Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections

Abstract

Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as ‘test-of-cure’ tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.

An Unbiased Immunization Strategy Results in the Identification of Enolase as a Potential Marker for Nanobody-Based Detection of Trypanosoma evansi

“…Here, we present the application of such an “unbiased” approach to the immunization of camelids with the aim of generating Nbs that can be employed for (i) the development of parasite-specific antigen-based immunoassays and (ii) the identification of the target antigen via Nb-mediated immunocapturing followed by MS analysis. Our group has previously adopted this strategy for the identification of novel biomarkers for the detection of active T. congolense infections [ 24 , 39 ]. The immunization of alpacas with soluble proteome and secretome fractions from T. congolense yielded highly specific Nbs targeting the glycolytic enzymes T. congolense aldolase ( Tco ALD) and T. congolense pyruvate kinase ( Tco PYK), respectively.…”

“…The identified Nbs are then used to determine the nature of their target antigens via immunocapturing followed by mass spectrometry (MS). The approach is “unbiased” given that Nb library construction is performed without prior knowledge of the target antigen(s) recognized by the identified binders [ 24 , 39 ]. Here, we report the identification of a single anti- T. evansi secretome Nb (Nb11) after immunizing an alpaca with soluble lysate preparations from different T. evansi strains followed by panning and screening against the T. evansi secretome.…”

Trypanosoma vivax infection in sheep: Different patterns of virulence and pathogenicity associated with differentially expressed proteomes

“…Pharmacological inhibition or RNAi silencing of this enzyme causes ATP depletion, cell growth arrest, and parasite death ( Albert et al, 2005 , Coustou et al, 2003 , Worku et al, 2015 ). Additionally, the antigenic properties of PK in T. evansi and T. congolense are known ( Pinto Torres et al, 2018 , Yadav et al, 2017 ), and PK has been identified as a diagnostic biomarker in T. congolense ( Pinto Torres et al, 2018 ).…”

Product Development Partnerships: Delivering Innovation for the Elimination of African Trypanosomiasis?

“…Currently, the only way to confirm a diagnosis in infected animals is to demonstrate and identify the parasites in blood and other bodily fluids (which is in itself difficult as parasite numbers are generally low). Most treatment of AAT is therefore presumptive, which has costs for farmers and their perception of the efficacy of treatment [54,55]. The lack of a simple diagnostic test similarly makes it difficult to identity and deal with parasitic reservoirs within livestock populations.…”