Generation of a Peptide Vaccine Candidate Against Falciparum Placental Malaria Based on a Discontinuous Epitope

Communication Generation of a Peptide Vaccine Candidate against Falciparum Placental Malaria Based on a Discontinuous Epitope

Catherine J. Mitran 1, Lauren M. Higa 1, Michael F. Good 2,3 and Stephanie K. Yanow 1,3,* 1 School of Public Health, University of Alberta, Edmonton, AB T6G 2R3, Canada; [email protected] (C.J.M.); [email protected] (L.M.H.) 2 Institute for Glycomics, Griﬃth University, Southport, Queensland 4215, Australia; michael.good@griﬃth.edu.au 3 Department of Medical Microbiology & Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB T6G 2R3, Canada * Correspondence: [email protected]

Received: 26 May 2020; Accepted: 10 July 2020; Published: 18 July 2020

Abstract: In pregnant women, Plasmodium falciparum-infected red blood cells adhere to the placenta via the parasite protein VAR2CSA. Two vaccine candidates based on VAR2CSA are currently in clinical trials; however, these candidates failed to elicit strain-transcending antibody responses. We previously showed that a cross-reactive monoclonal antibody (3D10) raised against the P. vivax antigen PvDBP targets epitopes in VAR2CSA. We now aim to design a peptide vaccine against VAR2CSA based on the epitope that generated 3D10. We mapped the epitope to subdomain 1 (SD1) of PvDBP and identified a peptide that contained the minimal sequence. However, this peptide did not elicit cross-reactive VAR2CSA antibodies in mice. When tested against a broader, overlapping peptide array spanning SD1, 3D10 in fact recognized a discontinuous epitope consisting of three segments of SD1. These findings presented the challenge to generate this larger structural epitope as a synthetic peptide since it is stabilized by two pairs of disulfide bonds. We overcame this using a synthetic scaffold to conformationally constrain the SD1 peptide and coupled it to keyhole limpet hemocyanin (KLH). The SD1-KLH conjugate elicited antibodies in mice that cross-reacted with VAR2CSA. This strategy successfully recapitulated a discontinuous epitope with a synthetic peptide and represents the first heterologous vaccine candidate against VAR2CSA.

Keywords: Plasmodium falciparum; Plasmodium vivax; vaccine; placental malaria; peptide; PvDBP; VAR2CSA

1. Introduction Pregnancy-associated malaria caused by infection with the parasite Plasmodium falciparum can result in preterm birth, low birth weight babies, spontaneous abortion, and infant and maternal death [1]. Despite ongoing efforts to develop a vaccine to prevent malaria in pregnancy, no effective vaccine exists. The leading vaccine candidates are based on VAR2CSA, a P. falciparum protein that mediates sequestration of infected red blood cells to the placenta [2–7]. While these vaccines show promise by eliciting strong antibodies to the homologous VAR2CSA allele, they failed to elicit broadly neutralizing antibodies against heterogeneous parasite strains due to extensive natural polymorphisms within VAR2CSA [6–8]. Polyvalent vaccines that include multiple alleles of VAR2CSA or new vaccines that target conserved epitopes are urgently needed. We discovered an alternate source of antibodies to VAR2CSA that can be exploited for vaccine design. The source of these antibodies is an epitope shared between the Duffy binding-like (DBL)

Vaccines 2020, 8, 392; doi:10.3390/vaccines8030392 www.mdpi.com/journal/vaccines Vaccines 2020, 8, 392 2 of 13 domain of the P. vivax Duffy binding protein (PvDBP), an invasion protein expressed by P. vivax merozoites, and the DBL domains of VAR2CSA [9]. Despite sharing only 16–21% sequence homology, the DBL domains of VAR2CSA and PvDBP have shared epitopes that are targeted by cross-reactive antibodies elicited by natural exposure to P. vivax infection or through immunization with PvDBP [9,10]. A mouse monoclonal antibody (3D10 mAb) raised against the DBL domain of PvDBP recognized VAR2CSA and blocked parasite adhesion in an in vitro assay of placental malaria [9]. When we investigated the cross-reactive target of 3D10, we found that it recognized epitopes that were cryptic in VAR2CSA [10]. Given the cryptic nature of these epitopes, they are unlikely to be under the same immune pressure as the more immunodominant epitopes in the protein. Therefore, identifying and targeting these epitopes in VAR2CSA may present a viable vaccine strategy against malaria in pregnancy. Targeting cryptic or subdominant epitopes has also been employed in the development of vaccine candidates for group A streptococcus [11], Ebola [12], and influenza [13–15]. Here, we designed an epitope-focused vaccine candidate against VAR2CSA based on the epitope that generated the 3D10 mAb. This epitope has been localized to subdomain 1 (SD1) of PvDBP region II (DBPII) [10,16,17], and we used peptide arrays to refine the epitope to three discontinuous segments of SD1. Using a synthetic scaffold, we recapitulated this discontinuous epitope within a conformationally constrained peptide. Importantly, this peptide elicited antibodies in mice and a rabbit that recognized DBPII and cross-reacted with VAR2CSA.

2. Materials and Methods

2.1. Synthetic Peptide Design and Conjugation Peptides representing different regions of SD1 in DBPII (Figure1) were synthesized (Synpeptides Co., Shanghai, China) based on the sequence from the Sal 1 allele of PvDBP. The SD1ss peptide was designed to cover the entire SD1 region, with one pair of cysteines (C9 and C38) mutated to serine to control disulfide bond formation. N10-C22 was conjugated to diphtheria toxoid (DT) using 6-maleimido-caproyl n-hydroxy succinimide (MCS) (Sigma, Oakville, Canada) [18]. Briefly, MCS dissolved in dimethylformamide (DMF) (33.3 mg/mL) was added to a solution of DT in 0.1 M phosphate buffer (10 mg/mL) and mixed slowly at room temperature for 1 h. The modified carrier protein was then dialyzed against 0.1 M phosphate buffer containing 0.1 M ethylenediaminetetraacetic acid (EDTA) before mixing with the lyophilized N10-C22 peptide (1.2 M excess of peptide). The conjugate was dialyzed overnight against 1X phosphate-buffered saline (PBS) and coupling was confirmed using SDS-PAGE analysis.

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N10-C29: NYKRKRRERDWDCNTKKDVC

C22-C29: CNTKKDVC

C22-K40: CNTKKDVCIPDRRYQLCMK

C29-K40: CIPDRRYQLCMK

Figure 1. Synthetic peptides were designed to cover the subdomain 1 (SD1) region of DBPII. The subscript numbers indicate the amino acid position in the parent SD1 peptide. Disulﬁde bonds are noted with horizontal lines.

Thirty-seven overlapping 10-mer linear peptides were designed (Pepscan, Amsterdam, Netherlands) to span the SD1 region of DBPII (Table S1). Chemically Linked Peptides on Scaﬀolds (CLIPS) technology was used to synthesize the SD1CLIPS peptide (C(T2-013)NYKRKRRERDWDCNT Vaccines 2020, 8, 392 3 of 13

KKDVCIPDRRYQLC(T2-013)K(Aoa)), as previously described [19]. The conformation of SD1CLIPS was constrained by first conjugating the outermost pair of cysteines (residues 1 and 30) using the T2-013 scaffold. The two interior cysteines (residues 14 and 21) were then deprotected and oxidized to form a disulfide bond. This peptide was conjugated to keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) using a N-succinimidyl 4-formylbenzoate (S-4FB) linker via the C-terminal N-epsilon-aminooxyacetyl-L-lysine residue on the peptide (Pepscan, Amsterdam, Netherlands). Conjugation was monitored by adding 2-hydrazinpyridine to the carrier protein/peptide mixture. This reagent reacted with free S-4FB linkers on the carrier protein, producing a colored product, which allowed the reaction to be monitored over time. Excess peptide was removed by filtration.

2.2. Animal Immunizations

For the (N10-C22)-diphtheria toxoid (DT) immunizations, five female BALB/c mice (6 to 8 weeks old) were immunized subcutaneously with (N10-C22)-DT or DT alone (30 µg/mouse) emulsified in Complete Freund’s Adjuvant (CFA) (catalogue no. F5881; Sigma, Oakville, Canada) on day 1. On days 21 and 31, mice were boosted with the immunogen (10 µg/mouse) emulsified in Incomplete Freund’s Adjuvant (IFA) (catalogue no. F5506; Sigma, Oakville, Canada) and the final sera samples were collected on day 45 via cardiac puncture. For the SD1CLIPS-KLH immunizations, five female BALB/c mice (6 to 8 weeks old) were immunized subcutaneously with SD1CLIPS-KLH or KLH alone (30 µg/mouse) emulsified in CFA on day 1. On days 21, 31, and 41, mice were boosted with the immunogen (10 µg/mouse on days 21 and 31, and 5 µg/mouse on day 41) emulsified in IFA. The final sera samples were collected on day 55 via cardiac puncture. All procedures were approved by the University of Alberta Animal Care and Use Committee (approval number: AUP00002124), and mice were handled in accordance with the Canadian Council on Animal Care Guidelines. Rabbit antisera were generated commercially by ProSci Inc. (Poway, CA, USA). One rabbit was immunized with 160 µg of SD1CLIPS-BSA in CFA followed by three boosts with 80 µg in IFA on days 14, 28, and 43 after the first immunization. The final bleed was collected on day 56.

2.3. ELISAs Indirect ELISAs were performed by coating 96-well plates (catalogue no. 439454; Thermo Fisher Scientiﬁc, Edmonton, Canada) with antigen diluted in 1X PBS, incubated overnight at 4 ◦C. Recombinant protein antigens were coated at 0.5 µg/mL, SD1ss was coated at 1.0 µg/mL, and all other synthetic peptides were coated at 5.0 µg/mL. Plates were blocked with 4% BSA (catalogue no. A7906; Sigma, Oakville, Canada) in 1X PBS for 1 h at 37 C, then washed once with 1 PBST (0.1% Tween 20). ◦ × Primary antibody samples were diluted in 2% BSA, added to wells, and incubated for 1 h at room temperature (RT). Plates were washed four times with 1 PBST, and 100 µL of horseradish peroxidase × (HRP)-conjugated goat anti-mouse secondary antibody (1/3000) (catalogue no. 170-6516, Bio-Rad, Mississauga, Canada) was added to each well. After incubation for 1 h at RT, the plate was washed four times with 1X PBST, and 100 µL of 3,30,5,50-tetramethylbenzidine (TMB) (catalogue no. T0440; Sigma, Oakville, ON, Canada) was added to each well. After incubation at RT for 30 min, the reaction was stopped by adding an equal amount of H2SO4 (0.5 N) to each well, and the optical density (OD) of individual wells was read at 450 nm. All samples were run in duplicate and the mean OD for each antigen alone plus secondary was subtracted from the OD of each sample. Competition ELISAs were performed as above, except that primary antibodies were incubated with test peptides for 30 min at RT before addition to wells. To measure antibody avidity, ELISAs were performed using the same protocol for indirect ELISAs, except that 100 µL of 1 M NaSCN (catalogue no. 251410; Sigma, Oakville, Canada) or 1 PBS for control wells was added to each well following the × primary antibody incubation. After a 10 min incubation at RT, the plates were washed four times with 1 PBST (0.1% Tween 20). The remaining steps were carried out as outlined for the indirect ELISA. × Data represent samples tested in duplicate in at least two independent experiments. Vaccines 2020, 8, 392 4 of 13 Vaccines 2020, 8, x 4 of 13

structure using a synthetic peptide, but based on mass spectrometry, the disulfide bonds did not form The peptide library was screened by Pepscan-based ELISA (Pepscan, Amsterdam, The in the correct configuration (data not shown). Therefore, we designed a peptide called SD1ss, in µ Netherlands).which two Briefly, of the cysteine the peptide residues array were was mutated incubated to serines with 3D10(‘ss’), (0.2whichg allowed/mL) overnight us to control at 4 ◦theC then washedintramolecular with 1X PBST. disulfide The arraybonding was pat thentern. incubatedWhen we screened with HRP-conjugated these peptides by rabbit ELISA anti-mouse; the regions IgG (cataloguenearest no. the 6175-05, amino terminus Southern of Biotech, SD1 (N10 Birmingham,-C29 and N10-C AL,22) were USA) strongly and incubated recognized for by 1 3D10 h at 25(Figure◦C. After washing,2A). 2,2The0-azino-di-3-ethylbenzthiazoline minimal epitope recognized by 3D10 sulfonate was a (ABTS)12 amino was acid added peptide with called 20 µ NL10/mL-C22 ofthat H 2wasO2 (3%) and therec colorognized development with an endpoint was measuredtiter of 2.7 afterng/mL, 1 h.similar to the reactivity against the parent protein DBPII. This finding is supported by previous reports that 3D10 recognizes the NXXRKR motif within 2.4. Statisticalthis peptide Analysis [16,20]. Using a competition ELISA, this peptide was sufficient to block heterologous recognition of VAR2CSA by 3D10 (Figure 2B). When 3D10 was incubated with increasing Data were plotted using Prism software (version 8; GraphPad, San Diego, CA, USA). concentrations of this peptide, N10-C22 blocked recognition of VAR2CSA to a similar degree as SD1ss. The C29-K40 peptide was included as a negative control for inhibition because 3D10 did not recognize 3. Results this peptide (Figure 2A). Based on these results, we concluded that N10-C22 represents the minimal epitope for homologous 3D10 recognition of DBPII and mediates heterologous recognition of 3.1. N10-C22 Is the Minimal Epitope for 3D10 Recognition, but Does Not Elicit Cross-Reactive Antibodies VAR2CSA. In order to identify the minimal epitope for 3D10 recognition, we designed a number of synthetic peptides spanning theSD1: SD1 regionASNTVMKNCNYKRKRRERDWDCNTKKDVCIPDRRYQLCMK of DBPII (Figure1). In the native protein, this region contains four cysteine residues that formSD1ss: twoASNTVMKNSNYKRKRRERDWDCNTKKDVCIPDRRYQLSMK disulfide bonds. We previously tried to recapitulate the native structure using a synthetic peptide, but based on mass spectrometry, the disulfide bonds did not form in the N10-C22: NYKRKRRERDWDC correct configuration (data not shown). Therefore, we designed a peptide called SD1ss, in which two N10-C29: NYKRKRRERDWDCNTKKDVC of the cysteine residues were mutated to serines (‘ss’), which allowed us to control the intramolecular C22-C29: CNTKKDVC disulfide bonding pattern. When we screened these peptides by ELISA; the regions nearest the amino N22-K40: CNTKKDVCIPDRRYQLCMK terminus of SD1 (N10-C29 and N10-C22) were strongly recognized by 3D10 (Figure2A). The minimal C29-K40: CIPDRRYQLCMK epitope recognized by 3D10 was a 12 amino acid peptide called N10-C22 that was recognized with an endpoint titer of 2.7 ng/mL, similar to the reactivity against the parent protein DBPII. This finding is Figure 1. Synthetic peptides were designed to cover the subdomain 1 (SD1) region of DBPII. The supportedsubscript by previous numbers reports indicate that the amino 3D10 acid recognizes position in the the NXXRKR parent SD1 motifpeptide. within Disulfide this bonds peptide are [16,20]. Using a competitionnoted with horizontal ELISA, thislines. peptide was sufficient to block heterologous recognition of VAR2CSA by 3D10 (Figure2B). When 3D10 was incubated with increasing concentrations of this peptide, N 10-C22 We next immunized five BALB/c mice with the N10-C22 peptide conjugated to DT. The mice blocked recognition of VAR2CSA to a similar degree as SD1ss. The C29-K40 peptide was included as a negativeproduced control high for levels inhibition of antibodies because against 3D10 did N10- notC22; recognizehowever, the this antibodies peptide (Figure did not2 crossA). Based-react onwith these VAR2CSA by ELISA (Figure 2C). This lack of cross-reactivity is not surprising, considering these results, we concluded that N -C represents the minimal epitope for homologous 3D10 recognition antibodies also failed to recognize10 22 DBPII, the cognate protein from which the peptide was derived of DBPII(Figure and 2C). mediates heterologous recognition of VAR2CSA.

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and after immunization with (N10-C22)-DT were tested against N10-C22, VAR2CSA, and DBPII by ELISA. Data are mean standard deviation (SD). ± VaccinesVaccines2020, 20208, 392, 8, x 5 of 13 5 of 13

3D10 was incubated alone (black bar) or with increasing concentrations of SD1ss, N10-C22, or C29-K40, We nextthen added immunized to wells coated five BALB with recombinant/c mice with full-length the N VAR2CSA.10-C22 peptide (C) Pooled conjugated sera from mice to DT. (n = The mice 5) before and after immunization with (N10-C22)-DT were tested against N10-C22, VAR2CSA, and DBPII produced high levels of antibodies against N10-C22; however, the antibodies did not cross-react with VAR2CSAby byELISA. ELISA Data (Figureare mean2 ±C). standard This deviation lack of cross-reactivity(SD). is not surprising, considering these antibodies3.2. 3D10 also Recognizes failed to a Discontinuous recognize DBPII, Epitope the in SD1 cognate protein from which the peptide was derived (Figure2C). To map the recognition site of 3D10, we designed an array of 37 overlapping linear peptides 3.2. 3D10spanning Recognizes SD1 of a DBPII Discontinuous (Table S1) Epitope. We used in SD1 a Pepscan-based ELISA for this screen because the peptides can be coated at a high concentration, which allows for identification of minor parts of the epitopeTo map that the may recognition not be detected site of using 3D10, a weconventional designed ELISA. an array We ofscreened 37 overlapping this library linearwith 3D10 peptides spanningand found SD1 ofthat DBPII it recognized (Table S1). peptides We used from a three Pepscan-based regions of SD1 ELISA (Figure for this3A). screenThese results because suggested the peptides can bethat coated the epitope at a high is discontinuous. concentration, We which then screened allows fora library identification of these peptides of minor w partshere two of theresidues epitope in that mayeach not be peptide detected were using mutated a conventional to alanine (Figure ELISA. 3B). We Several screened of these this mutations library with resulted 3D10 in anda significant found that it recognizeddecrease peptides in 3D10 from recognition. three regions These of data SD1 further (Figure confirm3A). These that results sequences suggested within thethat two the epitopeouter is discontinuous.segments in Weparticular then screened are critical a libraryfor 3D10 of recognition. these peptides where two residues in each peptide were In the native DBPII structure, there are two disulfide bonds that bring these regions in close mutated to alanine (Figure3B). Several of these mutations resulted in a significant decrease in 3D10 proximity, allowing them to form the discontinuous epitope (Figure 3C). This presents a significant recognition. These dataA further confirm that sequences within the two outer segments in particular are challenge for vaccine2500 development as conformational epitopes are notoriously difficult to critical for 3D10 recognition.

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FigureFigure 3. 3D10 3. 3D10 recognizes recognizes a discontinuousa discontinuous epitopeepitope in in SD1. SD1. (A (A) An) An overlapping overlapping peptide peptide array array spanning spanning the SD1the region SD1 region of DBPII of DBPII was screened was screened with 3D10. with 3D10. The colors The colors of the of lines the above lines abovethe bar th graphe bar correspond graph to thecorrespond region of to SD1 the mapped region of toSD1 the mapped structure to the in structure (C). (B) Thein (C). intensity (B) The ofintensity 3D10 recognitionof 3D10 recognition of peptides with alanine substitutions (bolded) was subtracted from that of the native sequence. Negative values indicate that 3D10 recognition was decreased following alanine substitution. (C) The structure of SD1 was determined from a published crystal structure of DBPII that was adapted using PyMOL (PDB ID 4NUU). The cysteines involved in disulﬁde bonds are shown as space-ﬁlling spheres. Vaccines 2020, 8, 392 6 of 13

In the native DBPII structure, there are two disulfide bonds that bring these regions in close proximity, allowing them to form the discontinuous epitope (Figure3C). This presents a significant challenge for vaccine development as conformational epitopes are notoriously difficult to recapitulate with synthetic peptides [21].

3.3. Immunization with SD1CLIPS Elicited Cross-Reactive VAR2CSAAntibodies To recapitulate the immunogen that elicited the cross-reactive 3D10 mAb in DBPII, we designed a 31 amino acid peptide called SD1CLIPS that uses CLIPS technology to control bond formation and conformationally constrain the peptide to its native structure. Specifically, a synthetic scaffold was inserted in place of one of the disulfide bonds to ensure that the disulfide bond formed between the correct pair of cysteines (Figure4A). After confirming that 3D10 recognized this peptide by ELISA (endpoint titer: 20.5 ng/mL), we immunized five BALB/c mice with SD1CLIPS conjugated to keyhole limpet hemocyanin (KLH). All mice produced high levels of antibodies against SD1CLIPS and 60% (3/5) of the mice also made antibodies that cross-reacted with VAR2CSA (Figure4B). Although the titers of the anti-VAR2CSA antibodies were relatively low compared to those against SD1CLIPS, these results demonstrate that a peptide immunogen successfully recapitulated a cross-reactive conformational epitope. When we measured the avidity of the cross-reactive antibodies against VAR2CSA, the avidity was low compared to the avidity of the antibodies for their immunogen. Incubation with 1 M NaSCN significantly reduced antibody binding to VAR2CSA across a range of dilutions (Figure4C–E), whereas there was little reduction in antibody binding to SD1CLIPS (Figure S1A–E). It is not uncommon for cross-reactive antibodies to have a lower avidity for the heterologous antigen. However, this does not negate the potential functional relevance of these antibodies. In fact, the avidity of these antibodies for VAR2CSA is similar to that of 3D10 (Figure4F), which blocked VAR2CSA-expressing P. falciparum parasites from binding to the placental ligand in vitro [9].

3.4. Fine Speciﬁcity and Avidity of Anti-SD1CLIPS Antibodies in Individual Mice

Despite making similar levels of antibodies against the SD1CLIPS immunogen, only three out of the five mice made cross-reactive antibodies that recognized VAR2CSA. This prompted us to evaluate the fine specificity of the individual immune responses of each mouse for DBPII and the smaller peptides within the SD1 region. Sera from all of the mice recognized DBPII, N10-C29,C22-K40, and C22-C29 at similarly high levels (Figure5A). However, the mice had varying levels of antibodies against the shorter N10-C22 and C29-K40 peptides. For instance, serum from mouse M0 (black circles in Figure5A) had an endpoint titer of 1/400 against N10-C22 and did not recognize C29-K40, whereas serum from mouse M2 (light purple circles in Figure5A) had an endpoint titer of over 1 /200,000 against N10-C22 and over 1/800,000 against C29-K40. Despite being of the same inbred strain of mouse, each of the five mice had a unique immune response to different regions of the SD1CLIPS immunogen. There was no discernable difference in the recognition patterns of sera that cross-reacted with VAR2CSA (mice M2, M3, and M4) and sera that did not (mice M0 and M1). However, the cross-reactive sera had much lower avidity for DBPII than the non-cross-reactive sera (Figure5B–F). This result is consistent with the avidity of 3D10 for DBPII, which is significantly lower compared to another DBPII monoclonal antibody, 2D10, which we showed previously does not cross-react with VAR2CSA (Figure5G,H) [ 9]. Together, these results suggest that antibodies against DBPII that cross-react with VAR2CSA exhibit low avidity against both of these proteins. Vaccines 2020, 8, 392 7 of 13 Vaccines 2020, 8, x 7 of 13

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FigureFigure 4. 4.A A conformationally conformationally constrainedconstrained peptide peptide encompassing encompassing the the SD1 SD1region region of of DBPII DBPII elicited elicited antibodiesantibodies that that cross-react cross-react with with VAR2CSA. VAR2CSA. ( A(A)) A A synthetic synthetic 31 31 amino amino acid acid peptide peptide of of the the SD1 SD1 region region waswas designed designed using using a a chemical chemical sca scaffoldffold in in place place of of one one of of the the disulfide disulfide bonds. bonds. The The cysteine cysteine residues residues thatthat formed formed a a disulfide disulfide bond bond are are shown shown in in green, green, and and those those involved involved in in the the chemical chemical linkage linkage are are shown in yellow. (B) Endpoint titers for the individual mice immunized with SD1 -keyhole limpet shown in yellow. (B) Endpoint titers for the individual mice immunized with SD1CLIPSCLIPS-keyhole limpet hemocyanin (KLH) were measured against SD1 -bovine serum albumin (BSA) and VAR2CSA by hemocyanin (KLH) were measured against SD1CLIPSCLIPS-bovine serum albumin (BSA) and VAR2CSA by ELISA.ELISA. Data Data fromfrom each individual mouse mouse (M#) (M#) are are represented represented by by a unique a unique color. color. Endpoint Endpoint titer titerss were were calculated relative to the mean optical density (OD) of the matching preimmune serum plus two calculated relative to the mean optical density (OD) of the matching preimmune serum plus two standard deviations. (C–F) The avidity of the cross-reactive sera (C–E) and 3D10 (F) for VAR2CSA was standard deviations. (C–F) The avidity of the cross-reactive sera (C–E) and 3D10 (F) for VAR2CSA determined by titrating the individual serum samples or 3D10 against VAR2CSA with and without the was determined by titrating the individual serum samples or 3D10 against VAR2CSA with and addition of 1 M NaSCN. Data are mean SD. without the addition of 1 M NaSCN. Data± are mean ± SD.

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Vaccines 2020, 8, x 8 of 13

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FigureFigure 5. 5.Cross-reactive Cross-reactive serasera thatthat recognizedrecognized VAR2CSAVAR2CSA had had low low avidity avidity against against DBPII. DBPII.( A(A)) Endpoint Endpoint

titerstiters forfor the the individual individual mice mice immunized immunized with with SD1 SD1CLIPSCLIPS-KLH-KLH werewere measuredmeasured againstagainst DBPIIDBPII andand thethe syntheticsynthetic peptides peptides spanning spanning the the SD1 SD1 region. region. Endpoint Endpoint titers titer weres were calculated calculated relative relative to theto the mean mean OD OD of theof the matching matching preimmune preimmune serum serum plus plus two standardtwo standard deviations. deviations. Data Data from from each individualeach individual mouse mouse (M#) are(M#) represented are represented by a unique by a unique color. color. (B–F) ( TheB–F avidity) The avidity of the of individual the individual serum serum samples samples for DBPII for DBPII was determinedwas determined by titrating by titrating against against DBPII DBPII with and with without and without the addition the addition of 1 M NaSCN.of 1 M NaSCN. (G–H) The (G– avidityH) The ofavidity 3D10 andof 3D10 2D10 (aand non-cross-reactive 2D10 (a non-cross DBPII-reactive monoclonal DBPII antibody) monoclonal against antibody) DBPII wasagainst determined DBPII was by titratingdetermined the monoclonalby titrating the antibodies monoclonal against antibodies DBPII with against or without DBPII with the additionor without of the 1 M addition NaSCN. of Data 1 M areNaSCN. mean DataSD. are mean ± SD. ±

3.5. SD1CLIPS Elicits Cross-Reactive Antibodies in a Rabbit

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Vaccines 2020, 8, x 9 of 13 3.5. SD1CLIPS Elicits Cross-Reactive Antibodies in a Rabbit

To Todetermine determine if antibodies if antibodies cross cross-reactive-reactive with with VAR2CSA VAR2CSA can can be elicited be elicited in a in different a diﬀerent animal, animal, wewe immunized immunized a rabbit a rabbit with with the the same same SD1 SD1CLIPSCLIPS peptide,peptide, only only conjugated conjugated to BSA. to BSA. This This conjugate conjugate was was highlyhighly immunogenic immunogenic in the in therabbit, rabbit, with with an anendpoint endpoint titer titer against against the thepeptide peptide of 1/256,000 of 1/256,000 and and very very highhigh avidity avidity (Figure (Figure 6A).6A). These These antibodies antibodies also also strongly strongly recognized recognized DBPII DBPII and and,, unlike unlike the the mouse mouse serum,serum, their their avidity avidity for for DBPII was was high high (Figure (Figure6B). 6B). Importantly, Importantly, the serum the serum also recognized also recognized VAR2CSA VAR2CSA(Figure6 (FiC),gure and 6 consistentC), and consistent with the with results the from results mice, from these mice, antibodies these antibodies were not were as strong not as (endpoint strong (endpointtiter of 1titer/1600) of 1/1600 and were) and of were lowavidity. of low avidity.

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Figure 6. SD1CLIPS elicits cross-reactive antibodies in a rabbit. The avidity of the anti-SD1CLIPS rabbit Figure 6. SD1CLIPS elicits cross-reactive antibodies in a rabbit. The avidity of the anti-SD1CLIPS rabbit serum against (A) SD1CLIPS, (B) DBPII, and (C) VAR2CSA was determined by titrating the serum serum against (A) SD1CLIPS,(B) DBPII, and (C) VAR2CSA was determined by titrating the serum againstagainst the theantigen antigen with with and and without without the theaddition addition of 1 of M 1 NaSCN. M NaSCN. Data Data are aremean mean ± SD.SD. ± 4. Discussion4. Discussion In thisIn this study, study, we we used used an an epitope epitope-focused-focused vaccine toto enhanceenhance the the intrinsic intrinsic cross-reactivity cross-reactivity that that exists existsbetween between DBPII DBPII and and VAR2CSA. VAR2CSA. We We identiﬁed identified a conformationally a conformationally constrained constrained synthetic synthetic peptide peptide that thatelicited elicited an an antibody antibody response response against against not only not its only parent its parent protein, protein, DBPII, but DBPII, also against but also a heterologous against a heterologousantigen, VAR2CSA. antigen, VAR2CSA. This immunogen This immunogen was based onwas the based recognition on the recognition site of a monoclonal site of a monoclonal antibody that antibodyis cross-reactive that is cross against-reactive VAR2CSA against [VAR2CSA9]. We initially [9]. We mapped initially the mapped minimal the epitope minimal to a epitope 12 amino to acida 12 aminolinear peptide acid linear within peptide SD1. However,within SD1. immunization However, immunization with this peptide with failed this to peptide elicit antibodies failed to toelicit DBPII antibodiesand, not to surprisingly, DBPII and,the not antibodies surprisingly, did the not antibodies cross-react withdid not VAR2CSA. cross-react These with results VAR2CSA. suggest These that the resultslinear suggest N10-C 22thatpeptide the linear is su ﬃNcient10-C22 as peptide an antigen is sufficient for 3D10 as recognition an antigen but for is 3D10 not a recognition suitable immunogen but is notto a generatesuitable immunogen antibodies to to the generate desired antibodies epitope in to DBPII. the desired It is also epitope possible in thatDBPII. immunization It is also possible with this thatpeptide immunization elicited antibodieswith this peptide that recognized elicited antibodies an unnatural that epitope recognized formed an unnatural by the synthetic epitop peptidee formed that by isthe not synthetic present peptide in DBPII. that Consistent is not present with in our DBPII. results, Consistent others reported with our that results, 3D10 others recognized reported a peptide that 3D10from recognized the same a region peptide of DBPIIfrom the that same overlapped region of with DBPII N10 -Cthat22 overlappedby seven amino with acidsN10-C [2216 by]. However,seven aminothis acids peptide [16] also. However, failed to this elicit peptide anti-DBPII also failed antibodies. to elicit anti-DBPII antibodies. The results of our peptide library screen expanded the minimal epitope to encompass three distinct segments of SD1 that are discontinuous yet constrained in the native protein by two disulfide bonds. To recapitulate this epitope, we designed the SD1CLIPS peptide that contains all regions of the

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The results of our peptide library screen expanded the minimal epitope to encompass three distinct segments of SD1 that are discontinuous yet constrained in the native protein by two disulfide bonds. To recapitulate this epitope, we designed the SD1CLIPS peptide that contains all regions of the discontinuous epitope and replaced one of the disulfide bonds with a synthetic scaffold. This allowed for control over bond formation in the peptide, constraining the bonding pattern to the structure in the parent protein. With this approach, we succeeded in generating the discontinuous epitope as it was in DBPII, overcoming a common challenge in peptide vaccine development. While all mice immunized with SD1CLIPS-KLH had high endpoint titers against the immunogen (over 1/2.0 million), only three out of five mice made cross-reactive antibodies that recognized VAR2CSA. To better understand this, we evaluated the immune responses of the five mice to peptides from different regions of SD1 and found that no two mice had the same recognition profile. Certain peptides were recognized very strongly (endpoint titers over 1/1.0 million) by sera from some mice but not others, revealing significant variation in the fine specificity of immune responses among the five mice. However, there was no discernable difference in the recognition patterns of the SD1 peptides between the sera samples that cross-reacted with VAR2CSA and those that did not. In both the mice and the rabbit, the cross-reactive antibodies had low avidity for VAR2CSA. These experiments highlight a common feature of cross-reactive antibodies that lower avidity antibodies may be multispecific for similar but not identical epitopes in different antigens [21]. There is evidence that people living in areas of high malaria transmission intensity develop lower avidity antibodies to some P.falciparum antigens, compared to those living in areas of low transmission intensity who develop relatively low titers of higher avidity antibodies [22,23]. This has been attributed to increased exposure to a highly diverse population of parasites in high transmission settings [22,24] and/or impaired affinity maturation as a result of infection [25–27]. Alternatively, lower avidity antibodies may reflect an adaptive advantage that allows the immune system to respond to antigens from different P. falciparum strains, as has been suggested for other pathogens [28]. However, several studies demonstrated that higher avidity antibodies against blood stage P. falciparum antigens provided greater protection from severe [29] or clinical malaria [30]. Importantly, in a retrospective case-control study and a longitudinal study in Cameroon, higher avidity anti-VAR2CSA antibodies were associated with protection from malaria in pregnancy [31,32]. Thus, it is essential to explore ways to increase the avidity of these cross-reactive antibodies against VAR2CSA. Indeed, the low-avidity 3D10 antibody can block parasite binding to chondroitin sulfate A (CSA), but only when used at a high concentration that is unlikely to be elicited through immunization [9]. In order to further develop this immunogen as a vaccine candidate, it is crucial to increase not only the avidity of the antibodies for VAR2CSA, but also the titers. This will facilitate the evaluation of the functional activity of these cross-reactive antibodies, since a high concentration of anti-VAR2CSA antibodies is required to block parasite adhesion to CSA in vitro [9,10,33,34]. We will use different methods to increase both the titer and avidity of the cross-reactive antibodies. For instance, we will employ different boosting schedules and/or adjuvant strategies that have been shown to increase the avidity of some antibody responses [35,36]. We will also further investigate the VAR2CSA target of these cross-reactive antibodies to inform optimal peptide design.

5. Conclusions Ultimately, our goal is to exploit the cross-reactive epitope in PvDBP that generates antibodies to shared epitopes in VAR2CSA. The structural peptide reported here provides a template for the synthesis of a discontinuous conformational epitope from PvDBP that elicits cross-reactive antibodies to VAR2CSA. With further optimization to enhance immunogenicity and avidity, this peptide could be developed as a new vaccine candidate against placental malaria.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-393X/8/3/392/s1. Table S1: Peptides used for the Pepscan-based ELISA screen in Figure3A,B. Figure S1: The avidity of individual mouse sera against SD1CLIPS-BSA. Vaccines 2020, 8, 392 11 of 13

Author Contributions: C.J.M. and L.M.H. performed experiments. C.J.M., M.F.G., and S.K.Y. conceived and designed the experiments. C.J.M. and S.K.Y. wrote the manuscript. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by funding from the Li Ka Shing Institute of Virology at the University of Alberta (2018–2019) and the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2017-04179). Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01AI150944. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. C.J.M. was supported by scholarships from NSERC, the Women and Children’s Health Research Initiative, and Alberta Innovates. M.F.G. was supported by the National Health and Medical Research Council of Australia. Acknowledgments: Thank you to Istvan Toth and Mariusz Skwarczynski for their contribution to peptide design and supervision of C.J.M. as a visiting student to the University of Queensland. We thank Ali Salanti for sharing the recombinant full-length VAR2CSA protein, John Adams for the 3D10 and 2D10 monoclonal antibodies, and Angie Mena for producing the recombinant DBPII. We thank Irina Dinu for statistical advice. We also thank the Health Sciences Laboratory Animal Services staff at the University of Alberta for their assistance with the animal experiments in this study. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Fried, M.; Duffy, P.E. Maternal malaria and parasite adhesion. J. Mol. Med. 1998, 76, 162–171. [CrossRef] 2. Fried, M.; Domingo, G.J.; Gowda, C.D.; Mutabingwa, T.K.; Duffy, P.E. Plasmodium falciparum: Chondroitin sulfate A is the major receptor for adhesion of parasitized erythrocytes in the placenta. Exp. Parasitol. 2006, 113, 36–42. [CrossRef][PubMed] 3. Gamain, B.; Trimnell, A.R.; Scheidig, C.; Scherf, A.; Miller, L.H.; Smith, J.D. Identification of multiple chondroitin sulfate A (CSA)-binding domains in the var2CSA gene transcribed in CSA-binding parasites. J. Infect. Dis. 2005, 191, 1010–1013. [CrossRef] 4. Salanti, A.; Dahlback, M.; Turner, L.; Nielsen, M.A.; Barfod, L.; Magistrado, P.; Jensen, A.T.; Lavstsen, T.; Ofori, M.F.; Marsh, K.; et al. Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. J. Exp. Med. 2004, 200, 1197–1203. [CrossRef][PubMed] 5. Salanti, A.; Staalsoe, T.; Lavstsen, T.; Jensen, A.T.; Sowa, M.P.; Arnot, D.E.; Hviid, L.; Theander, T.G. Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria. Mol. Microbiol. 2003, 49, 179–191. [CrossRef] [PubMed] 6. Mordmuller, B.; Sulyok, M.; Egger-Adam, D.; Resende, M.; de Jongh, W.A.; Jensen, M.H.; Smedegaard, H.H.; Ditlev, S.B.; Soegaard, M.; Poulsen, L.; et al. First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria. Clin. Infect. Dis. 2019, 69, 1509–1516. [CrossRef][PubMed] 7. Sirima, S.B.; Richert, L.; Chene, A.; Konate, A.T.; Campion, C.; Dechavanne, S.; Semblat, J.P.; Benhamouda, N.; Bahuaud, M.; Loulergue, P.; et al. PRIMVAC vaccine adjuvanted with Alhydrogel or GLA-SE to prevent placental malaria: A first-in-human, randomised, double-blind, placebo-controlled study. Lancet Infect. Dis. 2020, 20, 585–597. [CrossRef] 8. Chene, A.; Gangnard, S.; Guadall, A.; Ginisty, H.; Leroy, O.; Havelange, N.; Viebig, N.K.; Gamain, B. Preclinical immunogenicity and safety of the cGMP-grade placental malaria vaccine PRIMVAC. EBioMedicine 2019, 42, 145–156. [CrossRef][PubMed] 9. Gnidehou, S.; Mitran, C.J.; Arango, E.; Banman, S.; Mena, A.; Medawar, E.; Lima, B.A.S.; Doritchamou, J.; Rajwani, J.; Jin, A.; et al. Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA. J. Infect. Dis. 2019, 219, 110–120. [CrossRef] 10. Mitran, C.J.; Mena, A.; Gnidehou, S.; Banman, S.; Arango, E.; Lima, B.A.S.; Lugo, H.; Ganesan, A.; Salanti, A.; Mbonye, A.K.; et al. Antibodies to Cryptic Epitopes in Distant Homologues Underpin a Mechanism of Heterologous Immunity between Plasmodium vivax PvDBP and Plasmodium falciparum VAR2CSA. mBio 2019, 10.[CrossRef] Vaccines 2020, 8, 392 12 of 13

11. Pandey, M.; Ozberk, V.; Langshaw, E.L.; Calcutt, A.; Powell, J.; Batzloff, M.R.; Rivera-Hernandez, T.; Good, M.F. Skin infection boosts memory B-cells specific for a cryptic vaccine epitope of group A streptococcus and broadens the immune response to enhance vaccine efficacy. NPJ Vaccines 2018, 3, 15. [CrossRef][PubMed] 12. Mitchell, D.A.J.; Dupuy, L.C.; Sanchez-Lockhart, M.; Palacios, G.; Back, J.W.; Shimanovskaya, K.; Chaudhury, S.; Ripoll, D.R.; Wallqvist, A.; Schmaljohn, C.S. Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice. Hum. Vaccines Immunother. 2017, 13, 2883–2893. [CrossRef][PubMed] 13. Bajic, G.; Maron, M.J.; Adachi, Y.; Onodera, T.; McCarthy, K.R.; McGee, C.E.; Sempowski, G.D.; Takahashi, Y.; Kelsoe, G.; Kuraoka, M.; et al. Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope. Cell Host Microbe 2019, 25, 827–835. [CrossRef][PubMed] 14. Bangaru, S.; Lang, S.; Schotsaert, M.; Vanderven, H.A.; Zhu, X.; Kose, N.; Bombardi, R.; Finn, J.A.; Kent, S.J.; Gilchuk, P.; et al. A Site of Vulnerability on the Influenza Virus Hemagglutinin Head Domain Trimer Interface. Cell 2019, 177, 1136–1152. [CrossRef] 15. Watanabe, A.; McCarthy, K.R.; Kuraoka, M.; Schmidt, A.G.; Adachi, Y.; Onodera, T.; Tonouchi, K.; Caradonna, T.M.; Bajic, G.; Song, S.; et al. Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism. Cell 2019, 177, 1124–1135. [CrossRef] 16. George, M.T.; Schloegel, J.L.; Ntumngia, F.B.; Barnes, S.J.; King, C.L.; Casey, J.L.; Foley, M.; Adams, J.H. Identification of an Immunogenic Broadly Inhibitory Surface Epitope of the Plasmodium vivax Duffy Binding Protein Ligand Domain. mSphere 2019, 4.[CrossRef][PubMed] 17. Chen, E.; Salinas, N.D.; Huang, Y.; Ntumngia, F.; Plasencia, M.D.; Gross, M.L.; Adams, J.H.; Tolia, N.H. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein. Proc. Natl. Acad. Sci. USA 2016, 113, 6277–6282. [CrossRef] 18. Jones, G.L.; Edmundson, H.M.; Spencer, L.; Gale, J.; Saul, A. The use of maleimidocaproyloxysuccinimide to prepare malarial peptide carrier immunogens. Immunogenicity of the linking region. J. Immunol. Methods 1989, 123, 211–216. [CrossRef] 19. Timmerman, P.; Puijk, W.C.; Meloen, R.H. Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology. J. Mol. Recognit. 2007, 20, 283–299. [CrossRef] 20. Chen, S.W.; Van Regenmortel, M.H.; Pellequer, J.L. Structure-activity relationships in peptide-antibody complexes: Implications for epitope prediction and development of synthetic peptide vaccines. Curr. Med. Chem. 2009, 16, 953–964. [CrossRef] 21. Van Regenmortel, M.H. What is a B-cell epitope? Methods Mol. Biol. 2009, 524, 3–20. [CrossRef][PubMed] 22. Amoah, L.E.; Abagna, H.B.; Akyea-Mensah, K.; Lo, A.C.; Kusi, K.A.; Gyan, B.A. Characterization of anti-EBA175RIII-V in asymptomatic adults and children living in communities in the Greater Accra Region of Ghana with varying malaria transmission intensities. BMC Immunol. 2018, 19, 34. [CrossRef][PubMed] 23. Ssewanyana, I.; Arinaitwe, E.; Nankabirwa, J.I.; Yeka, A.; Sullivan, R.; Kamya, M.R.; Rosenthal, P.J.; Dorsey, G.; Mayanja-Kizza, H.; Drakeley, C.; et al. Avidity of anti-malarial antibodies inversely related to transmission intensity at three sites in Uganda. Malar. J. 2017, 16, 67. [CrossRef][PubMed] 24. Adjah, J.; Fiadzoe, B.; Ayanful-Torgby, R.; Amoah, L.E. Seasonal variations in Plasmodium falciparum genetic diversity and multiplicity of infection in asymptomatic children living in southern Ghana. BMC Infect. Dis. 2018, 18, 432. [CrossRef] 25. Anders, R.F. Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol. 1986, 8, 529–539. [CrossRef] 26. Cadman, E.T.; Abdallah, A.Y.; Voisine, C.; Sponaas, A.M.; Corran, P.; Lamb, T.; Brown, D.; Ndungu, F.; Langhorne, J. Alterations of splenic architecture in malaria are induced independently of Toll-like receptors 2, 4, and 9 or MyD88 and may affect antibody affinity. Infect. Immun. 2008, 76, 3924–3931. [CrossRef] 27. Ibison, F.; Olotu, A.; Muema, D.M.; Mwacharo, J.; Ohuma, E.; Kimani, D.; Marsh, K.; Bejon, P.; Ndungu, F.M. Lack of avidity maturation of merozoite antigen-specific antibodies with increasing exposure to Plasmodium falciparum amongst children and adults exposed to endemic malaria in Kenya. PLoS ONE 2012, 7, e52939. [CrossRef] 28. Jain, D.; Salunke, D.M. Antibody specificity and promiscuity. Biochem. J. 2019, 476, 433–447. [CrossRef] 29. Leoratti, F.M.; Durlacher, R.R.; Lacerda, M.V.; Alecrim, M.G.; Ferreira, A.W.; Sanchez, M.C.; Moraes, S.L. Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria. Malar. J. 2008, 7, 186. [CrossRef] Vaccines 2020, 8, 392 13 of 13

30. Ferreira, M.U.; Kimura, E.A.; De Souza, J.M.; Katzin, A.M. The isotype composition and avidity of naturally acquired anti-Plasmodium falciparum antibodies: Differential patterns in clinically immune Africans and Amazonian patients. Am. J. Trop. Med. Hyg. 1996, 55, 315–323. [CrossRef] 31. Tutterrow, Y.L.; Salanti, A.; Avril, M.; Smith, J.D.; Pagano, I.S.; Ako, S.; Fogako, J.; Leke, R.G.; Taylor, D.W. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria. PLoS ONE 2012, 7, e40049. [CrossRef] 32. Babakhanyan, A.; Fang, R.; Wey, A.; Salanti, A.; Sama, G.; Efundem, C.; Leke, R.J.; Chen, J.J.; Leke, R.G.; Taylor, D.W. Comparison of the specificity of antibodies to VAR2CSA in Cameroonian multigravidae with and without placental malaria: A retrospective case-control study. Malar. J. 2015, 14, 480. [CrossRef] [PubMed] 33. Saveria, T.; Oleinikov, A.V.; Wiliamson, K.; Chaturvedi, R.; Lograsso, J.; Keitany, G.J.; Fried, M.; Duffy, P. Antibodies to Escherichia coli-expressed C-terminal domains of Plasmodium falciparum variant surface antigen 2-chondroitin sulfate A (VAR2CSA) inhibit binding of CSA-adherent parasites to placental tissue. Infect. Immun. 2013, 81, 1031–1039. [CrossRef] 34. Doritchamou, J.Y.; Herrera, R.; Aebig, J.A.; Morrison, R.; Nguyen, V.; Reiter, K.; Shimp, R.L.; MacDonald, N.J.; Narum, D.L.; Fried, M.; et al. VAR2CSA Domain-Specific Analysis of Naturally Acquired Functional Antibodies to Plasmodium falciparum Placental Malaria. J. Infect. Dis. 2016, 214, 577–586. [CrossRef] [PubMed] 35. Thompson, E.A.; Ols, S.; Miura, K.; Rausch, K.; Narum, D.L.; Spangberg, M.; Juraska, M.; Wille-Reece, U.; Weiner, A.; Howard, R.F.; et al. TLR-adjuvanted nanoparticle vaccines differentially influence the quality and longevity of responses to malaria antigen Pfs25. JCI Insight 2018, 3.[CrossRef][PubMed] 36. Phillips, B.; Van Rompay, K.K.A.; Rodriguez-Nieves, J.; Lorin, C.; Koutsoukos, M.; Tomai, M.; Fox, C.B.; Eudailey, J.; Dennis, M.; Alam, S.M.; et al. Adjuvant-Dependent Enhancement of HIV Env-Specific Antibody Responses in Infant Rhesus Macaques. J. Virol. 2018, 92.[CrossRef]

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