Enhanced Staphylococcus aureus protection by uncoupling of the α-toxin-ADAM10 interaction during murine neonatal vaccination

Abstract

Staphylococcus aureus remains a leading global cause of bacterial infection-associated mortality and has eluded prior vaccine development efforts. S. aureus α-toxin (Hla) is an essential virulence factor in disease, impairing the T cell response to infection. The anti-Hla antibody response is a correlate of human protective immunity. Here we observe that this response is limited early in human life and design a vaccine strategy to elicit immune protection against Hla in a neonatal mice. By targeted disruption of the interaction of Hla with its receptor ADAM10, we identify a vaccine antigen (Hla_{H35L/R66C/E70C}, Hla_HRE) that elicits an ~100-fold increase in the neutralizing anti-Hla response. Immunization with Hla_HRE enhances the T follicular helper (T_FH) cell response to S. aureus infection, correlating with the magnitude of the neutralizing anti-toxin response and disease protection. Furthermore, maternal Hla_HRE immunization confers protection to offspring. Together, these findings illuminate a path for S. aureus vaccine development at the maternal-infant interface.

Fig. 1. The neutralizing anti-Hla response is a vaccine target early in life.

a Half-maximal effective concentration of anti-Hla reactive antibodies in sera from children within the designated age groups (Log EC50). b Serum-based protection against Hla-mediated (2 nM) rRBC lysis. Protection noted as percent compared to toxin-only control. For a, b, 75th and 25th (black) and 50th (red) percentiles noted. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 2. Analysis of Hla-targeting vaccine candidate antigens.

a OT-II CD4⁺ T cell subset analysis of OT-II CD4⁺ T cells harvested from the draining lymph nodes of mice immunized with adjuvant alone (sham) or candidate Hla vaccines Hla_H, Hla_HDY, Hla_HRE. Tissue harvest occurred 7 days following subcutaneous infection with 1 × 10⁸ CFU of wild-type S. aureus USA300/LAC strain compared by non-parametric one-way ANOVA with Dunnette’s multiple comparisons test. Data represents biological replicates (n = 5 per group) with mean ± SD. b Correlation between protection against rRBC lysis upon treatment with 2 nM Hla and serologic anti-Hla titer (Log EC50) from mice following immunization with Hla_H35L (purple), Hla_HDY (orange), Hla_HRE (blue). Data represents the mean of biological replicates (n = 5) c Vaccine antigen Hla variant binding to rRBC as compared to wild-type Hla by non-parametric one-way ANOVA with Tukey’s multiple comparisons test, Hla_HDY p = 0.0005, Hla_HRE p < 0.0001. Data represents the median of technical replicates (n = 3 per group). d Melting curve analysis of Hla_H, Hla_HDY, and Hla_HRE compared to wild-type Hla. e SDS-PAGE analysis of ³⁵S-methionine labeled Hla, Hla_H, Hla_HDY, and Hla_HRE produced by coupled in vitro transcription and translation. f Analysis of heptamer assembly and stability for Hla, Hla_H, Hla_HDY, and Hla_HRE, evaluated following incubation of radiolabeled toxin with rRBCs for one hour at room temperature followed by incubation at 37, 62, or 80 °C for 10 min. g Analysis of Hla_HRE heptamer capability with increasing concentrations of radiolabeled toxin. The radiolabeled toxin was incubated with rRBCs for one hour at room temperature, followed by incubation at 37 °C. Relative densitometric units of monomeric and heptameric toxin was quantified using ImageJ software. SDS-PAGE separation was followed by phosphor-image detection of labeled toxin; for e–g, toxin monomers (Hla₁) and oligomers (Hla₇) are noted. h Disulfide bond identified in antigen Hla_HRE by mass spectrometry. Disulfide bond (S-S) peptide linking of Cys66 and Cys70 within the Hla_HRE protein was identified by LC-MS/MS analysis and validated by manual alignment of y or b-ion matching. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 3. Neonatal vaccination model demonstrates protection against S. aureus skin and soft tissue infection.

a Schematic showing the timeline of neonatal vaccination and SSTI modeling (BioRender). Skin abscess (left) and dermonecrosis (right) area following primary (b) (n = 10 per group) and re-infection (c) of immunized mice (n = 10 for sham and Hla_H, and n = 9 for Hla_HDY and Hla_HRE) with 1 × 10⁸ CFU S. aureus, noting the statistical significance of Hla_HRE compared to each vaccine condition designated by color. Data analyzed by two-way ANOVA with Tukey’s multiple comparisons test, mean ± SD. Independent experiments were repeated twice. d Serum dilution from mice in (b, c) that affords 50% protection against rRBC lysis (IC50) when exposed to 0.8 pM recombinant Hla, noting the statistical significance of Hla_HRE compared to Hla_H by one-way ANOVA with Tukey’s multiple comparisons test. Data represents the median of biological replicates, n = 5 per group for all antigen-adjuvant combinations, except n = 4 for CpG-ODN1585 Hla_HRE. e Correlation between serum dilution that affords 50% protection against Hla-mediated (0.8 pM) rRBC lysis and anti-Hla titer from mice following immunization. Data represents the mean from biological replicates in (d). Independent experiments were repeated twice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file. Figure 3a Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Fig. 4. Hla_HRE amplifies the T_FH response.

a Serum dilution from immunized mice that affords 50% protection against rRBC lysis (IC50) by 0.8 pM Hla, noting statistical significance of Hla_HRE versus Hla_H by two-tailed unpaired t-test. Data represents the median of biological replicates (1 week, n = 8/group, 3 weeks, n = 7/group). b Serum neutralization of Hla binding to rRBC comparing sera from mice in (a), noting statistical analysis by two-tailed unpaired t-test. Data represented as the median of biological samples (n = 8) represented as the mean of two technical replicates/samples. c Densitometric analysis of Hla oligomer formation on rRBC comparing immunized sera from mice in (a). Data represents the median of biological replicates (n = 5/group), analyzed by a two-tailed unpaired t-test. Analysis of left inguinal germinal center cells 1-week following immunization of mice with Hla_H (n = 15) or Hla_HRE (n = 15) comparing: d GL7⁺Fas⁺ B cells, e Ki67⁺GL7⁺Fas⁺ B cells, f CD4⁺ T cells, g CXCR5 median fluorescence intensity (MFI) on CD4⁺ T cells, and h CXCR5 median fluorescence intensity (MFI) on CD4⁺Ki67⁺ T cells. Data represents the median of biological replicates, analyzed by a two-tailed paired t-test. i Gross pathology of skin lesions (2 days post-infection) in mice vaccinated as neonates with sham, Hla_H, or Hla_HRE (n = 5). Analysis of CXCR5^hiBcl6^hi in Ki67⁺⁺T_FH cell compartment in mice in (i), data represents the median of biological replicates (n = 6). k Analysis of CXCR5^hiBcl6^hi T_FH cell compartment in mice subjected to primary infection with wild-type (WT) (n = 6), Δhla (n = 6), or Δhla::phla (n = 3) variants of S. aureus. Statistical analysis by paired, two-tailed t-tests (d–h) and two-way ANOVA with Tukey’s multiple comparisons test (j, k) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 5. Hla_HRE vaccine exhibits protective efficacy following maternal immunization and prior S. aureus exposure.

a Schematic showing the timeline for maternal and neonatal vaccination followed by SSTI. b Skin abscess (left) and dermonecrosis (right) area of mice following primary infection with 1 × 10⁸ CFU S. aureus. Mice were born to dams that received Alum or the Alum-Hla_HRE vaccine; pups were either unimmunized (black n = 4 and blue n = 8, respectively), Alum immunized (gray, n = 5), or Alum-Hla_HRE immunized (green, n = 5), noting statistical significance of Alum maternal immunization compared to each vaccine condition designated by color by two-way ANOVA with Tukey’s multiple comparisons test. Data represented as the mean ± SD. c Serum dilution from mice in (b), that affords 50% protection against rRBC lysis (IC50) when exposed to 0.8 pM Hla. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test. Data represents the median of biological replicates (black n = 5, blue n = 5, gray n = 3, and green n = 3). d Schematic showing the timeline for a model in which juvenile mice are exposed to S. aureus infection prior to vaccination. e Skin abscess (left) and dermonecrosis (right) area of mice (sham n = 13, Hla_HRE n = 14) following secondary infection with 1 × 10⁸ CFU S. aureus. Data represents mean ± SD, analyzed by two-way ANOVA with Sidak’s multiple comparisons test. f Serum dilution from mice in (e), that affords 50% protection against rRBC lysis (IC50) when exposed to 0.8 pM Hla. Data represents the median, analzyed by one-way ANOVA with Tukey’s multiple comparisons test (primary infection group n = 7, sham n = 6, and Hla_HRE n = 6). Source data are provided as a Source Data file. Figure 5a, d Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Fig. 6. Hla_HRE vaccine formulation influences the T_FH response.

a Inverse correlation of T_FH cell recovery and clinical abscess size from immunized mice after S. aureus SSTI infection. b Direct correlation between the anti-Hla neutralizing IC50 and T_FH response from immunized mice after S. aureus SSTI infection. All mice were subjected to priming immunization as neonates with a boost at the time of weaning. Data represents the mean of biological replicates (n = 7 per group for T_FH cell analysis and Alum-Hla_HRE n = 11, AddaS03-Hla_HRE n = 10, and n = 12 for MPLA+Alum-Hla_HRE and CpG-ODN1585-Hla_HRE groups). Data and R² values were analyzed by non-linear regression, semi-log line. Source data are provided as a Source Data file.