Anti-tick vaccine development

1. 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)
Dr. Binod Kumar
Roll No.1374
Ph.D. Scholar
Division Of Parasitology
IVRI
1

2. INTRODUCTION

3. 106 species
Ghosh et al., 2005,2007

4. TICKS
Major tick species in INDIA infesting livestock
Hyalomma anatolicum anatolicum
Cattle, Buffalo and small ruminants
Three hosts tick
Rhipicephalus (Boophilus) microplus
Cattle, Buffalo, Horse, donkeys, goat, Sheep, Deer, Pig, Dog, and some
wild animals
One host tick
4

5. Direct and Indirect effects
Tick attachment
Deep bite wound
Annoyance
Predispose to myasis
Reduced hide value
Tick secretions
Tick toxicosis
Tick paralysis
Transmission of tick borne pathogens
Blood feeding
Anemia
Production and reproduction losses
Control
High cost of acaricide treatment
Ecological damage
Human health

6. Vectorial capacity
H. a. anatolicum
•Theileria annulata
•T. buffeli
•T. lestocardi
•CCHF (????)
R. (B.) microplus
Babesia bigemina
B. bovis
Anaplasma marginale
6

7. Economic impact of tick infestation on
livestock industry
In million US $
De Castro, 1997;
Minjauw and Mc
Leod, 2003;
Dayton et al., 1991;
Horn, 1987;
Mukhebi et al., 1999
*
* Control cost
7

8. Ticks control strategies
Methods of Control Major Control Side effects of chemical
Method control method
Selection of resistant
• Chemical control (Acaricides) ticks
• Biological control Environmental pollution
• Genetic transformation Acaricides Residues in livestock
• Immunological control products
• Herbal formulation as High cost of repeated
acaricide application
• Genetically resistance breeds Development of new
generation acaricide
Sustainable control ????????
8

9. 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

10. 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

11. Major areas of target
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

12. Targets selected for study
Subolesin
(Expressed in all
organs and tissues)
Function as
transcription
factors in the
Calreticulin regulation of gene
Anti-thrombotic and Cathepsin L expression
Part of a gut-associated multi-
complement-inhibition
peptidase complex.
activities in host
Its endopeptidase
activity is important in the
initial phase of
haemoglobinolysis.

13. •
..
Objectives
• Cloning and sequencing of Subolesin, Calreticulin and Cathepsin L
genes of Hyalomma anatolicum anatolicum and Rhipicephalus
(Boophilus) microplus
• Evaluation of Conservation of target genes in different isolates of H. a.
anatolicum and R. (B.) microplus
• Characterization of target genes of H. a. anatolicum
i). Through RNA interference
ii). In-vivo immunization trial using recombinant protein(s)

14. REVIEW OF LITERATURE
14

15. Native proteins as Vaccine targets
Immunogen Challenge dose Percentage protection
Larvae Nymphs Adults Immediate rejection (%)a Overall decrease in Reduction in
successive stageb egg massesc
AFF-TLE (L), 39kDa 4000 200 - 60.0 (L),44.0 (N) 34.0 (N),43.2 (A)
Aff-GHLAg (Gut), 34 2000 140 40 pairs 24.2 (L),22.4 (N),32.2 (A) 31.2 (N), 25.2 (A), 15.0
kDa)
Aff-HNAg (Nymph, 1600 140 40 pairs 38.0 (L), 25.0 (N), 32.2 (A) 32.7 (N), 28.7 (A), 20.0
39kDa
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) -
a
Mean differences in immediate rejection between immunized and control groups of animal.
b
Mean differences in the development of successive stage of the ticks fed on immunized and control group of
animals.
c
Mean differences in the egg masses laid by the ticks fed on immunized and control group of animals.
* p < 0.01. 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;
** p < 0.05 Indian J Anim. Sci., Exp. Appl. Acarol., 2003, Parasitology Res., 2005, Trop Anim Hlth
Prod., 2005; J Vet Parasitol., 2007; Vaccine 2008

16. 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)
16

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

18. 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

19. 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.

20. Cross-protection
Vaccine Tick species Effect Reference
molecul
es
Bm86 Hyalomma dromedarii DT%-27; DR%- 31; DO%- Rodríguez-Valle et
32 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%- Odongo et al., 2007
61
Bm86 Rhipicephalus sanguineus Reduction in Larvae, Perez-Perez et al.,
Nymph and Adults, 38%, 2010
29% and 31%,
respectively
Bm86 R. appendiculatus No effect de Vos et al., 2001
Bm86 Hyalomma anatolicum E%- 25 Kumar et al., 2012
anatolicum

21. 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- Azhahianambi et al.,
60% 2009; Jeyabal et al.,
Adults - 40-80% 2010; Kumar et al., 2012
Haa86 R. (B.) microplus 25% Kumar et al., 2012

22. Some other recently identified Vaccine targets-
Targeted molecules Species of Ticks Experimen Vaccine Efficacy Reference
tal animal
Acid phosphatase (HL-3) Haemaphysalis longicornis Rabbit DR% = 10.6 Zhang et al., 2011
Mortality (%) = 28.0
41.0 kDa
Hc-23 Haemaphysalis concinna Rabbit DR% = 11 Bian et al., 2011
DO% = 62
43 kDa
Cathepsin L (IrCL1) Ixodes ricinus Not tested Franta et al., 2011
35kDa
P- selectin-binding Ornithodorous moubata Pigs Reduction in fecundity- Garcia-Varas et al.,
44%; feeding inhibition 2010
protein
50%
(Om44), 44kDa
Calreticulins Haemaphysalis longicornis Mice and calves Immunogenicity tested Parizi et al., 2009
and proposed as good
55-60kDa target
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

23. 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)

24. Immunization with recombinant Subolesin
Tick species Vaccine efficay Reference
(against adults)
Ixodes scapularis 71% Canales et al., 2009
Amblyomma 66% de la Fuente et al.,
americanum 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.

25. 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)

26. • 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

27. Cathepsin L
Cathepsin 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

28. • 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

29. 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

30. Nucleus mRNA
RNA gene tRNA
s
NA
ncR
miRNA
shRNA
Cytoplasm
siRNA
Exogenous dsRNA
Long ncRNA
Dicer
RISC
RISC
RISC

32. Tick species Target gene Phenotype References
A.americanum Histamine-binding Reduction of Aljamali et al., 2002;
protein (HBP) histamine-binding 2003
activity in salivary
glands and aberrant
tick feeding pattern
A.americanum Salivary Cystatin ~80% decrease in Karim et al., 2005
transcript level, 32%
reduction in body
weight, only 20%
ticks was able to feed
on host after injection
H. longicornis Leucine Delay onset of egg- Hatta et al., 2007
aminopeptidase laying and reduced
oviposition

33. Tick species Target gene Phenotype References
R. (B.) microplus Subolesin reduction of 75% Nijhof et al., 2007;
and 99% in tick De la Fuente et al.,
weight and egg 2005
mass, respectively
and 46% mortality
compare to control
H. longicornis Ribosomal protein P0 Low body weight, Gong et al., 2008
lower rate of
engorgement, high
mortality
R. (B.) microplus Ferritin 2 42% rejection, 50% Hajdusek et al.,
reduction in weight, 2009
53% reduction in
oviposition

34. Tick species Target gene Phenotype References
Amblyomma CD147 receptor ~69% ticks are not Mulenga and
americanum homologue able to feed Khumthong, 2010a
properly, tick
morphology was
changed
A. americanum Insulin like growth Reduction in blood Mulenga and
factor meal size, tick Khumthong, 2010b
mortality, fail to lay
eggs.
R. (B.) microplus Metzincin Affects average egg Barnard et al., 2011
metalloproteases weight and
oviposition rates
R. (B.) microplus Ubiquitin-63E Knockdown of gene Lew-Tabor et al.,
associated with 2011
Ubiquitin-63E

35. Technical Program
• Cloning and sequencing of targeted genes (Subolesin,
Calreticulin and Cathepsin L) of Hyalomma
anatolicum anatolicum and Rhipicephalus (Boophilus)
microplus

36. 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.

37. 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

38. D. Charecterization of targeted genes of H. a.
anatolicum
3. Through gene silencing
4. In-vivo immunization study of recombinant
protein(s)

39. 1. Gene silencing by RNA interference
III.Preparation of dsRNA (200-500bp) using standard protocol
IV.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.

40. 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.

41. 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

42. 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.

43. • 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

44. 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
44

45. 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,

46. 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. 47