Non-Native Amino Acid Click Chemistry-Based Technology for Site-Specific Polysaccharide Conjugation to a Bacterial Protein Serving as Both Carrier and Vaccine Antigen.

Surface-expressed bacterial polysaccharides are important vaccine antigens but must be conjugated to a carrier protein for efficient antigen presentation and development of strong memory B cell and antibody responses, especially in young children. The commonly used protein carriers include tetanus toxoid (TT), diphtheria toxoid (DT), and its derivative CRM197, but carrier-induced epitopic suppression and bystander interference may limit the expanded use of the same carriers in the pediatric immunization schedule. Recent efforts to develop a vaccine against the major human pathogen group A Streptococcus (GAS) have sought to combine two promising vaccine antigens-the universally conserved group A cell wall carbohydrate (GAC) with the secreted toxin antigen streptolysin O (SLO) as a protein carrier; however, standard reductive amination procedures appeared to destroy function epitopes of the protein, markedly diminishing functional antibody responses. Here, we couple a cell-free protein synthesis (CFPS) platform, allowing the incorporation of non-natural amino acids into a C-terminally truncated SLO toxoid for the precise conjugation to the polyrhamnose backbone of GAC. The combined immunogen generated functional antibodies against both conserved GAS virulence factors and provided protection against systemic GAS challenges. CFPS may represent a scalable method for generating pathogen-specific carrier proteins for multivalent subunit vaccine development.

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation.

Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

A Porphyromonas gingivalis Capsule-Conjugate Vaccine Protects From Experimental Oral Bone Loss.

Periodontal diseases are chronic inflammatory diseases of the periodontium that result in progressive destruction of the soft and hard tissues supporting the teeth, and it is the most common cause of tooth loss among adults. In the US alone, over 100 million individuals are estimated to have periodontal disease. Subgingival bacteria initiate and sustain inflammation, and, although several bacteria have been associated with periodontitis, Porphyromonas gingivalis has emerged as the key etiological organism significantly contributing to the disease. Currently, intensive clinical maintenance strategies are deployed to mitigate the further progression of disease in afflicted individuals; however, these treatments often fail to stop disease progression, and, as such, the development of an effective vaccine for periodontal disease is highly desirable. We generated a conjugate vaccine, comprising of the purified capsular polysaccharide of P. gingivalis conjugated to eCRM®, a proprietary and enhanced version of the CRM197 carrier protein with predetermined conjugation sites (Pg-CV). Mice immunized with alum adjuvanted Pg-CV developed robust serum levels of whole organism-specific IgG in comparison to animals immunized with unconjugated capsular polysaccharide alone. Using the murine oral bone loss model, we observed that mice immunized with the capsule-conjugate vaccine were significantly protected from the effects of P. gingivalis-elicited oral bone loss. Employing a preclinical model of infection-elicited oral bone loss, our data support that a conjugate vaccine incorporating capsular polysaccharide antigen is effective in reducing the main clinical endpoint of periodontal disease-oral bone destruction. Further development of a P. gingivalis capsule-based conjugate vaccine for preventing periodontal diseases is supported.