VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation

Abstract

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection. Of the four agr alleles (agr types I-IV and corresponding AIPs1-4), agr type I isolates are most frequently associated with invasive infection. Cyclization via a thiolactone bond is essential for AIP function; therefore, recognition of the cyclic form of AIP1 may be necessary for antibody-mediated neutralization. However, the small sizes of AIPs and labile thiolactone bond have hindered vaccine development. To overcome this, we used a virus-like particle (VLP) vaccine platform (PP7) for conformationally-restricted presentation of a modified AIP1 amino acid sequence (AIP1S). Vaccination with PP7-AIP1S elicited AIP1-specific antibodies and limited agr-activation in vivo. Importantly, in a murine SSTI challenge model with a highly virulent agr type I S. aureus isolate, PP7-AIP1S vaccination reduced pathogenesis and increased bacterial clearance compared to controls, demonstrating vaccine efficacy. Given the contribution of MRSA agr type I isolates to human disease, vaccine targeting of AIP1-regulated virulence could have a major clinical impact in the fight against antibiotic resistance.

Figure 1

Design and preparation of PP7-AIP1S VLPs. (a) Schematic of AIP1 and amino acid sequence of AIP1-C4S (AIP1S). (b) Ribbon representation of the PP7 coat protein dimer (one monomer is shown in green and the other in magenta) which can be expressed as a single-chain dimer. Depicted is the first AB loop (indicated by arrow) and the AIP1S sequence (spheres) modeled into the second AB loop (PDB ID 2QUD) using GalaxyWeb^, . Image prepared using PyMol (PyMOL molecular graphics system, version 1.5.0.4; Schrodinger, LLC). (c) Schematic of the site of AIP1S insertion into the second AB loop of the PP7 single chain dimer. (d) Coomassie-stained 16% SDS-PAGE showing the relative size of the PP7 single-chain dimer compared to PP7 with the AIP1S insert.

Figure 2

PP7-AIP1S vaccination induces antibodies which recognize soluble AIP1. BALB/c mice were vaccinated twice (i.m.) at 4 week intervals with 10 µg of PP7-AIP1S or PP7 wild-type (control). (a) Serum was collected at the indicated time points after the second vaccination. Serum was then pooled (n = 3 mice per group), treated as described in Materials and Methods, and relative binding to PP7-AIP1S determined by ELISA. (b) PP7-AIP1S antiserum collected at eight weeks after the second vaccination was prepared as in (a), and relative AIP1S binding determined in the presence and absence of the indicated concentrations of AIP1 or AIP2 (n = 3 mice per group; duplicate experiments performed in triplicate). Data are mean ± s.e.m. Kruskal-Wallis ANOVA p < 0.0001 with Dunn’s post-test: *p < 0.05; ***p < 0.001.

Figure 3

PP7-AIP1S vaccination limits the severity of S. aureus skin infection in a mouse model of dermonecrosis. BALB/c mice were vaccinated twice (i.m.) at 4 week intervals with 10 µg of the indicated VLPs or PBS control. Eight weeks after the second vaccination, mice were challenged by subcutaneous infection with 4 × 10⁷ CFU of USA300 LAC. Representative (a) day 3 images of infection site and (b) daily measures of abscess area and dermonecrosis. Calculated area under the curve (AUC) values for (c) abscess area (ANOVA p < 0.0042), (d) dermonecrosis (p = 0.0177) and (e) percent weight change over the six day infection, as well as (f) day 6 bacterial burden at the site of infection (p = 0.0001) (representative of two independent experiments of n = 6 mice per group). (g) Cytokine levels in clarified abscess tissue homogenate on day 6 post-infection (ANOVA IL-1β, p = 0.0587; TNFα, p = 0.0358; IL-1α, p = 0.0171; IL-17, p = 0.0322; IL-6, p = 0.0010) (n = 6 mice per group). Data are mean ± s.e.m. Newman-Keuls post test: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

PP7-AIP1S vaccination limits agr function at the site of S. aureus infection. BALB/c mice were vaccinated twice (i.m.) at 4 week intervals with 10 µg of the indicated VLPs or PBS control. Eight weeks after the second vaccination, mice were challenged by subcutaneous infection with 4 × 10⁷ CFU of USA300 LAC. (a) Local RNAIII transcription on day 1 post-infection measured by qPCR (n = 4 mice per group, Kruskal-Wallis ANOVA p = 0.0029). (b) Representative immunoblot (showing recombinant Hla, MW marker and 3 mice per group) and (c) quantification of Hla levels (relative to PBS control) in clarified abscess tissue homogenate on day 6 post-infection (n = 6 mice per group) (Kruskal-Wallis ANOVA p = 0.0025) with Dunn’s post-test: ns, not significant; *p < 0.05; **p < 0.01.