A New Influenza Vaccine for Heterosubtypic Immunity

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A New Influenza Vaccine forHeterosubtypic
Immunity

• The risk of a major global pandemic of avian
  influenza has created widespread and justified
  concern.
• Vaccines designed to induce antibodies against H5
  haemagglutinin present an obvious potential
  control measure but the current high rate of
  diversification of H5N1 strains suggests that
  vaccines made now may differ so much in their H5
  sequence from any pandemic strain that emerges
  that these vaccines would have little or no
  efficacy.

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• However, it has been known for more than 20 years
  that cytotoxic T cells specific for the internal
  proteins NP and M1 show high cross-reactivity
  between strains and between subtypes, reflecting
  the 92-94 conservation of the internal proteins.
  Protective T cells are induced by influenza
  infection, and 90 of the adult UK population has
  memory T cells to flu antigens, but the level of
  effector cells quickly declines below that needed
  for protection.
• We will conduct a clinical trial using Modified
  Vaccinia virus Ankara (MVA) expressing internal
  flu antigens to boost pre-existing memory
  responses back to protective levels. Earlier
  clinical trials with MVA expressing malaria and
  tuberculosis antigens have demonstrated that it
  is safe and highly efficient at boosting T cell
  responses in humans. During the trial,
  cross-reactive responses will be assessed to
  determine the potential of the new vaccine to
  protect against H5N1 as well as H3N2 and H1N1.
• This work is supported by grant 081865 from the
  Wellcome Trust

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Schematic Diagram of an Influenza A Virus Virion
Two surface glycoproteins, haemagglutinin (HA)
and neuraminidase (NA), and the M2 ion-channel
protein are embedded in the viral envelope, which
is derived from the host plasma membrane. The
ribonucleoprotein complex comprises a viral RNA
segment associated with the nucleoprotein (NP)
and three polymerase proteins (PA, PB1 and PB2).
The matrix (M1) protein is associated with both
ribonucleoprotein and the viral envelope.
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Immunity to Influenza
Antibody-mediated immunity to the surface
antigens, especially HA and NA, reduces the
likelihood of infection and severity of disease
if infection occurs by preventing the virus from
infecting cells in the upper respiratory tract
after exposure to the virus. Antibody against one
influenza virus type or subtype confers limited
or no protection against another. Furthermore,
antibody to one antigenic variant of influenza
virus might not protect against a new antigenic
variant of the same type or subtype. Frequent
development of antigenic variants through
antigenic drift is the virologic basis for
seasonal epidemics. In contrast, cytotoxic T
cells specific for the internal proteins NP and
M1 show full or almost full cross-reactivity
between strains and between subtypes. In a study
published in 1983 (McMichael, Gotch et al. 1983),
cytotoxic T cell memory was measured in
volunteers who were then challenged with
infectious virus. All subjects with demonstrable
T cell memory cleared the challenge virus
effectively. This included the younger volunteers
in the study who were unlikely to have been
naturally exposed to virus of the same subtype of
the challenge strain, and who had no antibodies
specific for the challenge subtype. Protection
mediated by T cells will not prevent initial
infection of the upper respiratory tract by
influenza, which antibody-dependent protection
can do. However the memory T cell response is
capable of rapid expansion to produce effector
cells which destroy virus infected cells, prevent
the spread of infection and limit the disease
symptoms. Flu challenge studies showed that
people with a significant T cell response
(greater than 10 lysis at an ET ration of 501
in a Cr51 release lysis assay) shed minimal virus
after infection, compared to those without a T
cell response.
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CTL protects against flu infection (McMichael
1983)Challenge with H1N1
CTL without antibody
CTL and antibody
Open circles, born before 1957 Closed circles,
born after 1957 (probably exposed to H3N2 only)
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Polymorphism in External and Internal Proteins
External proteins of influenza exhibit much
greater polymorphism than internal proteins. HA
and NP genes from 13 human A/H3N2 isolates from
China over the period 1968 to 1994 are
shown. Maximum sequence divergence HA 11.9 NP
3.9

• Influenza A Virus A/Beijing/1/68
• Influenza A Virus A/Guandong/243/72
• Influenza A Virus A/Beijing/39/75
• Influenza A Virus A/Nanjing/49/77
• Influenza A Virus A/Nanjing/2/82
• Influenza A Virus A/Nanjing/36/83
• Influenza A Virus A/Nanjing/28/84
• Influenza A Virus A/Beijing/353/89
• Influenza A Virus A/Beijing/353/1989
• Influenza A Virus A/Nanchang/58/93
• Influenza A Virus A/Nanchang/12/93
• Influenza A Virus A/Nanchang/A1/94
• Influenza A Virus A/Nanchang/A2/94

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Polymorphism in HA
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Polymorphism in NP
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The Vector Modified Vaccinia Virus Ankara

• Recombinant viral vectors, such as poxviruses and
  adenoviruses, are a particularly effective way of
  boosting strong T cell responses to the antigen
  encoded within them. In our tuberculosis vaccine
  studies we reported exceptionally high T cell
  responses in BCG-naïve individuals who were
  immunised with a single low dose of intradermal
  Modified Vaccinia virus Ankara (MVA) expressing
  Antigen 85A (McShane, H., et al., Nat Med, 2004.
  10 1240-4). In clinical studies with new malaria
  vaccines, recombinant MVA was found to boost T
  cell responses in malaria-naïve subjects who had
  been primed with either a DNA vaccine or
  recombinant fowlpox expressing the same antigen
  (McConkey, S.J., et al.,. Nat Med, 2003. 9
  729-35, Webster, D.P., et al., Proc Natl Acad Sci
  U S A, 2005. 102 4836-41.)
• MVA (Mayr et al., 1978, Zentralbl. Bakteriol
  167, 375-390) is a highly attenuated strain of
  vaccinia virus that underwent multiple, fully
  characterised deletions during more than 570
  passages in CEF cells. These included host range
  genes and genes encoding cytokine receptors. The
  virus is unable to replicate efficiently in human
  and most other mammalian cells (Carroll and Moss,
  1997, Virology 238, 198-211) but the replication
  defect occurs at a late stage of virion assembly
  such that viral and recombinant gene expression
  is unimpaired (Sutter and Moss, 1992, Proc. Natl.
  Acad. Sci. USA 89, 1084710851) making MVA an
  efficient single round expression vector
  incapable of causing infection in mammals. MVA
  has been used to immunise some 120,000 humans
  during the smallpox eradication campaign and has
  an excellent safety record (Mayr et al., 1978,
  Zentralbl. Bakteriol 167, 375-390). The entire
  DNA sequence of MVA has been published (Antoine
  et al., 1998, Virology 24 365-396) and recent
  work in our own laboratory has confirmed that the
  virus stock used to prepare MVA-NPM1 has the
  same sequence as that published earlier.

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The Vaccine Antigen NPM1 Fusion

• The insert in the recombinant MVA consists of the
  coding sequence for NP fused to that of M1 to
  express both antigens as a single fusion protein.
• The recombinant MVA was produced using a
  transient dominant selection technique with
  different fluorescent markers, using only primary
  CEF cells for virus production and no drug
  selection of recombinant viruses. The final
  recombinant virus contains no marker gene.
• The insert was sequenced prior to commencing
  immunogenicity studies.

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Pre-clinical Immunogenicity Testing

• Groups of four mice BALB/c mice were immunized
  with either 1 x 106 pfu MVA-NPM1 intradermally,
  or 50 mg of a DNA vaccine expressing the same
  NPM1 insert intramuscularly followed by 1 x 106
  pfu MVA-NPM1 intradermally two weeks later. Two
  weeks after the MVA immunisation the spleens were
  tested for T cell responses to the immunodominant
  peptide TYQRTRALV (NP amino acid residues
  147-155) in an interferon-gamma Elispot assay (as
  described in Schneider, J., et al., (1998) Nat
  Med 4, 397).
• Results are plotted as the mean of four mice, and
  the response generated by vaccination to peptide
  TYQRTRALV are shown compared to the non-specific
  background response (no peptide). In these mice
  which have not been previously exposed to
  influenza, MVA is able to prime a T cell response
  to NP, as shown by the MVA alone group. In mice
  which are first primed using a DNA vaccine, the
  response is boosted to a higher level by
  MVA-NPM1.

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Pre-clinical Immunogenicity Results
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Clinical Trial Plans

• A phase I safety and immunogenicity study with
  dose escalation in healthy adult volunteers will
  commence in Oxford in early 2008.
• T cell responses to influenza antibodies will be
  measured before and after immunisation with a
  single dose of the vaccine
• If successful, influenza challenge studies will
  follow.