IAI Accepts, published online ahead of print on 14 October 2013 Infect. Immun. doi:10.1128/IAI.00970-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Classification: Microbial Immunity & Vaccines
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A bacterially expressed full-length recombinant Plasmodium falciparum RH5 protein binds
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erythrocytes and elicits potent strain-transcending parasite neutralizing antibodies
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K. Sony Reddy§, Alok K. Pandey§, Hina Singh, Tajali Sahar, Amlabu Emmanuel,
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Chetan E. Chitnis, Virander S. Chauhan, Deepak Gaur*
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Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
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*Corresponding Author
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Please send correspondence to:
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Deepak Gaur, Ph.D. Malaria Group International Centre for Genetic Engineering and Biotechnology (ICGEB) Aruna Asaf Ali Marg New Delhi, India Tel: +91-11-26741358 Fax: +91-11-26742316 E-mail:
[email protected]
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§ These authors contributed equally to the work
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Running Title: Recombinant PfRH5 elicits neutralizing antibodies
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Keywords: Malaria, Plasmodium, erythrocyte, invasion, ligand, receptors, vaccine, host pathogen
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interaction.
30 31 1
34 32 Abstract 33 35 Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an essential 36
merozoite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is an attractive malaria
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vaccine candidate as it is expressed by a wide number of P. falciparum strains, cannot be
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genetically disrupted and exhibits limited sequence polymorphisms. Viral vector induced PfRH5
39
antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate
40
full-length recombinant PfRH5 in a bacterial cell based expression system. Here, we have
41
produced full-length recombinant PfRH5 in Escherichia coli that exhibits specific erythrocyte
42
binding similar to that of the native PfRH5 parasite protein and also importantly elicits potent
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invasion inhibitory antibodies against a number of P. falciparum strains. Anti-Basigin antibodies
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blocked the erythrocyte binding of both native and recombinant PfRH5 further confirming that
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they bind with Basigin. We have thus successfully produced full-length PfRH5 as a functionally
46
active erythrocyte binding recombinant protein with a conformational integrity that mimics the
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native parasite protein and elicits potent strain-transcending parasite neutralizing antibodies. P.
48
falciparum has the capability to develop immune escape mechanisms and thus blood-stage
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malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In
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this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced
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potent additive inhibition against multiple worldwide P. falciparum strains. PfRH5 was
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immunogenic when immunized with other antigens eliciting potent invasion inhibitory antibody
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responses with no immune interference. Our results strongly support the development of PfRH5
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as a component of a combination blood-stage malaria vaccine.
55 56 2
57
Introduction
58
Malaria is a global infectious disease that accounts for around one million deaths across
59
the world primarily in young children below the age of five years (1). The causative agent of the
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most severe form of malaria that is responsible for maximum mortality is the parasite,
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Plasmodium falciparum. Invasion of human erythrocytes by P. falciparum is a critical process
62
during the parasite’s life cycle that leads to the development of blood stage parasites, which are
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primarily responsible for malaria pathogenesis. P. falciparum has evolved a complex, multistep
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process of erythrocyte invasion that involves numerous ligand-receptor interactions (2-4). This
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molecular redundancy allows the parasite to use many alternate pathways for invasion, thus
66
ensuring that the pathogen gains entry into its host erythrocyte (2-4).
67
The quest for developing a vaccine that targets blood-stage parasites has involved
68
extensive studies on identifying and characterizing key parasite molecules that mediate
69
erythrocyte invasion. Early efforts have focused on two leading candidates – MSP-142 and AMA-
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1, which play an essential role in erythrocyte invasion (2-4) but have unfortunately not generated
71
optimal protection in field efficacy trials (5-7). Recently, the family of P. falciparum reticulocyte
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binding-like homologous proteins (PfRH) has attracted most attention as key determinants of
73
merozoite invasion (2-4, 8, 9). The PfRH family comprises of five members - PfRH1, PfRH2a,
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PfRH2b, PfRH4 and PfRH5 that bind with either sialic acid dependent or sialic acid independent
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erythrocyte receptors (10-22). However, most of these proteins are not essential for erythrocyte
76
invasion and can be genetically disrupted (4, 8, 9) with the exception of PfRH5 (22).
77
PfRH5 (Accession Number: XP_001351544; PlasmoDB ID: PF3D7_0424100) was first
78
identified by genetic mapping as a key determinant of species specific erythrocyte invasion (21). 3
79
Genetic analysis of the progeny of a P. falciparum cross between two parental clones 7G8 x GB4
80
had mapped the PfRH5 gene on chromosome 4 as the locus responsible for mediating invasion
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of Aotus nancymaae erythrocytes as well as infectivity of Aotus monkeys (21). It was also
82
demonstrated that PfRH5 is an erythrocyte binding ligand in which single point mutations
83
critically affected the specificity of its binding with Aotus erythrocytes (21). Recently, PfRH5
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has also been shown to play a role in the invasion of both owl monkey and rat erythrocytes by P.
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falciparum (23). Further, PfRH5 was found to be unique in being the only erythrocyte binding
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ligand among the EBA/PfRH families that is essential for the parasite as it cannot be genetically
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knocked out (22), suggesting a crucial role in erythrocyte invasion. PfRH5 is also exceptional
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compared to other PfRH homologues as it is smaller in size (63 kDa) and lacks a transmembrane
89
domain (21, 22). PfRH5 has been shown to be localized on the merozoite surface in association
90
with another parasite molecule, PfRipr (P. falciparum PfRH5 interacting protein) (24). While,
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PfRH proteins are differentially expressed among different P. falciparum clones that exhibit
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phenotypic variation in their invasion properties (11, 13, 16-19), the expression of PfRH5 was
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found to be consistent among these parasite clones (21, 22).
94
Recently, PfRH5 was reported to bind with the CD147 IgG super family member,
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Basigin (BSG) on the erythrocyte surface (25). For this study, a mutated version of PfRH5 was
96
produced in mammalian HEK293 cells as a biotinylated fusion protein with the CD4 domains
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3+4(CD4d3+4) of rat origin, which was found to bind with a pentamer of Basigin (25). The
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mutations were necessary to produce a non-glycosylated protein in the mammalian cells similar
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to that of the native parasite protein, which like P. falciparum native proteins remains essentially
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unglycosylated. The significance of the PfRH5-BSG interaction was highlighted by the 4
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demonstration that anti-BSG antibodies blocked erythrocyte invasion by a large number of P.
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falciparum clones that were known to exhibit different invasion phenotypes (25). However, no
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data was reported in this elegant study on the interaction of native PfRH5 with Basigin.
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A heterologous prime-boost strategy based on the adenoviral/MVA viral vector platform
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was used to generate anti-PfRH5 antibodies that efficiently inhibited erythrocyte invasion by
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multiple heterologous P. falciparum clones (26). It has also been recently reported that
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antibodies against a fusion protein comprising of a mutated PfRH5 with the (d3+4) domains of
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the rat CD4 protein exhibited potent inhibition of erythrocyte invasion (27). This study further
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substantiated the importance of the PfRH5-BSG interaction during erythrocyte invasion and
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strongly supported PfRH5 as a blood stage vaccine candidate. However, polymorphisms in
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PfRH5 as well as other P. falciparum adhesins have been shown to induce changes in their
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receptor specificity (2, 3, 21, 23) and could also possibly alter native structure. Thus, the
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production of a functionally active recombinant wild-type full-length PfRH5 protein, which
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would elicit similar potent invasion inhibitory antibodies, in a cell based expression system that
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could be scalable for mass production still remained a challenge not only from a vaccine
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perspective but also to facilitate the basic structure-function analysis of PfRH5.
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Also considering that the target population of a prophylactic malaria vaccine is young
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infants and children, it is important to test different expression platforms so as to identify the
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safest and most efficacious antigen or delivery mechanism that would be most feasible to
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administer as a vaccine for mass immunization. In this regard, the subunit vaccine approach
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based on formulations of recombinant proteins and adjuvant pose a safe and effective platform
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for administering a vaccine for large masses. However, the test here lies in being able to produce 5
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the recombinant protein with a structural integrity that yields potent neutralizing antibodies
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against the respective pathogens.
125
Expression in Escherichia coli provides a cost-effective method for production of
126
recombinant proteins for use as biologics and vaccines. Previous reports of production of
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recombinant proteins against PfRH5 in E. coli have focused on expressing smaller fragments of
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143-168 amino acids (22, 28). Both these recombinant fragments failed to elicit invasion
129
inhibitory antibodies (22, 28). This is consistent with the recent report using the Adeno-MVA
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prime boost approach that demonstrated potent invasion inhibitory antibodies only against full-
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length PfRH5 and not against the 168 amino acid fragment of PfRH5 (26).
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In light of these reports, in our study we have demonstrated that the full-length wild-type
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PfRH5 recombinant protein produced in E. coli was efficacious in eliciting potent invasion
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inhibition consistent with that observed with the viral vector delivery platform (26) or with the
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ratCD4(d3+4) fusion construct (27). In the current report, we have successfully produced full-
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length PfRH5 as a recombinant protein in E. coli that exhibits specific erythrocyte binding
137
activity similar to that of the native PfRH5 parasite protein. We also demonstrated that anti-
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Basigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5
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proteins, further confirming that Basigin acts as their erythrocyte receptor. Our recombinant
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PfRH5 elicited potent strain-transcending invasion inhibitory antibodies that blocked a number
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of heterologous parasite clones. Importantly, the PfRH5 antibodies produced additive invasion
142
inhibition in combination with antibodies against other key merozoite antigens. Thus, our study
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strongly supports the development of PfRH5 based antigen combinations as malaria vaccine
144
candidates. 6
145
Materials and Methods:
146 147
Ethics statement
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The animal studies described below were approved by the International Centre for
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Genetic Engineering and Biotechnology (ICGEB) Institutional Animal Ethics Committee
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(IAEC) (reference no.MAL-51) according to the guidelines of the Department of Biotechnology,
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Government of India.
152 153
Cloning, expression and purification of the full-length recombinant PfRH5
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The 1500 bp PfRH5 gene that encodes the 500 amino acid full length parasite protein
155
excluding the signal sequence (rPfRH563, Asp27-Gln526) was PCR amplified from the genomic
156
DNA of the P. falciparum clone 3D7 using the following primers, RH5-Fwd: 5’-
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ATATATAATTCATATGAATGCAATAAAAAAAACGAAGAAT-3’
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AGCACTCGAGTTGTGTAAGTGGTTTATTTTTTT-3’. The PCR product encoding rPfRH563
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were digested with Nde I and Xho I (New England Biolabs, Beverly, MA) and inserted
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downstream of the T7 promoter in the E. coli expression vector, pET-24b (Novagen, San Diego,
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CA) with a C-terminal 6-histidine (6-His) tag to obtain the plasmid pPfRH5-pET24b.
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Sequencing of the ligated plasmid confirmed the correct sequence of the PfRh5 gene and that the
163
insertions were in the correct reading frame.
and
RH5-Rev:
5’-
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E. coli BL21(DE3) was transformed with pPfRH5-pET24b and was used to produce the
165
recombinant protein rPfRH563. Transformed E. coli BL21(DE3) were cultured in superbroth at
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37°C and later were induced with 1mM IPTG when the OD600 was around 0.8-0.9. Cells were 7
167
harvested by centrifugation at 3000g, after 4 hours of induction at 37°C. Cell pellets were lyzed
168
by sonication and rPfRH563 was found to be expressed as inclusion bodies. The inclusion bodies
169
were washed, collected by centrifugation at 15000g and solubilized in a buffer containing 20
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mM Tris pH 8.0, 6 M Guanidium-HCl, 300 mM NaCl, 10 mM Imidazole and 5 mM beta
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mercaptoethanol. rPfRH563 was purified from solubilized inclusion bodies by metal affinity
172
chromatography using the Ni-NTA (nitrilotriacetic acid) resin. Metal affinity purified rPfRH563
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was refolded in redox conditions by rapid dilution (30-fold) in an MES based buffer pH 6.5
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comprising of 1 mM GSH (reduced Glutathione), 0.1 mM GSSG (oxidized Glutathione) and 440
175
mM sucrose. The refolded protein was dialyzed against 25 mM MES (pH 6.5), 200mM sucrose
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and further purified to homogeneity by cation exchange chromatography using an SP-sepharose
177
column (GE Healthcare, Piscataway, NJ). The dialyzed protein was loaded on the SP-sepharose
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column and eluted with an increasing concentration of NaCl (0-1 M) in the MES based buffer,
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pH 6.5. The purified recombinant rPfRH563 protein was characterized by SDS-PAGE, Western
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blotting, Edman degradation, Mass spectrometric (LC-MS) analysis (Orbitrap VELOS PRO,
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Thermofisher Scientific), Reverse phase RP-HPLC (C8 column; Waters) and Size exclusion
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chromatography (Superdex 75 10/300 GL; GE Healthcare).
183 184
Animal Immunization and antibody generation
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Animal immunizations and total IgG purification were done as reported previously (29).
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Briefly, five rats and three rabbits were immunized intramuscularly with 50 µg and 100 µg
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rPfRH563, respectively. The rPfRH563 protein was emulsified with complete Freund’s adjuvant
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(Sigma, St. Louis, MO) for immunization on day 0 followed by two boosts emulsified with 8
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incomplete Freund’s Adjuvant on day 28 and 56. The sera were collected on day 70. Antibody
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levels were measured by ELISA.
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For co-immunogenicity, a group of six BALB/c mice were immunized with antigen
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mixtures PfF2+PfRH5+PfAARP, PfRH2+PfRH5+PfAARP as well as individual antigens
193
emulsified with complete Freund’s adjuvant on day 0 followed by two boosts emulsified with
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incomplete Freund’s adjuvant on days 28 and 56. 17 µg of each antigen was immunized in each
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mouse, whether used alone or as a co-immunization triple antigen mixture (total 51 µg).
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Terminal bleeds were collected on Day 70. Sera were tested for antibody titers and specific
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recognition of each recombinant protein by ELISA. For the GIA, the sera from the six mice in
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each group were pooled for IgG purification.
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Total IgG was purified from the mice, rat or rabbit sera using Protein G affinity column
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(GE Healthcare, Uppsala, Sweden), dialyzed with RPMI medium and further tested in invasion
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inhibition assays as described below. As an adjuvant negative control, we also had raised
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antibodies in mice, rats and rabbits against a non-related peptide (KESRAKKFQR
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KHITNTRDVD from human pancreatic RNase) that was also formulated with the same adjuvant
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(CFA/IFA) and injected in animals with the same schedule used for raising the PfRH5 antibodies
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as described above.
206 207
Erythrocytes and enzymatic treatment:
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Packed RBCs were procured from the Rotary Blood Bank (Tughlaqabad), New Delhi,
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India. Erythrocytes were washed in RPMI, and stored at 50% haematocrit. Enzymatic treatments
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of erythrocytes were done as stated previously (16, 20, 29). 9
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Erythrocyte binding assays:
212
Soluble parasite proteins were obtained from 3D7 culture supernatants as described
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previously (16, 20, 29). Briefly, 500µl of culture supernatant were incubated with 100µl packed
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volume of human erythrocytes at 37 C, following which the suspension was centrifuged through
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Dibutyl phthalate (Sigma). The supernatant and oil were removed by aspiration and bound
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parasite proteins were eluted using 1.5M NaCl. For rPfRH563 binding, 0.04µM of rPfRH5 was
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incubated with 100µl packed volume of human erythrocytes at 37 C in similar erythrocyte
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binding assays as described above. The eluate fractions were analyzed for the presence of native
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PfRH5 and rPfRH563 by immunoblotting using anti-rPfRH563 antibodies.
220 221
Invasion inhibition assays
222
Invasion inhibition assays were performed as described previously (16, 29) using total
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IgG purified from the sera of different animals immunized with PfRH5. Briefly, schizont-stage
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parasites at an initial parasitemia of 0.3% at 2% hematocrit were incubated with purified total
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IgG for one cycle of parasite growth (40 h post invasion). The parasite-infected erythrocytes
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were stained with ethidium bromide dye and measured by FACS as described previously (16,
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29). The percent invasion inhibition for each immune IgG was calculated with respect to the
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control pre-immune IgG from the same animal. As another negative control, we used immune
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IgG raised against a non-related peptide from human pancreatic RNase that was also immunized
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with the same freund’s adjuvant (CFA/IFA) used for raising the PfRH5 antibodies. The results
231
represent the average of three independent experiments performed in duplicate and the error bars
232
represent the standard error of the mean. Statistical significance was calculated using the 10
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Student’s t- test (Graph Pad Prism software, version 6.03). P values < 0.05 were considered
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statistically significant.
235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 11
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Results
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Expression of the full-length recombinant PfRH5 protein and generation of specific PfRH5
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antibodies
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A 500 amino acid sequence (Asn27-Gln526) of PfRH5 from the P. falciparum clone
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3D7, comprising of the full-length protein excluding the signal peptide, was chosen for
260
recombinant protein expression in E. coli (Fig. 1A). The recombinant 63kDa PfRH5 (rPfRH563)
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was expressed with a C-terminal 6-His tag. In E. coli, the rPfRH563 protein got expressed in
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inclusion bodies, which were then refolded after purification under denaturing conditions by
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metal affinity chromatography. rPfRH563 was further purified to homogeneity using ion-
264
exchange chromatography (Fig. 1B).
265
Recombinant rPfRH563 comprises of 6 cysteines that could lead to three potential
266
disulphide linkages. Initial SDS-PAGE analysis of the purified protein on a 12% gel under both
267
reducing and non-reducing conditions did not reflect a mobility shift (Fig. 1B). However, when
268
the rPfRH563 protein was run on the same gel for a longer period of time such that the 35 KDa
269
pre-stained marker protein reached the end of the gel, a mobility shift could be detected (Fig.
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S1A). The SDS-PAGE analysis was repeated along with a highly cysteine rich recombinant
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protein (rEBA-175 RII) that has 24 cysteines leading to 12 potential disulphide bonds (Fig. S1B,
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S1C). rEBA-175 RII exhibited a significant mobility shift compared to rPfRH563 that showed no
273
shift when run normally on the 12% SDS-PAGE gel (Fig. S1B). On running the gel for a longer
274
time, the mobility shift increased for rEBA-175 RII and became detectable for rPfRH563 (Fig.
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S1C). Edman degradation analysis of the full-length rPfRH563 protein yielded an N-terminal
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sequence (MNAIKK) that matched with the protein sequence of the PfRH5 native parasite 12
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protein after the signal sequence from amino acid Asn 27 onwards (Fig. S1C). Recombinant
278
rPfRH563 was identified in immunoblots using a specific anti-His tag antibody (Fig. 1C)
279
confirming expression of the full-length 500 amino acid protein with the C-terminal His-tag.
280
While, the SDS-PAGE analysis of the rPfRH563 protein showed a highly pure protein preparation
281
with the predominant band at 63 kDa, traces of a smaller 45 kDa protein were also faintly
282
visible. These two protein bands were excised from the gel and subjected to trypsin digestion
283
followed by LC-MS (liquid chromatography-mass spectrometry) analysis (Orbitrap VELOS
284
PRO, Thermofisher scientific). The LC-MS analysis revealed a large number of unique high
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scoring peptides for both proteins (32 peptides for 63 kDa protein; 31 peptides for 45 kDa
286
protein) that confirmed the identity of both proteins to be PfRH5 (Table S1, S2). The detection of
287
a smaller 45 kDa protein is consistent with previous reports on the production of recombinant
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PfRH5 in HEK293 cells (27) and that observed in the parasite lysate (22). We further analyzed
289
the recombinant protein on Size exclusion chromatography (SEC) and confirmed that our
290
recombinant protein eluted at the expected molecular size with respect to the BSA standard
291
protein, which also has a similar molecular weight (66 kDa) (Fig. S1D). The SEC profile also
292
showed that our recombinant protein was primarily in a monomeric state. The recombinant
293
rPfRH563 protein was further analyzed using Reverse Phase HPLC (RP-HPLC) that showed a
294
single symmetrical peak reflecting a highly pure protein preparation (Fig. S1E).
295
Rats and rabbits were immunized with rPfRH563 to raise PfRH5-specific antibodies. High
296
titer antibodies against rPfRH563 were detected in both rats and rabbits with end points observed
297
at dilutions of 1:320,000 (data not shown). The specificity of the PfRH5 antibodies was analyzed
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by immunoblotting studies to detect the PfRH5 native protein in parasite lysates and localization 13
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studies in merozoites by immunofluorescence super resolution confocal microscopy (N-SIM,
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Nikon, Japan). Consistent with previous reports, native parasite PfRH5 was detected in
301
immunoblots at the expected size for the full-length protein (63kDa) only in the schizont stages
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and not in early rings or trophozoite stages (Fig. S2A, S2B). PfRH5 has been reported to be
303
localized in the rhoptry bulb by immunoelectron microscopy (22). With our antibodies, PfRH5
304
was also found to co-localize with the known rhoptry bulb protein, PTRAMP (30) (Fig. S3A).
305
On the other hand, there was no co-localization observed with the rhoptry neck protein, PfRH2
306
(Fig. S3B) or the micronemal protein, PfEBA-175 (Fig. S3C). Our data was consistent with
307
previous reports (22) and confirmed the specificity of our PfRH5 antibodies.
308 309
Recombinant PfRH5 exhibits specific erythrocyte binding activity
310
Standard erythrocyte binding assays were performed with parasite culture supernatant
311
(3D7) incubated with human erythrocytes as described previously (16, 20, 29). Native PfRH5
312
parasite protein (63kDa) has been reported to be processed into smaller fragments of 45 kDa and
313
28 kDa (21, 22). The full-length native PfRH5 and its processed fragments bind erythrocytes
314
with the same specificity (21, 22, 28). In our assay, we prepared 3D7 culture supernatants and
315
observed that both the native 63 kDa full-length PfRH5 protein and the 45 kDa processed protein
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bound erythrocytes in a sialic acid independent, trypsin and chymotrypsin resistant manner (Fig.
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2A), which is consistent with previous reports (21, 22). While, in this culture supernatant we did
318
not observe the 28 kDa fragment, in another culture supernatant preparation in which the
319
parasites were incubated for a longer period of time, we detected the 28 kDa fragment and
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observed it to bind erythrocytes with the same specificity (Fig. S2C). Culture supernatants are 14
321
prepared in the absence of any protease inhibitors as they would impede parasite egress itself and
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this does lead to the possibility of parasite proteins undergoing proteolytic cleavage yielding
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fragments of different sizes (21, 22, 28).
324
The full-length, wild type PfRH5 recombinant protein, rPfRH563, expressed in E. coli
325
also exhibited an erythrocyte binding specificity that matched with that of the native parasite
326
protein (Fig. 2B). rPfRH563 specifically bound erythrocytes in a sialic acid independent, trypsin
327
and chymotrypsin resistant manner (Fig. 2B). Since, the native and recombinant PfRH5 proteins
328
bound erythrocytes treated with each of the three enzymes (neuraminidase, trypsin,
329
chymotrypsin), we also tested the effect of proteinase K treatment and found that binding of both
330
native and recombinant PfRH5 was sensitive to proteinase K (Fig.2A, 2B, S2C)
331
As controls, both native PfEBA-175 from the parasite culture supernatant and
332
recombinant PfF2, the receptor-binding domain of PfEBA-175, were found to bind erythrocytes
333
in a sialic acid dependent, trypsin sensitive and chymotrypsin resistant manner (Fig. 2C, 2D,
334
S2D) consistent with previous reports (31-33). In addition, no bound proteins were detected
335
when the erythrocytes were incubated with PBS alone (Fig. 2), thus confirming that no non-
336
specific erythrocyte protein was being detected in our assay and that the binding of PfRH5 or
337
PfEBA-175 was specific.
338 339
Antibodies against recombinant PfRH5 and Basigin block the erythrocyte binding activity
340
of native PfRH5
341
After demonstrating that rPfRH563 specifically bound erythrocytes, we determined
342
whether anti-rPfRH563 antibodies could block the erythrocyte binding of native PfRH5 parasite 15
343
protein. We demonstrated that total IgG purified from the sera of rabbits immunized with
344
rPfRH563 blocked the erythrocyte binding of native PfRH5 (Fig. 3A). Total IgG containing anti-
345
PfRH5 antibodies blocked binding of both the native and recombinant PfRH5 proteins with
346
erythrocytes in a dose-dependent manner (Fig. 3A, 3B). At a total IgG concentration of 200
347
µg/ml, the anti-PfRH5 IgG potently blocked the erythrocyte binding of both the native and
348
recombinant PfRH5 protein, whereas even at a concentration of 800 µg/ml the PfRH5 IgG had
349
no effect on the erythrocyte binding of another parasite ligand, PfEBA-175 (Fig. 3C) or its
350
recombinant receptor binding domain, PfF2 (Fig. 3D). This result clearly demonstrated that
351
PfRH5 antibodies specifically recognized only PfRH5 and further abrogated its interaction with
352
the erythrocyte surface.
353
As described earlier, the erythrocyte receptor of PfRH5 was recently identified as the
354
CD147 erythrocyte surface molecule, Basigin (BSG) (25). However, this elegant study had
355
demonstrated this interaction with recombinant PfRH5 and not the native parasite protein. We
356
tested the invasion inhibitory activity of the anti-BSG monoclonal antibodies (TRA-1-85; R&D
357
Systems, USA) and found them to potently inhibit invasion with 90% inhibition observed at a
358
concentration of 2.5µg/ml (Fig. 4A) consistent with the previous study (25). Further, we tested
359
the ability of the anti-BSG monoclonal TRA-1-85 antibodies to block the erythrocyte binding of
360
recombinant protein, rPfRH563 and native PfRH5 from parasite culture supernatants. Anti-BSG
361
monoclonal TRA-1-85 antibodies potently blocked erythrocyte invasion from a minimum
362
concentration of 2.5µg/ml and from the same concentration, the binding of the native PfRH5
363
protein was also observed to be significantly reduced in a dose dependent manner (Fig. 4B). 10
364
µg/ml of the anti-BSG monoclonal TRA-1-85 antibody completely abrogated the binding of both 16
365
the native PfRH5 protein (Fig. 4B) and rPfRH563 (Fig. 4C). As a control, anti-glycophorin A
366
monoclonal antibodies (Sigma-Aldrich) had no effect on the binding of the PfRH5 parasite
367
protein (Fig. 4B, 4C) suggesting that the anti-BSG TRA-1-85 monoclonal antibodies were acting
368
in a specific manner. This result substantiates the previous finding on PfRH5-BSG and
369
demonstrates for the first time the interaction between BSG with the native PfRH5 parasite
370
protein.
371 372
Antibodies against recombinant PfRH5 potently block erythrocyte invasion by multiple P.
373
falciparum clones
374
PfRH5 is the only parasite ligand among the EBA/PfRH families that is essential for
375
erythrocyte invasion (21, 22). We thus, compared the invasion inhibitory activity of PfRH5
376
antibodies with that of antibodies raised against five other parasite ligands from our antigen
377
portfolio – PfRH1, PfRH2, PfRH4, PfAARP (P. falciparum Apical Asparagine Rich Protein),
378
PfF2 (F2: receptor binding domain of PfEBA-175) as described previously (16, 20, 29, 32, 34).
379
The total rabbit IgGs (0.5-10 mg/ml) purified from the sera of rabbits individually immunized
380
with one of the six antigens were tested in standard one-cycle in vitro invasion inhibition assays
381
(Fig. 5A) as described in our previous report (29). The invasion inhibition of the six antibodies
382
was tested against the P. falciparum clone 3D7, which invades using both sialic acid dependent
383
and independent pathways (Fig. 5A). All antibodies exhibited a dose dependent inhibition that
384
suggested a specific effect (Fig.5A). The invasion inhibition for each immune IgG was
385
calculated with respect to the pre-immune IgG obtained from the same rabbit. In addition, as
386
another negative control, we tested the invasion inhibitory activity of purified rabbit total IgG 17
387
against a non-related peptide from a human pancreatic ribonuclease (HPR) that was also
388
formulated with the same freund’s adjuvant. The anti-HPR IgG failed to exhibit any invasion
389
inhibition at the maximum concentration of 10 mg/ml (Fig. 5A). The anti-HPR negative control
390
has been tested in each assay reported in the current study.
391
Among all six antibodies, PfRH5 total IgG was found to elicit maximum inhibition of
392
erythrocyte invasion with ~ 83% inhibition at 10 mg/ml followed by PfAARP IgG (51%, 10
393
mg/ml) and PfRH2 IgG (49%, 10 mg/ml) (Fig. 5A). PfRH5 total IgG exhibited an invasion
394
inhibition of 63% at 5 mg/ml and 52% at 3.3 mg/ml (Fig. 5A). The variation in inhibitory
395
activity among the different antibodies could not be attributed to any disparity in their antibody
396
titers. The end point antibody titers against all the six proteins immunized in rabbits were in the
397
range of 1:320,000 (data not shown). As mentioned above, we had raised antibodies against
398
PfRH5 in three rabbits that had shown equivalent end point titers in the range of 1:320,000. The
399
purified total IgG from the other two rabbits also exhibited a potent invasion inhibitory activity
400
against the P. falciparum clone 3D7 (Fig. S4A) similar to that observed with the anti-PfRH5 IgG
401
from rabbit 1 (Fig. 5A).
402
We further analyzed the strain-transcending invasion inhibitory activity of the PfRH5
403
antibodies (rabbit 1) against five P. falciparum strains that originate from different regions of
404
the world and express different polymorphic variants of the PfRH5 parasite protein (Table S3)
405
(21). In addition, these P. falciparum strains exhibit a variation in their invasion phenotype by
406
utilizing different ligand receptor interactions and pathways for invading human erythrocytes
407
(Table S3) (2-4,29 33,35). The invasion phenotypes of the P. falciparum clones are classified in
408
literature on the basis of their invasion sensitivity to enzymatic treatments of the target 18
409
erythrocytes (2-4, 29, 33, 35). Thus, the five parasite clones represent diversity both at the level
410
of PfRH5 antigenic polymorphisms as well as the phenotypic variation in the invasion properties
411
of the parasite clones (Table S3). The strains 3D7, HB3 and 7G8 are known to invade through
412
sialic acid independent pathways (3, 33, 35), whereas Dd2, MCamp are completely dependent on
413
sialic acids for erythrocyte invasion (3, 33, 35). The anti-PfRH5 antibodies (rabbit 1) were found
414
to potently inhibit erythrocyte invasion of all five P. falciparum clones (Fig. 5B). The purified
415
PfRH5 total IgG exhibited 81-85% invasion inhibition among the five parasite clones at a
416
concentration of 10 mg/ml (Fig.5B), which is comparable with the maximum inhibition that we
417
recently reported with an antibody combination against three antigens, PfAARP+PfRH2+PfF2
418
(29). The invasion inhibitory activity was dose dependent as reported earlier with an inhibition of
419
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
420
strain-transcending invasion inhibition was also observed with total IgG purified from the sera of
421
the other two rabbits immunized with rPfRH563 (data not shown). Our PfRH5 antibodies
422
exhibited a 50% inhibition of erythrocyte invasion at a total IgG concentration (EC50) of ~3
423
mg/ml, which is consistent with previous studies that have reported EC50 values for anti-PfRH5
424
IgG obtained from different rabbits within a concentration range of 0.7-4 mg/ml against multiple
425
P. falciparum strains (26, 27, 36).
426
The purified total IgG from five individual rats also exhibited a potent dose dependent
427
invasion inhibition of the P. falciparum clone 3D7 with the maximum inhibition of around 72-
428
77% at 10 mg/ml concentration (Fig. S4B). The specificity of the invasion inhibition by the
429
PfRH5 rat IgG was validated by reversing it with the addition of the recombinant rPfRH563 in the
430
assay. Addition of rPfRH563 at a concentration of 50µg/ml significantly reduced the invasion 19
431
inhibition observed with 5 mg/ml PfRH5 IgG by 80% (Fig. S4C). On the other hand, addition of
432
PfF2 at the same concentration had no effect on the invasion inhibitory activity of the PfRH5
433
antibodies (Fig. S4C).
434
Thus, not only within the PfRH family but also compared to other major parasite ligands,
435
PfRH5 is a potent target of specific antibody mediated blockade of erythrocyte invasion and
436
appears to have a dominant role in the erythrocyte invasion process. Therefore, antibodies raised
437
against a functional recombinant protein expressed in E. coli representing the wild-type full-
438
length sequence of PfRH5 have proven to be as potent in blocking parasite invasion as reported
439
earlier with antibodies raised by adenoviral vectors (26,36) or the mutated PfRH5-ratCD4(d3+4)
440
fusion protein (27).
441 442
PfRH5 based antibody combinations produce an additive inhibition of erythrocyte invasion
443
by P. falciparum
444
In a recent report, we identified a potent antigen combination (PfAARP+PfRH2+PfF2)
445
that elicits strain-transcending invasion inhibitory antibodies (29). Through this study, we had
446
demonstrated that triple antibody combinations were more efficacious in inhibiting erythrocyte
447
invasion compared to double antibody combinations (29). This study did not include PfRH5, so
448
we wanted to now analyze whether PfRH5 based triple antibody combinations would also be
449
effective in producing additive inhibition of invasion at lower individual IgG concentrations. In
450
this regard, we tested the invasion inhibition of ten PfRH5 based triple antibody combinations
451
that comprised of purified total IgG (3.3 mg/ml each antigen; Total: 10 mg/ml) against PfRH5
452
(rabbit 1) and two other antigens from our portfolio (PfRH1, PfRH2, PfRH4, PfAARP, PfF2) 20
453
generated previously (29). The invasion inhibition was first assessed against two P. falciparum
454
strains (3D7, Dd2) (Fig. 6) and then the six most efficacious antibody combinations were further
455
analyzed against a total of five P. falciparum strains (3D7, Dd2, 7G8, MCamp, HB3) (Fig. 7) to
456
ascertain whether the antibody combinations elicited strain-transcending activity.
457
Individually, the six antibodies at 3.3 mg/ml exhibited a broad range of invasion
458
inhibition of 3-54% against all five parasite clones (Fig. 6, 7). As described above, anti-PfRH5
459
IgG displayed maximum inhibition against all strains (44-54%), followed by anti-PfAARP IgG
460
(17-33%) (Fig. 6, 7). Consistent with previous findings, anti-PfRH1 and anti-PfF2 IgG were
461
more efficacious in blocking the sialic acid dependent clones- Dd2 (24-36%), MCamp (15-17%)
462
and not the sialic acid independent clones – 3D7 (8-12%), 7G8 (5-10%) and HB3 (5-10%)
463
(Fig.6, 7). Similarly, PfRH2 IgG and PfRH4 IgG efficiently blocked only the sialic acid
464
independent clones and not the sialic acid dependent clones, in which they are poorly expressed.
465
The different anti-PfRH5 based antibody combinations displayed potent inhibition of
466
erythrocyte invasion by the 3D7 parasite clone (Fig. 6A) with the maximum inhibition observed
467
with three antibody combinations, PfAARP+PfRH2+PfRH5 (82%), PfF2+PfRH5+PfAARP
468
(82%) and PfF2+PfRH2+PfRH5 (79%). In line with the utilization and expression of the
469
different PfRH ligands, we observed that the maximum inhibition with the Dd2 parasite clone
470
was by the antibody combinations, PfRH5+PfF2+PfAARP (81%), PfRH5+PfRH1+PfAARP
471
(79%) and PfRH5+PfRH1+PfF2 (78%) (Fig. 6B). This is consistent with the higher expression
472
and utilization of the sialic acid binding ligands (PfRH1, PfF2 and PfAARP) for erythrocyte
473
invasion by sialic acid dependent parasite clones such as Dd2. We further tested invasion
474
inhibition of the six best PfRH5 based antibody combinations against five P. falciparum clones 21
475
that originate from diverse regions of the world and exhibit different invasion phenotypes as
476
well.
477
The six antibody combinations were potent in blocking invasion of all five parasite
478
clones
with
the
antibody
combinations,
PfRH5+PfF2+PfAARP
(77-82%),
479
PfRH5+PfRH2+PfAARP (71-83%), and PfF2+PfRH2+PfRH5 (72-79%) eliciting the most
480
efficacious strain-transcending invasion inhibitory activity (Fig. 7). We also tested as a control
481
the PfAARP+PfF2+PfRH2 antibody combination, which we had previously reported (29) as our
482
most efficacious antibody combination (Fig. 7). While this antibody combination does elicit
483
strain-transcending activity, the combination PfRH5+PfF2+PfAARP appeared to be slightly
484
more efficient in its overall strain-transcending activity against all the five P. falciparum clones,
485
which is consistent with the observation that PfRH5 antibodies individually were observed to be
486
most efficient among the different parasite ligands in blocking erythrocyte invasion. However,
487
the difference in invasion inhibitory activity between the PfRH5+PfF2+PfAARP and
488
PfAARP+PfF2+PfRH2 antibody combinations was not statistically significant (p>0.05).
489 490
Co-immunized antigen mixtures also elicit potent strain-transcending invasion inhibitory
491
antibodies
492
After evaluating antibody combinations that were mixed in vitro for their invasion
493
inhibitory activity, we wanted to test whether the most potent combination identified would elicit
494
similar invasion inhibitory antibodies when co-immunized together as a single formulation. In
495
this next step, the PfRH5 based triple antigen mixtures (PfRH5+PfRH2+PfAARP and
496
PfRH5+PfF2+PfAARP) were used to immunize mice (BALB/c). The individual antigens in each 22
497
combination were also used to immunize mice separately. All antigens were formulated with the
498
adjuvant, CFA/IFA. The ELISA results (OD492) showed that the antibody titers (end point
499
1:320,000) against each protein immunized individually were not significantly altered when
500
immunized as a mixture with the two other antigens (Fig. S5). The immunogenicity curves for
501
PfRH5, PfRH2, PfF2 and PfAARP were identical and overlapping whether the antigens were
502
immunized alone or in their respective combinations (Fig. S5). Thus, our recombinant antigens
503
including rPfRH563 were immunogenic and did not elicit any significant immune interference
504
when immunized in combination.
505
Consistent with the invasion inhibition observed with the antibody combinations
506
physically added in vitro, the antibodies raised against the antigen mixtures were highly potent
507
and equally efficient in inhibiting erythrocyte invasion (Fig. 8, S6). The antibodies against the
508
two antigen mixtures displayed 71-79% and ~86% inhibition of the P. falciparum clones (3D7,
509
Dd2) at concentrations of 5 and 10 mg/ml, respectively (Fig. 8A, 8B). The antibodies displayed a
510
similar potent inhibition of three more parasite clones at an IgG concentration of 10 mg/ml (Fig.
511
S6). Thus, antibodies raised against co-immunized antigen mixtures were as potent in efficiently
512
blocking erythrocyte invasion as antibodies physically combined (Fig. 6, 7) in the in vitro
513
invasion inhibition assays.
514 515 516 517 518 23
519
Discussion
520
Parasite neutralizing antibodies that block P. falciparum erythrocyte invasion is one of
521
the key effector mechanisms known to mediate immunity against blood-stage malaria parasites
522
and is the fundamental basis for the development of blood-stage malaria vaccines. The ability to
523
impede erythrocyte invasion by P. falciparum merozoites can be quantitated in an in vitro
524
invasion inhibition or growth inhibition assay (GIA) that has been widely reported in the field. A
525
significant association of invasion inhibition measured in vitro with a reduced risk of malaria has
526
also been reported (37, 38) and thus, in vitro invasion-inhibition appears to be a useful surrogate
527
marker to predict the functional efficacy of antibodies induced by a blood-stage vaccine. In spite
528
of the extensive research on the parasite biology of P. falciparum, it has been difficult to
529
demonstrate potent invasion inhibitory activity with the exception of antibodies against Apical
530
Membrane Antigen-1, AMA-1 (39, 40). The challenge in generating potent invasion inhibitory
531
antibodies that have hindered the development of blood-stage malaria vaccines against P.
532
falciparum could be attributed to the enormous complexity of the parasite, which has evolved
533
redundancy in parasite ligands that enables invasion of diverse types of human erythrocytes (2-
534
4). As a result, no human erythrocyte is known to be totally refractory to invasion by P.
535
falciparum. Another level of complexity is the high level of antigenic polymorphisms that
536
mediates immune escape, which has proven to be a major impediment in developing leading
537
antigens such as AMA-1 and MSP-142 as blood stage vaccines (5-7). Both antigens have failed to
538
elicit optimal efficacy in field trials (5, 6),
539
Thus, to fully realize the potential of a candidate blood-stage antigen, it is not sufficient
540
to just show that the antigen elicits strong invasion inhibitory antibodies that block the parasite in 24
541
vitro. A good example is AMA-1, an essential parasite protein involved in the critical step of
542
junction formation during erythrocyte invasion (41, 42) , which has been demonstrated in vitro to
543
induce potent invasion inhibitory antibodies only against homologous parasite strains and not
544
against heterologous strains (39, 40). This inability has been attributed to high levels of antigenic
545
polymorphisms in AMA-1 among different parasite strains that render it’s antibodies to be
546
ineffective against heterologous P. falciparum strains (39, 43, 44). Unfortunately due to the
547
problem of inducing allele-specific immunity, AMA-1 has not yielded protection against malaria
548
in vaccine efficacy trials (7, 45).
549
Therefore, it is crucial that potent strain-transcending invasion inhibition is demonstrated
550
with the antibodies against a particular antigen that is being considered for clinical development
551
of a malaria vaccine. This problem has been reported to be overcome by targeting combinations
552
of different conserved parasite antigens or a number of different AMA-1 allelic proteins that
553
produce strong efficacious invasion inhibition in a strain-transcending manner (29, 46).
554
The disappointing results of a number of blood-stage vaccine trials has raised the concern
555
whether the in vitro invasion inhibition assay or GIA assay has any correlation with clinical
556
protection or could predict vaccine efficacy in humans (47). Unfortunately, only a few blood-
557
stage antigens have been tested in field efficacy trials. Due to the problems cited above, more
558
studies with parasite antigens that induce potent strain-transcending invasion inhibition are thus
559
required to validate the correlation of in vitro invasion inhibitory activity with clinical protection
560
in humans. It would thus be beneficial to validate the in vitro invasion inhibitory assay or GIA
561
with in vivo CHMI (controlled human malaria infection) studies using either a sporozoite or
562
blood-stage challenge model (47). However, the in vitro invasion inhibitory assay or GIA 25
563
remains as the only currently available laboratory assay to measure the functionality of
564
antibodies to inhibit erythrocyte invasion by Plasmodium merozoites. In this regard, this assay
565
has been successful in differentiating between invasion inhibitory and non-inhibitory antibodies
566
against a number of P. falciparum antigens as not all antibodies exhibit potent invasion
567
inhibitory activity. Hence, the in vitro invasion inhibition or GIA assay clearly appears to be an
568
informative assay, which in pre-clinical studies can identify and validate novel, efficacious P.
569
falciparum blood-stage targets that elicit strain-transcending invasion-inhibitory antibodies.
570
The past decade has seen a huge body of research conducted on understanding the
571
molecular basis of erythrocyte invasion and characterizing numerous antigens from the large
572
repertoire developed by P. falciparum (2-4, 8, 9). With respect to vaccine development, the goal
573
to search for promising antigens has shifted to the identification of relatively conserved antigens
574
that elicit potent strain-transcending invasion inhibitory antibodies. Among the parasite’s large
575
molecular arsenal, the PfRH proteins have been identified as key determinants of different
576
invasion pathways used by P. falciparum (2-4, 8, 9), of which PfRH5 is the only erythrocyte
577
binding ligand known to be essential for the parasite as its gene is refractory for disruption (22).
578
The importance of PfRH5 has been substantiated by the fact that antibodies against both PfRH5
579
and its erythrocyte receptor, Basigin, potently inhibit erythrocyte invasion by a number of P.
580
falciparum strains from diverse worldwide locations that also exhibit different invasion
581
phenotypes (25-27). The efficacious neutralization of heterologous parasite clones by PfRH5
582
antibodies raised first through a viral vector based prime boost strategy had demonstrated that
583
PfRH5 is a highly promising blood stage vaccine candidate (26).
584
the production of a potent functional wild-type full-length PfRH5 recombinant antigen that could 26
Our goal was to demonstrate
585
be used for the development of a subunit blood-stage malaria vaccine, which would have the
586
potential to be tested individually as well as in combination with other blood stage antigens and
587
possibly even pre-erythrocytic malaria vaccines such as RTS,S (48). RTS,S
588
advanced malaria vaccine currently being tested in a Phase III clinical trial in Africa (49, 50). It
589
is a subunit recombinant vaccine based on the CSP-HbSAg fusion protein that assembles into
590
virus-like particles (48-50).
is the most
591
In this regard, we have produced the full-length, wild type PfRH5 recombinant protein
592
without making any alterations to its sequence in a bacterial organism, E. coli. The recombinant
593
protein binds with erythrocytes with the same specificity as that of the native parasite protein and
594
anti-Basigin monoclonal antibodies specifically blocked the binding of the recombinant protein.
595
This binding was unaffected by other monoclonal antibodies against another major erythrocyte
596
receptor, glycophorin A. All these results strongly suggest that the recombinant PfRH5 was
597
produced with a conformational integrity that mimics the native parasite protein. This work
598
paves the way for a more detailed structure-function analysis of PfRH5 that would not only
599
improve our understanding of its role in the basic biology of the parasite but also help in
600
designing a more efficacious PfRH5 immunogen for vaccine development.
601
While, it was very elegantly demonstrated that PfRH5 binds with Basigin (25), this report
602
did not show any data with regard to the interaction of the native PfRH5 parasite protein and
603
based its inferences on the interaction with a pentamer of the biotinylated recombinant PfRH5-
604
ratCD4(d3+4) fusion protein. In the present study, we have demonstrated that the Basigin
605
monoclonal antibodies at the same concentration at which they blocked erythrocyte invasion also
606
completely inhibited the erythrocyte binding of the native PfRH5 parasite protein obtained from 27
607
culture supernatants. Our data strongly supports the previous study that the native PfRH5
608
parasite protein binds with the erythrocyte surface molecule, Basigin.
609
Further, most importantly, the antibodies raised against recombinant PfRH5 specifically
610
inhibited erythrocyte invasion of heterologous P. falciparum strains with a potent efficiency as
611
high as 85% and 71% at total IgG concentrations of 10 mg/ml and 5 mg/ml, respectively. PfRH5
612
antibodies were observed to be most potent among a pool of antibodies (0.5-10 mg/ml total IgG)
613
tested against six antigens in our portfolio that included four PfRH proteins (PfRH1, PfRH2,
614
PfRH4, PfRH5), PfEBA-175 (PfF2) and PfAARP (29). The normal physiological total IgG
615
concentration in human serum is in the range of 10-14 mg/ml (51). Thus, we have analyzed our
616
antibodies at a maximum total IgG concentration of 10 mg/ml, which is on the lower end of the
617
human physiological concentration. The specific component of the anti-PfRH5 IgG within the
618
total serum IgG (10 mg/ml) would be a much smaller fraction and should be achievable through
619
a human vaccine. In addition, PfRH5 antibodies exhibited the maximum cross-strain neutralizing
620
activity as they were efficacious in blocking a number of P. falciparum clones that display
621
phenotypic variation in their invasion pathways. Our invasion inhibition results are consistent
622
with previous reports of PfRH5 antibodies raised through viral vectors (26) or against the
623
PfRH5-ratCD4(d3+4) fusion protein (27). In our assays, the rPfRH563 antibodies exhibited an
624
EC50 of ~ 3 mg/ml, whereas previous studies with viral vectors and PfRH5-ratCD4(d3+4) fusion
625
protein have shown an EC50 with anti-PfRH5 IgG obtained from different rabbits at a broad
626
concentration range of 0.7-4 mg/ml against multiple P. falciparum strains (26, 27, 36). The
627
difference in invasion inhibitory activity could be attributed to the different nature of the PfRH5
628
immunogen used in the studies as well as the fact that different animals were used in the studies 28
629
to raise the PfRH5 specific antibodies. Outbred animals are known to exhibit large differences in
630
their immune responses and especially when located in different geographical locations.
631
As stated previously, achieving highly potent strain-transcending invasion inhibitory
632
antibodies against conserved functional domains of parasite antigens involved in invasion may
633
be the key to the development of an effective blood stage malaria vaccine. This has been difficult
634
to achieve by targeting single antigens with the exception of PfRH5 as reported here and in
635
previous reports. Earlier potent strain-transcending invasion inhibitory antibodies were
636
demonstrated only by targeting combinations of key merozoite antigens involved in erythrocyte
637
invasion (24, 29, 36, 46, 52).
638
In line with these reports, we feel that targeting single antigens may not be an effective
639
long-term strategy for malaria vaccines as the parasite has the strong ability to develop escape
640
mechanisms to counter the immune pressure. This capability is well displayed in the rapid
641
development of resistance in the parasite under drug pressure that has prompted only
642
combinatorial anti-malarial drug therapies. More so in combination, antibodies neutralize the
643
parasite at lower individual IgG concentrations (3.3 mg/ml total IgG each) that would be easier
644
to achieve using human compatible adjuvants or delivery platforms. PfRH5 antibodies in
645
combination with those against PfRH2, PfAARP and PfF2 produced an additive inhibition of
646
erythrocyte invasion. Our data is consistent with a previous study that has reported a potent
647
synergy in invasion inhibition by adenoviral vector induced antibodies against PfRH5 and other
648
EBA/PfRH antigens (36). We have also demonstrated that these PfRH5 based co-immunized
649
antigen mixtures induce balanced antibody responses against all three antigens with no immune
29
650
interference. Purified total IgG against the antigen mixtures were as potent in inhibiting
651
erythrocyte invasion as the antibodies combined in vitro.
652
Thus, our study establishes a proof of principle for the production of full-length wild-type
653
recombinant PfRH5 in a bacterial expression system that is known to be scalable for mass
654
vaccine production and has the potential to be taken forward for its development as a component
655
of a subunit blood-stage combination malaria vaccine. However, we have demonstrated potent
656
invasion inhibitory antibodies against PfRH5 raised through freund’s formulations that are
657
known to induce very strong immune responses but are not safe for human vaccine applications.
658
Thus, it is imperative for the clinical development of recombinant PfRH5 as a malaria vaccine to
659
identify human compatible adjuvants or delivery platforms that would also elicit similar highly
660
potent neutralizing antibodies. Nevertheless, the production of the wild type full length
661
recombinant PfRH5 protein with no amino acid modifications further enables the structure-
662
function analysis of this highly efficacious and attractive blood-stage malaria vaccine candidate.
663 664 665 666 667 668 669 670 671 672 673 30
674
Acknowledgements
675
We are grateful to Dr Louis Miller (NIH) for providing the P. falciparum clones used in
676
the study and the rPfRH430 expression plasmid. We also wish to thank Dr Lee Hall and Dr Annie
677
Mo from the Parasitology and International Programs Branch (PIPB), NIAID, NIH for providing
678
the recombinant EBA-175 RII protein. The technical assistance of Dr Alka Galav, Rakesh
679
Kumar Singh and Ashok Das from the ICGEB animal facility in performing the animal
680
experiments is deeply appreciated. We wish to thank Dr Inderjeet Kaur at the mass spectrometry
681
facility of the Malaria group, ICGEB for helping us in the LC-MS analysis. We appreciate Ms
682
Surbhi Dabral at the super resolution imaging facility of the Malaria group, ICGEB for technical
683
help in our imaging study.
684
Deepak Gaur is the recipient of the Ramalingaswami Fellowship from the Department of
685
Biotechnology, Government of India. Deepak Gaur is also the recipient of the Grand Challenges
686
Exploration Grant from the Bill and Melinda Gates Foundation. This work was supported by the
687
Bill & Melinda Gates Foundation through the Grand Challenges Explorations Initiative [GCE
688
OPP1007027 to D.G.]; Department of Biotechnology (DBT), Government of India through the
689
Ramalingaswami fellowship program [BT/HRD/35 /02/14/2008 to D.G.], Rapid Grant Scheme
690
for Young Investigators [BT/PR13376/GBD/27/ 260/2009 to D.G.], Vaccine Grand Challenges
691
Program [ND/DBT/12/040 to D.G., C.E.C., V.S.C.]. K.S.R., T.S. are recipients of Senior
692
Research Fellowships of the Council of Scientific and Industrial Research, Government of India;
693
A.K.P is a recipient of a Post-doctoral Research Associateship of DBT; H.S. is the recipient of
694
the Senior Research Fellowship of DBT. A.E. is the recipient of the ICGEB International PhD
31
695
Pre-doctoral fellowship. The funders had no role in study design, data collection and analysis,
696
decision to publish, or preparation of the manuscript.
697
This work has been previously presented at the 24th National Congress of Parasitology in
698
Jabalpur, Madhya Pradesh, India (April 27-29, 2013) and the 2013 Malaria Gordon Research
699
Conference in Tuscany, Italy (August 4-9, 2013).
700 701 702
Conflict of Interest: D.G., V.S.C., C.E.C are named on patent applications relating to PfRH5
703
and/or other malaria vaccines. This does not alter our adherence to all Infection and Immunity
704
policies on sharing data and material.
705 706 707 708 709 710 711 712 713 714 715 716 717 32
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in healthy children and adults. Clin. Exp. Immunol. 4:101–112.
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903
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905 906 907 908 909 910 911 912 41
913
Figure Legends
914 915
Figure 1: Production of the recombinant rPfRH563 protein. (A) Schematic
916
representation of the PfRH5 parasite protein. Region in black denotes the signal peptide
917
(residues 1-26) and region in green (residues 27-526) was expressed in E. coli. (B)
918
Purified rPfRH563 protein analyzed on 12% SDS-PAGE, stained with coomassie blue.
919
2µg of rPfRH563 has been loaded in each well. (C) Recombinant rPfRH563 protein
920
detected in immunoblots using the anti-His tag antibody. R and NR represents -reducing
921
and non-reducing conditions, respectively.
922 923
Figure 2: Erythrocyte binding activity of the native PfRH5 parasite protein and
924
recombinant rPfRH563 protein. Anti-PfRH5 antibodies in immunoblots detected both
925
native PfRH5 and rPfRH563 among the proteins that bound with the erythrocyte surface
926
and got eluted off by 1.5 M NaCl. (A) Native PfRH5 from the 3D7 culture supernatant
927
bound untreated (U) and all three enzymatically treated human erythrocytes
928
[neuraminidase (N), trypsin (T), chymotrypsin (C)] but failed to bind with proteinase K
929
(PK) treated erythrocytes. Thus, PfRH5 binds erythrocytes in a sialic acid independent,
930
trypsin/chymotrypsin resistant, proteinase K sensitive manner. (B) rPfRH563 bound
931
human erythrocytes with the same specificity as that of native PfRH5. (C) Native PfEBA-
932
175, as well as (D) PfF2, (recombinant receptor binding domain of PfEBA-175) were
933
analyzed as a control with the same set of enzymatically treated erythrocytes. Both native
934
PfEBA-175 and PfF2 bound erythrocytes in neuraminidase/trypsin/proteinase K 42
935
sensitive, chymotrypsin resistant manner. PBS denotes Phosphate buffer saline, pH 7.4
936
that contained no protein. No specific protein was detected in the eluate fractions with the
937
PBS control suggesting that no non-specific erythrocyte protein was being detected in the
938
assay. (* U= Untreated; N= Neuraminidase; T= Trypsin; and C= Chymotrypsin; PK =
939
Proteinase K)
940 941
Figure 3: Antibodies against recombinant rPfRH563 blocked erythrocyte binding of
942
both the native PfRH5 and rPfRH563 proteins. Purified total rabbit IgG against
943
rPfRH563 blocked binding of (A) native PfRH5 and (B) recombinant rPfRH563 in a dose
944
dependent manner. The PfRH5 antibodies had no effect on the erythrocyte binding of (C)
945
native EBA-175 or (D) recombinant PfF2 even at the maximum IgG concentration of 800
946
µg/ml.
947 948
Figure 4: Anti-Basigin monoclonal antibodies blocked the erythrocyte binding of
949
native and recombinant PfRH5. (A) Anti-Basigin TRA-1-85 monoclonal antibodies
950
potently inhibited invasion of human erythrocytes by the P. falciparum clone 3D7, with
951
complete blockade of invasion observed at 5µg/ml. At the same invasion inhibitory IgG
952
concentrations, the TRA-1-85 monoclonal antibodies potently blocked the erythrocyte
953
binding of both the (B) native PfRH5 protein and (C) recombinant rPfRH563. Anti-
954
glycophorin A monoclonal antibodies had no effect on the binding of both (B) native and
955
(C) recombinant PfRH5.
956 43
957
Figure 5: Invasion inhibitory activity of anti-PfRH5 rabbit antibodies. (A) Invasion
958
inhibitory activity of purified rabbit total IgG (0.5-10 mg/ml) against rPfRH563 and the
959
receptor binding domains of other key merozoite ligands (PfRH1, PfRH2, PfRH4,
960
PfAARP, PfF2) against the P. falciparum clone 3D7. AMA-1 IgG (5 mg/ml) was used as
961
a positive control. (B) Strain-transcending parasite neutralization activity of anti-PfRH5
962
total rabbit IgG (0.5-10 mg/ml) against five diverse P. falciparum clones. The control
963
anti-HPR total IgG failed to exhibit any invasion inhibition at the maximum
964
concentration of 10 mg/ml. The results represent the average of three independent
965
experiments performed in duplicate. The error bars represent the standard error of the
966
mean.
967 968
Figure 6: Invasion inhibitory efficacy of PfRH5 based antibody combinations
969
against P. falciparum clones (3D7, Dd2). Purified rabbit total IgG against the six
970
individual antigens (PfRH1, PfRH2, PfRH4, PfRH5, PfAARP, PfF2) were evaluated
971
individually (3.3 mg/ml) as well as in all ten possible PfRH5 based triple antibody
972
combinations (3.3 mg/ml each; total 10 mg/ml) against (A) the sialic acid independent
973
clone 3D7 and (B) the sialic acid dependent clone Dd2. The control anti-HPR total IgG
974
failed to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml. The
975
results represent the average of three independent experiments performed in duplicate.
976
The error bars represent the standard error of the mean.
977 978 44
979
Figure 7: Strain-transcending parasite neutralization of PfRH5 based antibody
980
combinations. Purified rabbit total IgG against the six individual antigens (PfRH1,
981
PfRH2, PfRH4, PfRH5, PfAARP, PfF2) were evaluated individually and in the six most
982
potent PfRH5 based antibody combinations (identified from Fig. 6) against five diverse
983
P. falciparum clones. The invasion inhibitory activity of the antibody combination,
984
AARP+RH2+PfF2, was also analyzed in the assay. The control anti-HPR total IgG failed
985
to exhibit any invasion inhibition at the maximum concentration of 10 mg/ml. Three
986
independent assays were performed in duplicate. The error bars represent the standard
987
error of the mean.
988 989
Figure 8: Invasion inhibitory activity of antibodies raised against the two co-
990
immunized antigen formulations: Total IgG purified from mice sera raised against the
991
immunogens (RH5, RH2, PfF2, AARP, PfF2+RH5+AARP, RH2+RH5+AARP) were
992
evaluated for its invasion inhibitory activity (at concentrations of 1, 3.3, 5 and 10 mg/ml)
993
against the (A) sialic acid independent clone 3D7 and the (B) sialic acid dependent clone
994
Dd2. The control anti-HPR total IgG failed to exhibit any invasion inhibition at the
995
maximum concentration of 10 mg/ml. Two independent assays were performed in
996
duplicate. The error bars show the standard error of the mean. * denotes statistical
997
significance between the invasion inhibition produced by the antibodies against the two
998
antigen mixtures (PfF2+RH5+AARP, RH2+RH5+AARP) with respect to the individual
999
PfRH5 antibodies at the same concentrations of purified total IgG, 3.3 mg/ml and 5 mg/ml
1000
(p values < 0.05). 45
