Implementation of a novel projector-splitting integrator for the multi-configurational time-dependent Hartree approach.

The variational equations of motion of the multi-configuration time-dependent Hartree (MCTDH) approach contain the inverse of reduced density matrices which are typically ill-conditioned and therefore lead to small stepsizes for numerical time integration. This problem is usually dealt with via regularization of the density matrices, which works well in many cases but still calls for systematic improvement schemes. Recently this problem, its implications and possible solutions have become the subject of increased interest. Notably, a projector splitting integrator for the MCTDH approach that does not require the inversion of reduced density matrices has been proposed [C. Lubich, Appl. Math. Res. Express 2015, 311]. Here, we present the first implementation of this integration scheme. Results for low-dimensional benchmark systems are presented, and the case of initially unoccupied single-particle functions is discussed.

The Mystery of Antibodies Against Polyethylene Glycol (PEG) - What do we Know?

PURPOSE: Recent findings demonstrated anti-PEG antibody formation in some healthy individuals and patients who have not received PEGylated biotherapeutics. Some of these findings evoked criticism because of shortcomings in the antibody assays used. To better understand this topic, we established robust antibody analytics and screened two cohorts of healthy individuals and one cohort of hemophilia patients for the expression of anti-PEG antibodies. METHODS: A flow cytometry approach and a fully validated ELISA platform were established to detect specific anti-PEG antibodies. Immunohistochemistry was used to test for potential binding of anti-PEG antibodies to human tissues. RESULTS: IgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia who have not received PEGylated biotherapeutics. These antibodies can be either transient or persistent and recognize PEGs of different sizes with or without terminal methoxy groups. Age and location of healthy individuals influence the prevalence of IgG but not of IgM antibodies. Anti-PEG antibodies do not cross-react with human tissues supporting the safety of the antibodies. CONCLUSION: We confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.

A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions.

PURPOSE: Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions. METHODS: We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 µM. We applied a combination of Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles. RESULTS: The FACS technology allows the analysis of particles between 0.75 and 10 µm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-ß-sheet structures contained in protein particles can be identified. CONCLUSIONS: The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 µm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.

CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice.

Today it is generally accepted that B cells require cognate interactions with CD4(+) T cells to develop high-affinity antibodies against proteins. CD4(+) T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4(+) T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4(+) T-cell epitopes presented by HLA-DRB1*1501 to CD4(+) T cells during immune responses against FVIII. CD4(+) T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells.

Background and aims: Hemophilia A is a severe bleeding disorder caused by the deficiency of functionally active coagulation factor VIII (FVIII). The induction of neutralizing anti-drug antibodies is a major complication in the treatment of hemophilia A patients with FVIII replacement therapies. Why some patients develop neutralizing antibodies (FVIII inhibitors) while others do not is not well understood. Previous studies indicated that the induction of FVIII inhibitors requires cognate interactions between FVIII-specific B cells and FVIII-specific CD4+ T cells in germinal center reactions. In this study, we investigated the FVIII peptide repertoire presented by antigen-presenting cells (APCs) under different microenvironment conditions that are expected to alter the uptake of FVIII by APCs. The aim of this study was to better understand the association between different microenvironment conditions during FVIII uptake and the FVIII peptide patterns presented by APCs. Methods: We used a FVIII-specific CD4+ T cell hybridoma library derived from humanized HLA-DRB1*1501 (human MHC class II) hemophilic mice that were treated with human FVIII. APCs obtained from the same mouse strain were preincubated with FVIII under different conditions which are expected to alter the uptake of FVIII by APCs. Subsequently, these preincubated APCs were used to stimulate the FVIII-specific CD4+ T cell hybridoma library. Stimulation of peptide-specific CD4+ T-cell hybridoma clones was assessed by analyzing the IL-2 release into cell culture supernatants. Results: The results of this study indicate that the specific microenvironment conditions during FVIII uptake by APCs determine the peptide specificities of subsequently activated FVIII-specific CD4+ T cell hybridoma clones. Incubation of APCs with FVIII complexed with von Willebrand Factor, FVIII activated by thrombin or FVIII combined with a blockade of receptors on APCs previously associated with FVIII uptake and clearance, resulted in distinct peptide repertoires of subsequently activated hybridoma clones. Conclusion: Based on our data we conclude that the specific microenvironment during FVIII uptake by APCs determines the FVIII peptide repertoire presented on MHC class II expressed by APCs and the peptide specificity of subsequently activated FVIII-specific CD4+ T cell hybridoma clones.

Modulation of the liver immune microenvironment by the adeno-associated virus serotype 8 gene therapy vector.

Adeno-associated viruses (AAVs) are emerging as one of the vehicles of choice for gene therapy. However, the potential immunogenicity of these vectors is a major limitation of their use, leading to the necessity of a better understanding of how viral vectors engage the innate immune system. In this study, we demonstrate the immune response mediated by an AAV vector in a mouse model. Mice were infected intravenously with 4 × 1012 copies (cp)/kg of AAV8, and the ensuing immune response was analyzed using intravital microscopy during a period of weeks. Administration of AAV8 resulted in the infection of hepatocytes, and this infection led to a moderate, but significant, activation of the immune system in the liver. This host immune response involved platelet aggregation, neutrophil extracellular trap (NET) formation, and the recruitment of monocytes, B cells, and T cells. The resident liver macrophage population, Kupffer cells, was necessary to initiate this immune response, as its depletion abrogated platelet aggregation and NET formation and delayed the recruitment of immune cells. Moreover, the death of liver cells produced by this AAV was moderate and failed to result in a robust, sustained inflammatory response. Altogether, these data suggest that AAV8 is a suitable vector for gene therapy approaches.