Protein glycosylation is a common post-translational modification that influences the functions and properties of proteins. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of site-specifically controlling glycosylation at each of several positions within a protein. Here, we address this limitation by discovering and exploiting the unique, conditionally orthogonal peptide acceptor specificities of N-glycosyltransferases (NGTs). We used cell-free protein synthesis and mass spectrometry of self-assembled monolayers to rapidly screen 41 putative NGTs and rigorously characterize the unique acceptor sequence preferences of four NGT variants using 1254 acceptor peptides and 8306 reaction conditions. We then used the optimized NGT-acceptor sequence pairs to sequentially install monosaccharides at four sites within one target protein. This strategy to sitespecifically control the installation of N-linked monosaccharides for elaboration to a variety of functional N-glycans overcomes a major limitation in synthesizing defined glycoproteins for research and therapeutic applications.

Effective SARS-CoV-2 vaccines are urgently needed. While most vaccine strategies have focused on systemic immunization, here we compared the protective efficacy of two adjuvanted subunit vaccines with spike protein S1: an intramuscular (IM)-primed /boosted vaccine and an IM-primed/intranasal (IN)-boosted mucosal vaccine, in rhesus macaques. The IM-alum-only vaccine induced robust binding and neutralizing antibody and persistent cellular immunity systemically and mucosally, while IN boosting with nanoparticles including IL-15 and TLR agonists elicited weaker T-cell and antibody responses, but higher dimeric IgA and IFNa.Nevertheless, following SARS-CoV-2 challenge, neither group showed detectable subgenomic RNA in upper or lower respiratory tracts vs naïve controls, indicating full protection against viral replication. Though mucosal and systemic protective mechanisms may differ, results demonstrate both vaccines can protect against respiratory SARS-CoV-2 exposure. The mucosal vaccine was safe after multiple doses and cleared the input virus more efficiently in the nasal cavity, and thus may act as a potent complementary reinforcing boost for conventional systemic vaccines to provide overall better protection.

Site-specific labeling and conjugation of antibodies are highly desirable for fundamental research and for developing more efficient diagnostic and therapeutic methods. We report here a general and robust chemoenzymatic method that permits a one-pot site-specific functionalization of antibodies. A series of selectively modified disaccharide oxazoline derivatives were designed, synthesized, and evaluated as donor substrates of different endoglycosidases for antibody Fc glycan remodeling. We found that among several endoglycosidases tested, wild-type endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) exhibited remarkable activity in transferring the functionalized disaccharides carrying site-selectively modified azide, biotin, or fluorescent tags to antibodies without hydrolyzing the resulting transglycosylation products. This discovery, together with the excellent Fc deglycosylation activity of Endo-S2 on recombinant antibodies, allowed direct labeling and functionalization of antibodies in a one-pot manner without the need of intermediate and enzyme separation. The site-specific introduction of varied numbers of azide groups enabled a highly efficient synthesis of homogeneous antibody–drug conjugates (ADCs) with a precise control of the drug-to-antibody ratio (DAR) ranging from 2 to 12 via a copper-free strain-promoted click reaction. Cell viability assays showed that ADCs with higher DARs were more potent in killing antigen-overexpressed cells than the ADCs with lower DARs. This new method is expected to find applications not only for antibody–drug conjugation but also for cell labeling, imaging, and diagnosis.

Emerging of SARS-CoV-2 variants and waning of vaccine/infection-induced immunity poses threats to curbing the COVID-19 pandemic. Effective, safe, and convenient booster vaccines are in need. We hypothesized that a variant-modified mucosal booster vaccine might induce local immunity to prevent SARS-CoV-2 infection at the port of entry. The beta-variant is one of the hardest to cross-neutralize. Herein we assessed the protective efficacy of an intranasal booster composed of beta variant-spike protein S1 with IL-15 and TLR agonists in previously immunized macaques. The macaques were first vaccinated with Wuhan strain S1 with the same adjuvant. One year later, negligibly detectable SARS-CoV-2-specific antibody remained. Nevertheless, the booster induced vigorous humoral immunity including serum- and bronchoalveolar lavage (BAL)-IgG, secretory nasal- and BAL-IgA, and neutralizing antibody against the original strain and/or beta variant. Beta-variant S1-specific CD4+ and CD8+ T cell responses were also elicited in PBMC and BAL. Following SARS-CoV-2 beta variant challenge, the vaccinated group demonstrated significant protection against viral replication in the upper and lower respiratory tracts, with almost full protection in the nasal cavity. The fact that one intranasal beta-variant booster administrated one year after the first vaccination provoked protective immunity against beta variant infections may inform future SARS-CoV-2 booster design and administration timing.

Glycosylation plays important roles in SARS-CoV-2 infection. We describe here a facile chemoenzymatic synthesis of core-fucosylated N-glycopeptides derived from the SARS-CoV-2 Spike protein and their binding with glycan-dependent neutralizing antibody...