Nanoparticle-detained toxins for safe and effective vaccination

Abstract

Toxoid vaccines--vaccines based on inactivated bacterial toxins--are routinely used to promote antitoxin immunity for the treatment and prevention of bacterial infections. Following chemical or heat denaturation, inactivated toxins can be administered to mount toxin-specific immune responses. However, retaining faithful antigenic presentation while removing toxin virulence remains a major challenge and presents a trade-off between efficacy and safety in toxoid development. Here, we show a nanoparticle-based toxin-detainment strategy that safely delivers non-disrupted pore-forming toxins for immune processing. Using erythrocyte membrane-coated nanoparticles and staphylococcal α-haemolysin, we demonstrate effective virulence neutralization via spontaneous particle entrapment. Compared with vaccination with heat-denatured toxin, mice vaccinated with the nanoparticle-detained toxin showed superior protective immunity against toxin-mediated adverse effects. We find that the non-disruptive detoxification approach benefited the immunogenicity and efficacy of toxoid vaccines. We anticipate that this study will open new possibilities in the preparation of antitoxin vaccines against the many virulence factors that threaten public health.

Figure 1. Schematic and in vitro characterizations

(a) Schematic preparation of nanoparticle-detained toxins, denoted as nanotoxoid, consisting of substrate-supported RBC membranes into which pore-forming toxins (PFTs) can spontaneously incorporate. (b) TEM visualization of the particle vectors with uranyl acetate staining (scale bar = 80 nm). (c) Western blotting results to verify the retention of 3 µg of staphylococcal α-hemolysin (Hla) by varying amounts of the particle vectors using 3 µg of free Hla as a standard (SD). (d) Release of toxin from the Hla-loaded nanotoxoids, denoted as nanotoxoid(Hla), over time in PBS buffer. Red circles indicate nanotoxoid(Hla) and black squares indicate free Hla. Error bars represent standard deviations of the mean. (e) Uptake of nanotoxoid(Hla) by a mouse dendritic cell (scale bar = 10 µm). The cell is membrane stained with DMPE-rhodamine B (red) and nuclei stained with DAPI (blue). FITC-labelled Hla (green) was used to monitor the toxin uptake. (f) Live, whole-body fluorescent imaging of nanotoxoid(Hla) at 1 h after subcutaneous administration.

Figure 2. Nanotoxoid(Hla) neutralises Hla virulence

(a) Free Hla, heat-treated Hla (30 min), heat-treated Hla (60 min), and nanotoxoid(Hla) were injected into the superficial dorsal skin of mice. 24 h following the injections, the skin was removed and examined for apoptosis using a TUNEL assay. Histological analyses were performed with H&E stained skin 48 h following the injections (Scale bar = 400 µm). (b) Toxicity of different Hla formulations against dendritic cells derived from mice. The cells were incubated for 48 h with Hla, heat-treated Hla (60 min) and nanotoxoid(Hla) at 15 µg/mL Hla concentration. Cellular viability was assessed using an MTT assay (n=6). (c) Induction of dendritic cell apoptosis by nanotoxoid(Hla) at 60 µg/mL Hla concentration 72 h after initial incubation. Propidium iodide and Annexin V staining were analysed by flow cytometry (n=6). All error bars represent standard deviations of the mean.

Figure 3. Nanotoxoid(Hla) vaccinations elicit strong Hla-specific antibody responses

(a) Hla-specific antibody responses were verified in the nanotoxoid(Hla)-vaccinated mice through coomassie staining (left panel) and western blotting (right panel). Blank particle vector, denoted as nanotoxoid(-), was used as a control. (b) RBC counts of mice immunized with nanotoxoid(Hla) (n=6). (c) Anti-Hla IgG titres at day 21 (n=7). Black lines indicate geometric means. Anti-Hla titres from mice vaccinated with non-toxin loaded particle vectors (nanotoxoid(-)) were monitored as controls (triangle). (d) Time course of anti-Hla IgG titres in unvaccinated mice (black triangle) and mice immunized with nanotoxoid(Hla) (prime + boost; red circle) or nanotoxoid(Hla) (prime only; blue circle) (n=7). (e) Avidity index of the anti-sera from immunized mice binding to Hla toxin was quantified (n=7). (f) An RBC haemolysis assay was performed to verify the presence of functional titres (n=7). All error bars represent standard deviations of the mean.

Figure 4. Nanotoxoid(Hla) vaccinations bestow strong protective immunity

Unvaccinated mice (black) and mice vaccinated with heat-treated Hla (prime; blue square), nanotoxoid(Hla) (prime; blue circle), heat-treated Hla (prime + boost; red square), or nanotoxoid(Hla) (prime + boost; red circle) received intravenous or subcutaneous administration of Hla. (a) Survival rates of mice over a 15-day period following intravenous injections of 120 µg/kg Hla on day 21 via the tail vein (n=10). The unvaccinated mice were used as a negative control and mice vaccinated with heat-treated Hla served as positive controls. Both the prime only schedule and the prime-boost schedule were conducted. (b) Skin lesion size comparison following subcutaneous injections of 5 µg of Hla on day 21. The lesion size was measured for 14 days following the challenge. The means ± SD for each vaccination group were plotted (n=6).