Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed inhibitor of RdRp activity

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nn-ITN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)(4) spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nn-ITN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.Peer reviewe

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein

Rissanen, Ilona Stass, Robert Krumm, Stefanie A Seow, Jeffrey Hulswit, Ruben Jg Paesen, Guido C Hepojoki, Jussi Vapalahti, Olli Lundkvist, Ake Reynard, Olivier Volchkov, Viktor Doores, Katie J Huiskonen, Juha T Bowden, Thomas A eng MR/L009528/1/Medical Research Council/United Kingdom MR/S007555/1/Medical Research Council/United Kingdom MR/N002091/1/Medical Research Council/United Kingdom MR/K024426/1/Medical Research Council/United Kingdom 309605/Academy of Finland 649053/H2020 European Research Council 203141/Z/16Z/Wellcome Trust/United Kingdom 060208/Z/00/Z/Wellcome Trust/United Kingdom 093305/Z/10/Z/Wellcome Trust/United Kingdom England Elife. 2020 Dec 22;9. pii: 58242. doi: 10.7554/eLife.58242.The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.Peer reviewe

HIV-1 glycan density drives the persistence of the mannose patch within an infected individual.

CAPRISA, 2016.Abstract available in pdf

A Protective Monoclonal Antibody Targets a Site of Vulnerability on the Surface of Rift Valley Fever Virus

Summary: The Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally. : Allen et al. reveal a molecular basis of antibody-mediated neutralization of Rift Valley fever virus, an important human and animal pathogen. They isolate and demonstrate the protective efficacy of a monoclonal antibody in a murine model of virus infection, providing a blueprint for rational therapeutic and vaccine design. Keywords: phlebovirus, Rift Valley fever virus, antibody, structure, bunyavirus, virus-host interactions, immune response, vaccine, antiviral, neutralizatio

Cross-reactivity of glycan-reactive HIV-1 broadly neutralizing antibodies with parasite glycans

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.This project has received funding from the European Union’s Horizon 2020 Research and Innovation program under grant agreement 681137 (to K.J.D. and I.H.), the Medical Research Council (MRC) (to K.J.D. [MR/K024426/1]), The Rosetrees Trust (to K.J.D. [M686]) and Fondation Dormeur, Vaduz (to K.J.D). This research was funded or supported by the National Institute for Health Research Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London and/or the NIHR Clinical Research Facility. The views expressed are those of the authors and not necessarily those of the National Health Service (NHS), the National Institute for Health Research (NIHR), or the Department of Health. N.R. acknowledges funding from Ministry of Science and Education grants CTQ2017-90039-R, RTC-2017-6126-1, and CTQ2011-27874 (fellowship to K.B.) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (grant MDM-2017-0720). F.A. was funded by the Wellcome Trust (104958/Z/14/Z). J.A. was supported by the Spanish Ministry of Science, Innovation and Universities through the grant PID2019-109395GB-I00. J.A. and S.M. acknowledge support of BBSRC (grant BB/P010660/1). T.H. and S.W. were funded by Biotechnology and Biological Sciences Research Council (BBSRC) Norwich Research Park Doctoral Training Grant BB/M011216/1. IAVI’s work is made possible by generous support from many donors, including the Bill & Melinda Gates Foundation, the Ministry of Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan in partnership with The World Bank, the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency for Development Cooperation, the United Kingdom Department for International Development (DFID), and the United States Agency for International Development. The full list of IAVI donors is available at www.iavi.org. Brendan McAtarsney and Jonathan Hare from the IAVI Human Immunology Lab (HIL) for coordinating the samples transfers and shipments. Monica Agromayor and the KCL Nikon Centre for assistance and advice on confocal microscopy. NMRI strain Schistosoma mansoni-infected Biomphalaria glabrata snails were provided by the NIAID Schistosomiasis Resource Center, Rockville, USA.Peer reviewe

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease

Cross-reactivity of glycan-reactive HIV-1 broadly neutralizing antibodies with parasite glycans

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development

Protein-Protein und Protein-Kleinmolekül-Inhibitor Interaktionen im Masern Virus Replikationskomplex

The disease measles is caused by the highly contagious measles virus (MeV). MeV belongs to the paramyxovirus family together with respiratory syncytial virus, human parainfluenza viruses and metapneumovirus. Paramyxoviruses are responsible for major pediatric morbidity and mortality. Despite the availability of an effective MeV vaccine, measles case numbers increased alarmingly in the past few years especially in Europe. The return of endemic measles in the European population can directly be linked to the decrease in acceptance/use of the measles-mumps-rubella (MMR) vaccine. The Measles Initiative has set a goal to eliminate measles by 2015. The addition of an effective antiviral to quickly treat sporadic outbreaks and the surrounding communities would greatly aid in the measles eradication efforts. Fundamental understanding of the viral replication mechanism at the molecular level will be critical for the successful development of antivirals. Therefore the following dissertation examined the protein-protein interactions in the measles virus polymerase complex to understand the events taking place at the molecular level. Additionally, it engaged in protein-small-molecule interactions to identify small-molecule inhibitors of viral replication and their targets. The first part of the thesis focused on molecular interactions in the viral replication complex. The viral replication complex is an attractive target for antiviral therapy since it possesses unique features and is expressed and functions in a sub cellular compartment distinct from host cell polymerases. The polymerase complex consists of the phosphoprotein (P) and the polymerase (L) protein. The P-L complex only interacts with nucleoprotein (N) encapsidated RNA (RNP) for transcription and replication. MeV N contains a core domain involved in RNA encapsidation and a 125-residue carboxy (C)-terminal tail (Ntail) considered to mediate P-L binding to RNP for polymerization. Ntail of MeV is largely unstructured, but a terminal microdomain is implicated in P binding. C-terminal tail truncated N mutant proteins progressively eliminating this microdomain and upstream tail sections demonstrated that the interaction of the Ntail microdomain with a C-terminal domain in P is not required for polymerase recruitment and initial binding of L to its template. Additional investigations showed that disrupting the domain organization by insertion of an epitope tag in the Ntail did not affect polymerase activity, but rather affected particle assembly. Cell free virions contained reduced levels of envelope proteins which did not affect cell-to-cell fusion kinetics. However, the N-mutant virus was observed to have a kinetic delay of viral mRNA and genome production. Studies to identify and characterize small-molecule antiviral compounds and their targets were conducted in the second part of this thesis. Non-nucleoside small-molecules are suitable antiviral therapeutics. There are two main approaches in identifying antivirals. First, compounds that target the virus, for example the RNA replication machinery, can be assayed for. Alternatively, compounds that target a host factor that the virus requires can also be a viable strategy. Cellular factors may also be necessary for the entire family of viruses and therefore compounds aiming for host factors may be more likely to be broadly active inhibitors. A potent pathogen-directed small-molecule compound class had been identified in a high-throughput screen. Hit-to-lead chemistry yielded a highly potent and water soluble compound ERDRP-0519. It targets the L subunit of the morbillivirus polymerase complex directly, since resistance-mediating mutations were exclusively located in the L protein. Unparalleled efficacy of this orally available small-molecule inhibitor was demonstrated and pioneered a path towards an effective morbillivirus therapy that can support measles eradication efforts. Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. JMN3-003 is a potent broadly active inhibitor of viral RdRp activity with a host factor mediated profile. It inhibited a wide range of different viral targets. Its antiviral activity was host cell species dependent and induced a temporary cell cycle arrest. While the compound inhibited viral mRNA and genome production, it left host cell mRNA and protein production unaffected. Taken together, this PhD studies changed the prevailing paradigm in polymerase recruitment and provided strong proof of concept for the potential of the development of pathogen- and host-directed antiviral therapy. These studies demonstrated how basic molecular research of protein-protein interactions critical for virus replication can complement a translational approach to identify, characterize, and improve novel antiviral candidates.Die Masern, eine hochansteckende Erkrankung, werden durch den Masernvirus verursacht. Dieser gehört zur Familie der Paramyxoviren. Paramyxoviren sind hauptverantwortlich für Morbidität und Mortalität bei Kindern. Obwohl ein wirksamer Impfstoff verfügbar ist, hat sich die Anzahl der Masernerkrankungen in den letzten Jahren drastisch erhöht. Die Rückkehr endemischer Masernausbrüche in der europäischen Bevölkerung kann direkt auf eine geringere Anwendung der Masern-Mumps-Röteln-Impfung (MMR) zurückgeführt werden. Die „Measles Initiative“ hat sich die Masernausrottung bis 2015 zum Ziel gesetzt. Eine Maßnahme zur Unterstützung der Masernausrottung wäre die Verwendung wirkungsvoller antiviraler Therapeutika. Für die erfolgreiche Entwicklung von antiviralen Medikamenten ist es von fundamentaler Bedeutung, den genauen Replikationsmechanismus des Virus auf molekularer Ebene zu verstehen. Daher beschäftigte sich diese Dissertation zum einen mit Protein-Protein-Wechselwirkungen im viralen Polymerasekomplex, um die Ereignisse während einer viralen Infektion genau zu verstehen und zum anderen mit Protein-Wirkstoff-Wechselwirkungen, um kleine Moleküle, die die virale Replikation verhindern, zu identifizieren und deren Zielproteine zu finden. Der erste Teil der Dissertation konzentrierte sich auf die molekularen Interaktionen im viralen Replikationskomplex. Dieser ist ein attraktives Ziel für eine antivirale Therapie, da er einzigartige Eigenschaften besitzt und in einem subzellulären Kompartiment getrennt von Polymerasen der Wirtszelle exprimiert wird. Er besteht aus dem Phosphoprotein (P) und der Polymerase (L). Der P-L Komplex erkennt die virale RNA für Transkription und Replikation nur, wenn sie von dem Nukleoprotein (N) umschlossen ist und als Ribonukleoproteinkomplex (RNP) vorliegt. Das N Protein besteht aus einer aminoterminalen Domäne, die mit der RNA assoziiert (Ncore) und einer carboxyterminalen Domäne (Ntail), die für die Bindung von P und L an die RNP verantwortlich ist. Die Ntail Domäne ist weitgehend unstrukturiert, aber eine Mikrodomäne im Ntail ist an der Bindung zu P beteiligt. Es wurden N Mutanten hergestellt, die schrittweise diese Mikrodomäne und Sequenzen davor nicht mehr exprimierten. Die Wechselwirkungen von der Mikrodomäne im Ntail mit einer carboxyterminalen Domäne in P nicht benötigt werden, um die Polymerase zu rekrutieren und an die RNP zu binden. Weitere Untersuchungen zeigten, dass eine Störung des Domänenaufbaus im Ntail durch Einfügung eines HA-Tags nicht die Polymeraseaktivität beeinflusst. Vielmehr war die Produktionskinetik von viraler mRNA und Genom beim mutierten Virus zeitlich verzögert. Im zweiten Teil der Dissertation wurden antivirale kleinmolekulare Wirkstoffe durch Protein-Wirkstoff-Wechselwirkungen identifiziert und charakterisiert. Zum einen kann man nach Wirkstoffen suchen, die den Erreger direkt inhibieren. Eine alternative Strategie zielt darauf Wirkstoffe zu finden, die wichtige Faktoren für den Virus in der Wirtszelle blockieren. Diese Zellfaktoren können auch von anderen Mitgliedern der gleichen Virusfamilie beansprucht werden und so können Wirkstoffe, die einen dieser Faktoren blockieren, ein breites Spektrum verschiedener Viren hemmen. Eine Molekülklasse, die sich direkt gegen den Erreger richtet, wurde in einem High-Throughput-Experiment identifiziert. Durch eine Hit-to-Lead-Optimierung wurde der hochwirksame und wasserlösliche kleinmolekulare Wirkstoff ERDRP-0519 entwickelt. Dieser inhibierte direkt die Morbillivirus Polymerase, da resistenzvermittelnde Mutationen ausschließlich im L Protein entdeckt wurden. Es konnte die antivirale Effektivität eines oral verabreichbaren Wirkstoffs in einem Kleintiermodell gezeigt werden. Dieses Ergebnis hat somit den Grundstein für die Entwicklung einer effektiven Morbillivirus Therapie gelegt. Eine zweite Molekülklasse griff in der Wirtszelle einen Faktor an, der für die virale Replikation von Bedeutung ist und blockierte ein breites Spektrum von Viren. JMN3-003 ist ein potenter Wirkstoff, der die virale Replikation hemmte, indem er einen dafür notwendigen Wirtszellfaktor inhibierte. Er unterband die Replikation vieler verschiedener positiv und negativ RNA-Viren sowie DNA-Viren. Außerdem bewirkte JMN3-003 einen temporären Stillstand des Zellzyklus. Während er die virale mRNA und Genom-Produktion hemmte, ließ er die mRNA- und Protein-Produktion der Wirtszelle unberührt. Diese Dissertation hat das vorherrschende Model zur Rekrutierung der viralen MeV Polymerase modifiziert und einen guten Beweis dafür geliefert, dass Wirkstoffe, die sich direkt gegen den Erreger richten bzw. die Faktoren der Wirtszelle als Zielgruppe haben, sich für eine antivirale Therapie eignen. Sie hat gezeigt, in welcher Weise grundlegende molekulare Untersuchungen der Protein-Protein-Wechselwirkungen und Methoden zur Identifizierung, Charakterisierung und Verbesserung neuer therapeutischer Wirkstoffe ineinandergreifen und sich ergänzen können