A Multivalent Peptide Vaccine Against Malaria, Targeting Plasmodium Circumsporozoite Protein and Mosquito AgTRIO

Abstract

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the host skin. The immune response against a mosquito saliva protein, AgTRIO, reduces Plasmodium infection and can work in combination with antibody against the Plasmodium circumsporozoite protein (CSP). We have now developed a chimeric peptide, PfAg, containing regions from Plasmodium falciparum CSP (PfCSP) and AgTRIO. Mice administered PfAg generated robust humoral responses against both PfCSP and AgTRIO. After exposure to PfCSP-expressing Plasmodium berghei–infected mosquitoes, PfAg-immunized inbred C57BL/6 and outbred CD-1 mice had significantly improved survival compared with control animals. These data will aid in the development of a new malaria vaccine.

Malaria begins when infected mosquitos inject saliva with Plasmodium sporozoites. We developed a chimeric peptide vaccine, PfAg, containing epitopes from Plasmodium falciparum circumsporozoite protein and a mosquito saliva protein, AgTRIO. PfAg generated robust humoral responses and provided protection in mice models.

Malaria is among the deadliest global infectious diseases, causing almost 600 000 deaths in 2024 according to the World Health Organization malaria report. Vaccine development remains an urgent need because the current World Health Organization–approved malaria vaccine Mosquirix (RTS,S) provides suboptimal protection that wanes over time [1]. Another vaccine, R21/Matrix-M, is similar to RTS,S but expresses circumsporozoite protein (CSP) epitopes at a higher CSP–hepatitis B surface antigen ratio, thus lowering the number of doses needed for immunogenicity from 4 to 3 doses [2]. Early results with R21 suggest higher efficacy than RTS,S and the potential for longer-lasting protection [2]. The challenges for all vaccine candidates to date, in addition to a lack of a high degree of efficacy, include short-lived protection and difficulties in production, cost, and logistics [3, 4]. Therefore, additional vaccine targets based on different mechanisms of action are needed to induce robust and long-lasting protection in combination with existing Plasmodium falciparum CSP (PfCSP)–based vaccines.

When an infected female Anopheles mosquito probes for a blood meal on a human, it injects saliva containing a small inoculum of Plasmodium sporozoites into the skin. Sporozoites enter individuals at the cutaneous bite site, migrate to a blood vessel, and then travel to the liver where a systemic infection begins. Mosquito saliva contains biologically active molecules that modulate the host's immune and hemostatic responses, some of which may also influence Plasmodium migration from the mosquito. One antigen in saliva, AgTRIO, elicits an antibody response that reduces both P. falciparum and Plasmodium berghei infection in murine and humanized mouse models of infection—including infection transmitted by both Anopheles gambiae and Anopheles stephensi mosquitoes [5]. This suggests the potential for a vaccine antigen that is effective against different Plasmodium species associated with different mosquito species [5].

Anti-CSP antibody can directly block the activities of sporozoites at the pre-erythrocytic stage. It has been reported that a neutralizing human monoclonal antibody (mAb), L9, preferentially bound the junction region of PfCSP with high affinity and that it offered more potent protection than other human PfCSP mAbs in mice exposed to infectious mosquitoes [6, 7]. Furthermore, an L9 peptide viruslike particle-based vaccine can lead to a humoral response against CSP and offer protection [8]. Based on all of this information, targeting both CSP and AgTRIO may be effective in combatting Plasmodium infection. To achieve the goal of inducing antibodies against both CSP and AgTRIO, we designed a multivalent peptide, named PfAg (for P. falciparum and A. gambiae). This novel peptide consists of the L9 epitope of CSP and an AgTRIO epitope that is recognized by an AgTRIO mAb that helps prevents mosquito-borne Plasmodium infection in mice [9]. Our results demonstrate that PfAg elicits robust antibody responses against PfCSP and AgTRIO proteins and affords significant protection against mosquito-borne Plasmodium in both inbred C57BL/6 and outbred CD-1 mice.

METHODS

Animals

A. gambiae (4arr strain) mosquitoes were raised at 27°C and 80% humidity, under a 12/12-hour light/dark cycle and maintained with 10% sucrose under standard laboratory conditions in the insectary at Yale University. Swiss Webster, CD-1, and C57BL/6 mice were purchased from Charles River Laboratories. All animal experiment protocols were approved by the Yale University Institutional Animal Care & Use Committee (protocol 2023-07941). All Plasmodium infections were performed in biosafety level 2 animal facilities. All animal experiments followed the Guide for the Care and Use of Laboratory Animals by the National Research Council. In all experiments, mice were housed and cared for in the animal facilities in Yale University, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. For retro-orbital blood collection, mosquito feeding, or active immunization, mice were anesthetized with intraperitoneal injection of ketamine/xylazine (100 mg/10 mg per kg body weight). When the experiments were completed, mice were euthanized using a carbon dioxide chamber in a manner consistent with American Veterinary Medical Association guidelines.

PfAg Synthesis

Lys residues were used as the scaffolding core to support the formation of 4 branches of the CSP L9 peptide sequence (NANPNVDPNANPNVD) in linear combination with the AgTRIO peptide sequence (DLQQSVDDLMAKF) (Figure 1A). The peptides were synthesized by Lifetein using Fmoc and Boc chemistry, purified by high-performance liquid chromatography, and characterized by mass spectrometry with approximately 95% purity.

Figure 1.

Immunization with PfAg offers protection in C57BL/6 mice. A, Design of multivalent PfAg, including an L9 epitope and an AgTRIO epitope. B, Groups of C57BL/6 mice were immunized with PfAg or a control protein, ovalbumin (OVA). Two weeks after the last boost, serum samples were collected from each group of mice, and the AgTRIO-specific and Plasmodium falciparum circumsporozoite protein (PfCSP)­–specific antibody titers were determined at a 1:2500 dilution, using enzyme-linked immunosorbent assay. C, The immunized mice were exposed to 9 PfCSP-expressing Plasmodium berghei–infected mosquitoes. After 40 hours, mice were euthanized and their livers dissected, and the Plasmodium infection level was determined by Plasmodium berghei housekeeping gene, Pbs18 after normalized with liver housekeeping gene hnf4α in reverse-transcription polymerase chain reaction (levels shown as medians with interquartile range; P < .05 by Mann-Whitney U test). D, In a separate experiment, parasitemia levels were determined with Giemsa staining; 1% parasitemia was used as the initial stage of terminal infection, and then the infected mice were euthanized. Kaplan-Meier survival curves were used to present the percentage of survival. Survival was defined as no significant parasitemia at 2 weeks after infection (P = .02 by log-rank test comparing the 2 groups). Abbreviation: OD₄₅₀, optical density at 450 nm.

Active Immunization

Five-week-old female C57BL/6 mice or CD-1 mice were immunized by subcutaneous injection with 100 μg of PfAg, adjuvanted with complete Freund adjuvant for the prime immunization, followed by two 100-μg boosters at days 14 and 28, adjuvanted with incomplete Freund adjuvant. The control groups received 10 μg of ovalbumin protein with complete Freund adjuvant via subcutaneous injection as the prime and then incomplete Freund adjuvant as the booster. Two weeks after each immunization, serum was collected from each mouse, and the immunoglobulin G (IgG) responses were tested using an enzyme-linked immunosorbent assay (ELISA) to confirm antigen-specific antibodies.

AgTRIO Protein Expression and Purification and ELISAs

Protein expression and purification details are presented in the Supplementary Methods, as are ELISA details.

P. berghei Infection

PfCSP expressing P. berghei (a gift from Fidel Zavala, MD, Johns Hopkins University) were maintained by serial passage in 6–8 week old female Swiss Webster or C57BL/6 mice. Briefly, Swiss Webster or C57BL/6 mice were challenged with intraperitoneally injected P. berghei–infected red blood cells. A. gambiae then took a blood meal from the infected mice, when the parasitemia was approximately 5%. Three weeks after the blood meals, mosquitoes were used for transmission studies.

Plasmodium Burden

Plasmodium burdens after infected mosquito bites are presented in the Supplementary Methods.

Statistical Analysis

Data from ≥3 biological replicates were used to calculate medians for graphing purposes. Statistical analyses used the Mann-Whitney test for unrelated groups. Differences and data were presented as medians with interquartile range, and differences were considered statistically significant at P < .05. Prism 10.0 software (GraphPad Software) was used for all graphs and statistical analysis of data.

RESULTS

Design of the Multivalent PfAg Peptide Vaccine

A neutralizing human mAb, L9, preferentially binds the junction region of PfCSP (NANPNVDPNANPNVD) [6, 7], and this linear epitope was therefore incorporated into PfAg (Supplementary Figure 1A). mAbs against AgTRIO—previously generated to delineate the epitopes of AgTRIO associated with protective immunity and a protective AgTRIO mAb, 13F-1—recognized the sequence within the VDDLMAKFN domain located near the carboxyl terminus of AgTRIO [9]. AlphaFold protein structural modeling predicts that this sequence is part of a short α-helix in the protein (DLQQSVDDLMAKF), and we therefore incorporated this sequence into PfAg as well (Supplementary Figure 1B). To generate a multivalent PfAg target, we used Lys residues as the scaffolding core to support the formation of 4 branches of the CSP L9 peptide sequence (NANPNVDPNANPNVD) in linear combination with the AgTRIO peptide sequence (DLQQSVDDLMAKF) (Figure 1A).

Immunogenicity of PfAg

To determine whether subcutaneous immunization with PfAg can generate humoral immune responses in mice, we collected serum samples 2 weeks after prime injection, the first boost, and the second boost. ELISAs demonstrated that PfAg generated significant IgG responses against both AgTRIO and PfCSP proteins after the first boost, and the levels increased after the second boost (Figure 1B).

Protection of Mice Against Mosquito-Borne Plasmodium Transmission

In our previous study [9], active immunization with AgTRIO or passive transfer with AgTRIO antiserum offered partial protection against mosquito-borne Plasmodium infection in mice [5]. To determine whether PfAg offered protection against mosquito-borne Plasmodium infection, C57BL/6 mice were immunized with PfAg, as mentioned above. Three weeks after the last immunization, we challenged the mice by exposing each animal to the bites of 9 PfCSP-expressing P. berghei–infected A. gambiae mosquitoes. After 40 hours, mice were euthanized, their livers were collected, and the Plasmodium liver burden was determined by means of quantitative reverse-transcription polymerase chain reaction. Mice immunized with PfAg had median hepatic levels of P. berghei reduced by 70% compared with control animals (Figure 1C; P = .04).

We then performed additional studies to determine whether PfAg vaccination could influence blood-stage infection with Plasmodium after transmission. We allowed 9 PfCSP-expressing P. berghei–infected A. gambiae mosquitoes to engorge on PfAg-immunized mice and monitored blood-stage parasite infection using blood smears for 2 weeks. The kinetics of survival was analyzed using Kaplan-Meier survival curves. When the parasitemia reaches 1%, neurologic symptoms developed shortly thereafter, and the mice are euthanized due to animal welfare and ethical considerations. Figure 1D shows that only 10% of C57BL/6 (1 of 10) mice that received PfAg reached a 1% parasitemia level after challenge, compared with 60% (6 of 10) in the control group (P = .02; log-rank test). These data demonstrate that vaccination with PfAg can markedly reduce both the initial hepatic Plasmodium burden and blood-stage parasitemia in C57BL/6 mice following mosquito-borne challenge with Plasmodium.

To determine whether the PfAg peptide vaccine can induce an immune response in diverse genetic backgrounds, we immunized outbred CD-1 mice, in a similar fashion as in the previous studies with C57BL/6 animals. Two weeks after the last boost, PfAg elicited antibody responses to both AgTRIO and CSP proteins by ELISA (Figure 2A). 9 PfCSP-expressing P. berghei–infected A. gambiae mosquitoes were then allowed to feed on the PfAg-immunized and control mice and monitored for blood-stage parasite infection by blood smears for 2 weeks. The kinetics of survival, including parasitemia that was undetectable or <1%, was analyzed using Kaplan-Meier survival curves. Figure 2B shows that none of the 7 CD-1 mice that received PfAg reached a 1% parasitemia level after challenge, compared with 66% of the control animals (4 of 6) (P = .01; log-rank test).

Figure 2.

Immunization with PfAg offers protection in outbred CD-1 mice. CD-1 mice were immunized with PfAg or ovalbumin (OVA; control). Two weeks after the last boost, serum samples were collected and the AgTRIO- and Plasmodium falciparum circumsporozoite protein (PfCSP)–specific antibody titers were determined at a 1:2500 dilution of serum, using enzyme-linked immunosorbent assay. B, At 21 days after the last boost, the immunized mice were exposed to 9 PfCSP-expressing Plasmodium berghei­–infected mosquitoes. Parasitemia levels were determined by Giemsa staining; 1% parasitemia was used as the initial stage of terminal infection, and the infected mice were then euthanized. Kaplan-Meier survival curves were used to present the percentage of animals that survived. Survival was defined as no significant parasitemia at 2 weeks after infection (P = .01 by log-rank test comparing the 2 groups). Abbreviation: OD₄₅₀, optical density at 450 nm.

DISCUSSION

When mosquitoes probe for blood, they secrete saliva into the host and Plasmodium sporozoites are simultaneously released into the skin. The immune response against one specific salivary protein, AgTRIO, offers partial protection against P. berghei or P. falciparum transmitted by A. gambiae or A. stephensi mosquito bites [5]. Furthermore, AgTRIO can enhance the efficacy of a PfCSP mAb and offer additive effects in mice [5]. AgTRIO also has a role in the ability of mosquitoes to probe on a host [5, 10]. Collectively, these factors may make AgTRIO a reasonable candidate mosquito protein for inclusion in a multicomponent vaccine to prevent malaria. In the current study, PfAg induced antibodies that recognized PfCSP and AgTRIO and significantly reduced Plasmodium infection in mice. Therefore, PfAg can serve as a prototypic combinatorial vaccine, incorporating arthropod and pathogen epitopes.

It has been demonstrated that specific amounts of a combination of AgTRIO IgG and CSP mAb provide better protection than either antibody administered alone [5]. However, the synergic effects of combination of 2 viruslike particle-based vaccines against AgTRIO and PfCSP are not obvious with high infection burdens [11]. In our study, even though 2 PfAg immunized CD-1 mice had relatively low titers of anti-PfCSP antibody, all of the animals were protected after exposure to infected mosquito bites, which may indicate that there were additive effects from the immune response to both AgTRIO and PfCSF. Further studies including a comparison between PfAg and vaccines solely targeting CSP will demonstrate the relative efficacy of these targets, and the importance of different adjuvants suitable for human use, will need to be assessed. After immunization or infection, waning of protective immune responses occurs over time and influences the longevity of protection and the need for boosters.

The approved PfCSP protein-based vaccines exhibit protection that diminishes over time [12, 13]. While the IgG response against AgTRIO gradually declined after immunization, immune responses to AgTRIO may be boosted by naturally occurring mosquito bites [14]—suggesting that protection mediated by AgTRIO could be longer lasting than with traditional vaccines that only target the pathogen. This would be a unique advantage for a vaccine that incorporates a mosquito-specific target, and further studies will be needed to characterize the kinetic changes of AgTRIO and PfCSP IgG over time. Moreover, since PfAg is based on selected epitopes, this design offers a platform where additional targets, such as Plasmodium vivax CSP, could be added to offer comprehensive protection against different Plasmodium species. Overall, PfAg targets both an arthropod protein and pathogen antigen and may lead to a new generation of vaccines against malaria.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.