Patients with naproxen-induced liver injury display T-cell memory responses toward an oxidative (S)-O-desmethyl naproxen metabolite but not the acyl glucuronide.

BACKGROUND: Exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen (IBU) and naproxen (NAP) is associated with idiosyncratic drug-induced liver injury (DILI). Carboxylate bioactivation into reactive metabolites (e.g., acyl glucuronides, AG) and resulting T-cell activation is hypothesized as causal for this adverse event. However, conclusive evidence supporting this is lacking. METHODS: In this work, we identify CD4+ and CD8+ T-cell hepatic infiltration in a biopsy from an IBU DILI patient. Lymphocyte transformation test and IFN-γ ELIspot, conducted on peripheral blood mononuclear cells (PBMCs) of patients with NAP-DILI, were used to explore drug-specific T-cell activation. T-cell clones (TCC) were generated and tested for drug specificity, phenotype/function, and pathways of T-cell activation. Cells were exposed to NAP, its oxidative metabolite 6-O-desmethyl NAP (DM-NAP), its AG or synthesized NAP-AG human-serum albumin adducts (NAP-AG adduct). RESULTS: CD4+ and CD8+ T-cells from patients expressing a range of different Vß receptors were stimulated to proliferate and secrete IFN-γ and IL-22 when exposed to DM-NAP, but not NAP, NAP-AG or the NAP-AG adduct. Activation of the CD4+ TCC was HLA-DQ-restricted and dependent on antigen presenting cells (APC); most TCC were activated with DM-NAP-pulsed APC, while fixation of APC blocked the T-cell response. Cross-reactivity was not observed with structurally-related drugs. CONCLUSION: Our results confirm hepatic T-cell infiltrations in NSAID-induced DILI, and show a T-cell memory response toward DM-NAP indicating an immune-mediated basis for the adverse event. Whilst bioactivation at the carboxylate group is widely hypothesized to be pathogenic for NSAID associated DILI, we found no evidence of this with NAP.

Modular and tunable alternative surfactants for biopharmaceuticals provide insights into Surfactant's Structure-Function relationship.

Surface-induced aggregation of protein therapeutics is opposed by employing surfactants, which are ubiquitously used in drug product development, with polysorbates being the gold standard. Since poloxamer 188 is currently the only generally accepted polysorbate alternative, but cannot be ubiquitously applied, there is a strong need to develop surfactant alternatives for protein biologics that would complement and possibly overcome known drawbacks of existing surfactants. Yet, a severe lack of structure-function relationship knowledge complicates the development of new surfactants. Herein, we perform a systematic analysis of the structure-function relationship of three classes of novel alternative surfactants. Firstly, the mode of action is thoroughly characterized through tensiometry, calorimetry and MD simulations. Secondly, the safety profiles are evaluated through cell-based in vitro assays. Ultimately, we could conclude that the alternative surfactants investigated possess a mode of action and safety profile comparable to polysorbates. Moreover, the biophysical patterns elucidated here can be exploited to precisely tune the features of future surfactant designs.

In vaccinated individuals serum bactericidal activity against B meningococci is abrogated by C5 inhibition but not by inhibition of the alternative complement pathway.

Introduction: Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. Methods: Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. Results: While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. Discussion: Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition.

An Intra-Company Analysis of Inherent Particles in Biologicals Shapes the Protein Particle Mitigation Strategy Across Development Stages.

To better understand protein aggregation and inherent particle formation in the biologics pipeline at Novartis, a cross-functional team collected and analyzed historical protein particle issues. Inherent particle occurrences from the past 10 years were systematically captured in a protein particle database. Where the root cause was identified, a number of product attributes (such as development stage, process step, or protein format) were trended. Several key themes were revealed: 1) there was a higher propensity for inherent particle formation with non-mAbs than with mAbs; 2) the majority of particles were detected following manufacturing at scale, and were not predicted by the small-scale studies; 3) most issues were related to visible particles, followed by subvisible particles; 4) 50% of the issues were manufacturing related. These learnings became the foundation of a particle mitigation strategy across development and technical transfer, and resulted in a set of preventive actions. Overall, this study provides further insight into a recognized industry challenge and hopes to inspire the biopharmaceutical industry to transparently share their experiences with inherent particles formation.

Inhibition of the different complement pathways has varying impacts on the serum bactericidal activity and opsonophagocytosis against Haemophilus influenzae type b.

Defense against Haemophilus influenzae type b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but vaccination significantly enhanced SBA titers. Independently of the vaccination status, both C3 and C5 inhibition abrogated SBA, whereas inhibition of the LP had no effect. AP inhibition had a major inhibitory effect on SBA of pre- vaccination serum, but vaccination mitigated this inhibition for all disease isolates tested. Despite this, SBA-mediated killing of some Hib isolates remained retarded. Even for the most serum-resistant isolate, SBA was the dominating defense mechanism in reconstituted whole blood, as addition of blood cells to the serum did not enhance bacterial killing. Limited Fc receptor-mediated opsonophagocytosis was unmasked when bacterial killing by the membrane attack complex was blocked. In the presence of C3 or C5 inhibitors, addition of post-vaccination, but not of pre-vaccination serum to the blood cells triggered opsonophagocytosis, leading to suppression of bacterial multiplication. Taken together, our data indicate that for host defense against Hib, killing by SBA is more efficient than by blood cell opsonophagocytosis. However, additional defense mechanisms, such as bacterial clearance by spleen and liver, may play an important role in preventing Hib-mediated sepsis, in particular for Hib isolates with increased serum-resistance. Results indicate potentially improved safety profile of AP inhibitors over C3 and C5 inhibitors as alternative therapeutic agents in patients with increased susceptibility to Hib infection.

Therapeutic antibody glycosylation impacts antigen recognition and immunogenicity.

In this study we show that glycosylation is relevant for immune recognition of therapeutic antibodies, and that defined glycan structures can modulate immunogenicity. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non-human cell lines. While non-human glycosylation is thought to cause hypersensitivity reactions and immunogenicity, less is known about effects of Fc-associated glycan structures on immune cell responses. We postulated that glycosylation influences antigen recognition and subsequently humoral responses to therapeutic antibodies by modulating 1) recognition and uptake by dendritic cells (DCs), and 2) antigen routing, processing and presentation. Here, we compared different glycosylation variants of the antibody rituximab (RTX) in in vitro assays using human DCs and T cells as well as in in vivo studies. We found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan-specific receptors. Furthermore, we show that DC-uptake of RTX increases or decreases if glycosylation is selectively modified to recognize activating (by mannosylation) or inhibitory lectin receptors (by sialylation). Moreover, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, we demonstrate that antibody glycosylation impacts anti-drug antibody (ADA) responses to RTX in vivo. Hence, defined glycan structures can modulate immune recognition and alter ADA responses. Glyco-engineering may help to decrease clinical immunogenicity and ADA-associated adverse events such as hypersensitivity reactions.

Alternative Complement Pathway Inhibition Does Not Abrogate Meningococcal Killing by Serum of Vaccinated Individuals.

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors.

Alternative Complement Pathway Inhibition Abrogates Pneumococcal Opsonophagocytosis in Vaccine-Naïve, but Not in Vaccinated Individuals.

To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independent of amplification by the alternative pathway, even low capsule-specific antibody concentrations were sufficient to efficiently trigger classical pathway mediated opsonophagocytosis. In heat-inactivated or C3-inhibited serum, high concentrations of capsule-specific antibodies were required to trigger complement-independent opsonophagocytosis. Our findings suggest that treatment with alternative complement pathway inhibitors will increase susceptibility for invasive pneumococcal infection in non-immune subjects, but it will not impede pneumococcal clearance in vaccinated individuals.

Extracellular Matrix-Induced GM-CSF and Hypoxia Promote Immune Control of Mycobacterium tuberculosis in Human In Vitro Granulomas.

Several in vitro cellular models have been developed with the aim to reproduce and dissect human granulomatous responses, the hallmark of tuberculosis (TB) immunopathogenesis. In that context, we compared two- (2D) versus three-dimensional (3D) granuloma models resulting from infection of human peripheral blood mononuclear cells with M. tuberculosis (Mtb) in the absence or presence of a collagen-based extracellular matrix (ECM). Granuloma formation was found to be significantly enhanced in the 2D model. This feature was associated with an earlier chemokine production and lymphocyte activation, but also a significantly increased bacterial burden. Remarkably, the reduction in Mtb burden in the 3D model correlated with an increase in GM-CSF production. GM-CSF, which is known to promote macrophage survival, was found to be inherently induced by the ECM. We observed that only 3D in vitro granulomas led to the accumulation of lipid inclusions within Mtb. Our data suggest that a hypoxic environment within the ECM could be responsible for this dormant-like Mtb phenotype. Furthermore, exposure to a TNF-α antagonist reverted Mtb dormancy, thereby mimicking the reactivation of TB observed in rheumatic patients receiving this therapy. To conclude, we showed that only in vitro granulomas generated in the presence of an ECM could recapitulate some clinically relevant features of granulomatous responses in TB. As such, this model constitutes a highly valuable tool to study the interplay between immunity and Mtb stress responses as well as to evaluate novel treatment strategies.

Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

The Emerging Jamboree of Transformative Therapies for Autoimmune Diseases.

Standard treatments for autoimmune and autoinflammatory disorders rely mainly on immunosuppression. These are predominantly symptomatic remedies that do not affect the root cause of the disease and are associated with multiple side effects. Immunotherapies are being developed during the last decades as more specific and safer alternatives to small molecules with broad immunosuppressive activity, but they still do not distinguish between disease-causing and protective cell targets and thus, they still have considerable risks of increasing susceptibility to infections and/or malignancy. Antigen-specific approaches inducing immune tolerance represent an emerging trend carrying the potential to be curative without inducing broad immunosuppression. These therapies are based on antigenic epitopes derived from the same proteins that are targeted by the autoreactive T and B cells, and which are administered to patients together with precise instructions to induce regulatory responses capable to restore homeostasis. They are not personalized medicines, and they do not need to be. They are precision therapies exquisitely targeting the disease-causing cells that drive pathology in defined patient populations. Immune tolerance approaches are truly transformative options for people suffering from autoimmune diseases.

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

TNF-α antagonists differentially induce TGF-ß1-dependent resuscitation of dormant-like Mycobacterium tuberculosis.

TNF-α- as well as non-TNF-α-targeting biologics are prescribed to treat a variety of immune-mediated inflammatory disorders. The well-documented risk of tuberculosis progression associated with anti-TNF-α treatment highlighted the central role of TNF-α for the maintenance of protective immunity, although the rate of tuberculosis detected among patients varies with the nature of the drug. Using a human, in-vitro granuloma model, we reproduce the increased reactivation rate of tuberculosis following exposure to Adalimumab compared to Etanercept, two TNF-α-neutralizing biologics. We show that Adalimumab, because of its bivalence, specifically induces TGF-ß1-dependent Mycobacterium tuberculosis (Mtb) resuscitation which can be prevented by concomitant TGF-ß1 neutralization. Moreover, our data suggest an additional role of lymphotoxin-α-neutralized by Etanercept but not Adalimumab-in the control of latent tuberculosis infection. Furthermore, we show that, while Secukinumab, an anti-IL-17A antibody, does not revert Mtb dormancy, the anti-IL-12-p40 antibody Ustekinumab and the recombinant IL-1RA Anakinra promote Mtb resuscitation, in line with the importance of these pathways in tuberculosis immunity.

Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-Host Interaction.

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma's environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the singular advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments, which have not been reported in other published protocols to generate in vitro granulomas.

Inhibition of IL-17A by secukinumab shows no evidence of increased Mycobacterium tuberculosis infections.

Secukinumab, a fully human monoclonal antibody that selectively neutralizes interleukin-17A (IL-17A), has been shown to have significant efficacy in the treatment of moderate to severe psoriasis, psoriatic arthritis and ankylosing spondylitis. Blocking critical mediators of immunity may carry a risk of increased opportunistic infections. Here we present clinical and in vitro findings examining the effect of secukinumab on Mycobacterium tuberculosis infection. We re-assessed the effect of secukinumab on the incidence of acute tuberculosis (TB) and reactivation of latent TB infection (LTBI) in pooled safety data from five randomized, double-blind, placebo-controlled, phase 3 clinical trials in subjects with moderate to severe plaque psoriasis. No cases of TB were observed after 1 year. Importantly, in subjects with a history of pulmonary TB (but negative for interferon-γ release and receiving no anti-TB medication) or positive for latent TB (screened by interferon-γ release assay and receiving anti-TB medication), no cases of active TB were reported. Moreover, an in vitro study examined the effect of the anti-tumor necrosis factor-α (TNFα) antibody adalimumab and secukinumab on dormant M. tuberculosis H37Rv in a novel human three-dimensional microgranuloma model. Auramine-O, Nile red staining and rifampicin resistance of M. tuberculosis were measured. In vitro, anti-TNFα treatment showed increased staining for Auramine-O, decreased Nile red staining and decreased rifampicin resistance, indicative of mycobacterial reactivation. In contrast, secukinumab treatment was comparable to control indicating a lack of effect on M. tuberculosis dormancy. To date, clinical and preclinical investigations with secukinumab found no evidence of increased M. tuberculosis infections.

T-cell assays confirm immunogenicity of tungsten-induced erythropoietin aggregates associated with pure red cell aplasia.

Immunogenicity of biotherapeutics and the elicitation of anti-drug antibodies are a key concern for their efficacy, pharmacokinetics, and safety. A particularly severe consequence of immunogenicity of a biotherapeutic is the rare development of antibody-mediated pure red cell aplasia (PRCA) in anemic patients treated with aggregated forms of recombinant human erythropoietin (rhEPO). Here, we investigated in vitro T-cell responses to experimentally heat-induced rhEPO aggregates, and to tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA. Heat-stressed rhEPO elicited T-cell responses only in blood obtained from healthy individuals identified as responders, whereas nonstressed rhEPO overall did not induce reactions neither in responders nor nonresponders. Tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA could induce in vitro T-cell responses in blood obtained from healthy donors, in contrast to rhEPO from low tungsten syringes. Importantly, ex vivo T-cell recall responses of patients treated with rhEPO without PRCA showed no T-cell responses, whereas T cells of a patient who developed PRCA after treatment with a clinical batch with elevated levels of tungsten and rhEPO aggregates showed a clear response to rhEPO from that clinical batch. To our knowledge, this is the first time that T-cell assays confirm the root cause of increased rhEPO immunogenicity associated with PRCA.

New Approaches to Investigate Drug-Induced Hypersensitivity.

The workshop on "New Approaches to Investigate Drug-Induced Hypersensitivity" was held on June 5, 2014 at the Foresight Center, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical, and chemical basis of small molecule drug hypersensitivity, (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, and (3) identify the limitations, knowledge gaps, and challenges that limit the use of these assays and utilize the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective reviews the clinical and immunological bases of drug hypersensitivity and summarizes various experts' views on the different topics covered during the meeting.

Controlled Mycobacterium tuberculosis infection in mice under treatment with anti-IL-17A or IL-17F antibodies, in contrast to TNFα neutralization.

Antibodies targeting IL-17A or its receptor IL-17RA show unprecedented efficacy in the treatment of autoimmune diseases such as psoriasis. These therapies, by neutralizing critical mediators of immunity, may increase susceptibility to infections. Here, we compared the effect of antibodies neutralizing IL-17A, IL-17F or TNFα on murine host responses to Mycobacterium tuberculosis infection by evaluating lung transcriptomic, microbiological and histological analyses. Coinciding with a significant increase of mycobacterial burden and pathological changes following TNFα blockade, gene array analyses of infected lungs revealed major changes of inflammatory and immune gene expression signatures 4 weeks post-infection. Specifically, gene expression associated with host-pathogen interactions, macrophage recruitment, activation and polarization, host-antimycobacterial activities, immunomodulatory responses, as well as extracellular matrix metallopeptidases, were markedly modulated by TNFα blockade. IL-17A or IL-17F neutralization elicited only mild changes of few genes without impaired host resistance four weeks after M. tuberculosis infection. Further, the absence of both IL-17RA and IL-22 pathways in genetically deficient mice did not profoundly compromise host control of M. tuberculosis over a 6-months period, ruling out potential compensation between these two pathways, while TNFα-deficient mice succumbed rapidly. These data provide experimental confirmation of the low clinical risk of mycobacterial infection under anti-IL-17A therapy, in contrast to anti-TNFα treatment.

Minipigs in Translational Immunosafety Sciences: A Perspective.

The porcine immune system has been studied especially with regard to infectious diseases of the domestic pig, highlighting the economic importance of the pig in agriculture. Recently, in particular, minipigs have received attention as alternative species to dogs or nonhuman primates in drug safety evaluations. The increasing number of new drug targets investigated to modulate immunological pathways has triggered renewed interest to further explore the porcine immune system. Comparative immunological studies of minipigs with other species broaden the translational models investigated in drug safety evaluations. The porcine immune system overall seems functionally similar to other mammalian species, but there are some anatomical, immunophenotypical, and functional differences. Here, we briefly review current knowledge of the innate and adaptive immune system in pigs and minipigs. In conclusion, more systematic and cross-species comparisons are needed to assess the significance of immunological findings in minipigs in the context of translational safety sciences.

Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis.

Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency.