Repeated exposure to 31 kDa protein fraction from the salivary gland of Aedes aegypti modulate humoral and cellular immune response in mouse model

Exposição repetida à fração proteica de 31 kDa da glândula salivar de Aedes aegypti modula a resposta imune humoral e celular em modelo murino

Abstract

Aedes (Ae) aegypti is the primary mosquito vector responsible for transmitting the pathogens that cause Dengue fever and Dengue hemorrhagic fever. We have previously identified 31 kDa and 56 kDa immunogenic proteins from the salivary glands of Ae. aegypti. The 31 kDa fraction primarily contains D7 protein, which has immunomodulatory properties that influence the host's immune response. This study aims to analyze the humoral (IgG) and specific immune responses (represented by IFN-γ and IL-4 cytokines) in a mouse model (Mus musculus) after exposure to the 31 kDa immunogenic protein from the salivary glands of Ae. aegypti. The mice were divided into three treatment groups: group A (elution buffer control), group B (adjuvant control), and group C (31 kDa 0.2 µg/µL + adjuvant). Injections were administered bi-weekly over six weeks. IgG levels and cytokine (IFN-γ and IL-4) concentrations were analyzed using the Enzyme-Linked Immunosorbent Assay (ELISA) method. The results showed that repeated exposure to the 31 kDa immunogenic protein elevated the humoral immune response (IgG) and modulated the host's immune response from the Th1 subset to the Th2 subset, characterized by a decrease in IFN-γ and an increase in IL-4 cytokine concentrations. This modulation is important in developing the host immune response against the transmitted dengue pathogen. This finding confirms that the 31 kDa protein is both immunogenic and immunomodulatory, inducing and modulating the immune response in mice. This study recognizes the 31 kDa protein from the salivary gland of Ae. aegypti as a potential target for the development of a vector-based dengue transmission-blocking vaccine.

Keywords:
Aedes aegypti; salivary gland; immunomodulator; dengue

Resumo

Aedes aegypti é o principal vetor de mosquito responsável pela transmissão dos patógenos que causam a dengue e a febre hemorrágica da dengue. Anteriormente, identificamos proteínas imunogênicas de 31 kDa e 56 kDa nas glândulas salivares de Ae. aegypti. A fração de 31 kDa contém principalmente a proteína D7, que possui propriedades imunomoduladoras que influenciam a resposta imune do hospedeiro. Este estudo visa analisar as respostas imunológicas humorais (IgG) e específicas (representadas pelas citocinas IFN-γ e IL-4) em um modelo murino (Mus musculus) após a exposição à proteína imunogênica de 31 kDa das glândulas salivares de Ae. aegypti. Os camundongos foram divididos em três grupos de tratamento: grupo A (controle com tampão de eluição), grupo B (controle com adjuvante) e grupo C (31 kDa 0,2 µg/µL + adjuvante). As injeções foram administradas quinzenalmente durante seis semanas. Os níveis de IgG e as concentrações das citocinas (IFN-γ e IL-4) foram analisados utilizando o método ELISA (Enzyme-Linked Immunosorbent Assay). Os resultados mostraram que a exposição repetida à proteína imunogênica de 31 kDa elevou a resposta imune humoral (IgG) e modulou a resposta imune do hospedeiro do subconjuntoTh1 para o subconjunto Th2, caracterizada por uma diminuição na IFN-γ e um aumento nas concentrações da citocina IL-4. Essa modulação é importante no desenvolvimento da resposta imune do hospedeiro contra o patógeno transmitido pelo dengue. Esta descoberta confirma que a proteína de 31 kDa é tanto imunogênica quanto imunomoduladora, induzindo e modulando a resposta imune em camundongos. Este estudo reconhece a proteína de 31 kDa da glândula salivar de Ae. aegypti como um alvo potencial para o desenvolvimento de uma vacina bloqueadora da transmissão do dengue baseada no vetor.

Palavras-chave:
Aedes aegypti; glândula salivar; imunomodulador; dengue

1. Introduction

Dengue Hemorrhagic Fever (DHF) is an infectious disease prevalent in tropical and subtropical regions caused by Dengue Virus (DENV). This virus has four different serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 (Roy and Bhattacharjee, 2021). The infection of dengue is primarily transmitted by Aedes mosquitoes, particularly Aedes aegypti and Aedes albopictus. While mosquito vector control measures such as insecticide spraying, larval source reduction, and environmental management have been widely implemented, they have not been sufficient to reduce dengue transmission effectively (Prompetchara et al., 2019). DHF cases remain relatively high in Indonesia. In 2023, there were 57,884 cases, including 422 deaths by the 33rd week (Kemenkes, 2023). This highlights the urgency for developing a dengue vaccine. Safe and effective dengue vaccines can significantly reduce global dengue infections (Deng et al., 2020). Pathogen-based vaccines have been developed to reduce dengue infections. However, concerns about their safety, particularly antibody-dependent enhancement (ADE), have posed challenges to their widespread use in dengue control (Katzelnick et al., 2017). Given these limitations, alternative approaches that do not rely on direct pathogen exposure are being explored. One promising strategy is targeting the mosquito vector itself, as disrupting pathogen transmission at its source could provide a safer and more sustainable method for disease control. Therefore, innovations in mosquito vector-based vaccines, particularly those targeting salivary proteins, are necessary to control dengue fever (Wang et al., 2023).

The dengue virus is transmitted when female Aedes mosquitoes blood-feed on a host. Blood-feeding aims to obtain nutrients useful for egg maturation (Talyuli et al., 2015). During blood-feeding, the virus from the infected host enters the mosquito's body, replicates, and infects midgut epithelial cells before migrating to the salivary glands. Viruses in the salivary glands are then transmitted back to humans or other hosts during subsequent blood-feedings (Lessa et al., 2023). Successful transmission of the dengue virus is facilitated by protein components in the salivary glands that aid the blood-feeding process, acting as anticoagulants, vasodilators, platelet anti-aggregants and immunomodulators (Marín-López et al., 2023).

Our previous research has identified immunogenic proteins in the salivary glands of Ae. aegypti, specifically 31 kDa and 56 kDa fractions (Oktarianti et al., 2014). Further analysis using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) revealed that the D7 protein is the main component of 31 kDa fraction, while the 56 kDa fraction is apyrase (Oktarianti et al., 2015). D7 protein possesses immunomodulatory properties and thus can modulate the host's immune system (Guo et al., 2025) This modulation can increase pathogen transmission by modulating the host's immune response, inducing an adaptive immune response, and resulting in antibody production (Fontaine et al., 2011). Thus, this protein can alter both innate and adaptive immune responses (Marín-López et al., 2023). Altering the adaptive immune response is indicated by a shift from the Th1 subset to the Th2 subset, characterized by a decrease in IL-2 and IFN-γ and an increase in Th2 cytokines such as IL-4 and IL-10. An increase in Th2 will activate B cells to produce antibodies, including IgG, IgM, and IgE (Fontaine et al., 2011; Guerrero et al., 2020; Marín-López et al., 2023).

The objective of this research is to analyze the immunomodulatory function of a 31 kDa immunogenic protein fraction from the salivary gland of Ae. aegypti. The investigation involved analyzing the immune response in the host by measuring IgG and cytokine levels (IFN-γ and IL-4) in a mouse model. This study emphasizes the important need to explore the potential use of D7 protein in mosquito salivary glands as a target for developing dengue vaccines based on salivary gland proteins of the mosquito vector Ae. aegypti.

2. Material and Methods

2.1. Rearing of Ae. aegypti

Mosquito larvae were collected from water reservoirs in households 15 times, with approximately 50–100 larvae per collection event. The larvae were reared in the laboratory, and experiments were conducted using the F1, F2, or F3 generations. Rearing of Ae. aegypti was carried out on laboratory scale under controlled conditions at 27°C ± 2°C and relative humidity at 75% ± 5% (Imam et al., 2014). The collected larvae were reared until they matured into adult mosquitoes. Initially, the larvae were kept in a container filled with clean water and allowed to develop into pupae. Once they reached the pupal stage, they were transferred to a 1x1x1 m³ cage, where they were maintained until they emerged as adult mosquitoes. Female mosquitoes required a blood meal from Wistar rats, while male mosquitoes were fed on a 10% sucrose solution.

2.2. Species identification and salivary glands isolation of Ae. aegypti

The mosquitoes were identified based on morphological properties, including the thorax, mesepimeron, and anterior. Species identification of Ae. aegypti followed the book Zootaxa: Pictorial Keys For The Identification of Mosquitoes (Diptera: Culicidae) Associated With Dengue Virus Transmission. This research used female Ae. aegypti (Rueda, 2004). Ae. aegypti salivary glands were obtained through a microdissection technique with a 0.5% NaCl solution. The extracted salivary glands were transferred into a microtube containing 1 mM Phenyl Methyl Sulfonyl Fluoride (PMSF) solution in a 1:1 (v/v) Phosphate-Buffered Saline (PBS). The salivary gland samples were stored in -20°C until for use.

2.3. Isolation and purification of 31 kDa protein fraction from Ae. aegypti Salivary Glands

Protein samples, mixed with loading buffer, were separated by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) using a 12% separating gel and 4% stacking gel. The electrophoresis was carried out at room temperature at 150 V for 60 minutes in an electrode buffer (pH 8.3). The gel was then stained with Coomassie Blue for 1 hour, followed by three washes in a destaining solution, each for 15 minutes. Following the destaining process, the target protein at 31 kDa was identified, and the corresponding band from the sample was excised aseptically in Laminar Air Flow (LAF). This study used 150 bands of 31 kDa protein. Electroelution was performed to purify the 31 kDa protein from Ae. aegypti salivary glands.

2.4. In-vivo assay

The protocol in this in-vivo assay was approved by Ethic Committee of Medical Research Faculty-Universitas Jember number: 2068/UN25.8/KEPK/DL/2023. In-vivo assay was performed using 24 male BALB/c mice (Mus musculus) 2-3 months old with 30 gr of weight. The experimental mice had free access to feed and water, and animal experiments were performed according to guidelines (Jones-Bolin, 2012). The 31 kDa protein was administered by subcutaneous injection every two weeks over a period of six weeks (3 times). The 31 kDa protein fraction used in this study was excised from SDS-PAGE after separating total salivary gland protein extracts from Aedes aegypti. The mice were divided into three treatment groups, each consisting of eight mice: Group A (elution buffer control, receiving only the electrode buffer), Group B (adjuvant control, receiving only the adjuvant), and Group C (31 kDa Ae. aegypti salivary gland protein + adjuvant). In Groups B and C, Complete Freund’s Adjuvant (CFA) was used for the initial injection, while Incomplete Freund’s Adjuvant (IFA) was used for subsequent injections.

2.5. Analysis of IgG and Cytokine (IFN-γ and IL-4) by ELISA

IgG levels were measured using an indirect ELISA method. A 96-well ELISA plate (Corning, USA) was coated with 50 µL per well of a 31 kDa protein fraction (4 µg/mL) in coating buffer and incubated overnight at 4°C to allow antigen binding. The wells were then washed with PBST (Phosphate Buffered Saline with 0.05% Tween-20) and blocked with 200 µL of blocking buffer (1% BSA in PBST) for 2 hours at room temperature to prevent non-specific binding. After washing with 250 µL of PBST, 50 µL of diluted primary antibody (mice blood serum, 1:100 in blocking buffer) was added to each well and incubated at 37°C for 1 hour. The wells were washed again before adding 50 µL of secondary antibody (anti-mouse IgG-HRP, 1:5000 in blocking buffer), followed by incubation for another hour. After final washes, 50 µL of TMB substrate was added to each well and incubated in a dark room at room temperature for 30 minutes until a color change was observed. The reaction was then stopped by adding 50 µL of 1 M sulfuric acid, and absorbance was measured at 450 nm using an ELISA reader.

The Cytokine analysis was performed using sandwich ELISA to detect shifts in the adaptive immune response. The research procedures complied with the manual of Bioassay Technology Laboratory (BT-Lab) Mouse IFN-γ, IL-4 ELISA Kit (Shanghai Korain Biotech Co., Ltd.). The measurement of cytokine concentrations began by generating standard curves. Microplate wells were washed using washing buffer four times, followed by the addition of 50 μl assay buffer A to each well. Afterward, 50 μl of standard and 40 μl of sample were added. To measure the concentration of IFN-γ and IL-4 cytokines, a total of 10 μl of IFN-γ and IL-4 antibody from mouse was added to the sample well. A 50 μl solution of streptavidin-HRP was added to well samples and standards. The treated plate was sealed and incubated for 60 minutes at 37°C followed by washing using 300 μl of buffer five times for 30-60 seconds. 50 μl substrate solution A as well as solution B, were added to each well. This well was then incubated in a dark room for 10 minutes at 37°C. Once the solution demonstrated a color change, the reaction was terminated by adding 50 μl stop solution to each well. The absorbance value of the sample was then measured using an ELISA Reader at a wavelength of 450 nm.

2.6. Data analysis

IgG and cytokine levels were statistically analyzed using One-Way ANOVA with p = 0.05 as the basis to confirm significant differences. Once significance was assured, a DMRT (Duncan Multiple Range Test) with α= 0.05 was performed.

3. Results

3.1. Characteristics of Ae. aegypti and its salivary gland

Ae. aegypti can be recognized by morphological properties such as the mesonotum, mesepimeron, and anterior mid-femur. The results of morphological identification are presented in Figure 1. The mesonotum of Ae. aegypti exhibits two straight lines (median-longitudinal) and two white curved lines on the right and left sides (submedian-longitudinal). Additionally, the mesepimeron of Ae. aegypti has separate patches of white scales. The legs have a longitudinal white line in the mid-femur. This result aligns with Rueda (2004) and Supriyono et al. (2023), explaining that Ae. aegypti is characterized by its unique mesonotum, mesepimeron, and anterior mid-femur.

Figure 1
Morphological properties of Ae. aegypti (50x magnification). (A) mesonotum; (B) mesepimeron; (C) anterior mid-femur (amf); median-longitudinal (ml); submedian-longitudinal (sl); white scale patches (wsp).

Only female Ae. aegypti was used for this study. Male and female Ae. aegypti have morphological differences in their body size and antennae. Female Ae. aegypti are larger in body size and have sparse, tiny hairs on their antennae. Male Ae. aegypti have smaller bodies and a pair of plumose antennae with much denser hairs compared to females.The proboscis of female Ae. aegypti is longer than the maxillary palpus. The female’s maxillary palpus is shorter compared to the male’s maxillary palpus. The length of the proboscis and maxillary palpus in male Ae. aegypti is relatively similar.The identification results of female and male Ae. aegypti are displayed in Figure 2. These identification results are consistent with Supriyono et al. (2023), who observed that antenna density is a distinguishing feature between male and female mosquitoes.

Figure 2
Morphological differences between male and female Ae. aegypti (40x magnification) and (A) Male; (B) Female; palpus (pl); probocis (pr); antenna (an).

We have succesfully isolated salivary glands from 1500 Ae. Aegypti mosquitos. The salivary glands consist of three lobes, one medial lobe and two lateral lobes, connected by a salivary duct. The medial lobe is located in the middle, while the lateral lobes are located on the sides. The lateral lobes are of equal size and composed of lateral proximal and lateral distal parts (Soohoo-Hui et al., 2021). The structure of female Ae. aegypti salivary glands is shown in Figure 3. The protein profile are shown in Figure 4 (A) which are consisted of protein fractions ranging from 255 to 27 kDa. The desired 31 kDa protein fraction were excised from the band in the SDS-PAGE gel, followed by the electroelution method to purify proteins and separating the desired 31 kDa protein from other components. The success of the electroelution was verified using SDS-PAGE, as shown in Figure 4 (B), which revealed a single band corresponding to 31 kDa.

Figure 3
The structure of the salivary glands Ae. aegypti (50x magnification) salivary duct (sd), proximal lateral (pl), lobus medial (m), distal lateral (dl), dan lobus lateral (lt).

Figure 4
The profile of protein from Ae. aegypti salivary gland (A); Purification results (B) Salivary gland extract of Ae. aegypti (SG); BlueElf Prestained Protein Marker (M); (S) Protein fraction 31 kDa.

3.2. Humoral (IgG) and cellular immune responses (IFN-γ and IL-4) in Mice (Mus musculus)

The results of quantifying IgG concentrations in individual and populations sample at weeks 0, 2, 4, and 6 are presented in Figure 5 and Tables 1 and 2. Figure 5 showed that IgG levels in all groups (A, B, and C) were relatively similar at weeks 0 and 2. However, at weeks 4 and 6, differences in IgG levels were observed between the control groups (A - elution and B - adjuvant) and the treatment group (C - 31 kDa protein + adjuvant). The IgG levels in the treatment group generally had a higher average compared to groups A (buffer) and B (adjuvant). The statistical test results of individual samples revealed no significant difference at week 0 (p > 0.05) (Table 1). However, the test results for individual sample IgG levels at weeks 2, 4, and 6 showed a significant difference. The one-way ANOVA test at week 2 yielded p = 0.020, while the tests at weeks 4 and 6 indicated a significant difference with p = 0.000 < 0.05. The results of the DMRT test revealed a significant difference in IgG concentration between control groups A (buffer) and B (adjuvant) and the treatment group C (protein 31 kDa + adjuvant). The test results for the population sample (Table 2) also indicated a significant difference, verified by p = 0.025 < 0.05. Subsequent DMRT tests confirmed that the control groups (A and B) showed no significant difference, while the treatment group exhibited significant differences. Both individual and population sample (8 mice for each treatment) tests consistently confirmed the highest IgG concentration in the treatment group.

Figure 5
Humoral immune response (IgG) of individuals and mouse populations at week 0, 2, 4, and 6; control group A (buffer); control group B (adjuvant); treatment group C (31 kDa protein + adjuvant).

Table 1
Statistical Analysis of IgG Concentration (triplicate) for Each Group (8 Mice per Group) before and after exposure to 31 kDa protein from Aedes aegypti salivary gland.

Treatment	IgG Concentration ($\overline{X}\pm$ SD)
	Week-0	Week-2	Week-4	Week-6
Group A	0.260$\pm$0.005a	0.263$\pm$0.016a	0.207$\pm$0.022a	0.182$\pm$0.021a
Group B	0.257$\pm$0.014a	0.255$\pm$0.019ab	0.266$\pm$0.022b	0.257$\pm$0.024b
Group C	0.255$\pm$0.009a	0.281$\pm$0.017b	0.306$\pm$0.015c	0.329$\pm$0.011c

• Analysis using Duncan's Multiple Range Test (DMRT): Numbers in the same column followed by different letters (a, b, c) represent significant differences between the groups (week 4 and week 6). Groups with the same letter show no significant difference. Control group A (buffer); control group B (adjuvant); treatment group C (31 kDa protein + adjuvant).

Table 2
Statistical Analysis of IgG Concentration (triplicate) for population in mice (Mus musculus) after exposure to 31 kDa protein of Ae. aegypti salivary gland.

Treatment	IgG Concentration ( $\overline{X}\pm$ SD)
Group A	0.390$\pm$0.062a
Group B	0.391$\pm$0.014a
Group C	0.410$\pm$0.015b

• Analysis using Duncan's Multiple Range Test (DMRT): Numbers in the same column followed by different letters (a, b, c) represent significant differences between the groups. Control group A (buffer); control group B (adjuvant); treatment group C (31 kDa protein + adjuvant).

Further quantitative analysis for IFN-γ and IL-4 was shown in Tables 3 and 4 as well as Figure 6. The statistical analysis of IFN-γ and IL-4 concentrations at week 0 exhibited no significant difference (p > 0.05). However, a significant difference was observed at weeks 2, 4, and 6, as verified by p < 0.05. This finding is supported by the data presented in Figure 6. The IFN-γ concentrations from week 0 to week 6 demonstrated a declining trend, although the control group showed an insignificant decrease. In contrast, the IFN-γ cytokine concentration in the treatment group (31 kDa Ae. aegypti + adjuvant) decreased from week 0 (339.06 ng/L) to week 6 (307.94 ng/L). The concentration of IL-4 cytokines increased from week 0 to week 6. The concentration of IL-4 cytokines in the treatment group (31 kDa Ae. aegypti + adjuvant) increased from week 0 (226.83 ng/L) to week 6 (270.69 ng/L), reaching the highest concentration observed in the final week.

Table 3
Statistical Analysis of IFN-γ cytokines (triplicate) for population in mice (Mus musculus) before and after exposure to 31 kDa protein of Ae. aegypti salivary gland.

Treatment	IFN-γ Concentration (ng/L)
	Week-0	Week-2	Week-4	Week-6
Group A	421.53a	413.65a	407.89a	400.57a
Group B	421.92a	419.03a	410.96a	399.61a
Group C	470.57a	462.88b	446.92b	422.49b

• Analysis using Duncan's Multiple Range Test (DMRT): Numbers in the same column followed by different letters (a, b, c) represent significant differences between the groups. Control group A (buffer); control group B (adjuvant); treatment group C (31 kDa protein + adjuvant).

Table 4
Statistical Analysis of IL-4 cytokines (triplicate) for population in mice (Mus musculus) before and after exposure to 31 kDa protein of Ae. aegypti salivary gland.

Treatment	IL-4 Concentration (ng/L)
	Week-0	Week-2	Week-4	Week-6
Group A	192,66a	202,06a	201,75a	195,84a
Group B	181,44a	203,11a	213,27b	216,29b
Group C	198,72a	220,24b	242,51c	245,23c

• Analysis using Duncan's Multiple Range Test (DMRT): Numbers in the same column followed by different letters (a, b, c) represent significant differences between the groups. Control group A (buffer); control group B (adjuvant); treatment group C (31 kDa protein + adjuvant).

Figure 6
Cellular immune response represented by IFN-γ and IL-4 Concentrations in Mice Model (Mus musculus).

4. Discussion

The quantification results and statistical tests of IgG concentration in individual and population samples exhibited increasing trends, particularly in the treatment group. This suggested that the 31 kDa protein could induce the host's immune response. Prolonged exposure led to the production of antibodies, including IgG, during the secondary immune response, characterized by a faster response with high antibody titers (Giesker and Hensel, 2014).

The 31 kDa protein had a main component in the form of D7 (Oktarianti et al., 2015). D7 is a protein that plays a role in the hemostatic process of blood feeding, acting as an anti-hemostatic, anti-inflammatory, and immunomodulator (Marín-López et al., 2023; Visser et al., 2023). D7 could inhibit inflammation in host blood vessels by binding to host leukotrienes. It also had a high affinity for ADP, enabling it to inhibit hemostasis and platelet aggregation during the blood-feeding process (Marín-López et al., 2023). The significance of this protein led to an increase in the amount of D7 protein during blood feeding (Visser et al., 2023).

When the host was first exposed to mosquito salivary gland proteins, the innate immune response was activated as the first line of defense (Marín-López et al., 2023). These proteins also activated leukocytes, mast cells, and neutrophils, which then secreted histamine, inflammatory proteins (MIP-1a), and leukotrienes that triggered the inflammatory process. The salivary gland proteins increased the production of antibodies and memory cells, enhancing the immune response upon repeated exposure. Repeated exposure resulted in the formation of adaptive immunity through Antigen Presenting Cells (APCs), which produced IL-12. IL-12 aided the differentiation of Th0 cells into Th1 and Th2 cells (Fontaine et al., 2011).

Based on the data from Tables 3 and 4, as well as Figure 6, repeated exposure to the 31 kDa protein in mice reduced T helper 1 (Th1), i.e., IFN-γ concentration, while increasing T helper 2 (Th2), i.e., IL-4 concentration. Increased IL-4 production activated B cells, which then formed antibodies in the form of IgG. This was consistent with the results of IgG measurements, which showed an increase along with the length of exposure. This finding corresponded to the research of Guerrero et al. (2020), which stated that the salivary gland protein of the Aedes mosquito shifted the humoral immune response from the T helper 1 (Th1) subset to T helper 2 (Th2). These results demonstrated that the 31 kDa protein also acted as an immunomodulator, modulating the immune response in the host. This aligned with a study reporting that the 31 kDa protein mainly consisted of D7, which is an immunomodulatory protein.

The detailed mechanism was that repeated exposure stimulated the formation of adaptive immunity through Antigen Presenting Cells (APCs), which produced IL-12 to convert Th0 into Th1 and Th2. This change was characterized by a decrease in IFN-γ and IL-2, accompanied by an increase in IL-4 and IL-10. Increased cytokines, in the form of IL-4 and IL-10, promoted Th2, leading to higher differentiation and polarization of B cells into plasma B cells that produced more antibodies. Antibody formation began when BCRs bound to specific antigens and B cells interacted with T0 cells through MHC II. This interaction prompted B cells to differentiate into plasma B cells and memory B cells (Fontaine et al., 2011; Guerrero et al., 2020; Manning et al., 2018). Plasma B cells produced various types of antibodies, while memory B cells formed memory responses to antigens (Fontaine et al., 2011). Memory B cells could enhance the immune response after repeated exposure to the same antigen (Rastogi et al., 2022).

These results prove that the 31 kDa protein of Ae. aegypti salivary gland is able to cause polarization of T helper 1 (Th1) response to T helper 2 (Th2) response so as to increase the proliferation of B cells into plasma cells and memory cells that cause the production of immunoglobulin G (IgG) in experimental animals. The immune response assessed in this study reflects specific activation, as evidenced by the presence of IgG antibodies targeting the 31 kDa protein and cytokine shifts (IFN-γ and IL-4), which indicate adaptive immune modulation. Thus, protein 31 has the potential to be developed into Transmission Blocking Vaccines (TBV). TBV works by interrupting or blocking the development of the pathogen in the vector so as to prevent its transmission to the body of an uninfected host (Olajiga et al., 2021). The TBV development can be done by utilizing molecules derived from the body of the vector that are immunized in the host body. The molecule is antigenic so that it can induce the host immune system to produce specific antibodies (Coutinho-Abreu and Ramalho-Ortigao, 2010).

5. Conclusion

Repeated exposure to the 31 kDa protein from Aedes aegypti salivary glands modulated the humoral and cellular immune responses in a mouse model. This was indicated by the increasing levels of IgG and the shifting of the cellular immune response from the Th1 subset to the Th2 subset. This shift was marked by a decrease in IFN-γ cytokine and an increase in IL-4 cytokine concentration. These findings demonstrate that the 31 kDa protein from the salivary glands of Aedes aegypti is an immunomodulatory protein. This modulation is an important factor for the immune response against Dengue. This research highlights the potential use of the 31 kDa protein as a novel target for the development of a transmission-blocking vaccine against Dengue.

Acknowledgements

This research is supported by the National Research and Innovation Agency (BRIIN) through the Research and Innovation Program for Advanced Indonesia, Batch 3, under reference number 12/II.7/HK/2023.

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Publication Dates

• Publication in this collection
  09 May 2025
• Date of issue
  2025

History

• Received
  12 Oct 2024
• Accepted
  14 Mar 2025