Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that emerged in the late 1980s in both Europe and North America as the causative agent of porcine reproductive and respiratory syndrome (PRRS), now the most important disease of swine worldwide. Despite extensive characterization of PRRSV proteins by direct analysis and comparison with other arteriviruses, determinants of virulence, pathogenesis and protective immune recognition remain poorly understood. Thus, we hypothesized that additional ORFs are present in the PRRSV genome that may contribute to its biological properties, and so we screened highly purified virions of strain VR2332, the prototype type 2 PRRSV, for evidence of novel polypeptides. A 51 aa polypeptide was discovered that is encoded by an alternative ORF of the subgenomic mRNA encoding the major envelope glycoprotein, GP5, and which is incorporated into virions. The protein, referred to as ORF5a protein, is expressed in infected cells, and pigs infected with PRRSV express anti-ORF5a protein antibodies. A similar ORF is present as an alternative reading frame in all PRRSV subgenomic RNA5 genes and in all other arteriviruses, suggesting that this ORF5a protein plays a significant role in arterivirology. Its discovery also provides a new potential target for immunological and pharmacological intervention in PRRS.

Purpose: Recombinant Listeria vaccines induce tumor-specific T-cell responses that eliminate established tumors and prevent metastatic disease in murine cancer models. We used dogs with HER2/neu þ appendicular osteosarcoma, a well-recognized spontaneous model for pediatric osteosarcoma, to determine whether a highly attenuated, recombinant Listeria monocytogenes expressing a chimeric human HER2/neu fusion protein (ADXS31-164) could safely induce HER2/neu-specific immunity and prevent metastatic disease. Experimental Design: Eighteen dogs that underwent limb amputation or salvage surgery and adjuvant chemotherapy were enrolled in a phase I dose escalation clinical trial and received either 2 Â 10 8 , 5 Â 10 8 , 1 Â 10 9 , or 3.3 Â 10 9 CFU of ADXS31-164 intravenously every 3 weeks for 3 administrations.Results: Only low-grade, transient toxicities were observed. ADXS31-164 broke peripheral tolerance and induced antigenspecific IFNg responses against the intracellular domain of HER2/neu in 15 of 18 dogs within 6 months of treatment. Furthermore, ADXS31-164 reduced the incidence of metastatic disease and significantly increased duration of survival time and 1-, 2-, and 3-year survival rates when compared with a historical control group with HER2/neu þ appendicular osteosarcoma treated with amputation and chemotherapy alone.Conclusions: These findings demonstrate that ADXS31-164 administered in the setting of minimal residual disease can induce HER2/neu-specific immunity and may reduce the incidence of metastatic disease and prolong overall survival in a clinically relevant, spontaneous, large animal model of cancer. These findings, therefore, have important translational relevance for children with osteosarcoma and adults with other HER2/neu þ cancers.

Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.

To further characterize the humoral immune response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV), direct enzyme-linked immunosorbent assays (ELISA) were used to study the kinetics of antibody responses directed against PRRSV nonstructural proteins in pigs experimentally exposed to the virus. The highest immunoreactivities were against nsp1, nsp2, and nsp7. Using the recombinant nsp7 as an antigen, we validated a dual ELISA for the simultaneous detection and differentiation of serum antibodies against type I and type II PRRSV. Receiver operating characteristic analysis based on 1,334 known-positive and 1,357 known-negative samples showed good specificity (98.3% to type I and 99.3% to type II) and sensitivity (97.4% for type I and 99.8% for type II). To differentiate type I and type II PRRSV, 470 sera originating from experimentally inoculated pigs were tested, and positive sera were correctly differentiated in 469 of 470 samples. The capability of the nsp7 dual ELISA to detect serum antibody responses from pigs infected with various genetically different field strains was determined. The nsp7 dual ELISA possessed 97.6% agreement with the Idexx HerdChek PRRS 2XR ELISA. In further testing of Idexx ELISA suspected false-positive samples, the nsp7 dual ELISA resolved 98% of the samples as negative. Taken together, these results indicate that the nsp7 dual ELISA can be used as a differential test for PRRSV serology with high levels of sensitivity and specificity. This ELISA offers an additional tool for routine or follow-up diagnostics, as well as having substantial value in epidemiological surveys and outbreak investigations.

The incidence of calcium oxalate (CaOx) urolithiasis in dogs has increased steadily over the last two decades. A potential mechanism to minimize CaOx urolithiasis is to reduce enteric absorption of dietary oxalate by oxalate-metabolizing enteric bacteria. Enteric colonization of Oxalobacter formigenes, an anaerobe which exclusively relies on oxalate metabolism for energy, is correlated with absence of hyperoxaluria or CaOx urolithiasis or both in humans and laboratory animals. We thus hypothesized that decreased enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis in dogs. Fecal samples from dogs with CaOx uroliths, clinically healthy, age-, breed- and gender-matched dogs, and healthy non-stone forming breed dogs were screened for the presence of O. formigenes by quantitative PCR to detect the oxalyl CoA decarboxylase (oxc) gene, and by oxalate degrading biochemical activity in fecal cultures. Prevalence of O. formigenes in dogs with CaOx uroliths was 25%, compared to 50% in clinically healthy, age-, breed- and gender-matched dogs, and 75% in healthy non-stone forming breeds. The presence of oxc genes of O. formigenes was significantly higher in healthy non-stone forming breed dogs than in the dogs with CaOx stones. Further, dogs with calcium oxalate stones and the stone-forming breed-matched controls showed comparable levels of biochemical oxalate degrading activity. We conclude that the absence of enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis.

The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model.

Aims The absence of enteric oxalate‐metabolizing bacterial species (OMBS) increases the likelihood of calcium oxalate (CaOx) urolithiasis in humans and dogs. The goal of this study was to compare the gut microbiota of healthy dogs and CaOx stone formed dogs (CaOx‐dogs), especially with respect to OMBS. Methods and Results Faecal samples from healthy and CaOx‐dogs were obtained to analyse the hindgut microbiota by sequencing the V3 region of bacterial 16S rDNA. In total, 1223 operational taxonomic units (OTUs) were identified at 97% identity. Only 38% of these OTUs were shared by both groups. Significant differences in the relative abundance of 152 OTUs and 36 genera were observed between the two groups of dogs. Conclusions The faecal microbiota of healthy dogs is distinct from that of CaOx‐dogs, indicating that the microbiota is altered in CaOx‐dogs. Significance and Impact of the Study This is the first study that has compared the gut microbial diversity in healthy and CaOx‐dogs. Results of this study indicate the future need for functional and comparative analyses of the total array of oxalate‐metabolizing genes between healthy and CaOx stone formers, rather than focusing on specific bacterial species, to understand the critical role of OMBS in CaOx urolithiasis.

Development of a vaccine against congenital infection with human cytomegalovirus (HCMV) is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC) of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV), consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.