Sebastian Bunk

Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Internalization and Coreceptor Expression Are Critical for TLR2-Mediated Recognition of Lipoteichoic Acid in Human Peripheral Blood

Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Δlgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Δlgt-LTA by human peripheral blood cells. In this study, we demonstrate that Δlgt-LTA–induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Δlgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA–mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Δlgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Δlgt-LTA–mediated immune activation and appear to induce the phagocytic uptake of Δlgt-LTA via engagement of FcγRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.

Apolipoprotein B100 is a suppressor of Staphylococcus aureus-induced innate immune responses in humans and mice.

Plasma lipoproteins such as LDL (low‐density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram‐positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size‐exclusion chromatography and solid‐phase‐binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA‐induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100‐directed immuno‐apheresis confirmed that ApoB100 inhibited LTA‐induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.

Antigens of persistent Chlamydia pneumoniae within coronary atheroma from patients undergoing heart transplantation

Aims In order for Chlamydia pneumoniae to play a causative role in chronic human disease, it would need to persist within infected tissue for extended periods of time. Current theory suggests that C pneumoniae may persist at the site of infection via an alternative replicative form, known as an aberrant body. Methods A panel of C pneumoniae-specific antibodies upregulated by the aberrant body was used to probe tissue specimens from the coronary atheroma from 13 explanted hearts to identify patterns of reactivity in these tissues, as well as to determine the presence and prevalence of C pneumoniae aberrant bodies. Results Six of 13 patients had an ischaemic cardiomyopathy secondary to coronary atherosclerosis, while another six patients had an idiopathic, dilated cardiomyopathy. One additional patient, a young (24 years) woman with cardiomyopathy, had no history of atherosclerotic disease. Eleven patients were positive by immunohistochemistry with at least one antibody. Coronary arteries of the two other patients were negative by immunohistochemistry with all antibodies. One of these patients was the 24-year-old woman with grade I disease and no risk factors for coronary artery disease. Conclusions The protein antigens of persistent C pneumoniae infection found in the atheromatous lesions from patients in this study could potentially be used as markers to detect such infections and some may be virulence factors or immunogens specific to C pneumoniae, thus serving as target molecules for diagnostic use or therapeutic intervention.

Chlamydia pneumoniae-Induced Memory CD4 T-Cell Activation in Human Peripheral Blood Correlates with Distinct Antibody Response Patterns

ABSTRACT Chlamydia pneumoniae is a frequent pathogen of the respiratory tract, and persistent infections with this obligate intracellular bacterium have been associated with different severe sequelae. Although T-cell activation during acute C. pneumoniae infections has been described, little is known about the frequency or the role of the C. pneumoniae-specific memory T cells that reside in the human body after the resolution of the infection. In the present study, the C. pneumoniae-induced T-cell responses in peripheral blood mononuclear cells of 56 healthy volunteers were analyzed and compared to the donors serum antibody reactivity toward whole C. pneumoniae as well as recombinant C. pneumoniae antigens. Following short-term stimulation with C. pneumoniae, both gamma interferon (IFN-γ)- and interleukin-2 (IL-2)-producing CD4+ T-cell responses could be detected in 16 of 56 healthy individuals. C. pneumoniae-activated CD4+ T cells expressed CD154, a marker for T-cell receptor-dependent activation, and displayed a phenotype of central memory T cells showing dominant IL-2 production but also IFN-γ production. Interestingly, individuals with both IFN-γ- and IL-2-producing responses showed significantly decreased immunoglobulin G reactivity toward C. pneumoniae RpoA and DnaK, antigens known to be strongly upregulated during chlamydial persistence, compared to IgG reactivity of seropositive individuals with no T-cell response or CD4+ T-cell responses involving the production of a single cytokine (IFN-γ or IL-2). Our results demonstrate that memory CD4+ T cells responding to C. pneumoniae stimulation can be detected in the circulation of healthy donors. Furthermore, among seropositive individuals, the presence or the absence of dual IFN-γ- and IL-2-producing T-cell responses was associated with distinct patterns of antibody responses toward persistence-associated C. pneumoniae antigens.

IL-10 release requires stronger toll-like receptor 4-triggering than TNF: A possible explanation for the selective effects of heterozygous TLR4 polymorphism Asp(299)Gly on IL-10 release

The toll-like receptor 4 Asp(299)Gly polymorphism results in an inactive receptor. Heterozygosis is associated with reduced LPS-inducible IL-10 protein and IL-10 mRNA from blood leukocytes and isolated monocytes, while numerous other mediators are not affected. We could exclude that this effect is due to the differences in the kinetics of IL-10 release, in the expression of total surface TLR4 or in LPS-binding to monocytes between subjects heterozygous for the Asp(299)Gly polymorphism or homozygous carriers of the wild-type allele. Furthermore, we could show that IL-10 induction in general requires stronger LPS-triggering than TNF and is more sensitive to LPS inhibitors. The lower number of responsive wild-type TLR4 receptors on monocytes of heterozygotes may explain why only IL-10 release is affected.

Identification of Borrelia Burgdorferi Ribosomal Protein L25 by the Phage Surface Display Method and Evaluation of the Protein's Value for Serodiagnosis

ABSTRACT The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.

Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

Abstract 3753: Identification of antibodies against a novel tumor-associated MHC/peptide-target and generation of highly specific and potent HLA-A*02MMP1-003/CD3 TandAbs

Tumor-associated antigens for effective and safe T-cell engagement are very limited, leaving a need to open up the therapeutic target space. Targeting disease-specific MHC/peptide complexes with bispecific T-cell-recruiting antibodies is a highly attractive strategy to address this need, but so far, generation of antibodies against these peptides has been reported to be challenging. Immatics’ unique target discovery engine XPRESIDENT ® holds the promise of identifying novel tumor-associated MHC/peptide complexes by providing direct and quantitative evidence for their presence on a large collection of primary human tumor and normal tissue specimens. By this approach, MMP1-003, an HLA-A*02-binding peptide originating from matrix metallopeptidase 1 (MMP1), was identified as a promising therapeutic target presented by several tumor types, including colorectal and lung cancer, but absent on normal tissues. These findings are underlined by RNAseq analysis of the source antigen which also points to MMP1 being a highly attractive tumor-associated target. Consequently, a fully human antibody phage display library was screened to identify highly specific single chain antibodies, which were shown to recognize the purified HLA-A*02/MMP1-003-complex in ELISA assays as well as on peptide-pulsed HLA-A*02 + T2 cells. The best candidates were reformatted into bispecific tetravalent TandAbs ® through Affimed´s proprietary platform using a human/cyno-cross-reactive CD3-binding domain for T-cell engagement. Specific target recognition was confirmed for the TandAbs in binding and cytotoxicity assays on peptide-pulsed T2 cells. HLA-A*02/peptide-complexes selected from the broad normal tissue immunopeptidome with a high degree of sequence similarity to the HLA-A*02/MMP1-003-complex served as controls to confirm the specificity and hence the low risk of off-target binding. The most promising candidates were tested on a panel of endogenously target-expressing cancer cell lines covering MMP1 +/- and HLA-A*02 +/- expression profiles, as well as the source proteins for the most closely related control peptides. The lead TandAb showed excellent target specificity across a wide range of peptide-pulsed and endogenously expressing cell lines as well as potent cytotoxicity with picomolar EC 50 . In summary, we have identified a tumor-associated MMP1-derived peptide in an HLA-A*02 context by exploiting the knowledge of tumor and healthy tissue immunopeptidomes using XPRESIDENT ® . Overcoming the existing barrier of developing antibodies targeting specific MHC/peptide complexes, we generated and characterized highly specific and potent T-cell-recruiting TandAbs. These hold the potential to open up the therapeutic target space for T-cell engagement by providing access to intracellular proteins that are presented in a disease-specific manner as MHC/peptide complexes. Citation Format: Toni Weinschenk, Erich Rajkovic, Uwe Reusch, Michael Weichel, Kristina Ellwanger, Ivica Fucek, Michael Tesar, Dominik Hinz, Vera Molkenthin, Sebastian Bunk, Norbert Hilf, Oliver Schoor, Dominik Maurer, Kerstin Mock, Carsten Reinhardt, Martin Treder. Identification of antibodies against a novel tumor-associated MHC/peptide-target and generation of highly specific and potent HLA-A*02 MMP1-003 /CD3 TandAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3753. doi:10.1158/1538-7445.AM2017-3753