Structural analysis of the N-terminal domain of Lister VSG 427-2 (VSG221 or MITat1.2), revealed a compact homodimeric structure (Blum et al., 1993
). The fact that the loops are well defined in the structure as well as their resistance to proteolytic cleavage indicates that they contribute to the overall structural integrity and function of this molecule. To assess whether insertions of a short peptide sequence such as the FLAG epitope (DYKDDDDK) within these loops would be tolerated, we designed a series of internally tagged VSGs using VSG427-2 as a backbone (termed FLAG-VSGs; ) (Blum et al., 1993
). In conjunction, we also created a series of internally tagged VSG427-2 constructs where we deleted portions of the VSG loop coding regions and replaced them with the short peptide sequences.
Linear epitopes inserted at multiple positions within VSG loops can be displayed on the surface of T.brucei
T.brucei parasites require surface expression of the VSG in order to survive. We therefore expressed the tagged VSGs from an internal (ectopic) location, while leaving the endogenous VSG intact. In so doing, issues of potential lethality (e.g. should the tagged protein hinder proper VSG assembly) were circumvented and an analysis of live trypanosomes which expressed FLAG-VSG, the endogenous VSG alone or a mixture of both could be carried out. We generated 2–5 clones per construct and tested their expression in whole trypanosome extract by dot blot (not shown) and FACS analysis to ascertain surface expression (). Most of the T.brucei clones transfected with constructs where the FLAG sequence was inserted within VSG loops were well expressed, and some of those were also displayed on the surface (, clones 1,3,5,6,7). Surprisingly, none of the clones transfected with constructs where the FLAG sequence replaced a portion of a VSG loop, were expressed (not shown), suggesting that these sequences are structurally important to the organism, and are not simply providing targets to the humoral immune system.
Overall, our chimeric clones fell into three categories, with regard to surface expression: (a) clones that displayed only VSG427-2 but not FLAG-VSG on the surface (e.g. , clones 2, 4, 8, 9), (b) clones that displayed VSG427-2 as well as low levels of FLAG-VSG on the surface (which were further binned into clones expressing low levels of the chimeric receptor vs. those containing a population with no surface expression and a subpopulation with low surface expression, (, clones 1 and 6) and finally (c) clones that prominently displayed the FLAG-VSG chimera on the surface (, clones 3, 5, 7). Since the FLAG-VSG coat was expressed ectopically, all our T.brucei clones also co-expressed VSG427-2 from the endogenous locus (a representative experiment for clone 7 is shown in ). We conclude that certain locations within the exposed VSG loops on the surface of T.brucei are not only tolerant to peptide insertions, but can also display exogenous peptides with high efficiency.
To assess whether the humoral immune system could respond to the FLAG-VSG coat by producing anti-FLAG antibodies, we injected a FLAG-VSG clone into C57BL/6 mice (clone 3, displaying high levels of FLAG on the surface – ). We confirmed infection by assessing the level of parasitemia through a tail nick five days after injection, and then drug cleared the trypanosomes from the bloodstream the next day (Murphy et al., 1993
). We then collected small amounts of sera at several timepoints after clearance and assessed the antibody affinity maturation response by ELISA. As a comparison, we also injected untransfected T.brucei (ZM control)
, as well as FLAG peptide conjugated to KLH (not shown).
As expected, infected mice mounted a robust and specific immune response against the FLAG peptide that was prominently displayed on the surface of the chimeric trypanosomes (). Additionally, this antibody response could be amplified after a single boost, with day 42 representing 21 days post boost (). We conclude that chimeric trypanosomes can elicit a specific antibody response against an exogenous peptide displayed on their surface coat.
Inoculation of mice with chimeric trypanosomes elicits specific anti-epitope antibodies
In the aforementioned experiments, live trypanosomes were injected into the host. To address whether live trypanosomes are required for stimulation of an immune response against an epitope in our tagged VSG, we generated freshly formalin fixed organisms. Trypanosomes can be fixed after a short treatment with formalin, however formalin fixation is likely to interfere with any epitope that contains lysines (such as FLAG). To assess whether fixed chimeric trypanosomes can elicit potent antibody responses such as those observed with the live organisms, we engineered trypanosomes to carry a chimeric VSG coat that displayed the HA peptide (YPYDVPDYA) on the surface (, same location as clone 3), a peptide which does not contain lysines. We were able to select a number of clones expressing HA-VSG on the surface ( and not shown). We then fixed one of the HA-VSG clones and the equivalent FLAG-VSG clone ( clone 3), and injected 107 fixed cells into C57BL/6 mice. These mice did not develop parasitemia, as expected (data not shown). However, they did mount a specific antibody response against HA (), though not against FLAG (not shown). We conclude that fixed trypanosomes can also be used to elicit peptide-specific immune responses.