Classification: Microbial Immunity & Vaccines

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1 IAI Accepts, published online ahead of print on 14 October 2013 Infect. Immun. doi: /iai Copyright 2013, American Society for Microbiology. All Rights Reserved Classification: Microbial Immunity & Vaccines A bacterially expressed full-length recombinant Plasmodium falciparum RH5 protein binds erythrocytes and elicits potent strain-transcending parasite neutralizing antibodies K. Sony Reddy, Alok K. Pandey, Hina Singh, Tajali Sahar, Amlabu Emmanuel, Chetan E. Chitnis, Virander S. Chauhan, Deepak Gaur* Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. *Corresponding Author Please send correspondence to: Deepak Gaur, Ph.D. Malaria Group International Centre for Genetic Engineering and Biotechnology (ICGEB) Aruna Asaf Ali Marg New Delhi, India Tel: Fax: dgaur@icgeb.res.in These authors contributed equally to the work Running Title: Recombinant PfRH5 elicits neutralizing antibodies Keywords: Malaria, Plasmodium, erythrocyte, invasion, ligand, receptors, vaccine, host pathogen interaction. 1

2 Abstract 34 Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is an attractive malaria vaccine candidate as it is expressed by a wide number of P. falciparum strains, cannot be genetically disrupted and exhibits limited sequence polymorphisms. Viral vector induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial cell based expression system. Here, we have produced full-length recombinant PfRH5 in Escherichia coli that exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also importantly elicits potent invasion inhibitory antibodies against a number of P. falciparum strains. Anti-Basigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5 further confirming that they bind with Basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics the native parasite protein and elicits potent strain-transcending parasite neutralizing antibodies. P. falciparum has the capability to develop immune escape mechanisms and thus blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwide P. falciparum strains. PfRH5 was immunogenic when immunized with other antigens eliciting potent invasion inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine

3 Introduction Malaria is a global infectious disease that accounts for around one million deaths across the world primarily in young children below the age of five years (1). The causative agent of the most severe form of malaria that is responsible for maximum mortality is the parasite, Plasmodium falciparum. Invasion of human erythrocytes by P. falciparum is a critical process during the parasite s life cycle that leads to the development of blood stage parasites, which are primarily responsible for malaria pathogenesis. P. falciparum has evolved a complex, multistep process of erythrocyte invasion that involves numerous ligand-receptor interactions (2-4). This molecular redundancy allows the parasite to use many alternate pathways for invasion, thus ensuring that the pathogen gains entry into its host erythrocyte (2-4). The quest for developing a vaccine that targets blood-stage parasites has involved extensive studies on identifying and characterizing key parasite molecules that mediate erythrocyte invasion. Early efforts have focused on two leading candidates MSP-1 42 and AMA- 1, which play an essential role in erythrocyte invasion (2-4) but have unfortunately not generated optimal protection in field efficacy trials (5-7). Recently, the family of P. falciparum reticulocyte binding-like homologous proteins (PfRH) has attracted most attention as key determinants of merozoite invasion (2-4, 8, 9). The PfRH family comprises of five members - PfRH1, PfRH2a, PfRH2b, PfRH4 and PfRH5 that bind with either sialic acid dependent or sialic acid independent erythrocyte receptors (10-22). However, most of these proteins are not essential for erythrocyte invasion and can be genetically disrupted (4, 8, 9) with the exception of PfRH5 (22). PfRH5 (Accession Number: XP_ ; PlasmoDB ID: PF3D7_ ) was first identified by genetic mapping as a key determinant of species specific erythrocyte invasion (21). 3

4 Genetic analysis of the progeny of a P. falciparum cross between two parental clones 7G8 x GB4 had mapped the PfRH5 gene on chromosome 4 as the locus responsible for mediating invasion of Aotus nancymaae erythrocytes as well as infectivity of Aotus monkeys (21). It was also demonstrated that PfRH5 is an erythrocyte binding ligand in which single point mutations critically affected the specificity of its binding with Aotus erythrocytes (21). Recently, PfRH5 has also been shown to play a role in the invasion of both owl monkey and rat erythrocytes by P. falciparum (23). Further, PfRH5 was found to be unique in being the only erythrocyte binding ligand among the EBA/PfRH families that is essential for the parasite as it cannot be genetically knocked out (22), suggesting a crucial role in erythrocyte invasion. PfRH5 is also exceptional compared to other PfRH homologues as it is smaller in size (63 kda) and lacks a transmembrane domain (21, 22). PfRH5 has been shown to be localized on the merozoite surface in association with another parasite molecule, PfRipr (P. falciparum PfRH5 interacting protein) (24). While, PfRH proteins are differentially expressed among different P. falciparum clones that exhibit phenotypic variation in their invasion properties (11, 13, 16-19), the expression of PfRH5 was found to be consistent among these parasite clones (21, 22). Recently, PfRH5 was reported to bind with the CD147 IgG super family member, Basigin (BSG) on the erythrocyte surface (25). For this study, a mutated version of PfRH5 was produced in mammalian HEK293 cells as a biotinylated fusion protein with the CD4 domains 3+4(CD4d3+4) of rat origin, which was found to bind with a pentamer of Basigin (25). The mutations were necessary to produce a non-glycosylated protein in the mammalian cells similar to that of the native parasite protein, which like P. falciparum native proteins remains essentially unglycosylated. The significance of the PfRH5-BSG interaction was highlighted by the 4

5 demonstration that anti-bsg antibodies blocked erythrocyte invasion by a large number of P. falciparum clones that were known to exhibit different invasion phenotypes (25). However, no data was reported in this elegant study on the interaction of native PfRH5 with Basigin. A heterologous prime-boost strategy based on the adenoviral/mva viral vector platform was used to generate anti-pfrh5 antibodies that efficiently inhibited erythrocyte invasion by multiple heterologous P. falciparum clones (26). It has also been recently reported that antibodies against a fusion protein comprising of a mutated PfRH5 with the (d3+4) domains of the rat CD4 protein exhibited potent inhibition of erythrocyte invasion (27). This study further substantiated the importance of the PfRH5-BSG interaction during erythrocyte invasion and strongly supported PfRH5 as a blood stage vaccine candidate. However, polymorphisms in PfRH5 as well as other P. falciparum adhesins have been shown to induce changes in their receptor specificity (2, 3, 21, 23) and could also possibly alter native structure. Thus, the production of a functionally active recombinant wild-type full-length PfRH5 protein, which would elicit similar potent invasion inhibitory antibodies, in a cell based expression system that could be scalable for mass production still remained a challenge not only from a vaccine perspective but also to facilitate the basic structure-function analysis of PfRH5. Also considering that the target population of a prophylactic malaria vaccine is young infants and children, it is important to test different expression platforms so as to identify the safest and most efficacious antigen or delivery mechanism that would be most feasible to administer as a vaccine for mass immunization. In this regard, the subunit vaccine approach based on formulations of recombinant proteins and adjuvant pose a safe and effective platform for administering a vaccine for large masses. However, the test here lies in being able to produce 5

6 the recombinant protein with a structural integrity that yields potent neutralizing antibodies against the respective pathogens. Expression in Escherichia coli provides a cost-effective method for production of recombinant proteins for use as biologics and vaccines. Previous reports of production of recombinant proteins against PfRH5 in E. coli have focused on expressing smaller fragments of amino acids (22, 28). Both these recombinant fragments failed to elicit invasion inhibitory antibodies (22, 28). This is consistent with the recent report using the Adeno-MVA prime boost approach that demonstrated potent invasion inhibitory antibodies only against fulllength PfRH5 and not against the 168 amino acid fragment of PfRH5 (26). In light of these reports, in our study we have demonstrated that the full-length wild-type PfRH5 recombinant protein produced in E. coli was efficacious in eliciting potent invasion inhibition consistent with that observed with the viral vector delivery platform (26) or with the ratcd4(d3+4) fusion construct (27). In the current report, we have successfully produced fulllength PfRH5 as a recombinant protein in E. coli that exhibits specific erythrocyte binding activity similar to that of the native PfRH5 parasite protein. We also demonstrated that anti- Basigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5 proteins, further confirming that Basigin acts as their erythrocyte receptor. Our recombinant PfRH5 elicited potent strain-transcending invasion inhibitory antibodies that blocked a number of heterologous parasite clones. Importantly, the PfRH5 antibodies produced additive invasion inhibition in combination with antibodies against other key merozoite antigens. Thus, our study strongly supports the development of PfRH5 based antigen combinations as malaria vaccine candidates. 6

7 145 Materials and Methods: Ethics statement The animal studies described below were approved by the International Centre for Genetic Engineering and Biotechnology (ICGEB) Institutional Animal Ethics Committee (IAEC) (reference no.mal-51) according to the guidelines of the Department of Biotechnology, Government of India Cloning, expression and purification of the full-length recombinant PfRH5 The 1500 bp PfRH5 gene that encodes the 500 amino acid full length parasite protein excluding the signal sequence (rpfrh5 63, Asp27-Gln526) was PCR amplified from the genomic DNA of the P. falciparum clone 3D7 using the following primers, RH5-Fwd: 5 - ATATATAATTCATATGAATGCAATAAAAAAAACGAAGAAT-3 and RH5-Rev: 5 - AGCACTCGAGTTGTGTAAGTGGTTTATTTTTTT-3. The PCR product encoding rpfrh5 63 were digested with Nde I and Xho I (New England Biolabs, Beverly, MA) and inserted downstream of the T7 promoter in the E. coli expression vector, pet-24b (Novagen, San Diego, CA) with a C-terminal 6-histidine (6-His) tag to obtain the plasmid ppfrh5-pet24b. Sequencing of the ligated plasmid confirmed the correct sequence of the PfRh5 gene and that the insertions were in the correct reading frame. E. coli BL21(DE3) was transformed with ppfrh5-pet24b and was used to produce the recombinant protein rpfrh5 63. Transformed E. coli BL21(DE3) were cultured in superbroth at 37 C and later were induced with 1mM IPTG when the OD 600 was around Cells were 7

8 harvested by centrifugation at 3000g, after 4 hours of induction at 37 C. Cell pellets were lyzed by sonication and rpfrh5 63 was found to be expressed as inclusion bodies. The inclusion bodies were washed, collected by centrifugation at 15000g and solubilized in a buffer containing 20 mm Tris ph 8.0, 6 M Guanidium-HCl, 300 mm NaCl, 10 mm Imidazole and 5 mm beta mercaptoethanol. rpfrh5 63 was purified from solubilized inclusion bodies by metal affinity chromatography using the Ni-NTA (nitrilotriacetic acid) resin. Metal affinity purified rpfrh5 63 was refolded in redox conditions by rapid dilution (30-fold) in an MES based buffer ph 6.5 comprising of 1 mm GSH (reduced Glutathione), 0.1 mm GSSG (oxidized Glutathione) and 440 mm sucrose. The refolded protein was dialyzed against 25 mm MES (ph 6.5), 200mM sucrose and further purified to homogeneity by cation exchange chromatography using an SP-sepharose column (GE Healthcare, Piscataway, NJ). The dialyzed protein was loaded on the SP-sepharose column and eluted with an increasing concentration of NaCl (0-1 M) in the MES based buffer, ph 6.5. The purified recombinant rpfrh5 63 protein was characterized by SDS-PAGE, Western blotting, Edman degradation, Mass spectrometric (LC-MS) analysis (Orbitrap VELOS PRO, Thermofisher Scientific), Reverse phase RP-HPLC (C8 column; Waters) and Size exclusion chromatography (Superdex 75 10/300 GL; GE Healthcare) Animal Immunization and antibody generation Animal immunizations and total IgG purification were done as reported previously (29). Briefly, five rats and three rabbits were immunized intramuscularly with 50 µg and 100 µg rpfrh5 63, respectively. The rpfrh5 63 protein was emulsified with complete Freund s adjuvant (Sigma, St. Louis, MO) for immunization on day 0 followed by two boosts emulsified with 8

9 incomplete Freund s Adjuvant on day 28 and 56. The sera were collected on day 70. Antibody levels were measured by ELISA. For co-immunogenicity, a group of six BALB/c mice were immunized with antigen mixtures PfF2+PfRH5+PfAARP, PfRH2+PfRH5+PfAARP as well as individual antigens emulsified with complete Freund s adjuvant on day 0 followed by two boosts emulsified with incomplete Freund s adjuvant on days 28 and µg of each antigen was immunized in each mouse, whether used alone or as a co-immunization triple antigen mixture (total 51 µg). Terminal bleeds were collected on Day 70. Sera were tested for antibody titers and specific recognition of each recombinant protein by ELISA. For the GIA, the sera from the six mice in each group were pooled for IgG purification. Total IgG was purified from the mice, rat or rabbit sera using Protein G affinity column (GE Healthcare, Uppsala, Sweden), dialyzed with RPMI medium and further tested in invasion inhibition assays as described below. As an adjuvant negative control, we also had raised antibodies in mice, rats and rabbits against a non-related peptide (KESRAKKFQR KHITNTRDVD from human pancreatic RNase) that was also formulated with the same adjuvant (CFA/IFA) and injected in animals with the same schedule used for raising the PfRH5 antibodies as described above Erythrocytes and enzymatic treatment: Packed RBCs were procured from the Rotary Blood Bank (Tughlaqabad), New Delhi, India. Erythrocytes were washed in RPMI, and stored at 50% haematocrit. Enzymatic treatments of erythrocytes were done as stated previously (16, 20, 29). 9

10 Erythrocyte binding assays: Soluble parasite proteins were obtained from 3D7 culture supernatants as described previously (16, 20, 29). Briefly, 500µl of culture supernatant were incubated with 100µl packed volume of human erythrocytes at 37 C, following which the suspension was centrifuged through Dibutyl phthalate (Sigma). The supernatant and oil were removed by aspiration and bound parasite proteins were eluted using 1.5M NaCl. For rpfrh5 63 binding, 0.04µM of rpfrh5 was incubated with 100µl packed volume of human erythrocytes at 37 C in similar erythrocyte binding assays as described above. The eluate fractions were analyzed for the presence of native PfRH5 and rpfrh5 63 by immunoblotting using anti-rpfrh5 63 antibodies Invasion inhibition assays Invasion inhibition assays were performed as described previously (16, 29) using total IgG purified from the sera of different animals immunized with PfRH5. Briefly, schizont-stage parasites at an initial parasitemia of 0.3% at 2% hematocrit were incubated with purified total IgG for one cycle of parasite growth (40 h post invasion). The parasite-infected erythrocytes were stained with ethidium bromide dye and measured by FACS as described previously (16, 29). The percent invasion inhibition for each immune IgG was calculated with respect to the control pre-immune IgG from the same animal. As another negative control, we used immune IgG raised against a non-related peptide from human pancreatic RNase that was also immunized with the same freund s adjuvant (CFA/IFA) used for raising the PfRH5 antibodies. The results represent the average of three independent experiments performed in duplicate and the error bars represent the standard error of the mean. Statistical significance was calculated using the 10

11 Student s t- test (Graph Pad Prism software, version 6.03). P values < 0.05 were considered statistically significant

12 Results Expression of the full-length recombinant PfRH5 protein and generation of specific PfRH5 antibodies A 500 amino acid sequence (Asn27-Gln526) of PfRH5 from the P. falciparum clone 3D7, comprising of the full-length protein excluding the signal peptide, was chosen for recombinant protein expression in E. coli (Fig. 1A). The recombinant 63kDa PfRH5 (rpfrh5 63 ) was expressed with a C-terminal 6-His tag. In E. coli, the rpfrh5 63 protein got expressed in inclusion bodies, which were then refolded after purification under denaturing conditions by metal affinity chromatography. rpfrh5 63 was further purified to homogeneity using ionexchange chromatography (Fig. 1B). Recombinant rpfrh5 63 comprises of 6 cysteines that could lead to three potential disulphide linkages. Initial SDS-PAGE analysis of the purified protein on a 12% gel under both reducing and non-reducing conditions did not reflect a mobility shift (Fig. 1B). However, when the rpfrh5 63 protein was run on the same gel for a longer period of time such that the 35 KDa pre-stained marker protein reached the end of the gel, a mobility shift could be detected (Fig. S1A). The SDS-PAGE analysis was repeated along with a highly cysteine rich recombinant protein (reba-175 RII) that has 24 cysteines leading to 12 potential disulphide bonds (Fig. S1B, S1C). reba-175 RII exhibited a significant mobility shift compared to rpfrh5 63 that showed no shift when run normally on the 12% SDS-PAGE gel (Fig. S1B). On running the gel for a longer time, the mobility shift increased for reba-175 RII and became detectable for rpfrh5 63 (Fig. S1C). Edman degradation analysis of the full-length rpfrh5 63 protein yielded an N-terminal sequence (MNAIKK) that matched with the protein sequence of the PfRH5 native parasite 12

13 protein after the signal sequence from amino acid Asn 27 onwards (Fig. S1C). Recombinant rpfrh5 63 was identified in immunoblots using a specific anti-his tag antibody (Fig. 1C) confirming expression of the full-length 500 amino acid protein with the C-terminal His-tag. While, the SDS-PAGE analysis of the rpfrh5 63 protein showed a highly pure protein preparation with the predominant band at 63 kda, traces of a smaller 45 kda protein were also faintly visible. These two protein bands were excised from the gel and subjected to trypsin digestion followed by LC-MS (liquid chromatography-mass spectrometry) analysis (Orbitrap VELOS PRO, Thermofisher scientific). The LC-MS analysis revealed a large number of unique high scoring peptides for both proteins (32 peptides for 63 kda protein; 31 peptides for 45 kda protein) that confirmed the identity of both proteins to be PfRH5 (Table S1, S2). The detection of a smaller 45 kda protein is consistent with previous reports on the production of recombinant PfRH5 in HEK293 cells (27) and that observed in the parasite lysate (22). We further analyzed the recombinant protein on Size exclusion chromatography (SEC) and confirmed that our recombinant protein eluted at the expected molecular size with respect to the BSA standard protein, which also has a similar molecular weight (66 kda) (Fig. S1D). The SEC profile also showed that our recombinant protein was primarily in a monomeric state. The recombinant rpfrh5 63 protein was further analyzed using Reverse Phase HPLC (RP-HPLC) that showed a single symmetrical peak reflecting a highly pure protein preparation (Fig. S1E). Rats and rabbits were immunized with rpfrh5 63 to raise PfRH5-specific antibodies. High titer antibodies against rpfrh5 63 were detected in both rats and rabbits with end points observed at dilutions of 1:320,000 (data not shown). The specificity of the PfRH5 antibodies was analyzed by immunoblotting studies to detect the PfRH5 native protein in parasite lysates and localization 13

14 studies in merozoites by immunofluorescence super resolution confocal microscopy (N-SIM, Nikon, Japan). Consistent with previous reports, native parasite PfRH5 was detected in immunoblots at the expected size for the full-length protein (63kDa) only in the schizont stages and not in early rings or trophozoite stages (Fig. S2A, S2B). PfRH5 has been reported to be localized in the rhoptry bulb by immunoelectron microscopy (22). With our antibodies, PfRH5 was also found to co-localize with the known rhoptry bulb protein, PTRAMP (30) (Fig. S3A). On the other hand, there was no co-localization observed with the rhoptry neck protein, PfRH2 (Fig. S3B) or the micronemal protein, PfEBA-175 (Fig. S3C). Our data was consistent with previous reports (22) and confirmed the specificity of our PfRH5 antibodies Recombinant PfRH5 exhibits specific erythrocyte binding activity Standard erythrocyte binding assays were performed with parasite culture supernatant (3D7) incubated with human erythrocytes as described previously (16, 20, 29). Native PfRH5 parasite protein (63kDa) has been reported to be processed into smaller fragments of 45 kda and 28 kda (21, 22). The full-length native PfRH5 and its processed fragments bind erythrocytes with the same specificity (21, 22, 28). In our assay, we prepared 3D7 culture supernatants and observed that both the native 63 kda full-length PfRH5 protein and the 45 kda processed protein bound erythrocytes in a sialic acid independent, trypsin and chymotrypsin resistant manner (Fig. 2A), which is consistent with previous reports (21, 22). While, in this culture supernatant we did not observe the 28 kda fragment, in another culture supernatant preparation in which the parasites were incubated for a longer period of time, we detected the 28 kda fragment and observed it to bind erythrocytes with the same specificity (Fig. S2C). Culture supernatants are 14

15 prepared in the absence of any protease inhibitors as they would impede parasite egress itself and this does lead to the possibility of parasite proteins undergoing proteolytic cleavage yielding fragments of different sizes (21, 22, 28). The full-length, wild type PfRH5 recombinant protein, rpfrh5 63, expressed in E. coli also exhibited an erythrocyte binding specificity that matched with that of the native parasite protein (Fig. 2B). rpfrh5 63 specifically bound erythrocytes in a sialic acid independent, trypsin and chymotrypsin resistant manner (Fig. 2B). Since, the native and recombinant PfRH5 proteins bound erythrocytes treated with each of the three enzymes (neuraminidase, trypsin, chymotrypsin), we also tested the effect of proteinase K treatment and found that binding of both native and recombinant PfRH5 was sensitive to proteinase K (Fig.2A, 2B, S2C) As controls, both native PfEBA-175 from the parasite culture supernatant and recombinant PfF2, the receptor-binding domain of PfEBA-175, were found to bind erythrocytes in a sialic acid dependent, trypsin sensitive and chymotrypsin resistant manner (Fig. 2C, 2D, S2D) consistent with previous reports (31-33). In addition, no bound proteins were detected when the erythrocytes were incubated with PBS alone (Fig. 2), thus confirming that no nonspecific erythrocyte protein was being detected in our assay and that the binding of PfRH5 or PfEBA-175 was specific Antibodies against recombinant PfRH5 and Basigin block the erythrocyte binding activity of native PfRH5 After demonstrating that rpfrh5 63 specifically bound erythrocytes, we determined whether anti-rpfrh5 63 antibodies could block the erythrocyte binding of native PfRH5 parasite 15

16 protein. We demonstrated that total IgG purified from the sera of rabbits immunized with rpfrh5 63 blocked the erythrocyte binding of native PfRH5 (Fig. 3A). Total IgG containing anti- PfRH5 antibodies blocked binding of both the native and recombinant PfRH5 proteins with erythrocytes in a dose-dependent manner (Fig. 3A, 3B). At a total IgG concentration of 200 µg/ml, the anti-pfrh5 IgG potently blocked the erythrocyte binding of both the native and recombinant PfRH5 protein, whereas even at a concentration of 800 µg/ml the PfRH5 IgG had no effect on the erythrocyte binding of another parasite ligand, PfEBA-175 (Fig. 3C) or its recombinant receptor binding domain, PfF2 (Fig. 3D). This result clearly demonstrated that PfRH5 antibodies specifically recognized only PfRH5 and further abrogated its interaction with the erythrocyte surface. As described earlier, the erythrocyte receptor of PfRH5 was recently identified as the CD147 erythrocyte surface molecule, Basigin (BSG) (25). However, this elegant study had demonstrated this interaction with recombinant PfRH5 and not the native parasite protein. We tested the invasion inhibitory activity of the anti-bsg monoclonal antibodies (TRA-1-85; R&D Systems, USA) and found them to potently inhibit invasion with 90% inhibition observed at a concentration of 2.5µg/ml (Fig. 4A) consistent with the previous study (25). Further, we tested the ability of the anti-bsg monoclonal TRA-1-85 antibodies to block the erythrocyte binding of recombinant protein, rpfrh5 63 and native PfRH5 from parasite culture supernatants. Anti-BSG monoclonal TRA-1-85 antibodies potently blocked erythrocyte invasion from a minimum concentration of 2.5µg/ml and from the same concentration, the binding of the native PfRH5 protein was also observed to be significantly reduced in a dose dependent manner (Fig. 4B). 10 µg/ml of the anti-bsg monoclonal TRA-1-85 antibody completely abrogated the binding of both 16

17 the native PfRH5 protein (Fig. 4B) and rpfrh5 63 (Fig. 4C). As a control, anti-glycophorin A monoclonal antibodies (Sigma-Aldrich) had no effect on the binding of the PfRH5 parasite protein (Fig. 4B, 4C) suggesting that the anti-bsg TRA-1-85 monoclonal antibodies were acting in a specific manner. This result substantiates the previous finding on PfRH5-BSG and demonstrates for the first time the interaction between BSG with the native PfRH5 parasite protein Antibodies against recombinant PfRH5 potently block erythrocyte invasion by multiple P. falciparum clones PfRH5 is the only parasite ligand among the EBA/PfRH families that is essential for erythrocyte invasion (21, 22). We thus, compared the invasion inhibitory activity of PfRH5 antibodies with that of antibodies raised against five other parasite ligands from our antigen portfolio PfRH1, PfRH2, PfRH4, PfAARP (P. falciparum Apical Asparagine Rich Protein), PfF2 (F2: receptor binding domain of PfEBA-175) as described previously (16, 20, 29, 32, 34). The total rabbit IgGs ( mg/ml) purified from the sera of rabbits individually immunized with one of the six antigens were tested in standard one-cycle in vitro invasion inhibition assays (Fig. 5A) as described in our previous report (29). The invasion inhibition of the six antibodies was tested against the P. falciparum clone 3D7, which invades using both sialic acid dependent and independent pathways (Fig. 5A). All antibodies exhibited a dose dependent inhibition that suggested a specific effect (Fig.5A). The invasion inhibition for each immune IgG was calculated with respect to the pre-immune IgG obtained from the same rabbit. In addition, as another negative control, we tested the invasion inhibitory activity of purified rabbit total IgG 17

18 against a non-related peptide from a human pancreatic ribonuclease (HPR) that was also formulated with the same freund s adjuvant. The anti-hpr IgG failed to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml (Fig. 5A). The anti-hpr negative control has been tested in each assay reported in the current study. Among all six antibodies, PfRH5 total IgG was found to elicit maximum inhibition of erythrocyte invasion with ~ 83% inhibition at 10 mg/ml followed by PfAARP IgG (51%, 10 mg/ml) and PfRH2 IgG (49%, 10 mg/ml) (Fig. 5A). PfRH5 total IgG exhibited an invasion inhibition of 63% at 5 mg/ml and 52% at 3.3 mg/ml (Fig. 5A). The variation in inhibitory activity among the different antibodies could not be attributed to any disparity in their antibody titers. The end point antibody titers against all the six proteins immunized in rabbits were in the range of 1:320,000 (data not shown). As mentioned above, we had raised antibodies against PfRH5 in three rabbits that had shown equivalent end point titers in the range of 1:320,000. The purified total IgG from the other two rabbits also exhibited a potent invasion inhibitory activity against the P. falciparum clone 3D7 (Fig. S4A) similar to that observed with the anti-pfrh5 IgG from rabbit 1 (Fig. 5A). We further analyzed the strain-transcending invasion inhibitory activity of the PfRH5 antibodies (rabbit 1) against five P. falciparum strains that originate from different regions of the world and express different polymorphic variants of the PfRH5 parasite protein (Table S3) (21). In addition, these P. falciparum strains exhibit a variation in their invasion phenotype by utilizing different ligand receptor interactions and pathways for invading human erythrocytes (Table S3) (2-4,29 33,35). The invasion phenotypes of the P. falciparum clones are classified in literature on the basis of their invasion sensitivity to enzymatic treatments of the target 18

19 erythrocytes (2-4, 29, 33, 35). Thus, the five parasite clones represent diversity both at the level of PfRH5 antigenic polymorphisms as well as the phenotypic variation in the invasion properties of the parasite clones (Table S3). The strains 3D7, HB3 and 7G8 are known to invade through sialic acid independent pathways (3, 33, 35), whereas Dd2, MCamp are completely dependent on sialic acids for erythrocyte invasion (3, 33, 35). The anti-pfrh5 antibodies (rabbit 1) were found to potently inhibit erythrocyte invasion of all five P. falciparum clones (Fig. 5B). The purified PfRH5 total IgG exhibited 81-85% invasion inhibition among the five parasite clones at a concentration of 10 mg/ml (Fig.5B), which is comparable with the maximum inhibition that we recently reported with an antibody combination against three antigens, PfAARP+PfRH2+PfF2 (29). The invasion inhibitory activity was dose dependent as reported earlier with an inhibition of 60-71% at 5 mg/ml, 49-59% at 3.3 mg/ml and 34-46% at 2.5 mg/ml (Fig. 5B). Similar potent strain-transcending invasion inhibition was also observed with total IgG purified from the sera of the other two rabbits immunized with rpfrh5 63 (data not shown). Our PfRH5 antibodies exhibited a 50% inhibition of erythrocyte invasion at a total IgG concentration (EC 50 ) of ~3 mg/ml, which is consistent with previous studies that have reported EC 50 values for anti-pfrh5 IgG obtained from different rabbits within a concentration range of mg/ml against multiple P. falciparum strains (26, 27, 36). The purified total IgG from five individual rats also exhibited a potent dose dependent invasion inhibition of the P. falciparum clone 3D7 with the maximum inhibition of around 72-77% at 10 mg/ml concentration (Fig. S4B). The specificity of the invasion inhibition by the PfRH5 rat IgG was validated by reversing it with the addition of the recombinant rpfrh5 63 in the assay. Addition of rpfrh5 63 at a concentration of 50µg/ml significantly reduced the invasion 19

20 inhibition observed with 5 mg/ml PfRH5 IgG by 80% (Fig. S4C). On the other hand, addition of PfF2 at the same concentration had no effect on the invasion inhibitory activity of the PfRH5 antibodies (Fig. S4C). Thus, not only within the PfRH family but also compared to other major parasite ligands, PfRH5 is a potent target of specific antibody mediated blockade of erythrocyte invasion and appears to have a dominant role in the erythrocyte invasion process. Therefore, antibodies raised against a functional recombinant protein expressed in E. coli representing the wild-type fulllength sequence of PfRH5 have proven to be as potent in blocking parasite invasion as reported earlier with antibodies raised by adenoviral vectors (26,36) or the mutated PfRH5-ratCD4(d3+4) fusion protein (27) PfRH5 based antibody combinations produce an additive inhibition of erythrocyte invasion by P. falciparum In a recent report, we identified a potent antigen combination (PfAARP+PfRH2+PfF2) that elicits strain-transcending invasion inhibitory antibodies (29). Through this study, we had demonstrated that triple antibody combinations were more efficacious in inhibiting erythrocyte invasion compared to double antibody combinations (29). This study did not include PfRH5, so we wanted to now analyze whether PfRH5 based triple antibody combinations would also be effective in producing additive inhibition of invasion at lower individual IgG concentrations. In this regard, we tested the invasion inhibition of ten PfRH5 based triple antibody combinations that comprised of purified total IgG (3.3 mg/ml each antigen; Total: 10 mg/ml) against PfRH5 (rabbit 1) and two other antigens from our portfolio (PfRH1, PfRH2, PfRH4, PfAARP, PfF2) 20

21 generated previously (29). The invasion inhibition was first assessed against two P. falciparum strains (3D7, Dd2) (Fig. 6) and then the six most efficacious antibody combinations were further analyzed against a total of five P. falciparum strains (3D7, Dd2, 7G8, MCamp, HB3) (Fig. 7) to ascertain whether the antibody combinations elicited strain-transcending activity. Individually, the six antibodies at 3.3 mg/ml exhibited a broad range of invasion inhibition of 3-54% against all five parasite clones (Fig. 6, 7). As described above, anti-pfrh5 IgG displayed maximum inhibition against all strains (44-54%), followed by anti-pfaarp IgG (17-33%) (Fig. 6, 7). Consistent with previous findings, anti-pfrh1 and anti-pff2 IgG were more efficacious in blocking the sialic acid dependent clones- Dd2 (24-36%), MCamp (15-17%) and not the sialic acid independent clones 3D7 (8-12%), 7G8 (5-10%) and HB3 (5-10%) (Fig.6, 7). Similarly, PfRH2 IgG and PfRH4 IgG efficiently blocked only the sialic acid independent clones and not the sialic acid dependent clones, in which they are poorly expressed. The different anti-pfrh5 based antibody combinations displayed potent inhibition of erythrocyte invasion by the 3D7 parasite clone (Fig. 6A) with the maximum inhibition observed with three antibody combinations, PfAARP+PfRH2+PfRH5 (82%), PfF2+PfRH5+PfAARP (82%) and PfF2+PfRH2+PfRH5 (79%). In line with the utilization and expression of the different PfRH ligands, we observed that the maximum inhibition with the Dd2 parasite clone was by the antibody combinations, PfRH5+PfF2+PfAARP (81%), PfRH5+PfRH1+PfAARP (79%) and PfRH5+PfRH1+PfF2 (78%) (Fig. 6B). This is consistent with the higher expression and utilization of the sialic acid binding ligands (PfRH1, PfF2 and PfAARP) for erythrocyte invasion by sialic acid dependent parasite clones such as Dd2. We further tested invasion inhibition of the six best PfRH5 based antibody combinations against five P. falciparum clones 21

22 that originate from diverse regions of the world and exhibit different invasion phenotypes as well. The six antibody combinations were potent in blocking invasion of all five parasite clones with the antibody combinations, PfRH5+PfF2+PfAARP (77-82%), PfRH5+PfRH2+PfAARP (71-83%), and PfF2+PfRH2+PfRH5 (72-79%) eliciting the most efficacious strain-transcending invasion inhibitory activity (Fig. 7). We also tested as a control the PfAARP+PfF2+PfRH2 antibody combination, which we had previously reported (29) as our most efficacious antibody combination (Fig. 7). While this antibody combination does elicit strain-transcending activity, the combination PfRH5+PfF2+PfAARP appeared to be slightly more efficient in its overall strain-transcending activity against all the five P. falciparum clones, which is consistent with the observation that PfRH5 antibodies individually were observed to be most efficient among the different parasite ligands in blocking erythrocyte invasion. However, the difference in invasion inhibitory activity between the PfRH5+PfF2+PfAARP and PfAARP+PfF2+PfRH2 antibody combinations was not statistically significant (p>0.05) Co-immunized antigen mixtures also elicit potent strain-transcending invasion inhibitory antibodies After evaluating antibody combinations that were mixed in vitro for their invasion inhibitory activity, we wanted to test whether the most potent combination identified would elicit similar invasion inhibitory antibodies when co-immunized together as a single formulation. In this next step, the PfRH5 based triple antigen mixtures (PfRH5+PfRH2+PfAARP and PfRH5+PfF2+PfAARP) were used to immunize mice (BALB/c). The individual antigens in each 22

23 combination were also used to immunize mice separately. All antigens were formulated with the adjuvant, CFA/IFA. The ELISA results (OD 492 ) showed that the antibody titers (end point 1:320,000) against each protein immunized individually were not significantly altered when immunized as a mixture with the two other antigens (Fig. S5). The immunogenicity curves for PfRH5, PfRH2, PfF2 and PfAARP were identical and overlapping whether the antigens were immunized alone or in their respective combinations (Fig. S5). Thus, our recombinant antigens including rpfrh5 63 were immunogenic and did not elicit any significant immune interference when immunized in combination. Consistent with the invasion inhibition observed with the antibody combinations physically added in vitro, the antibodies raised against the antigen mixtures were highly potent and equally efficient in inhibiting erythrocyte invasion (Fig. 8, S6). The antibodies against the two antigen mixtures displayed 71-79% and ~86% inhibition of the P. falciparum clones (3D7, Dd2) at concentrations of 5 and 10 mg/ml, respectively (Fig. 8A, 8B). The antibodies displayed a similar potent inhibition of three more parasite clones at an IgG concentration of 10 mg/ml (Fig. S6). Thus, antibodies raised against co-immunized antigen mixtures were as potent in efficiently blocking erythrocyte invasion as antibodies physically combined (Fig. 6, 7) in the in vitro invasion inhibition assays

24 Discussion Parasite neutralizing antibodies that block P. falciparum erythrocyte invasion is one of the key effector mechanisms known to mediate immunity against blood-stage malaria parasites and is the fundamental basis for the development of blood-stage malaria vaccines. The ability to impede erythrocyte invasion by P. falciparum merozoites can be quantitated in an in vitro invasion inhibition or growth inhibition assay (GIA) that has been widely reported in the field. A significant association of invasion inhibition measured in vitro with a reduced risk of malaria has also been reported (37, 38) and thus, in vitro invasion-inhibition appears to be a useful surrogate marker to predict the functional efficacy of antibodies induced by a blood-stage vaccine. In spite of the extensive research on the parasite biology of P. falciparum, it has been difficult to demonstrate potent invasion inhibitory activity with the exception of antibodies against Apical Membrane Antigen-1, AMA-1 (39, 40). The challenge in generating potent invasion inhibitory antibodies that have hindered the development of blood-stage malaria vaccines against P. falciparum could be attributed to the enormous complexity of the parasite, which has evolved redundancy in parasite ligands that enables invasion of diverse types of human erythrocytes (2-4). As a result, no human erythrocyte is known to be totally refractory to invasion by P. falciparum. Another level of complexity is the high level of antigenic polymorphisms that mediates immune escape, which has proven to be a major impediment in developing leading antigens such as AMA-1 and MSP-1 42 as blood stage vaccines (5-7). Both antigens have failed to elicit optimal efficacy in field trials (5, 6), Thus, to fully realize the potential of a candidate blood-stage antigen, it is not sufficient to just show that the antigen elicits strong invasion inhibitory antibodies that block the parasite in 24

25 vitro. A good example is AMA-1, an essential parasite protein involved in the critical step of junction formation during erythrocyte invasion (41, 42), which has been demonstrated in vitro to induce potent invasion inhibitory antibodies only against homologous parasite strains and not against heterologous strains (39, 40). This inability has been attributed to high levels of antigenic polymorphisms in AMA-1 among different parasite strains that render it s antibodies to be ineffective against heterologous P. falciparum strains (39, 43, 44). Unfortunately due to the problem of inducing allele-specific immunity, AMA-1 has not yielded protection against malaria in vaccine efficacy trials (7, 45). Therefore, it is crucial that potent strain-transcending invasion inhibition is demonstrated with the antibodies against a particular antigen that is being considered for clinical development of a malaria vaccine. This problem has been reported to be overcome by targeting combinations of different conserved parasite antigens or a number of different AMA-1 allelic proteins that produce strong efficacious invasion inhibition in a strain-transcending manner (29, 46). The disappointing results of a number of blood-stage vaccine trials has raised the concern whether the in vitro invasion inhibition assay or GIA assay has any correlation with clinical protection or could predict vaccine efficacy in humans (47). Unfortunately, only a few bloodstage antigens have been tested in field efficacy trials. Due to the problems cited above, more studies with parasite antigens that induce potent strain-transcending invasion inhibition are thus required to validate the correlation of in vitro invasion inhibitory activity with clinical protection in humans. It would thus be beneficial to validate the in vitro invasion inhibitory assay or GIA with in vivo CHMI (controlled human malaria infection) studies using either a sporozoite or blood-stage challenge model (47). However, the in vitro invasion inhibitory assay or GIA 25

26 remains as the only currently available laboratory assay to measure the functionality of antibodies to inhibit erythrocyte invasion by Plasmodium merozoites. In this regard, this assay has been successful in differentiating between invasion inhibitory and non-inhibitory antibodies against a number of P. falciparum antigens as not all antibodies exhibit potent invasion inhibitory activity. Hence, the in vitro invasion inhibition or GIA assay clearly appears to be an informative assay, which in pre-clinical studies can identify and validate novel, efficacious P. falciparum blood-stage targets that elicit strain-transcending invasion-inhibitory antibodies. The past decade has seen a huge body of research conducted on understanding the molecular basis of erythrocyte invasion and characterizing numerous antigens from the large repertoire developed by P. falciparum (2-4, 8, 9). With respect to vaccine development, the goal to search for promising antigens has shifted to the identification of relatively conserved antigens that elicit potent strain-transcending invasion inhibitory antibodies. Among the parasite s large molecular arsenal, the PfRH proteins have been identified as key determinants of different invasion pathways used by P. falciparum (2-4, 8, 9), of which PfRH5 is the only erythrocyte binding ligand known to be essential for the parasite as its gene is refractory for disruption (22). The importance of PfRH5 has been substantiated by the fact that antibodies against both PfRH5 and its erythrocyte receptor, Basigin, potently inhibit erythrocyte invasion by a number of P. falciparum strains from diverse worldwide locations that also exhibit different invasion phenotypes (25-27). The efficacious neutralization of heterologous parasite clones by PfRH5 antibodies raised first through a viral vector based prime boost strategy had demonstrated that PfRH5 is a highly promising blood stage vaccine candidate (26). Our goal was to demonstrate the production of a potent functional wild-type full-length PfRH5 recombinant antigen that could 26

27 be used for the development of a subunit blood-stage malaria vaccine, which would have the potential to be tested individually as well as in combination with other blood stage antigens and possibly even pre-erythrocytic malaria vaccines such as RTS,S (48). RTS,S is the most advanced malaria vaccine currently being tested in a Phase III clinical trial in Africa (49, 50). It is a subunit recombinant vaccine based on the CSP-HbSAg fusion protein that assembles into virus-like particles (48-50). In this regard, we have produced the full-length, wild type PfRH5 recombinant protein without making any alterations to its sequence in a bacterial organism, E. coli. The recombinant protein binds with erythrocytes with the same specificity as that of the native parasite protein and anti-basigin monoclonal antibodies specifically blocked the binding of the recombinant protein. This binding was unaffected by other monoclonal antibodies against another major erythrocyte receptor, glycophorin A. All these results strongly suggest that the recombinant PfRH5 was produced with a conformational integrity that mimics the native parasite protein. This work paves the way for a more detailed structure-function analysis of PfRH5 that would not only improve our understanding of its role in the basic biology of the parasite but also help in designing a more efficacious PfRH5 immunogen for vaccine development. While, it was very elegantly demonstrated that PfRH5 binds with Basigin (25), this report did not show any data with regard to the interaction of the native PfRH5 parasite protein and based its inferences on the interaction with a pentamer of the biotinylated recombinant PfRH5- ratcd4(d3+4) fusion protein. In the present study, we have demonstrated that the Basigin monoclonal antibodies at the same concentration at which they blocked erythrocyte invasion also completely inhibited the erythrocyte binding of the native PfRH5 parasite protein obtained from 27

28 culture supernatants. Our data strongly supports the previous study that the native PfRH5 parasite protein binds with the erythrocyte surface molecule, Basigin. Further, most importantly, the antibodies raised against recombinant PfRH5 specifically inhibited erythrocyte invasion of heterologous P. falciparum strains with a potent efficiency as high as 85% and 71% at total IgG concentrations of 10 mg/ml and 5 mg/ml, respectively. PfRH5 antibodies were observed to be most potent among a pool of antibodies ( mg/ml total IgG) tested against six antigens in our portfolio that included four PfRH proteins (PfRH1, PfRH2, PfRH4, PfRH5), PfEBA-175 (PfF2) and PfAARP (29). The normal physiological total IgG concentration in human serum is in the range of mg/ml (51). Thus, we have analyzed our antibodies at a maximum total IgG concentration of 10 mg/ml, which is on the lower end of the human physiological concentration. The specific component of the anti-pfrh5 IgG within the total serum IgG (10 mg/ml) would be a much smaller fraction and should be achievable through a human vaccine. In addition, PfRH5 antibodies exhibited the maximum cross-strain neutralizing activity as they were efficacious in blocking a number of P. falciparum clones that display phenotypic variation in their invasion pathways. Our invasion inhibition results are consistent with previous reports of PfRH5 antibodies raised through viral vectors (26) or against the PfRH5-ratCD4(d3+4) fusion protein (27). In our assays, the rpfrh5 63 antibodies exhibited an EC 50 of ~ 3 mg/ml, whereas previous studies with viral vectors and PfRH5-ratCD4(d3+4) fusion protein have shown an EC 50 with anti-pfrh5 IgG obtained from different rabbits at a broad concentration range of mg/ml against multiple P. falciparum strains (26, 27, 36). The difference in invasion inhibitory activity could be attributed to the different nature of the PfRH5 immunogen used in the studies as well as the fact that different animals were used in the studies 28

29 to raise the PfRH5 specific antibodies. Outbred animals are known to exhibit large differences in their immune responses and especially when located in different geographical locations. As stated previously, achieving highly potent strain-transcending invasion inhibitory antibodies against conserved functional domains of parasite antigens involved in invasion may be the key to the development of an effective blood stage malaria vaccine. This has been difficult to achieve by targeting single antigens with the exception of PfRH5 as reported here and in previous reports. Earlier potent strain-transcending invasion inhibitory antibodies were demonstrated only by targeting combinations of key merozoite antigens involved in erythrocyte invasion (24, 29, 36, 46, 52). In line with these reports, we feel that targeting single antigens may not be an effective long-term strategy for malaria vaccines as the parasite has the strong ability to develop escape mechanisms to counter the immune pressure. This capability is well displayed in the rapid development of resistance in the parasite under drug pressure that has prompted only combinatorial anti-malarial drug therapies. More so in combination, antibodies neutralize the parasite at lower individual IgG concentrations (3.3 mg/ml total IgG each) that would be easier to achieve using human compatible adjuvants or delivery platforms. PfRH5 antibodies in combination with those against PfRH2, PfAARP and PfF2 produced an additive inhibition of erythrocyte invasion. Our data is consistent with a previous study that has reported a potent synergy in invasion inhibition by adenoviral vector induced antibodies against PfRH5 and other EBA/PfRH antigens (36). We have also demonstrated that these PfRH5 based co-immunized antigen mixtures induce balanced antibody responses against all three antigens with no immune 29

30 interference. Purified total IgG against the antigen mixtures were as potent in inhibiting erythrocyte invasion as the antibodies combined in vitro. Thus, our study establishes a proof of principle for the production of full-length wild-type recombinant PfRH5 in a bacterial expression system that is known to be scalable for mass vaccine production and has the potential to be taken forward for its development as a component of a subunit blood-stage combination malaria vaccine. However, we have demonstrated potent invasion inhibitory antibodies against PfRH5 raised through freund s formulations that are known to induce very strong immune responses but are not safe for human vaccine applications. Thus, it is imperative for the clinical development of recombinant PfRH5 as a malaria vaccine to identify human compatible adjuvants or delivery platforms that would also elicit similar highly potent neutralizing antibodies. Nevertheless, the production of the wild type full length recombinant PfRH5 protein with no amino acid modifications further enables the structurefunction analysis of this highly efficacious and attractive blood-stage malaria vaccine candidate

31 Acknowledgements We are grateful to Dr Louis Miller (NIH) for providing the P. falciparum clones used in the study and the rpfrh4 30 expression plasmid. We also wish to thank Dr Lee Hall and Dr Annie Mo from the Parasitology and International Programs Branch (PIPB), NIAID, NIH for providing the recombinant EBA-175 RII protein. The technical assistance of Dr Alka Galav, Rakesh Kumar Singh and Ashok Das from the ICGEB animal facility in performing the animal experiments is deeply appreciated. We wish to thank Dr Inderjeet Kaur at the mass spectrometry facility of the Malaria group, ICGEB for helping us in the LC-MS analysis. We appreciate Ms Surbhi Dabral at the super resolution imaging facility of the Malaria group, ICGEB for technical help in our imaging study. Deepak Gaur is the recipient of the Ramalingaswami Fellowship from the Department of Biotechnology, Government of India. Deepak Gaur is also the recipient of the Grand Challenges Exploration Grant from the Bill and Melinda Gates Foundation. This work was supported by the Bill & Melinda Gates Foundation through the Grand Challenges Explorations Initiative [GCE OPP to D.G.]; Department of Biotechnology (DBT), Government of India through the Ramalingaswami fellowship program [BT/HRD/35 /02/14/2008 to D.G.], Rapid Grant Scheme for Young Investigators [BT/PR13376/GBD/27/ 260/2009 to D.G.], Vaccine Grand Challenges Program [ND/DBT/12/040 to D.G., C.E.C., V.S.C.]. K.S.R., T.S. are recipients of Senior Research Fellowships of the Council of Scientific and Industrial Research, Government of India; A.K.P is a recipient of a Post-doctoral Research Associateship of DBT; H.S. is the recipient of the Senior Research Fellowship of DBT. A.E. is the recipient of the ICGEB International PhD 31

32 Pre-doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work has been previously presented at the 24 th National Congress of Parasitology in Jabalpur, Madhya Pradesh, India (April 27-29, 2013) and the 2013 Malaria Gordon Research Conference in Tuscany, Italy (August 4-9, 2013) Conflict of Interest: D.G., V.S.C., C.E.C are named on patent applications relating to PfRH5 and/or other malaria vaccines. This does not alter our adherence to all Infection and Immunity policies on sharing data and material

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41 Miura K, Herrera R, Diouf A, Zhou H, Mu J, Hu Z, MacDonald NJ, Reiter K, Nguyen V, Shimp RL Jr, Singh K, Narum DL, Long CA, Miller LH Overcoming allelic specificity by immunization with five allelic forms of Plasmodium falciparum apical membrane antigen 1. Infect Immun. 81(5): Duncan CJ, Hill AV, Ellis RD Can growth inhibition assays (GIA) predict 893 blood-stage malaria vaccine efficacy? Hum Vaccin Immunother. 8(6): Regules JA, Cummings JF, Ockenhouse CF The RTS,S vaccine candidate for 895 malaria. Expert Rev Vaccines. 10(5): White NJ A vaccine for malaria. N Engl J Med. 365(20): Daily JP Malaria vaccine trials--beyond efficacy end points. N Engl J Med (24): Stoop JW, Zegers BJ, Sander PC, Ballieux RE Serum immunoglobulin levels 900 in healthy children and adults. Clin. Exp. Immunol. 4: Lopaticki S, Maier AG, Thompson J, Wilson DW, Tham WH, Triglia T, Gout A, Speed TP, Beeson JG, Healer J, Cowman AF Reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites. Infect. Immun. 79:

42 913 Figure Legends Figure 1: Production of the recombinant rpfrh5 63 protein. (A) Schematic representation of the PfRH5 parasite protein. Region in black denotes the signal peptide (residues 1-26) and region in green (residues ) was expressed in E. coli. (B) Purified rpfrh5 63 protein analyzed on 12% SDS-PAGE, stained with coomassie blue. 2µg of rpfrh5 63 has been loaded in each well. (C) Recombinant rpfrh5 63 protein detected in immunoblots using the anti-his tag antibody. R and NR represents -reducing and non-reducing conditions, respectively Figure 2: Erythrocyte binding activity of the native PfRH5 parasite protein and recombinant rpfrh5 63 protein. Anti-PfRH5 antibodies in immunoblots detected both native PfRH5 and rpfrh5 63 among the proteins that bound with the erythrocyte surface and got eluted off by 1.5 M NaCl. (A) Native PfRH5 from the 3D7 culture supernatant bound untreated (U) and all three enzymatically treated human erythrocytes [neuraminidase (N), trypsin (T), chymotrypsin (C)] but failed to bind with proteinase K (PK) treated erythrocytes. Thus, PfRH5 binds erythrocytes in a sialic acid independent, trypsin/chymotrypsin resistant, proteinase K sensitive manner. (B) rpfrh5 63 bound human erythrocytes with the same specificity as that of native PfRH5. (C) Native PfEBA- 175, as well as (D) PfF2, (recombinant receptor binding domain of PfEBA-175) were analyzed as a control with the same set of enzymatically treated erythrocytes. Both native PfEBA-175 and PfF2 bound erythrocytes in neuraminidase/trypsin/proteinase K 42

43 sensitive, chymotrypsin resistant manner. PBS denotes Phosphate buffer saline, ph 7.4 that contained no protein. No specific protein was detected in the eluate fractions with the PBS control suggesting that no non-specific erythrocyte protein was being detected in the assay. (* U= Untreated; N= Neuraminidase; T= Trypsin; and C= Chymotrypsin; PK = Proteinase K) Figure 3: Antibodies against recombinant rpfrh5 63 blocked erythrocyte binding of both the native PfRH5 and rpfrh5 63 proteins. Purified total rabbit IgG against rpfrh5 63 blocked binding of (A) native PfRH5 and (B) recombinant rpfrh5 63 in a dose dependent manner. The PfRH5 antibodies had no effect on the erythrocyte binding of (C) native EBA-175 or (D) recombinant PfF2 even at the maximum IgG concentration of 800 µg/ml Figure 4: Anti-Basigin monoclonal antibodies blocked the erythrocyte binding of native and recombinant PfRH5. (A) Anti-Basigin TRA-1-85 monoclonal antibodies potently inhibited invasion of human erythrocytes by the P. falciparum clone 3D7, with complete blockade of invasion observed at 5µg/ml. At the same invasion inhibitory IgG concentrations, the TRA-1-85 monoclonal antibodies potently blocked the erythrocyte binding of both the (B) native PfRH5 protein and (C) recombinant rpfrh5 63. Antiglycophorin A monoclonal antibodies had no effect on the binding of both (B) native and (C) recombinant PfRH

44 Figure 5: Invasion inhibitory activity of anti-pfrh5 rabbit antibodies. (A) Invasion inhibitory activity of purified rabbit total IgG ( mg/ml) against rpfrh5 63 and the receptor binding domains of other key merozoite ligands (PfRH1, PfRH2, PfRH4, PfAARP, PfF2) against the P. falciparum clone 3D7. AMA-1 IgG (5 mg/ml) was used as a positive control. (B) Strain-transcending parasite neutralization activity of anti-pfrh5 total rabbit IgG ( mg/ml) against five diverse P. falciparum clones. The control anti-hpr total IgG failed to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml. The results represent the average of three independent experiments performed in duplicate. The error bars represent the standard error of the mean Figure 6: Invasion inhibitory efficacy of PfRH5 based antibody combinations against P. falciparum clones (3D7, Dd2). Purified rabbit total IgG against the six individual antigens (PfRH1, PfRH2, PfRH4, PfRH5, PfAARP, PfF2) were evaluated individually (3.3 mg/ml) as well as in all ten possible PfRH5 based triple antibody combinations (3.3 mg/ml each; total 10 mg/ml) against (A) the sialic acid independent clone 3D7 and (B) the sialic acid dependent clone Dd2. The control anti-hpr total IgG failed to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml. The results represent the average of three independent experiments performed in duplicate. The error bars represent the standard error of the mean

45 Figure 7: Strain-transcending parasite neutralization of PfRH5 based antibody combinations. Purified rabbit total IgG against the six individual antigens (PfRH1, PfRH2, PfRH4, PfRH5, PfAARP, PfF2) were evaluated individually and in the six most potent PfRH5 based antibody combinations (identified from Fig. 6) against five diverse P. falciparum clones. The invasion inhibitory activity of the antibody combination, AARP+RH2+PfF2, was also analyzed in the assay. The control anti-hpr total IgG failed to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml. Three independent assays were performed in duplicate. The error bars represent the standard error of the mean Figure 8: Invasion inhibitory activity of antibodies raised against the two coimmunized antigen formulations: Total IgG purified from mice sera raised against the immunogens (RH5, RH2, PfF2, AARP, PfF2+RH5+AARP, RH2+RH5+AARP) were evaluated for its invasion inhibitory activity (at concentrations of 1, 3.3, 5 and 10 mg/ml) against the (A) sialic acid independent clone 3D7 and the (B) sialic acid dependent clone Dd2. The control anti-hpr total IgG failed to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml. Two independent assays were performed in duplicate. The error bars show the standard error of the mean. * denotes statistical significance between the invasion inhibition produced by the antibodies against the two antigen mixtures (PfF2+RH5+AARP, RH2+RH5+AARP) with respect to the individual PfRH5 antibodies at the same concentrations of purified total IgG, 3.3 mg/ml and 5 mg/ml 1000 (p values < 0.05). 45

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