cord-factors and dimethyldioctadecylammonium

Synthetic mycobacterial cord factor analogues, e.g., trehalose 6,6'-dibehenate (TDB), are highly promising adjuvants due to their strong immunopotentiating capabilities, but their biophysical properties have remained poorly characterized. Here, we report the synthesis of an array of synthetic TDB analogues varying in acyl chain length, degree of acylation, and headgroup display, which was subjected to biophysical characterization of neat nondispersed self-assembled nanostructures in excess buffer and as aqueous dispersions with cationic dimethyldioctadecylammonium (DDA) bromide. The array comprised trehalose mono- (TMX) and diester (TDX) analogues with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), myristate (Myr), and laurate (L)] and an analogue with a short hydrophilic polyethylene glycol (PEG) linker inserted between the trehalose headgroup of TDS and the acyl chains (PEG-TDS). All dispersions were liposomes, but in contrast to the colloidally stable and highly cationic TDX-containing liposomes, the zeta-potential was significantly reduced for DDA/TMX and DDA/PEG-TDS liposomes, suggesting a charge-shielding effect, which compromises the colloidal stability. An increased d-spacing was observed for the lamellar phase of neat TDB analogues in excess buffer (TDS < TMS < PEG-TDS), confirming that the charge shielding is caused by an extended molecular configuration of the more flexible headgroup. Differential scanning calorimetry showed highly cooperative phase transitions for all tested dispersions albeit the monoesters destabilized the lipid bilayers. Langmuir experiments demonstrated that incorporation of TDXs and PEG-TDS stabilized DDA monolayers due to improved hydrogen bonding and reduced intermolecular repulsions. In conclusion, data suggest that the DDA/TDS dispersions exhibit favorable physicochemical properties rendering these DDA/TDS liposomes an attractive vaccine adjuvant, and they emphasize that not only the receptor binding and immune activation but also the biophysical properties of immunopotentiator formulations should be collectively considered when designing adjuvants with optimal safety, efficacy, and storage stability.

Topics: Adjuvants, Pharmaceutic; Calorimetry, Differential Scanning; Cord Factors; Glycolipids; Liposomes; Mycobacterium; Polyethylene Glycols; Quaternary Ammonium Compounds

Incorporation of the glycolipid trehalose 6,6'-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 degrees C and 25 degrees C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-gamma cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-gamma was identified as CD4 T cells.

Topics: Acyltransferases; Adjuvants, Immunologic; Animals; Antibody Formation; Antigens, Bacterial; Bacterial Proteins; Calorimetry, Differential Scanning; Cord Factors; Cryoelectron Microscopy; Female; Glycolipids; Immunity, Cellular; Immunoglobulin G; Interferon-gamma; Light; Liposomes; Mice; Microscopy, Electron, Transmission; Mycobacterium tuberculosis; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Scattering, Radiation; Th1 Cells; Tuberculosis Vaccines

Recombinant, immunodominant antigens derived from Mycobacterium tuberculosis can be used to effectively vaccinate against subsequent infection. However, the efficacy of these recombinant proteins is dependent on the adjuvant used for their delivery. This problem affects many potential vaccines, not just those for tuberculosis, so the discovery of adjuvants that can promote the development of cell-mediated immunity is of great interest. We have previously shown that the combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and the immunomodulator modified lipid A synergistically potentiates Th1 T-cell responses. Here we report a screening program for other adjuvants with reported Th1-promoting activity and identify a second novel adjuvant formulation that drives the development of Th1 responses with an extremely high efficacy. The combination of dimethyl dioctadecyl ammonium bromide and the synthetic cord factor trehalose dibehenate promotes strong protective immune responses, without overt toxicity, against M. tuberculosis infection in a vaccination model and thus appears to be a very promising candidate for the development of human vaccines.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Female; Interferon-gamma; Kinetics; Lipid A; Mice; Mice, Inbred C57BL; Quaternary Ammonium Compounds; Surface-Active Agents; Th1 Cells; Tuberculosis; Tuberculosis Vaccines; Vaccines, Subunit

The adjuvant effect of interleukin 6 (IL-6) entrapped in liposomes was evaluated using a 65 kDa heat shock protein as a model antigen. The secondary humoral immune response either to antigen alone, or incorporated into liposomes, and the effect of IL-6 entrapped in liposomes, on this response were studied in Balb/c mice. The adjuvanticity of these formulations was compared with that of potent adjuvants such as Ribi and dimethyldioctadecylammoniumbromide (DDA). The importance of IL-6 during adjuvant activity was supported by the observation that high serum IL-6 levels were induced in Balb/c mice by all members of a panel of adjuvants tested. Following incorporation into liposomes, IL-6 retained its full biological activity, as shown by its capacity to sustain growth of the IL-6-dependent B9 cell line. At antigen dosages where Ribi and DDA gave minimal or no secondary antibody titres, incorporation of antigen into liposomes resulted in measurable secondary antibody titres. Interestingly, this adjuvant activity was significantly enhanced when liposomes containing IL-6 were co-injected with the liposomal antigen formulation. These results illustrate the potential adjuvant properties of this formulation, which seem especially useful for vaccines containing weak or non-immunogenic antigens.

Topics: Adjuvants, Immunologic; Animals; BCG Vaccine; Cell Line; Cell Wall Skeleton; Cord Factors; Enzyme-Linked Immunosorbent Assay; Female; Immunoglobulin G; Interleukin-6; Lipid A; Liposomes; Mice; Mice, Inbred BALB C; Quaternary Ammonium Compounds

The adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA) alone or in combination with trehalose dimycolate (TDM) or muramyl dipeptide (MDP) on bovine humoral and cellular responses to a soluble protein extract of gamma irradiated Brucella abortus strain 19 (SPEBA) were investigated. Thirty-five beef steers were randomly allotted to nine groups. Three of these groups received SPEBA (2 mg protein per dose) subcutaneously in combination with adjuvants, one group received the reduced dose of B. abortus strain 19 (S19), and one group received SPEBA alone. Controls included groups receiving adjuvant preparations only or no vaccine. Immune responses to the various immunizations were assessed sequentially for 56 days using various in vitro and in vivo assays. The humoral response to B. abortus was measured using standard serologic tests, an enzyme-linked immunosorbent assay, and a quantitative fluorometric immunoassay. The cell-mediated immune (CMI) response was measured by antigen-specific lymphoproliferation (LP), interleukin 2 (IL 2) production, and soluble suppressor factor release. Skin testing at day 35 for delayed-type hypersensitivity (DTH) to SPEBA was also performed. Minimal humoral responses were induced with SPEBA alone. The highest and most sustained serum antibody responses to B. abortus antigens were elicited by the S19 vaccine. A combination of SPEBA with DDA + TDM induced higher antibody levels than SPEBA with DDA or SPEBA with DDA + MDP. Responses to DTH among the groups receiving SPEBA were most notable in the SPEBA with DDA + TDM groups. Increased IL 2 production was greatest in the S19 and SPEBA with DDA + TDM vaccinates. The results indicated that a combination of DDA + TDM best potentiated immune responses to a soluble B. abortus antigen preparation and may be useful as adjuvants for future vaccines.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antigens, Bacterial; Brucella abortus; Brucella Vaccine; Cattle; Cord Factors; Hypersensitivity, Delayed; Immunity, Cellular; Injections, Subcutaneous; Interleukin-2; Lipopolysaccharides; Lymphocyte Activation; Male; Quaternary Ammonium Compounds; Random Allocation; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory

The capacity of trehalose dimycolate (TDM), muramyl dipeptide (MDP), and dimethyl dioctadecyl ammonium bromide (DDA)--alone or in combination--to potentiate the immunogenicity of killed Brucella abortus 45/20 bacteria was studied in guinea pigs. Bacterins that contained TDM in oil droplet emulsion were as effective in the prevention of brucellosis as those emulsified in Freund complete adjuvant, wereas bacterins that contained a combination of TDM and MDP were most effective. Vaccinal emulsions of bacteria and MDP were ineffective in the prevention of splenic infections. Likewise, DDA was unable to potentiate acquired resistance to Brucella. Addition of DDA to 1% oil emulsions of bacteria, TDM, and MDP eliminated protection. Adjuvants without bacteria were not able to nonspecifically protect animals from infection, although TDM was able to significantly reduce the numbers of splenic Brucella. A positive correlation (P < 0.0001) between splenic infection and splenic weight was found.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Brucella abortus; Brucella Vaccine; Cord Factors; Female; Glycolipids; Glycopeptides; Guinea Pigs; Immunity, Innate; Quaternary Ammonium Compounds; Spleen