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The gamma-interferon inducible PA28 protein complex, or 11S regulator, which is composed of two homologous subunit forming a hexa- or heptameric ring structure, is known to associate with the 20S proteasome particle. The kinetics of product generation by 20S proteasomes with and without PA28 is studied. Results indicate that the PA28 regulator significantly alters the cleavage mechanism of the proteasome and optimizes the generation of dominant T cell epitopes.

Highlights: [•] The antigenic repertoire can be analyzed at DNA, RNA and protein levels. [•] Three analytical steps required: identification, specificity, validation. [•] Suitable antigens can be overexpressed (TAA) or tumor-specific (TSA). [•] Mutated antigens represent the best vaccination targets, but are most challenging. [•] Integrated and individualized strategy for vaccine composition in patients holds promise. [ABSTRACT FROM AUTHOR]

• Single-dose CoVac-1 induces long-lasting T cell responses in 100% of participants. • Long-term follow-up shows good tolerability and no immune-related side effects. • Magnitude of T cell responses correlates with severity of injection site reactions. • Most CoVac-1-induced peptide-specific immunoglobulin G antibodies peaked at month 3. • Very low SARS-CoV-2 infection rate (8.3 %) in the study population. T cell immunity is key for the control of viral infections including SARS-CoV-2, in particular with regard to immune memory and protection against arising genetic variants. We recently evaluated a peptide-based SARS-CoV-2 T cell activator termed CoVac-1 in a first-in-human trial in healthy adults. Here, we report on long-term safety and efficacy data of CoVac-1 until month 12. CoVac-1 is well tolerated without long-term immune-related side effects and induces long-lasting anti-viral T cell responses in 100% of study participants, with potent expandability of clusters of differentiation (CD4+) and CD8+ T cells targeting multiple different CoVac-1 T cell epitopes. T cell responses were associated with stronger injection site reaction. Beyond induction of T cell immunity, 89% of subjects developed CoVac-1-specific immunoglobulin G antibodies which associated with the intensity of the T cell response, indicating that CoVac-1-specific CD4+ T cells support the induction of B-cell responses. Vaccination with approved COVID-19 vaccines boosted CoVac-1-specific T cell responses. Overall, a low SARS-CoV-2 infection rate (8.3%) was observed. Together, a single application of CoVac-1 elicits long-lived and broad SARS-CoV-2-specific T cell immunity, which further supports the current evaluation of our T cell activator in patients with congenital or acquired B-cell defects. [ABSTRACT FROM AUTHOR]

Abstract: Tumor cells (over-) express specific antigens which allow them to be recognized and destroyed by the immune system. Triggering anti-tumor immunity in cancer patients by specific vaccination is foreseen as a safe and versatile method to control cancer. As a source of antigen, whole tumor cells, nucleic acids, proteins or derived peptides have been used. This review focuses on the utilization of vaccines based on plasmid DNA (pDNA) and messenger RNA (mRNA) coding for tumor associated antigens. Both vectors (pDNA and mRNA) are grouped under the designation “minimal nucleic acid vector” or MNAV. The current knowledge on anti-tumor vaccination based on MNAV-encoded tumor antigens, methods of delivery, principles of production and optimization is discussed. Furthermore, an up-to-date summary of published clinical trials using MNAV for the vaccination against solid tumors is given. Recent preclinical and early phase clinical trials demonstrate promising synergies between vaccination and other treatments such as chemotherapy or non-specific immune enhancement regimens. Combining optimized MNAV formulations and parallel adjuvant treatments could allow to turn MNAV-based vaccines into efficient anti-tumor immunotherapies in humans. [Copyright &y& Elsevier]

The F pocket region of MHC class I molecules is highly flexible in the absence of peptides, leading to instability and denaturation. We present here the stabilization of class I molecules by a disulfide bond between residules 84 and 139. Disulfide-stabilized class I molecules can be folded in vitro with the help of small molecules with very low affinity such as dipeptides. Once the small molecules are removed, folded disulfide-stabilized class I molecules bind exogenous peptide rapidly and stoichiometrically. This allows for the production of peptide-free MHC multimers that may be converted into the desired peptide complex within minutes. Crystal structures, binding affinity measurements, and staining data show equivalence of TCR binding to wild type class I. The stabilization is principally accessible for all class I allotypes. [ABSTRACT FROM AUTHOR]

Background Heat shock proteins (HSP) are highly conserved immunogenic proteins serving as potent danger signals. They are upregulated under stress conditions like fever and hypoxia. Extracellular HSP are involved in antigen presentation, cytokine release and maturation of antigen presenting cells. Methods The release of the inducible members of the HSP70 family, Hsp72 and Hsp70B′, into the serum of 20 patients undergoing allogeneic hematopoietic cell transplantation and 20 healthy donors was evaluated using enzyme linked immunosorbent assay (ELISA) kits. Results Eight patients (40%) did not receive anti-thymocyte globulin (ATG) for prophylaxis of graft versus host disease (GvHD). These patients had no detectable or low serum levels of Hsp72 (n = 3, 0.03 to 1.92 ng/ml) which were in line with levels detected in 20 healthy individuals (p = 0.07). Measurable HSP was not associated with any medication or transplantation-related procedures. In twelve patients (60%) receiving ATG, detected high levels of HSP reflected cross-reactivity of the rabbit-derived ATG with the anti-rabbit antibody used in the ELISA. Conclusions Assumed HSP70 expression detected such ELISA has to be regarded carefully after ATG application. Neither radiochemotherapy, nor inflammation or sepsis during aplasia induced HSP70 release into the serum. Thus, soluble HSP70 may not be involved in the pathogenesis of acute GvHD. [ABSTRACT FROM AUTHOR]

FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies, contains the FLT3-antibody 4G8 and the CD3-antibody UCHT1. The 4G8 X UCHT1 Fabsc-antibody was found to be superior to a bssc-antibody with identical specificities with respect to (i) affinity to the target antigen FLT3, (ii) production yield by transfected cells, and (iii) the diminished formation of aggregates. T-cell activation in the presence and absence of cultured leukemic cells and killing of these cells was comparable for both molecules. In addition, the 4G8 X UCHT1 Fabsc-antibody was found to induce T-cell activation and efficient killing of leukemic blasts in primary peripheral blood mononuclear cell (PBMC) cultures of acute myeloid leukemia (AML) patients. In these experiments, the bispecific molecule was clearly superior to an Fc-optimized monospecific FLT3-antibody described previously, indicating that within PBMC of AML patients the recruitment of T cells is more effective than that of natural killer cells. [ABSTRACT FROM AUTHOR]

Abstract: The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity. [Copyright &y& Elsevier]

Abstract: Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic–polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC. [Copyright &y& Elsevier]

Abstract: Allogenic stem cell transplantation has shown considerable success in a number of hematological malignancies, in particular in leukemia. The beneficial effect is mediated by donor T cells recognizing patient-specific HLA-binding peptides. These peptides are called minor histocompatibility antigens (miHAs) and are typically caused by single nucleotide polymorphisms. Tissue-specific miHAs have successfully been used in anti-tumor therapy without causing unspecific graft-versus-host reactions. However, only a small number of miHAs have been identified to date, limiting the clinical use. Here we present an immunoinformatics pipeline for the identification of miHAs. The pipeline can be applied to large-scale miHA screening, for example, in the development of diagnostic tests. Another interesting application is the design of personalized miHA-based cancer therapies based on patient–donor pair-specific miHAs detected by this pipeline. The suggested method covers various aspects of genetic variant detection, effects of alternative transcripts, and HLA-peptide binding. A comparison of our computational pipeline and experimentally derived datasets shows excellent agreement and coverage of the computationally predicted miHAs. [Copyright &y& Elsevier]

The aim of this phase I/II nonrandomized trial was to assess feasibility, safety as well as immunological and clinical responses of a mRNA-based vaccination in patients with stage IV renal cell cancer using granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvant. Intradermal injections of in vitro transcribed naked mRNA, which was generated using plasmids coding for the tumor-associated antigens mucin 1(MUC1), carcinoembryonic (CEA), human epidermal growth factor receptor 2 (Her-2/neu), telomerase, survivin, and melanoma-associated antigen 1 (MAGE-A1) were performed in 30 enrolled patients. In the first 14 patients (cohort A) vaccinations were administered on days 0, 14, 28, and 42 (20 µg/antigen) while in the consecutive 16 patients (cohort B) an intensified protocol consisting of injections at days 0-3, 7-10, 28, and 42 (50 µg/antigen) was used. In both cohorts, after this induction period, vaccinations were repeated monthly until tumor progression analyzed by Response Evaluation Criteria In Solid Tumors criteria (RECIST). Vaccinations were well tolerated with no severe side effects and induced clinical responses [six stable diseases (SD) and one partial response in cohort A and nine SD in cohort B]. In cohort A, 35.7% survived 4 years (median survival 24 months) compared to 31.25% in cohort B (median survival 29 months). Induction of CD4+ and CD8+ T cell responses was shown for several tumor-associated antigens (TAA) using interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and Cr-release assays. [ABSTRACT FROM AUTHOR]

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (≥50 μg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses. [ABSTRACT FROM AUTHOR]

: Objectives and methodsIn an attempt to define HCMV IE1-derived, HLA-A&ast;0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed.: ResultsThis prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8+ T cells were detectable in 18/24 HCMV seropositive HLA-A&ast;0201-expressing individuals (median frequency 0.58%; range 0.1%–4.7%), and IE1 VLEETSVML-specific CD8+ T cells in 5/24 (median frequency 2.1%; range 0.1%–4.3%), respectively (p<0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8+ T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8+ T cells was higher (p = 0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8+ T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%–98.6% of all CD3+/CD8+ T cells) were successfully generated after 2–4 weeks of culture using the IFN-γ secretion assay.: ConclusionIn conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope. [Copyright &y& Elsevier]

Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. It has been suggested that vitiligo is caused by an autoimmune-mediated destruction of melanocytes. Recently, the presence of a high frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in the peripheral blood of patients with vitiligo was reported. Our study examines the frequency of melanocyte-specific cytotoxic T lymphocytes in vitiligo patients and its relationship to disease activity. Thirty-two patients with moderate to active vitiligo and 17 control subjects were included. Melanocyte specific reactive CD8+ T cells were identified by enzyme-linked immunospot assay after stimulation with five peptides from gp100, four peptides from MelanA/MART1, and two peptides from tyrosinase. In selected patients, intracellular interferon-γ staining for the detection of specific reactive CD8+ T cells was additionally performed. In seven of 10 patients (70%) with actively progressive disease CD8+ T cells directed against melanocyte epitopes were detected, whereas only in four of 22 patients (18%) with moderate disease activity such specific reactivity was found. MelanA/MART1 peptides were immunodominant in nine patients reacting against EAAGIGILTV and three patients reacting against ILTVILGVL. Intracellular interferon-γ staining confirmed the findings obtained by the enzyme-linked immunospot technique. The present study supports the hypothesis that vitiligo is a cytotoxic T lymphocyte-mediated autoimmune disease. The presence of melanocyte-specific reactive CD8+ T cells seems to be closely related to disease activity. [ABSTRACT FROM AUTHOR]