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

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.

Molecular Signature of Monocytes Shaped by the Shigella sonnei 1790-Generalized Modules for Membrane Antigens Vaccine

“…After mapping the assessed genes and filtering for those BTMs containing ≥ 25% modulated genes, we obtained 18 BTMs that represent the transcriptional fingerprint of 1790-GMMA-treated monocytes (Figure 5). These findings are consistent with our previous findings [31], because a closer analysis of DEGs within these modules points towards the upregulation of regulatory pathways involved in the production of cytokines and chemokines (such as IL1β, IL6, TNF, IL1α, CCL2, and CCL3) as well as the regulation of genes involved in cellular activation (e.g., CD40). Notably, our analysis revealed the capacity of the 1790-GMMA to regulate the expression of genes associated with additional modules, such as macrophage differentiation (M37.12) and T cell activation/regulation (M27.3).…”

“…The hypothesis that S. sonnei GMMA containing modified LPS (1790-GMMA) are likely to orchestrate the immune response by activating various immune cell populations was reinforced by our previous findings, which showed that this candidate vaccine activates APCs (monocytes and dendritic cells) and B and γδ T lymphocytes and promotes the production of pro-inflammatory cytokines, including IFNα, IL-10, IL-12p70, IL-1α, IL-6, TNFα, and IL-8, albeit in lower quantities compared to the GMMA variant with the wild-type lipid A [31]. Additionally, an increase in levels of IL-2, IL-4, Eotaxin, MCP-1, MIP-1α, MIP-1β, GM-CSF, IL-12p40, and IL-1β was detected [31].…”

“…The cytokine profile induced is similar in terms of quality but to a lesser extent compared with the wild-type counterpart. Interestingly, we also documented a 1790-GMMA-dependent activation of monocytes, dendritic cells, B cells, NK cells, and γδ T cells within hPBMCs [31]. Because single cell population among hPBMCs may be activated either through direct interaction with GMMA or through a bystander effect due to the nearby activated cells, we decided to identify the directly targeted cell population by using fluorescent 1790-GMMA.…”

“…Besides the ability of S. sonnei GMMA vaccine candidates to elicit bactericidal anti-OAg antibodies, their mode of action (MoA) at the cellular level is sparsely addressed. Previous in vitro studies conducted on human peripheral blood mononuclear cells (hPBMCs) demonstrated that lipid A mutation reduced the TLR4-dependent activation pathway and the release of pro-inflammatory cytokines [26] but still maintained the ability to activate monocytes, myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), B cells, NK cells, and γδ T cells [31]. Whether all of these immune cells are activated by a direct interaction with GMMA or become activated through a bystander effect remains unknown.…”

Outer Membrane Vesicle Vaccine Platforms

“…This is usually achieved through modification of the lipid A structure, in particular reducing the number of acyl chains and phosphate groups of lipid A, impacting its ability to recognize/trigger toll-like receptor (TLR) 4 [ 30 – 32 ] and decreasing the inflammatory response associated with lipid A [ 11 ]. Deletion of acyltransferases such as M sbB ( lpxM ), htrB ( lpxL ) and pagP (adding a myristoyl, lauroyl or palmitoyl chain, respectively, to the lipid A) has been used in E. coli , Shigella and Salmonella [ 33 – 35 ]; deletion of lpxL 1 and lpxL 2 has been used in Neisseria meningitidis [ 36 – 38 ].…”