Inactivated Porcine Reproductive And Respiratory Syndrome Virus Research Articles

Integrated microbiome and metabolome analysis reveals that new insight into Radix pseudostellariae polysaccharide enhances PRRSV inactivated vaccine.

In this study, we investigated how Radix pseudostellariae polysaccharide (RPP) enhances the immune response of the inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine through interactions with the microbiome and metabolome. We pretreated sows with 10 mg/kg body weight of RPP via drinking water for 7 days prior to intramuscular injection of the PRRSV vaccine. This significantly increased the concentrations of PRRSV GP5 protein antibody, interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ. Oral administration of RPP also significantly improved the abundance of beneficial bacteria in the stool, such as Parabacteroides distasonis, Prevotella_copri, Eubacterium_sp., and Clostridium_sp._CAG:226, and decreased the levels of potentially pathogenic bacteria, such as Paraeggerthella and [Clostridium] innocuum, compared to the vaccine alone. These bacterial changes were confirmed using quantitative real-time polymerase chain reaction (Q-PCR). Moreover, RPP treatment significantly increased the blood concentrations of L-theanine, taurodeoxycholic acid (TDCA), and N-arachidonoyl proline, and decreased the levels of L-glutamine, oclacitinib, lipoxin C4, and leukotriene C5 in sows after immunization (p< 0.05). The concentrations of various blood metabolites were validated using sandwich enzyme-linked immunosorbent assay (ELISA), confirming the accuracy of the metabolomics data. Intriguingly, the integration of microbiome and metabolome analyses highlighted the significance of Prevotella_copri and TDCA. We consequently developed a mouse immunity model using GP5 protein and discovered that oral administration of RPP significantly enhanced the levels of GP5 protein antibodies, IL-2, IL-4, IL-10, and IFN-γ in mouse serum. It also increased the number of CD3+ and CD3+CD4+ cells in the spleen. Additionally, Prevotella_copri was administered into the large intestine via the anus for 7 days prior to the intramuscular injection of the PRRSV GP5 protein. The results demonstrated a significant increase in TDCA and GP5 antibody concentration in the mouse serum, indicating that RPP modulates Prevotella_copri to elevate its metabolite TDCA, thereby enhancing the GP5 antibody level. In conclusion, oral administration of 10 mg/kg RPP optimizes gut flora diversity and blood metabolites, particularly Prevotella_copri and TDCA, thereby improving the immune response to the inactivated PRRSV vaccine.

Development of a Ferritin Protein Nanoparticle Vaccine with PRRSV GP5 Protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit nanovaccines is a promising strategy for preventing PRRSV variant infections. In this study, two different types of ferritin (Ft) nanovaccines targeting the major glycoprotein GP5, named GP5m-Ft and (Bp-IVp)3-Ft, were constructed and evaluated as vaccine candidates for PRRSV. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) demonstrated that both purified GP5m-Ft and (Bp-IVp)3-Ft proteins could self-assemble into nanospheres. A comparison of the immunogenicity of GP5m-Ft and (Bp-IVp)3-Ft with an inactivated PRRSV vaccine in BALB/c mice revealed that mice immunized with GP5m-Ft exhibited the highest ELISA antibody levels, neutralizing antibody titers, the lymphocyte proliferation index, and IFN-γ levels. Furthermore, vaccination with the GP5m-Ft nanoparticle effectively protected piglets against a highly pathogenic PRRSV challenge. These findings suggest that GP5m-Ft is a promising vaccine candidate for controlling PRRS.

An attenuated herpesvirus vectored vaccine candidate induces T-cell responses against highly conserved porcine reproductive and respiratory syndrome virus M and NSP5 proteins that are unable to control infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response.

Intranasal delivery of inactivated PRRSV loaded cationic nanoparticles coupled with enterotoxin subunit B induces PRRSV-specific immune responses in pigs

This study was conducted to evaluate the induction of systemic and mucosal immune responses and protective efficacy following the intranasal administration of inactivated porcine reproductive and respiratory syndrome virus (PRRSV) loaded in polylactic acid (PLA) nanoparticles coupled with heat-labile enterotoxin subunit B (LTB) and dimethyldioctadecylammonium bromide (DDA). Here, 42- to 3-week-old PRRSV-free pigs were randomly allocated into 7 groups of 6 pigs each. Two groups represented the negative (nonvaccinated pigs/nonchallenged pigs, NoVacNoChal) and challenge (nonvaccinated/challenged, NoVacChal) controls. The pigs in the other 5 groups, namely, PLA nanoparticles/challenged (blank NPs), LTB-DDA coupled with PLA nanoparticles/challenged (adjuvant-blank NPs), PLA nanoparticles-encapsulating inactivated PRRSV/challenged (KNPs), LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV/challenged pigs (adjuvant-KNPs) and inactivated PRRSV/challenged pigs (inactivated PRRSV), were intranasally vaccinated with previously described vaccines at 0, 7 and 14 days post-vaccination (DPV). Serum and nasal swab samples were collected weekly and assayed by ELISA to detect the presence of IgG and IgA, respectively. Viral neutralizing titer (VNT) in sera, IFN-γ-producing cells and IL-10 secretion in stimulated peripheral blood mononuclear cells (PBMCs) were also measured. The pigs were intranasally challenged with PRRSV-2 at 28 DPV and necropsied at 35 DPV, and then macro- and microscopic lung lesions were evaluated. The results demonstrated that following vaccination, adjuvant-KNP-vaccinated pigs had significantly higher levels of IFN-γ-producing cells, VNT and IgG in sera, and IgA in nasal swab samples and significantly lower IL-10 levels than the other vaccinated groups. Following challenge, the adjuvant-KNP-vaccinated pigs had significantly lower PRRSV RNA and macro- and microscopic lung lesions than the other vaccinated groups. In conclusion, the results of the study demonstrated that adjuvant-KNPs are effective in eliciting immune responses against PRRSV and protecting against PRRSV infections over KNPs and inactivated PRRSV and can be used as an adjuvant for intranasal PRRSV vaccines.

Efficacy of the Synergy Between Live-Attenuated and Inactivated PRRSV Vaccines Against a NADC30-Like Strain of Porcine Reproductive and Respiratory Syndrome Virus in 4-Week Piglets.

The NADC30-like strain of porcine reproductive and respiratory syndrome virus (PRRSV) is a novel strain responsible for substantial economic losses to swine production in China. This study evaluated the cross-protective efficacy of the synergy between live-attenuated and inactivated PRRSV vaccines compared with a single vaccination with PRRS modified-live virus (MLV) vaccine against challenge with NADC30-like strain, v2016/ZJ/09-03. A total of 45 PRRSV free pigs were randomly divided into five groups: (1) strict control (SC); (2) positive control (PC); (3) single MLV dose (M1); (4) primed intramuscularly with MLV and boosted with killed vaccine 3 weeks later (MK1); and (5) intramuscular prime MLV boosted subcutaneously with killed vaccine B 3 weeks later (MK2). Serological tests in MK groups revealed no differences in both anti-N and anti-GP protein antibodies compared with M1 group, and failed to provide further protection against clinical signs, virus shedding, and gross lesions. However, the viremic titer, gross lung lesions, and average daily weight gain were significantly improved in the MLV vaccinated groups, suggesting that MLV provides substantial cross-protection against the NADC30-like virus. Thus, as a booster, the killed vaccine confers minimal additional protection in NADC30-like infected piglets.

A porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM as a novel adjuvant for an inactivated PRRSV vaccine improves protection efficiency and enhances cell-mediated immunity against heterologous PRRSV challenge

Current strategies for porcine reproductive and respiratory syndrome (PRRS) control are inadequate and mainly restricted to immunization using different PRRS virus (PPRSV) vaccines. Although there are no safety concerns, the poor performance of inactivated PRRSV vaccines has restricted their practical application. In this research, we employed the novel PRRSV-specific IgM monoclonal antibody (Mab)-PR5nf1 as a vaccine adjuvant for the formulation of a cocktail composed of inactivated PRRSV (KIV) and Mab-PR5nf1 along with a normal adjuvant to enhance PRRSV-KIV vaccine-mediated protection and further compared it with a normal KIV vaccine and modified live virus vaccine (MLV). After challenge with highly pathogenic (HP)-PRRSV, our results suggested that the overall survival rate (OSR) and cell-mediated immunity (CMI), as determined by serum IFN-γ quantification and IFN-γ ELISpot assay, were significantly improved by adding PRRSV-specific IgM to the PRRSV-KIV vaccine. It was also notable that both the OSR and CMI in the Mab-PR5nf1-adjuvanted KIV group were even higher than those in the MLV group, whereas the CMI response is normally poorly evoked by KIV vaccines or subunit vaccines. Compared with those in piglets immunized with the normal KIV vaccine, viral shedding and serum neutralizing antibody levels were also improved, and reduced viral shedding appeared to be a result of enhanced CMI caused by the inclusion of IgM as an adjuvant. In conclusion, our data provide not only a new formula for the development of an effective PRRSV-KIV vaccine for practical use but also a novel method for improving antigen-specific CMI induction by inactivated vaccines and subunit vaccines.

Mannosylated gelatin nanoparticles enhanced inactivated PRRSV targeting dendritic cells and increased T cell immunity

The objective of the present work was to evaluate the efficacy of a novel antigen carrier using mannosylated gelatin nanoparticles with entrapped inactivated porcine reproductive and respiratory syndrome virus (PRRSV) in inducing T cell mediated immunity in vitro. Gelatin nanoparticles (GNP) were modified with mannose to form mannosylated gelatin nanoparticles (MnGNP), which can efficiently and specifically target monocyte derived dendritic cells (MoDCs). The inactivated PRRSV was encapsulated in the MnGNP and GNP, referred to as MnGNP-PRRSV and GNP-PRRSV, respectively. All these prepared nanometer particles were characterized for size, surface charge, drug encapsulation efficiency, and drug release. The efficacy of MnGNP in targeting MoDCs was investigated, as well as the subsequent MoDCs maturation and T cell mediated cytotoxicity. The developed MnGNP-PRRSV particle was characterized with a nanometric size of 302.67 ± 3.2 nm, surface charge of 23.81 ± 1.26 mV, and PRRSV encapsulation efficiency of 63.2 ± 1.85 %. The maximum uptake of MnGNP in MoDCs in vitro was 15.5 times higher than GNP with a shorter reaction time that peaked 4 h earlier. The uptake of MnGNP-PRRSV induced maturation of MoDCs and significantly enhanced expression of SWC-3a, CD80, CD1, SLA I, SLA II on MoDCs, compared to PRRSV (p < 0.001). The cytokine secretion of IL-1β, IL-6, IL-10, and IL-12 was also increased in MoDCs when treated with MnGNP-PRRSV, compared to PRRSV (p < 0.05). The matured MoDCs triggered T lymphocytes in autologous peripheral blood mononuclear cells (PBMCs) activation, proliferation, and differentiation into effector cytotoxic T lymphocyte, suggesting increased amount of activated T cells after MnGNP-PRRSV treatment. Additionally, the function of T cells to kill PRRSV infected cells was 83.98 ± 2.62 % when triggered by MnGNP-PRRSV, compared to 60 ± 4.7 % in PRRSV group (p < 0.001). These results indicate that MnGNP with entrapped inactivated PRRSV can effectively and specifically target dendritic cells for maturation and activation, and subsequently improve T cell activation, proliferation and function to kill PRRSV infected cells.

Escherichia coli Heat-Labile Enterotoxin B Subunit Combined with Ginsenoside Rg1 as an Intranasal Adjuvant Triggers Type I Interferon Signaling Pathway and Enhances Adaptive Immune Responses to an Inactivated PRRSV Vaccine in ICR Mice.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen that has threatened the global swine industry for almost 30 years. Because current vaccines do not provide complete protection, exploration of new preventive strategies is urgently needed. Here, we combined a heat-labile enterotoxin B subunit of Escherichia coli (LTB) and ginsenoside Rg1 to form an intranasal adjuvant and evaluated its enhancement of immune responses in mice when added to an inactivated-PRRSV vaccine. The combination adjuvant synergistically elicited higher neutralizing and non-neutralizing (immunoglobulin G and A) antibody responses in the circulatory system and respiratory tract, and enhanced T and B lymphocyte proliferation, CD4+ T-cell priming, and cytotoxic CD4+ T cell activities in mononuclear cells from spleen and lung tissues when compared to the PRRSV vaccine alone, and it resulted in balanced Th1/Th2/Th17 responses. More importantly, we observed that the combination adjuvant also up-regulated type I interferon signaling, which may contribute to improvement in adaptive immune responses. These results highlight the potential value of a combined adjuvant approach for improving the efficacy of vaccination against PRRSV. Further study is required to evaluate the efficacy of this combined adjuvant in swine.

Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in neonatal pigs using different adjuvants

Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Further research focused on the early immune response in draining lymph nodes is needed to elucidate the potential of TLR agonists in vaccines for neonatal pigs.

Immunization with a recombinant fusion of porcine reproductive and respiratory syndrome virus modified GP5 and ferritin elicits enhanced protective immunity in pigs

Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses in the swine industry worldwide. Live and inactivated vaccines have only been partially successful in generating protective immune responses. The PRRS virus (PRRSV) glycoprotein 5 (GP5) is a major viral antigenic target and is thus suitable for development of genetically engineered PRRSV vaccines. Here, a modified GP5 and ferritin were fused and expressed using a baculovirus system to generate a GP5m-ferritin nanoparticle vaccine. We demonstrated that the GP5m-ferritin vaccine elicited higher serum antibody titers in pigs than inactivated PRRSV. Moreover, immunization with GP5m-Ft promoted a Th1-dominant cellular immune response and enhanced specific T-lymphocyte immune responses. GP5m-ferritin-vaccinated pigs had significantly lower mean rectal temperatures, respiratory scores, viremia, and macroscopic and microscopic lung lesion scores post-challenge compared with unvaccinated pigs. These results indicated that GP5m-ferritin subunit vaccines can elicit specific protective immune responses and represent promising vaccine candidates.

The potential adjuvanticity of quaternized chitosan hydrogel based microparticles for porcine reproductive and respiratory syndrome virus inactivated vaccine

Infectious diseases possess a big threat to the livestock industry worldwide. Currently, inactivated veterinary vaccines have attracted much attention to prevent infection due to their safer profile compared to live attenuated vaccine. However, its intrinsic poor immunogenicity demands the incorporation of an adjuvant. Mineral oil based adjuvant (Montanide™ ISA206) was usually used to potentiate the efficacy of veterinary vaccines. However, ISA206 could not induce robust cellular immune responses, which was very important in controlling virus replication and clearing the infected cells. Moreover, mineral oil would result in severe side effects. To improve both the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (PRRSV) inactivated vaccine, we developed pH-sensitive and size-controllable quaternized chitosan hydrogel microparticles (Gel MPs) without using chemical cross linking agent. Gel MPs, ionic cross-linked with glycerophosphate (GP), were biocompatible and could efficiently adsorb the inactivated PRRSV vaccine with a loading capacity of 579.05μg/mg. After intramuscular immunization in mice, results suggested that Gel MPs elicited significantly higher cell-mediated immune responses and comparable humoral immune responses compared to ISA 206. Regarding the biocompatibility, safety and effectiveness, Gel MPs would be a promising candidate to enhance the efficacy of veterinary vaccine.

Inactivated porcine reproductive and respiratory syndrome virus vaccine adjuvanted with Montanide™ Gel 01 ST elicits virus-specific cross-protective inter-genotypic response in piglets

The efficacy of a novel BEI-inactivated porcine reproductive and respiratory syndrome virus (PRRSV) candidate vaccine in pigs, developed at RIBSP Republic of Kazakhstan and delivered with an adjuvant Montanide™ Gel 01 ST (D/KV/ADJ) was compared with a commercial killed PRRSV vaccine (NVDC-JXA1, C/KV/ADJ) used widely in swine herds of the Republic of Kazakhstan. Clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [ELISA and virus neutralizing (VN) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time PCR and cell culture assay) were assessed in vaccinated and both genotype 1 and 2 PRRSV challenged pigs. Our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, Kazakh strains of PRRSV type 1 and type 2 genotypes. In contrast, clinical protection, absence of viremia and lung lesions in D/KV/ADJ vaccinated pigs was associated with generation of VN antibodies in both homologous vaccine strain LKZ/2010 (PRRSV type 2) and a heterogeneous type 1 PRRSV strain (CM/08) challenged pigs. Thus, our data indicated the induction of cross-protective VN antibodies by D/KV/ADJ vaccine, and importantly demonstrated that an inactivated PRRSV vaccine could also induce cross-protective response across the viral genotype.

Downregulation of protein kinase PKR activation by porcine reproductive and respiratory syndrome virus at its early stage infection

The interferon-induced double-strand RNA activated protein kinase (PKR) plays an important role in antiviral response. The objective of this study was to assess the effect of porcine reproductive and respiratory syndrome virus (PRRSV) on PKR activation. Here we report that PRRSV inhibited PKR activation during its early stage infection of primary pulmonary alveolar macrophages (PAMs). PRRSV infection led to lower level of phosphorylated PKR in comparison with mock-infected cells. The PKR inhibition was sustained until 10h post infection in the presence of polyI:C, a synthetic analog of double-stranded RNA activating PKR. PKR-mediated phosphorylation of the eukaryotic translation initiation factor eIF2α was also lower in the PRRSV-infected PAMs during the early stage infection. Interestingly, inactivated PRRSV was capable to inhibit the PKR activation until 6h post infection. This suggests that structural components of PRRSV virions were responsible for the inhibition, although PRRSV replication was needed for longer inhibition. These results indicate that the downregulation of PKR activation during early infection stage should be essential for PRRSV to avoid the antiviral response to initiate replication. This finding contributes to our understanding on PRRSV interaction with host innate immune response and reveal a target for control of PRRSV infection.

Intracellular expression of an anti-idiotypic antibody single-chain variable fragment reduces porcine reproductive and respiratory syndrome virus infection in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome; it is one of the most economically important viral diseases affecting the swine industry worldwide. At present, neither live-attenuated nor inactivated PRRSV vaccines can provide sustainable disease control. Our previous studies have demonstrated that PRRSV infection can produce the auto-anti-idiotypic antibodies (aAb2s) specific to the idiotypic antibodies against PRRSV GP5, which plays an important role in the host immune responses to PRRSV infection. In the present study, a single-chain variable antibody fragment (scFv) from the monoclonal anti-idiotypic antibody specific for the idiotypic antibody against GP5 was expressed in MARC-145 cells and its effect on virus infection in vitro was evaluated. An scFv was constructed from the anti-idiotypic antibody (Mab2-5G2) and was named 5G2scFv. The lentiviral vector system was used as a vehicle to deliver 5G2scFv into MARC-145 cells. The effect of 5G2scFv expression in MARC-145 was analysed by determining the PRRSV N protein level and the virus titre in the supernatant. Virus attachment and the level of type I interferon (IFN) were determined to elucidate the mechanism of the scFv effect. 5G2scFv was delivered in MARC-145 cells using the lentiviral vector system as confirmed by the western blot and indirect immunofluorescence assays. The PRRSV challenge experiments demonstrated that expressed 5G2scFv in MARC-145 strongly reduced PRRSV infection and replication by inhibiting protein synthesis and progeny virus production. This effect was not due to the change of viability or virus binding, but increased IFN-α at messenger RNA and protein levels. The expression of the anti-idiotypic antibody 5G2scFv in MARC-145 cells has the interferential effect on PRRSV infection in the cells by induction of IFN-α, which provides a novel therapeutic approach for PRRSV infection.

Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry worldwide. Although inactivated and live vaccines are commercially available for the control of PRRS, both types of vaccine have not always proven successful in terms of generating a protective immune response, particularly in the case of inactivated vaccines. In this study, we tested whether an inactivated vaccine could induce a humoral immune response to PRRS during a homologous challenge. Amino acid substitutions were introduced into glycoprotein (GP) 5 of the FL12 strain of the PRRS virus (PRRSV) using site-directed mutagenesis with a pFL12 infectious clone. The substitutions led to double deglycosylation in the putative glycosylation moieties on GP5. The mutant virus was subsequently inactivated with binary ethylenimine. The efficacy of the inactivated mutant virus was compared with that of the inactivated wild-type PRRSV. Only the inactivated mutant PRRSV induced serum neutralizing antibodies at six weeks post-vaccination. The group that was administered the inactivated mutant virus twice exhibited a significantly increased neutralizing antibody titer after a challenge with the virulent homologous strain and exhibited more rapid clearing of viremia compared to other groups, including the groups that were administered either the inactivated mutant or wild-type virus only once and the group that was administered the inactivated wild-type virus twice. Histopathological examination of lung tissue sections revealed that the group that was administered the inactivated mutant virus twice exhibited significantly thinner alveolar septa, whereas the thickness of the alveolar septa of the other groups were markedly increased due to lymphocyte infiltration. These results indicated that the deglycosylation of GP5 enhanced the immunogenicity of the inactivated mutant PRRSV and that twice administrations of the inactivated mutant virus conferred better protection against the homologous challenge. These findings suggest that the inactivated PRRSV that expresses a hypo-glycosylated GP5 is a potential inactivated vaccine candidate and a valuable tool for controlling PRRS for the swine industry.

An innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating respiratory disease of pigs. The disease is caused by the PRRS virus (PRRSV), an Arterivirus which is a highly mutating RNA virus. Widely used modified live PRRSV vaccines have failed to prevent PRRS outbreaks and reinfections; moreover, safety of the live virus vaccines is questionable. Though poorly immunogenic, inactivated PRRSV vaccine is safe. The PRRSV infects primarily the lung macrophages. Therefore, we attempted to strengthen the immunogenicity of inactivated/killed PRRSV vaccine antigens (KAg), especially in the pig respiratory system, through: 1) entrapping the KAg in biodegradable poly(lactic-co-glycolic acid) nanoparticles (NP-KAg); 2) coupling the NP-KAg with a potent mucosal adjuvant, whole cell lysate of Mycobacterium tuberculosis (M. tb WCL); and 3) delivering the vaccine formulation twice intranasally to growing pigs. We have previously shown that a single dose of NP-KAg partially cleared the challenged heterologous PRRSV. Recently, we reported that NP-KAg coupled with unentrapped M. tb WCL significantly cleared the viremia of challenged heterologous PRRSV. Since PRRSV is primarily a lung disease, our goal in this study was to investigate lung viral load and various immune correlates of protection at the lung mucosal surfaces and its parenchyma in vaccinated heterologous PRRSV-challenged pigs. Our results indicated that out of five different vaccine-adjuvant formulations, the combination of NP-KAg and unentrapped M. tb WCL significantly cleared detectable replicating infective PRRSV with a tenfold reduction in viral RNA load in the lungs, associated with substantially reduced gross and microscopic lung pathology. Immunologically, strong humoral (enhanced virus neutralization titers by high avidity antibodies) and cell-mediated immune responses (augmented population of interferon-γ secreting CD4+ and CD8+ lymphocytes and reduced secretion of immunosuppressive cytokines) in the lungs were observed. In conclusion, combination of NP-KAg and soluble M. tb WCL elicits broadly cross-protective anti-PRRSV immunity in the pig respiratory system.

Adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is an economically devastating disease, causing daily losses of approximately $3 million to the US pork industry. Current vaccines have failed to completely prevent PRRS outbreaks. Recently, we have shown that poly(lactic-co-glycolic) acid (PLGA) nanoparticle-entrapped inactivated PRRSV vaccine (NP-KAg) induces a cross-protective immune response in pigs. To further improve its cross-protective efficacy, the NP-KAg vaccine formulation was slightly modified, and pigs were coadministered the vaccine twice intranasally with a potent adjuvant: Mycobacterium tuberculosis whole-cell lysate. In vaccinated virulent heterologous PRRSV-challenged pigs, the immune correlates in the blood were as follows: 1) enhanced PRRSV-specific antibody response with enhanced avidity of both immunoglobulin (Ig)-G and IgA isotypes, associated with augmented virus-neutralizing antibody titers; 2) comparable and increased levels of virus-specific IgG1 and IgG2 antibody subtypes and production of high levels of both T-helper (Th)-1 and Th2 cytokines, indicative of a balanced Th1–Th2 response; 3) suppressed immunosuppressive cytokine response; 4) increased frequency of interferon-γ+ lymphocyte subsets and expanded population of antigen-presenting cells; and most importantly 5) complete clearance of detectable replicating challenged heterologous PRRSV and close to threefold reduction in viral ribonucleic acid load detected in the blood. In conclusion, intranasal delivery of adjuvanted NP-KAg vaccine formulation to growing pigs elicited a broadly cross-protective immune response, showing the potential of this innovative vaccination strategy to prevent PRRS outbreaks in pigs. A similar approach to control other respiratory diseases in food animals and humans appears to be feasible.

Antibody response and maternal immunity upon boosting PRRSV-immune sows with experimental farm-specific and commercial PRRSV vaccines

The porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and respiratory disease in pigs of all ages. Despite the frequent use of vaccines to maintain PRRSV immunity in sows, little is known on how the currently used vaccines affect the immunity against currently circulating and genetically divergent PRRSV variants in PRRSV-immune sows, i.e. sows that have a pre-existing PRRSV-specific immunity due to previous infection with or vaccination against the virus. Therefore, this study aimed to assess the capacity of commercially available attenuated/inactivated PRRSV vaccines and autogenous inactivated PRRSV vaccines – prepared according to a previously optimized in-house protocol – to boost the antibody immunity against currently circulating PRRSV variants in PRRSV-immune sows.PRRSV isolates were obtained from 3 different swine herds experiencing PRRSV-related problems, despite regular vaccination of gilts and sows against the virus. In a first part of the study, the PRRSV-specific antibody response upon booster vaccination with commercial PRRSV vaccines and inactivated farm-specific PRRSV vaccines was evaluated in PRRSV-immune, non-pregnant replacement sows from the 3 herds. A boost in virus-neutralizing antibodies against the farm-specific isolate was observed in all sow groups vaccinated with the corresponding farm-specific inactivated vaccines. Use of the commercial attenuated EU type vaccine boosted neutralizing antibodies against the farm-specific isolate in sows derived from 2 farms, while use of the commercial attenuated NA type vaccine did not boost farm-specific virus-neutralizing antibodies in any of the sow groups. Interestingly, the commercial inactivated EU type vaccine boosted farm-specific virus-neutralizing antibodies in sows from 1 farm. In the second part of the study, a field trial was performed at one of the farms to evaluate the booster effect of an inactivated farm-specific vaccine and a commercial attenuated EU-type vaccine in immune sows at 60 days of gestation. The impact of this vaccination on maternal immunity and on the PRRSV infection pattern in piglets during their first weeks of life was evaluated. Upon vaccination with the farm-specific inactivated vaccine, a significant increase in farm-specific virus-neutralizing antibodies was detected in all sows. Virus-neutralizing antibodies were also transferred to the piglets via colostrum and were detectable in the serum of these animals until 5 weeks after parturition. In contrast, not all sows vaccinated with the commercial attenuated vaccine showed an increase in farm-specific virus-neutralizing antibodies and the piglets of this group generally had lower virus-neutralizing antibody titers. Interestingly, the number of viremic animals (i.e. animals that have infectious virus in their bloodstream) was significantly lower among piglets of both vaccinated groups than among piglets of mock-vaccinated sows and this at least until 9 weeks after parturition.The results of this study indicate that inactivated farm-specific PRRSV vaccines and commercial attenuated vaccines can be useful tools to boost PRRSV-specific (humoral) immunity in sows and reduce viremia in weaned piglets.

Immune responses in mice vaccinated with virus-like particles composed of the GP5 and M proteins of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces reproductive failure in sows and respiratory problems in pigs of all ages. Live attenuated and inactivated vaccines are used on swine farms to control PRRSV. However, their protective efficacy against field strains of PRRSV remains questionable. New vaccines have been developed to improve the efficacy of these traditional vaccines. In this study, virus-like particles (VLPs) composed of the GP5 and M proteins of PRRSV were developed, and the capacity of the VLPs to elicit antigen-specific immunity was evaluated. Serum antibody titers and production of cytokines were measured in BALB/C mice immunized intramuscularly three times with different doses (0.5, 1.0, 2.0, and 4.0 μg) of the VLP vaccine. A commercial vaccine consisting of inactivated PRRSV and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. IgG titers to GP5 were significantly higher in all groups of mice vaccinated with the VLPs than in control mice. Neutralizing antibodies were only detected in mice vaccinated with 2.0 and 4.0 μg of the VLPs. Cytokine levels were determined in cell culture supernatants after in vitro stimulation of splenocytes with the VLPs for 3 days. Mice immunized with 4.0 μg of the VLPs produced a significantly higher amount of interferon-gamma (IFN-γ) than mice immunized with the commercial inactivated PRRSV vaccine and PBS. In contrast, immunization with the commercial vaccine induced higher production of IL-4 and IL-10 in mice than mice vaccinated with VLPs. These data together demonstrate the capacity of VLPs to induce both neutralizing antibodies and IFN-γ in immunized mice. The VLP vaccine developed in this study could serve as a platform for the generation of improved VLP vaccines to control PRRSV.

Impact of a novel inactivated PRRS virus vaccine on virus replication and virus-induced pathology in fetal implantation sites and fetuses upon challenge

Preventing congenital infection is important for the control of porcine reproductive and respiratory syndrome (PRRS). Recently, in our laboratory, an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine has been developed. Promising results in young pigs encouraged us to test the vaccine potency to prevent congenital infection. In the present study, the performance of this experimental inactivated vaccine was investigated in pregnant gilts. An advanced protocol was used to test the PRRSV vaccine efficacy. This protocol is based on recent insights in the pathogenesis of congenital PRRSV infections. Three gilts were vaccinated with an experimental PRRSV 07V63 inactivated vaccine at 27, 55, and 83 days of gestation. Three unvaccinated gilts were included as controls. At 90 days of gestation, all animals were intranasally inoculated with 105 tissue culture infectious dose 50 (TCID50) of PRRSV 07V63. Twenty days postchallenge animals were euthanized and sampled. The vaccinated gilts quickly developed virus neutralizing (VN) antibodies starting from 3 to 7 days postchallenge (1.0 to 5.0 log2). In contrast, the unvaccinated gilts remained negative for VN antibodies after challenge. The vaccinated gilts had shorter viremia than the control gilts. Gross pathology (mummification) was observed in 8% of the fetuses from vaccinated gilts and in 15% of the fetuses from unvaccinated gilts. The number of fetuses with severe microscopic lesions in the fetal implantation sites (a focal detachment of the trophoblast from the uterine epithelium; a focal, multifocal, or full degeneration of the fetal placenta) was lower in the vaccinated (19%) versus unvaccinated (45%) gilts (P < 0.05). The number of PRRS-positive cells in the fetal placentae was higher in unvaccinated versus vaccinated gilts (P < 0.05). In contrast, the number of PRRS-positive cells in the myometrium/endometrium was higher in vaccinated versus unvaccinated gilts (P < 0.05). Fifty-seven percent of the fetuses from the vaccinated gilts and 75% of the fetuses from the unvaccinated gilts were PRRSV-positive. In conclusion, implementation of the novel experimental inactivated PRRSV vaccine primed the VN antibody response and slightly reduced the duration of viremia in gilts. It also reduced the number of virus-positive fetuses and improved the fetal survival, but was not able to fully prevent congenital PRRSV infection. The reduction of fetal infection and pathology is most probably attributable to the vaccine-mediated decrease of PRRSV transfer from the endometrium to the fetal placenta.