A pentavalent vaccine targeting adhesins and metal-binding lipoproteins confers robust protection against enterococcal infections

Infections caused by pathogenic enterococci are notoriously difficult to treat due to the genus intrinsically fastidious nature and high prevalence of multidrug-resistant strains. Recently, we developed a protein-based trivalent vaccine composed of the dual manganese and iron-binding lipoprotein EfaA and the zinc-binding lipoproteins AdcA and AdcAII that conferred significant protection against enterococcal infections in mouse infection models. In this study, we explored the potential of three enterococcal surface adhesins, namely Ace, EbpA, and Esp, as antigens of a novel pentavalent vaccine that included EfaA and AdcAII from the original trivalent vaccine. First, Ace, EbpA, and Esp were produced using a cell-free expression system and used either individually or in combination with EfaA and AdcAII to generate polyclonal antisera in rabbits. While monovalent and pentavalent (AdcAII, EfaA, Ace, EbpA, Esp) antisera exhibited comparable opsonizing activities, passive immunization with the pentavalent antiserum conferred greater protection in a peritoneal infection mouse model. Active immunization with the pentavalent vaccine formulation provided robust protection against both localized and systemic E. faecalis infections. When compared to the trivalent formulation, the pentavalent vaccine provided markedly enhanced protection against E. faecalis lethal infection. Finally, the pentavalent vaccine displayed robust cross-protection against E. faecium lethal infection, indicating its potential to serve as a broad anti-enterococcal vaccine.