Isolation of Naturally Released Gonococcal Outer Membrane Vesicles as Vaccine Antigens

The emergence and spread of fully antimicrobial resistant Neisseria gonorrhoeae (GC) highlights a clear need for next-generation antigonococcal therapeutics. A broadly reactive anti-GC vaccine would best address this global public health threat. Polyantigenic outer membrane vesicles (OMVs) derived from GC can overcome the challenges posed by GC's high rate of phase and antigen variation. In fact, GC OMVs have already shown promise as a vaccine antigen; however, all previous studies have utilized vesicles contaminated by RMP, a bacterioprotective antigen known to entirely abrogate vaccine-induced bactericidal activity in vivo. Additionally, these studies primarily utilized vesicles isolated through techniques like membrane disruption with detergents, which are known to increase contamination of cytoplasmic components as compared to naturally released OMVs (nOMVs). This chapter describes the isolation and characterization of naturally released nOMVs through sequential size and weight restrictive filtration. nOMVs are characterized by morphology, proteomics, and bioactivity via various methods. Herein we also describe methods for further evaluation of the innate and induced immunogenicity of rmp-deficient GC nOMVs by cell stimulation and murine vaccination. Per these methods, nOMVs are found to be largely homogenous spherical structures approximately 70 nm in diameter containing a consistent subset of GC outer membrane proteins. The rmp-deficient vesicles demonstrate a morphology and, with the exception of RMP, antigenic profile consistent with that of nOMVs derived from wild time N. gonorrhoeae. Additionally, vesicles lacking RMP are able to engage and strongly activate a diverse array of pattern recognition receptors in vitro. These methods lay the groundwork for future experiments examining the in vivo protective efficacy of the anti-GC response induced by these nOMVs as well as studies examining the mechanism of vaccine induced female genital tract immunity.