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 advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments

Assessing immunocompetence in minipigs in translational safety 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 non-human 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 and immunophenotypical differences. Here, we briefly review current knowledge of the innate and adaptive immune system in (mini)pigs. 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

Regulation of Allergic Responses to Chemicals: Possible Roles of Epigenetic Mechanisms

There is increasing evidence that epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune and allergic responses. Such regulatory mechanisms have potentially important implications for the acquisition of sensitization to chemical and drug allergens, and in determining the vigor, characteristics and longevity of allergic responses. Importantly, the discovery of long-lasting epigenetic alterations in specific immunoregulatory genes provides a mechanistic basis for immune cell memory, and thereby the potential of chemical allergens to influence the subsequent orientation of the adaptive immune system. In this article we consider the implications of epigenetic mechanisms for the development of sensitization to chemical allergens and the form that allergic reactions will take

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

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

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

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

Dysregulation of complement activation causes a number of diseases, which can be treated with inhibitors of the complement components C5 and 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 central complement component C3, the alternative pathway (fB and fD), the lectin pathway (MASP-2) and the terminal pathway (C5) on SBA against serogroup B meningococci. 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. While only a few pre-vaccination sera showed SBA against the B meningococcal isolates tested, 4CMenB vaccination induced potent complement activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed lipoprotein Factor H binding protein (fHbp). While SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on SBA of post-4CMenB vaccination sera. Compared to SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele, was thus remarkably resilient against alternative pathway inhibition

Investigative safety science as a competitive advantage for Pharma

Introduction: Following a US National Academy of Sciences report in 2007 entitled “Toxicity Testing of the 21st Century: a Vision and a Strategy”, significant advances within translational drug safety sciences promise to revolutionize drug discovery and development. Here we outline why investigative safety science is a competitive advantage for Pharma. Areas covered: Essential goals for modern investigative toxicologists are discussed including the cross-species target biology, molecular pathways of toxicity, and development of predictive tools, models and biomarkers that allow discovery researchers and clinicians to anticipate safety problems and plan ways to address them, earlier than ever before. Furthermore, we emphasize the importance of investigating unanticipated clinical safety signals through a combination of mechanistic pre-clinical studies and/or molecular characterization of clinical samples from affected organs. Expert Opinion: The traditional boundaries between Pharma industry teams focusing on safety/efficacy and preclinical/clinical development are rapidly disappearing in favor of translational safety science-centric organizations with a vision of bringing more effective medicines forward safely and quickly. Comparative biology and mechanistic toxicology approaches facilitate: 1) identifying translational safety biomarkers; 2) identifying new drug targets/indications; 3) mitigating off-target toxicities. These value-adding safety science contributions will change traditional toxicologists from side-effect identifiers to drug development enablers

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 (ADA) are a key concern for their efficacy, pharmacokinetics and safety. Prediction of clinical immunogenicity on the basis of quality attributes of biopharmaceuticals or by utilizing preclinical in vitro and in vivo screening remains challenging. Even fully human biotherapeutics have the potential for immunogenicity, and one important factor that might enhance potential immunogenicity is protein aggregation. 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 (i) heat-induced rhEPO aggregates, (ii) tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA, and (iii) ex vivo T cell recall responses of patients treated with rhEPO without PRCA, and of a patient who developed PRCA after treatment with a clinical batch with elevated levels of tungsten and aggregates. To our knowledge, this is the first time that T cell assays confirm the root cause of increased rhEPO immunogenicity associated with PRCA

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