Anthony F. Barbet

Recognition of conserved surface protein epitopes on Anaplasma centrale and Anaplasma marginale isolates from Israel, Kenya and the United States

Palmer G. H., Barbet A. F., Musoke A. J., Katende J. M., Rurangirwa F., Shkap V., Pipano E., Davis W. C. and Mcguire T. C. 1988. Recognition of conserved surface protein epitopes on Anaplasma centrale and Anaplasma marginale isolates from Israel, Kenya and the United States. International Journal for Parasitology18: 33–38. Field isolates of A. marginale from Israel, Kenya and the United States and Anaplasma centrale were shown to be antigenically distinct using a panel of 1:1 isolaterestricted monoclonal antibodies. One monoclonal antibody reacted with all A. marginale isolates but not A. centrale and may be species-specific. A second panel of seven antibodies detected epitopes common to all isolates and both species. These common epitopes were present on two major Anaplasma surface proteins of Mr 36,000 and Mr 105,000. Monoclonal antibodies capable of neutralizing heterologous A. marginale isolate infectivity recognized 100% of the organisms in each isolate including A. centrale. In addition, immune sera from cattle recovered from infection with each of the isolates contained high titer antibodies to the isolated surface proteins bearing the common epitopes. These results demonstrate that antigenically distinct Anaplasma isolates and species contain common surface epitopes that may be important in developing effective immunoprophylaxis.

Characterization of a neutralization-sensitive epitope on the Am 105 surface protein of Anaplasma marginale

Purified immunoglobulin from each of two hybridoma cell lines (ANA 15D2 and ANA 22B1) significantly neutralized the infectivity of 108Anaplasma marginale initial bodies for cattle. Both cell lines produce antibody to the same Am 105 epitope as they inhibited the binding of each other to Am 105 in a competition radioimmunoassay. Complete digestion of Am 105 with proteinase K, pronase, or trypsin prevented monoclonal antibody binding indicating that the epitope was protein in nature rather than surface polysaccharide. In addition, evidence that the neutralization-sensitive epitope was not membrane-protein-bound polysaccharide included: [1] 35S-methionine, but not 3H-glucosamine, was metabolically incorporated into Am 105 during short-term in vitro culture; [2] Am 105 was surface radiolabeled using 125I in a lactoperoxidase mediated reaction, but not labeled using a galactose oxidase-NaB[3H]4 mediated reaction with or without neuraminidase pretreatment; and [3] Am 105 did not bind to concanavalin A, Helix pomatia lectin, peanut lectin, soybean lectin, or wheat germ lectin.

Amino terminal sequence homology among variant surface glycoproteins of African trypanosomes

Amino terminal amino acid sequences were determined for eight variable surface glycoproteins purified from successive parasitemias of cloned Trypanosoma brucei. These sequences were compared by sequence alignment analysis with each other and with amino terminal sequences of variable surface glycoproteins from other clones of T. brucei, T. congolense and T. equiperdum. Contrary to previously held views, a remarkable degree of sequence homology was found among all sequences. In several cases the homology was greater between glycoproteins from different species than between glycoproteins isolated from successive parasite peaks, suggesting a common primordial gene for trypanosome variable surface glycoproteins. This amino terminal homology, taken together with homologies found in other regions of variant surface glycoproteins strongly suggests that their tertiary structures are conserved.

Structural features of antigenic determinants on variant surface glycoproteins from Trypanosoma brucei

The immunochemical structure of two variant surface glycoproteins (VSGs) from Trypanosoma brucei has been studied using monoclonal and polyclonal antibodies. These two VSGs, WaTat 1.1 and WaTat 1.12 have been shown to possess cross-reactive surface-exposed antigenic determinants [Barbet et al., Nature 300, 53-57 (1982)] and similar N-terminal amino acid sequences [Olafson et al., Molec. Biochem. Parasit. 12, 287-298 (1984)]. Monoclonal and polyclonal antibodies were raised against the soluble forms of the two VSGs and against their reduced, alkylated and cyanogen bromide (CNBr) cleaved forms. None of the monoclonal antibodies which bound to the surface of living trypanosomes bound to CNBr fragments of the VSGs nor to denatured VSGs. Polyclonal antibodies raised against denatured and cleaved VSG did not bind to the surface of the living trypanosomes. These results suggest that the variable surface exposed antigenic determinants of VSG are topographically assembled structures. It was also shown that the conserved amino terminal peptides of WaTat 1.1 and WaTat 1.12 do not contain antigenic determinants.

Shared surface epitopes among trypanosomes of the same serodeme expressing different variable surface glycoprotein genes.

African trypanosomes evade the immune response of the mammalian host by undergoing antigenic variation, caused by sequence changes in a variable surface glycoprotein (VSG). The majority of trypanosome clones analyzed thus far are not known to share surface exposed epitopes or express appreciably homologous VSGs. We show here that four clones of Trypanosoma brucei from the same serodeme express different VSGs and share exposed epitopes to varying degrees, as defined by monoclonal antibodies. Rabbit antiserum against any one of the four VSGs recognizes epitopes present on all four trypanosomes in live cell immunofluorescence assay. The expressed VSGs are partially homologous at the N-terminus with multiple point substitutions of amino acids which distinguish each of the four VSGs. The genes coding for these VSGs are members of one gene family and an expression-linked copy with a unique restriction map is present in each trypanosome. Analysis of the ontogeny of the expressed genes should reveal mechanisms of evolution in trypanosome variable antigen repertoires.

Conservation of structure detected in two trypanosome surface glycoproteins by amino acid sequence alignment

The predominant molecule exposed to antibody on the surface of Trypanosoma brucei is a glycoprotein of about 60 000 molecular weight which varies in amino acid sequence. The complete sequences of two such variable surface glycoproteins (VSGs) from randomly isolated, different antigenic types of trypanosomes were compared by amino acid sequence alignment. Homologous sequences were found distributed over various regions of the VSGs. Particularly good homology was observed between residues 16-34, 91-115, 177-194 and 254-345 from the N-terminus, in addition to the known conserved region close to the C-terminus. Homology was also demonstrated in the corresponding regions of the cDNA sequences by matrix analysis.

Trypanosoma brucei: A fluorescence and spin label study of the membranes of the bloodstream form

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.