Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype.

In many autoimmune diseases, autoantigen-specific Th17 cells play a pivotal role in disease pathogenesis. Th17 cells can transdifferentiate into other T cell subsets in inflammatory conditions, however, there have been no attempts to target Th17 cell plasticity using vaccines. We investigated if autoantigen-specific Th17 cells could be specifically targeted using a therapeutic vaccine approach, where antigen was formulated in all-trans retinoic acid (ATRA)-containing liposomes, permitting co-delivery of antigen and ATRA to the same target cell. Whilst ATRA was previously found to broadly reduce Th17 responses, we found that antigen formulated in ATRA-containing cationic liposomes only inhibited Th17 cells in an antigen-specific manner and not when combined with an irrelevant antigen. Furthermore, this approach shifted existing Th17 cells away from IL-17A expression and transcriptomic analysis of sorted Th17 lineage cells from IL-17 fate reporter mice revealed a shift of antigen-specific Th17 cells to exTh17 cells, expressing functional markers associated with T cell regulation and tolerance. In the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, vaccination with myelin-specific (MOG) antigen in ATRA-containing liposomes reduced Th17 responses and alleviated disease. This highlights the potential of therapeutic vaccination for changing the phenotype of existing Th17 cells in the context of immune mediated diseases.

Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01).

Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen-specific CD8+ T-cell and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen-specific humoral immune responses against subunit protein antigens.

Injection Vaccines Formulated with Nucleotide, Liposomal or Mineral Oil Adjuvants Induce Distinct Differences in Immunogenicity in Rainbow Trout.

Protection facilitated by the widespread use of mineral oil adjuvanted injection vaccines in salmonid fish comes with adverse effects of varying severity. In this study, we characterized the immunological profiles of two alternative vaccine formulations, both with proven efficacy and an improved safety profile in rainbow trout. Experimental injection vaccines were prepared on an identical whole-cell Aeromonas salmonicida bacterin platform and were formulated with CpG oligodeoxynucleotides, a liposomal (CAF01) or a benchmark mineral oil adjuvant, respectively. A naïve group, as well as bacterin and saline-injected groups were also included. Following administration, antigen-specific serum antibody titers, the tissue distribution of immune cell markers, and the expression of immune-relevant genes following the in vitro antigenic restimulation of anterior kidney leukocytes was investigated. Immunohistochemical staining suggested prolonged antigen presentation for the particulate formulations and increased mucosal presence of antigen-presenting cells in all immunized fish. Unlike the other immunized groups, the CAF01 group only displayed a transient elevation in specific antibody titers and immunohistochemical observations, and the transcription data suggest an increased role of cell-mediated immunity for this group. Finally, the transcription profile of the CpG formulation approached that of a TH1 profile. When compared to the benchmark formulation, CAF01 and CpG adjuvants induce slight, but distinct differences in the resulting protective immune responses. This is important, as it allows a broader immunological approach for the future development of safer vaccines.

Scale-Independent Microfluidic Production of Cationic Liposomal Adjuvants and Development of Enhanced Lymphatic Targeting Strategies.

Cationic liposomes prepared from dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6'-dibehenate (TDB) are strong liposomal adjuvants. As with many liposome formulations, within the laboratory DDAB:TDB is commonly prepared by the thin-film method, which is difficult to scale-up and gives high batch-to-batch variability. In contrast, controllable technologies such as microfluidics offer robust, continuous, and scale-independent production. Therefore, within this study, we have developed a microfluidic production method for cationic liposomal adjuvants that is scale-independent and produces liposomal adjuvants with analogous biodistribution and immunogenicity compared to those produced by the small-scale lipid hydration method. Subsequently, we further developed the DDAB:TDB adjuvant system to include a lymphatic targeting strategy using microfluidics. By exploiting a biotin-avidin complexation strategy, we were able to manipulate the pharmacokinetic profile and enhance targeting and retention of DDAB:TDB and antigen within the lymph nodes. Interestingly, redirecting these cationic liposomal adjuvants did not translate into notably improved vaccine efficacy.

Parenteral Vaccination With a Tuberculosis Subunit Vaccine in Presence of Retinoic Acid Provides Early but Transient Protection to M. Tuberculosis Infection.

Most microbes invading through mucosal surfaces cause disease and therefore strategies to induce mucosal immune responses are strongly needed. Vitamin A metabolites, such as retinoic acid (RA), play crucial roles in programming T and B cells to home to mucosal compartments, therefore we evaluated the capacity of RA to elicit mucosal immune responses against tuberculosis (TB) after parenteral vaccination. We found that mice immunized through subcutaneous injections with the TB subunit vaccine (CAF01+H56) in presence of RA show enhanced mucosal H56-specific IgA responses and enhanced Ag-specific CD4+ T lymphocytes homing to the lung as compared with control mice. Immunization with CAF01+H56 in presence of RA resulted in lower bacterial loads in the lungs of mice 14 days after challenge with virulent Mycobacterium tuberculosis (Mtb) as compared to mice immunized in the absence of RA or vaccinated with BCG. Higher amounts of IFNγ and IL-17 pro-inflammatory cytokines were found in lung homogenates of mice immunized with CAF01+H56 and RA 24 h after Mtb infection. However, 6 weeks after infection the protection was comparable in vaccinated mice with or without RA even though treatment with RA during immunization is able to better contain the inflammatory response by the host. Furthermore, at later stage of the infection a higher percentage of Mtb specific CD4+PD1+ T lymphocytes were found in the lungs of mice immunized with CAF01+H56 and RA. These data show that an enhanced mucosal immune response is generated during parenteral vaccination in presence of RA. Furthermore, RA treatment contained the bacterial growth at an early stage of the infection and limited the inflammatory response in the lung at later time points.

A Liposome-Based Adjuvant Containing Two Delivery Systems with the Ability to Induce Mucosal Immunoglobulin A Following a Parenteral Immunization.

Worldwide, enteric infections rank third among all causes of disease burdens, and vaccines able to induce a strong and long-lasting intestinal immune responses are needed. Parenteral immunization generally do not generate intestinal IgA. Recently, however, injections of retinoic acid (RA) dissolved in oil, administered multiple times before vaccination to precondition the vaccine-draining lymph nodes, enabled a parenteral vaccine strategy to induce intestinal IgA. As multiple injections of RA before vaccination is not an attractive strategy for clinical practice, we aimed to develop a "one injection" vaccine formulation that upon parenteral administration induced intestinal IgA. Our vaccine formulation contained two liposomal delivery systems. One delivery system, based on 1,2-distearoyl- sn-glycero-3-phosphocholine stabilized with PEG, was designed to exhibit fast drainage of RA to local lymph nodes to precondition these for a mucosal immune response before being subjected to the vaccine antigen. The other delivery system, based on the cationic liposomal adjuvant CAF01 stabilized with cholesterol, was optimized for prolonged delivery of the antigen by migratory antigen-presenting cells to the preconditioned lymph node. Combined we call the adjuvant CAF23. We show that CAF23, administered by the subcutaneous route induces an antigen specific intestinal IgA response, making it a promising candidate adjuvant for vaccines against enteric diseases.

Alternatives to mineral oil adjuvants in vaccines against Aeromonas salmonicida subsp. salmonicida in rainbow trout offer reductions in adverse effects.

In an effort to reduce the frequency and severity of adverse reactions seen from the use of mineral oil adjuvants in salmonid fish, the effects of two alternative adjuvants were assessed, focusing on the induction of adverse effects as well as protection. Using rainbow trout (Oncorhynchus mykiss) as recipients, injection vaccines based on formalin-inactivated Aeromonas salmonicida subspecies salmonicida were formulated with CpG oligodeoxynucleotides, the liposomal cationic adjuvant formulation 01 (CAF01) or with Freund's incomplete adjuvant and administered intraperitoneally. Control groups of unvaccinated, Tris-buffered saline-injected or bacterin-injected individuals were included, and each group included in the study held a total number of 240 individuals. Subsequently, individuals from each group were examined for differences in Fulton's condition factor, macro- and microscopic pathological changes, as well as protection against experimental infection with A. salmonicida. While adverse effects were not eliminated, reductions in microscopic and macroscopic adverse effects, in particular, were seen for both the nucleotide- and liposome-based vaccine formulations. Furthermore, the induced protection appears similar to that of the benchmark formulation, thus introducing viable, potential alternative types of adjuvants for use in future fish vaccines.

Increased humoral immunity by DNA vaccination using an α-tocopherol-based adjuvant.

DNA vaccines induce broad immunity, which involves both humoral and strong cellular immunity, and can be rapidly designed for novel or evolving pathogens such as influenza. However, the humoral immunogenicity in humans and higher animals has been suboptimal compared with that of traditional vaccine approaches. We tested whether the emulsion-based and α-tocopherol containing adjuvant Diluvac Forte® has the ability to enhance the immunogenicity of a naked DNA vaccine (i.e., plasmid DNA). As a model vaccine, we used plasmids encoding both a surface-exposed viral glycoprotein (hemagglutinin) and an internal non-glycosylated nucleoprotein in the Th1/Th2 balanced CB6F1 mouse model. The naked DNA (50 µg) was premixed at a 1:1 volume/volume ratio with Diluvac Forte®, an emulsion containing different concentrations of α-tocopherol, the emulsion alone or endotoxin-free phosphate-buffered saline (PBS). The animals received 2 intracutaneous immunizations spaced 3 weeks apart. When combined with Diluvac Forte® or the emulsion containing α-tocopherol, the DNA vaccine induced a more potent and balanced immunoglobulin G (IgG)1 and IgG2c response, and both IgG subclass responses were significantly enhanced by the adjuvant. The DNA vaccine also induced CD4+ and CD8+ vaccine-specific T cells; however, the adjuvant did not exert a significant impact. We concluded that the emulsion-based adjuvant Diluvac Forte® enhanced the immunogenicity of a naked DNA vaccine encoding influenza proteins and that the adjuvant constituent α-tocopherol plays an important role in this immunogenicity. This induction of a potent and balanced humoral response without impairment of cellular immunity constitutes an important advancement toward effective DNA vaccines.

Correlating liposomal adjuvant characteristics to in-vivo cell-mediated immunity using a novel Mycobacterium tuberculosis fusion protein: a multivariate analysis study.

OBJECTIVE: In this study, we have used a chemometrics-based method to correlate key liposomal adjuvant attributes with in-vivo immune responses based on multivariate analysis. METHODS: The liposomal adjuvant composed of the cationic lipid dimethyldioctadecylammonium bromide (DDA) and trehalose 6,6-dibehenate (TDB) was modified with 1,2-distearoyl-sn-glycero-3-phosphocholine at a range of mol% ratios, and the main liposomal characteristics (liposome size and zeta potential) was measured along with their immunological performance as an adjuvant for the novel, postexposure fusion tuberculosis vaccine, Ag85B-ESAT-6-Rv2660c (H56 vaccine). Partial least square regression analysis was applied to correlate and cluster liposomal adjuvants particle characteristics with in-vivo derived immunological performances (IgG, IgG1, IgG2b, spleen proliferation, IL-2, IL-5, IL-6, IL-10, IFN-γ). KEY FINDINGS: While a range of factors varied in the formulations, decreasing the 1,2-distearoyl-sn-glycero-3-phosphocholine content (and subsequent zeta potential) together built the strongest variables in the model. Enhanced DDA and TDB content (and subsequent zeta potential) stimulated a response skewed towards a cell mediated immunity, with the model identifying correlations with IFN-γ, IL-2 and IL-6. CONCLUSION: This study demonstrates the application of chemometrics-based correlations and clustering, which can inform liposomal adjuvant design.

Influence of trehalose 6,6'-diester (TDX) chain length on the physicochemical and immunopotentiating properties of DDA/TDX liposomes.

Linking physicochemical characterization to functional properties is crucial for defining critical quality attributes during development of subunit vaccines toward optimal safety and efficacy profiles. We investigated how the trehalose 6,6'-diester (TDX) chain length influenced the physicochemical and immunopotentiating properties of the clinically tested liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and analogues of trehalose-6,6'-dibehenate (TDB). TDB analogues with symmetrically shortened acyl chains [denoted X: arachidate (A), stearate (S), palmitate (P), myristate (Myr) and laurate (L)] were incorporated into DDA liposomes and characterized with respect to size, polydispersity index, charge, thermotropic phase behavior and lipid-lipid interactions. Incorporation of 11 mol% TDX into DDA liposomes significantly decreased the polydispersity index when TDA, TDS, TDP and TDMyr were incorporated, whereas both the initial size and the charge of the liposomes were unaffected. The long-term colloidal stability was only decreased when including TDL in DDA liposomes. The fatty acid length of TDX affected the phase transition of the liposomes, and for the DDA/TDP and DDA/TDS liposomes a homogeneous distribution of the lipids in the bilayer was indicated. The membrane packing was studied further by using the Langmuir monolayer technique. Incorporation of TDS improved the packing of the lipid monolayer, as compared to the other analogues, suggesting the most favorable stability. Finally, immunization of mice with the recombinant tuberculosis fusion antigen Ag85B-ESAT-6-Rv2660c (H56) and the physicochemically most optimal formulations (DDA/TDB, DDA/TDS and DDA/TDP) induced comparable T-cell responses. In conclusion, of the investigated TDB analogues, incorporation of 11 mol% TDS or TDP into DDA liposomes resulted in an adjuvant system with the most favorable physicochemical properties and an immunological profile comparable to that of DDA/TDB.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants.

OBJECTIVES: Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6'-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn-glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B-ESAT-6-Rv2660c, a multistage tuberculosis vaccine. METHODS: Liposomal were prepared at a 5 : 1 DDA-TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. KEY FINDINGS: As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. CONCLUSION: These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system.