the number of

adult females (NTV/NTC)

47