It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge. CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.

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.

The ability to mount accelerated and efficient mucosal immune responses is critically important to prevent the establishment of many infections. Secretion of immunoglobulin A (IgA) is a key component in this first line of defense, but the underlying cellular mechanisms are still not completely understood. We have evaluated different routes of immunization and examined the requirements for IgA induction in the airway mucosa. We demonstrate that subcutaneous priming with a recombinant antigen in a T helper (Th)17-inducing adjuvant followed by airway boosting promotes high and sustained levels of IgA in the lungs. This response is associated with germinal center formation in the lung-draining lymph nodes. The lung IgA response is dependent on Th17 cells and absent if interleukin (IL)-17 is depleted or when priming with vaccines inducing only Th1 or Th2 responses. We used intravascular staining to demonstrate that IgA+ B cells and chemokine receptor 6 (CCR6)+Th17 cells are recruited to the lung parenchyma after the airway booster immunization. Once recruited to the lung parenchyma, the Th17 cells transform into resident lymphocytes that persist in the lung tissue for at least 10 weeks. Here, they facilitate the accelerated recruitment of T and B cells resulting in an accelerated IgA recall response to a second airway booster immunization.

Summary: 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° 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.