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messenger RNA (mRNA)-based vaccines combine the positive attributes of both live-attenuated and subunit vaccines. In order for these to be applied for clinical use, they require to be formulated with delivery systems. However, there are limited in vivo studies which compare different delivery platforms. Therefore, we have compared four different cationic platforms: (1) liposomes, (2) solid lipid nanoparticles (SLNs), (3) polymeric nanoparticles (NPs) and (4) emulsions, to deliver a self-amplifying mRNA (SAM) vaccine. All formulations contained either the non-ionizable cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDA) and they were characterized in terms of physico-chemical attributes, in vitro transfection efficiency and in vivo vaccine potency. Our results showed that SAM encapsulating DOTAP polymeric nanoparticles, DOTAP liposomes and DDA liposomes induced the highest antigen expression in vitro and, from these, DOTAP polymeric nanoparticles were the most potent in triggering humoral and cellular immunity among candidates in vivo.

Background and Objectives Generalized modules for membrane antigens (GMMA) are outer membrane vesicles derived from gram‐negative bacteria that can be used to design affordable subunit vaccines. GMMA are highly immunogenic and capable to induce an optimal antigen‐specific humoral immune response both in animals and humans. Despite their potent immunogenicity, GMMA are usually formulated with aluminum salts (alum) to reduce their potential systemic reactogenicity. GMMA, in fact, contain agonists of toll‐like receptor 4 (TLR4) and TLR2 that possess pro‐inflammatory activity. The adsorption of GMMA onto alum is believed to reduce their systemic exposure. However, it has been found that GMMA formulated without alum did not induce concerning signs of systemic reactogenicity in the rabbit model. Here, we asked whether GMMA promote local reactogenicity. Methods We immunized mice intramuscularly with GMMA alone or adsorbed to alum and analyzed the injection site during 7 days after treatment. Results We found that GMMA alone promoted only mild inflammation within the muscle, whereas the presence of alum induced severe muscle inflammation, as expected. Thus, in the mouse model, GMMA demonstrated to possess mild local reactogenic potential, while alum is confirmed a major driver of local reactogenicity. Conclusion Our results further support the idea to investigate the reactogenicity of GMMA formulated without alum in clinical studies.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen–GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.

Group B Streptococcus (GBS) causes serious infection in neonates and is an important target of vaccine development. Zwitterionic polysaccharides (ZPS), obtained through chemical introduction of positive charges into anionic polysaccharides (PS) from GBS, have the ability to activate human and mouse antigen presenting cells (APCs) through toll-like receptor 2 (TLR2). To generate a polysaccharide vaccine with antigen (Ag) and adjuvant properties in one molecule, we have conjugated ZPS with a carrier protein. ZPS-glycoconjugates induce higher T-cell and Ab responses to carrier and PS, respectively, compared to control PS-glycoconjugates made with the native polysaccharide form. The increased immunogenicity of ZPS-conjugates correlates with their ability to activate dendritic cells (DCs). Moreover, protection of mothers or neonate offspring from lethal GBS challenge is better when mothers are immunized with ZPS-conjugates compared to immunization with PS-conjugates. In TLR2 knockout mice, ZPS-conjugates lose both their increased immunogenicity and protective effect after vaccination. When ZPS are coadministered as adjuvants with unconjugated tetanus toxoid (TT), they have the ability to increase the TT-specific antibody titer. In conclusion, glycoconjugates containing ZPS are potent vaccines. They target Ag to TLR2-expressing APCs and activate these APCs, leading to better T-cell priming and ultimately to higher protective Ab titers. Thus, rational chemical design can generate potent PS-adjuvants with wide application, including glycoconjugates and coadministration with unrelated protein Ags.

Microstructure patches provide an opportunity for simple, effective, and safe vaccine administration, while achieving the desired immune response. We have evaluated the MicroCor® transdermal system for cell culture-derived trivalent influenza vaccine administration. Influenza monovalent purified bulk vaccines (monobulks) (H1N1, H3N2, B) were concentrated by tangential flow filtration, lyophilized, and formulated with biocompatible excipients to form the microstructure array dissolvable tips. Standard single radial immunodiffusion (SRID) determined that the influenza antigens retained potency through the formulation and microstructure array fabrication processes. Array stability was evaluated for storage in both refrigerated and room temperature conditions. Microstructure mechanical strength was confirmed by application to excised pig skin, resulting in successful skin penetration and tip dissolution within 5min of microstructure insertion. Guinea pigs immunized with influenza vaccine-loaded microstructures had hemagglutinin inhibition (HI) and IgG titers comparable to those obtained by intramuscular injection. After two immunizations, serum HI titers for all immunized groups were greater than 40 (>4-fold higher than the untreated group). These data demonstrate the feasibility for the development of skin delivery technologies that are compatible with cell culture-derived influenza vaccines.

•This paper provides a novel and original strategy to develop subunit vaccines for sublingual delivery.•Sublingual administration of subunit influenza Ags adjuvanted with LTK63 elicited comparable antibody titers to intramuscular immunization.•The sublingual delivery elicited Ag-specific mucosal IgA with neutralizing activity, contributing to anti-influenza defenses.•The mucosal route in combination with LTK63 elicited an Ag-specific Th17 response that might play a role in protection against influenza.•Sublingual delivery of an adjuvanted subunit influenza vaccine could be an effective alternative to conventional intramuscular vaccines. Influenza is a vaccine-preventable disease that remains a major health problem world-wide. Needle and syringe are still the primary delivery devices, and injection of liquid vaccine into the muscle is still the primary route of immunization. Vaccines could be more convenient and effective if they were delivered by the mucosal route. Elicitation of systemic and mucosal innate and adaptive immune responses, such as pathogen neutralizing antibodies (including mucosal IgA at the site of pathogen entry) and CD4+ T-helper cells (especially the Th17 subset), have a critical role in vaccine-mediated protection. In the current study, a sublingual subunit influenza vaccine formulated with or without mucosal adjuvant was evaluated for systemic and mucosal immunogenicity and compared to intranasal and intramuscular vaccination. Sublingual administration of adjuvanted influenza vaccine elicited comparable antibody titers to those elicited by intramuscular immunization with conventional influenza vaccine. Furthermore, influenza-specific Th17 cells or neutralizing mucosal IgA were detected exclusively after mucosal immunization.

•Novel strategy to develop intradermally administered MenC glycoconjugate vaccine.•New glycoconjugate formulation was developed using a robust, scalable process.•Intradermal administration of MenC glycoconjugate vaccine is beneficial.•Appropriate adjuvants direct immune response to a more effective quality profile. Intradermal vaccine delivery is a promising alternative to the conventional intramuscular route. The skin layer is immunologically supported by a densely network of antigen presenting cells, while the skeletal muscle is loaded with a relatively sparse population of APCs. Nevertheless, the vaccine to be suitable for intradermal delivery needs a new formulation to facilitate either smaller injection volumes or the introduction into new delivery devises as micro-needles. This study presents a proof of concept for intradermal delivery of the MenC-CRM197 glycoconjugate vaccine using a mouse model. Tangential flow filtration allowed obtaining a 20-fold concentrated vaccine formulation suitable for intradermal injection. Importantly the intradermal delivery of non-adjuvanted MenC glycoconjugate vaccine showed a quicker on-set and superiority in terms of immunogenicity compared to intramuscular administration of the respective vaccine and comparable immunogenicity to the aluminum adjuvanted vaccine formulation given intramuscular. Subsequently, the use of adjuvants allowed to further increase the immunogenicity and to modulate the quality of the immune response towards a more beneficial Th1 response. As adjuvants two Toll like receptor agonists (TLR4a and TLR7a), a mutant of the heat-labile enterotoxin from Escherichia coli (LT), a α-GalactosylCeramide analogue and an oil in water emulsion were investigated in order to target skin-resident antigen-presenting cells. This approach has the potential to be extended to other meningococcal serogroups, representing a promising strategy for the development of dermally administered multivalent glycoconjugate vaccines.

Emulsions have been used to boost immunogenicity of antigens since the discovery of complete Freunds adjuvant. Optimization to reduce reactogenicity of emulsion adjuvants lead to the development of oil in water emulsions based on squalene. MF59 is an oil-in-water emulsion that is a component of an approved influenza product in Europe. Currently MF59 is manufactured from squalene derived from an animal source. Recently a high purity plant-derived squalene source has become available at an appropriate purity for a vaccine adjuvant. The purpose of this study was to evaluate and compare animal-derived squalene and plant-derived squalene for equivalency. Nanoemulsions were prepared and analyzed for size and viscosity prepared from each source. The two emulsions were administered in two separate animal studies, one focusing on Neisseria meningitidis B, and one focusing on influenza. Readouts were ELISA titers for each antigen and serum bactericidal activity for N. meningitidis B, and hemagglutinin inhibition for influenza to see the functionality of the antibodies produced. Results indicate that there are no differences between the antibodies elicited after immunization from an emulsion made with oil derived from either an animal or plant-source.