Targeting a malaria merozoite surface protein with mRNA vaccine generates multifunctional antibodies

Abstract

Introduction

Malaria is a leading health problem with high disease burden and mortality rates worldwide. Currently approved vaccines target the sporozoite form of Plasmodium falciparum that initially infects the liver, but only provide modest protection against malaria in young children. There is an urgent need to develop next-generation malaria vaccines that target multiple parasite developmental stages for greater efficacy. Antibodies to merozoites, which are involved in blood-stage replication, and are associated with clinical illness, have multiple functional activities and can protect against malaria. A promising merozoite vaccine candidate is Merozoite Surface Protein 2 (PfMSP2). Antibodies to PfMSP2 can promote multiple antibody Fc-mediated functional activities to clear merozoites.

Methods

We developed and evaluated monovalent and bivalent (3D7 and FC27 variants) PfMSP2-based mRNA vaccines. We designed and Codon-optimised mRNA, which was validated for in vitro expression in mammalian cells, and subsequently formulated as lipid nanoparticles for vaccination of mice in a 3-dose regimen. Vaccination with recombinant PfMSP2 protein with adjuvant was performed for comparison. We evaluated the induction of antibodies and functional activities relevant to protective immunity.

Results

mRNA vaccines induced prominent IgG responses using monovalent (3D7 allele) and bivalent (3D7 and FC27 alleles) vaccines encoding near full-length PfMSP2, and antibodies recognised the surface of whole merozoites. Vaccine responses were equivalent to, or superior to, a recombinant protein-based PfMSP2 vaccine. The bivalent vaccine induced equivalent antibodies to the two PfMSP2 alleles. Vaccination induced cytophilic IgG subclasses with multiple functional activities, including complement fixation, binding of human Fcγ-receptors I and IIa, and opsonic phagocytosis.

Conclusions

PfMSP2 is highly immunogenic using the mRNA vaccine platform and induces antibodies with multiple functional activities associated with protective immunity in humans. Combining PfMSP2 with other merozoite and sporozoite antigens is a promising strategy to develop highly efficacious vaccines to achieve malaria control and elimination goals.

Introduction

Malaria has a high global health burden spread across 83 countries and is transmitted by Anopheles mosquitoes, with the highest morbidity and mortality occurring in Africa. Plasmodium falciparum is the parasite species responsible for most deaths globally (1). The World Health Organization (WHO) estimated more than 280 million recorded cases and nearly 610,000 deaths in 2024, with the majority being children under 5 years. Progress in reducing malaria has stalled since 2015 (1), in part due to increasing resistance to antimalarial drugs and insecticides (2, 3), with risks that the burden will increase in coming years. Therefore, effective vaccines are required to complement current interventions to reduce the malaria burden and achieve and sustain malaria elimination in the long-term. Next-generation vaccines are required to achieve the Global Technical Strategy (GTS) targets of reduction in global malaria incidence and death rate of 90% by 2030 compared to the 2015 baseline (1).

The P. falciparum lifecycle is complex, whereby the sporozoite parasite form is transmitted to the skin from an infectious mosquito bite. The sporozoites establish infection in the liver and develop into merozoites, which are released into the blood and undergo asexual replication within red blood cells. This results in an exponential increase in parasitemia and clinical malaria symptoms. The only approved vaccines for malaria, RTS,S/AS01 and R21/Matrix-M, target the exact same sporozoite antigen and aim to prevent hepatic infection (4). However, several challenges remain for these vaccines: efficacy is modest in young children in the first year (56 to 75%), immunity wanes relatively quickly and annual boosters are needed, efficacy varies by population and is impacted by P. falciparum strain diversity, and efficacy is substantially lower in older children and adults (5–8). Next-generation vaccines targeting both the sporozoite and merozoite could be superior for achieving greater efficacy (>75%) and reducing malaria morbidity and mortality rates (>80%). This also includes improved durability of protection and vaccine accessibility and effectiveness across all risk populations. Merozoite antigens are attractive targets for vaccine development as they have the potential to reduce or prevent blood-stage replication of Plasmodium species thereby reducing or preventing clinical illness. Antibodies to merozoites can function by directly inhibiting merozoite invasion of erythrocytes and through antibody Fc-mediated functions including complement fixation and activation and interactions with Fc-receptors to promote opsonic phagocytosis and cellular cytotoxicity with monocytes, neutrophils and natural killer cells (9–13). Recently, a leading merozoite vaccine based on the PfRh5 antigen has shown modest efficacy (55% over 6 months) against malaria in young children (14), and functions by inhibition of merozoite invasion. Efficacy could potentially be improved by generating antibodies with multiple effector functions (13, 15, 16). Combining PfRh5 with another merozoite antigen that can generate antibodies to the merozoite surface to promote complement fixation, opsonic phagocytosis and cellular cytotoxicity is a potential strategy to achieve higher protective immunity.

P. falciparum Merozoite Surface Protein 2 (PfMSP2) is a promising candidate for blood-stage vaccine development (12, 17, 18). PfMSP2 is a highly abundant surface protein on the merozoite that is tethered to the membrane by a glycosyphophatidylinositol (GPI) anchor (19). Naturally acquired antibodies to PfMSP2 have been associated with protection against malaria across different populations (20). These antibodies can function via the antibody Fc region to mediate complement fixation and opsonic phagocytosis leading to inhibition of blood-stage replication and clearance (11, 12). Complement fixation by antibodies against merozoites can inhibit invasion and lead to merozoite killing (11, 12), and complement fixing antibodies to PfMSP2 have been associated with protection (21). Antibodies to PfMSP2 do not directly inhibit blood-stage replication in vitro on their own, but can fix and activate complement to inhibit merozoite invasion and parasite replication (11). A phase 2b trial of a PfMSP2-based vaccine in a malaria-endemic population showed evidence of strain-specific vaccine efficacy. PfMSP2 has a large variable region (~220 residues) flanked by conserved N- and C-terminal domains (22). These variable regions contain repetitive dimorphic sequences that give rise to two allelic families (3D7 and FC27) (22). Therefore, diversity in PfMSP2 can be covered by inclusion of the two major alleles in a vaccine to account for high sequence diversity and polymorphisms. A phase 1 clinical trial in malaria-naïve adults with a bivalent adjuvanted recombinant PfMSP2 vaccine containing both alleles showed significant induction of multifunctional IgG that targeted all P. falciparum strains (11, 12, 17).

Advances in molecular technologies offer new opportunities for next-generation vaccine development such as SARS-CoV-2 mRNA-LNP vaccines manufactured by Moderna and Pfizer that have been widely used globally (23). The mRNA platform has the advantage of efficient multi-antigen formulations and adaptability to emerging strains (24). The mRNA-LNP vaccine formulations induce robust antibody responses with small doses for sufficient immunogenicity to various infectious diseases (24–27). The mRNA-LNP platform has been evaluated for liver and transmission stages in both P. falciparum and P. vivax, indicating its feasibility for next-generation malaria vaccines (28–30). Here, we designed and evaluated PfMSP2 vaccines using the mRNA-LNP platform with the aim of generating multifunctional antibodies against merozoites. This builds on recombinant PfMSP2 clinical trials findings and naturally acquired immunity data from populations in malaria endemic regions. This work provides a proof-of-concept for including merozoite antigens that generate Fc-mediated functional immunity against merozoites in vaccine design, and the potential for including PfMSP2 in multi-antigen mRNA vaccines for malaria.

Materials and methods

Production of naked mRNA and mRNA-LNP vaccines

Codon-optimised P. falciparum MSP2 3D7 and FC27 sequences (GenBank accession number JN248383 and JN248384 without the C-terminal poly-Histidine tag) were synthesised as described previously (25). The mRNA vaccines in this study used Moderna’s LNP formulation (SM-102) in 8.8% (w/v) sucrose in 25 mM Tris buffer (pH 7.4). All mRNA synthesis, LNP formulation, and characterisation were performed by mRNA Core at the Monash Institute of Pharmaceutical Sciences, Australia (Supplementary Table 1).

Expression and validation of naked mRNA

The expression of naked mRNA was performed in Expi293F suspension cells (Gibco) using TransIT-mRNA (Mirus) transfection kit. Briefly, 5 μg of naked mRNA was added to 683 μL of Opti-MEM GlutaMAX (Gibco), then 7 μL of the Booster reagent was added and gently mixed, and finally 7 μL of the TransIT reagent was added and mixed. The transfection mix was incubated for 2 minutes at RT then added dropwise to 5 mL of Expi293F cells in a 6-well plate at density 1 × 10⁶ cell/mL. The cells were incubated shaking in high humidity at 37°C and 8% CO₂, and 1 mL samples were collected at multiple time-points for analysis via NuPAGE and western blot.

PfMSP2 production

Recombinant PfMSP2 3D7 and FC27 were manufactured at GroPep Pty Ltd (Adelaide, Australia) by expression in E. coli and purified from lysate by chelating, anion-exchange, and reverse-phase chromatography. The purified protein was stored at -80°C in 10 mM acetic acid to resolve amyloid-like fibrils formation (17).

Study design and mice immunisation

The evaluation of the PfMSP2 mRNA-LNP and adjuvanted recombinant PfMSP2 vaccines was conducted using female C57BL/6 mice aged 8–12 weeks (n=5 per group). As this is a descriptive study evaluating and describing mRNA vaccines for generating antibodies against P. falciparum merozoites, sample sizes were considered sufficient, balancing ethical considerations using animals in experimental research. The mice were immunised with two regimens for mRNA-LNP, fixed dose (3 × 5 μg; fix. group) and dose escalation (5 μg, 10 μg, 15 μg; esc. group). The recombinant protein vaccine followed a single regimen (3 × 5 μg PfMSP2 protein and 15 μg Quil-A adjuvant; Croda). All vaccine formulations were injected using 50 μL/dose. The mice were randomly assigned to each group, and were immunised at 28 days intervals with prior blood collection preceding the injection, and subsequent longevity bleeds for PfMSP2 (3D7) mRNA vaccines (fixed dose and dose escalation) were collected at days 120, 180, and 271. Mice received all immunisations via intramuscular injections to the calf muscle. The first two collections are retro-orbital bleeds, the subsequent collections are mandible bleeds, and the terminal collection is a cardiac bleed. Mice were anesthetically euthanised by an inhaled dose of isoflurane mixed with oxygen at a concentration of 4% at 1L/min flow rate for 1–2 minutes until unconscious to induce deep anesthesia. The mice remained anaesthetised throughout the procedure, and the depth of anesthesia was confirmed by loss of the pedal/toe-pinch reflex. The mice were anaesthetised and unresponsive until a secondary physical method of cervical dislocation was utilised to confirm death before performing cardiac puncture. All mice were housed, cared for, and handled (injection and bleeding) by the WEHI Antibody Facility in Bundoora.

Culture and isolation of P. falciparum merozoites

Parasite culture (Plasmodium falciparum 3D7) and merozoite isolation were performed as previously described (9, 43). Briefly, a laboratory-adapted P. falciparum 3D7 culture was grown in RPMI-HEPES, supplemented with 0.5% (v/v) Albumax and 0.18% (v/v) NaHCO₃. Cultures were synchronised by sorbitol treatment and maintained below 10% parasitemia. Merozoites were isolated from cultures by harvesting late-stage pigmented trophozoites using magnetic purification on columns. The purified trophozoites were cultured in protease-supplemented medium for eight hours, then the mature schizonts were collected and filtered (1.2 μm) to release and purify merozoites.

Culture of THP-1 monocytes and phagocytosis assay

These step-by-step methods were previously described and optimised (10, 12, 44, 45). Briefly, THP-1 cells were counted and resuspended to a density of 5 × 10⁶ cell/mL in THP-1 complete medium. Recombinant PfMSP2 3D7 was coupled with amine-modified fluorescent latex beads (Sigma-Aldrich) and used at 0.75 × 10⁶ beads/mL. For opsonisation, pooled sera from 5 mice were diluted to a 50 µL final volume using 1% (w/v) Bovine Serum Albumin (BSA; Bovogen) in 1 × PBS. The diluted sera were then incubated with the coated beads for 1 hour at RT. Following washing of beads, THP-1 cells were added and incubated at 37°C and 8% CO₂ for 40 minutes in the dark. Following washing and fixation, the cells were resuspended in 1% (w/v) BSA in 1 × PBS. Finally, quantification of phagocytosis was determined using flow cytometry in 96-well format on BD FACS Canto II (BD Biosiences). PBS only was used in negative controls.

NuPAGE expression analysis

To assess the expression of PfMSP2 mRNA, the transfection samples were run on a gel under reducing conditions. Briefly, whole cell samples were prepared to contain 100 mM dithiothreitol (Thermo Fisher Scientific), and 1 × NuPAGE LDS Sample Buffer (Invitrogen). Samples were incubated at 95 °C for 5 minutes, vortexed for 30 seconds, then loaded onto a 4-12% Bis-Tris gel (Invitrogen) with Precision Plus Protein Dual Color Standards (Bio-Rad) and ran at 135 V and 200 mA for 1.5 hours in 1 × MES running buffer (Invitrogen). The gel was then analysed by western blot.

Western blot analysis

The polyacrylamide gel was transferred onto a nitrocellulose (NC) membrane using iBlot 2 Transfer Stacks (Thermo Fisher Scientific) at a current of 20V for 7 minutes (default setting P3). The membrane was blocked with 1% (w/v) BSA in 0.05% (v/v) Tween20 (Sigma-Aldrich) in 1 × PBS (PBS-T) overnight at 4 °C. The membrane was probed with mouse anti-PfMSP2 3D7 primary antibodies (1/1000), and goat anti-mouse IgG horseradish peroxidase (Merck Millipore) secondary antibody (1/2000) in 1-2% (w/v) BSA at RT with extensive washing with PBS-T between steps. The SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) was used for detection. The membrane was then visualised using the ChemiDoc MP (Bio-Rad).

ELISA and plate-based assays

ELISA step-by-step protocols were performed as described previously for IgG, C1q fixation, and human Fc receptor binding (10, 12, 32, 40, 46, 47). All plate washing was performed between each step with 0.05% (v/v) Tween in 1 × PBS using the automated 405 LS microplate washer (Agilent Biotek). All assays were performed in duplicates (N = 2), and normalised across plates for individual experiments.

To measure IgG, 0.5 µg/mL of recombinant PfMSP2 3D7/FC27 was used to coat flat-bottom 96-well Nunc Maxisorp (Thermo Fisher Scientific) microplates overnight at 4 °C. The plates were then blocked with 0.1% (w/v) casein (Sigma-Aldrich) in 1 × PBS and incubated at 37 °C for 2 hours. Diluted mouse serum samples in 0.1% (w/v) casein in 1 × PBS were then added and incubated for 2 hours at RT, followed by HRP-conjugated antibodies (goat anti-mouse IgG HRP, Merck Millipore; anti-mouse IgG subclasses HRP, Southern BioTech) diluted in 0.1% (w/v) casein in 1 × PBS and incubated for 1 hour at RT. Finally, the plates were incubated with TMB liquid substrate (Life Technologies) and incubated in the dark for up to 1 hour at RT while the colour-change developed. The reaction was stopped using 1 M H₂SO₄ and OD values were immediately read at 450nm using a Multiskan GO plate reader (Thermo Fisher Scientific).

To measure vaccine-induced IgG in mouse sera binding to merozoites, the standard IgG ELISA protocol was followed with the following modifications: coating wells with 5 × 10⁴ merozoites/mL diluted in 5% (w/v) skim milk in PBS-T at 37 °C for 2 hours. All dilution steps of serum and antibodies were performed using 5% (w/v) skim milk in PBS-T, and plate blocking using 10% (w/v) skim milk in PBS-T.

To measure C1q fixation, modified ELISA protocols were performed as described previously. The plates were coated with 1 µg/mL of recombinant antigen and sera was incubated following the previous steps. Human C1q (Merck Millipore) was diluted to 10 µg/mL with 0.1% (w/v) casein in 1 × PBS and incubated for 30 minutes at RT, then rabbit anti-C1q IgG (Burnet Institute) (48) diluted with 0.1% (w/v) casein in 1 × PBS and incubated for 1 hour at RT. The goat anti-rabbit IgG HRP (Bio-Rad) was diluted with 0.1% (w/v) casein in 1 × PBS and incubated for a further 1 hour at RT before the substrate was added, reaction stopped, and absorbance measured.

To measure IgG binding to human FcγRI and FcγRIIa, the same standard IgG ELISA steps were followed with the exception of using 1% (w/v) BSA in 1 × PBS for blocking, and sample and reagent dilution. Following sera incubation, 0.125 µg/mL of biotinylated FcγRI 10256-H27H-B (Sino Biological) or 0.2 µg/mL dimeric FcγRIIa (Burnet Institute) (49) was added and incubated at 37 °C for 1 hour. For detection of FcγRs, Streptavidin-HRP (ThermoFisher Scientific) diluted 1/10000 was added and incubated at 37 °C for an additional hour, followed by substrate reaction and absorbance measurement.

Statistical analysis

All statistical analyses and graph generation for this study were conducted using GraphPad Prism 11 (MacOS) software. Mann-Whitney test was used to compare two groups, while the Kruskal-Wallis test was used for multiple groups (more than two) comparisons. A two-way ANOVA test with mixed effects was applied to account for the dependence of data points over time. Spearman’s rho was calculated to assess correlations between parameters.

Results

PfMSP2 mRNA vaccine shows high immunogenicity in mice

We first designed an mRNA vaccine construct based on the 3D7 allele of PfMSP2, which was codon-optimised and truncated (Δ19N and Δ23C) to remove the N-terminal secretory signal and C-terminal GPI anchor sites, respectively (Figure 1A). This design was identical to the recombinant PfMSP2 used in a previous human clinical trial, and was used in this study as a control regimen (17). To confirm protein expression, we transfected Expi293F (HEK293) suspension cells with the naked mRNA and samples were collected and processed under reducing conditions for western blot analysis. The PfMSP2 3D7 mRNA showed increasing protein expression over time that was detected using antigen-specific antibodies (Figure 1B). We then formulated our construct as lipid nanoparticles (LNPs) for preclinical testing and effective dose evaluation in female C57BL/6 mice (n=5 per group) which received fixed doses (3 × 5 μg) or dose escalation (5, 10, 15 μg sequentially) in a 50 μL formulation on days 0, 28 and 56 (Figure 1C); dose escalation enabled us to assess whether the fixed 5 μg dosing was non-inferior to a higher dose. We also included a third group of mice which received an adjuvanted recombinant PfMSP2 vaccine (17) as a reference for the mRNA PfMSP2 vaccine performance and dose optimisation following the same regimens and sampling schedule. Sera were collected at each vaccine dose and two weeks after the final dose on day 70 (peak vaccine response), and were assessed for vaccine-induced antibodies by enzyme-linked immunosorbent assay (ELISA). Vaccine-induced IgG to PfMSP2 3D7 increased after each dose in all vaccine groups (Figure 1D). Interestingly, there were minimal differences between the two mRNA PfMSP2 regimens indicating higher mRNA doses were not required to achieve optimal IgG induction. Additionally, IgG levels declined at a similar rate over 7 months for both mRNA vaccine regimens (Supplementary Figure 1). Next, the sera from the individual mouse groups were pooled and used to detect recombinant PfMSP2 3D7 in western blots, and the groups showed comparable detection (Figure 1E). To establish that vaccine-induced antibodies recognise native PfMSP2 on the merozoite surface, whole intact merozoites were isolated from P. falciparum blood-stage culture and used in ELISA. Both recombinant and mRNA PfMSP2 vaccines induced IgG at peak (day 70) that binds native PfMSP2 on the merozoite surface without a significant difference (31), indicating recognition of key native epitopes (Figure 1F).

Figure 1.

PfMSP2 3D7 mRNA vaccine evaluation. (A) Schematics representing the full-length PfMSP2 3D7 and FC27 alleles, and the truncated mRNA constructs with the deletion of the N-terminal signal peptide (SP) and C-terminal GPI anchor motif. PfMSP2 FC27 mRNA construct contained a Myc tag for mammalian expression detection in western blot. (B) Expi293F cells were transfected with naked PfMSP2 3D7 mRNA and cell lysate was collected at multiple time-point post-transfection. The samples were processed under reducing conditions for western blot, and protein expression was detected using mouse α-PfMSP2 polyclonal IgG (1/1000 dilution). (C) Schematic of mouse immunisation regimens and timeline for blood collection (recombinant PfMSP2: rMSP2; mRNA PfMSP2 fixed dose: mMSP2 fix; mRNA PfMSP2 dose escalation: mMSP2 esc). (D) Adjuvanted recombinant PfMSP2 (rMSP2) and mRNA-LNP PfMSP2 (fixed dose mMSP2 fix, and dose escalation mMSP2 esc) immunogenicity ELISA analysis of IgG in mouse sera at 1/25600 dilution (n=5 per group). Error bars represent median ± IQR and assays performed in duplicates. Statistical analysis was performed using Kruskal-Wallis test; ns is not significant (p=0.9676). (E) Western blot detection of 250 ng of recombinant PfMSP2 loaded into each lane and detected by pool mouse sera polyclonal IgG from immunisation regimens (1/1000 dilution). (F) ELISA analysis of mouse sera IgG from day 70 at 1/100 dilution (n=5 per group; recombinant PfMSP2 rMSP2, and fixed dose mRNA PfMSP2 mMSP2 fix) detecting native PfMSP2 on whole merozoites. Error bars represent median ± IQR and assays performed in duplicates. Statistical analysis was performed using Mann-Whitney test; ns is not significant (p=0.6905).

PfMSP2 mRNA vaccines induce IgG with Fc-mediated functional activity

The profiles of murine IgG subclasses induced by recombinant and mRNA PfMSP2 vaccines were examined to understand the functional or protective potential of vaccine responses (32). The cytophilic IgG subclasses in C57BL/6 mice with human analogues are IgG1 (huIgG4), IgG2b (huIgG3), IgG2c (huIgG1), and IgG3 (huIgG2). All murine IgG subclasses confer protective functions, however IgG1 exhibits the lowest Fc-mediated functional activity relative to its high abundance in serum (22, 33, 34). Testing IgG at the peak response point (day 70), the three vaccine regimens showed comparable subclass profiles with IgG1 at higher magnitude, and moderate IgG2b/c induction, but low IgG3 levels (Figure 2A). In studies of human immunity, it has been shown that antibodies to PfMSP2 have Fc-mediated functions including complement fixation and activation, which can lyse merozoites and inhibit erythrocyte invasion, antibody-dependent cellular inhibition (ADCI) and opsonic phagocytosis of merozoites, mediated by IgG interactions with Fcγ receptors (9–11, 17, 32, 35, 36). Therefore, we assessed similar Fc-mediated functions of vaccine-induced mouse antibodies. There were multiple functional antibody activities induced by vaccination among all mouse groups. Notably, the murine IgG showed prominent binding to human Fcγ receptors (I and IIa), which are activating receptors promoting phagocytosis and other functions. We did not assess FcγRIII binding because mouse IgG has weak affinity to human FcγRIII (37). The mouse antibodies also demonstrated prominent fixation of human C1q, which is the first essential step in classical complement activation. All functional activities were broadly comparable between groups, with mRNA PfMSP2 fixed dose sera tending to have the highest activity, although this was not significant due to biological variation (Figure 2B). Additionally, a pool of serum from mice vaccinated with mRNA PfMSP2 was used to opsonise PfMSP2 3D7 coated fluorescent beads and examine IgG-mediated phagocytosis in vitro using the human monocytic cell line, THP-1. There was substantial phagocytosis of opsonised beads using serum from vaccinated mice relative to the negative control (PBS only) indicating Fc receptor recognition and function (Figure 2C). THP-1 cells express FcγRI and FcγRIIa which we used in this study (Figure 2B). This supports the cross-reactivity of murine IgG and human Fc receptors, consistent with the in vitro binding to recombinant human Fcγ receptors observed by vaccine-induced antibodies, and the strong correlation between IgG subclasses and Fcγ receptor binding (Figure 2D).

Figure 2.

Functional activity of PfMSP2 3D7 monovalent mRNA vaccine IgG. (A) ELISA analysis of mice IgG subclasses in day 70 sera (n=5 per group) of vaccine regimens (recombinant PfMSP2: rMSP2; mRNA PfMSP2 fixed dose: mMSP2 fix; mRNA PfMSP2 dose escalation: mMSP2 esc). Assays were performed in duplicates (1/6400 dilution) and error bars represent median ± IQR. Statistical analysis was performed using two-way ANOVA; ns is not significant (p>0.8). (B) Functional assays of murine IgG from day 70 sera for human FcγRI binding (1/12800 dilution), human FcγRIIa binding (1/400 dilution), and human C1q engagement (1/100 dilution) of vaccine regimens (recombinant PfMSP2: rMSP2; mRNA PfMSP2 fixed dose: mMSP2 fix; mRNA PfMSP2 dose escalation: mMSP2 esc). Assays were performed in duplicates and error bars represent median ± IQR; two-way ANOVA statistical test showed no significant differences between regimens for all functional assays (p>0.05). (C) Phagocytosis of PfMSP2-coated fluorescent beads by THP-1 monocytes using mRNA-LNP PfMSP2 fixed dose (mMSP2 fix) pooled sera samples (n=5 mice) and 1/60 dilution with PBS for negative controls. Assays performed in duplicates and error bars represent median + 95% CI. (D) Correlation matrix (values are Spearman’s rho) of mouse IgG subclasses at peak and function (day 70; n=15 mice) with +1 indicating strong positive correlation, 0 indicating no correlation, and -1 indicating strong negative correlation. All correlations were p<0.05 except IgG3.

Bivalent PfMSP2 mRNA vaccine incorporating the two major alleles elicits high functional IgG responses

In P. falciparum populations, PfMSP2 occurs as two major allelic types and antibodies to PfMSP2 typically have a degree of allele-specific reactivity. Therefore, an important property for a protective PfMSP2 vaccine is the ability to generate an immune response to both major alleles (3D7 and FC27) (17, 36). To achieve this, the PfMSP2 FC27 allele was codon-optimised and truncated (Δ19N and Δ19C) to remove the N-terminal leader sequence and GPI anchor respectively, with the addition of a C-terminal Myc tag (Figure 1A). Following expression validation of the PfMSP2 FC27 allele (Supplementary Figure 2), the two alleles were encoded by mRNA and formulated as mRNA-LNPs individually then mixed in a 1:1 ratio and used to immunise mice (n=5). The regimen in this study included 3 × 10 μg doses (5 μg per allele) in a 50 μL formulation and sera collection at regular intervals for analysis (Figure 3A). The vaccine induced high and equivalent IgG responses against both alleles with vaccine-induced IgG highest titre after the third dose (Figure 3B). Additionally, there was clear induction of the functional murine subclasses in the bivalent mRNA PfMSP2 vaccine and high functional activity by binding to human Fcγ receptors and human C1q (Figures 3C, D). This activity was measured using recombinant PfMSP2 3D7 and FC27, and these antibodies are expected to bind native PfMSP2 on the parasite surface as observed in the monovalent mRNA vaccine. These results suggest the vaccine could generate potent and strain-transcending PfMSP2 antibodies capable of protective immunity (12, 22).

Figure 3.

PfMSP2 bivalent (3D7 and FC27) mRNA vaccine evaluation. (A) Schematic of mice immunisation regimens and timeline of blood collection for bivalent PfMSP2 formulations (3D7 and FC27 alleles). (B) Bivalent mRNA-LNP (3D7 and FC27 alleles) immunogenicity ELISA analysis of IgG in total mouse sera at 1/6400 dilution (n=5). Error bars represent median ± IQR and assays performed in duplicates. Statistical analysis was performed using Mann Whitney test; ns is not significant (p>0.05). (C) ELISA analysis of murine IgG subclasses in sera at day 70 (n=5). Assays were performed in duplicates (1/3200 dilution) and error bars represent median ± IQR. (D) Functional assays of mouse IgG from day 70 sera for human FcγRI binding (1/12800 dilution), human FcγRIIa binding (1/400 dilution), and human C1q engagement (1/800 dilution). Assays were performed in duplicates and error bars represent median ± IQR. Statistical analysis was performed using Kruskal-Wallis test; ns is not significant (p>0.9999).

Discussion

In this study, we demonstrate a strategy to target the merozoite surface using mRNA vaccines to generate antibodies with Fc-mediated functional activity. We designed mRNA vaccines based on PfMSP2 because it is an abundant merozoite surface antigen and a known target of antibodies that promote complement activation to inhibit blood-stage replication and mediate opsonic phagocytosis of merozoites (9–12). Furthermore, antibodies to PfMSP2 have been associated with protection in studies of naturally-acquired and vaccine-induced immunity (10, 12). We formulated monovalent and bivalent vaccines including the two major allelic isoforms of PfMSP2 and found the vaccine effectively generated antibodies with Fc-mediated functional activities to both alleles in preclinical studies.

Validating the expression of PfMSP2 in mammalian cells was essential for progressing the mRNA vaccine into preclinical animal studies. The mRNA encoding PfMSP2 3D7 and FC27 was codon-optimised for mammalian translation, and expression of both antigens was detected indicating compatibility of PfMSP2 with the mRNA platform. The mRNA-LNP formulation showed immunogenicity for both monovalent (3D7) and bivalent (3D7 and FC27) vaccines in mice, with comparable profiles between the monovalent (3D7) adjuvanted recombinant antigen and mRNA vaccines. The efficiency of mRNA vaccine translation in vivo has been reported to be 1-2% due to endosomal escape limitations, which equates to an estimated ≥100 ng of PfMSP2 for the 5 μg mRNA-LNP vaccine dose compared to the 5 μg of PfMSP2 when given as a recombinant protein formulated with adjuvant (38). The immunogenicity of the mRNA vaccine was comparable to the recombinant protein vaccine, and there was no improved immunogenicity using escalating mRNA doses indicating dose-sparing advantages. However, further dose optimisation studies and larger sample sizes would be needed to more formally evaluate dosing, compare the mRNA and recombinant protein platforms, and examine biological variability within and between regimens.

The mRNA vaccine-induced antibodies by monovalent and bivalent formulations showed substantial functional activity, including complement fixation, interactions with FcγRI and FcγRIIa, and opsonic phagocytosis by the monovalent vaccines (37, 39). This functional antibody profile is similar to what has been reported for antibodies in naturally-acquired immunity and clinical trials of a recombinant PfMSP2 protein vaccine in humans (11, 12). These Fcγ receptor engagement and complement activation functions are predicted to be important in parasite inhibition and clearance in vitro and in vivo (9, 11, 12, 40). The IgG2c and IgG2b subclasses of C57BL/6 mice, which have Fc-mediated functional activities, were collectively higher than murine IgG1, which can conversely confer inhibitory activity of downstream immune functions (41). Further, it was promising that the bivalent formulation showed the same induction of antibodies to both allelic forms. In our study, we quantified the functional activity of IgG generated by vaccination of mice, which may be different to the activities of vaccine-induced human antibodies. Prior analysis of a PfMSP2 vaccine, based on a recombinant protein with an adjuvant, in a clinical trial showed that vaccine-induced human antibodies had similar Fc-mediated functional antibodies as observed in the present study (17).

Both cross-reactive and allele-specific antibodies to PfMSP2 have been observed in naturally-acquired and vaccine-induced immunity in humans and cross-reactivity may be partly attributed to antibodies against the conserved C-terminal region (12). Naturally-acquired antibodies were reported to more prominently target the variable allele-specific regions of PfMSP2 than vaccine-induced antibodies (12, 36). Further investigation of responses induced by the bivalent vaccine to map key epitopes targeted by functional and cross-reactive antibodies may be valuable to fine-tune PfMSP2 construct design. It is also important to understand the kinetics of cellular immune responses that may provide insights into vaccine longevity and potential protection. This includes analysis of B cell memory and activation of CD4+ T-follicular helper cells that support antibody generation (42). There are limitations of performing these analyses in mouse models because of differences from human immunity. Exploring humanised mice and human organoid models to analyse vaccine responses may be valuable.

The findings that mRNA vaccines encoding PfMSP2 generate antibodies that target the merozoite surface and have functional activities associated with protective immunity in humans is encouraging, but future in vivo studies will be needed to confirm that vaccine-induced antibodies are protective. Evaluating the protective efficacy of PfMSP2-based vaccines is not possible with current mouse models of malaria because PfMSP2 or an orthologue is not present in rodent malaria species, such as Plasmodium berghei, and P. falciparum does not infect rodents. Furthermore, the very low activity of complement fixation in laboratory mice and different use of Fc-receptors and immune cells by mouse antibodies compared to humans pose further challenges (4). Therefore, evaluating PfMPS2 vaccines will require the use of non-human primate studies or controlled human malaria infection (CHMI).

Conclusions

In our study we showed that the mRNA-LNP platform can be used for monovalent and bivalent formulations that induce functional IgG against PfMSP2, further supporting PfMSP2 as a vaccine candidate and the broader concept of targeting merozoite surface proteins in malaria vaccine strategies. We showed that a 3-dose regimen induced peak immunity comparable to the adjuvanted recombinant protein. Another lead merozoite vaccine based on PfRh5 induces growth-inhibitory antibodies and shows modest efficacy in malaria endemic populations. However, studies of naturally-acquired immunity point to the importance of antibody Fc-mediated functions in protective immunity. Therefore, combining PfRh5 with PfMSP2, or other antigen combinations with PfMSP2, may be an effective strategy to induce antibodies with both growth-inhibitory and Fc-mediated functional activities for greater protective efficacy. Further, a highly effective blood-stage vaccine combined with the leading sporozoite antigen, PfCSP, could reduce the risk of initial infection and subsequent disease to achieve high-level protection against malaria.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the State of Victoria mRNA Activation Program grant (Burnet Institute Vaccine Initiative) awarded to JB in collaboration with CP, National Health and Medical Research Council of Australia grant awarded to JB (2033320), a Monash University postgraduate scholarship awarded to TH, and a DAAD PROMOS scholarship from the University of Bonn awarded to TR. Burnet Institute is supported by an Institute Operational Support grant from the Victorian State Government and the NHMRC Independent Institutes Infrastructure Support Scheme.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The animal studies received the following approvals: Burnet Institute malaria antigens mRNA handling (IBC 40702); WEHI Animal Ethics Committee and Antibody Facility (2020.019 and 2023.012); and Scientific Procedure Premises Licence (SPPL201904). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

AT: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing. TR: Formal analysis, Investigation, Methodology, Writing – review & editing. TH: Formal analysis, Investigation, Writing – review & editing. SF: Methodology, Resources, Writing – review & editing. CL: Methodology, Resources, Writing – review & editing. SC: Methodology, Writing – review & editing. RM: Project administration, Resources, Writing – review & editing. LR: Supervision, Writing – review & editing. LK: Methodology, Writing – review & editing. CD’S: Project administration, Resources, Writing – review & editing. CP: Funding acquisition, Resources, Writing – review & editing. JB: Conceptualization, Funding acquisition, Investigation, Resources, Supervision, Writing – review & editing.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer RT-L declared a past co-authorship with the author JB to the handling editor.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1840217/full#supplementary-material