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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.

SUBMITTER: Xin H

PROVIDER: S-EPMC1489732 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Hybridoma passage in vitro may result in reduced ability of antimannan antibody to protect against disseminated candidiasis.

Xin Hong H Cutler Jim E JE

Infection and immunity 20060701 7

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 th ...[more]

PMID: 16790805

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Project description:Our research on pathogenesis of disseminated candidiasis led to the discovery that antibodies specific for Candida albicans cell surface ?-1, 2-mannotriose [?-(Man)(3)] protect mice. A 14 mer peptide Fba, which derived from the N-terminal portion of the C. albicans cytosolic/cell surface protein fructose-bisphosphate aldolase, was used as the glycan carrier and resulted in a novel synthetic glycopeptide vaccine ?-(Man)(3)-Fba. By a dendritic cell-based immunization approach, this conjugate induced protective antibody responses against both the glycan and peptide parts of the vaccine. In this report, we modified the ?-(Man)(3)-Fba conjugate by coupling it to tetanus toxoid (TT) in order to improve immunogenicity and allow for use of an adjuvant suitable for human use. By new immunization procedures entirely compatible with human use, the modified ?-(Man)(3)-Fba-TT was administered either alone or as a mixture made with alum or monophosphoryl lipid A (MPL) adjuvants and given to mice by a subcutaneous (s.c.) route. Mice vaccinated with or, surprisingly, without adjuvant responded well by making robust antibody responses. The immunized groups showed a high degree of protection against a lethal challenge with C. albicans as evidenced by increased survival times and reduced kidney fungal burden as compared to control groups that received only adjuvant or DPBS buffer prior to challenge. To confirm that induced antibodies were protective, sera from mice immunized against the ?-(Man)(3)-Fba-TT conjugate transferred protection against disseminated candidiasis to naïve mice, whereas C. albicans-absorbed immune sera did not. Similar antibody responses and protection induced by the ?-(Man)(3)-Fba-TT vaccine was observed in inbred BALB/c and outbred Swiss Webster mice. We conclude that addition of TT to the glycopeptide conjugate results in a self-adjuvanting vaccine that promotes robust antibody responses without the need for additional adjuvant, which is novel and represents a major step forward in vaccine design against disseminated candidiasis.

Project description:Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality(6). Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia(7-9). Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils(10-14). In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts(10, 15-18). To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses(2, 19-21), simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection(22-24). Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection(5). In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection(3, 25). However, only recently have much larger pathogens such as fungi been used to infect larva(5, 23, 26), and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim(25) zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish(5).

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Hybridoma passage in vitro may result in reduced ability of antimannan antibody to protect against disseminated candidiasis.

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Hybridoma passage in vitro may result in reduced ability of antimannan antibody to protect against disseminated candidiasis.

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