Expression in Escherichia coli of a high-molecular-weight protective surface antigen found in nontypeable and type b Haemophilus influenzae

Abstract

An Escherichia coli clone producing a high-molecular-weight surface antigen of Haemophilus influenzae type b (Hib) was isolated from a library of Hib DNA fragments cloned as lysogens in a lambda replacement vector. The antigen is found in sarcosyl-insoluble outer membrane protein preparations and was produced by all 36 H. influenzae isolates tested. Absorption studies indicated that the antigen is a surface determinant on all isolates tested. Antibodies to the antigen (D15) were found in eight of nine convalescent-phase sera from children with invasive Hib infection. Affinity-purified antibodies prepared against the cloned antigen gave protection against the development of bacteremia in a rat pup model.

Identification of membrane‐associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

“…This is the first study to describe Omp50 as an antigen in humans. Omp85 homologs have been shown to confer protective immunity against Hemophilus influenzae [47] and Pasteurella multocida [48], however, we did not detect Cj0129c as an immunogen using convalescent serum. Cj1279c encodes a protein with sequence similarity to fibronectin-binding proteins (Fn-BP) and is one of three Fn-BP (including CadF and Cj1349c) predicted within the C. jejuni NCTC 11168 and RM1221 genomes [6,7].…”

A Toc75‐like protein import channel is abundant in chloroplasts

“…These usher proteins most likely facilitate the export of hemolysins. At the same time, they represent prominent surface antigens, which provoke the immune response upon infection (Thomas et al, 1990;Flack et al, 1995). We conclude that atToc75-V represents the most ancestral or earliest form of a Toc75-like channel, which was further modified during endosymbiosis.…”

T cell cytokine responses to outer membrane proteins of Haemophilus influenzae and the house dust mite allergens Der p 1 in allergic and non‐allergic subjects

“…A standard curve of pooled serum known to bind the recombinant protein was also set up, with serum concentrations defined by pilot studies. Pooled serum was absorbed with an E. coli lysate [16]. After washing, 50 µL of 1 : 10 000 dilution anti‐human IgG conjugated to horseradish peroxidase (Silenus Laboratories Pty Ltd, Boronia, Victoria, Australia) was added to each well for 1 h. Fifty microlitres of K‐Blue tetramethylbenzidine (TMB) substrate (Neogen Corporation, Lansing, MI, USA) was added to each well and the reaction then stopped with 50 µL 1 m H 2 SO 4 .…”

“…The measurement of immune responses to H. influenzae has been complicated by the fact that the major outer membrane protein, the porin P2, has extensive sequence and antigenic variation from isolate to isolate [10, 11]. Other membrane proteins including D15 and OMP6 have highly conserved sequences and antigenicity [12–16] and can be used to study the recall responses induced by infection with a wide range of isolates. Both antigens elicit protective immune responses [13, 16, 17] and both can be produced as recombinant polypeptides [13, 15, 17] so in vitro cytokine responses can be induced without inflammatory microbial products such as lipo‐oligosaccharide.…”

“…Other membrane proteins including D15 and OMP6 have highly conserved sequences and antigenicity [12–16] and can be used to study the recall responses induced by infection with a wide range of isolates. Both antigens elicit protective immune responses [13, 16, 17] and both can be produced as recombinant polypeptides [13, 15, 17] so in vitro cytokine responses can be induced without inflammatory microbial products such as lipo‐oligosaccharide. The whole OMP6 molecule can be produced as a soluble polypeptide and while this cannot be accomplished for D15, the peptide 6–201 is a large portion of the molecule which is soluble and can induce protective immunity [17].…”