Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

A multivalent Plasmodium falciparum circumsporozoite protein-based nanoparticle malaria vaccine elicits a robust and durable antibody response against the junctional epitope and the major repeats.

Plasmodium falciparum (Pf) malaria continues to cause considerable morbidity and mortality worldwide. The circumsporozoite protein (CSP) is a particularly attractive candidate for designing vaccines that target sporozoites-the first vertebrate stage in a malaria infection. Current PfCSP-based vaccines, however, do not include epitopes that have recently been shown to be the target of potent neutralizing antibodies. We report the design of a SpyCatcher-mi3-nanoparticle-based vaccine presenting multiple copies of a chimeric PfCSP (cPfCSP) antigen that incorporates these important "T1/junctional" epitopes as well as a reduced number of (NANP)n repeats. cPfCSP-SpyCatcher-mi3 was immunogenic in mice eliciting high and durable IgG antibody levels as well as a balanced antibody response against the T1/junctional region and the (NANP)n repeats. Notably, the antibody concentration elicited by immunization was significantly greater than the reported protective threshold defined in a murine challenge model. Refocusing the immune response toward functionally relevant subdominant epitopes to induce a more balanced and durable immune response may enable the design of a more effective second generation PfCSP-based vaccine.

Nanopatterning protein antigens to refocus the immune response.

Vaccines for many important diseases remain elusive, and those for others need to be updated frequently. Vaccine efficacy has been hindered by existing sequence diversity in proteins and by newly-acquired mutations that enable escape from vaccine-induced immune responses. To address these limitations, we developed an approach for nanopatterning protein antigens that combines the site-specific incorporation of non-canonical amino acids with chemical modification to focus the immune response on conserved protein regions. We demonstrated the approach using green fluorescent protein (GFP) as a model antigen and with a promising malaria vaccine candidate, Merozoite surface protein 119 (MSP119). Immunization of mice with nanopatterned MSP119 elicited antibodies that recognized MSP119 from heterologous strains, differing in sequence at as many as 21 of 96 residues. Nanopatterning should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.