The GMMA-IgG complexes allow B cell receptor crosslinking with the CR2 complex thus lowering the affinity threshold for B cell activation (61). mimics the bacterial environment, as well as to their intrinsic self-adjuvanticity. However, GMMA Rigosertib sodium mechanism of action and the role in adjuvanticity are still unclear and need further investigation. In this review, we discuss progresses in the development of the GMMA vaccine platform, highlighting successful applications and identifying knowledge gaps and potential difficulties. (4, 5, 7, 9, 11C13), (8, 14, 15), and (16, 18) species have been already generated using this approach, which is usually shown to be flexible enough to be potentially extended with minimal adjustments to any Gram-negative bacterial species. Indeed multiple industrial (17C19) and research (20C22) approaches based on genetic engineering of Rigosertib sodium bacteria for hyper-vesiculating and surface-expression of a variety of homologous and heterologous antigens, including bacterial (20C22), viral (23), parasitic (24) and even malignancy antigens (25) have been described. In this review, we will refer to genetically altered OMV as GMMA, and will focus our attention on GMMA-based vaccines that are in an advanced stage of the development and already relocated or Rigosertib sodium are approaching to move in clinical trials rather than on research vaccines. We will discuss progress in the development of the GMMA vaccine platform, highlighting successful applications, gaps and potential difficulties. GMMA LIKE A Vaccine Platform GMMA constitute a straightforward technology based on low-cost of production and high purification yields and is therefore suitable for the development of vaccines against bacterial pathogens and particularly of affordable vaccines targeting low- and middle-income countries (LMICs). GMMA resemble faithfully the outer membrane of the bacterial pathogen they shed from but lack the ability to cause the associated disease. They present to the immune system key antigens in their natural environment and conformation, facilitating uptake by immune cells and inducing strong immune response (26). The GMMA outer membrane also displays several Pathogen Associated Molecular Patterns (PAMPs), small molecular motifs well conserved in bacteria which are recognized by Patter Acknowledgement Receptors (PRRs) expressed on mammalian cells (27). PAMPs conversation with PRRs rapidly activates the complex signaling pathway, with the induction of pro-inflammatory cytokines and chemokines, and that may be the basis of GMMA self-adjuvanticity. However, while activation of the innate immune system can result in a high immune response to an antigen, it may also induce local and severe adverse effects in humans, from febrile response to septic shock (28). Thus, fine tuning the balance between immune activation and reactogenicity is usually important for an acceptable GMMA-based vaccine. LPS, the most abundant component of GMMA, is usually a key antigen in Gram-negative bacteria, but it is usually also the main component for systemic reactogenicity (29). Intrinsic LPS endotoxicity can be reduced by genetically modifying the lipid A structure. Lipid A is the endotoxic component of LPS which mediates the binding to toll like receptor (TLR) 4 inducing innate immune activation. TLR4 acknowledgement of LPS is usually strongly influenced by the structure of its lipid A component which, in most Gram-negative bacteria (i.e., (4, 5, 7, 9), MsbB and PagP in (8), or LpxL1 in (16, 17, 35) have been mutated to generate GMMA with different penta-acylated lipid A forms. In addition to LPS, other molecules contained in GMMA, like lipoproteins, are ACVRLK4 able to stimulate the innate immune system through the activation of TLR2. Indeed, GMMA from and were able to stimulate peripheral blood mononuclear cells (PBMCs) and induce interleukin (IL)-6 release, which was almost completely abolished when a combination of antibodies blocking TLR4 and TLR2 was used (7, 8), indicating that they are the PRRs mostly involved in GMMA mediated cytokine release. GMMA act as effective adjuvant and carrier, thus having an intrinsic ability to improve immunogenicity of protein and carbohydrate antigens. The glycoconjugate approach is the gold standard for enhancing immunogenicity, particularly for polysaccharides (36). Bacterial polysaccharides are T-cell-independent antigens which are generally not able to elicit germinal center (GC) formation and therefore immunological memory, persistence of antibody response, and affinity maturation of B cell receptors. Covalent linkage to a suitable carrier protein confers to saccharide antigens the ability to elicit a T-cell-dependent response, overcoming the limitation listed above. Consequently, vaccination with conjugates enhances polysaccharide immunogenicity and protective efficacy, especially in infants and children under 2 years of age (37, 38). As service providers for polysaccharides, GMMA can be somehow considered multi-valent antigens, as they may present multiple polysaccharide molecules and proteins in natural conformation (39). GMMA was shown to be superior to traditional glycoconjugate vaccines in animal models, likely due to the.