Hélène-Marie Thérien

Liposome adjuvanticity: Influence of dose and protein: Lipid ratio on the humoral response to encapsulated and surface-linked antigen

The humoral response to bovine serum albumin either encapsulated in or surface-linked to liposomes was studied as a function of dose and protein:lipid ratio. Total immunoglobulin, total IgG, IgM, and the G isotypes, IgG1, IgG2a, and IgG3 were measured during the plateau phase of production after a boosting injection. Although the adjuvant character of liposomes was confirmed regardless of the mode of antigen association, important differences in the response to the two types of liposomal formulations were observed. Our results suggest that surface-linked antigen stimulates the immune system at lower doses than its encapsulated counterpart, is more sensitive to the protein:lipid ratios, and can stimulate the production of particular immunoglobulin isotypes in controlled conditions. Our data support the idea that different pathways of processing are utilized by the two forms of liposomal antigen.

Liposomal vaccine: influence of antigen association on the kinetics of the humoral response

The kinetics of the primary and secondary humoral responses to either encapsulated or surface-linked bovine serum albumin have been compared by measuring the production of specific total immunoglobulin, IgG and IgM at various times postimmunization. From our data it can be concluded that surface linkage is the best way to induce a rapid, intense and prolonged response which, in contrast to that induced by encapsulated BSA, is characterized by a low IgG/IgM ratio. The results are discussed in relation to the possible routes followed by the antigen depending on its mode of association with liposomes in the initiation of the humoral response. Our results suggest that liposomal vaccine may be designed to activate specific pathways of the immune network preferentially.

Mechanism of liposome adjuvanticity : an in vivo approach

The reputation of liposomes as adjuvant of the immune response is now firmly established despite the lack of information on the mechanisms involved in their immunopotentiating properties. The rapid targeting of massive doses of antigenic material to antigen-presenting cells, especially macrophages has, however, often been invoked as the principal source of liposomal adjuvanticity. In order to test this hypothesis, we analyzed the humoral response to antigen encapsulated in liposomes containing increasing amounts of surface-exposed mannose residues, ligand specific of an exclusive macrophagic receptor. Using BSA as a model antigen, we demonstrated that the humoral response is profoundly affected by mannosylation, being of prolonged duration and either inhibited or activated depending on the immunizing doses. These results suggest that the rapidity of antigen targeting is not the sole reason to liposome adjuvanticity and that the role of liposomes as antigenic depot is probably important to sustain substantial activation through successive restimulations. In this context, the increased rapidity in antigen targeting which favors the concentration of activation signals in time, results in an under-optimization of the response at high immunizing doses and in an optimization of this response at doses that would otherwise give rise to signal of sub-threshold intensity albeit during a longer period of time.

Importance of physical association between antigen and liposomes in liposomes adjuvanticity

The importance of a physical association between antigen and liposomes in liposomes adjuvanticity has been evaluated by comparing the anti-BSA secondary humoral response induced by different BSA formulations including free antigen, free antigen + liposomes, or encapsulated antigen. Our results demonstrate that a physical association of antigen with liposomes is required for a potentiation of the humoral response to be observed and that empty liposomes, when used for priming together with free antigen, restrain the induced response, as shown by a slower triggering, a reduced intensity and a more rapid decay rate. Free BSA has an intermediate behavior and, in contrast to what is observed with BSA/liposomes preparations, its effect appears to be dose-dependent. Each of the three BSA formulations also differs in the relative production of IgG and IgM they induced during the response.

Correlation between in vitro and in vivo behaviour of liposomal antigens

Using conalbumin as a model antigen, we demonstrate in this paper that liposomal antigen differently influences the activation of the immune system depending on the mode of association of the antigen with the liposomal vehicle whether it is by encapsulation or surface linkage. This conclusion is based on in vivo data showing that encapsulated antigen induces a short-lasting response dominated by IgG1 production while surface-linked antigen has a longer-lasting effect characterized by increased production of IgM, IgG2a, IgG3 as well as of IgG1. The in vivo data were complemented by in vitro proliferation studies carried out on spleen cells or macrophage-depleted spleen cells obtained from mice sensitized in vivo and rechallenged in vitro on day 4 following sensitization. Rechallenge was carried out in the absence or presence of anti-IL1. The data indicate that, in contrast to what is generally observed in vivo, liposomes alone potentiate spleen cell proliferative response in a dose-dependent manner. This liposomal effect totally obscures the antigen-specific proliferation that was expected with encapsulated antigen without masking that induced by surface-linked antigen. The mode of antigen association also influences anti-cytokine responsiveness as demonstrated by the insensitivity of the surface-linked antigen response to the presence of anti-IL1 and the significantly decreased response observed with encapsulated antigen under identical conditions. The response to both liposomal antigenic formulations was almost totally abolished in adherent cell-depleted cultures. The overall results therefore suggest that encapsulated and surface-linked antigens activated different immune pathways.

Immunopotentiation of the humoral response by liposomes: Effect of a T cell polyclonal activator

In this paper, we analyzed the influence of surface-linked Con A on the secondary response to liposome-associated antigen via either encapsulation or covalent linkage at the liposomal surface. The study was carried out on BALB/c mice using bovine serum albumin as antigen. The humoral response was evaluated by measurements of antibody-producing cells (total, IgM, and IgG) and serum antibody titers. The results indicate that Con A at submitogenic concentrations does not potentiate the overall effect of liposomes but drastically changes the isotype distribution pattern obtained in response to encapsulated antigen without however affecting that obtained in response to surface-linked antigen. In all situations where Con A and/or BSA was covalently linked, IgG and IgM isotypes were produced in equal quantity, while in response to encapsulated BSA, IgG was by far the dominant isotype produced as expected for a thymo-dependent antigen. These results suggest that the quality of an immune response and the mechanisms of activation may be profoundly influenced by the nature of antigen association with liposomes as well as by the presence at the liposomal surface of immunomodulators such as Con A.

Comparative effect on humoral response of four different proteins covalently linked on BSA-containing liposomes

The humoral response to encapsulated BSA appears to be a classical TD antigen response with a high ratio of IgG to IgM, whereas that to covalently-linked antigen is more complex, characterized by an enhanced synthesis of IgM, leading to an equal production of IgM and IgG. In a recent paper, we observed that surface-linked Con A on BSA-containing liposomes changed the isotype distribution to encapsulated BSA so as to mimic the response to surface-linked antigen. In the present study, we compared the immune response to BSA in BALB/c mice immunized with the antigen encapsulated into liposomes coated with one of four different proteins: Con A, Myo, MSA, or PWM. The humoral response was analyzed by measurements of antibody production (total Ig, IgM, and IgG isotypes) on serum samples obtained by cardiac puncture. It can be concluded from our results that any surface-linked protein may affect the interaction between liposome-associated antigen and immunocompetent cells.

The synthesis and characterization of new triphenylethylene platinum(II) complexes

Abstract In our search for a chemotherapeutic agent with a better therapeutic index and selectivity for the treatment of breast cancer, we have synthesized a series of cytotoxic analogs of tamoxifen. The new triphenylethylene platinum(II) derivatives were designed to possess binding affinity for the estrogen receptor. The synthesis of this type of compound is straightforward and efficient. The new complexes were fully characterized by their IR, 1H NMR and 13C NMR spectra as well as their elemental analysis. The estrogen receptor binding affinity (RBA) of some of these triphenylethylenes was also evaluated and was found to be comparable to the affinity of the reference drug, i.e. tamoxifen, RBA=1.3%.

Effect of liposomal antigens on the priming and activation of the immune system by dendritic cells

Dendritic cells (DCs) are recognized as the sole professional antigen-presenting cells capable of priming naive T cells of the helper and cytotoxic phenotypes. This property is presently exploited with success in vaccinal strategies against pathogens or tumor cells that otherwise escape immune recognition, but the repeated infusions of ex vivo expanded and sensitized DCs are usually required to achieve protection. In this paper, we demonstrate that liposomal antigens can efficiently relay and propagate the action of DCs, inducing a strong long-term response against their associated antigen. Their effect is mainly achieved by improving the ex vivo loading of DCs and by efficiently channeling the activation stimulus into the induction of effector function. This is demonstrated by the sustained immunoglobulin production as well as by the sustained lymphoproliferation and the increased cytokine secretion that can be achieved upon restimulation of DC-primed immune cells with limited amount of liposomal antigenic material. Being well-tolerated and easily prepared, liposomal antigens could therefore be expected to significantly contribute to the efficiency and to a more general utilization of the highly promising but rather cumbersome DC-based immunotherapies.

Trafficking of Surface-linked and Encapsulated Liposomal Antigens in Macrophages: an Immunocytochemical Study

Liposomal antigens are potent adjuvants of humoral and cell-mediated immunity. Although this property requires as an essential condition a physical association between the antigen and the phospholipid vehicle, the nature of the association, i.e., encapsulation or surface linkage, markedly influences the outcome of the elicited response. Available evidence suggests that macrophages are involved in this fine tuning of the immune response in a manner that is not yet clearly established. It is postulated that this might be related to their capacity to interact differently with surface-linked and encapsulated formulations. Using conalbumin as a model antigen, we address the question by analyzing the movements of encapsulated and surface-linked antigen as well as those of MHC-II molecules in macrophages in a pulse-chase immunoelectron microscopic study carried out over a 24-hr period. The antigen was followed using a polyclonal serum specifically raised against fragmented conalbumin (fCA) that allows the detection of processed antigen and of some MHC-peptide complexes. The results indicate that, in macrophages, the two liposomal formulations affect macrophage morphology in distinct ways and circulate through the various subcellular compartments with different kinetics. On the basis of the overall results, we conclude that surface-linked antigen gains access less readily to the endogenous presentation pathway than encapsulated antigen but can favor a more sustained activation of the immune system through its production of exosome-like structures and its more thorough utilization of the MHC-II pathway.