Neue antigene Domänen für die Induktion von neutralisierenden Antikörpern auf Glykoprotein B des Humanen Cytomegalovirus

Das virale Oberflächenprotein Glykoprotein (g) B des humanen Cytomegalovirus (HCMV) spielt eine bedeutende Rolle bei der Induktion von neutralisierenden Antikörpern und wurde bereits mehrfach als experimenteller Impfstoff in klinischen Studien eingesetzt. Für die Herstellung prophylaktischer und/oder therapeutischer Wirkstoffe gegen HCMV bedingte Erkrankungen, ist ein tieferes Verständnis der humoralen Immunantwort gegen gB von höchster Bedeutung. Aus diesem Anlass wurde eine umfassende Analyse des gB-spezifischen Gedächtnis-B-Zell (GBZ)-Repertoires von mehreren gesunden, HCMV-infizierten Personen durchgeführt. Fluoreszenz-markiertes gB wurde dazu verwendet, mittels Durchflusszytometrie, gB-spezifische GBZ aus dem peripheren Blut zu isolieren, welche anschließend zu anti-gB Antikörper produzierenden Zelllinien stimuliert wurden. Die Frequenz von gB-spezifischen, IgG-positiven GBZ unter allen IgG-positiven GBZ lag zwischen 0,3 und 1,4% (Durchschnitt 0,7%) bei 15 gesunden, HCMV-positiven Personen. Die gB-produzierenden B Zelllinien wurden hinsichtlich ihrer Bindespezifität gegen die bekannten antigenen Domänen (AD, AD-1 und AD-2) und ihrer Neutralisations-Kapazität untersucht. AD-1-spezifische GBZ konnten aus jedem analysierten Spender (n=7) mit unterschiedlicher Frequenz (12-58%) isoliert werden, wohingegen AD-2-spezifische GBZ nur bei 3 von 7 Spendern mit sehr geringer Frequenz (1-2%)isoliert werden konnte. Nur ein geringer Anteil aller gB-spezifischen GBZ produzierte neutralisierende Antikörper (0-11%, Durschnitt 3%). Die Frequenz an AD-1-spezifischen GBZ mit neutralisierender Aktivität lag bei 2% und unter den AD-2-spezifischen GBZ konnten keine neutralisierenden Antikörper identifiziert werden. Diese Ergebnisse wiesen darauf hin, dass die Mehrzahl der gB-spezifischen neutralisierenden Antikörper an noch unbekannte Zielstrukturen auf gB binden. Western-Blot Analysen und Experimente mit gB-Deletionsmutanten deuteten darauf hin, dass die Bindung der Antikörper an die besagten Zielstrukturen konformationsabhängig ist und darüberhinaus der erkannte Bereich zwischen Aminosäure (AS) 100 und 447 von gB liegt. Um oberflächenexponierte Strukturen in diesem Bereich bestimmen zu können, wurde ein 3D-Strukturmodel von HCMV gB angefertigt. Zwei strukturelle Domänen auf gB wurden auf diese Weise und mittels rekombinanter Expression als Zielstrukturen der neutralisierenden Antikörper identifiziert. Domäne I liegt zwischen AS 132-343 auf gB und Domäne II, eine diskontinuierliche Domäne, erstreckt sich zwischen AS 121-131 und 344-437. Mittels ELISA mit einer Vielzahl an HCMV-positiven Seren konnte gezeigt werden, dass beide Domänen immunogen sind, bei 17% der analysierten Seren wurden IgG Antikörper gegen die Domäne I-nachgewiesen, 73% der Seren wiesen Antikörper gegen Domäne II auf. Interessanterweise erkennen alle neutralisierenden Antikörper, die nicht AD-1 oder -2-spezifisch sind die Domänen I oder II auf gB. Alle Domäne I- und 88% der Domäne II-spezifischen Antikörper, welche isoliert werden konnten, zeigten effektive Neutralisationsaktivitäten im nanomolaren Bereich. Hieraus lässt sich folgern, dass die Induktion einer Domäne I und II-spezifischen Immunantwort für eine gB-basierte Vakzinierung von großer Bedeutung sein könnte und darüberhinaus, dass Domäne I und II-spezifische Antikörper für den prophylaktisch/therapeutischen Einsatz, zum Beispiel bei Patienten nach Stammzelltransplantation geeignet wären.The humoral immune response against the human cytomegalovirus (HCMV) plays an important role during natural infection. Glycoprotein (g) B is a dominant viral antigenic determinant for the induction of neutralizing antibodies and is also a component in several experimental HCMV vaccines currently used in humans. In order to design optimal antiviral biologicals for the prophylaxis and/or therapy of HCMV induced disease, it is crucial to understand the humoral immune response against gB. To this end, the human anti-gB memory B cell (MBC) repertoire established by healthy, HCMV seropositive, individuals was comprehensively analyzed in an unbiased fashion. Fluorescence labeled gB was used for flow cytometric cell sorting of gB-specific MBC followed by polyclonal stimulation of MBC to gB-producing cell lines. Frequencies of gB-binding, IgG-positive MBC ranged from 0,3 to 1,4% (mean 0,7%) among all IgG-positive MBC (n=15 HCMV-positive individuals). The gB-producing B cell lines were analyzed in terms of antibody target structures on gB as well as virus-neutralizing activity. With respect to previously characterized target structures on gB (antigenic domain (AD)-1 and 2), we found that antibodies recognizing AD-1 were produced by all donors (n=7) with varying frequency (12-58% of all gB producing lines). AD-2-specific MBC were only isolated from 3 of 7 donors with low frequency (1-2 %). B cell clones producing HCMV-neutralizing antibodies represented a small fraction of all anti-gB lines (0-11%, mean 3%). Only a small percentage of AD-1-specific antibodies (2%)showed neutralizing capacity and no AD-2-specific antibodies had neutralizing potential. These results indicated that the majority of gB-specific, neutralizing antibodies bound to previously unknown binding sites on gB. Further experiments, using Western Blot and gB deletion mutants, revealed that protein conformation was crucial for the binding of these neutralizing antibodies and furthermore, narrowed down the binding site to a region between aa 100 to 447 of gB. To identify surface exposed protein domains in this region, we generated a 3D molecular model of HCMV gB. Upon recombinant expression, two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I is located between aa 132-343 of gB and domain II, a discontinuous domain, consists of residues 121-131 and 344-437. Analyzing a large panel of HCMV-positive sera for their reactivity to either domain, revealed a high antigenicity of domain II (73% seropositivity rate)and a weaker antigenicity of domain I (17% seropositivity rate). Strikingly, all neutralizing antibodies which bound to epitopes outside of AD-1 and -2, bound to either domain I or domain II and what is more, 100% of domain I antibodies and 88% of domain II antibodies, which could be tested, showed neutralizing activity. Most interestingly, the majority of antibodies against the two newly identified domains I and II show high neutralizing potency in the nanomolar range. Therefore, we propose that the induction of an optimal domain I and II-specific antibody response should be a major goal in vaccination trials using gB-containing vaccines and moreover, domain I and II-specific antibodies show promising potential as therapeutic antibody candidates

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies.

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization

Use of Fondaparinux Off-Label or Approved Anticoagulants for Management of Heparin-Induced Thrombocytopenia.

BACKGROUND Life-threatening heparin-induced thrombocytopenia (HIT) is treated with the alternative nonheparin anticoagulants argatroban, lepirudin, or danaparoid. Frequently, the pentasaccharide fondaparinux is used off-label. OBJECTIVES The authors sought to investigate the safety and efficacy of the different anticoagulants for treating HIT. METHODS In a national, multicenter registry study, hospitalized patients who were diagnosed with HIT, an at least intermediate clinical HIT-risk (4Ts score ≥4 points), and received treatment with ≥1 dose of the aforementioned anticoagulants were included. Main outcome measures were the incidences of HIT-specific complications (thromboembolic venous/arterial events, amputations, recurrent/persistent thrombocytopenia, skin lesions) and bleedings. RESULTS Of 195 patients, 46 (23.6%), 4 (2.1%), 61 (31.3%), and 84 (43.1%) had been treated first-line with argatroban, lepirudin, danaparoid, and fondaparinux, respectively. The composite endpoint of HIT-specific complications (thromboembolic events, amputation, skin necrosis) occurred in 11.7% of patients treated with approved alternative anticoagulation and in 0.0% of fondaparinux-treated patients. The all-cause in-hospital mortality rates were 14.4% during approved alternative anticoagulation and 0.0% during fondaparinux treatment. Bleeding complications occurred in alternatively anticoagulated patients and in fondaparinux-treated patients in 6.3% and 4.8%, respectively. Post hoc analysis of clinical and laboratory features confirmed "true" HIT in at least 74 of 195 (38.0%) patients; 35 of 74 (47.3%) were treated with fondaparinux. CONCLUSIONS Fondaparinux is effective and safe in suspected acute HIT; no HIT-specific complications occurred in the fondaparinux-treated patients, even among those with a high clinical HIT probability. Further data from randomized controlled trials are urgently needed because lepirudin was recalled from the market; danaparoid access has been limited and is not approved in the United States; and argatroban is contraindicated in patients with impaired liver function, and activated partial thromboplastin time confounding may interfere with monitoring. (Retrospective Registry of Patients With Acute Heparin-induced Thrombocytopenia Type II; NCT01304238)