Feasibility of outer membrane protein C as a protein presenting platform targeting antigen to surface of outer membrane vesicle / 中国生物制品学杂志

@#Abstract：Objective To investigate the feasibility of outer membrane protein C（OmpC）as a protein presenting platform targeting antigen to the surface of outer membrane vesicle（OMV）. Methods The recombinant expression plasmid containing ompC gene fragment and Staphylococcus aureus EsxA antigen gene（esxA gene）was constructed，transformed to competent E. coli BL21（DE3），inducedbyIPTG，andanalyzedforexpressedproductby 12%SDS⁃PAGE. Thetotalproteinofrecombinant strain OMV was analyzed by 12% SDS⁃PAGE，and the localization of fusion protein on the surface of OMV was detected by Western blot and Flow NanoAnalyzer. Results The recombinant expression plasmid containing ompC gene and esxA gene was constructed correctly as proved by sequencing. 12% SDS⁃PAGE showed that the fusion protein OmpC⁃EsxA had a relative molecular mass of about 57 000，which was consistent with the expected size，while the total protein of OMV showed multiple target protein bands，indicating that recombinant strain OMV was successfully extracted. The fusion protein OmpC⁃ EsxA on the surface of recombinant strain OMV specifically bound to mouse antibody against His⁃Tag，and OMVs labeled with fluorescent antibody were detected by Flow NanoAnalyzer. Conclusion OmpC may be used as a protein presenting plat⁃ form to locate antigen to OMV surface，which was expected to be applied in the development of antigen presentation vaccine. Keywords：Outer membrane protein C（OmpC）；Protein presentation；Outer membrane vesicle（OMV）

Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease

BACKGROUND Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.

Bacterial outer membrane vesicles: a new approach to diseases therapy / 药学学报

Outer membrane vesicles (OMVs) are nano-sized spherical vehicles, with a size range between 20-250 nm. OMVs are spontaneously secreted from Gram-negative bacteria and formed by lipid bilayer membranes, comprising multiple parent bacteria-derived components including bacterial antigens, pathogen-associated molecular patterns, proteins and lipids. OMVs have shown multiple potentials for the treatment of various diseases, including cancer therapy and bacterial infection. In this review, the structure, composition and methods for isolating and characterizing of OMVs were introduced. The applications of OMVs for diseases therapy were summarized and future perspectives were discussed.

Preparation of bacterial outer membrane vesicle coated nanoparticle loaded with drug and evaluation of its nasal immune effect in mice / 上海交通大学学报（医学版）

Objective: To prepare a bacterial outer membrane vesicle (OMV) coated poly (lactic-co-glycolic acid) copolymer (PLGA) nanoparticle loaded with ovalbumin (OVA) and evaluate its intranasal immune effect in mice. Methods: OMV was prepared by ultrafiltration concentration method. OVA loaded PLGA nanoparticle (NP) was prepared by emulsion-solvent evaporation method. OMV coated PLGA nanoparticle (OMV-PLGA NP) loaded with OVA was prepared by extrusion method and characterized. BALB/c mice were intranasally immunized and specific sIgA levels in nasal wash, jejunum and fecal pellet were determined by ELISA. Results: Size of OVA loaded OMV-PLGA NP was (234.4±22.9) nm. The shell-core structure of OVA loaded OMV-PLGA NP was proved by transmission electron microscope. After 14 d of administration, sIgA antibody levels in nasal wash, jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group were the highest in all treated groups. Compared with the group treated with OMV and OVA, OVA-specific sIgA antibody level in nasal wash, jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group was increased 1.6, 2.1 and 1.7 times, respectively. Compared with the group treated with OMV and OVA, OMV-specific sIgA antibody level in nasal wash, jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group was all increased 1.5 times. Conclusion: This novel nanoparticle drug delivery system can simultaneously delivery OVA and OMV to antigen presenting cells, resulting in stronger mucosal immune response in mice.

Preparation of bacterial outer membrane vesicle coated nanoparticle loaded with drug and evaluation of its nasal immune effect in mice / 上海交通大学学报（医学版）

Objective·To prepare a bacterial outer membrane vesicle (OMV) coated poly (lactic-co-glycolic acid) copolymer (PLGA) nanoparticle loaded with ovalbumin (OVA) and evaluate its intranasal immune effect in mice.Methods· OMV was prepared by ultrafiltration concentration method.OVA loaded PLGA nanoparticle (NP) was prepared by emulsion-solvent evaporation method.OMV coated PLGA nanoparticle (OMV-PLGA NP) loaded with OVA was prepared by extrusion method and characterized.BALB/c mice were intranasally immunized and specific sIgA levels in nasal wash,jejunum and fecal pellet were determined by ELISA.Results· Size of OVA loaded OMV-PLGA NP was (234.4±22.9) nm.The shell-core structure of OVA loaded OMV-PLGA NP was proved by transmission electron microscope.After 14 d of administration,sIgA antibody levels in nasal wash,jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group were the highest in all treated groups.Compared with the group treated with OMV and OVA,OVA-specific sIgA antibody level in nasal wash,jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group was increased 1.6,2.1 and 1.7 times,respectively.Compared with the group treated with OMV and OVA,OMV-specific sIgA antibody level in nasal wash,jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group was all increased 1.5 times.Conclusion· This novel nanoparticle drug delivery system can simultaneously delivery OVA and OMV to antigen presenting cells,resulting in stronger mucosal immune response in mice.

Roles of Outer Membrane Vesicles (OMVs) in Bacterial Virulence

Outer membrane vesicles (OMVs) are ubiquitous membranous structures in all Gram-negative bacteria, including pathogens and non-pathogens. Gram-positive bacteria also release membrane-derived vesicles (MV). Originating from the cell envelope, OMVs are enriched with bacterial antigen molecules that conduct multiple functions as decoys to manipulate the host immune system. Besides, OMVs and their components play diverse roles in nutrient acquisition, biofilm formation, and resistance to antibiotics. Despite the diverse benefits ascribed to OMVs, many questions remain unanswered with regard to OMV biogenesis and cargo selectivity. In this report, we review the advantages of vesiculation in the context of all bacteria and then focus on additional benefits acquired by OMVs in pathogenic bacteria.

New versus old meningococcal Group B vaccines: How the new ones may benefit infants & toddlers.

Invasive disease caused by Neisseria meningitidis is associated with high mortality and high disability rates and mainly affects children under one year of age. Vaccination is the best way to prevent meningococcal disease, especially in infants and toddlers. The introduction of massive meningococcal serogroup C vaccination has drastically reduced the incidence of disease caused by this serogroup, and serogroup B has now become the main causative agent in several industrialized countries. The first serogroup B vaccines, which were used for more than two decades, were based on outer membrane vesicles and proved to be protective only against specific epidemic strains in Cuba, Norway, Brazil and New Zealand. Moreover, these often elicited a scant immune response in young children. Innovative genomics-based reverse vaccinology subsequently enabled researchers to identify genes encoding for surface proteins that are able to elicit a strong immune response against several B strains. This important discovery led to the development and recent approval in Europe of the four-component meningococcal serogroup B (4CMenB) vaccine. Large clinical trials have shown high immunogenicity and tolerability and acceptable safety levels of 4CMenB in infants and toddlers. This vaccine is expected to cover a large number of circulating invasive strains and may also be efficacious against other serogroups. Young children are particularly vulnerable to the devastating consequences of meningococcal disease. Given the high performance of 4CMenB and its non-interference with routine vaccinations, this age-group will be the first to benefit from the introduction of this vaccine.

Cinética do cultivo de Neisseria lactamica em biorreator / Bioreactor cultivation kinetics of Neisseria lactamica

Neisseria lactamica está envolvida na aquisição da imunidade natural contra Neisseria meningitidis, causadora da doença meningocócica. Vesículas de membrana externa (OMV) de N. lactamica são antígenos potenciais contra N. meningitidis. Analisou-se a cinética de biomassa, de produção de OMV, da fonte de carbono (lactato), e dos metabólitos, para maximizar a produção de OMV. Realizaram-se ensaios em biorreator, em batelada simples, batelada alimentada com lactato, com ou sem pulsos de aminoácidos e extrato de levedura (YE). Utilizou-se o meio de Catlin (MC) como meio mínimo, e analisaram-se efeitos das concentrações do lactato, aminoácidos e YE. Lactato foi consumido e citrato e acetato produzidos. Os melhores resultados obtidos foram no meio com adição de 2 g/L de YE e concentrações dobradas de lactato e 5 aminoácidos constitutivos do MC, em batelada alimentada com pulsos. O lactato apresentou efeito positivo sobre o rendimento de OMV e o YE sobre a biomassa. Os 5 aminoácidos constitutivos do MC foram necessários para obtenção de biomassa e rendimento de OMV.

Neisseria lactamica is involved with the acquisition of natural immunity to Neisseria meningitidis. N. lactamica outer membrane vesicles (OMV) are potential antigens against N. meningitidis, the pathogen of meningococcal disease. The objective of this work was to analyze the kinetics of bacterial growth, OMV production, the carbon source (lactate), and products of metabolism, to improve growing conditions and OMV production. Groups were studied in batch process, fed-batch process with lactate, fed-batch process with pulses of amino acids and YE. MC was considered as minimal medium and it was analyzed the effect of lactate, amino acids and YE. Lactate was consumed and citrate and acetate increased in the medium. The best results were in fed-batch with pulses, in MC with the double concentrations of lactate and amino acids, added with 2 g/L of YE. The lactate had a positive effect over OMV yield and YE had a positive effect over biomass. 5 amino acids of MC were necessary to obtain biomass and OMV yield.