In vitro trypanocidal activity of antibodies to bacterially expressed Trypanosoma brucei tubulin

There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth. T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450micro g each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera. Low antisera dilutions [25%] from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions. Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes

Continuous growth of bloodstream forms of Trypanosoma brucei brucei in an axenic culture system containing a low concentration of serum.

An effective axenic culture system for bloodstream forms of Trypanosoma brucei brucei GUT at 3.1 containing a low concentration of serum is described. Bloodstream forms routinely maintained in Iscove's modification of Dulbecco's medium supplemented with 100 microM hypoxanthine, 30 microM thymidine, 40 microM adenosine, 1 mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% FBS for more than one year were grown in the same medium supplemented with 5% FBS without reducing their growth rate. Then culture adapted trypanosomes in the culture medium containing 5% FBS were transferred into the modified medium supplemented with 0.5% FBS. For the constant growth of bloodstream forms in the medium containing 0.5% FBS, the culture medium was further supplemented with 200 microM L-alanine, 100 microM glycine, 10 microM L-oruithine hydrochloride and 10 microM L-citrullin. The trypanosomes propagated in this culture system for one year retained their infectivity for mice. This culture system was also shown to be useful for cloning of T.b. brucei GUT at 3.1 which is important for separation of mutants.

An axenic culture system for the transformation of bloodstream forms to procyclic forms of Trypanosoma brucei brucei in vitro.

An axenic culture system for continuous cultivation of bloodstream forms of Trypanosoma brucei GUT at 3.1 and subsequent transformation of bloodstream forms to procyclic forms is described. Bloodstream forms were continuously grown at 37 degrees C in Iscove's modification of Dulbecco's medium (M-DMEM, with bovine serum albumin, transferrin and soybean lecithin supplemented with 100 microM hypoxanthine, 30 microns thymidine, 40 microM adenosine, 1mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% (v/v) heat-inactivated fetal bovine serum. In this system, 2-mercaptoethanol (2-ME) was essential and in the absence of 2-ME, 100 microM L-cysteine and 10 microM bathocuproine sulfonate could not be substituted for 2-ME. This culture system was useful for long-term culture of bloodstream forms of this clone. Axenic cultivation of bloodstream forms at 27 degrees C resulted in transformation to procyclic forms within 5 days in the same medium supplemented with 5 mM L-proline, 8 micrograms/ml hemin and 4 micrograms/ml hematin, respectively and, instead of FBS, 20% (v/v) hemoglobin-poor fraction of fetal bovine serum.