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. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.

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. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.

Self-assembling protein nanoparticles and virus like particles correctly display ß-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. In this work we demonstrated the successful repetitive antigen display of the C-terminal ß-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the ß-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.

Moraxella catarrhalis evades neutrophil oxidative stress responses providing a safer niche for nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae (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 M. catarrhalis induces less ROS and NET production in differentiated HL-60 cells compared to NTHi. It is also able to actively interfere with these responses in chemically activated cells in a phagocytosis and opsonin-independent and contact-dependent manner, possibly by engaging host immunosuppressive receptors. M. catarrhalis subverts the autophagic pathway of the phagocytic cells and survives intracellularly. It also promotes the survival of NTHi which is otherwise susceptible to the host antimicrobial arsenal. In-depth understanding of the immune evasion strategies exploited by these two human pathogens could suggest medical interventions to tackle COPD and potentially other diseases in which they co-exist.

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. AVAILABILITY AND IMPLEMENTATION: Publicly available at https://tmquery.gsk.com/.

Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella 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. Herein, the Neisseria meningitidis factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg-) Salmonella Typhimurium GMMA. Only the OAg- GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg- GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg- GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface.

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. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.

Proteome-minimized outer membrane vesicles from Escherichia coli 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 Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes-specific antibody titers and high frequencies of epitope-specific IFN-γ-producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV-based vaccines.

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. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.

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. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.

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. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG) was used. The cationic lipids investigated included 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) and N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ). Whilst all cationic LNP (cLNP) formulations promoted high association with cells in vitro, those formulations containing the fusogenic lipid 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) in combination with DOTAP or DDA were the most efficient at inducing antigen expression. Therefore, DOTAP and DDA formulations were selected for further in vivo studies and were compared to benchmark ionizable LNPs (iLNPs). Biodistribution studies revealed that DDA-cLNPs remained longer at the injection site compared to DOTAP-cLNPs and iLNPs when administered intramuscularly in mice. Both the cLNP formulations and the iLNPs induced strong humoral and cellular-mediated immune responses in mice that were not significantly different at a 1.5 µg SAM dose. In summary, cLNPs based on DOTAP and DDA are an efficient alternative to iLNPs to deliver SAM vaccines.

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 in vitro enhanced uptake in comparison to unglycosylated LNP from bone marrow-derived dendritic cells, and in vivo more rapid onset of the antibody response, independent of the route. The increased binding antibody levels also translated into higher functional hemagglutinin inhibition titers, particularly following intradermal administration. T cell assay on splenocytes from immunized mice also showed an increase in antigen specific CD8+ T responses, following intradermal administration of MLNP SAM vaccines. Induction of enhanced antigen specific CD4+ T cells, correlating with higher IgG2a antibody responses, was also observed. Hence, the present work illustrates the benefit of mannosylation of LNPs to achieve a faster immune response with SAM vaccines and these observations could contribute to the development of novel skin delivery systems for SAM vaccines.

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 Neisseria meningitidis 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. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy.

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). Previously described as a sortase substrate positively regulated by c-di-GMP, CD2831 was predicted to be a collagen-binding protein and thus potentially involved in sessility. By overexpressing CD2831 in C. difficile and heterologously expressing it on the surface of Lactococcus lactis, here we further demonstrated that CD2831 is a collagen-binding protein, able to bind to immobilized collagen types I, III and V as well as native collagen produced by human fibroblasts. We also observed that the overexpression of CD2831 raises the ability to form biofilm on abiotic surface in both C. difficile and L. lactis. Notably, we showed that CD2831 binds to the collagen-like domain of the human complement component C1q, suggesting a role in preventing complement cascade activation via the classical pathway. This functional characterization places CD2831 in the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMMs) family, a class of virulence factors with a dual role in adhesion to collagen-rich tissues and in host immune evasion by binding to human complement components.

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 kif18b gene (B16-M30) were used to decorate OMVs either alone or in combination. C57BL/6 were immunized with the OMVs and then challenged with B16F10EGFRvIII cells. Immunogenicity and protective activity was followed by measuring anti-EGFRvIII antibodies, M30-specific T cells, tumor-infiltrating cell population, and tumor growth. RESULTS: Immunization with engineered EGFRvIII-OMVs induced a strong inhibition of tumor growth after B16F10EGFRvIII challenge. Furthermore, mice immunized with engineered OMVs carrying both EGFRvIII and M30 epitopes were completely protected from tumor challenge. Immunization was accompanied by induction of high anti-EGFRvIII antibody titers, M30-specific T cells, and infiltration of CD4+ and CD8+ T cells at the tumor site. CONCLUSION: OMVs can be decorated with tumor antigens and can elicit antigen-specific, protective antitumor responses in immunocompetent mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs makes the OMV platform particularly attractive for cancer immunotherapy.

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. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

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. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

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. Here we discuss three key breakthrough approaches that have potentiated structural and computational vaccine design. Firstly, genomic sciences gave birth to the field of reverse vaccinology, which has enabled the rapid computational identification of potential vaccine antigens. Secondly, major advances in structural biology, experimental epitope mapping, and computational epitope prediction have yielded molecular insights into the immunogenic determinants defining protective antigens, enabling their rational optimization. Thirdly, and most recently, computational approaches have been used to convert this wealth of structural and immunological information into the design of improved vaccine antigens. This review aims to illustrate the growing power of combining sequencing, structural and computational approaches, and we discuss how this may drive the design of novel immunogens suitable for future vaccines urgently needed to increase the global prevention of infectious disease.

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. In this study, we produced influenza lentiviral pseudoparticles (PPs) containing only the NA antigen (NA-PPs) with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI) assay. Both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.

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. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).