Nanoparticle-Based Antivirulence Vaccine for the Management of Methicillin-Resistant Staphylococcus aureus Skin Infection

Abstract

With the rising threat of antibiotic-resistant bacteria, vaccination is becoming an increasingly important strategy to prevent and manage bacterial infections. Made from deactivated bacterial toxins, toxoid vaccines are widely used in the clinic as they help to combat the virulence mechanisms employed by different pathogens. Herein, the efficacy of a biomimetic nanoparticle-based anti-virulence vaccine is examined in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Vaccination with nanoparticle-detained staphylococcal α-hemolysin (Hla) effectively triggers the formation of germinal centers and induces high anti-Hla titers. Compared to mice vaccinated with control samples, those vaccinated with the nanoparticle toxoid show superior protective immunity against MRSA skin infection. The vaccination not only inhibits lesion formation at the site of bacterial challenge, but also reduces the invasiveness of MRSA, preventing dissemination into other organs. Overall, this biomimetic nanoparticle-based toxin detainment strategy is a promising method for the design of potent anti-virulence vaccines for managing bacterial infections.

Keywords: MRSA infection; anti-virulence vaccination; biomimetic nanoparticle; toxoid; α-hemolysin.

Figure 1. Schematic of nanotoxoid(Hla) protection against MRSA infection

(a) Under normal conditions, MRSA bacteria employ Hla to help them colonize the site of challenge, resulting in significant skin lesion formation and systemic invasiveness. (b) After vaccination with the nanotoxoid(Hla) formulation, anti-Hla titers are induced. These antibodies neutralize the toxin produced by the MRSA bacteria at the site of challenge, reducing the ability of the pathogen to colonize and enter into systemic circulation.

Figure 2. Nanotoxoid(Hla) characterization

(a) Size and (b) zeta potential of nanotoxoid(−) [denoted “NT(−)”] and nanotoxoid(Hla) [denoted “NT(Hla)”] (n=3). Error bars represent standard deviation. (c) TEM image of nanotoxoid(Hla) after negative staining with uranyl acetate. Scale bar = 100 nm. (d) TEM images of immunogold-stained NT(−) and NT(Hla) with anti-Hla as the primary immunostain and gold-labeled anti-IgG as the secondary stain. The gold (~10 nm) appears as dark punctates on the images. Scale bar = 100 nm. (e) Dot blotting results using anti-Hla as the primary immunostain. Quantification by image analysis revealed that 95.2% of the Hla input was retained on the final NT(Hla) formulation.

Figure 3. Germinal center formation and antibody production induced by nanotoxoid(Hla) vaccination

(a,b) Mice were vaccinated with PBS, nanotoxoid(−) [NT(−)], or nanotoxoid(Hla) [NT(Hla)] (n=3). The draining lymph nodes were collected 21 days later for the analysis of B220 (blue), IgD (green), and GL-7 (red) expression by either immunohistochemistry (a) or flow cytometry (b). Scale bars = 250 μm. For flow cytometric analysis, cells were first gated on B220⁺IgD^low and the numbers reported are the percentage GL-7⁺ cells within that population. Error bars represent standard error. Statistical significance determined by one-way ANOVA (**P < 0.01). (c–e) Mice were vaccinated with PBS, NT(−), or NT(Hla) on day 0 with a boost on day 14 (n=6). On days 0 (c), 14 (d), and 35 (e), serum was collected and the anti-Hla IgG titers were quantified by ELISA. Lines represent geometric means.

Figure 4. Effect of nanotoxoid (Hla) vaccination on MRSA skin colonization

Mice vaccinated with PBS, nanotoxoid(−) [NT(−)], or nanotoxoid(Hla) [NT(Hla)] on days 0 and 14 were challenged subcutaneously with 1 x 10⁹ CFU of MRSA bacteria on day 35. (a) The skin lesions were monitored over the course of 6 days (n=6). Lesion size is reported as the product of the largest and smallest dimensions. Error bars represent standard error. (b) Images of skin lesions on day 6 post-infection. Scale bar = 1 cm. (c) On day 6 post-infection, the affected skin and underlying tissue were collected and the bacterial burden enumerated (n=6). Lines represent geometric mean. Statistical significance determined by one-way ANOVA (**P < 0.01).

Figure 5. Effect of nanotoxoid(Hla) vaccination on MRSA invasiveness

Mice vaccinated with PBS, nanotoxoid(−) [NT(−)], or nanotoxoid(Hla) [NT(Hla)] on days 0 and 14 were challenged subcutaneously with 1 x 10⁹ CFU of MRSA bacteria on day 35. On day 6 post-infection, the major organs, including the heart (a), kidneys (b), spleen (c), lungs (d), and liver (e) were collected and the bacterial burden of each was enumerated (n=6). Lines represent geometric means. Statistical significance determined by one-way ANOVA (**P < 0.01, ***P < 0.001 and ****P < 0.0001).