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

I. Christiana; R. Aparnaa; R. Rohit; D. Nageswara Rao; P. Kaliraj

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

| 22 jul 2016

Volumen 53 (2016): Edición 3 (September 2016)

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Article Category: Research Article

Publicado en línea: 22 jul 2016

Páginas: 224 - 232

Recibido: 09 nov 2014

Aceptado: 01 jun 2015

DOI: https://doi.org/10.1515/helmin-2016-0022

Palabras clave
Filariasis, Thioredoxin, Transglutaminase, Multi Antigen Peptide (MAP), Muramyl dipeptide (MDP), Adjuvant

© 2016 Institute of Parasitology, SAS, Košice

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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 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

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)

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)

Antigens	Epitope Sequences
Transglutaminase peptide	KEFLLHETNGLVGIRTSENRYQFD
Thioredoxin peptide 1	ADLLANINLKKADGTVKKGSDALANK
Thioredoxin peptide 2	SEIEKLKNKYEVAGIP

Cytokine levels

Groups		IL2	IFN-G	IL-4	IL-5	IL-10
TRX	Alum	98 ± 10.6	160.81 ± 12.21a = P<0.005, Comparison of individual groups of alum and MDP	321 ± 3.11a = P<0.005, Comparison of individual groups of alum and MDP	230.33 ± 6.55a = P<0.005, Comparison of individual groups of alum and MDP	89.27 ± 2.29
TRX	MDP	144.25 ± 1.76a = 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 ± 1.45^a
TGA	Alum	177.35 ± .88	375.81 ± 6.423	509 ± 2.49	216.85 ± 6.766	61.11 ± .66
TGA	MDP	199.25 ± 5.3a = P<0.005, Comparison of individual groups of alum and MDP	367.45 ± 3.21	547.38 ± 1.35	336.30 ± 3.9a = P<0.005, Comparison of individual groups of alum and MDP	90.64 ± 0.06a = P<0.005, Comparison of individual groups of alum and MDP
TT Protein	Alum	127.37 ± 0.88	230.54 ± 1.28a = P<0.005, Comparison of individual groups of alum and MDP	329.88 ± 6.66a = P<0.005, Comparison of individual groups of alum and MDP	283.88 ± 0.922	94.44 ± .54
TT Protein	MDP	159.25 ± 1.78a = P<0.005, Comparison of individual groups of alum and MDP	150.81 ± 5.78	293.11 ± 4.57b = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups	263.95 ± 6.55	132.62 ± 7.25a = 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 MAP	Alum	149.25 ± 8.83	214 ± 2.86	341 ± 3.26a = P<0.005, Comparison of individual groups of alum and MDP	293 ± 5.48	85 ± 4.32a = P<0.005, Comparison of individual groups of alum and MDP
TT MAP	MDP	221 ± 8.87a = 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 ± 2.86a = 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 ± 6.86	309.92 ± 5.09b = P<0.005, Comparison of TT Protein MDP and TT MAP MDP groups	44.4 ± 0.2

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

Article Category: Research Article

Publicado en línea: 22 jul 2016

Páginas: 224 - 232

Recibido: 09 nov 2014

Aceptado: 01 jun 2015

DOI: https://doi.org/10.1515/helmin-2016-0022

Palabras clave
Filariasis, Thioredoxin, Transglutaminase, Multi Antigen Peptide (MAP), Muramyl dipeptide (MDP), Adjuvant

© 2016 Institute of Parasitology, SAS, Košice

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

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

Cytokine levels

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

Article Category: Research Article

Publicado en línea: 22 jul 2016

Páginas: 224 - 232

Recibido: 09 nov 2014

Aceptado: 01 jun 2015

DOI: https://doi.org/10.1515/helmin-2016-0022

Palabras claveFilariasis, Thioredoxin, Transglutaminase, Multi Antigen Peptide (MAP), Muramyl dipeptide (MDP), Adjuvant

© 2016 Institute of Parasitology, SAS, Košice

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

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

Cytokine levels

Palabras clave
Filariasis, Thioredoxin, Transglutaminase, Multi Antigen Peptide (MAP), Muramyl dipeptide (MDP), Adjuvant