U.S. patent application number 11/487769 was filed with the patent office on 2007-06-21 for influenza vaccine.
Invention is credited to Erik D'Hondt, Emmanuel Jules Hanon, Norbert Hehme, Jean Stephenne.
Application Number | 20070141078 11/487769 |
Document ID | / |
Family ID | 10861908 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141078 |
Kind Code |
A1 |
D'Hondt; Erik ; et
al. |
June 21, 2007 |
Influenza vaccine
Abstract
The present invention relates to monovalent influenza vaccine
formulations and vaccination regimes for immunising against
influenza disease, their use in medicine, in particular their use
in augmenting immune responses to various antigens, and to methods
of preparation. In particular, the invention relates to monovalent
influenza immunogenic compositions comprising an influenza antigen
or antigenic preparation thereof from an influenza virus strain
being associated with a pandemic outbreak or having the potential
to be associated with a pandemic outbreak, in combination with an
oil-in-water emulsion adjuvant comprising a metabolisable oil, a
sterol or a tocopherol such as alpha tocopherol, and an emulsifying
agent.
Inventors: |
D'Hondt; Erik; (Bazel,
BE) ; Hehme; Norbert; (Dresden, DE) ; Hanon;
Emmanuel Jules; (Rixensart, BE) ; Stephenne;
Jean; (Rixensart, BE) |
Correspondence
Address: |
GLAXOSMIHKLINE;Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Family ID: |
10861908 |
Appl. No.: |
11/487769 |
Filed: |
July 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10088632 |
Jul 30, 2002 |
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PCT/EP00/09509 |
Sep 27, 2000 |
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11487769 |
Jul 17, 2006 |
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Current U.S.
Class: |
424/204.1 ;
424/130.1 |
Current CPC
Class: |
A61K 2039/54 20130101;
A61K 2039/5252 20130101; A61P 31/16 20180101; C12N 2760/16222
20130101; A61P 37/04 20180101; C12N 7/00 20130101; A61K 39/145
20130101; C12N 2760/16234 20130101; C12N 2760/16251 20130101; C12N
2760/16122 20130101; A61K 2039/55505 20130101; C07K 14/005
20130101; A61P 31/12 20180101; C12N 2760/16151 20130101; A61K 39/12
20130101; A61K 2039/543 20130101 |
Class at
Publication: |
424/204.1 ;
424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/12 20060101 A61K039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 1999 |
GB |
9923176.3 |
Claims
1. A monovalent influenza vaccine composition comprising an
influenza virus component which is a low dose of influenza virus
antigen from an influenza virus strain that is associated with a
pandemic outbreak or has the potential to be associated with a
pandemic outbreak, in combination with a suitable adjuvant, wherein
said low antigen dose is less than 15 .mu.g of haemagglutinin per
dose or no more than 15 .mu.g per combined dose of vaccine, and
wherein said adjuvant is an oil-in-water emulsion carrier
comprising a metabolisable oil, alpha tocopherol and an
emulsifier.
2. A monovalent influenza vaccine composition comprising an
influenza virus component which is a low dose of influenza virus
antigen from an influenza virus strain that is associated with a
pandemic outbreak or has the potential to be associated with a
pandemic outbreak, in combination with a suitable adjuvant, wherein
said low antigen dose is less than 15 .mu.g of haemagglutinin per
dose or no more than 15 .mu.g per combined dose of vaccine, and
wherein said adjuvant is an oil-in-water emulsion carrier
comprising squalene and alpha tocopherol in a ratio which is equal
or less than 1.
3. A vaccine composition according to claim 1 wherein the influenza
virus antigen is in the form of purified whole or in the form of a
split influenza virus.
4. A vaccine composition according to claim 1 wherein the said
metabolisable oil is squalene.
5. A vaccine composition according to claim 1 wherein said
emulsifier is Tween 80.TM..
6. A vaccine composition according to claim 1, wherein said oil in
water emulsion comprise from 2 to 10% squalene, from 2 to 10% alpha
tocopherol and from 0.3 to 3% Tween 80.
7. A vaccine composition according to claim 1, wherein said oil in
water emulsion additionally comprise 3 De-O-acylated monophosphoryl
lipid A (3D-MPL), or QS21.
8. A vaccine composition according to claim 7 wherein 3D-MPL and
QS-21 are present at a range of 1 .mu.g -100 .mu.g, preferably 10
.mu.g -50 .mu.g per dose.
9. A vaccine composition according to claim 7 wherein 3D-MPL and
QS-21 are both present and wherein the ratio of 3D-MPL: QS21 is
2.5:1 to 1:1.
10. A vaccine composition according to claim 1 wherein the low
antigen dose is less than 15 .mu.g of haemagglutinin per dose.
11. A vaccine composition according to claim 10 in which the low
antigen dose is less than 10 .mu.g of haemagglutinin per dose or
per combined dose of vaccine.
12. A vaccine composition according to claim 11 in which the
antigen dose is between 0.1 and 7.5 .mu.g, or between 1 and 5 .mu.g
of haemagglutinin per dose or per combined dose of vaccine.
13. A vaccine composition of claim 1 which is egg-derived or cell
culture-derived.
14. A vaccine composition according to claim 1 wherein the
influenza virus antigen is substantially free of host cell
contamination.
15. A vaccine composition according to claim 1 wherein the
influenza virus component is purified by a method which includes a
protease incubation step to digest non-influenza virus
proteins.
16. A kit comprising: (i) a low dose of influenza virus antigen
formulated with an adjuvant as defined in claim 1 for parenteral
administration; and (ii) a low dose of influenza virus antigen for
mucosal administration, in a mucosal delivery device such as an
intranasal spray device, wherein the influenza virus component
which is an influenza virus antigen from an influenza virus strain
that is associated with a pandemic outbreak, or has the potential
to be associated with a pandemic outbreak, and wherein the low
antigen dose is less than 15 .mu.g of haemagglutinin per dose or no
more than 15 .mu.g per combined dose of vaccine.
17. The kit according to claim 16, wherein the combined antigen
dose of the parenteral and mucosal formulations is no more than 15
.mu.g haemagglutinin.
18. The kit according to claim 17 wherein the combined antigen dose
is less than 10 .mu.g haemagglutinin.
19. The kit according to claim 17 wherein the influenza antigen in
(i) is inactivated whole virus and the influenza antigen in (ii) is
split virus.
20. A method for the production of an influenza vaccine for a
pandemic situation which method comprises admixing a influenza
virus antigen from a single influenza virus strain that is
associated with a pandemic outbreak or has the potential to be
associated with a pandemic outbreak, with an adjuvant as defined in
claim 1 and providing vaccine lots or vaccine kits which contain
less than 10 .mu.g influenza haemagglutinin antigen per dose or no
more than 15 .mu.g haemagglutinin per combined dose.
21. A method according to claim 20 wherein the antigen is highly
purified.
22. A method according to claim 20 wherein the influenza virus
antigen is in the form of whole or split influenza virus
particles.
23. The vaccine composition or kit or method according to claim 1
wherein the influenza antigen is selected from an H2 antigen such
as H2N2 and an H5 antigen such as H5N1.
24. A method for producing influenza virus antigen for use in a
vaccine, which method is according to claim 20, and comprises the
step of incubating a mixture containing influenza virus particles
with a protease to digest non-influenza virus proteins.
25. A method according to claim 24 wherein the protease digestion
step is performed after the influenza virus antigen has been
partially purified by one or more physical separation steps.
26. A method according to claim 24 wherein the protease digestion
step is performed prior to a virus inactivation step.
27. A method according to claim 26 wherein the purification process
comprises the steps of: (i) providing a harvested mixture of
cultured influenza virus and host proteins from a culture; (ii)
partially purifying the influenza virus in the mixture by one or
more physical purification steps; (iii) performing a protease
digestion step on the partially purified mixture to digest host
proteins; (iv) inactivating the influenza virus; (iv) further
purifying the influenza virus by at least one filtration step.
28. A method of manufacturing a vaccine lot or a vaccine kit for
protection against influenza virus infection comprising the step of
using below 10 .mu.g, or below 8 .mu.g, or from 1-7.5 .mu.g, or
from 1-5 .mu.g of egg-derived influenza virus haemagglutinin
antigen from a single strain of influenza associated with a
pandemic outbreak or having the potential to be associated with a
pandemic outbreak in combination with an adjuvant as defined in
claim 1.
29. The method according to claim 28 for administration as a single
dose or in more than one dose such as two doses.
30. The method of no more than 15 .mu.g, or below 10 .mu.g, or
below 8 .mu.g, or from 1-7.5 .mu.g, or from 1-5 .mu.g of
egg-derived influenza virus haemagglutinin antigen from a single
strain of influenza associated with a pandemic outbreak or having
the potential to be associated with a pandemic outbreak, in the
manufacture of a two-dose vaccine for simultaneous parenteral and
mucosal administration, wherein the haemaggutinin for parenteral
administration has been formulated with an adjuvant as defined in
claim 1.
31. A monovalent influenza vaccine composition comprising a low
amount of influenza virus antigen or antigenic preparation from an
influenza virus strain that is associated with a pandemic or has
the potential to be associated with a pandemic, in combination with
an adjuvant, wherein said adjuvant is an oil-in-water emulsion
comprising a metabolisable oil, a sterol or a tocopherol, and an
emulsifying agent.
32. A composition according to claim 31 wherein said tocopherol is
alpha tocopherol.
33. A composition according to claim 31 wherein said metabolisable
oil is squalene.
34. A composition according to claim 31 wherein said metabolisable
oil is present in an amount of 0.5% to 20% of the total volume of
said immunogenic composition.
35. A composition according to claim 31 wherein said metabolisable
oil is present in an amount of 1.0% to 10% of the total volume of
said immunogenic composition.
36. A composition according to claim 31 wherein said metabolisable
oil is present in an amount of 2.0% to 6.0% of the total volume of
said immunogenic composition.
37. A composition according to claim 31 wherein said
alpha-tocopherol is present in an amount of 1.0% to 20% of the
total volume of said immunogenic composition.
38. A composition according to claim 31 wherein said
alpha-tocopherol is present in an amount of 1.0% to 5.0% of the
total volume of said immunogenic composition.
39. A composition according to claim 31 wherein the ratio of
squalene: alpha tocopherol is equal or less than 1.
40. A composition according to claim 31 wherein said emulsifying
agent is Tween 80.
41. A composition according to claim 31 wherein said emulsifying
agent is present at an amount of 0.01 to 5.0% by weight (w/w) of
said immunogenic composition.
42. A composition according to claim 31 wherein said emulsifying
agent is present at an amount of 0.1 to 2.0% by weight (w/w) of
said immunogenic composition.
43. A composition according to claim 31 wherein said antigen or
antigenic composition contains a low amount of haemagglutinin (HA)
antigen.
44. A composition according to claim 43 wherein the amount of HA
antigen does not exceed 15 .mu.g per dose.
45. A composition according to claim 44 wherein the amount of HA
antigen does not exceed 10 .mu.g, or 8 .mu.g, or 4 .mu.g, or 2
.mu.g per dose.
46. A composition according to claim 43 wherein the amount of HA
antigen is between 1-7.5 .mu.g, or from 1-5 .mu.g per dose.
47. A composition according to claim 46 wherein the amount of HA
antigen contains between 2.5 to 7.5 .mu.g of HA per strain.
48. A composition according to claim 31 wherein said pandemic
influenza virus strain is selected from the list consisting of:
H5N1, H9N2, H7N7, H2N2 and H1N1.
49. A composition according to claim 48 wherein said pandemic
influenza virus strain is selected from the list consisting of:
H5N1, H9N2, H7N7, H2N2 and H1N1.
50. A composition according to claim 31, wherein the antigen or
antigen composition is in the form of: a purified whole influenza
virus, a non-live influenza virus, or sub-unit component(s) of
influenza virus.
51. A composition according to claim 50 wherein said non-live
influenza virus is a split influenza virus.
52. A composition as claimed in claim 31 wherein said influenza
antigen or antigenic composition is derived from cell culture or
produced in embryonic eggs.
53. A composition as claimed in claim 31 for use in medicine.
54. A kit comprising a low amount of influenza virus antigen or
antigenic preparation and an oil-in-water adjuvant as defined in
claim 31.
55. A kit according to claim 54 wherein said antigen is HA.
56. A kit according to claim 55 wherein said amount of HA is as
defined in claims 14 to 17.
57. A method for the production of an influenza vaccine composition
for a pandemic situation or a pre-pandemic situation which method
comprises admixing an egg-derived or a cell culture-derived
influenza virus antigen from a single influenza virus strain that
is associated with a pandemic or has the potential to be associated
with a pandemic, with an an oil-in-water emulsion adjuvant and
providing vaccine lots or vaccine kits which contain no more than
15 .mu.g influenza haemagglutinin antigen per dose.
58. A method as claimed in claim 57 wherein the oil-in-water
emulsion adjuvant is as defined in claim 31.
59. A method of manufacturing an immunogenic composition as claimed
in claim 31 for inducing at least one of i) an improved CD4 T-cell
immune response, ii) an improved B cell memory response, against
said virus or antigenic composition in a human, iii) an improved
humoral response comprising the step of using (a) a low amount of
an influenza virus antigen or antigenic preparation thereof from a
single strain of influenza associated with a pandemic or having the
potential to be associated with a pandemic, and (b) an oil-in-water
emulsion adjuvant.
60. The method according to claim 59 wherein said CD4 T-cell immune
response involves the induction of a cross-reactive CD4 T helper
response or the induction of a cross-reactive humoral immune
response.
61. The method of (a) a low amount of a pandemic influenza virus
antigen or antigenic preparation thereof influenza virus
haemagglutinin antigen from a single strain of influenza associated
with a pandemic or having the potential to be associated with a
pandemic, and (b) an oil-in-water emulsion adjuvant as defined in
claim 31 in the manufacture of a vaccine lot or a vaccine kit for
protection against influenza virus infection.
62. The method according to claim 59 wherein said immune response
or protection meets all three EU regulatory criteria for influenza
vaccine efficacy.
63. The method according to claim 59 wherein said immune response
or protection meets is obtained after one or two doses of
vaccine.
64. The method according to claim 59 wherein said vaccine is
administered parenterally.
65. A method as claimed in claim 57 or use as claimed in any of
claims 29 to 34 wherein said HA antigen amount is as defined in
claim 44.
66. The method of an influenza virus or antigenic preparation
thereof in the manufacture of an immunogenic composition for
revaccination of humans previously vaccinated with an immunogenic
composition as claimed in claim 1.
67. The method according to claim 65 wherein the composition used
for the revaccination contains an adjuvant.
68. The method according to claim 67 wherein said adjuvant is an
oil-in-water emulsion adjuvant.
69. The method according to claim 66 wherein said immunogenic
composition for revaccination contains an influenza virus or
antigenic preparation thereof which is associated with a pandemic
or has the potential to be associated with a pandemic.
70. The method according to claim 69 wherein said pandemic strain
is selected from the list consisting of: H5N1, H9N2, H7N7, H2N2 and
H1N1.
71. The method according to claim 69 wherein said immunogenic
composition for revaccination contains an influenza virus or
antigenic preparation thereof which shares common CD4 T-cell
epitopes or common B cell epitopes with the influenza virus or
antigenic preparation thereof used for the first vaccination.
72. The method according to claim 66 wherein the first vaccination
is made with an influenza composition containing an influenza
strain that could potentially cause a pandemic outbreak and the
re-vaccination is made with an influenza composition containing a
circulating pandemic strain.
73. The method of an antigen or antigenic preparation from a first
influenza strain in the manufacture of an immunogenic composition
as claimed in claim 1 for protection against influenza infections
caused by a variant influenza strain.
74. The method according to claim 73 wherein the first influenza
strain is associated with a pandemic outbreak or has the potential
to be associated with a pandemic outbreak.
75. The method according to claim 73 wherein the variant influenza
strain is associated with a pandemic outbreak or has the potential
to be associated with a pandemic outbreak.
Description
TECHNICAL FIELD
[0001] The present invention relates to influenza vaccine
formulations and vaccination regimes for immunising against
influenza disease, their use in medicine, in particular their use
in augmenting immune responses to various antigens, and to methods
of preparation. In particular, the invention relates to monovalent
influenza immunogenic compositions comprising an influenza antigen
or antigenic preparation thereof from an influenza virus strain
that is associated with a pandemic outbreak or has the potential to
be associated with a pandemic outbreak, in combination with an
oil-in-water emulsion adjuvant.
TECHNICAL BACKGROUND
[0002] Influenza viruses are one of the most ubiquitous viruses
present in the world, affecting both humans and livestock.
Influenza results in an economic burden, morbidity and even
mortality, which are significant.
[0003] The influenza virus is an RNA enveloped virus with a
particle size of about 125 nm in diameter. It consists basically of
an internal nucleocapsid or core of ribonucleic acid (RNA)
associated with nucleoprotein, surrounded by a viral envelope with
a lipid bilayer structure and external glycoproteins. The inner
layer of the viral envelope is composed predominantly of matrix
proteins and the outer layer mostly of host-derived lipid material.
Influenza virus comprises two surface antigens, glycoproteins
neuraminidase (NA) and haemagglutinin (HA), which appear as spikes,
10 to 12 nm long, at the surface of the particles. It is these
surface proteins, particularly the haemagglutinin that determine
the antigenic specificity of the influenza subtypes. Virus strains
are classified according to host species of origin, geographic site
and year of isolation, serial number, and, for influenza A, by
serological properties of subtypes of HA and NA. 16 HA subtypes
(H1-H16) and nine NA subtypes (N1-N9) have been identified for
influenza A viruses [Webster R G et al. Evolution and ecology of
influenza A viruses. Microbiol. Rev. 1992; 56:152-179; Fouchier RA
et al. Characterization of a Novel Influenza A Virus Hemagglutinin
Subtype (H16) Obtained from Black-Headed Gulls. J. Virol. 2005;
79:2814-2822). Viruses of all HA and NA subtypes have been
recovered from aquatic birds, but only three HA subtypes (H1, H2,
and H3) and two NA subtypes (N1 and N2) have established stable
lineages in the human population since 1918. Only one subtype of HA
and one of NA are recognised for influenza B viruses.
[0004] Influenza A viruses evolve and undergo antigenic variability
continuously [Wiley D, Skehel J. The structure and the function of
the hemagglutinin membrane glycoprotein of influenza virus. Ann.
Rev. Biochem. 1987; 56:365-394]. A lack of effective proofreading
by the viral RNA polymerase leads to a high rate of transcription
errors that can result in amino-acid substitutions in surface
glycoproteins. This is termed "antigenic drift". The segmented
viral genome allows for a second type of antigenic variation. If
two influenza viruses simultaneously infect a host cell, genetic
reassortment, called "antigenic shift" may generate a novel virus
with new surface or internal proteins. These antigenic changes,
both `drifts` and `shifts` are unpredictable and may have a
dramatic impact from an immunological point of view as they
eventually lead to the emergence of new influenza strains and that
enable the virus to escape the immune system causing the well
known, almost annual, epidemics. Both of these genetic
modifications have caused new viral variants responsible for
pandemic in humans.
[0005] HA is the most important antigen in defining the serological
specificity of the different influenza strains. This 75-80 kD
protein contains numerous antigenic determinants, several of which
are in regions that undergo sequence changes in different strains
(strain-specific determinants) and others in regions which are
common to many HA molecules (common to determinants).
[0006] Influenza viruses cause epidemics almost every winter, with
infection rates for type A or B virus as high as 40% over a
six-week period. Influenza infection results in various disease
states, from a sub-clinical infection through mild upper
respiratory infection to a severe viral pneumonia. Typical
influenza epidemics cause increases in incidence of pneumonia and
lower respiratory disease as witnessed by increased rates of
hospitalization or mortality. The severity of the disease is
primarily determined by the age of the host, his immune status and
the site of infection.
[0007] Elderly people, 65 years old and over, are especially
vulnerable, accounting for 80-90% of all influenza-related deaths
in developed countries. Individuals with underlying chronic
diseases are also most likely to experience such complications.
Young infants also may suffer severe disease. These groups in
particular therefore need to be protected. Besides these `at
risk`-groups, the health authorities are also recommending to
vaccinate healthy adults who are in contact with elderly
persons.
[0008] Vaccination plays a critical role in controlling annual
influenza epidemics. Currently available influenza vaccines are
either inactivated or live attenuated influenza vaccine.
Inactivated flu vaccines are composed of three possible forms of
antigen preparation: inactivated whole virus, sub-virions where
purified virus particles are disrupted with detergents or other
reagents to solubilise the lipid envelope (so-called "split"
vaccine) or purified HA and NA (subunit vaccine). These inactivated
vaccines are given intramuscularly (i.m.) or intranasaly
(i.n.).
[0009] Influenza vaccines for interpandemic use, of all kinds, are
usually trivalent vaccines. They generally contain antigens derived
from two influenza A virus strains and one influenza B strain. A
standard 0.5 ml injectable dose in most cases contains 15 .mu.g of
haemagglutinin antigen component from each strain, as measured by
single radial immunodiffusion (SRD) (J. M. Wood et al.: An improved
single radial immunodiffusion technique for the assay of influenza
haemagglutinin antigen: adaptation for potency determination of
inactivated whole virus and subunit vaccines. J. Biol. Stand. 5
(1977) 237-247; J. M. Wood et al., International collaborative
study of single radial diffusion and immunoelectrophoresis
techniques for the assay of haemagglutinin antigen of influenza
virus. J. Biol. Stand. 9 (1981) 317-330).
[0010] Interpandemic influenza virus strains to be incorporated
into influenza vaccine each season are determined by the World
Health Organisation in collaboration with national health
authorities and vaccine manufacturers. Interpandemic Influenza
vaccines currently available are considered safe in all age groups
(De Donato et al. 1999, Vaccine, 17, 3094-3101). However, there is
little evidence that current influenza vaccines work in small
children under two years of age. Furthermore, reported rates of
vaccine efficacy for prevention of typical confirmed influenza
illness are 23-72% for the elderly, which are significantly lower
than the 60-90% efficacy rates reported for younger adults
(Govaert, 1994, J. Am. Med. Assoc., 21, 166-1665; Gross, 1995, Ann
Intern. Med. 123, 523-527). The effectiveness of an influenza
vaccine has been shown to correlate with serum titres of
hemagglutination inhibition (HI) antibodies to the viral strain,
and several studies have found that older adults exhibit lower HI
titres after influenza immunisation than do younger adults
(Murasko, 2002, Experimental gerontology, 37, 427-439). However,
persons at risk in case of an influenza pandemic may be different
from the defined risk-groups for complications due to seasonal
influenza. According to the WHO, 50% of the human cases cuased by
the avian influenza strain H5N1 occurred in people below 20 years
of age, 90% occurred among those aged <40. (WHO, weekly
epidemiological record, 30 June 2006)
[0011] A sub-unit influenza vaccine adjuvanted with the adjuvant
MF59, in the form of an oil-in-water emulsion is commercially
available, and has demonstrated its ability to induce a higher
antibody titer than that obtained with the non-adjuvanted sub-unit
vaccine (De Donato et al. 1999, Vaccine, 17, 3094-3101). However,
in a later publication, the same vaccine has not demonstrated its
improved profile compared to a non-adjuvanted split vaccine
(Puig-Barbera et al., 2004, Vaccine 23, 283-289).
[0012] By way of background, during inter-pandemic periods,
influenza viruses circulate that are related to those from the
preceding epidemic. The viruses spread among people with varying
levels of immunity from infections earlier in life. Such
circulation, over a period of usually 2-3 years, promotes the
selection of new strains that have changed enough to cause an
epidemic again among the general population; this process is termed
`antigenic drift`. `Drift variants` may have different impacts in
different communities, regions, countries or continents in any one
year, although over several years their overall impact is often
similar. In other words, an influenza pandemics occurs when a new
influenza virus appears against which the human population has no
immunity. Typical influenza epidemics cause increases in incidence
of pneumonia and lower respiratory disease as witnessed by
increased rates of hospitalisation or mortality. The elderly or
those with underlying chronic diseases are most likely to
experience such complications, but young infants also may suffer
severe disease.
[0013] At unpredictable intervals, novel influenza viruses emerge
with a key surface antigen, the haemagglutinin, of a totally
different subtype from strains circulating the season before. Here,
the resulting antigens can vary from 20% to 50% from the
corresponding protein of strains that were previously circulating
in humans. This can result in virus escaping `herd immunity` and
establishing pandemics. This phenomenon is called `antigenic
shift`. It is thought that at least in the past pandemics have
occurred when an influenza virus from a different species, such as
an avian or a porcine influenza virus, has crossed the species
barrier. If such viruses have the potential to spread from person
to person, they may spread worldwide within a few months to a year,
resulting in a pandemic. For example, in 1957 (Asian Flu pandemic),
viruses of the H2N2 subtype replaced H1 N1 viruses that had been
circulating in the human population since at least 1918 when the
virus was first isolated. The H2 HA and N2 NA underwent antigenic
drift between 1957 and 1968 until the HA was replaced in 1968
(Hong-Kong Flu pandemic) by the emergence of the H3N2 influenza
subtype, after which the N2 NA continued to drift along with the H3
HA (Nakajima et al., 1991, Epidemiol. Infect. 106, 383-395).
[0014] The features of an influenza virus strain that give it the
potential to cause a pandemic outbreak are: it contains a new
haemagglutinin compared to the haemagglutinin in the currently
circulating strains, which may or not be accompanied by a change in
neuraminidase subtype; it is capable of being transmitted
horizontally in the human population; and it is pathogenic for
humans. A new haemagglutinin may be one which has not been evident
in the human population for an extended period of time, probably a
number of decades, such as H2. Or it may be a haemagglutinin that
has not been circulating in the human population before, for
example H5, H9, H7 or H6 which are found in birds. In either case
the majority, or at least a large proportion of, or even the entire
population has not previously encountered the antigen and is
immunologically naive to it.
[0015] Several clinical studies have been performed to evaluate
safety and immunogenicity in unprimed populations, with monovalent
candidate vaccines containing a pandemic strain such as the
non-circulating H2N2 or H9N2 strains. These studies have
investigated split or whole virus formulations of various HA
concentrations (1.9, 3.8, 3.8, 7.5 or 15 .mu.g HA per dose), with
or without alum adjuvantation. Influenza viruses of the H2N2
subtype circulated from 1957 until 1968 when they were replaced by
H3N2 strains during the `Hong Kong pandemic`. Today, individuals
that were born after 1968 are immunologically naive to H2N2
strains. These vaccine candidates have been shown to be immunogenic
and well tolerated. Results are reported in Hehme, N et al. 2002,
Med. Microbiol. Immunol. 191, 203-208; in Hehme N. et al. 2004,
Virus Research 103, 163-171; and two studies were reported with
H5N1 (Bresson J L et al. The Lancet. 2006:367 (9523):1657-1664;
Treanor J J et al. N Engl J Med. 2006; 354:1343-1351).
[0016] During a pandemic, antiviral drugs may not be sufficient to
cover needs and the number of individuals at risk of influenza will
be greater than in interpandemic periods, therefore the development
of a suitable vaccine with the potential to be produced in large
amounts and with efficient distribution and administration
potential is essential. For these reasons, monovalent instead of
trivalent vaccines are being developed for pandemic purposes in an
attempt to reduce vaccine volume, primarily as two doses of vaccine
may be necessary in order to achieve protective antibody levels in
immunologically naive recipients (Wood J M et al. Med Mircobiol
Immunol. 2002; 191:197-201. Wood J M et al. Philos Trans R Soc Lond
B Biol Sci. 2001 ;356:1953-1960).
[0017] This problem may be countered by adjuvantation, the aim of
which is to increase immunogenicity of the vaccine in order to
decrease the antigen content (antigen sparing). In addition, the
use of an adjuvant may overcome the potential weak immunogenicity
of the antigen in a naive population. Clinical trials with plain
subvirion H5N1 vaccine or aluminium hydroxide adjuvanted split
virus H5N1 vaccine have already been performed. The results of
these trials indicate that both plain and adjuvanted H5N1 virus
vaccines are safe up to an antigen dose of 90 pg (tested only as
plain subvirion vaccine) (Bresson J L et al. The Lancet. 2006:367
(9523):1657-1664; Treanor J J et al. N Engl J Med. 2006;
354:1343-1351.)
[0018] New vaccines with an improved immunogenicity, in particular
against weakly or non-immunogenic pandemic strains or for the
immuno-compromised individuals such as elderly population, are
therefore still needed. Formulation of vaccine antigen with potent
adjuvants is a possible approach for enhancing immune responses to
subvirion antigens. Novel adjuvant formulations are hereby provided
which allow a antigen sparing formulation affording sufficient
protection (seroconversion of previously seronegative subjects to a
HI titer considered as protective, 1.sub.--40 or fourfold increase
in titer) of all age groups.
STATEMENT OF THE INVENTION
[0019] In first aspect of the present invention, there is provided
an influenza immunogenic composition, in particular a vaccine,
comprising a low amount of influenza virus antigen or antigenic
preparation from an influenza virus strain that is associated with
a pandemic or has the potential to be associated with a pandemic,
in combination with an adjuvant, wherein the low antigen amount
does not exceed 15 .mu.g of haemagglutinin (HA) per dose, and
wherein said adjuvant is an oil-in-water emulsion comprising a
metabolisable oil, a sterol or a tocopherol, such as alpha
tocopherol, and an emulsifying agent. Suitably the vaccine
composition is a monovalent composition.
[0020] Throughout the document it will be referred to a pandemic
strain as an influenza strain being associated or susceptible to be
associated with an outbreak of influenza disease, such as a
pandemic Influenza A strains. Suitable pandemic strains are, but
not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1. Others suitable
pandemic strains in human are H7N3 (2 cases reported in Canada),
H10N7 (2 cases reported in Egypt) and H5N2 (1 case reported in
Japan).
[0021] In another aspect the invention provides a method for the
production of an influenza immunogenic composition, in particular a
vaccine, for a pandemic situation or a pre-pandemic situation which
method comprises admixing an influenza virus antigen or antigenic
preparation thereof from a single influenza virus strain that is
associated with a pandemic or has the potential to be associated
with a pandemic, with an oil-in-water emulsion adjuvant as herein
above defined, and providing vaccine units which contain no more
than 15 .mu.g influenza haemagglutinin antigen per dose. Suitably
the influenza virus antigen is egg-derived or cell
culture-derived.
[0022] In a third aspect there is provided an immunogenic
composition as herein defined for use in medicine.
[0023] In another aspect there is provided the use of (a) a low
amount, as herein defined, of influenza virus antigen or antigenic
preparation thereof from a single strain of influenza associated
with a pandemic or having the potential to be associated with a
pandemic, and (b) an oil-in-water emulsion adjuvant, in the
manufacture of an immunogenic composition, or a kit, for inducing
at least one of i) an improved CD4 T-cell immune response, ii) an
improved B cell memory response, iii) an improved humoral response,
against said virus antigen or antigenic composition in a human.
Said immune response is in particular induced in an
immuno-compromised individual or population, such as a high risk
adult or an elderly. Suitably the immunogenic composition is as
herein defined.
[0024] There is also provided the use of an influenza virus or
antigenic preparation thereof and an oil-in-water emulsion adjuvant
in the preparation of an immunogenic composition as herein defined
for vaccination of human elderly against influenza.
[0025] In a specific embodiment, the immunogenic composition is
capable of inducing both an improved CD4 T-cell immune response and
an improved B-memory cell response compared to that obtained with
the un-adjuvanted antigen or antigenic composition. In another
specific embodiment, the immunogenic composition is capable of
inducing both an improved CD4 T-cell immune response and an
improved humoral response compared to that obtained with the
un-adjuvanted antigen or antigenic composition. In particular, said
humoral immune response or protection meets all three EU regulatory
criteria for influenza vaccine efficacy. Suitably, said immune
response(s) or protection is obtained after one, suitably two,
doses of vaccine. Efficacy criteria for the composition according
to the present invention are further detailed below (see Table 1
and below under "efficacy criteria"). Suitably said composition is
administered parenterally, in particular via the intramuscular or
the sub-cutaneous route.
[0026] In a further embodiment, there is provided the use of a low
amount of an influenza virus or antigenic preparation thereof in
the manufacture of an immunogenic composition for revaccination of
humans previously vaccinated with a monovalent influenza
immunogenic composition comprising an influenza antigen or
antigenic preparation thereof from a single influenza virus strain
which is associated with a pandemic or has the potential to be
associated with a pandemic, in combination with an oil-in-water
emulsion adjuvant as herein defined.
[0027] In a specific embodiment, the composition used for the
revaccination may be un-adjuvanted or may contain an adjuvant, in
particular an oil-in-water emulsion adjuvant. In another specific
embodiment, the immunogenic composition for revaccination contains
an influenza virus or antigenic preparation thereof which shares
common CD4 T-cell epitopes with the influenza virus or virus
antigenic preparation thereof used for the first vaccination. The
immunogenic composition for a revaccination may contain a classical
amount (i.e. about 15 pg of HA) of said variant pandemic
strain.
[0028] Preferably the revaccination is made in subjects who have
been vaccinated the previous season against influenza. Typically
revaccination is made at least 6 months after the first
vaccination, preferably 8 to 14 months after, more preferably at
around 10 to 12 months after or even longer.
[0029] Suitably said oil-in-water emulsion comprises a
metabolisable oil, a tocopherol, such as alpha tocopherol, and an
emulsifying agent. In a another specific embodiment, said
oil-in-water emulsion adjuvant comprises at least one metabolisable
oil in an amount of 0.5% to 20% of the total volume, and has oil
droplets of which at least 70% by intensity have diameters of less
than 1 .mu.m. Suitably said a tocopherol, such as alpha tocopherol,
is present in an amount of 1.0% to 20%, in particular in an amount
of 1.0% to 5% of the total volume of said immunogenic
composition.
[0030] In a further aspect of the present invention, there is
provided the use of an antigen or antigenic preparation from a
first pandemic influenza strain in the manufacture of an
immunogenic composition as herein defined for protection against
influenza infections caused by a variant influenza strain.
[0031] In a specific aspect, there is provided a method of
vaccination of an immuno-compromised human individual or population
such as high risk adults or elderly, said method comprising
administering to said individual or population an influenza
immunogenic composition comprising a low amount of an influenza
antigen or antigenic preparation thereof from a single pandemic
influenza virus strain in combination with an oil-in-water emulsion
adjuvant as herein defined.
[0032] In still another embodiment, the invention provides a method
for revaccinating humans previously vaccinated with a monovalent
influenza immunogenic composition comprising an influenza antigen
or antigenic preparation thereof from a single pandemic influenza
virus strain, in combination with an oil-in-water emulsion
adjuvant, said method comprising administering to said human an
immunogenic composition comprising an influenza virus, either
adjuvanted or un-adjuvanted.
[0033] In a further embodiment there is provided a method for
vaccinating a human population or individual against one pandemic
influenza virus strain followed by revaccination of said human or
population against a variant influenza virus strain, said method
comprising administering to said human (i) a first composition
comprising an influenza virus or antigenic preparation thereof from
a first pandemic influenza virus strain and an oil-in-water
emulsion adjuvant, and (ii) a second immunogenic composition
comprising a influenza virus strain variant of said first influenza
virus strain. In a specific embodiment said variant strain is
associated with a pandemic or has the potential to be associated
with a pandemic. In another specific embodiment said variant strain
is part of a multivalent composition which comprises, in addition
to said pandemic influenza virus variant, at least one circulating
(seasonal) influenza virus strain. In particular, said pandemic
influenza virus strain is part of a bivalent, or a trivalent, or
tetravalent composition additionally comprising one, two or three
seasonal strains, respectively.
[0034] Throughout the document, the use of a low amount of pandemic
influenza virus antigen in the manufacture of a composition as
herein defined for prevention of influenza infection or disease,
and a method of treatment of humans using the claimed composition
will be interchangeably used.
[0035] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
LEGEND TO FIGURES
[0036] FIG. 1: Oil droplet particle size distribution in SB62
oil-in-water emulsion as measured by PCS. FIG. 1 A shows SB62 lot
1023 size measurements with the Malvern Zetasizer 3000HS:
A=dilution 1/10000 (Rec22 to Rec24) (Analysis in Contin and adapted
optical model 1.5/0.01); B=Dilution 1/20000 (Rec28 to Rec30)
(Analysis in Contin and adapted optical model 1.5/0.01). FIG. 1B
shows a schematic illustration of record 22 (upper part) and record
23 (lower part) by intensity.
[0037] FIG. 2: Overview of the manufacture of influenza monovalent
bulks.
[0038] FIG. 3: Formulation flow sheet for final bulk of antigen
[0039] FIG. 4: Human clinical trial with a dose-range of H5N1 split
virus antigen, adjuvanted or not with AS03. GMT's (with 95%CI) for
anti-HA antibody at time-points days 0, 21 and 42.
[0040] FIG. 5: Human clinical trial with a dose-range of H5N1 split
virus antigen, adjuvanted or not with AS03. Seroconversion rates
(with 95% Cl) for anti-HA antibody at post-vaccination day 21 and
day 42.
[0041] FIG. 6: Human clinical trial with a dose-range of H5N1 split
virus antigen, adjuvanted or not with AS03. Seroprotection rates
(with 95% Cl) for anti-HA antibody at each time-points (Day 0, Day
21 and (Day 42).
[0042] FIG. 7: Human clinical trial with a dose-range of H5N1 split
virus antigen, adjuvanted or not with AS03. Seroconversion factor
(with 95% Cl) for anti-HA antibody at post-vaccination (day 21 and
42)
DETAILED DESCRIPTION
[0043] The present inventors have discovered that an influenza
formulation comprising low amount of an influenza virus or
antigenic preparation thereof associated with a pandemic or
susceptible to be associated with a pandemic, together with an
oil-in-water emulsion adjuvant comprising a metabolisable oil, a
sterol or a tocopherol, such as alpha tocopherol, and an
emulsifying agent, was capable of improving the humoral immune
response, and/or the CD4 T-cell immune response and/or B cell
memory response against said antigen or antigenic composition in a
human or population, compared to that obtained with the
un-adjuvanted virus or antigenic preparation thereof. The
formulations adjuvanted with an oil-in-water emulsion adjuvant as
herein defined will advantageously be used to induce anti-influenza
CD4-T cell response capable of detection of influenza epitopes
presented by MHC class 11 molecules. The formulations adjuvanted
with an oil-in-water emulsion adjuvant as herein defined will
advantageously be used to induce a cross-reactive immune response.
The adjuvanted formulations will advantageously be effective to
target the humoral and/or the cell-mediated immune system in order
to increase responsiveness against homologous and drift influenza
strains (upon vaccination and infection).
[0044] The adjuvanted influenza compositions according to the
invention have several advantages: [0045] 1) An improved
immunogenicity: they will allow to improve weak immune response to
less immunogenic influenza strains to level higher than those
obtained with the un-adjuvanted formulations; [0046] 2) The use of
adjuvants can overcome the potential weak immunogenicity of the
antigen in a naive population; [0047] 3) They may lead to an
improved immunogenicity in specific populations such as in the
elderly people (typically over 60 years of age) to levels seen in
younger people aged 18 to 60 (antibody and/or T cell responses);
[0048] 4) They may lead to an improved cross-protection profile:
increased cross-protection against variant (drifted) influenza
strains; [0049] 5) By reaching any or all of these further
advantages with a reduced antigen dosage, they will ensure an
increased capacity in case of emergency (antigen sparing in the
pandemic situation).
[0050] The compositions for use in the present invention may be
able to provide better sero-protection against influenza following
revaccination, as assessed by the number of human subjects meeting
the influenza correlates of protections. Furthermore, the
composition for use in the present invention may also be able to
induce a higher humoral response or B cell memory response
following the first vaccination of a human subject, and a higher
response following revaccination, compared to the un-adjuvanted
composition.
[0051] The claimed adjuvanted compositions may also be able not
only to induce but also maintain protective levels of antibodies
against the influenza strain present in the vaccine, in more
individuals than those obtained with the un-advanted
composition.
[0052] Thus, in still another embodiment, the claimed composition
is capable of ensuring a persistent immune response against
influenza related disease. In particular, by persistence it is
meant an HI antibody immune response which is capable of meeting
regulatory criteria after at least three months, preferably after
at least 6 months after the vaccination. In particular, the claimed
composition is able to induce protective levels of antibodies in
>70% of individuals, suitably in >80% of individuals or
suitably in >90% of individuals for the pandemic influenza
strain present in the vaccine, after at least three months. In a
specific aspect, protective levels of antibodies of >90% are
obtained at least 6 months post-vaccination against the influenza
strain of the vaccine composition.
[0053] According to further aspects of the present invention, the
claimed composition is capable to induce seroprotection and
seroconversion to a higher degree than that provided for by the EU
requirements for vaccine influenza strains. This will be further
detailed below (see Table 1 and below under "efficacy
criteria").
Influenza Viral Strains and Antigens
[0054] In one embodiment, an influenza virus or antigenic
preparation thereof for use according to the present invention may
be a split influenza virus or split virus antigenic preparation
thereof. In an alternative embodiment the influenza preparation may
contain another type of inactivated influenza antigen, such as
inactivated whole virus or purified HA and NA (subunit vaccine), or
an influenza virosome. In a still further embodiment, the influenza
virus may be a live attenuated influenza preparation.
[0055] A split influenza virus or split virus antigenic preparation
thereof for use according to the present invention is suitably an
inactivated virus preparation where virus particles are disrupted
with detergents or other reagents to solubilise the lipid envelope.
Split virus or split virus antigenic preparations thereof are
suitably prepared by fragmentation of whole influenza virus, either
infectious or inactivated, with solubilising concentrations of
organic solvents or detergents and subsequent removal of all or the
majority of the solubilising agent and some or most of the viral
lipid material. By split virus antigenic preparation thereof is
meant a split virus preparation which may have undergone some
degree of purification compared to the split virus whilst retaining
most of the antigenic properties of the split virus components. For
example, when produced in eggs, the split virus may be depleted
from egg-contaminating proteins, or when produced in cell culture,
the split virus may be depleted from host cell contaminants. A
split virus antigenic preparation may comprise split virus
antigenic components of more than one viral strain. Vaccines
containing split virus (called `influenza split vaccine`) or split
virus antigenic preparations generally contain residual matrix
protein and nucleoprotein and sometimes lipid, as well as the
membrane envelope proteins. Such split virus vaccines will usually
contain most or all of the virus structural proteins although not
necessarily in the same proportions as they occur in the whole
virus.
[0056] Alternatively, the influenza virus may be in the form of a
whole virus vaccine. This may prove to be an advantage over a split
virus vaccine for a pandemic situation as it avoids the uncertainty
over whether a split virus vaccine can be successfully produced for
a new strain of influenza virus. For some strains the conventional
detergents used for producing the split virus can damage the virus
and render it unusable. Although there is always the possibility to
use different detergents and/or to develop a different process for
producing a split vaccine, this would take time, which may not be
available in a pandemic situation. In addition to the greater
degree of certainty with a whole virus approach, there is also a
greater vaccine production capacity than for split virus since
considerable amounts of antigen are lost during additional
purification steps necessary for preparing a suitable split
vaccine.
[0057] In another embodiment, the influenza virus preparation is in
the form of a purified sub-unit influenza vaccine. Sub-unit
influenza vaccines generally contain the two major envelope
proteins, HA and NA, and may have an additional advantage over
whole virion vaccines as they are generally less reactogenic,
particularly in young vaccinees. Sub-unit vaccines can produced
either recombinantly or purified from disrupted viral
particles.
[0058] In another embodiment, the influenza virus preparation is in
the form of a virosome. Virosomes are spherical, unilamellar
vesicles which retain the functional viral envelope glycoproteins
HA and NA in authentic conformation, intercalated in the virosomes'
phospholipids bilayer membrane.
[0059] Said influenza virus or antigenic preparation thereof may be
egg-derived or tissue-culture derived. They may also be produced in
other systems such as insect cells, yeast or bacteria.
[0060] For example, the influenza virus antigen or antigenic
preparations thereof according to the invention may be derived from
the conventional embryonated egg method, by growing influenza virus
in eggs and purifying the harvested allaritoic fluid. Eggs can be
accumulated in large numbers at short notice. Alternatively, they
may be derived from any of the new generation methods using tissue
culture to grow the virus or express recombinant influenza virus
surface antigens. Suitable cell substrates for growing the virus
include for example dog kidney cells such as MDCK or cells from a
clone of MDCK, MDCK-like cells, monkey kidney cells such as AGMK
cells including Vero cells, suitable pig cell lines, or any other
mammalian cell type suitable for the production of influenza virus
for vaccine purposes. Suitable cell substrates also include human
cells e.g. MRC-5 cells. Suitable cell substrates are not limited to
cell lines; for example primary cells such as chicken embryo
fibroblasts and avian cell lines are also included.
[0061] The influenza virus antigen or antigenic preparation thereof
may be produced by any of a number of commercially applicable
processes, for example the split flu process described in patent
no. DD 300 833 and DD 211 444, incorporated herein by reference.
Traditionally split flu was produced using a solvent/detergent
treatment, such as tri-n-butyl phosphate, or diethylether in
combination with Tween.TM. (known as "Tween-ether" splitting) and
this process is still used in some production facilities. Other
splitting agents now employed include detergents or proteolytic
enzymes or bile salts, for example sodium deoxycholate as described
in patent no. DD 155 875, incorporated herein by reference.
Detergents that can be used as splitting agents include cationic
detergents e.g. cetyl trimethyl ammonium bromide (CTAB), other
ionic detergents e.g. laurylsulfate, taurodeoxycholate, or
non-ionic detergents such as the ones described above including
Triton X-100 (for example in a process described in Lina et al,
2000, Biologicals 28, 95-103) and Triton N-101, or combinations of
any two or more detergents.
[0062] The preparation process for a split vaccine may include a
number of different filtration and/or other separation steps such
as ultracentrifugation, ultrafiltration, zonal centrifugation and
chromatography (e.g. ion exchange) steps in a variety of
combinations, and optionally an inactivation step eg with heat,
formaldehyde or .beta.-propiolactone or U.V. which may be carried
out before or after splitting. The splitting process may be carried
out as a batch, continuous or semi-continuous process. A preferred
splitting and purification process for a split immunogenic
composition is described in WO 02/097072.
[0063] Preferred split flu vaccine antigen preparations according
to the invention comprise a residual amount of Tween 80 and/or
Triton X-100 remaining from the production process, although these
may be added or their concentrations adjusted after preparation of
the split antigen. Preferably both Tween 80 and Triton X-100 are
present. The preferred ranges for the final concentrations of these
non-ionic surfactants in the vaccine dose are: [0064] Tween 80:
0.01 to 1%, more preferably about 0.1% (v/v) [0065] Triton X-100:
0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02% (w/v).
[0066] In a specific embodiment, the final concentration for Tween
80 ranges from 0.045%-0.09% w/v. In another specific embodiment,
the antigen is provided as a 2 fold concentrated mixture, which has
a Tween 80 concentration ranging from 0.045%-0.2% (w/v) and has to
be diluted two times upon final formulation with the adjuvanted (or
the buffer in the control formulation).
[0067] In another specific embodiment, the final concentration for
Triton X-100 ranges from 0.005%-0.017% w/v. In another specific
embodiment, the antigen is provided as a 2 fold concentrated
mixture, which has a Triton X-100 concentration ranging from
0.005%-0.034% (w/v) and has to be diluted two times upon final
formulation with the adjuvanted (or the buffer in the control
formulation).
[0068] Preferably the influenza preparation is prepared in the
presence of low level of thiomersal, or preferably in the absence
of thiomersal. Preferably the resulting influenza preparation is
stable in the absence of organomercurial preservatives, in
particular the preparation contains no residual thiomersal. In
particular the influenza virus preparation comprises a
haemagglutinin antigen stabilised in the absence of thiomersal, or
at low levels of thiomersal (generally 5 .mu.g/ml or less).
Specifically the stabilization of B influenza strain is performed
by a derivative of alpha tocopherol, such as alpha tocopherol
succinate (also known as vitamin E succinate, i.e. VES). Such
preparations and methods to prepare them are disclosed in WO
02/097072.
[0069] Alternatively, especially for multi dose containers,
thiomersal is present in order to reduce the contamination risks.
This is particularly of relevance for pandemic vaccines, designed
to vaccinate as many people as possible in the shortest possible
time.
[0070] A preferred composition for re-vaccination contains three
inactivated split virion antigens prepared from the WHO recommended
strains of the appropriate influenza season, in addition to a
pandemic influenza strain.
[0071] In one embodiment the influenza virus or antigenic
preparation thereof and the oil-in-water emulsion adjuvant are
contained in the same container. It is referred to as `one vial
approach`. Suitably the vial is a pre-filled syringe. In an
alternative embodiment, the influenza virus or antigenic
preparation thereof and the oil-in-water emulsion adjuvant are
contained in separate containers or vials or units and admixed
shortly before or upon administration into the subject. It is
referred to as `two vials approach`. By way of example, when the
vaccine is a 2 components vaccine for a total dose volume of 0.7
ml, the concentrated antigens (for example the concentrated
inactivated split virion antigens) may be presented in one vial
(335 .mu.l) (antigen container) and a pre-filled syringe contains
the adjuvant (360 .mu.l) (adjuvant container). At the time of
injection, the content of the vial containing the concentrated
inactivated split virion antigens is removed from the vial by using
the syringe containing the adjuvant followed by gentle mixing of
the syringe. Prior to injection, the used needle is replaced by an
intramuscular needle and the volume is corrected to 530 .mu.l. One
dose of the reconstituted adjuvanted influenza vaccine candidate
corresponds to 530 .mu.l.
[0072] Suitably the AS03-adjuvanted pandemic influenza candidate
vaccine is a 2 component vaccine consisting of 0.5 ml of
concentrated inactivated split virion antigens presented in a type
I glass vial and of a pre-filled type I glass syringe containing
0.5 ml of the AS03 adjuvant. At the time of injection, the content
of the prefilled syringe containing the adjuvant is injected into
the vial that contains the concentrated trivalent inactivated split
vrion antigens. After mixing the content is withdrawn into the
syringe and the needle is replaced by an intramuscular needle. One
dose of the reconstituted the AS03-adjuvanted influenza candidate
vaccine corresponds to 0.5 ml. Each vaccine dose of 0.5 ml contains
3.8 .mu.g, 7.5 .mu.g, 15.mu. or 30 .mu.g haemagglutinin (HA) or any
suitable amount of HA which would have be determined such that the
vaccine composition meets the efficcacy criteria as defined herein.
A vaccine dose of 1 ml (0.5 ml adjuvant plus 0.5 ml antigen
preparation) is also suitable.
[0073] According to the present invention, the influenza strain in
the monovalent immunogenic composition as herein defined is
associated with a pandemic or has the potential to be associated
with a pandemic. Such strain may also be referred to as `pandemic
strains` in the text below. In particular, when the vaccine is a
multivalent vaccine for re-vaccination, such as a bivalent, or a
trivalent or a quadrivalent vaccine, at least one strain is
associated with a pandemic or has the potential to be associated
with a pandemic. [0074] Suitable pandemic strains [0075] Suitable
pandemic strains [0076] Suitable pandemic strains are, but not
limited to: H5N1, H9N2, H7N7, H2N2 and H1N1. [0077] Others pandemic
strains in human: H7N3 (2 cases reported in Canada), H10N7 (2 cases
reported in Egypt) and H5N2 (1 case reported in Japan). are, but
not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.
[0078] Said influenza virus or antigenic preparation thereof for
re-vaccination is suitably multivalent such as bivalent or
trivalent or quadrivalent or contain even more influenza strains.
Preferably the influenza virus or antigenic preparation thereof for
re-vaccination is trivalent or quadrivalent, having an antigen from
three different influenza strains, at least one strain being
associated with a pandemic outbreak or having the potential to be
associated with a pandemic outbreak. Suitably the re-vaccination
composition comprises a pandemic strain, which may be a variant of
the pandemic strain present in the composition for the first
vaccination, and three other strains, typically the classical
circulating strains.
[0079] The features of an influenza virus strain that give it the
potential to cause a pandemic or an outbreak of influenza disease
associated with pandemic influenza strains are: it contains a new
haemagglutinin compared to the haemagglutinin in the currently
circulating strains; it is capable of being transmitted
horizontally in the human population; and it is pathogenic for
humans. A new haemagglutinin may be one which has not been evident
in the human population for an extended period of time, probably a
number of decades, such as H2. Or it may be a haemagglutinin that
has not been circulating in the human population before, for
example H5, H9, H7 or H6 which are found in birds. In either case
the majority, or at least a large proportion of, or even the entire
population has not previously encountered the antigen and is
immunologically naive to it. At present, the influenza A virus that
has been identified by the WHO as one that potentially could
reassort with human viruses and cause a pandemic in humans is the
highly pathogenic H5N1 avian influenza virus. Therefore, the
pandemic vaccine according to the invention will suitably comprise
H5N1 virus.
[0080] Certain parties are generally at an increased risk of
becoming infected with influenza in a pandemic situation. The
elderly, the chronically ill and small children are particularly
susceptible but many young and apparently healthy people are also
at risk. For H2 influenza, the part of the population born after
1968 is at an increased risk. It is important for these groups to
be protected effectively as soon as possible and in a simple
way.
[0081] Another group of people who are at increased risk are
travelers. People travel more today than ever before and the
regions where most new viruses emerge, China and South East Asia,
have become popular travel destinations in recent years. This
change in travel patterns enables new viruses to reach around the
globe in a matter of weeks rather than months or years.
[0082] Thus for these groups of people there is a particular need
for vaccination to protect against influenza in a pandemic
situation or a potential pandemic situation. Suitable pandemic
strains [0083] Suitable pandemic strains [0084] Suitable pandemic
strains are, but not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.
[0085] Others pandemic strains in human: H7N3 (2 cases reported in
Canada), H10N7 (2 cases reported in Egypt) and H5N2 (1 case
reported in Japan). [0086] which have caused or could potential
cause a pandemic are, but not limited to: H5N1, H9N2, H7N7, H2N2
and H1N1. Oil-in-water Emulsion Adjuvant
[0087] The adjuvant composition of the invention contains an
oil-in-water emulsion adjuvant, preferably said emulsion comprises
a metabolisable oil in an amount of 0.5% to 20% of the total
volume, and having oil droplets of which at least 70% by intensity
have diameters of less than 1 .mu.m.
[0088] In order for any oil in water composition to be suitable for
human administration, the oil phase of the emulsion system has to
comprise a metabolisable oil. The meaning of the term metabolisable
oil is well known in the art. Metabolisable can be defined as
`being capable of being transformed by metabolism` (Dorland's
Illustrated Medical Dictionary, W. B. Sanders Company, 25th edition
(1974)). The oil may be any vegetable oil, fish oil, animal oil or
synthetic oil, which is not toxic to the recipient and is capable
of being transformed by metabolism. Nuts, seeds, and grains are
common sources of vegetable oils. Synthetic oils are also part of
this invention and can include commercially available oils such as
NEOBEE.RTM. and others. A particularly suitable metabolisable oil
is squalene. Squalene
(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an
unsaturated oil which is found in large quantities in shark-liver
oil, and in lower quantities in olive oil, wheat germ oil, rice
bran oil, and yeast, and is a particularly preferred oil for use in
this invention. Squalene is a metabolisable oil by virtue of the
fact that it is an intermediate in the biosynthesis of cholesterol
(Merck index, 10 th Edition, entry no.8619).
[0089] Oil in water emulsions per se are well known in the art, and
have been suggested to be useful as adjuvant compositions (EP
399843; WO 95/17210).
[0090] Suitably the metabolisable oil is present in an amount of
0.5% to 20% (final concentration) of the total volume of the
immunogenic composition, preferably an amount of 1.0% to 10% of the
total volume, preferably in an amount of 2.0% to 6.0% of the total
volume.
[0091] In a specific embodiment, the metabolisable oil is present
in a final amount of about 0.5%, 1%, 3.5% or 5% of the total volume
of the immunogenic composition. In another specific embodiment, the
metabolisable oil is present in a final amount of 0.5%,1%, 3.57% or
5% of the total volume of the immunogenic composition. A suitable
amount of squalene is about 10.7 mg per vaccine dose, suitably from
10.4 to 11.0 mg per vaccine dose.
[0092] Preferably the oil-in-water emulsion systems of the present
invention have a small oil droplet size in the sub-micron range.
Suitably the droplet sizes will be in the range 120 to 750 nm, more
preferably sizes from 120 to 600 nm in diameter. Most preferably
the oil-in water emulsion contains oil droplets of which at least
70% by intensity are less than 500 nm in diameter, more preferably
at least 80% by intensity are less than 300 nm in diameter, more
preferably at least 90% by intensity are in the range of 120 to 200
nm in diameter.
[0093] The oil droplet size, i.e. diameter, according to the
present invention is given by intensity. There are several ways of
determining the diameter of the oil droplet size by intensity.
Intensity is measured by use of a sizing instrument, suitably by
dynamic light scattering such as the Malvern Zetasizer 4000 or
preferably the Malvern Zetasizer 3000HS. A detailed procedure is
given in Example II.2. A first possibility is to determine the z
average diameter ZAD by dynamic light scattering (PCS-Photon
correlation spectroscopy); this method additionally give the
polydispersity index (PDI), and both the ZAD and PDI are calculated
with the cumulants algorithm. These values do not require the
knowledge of the particle refractive index. A second mean is to
calculate the diameter of the oil droplet by determining the whole
particle size distribution by another algorithm, either the Contin,
or NNLS, or the automatic "Malvern" one (the default algorithm
provided for by the sizing instrument). Most of the time, as the
particle refractive index of a complex composition is unknown, only
the intensity distribution is taken into consideration, and if
necessary the intensity mean originating from this
distribution.
[0094] The oil in water emulsion according to the invention
comprises a sterol or a tocopherol, such as alpha tocopherol,.
Sterols are well known in the art, for example cholesterol is well
known and is, for example, disclosed in the Merck Index, 11th Edn.,
page 341, as a naturally occurring sterol found in animal fat.
Other suitable sterols include .beta.-sitosterol, stigmasterol,
ergosterol and ergocalciferol. Said sterol is suitably present in
an amount of 0.01% to 20% (w/v) of the total volume of the
immunogenic composition, preferably at an amount of 0.1% to 5%
(w/v). Preferably, when the sterol is cholesterol, it is present in
an amount of between 0.02% and 0.2% (w/v) of the total volume of
the immunogenic composition, more preferably at an amount of 0.02%
(w/v) in a 0.5 ml vaccine dose volume, or 0.07% (w/v) in 0.5 ml
vaccine dose volume or 0.1% (w/v) in 0.7 ml vaccine dose
volume.
[0095] Suitably alpha-tocopherol or a derivative thereof such as
alpha-tocopherol succinate is present. Preferably alpha-tocopherol
is present in an amount of between 0.2% and 5.0% (v/v) of the total
volume of the immunogenic composition, more preferably at an amount
of 2.5% (v/v) in a 0.5 ml vaccine dose volume, or 0.5% (v/v) in 0.5
ml vaccine dose volume or 1.7-1.9% (v/v), preferably 1.8% in 0.7 ml
vaccine dose volume. By way of clarification, concentrations given
in v/v can be converted into concentration in w/v by applying the
following conversion factor: a 5% (v/v) alpha-tocopherol
concentration is equivalent to a 4.8% (w/v) alpha-tocopherol
concentration. A suitable amount of alpha-tocopherol is about 11.9
mg per vaccine dose, suitably from 11.6 to 12.2 mg per vaccine
dose.
[0096] The oil in water emulsion comprises an emulsifying agent.
The emulsifying agent may be present at an amount of 0.01 to 5.0%
by weight of the immunogenic composition (w/w), preferably present
at an amount of 0.1 to 2.0% by weight (w/w). Preferred
concentration are 0.5 to 1.5% by weight (w/w) of the total
composition.
[0097] The emulsifying agent may suitably be polyoxyethylene
sorbitan monooleate (Tween 80). In a specific embodiment, a 0.5 ml
vaccine dose volume contains 1% (w/w) Tween 80, and a 0.7 ml
vaccine dose volume contains 0.7% (w/w) Tween 80. In another
specific embodiment the concentration of Tween 80 is 0.2% (w/w). A
suitable amount of polysorbate 80 is about 4.9 mg per vaccine dose,
suitably from 4.6 to 5.2 mg per vaccine dose.
[0098] Suitably a vaccine dose comprises alpha-tocopherol in an
amount of about 11.9 mg per vaccine dose, squalene in an amount of
10.7 mg per vaccine dose, and polysorbate 80 in an amount of about
4.9 mg per vaccine dose.
[0099] The oil in water emulsion adjuvant may be utilised with
other adjuvants or immuno-stimulants and therefore an important
embodiment of the invention is an oil in water formulation
comprising squalene or another metabolisable oil, a tocopherol,
such as alpha tocopherol, and tween 80. The oil in water emulsion
may also contain span 85 and/or Lecithin. Typically the oil in
water will comprise from 2 to 10% squalene of the total volume of
the immunogenic composition, from 2 to 10% alpha tocopherol and
from 0.3 to 3% Tween 80, and may be produced according to the
procedure described in WO 95/1721 0. Preferably the ratio of
squalene: alpha tocopherol is equal or less than 1 as this 20
provides a more stable emulsion. Span 85 (polyoxyethylene sorbitan
trioleate) may also be present, for example at a level of 1%.
Immunogenic Properties of the Immunogenic Composition Used for the
First Vaccination of the Present Invention
[0100] In the present invention the monovalent influenza
composition is capable of inducing an improved CD4 T-cell immune
response against at least one of the component antigen(s) or
antigenic composition compared to the CD4 T-cell immune response
obtained with the corresponding composition which in un-adjuvanted,
i.e. does not contain any exogeneous adjuvant (herein also referred
to as `plain composition`). In a specific embodiment, said improved
CD4 T-cell immune response is against the pandemic influenza
strain.
[0101] By `improved CD4 T-cell immune response is meant that a
higher CD4 response is obtained in a human patient after
administration of the adjuvanted immunogenic composition than that
obtained after administration of the same composition without
adjuvant. For example, a higher CD4 T-cell response is obtained in
a human patient upon administration of an immunogenic composition
comprising an influenza virus or antigenic preparation thereof
together with an oil-in-water emulsion adjuvant comprising a
metabolisable oil, a tocopherol, such as alpha tocopherol, and an
emulsifying agent, compared to the response induced after
administration of an immunogenic composition comprising an
influenza virus or antigenic preparation thereof which is
un-adjuvanted. Such formulation will advantageously be used to
induce anti-influenza CD4-T cell response capable of detection of
influenza epitopes presented by MHC class 11 molecules.
[0102] Suitably said immunological response induced by an
adjuvanted split influenza composition for use in the present
invention is higher than the immunological response induced by any
other un-adjuvanted influenza conventional vaccine, such as
sub-unit influenza vaccine or whole influenza virus vaccine.
[0103] In particular but not exclusively, said `improved CD4 T-cell
immune response` is obtained in an immunologically unprimed
patient, i.e. a patient who is seronegative to said influenza virus
or antigen. This seronegativity may be the result of said patient
having never faced such virus or antigen (so-called `naive`
patient) or, alternatively, having failed to respond to said
antigen once encountered. Preferably said improved CD4 T-cell
immune response is obtained in an immunocompromised subject such as
an elderly, typically at least 50 years of age, typically 65 years
of age or above, or an adult below 65 years of age with a high risk
medical condition (`high risk` adult), or a child under the age of
two.
[0104] The improved CD4 T-cell immune response may be assessed by
measuring the number of cells producing any of the following
cytokines: [0105] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0106] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0107] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0108] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0109] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
[0110] There will be improved CD4 T-cell immune response when cells
producing any of the above cytokines will be in a higher amount
following administration of the adjuvanted composition compared to
the administration of the un-adjuvanted composition. Typically at
least one, preferably two of the five conditions mentioned herein
above will be fulfilled.
[0111] In a particular embodiment, the cells producing all four
cytokines will be present at a higher amount in the adjuvanted
group compared to the un-adjuvanted group.
[0112] In a specific embodiment, an improved CD4 T-cell immune
response may be conferred by the adjuvanted influenza composition
of the present invention and may be ideally obtained after one
single administration. The single dose approach will be extremely
relevant for example in a rapidly evolving outbreak situation. In
certain circumstances, especially for the elderly population, or in
the case of young children (below 9 years of age) who are
vaccinated for the first time against influenza, or in the case of
a pandemics, it may be beneficial to administer two doses of the
same composition for that season. The second dose of said same
composition (still considered as `composition for first
vaccination`) may be administered during the on-going primary
immune response and is adequately spaced. Typically the second dose
of the composition is given a few weeks, or about one month, e.g. 2
weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the first dose,
to help prime the immune system in unresponsive or poorly
responsive individuals.
[0113] In a specific embodiment, the administration of said
immunogenic composition alternatively or additionally induces an
improved B-memory cell response in patients administered with the
adjuvanted immunogenic composition compared to the B-memory cell
response induced in individuals immunized with the un-adjuvanted
composition. An improved B-memory cell response is intended to mean
an increased frequency of peripheral blood B lymphocytes capable of
differentiation into antibody-secreting plasma cells upon antigen
encounter as measured by stimulation of in-vitro differentiation
(see Example sections, e.g. methods of Elispot B cells memory).
[0114] In a still further specific embodiment, the vaccination with
the composition for the first vaccination, adjuvanted, has no
measurable impact on the CD8 response.
[0115] Suitably, the claimed composition comprising an influenza
virus or antigenic preparation thereof formulated with an
oil-in-water emulsion adjuvant, in particular an oil-in-water
emulsion adjuvant comprising a metabolisable oil, a sterol or a
tocopherol, such as alpha tocopherol, and an emulsifying agent,
will be effective in promoting T cell responses in an
immuno-compromised human population. Suitably, the administration
of a single dose of the immunogenic composition for first
vaccination, as described in the invention will be capable of
providing better sero-protection, as assessed by the correlates of
protection for influenza vaccines, following revaccination against
influenza in a human elderly population, than does the vaccination
with an un-adjuvanted influenza vaccine. The claimed adjuvanted
formulation will also induce an improved CD4 T-cell immune response
against influenza virus compared to that obtained with the
un-adjuvanted formulation. This property can be associated with an
increased responsiveness upon vaccination or infection vis-a-vis
influenza antigenic exposure. Furthermore, this may also be
associated with a cross-responsiveness, i.e. a higher ability to
respond against variant influenza strains. This improved response
may be especially beneficial in an immuno-compromised human
population such as the elderly population (65 years of age and
above) and in particular the high risk elderly population. This may
result in reducing the overall morbidity and mortality rate and
preventing emergency admissions to hospital for pneumonia and other
influenza-like illness. This may also be of benefit to the infant
population (below 5 years, preferably below 2 years of age).
Furthermore it allows inducing a CD4 T cell response which is more
persistent in time, e.g. still present one year after the first
vaccination, compared to the response induced with the
un-adjuvanted formulation.
[0116] Preferably the CD4 T-cell immune response, such as the
improved CD4 T-cell immune response obtained in an unprimed
subject, involves the induction of a cross-reactive CD4 T helper
response. In particular, the amount of cross-reactive CD4 T cells
is increased. By `cross-reactive` CD4 response is meant CD4 T-cell
targeting shared epitopes between influenza strains.
[0117] Usually, available influenza vaccines are effective only
against infecting strains of influenza virus that have
haemagglutinin of similar antigenic characteristics. When the
infecting (circulating) influenza virus has undergone minor changes
(such as a point mutation or an accumulation of point mutations
resulting in amino acid changes in the for example) in the surface
glycoproteins in particular haemagglutinin (antigenic drift variant
virus strain) the vaccine may still provide some protection,
although it may only provide limited protection as the newly
created variants may escape immunity induced by prior influenza
infection or vaccination. Antigenic drift is responsible for annual
epidemics that occur during interpandemic periods (Wiley &
Skehel, 1987, Ann. Rev. Biochem. 56, 365-394). The induction of
cross-reactive CD4 T cells provides an additional advantage to the
composition of the invention, in that it may provide also
cross-protection, in other words protection against heterologous
infections, i.e. infections caused by a circulating influenza
strain which is a variant (e.g. a drift) of the influenza strain
contained in the immunogenic composition. This may be advantageous
when the circulating strain is difficult to propagate in eggs or to
produce in tissue culture, rendering the use of a drifted strain a
working alternative. This may also be advantageous when the subject
received a first and a second vaccination several months or a year
apart, and the influenza strain in the immunogenic composition used
for a second immunization is a drift variant strain of the strain
used in the composition used for the first vaccination.
[0118] The adjuvanted influenza immunogenic composition as herein
defined has therefore a higher ability to induce sero-protection
and cross-reactive CD4 T cells in vaccinated elderly subjects. This
characteristic may be associated with a higher ability to respond
against a variant strain of the strain present in the immunogenic
composition. This may prove to be an important advantage in a
pandemic situation. For example a monovalent influenza immunogenic
composition comprising any of H5, a H2, a H9, H7 or H6 strain(s)
may provide a higher ability to respond against a pandemic variant,
i.e. a drift strain of said pandemic strain(s), either upon
subsequent vaccination with or upon infection by said drift
strain.
Detection of Cross-reactive CD4 T-cells Following Vaccination with
Influenza Vaccine
[0119] Following classical trivalent Influenza vaccine
administration (3 weeks), there is a substantial increase in the
frequency of peripheral blood CD4 T-cells responding to antigenic
strain preparation (whole virus or split antigen) that is
homologous to the one present in the vaccine (H3N2:
A/Panama/2007/99, H1N1: A/New Caledonia/20/99, B: B/Shangdong/7/97)
(see Example III). A comparable increase in frequency can be seen
if peripheral blood CD4 T-cells are restimulated with influenza
strains classified as drifted strains (H3N2: A/Sydney/5/97, H1N1:
A/Beijing/262195, B: B/Yamanashi/166/98).
[0120] In contrast, if peripheral blood CD4 T-cells are
restimulated with influenza strains classified as shift strains
(H2N2: A/Singapore/l/57, H9N2: A/Hongkong/1073/99) by expert in the
field, there is no observable increase following vaccination.
[0121] CD4 T-cells that are able to recognize both homologous and
drifted Influenza strains have been named in the present document
"cross-reactive". The adjuvanted influenza compositions as
described herein have been capable to show heterosubtypic
cross-reactivity since there is observable cross-reactivity against
drifted Influenza strains. As said above, the ability of a pandemic
vaccine formulation to be effective against drift pandemic strains
may prove to be an important characteristic in the case of
pandemics.
[0122] Consistently with the above observations, CD4 T-cell
epitopes shared by different Influenza strains have been identified
in human (Gelder C et al. 1998, Int Immunol. 10 (2):211-22; Gelder
C M et al. 1996 J Virol. 70 (7):4787-90; Gelder C M et al. 1995 J
Virol. 1995 69 (12):7497-506).
[0123] In a specific embodiment, the adjuvanted composition may
offer the additional benefit of providing better protection against
circulating strains which have undergone a major change (such as
gene recombination for example, between two different species) in
the haemagglutinin (antigenic shift) against which currently
available vaccines have no efficacy.
Other Adjuvants
[0124] The composition may comprise an additional adjuvant, in
particular a TRL-4 ligand adjuvant, suitably a non-toxic derivative
of lipid A. A suitable TRL-4 ligand is 3 de-O-acylated
monophosphoryl lipid A (3D-MPL). Other suitable TLR-4 ligands are
lipopolysaccharide (LPS) and derivatives, MDP (muramyl dipeptide)
and F protein of RSV.
[0125] In one embodiment the composition may additionally include a
Toll like receptor (TLR) 4 ligand, such as a non-toxic derivative
of lipid A, particularly monophosphoryl lipid A or more
particularly 3-Deacylated monophoshoryl lipid A (3D-MPL).
[0126] 3D-MPL is sold under the trademark MPL.RTM. by Corixa
corporation (herein MPL) and primarily promotes CD4+ T cell
responses with an IFN-.gamma. (Th1) phenotype. It can be produced
according to the methods disclosed in GB 2 220 211 A. Chemically it
is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or
6 acylated chains. Preferably in the compositions of the present
invention small particle 3 D- MPL is used. Small particle 3D -MPL
has a particle size such that it may be sterile-filtered through a
0.22 .mu.m filter. Such preparations are described in WO94/21292
and in Example II.
[0127] 3D-MPL can be used, for example, at an amount of 1 to 100
.mu.g (w/v) per composition dose, preferably in an amount of 10 to
50 .mu.g (w/v) per composition dose. A suitable amount of 3D-MPL is
for example any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 .mu.g (w/v) per composition dose. More preferably, 3D-MPL
amount ranges from 25 to 75 .mu.g (w/v) per composition dose.
Usually a composition dose will be ranging from about 0.5 ml to
about 1 ml. A typical vaccine dose are 0.5 ml, 0.6 ml, 0.7 ml, 0.8
ml, 0.9 ml or 1 ml. In a preferred embodiment, a final
concentration of 50 .mu.g of 3D-MPL is contained per ml of vaccine
composition, or 25 .mu.g per 0.5 ml vaccine dose. In other
preferred embodiments, a final concentration of 35.7 .mu.g or 71.4
.mu.g of 3D-MPL is contained per ml of vaccine composition.
Specifically, a 0.5 ml vaccine dose volume contains 25 .mu.g or 50
.mu.g of 3D-MPL per dose.
[0128] The dose of MPL is suitably able to enhance an immune
response to an antigen in a human. In particular a suitable MPL
amount is that which improves the immunological potential of the
composition compared to the unadjuvanted composition, or compared
to the composition adjuvanted with another MPL amount, whilst being
acceptable from a reactogenicity profile.
[0129] Synthetic derivatives of lipid A are known, some being
described as TLR-4 agonists, and include, but are not limited to:
[0130] OM174
(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phos-
phono-.beta.-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.alpha.-
-D-glucopyranosyldihydrogenphosphate), (WO 95/14026) [0131] OM 294
DP (3S, 9 R)
-3--[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-
-3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate-
) (WO99/64301 and WO 00/0462) [0132] OM 197 MP-Ac DP (3S-, 9R)
-3-[(R)
-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecano-
ylamino]decan-1,10-diol,1-dihydrogenophosphate
10-(6-aminohexanoate) (WO 01/46127)
[0133] Other suitable TLR-4 ligands are, for example,
lipopolysaccharide and its derivatives, muramyl dipeptide (MDP) or
F protein of respiratory syncitial virus.
[0134] Another suitable immunostimulant for use in the present
invention is Quil A and its derivatives. Quil A is a saponin
preparation isolated from the South American tree Quilaja Saponaria
Molina and was first described by Dalsgaard et al. in 1974
("Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol.
44, Springer Verlag, Berlin, p 243-254) to have adjuvant activity.
Purified fragments of Quil A have been isolated by HPLC which
retain adjuvant activity without the toxicity associated with Quil
A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and
QA21). QS-21 is a natural saponin derived from the bark of Quillaja
saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1
cells and a predominant IgG2a antibody response and is a preferred
saponin in the context of the present invention.
[0135] Particular formulations of QS21 have been described which
are particularly preferred, these formulations further comprise a
sterol (W096/33739). The saponins forming part of the present
invention may be in the form of an oil in water emulsion (WO
95/17210).
Revaccination and Composition Used for Revaccination (Boosting
Composition)
[0136] An aspect of the present invention provides the use of an
influenza antigen in the manufacture of an influenza immunogenic
composition for revaccination of humans previously vaccinated with
an monovalent influenza composition as claimed herein or with said
monovalent influenza composition comprising a variant influenza
strain, formulated with an oil-in-water emulsion adjuvant as herein
defined.
[0137] Typically revaccination is made at least 6 months after the
first vaccination(s), preferably 8 to 14 months after, more
preferably at around 10 to 12 months after.
[0138] The immunogenic composition for revaccination (the boosting
composition) may contain any type of antigen preparation, either
inactivated or live attenuated. It may contain the same type of
antigen preparation i.e. split influenza virus or split influenza
virus antigenic preparation thereof, a whole virion, a purified HA
and NA (sub-unit) vaccine or a virosome, as the immunogenic
composition used for the first vaccination. Alternatively the
boosting composition may contain another type of influenza antigen,
i.e. split influenza virus or split influenza virus antigenic
preparation thereof, a whole virion, a purified HA and NA
(sub-unit) vaccine or a virosome, than that used for the first
vaccination. Preferably a split virus or a whole virion vaccine is
used. The boosting composition may be adjuvanted or un-adjuvanted.
The un-adjuvanted boosting composition may be
Fluarix.TM./-Rix.RTM./Influsplit.RTM. given intramuscularly. The
formulation contains three inactivated split virion antigens
prepared from the WHO recommended strains of the appropriate
influenza season.
[0139] Accordingly, in a preferred embodiment, the invention
provides for the use of an influenza virus or antigenic preparation
thereof in the manufacture of an immunogenic composition for
revaccination of humans previously vaccinated with a monovalent
pandemic immunogenic composition as claimed herein.
[0140] The boosting composition may be adjuvanted or un-adjuvanted.
In a preferred embodiment, the boosting composition comprises an
oil-in-water emulsion adjuvant, in particular an oil-in-water
emulsion adjuvant comprising a metabolisable oil, a sterol or a
tocopherol, such as alpha tocopherol, and an emulsifying agent.
Preferably, said oil-in-water emulsion adjuvant preferably
comprises at least one metabolisable oil in an amount of 0.5% to
20% of the total volume, and has oil droplets of which at least 70%
by intensity have diameters of less than 1 .mu.m.
[0141] In one embodiment, the first vaccination is made with a
pandemic influenza composition, preferably a split influenza
composition, as herein defined and the re-vaccination is made as
follows.
[0142] In a specific embodiment, the immunogenic composition for
revaccination (also called herein below the `boosting composition`)
contains an influenza virus or antigenic preparation thereof which
shares common CD4 T-cell epitopes with the influenza virus or
antigenic preparation thereof used for the first vaccination. A
common CD4 T cell epitope is intended to mean
peptides/sequences/epitopes from different antigens which can be
recognised by the same CD4 cell (see examples of described epitopes
in: Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder CM et
al. 1996 J Virol. 70(7):4787-90; Gelder CM et al. 1995 J Virol.
1995 69(12):7497-506).
[0143] In an embodiment according to the invention, the boosting
composition is a monovalent influenza composition comprising an
influenza strain which is associated with a pandemic outbreak or
has the potential to be associated with a pandemic outbreak.
Suitable strains are, but not limited to: H5N1, H9N2, H7N7, H2N2
and H1 N1. Said strain may be the same as that, or one of those,
present in the composition used for the first vaccination. In an
alternative embodiment said strain may be a variant strain, i.e. a
drift strain, of the strain present in the composition used for the
first vaccination.
[0144] In another specific embodiment, the boosting composition is
a multivalent influenza vaccine. In particular, when the boosting
composition is a multivalent vaccine such as a bivalent, trivalent
or quadrivalent vaccine, at least one strain is associated with a
pandemic or has the potential to be associated with a pandemic. In
a specific embodiment, two or more strains in the boosting
composition are pandemic strains. In another specific embodiment,
the at least opne pandemic strain in the boosting composition is of
the same type as that, or one of those, present in the composition
used for the first vaccination. In an alternative embodiment the at
least one strain may be a variant strain, i.e. a drift strain, of
the at least one pandemic strain present in the composition used
for the first vaccination.
[0145] Accordingly, in another aspect of the present invention,
there is provided the use of an influenza virus or antigenic
preparation thereof, from a first pandemic influenza strain, in the
manufacture of an immunogenic composition as herein defined, for
protection against influenza infections caused by a influenza
strain which is a variant of said first influenza strain.
[0146] Accordingly, in another aspect of the present invention,
there is provided the use of: [0147] (a) an influenza virus or
antigenic preparation thereof, from a first influenza strain, and
[0148] (b) an oil-in-water emulsion adjuvant as herein defined in
the manufacture of an immunogenic composition as herein defined,
for protection against influenza infections caused by a influenza
strain which is a variant of said first influenza strain.
[0149] The boosting composition may be adjuvanted or not.
[0150] Typically a boosting composition, where used, is given at
the next influenza season, e.g. approximately one year after the
first immunogenic composition. The boosting composition may also be
given every subsequent year (third, fourth, fifth vaccination and
so forth). The boosting composition may be the same as the
composition used for the first vaccination. Suitably, the boosting
composition contains an influenza virus or antigenic preparation
thereof which is a variant strain of the influenza virus used for
the first vaccination. In particular, the influenza viral strains
or antigenic preparation thereof are selected according to the
reference material distributed by the World Health Organisation
such that they are adapted to the influenza strain which is
circulating on the year of the revaccination.
[0151] The influenza antigen or antigenic composition used in
revaccination preferably comprises an adjuvant or an oil-in-water
emulsion, suitably as described above. The adjuvant may be an
oil-in-water emulsion adjuvant as herein above described, which is
preferred, optionally containing an additional adjuvant such as
TLR-4 ligand such as 3D-MPL or a saponin, or may be another
suitable adjuvant such as alum or alum alternatives such as
polyphosphazene for example.
[0152] Preferably revaccination induces any, preferably two or all,
of the following: (i) an improved CD4 response against the
influenza virus or antigenic preparation thereof, or (ii) an
improved B cell memory response or (iii) an improved humoral
response, compared to the equivalent response induced after a first
vaccination with the un-adjuvanted influenza virus or antigenic
preparation thereof. Preferably the immunological responses induced
after revaccination with the adjuvanted influenza virus or
antigenic preparation thereof as herein defined, are higher than
the corresponding response induced after the revaccination with the
un-adjuvanted composition. Preferably the immunological responses
induced after revaccination with an un-adjuvanted, preferably
split, influenza virus are higher in the population first
vaccinated with the adjuvanted, preferably split, influenza
composition than the corresponding response in the population first
vaccinated with the un-adjuvanted, preferably split, influenza
composition.
[0153] In one aspect according to the invention, the revaccination
of the subjects with a boosting composition comprising an influenza
virus and an oil-in-water emulsion adjuvant comprising a
metabolisable oil, a sterol or a tocopherol, such as alpha
tocopherol, and an emulsifying agent, as defined herein above, will
show higher antibody titers than the corresponding values in the
group of people first vaccinated with the un-adjuvanted composition
and boosted with the un-adjuvanted composition. The effect of the
adjuvant in enhancing the antibody response to revaccination is
especially of importance in the elderly population which is known
to have a low response to vaccination or infection by influenza
virus. In particular, the adjuvanted composition-associated benefit
will also be marked in terms of improving the CD4 T-cell response
following revaccination.
[0154] The adjuvanted composition of the invention will be capable
of inducing a better cross-responsiveness against drifted strain
(the influenza strain from the next influenza season) compared to
the protection conferred by the control vaccine. Said
cross-responsiveness has shown a higher persistence compared to
that obtained with the un-adjuvanted formulation. The effect of the
adjuvant in enhancing the cross-responsiveness against drifted
strain is of important in a pandemic situation.
[0155] In a further embodiment the invention relates to a
vaccination regime in which the first vaccination is made with an
influenza composition, preferably a split influenza composition,
containing an influenza strain that could potentially cause a
pandemic and the revaccination is made with a composition, either
monovalent or multivalent, comprising at least one circulating
strain, either a pandemic strain or a classical strain.
CD4 Epitope in HA
[0156] This antigenic drift mainly resides in epitope regions of
the viral surface proteins haemagglutinin (HA) and neuraminidase
(NA). It is known that any difference in CD4 and B cell epitopes
between different influenza strains, being used by the virus to
evade the adaptive response of the host immune system, will play a
major role in influenza vaccination.
[0157] CD4 T-cell epitopes shared by different Influenza strains
have been identified in human (see for example: Gelder C et al.
1998, Int Immunol. 10(2):211-22; Gelder CM et al. 1996 J Virol.
70(7):4787-90; and Gelder CM et al. 1995 J Virol. 1995
69(12):7497-506).
[0158] In a specific embodiment, the revaccination is made by using
a boosting composition which contains an influenza virus or
antigenic preparation thereof which shares common CD4 T-cell
epitopes with the influenza virus antigen or antigenic preparation
thereof used for the first vaccination. The invention thus relates
to the use of the immunogenic composition comprising a pandemic
influenza virus or antigenic preparation thereof and an
oil-in-water emulsion adjuvant, in particular an oil-in-water
emulsion adjuvant comprising a metabolisable oil, a sterol or a
tocopherol, such as alpha tocopherol, and an emulsifying agent, in
the manufacture of a first vaccination-component of a multi-dose
vaccine, the multi-dose vaccine further comprising, as a boosting
dose, an influenza virus or antigenic preparation thereof which
shares common CD4 T-cell epitopes with the pandemic influenza virus
antigen or virus antigenic preparation thereof of the dose given at
the first vaccination.
Vaccination Means
[0159] The composition of the invention may be administered by any
suitable delivery route, such as intradermal, mucosal e.g.
intranasal, oral, intramuscular or subcutaneous. Other delivery
routes are well known in the art.
[0160] The intramuscular delivery route is particularly suitable
for the adjuvanted influenza composition. The composition according
to the invention may be presented in a monodose container, or
alternatively, a multidose container, particularly suitable for a
pandemic vaccine. In this instance an antimicrobial presentaive
such a thiomersal is typically present to present contamination
during use. A thiomersal concentration of 5 or 10 .mu.g/dose is
suitably present.
[0161] Intradermal delivery is another suitable route. Any suitable
device may be used for intradermal delivery, for example short
needle devices such as those described in US 4,886,499, US
5,190,521, US 5,328,483, US 5,527,288, US 4,270,537, US 5,015,235,
US 5,141,496, US 5,417,662. Intradermal vaccines may also be
administered by devices which limit the effective penetration
length of a needle into the skin, such as those described in
WO99/34850 and EP1092444, incorporated herein by reference, and
functional equivalents thereof. Also suitable are jet injection
devices which deliver liquid vaccines to the dermis via a liquid
jet injector or via a needle which pierces the stratum corneum and
produces a jet which reaches the dermis. Jet injection devices are
described for example in US 5,480,381, US 5,599,302, US 5,334,144,
US 5,993,412, US 5,649,912, US 5,569,189, US 5,704,911, US
5,383,851, US 5,893,397, US 5,466,220, US 5,339,163, US 5,312,335,
US 5,503,627, US 5,064,413, US 5,520, 639, US 4,596,556US
4,790,824, US 4,941,880, US 4,940,460, WO 97/37705 and WO 97/13537.
Also suitable are ballistic powder/particle delivery devices which
use compressed gas to accelerate vaccine in powder form through the
outer layers of the skin to the dermis. Additionally, conventional
syringes may be used in the classical mantoux method of intradermal
administration.
[0162] Another suitable administration route is the subcutaneous
route. Any suitable device may be used for subcutaneous delivery,
for example classical needle. Preferably, a needle-free jet
injector service is used, such as that published in WO 01/05453, WO
01/05452, WO 01/05451, WO 01/32243, WO 01/41840, WO 01/41839, WO
01/47585, WO 01/56637, WO 01/58512, WO 01/64269, WO 01/78810, WO
01/91835, WO 01/97884, WO 02/09796, WO 02/34317. More preferably
said device is pre-filled with the liquid vaccine formulation.
[0163] Alternatively the vaccine is administered intranasally.
Typically, the vaccine is administered locally to the
nasopharyngeal area, preferably without being inhaled into the
lungs. It is desirable to use an intranasal delivery device which
delivers the vaccine formulation to the nasopharyngeal area,
without or substantially without it entering the lungs.
[0164] Preferred devices for intranasal administration of the
vaccines according to the invention are spray devices. Suitable
commercially available nasal spray devices include Accuspray.TM.
(Becton Dickinson). Nebulisers produce a very fine spray which can
be easily inhaled into the lungs and therefore does not efficiently
reach the nasal mucosa. Nebulisers are therefore not preferred.
[0165] Preferred spray devices for intranasal use are devices for
which the performance of the device is not dependent upon the
pressure applied by the user. These devices are known as pressure
threshold devices. Liquid is released from the nozzle only when a
threshold pressure is applied. These devices make it easier to
achieve a spray with a regular droplet size. Pressure threshold
devices suitable for use with the present invention are known in
the art and are described for example in WO 91/13281 and EP 311 863
B and EP 516 636, incorporated herein by reference. Such devices
are commercially available from Pfeiffer GmbH and are also
described in Bommer, R. Pharmaceutical Technology Europe, September
1999.
[0166] Preferred intranasal devices produce droplets (measured
using water as the liquid) in the range 1 to 200 .mu.m, preferably
10 to 120 .mu.m. Below 10 .mu.m there is a risk of inhalation,
therefore it is desirable to have no more than about 5% of droplets
below 10 .mu.m. Droplets above 120 .mu.m do not spread as well as
smaller droplets, so it is desirable to have no more than about 5%
of droplets exceeding 120 .mu.m.
[0167] Bi-dose delivery is a further preferred feature of an
intranasal delivery system for use with the vaccines according to
the invention. Bi-dose devices contain two sub-doses of a single
vaccine dose, one sub-dose for administration to each nostril.
Generally, the two sub-doses are present in a single chamber and
the construction of the device allows the efficient delivery of a
single sub-dose at a time. Alternatively, a monodose device may be
used for administering the vaccines according to the invention.
[0168] Alternatively, the epidermal or transdermal vaccination
route is also contempletd in the present invention.
[0169] In one aspect of the present invention, the adjuvanted
immunogenic composition for the first administration may be given
intramuscularly, and the boosting composition, either adjuvanted or
not, may be administered through a different route, for example
intradermal, subcutaneous or intranasal. In a specific embodiment,
the composition for the first administration contains a HA amount
of less than 15 .mu.g for the pandemic influenza strain, and the
boosting composition may contain a standard amount of 15 .mu.g or,
suitably a low amount of HA, i.e. below 15 .mu.g, which, depending
on the administration route, may be given in a smaller volume.
Populations to Vaccinate
[0170] The target population to vaccinate is the entire population,
e.g. healthy young adults (e.g. aged 18-60), elderly (typically
aged above 60) or infants. The target population may in particular
be immuno-compromised human. Immuno-compromised humans generally
are less well able to respond to an antigen, in particular to an
influenza antigen, in comparison to healthy adults.
[0171] In one aspect according to the invention, the target
population is a population which is unprimed against influenza,
either being naive (such as vis a vis a pandemic strain), or having
failed to respond previously to influenza infection or vaccination.
Preferably the target population is elderly persons suitably aged
at least 60, or 65 years and over, younger high-risk adults (i.e.
between 18 and 60 years of age) such as people working in health
institutions, or those young adults with a risk factor such as
cardiovascular and pulmonary disease, or diabetes. Another target
population is all children 6 months of age and over, especially
children 6-23 months of age who experience a relatively high
influenza-related hospitalization rate. Preferably the target
population is elderly above 65 years of age.
Vaccination Regimes, Dosing and Efficacy Criteria
[0172] Suitably the immunogenic compositions according to the
present invention are a standard 0.5 ml injectable dose in most
cases, and contains less than 15 .mu.g of haemagglutinin antigen
component from a pandemic influenza strain, as measured by single
radial immunodiffusion (SRD) (J. M. Wood et al.: J. Biol. Stand. 5
(1977) 237-247; J. M. Wood et al., J. Biol. Stand. 9 (1981)
317-330). Suitably the vaccine dose volume will be between 0.5 ml
and 1 ml, in particular a standard 0.5 ml, or 0.7 ml vaccine dose
volume. Slight adaptation of the dose volume will be made routinely
depending on the HA concentration in the original bulk sample.
[0173] Suitably said immunogenic composition contains a low amount
of HA antigen--e.g any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
or 14 .mu.g of HA per influenza strain. Said low amount of HA
amount may be as low as practically feasible provided that it
allows to formulate a vaccine which meets the EU criteria for
efficacy, as detailed below (see Table 1 and the specific
parameters as set forth). A suitable low amount of HA is between 1
to 7.5 .mu.g of HA per influenza strain, suitably between 3.5 to 5
.mu.g such as 3.75 .mu.g of HA per influenza strain, typically
about 5 .mu.g of HA per influenza strain. Another suitable amount
of HA is between 0.1 and 5 .mu.g of HA per influenza strain,
suitably between 1.0 and 2 .mu.g of HA per influenza strain such as
1.9 .mu.g of HA per influenza strain.
[0174] Advantageously, a vaccine dose according to the invention,
in particular a low dose vaccine, may be provided in a smaller
volume than the conventional injected split flu vaccines, which are
generally around 0.5, 0.7 or 1 ml per dose. The low volume doses
according to the invention are preferably below 500 .mu.l, more
preferably below 300 .mu.l and most preferably not more than about
200 .mu.l or less per dose.
[0175] Thus, a preferred low volume vaccine dose according to one
aspect of the invention is a dose with a low antigen dose in a low
volume, e.g. about 15 .mu.g or about 7.5 .mu.g HA or about 3.0
.mu.g HA (per strain) in a volume of about 200 .mu.l.
[0176] The influenza medicament of the invention preferably meets
certain international criteria for vaccines. Standards are applied
internationally to measure the efficacy of influenza vaccines.
Serological variables are assessed according to criteria of the
European Agency for the Evaluation of Medicinal Products for human
use (CHMP/BWP/214/96, Committee for Proprietary Medicinal Products
(CPMP). Note for harmonization of requirements for influenza
vaccines., 1997. CHMP/BWP/214/96 circular N.degree.96-0666:1-22)
for clinical trials related to annual licensing procedures of
influenza vaccines (Table 1). The requirements are different for
adult populations (18-60 years) and elderly populations (>60
years) (Table 1). For interpandemic influenza vaccines, at least
one of the assessments (seroconversion factor, seroconversion rate,
seroprotection rate) should meet the European requirements, for all
strains of influenza included in the vaccine. The proportion of
titres equal or greater than 1:40 is regarded most relevant because
these titres are expected to be the best correlate of protection
[Beyer W et al. 1998. Clin Drug Invest.;15:1-12].
[0177] As specified in the "Guideline on dossier structure and
content for pandemic influenza vaccine marketing authorisation
application. (CHMP/VEG/4717/03, Apr. 5, 2004), in the absence of
specific criteria for influenza vaccines derived from non
circulating strains, it is anticipated that a pandemic candidate
vaccine should at least be able to elicit sufficient immunological
responses to meet all three of the current standards set for
existing vaccines in unprimed adults or elderly subjects.
[0178] The compositions of the present invention suitably meet at
least one such criteria for the pandemic strain included in the
composition, suitably at least two, or typically at least all three
criteria for protection as set forth in Table 1. TABLE-US-00001
TABLE 1 18-60 years >60 years Seroconversion rate* >40%
>30% Conversion factor** >2.5 >2.0 Protection rate***
>70% >60% *Seroconversion rate is defined as the proportion
of subjects in each group having a protective post-vaccination
titre .gtoreq.1:40. **Conversion factor is defined as the fold
increase in serum HI geometric mean titres (GMTs) after
vaccination, for each vaccine strain. ***Protection rate is defined
as the the proportion of subjects who were either seronegative
prior to vaccination and have a protective post-vaccination titre
of .gtoreq.1:40 or who were seropositive prior to vaccination and
have a significant 4-fold increase in titre post-vaccination; it is
normally accepted as indicating protection.
[0179] Accordingly, in one aspect of the invention, it is provided
for a composition, method or use as claimed herein wherein said
immune response or protection induced by the administration of the
contemplated pandemic composition meets all three EU regulatory
criteria for influenza vaccine efficacy. Suitably at least one,
suitably two, or three of following criteria are met for the
pandemic strain of the composition: [0180] a seroconversion rate of
>50%, of >60%, of >70%, suitably of >80% or >90% in
the adult population (aged 18-60), and/or suitably also in the
elderly population (aged >60 years); [0181] a protection rate of
>75%, of >80%, of >85%, suitably of >90% in the adult
population (aged 18-60), and/or suitably also in the elderly
population (aged >60 years); [0182] a conversion factor of
>4.0, of >5.0, of >6.0, of >7.0, of >8.0, of >9.0
or of 10 or above 10 in the adult population (aged 18-60), and/or
suitably also in the elderly population (aged >60 years).
[0183] In a specific embodiment the composition according to the
invention will meet both a seroconversion rate of >60%, or
>70%, or suitably >80% and a protection rate of >75%,
suitably of >80% in the adult population. In another specific
embodiment the composition according to the invention will meet
both a conversion factor of >5.0, or >7.0 or suitably
>10.0 and a seroconversion rate of >60%, or >70%, or
suitably >80% in the adult population. In another specific
embodiment, the composition according to the invention will meet
both a conversion factor of >5.0, or >7.0 or suitably
>10.0, and a protection rate of >75%, suitably >80% in the
adult population. In still another specific embodiment the
composition according to the invention will meet both a conversion
factor of 10.0 or above, a seroconversion rate of 80% or above, and
a protection rate of 80% or above.
[0184] Suitably any or all of such criteria are also met for other
populations, such as in children and in any immuno-compromised
population.
[0185] In respect of the composition for re-vaccination, when it is
a multivalent composition, at least two or all three of the
criteria will need to be met for all strains, particularly for a
new vaccine such as a new vaccine for delivery via a different
route. Under some circumstances two criteria may be sufficient. For
example, it may be acceptable for two of the three criteria to be
met by all strains while the third criterion is met by some but not
all strains (e.g. two out of three strains).
[0186] Suitably the above response(s) is(are) obtained after one
dose, or typically after two doses.
[0187] In a further aspect the invention provides a method of
designing a vaccine for diseases known to be cured or treated
through a CD4+ T cell activation, comprising [0188] 1) selecting an
antigen containing CD4+ epitopes, and [0189] 2) combining said
antigen with an oil-in-water emulsion adjuvant as defined herein
above, wherein said vaccine upon administration in said mammal is
capable of inducing an enhanced CD4 T cell response in said
mammal.
[0190] The teaching of all references in the present application,
including patent applications and granted patents, are herein fully
incorporated by reference. Any patent application to which this
application claims priority is incorporated by reference herein in
its entirety in the manner described herein for publications and
references.
[0191] For the avoidance of doubt the terms `comprising`,
`comprise` and `comprises` herein is intended by the inventors to
be optionally substitutable with the terms `consisting of`,
`consist of`, and `consists of`, respectively, in every
instance.
[0192] The invention will be further described by reference to the
following, non-limiting, examples: [0193] Example I describes
immunological read-out methods used ferret and human studies.
[0194] Example II describes the preparation and characterization of
the oil in water emulsion and adjuvant formulations used in the
studies exemplified. [0195] Example III shows a pre-clinical
evaluation of adjuvanted and un-adjuvanted influenza vaccines in
ferrets. [0196] Example IV describes a clinical trial in an adult
population aged 18-60 years with a vaccine containing a split
influenza antigen preparation from a panemic H5N1 strain and AS03
adjuvant.
EXAMPLE I
Immunological Read-out Methods
[0196] 1.1. Ferrets Methods
[0197] Suitable methods are given below which are routinely used
for experiments performed 30 with seasonal strains. The skilled
reader will understand that it may need some adaptation or
optimization depending on the influenza strain used.
[0198] I.2.1. Hemagglutination Inhibition Test (HI)
[0199] Test Procedure.
[0200] Anti-Hemagglutinin antibody titers to the influenza virus
strain are determined using the hemagglutination inhibition test
(HI). The principle of the HI test is based on the ability of
specific anti-Influenza antibodies to inhibit hemagglutination of
chicken red blood cells (RBC) by influenza virus hemagglutinin
(HA). After pre-treatment of sera (cholera, RDE, heat inactivation,
. . . ), two-fold dilutions of sera are incubated with 4
hemagglutination units of the influenza strain. Chicken
(adaptation: turkey, or horse) red blood cells are then added and
the inhibition of agglutination is scored. The titers are expressed
as the reciprocal of the highest dilution of serum that completely
inhibited hemagglutination. As the first dilution of sera was 1:10,
an undetectable level was scored as a titer equal to 5.
[0201] I.2.2. Body Temperature Monitoring
[0202] Individual temperatures are monitored during the challenge
period with the transmitters and by the telemetry recording. All
implants are checked and refurbished and a new calibration is
performed before placement in the intraperitoneal cavity.
[0203] Temperatures are recorded continually before and after the
challenge
[0204] I.2.3. Nasal Washes
[0205] The nasal washes are performed by administration of 5 ml of
PBS in both nostrils in awoke animals. The inoculum is collected in
a Petri dish and placed into sample containers on dry ice.
[0206] Viral Titration in Nasal Washes
[0207] All nasal samples are first sterile filtered through Spin X
filters (Costar) to remove any bacterial contamination. 50 p1 of
serial ten-fold dilutions of nasal washes are transferred to
microtiter plates containing 50 .mu.l of medium (10
wells/dilution). 100 .mu.l of MDCK cells (2.4.times.10.sup.5
cells/ml) were then added to each well and incubated at 35.degree.
C. for 5-7 days. The viral titration was read and visualized by
screening for cytopathic effects (CPE). The amount of infectious
virus in a culture supernatant can be titrated by determining the
dilution that causes CPE in 50% of the inoculated cell cultures.
The dilution is called the 50% tissue culture infectious dose
endpoint (TClD50) and can be calculated using the "Reed and Muench"
method and expressed as Log TClD50/ml.
1.2. Assays for Assessing the Immune Response in Humans
[0208] I.2.1. Hemagglutination Inhibition Assay
[0209] The immune response was determined by measuring HI
antibodies using the method described by the WHO Collaborating
Centre for influenza, Centres for Disease Control, Atlanta, USA
(1991).
[0210] Antibody titre measurements were conducted on thawed frozen
serum samples with a standardised and comprehensively validated
micromethod using 4 hemagglutination-inhibiting units (4 HIU) of
the appropriate antigens and a 0.5% fowl (or 0.5% fowl and horse
for H5N1) erythrocyte suspension. Non-specific serum inhibitors
were removed by heat treatment and receptor-destroying enzyme.
[0211] The sera obtained were evaluated for HI antibody levels.
Starting with an initial dilution of 1 :10, a dilution series (by a
factor of 2) was prepared up to an end dilution of 1:20480.
[0212] The titration end-point was taken as the highest dilution
step that showed complete inhibition (100%) of hemagglutination.
All assays were performed in duplicate. Adaptation for H5N1
[0213] Specific Description of HI Using Horse Eerythrocytes:
[0214] Glycoproteins (haemaglutinins) are located in the viral
envelope, and are able to agglutinate erythrocytes (red blood
cells) of many species e.g. chicken.
[0215] The haemagglutination inhibition test is carried out in two
steps: [0216] 1. Antigen-antibody reaction: the Influenza antigen
(DTA, dialysis test antigen) reacts with the antibodies of the
subject's serum. [0217] 2. Agglutination of excessive antigen:
excessive antigen reacts with added red blood cells
[0218] Erythrocytes of horses are used for the H5N1 Pandemic
strains.
[0219] 0.5% (end concentration) horse red blood cell suspension in
phosphate buffer containing 0.5% BSA (bovine serum albumin, end
concentration).
[0220] This suspension is prepared every day by washing red blood
cell with the same phosphate buffer and a subsequent centrifugation
step (10 min, 2000 rpm). This washing step has to be repeated
once.
[0221] After the addition of the horse red blood cells to the
reaction mix of patient/subject sera and virus suspension the
plates have to be incubated at room temperature (RT, 20.degree. C.
+/-2.degree. C.) for two hours due to the low sedimentation rate of
the horse red blood cells.
[0222] I.2.2. Neuraminidase Inhibition Assay
[0223] The assay was performed in fetuin-coated microtitre plates.
A 2-fold dilution series of the antiserum was prepared and mixed
with a standardised amount of influenza A H3N2, H1 N1 or influenza
B virus. The test was based on the biological activity of the
neuraminidase which enzymatically releases neuraminic acid from
fetuin. After cleavage of the terminal neuraminic acid
.beta.-D-glactose-N-acetyl-galactosamin was unmasked. Horseradish
peroxidase (HRP)-labelled peanut agglutinin from Arachis hypogaea,
which binds specifically to the galactose structures, was added to
the wells. The amount of bound agglutinin can be detected and
quantified in a substrate reaction with tetra-methylbenzidine (TMB)
The highest antibody dilution that still inhibits the viral
neuraminidase activity by at least 50% was indicated is the NI
titre.
[0224] I.2.3. Neutralising Antibody Assay
[0225] Neutralising antibody measurements were conducted on thawed
frozen serum samples. Virus neutralisation by antibodies contained
in the serum is determined in a microneutralization assay. The sera
are used without further treatment in the assay. Each serum is
tested in triplicate. A standardised amount of virus is mixed with
serial dilutions of serum and incubated to allow binding of the
antibodies to the virus. A cell suspension, containing a defined
amount of MDCK cells is then added to the mixture of virus and
antiserum and incubated at 33.degree. C. After the incubation
period, virus replication is visualised by hemagglutination of
chicken red blood cells. The 50% neutralisation titre of a serum is
calculated by the method of Reed and Muench (Am.J; Hyg.1 938, 27:
493-497).
[0226] I.2.4. Cell-mediated Immunity was Evaluated by Cytokine Flow
Cytometry (CFC)
[0227] Peripheral blood antigen-specific CD4 and CD8 T cells can be
restimulated in vitro to produce IL-2, CD40L, TNF-alpha and IFN if
incubated with their corresponding antigen. Consequently,
antigen-specific CD4 and CD8 T cells can be enumerated by flow
cytometry following conventional immunofluorescence labelling of
cellular phenotype as well as intracellular cytokines production.
In the present study, Influenza vaccine antigen are used as antigen
to restimulate Influenza-specific T cells. Results are expressed as
a frequency of cytokine(s)-positive CD4 or CD8 T cell within the
CD4 or CD8 T cell sub-population.
[0228] I.2.5. Memory B Cells by ELISPOT
[0229] The ELISPOT technology allows the quantification of memory B
cells specific to a given antigen. Memory B-cells can be induced to
differentiate into plasma cells in vitro following cultivation with
CpG for 5 days. In vitro generated antigen-specific plasma cells
can therefore be enumerated using the ELISPOT assay. Briefly, in
vitro generated plasma cells are incubated in culture plates coated
with antigen. Antigen-specific plasma cells form antibody/antigen
spots, which can be detected by conventional immuno-enzymatic
procedure. In the present study, influenza vaccine strains or
anti-human Immunoglobulins are used to coat culture plates in order
to enumerate influenza-specific antibody or IgG secreting plasma
cells, respectively. Results are expressed as a frequency of
influenza-specific antibody secreting plasma cells within the
IgG-producing plasma cells.
[0230] I.2.6. Statistical Methods
[0231] I.2.6. 1. Primary Endpoints [0232] Percentage, intensity and
relationship to vaccination of solicited local and general signs
and symptoms during a 7 day follow-up period (i.e. day of
vaccination and 6 subsequent days) after vaccination and overall.
[0233] Percentage, intensity and relationship to vaccination of
unsolicited local and general signs and symptoms during a 21 day
follow-up period (i.e. day of vaccination and 20 subsequent days)
after vaccination and overall. [0234] Occurrence of serious adverse
events during the entire study.
[0235] I.2.6.2. Secondary endpoints
[0236] For the Humoral Immune Response:
[0237] Observed variables: [0238] At days 0 and 21: serum
hemagglutination-inhibition (HI) and NI antibody titres, tested
separately against each of the three influenza virus strains
represented in the vaccine (anti-H1 N1, anti-H3N2 &
anti-B-antibodies). [0239] At days 0 and 21: neutralising antibody
titres, tested separately against each of the three influenza virus
strains represented in the vaccine
[0240] Derived variables (with 95% confidence intervals): [0241]
Geometric mean titres (GMTs) of serum HI antibodies with 95%
confidence intervals (95% Cl) pre and post-vaccination [0242]
Seroconversion rates* with 95% Cl at day 21 [0243] Conversion
factors** with 95% Cl at day 21 [0244] Seroprotection rates*** with
95% Cl at day 21 [0245] Serum NI antibody GMTs` (with 95%
confidence intervals) at all timepoints. *Seroconversion rate
defined as the percentage of vaccinees who have at least a 4-fold
increase in serum HI titres on day 21 compared to day 0, for each
vaccine strain. **Conversion factor defined as the fold increase in
serum HI GMTs on day 21 compared to day 0, for each vaccine strain.
***Protection rate defined as the percentage of vaccinees with a
serum HI titre =40 after vaccination (for each vaccine strain) that
usually is accepted as indicating protection.
[0246] For the cell mediated immune (CMI) response
[0247] Observed variable
[0248] At days 0 and 21: frequency of cytokine-positive CD4/CD8
cells per 10.sup.6 in different tests. Each test quantifies the
response of CD4/CD8 T cell to: [0249] Peptide Influenza (pf)
antigen (the precise nature and origin of these antigens needs to
be given/explained [0250] Split Influenza (sf) antigen [0251] Whole
Influenza (wf) antigen.
[0252] Derived variables: [0253] cells producing at least two
different cytokines (CD40L, IL-2, IFN.gamma., TNF.alpha.) [0254]
cells producing at least CD40L and another cytokine (IL-2,
TNF.alpha., IFN.gamma.) [0255] cells producing at least IL-2 and
another cytokine (CD40L, TNF.alpha., IFN.gamma.) [0256] cells
producing at least IFN.gamma. and another cytokine (IL-2,
TNF.alpha., CD40L) [0257] cells producing at least TNF.alpha. and
another cytokine (IL-2, CD40L, IFN.gamma.)
[0258] I.3.5.3. Analysis of Immunogenicity
[0259] The immunogenicity analysis was based on the total
vaccinated cohort. For each treatment group, the following
parameters (with 95% confidence intervals) were calculated: [0260]
Geometric mean titres (GMTs) of HI and NI antibody titres at days 0
and 21 [0261] Geometric mean titres (GMTs) of neutralising antibody
titres at days 0 and 21. [0262] Conversion factors at day 21.
[0263] Seroconversion rates (SC) at day 21 defined as the
percentage of vaccinees that have at least a 4-fold increase in
serum HI titres on day 21 compared to day 0. [0264] Protection
rates at day 21 defined as the percentage of vaccinees with a serum
HI titre=1:40. [0265] The frequency of CD4/CD8 T-lymphocytes
secreting in response was summarised (descriptive statistics) for
each vaccination group, at each timepoint (Day 0, Day 21) and for
each antigen (Peptide influenza (pf), split influenza (sf) and
whole influenza (wf)). [0266] Descriptive statistics in individual
difference between timepoint (Post-Pre) responses fore each
vaccination group and each antigen (pf, sf, and wf) at each 5
different tests. [0267] A non-parametric test (Kruskall-Wallis
test) was used to compare the location differences between the 3
groups and the statistical p-value was calculated for each antigen
at each 5 different tests. All significance tests were two-tailed.
P-values less than or equal to 0.05 were considered as
statistically significant.
EXAMPLE II
Preparation and Characterization of the Oil in Water Emulsion and
Adjuvant Formulations
[0268] Unless otherwise stated, the oil/water emulsion used in the
subsequent examples is composed an organic phase made of 2 oils
(alpha-tocopherol and squalene), and an aqueous phase of PBS
containing Tween 80 as emulsifying agent. Unless otherwise stated,
the oil in water emulsion adjuvant formulations used in the
subsequent examples were made comprising the following oil in water
emulsion component (final concentrations given): 2.5% squalene
(v/v), 2.5% alpha-tocopherol (v/v), 0.9% polyoxyethylene sorbitan
monooleate (v/v) (Tween 80), see WO 95/17210. This emulsion, termed
AS03 in the subsequent examples, was prepared as followed as a
two-fold concentrate.
II.1. Preparation of emulsion SB62
[0269] II.1.1. Lab-scale Preparation
[0270] Tween 80 is dissolved in phosphate buffered saline (PBS) to
give a 2% solution in the PBS. To provide 100 ml two-fold
concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of
squalene are vortexed to mix thoroughly. 90 m1 of PBS/Tween
solution is added and mixed thoroughly. The resulting emulsion is
then passed through a syringe and finally microfluidised by using
an M110S microfluidics machine. The resulting oil droplets have a
size of approximately 120-180 nm (expressed as Z average measured
by PCS). The other adjuvants/antigen components are added to the
emulsion in simple admixture.
[0271] II.1.2. Scaled-up preparation
[0272] The preparation of the SB62 emulsion is made by mixing under
strong agitation of an oil phase composed of hydrophobic components
(.alpha.-tocopherol and squalene) and an aqueous phase containing
the water soluble components (Tween 80 and PBS mod (modified), pH
6.8). While stirring, the oil phase (1/10 total volume) is
transferred to the aqueous phase (9/10 total volume), and the
mixture is stirred for 15 minutes at room temperature. The
resulting mixture then subjected to shear, impact and cavitation
forces in the interaction chamber of a microfluidizer (15000 PSI--8
cycles) to produce submicron droplets (distribution between 100 and
200 nm). The resulting pH is between 6.8.+-.0.1. The SB62 emulsion
is then sterilised by filtration through a 0.22 .mu.m membrane and
the sterile bulk emulsion is stored refrigerated in Cupac
containers at 2 to 8.degree. C. Sterile inert gas (nitrogen or
argon) is flushed into the dead volume of the SB62 emulsion final
bulk container for at least 15 seconds.
[0273] The final composition of the SB62 emulsion is as follows:
[0274] Tween 80: 1.8% (v/v) 19.4 mg/ml; Squalene: 5% (v/v) 42.8
mg/ml; a-tocopherol: 5% (v/v) 47.5 mg/ml; PBS-mod: NaCl 121 mM, KCl
2.38 mM, Na2HPO4 7.14 mM, KH2PO4 1.3 mM; pH 6.8.+-.0.1. II.2.
Measure of Oil Droplet Size Dynamic Light Scattering
[0275] II.2.1. Introduction
[0276] The size of the diameter of the oil droplets is determined
according to the following procedure and under the following
experimental conditions. The droplet size measure is given as an
intensity measure and expressed as z average measured by PCS.
[0277] II.2.2. Sample Preparation
[0278] Size measurements have been performed on the oil-in-water
emulsion adjuvant: SB62 prepared following the scaled-up method,
AS03 and AS03+MPL (50 .mu.g/ml), the last two being prepared just
before use. The composition of the samples is given below (see
section II.2.4). Samples were diluted 4000.times.8000.times. in PBS
7.4.
[0279] As a control, PL-Nanocal Particle size standards 100 nm (cat
n.degree. 6011-1015) was diluted in 10 mM NaCl.
[0280] II.2.3. Malvern Zetasizer 3000HS Size Measurements
[0281] All size measurements were performed with both Malvern
Zetasizer 3000HS.
[0282] Samples were measured into a plastic cuvette for Malvern
analysis at a suitable dilution (usually at a dilution of
4000.times. to 20000.times. depending on the sample concentration),
and with two optical models: [0283] either real particle refractive
index of 0 and imaginary one of 0. [0284] or real particle
refractive index of 1.5 and imaginary one of 0.01 (the adapted
optical model for the emulsion, according to the values found in
literature).
[0285] The technical conditions were: [0286] laser wavelength: 532
nm (Zeta3000HS). [0287] laser power: 50 mW (Zeta3000HS). [0288]
scattered light detected at 900 (Zeta3000HS). [0289] temperature:
25.degree. C., [0290] duration: automatic determination by the
soft, [0291] number: 3 consecutive measurements, [0292] z-average
diameter: by cumulants analysis [0293] size distribution: by the
Contin or the Automatic method.
[0294] The Automatic Malvern algorithm uses a combination of
cumulants, Contin and non negative least squares (NNLS)
algorithms.
[0295] The intensity distribution may be converted into volume
distribution thanks to the Mie theory.
[0296] II.2.4. Results (See Table 2)
[0297] Cumulants Analysis (Z Average Diameter): TABLE-US-00002
TABLE 2 Sample Dilution Record Count rate ZAD Polydispersity SB62
5000 1 7987 153 0.06 2 7520 153 0.06 3 6586 152 0.07 average 7364
153 0.06 SB62 8000 1 8640 151 0.03 (Example IV) 2 8656 151 0.00 3
8634 150 0.00 average 8643 151 0.01 SB62 + 8000 1 8720 154 0.03 MPL
25 .mu.g (*) 2 8659 151 0.03 3 8710 152 0.02 average 8697 152 0.02
(*) Prepared as follows: Water for injection, PBS 10x concentrated,
250 .mu.l of SB62 emulsion and 25 .mu.g of MPL are mixed together
to reach a final volume of 280 .mu.l. The z-average diameter (ZAD)
size is weighed by the amount of light scattered by each size of
particles in the sample. This value is related to a monomodal
analysis of the sample and is mainly used for reproducibility
purposes. The count rate (CR) is a measure of scattered light: it
corresponds to thousands of photons per second. The polydispersity
(Poly) index is the width of the distribution. This is a
dimensionless measure of the distribution broadness.
[0298] Contin and Automatic Analysis:
[0299] Two other SB62 preparations (2 fold concentrated AS03) have
been made and assessed according to the procedure explained above
with the following minor modifications: Samples were measured into
a plastic cuvette for Malvern analysis, at two dilutions determined
to obtain an optimal count rate values: 10000.times. and
20000.times. for the Zetasizer 3000HS, the same optical models as
used in the above example.
[0300] Results are shown in Table 3. TABLE-US-00003 TABLE 3
Analysis in Analysis in IR Contin Automatic Imag- (mean in nm)
(mean in nm) SB62 Dilution Real inary Intensity Volume Intensity
Volume 1022 1/10000 0 0 149 167 150 -- 1.5 0.01 158 139 155 143
1/20000 0 0 159 200 155 196 1.5 0.01 161 141 147 -- 1023 1/10000 0
0 158 198 155 -- 1.5 0.01 161 140 150 144 1/20000 0 0 154 185 151
182 1.5 0.01 160 133 154 -- "--" when the obtained values were not
coherent.
[0301] A schematic representation of these results is shown in FIG.
1 for formulation 1023. As can be seen, the great majority of the
particles (e.g. at least 80%) have a diameter of less than 300 nm
by intensity.
[0302] II.2.5. Overall Conclusion
[0303] SB62 formulation was measured at different dilutions with
the Malvern Zetasizer 3000HS and two optical models. The particle
size ZAD (i.e. intensity mean by cumulant analysis) of the
formulations assessed above was around 150-155 nm.
[0304] When using the cumulants algorithm, we observed no influence
of the dilution on the ZAD and polydispersity.
EXAMPLE III
Pre-clinical Evaluation of an Adjuvanted Pandemic Split Influenza
Vaccines (Comprising H5N1 Strain) in Ferrets
III.1. Rationale and Objectives
[0305] Influenza infection in the ferret model closely mimics human
influenza, with regards both to the sensitivity to infection and
the clinical response. The ferret is extremely sensitive to
infection with both influenza A and B viruses without prior
adaptation of viral strains. Therefore, it provides an excellent
model system for studies of protection conferred by administered
influenza vaccines.
[0306] This study investigated the efficacy of H5N1 Split vaccines
adjuvanted with AS03 to protect ferrets against a lethal challenge
with a H5N1 homologous strain. The objective of this experiment was
to demonstrate the efficacy of an adjuvanted influenza vaccine
compared to ferrets immunized with PBS or the adjuvant alone.
III.2. Experimental Design
[0307] III.2.1. Treatment/Group (Table 4)
[0308] Female ferrets (Mustela putorius furo) (6 ferrets/group)
aged 14-20 weeks were injected intramuscularly on days 0 and 21
with a full human dose (500 .mu.l vaccine dose) of a dose range of
H5N1 A/Vietnam/1194/2004 (0.6 to 15 .mu.g HA). Ferrets were then
challenged on day 49 by the intranasal route with an homotypic
strain (5 Log TClD.sub.50/ml). TABLE-US-00004 TABLE 4 Antigen +/-
Group adjuvant Dosage Route/schedule Other treatment 1 PBS IM
Challenge H5N1 Days 0 and 21 (A/Vietnam/1194/04) Day 49 2 H5N1 AS03
15 .mu.g IM Challenge H5N1 HA Days 0 and 21 (A/Vietnam/1194/04) Day
49 3 H5N1 AS03 5 .mu.g IM Challenge H5N1 HA Days 0 and 21
(A/Vietnam/1194/04) Day 49 4 H5N1 AS03 1.7 .mu.g IM Challenge H5N1
HA Days 0 and 21 (A/Vietnam/1194/04) Day 49 5 H5N1 AS03 0.6 .mu.g
IM Challenge H5N1 HA Days 0 and 21 (A/Vietnam/1194/04) Day 49 6
AS03 alone IM Challenge H5N1 Days 0 and 21 (A/Vietnam/1194/04) Day
49
[0309] III.2.2. Preparation of the Vaccine Formulations
[0310] III.2.2.2. Split H5N1 Adjuvanted with the Oil-in-water
Emulsion Adjuvant AS03A in a 500 .mu.l Dose
[0311] Version 1
[0312] A premixed buffer is previously prepared in the Final Bulk
Buffer (PBS pH 7.4) containing Thiomersal, Tween 80 and Triton X100
in quantities taking into account their concentrations in the
strain. The day of the immunizations 15-5-1.7 or 0.6 .mu.g of H5N1
strain are added to the premixed buffer. After 30 minutes stirring,
250 .mu.l of SB62 emulsion is added. The formulation is stirred for
30 minutes. Injections occur within the hour following the end of
the formulation.
[0313] Version 2
[0314] Suitably, the formulation is prepared as follows. Tween 80,
Triton X100 and Thiomersal are added to the Final Bulk Buffer in
quantities taking into account their concentrations in the strain.
After 5 min stirring, 15-5-1.7 or 0.6 .mu.g of H5N1 strain are
added. After 30 minutes stirring, 250 .mu.l of SB62 emulsion is
added. The formulation is stirred for 30 minutes. Injections occur
within the hour following the end of the formulation.
[0315] III.2.2.3. AS03A in a 500 .mu.l Dose
[0316] Version 1
[0317] 250 .mu.l SB62 emulsion is mixed with 250 .mu.l PBS pH6.8,
stirred for 5 minutes and stored at 4.degree. C. until its
administration.
[0318] Version 2
[0319] Suitably the formulation is prepared as follows. 250 .mu.l
SB62 emulsion is mixed with 250 .mu.l PBS pH6.8 and stirred for 5
minutes. Injections occur within the hour following the end of the
formulation.
[0320] Remark: In each formulation, PBS 10 fold concentrated is
added to reach isotonicity and is 1 fold concentrated in the final
volume. H2O volume is calculated to reach the targeted volume.
[0321] III.2.3. Read-outs (Table 5) TABLE-US-00005 TABLE 5 Readout
Timepoint Analysis method Protection D + 4 Post challenge %
protection (number of ferrets alive/total number ferrets per
group)
III.3. Results and Conclusions
[0322] Table 6 summarizes the protection data obtained in ferrets
after challenge with a homologous strain. TABLE-US-00006 TABLE 6
Immunization Dead Alive % protection PBS* 4 1 20.00 0.6 .mu.g H5N1
AS03 2 4 66.67 1.7 .mu.g H5N1 AS03* 1 4 80.00 5 .mu.g H5N1 AS03 0 6
100.00 15 .mu.g H5N1 AS03 0 6 100.00 AS03 alone 6 0 0.00 Pooled
controls 10 1 9.09 (PBS & AS03) *1/6 ferrets died in
vaccination phase due to Aleutian disease.
[0323] Compared to control groups (PBS and AS03 alone) for which
only 9.09% protection was observed after the challenge with a
homologous H5N1 strain, a dose dependent protection was observed
following immunization of naive ferrets with H5N1 split vaccine
adjuvanted with AS03.
[0324] The lowest doses resulted in 66.67 and 80.00% protection
against the homologous challenge in ferrets immunized with 0.6 and
1.7 .mu.g H5N1 split vaccine adjuvanted with AS03,
respectively.
[0325] All ferrets immunized with the highest doses (5 and 15
.mu.g) of H5N1 split vaccine adjuvanted with AS03 were alive
following the challenge with the homologous strain (1 00.00%
protection).
[0326] Whatever the dose, all H5N1 split vaccine adjuvanted with
AS03 were statistically significant different than the control
groups (PBS or AS03 alone).
[0327] A statistical analysis performed on these data led to the
following conclusions: [0328] all vaccine doses were statistically
different from controls [0329] P values (Fischer's exact test) were
ranging from 0.0276 for lowest dose to 0.0006 for highest dose
[0330] the estimated dose to induce 90% protection was estimated in
this model to be 2.9 .mu.g [0331] the lowest dose to induce 100%
protection was estimated in this model to be between 2.9 and 5
.mu.g.
[0332] In summary, a dose dependant protection of naive ferrets
against a homologous H5N1 challenge was observed following
immunization with H5N1 split vaccine adjuvanted with AS03. Highest
doses (5 and 15 .mu.g) resulted in a full protection against the
H5N1 pandemic strain.
EXAMPLE IV
Clinical Trial in an Adult Population Aged in Adults Aged Between
18 and 60 Years with a Vaccine Containing a Split Influenza Antigen
Preparation and AS03 Adjuvant
IV.1. Introduction
[0333] A phase I, observer-blind, randomized study is currently
conducted in an adult population aged 18 to 60 years in 2006 in
order to evaluate the reactogenicity and the immunogenicity of
GlaxoSmithKline Biologicals pandemic influenza candidate
administered at different antigen doses (3.8 .mu.g, 7.5 .mu.g, 15
.mu.g and 30 .mu.g HA) adjuvanted or not with the adjuvant ASO3.
The humoral immune response (i.e. anti-hemagglutinin, neutralising
and anti-neuraminidase antibody titres) and cell mediated immune
response (CD4 and/or CD8 T cell responses) are measured 21 days
after each of the two intramuscular administration of the candidate
vaccine formulations. The non-adjuvanted groups served as reference
for the respective adjuvanted group receiving the same antigen
content.
IV.2. Study Design
[0334] Eight groups of 50 subjects each (planned) received in
parallel the following vaccine intramuscularly: [0335] one group of
50 subjects received two administrations of the pandemic split
virus influenza vaccine containing 3.8 .mu.g HA [0336] one group of
51 subjects received two administrations of the pandemic split
virus influenza vaccine containing 3.8 .mu.g HA and adjuvanted with
AS03 [0337] one group of 50 subjects received two administrations
of the pandemic split virus influenza vaccine containing 7.5 .mu.g
HA [0338] one group of 50 subjects received two administrations of
the pandemic split virus influenza vaccine containing 7.5 .mu.g HA
and adjuvanted with AS03 [0339] one group of 50 subjects received
two administrations of the pandemic split virus influenza vaccine
containing 15 .mu.g HA [0340] one group of 50 subjects received two
administrations of the pandemic split virus influenza vaccine
containing 15 .mu.g HA and adjuvanted with AS03 [0341] one group of
50 subjects received two administrations of the pandemic split
virus influenza vaccine containing 30 .mu.g HA [0342] one group of
49 subjects received two administrations of the pandemic split
virus influenza vaccine containing 30 .mu.g HA and adjuvanted with
AS03
[0343] The enrolment was performed to ensure that half of subjects
from each group will be aged between 18 and 30 years.
[0344] Vaccination schedule: two injection of pandemic influenza
candidate vaccine at day 0 and day 21, blood sample collection,
read-out analysis at day 21 and 42 (HI antibody determination, NI
antibody determination, determination of neutralising antibodies,
and CMI analysis), study conclusion (day 51) and study end (180
days).
IV.3. Study Objectives
[0345] IV.3.1. Primary Objectives [0346] To evaluate the humoral
immune response induced by the study vaccines in term of
anti-haemagglutinin antibody titers. [0347] To evaluate the safety
and reactogenicity of the study vaccines in term of solicited local
and general adverse events, unsolicited adverse events and serious
adverse events.
[0348] For the Humoral Immune Response:
[0349] Observed variables at days 0, 21, 42 and 180: serum
Heamagglutination-inhibition antibody titers.
[0350] Derived variables (with 95% confidence intervals): [0351]
Geometric mean titers (GMTs) of serum antibodies at days 0, 21, 42
and 180 [0352] Seroconversion rates* at days 21, 42 and 180 [0353]
Conversion factors** at days 21, 42 and 180 [0354] Protection
rates*** at days 0, 21, 42 and 180 Seroconversion rate for
Haemagglutinin antibody response is defined as the percentage of
vaccinees who have either a prevaccination titer <1:10 and a
post-vaccination titer.gtoreq. 1:40 or a prevaccination titer
.gtoreq.1:10 and at least a fourfold increase in post-vaccination
titer Conversion factor defined as the fold increase in serum HI
GMTs post-vaccination compared to day 0; Protection rate defined as
the percentage of vaccinees with a serum HI titer .gtoreq.40 after
vaccination that usually is accepted as indicating protection.
[0355] For the Safety/Reactogenicity Evaluation: [0356] 1.
Percentage, intensity and relationship to vaccination of solicited
local and general signs and symptoms during a 7 day follow-up
period (i.e. day of vaccination and 6 subsequent days) after each
vaccination and overall. [0357] 2. Percentage, intensity and
relationship to vaccination of unsolicited local and general signs
and symptoms during a 21 days follow-up period after the first
vaccination, during 30 days follow-up period after the second
vaccination and overall. Occurrence of serious adverse events
during the entire study.
[0358] IV.3.2. Secondary Objectives [0359] To evaluate the humoral
immune response induced by the study vaccines in term of serum
neutralizing antibody titers [0360] To evaluate the cell-mediated
immune response induced by the study vaccines in term of frequency
of influenza-specific CD4/CD8 T lymphocytes
[0361] In addition, the impact of vaccination on Influenza-specific
memory B cells using the Elispot technology will be evaluated.
[0362] For the Humoral Immune Response:
[0363] Observed variables at days 0, 21, 42 and 180: serum
neutralizing antibody titers.
[0364] Derived variables (with 95% confidence intervals): [0365]
Geometric mean titers (GMTs) of serum antibodies at days 0, 21, 42
and 180 [0366] Seroconversion rates* at day s21, 42 and 180
Seroconversion rate for Neutralising antibody response is defined
as the percentage of vaccinees with a minimum 4-fold increase in
titer at post-vaccination.
[0367] For the CMI Response: [0368] 1. Frequency of cytokine
CD4/CD8 cells per 10.sup.6 in tests producing at least two
different cytokines (CD40L, IL-2, TNF-.alpha., IFN-.gamma.) [0369]
2. Frequency of cytokine-positive CD4/CD8 cells per 10.sup.6 in
tests producing at least CD40L and another signal molecule (IL-2,
IFN-.gamma., TNF-.alpha.) [0370] 3. Frequency of cytokine-positive
CD4/CD8 cells per 10.sup.6 in tests producing at least IL-2 and
another signal molecule (CD40L, IFN-.gamma., TNF-.alpha.) [0371] 4.
Frequency of cytokine-positive CD4/CD8 cells per 10.sup.6 in tests
producing at least TNF-.alpha. and another signal molecule (IL-2,
IFN-.gamma., CD40L) Frequency of cytokine-positive CD4/CD8 cells
per 10.sup.6 in tests producing at least IFN-.gamma. and another
signal molecule (CD40L, IL-2, TNF-.alpha.).
[0372] At days 0, 21, 42 and 180: frequency of influenza-specific
memory B cells per 10.sup.6 cells in test.
IV.4. Vaccine Composition and Administration (Table 7)
[0373] III.4.1. Vaccine Preparation
[0374] III.4.1.1. Composition of AS03 Adjuvanted Influenza
Vaccine
[0375] AS03 is contains the oil-in-water SB62 emulsion, consisting
of an oil phase containing DL-.alpha.-tocopherol and squalene, and
an aqueous phase containing the non-ionic detergent polysorbate
80.
[0376] The active substance of the pandemic influenza vaccine
candidate is a formaldehyde inactivated split virus antigen derived
from the vaccine virus strain A/VietNam/1194/2004 (H5N1) NIBRG-14.
The dose of HA antigen is ranging from 3.8 to 30 .mu.g per
dose.
[0377] The split virus monovalent bulks used to produce the AS03
adjuvanted influenza vaccine are manufactured following the same
procedure as used for GSK Biologicals licensed interpandemic
influenza vaccine Fluarix.TM./.alpha.-Rix.RTM.. For the purpose of
this clinical trial the virus strain used to manufacture the
clinical lots is the H5N1 vaccine strain A/Vietnam/1194/04 NIBRG-14
recombinant H5N1 prototype vaccine strain derived from the highly
pathogenic A/Vietnam/1194/04. This recombinant prototype strain has
been developed by NIBSC using reverse genetics (a suitable
reference is Nicolson et al. 2005, Vaccine, 23, 2943-2952)). The
reassortant strain combines the H5 and N1 segments to the A/PR/8/34
strain backbone, and the H5 was engineered to eliminate the
polybasic stretch of amino-acids at the HA cleavage site that is
responsible for high virulence of the original strains. This was
achieved by transfecting Vero cells with plasmids containing the HA
gene (modified to remove the high pathogenicity determinants) and
NA gene of the human isolate A/Viet Nam/1194/2004 (H5N1) and
plasmids containing the internal genes of PR8. The rescued virus
was passaged twice on eggs and was then designated as the reference
virus NIBRG-14. The attenuated character of this H5N1 reassortant
was extensively documented in a preclinical safety assessment
(performed by NIBSC), as is also done routinely for the classical
flu vaccine strains.
[0378] The AS03-adjuvanted pandemic influenza candidate vaccine
according to the invention is a 2 component vaccine consisting of
0.5 ml of concentrated inactivated split virion antigens presented
in a type I glass vial and of a pre-filled type I glass syringe
containing 0.5 ml of the AS03 adjuvant. At the time of injection,
the content of the prefilled syringe containing the adjuvant is
injected into the vial that contains the concentrated inactivated
split vrion antigens. After mixing the content is withdrawn into
the syringe and the needle is replaced by an intramuscular needle.
One dose of the reconstituted the AS03-adjuvanted influenza
candidate vaccine corresponds to 1 ml. Each vaccine dose of 1 ml
contains 3.8 .mu.g, 7.5 .mu.g, 15.mu. or 30 .mu.g haemagglutinin
(HA) or any suitable HA amount which would have be determined such
that the vaccine meets the efficacy criteria as detailed
herein.
[0379] Alternatively, the AS03-adjuvanted pandemic influenza
candidate vaccine according to the invention is a 2 component
vaccine consisting of 0.25 ml of concentrated inactivated split
virion antigens presented in a type I glass vial and of a
pre-filled type I glass syringe containing 0.25 ml of the AS03
adjuvant. At the time of injection, the content of the prefilled
syringe containing the adjuvant is injected into the vial that
contains the concentrated inactivated split vrion antigens. After
mixing the content is withdrawn into the syringe and the needle is
replaced by an intramuscular needle. One dose of the reconstituted
the AS03-adjuvanted influenza candidate vaccine corresponds to 0.5
ml. Each vaccine dose of 0.5 ml contains 3.8 .mu.g, 7.5 .mu.g,
15.mu. or 30 .mu.g haemagglutinin (HA) or any suitable HA amount
which would have be determined such that the vaccine meets the
efficacy criteria as detailed herein.
[0380] The vaccine excipients are polysorbate 80 (Tween 80),
octoxynol 10 (Triton X-100), sodium chloride, disodium hydrogen
phosphate, potassium dihydrogen phosphate, potassium chloride,
magnesium chloride hexahydrate and water for injection. Thiomersal
has been added as an antimicrobial preservative to prevent
contamination during use, since it is anticipated that when a
pandemic occurs the main presentation will be presented in a
multidose container (vials or ampoules), for which a preservative
is required. For this reason, the pandemic vaccine is formulated
with thiomersal at 5 .mu.g/dose as preservative. Suitably the
pandemic vaccine may be formulated with thiomersal at 10 .mu.g/dose
as preservative or a slightly higher dose, such as up to 25
.mu.g/dose of vaccine.
[0381] III.4.1.2. Production of Split Inactivated Influenza H5N1
Antigen Preparation
[0382] The virus monobulks are prepared by growing H5N1 working
seed in embryonated hen's eggs. The manufacturing process for the
monovalent bulks of split, inactivated influenza H5N1 strain,
illustrated in FIG. 2, is identical to the manufacturing process
for the monovalent bulks of .alpha.-Rix.TM..
[0383] Basically, the manufacturing process of the monovalent bulks
can be divided in four main parts: [0384] 1) Propagation of the
working seed in fertilized hen's eggs, harvesting and pooling of
infected allantoic fluids so as to obtain the "crude monovalent
whole virus bulk" (step 1). [0385] 2) Purification of each virus
strain leading to the "purified monovalent whole virus bulk" (steps
2-6). [0386] 3) Splitting of the purified monovalent whole virus
bulk with sodium deoxycholate resulting in the "purified monovalent
split virus bulk" (steps 7-8/1). [0387] 4) Inactivation of the
purified monovalent split virus bulk in two steps by incubation
with sodium deoxycholate and with formaldehyde, followed by
ultrafiltration and sterile filtration, in order to obtain the
"purified monovalent inactivated split virus bulk", or "Monovalent
Bulk" (steps 8/2-9).
[0388] 1) Production of Crude Monovalent Whole Virus Bulk
[0389] Preparation of the Virus Inoculum:
[0390] On the day of inoculation of the embryonated eggs, an
inoculum is prepared by mixing the working virus seed lot with
phosphate buffer containing 25 .mu.g/mL hydrocortisone, and 0.5
mg/mL gentamicin sulfate. The virus inoculum is kept at room
temperature until the inoculation.
[0391] Inoculation of Embryonated Eggs:
[0392] Eleven day-old pre-incubated embryonated eggs are used for
virus replication. The eggs are transferred into the production
rooms after formaldehyde fumigation of the shells. Approximately
120,000 eggs are inoculated with 0.2 mL of the virus inoculum each
using an automatic egg inoculation apparatus. The inoculated eggs
are incubated at 34.0.degree. C. for 72 hours.
[0393] At the end of the incubation period, the eggs are inspected
visually for the presence of living embryo and age-adequate blood
vessels. The embryos are killed by cooling the eggs and stored for
12-46 hours at 2-8.degree. C.
[0394] Harvest
[0395] The allantoic fluid (approximately 12 mL) from the chilled
embryonated eggs is harvested by egg harvesting machines. The
allantoic fluids are collected in a stainless steel tank
thermo-regulated at 2-8.degree. C. At this stage the product is
called the "crude monovalent whole virus bulk". The crude
monovalent whole virus bulk is not stored but immediately
transferred to the clarification step.
[0396] 2) Production of Purified Monovalent Whole Virus Bulk
[0397] All operations are performed at 2-8.degree. C., until the
flow through ultracentrifugation, which is performed at room
temperature.
[0398] Clarification:
[0399] The harvested allantoic fluid is clarified by continuous
moderate speed centrifugation. This step removes big particles that
could have been collected during the harvest of the allantoic fluid
(e.g. parts of egg shells).
[0400] Adsorption Step:
[0401] This step permits to clarify further the allantoic fluid
through a precipitation of virus material, by adsorption to a
dibasic calcium hydrogen phosphate gel.
[0402] To obtain the dibasic calcium hydrogen phosphate
(CaHPO.sub.4) gel, 0.5 mol/L disodium hydrogen phosphate
(Na.sub.2HPO.sub.4) and 0.5 mol/L calcium chloride (CaCl.sub.2) are
added to the clarified virus pool to reach a final concentration of
1.87 g CaHPO.sub.4 per L.
[0403] After sedimentation for at least 8 hours to maximum 36
hours, the supernatant is removed and the sediment containing the
influenza viruses is re-solubilized by the addition of an 8.7%
disodium EDTA solution.
[0404] Filtration:
[0405] The resuspended influenza sediment is filtered through a
6-.mu.m filter membrane to remove potential remaining pellets.
[0406] Flow through Ultracentrifugation:
[0407] The influenza virus is further purified (removal of proteins
and phospholipids) and concentrated by isopycnic
ultracentrifugation in a linear sucrose gradient (0-55%) at a flow
rate of 8-20 liters per hour. The gradient is formed using the
sucrose solution 55% (w/w) with 0.01% thimerosal, and a Phosphate
buffer pH 7.4 with 0.01% thimerosal. This is done in the presence
of 100.+-.15 .mu.g/mL thiomersal in order to control the process
bioburden, as the centrifugation is performed at room
temperature.
[0408] Four different fractions are recovered by measuring the
sucrose concentration via a refractometer: [0409] Fraction 4/1:
55-47% sucrose [0410] Fraction 4/2: 47-38% sucrose [0411] Fraction
4/3: 38-20% sucrose [0412] Fraction 4/4: 20-0% sucrose
[0413] The upper limit of fraction 4/2 is selected to balance
between a high purity coefficient HA/protein and a maximum recovery
of whole virus. The limit between fractions 4/2 and 4/3 is selected
to minimize the ovalbumin content in fraction 4/2. The lower limit
of fraction 4/3 is selected on the basis of the HA content found in
the low sucrose gradient range. Fractions 4/2 and 4/3 are used for
further preparations. Most of the virus is collected in Fraction
4/2. Fraction 4/3, which contains both virus and proteins, is
further purified. First, the sucrose concentration of Fraction 4/3
is reduced below 6% (necessary for the subsequent centrifugation
step) by ultrafiltration. Then, Fraction 4/3 is pelleted via
centrifugation to remove any soluble contaminants (proteins). The
pellet is re-suspended in phosphate buffer pH 7.4 and thoroughly
mixed to obtain a homogeneous suspension. The holding times are
maximum 36 hours for Fraction 4/3, maximum 60 hours for Fraction
4/2 and maximum 36 hours for the purified Fraction 4/3.
[0414] Dilution
[0415] Both fractions, the treated Fraction 4/3 and untreated
Fraction 4/2, are pooled and diluted by adding 60 L of phosphate
buffer pH 7.4.
[0416] At this stage, the pool of material corresponds to the
"purified monovalent whole virus bulk".
[0417] 3) Preparation of the Purified Monovalent Split Virus
Bulk
[0418] Flow through Ultracentrifugation in the Presence of Sodium
Deoxycholate:
[0419] The influenza virus is splitted and further purified by
centrifugation through a linear sucrose gradient (0-55%-formed with
sucrose solution S8a and buffer S6a) that contains 1.5% sodium
deoxycholate. Tween-80 is present at 0.1% in the gradient. The
virus is processed at a rate of 8 liters per hour. At the end of
the centrifugation, three different fractions are collected. The
range of the main fraction (Fraction 7/2) is selected based on
strain-dependent validation of splitting conditions, with as
objective to collect a fraction consisting of predominantly
disrupted influenza virus antigen, while minimizing as much as
possible remaining whole virus particles and phospholipids coming
from the virus membrane after splitting.
[0420] For A/Vietnam/1194/2004 NIBRG-145 the range of fraction 7/2
is set at 20-41% sucrose. The haemagglutinin antigen is
concentrated in Fraction 7/2, which contains approximately 1.2%
sodium deoxycholate. This material corresponds to the "purified
monovalent split virus bulk".
[0421] 4) Preparation of the Purified Final Monovalent Split,
Inactivated Virus Bulk
[0422] Filtration:
[0423] Fraction 7/2 is diluted threefold in phosphate buffer S7c,
which contains 0.025% Tween-80. Then, fraction 7/2 is gradually
filtered down to a 0.45 .mu.m filter membrane, briefly sonicated
(to facilitate filtration) and filtered through a 0.2 .mu.m
membrane. At the end of the filtration, the filters are rinsed with
phosphate buffer (S107c) containing 0.025% Tween-80. As a result of
the filtration and rinsing, the final volume of the filtrate is 5
times the original fraction 7/2 volume.
[0424] Sodium Deoxycholate Inactivation:
[0425] The resulting solution is incubated at 22.+-.2.degree. C.
for at least 84 hours. After completion of the first inactivation
step, the material is diluted with phosphate buffer S7c to reduce
the total protein content to a calculated concentration of 500
.mu.g/mL:
[0426] Formaldehyde Inactivation:
[0427] Formaldehyde is added to a calculated final concentration of
100 .mu.g/mL. Inactivation takes place in a single use low density
polyethylene 100 L bag at 20.+-.2.degree. C. for at least 72
hours.
[0428] Ultrafiltration:
[0429] The inactivated split virus material is ultrafiltered
through membranes with a molecular weight cut off of 30,000 Dalton,
using consecutively buffers S7b and S1b
[0430] After a volume reduction, the volume remains constant during
ultrafiltration (diafiltration) by adding phosphate buffer and
phosphate buffered saline (S1b) containing 0.01% Tween-80.
[0431] During ultrafiltration, the content of formaldehyde, NaDoc
and sucrose is reduced.
[0432] The material is concentrated to 15-25 liters and is
transferred immediately to the final filtration step.
[0433] Sterile Filtration:
[0434] After ultrafiltration, the split inactivated material is
gradually filtered down to a 0.2 .mu.m membrane.
[0435] The final sterile filtration through a 0.22 .mu.m sterile
grade membrane is performed in a Class 100 environment. At the end
of the filtration the filters are rinsed with phosphate buffered
saline solution S1b, containing 0.01% Tween-80. Herewith, the
filtrate is diluted to a protein concentration less than 1000
.mu.g/mL, to avoid aggregation during subsequent storage.
[0436] The resulting material is the "purified monovalent
inactivated split virus bulk" or "monovalent bulk".
[0437] Storage:
[0438] The final monovalent bulks of split inactivated influenza
H5N1 viruses are stored at 2-8.degree. C. for a maximum of 18
months in Type I glass bottles.
[0439] III.4.1.3. Preparation of the Vaccine Compositions with AS03
Adjuvanted H5N1
[0440] 1) Composition
[0441] The AS03 adjuvanted inactivated split virus pandemic
influenza candidate vaccine to be evaluated in the phase I clinical
trial H5N1-007 is intended for intramuscular administration. The
vaccine is a 2 component vaccine consisting of concentrated
inactivated split virion (H5N1) antigens presented in a type I
glass vial and of the AS03 adjuvant contained in a pre-filled type
I glass syringe.
[0442] One dose of reconstituted AS03-adjuvanted pandemic influenza
vaccine corresponds to 1 ml. The composition is given in Table 7.
Since study H5N1-007 is a dose finding study, the HA content per
dose is different for each of the clinical lots to be tested. One
dose contains 3.8, 7.5,15 or 30 .mu.g HA.
[0443] The vaccine contains the following residuals from the
manufacturing process of the drug substance: formaldehyde,
ovalbumin, sucrose, thiomersal and sodium deoxycholate.
TABLE-US-00007 TABLE 7 Composition of the reconstituted AS03
adjuvanted pandemic influenza candidate vaccine Component Quantity
per dose Active Ingredients Inactivated split virions 30/15/7.5/3.8
.mu.g HA A/VietNam/1194/2004 NIBRG-14 (H5N1) AS03 Adjuvant SB62
emulsion squalene 10.68 mg DL-.alpha.-tocopherol 11.86 mg
Polysorbate 80 (Tween 80) 4.85 mg Excipients Polysorbate 80 (Tween
80).sup.1 12.26 .mu.g/.mu.g HA Octoxynol 10 (Triton X-100).sup.2
1.16 .mu.g/.mu.g HA Thiomersal 5 .mu.g Sodium chloride 7.5 mg
Disodium hydrogen phosphate 1 mg Potassium dihydrogen phosphate
0.36 mg Potassium chloride 0.19 mg Magnesium chloride hexahydrate
2.38/12.84/18.07/20.65 .mu.g
[0444] 2) Formulation
[0445] The manufacturing of the AS03-adjuvanted pandemic influenza
vaccine consists of three main steps: [0446] (a) Formulation of the
split virus final bulk (2.times. concentrated) without adjuvant and
filling in the antigen container [0447] (b) Preparation of the AS03
adjuvant and filling in the adjuvant container [0448] (c)
Extemporaneous reconstitution of the AS03 adjuvanted split virus
vaccine
[0449] 1) Formulation of the Final Bulk without Adjuvant and
Filling in the Antigen Container.
[0450] The formulation flow diagram is presented in FIG. 2.
[0451] The volume of the monovalent bulk is based on the HA content
measured in the monovalent bulk prior to the formulation and on a
target volume of 4000 ml.
[0452] The final bulk buffer (Formulation buffer comprising: Sodium
chloride: 7.699 g/l; Disodium phosphate dodecahydrate: 2.600 g/l;
Potassium dihydrogen phosphate: 0.373 g/l; potassium chloride: 0.2
g/l; Magnesium chloride hexahydrate: 0.1 g/l) and the correct
volumes of Triton X-100 (5% Octoxynol 10 (Triton X-100) solution)
and thiomersal (0.9% Thiomersal stock solution) are mixed together
under continuous stirring. The monobulk H5N1 is then diluted in the
resulting bulk buffer-Triton X-100-thiomersal in order to have a
final concentration of 60/30/15/7.6 .mu.g H5N1 per ml of final bulk
per ml (30, 15, 7.5 or 3.8 .mu.g HA/500 .mu.l final bulk). The
mixture is stirred during 30-60 minutes. The pH is checked to be at
7.2.+-.0.3. There was no need to add Tween 80 because the
concentration of Tween 80 (822 .mu.g/ml) present in the monobulk
was sufficient to reach the concentration target (12.26 .mu.g/.mu.g
HA).
[0453] The final bulk is aseptically filled into 3-ml sterile Type
I (Ph. Eur.) glass vials. Each vial contains a volume of 0.65
ml.+-.0.05 ml.
[0454] 2) Preparation of the AS03 Sterile Adjuvant Bulk and Filling
in the Adjuvant Container.
[0455] The adjuvant AS03 is prepared by mixing of two components:
SB62 emulsion and phosphate buffer.
[0456] SB62 Emulsion
[0457] The preparation of the SB62 emulsion is realised by mixing
under strong agitation of an oil phase composed of hydrophobic
components (a-tocopherol and squalene) and an aqueous phase
containing the water soluble components (Tween 80 and
phosphate-saline buffer at pH 6.8). While stirring, the oil phase (
1/10 total volume) is transferred to the aqueous phase ( 9/10 total
volume), and the mixture is stirred for 15 minutes at room
temperature. The resulting mixture then subjected to shear, impact
and cavitation forces in the interaction chamber of a
microfluidizer (15000 PSI--8 cycles) to produce submicron droplets
(distribution between 100 and 200 nm). The resulting pH is between
6.8.+-.0.1. The SB62 emulsion is then sterilised by filtration
through a 0.22 .mu.m membrane and the sterile bulk emulsion is
stored refrigerated in Cupac containers at 2 to 8.degree. C.
Sterile inert gas (nitrogen) is flushed into the dead volume of the
SB62 emulsion final bulk container for at least 15 seconds.
[0458] The final composition of the SB62 emulsion is as follows
(Table 8): TABLE-US-00008 TABLE 8 Tween 80: 1.8% (v/v) 19.4 mg/ml
Squalene: 5% (v/v) 42.8 mg/ml .alpha.-tocopherol: 5% (v/v) 47.5
mg/ml Phosphate-saline buffer NaCl 121 mM KCl 2.38 mM
Na.sub.2HPO.sub.4 7.14 mM KH.sub.2PO.sub.4 1.3 mM pH 6.8 .+-.
0.1
[0459] AS03 Adjuvant System
[0460] The AS03 adjuvant system is prepared by mixing buffer (PBS
mod) with SB62 bulk. The mixture is stirred for 15-45 minutes at
room temperature, and the pH is adjusted to 6.8.+-.0.1 with NAOH
(0.05 or 0.5 M)/HCl (0.03 M or 0.3 M). After another stirring for
15-20 minutes at room temperature, the pH is measured and the
mixture is sterilised by filtration through a 0.22 .mu.m membrane.
The sterile AS03 adjuvant is stored at +2-8.degree. C. until
aseptical filling into 1.25-ml sterile Type I (Ph. Eur.) glass
syringes. Each syringe contains a volume overage of 720 .mu.l (500
.mu.l+220 .mu.l overfill)
[0461] The final composition of the AS03 adjuvant is as follows
(Table 9): TABLE-US-00009 TABLE 9 SB62 0.25 ml Squalene 10.68 mg
Tocopherol 11.86 mg Polysorbate 80 4.85 mg PBS-mod: NaCl 137 mM KCl
2.7 mM Na.sub.2HPO.sub.4 8.1 mM KH.sub.2PO.sub.4 1.47 mM pH 6.8
.+-. 0.1 Volume 0.5 ml
[0462] 3) Extemporaneous Reconstitution of the AS03 Adjuvanted
Split Virus Vaccine.
[0463] At the time of injection, the content of the prefilled
syringe containing the adjuvant is injected into the vial that
contains the concentrated trivalent inactivated split virion
antigens. After mixing the content is withdrawn into the syringe
and the needle is replaced by an intramuscular needle. One dose of
the reconstituted the AS03-adjuvanted influenza candidate vaccine
corresponds to 1 ml
[0464] III.4.2 Vaccine Preparation
[0465] The vaccines were administered intramuscularly in the
deltoid region of the non-dominant arm. The pandemic influenza
candidate vaccines are 2 components vaccines consisting of antigens
presented in a vial (antigen container) and a pre-filled syringe
containing either the adjuvant (adjuvant container) or the diluent.
At the time of injection, the content of the pre-filled syringe is
injected into the vial that contains the antigens. After mixing,
the content is withdrawn into the syringe. The used needle is
replaced by an intramuscular needle. One dose of the vaccine
corresponds to 1 ml.
IV.5 Study Population Results
[0466] A total of 400 subjects were enrolled in this study: 49 to
51 subjects in each of the 8 groups. The mean age of the total
vaccinated cohort at the time of vaccination was 34.3 years with a
standard deviation of 12.76 years. The mean age and gender
distribution of the subjects across the 8 vaccine groups was
similar.
IV.6 Safety Conclusions
[0467] The administration of the pandemic influenza candidate
vaccine adjuvanted with AS03 was safe and clinically well tolerated
in the study population, i.e. adult people aged between 18 and 60
years.
IV.7 Immunogenicity Results
[0468] Analysis of immunogenicity was performed on the ATP cohort
(394 subjects).
[0469] III.7.1. Humoral Immune Response
[0470] In order to evaluate the humoral immune response induced by
the pandemic influenza H5N1 candidate vaccine adjuvanted with ASO3,
the following parameters (with 95% confidence intervals) were
calculated for each treatment group: [0471] Geometric mean titres
(GMTs) of HI antibody titres at days 0, 21 and 42. [0472]
Seroconversion rates (SC) at days 21 and 42; [0473] Conversion
factors at day 21 and 42; [0474] Protection rates at day 21 and
42.
[0475] III.7.1.1 Anti-hemagglutinin Antibody Response
[0476] a) HI Geometric Mean titres (GMT)
[0477] The GMTs for HI antibodies with 95% Cl are shown in Table 10
(GMT for anti-HI antibody). Pre-vaccination GMTs of antibodies for
the H5N1 vaccination strain were within the same range in the eight
study groups. Following the first vaccination, in all
non-adjuvanted groups anti-haemagglutinin antibody levels increased
only very modestly in a dose dependent manner. In the adjuvanted
vaccination groups, a more prominent increase in
anti-haemagglutinin antibody levels was already observed after the
first vaccination, with the highest GMT in the group receiving the
highest antigen dose (HN30AD). Post second vaccination, GMTs in the
non adjuvanted groups increased slightly over the post-first
vaccination GMT. In comparison, significant higher GMTs were
observed after the second vaccination in all adjuvanted groups,
with a dose dependant increase observed from the 3.8 .mu.g to the
7.5 .mu.g to the 15 .mu.g group. For the 30 .mu.g group, a lower
GMT than for the 7.5 .mu.g group was observed. TABLE-US-00010 TABLE
10 Geometric mean titers (GMTs) for anti-HA antibody at different
timepoints (ATP cohort for immunogenicity) GMT 95% CI Antibody
Group Timing N value LL UL Min Max FLU A/VIET/04 AB HN30 PRE 49 5.2
4.8 5.6 <10.0 28.0 PI(D21) 49 14.1 8.9 22.6 <10.0 1280.0
PII(D42) 49 20.0 12.5 32.1 <10.0 905.0 HN15 PRE 49 5.3 4.8 5.9
<10.0 40.0 PI(D21) 49 10.4 6.9 15.6 <10.0 640.0 PII(D42) 49
14.7 9.6 22.4 <10.0 640.0 HN8 PRE 49 5.0 5.0 5.0 <10.0
<10.0 PI(D21) 49 6.8 5.4 8.7 <10.0 160.0 PII(D42) 49 8.5 6.3
11.5 <10.0 160.0 HN4 PRE 50 5.0 5.0 5.0 <10.0 <10.0
PI(D21) 50 5.1 4.9 5.4 <10.0 20.0 PII(D42) 50 6.2 5.3 7.4
<10.0 57.0 HN30AD PRE 48 5.1 4.9 5.5 <10.0 20.0 PI(D21) 48
36.7 22.7 59.3 <10.0 640.0 PII(D42) 48 187.5 116.2 302.7
<10.0 1280.0 HN15AD PRE 49 5.1 4.9 5.2 <10.0 10.0 PI(D21) 49
24.7 14.8 41.4 <10.0 1280.0 PII(D42) 49 306.7 218.4 430.8
<10.0 1810.0 HN8AD PRE 50 5.4 4.8 6.0 <10.0 40.0 PI(D21) 50
24.6 15.8 38.4 <10.0 640.0 PII(D42) 50 205.3 135.1 312.0
<10.0 1280.0 HN4AD PRE 50 5.4 4.8 6.0 <10.0 80.0 PI(D21) 50
12.9 8.9 18.7 <10.0 640.0 PII(D42) 50 149.3 93.2 239.1 <10.0
1280.0 HN30 = H5N1 30 .mu.g HN15 = H5N1 15 .mu.g HN8 = H5N1 7.5
.mu.g HN4 = H5N1 3.8 .mu.g HN30AD = H5N1 30 .mu.g + AS03 HN15AD =
H5N1 15 .mu.g + AS03 HN8AD = H5N1 7.5 .mu.g + AS03 HN4AD = H5N1 3.8
.mu.g + AS03 GMT = geometric mean antibody titre calculated on all
subjects N = number of subjects with available results n/% =
number/percentage of subjects with titre within the specified range
95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper
Limit MIN/MAX = Minimum/Maximum PRE = Pre-vaccination dose 1
PI(D21) = Post-vaccination at day 21 PII(D42) = Post-vaccination at
day 42 Data source = Appendix table IIIA
[0478] b) Conversion Factors of Anti-HI Antibody Titres,
Seroprotection Rates and Seroconversion Rates (Correlates for
Protection as Established for Influenza Vaccine in Humans)
[0479] Results are presented in Tables 11 (conversion factors), 12
(seroprotection rates) and 13 (seroconversion rates).
[0480] The conversion factors (Table 11, FIG. 7) represent the fold
increase in serum HI GMTs for the vaccine strain on day 21 and 42
compared to day 0. The conversion factor after the second
vaccination varies from 1.2 to 3.9 in the 4 non-adjuvanted groups
and from 27.9 to 60.5 in the adjuvanted groups. Conversion factors
in the AS03 adjuvanted groups are largely superior to the 2.5 fold
increase in GMT required by the European Authorities for
interpandemic vaccines for adults (set forth in Table 1).
Currently, for pandemic candidate vaccines the same criteria are
applied as utilized for annual licensure of interpandemic influenza
vaccine. Of note, all except the lowest antigen concentration
adjuvanted groups achieve a conversion factor of .gtoreq.2.5
already after the first vaccination.
[0481] The seroprotection rates (Table 12, FIG. 6) represent the
proportion of subjects with a serum HI titre .gtoreq.40 on day 21
and 42. Prior to vaccination, 3 of the subjects (1 in group HN15, 1
in group HN8AD and 1 in group HN4D) were found to have protective
levels of antibodies for vaccine strain H5N1 A/Vietnam/1194/2004.
For H5N1 a very low percentage of seroprotected individuals prior
to vaccination was obtained, confirming observation of previous
studies (Bresson J L et al. The Lancet. 2006:367 (9523):1657-1664;
Treanor J J et al. N Engl J Med. 2006; 354:1343-1351). At day 21,
the seroprotection rates in the non-adjuvanted groups ranged from
0.0% to 28.6% (Table 12), while in the adjuvanted groups 26.0% to
58.3% of subjects achieved a protective titer. After the second
dose of pandemic influenza candidate vaccine, 4.0 to 42.9% of
subjects in the non-adjuvanted groups and 84.0% to 95.9% in the
adjuvanted groups had a titer equal or above the threshold
considered as protective (i.e. HI titer .gtoreq.1:40).
Consequently, up to 95.9% of subjects (group 15HNAD) receiving an
adjuvanted pandemic candidate vaccine had a serum HI titre
.gtoreq.40 after 2 vaccinations and were deemed to be protected
against the H5N1 vaccination strain. All four candidate
formulations exceeded the seroprotection rate of 70% required in
the 18-60 year old population by the European Authorities--with a
substantial proportion of subjects already achieving a protective
tier after the first dose--while non of the non-adjuvanted
candidates vaccines reached this criterion.
[0482] The seroconversion rates (Table 13, FIG. 5) represent the
percentage of vaccinees that have either a prevaccination titer
<1:10 and a post-vaccination titer .gtoreq.1:40 or a
prevaccination titer .gtoreq.1:10 and at least a fourfold increase
in post-vaccination titer on day 21 and 42 as compared to day 0.
After the first vaccination, seroconversion rates in the
non-adjuvanted groups ranged from 0.0% to 14.9% (Table 13). In the
corresponding adjuvanted study groups, seroconversion rates between
24.0% and 58.3% were observed after the first vaccination,
exceeding already in 3 of the 4 adjuvanted groups receiving
different antigen contents the requirements of the EMEA
(seroconversion rate greater than 40% in the 18-60 year old
population required). After the second vaccination between 4.0% and
40.8% of subjects in the non-adjuvanted groups, but 82.0% to 95.9%
of subjects in the adjuvanted groups either achieved a
seroconversion or four-fold increase. Therefore, after two
vaccinations all four adjuvanted formulations of the candidate
vaccine fulfilled the criterion for licensure as set by the EMEA,
but only the highest dose of non-adjuvanted vaccine just achieved
(HN30: 40.8%) this threshold. TABLE-US-00011 TABLE 11
Seroconversion factor for HAI antibody titer at each
post-vaccination time point (ATP cohort for immunogenicity) 95% CI
Vaccine strain Timing Group N GMR LL UL FLU A/VIET/04 AB PI(D21)
HN30 49 2.7 1.7 4.3 HN15 49 1.9 1.3 2.8 HN8 49 1.4 1.1 1.7 HN4 50
1.0 1.0 1.1 HN30AD 48 7.1 4.3 11.7 HN15AD 49 4.9 2.9 8.1 HN8AD 50
4.6 3.0 7.0 HN4AD 50 2.4 1.7 3.5 PII(D42) HN30 49 3.9 2.4 6.2 HN15
49 2.8 1.9 4.1 HN8 49 1.7 1.3 2.3 HN4 50 1.2 1.1 1.5 HN30AD 48 36.4
22.7 58.5 HN15AD 49 60.5 42.8 85.5 HN8AD 50 38.1 24.8 58.4 HN4AD 50
27.9 17.2 45.2 HN30 = H5N1 30 .mu.g HN15 = H5N1 15 .mu.g HN8 = H5N1
7.5 .mu.g HN4 = H5N1 3.8 .mu.g HN30AD = H5N1 30 .mu.g + AS03 HN15AD
= H5N1 15 .mu.g + AS03 HN8AD = H5N1 7.5 .mu.g + AS03 HN4AD = H5N1
3.8 .mu.g + AS03 N = number of subjects with available results n/%
= number/percentage of subjects with titre within the specified
range PRE = Pre-vaccination PI(D21) = Post vaccination at day 21
PII(D42) = Post vaccination at day 42
[0483] TABLE-US-00012 TABLE 12 Seroprotection rates at days 0, day
21 and day 42 defined as the percentage of vaccinees with the serum
anti-HA titer .gtoreq.1:40 (ATP cohort for immunogenicity)
.gtoreq.40 1/DIL 95% CI Antibody Group Timing N n % LL UL FLU A/
HN30 PRE 49 0 0.0 0.0 7.3 VIET/04 AB PI(D21) 49 14 28.6 16.6 43.3
PII(D42) 49 21 42.9 28.8 57.8 HN15 PRE 49 1 2.0 0.1 10.9 PI(D21) 49
10 20.4 10.2 34.3 PII(D42) 49 17 34.7 21.7 49.6 HN8 PRE 49 0 0.0
0.0 7.3 PI(D21) 49 4 8.2 2.3 19.6 PII(D42) 49 8 16.3 7.3 29.7 HN4
PRE 50 0 0.0 0.0 7.1 PI(D21) 50 0 0.0 0.0 7.1 PII(D42) 50 2 4.0 0.5
13.7 HN30AD PRE 48 0 0.0 0.0 7.4 PI(D21) 48 28 58.3 43.2 72.4
PII(D42) 48 41 85.4 72.2 93.9 HN15AD PRE 49 0 0.0 0.0 7.3 PI(D21)
49 24 49.0 34.4 63.7 PII(D42) 49 47 95.9 86.0 99.5 HN8AD PRE 50 1
2.0 0.1 10.7 PI(D21) 50 25 50.0 35.5 64.5 PII(D42) 50 45 90.0 78.2
96.7 HN4AD PRE 50 1 2.0 0.1 10.7 PI(D21) 50 13 26.0 14.6 40.3
PII(D42) 50 42 84.0 70.9 92.8 HN30 = H5N1 30 .mu.g HN15 = H5N1 15
.mu.g HN8 = H5N1 7.5 .mu.g HN4 = H5N1 3.8 .mu.g HN30AD = H5N1 30
.mu.g + AS03 HN15AD = H5N1 15 .mu.g + AS03 HN8AD = H5N1 7.5 .mu.g +
AS03 HN4AD = H5N1 3.8 .mu.g + AS03 N = number of subjects with
available results n/% = number/percentage of subjects with titre
within the specified range PRE = Pre-vaccination PI(D21) = Post
vaccination at day 21 PII(D42) = Post vaccination at day 42
[0484] TABLE-US-00013 TABLE 13 Seroconversion rates for anti-HA
antibody titer at each post- vaccination at day 21 and day 42 (ATP
cohort for immunogenicity) Seroconversion 95% CI Vaccine strain
Timing Group N n % LL UL FLU A/VIET/ PI(D21) HN30 49 13 26.5 14.9
41.1 04 AB HN15 49 10 20.4 10.2 34.3 HN8 49 4 8.2 2.3 19.6 HN4 50 0
0.0 0.0 7.1 HN30AD 48 28 58.3 43.2 72.4 HN15AD 49 24 49.0 34.4 63.7
HN8AD 50 25 50.0 35.5 64.5 HN4AD 50 12 24.0 13.1 38.2 PII(D42) HN30
49 20 40.8 27.0 55.8 HN15 49 17 34.7 21.7 49.6 HN8 49 8 16.3 7.3
29.7 HN4 50 2 4.0 0.5 13.7 HN30AD 48 41 85.4 72.2 93.9 HN15AD 49 47
95.9 86.0 99.5 HN8AD 50 45 90.0 78.2 96.7 HN4AD 50 41 82.0 68.6
91.4 HN30 = H5N1 30 .mu.g HN15 = H5N1 15 .mu.g HN8 = H5N1 7.5 .mu.g
HN4 = H5N1 3.8 .mu.g HN30AD = H5N1 30 .mu.g + AS03 HN15AD = H5N1 15
.mu.g + AS03 HN8AD = H5N1 7.5 .mu.g + AS03 HN4AD = H5N1 3.8 .mu.g +
AS03 N = number of subjects with available results PI(D21) = Post
vaccination at 21 days PII(D42) = Post vaccination at 42 days Data
source = Appendix table IIIA n/% = number/percentage of subjects
with either a pre-vaccination titer <1:10 and post-vaccination
titre .gtoreq.1:40 or a pre-vaccination titer .gtoreq.1:10 and a
minimum 4-fold increase in pot-vaccination titer. 95% confidence
interval, LL = Lower Limit, UL = Upper Limit
[0485] In Conclusion:
[0486] In case of an influenza pandemic, large proportions of the
population will be naive towards the pandemic influenza strain and
will likely require 2 doses of vaccine to be protected. To reduce
the antigen content in the potential pandemic vaccine and therefore
increase vaccine supply, adjuvantation strategies are employed
after it has been shown that non-adjuvanted H5N1 candidates
vaccines (H5N1 is a leading candidate for causing the next
influenza pandemic) elicit a immune response only after large doses
of antigen (Treanor J J et al. N Engl J Med. 2006;
354:1343-1351).
[0487] In this first trial reported herein with a H5N1 pandemic
influenza candidate vaccine with AS03, the following results were
obtained: [0488] There is a clear benefit of the adjuvant AS03 in
comparison to the plain antigen formulations for all different
hemagglutinin doses tested. Post second vaccination, there was a
clear superiority of the adjuvanted groups in GMTs of HI antibody
observed: The GMT of the adjuvanted group receiving the lowest
antigen dose (3.8 .mu.g HA) tested was still 7.5 fold higher than
the highest GMT achieved in the non-adjuvanted groups, elicited by
the highest antigen dose (2 injections a 30 .mu.g of HA). There was
no overlap of 95% Cl between either of the adjuvanted groups with
either of the non-adjuvanted groups at day 42. [0489] The
seroconversion rates at day 42 were 82.0%, 90.0%, 95.9% and 85.6%
for the 3.8 .mu.g, 7.5 .mu.g, 15 .mu.g and 30 .mu.g plus adjuvant
groups, respectively. This is for all four antigen contents
adjuvanted with AS03 tested superior to the 40% required by the
European Authorities. Only one of the non adjuvanted groups, the
highest antigen dose group (30 .mu.g), was just able to accomplish
a percentage above the set threshold. [0490] At day 42, the
seroprotection rates in the four adjuvanted groups were 84.0%,
90.0%, 95.9% and 85.4% for the 3.8 .mu.g, 7.5 .mu.g, 15 .mu.g and
30 .mu.g plus adjuvant groups, respectively. The required
percentage by the EMEA for the adult age group below 60 years of
age is 70%, thereby all adjuvanted groups fulfilled this criterion,
while non of the plain non adjuvanted groups could achieved the
seroprotection rate required. [0491] In this study, after two
vaccinations with the different candidate vaccine formulations, the
seroconversion factor was greater than 27.9 for the four adjuvanted
groups, thereby exceeding largely the requirement set at 2.5. Also
for the non-adjuvanted groups, the 2 groups receiving the highest
antigen doses (15 .mu.g and 30 .mu.g) fulfilled the requirement
with 2.8 (HN15 group) and 3.9 (HN30 group).
[0492] Regarding the three criteria as set out by the EMEA which
are also applicable for the evaluation of pandemic influenza
candidate vaccines, all adjuvanted groups achieved after the second
dose of the respective H5N1 vaccine adjuvanted with AS03 all three
criteria defined for this age group:
IV.8. Overall Conclusions
[0493] IV.8.1. Reactogenicity and Safety Results
[0494] The leading candidate for the next influenza pandemic is the
avian virus H5N1, which has resulted in a high mortality rate in
cases of bird-to-human transmission, although efficient
human-to-human transmission has not been fully confirmed. Should
H5N1 demonstrate the ability to spread efficiently from person to
person combined with the global transport network, the outcome may
feasibly be a widespread influenza outbreak affecting a high
percentage of individuals, leading to increased mortality and
morbidity in all countries. Therefore, an immunologically effective
and antigen sparing approach to vaccination has to be established
to prevent potentially devastating effects of a pandemic. This can
be achieved by using a suitable adjuvant, and for the first time,
the immunogenicity enhancing effect of a novel adjuvant on a H5N1
candidate vaccine could be shown in this trial.
[0495] This study was designed to evaluate (1) the safety and
reactogenicity in healthy adults of an pandemic influenza candidate
vaccine adjuvanted or not with oil in water emulsion, i.e. AS03,
(2) the antibody and cell-mediated immune responses.
[0496] Reactogenicity data show that the adjuvanted pandemic
candidate vaccine induced (independent from antigen content) more
local and general symptoms than the non-adjuvanted groups. However,
the safety profile of all 4 adjuvanted groups was clinically
acceptable. No serious adverse event was reported.
[0497] From these results, it can be concluded that the
reactogenicity and safety profile of the pandemic candidate vaccine
adjuvanted with AS03 is satisfactory and clinically acceptable.
[0498] IV.8.2. Immunogenicity Results
[0499] Regarding the immune response, the pandemic influenza
candidate vaccine adjuvanted with AS03 exceeded with all antigen
contents tested (3.8 .mu.g, 7.5 .mu.g, 15 .mu.g and 30 .mu.g HA,
H5N1 A/Vietnam/1194/2004) the requirements of the European
authorities for annual registration of split virion influenza
vaccines ("Note for Guidance on Harmonisation of Requirements for
influenza Vaccines" for the immunological assessment of the annual
strain changes--CPMP/BWP/214/96), currently used as basis for
evaluation of pandemic candidate influenza vaccines (Guideline on
dossier structure and content for pandemic influenza marketing
authorization application, CPM PNEG/4717/03).
[0500] The four different antigen contents for a adjuvanted
pandemic influenza candidate vaccine tested in this trial were
immunogenic in the healthy adults, who developed a excellent
antibody response to influenza hemagglutinin as measured by HI
(Table 14). TABLE-US-00014 TABLE 14 EU standard for antibody
Variable response 30HNAD 15HNAD 7.5HNAD 3.8HNAD Con- >2.5 27.9
38.1 60.5 36.4 version factor Sero- >40% 85.4 95.9 90.0 82.0
conversion rate (%) Protection >70% 84.0 90.0 95.9 85.4 rate
(%)
[0501] In summary, 2 doses of a novel adjuvanted pandemic influenza
candidate vaccine induce at the lowest tested dose of 3.8 .mu.g a
protective titer against the vaccine strain H5N1
A/Vietnam/1194/2004 in a very high proportion of subjects,
exceeding all criteria established for evaluation of immunogenicity
of influenza vaccines.
* * * * *