Cover Page. The handle holds various files of this Leiden University dissertation

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1 Cover Page The handle holds various files of this Leiden University dissertation Author: Soema, Peter Title: Formulation of influenza T cell peptides : in search of a universal influenza vaccine Issue Date:

2 Chapter 1 General introduction and thesis outline 7

3 chapter 1 introduction Influenza The influenza virus is a negative-stranded ssrna virus of the Orthomyxoviridae family. Influenza viruses are able to infect numerous species, which include humans, birds, pigs, dogs and horses. In humans, influenza viruses infects via the respiratory tract. Symptoms of influenza infection include coughing, rhinitis, headache, fever, chills, muscle pain and fatigue. Severe cases of influenza infections may lead to primary viral pneumonia, secondary bacterial pneumonia and sinus infections, which are potentially lethal. Influenza epidemics occur annually in the northern (October through May) and southern (May through October) hemispheres. Estimations of the World Health Organization (WHO) indicate that annually, around 3 to 5 million influenza cases lead to severe illness and 0.5 million cases lead to influenza-associated death. Furthermore, each influenza season has considerable financial consequences, from hospitalization and treatment costs, to costs involved with sick personnel. Thus, for both health and economic reasons, it is essential that influenza infections are prevented. Influenza vaccination Vaccination is the only way to prevent influenza infections. Today, influenza vaccines are adapted each season to match circulating strains as predicted by worldwide epidemiological monitoring. These vaccines elicit antibodies specific to the main surface proteins of influenza, hemagglutinin and neuraminidase (Figure 1). However, these proteins are highly variable due to random genetic mutations (antigen drift) or genetic reassortment (antigenic shift). Antigenic drift is a continuous process that leads to small antigenic changes within an influenza subtype. However, antigenic shift is a complete genetic reassortment of the influenza subtypes, and vaccine-elicited immune responses are generally unable to prevent infection of influenza viruses that do not match with the vaccine strain. The greatest concern currently is the emergence of a pandemic influenza strain, which is created by recombination of influenza strains hosted by different species. Since current influenza vaccines lack cross reactivity, a pandemic can only be managed by antiviral drugs. While these antivirals are effective, increasing numbers of resistant influenza virus strains, including prepandemic H1N1, have been reported since 2007, casting serious doubt on the effectiveness of these antivirals during future influenza outbreaks. Increasing the cross reactivity of influenza vaccines is therefore an important goal in influenza vaccine development. T cell-based influenza vaccines To address the lack of cross reactivity of current influenza vaccines, several novel vaccine approaches are currently being pursued. One of these approaches is the induction of influenza-specific T cells that recognize conserved epitopes located on internal proteins. Such T cells have the potential to recognize any influenza virus, regardless of antigenic shift or drift. Peptide antigens derived from influenza proteins can be used to induce such T cells. However, peptides are poorly immunogenic. Multiple factors contribute to this poor immunogenicity: peptides are easily degraded, they are not adequately recognized and taken up by antigen presenting cells (APCs), and lack immunostimulatory 8

4 General introduction and thesis outline signals to activate APCs. Additional formulation of peptide antigens is thus needed to increase the immunogenicity of the peptides and to make an effective T cell-based influenza vaccine. In this thesis, several formulation strategies for peptide-based influenza vaccines were investigated. A B Hemagglutinin Matrix proteins Neuraminidase Viral RNA Figure 1. Schematic drawing of an influenza virus particle (A), and a cryogenic transmission electron microscopic image of inactivated influenza virus particles (B). 9

5 chapter 1 Thesis scope and outline The main objective of this thesis is to investigate and develop novel formulations for peptide-based influenza vaccines that ultimately could be used as universal influenza vaccines. Several formulation approaches are evaluated in this thesis. Furthermore, the current landscape of influenza vaccine research in general is assessed and the feasibility of T cell-based influenza vaccines in particular is reviewed. In Chapter 2 the status of current and future influenza vaccines is reviewed. The limitations of current seasonal vaccines and possible solutions to these limitations are addressed. Immunological and formulation aspects play important roles in these solutions, and are highlighted in this review. Finally, the production of current influenza vaccines and the production feasibility of future influenza vaccines are discussed. In Chapter 3 the development of peptide-loaded virosomes, a T cell-based influenza vaccine, is described. Peptide-loaded virosomes were produced from whole inactivated influenza virus (WIV) and synthetic T cell peptides. Physicochemical characteristics such as peptide loading efficiency, protein content of virosomes and membrane fusion capacity of the peptide-loaded virosomes were studied. The immunogenicity and protection against virus challenge of peptide-loaded virosomes with or without adjuvant was evaluated in HLA-A2.1 transgenic mice. In addition, the ability of virosomes to deliver peptides was assessed in a human dendritic cell (DC) model and in mice. Chapter 4 presents the use of WIV as an adjuvant for influenza T cell peptides. The adjuvant effect of WIV in combination with a T cell peptide was investigated in HLA-A2.1 transgenic mice in a proofof-principle study. Subsequently, a dose response study was carried out by varying both WIV and peptide doses. A DoE approach was used to identify potential synergistic effects between WIV and peptide doses. Moreover, the effect of WIV and peptide co-localization and membrane fusogenicity of WIV on the immunogenicity was investigated in the same transgenic mice. Finally, WIV was used as an adjuvant for mixtures of either three wild type influenza peptides or three chemically modified influenza peptides. A novel method to develop optimized liposomal formulations for peptide antigens is described in Chapter 5. Using a DoE approach, the influence of liposomal lipid composition on liposomal size, surface charge and liposome-induced DC maturation was investigated. Four different lipids were varied during the formulation of liposomes. The liposomes were tested in a human DC maturation model and the influence of the liposomal lipid composition on the expression of DC maturation factors was modeled. Finally, the accuracy of the generated prediction model was evaluated. In Chapter 6, the latest developments in T cell-based influenza vaccine research are reviewed. The immunological steps of T cell activation are presented and an overview of T cell-based vaccines in both preclinical and clinical development is given. Furthermore, the need for cellular correlates of 10

6 General introduction and thesis outline protection for influenza vaccines and the risks and limitations associated with T cell-based influenza vaccines are discussed. An alternative delivery system for influenza vaccines, the Bioneedle, is presented in Chapter 7. Bioneedles were filled with either subunit, split, virosomal or WIV influenza antigens, stabilized with trehalose. Antigen-filled Bioneedles were implanted in C57BL/6 mice and influenza-specific humoral and cellular responses were evaluated and compared to those induced by intramuscular or subcutaneous administered influenza vaccines. In addition, the thermostability of vaccine-filled Bioneedles and conventional liquid vaccines were assessed at temperatures up to 60 C. Chapter 8 summarizes the findings and conclusions of this thesis. The prospect of T cell-based influenza vaccines and alternative delivery systems for influenza vaccines are discussed. 11

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