Oliveira, D. S. L. d. | Search bioRxiv

bioRxiv2021

Hinge Influences in Murine IgG Binding to Cryptococcus neoformans Capsule

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One such case is that of the 3E5 antibodies, a family of monoclonal murine IgGs in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with a mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in binding of antibodies to the antigen could affect the design of therapeutic antibodies and our understanding of their function in immunity. Key pointsO_LIKey point 1- 2H1 antibodies recapitulate differences between mIgG isotypes observed with 3E5. C_LIO_LIKey point 2 - The hinge region is necessary but not sufficient for these differences. C_LIO_LIKey point 3 - The antibody constant region can also play a role in mIgG binding to antigen. C_LI

immunology↗

bioRxiv2020

Paracoccidioides HSP90 can be found in the cell surface and is a target for antibodies with therapeutic potential

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the hosts antifungal immunity. In this work we show the generation of and promising results with a mAb against HSP90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM. Author summaryParacoccidioidomycosis (PCM) is a severe disease caused by fungi, common in Latin America. It is treatable, but some of the drugs that are available are very toxic or not very effective, and the treatment can take as long as two years to clear the infection. To address the need for improved therapeutic alternatives, we have been developing drug candidates based on antibody technologies against Paracoccidioides brasiliensis and P. lutzii, which cause PCM. In this work, we produced monoclonal antibodies (mAbs) that bind to the fungal protein HSP90, which is essential for fungal cells to survive. One mAb, 4D11, recognized the HSP90 target on the surface of fungal cells. These antibody-covered cells were ingested more efficiently by immune cells called macrophages, suggesting they could improve the host resistance to infection by Paracoccidioides. Future improvements on these antibodies could thus lead to more effective and safer PCM treatments.

microbiology↗