The Occluded Epitope Residing in Spike Receptor-Binding Motif Is Essential for Cross-Neutralization of SARS-CoV-2 Delta Variant.

Concerns over vaccine efficacy after the emergence of the SARS-CoV-2 Delta variant prompted revisiting the vaccine design concepts. Monoclonal antibodies (mAbs) have been developed to identify the neutralizing epitopes on spike protein. It has been confirmed that the key amino acid residues in epitopes that induce the formation of neutralizing antibodies do not have to be on the receptor-binding domain (RBD)- angiotensin-converting enzyme 2 (ACE2) contact surface, and may be conformationally hidden. In addition, this epitope is tolerant to amino acid mutations of the Delta variant. The antibody titers against RBD in health care workers in Thailand receiving two doses of CoronaVac, followed by a booster dose of BNT162b2, were significantly increased. The neutralizing antibodies against the Delta variant suggest that the overall neutralizing antibody level against the Wuhan strain, using the NeutraLISA, was consistent with the levels of anti-RBD antibodies. However, individuals with moderate anti-RBD antibody responses have different levels of a unique antibody population competing with a cross-neutralizing mAb clone, 40591-MM43, determined by in-house competitive ELISA. Since 40591-MM43 mAb indicates cross-neutralizing activity against the Delta variant, this evidence implies that the efficiency of the vaccination regimen should be improved to facilitate cross-protective antibodies against Delta variant infections. The RBD epitope recognized by 40591-MM43 mAb is hidden in the close state.

Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli.

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E. coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.