Iris Delrue

The unfolded-protein-response sensor IRE-1α regulates the function of CD8α + dendritic cells

The role of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in homeostasis of the immune system is incompletely understood. Here we found that dendritic cells (DCs) constitutively activated the UPR sensor IRE-1α and its target, the transcription factor XBP-1, in the absence of ER stress. Loss of XBP-1 in CD11c+ cells led to defects in phenotype, ER homeostasis and antigen presentation by CD8α+ conventional DCs, yet the closely related CD11b+ DCs were unaffected. Whereas the dysregulated ER in XBP-1-deficient DCs resulted from loss of XBP-1 transcriptional activity, the phenotypic and functional defects resulted from regulated IRE-1α-dependent degradation (RIDD) of mRNAs, including those encoding CD18 integrins and components of the major histocompatibility complex (MHC) class I machinery. Thus, a precisely regulated feedback circuit involving IRE-1α and XBP-1 controls the homeostasis of CD8α+ conventional DCs.

Porcine Arterivirus Attachment to the Macrophage-Specific Receptor Sialoadhesin Is Dependent on the Sialic Acid-Binding Activity of the N-Terminal Immunoglobulin Domain of Sialoadhesin

ABSTRACT The sialic acid-binding lectin sialoadhesin (Sn) is a macrophage-restricted receptor for porcine reproductive and respiratory syndrome virus (PRRSV). To investigate the importance of pSn sialic acid-binding activity for PRRSV infection, an R116-to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSnRE, that could not bind sialic acids. PSn, but not pSnRE, allowed PRRSV binding and internalization. These data show that the sialic acid-binding activity of pSn is essential for PRRSV attachment to pSn and thus identifies the variable, N-terminal domain of Sn as a PRRSV binding domain.

Development of an experimental inactivated PRRSV vaccine that induces virus-neutralizing antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV) can induce reproductive disorders and is involved in the porcine respiratory disease complex, causing tremendous economic losses to the swine industry. Inactivated PRRSV vaccines are preferred over attenuated vaccines because of their safety and flexibility towards emerging virus strains, but the efficacy of current inactivated PRRSV vaccines is questionable. In this study, experimental inactivated PRRSV vaccines were developed, based on two formerly optimized inactivation procedures: UV irradiation and treatment with binary ethylenimine (BEI). In a first experiment, it was shown that vaccination with UV- or BEI-inactivated virus in combination with Incomplete Freunds Adjuvant induced virus-specific antibodies and strongly primed the virus-neutralizing (VN) antibody response. Subsequently, the influence of adjuvants on the immunogenicity of neutralizing epitopes on the inactivated virus was investigated. It was shown that vaccination with BEI-inactivated virus in combination with a commercial oil-in-water adjuvant induced high titers (3.4 log(2)) of VN antibodies in 6/6 pigs, instead of only priming the neutralizing antibody response. After challenge, neutralizing antibody titers in these vaccinated animals rose to a mean value of 5.5 log(2), and the duration of the viremia was reduced to an average of 1 week. This study shows that, by the use of an optimized inactivation procedure and a suitable adjuvant, inactivated PRRSV vaccines can be developed that induce VN antibodies and offer partial protection upon challenge.

Susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and CD163

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV.ResultsDetailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHOSn-CD163 and PK15Sn-CD163 cells. Initially, this occurred less efficiently for macrophage grown virus than for Marc-145 grown virus. Upon internalization, disassembly of the virus particles was observed. The two cell lines could be infected with PRRSV strains LV and VR-2332. However, it was observed that Marc-145 grown virus infected the cells more efficiently than macrophage grown virus. If the cells were treated with neuraminidase to remove cis-acting sialic acids that hinder the interaction of the virus with Sn, the amount of infected cells with macrophage grown virus increased. Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line. Only 2 out of 5 PRRSV strains replicated well in CHOSn-CD163 cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15Sn-CD163 cells was similar to the virus titer of those strains produced in Marc-145 cells. Analysis of the sequence of the structural proteins of original virus and virus grown for 5 passages on PK15Sn-CD163 cells showed either no amino acid (aa) changes (VR-2332 and 07V063), one aa (LV), two aa (08V194) or three aa (08V204) changes. None of these changes are situated in known neutralizing epitopes.ConclusionsA PRRSV susceptible cell line was constructed that can grow virus to similar levels compared to currently available cell lines. Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes. Together, the results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation.

Assessing the functionality of viral entry-associated domains of porcine reproductive and respiratory syndrome virus during inactivation procedures, a potential tool to optimize inactivated vaccines

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses in the pig industry worldwide. Currently, vaccines based on inactivated PRRSV provide limited protection of pigs against infection, most likely because viral epitopes associated with the induction of neutralizing antibodies are not or poorly conserved during inactivation. To analyze the effect of inactivation procedures on the interaction of PRRSV with receptors involved in virus entry, a new assay was set up in this study. Viral entry-associated domains are most likely important for the induction of neutralizing antibodies, since neutralizing antibodies block interaction of PRRSV with cellular receptors. To investigate the interaction of PRRSV with the cellular receptors upon different inactivation procedures, attachment to and internalization of inactivated PRRSV into macrophages were monitored. AT-2 could not inactivate PRRSV completely and is therefore not useful for vaccine development. PRRSV inactivated with ultraviolet light, binary ethyleneimine and gamma irradiation, which all mainly have an effect at the genomic level, showed no difference compared to control live virus at all levels of virus entry, whereas PRRSV treated with formaldehyde, glutaraldehyde and pH changes, which all have a modifying effect on proteins, was not able to internalize into macrophages anymore. These results suggest that inactivation with methods with a main effect on the viral genome preserve PRRSV entry-associated domains and are useful for future development of an effective inactivated vaccine against PRRSV. Although PRRSV incubation at 37 degrees C can completely inactivate PRRSV with preservation of entry-associated domains, this method is not recommended for vaccine development, since the mechanism is yet unknown.

Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges

The aim of this review is to make researchers aware of the benefits of an efficient quality control system for prediction of a developed vaccine’s efficacy. Two major goals should be addressed when inactivating a virus for vaccine purposes: first, the infectious virus should be inactivated completely in order to be safe, and second, the viral epitopes important for the induction of protective immunity should be conserved after inactivation in order to have an antigen of high quality. Therefore, some problems associated with the virus inactivation process, such as virus aggregate formation, protein crosslinking, protein denaturation and degradation should be addressed before testing an inactivated vaccine in vivo.

A real-time fluorometric method for the simultaneous detection of cell death type and rate

Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is ∼2 h.