Hybridoma Passage In Vitro May Result in Reduced Ability of Antimannan Antibody To Protect against Disseminated Candidiasis

Abstract

We previously reported the enhanced resistance of monoclonal antibodies B6.1 (an immunoglobulin M [IgM]) and C3.1 (an IgG3) against experimental candidiasis. Both MAbs recognize the same fungal epitope. We have since found that a highly passaged B6.1 hybridoma (hp-B6.1) resulted in antibody that has little protective potential. The potential clinical applicability of the antibody and our interest in understanding antibody protection against candidiasis led us to investigate an explanation for this phenomenon. Antibody genetic structure of hp-B6.1, the original hybridoma clone (ori-B6.1) stored frozen since 1995, a subclone of hp-B6.1 that produces protective antibody, the IgG3-producing hybridoma, and a nonprotective IgG1-producing hybridoma were compared. Variable region gene sequences of heavy (V H ) and light chains showed genetic instability of V H chains with only the hp-B6.1; the V H sequences from ori-B6.1 and the subclone were, however, identical. Activation-induced cytidine deaminase levels were greatest in the B6.1 hybridomas, which may explain the instability. The constant region CH3 domain remained unchanged, implying normal N-glycation and complement-fixing potential, and antibody binding affinities appeared unchanged. Complement fixation assays surprisingly showed that ori-B6.1 antibody fixes C3 more rapidly than does hp-B6.1 antibody. The V H region primary structure may affect complement activation, which could explain our result. Indeed, antibody from the hp-B6.1 subclone fixed complement like antibody from ori-B6.1. These results show that the greatest protection occurs when antimannan antibodies possess the dual abilities of recognizing the appropriate carbohydrate epitope and rapidly fixing complement; loss of the latter property results in the loss of protective potential by the antibody.

Advances in combating fungal diseases: vaccines on the threshold

“…On closer examination, however, in at least one case, T H 2-derived antibodies have a protective effect by somehow augmenting cell-mediated immunity 39 . In other cases, antibodies can function as opsonins, promoting fungal ingestion and even killing by phagocytes, and several antibodies are directly fungicidal [40][41][42][43][44][45][46][47] (TABLE 1). These kinds of activities are complementary, rather than exclusive, to cell-mediated mechanisms.…”

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

“…The increase in the number of cell subcultures performed over a time, or cell passages over a time (number of cell passages), could favor the growth of unproductive cell cultures, reduce productivity and reduce the mAb protective potential against infections (Xin and Cutler 2006;Zhu and Yang 2004).…”

“…For instance, it has been reported that murine hybridoma strain AB-1432 lost 50 % of mAb productivity when cultured in DMEM supplemented with 10 % (v/v) fetal bovine serum (FBS) as of P20 (Schmid et al 1990). Whereas hybridoma B6.1 cells maintained at high passages reduced the protective capacity of their secreted mAb (Xin and Cutler 2006). The production instability in recombinant mAb in CHO cells can occur through gene silencing mechanism, or else the progressive loss in the number of copies of the recombinant gene, reducing mAb yield as of P14 or P95 (Beckmann et al 2012;Kim et al 2011).…”

“…Considering cost-effectiveness, it is important to have a cell line secreting proteins or antibodies of interest for development of diagnostics kits (Barnes et al 2003;Schmid et al 1990;Xin and Cutler 2006;Beckmann et al 2012;Kim et al 2011;Li et al 2010;Lee et al 1991). …”

Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis

“…IgE may represent one example, but although a relationship between allergic hypersensitivity and recurrent vaginal candidiasis has been speculated (4), the role in systemic or disseminated mycotic diseases requires more investigation. We showed that certain IgM and IgG 1 antibodies with anti-mannan specificity may not be protective against disseminated candidiasis (5,6), but whether other antibody classes and specificity enhance disease progression is unknown. Nonetheless, in recent years, attention to these various details has resulted in solid evidence for antibody defense against candidiasis and cryptococcosis, and perhaps other mycoses (1).…”