Intradermal Vaccination with PLGA Nanoparticles via Dissolving Microneedles and Classical Injection Needles.

PURPOSE: A dissolving microneedle array (dMNA) is a vaccine delivery device with several advantages over conventional needles. By incorporating particulate adjuvants in the form of poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) into the dMNA, the immune response against the antigen might be enhanced. This study aimed to prepare PLGA-NP-loaded dMNA and to compare T-cell responses induced by either intradermally injected aqueous-PLGA-NP formulation or PLGA-NP-loaded dMNA in mice. METHODS: PLGA NPs were prepared with microfluidics, and their physicochemical characteristics with regard to encapsulation efficiencies of ovalbumin (OVA) and CpG oligonucleotide (CpG), zeta potentials, polydispersity indexes, and sizes were analysed. PLGA NPs incorporated dMNA was produced with three different dMNA formulations by using the centrifugation method, and the integrity of PLGA NPs in dMNAs was evaluated. The immunogenicity was evaluated in mice by comparing the T-cell responses induced by dMNA and aqueous formulations containing ovalbumin and CpG (OVA/CpG) with and without PLGA NP. RESULTS: Prepared PLGA NPs had a size of around 100 nm. The dMNA formulations affected the particle integrity, and the dMNA with poly(vinyl alcohol) (PVA) showed almost no aggregation of PLGA NPs. The PLGA:PVA weight ratio of 1:9 resulted in 100% of penetration efficiency and the fastest dissolution in ex-vivo human skin (< 30 min). The aqueous formulation with soluble OVA/CpG and the aqueous-PLGA-NP formulation with OVA/CpG induced the highest CD4 + T-cell responses in blood and spleen cells. CONCLUSIONS: PLGA NPs incorporated dMNA was successfully fabricated and the aqueous formulation containing PLGA NPs induce superior CD4+ and CD8+ T-cell responses.

Stabilin-1 is required for the endothelial clearance of small anionic nanoparticles.

Clearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery.

Systematic Investigation of the Role of Surfactant Composition and Choice of oil: Design of a Nanoemulsion-Based Adjuvant Inducing Concomitant Humoral and CD4+ T-Cell Responses.

PURPOSE: Induction of cell-mediated immune (CMI) responses is crucial for vaccine-mediated protection against difficult vaccine targets, e.g., Chlamydia trachomatis (Ct). Adjuvants are included in subunit vaccines to potentiate immune responses, but many marketed adjuvants stimulate predominantly humoral immune responses. Therefore, there is an unmet medical need for new adjuvants, which potentiate humoral and CMI responses. The purpose was to design an oil-in-water nanoemulsion adjuvant containing a synthetic CMI-inducing mycobacterial monomycoloyl glycerol (MMG) analogue to concomitantly induce humoral and CMI responses. METHODS: The influence of emulsion composition was analyzed using a systematic approach. Three factors were varied: i) saturation of the oil phase, ii) type and saturation of the applied surfactant mixture, and iii) surfactant mixture net charge. RESULTS: The emulsions were colloidally stable with a droplet diameter of 150-250 nm, and the zeta-potential correlated closely with the net charge of the surfactant mixture. Only cationic emulsions containing the unsaturated surfactant mixture induced concomitant humoral and CMI responses upon immunization of mice with a Ct antigen, and the responses were enhanced when squalene was applied as the oil phase. In contrast, emulsions with neutral and net negative zeta-potentials did not induce CMI responses. The saturation degree of the oil phase did not influence the adjuvanticity. CONCLUSION: Cationic, MMG analogue-containing nanoemulsions are potential adjuvants for vaccines against pathogens for which both humoral and CMI responses are needed.

Evaluation of PLGA, lipid-PLGA hybrid nanoparticles, and cationic pH-sensitive liposomes as tuberculosis vaccine delivery systems in a Mycobacterium tuberculosis challenge mouse model - A comparison.

Tuberculosis (TB) continues to pose a global threat for millennia, currently affecting over 2 billion people and causing 10.6 million new cases and 1.3 million deaths annually. The only existing vaccine, Mycobacterium Bovis Bacillus Calmette-Guérin (BCG), provides highly variable and inadequate protection in adults and adolescents. This study explores newly developed subunit tuberculosis vaccines that use a multistage protein fusion antigen Ag85b-ESAT6-Rv2034 (AER). The protection efficacy, as well as in vivo induced immune responses, were compared for five vaccines: BCG; AER-CpG/MPLA mix; poly(D,L-lactic-co-glycolic acid) (PLGA); lipid-PLGA hybrid nanoparticles (NPs); and cationic pH-sensitive liposomes (the latter three delivering AER together with CpG and MPLA). All vaccines, except the AER-adjuvant mix, induced protection in Mycobacterium tuberculosis (Mtb)-challenged C57/Bl6 mice as indicated by a significant reduction in bacterial burden in lungs and spleens of the animals. Four AER-based vaccines significantly increased the number of circulating multifunctional CD4+ and CD8+ T-cells producing IL-2, IFNγ, and TNFα, exhibiting a central memory phenotype. Furthermore, AER-based vaccines induced an increase in CD69+ B-cell counts as well as high antigen-specific antibody titers. Unexpectedly, none of the observed immune responses were associated with the bacterial burden outcome, such that the mechanism responsible for the observed vaccine-induced protection of these vaccines remains unclear. These findings suggest the existence of non-classical protective mechanisms for Mtb infection, which could, once identified, provide interesting targets for novel vaccines.

Understanding opalescence measurements of biologics - A comparison study of methods, standards, and molecules.

Opalescence measurements are broadly applied to assess the quality and stability of biopharmaceutical products at all stages of development and manufacturing. They appear to be simple and straight forward but detect complex light scattering phenomena. Despite a routine calibration step, opalescence values obtained with the same biopharmaceutical sample but on different instruments and/or with different methods may vary significantly. Since the reasons for this high variability are generally not well understood, comparison of opalescence results from different biopharmaceutical laboratories is difficult. Here, we characterized a comprehensive set of biopharmaceutically relevant samples with five opalescence methods to illustrate fundamental differences in method performance and explore the reasons for poor comparability. In addition, we developed a high-throughput method for measuring opalescence in a conventional light scattering plate reader that yields opalescence values in the same range as compendial methods. The presented results underline the impact of sample properties, instrument type, and calibration standards on the determined opalescence value. Based on our findings we provide recommendations for the appropriate application of each method during biopharmaceutical drug product development. Overall, our study contributes to an improved understanding of opalescence measurements in the biopharmaceutical field.

Atomic force microscopy measurements of anionic liposomes reveal the effect of liposomal rigidity on antigen-specific regulatory T cell responses.

Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigen-specific Treg responses. We hypothesized that altering the rigidity of these liposomes while maintaining their size and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes is affected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part by the presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of 4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL (1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa), DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG (494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-derived dendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation between liposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responses in vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measure liposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system.

Induction of Cytotoxic T-Lymphocyte Responses Upon Subcutaneous Administration of a Subunit Vaccine Adjuvanted With an Emulsion Containing the Toll-Like Receptor 3 Ligand Poly(I:C).

There is an unmet medical need for new subunit vaccines that induce cytotoxic T-lymphocyte (CTL) responses to prevent infection with a number of pathogens. However, stimulation of CTL responses via clinically acceptable subcutaneous (s.c.) and intramuscular (i.m.) injection is challenging. Recently, we designed a liposomal adjuvant [cationic adjuvant formulation (CAF)09] composed of the cationic lipid dimethyldioctadecylammonium (DDA) bromide, a synthetic monomycoloyl glycerol analog and polyinosinic:polycytidylic acid, which induce strong CTL responses to peptide and protein antigens after intraperitoneal administration. By contrast, CAF09 does not stimulate CTL responses upon s.c. or i.m. injection because the vaccine forms a depot that remains at the injection site. Hence, we engineered a series of nanoemulsions (CAF24a-c) based on the active components of CAF09. The oil phase consisted of biodegradable squalane, and the surface charge was varied systematically by replacing DDA with zwitterionic distearoylphosphoethanolamine. We hypothesized that the nanoemulsions drain to the lymph nodes to a larger extent than CAF09, upon s.c. co-administration with the model antigen chicken egg ovalbumin (OVA). This results in an increased dose fraction that reaches the draining lymph nodes (dLNs) and subsequently activates cross-presenting dendritic cells (DCs), which can prime CTL responses. Indeed, the nanoemulsions induced antigen-specific CD8+ T-cell responses, which were significantly higher than those stimulated by OVA adjuvanted with CAF09. We explain this by the observed rapid localization of CAF24a in the dLNs and the subsequent association with conventional DCs, which promotes induction of CTL responses. Uptake of CAF24a was not specific for DCs, because CAF24a was also detected with B cells and macrophages. No measurable dose fraction of CAF09 was detected in the dLNs within the study period, and CAF09 formed a depot at the site of injection. Importantly, s.c. vaccination with OVA adjuvanted with CAF24a induced significant levels of specific lysis of antigen-pulsed splenocytes were induced after, which was not observed for OVA adjuvanted with CAF09. Thus, CAF24a is a promising adjuvant for induction of CTL responses upon s.c. and i.m. immunization, and it offers interesting perspectives for the design of vaccines against pathogens for which CTL responses are required to prevent infection.