A Powassan virus domain III nanoparticle immunogen elicits neutralizing and protective antibodies in mice

Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C\u27 loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines

The role of the dynamic flavivirus surface in virus entry

Zu den Flaviviren zählen viele wichtige humanpathogene Erreger, wie das Frühsommer-Meningoenzephalitis- (FSME), West Nil-, Gelbfieber- und Zika-Virus sowie die Dengue-Viren. Diese kleinen umhüllten Viren werden von Arthropoden übertragen und können eine Vielzahl an Zellen von Vertebraten und Invertebraten infizieren. Das Hauptoberflächenprotein E spielt eine entscheidende Rolle während des Zelleintritts. Es vermittelt die Bindung des Virus an die Zelloberfläche und die von saurem pH induzierte Fusion der viralen und endosomalen Membran nach Rezeptor-vermittelter Endozytose. Eine Reihe von zellulären Oberflächenmolekülen wurden bereits als Anheftungsfaktoren identifiziert, aber ihre spezifische Rolle während der Virusaufnahme ist in den meisten Fällen noch ungeklärt. Außerdem können Flaviviren Fc-Rezeptor-positive Zellen durch die Aufnahme von infektiösen Virus-Antikörper-Komplexen infizieren, wobei sie keinen „echten“ Virus-spezifischen Rezeptor benötigen. Neueste Hinweise legen nahe, dass die E-Homodimere an der Virusoberfläche in ständiger dynamischer Bewegung („virus breathing“) sind, was zu einer vorübergehenden Exposition von verborgenen Strukturen führen kann, die in einer starren Virushülle nicht zugänglich wären. Diese „kryptischen“ Regionen könnten dann aufgrund der beweglichen Virushülle potentiell mit zellulären Liganden interagieren. In dieser Dissertation untersuchten wir die Rolle der dynamischen Oberfläche des FSME-Virus in Bezug auf die Zellbindung und deren Einfluss auf die Infektiösität. Insbesondere adressierten wir diese Frage durch Analysen von möglichen Effekten von E-spezifischen monoklonalen Antikörpern (mak) auf a) die Dynamik von Viruspartikeln, die zu Änderung in der Oligomerstruktur von E führen können, b) das Eindringen des Virus in die Zelle und c) die Infektion von primären bzw. permanenten Zellen. Eine Vorinkubation des FSME-Virus mit einem mak (mak A5), der ein Epitop in der E-Dimer-Grenzfläche („interface“) erkennt, erhöhte die Zellbindung, die auch zu einer verstärkten Infektion führte. Wie wir mittels biochemischer Analysen zeigen konnten, dissoziierte mak A5 das E-Dimer und führte zu einer Exposition des Fusionspeptids (FP), das normalerweise bei neutralem pH in der dimeren E-Proteinstruktur verborgen ist und nur in den Endosomen bei sauren pH exponiert wird, um die Membranfusion einzuleiten. Des Weiteren wiesen wir nach, dass die mak-induzierte FP-Exposition für die erhöhte Bindung an die Plasmamembran verantwortlich ist, aber nicht die Membranfusion auslösen kann. Aus diesen Daten schließen wir, dass die beobachtete Verstärkung der Infektion durch eine erhöhte Partikelaufnahme verursacht wurde. In dieser Arbeit beschreiben wir die Identifizierung eines neuen Mechanismus der Antikörper-induzierten Verstärkung der Infektion von Flaviviren, der unabhängig von Fc-Rezeptoren ist. Er beruht auf der Exposition des verborgenen FPs und dessen Interaktion mit der Wirtsmembran bereits in der Phase der Virusbindung. Flavivirus-Antikörper-Interaktionen mit ähnlichen Effekten könnten auch eine Rolle in polyklonalen Antikörperantworten spielen und daher potentiell zu einer Veränderung des Infektionsverlaufs führen, besonders wenn bereits Antikörper aus vorangegangen Infektionen mit verschiedenen Flaviviren vorhanden sind.Flaviviruses comprise a number of important human pathogens such as tick-borne encephalitis (TBE), West Nile, yellow fever, Zika and dengue viruses. They are small enveloped, arthropod-borne viruses and can infect a wide variety of cells from vertebrate and invertebrate host species. The major envelope protein E is crucial for the mechanisms of cell entry. It mediates binding to the cell surface as well as low-pH-induced fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Several cellular attachment factors have been identified, but their specific role in virus entry is still obscure in many instances. Flaviviruses can also infect Fc receptor-positive cells through the internalization of infectious virus-antibody complexes, thereby bypassing the requirement for a “true” virus-specific receptor. Recent evidence has suggested that the E homodimers on the virus surface are in dynamic motion (“virus breathing”), leading to the transient exposure of sites that would be cryptic in a rigid particle, but can potentially interact with cellular ligands due to the breathing of the viral envelope. In this PhD thesis, we investigated the role of the dynamic surface of TBE virus (TBEV) in virus-cell binding and its impact on infectivity. Specifically, we addressed this question by analyzing the possible effect of E-specific monoclonal antibodies (mabs) on particle dynamics that would affect the oligomeric structure of E, virus entry as well as infection of primary and permanent cell lines. Pre-incubation of TBEV with a mab (mab A5), recognizing an epitope at the E dimer interface, enhanced binding to cells, which also resulted in increased infectivity. As revealed by biochemical analyses, mab A5 dissociated the E dimer and led to the exposure of the fusion loop (FL), which is normally buried in the dimeric E protein structure at neutral pH and becomes exposed only at the low pH in endosomes to initiate membrane fusion. We could demonstrate that the mab-induced FL exposure was responsible for enhanced binding to the plasma membrane, without leading to membrane fusion. Therefore, we conclude that the observed enhancement of infectivity was caused by an increase in particle uptake. In summary, we have identified a novel mechanism of antibody-induced enhancement of infection of flaviviruses, which is independent of Fc receptors, but is mediated by the exposure of the otherwise cryptic FL and its insertion into the host membrane already at the stage of viral attachment. Flavivirus-antibody interactions with similar effects can also occur in the context of polyclonal antibody responses and therefore have the potential to modulate the course of infection, especially when pre-existing antibodies are present in sequential infections with different flaviviruses.submitted by Denise Angelika HaslwanterAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018OeBB(VLID)257757

A novel mechanism of antibody-mediated enhancement of flavivirus infection

<div><p>Antibody-dependent enhancement of viral infection is a well-described phenomenon that is based on the cellular uptake of infectious virus-antibody complexes following their interaction with Fcγ receptors expressed on myeloid cells. Here we describe a novel mechanism of antibody-mediated enhancement of infection by a flavivirus (tick-borne encephalitis virus) in transformed and primary human cells, which is independent of the presence of Fcγ receptors. Using chemical cross-linking and immunoassays, we demonstrate that the monoclonal antibody (mab) A5, recognizing an epitope at the interface of the dimeric envelope protein E, causes dimer dissociation and leads to the exposure of the fusion loop (FL). Under normal conditions of infection, this process is triggered only after virus uptake by the acidic pH in endosomes, resulting in the initiation of membrane fusion through the interaction of the FL with the endosomal membrane. Analysis of virus binding and cellular infection, together with inhibition by the FL-specific mab 4G2, indicated that the FL, exposed after mab A5- induced dimer-dissociation, mediated attachment of the virus to the plasma membrane also at neutral pH, thereby increasing viral infectivity. Since antibody-induced enhancement of binding was not only observed with cells but also with liposomes, it is likely that increased infection was due to FL-lipid interactions and not to interactions with cellular plasma membrane proteins. The novel mechanism of antibody-induced infection enhancement adds a new facet to the complexity of antibody interactions with flaviviruses and may have implications for yet unresolved effects of polyclonal antibody responses on biological properties of these viruses.</p></div

Fusion of pyrene-labeled TBEV with liposomes.

<p>Pyrene-labeled TBEV was allowed to interact with liposomes at pH 8.0 or pH 5.5 in the absence or presence of mab A5. The results are expressed as the ratio of pyrene excimer to monomer (E/M), which decreases upon fusion of the viral membrane with the liposomal membrane. Data represent the mean +/- SEM of three independent experiments.</p

Mab A5-enhanced TBEV binding to cells and liposomes.

<p>(A) TBEV was allowed to attach to HeLa cells for 1 hour at 4°C in the absence or presence of mabs (as indicated on the x-axis). The y-axis indicates percent bound virus relative to input virus using RNA copy numbers determined by qPCR. (B) TBEV was allowed to attach to liposomes for 20 minutes at 37°C in the absence or presence of mabs (as indicated on the x-axis). The y-axis indicates percent bound virus relative to input virus using the amounts of E protein determined by quantitative ELISA. Data represent the mean +/- SEM of at least three independent experiments. The amounts of bound TBEV-mab complexes were compared to those obtained with the control (ctrl, TBEV without mabs) with ANOVA followed by Dunnett’s multiple comparisons test. *, p < 0.05.</p

Schematic representation of different modes of virus entry.

<p>Three different modes of internalization are shown. Left: after binding to a specific receptor Middle: antibody-induced exposure of the FL and attachment to the plasma membrane Right: FcR-mediated uptake of antibody-virus complexes Color code E: domain I—red, domain II—yellow, domain III—blue, fusion loop—orange, stem-anchor region—gray. For simplification the M protein is not shown in the virus particles.</p

Characteristics of mabs used in this study.

<p>Characteristics of mabs used in this study.</p

Virus-cell binding assays.

<p>(A-B) Binding of virus to HeLa cells. (A) TBEV, (B) DENV. (C) Binding of TBEV (white columns) or DENV (grey columns) to human foreskin fibroblasts (HFFs). (D) Binding of RV-A2 to HeLa cells. (E) Binding of TBEV (white columns) or DENV (grey columns) to immature monocyte-derived dendritic cells (moDCs). Viruses were allowed to attach to cells for 1 hour at the temperatures as indicated on the x-axis. The y-axis indicates percent bound virus relative to input virus using RNA copy numbers determined by quantitative PCR (qPCR). Data represent the mean +/- SEM of at least three independent experiments. For each virus, the amounts of bound virus at 30°C and/or 37°C were compared to those obtained at 4°C with ANOVA followed by Dunnett’s multiple comparisons test (A,B,E) or Student’s t-test (C,D). *, p < 0.05.</p

Blocking of mab A5-enhanced cell binding and infectivity by the FL-specific mab 4G2.

<p>(A-D) Binding of TBEV to cells after incubation with different mabs (A5, A5+4G2, 4G2) for 1 hour at 4°C. (A) HeLa cells, (B) Vero cells, (C) HFFs, (D) immature moDCs. The y-axis indicates percent bound virus relative to input virus using RNA copy numbers determined by qPCR. (E) Infectivity of TBEV after incubation with different mabs (A5, A5+4G2, 4G2) on HeLa cells using focus formation assays. (F) TBEV production in Vero cells after infection in the presence of different mabs (A5, A5+4G2, 4G2). In contrast to HeLa cells, TBEV did not produce foci in Vero cells. Virus production was therefore measured by quantifying RNA copy numbers with qPCR. Data represent the mean +/- SEM of at least three independent experiments. The amounts of bound TBEV-mab complexes or their infectivity were compared to the corresponding values obtained with the control (ctrl, TBEV without mabs) with ANOVA followed by Dunnett’s multiple comparisons test. *, p < 0.05.</p