In a vaccine model, selected substitution of a highly stimulatory T cell epitope of hen's egg lysozyme into a Salmonella flagellin does not result in a homologous, specific, cellular immune response and may alter the way in which the total antigen is processed.

A 13 amino acid peptide corresponding to a potent BALB/c mouse T cell epitope of hen's egg lysozyme (HEL) was substituted singly at five sites in the d flagellin of Salmonella muenchen. The resulting chimeric proteins were unable to expand T cells capable of being stimulated by the HEL epitope and induced T cell populations which either failed to respond or responded at a low level to a normally highly stimulatory flagellin T cell epitope that was present in all chimeras. The results suggested that substitution of a T cell epitope in flagellin may alter the processing of the resulting immunogen.

Molecular analyses of the phase-2 antigen complex 1,2,.. of Salmonella spp.

The nucleotide sequences of the structural genes (fljB) for salmonellar flagellins representative of the phase-2 flagellar antigens 1,2.., 1,5.., 1,6.., and 1,7.. were determined. The results did not indicate linear epitopes for the antigen 1 subfactors, suggesting that conformational aspects are involved in determining these antigenic specificities.

Expression and immunogenicity of an 18-residue epitope of HIV1 gp41 inserted in the flagellar protein of a Salmonella live vaccine.

A synthetic oligonucleotide specifying residues 735-752 of the product of the env gene of HIV1 IIIB was inserted by blunt-end ligation at restriction sites in the hypervariable, antigenically determinant region IV of two flagellin genes. Its integration, in frame and correct orientation, into gene fliC(d) in plasmid pLS408 allowed production of functional flagella when the plasmid was placed in a flagellin-negative aroA live-vaccine Salmonella dublin strain, SL5928. Bacteria thus made motile were immobilized and agglutinated by anti-(735-752 peptide) serum; expression was also shown by immunoelectron-microscopy and by Western blot of whole-cell lysates. Enzyme immunoassay (EIA) of sera of mice given three doses by intraperitoneal injection of the live-vaccine strain producing chimeric flagellin, or of concentrated flagella from it, showed production of antibody with affinity for the peptide, and in some sera, also for r-gp160. Pooled serum from mice given five i.p. doses of the live vaccine strain expressing the gp41 epitope at the surface of its flagellar filaments had higher titres of anti-peptide and anti-r-gp160 antibody and weak neutralizing activity on the IIIB isolate (90% neutralization at 1/100). The sera of nine mice given two doses of the live vaccine by the oral route had either no or only very low titres of antibody to flagellar antigen d; they were therefore not tested for anti-peptide activity.

Molecular analyses of the Salmonella g. . . flagellar antigen complex.

Salmonella flagellar filaments are polymers of a highly antigenic protein, termed flagellin. Eight main subfactors have been identified in the Salmonella phase-1 g. . . series flagellar antigen. To determine the molecular basis for expression of the epitopes by which the g. . . family subfactors are distinguished, 10 members of this series were selected and their fliC (the structural gene for phase-1 flagellin) genes were sequenced. Comparative analyses of the inferred primary structures of these flagellins did not allow the identification of linear epitopes responsible for the antigen subfactors. This suggests that conformational aspects are involved in determining the antigenic specificity in these cases. A phylogenetic analysis of the flagellin sequences showed that members of the g. . . series do not form a single coherent unit.

Mapping of T-cell epitopes of flagellar antigen d of Salmonella muenchen.

Regions of the antigen d flagellin of Salmonella muenchen capable of causing the proliferation of repolymerized d flagellin-primed T cells were delineated by using dodecameric peptides. Three areas causing substantial DNA replication were identified, together with five areas eliciting less response. On the basis of known salmonella flagellin structure, the major areas were located in highly conserved regions of the molecule.

Epitope mapping of the d flagellar antigen of Salmonella muenchen.

Contiguous exposed antibody-binding regions of the antigen d flagellin of Salmonella muenchen were identified by using octameric peptides synthesized on polyethylene pins. Identification was confirmed by the serological activity of immunoglobulins recovered from specified pin peptides. Peptides equivalent to four regions of the d flagellin reacted with three different sera tested.

Epitope mapping in Salmonella flagellar protein.

The flagellar filaments of bacteria of the genus Salmonella are highly immunopotent and antigenically diverse. It is proposed to develop vaccines by replacing the flagella of live attenuated Salmonella strains with engineered flagellar filament proteins containing foreign epitopes of medical and agricultural importance. As an initial step in this process, the major linear epitope regions of one filament protein have been identified.

The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins.

In order to circumvent problems associated with direct chemical analysis of the phase-1 flagellar filament protein (flagellin) of Salmonella typhimurium, the covalent structure was determined by recombinant DNA procedures. The corresponding structural gene (H-1i) was cloned into plasmid pBR322 in a 4.3-kilobase fragment produced by EcoRI digestion of chromosomal DNA, and the nucleotide sequence of the region specifying the flagellar protein was determined. Comparison of the data obtained with the limited information available for other salmonellar flagellins supported the concept that both ends of the molecule are conserved in this genus. Additionally, a conservation of base sequence in the region of H-1 genes coding for the N-terminal end of flagellins was apparent, suggesting that this area may have an additional regulatory role. The i flagellin was found to be unrelated to proteins in the NBRF data base with the exception of other flagellins. The three flagellins which have been sequenced to date (those produced by Bacillus subtilis, Caulobacter crescentis, and phase-1 S. typhimurium) show homologies in amino acid sequence at both the N-terminal and C-terminal ends despite large differences in their total molecular weight, and comparison suggests that B. subtilis and Salmonella are more closely related to each other than either is to Caulobacter.

Covalent structure of three phase-1 flagellar filament proteins of Salmonella.

Using recombinant DNA techniques, the covalent structure was determined for three flagellar filament proteins produced by Salmonella serotypes with phase-1 antigens a, c and d. Comparison of the results obtained, together with previous results for antigen i, indicated an overall structure in which conservation of amino acid sequence was absolute at both ends of the molecule and proceeded inwards with progressively greater variation. Very few differences in nucleotide sequence were detected in regions of amino acid conservation, which suggested that these areas of the gene may be involved in regulatory functions.

The susceptibility to tryptic hydrolysis of peptide bonds involving epsilon-N-methyllysine.

Using isolation of soluble peptides as a means of comparing the efficiency of tryptic hydrolysis of various forms of the phase-1 flagellin of Salmonella typhimurium, it was found that peptides terminating in N-methyllysine required a longer period of exposure to enzyme for their detection than did equivalent peptides terminating in lysine.

The flagellar protein of Clostridium tetani.

Clostridium tetani ATCC 19406 was investigated with regard to the flagellar filaments produced by this anaerobic species. Flagellar filaments were removed from the cell bodies by hydrodynamic shear forces and purified by differential centrifugation. Exposure to acid was shown to result in disaggregation of the flagellar filaments into a preparation of flagellar protein containing 3.5% carbohydrate. The protein was judged homogeneous after examination by acrylamide gel electrophoresis in the presence of 4 M urea at several pH levels, and was shown to have a molecular weight of 35,000 daltons. Amino acid analyses indicated the absence of cysteine and tryptophan and a preponderance of acidic residues, epsilon-N-methyllysine was shown to be absent and the N-terminal amino acid was identified as alanine. Analysis of the C-terminal region indicated the sequence -Leu-Leu-Arg. These findings indicated that the obligate anaerobe C. tetani produced flagella filaments similar to previously studied filaments of aerobic and facultatively anaerobic bacteria.

Identification of an antibody binding site in the phase-1 flagellar protein of Salmonella typhimurium.

A synthesized pentapeptide equivalent to a region of the phase-1 flagellar protein of Salmonella typhimurium was found to be specifically bound by antisera possessing agglutinating activity against the phase-1 flagellar antigen i.