Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector.

Traditional virus infectivity titration methods for lymphocytic choriomeningitis virus (LCMV) are laborious, time-consuming, and low-throughput (e.g., focus forming unit (FFA) assay). In this report, we developed a high-throughput reverse transcription quantitative PCR (RT-qPCR)-based virus infectivity assay for relative quantitation of a live, recombinant replicating LCMV -based viral vector (TT1). This in vitro infectivity assay demonstrated a 4-log linear range for TT1 titer quantitation. A high positive Pearson correlation coefficient value (≥ 0.80) was obtained between the RT-qPCR vs. the "gold-standard" FFU assay when comparing the stability profiles of stressed TT1 vector samples. In addition to the RT-qPCR infectivity assay, the stability of the TT1 vector upon freeze-thaw stress was investigated further with complementary viral particle characterization techniques (e.g., TEM, NTA, MFI). Correlations between viral infectivity and particle measurements during forced degradation studies were observed to be specific to the TT1 vector and its various formulations and such results facilitated the rank-ordering of formulation conditions. Overall, this infectivity RT-qPCR method showed increased sample throughput and improved assay flexibility compared to traditional viral infectivity assays. These results are discussed in the context of enabling future TT1 vector formulation development work, and potential utilization as an in-process monitoring tool during TT1 vector manufacturing.

Investigations of a rabbit (Oryctolagus cuniculus) model of systemic lupus erythematosus (SLE), BAFF and its receptors.

B-cell activation factor belonging to the tumor necrosis factor family (BAFF) is a major contributor to survival of B lymphocytes during development and maturation. A relationship between circulating BAFF levels and disease activity has been reported in patients with the autoimmune disease Systemic Lupus Erythematosus (SLE). Clinical trials targeting BAFF or its receptors are currently in progress. In order to further characterize a rabbit (Oryctolagus cuniculus) model of SLE, we investigated the expression of BAFF and its receptors in non-inbred, pedigreed rabbits derived from breeding and selection based on autoantibody responses. We immunized rabbits related to previous groups that developed autoantibodies and inflammatory responses after immunizations with peptides synthesized on multiple antigen-branched polylysine backbones. Blood and sera collected before immunization and after boosts were used for health monitoring, analyses of serum autoantibody responses by ELISA and immunofluorescence. Peripheral blood mononuclear cells (PBMC) were studied by flow cytometry and were the source of mRNA for quantitative PCR analyses. We hypothesized that BAFF mRNA expression and serum BAFF levels measured indirectly through BAFF receptor binding might increase in autoantibody-producing rabbits. Immunized rabbits developed elevated levels of leucocyte populations, anti-nuclear, anti-dsDNA and other autoantibodies. BR3 mRNA levels in total PBMC decreased and BAFF levels remained low and unchanged in most immunized rabbits. By flow cytometry, percentages of BAFF positive cells decreased. Percentages of transmembrane activator and CAML interactor (TACI) decreased in most rabbits from all the immunized groups. The rabbit is an important model for human autoimmune and infectious diseases, and a high quality draft rabbit genome assembly was recently completed. Human disease models developed in non-inbred pedigreed animals are better able to reflect the complexities of diseases such as SLE with familial patterns of inheritance. Although no consistent pattern of elevated expression of BAFF mRNA or protein was found in the rabbits studied, the data collected and reported here build upon previous data to refine understanding of a rabbit model of SLE.

Characterization of rabbit CD5 isoforms.

Previously described polyclonal or monoclonal antibodies (mAb) to rabbit CD5, raised against expressed recombinant protein or peptides, recognize CD5 on most rabbit B cells. The mAb KEN-5 was originally reported to recognize rabbit CD5. However, KEN-5 binds almost exclusively to T cells and only to a minor population of B cells. We show here that by Enzyme-linked Immunosorbent Assay (ELISA), KEN-5 binds to recombinant rabbit CD5. This interaction is partially inhibited by polyclonal goat anti-CD5 antibody. In addition, immunoprecipitations from lysates of surface biotinylated rabbit lymphocytes with KEN-5 or our anti-CD5 mAb isolate molecules that migrate identically on gels with the same approximate relative molecular mass of 67,000 M(r). By flow cytometric analyses of individual cells from spleen, thymus and appendix, KEN-5 recognizes CD5-like molecules mainly on T cells and on 3-6% of IgM(+) B cells. Immunohistochemical staining of splenic and appendix tissues and confocal immunofluorescent imaging confirm and extend results from flow cytometric analyses. Quantitation of fluorescent colocalization indicates that staining by KEN-5 colocalizes with staining by anti-CD5 on small percentage lymphocytes in splenic tissue sections. As CD5 has both N- and O-linked glycosylation, we hypothesised that differential binding of KEN-5 to T cells and B-cells may be explained by different glycan structures on the CD5 present on T compared to B cells. This hypothesis is supported by ELISA data that show that deglycosylation diminishes the binding of KEN-5 to recombinant rabbit CD5. Screening KEN-5 on an array with 406 glycans was inconclusive. Although we did not identify a strongly binding glycan structure, the data are suggestive that the epitope recognized by KEN-5 may be influenced by glycan structures. The epitope this mAb recognizes may either be the glycan itself, or more likely, is influenced by neighboring glycan structure. Our findings suggest that development, selection and function of different B- and T-cell subsets or their preferential survival may be directly or indirectly dependent on different glycan structures associated with CD5 or CD5-like molecules expressed on T cells compared to B cells.

Expression and localization of rabbit B-cell activating factor (BAFF) and its specific receptor BR3 in cells and tissues of the rabbit immune system.

Rabbits are widely used for vaccine development, and investigations of human infectious and autoimmune diseases such as Systemic Lupus Erythematosus (SLE). For these applications, we cloned, sequenced and expressed rabbit B-cell Activating Factor (BAFF), and localized BAFF in cells and tissues of the rabbit immune system. The rabbit homolog of the human BAFF binding site (miniBR3 peptide) within the BAFF-specific receptor BR3 was synthesized. This 26-residue core domain binds to recombinant rabbit BAFF protein. Flow cytometric analyses using purified recombinant rabbit BAFF combined with real-time PCR findings revealed that BAFF detected on peripheral blood B-cells from normal rabbits is probably complexed to BAFF receptors rather than produced by the B-cells. BAFF was detected in developing appendix of young rabbits by immunohistochemical staining suggesting that BAFF plays a role during the period following birth when rabbit B-cell development and pre-immune antibody repertoire diversification and selection is occurring.

Biomarkers of Contaminant Exposure in Chub (Leuciscus cephalus L.) - Biomonitoring of Major Rivers in the Czech Republic.

Biochemical analysis of organisms to assess exposure to environmental contaminants is of great potential use. Biochemical markers, specifically liver enzymes of the first and the second phase of xenobiotic transformation - cytochrome P450 (CYP 450), ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST) and tripeptide reduced glutathione (GSH) - were used to assess contamination of the aquatic environment at 12 locations near the mouths of major rivers in the Czech Republic. These rivers were the Luznice, Otava, Sázava, Berounka, Vltava, Labe, Ohre, Svratka, Dyje, Morava and Odra. The indicator species selected was the Chub (Leuciscus cephalus L.). The highest levels of CYP 450 and EROD catalytic activity were found in livers of fish from the Labe (Obríství) (0.32±0.10 nmol mg-1 protein and 1061.38±545.51 pmol min-1 mg-1 protein, respectively). The highest levels of GST catalytic activity and GSH content were found in fish from the Otava (35.39±13.35 nmol min-1 mg-1 protein and 4.29±2.10 nmol GSH mg-1 protein, respectively). They were compared with levels of specific inductors of these biochemical markers in muscle. The results confirmed contamination of some river locations (Labe Obríství, Svratka).

CD5+ B cells are preferentially expanded in rabbit appendix: the role of CD5 in B cell development and selection.

Although only a small proportion of mouse and human B cells are CD5(+), most adult rabbit B cells express CD5. However, CD5 was not detectable on the majority of B cells in neonatal appendix 1 and 3days after birth. Cell trafficking studies demonstrated that CD5(+) and CD5(-) CD62L(+) B cells from bone marrow migrated into appendix. There, CD5(+) B cells were preferentially expanded and predominated by approximately 2weeks of age. In mutant ali/ali rabbits, VHa2(+) B cells develop through gene conversion-like alteration of rearranged VH genes upstream of deleted VH1a2. Correlated appearance of individual CD5(+) germinal centers and VHa2(+) B-cells in mutant appendix suggests that CD5 binding positively selects cells with a2(+) framework regions that bind CD5. Following negative and positive selection, cells with diversified rearranged heavy- and light-chain sequences exit appendix, migrate to peripheral tissues and constitute the preimmune repertoire of CD5(+) B cells that encounter foreign antigens.

Stable expression of the extracellular domains of rabbit recombinant CD5: development and characterization of polyclonal and monoclonal antibodies.

Previous studies in our laboratory suggested that there was positive selection of B cells during early development in the appendix of normal and V(H) mutant (ali/ali) rabbits. Preferential expansion and survival of B lymphocytes was affected by the Ig V(H) frameworks 1 and 3 sequences expressed on the cell surface. We demonstrated a specific interaction between rabbit CD5 and the V region of rabbit heavy chains and suggested that CD5 is a potential selecting ligand for B-cell surface immunoglobulin framework region sequences. To further investigate the role of CD5 in rabbit B-cell selection and survival we prepared recombinant constructs and obtained stable expression of the three scavenger receptor cysteine-rich (SRCR) extracellular domains of rabbit CD5. Here we describe the production and purification of this expressed recombinant CD5 protein, polyclonal antibody obtained by immunization of a goat and initial production and characterization of specific mAbs against peptides selected from each sequenced SRCR domain.