Inclusion of an optimized Plasmodium falciparum merozoite surface protein 2-based antigen in a trivalent, multi-stage malaria vaccine

Jacqueline S Eacret; Elizabeth M Parzych; Donna M Gonzales; James M Burns, Jr

doi:10.4049/jimmunol.2000927

. Author manuscript; available in PMC: 2022 Apr 15.

Published in final edited form as: J Immunol. 2021 Mar 31;206(8):1817–1831. doi: 10.4049/jimmunol.2000927

Inclusion of an optimized Plasmodium falciparum merozoite surface protein 2-based antigen in a trivalent, multi-stage malaria vaccine

Jacqueline S Eacret ^*, Elizabeth M Parzych ^*, Donna M Gonzales ^*, James M Burns Jr ^*,^†

PMCID: PMC8026686 NIHMSID: NIHMS1673293 PMID: 33789984

Abstract

Plasmodium falciparum merozoite surface protein 2 (PfMSP2) is a target of parasite neutralizing antibodies. Inclusion of rPfMSP2 as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we utilized the highly immunogenic PfMSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. Here, 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 dose and adjuvant. Immunization of mice with both rPfMSP2-based vaccines elicited high titer anti-PfMSP2 antibodies 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 antibody specificities induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. In contrast to Alum, formulation of rPfMSP2 vaccines with GLA-SE, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of antibodies to cytophilic IgG subclasses. The rPfMSP2/8 + GLA-SE formulation induced significantly higher antibody titers with superior durability and capacity to opsonize P. falciparum merozoites for phagocytosis. Immunization with a trivalent vaccine including PfMSP2/8, PfMSP1/8 and Pfs25/8 induced high levels of antibodies 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.

Introduction

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 (2–5). 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 antigen formulations encourage the evaluation of alternative approaches. Ultimately, it is expected that the development of a highly efficacious, multi-component 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 due to antigen 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 post-vaccination (7, 9). The approaches to improve immunogenicity and durability include testing of novel antigens and adjuvant platforms along with varying vaccine dose and immunization schedule (8, 10–15). These efforts still focus mainly on single antigen formulations (16–19). Concurrent immunization with multiple target antigens and/or multiple alleles of candidate antigens represents another significant opportunity to increase overall protective efficacy. Nevertheless, this presents the additional challenge of maintaining the immunogenicity of each component in a multi-antigen formulation.

With a focus on construct design, we established the use of P. falciparum merozoite surface protein 8 (PfMSP8) as a Plasmodium-specific carrier protein for leading blood-stage and sexual-stage vaccine candidate antigens. Genetic fusion of the 19 kDa fragment of P. falciparum merozoite surface protein 1 (PfMSP1₁₉) to the N-terminus of PfMSP8 resulted in production and purification of properly folded recombinant antigen in high yield. Immunization of mice and rabbits with the chimeric rPfMSP1/8 vaccine elicited high-titer, growth-inhibitory antibodies 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 antigens, PfMSP1₄₂ 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 to anti-PfMSP1₄₂ IgG (20). Importantly, immunization of non-human primates with rPfMSP1/8 was well tolerated, and induced high titer PfMSP1₁₉-specific antibodies that potently inhibited parasite growth in vitro (21).

For Pfs25, the 25 kDa sexual stage transmission-blocking candidate, fusion to the PfMSP8 carrier (rPfs25/8) significantly increased yield and quality of 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 antibodies 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 antibodies, prompted us to evaluate PfMSP8 as a fusion partner for another leading blood-stage vaccine candidate, P. falciparum merozoite surface protein 2 (PfMSP2).

Individuals living in endemic areas generate anti-PfMSP2 IgG1 and IgG3 antibodies that contribute to protection from malaria (24–26). Vaccine-induced PfMSP2-specific antibodies have also been shown to contribute to partial protection in one clinical trial (27–29). However, as with other blood-stage subunit vaccine candidates, attempts to advance a PfMSP2-based vaccine have faced several challenges, most notably are those related to the structure of recombinant PfMSP2 and the allele-specificity of protective anti-PfMSP2 antibodies. 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 antigens 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 antigen-specific IgG are maintained upon co-administration when formulated with Th1- or Th2- biasing adjuvants (23). Vaccines formulated with Alum as adjuvant typically drive Th2-associated antibody responses with IL-4 and IL-5 cytokine production by CD4+ T cells (31, 32). The adjuvant GLA-SE, composed of a synthetic TLR4 agonist, glucopyranosyl Lipid A (GLA) 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 (33–35). Here, we conducted comparative immunogenicity studies evaluating the influence of antigen, dose and adjuvant on PfMSP2 vaccine-induced immune responses with consideration of IgG isotype and functionality of elicited antibodies. Following down-selection, we evaluated the immunogenicity of a trivalent vaccine containing PfMSP2/8 in combination with PfMSP1/8 and Pfs25/8.

Materials and Methods

Mice and immunization protocols

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, Inc. (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 rGST-PfMSP1₁₉ have been previously reported (20, 22, 30). Four immunogenicity protocols were followed in which serum and/or splenocytes were collected three or four weeks following the final immunization.

(i). Experimental protocols 1 and 2

Groups of inbred CB6F1/J mice (n = 10) were immunized subcutaneously with rPfMSP2 (3D7), rPfMSP2/8, rPfMSP8, an admixture of rPfMSP2 (3D7) + rPfMSP8 or adjuvant alone. rPfMSP2 and rPfMSP2/8 antigens used for immunization were stored at −80^oC and administered within two years of purification. High (2.5 μg) or low (0.5 μg) antigen doses were formulated with either 2% Alhydrogel® (500 μg/dose; InvivoGen, San Diego, CA) or glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE, 5 μg/dose; Infectious Disease Research Institute, Seattle, WA) as adjuvant. Mice were immunized three times at 4-week intervals. Test bleeds were collected 3 weeks following the primary and secondary immunization. Terminal bleeds were conducted 4 weeks 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-week rest period, mice were boosted intraperitoneally, and spleens were harvested 3 weeks later for analysis of cellular responses.

(ii). Experimental protocol 3

To analyze the durability of vaccine-induced antibody responses, groups of mice (n = 5) were immunized subcutaneously 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 subcutaneously, boosted 4 weeks later and allowed to rest before receiving a delayed booster immunization 11 months following the secondary immunization. For antibody analysis, test bleeds were taken 3 weeks post-primary and 4 weeks post-secondary immunizations, and every four to eight weeks thereafter. Terminal sera were collected three weeks following the delayed third immunization.

(ii). Experiment protocol 4

Groups of outbred CD1 mice (n = 10; 5 males, 5 females) were immunized subcutaneously with a monovalent (rPfMSP2/8), bivalent (rPfMSP1/8, rPfs25/8) or trivalent (rPfMSP2/8, rPfMSP1/8, rPfs25/8) vaccine containing 2.5 μg of each antigen per dose and formulated with Alum or GLA-SE as adjuvant. Immunizations, test bleeds and terminal bleeds were conducted as in experiment protocol 1.

Analysis of cellular responses

(i). Generation of PfMSP2 overlapping peptides

rPfMSP2-associated fibrils have been shown to induce proliferation of naïve splenocytes in an antigen 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 (RP-HPLC purity >90%; GenScript, Piscataway, NJ), and reconstituted at a concentration of 1–2 mg/ml in dH₂0, dimethyl sulfoxide or 3% ammonia solution according to the manufacturer’s recommendation. To evaluate potential toxicity and/or non-specific 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-mercaptoethanol, 1X penicillin-streptomycin, 10% heat-inactivated Benchmark™ fetal bovine serum (FBS) and 10 μg/ml of Polymyxin B) to a concentration of 10 μg/ml and tested for the ability to stimulate proliferation of naïve mouse splenocytes in the presence or absence of Concanavalin A (Con A; 1 μg/ml), as described (30). Two peptides were eliminated due to toxicity and the remaining 23 peptides were grouped into 5 pools with 4–5 peptides/pool.

(ii). Antigen-specific splenocyte proliferation

Antigen-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/well and stimulated in triplicate. Splenocytes were stimulated with rPfMSP2 peptide pools (5 pools of 4–5 peptides, 3 μg/ml of each peptide/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 counts per minute of stimulated samples divided by the mean counts per minute of unstimulated samples.

(iii). Antigen-induced cytokine production

To quantify cytokine production by antigen-specific CD4+ T cells, splenocytes were isolated, plated at a concentration of 5 × 10⁵ cells/well and stimulated in vitro with rPfMSP2/8 (10 μg/ml) for 96 hours, as previously described (23). Following incubation, culture supernatants were collected and stored at −80^oC until analysis. Multiplex Luminex® assays (R&D Systems, Minneapolis, MN) for quantification of IL-5, IFNγ and TNFα 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 (pg/ml) of each analyte were calculated based on a standard curve, and corresponding unstimulated control values were subtracted as background.

Analysis of humoral responses

(i). Antigen-specific antibody titer

Antigen-specific antibody titers were determined by direct-binding ELISA as previously described (30). Briefly, two-fold serial dilutions of individual mouse sera were incubated (2 hrs., room temperature) on plates coated with recombinant antigen (0.25 μg/well). Bound antibodies were detected using horseradish peroxidase (HRP) conjugated rabbit-anti-mouse IgG (H+L) with ABTS [2,2’-azino-bis(ethylbenzothiazoline-6-sulfonic acid) diammonium salt] as substrate. A₄₀₅ values between 1.0 and 0.1 were plotted, and titer was calculated as reciprocal of the dilution yielding an A₄₀₅ of 0.5. High titer pooled sera included on each plate served as a standard for normalization of values across plates.

(ii). Antibody isotype profile

To determine IgG subtypes induced by immunization, terminal serum samples from experiments 1 and 4 were titered by ELISA on rPfMSP2/8- or rPfMSP8- coated plates, respectively, as described above. Bound antibodies were detected by HRP-conjugated rabbit antibodies specific for mouse IgG1, IgG2a, IgG2b, IgG2c and IgG3 (Southern Biotech, Inc., 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 ml (U/ml), where 1 U/ml is equivalent to 1 μg/ml of myeloma standard.

(iii). Avidity of antigen-specific antibodies

The avidity of vaccine-induced, antigen-specific antibodies was estimated by ELISA based on the resistance of antigen-antibody 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 approximately 1.2. Following binding of antigen-specific antibodies 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 minutes at room temperature. The plates were then washed and bound IgG detected as above. An avidity index for each serum was calculated as the concentration of ammonium thiocyanate required to reduce the binding of antigen-specific antibodies 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.

(iv). Competition ELISA

To analyze differences in epitope specificity of immunization-induced antibodies, 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^oC 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 antibodies, 1 μM of rPfMSP8 was included in all antigen-antibody mixtures. Samples were added to ELISA wells coated with rPfMSP2 (3D7) or rPfMSP2/8 coated wells, and binding of non-complexed antibodies was measured as described above. Percent residual binding for each sample was calculated as (A₄₀₅ of IgG in the presence of inhibitor/A₄₀₅ of IgG in the absence of inhibitor) x 100.

Opsonophagocytosis Assay

A pool of serum was generated from each group of mice immunized with rPfMSP2 or rPfMSP2/8 (2.5 μg, 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 (ATCC 30–2001) supplemented with 0.05 mM 2-mercaptoethanol and 10% FBS), and were plated at 30 μl/well in FBS-coated plates. For opsonization, merozoites were co-incubated with 0.1 mg/ml or 0.05 mg/ml of purified IgG for 1 hr. 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 co-incubated with THP-1 cells at a 5:1 ratio (merozoite:cells) for 10 minutes at 37^oC. Phagocytosis was stopped by the addition of cold PBS containing 3% FBS. Samples were washed, fixed and analyzed by flow cytometry (BD Biosciences Accuri™ C6 flow cytometer). Data are presented as percent phagocytosis, determined by the percentage of fluorescent-positive THP-1 cells. Percent phagocytosis of concentration-matched adjuvant control samples was subtracted as background from each test sample.

Statistical Analysis

All statistical analyses used nonparametric tests. For analysis of T cell proliferation (Figure 1) and cytokine production (Figure 4) in immunized versus adjuvant control groups, the Kruskal-Wallis test followed by an uncorrected Dunn’s test was used. To determine boosting of antigen-specific IgG over time (Figures 2, 6, 9), the Friedman test followed by Dunn’s test for multiple comparisons was used. In the comparison of two, unpaired samples, the Mann-Whitney test was used. To evaluate statistical differences in OPA functionality of IgG induced by GLA-SE versus Alum as adjuvant, mean percent phagocytosis for all samples from each adjuvant group were combined and compared by Mann-Whitney test (Table I). For all analyses, differences were considered significant with probability (p) values ≤ 0.05.

Figure 2. — Groups of CB6F1/J mice (n=5) were immunized with high dose (A, B 2.5 μg) or low dose (C, D 0.5 μg) vaccines containing rPfMSP2 and/or PfMSP8 (x-axis) formulated with Alum (A, C) or GLA-SE (B, D) as adjuvant. IgG specific for rPfMSP2 and rPfMSP8 in sera collected following primary, secondary and tertiary immunization were analyzed by ELISA. Antibody titers were calculated as the reciprocal of the dilution yielding an A₄₀₅ of 0.5. Mean antigen-specific IgG titer (± SD) for each group is shown. Asterisks indicate statistically significant boosting of titer over time within an immunization group (Friedman test followed by Dunn’s test for multiple comparisons; p<0.05).

Figure 6. — Groups of CB6F1/J mice (n=5) were immunized with 0.5 μg per dose of (A) rPfMSP2 or (B) rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. Prime and boost immunizations were given 4 weeks apart, followed by a delayed boost given 11 months 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. Asterisks indicate statistically significant differences in anti-PfMSP2 titer between adjuvant groups at each time point (Mann-Whitney test; p<0.05). Colored (#) indicate significant drop from peak titers within an immunization group (Friedman test followed by Dunn’s test for multiple comparisons; p<0.05).

Figure 9. — Primary, secondary and tertiary sera were collected from groups of outbred CD1 mice (5 male, 5 female) 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. Antigen-specific antibody 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). Asterisks indicate statistically significant boosting of titer over time within an immunization group (Friedman test followed by Dunn’s test for multiple comparisons; p<0.05). (C) Direct comparison of final anti-PfMSP2 (3D7), anti-PfMSP1₁₉ and anti-Pfs25 IgG titers (mean ± SD). Asterisks indicate statistically significant differences between groups over-lined by horizontal bars (Mann-Whitney test; p<0.05; ns, not significant).

Table I.

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

	IgG		Combined
Parasite Strain	Concentration	Adjuvant	% Phagocytosis	P value
	(mg/ml)		(mean ± SD)
		GLA-SE	49.0 ± 14.1
	0.1			<0.05
		Alum	15.7 ± 4.3
PfNF54
		GLA-SE	45.6 ± 15.6
	0.05			<0.05
		Alum	11.6 ± 4.0

		GLA-SE	31.9 ± 7.8
	0.1			<0.05
		Alum	8.5 ± 2.2
PfD10
		GLA-SE	31.8 ± 6.6
	0.05			<0.05
		Alum	6.7 ± 1.7

Open in a new tab

Statistical analysis of summary data (Figure 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 test; p<0.05).

Results

Immunization-induced T cells are specific for both PfMSP2 and PfMSP8 epitopes

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 antigen-specific proliferative responses in vitro via ³[H]-thymidine incorporation. Splenocytes from animals immunized with PfMSP2/8 proliferated in response to stimulation with a peptide pool spanning the first 54 amino acids of mature rPfMSP2 (Figure 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.

Splenocytes from all mice immunized with formulations containing rPfMSP8 strongly proliferated in response to in vitro stimulation with rPfMSP8 (Figure 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.

High levels of antigen-specific IgG are induced upon immunization with rPfMSP2-based vaccines

To quantify vaccine-induced, antigen-specific antibody 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 antibodies were detected following the primary immunization, which were significantly boosted to high levels by subsequent immunization (Figure 2A & C, left, p<0.05). Comparable anti-PfMSP2 antibodies were detected regardless of antigen dose. PfMSP8-specific antibody 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 (Figure 2A & C, 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.

Antigen-specific antibody responses induced by vaccines formulated with GLA-SE as adjuvant were similarly compared (Figure 2B & D). Following primary immunization with rPfMSP2-based vaccines at both high and low doses, high levels of PfMSP2-specific IgG were detected. These antibody titers were significantly boosted upon subsequent immunizations (Figure 2B & D, left; p<0.05). Similarly, upon immunization with high or low doses of rPfMSP8-containing vaccines, high levels of rPfMSP8-specific antibody were detected following primary immunization, which were also significantly boosted (Figure 2B & D, 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 antibody specific for the His₆-tag or linker domains common to recombinant vaccine antigens under study were detected in assays evaluating rPfMSP2- or rPfMSP8- induced antibodies against plate-bound rPfMSP8 or rPfMSP2, respectively. These antibodies were less than 10% of total P. falciparum-specific antibody titer.

When considering merozoite neutralization and potential efficacy, vaccine-induced PfMSP2-specific antibody titers are of primary interest. With most formulations, titers of PfMSP2-specific antibodies were maximal after a prime and boost, with marginal or no increase following a third immunization (Figure 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 (Figure 3). However, with rPfMSP2/8, significantly higher anti-PfMSP2 antibodies were elicited with GLA-SE as adjuvant, as compared to 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 antibodies than immunization with 2.5 μg of rPfMSP2 formulated with GLA-SE (Figure 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. — Direct comparison of final anti-PfMSP2 IgG titers induced by rPfMSP2 vs chimeric rPfMSP2/8 vaccines when formulated as indicated. Graphs depict the mean IgG titers (± SD). Asterisks indicate statistically significant differences between groups over-lined by horizontal bars (Mann-Whitney test; p<0.05).

Formulation of PfMSP2-based vaccines with GLA-SE versus Alum shifts cytokine production from a Th2 toward a Th1 profile

To assess cytokine production by antigen-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, IFNγ and TNFα 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 cells 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 (Figure 4A–B, 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 (Figure 4A–B, 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 (Figure 4A–B, left).

For PfMSP2-based vaccines formulated with Alum, IFNγ and TNFα 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 IFNγ production upon stimulation with rPfMSP2/8. These responses were significantly higher than responses from GLA-SE adjuvant control splenocytes and were significantly higher than IFNγ levels produced by splenocytes from mice immunized with comparable vaccines formulated with Alum (Figure 4A–B, middle, p<0.05). IFNγ 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 IFNγ, TNFα production by splenocytes from immunized animals was generally low. However, TNFα 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 TNFα by splenocytes from mice immunized with vaccines containing PfMSP8 (2.5 μg) formulated with GLA-SE was significantly higher in comparison to the same vaccines formulated with Alum (Figure 4A–B, right; p<0.05). At low vaccine doses, modest TNFα 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 IFNγ and TNFα production as compared to Th2-associated IL-5 production, which characterized the profile elicited by immunization with PfMSP2 vaccines formulated with Alum.

Use of GLA-SE as adjuvant drives class switching to cytophilic IgG subclasses

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 Figure 4C–D, immunization with rPfMSP2, rPfMSP2 + rPfMSP8, rPfMSP2/8 or rPfMSP8 induced high titers of IgG1 antibody, 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 antigen (Figure 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 (Figure 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 to vaccines formulated with Alum.

PfMSP2-based vaccine formulations induce antigen-specific antibodies of comparable avidity

The influence of antigen, dose, and/or adjuvant on the avidity of vaccine-induced antibodies was evaluated. Avidity indices were calculated based on resistance of antigen-antibody complexes to disruption by ammonium thiocyanate (Figure 5). Immunization with chimeric rPfMSP2/8 trended to induce PfMSP2-specific antibodies 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 antibodies. Comparison of high (2.5 μg) vs low (0.5 μg) dose formulations of the same vaccine antigen (PfMSP2, PfMSP8 or PfMSP2/8) formulated with the same adjuvant revealed no significant differences in the avidity of PfMSP2- or PfMSP8-specific antibodies. Similarly, the avidity of PfMSP2- or PfMSP8-specific antibodies induced by immunization with non-fused vaccines was not influenced by choice of Alum vs 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. While minor differences were noted, it does not appear that variation in vaccine antigen, dose or adjuvant had a noteworthy influence on the avidity of induced antibodies.

Figure 5. — Comparison of avidity indices of anti-PfMSP2 IgG titers induced by rPfMSP2 vs chimeric rPfMSP2/8 vaccines when formulated as indicated. Graphs depict the mean avidity index (± SD). Asterisks indicate statistically significant differences between groups over-lined by horizontal bars (Mann-Whitney test; p<0.05).

Fusion to PfMSP8 and formulation with GLA-SE promotes high and durable PfMSP2-specific antibody responses

The impact of antigen and/or adjuvant selection on durability of PfMSP2-specific antibody responses was also determined for rPfMSP2-based antigens. 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 weeks after the primary immunization followed by an 11-month period of monitoring. Subsequently, a delayed second boost (tertiary immunization) was administered and animals were sacrificed 3 weeks later. Test bleeds were taken three weeks after the primary immunization, four weeks after the secondary immunization, and every 4–8 weeks thereafter. Antigen-specific responses were monitored at each time point via ELISA. As shown in Figure 6A, high levels of PfMSP2-specific antibodies 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 three months post-boost. Anti-PfMSP2 titers induced by rPfMSP2 formulated with Alum never significantly dropped from peak levels; anti-PfMSP2 titers induced by antigen formulated with GLA-SE demonstrated a minor, but significant drop from peak titers at 9 months post-boost (Figure 6A, #). 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 antibody titers induced by rPfMSP2 with GLA-SE as adjuvant were significantly higher than titers induced by rPfMSP2 with Alum.

In contrast to rPfMSP2-based formulations, there were significant differences in anti-PfMSP2 IgG titers induced by rPfMSP2/8-based vaccines (Figure 6B). Initially, immunization with rPfMSP2/8 formulated with Alum induced significantly higher titers of anti-PfMSP2 antibodies 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-month duration of the experiment. In contrast, a significant drop in PfMSP2-specific titers was observed by 9 months post-immunization in animals immunized with rPfMSP2/8 formulated with Alum (Figure 6B, #). Furthermore, anti-PfMSP2 antibody 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 months post-boost, 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 antibody 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 Figure 1). These results indicate that immunization with rPfMSP2/8 formulated with GLA-SE induces high and durable levels of antigen-specific antibodies, superior to levels induced by immunization with rPfMSP2 or with rPfMSP2/8 formulated with Alum as adjuvant.

Structural differences between rPfMSP2 and rPfMSP2/8 impact antibody epitope specificity

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 (39–43). 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 antibodies induced upon immunization of rabbits (30). To determine if induction of allele-specific and/or cross-reactive anti-PfMSP2 antibodies was impacted by antigen structure, dose or adjuvant upon immunization of mice, rPfMSP2 vaccine-induced antibodies specific for rPfMSP2 (3D7) versus rPfMSP2 (FC27) were measured by ELISA (Figure 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 antigen dose. However, when GLA-SE was used as adjuvant, differences in PfMSP2 (3D7)- and PfMSP2 (FC27)- specific IgG titers were observed in an antigen-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, antigen-specific titers were significantly higher against the homologous PfMSP2 (3D7) antigen versus the heterologous rPfMSP2 (FC27) antigen (p<0.05).

Figure 7. — Groups of CB6F1/J mice (n=5) were immunized with rPfMSP2 or rPfMSP2/8 formulated with either Alum or GLA-SE as adjuvant. (A) Antigen-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. Asterisks indicate statistically significant differences between indicated groups (Mann-Whitney test; p<0.05). (B-E) For competitive binding assays, individual mouse sera diluted to yield an initial A₄₀₅ of ~1.2 were co-incubated with increasing concentrations of rPfMSP2 (fibril containing) or rPfMSP2/8 (non-fibrillar) and residual binding of antibody to plate-bound immunizing antigen (B, C – rPfMSP2); D, E – rPfMSP2/8) was measured by ELISA. Graphs depict percent residual binding (mean ± SD) at each concentration of soluble inhibitor. Asterisks indicated statistically significant differences between rPfMSP2 and rPfMSP2/8 as inhibitor (Mann-Whitney test; p<0.05).

To further analyze potential differences in the specificity of antibodies induced by fibril-containing rPfMSP2 or non-fibrillar rPfMSP2/8, competitive binding ELISAs were conducted (Figure 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 antigen in the presence of increasing concentrations of soluble rPfMSP2 (fibril containing) or rPfMSP2/8 (non-fibrillar) as inhibitor. To eliminate detection of PfMSP8-specific antibodies, all antibody + inhibitor cocktails 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 antibody binding to plate-bound rPfMSP2 (3D7) was measured. rPfMSP2 (3D7) inhibited binding of PfMSP2-specific antibody to plate-bound antigen, with a mean of 38% and 39% inhibition for the 2.5 μg Alum and GLA-SE groups (Figure 7B), respectively, and a mean of 33% and 40% inhibition for the 0.5 μg Alum and GLA-SE groups (Figure 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 antibody to plate-bound antigen, with a mean of 79% and 74% inhibition for the 2.5 μg Alum and GLA-SE groups (Figure 7B), respectively, and a mean of 67% and 72% inhibition for the 0.5 μg Alum and GLA-SE groups, respectively (Figure 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 antibody to plate-bound rPfMSP2/8 was measured. Similar to results for anti-PfMSP2 sera, rPfMSP2 (3D7) inhibited binding of rPfMSP2/8-specific antibody to plate-bound antigen with a mean of 52% and 42% inhibition for the 2.5 μg Alum and GLA-SE groups (Figure 7D), respectively, and a mean of 44% and 38% inhibition for the 0.5 μg Alum and GLA-SE groups (Figure 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 antibody to plate-bound antigen with a mean of 85% and 75% inhibition for the 2.5 μg Alum and GLA-SE groups (Figure 7D), respectively, and a mean of 80% and 78% inhibition for the 0.5 μg Alum and GLA-SE groups, respectively (Figure 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 (Figure 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 antibody recognition.

Cytophilic IgG generated by immunization with PfMSP2 vaccines formulated with GLA-SE demonstrates superior merozoite neutralizing activity

It is known that PfMSP2-specific antibodies function through Fc-dependent mechanisms, in particular, via opsonophagocytosis. To evaluate the functionality of antibodies induced by immunization with rPfMSP2-based vaccines, an in vitro opsonophagocytosis assay (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 (PfNF54) or heterologous (PfD10) merozoites for phagocytosis by THP-1 monocytes. As shown in Figure 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 of IgG, with a slight reduction when tested at 0.05 mg/ml of 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), while phagocytosis of merozoites following opsonization with anti-PfMSP2/8 IgG reached 65% and 62% at high and low concentrations, respectively (Figure 8A).

Figure 8. — The ability of rPfMSP2- or rPfMSP2/8- induced IgG to mediate phagocytosis of (A, B) homologous or (C, 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 mg/ml or 0.05 mg/ml of vaccine-induced IgG. Phagocytosis was tested in triplicate and quantified as the percent fluorescent THP-1 cells following co-incubation with opsonized merozoites (% phagocytosis, mean ± SD). Concentration-matched, adjuvant control-opsonized samples were subtracted as background.

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 (Figure 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 (Figure 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), while 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) (Figure 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 Figure 8C–D, 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 (Figure 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 (Figure 8D), anti-PfMSP2/8 IgG was highly active, with 36% and 35% phagocytosis observed at 0.1 mg/ml 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 antibodies 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 antibodies induced by comparable Alum-based formulations.

Induction of high levels of PfMSP2-specific antibodies is maintained following immunization with multivalent vaccine 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. Here, 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 to 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 antibodies that were significantly boosted to high levels following secondary and tertiary immunization (Figure 9A, left panel). Similarly, anti-PfMSP1₁₉ antibodies (Figure 9A, middle panel) and anti-Pfs25 antibodies (Figure 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 antibodies following immunization with monovalent, bivalent and trivalent vaccines formulated with GLA-SE as adjuvant (Figure 9B). In addition, high titers of anti-PfMSP8 antibodies were induced by all vaccine formulations (Supplemental Figure 2).

Titers of antigen-specific antibodies following the tertiary immunizations were compared to evaluate antigenic competition and the influence of adjuvant when vaccines were formulated in combination. As shown in Figure 9C and Supplemental Figure 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 antigen-specific responses were impacted by adjuvant choice. As shown in Figure 9C and Supplemental Figure 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 (Figure 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 antigen-specific IgG titers were noted with Alum-based formulations, but not with GLA-SE based formulations (Supplemental Figure 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 antibodies 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 Figure 10, all vaccines formulated with Alum and GLA-SE induced high and comparable levels of IgG1. As observed with inbred mice (Figure 4C–D), use of GLA-SE as adjuvant also drove production of antigen-specific antibodies of the IgG2a/c, IgG2b and IgG3 subclass. The induction of antigen-specific IgG of these three subclasses was significantly higher in comparison to 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 antigen-specific IgG with an increased diversity of antigen-specific IgG subtypes in comparison to Alum-based formulations.

Figure 10. — Outbred CD1 mice (5 male, 5 female) 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 antibodies were determined by ELISA using rPfMSP8-coated plates. Bound antibodies were detected by HRP-conjugated rabbit antibodies specific for the indicated mouse IgG subclass. Graphs depict mean IgG subclass concentration (± SD). Asterisks indicate statistically significant differences between groups over-lined by horizontal bars (Mann-Whitney test; p<0.05).

Discussion

For subunit malaria vaccine development, combining multiple vaccine components into a single formulation without loss of immunogenicity of individual candidate antigens is a major challenge (44–46). In a recent Phase IIb trial for example, upon co-administration of a ChAd63-MVA-vectored ME-TRAP with RTS,S/AS01B, both PfTRAP-specific T cell responses and PfCSP-specific B cell responses were impaired (46). Previously, we showed that PfMSP8 could serve as a Plasmodium-specific carrier protein for targeted antigens and/or domains to improve vaccine performance. Studies involving rPfMSP1₁₉ and rPfs25, demonstrated that their fusion to rPfMSP8 i) enhanced vaccine production and promoted proper folding of their structurally complex EGF-like domains; ii) enhanced immunogenicity of PfMSP1₁₉ and Pfs25 domains and the induction of high levels of parasite neutralizing antibodies; and importantly, iii) preserved immunogenicity of PfMSP1₁₉ and Pfs25 in multi-antigen vaccine formulations (20–23). 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 antibody (30). The major objective of the study was two-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 antibodies. 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 4 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 antigen. This is distinct from studies of PfMSP1/8 where T cell help for antibody production depended exclusively on T cell recognition of PfMSP8-specific epitopes. Furthermore, T and B cell responses to the non-chimeric 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, where 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 antibodies elicited by immunization with rPfMSP2-based vaccines, use of GLA-SE as adjuvant is preferred over Alum. Antibodies 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 FcRs 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 (IFNγ, TNFα) 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 antibodies, isotype is not critical for the functionality of antibodies 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 antibody 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 antibodies, better than those elicited by formulations containing Alum as adjuvant or non-chimeric PfMSP2. The durability of vaccine-induced antibody responses is influenced by many factors at the time of immunization including robust germinal center formation and strong T cell help in order 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 antibodies that persisted with no significant decline for a period of 11 months. 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, non-fibrillar structure of the PfMSP2 domain unmasks a subset of B cell epitopes. This was observable in competitive binding ELISAs where a portion of the anti-PfMSP2-specific antibodies induced by PfMSP2/8 could not bind to fibrillar rPfMSP2. Based on these and prior data (30), the enhanced functionality of antibodies induced by immunization with PfMSP2/8 + GLA-SE in the OPA was likely due to a combination of higher antibody 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 antibodies (47). Similar effects of GLA-SE on vaccine-induced immunity were observed for influenza and RSV vaccines in mice and non-human primates, including improved durability (48, 49). These data are encouraging for downstream evaluation of a multivalent, multi-stage vaccine, as they validate the potency of GLA-SE in both non-human 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 antibody 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 antibodies 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 (50–52) as well as the malaria vaccine RTS,S (53). Given the strength of the immune response to non-chimeric 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 antibody 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 antigen 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 antibodies induced by multivalent formulations in both blood-stage growth inhibition and opsonophagocytosis assays, utilizing 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 non-human 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.

Supplementary Material

NIHMS1673293-supplement-1.pdf^{(450.5KB, pdf)}

Key Points.

Construction of a PfMSP2/8 immunogen improved vaccine potential.
GLA-SE as adjuvant increased titer and functionality of vaccine-induced IgG.
Individual components of a trivalent malaria vaccine retained immunogenicity.

Acknowledgments

1. Grant support: This work was supported by NIH-NIAID Grant AI114292 (JMB). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

4. Non-standard abbreviations:

MSP: merozoite surface protein
GLA-SE: glucopyranosyl lipid adjuvant-stable emulsion
Pf: Plasmodium falciparum
OPA: opsonophagocytosis assay

References

1.Ghehreyesus T 2019. World Malaria Report 2019. World Health Organization, Geneva. [Google Scholar]
2.RTS SCTP 2015. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 386: 31–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.White MT, Verity R, Griffin JT, Asante KP, Owusu-Agyei S, Greenwood B, Drakeley C, Gesase S, Lusingu J, Ansong D, Adjei S, Agbenyega T, Ogutu B, Otieno L, Otieno W, Agnandji ST, Lell B, Kremsner P, Hoffman I, Martinson F, Kamthunzu P, Tinto H, Valea I, Sorgho H, Oneko M, Otieno K, Hamel MJ, Salim N, Mtoro A, Abdulla S, Aide P, Sacarlal J, Aponte JJ, Njuguna P, Marsh K, Bejon P, Riley EM, and Ghani AC 2015. Immunogenicity of the RTS,S/AS01 malaria vaccine and implications for duration of vaccine efficacy: secondary analysis of data from a phase 3 randomised controlled trial. Lancet Infect Dis 15: 1450–1458. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Ubillos I, Ayestaran A, Nhabomba AJ, Dosoo D, Vidal M, Jimenez A, Jairoce C, Sanz H, Aguilar R, Williams NA, Diez-Padrisa N, Mpina M, Sorgho H, Agnandji ST, Kariuki S, Mordmuller B, Daubenberger C, Asante KP, Owusu-Agyei S, Sacarlal J, Aide P, Aponte JJ, Dutta S, Gyan B, Campo JJ, Valim C, Moncunill G, and Dobano C 2018. Baseline exposure, antibody subclass, and hepatitis B response differentially affect malaria protective immunity following RTS,S/AS01E vaccination in African children. BMC Med 16: 197. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Gosling R, and von Seidlein L 2016. The Future of the RTS,S/AS01 Malaria Vaccine: An Alternative Development Plan. PLoS Med 13: e1001994. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Olotu A, Fegan G, Wambua J, Nyangweso G, Awuondo KO, Leach A, Lievens M, Leboulleux D, Njuguna P, Peshu N, Marsh K, and Bejon P 2013. Four-year efficacy of RTS,S/AS01E and its interaction with malaria exposure. N Engl J Med 368: 1111–1120. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.RTS SCTP 2014. Efficacy and safety of the RTS,S/AS01 malaria vaccine during 18 months after vaccination: a phase 3 randomized, controlled trial in children and young infants at 11 African sites. PLoS Med 11: e1001685. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Regules JA, Cicatelli SB, Bennett JW, Paolino KM, Twomey PS, Moon JE, Kathcart AK, Hauns KD, Komisar JL, Qabar AN, Davidson SA, Dutta S, Griffith ME, Magee CD, Wojnarski M, Livezey JR, Kress AT, Waterman PE, Jongert E, Wille-Reece U, Volkmuth W, Emerling D, Robinson WH, Lievens M, Morelle D, Lee CK, Yassin-Rajkumar B, Weltzin R, Cohen J, Paris RM, Waters NC, Birkett AJ, Kaslow DC, Ballou WR, Ockenhouse CF, and Vekemans J 2016. Fractional Third and Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria Parasite Infection and Immunogenicity Study. J Infect Dis 214: 762–771. [DOI] [PubMed] [Google Scholar]
9.Laurens MB, Thera MA, Coulibaly D, Ouattara A, Kone AK, Guindo AB, Traore K, Traore I, Kouriba B, Diallo DA, Diarra I, Daou M, Dolo A, Tolo Y, Sissoko MS, Niangaly A, Sissoko M, Takala-Harrison S, Lyke KE, Wu Y, Blackwelder WC, Godeaux O, Vekemans J, Dubois MC, Ballou WR, Cohen J, Dube T, Soisson L, Diggs CL, House B, Bennett JW, Lanar DE, Dutta S, Heppner DG, Plowe CV, and Doumbo OK 2013. Extended safety, immunogenicity and efficacy of a blood-stage malaria vaccine in malian children: 24-month follow-up of a randomized, double-blinded phase 2 trial. PLoS One 8: e79323. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Ouattara A, Barry AE, Dutta S, Remarque EJ, Beeson JG, and Plowe CV 2015. Designing malaria vaccines to circumvent antigen variability. Vaccine 33: 7506–7512. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Barry AE, and Arnott A 2014. Strategies for designing and monitoring malaria vaccines targeting diverse antigens. Front Immunol 5: 359. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Draper SJ, Sack BK, King CR, Nielsen CM, Rayner JC, Higgins MK, Long CA, and Seder RA 2018. Malaria Vaccines: Recent Advances and New Horizons. Cell Host Microbe 24: 43–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Asante KP, Abdulla S, Agnandji S, Lyimo J, Vekemans J, Soulanoudjingar S, Owusu R, Shomari M, Leach A, Jongert E, Salim N, Fernandes JF, Dosoo D, Chikawe M, Issifou S, Osei-Kwakye K, Lievens M, Paricek M, Möller T, Apanga S, Mwangoka G, Dubois MC, Madi T, Kwara E, Minja R, Hounkpatin AB, Boahen O, Kayan K, Adjei G, Chandramohan D, Carter T, Vansadia P, Sillman M, Savarese B, Loucq C, Lapierre D, Greenwood B, Cohen J, Kremsner P, Owusu-Agyei S, Tanner M, and Lell B 2011. Safety and efficacy of the RTS,S/AS01E candidate malaria vaccine given with expanded-programme-on-immunisation vaccines: 19 month follow-up of a randomised, open-label, phase 2 trial. Lancet Infect Dis 11: 741–749. [DOI] [PubMed] [Google Scholar]
14.Collins KA, Snaith R, Cottingham MG, Gilbert SC, and Hill AVS 2017. Enhancing protective immunity to malaria with a highly immunogenic virus-like particle vaccine. Sci Rep 7: 46621. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Genito CJ, Beck Z, Phares TW, Kalle F, Limbach KJ, Stefaniak ME, Patterson NB, Bergmann-Leitner ES, Waters NC, Matyas GR, Alving CR, and Dutta S 2017. Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013. Vaccine 35: 3865–3874. [DOI] [PubMed] [Google Scholar]
16.Thera MA, Doumbo OK, Coulibaly D, Laurens MB, Ouattara A, Kone AK, Guindo AB, Traore K, Traore I, Kouriba B, Diallo DA, Diarra I, Daou M, Dolo A, Tolo Y, Sissoko MS, Niangaly A, Sissoko M, Takala-Harrison S, Lyke KE, Wu Y, Blackwelder WC, Godeaux O, Vekemans J, Dubois MC, Ballou WR, Cohen J, Thompson D, Dube T, Soisson L, Diggs CL, House B, Lanar DE, Dutta S, Heppner DG, and Plowe CV 2011. A field trial to assess a blood-stage malaria vaccine. N Engl J Med 365: 1004–1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Otsyula N, Angov E, Bergmann-Leitner E, Koech M, Khan F, Bennett J, Otieno L, Cummings J, Andagalu B, Tosh D, Waitumbi J, Richie N, Shi M, Miller L, Otieno W, Otieno GA, Ware L, House B, Godeaux O, Dubois MC, Ogutu B, Ballou WR, Soisson L, Diggs C, Cohen J, Polhemus M, Heppner DG, Ockenhouse CF, and Spring MD 2013. Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP1(42)) administered intramuscularly with adjuvant system AS01. Malar J 12: 29. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Thera MA, Doumbo OK, Coulibaly D, Diallo DA, Sagara I, Dicko A, Diemert DJ, Heppner DG, Stewart VA, Angov E, Soisson L, Leach A, Tucker K, Lyke KE, Plowe CV, and Group MFW 2006. Safety and allele-specific immunogenicity of a malaria vaccine in Malian adults: results of a phase I randomized trial. PLoS Clin Trials 1: e34. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Ogutu BR, Apollo OJ, McKinney D, Okoth W, Siangla J, Dubovsky F, Tucker K, Waitumbi JN, Diggs C, Wittes J, Malkin E, Leach A, Soisson LA, Milman JB, Otieno L, Holland CA, Polhemus M, Remich SA, Ockenhouse CF, Cohen J, Ballou WR, Martin SK, Angov E, Stewart VA, Lyon JA, Heppner DG, Withers MR, and M.-M. V. W. Group. 2009. Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya. PLoS One 4: e4708. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Alaro JR, Partridge A, Miura K, Diouf A, Lopez AM, Angov E, Long CA, and Burns JM 2013. A chimeric Plasmodium falciparum merozoite surface protein vaccine induces high titers of parasite growth inhibitory antibodies. Infect Immun 81: 3843–3854. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Burns JM, Miura K, Sullivan J, Long CA, and Barnwell JW 2016. Immunogenicity of a chimeric Plasmodium falciparum merozoite surface protein vaccine in Aotus monkeys. Malar J 15: 159. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Parzych EM, Miura K, Ramanathan A, Long CA, and Burns JM 2018. Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant. Infect Immun 86. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Parzych EM, Miura K, Long CA, and Burns JM 2020. Maintaining immunogenicity of blood stage and sexual stage subunit malaria vaccines when formulated in combination. PLoS One 15: e0232355. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Osier FH, Murungi LM, Fegan G, Tuju J, Tetteh KK, Bull PC, Conway DJ, and Marsh K 2010. Allele-specific antibodies to Plasmodium falciparum merozoite surface protein-2 and protection against clinical malaria. Parasite Immunol 32: 193–201. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Reddy SB, Anders RF, Beeson JG, Farnert A, Kironde F, Berenzon SK, Wahlgren M, Linse S, and Persson KE 2012. High affinity antibodies to Plasmodium falciparum merozoite antigens are associated with protection from malaria. PLoS One 7: e32242. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Osier FH, Fegan G, Polley SD, Murungi L, Verra F, Tetteh KK, Lowe B, Mwangi T, Bull PC, Thomas AW, Cavanagh DR, McBride JS, Lanar DE, Mackinnon MJ, Conway DJ, and Marsh K 2008. Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 76: 2240–2248. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Genton B, Al-Yaman F, Betuela I, Anders RF, Saul A, Baea K, Mellombo M, Taraika J, Brown GV, Pye D, Irving DO, Felger I, Beck HP, Smith TA, and Alpers MP 2003. Safety and immunogenicity of a three-component blood-stage malaria vaccine (MSP1, MSP2, RESA) against Plasmodium falciparum in Papua New Guinean children. Vaccine 22: 30–41. [DOI] [PubMed] [Google Scholar]
28.Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF, Saul A, Rare L, Baisor M, Lorry K, Brown GV, Pye D, Irving DO, Smith TA, Beck HP, and Alpers MP 2002. A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1–2b trial in Papua New Guinea. J Infect Dis 185: 820–827. [DOI] [PubMed] [Google Scholar]
29.Genton B, Al-Yaman F, Anders R, Saul A, Brown G, Pye D, Irving DO, Briggs WR, Mai A, Ginny M, Adiguma T, Rare L, Giddy A, Reber-Liske R, Stuerchler D, and Alpers MP 2000. Safety and immunogenicity of a three-component blood-stage malaria vaccine in adults living in an endemic area of Papua New Guinea. Vaccine 18: 2504–2511. [DOI] [PubMed] [Google Scholar]
30.Eacret JS, Gonzales DM, Franks RG, and Burns JM 2019. Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody. Sci Rep 9: 9022. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Ulanova M, Tarkowski A, Hahn-Zoric M, and Hanson LA 2001. The Common vaccine adjuvant aluminum hydroxide up-regulates accessory properties of human monocytes via an interleukin-4-dependent mechanism. Infect Immun 69: 1151–1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Brewer JM, Conacher M, Hunter CA, Mohrs M, Brombacher F, and Alexander J 1999. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. J Immunol 163: 6448–6454. [PubMed] [Google Scholar]
33.Orr MT, Duthie MS, Windish HP, Lucas EA, Guderian JA, Hudson TE, Shaverdian N, O’Donnell J, Desbien AL, Reed SG, and Coler RN 2013. MyD88 and TRIF synergistic interaction is required for TH1-cell polarization with a synthetic TLR4 agonist adjuvant. Eur J Immunol 43: 2398–2408. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Baldwin SL, Shaverdian N, Goto Y, Duthie MS, Raman VS, Evers T, Mompoint F, Vedvick TS, Bertholet S, Coler RN, and Reed SG 2009. Enhanced humoral and Type 1 cellular immune responses with Fluzone adjuvanted with a synthetic TLR4 agonist formulated in an emulsion. Vaccine 27: 5956–5963. [DOI] [PubMed] [Google Scholar]
35.Desbien AL, Reed SJ, Bailor HR, Dubois Cauwelaert N, Laurance JD, Orr MT, Fox CB, Carter D, Reed SG, and Duthie MS 2015. Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ. Eur J Immunol 45: 407–417. [DOI] [PubMed] [Google Scholar]
36.Pullen GR, Fitzgerald MG, and Hosking CS 1986. Antibody avidity determination by ELISA using thiocyanate elution. Journal of immunological methods 86: 83–87. [DOI] [PubMed] [Google Scholar]
37.Lynch MM, Cernetich-Ott A, Weidanz WP, and Burns JM Jr. 2009. Prediction of merozoite surface protein 1 and apical membrane antigen 1 vaccine efficacies against Plasmodium chabaudi malaria based on prechallenge antibody responses. Clin Vaccine Immunol 16: 293–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Malkin EM, Diemert DJ, McArthur JH, Perreault JR, Miles AP, Giersing BK, Mullen GE, Orcutt A, Muratova O, Awkal M, Zhou H, Wang J, Stowers A, Long CA, Mahanty S, Miller LH, Saul A, and Durbin AP 2005. Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria. Infect Immun 73: 3677–3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Yang X, Adda CG, MacRaild CA, Low A, Zhang X, Zeng W, Jackson DC, Anders RF, and Norton RS 2010. Identification of key residues involved in fibril formation by the conserved N-terminal region of Plasmodium falciparum merozoite surface protein 2 (MSP2). Biochimie 92: 1287–1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Yang X, Adda CG, Keizer DW, Murphy VJ, Rizkalla MM, Perugini MA, Jackson DC, Anders RF, and Norton RS 2007. A partially structured region of a largely unstructured protein, Plasmodium falciparum merozoite surface protein 2 (MSP2), forms amyloid-like fibrils. J Pept Sci 13: 839–848. [DOI] [PubMed] [Google Scholar]
41.Chandrashekaran IR, Adda CG, Macraild CA, Anders RF, and Norton RS 2011. EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2. Arch Biochem Biophys 513: 153–157. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Chandrashekaran IR, Adda CG, MacRaild CA, Anders RF, and Norton RS 2010. Inhibition by flavonoids of amyloid-like fibril formation by Plasmodium falciparum merozoite surface protein 2. Biochemistry 49: 5899–5908. [DOI] [PubMed] [Google Scholar]
43.Adda CG, Murphy VJ, Sunde M, Waddington LJ, Schloegel J, Talbo GH, Vingas K, Kienzle V, Masciantonio R, Howlett GJ, Hodder AN, Foley M, and Anders RF 2009. Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils. Mol Biochem Parasitol 166: 159–171. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Pichyangkul S, Tongtawe P, Kum-Arb U, Yongvanitchit K, Gettayacamin M, Hollingdale MR, Limsalakpetch A, Stewart VA, Lanar DE, Dutta S, Angov E, Ware LA, Bergmann-Leitner ES, House B, Voss G, Dubois MC, Cohen JD, Fukuda MM, Heppner DG, and Miller RS 2009. Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys. Vaccine 28: 452–462. [DOI] [PubMed] [Google Scholar]
45.Forbes EK, Biswas S, Collins KA, Gilbert SC, Hill AV, and Draper SJ 2011. Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference. J Immunol 187: 3738–3750. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Rampling T, Ewer KJ, Bowyer G, Edwards NJ, Wright D, Sridhar S, Payne R, Powlson J, Bliss C, Venkatraman N, Poulton ID, de Graaf H, Gbesemete D, Grobbelaar A, Davies H, Roberts R, Angus B, Ivinson K, Weltzin R, Rajkumar BY, Wille-Reece U, Lee C, Ockenhouse C, Sinden RE, Gerry SC, Lawrie AM, Vekemans J, Morelle D, Lievens M, Ballou RW, Lewis DJM, Cooke GS, Faust SN, Gilbert S, and Hill AVS 2018. Safety and efficacy of novel malaria vaccine regimens of RTS,S/AS01B alone, or with concomitant ChAd63-MVA-vectored vaccines expressing ME-TRAP. NPJ Vaccines 3: 49. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Mordmüller B, Sulyok M, Egger-Adam D, Resende M, de Jongh WA, Jensen MH, Smedegaard HH, Ditlev SB, Soegaard M, Poulsen L, Dyring C, Calle CL, Knoblich A, Ibáñez J, Esen M, Deloron P, Ndam N, Issifou S, Houard S, Howard RF, Reed SG, Leroy O, Luty AJF, Theander TG, Kremsner PG, Salanti A, and Nielsen MA 2019. First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria. Clin Infect Dis 69: 1509–1516. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Coler RN, Baldwin SL, Shaverdian N, Bertholet S, Reed SJ, Raman VS, Lu X, DeVos J, Hancock K, Katz JM, Vedvick TS, Duthie MS, Clegg CH, Van Hoeven N, and Reed SG 2010. A synthetic adjuvant to enhance and expand immune responses to influenza vaccines. PLoS One 5: e13677. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Patton K, Aslam S, Shambaugh C, Lin R, Heeke D, Frantz C, Zuo F, Esser MT, Paliard X, and Lambert SL 2015. Enhanced immunogenicity of a respiratory syncytial virus (RSV) F subunit vaccine formulated with the adjuvant GLA-SE in cynomolgus macaques. Vaccine 33: 4472–4478. [DOI] [PubMed] [Google Scholar]
50.Klein SL, Jedlicka A, and Pekosz A 2010. The Xs and Y of immune responses to viral vaccines. Lancet Infect Dis 10: 338–349. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Voysey M, Barker CI, Snape MD, Kelly DF, Trück J, and Pollard AJ 2016. Sex-dependent immune responses to infant vaccination: an individual participant data meta-analysis of antibody and memory B cells. Vaccine 34: 1657–1664. [DOI] [PubMed] [Google Scholar]
52.Flanagan KL, Fink AL, Plebanski M, and Klein SL 2017. Sex and Gender Differences in the Outcomes of Vaccination over the Life Course. Annu Rev Cell Dev Biol 33: 577–599. [DOI] [PubMed] [Google Scholar]
53.Klein SL, Shann F, Moss WJ, Benn CS, and Aaby P 2016. RTS,S Malaria Vaccine and Increased Mortality in Girls. MBio 7: e00514–00516. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1673293-supplement-1.pdf^{(450.5KB, pdf)}

[R1] 1.Ghehreyesus T 2019. World Malaria Report 2019. World Health Organization, Geneva. [Google Scholar]

[R2] 2.RTS SCTP 2015. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 386: 31–45. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.White MT, Verity R, Griffin JT, Asante KP, Owusu-Agyei S, Greenwood B, Drakeley C, Gesase S, Lusingu J, Ansong D, Adjei S, Agbenyega T, Ogutu B, Otieno L, Otieno W, Agnandji ST, Lell B, Kremsner P, Hoffman I, Martinson F, Kamthunzu P, Tinto H, Valea I, Sorgho H, Oneko M, Otieno K, Hamel MJ, Salim N, Mtoro A, Abdulla S, Aide P, Sacarlal J, Aponte JJ, Njuguna P, Marsh K, Bejon P, Riley EM, and Ghani AC 2015. Immunogenicity of the RTS,S/AS01 malaria vaccine and implications for duration of vaccine efficacy: secondary analysis of data from a phase 3 randomised controlled trial. Lancet Infect Dis 15: 1450–1458. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Ubillos I, Ayestaran A, Nhabomba AJ, Dosoo D, Vidal M, Jimenez A, Jairoce C, Sanz H, Aguilar R, Williams NA, Diez-Padrisa N, Mpina M, Sorgho H, Agnandji ST, Kariuki S, Mordmuller B, Daubenberger C, Asante KP, Owusu-Agyei S, Sacarlal J, Aide P, Aponte JJ, Dutta S, Gyan B, Campo JJ, Valim C, Moncunill G, and Dobano C 2018. Baseline exposure, antibody subclass, and hepatitis B response differentially affect malaria protective immunity following RTS,S/AS01E vaccination in African children. BMC Med 16: 197. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Gosling R, and von Seidlein L 2016. The Future of the RTS,S/AS01 Malaria Vaccine: An Alternative Development Plan. PLoS Med 13: e1001994. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Olotu A, Fegan G, Wambua J, Nyangweso G, Awuondo KO, Leach A, Lievens M, Leboulleux D, Njuguna P, Peshu N, Marsh K, and Bejon P 2013. Four-year efficacy of RTS,S/AS01E and its interaction with malaria exposure. N Engl J Med 368: 1111–1120. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.RTS SCTP 2014. Efficacy and safety of the RTS,S/AS01 malaria vaccine during 18 months after vaccination: a phase 3 randomized, controlled trial in children and young infants at 11 African sites. PLoS Med 11: e1001685. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Regules JA, Cicatelli SB, Bennett JW, Paolino KM, Twomey PS, Moon JE, Kathcart AK, Hauns KD, Komisar JL, Qabar AN, Davidson SA, Dutta S, Griffith ME, Magee CD, Wojnarski M, Livezey JR, Kress AT, Waterman PE, Jongert E, Wille-Reece U, Volkmuth W, Emerling D, Robinson WH, Lievens M, Morelle D, Lee CK, Yassin-Rajkumar B, Weltzin R, Cohen J, Paris RM, Waters NC, Birkett AJ, Kaslow DC, Ballou WR, Ockenhouse CF, and Vekemans J 2016. Fractional Third and Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria Parasite Infection and Immunogenicity Study. J Infect Dis 214: 762–771. [DOI] [PubMed] [Google Scholar]

[R9] 9.Laurens MB, Thera MA, Coulibaly D, Ouattara A, Kone AK, Guindo AB, Traore K, Traore I, Kouriba B, Diallo DA, Diarra I, Daou M, Dolo A, Tolo Y, Sissoko MS, Niangaly A, Sissoko M, Takala-Harrison S, Lyke KE, Wu Y, Blackwelder WC, Godeaux O, Vekemans J, Dubois MC, Ballou WR, Cohen J, Dube T, Soisson L, Diggs CL, House B, Bennett JW, Lanar DE, Dutta S, Heppner DG, Plowe CV, and Doumbo OK 2013. Extended safety, immunogenicity and efficacy of a blood-stage malaria vaccine in malian children: 24-month follow-up of a randomized, double-blinded phase 2 trial. PLoS One 8: e79323. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Ouattara A, Barry AE, Dutta S, Remarque EJ, Beeson JG, and Plowe CV 2015. Designing malaria vaccines to circumvent antigen variability. Vaccine 33: 7506–7512. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Barry AE, and Arnott A 2014. Strategies for designing and monitoring malaria vaccines targeting diverse antigens. Front Immunol 5: 359. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Draper SJ, Sack BK, King CR, Nielsen CM, Rayner JC, Higgins MK, Long CA, and Seder RA 2018. Malaria Vaccines: Recent Advances and New Horizons. Cell Host Microbe 24: 43–56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Asante KP, Abdulla S, Agnandji S, Lyimo J, Vekemans J, Soulanoudjingar S, Owusu R, Shomari M, Leach A, Jongert E, Salim N, Fernandes JF, Dosoo D, Chikawe M, Issifou S, Osei-Kwakye K, Lievens M, Paricek M, Möller T, Apanga S, Mwangoka G, Dubois MC, Madi T, Kwara E, Minja R, Hounkpatin AB, Boahen O, Kayan K, Adjei G, Chandramohan D, Carter T, Vansadia P, Sillman M, Savarese B, Loucq C, Lapierre D, Greenwood B, Cohen J, Kremsner P, Owusu-Agyei S, Tanner M, and Lell B 2011. Safety and efficacy of the RTS,S/AS01E candidate malaria vaccine given with expanded-programme-on-immunisation vaccines: 19 month follow-up of a randomised, open-label, phase 2 trial. Lancet Infect Dis 11: 741–749. [DOI] [PubMed] [Google Scholar]

[R14] 14.Collins KA, Snaith R, Cottingham MG, Gilbert SC, and Hill AVS 2017. Enhancing protective immunity to malaria with a highly immunogenic virus-like particle vaccine. Sci Rep 7: 46621. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Genito CJ, Beck Z, Phares TW, Kalle F, Limbach KJ, Stefaniak ME, Patterson NB, Bergmann-Leitner ES, Waters NC, Matyas GR, Alving CR, and Dutta S 2017. Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013. Vaccine 35: 3865–3874. [DOI] [PubMed] [Google Scholar]

[R16] 16.Thera MA, Doumbo OK, Coulibaly D, Laurens MB, Ouattara A, Kone AK, Guindo AB, Traore K, Traore I, Kouriba B, Diallo DA, Diarra I, Daou M, Dolo A, Tolo Y, Sissoko MS, Niangaly A, Sissoko M, Takala-Harrison S, Lyke KE, Wu Y, Blackwelder WC, Godeaux O, Vekemans J, Dubois MC, Ballou WR, Cohen J, Thompson D, Dube T, Soisson L, Diggs CL, House B, Lanar DE, Dutta S, Heppner DG, and Plowe CV 2011. A field trial to assess a blood-stage malaria vaccine. N Engl J Med 365: 1004–1013. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Otsyula N, Angov E, Bergmann-Leitner E, Koech M, Khan F, Bennett J, Otieno L, Cummings J, Andagalu B, Tosh D, Waitumbi J, Richie N, Shi M, Miller L, Otieno W, Otieno GA, Ware L, House B, Godeaux O, Dubois MC, Ogutu B, Ballou WR, Soisson L, Diggs C, Cohen J, Polhemus M, Heppner DG, Ockenhouse CF, and Spring MD 2013. Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP1(42)) administered intramuscularly with adjuvant system AS01. Malar J 12: 29. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Thera MA, Doumbo OK, Coulibaly D, Diallo DA, Sagara I, Dicko A, Diemert DJ, Heppner DG, Stewart VA, Angov E, Soisson L, Leach A, Tucker K, Lyke KE, Plowe CV, and Group MFW 2006. Safety and allele-specific immunogenicity of a malaria vaccine in Malian adults: results of a phase I randomized trial. PLoS Clin Trials 1: e34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Ogutu BR, Apollo OJ, McKinney D, Okoth W, Siangla J, Dubovsky F, Tucker K, Waitumbi JN, Diggs C, Wittes J, Malkin E, Leach A, Soisson LA, Milman JB, Otieno L, Holland CA, Polhemus M, Remich SA, Ockenhouse CF, Cohen J, Ballou WR, Martin SK, Angov E, Stewart VA, Lyon JA, Heppner DG, Withers MR, and M.-M. V. W. Group. 2009. Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya. PLoS One 4: e4708. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Alaro JR, Partridge A, Miura K, Diouf A, Lopez AM, Angov E, Long CA, and Burns JM 2013. A chimeric Plasmodium falciparum merozoite surface protein vaccine induces high titers of parasite growth inhibitory antibodies. Infect Immun 81: 3843–3854. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Burns JM, Miura K, Sullivan J, Long CA, and Barnwell JW 2016. Immunogenicity of a chimeric Plasmodium falciparum merozoite surface protein vaccine in Aotus monkeys. Malar J 15: 159. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Parzych EM, Miura K, Ramanathan A, Long CA, and Burns JM 2018. Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant. Infect Immun 86. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Parzych EM, Miura K, Long CA, and Burns JM 2020. Maintaining immunogenicity of blood stage and sexual stage subunit malaria vaccines when formulated in combination. PLoS One 15: e0232355. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Osier FH, Murungi LM, Fegan G, Tuju J, Tetteh KK, Bull PC, Conway DJ, and Marsh K 2010. Allele-specific antibodies to Plasmodium falciparum merozoite surface protein-2 and protection against clinical malaria. Parasite Immunol 32: 193–201. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Reddy SB, Anders RF, Beeson JG, Farnert A, Kironde F, Berenzon SK, Wahlgren M, Linse S, and Persson KE 2012. High affinity antibodies to Plasmodium falciparum merozoite antigens are associated with protection from malaria. PLoS One 7: e32242. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Osier FH, Fegan G, Polley SD, Murungi L, Verra F, Tetteh KK, Lowe B, Mwangi T, Bull PC, Thomas AW, Cavanagh DR, McBride JS, Lanar DE, Mackinnon MJ, Conway DJ, and Marsh K 2008. Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 76: 2240–2248. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Genton B, Al-Yaman F, Betuela I, Anders RF, Saul A, Baea K, Mellombo M, Taraika J, Brown GV, Pye D, Irving DO, Felger I, Beck HP, Smith TA, and Alpers MP 2003. Safety and immunogenicity of a three-component blood-stage malaria vaccine (MSP1, MSP2, RESA) against Plasmodium falciparum in Papua New Guinean children. Vaccine 22: 30–41. [DOI] [PubMed] [Google Scholar]

[R28] 28.Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF, Saul A, Rare L, Baisor M, Lorry K, Brown GV, Pye D, Irving DO, Smith TA, Beck HP, and Alpers MP 2002. A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1–2b trial in Papua New Guinea. J Infect Dis 185: 820–827. [DOI] [PubMed] [Google Scholar]

[R29] 29.Genton B, Al-Yaman F, Anders R, Saul A, Brown G, Pye D, Irving DO, Briggs WR, Mai A, Ginny M, Adiguma T, Rare L, Giddy A, Reber-Liske R, Stuerchler D, and Alpers MP 2000. Safety and immunogenicity of a three-component blood-stage malaria vaccine in adults living in an endemic area of Papua New Guinea. Vaccine 18: 2504–2511. [DOI] [PubMed] [Google Scholar]

[R30] 30.Eacret JS, Gonzales DM, Franks RG, and Burns JM 2019. Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody. Sci Rep 9: 9022. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Ulanova M, Tarkowski A, Hahn-Zoric M, and Hanson LA 2001. The Common vaccine adjuvant aluminum hydroxide up-regulates accessory properties of human monocytes via an interleukin-4-dependent mechanism. Infect Immun 69: 1151–1159. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] 32.Brewer JM, Conacher M, Hunter CA, Mohrs M, Brombacher F, and Alexander J 1999. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. J Immunol 163: 6448–6454. [PubMed] [Google Scholar]

[R33] 33.Orr MT, Duthie MS, Windish HP, Lucas EA, Guderian JA, Hudson TE, Shaverdian N, O’Donnell J, Desbien AL, Reed SG, and Coler RN 2013. MyD88 and TRIF synergistic interaction is required for TH1-cell polarization with a synthetic TLR4 agonist adjuvant. Eur J Immunol 43: 2398–2408. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Baldwin SL, Shaverdian N, Goto Y, Duthie MS, Raman VS, Evers T, Mompoint F, Vedvick TS, Bertholet S, Coler RN, and Reed SG 2009. Enhanced humoral and Type 1 cellular immune responses with Fluzone adjuvanted with a synthetic TLR4 agonist formulated in an emulsion. Vaccine 27: 5956–5963. [DOI] [PubMed] [Google Scholar]

[R35] 35.Desbien AL, Reed SJ, Bailor HR, Dubois Cauwelaert N, Laurance JD, Orr MT, Fox CB, Carter D, Reed SG, and Duthie MS 2015. Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ. Eur J Immunol 45: 407–417. [DOI] [PubMed] [Google Scholar]

[R36] 36.Pullen GR, Fitzgerald MG, and Hosking CS 1986. Antibody avidity determination by ELISA using thiocyanate elution. Journal of immunological methods 86: 83–87. [DOI] [PubMed] [Google Scholar]

[R37] 37.Lynch MM, Cernetich-Ott A, Weidanz WP, and Burns JM Jr. 2009. Prediction of merozoite surface protein 1 and apical membrane antigen 1 vaccine efficacies against Plasmodium chabaudi malaria based on prechallenge antibody responses. Clin Vaccine Immunol 16: 293–302. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Malkin EM, Diemert DJ, McArthur JH, Perreault JR, Miles AP, Giersing BK, Mullen GE, Orcutt A, Muratova O, Awkal M, Zhou H, Wang J, Stowers A, Long CA, Mahanty S, Miller LH, Saul A, and Durbin AP 2005. Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria. Infect Immun 73: 3677–3685. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Yang X, Adda CG, MacRaild CA, Low A, Zhang X, Zeng W, Jackson DC, Anders RF, and Norton RS 2010. Identification of key residues involved in fibril formation by the conserved N-terminal region of Plasmodium falciparum merozoite surface protein 2 (MSP2). Biochimie 92: 1287–1295. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Yang X, Adda CG, Keizer DW, Murphy VJ, Rizkalla MM, Perugini MA, Jackson DC, Anders RF, and Norton RS 2007. A partially structured region of a largely unstructured protein, Plasmodium falciparum merozoite surface protein 2 (MSP2), forms amyloid-like fibrils. J Pept Sci 13: 839–848. [DOI] [PubMed] [Google Scholar]

[R41] 41.Chandrashekaran IR, Adda CG, Macraild CA, Anders RF, and Norton RS 2011. EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2. Arch Biochem Biophys 513: 153–157. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R42] 42.Chandrashekaran IR, Adda CG, MacRaild CA, Anders RF, and Norton RS 2010. Inhibition by flavonoids of amyloid-like fibril formation by Plasmodium falciparum merozoite surface protein 2. Biochemistry 49: 5899–5908. [DOI] [PubMed] [Google Scholar]

[R43] 43.Adda CG, Murphy VJ, Sunde M, Waddington LJ, Schloegel J, Talbo GH, Vingas K, Kienzle V, Masciantonio R, Howlett GJ, Hodder AN, Foley M, and Anders RF 2009. Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils. Mol Biochem Parasitol 166: 159–171. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R44] 44.Pichyangkul S, Tongtawe P, Kum-Arb U, Yongvanitchit K, Gettayacamin M, Hollingdale MR, Limsalakpetch A, Stewart VA, Lanar DE, Dutta S, Angov E, Ware LA, Bergmann-Leitner ES, House B, Voss G, Dubois MC, Cohen JD, Fukuda MM, Heppner DG, and Miller RS 2009. Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys. Vaccine 28: 452–462. [DOI] [PubMed] [Google Scholar]

[R45] 45.Forbes EK, Biswas S, Collins KA, Gilbert SC, Hill AV, and Draper SJ 2011. Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference. J Immunol 187: 3738–3750. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] 46.Rampling T, Ewer KJ, Bowyer G, Edwards NJ, Wright D, Sridhar S, Payne R, Powlson J, Bliss C, Venkatraman N, Poulton ID, de Graaf H, Gbesemete D, Grobbelaar A, Davies H, Roberts R, Angus B, Ivinson K, Weltzin R, Rajkumar BY, Wille-Reece U, Lee C, Ockenhouse C, Sinden RE, Gerry SC, Lawrie AM, Vekemans J, Morelle D, Lievens M, Ballou RW, Lewis DJM, Cooke GS, Faust SN, Gilbert S, and Hill AVS 2018. Safety and efficacy of novel malaria vaccine regimens of RTS,S/AS01B alone, or with concomitant ChAd63-MVA-vectored vaccines expressing ME-TRAP. NPJ Vaccines 3: 49. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R47] 47.Mordmüller B, Sulyok M, Egger-Adam D, Resende M, de Jongh WA, Jensen MH, Smedegaard HH, Ditlev SB, Soegaard M, Poulsen L, Dyring C, Calle CL, Knoblich A, Ibáñez J, Esen M, Deloron P, Ndam N, Issifou S, Houard S, Howard RF, Reed SG, Leroy O, Luty AJF, Theander TG, Kremsner PG, Salanti A, and Nielsen MA 2019. First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria. Clin Infect Dis 69: 1509–1516. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R48] 48.Coler RN, Baldwin SL, Shaverdian N, Bertholet S, Reed SJ, Raman VS, Lu X, DeVos J, Hancock K, Katz JM, Vedvick TS, Duthie MS, Clegg CH, Van Hoeven N, and Reed SG 2010. A synthetic adjuvant to enhance and expand immune responses to influenza vaccines. PLoS One 5: e13677. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Patton K, Aslam S, Shambaugh C, Lin R, Heeke D, Frantz C, Zuo F, Esser MT, Paliard X, and Lambert SL 2015. Enhanced immunogenicity of a respiratory syncytial virus (RSV) F subunit vaccine formulated with the adjuvant GLA-SE in cynomolgus macaques. Vaccine 33: 4472–4478. [DOI] [PubMed] [Google Scholar]

[R50] 50.Klein SL, Jedlicka A, and Pekosz A 2010. The Xs and Y of immune responses to viral vaccines. Lancet Infect Dis 10: 338–349. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R51] 51.Voysey M, Barker CI, Snape MD, Kelly DF, Trück J, and Pollard AJ 2016. Sex-dependent immune responses to infant vaccination: an individual participant data meta-analysis of antibody and memory B cells. Vaccine 34: 1657–1664. [DOI] [PubMed] [Google Scholar]

[R52] 52.Flanagan KL, Fink AL, Plebanski M, and Klein SL 2017. Sex and Gender Differences in the Outcomes of Vaccination over the Life Course. Annu Rev Cell Dev Biol 33: 577–599. [DOI] [PubMed] [Google Scholar]

[R53] 53.Klein SL, Shann F, Moss WJ, Benn CS, and Aaby P 2016. RTS,S Malaria Vaccine and Increased Mortality in Girls. MBio 7: e00514–00516. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Inclusion of an optimized Plasmodium falciparum merozoite surface protein 2-based antigen in a trivalent, multi-stage malaria vaccine

Jacqueline S Eacret

Elizabeth M Parzych

Donna M Gonzales

James M Burns Jr

Abstract

Introduction

Materials and Methods

Mice and immunization protocols

(i). Experimental protocols 1 and 2

(ii). Experimental protocol 3

(ii). Experiment protocol 4

Analysis of cellular responses

(i). Generation of PfMSP2 overlapping peptides

(ii). Antigen-specific splenocyte proliferation

(iii). Antigen-induced cytokine production

Analysis of humoral responses

(i). Antigen-specific antibody titer

(ii). Antibody isotype profile

(iii). Avidity of antigen-specific antibodies

(iv). Competition ELISA

Opsonophagocytosis Assay

Statistical Analysis

Figure 1. PfMSP2- and PfMSP8- specific T cell responses are induced by immunization.

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.

Figure 2. Impact of antigen, dose and adjuvant on vaccine-induced antigen-specific IgG titers.

Figure 6. Durability of PfMSP2-specific IgG titers differs based on antigen and adjuvant selection.

Figure 9. Multivalent formulations containing rPfMSP2/8, rPfMSP1/8 and rPfs25/8, irrespective of adjuvant, elicit strong antibody responses against each fusion partner with no indication of antigenic competition.

Table I.

Results

Immunization-induced T cells are specific for both PfMSP2 and PfMSP8 epitopes

High levels of antigen-specific IgG are induced upon immunization with rPfMSP2-based vaccines

Figure 3. Final anti-PfMSP2 IgG titers induced by immunization with rPfMSP2 and rPfMSP2/8 as a function of dose and adjuvant.

Formulation of PfMSP2-based vaccines with GLA-SE versus Alum shifts cytokine production from a Th2 toward a Th1 profile

Use of GLA-SE as adjuvant drives class switching to cytophilic IgG subclasses

PfMSP2-based vaccine formulations induce antigen-specific antibodies of comparable avidity

Figure 5. Avidity of anti-PfMSP2 IgG induced by immunization with rPfMSP2 and rPfMSP2/8 as a function of dose and adjuvant.

Fusion to PfMSP8 and formulation with GLA-SE promotes high and durable PfMSP2-specific antibody responses

Structural differences between rPfMSP2 and rPfMSP2/8 impact antibody epitope specificity

Figure 7. The fibrillar state of rPfMSP2 antigens impacts epitope availability and specificity of vaccine-induced IgG.

Cytophilic IgG generated by immunization with PfMSP2 vaccines formulated with GLA-SE demonstrates superior merozoite neutralizing activity

Figure 8. Immunization with rPfMSP2-based vaccines formulated with GLA-SE versus Alum enhances opsonophagocytosis of merozoites.

Induction of high levels of PfMSP2-specific antibodies is maintained following immunization with multivalent vaccine formulations

Figure 10. Adjuvant shifts the profile of IgG subclasses induced upon immunization with multivalent vaccine formulations.

Discussion

Supplementary Material

Key Points.

Acknowledgments

4. Non-standard abbreviations:

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases