Immunogenicity of dimorphic and C-terminal fragments of Plasmodium falciparum MSP2 formulated with different adjuvants in mice

Vaccine 34 (2016) 1566–1574

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Immunogenicity of dimorphic and C-terminal fragments of Plasmodium falciparum MSP2 formulated with different adjuvants in mice Saidou Balam a,∗ , Ali Jafarshad b , Catherine Servis a , Geraldine Frank a , Steve Reed c , Richard Pink a , Pierre Druilhe b , Franc¸ois Spertini d , Giampietro Corradin a a

Department of Biochemistry, University of Lausanne, Ch des Boveresses 155, Epalinges, Switzerland1 Pasteur Institut, Paris, France c IDRI, Seattle, USA d Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland b

a r t i c l e

i n f o

Article history: Received 14 September 2015 Received in revised form 22 January 2016 Accepted 2 February 2016 Available online 11 February 2016 Keywords: P. falciparum MSP2 Dimorphic region Constant region Immunogenicity Adjuvant

a b s t r a c t Background: Plasmodium falciparum MSP2 is a blood stage protein that is associated with protection against malaria. It was shown that the MSP2 dimorphic (D) and constant (C) regions were well recognized by immune human antibodies, and were characterized by major conserved epitopes in different endemic areas and age groups. These Abs recognized merozoite-derived proteins in WB and IFA. Here, the goal was to determine in mice the immunogenicity of the two allelic MSP2 D and C domains formulated with different adjuvants, for their possible use in future clinical studies. Method: Female A/J, C3H, and ICR mice were immunized subcutaneously 3 times at 3-week interval with a mixture of allelic and conserved MSP2 long synthetic peptides formulated with different adjuvants. One week after the third injection, sera from each group were obtained and stored at −20 ◦ C for subsequent testing. Results: Both domains of the two MSP2 families are immunogenic and the fine specificity and intensity of the Ab responses are dependent on mouse strains and adjuvants. The major epitopes were restricted to the 20-mer peptide sequences comprising the last 8 aa of D and first 12 aa of C of the two allelic families and the first 20 aa of the C region, this for most strains and adjuvants. Strong immune responses were associated with GLA-SE adjuvant and its combination with other TLR agonists (CpG or GDQ) compared to alhydrogel and Montanide. Further, the elicited Abs were also capable of recognizing Plasmodium-derived MSP2 and inhibiting parasite growth in ADCI. Conclusion: The data provide a valuable opportunity to evaluate in mice different adjuvant and antigen formulations of a candidate vaccine containing both MSP2 D and C fragments. The formulations with GLA-SE seem to be a promising option to be compared with the alhydrogel one in human clinical trials. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In a previous study the fine specificities of immune human antibody (Ab) responses specific for long synthetic peptides (LSPs) representing dimorphic (D) and constant (C) domains of the two

∗ Corresponding author. Current address: University Hospital Regensburg, FranzJosef-Strauss-Allee 11, 93053 Regensburg, Germany. Tel.: +49 941 944 6990/91. E-mail addresses: [email protected] (S. Balam), [email protected] (A. Jafarshad), [email protected] (C. Servis), [email protected] (G. Frank), [email protected] (S. Reed), [email protected] (R. Pink), [email protected] (P. Druilhe), [email protected] (F. Spertini), [email protected] (G. Corradin). 1 Tel.: +41 21 692 57 31; fax: +41 21 692 57 05. http://dx.doi.org/10.1016/j.vaccine.2016.02.013 0264-410X/© 2016 Elsevier Ltd. All rights reserved.

allelic MSP2 families (represented by 3D7 and FC27 parasite strains) were evaluated. These were performed using plasma and purified Abs (pAbs) from different age group donors and living in different malaria endemic areas. It was shown that the Ab prevalence against D and C regions of the two MSP2 allelic families was high in all age groups; however, 3D7-D region was the most recognized in endemic areas compared to FC27-D and C regions [1]. In addition, several shorter epitopes (20 mers) located in the two allelic families (3D7 and FC27) and C domains were delineated in malaria endemic populations. Thus, these data and the following considerations support the development of the two dimorphic and C regions as candidate vaccines: (a) in epidemiological studies, Abs to MSP2 constructs are associated with protection [2–5]; (b) polymorphism is limited in the D region; (c) D and C regions represent

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“intrinsically unstructured regions” and the synthetic peptides mimic such a structure [6,7]; and (d) human pAbs reactive against D+C constructs showed ADCI activity in vitro and were associated with protection [7,8]. However, very little is known on the capacity of adjuvants to precisely shape the B cell response against antigens. Preclinical determination of the B cell response to MSP2 fragments in animal models immunized with different adjuvant formulations may contribute to define the optimal MSP2 formulations to be used in humans [9,10]. In the current study, the first objective was to identify optimal adjuvant formulations that could be associated with high immune responses to D and C regions. The second objective was to define D and C epitopes relative to the adjuvants used. This task is facilitated by the fact that the MSP2 fragments used are “intrinsically unstructured”, and can be accomplished by using short overlapping peptides (20 mers). The third objective was to determine the Ab ability to recognize Plasmodium-derived MSP2 and inhibit parasite growth. To achieve these objectives, different strains of mice (inbred and outbred) were immunized with a mixture of D and C polypeptides in different combinations of adjuvants. Inbred and outbred mice were used in order to know whether there is variation of immunogenicity and epitope recognition among the various strains used. Various adjuvants were tested, either alone: alhydrogel (alum); glucopyranosyl-lipid A, as a stable oil-in-water emulsion (GLA-SE); and Montanide ISA720 (Mt), or in combination, with GLA-SE: GLA-CpG-SE (CpG, is a synthetic oligodeoxynucleotide agonist for toll-like receptor 9/TLR9), GLA-GDQ-SE (GDQ: gardiquimod, agonist specific for TLR7) and GLA-CpG-GDQ-SE [11–14]. The latter two-adjuvant combinations were used to see if engagement of additional TLR would increase the antibody response. The use of a mixture of the two LSPs and their compositions are dictated by the fact that MSP2-based vaccines must contain antigens from the two allelic families and by the optimization of the peptide synthesis and purification protocols. 2. Materials and methods 2.1. Peptides All peptides were synthesized by solid-phase Fmoc chemistry using Applied Biosystems 431A and 433A synthesizers (Foster City, CA) and a MultiRespep synthesizer (Bioanalytical Instrument, Intavis AG) as previously described [1]. Peptide purity was assessed by analytic C18 HPLC and mass spectrometry (MALDI-TOF, Applied Biosystems) and was higher than 80%. All the reagents used

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were purchased from Fluka (Buchs, Switzerland) or Novabiochem (Laufelfingen, Switzerland). The 3D7-D+C LSP (111–238) and FC27D+C LSP (143–230) cover the 3D7- and FC27-dimorphic (D) and constant (C) fragments, respectively. 3D7D+8aa C (111–206), 96 aa, includes 3D7-D plus 8 first aa from C and FC27-D+C (Table 1) were also synthesized by Almac Sciences, Craigavon, Northern Ireland (purity higher than 80%). The commercial and our constructs were used interchangeably. The overlapping 20-mer peptides (overlapping by 10 amino acids; Fig. 1A and B) cover the two allelic families and C region of MSP2. Thus, twelve 20-mer peptides (P11-P22) and eight 20-mer peptides (P23–P30) covered, respectively, 3D7 and FC27-MSP2 D+C region. The C region 20 mers, P20, P21 and P22 from 3D7 allelic family are, respectively, equal (sequences) to the P28, P29 and P30 from FC27 allelic family (Fig. 1). Peptides P19 (from 3D7 family) and P27 (from FC27 family) contained the 8 last aa of the D regions plus the 12 first aa of their C region. 2.2. Adjuvants The adjuvants used in the various immunization formulations were: Alum (Alhydrogel, Novartis, Siena, Italy); glucopyranosyllipid A in oil-in-water stable emulsion (GLA-SE, an agonist for toll-like receptor 4 (TLR4, IDRI, Seattle, WA), alone or in association with agonists for TLR 7/9, (gardiquimod (GPQ) and/or CpG); and Montanide (Mt) ISA720 (Seppic, Inc., Paris, France). 2.3. Mice and immunization Inbred A/J and C3H and outbred ICR mice were immunized with 20 ␮g of a peptide mixture, i.e. 10 ␮g of 3D7-D+8aaC plus 10 ␮g of FC27-D+C 3 (Table 1) 3 times at 3 week intervals. Formulations containing alum (500 ␮l) were injected ip, those containing GLASE and its combination with CpG and GDQ (50 ␮l at 20 ␮g/ml) and Montanide (50 ␮l) sc at the base of the tail. C3H mice were also immunized with 20 ␮g of allele-specific LSPs (3D7-D or FC27-D LSPs) or C LSP formulated with Montanide (50 ␮l, sc). Seven to 10 days after the third immunization mice were bled and sera were stored at −20 ◦ C. 2.4. Enzyme-linked immunosorbent assay (ELISA) Ab titers were determined in direct ELISA; antigens were diluted in PBS and used to coat ELISA plates. Antigen concentration for coating the 96-well flat plates (BD Biosciences, Allschwil, Switzerland) was 1 ␮g/ml for peptides longer than 40 residues, and 5 ␮g/ml for

Table 1 Sequences of LSPs covering D and C regions of the two MSP2 allelic families. LSP name

MSP2 allelic family of Pf

Sequences

aa number

Sequence number

3D7-D+C

3D7

128

111–238

3D7-D+8aaC

3D7

96

111–206

3D7-D

3D7

88

111–198

FC27-D+C

FC27

88

143–230

FC27-D

FC27

48

143–191

C

Common C-terminal

AEASTSTSSENPNHKNAETNPKGKGEVQEPNQAN KETQNNSNVQQDSQTKSNVPPTQDADTKSPTAQP EQAENSAPTAEQTESPELQSAPENKGTGQHGHM HGSRNNHPQNTSDSQKECTDGNKENCG AEASTSTSSENPNHKNAETNPKGKGEVQEPNQAN KETQNNSNVQQDSQTKSNVPPTQDADTKSPTAQP EQAENSAPTAEQTESPELQSAPENKGTG AEASTSTSSENPNHKNAETNPKGKGEVQEPNQAN KETQNNSNVQQDSQTKSNVPPTQDADTKSPTAQP EQAENSAPTAEQTESPELQS ESSSSGNAPNKTDGKGEESEKQNELNESTEEGPK APQEPQTAENENPAAPENKGTGQHGHMHGSRN NHPQNTSDSQKECTDGNKENCG ESSSSGNAPNKTDGKGEESEKQNELNESTEEGPK APQEPQTAENENPA APENKGTGQHGHMHGSRNNHPQNTSDSQKEC TDGNKENCG

40

198–238

The bold and underlined sequences correspond to the common C-terminal region sequence of MSP2-3D7 and FC27 families (198–238; 3D7 numbering). Pf: Plasmodium falciparum; LSP: Long synthetic peptide and aa: amino acid.

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Fig. 1. Schematic sequence of the two allelic families of MSP2 (3D7 and FC27) (cf: Balam et al. [1] and Flueck et al. [7]). (A) Represent full-length alignment of sequence of the two allelic families of MSP2 (3D7 and FC27). Non-repetitive family-dimorphic regions are shaded in light gray, conserved (N- and C-termini) sequences are underlined. The non-shaded and non-underlined sequences represent the polymorphic regions; (B) schematic representation of LSPs and overlapping 20-mers covering D and C regions of the two MSP2 allelic families. The D sequences are in black and conserved C sequences are in blue. LSP: long synthetic peptide; in parentheses: the aa sequence position.

peptides shorter or equal to 40 residues as previously described [1,15,16]. Secondary Ab (goat anti-mouse polyvalent immunoglobulin (G, A, M)-AP (Sigma® , 50 ␮l/well), was used at dilution 1:1000 in PBS-T plus 2.5% milk. Log of Geometric mean titer (GMT) of Ab response from each group were determined (titer is defined as the last dilution where OD sample is ≥OD control + 3SD. Or samples were considered positive if the ratio of OD of test samples divided by mean OD of negative control (naïve mouse serum, NMS) was ≥3. 2.5. Western blot (WB) WB was performed using reduced 3D7 and FC27 parasite lysates as previously described [16,17]. Immune mouse sera were used at dilution 1/200 in PBS-2.5% non-fat milk, then washed blots were incubated with horseradish peroxidase labeled goat anti-mouse IgG (Southern Biotech, Birmigham). Negative control (−C, normal mouse serum, NMS) and positive control (+C, serum from mice immunized with P27A + MSP2 LSP called FusN) were both used on 3D7 Plasmodium falciparum lysates. P27A is another intrinsically unstructured fragment derived from the P. falciparum blood stage protein PFF0165c as describedt by Olugbile et al. [17]. 2.6. Antibody dependent cellular inhibition (ADCI) The inhibition of P. falciparum (3D7 strain) growth in vitro in the presence of human monocytes (MN) plus antigen specific Abs was carried out by methods described elsewhere [18]. In brief, peripheral blood mononuclear cells (PBMC) isolated from healthy (malaria naïve) blood donors were separated by Ficoll-Paque (GE Healthcare, Frankfurt, Germany), and monocytes were further separated by adherence to plastic surfaces for 1 h at 37 ◦ C. Mature schizonts from a synchronized parasite culture were diluted at starting parasitemia of 0.5% in normal human AB+ type RBCs and the hematocrit was adjusted to 2% with RPMI 1640 culture medium. Duplicate assays were set up in preheated 96-wells flat bottom sterile plastic

plates (TPP, Trasadingen, Switzerland) containing 2 × 105 MN/well and by addition of 50 ␮l of parasite culture mixed with 50 ␮l of RPMI and various dilutions of each pool of sera at final concentration of 0.5–10%. Control wells with parasite culture and RPMI were done in parallel. The plates were incubated in a candle jar at 37 ◦ C, in a 5% CO2 incubator for 96 h. Thin blood smears for each well were fixed in methanol and stained in eosin and methylene blue. The parasitemia was determined by microscopic examination and counting of at least 5000 RBCs in duplicate. The Specific Growth Inhibitory index (SGI) which estimates the parasite growth inhibition due to the effect of test Abs cooperating with monocytes (MN) was calculated as follows: SGI = 100 × [1 − (% parasitemia with MN and test Abs/% parasitemia test Abs)/(% parasitemia with MN and N-IgG/% parasitemia N-IgG)]. For each antibody tested, duplicate wells included the following controls: (1) non-specific monocytic inhibition, both MN + parasite, and MN + normal (N)-IgG + parasites and (2) direct inhibition by control or test IgG, both N-IgG + parasites, and test Abs + parasites. PIAG (Pool of hyper-Immune African sera IgG) and N-IgG were used at a final concentration of 1 mg/ml and as positive and negative controls, respectively. Immunopurified test human Abs were used at 15 ␮g/ml while mouse sera were used at different dilutions. 3. Results 3.1. Immunogenicity and epitope mapping of D and C regions in mice immunized with MSP2 LSP mixture formulated in single adjuvants To optimize MSP2 vaccine candidates, sera from mice immunized with a mixture of the two allelic MSP2 LSPs (3D7-D+8aaC plus FC27-D+C; Table 1), formulated in alhydrogel, GLA-SE or Mt, were tested for Ab recognition in ELISA (Table 2). The results showed that the LSP mixture was immunogenic in all 3-mouse strains

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Table 2 Immunogenicity of D and C region in mice immunized with MSP2 LSP mixture formulated with single adjuvants. Mouse strain

Adjuvants

MSP2 peptides

GMT (log10)

SD (log 10)

Responders (OD ratio ≥3)

3.99 4.61 4.57 4.57 5.34 5.34 4.56 5.46 5.44 5.44 5.34 5.34 3.00 2.51 2.44

1.53 1.19 1.42 1.42

0.26 0.04 0.02

2/4 0/4 0/4

2.18 2.74 2.69 2.42 4.39 4.39 2.82 3.37 3.88 3.23 5.34 5.34 2.79 2.94 2.87

0.38 1.26 1.25 0.93

0/7 3/7 3/7 2/7

0.51 0.68 0.11

2/4 2/4 2/4

4.85 2.43 4.85 2.6 4.86 3.91 4.86 5.18 4.48 4.48 5.43 5.91 3.42 3.75 3.57

0.39 1.19 0.48 1.38

4/4 1/4 4/4 1/4

A

Alum

A/J GLA-SE

Montanide

3D7-D+8aaC FC27-D+C 3D7-D+C C Rec 3D7 Rec FC27 3D7-D+8aaC FC27-D+C 3D7-D+C C Rec 3D7 Rec FC27 3D7-D+8aaC FC27-D+C C

3/4 3/4 3/4 3/4 Done on pool Done on pool

0.83 0.24 0.6 0

4/4 4/4 4/4 4/4 Done on pool Done on pool

B

Alum

ICR GLA-SE

Montanide

3D7-D+8aaC FC27-D+C 3D7-D+C C Rec 3D7 Rec FC27 3D7-D+8aaC FC27-D+C 3D7-D+C C Rec 3D7 Rec FC27 3D7-D+8aaC FC27-D+C C

Done on pool Done on pool 1.30 1.31 1.25 1.35

2/7 4/7 5/7 2/7 Done on pool Done on pool

C

Alum

C3H GLA-SE

Montanide

3D7-D+8aaC FC27-D+C 3D7-D+C C Rec 3D7 Rec FC27 3D7-D+8aaC FC27-D+C 3D7-D+C C Rec 3D7 Rec FC27 3D7-D+8aaC FC27-D+C C

Done on pool Done on pool 0.00 0.28

4/4 4/4 Done on pool Done on pool Done on pool Done on pool

0.17 0.01 0.08

4/4 4/4 4/4

A/J (A), ICR (B) and C3H (C) (4 to 7 mice/group) were immunized with MSP2 LSP mixture formulated with different adjuvants as described in Section 2. GMT represents the geometric mean titer of Ab responses determined by ELISA. Rec is the full-length recombinant protein MSP2-3D7 or FC-27. “Done on pool” means sera from mice were pooled to perform ELISA.

(Table 2A–C), the highest responses being, in general, obtained with GLA-SE. In A/J and ICR strains, lower responses were associated with Montanide compared to the other adjuvants (Table 2A and B), and with Alum in C3H (Table 2C). Overall, outbred ICR mice seem to be the strain that gave the lowest immune responses. In addition, full-length recombinant proteins, Rec-3D7 and RecFC27 [8], were well recognized by serum pools from the 3 strains of mice (Table 2) immunized with alhydrogel or GLA-SE formulations (this was not done for Montanide groups). As expected, sera from all mouse strains recognized not only the immunogen 3D7-D+8aaC LSP but also the longer 3D7-D+C LSP as shown in GLASE and Alum formulation (Table 2). In addition, all immune sera tested recognized both 3D7 and FC27 parasites in IF assay (data not shown). To further characterize the immune response against D and C regions, epitope mapping using the overlapping 20-mers was performed on serum pools from the 3 mouse strains. The P19 20-mer

peptide, which contains the last 8 aa of the 3D7 D region and the first 12 aa from the C region (Table 3A), was the best recognized, mainly in association with GLA-SE in all of the 3 strains. For the FC27-D, the recognition of 20-mer peptides was mainly restricted to the A/J strain in the presence of alum with multiple epitope recognition (4 out of 5; Table 3B). The common recognized 20 mers were P23, P25 and P27 (which contains the 8 last aa of the FC27 D region and the first 12 aa from the C region; Table 3B). In the C region P28 appeared to be the most recognized sequence in the 3 mouse strains (Table 3B). 3.2. Immunogenicity and epitope mapping of D and C fragments in mice immunized with MSP2 LSP mixture formulated with different GLA-SE combinations In order to determine if addition of other TLR agonists to the GLA-SE adjuvant (TLR4 agonist) would have an effect on the

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Table 3 D and C epitope mapping in mice immunized with MSP2 LSP mixture formulated with single adjutants.

A

3D7 family peptides Mouse strains Adjuvants

D+C LSP

D-LSP

P11

P12

P13

P14

P15

P16

P17

P18

P19

Alum GLA-SE Montanide Alum GLA-SE Montanide Alum GLA-SE

A/J

C3H

ICR

Montanide

B

FC27 family peptides Mouse strains A/J

C3H

ICR

Adjuvants

D+C-LSP

D-LSP

P23

P24

C region peptides P25

P26

27

P28

P29

P30

CLSP

Alum GLA-SE Montanide Alum GLA-SE Montanide Alum GLA-SE Montanide

Code of ratio value

1-3 ≥3-5 ≥ 5 - 10 ≥ 10 A/J, C3H and ICR strains (5 mice/group) were immunized with MSP2 LSP mixture as indicated in Section 2. Serum pool from each strain and different adjuvant formulations was obtained to perform ELISA. A and B show Ab responses against D and C region LSPs and 20-mer peptides covering the two allelic families of MSP2, 3D7 and FC27, respectively. Results are expressed as ratio of mean of Ab OD of test sample/mean of OD of negative control, and are evaluated on a scale code.

immunogenicity of the MSP2 fragment mixture and fine specificity of the corresponding Abs, A/J strain mice were immunized with MSP2 LSP mixture in the presence of different combinations of TLR agonists as described in Section 2. The A/J strain was considered here because it generated, in general, the best Ab responses in formulation with GLA-SE as shown in Table 2. Individual sera from mice immunized with the MSP2 LSP mixture (3D7-D+8aaC plus FC27-D+C LSPs) formulated in Montanide, alum and in GLASE present in different combinations with CpG and/or GDQ were tested (Table 4). Results obtained with 3D7 allelic derived fragment showed that recognition was restricted to P19, with an increase of Ab response in the presence of CpG and GDQ compared to all other formulations (Table 4A). In the FC27 allelic family, responses to P25 and P27 were higher for the GLA-SE adjuvant associated with different CpG and/or GDQ (Table 4B). On the other hand in the case of the C region and epitope P28, the highest Ab levels were also associated with the presence of CpG and GDQ (Table 4B). Furthermore, nearly all of mice recognized the major epitopes of the two allelic family and C regions, as indicated by the number of responder mice (Table 4).

3.3. D and C immunogenicity in C3H mice immunized with single region specific LSP in formulation with Montanide ISA720 Here the aim was to determine whether immunization with single LSP covering each allelic family D or C regions of MSP2 could induce a vigorous Ab response, and thus obtain region specific immune sera. For that, C3H mice (5 mice/group) were immunized 3 times with allelic specific peptides (3D7-D or FC27-D LSPs) or with their common C LSP, all formulated in Mt adjuvant (50 ␮l, sc). The choice of this strain was motivated by the fact that the C3H mice were giving a good response to individual LSP immunization formulated with in Mt and no other adjuvants were tested (data not shown). In ELISA, pool of immune sera recognized only the peptide used for immunization (data not shown), unlikely in humans where a cross-reactivity was observed between the two allelic families in natural exposed populations [1]. Thus in WB, sera from allelic specific D-immunized mice recognized only the homologous MSP2 polypeptide (Fig. 2A). As expected, C region reactive sera recognized MSP2 protein of both allelic families (Fig. 2B) and confirmed data previously observed in humans [1,7,8].

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Table 4 D and C fragment immunogenicity and epitope mapping in A/J mice after immunization with MSP2 mixture formulated with different adjuvant combinations.

Code of ratio value

1-3 ≥3-5 ≥ 5 - 10 ≥ 10 Sera were obtained from A/J mice (3–4 mice/group) immunized with MSP2 LSP mixture formulated with different adjuvants as described in Section 2. (A) Shows the recognition of 3D7-D LSP and its 20 mers; (B) that of FC27-D and C regions and their respective 20 mers. The number of mouse sera whose ratio was equal or higher than 3 relative to the negative control determined responder mice.

3.4. MSP2 LSP mixture immune sera are active and inhibit growth of both allelic MSP2 parasites in ADCI assay Once the immunogenicity of MSP2 LSP mixture formulations was assessed it was important to determine if the immune sera from both inbred and outbred mouse strains or sera from GLA-SE and alum formulation groups could inhibit parasite growth in vitro. For that, sera from both A/J and ICR mice immunized with the

MSP2 LSP mixture formulated in GLA-SE or those from A/J immunized with the MSP2 mixture formulated with GLA-SE or alum were pooled. ADCI was then performed on both 3D7 and FC27 parasites at dilutions of sera 1:40, 1:200 and 1:1000. Growth of both allelic family parasites (3D7 and FC27) was inhibited whatever the mouse strains and adjuvants used (Fig. 3A and B). The data clearly showed that parasite inhibition was dependent on the dilution of immune sera: the less diluted the sera, the higher the inhibition of the two

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Fig. 2. D and C LSP immunized mice sera recognized merozoite-derived proteins in Western blot. Western blot shows 3D7 and FC27 merozoite-derived MSP2 binding. (A) Pooled sera from C3H mice (5 mice/group) immunized with 3D7-D or FC27-D fragments formulated with Montanide (Mt) were tested on both allele parasite proteins (lysates) at a dilution of 1/200; (B) results of pooled sera from immunization of C3H (5 mice/group) with C region LSP formulated with Mt and tested both on 3D7 and FC27 parasites (lysates) at a dilution of 1/100. All bands were visualized with enhanced chemiluminescence. Negative control (−C, NMS) and positive control (+C, serum from mice immunized with a recombinant product containing P27A and MSP2 called FusN) were both used on 3D7 P. falciparum lysates.

Fig. 3. Capability of MSP2 LSP mixture specific immune sera in P. falciparum parasite growth inhibition in ADCI. ICR and A/J strain immune sera were obtained after immunization with MSP2 LSP mixture (3D7-D+8aaC plus FC27-D+C) formulated with GLA-SE adjuvant. A/J were also immunized with MSLP2 LSP mixture formulated with GLA-SE or alum. Sera of each strain and adjuvants groups were then pooled to perform P. falciparum growth inhibition in ADCI. Different dilutions of sera were carried out. (A) Shows 3D7 parasite inhibition; (B) FC27 parasite inhibition. PIAG (purified immune adult globulin) from Africa endemic area was used at 10% as positive control for its highly inhibitory activity on both allelic parasites growth. MSP2 represents the MSP2 LSP mixture. GMT titers of serum pools are given in Table 2 using Rec-MSP2 proteins.

allelic parasites, suggesting that Ab levels were a limiting factor in elicitation of ADCI activity. However, if inhibition of 3D7 and FC27 parasites appeared to be similar at lower dilutions (1:40, 1:100) of sera, at dilution 1:1000 inhibition was less effective against the FC27 compared to 3D7 parasites. Overall, inhibition was similar between the two strains (outbred and inbred) at the same dilution for each allelic parasite. Furthermore, despite the relative high Ab titer GLA-SE (as shown in ELISA), ADCI effect on parasite growth

seems to be similar between the two adjuvants at the same dilution for each allelic parasite (Fig. 3A and B). 4. Discussion This preclinical immunogenicity assessment of MSP2 dimorphic and constant fragments in mice represents the continuity of our previous study in immune human populations [1]. The human

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data suggested that the two MSP2 allelic-derived LSPs would represent valuable vaccine candidates against blood-stage malaria. Since D and C regions of MSP2 exhibit an unstructured conformation, they are particularly suitable to compare the fine specificity of Ab response obtained in mice and humans using overlapping 20-mer peptides. Thus, immunogenicity evaluation of the D and C LSPs formulated with different human compatible adjuvants may provide indications to optimize MSP2-based vaccines. To this aim, immunization in different strains of mice with different adjuvants was performed to identify optimal formulations. Secondly, the fine specificity of Ab response to the MSP2 two allelic and constant regions was determined. Thirdly, the biological activity of specific immune sera in inhibiting P. falciparum growth in ADCI was assessed. As shown in the results section, immunization of mice with mixture of MSP2 LSPs formulated with different single adjuvants resulted in vigorous Ab responses against D and C domains, and recombinant MSP2 proteins (Tables 2–4). This supports the notion of a simultaneous inclusion of D allelic family and C regions in vaccine studies as previously suggested [1,7]. The current results are consistent with those obtained in humans where recombinant MSP2 proteins were recognized by D and C reactive human purified Abs [7,8,19–23]. In most of mouse strains the major epitopes were restricted to the sequences comprising the last 8 aa of 3D7-D and first 12 aa of C (P19) and the first 20 aa (P28) of the C region (Table 3). Interestingly, the level and the specificity of Ab response against these epitopes were dependent on the adjuvant used. Since using the single adjuvant formulations influenced Ab level and epitope recognition, the immunization was extended in combination of GLA-SE with other different TLR agonist adjuvants (CpG and GDQ). Thus, as shown in Table 4, immune responses were stronger after immunization with GLA-SE and its combination with other TLR agonists (CpG or GDQ) than alhydrogel or Mt, but without reaching a significant p value either for the immunizing fragment 3D7-D+8aaC or D alone. On the other hand recognition of peptide 19 is the highest with the GL-CpG-GDQ-SE. Similar results were obtained for FC-27 D region where the difference among results obtained with all combinations were not statistically significant. On the contrary a clear difference was observed for C fragment where all of the additional GLA-SE formulations were better than GLA-SE. The generally lower immunogenicity of alum-based formulations compared to GLA-SE was expected as observed with another unstructured fragment derived from blood stage protein PFF0165c [17]. Another interesting point as observed with single adjuvant formulations is the difference of the magnitude and the fine specificity of the response observed for the FC-27 fragment depending on the formulations used. The dependence of breadth of an antibody response upon use of specific TLR agonists was originally described by Wiley et al. [24]. Further studies are needed to confirm and expand these observations. Comparison between the immune response in humans and mice indicates that some major epitopes defined in natural exposed human population are also immunogenic in mice [1]. This similarity was mainly observed for Montanide formulation (P13 for 3D7; P25 for the FC27 and P29 for the C region), and P25 of FC27 family in alum and GLA-SE with its different combinations. Of interest is the also fact that allelic specific mouse immune sera recognized MSP2 only in allelic-specific manner unlike in humans where cross-reactivity is observed [1,8]. This suggests that the cross-reactivity between the two allelic families of MSP2 shown in humans is most likely acquired by prolonged multiple infection with both 3D7 and FC27 parasites. In addition this study also shows that the sera of immunized mice (Fig. 3) inhibit parasite growth in ADCI. Antibodies to MSP2 proteins have been recently implicated and associated with protection in two other assays, opsonisation of and complement fixation

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on merozoites [25,26]. It would be interesting to extend these studies to MSP2 fragments described here. 5. Conclusion These data offer the possibility to better understand the immune responses leading to a differential quantitative and potentially qualitative Ab response to MSP2 LSPs according to the adjuvant(s) used. The possibility to finely delineate in animals the immune responses to antigens may help to select optimal antigen/adjuvant combinations to be later tested in clinical trials. In particular this study indicates that the combination of the family specific and common MSP2 fragments formulated with GLA-SE (with or without CpG) represents a promising option to be tested and compared to the alum formulation in human clinical trials. Acknowledgements This work has been supported by the European Vaccine Initiative and EC LSHP-CT-2007-037506 grants. Competing interests The authors have declared that they have no competing interests. References [1] Balam S, Olugbile S, Servis C, Diakité M, D’Alessandro A, Frank G, et al. Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains. Malar J 2014;13:510. [2] Aucan C, Traoré Y, Tall F, Nacro B, Traoré-Leroux T, Fumoux F, et al. High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria. Infect Immun 2000;68(3):1252–8. [3] Metzger WG, Okenu DM, Cavanagh DR, Robinson JV, Bojang KA, Weiss HA, et al. Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria. Parasite Immunol 2003;25(6):307–12. [4] al-Yaman F, Genton B, Anders RF, Falk M, Triglia T, Lewis D, et al. Relationship between humoral response to plasmodjum falciparum merozoite surface antigen-2 and malaria morbidity in a highly endemic area of papua new guinea. Am J Trop Med Hyg 1994;51(5):593–602. [5] al-Yaman F, Genton B, Anders RF, Taraika J, Ginny M, Mellor S, et al. Assessment of the role of the humoral response to Plasmodium falciparum MSP2 compared to RESA and SPf66 in protecting Papua New Guinean children from clinical malaria. Parasite immunol 1995;17(9):493–501. [6] Wright PE, Dyson HJ. Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 1999;293(2):321–31. [7] Flueck C, Frank G, Smith T, Jafarshad A, Nebie I, Sirima SB, et al. Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates. Vaccine 2009;27(20):2653–61. [8] Stubbs J, Olugbile S, Balam S, Simpore J, Corradin G, Lanzavecchia A. Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies. Infect Immun 2011;79(3):1143–52. [9] Pye D, Vandenberg KL, Dyer SL, Irving DO, Goss NH, Woodrow GC, et al. Selection of an adjuvant for vaccination with the malaria antigen, MSA-2. Vaccine 1997;15(9):1017–23. [10] Mata E, Carcaboso AM, Hernández RM, Igartua M, Corradin G, Pedraz JL. Adjuvant activity of polymer microparticles and Montanide ISA 720 on immune responses to Plasmodium falciparum MSP2 long synthetic peptides in mice. Vaccine 2007;25(5):877–85. [11] Lombardi V, Van Overtvelt L, Horiot S, Moingeon P. Human dendritic cells stimulated via TLR7 and/or TLR8 induce the sequential production of Il-10, IFN-g, and IL-17A by naive CD4+ T cells. J Immunol 2009;182(6):3372–9. [12] Klinman DM. Adjuvant activity of CpG oligodeoxynucleotides. Int Rev Immunol 2006;25(3–4):135–54. [13] Weeratna RD, Makinen SR, McCluskie MJ, Davis HL. TLR agonists as vaccine adjuvants: comparison of CpG ODN and Resiquimod (R-848). Vaccine 2005;23(45):5263–70. [14] Coler RN, Baldwin SL, Shaverdian N, Bertholet S, Reed SJ, Raman VS, et al. A synthetic adjuvant to enhance and expand immune responses to influenza vaccines. PLoS ONE 2010;5(10):e13677. [15] Agak GW, Bejon P, Fegan G, Gicheru N, Villard V, Kajava AV, et al. Longitudinal analyses of immune responses to Plasmodium falciparum derived

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