Immunization with a tubulin-rich preparation from Trypanosoma brucei confers broad protection against African trypanosomosis

Abstract

Tubulin from Trypanosoma brucei was purified to near homogeneity using a protocol which involved treatment with urea with subsequent renaturation and was then used to immunize mice. Renatured tubulin further purified by SDS–PAGE (denatured), synthetic tubulin peptides (STP), and rat brain tubulin (RbTub) were also used. Immunized mice were challenged with T. brucei, Trypanosoma congolense or Trypanosoma rhodesiense. Renatured T. brucei tubulin (nTbTub) induced protection in all mice tested, of which 60–80% ( n=81) was complete and the remainder partial. Denatured T. brucei tubulin (dTbTub), STP, or RbTub induced lower antibody levels than nTbTub and did not offer protection. However, in culture, the antibodies against dTbTub or STP killed trypanosomes although at lower dilutions than nTbTub, but those against RbTub did not. In Western blots anti-trypanosome antibodies recognized the tubulin of all the trypanosome species investigated but not vertebrate tubulin, whereas the anti-RbTUB antibodies recognized both trypanosome and vertebrate tubulin. Of the five mice given passive immunity by the transfer of anti-nTbTub serum, four were completely protected and one partially protected. These data suggest that tubulin is the relevant immunogen in the preparation used and could therefore be a promising target for the development of a parasite-specific, broad spectrum vaccine. Index Descriptors and Abbreviations: Protozoa; Trypanosoma brucei; T. congolense; T. rhodesiense; African trypanosomosis; Tubulin; immunization; heterologous immunoprotection; Copy deoxyribonucleic acid (cDNA); Denatured Trypnosoma brucei tubulin (dTbTub); Diethylaminoethyl (DEAE); Enzyme-linked immunosorbent assay (ELISA); Ethylenediaminetetra acetic acid (EDTA); Ethyleneglycol bis(2-aminoethyl ether)- N, N, N-tetra acetic acid (EGTA); Freund’s complete adjuvant (FCA); Guanosine-5-triphosphate (GTP); Incomplete Freund’s adjuvant (IFA); International Scientific Council for Trypanosomiasis Research and Control (ISCTRC); Key Hole Limpet Haemocyanin (KLH); Microtubule Associated Protein (MAP); Renatured Trypanosoma brucei tubulin (nTbTub); Phenylmethylsulfonyl fluoride (PMSF); Phosphate buffered saline(PBS); Phosphate buffered saline glucose (PBS-G); Piperazine-N, N-bis[2-ethanesulfonic acid] (Pipes); R(-)-2-(4-morpholino)-ethane sulfonate (MES); Rat brain tubulin (RbTub); Sodium dodecyl sulphate–Polyacrylamide gel electrophoresis (SDS–PAGE); Standard error of the mean (SEM); Synthetic tubulin peptide (STP); Uganda Trypanosomiasis Research Organization (UTRO); Variable surface glycoprotein (VSG).