Les papillomavirus à « haut risque » (HR) sont les agents étiologiques du cancer du col de l’utérus. Les vaccins prophylactiques actuels assurent une protection contre les lésions induites par les génotypes 16 et 18, responsables de 70% de ces cancers. Les lésions induites par les treize autres HPV HR responsables de 30% des cancers ne sont potentiellement pas prévenues par ces vaccins. Les objectifs de ma thèse ont été d’évaluer la capacité du vaccin quadrivalent anti-HPV et des vaccins de seconde génération à induire une immunité cross-neutralisante contre les HPV HR. Tout d’abord, la capacité du vaccin quadrivalent anti-HPV à induire des anticorps neutralisant les HPV 31 et 58 a été analysée. L’induction d’anticorps neutralisant les HPV 31 et 58 a été mise en évidence, 1 mois après la dernière injection, chez 33% et 24% des femmes vaccinées, respectivement. Les pseudovirions (PsV) de l'HPV 58 produits en système cellulaire et utilisés pour cette étude présentent une capacité de transduction des cellules COS-7 10 fois supérieure à celle des PsV 31 et représente un vecteur de choix pour des applications en immunisation génique. La deuxième partie de ma thèse s’est orientée sur le développement et l’analyse de la réponse immune induite par des vaccins de seconde génération basés sur la protéine L2. L’utilisation de la protéine L2, capable d’induire des anticorps cross-neutralisants, est un candidat de choix pour développer un vaccin protégeant contre l'ensemble des papillomavirus HR. Afin d’augmenter la réponse anti-L2, trois systèmes reposant sur l'interaction streptavidine/biotine ont été évalués pour exposer la protéine L2 à de multiples copies à la surface de VLP.Enfin, une stratégie reposant sur l’immunisation avec des PsV, composés des protéines L1 et L2 de l’HPV 58 contenant le gène de la protéine L2 de l’HPV 31, a également été évaluée. Les résultats obtenus indiquent que les PsV58-31L2 représentent la meilleur formulation de la protéine L2 et permet d’induire des anticorps neutralisants les HPV 16, 31,58 et surtout l'HPV 18, phylogénétiquement éloigné des valences vaccinales. / High risk human papillomaviruses (HR HPV) are the etiologic agents of cervical cancer. Current prophylactic HPV vaccines have been shown to protect against natural infection and against the development of high-grade lesions associated with HPV 16 and 18, both responsible of 70% of cervical cancer. However, these vaccines have not the potential to protect against all the thirteen other HR HPV. The aims of my PhD thesis were to evaluate the quadrivalent anti HPV vaccine and second generation vaccine capacity to induce cross-neutralizing antibodies against HR HPV. First, we have investigated the neutralizing antibodies against HPV 58 and HPV 31 after immunization in a population of 65 vaccinated HPV 31 and HPV 58 DNA-negative Colombian women. The results have shown that 33% and 24% of the immunized women exhibited cross neutralizing antibodies against HPV 31 and HPV 58 one month after vaccination, respectively. HPV 58 pseudovirions generated in a cellular system and used in this study exhibit a 10 fold higher capacity to transduce COS-7 cells than those of the HPV 31 PsV and thus represent an interesting candidate for DNA immunization application. The second and third parts of my work were to develop and analyze the immune response induced by second generation vaccine based on L2 protein. In fact, because L2 protein is able to induce cross-neutralizing antibodies, it represents a good alternative in order to develop broad-spectrum HPV vaccine. In order to enhance the anti-L2 immune response, three systems based on the streptavidin/biotin interaction have been evaluated in order to decorate HPV 16 VLPs with multiple L2 protein.Finally, we have produced HPV58 pseudovirions encoding the HPV31 L2 protein. Results obtained indicated that cross-neutralizing antibodies against HPV 16, 31, 58 and a more distant type HPV 18 are only obtained after immunization with pseudovirions encoding the L2 protein.

La mucoviscidose est une maladie héréditaire monogénique grave en rapport avec une mutation du gène codant pour la protéine CFTR et dont la morbidité est due principalement aux atteintes pulmonaires. La greffe pulmonaire développée depuis quelques années semble être la seule thérapie curative efficace. Toutefois, la rareté des greffons disponibles, justifie de développer d’autres thérapies comme la thérapie génique. Les pseudovirions de papillomavirus sont capables de transférer des gènes dans de nombreuses lignées cellulaires. Cependant, le tropisme naturel des papillomavirus est l’épithélium malpighien et ces pseudovirions sont peu efficaces pour les cellules de l’épithélium pulmonaire. Afin de modifier le tropisme de nos pseudocapsides de papillomavirus, la première étape de mon travail a été d’identifier de nouveaux motifs de ciblage pulmonaire. La technique de phage display a permis l’identification des motifs PHPNRAQ et VDRLQQK par sélection sur les cellules épithéliales bronchique IB3-1 et S9. / Cystic fibrosis is an autosomal recessive genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Lung transplantation developed in recent years is the only effective curative treatment. However, due to the limitation in organ donors, it is necessary to develop other therapies such as gene therapy. Papillomavirus pseudovirions have the potential to deliver genes into different cells. However, the natural tropism of HPV is directed to the cervical epithelial cells and these pseudovirions have low transduction efficacy in pulmonary epithelial cells. The purpose of this thesis was to retarget and optimize the HPV-16 pseudovirions tropism to airway cells by insertion of short amino acid sequences within the major capsid protein L1.

The L1 and L2 capsid proteins of papillomaviruses are characterized by the ability to self- assemble into viral capsids, which can be divided into pseudovirions (PsVs) and virus-like particles (VLPs) by inner content. In addition to the fact that such particles can serve as "nano-containers" for diagnostic and therapeutic agents, it has also been shown that papillomaviruses, whether wild, PsVs or VLPs have a higher affinity for tumor tissue than non-tumor tissue. This thesis deals with relatively newly discovered (2011) mouse papillomavirus (MusPV) and nanoparticles derived from this virus. This papillomavirus has been chosen for its positives, including easy preparation of VLPs and PsVs, as well as an available model organism for possible testing. Furthermore, MusPV has the potential for use in gene therapy and cancer diagnosis, because there is no immune response in the human population. The aim of this diploma thesis is to prepare an expression system for the production of PsVs and VLPs. In additional it will also look at the quality and quantity of PsVs and VLPs, characterization of these particles and verification of existing postulates regarding higher affinity of papillomaviruses for tumor cells. Finally, it will also to verify whether the same effect is observed in MusPV. In the results of...

BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...

BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...

Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...