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The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the variant surface glycoprotein or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory; minor alterations within these exposed epitopes confer antigenically distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross-reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation. [Display omitted] • Mutated VSGs are identical in structure but elicit distinct antibody repertoires • Antibody repertoires elicited by the same VSGs are highly stereotyped between mice • Host immunity is focused on a restricted set of immunodominant epitopes per VSG Gkeka et al. analyze the antibody response to different surface proteins of the African trypanosome. They determine that it is restricted and targeted to specific surface epitopes. They also show that minor changes in these epitopes trigger a different response, allowing T. brucei to focus host immunity on those epitopes and prolonging immune evasion. [ABSTRACT FROM AUTHOR]

Poorly immunogenic small molecules pose challenges for the production of clinically efficacious vaccines and antibodies. To address this, we generate an immunization platform derived from the immunogenic surface coat of the African trypanosome. Through sortase-based conjugation of the target molecules to the variant surface glycoprotein (VSG) of the trypanosome surface coat, we develop VSG-immunogen array by sortase tagging (VAST). VAST elicits antigen-specific memory B cells and antibodies in a murine model after deploying the poorly immunogenic molecule fentanyl as a proof of concept. We also develop a single-cell RNA sequencing (RNA-seq)-based computational method that synergizes with VAST to specifically identify memory B cell-encoded antibodies. All computationally selected antibodies bind to fentanyl with picomolar affinity. Moreover, these antibodies protect mice from fentanyl effects after passive immunization, demonstrating the ability of these two coupled technologies to elicit therapeutic antibodies to challenging immunogens. [Display omitted] • Antibody elicitation platform based on the surface coat of Trypanosoma brucei • Adjuvant-free immunizations elicit antibodies against fentanyl • Memory B cell identification by RNA-seq facilitates high-affinity mAb discovery • Picomolar-affinity anti-fentanyl mAbs protect from fentanyl pharmacological effects Triller et al. exploit the unique immunogenicity of Trypanosoma brucei surface coats to generate an antibody elicitation platform tailored to difficult targets including small molecules. They use this platform to elicit high-affinity monoclonal antibodies to the synthetic opioid fentanyl, which could be used as overdose-preventing prophylactics. [ABSTRACT FROM AUTHOR]

The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells. [Display omitted] • Anti-VSG nanobodies (Nbs) can bind deep into the VSG coat of living trypanosomes • Higher-affinity Nbs are linked to larger surface areas of interaction • High-affinity binding of VSG Nbs can affect VSG diffusion and cell motility • Nb binding can lead to molecular crowding-mediated shedding of the cell surface Hempelmann et al. analyze effects of anti-VSG nanobodies (Nbs) on African trypanosomes. Employing techniques spanning structural biology and cellular dynamics, they identify one Nb that binds with high affinity and causes molecular crowding, resulting in a perturbation of protein diffusion and cell motility with shedding of the surface coat. [ABSTRACT FROM AUTHOR]