Effect of the host cell line on the vaccine efficacy of an attenuated porcine reproductive and respiratory syndrome virus.

The abilities of the modified-live Prime Pac (PP) strain of porcine reproductive and respiratory syndrome virus (PRRSV), propagated in either traditional simian cells (MARC-145) or in a novel porcine alveolar macrophage cell line (ZMAC), to confer pigs protection against subsequent PRRSV challenge were compared. Eight week-old pigs were injected with PP virus grown in one of the two cell types and then exposed 4 weeks later to the "atypical" PRRSV isolate NADC-20. Control animals were similarly challenged or remained PRRSV-naïve. While the average adjusted body weight (aabw) of the strict control group increased 22% by 10 days post challenge (pc), this value for the non-vaccinated, challenged group dropped 4%. In contrast, prior immunization with PP virus, regardless of its host cell source, ameliorated this effect by affording a >9% rise in aabw. Likewise, nearly equivalent protection was extended to both groups of vaccinates in regards to the temporal elimination of their pc clinical distress and viremia. However, the PP virus propagated in ZMAC cells appeared to be more efficacious since four of the six pigs receiving this biologic cleared the challenge virus from the their lungs by 10 days pc as compared to only one member of the other vaccinated group. Notably, the predominant quasispecies in the ZMAC cell-prepared PP virus stock contained a highly conserved N-glycosylation site at position 184 in its glycoprotein 2 while this entity was underrepresented in the MARC-145 cell grown biologic. Since glycoprotein 2 is involved in infectivity, such additional glycosylation may enhance virus replication in porcine alveolar macrophages.

North American porcine reproductive and respiratory syndrome viruses inhibit type I interferon production by plasmacytoid dendritic cells.

Although enveloped viruses typically trigger the prodigious secretion of alpha interferon (IFN-α) by plasmacytoid dendritic cells (pDC), porcine pDC remain quiescent when exposed to porcine reproductive and respiratory syndrome virus (PRRSV). This inactivity is likely due to virus-mediated interference since the typical IFN-α response by either purified or nonsorted porcine pDC to transmissible gastroenteritis virus (TGEV) or the Toll-like receptor 9 agonist, oligodeoxynucleotide (ODN) D19, was markedly reduced in the presence of PRRSV. Suppression occurred independently of virus viability and acidification of pDC early endosomes but correlated with diminished levels of IFN-α mRNA. This change was attributed to an abrogation of transcription resulting from a decrease in the otherwise enhanced amounts of the requisite interferon regulatory factor 7 (IRF-7), whose gene expression in turn was limited as a consequence of a lessened availability of nuclear-localized signal transducer and activator of transcription 1 (STAT1). While PRRSV also inhibited tumor necrosis factor alpha (TNF-α) synthesis by pDC responding to either agent, only the interleukin-2 (IL-2) and IL-6 production instigated by ODN D19 exposure was blocked. Likewise, PRRSV did not impact a specific TGEV-associated enhancement of IL-8 expression. Moreover, an augmented phosphorylation of NF-κB seen in activated pDC was not only unaffected by PRRSV but actually occurred in its presence. Thus, as supported by a demonstrated resilience of pDC to PRRSV infection, this pathogen may interact with a cell surface protein(s) to selectively impede the completion of cascades involved in cytokine production by stimulated pDC.

Immunologic responses and reproductive outcomes following exposure to wild-type or attenuated porcine reproductive and respiratory syndrome virus in swine under field conditions.

OBJECTIVE: To compare immunologic responses and reproductive outcomes in sows housed under field conditions following controlled exposure to a wild-type strain of porcine reproductive and respiratory syndrome virus (PRRSV strain WTV) or vaccination with a modified-live virus (MLV) vaccine. DESIGN: Randomized controlled trial. ANIMALS: 30 PRRSV-naïve 10-week-old female pigs. PROCEDURE: Humoral and cell-mediated immune responses were monitored while pigs were held in isolation for 84 days after inoculation with the WTV strain (n = 10), inoculation with the WTV strain and 42 days later vaccination with a killed-virus vaccine (10), or vaccination with an MLV vaccine (10). Reproductive outcomes were measured after pigs were released into the farm herd. RESULTS: Inoculation with the WTV strain, regardless of whether a killed-virus vaccine was subsequently administered, elicited faster and more substantial production of strain-specific neutralizing antibodies, as well as a more rapid generation of interferon-gamma secreting cells, than did vaccination with the MLV vaccine. Despite the enhanced immune responses in pigs inoculated with the WTV strain, animals vaccinated with the MLV vaccine produced a mean of 2.45 more pigs than did sows exposed to the WTV strain, mainly because of a lower rate for failure to conceive. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that current assays of immunity to PRRSV correlate only imperfectly with degree of clinical protection and that the practice of controlled exposure of sows to a circulating PRRSV strain should be reconsidered in light of negative clinical outcomes.

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences.

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.

Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination.

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus.

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.

Identification of key immune mediators regulating T helper 1 responses in swine.

This publication describes the cloning of full or partial length sequences for pig TBX21 (T-bet), MYD88, ICSBP1, CD8A (CD8alpha), CD8B (CD8beta), and CD28 cDNAs. Real-time PCR assays have been developed for the relative quantitation of these products as well as previously characterized transcripts that encode exon A-containing CD45, HLX1, IRF1, STAT1 and RPL32. When used for examining temporal immune gene expression in the liver of Toxoplasma gondii infected pigs, the positive regulators of Th1 responses, IRF1, MYD88, and STAT1, were found to be expressed prior to the simultaneous upregulation of interferon gamma (IFNG), HLX1 and TBX21 gene expression. In contrast, in the mesenteric lymph node (MLN), only expression of IRF1 and IFNG was significantly upregulated. Based on their demonstrated utility in establishing an immune response pathway, these PCR assays should be valuable additions to our swine immune toolkit.

Characterization of an avianpox virus isolated from an Andean condor (Vultur gryphus).

A novel pox virus, condorpox virus (CPV) isolated from the spleen of an Andean condor (Vultur gryphus) by inoculation of chorioallantoic membranes (CAM) of specific pathogen free (SPF) chicken embryos was compared biologically, antigenically and genetically with fowlpox virus (FPV), the type species of the genus Avipoxvirus. Susceptible chickens inoculated with CPV developed only mild localized lesions but were not protected against subsequent challenge with FPV. Based on Western blotting, in addition to the presence of cross-reacting antigens, distinct differences in antigenic profiles of CPV and FPV were observed. Sequence analysis of a 4.5 kb HindIII fragment of CPV genomic DNA revealed the presence of eight co-linear genes corresponding to FPV open reading frame (ORF)193-198, 201 and 203. Interestingly, reticuloendotheliosis virus (REV) sequences present in the genome of all FPV were absent in CPV. Although, the results of a phylogenic analysis suggested that CPV is a member of the genus Avipoxvirus, its unique antigenic, biologic and genetic characteristics distinguish it from FPV to be considered as a new member of this genus.

Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination.

Infection of swine with virulent porcine reproductive and respiratory syndrome (PRRS) virus induced a rapid, robust antibody response that comprised predominantly nonneutralizing antibodies and waned after approximately 3 months. In contrast, the initial onset of virus-specific interferon (IFN)-gamma-secreting cells (SC) in the pig lymphocyte population remained at a fairly low level during this period and then increased gradually in frequency, plateauing at 6 months postinfection. A similar polarization of the host humoral and cellular immune responses was also observed in pigs immunized with a PRRS-modified live virus (MLV) vaccine. Even coadministration of an adjuvant that enhanced the immune response to a pseudorabies (PR) MLV vaccine failed to alter the induction of PRRS virus-specific IFN-gamma SC (comprising predominantly CD4/CD8 alpha double positive memory T cells with a minority being typical CD4(-)/CD8 alpha beta(+) T cells) and the generation of neutralizing antibodies. Moreover, unlike inactivated PR virus, nonviable PRRS virus did not elicit virus-neutralizing antibody production. Presumably, an intrinsic property of this pathogen delays the development of the host IFN-gamma response and preferentially stimulates the synthesis of antibodies incapable of neutralization.

Reticuloendotheliosis virus sequences within the genomes of field strains of fowlpox virus display variability.

Nine field strains of fowlpox virus (FPV) isolated during a 24-year span from geographically diverse outbreaks of fowlpox in the United States were screened for the presence of reticuloendotheliosis virus (REV) sequences in their genomes by PCR. Each isolate appeared to be heterogeneous in that either a nearly intact provirus or just a 248- or 508-nucleotide fusion of portions of the integrated REV 5' and 3' long terminal repeats (LTRs) was exclusively present at the same genomic site. In contrast, four fowlpox vaccines of FPV origin and three originating from pigeonpox virus were genetically homogeneous in having retained only the 248-bp LTR fusion, whereas two other FPV-based vaccines had only the larger one. These remnants of integrated REV presumably arose during homologous recombination at one of the two regions common to both LTRs or during retroviral excision from the FPV genome. Loss of the provirus appeared to be a natural event because the tripartite population could be detected in a field sample (tracheal lesion). Moreover, the provirus was also readily deleted during propagation of FPV in cultured cells, as evidenced by the detection of truncated LTRs after one passage of a plaque-purified FPV recombinant having a "genetically marked" provirus. However, the deletion mutants did not appear to have a substantial replicative advantage in vitro because even after 55 serial passages the original recombinant FPV was still prevalent. As to the in vivo environment, retention of the REV provirus may confer some benefit to FPV for infection of poultry previously vaccinated against fowlpox.

Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus.

The spike 1 (S1) surface glycoprotein of infectious bronchitis virus (IBV) is the major inducer of the generation of virus neutralizing antibodies, and the administration of purified S1 has been shown to elicit a protective immune response against virulent virus challenge. On the basis of these observations, recombinant fowl poxvirus (rFPV) containing a cDNA copy of the S1 gene of IBV Mass 41 (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. Initially, rFPV-S1 was shown to express the S1 in vito by indirect immunofluorescence staining and western blot analyses. Later, in vivo expression was demonstrated by the detection of IBV-specific serum immunoglobulin G and neutralization antibodies in the sera of chickens immunized with rFPV-S1. That the recombinant virus elicited anti-IBV protective immunity was indicated by the manifested, relatively mild clinical signs of disease, decreased titers of recovered challenge virus, and less severe histologic changes of the tracheas in virulent IBV Mass 41-challenged chickens previously receiving rFPV-S1 as compared with parental fowl poxvirus (FPV)-vaccinated control birds. In contrast, chickens immunized with either recombinant or parental FPV were resistant to a subsequent virulent FPV challenge. As to a preferred method of immunization, wing web administration appeared to be superior to the subcutaneous route because a greater percentage of birds vaccinated by the former protocol exhibited an anti-IBV humoral immune response. Thus, rFPV-S1 has potential as a poultry vaccine against both fowl pox and infectious bronchitis.