Effective Multivalent Oriented Presentation of Meningococcal NadA Antigen Trimers by Self-Assembling Ferritin Nanoparticles.

The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity.

FHUSPA2/10 is a bactericidal monoclonal antibody targeting multiple repeated sequences of Moraxella catarrhalis UspA2.

Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs.

Self-assembling protein nanoparticles and virus like particles correctly display β-barrel from meningococcal factor H-binding protein through genetic fusion.

Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies.

evades neutrophil oxidative stress responses providing a safer niche for nontypeable .

and nontypeable (NTHi) are pathogenic bacteria frequently associated with exacerbation of chronic obstructive pulmonary disease (COPD), whose hallmark is inflammatory oxidative stress. Neutrophils produce reactive oxygen species (ROS) which can boost antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that induces less ROS and NET production in differentiated HL-60 cells compared to NTHi.

TMQuery: a database of precomputed template modeling scores for assessment of protein structural similarity.

Summary: Comparisons of protein structures are critical for developing novel protein designs, annotating protein functions and predicting protein structure. The template modeling score (TM-score) is a widely used but computationally expensive measure of protein similarity that is applicable to a wide variety of structural biology problems. We introduce TMQuery-a continuously updated database containing over eight billion pre-computed TM-score values for every pair of proteins in the Protein Data Bank, allowing researchers to quickly query and download TM-scores via a web interface.

Factor H Binding Protein Surface Exposure on Typhimurium GMMA Is Critical to Induce an Effective Immune Response against Both Diseases.

GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated.

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency.

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability.

Proteome-minimized outer membrane vesicles from as a generalized vaccine platform.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram-negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create BL21(DE3)Δ60, a strain releasing OMVs (OMVs) deprived of 59 endogenous proteins.

Rational design of adjuvants for subunit vaccines: The format of cationic adjuvants affects the induction of antigen-specific antibody responses.

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo.

Synergic complement-mediated bactericidal activity of monoclonal antibodies with distinct specificity.

The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen.

Delivery of self-amplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic lipid selection.

Self-amplifying RNA (SAM) represents a versatile tool that can be used to develop potent vaccines, potentially able to elicit strong antigen-specific humoral and cellular-mediated immune responses to virtually any infectious disease. To protect the SAM from degradation and achieve efficient delivery, lipid nanoparticles (LNPs), particularly those based on ionizable amino-lipids, are commonly adopted. Herein, we compared commonly available cationic lipids, which have been broadly used in clinical investigations, as an alternative to ionizable lipids.

Mannosylation of LNP Results in Improved Potency for Self-Amplifying RNA (SAM) Vaccines.

Mannosylation of Lipid Nanoparticles (LNP) can potentially enhance uptake by Antigen Presenting Cells, which are highly abundant in dermal tissues, to improve the potency of Self Amplifying mRNA (SAM) vaccines in comparison to the established unmodified LNP delivery system. In the current studies, we evaluated mannosylated LNP (MLNP), which were obtained by incorporation of a stable Mannose-cholesterol amine conjugate, for the delivery of an influenza (hemagglutinin) encoded SAM vaccine in mice, by both intramuscular and intradermal routes of administration. SAM MLNP exhibited enhanced uptake in comparison to unglycosylated LNP from bone marrow-derived dendritic cells, and more rapid onset of the antibody response, independent of the route.

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein.

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines.

Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis.

Clostridium difficile is a Gram-positive, anaerobic bacterium and the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile modulates its transition from a motile to a sessile lifestyle through a mechanism of riboswitches regulated by cyclic diguanosine monophosphate (c-di-GMP).

Synergistic Protective Activity of Tumor-Specific Epitopes Engineered in Bacterial Outer Membrane Vesicles.

Introduction: Bacterial outer membrane vesicles (OMVs) are naturally produced by all Gram-negative bacteria and, thanks to their plasticity and unique adjuvanticity, are emerging as an attractive vaccine platform. To test the applicability of OMVs in cancer immunotherapy, we decorated them with either one or two protective epitopes present in the B16F10EGFRvIII cell line and tested the protective activity of OMV immunization in C57BL/6 mice challenged with B16F10EGFRvIII.

Materials And Methods: The 14 amino acid B cell epitope of human epidermal growth factor receptor variant III (EGFRvIII) and the mutation-derived CD4+ T cell neo-epitope of gene (B16-M30) were used to decorate OMVs either alone or in combination.

Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain.

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein.

Structural and Computational Biology in the Design of Immunogenic Vaccine Antigens.

Vaccination is historically one of the most important medical interventions for the prevention of infectious disease. Previously, vaccines were typically made of rather crude mixtures of inactivated or attenuated causative agents. However, over the last 10-20 years, several important technological and computational advances have enabled major progress in the discovery and design of potently immunogenic recombinant protein vaccine antigens.

Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay.

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods.

An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies.

Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA) which relies on hemagglutinin (HA) mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials.

Ultrastructural Visualization of Vaccine Adjuvant Uptake In Vitro and In Vivo.

Adjuvants are substances that enhance adaptive immune responses when formulated in a vaccine. Alum and MF59 are two vaccine adjuvants licensed for human vaccination. Their mode of action has not been completely elucidated.

Streptococcus agalactiae capsule polymer length and attachment is determined by the proteins CpsABCD.

The production of capsular polysaccharides (CPS) or secreted exopolysaccharides is ubiquitous in bacteria, and the Wzy pathway constitutes a prototypical mechanism to produce these structures. Despite the differences in polysaccharide composition among species, a group of proteins involved in this pathway is well conserved. Streptococcus agalactiae (group B Streptococcus; GBS) produces a CPS that represents the main virulence factor of the bacterium and is a prime target in current vaccine development.

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine.

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS).

Structural vaccinology: a three-dimensional view for vaccine development.

The Structural Vaccinology (SV) approach is the logical evolution of Reverse Vaccinology: a genome-based approach combined with structural biology, with the idea that protective determinants can be used to selectively engineer the antigens that can be re-designed and simplified for inclusion in vaccine combinations. The final objectives of the rational structure-based antigen optimization are the facilitation of industrial-scale production of the antigens combination, obtain a greater immunogenicity and a greater safety profile and finally expand the breadth of protection. Structural Vaccinology is particularly powerful in case of antigenic variation between closely related strains and species.