M2e in Human and Avian Influenza

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M2e in Human and Avian Influenza

• The M2 protein is a tetrameric, type III
  transmembrane protein
• Relatively scarce on the viral surface but is
  abundant on the surface of virus-infected host
  cells
• The M2 protein is 97 amino acids long with 24
  amino
• acids at the N-terminus exposed outside the
  membrane surface (M2e), 54 amino acids at the
  C-terminus located on the cytoplasmic side of the
  membrane and 19 amino acids spanning the lipid
  bilayer.

Amino acid sequence of M2e (Liu, Li et al. 2003)
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The M2e sequence is highly conserved in human
(one type) and avian (three types) influenza
viruses. M2e is thus able to induce and confer
broad spectrum cross protection to diverse
strains of influenza encountered over several
years. We will deliver two critical M2e peptide
sequences (one human and one avian) fused to HBV
and WHV cores delivered by Salmonella vaccines
that lyse in vivo.
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We have engineered Salmonella to first escape a
membrane-bound state (the endosome) in infected
host cells and then to undergo lysis. This
sequence of events is highly regulated to occur
sequentially. Lysis is due to the inability to
synthesize the rigid layer of the bacterial cell
wall. This is accomplished by an induced
inability to synthesize two essential cell wall
constituents, diaminopimelic acid (DAP) and
muramic acid. Lysis results in liberation of
protective influenza antigens into the cell
cytoplasm and circular plasmid DNA vaccines that
cause the host to synthesize the hemagglutinin
protective antigen.
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DAP (meso-diaminopimelic acid) and
N-acetylmuramic acid are essential unique
constituents of the rigid layer of the bacterial
cell wall.
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DAP-less and muramic acid-less death in host
strain with DNA vaccine vectors
?8888
?8888(pYA4050)
LB agar 0.2Arabinose 50?g/ml DAP
LB agar
LB agar
LB agar 0.2 Arabinose
?8888 ?asdAaraC PBAD c2 ,?PmurAaraC PBAD
murA, ?araBAD, ?araE, ?endA,
?gmd-fcl, ?relA
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These RASV systems for multiple protective
antigen delivery of human and avian influenza
viruses are in the construction and early
evaluation phases. Features of this
RASV-influenza vaccine include Ease of rapid
annual revision of HA antigen(s) and manufacture
(4 to 6 weeks) Oral, needle-free delivery
Inexpensive to manufacture, distribute and use
(stable, reconstitute at time of delivery).
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Summary
New vaccines against influenza - cell culture
produced, bacterial and viral vectored - will
require some five to ten years for clinical
validation and licensure prior to availability
for distribution and use. Current vaccines
produced in egg embryos will require (in peak
vaccine production time) fertile eggs from some
six million layers (plus 300,000 roosters) to
yield sufficient vaccine to immunize the U.S.
population. If protection against avian flu
strains is necessary, this capacity might require
a ten- to fifty-fold escalation. Such capacity
does not now exist in the U.S.
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Salmonella cell lysing to release plasmid DNA
vaccine
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