Pv RON2, a new Plasmodium vivax rhoptry neck antigen

The search for a Pf RON2 homologous gene in P. vivax was carried out using the tBlastn tool in the P. vivax Sal-1 strain genome [30]. The sequence having the greatest score was selected as pvron2 putative gene. PlasmoDB and Sanger Institute [31] databases were scanned for pvron2 and pfron2 homologous genes in partial genomes from other Plasmodium species (Plasmodium knowlesi, Plasmodium chabaudi, Plasmodium yoelii and Plasmodium berghei). Identity and similarity values between P. falciparum - P. vivax and the other species were obtained with ALignX and ClustalW tools [32]. The presence of a signal peptide was assessed by using SignalP [33] and anchor regions were predicted using the PredGPI and TMHMM servers [34]. Repeat sequences and domains were predicted with the sequence tandem repeats extraction and architecture modelling software (XSTREAM, variable 'X'), the simple modular architecture research tool (SMART) and GlobPlot tools [35‐37]. Bepipred tool [38] and ANTHEPROT software [39] were used for linear B epitope selection.

The P. vivax Colombia Guaviare 1 (VCG-1) strain was used as DNA, RNA and protein source. The strain was cultured through successive passes in Aotus spp monkeys from FIDIC's Primate Station in Leticia, Amazonas, as previously described [40] and according to the conditions established by the Ministry of the Environment's official Institute, Corpoamazonía (resolution 00066, September 13^th 2006). Three to four mL of P. vivax VCG-1-infected monkey's blood were extracted; a schizont-rich sample was then obtained by discontinuous Percoll gradient (GE Healthcare, Uppsala, Sweden) according to a previously described protocol [41]. A Wizard genomic DNA purification kit (Promega, Wisconsin, USA) was used for genomic DNA extraction (gDNA) following the manufacturer's specifications. Total RNA was extracted by the Trizol method [42] and then treated with RQ1 RNase-free DNase (Promega, Wisconsin, USA). Five microlitres of RNA were used as cDNA synthesis template using the Superscript III enzyme (Invitrogen, Carlsbad CA) and oligo (dT) primers in a 5-min cycle at 65°C, followed by 60 minutes at 50°C and a final 15-min cycle at 70°C.

The pvron-2 nucleotide sequence (PVX_117880), reported in the PlasmoDB database, was used as template for designing three sets of primers with GeneRunner v3.05 software. PvRON2-pEXP-F1 5'-ATG ATA AGTA CAA GGG AGG CAA AA-3' and PvRON2-pEXP-R1 5'-ATA TCT TTT GTT TCT CGT CCT G-3' primers were used for amplifying region I, consisting of amino acids 18 to 742. PvRON2-pEXP-F2 5'-ATG AAC CCAT TAG TAT ATC ACG TG-3' and PvRON2-pEXP-R2 5'-CAG CAG TTT CAT CTTG GCC-3' were used for amplifying region II, consisting of amino acids 701 to 1560. Region III (amino acids 1517 to 2203) was amplified with PvRON2-pEXP-F3 5'-ATG ACC AGG GCT GAG AAA TTC G-3' and PvRON2-pEXP-R3 5'-CAC CTG TAT GCG GGC GTA-3'. Two primers were used for amplifying the PvAMA-1 ectodomain (43-487 amino acids): PvAMA-1D 5'-ATG CCT ACC GTT GAG AGA AGC A-3' and PvAMA-1R 5'-TAG TAG CAT CTG CTT GTT CG-3'.

PCR amplification was carried out using GoTaq Flexi DNA polymerase enzyme (Promega) in a 25 μL final reaction, according to manufacturer's instructions. Amplification conditions were as follows: a 7-min cycle at 95°C, followed by 35 cycles of 1 min at 58°C, 3 min at 72°C and 1 min at 95°C, and finally, a 10-min extension step at 72°C. Products were visualized on a 1% agarose gel and then purified with a Wizard PCR preps kit (Promega). PCR products obtained from cDNA were cloned in the pEXP5-CT/TOPO expression vector using TOPO TA cloning (Invitrogen, Carlsbad CA). Positive clones were analysed by enzymatic restriction and sequenced in an ABI PRISM 310 Genetic Analyser (PE Applied Biosystems).

Two linear B-cell epitope peptides were selected for producing polyclonal antibodies against the Pv RON2 protein based on the following parameters: (1) high average values for Parker's antigenicity, hydrophilicity and solvent accessibility obtained with Antheprot software [39], (2) high values in results obtained with the Bepipred tool (at default 0.35 threshold and 75% specificity) [38] and (3) selected peptides had to be located in different portions of the protein, with the aim of detecting different fragments in case the Pv RON2 protein was proteolytically processed. Selected peptides were synthesized by solid-phase peptide synthesis (SPPS) using the tert-butoxycarbonyl (t-Boc) strategy [43] and numbered according to our institute's serial numbering system as: 35519 (CG⁷³⁴YGRTRNKRYMHRNPGEKYKG⁷⁵³GC) and 35520 (CG¹⁶⁷⁴KLQQEQNELNEEKERQRQEN¹⁶⁹³GC).

Peptide 37870, derived from the N-terminal region of Pv AMA-1 protein (CG²³RNQKPSRLTRSANNVLLE⁴⁰GC), and 32416, derived from Pv RhopH3 protein (CG⁷⁹²SAGVGTVSTHSPATAARMGL⁸¹¹GC), were synthesized by SPPS. Peptide 37870 has been shown to be immunogenic in mice [44] and peptide 32416 has previously been used for polyclonal antibody production in rabbits, followed by localization experiments for the Pv RhopH3 protein [45]. Synthesized peptides were analysed by reverse phase high performance liquid chromatography (RP-HPLC) and MALDI-TOF mass spectrometry (Auoflex, Bruker Daltonics, Bremen, Germany). Cysteine and glycine were added to the N- and C-termini during synthesis to allow peptide polymerization. These peptides were inoculated in mice and the obtained sera were used for co-localization experiments as explained below.

Two New Zealand rabbits were selected (numbered 89 and 90) for obtaining polyclonal antibodies against Pv RON2 protein; they were negative for P. vivax-derived protein recognition by Western Blot. Each rabbit was subcutaneously inoculated with 500 μg of putative Pv RON2-derived peptide 35519 (rabbit 90) or peptide 35520 (rabbit 89), emulsified in Freund's complete adjuvant (FCA) on day 0. Booster immunizations on days 20 and 40 were administered using the same peptides emulsified in Freund's incomplete adjuvant (FCI). Rabbits' sera were collected on day 60 and used for further assays.

7-8 week old BALB/c strain mice were intraperitoneally (i.p.) immunized with 100 μg of peptide 37870 or peptide 32416, emulsified in FCA. Three boosters were given on days 30, 45 and 60 with 100 μg of FCI-emulsified peptide. These animals were bled 15 days after the last immunization and their sera were collected for further assays. Immunizations and animal bleeding were carried out following Colombian Ministry of Health recommendations for handling live animals used in research or experimentation.

Plasmodium vivax VCG-1 thick smears were used for immunofluorescence assays and fixed with 4% v/v formaldehyde for 10 min. The slides were then permeabilized for 10 min with 1% v/v Triton and blocked with a 1% BSA/PBS solution at 37°C. The slides were washed several times with PBS and incubated with 300 μL of anti-Pv RON2 polyclonal serum (primary antibody) in a 1:40 dilution with either anti-Pv AMA-1 in a 1:20 dilution or anti-Pv RhopH3 in the same dilution for 60 min. Fluorescein-labelled anti-rabbit IgG (FITC) (Vector Laboratories, Burlingame, CA, USA) and rhodamine-labelled anti-mouse IgG (Millipore, Billerica, MA, USA) were used as secondary antibody for 60 min, followed by three PBS washes. Parasite nuclei were stained with a 2 μg/mL solution of 4',6-diamidino-2-phenylindole (DAPI) for 20 minutes at room temperature and fluorescence was visualized in a fluorescence microscope (Olympus BX51) using an Olympus DP2 camera and Volocity software (Perkin Elmer, Waltham, MA, USA).

The Pf RON2 protein amino acid sequence (PF14_0495) was used as template for scanning the P. vivax complete genome, available in PlasmoDB (version 6.5), in the search for the homologous Pv RON2 encoding gene. tBlastn analysis revealed a nucleotide sequence having a high probability of containing the pvron2 gene located in reading frame -2 between 2,221,529-2,214,921 bp, contig CM000453. High similarity (61.7%) and identity (47.8%) values were found between Pf RON2 and Pv RON2 protein amino acid sequences, suggesting that these two proteins share a common origin. pvron2 neighbouring genes located upstream and downstream were also analysed, as well as their intron-exon organization; identity and similarity values were determined by comparing P. falciparum and P. vivax protein sequences (Figure 1). Similarity and identity values were found ranging from 60.4%-98.3% and 42.7-96.6%, respectively, in the analysed chromosomal region.

Pf RON2 and Pv RON2 orthologues were found in P. knowlesi (Pk RON2: PKH_125430), P. chabaudi (Pc RON2: PCAS_131900), P. berghei (PBANKA_131570) and P. yoelii (Py 06813) when the Pf RON2 amino acid sequence was used as template for Blastp analysis for some Plasmodium species partial genomes. Plasmodium species ron2 genes were located in homologous chromosomal regions, as shown by their high similarity and identity values (35%-88% and 16%-75%, respectively) at amino acid level, similar ORF orientation and intron-exon pattern. P. yoelii pyron2 downstream genes (Figure 1) were excluded from analysis, given that this genome has not been completely assembled.

Plasmodium falciparum transcriptome analysis revealed that Pf RON2 begins its transcription after 35 hours, reaching its maximum peak of expression 45 hours into the erythrocytic cycle [46]. PCR amplification of Pv RON2 encoding sequence confirmed the transcript's presence in P. vivax VCG-1 strain parasites during the blood stage (Figure 2A). This agreed with the results obtained from P. vivax transcriptome analysis which showed that Pv RON2 is transcribed between hour 35 (TP7) and 40 (TP8) in the intraerythrocytic cycle, similar to other proteins involved in invasion such as Pv MSP-1 [5]. When pvron2 gene gDNA and cDNA sequences were compared, obtained from the three amplification products overlapping by around 100 bp, both are identical, thus confirming that this gene consisted of a single 6,615bp exon. Recombinant clone sequences were analysed using CLC DNA Workbench (CLC bio) and the consensus sequence was deposited in the GenBank with the ID: HQ825321.

Two substitutions and the insertion of a nucleotide triplet were found when VCG-1 strain and Sal-1 reference strain nucleotide sequences were compared. Substitutions in positions 1,241 and 1,814 produced a change from valine to glycine (V 414G) and histidine to proline (H 605P), respectively. The addition of a glutamic acid (E)-encoding triplet (AAG) was found in position 1,487-1,489nt (residue 496). Interestingly, these changes were located in an interspecies variable region, spanning around residues 50 to 850 [21], suggesting that this region might be subjected to selective immune pressure.

The Pv RON2 complete protein sequence in the VCG-1 strain consists of 2,204 residues having a putative hydrophobic signal sequence within its first 17 amino acids and a transmembrane domain (TMD) towards the C-terminus between residues 2,087-2,109. The RON2 protein has similar lengths in other species, ranging from 1,990 amino acids in P. chabaudi to 2,232 in P. yoelii, as well as a similar domain organization, including a signal peptide, a TMD and containing eight conserved cysteines (Figure 2B) probably related to common protein structural features.

Pv RON2 contains two coiled coil α-helical motifs (residues 145-184 and 1,651-1,703) (Figure 2B), characterized by seven amino acid repeats (abcdefg) _n with hydrophobic residues located in positions a and d, and residues (generally polar) in the remaining sites which have been involved in protein-protein interactions. These coiled coil motifs have been identified in several important P. falciparum vaccine candidates such as LSA-1, MSP-3, MSP6 and MSP11 [47, 48]; such motifs are recognized by naturally-acquired antibodies and are also immunogenic in mice [49]. Interestingly, peptide 35520 (containing part of the second coiled coil α-helical motif) has induced an antibody response in rabbits. Additionally, Pv RON2 has two tandem repeat (TR) regions located within the interspecies variable sequence. Eight 11 amino acid long repeats (GADGKGYGPYG) are located between residues 258 and 345, and the second tandem (GGYGNGGHE) is located between residues 542-628, having 9 repeats (Figure 2B). TRs were mostly found in RON2 sequences from different Plasmodium species and, even though the DNA and protein sequences from the repeats varied widely amongst RON2 proteins, there was close to 40% similarity between Pv RON2 and Pk RON2 repeats. Such similarity between Pv and Pk was in agreement with a close evolutionary relationship between simian malarial parasites and the human P. vivax parasite. TRs have been identified in different malarial antigens such as the P. falciparum circumsporozoite protein (CSP), the ring-infected erythrocyte surface antigen (Pf RESA) and the knob-associated histidine rich protein (KAHRP). These TRs could downregulate antibody isotype maturation and high-affinity antibody production in the specific case of malaria by acting as B-cell superantigens, predominantly inducing a polyclonal thymus-independent humoral response. T-independent antibody responses are usually short lived, predominantly composed of IgM and IgG3 and have low affinity, suggesting that these repeats are used during invasion to distract the immune response, acting as decoys or "smokescreens", thereby masking the critical epitopes [50, 51]. Given all the above-mentioned data, it would be important to assess the functional and immunological implications of these repeat regions in Pv RON2.

Polyclonal antibodies were produced against the protein by immunizing rabbits with polymeric Pv RON2-derived peptides to assess Pv RON2 expression and cellular localization in P. vivax schizonts. Polyclonal antibodies detected two bands at around ~220 kDa and ~185 kDa (Figure 3A), suggesting that Pv RON2 can undergo proteolytic processing, by contrast with that reported for Pf RON2 [21]. The predicted size for Pv RON2 (240 kDa) is slightly larger than that obtained from mobility on SDS-PAGE (220 kDa). Interestingly, similar behaviour has been described for Tg RON2, suggesting anomalous migration [52].

In many cases, it has been found that rhoptry proteins are initially synthesized as pre-proteins and maturate during transport [53]. It could be hypothesized that such cleavage could serve to activate Pv RON2 by revealing a functional domain or releasing the protein of parasite surface or RBC membrane to allow successful invasion. Additionally, pulse-chase analysis has shown that Tg RON2 is expressed as a pro-protein (~150 kDa) which is cleaved to produce a ~120 kDa mature protein. Even though it is not known which specific proteases act in Tg RON2 maturation, it has been suggested that this protein can be cleaved by subtilisin 2 (Tg SUB2) [18]. Studies carried out with important P. falciparum adhesins located on merozoite membrane, micronemes or rhoptries, such as AMA-1, merozoite surface protein (MSP), EBL, RBL and thrombospondin-related anonymous protein (TRAP) families, contain a putative rhomboid cleavage site within their TMD and putative SUB-2 cleavage sites. COS-7 cell system studies have revealed that A1427 residue substitution in the EBA-175 protein has prevented Pf ROM4-mediated shedding, avoiding the release of EBA-175 from the merozoite surface [54]. Similarly, substituting the GA motif (residues which destabilize α-helices) closest to the TMD extracellular end abolished specific cleavage (also predicted as the site required for rhomboid recognition). Interestingly, Pv RON2 sequence analysis revealed a putative rhomboid cleavage site between 2,101-2,104; this agreed with the fact that sera recognized two fragments from the protein, but additional studies are needed for assessing the importance of such processing, as well as the identity of the responsible protease.

Immunofluorescence analysis of P. vivax schizonts showed that Pv RON2 had a dotted pattern typical of apical organelles, such as rhoptries and micronemes (Figure 3B). To examine their localization in detail, dual labelling was performed using mouse polyclonal antibodies against Pv AMA-1 and Pv RhopH3. It was found that there was no co-localization between the Pv AMA-1 protein (microneme marker) and Pv RON2 (Figure 3C), suggesting that Pv RON2 is not present in micronemes. By contrast, there was a small area of central localization between Pv RhopH3 (rhoptry bulb marker) and Pv RON2 suggesting that even though Pv RON2 is located in the rhoptries, it is not located in the rhoptry bulb, probably forming part of the rhoptry neck, as has been described for Pf RON2 and Tg RON2 proteins [18, 21]. Recently, a study that characterized the timing of expression and subcellular location of Plasmodium homologues in some T. gondii rhoptry proteins showed that P. berghei RON2 protein is located in merozoite and sporozoite rhoptries, and presents a timing of expression comparable to the one found in RAP2/3. These data strongly suggest an essential role of RON2 protein during the invasion and infection establishment in sporozoites [55].