Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases. The lack of diagnostic tests that are sensitive, affordable and user-friendly is the impediment to early detection and containment of infectious diseases. For these reasons, African trypanosomosis continues to pose serious threat to the communities that are unable to access laboratory services. Assays that are able to address above-mentioned challenges would greatly reduce the disease burden. Although few relatively sensitive agglutination assays for some forms of African trypanosomosis have been adapted to field conditions, the tests are not reliable indicators of active infections given the fact that they only detect antibodies. The barrier in the development of alternative tests capable of revealing ongoing infections through antigen detection is the lack of potent monoclonal antibodies that can compete with infection-induced host antibodies for the circulating parasite antigens. Using T. congolense as a model system, we demonstrate that Nanobodies (Nbs) targeting the parasite glycosomal aldolase can detect active infections. The strategy described addresses the technical shortcoming of conventional monoclonal antibody-based assay development by adopting an unbiased proteome screening approach combined with a phage panning strategy that is adapted to avoid interference of the infection-induced host antibody response. Hence, this study shows prospect for future development of Nb-based tests for other infectious diseases.

Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay’s high specificity. The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays. Sub-Saharan Africa is plagued by many diseases, which impede its socio-economical development. One of these diseases, Animal African Trypanosomosis, affects livestock and is caused by the parasites of the Trypanosoma genus (T. vivax and T. congolense). Animal African Trypanosomosis leads to considerable economic losses and renders sustainable livestock industry in Sub-Saharan Africa very difficult. In order to proceed with the selective treatment of infected animals, they need to be properly diagnosed. We recently described the use of an assay to specifically detect T. congolense infections in both experimentally and naturally infected animals. The diagnostic assay employs a Nanobody (Nb), which is the smallest antigen-binding unit derived from camelid heavy-chain only antibodies. Our previous results showed that the Nb-based diagnostic test specifically recognizes T. congolense fructose-1,6-bisphosphate aldolase, a glycolytic enzyme that is well conserved amongst other Trypanosoma species. In this paper, we studied the molecular mechanisms underlying the high specificity of the Nb-based diagnostic assay. The principles derived from this work may be important for the design and improvement of similar diagnostic tests.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection.

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly, laborious, time-consuming, complex, and require skilled personnel. Hence, utilisation of most diagnostics for AAT is impracticable in rural areas, where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection, without relying on expensive equipment, would facilitate AAT detection. In turn, early management, would reduce disease incidence and severity. Today, several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context, we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T. congolense infections, which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here, originated from a past study. Briefly, the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T. congolense glycosomal fructose-1,6-bisphosphate aldolase (TcoALD) was discovered as the cognate Ag of Nb474H. In this study, splenocytes were harvested from a mouse immunized with recombinant TcoALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on TcoALD retrieved a lone binder, designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native TcoALD released during infection by T. congolense parasites. Hitherto, development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) “hybrid” sandwich technology offers prospects for exploration, using the unique specificity of Nb as a key determinant in Ag capturing, while using the versatility of monoclonal Ab to adapt to various detection conditions.

The global surge in multi-drug resistant bacteria, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has led to a growing need for new antibacterial compounds. Despite being promising, the potential of fish-derived antimicrobial peptides (AMPs) in combating ESBL-producing E. coli is largely unexplored. In this study, native African catfish antimicrobial peptides (NACAPs) were extracted from the skin mucus of farmed African catfish, Clarias gariepinus, using a combination of 10% acetic acid solvent hydrolysis, 5 kDa ultrafiltration, and C₁₈ hydrophobic interaction chromatography. Peptides were then sequenced using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. The identified peptides were screened for potential antibacterial activity using Random Forest and AdaBoost machine learning algorithms. The most promising peptide was chemically synthesized and evaluated in vitro for safety on rabbit red blood cells and activity against ESBL-producing E. coli (ATCC 35218) utilizing spot-on-lawn and broth dilution methods. Eight peptides ranging from 13 to 22 amino acids with molecular weights between 968.42 and 2434.11 Da were identified. Peptide NACAP-II was non-hemolytic to rabbit erythrocytes (p > 0.05) with a zone of inhibition (ZOI) of 22.7 ± 0.9 mm and a minimum inhibitory concentration (MIC) of 91.3 ± 1.2 μg/mL. The peptide is thus a candidate antibacterial compound with enormous potential applications in the pharmaceutical industry. However, further studies are still required to establish an upscale production strategy and optimize its activity and safety in vivo.