A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2.

In this communication we present the detailed disulfide structure of IgG2 molecules. The consensus structural model of human IgGs represents the hinge region positioned as a flexible linker connecting structurally isolated Fc and Fab domains. IgG2 molecules are organized differently from that model and exhibit multiple structural isoforms composed of (heavy chain-light chain-hinge) covalent complexes. We describe the precise connection of all the disulfide bridges and show that the IgG2 C H1 and C-terminal C L cysteine residues are either linked to each other or to the two upper hinge cysteine residues specific to the IgG2 subclass. A defined arrangement of these disulfide bridges is unique to each isoform. Mutation of a single cysteine residue in the hinge region eliminates these natural complexes. These results show that IgG2 structure is significantly different from the conventionally accepted immunoglobulin structural model and may help to explain some of the unique biological activity attributed only to this subclass.

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins.

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