1. Human Babesiosis: The
development of a new line of
drugs
Azan Virji
STARS Research Fellow 2015
PI: Professor Ben Mamoun Choukri
Mentor: Lauren Lawres
Department of Infectious Diseases, Yale School of Medicine

2. What is Babesiosis?
• Babesia microti
• Northeast & Northern
Midwestern States
• Spread by ticks
http://www.cdc.gov/parasites/images/babesiosis/map_babesiosis_by_county_2013.jpg

3. Babesiosis is commonly
misdiagnosed as malaria
https://classconnection.s3.amazonaws.com/915/flashcards/2974915/png/babesia1326269959514-14663CD9F46086C79A4.png
http://blogs.cdc.gov/global/2014/02/24/dpdx-15-years-of-strengthening-laboratory-capacity-for-parasitic-disease-
diagnosis/b_microti_vs_p_falciparum-2/
Babesia microti Plasmodium falciparum

4. Current therapies against Babesiosis
Vannier, E. et al. (2012, New England Journal of Medicine)

5. Recrudescence:
Resistance against current therapies
Treatment
Pereira, M. E. S. et al. (1996, Memórias do Instituto Oswaldo Cruz)

6. Addressing the problem
with current therapies
Current therapies are less
effective than the new drug
Endochin-like Quinolones
(ELQ)
Why are current therapies
ineffective?
DNA
Sequencing
In vivo
study
SCID Mice
10 mg/kg by oral gavage
Light Microscope counting
Day 45
PCR- Cytochrome b gene
Yale Keck Sequencing Facility

7. Three of the four current therapies
are ineffective
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
%Parasitemia
Days post infection
Giemsa Counts
CONTROL
AZITHROMYCIN
QUININE
CLINDAMYCIN
Treatment

8. Atavaquone delays recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
%Parasitemia
Days post infection
Giemsa Counts
CONTROL
ATAVAQUONE
Treatment

9. Endochin-like Quinolones also
delay recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
%Parasitemia
Days post infection
Giemsa Counts
CONTROL
ELQ
Treatment

10. ATV + ELQ eliminates
recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
%Parasitemia
Days post infection
Giemsa Counts
CONTROL
ELQ + ATV
Treatment

11. ATV + ELQ eliminates
recrudescence
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0 5 10 15 20 25 30 35 40 45 50
%Parasitemia
Days post infection
Giemsa Counts
ELQ + ATV
Treatment

12. Why does a combination of
ATV and ELQ work better?
CL1
CL2
CL3
CL1
CL2
ELQV
Sarewicz, M. et al. (2015, jhjournal)
Atavaquone
ELQ
GCT GTT
Alanine Valine

13. Implications of this research
• Synergy
• Probability that mutation arises is lowered
10-7
ATV/ELQ
10-14
ATV+ELQ
• 2 mutations may lower fitness
• Next steps: Dosing, Clinical trials

14. Acknowledgements
• Laboratory Of Infectious Diseases
o PI: Professor Ben Mamoun Choukri
o Mentor: Lauren Lawres
o Isaline and Pierre
o Yale School of Medicine
• STARS Summer Research Program
o Dr. Moreno, Dr. Nelson and Dr. P And the TAs
o Howard Hughes Medical Institute (HHMI)
o Yale College

15. A%Parasitemia
C
o
n
tro
lQ
u
in
in
e
C
lin
d
a
m
y
c
in
A
zith
ro
m
y
c
in
A
to
v
o
q
u
o
n
eE
n
d
o
c
h
in
A
rte
s
u
n
a
te
0
2
4
6
8
*
*
*
B
FSH
3.04%Control
YOYO Quinine
Endochin
Atovoquone
Artesunate Azithromycin
3.99
Clindamycin
3.40
4.5
1.02
1.3
2.8
3.01
Control
FSC
YOYO-1Figure 1

16. Figure 2
%Parasitemia
- V 1 1 0 1 1 0 1 1 0
0
1
2
3
4
5
E L Q 2 7 1 E L Q 3 0 0 E L Q 3 1 6
V
4.66%
ELQ-1
1.50%
FSC
YOYO-1
FSC
YOYO-1
ELQ-2
1.52%
ELQ-3
1.28%
FSC
YOYO-1
FSC
YOYO-1
A
B
ELQ 1 ELQ 2 ELQ 3

17. Figure 3 A B
C D
ELQ 1 ELQ 2
ELQ 3 ELQ 4
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 2 7 1 (1 0 m g /K g X 7 )
m o n ito re d b y S m e a r
D a y s p o s t in fe c tio n
%Parasitemia
M o u se # 1 6
M o u se # 1 7
E L Q 2 7 1
C trl (F )
M o u se # 1
M o u se # 2
M o u se # 3
T r e a tm e n t
1 5 8 1 2 1 5 1 9 2 3
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 3 0 0 (1 0 m g /K g X 7 )
m o n ito re d b y S m e a r
D a y s p o s t in fe c tio n
%Parasitemia
M o u se # 1 6
M o u se # 1 7
E L Q 3 0 0
C trl (F )
T r e a tm e n t
M o u se # 4
M o u se # 5
M o u se # 6
1 5 8 1 2 1 5 1 9
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 3 1 6 (1 0 m g /K g X 7 )
m o n ito re d b y S m e a r
D a y s p o s t in fe c tio n
%Parasitemia
M o u se # 1 6
M o u se # 1 7
E L Q 3 1 6
C trl (F )
T r e a tm e n t
M o u se # 7
M o u se # 9
M o u se # 8
1 5 8 1 2 1 5 1 9 2 3
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 4 0 0 (1 0 m g /K g X 7 )
m o n ito re d b y S m e a r
D a y s p o s t in fe c tio n
%Parasitemia
E L Q 4 0 0
C trl (M )
T reatm ent
M o u se # 1 0
M o u se # 1 1
M o u se # 1 2
1
M o u se # 1 7
M o u se # 1 6
5 8 1 2 1 5 1 9
C o n tro l 3

18. Figure 4
A C
B
LabS1-S
+
V
LabS1-S
+
ELQ-1
LabS1-S
+
ELQ-3
CL1
CL2
CL3
CL1
CL2
CL3
CL1
CL2
ELQ-1ELQ-3V
V271-R
Parasitemia(%)26DPI
0 .0
0 .5
2 0
3 0
4 0
5 0
6 0
1
10
Parasitemia(%)39DPI
0 .0 0
0 .0 5
0 .1 0
2 0
3 0
4 0
5 0
6 0
10
LabS1-S
+
V
LabS1-S
+
ELQ-1
LabS1-S
+
ELQ-3
D

19. 19
Figure 5
Vannier, E. et al. (2012, New England Journal of Medicine)

20. What is currently unknown?
• Effective treatment: Are Endochin-like
Quinolones the solution?
• Pathophysiology: why does recrudescence
occur?

21. How to address the problem
of current therapies
• Prove that current therapies are ineffective and provide
evidence that new drug is significantly better than current
therapies
• Find out why current therapies do not work
• ELQs were considered potent due to previous studies

22. Methodology of infecting mice
• In vivo study
• SCID Mice/Rag2 knockout mice: Mice with weaker
immune system
• Mice from Dr. Ruslan Medzhitov’s Lab
• 107 parasites
• 10mg/ kg by oral gavage 4 to 11 days post infection (dpi)
• Control mice given vehicle with no drug

23. Methodology to test efficacy
• Blood collected from tail every 4 days
• Giesma Staining and Counting
• Flow cytometry to check counts
• DNA sequencing to identify mutations