Comparison of Three Antihapten VHH Selection Strategies for the Development of Highly Sensitive Immunoassays for Microcystins.

Owing to their reproducibility, stability, and cost-effective production, the recombinant variable domains of heavy-chain-only antibodies (VHHs) are becoming a salient option as immunoassay reagents. Recently, there have been several reports describing their application to the detection of small molecules (haptens). However, lacking the heavy-light chain interface of conventional antibodies, VHHs are not particularly apt to bind small analytes and failures are not uncommon. Here we describe the construction of a VHH phage display library against the cyanobacterial hepatotoxin microcystin LR and its selection using competitive panning and two novel panning strategies. The outcome of each strategy was evaluated by a large-scale screening using in vivo biotinylated nanobodies. The three methods selected for different nonoverlapping subsets of VHHs, allowing one to optimize the immunodetection of the toxin. The best results were obtained by promoting the isolation of VHHs with the slowest koff (off-rate selection). Among these, the biotinylated nanobody A2.3 performed in ELISA with excellent recovery and high sensitivity, IC50 = 0.28 µg/L, with a limit of detection that is well below the most rigorous guidelines for the toxin. While it may be case-specific, these results highlight the importance of exploring different panning strategies to optimize the selection of antihapten nanobodies.

Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells.

BACKGROUND: Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS: Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. RESULTS: The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS: This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE: This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.

Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

Highly pathogenic avian influenza H5N1 virus infections in pinnipeds and seabirds in Uruguay: Implications for bird-mammal transmission in South America.

The highly pathogenic avian influenza viruses of clade 2.3.4.4b have caused unprecedented deaths in South American wild birds, poultry, and marine mammals. In September 2023, pinnipeds and seabirds appeared dead on the Uruguayan Atlantic coast. Sixteen influenza virus strains were characterized by real-time reverse transcription PCR and genome sequencing in samples from sea lions (Otaria flavescens), fur seals (Arctocephalus australis), and terns (Sterna hirundinacea). Phylogenetic and ancestral reconstruction analysis showed that these strains have pinnipeds most likely as the ancestral host, representing a recent introduction of clade 2.3.4.4b in Uruguay. The Uruguayan and closely related strains from Peru (sea lions) and Chile (sea lions and a human case) carry mammalian adaptative residues 591K and 701N in the viral polymerase basic protein 2 (PB2). Our findings suggest that clade 2.3.4.4b strains in South America may have spread from mammals to mammals and seabirds, revealing a new transmission route.

Co-circulation of Hepatitis E Virus (HEV) Genotype 3 and Moose-HEV-Like Strains in Free-Ranging-Spotted Deer (Axis axis) in Uruguay.

Hepatitis E caused by hepatitis E virus (HEV) is considered an emerging foodborne zoonosis in industrialized, non-endemic countries. Domestic pigs and wild boars are considered the main reservoir of HEV. However, HEV can also infect an ever-expanding host range of animals, but they exact role in transmitting the virus to other species or humans is mostly unknown. In this work, we investigated the spread of HEV in free-living and captive spotted deer (Axis axis) from Uruguay in a 2-year period (2020-2022) and examined the role of this invasive species as a new potential reservoir of the virus. In addition, with the aim to gain new insights into viral ecology in the context of One Health, by using camera trapping, we identified and quantified temporal and spatial coexistence of spotted deer, wild boars, and cattle. In free-living animals, we detected an anti-HEV seropositivity of 11.1% (6/54). HEV infection and viral excretion in feces were assessed by RT-PCR. Thirteen of 19 samples (68.4%) had HEV RNA. Six samples were amplified using a broadly reactive RT-PCR and sequenced. No captive animal showed evidence of HEV infection. Additionally, HEV RNA was detected in a freshwater pond shared by these species. Phylogenetic and p-distance analysis revealed that zoonotic HEV genotype 3 strains circulate together with unclassified variants related to moose HEV whose potential risk of transmission to humans and other domestic and wild animals is unknown. The data presented here suggest that spotted deer (A. axis) may be a novel host for zoonotic HEV strains.

Spreading of the High-Pathogenicity Avian Influenza (H5N1) Virus of Clade 2.3.4.4b into Uruguay.

BACKGROUND: Avian influenza viruses (genus Alphainfluenzavirus, family Orthomyxoviridae) infect avian and mammal hosts. In 2022, the high pathogenicity avian influenza virus (H5N1) spread to South America, resulting in the loss of thousands of wild birds, including endangered species, and severely impacting the global poultry industry. OBJECTIVES: We analyzed the complete genomes of influenza viruses obtained from wild birds and backyard poultry in Uruguay between February and May 2023. METHODS: Twelve complete genomes were obtained in 2023 from cloacal swabs using Illumina sequencing. Genomes were phylogenetically analyzed with regional and global strains. FINDINGS: The identified strains have multiple basic amino acids at the hemagglutinin cleavage sites, which is typical for highly pathogenic strains. The Uruguayan viruses belonged to hemagglutinin clade 2.3.4.4b of the H5N1 subtype. A reassortment in North America has resulted in some segments of South American strains being of Eurasian or North American origins. The Uruguayan viruses shared a common ancestor with South American strains from Argentina and Chile. The influenza viruses displayed a spatiotemporal divergence pattern rather than being host-specific. MAIN CONCLUSIONS: The arrival of the 2.3.4.4b clade in Uruguay may have been mediated by birds that acquired the virus from Argentine and Chilean waterfowl migrating in the Pacific Flyway.

Hepatitis E Virus (HEV) infection in captive white-collared peccaries (Pecari tajacu) from Uruguay.

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Clinical presentation of hepatitis E mainly occurs as an acute and self-limited disease, though chronic cases are now being commonly reported in immunocompromised individuals. In high-income developed areas and non-endemic regions, HEV is mainly transmitted by the zoonotic route through direct contact with infected animals or by consumption of contaminated meat products. Although pigs and wild boars are the main reservoirs of the disease, HEV can also infect deer, camels, and rats and seems to have an ever-expanding host range. Peccaries (Tayassuidae family, superfamily Suoidea), the 'new world pigs', share susceptibility to several pathogens with domestic pigs and wild boars. Herein, we performed a serological and molecular survey of two captive populations of white-collared peccaries (Pecari tajacu) from Uruguay, with the aim to assess the role of the species as an HEV reservoir. One-hundred and one serum samples were analysed for anti-HEV antibodies. Further evidences of active HEV infection were investigated in stool by RT-nested PCR. Animals from both wildlife reserves were exposed to HEV with an overall prevalence of 24.7%. Moreover, HEV RNA could be detected in peccaries' stool samples from one of the reserves. Phylogenetic analysis clustered the strains within HEV-3, closely related to both human and swine isolates. Our work provides the first evidences supporting the notion that white-collared peccaries are susceptible to HEV. However, these data should not be overinterpreted. Further research is needed concerning the role of peccaries in the transmission of HEV.

Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA.

The Zika virus was introduced in Brazil in 2015 and, shortly after, spread all over the Americas. Nowadays, it remains present in more than 80 countries and represents a major threat due to some singularities among other flaviviruses. Due to its easy transmission, high percentage of silent cases, the severity of its associated complications, and the lack of prophylactic methods and effective treatments, it is essential to develop reliable and rapid diagnostic tests for early containment of the infection. Nonstructural protein 1 (NS1), a glycoprotein involved in all flavivirus infections, is secreted since the beginning of the infection into the blood stream and has proven to be a valuable biomarker for the early diagnosis of other flaviviral infections. Here, we describe the development of a highly sensitive nanobody ELISA for the detection of the NS1 protein in serum samples. Nanobodies were selected from a library generated from a llama immunized with Zika NS1 (ZVNS1) by a two-step high-throughput screening geared to identify the most sensitive and specific nanobody pairs. The assay was performed with a sub-ng/mL detection limit in the sera and showed excellent reproducibility and accuracy when validated with serum samples spiked with 0.80, 1.60, or 3.10 ng/mL of ZVNS1. Furthermore, the specificity of the developed ELISA was demonstrated using a panel of flavivirus' NS1 proteins; this is of extreme relevance in countries endemic for more than one flavivirus. Considering that the nanobody sequences are provided, the assay can be reproduced in any laboratory at low cost, which may help to strengthen the diagnostic capacity of the disease even in low-resource countries.

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain.

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (~30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.