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Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico. Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope 748YSNIGVCK755 from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).

Significance We report a vaccine platform to express vaccine antigens on the surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrate the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface of Escherichia coli to produce killed whole-cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates interferon-γ responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly protective coronavirus vaccine since a betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against alphacoronavirus porcine epidemic diarrhea virus. When using a vaccine-appropriate bacteria vector, this inexpensive vaccine platform offers the potential for use in developing countries., As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible Gram-negative autotransporter to express vaccine antigens on surface of genome-reduced bacteria to enhance interaction of vaccine antigen with immune system. As a proof of principle, we utilized genome-reducedE. colito express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as a killed whole cell vaccine. The FP sequence is highly conserved across coronaviruses; the 6 FP core amino acid residues along with the 4 adjacent residues upstream and the 3 residues downstream the core are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated IFN-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly-protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.Significance StatementWe report a new vaccine platform to express vaccine antigens on surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrated the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface ofE.colito produce killed whole cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates IFN-γ responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly-protective coronavirus vaccine since a Betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against Alphacoronavirus PEDV. When using a vaccine appropriate bacteria vector, this inexpensive new vaccine platform offers the potential for use in developing countries.

Hepatitis E virus (HEV) infects humans and more than a dozen other animal species. We previously showed that open reading frame 2 (ORF2) and ORF3 are apparently not involved in HEV cross-species infection, which infers that the ORF1 may contribute to host tropism. In this study, we utilize the genomic backbone of HEV-1 which only infects humans to construct a panel of intergenotypic chimeras in which the entire ORF1 gene or its functional domains were swapped with the corresponding regions from HEV-3 that infects both humans and pigs. We demonstrated that the chimeric HEVs were replication competent in human liver cells. Subsequently, we intrahepatically inoculated the RNA transcripts of chimeras into pigs to determine if the swapped ORF1 regions confer the chimeras' ability to infect pigs. We showed that there was no evidence of infectivity in pigs for any of the chimeras. We also investigated the role of human ribosome protein sequence S17, which expanded host range in cultured cells, in HEV cross-species infection. We demonstrated that S17 insertion in HEV ORF1 did not abolish HEV replication competency in vitro, but also did not expand HEV host tropism in vivo. The results highlight the complexity of the underlying mechanism of HEV cross-species infection.

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ–specific CD4+ T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α–specific CD8+ T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.

Immuno-stimulatory class I-restricted cytotoxic T lymphocytes (CTL) epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) are important for vaccine development. In this study we first determined the expression frequency of swine leukocyte antigen (SLA) class I alleles in commercial pigs in the United States. The SLA genotyping result allowed us to predict potential CTL epitopes from a contemporary strain of PRRSV (RFLP 1-7-4) by using bioinformatic tools. The predicted epitopes were then evaluated in an ex vivo stimulation assay with peripheral blood mononuclear cells isolated from pigs experimentally-infected with PRRSV. Using flow-cytometry analysis, we identified a number of immuno-stimulatory CTL epitopes, including two peptides from GP3 and two from Nsp9 that significantly improved both degranulation marker CD107a and IFN-γ production in cytotoxic CD4+CD8+ T cells, CD8+ T cells, and γδ T cells, and two peptides that inhibited IFN-γ production. These CTL epitopes will aid future vaccine development against PRRSV.

A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs.

CD137 is a costimulatory molecule transiently expressed on activated T cells after mitogen or antigen stimulation that can be exploited for isolating antigen-specific T cells as reported in mouse models. By utilizing an antiporcine CD137 monoclonal antibody (mAb, clone 3B9) developed in our laboratory, we isolated virus-specific CD8β T cells from peripheral blood of pigs experimentally infected with different porcine reproductive and respiratory syndrome virus (PRRSV) strains. Similar to mouse, porcine CD8β T cells also express CD137 transiently upon Concavalin A stimulation while the unstimulated cells did not. Most frequently, virus-specific CD8β T cells were isolated at low levels from peripheral blood of pigs experimentally infected with PRRSV strains VR2385, NADC20, and MN184B at 49 and 63 days postinfection. The results suggest that porcine CD137-specific mAb is a useful tool for isolating virus-specific CD8 T cells from peripheral blood and tissues of pigs after in vitro stimulation with viral antigen.

The quasispecies diversity of RNA viruses is mainly determined by the fidelity of RNA-dependent RNA polymerase (RdRp) during viral RNA replication. Certain amino acid residues play an important role in determining the fidelity, and such residues can be substituted with other amino acids to produce virus strains with higher fidelity. In this study, two amino acid substitutions (A283T and H421Y) in the RdRp of porcine reproductive and respiratory syndrome virus (PRRSV) were identified under the selection of ribavirin. Preliminary data showed that two substitutions were involved in conferring PRRSV with the properties of increased ribavirin resistance and restricted quasispecies diversity. The results indicated that these two amino acid residues (Ala283 and His421) play a crucial role in PRRSV replication by affecting the fidelity of its RdRp. The results have important implications for understanding the molecular mechanism of PRRSV evolution and pathogenicity, and developing a safer modified live-attenuated vaccine (MLV) against PRRSV.

Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with the porcine cytokine interleukin-15 (IL-15) or IL-18 gene fused to a glycosylphosphatidylinositol (GPI) modification signal that can anchor the cytokines to the cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophages after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or the parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lungs after heterologous challenge with the PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition of the NK cell response seen with Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the numbers of gamma interferon (IFN-γ)-producing cells in circulation at 49 days postvaccination (dpv), especially for IFN-γ-producing CD4 − CD8 + T cells and γδ T cells, compared to the Suvaxyn MLV and SUV-IL-18. Additionally, MLV SUV-IL-15-vaccinated pigs also had elevated levels of γδ T cell responses observed at 7 dpv, 49 dpv, and 7 days postchallenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination and confers improved heterologous protection, although this was not statistically significant compared to the parental MLV. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective against only homologous or closely related virus strains but are ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, in this study, we present a novel approach of using porcine IL-15 or IL-18 as an adjuvant by directly incorporating its encoding gene into a PRRSV MLV and expressing it as an adjuvant. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV virus expressing the membrane-bound IL-15 cytokine greatly enhanced NK cell and γδ T cell responses and also conferred improved protection against heterologous challenge with the PRRSV NADC20 strain.

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are three of the most economically important swine pathogens, causing immense economic losses to the global swine industry. Monovalent commercial vaccines against each of the three viruses are routinely used in pig farms worldwide. A trivalent vaccine against all three pathogens would greatly simplify the vaccination programme and reduce the financial burden to the swine industry. In this study, by using an attenuated strain of PRRSV (strain DS722) as a live virus vector, we generated a multi-component vaccine virus, DS722-SIV-PCV2, which expresses the protective antigens from SIV and PCV2. The DS722-SIV-PCV2 trivalent vaccine virus replicates well, and expresses PCV2 capsid and SIV HA proteins in vitro. A subsequent vaccination and challenge study in 48 pigs revealed that the DS722-SIV-PCV2-vaccinated pigs had significantly reduced lung lesions and viral RNA loads when challenged with PRRSV. Upon challenge with PCV2, the vaccinated pigs had partially reduced lymphoid lesions and viral DNA loads, and when challenged with SIV the vaccinated pigs had significantly reduced acute respiratory sign scores. The results from this study demonstrate the potential of DS722-SIV-PCV2 as a candidate trivalent vaccine, and also shed light on exploring PRRSV as a potential live virus vaccine vector.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of arguably the most economically important global swine disease. The extensive genetic variation of PRRSV strains is a major obstacle for heterologous protection of current vaccines. Previously, we constructed a panel of chimeric viruses containing only the ectodomain sequences of DNA-shuffled structural genes of different PRRSV strains in the backbone of a commercial vaccine, and found that one chimeric virus had an improved cross-protection efficacy. In this present study, to further enhance the cross-protective efficacy against heterologous strains, we constructed a novel chimeric virus VR2385-S3456 containing the full-length sequences of shuffled structural genes (ORFs 3-6) from 6 heterologous PRRSV strains in the backbone of PRRSV strain VR2385. We showed that the chimeric virus VR2385-S3456 induced a high level of neutralizing antibodies in pigs against two heterologous strains. A subsequent vaccination and challenge study in 48 pigs revealed that the chimeric virus VR2385-S3456 conferred an enhanced cross-protection when challenged with heterologous virus strain NADC20 or a contemporary heterologous strain RFLP 1-7-4. The results suggest that the chimera VR2385-S3456 may be a good PRRSV vaccine candidate for further development to confer heterologous protection.

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.

Current porcine reproductive and respiratory syndrome virus (PRRSV) vaccines sometimes fail to provide adequate immunity to protect pigs from PRRSV-induced disease. This may be due to antigenic differences among PRRSV strains. Rapid production of attenuated farm-specific homologous vaccines is a feasible alternative to commercial vaccines. In this study, attenuation and efficacy of a codon-pair de-optimized candidate vaccine generated by synthetic attenuated virus engineering approach (SAVE5) were tested in a conventional growing pig model. Forty pigs were vaccinated intranasally or intramuscularly with SAVE5 at day 0 (D0). The remaining 28 pigs were sham-vaccinated with saline. At D42, 30 vaccinated and 19 sham-vaccinated pigs were challenged with the homologous PRRSV strain VR2385. The experiment was terminated at D54. The SAVE5 virus was effectively attenuated as evidenced by a low magnitude of SAVE5 viremia for 1–5 consecutive weeks in 35.9% (14/39) of the vaccinated pigs, lack of detectable nasal SAVE5 shedding and failure to transmit the vaccine virus from pig to pig. By D42, all vaccinated pigs with detectable SAVE5 viremia also had detectable anti-PRRSV IgG. Anti-IgG positive vaccinated pigs were protected from subsequent VR2385 challenge as evidenced by lack of VR2385 viremia and nasal shedding, significantly reduced macroscopic and microscopic lung lesions and significantly reduced amount of PRRSV antigen in lungs compared to the non-vaccinated VR2385-challenged positive control pigs. The nasal vaccination route appeared to be more effective in inducing protective immunity in a larger number of pigs compared to the intramuscular route. Vaccinated pigs without detectable SAVE5 viremia did not seroconvert and were fully susceptible to VR2385 challenge. Under the study conditions, the SAVE approach was successful in attenuating PRRSV strain VR2385 and protected against homologous virus challenge. Virus dosage likely needs to be adjusted to induce replication and protection in a higher percentage of vaccinated pigs.

Highlights • Targeting PEDV spike protein domains to Langerin-expressing dendritic cells through a single chain antibody. • Transdermal Langerin-targeting of PEDV S antigen significantly enhanced CD4 T cell immunity. • Systemic Langerin-targeting of PEDV S antigen significantly augmented serum IgG and IgA responses., Porcine epidemic diarrhea virus (PEDV) first emerged in the United States in 2013 causing high mortality and morbidity in neonatal piglets with immense economic losses to the swine industry. PEDV is an alpha-coronavirus replicating primarily in porcine intestinal cells. PEDV vaccines are available in Asia and Europe, and conditionally-licensed vaccines recently became available in the United States but the efficacies of these vaccines in eliminating PEDV from swine populations are questionable. In this study, the immunogenicity of a subunit vaccine based on the spike protein of PEDV, which was directly targeted to porcine dendritic cells (DCs) expressing Langerin, was assessed. The PEDV S antigen was delivered to the dendritic cells through a single-chain antibody specific to Langerin and the targeted cells were stimulated with cholera toxin adjuvant. This approach, known as “dendritic cell targeting,” greatly improved PEDV S antigen-specific T cell interferon-γ responses in the CD4posCD8pos T cell compartment in pigs as early as 7 days upon transdermal administration. When the vaccine protein was targeted to Langerinpos DCs systemically through intramuscular vaccination, it induced higher serum IgG and IgA responses in pigs, though these responses require a booster dose, and the magnitude of T cell responses were lower as compared to transdermal vaccination. We conclude that PEDV spike protein domains targeting Langerin-expressing dendritic cells significantly increased CD4 T cell immune responses in pigs. The results indicate that the immunogenicity of protein subunit vaccines can be greatly enhanced by direct targeting of the vaccine antigens to desirable dendritic cell subsets in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is classified into two genotypes, type 1 and type 2, which share only about 60% genetic identity. Here, we report viable chimeric viruses in which the envelope protein genes from ORF2a to ORF5 of vSHE (type 1) were swapped into the genetic backbone of vAPRRS (type 2). We found that the envelope proteins of genotype 1 were fully functional in genotype 2 PRRSV, and the rescued chimeric progeny viruses showed robust genetic stability and similar replication properties to the parental strains in vitro. To our knowledge, this is the first study to report the substitution of complete ORFs between different genotypes of porcine arterivirus. These findings pave the way to further elucidate the structure–function relationship of PRRSV envelope proteins, and may enable the development of novel marker vaccines that can be used to differentiate vaccinated from infected animals.

We previously demonstrated that the C-terminus of the capsid gene of porcine circovirus type 2 (PCV2) is an immune reactive epitope displayed on the surface of virions. Insertion of foreign epitope tags in the C-terminus produced infectious virions that elicited humoral immune responses against both PCV2 capsid and the inserted epitope tags, whereas mutation in the N terminus impaired viral replication. Since the non-pathogenic porcine circovirus type 1 (PCV1) shares similar genomic organization and significant sequence identity with pathogenic PCV2, in this study we evaluated whether PCV1 can serve as a vaccine delivery virus vector. Four different antigenic determinants of porcine reproductive and respiratory syndrome virus (PRRSV) were inserted in the C-terminus of the PCV1 capsid gene, the infectivity and immunogenicity of the resulting viruses are determined. We showed that an insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not affect PCV1 replication. We successfully rescued and characterized four chimeric PCV1 viruses expressing PRRSV linear antigenic determinants (GP2 epitope II: aa 40-51, ASPSHVGWWSFA; GP3 epitope I: aa 61-72, QAAAEAYEPGRS; GP5 epitope I: aa 35-46, SSSNLQLIYNLT; and GP5 epitope IV: aa 187-200, TPVTRVSAEQWGRP). We demonstrated that all chimeric viruses were stable and infectious in vitro and three chimeric viruses were infectious in vivo. An immunogenicity study in pigs revealed that PCV1-VR2385EPI chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. The results have important implications for further evaluating PCV1 as a potential vaccine delivery vector.

The extensive genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains is a major obstacle for vaccine development. We previously demonstrated that chimeric PRRSVs in which a single envelope gene (ORF3, ORF4, ORF5 or ORF6) was shuffled via DNA shuffling had an improved heterologous cross-neutralizing ability. In this study, we incorporate all of the individually-shuffled envelope genes together in different combinations into an infectious clone backbone of PRRSV MLV Fostera(®) PRRS. Five viable progeny chimeric viruses were rescued, and their growth characteristics were characterized in vitro. In a pilot pig study, two chimeric viruses (FV-SPDS-VR2,FV-SPDS-VR5) were found to induce cross-neutralizing antibodies against heterologous strains. A subsequent vaccination/challenge study in 72 pigs revealed that chimeric virus FV-SPDS-VR2 and parental virus conferred partial cross-protection when challenged with heterologous strains NADC20 or MN184B. The results have important implications for future development of an effective PRRSV vaccine that confers heterologous protection.

Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly.

The nucleocapsid (N) proteins of the North American (type II) and European (type I) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) share only approximately 60% genetic identity, and the functionality of N in both genotypes, especially its role in virion assembly, is still poorly understood. In this study, we demonstrated that the ORF7 3′ untranslated region or ORF7 of type I is functional in the type II PRRSV background. Based on these results, we postulated that the cysteine at position 90 (Cys90) of the type II N protein, which corresponds to an alanine in the type I protein, is nonessential for virus infectivity. The replacement of Cys90 with alanine confirmed this hypothesis. We then hypothesized that all of the cysteines in the N protein could be replaced by alanines. Mutational analysis revealed that, in contradiction to previously reported findings, the replacement of all of the cysteines, either singly or in combination, did not impair the growth of either type II or type I PRRSV. Treatment with the alkylating agent N -ethylmaleimide inhibited cysteine-mediated N dimerization in living cells but not in released virions. Additionally, bimolecular fluorescence complementation assays revealed noncovalent interactions in living cells among the N and C termini and between the N-terminal and C-terminal regions of the N proteins of both genotypes of PRRSV. These results demonstrate that the disulfide linkages mediating the N dimerization are not required for PRRSV viability and help to promote our understanding of the mechanism underlying arterivirus particle assembly.

We selectively expressed protective Mycobacterium tuberculosis antigen ESAT-6 in recombinant strains Lm(esat-6) and LmΔactA/plcB(esat-6) to evaluate the capacity of Listeria monocytogenes to deliver antigens from M. tuberculosis, and we studied the pathogenicity and immunogenicity of these strains compared with virulent parental strain yzuLm4 and attenuated strain LmΔactA/plcB. The two recombinant strains retained listeriolysin O hemolytic activity, escaped into the cytosol niche and established replication in the macrophage-like RAW264.7 cell line; however, these strains showed decreased virulence in C57BL/6 mice. Histopathology revealed no obvious pathological changes following administration of the recombinant strains to mice, indicating that they were significantly safer than parental strains. Moreover, intravenous vaccination of mice with the recombinant strains elicited specific Th1-type cellular immunity, splenocyte proliferation and effective CTL activity in vivo. Thus, attenuated L. monocytogenes strains can be used as effective vectors for delivering M. tuberculosis ESAT-6 and inducing a cellular immune response, suggesting that such vectors may be effective as novel vaccines for preventing tuberculosis.

The subgenomic mRNA transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (PRRSV) are directed by the viral replicase. The replicase is expressed in the form of two polyproteins and is subsequently processed into smaller nonstructural proteins (nsps). nsp9, containing the viral replicase, has characteristic sequence motifs conserved among the RNA-dependent RNA polymerases (RdRp) of positive-strand (PS) RNA viruses. To test whether the conserved SDD motif can tolerate other conserved motifs of RNA viruses and the influence of every residue on RdRp catalytic activity, many amino acids substitutions were introduced into it. Only one nsp9 substitution, of serine by glycine (S3050G), could rescue mutant viruses. The rescued virus was genetically stable. Alteration of either aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, but did not eliminate virus replication. We also found that the SDD motif was essentially invariant for the signature sequence of PRRSV RdRp. It could not accommodate other conserved motifs found in other RNA viral polymerases, except the GDD motif, which is conserved in all the other PS RNA viruses. These findings indicated that nidoviruses are evolutionarily related to other PS RNA viruses. Our studies support the idea that the two aspartate residues of the SDD motif are critical and essential for PRRSV transcription and represent a sequence variant of the GDD motif in PS RNA viruses.

Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA - and plcB -deficient L. monocytogenes strain, yzuLM4Δ actA / plcB , and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD 50 ] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.

It has been proposed that the N-linked glycan of the minor proteins of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the production of infectious virus. In this study, we showed that N-linked glycosylation of GP2 is not essential for virus viability and none of the individual glycosylation sites in GP3 has a vital effect on the production of infectious virus. Moreover, mutations of single and double glycosylation sites in GP4 are not critically important for infectious virus recovery, triple and quadruple mutations are lethal. The bimolecular fluorescence complementation (BiFC) analysis also showed that GP4, but might be not GP2, is involved in interaction with cellular receptor CD163 and that glycosylation of GP4 might not play a vital role in the interaction with CD163. The study further revealed that none of the N-glycosylation sites in the minor proteins is critical for the susceptibility of mutants to neutralizing antibody.