Immunogenicity of a plasmid DNA vaccine encoding 42 kDa fragment of Plasmodium vivax merozoite surface protein-1

Accepted Manuscript Title: Immunogenicity of a plasmid DNA vaccine encoding 42 kDa fragment of Plasmodium vivax merozoite surface protein-1 Author: Inayat Hussain Sheikh Deep C. Kaushal Deepak Chandra Nuzhat A. Kaushal PII: DOI: Reference:

S0001-706X(16)30380-1 http://dx.doi.org/doi:10.1016/j.actatropica.2016.06.013 ACTROP 3959

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

13-2-2016 7-6-2016 10-6-2016

Please cite this article as: Sheikh, Inayat Hussain, Kaushal, Deep C., Chandra, Deepak, Kaushal, Nuzhat A., Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Immunogenicity of a plasmid DNA vaccine encoding 42 kDa fragment of Plasmodium vivax merozoite surface protein-1

Inayat Hussain Sheikha, c, Deep C. Kaushalb, Deepak Chandrac and Nuzhat A. Kaushala*

a

Division of Parasitology, CSIR- Central Drug Research Institute, Lucknow-226031, India. Amity University Uttar Pradesh, Lucknow Campus, Lucknow-226028, India. c Department of Biochemistry, Lucknow University, Lucknow, India b

*

Corresponding author: Former Senior Principal Scientist, Division of Parasitology, CSIRCentral Drug Research Institute, Lucknow-226031 E-mail addresses: [email protected] (I.H. Sheikh), [email protected] (D.C. Kaushal), [email protected] (D.Chandra), [email protected], [email protected](N.A. Kaushal)

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Graphical abstract

Highlights

   

Cloned and expressed 42 kDa fragment of P. vivax MSP-1 in eukaryotic vector pcDNA 3.1 Transfection of COS-1 cells with PvMSP-142 plasmid DNA construct showed expression of PvMSP-142 Plasmid DNA being less immunogenic when compared to recombinant protein PvMSP-142 Prime boosting of plasmid DNA immunized mice with recombinant protein significantly enhanced immunogenicity

ABSTRACT

Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms 2

of recombinant 42 kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42 kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42 kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses.

Key words: DNA vaccine, Malaria vaccine, Erythrocytic stage, P. vivax, PvMSP-142

1.

Introduction

Plasmodium vivax has been recognized as the second most important disease causing malaria parasite of humans after P. falciparum and is currently the leading causative agent of human malaria in Asia and Latin America. According to a recent WHO report an estimated 3.3 billion people worldwide were at risk of having malaria infection. Most of these cases were in African countries, while almost half of the malaria cases outside Africa were due to P. vivax (Carlton et al., 2011; WHO, 2014). The vivax malaria, unlike P. falciparum, is associated with dormant liver stages (hypnozoites) that reactivate in weeks to months time following the primary infection (Krotoski et al., 1989). P. vivax parasites have also been shown to cause severe disease related complications that have long been attributed only to P. falciparum. Several factors along with the emergence of drug-resistant P. vivax strains have emphasized the need for a vivax vaccine (Price et al, 2009; Petersen el al, 2011; Shanks et al., 2012).

Merozoite surface protein-1 of malaria parasites, present on the surface of merozoite and playing a key role in the invasion process, is one of the leading vaccine targets for both P.

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falciparum and P. vivax malaria (Blackman et al., 1990; Holder et al., 1992). MSP-1 occurs as a 200 kDa precursor and undergoes step-wise proteolytic processing resulting in a glycosyl-phosphatidylinositol-anchored 42 kDa protein fragment (MSP-142) on the surface of free merozoite (Blackman et al., 1991). A large number of studies have shown the protective potential of MSP-1 fragments (42 and 19 kDa) of P. falciparum and P. yoelii (Ling et al., 1994; Kumar et al., 2000; Shi et al., 2000; Ahlborg et al., 2002), however, few efforts have been made to evaluate the immunogenicity of P. vivax MSP-1 C-terminal conserved fragments (Yang et al., 1996; Perera et al., 1998; Dutta et al., 2001; 2005). Efforts towards the development of effective malaria vaccine including P. vivax vaccine have been seriously hampered by the presence of many disulfide-linked structures in 42 kDa fragment of merozoite surface protein-1 and it has been difficult to produce this protein in native conformation state (Birkholtz et al., 2008). Many other reasons ranging from diversity and variation in antigens of malaria parasites and the non-availability of suitable animal models have been reported for the unsuccessful efforts towards the development of P. vivax vaccine.

DNA vaccines have emerged as a promising approach for a variety of infectious agents including malaria parasites (Hoffman et al., 1994; Khan et al., 2013; Rosa et al., 2015). DNA based vaccines are particularly important for developing multistage/multiantigen vaccines for complex parasites like malaria thereby inducing specific and protective immunity against different life cycle stages (pre-erythrocytic, erythrocytic and sexual stages) of malaria (Doolan and Hoffman, 1997; Hoffman et al., 1998; Du et al., 2016). The advantages of DNA vaccines are the ease of their production and their ability to induce responses without any exogenous adjuvant, which is an absolute requirement for protein based vaccine formulations (Kumar et al., 2002).

DNA vaccines have been shown to induce strong humoral and cellular responses to malarial antigens in immunized small animals (Sedegah et al., 1994, Kang et al., 1998; Good et al. 4

2005). However, clinical trials of these candidate vaccines when used alone or in repeated homologous boosting regimens have been disappointing, with short lived low levels of induced specific T-cells responses. Sequential immunization with different plasmids/vectors known as heterologous prime-boosting appears to be a better approach and has been shown to induce enhanced and persistent levels of antibody and cell mediated immune responses against malaria (Dunachie and Hill, 2003; Dunachie et al., 2006; McConkey et al., 2003; Moore and Hill, 2004; Wang et al., 2004; Mehrizi et al., 2011). In heterologous prime-boosting strategy the priming was done with DNA followed by the booster immunization with a recombinant protein, a recombinant viral vaccine, or exposure to the live organism (Kumar et al., 2002). T helper type 1 (Th1) cells principally mediate cellular immune responses by secreting IL-2, IFN and TNF-α, and they promote IgG2a production. IFN- and IL-2 are correlated with the protection against malaria parasites as high levels of IFN- can inhibit the parasite development, prevent re-infection and provide protection against clinical malaria (Petritus and Burns, 2008; D’Ombrain et al., 2008; Roestenberg et al., 2009; McCall and Sauerwein, 2010, de Souza, 2014). Roles of IFN- and IL-12 cytokines have been studied in mouse malaria infection and the cytokine gene knockout mice were unable to control P. chabaudi infection (Stevenson et al., 1995; de Souza, 2014). IL-12 has been shown to play an important role in antibody mediated protective immunity against blood stages of P. chabaudi (Su and Stevenson, 2002) and in defence against P. falciparum infection in humans (Malaguarnera et al., 2002).

We have earlier studied the immunogenicity and protective efficacy of soluble and refolded proteins of P. vivax MSP-142 (PvMSP-142) using a closely related P. cynomolgi rhesus monkey model. Soluble and refolded forms of PcMSP-142 and PvMSP-142 proteins also appeared to have a similar partially protective effect (Dutta et al., 2005). In the present study we have evaluated the immunogenicity of a DNA vaccine encoding MSP-142 of P. vivax

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using prime-boosting strategy in BALB/c mice. We have also compared the immunogenicity of PvMSP-142 recombinant protein in BALB/c mice.

Materials and Methods

2.1. DNA vaccine constructs encoding P. vivax MSP-142 Expression of the recombinant P. vivax MSP-142 in E. coli using pTriEX4 vector has been reported by us previously (Sheikh et al., 2014). The PvMSP-142-pTriEX4 clone was used as a template for amplification of the PvMSP-142 by polymerase chain reaction (PCR), using 5’-CGCGGATCCGCCATGGACCAA GTAACAACGGGAGA-3’ as the forward primer and 5’-CGGTGCGGCCGCTTAGCTACAGAAAACTCC-3’ as the reverse primer for cloning in mammalian expression vector pcDNA 3.1(+) (Invitrogen-Life Technologies, USA ). PCR amplification was performed in 50 μl reaction mixture containing 5 μl of 10X PCR buffer, 2 μl of 50 mM MgCl2, 1 μl of 25 mM dNTPs mix, 1 μl each of 20 μM forward and reverse primers, 100 ng of PvMSP-142-pTriEX4- DNA and1 U of Taq DNA polymerase (5 U/μl, NEB, USA) in a programmable Thermocycler (Eppendorf, Germany). Reaction conditions comprised an initial denaturation of 5 min at 94 0C, followed by 35 cycles of 940C for 1 min, 480C for 1 min and 720C for 2 min, with a final extension at 720C for 10 min. Amplicon was analyzed on 1% agarose gel containing 0.5 μg/ml of ethidium bromide. PCR amplified product was purified from gel by the gel extraction kit (QIAGEN, USA). Purified PvMSP-142 PCR product was ligated into BamH1-Not1 site of pcDNA3.1 at 40C overnight using T4 DNA Ligase (Promega Corporation, USA). The PvMSP-142– pcDNA 3.1 plasmid construct was transformed into competent DH5α E. coli cells ( made competent using Bangalore Genie Kit, Bangalore, India) and plated on LB-ampicillin plates followed by incubation at 370C for about 16-18 h. Positive colonies were screened by colony PCR using the gene specific primers for the presence of insert. Plasmid DNA was purified from overnight culture using QIAGEN Plasmid Miniprep kit and the presence of insert was verified by colony PCR and by BamH1-Not1 restriction digestion of purified recombinant plasmid. One 6

clone was selected and sequenced from the DNA Sequencing Facility at the Department of Biochemistry, University of Delhi South Campus (UDSC), New Delhi, India.

2.2. In vitro transfection of mammalian cells with P. vivax MSP-142-pcDNA 3.1 Plasmid Plasmid DNA corresponding to PvMSP-142 -pcDNA 3.1 and pcDNA 3.1 vector were purified using QIAGEN DNA purification kit (QIAGEN, USA) and used for in vitro transfection of COS-1 mammalian cell line (Choudhury et al., 2009). Briefly, the COS-1 cells were maintained in in vitro culture using Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCOLife Technologies, USA) supplemented with 10% foetal calf serum (FCS, GIBCO-Life Technologies, USA). Cells were seeded in a 24-well tissue culture plate (Nunc, Denmark) at a density of 1x105 cells per well, a day prior to transfection. For transfection, varying amount of plasmid DNA (0.5–1.5 µg) was mixed at 1:2.5 ratio with lipofectamine (GIBCO-Life Technologies, USA) in Hanks Balanced Salt solution (HSSB, final reaction volume: 100 µl) and incubated at room temperature for 30 min. Plasmid DNA–lipofectamine mixture (PvMSP-142 -pcDNA 3.1 plasmid/ pcDNA 3.1vector) was added dropwise to the cells, and plate was incubated for 8 h at 37 0C in humidified atmosphere of 5% CO2. Subsequently, after 8 h, medium containing DNA-lipofectamine was removed and replaced with DMEM containing 10% FCS and cells were allowed to grow for 48 h. After that 300 µg/ml of Geneticin (GIBCO-Life Technologies, USA) was added and cells were allowed to grow for 3 weeks with change of medium every alternate day. After three weeks, the cells were processed for localization of PvMSP-142 by indirect immunofluorescence assay.

2.3. Monoclonal antibodies against P. vivax MSP-142 Monoclonal antibodies againstPvMSP-142, used in the present study, were produced earlier in our lab (Kaushal et al., under communication). Monoclonal antibody (Moab5) is against the conformational epitope specific to P. vivax MSP-142 and the other monoclonal (Moab8) against the linear epitope of P. vivax MSP-142.

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2.4. Localization of P. vivax MSP-142 by indirect immunofluorescence Briefly, for indirect immunofluorescence assay (IFA) cells were seeded onto cover slips in 6well plate at concentration of 1x104 cells per well and incubated for 48 h at 370C in humidified atmosphere of 5% CO2. The cells were then washed twice with 50 mM PBS (pH 7.4), fixed with chilled methanol (-200C) for 5 min, and blocked with 3% bovine serum albumin (BSA) in PBS for 2 h at 4 0C. For detection of PvMSP-142, a mouse monoclonal antibody (Moab5) generated against E. coli expressed rPvMSP-142 (Kaushal et al. under communication) was used at a dilution of 1:250 for 2 h at 4 0C. Cells were then washed five times with PBS and incubated for 1 h with 1:100 dilution of goat anti-mouse Ig-fluorescein isothiocyanate (FITC) conjugate (Sigma-Aldrich, USA) at 40C. After washing with PBS, cover slips with the cells were mounted in glycerol:PBS (9:1), and examined under an Optiphot fluorescent microscope (Nikon, Tokyo, Japan).

2.5. SDS–PAGE and immunoblotting Cells transformed with either PvMSP-142-pcDNA 3.1 plasmid DNA clone or pcDNA 3.1 vector DNA were analysed by SDS-PAGE (Laemmli, 1970) and immunoblotting as described elsewhere (Towbin et al., 1979) with slight modifications (Sheikh et al., 2014). Cells were solubilised by boiling for 5 min with 2X sample buffer (62.5 mM Tris, pH 6.8, 2% SDS (w/v), 10% glycerol (v/v), 5% -mercaptoethanol (v/v), and 0.001% bromophenol blue (w/v)). Proteins were resolved by 10%SDS- PAGE and processed for immunoblotting for the detection of PvMSP-142 using a 1:1000 dilution of anti-PvMSP142 monoclonal (Moab8).

2.6. Purification of recombinant P. vivax MSP-142 expressed in E. coli Recombinant P. vivax MSP-142 protein was expressed as polyhistidine fusion protein in E. coli and purified using nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, USA) as described previously (Sheikh et al., 2014).

2.7. Purification of plasmid for immunization 8

Plasmids corresponding to expressed clone PvMSP-142-pcDNA 3.1(+) and pcDNA3.1(+) vector were isolated from the cells using Midi prep Plasmid isolation kit (Endotoxin free, Sigma-Aldrich, USA) according to manufacturer’s instructions. Purified plasmid preparations were concentrated and used for the immunization of mice.

2.8. Immunization studies in mice Inbred male BALB/c mice (6–8 weeks of age), kept under the experimental Animal Facility, CSIR-Central Drug Research Institute, Lucknow, India, were used for the study. Immunization and bleeding of mice was conducted as per the guidelines approved by the Institutional Animal Ethics Committee. BALB/c mice were distributed into four groups consisting of five mice each group. Mice in the first (Group1) and second (Group2) group were immunized intramuscularly (i.m.) with PvMSP-142-pcDNA 3.1 plasmid DNA (equivalent to 100 µg DNA in PBS/mouse) without any adjuvant in the anterior tibialis muscle of the hind limbs (each limb receiving 50 µg), the third group (Group3) was immunized with recombinant PvMSP-142 protein (rPvMSP-142, 35 µg/mouse) intraperitoneally along with Freund’s complete adjuvant (FCA) and the fourth group (Group4) was given pcDNA 3.1 Plasmid DNA (Control). Mice in Group3 were given two booster injections each of 35 µg of rPvMSP-142 protein per mouse emulsified in Freund’s incomplete adjuvant (IFA) intraperitoneally on days 21 and 35. Similarly, two booster injections of 100 µg PvMSP-142-pcDNA 3.1 plasmid were administered intramuscularly on days 21 and 35 to the mice in Group1 and Group2 while mice in Group4 received the booster injections of pcDNA 3.1 plasmid. Mice in all the four groups were bled on day 42; serum was collected and kept at -20OC until used. On day 45, the mice were prime boosted either with recombinant PvMSP-142 protein or PvMSP-142 pcDNA 3.1 plasmid DNA such that Group2 and Group3 received the recombinant PvMSP142 protein of 35 µg/mouse intraperitoneally in IFA. Mice in Group1 received 100 µg /mouse i.m of PvMSP-142-PcDNA 3.1 plasmid DNA while Group4 received the pcDNA3.1 plasmid DNA (100 µg/mouse i.m) following the same immunization schedule as for other groups. Based on prime boosting antigen, four groups of mice were named as DNA/DNA (Group1), 9

DNA/Protein (Group2), Protein/Protein (Group3) and control (pcDNA 3.1, Group4). The animals were bled before initiating the immunization and on days 42 and 52 retro-orbitally. Details of immunization, prime boosting and bleeding of mice in all four groups are given in Table S1.

2.9. Enzyme linked immunosorbent assay The enzyme linked immunosorbent assay (ELISA) was performed in 96 well flat bottom microtitre plates by the method of Voller et al. (1976) with some modifications (Kaushal et al., 2007). Briefly, the wells of the microtitre plates were sensitized with appropriate concentration of purified recombinant PvMSP-142 protein diluted in phosphate buffered saline (PBS, pH 7.4) by incubation for 2 h at 37 0C and then overnight at 40C. The plate was washed three times with PBS to remove the unbound antigen and the wells were blocked with 3% non-fat dry milk at 370C for 2 h. After three washes with PBS-Tween (PBS containing 0.05% Tween-20), 100 μl of sera from the immunized and control groups of mice (appropriately diluted in 1% milk–PBS) were added to the wells, incubated for 2 h at 37 0C and then washed thrice with PBS-Tween. To this 100 μl of peroxidase conjugated secondary antibody (appropriately diluted in 1% milk–PBS) was added and the plate was allowed to incubate at 370C for 1.5 h. The plate was finally washed with PBS-Tween and the colour was developed by adding 100 μl of substrate solution (1 mg/ml O-phenylenediamine in citrate– phosphate buffer, pH 5.0, containing 1 μl/ml H2O2). The reaction was stopped after 10–15 min by adding 50 μl of 5N H2SO4 and the absorbance was read at 490 nm using UV 190 plus microplate ELISA reader (Molecular Devices, USA).

2.10. Determination of IgG subclasses IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) were determined in immune mice sera by ELISA method as described elsewhere (Kaushal et al., 1995). Briefly, the wells of flat bottom microtitre plates (Falcon, USA) were coated with the rPvMSP-142 (100 ng/well) prepared in phosphate saline buffer (pH 7.2). Appropriate dilutions of sera were added to the 10

wells followed by incubation for 2 h at 370C. After washing, the plate was probed with appropriately diluted class and sub-class specific antibodies and incubation for 1 h at 37 0C. This was followed by 1 h incubation with 1:1000 dilution of peroxidase labelled rabbit antimouse-IgG conjugate (Sigma-Aldrich, USA) at 370C. The plate was finally washed and developed as described for ELISA and the absorbance was read at 490 nm in an ELISA reader (Molecular Devices, USA).

2.11. Cell Proliferation Assay Proliferation of cells from the immunized and control groups of mice was measured in response to rPvMSP142 antigen by XTT cell proliferation assay as described elsewhere (Roehm et al., 1991). Briefly, the mice were sacrificed and spleens were removed aseptically and the splenocytes were isolated by mechanical disruption of the spleen in DMEM. The red blood cells were lyzed by exposing the cell pellet to 10x concentration of 50 mM PBS and immediately bringing the concentration to 1xPBS by addition of water. Cells were diluted to a final concentration of 1X10 6 cells/ml in DMEM supplemented with 10% FCS. Cells were stimulated with 10 µg/ml of rPvMSP-142 protein by incubating for 72 h at 370C in CO2 incubator with 5% of CO2. Concanavalin A (5 µg/ml; Sigma-Aldrich, USA) was used as positive control. After 72 h, the proliferation of the cells was measured by adding XTT (100 µl/well) containing 1% PMS and the absorbance was recorded at 450 nm. All assays were carried out in triplicates. Data were represented as stimulation index (SI) which is calculated by dividing the absorbance (OD) observed in presence of stimulating antigen by absorbance (OD) observed in unstimulated cells. Values are expressed as mean ± S.E. from three immunized mice.

2.12. Detection of cytokines IFN-, IL-2 and IL-12 by ELISA The levels of cytokine ( IL-2, IL-12 and IFN-) in the supernatants of antigen stimulated splenocytes from immunized and control groups of mice were measured using BD

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Bioscience Cytokine ELISA kit (BD Biosciences Pharmingen, USA) in triplicate, according to the manufacturer’s instructions and following the procedure described elsewhere (Mastroeni et al., 2000). Briefly, appropriately diluted anti-cytokine capture antibody (100 µl/well)was added to the well of flat bottom ELISA plate (Falcon, USA) and incubated at 40C overnight. Then unbound capture antibody was removed by washing three times with PBS containing 0.05% Tween-20 (PBS-Tween) and the wells were blocked with Blocking buffer (PBS with 10% FBS) at room temperature for 1h. After three washes with PBS-Tween,100 µl/well of standard and 100 µl/well of the culture supernatant were added and incubated at room temperature for 2 h. The plates were then washed three times with PBS-Tween and 100 µl of pre-mixed detection antibody plus Streptavidin-HRP was added and incubated for 1 h at room temperature. After three washes, 100 µl of 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate (BD Biosciences Pharmingen, USA) was added and incubated for 30 min at room temperature in the dark. The reaction was stopped by adding 50 μl of 2N H2SO4 and the absorbance was read at 450 nm using UV 190 plus microplate ELISA reader (Molecular Devices, USA).

2.13. Statistical analysis Results were presented as mean ±S.D. Data was analyzed in GraphPad Prism 5.0 using Newman-Keuls Multiple comparison test. The p-values <0.05 were considered significant.

3. Results

3.1. Construction of a P. vivax merozoite surface protein-142-pcDNA 3.1 plasmid DNA vaccine and its in vitro expression in COS1 cells DNA encoding 42 kDa fragment of P. vivax merozoite surface protein-1 (PvMSP-142) was cloned into BamHI-Not1 site downstream from the TPA leader sequence in the pcDNA 3.1(+) vector, which contains a CMV promoter, a bovine growth hormone poly(A) terminus, and a Neomycin resistance marker. PvMSP-142 was amplified using gene specific sense 12

and anti-sense primers and the PCR product of 1.2 kb was obtained (Fig. S1). PvMSP-142 PCR product was ligated into pcDNA 3.1 vector at BamH1 – Not1 sites. Purified PvMSP142-pcDNA 3.1 plasmid was digested with BamH1- Not1 restriction enzymes and 1.2 kb insert was observed (Fig. 1). Sequence of the PvMSP-142 insert was confirmed by sequencing analysis. Nucleotide sequences and the amino acid sequences of the PvMSP142 inserts were found to be identical to the PvMSP-142 gene sequence (Gen Bank accession number KF612323) published earlier from our group. Ability of the PvMSP-142-pcDNA 3.1 plasmid DNA construct to express PvMSP-142 protein was checked by in vitro transfection of COS-1 mammalian cells. The indirect immunofluorescence assay of transfected COS-1 cells with monoclonal antibody (Moab5, against the conformational epitope of PvMSP-142) revealed positive fluorescence (Fig. 2b) whereas no fluorescence was observed with COS-1 cells transfected with pcDNA 3.1 plasmid vector (Fig. 2a). This finding was further confirmed by western blot analysis of transfected cells. COS- 1 cells transfected with PvMSP-142pcDNA3.1 plasmid DNA exhibited prominent band of about 55 kDa when probed with the monoclonal antibody (Moab8) against the linear epitope of PvMSP-142 protein while COS- 1 cells transfected with pcDNA 3.1 plasmid vector failed to react with the monoclonal antibody (Fig. 3).

3.2.

Antibody responses in immune mice against P. vivax merozoite surface protein-142-

pcDNA3.1 DNA vaccine construct and recombinant PvMSP-142 protein In order to analyse the antibody responses induced by immunization of BALB/c mice with the PvMSP-142-pcDNA3.1 plasmid construct encoding PvMSP-142 and the recombinant protein PvMSP-142, immune mice sera from different groups of mice (DNA/DNA, DNA/Protein, Protein/Protein groups), collected 3 days before and 7 days after the booster injection, were tested in ELISA. Antibody titration curves are shown in Fig. 4a and the 1OD antibody titres of different groups of mice are shown in Fig. 4b. Antibody levels in sera collected at 42 days were significantly higher in the group of mice immunized with the recombinant protein (Protein/Protein group) compared to the immunized mice in DNA/DNA and DNA/Protein 13

groups. However, antibody titres in DNA /DNA group were still higher when compared to the control group of mice immunized with pcDNA 3.1 vector alone (p <0.05). There was significant increase in the antibody levels in sera of mice immunized with plasmid DNA group boosted with recombinant protein (Fig. 4b).

Immune serum pools from different groups of immunized mice (DNA/DNA, DNA/Protein, Protein/Protein group) were also tested in immunoblotting using the E. coli expressed PvMSP-142 protein. All the three serum pools (DNA/DNA, DNA/Protein, Protein/Protein group) detected the 55 kDa protein band. The serum pool from the control group (pcDNA3.1 vector) did not recognize any protein band (Fig. S2). These results confirmed that the mice immunized with plasmid DNA construct (PvMSP-142-pcDNA3.1 plasmid) produced antibodies against PvMSP-142.

3.3.

Measurement of P. vivax merozoite surface protein-142 specific IgG Isotypes in

immune mice sera Sera obtained from different groups of immunized mice were analyzed for IgG isotypes (IgG1, IgG2a, IgG2b and IgG3) against PvMSP-142 by ELISA. Sera from mice in all the three groups exhibited high antibody titres, with IgG1 being the predominant isotype, followed by IgG2b and IgG2a. The IgG3 levels were found to be lower in DNA/DNA and DNA/Protein groups as compared to Protein/Protein group. Mice sera from control group did not show any significant ELISA values (Fig. 5).

3.4.

In vitro T cell responses against P. vivax merozoite surface protein-142

Proliferation responses by the splenocytes from immunized groups of mice were studied in vitro after stimulation with recombinant PvMSP-142 protein. All the three immunized groups demonstrated significantly higher T cell proliferation (p<0.001) compared to the control group (mice immunized with pcDNA 3.1 vector alone).T cell proliferation of splenocytes from mice immunized with recombinant PvMSP-142 protein was the highest (Fig 6). Spleen cells from 14

mice immunized with DNA and boosted with protein showed higher T cell proliferation compared to the mice in DNA/DNA group.

3.5.

In vitro Cytokines production against P. vivax merozoite surface protein-142

To observe the cytokine production of the immunized groups of mice, three cytokines (IL-2, IL-12 and IFN-) were estimated in the culture supernatants of splenocytes from all groups of immunized mice in comparison to the control group (pcDNA 3.1 vector). Levels of all the three cytokines (IL-2, IL-12 and IFN-) were found to be significantly higher in culture supernatant of splenocytes of Protein/Protein group followed by DNA/Protein and DNA/DNA groups as compared to the control group of mice (Fig. 7).

4. Discussions

Asexual blood-stage (erythrocytic) vaccines are aimed to primarily protect against severe malaria disease (Carvalho et al., 2002; Kaushal et al., 2009; De Souza, 2014). The most important asexual blood stage vaccines are based on the use of merozoite surface protein-1 (MSP1), which is part of a complex molecule involved in red blood cell invasion (Holder et al., 1992; Ling et al., 1994; Good et al. 2005). Substantial efforts have been focussed on the 42- and 19-kDa C-terminal recombinant fragments of MSP1 (Kumar et al., 2000; Dutta et al., 2005; Holder et al., 2009). A number of earlier studies have testified to the powerful nature of the revolutionary approach of DNA vaccinology in malaria (Doolan and Hoffman, 1997; Hoffman et al., 1998; Du et al., 2016). DNA vaccination represents an ideal approach for developing malaria vaccine expressing antigens from different life cycle stages of malaria parasites particularly in the case of P. vivax, where resources for the production of multiple recombinant proteins are quite limited. In the present study, we have prepared a plasmid DNA construct encoding P. vivax PvMSP-142 antigen. The expression of PvMSP-142 protein

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was checked by in vitro transfection of COS-1 mammalian cells with PvMSP-142-pcDNA 3.1 plasmid DNA construct. Immunogenicity of PvMSP-142 DNA vaccine construct was investigated by priming the BALB/c mice either with PvMSP-142 plasmid DNA or with recombinant PvMSP-142 protein and boosting with recombinant PvMSP-142 protein.

Previous studies from our lab as well as by other investigators have shown that proper conformation and folding of the PvMSP-142 is critical for the induction of protective humoral immune responses (Dutta et al., 2005; Calvo et al., 1996; Kang et al., 1998). Therefore, it is important to determine initially, before immunization, whether the DNA vaccine construct could synthesize the encoded polypeptide correctly in the mammalian cells. Recognition of 55 kDa protein band in COS- 1 cells transfected with PvMSP-142-pcDNA3.1 plasmid DNA by the PvMSP1-42 specific monoclonal antibody (against linear epitope) in immunoblotting confirmed the expression of PvMSP-142 protein. Positive immunofluorescence observed with conformational anti-PvMSP-142 monoclonal antibody (Kaushal et al., under communication) demonstrated that PvMSP-142 expressed in transfected COS-1 cells was in a conformational form.

In the present study we have conducted DNA vaccination for P. vivax MSP-1 42 and compared the immunogenicity of DNA vaccine construct with corresponding protein vaccination in BALB/c mice. Immunization of mice with recombinant PvMSP-142 resulted in the induction of high titres of anti-rPvMSP-142 antibodies than that of DNA immunization. However, the antibody titres observed in PvMSP-142 plasmid DNA immunized mice were significantly higher than the mice in the control group (vector immunized). DNA vaccine plasmids encoding merozoite surface protein-1 from murine malaria has been shown to provide protection against malaria infection (Becker et al., 1998). Immunization of mice with a genetic vaccine of P. vivax MSP119-HBs viral particle induced significant high titres of antibody responses (Wunderlich and del Portillo, 2000; Wunderlich et al., 2000). Rogers et al. (1999) have shown that immunization of mice with DNA plasmids encoding SSP2, AMA1 16

and MSP142 of P. vivax induced high titre antibodies and these antibodies recognized the corresponding proteins in parasite extract.

The importance of heterologous prime-boost strategy in malaria infection has been demonstrated by a number of studies (Sedegah et al., 1998; Wang et al., 2004; Hill et al., 2010). In the present study, priming with P. vivax MSP-142 plasmid DNA and boosting with recombinant PvMSP-142 protein exhibited a significant increase in the antibody responses in DNA/protein group compared to DNA/DNA group as it was evident on comparing the 1OD titre as well as endpoint titre values of different immunized groups. These results suggest that the priming with plasmid DNA and boosting with recombinant protein is essential for the enhancement of immune responses. However, the antibody titres in Protein/Protein group were significantly higher than DNA/Protein group. This may be attributed to the use of FCA/IFA for protein immunization which resulted in high levels of antibody in Protein/Protein group. FCA/IFA has also been used by Mehrizi et al. (2011) in their prime boosting studies on co-immunization of mice with P. vivax and P. falciparum MSP119 DNA plasmids and proteins. They have obtained almost same levels of antibody titres in both Protein/Protein and DNA/protein groups. In their studies on prime boost immunization with Pf/Pv MSP1 19, priming of mice was done with single injection of DNA and boosting by giving two injections of protein, whereas in our studies we have primed the mice with two injection of PvMSP-142 plasmid DNA followed by boosting with single injection of PvMSP-142 recombinant protein.

Previous studies have shown that cell mediated immune responses, in addition to antibody responses, are required for protection against malaria (Hoffman et al., 1994; Sedegah et al., 1998; de Souza, 2014). Present study on measuring T cell responses in mice after DNA immunization and protein boosting exhibited that T cell proliferation was significantly enhanced in DNA/Protein and Protein/Protein groups. Elevated levels of cytokines (IL-2, IL12, and IFN-) and high titres of antigen specific IgG isotypes (IgG1 > IgG2b> IgG2a) indicated that both Th1 and Th2 subsets of T helper cells may be produced during immunization of mice in the present study. This type of responses, i.e., the activation of both Th1 and Th2 cells, is considered ideal for a blood-stage vaccine (Petritus and Burns, 2008; 17

de Souza, 2014). Our findings on DNA prime/protein boosting have also indicated that among the DNA immunized groups, priming with DNA and boosting with protein clearly elevated the levels of all the cytokine in DNA/Protein group suggesting that DNA can prime the mice but itself is not sufficient enough to enhance the immune responses.

5. Conclusions In the present study, we have cloned 42 kDa fragment of P. vivax merozoite surface protein1 (PvMSP-142) in eukaryotic expression vector pcDNA3.1. We checked the in vitro expression of PvMSP-142- pcDNA3.1 in COS-1 cell line. Indirect fluorescent assay and western blotting confirmed the expression of PvMSP-142 in COS-1 cells. In order to study the immunogenicity of recombinant vaccine plasmid, we immunized BALB/c mice with PvMSP-1 42-

pcDNA3.1 plasmid DNA and compared its immunogenicity with the purified recombinant

PvMSP-142 protein. PvMSP-142 plasmid DNA vaccine construct was immunogenic in mice, but the use of plasmid DNA alone may not be sufficient to elicit substantial immune responses. In order to increase the immunogenicity of PvMSP-142 plasmid DNA vaccine we used heterologous prime boosting with PvMSP-142 recombinant protein. Our results showed that the priming with DNA followed by boosting with recombinant protein significantly enhanced the immune responses of DNA/protein immunized mice. Conflict of interest statement The authors have declared that no competing interests exist. Contributors: NAK and DCK conceived and designed the experiments. IHS performed the experiments and wrote the manuscript. NAK, DCK and DC analyzed the data, provided valuable suggestions and modified the manuscript. Acknowledgements COS-1 cell line was a kind gift from Dr. Satish. K. Gupta, National Institute of Immunology, New Delhi, India. One of the authors (IHS) is grateful to the Council of Scientific and Industrial Research for providing fellowship during the course of study. This study was supported from the CSIR-CDRI network project grant (SPlenDID).

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Legends to Figures Fig. 1: Cloning of 42 kDa fragment of P. vivax MSP-1 in pcDNA 3.1. Lane 1 = BamH1Not1digested PvMSP-142-pcDNA 3.1; Lane 2 =PvMSP-142-pcDNA 3.1 vector; Lane 3 = 1 kb ladder

Fig. 2: Detection of P. vivax MSP-142 recombinant protein expression in COS-1 cells by Immunofluorescence assay using monoclonal antibody. COS-1 cells (1×105/well) grown in a 24-well tissue culture plate were transfected with either plasmid DNA encoding PvMSP142or pcDNA 3.1 vector as described in Material and Methods. After 48 h, transfected cells were fixed in chilled methanol and processed for detection of PvMSP-142 protein expression by indirect immunofluorescence assay using monoclonal antibody (Moab5) against conformational epitope of PvMSP-142 protein. Immunofluorescence patterns are shown: (a) COS-1 cells transfected with Vector pcDNA 3.1 (b) COS-1 cells transfected with PvMSP-142PcDNA plasmid DNA. Fig. 3: Immunoblotting of COS-1 cells transfected with P. vivax MSP-142-pcDNA3.1 plasmid using monoclonal antibody. Lane 1= protein molecular weight markers; Lane 2= whole cell lysate from COS-1 cells transfected with vector pcDNA3.1 plasmid. Lane 3= whole cell lysate from COS-1 cells transfected with PvMSP-142-pcDNA 3.1 plasmid DNA. The IgG fraction of anti-PvMSP-142 monoclonal antibody (Moab8, linear epitope) was used at 1:250 dilution.

Fig. 4: Humoral immune responses of different groups of immunized mice. (a) Titration curves and (b) 1OD titre of immune mice sera. The immunization schedule and prime boosting of four groups (DNA/DNA, DNA/Protein, Protein/Protein and control groups) of mice are described in Materials and Methods. The immune sera samples (days 42 and 52) were analyzed at different dilutions in ELISA against PvMSP-142 recombinant Protein. Data are expressed as mean±S.D. Fig. 5: Analysis of IgG subclass antibodies to P. vivax MSP-142 recombinant protein in sera of immunized mice of different groups. The immunization schedule and prime boosting of four groups (DNA/DNA, DNA/Protein, Protein/Protein and control groups) of mice are described in Materials and Methods. The IgG subclass levels were determined in sera of animals by ELISA as described in Materials and Methods.The bars represent mean ELISA OD490±S.D.of antibodies in each group of mice. Statistical significance (p < 0.05) was considered significant. 29

Fig.6: T cell proliferation responses of different groups of mice immunized P. vivax MSP-142 plasmid and recombinant protein. The immunization schedule and prime boosting of four groups (DNA/DNA, DNA/Protein, Protein/Protein and control groups) of mice are described in Materials and Methods. The immunized BALB/C mice were euthanized on day 52 and spleen cells harvested. Single cell suspensions of spleen cells (1×106 cells/well) were stimulated in vitro with recombinant protein PvMSP-142 (10 µg/ml) and processed for analysis of T cell proliferation as described in Material and Methods. The values are expressed as stimulation index (mean±S.E.) obtained from the analysis of three immunized mice.

Fig. 7: Cytokine levels in stimulated splenocytes supernatants from different groups of immunized mice. The immunization schedule and prime boosting of four groups (DNA/DNA, DNA/Protein, Protein/Protein and control groups) of mice are described in Materials and Methods. For measuring cytokine levels in the splenocytes cultured supernatants, 10 days after the last immunization, 3 mice fromeach group were sacrificed, their spleens removed and splenocytes were prepared, cultured for 72 h and supernatants were collected. The cytokines IFN- (a), IL-2 (b), IL-12 (c) were assayed using BD Bioscience mouse cytokine kit. Each bar represents the mean±SD of cytokine levels in triplicate wells. The p value (p< 0.05) was considered significant.

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