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«DISSERTATION INFLUENZA B NS1 TRUNCATION MUTANTS: A LIVE ATTENUATED VACCINE APPROACH Doktor/in der Naturwissenschaften (Dr. rer.nat.) Verfasserin / ...»

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Doktor/in der Naturwissenschaften (Dr. rer.nat.)

Verfasserin / Verfasser: Nina Wressnigg

Matrikel-Nummer: 9702618

Dissertationsgebiet (lt. Mikrobiologie/Genetik


Betreuerin / Betreuer: Dr. Thomas Muster Wien, am 16. Dezember 2008

1. Summary The aim of the thesis is to investigate the potential use of mutant replication deficient influenza B viruses with impaired interferon antagonistic function as live attenuated vaccines.

We generated several influenza B viruses containing either carboxyterminal truncated NS1 proteins of different length or completely lacking the NS1 ORF (∆NS1-B) employing reverse genetics on Vero cells. Due to a unique, single amino acid mutation M86V in the influenza B M1 protein viral growth in Vero cells was increased, enabling the rescue of a ∆NS1-B virus growing to titers of 8 logs. All viruses showed restricted growth in human alveolar epithelial cells (A549) and in 6 day old human macrophages. The attenuated phenotype of the viruses was associated with induction of antiviral (IFN-α) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) early after infection. All vaccine candidates were replication deficient, did not provoke any clinical symptoms and induced neutralizing antibody response in mice and ferrets. Complete protection against homologous challenge with wild-type virus was accomplished after a single intranasal immunization.

So far, the lack of a ∆NS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the major interferon antagonist. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-defective ∆NS1 intranasal influenza vaccine.

2. Acknowledgments Above all, this work owes a lot to my advisors Thomas Muster and Christian Kittel. I thank Thomas Muster for his guidance and financial support. I thank Christian Kittel for his creative thoughts, for his commitment and most of all for his continuous efforts to push me to finish my thesis. I thank Dmitrij Zamarin for advising me in New York.

Furthermore I would like to thank many current or former colleagues at Greenhills Biotechnology: especially Julia Romanova and Andrej Egorov for sharing their expertise with me and for their advice, Daniela Ribarits for technical support and interesting conversations during teatime. I thank for helpful impulses and discussions Sabine Nakowitsch, Brigitte Krenn and Bettina Kiefmann. I want to thank Helena Seper and Johannes Humer for being close friends since the first day of my thesis, for their advice and help.

I also want to thank other friends, many of whom I met during my studies, among them Anna Gieras, Andrea Hoelbl and Klaus Orlinger.

Countless hours of conversations, speed kings and discussions were highly appreciated and made this time so much easier. And I thank Raphael Auer for his support in all circumstances, his tolerance and patience.

Last but not least, I am very grateful to my family. I want to thank my mother Ingrid, my father Franz and my brother Thomas for their unconditional support and understanding.

3. Table of contents






4.1.1. Structure

4.1.2. Replication

4.1.3. Structure and function of the NS1 protein

4.1.4. NS1 combating the innate immune system

4.1.5. NS1 circumventing the adaptive immune system


4.2.1. RNP-transfection

4.2.2. Plasmid-only transfection


4.3.1. Inactivated vaccine (TIV)

4.3.2. Live vaccines Live cold-adapted vaccine (LAIV) Live recombinant vaccine NS1 live-attenuated vaccine

4.3.3. DNA vaccination







6.3.1. Cells, viruses and viral infections

6.3.2. Generation of NS1-truncated viruses

6.3.3. Isolation, generation and infection of immature monocyte-derived macrophages

6.3.4. Cytokine measurement in cell-culture supernatants

6.3.5. Immunization and challenge of mice

6.3.6. Influenza-specific IgG ELISA








7.3.1. Cells, viruses and viral infections

7.3.2. Generation of NS1-truncated viruses

7.3.3. Production of influenza virus in Vero cell culture-based micro-carrier fermenter

7.3.4. Immunization and challenge of ferrets

7.3.5. Hemagglutination inhibition (HAI) assay






4. Introduction

4.1. Influenza virus 4.1.1. Structure Influenza viruses belong to the virus family of Orthomyxoviridae, containing eight single-stranded genes in negative polarity. Three types of influenza virus (A, B and C) occur in nature, whereas only influenza virus A and B undergo antigenic drift (A and B) and antigenic shift (only A), defining the epidemiologic and pandemic nature of these viruses and causing significant morbidity and mortality in humans. In contrast to influenza A virus and influenza B virus, as recently discovered, influenza C virus lacks an animal reservoir and is therefore not of major interest concerning outbreaks in humans (Lamb, 1989, Osterhaus et al., 2000, Palese, 2007).

All influenza viruses’ posses a common structure (Fig.1). The virus is surrounded by a host derived lipid membrane in which the two viral surface glycoproteins haemaglutinin (HA) and neuraminidase (NA), and the ion channel protein (M2) are embedded. The matrix protein 1 (M1) underlies the lipid envelope forming a protein layer. In the core, each RNA segment is encapsulated by the nucleoprotein (NP) and the three viral encoded polymerase proteins (PB1, PB2 and PA) forming the ribonucleoprotein complex (RNP complex). These RNA segments of influenza A and B possess highly conserved 5’ and 3’ complementary non coding regions (NCR) of 16 & 15nt in length for influenza A and 10 & 10nt for influenza B viruses, respectively. The NCR are hybridizing with each other, forming a “panhandle” structure (Baudin et al., 1994, Flick & Hobom, 1999). At the same time they are recognized by the viral polymerase complex serving as a promoter controlling the transcription of vRNA. The RNA genome of Influenza A and B virus consists of eight genes coding for 11 proteins due to different open reading frames (ORF) (Compans RW, 1974). Whereas influenza A virus is coding for an additional protein termed PB1-F2, influenza B virus differs from A in expressing an NB protein from the NA ORF (Table 1) (Betakova et al., 1996, Chen et al., 2001, Imai et al., 2004, Imai et al., 2003). The nuclear export protein (NEP/NS2), formerly believed to be non structural, is also located in the virus particle in a very low copy number (Lamb & Choppin, 1979, Richardson & Akkina, 1991). The small non structural protein 1 (NS1) is only expressed in infected cells and is not being packaged into the virion.

Fig. 1: Schematic drawing of the influenza virus structure

Influenza B viruses are mostly indistinguishable from the A viruses by electron microscopy. In contrast to influenza A virus they have four viral proteins inserted in their lipid membrane, the HA, NA, NB and BM2, whereas the M1 and the RNP complexes make up the interior of the particle.

Table 1: Illustration of gene segments of Influenza A and B (Lamb, 2001)

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Influenza virus binds to N-acetyl-neuraminic acid (sialic acid) containing receptors on the host cell surface to initiate viral infection and replication via it’s HA protein as illustrated in Fig. 3 (Palese, 2007). Upon adsorption to the receptor the viral particle enters the cell by receptor mediated endocytosis and is internalized into endosomes. Due to the acidic environment in the endosomes, the HA protein undergoes a conformational change, leading to fusion of the viral and cellular membrane (Stegmann, 2000). The viral compartment also gets acidified by M2 protein acting as an ion channel resulting in uncoating (Pinto et al., 1992). This uncoating event leads to the dissociation of the M1 protein from the RNP complex and successively the release of the viral genome (in form of vRNPs) into the cytoplasm. The vRNPs, mediated by the nuclear localisation sequences (NLS) of the NP and polymerase proteins, are then imported into the nucleus for replication and transcription by the virus’ own RNA-dependent-RNA-polymerase. Replication occurs later in infection where vRNA is also transcribed into full length cRNA copies from which more copies of vRNA are made to be later on packaged into new virions. During transcription the subunit of PB2 recognizes and binds m7Gpppm-CAP structures of newly synthesized cellular mRNA. “CAP – snatching” occurs, where the endonuclease activity of PB1 cuts off the CAP and attaches it to the 3’ end of vRNA initiating transcription via the 3’ OH end of the CAP structure. The viral mRNA acquires the missing polyadenylation tail by the PB1 subunit which elongates the nucleotides until a stretch of five to seven uridines at the 5’ end of the vRNA. The RNA polymerase stutters at this uridine stretch and thereby adding the poly (A) tail. Positive sense viral mRNAs are transported out of the nucleus into the cytoplasm for protein synthesis by the ribosomes. After translation the membrane proteins (HA, NA and M2) are transported through the rough endoplasmatic reticulum (rER) and the golgi apparatus to the cellular membrane. During transport different post translational modifications like N-glycolsylation or palmitoylisation occur. The neuraminic acid residues of NA are deleted in order to prevent premature binding to HA.

Due to their NLS the other remaining proteins (PB2, PB1, PA, NP, M1, NS1 and NEP) are imported back into the nucleus to assist in viral replication and vRNP assembly. During late stages of infection, newly synthesized vRNPs are transported to the cytoplasm due to the nuclear export protein (NEP) interacting with the cellular nuclear export machinery. The BM2 protein of influenza B virus is synthesized in the late phase of infection and then incorporated into the virions as a subviral component and plays a critical role in production of infectious virus (Imai et al., 2008, Odagiri et al., 1999). Finally, progeny viruses assemble at the apical surface of the cell and lipid rafts and bud from the host cell plasma membrane.

Fig. 2: Influenza virus replication cycle

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The eleventh gene of the influenza virus, the NS1 protein, is a non structural protein, which is synthesized in the infected cell but is not incorporated into the progeny virus. NS1 is a dimeric, multifunctional protein expressed by all influenza strains. In the case of influenza A virus NS1-A contains 890nt and NS1-B 1096nt coding for two mRNAs (Lamb & Choppin, 1979, Lamb et al., 1980). The first one encoding the NS1 protein of 230 amino acids (aa) or 281aa in length for influenza B viruses and the other mRNA derived by splicing of the NS1 mRNA, encoding the nuclear export protein (NEP/NS2) of 121aa in length for influenza A or 122aa for influenza B viruses, respectively. NEP plays a major role in exporting the newly synthesized vRNPs out of the nucleus. Unspliced NS1 mRNA is exported very efficiently out of the nucleus, ensuring presence of NS1 protein already at early stages of infection and thereby counteracting the onset of the host’s antiviral immune response and ensuring prolongation of the viral replication cycle (Alonso-Caplen et al., 1992).

NS1 is a 26kDa protein involved in both protein-RNA as well as proteinprotein interactions. Each dimer consists of an N-terminal RNA-binding domain (RBD) and a C-terminal effector domain (ED) as shown in Fig. 3 (Palese, 2007). Due to its NLS in each of the two domains NS1 protein localizes mainly in the nucleus (Greenspan et al., 1988). In the cytoplasm the NS1 protein is found as a result of the NES (nuclear export signal) in the ED (Li et al., 1998). Although there is only 20% sequence homology between NS1-A and NS1-B protein, the function to counteract the hosts own immune system and modulate and regulate the immune response at several stages seems to be alike.

Fig. 3: Binding sites of cellular proteins on NS1 domains of Influenza A and B viruses One of the functions of the RBD domain of NS1-A is to prevent activation of the cellular Ser/Thr protein kinase R (PKR) (Bergmann et al., 2000, Hatada et al., 1999, Krug et al., 2003). The RBD of NS1-A sequesters dsRNA present in the nucleus away from PKR and/or directly binds to PKR by its ED and therefore prevents its autophosphorylation and activation (Li et al., 2006, Min et al., 2007). Activated PKR induces phosphorylation of its substrate the eucaryontic translation-inhibition factor (eIF-2α) leading to a blockage in cellular protein synthesis which would also affect viral protein synthesis in infected cells leading to inhibition of viral replication (Garcia et al., 2006). The RBD of NS1-B is also able to bind dsRNA and suffices to inhibit PKR activation as demonstrated in vitro (Dauber et al., 2006).

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