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The long‐term protective efficacy of neutralizing antibodies (Nabs) against Omicron subvariants after inactivated booster vaccines remains elusive. During the follow‐up study, 54 healthy volunteers aged 20–31 years received inactivated CoronaVac booster vaccinations and were monitored for 221 days. The dynamic efficacy and durability of Nab against Omicron subvariants BA.1, BA.2, BA.2.12.2, and BA4/5 were assessed using a pseudotyped virus neutralization assay at up to nine time points post immunization. The antibody response against Omicron subvariants was substantially weaker than D614G, with BA.4/5 being the least responsive. The geometric mean titer (GMT) of Nab against Omicron subvariants BA.1, BA.2, BA.2.12.1, and BA.4/5 was 2.2‐, 1.7‐, 1.8‐, and 2.2‐fold lower than that against D614G (ps < 0.0001). The gap in Nab response between Omicron subvariants was pronounced during the 2 weeks–2 months following booster vaccination (ps < 0.05). Seven months post booster, the antibody potency against D614G was maintained at 100% (50% for Nab titers ≥ 100 50% inhibitory dilution [EC50]), whereas at 77.3% for BA.1, 90.9% for BA.2, 86.4% for BA.2.12.1, and 86.4% for BA.4/5 (almost 20% for Nab titers ≥ 100 EC50). Despite the inevitable immune escape, Omicron subvariants maintained sustained and measurable antibody potency post‐booster vaccination during long‐term monitoring, which could help optimize immunization strategies. [ABSTRACT FROM AUTHOR]

To overcome limitations with respect to the low sensitivity of rapid immunoassay methods due to dilution of the extract in extraction solution, ultra-sensitive antibodies are needed. The linker-tethering site between the target and carrier protein, the length of the spacer arm, and the atomic charge of the linker-tethering site between the target and spacer arm in haptens design play a pivotal role in the preparation of ultra-sensitive antibodies. In this study, carbendazim (CBZ), a systemic broad-spectrum fungicidal pesticide, was used as model compound. 2-Aminobenzimidazole was chosen as the common skeletal structure and reacted with different groups, such as alkanes and urine groups, to form haptens H1 (previously reported), H2 (novel), and H3 (novel), respectively. Subsequently, eight monoclonal antibodies (mAbs) were generated using these three haptens, which exhibited notable variation in sensitivity. The half maximal inhibitory concentration (IC 50) value of mAb 4B11 based on hapten H3 against CBZ was 0.04 ng/mL, which was seven times better than that of mAb 5B10 (IC 50 value of 0.28 ng/mL) prepared using hapten H2 and 129 times better than that of mAb 6D5 (IC 50 value of 5.15 ng/mL) prepared using hapten H1. Finally, the optimal antigen–antibody combination was employed to establish a sensitive colloidal gold lateral flow immunoassay (CG-LFA) for CBZ detection in vegetables and fruits with convenient sample pretreatment. The developed CG-LFA shows promising practical utility and prospects for application due to its low limit of detection (0.10–0.18 ng/mL) and high recovery ratios (76.0%–96.1%) in six different samples. [Display omitted] • A novel hapten design strategy focuses on the link site and spacer arm length of the target compound. • An ultra-sensitive monoclonal antibody (mAb) 4B11 against carbendazim was prepared. • A sensitive colloidal gold lateral flow immunoassay in fruits and vegetables was developed. [ABSTRACT FROM AUTHOR]

Human calcium sensing receptor (CaSR) senses calcium ion concentrations in vivo and is an important class of drug targets. Mutations in the receptor can lead to disorders of calcium homeostasis, including hypercalcemia and hypocalcemia. Here, 127 CaSR-targeted nanobodies were generated from camels, and four nanobodies with inhibitory function were further identified. Among these nanobodies, NB32 can effectively inhibit the mobilization of intracellular calcium ions (Ca2+ i) and suppress the G 12/13 and ERK 1/2 signaling pathways downstream of CaSR. Moreover, it enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). We determined the structure of complex and found NB32 bound to LB2 (Ligand-binding 2) domain of CaSR to prevent the interaction of LB2 domains of two protomers to stabilize the inactive state of CaSR. • 127 CaSR-targeting nanobodies were screened from camels. • NB32 effectively inhibit the mobilization of intracellular calcium ions. • NB32 enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). • Cryo-EM revealed that NB32 binds to the LB2 domain of CaSR. [ABSTRACT FROM AUTHOR]

[Display omitted] • Crystal Structure of Two Non-competitive Nanobodies and SARS-CoV-2 RBD. • Two nanobodies neutralize SARS-CoV-2 including omicron variants by targeting ultra-conservative epitopes. • Developed a multivalent nanobody with high affinity and competitiveness. The evolving SARS-CoV-2 Omicron strain has repeatedly caused widespread disease epidemics, and effective antibody drugs continue to be in short supply. Here, we identified a batch of nanobodies with high affinity for receptor binding domain (RBD) of SARS-CoV-2 spike protein, separated them into three classes using high performance liquid chromatography (HPLC), and then resolved the crystal structure of the ternary complexes of two non-competing nanobodies (NB1C6 and NB1B5) with RBD using X-ray crystallography. The structures showed that NB1B5 and NB1C6 bind to the left and right flank of the RBD, respectively, and that the binding epitopes are highly conserved cryptic sites in all SARS-CoV-2 mutant strains, as well as that NB1B5 can effectively block the ACE2. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, and have a high affinity and neutralization potency for omicron, potentially inhibiting viral escape. The binding sites of these two nanobodies are relatively conserved, which help guide the structural design of antibodies targeting future variants of SARS-CoV-2 to combat COVID-19 epidemics and pandemics. [ABSTRACT FROM AUTHOR]

As one of the receptors of the TAM family, AXL plays a vital role in stem cell maintenance, angiogenesis, immune escape of viruses and drug resistance against tumors. In this study, the truncated extracellular segment containing two immunoglobulin-like domains of human AXL (AXL-IG), which has been confirmed to bind growth arrest specific 6 (GAS6) by structural studies [1], was expressed in a prokaryotic expression system and then purified. Immunizing camelid with the purified AXL-IG as antigen could lead to the production of unique nanobodies composed of only variable domain of heavy chain of heavy-chain antibody (VHH), which are around 15 kD and stable. We screened out a nanobody A-LY01 specific binding to AXL-IG. We further determined the affinity of A-LY01 to AXL-IG and revealed that A-LY01 could specifically recognize full-length AXL on the surface of HEK 293T/17 cells. Our study provides appropriate support for the development of diagnostic reagents and antibody therapeutics targeting AXL. • AXL-IG protein was expressed in E. coli WK6. • A nanobody phage library was constructed. • A nanobody A-LY01 specifically binds to AXL was screened out by phage display technology. [ABSTRACT FROM AUTHOR]

Because of the evolutionary variants of SARS-CoV-2, development of broad-spectrum neutralizing antibodies resilient to virus escape is urgently needed. We identified a group of high-affinity nanobodies from camels immunized with receptor-binding domain (RBD) of SARS-CoV-2 spike protein and resolved the structures of two non-competing nanobodies (NB1A7 and NB1B11) in complex with RBD using X-ray crystallography. The structures show that NB1A7 targets the highly conserved cryptic epitope shared by SARS-CoV-2 variants and some other coronaviruses and blocks ACE2 receptor attachment of the spike protein, and NB1B11 epitope overlaps with the contacting surface of ACE2 and is different from the binding site of NB1A7. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, which significantly improved the avidity and neutralization potency and may further inhibit viral escape. The results contribute to the structure-guided design of antibodies against future variants of SARS-CoV-2 virus to combat coronavirus epidemics and pandemics. [Display omitted] • Two neutralizing nanobodies are generated against the RBD of SARS-CoV-2 • The crystal structures reveal that the two nanobodies have different epitopes • One nanobody targets the highly conserved epitope • Bispecific nanobodies improve neutralization potency to SARS-CoV-2 variants Shi et al. identify two neutralizing nanobodies against SARS-CoV-2. Crystal structures reveal how they prevent the virus and its variants from entering cells. The combination of two nanobodies significantly improves neutralization potency, which is a potential therapeutic candidate to reduce the probability of the emergence of escape variants of SARS-CoV-2. [ABSTRACT FROM AUTHOR]