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Generation and evaluation of chimeric particles consisting of HPV16 L1 and p16INK4a for second generation HPV vaccines

Faulstich, Franziska

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The tumor suppressor p16INK4a is strongly expressed in HPV-transformed precursor lesions and cervical cancer, whereas in normal tissues barely any p16INK4a expression is detectable. It is thus used as a diagnostic marker since several years and it was speculated that targeting p16INKa-overexpressing cells with a therapeutic vaccine could have high benefit for patients suffering from HPV induced neoplasia and cancer. We designed chimeric capsomeres consisting of full-length p16INK4a and HPV16 L1, the major capsid protein of HPV and antigen of the available prophylactic HPV vaccines, with the aim of using the adjuvant-like effects of L1 particles to potentiate an effective p16INK4a immune response. For this purpose, three constructs were generated to evaluate the antigenic effect of different structural isoforms. The complete p16INK4a encoding cDNA sequence was cloned downstream (pGex-L1ΔN10Δh4ΔC29-p16INK4a) and upstream (pGex-p16INK4a-L1ΔN10Δh4ΔC29) of a modified HPV16 L1 sequence into a pGex-4T-2 expression vector. For the third construct (pGex-L1ΔN10Δh4-p16INK4a-L1ΔC29) the helix 4 region of L1 was replaced by p16INK4a. The proteins were inducible expressed in E. coli. Due to the low solubility of the chimeras, an inclusion body (IB) purification protocol was developed to purify the proteins in high amounts. After IB purification, the proteins were extracted under denaturing conditions with N-Lauroylsarcosine and refolded by dialysis. The capsomere preparations were found to be free of endotoxins after refolding from IBs. The produced particles were then evaluated for their structural properties and the in vivo immunogenicity of the capsomeres was tested in a C57BL/6 mouse model. Besides good stability characteristics, the capsomeres were found to be of rather heterogeneous structure and the immunological comparison of the three different constructs revealed different characteristics. GST-L1ΔN10Δh4-p16INK4a-L1ΔC29 induced highest L1-specific T cell numbers, GST-p16INK4a-L1ΔN10Δh4ΔC29 showed the best antibody response in the VLP-capture ELISA and GST-L1ΔN10Δh4ΔC29-p16INK4a seemed to induce the most efficient anti-p16 humoral immune response. Also the induction of p16-specific T cells could be demonstrated with the GST-L1ΔN10Δh4-p16INK4a-L1ΔC29 construct. The objective of this thesis was to generate, purify and evaluate a cost-effective second generation therapeutic vaccine based on chimeric capsomeres. The presented vaccine candidates can be cost-efficiently produced in bacteria and purified from inclusion bodies with high yields. The proteins were also found to be stable at room temperature and their immunogenicity was demonstrated in first in vivo mouse experiments. A protein-based vaccine with these properties could have substantial benefit, especially for unindustrialized countries where most of the cervical cancer cases occur. The possibility to generate an effective immune response to p16INK4a further opens new opportunities in the field of cancer immunotherapy. Not only patients with HPV-associated cancers would benefit from such a vaccine as many other cancers express high levels of p16INK4a, too. Also several precancerous neoplasias were shown to elicit increased p16INK4a expression and a p16INK4a-directed vaccine might even prevent development of invasive cancer.

Item Type: Dissertation
Supervisor: Müller, Prof. Dr. Martin
Place of Publication: Heidelberg
Date of thesis defense: 25 June 2014
Date Deposited: 23 Sep 2014 10:05
Date: 2014
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
Uncontrolled Keywords: human papillomavirus, p16INK4a, L1 protein, therapeutic vaccine, E. coli, inclusion bodies
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