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induces superior antimalarial immunity in C57BL/6 mice” is a compilation of original work
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 “Vaccination with late liver stage-arresting
GAP induces superior antimalarial immunity
in C57BL/6 mice”

A PROJECT REPORT

Submitted in partial fulfillment of the requirements for the award of

the degree of

Master of Science
in

Applied Microbiology and Biotechnology, 2023

Submitted by
Yuti Besain

Under the Supervision of

Dr Satish Mishra
(Principal Scientist)

Department of Bioscience and Biotechnology

Banasthali Vidyapith
Rajasthan – 304022

2023
Acknowledgement

I would like to express my sincere gratitude to all those who have supported me throughout the
journey of completing this project report.

Firstly, I would like to thank my supervisor, Dr. Satish Mishra, principal scientist, CSIR-CDRI
for his guidance, patience, and unwavering support throughout the research process. His
feedback and constructive criticism has been instrumental in shaping my ideas and improving
my research methodology.

I would also like to thank my mentor Miss Plabita Paul for her patience and guidance
throughout the tenure.

Furthermore, I extend my thanks to rest of the scholars in the laboratory, Miss Akancha Mishra,
Miss Bandita Nayak, Miss Himadri Shukla, Miss Raksha Devi, Mrs Aastha Varshney, Miss
Rohini Nandi, Miss Pragya Mehra and Mr Nirdosh Mishra who graciously helped me
whenever I was in need. Without their participation, this thesis would not have been feasible.

My co-trainees Monalisha Sahu, Chikesh Mishra, Simran Shukla, Sanjay Verma,

Aishwarya U, Claire Maria Dominic, and Sonal Dodiyar, who kept me sane throughout also
need a special mention.

Additionally, I am grateful to my parents and my siblings for their unwavering support,

encouragement, and understanding throughout this journey. Their belief in me has been a
constant source of motivation and inspiration.

Thank you all for your contributions to this project report.

Table of Contents

1. Table of Figures

2. List of Tables

3. Abbreviations

4. Introduction

4.1 Introduction to Malaria

4.2 Malaria Lifecycle

4.3 Introduction to Anopheles

4.4 Anopheles Lifecycle

5. Literary Review
6. Materials and Methods

6.1 Giemsa Staining and determination of parasite

6.2 Transfer of infection in mosquitoes

6.3 Isolation of salivary gland sporozoites

6.4 Serum collection from immunized mice

6.5 Immunisation in C57BL/6 mice

6.6 Drug cover after immunisation

6.7 In Vitro determination of Exo Erythrocytic Forms (EEF)

6.8 Immunoflorescence Assay

6.9 In vivo imaging

6.10 Statistical Analysis

6.11 Retro-orbital blood collection

 6.12 Perfusion and isolation of immune cells

6.13 Sample preparation for Fluorescence Acquired Cell Sorting

(FACS)

7. Results

8. Discussion

9. Conclusion

10. References
1. Table of Figures

Figure 1 An approximation of the parts of world where malaria occur

Figure 2 Life Cycle of Malaria

Figure 3 Transmission of Malarial Parasite

Merozoites develop into ring stage and then trophozoites, which undergo
Figure 4
further replication giving rise to schizonts
Figure 5 Larval Stage

Figure 6 Pupal Stage

Figure 7 Life cycle of Anopheles

Figure 8 Giemsa Staining

Figure 9 Anopheles mosquitoes feeding on the infected mice’s blood

Figure 10 Isolation of salivary gland from infected Anopheles mosquitoes

Figure 11 Blood and Sera separated

Figure 12 Immunisation by intravenous injection

Chloroquine Drug
Figure 13

Figure 14 In vitro determination of EEF by using intra vital microscope

Figure 15 In vivo imaging through in vivo imaging system

Figure 16 Immunised mice liver before and after perfusion

Figure 17 Flow cytometry System

Figure 18 Perfused liver of Immunised mice

2. List of Tables

Table 1 Antibody used in sample preparation for FACS

3. Abbrevations

% Percentage
°C Degree Centigrade/Celsius
Bp Base pairs
KO Knockout
GAP Genetically attenuated parasites
DNA Deoxyribonucleic Acid
RNA Ribonucleic acid
EEFs Exo- erythrocytic forms
WHO World Health Organisation
FP Forward Primer
RP Reverse primer
GKO General Knock Out
h hours/hour
i.p. intraperitoneally
i.v. intravenously
M Molarity
Mg Milligram
Min Minute(s)
mM Milli Molar
ng Nanogram
µg Microgram
o/n Overnight
rcf Relative Centrifugal Force
rpm Revolution per minute
sec Seconds(s)
BS Blood stage
LS Liver stage
MG Midgut
SG Salivary gland
4. Introduction to Malaria

Malaria is a potentially life-threatening disease caused by a parasite transmitted to humans

through the bite of infected female Anopheles mosquitoes. Malaria is a major global health
problem, causing an estimated 229 million cases and 409,000 deaths in 2019 alone, according to
the World Health Organization (WHO). It is prevalent in tropical and subtropical regions of the
world, with sub-Saharan Africa being the most affected region. The disease is especially
dangerous for young children, pregnant women, and people with weakened immune systems.
Symptoms include fever, headache, chills, and flu-like symptoms, which can progress to severe
illness and death if left untreated. Effective prevention and treatment measures are available,
including the use of insecticide-treated bed nets, antimalarial drugs, and mosquito control efforts.
Despite progress in recent years, malaria continues to be a significant public health challenge,
particularly in low-income countries with limited access to healthcare and resources.

Classification

Domain: Eukaryote
Kingdom: Protista
Sub Kingdom: Protozoa
Phylum: Apicomplexa
Class: Sporozoasida
Order: Eucoccidiorida
Family: Plasmodiidae
Genus: Plasmodium
Species: P. falciparum

4.1 Epidemiology of Malaria

According to the WHO's 2021 World Malaria Report, there were 241 million clinical episodes
reported in 2020 and an estimated 6, 27,000 fatalities from malaria, the majority of which were
young children from Sub-Saharan Africa. 98% of malaria deaths and 95% of cases in the region
occurred in 2020. 80% of deaths in the area were of children under the age of 5. With 227
million clinical cases and 4, 09,000 fatalities reported in 2019, the infection toll has significantly
increased. Only four African nations—Nigeria (31.9%), the Democratic Republic of the Congo
(13%), the United Republic of Tanzania (4.1%), and Mozambique (3.8%)—accounted for more
than half of all malaria deaths globally.

Geographic Distribution: Malaria is found in more than 90 countries, mainly in tropical and
subtropical regions of Africa, Asia, and Latin America. The distribution is determined by the
presence of suitable mosquito vectors, primarily Anopheles mosquitoes, and environmental
conditions that support their survival.

Figure 1: An approximation of the parts of world where malaria occur.

High-Risk Populations: Certain populations are particularly vulnerable to malaria, including

pregnant women, young children, and travelers from non-endemic regions. People living in rural
areas with limited access to healthcare and preventive measures are also at higher risk.

4.2 Malaria Life cycle

Malaria, a vector-borne infectious disease caused by the Plasmodium parasite, continues to pose
a significant public health challenge worldwide. Understanding the complex life cycle of the
malaria parasite is crucial for developing effective prevention strategies, diagnostic tools, and
therapeutic interventions.
 Figure 2: Life Cycle of Malaria
(Source: [Link])

Transmission to the Human Host:

The life cycle of malaria begins when an infected female Anopheles mosquito, belonging to the
family Culicidae capable of transmitting the disease, takes a blood meal from a human. Upon
entering dermis, sporozoites rapidly travel to the liver via the blood stream, where they invade
hepatocytes, a type of liver cell.
 Source: [Link]

Fig 3: Invasion path of sporozoites from skin to the liver. At the initial stage of the host
parasite interaction, the vector female Anopheles mosquito inject 20-200 sporozoites beneath the
dermis which enters into the blood stream via lymph nodes. Via the circulation it reaches to the
liver where it enters into the sinusoidal space through the fenestration of the capillary. The host-
parasite interaction happens when the surface molecules of the sporozoite interacts with hepatic
heparan sulphate proteoglycan (HSPG). Circum-sporozoite protein (CSP) and TRAP
(Thrombospondin related adhesive protein) of the sporozoite helps in the successful invasion.
Upon entering into the hepatocyte, sporozoites transform into bulb like structure withing 3hours
and develop further which is known as EEFs or exo-erythrocytic forms. 65-70 hours p.i.
merosome forms which contains thousands or hepatic merozoites. These merosomes bursts and
merozoites enters into the blood stream to infect the RBCs.
 Figure 4: Merozoites develop into ring stage and then trophozoites, which undergo further
replication giving rise to schizonts (Nature Journal).

Blood Stage infection:

Hepatic merozoites interacts with the surface molecules of RBC membrane for entering within it.
Upon entering it forms a ring like stricture which is known as ring stage. Ring stage transformed
into the trophozoites when it feed on the hemoglobin and pigmented hemozoin accumulates
around it. The trophozoites undergo further replication, giving rise to schizonts that contain
multiple merozoites (Chen et al., 2020). Within the bloodstream, a small percentage of the
merozoites differentiate into sexual forms known as gametocytes. Gametocytes, consisting of
male (microgametocytes) and female (macrogametocytes) counterparts can be taken up by
another feeding mosquito during a blood meal. Male gametocytes release microgametes, while
female gametocytes serve as receptive sites.

Transmission to the Mosquito Host:

When an infected mosquito takes a blood meal, it ingests gametocytes from the human host. In
the mosquito's midgut, the microgametes fertilize macrogametes, forming zygotes. The zygotes
develop into motile ookinetes, which traverse the midgut wall and form oocysts on the outer
surface. Inside oocysts, the sporogonic division occurs, leading to the production of thousands of
sporozoites. Sporozoites are released from mature oocysts and migrate to the salivary glands of
the mosquito, where they become infectious. When an infected mosquito takes a subsequent
blood meal, sporozoites are injected into the human host, completing the transmission cycle and
initiating a new infection.

4.3 Introduction to the Vector Anopheles

Anopheles mosquitoes are a type of mosquito that can transmit malaria, a serious and sometimes
fatal disease caused by a parasitic protozoan of the genus Plasmodium. Anopheles mosquitoes
are found in tropical and subtropical regions around the world, including Africa, Asia, South
America, and parts of North America. There are over 400 species of Anopheles mosquitoes, but
only about 30-40 of them are significant vectors of malaria. Anopheles mosquitoes are easily
recognized by their long, slender bodies and narrow wings. They are typically most active during
the evening and early morning hours, and are often found near bodies of water such as lakes,
rivers, and swamps where they lay their eggs. Anopheles mosquitoes can be controlled through a
variety of methods, including the use of insecticides, mosquito nets, and environmental
management strategies such as removing standing water where mosquitoes breed. However, the
spread of malaria remains a significant public health challenge, particularly in areas where
resources are limited.
The Anopheles mosquito is a genus within the Culicidae family, which comprises over 460
recognized species (Sinka et al., 2010). These mosquitoes possess distinguishing morphological
features that enable their identification. Key characteristics include long and slender legs,
elongated mouthparts known as proboscis, and dark-scaled wings.
Anopheles mosquitoes are found worldwide, inhabiting various ecological settings ranging from
tropical rainforests to arid regions. However, their distribution is more prominent in regions with
favorable environmental conditions for their survival and reproduction. Anopheles species are
prevalent in tropical and subtropical regions of Africa, Asia, and the Americas, where malaria
remains endemic (Sinka et al., 2010).
Of all mosquito species, Anopheles mosquitoes are of utmost importance due to their ability to
transmit malaria parasites. Female Anopheles mosquitoes acquire Plasmodium parasites when
they feed on the blood of an infected human host. Within the mosquito's midgut, the parasites
undergo a complex developmental process, eventually forming infective sporozoites. These
sporozoites migrate to the mosquito's salivary glands, ready to be transmitted to a new host
during subsequent blood feeding (Cohuet et al., 2010).

Malaria transmission occurs when an infected Anopheles mosquito bites a human, introducing
the sporozoites into the bloodstream. The parasites then invade hepatocytes, leading to the
development of liver-stage forms and subsequent release of merozoites into the bloodstream.
Merozoites infect red blood cells, causing the characteristic symptoms of malaria (Gupta et al.,
2020).

4.4 Life Cycle Anopheles

Introduction:
Anopheles mosquitoes play a critical role in the transmission of malaria, a devastating vector-
borne disease caused by the Plasmodium parasite.

Egg Stage:
The life cycle of Anopheles mosquitoes begins when a female mosquito lays eggs on the surface
of stagnant or slow-moving water bodies such as ponds, puddles, or ditches. These eggs are
usually laid in clusters or rafts, and they float on the water surface (Ogoma et al., 2012).

Larval Stage:
When conditions are suitable, the eggs hatch, giving rise to mosquito larvae or wigglers. The
larvae live in the water and primarily feed on microorganisms and organic matter. They go
through four larval instars, molting between each stage, and continue to grow in size (Mukabana
et al., 2006).
 Figure 5: Larval stage

Pupal Stage
After the fourth larval instar, the larvae enter the pupal stage, also known as tumblers. During
this stage, the mosquito undergoes significant internal transformations and does not feed. The
pupae are comma-shaped and have respiratory trumpets that allow them to breathe at the water
surface (Ogoma et al., 2012).

Figure 6: Pupal Stage

Adult Stage:
Once the pupal development is complete, adult mosquitoes emerge from the pupal case and float
on the water surface until their wings and bodies are fully expanded and hardened. Female
Anopheles mosquitoes require a blood meal to obtain the necessary nutrients for egg
development, while males primarily feed on plant nectar.

Figure 6. Mosquito cage

Mating and Blood Feeding:

During mating, males locate and court females using various sensory cues. After mating, female
Anopheles mosquitoes seek a blood meal, which is essential for the development of their eggs.
They locate hosts by detecting body heat, moisture, and chemical signals such as carbon dioxide
(Gillies and De Meillon, 1968).

Malaria Parasite Transmission:

If a female Anopheles mosquito feeds on a human infected with the Plasmodium parasite during
a blood meal, the mosquito becomes infected with the parasite. The parasites undergo further
development and multiplication within the mosquito's body, specifically in the midgut and
salivary glands (Vaughan et al., 2018).

Mosquito Lifespan and Repeat Infections:

Anopheles mosquitoes can live for several weeks to months, depending on environmental
conditions. Throughout their lifespan, infected mosquitoes can transmit the malaria parasite to
multiple human hosts through subsequent blood meals, contributing to the persistence and spread
of malaria (Vaughan et al., 2018).

Figure 7: Life Cycle of Anopheles

Understanding the life cycle of Anopheles mosquitoes is crucial for effective malaria control
strategies. By targeting different stages of the mosquito's life cycle, such as eliminating breeding
sites or using insecticide-treated bed nets, it is possible to reduce mosquito populations and
interrupt malaria transmission. Continued research and innovative interventions are essential to
combat malaria and reduce the burden of this global health threat.

Introduction to whole sporozoite immunization

Currently, there is no licensed vaccine for malaria that provides complete protection against the
disease. However, there are ongoing efforts to develop a highly effective vaccine. The most
advanced vaccine candidate is the RTS, S/AS01 vaccine, which has been shown to provide
partial protection against malaria in clinical trials. In addition to vaccination, other immunization
strategies include the use of antimalarial drugs for chemoprophylaxis, which involves the
administration of drugs to prevent the occurrence of malaria in individuals who are at high risk
of infection. This strategy is particularly important for travelers visiting malaria-endemic
regions. Another form of immunization is the use of passive immunization through the
administration of monoclonal antibodies that can neutralize the parasite. This approach is still in
the experimental stages, and more research is needed to determine its effectiveness in preventing
and treating malaria.

Whole sporozoite immunization aims to mimic natural infection by using attenuated Plasmodium
sporozoites as vaccine candidates. Attenuation can be achieved through various techniques such
as radiation, genetic modification, or drug-based approaches. These strategies render sporozoites
incapable of causing clinical disease while still retaining their ability to induce immune
responses. Immunization is typically administered through intravenous or intradermal injection,
or via controlled mosquito bites in clinical settings (Sissoko et al., 2017).

Whole sporozoite immunization triggers a cascade of immunological events leading to protective

immune responses against malaria. Key mechanisms involved include:

Cellular Immune Responses: CD8+ T cells play a crucial role in recognizing parasite-infected
hepatocytes. Sporozoites, after inoculation, migrate to the liver, infect hepatocytes, and produce
liver-stage forms. Attenuated sporozoites, however, fail to fully develop and are eliminated by
CD8+ T cells, resulting in reduced parasite burden (Hoffman et al., 2015).

Humoral Immune Responses: Antibodies induced by whole sporozoite immunization target

sporozoite and liver-stage antigens. These antibodies can inhibit sporozoite motility, prevent
hepatocyte invasion, and neutralize sporozoite infectivity (Ishizuka et al., 2016).
Innate Immune Activation: The initial encounter of sporozoites with the innate immune system
triggers the activation of antigen-presenting cells, secretion of cytokines, and recruitment of
immune cells, which shape the subsequent adaptive immune response (Yamauchi et al., 2007).

Whole sporozoite immunization is an experimental approach to malaria vaccination that involves

the injection of live, attenuated Plasmodium sporozoites into an individual's bloodstream to
induce an immune response. This approach is based on the idea that exposure to live parasites
will stimulate a robust immune response that will protect against future infections. Several
clinical trials have been conducted to evaluate the safety and efficacy of whole sporozoite
immunization, and the results have been promising. In particular, the use of intravenous
administration of sporozoites has shown to provide high levels of protection against malaria
infection in both controlled laboratory settings and in real-world field trials. One of the
advantages of whole sporozoite immunization is that it can provide long-lasting protection
against multiple strains of the malaria parasite. However, there are also some challenges
associated with this approach, including the need for careful attenuation of the parasite to prevent
the development of full-blown malaria in the vaccinated individual. Overall, whole sporozoite
immunization represents a promising approach to malaria vaccination, and ongoing research is
focused on further optimizing this approach to improve its safety and efficacy.
5. Literary Review

The researchers generated a genetically modified Plasmodium berghei parasite with a specific
gene deletion that disrupted parasite development during the late liver stage. C57BL/6 mice were
infected with the genetically attenuated parasite, and the immune responses, including T cell
responses and antibody production, were assessed. The researchers also measured the protective
efficacy of the vaccine by challenging vaccinated mice with wild-type parasites.
Immune Responses: Vaccination with the late liver stage-arresting GAP induced robust CD8+ T
cell responses and specific antibody production in C57BL/6 mice. The immune responses were
superior to those induced by vaccination with a wild-type parasite or an early liver stage-
arresting GAP.
Protective Efficacy: The late liver stage-arresting GAP vaccination conferred superior protection
against subsequent infection with wild-type parasites compared to other vaccine candidates.
Vaccinated mice exhibited reduced parasitemia and prolonged survival.

The growth of genetically attenuated malaria parasites (GAP), which halt during the liver stage,
is inhibited, are potent immunogens that provide complete and long-lasting protection from
sporozoite infection. When compared to early live stage-arresting attenuated parasites, late liver
stage-arresting GAP offers mice greater defense against sporozoite challenge. The liver-stage
infection of malaria can be prevented by immunizing with radiation (c)-attenuated Plasmodia
sporozoites (c-spz), which provides sterile and long-lasting protection. However, neither the
source of their induction nor the antigen-presenting cells (APC) that activate these CD8+ TEM
cells have been thoroughly studied. They have shown that frequent exposures of mice to Pb c-spz
cause a gradual and virtually simultaneous increase of both CD11c+NK1.12 DC and CD8+ TEM
cells in the liver but not the spleen. Liver CD11c+NK1.12 DC from Pb c-spz-immunized mice
induced protective immunity upon adoptive transfer against sporozoite challenge. Additionally,
liver cCD8a+ DC caused naive CD8+ T cells to show the CD8+ TEM phenotype and release
IFN-c in an in vitro system. Anti-MHC class I and anti-IL12 mAbs prevented cCD8a+ DC from
inducing functional CD8+ TEM cells in vitro. Through a variety of unique functions in T cell
activation and T cell contact with other immune cells, the co-stimulatory molecule CD40
improves immunity. The full maturation of liver dendritic cells, the increase of CD8+ T cells in
the liver, and the protective immunity brought on by immunization with the genetically
attenuated P. yoelii parasite were all dependent on CD40 in a mouse model of immunity against
liver stage Plasmodium infection. They compared the contributions of CD40 expressed on host
tissues, including antigen-presenting cells (APC), to CD8+ T cell immunity to CD40 expressed
on the CD8+ T cells themselves using mixed adoptive transfers of polyclonal wild type (WT)
and CD40-deficient (CD40/) CD8+ T cells into WT and CD40/ hosts. Plasmodium sporozoites
that have been radiation-damaged have been used to immunise people against malaria in order to
stimulate protective immunological responses. However, heat-killed, non-viable sporozoites do
not result in protection, highlighting the necessity of living parasites for the induction of efficient
immune responses. We examined the primary CD8+ T cell responses induced by heat-killed
inactivated sporozoites using an experimental system containing CD8+ T cells from T cell
receptor-transgenic mice. When immunising with attenuated sporozoites, we discovered that the
numbers of specific CD8+ T cells that were activated were significantly lower; nevertheless, the
kinetics of activation and the phenotype of these T cells were identical in both groups. High
numbers of certain CD8+ T cells were seen after boosting with a recombinant vaccinia virus,
despite their low frequency after priming. When either heat-killed or attenuated sporozoites were
employed for priming, the same level of protection was seen after the induction of the recall
[Link] contend that the development of memory T cell populations against the liver stages
of malaria does not need the presence of living parasites.
6. Materials and Methods

6.1 Giemsa Staining and determination of Parasite

A blood smear was made, air dried and fixed with methanol. The slides were incubated for 15 to
20 minutes with Giemsa solution prepared by mixing 1 ml Giemsa stain and 4 ml Giemsa buffer
and washed with tap water. The slides were again air-dried and observed under a light
microscope.

Figure 8: Giemsa Staining

The calculation of parasitemia, RBCs and parasites per field were counted and the parasitemia
was calculated using the following formula:

Percent Parasitemia = Total No. of parasites x 100

Average RBC’s

6.2 Retro-orbital Blood transfer

Blood smear of the mice was made, stained with Giemsa staining solution and then parasitemia
count of an average of 10 fields was performed. After the calculations the next step was to
collect blood from mice through its retro-orbital sinus using a heparinized Pasteur pipette and the
dilutions were made accordingly. After the dilutions were made the blood sample was injected
intravenously in three healthy mice, for each sample.
6.3 Transfer of Infection in Mosquitoes
Infected mice having optimal number of gametocytes were anesthetized and kept on the
mosquito cage for them to feed on the infected mice’s blood. The feeding procedure was
performed for 20 minutes by changing the positions of the mice every 5 minutes. Prior to
performing the feeding procedure, the mosquitoes are kept in a starving condition for 3-4 hours
the feeding is then performed twice, one in the evening after starvation and one in the next
morning. Cotton pads soaked in sucrose solution were used to maintain the nutrient conditions of
the mosquitoes. The infected mosquitoes are kept in environmental condition like 19ºC and 80%
relative humidity. The infection cycle was monitored further.

Figure 9: Anopheles mosquitoes feeding on the infected mice’s blood

6.4 Isolation of salivary gland sporozoites

Mosquitoes were collected and kept in -20ºC for 2 minutes after which they become
unconscious. It is then washed with the ethanol only for a few seconds and it is removed right
after. Dulbecco’s Modified Eagle’s Medium (DMEM) was used to wash the mosquitoes. Three
washes were performed, 4 minutes each kept in ice. After the three washings the mosquitoes
were transferred in an Eppendorf tube and dissected under the stereo-binocular microscope with
the help of needles to isolate the salivary glands.
 Figure 10: Isolation of salivary gland from infected Anopheles mosquitoes
6.5 Serum Collection from Immunized mice

Before the WT challenge of the immunized mice, whole blood was collected through the process
of retro orbital blood collection. The Eppendorf had no anti-coagulant present in it; hence the
plasma was separated easily. Clot was removed by centrifuging at 1000g for 10mins in a
refrigerated centrifuge machine. Supernatant was collected in a new tube and stored at -20֯ C
refrigerator.

Figure 11: Blood and Sera separated

6.6 Immunisation in C57BL/6 mice

The isolated salivary glands were collected in an Eppendorf tube and DMEM was poured in it.
Using an ethanol wiped plunger the salivary glands were plunged thrice with first a 4-minute
spin and the rest two as short spins respectively after every 10 to 15 plunging. To count the
number of sporozoites a 50X dilution was made and 10µl of it was transferred to a
hemocytometer to count the number of sporozoites. All the quadrants are counted and an average
was taken out.
After counting the number of sporozoites/µl by the formula:

Number of sporozoites/µl = Average x Dilution x Hemocytometer Factor

Amount of sporozoites to be injected in the mice was calculated using the following formula:

Amount of sporozoites to be injected /mice = No. of sporozoites x No. of mice

Sporozoites/µl

Figure 12: Immunisation by intravenous injection

6.7 Drug Cover for mice after Immunisation

The drug used for cover was Chloroquine (C18H26CIN3.2H3PO4). A diphosphate salt which is
already recognized to act upon blood stage parasites. It was prepared as per the number of mice
having each mouse to receive 0.8mg (28mg in 7ml).

Figure 13: Chloroquine Drug

6.8 In Vitro determination of Exo Erythrocytic Forms (EEF)
HepG2 cells were revived from the stock stored in liquid nitrogen, incubated at 37ºC and 5%
CO2. To determine EEFs the sporozoites were added to HepG2 cell culture to initiate the in vitro
culture. The cells were seeded a day before the infection. To prepare cells for infection the cells
were treated with trypsin-EDTA and then collected in complete DMEM medium. The cell
number is counted after making a dilution and using hemocytometer. Then they are seeded onto
the glass coverslips in a 48 well plate. Prior to seeding the cells, the surface of the well plate was
treated with collagen; Type I solution from rat tail, diluted 1:9 times in MQ water. The salivary
gland sporozoites were added to HepG2 culture and the plate was spun at 310xg for 4 min and
incubated in a CO2 incubator. After 1 h, cells were washed with a medium containing
antibiotic/antimycotic and amphotericin B for 5 min. The medium was changed every 12 h and
the coverslips were taken out at different time points and fixed in 4% paraformaldehyde for 20
min at room temperature.

Figure 14: In vitro determination of EEF by using intra vital microscope

6.9 Immunoflorescence Assay

For localization experiments, transgenic parasites in the blood and liver stages were fixed at
various times using 4% PFA for 20 min at room temperature. 1% BSA-PBS was used to block
the cells after they had been permeableized by cooled methanol at 4°C for 15 minutes. EEFs
were stained with Upregulated in Infectious Sporozoite 4 (UIS4, 1:1000, Rabbit) and sera (1:50)
gathered from the immunised mice in order to see the parasite. For sporozoite stain TRAP 1:200)
and sera (1:50) from the immunized mice was used. Nuclei were stained with Hoechst 33342 and
the coverslips were mounted on Prolong diamond antifade reagent (Invitrogen). The images were
acquired in a using FV1000 software on a confocal laser scanning microscope (Olympus
BX61WI) using an UPlanSAPO 100x/1.4 immersion oil.

6.10 In vivo Imaging

We injected 3mg luciferin in each mouse according to the body weight of 20g. With the help of
In Vivo Imaging System, we took images with an exposure of 120 seconds each time.

Figure 15: In vivo imaging through in vivo imaging system

6.11 Statistical analysis

The Luciferin assay was analyzed by spectral imaging software named AURA Imaging.

6.12 Perfusion and Isolation Immune Cells

Compositions:
1. Perfusion Buffer: 5-10 ml/ mouse
HBSS, 5mM HEPES, 0.5 mM EDTA
2. Wash Buffer: 50 ml/ mouse
PBS, 4% FBS, 0.5 mM EDTA
3. PBS Flow Buffer: 20 ml/mouse
1mM EDTA, 2% FBS
4. Collagen Solution: 5-10 ml/mouse
HBSS, 5mM HEPES, 0.5 mM CaCl2, 0.5 mg/ml collagenase.

Anesthetized the mouse by injecting the 100µl of ketamine xylazine. Placed the mouse’s belly on
gauze pad and secured footpads in an X orientation. Disinfected the mouse using 70% ethanol
and cut opened the skin to expose the peritoneal membrane. After exposing the inner side of the
mouse’s body, we gently moved the intestines and stomach to the right side and stuck the liver to
the diaphragm. Identified the portal vein and the vena cava and using sharp scissors we cut the
portal vein. Turned on the pump and dripped the perfusion buffer to the left side of the pump’s
pipe. Catheterized the vein after which the liver blanched. We cut the vena cava such as the
blood and the buffer visibly flew through the vena cava. The pump was stopped after the liver
was completely blanched. The pump was now stopped and the catheter was removed. Then the
line was shifted back for the perfusion of the next mouse.

Portal Vein
Venacava
Venacava

Fig 16: Before Perfusion After Perfusion

6.13 Sample preparation for Fluorescence Acquired Cell Sorting (FACS)

The liver was grabbed using forceps and the attachment between the liver and the diaphragm was
cut gently. The digested liver was transferred to the petri dish and 30ml cold wash buffer was
added to it. A rubber plunger was used to gently massage the liver from over the strainer the
disperse the cells through it. The cell suspension was filtered in a 50ml conical tube. Then it was
pelleted 600g for 10 minutes at 4ºC. It was then gently resuspended in 2.5 ml PBS. After
pelleting the cell suspension, the supernatant was discarded and the pellet was strained with the
help of using a strainer of pore size 70µm and PBS. The next step was to lyse the RBC with the
help of RBC lysis buffer for 2 minutes and centrifuged again. It was strained again with the help
of the same strainer and the volume make up was done by chilled PBS. The filtrate was then
spun at 600g for 10 minutes at 4ºC. The supernatant was discarded and the pellet was dissolved
in flow buffer.

Figure 17: Flow Cytometry System

An antibody cocktail was prepared using a multiple number of antibodies

Catalogue Antibody Fluorochrome Stock

Number Concentration

2265468 CD3e FITC (0.5 mg/ml)

2262331 CD4 PE-Cy5 (0.2 mg/ml)
2331993 CD8a PE-Cy7 (0.2 mg/ml)
2197878 CD44 Alexa 700 (0.2 mg/ml)
2282646 CD62L APC (0.2 mg/ml)
2392637 CD183(CXR3-173) FITC (0.5 mg/ml)
2284176 KLRG1 Per-CP-eFlour 710 (0.2 mg/ml)
2452842
CD69 Per-CP Cy 5.5 (0.5 mg/ml)
Table 1: Antibody used in sample preparation for FACS
The antibody cocktails made were as follows:

Cocktail 1: Lymphocytes
[CD3 + CD8 + CD4]
Cocktail 2: Effector Memory T cells
[CD3 + CD8 + CD44 + KLRG 1 + CD62L]
Cocktail 3: Regulator Memory T cells
[CD8 + CD44 + CD69 + CD183(CXR3-173)

Figure 18: Perfused liver of Immunised mice

7. Results

I Confirmation of Early and late attenuation of EA-GAP and LA-GAP parasite.

We cultured HepG2 in 48 well plate (in vitro) and infected them with the sporozoites
which were isolated from the salivary glands of the Anopheles mosquitoes at the 21 st day
of post feeding. The culture was terminated by formaldehyde fixing at different time
points and IFA was performed by UIS4 (1:1000; antibody against the PVM) and Hoechst
33342 (1:3000; nuclear stain). After quantifying the EEFs area we confirmed the
significant reduction of the size of EA-GAP EEFs which was comparable to that of the
30-36 hrs of WT EEFs and does not mature further. Whereas LA-GAP EEFs grew
normally but not all merozoites egressed from hepatocytes. We measured the area of
EEFs and observed that the area is significantly less in the case of EA-GAP.
A. GFP UIS4 Hoechst Merge
EA-GAP

Scale bar 10 μm
LA-GAP

B. 400 EA-GAP
EEFs area 65hr p.i. (μm2) LA-GAP
***
300

200

100

0
EA-GAP
LA-GAP
Fig 19: Comparison between In-vitro liver stage development of EA-GAP and LA-GAP.
(A) IFA using α-UIS4 and Hoechst 33342 was performed to visualize the hepatic development
of the GAPs which stains PVM and nuclei respectively. EEFs were also recognized by its own
GFP. Scale bar 10 μm. (B) Reduced EEFs size in the case of EA-GAP was also reflected by the
area quantification. Data represents mean ± SD. n=50 (P<0.0001).
II Late attenuated GAP provide superior protections in C57 BL/6 mice.
Batches of 15 C57BL/6 mice were immunized thrice with salivary gland sporozoites of
both of the GAPs and was kept under Chloroquine drug cover (0.8mg/mouse) for 7 days
after each immunization. Only the salivary gland extracts were injected for the mock
control to confirm the immunity acquired entirely by the sporozoites. The entire
immunized batch of mice was segregated into three groups, 10 days after the third
immunization one of the groups were challenged with 5000 WT GOMO
sporozoites/mouse and parasitemia was observed up to 15 days p.i. 2 out of 4 mice of
EA GAP immunized group were positive at 5-6 days, whereas all the mice of the LA
GAP immunized batch remained negative throughout the observation period. Another
group of mice were challenged in a similar manner. 55hour post challenge we injected
luciferin D (VivoGlo™ Luciferin, In Vivo Grade) 5-10min prior to visualize the liver
burden in vivo. Mice were imaged under IVIS spectrum in vivo imaging system and we
found that all the WT mice were showing significant bioluminescence signal while the
LA GAP immunized mice were negative for the same. In case of EA-GAP 2 out of 3
mice showed bioluminescence signal however it was significantly less as compared to
mock control.

WT sporozoite No of Sporozoites Mice Pre-Patent period

challenge injected positive/mice (Days)
injected
Mock 5000 4/4 3

EA-GAP 5000 2/4 5.5

LA-GAP 5000 0/4 - Up to

 15day p.i.
A.

B.
MOCK EA-GAP LA-GAP

55 hr

C. ***
40 **
Mock
5
Total flux (P/s) x 10 EA-GAP
LA-GAP
30

20

10 ns

0 Mock EA-GAP
LA-GAP

Fig 20: Visualization and quantification of the liver burden of immunized mice in-vivo
after WT challenge. (A) Immunized mice were challenged with 5000 WT GOMO sporozoites.
Blood smear was prepared every day post challenge and stained with Giemsa to monitor the
parasitemia. Parasitemia was monitored up to 15days p.i. (B) Liver burden of the immunized
mice was compared by the luciferase expression in the form of bioluminescence signal and
captured by IVIS system. As GOMO WT parasite constitutively expresses Luciferase it catalyzes
Luciferin provided externally by intraperitoneal injection 5-10 min prior to the imaging. (C)
Bioluminescence signal was quantified in the form of Total photon flux per sec (P/s) of same
ROI. Data represents mean ± SD, N=3 (Unpaired T-test, Mock vs EA-GAP P<0.001, Mock vs
LA-GAP P<0.0001, EA-GAP vs LA-GAP non-significance).

III Sera from immunized mice recognized different parasite stages.

Immune sera were collected from the both GAP immunized mice before WT challenge and
used for IFA to recognize different stages of the parasite. The P. berghei salivary gland
sporozoite was immuno-stained with sera (1:200 dil) and α -TRAP (anti-Rabbit; 1:250 dil)
antibody which is a recognized marker for microneme. It was observed that the sera stain the
membrane of the sporozoites indicating, both the GAP immunized mice produce sporozoite
specific antibody. Later, we also performed IFA with PBANKA WT infected hepatic EEFs after
fixing the culture at different time points. For the recognition of EEFs we have used α-UIS4
(anti-Rabbit; 1:1000 dil) which is a known marker against the PV membrane. It has been
observed that sera also have the capability to recognize up to 65hr p.i. EEFs.

A. Anti- TRAP Immunization Sera Hoechst Merge

EA-GAP

B. Anti-UIS4 Immune-Sera Hoechst Merge

EA-GAP

5 μm Scale bar
LA-GAP
24hr
LA-GAP

EA-GAP

LA-GAP 48hr
Fig 21: Sera from GAP immunized mice can recognize different stages of the
parasite. (A) PBANKA WT sporozoites were collected from salivary gland and fixed on
the glass slides by 4% paraformaldehyde. IFA was done by TRAP antibody and immune
sera collected from GAPs to recognize the sporozoites. (B) HepG2 cells were cultured in-
vitro and infection was given by PBANKA WT sporozoites. Culture was terminated at
24hr, 48hr and 65hr p.i. To observe EEFs UIS4 antibody was used along with the sera.
Scale bar 5 μm.

IV Mice immunized with GAP parasite show increased level of CD44 and
CD8 cell population.
To observe the immune parameters of the immunized mice we isolated single cell
suspension of the liver and stained with specific markers of different immune cells. Flow
cytometry was performed to segregate different cells and later quantified the percentage
of the cell populations. Here, we observed a significant increase of the lymphocytes in
both of the GAP immunized mice with comparison to the mock. Significant increase of
 CD8+ /CD44+ T cell population of LA-GAP implies its superiority than EA-GAP as a
vaccine candidate.
A.

Mock

EA-GAP

LA-GAP

B.
Mock

EA-GAP

LA-GAP
F

C.
* D. E.
*** *** ***
50 ** 70 90
** ***
60 ** % CD44+ T cells
40
% Lymphocyte
**
% CD8+
50 T cells 60
30 40

20 30
30
20
10
10
Mock
0 EA-GAP Mock
0 EA-GAP 0MockEA-GAP
LA-GAP LA-GAP LA-GAP

Fig 22: Increased level of CD8+ and CD44+ T lymphocytes in LA-GAP: (A) Outline
of the gating strategy in flow cytometry for lymphocytes (P3). P4 is the CD3+ cells and
P5 is the CD8+ T cells. (B) CD 44+ cells were gated from CD8+ cell population by
specific marker. (C) % lymphocytes (D) % CD8+ T cells (E) % CD44+ T cells. Data
represents ± SD, N=4. (Unpaired T test, * represents P<0.01; ** represents P<0.001; ***
represents P<0.0001).
V GAP vaccination induced increased level of TRM cells.

Regulatory memory T (CD 69+/CD 62Llo) cell population was also upregulated in LA-GAP.

Mock

EA-GAP

LA-GAP

**
ns
80
*
% T RM (CD69+/CD62Llo)
60

40

20

0Mock LA-GAP
EA-GAP
Fig 23: GAP immunized mice Induces T RM cell population: (A) Flow cytometry
gating outline for TRM (CD69+/CD62Llo) cell population. (B) Percentage of the
TRM cell population. Data represents ± SD, N=4. (Unpaired T test, * represents P<0.01; **
represents P<0.001; *** represents P<0.0001).

8. Discussion
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