Efficacy of TLR7 agonistic imidazoquinoline as immunochemotherapeutic agent against P. Berghei ANKA infected rodent host

Article abstract of DOI:10.1016/j.bmcl.2019.02.029

Malaria is a serious disease and is one of the most alarming public health issues. Plasmodium is being resistant to various antimalarials including Chloroquine (CQ) which was the first-line therapy for malaria treatment. WHO recommended several combination therapies but declining efficacy was reported to many of these therapies. Despite a great amount of research, efficient malaria vaccine still seems to be a distant dream. Immunochemotherapy could be an alternate strategy to deal with malaria. Based on the differential activity of various cytokines in the pathogenesis of and protection against malaria, the efficacy of highly active TLR7 agonistic imidazoquinoline (BBIQ) in combination with a suboptimal dose of CQ against P. berghei ANKA (PbA) in vivo was investigated. In mice treated with CQ alone, parasite appeared on Day 17 and all mice of this group died by Day 21. Whereas, mice treated with BBIQ along with CQ exhibited no appearance of parasite till Day 23. Frequencies of T cells (CD3⁺, CD4⁺and CD8⁺) and T regulatory cells (CD4⁺, CD25 ⁺and FoxP3⁺) were found to be lower in brain of BBIQ + CQ treated mice as compared to BBIQ alone and CQ alone treated mice on Day 10. Inhibition of infiltration of inflammatory T cells and activation of T helper and T cytotoxic cells against the parasite was observed in the mice treated with this combination therapy. Serum levels of IFN-γ and IL-12 were found to be higher on same day in mice treated with BBIQ + CQ which revealed the generation of strong Th1 immune response in mice against the infection. Overall, TLR7 agonist acted as an efficient partner when combined with potent antimalarial drug.

Full text of DOI:10.1016/j.bmcl.2019.02.029

Accepted Manuscript
Efficacy of TLR7 Agonistic Imidazoquinoline as Immunochemotherapeutic
Agent Against P. berghei ANKA Infected Rodent Host
Ruchika Saroa, Deepender Kaushik, Upma Bagai, Sukhbir Kaur, Deepak B.
Salunke
PII:
S0960-894X(19)30113-1
https://doi.org/10.1016/j.bmcl.2019.02.029
BMCL 26309
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 January 2019
23 February 2019
26 February 2019
Please cite this article as: Saroa, R., Kaushik, D., Bagai, U., Kaur, S., Salunke, D.B., Efficacy of TLR7 Agonistic
Imidazoquinoline as Immunochemotherapeutic Agent Against P. berghei ANKA Infected Rodent Host, Bioorganic
& Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.02.029
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Efficacy of TLR7 Agonistic Imidazoquinoline as Immunochemotherapeutic
Agent Against P. berghei ANKA Infected Rodent Host
Ruchika Saroa,a,† Deepender Kaushik,b,† Upma Bagai,a,‡ Sukhbir Kaur,a,* Deepak B. Salunkeb,*
aDepartment of Zoology, Panjab University, Chandigarh 160 014, India.
bDepartment of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University,
Chandigarh 160 014, India.
*Corresponding Authors
Dr. Deepak B. Salunke, Assistant Professor, Department of Chemistry and Centre of
Advanced Studies in Chemistry, Panjab University, Chandigarh-160 014, India. Email:
salunke@pu.ac.in
Prof. Sukhbir Kaur, Professor, Department of Zoology, Panjab University, Chandigarh-160
014, India. Email: sukhbir@pu.ac.in
†Both the authors contributed equally.
‡Deceased 8 September 2018.
ruchika.saroa@yahoo.com
1

kaushikdeepender@gmail.com
2

ABSTRACT
Malaria is a serious disease and is one of the most alarming public health issues. Plasmodium
is being resistant to various antimalarials including Chloroquine (CQ) which was the first-
line therapy for malaria treatment. WHO recommended several combination therapies but
declining efficacy was reported to many of these therapies. Despite a great amount of
research, efficient malaria vaccine still seems to be a distant dream. Immunochemotherapy
could be an alternate strategy to deal with malaria. Based on the differential activity of
various cytokines in the pathogenesis of and protection against malaria, the efficacy of highly
active TLR7 agonistic imidazoquinoline (BBIQ) in combination with a suboptimal dose of
CQ against P. berghei ANKA (PbA) in vivo was investigated. In mice treated with CQ alone,
parasite appeared on Day 17 and all mice of this group died by Day 21. Whereas, mice
treated with BBIQ along with CQ exhibited no appearance of parasite till Day 23.
Frequencies of T cells (CD3+, CD4+and CD8+) and T regulatory cells (CD4+, CD25 +and
FoxP3+) were found to be lower in brain of BBIQ+CQ treated mice as compared to BBIQ
alone and CQ alone treated mice on Day 10. Inhibition of infiltration of inflammatory T cells
and activation of T helper and T cytotoxic cells against the parasite was observed in the mice
treated with this combination therapy. Serum levels of IFN-γ and IL-12 were found to be
higher on same day in mice treated with BBIQ+CQ which revealed the generation of strong
Th1 immune response in mice against the infection. Overall, TLR7 agonist acted as an
efficient partner when combined with potent antimalarial drug.
Keywords: Cerebral malaria, Chloroquine, TLR7 agonist, Imidazoquinolines, Adjuvant,
PbA, Immunochemotherapy
3

Malaria is a serious disease and is one of the most alarming public health issues. P.
falciparum is the causative agent of malaria in humans and is responsible for 216 million
deaths in 2017.1 Cerebral malaria is one of the deadly complications of malaria causing
neurological syndrome in the infected host. Attachment of infected/non-infected RBCs and
leukocytes to cerebral vessels are the hallmark features of cerebral syndrome. Various cells
sequester into brain vasculature during cerebral malaria and create an obstruction in blood
flow which is mainly responsible for the breakdown of the blood brain barrier.2 P. berghei
ANKA is the accepted model for studying cerebral syndrome in rodents because of its
similarity with cerebral malaria in humans.3
Due to the mutations in genetic makeup of the parasite, Plasmodium is being resistant to
various antimalarials which is the main hurdle in controlling or eliminating malaria. WHO
recommended Artesunate Combination Therapy (ACT) as an effective treatment for malaria.
Drugs with longer life-span are co-administered with Artemisinin derivatives which kill the
parasite rapidly before getting metabolised from the host’s system.4 Declining efficacy of the
Artesunate/Mefloquine and Artesunate/Amodiaquine combination was reported in
Cambodia-Thailand border in Falciparum infected patients.5 Frequent failures of
Dihydroartemisinin/Piperaquine in some areas of Cambodia has also been reported.6
Artesunate-Sulfadoxine-Pyrimethamine (AS+SP) has been shown to be more efficacious
against uncomplicated malaria. It has long-acting sulfonamide and the antifolate
Pyrimethamine.7 In India, the combination of OZ277 (Arterolane) and Piperaquine is
available and known as Synriam™. OZ439 (Artefenomel) is currently being assessed either
alone or as a combination therapy in Phase II human clinical trials.8 Malaria is also known as
a disease of rural areas and due to the overpriced available drugs, its treatment is impossible
for underprivileged areas.
4

Manipulation of immune response is a proven strategy and provided vaccination as the most
important contributions to public health. Apart from several combination therapies, vaccines
are known to provide active acquired immunity against this disease and is a promising
approach to control malaria. RTS, S formulated with AS02 is in phase IIb trials and showed
35.5% efficacy in infants which persist over 6 months.9 During the follow-up study RTS,S
was found to be effective only 4 percent of the time and the interim poor results of this
vaccine trial in African children raised a question mark over its deployment.10 Colfavac, a
new synthetic vaccine, is another promising candidate being tested in various countries,
reported 81.7% efficacy in laboratory against P. falciparum malaria. Despite of great amount
of research, efficient malaria vaccine still seems to be a distant dream. The present scenario
clearly demands a need of new antimalarials or a novel approach to deal with this fatal
disease.
Immunochemotherapy could be an alternate strategy to deal with malaria. A combination
therapy with low doses of Interleukin 12 (IL12) and Chloroquine was promising which
completely cured blood-stage malaria. It also prevented severe anemia, and induced
immunity to reinfection.11 Immunotherapy as well as immunochemotherapy in visceral
leishmaniasis is also found to be a promising treatment.12
The role of Toll-like Receptors (TLR) in innate and adaptive immune responses and their
function in immune response regulation is very well documented.13 TLR7 is identified as an
important inducer of immune activation during Plasmodium infection.14 Topically applied
Imiquimod (TLR7 agonist) along with P. falciparum peptide vaccine elicited protective
humoral immunity against sporozoite challenge.15 The TLR7 agonist preferentially induced
secretion of IFNα and IFN-regulated chemokines, such as I-TAC (CXCL11) and IP-10
5

(CXCL10), while the TLR8 agonist predominantly induced proinflammatory cytokines and
chemokines including TNFα, IL-12 and MIP-1α (CCL3).16 Based on the differential activity
of various cytokines in the pathogenesis of and protection against malaria,17 the overall aim
of the present investigation was to study the efficacy of highly active TLR7 agonist in
combination with a suboptimal dose of Chloroquine (CQ) against cerebral malaria induced
by P. berghei ANKA (PbA). Another objective of this study was to carry out the flow
cytometric analysis of brain infiltrating and splenic lymphocytes to benchmark the T cell
differentiation and understand the serum levels of cytokines to plan the possible use of such
TLR7 agonist as vaccine adjuvant in malaria.
Design of Work Plan. Since 1946, Chloroquine (CQ) has been the first-line therapy for
malaria treatment. Plasmodium developed resistance to CQ due to the emerging changes in
genetic makeup possess a threat to millions of people. Incidence of CQ resistance occurred in
1950s and now it occurred globally.18 Plasmodium infecting rodents also showed resistance
against CQ due to the short-term protection in rodents. Adepiti et al.19 reported 58.3%
chemosuppression in mice treated with 10 mg/kg CQ in Peters 4-day test. Whereas,
chemosuppresion was 89.04% in mice against PbA infection treated with 25 mg/kg CQ.20
89.6% Chemosuppresion was also reported against P. yoelli infection in mice on treatment
with CQ at 3 mg/kg.21 Based upon the variable data available, present study was designed by
selecting a sub-optimal dose of CQ (10 mg/kg) against PbA infection.
Ten functional TLRs have been identified in humans which are known to detect a broad
range of pathogens and initiate innate and adaptive immune responses against the specific
infection. TLR3, -7, -8, and -9 located in the intracellular compartments are known to sense
viral and bacterial nucleic acids whereas, TLR1, -2, -4, -5, -6, and -11 expressed on the cell
6

surface are known to sense the outer membrane components of bacteria, fungi, and protozoan
organisms. Of the ten human and twelve mouse TLRs, TLR2, TLR4, TLR7 and TLR9 are
known to detect malarial antigens and induce anti-malarial immune responses.22 Among the
endolysosomal TLRs, TLR7 and TLR8, are belong to the same subfamily and are
phylogenetically similar.23 TLR7 and TLR8 agonists differ in their target cell selectivity and
cytokine induction profile. TLR7-selective agonists are known to be more effective than
TLR8-selective agonists at inducing IFN- and IFN-regulated chemokines such as IFN-
inducible protein and IFN-inducible T cell chemoattractant from human PBMC. In contrast,
TLR8 agonists are more effective than TLR7 agonists at inducing proinflammatory cytokines
and chemokines, such as TNF-, IL-12, and MIP-1.24 TLR8 is also known to be activated in
murine plasmacytoid DCs by vaccinia virus DNA, an A/T rich DNA virus.25
Based on these observations, selection of an appropriate and highly active TLR7 agonist for
the present immunochemotherapeutic study plan was an important task. Several small
molecule synthetic TLR7/8 agonists are reported in the literature (Figure 1) and TLR7
specific imidazoquinoline based Imiquimod (1) showed promising results when used along
with P. falciparum peptide vaccine which elicited protective humoral immunity against
sporozoite challenge.26 Considering the low potency of commercially available
imidazoquinoline based TLR7 ligands (Imiquimod, 1 and Gardiquimod, 2), a highly potent 1-
benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine 3 (BBIQ) was selected for this study.
BBIQ (3) is one of the most active known TLR7 agonist with its potency in the nanomolar
range. (EC50 = 59.1 nM in human TLR7 specific reporter gene assay).27 Compound 3 is also
known to induce IFN-α in human PBMCs (520 pg/mL at 5µg/mL)27 but because of its
commercial unavailability not explored for its possible applications in various diseases.
7

Figure 1. Pure TLR7 agonistic synthetic small molecules.
Synthesis of TLR7 Agonist. Synthesis of desired imidazoquinoline 3 was achieved from the
commercially available 2,4-quinolinediol (7) using the procedures as reported by Shukla et.
al. with minor modifications.27
Scheme 1. Synthesis of 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine.
o
o
Reagents and conditions: (i) HNO3, 75 C, 91%; (ii) (a) POCl3, 135 C, (b) benzylamine,
Et3N, CH2Cl2, reflux, 45 min., 46% (for 2 steps); (iii) (a) Pd/C, H2, EtOAc, Na2SO4, 3h, (b)
C4H9COCl, Et3N, THF, (c) NH3/MeOH, 150 oC, 8% (for 3 steps).
The selective nitration to synthesize 3-nitroquinoline-2,4-diol (8) was carried out using nitric
acid in good yield (Scheme 1). Quantitative yield was achieved for the chlorination reaction
using phosphorus oxychloride (POCl3) instead of relatively expensive phenyl
8

dichlorophosphate. The crude product obtained was used as it is for the regioselective
displacement of chloro functionality at C4 position using benzylamine in the presence of
triethylamine to afford trisubstituted quinoline 9 which was purified using silica gel column
chromatography. The subsequent nitro reduction was carried out using Pd/C under hydrogen
atmosphere at 55 psi and the residue obtained was treated with valeroyl chloride in the
presence of triethylamine to furnish intermediate amide derivative which on further exposure
to ammonia in methanol under microwave irradiation at 150 ºC resulted in a complex mixture
which on column chromatographic purification afforded desired BBIQ (3) as a white solid.
The overall yield for this process was found to be quite low but the product could be obtained
quickly with desired purity (>98% confirmed by HPLC-MS) required for the animal studies.
Study Design. Five groups having 6 mice each were used for present study. GP-I and GP-II
mice were administered with 0.2 mL phosphate buffer saline (PBS, pH-7.2) for D0-D3 orally.
Whereas, GP-II, III and IV were infected with 1x106 parasitized RBCs (i.p.) on D0. GP-III
and GP-IV were given TLR7 agonist BBIQ 3 (10 g) 0.2 mL/mouse/i.p. and CQ (10 mg/kg)
0.2 mL/mouse/orally from D0-D3 respectively. GP-V were given CQ (10 mg/kg) 0.2
mL/mouse/oral dose (OD) along with BBIQ 3 (10g) i.p. from D0-D3. Brain and spleen
from mice of these various groups were dissected out and teased into a single cell suspension.
Lymphocytes from brain cell suspension28 as well as spleen cell suspension29 were isolated
and analysed by FACS. The levels of IFN-, TNF-, IL-12, IL-10 and IL-27 in serum
samples of various groups on D0 and D10 were measured by sandwich ELISA.
Course of Infection. GP-I was not infected so smears were clear of any infection on all the
days. Mice of GP-II infected with 1x106 pRBCs showed regular course of infection (Figure 2)
and died due to infection by D15. In GP-III, only 5.7±0.9% infection was observed on D7
9

which cleared by D9. All mice in this group showed complete clearance of parasite but
became very weak due to weight loss and died by D15. Mice of GP-IV did not show any
parasite in blood smears till D15 but on D17 5.99±2.3% infection was recorded. Maximum
infection (10.76±3.5%) was observed in GP-IV on D21 after which all mice died due to
infection. In GP-V, the infection was delayed and parasite appeared on D25 and all mice died
by D30 with 11.65±5.6% infection.
Figure 2: Line graph showing the course of infection in PbA infected (GP-II), TLR7 agonist
(GP-III), CQ (GP-IV) and TLR7 agonist + CQ (GP-V) mice infected with 1×106P. berghei
ANKA. The parasitemia was determined by examining Giemsa- stained thin blood smears of
individual mice. Parasitemia (mean ± S.D.) of the infected and treated mice is plotted vs.
days post-infection.
Flow cytometric analysis of brain infiltrating lymphocytes. Sequestration of infected/non-
infected and various lymphocytes to cerebral vessels are the hallmark features of cerebral
malaria. Therefore, various T cells have been quantified in brain of mice in various groups
during present study (Figure 3). CD3+ is a common marker for T cells and was evaluated for
10

the presence of T cells in brain microvasculature of mice. Flow cytometric analysis of brain
of GP-V mice on D10 showed relatively lower percentage of CD3+ cells in comparison to
GP-III, whereas, the decline was substantial in brain of GP-V mice in comparison to GP-IV
on D10. On D21, an increase was recorded in level of CD3+ cells of GP-V mice which may
be due the clearance of drug and immunostimulant from host’s system by D21. An increase
was observed in percentage of CD3+ cells in brain vasculature of GP-II mice in comparison
to GP-I indicating the recruitment of T cells into brain due to infection in GP-II.
A
B
D0 D10 D21
D0 D10 D21
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50
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Figure 3: Histograms depicting the percentages of CD3+ (A), CD4+ (B), CD8+ (C) and
CD4+CD25+FoxP3+ T regulatory cells (D) isolated from brain of GP-I, GP-II, GP-III, GP-IV
and GP-V. Mean  S.D. of three mice were plotted.
Further, CD3+ cells were gated for CD4+ and CD8+ T cells which are specific for T helper
cells and cytotoxic cells respectively. CD4+ cells are crucial for the recruitment of various T
cells into brain during cerebral malaria. Significantly higher percentage was reported in brain
of P. berghei ANKA infected mice with rise in infection.30 In present study, higher
percentage of CD4+ cells were also observed in brain of GP-II mice with increase in
11

infection. Whereas, mice of GP-V showed decline in level of CD4+ cells in brain in
comparison to both GP-III and GP-IV mice. This indicates the inhibition of migration of
CD4+ cells to brain of GP-V mice due to administration of drug along with immunostimulant.
Swanson et al.31 reported an increase in frequency of CD8+ cells in brain of P. berghei
infected mice on D6 and D12 post infection. Present study showed similar observation with
increase in level of CD8+ cells on D10 post infection in GP-II in comparison to GP-I. Also, a
significant decline was observed in level of CD8+ cells of GP-V as compared to GP-III and
GP-IV.
T regulatory cells are the subsets of T cells and are crucial for the control of aggressive
immune responses during the infection. They can limit the proliferation of CD4+ and CD8+ T
cells against infection.32 It has also been seen in present study that during PbA infection, a
significant rise was recorded in level of Tregs in brain on D10. GP-III and GP-IV also
showed an increase in level of Tregs in comparison to GP-V.
All mice of GP-III and GP-IV died on D9 and D21 respectively. On D21, an increase was
recorded in percentages of CD3+, CD4+, CD8+ T cells as well as Tregs in brain of GP-V
mice. These elevated levels of various T cells and Tregs indicated the occurrence of cerebral
syndrome in mice. The infection appeared on D23 might be because of the clearance of both
CQ and immunostimulant (BBIQ) from the host system.
Flow Cytometric Analysis of Splenic Lymphocytes and Analysis of Blood Cytokine
Levels. Spleen is known as crucial organ in generation of immune response against malaria.
In present study, flow cytometric analysis were also performed on splenic lymphocytes to
12

differentiate between Th1 and Th2 immune response (Figure 4). significant increase in
percentage of CD3+ cells were observed in spleen of GP-V mice on D10 in comparison to
GP-III and GP-IV. This increase in level of CD3+ T cells indicate the activation of enough
number of T cells inside host to fight against infection.
D0 D10 D21
D0 D10 D21
B
A
90
80
70
60
50
40
30
20
10
0
45
40
35
30
25
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15
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GP-II
GP-III
GP-IV
GP-V
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Groups
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GP-I
GP-II
GP-III
GP-IV
GP-V
GP-I
GP-II
GP-III
GP-IV
GP-V
Groups
Groups
Figure 4: Histograms depicting the percentages of CD3+ (A), CD4+ (B), CD8+ (C) and
CD4+CD25+FoxP3+ T regulatory cells (D) isolated from spleen of GP-I, GP-II, GP-III, GP-IV
and GP-V. Mean  S.D. of three mice were plotted.
CD4+ T cells played a multiple role in protection and pathogenesis of the disease. They are
the major source of IFN-γ and TNF-α during experimental cerebral malaria.33 Together, IFN-
γ and TNF-α during malaria induce nitric oxide synthase in spleen to control parasite growth
in vivo.34 IFN-γ responses co-relate with anti-parasite immunity in human and generate
protective responses against the disease. During P. berghei ANKA infection in mice, IFN-
are involved in the development of cerebral pathology.35 It can directly influence the local
cerebral environment during infection and mediate the accumulation of various T cells in
brain microvasculature.36
13

A
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GP-II GP-III GP-IV GP-V
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C
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1000
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0
GP-I
GP-II GP-III GP-IV GP-V
Groups
Figure 5: Histograms depicting the sera levels of IFN-γ (A), TNF-α (B) and IL-10 (C)
collected from GP-I, GP-II, GP-III, GP-IV and GP-V. Cytokines were analysed using ELISA
in pooled sera samples of three mice in duplicate.
Present study showed increase in percentage of CD4+ T cells in spleen pointing towards the
generation of protection in mice of GP-V in comparison to GP-III and GP-IV. On the basis of
cytokines released, these CD4+ T cells were differentiated into Th1 and Th2 immune
response.37 CD4+ T cells mediates the production of IFN- which help B cells in controlling
and eliminating the infected erythrocytes. During our investigation, a high level of IFN- was
14

found in GP-V in comparison to GP-III and GP-IV (Figure 5). This signifies the generation
of effective Th1 immune response in host to control the infection in mice.
Inflammation to brain is the main characteristic feature of cerebral malaria. As IL-10 is
known as an anti-inflammatory cytokine, its sera levels have also been demonstrated in the
present study. IL-10 counter balance the inflammation and is crucial in protection from
inflammation in rodents as well as humans during malaria. It is mainly produced by CD4+ T
cells during Plasmodium infection.37 Sera of GP-V mice showed higher level of IL-10 on
D10 in comparison to GP-III and GP-IV. This increase in level of IL-10 was also significant
in comparison to GP-II mice, indicating that the inflammation to brain was also inhibited in
GP-V mice. Whereas, lower levels in other groups indicate the inflammation to host.
TNF-, a pro-inflammatory cytokine is produced by activated immune cells such as
macrophages, T cells, B cells and mast cells. During cerebral malaria, TNF- and IFN-, are
together known to upregulate the expression of adhesion molecules on endothelial cells
which mediates the attachment of various leukocytes to the cerebral vessels.38 In accordance
to this, present study showed an increase in sera levels of TNF-α in GP-II in comparison to
GP-V confirming the occurrence of inflammation in brain of the host and causing cerebral
malaria. Whereas, GP-III and GP-IV had lower levels of TNF-α in comparison to GP-V on
D10 but all mice in this group died because of the short-term protection.
Infected erythrocytes during blood stage infection induce Dendritic cells to produce IL-12
which further contributes to the activation of Natural killer cells (NK) and differentiation of
Th1 cells. Level of IL-12 was found to be extremely higher in GP-V mice in comparison to
15

GP-III and GP-IV signifies the genesis of Th1 response against the infection in the host
(Figure 6).
D
D0 D10
2500
2000
1500
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500
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GP-I GP-II GP-III GP-IV GP-V
Groups
E
D0 D10
3000
2500
2000
1500
1000
500
0
GP-I GP-II GP-III GP-IV GP-V
Groups
Figure 6: Histograms depicting the sera levels of IL-12 (D) and IL-27 (E) collected from GP-
I, GP-II, GP-III, GP-IV and GP-V. Cytokines were analysed using ELISA in pooled sera
samples of three mice in duplicate.
IL-27 also has anti-inflammatory responses and is involved in production of IL-10. GP-III
and GP-V had comparable levels of IL-27 on D10. Whereas, GP-V showed significant
increase in the level of IL-27 in comparison to GP-IV. An increase was also observed in level
of IL-27 in mice of GP-II in comparison to GP-V but this increase was not enough to protect
the host against infection and all the mice died of cerebral malaria.
CD8+ T cells provide protection against blood stages of parasite via IFN- secretion. During
present study, their frequencies were also found to be high in GP-V mice in comparison to
16

GP-III and GP-IV on D10 signifies the activation of cytotoxic cells against infection. On
D21, their level was found to be declined in GP-V due to the short-term protection provide by
the drug along with TLR agonist.
Tregs are T regulatory cells which regulate the functioning of T cells against infection. With
the help of Tregs parasite is capable for evading effective immune response and can promote
the parasite multiplication.39 In present study, no significant change was observed in splenic
Tregs in GP-V in comparison to GP-IV. Tregs were significantly increased in spleen of GP-
III mice in comparison to GP-V leading to the death of all mice in group. A sudden increase
was observed in frequencies of splenic Tregs of GP-V mice on D21 which was followed by
the death of all mice in GP-V.
TLR7-selective agonists are known to induce IFN- and IFN-regulated chemokines such as
IFN-inducible protein and IFN-inducible T cell chemoattractant from human PBMC. Even
though less effective than TLR8 agonists, TLR7 ligands are also known to induce the
secretion of pro-inflammatory cytokines. Due to the uncontrolled secretion of inflammatory
cytokines (IFN- and TNF-α) and establishment of PbA infection, early death of mice was
observed in a group treated with TLR7 agonist alone. TLR7 agonistic BBIQ played a
completely different role upon combining with Chloroquine (CQ). The migration of T cells in
the brain was inhibited. IFN-, IL-10 and IL-12 levels were upregulated in the blood sera as
well as higher levels of splenic T cells were noticed. T cells profiling in spleen of mice
treatment with BBIQ+CQ depicted the generation of strong humoral as well as cytotoxic
responses against the infection. Further cytokine profiling confirmed the generation of Th1
immune response in the host.
17

In brain of GP-V
In spleenof GP-V
In serum of GP-V
CD3
+ T
CD3+
T
IFN-,
IL-12
CD4
+ T
CD4+
CD8
+ T
IL-10
CD8+
T
T
TNF-,
IL-27
Tregs
Tregs
MICE SURVIVED
UPTO ONE MONTH
Figure 7: A diagrammatic representation showing the levels of various T (CD3+, CD4+,
CD8+ and T regs) cells in brain and spleen of mice along with the cytokines (IFN-, TNF-α,
IL-12, IL-10 and IL-27) level upon treatment with TLR7 agonist (BBIQ)+CQ (GP-V). A
strong but short lived Th1 mediated immune response was generated in mice mediating the
survival of mice up to one month.
Overall, a strong but short lived Th1 mediated immune response was observed and infection
appeared in peripheral blood of mice on D25. More research is required to improve this
short-term protection which can be achieved by varying the dose as well as dose profile.
Though, the effective Th1 immune response was for short term, it points out that the
immunochemotherapy approach combining TLR agonists along with an antimalaria drug has
potential to prevent cerebral syndromes as well as malaria infection in mice.
18

ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedure for the synthesis of target compound BBIQ. Materials and
methods for the biological studies. 1NMR spectroscopy and LC-MS data for the synthesized
intermediates and BBIQ (3).
Notes
The authors declare no competing financial or any conflict of interest.
ACKNOWLEDGMENTS
DBS is thankful to UGC New Delhi for start-up research grant, DBT New Delhi for the
award of Ramalingaswami Fellowship and PU Alumni Relations (contribution by Sh. S. K.
Munjal, Chairman of Hero Corporate Service Pvt. Ltd.) for the financial support. The support
from UGC-CAS, DST-PURSE, DST-SAIF and Panjab University Development fund is
gratefully acknowledged. The technical work of CSIF for immunophenotyping by flow
cytometer (FACS Aria B.D.) at PGIMER, Chandigarh. RS and DK are thankful to UGC for
the award of Research Fellowship.
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GRAPHICAL ABSTRACT
P. berghei ANKA
Chloroquine (CQ)
(10 mg/Kg)
TLR7 agonist (BBIQ)
(10 g/mouse)
T cells migration
in brain inhibited
Splenic T cells
IFN, IL-10 and IL-12
increased
upregulated
Generation of Th1 immune
response against infection
25