Significance: Conjugate vaccines have proven to be an effective and safe strategy for reducing the incidence of disease caused by bacterial pathogens. However, the manufacture of these vaccines is technically demanding, inefficient, and expensive, thereby limiting their widespread use. Here, we describe an alternative methodology for generating glycoconjugate vaccines whereby recombinant polysaccharide biosynthesis is coordinated with vesicle formation in nonpathogenic Escherichia coli , resulting in glycosylated outer membrane vesicles (glycOMVs) that can effectively deliver pathogen-mimetic glycotopes to the immune system. An attractive feature of our approach is the fact that different plasmid-encoded polysaccharide biosynthetic pathways can be readily transformed into E. coli , enabling a “plug-and-play” platform for the on-demand creation of glycOMV vaccine candidates that carry heterologous glycotopes from numerous pathogenic bacteria.

Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

Significance: A broad range of bacterial, fungal, and protozoan cells produce the surface polysaccharide poly- N -acetyl- d -glucosamine (PNAG), making this antigen an attractive target for vaccination against multiple human and economically important animal pathogens. While conjugate vaccines involving surface polysaccharides, such as PNAG, are a proven strategy for reducing the incidence of disease caused by bacterial pathogens, their manufacture is technically demanding, inefficient, and expensive, thereby limiting their widespread adoption. Here, we describe an alternative route to producing PNAG-containing glycoconjugates, whereby recombinant PNAG biosynthesis is coordinated with outer membrane vesicle formation in nonpathogenic Escherichia coli strains. The resulting glycosylated outer membrane vesicles effectively deliver PNAG antigens to the immune system while bypassing many of the drawbacks of conventional conjugate vaccines.

Significance: Asparagine-linked ( N -linked) protein glycosylation—the covalent attachment of complex sugars to the nitrogen atom in asparagine side chains—is the most widespread posttranslational modification to proteins and also the most complex. N- glycosylation affects a significant number of cellular proteins and can have profound effects on their most important attributes such as biological activity, chemical solubility, folding and stability, immunogenicity, and serum half-life. Accordingly, the strategic installation of glycans at naïve sites has become an attractive means for endowing proteins with advantageous biological and/or biophysical properties. Here, we describe a glycoprotein engineering strategy that enables systematic investigation of the structural and functional consequences of glycan installation at every position along a protein backbone and provides a new route to bespoke glycoproteins.

Diarrheal disease is a global public health threat. Cases of enteric disease caused by bacterial enteropathogens result in a significant health burden and can lead to antimicrobial resistance patterns that increase and evolve over time and geography, posing serious health challenges for local populations as well as civilian travelers and military personnel deployed to endemic regions. This review describes the prevalence of the most common bacterial agents of diarrheal disease in South America, as well as the distribution patterns of antibiotic resistance and predominant strains that are present in the region. Furthermore, the development of relevant prevention and treatment strategies are described, and United States Naval Medical Research Unit SOUTH disease surveillance and enteric disease research efforts in this regional context are highlighted.