Self-assembled monovalent lipidated mannose ligand as a standalone nanoadjuvant.

Subunit vaccines require an immunostimulant (adjuvant) and/or delivery system to induce immunity. However, currently, available adjuvants are either too dangerous in terms of side effects for human use (experimental adjuvants) or have limited efficacy and applicability. In this study, we examined the capacity of mannose-lipopeptide ligands to enhance the immunogenicity of a vaccine consisting of polyleucine(L15)-antigen conjugates anchored to liposomes. The clinically tested Group A Streptococcus (GAS) B-cell epitope, J8, combined with universal T helper PADRE (P) was used as the antigen. Six distinct mannose ligands were incorporated into neutral liposomes carrying L15PJ8. While induced antibody titers were relatively low, the ligand carrying mannose, glycine/lysine spacer, and two palmitic acids as liposomal membrane anchoring moieties (ligand 3), induced significantly higher IgG titers than non-mannosylated liposomes. The IgG titers were significantly enhanced when positively charged liposomes were employed. Importantly, the produced antibodies were able to kill GAS bacteria. Unexpectedly, the physical mixture of only ligand 3 and PJ8 produced self-assembled nanorods that induced antibody titers as high as those elicited by the lead liposomal formulation and antigen adjuvanted with the potent, but toxic, complete Freund's adjuvant (CFA). Antibodies produced upon immunization with PJ8 + 3 were even more opsonic than those induced by CFA + PJ8. Importantly, in contrast to CFA, ligand 3 did not induce observable adverse reactions or excessive inflammatory responses. Thus, we demonstrated that a mannose ligand, alone, can serve as an effective vaccine nanoadjuvant.

Cholesterol as an inbuilt immunoadjuvant for a lipopeptide vaccine against group A Streptococcus infection.

Peptide-based vaccines can trigger highly specific immune responses, although peptides alone are usually unable to confer strong humoral or cellular immunity. Consequently, peptide antigens are administered with immunostimulatory adjuvants, but only a few are safe and effective for human use. To overcome this obstacle, herein a peptide antigen was lipidated to effectively anchor it to liposomes and emulsion. A peptide antigen B cell epitope from Group A Streptococcus M protein was conjugated to a universal T helper epitope, the pan DR-biding epitope (PADRE), alongside a lipidic moiety cholesterol. Compared to a free peptide antigen, the lipidated version (LP1) adopted a helical conformation and self-assembled into small nanoparticles. Surprisingly, LP1 alone induced the same or higher antibody titers than liposomes or emulsion-based formulations. In addition, antibodies produced by mice immunized with LP1 were more opsonic than those induced by administering the antigen with incomplete Freund's adjuvant. No side effects were observed in the immunized mice and no excessive inflammatory immune responses were detected. Overall, this study demonstrated how simple conjugation of cholesterol to a peptide antigen can produce a safe and efficacious vaccine against Group A Streptococcus - the leading cause of superficial infections and the bacteria responsible for deadly post-infection autoimmune disorders.

Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

The influence of component structural arrangement on peptide vaccine immunogenicity.

Peptide-based subunit vaccines utilise minimal immunogenic components (i.e. peptides) to generate highly specific immune responses, without triggering adverse reactions. However, strong adjuvants and/or effective delivery systems must be incorporated into such vaccines, as peptide antigens cannot induce substantial immune responses on their own. Unfortunately, many adjuvants are too weak or too toxic to be used in combination with peptide antigens. These shortcomings have been addressed by the conjugation of peptide antigens with lipidic/ hydrophobic adjuvanting moieties. The conjugates have shown promising safety profiles and improved immunogenicity without the help of traditional adjuvants and have been efficient in inducing desired immune responses following various routes of administration, including subcutaneous, oral and intranasal. However, not only conjugation per se, but also component arrangement influences vaccine efficacy. This review highlights the importance of influence of the vaccine chemical structure modification on the immune responses generated. It discusses a variety of factors that affect the immunogenicity of peptide conjugates, including: i) self-adjuvanting moiety length and number; ii) the orientation of epitopes and self-adjuvanting moieties in the conjugate; iii) the presence of spacers between conjugated components; iv) multiepitopic arrangement; and v) the effect of chirality on vaccine efficacy.