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Recombinant NcSRS2, a major immunodominant surface antigen of the intracellular protozoan parasite Neospora caninum, was used as a model antigen to compare the immunogenicity of iscoms prepared according to three different methods. Two NcSRS2 fusion proteins were used, one that was biotinylated upon expression in Escherichia coli and linked to Ni2+-loaded iscom matrix (iscom without any protein) via a hexabistidyl (HiS(6)-tagged streptavidin fusion protein, and another that contained both a HiS(6)-tag and streptavidin (HiS(6)-SA-SRS2') and was coupled to either Ni2+-loaded or biotinylated matrix. While all three iscom preparations induced N. caninum specific antibodies at similar levels, HiS(6)-SA-SRS2' coupled to biotinylated matrix generated the strongest cellular responses measured as in vitro proliferation and production of interferon-gamma and interleukin-4 after antigen stimulation of spleen cells. However, the relationship between the levels of these cytokines as well as between IgG1 and IgG2a titres in serum induced by the three iscom preparations were similar, indicating that the balance between Th1 and Th2 responses did not differ. After challenge infection, mice immunised with His(6)-SA-SRS2' coupled to biotinylated matrix had significantly lower amounts of parasite DNA in their brains compared to the other immunised groups. Possible reasons for the performance of the different iscom formulations are discussed., QC 20100525

In recent years, several studies have been reported with the common aim of generating general expression systems for straightforward production and subsequent coupling of expressed antigens to an adjuvant system. Here, we describe a series of such efforts with a common theme of using gene fusion technology for association of recombinant antigens to immunostimulating complexes (iscoms). In the early stages of vaccine development, uniform antigen preparations are crucial to allow the comparison of immune responses to different antigens, or even subdomains thereof, and we believe that the described systems constitute an important development in this context., QC 20100525

The coccidian parasite Neospora caninum is an intracellular protozoan, causing abortion in cattle in many countries around the world. In this study, the protective potential of the major N. caninum surface antigen NcSRS2, expressed in Escherichia coli and formulated into immunostimulating complexes (iscoms), was investigated in an experimental mouse model. The recombinant protein was specially designed for binding to iscoms via biotin-streptavidin interaction. Two groups of 10 BALB/c mice were immunised twice, on days 0 and 28 with iscoms containing either the recombinant NcSRS2 (NcSRS2 iscoms) or similar iscoms with NcSRS2 substituted by an unrelated recombinant malaria peptide (M5) as a control (M5 iscoms). A third group of 10 age-matched BALB/c mice served as an uninfected control group. Immunisation with recombinant NcSRS2 iscoms resulted in production of substantial antibody titres against N. caninum antigen, while the mice immunised with M5 iscoms produced only very low levels of antibodies reacting with N. caninum antigen. After challenge infection with N. caninum tachyzoites on day 69, mice immunised with NcSRS2 iscoms showed only mild and transient symptoms, whereas the group immunised with M5 iscoms showed clinical symptoms until the end of the experiment at 31 days post inoculation. A competitive PCR assay detecting Nc5-repeats was applied to evaluate the level of parasite DNA in the brain. The amount of Nc5-repeats in the group vaccinated with NcSRS2 iscoms was significantly lower than in the control group given M5 iscoms. In conclusion, it was found that the recombinant NcSRS2 iscoms induced specific antibodies to native NcSRS2 and immunity sufficient to reduce the proliferation of N. caninum in the brains of immunised mice., QC 20100917

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptides or lipid tags to improve their capacity to be incorporated into an adjuvant formulation, e.g., immunostimulating complexes (iscoms). Recently, we also explored the strong interaction between biotin and streptavidin to achieve iscom association of recombinant immunogens. Plasmodium falciparum, Toxoplasma gondii and Neospora caninum antigens have served as model immunogens in the different studies. Generated fusion proteins have been found to be successfully incorporated into iscoms and high-titer antigen-specific antibody responses have been obtained upon immunization of mice. We believe that the different concepts presented, utilizing either hydrophobic peptide or lipid tags, or the recently explored biotin-streptavidin principle, offer convenient methods to achieve efficient adjuvant incorporation of recombinant immunogens., QC 20100525 QC 20111012. 4th World Congress on Vaccines and Immunisation (WCVI). Tokyo, JAPAN. SEP 30-OCT 03, 2004

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K-D approximate to 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni2+-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M), derived from the central repeat region of the Plasmodium faiciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen, QC 20111005

BACKGROUND & OBJECTIVES: The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4. METHODS: Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes. RESULTS: Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P<0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P<0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%). INTERPRETATION & CONCLUSION: These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

BACKGROUND & OBJECTIVES: The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4. METHODS: Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes. RESULTS: Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P<0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P<0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%). INTERPRETATION & CONCLUSION: These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

BACKGROUND & OBJECTIVES: The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4. METHODS: Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes. RESULTS: Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P<0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P<0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%). INTERPRETATION & CONCLUSION: These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

The protection induced by immunostimulating complexes (ISCOMs) against bovine respiratory syncytial virus (BRSV) was evaluated and compared to that of a commercial inactivated vaccine (CV) in calves with BRSV-specific maternal antibodies. Following experimental challenge, controls (n = 4) and animals immunized with CV (n = 5) developed moderate to severe respiratory disease, whereas calves immunized with ISCOMS (17 = 5) remained clinically healthy. BRSV was re-isolated from the nasopharynx of all controls and from all calves immunized with CV, but from none of the calves immunized with ISCOMs. BRSV-RNA was detected by real-time PCR from a single animal in this group. Significantly higher BRSV-specific nasal IgG, serum IgG(1) and IgG(2) titers were detected before and after challenge in animals immunized with ISCOMs versus CV. In conclusion, the ISCOMs overcame the suppressive effect of maternal antibodies in calves and induced strong clinical and virological protection against a BRSV challenge.

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 r, QC 20100917

Immune-stimulating complexes (ISCOMs) are stable colloidal complexes of the adjuvant Quil A, cholesterol and phospholipid, which are effective carriers for subunit vaccines. The techniques currently available for the preparation of ISCOMs from the constituent components are rather complex and are based on either centrifugation or dialysis. This note reports a new simple procedure for the preparation of ISCOM matrices based on hydration of a cholesterol/phospholipid film with an aqueous solution of Quil A. It is demonstrated that ISCOM matrices do not form in the absence of phospholipid when prepared by this method. Further, the ratio by weight of phospholipid to either cholesterol or Quil A must be greater than that required for preparation by either dialysis or centrifugation. Photon correlation spectroscopy, negative stain transmission electron microscopy and centrifugation through a sucrose gradient demonstrate that ISCOM matrices can be prepared from cholesterol/lipid films by hydration with an aqueous solution of Quil A when the ratio of phospholipid:cholesterol:Quil A by weight is 6:1:4, respectively. Lower ratios of phospholipid:cholesterol reduce the efficiency of ISCOM formation while higher ratios produce systems containing a mixture of ISCOMs together with liposomes. Copyright (C) 2000 Elsevier Science B.V.

The immunogenicity and efficacy of an inactivated whole SIVmac (32H) preparation adjuvanted with muramyl dipeptide (SIV-MDP) and a gp120-enriched SIVmac (32H) ISCOM preparation (SIV-ISCOM), were compared by immunizing four rhesus macaques (Macaca mulatta) four times with SIV-MDP and four others in the same way with SIV-ISCOM. Two monkeys immunized with whole inactivated measles virus (MV) adjuvanted with MDP (MV-MDP) and two monkeys immunized with MV-ISCOM served as controls. In the SIV-ISCOM-immunized monkeys higher SIV-specific serum antibody titres were found than in the SIV-MDP-immunized monkeys. In contrast to the MV-immunized monkeys all SIV-MDP- and SIV-ISCOM-immunized monkeys were protected against intravenous challenge 2 weeks after the last immunization with 10 median monkey infectious doses (MID50) of a cell-free SIVmac (32H) challenge stock propagated in the human T-cell line C8166. After 43 weeks the protected monkeys were reboosted and 2 weeks later rechallenged with 10 MID50 of the same virus produced in peripheral blood mononuclear cells (PBMC) from a rhesus macaque. None of these animals proved to be protected against this challenge. In a parallel experiment in which the same numbers of monkeys were immunized in the same way, the animals