Development of a new inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for reproduction disorders in sows, which leads to tremendous economic losses in the pig industry worldwide. It is also associated with porcine respiratory disease complex (PRDC), which supports massive use of antibiotics. Although several modified live virus (MLV) and killed virus (KV) vaccines are on the market, PRRSV remains difficult to control in the field. The major aim of this thesis was to develop a new experimental inactivated PRRSV vaccine. The different processes to develop a vaccine are virus production, virus inactivation or attenuation and vaccine formulation. In this thesis, the virus production and the virus inactivation of PRRSV were optimized to develop an efficient KV vaccine. The first section of chapter 1 gives an introduction on PRRSV, more particular its history, classification, virus structure, genomic organization, viral proteins, replication cycle, clinical signs, pathogenesis, innate and adaptive immune response to PRRSV and the available vaccines. A second section of chapter 1 gives an overview of different viral inactivation procedures, such as cross-linkers (formaldehyde, glutaraldehyde and AT-2), denaturing agents (pH and temperature), irradiation (ultraviolet (UV) and gamma irradiation) and alkylating agents (binary ethyleneimine (BEI)), and their effect on viruses

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 Freund's 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

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. <it>In vivo</it>, the virus infects a subpopulation of tissue macrophages. <it>In vitro</it>, 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 (CHO^Sn-CD163and PK15^Sn-CD163) and evaluated their potential for production of PRRSV.</p> <p>Results</p> <p>Detailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHO^Sn-CD163and PK15^Sn-CD163cells. 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 PK15^Sn-CD163cell line gave in general better results than the CHO^Sn-CD163cell line. Only 2 out of 5 PRRSV strains replicated well in CHO^Sn-CD163cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15^Sn-CD163cells 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 PK15^Sn-CD163cells 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.</p> <p>Conclusions</p> <p>A 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.</p

Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity

SummarySuccessful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. We have developed a genetic model in which direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release damage-associated molecular patterns and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy

Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages

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 $^{\circ}$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

Improvement of malt modification by use of Rhizopus VII as starter culture

The development of a selected starter culture on malting barley and its effects on malt quality aspects were studied. Application of Rhizopus sporangiospores in a malting process resulted in increased beta-glucanase and xylanase contents of the malting barley and improved starchy endosperm cell-wall degradation. Activation of the sporangiospores and optimization of the inoculation procedure led to a further increase in enzyme levels and to larger and more consistent impacts on cell-wall modification. Whereas the main effect of the starter culture on beta-glucan degradation was observed during malting, a further decrease in beta-glucan during mashing suggests that the microbial enzymes that survived the kilning step were active during mashing. Other quality aspects that were influenced by the starter culture activity were protein modification, wort color, and wort pH. The level of microbial enzymes produced was related to the amount of barley kernels infected with the starter culture.status: publishe