Quillaja saponin formulations that stimulate proinflammatory cytokines elicit a potent acquired cell-mediated immunity.

We examined the ability of various Quillaja saponins in iscom-matrix formulations to induce proinflammatory cytokines, such as interleukin (IL)-1alpha and IL-6, and to stimulate acquired immune responses to influenza virus envelope proteins. The A-fraction of Quillaja saponins (QH-A) was shown to stimulate antigen-presenting cells (APC) to produce proinflammatory cytokines, and elicited a high primary antigen-specific antibody response and potent cell-mediated responses, as measured by T-cell proliferation, production of cytokines and cytotoxic T-lymphocyte (CTL) activity. The C-fraction of Quillaja saponins (QH-C) was shown to have a low capacity to stimulate proinflammatory cytokines and elicited low primary antibody and T-cell responses. However, the QH-C iscom-matrix mediated a potent booster effect, resulting in a high secondary antibody response. The ability of APC to discriminate and to respond to QH-A formulations more efficiently than to QH-C with release of proinflammatory cytokines, which precedes a potent acquired immune response, identifies an important mechanism through which some adjuvants may exert their immunoenhancing activities.

An immunoaffinity-purified Trypanosoma cruzi antigen suppresses cellular proliferation through a TGF-beta-mediated mechanism.

Two subfractions with opposite immunological properties were obtained from the flagellar antigens (FF) of Trypanosoma cruzi epimastigotes by immunoaffinity chromatography. The ligand-bound material (Ag 123) contained four polypeptide bands of 97, 55, 38 and 14 kDa. The nonretained flow-through (FT), induced a potent proliferation of murine naive splenocytes. In contrast, Ag 123 inhibited the proliferative capacity of the FT as well as the proliferation mediated by the mitogen Concanavalin A (Con A). The suppressive effect of Ag 123 on the Con A-mediated proliferation was neutralized by an anti-TGF-beta monoclonal antibody. Both Ag 123 and FF stimulated high serum levels of TGF-beta in injected mice. Ag 123 also induced in vitro secretion of TGF-beta by murine splenocytes. These results demonstrate that Ag 123 is a potent stimulator of TGF-beta both in vivo and in vitro. Oligopeptides derived from the 38 kDa protein present in Ag 123 showed homology with human and rat alpha-fetoproteins (AFP). Ag 123 seems to have a key role in the immunosuppression that develops during early stages in the infection with T. cruzi.

Iscom, a delivery system for parenteral and mucosal vaccination.

The iscom is a supramolecular spherical structure, about 40nm in diameter, built up by structure-forming and immunomodulating quillaja triterpenoids, lipids and antigens. Iscoms with a defined quillaja triterpenoid formulation named QH 703 are in human trials. The advantages of using the particulate iscom form of quillaja components are (i) that local reactions at the site of injection can be avoided; a manifold higher dose of quillaja components in iscoms than in free form can be injected without causing side effects; (ii) considerably lower doses of both quillaja components and antigens are required to obtain a certain level of immune response. The iscom particle targets the antigen and adjuvant components to both the endosomal and cytosolic pathways for antigen presentation, resulting in both MHC class I and class II restricted immune responses. Further, iscoms induce APC to produce IL-1, IL-6 and IL-12 and a TH1 type of response with enhanced IL-2 and IFN-gamma production. Iscoms are now constructed to target the mucosal lymphatic systems. Iscoms administered intranasally induce secretory IgA responses in lungs and distant mucosal membranes e.g. in the genital tract.

In vivo and in vitro induction of IL-6 by Quillaja saponaria molina triterpenoid formulations.

Quillaja saponaria Molina and some of the defined Quillaja components are potent adjuvants. An important function of adjuvants is the activation of antigen-presenting cells (APC), a prerequisite for the development of immune responses. Interleukin 6 (IL-6) has been identified as a key factor in the generation of cytolytic T lymphocytes, which constitute an important effector mechanism elicited by immuno-stimulatory complex (iscom)-borne antigens. To identify factors relevant to the unique property of iscoms to mediate CTL responses, we analysed the capacity of different defined Quillaja triterpenoid components in various formulations to stimulate production of IL-6 by APC in vitro and in vivo. The iscom formed with Quillaja adjuvant and incorporated influenza virus envelope proteins elicited the highest secretion of IL-6. The production of IL-6 was also stimulated by the antigen free matrix of the iscom and even by the Quillaja triterpenoids as free components albeit to a significantly lesser extent. Among the various combinations of QH-A and QH-C tested and also the original semipurified spikoside, the QH 7.0.3 matrix was the most efficient formulation for activation of IL-6 production by APC. In general, an increasing proportion of QH-A vs QH-C increases the capacity to activate APC. The results demonstrate that the incorporated antigen and the adjuvant component in the same particle have the synergistic effects on immunogenicity.

Intranasal immunization of mice with Echinococcus granulosus surface antigens iscoms evokes a strong immune response, biased towards glucidic epitopes.

The present work describes the preparation and characterization of iscoms from tegumental antigens of Echinococcus granulosus protoscoleces, and their use as immunogens in mice by intranasal and subcutaneous routes. Iscoms given at 10 mg doses evoke a significant antibody response when administered i.n. and a strong and long lasting antibody response by s.c. route. The Ag administered i.n. in a non-adjuvanted form as a booster was not immunogenic. The i.n. route of immunization induced higher IgA serum titre in relation to IgG than the s.c. route and antisera of slightly higher avidity. Also, ten fold lower IgG2a/IgG1 and ten fold higher IgG3/IgG1 ratios were observed for the i.n. protocol compared to the s.c. one. Moreover, the mucosal route of immunization induced more efficiently antibodies of both isotypes directed to carbohydrate epitopes. The differences between immune response generated by i.n. and parenteral immunizations should be taken into consideration, particularly in those cases in which protective antigens are glycosylated.

Influence of Quillaja saponaria triterpenoid content on the immunomodulatory capacity of Epstein-Barr virus iscoms.

The immune responses to immunostimulating complexes (iscoms) containing recombinant Epstein-Barr virus (EBV) gp340 envelope protein was evaluated in BALB/c (H-2(d)) and CBA (H-2(k)) mice. Gp340-iscoms were used either with a low content of Quillaja triterpenoid adjuvant (L-iscoms) or supplemented with additional Quillaja adjuvant in the form of iscomatrix (S-iscoms). Class and subclass distribution of anti-gp340 antibodies, EBV-neutralizing antibodies, antigen-specific T cell proliferation and cytokine production were determined and these results compared to those obtained by immunization with non-adjuvated gp340. The H-2(d) and H-2(k) mice were characterized as low or high responders in respect to the level of specific anti-gp340 antibodies, secretion of IgG2a isotype, antigen-specific lymphoproliferative capacity, interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) production in the basic immunizations with gp340. While presentation of the antigen in iscom formulations with low levels of Quillaja triterpenoids induces a moderate enhancement of the immune responses in the low responder H-2(d) mice, supplementation with high levels of iscomatrix immunomodulator was required to enhance the immune responses in the high responder H-2(k) mice. In both mouse strains subcutaneous immunization with S-iscoms resulted in a significant increase of IgG1- and IgG2a-specific antibodies, as well as in strong antigen-specific proliferative response confirmed by the simultaneous cytokine production. The enhanced antigen-specific secretion of IL-2 and IFN-gamma together with the abrogation of IL-10 and the absence of IL-4 indicates that the responses were driven towards a Th1-type rather than Th2-type immune response. The S-iscom formulations minimized the differences in immune responses between the two mouse strains, but the capacity of immune sera to neutralize EBV transformation in vitro remained completely strain-dependent. These data indicate that immune responses generated by iscoms can be manipulated by altering the triterpenoid composition of the iscoms and that the levels of triterpenoids can determine whether or not a Th1-type response is made.

Immunomodulation by Quillaja saponaria adjuvant formulations: in vivo stimulation of interleukin 12 and its effects on the antibody response.

The capacity of adjuvants to activate Ag-presenting cells during the induction of the primary immune response is of critical importance for the development of protective immunity to a number of pathogens. In this context, interleukin 12 (IL-12) has a key role by controlling the differentiation of T helper cells and favouring the expansion of Th1 cells. The capacity of iscoms with influenza virus Ag (flu-iscoms) and iscom matrix with EBV gp340 Ag to induce IL-12 was analysed in mice. The flu-iscom drives the immune response towards a Th1 type subsequent to IL-12 induction as measured in the serum of H2b, H2d and H2k mice. The iscom presenting the Ag and adjuvant in the same particle was considerably more efficient than the formulation of matrix and Ag in separate particles. Inhibition experiments with mAb neutralizing IL-12, interferon gamma (IFN-gamma) or IL-4, the latter two cytokines representing the Th1 and Th2 type of responses, showed that iscoms induce a broader immune response than that involving IL-12. This was shown by the additional effect that IL-4 neutralization had on the immune response to iscoms. Anti-IL 12 reduced the specific total Ab as well as IgG1, IgG2a and IgG2b while anti-IL 4 influenced the response to iscom by decreasing IgG2a and increasing IgG1. Further, the neutralization experiments indicate that IL-12 has a broader effect than IFN-gamma on the Ab response by influencing the production of IgG1, IgG2a and IgG2b.

The flagellar fraction of Trypanosoma cruzi depleted of an immunosuppressive antigen enhances protection to infection and elicits spontaneous T cell responses.

The flagellar fraction (FF) of Trypanosoma cruzi can be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF-beta (Hansen et al., submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom-matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice. In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL-1 and IL-6. In immunized mice, the FT elicited higher levels of antigen-specific IgG2a than the FF as well as broader recognition of T. cruzi antigens. Splenocytes from mice immunized with FT proliferated spontaneously in vitro and secreted TH1 and TH2 cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN-gamma. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN-gamma through a TGF-beta dependent mechanism.

Novel adjuvants and vaccine delivery systems.

Conventionally the efficiency of an adjuvant is measured by the capacity to induce enhanced antibody serum titres and cell mediated immunity (CMI) to a given antigen. Nowadays the capacity of an adjuvant is also measured by the quality as well as the magnitude of the induced immune response, guided by the protective immune response required. Quality includes isotype and IgG subclass responses, T-helper cell responses characterized by the cytokine profile and cytotoxic T cells (CTL). In the early phase of immunization some adjuvants influence the antigen administration and uptake by a so-called depot effect exemplified by aluminium hydroxide gel and oil adjuvants, which possibly is not as desired as alledged. A modern depot is exerted by slow release formulations continuously releasing the antigen over a period of time or by pulses at intervals aiming at 'single injection' vaccine. Great efforts are made to formulate efficient delivery formulations targeting the antigens from the site of administration, to draining lymph nodes or distant lymphatic tissue or to mucosal surfaces by parenteral or mucosal administrations. Nowadays, non-replicating carriers besides replicating vaccines are formulated to induce mucosal immune responses encompassing secretory IgA and CMI. Efforts to evoke immune responses on mucosal membranes distant from the site of administration have resulted mostly in little success. For a long time it was considered that CTL under the restriction of MHC Class I only could be evoked by replicating viruses or intracellular parasites. However, novel adjuvant delivery systems readily induce CTL by delivering the antigen to the APC resulting in intracellular transport to the cytosol for the MHC Class I presentation system, as well as to the endosomal pathway for the MHC Class II presentation.

In vitro activation of antigen-presenting cells (APC) by defined composition of Quillaja saponaria Molina triterpenoids.

The capacity of adjuvants to stimulate cytokine production by APC is important for the initiation of the immune response. Novel adjuvant formulations based on the iscom technology have been developed using selected triterpenoid components from Quillaja saponaria Molina. Five of these new Quillaja formulations were used to prepare matrix (an antigen-free particle) and tested for their capacity to stimulate IL-1 secretion by murine peritoneal cells in vitro. The formulation denominated QH 7.0.3 was superior to the other matrix formulations, including the original spikoside matrix. The QH 7.0.3 formulation in iscoms containing influenza virus envelope antigens induced IL-1 secretion more efficiently than the antigen-free matrix, or a mixture of matrix and viral antigens, or the free Quillaja components of similar composition. Compared with adjuvants known as IL-1 inducers, QH 7.0.3 flu-iscoms were as efficient as the most prominent IL-1 inducer, i.e. lipopolysaccharide (LPS) and superior to cholera toxin (CT) and muramyl dipeptide (MDP). These results indicate that the composition per se of triterpenoids included in iscoms or matrix has a prominent influence on the level of APC activation which may result in qualitatively different immune responses in vivo.

Antigen presentation by naive macrophages, dendritic cells and B cells to primed T lymphocytes and their cytokine production following exposure to immunostimulating complexes.

Influenza virus envelope proteins incorporated into immunostimulating complexes (iscoms) are taken up and processed by various kinds of antigen-presenting cells (APC), encompassing peritoneal cells (PEC), unfractionated splenocytes, splenic dendritic cells (DC) or B cells. The iscom-pulsed naive APC stimulated primed T cells to proliferate and produce cytokine in vitro. In contrast, only DC and B cells pulsed with the same antigen (Ag) in the micelle form functioned as accessory cells stimulating the primed T cells to proliferate and produce cytokine. In general, iscoms were better inducers of cell proliferation than micelles. Iscoms stimulated more secretion of IL-2 and interferon-gamma (IFN-gamma) than the micelles, but both antigenic forms stimulated secretion of IL-4. DC and B cells pulsed with iscoms stimulated most efficiently the secretion of IL-2 and IFN-gamma. DC were superior to the other APC in stimulating primed T cells to secrete IFN-gamma. On the other hand, micelles stimulated more efficiently than iscoms splenic T cells from micelle-primed as well as iscom-primed mice to secrete IL-10. These data indicate that influenza virus envelope proteins incorporated in iscoms stimulate a broad T cell response, possibly emphasizing a Th1 type of response. The same Ag in a micelle form induce a more prominent Th2 type of T cell response. The results indicate that the administration of an Ag in an adjuvant formulation can superimpose a different cytokine profile on the immune response than that induced by the protein Ag alone.

Influence of oestradiol-17 beta on interleukin-1 secretion by endotoxin-stimulated porcine mononuclear cells in vitro.

A bioassay for porcine interleukin-1 (IL-1) was validated. By use of this bioassay, the influence of increasing concentrations of oestradiol-17 beta on IL-1 secretion by endotoxin-stimulated porcine mononuclear cells (MNC) in vitro was studied. Supernatants from cultures of porcine MNC stimulated with LPS (0.1-10 micrograms/ml) induced a proliferative response in the IL-1 and IL-2 dependent cell line D10.G4.1 (D-10) in a dose-dependent manner with respect to LPS. Supernatants of LPS stimulated porcine MNC did not show any IL-2 activity in an bioassay for IL-2. Porcine MNC were cultured in the presence of increasing concentrations of oestradiol-17 beta (42-1500 pmol/l) and stimulated with LPS (10 micrograms/ml) or in the absence of oestradiol and LPS. The IL-1 secretion by the MNC, as determined by the D-10 bioassay, was significantly (P < 0.01) suppressed by the highest oestradiol concentration tested (1500 pmol/l). The results indicate that high physiological concentrations of oestradiol-17 beta may affect the secretion of IL-1 by porcine MNC.

The induction of cell-associated and secreted IL-1 by iscoms, matrix or micelles in murine splenic cells.

The kinetics of the expression of membrane-associated IL-1 (mIL-1) and soluble IL-1 (sIL-1) was studied in in vitro stimulated spleen cells from non-primed mice or from mice primed with influenza virus antigens incorporated in the immuno-stimulating complexes (iscoms) or as micelles. Matrix, which is the carrier structure for the antigens in the iscom, was used as a non-antigen stimulus. The IL-1 produced was assayed in an IL-1-dependent cell line and the specificity was demonstrated in a blocking experiment with antiserum to IL-1 alpha. Soluble IL-1 alpha was also quantified in ELISA. Iscoms and matrix induced production of mIL-1 and sIL-1 in cultures from non-treated mice as well as from mice primed 4 days before with iscoms or micelles. Micelles were a less strong stimulus and did not induce production of sIL-1. Micelles induced production of mIL-1 in cultures from non-primed mice or from mice which were recently immunized with micelles. No mIL-1 expression was induced by micelles if the spleen cells originated from mice immunized shortly before with iscoms. Depletion experiments demonstrated that sIL-1 was produced by adherent cells upon stimulation with iscoms or matrix. However, factor(s) from the non-adherent cells seem to be necessary for optimal secretion of sIL-1.

Involvement of interleukin-2 and interferon-gamma in the immune response induced by influenza virus iscoms.

Splenocytes from mice primed with influenza virus envelope proteins incorporated in iscoms, as micelles or as infectious virus, were restimulated in vitro with the same antigen. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were assayed in the supernatants of such cultures. Influenza virus iscoms induced IL-2 and IFN-gamma responses in restimulation experiments that were antigen specific and significantly higher than those induced by micelles or infectious virus. Serum samples collected at the end of the experiments were analysed for the antibody response and profile. The antibody titres induced by iscoms were of a similar order of magnitude as those induced by infectious virus, and were about 18 times higher than the titres induced by micelles. In mice immunized with iscoms or infectious virus the most abundant antibodies were of the IgG1 and IgG2a isotype, and the IgE response was low. We conclude that immunization with iscoms stimulates the Th1-like subtype of murine T lymphocytes.

Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gp120.

Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought. Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120. Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1. One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120. These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization. Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well. In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin. The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region. The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs. Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120. These MAbs did not neutralize HIV-1IIIB.