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.

Fig 1. The design of epitope-scaffold DTNF proteins.

(A) The crystal structure of the mTNF-α from PDB 2tnf. The neutralizing epitope-containing peptide was green colored. (B) The crystal structure of DTT from PDB 1MDT. The transplantation site is highlighted in green. The position of Th epitopes is indicated in red. The position of conformational perturbation in DTNF2, 5, 6 is colored in yellow, and that in DTNF4 is colored in blue. (C) The circular dichroism spectra of DTT and DTNF proteins. (D) Generation of epitope-scaffold DTNF proteins. Two to eight amino acid residues of DTT at indicated positions (red hyphens) were replaced by the TNF-α epitope peptide (in black and bold). TD (Å) stands for the interatomic distance between Valine 80 to Serine 96 or Valine 97 of TNF-α. DD (Å) stands for the interatomic distance between end residues of the replaced segment.

Fig 2. Antibody responses induced by DTT and DTNF proteins.

(A) Anti-DTT antibody titers in sera of mice immunized with DTT or DTNF proteins on day 49. (B) Anti-mTNF-α antibody titers in sera of mice immunized with DTT or DTNF proteins on day 49, serum samples were diluted 1:100. (C) Serum anti-mTNF-α and anti-hTNF-α titers measured by ELISA. Serum samples were diluted 1:100. Data are presented as the mean ± SD. (D) Average anti-mTNF-α antibody titers in sera of mice immunized with DTNF7 on day 49. (E) Analysis of antibody subclass in the sera of DTNF7 immunized mice. (F) The persistent antibody response against mTNF-α and DTT in mice immunized with DTNF7. Data are presented as the mean ± SD.

Fig 3. The stability of DTT and DTNF proteins.

(A) Thermal stability of DTT and DTNF proteins measured by DSC. Left panel, temperature dependent heat capacity of DTT and DTNF proteins. Right panel: melting temperatures of DTT and DTNF proteins. (B) SDS-PAGE analysis of the soluble fraction of DTT and DTNF proteins stored at 4°C for 2 days (Top) and 10 days (Bottom).

Fig 4. The molecular dynamics of DTNF proteins.

(A) Left panel: Molecular dynamic simulation of DTNF proteins. The secondary structure conformation was shown for residues belong to TNF-α epitope and DTT at positions 97–111. Right panel: Pie charts showing fraction of time the amino acid residue adopting indicated conformation during 300 ns simulation. (B)(C)(D) The RMSF trajectories of DTT and DTNF proteins. (E) Molecular dynamic simulation of the tertiary structure of DTNF7. The dashed circle highlights the positions of TNF-α epitope peptide at indicated time.

Fig 5. DTNF7 suppresses the development of collagen-induced arthritis in mice.

(A) Mice were immunized with chicken type II collagen (CII) emulsified with CFA on day1, and immunized with alum formulated DTT or DTNF7 on day 14, 28, and 42. (B) Arthritis incidence. (C) The average clinical scores of mice immunized with DTT or DTNF7. Data are presented as the mean clinical score ± SEM (DTT, n = 10; DTNF7, n = 9). (D) The titers of anti-mTNF-α in mice sera on day 56 measured by ELISA. Sera samples were diluted 1:200. Data are presented as the mean ± SD. (E) Examples of front and hind paws on day 28 and 56. (F) Mice body weights recorded during the entire experiments.

Fig 6. DTNF7 inhibits joint inflammation.

Tissue sections of ankle joints from DTNF7 and DTT immunized mice on day 56 were stained with hematoxylin-eosin staining (A) and Safranin O staining (B). The arrows indicate synovial hyperplasia (green) cell infiltration (black), cartilage destruction (orange), and bone erosion (blue).

Fig 7. The cytokine levels in mice sera immunized with DTT or DTNF7.

Serum samples were collected from CIA mice on day 28 and 56 after the first injection with chicken type II collagen. The cytokine concentrations in sera were determined by Bio-Plex assay. The average of four mice with standard variation was plotted for each cytokine. Data are presented as the mean ± SD.