Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential

Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential

Vaccine xxx (2017) xxx–xxx Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Recombinant Calponin...

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Vaccine xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential Shiv K. Verma a,1, Ashish Arora b, P. Kalpana Murthy a,⇑ a b

Division of Parasitology, CSIR-Central Drug Research Institute, New Campus, BS 10/1, Sector 10, Jankipuram Extension, Lucknow 226 031, India Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, New Campus, BS 10/1, Sector 10, Jankipuram Extension, Lucknow 226 031, India

a r t i c l e

i n f o

Article history: Received 24 March 2017 Received in revised form 9 July 2017 Accepted 27 July 2017 Available online xxxx Keywords: Brugia malayi Calponin iNOS COX-2 Cytokines IgG and its subclasses LTT assay

a b s t r a c t In the search for potential vaccine candidates for the control of human lymphatic filariasis, we recently identified calponin-like protein, that regulates actin/myosin interactions, in a proinflammatory fraction F8 (45.24–48.64 kDa) of Brugia malayi adult worms. In the present study, the gene was cloned, expressed, and the recombinant Calponin of B. malayi (r-ClpBm) was prepared and characterized. r-ClpBm bears homology with OV9M of Onchocerca volvulus, a non-lymphatic filariid that causes loss of vision and cutaneous pathology. r-ClpBm was found to be a 45 kDa protein that folds into a predominantly a-helix conformation. The protective efficacy of r-ClpBm against B. malayi infection in Mastomys coucha was investigated by assessing the course of microfilaraemia and adult worm burden in the host immunized with r-ClpBm and subsequently infected with infective third stage larvae (L3). Expression of the Calponin was detected in all life stages (microfilariae, L3, L4, L5 and adults) of the parasite and immunization with r-ClpBm partially protected M. coucha against establishment of infection as inferred by 42% inhibition in parasite burden. Upregulated cellular proliferation, TNF-a, IFN-c, IL-1b, IL-4, nitric oxide (NO) release, expression of iNOS, and specific IgG, IgG1 and IgG2b in immunized animals correlated with parasitological findings. r-ClpBm immunization caused degranulation in majority of mast cells indicating possible involvement of mast cell products in reducing the parasite survival. It appears that complex mechanisms including Th1, Th2, NO and mast cells are involved in the clearance of infection. To the best of our knowledge this is the first report on cloning, expression of the gene and purification of r-ClpBm, determination of its secondary structure and its ability to partially prevent establishment of B. malayi infection. Thus, r-ClpBm may further be studied and developed in combination with other protective molecules of B. malayi as a component of potential filarial cocktail vaccine candidate. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Lymphatic filariasis (LF) caused by the nematode parasites, Wuchereria bancrofti, Brugia malayi and B. timori is one of the neglected tropical diseases transmitted by mosquitoes. LF is an incapacitating disease of tropical and subtropical areas affecting 120 million people in 73 countries and 1.4 billion people are at risk of acquiring the disease [1].

⇑ Corresponding author at: CSIR-Emeritus Scientist, Department of Zoology, Lucknow University, University Road, Lucknow 226007, India. E-mail addresses: [email protected], [email protected] (S.K. Verma), [email protected], [email protected], [email protected] (P.K. Murthy). 1 Present address: United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA.

Although mass drug administration of diethylcarbamazine/ ivermectin and albendazole in the endemic countries showed significant impact on morbidity control, the infection continues to spread in endemic countries [2]. An alternative strategy is development of a vaccine. Towards this direction several parasite molecules have been identified [3–6]. Vaccination with irradiated third stage larvae (L3) [7], some recombinant antigens [8] and a DNA vaccine [9,10] are being tried, but none could give a success story. Filarial parasites elicit a broad spectrum of immune and inflammatory responses in their hosts [11] and cytokine network plays an important role in the outcome of host’s responses [12]. We have shown that parasite molecules that stimulate pro-inflammatory cytokine release protected the host from filarial infection [5,13,14] and even leishmanial infections [15,16] via Th1/Th2 type responses. One of the pro-inflammatory fractions F8 (45.24– 48.64 kDa) of B. malayi also stimulated NO release and protected

http://dx.doi.org/10.1016/j.vaccine.2017.07.105 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105

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S.K. Verma et al. / Vaccine xxx (2017) xxx–xxx

the host against filarial infections via IFN-c induced iNOS induction [3]. 2DE MALDI-TOF-MS analysis of F8 revealed 3 proteins: Tropomyosin 1 (TM1), De novo peptides, and Calponin homolog OV9M [3]. TM1 are integral components of actin filaments that play a critical role in regulating the function of actin in muscle and nonmuscle cells while Calponin is a calcium binding protein involved in the regulation of muscle contraction and these proteins are immunogenic [17–19]. OV9M protein (Calponin) is present in patent and non-patent O. volvulus infections [17]. However, nothing is known about Calponin-like proteins of the B. malayi and whether it has any role in modulating the infection. In the present study we cloned the gene of Calponin homolog OV9M, expressed it and characterized the recombinant Calponin of B. malayi (r-ClpBm) by CD spectroscopy. We also investigated immunological responses of M. coucha to r-ClpBm and its effect on the infection establishment. 2. Materials and methods 2.1. Animals Healthy male M. coucha and jirds (Meriones unguiculatus) bred and maintained at National Laboratory Animal Centre (NLAC) of CSIR-Central Drug Research Institute (CDRI), Lucknow, India, were used in the present study in compliance with the Institutional Animal Ethics Committee guidelines (approval # IAEC/2010/140/ Renew 02 dated 16.5.2012). 2.2. Parasite B. malayi infection was cyclically maintained in the above two rodents using laboratory bred black eyed susceptible strain of Aedes aegypti mosquito as vector [20,21]. L3 was isolated from the infected mosquitoes whereas mf, L4, L5 and adults were harvested from peritoneal cavity of the infected jirds [22].

gene from TA vector was cloned in expression vector pTriEx-4 followed by isolation of plasmid using Quick Plasmid Mini Prep Kit (INVITROGEN, USA). The gene was transformed and expressed in competent cells of E. coli BL21-DE3. Positive colonies were screened by colony PCR and protein isolated from E. coli BL21-DE3. 2.4.1. Protein expression Five ml of overnight incubated primary culture of transformed E. coli BL21-DE3 cells containing pTriEx-4-Clp gene was added to 250 ml LB broth containing ampicillin and incubated at 37 °C in a shaker incubator (200 rpm). When the cell density reached an absorbance of 0.5–0.6 (mid-log phase) at 600 nm (A600), the culture was induced by isopropyl b-D-thiogalactoside (IPTG; 1.0 mM) and incubated for 16 h at 25 °C. The cells centrifuged at 3500g for 20 min and pellet obtained was suspended in lysis buffer (50 mM Tris, 250 mM NaCl, 10 mM imidazole, pH 8.0) containing 2 mM phenylmethyl sulfonylfluoride (PMSF; Sigma–Aldrich, St. Louis, USA) followed by incubation with 1 mg/ml lysozyme (Sigma–Aldrich, St. Louis, USA) for 30 min in ice. The suspension was pulse-sonicated in ice for 10  20 s at 30 s intervals and centrifuged as above. The protein in supernatant was confirmed by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [4]. 2.4.2. Protein purification The cell lysate obtained was applied to Ni-NTA Sepharose column and washed with 20, 50 and 80 mM imidazole prepared in 50 mM NaH2PO4, 300 mM NaCl, pH 7.8. No protein of interest was detected in the flow through. The histidine tagged r-ClpBm was eluted at 300 mM imidazole, desalted by dialysis (cut off: 10 kDa) and stored in 20% glycerol. The purity of r-ClpBm protein was checked in SDS-PAGE and transferred to nitrocellulose paper (NCP) [4]. r-ClpBm with anti-His antibody in western blot showed a single band (45 kDa). The purified r-protein was free from endotoxin [4].

2.3. RNA isolation and first-strand cDNA synthesis cDNA was synthesized from RNA extracted from B. malayi adult worms using RNA isolation kit (Sigma–Aldrich, St. Louis, USA) and cDNA synthesis Kit (Fermentas, USA) [22]. 2.4. Cloning, sub-cloning and sequencing of B. malayi cDNA Primer for Calponin gene consisting of EcoRI and SalI restriction enzyme site was designed using OLIGO analyzer program, according to the gene sequence submitted in B. malayi draft genome database of TIGR, J. Craig Venter Institute, and the complete Calponin protein gene was amplified by PCR [23]. Primers for amplification were: Forward:

50

CCCGAATTCAATGGAAACTCGAGTTGCAGGAC 0

30

(With EcoRI site +1 insertions); Reverse: 5 CCCGTCGACCTAAAGTCGATCAAGTGAAGCTTC 30 (With SalI site) (restricted sites underlined). PCR was carried out in thermo cycler (Bio-Rad, USA) under optimized conditions: denaturation (95 °C for 5 min), 30 cycles of amplification (95 °C for 30 s, 56.6 °C for 1 min, and 72 °C for 60 s), final extension at 72 °C for 10 min. Purified PCR products were ligated in TA vector pTZ57 R/T using Ins TA clone PCR kit (Fermentas, USA). Escherichia coli DH5-a competent cells were transformed by the TA-Calponin gene construct and plated on Luria Bertani (LB)-Ampicillin plates treated with X-gal. White colonies were screened and the sequence was confirmed (Xcelris Labs Ltd., Ahmedabad, India). Plasmid was isolated from TA clone using Quick Plasmid Mini Prep Kit (INVITROGEN, USA). Subsequently,

2.5. CD spectroscopy Secondary structure of r-ClpBm was determined by Far-UV CD measurements. The experiments were performed on a Jasco Spectropolarimeter model J-810. The instrument was calibrated with (+)-10-camphorsulfonic acid. The protein (5 lM) spectra were recorded with the protein samples in buffer (20 mM NaH2PO4, 50 mM NaCl, pH 6.5). Three scans were averaged for each spectrum. Isothermal wavelength scans were recorded in the range of 250–200 nm, with a path length of 2 mm, response time of 1 s, scan seed of 20 nm/min, and data pitch of 0.5. 2.6. Parasite extract preparations, fractionation by SDS-PAGE and western blotting Soluble somatic extracts of various life stages of the parasite were prepared [22], protein content estimated [24] and 20 mg protein of each extract was resolved by SDS PAGE in 10% acrylamide [25]. Prestained molecular weight marker (SDS7B; Sigma-Aldrich, St. Louis, USA) was run simultaneously. The resolved components were transferred to NCP (porosity: 0.22 lm; Millipore, India) [26]. 2.6.1. Immunoreactivity Immunoblot reactivity of Calponin in resolved components of various life stages of the parasite was ascertained using pooled serum of r-ClpBm immunized M. coucha [22].

Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105

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2.7. Immunization and challenge infection A total of 32 male M. coucha (8–10-week-old) were used. In the first experiment 12–16 animals in two groups were used, each group consisted of 6–8 animals. The method of immunization of animals was as described by Verma et al. [3]. Briefly, each animal received r-ClpBm (50 lg/animal) with Freund’s complete adjuvant (FCA) followed by two booster doses each having half the protein content (25 lg/animal) in FIA on days 14 and 21 post first immunization (p.f.i.). Control (non-immunized) animals received PBS +FCA/FIA only. Both the groups received 100 L3/animal on day 28/29 p.f.i. All the injections were given subcutaneously (s.c.) on the back below the nape of the neck. The animals were killed on day 139 post larval inoculation (p.l.i.) and parasite burden assessed. In the 2nd experiment, two groups (6–8 animals/group) received r-ClpBm or PBS as described above but the animals were not challenged with L3 infection. These animals were killed on day 28/29 p.f.i. and various immune parameters (cellular proliferation, Th1 and Th2 cytokines, NO release, and expression of iNOS and COX-2, serum IgG and its subclasses) and histological status of lymph node mast cells were analyzed. All the animals were killed by an overdose of ether anesthesia. 2.7.1. Estimation of microfilaraemia, worm recovery and fecundity Microfilaraemia was assessed in 10 ll tail blood on day 90 p.l.i. and thereafter at weekly interval till termination of the experiment. At autopsy (day 139 p.l.i.) adult worms isolated from heart, lungs, testes, lymph node and lymphatic vessels were examined under microscope. Percent change in mf count at each time point, adult worm-recovery and fecundity of female worms were calculated [27]. 2.8. Immune parameters 2.8.1. Preparation and culture of splenocytes/macrophages Suspension of splenocytes/peritoneal macrophages of M. coucha was prepared [14,28]. The cells were incubated with the r-ClpBm or LPS (1 lg/ml) or Con A (10 lg/ml) in vitro for 48/72 h at 37 °C in 5% CO2 atmosphere. 2.8.2. Nitric oxide determination NO in the form of nitrite was quantified in r-ClpBm or LPS stimulated culture supernatants of macrophages [29]. Triplicates were used for each sample. 2.8.3. Lymphocyte transformation test (LTT) Cellular proliferative response using splenocytes of M. coucha was assessed by LTT. The cells were stimulated in vitro with rClpBm or Con A for 72 h [14]. 2.8.4. Cytokine release assay Six paired mouse monoclonal antibodies to TNF-a, IL-4, IL-10, TGF-b (BD Pharmingen), IFN-c and IL-1b (Pierce Endogen, Rockford, IL, USA) and their respective standards were used. Cytokine release in 48 h culture supernatants of splenocytes stimulated with r-ClpBm or LPS was determined by sandwich ELISA following the manufacturer’s instructions. Triplicates were run for each sample. The concentration of the cytokines was calculated using O. D. readings for standards. TGF-b was expressed in O. D. as its standard was not commercially available.

2.8.5. Determination of IgG and its subclasses The specific IgG and its 4 subclasses were determined by indirect ELISA [5] using optimum concentration/dilution of r-ClpBm (0.1 lg/ml), sera (IgG: 1:200; subclasses: 1:50) and HRPconjugated rabbit anti-mouse-IgG (1:1000, Sigma-Aldrich, USA) 2.8.6. Determination of iNOS and COX-2 expression Semi quantitative reverse transcriptase (RT)-PCR was performed to assess expression of iNOS and COX-2 in lymph node (superficial, inguinal, axillary and retroperitoneal) cells of animals [30]. The primer sequences of mouse for iNOS and COX-2 [31,32] given in Table 1 were used to amplify the respective cDNA keeping HGPRT as housekeeping gene. The final products were visualized in gel documentation system and analyzed by Quantity oneÒ software (Bio-Rad, USA). 2.9. Histopathology Superficial, inguinal, axillary and retroperitoneal lymph nodes from animals immunized with r-ClpBm only were isolated, fixed and processed for paraffin sectioning and sections (3 lm) were stained with toluidine blue for mast cells. The numerical density and granularity status of mast cells were determined [13]. 2.10. Statistical analysis Results were presented as mean ± SD of data from 6 to 8 animals/group in two experiments and analyzed in GraphPad Prism 6.0 using Student’s‘t’ tests. Differences with P < 0.05 were considered significant. 3. Results 3.1. Cloning, expression and purification The protein was successfully cloned (Fig. 1A), expressed (Fig. 1B) and purified r-protein. A single band of 45 kDa represented r-ClpBm protein together with polyhistidine-tag (Fig. 1C). 3.2. Characteristics of secondary structure element in r-ClpBm (Fig. 2) The Far-UV CD spectrum is characterized by the presence of two minima at 208 nm and 222 nm, which indicate a predominant a-helical conformation. The folding state of r-ClpBm is predominantly a-helical and this closely matches to that of the NMR structure of natively folded Calponin homology (CH) domain [33]. 3.3. Expression of Calponin in different life stages of B. malayi Expression of the Calponin (42 kDa) was detected in all the parasite life stages (Fig. 3).

Table 1 Primer sequences of mouse for expression of iNOS and COX-2 by semi quantitative reverse transcriptase-PCR amplification. Gene (Accession No.)

Primer

iNOS (gi|146134510)

Forward: TCCTCACTGGGACAGCACAGAATG Reverse: GTGTCATGCAAAATCTCTCCACTGCC

COX-2 (gi|118130137)

Forward: GGAGAGACTATCAAGATAGTGATC Reverse: ATGGTCAGTAGACTTTTACAGCTC

HGPRT (gi|96975137)

Forward: CTCATGGACTGATTATGGACAGGAC Reverse: GCAGGTCAGCAAAGAACTTATAGCC

Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105

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Fig. 1. Cloning, expression and purification of recombinant Calponin. (A) SDS-PAGE of transform BL21-DE3 cells for determining the expression of protein. Lanes 1 and 2: cells of clone 1 and 2 respectively, transformed with recombinant plasmid and induced with 1.0 mM IPTG at 37 °C; Lane 3: Non-induced cells transformed with recombinant plasmid, (B) Chemiblot shows over expression of BL21-DE3 cells. Lane 1 and Lane 2: cells transformed with recombinant plasmid and induced with 1.0 mM IPTG at 37 °C, (C) SDS PAGE of affinity purified r-Calponin; Lanes 1–5 represents different purified fraction 1, 2, 3, 4 and 5 respectively; Lane 6: whole induced cell lysate; MW (kDa): Molecular weight marker. The best expression was observed in BL21-DE3 soluble fraction at 1.0 mM IPTG induced for 4 h at 37 °C. r-Calponin was purified successfully and represented a single band of 45 kDa. Abbreviation: r-Calponin (recombinant Calponin).

Fig. 2. Far-UV spectra of recombinant Calponin. The CD measurements were performed and determined the secondary structure of r-Calponin. Each spectrum was average of three scans. The protein was folded into a predominantly a-helix conformation.

3.4. Effect of immunization with r-ClpBm on responses of the host 3.4.1. Parasite burden r-ClpBm caused: 1. inhibition (67.60–83.62%; P < 0.01; Fig. 4A) in microfilaraemia till the day 111 p.l.i. but thereafter mf counts were comparable to that of control animals, 2. 42% reduction in worm recovery (P < 0.01; Fig. 4B) and 3. no alteration in fecundity.

3.4.2. Cellular proliferative response Con A-induced proliferative response of splenocytes from r-ClpBm-immunized animals was greater (>3.4 times) than r-ClpBm-induced response (Fig. 5A). 3.4.3. Cytokine release Immunization resulted in upregulated r-ClpBm-induced IFN-c (P < 0.05; Fig. 5B), TNF-a (P < 0.01; Fig. 5C), IL-1b (P < 0.001;

Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105

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Fig. 3. Immunoreactivity of Calponin of various life stages of Brugia malayi. Immunoreactivity of NCP bound fractionated antigens derived from various life stages of B. malayi with pooled serum of (Lane 1) non-immunized and (Lane 2) rClpBm immunized Mastomys coucha. Mf: microfilariae; L3: Infective third larval stage; L4: Fourth larval stage; L5: Fifth larval stage; BmA: B. malayi adult stage, MW: molecular weight marker. 42 kDa band represents Calponin protein expression in different life stages of the parasite.

Fig. 5D) and IL-4 (P < 0.01; Fig. 5E) but downregulated IL-10 (P < 0.01; Fig. 5F) and TGF-b (P < 0.001; Fig. 5G) release. 3.4.4. IgG and subclasses Specific IgG1 level (P < 0.001) was the highest followed by IgG (P < 0.001) and IgG2b (P < 0.01), IgG2a level was low (P < 0.01) and IgG3 remained unaltered (Fig. 5H). 3.4.5. NO generation and expression of iNOS and COX-2 r-ClpBm significantly stimulated NO production (P < 0.001; Fig. 5I). iNOS expression in immunized animal cells was significantly high but COX-2 was suppressed (Fig. 5L). 3.4.6. Histopathology Lymph nodes of non-immunized animals showed richly granulated mast cells whereas nodes of immunized-uninfected animals demonstrated large number of degranulated mast cells (Fig. 5J). However, there was no change in mast cell numerical density (Fig. 5K). 4. Discussion Protective immunity of the host against filarial parasites or their molecules is a complex process involving several components of immune bodies including a variety of mediators/effector molecules [13,34]. Our recent study showed that F8, a proinflammatory fraction of B. malayi which contains tropomyosin, Calponin and de novo peptides protected the host against L3 establishment via IFN-c mediated iNOS induction [3]. In this study we intended to find out whether r-ClpBm can also adversely affect the parasites. The major findings of the present study are: (i) The protein was successfully cloned, expressed, and purified r-ClpBm represented a single band of 45 kDa, (ii) r-ClpBm was found to be folded into a predominantly a-helix conformation, (iii) the protein was expressed in all life stages of the parasite, (iv) r-ClpBm partially prevented establishment of infection, (v) r-ClpBm immunization resulted in upregulated responses of cellular proliferation, TNF-a, IFN-c, IL-1b, IL-4, IgG1, IgG2b, NO, and iNOS expression and downregulated IL-10, TGF-b and IgG2a and COX-2 expression, and (vi) rClpBm immunization caused degranulation in majority of mast cells. Host’s CMI responses play a major role in containing or eliminating filarial parasite [35]. In this study we found that increase in cell proliferation in immunized animals was relatable to parasite

Fig. 4. Effect of immunization with r-ClpBm on parasite burden in Mastomys coucha. A total of 12–16 animals (8–10-week-old) in two groups were used, each group consisted of 6–8 animals. The 1st group was immunized with 3 doses of rprotein, the first dose (50 lg protein/animal) in Freund’s complete adjuvant (FCA) and the rest two doses (25 lg protein/animal) in Freund’s incomplete adjuvant (FIA) on days 14 and 21 post first immunization (p.f.i.). The 2nd group (nonimmunized/control) received PBS + FCA/FIA only. On day 28/29 p.f.i. both the groups received 100 L3/animal. All the injections were given through subcutaneous (s.c.) route on the back below the nape of the neck. (A) Course of microfilaraemia. Microfilariae (mf) count in 10 ll tail blood was assessed on day 90 post L3 inoculation (p.l.i.) and thereafter at 7 days intervals till day 139 p.l.i. All the animals were killed on day 139 p.l.i. by an overdose of ether anesthesia. (B) Adult worm burden. The parasite recovered from different sites (heart, lungs, testes, lymph nodes and lymphatics) were counted and percent recovery of the worms was calculated. Abbreviations: r-ClpBm (r-Calponin of B. malayi), Non-Im + L3 (NonImmunized + L3 infection), r-ClpBm-Im + L3 (r-Calponin-Immunized + L3 infection). Values are mean ± SD of 6–8 animals/group from two experiments. Statistics: Student’s ‘t’ test. **P < 0.01 (vs Non-Im + L3).

elimination. Activation of macrophages by IFN-c leads to either antibody dependent cytotoxicity, resulting in the release of reactive oxygen intermediates or directly by production of reactive nitrogen intermediates. NO has been shown to be involved in killing of mf in vitro and L3 in vivo [36,37]. Although we did not find a strong increase in IFN-c release from immunized animals but IFNc in combination with upregulated TNF-a and IL-1b appears effective in inducing NO production and suggesting the role of Th1 cells in iNOS induction. This clearly suggests that IFN-c mediated NO response may be responsible for reduced parasite burden. Endemic normal and chronic filarial cases have shown elevated levels of IFN-c and IL-2 [38,39]. Parasite products increased the expression of enzymes related to inflammation, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The synthesis of COX-2 was coordinated with induction of nitric oxide synthase (NOS)-2 and NO production [40]. Swierkosz et al. [41] have shown that iNOS and COX2 co-induced in vitro using LPS activated J774.2 macrophage cells produced an enormous amount of NO which inhibited COX-2 expression and activity.

Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105

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Fig. 5. Effect of immunization with r-ClpBm on immune parameters and histological status of lymph node mast cells in M. coucha. Two groups (6–8 animals/group) of male M. coucha of 8–10-week-old were immunized with r-ClpBm or PBS. The 1st and 2nd groups received 3 doses of r-ClpBm and PBS, respectively, as described above, but the animals were not challenged with L3 infection. All the animals were killed on day 28/29 p.f.i. (A) Cellular proliferative response. Proliferative response of splenocytes from the two groups to r-ClpBm (1 lg/ml) or Con A (10 lg/ml) for 72 h at 37 °C in 5% CO2 atmosphere, was determined by LTT assay. The cells were pulse labeled with [3H]-thymidine (1 mCi/well) and incubated for 18 h followed by counting of thymidine incorporated cells in scintillation b counter and results were expressed as cpm. Release of cytokines: Splenocytes of animals were stimulated with r-ClpBm (1 lg/ml) or LPS (1 lg/ml) for 48 h in vitro as stated above and the release of (B) IFN-c, (C) TNF-a, (D) IL-1b, (E) IL-4, (F) IL-10, and (G) TGF-b was assessed by sandwich ELISA. (H) Specific IgG and its subclasses in sera of animals. IgG and its subclasses were assayed in an indirect ELISA using rClpBm protein (0.1 mg/ml), primary antibody (IgG: 1:200; IgG subclasses: 1:50) and HRP-conjugated secondary antibody and rabbit anti-mouse-IgG/subclasses (1:1000). (I) NO production. Splenocytes from animals of the two groups were incubated with r-ClpBm (1 lg/ml) or LPS (1 lg/ml) for 48 h at 37 °C in 5% CO2 atmosphere. NO release was quantitated as nitrite in the culture supernatant. (J) Percent granulated and degranulated mast cells in lymph nodes: Superficial, inguinal, axillary and retroperitoneal lymph nodes of animals were fixed and processed for paraffin sectioning and stained with toluidine blue and the number of cells that were fully granulated and those showing at least 50% loss in granularity were counted to calculate% degranulated cells. (K) Numerical density of mast cells: The total number of mast cells in lymph node sections was determined and expressed as no. of cells/mm2. (L) Expression iNOS and COX-2 in cells of animals. Cells from lymph nodes (superficial, inguinal, axillary and retroperitoneal) of r-ClpBm immunized/non-immunized animals were isolated and mRNA expression of iNOS and COX-2 was determined by semi quantitative RT-PCR. HGPRT was used as housekeeping gene (control). Abbreviations: US (unstimulated), r-ClpBm (r-Calponin of B. malayi), Non-Im (Non-Immunized), r-ClpBm-Im (r-Calponin-Immunized). Values are mean ± SD of 6–8 animals from two experiments; Statistics: Student’s ‘t’ test. *P < 0.05, **P < 0.01, ***P < 0.001 (vs Non-Im).

In murine system, IgG2a antibody isotype is indicative of Th1 response, whereas IgG1 and IgG2b isotypes are associated with Th2 response [42]. In this study, of the three Th2 cytokines only IL-4 (a signature cytokine of Th2) was increased in r-ClpBmimmumized group. IL-4 is known to induce IgG1 [43] and the increase in IgG1 levels in this group is thus relatable to IL-4. In addition, the increased IgG2b (Th2 type) and decreased IgG2a (Th1 type) further fortified the Th2 milieu. During high cellproliferative responses, lower levels of antibodies are produced and thereby the antibody responses during Th1 response synergize the cellular components for more effective clearance of the pathogen [44]. Increased IgG1 and IgG2b may be involved in parasite

elimination via ADCC mechanism. Therefore, we presume that parasite removal may have occurred due to iNOS induction or ADCC or both the mechanisms. Together these findings apparently suggest that both Th1 and Th2 and NO responses are involved in the clearance of infection. However, the% parasite survival was higher in rClpBm-immumized group compared to F8-immunized animals [3] and this could be due to other immunogenic proteins in F8. Mast cell products play an important role in protection of host against filarial parasite [13]. r-ClpBm immunization-induced degranulation of mast cells suggests that mast cell products may have affected survival of the parasite. Taken together, the present study shows that ClpBm is an interesting immunoprophylactic protein,

Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105

S.K. Verma et al. / Vaccine xxx (2017) xxx–xxx

but as its efficacy is only moderate, it may best be explored as a component of ‘cocktail’ vaccine candidates. 5. Conclusion In conclusion, ClpBm was successfully cloned, expressed, and the recombinant protein purified. A single band at 45 kDa represented the r-protein. It was folded into a native-like, predominantly a-helix conformation and ClpBm gene expression was detected in all the life stages of the parasite. Immunization with r-ClpBm partially prevented (42%) infection establishment in M. coucha. Increased cellular proliferation, TNF-a, IFN-c, IL-1b, IL-4, NO release, iNOS expression, and specific IgG, IgG1, IgG2b in immunized animals correlated with parasitological findings. Immunization with r-ClpBm caused degranulation in majority of mast cells indicating possible involvement of mast cell products in reducing the parasite survival. It appears that complex mechanisms including Th1, Th2, NO and mast cells are involved in the clearance of infection. This is the first report on cloning, expression, and purification of r-ClpBm, its secondary structure and its ability to partially prevent establishment of B. malayi infection. Thus, r-ClpBm may further be studied and developed in combination with other protective molecules of B. malayi as a component of potential filarial cocktail vaccine candidate. Acknowledgements Thanks are due to Dr. Madhu Dikshit, Director, CSIR-CDRI, Lucknow, for her encouragement and providing facilities. This study was supported in part by the Network project SPLENDID of Council of Scientific and Industrial Research, New Delhi, India. Thanks are due to CSIR, New Delhi for award of Emeritus Scientist scheme to PKM. Thanks are also due to Vice Chancellor, Lucknow University, Lucknow, for providing office/laboratory space and computational facilities to corresponding author (PKM) for carrying out part of the study and manuscript preparation at Department of Zoology. Authors thank UGC (SKV) for receiving Senior Research Fellowship. This manuscript bears CDRI communication number 9535. Author Contributions Conceived and designed the experiments: PKM, AA. Performed the animal experiment: SKV. Biophysical study carried out: AA. Analyzed the data: PKM, AA, SKV. Wrote the paper: PKM, AA, SKV. Conflict of Interest The authors have declared that no conflict of interests exists. References [1] Rebollo MP, Bockarie MJ. Toward the elimination of lymphatic filariasis by 2020: treatment update and impact assessment for the endgame. Expert Rev Anti Infect Ther 2013;11:723–31. [2] Addiss DG, Brady MA. Morbidity management in the global programme to eliminate lymphatic filariasis: a review of the scientific literature. Filaria J 2007;6:2. [3] Verma SK, Joseph SK, Verma R, Kushwaha V, Parmar N, Yadav PK, et al. Protection against filarial infection by 45–49 kDa molecules of Brugia malayi via IFN-gamma-mediated iNOS induction. Vaccine 2015;33:527–34. [4] Kushwaha V, Kumar V, Verma SK, Sharma R, Siddiqi MI, Murthy PK. Disorganized muscle protein-1 (DIM-1) of filarial parasite Brugia malayi: cDNA cloning, expression, purification, structural modeling and its potential as vaccine candidate for human filarial infection. Vaccine 2014;32:1693–9. [5] Sahoo MK, Sisodia BS, Dixit S, Joseph SK, Gaur RL, Verma SK, et al. Immunization with inflammatory proteome of Brugia malayi adult worm induces a Th1/Th2-immune response and confers protection against the filarial infection. Vaccine 2009;27:4263–71.

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Please cite this article in press as: Verma SK et al. Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.07.105