Fig 1.
Immunization with R-P4 induces antibodies that mediate GBS opsonophagocytic killing in vitro.
(A) Structure of GBS hemolysin or Granadaene which contains a rhamnose sugar, a polyene chain with 12 double bonds, and a terminal ornithine amino acid; generated using the software ChemDraw. (B) Structure of R-P4, which is composed of a rhamnose sugar, a polyene chain with 4 double bonds, and a terminal alanine; generated using the software ChemDraw. (C) Schematic created using BioRender.com displaying the experimental design wherein mice were immunized intraperitoneally (i.p). with an emulsion of R-P4 and complete Freund’s adjuvant and boosted 14 days later with an emulsion of R-P4 and incomplete Freund’s adjuvant. After 21 days, serum was collected for opsonophagocytosis assay. (D) Opsonophagocytic killing of HH GBS (WT NCTC10/84) by human neutrophils in the presence of R-P4 immune serum. HH GBS was pre-treated with inactivated serum from either R-P4 immunized or adjuvant-treated mice or with inactivated pre-immune serum for 30 minutes and then incubated with neutrophils for 60 minutes. Surviving CFU were enumerated by dilution plating onto TSA and percent killing of GBS was calculated. Treatment groups were compared using a one-way ANOVA test with Tukey’s post-test. Mean and SEM from at least three independent experiments performed in triplicate are shown. *Indicates p < 0.05, ns indicates not significant, or p ≥ 0.05. (E) Opsonophagocytic killing of HH GBS (WT NCTC10/84) by mouse macrophages in the presence of R-P4 immune serum. HH GBS was opsonized with inactivated serum from R-P4 immunized or adjuvant-treated mice or with inactivated pre-immune serum and then incubated with macrophages for 60 minutes. Surviving CFU were enumerated by dilution plating onto TSA and percent killing of GBS was calculated. Treatment groups were compared using a one-way ANOVA test with Tukey’s post-test. Mean and SEM are shown from three independent samples performed in duplicate are shown. ** Indicates p < 0.01, *indicates p < 0.05, ns indicates not significant or p ≥ 0.05.
Fig 2.
R-P4 immunization generates antibody responses that are protective in vivo.
(A) Schematic created using BioRender.com displaying the serum transfer schedule. Mice were immunized i.p with an emulsion of R-P4 and complete Freund’s adjuvant and boosted 14 days later with an emulsion of R-P4 and incomplete Freund’s adjuvant. Adjuvant control mice received the adjuvant on the same schedule. After 21 days, blood was collected from the R-P4 immunized or adjuvant-treated mice and serum from the blood was transferred intravenously (i.v) to naïve recipient mice, respectively. One day post serum transfer, the recipient mice were challenged i.p with HH GBS (WT NCTC10/84). (B) At 24 hours post GBS infection, mice were euthanized, and spleen, lungs and brains were collected to enumerate GBS CFU in each tissue. Sample size for each treatment group: n = 23 for R-P4 immune serum recipients and n = 20 for adjuvant serum recipients. Data points represent individual mice, with the horizontal line representing the median. Mann-Whitney test was used to compare treatment groups. * Indicates p < 0.05.
Fig 3.
R-P4 vaccination induces IgG and IgM responses in mice.
(A) Antibody isotype responses following R-P4 vaccination. Approx. 5, 2.5 and 1 μg of purified Granadaene was spotted on PVDF membranes and probed with either R-P4 serum or adjuvant serum (1:62.5 dilution). Membranes were blocked, washed, and probed overnight at RT with either anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgA, IgD or IgM (1:2500 dilution). Immunoreactive spots were visualized using an infrared imager (LI-COR Biosciences) at 680 nm and images analyzed using Image Studio v5.2.5 software. (B) IgG and IgM endpoint titers induced from R-P4 vaccination were determined by incubation of Granadaene (1μg, spotted on PVDF membranes as above) with serial dilutions of R-P4 vaccinated mouse sera, followed by probing with either mouse anti-IgG or IgM antibodies. Endpoint titers were determined as the serum dilution required to exhibit a similar reactivity to Granadaene as that observed with adjuvant control serum. C) Endpoint titers mean ± standard deviation (SD) of IgM and IgG from R-P4 vaccinated mice (n = 3) are shown. ns indicates not significant or p ≥ 0.05, unpaired t test.
Fig 4.
CD4+ memory T cells are induced by R-P4 immunization.
(A) CD4+ T cells isolated from WT mice immunized with R-P4 or adjuvant only, were stained with CellTraceViolet (CTV) and co-cultured with R-P4 pulsed WT DCs. As a negative control, T cells were co-cultured with DCs without antigen stimulation. As a positive control for T cell proliferation, T cells were stimulated with anti-CD3 and PMA. After 5–7 days of co-culture, cells were stained for CD4 and assessed for proliferation (as measured by dilution of CTV dye) by flow cytometry. Sidak’s multiple comparison test following 2way ANOVA was used to compare proliferation of T cells between R-P4 vs adjuvant T cell groups. Data represent mean ± SEM from five independent experiments. * Indicates p < 0.05, ns indicates not significant, or p ≥ 0.05. (B) CD4+ T cells isolated from WT mice immunized with R-P4 or adjuvant only, were stained with CTV and co-cultured with R-P4 pulsed WT DCs or CD1d-/- DCs. As a negative control, T cells were co-cultured with DCs without antigen stimulation. As a positive control for T cell proliferation, T cells were stimulated with anti-CD3 and PMA. After 5–7 days of co-culture, cells were stained for CD4 and assessed for proliferation (as measured by dilution of CTV dye) by flow cytometry. Data represent mean ± SEM from three independent experiments. Sidak’s multiple comparison test following 2way ANOVA was used to compare T cell proliferation between R-P4 vs adjuvant groups. ** Indicates p < 0.01, *** indicates p < 0.001, ns indicates not significant, or p ≥ 0.05.
Fig 5.
CD4+ memory T cells are not induced by R-P4 immunization of CD1d-/- and Traj18-/- mice.
CD4+ T cells isolated from WT, CD1d-/-, and Traj18-/- mice immunized with R-P4 (A) or adjuvant control (B) were stained with CTV, and co-cultured with R-P4 pulsed WT DCs. As a positive control for T cell proliferation, T cells were stimulated with anti-CD3 and PMA. As negative controls, treatment groups included unstimulated DCs. After 5–7 days of co-culture, cells were stained for CD4 and assessed for proliferation (dilution of CTV dye) by flow cytometry. Data represent mean ± SEM from at least three independent experiments. Sidak’s multiple comparison test following 2way ANOVA was used to compare proliferation between treatment groups. * Indicates p < 0.05, ns indicates not significant, or p ≥ 0.05.
Fig 6.
CD1d-restricted iNKT cells contribute to protection against GBS infection in vivo.
(A) Schematic created using BioRender.com displaying the experimental design. WT, CD1d-/-, and Traj18-/- mice were immunized with R-P4 and boosted 14 days later. At day 21, mice were challenged with HH GBS (WT NCTC10/84). After 24 hours post-infection, mice were euthanized and GBS recovered from spleen, lungs and brain were enumerated using serial dilution and plating on TSA. (B) Bacterial burden in R-P4 immunized mice. Sample sizes are as follows: n = 27 WT; n = 23 CD1d-/-; n = 26 Traj18-/-. Adjuvant only control mice were also included. Sample sizes are as follows: n = 26 WT; n = 19 CD1d-/-;n = 25 Traj18-/-. Data points represent individual mice, with the horizontal line representing the median. Bacterial burden was compared using Kruskal-Wallis test with Dunn’s multiple comparison test. * Indicates p < 0.05, **p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.
Fig 7.
iNKT cells from R-P4 vaccinated mice diminishes GBS infection in vivo.
(A) Schematic created using BioRender.com displaying the experimental design for the isolation and adoptive transfer of iNKT cells. Mice were immunized i.p with an emulsion of R-P4 and complete Freund’s adjuvant and boosted 14 days later with an emulsion of R-P4 and incomplete Freund’s adjuvant. Adjuvant control mice received the adjuvant on the same schedule. On day 21, iNKT cells were isolated from the livers of R-P4 immunized or adjuvant control mice and the cells were adoptively transferred i.v (retro-orbital route) to naive recipient mice (~1–3 x 106 cells/mouse, n = 14/group). One day post transfer, the recipient mice were challenged i.p. with HH GBS (WT NCTC10/84, 1 x 107 CFU, i.p) and mice were monitored for symptoms of morbidity for 24 hrs. (B) Kaplan–Meier survival curve shows that mice that received iNKT cells from R-P4 immunized mice exhibited better survival compared to mice that received iNKT cells from adjuvant-treated mice. * Indicates p < 0.05, Long-rank (Mantel-Cox) test. (C) At 24 hrs post GBS infection or earlier if morbidity was seen, blood, peritoneal fluid, spleen, lungs, and brain were harvested, tissues were homogenized and GBS CFU enumerated using serial dilution and plating. Data points represent individual mice, with the horizontal line representing the median. * Indicates p < 0.05, Mann-Whitney test.
Fig 8.
iNKT cells exhibit cytokine responses to R-P4 antigen and GBS stimulation.
(A) iNKT cells isolated from WT mice immunized with R-P4 or adjuvant were co-cultured with R-P4 pulsed WT DCs. As controls, T cells were co-cultured with DCs without antigen stimulation or DCs were cultured alone. After 5 days of co-culture, cytokine concentrations in supernatants were determined using multiplex assays. Data represent mean ± SEM. Tukey’s multiple comparison test following 2-way ANOVA. * Indicates p<0.05. (B) iNKT cells isolated from WT mice immunized with R-P4 or adjuvant were co-cultured with WT DCs pulsed with UV-killed WT GBS (NCTC10/84). As controls, T cells were co-cultured with DCs pulsed with UV-killed isogenic pigment/hemolysin deficient GBS (ΔcylE) or DCs were cultured alone. After 5 days of co-culture, cytokine concentrations in supernatants were determined using multiplex assays. Data represent mean ± SEM. Tukey’s multiple comparison test following 2way ANOVA. * Indicates p<0.05, ** indicates p < 0.01, *** indicates p<0.001, *** indicates p<0.0001, ns indicates not significant, or p ≥ 0.05.
Fig 9.
Decreased hemolysis inhibition by R-P4-immunized CD1d-/- and Traj18-/- mice.
Schematic created using BioRender.com displaying the experimental design wherein diluted serum from R-P4 immunized or adjuvant control WT, CD1d-/- and Traj18-/- mice was incubated with Granadaene (1.25mM) prior to hemolysis assays with human red blood cells. Hemoglobin release in cell supernatants was measured (absorbance at 420 nm), and percent hemolysis relative to Triton X-100 (0.1%) control (100% hemolysis) and PBS-treated negative controls (0% hemolysis) was calculated. Data represent mean ± SEM from five samples/group. Tukey’s multiple comparison test following ANOVA. * Indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
Fig 10.
Maternal R-P4 immunization diminished GBS ascending infection.
(A) Schematic created using BioRender.com displaying the timeline for pregnancy-associated vaginal GBS challenge following R-P4 immunization. After their final vaccine boost, mice were mated and monitored for pregnancy. On day 14 of pregnancy, mice were intravaginally challenged with 108 CFU of GBS (WT NCTC10/84). (B) Dams were euthanized at ~72 hours post-infection and blood, lower genital tract (LGT), uterus, proximal and distal placentas and their pups were collected, homogenized, and plated for CFU enumeration. Medians are indicated with circles representing values from individual mice. Statistical differences were determined by Mann-Whitney test (sample sizes: n = 14 adjuvant, n = 18 adjuvant + R-P4). * Indicates p < 0.05, ** indicates p < 0.01, ns indicates not significant, or p ≥ 0.05.
Fig 11.
Model demonstrating the importance of antibody and T cell responses in R-P4 mediated immune protection against GBS infection.
Model created using BioRender.com showing humoral and cellular immunity in R-P4-mediated protection against GBS infection. R-P4 presentation by dendritic cells requires CD1d, which promotes T cell proliferation resulting in antibody production (IgG and IgM) via B cells. CD1d restricted T cells such as iNKT cells also are important for R-P4 mediated protection, which can provide noncognate help through production of inflammatory cytokines such as IL-18, IL-22 and IL-17 resulting in recruitment of phagocytes that dimmish GBS burden. Alternatively, iNKT cells can provide cognate help via IL-4 and INF-γ promoting direct interaction between iNKT follicular helper cells (iNKTFH) with T cells resulting in antibody production that dimmish cytotoxicity and GBS dissemination.