Effect of muramyl dipeptide and alum adjuvants on immunization with Filarial multi antigen peptide vaccine in mice model

The chimeric MAP is a grouping of heteromeric/homomeric immunodominant peptide sequences. The synthesis is done using standard Fmoc chemistry. Briefly, the Fmoc-Gly-HMP-TentaGel resin, substitution factor 0.16 mmol/g, was pre-swelled in dichloromethane (DCM) for 10 min as per the manufacturer’s instructions and kept for swelling in dimethylformamide (DMF) overnight. The F-moc group was removed using 20 % piperidine in DMF. To this, F-moc-Lys-(ivDde) was attached. The N-terminal F-moc is removed and the peptide was synthesized. At the end of the sequence, the last amino acid used was with a different protecting group, t-Boc, in order to prevent its removal while deprotecting F-moc groups during attachment of different sequences and to prevent unwanted chain elongation thus end capping the synthesized peptide sequences. In the next step, the ξ-NH₂ protecting group, ivDde is removed using 2 % hydrazine in DMF and another Fmoc-Lys-(ivDde) was attached. The cycle was repeated till the completion of all the sequences. The epitopic sequences are given in Table 1.

The peptide sequences were synthesized by solid phase technique, using f-moc chemistry and assembled on Tentagel resin (Sigma-Aldrich) as TRX-TGA MAP (TT MAP) constructs. The MAP construct were purified and the purity was analyzed by high performance liquid chromatography.

The partial sequence of Wb-TGA was cloned in T7 expression vector pRSETB (Invitrogen, Carlsbad, CA), with an N-terminal histidine tag, were expressed in E.coli GJ-1158. Bacterial cell cultures were induced for 4 hours with 150 mM NaCl at an O.D of 0.6. The recombinant protein were analysed on 12 % SDS-PAGE and the electro-transferred protein bands on to the nitro cellulose membrane were further analysed by Western blot analysis. Anti-TGA antibody raised in mice (1:10000 dilutions) and alkaline phosphatase labelled goat anti-mouse antibody were used as primary and secondary antibodies, respectively, for the immunoblot analysis. Bacterial culture was subjected to cell lysis by sonication. The recombinant protein was present in the insoluble fraction revealing its property to form inclusion body. The insoluble fraction was washed twice with cell lysis buffer (50 mM Tris-Cl, 100 mM NaCl, 0.5 % (v/v) Triton X-100, 1 M urea) pH8.0, centrifuged at 12000 g for 15 min. The pellets were then resuspended in 3 M urea in buffer (50 mM Tris, 200 mM NaCl, pH 8.2) overnight, centrifuged at 15000 g for 15 min. The solubilised protein in supernatant was purified using chelating sepharose fast flow (Amersham Biosciences, Uppasala, Sweden). After washing this column with the same buffer containing urea, the recombinant proteins were eluted with the same buffer containing 150mM imidazole. The eluted proteins were loaded on to a desalting column (GE Healthcare-Lifesciences) for further purification. The Bm-TRX-1 gene in pRSET B vector was expressed in E. coli (GJ1158) using 100 mM NaCl at 0.6 O.D. for 3 h. The soluble protein was purified in non-denaturing conditions by ion exchange chromatography using DEAE Sepharose and analyzed on 12 % SDS-PAGE. The protein was further analyzed using mouse anti-TRX sera.

Protein concentrations were estimated by Bradford assay (Biorad, CA, USA) and then used for immunization.

Six to eight weeks old female BALB/c (H-2^d) were procured from TamilNadu Veterinary and Animal Sciences University, Chennai. All experiments were performed in accordance with ‘Indian Animal Ethics Committee’ regulations. A dosage of 50 μg of TT MAP/100 pil PBS was given for each mouse in TT MAP groups. TT protein groups received 12.5 μg each of rTRX & rTGA (total 25 μg/100μl PBS for each mouse) as cocktail vaccine. Individual proteins TRX and TGA (25 μg/100μl PBS for each mouse) were immunised with MDP/Alum as separate groups which served as whole protein controls. Aluminium hydroxide gel & MDP (Sigma Aldrich, USA)) were admixed to each dose. 5 μg of MDP/mice was given for each dose. In case of alum, a 1:1 ratio was given. Four doses at weekly intervals were administered intra muscularly for MDP groups and intra peritoneally for alum groups. Intramuscular immunizations were carried out by injection of 100 μl volumes in the hind right leg ventral muscle. The adjuvant control group received Phosphate Buffered Saline alone in MDP/alum. Sera collected periodically after immunization in mice was used to check the antibody titer by ELISA. Immunizations of all the groups were performed during the same period. Booster doses were given on days 14, 21 and 28.

The sera were collected periodically after immunization and stored at -80 °C and the antibody titer was determined by ELISA, in which 96-well microtiter plates (Immulon2, Dynatech, VA, USA) were coated with 100 ng/well of respective antigen. The plates were blocked with 5 % skimmed milk, after washing with PBST and PBS, a 2 fold serial dilution from 1:500 to 1:64000 were performed using respective antisera. The antibody titer was determined by fixing a cut-off value which was obtained by the mean plus 3SD of the OD value of pre-immune serum. The highest dilution of the antiserum that showed an OD value above the cut-off value was taken as the antibody titer. The colour developed using mouse antibody conjugated with alkaline phosphatase/p-nitrophenyl phosphate as substrate (1 mg/ml) in substrate buffer (100 mM Tris-Cl, pH 9.5, 100 mM Nacl, 5 mM MgCl₂) and the absorbance was read at 405 nm.

For determination of antibody isotypes, the sera (dilution1:500) from different immunization groups of mice were incubated for 1 h at 37 °C, with respective proteins (100 ng of TRX/TGA for whole protein groups and 50 ng of TRX and 50 ng of TGA for TT protein groups and 100 ng of TT MAP for MAP immunised groups) coated on ELISA plates. The IgG isotype binding was detected using secondary rabbit anti-mouse IgG antibody specific for each subclass (Sigma, USA) as per the manufacturer instructions. The absorbance was read at 405 nm.

Immunized mice were splenectomised aseptically and the splenocytes were separated and washed twice with fresh culture medium (RPMI 1640 from Gibco BRL, USA). Lysis buffer (0.1 % ammonium chloride) was added to the pellet to remove the red blood cells after which the lymphocytes were counted. The single cell suspension was cultured in triplicate in 96 well plates at 0.2×10⁶ cells/ml in RPMI 1640 medium (100 μl/well) supplemented with gentamycin (80 μg/ml), 25 mM HEPES, 2 mM glutamine and 10 % fetal bovine serum. The cells were then stimulated in vitro with respective antigen, a positive control Concanavalin (Con A) (1 μg/ well each). Wells with medium alone were used as unstimulated controls. After growth at 37 °C in a CO₂ incubator for 72h, the cultures were pulsed with (H₃) thymidine (USB, Amersham Pharmacia, UK) at 0.5 μCi/well. The cells were incubated for 18h and harvested onto glass fiber disks and thymidine incorporation was measured by a liquid scintillation counter. All cultures were set up in triplicate and the results were expressed as mean stimulation index (SI) = (counts per minute of stimulated cultures/ counts per minute of unstimulated cultures) ± SD.

Cytokine levels of the various immunised groups of mice were analysed. 5x10⁶ cells (lymphocytes obtained from immunised mice)/ ml were stimulated with respective antigens. The culture supernatants were centrifuged at 5000 rpm for 15 min, filtered through 0.22-μm pores and assayed for cytokine levels using sandwich ELISA for anti-mouse IL-2, IL-4, IL-5, IL-10 and IFN-ɤ (e-Biosciences) according to the manufacturer’s instructions. All concentrations were derived from standard curves and data expressed in picograms/ml.

The statistical analysis of the data by One-way or Two-way ANOVA with Bonferroni post-test were performed using GraphPad Prism software (version 5.03). Probability (p) value of <0.05 was considered statistically significant.

The Thioredoxin-Transglutaminase Multi Antigen Peptide (TT MAP) used in this study comprises of a tri-peptide, one epitope from Brugia malayi Transglutaminase (TGA) (amino acids 203-226 named as TGA P1) and two epitopic portions from Brugia malayi Thioredoxin (TRX) (amino acids 2-27 named as TRXP1 and amino acids 94-107 named as TRX P2) attached to an inert lysine core. The three peptides were chosen to be synthesised as MAP based on previous studies in mice models (Madhumathi et al., 2010) with individual peptides (Data not shown). All the 3 peptides are bonded to the inert lysine core. The chemically synthesised TT MAP has a molecular weight of 7.8kDa which is shown in Tricine SDS-PAGE (Fig. 1a) and confirmed by immunoblot (Fig. 1b).

Fig. 1

Expression and Purification of recombinant proteins TRX and TGA The rTGA was expressed as 47kDa protein and rTRX was expressed as a 20 kDa protein without histidine tag (Fig. 2a, 2c). Both the proteins were expressed in E. coli (GJ1158) Recombinant TGA was obtained in the insoluble fraction and hence the lysate was subjected to urea solubilisation which was then purified by Immobilised Metal Affinity Chromatography (Fig. 2b). Recombinant TRX was purified in the non- denatured form by Anion exchange chromatography (Fig. 2d). The purified proteins were then confirmed by mouse anti TGA and anti TRX sera (Fig. 2e).

Fig. 2

Sera from mice were collected after each immunization and were used to measure the total IgG responses by ELISA. The antibody response was measured in terms of peak titers. Although the peak titers for TT MAP with MDP and alum were the same (1 in 64000) MDP group immunised with Individual proteins (TRX/TGA) showed a higher titer when compared with alum. TGA alone elicited a higher titre with MDP (1 in 1, 28,000) whereas TGA+alum elicited a peak titre of 1 in 32,000. MDP in combination with TRX elicited a peak titre of 1 in 32,000 which eventually dropped to 16,000 on 42^nd day (Fig. 3a, 3b). The isotype profile of TT MAP with alum/MDP showed a similar pattern with a higher IgG1 response. TT MAP with alum shows a significant increase of all isotypes as compared to the individual and TT Protein groups immunised with alum (a***, P<0.001 in Fig. 4a). In case of MDP as adjuvant, TGA immunised group shows a significant increase in IgG2b levels as compared to other groups (b*** in Fig 4b). The IgG1 levels of TGA-MDP was in par with TT MAP immunised with MDP. The isotype levels of TT MAP was significant (c**, P<0.01) as compared to TT protein group immunised with MDP.

Fig. 3

Fig. 4

The cell-mediated lymphoproliferative responses when examined using the splenocytes from each animal in proliferation assays, the maximum proliferation was observed for TT MAP with MDP. (Fig. 5). The difference in splenocyte proliferation was well pronounced between alum and MDP immunised groups (P < 0.001). The levels of the cytokines IL-2, IL-4, IL-5, IL-10, and IFN-ɤ were measured, to understand the cytokines involved in the cell-mediated pathway (Table 2).The IL-2 and IFN-ɤ levels in TT MAP with MDP immunized groups is higher than all the alum immunized groups. IL-4 levels in TT MAP with alum immunised groups were significantly higher than TT MAP with MDP group whereas there existed no significance in IL-5 levels of TT MAP groups. IL-10 level in TT MAP with MDP is significantly lower in comparison with alum immunised groups as wells as protein immunised groups with MDP/alum.

Fig. 5