Immunological assessment of NSFu1: A novel fusion molecule constructed from structural proteins of SARS-CoV-2 for improving COVID-19 antibody detection

The SARS-CoV-2 outbreak has claimed millions of lives and caused significant clinical challenges. The availability of a rapid, cost-effective, and sensitive test to detect antibodies at different stages of COVID-19 is crucial for effective clinical management, epidemiological studies, and public health surveillance. Four novel peptides (SF1, SF2, SF4, SF6) and two multi-epitope fusion proteins (SFu1 and NSFu1) from less variable regions of the spike and nucleocapsid proteins were developed. After detailed in silico structural validation, all the proteins were expressed in E. coli (BL21), purified by Ni²⁺ affinity chromatography, and CD spectroscopy was also executed for secondary structural analysis. The serological potential was assessed by screening 462 plasma samples from symptomatic, asymptomatic, recovered, follow-up COVID-19 cases, and 212 healthy controls. The recombinant antigens SF1, SF2, SF4, SF6, NC, SFu1, and NSFu1 showed ELISA sensitivities of 32.9%, 41.5%, 37.3%, 28.8%, 30.7%, 65.8%, and 82.0%, respectively with specificities ranging from 97 to 99% for symptomatic and asymptomatic COVID-19 cases. The sensitivities for the fusion proteins were nearly equivalent to the combined sensitivities of their constituent antigens. In conclusion, the NSFu1 fusion protein showing 82% sensitivity and 99% specificity could be a potential antigen for developing new molecules to achieve higher sensitivity for COVID-19 antibody detection.