In a recent study published in bioRxiv*, researchers demonstrated a new strategy to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using chitin-immobilized nanobodies.
Study: Efficient detection of SARS-CoV-2 using chitin-immobilized nanobodies synthesized in Ustilago maydis. Image credit: CROCOTHERY/Shutterstock
background
Nanobodies derived from sharks and camelids are emerging alternatives to traditional antibodies. Nanobodies bind ligands in the nanomolar range and remain stable under heat/chemical induced stress conditions, making them promising candidates for widespread antigen testing. Numerous anti-SARS-CoV-2 nanobodies have been engineered by phage display or immunization of llamas, sharks, and alpacas.
The microbial form of Ustilago maydis (corn fungus) can be exploited to synthesize heterologous proteins such as nanobodies. Recently, the authors established a proof of principle for the synthesis of anti-SARS-CoV-2 nanobodies. Furthermore, the unconventional secretion mechanism used by U. maydis to export the Cts1 chitinase can be exploited for the secretion of heterologous target proteins. Cts1 has chitin-binding activity and can therefore be exploited as an intrinsic tag for immobilization and purification. Jps1, the anchoring factor required for Cts1 secretion, may be an alternative carrier.
The study and conclusions
In the present study, the researchers established a new approach to detect SARS-CoV-2 using chitin-immobilized nanobodies. First, they screened different nanobody-Cts1 fusion proteins for expression, unconventional secretion, and binding activity against the SARS-CoV-2 spike receptor-binding domain (RBD). Four nanobody-Cts1 fusion constructs were synthesized using two llama-derived nanobodies (VHHE and VHHV) and two synthetic nanobodies (Sy15 and Sy68).
Furthermore, a bivalent nanobody was generated by pairing VHHV with VHHE to produce the VHHVE nanobody. In addition, the bivalent VHHEE nanobody was produced to test the binding ability of the dimers. The published nanobody versions Sy68/15-Jps1 and Sy68/15-Cts1 were controls. Expression/secretion of target fusion proteins was examined using western blots.
Culture supernatants showed sufficient secretion of Sy15-Cts1, VHHV-Cts1, VHHE-Cts1, Sy68/15-Jps1, and VHHEE-Cts1 fusion proteins. RBD binding activity was assessed by enzyme-linked immunosorbent assay (ELISA) of cell extracts containing nanobody-Cts1 fusion proteins. VHHEE-Cts1 and Sy68/15-Jps1 showed the strongest binding to RBD, while VHHE-Cts1 showed approximately half the signal intensity. The remaining fusion proteins had no clear binding activity.
In addition, these three fusion proteins were purified and evaluated in a direct ELISA against the full-length SARS-CoV-2 S1 protein. All three fusion proteins showed significant binding activity; VHHEE-Cts1 and Sy68/15-Jps1 had a twofold increase in binding compared to VHHE-Cts1. The researchers performed tailored neutralization assays to determine whether in vitro activity translated into binding or neutralization in vivo.
VHHE-Cts1 had no neutralizing activity, whereas VHHEE-Cts1 and Sy68/15-Jps1 exhibited virus neutralizing activity. Since Cts1 can bind to chitin-coated surfaces such as magnetic chitin beads, this feature could be exploited to develop a new antigen-testing strategy. Chitin binding was recapitulated in chitin beads using purified recombinant Cts1. The chitin beads were mixed with recombinant Cts1, which confirmed binding of the recombinant protein to chitin.
The β-glucuronidase (Gus)-Cts1 fusion protein was used to quantify the results. The Gus-Jps1 fusion protein, which was predicted not to bind chitin, served as a negative control. Chitin beads were coated with Gus-Cts1 or Gus-Jps1 fusion proteins. Only the Gus-Cts1 fusion protein bound to the chitin beads, corroborating the binding capacity of N-terminal Cts1 fusion proteins.
Quantification of signal intensities revealed that 44% of the recombinant Cts1 protein and 68% of the Gus-Cts1 fusion protein were captured on the bead. Functionality of the fusion protein was assessed after immobilization (in beads). Specifically, Gus activity was detected in beads incubated with cell extracts containing Gus-Cts1, implying retention of functional (enzymatic) activity despite immobilization on beads.
Finally, sandwich immunosorbent assays were performed on ELISA plates and chitin beads to evaluate the ability of VHHEE-Cts1 and VHHE-Cts1 nanobody fusion proteins. Sy68/15-Jps1 was the control in both assays, as it should show activity in ELISA but not in chitin beads. Purified fusion proteins were coated onto ELISA plates, incubated with serially diluted recombinant RBD, and detected using an anti-RBD antibody and horseradish peroxidase (HRP) conjugate.
Although all three nanobody fusion proteins could capture RBD on ELISA plates, only Sy68/15-Jps1 and VHHEE-Cts1 showed volumetric activity for serial dilutions of RBD. VHHEE-Cts1 showed the strongest binding event at the lowest concentration of RBD. Chitin beads were incubated separately with the three nanobody fusion proteins and mixed with RBD. Both VHHEE-Cts1 and VHHE-Cts1 retained binding activity, whereas Sy68/15-Jps1 had no activity on chitin beads. Similar to previous findings, VHHEE-Cts1 showed two-fold more potent activity than VHHE-Cts1.
The RBD capture ability of this chitin-based sensing system was further characterized by determining the volumetric binding activity using the most potent nanobody fusion protein (VHHEE-Cts1). Chitin beads were loaded with VHHEE-Cts1 and incubated with serially diluted recombinant RBD. Activity was detected with a commercial antibody sandwich. The researchers observed a colorimetric reaction within two minutes, with its intensity proportional to the concentrations of RBD.
Conclusions
In summary, the study achieved Cts1-mediated secretion of mono- and bivalent nanobodies against SARS-CoV-2. It provided a proof-of-principle for a chitin-based SARS-CoV-2 antigen assay, facilitated by the unconventional secretion mechanism of Cts1 in U. maydis. The authors verified the applicability of nanobody-Cts1 fusion proteins in the detection and neutralization of viruses in vivo. This confirmed that the nanobody could bind to the infectious virus in addition to the RBD spike. The authors believe that this strategy could be transformed into a lab-on-a-chip method for SARS-CoV-2 antigen testing.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.