Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Utility Patent
1997-08-06
2001-01-02
Park, Hankyel (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S009340, C424S209100, C435S069300, C435S252330, C435S325000, C435S320100
Utility Patent
active
06169175
ABSTRACT:
BACKGROUND OF THE INVENTION
The prior art illustrates the current strategy for control of influenza by yearly vaccination with whole-virus or subunit vaccines. The currently-licensed vaccines are designed to stimulate neutralizing antibodies against hemagglutinin (HA) and/or neuraminidase (NA), the major surface antigens of the influenza virus. However, due to frequent and unpredictable structural variation of HA and NA, influenza vaccines must be seasonally customized to circulating virus strains, a process which is deficient in providing protective immunity against all but closely matched viral strains.
There is a need for a vaccine subunit component capable of inducing broader, more cross-reactive immunity to type A influenza viruses. One such component may be M2, a structurally conserved influenza A viral surface protein (Slepushkin et al., 1995; to et al., 1991). Antibody to M2 has been shown to restrict influenza virus replication in cell culture and in infected mice (Zebedee and Lamb (1988) and Treanor et al., (1990). Full length M2 expressed in baculovirus has been shown to raise serum titers and stimulate T-cell responses in immunized animals (Katz, et al, 1996). Further, vaccination of mice with recombinant full-length M2 has been shown to enhance viral clearance from infected lungs and to provide protection from lethal challenge with heterologous influenza A virus (Slepushkin et al., 1995).
Since M2 is not expressed to any extent in virions (Zebedee & Lamb, 1988), the current whole virus or split-product influenza vaccine contains only minimal amounts of M2. To be useful as a component of a vaccine, M2 must be expressed and purified as a recombinant product. However, expression of full-length M2 has been shown to be detrimental to cell culture in prokaryotic and eukaryotic expression systems (Guinea and Carrasco, 1996; Black et al., 1993). To date, expression of sufficient quantities of recombinant M2 for use in experimental studies can only be accomplished by culturing eukaryotic host cells in the presence of the irreversible M2 inhibitor, amantadine.
Wholly apart from the challenges in expression of recombinant M2, the hydrophobic nature of full-length M2 compromises the yield and purity of M2 preparations and necessitates the use of detergents or other agents to maintain M2 in a soluble form. Certain such solubilizing agents are not desirable constituents of vaccine formulations. The present invention solves this shortcoming in the prior art by providing a variant M2 protein with reduced hydrophobicity and concomitantly enhanced solubility characteristics relative to full-length M2.
SUMMARY OF THE INVENTION
The present invention solves the problems of the prior art approaches to recombinant M2 production by providing new recombinant forms of M2 whose structure has been modified to allow simple prokaryotic expression as a soluble, readily purified variant protein which retains antigenic and immunogenic properties. A preferred embodiment of the present invention involves a recombinant construct in which the entire or a significant portion of the transmembrane domain has been deleted. Alternatively, residues within the transmembrane domain may simply be altered, for example by substitution of hydrophilic or neutral amino acid residues for hydrophobic residues in such a way as to inactivate the ion channel activity of the variant M2 polypeptide. Additional hydrophobic residues may also be deleted, resulting in a less hydrophobic molecule which can be readily solubilized and efficiently purified. The present invention further relates to vaccines comprised of these new recombinant forms of M2, and to methods of prevention and treatment of influenza A virus infections.
Other objects, features and advantages of the present invention will become apparent more fully by the following description.
Further, the present invention can more fully be understood from the following description in conjunction with the accompanying drawings in which:
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Frace A. Michael
Katz Jacqueline M.
Klimov Alexander I.
Centers for Disease Control and Prevention
McDonnell & Boehnen Hulbert & Berghoff
Park Hankyel
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