Jurnal Profesi Medika : Jurnal Kedokteran dan Kesehatan

DOI: https://doi.org/10.33533/jpm.v18i1.7451
Vol 18 No 1 (2024) ISSN 0216-3438 (print); ISSN 2621-1122 (online)

ARTICLE

INCREASED OF SPLEEN WHITE PULP DIAMETER POST DBL2β-PfEMP1 RECOMBINANT

PROTEIN INJECTION IN WISTAR RATS: PRE-CLINICAL STUDY FOR MALARIA VACCINE
DEVELOPMENT

Erma Sulistyaningsih1*, Nindya Audatus Sa’diyah2, Irawan Fajar Kusuma1, Rosita Dewi1, Sheilla
Rachmania1
1Departemen Bioteknologi, Fakultas Kedokteran, Universitas Jember, Jember, Indonesia
2FakultasKedokteran, Universitas Jember, Jember, Indonesia

*Correspondence email : [email protected]

ABSTRACT

Malaria is a major infectious disease worldwide, and vaccination is essential for disease control. The Plasmodium
falciparum Erythrocyte Membrane Protein-1 (PfEMP1) is a potential malaria vaccine candidate due to its
involvement in pathogenesis. Injection of DBL2β-PfEMP1 recombinant protein in animal models induces IgG and
CD4+ production and inhibits binding with host endothelial receptors. This study aimed to analyze the spleen
immune response by measuring the white pulp diameter. The experimental study used Wistar rats (Rattus
norvegicus) that were divided into four groups, a control group and three treatment groups, which were injected
with 100, 150, and 200 μg of DBL2β-PfEMP1 protein. Injection was done three times with three-week intervals
(days 0, 21, and 42). On day 56, rats were euthanized, and spleens were prepared for histology examination. The
white pulp diameter increased along with increasing the dose of protein. The ANOVA test showed a significant
difference between groups (p=0.001). The post-hoc Bonferroni test showed a significant difference between the
control and the 150 and 200 μg groups (p=0.013, p=0.002) and the 100 μg and 200 μg groups (p=0.027). In
conclusion, the DBL2β-PfEMP1 recombinant protein injection increased the spleen white pulp diameter in Wistar
rats, and the 200 μg dose resulted in the highest increase.

Keywords: DBL2β; Malaria; Plasmodium falciparum; PfEMP1; Spleen-white pulp

АБСТРАКТ
Малярия является одним из основных инфекционных заболеваний во всем мире, и вакцинация необходима
для контроля заболевания. Мембранный белок эритроцитов Plasmodium falciparum Erythrocyte Membrane
Protein-1 (PfEMP1) является потенциальным кандидатом в малярийные вакцины благодаря его участию в
патогенезе. Инъекция рекомбинантного белка DBL2β-PfEMP1 на животных моделях индуцирует
выработку IgG и CD4+ и ингибирует связывание с рецепторами эндотелия хозяина. Целью данного
исследования был анализ иммунного ответа селезенки путем измерения диаметра белой пульпы. В
экспериментальном исследовании использовались крысы Вистар (Rattus norvegicus), которые были
разделены на четыре группы, контрольную и три лечебные группы, которым вводили 100, 150 и 200 мкг
белка DBL2β-PfEMP1. Инъекции проводили три раза с трехнедельными интервалами (дни 0, 21 и 42). На 56-
й день крыс умерщвляли, а селезенки готовили для гистологического исследования. Диаметр белой
пульпы увеличивался по мере увеличения дозы белка. Тест ANOVA показал значительную разницу между
группами (p=0,001). Пост-хок тест Бонферрони показал значительную разницу между контролем и
группами 150 и 200 мкг (p=0,013, p=0,002) и группами 100 и 200 мкг (p=0,027). В заключение следует
отметить, что введение рекомбинантного белка DBL2β-PfEMP1 увеличивало диаметр белой пульпы
селезенки у крыс Вистар, причем доза 200 мкг приводила к наибольшему увеличению.

Ключевые слова: DBL2β; Малярия; Plasmodium falciparum; PfEMP1; Белая пульпа селезенки

Copyright © 2024 Jurnal Profesi Medika : Jurnal Kedokteran dan Kesehatan 51

Submitted: 29 January 2024 ; Received in revised form: 7 February 2024 Accepted: 14 February 2024 ; Available online:15 June 2024;
Published regularly: June 2024
 E. Sulistyaningsih , N.A. Sa’diyah , I.F. Kusuma , R. Dewi , S. Rachmania

INTRODUCTION (EPCR), and is divided into α, β, and γ subtypes.

Malaria is an infectious disease caused by The DBL domain is found in almost all parts of
Plasmodium sp. and a major global health the PfEMP1 head. It has subtypes α, β, γ, δ, ε,
problem. World Health Organization (WHO) and χ. The DBL2β domain specifically binds the
reported 247 million malaria cases in 2021, ICAM-1 receptor. Injection of PfEMP1 could
resulting in 619,000 deaths. Indonesia is induce IgG and CD4+ production, which
second-ranked in Asia in terms of the highest reduces the risk of severe malaria by 37% and
malaria cases, with a total of 399,666 cases in inhibits merozoite invasion, cytoadherence,
2021. The number of malaria cases in and infection.7 It supports the evidence for the
Indonesia has increased by 30% compared to development of the DBL2β-PfEMP1
the previous year, rising from 304,607 cases to recombinant protein as a malaria vaccine.8
400,253 cases in 2022.1 Vaccine development involves two main
Plasmodium falciparum, P. ovale, P. vivax, P. stages, i.e., pre-clinical and clinical studies. The
malariae, and P. knowlesi are the species that pre-clinical study uses animal models to
cause malaria infection in humans. 90% of determine the immunogenicity, safety, and
malaria deaths in the world are caused by effectiveness of vaccine candidates in a
Plasmodium falciparum.2,3 Severe malaria can laboratory and in vivo setting. Previous studies
occur at any age in areas with low transmission reported the immunogenicity of the DBL2β-
rates, as well as in individuals with a history of PfEMP1 recombinant protein by inducing IgG
traveling to malaria-endemic areas. The initial and CD4+ production. This study evaluated
symptoms of malaria are malaise, headache, further immune response by the spleen-white
muscle pain, fever, nausea, vomiting, diarrhea, pulp diameter. The spleen is a secondary
and orthostatic hypotension. Malaria can cause lymphoid organ and has a crucial role in
severe clinical manifestations such as cerebral regulating immune response.9 The spleen has
malaria, acidosis, severe anemia, acute renal two main structures: the white pulp and the
failure, and hypoglycemia.4 red pulp. The white pulp is the part
The manifestation of severe P. falciparum surrounding the central artery and is
malaria is due to cytoadherence and rosetting constituted by three regions; the periarteriolar
mechanism. Cytoadherence is the attachment lymphocyte sheath (PALS, T-cell area),
of infected red blood cells (IRBCs) to the lymphoid follicles (B-cell area), and marginal
Intracellular Adhesion Molecule 1 (ICAM-1) zones (MZ, B-cell area). It consists of T and B
receptor in the host endothelium. While cells, contains white blood cells, and is
rosetting is an attachment of infected red involved in the adaptive immune system
blood cells (IRBCs) and uninfected red blood activation response. The whole white pulp
cells (URBCs), forming a flower-like structure. contains about a quarter of the total number of
Cytoadherence and rosetting are mediated by lymphocytes in the body. It allows the
the antigen protein Plasmodium falciparum formation of specific antigen immune
Erythrocyte Membrane Protein-1 (PfEMP1). responses that protect the body from disease.
This process leads to microvascular blockage, Changes in the cellular composition occur in
ischemia, and hypoxia and causes organ the area of the white pulp after exposure to
damage.5 immunomodulatory agents.9,10 The study
PfEMP1 is a protein that binds to the aimed to analyze the effect of DBL2β-PfEMP1
membrane surface of host IRBCs. The outer recombinant protein on the spleen-white pulp
part of the PfEMP1 consists of the N-terminal diameter.
segment (NTS), the cysteine-rich interdomain
region (CIDR), and the Duffy binding-like MATERIAL AND METHODS
(DBL).6 The CIDR domain binds to multiple This study has received ethical approval
receptors, including Cluster of Differentiation from the Research Ethics Committee of the
36 (CD36) and Endothelial Protein C Receptor

52 | DOI: https://doi.org/10.33533/jpm.v18i1.7451 Vol 18 No 1 (2024)

 E. Sulistyaningsih , N.A. Sa’diyah , I.F. Kusuma , R. Dewi , S. Rachmania

Faculty of Medicine, University of Jember, No. diameter, then divided into two. Two
1830/H25.1.11/KE/2023. observers performed the examination.
The study was a true experimental study The reliability of data was determined by
with a post-test-only control group design. The the Cronbach alpha test.
samples were Wistar rats (Rattus norvegicus) The data were analyzed using SPSS.
aged 2-3 months, weighing 150–250 g, and Statistical analysis was performed using the
divided into a control group and three Saphiro-Wilk normality test, Levene
treatment groups. Rats were acclimatized for homogeneity test, and One-way ANOVA test
two weeks in optimal condition before followed by the posthoc Bonferroni test. This
receiving treatment with injection of NaCl study uses a confidence interval of 95%.
0.9% for the control group and DBL2β-PfEMP1
recombinant protein at doses of 100, 150, and RESULT
200 μg for treatment groups. The features of spleen histology are
The DBL2β-PfEMP1 recombinant protein presented in Figure 1.
was produced in Escherichia coli BL21(DE3). It
started by culturing the clone in Luria-Bertani
media at 37 °C with a shaking incubator until
reaching an optical density of 0.6-0.8 at 595 nm
wavelength. The culture was inducted using
isopropyl β-d-1-thiogalactopyranoside (IPTG),
and recombinant proteins were extracted
using a sonicator by adding lysozyme. Then,
they were purified using NI-NTA resin with
elution buffers containing imidazole 60 and
100 mM. The purified protein was then
quantitatively analyzed using Bradford protein
assay and qualitatively by SDS-PAGE.
The treatment was done by subcutaneous
injection on days 0, 21, and 42. The DBL2β-
PfEMP1 recombinant protein was mixed with
the complete Freund’s adjuvant (CFA) in the
primary injection (day 0) and the incomplete
Freund's adjuvant (IFA) in the secondary
injections (day 21 and 42) in a 1:1 ratio. On day Figure 1. Histology appearance of the spleen
56, the rats were euthanized with ketamine- with white pulp in all groups stained with
xylazine, and spleens were taken for hematoxylin-eosin (HE) and observed by
histopathology preparation. The slides were microscope at 100x magnification. (A) control
prepared using a microtome and stained using group, (B) treatment group 100 μg (C)
hematoxyline-eosin (HE). treatment group 150 μg, and (D) treatment
The white pulp diameter was observed group 200 μg.
using the Olympus CX23LED microscope with a In Figure 2, the average number of spleen-
100× magnification. Five visual fields were white pulp diameters was displayed for your
selected using the zig-zag method and consumption. The fact that the data were
documented using the Optilab camera. reliable was demonstrated by the fact that the
Diameter measurements were done using the Cronbach alpha test yielded a value of α=0.985
ImageJ application. The white pulp diameter (Sig.>0.7). The observation was carried out by
was calculated by adding the longest diameter two persons.
(transversal diameter) and the perpendicular

53 | DOI: https://doi.org/10.33533/jpm.v18i1.7451 Vol 18 No 1 (2024)

 I . A. P. D. J. Sari , E. Setiawati , L. Nurhasanah

Figure 2. The average number of spleen-white pulp diameter in all groups. The white pulp
diameter is increased with the increasing DBL2β-PfEMP1 recombinant protein dose.

The Saphiro-Wilk and Levene tests for each groups. Further analysis using a posthoc
group resulted in p>0.05, meaning all data Bonferroni test resulted in a significant
were normally distributed and homogenous. difference between a control group and
The one-way ANOVA test showed p=0.001, treatment group 150 μg and 200 μg, and a
indicating a significant difference between group 100 μg and 200 μg (Table 1).

Table 1. The post hoc Bonferroni test results

Control Dose 100 μg Dose 150 μg Dose 200 μg

Control 0.306 0.013* 0.002*
Dose 100 μg 0.306 0.114 0.027*
Dose 150 μg 0.013* 0.114 0.527
Dose 200 μg 0.002* 0.027* 0.527
*= significantly different groups (p<0.05)

DISCUSSION immunogenicity of the protein by inducing

The study aimed to determine the effect of IgG and CD4+.16
DBL2β-PfEMP1 recombinant protein There was an increase in white pulp
injection on the spleen-white pulp diameter. diameter post-protein injection, along with the
An established approach for the manufacture increasing protein dose, and the widest
of the recombinant protein DBL2β-PfEMP1 diameter was observed in a dose of 200 μg. The
has been established through previous results were supported by a posthoc
studies. The use of recombinant protein has Bonferroni test, which showed significant
some advantages regarding the process and differences between this group and control
cost. Furthermore, studies have reported the and a group dose of 100 μg.

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 E. Sulistyaningsih , N.A. Sa’diyah , I.F. Kusuma , R. Dewi , S. Rachmania

The DBL2β-PfEMP1 recombinant protein is The antigen-presenting cell (APC) can

a protein with a molecular weight of 72 kDa8, recognize the recombinant protein DBL2β-
while a protein with a molecular weight of PfEMP1 acting as an antigen. APC will present
more than 8 kDa is potentially antigenic and antigen in major histocompatibility class I
immunogenic. In this study, the DBL2β- (MHC I) and major histocompatibility class II
PfEMP1 recombinant protein is (MHC II) and stimulate cytokines to call other
subcutaneously injected, which further immune cells. Th1 produces cytokines like
reaches the spleen through the circulatory interferon-gamma (IFN-γ) and tumor necrosis
system and induces the immune system. The factor-alpha (TNF-α) that activate
increase in spleen-white pulp diameter macrophages, monocytes, and NK cells in the
indicated an induction of immune response. An cellular immune response. Th2 produces
acute immune response to antigens may cytokines such as IL-4, IL-5, IL-6, and IL-10 that
increase cellularity in the B-cell areas and activate the humoral immunity response. IL-4
increase secondary follicles with prominent and IL-5 stimulate B cell proliferation and
germinal centers. Immature B cells, or differentiation into plasma cells to produce
immunoblasts, will proliferate in response to specific antibodies to the PfEMP1 antigen.
antigenic stimuli. They will mature into When an immune response occurs, APC
immunoglobulin-producing plasma cells and releases various pro-inflammatory and anti-
migrate into the red pulp.11,12 The proliferation inflammatory cytokines that can trigger effects
of B cells will be observed as the increased on various organs.14,15 Antibodies to the
white pulp diameter. Furthermore, the DBL2β- DBL2β-PfEMP1 recombinant protein have a
PfEMP1 recombinant protein could activate crucial role in preventing cytoadherence,
central memory T cell and T-dependent B cell which is a factor causing severe malaria.
germinal center resulting in antibody Previous research has shown that the
production in response to serial recombinant recombinant proteins DBLβ2-PfEMP1 have
protein injection. immunogenic properties that can stimulate the
Spleen is a remarkable lymphoid organ that production of IgG-specific antibodies and
integrates innate and adaptive immunity by CD4+ T lymphocytes in Wistar rats.16
facilitating the interaction between antigen- The finding of the study reinforces the
presenting cells (APCs) and lymphocytes and is immunogenicity of the DBL2β-PfEMP1
responsible for removing blood-borne recombinant protein as the key requirement of
pathogens and eliminating old and abnormal vaccine candidates. Previous studies showed
erythrocytes from circulation.11,12 The white the outcome of an increase of IgG and CD4+
pulp, which contains nodular and diffused B cells after recombinant protein injection, and
and T lymphocytes, macrophages, and this study indicated the possible mechanism of
dendritic cells, allows the formation of specific recombinant protein to induce immune
immune responses to protect the body from response by increasing the spleen-white pulp
disease, making it a vital role in the normal diameter. However, the study has a limitation
immune response to infection. Changes in the in its examination scope, as it only covers a
cellular composition in this area after exposure morphological parameter, particularly splenic-
to immunomodulatory agents such as the white pulp, and does not incorporate
recombinant protein DBL2β-PfEMP1, which histochemical markers. Therefore, future
causes activation of T lymphocytes and, in studies on the effect of DBL2β-PfEMP1
turn, will activate B lymphocytes in the follicle, recombinant protein on the spleen could
converting them to plasma cells, which then include more comprehensive morphological
produce IgM and further IgG antibodies. The parameters and biochemical markers.
activation also implied T and B cell Vaccine development is a long way to go,
proliferation. This prolifération can increase especially for malaria vaccines with the
the diameter of the white pulp.13 complexity of Plasmodium spp life cycle as the

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 E. Sulistyaningsih , N.A. Sa’diyah , I.F. Kusuma , R. Dewi , S. Rachmania

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