SBIR-STTR Award

An influenza pandemic remains an acute threat to world health; and stockpiling a universally protective influenza vaccine provides a strong defense against this potential catastrophe. The hemagglutinin (HA) protein is the primary target of humoral Ab responses to Influenza. Vaccines based on full-length HA give rise predominantly to strain-specific Ab responses to the immunodominant, and highly variable head domain, and lack breadth of protection. However, the majority of the broadly neutralizing antibodies (bnAbs) that have been isolated to date target HA's highly conserved stalk domain, where they recognize trimer-specific quaternary neutralizing epitopes (QNEs). An HA immunogen from which the head domain has been removed is expected to focus immune responses on these conserved stalk QNEs, resulting in broadly protective Ab responses. But current "Headless HA" constructs do not fold correctly or display only part of the stalk's key QNE(s), and thus have only shown limited heterologous protection. Avatar has developed a method of locking protein immunogens in their native, quaternary conformation, in order to better present broadly protective QNEs. Targeted dityrosine (DT) crosslinks are engineered into fully folded, native proteins by (i) introducing conservative Tyr substitutions, and then (ii) catalyzing DT bond formation, in order to conformationally lock the proteins. DT bonds are catalyzed enzymatically, and only form between Tyr side-chains in very close structural proximity and - unlike engineered disulfide bonds - are introduced after the protein has fully folded. DT bonds are safe and irreversible, and because the bonds are zero-length, this approach fully preserves protein structure, and furthermore avoids aggregation because DT bonds do not form spontaneously. We will engineer DT bonds in the stalk of the intact HA trimer to lock it in its native conformatio. This will allow us to subsequently remove the head by proteolysis, while maintaining the stalk's native, trimeric structure, generating a conformationally locked "DT-Headless HA". To this end, we will introduce both Tyr substitutions at targeted positions into the stalk, and proteolytic cleavage sites into the head domain of the same HA molecule. Following crosslinking of the stalk, and proteolytic removal of the head, we will analyze the DT-locked stalk trimer antigenically, by comparison to uncrosslinked and wild-type controls (AIM 1). Then we will perform lethal viral challenge studies in mice, to confirm that conformationally locked DT-Headless HA elicits robust homologous protection, and improved heterospecific neutralization responses, compared to full- length WT HA (AIM 2) By focusing Ab responses on the conserved stalk QNEs, and away from the immunodominant head of HA, the DT-Headless HA immunogen is expected to give rise to broadly neutralizing Abs that protect from homologous and drift variants, as well as group 1 heterologous, and perhaps even group 2 heterologous challenges.

Public Health Relevance Statement:


Public Health Relevance:
Influenza pandemic outbreaks remain an acute threat to world health; and stockpiling a universally protective influenza vaccine would defend against such a potential catastrophe. We propose to apply our protein engineering approach to the design of a universal influenza vaccine immunogen that will protect against all strains of the influenza virus, including pandemic strains influenza, by specifically triggering the production of antibodies in vaccinated individuals that will bind to, and neutralize the virus when it enters the body.

Project Terms:
Acute; Antibody Formation; Antigens; base; Binding (Molecular Function); Biological Assay; C-terminal; Cleaved cell; crosslink; design; Disease Outbreaks; disulfide bond; dityrosine; Engineering; Ensure; Epitopes; Excision; Ferrets; Goals; Head; Hemagglutinin; Immune response; Immunodominant Epitopes; improved; Individual; Influenza; Influenza A Virus, H1N1 Subtype; influenza virus strain; Influenza virus vaccine; innovation; Length; manufacturing process; Marketing; Methods; Molecular Conformation; Mus; National Institute of Allergy and Infectious Disease; neutralizing antibody; pandemic disease; pandemic influenza; Peptide Hydrolases; Persons; Phase; Positioning Attribute; protein complex; Protein Engineering; protein structure; Proteins; Proteolysis; public health relevance; Reaction; Recombinants; research clinical testing; response; Rodent; safety study; scale up; Serum; Side; single molecule; Site; Structure; Subunit Vaccines; swine flu; Technology; Vaccinated; Vaccines; Variant; Viral; Virulent; Virus; World Health

An Influenza pandemic remains an acute threat to world health; and stockpiling a universally protective Influenza vaccine provides a strong defense against this potential catastrophe. The hemagglutinin (HA) protein is the primary target of humoral Ab responses to Influenza; the majority of broadly protective mAbs (bnAbs) against influenza isolated from humans recognize conserved and conformation-specific epitopes in the HA Stalk. But, strain-specific, immunodominant epitopes in the Head of HA overwhelm immune responses to the Stalk. An HA immunogen from which the Head domain has been removed will elicit anti-Stalk antibodies that protect broadly against seasonal, as well as pandemic, Influenza. Despite significant progress, it has so far not proved possible to design a stable Headless HA that assumes its fully native conformation and thereby elicits universally protective Ab responses. None of the most promising, recent designs have progressed to clinical development. Avatar has developed a strategy to produce a conformationally intact Headless HA that overcomes these limitations. This strategy involves first locking the structure of the conserved Stalk with target dityrosine (DT) crosslinks, so that it can no longer lose its native conformation. Then we remove the variable and immunodominant Head domain with a site-specific protease, using engineered recognition sites. This DT- Headless HA immunogen is in its fully native conformation, and responses to this more perfect immunogen will improve Ab titers and affinities to conserved epitopes, and thus protect broadly against all strains of Influenza. In Phase I, we successfully designed and characterized our DT-Headless HA (AI118087). In Phase II we will demonstrate heterologous protection in Balb/c mouse challenge studies, and confirm efficacy of this product in ferret challenge studies (Aims 1 & 2). We will also transfer our design into a Group II HA and test its heterologous protection in mouse and ferret lethal challenge studies (Aim 3 & 4). By inducing higher avidity, higher titer Ab responses to the conserved Stalk, Avatar's Headless HA immunogen will give rise to broad protection against homologous and drift variants, as well as Group 1 & 2 heterologous challenge. We will compare the results obtained with our Group I and II Headless HA immunogens and select a product candidate for preclinical and clinical development.

Public Health Relevance Statement:
Influenza pandemic outbreaks remain an acute threat to world health; and stockpiling a broadly protective influenza vaccine would defend against such a potential catastrophe. We propose to confirm that our highly innovative Influenza vaccine immunogen protects against all strains of the Influenza virus, including pandemic strains Influenza, by specifically triggering the production of antibodies in vaccinated individuals that bind to, and inactivate the virus when it enters the body.

Project Terms:
Acute; Affinity; Antibodies; Antibody Formation; Antigens; Avian Influenza; Avidity; Baculovirus Expression System; base; Binding; Cells; clinical development; Collaborations; crosslink; Dangerousness; design; Development; Disease Outbreaks; dityrosine; Engineering; Epitopes; Fatality rate; Ferrets; flu; Goals; Head; Hemagglutinin; Human; Immune response; Immunize; Immunodominant Epitopes; improved; Inbred BALB C Mice; Individual; Influenza; influenza virus strain; Influenza virus vaccine; innovation; interest; Lead; Molecular Conformation; Monoclonal Antibodies; Mus; pandemic disease; pandemic influenza; pathogen; Peptide Hydrolases; Persons; Phase; Population; preclinical development; prevent; Process; Production; Proteins; Proteolysis; Reaction; Research; response; Rodent; scale up; Science; Site; Structure; swine flu; System; Testing; tool; Toxicology; universal influenza vaccine; Vaccinated; Variant; Virus; World Health