NGS method by library enrichment for rapid pestivirus purity testing in biologics.

NGS sequencing was evaluated to understand its added value for animal health vaccine candidates. We have previously established the proof of concept for its application in purity testing on several Master Seeds. Here we evaluate the NGS method after enrichment to detect pestiviruses. To achieve this, we conducted a spiking study using 6 viruses, consisting of 3 pestiviruses and 3 other RNA-viruses at different concentrations into cell suspension. A deep Illumina random sequencing of all nucleic acids (DNA and RNA) was performed. The bioinformatics analysis including both assembly into contigs and annotation were processed using viral public databases for the spiked viruses' identification. Here we present the results of spiking experiments for the simultaneous spike of 6 viruses at 100-10 and 1 TCID50/ml. Using Illumina sequencing, the 3 pestiviruses were all detected at the highest concentration, and even at the lowest one such as 1 TCID50/ml for CSFV. Regarding the other viruses, they were not detected at all. Overall, the study showed consistent results for specific detection of pestiviruses with an increase of sensitivity after enrichment. The sensitivity of NGS evaluated by virus spiking experiments of cells demonstrated that NGS method is a valuable and sensitive tool for specific agent detection required in purity testing during vaccine development. This NGS method should be considered as an alternative tool of current purity testing for the prospective testing of biological products.

Involvement of the Wnt pathway in BVDV cytopathogenic strain replication in primary bovine cells.

BACKGROUND: Bovine viral diarrhea virus 1 (BVDV-1) of the pestivirus genus is an economically crippling virus in the cattle industry; this positive RNA virus causes mucosal disease resulting in reproductive losses and other disease syndromes. The pathogenesis mechanism of the disease caused by BVDV infection is not well understood; for a better understanding of in vivo host BVDV-1 interactions, we conducted a transcriptomic study of infected cells at different times post-infection. METHODS: We compared the permissiveness and cellular response of a BVDV-1 cytopathogenic strain on Madin-Darby Bovine Kidney cells (MDBK) and bovine lung primary cells, a model closer to in vivo infection. Then a RNAseq analysis was realized on the infected bovine lung primary cells, at 10 hpi and 30 hpi (hours post-infection), to identify transcriptomic signatures. RESULTS: RNAseq analysis on BVDV-1 infected bovine primary cells showed 2,759 and 5,376 differentially expressed genes at respectively 10 hpi and 30 hpi with an absolute Fold Change ≥ 2. Among the different pathways deregulated, data analysis revealed a deregulation of Wnt signaling pathway, a conserved process that play a critical role in embryogenesis, cellular proliferation, and differentiation as well as in viral responses against viruses such as Influenza or Hepatitis C. We demonstrated here that the deregulation of the Wnt/ßcatenin signaling pathway plays a role in viral replication of BVDV cp strain. Interestingly, we showed that the inhibition of this Wnt pathway using two inhibitors, FZM1 and iCRT14, induced a delay in onset of the establishment of a cytopathic effect of primary cells. CONCLUSIONS: Thereby, this study highlighted a role of the Wnt signaling pathway in the BVDV-1 viral replication in bovine cells, suggesting an interesting option to explore as a new therapeutic target.

Identification of differentially expressed gene pathways between cytopathogenic and non-cytopathogenic BVDV-1 strains by analysis of the transcriptome of infected primary bovine cells.

The bovine viral diarrhea virus 1 (BVDV-1), belonging to the Pestivirus genus, is characterized by the presence of two biotypes, cytopathogenic (cp) or non-cytopathogenic (ncp). For a better understanding of the host pathogen interactions, we set out to identify transcriptomic signatures of bovine lung primary cells (BPCs) infected with a cp or a ncp strain. For this, we used both a targeted approach by reverse transcription droplet digital PCR and whole genome approach using RNAseq. Data analysis showed 3571 differentially expressed transcripts over time (Fold Change >2) and revealed that the most deregulated pathways for cp strain are signaling pathways involved in responses to viral infection such as inflammatory response or apoptosis pathways. Interestingly, our data analysis revealed a deregulation of Wnt signaling pathway, a pathway described in embryogenesis, that was specifically seen with the BVDV-1 cp but not the ncp suggesting a role of this pathway in viral replication.

Quantitative Real-Time PCR Assays for the Detection of Pathogenic Leptospira Species in Urine and Blood Samples in Canine Vaccine Clinical Studies: a Rapid Alternative to Classical Culture Methods.

Leptospirosis is a vaccine-preventable bacterial zoonotic disease caused by pathogenic Leptospira species. The efficacy of Leptospira canine vaccines is assessed by challenging vaccinated and control dogs with virulent serovars of Leptospira, followed by detection of Leptospira in blood and urine. We assessed the consistency between results obtained for urine and blood samples from clinical studies with species-specific real-time quantitative PCR (qPCR) targeting the lipL32 gene and those obtained with the reference culture method. The specificity of the qPCR assay was confirmed by negative results for nonpathogenic Leptospira and for several canine viruses, bacteria, and parasites. The results from the two methods were compared using McNemar's test, kappa coefficient (κ), and percentage of agreement analyses. The results for numbers of positive and negative dogs were similar, with no false-negative results with the qPCR assay. For both blood and urine, there was strong agreement between the culture method and qPCR results (κ = 0.68 [95% confidence interval (CI), 0.62 to 0.74] and κ = 0.65 [95% CI, 0.59 to 0.71], respectively). However, there was a statistically significant difference between blood samples (P < 0.001) and urine samples (P = 0.028). The negative percentage agreements were 97% and 84% and the positive percentage agreements were 68% and 83% for blood and urine samples, respectively. Although the cell culture method is the recommended gold standard, our results show that qPCR assay is a valid alternative method for the rapid and specific detection of pathogenic Leptospira spp. in urine and blood samples during vaccine efficacy studies, without loss of sensitivity.

Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine.

Mycoplasmas are potential contaminants that introduce undesirable changes in mammalian cell cultures. They frequently contaminate cell substrates and other starting materials used for manufacturing cell-derived biologics, such as vaccines and pharmaceutical products. Mycoplasma purity testing of live vaccines, active ingredients, raw material, and seed lots is required during vaccine production. Previously, testing using a time-consuming, costly 28-day culture assay, which lacks sensitivity for species that do not grow in culture, was required in the European Pharmacopoeia (Ph. Eur). But now nucleic acid amplification techniques (NATs) can be used. NATs provide rapid results and are sensitive. We evaluated the sensitivity and specificity of a commercially-available NAT to detect individual mycoplasma DNA in a veterinary modified live vaccine using five reference strains recommended by the Ph. Eur. Our results showed that this NAT-based method can be used to detect mycoplasma in spiked live vaccine, without interference from the vaccine components, with a limit of detection of 10 CFU/mL, as required by the Ph. Eur. Its specificity was demonstrated since no mycoplasmas were detected in non-spiked vaccine. This method is undergoing validation as a replacement for the conventional culture method in the production of veterinary live vaccines.

Human Langerhans cells express a specific TLR profile and differentially respond to viruses and Gram-positive bacteria.

Dendritic cells (DC) are APCs essential for the development of primary immune responses. In pluristratified epithelia, Langerhans cells (LC) are a critical subset of DC which take up Ags and migrate toward lymph nodes upon inflammatory stimuli. TLR allow detection of pathogen-associated molecular patterns (PAMP) by different DC subsets. The repertoire of TLR expressed by human LC is uncharacterized and their ability to directly respond to PAMP has not been systematically investigated. In this study, we show for the first time that freshly purified LC from human skin express mRNA encoding TLR1, TLR2, TLR3, TLR5, TLR6 and TLR10. In addition, keratinocytes ex vivo display TLR1-5, TLR7, and TLR10. Accordingly, highly enriched immature LC efficiently respond to TLR2 agonists peptidoglycan and lipoteichoic acid from Gram-positive bacteria, and to dsRNA which engages TLR3. In contrast, LC do not directly sense TLR7/8 ligands and LPS from Gram-negative bacteria, which signals through TLR4. TLR engagement also results in cytokine production, with marked differences depending on the PAMP detected. TLR2 and TLR3 ligands increase IL-6 and IL-8 production, while dsRNA alone stimulates TNF-alpha release. Strikingly, only peptidoglycan triggers IL-10 secretion, thereby suggesting a specific function in tolerance to commensal Gram-positive bacteria. However, LC do not produce IL-12p70 or type I IFNs. In conclusion, human LC are equipped with TLR that enable direct detection of PAMP from viruses and Gram-positive bacteria, subsequent phenotypic maturation, and differential cytokine production. This implies a significant role for LC in the control of skin immune responses.

The class 6 semaphorin SEMA6A is induced by interferon-gamma and defines an activation status of langerhans cells observed in pathological situations.

Originally implicated in axon guidance, semaphorins represent a large family of molecules that are now known to be expressed in the immune system. Among different semaphorins tested by reverse transcriptase-polymerase chain reaction in human immune cells, the expression of class 6 transmembrane semaphorin SEMA6A was restricted to dendritic cells (DCs). Using in-house generated monoclonal antibodies, SEMA6A expression appeared further restricted to Langerhans cells (LCs). In vivo, SEMA6A mRNA was expressed in freshly isolated skin LCs but SEMA6A protein was not detectable on normal skin and tonsillar epithelium. Of interest, SEMA6A protein was strongly expressed on skin and bone LCs and on LCs in draining lymph nodes from patients with LC histiocytosis or dermatopathic lymphadenitis, respectively, representing two inflammatory conditions in which LCs display an immature DC-LAMP(low), CD83(low), and CCR7+ phenotype. SEMA6A expression was low in resting LCs generated in vitro and was enhanced by interferon (IFN)-gamma but not by interleukin-4, interleukin-10, IFN-alpha/beta, or lipopolysaccharide. Most IFN-gamma-induced SEMA6A-positive cells remained immature with low CD83 and DC-LAMP/CD208 expression, but they expressed CCR7 and responded to macrophage inflammatory protein-3beta (MIP-3beta/CCL19). The expression of SEMA6A, for which the ligand and function remain unknown, may therefore identify an alternative IFN-gamma-dependent activation status of LCs in vivo.

Fc receptor gamma-chain activation via hOSCAR induces survival and maturation of dendritic cells and modulates Toll-like receptor responses.

We previously reported the characterization of human osteoclast-associated receptor (hOSCAR), a novel Fc receptor gamma-chain (FcRgamma)-associated receptor expressed by myeloid cells. Here we show that ligation of hOSCAR by specific antibodies promotes dendritic cell (DC) survival by an extracellular signal-regulated kinase (ERK)- and phosphatidylinositol 3-kinase (PI3K)-dependent pathway, linked to expression of the Bcl-2 and Bcl-x(L) antiapoptotic molecules. Crosslinking of hOSCAR leads to maturation of DCs, as demonstrated by up-regulation of maturation markers, decrease in dextran uptake capacity, and secretion of immunesystem effectors such as interleukin-8 (IL-8)/CXC chemokine ligand 8 (CXCL8), IL-12 p40, monocyte chemoattractant protein-1 (MCP-1)/chemokine receptor ligand 2 (CCL2) and macrophage-derived chemokine (MDC)/CCL22. Stimulation of hOSCAR acts in conjunction with the Toll-like receptor (TLR) ligands, lipopolysaccharide (LPS), R-848, and polyinosinic-polycytidylic acid (poly(I:C)), to increase the expression of maturation markers, and to modulate cytokine release. A PI3K-dependent up-regulation of IL-10 release is observed with all the TLR ligands used, whereas regulation of IL-12 production is variable depending on the TLR stimulated. hOSCAR engagement on DCs did not significantly increase the proliferation of naive T cells; however, when co-incubated with TLR ligands, an enhanced proliferation was observed. The percentage of interferon (IFN)-gamma-producing T cells is decreased when hOSCAR engagement is combined with LPS stimulation. Altogether, these data suggest that hOSCAR may modulate the responses of both innate resistance and adaptive immunity.

CD208/dendritic cell-lysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes.

Dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas.

Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.

Human dendritic cells express neuronal Eph receptor tyrosine kinases: role of EphA2 in regulating adhesion to fibronectin.

Eph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+ progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a beta1 integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking.

MHC class II compartments in human dendritic cells undergo profound structural changes upon activation.

Immature dendritic cells efficiently capture exogenous antigens in peripheral tissues. In an inflammatory environment, dendritic cells are activated and become highly competent antigen-presenting cells. Upon activation, they lose their ability for efficient endocytosis and gain capability to migrate to secondary lymphoid organs. In addition, peptide loading of MHC class II molecules is enhanced and MHC class II/peptide complexes are redistributed from an intracellular location to the plasma membrane. Using immuno-electron microscopy, we show that activation of human monocyte-derived dendritic cells induced striking modifications of the lysosomal multilaminar MHC class II compartments (MIICs), whereby electron-dense tubules and vesicles emerged from these compartments. Importantly, we observed that MHC class II expression in these tubules/vesicles transiently increased, while multilaminar MIICs showed a strongly reduced labeling of MHC class II molecules. This suggests that formation of the tubules/vesicles from multilaminar MIICs could be linked to transport of MHC class II from these compartments to the cell surface. Further characterization of endocytic organelles with lysosomal marker proteins, such as the novel dendritic cell-specific lysosomal protein DC-LAMP, HLA-DM and CD68, revealed differential sorting of these markers to the tubules and vesicles.

Subtractive hybridization reveals the expression of immunoglobulin-like transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells.

Recent studies in humans have highlighted the importance of a distinct cellular entity, the plasmacytoid dendritic cell (PDC). To identify genes for which expression is restricted to human PDCs, a cDNA subtraction technique was applied using cDNA from activated monocyte-derived DCs (MDDCs) as competitor. In the 650 sequences analyzed, 25% were for B-cell transcripts. We also found lymphoid-related genes, immunoglobulinlike transcript 7 (ILT7), granzyme B (GrB), Spi-B, and the receptor tyrosine kinase Eph-B1. Granzyme B was up-regulated on activation, and protein was detected only in PDCs. Eph-B1 protein was expressed in the cytoplasm and the nuclei of PDCs and MDDCs, respectively. Interestingly, several novel molecules have been identified that were predicted to encode for a type 2 transmembrane protein (BRI(3)), a putative cytokine (C-15, a cysteine-rich-secreted protein), and a type 1 leucine-rich repeat protein (MAPA). The identification of genes expressed in PDCs provides new insights into their function and origin.