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.