Research ArticleHEALTH AND MEDICINE

Antibodies to the conserved region of the M protein and a streptococcal superantigen cooperatively resolve toxic shock-like syndrome in HLA-humanized mice

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Science Advances  04 Sep 2019:
Vol. 5, no. 9, eaax3013
DOI: 10.1126/sciadv.aax3013
  • Fig. 1 BALB/c mice as a model for STSS.

    (A and B) Virulence of human isolates in murine skin infection model. Cohorts of BALB/c mice (n = 5 per group) were infected with SN1 or NS33 strain via the skin route of infection. After day 3, 6, or 9 of challenge, the mice were culled and skin biopsy (A) and spleen (B) samples were harvested, processed, and plated to determine the bacterial burden. The results are shown as box and whisker plot, where the line in the box indicates the median, the box extremities indicate the upper and lower quartiles, and the whiskers show the minimum to maximum values. Statistical analysis was performed using nonparametric, unpaired Mann-Whitney U test to compare the two groups at each time point. **P < 0.01 and ***P < 0.001. (C and D) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot profile of SpeC in serum samples collected at various time points after infection. Serum samples from SN1- or NS33-infected mice were collected on days 3, 6, and 9 after infection and run on 4 to 12% SDS-PAGE gels (C). Following protein transfer from the gel, the membrane was probed with rabbit anti-SpeC immunoglobulin G (IgG) primary antibody, followed by detection with sheep anti-rabbit IgG-AP (alkaline phosphatase), and developed using SIGMAFAST BCIP/NBT (bromochloroindolyl phosphate–nitro blue tetrazolium) substrate. rSpeC protein was also run as a positive control. Detection of SpeC is shown (D). MW, molecular weight. (E and F) SpeC detection in individual mouse serum samples from the day 6 collection. Serum samples from each individual mouse on day 6 following SN1/NS33 infection were also assessed for the presence of SpeC as described. A representative image of SDS-PAGE (E) and Western blot (F) is shown. The symbol “#” indicates the mice that had positive spleen culture.

  • Fig. 2 Mitogenic and inflammatory activity of SN1-infected sera in vitro.

    (A) Splenocyte proliferation in response to serum from SN1-infected mice and rSpeC. Splenocytes from HLA-B6 and B6 mice were stimulated in vitro either with 20 μl of sterile-filtered serum from SN1-infected BALB/c mice or with rSpeC. As controls, sterile-filtered serum from mice infected with a SAg-negative GCS isolate (NS33) and Concanavalin A (ConA) were also included. Proliferation of splenocytes was assessed after 72 hours by measuring incorporation of tritiated [3H]thymidine, and data are represented as stimulation indices (see below). The specificity of the response was confirmed by addition of 20 μl of anti-rSpeC serum. Ab, antibody. (B and C) Cytokine profiles following splenocyte proliferation. Cytokine responses of splenocytes from HLA-B6 and B6 mice were measured at 72 hours after incubation with various stimulants. Concentrations of TNF (B) and IFN-γ (C) in the culture supernatants were measured using a TH1/TH2/TH17 cytometric bead array (CBA) kit (BD Biosciences). The specificity of the responses was confirmed by addition of rSpeC antiserum. (D to F) Proliferation of human PBMCs in response to stimulation with serum from SN1- or NS33-infected mice. PBMCs from three different individuals were cultured in the presence of serum collected at various time points following infection with SN1 or NS33. An optimized amount of serum (20 μl) was used for PBMC stimulation. Proliferation was measured by [3H]thymidine uptake after 72 hours. Data are mean ± SEM of three replicates in each experiment, with experiments repeated twice. Stimulation index was defined as counts per minute in the presence of antigen/counts per minute in the absence of antigen. One-way analysis of variance (ANOVA) with Tukey’s post hoc method was used to calculate significance between various groups. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Infectivity of S. pyogenes SN1 in HLA-B6 mice following intraperitoneal and skin infection.

    (A) Western blot analysis of serum from SN1-infected HLA-B6 and B6 mice following intraperitoneal infection. Naïve HLA-B6 and B6 mice (n = 10 per group) were infected with 106 SN1 via the intraperitoneal route of infection. Serum samples were collected at 24 hours after infection and analyzed to detect the toxin. The samples were run on 4 to 12% SDS-PAGE gel. Following protein transfer from the gel, the membrane was probed with rabbit anti-SpeC IgG primary antibody, followed by detection with sheep anti-rabbit IgG-AP, and developed using SIGMAFAST BCIP/NBT substrate. rSpeC protein was also run as a positive control. (B to D) Serum cytokine profile of HLA-B6 and B6 mice following intraperitoneal infection with SN1. The mice infected with SN1 were culled at 24 hours after infection. Blood cytokine levels were measured from the cohort that received 106 SN1 using a CBA kit. TNF, IFN-γ, and IL-2 responses are shown. Statistical analysis was performed using nonparametric, unpaired Mann-Whitney U test to compare the two cohorts. ***P < 0.001. (E and F) Bacterial burden in HLA-B6 mice after skin infection with SN1. Naïve HLA-B6 and B6 mice (n = 10 per group) were infected with SN1 or NS33 via skin. On day 6 after infection, mice were culled and skin bacterial burdens were assessed (E). Mann-Whitney U test used to compare the two cohorts. ***P < 0.001. The presence of systemic infection was assessed by plating blood samples at days 3 to 6 after infection (F). The results are shown as box and whisker plot, where the line in the box indicates the median, the box extremities indicate the upper and lower quartiles, and the whiskers show the minimum to maximum values. (G) Western blot analysis of serum from SN1- or NS33-infected mice. Serum samples collected from SN1- or NS33-infected HLA-B6 and B6 mice were analyzed to detect the presence of SpeC in their serum. The samples were run on 4 to 12% SDS-PAGE gel. Following protein transfer from the gel, the membrane was probed with rabbit anti-SpeC IgG primary antibody, followed by detection with sheep anti-rabbit IgG-AP, and developed using SIGMAFAST BCIP/NBT substrate. The band at 26 kDa in the serum sample from SN1-infected mice corresponds to rSpeC in the positive control sample. (H to J) Cytokine responses in the serum of HLA-B6 and B6 mice following skin infection. Cytokine responses in the serum of HLA-B6 and B6 mice were measured at day 6 after infection with SN1 or NS33. Concentrations of TNF (H), IFN-γ (I), and IL-2 (J) were measured using a CBA kit. One-way ANOVA with Tukey’s post hoc method was used to calculate significance between various groups. ***P < 0.001.

  • Fig. 4 Abolition of mitogenic and inflammatory effects after J8-DT vaccination.

    (A) Protective efficacy of J8-DT vaccine against S. pyogenes SN1 infection. HLA-B6 mice were vaccinated with 30 μg of J8-DT/alum or PBS/alum on days 0, 21, and 28. Two weeks after immunization, mice were infected with SN1 via the skin. On day 6 after infection, mice were culled and bacterial burden in skin (CFU/lesion), blood (CFU/ml), and spleen (CFU/spleen) is shown. Statistical analysis was performed using nonparametric, unpaired Mann-Whitney U test to compare the two cohorts. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Western blot analysis to detect SpeC in infected mice serum. Pooled serum samples from vaccinated and control cohorts collected at day 6 after SN1 infection were run on 4 to 12% SDS-PAGE gels. Following protein transfer from the gel, the membrane was probed with rabbit anti-SpeC IgG primary antibody, followed by detection with sheep anti-rabbit IgG-AP, and developed using SIGMAFAST BCIP/NBT substrate. The band at 26 kDa in the serum sample from PBS-treated (control) mice corresponds to rSpeC in the positive control sample. (C and D) Cytokine responses in the serum of HLA-B6 mice following skin infection. Cytokine responses in the serum of vaccinated and control HLA-B6 mice were measured at day 6 after infection with SN1. Concentration of IL-4 and IL-10 (C) and TNF and IFN-γ (D) in serum was measured using a CBA kit. One-way ANOVA with Tukey’s post hoc method was used to calculate significance between various groups. ***P < 0.001. (E to G) Assessment of PBMC proliferation induced by serum from vaccinated/control-infected mice. PBMCs from three different individuals were stimulated with pre-optimized concentration of serum from J8-DT–vaccinated SN1-infected (V-infected sera) or nonvaccinated SN1-infected (NV-infected sera) mice. rSpeC was used as controls for stimulation. The specificity of response was assessed by addition of various amounts of rSpeC antisera. PBMCs in the presence of naïve sera were used as a control for specificity of neutralization. Proliferation was measured by [3H]thymidine uptake after 72 hours. Data are mean ± SEM of three replicates in each experiment, with experiments repeated twice. Representative data from two individuals are shown. One-way ANOVA with Tukey’s post hoc method was used to calculate significance. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

  • Fig. 5 Therapeutic potential of combination immunotherapy.

    (A) Timeline of infection and treatment protocol. (B) Clinical scores for SN1-infected mice before and after antisera treatment. Four cohorts of HLA-B6 mice (n = 3 to 5 per group) were infected intraperitoneally (IP) with 106 S. pyogenes SN1. Eighteen hours after infection (0 hours), mice were scored for clinical symptoms and intravenously administered 200 μl of either anti–J8-DT, anti-rSpeC, a combination of anti–J8-DT and anti-rSpeC, or PBS-treated (control) sera. All mice were again scored for clinical symptoms after treatment to assess disease severity. The scoring system included appearance, level of consciousness, activity, response to stimulus, eyes, respiration rate, and respiration quality. The mice were scored from 0 to 4. The clinical scores for all cohorts before (0 hours) and after (24 hours) antisera treatment are shown. At 24 hours after treatment (42 hours after infection), mice were culled. (C) Bacterial burden in SN1-infected mice before and after antisera treatment. Blood and spleen samples were harvested, processed, and plated for quantification of bacteria. The bacterial burdens in blood and spleen of mice are shown. Mann-Whitney U test was performed to compare each group with the PBS-treated (control) group. *P < 0.05; **P < 0.01; ***P < 0.001; and ns, P > 0.05. (D to G) Assessment of SpeC in serum samples from mice before and after treatments. To assess SpeC neutralization in vivo, sera samples from all cohorts were collected before (0 hours) and then at 24 hours after antisera administration. Pooled serum samples from treated and untreated cohorts were run on 4 to 12% SDS-PAGE gels. Following protein transfer from the gel, the membrane was probed with rabbit anti-SpeC IgG primary antibody, followed by detection with sheep anti-rabbit IgG-AP, and developed using SIGMAFAST BCIP/NBT substrate. The presence of SpeC in HLA-B6 mice treated with J8-DT antiserum (D), rSpeC antiserum (E), J8-DT + rSpeC antiserum (F), or control serum (G) sera before and after treatment is shown. “N” represents pooled serum from naive mice. (H to J) Cytokine profile in SN1-infected mice before and after treatment. Following treatment with various antisera, at 24 hours, mice were culled and cytokine responses in the pre- and post-treatment serum were measured using a CBA kit. Concentration of TNF (H), IFN-γ (I), and IL-2 (J) in serum is shown. Statistical analysis was carried out using one-way ANOVA with Tukey’s post hoc method to calculate significance between various groups, with color of asterisk (*) denoting the groups being compared. *P < 0.05, **P < 0.01; ***P < 0.001; and ns, P > 0.05. (K and L) Splenocyte proliferation and inhibition in response to S. pyogenes antigens and various antisera. (K) Splenocytes from HLA-B6 mice (n = 3) were stimulated with 20 μl of SN1-infected serum. Proliferation was measured in the presence or absence of J8-DT, rSpeC, J8-DT + rSpeC, or PBS-treated (control) sera by [3H]thymidine uptake after 72 hours. Statistical analysis was performed using nonparametric, unpaired Mann-Whitney U test to compare the two groups. ns, P > 0.05; **P < 0.01; and ***P < 0.001. (L) Splenocytes from HLA-B6 mice were stimulated with pre-optimized concentration (5 μg/ml) of rSpeC, rM1, or rSpeC + rM1. To assess inhibition by various sera, 20 μl of J8-DT, rSpeC, or J8-DT + rSpeC antisera was added to each well. Nonimmune sera were used as control. Proliferation of splenocytes in the presence or absence of various antisera was assessed after 72 hours by [3H]thymidine uptake. Data are represented as stimulation indices, which were defined by comparing the proliferation in the presence or absence of antisera. One-way ANOVA with Tukey’s post hoc method was used to calculate significance between various groups. **P < 0.01; ***P < 0.001; and ns, not significant.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax3013/DC1

    Fig. S1. SAg profile of streptococcal isolates.

    Fig. S2. Cytokine response of PBMCs following stimulation with vaccinated and control sera.

    Fig. S3. Cytokine profile in HLA-B6 mice after stimulation with rM1.

    Fig. S4. Immunological cross-reactivity between J14 and J14.1.

    Table S1. Clinical isolates and medical history of patients.

    Table S2. Primers used for SAg gene profiling.

    Table S3. Distribution of spec and spea genes in ISD isolates from Canada.

    Reference (46)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. SAg profile of streptococcal isolates.
    • Fig. S2. Cytokine response of PBMCs following stimulation with vaccinated and control sera.
    • Fig. S3. Cytokine profile in HLA-B6 mice after stimulation with rM1.
    • Fig. S4. Immunological cross-reactivity between J14 and J14.1.
    • Table S1. Clinical isolates and medical history of patients.
    • Table S2. Primers used for SAg gene profiling.
    • Table S3. Distribution of spec and spea genes in ISD isolates from Canada.
    • Reference (46)

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