Exploring the use of Circumsporozoite Protein (CSP) as Diagnostic Biomarker for Specific Detection of Plasmodium Knowlesi

doi:10.21203/rs.3.rs-429142/v1

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Exploring the use of Circumsporozoite Protein (CSP) as Diagnostic Biomarker for Specific Detection of Plasmodium Knowlesi

https://doi.org/10.21203/rs.3.rs-429142/v1

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Background

Malaria has been a public health concern for many years. People living in Southeast Asia are now threatened by Plasmodium knowlesi infection which can cause severe complications if not treated immediately. Rapid diagnostic tests (RDTs) targeting at P. knowlesi infection is essential for rapid diagnosis especially in resource limited settings. In this study, circumsporozoite protein (CSP) was assessed using in silico analysis and antibody detection approach to act as a P. knowlesi diagnostic biomarker in RDTs.

Methods

Animal ethics approval was obtained for animal immunization work. Two peptide epitopes specific for P. knowlesi CSP (PkCSP) were identified from amino acid sequence alignment and were used as immunogen to raise antibodies in animal models. The reactivity of the anti-peptide antiserums was evaluated against recombinant PkCSP using Western Blot assays.

Results

Mouse anti-peptide antiserum generated was able to react with recombinant PkCSP. Western blot assay showed that the mouse anti-peptide antiserum did not probe on any protein in P. knowlesi total protein extract, indicating the absence of PkCSP in the erythrocytic stage. This finding was further confirmed by using anti-recombinant PkCSP antiserum raised in mice models.

Conclusion

Our study has demonstrated that CSP is not suitable to be used as a diagnostic biomarker for P. knowlesi specific detection. More studies should be done to screen for suitable biomarkers for specific P. knowlesi detection in RDT to allow prompt disease management and epidemiological surveillance.

Infectious Diseases

Plasmodium knowlesi

Circumsporozoite protein

Rapid diagnostic test

Biomarker

Malaria, a life threatening disease caused by protozoan of Plasmodium genus was reported with a gradual decline in the number of cases worldwide with the efforts implemented based on the Global Malaria Program. This is especially significant in the Southeast Asia region whereby the number of malaria cases reported in 2018 is 69% lesser compared to 2010 [1]. Yet, this region now faces an increased incidence of Plasmodium knowlesi malaria infection. P. knowlesi is a simian malaria in which its natural hosts are the long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) which inhabit forested areas throughout Southeast Asia countries. The parasite was thought to rarely cause malaria infection in humans until Singh et al. (2004) identified P. knowlesi infection in a large number of human samples that were misdiagnosed as Plasmodium malariae in Kapit Division of Malaysian Borneo [2]. Since then, many other Southeast Asia countries have reported knowlesi malaria infection [3–6].

Knowlesi malaria can develop into potentially fatal complications if not treated immediately [7]. Hence, accurate diagnosis is required for prompt management of the disease. Studies have demonstrated the limitations of microscopy examination in distinguishing P. knowlesi from Plasmodium falciparum, Plasmodium vivax, and P. malariae especially in region co-endemic with these parasite species [8]. This is due to morphological similarities of P. knowlesi with other human Plasmodium species in the erythrocytic cycle such as ring form at early trophozoite stage that resembles distinct characteristics of P. falciparum, and band form at late trophozoite stage which is difficult to distinguish from P. malariae [9]. Molecular technique like nested PCR is highly sensitive and specific for the identification of Plasmodium species [10] yet, the method is costly for daily routine diagnosis and screening purposes.

Current available rapid diagnostic tests (RDTs) have shown to be insensitive for the detection of P. knowlesi as non-specific cross-reactivity of antibodies with P. knowlesi-infected blood was observed [11,12]. Nonetheless, RDT is rather a suitable tool for point-of-care diagnosis and screening in rural and suburban areas where microscopy and PCR are inaccessible. Literature showed that current malaria RDTs commonly target P. falciparum histidine-rich protein 2 (PfHRP2), plasmodial lactate dehydrogenase (pLDH), and plasmodial aldolase [13]. Antibodies targeting PfHRP2 are used for specific detection of P. falciparum whereas pLDH and plasmodial aldolase antibodies are used for pan-malarial detection. Species-specific epitopes were also identified from pLDH for the detection of P. falciparum and P. vivax. However, antibodies targeting these proteins were shown to cross-react with P. knowlesi-infected blood [11]. This was supported by McCutchan et al. (2008) in which pLDH antibodies that detected P. falciparum and P. vivax were also able to detect P. knowlesi. Thus, these antibodies cannot distinguish P. knowlesi infection from a mixed infection with P. vivax and P. falciparum [14]. On a side note, P. knowlesi does not react with pLDH antibodies specific for Plasmodium ovale and P. malariae.

Efforts to look for other target biomarkers suitable for use in RDT are on-going. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme present in Plasmodium life cycle was shown to be an alternative biomarker to LDH for use in RDTs [15]. The study noted an epitope from GAPDH that is specific for P. knowlesi. However, the percentage identity of this peptide epitope is 79% when compared to P. vivax orthologue. Thus, the specificity of the antibodies targeting this epitope would require further analysis. In Krause and Goldring (2018), it was demonstrated that phosphoethanolamine-N-methyltransferase (PMT) may be able to be used as a target biomarker in malaria RDT [16]. The researchers identified PMT epitopes that are specific to P. knowlesi, P. vivax, and P. falciparum in in silico analysis. However, antibodies produced against the selected PkPMT epitope detected both recombinant PkPMT and PvPMT orthologues.

In this study, we investigated the use of circumsporozoite protein (CSP) as a biomarker for specific detection of P. knowlesi. Circumsporozoite protein is a major surface protein found in all Plasmodium species. The amino acid sequences of CSP across Plasmodium species have well-conserved overall domain structure, with two non-repeat end regions, N- and C-terminal, which flanks a central repeat region [17]. Even though CSP is known to be a sporozoite stage-specific protein, a study carried out by Jesuíno et al. (2006) suggested that CSP might play a role during the erythrocytic development of parasites [18]. In addition to that, Cochrane et al. (1989) described the presence of circumsporozoite-like protein at the erythrocytic stage of malaria [19]. In this study we cloned and expressed PkCSP as well as evaluated its potential as a diagnostic biomarker for the specific detection of P. knowlesi.

Animal ethics

The animal use protocol was reviewed and approved by the Faculty of Medicine, Institutional Animal Care and Use Committee, University of Malaya (2019-201010/PARA/R/TJH).

In silico analysis on CSP and selection of target peptides

Amino acid sequences of CSP of P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi were obtained from GenBank with the accession number: AAN87622 (PfCSP), AGN05254 (PvCSP), ARF20132 (PmCSP), SBT00176 (PoCSP), and XP_002259002 (PkCSP), respectively. Two peptide sequences that were specific to PkCSP were selected based on sequence alignment using Clustal Omega, namely PkCSP peptide I and PkCSP peptide II. Percentage of identity of both selected peptides with other human Plasmodium species were determined using NCBI global alignment. Relative surface accessibility and solubility of the peptides were assessed using NetSurfP-2.0 and PepCalc.com respectively. PkCSP peptide I sequence was synthesized and used as immunogen in monoclonal antibody production by ABclonal, Wuhan, China. PkCSP peptide II was synthesized by Bio Basic Canada Inc, Canada and used as immunogen for generation of polyclonal antibody in rabbit. Both peptide epitopes were synthesized with an additional cysteine in N-terminal for conjugation to keyhole limpet hemocyanin (KLH) as the carrier protein.

Construction of recombinant PkCSP plasmid

Plasmodium knowlesi genomic DNA was extracted from P. knowlesi strain A1H1 culture using blood extraction kit (QIAGEN, Hilden, Germany). Primers for PkCSP were designed based on the PkCSP gene sequence from P. knowlesi strain H genome assembly (NCBI reference sequence: NC_011909). DNA encoding for the signal peptide of PkCSP was excluded during the design of primers. The CSP gene was amplified using polymerase chain reaction (PCR) with the primer pair PkCSP FP: 5’-GGATCCACACACTTCGAACATAATG-3’ and PkCSP RP: 5’-GGATCCTTAATTGAATAATGCTAGGAC-3’. The PCR conditions were as follows: initial denaturing step at 95°C for 4 minutes; 35 cycles at 95°C for 30 seconds, 51°C for 45 seconds, and 72°C for 80 seconds; final elongation step at 72°C for 10 minutes. PCR product was cloned into pGEM®-T plasmid vector (Promega Corporation, USA). Then, the recombinant plasmid was digested using BamHI restriction enzyme (New England Biolabs, Inc., USA) and subsequently cloned into Novagen® pET-30a(+) (Merck KGaA, Germany), USA). Recombinant plasmid was transformed into Escherichia coli expression host T7 Express lysY/I^q (New England Biolabs, Inc., USA). All the recombinant plasmids constructed were sent for sequencing to verify the gene identity.

Expression of PkCSP

Single colony T7 host cell containing recombinant PkCSP plasmid was propagated overnight in Luria-Bertani broth containing kanamycin (30 µg/ml) and chloramphenicol (35 µg/ml) at 37°C with constant shaking at 250 rpm. Next day, the overnight culture was sub-cultured until the optical density at 600 nm (OD₆₀₀) reached 0.4-0.6. The culture was induced with 1 mM isopropyl β-D-1-thiogalactopyanoside (IPTG) and allowed to propagate for another 4 hours. Cell pellets were harvested by centrifugation at 6500 rpm for 10 minutes and stored at -80°C.

Purification, dialysis and quantification of recombinant PkCSP

Recombinant PkCSP (rPkCSP) was affinity purified under hybrid condition of the ProBond^TM purification system (Invitrogen, USA). Cell pellet was resuspended in 6 M guanidinium lysis buffer (pH 7.8) and sonicated on ice to break the cells. Purification column containing nickel-NTA agarose resin was prepared under denaturing condition. Protein lysate was added to the column and incubated on ice for 2 hours. Then, the column was washed with denaturing binding buffer (pH 7.8), denaturing wash buffer (pH 6.0) and native wash buffer (pH8.0) according to the manufacturer’s instruction. The purified rPkCSP was eluted with native elution buffer (pH 8.0). Purified protein was dialysed and the concentration of purified rPkCSP was determined using Quick Start^TM Bradford Protein Assay (Bio-Rad Laboratories, USA).

SDS-PAGE, Coomassie brilliant blue staining and Western blot

Crude protein lysate and purified rPkCSP were resolved in 12% SDS-PAGE under reducing conditions. The gels were stained with coomassie brilliant blue to reveal the protein bands. Separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked overnight in 5% blocking buffer (Tris-buffered saline (TBS) containing 5% skimmed milk) at 4°C. The membranes were probed with His-Tag® monoclonal antibody (EMD Millipore Corp., USA) diluted with 2.5% blocking buffer (1:2500 dilution) for one hour at room temperature. The membranes were washed three times with 0.2% TBS-T (TBS containing 0.2% Tween-20) and incubated with biotin-labelled goat anti-mouse IgG (1:2500 dilution) for one hour, followed by alkaline phosphatase (AP)-conjugated streptavidin (1:2500 dilution) for one hour. Finally, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) was added in the dark to develop colour. Purified rPkCSP was excised from SDS-PAGE gel and sent for protein identification using MALDI/TOF.

Rabbit immunization

The synthesized PkCSP peptide II was conjugated to mariculture KLH via carboxyl-reactive carbodiimide crosslinker (EDC) using Thermo Scientific^TM Imject^TM EDC mcKLH spin kit. A 12-week old female New Zealand white rabbit was used for immunization. Pre-immune serum was collected on day 0. On the same day, KLH-conjugated PkCSP peptide II, 200 µg was mixed with complete Freund’s adjuvant (CFA) (Sigma-Aldrich, USA) at 1:1 ratio and injected into the rabbit subcutaneously at four different sites (50 µg each site) to minimize adverse reactions post-immunization. Four boosters prepared in incomplete Freund’s adjuvant (IFA) (200 µg each booster) were administered using the same administration route as primary immunization at week 2, 4, 6 and 8. Endpoint titre assay was carried out using pre-immune serum as negative control. Maximum amount of blood were collected from the rabbit when the endpoint antibody titre is higher than 1:51200. Antiserum was collected by centrifuging the blood at 2,000 rpm for 10 mins and was stored in aliquots at -80°C until use.

Evaluation of reactivity of mice and rabbit antiserums against rPkCSP and detection of PkCSP in P. knowlesi total protein extract

A total of five mice were immunized with KLH-conjugated PkCSP peptide I using the company’s protocol for monoclonal antibody production. The reactivity of the rabbit antiserum and most reactive mouse antiserum against rPkCSP was evaluated using western blot assay. rPkCSP protein (100 ng) was resolved in 12% SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked overnight with 5% blocking buffer at 4°C. On the next day, the membrane was cut into strips and treated individually with pre-immune serum and antiserum (1:250 dilution) of both rabbit and mouse for 2 hours at room temperature. The strips probed with rabbit serum were incubated in horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:2500 dilution) for one hour. TMB 1-component membrane peroxidase substrate was added in dark to develop colour.

For strips that were probed with mouse serum, biotin-labelled goat anti-mouse IgG + IgM (1:1000 dilution) was added and incubated for one hour. Following this, the strips were incubated in AP-conjugated streptavidin (1:2500 dilution) for one hour. Lastly, colour was developed using NBT/BCIP in dark.

Plasmodium knowlesi total protein extract was extracted from P. knowlesi strain A1H1 culture (5% haematocrit, 5% parasitaemia) according to Cooper (2002) [20]. Western blot assay was carried out as mentioned previously by probing anti-PkCSP peptide I antiserum (1:250 dilution) against P. knowlesi total protein extract. Recombinant PkCSP was included as a control of this assay.

Mice immunization and evaluation of PkCSP mice antisera reactivity

Six to eight-week old female BALB/c mice were used for immunization (n=3). Purified PkCSP, 30 µg, was mixed with CFA at 1:1 ratio and the mixture was injected into mice as primary immunization (day 0). For booster injections, 30 µg were mixed with IFA and administered on day 14 and 21. All injections were given subcutaneously. Serum of each mouse was collected on day 0, 14, 21 and 31. Endpoint titre assay was carried out using day 31 mice antiserum. Antiserum from the most reactive mouse was used in western blot assay as described in the previous method. For this assay, purified rPkCSP and P. knowlesi total protein extract were resolved on 12% SDS-PAGE. pET-30a(+) lysate and crude rPkCSP lysate were included as controls. Dilution of biotin-labelled goat anti-mouse IgG + IgM used was increased to 1:2500 to reduce nonspecific background.

Selection of CSP peptide epitopes for P. knowlesi specific detection

Two peptide epitopes that are specific for P. knowlesi were selected from the sequence alignment using Clustal Omega (Additional file 1). The selected peptide sequences are PkCSP peptide I “VVNDYLHKIRSSVT” and PkCSP peptide II “AEDLTMDDLEVEACV” with amino acid length of 14 and 15 respectively. Percentage identities of both peptide epitopes when compared to corresponding amino acid sequences of other human Plasmodium species are less than 60% as shown in Table 1. Analysis from PepCalc.com showed both peptide sequences have good water solubility. NetSurfP-2.0 also predicted that the relative surface accessibility of both peptide sequences are “exposed”, which allow antibody interaction.

Table 1 Global alignment of selected CSP peptide epitopes.

Species	PkCSP peptide I	PkCSP peptide II
P. knowlesi	VVNDYLHKIRSSVT (100%)	AEDLT-MDDLEVEACV (100%)
P. falciparum	HIEQYLKKIQNSLS (36%)	KDELDYENDIEKKICK (25%)
P. vivax	SVKEYLDKVRATVG (43%)	PEDLT-LNDLETDVCT (53%)
P. malariae	HIKNYLESIRNSIT (43%)	PAELV-LSDLETEICS (40%)
P. ovale	DIKKYIDKIRNDIT (36%)	AQELT-LSDLETEICK (53%)
Overall identity	::.*:..:: :	:* .: . *

Note: The underlined peptide sequences were used as the query sequences in the global alignment with the corresponding amino acid sequences.

Cloning, expression and purification of rPkCSP

The amplified PkCSP fragment has a size of 1047 bp and the identity was confirmed as P. knowlesi CSP gene through sequencing result. The predicted molecular mass of the rPkCSP protein is ~40 kDa. Under SDS-PAGE, the resolved rPkCSP protein appeared as doublets at ~55 kDa (Additional file 2). The bands at ~55 kDa was excised from purified rPkCSP and was confirmed to be PkCSP by MALDI/TOF.

Reactivity of rabbit and mice antiserums against rPkCSP and detection of PkCSP in P. knowlesi total protein extract

Western blot assay strips showed that mouse anti-PkCSP peptide I antiserum was able to bind to rPkCSP (Figure 1A, arrow). For rabbit anti-PkCSP peptide II antiserum, there was no band observed in western blot strip against the rPkCSP. All the western blot strips treated with pre-immune serums showed no band.

The same mouse antiserum was used for the detection of PkCSP in P. knowlesi total protein extract. As the previous assay, mouse anti-PkCSP peptide I antiserum was able to probe on rPkCSP. Yet, no band was observed on the strip blotted with P. knowlesi total protein extract (Figure 1C).

Detection of PkCSP in P. knowlesi total protein extract using mouse anti-rPkCSP antiserum

The western blot assay showed that mouse anti-rPkCSP antiserum bound to the rPkCSP at ~55 kDa (Figure 2B, lane 2 and 3, arrows). The antiserum did not bind to E. coli proteins (Figure 2B, lane 1) with only minimal nonspecific bands observed. There was no band seen in the P. knowlesi total protein extract.

In RDT, both capture and detection antibodies against the same protein are required, and these antibodies should bind to different epitopes of the protein to avoid competition that affects binding efficiency. It has been shown that antibodies raised against peptides are able to recognize the native proteins. This was demonstrated by Verma et al. (2013) in which the antibodies raised against PfHRP2 and pLDH peptides were able to recognize the parent proteins in ELISA-based diagnostic assay [21]. Besides, Hurdayal et al. (2010) had shown that the species-specific anti-peptide antibodies raised against pLDH were able to differentiate P. falciparum and P. vivax infection [22].

In this study, species-specific peptide epitopes were selected based on CSP sequence alignment. Selection of candidate peptides from PkCSP central repeat region was impossible as the region is hyperpolymorphic [23]. Hence, two different P. knowlesi specific peptides were selected from the C-terminal of CSP protein. The range of percentage identities of PkCSP peptide I and PkCSP peptide II are within 36-43% and 25-53% when compared to the other Plasmodium species, respectively (Table 1). The selected PkCSP peptides have the highest similarity when compared to P. vivax CSP (43% and 53%). This may be due to the close relationship of the two species, sharing a common ancestor [24]. Nevertheless, in the study by Verma et al. (2013), a difference of four to six amino acids of the selected peptides were able to generate pLDH antibodies that are specific to their respective species [21]. Hence, in our study, the percentage identities of the selected peptides were believed to be sufficient to generate antibodies specific for P. knowlesi detection.

Signal peptide of PkCSP was removed in the construction of recombinant PkCSP plasmids as it may affect protein expression [25]. Purification of protein was performed in hybrid condition, thereby exposing the protein to denaturing conditions and then to native conditions. The expressed PkCSP appeared to be ~15 kDa larger than its theoretical mass when resolved in SDS-PAGE. This may be due to the presence of protein complexes that are not fully disrupted even in the presence of reducing agents. A similar scenario was observed by Plassmeyer et al. (2009) in which the recombinant P. falciparum CSP expressed had a molecular mass larger than the theoretical mass due to the incomplete separation of the protein complexes [26].

Western blot assay was performed to analyse the ability of anti-PkCSP peptides antiserum to bind to the rPkCSP. Only mouse anti-PkCSP peptide I antiserum was able to detect the rPkCSP in western blot assay. It is important to note that antibodies produced against linear epitopes should be able to recognize denatured proteins under SDS-PAGE gel [27]. However, the rabbit anti-PkCSP peptide II antiserum did not detect the rPkCSP despite optimizing multiple times the amount of protein loaded, antiserum dilution, and enzyme conjugate dilution. This is likely due to the PkCSP peptide II was presented in folded form when immunized in the rabbit. Hence, the antibodies raised were only able to recognize conformational epitopes but not linearized protein under SDS-PAGE [28].

The mouse anti-PkCSP peptide I antiserum was then used to detect PkCSP from P. knowlesi total protein extract. However, no distinct band was observed in P. knowlesi total protein extract (Figure 1). The multiple unspecific bands are likely due to high background caused by the secondary antibody. This may indicate that no PkCSP was produced in the erythrocyte stage of the parasite life cycle despite Cochrane et al. (1989) describing presence of CS-like protein in the merozoite stage [19]. To further validate this, anti-rPkCSP antibodies were produced in mice models. This was carried out to determine the ability of anti-rPkCSP antibodies, which target multiple epitopes, in detecting PkCSP in the parasite total protein extract. As shown in Figure 2, the anti-rPkCSP antiserum did not detect PkCSP in P. knowlesi total protein extract and no background was observed as the dilution of secondary antibody was optimized to a higher dilution. In a literature by Herman et al., (2018), it was demonstrated that PkCSP was not amplified from RNA extracted from P. knowlesi strain A1H1 culture using reverse-transcription PCR [29]. Thus, our study has validated that PkCSP is absent in the parasite blood stage with the use of antibody detection.

From this study, we conclude that CSP was not expressed in P. knowlesi erythrocytic stage by using antibody detection approach. Thus, it is not a suitable candidate to be used as a target biomarker for specific detection of P. knowlesi despite species-specific epitopes being identified. Further study will be done to screen for other protein candidates for P. knowlesi specific detection in rapid diagnostic tests.

Acknowledgements

The authors would like to thank Shahhaziq Shahari from malaria culture laboratory, Parasitology Department, University of Malaya for providing P. knowlesi strain A1H1 culture. The authors also like to thank Ummi Wahidah Azlan for the mice immunization work.

Funding

This study is funded by the Long Term Research Grant Scheme (LRGS) provided by the Ministry of Education Malaysia (LR002D-2018).

Declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

All relevant data are provided in the manuscript or available from published materials as cited.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JHT carried out the expression, animal immunization and assays, and drafted the manuscript. YLL was involved in experimental design and reviewed the manuscript. All authors read and approved the final manuscript.

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Exploring the use of Circumsporozoite Protein (CSP) as Diagnostic Biomarker for Specific Detection of Plasmodium Knowlesi

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