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PubMed2000

Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation.

We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce M5-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.

Adjuvants, Immunologic↗

PubMed2001

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Amino Acid Sequence↗

PubMed1999

General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation.

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.

Amino Acid Sequence↗

PubMed1997

Influenza (H1N1)-ISCOMs enhance immune responses and protection in aged mice.

Aging is associated with a decline in immune function and the elderly are therefore more susceptible to infectious disease and less responsive to vaccination. Influenza antigens complexed as immunostimulatory complexes (ISCOMs) generate more potent protective immune responses compared with non-adjuvanted flu antigens in young adult mice. We report on the protective efficacy of flu-ISCOMs compared with the current split flu vaccine in an aged mouse model. DBA/2 mice aged 2 or 18 months were immunized with flu vaccine, ISCOMs or live virus, prior to challenge with the homologous virus. In aged mice, flu-ISCOMs induced significantly higher serum hemagglutination inhibition (HAI) titers compared to vaccine, similar to the levels obtained in young adult mice that received the split vaccine. Flu-ISCOMs but not vaccine induced cytotoxic T lymphocyte (CTL) responses in young and to a lesser degree in aged mice. In aged mice flu-ISCOMs significantly reduced illness and enhanced recovery from viral infection compared with vaccine. Our data suggests that flu-ISCOMs may offer an improved vaccine strategy for protection of elderly humans against the complications of influenza infection.

Aging↗

PubMed1999

Direct biophysical characterization of human apolipoprotein A-1 in ISCOMs.

Human apolipoprotein A-1 was formulated in "Immune Stimulating Complexes" (ISCOMs). The structure of the protein in ISCOMs was examined directly using several biophysical techniques including Fourier transform infrared (FTIR) spectroscopy, near UV circular dichroism (CD), and fluorescence spectroscopy. Amide I FTIR data indicate that human apolipoprotein A-1 displays a slightly increased alpha-helical content after its incorporation into ISCOMs. Near UV CD and tryptophan fluorescence data suggest that association with ISCOMs results in the tryptophan residues of the protein experiencing a relatively hydrophobic environment, motional restriction, and local electrostatic interactions. These observations are consistent with an increased order in the protein structure upon incorporation in ISCOMs. In addition, biomolecular interaction analysis (BIA), based on surface plasmon resonance (SPR) measurements, suggests that the binding affinity of human apolipoprotein A-1 to a monoclonal anti-human apolipoprotein A-1 antibody is moderately decreased (by 20%) after its incorporation into ISCOMs. This study demonstrates that these biophysical techniques can be used to noninvasively monitor integrity of or changes in secondary and tertiary structure of proteins within the ISCOM particles without the need for protein extraction.

Apolipoprotein A-I↗

PubMed1996

Kinetics, localization and isotype profile of antibody responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins.

The immune stimulating complex (iscom) is a particulate adjuvant formulation combining multimeric presentation of antigen with a built-in adjuvant, Quillaja saponin. Iscoms induce strong serum antibody responses that are readily boosted. To further characterize this property of iscoms, the development and maturation of primary and secondary antibody responses to iscoms containing influenza virus antigen were investigated, in serum by ELISA and on single B-cell level by ELISPOT. After a single subcutaneous injection, B cells secreting antigen-specific IgG (IgG-SC) were primarily observed in the draining lymph nodes (LN), showing peak numbers at day 7 which then declined rapidly. Serum IgG levels, as well as IgG-SC in the spleen, persisted for several weeks and, with time, IgG-SC cells also appeared in the bone marrow (BM). These results suggest that the IgG response to iscoms initially is located to the LN but that IgG-SC are redistributed with time and may persist for a long time in other organs, including the spleen and BM. Moreover, a booster dramatically enhanced the frequency of IgG-SC in LN, spleen and BM suggesting that iscoms induce a potent B-cell memory. Comparisons of antibody responses to iscoms with those to influenza virus antigen in Freund's complete adjuvant, TiterMax or aluminium hydroxide suggest that the choice of adjuvant influences both the magnitude, kinetics, localization and isotype profile of antibody responses.

Adjuvants, Immunologic↗

PubMed2006

Saponins from Quillaja saponaria Molina: isolation, characterization and ability to form immuno stimulatory complexes (ISCOMs).

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann-Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from approximately 1200 to approximately 2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

ISCOMs↗

PubMed1990

The iscom: an immunostimulating system.

To make purified antigens highly immunogenic, they have to be presented in several copies in the form of a microscopic or submicroscopic particle. This is the case, regardless of whether the antigens are obtained by isolation from conventional microorganisms, or from gene-manipulated cells, or synthesized. In the iscom, the antigens are attached as multimers to a 40-nm cage-like particle with a built-in adjuvant. The antigens in iscoms are rapidly transported from the injection site to the draining lymphatic organ. Iscom-borne antigens induced a 10-fold higher antibody response than the same amount of antigen in micelle form. One intranasal immunization with influenza virus iscoms induced protection to intranasal challenge infection in mice. Besides a strong antibody response in all Ig classes and isotypes, cytotoxic T cells were induced. With iscoms containing gp160 of HIV-1, cytotoxic T cells (CD8+ CD4-) were induced under restriction of class I MHC antigen. Iscoms containing the fusion protein of measles virus induced T cell clones in mice whereof one, after adoptive transfer, protected mice against intracerebral challenge infection. Protective immunity against Epstein-Barr virus (EBV)-induced tumor formation by iscoms containing gp350 of EBV has been elicited in cotton-top Tamerin monkeys. Protective immunity has also been induced against several virus infections including feline leukemia virus and against parasites, i.e., Trypanosoma cruzi, in mice.

Adjuvants, Immunologic↗

PubMed1989

Formation and characterization of FeLV ISCOMs.

Immunostimulating complexes (ISCOMs) have been prepared from feline leukaemia virus (FeLV) envelope proteins. The ISCOMs were characterized biochemically in SDS-polyacrylamide gel electrophoresis showing the presence of proteins of estimated molecular weights of 15,000, 27,000 and 70,000. Immunoblotting showed that both the transmembrane protein p15E and the external glycoprotein gp70 (making up the gp85 protein) were present in the ISCOM. Furthermore, a degradation product of gp70 with an estimated molecular weight of 32,000 was identified in the immunoblot. The FeLV ISCOM was shown by electron microscopy to have the characteristic cage-like structure of an ISCOM with a mean diameter of 37 nm. About 10% of the total amount of gp70 in the culture fluid was recovered in the ISCOMs. The largest loss was encountered during the sedimentation of the virus. In a preliminary immunization experiment in mice the FeLV ISCOMs elicited after a booster gave a clear-cut immune response against gp70.

Animals↗

PubMed1991

Efficacy of HSV-1 ISCOM vaccine in the guinea-pig model of HSV-2 infection.

The capability of a herpes simplex virus (HSV)-1 ISCOM vaccine to protect against intravaginal HSV-2 challenge infection in guinea-pigs is described. The protective efficacy of the HSV-1 ISCOM vaccine is compared with that of a purified, aqueous HSV-1 antigen preparation administered using a similar immunization schedule. The results show that female guinea-pigs immunized with two doses of HSV-1 ISCOM vaccine, each consisting of 20 micrograms of protein given 2 weeks apart responded with high ELISA and neutralization antibody titres, and are almost completely protected against the clinical effects of intravaginal challenge with 10(5.2) TCID50 of HSV-2. This cross-protection is significantly greater than that observed in guinea-pigs immunized with a single dose of HSV-1 ISCOM vaccine, two doses of aqueous HSV-1 antigen preparation or two doses of a mock ISCOM vaccine. However, none of the vaccine preparations completely prevented HSV-2 replication following challenge. Western blot and radioimmunoprecipitation of sera from immunized guinea-pigs show the HSV-1 ISCOM vaccine preparation to contain the major HSV-1 glycoproteins. These findings are discussed in relation to the value and potential use of HSV-1 ISCOM vaccine in humans.

Animals↗

PubMed1988

Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes (iscoms): chemical analysis, some structural features, and comparison of their immunogenicity with three other antigen delivery systems.

We incorporated the major outer membrane protein (PI) of Neisseria gonorrhoeae into immunostimulating complexes (iscoms) and examined some analytical, physicochemical, and immunological properties of these structures. The immunogenicity was compared with that of three other PI-containing structures, i.e., liposomes, outer membrane complexes produced by the bacterium, and protein-detergent-adjuvant complexes. AIPO4 and dioctadecyldimethylammonium bromide were used as adjuvants. Our results show that iscoms are much more immunogenic than liposomes and protein-detergent complexes but are also much more toxic. The localization of PI in iscoms was investigated. Therefore, the chymotrypsin susceptibility of PI in iscoms was tested, and the incorporation of fragments of PI was determined. Amphiphilic fragments of PI were incorporated in iscoms, but hydrophilic and hydrophobic fragments were not. Chymotrypsin degradation of PI in iscoms indicated that the protein is exposed to the environment in a similar manner as PI in outer membrane complexes, i.e., with both termini anchored in the iscom.

Adjuvants, Immunologic↗

PubMed1995

Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS.

We examined the ability of oral or parenteral immunization with immune stimulating complexes containing ovalbumin (ISCOMS-OVA) to prime T cell proliferative and cytokine responses. A single subcutaneous immunization with ISCOMS-OVA primed potent antigen-specific proliferative responses in the draining popliteal lymph node, which were entirely dependent on the presence of CD4+ T cells. CD8+ T cells did not proliferate in vitro even in the presence of the appropriate peptide epitope and exogenous interleukin (IL)-2. Primed popliteal lymph node cells produced IL-2, IL-5 and interferon (IFN)-gamma, but not IL-4 when restimulated with OVA in vitro. Serum antigen-specific IgG1 and IgG2a antibody responses were also primed by subcutaneous immunization with ISCOMS-OVA, confirming the stimulation of both Th1 and Th2 cells in vivo. Spleen cells from subcutaneously primed mice produced a similar pattern of cytokines, indicating that disseminated priming had occurred. Oral immunization with ISCOMS-OVA also primed local antigen-specific proliferative responses in the mesenteric lymph node and primed an identical pattern of systemic cytokine responses in the spleen. The ability of ISCOMS to prime both Th1 and Th2 CD4+ T cell responses may be central to their potent adjuvant activities and confirm the potential of ISCOMS as future oral vaccine vectors.

Administration, Oral↗

PubMed1991

Characterization of purified gp 51 from bovine leukemia virus integrated into iscom. Physicochemical properties and serum antibody response to the integrated gp51.

It is proposed that the envelope glycoprotein, gp 51, is the protective antigen of bovine leukemia virus (BLV). An experimental iscom vaccine has been prepared from immunoaffinity purified gp 51. To overcome the problem of integrating a nonamphipathic protein, gp 51 was partially denatured at pH 2.4 before integration into the iscom. The recovery of gp 51 into the iscom was calculated to be 85%. The gp 51 incorporated into iscom retained its physicochemical properties and the neutralizing epitopes F, G and H were found to be intact. The iscom preparation was shown to induce a specific immune response to gp 51 after inoculation into mice and calves, as tested by ELISA and Western blotting. Sera from the immunized calves specifically inhibited the VSV-(BLV) pseudotypes. Thus the gp 51-iscom preparations appear to be highly immunogenic and to induce a gp 51 specific response.

Animals↗

PubMed1998

Application of iscom antigen preparations in ELISAs for diagnosis of Neospora and Toxoplasma infections.

Immunostimulating complexes (iscoms) are cage-like structures of about 40 nm composed of Quil A, cholesterol, phospholipids and antigen. Their main area of use has been as adjuvants and carriers of immunogens in vaccines. Iscoms can also be used for selection of surface membrane proteins of micro-organisms for use in immunoassays, thus decreasing the number of internal proteins that might cause problems with non-specific binding and cross-reactivity. Enzyme-linked immunoassays (ELISAs) utilising parasite antigens incorporated into iscoms have been developed for demonstration of antibodies directed to the intracellular coccidian parasites Toxoplasma gondii and Neospora caninum. These iscom ELISAs have proved very reliable, with high sensitivity and specificity. The preparation of T. gondii and N. caninum iscoms is described, and ELISAs based on iscom antigen preparations that have so far been used for diagnosis of protozoal infections are reviewed.

Animals↗

PubMed1998

Characterization of immune responses to experimental polyvalent subunit vaccines assembled in iscoms.

Immune responses to experimental polyvalent subunit vaccines assembled in a particulate adjuvant/delivery system, iscoms, are described. The fusion protein ZZ-M5 comprises structures of staphylococcal protein A (ZZ) and the Plasmodium falciparum malaria antigen Pf155/RESA (M5). MHC congenic mice were immunized with ZZ-M5 conjugated to iscoms containing human influenza virus antigen (flu ag, M5-flu-isc) or to iscom matrix (iscom particles without flu ag, M5-isc). Comparison of antibody and T-cell responses to M5-isc and M5-flu-isc demonstrated that the flu ag in M5-flu-isc exhibits carrier-related helper functions and that the assembly of immunogens in M5-flu-isc did not result in any apparent antigenic competition. In addition, assembly of ZZ-M5 and flu ag in iscoms induced an alteration of the IgG subclass profile of the antibody response to M5. The results suggest that assembly of immunogens in iscoms may be a useful approach to the design of subunit vaccines but that both quantitative and qualitative aspects of the immunogenic properties of such constructs should be scrutinized.

Amino Acid Sequence↗

PubMed2002

Induction of local innate immune responses and modulation of antigen uptake as mechanisms underlying the mucosal adjuvant properties of immune stimulating complexes (ISCOMS).

Lipophilic immune stimulating complexes (ISCOMS) containing the Quil A adjuvant can induce local and systemic immune responses against orally delivered protein antigens. Here, we have examined the possibility that ISCOMS act by modulating local innate immune responses and antigen uptake in the intestine. Feeding ISCOMS to mice induced recruitment of dendritic cells (DCs), activated macrophages (mphi) and lymphocytes into the mesenteric lymph node (MLN), as well as recruitment of macrophages and B cells into the PP. Feeding ISCOMS also enhanced the absorption into circulation of a test feed of ovalbumin (OVA), with accelerated kinetics and increased peak levels. Serum taken 1h after feeding a tolerogenic dose of OVA to mice given ISCOMS orally did not induce the tolerance of systemic delayed type hypersensitivity (DTH) and antibody responses found in mice receiving serum from donors fed OVA alone. Thus, ISCOMS may increase the immunogenicity of intestinal antigen via a combination of effects on antigen uptake and on local accessory cells.

Adjuvants, Immunologic↗

PubMed1996

Preparation and characterization of immunostimulating complexes (ISCOMs) of Japanese encephalitis virus.

ISCOMs (immunostimulating complexes) were prepared from envelope glycoprotein (Egp) of Japanese encephalitis (JE) virus. ISCOMs showed a single band of the viral Egp in SDS-PAGE, which reacted with polyclonal and monoclonal antibody (MAb) raised against Egp. Comparison between the epitopes exposed on JE virion and JE ISCOMs, by antigen capture ELISA, utilizing a panel of domain-specific MAbs, revealed identical epitopes exposed on the Egp incorporated in ISCOMs and the whole virion. Electron micrographs of ISCOMs showed spherical cage-like structures of 35 nm. ISCOMs with Egp were good immunogenes, which stimulated high titres of neutralizing antibodies, both in mice and rabbits.

Adjuvants, Immunologic↗

PubMed1998

Functional aspects of iscoms.

The iscom is a delivery system, designed for both parenteral and mucosal modes of administration, for both antigens and adjuvants, components which are interchangeable. By the parenteral route a prominent systemic Th1 type of response is evoked, but the mucosal immunoglobulin A (IgA) response was insignificant. Intranasal (i.n.) immunization with iscoms evoked potent mucosal IgA response and serum IgG which was much higher than that induced by i.n. administration of the B subunit of cholera toxin (rCTB), both to rCTB itself as well as to co-administered antigen. The immunomodulatory effect on rCTB or co-administered antigens imposed by the iscom was demonstrated by a potent mucosal IgA switch and an enhanced IgG2a serum response. The incorporation of a targeting molecule in the iscom enhanced the remote IgA response in the genital tract mucosa. The capacity to induce CD8-restricted cytotoxic T lymphocytes (CTL) is unique for the iscom as a nonreplicating system, which is facilitated by the delivery of antigens to the cytosol. The immunomodulatory capacity of iscoms also paved the way to override the inhibitory effect of maternally derived antibodies and the relative unresponsiveness of an immature neonatal immune system.

Animals↗