Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.Fil: Roman Sosa, Gleyder. Ulm University Hospital; AlemaniaFil: Leske, Anne. Friedrich-Loeffler-Institut; AlemaniaFil: Ficht, Xenia. Ulm University Hospital; AlemaniaFil: Dau, Tung Huy. Friedrich-Loeffler-Institut; AlemaniaFil: Holzerland, Julia. Friedrich-Loeffler-Institut; AlemaniaFil: Hoenen, Thomas. Friedrich-Loeffler-Institut; AlemaniaFil: Beer, Martin. Friedrich-Loeffler-Institut; AlemaniaFil: Kammerer, Robert. Friedrich-Loeffler-Institut; AlemaniaFil: Schirmbeck, Reinhold. Friedrich-Loeffler-Institut; AlemaniaFil: Rey, Felix A.. Friedrich-Loeffler-Institut; AlemaniaFil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Groseth, Allison. Friedrich-Loeffler-Institut; Alemani

Identification of molecular detection mechanisms associated with apoptosis induction in response to arenavirus infection

New World arenaviruses represent an important group of zoonotic pathogens that pose a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell responses to infection, or how they contribute to virulence; however, TCRV strongly induces caspase-dependent apoptosis (i.e. non-inflammatory programmed cell death) in infected cells, whereas JUNV does not. In order to better understand the connection between apoptosis and pathogenesis, we sought to unravel the regulation of pro- and anti-apoptotic signaling in response to arenavirus infection. We demonstrated that apoptosis induced by TCRV proceeds over the mitochondrial-regulated intrinsic pathway and involves activation of p53 (accumulation and phosphorylation), activation of the pro-apoptotic BH3-only factors Puma and Noxa (accumulation), as well as inactivation of another pro-apoptotic factor called Bad (phosphorylation). The regulation of these factors in response to TCRV infection is accompanied by other classical hallmarks of intrinsic apoptosis, such as disorganization of the mitochondrial network, cytochrome c release, PS flipping, caspase cleavage and nuclear condensation. The involvement of the BH3-only factors as key players in regulating TCRV-induced apoptosis could also be validated in knockout cells, which showed either suppressed or increased apoptosis depending on the respective activation (i.e. Puma and Noxa) or inactivation (i.e. Bad) status of the respective BH3 protein. Interestingly, while JUNV does not trigger late stages of apoptosis induction (i.e. caspase activation, nuclear condensation and cell death), we could show that it activates similar upstream pro-apoptotic signaling events including activation of p53, Puma and Noxa. This supports the current hypothesis that JUNV actively evades the induction of apoptosis through the involvement of a mechanism targeting late steps in the apoptotic cascade. Specifically, this model proposes that intrinsic activation is suppressed at the level of caspase activation by JUNV NP, which serves as an alternative substrate for caspase cleavage. Additionally, in order to identify viral factors associated with the induction of apoptosis, a full genome sequencing of TCRV was performed and contributed to the validation and correction of substantial errors reported in existing sequences for TCRV. With the help of this sequence, correct expression plasmids containing the viral genes for NP, GP and Z were constructed and tested for their ability to induce apoptosis in vitro. This revealed that both TCRV and JUNV Z are triggers for apoptosis, which further supports our finding that JUNV also induces activation of pro-apoptotic factors. Again, consistent with a model where JUNV NP blocks caspase activation directly, co-expression of JUNV Z and NP abrogated caspase activation, while simultaneous expression of TCRV NP and Z still resulted in cell death. Finally, identification of the specific apoptotic factors involved in regulating TCRV-induced apoptosis (i.e. Bad, Puma and Noxa) and the generation of the respective knockout cell lines allowed us to investigate what influence apoptosis induction has on virus infection. Interestingly, knockout of these factors showed no direct impact on virus growth in Vero cells. However, TCRV particles produced in cells with the individual pro-apoptotic (i.e. Puma and Noxa) or anti-apoptotic (i.e. Bad) factors knocked out showed altered infectivity in primary human monocytes and macrophages, which represent important target cells for arenaviruses. Since TCRV particles that originate from the different knockout cells would be expected to contain different amounts of PS in their envelope (depending on the level of apoptosis taking place), this suggests a role of apoptosis in facilitating PS-receptor-mediated entry and/or PS-receptor signaling through downstream kinases, either of which could be contributing to successful infection in professional phagocytic cells. In particular, phosphorylation of some of the identified factors involved in regulating TCRV-induced apoptosis indicates the involvement of upstream kinases from diverse signaling pathways, some of which also play a role in regulating cytokine production – another host cell reaction that differs significantly between TCRV- and JUNV-infected monocytes and macrophages. As such, these findings represent an exciting basis for a possible connection between apoptotic responses and the regulation of pro- and anti-inflammatory cytokine responses via their associated upstream signaling processes and provide a starting point for future studies that will help us to better understand how these processes contribute to arenavirus pathogenicity.Neuwelt-Arenaviren sind eine wichtige Gruppe zoonotischer Erreger, die eine ernstzunehmende Bedrohung für die Bevölkerung darstellen. Während einige Viren dieser Familie schwerwiegende Krankheitsverläufe im Menschen auslösen können, wie beispielsweise hämorrhagisches Fieber oder neurologischen Symptomen, weisen eng verwandte Arenaviren keine derartige Pathogenität auf. Ein Beispiel hierfür sind das Junín-Virus (JUNV), der Auslöser für Argentinisches hämorrhagisches Fieber und der eng verwandte Prototyp, das Tacaribe-Virus (TCRV), welcher als apathogen für den Menschen gilt. Die zugrundeliegenden Mechanismen für diese fundamentalen Unterschiede im Pathogenitätspotential eng verwandter Arenaviren sind derzeit wenig verstanden. Es konnte jedoch gezeigt werden, dass TCRV eine starke Caspase-abhängige Apoptose-Antwort (nicht-entzündlicher programmierter Zelltod) in infizierten Zellen induziert, wohingegen JUNV diese Reaktion nicht auslöst. Um ein besseres Verständnis über den Zusammenhang zwischen Apoptose-Induktion und der Pathogenese dieser Arenaviren zu erlangen, wurde in dieser Arbeit die Regulation der pro- und anti-apoptotischen Signaltransduktion als Antwort auf eine Infektion mit dem avirulenten TCRV und dem virulenten JUNV näher beleuchtet. Es konnte gezeigt werden, dass die TCRV-vermittelte Apoptose über den mitochondrialen intrinsischen Apoptose-Weg, mit der Aktivierung von p53 (Akkumulation und Phosphorylierung), den pro-apoptotischen BH3-only Faktoren Puma und Noxa (Akkumulation), sowie mit der Inaktivierung eines weiteren pro-apoptotischen Faktors namens Bad (Phosphorylierung), verläuft. Zusätzlich konnte neben der Regulierung dieser Faktoren das Auftreten charakteristischer Merkmale der intrinsische Apoptose-Induktion, wie beispielsweise die Störung des mitochondrialen Netzwerks mit Zytochrom C Ausschüttung, Umverteilung von Phosphatidylserin (PS), Caspase-Spaltung und nukleäre Kondensation, detektiert werden. Die Beteiligung der BH3-only Proteine als Schlüsselfaktoren für die Regulierung der TCRV-induzierten Apoptose konnte ebenfalls in Knockout Zellen bestätigt werden, wobei in den jeweiligen Knockouts entweder eine erhöhte oder reduzierte Apoptose-Antwort gemessen werden konnte, in Abhängigkeit von der entsprechenden Aktivierung (Puma und Noxa) oder Inaktivierung (Bad) der individuellen BH3-only Proteine. Obwohl JUNV interessanterweise keine Aktivierung der spätgeschalteten Apoptose-Schritte (z.B. Caspase-Aktivierung) auslöst, konnte dennoch nachgewiesen werden, dass JUNV eine ähnliche pro-apoptotische Signaltransduktion mit Aktivierung von p53, Puma und Noxa induziert. Dies stützt wiederum eine aktuelle Hypothese in der JUNV einen Mechanismus entwickelt hat um die Induktion der Apoptose aktiv zu umgehen, wobei es gezielt späte Schritte in der Apoptose-Signalkaskade blockieren kann. Konkret beschreibt dieses Modell die Hemmung der Apoptose während der JUNV Infektion auf der Ebene der Caspasen durch die Verwendung und Spaltung des viralen NP, welches als alternatives Substrat für die Caspasen dient um diese abzulenken. Um zusätzlich die viralen Faktoren, die für die Apoptose-Induktion verantwortlich sind ausfindig zu machen wurde eine vollständige Genomsequenzierung für TCRV durchgeführt, welche als Grundlage für molekularbiologische Analysen der Proteinfunktionen diente und dazu beitrug bestehende Fehler in bereits existierenden Sequenzen für TCRV zu validieren und zu korrigieren. Unter Zuhilfenahme der von uns generierten Sequenz konnten korrekte Expressions-Plasmide, welche die viralen Gene für NP, GP und Z beinhalten generiert und getestet werden, wobei die Apoptose-Induktion anschließend gemessen wurde. Diese Transfektions-Experimente enthüllten das Matrixprotein Z von TCRV und JUNV als Auslöser für die Apoptose, was wiederrum die zuvor aufgedeckte Fähigkeit beider Viren, den intrinsischen Weg zu induzieren, bestätigt. In Übereinstimmung mit dem beschriebenen Modell, in dem JUNV Caspase Aktivität mit NP blockieren kann, konnte die Caspase-Spaltung durch die Ko-Expression von JUNV NP und Z aufgehoben werden, während eine Ko-Expression von TCRV NP und Z weiterhin Zelltod auslöste. Letztendlich konnte durch die Identifizierung der spezifischen apoptotischen Faktoren (Bad, Puma und Noxa), die an der TCRV-induzierten Apoptose beteiligt sind und durch die Generierung jeweiliger Knockout-Zellen der Einfluss der Apoptose auf die virale Replikation untersucht werden. Wachstums-Kinetiken zeigten, dass weder eine verstärkte noch eine abgeschwächte Apoptose-Induktion in Vero-Zellen einen Einfluss auf das Wachstum von TCRV hat. Interessanterweise wiesen jedoch TCRV Partikel, die auf den unterschiedlichen Knockout-Zellen angezogen wurden, eine veränderte Infektiosität in primären humanen Monozyten und Makrophagen auf, die wiederum wichtige Zielzellen für Arenaviren darstellen. Da zu erwarten ist, dass TCRV-Partikel, die auf Bad-, Puma- oder Noxa-Knockout Zellen angezogen wurden, unterschiedliche Mengen an PS in ihrer Hülle eingebaut haben (abhängig vom Ausmaß der stattfindenden Apoptose), deutet dies auf eine mögliche Rolle für PS-Rezeptoren und/oder PS-Rezeptor-Signalweiterleitung vermittelt durch diverse nachgeschaltete Kinasen hin, die eine wichtige Rolle während des Eintritts-Mechanismus spielen könnten. Auch die in dieser Arbeit identifizierten Phosphorylierungen der apoptotischen Faktoren deuten auf die Beteiligung vorgeschalteter Kinasen verschiedener Signalwege hin, von denen einige auch eine Rolle bei der Regulierung der Zytokinproduktion spielen - eine weitere Wirtszellreaktion, die sich zwischen TCRV- und JUNV-infizierten Monozyten und Makrophagen deutlich unterscheidet. Demzufolge stellen diese Ergebnisse eine spannende Grundlage für eine mögliche Verbindung zwischen der Regulation der pro- oder anti-inflammatorischen Zytokin-Antwort, und der apoptotischen Signaltransduktion über der damit verbundenen mechanistisch vorgeschalteten Signalkaskade dar, und liefern somit wichtige Anhaltspunkte für weiterführende Studien, die dabei helfen sollen die Prozesse der Arenavirus-vermittelten Pathogenität besser verstehen zu können

Regulation of Stress-Activated Kinases in Response to Tacaribe Virus Infection and Its Implications for Viral Replication

Arenaviruses include important zoonotic pathogens that cause hemorrhagic fever (e.g., Junín virus; JUNV) as well as other viruses that are closely related but apathogenic (e.g., Tacaribe virus; TCRV). We have found that, while TCRV and JUNV differ in their ability to induce apoptosis in infected cells, due to active inhibition of caspase activation by the JUNV nucleoprotein, both viruses trigger similar upstream pro-apoptotic signaling events, including the activation/phosphorylation of p53. In the case of TCRV, the pro-apoptotic factor Bad is also phosphorylated (leading to its inactivation). These events clearly implicate upstream kinases in regulating the induction of apoptosis. Consistent with this, here we show activation in TCRV-infected cells of the stress-activated protein kinases p38 and JNK, which are known to regulate p53 activation, as well as the downstream kinase MK2 and transcription factor c-Jun. We also observed the early transient activation of Akt, but not Erk. Importantly, the chemical inhibition of Akt, p38, JNK and c-Jun all dramatically reduced viral growth, even though we have shown that inhibition of apoptosis itself does not. This indicates that kinase activation is crucial for viral infection, independent of its downstream role in apoptosis regulation, a finding that has the potential to shed further light on the determinants of arenavirus pathogenesis, as well as to inform future therapeutic approaches

BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis.

Pathogenicity often differs dramatically among even closely related arenavirus species. For instance, Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF), is closely related to Tacaribe virus (TCRV), which is normally avirulent in humans. While little is known about how host cell pathways are regulated in response to arenavirus infection, or how this contributes to virulence, these two viruses have been found to differ markedly in their ability to induce apoptosis. However, details of the mechanism(s) governing the apoptotic response to arenavirus infections are unknown. Here we confirm that TCRV-induced apoptosis is mitochondria-regulated, with associated canonical hallmarks of the intrinsic apoptotic pathway, and go on to identify the pro- and anti-apoptotic Bcl-2 factors responsible for regulating this process. In particular, levels of the pro-apoptotic BH3-only proteins Noxa and Puma, as well as their canonical transcription factor p53, were strongly increased. Interestingly, TCRV infection also led to the accumulation of the inactive phosphorylated form of another pro-apoptotic BH3-only protein, Bad (i.e. as phospho-Bad). Knockout of Noxa or Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad increased apoptosis, confirming that these factors are key regulators of apoptosis induction in response to TCRV infection. Further, we found that while the highly pathogenic JUNV does not induce caspase activation, it still activated upstream pro-apoptotic factors, consistent with current models suggesting that JUNV evades apoptosis by interfering with caspase activation through a nucleoprotein-mediated decoy function. This new mechanistic insight into the role that individual BH3-only proteins and their regulation play in controlling apoptotic fate in arenavirus-infected cells provides an important experimental framework for future studies aimed at dissecting differences in the apoptotic responses between arenaviruses, their connection to other cell signaling events and ultimately the relationship of these processes to pathogenesis

Complete genome sequence of Tacaribe virus

Tacaribe virus (TCRV) is the prototype of the New World arenaviruses (also known as TCRV serocomplex viruses). While TCRV is not itself a human pathogen, many closely related members of this group cause hemorrhagic fever, and thus TCRV has long served as an important BSL2 system for research into diverse areas of arenavirus biology. Due to its widespread use, a coding-complete sequence for both the S and L segments of the bipartite genome has been publically available for almost 30 years. However, more recently, this sequence has been found to contain significant discrepancies compared to other samples of the same original strain (i.e., TRVL-11573). Further, it is incomplete with respect to the genome ends, which contain critical regulatory elements for RNA synthesis. In order to rectify these issues we now present the first complete genome sequence for this important prototype arenavirus. In addition to completing the S segment 5’ end, we identified an apparent error in the L segment 3’ end as well as substantial discrepancies in the S segment intergenic region likely to affect folding. Comparison of this sequence with existing partial sequences confirmed a 12-amino-acid deletion in GP, including putative glycosylation sites, and a 4-amino-acid exchange flanking the exonuclease domain of NP. Accounting for these corrections, the TRVL-11573 strain appears to be nearly identical to that isolated in Florida in 2012. The availability of this information provides a solid basis for future molecular and genetic work on this important prototype arenavirus

Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species.Fil: Leske, Anne. Friedrich Loeffler Institut; AlemaniaFil: Waßmann, Irke. Friedrich Loeffler Institut; AlemaniaFil: Schnepel, Kevin. Friedrich Loeffler Institut; AlemaniaFil: Shifflett, Kyle. National Institutes of Health; Estados UnidosFil: Holzerland, Julia. Friedrich Loeffler Institut; AlemaniaFil: Bostedt, Linus. Friedrich Loeffler Institut; AlemaniaFil: Bohn, Patrick. Friedrich Loeffler Institut; AlemaniaFil: Mettenleiter, Thomas C.. Friedrich Loeffler Institut; AlemaniaFil: Briggiler, Ana Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Brignone, Julia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Enria, Delia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Hoenen, Thomas. Friedrich Loeffler Institut; Alemania. National Institutes of Health; Estados UnidosFil: Groseth, Allison. Friedrich Loeffler Institut; Alemania. National Institutes of Health; Estados Unido