A new epitope on swine CD5 molecule detected by monoclonal antibody 5F12/9.

This paper describes the production and characterization of a monoclonal antibody (MAb), 5F12/9, that recognizes a new epitope on porcine CD5. Conformation of its CD5 specificity was obtained by means of sequential immunoprecipitation and Western blot experiments in combination with anti-CD5 MAb 1H6/8, whereas cross-blocking experiments with both MAbs showed that they reacted with different epitopes.

Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs.

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.

A porcine cell surface receptor identified by monoclonal antibodies to SWC3 is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase SHP-1.

SWC3 was defined at the First International Swine CD Workshop as a specific myelomonocytic antigen of 230 kDa with mAbs 74-22-15, 6F3 and DH59B. In this report, we describe two new mAbs (BL1H7 and BA1C11) that react selectively with granulocytes, monocytes and macrophages. These monoclonal antibodies (mAbs) recognize a molecule in the range of 90-115 kDa in immunoprecipitation and/or Western blotting analyses. Two-colour FACS analyses showed that the distribution of BL1H7 and BA1C11 antigens was identical to that of SWC3. Moreover, in this assay, mAb 74-22-15 appeared to partially block the binding of mAbs BL1H7 and BA1C11, suggesting that all these mAbs reacted with the same or spatially close epitopes. Cross-blocking analyses indicated that it was the case with mAbs 74-22-15 and BL1H7. Immunoprecipitation experiments with mAbs 74-22-15, BL1H7 and BA1C11, followed by immunoblotting with mAb BL1H7 confirmed that all three mAbs recognize the same molecule. Analysis of the N-terminal sequence carried out on the affinity purified protein revealed homology with members of signal regulatory protein (SIRP) family. Like other members of this family, after treatment with sodium pervanadate, SWC3 became phosphorylated in tyrosines, and associated with the protein-tyrosine phosphatase SHP-1.

Induction of aggregation in porcine lymphoid cells by antibodies to CD46.

CD46 is a major transmembrane glycoprotein that belongs to the regulator of complement activation (RCA) family. Recently, mAbs to human CD46 were shown to suppress IL-12 production. Here, we describe that mAbs against different porcine CD46 epitopes induced a marked adhesion of normal lymphocytes. Addition of low amounts of antibody to freshly isolated lymphocytes or thymocytes resulted in the clustering of the cells. Cross-linking of CD46 molecules seems essential since Fab fragments failed to induce aggregation. This aggregation was dependent on active cell metabolism and on the presence of divalent cations and required a functional cytoskeleton. It was not inhibited by antibodies to CD18, CD29, CD2, CD11a and CD11b. Staurosporine, an inhibitor of protein kinases, partially blocked the aggregation. This finding is indicative of a role of protein kinases in the transduction of the signal generated by CD46 engagement.

Several protein regions contribute to determine the nuclear and cytoplasmic localization of the influenza A virus nucleoprotein.

A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the transport of influenza virus ribonucleoprotein complexes into and out of the nucleus.

Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP).

Pig membrane cofactor protein (MCP; CD46) is a 50 000-60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals - a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I.

Monoclonal antibodies 2F6/8 and 2A10/8 recognize a porcine antigen (SWC7) expressed on B cells and activated T cells.

This report describes the production and characterization of two monoclonal antibodies (mAbs), 2F6/8 and 2A10/8, that recognize a porcine antigen (SWC7) expressed on B cells in lymphoid tissues. The antigen was not detectable on resting PBMC but its expression could be induced after treatment with phorbol esters (PMA) but not by ConA, PWM, LPS or Ca ionophore. Kinetic studies showed that the antigen was expressed 24 h after PMA treatment, peaked at day 2 or 3 and slightly declined by day 6. Interestingly, the antigen was also found on a subset of CD3 + T cells, with levels of expression similar to those of B cells. By immunohistochemistry, the antigen was detected on follicular dendritic cells of germinal centers in tonsils, spleen, lymph nodes and Peyer's patches. MAb 2F6/8 precipitates a molecule of approximately 40 kDa under non-reducing conditions, and 24 kDa under reducing conditions. The restricted and tightly regulated expression of this antigen may reflect an important role in B cell differentiation within the germinal center. These mAbs will be useful reagents for phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.

Analyses of monoclonal antibodies reacting with porcine CD5: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies analyzed within the T-cell group, three mAbs fell within cluster T13 including the CD5a standard b53b7 (No. 174). The two new mAbs 1H6/8 (No. 058) and BB6-9G12 (No. 166) both precipitated 55 and 60 kDa proteins that were of similar molecular weights as the standard. Staining patterns on the various cell types were similar. Both new antibodies inhibited the binding of the CD5a reference mAbs b53b7 to peripheral lymphocytes. These mAbs, therefore both react with the CD5a epitope bringing the number of anti-porcine CD5 mAbs to eight, all of which appear to recognize the same epitope.

Analyses of monoclonal antibodies reacting with porcine wCD6: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9.

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.

Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus.

Antibody linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salmonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout antibodies. Among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa & Coll 1996; Virology 216:60-70) and leucocyte stimulating peptides (Lorenzo et al. 1995; Virology 212:348-355). Among 17 monoclonal antibodies (MAbs), only 2 non-neutralizing MAbs, 110 (aa 139-153) and IP1H3 (aa 399-413), could be mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences. None of the neutralizing MAbs tested reacted with any of the gpG peptides. Previously mapped MAb resistant mutants in the gpG did not coincide with any of the linear epitopes defined by the pepscan strategy, suggesting the complementarity of the 2 methods for the identification of antibody recognition sites.

Monoclonal antibodies to a high molecular weight isoform of porcine CD45: biochemical and tissue distribution analyses.

This report describes the obtention and characterization of two monoclonal antibodies (mAbs), 6E3/7 [mAb 6E3/7 was submitted to the Second International Swine CD Workshop, where it has been assigned to CD45R] and 3C3/9, which recognize the isoform of highest molecular weight of porcine CD45. This conclusion is based on their cell reactivity and tissue distribution, identical to that reported for the human high molecular weight isoform of CD45, and on data from immunoprecipitation and immunoblotting analyses which show that these mAbs react with the largest polypeptide of those precipitated by mAb 2A5, that recognizes an epitope shared by all CD45 isoforms. These mAbs react with 60% of peripheral blood mononuclear cells (PBMC) but not with alveolar macrophages, granulocytes, platelets or erythrocytes. Antigen expression on PBMC is heterogeneous and is reduced after in vitro activation with mitogens. B cells and CD8+ T cells express more antigen than CD4+ T cells. Using immunoperoxidase techniques, the antigen was detected on B cell areas of lymph nodes and Peyer's patches, and on a subpopulation of medullary thymocytes. These mAbs will be useful reagents for functional and phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.

Characterization of five monoclonal antibodies specific for swine class II major histocompatibility antigens and crossreactivity studies with leukocytes of domestic animals.

A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized. These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation. By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules. mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattle but not those of human, dog and horse. These mAbs effectively blocked the mixed lymphocyte reaction and the proliferative response to viral antigens (African swine fever virus) and to staphylococcal enterotoxin B. Therefore, these mAbs can be useful reagents for studying MHC class II molecules of pig and crossreactive species, and the immunological processes where they are involved.

Monoclonal antibodies specific for porcine monocytes/macrophages: macrophage heterogeneity in the pig evidenced by the expression of surface antigens.

Macrophages are widely distributed in most tissues of the body, where they play important roles in host defense and repair of tissue damage. In this report we describe the production and characterization of a panel of six monoclonal antibodies (mAb) against porcine macrophages and their use for phenotyping tissue macrophages. All mAbs were produced by immunizing mice with porcine alveolar macrophages. Three of them (2A10/11, 3B11/11 and 3F7/11) react mainly with macrophages and, at a lower extent, blood monocytes, whereas the others (1E12/11, 2C12/10 and 4E9/11) also recognize granulocytes. Antigens recognized by these antibodies could be characterized by Western blot and/or immunoprecipitation, with the exception of that one recognized by 2C12/10. By their behavior in SDS-PAGE under reducing and nonreducing conditions, all seem to be single polypeptides, whose apparent molecular weight under reducing conditions are: 1E12/11 and 3B11/11 larger than 204 kDa; 2A10/11, 150 kDa; 4E9/11, 125-170 kDa; and 3F7/11, 135 kDa. Immunohistochemical analyses of both lymphoid and non-lymphoid organs using these mAbs reveal important antigenic heterogeneity among tissue macrophages. These mAbs are, therefore, useful tools for the study of porcine macrophage maturation and differentiation and for determining their heterogeneity both in normal and pathological conditions.

Monoclonal antibody 2F4/11 recognizes the alpha chain of a porcine beta 2 integrin involved in adhesion and complement mediated phagocytosis.

The characterization of a new mAb, named 2F4/11, specific for porcine myelomonocytic cells is described. This mAb immunoprecipitates a non-covalently linked heterodimer of 155,000/95,000, which is expressed by granulocytes, monocytes and tissue macrophages but not by lymphocytes, erythrocytes or platelets. Immunoblot analysis localizes the 2F4/11 epitope on the largest subunit of the heterodimer. Mab 2F4/11 is able to block phagocytosis of complement-opsonized zymosan particles by PMN granulocytes and alveolar macrophages, as well as adherence to plastic surfaces of PMA-activated PMN. Together, these results suggest that mAb 2F4/11 recognizes the CD11b or alpha chain of the porcine complement type 3 receptor (CR3).