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. 2022 Jan 25;90(1):e00359-21. doi: 10.1128/IAI.00359-21

A Mosquito AgTRIO Monoclonal Antibody Reduces Early Plasmodium Infection of Mice

Yu-Min Chuang a,✉, Xu-Dong Tang b, Erol Fikrig a
Editor: De’Broski R Herbertc
PMCID: PMC8788779  PMID: 34724388

ABSTRACT

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. Passive immunization of mice with antiserum against the Anopheles gambiae mosquito saliva protein TRIO (AgTRIO) offers significant protection against Plasmodium infection of mice. Furthermore, passive transfer of both AgTRIO antiserum and an anti-circumsporozoite protein monoclonal antibody provides synergistic protection. In this study, we generated monoclonal antibodies against AgTRIO to delineate the regions of AgTRIO associated with protective immunity. Monoclonal antibody 13F-1 markedly reduced Plasmodium infection in mice and recognized a region (VDDLMAKFN) in the carboxyl terminus of AgTRIO. 13F-1 is an IgG2a isotype monoclonal antibody, and the Fc region is required for protection. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

KEYWORDS: Plasmodium, malaria, mosquito saliva protein, AgTRIO, monoclonal antibodies

INTRODUCTION

Malaria is one of the deadliest global infectious diseases and can be caused by at least 5 different species of Plasmodium (1). Transmission begins when a Plasmodium-infected Anopheles mosquito takes a blood meal. While probing for blood, the mosquito injects saliva together with Plasmodium sporozoites into the skin of the vertebrate host. To date, a highly effective, safe, and FDA-approved human vaccine has not been developed. The most established malaria vaccine candidate, RTS,S/AS01, is based on the circumsporozoite protein of Plasmodium falciparum (PfCSP) and confers modest protection that wanes over time (2–5). For travelers to areas where malaria is endemic, current prophylaxis is based on various antimalarial drugs, and issues of drug resistance, patient compliance, and adverse side effects are substantial (6). To overcome some of the obstacles associated with active immunization, including the time required to achieve protection following multiple immunizations and natural variations of the human immune response, there is an ongoing clinical trial on the efficacy of a PfCSP monoclonal antibody for protection against malaria in humans (7). Testing the efficacy of antibodies directed against different targets would provide additional information to aid in the prevention of Plasmodium infection.

Passive immunization with antiserum against the Anopheles gambiae mosquito saliva protein TRIO (AgTRIO) offers partial protection against Plasmodium berghei or P. falciparum transmitted by Anopheles gambiae or Anopheles stephensi in murine models (8). Passive immunization of mice with AgTRIO antiserum and an anti-CSP antibody afforded synergistic protection (8). Consistent with these findings, silencing AgTRIO reduces the ability of mosquitoes to transmit Plasmodium to the vertebrate host, and AgTRIO can modulate the host immune response in the skin (9). In this study, we generated monoclonal antibodies against AgTRIO and examined their protective effect against malaria in mice as an important step toward developing new strategies to prevent Plasmodium infection.

RESULTS

Monoclonal antibodies against AgTRIO.

To produce monoclonal antibodies against AgTRIO, we generated hybridomas by immunizing C57BL/6 mice with purified AgTRIO and then fusing the splenocytes with a mouse myeloma cell line. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for binding to purified AgTRIO derived from baculovirus (data not shown). Five hybridoma clones (3B-1, 3B-2, 10H-9,13F-1, and 14E-9) were selected for further screening by immunoblot assays, and all showed reactivity with AgTRIO protein (Fig. S1 in the supplemental material). Clone 13F-1 is IgG2a, and all the others are IgG1. All these AgTRIO monoclonal antibodies also recognized purified E. coli-derived AgTRIO, which was used in our previous study on protective immunity (8) (Fig. 1A). In the immunoblot assays, all the monoclonal antibodies also recognized TRIO (VectorBase accession number AGAP001374; molecular weight, 43.79 kDa) in A. gambiae salivary gland extracts and TRIO (VectorBase accession number ASTE008872; molecular weight:, 43.76 kDa) in A. stephensi salivary gland extracts (Fig. 1B, Fig. S2).

FIG 1.

FIG 1

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Mouse monoclonal antibodies recognize AgTRIO. (A) All AgTRIO monoclonal antibodies recognized Escherichia coli-derived AgTRIO protein in ELISA. (B) Immunostaining with AgTRIO monoclonal antibody 13F-1 demonstrated that this monoclonal antibody recognized TRIO protein from salivary gland extracts from A. gambiae (Ag) or A. stephensi (As).

AgTRIO monoclonal antibody 13F-1 offers protection against Plasmodium infection.

In our previous study, passive immunization with AgTRIO antiserum offered partial protection against Plasmodium infection in mice (8). To determine whether immunization with individual monoclonal antibodies against AgTRIO can influence Plasmodium infection, we harvested monoclonal antibodies from cultures of individual hybridomas. Groups of C57BL/6 mice each received 100 μg of an individual AgTRIO monoclonal antibody. Irrelevant mouse monoclonal antibodies were used as negative controls (MOPC-21 [mouse IgG1] or C1.18.4 [mouse IgG2a]). Then, we challenged the mice by the intradermal injection of 300 P. berghei sporozoites. Mice receiving monoclonal antibody 3B-2 or 13F-1 had significantly reduced hepatic levels of P. berghei compared to the levels in mice passively immunized with the control antibody (Fig. 2A). 13F-1 offered more effective protection, and thus, we further determined whether the 13F-1 monoclonal antibody affects the transmission of Plasmodium by mosquito bites. After C57BL/6 mice were passively immunized with 100 μg of 13F-1 or control IgG2a (C1.18.4), we challenged the mice using three P. berghei-infected mosquitoes for each mouse. Mice passively immunized with monoclonal antibody13F-1 had reduced hepatic burdens of P. berghei compared to the hepatic burdens in mice passively immunized with the control IgG2a monoclonal antibody (Fig. 2B). To further examine the effects of this AgTRIO monoclonal antibody on the development of malaria, we passively immunized mice with either 100 μg of 13F-1 or control IgG2a (C1.18.4) and then exposed the animals to three P. berghei-infected mosquitoes. Blood-stage parasite infection was determined by blood smear, and the kinetics of 1% parasitemia detection was shown using Kaplan-Meier survival curves (P = 0.034 by Log rank test) (Fig. 3). Mice were followed for 2 weeks after exposure to mosquito bites. In all, 31% (4/13) of mice that received 13F-1 had 1% parasitemia, compared to 75% (9/12) of the control group. All the results show that passive immunization with AgTRIO monoclonal antibody 13F-1 offers significant protection against mosquito-borne P. berghei infection in mice.

FIG 2.

FIG 2

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Passive immunization with AgTRIO monoclonal antibody 13F-1 reduces the initial Plasmodium berghei infection level in mice. (A) Each C57BL/6 mouse received 100 μg of each AgTRIO monoclonal antibody or control isotype IgG1 or IgG2a monoclonal antibody. The next day, 300 P. berghei sporozoites, collected from infected-mosquito salivary glands, were intradermally injected into the left ear of individual C57BL/6 mice. Forty hours later, the livers were dissected and the Plasmodium burdens were determined by RT-qPCR. (B) C57BL/6 mice received 100 μg of AgTRIO monoclonal antibody 13F-1 or a control isotype IgG2a monoclonal antibody. The following day, each mouse was exposed to three P. berghei-infected mosquitoes, and the liver burden was determined 40 h later by RT-qPCR. Each dot represents the value for one mouse, and bars and whiskers show median ± IQR. Significance was determined at P < 0.05 using the Mann-Whitney U-test.

FIG 3.

FIG 3

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Passive immunization with AgTRIO monoclonal antibody 13F-1 offers protection in mice. Each C57BL/6 mouse received 100 μg of AgTRIO monoclonal antibody 13F-1 or control isotype IgG2a monoclonal antibody. The next day, each mouse was exposed to three P. berghei-infected mosquitoes. After infection, parasitemia was determined on a blood smear using Giemsa staining. The kinetics of 1% parasitemia detection was depicted using Kaplan-Meier survival curves after infection. P = 0.034 using the Log rank test.

AgTRIO monoclonal antibodies recognize the carboxyl terminus of AgTRIO.

To determine the regions of AgTRIO recognized by the monoclonal antibodies, we expressed five overlapping segments of the AgTRIO protein in 293T cells (Fig. 4A). Each segment had 50 to 100 amino acids and a His tag (Fig. 4B). In the immunoblot assay, all monoclonal antibodies and polyclonal antisera that we generated in our previous study (8) recognized the carboxyl terminus of AgTRIO (Fig. 4C, Fig. S3). To further characterize the binding specificity, overlapping peptides (20-22 amino acids in length with 9-amino-acid overlaps) spanning the carboxyl terminus of AgTRIO were used to localize the precise epitopes recognized by each monoclonal antibody (Fig. 5A). Using dot blot assays, monoclonal antibodies 13F-1 and 10H-9 recognized peptides 2 and 3, indicating that the epitope is contained within the sequence VDDLMAKFN (Fig. 5B, Fig. S4). Monoclonal antibodies 3B-1, 3B-2, and 14E-9 only recognized peptide 3 (Fig. S4), demonstrating that they recognize an epitope within VDDLMAKFNTPIDGKTLQYF.

FIG 4.

FIG 4

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AgTRIO monoclonal antibodies recognize the carboxyl terminus of AgTRIO. (A) Illustration of gene positions encoding individual segments of AgTRIO. Each segment was expressed, with a His tag, by 293T cells. (B) The expression of each segment was confirmed by using immunoblots probed with an anti-His tag antibody. (C) AgTRIO monoclonal antibody 13F-1 recognized segment E in an immunoblot assay.

FIG 5.

FIG 5

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AgTRIO monoclonal antibody 13F-1 recognizes a region in the carboxyl terminus of AgTRIO. (A) Illustration of individual, overlapping peptides corresponding to the carboxyl terminus of AgTRIO (segment E). Each peptide was synthesized and probed with each monoclonal antibody. (B) AgTRIO monoclonal antibody 13F-1 recognized peptides E2 and E3 in a dot blot assay.

The protection effect depends on the Fc region.

As monoclonal antibodies 13F-1 and 10H-9 recognized similar regions of AgTRIO and the influence of these two monoclonal antibodies on the hepatic burden of Plasmodium was different (Fig. 2A), we assessed the protective effects further. 13F-1 is an IgG2a monoclonal antibody and 10H-9 is IgG1, which suggests that the IgG subclass may contribute to protection in the murine model. We therefore examined whether the protective effect was at least partially on the Fc region. The variable region portion of antibody, F(ab′)2, directly binds antigen, and the constant region of antibody, Fc, can generate downstream responses through interaction with the Fc receptor (10–12). To diminish the effector effects of IgG, we generated F(ab′)2 from 13F-1 and the isotype control IgG2a (clone C1.18.4) by pepsin digestion. To test whether F(ab′)2 still offered protection, we administered F(ab′)2 fragments of 13F-1 or F(ab′)2 fragments of control IgG2a to C57BL/6 mice. One day later, each mouse was challenged by three P. berghei-infected mosquitoes. In contrast to the monoclonal antibody 13F-1, F(ab′)2 of 13F-1 did not diminish the Plasmodium liver burden in mice (Fig. 6), suggesting that protection depends, at least in part, on the biologic responses associated with the complete antibody.

FIG 6.

FIG 6

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F(ab′)2 of AgTRIO MAb does not offer protection against parasitemia in mice. C57BL/6 mice received 90 μg of F(ab′)2 of AgTRIO monoclonal antibody 13F-1 or F(ab′)2 of control isotype IgG2a 1 day before infection. The next day, each mouse was challenged by three P. berghei-infected mosquitoes. The liver burden was determined 40 h later by RT-qPCR. Each dot represents the value for one mouse, and bars and whiskers show median ± IQR. ns, no significant difference was found using the Mann-Whitney U-test.

DISCUSSION

Mosquito saliva proteins that are injected with Plasmodium into the bite site can influence infection and constitute targets for disease prevention (8, 13). In our previous study, we showed that passive immunization with antiserum against the salivary protein AgTRIO offers significant protection against P. berghei and P. falciparum transmitted by A. gambiae or A. stephensi mosquito bites (8). AgTRIO is secreted at high levels in mosquito saliva and enhances sporozoite infection in the skin (8, 9), which makes AgTRIO a suitable candidate for a transmission-blocking vaccine. In this study, we generated an AgTRIO monoclonal antibody, 13F-1, that significantly reduces Plasmodium infection in mice. Our finding affirms the concept that humoral immunity against a mosquito saliva protein can alter the transmission of Plasmodium from the arthropod vector to a vertebrate host. To develop a clinically useful monoclonal antibody, 13F-1 would need to be humanized to minimize the risk of cross-species sensitization and immunogenicity (14, 15). In addition, protection against infection with Plasmodium species that cause human infection in either humanized mice or nonhuman primates would be required. As the half-life of the monoclonal antibody will limit the period of protection, the monoclonal antibody would likely be an alternative prophylaxis strategy for travelers, short-term visitors, or pregnant women (7).

In our previous study, rabbit polyclonal antiserum against AgTRIO recognized TRIO from saliva gland extracts of A. stephensi and offered protection against Plasmodium infection delivered by A. stephensi (8). Since the site of A. stephensi TRIO that is recognized by antiserum is alternated or partially absent, further studies will be needed to determine if an AgTRIO monoclonal antibody offers protection against Plasmodium infection transmitted by diverse Anopheles species. It is also possible that different doses or multiple injections of the monoclonal antibody can maximize protection. Furthermore, an AgTRIO monoclonal antibody may potentially offer synergistic protection with PfCSP monoclonal antibody, which has been used in a preclinical trial (7).

Antibody subclasses can influence protection against infection (16, 17). During fungal infection, murine IgG2a and IgG2b promote better phagocytosis than IgG1 (16). In contrast, IgG3 offers improved protection against carbapenem-resistant Klebsiella pneumonia infection in neutropenic mice (17). There is also an association between IgG3 antibodies against conserved regions of MSP3 and protection against malaria (18). In our study, AgTRIO monoclonal antibody 13F-1 reduced Plasmodium infection levels in mice, and the protection depended on the presence of the Fc region. This suggests that the downstream effects of antigen-binding antibodies contribute to the protection when sporozoites are injected into the skin. Cytokine production following antibody binding to antigen partly depends on Fcγ receptor (FcγR) activation by the different IgG subclasses (19, 20). In addition, distinct subclasses of IgG can differentially influence antibody-dependent phagocytosis or cytotoxicity and complement activation (12, 21). Further studies are required to clarify the mechanism that contributes to the protective effects of different IgG subclasses against AgTRIO on the transmission of Plasmodium, especially when sporozoites are deposited in the skin.

In summary, passive immunization of mice with 13F-1, a monoclonal antibody against a mosquito saliva AgTRIO protein, is able to reduce early Plasmodium infection levels in mice. Murine monoclonal antibodies like 13F-1 can also be humanized and optimized to potentially serve as prophylaxis to afford rapid protection to travelers to areas where malaria is endemic. Our study offers important information for the generation of the next new vaccines against malaria that will target the first step of Plasmodium transmission and may be used in conjunction with traditional, pathogen-based vaccines.

MATERIALS AND METHODS

Hybridoma synthesis.

Monoclonal antibodies against AgTRIO were generated by GenScript (Piscataway, NJ) using 10 μg purified AgTRIO expressed by baculovirus (9). Individual hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for binding to the native AgTRIO protein from saliva, to confirm specificity. Five hybridoma clones were selected after recognizing AgTRIO in Western blot analysis (Fig. S1) and ELISA (data not shown). Hybridoma clones were maintained in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. For monoclonal antibody purification, cells were grown in serum-free hybridoma medium (Thermo Fisher Scientific) for 5 days at 37°C and 5% CO2. The supernatant was diluted with protein A/G binding buffer (Thermo Fisher Scientific), and the monoclonal antibody was purified using protein A/G agarose (Thermo Fisher Scientific) and concentrated in PBS with a 10-kDa Amicon filter (22). The control mouse monoclonal antibodies MOPC-1 (IgG1) and C1.18.4 (IgG2a) were purchased from BioXCell (Lebanon, NH).

Plasmodium burdens.

TRIzol reagent (Thermo Fisher Scientific) was used to purify total RNA from murine livers. All extraction followed the manufacturer’s protocols. The iScript real-time quantitative PCR (RT-qPCR) kit (Bio-Rad, CA) was used to generate cDNA from RNA. Using iTaq SYBR green supermix (Bio-Rad, CA), real-time PCR was performed on a CFX96 real-time platform (Bio-Rad). The PCR involved an initial denaturation at 95°C for 2 min and 50 cycles of 15 s at 95°C, 15 s at 60°C, and 20 s at 72°C. Fluorescence readings were taken at 72°C after each cycle. At the end of each reaction, a melting curve (60 to 95°C) was checked to confirm the identity of the PCR product. The burden of Plasmodium in liver after sporozoite infection was determined by assessing the expression level of P. berghei 18S rRNA, normalized to the Mus musculus hepatocyte nuclear factor 4 alpha gene, hnf4α, which has been used as housekeeping gene for liver tissue (8, 13). These data are presented as the copy number of the target gene per 10,000 housekeeping genes. The primers used for the expression of sporozoite genes are listed in Table S1.

ELISAs.

Antigen-specific antibody responses were measured by ELISA as described previously (13, 23), with minor modifications in coating and serum incubation. The 96-well microplates were coated with purified AgTRIO (1 μg/ml) overnight. After blocking, different concentrations of AgTRIO monoclonal antibodies were diluted in PBS, added to the wells, and incubated at room temperature for 2 h. Horseradish peroxidase (HRP)–goat anti-mouse antibody (Thermo Fisher Scientific) was used for detection.

Western blotting.

Each salivary gland from uninfected female mosquitoes was separated by SDS-PAGE using 4 to 20% Mini-Protean TGX gels (Bio-Rad). Proteins were transferred onto a 0.45-μm polyvinylidene difluoride (PVDF) membrane and then probed with AgTRIO monoclonal antibody (1 μg/5 ml). HRP–goat anti-mouse antibody (Thermo Fisher Scientific) was used as the secondary antibody (1:5,000), and the images were developed with a LI-COR Odyssey imaging system.

Segments of AgTRIO for expression.

The full length of AgTRIO was divided into 5 segments (Fig. 4A, segments A to E), and each segment had 50 to 100 amino acids. Each segment was amplified by PCR with specific primers (Table S1) and cloned into the pEZT D-lux plasmid with a His tag (24). Each insertion was confirmed by sequencing. The Expi293F expression system (Thermo Fisher Scientific) was used to express each segment of AgTRIO.

Dot blot assays of peptides.

Each of the peptides corresponding to different regions of the carboxyl terminus of AgTRIO (Fig. 5A) was synthesized by GenScript (Piscataway, NJ). Two micrograms of each peptide or AgTRIO protein or bovine serum albumin (BSA) protein was blotted onto a nitrocellulose membrane. Peptides or proteins were allowed to dry on the membrane. After blocking with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBS-T) for 1 h, the membrane was incubated with primary antibody (0.5 μg/ml) in 5% nonfat milk in TBS-T overnight at 4°C. The membrane was washed three times with TBS-T and then incubated with goat anti-mouse antibody in 5% nonfat milk in TBS-T for 1 h at room temperature. Then, the membrane was washed three times with TBS-T and developed with enhanced chemiluminescence (ECL) solution (GE Healthcare).

Animals.

A. gambiae (4arr strain) mosquitoes were raised at 27°C, 80% humidity, under a 12-h/12-h light/dark cycle and maintained with 10% sucrose under standard laboratory conditions in the insectary at Yale University. Swiss Webster and C57BL/6 mice were purchased from Charles River Laboratories. All animal experiment protocols were approved by the Yale University Institutional Animal Care and Use Committee (protocol number 2017-07941).

P. berghei infection.

P. berghei mosquitoes (ANKA GFPcon 259cl2, MRA-865, or NK65 RedStar, MRA-905; ATCC) were maintained by serial passage in 6- to 8-week-old female Swiss Webster or C57BL/6 mice as previously described (8). Briefly, Swiss Webster or C57BL/6 mice were challenged with P. berghei-infected red blood cells by intraperitoneal injection. A. gambiae mosquitoes then took a blood meal from the infected mice, when the parasitemia was approximately 5%. Seventeen to 24 days after P. berghei infection, the mosquitoes were sorted using the fluorescent signal of the salivary glands. The salivary glands from infected mosquitoes were used to harvest the sporozoites.

For passive immunization studies, C57BL/6 mice received 100 μg of each AgTRIO monoclonal antibody or control isotype IgG, IgG1, or IgG2a 1 day before infection. Mosquitoes with similar salivary gland fluorescence intensities were used for challenge studies. Three infected mosquitoes were randomly put in a paper cup, and a mesh was used to cover the opening. After overnight starvation, each mouse was randomly put on a cup, and then the infected mosquitoes could freely feed on the mice (8).

In the studies, protection was defined as the lack of blood-stage parasite detection 2 weeks following challenge. Parasitemia was observed by light microscopy using air-dried blood smears that were methanol fixed and stained with 10% Giemsa stain. After exposure to infected-mosquito bites, mice were monitored for 2 weeks. The absence of detectable parasitemia after 14 days of infection indicated that the animals were free of disease (25, 26).

Intradermal injection of sporozoites.

The sporozoites were collected from salivary glands and then passed through 30-gauge syringes 10 to 15 times. The cell debris was removed by filtering through a 40-μm filter mesh. The number of sporozoites was counted using a hemocytometer. C57BL/6 mice were anesthetized with 100 mg/kg of body weight of ketamine and 10 mg/kg of xylazine. Three hundred sporozoites were injected into the left ear of each mouse using glass micropipettes with an 80-μm diameter beveled opening and a Nanoject II auto-nanoliter injector (Drummond). To determine the burden of Plasmodium infection, the mice were euthanized 40 h after injection and the livers were collected for RNA extraction.

Generation of F(ab′)2.

AgTRIO monoclonal antibody or isotype mouse IgG was incubated with pepsin agarose resin (Thermo Fisher Scientific) to remove Fc regions. After digestion, F(ab′)2 segments were collected and concentrated using a 30-kDa Amicon filter.

Statistical analysis.

Data from at least three biological replicates were used to calculate median values for graphing purposes. Statistical analyses employed the Mann-Whitney test to determine the differences, and the data were presented as median value with interquartile ranges (IQR). A P value of <0.05 was considered statistically significant. To assess the kinetics of 1% parasitemia detection, Kaplan-Meier survival curves were used to present the time to 1% parasitemia after infection, and the Log rank test was used to compare the differences. The analysis, graphing, and statistical analysis of all data were performed using Prism 9.0 software (GraphPad Software).

ACKNOWLEDGMENTS

We thank Kathleen DePonte and Ming-Jie Wu for their technical assistance.

These studies were supported by the National Institute of Allergy and Infectious Diseases (R01 grant number AI158615, R21 grant number AI142708 and R41 grant number AI145779 to E.F.).

Erol Fikrig has equity in and consults for L2 Diagnostics, LLC.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Supplemental material. Download IAI.00359-21-s0001.pdf, PDF file, 0.3 MB (320.6KB, pdf)

Contributor Information

Yu-Min Chuang, Email: yu-min.chuang@yale.edu.

De’Broski R. Herbert, University of Pennsylvania

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