Design of vaccine nanotechnology-based delivery systems, the effect of CpGODN TLR9 agonist-protein antigen conjugates anchored to liposomes

Incorrect date on title page, 2019. Date of award is 2020.The efficiency of CpG oligonucleotides as Toll like receptor (TLR) 9 agonist has been well established along the last few years. Although CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity have generated concern for the use of CpGODN. In an effort to increase stability, localise action and reduce dosage, different strategies have been approached, such as conjugation of CpGODN with immunogenic agents or encapsulation/adsorption of CpGODN into/onto liposomes resulting in enhanced immunopotency compared to coadministration of free CpGODN and antigen. Despite the advances in the field, the effect of conjugation of TLR9 to antigen in combination with liposomes on the immunogenicity of protein-based vaccines has not been explored yet.In this present study, thiol-maleimide chemistry was utilised for the covalent ligation between protein antigen and CpGODN TLR9 agonist, which did not alter protein's ability to be recognised by specific antibodies or activation of receptor by TLR9 agonist. Thanks to its negative charge, protein conjugate was electrostatically bound to cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The designed system GBS67-CpGODN+L shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following immunisation through the intramuscular (i.m.) route, cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) formed a vaccine depot at the injection site, which translated into notable increase of functional immune responses compared to the simple coadministration of GBS67, CpGODN and liposomes (GBS67+CpGODN+L). This effect seems due to increased total IgG level and specifically of IgG2a subtype, although no specific Th1/Th2-driven response was found.This work demonstrates that the conjugation of TLR9 agonist to GBS67 in conjunction with adsorption on cationic liposomes, can promote codelivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. The findings of this study highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms achieving therapeutic effect at lower doses.The efficiency of CpG oligonucleotides as Toll like receptor (TLR) 9 agonist has been well established along the last few years. Although CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity have generated concern for the use of CpGODN. In an effort to increase stability, localise action and reduce dosage, different strategies have been approached, such as conjugation of CpGODN with immunogenic agents or encapsulation/adsorption of CpGODN into/onto liposomes resulting in enhanced immunopotency compared to coadministration of free CpGODN and antigen. Despite the advances in the field, the effect of conjugation of TLR9 to antigen in combination with liposomes on the immunogenicity of protein-based vaccines has not been explored yet.In this present study, thiol-maleimide chemistry was utilised for the covalent ligation between protein antigen and CpGODN TLR9 agonist, which did not alter protein's ability to be recognised by specific antibodies or activation of receptor by TLR9 agonist. Thanks to its negative charge, protein conjugate was electrostatically bound to cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The designed system GBS67-CpGODN+L shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following immunisation through the intramuscular (i.m.) route, cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) formed a vaccine depot at the injection site, which translated into notable increase of functional immune responses compared to the simple coadministration of GBS67, CpGODN and liposomes (GBS67+CpGODN+L). This effect seems due to increased total IgG level and specifically of IgG2a subtype, although no specific Th1/Th2-driven response was found.This work demonstrates that the conjugation of TLR9 agonist to GBS67 in conjunction with adsorption on cationic liposomes, can promote codelivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. The findings of this study highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms achieving therapeutic effect at lower doses

Lipid-based nanoparticles for delivery of vaccine adjuvants and antigens : toward multicomponent vaccines

Despite the many advances that have occurred in the field of vaccine adjuvants, there are still unmet needs that may enable the development of vaccines suitable for more challenging pathogens (e.g., HIV and tuberculosis) and for cancer vaccines. Liposomes have already been shown to be highly effective as adjuvant/delivery systems due to their versatility and likely will find further uses in this space. The broad potential of lipid-based delivery systems is highlighted by the recent approval of COVID-19 vaccines comprising lipid nanoparticles with encapsulated mRNA. This review provides an overview of the different approaches that can be evaluated for the design of lipid-based vaccine adjuvant/delivery systems for protein, carbohydrate, and nucleic acid-based antigens and how these strategies might be combined to develop multicomponent vaccines

Synthesis of protein conjugates adsorbed on cationic liposomes surface

The well-known Toll like receptor 9 (TLR9) agonist CpG ODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, however its in vivo stability and potential systemic toxicity remain a concern. In an effort to overcome these issues, different strategies have been explored including conjugation of CpG ODN with proteins or encapsulation/adsorption of CpG ODN into/onto liposomes. Although these methods have resulted in enhanced immunopotency compared to co-administration of free CpG ODN and antigen, we believe that this effect could be further improved. Here, we designed a novel delivery system of CpG ODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate model protein with the CpG ODN 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) in a very high degree. The novel cationic liposomes-protein conjugate complex shared similar vesicle characteristics (size and charge) compared to free liposomes. The conjugation of CpG ODN to protein in conjunction with adsorption on cationic liposomes, could promote co-delivery leading to the induction of immune response at low antigen and CpG ODN doses. • The CpG ODN Toll-like receptor (TLR) 9 agonist was conjugated to protein antigens via thiol-maleimide chemistry. • Due to their negative charge, protein conjugates readily electrostatically bound cationic liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA) resulting to the design of novel cationic liposomes-protein conjugate complexes. • The method is suited for the liposomal delivery of a variety of adjuvant-protein conjugates

Development and in vivo validation of phospholipid-based depots for the sustained release of bupivacaine.

By direct deposition of the drug at the local site of action, injectable depot formulations - intended for treatment of a local disease or for local intervention - are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to neutral ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP

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