Plasmodium falciparum merozoite surface protein (PfMSP)2 is a target of parasite-neutralizing Abs. Inclusion of recombinant PfMSP2 (rPfMSP2) as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we used the highly immunogenic PfMSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. In this study, we exploited the benefits of rPfMSP8 as a carrier to optimize a rPfMSP2-based subunit vaccine. rPfMSP2 and chimeric rPfMSP2/8 vaccines produced in Escherichia coli were evaluated in comparative immunogenicity studies in inbred (CB6F1/J) and outbred (CD1) mice, varying the dose and adjuvant. Immunization of mice with both rPfMSP2-based vaccines elicited high-titer anti-PfMSP2 Abs that recognized the major allelic variants of PfMSP2. Vaccine-induced T cells recognized epitopes present in both PfMSP2 and the PfMSP8 carrier. Competition assays revealed differences in Ab specificities induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. In contrast to aluminum hydroxide (Alum) as adjuvant, formulation of rPfMSP2 vaccines with glucopyranosyl lipid adjuvant–stable emulsion, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of Abs to cytophilic IgG subclasses. The rPfMSP2/8 + glucopyranosyl lipid adjuvant–stable emulsion formulation induced significantly higher Ab titers with superior durability and capacity to opsonize P. falciparum merozoites for phagocytosis. Immunization with a trivalent vaccine including PfMSP2/8, PfMSP1/8, and the P. falciparum 25 kDa sexual stage antigen fused to PfMSP8 (Pfs25/8) induced high levels of Abs specific for epitopes in each targeted domain, with no evidence of antigenic competition. These results are highly encouraging for the addition of rPfMSP2/8 as a component of an efficacious, multivalent, multistage malaria vaccine.

In recent years, great progress has been made toward global malaria elimination with the widespread implementation of a number of prevention and control programs (1). However, recent evidence of a plateau or a rebound in the overall number of clinical cases and malaria-related deaths raises significant concerns. The development of a highly efficacious malaria vaccine is needed to enhance current malaria control efforts and move the field closer to achieving the goals of malaria elimination and eradication.

The most advanced malaria vaccine, RTS,S, has demonstrated suboptimal immunogenicity and efficacy in phase III clinical trials (25). Follow-up studies have revealed that rapidly waning protective responses result in a loss in vaccine efficacy over time (3, 6, 7), and efforts to improve the efficacy of RTS,S/AS01 are ongoing (8). However, the requirement for sterilizing immunity and the limitations of single-Ag formulations encourage the evaluation of alternative approaches. Ultimately, it is expected that the development of a highly efficacious, multicomponent vaccine that potently blocks the parasite at multiple stages will be necessary to protect individuals and reduce transmission within populations.

More generally, success with subunit malaria vaccines has been limited because of Ag polymorphism, poor immunogenicity, and the challenges associated with large-scale production of structurally complex vaccine candidates. Like the RTS,S vaccine, many malaria vaccine candidates fail to induce highly protective immune responses that are sustained for an extended period of time postvaccination (7, 9). The approaches to improve immunogenicity and durability include testing of novel Ags and adjuvant platforms, along with varying vaccine dose and immunization schedule (8, 1015). Thessnie efforts still focus mainly on single-Ag formulations (1619). Concurrent immunization with multiple target Ags and/or multiple alleles of candidate Ags represents another significant opportunity to increase overall protective efficacy. Nevertheless, this presents the additional challenge of maintaining the immunogenicity of each component in a multiantigen formulation.

With a focus on construct design, we established the use of Plasmodium falciparum merozoite surface protein (PfMSP)8 as a Plasmodium-specific carrier protein for leading blood-stage and sexual-stage vaccine candidate Ags. Genetic fusion of the 19-kDa fragment of PfMSP1 (PfMSP119) to the N terminus of PfMSP8 resulted in production and purification of properly folded rAg in high yield. Immunization of mice and rabbits with the chimeric recombinant PfMSP1/8 (rPfMSP1/8) vaccine elicited high-titer, growth-inhibitory Abs specific for conformation-dependent, protective epitopes of PfMSP119. Robust PfMSP8-restricted CD4+ T cell responses were able to provide sufficient help for PfMSP119-specific B cells (20). Fusion to PfMSP8 alleviated the impact of antigenic competition on PfMSP119-specific B cell responses that was evident when animals were immunized with an admixture of two Ags, 42-kDa fragment of PfMSP1 and PfMSP8. Significantly, PfMSP1/8-induced IgG demonstrated superior activity against homologous (FVO) and heterologous (3D7) parasites in the standard in vitro blood-stage growth inhibition assay, as compared with anti–42-kDa fragment of PfMSP1 IgG (20). Importantly, immunization of nonhuman primates with rPfMSP1/8 was well tolerated and induced high-titer PfMSP119-specific Abs that potently inhibited parasite growth in vitro (21).

For the 25-kDa sexual stage transmission blocking candidate (Pfs25) fusion to the PfMSP8 carrier (recombinant Pfs25/8 [rPfs25/8]) significantly increased yield and quality of recombinant Pfs25 (rPfs25) and eliminated the need for refolding procedures during purification to achieve proper conformation of its four EGF-like domains. Immunization with chimeric rPfs25/8 induced high titers of Abs against conformational epitopes of Pfs25 that exhibited potent transmission-reducing activity as measured in the standard mosquito membrane feeding assay (22). Furthermore, the PfMSP8 carrier enhanced immunogenicity and prevented antigenic competition when Pfs25/8 was formulated in combination with PfMSP1/8 (23). The success with chimeric rPfMSP1/8 and rPfs25/8 vaccines with respect to production, folding, immunogenicity, and induction of functional Abs prompted us to evaluate PfMSP8 as a fusion partner for another leading blood-stage vaccine candidate, PfMSP2.

Individuals living in endemic areas generate anti-PfMSP2 IgG1 and IgG3 Abs that contribute to protection from malaria (2426). Vaccine-induced, PfMSP2-specific Abs have also been shown to contribute to partial protection in one clinical trial (2729). However, as with other blood-stage subunit vaccine candidates, attempts to advance a PfMSP2-based vaccine have faced several challenges, most notable are those related to the structure of recombinant PfMSP2 and the allele-specificity of protective anti-PfMSP2 Abs. We have shown that for PfMSP2, fusion to PfMSP8 facilitated production and purification while preventing formation of PfMSP2-associated fibrils. The more open conformation of chimeric rPfMSP2/8 increased PfMSP2 epitope accessibility, resulting in enhanced functionality of immunization-induced rabbit IgG in the opsonophagocytosis of P. falciparum merozoites (30).

In this paper, we continue with this systematic approach to select Ags and formulations to build a highly efficacious, multivalent malaria vaccine capable of reducing clinical disease and interrupting transmission. Initial studies evaluating PfMSP1/8 and Pfs25/8 as a bivalent formulation showed that high levels of Ag-specific IgG are maintained upon coadministration when formulated with Th1- or Th2-biasing adjuvants (23). Vaccines formulated with aluminum hydroxide (Alum) as adjuvant typically drive Th2-associated Ab responses with IL-4 and IL-5 cytokine production by CD4+ T cells (31, 32). The glucopyranosyl lipid adjuvant–stable emulsion (GLA-SE), composed of a synthetic TLR4 agonist, glucopyranosyl lipid A in a squalene-oil stable emulsion, can shift responses toward a Th1 profile with class switching to the cytophilic IgG subtypes desirable for PfMSP2-based vaccines (3335). In this study, we conducted comparative immunogenicity studies evaluating the influence of Ag, dose, and adjuvant on PfMSP2 vaccine-induced immune responses with consideration of IgG isotype and functionality of elicited Abs. Following downselection, we evaluated the immunogenicity of a trivalent vaccine containing PfMSP2/8 in combination with PfMSP1/8 and Pfs25/8.

Five- to six-week-old male CB6F1/J (BALB/cJ X C57BL/6J) mice or male and female CD1 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and Charles River Laboratories (Wilmington, MA), respectively. All animals were housed under specific pathogen–free conditions in the Animal Care Facility of Drexel University College of Medicine. Animal studies were reviewed, approved, and conducted in compliance with Drexel University’s Institutional Animal Care and Use Committee (protocol no. 20645). Production and purification of rPfMSP2 (3D7), rPfMSP2 (FC27), rPfMSP2/8, rPfs25, rPfs25/8, rPfMSP1/8 and recombinant GST fused to PfMSP119 (rGST-PfMSP119) have been previously reported (20, 22, 30). Four immunogenicity protocols were followed in which serum and/or splenocytes were collected 3 or 4 wk following the final immunization.

Experimental protocols 1 and 2

Groups of inbred CB6F1/J mice (n = 10) were immunized s.c. with rPfMSP2 (3D7), rPfMSP2/8, rPfMSP8, an admixture of rPfMSP2 (3D7) + rPfMSP8, or adjuvant alone. rPfMSP2 and rPfMSP2/8 Ags used for immunization were stored at −80°C and administered within 2 y of purification. High (2.5 μg) or low (0.5 μg) Ag doses were formulated with either 2% Alhydrogel (500 μg/dose; InvivoGen, San Diego, CA) or GLA-SE (5 μg/dose; Infectious Disease Research Institute, Seattle, WA) as adjuvant. Mice were immunized three times at 4-wk intervals. Test bleeds were collected 3 wk following the primary and secondary immunization. Terminal bleeds were conducted 4 wk following the third immunization for analysis of humoral responses (n = 5). For analysis of T cell responses, mice (n = 5) were immunized three times, as described above. Following a 9-wk rest period, mice were boosted i.p., and spleens were harvested 3 wk later for analysis of cellular responses.

Experimental protocol 3

To analyze the durability of vaccine-induced Ab responses, groups of mice (n = 5) were immunized s.c. with 0.5 μg of rPfMSP2 (3D7) or rPfMSP2/8 formulated with Alhydrogel or GLA-SE, as described above. Corresponding control groups received adjuvant alone. Mice were immunized s.c., boosted 4 wk later, and allowed to rest before receiving a delayed booster immunization 11 mo following the secondary immunization. For Ab analysis, test bleeds were taken 3 wk postprimary and 4 wk postsecondary immunizations and every 4–8 wk thereafter. Terminal sera were collected 3 wk following the delayed third immunization.

Experiment protocol 4

Groups of outbred CD1 mice (n = 10; five males and five females) were immunized s.c. with a monovalent (rPfMSP2/8), bivalent (rPfMSP1/8 and rPfs25/8), or trivalent (rPfMSP2/8, rPfMSP1/8, and rPfs25/8) vaccine containing 2.5 μg of each Ag per dose and formulated with Alum or GLA-SE as adjuvant. Immunizations, test bleeds, and terminal bleeds were conducted as in experiment protocol 1.

Generation of PfMSP2 overlapping peptides

rPfMSP2-associated fibrils have been shown to induce proliferation of naive splenocytes in an Ag receptor–independent, TLR2-dependent manner (30). To avoid activation of cells in this manner, 18-mer overlapping peptides (9-mer overlap) spanning the length of mature PfMSP2 were generated. The resulting 25 peptides were synthesized (reversed-phase HPLC purity >90%; GenScript, Piscataway, NJ) and reconstituted at a concentration of 1–2 mg/ml in dH2O, dimethyl sulfoxide, or 3% ammonia solution according to the manufacturer’s recommendation. To evaluate potential toxicity and/or nonspecific activation of T cells, each peptide was diluted in complete T cell media (RPMI 1640 supplemented with 2 mM l-glutamine, 0.5 mM sodium pyruvate, 50 μM 2-ME, 1× penicillin/streptomycin, 10% heat-inactivated BenchMark FBS, and 10 μg/ml of polymyxin B) to a concentration of 10 μg/ml and tested for the ability to stimulate proliferation of naive mouse splenocytes in the presence or absence of Con A (1 μg/ml), as described (30). Two peptides were eliminated because of toxicity, and the remaining 23 peptides were grouped into five pools with four to five peptides per pool.

Ag-specific splenocyte proliferation

Ag-specific proliferation was measured by [3H]-thymidine incorporation as previously described (30). Briefly, a single-cell suspension of splenocytes was generated, plated at 1 × 105 cells per well, and stimulated in triplicate. Splenocytes were stimulated with rPfMSP2 peptide pools (five pools of four to five peptides, 3 µg/ml of each peptide per pool) or with rPfMSP8 (10 μg/ml). Con A (1 μg/ml) or unstimulated wells served as positive and negative controls, respectively. A stimulation index for each condition was calculated as the mean cpm of stimulated samples divided by the mean cpm of unstimulated samples.

Ag-induced cytokine production

To quantify cytokine production by Ag-specific CD4+ T cells, splenocytes were isolated, plated at a concentration of 5 × 105 cells per well and stimulated in vitro with rPfMSP2/8 (10 µg/ml) for 96 h, as previously described (23). Following incubation, culture supernatants were collected and stored at −80°C until analysis. Multiplex Luminex assays (R&D Systems, Minneapolis, MN) for quantification of IL-5, IFN-γ, and TNF-α were conducted according to the manufacturer’s protocol. Data were collected on a Luminex 200 system and analyzed using the xPONENT3.1 software (R&D Systems). Final concentrations (picograms per milliliter) of each analyte were calculated based on a standard curve, and corresponding unstimulated control values were subtracted as background.

Ag-specific Ab titer

Ag-specific Ab titers were determined by direct-binding ELISA as previously described (30). Briefly, 2-fold serial dilutions of individual mouse sera were incubated (for 2 h at room temperature) on plates coated with rAg (0.25 μg/well). Bound Abs were detected using HRP-conjugated rabbit anti-mouse IgG (H+L chains) with ABTS as substrate. A405 values between 1.0 and 0.1 were plotted, and titer was calculated as reciprocal of the dilution, yielding an absorbance at 405 nm (A405) of 0.5. High-titer pooled sera included on each plate served as a standard for normalization of values across plates.

Ab isotype profile

To determine IgG subtypes induced by immunization, terminal serum samples from experiments 1 and 4 were titrated by ELISA on rPfMSP2/8- or rPfMSP8-coated plates, respectively, as described above. Bound Abs were detected by HRP-conjugated rabbit Abs specific for mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3 (SouthernBiotech, Birmingham, AL), followed by ABTS as substrate. A standard curve generated by detection of isotype-specific mouse myeloma proteins was used to calculate the concentration of each isotype. IgG isotype concentration was expressed as units per milliliter, in which 1 U/ml is equivalent to 1 μg/ml myeloma standard.

Avidity of Ag-specific Abs

The avidity of vaccine-induced, Ag-specific Abs was estimated by ELISA based on the resistance of Ag–Ab complexes to dissociation with increasing concentrations of ammonium thiocyanate, as previously described (36, 37). Individual serum samples from immunized mice were diluted to yield an A405 of ∼1.2. Following binding of Ag-specific Abs to rPfMSP2- or rPfMSP8-coated wells, ammonium thiocyanate was added at molar concentration of 0, 0.5, 1.0, 2.0, 3.0, or 4.0 and incubated for 15 min at room temperature. The plates were then washed, and bound IgG was detected as above. An avidity index for each serum was calculated as the concentration of ammonium thiocyanate required to reduce the binding of Ag-specific Abs by 50%. Data points were fitted to a linear trend line, and only equations with an r2 value ≥0.9 were used for calculation of avidity indices.

Competition ELISA

To analyze differences in epitope specificity of immunization-induced Abs, competitive binding ELISAs were performed as described previously (30). Individual serum samples from animals immunized with rPfMSP2 or rPfMSP2/8 were diluted to yield an A405 of ∼1.2 and then incubated overnight at 4°C with 10-fold increasing concentrations of rPfMSP2 (3D7) or rPfMSP2/8 (0.01–10 nM) as inhibitor diluted in 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, and 0.5% deoxycholate. To ensure complete adsorption of anti-PfMSP8–specific Abs, 1 μM of rPfMSP8 was included in all Ag–Ab mixtures. Samples were added to ELISA wells coated with rPfMSP2 (3D7)– or rPfMSP2/8-coated wells, and binding of noncomplexed Abs was measured as described above. Percentage of residual binding for each sample was calculated as (A405 of IgG in the presence of inhibitor/A405 of IgG in the absence of inhibitor) × 100.

A pool of serum was generated from each group of mice immunized with rPfMSP2 or rPfMSP2/8 (2.5 or 0.5 μg; Alum or GLA-SE) and IgG purified by protein A/G affinity chromatography (38). The ability of IgG to opsonize merozoites for uptake by THP-1 cells was evaluated using a previously described phagocytosis assay (30). Briefly, P. falciparum merozoites (3D7 or D10) were isolated, stained with Nuclear Red LCS1 (AAT Bioquest, Sunnyvale, CA), and fixed with 2% formaldehyde prior to counting by flow cytometry. Merozoites were resuspended at 1 × 107 merozoites/ml in THP-1 culture media (RPMI 1640 (30-2001; American Type Culture Collection), supplemented with 0.05 mM 2-ME and 10% FBS, and were plated at 30 μl/well in FBS-coated plates. For opsonization, merozoites were coincubated with 0.1 or 0.05 mg/ml purified IgG for 1 h at room temperature. Unopsonized merozoites and merozoites opsonized with adjuvant control IgG served as negative controls. All samples were tested in triplicate. For phagocytosis, opsonized merozoites were washed and coincubated with THP-1 cells at a 5:1 ratio (merozoite/cells) for 10 min at 37°C. Phagocytosis was stopped by the addition of cold PBS containing 3% FBS. Samples were washed, fixed, and analyzed by flow cytometry (BD Accuri C6 flow cytometer; BD Biosciences). Data are presented as percentage of phagocytosis, determined by the percentage of fluorescent-positive THP-1 cells. Percentage of phagocytosis of concentration-matched adjuvant control samples was subtracted as background from each test sample.

All statistical analyses used nonparametric tests. For analysis of T cell proliferation (Fig. 1) and cytokine production (Fig. 4) in immunized versus adjuvant control groups, the Kruskal–Wallis test followed by an uncorrected Dunn test was used. To determine boosting of Ag-specific IgG over time (Figs. 2, 6, 9), the Friedman test followed by Dunn test for multiple comparisons was used. In the comparison of two unpaired samples, the Mann–Whitney U test was used. To evaluate statistical differences in opsonophagocytosis assay (OPA) functionality of IgG induced by GLA-SE versus Alum as adjuvant, mean percentage of phagocytosis for all samples from each adjuvant group was combined and compared by Mann–Whitney U test (Table I). For all analyses, differences were considered significant with p values ≤0.05.

Table I.

IgG induced by GLA-SE–formulated vaccines demonstrates superior capacity to opsonize merozoites for phagocytosis

Parasite StrainIgG Concentration (mg/ml)AdjuvantCombined Phagocytosis (%) (Mean ± SD)p Value
PfNF54 0.1 GLA-SE 49.0 ± 14.1 <0.05 
  Alum 15.7 ± 4.3  
 0.05 GLA-SE 45.6 ± 15.6 <0.05 
  Alum 11.6 ± 4.0  
PfD10 0.1 GLA-SE 31.9 ± 7.8 <0.05 
  Alum 8.5 ± 2.2  
 0.05 GLA-SE 31.8 ± 6.6 <0.05 
  Alum 6.7 ± 1.7  
Parasite StrainIgG Concentration (mg/ml)AdjuvantCombined Phagocytosis (%) (Mean ± SD)p Value
PfNF54 0.1 GLA-SE 49.0 ± 14.1 <0.05 
  Alum 15.7 ± 4.3  
 0.05 GLA-SE 45.6 ± 15.6 <0.05 
  Alum 11.6 ± 4.0  
PfD10 0.1 GLA-SE 31.9 ± 7.8 <0.05 
  Alum 8.5 ± 2.2  
 0.05 GLA-SE 31.8 ± 6.6 <0.05 
  Alum 6.7 ± 1.7  

Statistical analysis of summary data (Fig. 7) comparing phagocytosis in the presence of IgG from GLA-SE–based vaccine formulations with phagocytosis in the presence of IgG from Alum-based vaccine formulations (Mann–Whitney U test; p < 0.05). PfNF54, P. falciparum strain NF54; PfD10, P. falciparum strain D10.

Groups of CB6F1/J mice were immunized with low (0.5 µg) and high (2.5 µg) doses of rPfMSP2, an admixture rPfMSP2 + rPfMSP8 or chimeric rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. Control groups were immunized with the PfMSP8 carrier or with adjuvant alone. Three weeks following the final boost, splenocytes were harvested, and the specificity of vaccine-induced T cell responses was determined by measuring Ag-specific proliferative responses in vitro via [3H]-thymidine incorporation. Splenocytes from animals immunized with PfMSP2/8 proliferated in response to stimulation with a peptide pool spanning the first 54 aa of mature rPfMSP2 (Fig. 1, left panel). These PfMSP2-specific responses were significantly higher than in adjuvant controls or in animals immunized with rPfMSP8 carrier (p < 0.05) and were maintained when dose and adjuvant varied. Splenocytes from mice immunized with PfMSP2 or rPfMSP2 + rPfMSP8 also proliferated upon stimulation with PfMSP2 peptides but with some variability with respect to vaccine dose and adjuvant formulation.

FIGURE 1.

PfMSP2- and PfMSP8-specific T cell responses are induced by immunization. Groups of CB6F1/J mice (n = 5) were immunized with high-dose (A, 2.5 µg) or low-dose (B, 0.5 µg) vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum or GLA-SE as adjuvant. Splenocytes (1 × 105/well) were stimulated in triplicate with a pool of four PfMSP2 peptides (12 μg/ml) or rPfMSP8 (10 μg/ml). Con A (1 μg/ml) stimulated or unstimulated wells served as positive and negative controls, respectively. Proliferation was quantified by [3H]-thymidine incorporation, and a stimulation index was calculated for each sample relative to unstimulated controls. Mean stimulation index (±SD) for each group is shown. *p < 0.05, relative to corresponding unstimulated controls (Kruskal–Wallis test followed by uncorrected Dunn test).

FIGURE 1.

PfMSP2- and PfMSP8-specific T cell responses are induced by immunization. Groups of CB6F1/J mice (n = 5) were immunized with high-dose (A, 2.5 µg) or low-dose (B, 0.5 µg) vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum or GLA-SE as adjuvant. Splenocytes (1 × 105/well) were stimulated in triplicate with a pool of four PfMSP2 peptides (12 μg/ml) or rPfMSP8 (10 μg/ml). Con A (1 μg/ml) stimulated or unstimulated wells served as positive and negative controls, respectively. Proliferation was quantified by [3H]-thymidine incorporation, and a stimulation index was calculated for each sample relative to unstimulated controls. Mean stimulation index (±SD) for each group is shown. *p < 0.05, relative to corresponding unstimulated controls (Kruskal–Wallis test followed by uncorrected Dunn test).

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Splenocytes from all mice immunized with formulations containing rPfMSP8 strongly proliferated in response to in vitro stimulation with rPfMSP8 (Fig. 1, middle panel; p < 0.05). PfMSP8-specific proliferation by splenocytes from mice immunized with PfMSP2 + PfMSP8 or PfMSP2/8 formulated with Alum were also consistently higher than the adjuvant control (p < 0.05). Splenocytes from mice immunized with PfMSP2 + PfMSP8 or PfMSP2/8 formulated with GLA-SE proliferated upon stimulation with PfMSP8 to varying degrees relative to adjuvant controls. This variation in PfMSP8-specific responses in these vaccine groups was balanced by the consistent PfMSP2-specific responses noted above. Together, these data demonstrate that immunization with an admixture rPfMSP2 + rPfMSP8 or chimeric rPfMSP2/8 elicits T cell responses specific for epitopes present in both the PfMSP2 component as well as the PfMSP8 carrier.

To quantify vaccine-induced, Ag-specific Ab responses, sera from immunized animals were analyzed by ELISA using rPfMSP2- or rPfMSP8-coated plates. In animals immunized with rPfMSP2, rPfMSP2 + rPfMSP8, or rPfMSP2/8 with Alum as adjuvant, modest levels of PfMSP2-specific Abs were detected following the primary immunization, which were significantly boosted to high levels by subsequent immunization (Fig. 2A, 2C, left; p < 0.05). Comparable anti-PfMSP2 Abs were detected regardless of Ag dose. PfMSP8-specific Ab titers measured for animals immunized with 2.5 μg of PfMSP8-containing vaccines with Alum as adjuvant were detected at high levels following primary immunization, which were significantly boosted by additional immunization (Fig. 2A, 2C, right; p < 0.05). Similar results were obtained for PfMSP8-specific IgG from animals immunized with the 0.5-μg dose. Of significance, there was no evidence of antigenic competition in the rPfMSP2 + rPfMSP8 admixture group at either high or low dose with Alum as adjuvant.

FIGURE 2.

Impact of Ag, dose, and adjuvant on vaccine-induced, Ag-specific IgG titers. Groups of CB6F1/J mice (n = 5) were immunized with high-dose (A and B, 2.5 µg) or low-dose (C and D, 0.5 µg) vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum (A and C) or GLA-SE (B and D) as adjuvant. IgG specific for rPfMSP2 and rPfMSP8 in sera collected following primary, secondary, and tertiary immunization were analyzed by ELISA. Ab titers were calculated as the reciprocal of the dilution yielding an A405 of 0.5. Mean Ag-specific IgG titer (± SD) for each group is shown. *p < 0.05, boosting of titer over time within an immunization group (Friedman test followed by Dunn test for multiple comparisons).

FIGURE 2.

Impact of Ag, dose, and adjuvant on vaccine-induced, Ag-specific IgG titers. Groups of CB6F1/J mice (n = 5) were immunized with high-dose (A and B, 2.5 µg) or low-dose (C and D, 0.5 µg) vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum (A and C) or GLA-SE (B and D) as adjuvant. IgG specific for rPfMSP2 and rPfMSP8 in sera collected following primary, secondary, and tertiary immunization were analyzed by ELISA. Ab titers were calculated as the reciprocal of the dilution yielding an A405 of 0.5. Mean Ag-specific IgG titer (± SD) for each group is shown. *p < 0.05, boosting of titer over time within an immunization group (Friedman test followed by Dunn test for multiple comparisons).

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Ag-specific Ab responses induced by vaccines formulated with GLA-SE as adjuvant were similarly compared (Fig. 2B, 2D). Following primary immunization with rPfMSP2-based vaccines at both high and low doses, high levels of PfMSP2-specific IgG were detected. These Ab titers were significantly boosted upon subsequent immunizations (Fig. 2B, 2D, left; p < 0.05). Similarly, upon immunization with high or low doses of rPfMSP8-containing vaccines, high levels of rPfMSP8-specific Ab were detected following primary immunization, which were also significantly boosted (Fig. 2B, 2D, right; p < 0.05). As with Alum-based formulations, there was no evidence of antigenic competition observed in the rPfMSP2 + rPfMSP8 admixture group at either dose. In all immunization groups, low levels of Ab specific for the His6-tag or linker domains common to vaccine rAgs under study were detected in assays evaluating rPfMSP2- or rPfMSP8-induced Abs against plate-bound rPfMSP8 or rPfMSP2, respectively. These Abs were <10% of total P. falciparum–specific Ab titer.

When considering merozoite neutralization and potential efficacy, vaccine-induced, PfMSP2-specific Ab titers are of primary interest. With most formulations, titers of PfMSP2-specific Abs were maximal after a prime and boost, with marginal or no increase following a third immunization (Fig. 2A–D). In direct comparison of PfMSP2-specific IgG titers induced by immunization with rPfMSP2 at high and low dose, there were no significant differences when Alum or GLA-SE was used as adjuvant (Fig. 3). However, with rPfMSP2/8, significantly higher anti-PfMSP2 Abs were elicited with GLA-SE as adjuvant, as compared with Alum. This was true at both the high (2.5 μg) and low (0.5 μg) doses (p < 0.05). In addition, immunization with 2.5 μg of rPfMSP2/8 with GLA-SE as adjuvant induced significantly higher titers of PfMSP2-specific Abs than immunization with 2.5 μg of rPfMSP2 formulated with GLA-SE (Fig. 3; p < 0.05). Overall, the highest average anti-PfMSP2–specific titers were achieved when the chimeric rPfMSP2/8 was formulated with GLA-SE (high and low dose).

FIGURE 3.

Final anti-PfMSP2 IgG titers induced by immunization with rPfMSP2 and rPfMSP2/8 as a function of dose and adjuvant. Direct comparison of final anti-PfMSP2 IgG titers induced by rPfMSP2 versus chimeric rPfMSP2/8 vaccines when formulated as indicated. Graphs depict the mean IgG titers (±SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

FIGURE 3.

Final anti-PfMSP2 IgG titers induced by immunization with rPfMSP2 and rPfMSP2/8 as a function of dose and adjuvant. Direct comparison of final anti-PfMSP2 IgG titers induced by rPfMSP2 versus chimeric rPfMSP2/8 vaccines when formulated as indicated. Graphs depict the mean IgG titers (±SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

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To assess cytokine production by Ag-specific T cells induced by PfMSP2-based vaccines formulated with Alum or GLA-SE as adjuvant, culture supernatants were collected following stimulation of splenocytes in vitro and analyzed for production of IL-5, IFN-γ, and TNF-α via Luminex multiplex assay. To measure cytokines produced by both PfMSP8- and PfMSP2-specific T cells, splenocytes were stimulated with rPfMSP2/8. Con A–stimulated and –unstimulated cells served as positive and negative controls, respectively. Splenocytes from mice immunized with high- and low-dose PfMSP2 vaccines formulated with Alum produced high levels of IL-5 in response to rPfMSP2/8 stimulation relative to adjuvant controls (Fig. 4A, 4B, left; p < 0.05). In contrast, splenocytes from animals immunized with PfMSP2-containing vaccines formulated with GLA-SE produced lower levels of IL-5 upon rPfMSP2/8 stimulation (Fig. 4A, 4B, left). In most cases, IL-5 levels from animals immunized with Alum-based formulations were significantly higher than those from animals immunized with GLA-SE–based formulations (p < 0.05). In one notable exception, comparable levels of IL-5 were produced by splenocytes from animals immunized with the rPfMSP8 carrier formulated with Alum and GLA-SE (Fig. 4A, 4B, left).

FIGURE 4.

Formulation of PfMSP2-based vaccines with GLA-SE shifts cytokine production and IgG subclass from a Th2 (Alum) to a Th1 (GLA-SE) phenotype. Groups of CB6F1/J mice (n = 5) were immunized with high- and low-dose subunit vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum or GLA-SE as adjuvant. (A and B) Splenocytes (5 × 105 per well) were stimulated in vitro with rPfMSP2/8 (10 μg/ml) in triplicate for 96 h. Following incubation, culture supernatants were collected and levels of IL-5, IFN-γ, and TNF-α were quantified via Luminex multiplex assay. Final concentrations (mean ± SD, picograms per milliliter) of each analyte were calculated, and corresponding unstimulated control values were subtracted as background. *p < 0.05, colored asterisks indicate statistically significant differences relative to the corresponding adjuvant control (Kruskal–Wallis test followed by uncorrected Dunn test); *p < 0.05, black bars with a black asterisk indicate statistically significant differences between immunization groups (Mann–Whitney U test). (C and D) IgG subclass profiles were determined by ELISA using rPfMSP2/8-coated plates. Bound Abs were detected by HRP-conjugated rabbit Abs specific for the indicated mouse IgG subclass. Graphs depict mean IgG subclass concentration (±SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

FIGURE 4.

Formulation of PfMSP2-based vaccines with GLA-SE shifts cytokine production and IgG subclass from a Th2 (Alum) to a Th1 (GLA-SE) phenotype. Groups of CB6F1/J mice (n = 5) were immunized with high- and low-dose subunit vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum or GLA-SE as adjuvant. (A and B) Splenocytes (5 × 105 per well) were stimulated in vitro with rPfMSP2/8 (10 μg/ml) in triplicate for 96 h. Following incubation, culture supernatants were collected and levels of IL-5, IFN-γ, and TNF-α were quantified via Luminex multiplex assay. Final concentrations (mean ± SD, picograms per milliliter) of each analyte were calculated, and corresponding unstimulated control values were subtracted as background. *p < 0.05, colored asterisks indicate statistically significant differences relative to the corresponding adjuvant control (Kruskal–Wallis test followed by uncorrected Dunn test); *p < 0.05, black bars with a black asterisk indicate statistically significant differences between immunization groups (Mann–Whitney U test). (C and D) IgG subclass profiles were determined by ELISA using rPfMSP2/8-coated plates. Bound Abs were detected by HRP-conjugated rabbit Abs specific for the indicated mouse IgG subclass. Graphs depict mean IgG subclass concentration (±SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

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For PfMSP2-based vaccines formulated with Alum, IFN-γ and TNF-α were detected at low levels upon stimulation of splenocytes with rPfMSP2/8. In marked contrast, PfMSP2-based vaccines formulated with GLA-SE elicited T cell responses characterized by high levels of IFN-γ production upon stimulation with rPfMSP2/8. These responses were significantly higher than responses from GLA-SE adjuvant control splenocytes and were significantly higher than IFN-γ levels produced by splenocytes from mice immunized with comparable vaccines formulated with Alum (Fig. 4A, 4B, middle; p < 0.05). IFN-γ production by splenocytes from animals immunized with high and low doses of PfMSP2 vaccines formulated with GLA-SE was comparable. Relative to IL-5 and IFN-γ, TNF-α production by splenocytes from immunized animals was generally low. However, TNF-α was produced at significantly higher levels in animals immunized with rPfMSP2 + rPfMSP8, rPfMSP2/8, or rPfMSP8 at the 2.5-μg dose relative to adjuvant controls. Furthermore, production of TNF-α by splenocytes from mice immunized with vaccines containing PfMSP8 (2.5 µg) formulated with GLA-SE was significantly higher in comparison with the same vaccines formulated with Alum (Fig. 4A, 4B, right; p < 0.05). At low vaccine doses, modest TNF-α production was only observed in splenocytes from rPfMSP2/8-immunized animals. Collectively, these data demonstrate that immunization with PfMSP2 vaccines formulated with GLA-SE as adjuvant shifted responses toward Th1-associated IFN-γ and TNF-α production as compared with Th2-associated IL-5 production, which characterized the profile elicited by immunization with PfMSP2 vaccines formulated with Alum.

The impact of differences in Th1/Th2 cytokine production on the subtype of IgG elicited by PfMSP2 vaccines formulated with GLA-SE versus Alum as adjuvant was measured. Terminal sera from immunized mice were analyzed for production of IgG1, IgG2a/c, IgG2b, and IgG3 subtypes via ELISA using rPfMSP2/8-coated plates. As shown in Fig. 4C, 4D, immunization with rPfMSP2, rPfMSP2 + rPfMSP8, rPfMSP2/8, or rPfMSP8 induced high titers of IgG1 Ab, regardless of adjuvant or dose. Consistent with the shift in cytokine profile toward a Th1 phenotype, immunization with vaccines formulated with GLA-SE resulted in a diversified profile of IgG subtypes, characterized by significantly higher levels of IgG2a/c, IgG2b, and IgG3 at the high dose, regardless of immunizing Ag (Fig. 4C). This was also observed with vaccines given at the low dose, with one exception; there was no significant difference in concentration of IgG2a/c induced by PfMSP2 alone when formulated with either GLA-SE or Alum (Fig. 4D). Together, these results demonstrate that vaccines formulated with GLA-SE result in a more diverse profile of IgG subtypes, including elevated cytophilic IgG2a/c desired for PfMSP2-based vaccines, as compared with vaccines formulated with Alum.

The influence of Ag, dose, and/or adjuvant on the avidity of vaccine-induced Abs was evaluated. Avidity indices were calculated based on resistance of Ag–Ab complexes to disruption by ammonium thiocyanate (Fig. 5). Immunization with chimeric rPfMSP2/8 tended to induce PfMSP2-specific Abs of slightly higher avidity relative to unfused rPfMSP2 but with a statistically significant difference noted with only the low dose + Alum formulation. This trend was not observed with PfMSP8-specific Abs. Comparison of high (2.5 µg)– versus low (0.5 µg)–dose formulations of the same vaccine Ag (PfMSP2, PfMSP8, or PfMSP2/8) formulated with the same adjuvant revealed no significant differences in the avidity of PfMSP2- or PfMSP8-specific Abs. Similarly, the avidity of PfMSP2- or PfMSP8-specific Abs induced by immunization with nonfused vaccines was not influenced by choice of Alum versus GLA-SE as adjuvant. Use of GLA-SE as adjuvant trended to induce higher avidity anti-PfMSP8 following immunization with chimeric PfMSP2/8 with a statistically significant difference noted with the 0.5-µg dose. Although minor differences were noted, it does not appear that variation in vaccine Ag, dose, or adjuvant had a noteworthy influence on the avidity of induced Abs.

FIGURE 5.

Avidity of anti-PfMSP2 IgG induced by immunization with rPfMSP2 and rPfMSP2/8 as a function of dose and adjuvant. Comparison of avidity indices of anti-PfMSP2 IgG titers induced by rPfMSP2 versus chimeric rPfMSP2/8 vaccines when formulated as indicated. Graphs depict the mean avidity index (±SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

FIGURE 5.

Avidity of anti-PfMSP2 IgG induced by immunization with rPfMSP2 and rPfMSP2/8 as a function of dose and adjuvant. Comparison of avidity indices of anti-PfMSP2 IgG titers induced by rPfMSP2 versus chimeric rPfMSP2/8 vaccines when formulated as indicated. Graphs depict the mean avidity index (±SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

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The impact of Ag and/or adjuvant selection on durability of PfMSP2-specific Ab responses was also determined for rPfMSP2-based Ags. Groups of mice were immunized with 0.5 μg of rPfMSP2 or rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. A booster immunization was given 4 wk after the primary immunization, followed by an 11-mo period of monitoring. Subsequently, a delayed second boost (tertiary immunization) was administered, and animals were sacrificed 3 wk later. Test bleeds were taken 3 wk after the primary immunization, 4 wk after the secondary immunization, and every 4–8 wk thereafter. Ag-specific responses were monitored at each time point via ELISA. As shown in Fig. 6A, high levels of PfMSP2-specific Abs were induced by immunization with rPfMSP2 when formulated with Alum or GLA-SE. PfMSP2-specific titers were boosted following secondary immunization, reaching peak levels that were maintained for up to 3 mo postboost. Anti-PfMSP2 titers induced by rPfMSP2 formulated with Alum never significantly dropped from peak levels; anti-PfMSP2 titers induced by Ag formulated with GLA-SE demonstrated a minor, but significant, drop from peak titers at 9 mo postboost (Fig. 6A, pound symbol [#]). Throughout the course of the experiment, PfMSP2-specific IgG titers induced by Alum or GLA-SE were not significantly different. Following the delayed second boost, final PfMSP2-specific Ab titers induced by rPfMSP2 with GLA-SE as adjuvant were significantly higher than titers induced by rPfMSP2 with Alum.

FIGURE 6.

Durability of PfMSP2-specific IgG titers differs based on Ag and adjuvant selection. Groups of CB6F1/J mice (n = 5) were immunized with 0.5 μg/dose (A) rPfMSP2 or (B) rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. Prime and boost immunizations were given 4 wk apart, followed by a delayed boost given 11 mo later. Immunizations are indicated by arrows on the x-axis. At each time point, PfMSP2-specific IgG titers were quantified by ELISA. High-titer pooled sera served as an assay standard, and titer was calculated as the reciprocal of the dilution yielding an A405 of 0.5. *p < 0.05, in anti-PfMSP2 titer between adjuvant groups at each time point (Mann–Whitney U test); #p < 0.05, significant drop from peak titers within an immunization group (Friedman test followed by Dunn test for multiple comparisons).

FIGURE 6.

Durability of PfMSP2-specific IgG titers differs based on Ag and adjuvant selection. Groups of CB6F1/J mice (n = 5) were immunized with 0.5 μg/dose (A) rPfMSP2 or (B) rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. Prime and boost immunizations were given 4 wk apart, followed by a delayed boost given 11 mo later. Immunizations are indicated by arrows on the x-axis. At each time point, PfMSP2-specific IgG titers were quantified by ELISA. High-titer pooled sera served as an assay standard, and titer was calculated as the reciprocal of the dilution yielding an A405 of 0.5. *p < 0.05, in anti-PfMSP2 titer between adjuvant groups at each time point (Mann–Whitney U test); #p < 0.05, significant drop from peak titers within an immunization group (Friedman test followed by Dunn test for multiple comparisons).

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In contrast to rPfMSP2-based formulations, there were significant differences in anti-PfMSP2 IgG titers induced by rPfMSP2/8-based vaccines (Fig. 6B). Initially, immunization with rPfMSP2/8 formulated with Alum induced significantly higher titers of anti-PfMSP2 Abs than rPfMSP2/8 formulated with GLA-SE. Upon boosting, however, anti-PfMSP2–specific IgG reached significantly higher levels when rPfMSP2/8 was formulated with GLA-SE versus Alum as adjuvant. PfMSP2-specific IgG titers reached peak levels following the secondary immunization with rPfMSP2/8 and were maintained at high levels for the 12-mo duration of the experiment. In contrast, a significant drop in PfMSP2-specific titers was observed by 9 mo postimmunization in animals immunized with rPfMSP2/8 formulated with Alum (Fig. 6B, pound symbol [#]). Furthermore, anti-PfMSP2 Ab titers induced by rPfMSP2/8 formulated with GLA-SE as adjuvant were significantly higher than titers induced by rPfMSP2/8 formulated with Alum throughout the duration of the experiment. Following the delayed tertiary immunization at 11 mo postboost, PfMSP2-specific titers were boosted by immunization with rPfMSP2/8 formulated with either adjuvant. However, final PfMSP2-specific IgG levels induced by rPfMSP2/8 formulated with GLA-SE remained significantly higher than those induced by rPfMSP2/8 formulated with Alum as adjuvant. Similarly, the titers of Ab to the PfMSP8 carrier induced by immunization with rPfMSP2/8 formulated with GLA-SE were higher than that induced by the comparable Alum formulation and persisted over time (Supplemental Fig. 1). These results indicate that immunization with rPfMSP2/8 formulated with GLA-SE induces high and durable levels of Ag-specific Abs, superior to levels induced by immunization with rPfMSP2 or with rPfMSP2/8 formulated with Alum as adjuvant.

Through various biochemical assays as well as electron microscopy, rPfMSP2 has been shown to form amyloid-like fibrils in vitro, with the proportion of fibrils changing over time (3943). Previous work demonstrated that fusion of rPfMSP2 to rPfMSP8 prevented the fibrillization of rPfMSP2 and that the structural differences between rPfMSP2 and rPfMSP2/8 influenced the specificity of Abs induced upon immunization of rabbits (30). To determine if induction of allele-specific and/or cross-reactive anti-PfMSP2 Abs was impacted by Ag structure, dose, or adjuvant upon immunization of mice, rPfMSP2 vaccine-induced Abs specific for rPfMSP2 (3D7) versus rPfMSP2 (FC27) were measured by ELISA (Fig. 7A). When rPfMSP2 or rPfMSP2/8 was formulated with Alum, there was no significant difference in PfMSP2 (3D7)– or PfMSP2 (FC27)–specific IgG titers, regardless of Ag dose. However, when GLA-SE was used as adjuvant, differences in PfMSP2 (3D7)– and PfMSP2 (FC27)–specific IgG titers were observed in an Ag-dependent manner. When rPfMSP2 was formulated with GLA-SE, there was no significant difference in anti-PfMSP2 (3D7) or anti-PfMSP2 (FC27) IgG at low or high dose. However, when chimeric rPfMSP2/8 was formulated with GLA-SE at either high or low dose, Ag-specific titers were significantly higher against the homologous PfMSP2 (3D7) Ag versus the heterologous rPfMSP2 (FC27) Ag (p < 0.05).

FIGURE 7.

The fibrillar state of rPfMSP2 Ags impacts epitope availability and specificity of vaccine-induced IgG. Groups of CB6F1/J mice (n = 5) were immunized with rPfMSP2 or rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. (A) Ag-specific IgG titers were determined by ELISA using rPfMSP2 (3D7)– and rPfMSP2 (FC27)–coated plates. Assays included high-titer pooled sera for normalization between plates and titer was calculated as the reciprocal of the dilution yielding an A405 of 0.5. *p < 0.05, between indicated groups (Mann–Whitney U test). (BE) For competitive binding assays, individual mouse sera diluted to yield an initial A405 of ∼1.2 were coincubated with increasing concentrations of rPfMSP2 (fibril containing) or rPfMSP2/8 (nonfibrillar) and residual binding of Ab to plate-bound immunizing Ag (B and C; rPfMSP2); (D) and (E) rPfMSP2/8 was measured by ELISA. Graphs depict percentage of residual binding (mean ± SD) at each concentration of soluble inhibitor. *p < 0.05, between rPfMSP2 and rPfMSP2/8 as inhibitor (Mann–Whitney U test).

FIGURE 7.

The fibrillar state of rPfMSP2 Ags impacts epitope availability and specificity of vaccine-induced IgG. Groups of CB6F1/J mice (n = 5) were immunized with rPfMSP2 or rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. (A) Ag-specific IgG titers were determined by ELISA using rPfMSP2 (3D7)– and rPfMSP2 (FC27)–coated plates. Assays included high-titer pooled sera for normalization between plates and titer was calculated as the reciprocal of the dilution yielding an A405 of 0.5. *p < 0.05, between indicated groups (Mann–Whitney U test). (BE) For competitive binding assays, individual mouse sera diluted to yield an initial A405 of ∼1.2 were coincubated with increasing concentrations of rPfMSP2 (fibril containing) or rPfMSP2/8 (nonfibrillar) and residual binding of Ab to plate-bound immunizing Ag (B and C; rPfMSP2); (D) and (E) rPfMSP2/8 was measured by ELISA. Graphs depict percentage of residual binding (mean ± SD) at each concentration of soluble inhibitor. *p < 0.05, between rPfMSP2 and rPfMSP2/8 as inhibitor (Mann–Whitney U test).

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To further analyze potential differences in the specificity of Abs induced by fibril-containing rPfMSP2 or nonfibrillar rPfMSP2/8, competitive binding ELISAs were conducted (Fig. 7B–E). For each rPfMSP2 or rPfMSP2/8 immunization group, individual mouse sera were tested by ELISA for the ability to bind to wells coated with immunizing Ag in the presence of increasing concentrations of soluble rPfMSP2 (fibril containing) or rPfMSP2/8 (nonfibrillar) as inhibitor. To eliminate detection of PfMSP8-specific Abs, all Ab + inhibitor mixtures included 1 μM of rPfMSP8.

In the first set of assays, sera from animals immunized with rPfMSP2 + Alum or rPfMSP2 + GLA-SE were analyzed. The ability of rPfMSP2 (3D7) versus rPfMSP2/8 to inhibit Ab binding to plate-bound rPfMSP2 (3D7) was measured. rPfMSP2 (3D7) inhibited binding of PfMSP2-specific Ab to plate-bound Ag, with a mean of 38 and 39% inhibition for the 2.5 μg Alum and GLA-SE groups (Fig. 7B), respectively, and a mean of 33 and 40% inhibition for the 0.5 μg Alum and GLA-SE groups (Fig. 7C), respectively, at the highest concentration of inhibitor tested. However, when these same sera from rPfMSP2-immunized animals were similarly evaluated with rPfMSP2/8 as inhibitor, a consistently higher level of inhibition was observed. At the highest concentration of inhibitor tested, rPfMSP2/8 inhibited binding of PfMSP2-specific Ab to plate-bound Ag, with a mean of 79 and 74% inhibition for the 2.5-μg Alum and GLA-SE groups (Fig. 7B), respectively, and a mean of 67 and 72% inhibition for the 0.5-μg Alum and GLA-SE groups, respectively (Fig. 7C; p < 0.05). These differences were observed at multiple concentrations of inhibitor and were consistent among all anti-PfMSP2 serum samples tested.

In the second set of assays, the ability of rPfMSP2 (3D7) versus rPfMSP2/8 to inhibit binding of rPfMSP2/8-induced Ab to plate-bound rPfMSP2/8 was measured. Similar to results for anti-PfMSP2 sera, rPfMSP2 (3D7) inhibited binding of rPfMSP2/8-specific Ab to plate-bound Ag with a mean of 52 and 42% inhibition for the 2.5 μg Alum and GLA-SE groups (Fig. 7D), respectively, and a mean of 44 and 38% inhibition for the 0.5 μg Alum and GLA-SE groups (Fig. 7E), respectively, at the highest concentration of inhibitor tested. When these same sera from rPfMSP2/8-immunized animals were similarly evaluated with rPfMSP2/8 as inhibitor, a consistently higher level of inhibition was again observed. At the highest concentration of inhibitor tested, rPfMSP2/8 inhibited binding of PfMSP2/8-specific Ab to plate-bound Ag with a mean of 85 and 75% inhibition for the 2.5-μg Alum and GLA-SE groups (Fig. 7D), respectively, and a mean of 80 and 78% inhibition for the 0.5-μg Alum and GLA-SE groups, respectively (Fig. 7E). Parallel to results from the first set of assays, the differences in inhibition of anti-PfMSP2/8 binding by rPfMSP2/8 were significantly higher than levels of inhibition achieved with rPfMSP2 (3D7) as inhibitor (Fig. 7D; p < 0.05). Together, these data confirm prior studies in rabbits (30) and indicate that rPfMSP2-associated fibrils can mask relevant B cell epitopes and that in the open conformation of chimeric rPfMSP2/8, these PfMSP2 epitopes are accessible for Ab recognition.

It is known that PfMSP2-specific Abs function through Fc-dependent mechanisms, in particular via opsonophagocytosis. To evaluate the functionality of Abs induced by immunization with rPfMSP2-based vaccines, an in vitro OPA was employed. Serum samples from groups of immunized mice were pooled, and purified IgG from each pool was tested for the ability to opsonize homologous (P. falciparum strain NF54 [PfNF54]) or heterologous (P. falciparum strain D10 [PfD10]) merozoites for phagocytosis by THP-1 monocytes. As shown in Fig. 8A, PfMSP2- or rPfMSP2/8-specific IgG induced by Alum-formulated vaccines (2.5-μg dose) mediated low levels of phagocytosis of homologous merozoites, with a maximum of ∼20% phagocytosis at 0.1 mg/ml IgG, with a slight reduction when tested at 0.05 mg/ml IgG. In stark contrast, PfMSP2- or rPfMSP2/8-specific IgG induced by GLA-SE–formulated vaccines (2.5-μg dose) enhanced phagocytosis of merozoites by 2.5–3-fold. With GLA-SE formulations, phagocytosis mediated by anti-PfMSP2 IgG reached 53% (0.1 mg/ml IgG) and 49% (0.05 mg/ml), whereas phagocytosis of merozoites following opsonization with anti-PfMSP2/8 IgG reached 65 and 62% at high and low concentrations, respectively (Fig. 8A).

FIGURE 8.

Immunization with rPfMSP2-based vaccines formulated with GLA-SE versus Alum enhances opsonophagocytosis of merozoites. The ability of rPfMSP2- or rPfMSP2/8- induced IgG to mediate phagocytosis of (A and B) homologous or (C and D) heterologous P. falciparum merozoites was measured using THP-1 monocytes. Serum samples from each immunization group were pooled prior to IgG purification. Merozoites were opsonized with 0.10 or 0.05 mg/ml of vaccine-induced IgG. Phagocytosis was tested in triplicate and quantified as the percentage of fluorescent THP-1 cells following coincubation with opsonized merozoites (percentage of phagocytosis, mean ± SD). Concentration-matched, adjuvant, control-opsonized samples were subtracted as background.

FIGURE 8.

Immunization with rPfMSP2-based vaccines formulated with GLA-SE versus Alum enhances opsonophagocytosis of merozoites. The ability of rPfMSP2- or rPfMSP2/8- induced IgG to mediate phagocytosis of (A and B) homologous or (C and D) heterologous P. falciparum merozoites was measured using THP-1 monocytes. Serum samples from each immunization group were pooled prior to IgG purification. Merozoites were opsonized with 0.10 or 0.05 mg/ml of vaccine-induced IgG. Phagocytosis was tested in triplicate and quantified as the percentage of fluorescent THP-1 cells following coincubation with opsonized merozoites (percentage of phagocytosis, mean ± SD). Concentration-matched, adjuvant, control-opsonized samples were subtracted as background.

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Parallel assays conducted with IgG from mice immunized with PfMSP2 vaccines at the 0.5-μg dose revealed similar patterns of phagocytosis, but at a slightly lower magnitude (Fig. 8B). Anti-PfMSP2 IgG or anti-PfMSP2/8 IgG from Alum-immunized animals were low and similar at both concentrations tested, with a maximum of 14% phagocytosis at 0.1 mg/m of IgG and <10% phagocytosis at 0.05 mg/ml of IgG (Fig. 8B). Again, IgG from the mice immunized with GLA-SE–formulated vaccines exhibited higher levels of phagocytosis at both IgG concentrations tested. In addition, there was a notable difference in the functionality of anti-PfMSP2 and anti-PfMSP2/8 IgG. With GLA-SE formulations, phagocytosis mediated by anti-PfMSP2 IgG reached 32% (0.1 mg/ml IgG) and 24% (0.05 mg/ml), whereas a higher, stable level of phagocytosis of merozoites was achieved following opsonization with anti-PfMSP2/8 IgG at 46% (0.1 mg/ml IgG) and 47% (0.05 mg/ml) (Fig. 8B).

The second set of experiments measured the ability of PfMSP2- and PfMSP2/8-specific IgG to opsonize heterologous merozoites (PfD10) for uptake by THP-1 cells. As shown in Fig. 8C, 8D, only low levels of phagocytosis (range 5–12%) were observed following opsonization with IgG from mice immunized with high or low dose of rPfMSP2 or rPfMSP2/8 formulated with Alum. As anticipated, higher levels of phagocytosis were observed following opsonization of PfD10 merozoites with IgG from animals immunized with GLA-SE–adjuvanted formulations. With the high-dose vaccine groups (Fig. 8C), phagocytosis in the presence of PfMSP2/8-specific IgG was 41%, but only 26% in the presence of PfMSP2-specific IgG, when tested at 0.1 mg/ml IgG. When assayed at 0.05 mg/ml, this difference was not apparent, as anti-PfMSP2 and anti-PfMSP2/8 IgG mediated comparable levels of PfD10 merozoite uptake with phagocytosis at 35–36%. With the low-dose GLA-SE vaccine formulations (Fig. 8D), anti-PfMSP2/8 IgG was highly active, with 36 and 35% phagocytosis observed at 0.1 and 0.05 mg/ml of IgG, respectively. Consistent but lower opsonophagocytosis activity was observed with anti-PfMSP2 IgG, with 25 and 22% phagocytosis at high and low IgG concentrations, respectively. To focus primarily on the effect of adjuvant, phagocytosis in the presence of IgG from all GLA-SE–based vaccine formulations was compared with phagocytosis in the presence of IgG from all Alum-based vaccine formulations. As shown in Table I, the use of GLA-SE as adjuvant significantly enhanced the functionality of vaccine-induced IgG in the OPA. Overall, the key finding from this analysis is that cytophilic Abs induced by PfMSP2-based vaccines formulated with GLA-SE exhibit greater ability to opsonize merozoites from homologous and heterologous strains of P. falciparum for uptake by THP-1 monocytes, relative to Abs induced by comparable Alum-based formulations.

Considering the data on immunogenicity and conformation, rPfMSP2/8 was selected for evaluation as a component of a multivalent malaria vaccine. Previous work demonstrated success with rPfMSP1/8 and rPfs25/8 as a bivalent formulation. In this study, groups of outbred CD1 mice of both sexes were immunized with a trivalent vaccine containing rPfMSP2/8 + rPfMSP1/8 + rPfs25/8 formulated with either Alum or GLA-SE as adjuvant. Immunogenicity was additionally compared with groups of mice immunized with monovalent PfMSP2/8 or bivalent rPfMSP1/8 + rPfs25/8 vaccines. Upon primary immunization with the rPfMSP2/8 monovalent vaccine or the trivalent vaccine formulated with Alum, mice produced detectable levels of PfMSP2-specific Abs that were significantly boosted to high levels following secondary and tertiary immunization (Fig. 9A, left panel). Similarly, anti-PfMSP119 Abs (Fig. 9A, middle panel) and anti-Pfs25 Abs (Fig. 9A, right) were induced after the primary immunization and were significantly boosted upon secondary and tertiary immunization with both bivalent and trivalent formulations. Similar results were obtained with respect to priming and boosting of PfMSP2-, PfMSP119-, and Pfs25-specific Abs following immunization with monovalent, bivalent, and trivalent vaccines formulated with GLA-SE as adjuvant (Fig. 9B). In addition, high titers of anti-PfMSP8 Abs were induced by all vaccine formulations (Supplemental Fig. 2).

FIGURE 9.

Multivalent formulations containing rPfMSP2/8, rPfMSP1/8, and rPfs25/8, irrespective of adjuvant, elicit strong Ab responses against each fusion partner with no indication of antigenic competition. Primary, secondary, and tertiary sera were collected from groups of outbred CD1 mice (five males and five females) immunized with monovalent, bivalent, or trivalent vaccines containing PfMSP2/8, PfMSP1/8, and Pfs25/8 (x-axis) formulated with (A) Alum or (B) GLA-SE as adjuvant. Ag-specific Ab titers were determined by ELISA using plates coated with rPfMSP2 (3D7) (left), rPfMSP119 (middle), or rPfs25 (right). Assays were standardized using high-titer pooled sera, and IgG titers were calculated as the reciprocal of the dilution yielding an A405 of 0.5. Graphs depict the mean IgG titers (±SD). *p < 0.05, boosting of titer over time within an immunization group (Friedman test followed by Dunn test for multiple comparisons). (C) Direct comparison of final anti-PfMSP2 (3D7), anti-PfMSP119 and anti-Pfs25 IgG titers (mean ± SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test. ns, not significant.

FIGURE 9.

Multivalent formulations containing rPfMSP2/8, rPfMSP1/8, and rPfs25/8, irrespective of adjuvant, elicit strong Ab responses against each fusion partner with no indication of antigenic competition. Primary, secondary, and tertiary sera were collected from groups of outbred CD1 mice (five males and five females) immunized with monovalent, bivalent, or trivalent vaccines containing PfMSP2/8, PfMSP1/8, and Pfs25/8 (x-axis) formulated with (A) Alum or (B) GLA-SE as adjuvant. Ag-specific Ab titers were determined by ELISA using plates coated with rPfMSP2 (3D7) (left), rPfMSP119 (middle), or rPfs25 (right). Assays were standardized using high-titer pooled sera, and IgG titers were calculated as the reciprocal of the dilution yielding an A405 of 0.5. Graphs depict the mean IgG titers (±SD). *p < 0.05, boosting of titer over time within an immunization group (Friedman test followed by Dunn test for multiple comparisons). (C) Direct comparison of final anti-PfMSP2 (3D7), anti-PfMSP119 and anti-Pfs25 IgG titers (mean ± SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test. ns, not significant.

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Titers of Ag-specific Abs following the tertiary immunizations were compared to evaluate antigenic competition and the influence of adjuvant when vaccines were formulated in combination. As shown in Fig. 9C and Supplemental Fig. 2, final titers of PfMSP2-, PfMSP119-, Pfs25-, and PfMSP8-specific IgG induced by immunization with the trivalent vaccines were comparable to the titers induced by the corresponding monovalent or bivalent vaccine with the same adjuvant. As such, there was no evidence of antigenic competition or impaired response to any vaccine component in the trivalent formulation. However, differences in the magnitude of the Ag-specific responses were impacted by adjuvant choice. As shown in Fig. 9C and Supplemental Fig. 2, final titers of PfMSP2-, PfMSP119-, and PfMSP8-specific IgG induced by immunization with vaccines formulated with GLA-SE as adjuvant were significantly higher than those induced by corresponding vaccines formulated with Alum as adjuvant (p < 0.05). For anti-Pfs25 responses (Fig. 9C, right), titers were also significantly higher in the group immunized with the bivalent vaccine formulated with GLA-SE versus Alum as adjuvant (p < 0.05). Final anti-Pfs25 titers induced by the trivalent vaccine formulated with GLA-SE and Alum were high and comparable. When comparing responses of male versus female mice in each vaccine group, some differences in Ag-specific IgG titers were noted with Alum-based formulations, but not with GLA-SE–based formulations (Supplemental Fig. 3). In addition, the avidity of anti-PfMSP2 IgG and anti-PfMSP8 IgG induced by monovalent versus trivalent vaccines formulated with Alum was comparable. Trivalent vaccines formulated with GLA-SE as adjuvant induced slightly higher avidity Abs relative to the corresponding monovalent PfMSP2/8 formulation (Supplemental Table I).

Finally, the impact of adjuvant choice on the profile of IgG subclasses induced upon immunization with multivalent vaccine formulations was confirmed. The subclasses of anti-PfMSP8 IgG induced by vaccines formulated with Alum and GLA-SE were analyzed by ELISA. As shown in Fig. 10, all vaccines formulated with Alum and GLA-SE induced high and comparable levels of IgG1. As observed with inbred mice (Fig. 4C, 4D), use of GLA-SE as adjuvant also drove production of Ag-specific Abs of the IgG2a/c, IgG2b, and IgG3 subclasses. The induction of Ag-specific IgG of these three subclasses was significantly higher in comparison with Alum-based formulations (p < 0.05). Combined, these results demonstrate that use of GLA-SE as adjuvant for this trivalent malaria vaccine formulation results in the induction of higher titers of Ag-specific IgG with an increased diversity of Ag-specific IgG subtypes in comparison with Alum-based formulations.

FIGURE 10.

Adjuvant shifts the profile of IgG subclasses induced upon immunization with multivalent vaccine formulations. Outbred CD1 mice (five males and five females) were immunized with monovalent, bivalent, or trivalent vaccines containing PfMSP2/8, PfMSP1/8, and Pfs25/8 (x-axis) formulated with Alum or GLA-SE as adjuvant. IgG subclass profiles of vaccine-induced Abs were determined by ELISA using rPfMSP8-coated plates. Bound Abs were detected by HRP-conjugated rabbit Abs specific for the indicated mouse IgG subclass. Graphs depict mean IgG subclass concentration (± SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

FIGURE 10.

Adjuvant shifts the profile of IgG subclasses induced upon immunization with multivalent vaccine formulations. Outbred CD1 mice (five males and five females) were immunized with monovalent, bivalent, or trivalent vaccines containing PfMSP2/8, PfMSP1/8, and Pfs25/8 (x-axis) formulated with Alum or GLA-SE as adjuvant. IgG subclass profiles of vaccine-induced Abs were determined by ELISA using rPfMSP8-coated plates. Bound Abs were detected by HRP-conjugated rabbit Abs specific for the indicated mouse IgG subclass. Graphs depict mean IgG subclass concentration (± SD). *p < 0.05, between groups overlined by horizontal bars (Mann–Whitney U test).

Close modal

For subunit malaria vaccine development, combining multiple vaccine components into a single formulation without loss of immunogenicity of individual candidate Ags is a major challenge (4446). In a recent phase IIb trial, for example, upon coadministration of a chimpanzee adenovirus 63-modified vaccinia virus ANKA (ChAd63-MVA)–vectored multiple epitope string with thrombospondin-related adhesion protein (ME-TRAP) with RTS,S/AS01B, both P. falciparum TRAP–specific T cell responses and P. falciparum circumsporozoite protein–specific B cell responses were impaired (46). Previously, we showed that PfMSP8 could serve as a Plasmodium-specific carrier protein for targeted Ags and/or domains to improve vaccine performance. Studies involving rPfMSP119 and rPfs25 demonstrated that their fusion to rPfMSP8 1) enhanced vaccine production and promoted proper folding of their structurally complex EGF-like domains; 2) enhanced immunogenicity of PfMSP119 and Pfs25 domains and the induction of high levels of parasite-neutralizing Abs; and importantly, 3) preserved immunogenicity of PfMSP119 and Pfs25 in multiantigen vaccine formulations (2023). Structurally, PfMSP2 presented a different challenge. We have reported that fusion of PfMSP2 to the PfMSP8 carrier prevented rPfMSP2 associated fibril formation, increasing accessibility of allele-specific and conserved B cell epitopes that induced functional Ab (30). The major objective of the study was 2-fold. We completed a series of comparative immunogenicity studies with PfMSP2 and PfMSP2/8 to assess the specificity of T and B cell responses along with the magnitude, durability, and functionality of vaccine-induced Abs. Based on these data, we introduced a PfMSP2 component into a multivalent formulation with PfMSP1/8 and Pfs25/8 to assess impact on immunogenicity when combined. We discuss our findings in four key areas.

First, the assessment of vaccine-induced T cell responses indicated that mice respond to epitopes present in both the PfMSP2 and PfMSP8 domains of the chimeric Ag. This is distinct from studies of PfMSP1/8 in which T cell help for Ab production depended exclusively on T cell recognition of PfMSP8-specific epitopes. Furthermore, T and B cell responses to the nonchimeric PfMSP2 were strong, and immunogenicity of PfMSP2 was not impaired in mice immunized with an admixture of PfMSP2 and PfMSP8. Again, this is in contrast to earlier studies with both PfMSP1 and Pfs25 constructs in which genetic fusion to PfMSP8 was necessary to avoid antigenic competition upon immunization with admixtures. This could be related to the strength of the PfMSP2-specific T cell response in inbred mice noted above. When considering immunization of human subjects with a PfMSP2-based vaccine, the great diversity of MHC class II alleles must not be overlooked. PfMSP2 is a relatively small protein of ∼28 kDa, and its sequence is characterized by a large, central repeat domain. As such, fusion of PfMSP2 to PfMSP8 may still be of value by providing an additional set of CD4+ T cell epitopes to promote broad immune recognition and strong B cell responses. We were encouraged by these results, as they suggested that PfMSP2 can be added to a multivalent vaccine without reduction in immunogenicity.

Second, considering the magnitude, IgG subclass profile, and functionality of Abs elicited by immunization with rPfMSP2-based vaccines, use of GLA-SE as adjuvant is preferred over Alum. Abs to PfMSP2 contribute to parasite clearance by opsonizing extracellular merozoites to enhance their uptake and killing by phagocytes. As such, cytophilic subclasses of IgG with the ability to engage Fc receptors on phagocytes are needed. Relative to Alum, use of GLA-SE as adjuvant shifted the cytokine responses from a Th2 (IL-5) to a Th1 (IFN-γ and TNF-α) profile, affecting class switching, increasing production of cytophilic IgG2a/c, and ultimately resulting in enhanced merozoite neutralization in the OPA. We previously reported a similar shift in IgG subclass profile with PfMSP1/8 and Pfs25/8 vaccines adjuvanted with Alum versus GLA-SE. However, for PfMSP119- and Pfs25-specific Abs, isotype is not critical for the functionality of Abs in the blood-stage growth inhibition assays or the standard membrane feeding assays. Nevertheless, GLA-SE was still the preferred adjuvant for PfMSP1/8 and Pfs25/8 vaccines considering the magnitude of the Ab responses induced. As such, GLA-SE is an excellent adjuvant choice for a multivalent combination of PfMSP2/8, PfMSP1/8, and Pfs25/8.

Third, the combination of PfMSP2/8 with GLA-SE as adjuvant elicited high and sustained production of anti-PfMSP2–specific Abs, better than those elicited by formulations containing Alum as adjuvant or nonchimeric PfMSP2. The durability of vaccine-induced Ab responses is influenced by many factors at the time of immunization including robust germinal center formation and strong T cell help to generate long-lived plasma cells and memory B cell populations. With PfMSP2/8 formulated with GLA-SE adjuvant, a prime and boost elicited a high level of anti-PfMSP2 Abs that persisted with no significant decline for a period of 11 mo. Again, this is a very important finding, as waning of malaria vaccine-induced protection has been an obstacle. Furthermore, we confirmed that with the PfMSP2/8 + GLA-SE formulation, the open, nonfibrillar structure of the PfMSP2 domain unmasks a subset of B cell epitopes. This was observable in competitive binding ELISAs in which a portion of the anti-PfMSP2–specific Abs induced by PfMSP2/8 could not bind to fibrillar rPfMSP2. Based on these and prior data (30), the enhanced functionality of Abs induced by immunization with PfMSP2/8 + GLA-SE in the OPA was likely due to a combination of higher Ab titers, recognition of a more diverse set of B cell epitopes, and class switching to production of cytophilic IgG.

Our findings with GLA-SE as adjuvant are consistent with recent reports in the literature. Human trials of a malaria vaccine candidate that aims to prevent pregnancy-associated malaria demonstrated that vaccines formulated with GLA-SE were highly immunogenic and contributed to induction of functional and cross-reactive Abs (47). Similar effects of GLA-SE on vaccine-induced immunity were observed for influenza and respiratory syncytial virus vaccines in mice and nonhuman primates, including improved durability (48, 49). These data are encouraging for downstream evaluation of a multivalent, multistage vaccine, as they validate the potency of GLA-SE in both nonhuman primates and human subjects.

Fourth, the above results from these comparative immunogenicity studies provided a clear rationale to pursue evaluation of rPfMSP2/8 in a multivalent vaccine targeting multiple parasite stages. The results were very encouraging. When tested in a trivalent formulation with PfMSP1/8 and Pfs25/8, the immunogenicity of PfMSP2/8 was not impaired. With both Alum and GLA-SE as adjuvant, the PfMSP2-, PfMSP119-, and Pfs25- specific Ab responses were induced and subsequently boosted to high levels, comparable to monovalent and bivalent control groups. Consistent with our initial comparison of low- and high-dose formulations, the increased concentration of PfMSP8 in the trivalent formulations did not reduce the avidity of anti-PfMSP2 or anti-PfMSP8 Abs elicited. Importantly, these immunogenicity studies were conducted in outbred mice of both sexes. Some differences were noted in the response of male versus female mice with Alum-based formulations. However, with the use of GLA-SE as adjuvant, sex was not a factor. These results address an important question for vaccine development, as sex differences have been reported for vaccines targeting bacterial and viral pathogens (5052) as well as the malaria vaccine RTS,S (53). Given the strength of the immune response to nonchimeric rPfMSP2, it may not be a surprise that PfMSP2-specific responses were maintained in the trivalent formulation. The flip side is equally important. In the trivalent formulation and in the presence of a strong PfMSP2/8 immunogen, the Ab responses to both PfMSP119 and Pfs25 were similarly maintained.

Use of PfMSP8 as a malaria vaccine carrier protein provides advantages for malaria vaccine development related to production and purification, maintenance of proper Ag conformation, and immunogenicity, all of which depend, to a degree, on the specific vaccine target. To date, we have had success with a PfMSP8-based approach with a trivalent formulation incorporating PfMSP1, PfMSP2, and Pfs25 components. Moving forward, studies will need to be expanded to assess additive and/or synergistic effects of anti-PfMSP119 and anti-PfMSP2 Abs induced by multivalent formulations in both blood-stage growth inhibition and OPAs, using a panel of P. falciparum strains. Further work is also needed to determine if additional components can be added without encountering problems with antigenic completion. Of high importance, we expect that moving preclinical vaccine testing from mice to nonhuman primates will give us a better indication of how a trivalent formulation containing PfMSP2/8, PfMSP1/8, and Pfs25/8 formulated with GLA-SE as adjuvant will perform in human subjects.

This work was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health Grant AI114292 (to J.M.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article:

Alum

aluminum hydroxide

GLA-SE

glucopyranosyl lipid adjuvant–stable emulsion

OPA

opsonophagocytosis assay

PfMSP

Plasmodium falciparum merozoite surface protein

PfMSP119

19-kDa fragment of PfMSP1.

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J.M.B. is an inventor listed on United States patent no. 7931908 entitled “Chimeric MSP-based malaria vaccine.” The other authors have no financial conflicts of interest.

Supplementary data