Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction

Within a few years of its emergence in the late 1980s, the PRRS virus had spread globally to become the foremost infectious disease concern for the pork industry. Since 1994, modified live-attenuated vaccines against porcine reproductive and respiratory syndrome virus (PRRSV-MLV) have been widely used, but have failed to provide complete protection against emerging and heterologous field strains of the virus. Moreover, like many other MLVs, PRRSV-MLVs have safety concerns including vertical and horizontal transmission of the vaccine virus and several documented incidences of reversion to virulence. Thus, the development of efficacious inactivated vaccines is warranted for the control and eradication of PRRS. Since the early 1990s, researchers have been attempting to develop inactivated PRRSV vaccines, but most of the candidates have failed to elicit protective immunity even against homologous virus challenge. Recent research findings relating to both inactivated and subunit candidate PRRSV vaccines have shown promise, but they need to be pursued further to improve their heterologous efficacy and cost-effectiveness before considering commercialization. In this comprehensive review, we provide information on attempts to develop PRRSV inactivated and subunit vaccines. These includes various virus inactivation strategies, adjuvants, nanoparticle-based vaccine delivery systems, DNA vaccines, and recombinant subunit vaccines produced using baculovirus, plant, and replication-deficient viruses as vector vaccines. Finally, future directions for the development of innovative non-infectious PRRSV vaccines are suggested. Undoubtedly there remains a need for novel PRRSV vaccine strategies targeted to deliver cross-protective, non-infectious vaccines for the control and eradication of PRRS.     

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.     

Purifying selection in porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

Porcine reproductive and respiratory syndrome virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3466 type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS.     

Vaccination with porcine reproductive and respiratory syndrome killed virus vaccine and immunostimulatory oligodeoxynucleotides induces specific immunity in piglets

A large number of studies demonstrated the immunostimulatory effects of CpG oligonucleotides (ODN), particularly in mice. In present study, the objective was to investigate the immunoadjuvant effects of CpG ODN to porcine reproductive and respiratory syndrome (PRRS) killed virus vaccine (PRRSV) and its protective effects against PRRS virus in piglets. The PRRSV-specific antibodies titres and serum IgG1/IgG2 titres, the proliferation of lymphocytes, PRRSV-specific IFN-gamma, the expression of major histocompatibility complex class II (MHCII) of peripheral blood mononuclear cells (PBMCs) and post-challenge clinical protection were examined to identify the immune responses of the piglets. The results were found that the above-mentioned immune responses of the piglets inoculated with CpG ODN plus PRRSV were significantly stronger than those indued by PRRSV or PBS control groups. All these data suggested that CpG ODN could be employed as effective immunoadjuvant to raise the humoral and cellular responses of the piglets to PRRSV.     

Enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) continues to cause substantial economic losses to the global swine industry. PRRSV appears to inhibit synthesis of type I interferons (IFNs), such as IFN-α and -β, which are critical for the innate immunity and play an important role in the modulation of adaptive immunity. An atypical PRRSV strain, A2MC2, is able to induce type I IFNs in vitro. In this study, A2MC2 induction of neutralizing antibodies in vivo was compared with the Ingelvac PRRS modified live virus (MLV) vaccine strain and VR-2385 (a moderate virulent strain). Three-week-old pigs were exposed to these PRRSV strains via intranasal or intramuscular routes to also account for a possible effect of inoculation routes. The interferon-inducing A2MC2 resulted in earlier onset and significantly higher levels of PRRSV neutralizing antibodies than the MLV. In addition, the A2MC2-induced neutralizing antibodies were capable of neutralizing VR-2385, a heterologous strain. The pigs exposed via intranasal route had higher titers of neutralizing antibodies than those injected via intramuscular route. Macroscopic and microscopic lung lesions 14 days post-exposure indicated that A2MC2 had similar virulence in vivo as VR-2385. Pulmonary alveolar macrophages (PAMs) collected during the necropsy 14 days post-exposure in the A2MC2 group had higher level expression of IFN-γ than the MLV group. These results indicate that A2MC2 can be further explored for development of an improved vaccine against PRRS.     

Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) was reported in the late 1980s. PRRS still is a huge economic concern to the global pig industry with a current annual loss estimated at one billion US dollars in North America alone. It has been 20 years since the first modified live-attenuated PRRSV vaccine (PRRSV-MLV) became commercially available. PRRSV-MLVs provide homologous protection and help in reducing shedding of heterologous viruses, but they do not completely protect pigs against heterologous field strains. There have been many advances in understanding the biology and ecology of PRRSV; however, the complexities of virus-host interaction and PRRSV vaccinology are not yet completely understood leaving a significant gap for improving breadth of immunity against diverse PRRS isolates. This review provides insights on immunization efforts using infectious PRRSV-based vaccines since the 1990s, beginning with live PRRSV immunization, development and commercialization of PRRSV-MLV, and strategies to overcome the deficiencies of PRRSV-MLV through use of replicating viral vectors expressing multiple PRRSV membrane proteins. Finally, powerful reverse genetics systems (infectious cDNA clones) generated from more than 20 PRRSV isolates of both genotypes 1 and 2 viruses have provided a great resource for exploring many innovative strategies to improve the safety and cross-protective efficacy of live PRRSV vaccines. Examples include vaccines with diminished ability to down-regulate the immune system, positive and negative marker vaccines, multivalent vaccines incorporating antigens from other porcine pathogens, vaccines that carry their own cytokine adjuvants, and chimeric vaccine viruses with the potential for broad cross-protection against heterologous strains. To combat this devastating pig disease in the future, evaluation and commercialization of such improved live PRRSV vaccines is a shared goal among PRRSV researchers, pork producers and biologics companies.     

DNA vaccination of pigs with open reading frame 1-7 of PRRS virus

We cloned all open reading frames of a Danish isolate of porcine reproductive and respiratory syndrome (PRRS) virus in DNA vaccination vectors. Pigs were vaccinated using a gene gun with each single construct (ORF1, ORF2, ORF3, ORF4, ORF5, ORF6, or ORF7) or combinations thereof. Vaccination with ORF7 consistently induced antibodies after three vaccinations, while antibodies were only sporadically detected in the remaining groups. After six vaccinations, all pigs were inoculated with PRRS virus and the post-inoculation antibody response was studied. Pigs vaccinated with ORF1 or ORF4 were primed for antibody response against NSP2 or GP4, respectively. Neutralising antibodies were detected in all pigs, with ORF5 vaccinated pigs showing the highest titres.     

Immune response of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus

Pigs were immunised with recombinant BCG (rBCG) expressing a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) (rBCGGP5) and M protein (rBCGM) of porcine reproductive and respiratory syndrome virus (PRRSV). At 30 days post-inoculation (dpi), pigs inoculated with rBCGGP5 and rBCGM developed a specific humoral immune response against the viral proteins, as detected by commercial ELISA and Western blot tests, and at 60 dpi, three out of five animals developed neutralizing antibodies with titers ranging from 1:4 to 1:8. At 67 dpi, an IFN-gamma response against BCG antigens, but not against the viral proteins, was detected by ELISPOT in inoculated pigs. Following challenge with a pathogenic strain of PRRSV, pigs inoculated with rBCG showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection.     

Chlorine dioxide inhibits the replication of porcine reproductive and respiratory syndrome virus by blocking viral attachment

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a great economic loss to the swine industry globally. Current prevention and treatment measures are not effective to control the outbreak and spread of porcine reproductive and respiratory syndrome (PRRS). In other words, new antiviral strategies are urgently needed. Chlorine dioxide (ClO₂) is regarded as a broad-spectrum disinfectant with strong inhibitory effects on microbes and parasites. The purpose of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of ClO₂ against PRRSV infection in vitro. Here, we identified ClO₂ (the purity is 99%) could inhibit the infection and replication of PRRSV in both Marc-145 cells and porcine alveolar macrophages (PAMs). ClO₂ could block PRRSV binding to cells rather than internalization and release, suggesting that ClO₂ blocks the first stage of the virus life cycle. We also demonstrated that the inhibition exerted by ClO₂ was attributed to the degradation of PRRSV genome and proteins. Moreover, we confirmed that ClO₂ could decrease the expression of inflammatory cytokines induced by PRRSV. In summary, ClO₂ is an efficient agent and potently suppressed PRRSV infection in vitro.     

Cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.     

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.     

Co-infection status of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018

Classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) are economically important swine viruses that cause reproductive failure and/or respiratory symptoms in pigs. However, the co-infection status of these viruses in Chinese swine herds is not well clarified. In this study, we evaluated the co-infection of these four viruses in 159 pigs collected from 63 herds in eight regions of China from 2016 to 2018. CSFV, PRRSV, PCV2 and PCV3 were detected in 14, 56, 43 and 4 of the pigs, respectively. The percentage of singular infections was 32.71%, while the percentages of dual infections and multiple infections were 15.72% and 3.15%, respectively. The E2 of CSFV, ORF5 of PRRSV, ORF2s of PCV2 and PCV3 from all positive samples were determined and used for phylogenetic analyses. E2-based phylogenetic tree showed that all 14 CSFVs identified in this study belong to 2.1b subtype. ORF5-based phylogenetic tree showed that PRRSV2 is predominant in China while PRRSV1 can also be detected. In addition, 35, 16, 4 and 1 of our PRRSVs are clustered with highly pathogenic PRRSV2, NADC30-like PRRSV2, classical PRRSV2 and PRRSV1, respectively. ORF2-based phylogenetic trees showed that our PCVs are grouped with 2 PCV2 subtypes (PCV2d and PCV2b) and 3 PCV3 subtypes (PCV3a, PCV3b and PCV3c), respectively. Our results provide the latest co-infection status and the diversity of four important swine viruses in Chinese swine herds, which is beneficial for understanding the epidemiology of these viruses.     

Cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection

Because porcine reproductive and respiratory syndrome virus (PRRSV) exhibits extensive genetic variation among field isolates, characterizing the extent of cross reactivity of immune responses, and most importantly cell-mediated immunity (CMI), could help in the development of broadly cross-protective vaccines. We infected 12 PRRSV-naïve pigs with PRRSV strain FL12 and determined the number of interferon (IFN)-γ secreting cells (SC) by ELISpot assay using ten type 2 and one type 1 PRRSV isolates as recall antigens. The number of IFN-γ SC was extremely variable among animals, and with exceptions, late to appear. Cross reactivity of IFN-γ SC among type 2 isolates was broad, and we found no evidence of an association between increased genetic distance among isolates and the intensity of the CMI response. Comparable to IFN-γ SC, total antibodies evaluated by indirect immunofluorescence assay (IFA) were cross reactive, however, neutralizing antibody titers could only be detected against the strain used for infection. Finally, we observed a moderate association between homologous IFN-γ SC and neutralizing antibodies.     

Intranasal delivery of whole cell lysate of Mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease to pork producers worldwide. Commercially, both live and killed PRRSV vaccines are available to control PRRS, but they are not always successful. Based on the results of mucosal immunization studies in other viral models, a good mucosal vaccine may be an effective way to elicit protective immunity to control PRRS outbreaks. In the present study, mucosal adjuvanticity of Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was evaluated in pigs administered a modified live PRRS virus vaccine (PRRS-MLV) intranasally. A Mtb WCL mediated increase in the frequency of NK cells, CD8⁺and CD4⁺ T cells, and γδ T cells in pig lungs were detected. Importantly, an increased and early generation of PRRSV specific neutralizing antibodies were detected in PRRS-MLV+ Mtb WCL compared to pigs inoculated with vaccine alone. In addition, there was an increased secretion of Th1 cytokines (IFNγ and IL-12) that correlated with a reciprocal reduction in the production of immunosuppressive cytokines (IL-10 and TGFβ) as well as T-regulatory cells in pigs vaccinated with PRRS-MLV+ Mtb WCL. Further, a complete rescue in arginase levels in the lungs mediated through Mtb WCL was observed in pigs inoculated with PRRS-MLV. In conclusion, Mtb WCL may be a potent mucosal adjuvant for PRRS-MLV in order to potentiate the anti-PRRSV specific immune responses to control PRRS effectively.     

Recombination in lineage 1, 3, 5 and 8 of porcine reproductive and respiratory syndrome viruses in China

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important viral swine diseases, resulting in immense economic losses in Chinese pig industry. Currently, four major lineages: lineage 1 (NADC30-like), 3 (QYYZ-like), 5.1 (VR2332-like) and 8.7 (JXA1-like) of type 2 PRRSV (North American type) have been circulating in China based on classification system, which have caused concern about the potential of virus recombination. In the present study, a novel variant of PRRSV strain named FJLIUY-2017 was isolated from abortion rate (25%) in pregnant gilts in Fujian Province in China in 2017. To further our knowledge about the novel virus strain, we characterized the complete genome of FJLIUY-2017. Comparison to PRRS sequences in GenBank confirmed the absence of close relatives (<92%), but indicated FJLIUY-2017 belonged to NADC30-like PRRSV. The full length of FJLIUY-2017 was determined to be 15017 nucleotides (nt), excluding the poly(A) tail, shared 86.2–86.6% identity with JXA1-like strains (JXA1, TJ and FJYR), 88.9–90.6% with NADC30-like PRRSVs (NADC30, FJZ03 and CHsx1401), 86.4–86.5% with VR2332-like (VR2332, RespPRRS MLV and BJ-4) and only 60.8% with LV (European type). Recombination analyses revealed genomic breakpoints in structural (ORF3, ORF4 and ORF7) and nonstructural (Nsp1, Nsp2, Nsp6, Nsp9, Nsp11 and Nsp12) regions of the genomes with evidence for recombination events between lineages 1, 3, 5.1 and 8.7. Taken altogether, the results of our study provide further confirmation that PRRSV is prone to undergo recombination events. Thus, it is critical to monitor PRRSV evolution in China and establish an effective strategy for the control of PRRS.     

Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus

Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.     

Identification of immunodominant T-cell epitopes present in glycoprotein 5 of the North American genotype of porcine reproductive and respiratory syndrome virus

Ninty-six pentadecapeptides spanning glycoprotein 5 (GP5) of porcine reproductive and respiratory virus (PRRSV) were screened for their ability to elicit a recall interferon-gamma response from peripheral blood mononuclear cells isolated from 22 pigs infected with up to two genetically divergent PRRSV strains. Two distinct regions (amino acid residues 117-131, LAALICFVIRLAKNC, and 149-163, KGRLYRWRSPVII/VEK) appeared to contain immunodominant T-cell epitopes based on their ability to stimulate above average numbers of interferon-gamma secreting cells as compared to other GP5 peptides. A survey of PRRSV isolates indicated that these two sites are relatively conserved with at most a two amino acid variation and thus should be considered for incorporation into a multi-valent vaccine against PRRS.     

Pigs immunized with Chinese highly pathogenic PRRS virus modified live vaccine are protected from challenge with North American PRRSV strain NADC-20

Modified live virus (MLV) vaccines developed to protect against PRRSV circulating in North America (NA) offer limited protection to highly pathogenic (HP) PRRSV strains that are emerging in Asia. MLV vaccines specific to HP-PRRSV strains commercially available in China provide protection to HP-PRRSV; however, the efficacy of these HP-PRRSV vaccines to current circulating NA PRRS viruses has not been reported. The aim of this study is to investigate whether pigs vaccinated with attenuated Chinese HP-PRRSV vaccine (JXA1-R) are protected from infection by NA PRRSV strain NADC-20. We found that pigs vaccinated with JXA1-R were protected from challenges with HV-PRRSV or NADC-20 as shown by fewer days of clinical fever, reduced lung pathology scores, and lower PRRS virus load in the blood. PRRSV-specific antibodies, as measured by IDEXX ELISA, appeared one week after vaccination and virus neutralizing antibodies were detected four weeks post vaccination. Pigs vaccinated with JXA1-R developed broadly neutralizing antibodies with high titers to NADC-20, JXA1-R, and HV-PRRSV. In addition, we also found that IFN-α and IFN-β occurred at higher levels in the lungs of pigs vaccinated with JXA1-R. Taken together, our studies provide the first evidence that JXA1-R can confer protection in pigs against the heterologous NA PRRSV strain NADC-20.