Lymphatic filariasis (LF) is a mosquito-borne parasitic infection responsible for long-term chronic morbidity in the form of lymphoedema, genital pathology (especially hydroceles), recurrent disabling fevers (lymphangitis) and elephantiasis in over 40 million people around the world (Ottesen
A new approach in vaccine technology is using recombinant protein subunits as peptide vaccines. Synthetic peptide vaccines and B–T cell chimera’s containing immunodominant regions from antigens have been developed for malaria (Nardin
Adjuvants are usually defined as compounds that can increase and/or modulate the intrinsic immunogenicity of an antigen. Adjuvants are molecules, compounds or macromolecular complexes that boost the potency and longevity of specific immune response to antigens, but cause minimal toxicity or long lasting immune effects on their own (Wack, 2005). Alum is the universally licensed adjuvant so far used in humans approved by the Food and Drug Administration (FDA). Aluminum adjuvants selectively enhance the type 2 immune response. Although alum is able to induce a good antibody (TH2) response, it has little capacity to stimulate cellular (TH1) immune responses which are important for protection against many pathogens. Alum has potential to cause severe local and more serious systemic side- effects. (Petrovsky & Aguilar, 2004). Hence, there is demand for safe and non-toxic adjuvants which are able to stimulate cellular (TH1) immunity.
N-acetylmuramyl-L-alanyl-D-glutamic acid (AcMur-L-Ala-D-Glu), a synthetic analog of Muramyl Di Peptide (MDP) increases the humoral immune response when given in aqueous media instead of the usual water-in-oil emulsion (Audibert
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.
Epitope sequences in Thioredoxin Transglutaminase Multi Antigen Peptide (TT MAP)
Antigens | Epitope Sequences |
---|---|
Transglutaminase peptide | KEFLLHETNGLVGIRTSENRYQFD |
Thioredoxin peptide 1 | ADLLANINLKKADGTVKKGSDALANK |
Thioredoxin peptide 2 | SEIEKLKNKYEVAGIP |
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
Protein concentrations were estimated by Bradford assay (Biorad, CA, USA) and then used for immunization.
Six to eight weeks old female BALB/c (H-2d) 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
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
Thioredoxin-Transglutaminase Multi Antigen Peptide (TT MAP):
1a. SDS-PAGE analysis of the synthetic TT MAP antigen: Lane 1 - Molecular marker, Lane 2 -TT MAP.
1b. Immunoblot analysis of TT MAP peptide: Lane M - Molecular marker, Lane 1-3 - Cross-reactivity of TT MAP with antibodies raised in mice against individual peptides (Bm TGA P1, Bm TRX P1 and Bm TRX P2, respectively)
Expression and purification of recombinant proteins:
(2a) Expression of the rTGA analyzed by 12 % SDS gel: Lane 1- Molecular marker, Lane 2 - pRSETB TGA uninduced, Lane 3 - pRSETB TGA induced
(2b) Purification of rTGA analyzed by 12 % SDS gel: Lane 1 - Molecular marker, Lanes 2-4 Pure TGA elutes
(2c) Expression of rTRX analyzed by 12 % SDS gel. Lane 1 - Molecular marker, Lane 2 - Expression of the 20 kDa rTRX produced from
(2d) Purification of rTRX analyzed by 12 % SDS gel: Lane 1- Molecular marker, Lane 2- Flow through elute of TRX, Lanes 3-7 - Pure TRX elutes
(2e) Immunoblot cross-reactivity of purified rTRX and TGA with anti filarial mouse serum: Lane 1 - Molecular marker, Lane 2 - immunoblot with anti-TRX sera, Lane 3 - immunoblot with anti-TGA sera
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 42nd 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.
Humoral immune response of TT MAP with Alum and MDP
3a. Antigen specific antibody titers (in thousands) in mice immunised with alum as adjuvant at day 14, 21, 35 and 42 post-immunization.
3b. Antigen specific antibody titers (in thousands) in mice immunised with MDP as adjuvant at day 14, 21, 35 and 42 post-immunization.
3c. Comparison of peak antibody titers followed by immunization with TRX, TGA, TT protein and TT MAP with alum/MDP as adjuvants. Individual points represent mean peak titers determined on day 35 (n=5 mice per experimental group)
Immunglobulin isotype profiles detected after the immunizations with Alum or MDP adjuvant
4a. Immunoglobulin isotype profile (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) followed by immunization with TRX, TGA, TT protein and TT MAP with alum as adjuvant.
4b. Immunoglobulin isotype profile (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) followed by immunization with TRX, TGA, TT protein, TT MAP with MDP as adjuvant. The antibody isotypes measured in 1:500 diluted sera are shown in bars. Data represents mean titer ± SD (n=5 mice per experimental group). Asterisks represents level of significance between control (adjuvant) and immunised groups (*** P < 0.001; ** P<0.01)
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.
Splenocyte proliferation
Splenocyte proliferation in different mice groups stimulated with corresponding antigens TRX, TGA, TT protein, TT MAP, compared to MDP/Alum controls. Data are presented as mean (± SD) Stimulation Index (S.I.) of five mice per experimental group. Asterisks represents level of significance between the TGA and TT MAP group where the MDP was used as adjuvant (*** P < 0.001)
Cytokine levels
Groups | IL2 | IFN-G | IL-4 | IL-5 | IL-10 | |
---|---|---|---|---|---|---|
TRX | Alum | 98 ± 10.6 | 160.81 ± 12.21 = P<0.005, Comparison of individual groups of alum and MDP | 321 ± 3.11 = P<0.005, Comparison of individual groups of alum and MDP | 230.33 ± 6.55 = P<0.005, Comparison of individual groups of alum and MDP | 89.27 ± 2.29 |
MDP | 144.25 ± 1.76 = P<0.005, Comparison of individual groups of alum and MDP | 100.81 ± 3.21 | 259.29 ± 0.41 | 116.53 ± 3.68 | 110.47 ± 1.45a | |
TGA | Alum | 177.35 ± .88 | 375.81 ± 6.423 | 509 ± 2.49 | 216.85 ± 6.766 | 61.11 ± .66 |
MDP | 199.25 ± 5.3 = P<0.005, Comparison of individual groups of alum and MDP | 367.45 ± 3.21 | 547.38 ± 1.35 | 336.30 ± 3.9 = P<0.005, Comparison of individual groups of alum and MDP | 90.64 ± 0.06 = P<0.005, Comparison of individual groups of alum and MDP | |
TT Protein | Alum | 127.37 ± 0.88 | 230.54 ± 1.28 = P<0.005, Comparison of individual groups of alum and MDP | 329.88 ± 6.66 = P<0.005, Comparison of individual groups of alum and MDP | 283.88 ± 0.922 | 94.44 ± .54 |
MDP | 159.25 ± 1.78 = P<0.005, Comparison of individual groups of alum and MDP | 150.81 ± 5.78 | 293.11 ± 4.57 = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups | 263.95 ± 6.55 | 132.62 ± 7.25 = P<0.005, Comparison of individual groups of alum and MDP = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups | |
TT MAP | Alum | 149.25 ± 8.83 | 214 ± 2.86 | 341 ± 3.26 = P<0.005, Comparison of individual groups of alum and MDP | 293 ± 5.48 | 85 ± 4.32 = P<0.005, Comparison of individual groups of alum and MDP |
MDP | 221 ± 8.87 = P<0.005, Comparison of individual groups of alum and MDP = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups | 412 ± 2.86 = P<0.005, Comparison of individual groups of alum and MDP = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups | 226.91 ± 6.86 | 309.92 ± 5.09 = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups | 44.4 ± 0.2 |
Cytokine levels (pg/ml) in culture supernatants of spleen cells, from mice, immunized with the TRX, TGA, TT Protein or TT MAP with MDP as adjuvant and stimulated
Filariasis is considered one of the most complex infections of humans, and sufficient immunoprophylactic studies for lymphatic filariasis has not been carried out. The widespread chronic infection and the morbidity are due to the spectrum of immune evasion strategy developed by the filarial nematode. Mouse models provide us with the insight into the possible mechanism by which the filarial nematode can be destroyed (Lawrence & Devaney, 2001).
Metabolically active enzymes are highly conserved and hence they share homologous regions with host proteins which may show cross reactivity when developed as vaccines (Madhumathi
The present study which focuses on the immune responses of TT MAP formulated with MDP is an attempt to improve the vaccine efficacy of TT MAP. MDP, the synthetic analogue of the bacterial peptidoglycan moiety has been reported as safe and potent adjuvant in various immunisation studies (Parant, 1979; Moschos
In conclusion, our findings with MDP as adjuvant administered with TT MAP via intramuscular route in Balb/c mice model was safe and the formulation elicited a higher antibody response with a balanced TH1/TH2 response. However the assessment of this formulation capable of contributing to protective immune response could be confirmed only after immunisation and challenge studies carried out in permissive animal models.