Characterization of transfusion-relevant bacteria reference strains in a lyophilized format.

BACKGROUND AND OBJECTIVES: Blood safety measures used by blood establishments to increase blood component safety can be validated using Transfusion-Relevant Bacterial Reference Strains (TRBRS). Ultra-cold storage conditions and manual preparation of the current TRBRS may restrict their practical use. To address this issue, the ISBT Transfusion-Transmitted Infectious Diseases Working Party's Bacterial Subgroup organized an international study to validate TRBRS in a user-friendly, lyophilised format. MATERIALS AND METHODS: Two bacterial strains Klebsiella pneumoniae PEI-B-P-08 and Staphylococcus aureus PEI-B-P-63 were manufactured as lyophilised material. The lyophilised bacteria were distributed to 11 different labs worldwide to assess the robustness for enumeration, identification and determination of growth kinetics in platelet concentrates (PCs). RESULTS: Production of lyophilised TRBRS had no impact on the growth properties compared with the traditional format. The new format allows a direct low-quantity spiking of approximately 30 bacteria in PCs for transfusion-relevant experiments. In addition, the lyophilised bacteria exhibit long-term stability across a broad temperature range and can even be directly rehydrated in PCs without losing viability. Interlaboratory comparative study demonstrated the robustness of the new format as 100% of spiked PC exhibited growth. CONCLUSION: Lyophilised TRBRS provide a user-friendly material for transfusion-related studies. TRBRS in the new format have improved features that may lead to a more frequent use in the quality control of transfusion-related safety measures in the future.

Expert workshop summary: Advancing toward a standardized murine model to evaluate treatments for antimicrobial resistance lung infections.

The rise in antimicrobial resistance (AMR), and increase in treatment-refractory AMR infections, generates an urgent need to accelerate the discovery and development of novel anti-infectives. Preclinical animal models play a crucial role in assessing the efficacy of novel drugs, informing human dosing regimens and progressing drug candidates into the clinic. The Innovative Medicines Initiative-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium is establishing a validated and globally harmonized preclinical model to increase reproducibility and more reliably translate results from animals to humans. Toward this goal, in April 2021, COMBINE organized the expert workshop "Advancing toward a standardized murine model to evaluate treatments for AMR lung infections". This workshop explored the conduct and interpretation of mouse infection models, with presentations on PK/PD and efficacy studies of small molecule antibiotics, combination treatments (ß-lactam/ß-lactamase inhibitor), bacteriophage therapy, monoclonal antibodies and iron sequestering molecules, with a focus on the major Gram-negative AMR respiratory pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Here we summarize the factors of variability that we identified in murine lung infection models used for antimicrobial efficacy testing, as well as the workshop presentations, panel discussions and the survey results for the harmonization of key experimental parameters. The resulting recommendations for standard design parameters are presented in this document and will provide the basis for the development of a harmonized and bench-marked efficacy studies in preclinical murine pneumonia model.

Variability of murine bacterial pneumonia models used to evaluate antimicrobial agents.

Antimicrobial resistance has become one of the greatest threats to human health, and new antibacterial treatments are urgently needed. As a tool to develop novel therapies, animal models are essential to bridge the gap between preclinical and clinical research. However, despite common usage of in vivo models that mimic clinical infection, translational challenges remain high. Standardization of in vivo models is deemed necessary to improve the robustness and reproducibility of preclinical studies and thus translational research. The European Innovative Medicines Initiative (IMI)-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium, aims to develop a standardized, quality-controlled murine pneumonia model for preclinical efficacy testing of novel anti-infective candidates and to improve tools for the translation of preclinical data to the clinic. In this review of murine pneumonia model data published in the last 10 years, we present our findings of considerable variability in the protocols employed for testing the efficacy of antimicrobial compounds using this in vivo model. Based on specific inclusion criteria, fifty-three studies focusing on antimicrobial assessment against Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were reviewed in detail. The data revealed marked differences in the experimental design of the murine pneumonia models employed in the literature. Notably, several differences were observed in variables that are expected to impact the obtained results, such as the immune status of the animals, the age, infection route and sample processing, highlighting the necessity of a standardized model.

A 5-year look-back at the notification and management of vaccine supply shortages in Germany.

BackgroundUnavailability of vaccines endangers the overall goal to protect individuals and whole populations against infections.MethodsThe German notification system includes the publication of vaccine supply shortages reported by marketing authorisation holders (MAH), information on the availability of alternative vaccine products, guidance for physicians providing vaccinations and an unavailability reporting tool to monitor regional distribution issues.AimThis study provides a retrospective analysis of supply issues and measures in the context of European and global vaccine supply constraints.Resultsbetween October 2015 and December 2020, the 250 notifications concerned all types of vaccines (54 products). Most shortages were caused by increased demand associated with immigration in Germany in 2015 and 2016, new or extended vaccine recommendations, increased awareness, or changes in global immunisation programmes. Shortages of a duration up to 30 days were mitigated using existing storage capacities. Longer shortages, triggered by high demand on a national level, were mitigated using alternative products and re-allocation; in a few cases, vaccines were imported. However, for long lasting supply shortages associated with increased global demand, often occurring in combination with manufacturing issues, few compensatory mechanisms were available. Nevertheless, only few critical incidents were identified: (i) shortage of hexavalent vaccines endangering neonatal immunisation programmes in 2015;(ii) distribution issues with influenza vaccines in 2018; and (iii) unmet demand for pneumococcal and influenza vaccines during the coronavirus disease (COVID)-19 pandemic.ConclusionVaccine product shortages in Germany resemble those present in neighbouring EU states and often reflect increased global demand not matched by manufacturing capacities.

Prevalence and Antibiotic Susceptibility Trends of Selected Enterobacteriaceae, Enterococci, and Candida albicans in the Subgingival Microbiota of German Periodontitis Patients: A Retrospective Surveillance Study.

The periodontal microbiota is ecologically diverse and may facilitate colonization by bacteria of enteric origin (Enterobacteriaceae, Enterococci) and co-infections with Candida albicans, possibly producing subgingival biofilms with high antimicrobial tolerance. This retrospective surveillance study followed periodontitis-associated superinfection profiles in a large patient sample. From 2008 to 2015, biofilm samples from deep periodontal pockets were collected from a total of 16,612 German adults diagnosed with periodontitis. The presence of selected Enterobacteriaceae, Enterococci, and Candida albicans was confirmed in overnight cultures. Antimicrobial susceptibility of these clinical isolates was tested by disk diffusion with antibiotics routinely used for treatment of oral infections, e.g., amoxicillin (AML), amoxicillin/clavulanic acid (AMC), doxycycline (DO), and ciprofloxacin (CIP). The mean annual prevalence of patients harboring Enterobacteriaceae in periodontal plaques was 11.5% in total and ranged from 2.5% for Enterobacter cloacae to 3.6% for Klebsiella oxytoca, 1.1% for Klebsiella pneumoniae, 2.8% for Serratia marcescens, and 1.5% for Serratia liquefaciens. In comparison, the mean detection rates for microbiota typically found in the oral cavity were higher, e.g., 5.6% for Enterococcus spp. and 21.8% for Candida albicans. Among the Enterobacteriaceae, species harboring intrinsic resistance to AML (Enterobacter spp., Klebsiella spp., Serratia spp.) were predominant. Non-susceptibility to AMC was observed for Serratia spp. and Enterobacter cloacae. By contrast, Enterococcus spp. only showed non-susceptibility to DO and CIP. Trends for increasing resistance were found to AML in Serratia liquefaciens and to DO in Enterococcus spp. Trend analysis showed decreasing resistance to AMC in Serratia liquefaciens and Klebsiella oxytoca; and to DO in Serratia marcescens, liquefaciens, and Enterobacter cloacae. This study confirms the low but consistent presence of Enterobacteriaceae and Enterococci among the subgingival microbiota recovered from periodontitis specimen. Although their pathogenetic role in periodontal lesions remains unclear, their presence in the oral cavity should be recognized as a potential reservoir for development and spread of antibiotic resistance in light of antibiotic usage in oral infections.

Controlled Human Infection Studies: Proposals for guidance on how to design, develop and produce a challenge strain.

There is an increasing need to establish quality principles for designing, developing and manufacturing challenge agents as currently these agents are classified differently by various jurisdictions. Indeed, considerations for challenge agent manufacturing vary between countries due to differences in regulatory oversight, the categorization of the challenge agent and incorporation into medicinal/vaccine development processes. To this end, a whitepaper on the guidance has been produced and disseminated for consultation to researchers, regulatory experts and regulatory or advisory bodies. This document is intended to discuss fundamental principles of selection, characterization, manufacture, quality control and storage of challenge agents for international reference. In the development phase, CMC documentation is needed for a candidate challenge agent, while standard operating procedure documentation is needed to monitor and control the manufacturing process, followed by use of qualified methods to test critical steps in the manufacturing process, or the final product itself. These activities are complementary: GMP rules, which intervene only at the time of the routine manufacturing of batches, do not contribute to the proper development and qualification of the candidate product. Some considerations regarding suitability of premises for challenge manufacturing was discussed in the presentation dedicated to "routine manufacturing".

A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen.

Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines.

Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations.

Cryopreservation of human peripheral blood mononuclear cells (PBMC) is used in many clinical and research applications to avoid direct and on-site analysis of samples. Storage of PBMC further allows prequalification of donor cells for routine laboratory methods involving the evaluation of immune responses. Previous studies reported changes in cellular composition and phenotype of PBMC following the freezing procedure. In our 12-month follow-up study, we focused on B cells and proportional representation of B cell subpopulations during long-term storage at -80 °C. Over the 12-month period, we observed a gradual decline in B cell viability and recovery. Notably, no changes in the proportional representation of human B cell subpopulations occurred in this period and the functional response elicited by antigen and TLR9 ligand CpG remained comparable to that observed after short-term storage for one month.

Prevalence and antibiotic susceptibility trends of periodontal pathogens in the subgingival microbiota of German periodontitis patients: A retrospective surveillance study.

OBJECTIVE: This retrospective surveillance study aimed to follow periodontitis-associated bacterial profiles and to identify time-dependent changes in antibiotic susceptibility patterns. MATERIALS AND METHODS: From 2008 to 2015, bacterial specimen from deep periodontal pockets were collected from a total of 7804 German adults diagnosed with periodontitis. Presence of selected bacteria was confirmed by anaerobic culture and nucleic acid amplification. Antimicrobial susceptibility of clinical isolates was tested by disc diffusion with antibiotics used for the treatment of periodontitis and oral infections. The prevalences of periodontal pathogens were calculated and temporal evolution of antimicrobial susceptibility towards amoxicillin, amoxicillin/clavulanic acid, metronidazole, doxycycline, clindamycin, azithromycin, ciprofloxacin and ampicillin was analysed with logistic regression. RESULTS: The prevalence of patients harbouring bacteria was 95.9% Fusobacterium nucleatum, 88.0% Tannerella forsythia, 76.4% Treponema denticola, 76.5%, Campylobacter rectus, 76.0% Eikenella corrodens, 75.0% Capnocytophaga spp., 68.2% Porphyromonas gingivalis, 57.7% Peptostreptococcus micros, 43.1% Prevotella intermedia, 30.4% Eubacterium nodatum and 21.5% Aggregatibacter actinomycetemcomitans. In 63.5% of patients, one or more isolates were not susceptible to at least one of the antibiotics tested. The data further revealed a trend towards decreasing susceptibility profiles (p < 0.05) with antibiotic non-susceptibilities in 37% of patients in 2008 and in 70% in 2015. CONCLUSIONS: The present study confirmed a high prevalence of periodontal pathogens in the subgingival microbiota of German periodontitis patients. The data revealed an incremental increase in isolates displaying resistance to some antibiotics but no relevant change in susceptibility to amoxicillin and metronidazole.

Human plasmacytoid dendritic cells at the crossroad of type I interferon-regulated B cell differentiation and antiviral response to tick-borne encephalitis virus.

The Tick-borne encephalitis virus (TBEV) causes different disease symptoms varying from asymptomatic infection to severe encephalitis and meningitis suggesting a crucial role of the human host immune system in determining the fate of the infection. There is a need to understand the mechanisms underpinning TBEV-host interactions leading to protective immunity. To this aim, we studied the response of human peripheral blood mononuclear cells (PBMC) to the whole formaldehyde inactivated TBEV (I-TBEV), the drug substance of Encepur, one of the five commercially available vaccine. Immunophenotyping, transcriptome and cytokine profiling of PBMC revealed that I-TBEV generates differentiation of a sub-population of plasmacytoid dendritic cells (pDC) that is specialized in type I interferon (IFN) production. In contrast, likely due to the presence of aluminum hydroxide, Encepur vaccine was a poor pDC stimulus. We demonstrated I-TBEV-induced type I IFN together with Interleukin 6 and BAFF to be critical for B cell differentiation to plasmablasts as measured by immunophenotyping and immunoglobulin production. Robust type I IFN secretion was induced by pDC with the concerted action of both viral E glycoprotein and RNA mirroring previous data on dual stimulation of pDC by both S. aureus and influenza virus protein and nucleic acid that leads to a type I IFN-mediated sustained immune response. E glycoprotein neutralization or high temperature denaturation and inhibition of Toll-like receptor 7 signalling confirmed the importance of preserving the functional integrity of these key viral molecules during the inactivation procedure and manufacturing process to produce a vaccine able to stimulate strong immune responses.

How to draw the line - Raman spectroscopy as a tool for the assessment of biomedicines.

Biomedicines are complex biochemical formulations with multiple components that require extensive quality control during manufacturing and in subsequent batch testing. A proof-of-concept study has shown that an application of Raman spectroscopy can be beneficial for a classification of vaccines. However, the complexity of biomedicines introduces new challenges to spectroscopic methodology that require advanced experimental protocols. We further show the impact of analytical protocols on vaccine classification using R as an Open Source data analysis platform. In conclusion, we advocate for standardized and transparent experimental and analytical procedures and discuss current findings and open challenges.

Systematic memory B cell archiving and random display shape the human splenic marginal zone throughout life.

Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging.

TNFR2 expression is a hallmark of human memory B cells with suppressive function.

Tumor Necrosis Factor Receptor 2 (TNFR2) expression is increasingly being linked to tolerogenic immune reactions and cells with suppressor function including a subset of T-regulatory cells. B-regulatory cells play an important role in control of T-cell responses and inflammation. Recently, we described TNFR2 as a marker for IL-10-producing B cells, a hallmark of this cell subset. Here, we demonstrate that proliferation of T cells is reduced in the presence of TNFR2 positive human memory B cells generated with TLR9 ligand, while TNFR2- and TNFR2+CD27- B cells display costimulatory activity. Our data further reveal that IL-10 secretion is characteristic of IgM+ naïve and memory B cells but suppressive activity is not restricted to IL-10: (i) the inhibitory effect of TNFR2+ switched memory B cells was comparable to that exerted by TNFR2+ IgM+ memory B cells although IL-10 secretion levels in the cocultures were lower; (ii) supernatants from TNFR2+ memory B cells failed to suppress T-cell proliferation. Based on our findings, we propose that formation of Breg is a specific characteristic of human memory B cells undergoing terminal differentiation. Our data further corroborate that TNFR2 represents a viable marker for identification of memory B cells with regulatory function.

Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Staphylococcus aureus Protein A Induces Human Regulatory T Cells Through Interaction With Antigen-Presenting Cells.

Despite continuous exposure and development of specific immunity, Staphylococcus aureus (Sa) remains one of the leading causes of severe infections worldwide. Although innate immune defense mechanisms are well understood, the role of the T cell response has not been fully elucidated. Here, we demonstrate that Sa and one of its major virulence factors protein A (SpA) induce human regulatory T cells (Tregs), key players in immune tolerance. In human PBMC and MoDC/T cell cocultures CD4+CD25+CD127dim Tregs were induced upon stimulation with Sa and to a lower extent with SpA alone. Treg induction was strongly, but not exclusively, dependent on SpA, and independent of antigen presentation or T cell epitope recognition. Lastly, soluble factors in the supernatant of SpA-stimulated MoDC were sufficient to trigger Treg formation, while supernatants of MoDC/T cell cocultures containing Sa-triggered Tregs displayed T cell suppressive activity. In summary, our findings identify a new immunosuppressory function of SpA, which leads to release of soluble, Treg-inducing factors and might be relevant to establish colonization.

Challenges for Clinical Development of Vaccines for Prevention of Hospital-Acquired Bacterial Infections.

Increasing antibiotic resistance in bacteria causing endogenous infections has entailed a need for innovative approaches to therapy and prophylaxis of these infections and raised a new interest in vaccines for prevention of colonization and infection by typically antibiotic resistant pathogens. Nevertheless, there has been a long history of failures in late stage clinical development of this type of vaccines, which remains not fully understood. This article provides an overview on present and past vaccine developments targeting nosocomial bacterial pathogens; it further highlights the specific challenges associated with demonstrating clinical efficacy of these vaccines and the facts to be considered in future study designs. Notably, these vaccines are mainly applied to subjects with preexistent immunity to the target pathogen, transient or chronic immunosuppression and ill-defined microbiome status. Unpredictable attack rates and changing epidemiology as well as highly variable genetic and immunological strain characteristics complicate the development. In views of the clinical need, re-thinking of the study designs and expectations seems warranted: first of all, vaccine development needs to be footed on a clear rationale for choosing the immunological mechanism of action and the optimal time point for vaccination, e.g., (1) prevention (or reduction) of colonization vs. prevention of infection and (2) boosting of a preexistent immune response vs. altering the quality of the immune response. Furthermore, there are different, probably redundant, immunological and microbiological defense mechanisms that provide protection from infection. Their interplay is not well-understood but as a consequence their effect might superimpose vaccine-mediated resolution of infection and lead to failure to demonstrate efficacy. This implies that improved characterization of patient subpopulations within the trial population should be obtained by pro- and retrospective analyses of trial data on subject level. Statistical and systems biology approaches could help to define immune and microbiological biomarkers that discern populations that benefit from vaccination from those where vaccines might not be effective.

Kinetic Fingerprinting Links Bacteria-Phage Interactions with Emergent Dynamics: Rapid Depletion of Klebsiella pneumoniae Indicates Phage Synergy.

The specific temporal evolution of bacterial and phage population sizes, in particular bacterial depletion and the emergence of a resistant bacterial population, can be seen as a kinetic fingerprint that depends on the manifold interactions of the specific phage-host pair during the course of infection. We have elaborated such a kinetic fingerprint for a human urinary tract Klebsiella pneumoniae isolate and its phage vB_KpnP_Lessing by a modeling approach based on data from in vitro co-culture. We found a faster depletion of the initially sensitive bacterial population than expected from simple mass action kinetics. A possible explanation for the rapid decline of the bacterial population is a synergistic interaction of phages which can be a favorable feature for phage therapies. In addition to this interaction characteristic, analysis of the kinetic fingerprint of this bacteria and phage combination revealed several relevant aspects of their population dynamics: A reduction of the bacterial concentration can be achieved only at high multiplicity of infection whereas bacterial extinction is hardly accomplished. Furthermore the binding affinity of the phage to bacteria is identified as one of the most crucial parameters for the reduction of the bacterial population size. Thus, kinetic fingerprinting can be used to infer phage-host interactions and to explore emergent dynamics which facilitates a rational design of phage therapies.

Human challenge trial workshop: Focus on quality requirements for challenge agents, Langen, Germany, October 22, 2019.

Controlled human infection models can be helpful to study pathogenesis and immune responses as a basis for the development of vaccines. In controlled human infection models, human challenge agents are used to infect healthy volunteers, therefore, ethical considerations include that the exposure studies need to be safe and results should be meaningful, e.g. contribute to a better cure. Both in the US and in Europe, the level of Good Manufacturing Practice required is related to the phase of the study ('sliding scale Good Manufacturing Practice'), and, hence, is much more open to speedy drug development than anticipated. Recommendations included: the development of guidelines for human challenge agents; a focus on strain selection, in particular with regard to strain infectivity, stability and purity; the use of whole genome sequencing; a reference repository of challenge agents, the need for early exchange with regulators to ensure acceptability of strain selection and manufacturing for later drug development; sharing of models and challenge agents.