Design of a novel vaccine nanotechnology-based delivery system comprising CpGODN-protein conjugate anchored to liposomes

Although the well-known Toll like receptor 9 (TLR9) agonist CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity remain a concern. In an effort to circumvent these issues, different strategies have been employed to increase its stability, localise action and reduce dosage. These include conjugation of CpGODN with proteins or encapsulation/adsorption of CpGODN into/onto liposomes, and have resulted in enhanced immunopotency compared to co-administration of free CpGODN and antigen. Here, we designed a novel delivery system of CpGODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate the Group B Streptococcus (GBS) GBS67 protein antigen with the CpGODN TLR9 agonist. This treatment did not alter protein's ability to be recognised by specific antibodies or the CpGODN to function as a TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The novel cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following intramuscular immunisation, GBS67-CpGODN+L formed a vaccine depot at the injection site and induced a remarkable increase of functional immune responses against GBS compared to the simple co-administration of GBS67, CpGODN and liposomes. This work demonstrates that the conjugation of CpGODN to GBS67 in conjunction with adsorption on cationic liposomes, can promote co-delivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. Our findings highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms

Functional characterization of the gonococcal polyphosphate pseudo-capsule.

Neisseria gonorrhoeae is an exclusively human pathogen able to evade the host immune system through multiple mechanisms. Gonococci accumulate a large portion of phosphate moieties as polyphosphate (polyP) on the exterior of the cell. Although its polyanionic nature has suggested that it may form a protective shield on the cell surface, its role remains controversial. Taking advantage of a recombinant His-tagged polyP-binding protein, the presence of a polyP pseudo-capsule in gonococcus was demonstrated. Interestingly, the polyP pseudo-capsule was found to be present in specific strains only. To investigate its putative role in host immune evasion mechanisms, such as resistance to serum bactericidal activity, antimicrobial peptides and phagocytosis, the enzymes involved in polyP metabolism were genetically deleted, generating mutants with altered polyP external content. The mutants with lower polyP content on their surface compared to the wild-type strains, became sensitive to complement-mediated killing in presence of normal human serum. Conversely, naturally serum sensitive strains that did not display a significant polyP pseudo-capsule became resistant to complement in the presence of exogenous polyP. The presence of polyP pseudo-capsule was also critical in the protection from antibacterial activity of cationic antimicrobial peptide, such as cathelicidin LL-37. Results showed that the minimum bactericidal concentration was lower in strains lacking polyP than in those harboring the pseudo-capsule. Data referring to phagocytic killing resistance, assessed by using neutrophil-like cells, showed a significant decrease in viability of mutants lacking polyP on their cell surface in comparison to the wild-type strain. The addition of exogenous polyP overturned the killing phenotype of sensitive strains suggesting that gonococcus could exploit environmental polyP to survive to complement-mediated, cathelicidin and intracellular killing. Taken together, data presented here indicate an essential role of the polyP pseudo-capsule in the gonococcal pathogenesis, opening new perspective on gonococcal biology and more effective treatments

Proof of concept for a single-dose Group B Streptococcus vaccine based on capsular polysaccharide conjugated to Qβ virus-like particles

Abstract A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine