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1.
FEMS Immunol Med Microbiol ; 52(1): 78-87, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17995960

ABSTRACT

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.


Subject(s)
Antibodies, Bacterial , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Burkholderia pseudomallei/immunology , Carrier Proteins/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Monoclonal , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Burkholderia mallei/genetics , Burkholderia pseudomallei/genetics , Carrier Proteins/genetics , Conserved Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Melioidosis/immunology , Melioidosis/prevention & control , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Shigella/genetics , Survival Analysis , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology
2.
Immunogenetics ; 57(10): 730-8, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16215733

ABSTRACT

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5' rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V(H) repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V(H)3 (41%), V(H)4 (39%), and V(H)1 (14%) subgroups used more frequently than the V(H)5 (3.9%) or V(H)7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5beta gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80-89% for IGHV and 72-92% for IGHJ. M. fascicularis and human V(H) genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Ebolavirus/immunology , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Variable Region/biosynthesis , Macaca fascicularis/immunology , Viral Envelope Proteins/immunology , Amino Acid Sequence , Animals , Antibody Diversity , Base Sequence , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Macaca mulatta , Molecular Sequence Data , Peptide Library
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