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

Helminthologia
Volume 53 (2016): Issue 3 (September 2016)
By:
Open Access
|Jul 2016

Figures & Tables

Fig. 1

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)
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)

Fig. 2

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 E. coli (GJ1158)(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
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 E. coli (GJ1158)(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

Fig. 3

Humoral immune response of TT MAP with Alum and MDP3a. 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)
Humoral immune response of TT MAP with Alum and MDP3a. 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)

Fig. 4

Immunglobulin isotype profiles detected after the immunizations with Alum or MDP adjuvant4a. 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)
Immunglobulin isotype profiles detected after the immunizations with Alum or MDP adjuvant4a. 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)

Fig. 5

Splenocyte proliferationSplenocyte 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)
Splenocyte proliferationSplenocyte 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)

Epitope sequences in Thioredoxin Transglutaminase Multi Antigen Peptide (TT MAP)

AntigensEpitope Sequences
Transglutaminase peptideKEFLLHETNGLVGIRTSENRYQFD
Thioredoxin peptide 1ADLLANINLKKADGTVKKGSDALANK
Thioredoxin peptide 2SEIEKLKNKYEVAGIP

Cytokine levels

GroupsIL2IFN-GIL-4IL-5IL-10
TRXAlum98 ± 10.6160.81 ± 12.21
a

= P<0.005, Comparison of individual groups of alum and MDP

321 ± 3.11
a

= P<0.005, Comparison of individual groups of alum and MDP

230.33 ± 6.55
a

= P<0.005, Comparison of individual groups of alum and MDP

89.27 ± 2.29
MDP144.25 ± 1.76
a

= P<0.005, Comparison of individual groups of alum and MDP

100.81 ± 3.21259.29 ± 0.41116.53 ± 3.68110.47 ± 1.45a
TGAAlum177.35 ± .88375.81 ± 6.423509 ± 2.49216.85 ± 6.76661.11 ± .66
MDP199.25 ± 5.3
a

= P<0.005, Comparison of individual groups of alum and MDP

367.45 ± 3.21547.38 ± 1.35336.30 ± 3.9
a

= P<0.005, Comparison of individual groups of alum and MDP

90.64 ± 0.06
a

= P<0.005, Comparison of individual groups of alum and MDP

TT ProteinAlum127.37 ± 0.88230.54 ± 1.28
a

= P<0.005, Comparison of individual groups of alum and MDP

329.88 ± 6.66
a

= P<0.005, Comparison of individual groups of alum and MDP

283.88 ± 0.92294.44 ± .54
MDP159.25 ± 1.78
a

= P<0.005, Comparison of individual groups of alum and MDP

150.81 ± 5.78293.11 ± 4.57
b

= P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups

263.95 ± 6.55132.62 ± 7.25
a

= P<0.005, Comparison of individual groups of alum and MDP

,
b

= P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups

TT MAPAlum149.25 ± 8.83214 ± 2.86341 ± 3.26
a

= P<0.005, Comparison of individual groups of alum and MDP

293 ± 5.4885 ± 4.32
a

= P<0.005, Comparison of individual groups of alum and MDP

MDP221 ± 8.87
a

= P<0.005, Comparison of individual groups of alum and MDP

,
b

= P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups

412 ± 2.86
a

= P<0.005, Comparison of individual groups of alum and MDP

,
b

= P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups

226.91 ± 6.86309.92 ± 5.09
b

= P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups

44.4 ± 0.2
DOI: https://doi.org/10.1515/helmin-2016-0022 | Journal eISSN: 1336-9083 | Journal ISSN: 0440-6605
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Language: English
Page range: 224 - 232
Submitted on: Nov 9, 2014
Accepted on: Jun 1, 2015
Published on: Jul 22, 2016
Published by: Slovak Academy of Sciences, Mathematical Institute
In partnership with: Paradigm Publishing Services
Publication frequency: 4 times per year
Keywords:
Filariasis,
Thioredoxin,
Transglutaminase,
Multi Antigen Peptide (MAP),
Muramyl dipeptide (MDP),
Adjuvant
Related subjects:
Life sciences,
Zoology,
Ecology,
Life sciences, other,
Medicine,
Clinical medicine,
Microbiology, virology and infection epidemiology

© 2016 I. Christiana, R. Aparnaa, R. Rohit, D. Nageswara Rao, P. Kaliraj, published by Slovak Academy of Sciences, Mathematical Institute
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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