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.

Prevalence of Mycobacterium tuberculosis infection as measured by the QuantiFERON-TB Gold assay and ESAT-6 free IGRA among adolescents in Mwanza, Tanzania.

BACKGROUND: The prevalence of latent tuberculosis infection (LTBI) is vastly higher than that of tuberculosis (TB) disease and this enormous reservoir of individuals with LTBI impacts the global TB control strategy. Adolescents are at greatest risk of TB infection and are thus an ideal target population for a potential effective TB vaccine to be added to the current BCG programme as it could reduce the number of latent infections and consequently the number of adults with TB disease. However, LTBI rates are often unknown for this population. This study aims to estimate the magnitude of LTBI and to determine if Tanzanian adolescents would be a good population for a prevention of TB infection trial. METHODS: This was a descriptive cross-sectional study that recruited 193 adolescents aged 12 and 16 years from government schools and directly from the community in Mwanza Region, Tanzania. Socio-demographic characteristics were collected for all enrolled participants. Blood was drawn and tested using QuantiFERON-TB Gold In-Tube (QFT-GIT), and Early Secretory Antigenic Target-6-Free Interferon-gamma Release Assay (ESAT-6 free IGRA). Concordance between QFT-GIT and ESAT-6 free IGRA was evaluated using the McNemar's test. RESULTS: Overall estimates of LTBI prevalence were 19.2% [95%CI, 14.1; 25.2] and 18.6% [95%CI, 13.6; 24.6] as measured by QFT-GIT IGRA and ESAT-6 free IGRA, respectively. The 16-year-old cohort had a higher LTBI prevalence (23.7% [95%CI, 16.1; 32.9]) as compared to 12-year-old cohort (14.6% [95%CI, 8.6; 22.7]) as measured by QFT-GIT IGRA. When measured by ESAT-6 Free IGRA, LTBI prevalence was 24.7% (95%CI, 16.9; 34.0) for the 16-year-old cohort and 12.5% (95%CI, 7.0; 20.3) among the 12-year-old cohort. According to both tests the prevalence of TB infection and the corresponding annual risk of tuberculosis infection (ARTI) and force of infection were high and increased with age. Of all enrolled participants, 97.4% had concordant results for QFT-GIT IGRA and ESAT-6 free IGRA (p = 0.65). CONCLUSIONS: The prevalence of LTBI and the associated ARTI and force of infection among adolescents is high and increases with age in Mwanza Region. There was a high concordance between the QFT-GIT and the novel ESAT-6 free IGRA assays. These findings suggest Mwanza is a promising area to conduct novel TB vaccine research prevention of infection (POI) studies targeting adolescents.

T-helper 1 and T-helper 2 adjuvants induce distinct differences in the magnitude, quality and kinetics of the early inflammatory response at the site of injection.

Vaccine adjuvants activate the innate immune system and thus influence subsequent adaptive T-cell responses. However, little is known about the initial immune mechanisms preceding the adjuvant-induced differentiation of T-helper (Th) cells. The effect of a T-helper 1 (Th1) adjuvant, dimethyldioctadecylammonium liposomes with monophosphoryl lipid-A (DDA/MPL), and a T-helper 2 adjuvant, aluminium hydroxide [Al(OH)(3)], on early, innate chemotactic signals and inflammatory cell influx at the site of injection was therefore investigated. Injection of the adjuvants into the peritoneal cavity of mice demonstrated distinct differences in the magnitude, quality and kinetics of the response. The inflammatory response to DDA/MPL was prominent, inducing high local levels of pro-inflammatory cytokines, chemokines and a pronounced inflammatory exudate consisting of neutrophils, monocytes/macrophages and activated natural killer cells. This was in contrast to the response induced by Al(OH)(3), which, although sharing some of the early chemokine signals, was more moderate and consisted almost exclusively of neutrophils and eosinophils. Notably, Al(OH)(3) specifically induced the release of a significant amount of interleukin (IL)-5, whereas DDA/MPL induced high amounts of tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-6. Finally, a microarray analysis confirmed that the effect of DDA/MPL was broader with more than five times as many genes being specifically up-regulated after injection of DDA/MPL compared with Al(OH)(3). Thus, the adjuvants induced qualitatively distinct local inflammatory signals early after injection.

Comparison of two different PEGylation strategies for the liposomal adjuvant CAF09: Towards induction of CTL responses upon subcutaneous vaccine administration.

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG1000 moieties, resulting in mono- and bis-PEG-peptides of different lengths (10, 15 and 20 amino acid residues). For comparison, CAF09 was also grafted by inclusion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG2000) in the bilayer structure during preparation. Grafting of CAF09 with either type of PEG resulted in charge shielding, evident from a reduced surface charge. Upon s.c. immunization of mice with the model antigen ovalbumin (OVA) adjuvanted with PEGylated CAF09, stronger CTL responses were induced as compared to immunization of mice with unadjuvanted OVA. Biodistribution studies confirmed that grafting of CAF09 with DSPE-PEG2000 improved the passive drainage of the vaccine to LNs, because a higher dose fraction was recovered in DCs present in the draining LNs, as compared to the dose fraction detected for non-PEGylated CAF09. In conclusion, PEGylation of CAF09 may be a useful strategy for the design of an adjuvant, which induces CTL responses after s.c. and i.m. administration. In the present studies, CAF09 grafted with 10 mol% DSPE-PEG2000 is the most promising of the tested adjuvants, but additional studies are required to further elucidate the potential of the strategy.

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.

Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators.

The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR expression profile of the target APCs. Here, we review state-of-the-art formulation approaches employed for the inclusion of immunostimulators and subunit antigens into liposome dispersion and their optimization towards robust vaccine formulations.

Immunogenicity of HLA Class I and II Double Restricted Influenza A-Derived Peptides.

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1*01:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.

The administration route is decisive for the ability of the vaccine adjuvant CAF09 to induce antigen-specific CD8(+) T-cell responses: The immunological consequences of the biodistribution profile.

A prerequisite for vaccine-mediated induction of CD8(+) T-cell responses is the targeting of dendritic cell (DC) subsets specifically capable of cross-presenting antigen epitopes to CD8(+) T cells. Administration of a number of cationic adjuvants via the intraperitoneal (i.p.) route has been shown to result in strong CD8(+) T-cell responses, whereas immunization via e.g. the intramuscular (i.m.) or subcutaneous (s.c.) routes often stimulate weak CD8(+) T-cell responses. The hypothesis for this is that self-drainage of the adjuvant/antigen to the lymphoid organs, which takes place upon i.p. immunization, is required for the subsequent activation of cross-presenting lymphoid organ-resident CD8α(+) DCs. In contrast, s.c. or i.m. immunization usually results in the formation of a depot at the site of injection (SOI), which hinders the self-drainage and targeting of the vaccine to cross-presenting CD8α(+) DCs. We investigated this hypothesis by correlating the biodistribution pattern and the adjuvanticity of the strong CD8(+) T-cell inducing liposomal cationic adjuvant formulation 09 (CAF09), which is composed of dimethyldioctadecylammonium bromide/monomycoloyl glycerol liposomes with polyinosinic:polycytidylic acid electrostatically adsorbed to the surface. Biodistribution studies with radiolabeled CAF09 and a surface-adsorbed model antigen [ovalbumin (OVA)] showed that a significantly larger fraction of the vaccine dose localized in the draining lymph nodes (dLNs) and the spleen 6h after i.p. immunization, as compared to after i.m. immunization. Studies with fluorescently labelled OVA+CAF09 demonstrated a preferential association of OVA+CAF09 to DCs/monocytes, as compared to macrophages and B cells, following i.p. immunization. Administration of OVA+CAF09 via the i.p. route did also result in DC activation, whereas no DC activation could be measured within the same period with unadjuvanted OVA and OVA+CAF09 administered via the s.c. or i.m. routes. In the dLNs, the highest level of activated, cross-presenting CD8α(+) DCs was detected at 24h post immunization, whereas an influx of activated, migrating and cross-presenting CD103(+) DCs to the dLNs could be measured after 48h. This suggests that the CD8α(+) DCs are activated by self-draining OVA+CAF09 in the lymphoid organs, whereas the CD103(+) DCs are stimulated by the OVA+CAF09 at the SOI. These results support the hypothesis that the self-drainage of OVA+CAF09 to the draining LNs is required for the activation of CD8α(+) DCs, while the migratory CD103(+) DCs may play a role in sustaining the subsequent induction of strong CD8(+) T-cell responses.

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.

A stable nanoparticulate DDA/MMG formulation acts synergistically with CpG ODN 1826 to enhance the CD4⁺ T-cell response.

AIM: To combine the dimethyldioctadecyl ammonium/monomycoloyl glycerol (DDA/MMG) liposomal vaccine adjuvant with the Toll-like receptor (TLR) ligands poly(I:C) (TLR3), flagellin (TLR5) or CpG oligodeoxynucleotide 1826 (TLR9) and investigate their physicochemical properties as well as their CD4(+) T-cell-inducing capacity. MATERIALS & METHODS: Formulations were investigated by dynamic light scattering and differential scanning calorimetry. Their CD4(+) T-cell induction with a tuberculosis antigen was analyzed by multiplex cytokine analysis, ELISA and intracellular cytokine staining. RESULTS: DDA/MMG/CpG was the best combination for obtaining increased CD4(+) T-cell responses. However, coformulating CpG and DDA/MMG liposomes led to instability and the formulation was therefore optimized systematically using a design of experiment. CONCLUSION: The nanoparticulate DDA/MMG/CpG adjuvant can be stabilized and synergistically enhances CD4(+) T-cell responses compared with DDA/MMG liposomes.

Induction of CD8+ T-cell responses against subunit antigens by the novel cationic liposomal CAF09 adjuvant.

Vaccines inducing cytotoxic T-cell responses are required to achieve protection against cancers and intracellular infections such as HIV and Hepatitis C virus. Induction of CD8+ T cell responses in animal models can be achieved by the use of viral vectors or DNA vaccines but so far without much clinical success. Here we describe the novel CD8+ T-cell inducing adjuvant, cationic adjuvant formulation (CAF) 09, consisting of dimethyldioctadecylammonium (DDA)-liposomes stabilized with monomycoloyl glycerol (MMG)-1 and combined with the TLR3 ligand, Poly(I:C). Different antigens from tuberculosis (TB10.3, H56), HIV (Gag p24), HPV (E7) and the model antigen ovalbumin were formulated with CAF09 and administering these vaccines to mice resulted in a high frequency of antigen-specific CD8+ T cells. CAF09 was superior in its ability to induce antigen-specific CD8+ T cells as compared to other previously described CTL-inducing adjuvants, CAF05 (DDA/trehalose dibehenate (TDB)/Poly(I:C)), Aluminium/monophosphoryl lipid-A (MPL) and Montanide/CpG/IL-2. The optimal effect was obtained when the CAF09-adjuvanted vaccine was administered by the i.p. route, whereas s.c. administration primed limited CD8+ T-cell responses. The CD4+ T cells induced by CAF09 were mainly of an effector-memory-like phenotype and the CD8+ T cells were highly cytotoxic. Finally, in a mouse therapeutic skin tumor model, the HPV-16 E7 antigen formulated in CAF09 significantly reduced the growth of already established subcutaneous E7-expressing TC-1 tumors in 38% of the mice and in a corresponding prophylactic model 100% of the mice were protected. Thus, CAF09 is a potent new adjuvant which is able to induce CD8+ T-cell responses against several antigens and to enhance the protective efficacy of an E7 vaccine both in a therapeutic and in a prophylactic tumor model.

Fast vaccine design and development based on correlates of protection (COPs).

New and reemerging infectious diseases call for innovative and efficient control strategies of which fast vaccine design and development represent an important element. In emergency situations, when time is limited, identification and use of correlates of protection (COPs) may play a key role as a strategic tool for accelerated vaccine design, testing, and licensure. We propose that general rules for COP-based vaccine design can be extracted from the existing knowledge of protective immune responses against a large spectrum of relevant viral and bacterial pathogens. Herein, we focus on the applicability of this approach by reviewing the established and up-coming COPs for influenza in the context of traditional and a wide array of new vaccine concepts. The lessons learnt from this field may be applied more generally to COP-based accelerated vaccine design for emerging infections.

Cationic liposomal vaccine adjuvants in animal challenge models: overview and current clinical status.

Cationic liposome formulations can function as efficient vaccine adjuvants. However, due to the highly diverse nature of lipids, cationic liposomes have different physical-chemical characteristics that influence their adjuvant mechanisms and their relevance for use in different vaccines. These characteristics can be further manipulated by incorporation of additional lipids or stabilizers, and inclusion of carefully selected immunostimulators is a feasible strategy when tailoring cationic liposomal adjuvants for specific disease targets. Thus, cationic liposomes present a plasticity, which makes them promising adjuvants for future vaccines. This versatility has also led to a vast amount of literature on different experimental liposomal formulations in combination with a wide range of immunostimulators. Here, we have compiled information about the animal challenge models and administration routes that have been used to study vaccine adjuvants based on cationic liposomes and provide an overview of the applicability, progress and clinical status of cationic liposomal vaccine adjuvants.

One does not fit all: new adjuvants are needed and vaccine formulation is critical.

In recent years, adjuvant research has moved from empirical trial and error to a more rational approach engaging the multidisciplinary fields of immunology, biochemistry, pharmacy and physical chemistry. At the Modern Vaccines/Adjuvants Formulation meeting held in Cannes, France in October 2010, scientists from this broad field met to discuss recent progress in adjuvant research and development. The focus of the meeting was on formulation and as a result clinically and pharmaceutically relevant aspects of how to design and optimize vaccines and adjuvants were also addressed.

Liposomal vaccine delivery systems.

INTRODUCTION: Liposomes remain at the forefront of drug and vaccine design owing to their well-documented abilities to act as delivery vehicles. Nevertheless, the concept of liposomes as delivery vehicles is not a new one, with most works focusing on their use for the delivery of genes and drugs. However, in the last 10 years a significant amount of research has focused on using liposomes as vaccine adjuvants, not only as an antigen delivery vehicle but also as a tool to increase the immunogenicity of peptide and protein antigens. AREAS COVERED: This paper reviews liposomal adjuvants now in vaccine development, with particular emphasis on their adjuvant mechanism and how specific physicochemical characteristics of liposomes affect the immune response. The inclusion of immunomodulators is also discussed, with prominence given to Toll-like receptor ligands. EXPERT OPINION: The use of liposomes as vaccine delivery systems is evolving rapidly owing to the combined increase in technological advances and understanding of the immune system. Liposomes that contain and deliver immunostimulators and antigens are now being developed to target diseases that require stimulation of both humoral and cell-mediated immune responses. The CAF liposomal system, described in detail in this review, is one liposomal model that shows such flexibility.

Cationic liposomes as vaccine adjuvants.

The application of cationic liposomes as vaccine delivery systems and adjuvants has been investigated extensively over the last few decades. However, cationic liposomes are, in general, not sufficiently immunostimulatory, which is why the combination of liposomes with immunostimulating ligands has arisen as a strategy in the development of novel adjuvant systems. Within the last 5 years, two novel adjuvant systems based on cationic liposomes incorporating Toll-like receptor or non-Toll-like receptor immunostimulating ligands have progressed from preclinical testing in smaller animal species to clinical testing in humans. The immune responses that these clinical candidates induce are primarily of the Th1 type for which there is a profound unmet need. Furthermore, a number of new cationic liposome-forming surfactants with notable immunostimulatory properties have been discovered. In this article we review the recent progress on the application of cationic liposomes as vaccine delivery systems/adjuvants.

Incorporation of a synthetic mycobacterial monomycoloyl glycerol analogue stabilizes dimethyldioctadecylammonium liposomes and potentiates their adjuvant effect in vivo.

The combination of delivery systems such as cationic liposomes and immunopotentiating molecules is a promising approach for the rational design of vaccine adjuvants. In this study, a synthetic analogue of the mycobacterial lipid monomycoloyl glycerol (MMG), referred to as MMG-1, was synthesized and combined with the cationic surfactant dimethyldioctadecylammonium (DDA). The purpose of the study was to provide a thorough pharmaceutical characterization of the resulting DDA/MMG-1 binary system and to evaluate how incorporation of MMG-1 affected the adjuvant activity of DDA liposomes. Thermal analyses demonstrated that MMG-1 was incorporated into the DDA lipid bilayers, and cryo-transmission electron microscopy (TEM) confirmed that liposomes were formed. The particles had a polydisperse size distribution and an average diameter of approximately 400 nm. Evaluation of the colloidal stability indicated that at least 18 mol% MMG-1 was required to stabilize the DDA liposomes as the average particle size remained constant during storage for 6 months. The improved colloidal stability is most likely caused by increased hydration of the lipid bilayer. This was demonstrated by studying Langmuir-Blodgett monolayers of DDA and MMG-1 which revealed an increased surface pressure in the presence of high concentrations of MMG-1 when the DDA/MMG-1 monolayers were fully compressed, indicating an increased interaction with water due to enhanced hydration of the lipid head groups. Finally, immunization of mice with the tuberculosis fusion antigen Ag85B-ESAT-6 and DDA/MMG-1 liposomes induced a strong cell-mediated immune response characterized by a mixed Th1/Th17 profile and secretion of IgG1 and IgG2c antibodies. The Th1/Th17-biased immunostimulatory effect was increased in an MMG-1 concentration-dependent manner with maximal observed effect at 31 mol% MMG-1. Thus, incorporation of 31 mol% MMG-1 into DDA liposomes results in an adjuvant system with favorable physical as well as immunological properties.

Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C(60) fullerenes in the FE1-Mutatrade markMouse lung epithelial cells.

Viability, cell cycle effects, genotoxicity, reactive oxygen species production, and mutagenicity of C(60) fullerenes (C(60)) and single-walled carbon nanotubes (SWCNT) were assessed in the FE1-Mutatrade markMouse lung epithelial cell line. None of these particles induced cell death within 24 hr at doses between 0 and 200 microg/ml or during long-term subculture exposure (576 hr) at 100 microg/ml, as determined by two different assays. However, cell proliferation was slower with SWCNT exposure and a larger fraction of the cells were in the G1 phase. Exposure to carbon black resulted in the greatest reactive oxygen species generation followed by SWCNT and C(60) in both cellular and cell-free particle suspensions. C(60) and SWCNT did not increase the level of strand breaks, but significantly increased the level of FPG sensitive sites/oxidized purines (22 and 56%, respectively) determined by the comet assay. The mutant frequency in the cII gene was unaffected by 576 hr of exposure to either 100 microg/ml C(60) or SWCNT when compared with control incubations, whereas we have previously reported that carbon black and diesel exhaust particles induce mutations using an identical exposure scenario. These results indicate that SWCNT and C(60) are less genotoxic in vitro than carbon black and diesel exhaust particles.

The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes.

Cationic liposomes are being used increasingly as efficient adjuvants for subunit vaccines but their precise mechanism of action is still unknown. Here, we investigated the adjuvant mechanism of cationic liposomes based on the synthetic amphiphile dimethyldioctadecylammonium (DDA). The liposomes did not have an effect on the maturation of murine bone-marrow-derived dendritic cells (BM-DCs) related to the surface expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86. We found that ovalbumin (OVA) readily associated with the liposomes (> 90%) when mixed in equal concentrations. This efficient adsorption onto the liposomes led to an enhanced uptake of OVA by BM-DCs as assessed by flow cytometry and confocal fluorescence laser-scanning microscopy. This was an active process, which was arrested at 4 degrees and by an inhibitor of actin-dependent endocytosis, cytochalasin D. In vivo studies confirmed the observed effect because adsorption of OVA onto DDA liposomes enhanced the uptake of the antigen by peritoneal exudate cells after intraperitoneal injection. The liposomes targeted antigen preferentially to antigen-presenting cells because we only observed a minimal uptake by T cells in mixed splenocyte cultures. The adsorption of antigen onto the liposomes increased the efficiency of antigen presentation more than 100 times in a responder assay with MHC class II-restricted OVA-specific T-cell receptor transgenic DO11.10 T cells. Our data therefore suggest that the primary adjuvant mechanism of cationic DDA liposomes is to target the cell membrane of antigen-presenting cells, which subsequently leads to enhanced uptake and presentation of antigen.

Protective immunity to tuberculosis with Ag85B-ESAT-6 in a synthetic cationic adjuvant system IC31.

In this study, we evaluated the potential of a novel synthetic adjuvant designated IC31 for the ability to augment the immune response and protective efficacy of the well-known mycobacterial vaccine antigen, Ag85B-ESAT-6. The IC31 adjuvant, consisting of a vehicle based on the cationic peptide KLKL(5)KLK and the immunostimulatory oligodeoxynucleotide ODN1a signalling through the TLR9 receptor, was found to promote highly efficient Th1 responses. The combination of Ag85B-ESAT-6 and IC31 exhibited significant levels of protection in the mouse aerosol challenge model of tuberculosis and a detailed analysis of the immune response generated revealed the induction of CD4 T cells giving rise to high levels of IFN-gamma secretion. Furthermore, the combination of Ag85B-ESAT-6/IC31 was found to confer efficient protection in the guinea pig aerosol model of tuberculosis infection and is at present moving towards clinical testing.