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Plant-produced STI vaccine antigens with special emphasis on HIV-1 p24
Örebro University, School of Science and Technology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Objective: To establish stable transgenic non-toxic plants as a platform for plant-based vaccine production as well as potential oral delivery system of vaccine antigens for sexually transmitted infections (STIs). The concept is to immunize the mucosal immune system present in the gut-associated lymphoid tissues (GALT). HIV-1 p24 subtype C protein has been used as the main antigen model, in parallel with an engineered unique chimeric MOMP antigen from Chlamydia trachomatis serovar E.

Methods: Chimeric MOMP and p24 vaccine antigens were successfully inserted into the nuclear genomes of Arabidopsis thaliana and Daucus carota via Agrobacterium-mediated gene transfer. The characteristics of the genetic inserts and corresponding mRNAs and recombinant proteins in planta were described using several methods, including northern, Southern, and western blotting, ELISA, and a commercial HIV Ag/Ab combination assay. Immunogenicity of the antigens was studied in mice models.

Results: Transgenes of both plant species expressing p24 or chimeric MOMP were successfully generated. Additional HIV-1 vaccine antigen candidates were introduced and the genetic inserts have been confirmed in Arabidopsis thaliana. The Arabidopsis thaliana expressing p24 and chimeric MOMP were demonstrated to be stable over generations and antigenicity analyses showed that plant-derived HIV-1 p24 and chimeric MOMP retained immunological epitopes when they were expressed in planta. Oral administration of transgenic plant material generated a priming effect of the immune competent cells present in the GALT, shown by the presence of antigen-specific-IgG in mice sera after boosting. Mice immunized with plant-derived HIV-1 p24 antigen were also analyzed for antigen-specific faecal IgA as well as cellular immune responses. However, detectable levels of the two latter immune responses were not observed. The Chlamydia trachomatis chimeric MOMP antigen was further evaluated for its potential as a vaccine antigen candidate, with positive results indicating a more rapid clearance of the Chlamydia trachomatis infection post immunization.

Conclusion: Stable non-toxic transgenic plants expressing either HIV-1 p24 or a novel  Chlamydia trachomatis chimeric MOMP antigens have successfully been developed. The two plant-produced STI vaccine antigens have in initial mice feeding studies provided important proof-of-concept for the oral vaccination approach. Now, immunization studies to expand, en-hance, and improve knowledge of the immune responses generated by the orally delivered transgenic plants are of high priority.

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2011. , 86 p.
Series
Örebro Studies in Life Science, 8
Keyword [en]
Plant-based vaccines, Arabidopsis thaliana, HIV-1, p24, GALT, mucosal immunity, Chlamydia trachomatis, MOMP, Daucus carota
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:oru:diva-17242ISBN: 978-91-7668-817-5 (print)OAI: oai:DiVA.org:oru-17242DiVA: diva2:440739
Public defence
2011-10-28, Hörsal M, Örebro universitet, Fakultetsgatan 1, Örebro, 10:15 (English)
Opponent
Supervisors
Projects
Kemi/biokemi
Available from: 2011-09-13 Created: 2011-09-13 Last updated: 2017-10-17Bibliographically approved
List of papers
1. Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses
Open this publication in new window or tab >>Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses
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2008 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 116, no 11, 985-994 p.Article in journal (Refereed) Published
Abstract [en]

Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not the least for use in developing countries. Therefore, we used Agrobacterium tumefaciens-mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV-1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR, four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by western blot analysis. The plants were shown to contain between 0.2 µg and 0.5 µg p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2-3, 2 and 1 T-DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T1 to the T4 generation and gave rise to the p24 protein in all cases, as verified by western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti-gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

Place, publisher, year, edition, pages
Oxford: Blackwell, 2008
National Category
Medical and Health Sciences Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:oru:diva-4631 (URN)10.1111/j.1600-0463.2008.00900.x (DOI)
Available from: 2008-10-14 Created: 2008-10-14 Last updated: 2017-12-14Bibliographically approved
2. Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs
Open this publication in new window or tab >>Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs
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2009 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 1, 46-51 p.Article in journal (Refereed) Published
Abstract [en]

An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein 5-fold in the Arabidopsis thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

Place, publisher, year, edition, pages
St. Louis: Academic Press, 2009
National Category
Natural Sciences Chemical Sciences Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:oru:diva-5166 (URN)10.1016/j.pep.2008.12.015 (DOI)000265346000008 ()19167502 (PubMedID)2-s2.0-63649152546 (Scopus ID)
Available from: 2009-11-05 Created: 2009-01-29 Last updated: 2017-12-14Bibliographically approved
3. Plant-based production of HIV antigens: towards a candidate for an edible vaccine
Open this publication in new window or tab >>Plant-based production of HIV antigens: towards a candidate for an edible vaccine
2010 (English)In: In Vivo, ISSN 0258-851X, E-ISSN 1791-7549, Vol. 24, no 3, 368-370 p.Article in journal, Meeting abstract (Refereed) Published
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:oru:diva-14224 (URN)
Projects
Ätbara vacciner
Available from: 2012-08-06 Created: 2011-01-25 Last updated: 2017-10-02Bibliographically approved
4. Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses
Open this publication in new window or tab >>Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4+ T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 p24 antigen, which is released by immune competent cells in the GALT during oral administration, are interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific-IgG in sera after an intramuscular p24 protein boost. Initial dose-dependent antigen analyses using transgenic Arabidopsis thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses. No detectable levels of faecal IgA antibodies or cellular immune responses were observed.

National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:oru:diva-17314 (URN)
Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2017-10-17Bibliographically approved
5. A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate
Open this publication in new window or tab >>A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate
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2011 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 80, no 2, 194-202 p.Article in journal (Refereed) Published
Abstract [en]

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.

Place, publisher, year, edition, pages
Academic Press, 2011
National Category
Natural Sciences Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:oru:diva-17313 (URN)10.1016/j.pep.2011.08.010 (DOI)000296225800005 ()21903168 (PubMedID)2-s2.0-80052854898 (Scopus ID)
Projects
Ätbara vacciner
Available from: 2012-08-06 Created: 2011-09-22 Last updated: 2017-12-08Bibliographically approved

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