Influenzaviren müssen in die Wirtszelle aufgenommen werden, um dort ihr Genom freizusetzen und ihre Replikation mit Hilfe des Reproduktionsapparats der Zelle einzuleiten. Der komplexe Replikationszyklus der Influenza A Viren ist noch nicht vollständig verstanden. Er beginnt mit der Bindung des viralen Hämagglutinins (HA) an Sialinsäure (SA) auf der Zelloberfläche der Wirtszelle. In dieser Arbeit wurde die Virusbindung an Zellen mit unterschiedlicher Rezeptorkomposition verglichen. Dabei konnte gezeigt werden, dass für die zelluläre Spezifität die Präsentation des Rezeptors innerhalb der Plasmamembran der Zelle eine größere Rolle spielt als die Struktur des Rezeptorglykans selbst. Des Weiteren deuten die Beobachtung sehr kleiner Kräfte und ein stufenweises Öffnen von Bindungen auf eine multivalente Interaktion hin. Multivalenz wird oft in biologischen Bindungsprozessen beobachtet und kann Bindungskräfte enorm verstärken. Basierend auf diesen Ergebnissen wurden inhibitorische Nanopartikel entwickelt, die die natürliche Zelloberfläche als hochaffine Bindungsalternative imitieren. Verschiedenartige Nanopartikel wurden evaluiert und konnten die Virusaktivität um mehr als 80 % hemmen. Nach der Bindung wird das Virus durch Endozytose in die Zelle aufgenommen. Durch spezifische Virusmarkierung und gleichzeitiger Expression von zellulären Markerproteinen wurde der Transport einzelner Viren in lebenden Zellen verfolgt. Dabei konnte gezeigt werden, dass das Virus sowohl durch frühe, als auch durch späte Endosomen wandern muss, um sein Genom erfolgreich in das Zytoplasma zu entlassen. Außerdem verzögert das Virus die endosomale Ansäuerung um eine optimale Aufenthaltsdauer im Endosom und die lokalisierte Fusion in der Nähe des Zellkerns zu gewährleisten. Pharmakologisches Eingreifen in diese Prozesse konnte zudem weitere kritische Faktoren identifizieren, die die Effizienz der Virusinfektion stark beeinflussen. / Influenza virus must enter a host cell to deliver its genome, use the cells reproductive machinery and eventually initiate its replication. The replication cycle of influenza A virus is very complex and still not fully understood. It generally starts with binding of the viral protein hemagglutinin (HA) to its cellular receptor sialic acid (SA). In this work, virus-cell attachment forces were investigated at the single molecule level using intact virus binding to living cells, a set-up that closely mimics the in vivo situation. Cells of different surface SA composition were compared. It could be shown that the unique presentation of the ligand within the cells plasma membrane, rather than the structure of the receptor-glycan itself, strongly affects cellular specificity. The low binding forces as well as the observation of stepwise unbinding events suggest a multivalent interaction type. Based on this finding, inhibitory nanoparticles mimicking the cell surface were constructed. Different particles were evaluated and shown to efficiently inhibit virus infection by ≥ 80 %. Since many molecular details of multivalent interactions remain poorly understood parameters such as ligand spacing and presentation were varied and revealed that the density of ligands as well as the interacting surface plays critical roles for virus inhibition. Upon attachment, the virus enters the cell by endocytosis. Virus trafficking was followed at the single-virus level in living cells. The kinetics of virus transport were visualized using fluorescent marker proteins in combination with specific virus labeling. It was found that the virus needs to progress through early and late endosomal compartments in order to efficiently uncoat and release its genome. Further, the virus delays the endosomal acidification to ensure optimal residence time and fusion in the region close to the host cell nucleus. Drug treatment furthermore unraveled critical factors influencing viral infection efficiency.

Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.

The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened. The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected. We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic. We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay. Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants. We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.

The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Neutralising antibodies and antigen-specific B cells are important for protection against influenza A virus. However, the antigenic evolution of influenza A viruses has made a continuing challenge to the design of vaccine and the public health. The ability to generate cross-reactive response against influenza remains unclear in human. It is important to explore the antibody and B cell repertoire at single cell level. The pandemic H1N1 and seasonal influenza vaccine induced robust antibody response in adults. However, pre- or co-vaccination with the seasonal vaccine led to a significantly reduced antibody response to pandemic H1N1 virus. Whether this interference has impact on subsequent infection rates remains undetermined. There observed substantial cross-reactive antibody response upon vaccination, as measured by HI, MN and B cell ELISpot assays. The antibody recognizing conserved proteins could be the main component of cross-reactivity against influenza A strains and subtypes. A significant expansion of influenza-specific MBC was observed after infection. Crossreactive response was also noted in the MBC response. Importantly, a robust early-phase ASC response was detected in the peripheral blood upon influenza vaccination or infection. The size of ASC response significantly correlated with serum HI, MN and anti-HA IgG titre three weeks after vaccination. The sequence analysis revealed that early-phase ASC accumulated high level of somatic mutations on Ig variable region and affinity maturation, as well as anti-influenza mAb, which suggested their origin from pre-existing MBC. Eight anti-influenza mAb were made from early-phase ASC, including one high-titre virus-neutralising HA1-specific, two other HA1-specific, one cross-reactive HA2-specific, and four cross-reactive NP-specific antibodies, indicating of the broad diversity of ASC repertoire. In conclusion, this study demonstrated the properties of antibody and B cell responses to influenza A virus at serological, cellular and sequence level. The virus-neutralising and cross-reactive mAb derived from ASC could have therapeutic potential and their analysis might direct the vaccine design in the near future.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Juergen A. Richt / Influenza A virus is an important respiratory pathogen with the potential to affect both humans and animals, thereby creating the conditions for public health disasters, especially during pandemic episodes. At present, two primary strategies to combat influenza are vaccination and antiviral drugs. Since influenza viruses mutate rapidly and constantly via antigenic drift and shift, vaccines can become quickly outdated; and resistance to antiviral drugs can readily result. Interferon alpha (IFN-[alpha]) plays an important role as a first line of innate antiviral immunity. To investigate the antiviral potential of exogenously applied IFN-[alpha] on the replication of different subtypes of influenza A viruses, three subtypes of influenza A virus, i.e. swine H3N2, pandemic H1N1 and avian H9N2 were chosen. Their replication kinetics in the presence of Alferon N (human Interferon alpha) on human epithelium (A549) cells and swine testis (ST) cells was evaluated. In these tests of the three subtypes of influenza A viruses, it was found that the replication ability of all three viruses was inhibited when ST cells were treated with Alferon for four hours before infection. The ability of Alferon to inhibit influenza A viruses replication was found to be dose-dependent. Similar results were obtained when A549 cells were used; however, pretreatment of A549 cells with Alferon for more than 16 hours was necessary before infection. Furthermore, the expression of some ISGs (Interferon stimulated genes) between ST and A549 cells was also investigated. The differences in response of the ISGs between the two cell lines provided an explanation of the disparity towards exogenous interferon treatment. In summary, these results demonstrated that Alferon N has the ability to inhibit replication of different subtypes of influenza A viruses in cell cultures. This study provides a foundation for future in vivo studies using exogenous IFN-[alpha] treatment as an alternative approach to combat influenza A virus infection.