Active immunotherapy for TNF-mediated inflammation using self-assembled peptide nanofibers

Abstract

Active immunotherapies raising antibody responses against autologous targets are receiving increasing interest as alternatives to the administration of manufactured antibodies. The challenge in such an approach is generating protective and adjustable levels of therapeutic antibodies while at the same time avoiding strong T cell responses that could lead to autoimmune reactions. Here we demonstrate the design of an active immunotherapy against TNF-mediated inflammation using short synthetic peptides that assemble into supramolecular peptide nanofibers. Immunization with these materials, without additional adjuvants, was able to break B cell tolerance and raise protective antibody responses against autologous TNF in mice. The strength of the anti-TNF antibody response could be tuned by adjusting the epitope content in the nanofibers, and the T-cell response was focused on exogenous and non-autoreactive T-cell epitopes. Immunization with unadjuvanted peptide nanofibers was therapeutic in a lethal model of acute inflammation induced by intraperitoneally delivered lipopolysaccharide, whereas formulations adjuvanted with CpG showed comparatively poorer protection that correlated with a more Th1-polarized response. Additionally, immunization with peptide nanofibers did not diminish the ability of mice to clear infections of Listeria monocytogenes. Collectively this work suggests that synthetic self-assembled peptides can be attractive platforms for active immunotherapies against autologous targets.

Keywords: Active immunotherapy; Adjuvant-free; Supramolecular; TNF; Vaccine.

Fig 1. TEM and schematic of co-assembled peptide nanofibers

Negative-stained TEM images of nanofibers (prepared at 2 mM total peptide concentration and diluted to 0.2 mM for imaging) (a) TNFQ11/Q11 (1:1 molar ratio), (b) PADREQ11/Q11 (1:1), (c) VACQ11/Q11 (1:1), (d) TNFQ11/PADREQ11/Q11 (1:0.05:0.95), (e) TNFQ11/VACQ11/Q11 (1:1.25:0.25). (f) schematic of co-assembled peptide nanofibers containing B-cell epitopes and T-cell epitopes.

Fig 2. Co-assembled peptide nanofibers raise anti-TNF antibodies

(a) TNFQ11 peptide-specific IgG responses in the sera of mice immunized with formulations with or without the self-assembling T-cell epitope PADREQ11, evaluated over time by ELISA. (b) Reactivity of immune sera against TNF cytokine (mouse), TNF_4–23 peptide, and PADRE peptide. (c) TNFQ11 peptide-specific IgG responses in the sera of mice immunized with formulations with or without the self-assembling T-cell epitope VACQ11, evaluated over time by ELISA. (d) Reactivity of immune sera against TNF cytokine, TNF_4–23 peptide, and VAC peptide. Only T-cell epitope-containing assemblies raised significant IgG responses against TNF peptide and TNF cytokine, and there was minimal antibody development against the T-cell epitopes PADRE or VAC. Formulations were 1 mM TNFQ11/0.05 mM PADREQ11/ 0.95 mM Q11 (a–b), 1mM TNFQ11/0.25 mM VACQ11/0.75 mM VACQ11 (c), and 1mM TNFQ11/1.25 mM VACQ11/0.25 mM Q11 (d). In (a, c) mice were immunized at week 0 and boosted at week 4 with a half dose. In (b, d), titers were measured after priming and two boosts at weeks 4 and 8. Mean ± SD is shown. n=4 for each group in a and c. n=5 for each group in b and d. Statistical significance was confirmed by t-test at each time point (a and c) or for each antigen (b and d). * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. The strength and phenotype of anti-TNF antibody responses can be modulated by the amount of T-cell epitopes within nanofibers

C57BL/6 mice were immunized with peptide formulations consisting of a fixed molar ratio of 1 mM TNFQ11 peptide (B-cell epitope) and progressively increasing amounts of PADREQ11 (a, b) or VACQ11 (c, d). Total peptide in the nanofibers was brought to 2 mM with a balance of unmodified Q11 peptide. Nanofibers containing PADREQ11 showed a clear maximum in anti-TNF IgG titers at 0.05 mM PADREQ11 by ELISA probed against whole mouse TNF (a), whereas nanofibers containing VACQ11 showed progressively increasing titers with increasing T-cell epitope content (c). Immunization with PADRE-containing nanofibers elicited predominantly IgG1 antibodies (b) whereas immunization with VAC-containing nanofibers elicited IgG responses more balanced between IgG1, IgG2b, IgG2c, and IgG3 (d). In a-b, for the various PADREQ11 contents (0, 0.002, 0.01, 0.05, 0.25, and 0.75 mM) group sizes were 3, 4, 4, 4, 4, and 4 respectively. c-d represent a combination of three independently conducted experiments, where sample sizes for the various groups were 4, 0, and 6 (0 mM); 5, 4, and 6 (0.002 mM); 4, 5, and 6 (0.01 mM); 3, 5, and 7 (0.05 mM); 4, 4, and 6 (0.25 mM); and 0, 4, and 6 (1.25 mM). Mean ± SD is shown. Statistical significance was confirmed by ANOVA followed by Tukey’s multiple comparison test. * p<0.05, *** p<0.001 compared to 0 mM T-cell epitope.

Figure 4. Co-assembled nanofibers elicited dose-dependent T-cell responses focused on the foreign CD4⁺ T-cell epitope, not the TNF B-cell epitope

Cytokine-secreting cells from mice immunized with TNFQ11 combined with the indicated doses of T-cell epitope (PADREQ11 or VACQ11) were quantified ex vivo by ELISPOT after re-stimulation of lymph node cell suspensions with peptide TNF, peptide PADRE (a, b) or peptide VAC (c, d). In a–b, for the various PADREQ11 contents (0, 0.002, 0.01, 0.05, 0.25, and 0.75 mM) group sizes were 2, 3, 4, 3, 4, and 4 mice, respectively. c–d represent a combination of two independently conducted experiments, where sample sizes for each group were 3 mice (data in the figure shows these two experiments together, with a total sample size of 6 mice per group). Mean ± SD is shown. Statistical significance was tested by two way ANOVA with Tukey’s multiple comparison test. Significance found between T and B-cell epitope stimulation at each T-cell epitope concentration is denoted. *p<0.05, **p<0.01, and ***p<0.001.

Figure 5. Immunization with peptide nanofibers protected mice from LPS-induced inflammation

Mice were challenged intraperitoneally with 10mg/kg LPS one week after final booster immunizations, and body temperature (a) and overall survival (b) were monitored. Humane endpoints of 32 °C (red line in a) or 20% body weight loss were employed. Group A, PADRE formulation 1: 1mM TNFQ11/0.05mM PADREQ11/0.95mM Q11; Group B, PADRE formulation 2: 0.2 mM TNFQ11/0.05 mM PADREQ11/1.75 mM Q11; Group C, VAC formulation: 1 mM TNFQ11/1.25 mM VACQ11/0.25 mM Q11; Group D, same VAC formulation as Group C, plus 10 µg CpG; Group E, negative control, missing any T-cell epitope; Group F, negative control, unvaccinated mice receiving LPS challenge; Group G, positive control: unvaccinated mice not receiving LPS challenge. Formulations containing TNFQ11 and either T-cell epitope offered significant protection, whereas unimmunized mice, vaccinations without T-cell epitopes, and CpG-containing vaccinations had poor survival. Data points in (a) represent individual mice. Box-and-whisker plots indicate medians and 25%/75% quartiles (horizontal line and box) and max/min values (whiskers). For Group D, quartile boxes are omitted where the number of surviving mice drops below 4. Statistical comparisons of survival between all groups in (b) were made using logrank test, *p<0.05, ns p>0.05. Individual p-values between all groups and descriptions of experimental replicates are provided in Table S2. All experiments shown in (a) were performed at Duke University, and (b) shows overall survival curves for the mice shown in (a) plus replicate experiments performed at the University of Chicago. ELISPOT analysis (c–d) of Th1 (IFNγ) and Th2 (IL-4)-secreting lymph node cells from unadjuvanted nanofiber immunizations (Group C), compared with nanofibers adjuvanted with CpG (Group D). Shown are total numbers of spots (c) and ratios of IFNγ/IL-4 spots (d). Mean ± SD is shown. *p<0.05 by ANOVA with Tukey’s multiple comparison (c) or two-tailed t-test (d).

Figure 6. Immunization with peptide assemblies did not increase susceptibility to infection by Listeria monocytogenes

Unimmunized mice and mice immunized with the indicated nanofiber formulations were challenged intraperitoneally with 1 × 10⁵ Listeria colony-forming units (CFU). 48 hours later, CFU were counted in the spleen and liver. Mice injected with 500 µg of anti-TNF antibody 3h prior to intraperitoneal Listeria challenge had elevated Listeria CFU 48 hours later (a). Conversely, there were no significant differences in total CFU counts per organ between vaccinated mice, unvaccinated mice, and mice vaccinated with nanofibers lacking T-cell epitopes (b). n=5 for all groups, data points represent individual mice. Mean ± SD is shown. Statistical significance was tested using two-way ANOVA with Tukey’s multiple comparison test. ns: not significant, * p<0.05, ***p<0.001.