Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection

Abstract

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.

Keywords: Staphylococcus aureus; Th17; Th22; skin; vaccine.

Conflict of interest statement

Conflict of interest statement: M.R.Y., S.G.F., J.P.H., Y.F., C.S.S., J.E.E., and A.S.I. are shareholders of NovaDigm Therapeutics, Inc., which is developing new vaccines including NDV-3 targeting Staphylococcus aureus and other pathogens.

Figures

Fig. 1.

NDV-3 vaccine-mediated restriction of abscess magnitude and progression due to MRSA Xen30. (A) Restriction of abscess volume at day 7 of the infection model. Each circle represents an individual lesion (two lesions per mouse). †P < 0.05 or ††P < 0.01 versus control mice receiving adjuvant alone. Bars represent means of the respective datasets. (B) Restriction of dermonecrosis area and severity at day 7 of the infection model. Visualization of dermonecrosis areas in control and NDV-3–vaccinated mice representing mean results. Note the smaller areas of dermonecrosis and greater degrees of lesion healing in NDV-3–immunized mice, compared with controls, in which lesions remain exudative and poorly contained at this time point. (C) Progression of mean dermonecrosis area and mean abscess volume over a 14-d infection model. Dermonecrosis area was significantly attenuated in NDV-3–immunized mice compared with controls at all time points subsequent to day 1. Abscess volume was significantly attenuated in NDV-3–immunized mice compared with controls at all time points subsequent to day 1. Key: ○, control; □, NDV-3 dose regimen 3 μg; Δ, NDV-3 dose regimen 100 μg; ◆, NDV-3 dose regimen 300 μg. For C, †P < 0.05 for all NDV-3 regimens versus control mice receiving adjuvant alone; ††P < 0.01 for NDV-3 regimens Δ or ◆ versus control mice receiving adjuvant alone.

Fig. 2.

NDV-3 vaccine-mediated suppression of MRSA Xen30 proliferation in SSSI abscesses in vivo. (A) Reduction in luminescent signal at day 7 of the infection model as a function of NDV-3 dose regimen. †P < 0.05 or ††P < 0.01 versus control mice receiving adjuvant alone. Bars represent means of the respective datasets. (B) Visualization of NDV-3–mediated (100 μg dose regimen) suppression of MRSA signal within abscesses in vivo. Imaging was performed and luminescence flux quantified (scale at right) at selected time points over a 7-d infection model. Note the significant reductions in proliferation signal of MRSA strain Xen30 at each time point, as is consistent with restriction of lesion magnitude (Fig. 1) and reduction in abscess CFU burden (Fig. 3) compared with control mice receiving adjuvant alone.

Fig. 3.

NDV-3 vaccine-mediated reduction in MRSA Xen30 abscess CFU burden. Distributions of CFU densities of abscesses were determined by quantitative culture at day 7 of the infection model. Substantial reductions in CFU associated with NDV-3 immunization are congruent with the observed restrictions in abscess magnitude (Fig. 1) and suppression of MRSA proliferation (Fig. 2). Each circle represents quantitative culture results from an individual lesion (two lesions per mouse, 10 mice per arm). †P < 0.05 versus control mice receiving adjuvant alone. Bars represent means of the respective datasets.

Fig. 4.

Histopathology of abscesses due to MRSA Xen30 at day 7 of the infection model. Low-power fields (LPFs) of the epidermal surface (Top) are shown in images 1 and 4 of each panel. Relative tissue regions are shown in HPFs (Insets) compared with equivalent representative regions in control mice (adjuvant alone; images 2 and 3) versus NDV-3–immunized mice (images 5 and 6) of each panel. (A) Hematoxylin and eosin (H&E) staining of MRSA abscesses. Arrows indicate the presence of high-density MRSA microabscesses lacking granulocyte intensification in cutaneous tissues of control mice (images 1–3). The number and density of microabscesses are reduced in NDV-3–immunized mice (images 4–6). (B) Anti-CD3+ staining (brown) of CD3+ lymphocyte infiltration into abscess lesions. Note the high density of CD3+ cells and germinal centers amid intensive areas of neutrophil infiltration in NDV-3–immunized mice (images 4–6) compared with lesions from control mice (images 1–3). Arrows (image 1) indicate S. aureus microabscesses. (C) Anti-Ly6G+ staining (brown) of granulocyte infiltration into abscess lesions. Note the high density of Ly6G+ multilobar nucleated neutrophils engaging microabscess sites in NDV-3–immunized mice (images 5 and 6). In contrast, MRSA microabscesses relatively free of neutrophil infiltration are observed in epidermal and hypodermal regions in lesions of control nonvaccinated mice (images 2 and 3). Arrows (plate 1) indicate high-density S. aureus microabscesses devoid of neutrophil access. (D) Anti–IL-17A staining (brown) in abscess lesions. Note the homogenous, high-level expression of IL-17A expression particularly in mesodermal and hypodermal tissues of NDV-3–immunized mice (images 5 and 6) compared with a relative paucity of IL-17 expression in lesions of control mice (images 2 and 3). Induction of IL-17A is consistent with the recruitment of CD3+ T cells (Fig. 4B) and Ly6G+ neutrophils (Fig. 4C) and reduced MRSA burden correlating with NDV-3 immunization. Arrows (image 1) indicate S. aureus microabscesses. (E) Anti–IL-22 staining (brown) of IL-22 expression in abscess lesions. Note the inverse correlation between IL-22 expression and MRSA invasion into the mesodermal and hypodermal regions of NDV-3–immunized mice (images 5 and 6) compared with low-level IL-22 expression and abundant MRSA microabscesses in lesions of control mice (images 2 and 3). (F) Anti–mβd-3 staining (brown) of mβd-3 expression in abscess lesions. Note the broad intensification of mβd-3 expression in surface epithelium and cutaneous gland structures of NDV-3–immunized mice (images 5 and 6) compared with baseline-level expression in lesions of control mice (images 2 and 3). The up-regulation of antimicrobial peptides such as mβd-3 supports the concept that NDV-3 immunization stimulates activation of innate and adaptive immune mechanisms to protect against disease due to MRSA.

Fig. 5.

Coordinated immune response to MRSA challenge induced by NDV-3 at day 7 of the infection model. Immunofluorescence and confocal microscopy were used to generate composite images visualizing complete abscess columns [epidermis (Top) to hypodermis (Bottom)] in lesions representing NDV-3–immunized (A) versus control mice (B). NDV-3 immunization (A) induces a coordinated and intensive CD3+ T-cell (green) and Ly6G+ neutrophil (red) infiltrative response that extends from the hypodermis into the epidermis and is associated with a paucity of MRSA organisms or microabscesses (blue). In contrast, lesions from control animals (B) exhibit low-density T-cell and neutrophil responses that are limited to the hypodermal and mesodermal regions, allowing MRSA invasion into hypodermal tissue and dense MRSA microabscesses to proliferate extensively.

Fig. 6.

Impact of IL-17A and/or IL-22 neutralization on parameters of MRSA infection in the setting of NDV-3 vaccination. The kinetics of abscess volume (A) and in situ proliferation of MRSA (as measured by luminescence of lux+ strain Xen30) (B) are shown over the 7-d study period. The actual CFUs recovered at the study endpoint (day 7) from skin abscesses (Top, C) and kidneys (Bottom, C) are also shown. *P < 0.05 comparing NDV-3–immunized versus control mice receiving adjuvant alone; †P < 0.05 comparing NDV-3–immunized and anti-cytokine–treated mice versus NDV-3–immunized but untreated controls. Bars, SEM.

Fig. 7.

Integrated model of NDV-3–mediated protective efficacy versus MRSA in murine SSSI. We hypothesize that the NDV-3 immunogen (rAls3p-N) is recognized through toll-like receptors (TLRs) or like sensors for pathogen-associated molecular patterns (PAMPs) on antigen-presenting cells (APCs; 1). Presentation of processed rAls3 to naïve T cells in the context of a TGF-β1, IL-6, and TNF-α cytokine milieu (2) stimulates immune polarization bias to Th22/Th17 via the signal transduction activator of transcription-3 (STAT3; 3). Supporting the Th17 pathway is the elaboration of CC chemokines and IL-23 that recruit/expand antigen-specific Th17 T cells to sites of infection (4). There, immunogen-stimulated Th22 and Th17 cells elaborate IL-17A and IL-22 (5), which serve to activate two parallel arms of immune effector function—induction of β-defensins and other cutaneous host defense peptides (6) as prompted by IL-22—and CXC chemokine ligand generation (e.g., keratinocyte chemoattractant in mice or its ortholog CXCL8 in humans) from fibroblasts or epithelial cells (7), in turn recruiting/activating neutrophils or other leukocytes to foci of infection (8). Thus, the current findings support the hypothesis that NDV-3 stimulates an antigen-specific and highly coordinated immune response that is protective against SSSI due to MRSA.