Poly(amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines

Abstract

To be optimally effective, peptide-based vaccines need to be administered with adjuvants. Many currently available adjuvants are toxic, not biodegradable; they invariably invoke adverse reactions, including allergic responses and excessive inflammation. A nontoxic, biodegradable, biocompatible, self-adjuvanting vaccine delivery system is urgently needed. Herein, we report a potent vaccine delivery system fulfilling the above requirements. A peptide antigen was coupled with poly-hydrophobic amino acid sequences serving as self-adjuvanting moieties using solid-phase synthesis, to produce fully defined single molecular entities. Under aqueous conditions, these molecules self-assembled into distinct nanoparticles and chain-like aggregates. Following subcutaneous immunization in mice, these particles successfully induced opsonic epitope-specific antibodies without the need of external adjuvant. Mice immunized with entities bearing 15 leucine residues were able to clear bacterial load from target organs without triggering the release of soluble inflammatory mediators. Thus, we have developed a well-defined and effective self-adjuvanting delivery system for peptide antigens.

Fig. 1. Schematic structures of compounds 1 to 5.

The vaccine candidates were constructed from three building blocks: the B cell epitope (J8), the T helper epitope (PADRE), and a poly-hydrophobic amino acid (pHAA) unit.

Fig. 2. Physicochemical characterization of compounds 1 to 5.

TEM photographs of the vaccine compounds (A) 2, (B) 3, (C) 4, and (D) 5 (scale bars, 200 nm). (E) Circular dichroism (CD) spectra of compounds 1 to 5.

Fig. 3. J8-specific antibody titer and average opsonization percentage of different GAS strains.

Experimental mice (C57BL/6, n = 10) were vaccinated subcutaneously (on days 0, 21, 28, and 35) with compounds 1 to 5. Each triangle in (A) to (C) represents an individual mouse; the mean J8-specific titers are presented as a bar. (A) Titers of J8-specific immunoglobulin G (IgG) in serum, (B) titers of J8-specific IgA in saliva, and (C) titers of J8-specific IgG in saliva, collected after final immunization. (D) Average percentage opsonization of GAS strain 2002, (E) D2612, (F) D3840, (G) GC2 203, (H) 5203, and (I) 5199. PBS: negative control group, mice immunized with PBS; 1/CFA: positive control group, compound 1 emulsified with CFA. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test compared with PBS indicated as ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4. Bacterial burden after intranasal challenge with M1 GAS strain in C57BL/6 mice (n = 10 per group).

Bacterial burden results are represented as the mean CFU ± SEM for each group. (A) Nasal shedding, (B) throat swabs, (C) colonization of nasal-associated lymphoid tissue (NALT), and (D) colonization of spleen tissue. PBS: negative control group, mice immunized with PBS; 1/CFA: positive control group, compound 1 emulsified with CFA. On day 1, 10 mice were examined; on day 2, 5 mice were examined and euthanized for spleen and NALT organ harvesting; and the remaining 5 mice were processed on day 3. Statistical analysis was performed using a two-way ANOVA followed by Tukey’s post hoc test compared with PBS indicated as ns, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 5. Cytokine production following stimulation with 5 and 1 with or without costimulation of LPS.

Cytokine production is expressed as pg/mL ± SD. (A) Production of IL-1β stimulated by LPS (0.1 μg/ml), silica (125 μg/ml), peptide 1 (0.1 mg/ml), and compound 5 (0.1 mg/ml). (B) Production of TNF stimulated by LPS (0.001 μg/ml), peptide 1 (0.1 mg/ml), and compound 5 (0.1 mg/ml). Positive control: cells stimulated with LPS alone or in combination with silica. Negative control: untreated cells. Cells were prestimulated with LPS for 3 hours followed by either 6 hours of stimulation with silica or 4 hours of stimulation with 5 and control peptide 1. ND, not detectable.