• No results found

The next two chapters of this thesis [Chapter 2 and 3] focus on the use of rSFV for immunization and the mechanism of immune response induction by this vector. Chapter 2 describes the construction and characterization of rSFV encoding the early proteins E6 and E7 of HPV16 followed by immunization and tumor challenge studies with this rSFVE6E7 virus in mice. The induction of CTL responses against HPV16 E6 and E7 in mice by immunization with rSFVE6E7 turned out to be quite effective. In mice immunized with rSFVE6E7 particles, 40% was protected from tumor challenge with tumor cells expressing the HPV16 antigens E6 and E7. Next, the mechanism of rSFV-mediated induction of CTL responses was investigated [Chapter 3]. Infection studies with murine and human DC revealed that direct infection of DC is very inefficient.

Further infection and immunization experiments were performed using three different rSFV constructs encoding the influenza virus nucleoprotein (NP) as a model antigen: a standard NP construct, a construct encoding enhanced NP resulting in higher production of the protein and a construct encoding an unstable NP variant. The induction of CTL activity against NP was most efficient in mice that received the rSFV construct with enhanced NP. Mice that received a construct encoding an unstable variant of NP had the lowest levels of NP-positive T cells. These results point towards a mechanism of cross-priming for rSFV-mediated CTL induction.

The second part of this thesis [Chapter 4 to 6] focuses on induction of immune responses against protein antigens delivered by influenza virosomes. First, we studied delivery of the model antigen ovalbumin (OVA) to antigen-presenting cells, especially dendritic cells (DC) in vitro [Chapter 4]. Murine DC were incubated with fusion-active or fusion-inactivated OVA virosomes and MHC class I and II presentation of OVA peptides by DC was monitored. OVA virosomes were capable of inducing MHC class I and II presentation of OVA peptide at very low concentrations of antigen. The induction of MHC class I presentation of OVA peptides was dependent on the fusion-activity of the virosomes, in contrast to the induction of MHC class II presentation of OVA peptide.

Next, the immune response to OVA virosomes was studied in mice [Chapter 5].

Immunization with as little as 0.75 µg of virosomal OVA turned out to be sufficient for induction of a powerful CTL response. All of the immunization routes tested were effective, intramuscular and intraperitoneal immunizations being slightly superior to subcutaneous injection. Finally, immunization and tumor challenge studies were performed in mice with influenza virosomes containing the HPV16 early protein E7 [Chapter 6]. For these experiments E7 was produced in E. coli and purified. Influenza virosomes containing the purified E7 protein were injected into mice and CTL and antibody responses to the protein were measured. Immunization with E7 virosomes resulted in strong HPV16 E7-specific CTL responses. In addition, most mice immunized with E7 virosomes mounted an E7-specific IgG response. The induced E7-specific CTL

were capable of protecting 70% of mice against a tumor challenge with HPV16 E6 and E7-expressing tumor cells.

In Chapter 7 the two immunization strategies are compared and their potential application for therapeutic vaccination against CIN lesions and cervical cancer is discussed. Chapter 8 contains a summary of this thesis.


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