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Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
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2005 (English)In: Veterinary parasitology, ISSN 0304-4017, E-ISSN 1873-2550, Vol. 129, no 1-2, 25-34 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2005. Vol. 129, no 1-2, 25-34 p.
Keyword [en]
Neospora caninum, subunit vaccine, recombinant immunogen, NcSRS2, iscom, antibody response, competetive PCR
National Category
Microbiology in the medical area
URN: urn:nbn:se:kth:diva-5176DOI: 10.1016/j.vetpar.2004.12.004ISI: 000228600600004ScopusID: 2-s2.0-16244380770OAI: diva2:7984
QC 20100917Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2010-09-17Bibliographically approved
In thesis
1. Rational and combinatorial protein engineering for vaccine delivery and drug targeting
Open this publication in new window or tab >>Rational and combinatorial protein engineering for vaccine delivery and drug targeting
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting.

In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines.

In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

Biomedicine, affibody, affinity chromatography, biotin, combinatorial, delivery, phage display, Biomedicin
National Category
Microbiology in the medical area
urn:nbn:se:kth:diva-231 (URN)91-7178-003-3 (ISBN)
Public defence
2005-06-03, Sal FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00
Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2012-03-22

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Wikman, MariaFriedman, MikaelaStåhl, Stefan
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