A Rationally Designed TNF-α Epitope-Scaffold Immunogen Induces Sustained Antibody Response and Alleviates Collagen-Induced Arthritis in Mice

Abstract

The TNF-α biological inhibitors have significantly improved the clinical outcomes of many autoimmune diseases, in particular rheumatoid arthritis. However, the practical uses are limited due to high costs and the risk of anti-drug antibody responses. Attempts to develop anti-TNF-α vaccines have generated encouraging data in animal models, however, data from clinical trials have not met expectations. In present study, we designed a TNF-α epitope-scaffold immunogen DTNF7 using the transmembrane domain of diphtheria toxin, named DTT as a scaffold. Molecular dynamics simulation shows that the grafted TNF-α epitope is entirely surface-exposed and presented in a native-like conformation while the rigid helical structure of DTT is minimally perturbed, thereby rendering the immunogen highly stable. Immunization of mice with alum formulated DTNF7 induced humoral responses against native TNF-α, and the antibody titer was sustained for more than 6 months, which supports a role of the universal CD4 T cell epitopes of DTT in breaking self-immune tolerance. In a mouse model of rheumatoid arthritis, DTNF7-alum vaccination markedly delayed the onset of collagen-induced arthritis, and reduced incidence as well as clinical score. DTT is presumed safe as an epitope carrier because a catalytic inactive mutant of diphtheria toxin, CRM197 has good clinical safety records as an active vaccine component. Taken all together, we show that DTT-based epitope vaccine is a promising strategy for prevention and treatment of autoimmune diseases.

The Immunogenicity and Anti-Tumor Efficacy of a Rationally Designed EGFR Vaccine

“…Previous studies have shown that DTT can help break immune self-tolerance when an immunogen is fused to its C-terminal [16]. For immunogen specific against mEGFR, both ECD domain II (aa190-334) and domain III (aa335-505) are considered as they are functionally important for the receptor activation.…”

“…In order to mimic the native conformation of the epitope peptide, we grafted the peptide onto the DTT at positions aa 88-94 that has been shown an ideal site for epitope presentation [16]. EGFR 265-283 is grafted to minimize structural perturbation of the carrier DTT, according to the method of Li Zhang et al [16]. To achieve higher anti-tumor efficacy of the vaccine, we considered fusion of ECD subdomain III to the C-terminal of DEGFR p .…”

“…Finally, the amino acids 88-94 of DTT was replaced by EGFR 265-283 (NH2-PLMLYNPTTYQMDVNPEG-COOH) using Discovery studio 3.5. Homology model of fusion protein was built based on the X-ray structure of DTT (PDB entry 1MDT, 2.3Å) using Modeller9V7 [16]. …”

“…The DTT expression vector was described previously [16]. The recombinant DNA fragment encoding DTT 1-87 -EGFR 265-283 -DTT 95-176 -EGFR 334-505 was chemically synthesized, cloned into pET-32a after BamHI and Xho I digestion.…”

“…In our previous studies, we show that the translocation domain of diphtheria toxin (DTT) is a safe antigen carrier capable of breaking immune self-tolerance by virtue of its universal help T cell epitopes [16, 17]. Moreover, DTT-based antigen is immunogenic when formulated with Alum, a widely used adjuvant in human vaccination.…”