Influenza virus recombinant proteins
Inventors
Khurana, Surender • Golding, Hana
Assignees
Publication Number
US-9163068-B2
Publication Date
2015-10-20
Expiration Date
2030-11-02
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Abstract
The present invention includes influenza Hemagglutinin protein fragments that fold properly when expressed in bacteria.
Core Innovation
The invention provides isolated influenza Hemagglutinin protein fragments, specifically portions of the Hemagglutinin-1 (HA-1) domain, which fold properly when expressed in bacteria. These polypeptides comprise amino acid sequences corresponding to certain positions of influenza Hemagglutinin (e.g., positions 1-259, 1-320, or 1-330 of various SEQ ID NOS), and notably lack the Hemagglutinin-2 (HA-2) domain and the transmembrane domain. By expressing only the HA-1 domain without the HA-2 and transmembrane domains, the proteins achieve proper folding and functional activity despite being produced in prokaryotic systems.
The problem addressed is the longstanding difficulty in influenza vaccine production related to the requirement to generate vaccine active ingredients in chicken eggs and the inability to produce properly folded trimeric functional Hemagglutinin proteins in bacterial systems. Traditional vaccine production is time-consuming, dependent on egg supply, and has limitations in scalability. The invention overcomes these issues by producing properly folded HA-1 domain proteins in bacteria, which retain important functional properties such as receptor binding, hemagglutination, and recognition by neutralizing antibodies.
The invention demonstrates that bacterially expressed HA-1 domain polypeptides form structurally stable trimers and oligomers capable of binding to conformation-sensitive neutralizing antibodies, binding to sialic acid receptors, and agglutinating red blood cells. These polypeptides are immunogenic, eliciting potent neutralizing antibodies and protective immune responses in animal models (rabbits, ferrets). Additionally, the HA-1 proteins serve as reagents for potency assays such as single radial immunodiffusion (SRID). The approach applies broadly to multiple influenza virus strains, including pandemic H1N1, H5N1, H7N7, and H3N2.
Claims Coverage
The patent presents 18 inventive features mainly directed to compositions comprising bacterially expressed, unglycosylated influenza HA-1 polypeptides lacking HA-2 and transmembrane domains, physiological vaccine compositions, methods of inducing immune responses, and specific sequence or structural features associated with oligomerization and functionality.
Compositions of oligomeric unglycosylated HA-1 polypeptides lacking HA-2 and transmembrane domains that generate neutralizing antibodies
A composition comprising oligomers of unglycosylated polypeptides comprising at least a portion of an influenza HA-1 domain and lacking an HA-2 domain or both HA-2 and transmembrane domains, wherein administration induces neutralizing antibodies against influenza.
Physiological vaccine compositions including the oligomeric HA-1 polypeptides
A physiological vaccine composition comprising the aforementioned oligomeric HA-1 polypeptide compositions and a physiological excipient, optionally further comprising an adjuvant.
Methods of inducing immune response using vaccine compositions
A method for inducing an immune response against influenza Hemagglutinin in an animal by administering an effective amount of the physiological vaccine composition containing oligomeric HA-1 polypeptides.
Specific amino acid sequence features in HA-1 associated with oligomerization and functionality
Polypeptides wherein the HA-1 portion comprises amino acids isoleucine or leucine at position 3, cysteine at position 4, isoleucine, leucine, or valine at position 5 and glycine at position 6 of SEQ ID NO:2, and optionally include sequences at positions 1-259, 1-320 or 1-330 of various SEQ ID NOS.
Binding to conformation sensitive influenza neutralizing antibodies
Polypeptides comprising HA-1 sequences that bind to conformation sensitive influenza neutralizing antibodies.
Sequence identity requirements in HA-1 polypeptides
Polypeptides comprising HA-1 sequences that are at least 80% identical to corresponding positions 1-259 or 1-320 or 1-330 of SEQ ID NOS:1, 2, 3, 4, 5, 6, or 7, with respective coverage of these positions.
Substitutions in N-terminal amino acids affect oligomerization
Polypeptides comprising substitutions of amino acids corresponding to positions 2 to 7 of SEQ ID NO:2 into heterologous HA1 polypeptides from sequence groups specified, affecting oligomerization and function.
The claims focus on compositions and methods involving bacterially produced HA-1 polypeptides lacking HA-2 and transmembrane domains that form oligomers, induce neutralizing immune responses, and exhibit specific sequence features essential for oligomerization and biological function. The coverage extends to vaccine compositions and immunization methods using these polypeptides.
Stated Advantages
Bacterially expressed HA-1 domain polypeptides fold properly into functional trimers and oligomers, overcoming the need for egg-based vaccine production.
The proteins bind conformation-sensitive neutralizing antibodies and receptors, maintaining biological activity without post-translational glycosylation.
The approach enables rapid and economical large-scale production of influenza vaccines, reducing manufacturing timelines by several months.
The HA-1 polypeptides serve as reagents for potency assays such as SRID, improving vaccine lot release testing.
Vaccination with these polypeptides induces strong neutralizing antibody responses and provides protective immunity in animal models including ferrets and rabbits.
Documented Applications
Production of influenza vaccines using bacterially expressed HA-1 polypeptides that elicit protective immune responses against influenza infection.
Use of the HA-1 polypeptides as reagents in potency assays (e.g., SRID) for quantifying Hemagglutinin in vaccine lots.
Detection of influenza-specific antibodies in biological samples by assays using the bacterially expressed HA-1 proteins.
Immunization methods involving administration of compositions comprising the HA-1 polypeptides to animals to induce immune responses.
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