Peter van der Ley’s research while affiliated with Intravacc and other places

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Publications (161)


Overview of TRANSVAC projects.
Pillars of the TRANSVAC vaccine infrastructure.
Distribution of TRANSVAC service projects. (A) Countries of residence of applicants awarded TRANSVAC projects. (B) Disease targets of TRANSVAC service projects. * Diseases not included in Vaccines Europe’s 2023 pipeline review [5]. ‡ Diseases with no approved vaccine currently available. (C) Delivery systems employed by TRANSVAC-supported vaccine candidates. Fifteen projects were omitted from the figure due to being too early stage (e.g., antigen identification), or platform-based, and three projects included multiple candidate delivery systems. (D) Intended administration route of TRANSVAC vaccine projects. Twenty-nine projects were not included as an administration route was not decided or unclear, and five projects involved hybrid protocols (e.g., prime-pull strategies) or compared multiple administration routes.
TRANSVAC scientific services provided along vaccine development pipeline.
Results of the TRANSVAC scientific services user survey.

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Boosting Vaccine Research: The 16-Year Journey of TRANSVAC Vaccine Infrastructure
  • Literature Review
  • Full-text available

December 2024

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31 Reads

William Martin

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Catarina Luís

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Stefan Jungbluth

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[...]

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TRANSVAC represents a long-running effort to accelerate the development of novel vaccines by integrating institutions from across Europe under a single collaborative framework. This initiative has empowered the global vaccine community since 2009 including contributing toward the development and optimization of vaccine candidates as well as the provision of new adjuvants, research protocols, and technologies. Scientific services were provided in support of 88 different vaccine development projects, and 400 professionals attended TRANSVAC training events on various vaccine-related topics. Here, we review the accomplishments of the TRANSVAC consortia and analyze the continued needs of academic and industrial vaccine developers in Europe. The findings highlight the benefits of coordination across different sectors, both through research infrastructures such as TRANSVAC and other mechanisms, to address the current and future global health challenges and ensure that European vaccine developers have the support required to successfully compete in the global market.

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Outer membrane vesicle-based intranasal vaccines

August 2023

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19 Reads

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10 Citations

Current Opinion in Immunology

Delivery of vaccines via the mucosal route is regarded as the most effective mode of immunization to counteract infectious diseases that enter via mucosal tissues, including oral, nasal, pulmonary, intestinal, and urogenital surfaces. Mucosal vaccines not only induce local immune effector elements, such as secretory Immunoglobulin A (IgA) reaching the luminal site of the mucosa, but also systemic immunity. Moreover, mucosal vaccines may trigger immunity in distant mucosal tissues because of the homing of primed antigen-specific immune cells toward local and distant mucosal tissue via the common mucosal immune system. While most licensed intramuscular vaccines induce only systemic immunity, next-generation mucosal vaccines may outperform parenteral vaccination strategies by also eliciting protective mucosal immune responses that block infection and/or transmission. Especially the nasal route of vaccination, targeting the nasal-associated lymphoid tissue, is attractive for local and distant mucosal immunization. In numerous studies, bacterial outer membrane vesicles (OMVs) have proved attractive as vaccine platform for homologous bacterial strains, but also as antigen delivery platform for heterologous antigens of nonbacterial diseases, including viruses, parasites, and cancer. Their application has also been extended to mucosal delivery. Here, we will summarize the characteristics and clinical potential of (engineered) OMVs as vaccine platform for mucosal, especially intranasal delivery.


Lipid A heterogeneity and its role in the host interactions with pathogenic and commensal bacteria

June 2022

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67 Reads

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8 Citations

microLife

Lipopolysaccharide (LPS) is for most but not all Gram-negative bacteria an essential component of the outer leaflet of the outer membrane. LPS contributes to the integrity of the outer membrane, which acts as an effective permeability barrier to antimicrobial agents and protects against complement-mediated lysis. In commensal and pathogenic bacteria LPS interacts with pattern recognition receptors (e.g LBP, CD14, TLRs) of the innate immune system and thereby plays an important role in determining the immune response of the host. LPS molecules consist of a membrane-anchoring lipid A moiety and the surface-exposed core oligosaccharide and O-antigen polysaccharide. While the basic lipid A structure is conserved among different bacterial species, there is still a huge variation in its details, such as the number, position and chain length of the fatty acids and the decoration of the glucosamine disaccharide with phosphate, phosphoethanolamine or amino sugars. New evidence has emerged over the last few decades on how this lipid A heterogeneity confers distinct benefits to some bacteria because it allows them to modulate host responses in response to changing host environmental factors. Here we give an overview of what is known about the functional consequences of this lipid A structural heterogeneity. In addition, we also summarize new approaches for lipid A extraction, purification and analysis which have enabled analysis of its heterogeneity.



Schematic presentation of the spike molecule and OMV decoration with spike. (A) Schematic of 2019-nCoV S primary structure colored by domain. S, signal sequence; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1–HR2, heptad repeat 1 and 2; FC, disrupted S1/S2 furin cleavage site (R682G, R684S, R685S). Features that were added to the ectodomain (amino acids 1–1,208) expression construct are colored white gray gradient (amino acid 1,209–1,333). From light to dark gray: Foldon trimerization motif (F); HRV 3C site; 8xHis tag; Twin Strep tag; 3x GGGS repeat; mCramp (mC). Not shown: six stabilizing prolines in S2 at positions F817P, A892P, A899P, A942P, K986P, and V987P (27). The aspartic acid at position 614 in the original HexaPro (27) spike protein was replaced with a glycine (D614G). (B) Schematic overview of how antigens can associate with the OMVs. In the spike protein, an mCRAMP (antimicrobial peptide) motif was included which is depicted in light blue. This peptide associates spontaneously to the LPS of the OMV thereby attaching the spike protein (dark blue) to the OMV.
Mouse immunogenicity study. Balb/C mice were immunized intranasally or intramuscular on day 0 and day 21 with 15 µg OMV (control group) or 15 µg OMV combined with 15 µg Spike with the presence of mCRAMP or without mCRAMP (OMV+Spike). Sera were collected from all animals at day 35. (A) Experimental setup and timeline of the mouse immunogenicity experiment. (B) Total IgG antibody levels were measured in sera diluted at 1:50,000. (C) IgA levels were measured in nasal washes (1:1 dilution), lung (1:50 dilution), and serum (1:200 dilution). (D) Virus neutralization titers were determined in sera. Data are depicted as mean ± SD and are representative results of two independent experiments. Significance (E) is depicted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance of the difference was evaluated by a 2-way ANOVA test followed by the non-parametric Mann whitney test.
Hamster challenge study. Animals were immunized intranasally or intramuscularly on day 0 and day 21 with 15 µg OMV, 15 µg spike or 15 µg OMV, and 15 µg Spike combined with mCRAMP. In the control group, animals were immunized with 10 mM Tris-3% sucrose, which is the OMV buffer. Sera were collected from all hamsters at experimental days 0, 21, 42, 46, and 49. At day 42, all hamsters were challenged intranasally with 10^4.0 TCID50 SARS-CoV-2, strain BetaCoV/Munich/BavPat1/2020. At day 46, half of the animals per group (4 out of 8) were sacrificed and at day 49 the remaining 4 animals were sacrificed. (A) Experimental setup and timeline of the hamster challenge experiment. (B) Total IgG anti-spike antibody levels were measured in sera (dilution 1:4,000) from day 42 in an ELISA. (C) Virus neutralization was determined in sera from day 42. (D) When animals were sacrificed at day 46 (day 4 post challenge) and day 49 (day 7 post challenge), the percentage of the lung that presented lung lesions was quantified. The viral load was determined in throat swabs (E), lungs, and nasal turbinates (F). Data are depicted as mean ± SD. Significance (E) is depicted as *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance of the difference was evaluated by a 2-way ANOVA test followed by the non-parametric Mann whitney test.
An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection

December 2021

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150 Reads

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88 Citations

The development of more effective, accessible, and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector, and whole inactivated virus vaccines is essential to curtailing the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six-proline-stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to outer membrane vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells, and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum-neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination, hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs 7 days after challenge in OMV-mC-Spike-vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.



Characterization of OMVs
An Intranasal OMV-based vaccine induces high mucosal and systemic protecting immunity against a SARS-CoV-2 infection

August 2021

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188 Reads

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3 Citations

The development of more effective, accessible and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector and whole inactivated vaccines, is essential to curtain the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six proline stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to Outer Membrane Vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs seven days after challenge in OMV-mC-Spike vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.


Figure 1. Humoral and cell-mediated adaptive immune responses. Adaptive immunity involves the activation of lymphocytes and develops following exposure to diseases or immunization against diseases through vaccination. Pathogen, tumor or vaccine antigens are recognized by antigen-presenting cells (APCs). Once processed, the antigenic peptides are presented on the surface of major histocompatibility complex (MHC)-II, which are recognized by T cell receptors (TCR) on naïve CD4 + T cells. Upon TCR activation by APCs, naïve CD4 + T cells differentiate into subpopulations: T helper (Th)1, Th2, Th9, Th17, Th22, T follicular helper (Tfh), and T regulatory cells (Treg) under unique cytokine-polarized environments. B-cells, following co-stimulation from cytokines produced by CD4 + T cells, transform into plasma cells that secrete antibodies which circulate in blood and extracellular fluid. A humoral immune response is mediated by secreted antibodies produced by B-cells, which are specific for an individual antigen. CD8 + T cells recognize and bind to intracellularly processed antigenic peptides through their TCR, which are presented on MHC-I molecules on the surface of APCs and infected cells. Cytokines released by CD4 + T cells also stimulate cytotoxic CD8 + T cells, which release effector molecules such as granzyme, perforin, and IFN-γ that destroy infected host cell. A subset of memory B and T cells confer future immunity to the cognate pathogen or antigen. IFN-γ, interferon gamma; IL, interleukin; TGF-β, transforming growth factor β; TNF-α, tumor necrosis factor α.
Figure 2. Methodology using dendritic cells (DCs) and adjuvants to select vaccine antigens for cell- mediated immunity. (1) DC loaded with peptides, good Major histocompatibility (MHC)-I and MHC-II binders and a Th1 adjuvant are checked for Th1 and Th2 cytokine releases. (2) Peptides inducing Th1 cytokines are selected and validated in mice vaccinated with DCs loaded with the peptides and inoculated into the footpads to measured delayed hypersensitivity (DTH) reactions. Peptides inducing strong DTH reactions are considered good candidates for future vaccines.
Figure 3. Monophpsphoryl lipids (MPLs)Ls and synthetic lipid A mimetics as promising vaccine adjuvant candidates.
Figure 5. (a) Chemical structure of QS-21 saponin natural product adjuvant. (b-d) Synthetic saponin variants based on QS-21.
Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation

April 2021

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10,309 Reads

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31 Citations

Pharmaceutics

Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases.


Lipid A structures of Escherichia coli and Bordetella pertussis (A) and those expected after replacing the acyltransferases in B. pertussis (B). The altered acyl chains that are introduced after replacing the acyltransferases of the wild type by the heterologous enzymes are shown in red. The heterologous enzymes expressed are indicated below the structures. Pa, Pseudomonas aeruginosa; Nm, Neisseria meningitidis; Pg, Porphyromonas gingivalis. 3OH-C14, 3OH-C12, and 3OH-C10 acyl chains are indicated as C14OH, C12OH, and C10OH, respectively.
Structural analysis of lipid A by ESI-MS. Negative-ion lipid A mass spectra were obtained by in-source collision-induced dissociation nano-ESI-MS of intact LPS isolated from cells of (A) B213, (B) B213 expressing lpxAPa (B213-pLpxAPa), (C) ΔlpxA mutant of B213 expressing lpxAPa (B213 ΔlpxA-pLpxAPa), (D) B213 expressing lpxLNm (B213-pLpxLNm), (E) B213 expressing lpxLPg (B213-pLpxLPg), (F) B213 expressing lpxDPa (B213-pLpxDPa), (G) B1917 with the chromosomal lpxA replaced by lpxAPa (B1917 lpxAPa), and (H) B1917 with the chromosomal lpxD replaced by lpxDPa (B1917 lpxDPa). A major singly-deprotonated ion at m/z 1557.97 was interpreted as the typical B. pertussis lipid A structure: a diglucosamine (2 GlcN), penta-acylated (three 3OH-C14, one 3OH-C10, and one C14) with two phosphates residues (2 P) as illustrated in Figure 1A. Additional singly-deprotonated lipid A ions were detected in different derivatives and their interpretations are also indicated. Only the m/z range covering lipid A ions is shown.
Stimulation of HEK293-Blue cells expressing hTLR4 (A,C) or mTLR4 (B,D) with purified LPS (A,B) or whole-cell preparations of B213 and derivatives (C,D). LPS preparations and bacterial suspensions were serially diluted. After incubation for 2 h with HEK293-Blue cells expressing mTLR4 or for 4 h with HEK293-Blue cells expressing hTLR4, alkaline phosphatase activity was determined. Graphs show the mean and standard deviation from a representative experiment of three repeats in duplicate.
Pyrogenicity assays in rabbits. Groups of five animals were injected with saline solution (control) or with 10 µg of B. pertussis B1917 wild-type LPS, or mutant LPS derivatives resulting from the expression of lpxAPa or lpxDPa. The temperature of each animal was monitored at different time intervals after injection. (A) Mean body temperature and standard deviation for each group before injection (0 h) and at different times post-injection are shown (1, 2, 4, 6, 24, 48 h). (B) The differences in body temperatures within groups at 4 h post-injection. Statistically significant differences were determined using ANOVA and Dunnett tests and are indicated with two asterisks (p < 0.001). ns, not significant. (C) The mean of area under the curve and standard deviation of all animals of a group were calculated between 0 and 6 h using as baseline the mean of each group. Statistically significant differences between the two groups are indicated with one asterisk (p < 0.05) using an unpaired t-test. Ns, not significant.
The proposed mode of interaction of LPS with hTLR4/MD-2. The proposed binding modes of LPS of E. coli K-12 (PDB code: 3FXI) (A), B. pertussis B213/B1917 (B), and several mutant LPS derivatives generated in this study (C–E) are shown. The binding poses (orientation and affinity) with MD-2 are displayed below the figures.
Shortening the Lipid A Acyl Chains of Bordetella pertussis Enables Depletion of Lipopolysaccharide Endotoxic Activity

October 2020

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111 Reads

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18 Citations

Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis. Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines. The reactogenicity of whole-cell pertussis vaccines is, to a large extent, derived from the lipid A moiety of the lipopolysaccharides (LPS) of the bacteria. Here, we engineered B. pertussis strains with altered lipid A structures by expressing genes for the acyltransferases LpxA, LpxD, and LpxL from other bacteria resulting in altered acyl-chain length at various positions. Whole cells and extracted LPS from the strains with shorter acyl chains showed reduced or no activation of the human Toll-like receptor 4 in HEK-Blue reporter cells, whilst a longer acyl chain increased activation. Pyrogenicity studies in rabbits confirmed the in vitro assays. These findings pave the way for the development of a new generation of whole-cell pertussis vaccines with acceptable side effects.


Figure 2. Schematic representation of the Raetz pathway for the synthesis of Kdo 2 -lipid A. Shown is the pathway as operative in E. coli K-12 according to [1]. The pathway is constituted by nine conserved enzymes located in the cytoplasm or the cytoplasmic membrane. The chemical structure of substrates and products of the reactions is indicated. From the top left, arrows show the order of reactions, and the corresponding enzymes are colored according with the resulting structural modification of the product. Where appropriate, the names of substrates or products are specified.
Figure 3. Structural analysis of lipid A. Negative-ion lipid A mass spectra were obtained by in-source CID nano-ESI-FT-MS of intact LPS isolated from cells of (A) B213, (B) B213 expressing lpxH Nm , and (C) B213 expressing lpxH Nm and lpxA Nm . Bacteria were grown for 12 h in Verwey medium in the presence of IPTG. A major singly-deprotonated ion at m/z 1557.97 was interpreted as the typical B. pertussis lipid A structure: a diglucosamine (2 GlcN), penta-acylated (three 3OH-C14, one 3OH-C10 and one C14) with two phosphate residues (2 P) as illustrated in Figure 1B. Additional singly-deprotonated lipid A ions were detected in different derivatives and their interpretations are indicated. Only the m/z range covering lipid A ions is shown.
Figure 5. Stimulation of HEK293-Blue reporter cells expressing hTLR4 (A) or mTLR4 (B) with purified LPS of strain B213 or mutant derivatives. After incubation of the HEK293-Blue cells with the LPS variants indicated for 17 h, alkaline phosphatase activity was determined. Grafts show the log response ratio from three independent experiments with average and standard deviation (error bars). Statistically significant differences were analyzed with GraphPad Prism 6 and are indicated with one asterisk (p < 0.05).
Figures
Substrate specificity of the Pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis Lipid A

March 2019

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171 Reads

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5 Citations

Journal of Biological Chemistry

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3′ positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in , has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in . This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of lipid A. Expression of meningococcal LpxH in indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3′ positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in lipid A. We conclude that the asymmetry of lipid A is determined by the acyl chain length specificity of LpxH.


Citations (80)


... In the same line of consideration, the possibility to create drug delivery systems by loading OMVs with specific cargo emerged [17,18]. Within the last 25 years, OMVs were tested by various research groups as cheap alternatives for vaccines, and promising results were obtained in animal test models [19,20]. Since OMVs are non-replicative as compared to their Within the last 25 years, OMVs were tested by various research groups as cheap alternatives for vaccines, and promising results were obtained in animal test models [19,20]. ...

Reference:

Immune Responses Elicited by Outer Membrane Vesicles of Gram-Negative Bacteria: Important Players in Vaccine Development
Outer membrane vesicle-based intranasal vaccines
  • Citing Article
  • August 2023

Current Opinion in Immunology

... On the other hand, the LPS also leads to beneficial immunostimulatory effects in the TME. Therefore, a balance has to be reached between these two effects [212]. ...

Lipid A heterogeneity and its role in the host interactions with pathogenic and commensal bacteria

microLife

... The intramuscularinjection vaccine could effectively induce serum IgG antibody responses to protect the lower respiratory tract but could not provoke the epithelial IgA antibody that safeguards the upper respiratory tract [23]. In contrast, aerosolized inhaled vaccines not only induce a robust local immune response in mucosal sites, including secretory IgA antibodies, mucosal IgG antibodies, and the expression of cytokines in T cells, which serve as the frontline of defense by blocking infection entrance [40] but also robustly induce robust systemic humoral immunity that eradicates any virus particle escaping from the immune response generated at the mucosal site, as suggested by animal models and clinical trials [24,[41][42][43]. The longterm profile for the magnitude of the immune response provoked by inhaled Ad5-nCoV is warranted for further investigation. ...

An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection

... Our ultimate goal is to directly analyze the phenotype and sialylation of LOS on gonococci isolated from infected humans and their cells, which would require methods enabling highly sensitive purification and detection. A method was described recently enabling LC-MS/MS of multiple LOS phenotypes from N. meningitidis with less than 1-µg LOS (Pupo et al., 2021). Herein, we evaluated a method for purification of the N. gonorrhoeae LOS using microwaveenhanced enzymatic (ME) digestions that was described for microscale purification of LOS from Campylobacter jejuni LOS and that shortened the time for LOS purification from 3-4 days to a few hours (Dzieciatkowska et al., 2008). ...

Nanoflow LC–MS Method Allowing In-Depth Characterization of Natural Heterogeneity of Complex Bacterial Lipopolysaccharides
  • Citing Article
  • November 2021

Analytical Chemistry

... The earliest vaccines were attenuated versions of the target pathogens such as smallpox and poliovirus. While these vaccines are effective, safety concerns such as reports of poliomyelitis developing after vaccine administration have led to the development of other types of vaccines such as killed/inactivated formulations [2]. ...

Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation

Pharmaceutics

... Outer membrane vesicles (OMVs) are spherical buds produced by the outer membrane of Gram-negative bacteria with diameters ranging from 20 to 250 nm [1]. It has been found that OMVs are mainly composed of lipopolysaccharides, proteins (including various enzymes), peptidoglycan, DNA, RNA, and other small molecules [2]. ...

Shortening the Lipid A Acyl Chains of Bordetella pertussis Enables Depletion of Lipopolysaccharide Endotoxic Activity

... Leptospiral LPS was extracted from the whole-inactivated bacterial preparations mentioned above by the hot phenol/ water method, as described previously (33,34). Then, the aqueous phase of phenol/water extractions was selected for purification of leptospiral LPS by solid phase extraction on C8 reversed-phase cartridges (34). ...

Substrate specificity of the Pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis Lipid A

Journal of Biological Chemistry

... For the optimization of the MAT an OMV vaccine against B. pertussis (omvPV(WT LPS)) was used that was still in early development with respect to dosing, production process and modification to the LPS. An estimated dosing of 100 µg/mL total protein content in the OMV was used as starting dose based on immunogenicity data in mice 25 . Bexsero (OMV based vaccine against Neisseria meningitidis serogroup B (NmB)) was used as a reference product since the omvPV(WT LPS) vaccine was still in development and thus a reference lot was not yet available. ...

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination

Mucosal Immunology

... vesicle (OMV) vaccines are in development. Pulmonary or intranasal immunization of mice with pertussis OMV vaccine promoted mucosal IgA, Th1/Th17 responses in the spleen and lungs, and protected against infection of lungs, trachea, and nasal cavity following challenge with B. pertussis [42,43]. The pertussis OMV vaccine induced stronger T-cell responses, including respiratory CD4 T RM cells and better protection when delivered intranasally compared with the subcutaneous route [43] [44]. ...

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination

Mucosal Immunology

... 13,14 Previously, we engineered the "display" E. coli hemoglobin protease (HbpD) autotransporter to present multiple heterologous antigens at the surface of LPS-detoxified Salmonella Typhimurium OMVs. 15,16 In essence, domains that protrude from the core β-helix structure of HbpD were replaced by antigenic sequences through translational fusion. In previous proof-of-concept studies, we have shown that epitopes of the model antigen ovalbumin (OVA) presented in this context, induced maturation of professional dendritic cells (DCs) allowing for a tailored induction of immunity. ...

Th17-Mediated Cross Protection against Pneumococcal Carriage by Vaccination with a Variable Antigen