Stimulation of human B cells specific for Candida albicans for monoclonal antibody production

Davenport, Claire

doi:10.1016/0378-1097(92)90274-r

Cited by 3 publications

(3 citation statements)

References 26 publications

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“…Generation of EBV-transformed B-cell lines. B-cell lines were generated, as described previously (12), to serve as APCs in the T-cell proliferation assay and for maintaining the T-cell lines. PBMCs were incubated with Epstein-Barr virus (EBV) produced by the B95-8 cell line (provided by C. Davenport, Division of Immunology, University of Nottingham) and then cultured in supplemented RPMI 1640 containing 10% fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

et al. 1999

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In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis (strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing fromN. meningitidis infection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients’ T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2,778 bp). The strong CD4+ T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients’ serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspA failed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeae genome sequences, suggesting that tspA is unique to the genusNeisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimurium andEscherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4+T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.

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Section: Methodsmentioning

confidence: 99%

Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

et al. 1999

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“…Invasive infections caused by Candida albicans , a constituent of human amphibiotic microbiota, have been revealed as an increasing problem especially in immunocompromised patients. In this context, the use of the Elispot assay and variations of the technique in mycology started in 1992 with a study of candidiasis, the most frequent mycosis using Elispot assay as a tool for analyses [ 109 ]. The authors investigated the stimulation and immortalization of human peripheral blood B lymphocytes specific for Candida albicans antigen as a strategy for the generation of human monoclonal antibodies to infectious agents.…”

Section: Mycosismentioning

confidence: 99%

How Can Elispot Add Information to Improve Knowledge on Tropical Diseases?

Lima-Junior

Morgado

Conceição-Silva

2017

Cells

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Elispot has been used as an important tool for detecting immune cells’ products and functions and has facilitated the understanding of host-pathogen interaction. Despite the incredible diversity of possibilities, two main approaches have been developed: the immunopathogenesis and diagnosis/prognosis of infectious diseases as well as cancer research. Much has been described on the topics of allergy, autoimmune diseases, and HIV-Aids, however, Elispot can also be applied to other infectious diseases, mainly leishmaniasis, malaria, some viruses, helminths and mycosis usually classified as tropical diseases. The comprehension of the function, concentration and diversity of the immune response in the infectious disease is pointed out as crucial to the development of infection or disease in humans and animals. In this review we will describe the knowledge already obtained using Elispot as a method for accessing the profile of immune response as well as the recent advances in information about host-pathogen interaction in order to better understand the clinical outcome of a group of tropical and neglected diseases.

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“…These T-cell lines were used to screen the genomic expression library (see below). Antigen presenting feeder cells were Epstein±Barr virus-transformed B-cell lines (EBVBs) generated as described previously (Davenport et al, 1992;Kizil et al, 1999).…”

Section: Generation Of T-cell and Epstein-barr Virus-transformed B-cementioning

confidence: 99%

Auto‐transporter A protein of Neisseria meningitidis: a potent CD4⁺ T‐cell and B‐cell stimulating antigen detected by expression cloning

Ait-Tahar

Wooldridge

Turner

et al. 2000

Molecular Microbiology

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A meningococcal genomic expression library was screened for potent CD4+ T‐cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto‐transporter A) as its peptide sequence shares molecular characteristics of the auto‐transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T‐cell responses in PBLs of patients and some healthy donors, and induced strong primary T‐cell responses in healthy donors. The human B‐cell immunogenicity and cross‐reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross‐reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB‐specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross‐react with AutA. Thus, AutA, being a potent CD4+ T‐cell and B‐cell‐stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.

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Stimulation of human B cells specific for Candida albicans for monoclonal antibody production

Cited by 3 publications

References 26 publications

Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

How Can Elispot Add Information to Improve Knowledge on Tropical Diseases?

Auto‐transporter A protein of Neisseria meningitidis: a potent CD4⁺ T‐cell and B‐cell stimulating antigen detected by expression cloning

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Stimulation of human B cells specific for Candida albicans for monoclonal antibody production

Cited by 3 publications

References 26 publications

Identification and Characterization of TspA, a Major CD4+T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

Identification and Characterization of TspA, a Major CD4+T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

How Can Elispot Add Information to Improve Knowledge on Tropical Diseases?

Auto‐transporter A protein of Neisseria meningitidis: a potent CD4+ T‐cell and B‐cell stimulating antigen detected by expression cloning

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Product

Resources

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Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

Auto‐transporter A protein of Neisseria meningitidis: a potent CD4⁺ T‐cell and B‐cell stimulating antigen detected by expression cloning