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. 2022 Feb 3:12:767153.
doi: 10.3389/fcimb.2022.767153. eCollection 2022.

Dissecting in Vitro the Activation of Human Immune Response Induced by Shigella sonnei GMMA

Serena Tondi  1   2 , Bruna Clemente  1 , Carmen Esposito  1 , Chiara Sammicheli  1 , Simona Tavarini  1 , Laura B Martin  3 , Omar Rossi  3 , Francesca Micoli  3 , Erika Bartolini  1 , Michela Brazzoli  1 , Cristina Ulivieri  2 , Christoph J Blohmke  4 , Francesca Schiavetti  1
Affiliations

Dissecting in Vitro the Activation of Human Immune Response Induced by Shigella sonnei GMMA

Serena Tondi et al. Front Cell Infect Microbiol. .

Abstract

Generalized Modules for Membrane Antigens (GMMA) are outer membrane exosomes purified from Gram-negative bacteria genetically mutated to increase blebbing and reduce risk of reactogenicity. This is commonly achieved through modification of the lipid A portion of lipopolysaccharide. GMMA faithfully resemble the bacterial outer membrane surface, and therefore represent a powerful and flexible platform for vaccine development. Although GMMA-based vaccines have been demonstrated to induce a strong and functional antibody response in animals and humans maintaining an acceptable reactogenicity profile, the overall impact on immune cells and their mode of action are still poorly understood. To characterize the GMMA-induced immune response, we stimulated human peripheral blood mononuclear cells (hPBMCs) with GMMA from Shigella sonnei. We studied GMMA both with wild-type hexa-acylated lipid A and with the corresponding less reactogenic penta-acylated form. Using multicolor flow cytometry, we assessed the activation of immune cell subsets and we profiled intracellular cytokine production after GMMA stimulation. Moreover, we measured the secretion of thirty cytokines/chemokines in the cell culture supernatants. Our data indicated activation of monocytes, dendritic, NK, B, and γδ T cells. Comparison of the cytokine responses showed that, although the two GMMA have qualitatively similar profiles, GMMA with modified penta-acylated lipid A induced a lower production of pro-inflammatory cytokines/chemokines compared to GMMA with wild-type lipid A. Intracellular cytokine staining indicated monocytes and dendritic cells as the main source of the cytokines produced. Overall, these data provide new insights into the activation of key immune cells potentially targeted by GMMA-based vaccines.

Keywords: GMMA; OMV; Shigella sonnei; hPBMCs; immune response; in vitro; vaccines.

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Conflict of interest statement

STo is a student at the University of Siena and participated in a post graduate studentship program at GSK. BC, CE, CS, STa, MB, CB, and FS are employees of GSK group of companies. OR, FM, and LM are employees of the GSK Vaccines Institute for Global Health Srl, an affiliate of GlaxoSmithKline Biologicals SA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was sponsored by GlaxoSmithKline Biologicals SA which was involved in all stages of the study conduct and analysis.

Figures

Figure 1
Figure 1
Cytokine levels in the supernatants of hPMBCs stimulated with S. sonnei GMMA. (A, B) Cytokine expression profile of GMMA Hexa, GMMA Penta, and LPS after 4 hours (A) and 22 hours (B) incubation. Results are expressed as log10 of the median fold change (log10FC) over the six donors compared to the non-stimulated samples. Heatmaps show the relative cytokine levels. Line plots further illustrate the comparison between GMMA Hexa (dark cyan) and GMMA Penta (light cyan). Lines mark the median log10FC, while error bars represent the standard deviation over the donors. Dark gray and light gray areas indicate cytokines whose expression falls respectively above/below the upper/lower limit of detection (ULOD/LLOD) for more than 50% of the donors. For these cytokines, values were substituted respectively with ULOD or LLOD. (C, D) Comparison of cytokine concentrations after 4 (C) and 22 hours (D) incubation with GMMA Hexa (dark cyan), GMMA Penta (light cyan), or LPS (green). Non-stimulated samples are shown as reference in gray (MED). Dots show the results for each of the donors. Concentrations out of the detection range were replaced with the detection limits. Dashed black lines indicate the upper and lower limits of detection. The Mann-Whitney test with Benjamini-Hochberg correction was used to determine the significance of the observed differences (*p-value < 0,05, **p-value<0.005; ns stands for nonsignificant, p-value > 0.05).
Figure 2
Figure 2
Flow cytometry analysis for activation marker upregulation in different cell populations after 22 hours of stimulation. (A) t-SNE projection of the FlowSOM clustering results. Clusters were assigned to cellular subpopulations according to the expression profile of specific population markers (right panel). (B) t-SNE plots showing the expression of two different activation markers among different conditions. (C) Upregulation levels of CD40 and CD69 in different cellular populations after hPBMCs stimulation with GMMA Hexa, GMMA Penta and specific positive controls. LPS was used as positive control for monocytes and mDC; CpG for B cells and pDC; and SEB for γδ T cells and NK cells. Marker upregulation is expressed as fold increase (FI) of the geoMFI with respect to negative control (MED). Dotted lines indicate the reference value of 1 for the control samples. The different dots represent the six different donors. Significance was estimated using the paired Wilcoxon test (*p < 0.05). Significant differences with respect to the negative control are indicated on top of each box, instead significant differences among stimuli are shown explicitly. Non-significant differences are not shown.
Figure 3
Figure 3
Intracellular cytokines production after 22 hours of stimulation with GMMA. (A–C) t-SNE projections of two different cell populations, i.e. monocytes and mDC, showing intracellular cytokines production after stimulation with GMMA Hexa and GMMA Penta. Medium and MED are used as synonyms for control samples. (B–D) Percentages of cells producing the analyzed cytokines. Percentage were calculated on the manually gated cell populations. The different dots represent the six different donors. Significance was estimated using the paired Wilcoxon test (*p < 0.05). Significant differences with respect to the negative control are indicated on top of each box, instead significant differences among stimuli are shown explicitly. Non-significant differences are not shown.
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