| 1. |
- Andersson, Christin, et al.
(författare)
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In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation
- 2001
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Ingår i: Journal of Immunological Methods. - 0022-1759. ; 255:1-2, s. 135-148
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Tidskriftsartikel (refereegranskat)abstract
- 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 ΔSAG1, 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-ΔSAG1 and His6-ABP-ΔSAG1, 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-ΔSAG1 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, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.
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| 2. |
- Johansson, Jeannette, et al.
(författare)
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Identification of Adjuvants that Enhance the Therapeutic Antibody Response to Host IgE
- 2004
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 22:21-22, s. 2873-2880
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Tidskriftsartikel (refereegranskat)abstract
- In the development of a novel vaccine against atopic allergies, we have screened for adjuvants that enhance the therapeutic antibody response against self immunoglobulin E (IgE). The response against self IgE is induced by administration of a vaccine antigen, which contains both self and non-self IgE regions, together with an adjuvant. We evaluated five commonly used adjuvants; Freund's, aluminium hydroxide, ISCOMs, Montanide ISA 51 and Montanide ISA 720, and found that the mineral oil-based adjuvants; Montanide ISA 51 and Freund's induced at least 5-10-fold higher anti-self IgE titers than any of the other candidates. However, with one exception, Alum, the immune responses against the carrier, i.e. the non-self regions, were similar for all adjuvants, indicating that the ability to induce responses against self and non-self antigens differ among adjuvants. The responses against non-self IgE were more than 50-fold higher than antibody responses against self IgE in both the Freund's and Montanide 51-administered animals, indicating that the response against self molecules is markedly inhibited by tolerance-inducing mechanisms. Co-administration of Montanide ISA 51 with immuno-stimulatory substances from bacteria; muramyldipeptide (MDP), monophosphoryl-lipid A (MPL) or a formyl-methionine-containing tripeptide (fMLP), did not elevate the anti-self IgE response. Hence, adjuvants based on pure mineral oil without additional immuno-stimulatory substances appear to be the best adjuvant candidates in therapeutic vaccines aimed at regulating the in vivo levels of self-proteins.
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