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Immune Modulation and Prevention of Autoimmune Disease by Repeated Sequences from Parasites Linked to Self Antigens

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Abstract

Parasite proteins containing repeats are essential invasion ligands, important for their ability to evade the host immune system and to induce immunosuppression. Here, the intrinsic suppressive potential of repetitive structures within parasite proteins was exploited to induce immunomodulation in order to establish self-tolerance in an animal model of autoimmune neurological disease. We tested the tolerogenic potential of fusion proteins containing repeat sequences of parasites linked to self-antigens. The fusion constructs consist of a recombinant protein containing repeat sequences derived from the S-antigen protein (SAg) of Plasmodium falciparum linked to a CD4 T cell epitope of myelin. They were tested for their efficacy to control the development of experimental autoimmune encephalomyelitis (EAE), In addition, we used the DO11.10 transgenic mouse model to study the immune mechanisms involved in tolerance induced by SAg fusion proteins. We found that repeated sequences of P. falciparum SAg protein linked to self-epitopes markedly protected mice from EAE. These fusion constructs were powerful tolerizing agents not only in a preventive setting but also in the treatment of ongoing disease. The tolerogenic effect was shown to be antigen-specific and strongly dependent on the physical linkage of the T cell epitope to the parasite structure and on the action of anti-inflammatory cytokines like IL-10 and TGF-β. Other mechanisms include down-regulation of TNF-α accompanied by increased numbers of FoxP3+ cells. This study describes the use of repetitive structures from parasites linked to defined T cell epitopes as an effective method to induce antigen-specific tolerance with potential applicability for the treatment and prevention of autoimmune diseases.

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Acknowledgments

This work was supported by a Deutsche Forschungsgemeinschaft Grant SFB650. We thank Jorge Enrique Pineda for reviewing the manuscript.

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Authors and Affiliations

  1. Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany

    Fabiola Puentes, Katharina Dickhaut, Maria Hofstätter, Kirsten Falk & Olaf Rötzschke

  2. Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Neuroimmunology Unit. 4 Newark Street, London, E1 2AT, UK

    Fabiola Puentes

  3. Experimental Rheumatology, Deutsches Rheuma-Forschungszentrum, Berlin and Department of Rheumatology and Clinical Immunology, Charité–Universitätsmedizin Berlin, Berlin, Germany

    Jennifer Pfeil, Uta Lauer, Alf Hamann & Ute Hoffmann

  4. Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Olaf Rötzschke

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  1. Fabiola Puentes
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  2. Katharina Dickhaut
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Correspondence to Fabiola Puentes.

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Human and Animal Rights

All procedures performed in studies involving animals were in accordance with ethical standards of the Directive 86/609/EEC of the European Community Council and of the institutional, state and federal guidelines. All animal protocols were approved by the ethics committee of the Landesamt für Gesundheit und Soziales (LAGeSo, Berlin, Germany) with registration numbers G0140–06 and G0331–08.

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This work was supported by a Deutsche Forschungsgemeinschaft Grant SFB650. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Fabiola Puentes and Katharina Dickhaut contributed equally to the study as shared first author.

Electronic supplementary material

Fig. S1

Generation of parasite-derived fusion proteins. (A) Schematic diagram of fusion proteins. Parasite repetitive structures were fused through a linker to CD4+ T cell antigens and expressed in E.coli. Fusion proteins containing repetitive sequences of the S-antigen (SAg) of P. falciparum were designed: 24 repeats of a 8-mer unit derived from the NF7 isolate (A(L/R)KSDEAE) were linked to the N-terminus of the respective CD4+ T cell epitopes: MOG38–51 (SAg-MOG38–51), PLP139–151 (C140S) (SAg-PLP139–151) and OVA323–339 (SAg-OVA323–339). Since the natural units contained either L or R in their repeats, building blocks with alternating amino acids were used in tandem repeats. (B) Analysis of the expression of epitope fusion-constructs containing S-Antigen repeats fused to CD4 T cell epitopes in E. coli. Lane 1: SAg 12-mer (22 KDa); lane 2: SAg 12-mer MOG38–51 S3 (24Kda); lane 3: SAg 12-mer PLP139–151 S3 (24 KDa) and lane M: molecular weight markers. The weights calculated from the DNA sequence are lower compared to those estimated from the SDS gel due to abnormal SDS binding (SAg sequence is 38 % ASP and GLU) (Matagne et al. 1991). (GIF 6 kb)

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Fig. S2

Therapeutic effect of SAg fusion proteins. SJL/J mice (n = 8) were individually treated once the first clinical signs of EAE appeared. 50 μg of SAg-PLP fusion protein was given intravenously to diseased mice. Administration of fusion protein inhibits the evolution of EAE. The curve shows the mean ± SEM daily clinical score. Statistical significance between groups was determined by Mann-Whitney U test. **P < 0.01 and *P < 0.05. (GIF 23 kb)

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Fig. S3

Antigen-specific EAE protection after vaccination with SAg-PLP fusion proteins. EAE was induced in SJL/J mice using 50 μg of the encephalitogenic PLP139–151 peptide in CFA. The following day, animals received 200 ng of Pertussis toxin. Mice were vaccinated 7 days prior disease induction with 50 μg of SAg-PLP139–151 or unrelated SAg-OVA323–339 fusion proteins. Mice were monitored daily and the mean clinical score of five mice per group was plotted. One representative experiment of two performed is shown. (GIF 31 kb)

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Fig. S4

SAg repeat unit alone does not induce protection in the MOG-EAE model. EAE was induced in C57BL/6 mice by priming with 50 μg of MOG35–55 peptide and injection of 500 ng of Pertussis toxin one day after priming. Mice were vaccinated 7 days before disease induction with 70 μg of SAg-MOG fusion protein or SAg only. Mice were monitored daily and the average ± SEM of the clinical score of five mice per group was calculated. (GIF 30 kb)

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Fig. S5

T cell proliferation induced by SAg fusion protein. To compare the proliferative capacity of free peptide and SAg fusion constructs in vitro, [3H]Thymidine proliferation assay was performed. Lymph node cells were harvested from DO11.10 mice and 5x105 cells were incubated for 96 h with titrated amounts of SAg-OVA, free OVA323–339 and MOG38–51 as negative control. [3H]Thymidine was added after 72 h. Counts per minute (cpm) mean values and standard deviation of triplicates are shown. (GIF 21 kb)

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Fig. S6

Impact of in vivo neutralization of TGF-β on the protective effect induced by SAg fusion proteins. EAE was induced in SJL/J mice with PLP139–151 peptide. On day 7 after EAE induction, mice received intravenously 50 μg of SAg-PLP or in combination with 0.5 mg of αTGF-β antibody. Control groups were left untreated with or without αTGF-β antibody treatment. Neutralizing antibodies were administered intraperitoneally on days 7, 9 and 11. Animals were scored daily for the development of clinical signs of the disease. Mean clinical score from the groups (n = 5) are presented. (GIF 30 kb)

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Fig. S7

Suppressive capacity of SAg constructs is independent of B cells. EAE was induced in JHT+/− and JHT−/− mice after subcutaneous immunization with MOG35–55 peptide emulsified in adjuvant containing Mycobacterium tuberculosis and injection of Pertussis toxin. Mice were treated with SAg-MOG fusion protein and monitored daily. Averages of the clinical scores were calculated. The plot shows that SAg constructs can also induce tolerogenic effect in B cell deficient JHT−/− mice. (GIF 26 kb)

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Fig. S8

Antigen-specific suppression of pro-inflammatory TNF-α following treatment with SAg fusion proteins in B cell deficient mice. To measure the production of TNF-α in JHT−/− mice, OTII (OVA323–339 specific) cells labeled with CFSE were transferred into OVA323–339 primed JHT+/− and JHT−/− mice and treated with SAg-OVA. Lymph node cells were isolated from treated and untreated mice and activated for 6 h with OVA323–339 peptide and α-CD28. Cytokine secretion was blocked by the addition of Brefeldin A for the last 4 h of stimulation. Cells were stained for surface CD4 followed by intracellular staining with αTNF-α. For analysis of TNF-α in antigen-specific activated T cells; combined intracellular α-CD154 staining was performed. The frequency of CD154 + TNF-α + double positive in OTII CD4+ T cells was determined and analyzed by FACSDivaTM software (BD Bioscience). (GIF 25 kb)

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Fig. S9

Suppressive capacity of SAg-OVA in a DTH model. OVA323–339 specific T cells were isolated from DO11.10 mice and 3x106 CD4+ T cell were adoptively transferred intravenously into Balb/c mice. One day later, mice were tolerized by intravenous administration of 5 μg free OVA323–339 or 60 μg SAg-OVA (equimolar amounts, based on OVA-peptide amount). DTH reaction was induced on day 7 by adoptive transfer of OVA-specific cells that were cultured for 6 days under Th1 polarizing conditions. One day later mice were immunized into the footpad by intradermal injection of 250 ng OVA323–339 in IFA or PBS/ IFA. DTH reaction was measured 8 days post immunization by determining footpad swelling compared to days zero. (GIF 21 kb)

High resolution image (TIFF 116 kb)

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Puentes, F., Dickhaut, K., Hofstätter, M. et al. Immune Modulation and Prevention of Autoimmune Disease by Repeated Sequences from Parasites Linked to Self Antigens. J Neuroimmune Pharmacol 11, 749–762 (2016). https://doi.org/10.1007/s11481-016-9701-x

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