Influenza is an important respiratory pathogen that infects several million people each year. Currently available flu vaccines have to be updated regularly in order to be effective as the virus changes its composition by antigenic drift and shift. Most of the antibody response generated by these vaccines is strain specific as it is directed against the head domain (HA1) of HA. The HA2 subunit of hemagglutinin is highly conserved and immunogens designed from this subunit are likely to provide protection against multiple strains of the virus. However, expression of HA2 alone in the absence of HA1 resulted in a protein that took up the low pH conformation of HA. Our goal was to design immunogens from HA2 that would fold into the neutral pH form. Sequence analysis of a large number of HA protein sequences was carried out to identify conserved and exposed regions on HA. Several peptide and protein constructs were designed from the stem region of HA. These proteins were expressed in bacteria and purified proteins were used to immunize mice. Immunized mice were challenged with a lethal dose of virus to test for efficacy of the immunogen. Using this approach, stem domain constructs of HA were successfully designed and shown to take up the neutral pH form. These immunogens were also shown to be capable of providing broad range protection. Residues involved in the low pH induced conformational change of HA were identified from studies on HA2 derived peptides.

Die Fusion von Membranen erfordert die Verschmelzung von zwei Phospholipiddoppel-schichten, wobei dies über dieselben Zwischenschritte abzulaufen scheint. Eine lokale Störung (‚Stalk’) stellt eine erste Verbindung der äußeren Membranhälften dar, die anschließend lateral expandiert und ein Hemifusionsdiaphragma (HD) bildet. Das Öffnen einer Fusionspore im HD führt zur vollständigen Fusion. Mittels konfokaler Mikroskopie wurde die Fusion von Giant unilamellar vesicles (GUVs) mit negativ geladenen Lipiden und transmembranen (TM) Peptiden in Anwesenheit von zweiwertigen Kationen beobachtet, wobei die Peptide bei der HD Entstehung völlig verdrängt wurden. Eine detaillierte Analyse zeigte, dass es sich bei diesem Mikrometer-großen Bereich um ein HD handelt, dessen Größe von der Lipidzusammensetzung und Peptidkonzentration in den GUVs abhängt. Laterale Lipiddomänen gelten als entscheidend für Signal- und Sortierungsprozesse in der Zelle. Liquid ordered (Lo) Domänen in Modellsystemen wie GUVs ähneln den mit Sphingo-lipiden und Cholesterol angereicherten biologischen Raft-Domänen, allerdings scheinen Membraneigenschaften wie die Lipidpackung sich von biologischen Membranen zu unterscheiden. In diesem Zusammenhang wird die Sortierung des TM-verankerten Hemag-glutinin (HA) des Influenzavirus und von lipidverankerten Ras-Proteinen in GUVs wie auch in abgelösten Plasmamembran-Ausstülpungen (GPMVs) untersucht. HA Protein und TM-Pepitde von HA wurden ausschließlich (GUVs) bzw. vorwiegend (GPMVs) in der liquid disordered (Ld) Domäne gefunden. K-Ras wurde inmitten der Ld detektiert, während N-Ras zur Lo/Ld Grenzlinie diffundierte. Diese Ergebnisse werden im Zusammenhang mit den Unterschieden der Lipidpackung innerhalb der verschiedenen membranverankerten Systeme diskutiert. Es ist wahrscheinlich, dass die Bildung, Größe und Stabilität sowie die physikalischen Eigenschaften der Lipiddomänen in biologischen Membranen stark von Protein-Lipid-Wechsel-wirkungen beeinflusst werden. / Membrane fusion is ubiquitous in life and requires remodelling of two phospholipid bilayers. Fusion likely proceeds through similar sequential intermediates. A stalk between the contacting leaflets forms and radially expands into a hemifusion diaphragm (HD) wherein finally a fusion pore opens up. Direct experimental verification of this key structure is difficult due to its transient nature. Confocal microscopy was used to visualize the fusion of giant unilamellar vesicles (GUVs) comprising negatively charged phosphatidylserine and fluorescent transmembrane (TM) entities in the presence of divalent cations. A complete displacement of TM peptides preceded full fusion. This is consistent with HD formation. Detailed analysis provided proof that the micrometer sized structures are in fact HDs. HD size is dependent on lipid composition and peptide concentration. Lateral lipid domain formation is believed to be essential for sorting and signalling processes in the cell. Liquid ordered (Lo) domains in model systems like GUVs resemble biological rafts enriched in sphingolipids and cholesterol, but their physical properties seem distinct from biological membranes as judged by e.g. lipid order and packing. In this context the sorting of TM anchored influenza virus hemagglutinin (HA) and different lipid anchored Ras proteins is studied in GUVs and giant plasma membrane derived vesicles (GPMVs). Authentic HA or the TM domain peptides were sorted exclusively (GUVs) or predominantly (GPMVs) to the liquid disordered (Ld) domains. Whereas K-Ras was found in the bulk Ld domains, N-Ras diffuses to the Lo/Ld interface. These results are discussed with respect to differences in lipid packing in the different membrane systems and regarding the membrane anchors and their hydrophobic matching. The results suggest that the formation, size and stability as well as the physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

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.

Influenza virus is a pleiomorphic human pathogen which causes self-limiting respiratory illness lasting one-two weeks in most individuals. However, in immunologically compromised individuals, influenza infection may lead to severe morbidity and fatality. Annual epidemics cause 250,000-500,000 deaths worldwide and remain a major public health threat. The virus has evolved mechanisms of antigenic ‘drift’ and ‘shift’ to evade the host immune response. Hence, current influenza vaccines need to be updated every few years. Moreover, the currently available inactivated/live attenuated vaccines entail virus culture in embryonated chicken eggs hindering rapid scale-up. The aforementioned limitations of the current vaccines has had debilitating effect when strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past, despite intensive monitoring. Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift, facilitating a potential pandemic outbreak. These concerns have expedited efforts towards developing a ‘universal’ flu vaccine. Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the mature, disulfide linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit while the stem domain predominantly comprises of the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH induced conformational rearrangement in the host-cell endosomes to adopt the virus-host membrane fusion competent state. The ‘antigenic sites’ on the immunodominant globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed neutralizing antibodies (nAbs). As opposed to the highly-variable head domain, the HA stem is conserved and targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes. Although several bnAbs bind to the conserved HA stem, focusing the immune response to this conserved, subdominant stem domain in presence of the variable head domain of HA has been challenging. Alternatively, mimicking the epitope of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogen capable of eliciting a broadly protective immune response has been difficult because of the metastable conformation of HA. Addressing the aforementioned challenges, we describe the design and characterization of novel influenza immunogens by HA protein minimization. Chapter 1 gives an overview of the influenza virus life cycle, and outlines the structural organization and function of viral proteins. The conventional vaccines that are currently used and their limitations are described in this chapter. Recent improvements in influenza vaccine production focusing on recombinant HA as an alternate solution are discussed. Painstaking efforts of several groups in the recent past has led to the isolation of bnAbs that recognize novel ‘antigenic signatures’ within the globular head and the HA stem domains. Attempts to focus the immune response to these ‘cross-protective’ epitopes are described. The design and characterization of trimeric HA stem-fragment immunogens from influenza A Group-1 viruses which mimic the native, pre-fusion conformation of HA are described in Chapter 2. We engineered ‘headless’ HA stem immunogens based on influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation sensitive stem-directed bnAbs such as CR6261, F10 and FI6v3 with high affinity (equilibrium dissociation constant [KD] of 10-50nM). The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both Group-1 (H1, H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD90) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens conferred robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous HA stem domain immunogens, the designed immunogens described here were purified from the soluble fraction in E.coli. These HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks. The detailed mechanism(s) by which our ‘headless’ stem immunogens provide protection need further investigation. The long central α-helices (LAH) located in the HA stem assemble together into a parallel, trimeric coiled-coil. Immunization with the wt-LAH (76-130 of HA2) derived synthetic peptide designed from an H3 subtype (H3N2 A/Hong Kong/1/68) and conjugated to keyhole limpet hemocyanin (KLH) was shown previously to elicit antibodies reactive in ELISA with multiple hemagglutinin subtypes and to confer protection against challenge with H3N2, H1N1 and H5N1 virus strains. The LAH peptide sequence was chosen based on maximal binding to the monoclonal antibody (MAb), 12D1, which has broad neutralizing activity against influenza viruses of the H3 subtype. These results motivated us to rationally design stabilized derivatives of wt-LAH and test their protective capacity in a mouse challenge model of influenza. This work is described in Chapter 3. Additionally, to understand the contribution towards protection conferred by the two distinct surface exposed patches on LAH, we designed constructs spanning different stretches of LAH. The biophysical characterization of the LAH-derived constructs indicates that most of them were well-folded. All these constructs were moderately immunogenic in mice but at best, conferred limited protection from lethal viral challenge. In contrast to previously reported results, our data suggests that the LAH in the absence of other regions of HA may require not only strong, but also specific adjuvantation to induce a robust and functional immune response in vivo. Chapter 4 describes an immunogen design (H1pHA9) based on the globular head domain of pandemic H1N1 HA which can be produced using a prokaryotic expression system. The HA-fragment, H1pHA9, stably refolds to mimic the conformation sensitive neutralizing epitopes in the globular head domain of HA. We have also successfully engineered the HA head domain to delineate the epitope of antibodies neutralizing the pandemic H1N1 virus using a yeast cell-surface display platform. In this direction, we report the isolation of a novel, neutralizing murine MAb, MA2077, against the pandemic H1N1 virus. The epitope of this MAb has been mapped onto the ‘Sa’ antigenic site. The ability of the head domain fragment, H1pHA9, which binds MA2077 with high affinity to elicit such neutralizing antibodies in vivo needs to be further explored. Structural analysis has shown that elements of the HA stem diverge between the two phylogenetic groups. Therefore, to mitigate the threat of circulating influenza A viruses from these distinct structural classes (H1 and H3 belonging to Groups 1 and 2 respectively), in lieu of a ‘universal’ vaccine, a combination of immunogens derived from both the groups is a practical alternative. In Chapter 5 we describe the design of stem-fragment immunogens from an influenza A Group-2 virus strain. We report the characterization of engineered ‘headless’ HA stem immunogens based on influenza A/Hong Kong/1/68 (H3N2) subtype. The designed immunogens were expressed in E.coli and purified from the soluble fraction with abundant yields (~15mg/lt). The HA stem-fragment immunogens could be concentrated to high concentrations without aggregation. While, H3HA10-IZ and H3HA10-Foldon, the trimeric derivatives of our parent construct (H3HA10) which were folded, conferred modest protection against a lethal homologous virus challenge in mice, there is considerable scope to improve our immunogen design. Analyzing the results from our previous work (Chapter 2), we speculate that structural elements at the N-terminus of A-helix are critical for helix initiation. We therefore extended the design to include residues from the start of the A-helix. We designed the extended stem immunogens from both H3 and H7 subtypes. The proteins were purified from the soluble fraction of the E.coli cell culture lysate. Preliminary studies suggest that extension of the A-helix has aided proper folding. These proteins need to be further characterized and evaluated in an animal model.