Continuous production of Neisseria meningitidis outer membrane vesicles.

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.

High dissolved oxygen tension triggers outer membrane vesicle formation by Neisseria meningitidis.

BACKGROUND: Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria and can be used as vaccines. Often, detergents are used to promote release of OMVs and to remove the toxic lipopolysaccharides. Lipopolysaccharides can be detoxified by genetic modification such that vesicles spontaneously produced by bacteria can be directly used as vaccines. The use of spontaneous OMVs has the advantage that no separate extraction step is required in the purification process. However, the productivity of spontaneous OMVs by bacteria at optimal growth conditions is low. One of many methods for increasing OMV formation is to reduce the linkage of the outer membrane to the peptidoglycan layer by knocking out the rmpM gene. A previous study showed that for Neisseria meningitidis this resulted in release of more OMVs. Furthermore, cysteine depletion was found to trigger OMV release and at the same time cause reduced growth and oxidative stress responses. Here we study the effect of growth rate and oxidative stress on OMV release. RESULTS: First, we identified using chemostat and accelerostat cultures of N. meningitidis that increasing the growth rate from 0.03 to 0.18 h-1 has a limited effect on OMV productivity. Thus, we hypothesized that oxidative stress is the trigger for OMV release and that oxidative stress can be introduced directly by increasing the dissolved oxygen tension of bacterial cultures. Slowly increasing oxygen concentrations in a N. meningitidis changestat showed that an increase from 30 to 150% air saturation improved OMV productivity four-fold. Batch cultures controlled at 100% air saturation increased OMV productivity three-fold over batch cultures controlled at 30% air saturation. CONCLUSION: Increased dissolved oxygen tension induces the release of outer membrane vesicles in N. meningitidis cultures. Since oxygen concentration is a well-controlled process parameter of bacterial cultures, this trigger can be applied as a convenient process parameter to induce OMV release in bacterial cultures. Improved productivity of OMVs not only improves the production costs of OMVs as vaccines, it also facilitates the use of OMVs as adjuvants, enzyme carriers, or cell-specific drug delivery vehicles.

A Synthetic Carbohydrate Conjugate Vaccine Candidate against Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity.

Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.

Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²âº and Mn²âº ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases.

The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.

Microencapsulated IL-12 Drives Genital Tract Immune Responses to Intranasal Gonococcal Outer Membrane Vesicle Vaccine and Induces Resistance to Vaginal Infection with Diverse Strains of Neisseria gonorrhoeae.

An experimental gonococcal vaccine consisting of outer membrane vesicles (OMVs) and microsphere (ms)-encapsulated interleukin-12 (IL-12 ms) induces Th1-driven immunity, with circulating and genital antibodies to Neisseria gonorrhoeae, after intravaginal (i.vag.) administration in female mice, and generates resistance to vaginal challenge infection. Because i.vag. administration is inapplicable to males and may not be acceptable to women, we determined whether intranasal (i.n.) administration would generate protective immunity against N. gonorrhoeae. Female and male mice were immunized i.n. with gonococcal OMVs plus IL-12 ms or blank microspheres (blank ms). Responses to i.n. immunization were similar to those with i.vag. immunization, with serum IgG, salivary IgA, and vaginal IgG and IgA antigonococcal antibodies induced when OMVs were administered with IL-12 ms. Male mice responded with serum IgG and salivary IgA antibodies similarly to female mice. Gamma interferon (IFN-γ) production by CD4+ T cells from iliac lymph nodes was elevated after i.n. or i.vag. immunization with OMVs plus IL-12 ms. Female mice immunized with OMVs plus IL-12 ms by either route resisted challenge with N. gonorrhoeae to an equal extent, and resistance generated by i.n. immunization extended to heterologous strains of N. gonorrhoeae. Detergent-extracted OMVs, which have diminished lipooligosaccharide, generated protective immunity to challenge similar to native OMVs. OMVs from mutant N. gonorrhoeae, in which genes for Rmp and LpxL1 were deleted to eliminate the induction of blocking antibodies against Rmp and diminish lipooligosaccharide endotoxicity, also generated resistance to challenge infection similar to wild-type OMVs when administered i.n. with IL-12 ms. IMPORTANCE We previously demonstrated that female mice can be immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microsphere (ms)-encapsulated interleukin-12 (IL-12 ms) to induce antigonococcal antibodies and resistance to genital tract challenge with live Neisseria gonorrhoeae. However, this route of vaccination may be impractical for human vaccine development and is inapplicable to males. Because intranasal immunization has previously been shown to induce antibody responses in both male and female genital tracts, we have evaluated this route of immunization with gonococcal OMVs plus IL-12 ms. In addition, we have refined the composition of gonococcal OMVs to reduce the endotoxicity of lipooligosaccharide and to eliminate the membrane protein Rmp, which induces countereffective blocking antibodies. The resulting vaccine may be more suitable for ultimate translation to human application against the sexually transmitted infection gonorrhea, which is becoming increasingly resistant to treatment with antibiotics.

An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential.

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.

Secondary structure of antisense RNAß, an internal transcriptional terminator of the plasmid-encoded iron transport-biosynthesis operon of Vibrio anguillarum.

RNAß affects the transcription process of the iron transport-biosynthesis operon encoded in the pJM1 plasmid of Vibrio anguillarum at a stem-loop structure located in the intergenic region between the fatA and angR genes. The net result is a higher level of the fatD, fatC, fatB, and fatA moiety as compared with the longer transcript encoding those genes as well as the angR and angT genes. In this work we report the secondary structure of RNAß determined by treatment with single and double strand specific ribonucleases as well as lead acetate followed by sequencing. The generated in vitro structural data indicated that three of the four previously described loops are in agreement with the original model, however, the alteration of loop IV as well as several other structural differences in the overall shape of the molecule led to the necessity of creating a new in silico model. Using the sites of mutations in the various loops we modeled the change in the RNAß secondary structure induced by those mutations. Mutations of loops III and IV to their complementary bases alter the overall structure of the RNAß significantly and increase its function while mutations in loops I and II have the opposite effect, the structure is unchanged but the activity of RNAß decreases. This indicates that loops I and II are necessary for interaction with the target mRNA. It is possible that the structural rearrangement introduced by mutations in loops III and IV promote activity and binding in loops I and II through reducing steric hindrance or increased binding to the target. This result also indicates that the exact relative positions of the critical loops are unimportant for activity.

The First-in-Human Synthetic Glycan-Based Conjugate Vaccine Candidate against Shigella.

Shigella, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting Shigella flexneri 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.

Large-scale production of extracellular vesicles: Report on the "massivEVs" ISEV workshop.

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, "The Extracellular Vesicle Foundry" (evFOUNDRY) and "Extracellular vesicles from a natural source for tailor-made nanomaterials" (VES4US), organized a workshop entitled "massivEVs" to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during "massivEVs", the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.

Role of the periplasmic chaperones Skp, SurA, and DegQ in outer membrane protein biogenesis in Neisseria meningitidis.

The periplasmic chaperones Skp, SurA, and DegP are implicated in the biogenesis of outer membrane proteins (OMPs) in Escherichia coli. Here, we investigated whether these chaperones exert similar functions in Neisseria meningitidis. Although N. meningitidis does not contain a homolog of the protease/chaperone DegP, it does possess a homolog of another E. coli protein, DegQ, which can functionally replace DegP when overproduced. Hence, we examined whether in N. meningitidis, DegQ acts as a functional homolog of DegP. Single skp, surA, and degQ mutants were easily obtained, showing that none of these chaperones is essential in N. meningitidis. Furthermore, all combinations of double mutants were generated and no synthetic lethality was observed. The absence of SurA or DegQ did not affect OMP biogenesis. In contrast, the absence of Skp resulted in severely lower levels of the porins PorA and PorB but not of other OMPs. These decreased levels were not due to proteolytic activity of DegQ, since porin levels remained low in a skp degQ double mutant, indicating that neisserial DegQ is not a functional homolog of E. coli DegP. The absence of Skp resulted in lower expression of the porB gene, as shown by using a P(porB)-lacZ fusion. We found no cross-species complementation when Skp of E. coli or N. meningitidis was heterologously expressed in skp mutants, indicating that Skp functions in a species-specific manner. Our results demonstrate an important role for Skp but not for SurA or DegQ in OMP biogenesis in N. meningitidis.

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775.

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

Spontaneously released Neisseria meningitidis outer membrane vesicles as vaccine platform: production and purification.

Outer membrane vesicles (OMVs) are nanoparticles produced by Gram-negative bacteria that can be used as vaccines. The application of OMVs as vaccine component can be expanded by expressing heterologous antigens on OMVs, creating an OMV-based antigen presenting platform. This study aims to develop a production process for such OMV-based vaccines and studies a production method based on meningococcal OMVs that express heterologous antigens on their surface. As a proof of concept, the Borrelia burgdorferi antigens OspA and OspC were expressed on Neisseria meningitidis OMVs to create a concept anti-Lyme disease vaccine. Production of OMVs released in the culture supernatant was induced by high dissolved oxygen concentrations and purification was based on scalable unit operations. A crude recovery of 90 mg OMV protein could be obtained per liter culture. Expressing heterologous antigens on the OMVs did result in minor reduction of bacterial growth, while OMV production remained constant. The antigen expression did not alter the OMV characteristics. This study shows that production of well characterized OMVs containing heterologous antigens is possible with high yields by combining high oxygen concentrations with an optimized purification process. It is concluded that heterologous OMVs show potential as a vaccine platform.

Sulfate depletion triggers overproduction of phospholipids and the release of outer membrane vesicles by Neisseria meningitidis.

Outer membrane vesicles (OMVs) produced by bacteria are interesting vaccine candidates. OMVs are nanoparticles that contain many immunogenic components, are self-adjuvating, and non-replicative. Despite recent insights in the biogenesis of OMVs, there is no consensus on a conserved mechanism of OMV release and the OMV yield from bacterial cultures remains low. For Neisseria meningitidis, a Gram-negative human pathogen causing meningitis and sepsis, a feasible OMV production method based on triggering OMV release by cysteine depletion has been described. In this study, we investigated the mechanism behind this external trigger for OMV release to improve the production process. Since enhanced OMV release upon cysteine depletion was associated with oxidative stress and redox responses, we investigate the influence of more oxidized sulfur sources on OMV release. We show that N. meningitidis grows similarly on sulfate, the most oxidized sulfur source, and OMV release is triggered by sulfur depletion in general. Sulfate depletion induced increased release of OMVs over cysteine depletion. Proteomics showed that sulfur depletion resulted in oxidative stress responses and upregulated phospholipid and LPS biosynthesis. Furthermore, OMVs produced by sulfur depletion were enriched in phospholipids. Mechanistically, we hypothesize that sulfur depletion results in overproduction of phospholipids causing increased bulging of the outer membrane and subsequent OMV release.

Pbp, a cell-surface exposed plasminogen binding protein of Bacteroides fragilis.

The Gram-negative anaerobic bacterium B. fragilis is a member of the commensal flora of the human intestine, but is also frequently found in severe intra-abdominal infections. Several B. fragilis virulence factors have been implicated in the development of these infections. A B. fragilis protein of circa 60-kDa was identified as a putative plasminogen binding protein (Pbp). The corresponding gene was located, cloned, sequenced and the subcellular localization of the protein was investigated. Pbp was both determined in the outer membrane of B. fragilis and of E. coli that expressed the cloned protein. Protease accessibility studies showed that the protein is expressed at the cell surface. Importantly, we demonstrated that Pbp is sufficient and required for plasminogen binding to whole cells in both E. coli and B. fragilis. Pbp-like proteins were also detected in some other Bacteroides subspecies. The role of this potential B. fragilis virulence factor in pathogenicity is discussed.

Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum.

Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa predicted protein, AngN. In this work we show that AngN is essential for anguibactin biosynthesis and possesses two domains with homology to cyclization (Cy) domains of NRPSs. Substitution by alanine of the aspartic acid residues within a conserved motif of either Cy1 or Cy2 domain demonstrated the importance of these two domains in AngN function during siderophore biosynthesis. Site-directed mutations in both domains (D133A/D575A and D138A/D580A) resulted in anguibactin-deficient phenotypes while mutations in each domain did not abolish siderophore production but caused a reduction in the amounts produced. The mutations D133A/D575A and D138A/D580A also resulted as expected in a dramatic attenuation of the virulence of V. anguillarum 775 highlighting the importance of this gene for the biosynthesis of anguibactin within the vertebrate host. Regulation of the angN gene follows the patterns observed at the iron transport-biosynthesis promoter with angN transcription repressed in the presence of iron and enhanced by AngR and trans-acting factor (TAF) under iron limitation.

High throughput nanoparticle tracking analysis for monitoring outer membrane vesicle production.

Outer membrane vesicles (OMVs) are spherical membrane nanoparticles released by Gram-negative bacteria. OMVs can be quantified in complex matrices by nanoparticle tracking analysis (NTA). NTA can be performed in static mode or with continuous sample flow that results in analysis of more particles in a smaller time-frame. Flow measurements must be performed manually despite the availability of a sample changer on the NanoSight system. Here we present a method for automated measurements in flow mode. OMV quantification in flow mode results in lower variance in particle quantification (coefficient of variation (CV) of 6%, CV static measurements of 14%). Sizing of OMVs was expected to be less favorable in flow mode due to the increased movement of the particles. However, we observed a CV of 3% in flow mode and a CV of 8% in static measurements. Flow rates of up to 5 µL/min displayed correct size and particle measurements, however, particle concentration was slightly lower than in static measurements. The automated method was used to assess OMV release of batch cultures of Neisseria meningitidis. The bacteria released more OMVs in stationary growth phase, while the size of the vesicles remained constant throughout the culture. Taken together, this study shows that automated measurements in flow mode can be established with advanced scripting to reduce the workload for the user.

Bioengineering bacterial outer membrane vesicles as vaccine platform.

Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.

Surface display of a borrelial lipoprotein on meningococcal outer membrane vesicles.

Outer Membrane Vesicles (OMVs) are gaining attention as vaccine candidates. The successful expression of heterologous antigens in OMVs, with the OMV functioning both as adjuvant and delivery vehicle, has greatly enhanced their vaccine potential. Since there are indications that surface exposed antigens might induce a superior immune response, targeting of heterologous antigens to the OMV surface is of special interest. Several systems for surface display of heterologous antigens on OMVs have been developed. However, these systems have not been used to display lipidated membrane-associated proteins known as lipoproteins, which are emerging as key targets for protective immunity. We were therefore interested to see whether we could express a foreign lipoprotein on the outer surface of OMVs. When outer surface protein A (OspA), a borrelial surface-exposed lipoprotein, was expressed in meningococci, it was found that although OspA was present in OMVs, it was no longer surface-exposed. Therefore, a set of fusions of OspA to different regions of factor H binding protein (fHbp), a meningococcal surface-exposed lipoprotein, were designed and tested for their surface-exposure. An N-terminal part of fHbp was found to be necessary for the successful surface display of OspA on meningococcal OMVs. When mice were immunized with this set of OMVs, an OspA-specific antibody response was only elicited by OMVs with clearly surface-exposed OspA, strengthening the idea that the exact positioning of an antigen in the OMV affects the immune response. This method for the surface display of heterologous lipoproteins on OMVs is a step forward in the development of OMVs as a vaccine platform.

Outer membrane vesicles as platform vaccine technology.

Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram-negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self-adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV-containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV-producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well-defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications.