An Equine Herpesvirus Type 1 (EHV-1) Expressing VP2 and VP5 of Serotype 8 Bluetongue Virus (BTV-8) Induces Protection in a Murine Infection Model

Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economic losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) started in northern Europe that caused significant disease in cattle and sheep before comprehensive vaccination was introduced two years later. Here, we evaluate the potential of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus, as a novel vectored DIVA (differentiating infected from vaccinated animals) vaccine expressing VP2 of BTV-8 alone or in combination with VP5. The EHV-1 recombinant viruses stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferon-receptor-deficient (IFNAR−/−) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a DIVA marker. In summary, we show that EHV-1 expressing BTV-8 VP2 and VP5 is capable of eliciting a protective immune response that is distinguishable from that after infection and as such may be an alternative for BTV vaccination strategies in which DIVA compatibility is of importance

Immunevasion und Vektorentwicklung

Equine herpesvirus type 1 (EHV-1) remains a severe threat to horse industry despite widespread vaccination. The control of EHV-1 infection is mainly dependent on cellular immunity that is mediated by CD8+ cytotoxic T lymphocytes (CTLs). EHV-1, as its relatives in the large Herpesviridae family, has evolved strategies to evade CTL immunity by interfering with the major histocompatibility complex class I (MHC-I) antigen presentation pathway. In the first part of this thesis, we identified a novel immunomodulatory protein involved in the downregulation of MHC-I from the cell surface. The responsible viral protein, pUL56, which is encoded by EHV-1 open reading frame 1 (ORF1), is a phosphorylated early protein, which is expressed as different forms and predominantly localizes to Golgi-derived membranes. In addition, the transmembrane (TM) domain of pUL56 was shown to be indispensable for correct subcellular localization and proper function. The function of pUL56 was independent of that mediated by pUL49.5, a viral protein known to inhibit the transporter associated with antigen processing (TAP) and encoded by EHV-1 and related viruses. Surprisingly, pUL56 by itself was not capable of downregulating MHC-I and likely needs (an)other unidentified viral protein(s) to perform this action. EHV-1 has recently been demonstrated to be a promising viral vehicle for delivery of foreign antigens. In the second part of the thesis, we utilized ORF1 as the locus for the insertion of foreign genes, more specifically the VP2 and/or VP5 genes of bluetongue virus serotype 8 (BTV-8). BTV-8 can infect most domestic and wild ruminants species and was responsible for an epizootic in northern Europe in 2006. The EHV-1 recombinant viruses generated stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferonreceptor- deficient (IFNAR-/-) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a marker for differentiating infected from vaccinated animals (DIVA). In conclusion, EHV-1 ORF1-encoded pUL56 is a novel immune evasion protein involved in the interference of MHC-I surface expression, but is unable to perform its function outside of the context of viral infection. An EHV-I recombinant carrying VP2 and VP5 of BTV-8 in the ORF1 locus is capable of eliciting protective immunity in a murine infection model and as such may be an alternative for BTV vaccination strategies.Das equine Herpesvirus 1 (EHV-1) ist, trotz routinemäßiger Impfung, auch heutzutage ein großes Problem in der Pferdehaltung. EHV-1-Infektionen werden hauptsächlich durch die zelluläre Immunantwort kontrolliert, getragen durch CD8+ zytotoxische T-Lymphozyten (cytotoxic T lymphocytes, CTL). EHV-1, wie auch weitere Vertreter der großen Herpesviridae-Familie, hat Strategien entwickelt, diese CTL-vermittelte Immunantwort zu umgehen, indem es die Antigenpräsentation durch den Hauptgewebeverträglichkeitskomplex Klasse I (major histocompatibility complex class I, MHC-I) stört. Im Rahmen dieser Doktorarbeit wurde ein immunmodulatorisches Protein identifiziert, welches an der Unterdrückung der MHC-I-Lokalisierung an der Zellmembran beteiligt ist. Das verantwortliche virale Protein pUL56, kodiert im offenen Leserahmen (open reading frame, ORF) EHV-1 ORF1, ist ein phosphoryliertes frühes Protein, mit unterschiedlichen Translationsvarianten, das vornehmlich an Membranen des Golgi-Apparates und davon gebildeten Vesikeln lokalisiert ist. Außerdem wurde gezeigt, daß die Transmembrandomäne von pUL56 essentiell für die korrekte Funktionalität und subzelluläre Lokalisierung des Proteins ist. Unabhängig davon wird die Funktion von pUL56 auch von pUL49.5 vermittelt, einem viralen Protein, das den Transporter der Antigenprozessierung (TAP) inhibiert und von EHV-1 und verwandten Viren kodiert wird. Überraschenderweise ist pUL56 allein nicht in der Lage, MHC-I herunterzuregulieren, daher benötigt es wahrscheinlich weitere, bisher unidentifizierte, virale Proteine für diese Funktion. EHV-1 ist, wie kürzlich demonstriert, ein vielversprechender viraler Vektor für fremde Antigene. Im zweiten Teil dieser Doktorarbeit wurde der ORF1-Genort benutzt, um dort fremde Gene einzufügen, spezifisch die VP2- und/oder VP5-Gene des Blauzungenkrankheitvirus (blue tongue virus, BTV) Serotyp 8. BTV-8 infiziert die meisten domestizierten und freilebenden Wiederkäuer und war für einen Seuchenausbruch in Nordeuropa 2008 verantwortlich. Die konstruierten EHV-1-Rekombinanten exprimieren stabil die entsprechenden Transgene und zeigen mit dem Wildtyp identische Wachstumskurven in vitro. Immunisierung von Mäusen mit transgenem Virus führte zur Bildung BTV-8-spezifischer Antikörper. In einem Infektionsversuch mit einer tödlichen Dosis BTV-8 an Interferon-A-Rezeptor-defizienten (IFNAR-/-) Mäusen überlebten 100% der mit VP2/VP5-doppelttransgenem EHV-1 geimpften Tiere, aber keines der mit VP2- einfachtransgenem EHV-1 geimpften. VP7 wurde in den transgenen Impfstoffen nicht verwendet und ist erfolgreich als Unterscheidungsmerkmal infizierter und geimpfter Tiere (marker for differentiating infected from vaccinated animals, DIVA) benutzt worden. Zusammengefaßt kann gesagt werden, daß das vom EHV-1 ORF1 kodierte Protein pUL56 ein bisher unbekannter Faktor ist, mit dem das Virus der Immunantwort ausweicht. pUL56 ist an der Störung der MHC-I-Oberflächenexpression beteiligt, kann aber seine Funktion nicht außerhalb des viralen Kontextes ausüben. Ein rekombinantes EHV-1, das VP2 und VP5 vom BTV-8 im ORF1-Genort trägt, löst im Mausmodell protektive Immunität aus und kann daher eine mögliche Alternative zu bisherigen BTV-8-Impfstrategien sein

Identification and characterization of equine herpesvirus type 1 (EHV-1) pUl56 and its role in virus-induced downregulation of major histocompatibility complex class I

Major histocompatibility complex class I (MHC- I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the Herpesviridae have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by ORF1 of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein which is expressed as different forms and predominantly localizes to Golgi membranes. In addition, the transmembrane (TM) domain of the type II membrane protein was shown to be indispensable for correct subcellular localization and a proper function. pUL56 by itself is not functional with respect to interference with MHC- I and likely needs another unidentified viral protein(s) to perform this action. Surprisingly, pUL49.5, an inhibitor of the transporter associated with antigen processing (TAP) and encoded by EHV-1 and related viruses, appeared not to be required for pUL56-induced early MHC-I downmodulation in infected cells. In conclusion, our data identify a new immunomodulatory protein, pUL56, involved in MHC- I downregulation which is unable to perform its function outside the context of viral infection

An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep

Abstract Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause serious and fatal disease in humans and animals. RVFV is a negative-sense RNA virus of the Phlebovirus genus in the Bunyaviridae family. The main envelope RVFV glycoproteins, Gn and Gc, are encoded on the M segment of RVFV and known inducers of protective immunity. In an attempt to develop a safe and efficacious RVF vaccine, we constructed and tested a vectored equine herpesvirus type 1 (EHV-1) vaccine that expresses RVFV Gn and Gc. The Gn and Gc genes were custom-synthesized after codon optimization and inserted into EHV-1 strain RacH genome. The rH_Gn-Gc recombinant virus grew in cultured cells with kinetics that were comparable to those of the parental virus and stably expressed Gn and Gc. Upon immunization of sheep, the natural host, neutralizing antibodies against RVFV were elicited by rH_Gn-Gc and protective titers reached to 1:320 at day 49 post immunization but not by parental EHV-1, indicating that EHV-1 is a promising vector alternative in the development of a safe marker RVFV vaccine

The studies on gas adsorption properties of MIL-53 series MOFs materials

Molecular dynamics (MD), grand canonical Monte Carlo (GCMC) and ideal adsorbed solution theory (IAST) were used to study the structures and gas adsorption properties of MIL-53(M)[M=Cr, Fe, Sc, Al] metal organic framework (MOF) materials. The results show that the volumes of those MOF materials increase significantly at high temperature. By analyzing the adsorption isotherms, we found that the temperature had a paramount effect on the gas adsorption behaviors of these MOF materials. For MIL-53(Cr), the orders of the quantities of adsorbed gases were CH4>N2>CO2>H2S, CH4>H2S>CO2>N2 and CH4>CO2>H2S>N2 at 100K, 293K and 623K, respectively. We also calculated the adsorption of several combinations of two gases by MIL-53(Cr) at 293K, the results indicate that the material had selective adsorption of CH4 over CO2, H2S and N2. Our calculations provide microscopic insights into the gas adsorption performances of these MOFs and may further guide the practice of gas separation

Self-assembled structure of sulfonic gemini surfactant solution

Sulfonate gemini surfactant is a new type of anionic gemini surfactant. The unique structure of double sulfonate endows the sulfonate gemini surfactant with superior surfactant properties, including lower critical micelle concentration (CMC), unusual decontamination ability, excellent stability in strong acid/alkali solution. In this paper, the self-assembled structure of gemini dodecyl sulfonate sodium, abbreviated as 12-2-12(SO3Na)2, is studied by using of dissipative particle dynamics (DPD) method. We have constructed a spring structure model of surfactant molecules, and the effect of length hydrophobic chain, the concentration of surfactants, ethanol addictive on the self-assembly behavior and critical micelle concentration (CMC) was investigated. The results show that with the increase of the concentration of surfactants in aqueous solution, spherical, wormlike and layered micelles appear in turn. With the increase of the length of the hydrophobic chain, the clusters of the surfactants become tighter and the larger clusters are presented at the lower concentration. It was found that the addition of ethanol molecule can enhance the solubility of hydrophobic group and thus inhibit the formation of the micelles

Schematic illustration of the construction strategies of the recombinant viruses.

<p>(A) The organization of the left terminus of the EHV-1 RacH genome showing that ORF1 and ORF2 are absent. UL: unique long; US: unique short; IR: internal repeat; TR: terminal repeat. (B) A fragment released from transfer plasmid pEP-VP2 by <i>I-Ceu</i>I digestion was used to recombine with RacH genome, resulting in an intermediate kanamycin (<i>aphAI</i> cassette)-resistant BAC clone. After <i>I-Sce</i>I digestion, kanamycin was removed in the following step of <i>en passant</i> mutagenesis (in box) to generate VP2-expressing virus. (C) With another round of <i>en passant</i> mutagenesis, VP5 gene with an IRES sequence upstream were inserted in between VP2 and BGH polyA, and a final construct expressing both VP2 and VP5 (D) was generated.</p