Immunization with a multisubunit vaccine considerably reduces establishment of infective larvae in a rodent model of Brugia malayi

Comparative Immunology, Microbiology and Infectious Diseases 36 (2013) 507–519

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Immunization with a multisubunit vaccine considerably reduces establishment of infective larvae in a rodent model of Brugia malayi Nidhi Shrivastava 1 , Prashant Kumar Singh 1 , Jeetendra Kumar Nag, Susheela Kushwaha, Shailja Misra-Bhattacharya ∗ Divisions of Parasitology, Central Drug Research Institute (CSIR), Jankipuram Extension BS10/1, Sector 10, Sitapur Road, Lucknow 226021, UP, India

a r t i c l e

i n f o

Article history: Received 7 June 2012 Received in revised form 20 February 2013 Accepted 3 May 2013 Keywords: BmAF-Myo Bm-TPP Recombinant protein Expression

a b s t r a c t Although recombinant vaccines have several advantages over conventional vaccines, protection induced by single antigen vaccines is often inadequate for a multicellular helminth parasite. Therefore, immunoprophylactic efficacy of cocktail antigen vaccines comprised of several combinations of three Brugia malayi recombinant proteins BmAF-Myo, Bm-iPGM and Bm-TPP were evaluated. Myosin + TPP and iPGM + TPP provided the best protection upon B. malayi infective larval challenge with ∼70% reduction in adult worm establishment over non-vaccinated animals that was significantly higher than the protection achieved by any single antigen vaccine. Myosin + iPGM, in contrast did not provide any enhance protection over the single recombinant protein vaccines. Specific IgG, IgM level, IgG antibody subclasses levels (IgG1, IgG2a, IgG2b, IgG3), lymphocyte proliferation, reactive oxygen species level and cytokines level were also determined to elucidate the characteristics of the protective immune responses. Thus the study undertaken provided more insight into the cocktail vaccination approach to combat LF. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Lymphatic filariasis (LF) is a mosquito-borne neglected tropical disease caused by Wuchereria bancrofti, Brugia malayi and Brugia timori. More than 1.3 billion people in 81 countries worldwide are threatened by LF. Disease pathogenesis is linked to host inflammation invoked by the death of the parasite, causing hydrocele, lymphoedema, and elephantiasis [1]. The current efforts for controlling LF depend

∗ Corresponding author. Tel.: +91 522 2612411 18x4221/4; fax: +91 522 2623405/3938/9504. E-mail addresses: shailja [email protected], [email protected] (S. Misra-Bhattacharya). 1 Both these authors have equal authorship. 0147-9571/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cimid.2013.05.003

mainly on the use of drugs diethylcarbamazine, albendazole and ivermectin which are principally microfilaricidal without much affecting the adult worms, therefore necessitating repeated administrations. Furthermore, signs of emerging drug resistance are becoming increasingly apparent, especially against albendazole and ivermectin [2–4]. The antiwolbachial targeting with antibiotic is also not suitable for mass administration since macrofilaricidal activity requires continuous weeks’ long treatment. Therefore, discovery of a new macrofilaricidal drug or a potent vaccine would be the appropriate complementary approaches. In LF, each parasite stage in the mammalian host interacts with an immunologically distinct compartments, therefore, immune response elicited by each stage has its own distinctive features. Exposure to microfilariae (mf) induces an inflammatory type 1 response whereas infective

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larve (L3) and adults induce primarily anti-inflammatory type 2 responses. Aggressive immune responses to filarial nematodes occur in some individuals, resulting in chronic pathology, such as elephantiasis. However, the majority of individuals, although having detectable microfilariae in their bloodstreams, are otherwise apparently asymptomatic and have been described as being immunologically tolerant to the parasite. This is thought to be the result of immunomodulation to achieve a situation conducive to both parasite survival and host health. Such people often have dramatically increased levels of immunoglobulin G4 (IgG4) and interleukin-10 (IL-10), with reduced interferon␥ (IFN-␥) production demonstrating the Th2 response. Endemic normal individuals have been reported to mount a Th1-like antifilarial immune response. These observations had led to the conclusion that both the Th1- and the Th2type responses contribute to effector mechanisms, thus limiting parasite loads, whereas the concomitant suppression of both these responses has to be achieved in order to allow for high parasite loads associated with a low degree of damage to the host. Therefore, developing preventive strategies based on both Th1 and Th2 arms of the immune system appears reasonable. Attempts have been made to use single recombinant protein [5–8]. However, even in the most successful individual cases, the protection levels obtained were much inferior to that of live attenuated vaccines. Filarial parasites are antigenically complex organism and the immune responses induced by single antigen immunization may not be sufficient to combat the challenge infection. This suggests that development of a cocktail antigen vaccine may be the way forward. Vaccination studies using more than one antigen showed promising results in parasitic diseases such as malaria, leishmaniasis, schistosomiasis and onchocerciasis [9–12]. Few studies have been also reported in the case of LF where cocktail of B. malayi recombinant proteins were used. One of these was reported by Vanam et al. [13] where three B. malayi recombinant proteins, viz. Bm-TPX (thioredoxin), Bm-TGA (transglutaminase) and Bm-ALT-2 (abundant larval transcript) were administered in various combinations and BmTPX + BmTGA provided better degree of protection as compared to the single antigen immunization. Similarly, a recent study by Anand et al. [14] showed that BmALT-2 and BmVAH (Vespid Allergen Homologue) when given as a cocktail vaccine can confer significant protection (80%) as compared to either of these single protein. Therefore, in the present study, an attempt has been made to investigate the immunoprophylactic potential of B. malayi recombinant proteins in cocktails comprised of B. malayi adult female heavy chain myosin (BmAF-Myo), independent phosphoglycerate mutase (BmiPGM) and trehalose-6-phosphate phosphatase (Bm-TPP). B. malayi myosin was selected on the basis of its high reactivity with endemic normal human sera from bancroftian endemic area [15]. It is a body wall muscle protein and recently its presence has been shown in excretory secretary product of adult worms [16]. Besides, myosin has been investigated as a vaccine candidate in a number of nematode parasite [17,18]. Excretory-secretary molecules being secreted on host parasite interface are entirely accessible to the host immune system. Bm-iPGM is an enzyme

involved in interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic/gluconeogenic pathway. It is not reported from mammals and has a sequence and structure different from the 2,3-bisphosphoglyceratedependent phosphoglycerate mutase (dPGM) found in mammals. Bm-iPGM is also secreted by adult Brugia worms [19]. Thus, these two immunogenic and secretary proteins of B. malayi were included as effective recombinant proteins in a multivalent subunit vaccination approach. In addition, Bm-TPP was also included as a component of protein cocktail. TPP is a crucial enzyme in trehalose metabolism. Trehalose disaccharide is an abundant storage sugar in the filarial nematodes that serves as an energy reserve as well as a stress protectant. TPP is also absent in mammals. The present study aims at investigating the possibility of using a cocktail vaccine against lymphatic filariasis using three recombinant proteins of B. malayi, BmAF-Myo, Bm-iPGM and Bm-TPP. 2. Materials and methods 2.1. Parasite–host The experimental animals used in the current study were Mastomys coucha, which were maintained under appropriate housing conditions at Laboratory Animal Facility of our Institute. All the animals and experimental protocols involving animal handling were duly approved by the Institutional Animal Ethics Committee (IAEC). B. malayi infective larvae (L3) for challenge experiments were recovered from the laboratory bred vector mosquitoes (Aedes aegypti) fed on donor M. coucha 9 ± 1 day back [20]. 2.2. Overexpression and purification of recombinant BmAF-Myo cDNA coding for B. malayi BmAF-Myo was picked up earlier [15] by immunoscreening of adult female B. malayi cDNA expression library with human bancroftian sera and subsequently subcloned in pET28b expression vector followed by transformation into DH5␣ Escherichia coli cells (Qiagen). The recombinant plasmid was further transformed into BL21 (DE3) E. coli cells (Qiagen) and grown in 5 ml Luria–Bertani (LB) medium overnight (O/N) in the presence of 50 ␮g/ml of kanamycin at 37 ◦ C with 220 rpm shaking. This O/N grown culture was inoculated in 500 ml of LB medium. The culture was grown in a shaker incubator with constant stirring till A600 reached ∼0.6. The culture was induced with 0.5 mM isopropyl␤-d-thiogalactopyranoside (IPTG; Sigma) and cells were re-incubated for another 3 h at 30 ◦ C for overexpression of protein containing His tag at the N-terminal. Induced bacterial cells were harvested and disrupted in buffer A (50 mM NaH2 PO4 , 300 mM NaCl, pH 6.5) containing 10 mM imidazole and 1.0 mM phenylmethylene sulfonate (PMSF) as protease inhibitor. Cells were disrupted by sonication and pelleted at 12,000 × g for 30 min. Subsequently, the supernatant containing the histidine-tagged recombinant protein was subjected to affinity purification through nickel nitrilotriacetic acid agarose affinity column (Ni-NTA Column, Qiagen). After washing with buffer-A containing

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10 mM, 20 mM or 50 mM imidazole, protein was eluted by buffer-A containing 250 mM imidazole. Eluates were collected as 1 ml fractions which were then analyzed on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing purified protein were pooled and dialyzed O/N against 50 mM NaH2 PO4 at 4 ◦ C and the protein concentration was assayed by the Bradford method. 2.3. Overexpression and purification of recombinant Bm-iPGM For overexpression of recombinant Bm-iPGM, BL21(DE3) E. coli cells containing Bm-iPGM-pET28a construct were grown in 5 ml LB medium O/N in the presence of 50 ␮g/ml of kanamycin at 37 ◦ C with 220 rpm shaking. This O/N grown culture was inoculated into 500 ml of LB medium and grown till A600 reached ∼0.6. 1.0 mM IPTG (Sigma, USA) was added and incubated for 5 h at 37 ◦ C. The cells were pelleted, resuspended in 50 mM NaH2 PO4 (pH 8.0) containing 300 mM NaCl, 1.0 mM PMSF and 10 mM imidazole. Cells were disrupted by sonication and centrifuged at 12,000 × g for 30 min. The supernatant was aspirated and loaded on the Ni-NTA agarose affinity column. The column was subsequently washed with the same buffer containing 25 mM and 50 mM imidazole and the recombinant protein was finally eluted with 250 mM imidazole [21]. The purity of eluted protein was analyzed on 10% SDS-PAGE. Purified protein was dialyzed and protein concentration evaluated as described for BmAF-Myo. 2.4. Overexpression and purification of recombinant Bm-TPP Bm-TPP gene was PCR amplified using cDNA prepared from the adult worms of B. malayi, subcloned in pET28a(+) expression vector, transformed in BL21 E. coli cells. Cells were grown, recombinant protein over-expressed and purified as published earlier [22]. 2.5. Antigenicity and epitope prediction in silico The antigenicity of all the three proteins were determined using Kolaskar’s method; available at http://imed.med.ucm.es/Tools/antigenic.pl [23]. The ProPred algorithm (www.imtech.res.in/raghava/propred/) was used to predict the presence of promiscuous human major histocompatibility complex (MHC) class II-restricted epitopes [24]. Promiscuous epitopes are defined as peptides that are predicted to bind to at least 80% of the MHC class II molecules expressed by the 51 human leukocyte antigen (HLA) subregion DR alleles available for analysis in the ProPred algorithm. The threshold for the ProPred analyses was set at a relatively high stringency of 3%. B cell epitopes were identified by BcelPred [25].

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Table 1 Summarized table for the adult worm recovery, female worm sterilization in various immunized and control groups. S.No.

Immunization groups

Percentage reduction in adult worm

Percentage female worm sterilization (mean ± SE)

1 2 3 4 5 6 7 8

Myosin iPGM TPP Myosin + TPP iPGM + TPP Myosin + iPGM PBS Adjuvant

50.0% 52.1% 37.5% 70.8% 69.8% 51.8%

57.81 37.55 50.06 57.33 56.43 74.86 13.93 15.15

a b

± ± ± ± ± ± ± ±

8.5a 10.3 9.0a 6.2a 4.9a 2.9a , b 6.4 2.7

p < 0.01 values significantly different from PBS and adjuvant controls. p < 0.05 values significantly different from iPGM immunized group.

6- to 8-week-old animals (∼50 g) were divided into eight groups each consisting of five mastomys. Three groups of rodents were immunized with recombinant BmAFMyo, Bm-TPP and Bm-iPGM, respectively, while other 3 groups received various combinations of these proteins as summarized in Table 1. Each protein was administered at 12.5 ␮g/dose/animal either as a single recombinant or in combination with other recombinant protein. Proteins were administered with Freund’s complete adjuvant (FCA) in the first dose (day 0) and two subsequent booster doses (days 14 and 21) were administered with Freund’s incomplete adjuvant (FIA) via subcutaneous (s.c.) route. The two groups of animals served as phosphate buffer saline (PBS) and adjuvant control groups. One week following final booster, all groups of mastomys were challenged subcutaneously with 100 L3. The experiment was repeated using the same number of mastomys and groups. 2.7. Effect of immunization on microfilaraemia, adult parasite recovery and female worm fecundity The observation of microfilaraemia in 10 ␮l tail blood was initiated from day 90 post-challenge (p.c.) and followed till day 180. Mastomys were euthanized on day 180 p.c. for assessing the recovery of adult worms and investigating the immune response generated against vaccine antigens in the immunized host. Various tissues, viz. heart, lungs, testes were taken out and gently teased in 0.85% saline to isolate and innumerate the adult worms. Female worms were teased and observed microscopically to assess the effect of immunization on worm fecundity. Arithmetic means were calculated for the total worm burden and the percentage protection was calculated as [(u − v)/u × 100], where u is the mean value for the control group and v is the mean value for the experimental group. Female worm sterilization was calculated as number of sterile worms/total worm teased × 100. 2.8. Measurement of specific IgG, IgM levels and IgG antibody subclasses in the sera by enzyme-linked immunosorbent assay (ELISA)

2.6. Immunization and challenge M. coucha is a susceptible host for B. malayi infection and therefore used in the immunization experiment.

Blood for sera was collected from the retro-orbital plexus of animals on days 0, 15, 30 prechallenge and at 1 month, 3 months and 6 months p.c. to measure serum IgG

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[18]. In brief, ELISA plates were coated with 1.0 ␮g/ml solution of protein by adding 100 ␮l/well and blocked with 1% gelatin in PBS–T (0.05% Tween-20 in PBS). Serum samples of mastomys immunized with individual as well as cocktail of proteins were used as primary antibody and anti mouseIgG-HRP as secondary antibody (Sigma). The absorbance was read at 492 nm by a multiplate reader (Tecan T200, Switzerland) after adding substrate, orthophenylenediamine (OPD) and stopping the reaction by addition of 20 ␮l of H2 SO4 . The subclass distribution of the IgG reflects the functional characteristics of the induced humoral immunity [26]. Antigen specific IgG1, IgG2a, IgG2b, IgG3 and IgM level were also measured in the mastomys sera as mentioned earlier [12]. Briefly, ELISA plates were coated with 0.1 ␮g/ml of proteins followed by blocking with 1% gelatin in PBS–T. Mastomys sera were added at 1:100 dilution, followed by addition of 1:1000 dilution of monoclonal antibodies to IgG subclasses (goat anti-mouse IgG1, IgG2a, IgG2b, IgG3) and IgM. Finally, rabbit anti goat-IgG-HRP were added at 1:5000 dilution.

2.9. Measurement of Th1/Th2 cytokines Intracellular cytokines in the spleen cells were estimated as described earlier [27]. Briefly, splenocytes from various immunized and control groups (4 × 106 /ml) were incubated with Brefeldin A (10 ␮g/ml) in dark for 4 h in CO2 incubator at 37 ◦ C and after washing, cells were incubated in dark at 4 ◦ C in the presence of fluorescein isothiocynate (FITC) labelled CD4+ antibody and washed with PBS. The cells were fixed with Leucoperm A, permeabilized in the presence of Leucoperm B for 15 min and dispensed in four tubes each containing 1 × 106 cells/100 ␮l. Phycoerythrin (PE) labelled rat antimouse monoclonal antibodies to various cytokines IL-2, IL-4, IL-10 and IFN-␥ were added and cells were incubated for 15 min in dark. Cells were washed and suspended in 250 ␮l of 0.5% paraformaldehyde for fluorescence activated cell sorter (FACS) readings.

2.10. Reactive oxygen species content in peritoneal macrophages Peritoneal exudate cells from all the immunized and control groups of mastomys were collected after introducing cold RPMI medium (Gibco) in the peritoneal cavity through sterile syringe. Cells were pelleted and macrophages were allowed to adhere at the bottom of the wells in 96-well culture plate as mentioned earlier [28]. A real-time monitoring of intracellular reactive oxygen species (ROS) was carried out by fluorometric assay using 2 ,7 -dichlorofluorescin diacetate (DCF-DA) as described earlier [18,29] by probing 1 × 106 cells with DCF-DA at 1 ␮M followed by incubation for 15 min at 37 ◦ C. ROS levels in individual living cells were determined on FACS Calibur (Becton Dickinson, USA) and data was analyzed by CellQuest Software (Becton Dickinson, USA) to obtain mean ROS values.

2.11. In vitro proliferation of splenic lymphocytes The proliferation of lymphocytes was assessed using single cell suspension of splenocytes from all the immunized and control groups in response to BmAF-Myo, Bm-TPP or Bm-iPGM antigens, either tested singly or as a different cocktail used in the study. Concanavalin A (ConA, T cell mitogen; Sigma) and Lipopolysaccharide (LPS, B cell mitogen; Sigma) were used as a positive control of lymphocyte proliferation. Spleens were isolated aseptically and single cell suspensions were prepared as mentioned earlier [30]. Splenocyte suspension (100 ␮l/well) from stock (5 × 106 cells/ml) was plated in 96 well Nunc culture plate in triplicate. The splenocytes were proliferated with three different conditions—unstimulated (medium (RPMI1640 + 10% foetal calf serum; Gibco), ConA (2.5 ␮g/ml; Sigma) stimulated, LPS stimulated (2.5 ␮g/ml; Sigma) and protein stimulated (2.5 ␮g in case of single antigen or 1.25 + 1.25 ␮g in case of dual antigen), and incubated for 72 hours at 37 ◦ C in a CO2 incubator and pulsed with 1.0 ␮Ci/well of [3 H]thymidine (specific activity 18 Ci/mmol; BARC, India) for 18 h preceding harvest. Radioactive incorporation was measured by standard liquid scintillation counting (Beckman Instruments, Palo Alto, CA) and stimulation index (SI) was calculated. 2.12. Statistical analysis Data were analyzed by Dunnett’s multiple comparison test using one-way analysis of variance (ANOVA) with the help of statistical software PRISM 3.0. p < 0.05 was considered as significant and p < 0.01 as highly significant. 3. Results 3.1. All the three recombinant proteins were purified by Ni-NTA column All the three recombinant proteins were overexpressed by IPTG and subsequently purified by Ni-NTA column. BmAF-Myo, Bm-TPP and Bm-iPGM was eluted in phosphate buffer having 250 mM imidazole. The purified recombinant proteins were obtained as a single band on 10% SDS-PAGE with molecular masses of ∼73 kDa for BmAF-Myo, ∼60 kDa for Bm-TPP and Bm-iPGM (Fig. 1A–C). 3.2. Myosin + TPP and iPGM + TPP protein cocktails offered best protection against L3 challenge Adult worm burden in different groups of mastomys is shown in Fig. 2. Adult worm burden in all the protein immunized groups were significantly lower (p < 0.01) as compared to the PBS or adjuvant controls. Myosin + TPP and iPGM + TPP produced maximum protective effect in terms of adult worm establishment. Adult worm burden in these groups were significantly lower (p < 0.01) than PBS or adjuvant controls as well as Bm-TPP immunized group (p < 0.05). A reduction of 70.8% and 69.8% in the adult parasite recovery was noticed in these two groups respectively as compare to the PBS control. Immunization with single proteins in contrast had moderate adverse effect on

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Fig. 1. Overexpression and purification of recombinant BmAF-Myo, Bm-iPGM, Bm-TPP by affinity chromatography using nickel column. Coomassie blue stained SDS-polyacrylamide gel containing (A) lane 1, insoluble lysates of E. coli cells following induction with 0.5 mM IPTG at 30 ◦ C for 3 h; lane 2, soluble E. coli lysates; lane 3, flow through from the nickel column; lane 4, wash through from column prior to elution; lane 5, His-tagged purified recombinant BmAF-Myo; lane M, standard protein molecular weight marker. (B) Lane M, standard protein molecular weight marker; lane 1, E. coli lysates of uninduced cells, lane 2, E. coli lysates of induced cells; lane 3, flow through, lane 4, wash through; lanes 5 and 6 purified recombinant Bm-iPGM. (C) Lane 1, E .coli lysates of uninduced cells; lane 2, E. coli lysates of induced cells; lane 3, flow through; lane 4, wash through from column prior to elution; lane 5, elution of purified recombinant Bm-TPP; lane M, standard protein molecular weight marker.

the worm burden resulting into 50.0%, 52.1% and 37.5% reductions in adult worm establishment in BmAF-Myo, Bm-iPGM and Bm-TPP groups respectively. Myosin + iPGM was unable to improve protection (51.8%) achieved with any of these two proteins individually (Table 1).

74.86 ± 2.9% respectively) as compared to the controls (p < 0.01). However, cocktail of Myosin + TPP showed more or less similar (57.33 ± 6.2%) female worm sterilization as compared to these two individual proteins. 3.4. Effect on microfilarial density

3.3. Female worm sterilization As shown in Table 1 single recombinant immunization with Bm-iPGM produced 37.55 ± 10.3% female worm sterilization that was not significantly higher than either of the two controls (PBS, adjuvant) but in the combination with either Bm-TPP or BmAF-Myo, it produced significantly higher female worm sterilization (56.43 ± 4.9%,

Microfilarial density remained low in all the immunized groups as compared to the PBS, adjuvant control groups throughout the observation period (Fig. 3A–C). Myosin + iPGM immunized group had profoundly reduced (72.7 ± 4.9%) microfilaraemia as compared to 63.74 ± 12.7% and 35.19 ± 7.8% in Bm-iPGM and BmAF-Myo groups respectively. iPGM + TPP group exhibited 67.72 ± 8.8% mf reduction that was much higher than any of these two proteins singly (Bm-iPGM 35.19 ± 7.8%) or Bm-TPP (40.99 ± 7.6%). In Myosin + TPP group reduction in mf density was moderate (57.25 ± 9.8%) and could be compared with either BmAF-Myo or Bm-TPP group. 3.5. Immunogenicity of the recombinant proteins

Fig. 2. Evaluation of the protective efficacy of cocktail protein vaccines in terms of adult worm establishment as compared to single protein vaccines. In Myosin + TPP and iPGM + TPP immunized group the adult worm recovery was lower as compared to their individual protein myosin, iPGM or TPP. In Myosin + iPGM group the adult worm recovery was comparable to that of either myosin or iPGM immunized group. Bar represents mean ± SE. Asterisks (*) on top of the bars represents statistical significance with respect to controls and just above a line showed statistical significance with respect to single recombinant protein: *p < 0.05 and ** p < 0.01.

The B. malayi proteins used in the current study were highly immunogenic as evidenced by their antigenic index which were 0.9917 (BmAF-Myo), 1.0221(Bm-iPGM), 1.0223 (Bm-TPP) based on the presence of antigenic peptides. Antigenic peptides predictions by the method of Kolaskar and Tongaonkar [23] are based the occurrence of amino acid residues in experimentally known segmental epitopes. BmAF-Myo, Bm-iPGM, Bm-TPP have 20, 13 and 12 promiscuous MHC II binding peptides respectively as predicted by Propred (Table 2A). Number of distinct B cell epitopes in BmAF-Myo is 5, Bm-iPGM 15 and Bm-TPP 12 as determined by BcelPred online algorithms (Table 2B). 3.6. Humoral immune response triggered by single and cocktail antigen vaccine Indirect ELISA was performed to detect specific IgG antibody against each antigen in the sera of the rodents immunized with single and cocktail recombinant

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Fig. 3. Microfilarial kinetics in different cocktails and single immunized group of Mastomys coucha. Blood microfilaraemia was determined after 12–24 weeks post challenge. In all the experimental groups microfilarial density was low during initial monitoring but increased subsequently in the following weeks but in different immunized groups level remained low throughout the observation period. (A) Comparison of microfilarial density in Myosin + TPP, myosin and TPP immunized groups. In Myosin + TPP group the microfilarial density remain high as compared to the myosin immunized group up to 180 days however it was low as compared to the TPP immunized group. (B) Comparison of microfilarial density in iPGM + TPP, iPGM and TPP immunized groups. The microfilarial density in cocktail of iPGM + TPP was low as compared to the iPGM or TPP immunized group separately. (C) Comparison of microfilarial density in Myosin + iPGM, myosin and iPGM. Microfilarial density in Myosin + iPGM immunized group was more or less similar to the myosin but low as compared to the iPGM immunized group. Each point represents mean ± SE.

Table 2A Prediction of promiscuous human major histocompatibility complex (MHC) class II-restricted epitopes in BmAF-Myo, Bm-iPGM, Bm-TPP proteins by ProPred. The threshold for the ProPred analyses was set at a 3%. S. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

TPP

iPGM

Myosin

Peptide sequence

No. of MHC II alleles binding to epitopes

Peptide sequence

No. of MHC II alleles binding to epitopes

Peptide sequence

No. of MHC II alleles binding to epitopes

IRREEPNLL YKMQETRRA LKYLDALMN VKFLKTFIS YQMAVKERM LFSILANYH VVAHNSGII MVRQAVKQN VLTAGPLRG MHRVDPNSQ LRGPGILDL IVFALLNEK

42

LVVIDGWGI VRINLAVKN VVYQDIVRI LVPFTCSSM YILMVTADH LKQLKVPKL VMNSHPIQA FALITALKQ MRTLHSSSN ILNAKTPVM LKVMGVPLP YKFMDKLPD IVRINLAVK

46 51 51 51 48 48 44 44 41 48 46 47 51

VQISQSSIL YLNMEKKAT VERANSYAS FEMQKIIRR LNMEKKATL MKQLSRANA IRRLEVEKD LRSSSRVQF LRRENKELA FRQIQHQLD VLRAQVEVS MRLKKKLES LRAQVEVSQ LQKFRQIQH FESEGLLKS ILRSSSRVQ LMRLKKKLE ISSLEKTKS MRAKSRSGM MQKIIRRLE

50 44 48 47 45 48 51 48 46 41 51 51 41 46 41 50 44 43 42 44

49 51 41 41 45 44 45 45 46 49

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Table 2B Predicted B-cell epitopes in Bm-TPP, Bm-iPGM and BmAF-Myo protein. S. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

B-cell epitopes TPP

iPGM

Myosin

QNSSLLVGQPDVI LTTKLSV RVDLFSIL NLQPVYS LSGKRVVHQDGI LVGSGVQ TLGVQTVCHHVTSEL PNSQILVFDPS ELEVEVV PGKILICGDTL VKQVIGDESRCCFVSCPDVIH ILNEHCI

KNRVCLVVIDGW PVMDELCVMNSH HGLHVGLPE RVVYQDIVR MHLCGLVSDGGVHSHIDH LKQLKVPKLYIQFFG VGFLQQLID ERIRVCYD FLKPIILS EERCLVVSPKV KVIKQLHM KHPFVMCNF VGHTGVY HTCNLVPFTCSS PTVLKVMGVPLP

SRLVSDL QVEVSQIR LQQQVEI HVQCNELS ISQSSIL

proteins to investigate which combination elicits improved antibody response over the single recombinant protein. Single protein immunization with BmAF-Myo, Bm-iPGM and Bm-TPP elicited significant (p < 0.01) anti-BmAF-Myo, anti-Bm-iPGM and anti-Bm-TPP IgG antibodies in the sera of rodents compared to the controls groups. Each subsequent antigen booster led to successive increase in the IgG titre. Immunization with the cocktail vaccine formulations, viz. Myosin + TPP and iPGM + TPP elicited much higher IgG antibody level against each protein (p < 0.05) as compared to the single antigen immunization especially after third immunization (Fig. 4A and B). After challenging these animals with the infective larvae, IgG level initially increased, but started declining once the animals developed patent microfilaraemia, however it remain significantly high (p < 0.01/p < 0.05) in both the cocktail immunized groups up to the day of autopsy with respect to single antigen immunized group (Fig. 4A and B ). Myosin + iPGM protein cocktail also triggered profound IgG production (p < 0.01) in comparison to control groups, however, the increase was statistically not significant over BmAF-Myo or Bm-iPGM immunized groups (Fig. 4C and C ). 3.7. IgG subclass and IgM response to immunization All the three recombinant protein as single antigen vaccine triggered considerable generation of specific IgG1, IgG2a, IgG2b and IgG3 isotypes. However, the isotype response was significantly up-regulated (p < 0.01) when BmAF-Myo and Bm-TPP were immunized together. A predominance of IgG1, IgG2a, IgG2b and IgG3 antibody isotypes indicated production of a mixed Th1/Th2 immune response (Fig. 5A). iPGM + TPP also showed significant increase in the IgG1, IgG2a, IgG2b (p < 0.01) as compared to either of the two protein. However, IgG3 level was significantly high with respect to only Bm-TPP immunized group (p < 0.01). Bm-iPGM and Bm-TPP alone or in combination produced mixed Th1/Th2 response with marginal Th2 biasness when IgG1 and IgG2a ratio was considered (Fig. 5B). Myosin + iPGM immunization did not show noticeable

advantage over individual protein (Fig. 5C). Myosin alone induced significant increase (p < 0.01) in IgG2a and IgG2b followed by IgG1 and IgG3 with low IgG1:IgG2a indicating marginal Th1 skewing. IgM level was significantly elevated in Myosin + TPP and iPGM + TPP immunized group (p < 0.01/p < 0.05) with respect to immunization with single recombinant protein. However, Myosin + iPGM group failed to provide such response although the IgM level was elevated in comparison to the control groups (p < 0.01).

3.8. Cytokine response in the immunized animals The levels of both pro-inflammatory and antiinflammatory cytokines were determined intracellularly in the splenic cell population of different groups of immunized and control animals. As shown in Fig. 6A–D rodents immunized with Myosin + TPP elicited stronger mixed Th1/Th2 type immune response with enhanced pro-inflammatory (IL-2, IFN-␥) and anti-inflammatory (IL-4, IL-10) cytokine production as compared to those of unimmunized controls (p < 0.01). The level of these cytokines except IL-10 was also significantly higher than either BmAF-Myo or Bm-TPP immunized groups (p < 0.01/p < 0.05). iPGM + TPP group showed elevated level of anti-inflammatory cytokines followed by proinflammatory cytokines as compared to controls (p < 0.01) indicating mixed Th1/Th2 response with biasness towards Th2 response. IFN-␥, IL-4 level in iPGM + TPP group were also significantly higher (p < 0.05) than either Bm-TPP or Bm-iPGM immunized group separately. However, Myosin + iPGM cocktail triggered a mixed Th1/Th2 response, there was not much difference in cytokine level between this cocktail immunized group and single antigen immunized groups of BmAF-Myo or Bm-iPGM. Rodents immunized with BmAF-Myo alone showed significantly elevated level of pro-inflammatory cytokines, viz. IFN-␥, IL-2 (p < 0.01) as compared to the control groups showing biasness towards Th1 response. Bm-TPP and Bm-iPGM protein individually resulted in mixed Th1/Th2 response

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Fig. 4. IgG response in various immunized and control groups. (A) Immunization induced myosin and TPP specific IgG responses at the indicated day in the sera of myosin, TPP and Myosin + TPP immunized groups. IgG in all the immunized group was high as compared to the PBS and adjuvant control groups. IgG in cocktail immunized group was also significantly higher than either myosin or TPP immunized group that remain high even after infective larve challenge. (B) Immunization-induced iPGM and TPP specific IgG responses at the indicated day in the sera of iPGM, TPP and iPGM + TPP immunized groups. Total IgG in cocktail immunized group was significantly higher than either myosin or TPP immunized group that remain high after challenge with infective larve. (C) Immunization induced myosin and iPGM specific IgG responses at the indicated day in the sera of myosin, iPGM, and Myosin + iPGM groups. IgG in all the immunized group was significantly higher as compared to the control groups. However, in cocktail immunized group the level was not significantly higher as compared to the either myosin or iPGM immunized groups. OD values (at 492 nm) are shown as mean ± SE. Asterisks (*) on top of the bars represents statistical significance with respect to controls and above a line showed statistical significance with respect to single recombinant protein: *p < 0.05 and ** p < 0.01.

with significant increase in IFN-␥, IL-2, IL-10 or IL-4 (p < 0.05/p < 0.01). 3.9. Reactive oxygen species generation in immunized groups There was significant increase in the oxidative burst in peritoneal macrophages isolated from the animals administered with single or cocktail recombinant proteins over

unimmunized controls (p < 0.01/p < 0.05). Myosin + TPP maximally incited the macrophages to produce ROS that was significantly higher as compared to either BmAFMyo (p < 0.05) or Bm-TPP (p < 0.01) immunized groups. In contrast, there was no significant difference in ROS level between iPGM + TPP combination and their respective individual proteins (Bm-iPGM or Bm-TPP immunized groups). However this combination produced significantly high ROS level as compared to the control groups (p < 0.01).

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Fig. 5. IgG subclass and IgM response in various immunized and control groups. (A) IgG subclass and IgM response in Myosin + TPP, myosin or TPP immunized groups. IgG1, IgG2a, IgG2b, IgG3 and IgM level were significantly high as compared to myosin or TPP immunized group. (B) IgG subclass and IgM response in iPGM + TPP, iPGM or TPP immunized groups. IgG1, IgG2a, IgG2b, and IgM level were significantly high as compared to Myosin or TPP immunized group. (C) IgG subclass and IgM response in Myosin + iPGM, myosin or iPGM immunized groups. IgG1, IgG2a, IgG2b, IgG3 and IgM level were significantly high as compared to PBS or adjuvant control groups however there was not much significant difference from myosin or iPGM immunized groups.

Myosin + iPGM cocktail vaccine on the other hand significantly suppressed the ROS level when compared with that of BmAF-Myo immunization alone (p < 0.05), however, it remained unchanged when compared with that of BmiPGM alone immunization (p > 0.05) (Fig. 7). 3.10. Mitogen specific and recombinant specific lymphoproliferation in vitro

presence of respective proteins over that of TPP vaccinated group, however, the increase by iPGM + TPP was not significant with respect to iPGM immunized animals (p > 0.05).

Table 3A The in vitro lymphoproliferative stimulation index (SI) in immunized and control groups of mastomys in the presence of mitogens (mean ± SE). S. No.

The splenocyte proliferation in different immunized and control groups are shown in Tables 3A and 3B. In vitro stimulation of splenocyte isolated from all the six immunized groups of mastomys exhibited profoundly higher proliferation in the presence of ConA, LPS or respective proteins over that of two unimmunized control groups (p < 0.05/p < 0.01). The enhancement in the cellular proliferation between Myosin + TPP, Bm-Myo and Bm-TPP immunized groups was significant too (p < 0.05) whether stimulated with LPS or recombinant proteins on contrary to Myosin + iPGM vaccination where proliferative increase was not significant (p > 0.05). The third cocktail recombinant vaccine, iPGM + TPP stimulated cell proliferation (p < 0.05) in the

Animal groups

Stimulatory index (mean ± SE) ConA stimulated

1 2 3 4 5 6 7 8 c

Myosin iPGM TPP Myosin + TPP iPGM + TPP Myosin + iPGM PBS Adjuvant

3.05 2.58 2.34 3.49 2.93 2.90 1.08 1.18

± ± ± ± ± ± ± ±

c

0.38 0.34c 0.23d 0.22c 0.38c 0.54c 0.28 0.17

LPS stimulated 2.76 3.59 2.85 4.85 3.96 2.88 0.77 1.08

± ± ± ± ± ± ± ±

0.73d 0.41c 0.31d 0.23c , d , e 0.31c 0.29c 0.21 0.18

p < 0.01 values highly significant than PBS or adjuvant control group. p < 0.05 values significant with respect to PBS or adjuvant control group. e p < 0.05 values significant with respect to Myosin or TPP immunized group. d

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Table 3B The in vitro lymphoproliferative stimulation index (SI) in immunized and control groups of mastomys in the presence of specific recombinant protein either singly or in cocktail (mean ± SE). S. No.

Recombinant protein used for in vitro stimulation

SI (mean ± SE) with statistical significance in parenthesis PBS control group

1 2 3 4 5 6 f g h i

Myosin iPGM TPP Myosin + TPP iPGM + TPP Myosin + iPGM

1.165 1.008 1.044 1.165 1.610 1.845

± ± ± ± ± ±

0.25 0.25 0.43 0.25 0.25 0.20

Adjuvant control group 1.357 1.158 1.620 1.323 1.525 2.065

± ± ± ± ± ±

0.32 0.23 0.33 0.46 0.34 0.21

Recombinant protein immunized group 2.432 2.923 2.420 3.923 3.091 2.620

± ± ± ± ± ±

0.30f 0.15f 0.22f 0.12f , g , h 0.28f , i 0.53f

p < 0.01 values highly significant with respect to PBS or adjuvant control group. p < 0.01 values highly significant with respect to Myosin immunized group. p < 0.01 values significant with respect to the TPP immunized group. p < 0.05 values significant with respect to the TPP immunized group.

Fig. 6. Cytokine responses in rodents immunized with different single and cocktail proteins. The levels of both pro-inflammatory (IL-2, IFN-␥) and antiinflammatory cytokines (IL-4, IL-10) were determined intracellularly in the splenic cell population of different immunized animals. (A) IFN-␥ level in various experimental groups. All single and multiple proteins immunized groups showed elevated level of IFN-␥ as compared to control groups. Myosin + TPP and iPGM + TPP immunized groups had significantly high level of IFN-␥ over either of the individual proteins. (B) IL-2 level in various experimental groups. All single and multiple proteins immunized groups showed statistically high level of IL-2 as compared to control groups. Myosin + TPP immunized group had significant high level of IL-2 over either of the individual proteins. (C) IL-10 level in various groups. All single and multiple proteins immunized groups showed significantly high level of IL-10 as compared to controls except myosin immunized group. However, there was no statistical difference in IL-10 level between single and multiple antigen immunized groups. (D) IL-4 level in various experimental groups. All single and multiple proteins immunized groups showed statistically high level of IL-4 as compared to controls except myosin immunized group. Myosin + TPP and iPGM + TPP immunized group had significant high level of IL-4 over either of the individual protein. Bar represents the mean ± SE. Asterisks (*) on top of the bars represents statistical significance with respect to controls and above a line showed statistical significance with respect to single recombinant protein: *p < 0.05 and **p < 0.01.

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Fig. 7. Determination of reactive oxygen species level in different immunized groups. All the immunized groups had shown significant increase in the ROS level as compared to the PBS or adjuvant control groups. In Myosin + TPP immunized group the ROS level was statistically high as compared to either myosin or TPP. In iPGM + TPP immunized group the ROS level was more or less similar to either iPGM or TPP immunized groups. In Myosin + iPGM group the ROS level was comparable to the iPGM immunized group but low than Myosin immunized group. Each bar represents mean ± SD. Asterisks (*) on top of the bars represent statistical significance with respect to controls and above a line showed statistical significance with respect to single recombinant protein: *p < 0.05 and **p < 0.01.

4. Discussion Developing a vaccine against LF has been challenging due to the complex life cycle of the parasite involving several life-stages with different antigenic make up. Several vaccine candidates including the live parasites, crude extracts, semi-purified antigens as well as recombinant proteins have been tried in the past with partial success [30–35]. Multiple subunit vaccine candidates may permit immune targeting against multiple epitopes of specific immunogens together with more likelihood of stimulation of different layers of the immune responses at various levels. We earlier demonstrated the production of substantial protection against infective larval challenge by heavy chain myosin of B. malayi in a rodent model of brugian filariasis [18]. Two other B. malayi recombinant proteins, trehalose-6-phosphate phosphatase (TPP) or independent phosphoglycerate mutase (iPGM) also provided the protection (unpublished). The three recombinant proteins of B. malayi, viz. myosin (BmAF-Myo), iPGM (Bm-iPGM) and TPP (Bm-TPP) were investigated for their ability to elicit protective immunity as protein cocktails in B. malayi susceptible rodent, M. coucha. The findings revealed that of the three formulations, viz. Myosin + TPP, iPGM + TPP and Myosin + iPGM, former two protein cocktails performed better in terms of the extent of immunoprotection by enhancement of the efficacy of individual recombinant protein. Myosin + iPGM did not provide better protection in terms of adult worm establishment as compared to the single protein immunization.

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The measurement of humoral and cellular immune responses generated by Myosin + TPP or iPGM + TPP protein cocktails indicated that antigen specific IgG, IgM production was largely improved in combinations over that of respective single proteins. Antibodies are known to play a major role in the protection in helminth infections [36–39]. This was further substantiated by the failure to increase antibody production when myosin and iPGM were used together in the form of cocktail antigen vaccine with no noticeable increase in IgG over single recombinant proteins. This was accompanied with unchanged degree of protection against larval challenge. On the other hand, Myosin + TPP also produced significantly higher cellular proliferation as compared to the single protein immunization. However, Myosin + iPGM cocktail did not respond in the same manner. It appears that neutralization of lymphocyte hyporesponsiveness associated with both B and T cells facilitated development of protection against L3 invasion [40,41]. Antibody subclasses in the sera of immunized mastomys gave further insight into protective mechanism involving humoral response. Myosin + TPP and iPGM + TPP cocktails led to predominant generation of IgG1, IgG2a, IgG2b antibody subclasses that were significantly higher than any of the single proteins. IgG1 and IgG2a antibodies are reported to offer adequate protection against challenged filarial larvae post irradiated L3 vaccination [42,43]. The results obtained from cytokines analysis which are better indicator of Th1 or Th2 response, were also in accordance with the antibody subclasses pattern mentioned above. Single antigen vaccine (iPGM or TPP) and cocktail antigen vaccine comprising both of them (iPGM + TPP) generated a generalized mixed Th1/Th2 immunity with Th2 biasness. However, IL-4 and IFN-␥ was significantly high in combination (iPGM + TPP) as compared to single protein immunization. The recombinant myosin showed Th1 biasness unlike other recombinant proteins that changed to a mixed Th1/Th2 pattern when myosin was used in combination either with TPP or iPGM. This mixed response in Myosin + TPP was considerably higher as compared to that of myosin or TPP alone which resulted into profound adverse effect on the establishment of challenged larvae resulting into increased protection since both proinflammatory and anti-inflammatory cytokines have roles in host defense against the human filarial parasite B. malayi [44]. In contrast, Myosin + iPGM could not produce enhance immune effect resulting into non altered cytokine parameters over each of the two single recombinant proteins. Th1 responses are usually cellular mediated and it is evident that Th1 cytokine, IFN-␥ activates the macrophages which are involved in parasite killing indirectly by antibody dependent cytotoxicity resulting in the release of reactive oxygen intermediates. Earlier studies have demonstrated the role of reactive oxygen species in effector protective mechanism against B. malayi [45]. In the current study, ROS level was significantly elevated in Myosin + TPP group than either myosin or TPP alone groups. In iPGM + TPP, oxidative burst remained same as immunization with single protein, and did not increase as in case of Myosin + TPP, however, the protection achieved was comparable to that of Myosin + TPP. It shows that only ROS cannot be

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responsible for protection, but other factors do play a significant role. Myosin developed a predominant proinflammatory response with increased oxidative burst, but this effect was antagonized when iPGM was added to this protein for immunization. Different immune effector mechanisms generated in response to different antigens require recognition by T and B cell of every different epitopes. Myosin, iPGM and TPP consisted of MHC 21, 13, 12 class II restricted promiscuous T cell epitopes respectively. Combining myosin with TPP or iPGM with TPP would enhance stimulation of T helper cells due to increase in number of MHC II binding peptides with subsequent activation of increased number of B cells due to exposure to cumulative B cell epitopes coming from individual proteins. It has earlier been well established that administration of a single vaccine carrying highly complex combination of appropriately selected epitopes from different antigens permits the prevention of many different diseases in a practical way with single preparation. These are referred to as poly topic vaccines. A second possible explanation for involvement of different immune cells in different combinations is the excretory secretary nature of myosin [16] and iPGM [19] which are easily accessible to the host immune system. On the other hand, TPP is a non secretory internal protein [22] which becomes exposed to immune system when B. malayi parasites die as a result of natural attrition in TPP immunized animal or during an inflammatory response elicited as a result of immunization with iPGM or myosin as a result of multiple protein immunization that could trap and/or kill bystander parasites that normally would survive in an unimmunized rodents. Thus in iPGM + TPP and Myosin + TPP immunized animals immune response functions at different layers. This is similar to HIV-1 infection where an attractive vaccine strategy is to design a composition of immunogens that targets several viral components including the full structural and regulatory proteins as well as the viral enzymes [46]. However, combination of myosin and iPGM could neither provoke enhanced immune response nor could noticeably protect the immunized animals against L3 challenge. It is possible that the simultaneous induction of immune response against two ES proteins might have antagonized the effect of each individual protein due to antigenic interference. It is the phenomenon where strong immune responses to one of the proteins generated, result in to decreased immunity to other antigens in the same mixture because of competition for the effector immune cells or presentation pathways resulting in biased display of immunogenic epitopes. Therefore, the present study gave an insight into cocktail vaccination strategy to combat LF. The present findings illustrate that cocktail antigen vaccine comprised of one excretory secretory protein and one non secretory internal protein, such as Myosin + TPP or iPGM + TPP is much more immunogenic than a single antigen vaccine. Thus the combination of BmAF-Myo and Bm-iPGM, Bm-iPGM and Bm-TPP is an excellent choice for further vaccine development. It may also be surmized that different combinations of proteins stimulate different protective mechanisms to attain similar level of protection depending on the nature of different proteins used in cocktail antigen vaccine. In future studies, epitope-based vaccine containing validated T-cell

epitopes of the selected vaccine candidates can be focused since polytopic vaccine appear to be capable of inducing more potent responses than whole recombinant protein and overcomes potential safety concerns associated with whole recombinant proteins. Conflict of interest None. Acknowledgements This research was supported by the Council of Scientific and Industrial Research (CSIR), Delhi, India in the form of senior research fellowships to Nidhi Shrivastava, Jeetendra Kumar Nag and Prashant Kumar Singh. We also acknowledge University Grant Commission, India for providing fellowship to Susheela Kushwaha. The technical assistance rendered by Mr A.K. Roy and R.N. Lal in experimental maintenance of infection is gratefully acknowledged. This article bears CDRI communication number 8478. References [1] Taylor MJ, Hoerauf A, Bockarie M. Lymphatic filariasis and onchocerciasis. Lancet 2010;376(9747):1175–85. [2] Wolstenholme AJ, Fairweather I, Prichard R, Von SamsonHimmelstjerna G, Sangster NC. Drug resistance in veterinary helminths. Trends in Parasitology 2004;20:469–76. [3] Bourguinat C, Pion SD, Kamgno J, Gardon J, Duke BO, Boussinesq M, et al. Genetic selection of low fertile Onchocerca volvulus by ivermectin treatment. PLoS Neglected Tropical Disease 2007;1(1):e72. [4] Lustigman S, McCarter JP. Ivermectin resistance in Onchocerca volvulus: toward a genetic basis. PLoS Neglected Tropical Disease 2007;1(1):e76. [5] Dabir S, Dabir P, Goswami K, Reddy MV. Prophylactic evaluation of recombinant extracellular superoxide dismutase of Brugia malayi in jird model. Vaccine 2008;26(29–30):3705–10. [6] Rathaur S, Yadav M, Gupta S, Anandharaman V, Reddy MV. Filarial glutathione-S-transferase: a potential vaccine candidate against lymphatic filariasis. Vaccine 2008;26(32):4094–100. [7] Sharmila S, Christiana I, Kiran P, Reddy MV, Kaliraj P. The adjuvantfree immunoprotection of recombinant filarial protein Abundant Larval Transcript-2 (ALT-2) in Mastomys coucha and the immunoprophylactic importance of its putative signal sequence. Experimental Parasitology 2011;129(3):247–53. [8] Dakshinamoorthy G, Samykutty AK, Munirathinam G, Shinde GB, Nutman T, Reddy MV, et al. Biochemical characterization and evaluation of a Brugia malayi small heat shock protein as a vaccine against lymphatic filariasis. PLoS ONE 2012;7(4):e34077. [9] Kedzierski L, Black CG, Goschnick MW, Stowers AW, Coppel RL. Immunization with a combination of merozoite surface proteins 4/5 and 1 enhances protection against lethal challenge with Plasmodium yoelii. Infection and Immunity 2002;70(12):6606–13. [10] Burns Jr JM, Flaherty PR, Romero MM, Weidanz WP. Immunization against Plasmodium chabaudi malaria using combined formulations of apical membrane antigen-1 and merozoite surface protein-1. Vaccine 2003;21(17–18):1843–52. ˜ [11] Moreno J, Nieto J, Masina S, Canavate C, Cruz I, Chicharro C, et al. Immunization with H1, HASPB1 and MML leishmania proteins in a vaccine trial against experimental canine leishmaniasis. Vaccine 2007;25(29):5290–300. [12] Shi Q, Lynch MM, Romero M, Burns Jr JM. Enhanced protection against malaria by a chimeric merozoite surface protein vaccine. Infection and Immunity 2007;75(3):1349–58. [13] Vanam U, Pandey V, Prabhu PR, Dakshinamurthy G, Reddy MV, Kaliraj P. Evaluation of immunoprophylactic efficacy of Brugia malayi transglutaminase (BmTGA) in single and multiple antigen vaccination with BmALT-2 and BmTPX for human lymphatic filariasis. American Journal of Tropical Medicine and Hygiene 2009;80(2):319–24. [14] Anand SB, Kodumudi KN, Reddy MV, Kaliraj P. A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and

N. Shrivastava et al. / Comparative Immunology, Microbiology and Infectious Diseases 36 (2013) 507–519

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30]

BmVAH) enhances immune responses and protection in jirds. Journal of Helminthology 2011;85(4):442–52. Verma S, Bansal I, Vedi S, Saxena JK, Katoch VM, Misra-Bhattacharya S. Molecular cloning, purification and characterisation of myosin of human lymphatic filarial parasite Brugia malayi. Parasitology Research 2008;102(3):481–90. Bennuru S, Semnani R, Meng Z, Ribeiro JM, Veenstra TD, Nutman TB. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling. PLoS Neglected Tropical Disease 2009;3(4):e410. Obwaller A, Duchêne M, Bruhn H, Steipe B, Tripp C, Kraft D, et al. Recombinant dissection of myosin heavy chain of Toxocara canis shows strong clustering of antigenic regions. Parasitology Research 2001;87(5):383–9. Vedi S, Dangi A, Hajela K, Misra-Bhattacharya S. Vaccination with 73 kDa recombinant heavy chain myosin generates high level of protection against Brugia malayi challenge in jird and mastomys models. Vaccine 2008;26(47):5997–6005. Moreno Y, Geary TG. Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products. PLoS Neglected Tropical Disease 2008;2(10):e326. Singh U, Misra S, Murthy PK, Katiyar JC, Agrawal A, Sircar AR. Immunoreactive molecules of Brugia malayi and their diagnostic potential. Serodiagnosis and Immunotherapy in Infectious Disease 1997;8:207–12. Zhang Y, Foster JM, Kumar S, Fougere M, Carlow CK. Cofactor independent phosphoglycerate mutase has an essential role in Caenorhabditis elegans and is conserved in parasitic nematodes. Journal of Biological Chemistry 2004;279: 37185–90. Kushwaha S, Singh PK, Rana AK, Misra-Bhattacharya S. Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi. Acta Tropica 2011;119(2–3):151–9. Kolaskar AS, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Letters 1990;276:172–4. Singh H, Raghava GP. ProPred: prediction of HLA-DR binding sites. Bioinformatics 2001;17(12):1236–7. Saha S, Raghava GPS. BcePred: Prediction of Continuous B-cell Epitopes in Antigenic Sequences Using Physico-chemical Properties. LNCS; 2004. p. 3239197–204. Maguire GA, Kumararatne DS, Joyce HJ. Are there any clinical indications for measuring IgG subclass? Annals of Clinical Biochemistry 2002;39:374–7. Singh M, Shakya S, Soni VK, Dangi A, Kumar N, Bhattacharya SM. The n-hexane and chloroform fractions of Piper betle L. trigger different arms of immune responses in BALB/c mice and exhibit antifilarial activity against human lymphatic filarid Brugia malayi. International Immunopharmacology 2009;9(6):716–28. Zurgil N, Shafran Y, Afrimzon E, Fixler D, Shainberg A, Deutsch M. Concomitant real-time monitoring of intracellular reactive oxygen species and mitochondrial membrane potential in individual living promonocytic cells. Journal of Immunological Methods 2006;316(1–2):27–41. Misra S, Mukherjee M, Dikshit M, Chaterjee RK. Cellular response of Mastomys and gerbil in experimental filariasis. Tropical Medicine and International Health 1998;3(2):124–9. Yates JA, Higashi GI. Brugia malayi: vaccination of jirds with 60 cobalt-attenuated infective stage larvae protects against homologous challenge. American Journal of Tropical Medicine and Hygiene 1985;34(6):1132–7.

519

[31] Chenthamarakshan V, Reddy MV, Harinath BC. Immunoprophylactic potential of a 120 kDa Brugia malayi adult antigen fraction, BmA-2, in lymphatic filariasis. Parasite Immunology 1995;17(6):277–85. [32] Dixit S, Gaur RL, Sahoo MK, Joseph SK, Murthy PS, Murthy PK. Protection against L3 induced Brugia malayi infection in Mastomys coucha pre-immunized with BmAFII fraction of the filarial adult worm. Vaccine 2006;24(31–32):5824–31. [33] Thirugnanam S, Pandiaraja P, Ramaswamy K, Murugan V, Gnanasekar M, Nandakumar K, et al. Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model. Experimental Parasitology 2007;116(4):483–91. [34] Shakya S, Singh PK, Kushwaha S, Misra-Bhattacharya S. Adult Brugia malayi approximately 34 kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha. Parasitology International 2009;58(4):346–53. [35] Anand SB, Rajagopal V, Kaliraj P. Brugia malayi thioredoxin peroxidase as a potential vaccine candidate antigen for lymphatic filariasis. Applied Biochemistry and Biotechnology 2012 [Epub ahead of print]. [36] Ravindran BAK, Satapathy PK, Sahoo JJ, Geddam B. Protective immunity in human Bancroftian filariasis: inverse relationship between antibodies to microfilarial sheath and circulating filarial antigens. Parasite Immunology 2000;22:633–7. [37] Martin C, Saeftel M, Vuong PN, Babayan S, Fischer K, Bain O, et al. B-cell deficiency suppresses vaccine-induced protection against murine filariasis but does not increase the recovery rate for primary infection. Infection and Immunity 2001;69(11):7067–73. [38] Bethony J, Loukas A, Smout M, Brooker S, Mendez S, Plieskatt J, et al. Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals. FASEB Journal 2005;19(12):1743–5. [39] Rajan B, Ramalingam T, Rajan TV. Critical role for IgM in host protection in experimental filarial infection. Journal of Immunology 2005;175:1827–33. [40] Babu S, Blauvelt CP, Kumaraswami V, Nutman TB. Cutting edge: diminished T cell TLR expression and function modulates the immune response in human filarial infection. Journal of Immunology 2006;176(7):3389–885. [41] Harnett W, Harnett MM. What causes lymphocyte hyporesponsiveness during filarial nematode infection? Trends in Parasitology 2006;22(3):105–10. [42] El Bouhdidi A, Truyens C, Rivera MT, Bazin H, Carlier Y. Trypanosoma cruzi infection in mice induces a polyisotypic hypergammaglobulinemia and parasite-specific response involving high IgG2a concentrations and highly avid IgG1 antibodies. Parasite Immunology 1994;16(2):69–76. [43] Babayan SA, Attout T, Harris A, Taylor MD, Le Goff L, Vuong PN, et al. Vaccination against filarial nematodes with irradiated larvae provides long-term protection against the third larval stage but not against subsequent life cycle stages. International Journal for Parasitology 2006;36(8):903–14. [44] Babu S, Ganley LM, Klei TR, Leonard D, Shultz LD, Rajan TV. Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi. Infection and Immunity 2000;68(5):3034–5. [45] Gupta R, Bajpai P, Tripathi LM, Srivastava VML, Jain SK, MisraBhattacharya S. Macrophages in the development of protective immunity against experimental Brugia malayi infection. Parasitology 2004;129(3):311–23. [46] Rollman E, Bråve A, Boberg A, Gudmundsdotter L, Engström G, Isaguliants M, et al. The rationale behind a vaccine based on multiple HIV antigens. Microbes and Infection 2005;7(14):1414–23.