Osmotic properties of T cells determined by flow imaging microscopy in comparison to electrical sensing zone analysis.

To develop cryopreservation methods for cell-based medicinal products it is important to understand osmotic responses of cells upon immersion into solutions with cryoprotective agents (CPAs) and during freezing. The aim of this study was to assess the osmotic response of T cells by using flow imaging microscopy (FIM) as a novel cell-sizing technique, and to corroborate the findings with electrical impedance measurements conducted on a Coulter counter. Jurkat cells were used as a potential model cell line for primary T cells. Cell volume responses were used to derive important cell parameters for cryopreservation such as the osmotically inactive cell volume Vb and the membrane permeability towards water and various CPAs. Unlike Coulter counter measurement, FIM, combined with Trypan blue staining can differentiate between viable and dead cells, which yields a more accurate estimation of Vb. Membrane permeabilities to water, dimethyl sulfoxide (Me2SO) and glycerol were measured for Jurkat cells at different temperatures. The permeation of Me2SO into the cells was faster in comparison to glycerol. CPA permeation decreased with decreasing temperature following Arrhenius behavior. Moreover, membrane permeability to water decreased in the presence of CPAs. Vb of Jurkat cells was found to be 49% of the isotonic volume and comparable to that of primary T cells. FIM proved to be a valuable tool to determine the membrane permeability parameters of mammalian cells to water and cryoprotective agents, which in turn can be used to rationally design CPA loading procedures for cryopreservation.

Novel Formaldehyde-Induced Modifications of Lysine Residue Pairs in Peptides and Proteins: Identification and Relevance to Vaccine Development.

Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.

Systems vaccinology and big data in the vaccine development chain.

Systems vaccinology has proven a fascinating development in the last decade. Where traditionally vaccine development has been dominated by trial and error, systems vaccinology is a tool that provides novel and comprehensive understanding if properly used. Data sets retrieved from systems-based studies endorse rational design and effective development of safe and efficacious vaccines. In this review we first describe different omics-techniques that form the pillars of systems vaccinology. In the second part, the application of systems vaccinology in the different stages of vaccine development is described. Overall, this review shows that systems vaccinology has become an important tool anywhere in the vaccine development chain.

Coated and Hollow Microneedle-Mediated Intradermal Immunization in Mice with Diphtheria Toxoid Loaded Mesoporous Silica Nanoparticles.

PURPOSE: To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. METHODS: DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls. RESULTS: LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles. CONCLUSION: Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.

Antigenic fingerprinting of diphtheria toxoid adsorbed to aluminium phosphate.

The antigenicity of alum-adsorbed diphtheria toxoid (DTd) was determined in combination vaccines, containing DTd, tetanus toxoid and inactivated poliovirus. A panel of monoclonal antibodies was used, covering five epitopes, distributed over the antigen. The resulting antigenic fingerprint of DTd demonstrates consistency of adsorption at antigen level in final product combination vaccines. The antigenic quality of DTd alone, adsorbed to aluminium phosphate, was also determined and compared with pre-adsorbed toxoid (starting material as well as toxoid desorbed from aluminium phosphate). Some epitopes became less accessible after adsorption, while others became relatively better exposed. Some epitopes disappeared almost completely upon adsorption, but were re-established after desorption of the antigen. The results indicate that DTd is adsorbed to aluminium phosphate in a preferred orientation and not randomly.

Determination of Depth-Dependent Intradermal Immunogenicity of Adjuvanted Inactivated Polio Vaccine Delivered by Microinjections via Hollow Microneedles.

PURPOSE: The aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT). METHODS: A novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 µm, or at 400 µm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization. RESULTS: The applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 µm), -volumes (1-100 µL) and -rates (up to 60 µL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold). CONCLUSION: Intradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.

Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity.

The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.

Layer-by-Layer Assembly of Inactivated Poliovirus and N-Trimethyl Chitosan on pH-Sensitive Microneedles for Dermal Vaccination.

The aim of this work was to coat pH-sensitive microneedle arrays with inactivated polio vaccine (IPV) particles and N-trimethyl chitosan chloride (TMC) via electrostatic interactions, and assess the immunogenicity of the vaccine after topical application of the coated microneedles in rats. The surface of 200 µm long microneedles was first chemically modified with pH-sensitive (pyridine) groups and then coated with negatively charged IPV and a positively charged polymer (TMC). To obtain a sufficient high antigen dose, 10 layers of IPV were alternately coated with TMC. The binding of IPV and TMC onto pH-sensitive microneedles was quantified and visualized by using fluorescently labeled TMC and IPV. The release of IPV and TMC from the microneedles was evaluated in ex vivo human skin by fluorescence and the immunogenicity of (unlabeled) IPV was assessed after topical application of the coated microneedles in rats. pH-sensitive microneedles were homogeneously coated with 10 layers of both IPV and TMC, resulting in 45 D antigen units IPV and 700 ng TMC per microneedle array. Fluorescence microscopy imaging revealed that both IPV and TMC were released into ex vivo human skin upon application of the coated microneedles. Finally, in vivo application of IPV-TMC-coated pH-sensitive microneedles in rats led to the induction of IPV specific antibody responses, illustrating that they are practically applicable. Topical administration of pH-sensitive microneedles coated with polyelectrolyte multinanolayers of antigens and oppositely charged polymers may be a useful approach for microneedle-based vaccination.

Influenza T-cell epitope-loaded virosomes adjuvanted with CpG as a potential influenza vaccine.

PURPOSE: Influenza CD8(+) T-cell epitopes are conserved amongst influenza strains and can be recognized by influenza-specific cytotoxic T-cells (CTLs), which can rapidly clear infected cells. An influenza peptide vaccine that elicits these CTLs would therefore be an alternative to current influenza vaccines, which are not cross-reactive. However, peptide antigens are poorly immunogenic due to lack of delivery to antigen presenting cells, and therefore need additional formulation with a suitable delivery system. In this study, the potential of virosomes as a delivery system for an influenza T-cell peptide was investigated. METHODS: The conserved human HLA-A2.1 influenza T-cell epitope M158-66 was formulated with virosomes. The immunogenicity and protective effect of the peptide-loaded virosomes was assessed in HLA-A2 transgenic mice. Delivery properties of the virosomes were studied in mice and in in vitro dendritic cell cultures. RESULTS: Immunization of HLA-A2.1 transgenic C57BL/6 mice with peptide-loaded virosomes in the presence of the adjuvant CpG-ODN 1826 increased the number of peptide-specific CTLs. Vaccination with adjuvanted peptide-loaded virosomes reduced weight loss in mice after heterologous influenza infection. Association with fusion-active virosomes was found to be crucial for antigen uptake by dendritic cells, and subsequent induction of CTLs in mice. CONCLUSIONS: These results show that influenza virosomes loaded with conserved influenza epitopes could be the basis of a novel cross-protective influenza vaccine.

Development of thermostable lyophilized inactivated polio vaccine.

PURPOSE: The aim of current study was to develop a dried inactivated polio vaccine (IPV) formulation with minimal loss during the drying process and improved stability when compared with the conventional liquid IPV. METHODS: Extensive excipient screening was combined with the use of a Design of Experiment (DoE) approach in order to achieve optimal results with high probability. RESULTS: Although it was shown earlier that the lyophilization of a trivalent IPV while conserving its antigenicity is challenging, we were able to develop a formulation that showed minimal loss of potency during drying and subsequent storage at higher temperatures. CONCLUSION: This study showed the potential of a highly stable and safe lyophilized polio vaccine, which might be used in developing countries without the need of a cold-chain.

Novel hollow microneedle technology for depth-controlled microinjection-mediated dermal vaccination: a study with polio vaccine in rats.

PURPOSE: The aim of the study was to develop a cheap and fast method to produce hollow microneedles and an applicator for injecting vaccines into the skin at a pre-defined depth and test the applicability of the system for dermal polio vaccination. METHODS: Hollow microneedles were produced by hydrofluoric acid etching of fused silica capillaries. An electromagnetic applicator was developed to control the insertion speed (1-3 m/s), depth (0-1,000 µm), and angle (10°-90°). Hollow microneedles with an inner diameter of 20 µm were evaluated in ex vivo human skin and subsequently used to immunize rats with inactivated poliovirus vaccine (IPV) by an intradermal microinjection of 9 µL at a depth of 300 µm and an insertion speed of 1 m/s. Rat sera were tested for IPV-specific IgG and virus-neutralizing antibodies. RESULTS: Microneedles produced from fused silica capillaries were successfully inserted into the skin to a chosen depth, without clogging or breakage of the needles. Intradermal microinjection of IPV induced immune responses comparable to those elicited by conventional intramuscular immunization. CONCLUSIONS: We successfully developed a hollow microneedle technology for dermal vaccination that enables fundamental research on factors, such as insertion depth and volume, and insertion angle, on the immune response.

Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.

A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products.

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid.

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.

Mass Spectrometry-Based Quantification of the Antigens in Aluminum Hydroxide-Adjuvanted Diphtheria-Tetanus-Acellular-Pertussis Combination Vaccines.

Vaccines undergo stringent batch-release testing, most often including in-vivo assays for potency. For combination vaccines, such as diphtheria-tetanus-pertussis (DTaP), chemical modification induced by formaldehyde inactivation, as well as adsorption to aluminum-based adjuvants, complicates antigen-specific in-vitro analysis. Here, a mass spectrometric method was developed that allows the identification and quantitation of DTaP antigens in a combination vaccine. Isotopically labeled, antigen-specific internal standard peptides were employed that permitted absolute quantitation of their antigen-derived peptide counterparts and, consequently, the individual antigens. We evaluated the applicability of the method on monovalent non-adjuvanted antigens, on final vaccine lots and on experimental vaccine batches, where certain antigens were omitted from the drug product. Apart from the applicability for final batch release, we demonstrated the suitability of the approach for in-process control monitoring. The peptide quantification method facilitates antigen-specific identification and quantification of combination vaccines in a single assay. This may contribute, as part of the consistency approach, to a reduction in the number of animal tests required for vaccine-batch release.

Particles in Biopharmaceutical Formulations, Part 2: An Update on Analytical Techniques and Applications for Therapeutic Proteins, Viruses, Vaccines and Cells.

Particles in biopharmaceutical formulations remain a hot topic in drug product development. With new product classes emerging it is crucial to discriminate particulate active pharmaceutical ingredients from particulate impurities. Technical improvements, new analytical developments and emerging tools (e.g., machine learning tools) increase the amount of information generated for particles. For a proper interpretation and judgment of the generated data a thorough understanding of the measurement principle, suitable application fields and potential limitations and pitfalls is required. Our review provides a comprehensive overview of novel particle analysis techniques emerging in the last decade for particulate impurities in therapeutic protein formulations (protein-related, excipient-related and primary packaging material-related), as well as particulate biopharmaceutical formulations (virus particles, virus-like particles, lipid nanoparticles and cell-based medicinal products). In addition, we review the literature on applications, describe specific analytical approaches and illustrate advantages and drawbacks of currently available techniques for particulate biopharmaceutical formulations.

Immunochemical and biophysical characterization of inactivated Sabin poliovirus products: Insights into rapid quality assessment tools.

The aim of this study was to demonstrate the strength of combining immunochemical and biophysical analysis tools for assessing the quality of Sabin inactivated poliovirus vaccine (Sabin-IPV) bulk products. We assessed Sabin-IPV serotypes 1, 2 and 3 from six different manufacturers and evaluated their comparability through biosensor analysis and biophysical characterization methods, including tryptophan fluorescence and asymmetrical flow field-flow fractionation - multi-angle light scattering analysis. These methods enabled us to assess antigenic as well as conformational and structural integrity profiles, respectively. Based on Sabin-IPV samples that were subjected to accelerated storage conditions, we revealed that existing immunochemical methods exhibit remarkably similar trends to the results obtained by the biophysical characterization methods. While the results underpin that the comparability of Sabin-IPV bulk products of different manufacturers is weak, information about their quality can rapidly be obtained by using both immunochemical and biophysical methods. Furthermore, the study highlights that quality assessment of Sabin-IPV can be obtained through biophysical techniques can complement the assessments performed with monoclonal antibodies and suggests that similar techniques could be employed to characterize other enteroviruses.

Aluminum Hydroxide And Aluminum Phosphate Adjuvants Elicit A Different Innate Immune Response.

Aluminum hydroxide (Al(OH)3) and aluminum phosphate (AlPO4) are widely used adjuvants in human vaccines. However, a rationale to choose one or the other is lacking since the differences between molecular mechanisms of action of these adjuvants are unknown. In the current study, we compared the innate immune response induced by both adjuvants in vitro and in vivo. Proteome analysis of human primary monocytes was used to determine the immunological pathways activated by these adjuvants. Subsequently, analysis of immune cells present at the site of injection and proteome analysis of the muscle tissue revealed the differentially regulated processes related to the innate immune response in vivo. Incubation with Al(OH)3 specifically enhanced the activation of antigen processing and presentation pathways in vitro. In vivo experiments showed that only intramuscular (I.M.) immunization with Al(OH)3 attracted neutrophils, while I.M. immunization with AlPO4 attracted monocytes/macrophages to the site of injection. In addition, only I.M. immunization with Al(OH)3 enhanced the process of hemostasis after 96 hours, possibly related to neutrophilic extracellular trap formation. Both adjuvants differentially regulated various immune system-related processes. The results show that Al(OH)3 and AlPO4 act differently on the innate immune system. We speculate that these different regulations affect the interaction with cells, due to the different physicochemical properties of both adjuvants.

Transcutaneous immunization studies in mice using diphtheria toxoid-loaded vesicle formulations and a microneedle array.

PURPOSE: To determine the immunogenicity of diphtheria toxoid (DT) formulated in two types of vesicles following transcutaneous immunization (TCI) of mice onto microneedle array-treated skin. METHODS: DT-containing cationic liposomes or anionic surfactant-based vesicles were prepared by extrusion and sonication. The physicochemical properties were characterized in terms of size, ζ-potential, vesicle elasticity and antigen association. TCI was performed by applying formulations onto intact or microneedle array-pretreated mice skin, using cholera toxin as an adjuvant. Subcutaneous and intradermal immunizations were as control. Immune responses were evaluated by IgG and neutralizing antibody titers, and the immune-stimulatory properties were assessed using cultured dendritic cells. RESULTS: Stable DT-containing cationic liposomes (∼150 nm) and anionic vesicles (∼100 nm) were obtained. Incorporation of Span 80 increased liposome elasticity. About 90% and 77% DT was associated with liposomes and vesicles, respectively. TCI of all formulations resulted in substantial antibody titers only if microneedle pretreatment was applied. Co-administration of cholera toxin further augmented the immune responses of TCI. However, vesicle formulations didn't enhance the immunogenicity on either intact or microneedle-treated skin and showed low stimulatory activity on dendritic cells. CONCLUSIONS: Microneedle pretreatment and cholera toxin, but not antigen association to vesicles, enhances the immunogenicity of topically applied DT.

mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability.

A drawback of the current mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they have to be stored at (ultra)low temperatures. Understanding the root cause of the instability of these vaccines may help to rationally improve mRNA-LNP product stability and thereby ease the temperature conditions for storage. In this review we discuss proposed structures of mRNA-LNPs, factors that impact mRNA-LNP stability and strategies to optimize mRNA-LNP product stability. Analysis of mRNA-LNP structures reveals that mRNA, the ionizable cationic lipid and water are present in the LNP core. The neutral helper lipids are mainly positioned in the outer, encapsulating, wall. mRNA hydrolysis is the determining factor for mRNA-LNP instability. It is currently unclear how water in the LNP core interacts with the mRNA and to what extent the degradation prone sites of mRNA are protected through a coat of ionizable cationic lipids. To improve the stability of mRNA-LNP vaccines, optimization of the mRNA nucleotide composition should be prioritized. Secondly, a better understanding of the milieu the mRNA is exposed to in the core of LNPs may help to rationalize adjustments to the LNP structure to preserve mRNA integrity. Moreover, drying techniques, such as lyophilization, are promising options still to be explored.

Common Reference-Based Tandem Mass Tag Multiplexing for the Relative Quantification of Peptides: Design and Application to Degradome Analysis of Diphtheria Toxoid.

Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids.