U.S. patent application number 13/096180 was filed with the patent office on 2011-11-24 for multivalent adjuvanted influenza virus compositions.
This patent application is currently assigned to GlaxoSmithKline Biologicals, s.a.. Invention is credited to Emmanuel Jules HANON, Jean Stephenne.
Application Number | 20110287054 13/096180 |
Document ID | / |
Family ID | 36441219 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110287054 |
Kind Code |
A1 |
HANON; Emmanuel Jules ; et
al. |
November 24, 2011 |
MULTIVALENT ADJUVANTED INFLUENZA VIRUS COMPOSITIONS
Abstract
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 multivalent influenza
immunogenic compositions comprising an influenza antigen or
antigenic preparation thereof from at least two influenza virus
strains, at least one 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.
Inventors: |
HANON; Emmanuel Jules;
(Rixensart, BE) ; Stephenne; Jean; (Rixensart,
BE) |
Assignee: |
GlaxoSmithKline Biologicals,
s.a.
|
Family ID: |
36441219 |
Appl. No.: |
13/096180 |
Filed: |
April 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11909388 |
Sep 21, 2007 |
|
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PCT/EP2006/002837 |
Mar 21, 2006 |
|
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13096180 |
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Current U.S.
Class: |
424/209.1 |
Current CPC
Class: |
A61K 2039/55572
20130101; A61K 2039/55511 20130101; A61K 2039/55 20130101; A61P
31/16 20180101; C12N 2760/16134 20130101; A61K 39/39 20130101; A61P
43/00 20180101; A61K 39/12 20130101; C12N 7/00 20130101; A61K
2039/57 20130101; A61P 37/04 20180101; C12N 2760/16034 20130101;
A61P 31/12 20180101; A61P 31/14 20180101; A61K 2039/55566 20130101;
A61K 39/145 20130101; A61P 31/00 20180101; C12N 2760/16234
20130101; A61K 2039/70 20130101 |
Class at
Publication: |
424/209.1 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61P 31/16 20060101 A61P031/16 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 23, 2005 |
GB |
0505989.4 |
Mar 23, 2005 |
GB |
0505998.5 |
Mar 23, 2005 |
GB |
0506000.9 |
Mar 23, 2005 |
GB |
0506001.7 |
Mar 23, 2005 |
GB |
0506004.1 |
May 24, 2005 |
GB |
0510589.5 |
May 24, 2005 |
GB |
0510591.1 |
May 24, 2005 |
GB |
0510593.7 |
May 24, 2005 |
GB |
0510596.0 |
May 24, 2005 |
GB |
0510598.6 |
Feb 24, 2006 |
GB |
0603788.1 |
Feb 24, 2006 |
GB |
0603789.9 |
Feb 24, 2006 |
GB |
0603790.7 |
Claims
1. A multivalent influenza immunogenic composition comprising: a
plurality of influenza antigens or antigenic preparations, wherein
at least one antigen is selected from a strain associated with a
pandemic outbreak; and an oil-in-water emulsion.
2. The multivalent influenza immunogenic composition of claim 1,
wherein the oil-in-water emulsion comprises a metabolisable
oil.
3. The multivalent influenza immunogenic composition of claim 2,
wherein the metabolisable oil is squalene.
4. The multivalent influenza immunogenic composition of claim 1,
wherein the oil-in-water emulsion comprises a metabolisable oil in
an amount of 0.5% to 20% of the total volume.
5. The multivalent influenza immunogenic composition of claim 1,
wherein at least 70% by intensity of the droplets of the
oil-in-water emulsion have a diameter of less than 1 .mu.m.
6. The multivalent influenza immunogenic composition of claim 1,
wherein the oil-in-water emulsion further comprises an emulsifying
agent.
7. The multivalent influenza immunogenic composition of claim 6,
wherein the emulsifying agent is selected from: polyoxyethylene
sorbitan monooleate (TWEEN); polyoxyethylene sorbitan trioleate
(SPAN); octylphenyl ethoxylate (TRITON); and lecithin, or a
combination thereof.
8. The multivalent influenza immunogenic composition of claim 7,
wherein the emulsifying agent is present at an amount of from about
0.01% to about 5.0% by weight (w/w) of said immunogenic
composition.
9. The multivalent influenza immunogenic composition of claim 1,
wherein the multivalent influenza immunogenic composition is a
trivalent composition or a quadrivalent composition.
10. The multivalent influenza immunogenic composition of claim 1,
wherein the pandemic influenza virus strain is selected from the
group of: H5N1, H9N2, H7N7, H2N2, and H1N1.
11. The multivalent influenza immunogenic composition of claim 10,
wherein the pandemic influenza virus strain is H1N1.
12. The multivalent influenza immunogenic composition of claim 1,
wherein the antigen or antigenic preparation is selected from the
group of: a purified whole influenza virus, a non-live influenza
virus, and at least one sub-unit component of influenza virus.
13. The multivalent influenza immunogenic composition of claim 1,
wherein the influenza antigen or antigenic preparation comprises
between 1 and 15 .mu.g of HA per influenza strain.
14. The multivalent influenza immunogenic composition of claim 1,
wherein the influenza antigen or antigenic preparation comprises
between 2.5 and 7.5 .mu.g HA per influenza strain.
15. A method for producing an influenza immunogenic composition
comprising the steps of admixing an influenza antigen or antigenic
preparation from at least two influenza virus strains, wherein at
least one of the influenza strains is associated with a pandemic
outbreak, with an oil-in-water emulsion.
16. The method of claim 15, wherein the oil-in-water emulsion
comprises a metabolisable oil.
17. The method of claim 15, wherein the oil-in-water emulsion
further comprises an emulsifying agent.
18. The method of claim 15, wherein the pandemic influenza virus
strain is selected from the group of: H5N1, H9N2, H7N7, H2N2, and
H1N1.
19. The method of claim 15, wherein the influenza antigen or
antigenic preparation comprises between 1 and 15 .mu.g of HA per
influenza strain.
20. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of pending U.S.
application Ser. No. 11/909,388, which is the 371 Application of
the National Stage of PCT Application No. PCT/EP2006/002837, filed
21 Mar. 2006, the disclosure of which is incorporated herein by
reference. This application also claims benefit of the filing dates
of the Great Britain Applications No. 0505998.5, filed 23 Mar.
2005, No. 0506000.9, filed 23 Mar. 2005, No. 0506001.7, filed 23
Mar. 2005, No. 0505989.4, filed 23 Mar. 2005, No. 0506004.1, filed
23 Mar. 2005, No. 0510589.5, filed 24 May 2005, No. 0510591.1,
filed 24 May 2005, No. 0510593.7, filed 24 May 2005, No. 0510596.0,
filed 24 May 2005, No. 0510598.6, filed 24 May 2005, No. 0603789.9,
filed 24 Feb. 2006, No. 0603788.1, filed 24 Feb. 2006, and No.
0603790.7, filed 24 Feb. 2006.
TECHNICAL FIELD
[0002] 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 multivalent
influenza immunogenic compositions comprising an influenza antigen
or antigenic preparation thereof from at least two influenza virus
strains, at least one 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.
TECHNICAL BACKGROUND
[0003] 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.
[0004] 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.
[0005] These surface antigens progressively, sometimes rapidly,
undergo some changes leading to the antigenic variations in
influenza. These antigenic changes, called `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.
[0006] The 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.
[0007] 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).
[0008] 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.
[0009] 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.
[0010] 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.).
[0011] Influenza vaccines, 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).
[0012] 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).
[0013] New vaccines with an improved immunogenicity are therefore
still needed. Formulation of vaccine antigen with potent adjuvants
is a possible approach for enhancing immune responses to subvirion
antigens.
[0014] 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).
[0015] 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. 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 H1N1 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
(HongKong 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).
[0016] 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.
[0017] There is still a need for improved influenza vaccines,
especially in the case of influenza pandemics and for the elderly
population.
STATEMENT OF THE INVENTION
[0018] In first aspect of the present invention, there is provided
a multivalent influenza immunogenic composition comprising an
influenza virus or antigenic preparation thereof from at least two
influenza virus strains, at least one 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, wherein said oil-in-water emulsion adjuvant comprises a
metabolisable oil, a sterol and an emulsifying agent. Suitably said
sterol is alpha-tocopherol.
[0019] Suitable pandemic strains are, but not limited to: H5N1,
H9N2, H7N7, H2N2 and H1N1.
[0020] In another aspect the invention provides a method for the
production of an influenza immunogenic composition for a pandemic
situation which method comprises admixing influenza virus antigen
or antigenic preparation from at least two influenza virus strains,
at least one of which is associated with a pandemic outbreak or has
the potential to be associated with a pandemic outbreak, with an
oil-in-water emulsion as herein above defined.
[0021] In a third aspect there is provided an immunogenic
composition as herein defined for use in medicine.
[0022] In another aspect there is provided the use of (a) an
influenza virus antigen or antigenic preparation thereof, and (b)
an oil-in-water emulsion adjuvant in the manufacture of an
immunogenic composition for inducing at least one of i) an improved
CD4 T-cell immune response, ii) an improved B cell memory response,
against said virus antigen or antigenic composition in a human,
preferably in an immuno-compromised individual or population, such
as a high risk adult or an elderly, which is preferred. Preferably
the immunogenic composition is as herein defined.
[0023] 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.
[0024] 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.
[0025] In a further embodiment, there is provided 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 multivalent influenza immunogenic
composition comprising an influenza antigen or antigenic
preparation thereof from at least two influenza virus strains, at
least one 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 as herein
defined.
[0026] 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.
[0027] 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.
[0028] Preferably said oil-in-water emulsion comprises a
metabolisable oil, 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.
[0029] In a further aspect of the present invention, there is
provided the use of an antigen or antigenic preparation from a
first influenza strain in the manufacture of an immunogenic
composition as herein defined for protection against influenza
infections caused by a variant influenza strain.
[0030] In another aspect, there is provided a method of vaccination
an immuno-compromised human individual or population such as high
risk adults or elderly, influenza immunogenic composition
comprising an influenza antigen or antigenic preparation thereof
from at least two influenza virus strains, at least one 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 as herein defined.
[0031] In still another embodiment, the invention provides a method
for revaccinating humans previously vaccinated with a multivalent
influenza immunogenic composition comprising an influenza antigen
or antigenic preparation thereof from at least two influenza virus
strains, at least one 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,
said method comprising administering to said human an immunogenic
composition comprising an influenza virus, either adjuvanted or
un-adjuvanted.
[0032] In a further embodiment there is provided a method for
vaccinating a human population or individual against one 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
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 first strain is associated with a pandemic outbreak
or has the potential to be associated with a pandemic outbreak. In
another specific embodiment said variant strain is associated with
a pandemic outbreak or has the potential to be associated with a
pandemic outbreak.
[0033] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
LEGEND TO FIGURES
[0034] FIG. 1: Oil droplet particle size distribution in SB62
oil-in-water emulsion as measured by PCS. FIG. 1A 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.
[0035] FIG. 2: Schematic illustration of the preparation of MPL
bulk.
[0036] FIG. 3: Schematic illustration of the preparation of
AS03+MPL adjuvant.
[0037] FIG. 4: Explo Flu-001 clinical trial. CD4 T cell response to
split influenza antigen (Q1=first quartile, Q3=third quartile).
[0038] FIG. 5: Explo Flu-001 clinical trial. CD8 T cell response to
split influenza antigen (Q1=first quartile, Q3=third quartile).
[0039] FIG. 6: Explo Flu-001 clinical trial. Cross-reactive CD4
T-cell response to split influenza virus antigen after vaccination
with FLUARIX.RTM./.alpha.-RIX.RTM./INFLUSPLIT.RTM. (Influenza
vaccine)+AS03.
[0040] FIG. 7: Explo Flu-001 clinical trial. B cell memory response
post vaccination.
[0041] FIG. 8: Explo Flu-002 clinical trial. CD4 T cell response
against split influenza antigen following revaccination.
[0042] FIG. 9: Explo Flu-002 clinical trial. Anti-HI titers
following revaccination.
[0043] FIG. 10: Ferret study I. Temperature monitoring (priming and
challenge). FIG. 10A is priming, FIG. 10B is challenge.
[0044] FIG. 11: Ferret study I. Viral shedding.
[0045] FIG. 12: Ferret study II. Temperature monitoring (priming
and challenge). FIG. 12A is priming, FIG. 12B is challenge.
[0046] FIG. 13: Ferret study II. Viral shedding.
[0047] FIG. 14: Ferret study II. HI titers to H3N2 A/Panama
(vaccine strain) (FIG. 14A) and to H3N2 A/Wyoming (challenge
strain) (FIG. 14B).
[0048] FIG. 15: Mice study. Frequencies of CD4 T cells in C57BI/6
primed mice using whole inactivated virus as re-stimulating antigen
(day 7 post-immunisation).
[0049] FIG. 16: Mice study. Frequencies of CD8 T cells in C57BI/6
primed mice using whole inactivated virus as re-stimulating antigen
(day 7 post-immunisation).
[0050] FIG. 17: Mice study. Frequencies of CD4 (upper part) and CD8
(lower part) T cells in C57BI/6 mice primed with heterologous
strains, using whole inactivated virus as re-stimulating antigen
(day 7 post-immunisation).
[0051] FIG. 18: Human clinical trial. B cell memory response
post-vaccination of elderly with FLUARIX.RTM., FLUARIX.RTM.+ AS03,
Fluarix FLUARIX.RTM.+ AS03+MPL (difference between pre- and
post-).
[0052] FIG. 19: Ferret study III. Temperature monitoring before and
after challenge.
[0053] FIG. 20: Ferret study III. Viral shedding before and after
challenge.
[0054] FIG. 21: Ferret study III. HI titers to H3N2 A/Wyoming
(vaccine strain).
[0055] FIG. 22: Ferret study III. HI titers to H3N2 A/Panama
(challenge strain).
[0056] FIG. 23: Human clinical trial. HI titers (GMTs) at days 21,
90 and 180 post vaccination (persistence).
[0057] FIG. 24: Human clinical trial. CD4 response--all double
test--Pool antigen at days 21, 90 and 180 post vaccination
(persistence).
[0058] FIG. 25: Human clinical trial. HI titers in a revaccination
clinical trial with AS03+MPL compared to FLUARIX.RTM..
[0059] FIG. 26: Human clinical trial. CMI for CD4 response--all
double test--Pool antigen at days 0 and 21.
[0060] FIG. 27: Human clinical trial with AS03+MPL at two
concentrations. HI titers at days 0 and
[0061] FIG. 28: Human clinical trial with AS03+MPL at two
concentrations. Reactogenicity.
DETAILED DESCRIPTION
[0062] The present inventors have discovered that an influenza
formulation comprising an influenza virus or antigenic preparation
thereof together with an oil-in-water emulsion adjuvant comprising
a metabolisable oil, a sterol such as alpha tocopherol and an
emulsifying agent, was capable of improving the CD4 T-cell immune
response and/or B cell memory response against said antigen or
antigenic composition in a human 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 II molecules. The present Applicant has now
found that it is effective to target the cell-mediated immune
system in order to increase responsiveness against homologous and
drift influenza strains (upon vaccination and infection).
[0063] The adjuvanted influenza compositions according to the
invention have several advantages: [0064] 1) An improved
immunogenicity: they will allow to restore weak immune response in
the elderly people (over 50 years of age, typically over 65 years
of age) to levels seen in young people (antibody and/or T cell
responses); [0065] 2) An improved cross-protection profile:
increased cross-protection against variant (drifted) influenza
strains; [0066] 3) They will also allow an reduced antigen dosage
to be used for a similar response, thus ensuring an increased
capacity in case of emergency (pandemics for example).
[0067] The compositions for use in the present invention have been
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 have also been able to
induce a trend for a higher B cell memory response following the
first vaccination of a human subject, and a higher humoral response
following revaccination, compared to the un-adjuvanted
composition.
[0068] The Inventors have also been capable of demonstrating that
the claimed adjuvanted composition was able to not only induce but
also maintain protective levels of antibodies against all three
strains present in the vaccine, in more individuals than those
obtained with the un-advanted composition (see Table 43 for
example).
[0069] 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 at least one influenza
strain, preferably for all strains 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 at least one, suitably two, or all strains
present in the vaccine composition.
Influenza Viral Strains and Antigens
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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 be produced
either recombinantly or purified from disrupted viral
particles.
[0074] 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.
[0075] Said influenza virus or antigenic preparation thereof may be
egg-derived or tissue-culture derived.
[0076] 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 allantoic 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.
[0077] 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.RTM. (known as "Tween-ether" splitting)
(Polyoxyethylenesorbitan monooleate; Sorbitan monooleate
ethoxylate) 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 X100.TM. (Octylphenol ethoxylate)
(for example in a process described in Lina et al, 2000,
Biologicals 28, 95-103) and TRITON N-101.TM., or combinations of
any two or more detergents.
[0078] 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.
[0079] Preferred split flu vaccine antigen preparations according
to the invention comprise a residual amount of TWEEN.RTM. 80 and/or
TRITON X100.TM. remaining from the production process, although
these may be added or their concentrations adjusted after
preparation of the split antigen. Preferably both TWEEN.RTM. 80 and
TRITON X100.TM. are present. The preferred ranges for the final
concentrations of these non-ionic surfactants in the vaccine dose
are:
TWEEN.RTM. 80: 0.01 to 1%, more preferably about 0.1% (v/v) TRITON
X100.TM.: 0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02%
(w/v).
[0080] In a specific embodiment, the final concentration for
TWEEN.RTM. 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.RTM. 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).
[0081] In another specific embodiment, the final concentration for
TRITON X-100.TM. 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 X100.TM. 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).
[0082] 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.
[0083] A preferred composition contains three inactivated split
virion antigens prepared from the WHO recommended strains of the
appropriate influenza season.
[0084] Preferably 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`.
Preferably 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 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 trivalent inactivated split
virion antigens) are 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 trivalent 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.
[0085] According to the present invention, at least one influenza
strain in the multivalent immunogenic composition as herein defined
is associated with a pandemic outbreak or have the potential to be
associated with a pandemic outbreak. Such strain may also be
referred to as `pandemic strains` in the text below. In particular,
when the vaccine is a multivalent vaccine such as a bivalent, or a
trivalent or a quadrivalent vaccine, at least one strain 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 H1N1.
[0086] Said influenza virus or antigenic preparation thereof is
suitably multivalent such as bivalent or trivalent or quadrivalent.
Preferably the influenza virus or antigenic preparation thereof 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.
[0087] 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; 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.
[0088] 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.
[0089] 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.
[0090] 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 strains are,
but not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.
[0091] Optionally the composition may contain more than three
valencies, for example two non pandemic strains plus a pandemic
strain. Alternatively the composition may contain three pandemic
strains. Preferably the composition contains three pandemic
strains.
Oil-in-Water Emulsion Adjuvant
[0092] 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.
[0093] 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, 10th Edition, entry no. 8619).
[0094] 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).
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] The oil in water emulsion according to the invention
comprises a sterol. 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, alpha-tocopherol 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.
[0100] Suitably the sterol is alpha-tocopherol or a derivative
thereof such as alpha-tocopherol succinate. 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.
[0101] The oil in water emulsion may further comprise 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.
[0102] The emulsifying agent may suitably be polyoxyethylene
sorbitan monooleate (TWEEN.RTM. 80). In a specific embodiment, a
0.5 ml vaccine dose volume contains 1% (w/w) TWEEN.RTM. 80, and a
0.7 ml vaccine dose volume contains 0.7% (w/w) TWEEN.RTM. 80. In
another specific embodiment the concentration of TWEEN.RTM. 80 is
0.2% (w/w).
[0103] 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, alpha tocopherol,
and TWEEN.RTM. 80. The oil in water emulsion may also contain SPAN
85.TM. (sorbitan trioleate) 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.RTM. 80, and may be produced according to the
procedure described in WO 95/17210. Preferably the ratio of
squalene:alpha tocopherol is equal or less than 1 as this 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
[0104] In the present invention the multivalent 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.
[0105] 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, 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
II molecules.
[0106] Preferably 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.
[0107] 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.
[0108] The improved CD4 T-cell immune response may be assessed by
measuring the number of cells producing any of the following
cytokines: [0109] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0110] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0111] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0112] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0113] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
[0114] 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. 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.
[0115] The improved CD4 T-cell immune response conferred by the
adjuvanted influenza composition of the present invention 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.
[0116] 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).
[0117] In a still further specific embodiment, the vaccination with
the composition for the first vaccination, adjuvanted, has no
measurable impact on the CD8 response.
[0118] The Applicants have surprisingly found that a 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 such as alpha tocopherol and an emulsifying agent, is
effective in promoting T cell responses in an immuno-compromised
human population. As the Applicants have demonstrated, the
administration of a single dose of the immunogenic composition for
first vaccination, as described in the invention is 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 has also been able to induce an improved CD4 T-cell
immune response against influenza virus compared to that obtained
with the un-adjuvanted formulation. This finding 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.
[0119] 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.
[0120] 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.
[0121] 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 multivalent influenza immunogenic
composition comprising any or several 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
[0122] 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/262/95, B: B/Yamanashi/166/98).
[0123] In contrast, if peripheral blood CD4 T-cells are
restimulated with influenza strains classified as shift strains
(H2N2: A/Singapore/1/57, H9N2: A/Hongkong/1073/99) by expert in the
field, there is no observable increase following vaccination.
[0124] 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.
[0125] 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).
[0126] 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
[0127] 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.
[0128] 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).
[0129] 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.
[0130] 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.
[0131] 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.
[0132] Synthetic derivatives of lipid A are known, some being
described as TLR-4 agonists, and include, but are not limited
to:
OM174
(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4--
o-phosphono-[3-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.alph-
a.-D-glucopyranosyldihydrogenphosphate), (WO 95/14026) OM 294 DP
(3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-h-
ydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)
(WO99/64301 and WO 00/0462) OM 197 MP-Ac DP
(3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hyd-
roxytetradecanoylamino]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 (WO96/33739). 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 multivalent influenza composition as claimed herein or with said
multivalent 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./.alpha.-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 an 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 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 a preferred embodiment, the first vaccination is made
with an influenza composition, preferably a split influenza
composition, containing at least one influenza strain that could
potentially cause a pandemic outbreak and the re-vaccination is
made with an influenza composition comprising at least one strain
which is a circulating pandemic strain.
[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 C M et
al. 1996 J. Virol. 70(7):4787-90; Gelder C M 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 H1N1. 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 outbreak or has the potential to be associated with a
pandemic outbreak. In a specific embodiment, two or more strains in
the boosting composition are pandemic strains. In another specific
embodiment, the at least one 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 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 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. As the Applicants have demonstrated, 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 such as alpha tocopherol and an
emulsifying agent, as defined herein above, shows 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. The adjuvanted
composition-associated benefit was also marked in terms of
improving the CD4 T-cell response following revaccination.
[0153] The adjuvanted composition of the invention is 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.
[0154] Preclinical data given in Example 3 for example show the
ability of the composition of the invention to protect against
heterotypic influenza infection and disease as assessed by body
temperature readouts. The same conclusion holds true for the
clinical trials data obtained in revaccination studies.
[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 at least one influenza strain that could potentially
cause a pandemic outbreak and the revaccination is made with a
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 and is.
[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 C M et al. 1996 J. Virol.
70(7):4787-90; and Gelder C M 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 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 preferred for the
adjuvanted influenza composition.
[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 U.S. Pat. No. 4,886,499,
U.S. Pat. No. 5,190,521, U.S. Pat. No. 5,328,483, U.S. Pat. No.
5,527,288, U.S. Pat. No. 4,270,537, U.S. Pat. No. 5,015,235, U.S.
Pat. No. 5,141,496, U.S. Pat. No. 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 U.S. Pat. No. 5,480,381, U.S.
Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No.
5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S.
Pat. No. 5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No.
5,893,397, U.S. Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S.
Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No.
5,064,413, U.S. Pat. No. 5,520,639, U.S. Pat. No. 4,596,556 U.S.
Pat. No. 4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat. No.
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 BD ACCUSPRAY
SCF.TM. (nasal spray system). 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 contemplated in the present invention.
[0169] In a specific 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 another
specific embodiment, the composition for the first administration
may contain a standard HA content of 15 .mu.g per influenza strain,
and the boosting composition may contain a low dose of HA, i.e.
below 15 .mu.g, and depending on the administration route, may be
given in a smaller volume.
Populations to Vaccinate
[0170] The target population to vaccinate may 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] Preferably 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 50, typically
at least 55, or at least 60, or 65 years and over, younger
high-risk adults (i.e. between 18 and 64 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 Additional 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 15 .mu.g of haemagglutinin antigen component
from the or each 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 dose 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. A suitable low dose 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.
[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.
[0177] Standards are applied internationally to measure the
efficacy of influenza vaccines. The European Union official
criteria for an effective vaccine against influenza are set out in
the Table 1 below. Theoretically, to meet the European Union
requirements, an influenza vaccine has to meet only one of the
criteria in the table, for all strains of influenza included in the
vaccine. The compositions of the present invention suitably meet at
least one such criteria.
[0178] However in practice, 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). The requirements are
different for adult populations (18-60 years) and elderly
populations (>60 years).
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 percentage of vaccinees who have at least a 4-fold increase
in serum haemagglutinin inhibition (HI) titres after vaccination,
for each vaccine strain. **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 percentage of vaccinees with a serum HI titre equal to or
greater than 1:40 after vaccination (for each vaccine strain) and
is normally accepted as indicating protection.
[0179] 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 [0180] 1) selecting an
antigen containing CD4+ epitopes, and [0181] 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.
[0182] The teaching of all references in the present application,
including patent applications and granted patents, are herein fully
incorporated by reference.
[0183] 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.
[0184] The invention will be further described by reference to the
following, non-limiting, examples:
Example I describes immunological read-out methods used in mice,
ferret and human studies. Example II describes the preparation and
characterization of the oil in water emulsion and adjuvant
formulations used in the studies exemplified. Example III describes
a clinical trial in an elderly population aged over 65 years with a
vaccine containing a split influenza antigen preparation and AS03
adjuvant Example IV describes a second clinical
trial--revaccination trial--in an elderly population aged over 65
years with a vaccine containing a split influenza antigen
preparation and AS03 adjuvant. Example V shows a pre-clinical
evaluation of adjuvanted and un-adjuvanted influenza vaccines in
ferrets (study I and study II). The temperature monitoring, viral
shedding and CD4 T-cell response were measured. Example VI shows a
pre-clinical evaluation of adjuvanted and un-adjuvanted influenza
vaccines in C57BI/6 naive and primed mice. Example VII shows a
pre-clinical evaluation of adjuvanted and un-adjuvanted split and
sub-unit influenza vaccines in C57BI/6 mice primed with
heterologous strains. Example VIII describes a clinical trial in an
elderly population aged over 65 years with a vaccine containing a
split influenza antigen preparation containing AS03 adjuvant,
AS03+MPL adjuvant, or no exogeneous adjuvant. Example IX shows a
pre-clinical evaluation of adjuvanted and un-adjuvanted influenza
vaccines in ferrets (study III). The temperature monitoring, viral
shedding and HI titers were measured. Example X shows a clinical
trial in an elderly population aged over 65 years with a vaccine
containing a split influenza antigen preparation containing AS03
with or without MPL adjuvant: immunogenicity persistence data at
day 90 and day 180. Example XI shows a clinical trial in an elderly
population aged over 65 years with a vaccine containing a split
influenza antigen preparation containing AS03 with MPL adjuvant.
Example XII shows a clinical trial in an elderly population aged
over 65 years with a vaccine containing a split influenza antigen
preparation containing AS03 with MPL adjuvant at two
concentrations.
Example I
Immunological Read-Out Methods
[0185] I.1. Mice methods
I.1.1. Hemagglutination Inhibition Test
Test Procedure
[0186] Anti-Hemagglutinin antibody titers to the three influenza
virus strains were 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). Heat inactivated sera were previously treated by Kaolin and
chicken RBC to remove non-specific inhibitors. After pretreatment,
two-fold dilutions of sera were incubated with 4 hemagglutination
units of each influenza strain. Chicken red blood cells were then
added and the inhibition of agglutination was scored. The titers
were expressed as the reciprocal of the highest dilution of serum
that completely inhibited hemagglutination. As the first dilution
of sera was 1:20, an undetectable level was scored as a titer equal
to 10.
Statistical Analysis
[0187] Statistical analysis were performed on post vaccination HI
titers using UNISTAT. The protocol applied for analysis of variance
can be briefly described as follow: [0188] Log transformation of
data [0189] Shapiro-Wilk test on each population (group) in order
to verify the normality of groups distribution [0190] Cochran test
in order to verify the homogenicity of variance between the
different populations (groups) [0191] Two-way Analysis of variance
performed on groups [0192] Tukey HSD test for multiple
comparisons
I.1.2. Intracellular Cytokine Staining
[0193] This technique allows a quantification of antigen specific T
lymphocytes on the basis of cytokine production: effector T cells
and/or effector-memory T cells produce IFN-.gamma. and/or central
memory T cells produce IL-2. PBMCs are harvested at day 7
post-immunization.
[0194] Lymphoid cells are re-stimulated in vitro in the presence of
secretion inhibitor (Brefeldine). These cells are then processed by
conventional immunofluorescent procedure using fluorescent
antibodies (CD4, CD8, IFN-.gamma. and IL-2). Results are expressed
as a frequency of cytokine positive cell within CD4/CD8 T cells.
Intracellular staining of cytokines of T cells was performed on
PBMC 7 days after the second immunization. Blood was collected from
mice and pooled in heparinated medium RPMI+Add. For blood,
RPMI+Add-diluted PBL suspensions were layered onto a
Lympholyte--Mammal gradient according to the recommended protocol
(centrifuge 20 min at 2500 rpm and R.T.). The mononuclear cells at
the interface were removed, washed 2.times. in RPMI+Add and PBMCs
suspensions were adjusted to 2.times.10.sup.6 cells/ml in RPMI 5%
fetal calf serum.
[0195] In vitro antigen stimulation of PBMCs was carried out at a
final concentration of 1.times.10.sup.7 cells/ml (tube FACS) with
Whole FI (1 .mu.gHA/strain) and then incubated 2 hrs at 37.degree.
C. with the addition of anti-CD28 and anti-CD49d (1 .mu.g/ml for
both).
[0196] Following the antigen restimulation step, PBMC are incubated
overnight at 37.degree. C. in presence of Brefeldin (1 .mu.g/ml) at
37.degree. C. to inhibit cytokine secretion.
[0197] IFN-.gamma./IL-2/CD4/CD8 staining was performed as follows:
Cell suspensions were washed, resuspended in 50 .mu.l of PBS 1% FCS
containing 2% Fc blocking reagent (1/50; 2.4G2). After 10 min
incubation at 4.degree. C., 50 .mu.l of a mixture of anti-CD4-PE
(2/50) and anti-CD8 perCp (3/50) was added and incubated 30 min at
4.degree. C. After a washing in PBS 1% FCS, cells were
permeabilized by resuspending in 200 .mu.l of Cytofix-Cytoperm (Kit
BD) and incubated 20 min at 4.degree. C. Cells were then washed
with Perm Wash (Kit BD) and resuspended with 50 .mu.l of a mix of
anti-IFN-.gamma. APC (1/50)+anti-IL-2 FITC (1/50) diluted in Perm
Wash. After an incubation min 2 h max overnight at 4.degree. C.,
cells were washed with Perm Wash and resuspended in PBS 1% FCS+1%
paraformaldehyde. Sample analysis was performed by FACS. Live cells
were gated (FSC/SSC) and acquisition was performed on .about.20,000
events (lymphocytes) or 35,000 events on CD4+T cells. The
percentages of IFN-.gamma.+ or IL2+ were calculated on CD4+ and
CD8+ gated populations.
1.2. Ferrets Methods
1.2.1. Hemagglutination Inhibition Test (HI)
Test Procedure.
[0198] Anti-Hemagglutinin antibody titers to the three influenza
virus strains were 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). Sera were first treated with a 25% neuraminidase solution
(RDE) and were heat-inactivated to remove non-specific inhibitors.
After pre-treatment, two-fold dilutions of sera were incubated with
4 hemagglutination units of each influenza strain. Chicken red
blood cells were then added and the inhibition of agglutination was
scored. The titers were 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.
Statistical Analysis.
[0199] Statistical analysis were performed on HI titers (Day 41,
before challenge) using UNISTAT. The protocol applied for analysis
of variance can be briefly described as followed: [0200] Log
transformation of data. [0201] Shapiro-wilk test on each population
(group) in order to verify the normality of groups distribution.
[0202] Cochran test in order to verify the homogenicity of variance
between the different populations (groups). [0203] Test for
interaction of one-way ANOVA. [0204] Tuckey-HSD Test for multiple
comparisons.
I.2.2. Body Temperature Monitoring
[0205] Individual temperatures were monitored during the challenge
period with the transmitters and by the telemetry recording. All
implants were checked and refurbished and a new calibration was
performed by DSI (Data Sciences International, Centaurusweg 123,
5015 TC Tilburg, The Netherlands) before placement in the
intraperitoneal cavity. All animals were individually housed in
single cage during these measurements.
[0206] Temperatures were recorded every 15 minutes 4 days before
challenge until 7 days Post-challenge.
I.2.3. Nasal Washes
[0207] The nasal washes were performed by administration of 5 ml of
PBS in both nostrils in awoke animals. The inoculum was collected
in a Petri dish and placed into sample containers on dry ice.
Viral Titration in Nasal Washes
[0208] All nasal samples were first sterile filtered through Spin X
filters (Costar) to remove any bacterial contamination. 50 .mu.l of
serial ten-fold dilutions of nasal washes were 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.
[0209] After 5-7 days of incubation, the culture medium is gently
removed and 100 .mu.l of a 1/20 WST-1 containing medium is added
and incubated for another 18 hrs.
[0210] The intensity of the yellow formazan dye produced upon
reduction of WST-1 by viable cells is proportional to the number of
viable cells present in the well at the end of the viral titration
assay and is quantified by measuring the absorbance of each well at
the appropriate wavelength (450 nanometers). The cut-off is defined
as the OD average of uninfected control cells--0.3 OD (0.3 OD
correspond to +/-3 StDev of OD of uninfected control cells). A
positive score is defined when OD is <cut-off and in contrast a
negative score is defined when OD is >cut-off. Viral shedding
titers were determined by "Reed and Muench" and expressed as Log
TCID50/ml.
I.3. Assays for Assessing the Immune Response in Humans
I.3.1. Hemagglutination Inhibition Assay
[0211] 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).
[0212] 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 erythrocyte suspension.
Non-specific serum inhibitors were removed by heat treatment and
receptor-destroying enzyme.
[0213] 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.
[0214] The titration end-point was taken as the highest dilution
step that showed complete inhibition (100%) of hemagglutination.
All assays were performed in duplicate.
I.3.2. Neuraminidase Inhibition Assay
[0215] 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, H1N1 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.
I.3.3. Neutralising Antibody Assay
[0216] Neutralising antibody measurements were conducted on thawed
frozen serum samples. Virus neutralisation by antibodies contained
in the serum was determined in a microneutralization assay. The
sera were used without further treatment in the assay. Each serum
was tested in triplicate. A standardised amount of virus was 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 was then added to the mixture of virus
and antiserum and incubated at 33.degree. C. After the incubation
period, virus replication was visualised by hemagglutination of
chicken red blood cells. The 50% neutralisation titre of a serum
was calculated by the method of Reed and Muench.
I.3.4. Cell-Mediated Immunity was Evaluated by Cytokine Flow
Cytometry (CFC)
[0217] 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 as well as peptides
derived from specific influenza protein were used as antigen to
restimulate Influenza-specific T cells. Results were expressed as a
frequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4
or CD8 T cell sub-population.
I.3.5. Statistical Methods
[0218] I.3.5.1. Primary endpoints [0219] 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.
[0220] 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. [0221] Occurrence of serious adverse
events during the entire study.
I.3.5.2. Secondary Endpoints
For the Humoral Immune Response:
Observed Variables:
[0221] [0222] 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-H1N1, anti-H3N2 & anti-B-antibodies). [0223] At days 0
and 21: neutralising antibody titres, tested separately against
each of the three influenza virus strains represented in the
vaccine Derived Variables (with 95% Confidence Intervals): [0224]
Geometric mean titres (GMTs) of serum HI antibodies with 95%
confidence intervals (95% CI) pre and post-vaccination [0225]
Seroconversion rates* with 95% CI at day 21 [0226] Conversion
factors** with 95% CI at day 21 [0227] Seroprotection rates*** with
95% CI at day 21 [0228] Serum NI antibody GMTs' (with 95%
confidence intervals) at all timepoints. [0229] *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.
[0230] **Conversion factor defined as the fold increase in serum HI
GMTs on day 21 compared to day 0, for each vaccine strain.
[0231] ***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.
For the Cell Mediated Immune (CMI) Response
Observed Variable
[0232] 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: [0233] Peptide Influenza (pf)
antigen (the precise nature and origin of these antigens needs to
be given/explained [0234] Split Influenza (sf) antigen [0235] Whole
Influenza (wf) antigen.
Derived Variables:
[0235] [0236] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0237] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0238] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0239] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0240] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
I.3.5.3. Analysis of Immunogenicity
[0241] The immunogenicity analysis was based on the total
vaccinated cohort. For each treatment group, the following
parameters (with 95% confidence intervals) were calculated: [0242]
Geometric mean titres (GMTs) of HI and NI antibody titres at days 0
and 21 [0243] Geometric mean titres (GMTs) of neutralising antibody
titres at days 0 and 21. [0244] Conversion factors at day 21.
[0245] 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. [0246] Protection
rates at day 21 defined as the percentage of vaccinees with a serum
HI titre=1:40. [0247] 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)). [0248] 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. [0249] 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
[0250] 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.RTM. 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.RTM. 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
II.1.1. Lab-Scale Preparation
[0251] TWEEN.RTM. 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 ml of PBS/TWEEN.RTM.
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.
II.1.2. Scaled-Up Preparation
[0252] 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.RTM. 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.
[0253] The final composition of the SB62 emulsion is as
follows:
[0254] TWEEN.RTM. 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; 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
II.2.1. Introduction
[0255] 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.
II.2.2. Sample Preparation
[0256] 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.
[0257] As a control, PL-Nanocal Particle size standards 100 nm (cat
n.sup.o 6011-1015) was diluted in 10 mM NaCl.
II.2.3. Malvern Zetasizer 3000HS Size Measurements
[0258] All size measurements were performed with both Malvern
Zetasizer 3000HS. 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: [0259] either real particle refractive
index of 0 and imaginary one of 0. [0260] 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).
[0261] The technical conditions were: [0262] laser wavelength: 532
nm (Zeta3000HS). [0263] laser power: 50 mW (Zeta3000HS). [0264]
scattered light detected at 90.degree. (Zeta3000HS). [0265]
temperature: 25.degree. C., [0266] duration: automatic
determination by the soft, [0267] number: 3 consecutive
measurements, [0268] z-average diameter: by cumulants analysis
[0269] size distribution: by the Contin or the Automatic
method.
[0270] The Automatic Malvern algorithm uses a combination of
cumulants, Contin and non negative least squares (NNLS)
algorithms.
[0271] The intensity distribution may be converted into volume
distribution thanks to the Mie theory.
II.2.4. Results (See Table 2)
Cumulants Analysis (Z Average Diameter):
TABLE-US-00002 [0272] TABLE 2 Count Sample Dilution Record 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 10.times. 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.
[0273] 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.
[0274] The count rate (CR) is a measure of scattered light: it
corresponds to thousands of photons per second.
[0275] The polydispersity (Poly) index is the width of the
distribution. This is a dimensionless measure of the distribution
broadness.
Contin and Automatic Analysis:
[0276] 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. Results are shown in Table 3.
TABLE-US-00003 TABLE 3 Analysis in Contin Analysis in Automatic IR
(mean in nm) (mean in nm) SB62 Dilution Real Imaginary 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.
[0277] 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.
II.2.5. Overall Conclusion
[0278] 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.
[0279] When using the cumulants algorithm, we observed no influence
of the dilution on the ZAD and polydispersity.
II.3. Preparation of AS03 Comprising MPL
II.3.1. Preparation of MPL Liquid Suspension
[0280] The MPL (as used throughout the document it is an
abbreviation for 3D-MPL, i.e. 3-O-deacylated monophosphoryl lipid
A) liquid bulk is prepared from MPL.RTM. lyophilized powder. MPL
liquid bulk is a stable concentrated (around 1 mg/ml) aqueous
dispersion of the raw material, which is ready-to-use for vaccine
or adjuvant formulation. A schematic representation of the
preparation process is given in FIG. 2.
[0281] For a maximum batch size of 12 g, MPL liquid bulk
preparation is carried over in sterile glass containers. The
dispersion of MPL consists of the following steps: [0282] suspend
the MPL powder in water for injection [0283] disaggregate any big
aggregates by heating (thermal treatment) [0284] reduce the
particle size between 100 nm and 200 nm by microfluidization [0285]
prefilter the preparation on a SARTOCLEAN.RTM. Pre-filter unit,
0.8/0.65 .mu.m [0286] sterile filter the preparation at room
temperature (SARTOBRAN.RTM. P unit, 0.22 .mu.m)
[0287] MPL powder is lyophilized by microfluidisation resulting in
a stable colloidal aqueous dispersion (MPL particle size smaller
than 200 nm). The MPL lyophilized powder is dispersed in water for
injection in order to obtain a coarse 10 mg/ml suspension. The
suspension then undergoes a thermal treatment under stirring. After
cooling to room temperature, the microfluidization process is
started in order to decrease the particle size. Microfluidization
is conducted using Microfluidics apparatus M110EH, by continuously
circulating the dispersion through a microfluidization interaction
chamber, at a defined pressure for a minimum amount of passages
(number of cycles: n.sub.min). The microfluidization duration,
representing the number of cycles, is calculated on basis of the
measured flow rate and the dispersion volume. On a given equipment
at a given pressure, the resulting flow rate may vary from one
interaction chamber to another, and throughout the lifecycle of a
particular interaction chamber. In the present example the
interaction chamber used is of the type F20Y Microfluidics. As the
microfluidization efficiency is linked to the couple pressure-flow
rate, the processing time may vary from one batch to another. The
time required for 1 cycle is calculated on basis of the flow rate.
The flow rate to be considered is the flow rate measured with water
for injection just before introduction of MPL into the apparatus.
One cycle is defined as the time (in minutes) needed for the total
volume of MPL to pass once through the apparatus. The time needed
to obtain n cycles is calculated as follows: [0288]
n.times.quantity of MPL to treat (ml)/flow rate (ml/min)
[0289] The number of cycles is thus adapted accordingly. Minimum
amount of cycles to perform (n.sub.min) are described for the
preferred equipment and interaction chambers used. The total amount
of cycles to run is determined by the result of a particle size
measurement performed after n.sub.min cycles. A particle size limit
(d.sub.lim) is defined, based on historical data. The measurement
is realized by photon correlation spectroscopy (PCS) technique, and
d.sub.lim is expressed as an unimodal result (Z.sub.average). Under
this limit, the microfluidization can be stopped after n.sub.min
cycles. Above this limit, microfluidization is continued until
satisfactory size reduction is obtained, for maximum another 50
cycles.
[0290] If the filtration does not take place immediately after
microfluidization, the dispersed MPL is stored at +2 to +8.degree.
C. awaiting transfer to the filtration area. After
microfluidization, the dispersion is diluted with water for
injection, and sterile filtered through a 0.22 .mu.m filter under
laminal flow. The final MPL concentration is 1 mg/ml (0.80-1.20
mg/ml).
II.3.2. Preparation of AS03+MPL Adjuvanted Vaccine: 1 Vial
Approach
[0291] To the AS03 adjuvant formulation, MPL is added at a final
concentration of between 10 and 50 .mu.g per vaccine dose.
[0292] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a SB62 mixture containing TWEEN.RTM., TRITON X-100.TM.
and VES (vitamin E succinate) is added to water for injection. The
quantities take into account the detergent present in the influenza
strains so as to reach a target final concentration of 750 .mu.g/ml
TWEEN.RTM. 80, 110 .mu.g/ml TRITON X100.TM. and 100 .mu.g/ml VES.
After 5 min stirring, 15 .mu.g of each influenza strain of interest
(for example strain H1N1, H3N2 and B in a classical tri-valent
vaccine) are added. After 15 min stirring, 250 .mu.l of SB62
emulsion is added and then 25 .mu.g or 50 .mu.g of MPL.
[0293] A schematic representation of the preparation process is
given in FIG. 3. The final composition of AS03 comprising MPL per
human dose is given the Table 4.
TABLE-US-00004 TABLE 4 Ingredients Concen- Per human dose Name
Component tration Quantity Other SB62 Squalene 781 .mu.l/ml 250
.mu.l (solution 10.68 mg 43 mg/ml) 11.86 mg Tocopherol (solution 48
mg/ml) TWEEN .RTM. 80 4.85 mg (solution 20 mg/ml) MPL** (solution
78 .mu.g/ml or 25 .mu.g or 1 mg/ml) 156 .mu.g/ml 50 .mu.g PBS mod*
NaCl 137 mM 2.56 mg KCl 2.7 mM 0.064 mg Na2HPO4 8.1 mM 0.368 mg
KH2PO4 1.47 mM 0.064 mg Water Ad for in- 320 .mu.l jection pH 6.8
+/- 0.1 *PBS mod 10.times. concentrated pH 6.8 = KH2PO4, Na2HPO4,
NaCl, KCl--HCl **MPL is either 25 .mu.g or 50 .mu.g per dose
II.3.3. Preparation of AS03+MPL Adjuvanted Vaccine: 2 Vials
Approach
[0294] The same formulation can be prepared from a 2 vials approach
by mixing 2 fold concentrated antigen or antigenic preparation with
the AS03 (SB62 250 .mu.l) or the AS03+MPL (SB62 250 .mu.l+25 .mu.g
or 50 .mu.g MPL) adjuvant. In this instance it is proceeded as
follows. The manufacturing of the AS25-adjuvanted influenza vaccine
consists of three main steps:
1) Formulation of the trivalent final bulk (2.times. concentrated)
without adjuvant and filling in the antigen container 2)
Preparation of the AS03+MPL adjuvant 3) Extemporaneous
reconstitution of the AS03+MPL adjuvanted split virus vaccine. 1)
Formulation of the Trivalent Final Bulk without Adjuvant and
Filling in the Antigen Container
[0295] The volumes of the three monovalent bulks are based on the
HA content measured in each monovalent bulk prior to the
formulation and on a target volume of 1100 ml. Concentrated
phosphate buffered saline and a pre-mixture of TWEEN.RTM. 80,
TRITON X-100.TM. and .alpha.-tocopheryl hydrogen succinate are
diluted in water for injection. The three concentrated monobulks
(A/New Caledonia, A/New York, B/Jiangsu) are then successively
diluted in the resulting phosphate buffered saline/TWEEN.RTM.
80--TRITON X100.TM. --.alpha.-tocopheryl hydrogen succinate
solution (pH 7.4, 137 mM NaCl, 2.7 mMKCl, 8.1 mM Na2HPO4, 1.47 mM
KH2PO4, 990 .mu.g/ml TWEEN.RTM. 80, 150 .mu.g/ml TRITON X100.TM.
and 130 .mu.g/ml .alpha.-tocopheryl hydrogen succinate) in order to
have a final concentration of 39.47 .mu.g HA of A strains (H1N1,
H3N2) per ml of trivalent final bulk (15 .mu.g HA/A strain/380
.mu.l trivalent final bulk) and 46 .mu.g HA of B strain (17.5 .mu.g
HA/B strain/380 .mu.l trivalent final bulk). Between addition of
each monovalent bulk, the mixture is stirred for 10-30 minutes at
room temperature. After addition of the last monovalent bulk and
15-30 minutes of stirring, the pH is checked and adjusted to
7.2.+-.0.2 with HCl or NaOH. The trivalent final bulk of antigens
is aseptically filled into 3-ml sterile Type I (Ph. Eur.) glass
vials. Each vial contains a volume of 470 .mu.l (380 .mu.l+90 .mu.l
overfill).
2) Preparation of AS03/MPL Adjuvant Bulk and Filling in the
Adjuvant Container.
[0296] The adjuvant AS03/MPL is prepared by mixing of two
components: SB62 emulsion (method in section II.1.2) and MPL
(method in section II.3.1). One-fold concentrated PBS mod (prepared
by diluting 10.times. concentrated PBS mod in water for injection)
with SB62 bulk and MPL liquid bulk at 1 mg/ml. MPL concentration
will be determined so as to reach a final content of between 10 to
50 .mu.g, suitably around 25 .mu.g per final human vaccine dose.
The mixture is stirred for 5-30 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 5-30 minutes at room
temperature the mixture is sterilised by filtration through a 0.22
.mu.m membrane. Sterile inert gas (nitrogen) flushing is performed
to produce inert head space in the filled containers during minimum
1 minute. The sterile AS03+MPL 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 80 .mu.l
(320 .mu.l+80 .mu.l overfill).
[0297] 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 AS25-adjuvanted influenza candidate vaccine
corresponds to 0.7 mL.
II.4. Preparation of Immunogenic Compositions Comprising an
Influenza Antigen and Optionally MPL in an Oil in Water Emulsion
Formulation
[0298] To the SB62 emulsion of II.1 an equal volume of twice
concentrated split influenza antigen (Fluarix.TM.) (15 .mu.g HA per
strain) was added and mixed. This was combined, when appropriate,
with 50 .mu.g/ml of MPL to give the final formulation.
Example III
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine Containing a Split Influenza Antigen Preparation and AS03
Adjuvant (Explo-Flu-001)
[0299] A phase I, open, randomised study was conducted in an
elderly population aged over 65 years in 2003 in order to evaluate
the reactogenicity and the immunogenicity of GlaxoSmithKline
Biologicals influenza candidate vaccine containing the adjuvant
AS03. 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) was
measured 21 days after intramuscular administration of one dose of
an AS03 adjuvanted vaccine or a WV vaccine. Fluarix.TM. was used as
reference.
III.1. Study Design
[0300] Three groups of subjects in parallel received the following
vaccine intramuscularly: [0301] one group of 50 subjects receiving
one dose of the reconstituted and adjuvanted SV influenza vaccine
(FluAS03) [0302] one group of 50 subjects receiving one dose of
whole virus influenza vaccine (FIuWVV) [0303] one group of 50
subjects receiving one dose of FLUARIX.RTM.=control
[0304] Vaccination schedule: one injection of influenza vaccine at
day 0, blood sample collection, read-out analysis at day 21 (HI
antibody determination, NI antibody determination, determination of
neutralising antibodies, and CMI analysis) and study
conclusion.
[0305] The standard trivalent split influenza vaccine--FLUARIX.RTM.
used in this study, is a commercial vaccine from the year 2003
developed and manufactured by GlaxoSmithKline Biologicals.
III.2. Vaccine Composition and Administration (Table 5)
III.2.1. Vaccine Preparation
AS03 Adjuvanted Influenza Vaccine
[0306] The AS03-adjuvanted influenza vaccine candidate is a 2
components vaccine consisting of a concentrated trivalent
inactivated split virion antigens presented in a type I glass vial
(335 .mu.l) (antigen container) and of a pre-filled type I glass
syringe containing the SB62 emulsion (335 .mu.l) (adjuvant
container). At the time of injection, the content of the antigen
container is removed from the with the help of the SB62 emulsion
pre-filled syringe, followed by gently mixing of the syringe.
Mixing of the SB62 emulsion with the vaccine antigens reconstitute
the AS03 adjuvant. Prior to injection, the used needle is replaced
by an intramuscular needle and the volume is corrected to 500
.mu.l.
[0307] One dose of the reconstituted AS03-adjuvanted influenza
vaccine corresponds to 0.5 ml, contains 15 .mu.g HA of each
influenza virus strain as in the registered
FLUARIX.RTM./.alpha.-RIX.RTM. vaccine and contains 10.68 mg
squalene, 11.86 mg DL-alpha tocopherol, and 4.85 mg polysorbate 80
(TWEEN.RTM. 80).
Preparation
[0308] The manufacturing of the AS03-adjuvanted influenza vaccine
consists of three main steps:
1) Formulation of the Trivalent Final Bulk without Adjuvant and
Filling in the Antigen Container.
[0309] The volumes of the three monovalent bulks are based on the
HA content measured in each monovalent bulk prior to the
formulation and on a target volume of 800 ml. Concentrated
phosphate buffered saline and a pre-mixture of TWEEN.RTM. 80,
TRITON X-100.TM. and .alpha.-tocopheryl hydrogen succinate are
diluted in water for injection. The three concentrated monobulks
(strain A/New Caledonia--, strain A/Panama--and strain
B/Shangdong--) are then successively diluted in the resulting
phosphate buffered saline/TWEEN.RTM. 80--TRITON X100.TM.
--.alpha.-tocopheryl hydrogen succinate solution (pH 7.4, 137 mM
NaCl, 2.7 mM KCl, 8.1 mM Na.sub.2HPO.sub.4, 1.47 mM KH2PO.sub.4,
1500 .mu.g/ml TWEEN.RTM. 80, 220 .mu.g/ml TRITON X100.TM. and 200
.mu.g/ml .alpha.-tocopheryl hydrogen succinate) in order to have a
final concentration of 60 .mu.g HA of A strains per ml of trivalent
final bulk (15 .mu.g HA/A strain/250 .mu.l trivalent final bulk)
and 70 .mu.g HA of B strain (17.5 .mu.g HA/B strain/250 .mu.l
trivalent final bulk). Between addition of each monovalent bulk,
the mixture is stirred for 10 minutes at room temperature. After
addition of the last monovalent bulk and 15 minutes of stirring,
the pH is checked and adjusted to 7.2.+-.0.1 with HCl or NaOH.
[0310] The trivalent final bulk of antigens is aseptically filled
into 3-ml sterile Type I glass vials. Each vial contains a 34%
volume overage (335 .mu.l total volume).
2) Preparation of the SB62 Emulsion Sterile Bulk and Filling in the
Adjuvant Container.
[0311] Aqueous phase: while stirring, 902 ml of TWEEN.RTM. 80 is
mixed with 44105 ml of PBS-mod buffer (pH=6.8 after adjustment with
HCl). [0312] Oil phase: while stirring, 2550 ml of squalene is
added to 2550 ml of .alpha.-tocopherol. [0313] Mixing of the
aqueous and oil phases: while stirring, 5000 ml of oil phase (1/10
total volume) is transferred to 45007 ml of aqueous phase (9/10
total volume). The mixture is stirred for 15 minutes at room
temperature. [0314] Emulsification: the resulting mixture is
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. [0315] Sterile filtration: the
SB62 emulsion is 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.
[0316] All quantities of ingredients given are for the preparation
of 50 L of emulsion and are given in volumes. In practice, amounts
are weighed taking into account the densities of the ingredients.
Density of PBS is considered equal to 1.
[0317] The final composition of the SB62 emulsion is as
follows:
TABLE-US-00005 TABLE 5 TWEEN .RTM. 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
PBS-mod: 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
[0318] The sterile SB62 bulk emulsion is then aseptically filled
into 1.25-ml sterile Type I glass syringes. Each syringe contains a
34% volume overage (335 .mu.l total volume).
3) Extemporaneous Reconstitution of the AS03 Adjuvanted Split Virus
Vaccine.
[0319] At the time of injection, the content of the vial containing
the concentrated trivalent inactivated split virion antigens is
removed from the vial with the help the syringe containing the SB62
emulsion followed by gently mixing of the syringe. Mixing of the
SB62 emulsion with the vaccine antigens reconstitutes the AS03
adjuvant.
III.2.2. Vaccine Composition (Table 6) and Administration
TABLE-US-00006 [0320] TABLE 6 Vaccine Formulation Group FLUARIX
.RTM. HA from 3 influenza strains (total HA = 45 .mu.g) FLUARIX
.RTM. A/New Caledonia/20/99 (IVR-116): 15 .mu.g A/Panama/2007/99
(RESVIR-17): 15 .mu.g B/Shangdong/7/97: 15 .mu.g Thiomersal
content: 5 .mu.g In pre-filled syringes of 0.5 ml WVV HA from 3
influenza strains (total HA = 45 .mu.g) FluWVV A/New
Caledonia/20/99 (IVR-116): 15 .mu.g A/Panama/2007/99 (RESVIR-17):
15 .mu.g B/Shangdong/7/97: 15 .mu.g Thiomersal content: 5 .mu.g In
vials of 0.5 ml FLUARIX .RTM. + HA from 3 influenza strains (total
HA = 45 .mu.g) Flu-AS03 AS03 A/New Caledonia/20/99 (IVR-116): 15
.mu.g A/Panama/2007/99 (RESVIR-17): 15 .mu.g B/Shangdong/7/97: 15
.mu.g Thiomersal content: 5 .mu.g In vial of 0.335 ml (2 times
concentrated) + syringe (0.335 ml) containing oil-in-water SB62
emulsion (scaled-up preparation)
[0321] The vaccines were administered intramuscularly in the
deltoid region of the non-dominant arm. The vaccinees were observed
closely for at least 30 minutes, with appropriate medical treatment
readily available in case of a rare anaphylactic reaction following
the administration of vaccine.
III.3. Study Population Results
[0322] A total of 148 subjects were enrolled in this study: 49
subjects in the FluAS03 group, 49 subjects in the FLUARIX.RTM.
group and 50 subjects in the FIuWVV group. The mean age of the
total vaccinated cohort at the time of vaccination was 71.8 years
with a standard deviation of 6.0 years. The mean age and gender
distribution of the subjects across the three vaccine groups was
similar.
III.4. Safety Conclusions
[0323] The administration of the influenza vaccine adjuvanted with
AS03 was safe and clinically well tolerated in the study
population, i.e. elderly people aged over 65 years.
III.5. Immunogenicity Results
[0324] Analysis of immunogenicity was performed on the total
vaccinated cohort.
III.5.1. Humoral Immune Response
[0325] In order to evaluate the humoral immune response induced by
the AS03 adjuvanted vaccine, the following parameters (with 95%
confidence intervals) were calculated for each treatment group:
[0326] Geometric mean titres (GMTs) of HI and NI antibody titres at
days 0 and 21 [0327] Geometric mean titres (GMTs) of neutralising
antibody titres at days 0 and 21. [0328] 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.
[0329] Conversion factors at day 21 defined as the fold increase in
serum HI GMTs on day 21 compared to day 0, for each vaccine strain.
[0330] Protection rates at day 21 defined as the percentage of
vaccinees with a serum HI titre=1:40. III.5.1.1 Anti-hemagglutinin
antibody response
a) HI Geometric Mean Titres (GMT)
[0331] The GMTs for HI antibodies with 95% CI are shown in Table 7
(GMT for anti-HI antibody). Pre-vaccination GMTs of antibodies for
all vaccine strains were within the same range in the three groups.
After vaccination, anti-haemagglutinin antibody levels increased
significantly. Post vaccination, there was a trend for higher GMTs
of HI antibody for all three vaccine strains in the FluAS03 and
FLUARIX.RTM. groups although there was some overlap of 95% CI
between the FLUARIX.RTM. group and the FIuWVV group.
TABLE-US-00007 TABLE 7 GMT 95% CI Antibody Group Timing N Value LL
UL A/New FluAS03 Pre 49 25.6 17.3 37.9 Caledonia FLUARIX .RTM.
PI(day 21) 49 317.7 219.1 460.7 FluWVV Pre 49 26.3 18.1 38.4 PI(day
21) 49 358.5 244.2 526.4 Pre 50 19.7 13.6 28.6 PI(day 21) 50 138.2
90.3 211.7 A/Panama FluAS03 Pre 49 52.3 35.4 77.4 FLUARIX .RTM.
PI(day 21) 49 366.1 264.5 506.6 FluWVV Pre 49 40.9 28.1 59.5 PI(day
21) 49 296.0 205.4 426.6 Pre 50 25.8 18.0 37.1 PI(day 21) 50 165.6
116.0 236.5 B/Shangdong FluAS03 Pre 49 27.5 19.0 39.8 FLUARIX .RTM.
PI(day 21) 49 317.7 226.9 444.9 FluWVV Pre 49 26.0 17.2 39.2 PI(day
21) 49 270.0 187.0 389.7 Pre 50 32.0 20.8 49.3 PI(day 21) 50 195.6
135.2 282.9 N = number of subjects with available results 95% CI =
95% confidence interval; LL = Lower Limit; UL = Upper Limit MIN/MAX
= Minimum/Maximum PRE = Prevaccination at Day 0 PI(D21) =
Post-vaccination at Day 21)
b) Conversion Factors of Anti-HI Antibody Titres, Seroprotection
Rates and Seroconversion Rates (Correlates for Protection in
Human)
[0332] Results are presented in Table 8.
[0333] The conversion factors represent the fold increase in serum
HI GMTs for each vaccine strain on day 21 compared to day 0. The
conversion factor varies from 6.1 to 13.6 according to the virus
strain and the vaccine. This conversion factor is largely superior
to the 2.0 fold increase in GMT required by the European
Authorities.
[0334] The seroprotection rates represent the proportion of
subjects with a serum HI titre 40 on day 21. At the outset of the
study, half of the subjects (range 34.0%-69.4%) in all groups had
protective levels of antibodies for all strains At day 21, the
seroprotection rates in the three groups ranged from 88.0% to 100%
for the different virus strains. In terms of protection, this means
that more than 88% of the subjects had a serum HI titre 40 after
vaccination and were deemed to be protected against the three
strains. This rate is largely superior to the seroprotection rate
of 60% required in the 60 years old population, by the European
Authorities.
[0335] The seroconversion rates represent the proportion of
subjects with at least a four-fold increase in serum HI titres on
day 21 as compared to day 0. Overall response rates for the three
strains were essentially equal in the three groups. To be deemed
effective and according to European Union, a vaccine should induce
a seroconversion rate greater than 30% in the =60 years old
population. In this study, the seroconversion rate was greater than
50% for the three groups.
TABLE-US-00008 TABLE 8 Seroprotection Seroconversion Conversion
rate rate factor EU standard (>60 years) >60% >30% >2.0
Strains Group N % [95% CI] % [95% CI] GMR [95% CI] A/New Flu AS03
49 98.0 [89.1-99.9] 69.4 [54.6-81.7] 12.4 [7.3-21.0] Caledonia
FLUARIX .RTM. 49 98.0 [89.1-99.9] 69.4[54.6-81.7] 13.6 [8.0-23.2]
Flu WVV 50 88.0 [75.7-95.5] 52.0 [37.4-66.3] 7.0 [4.0-12.2]
A/Panama Flu AS03 49 100.0 [92.7-100.0] 55.1 [40.2-69.3] 7.0
[4.2-11.6] FLUARIX .RTM. 49 91.8 [80.4-97.7] 65.3 [50.4-78.3] 7.2
[4.7-11.3] Flu WVV 50 90.0 [78.2-96.7] 56.0 [41.3-70.0] 6.4
[3.9-10.4] B/shangdong Flu AS03 49 100.0 [92.7-100.0] 73.5
[58.9-85.1] 11.6 [7.2-18.6] FLUARIX .RTM. 49 95.9 [86.0-99.5] 69.4
[54.6-81.7] 10.4 [6.5-16.5] Flu WVV 50 90.0 [78.2-96.7] 50.0
[35.5-64.5] 6.1 [3.6-10.3] N = total number of subjects
In conclusion: [0336] vaccination, there was a trend for higher
GMTs of HI antibody for all three vaccine strains in the FluAS03
and--FLUARIX.RTM. groups although there was some overlap of 95% CI
between the FLUARIX.RTM. group and the FIuWVV group. [0337] The
conversion factor varies from 6.1 to 13.6 according to the virus
strain and the vaccine. This conversion factor is largely superior
to the 2.0 fold increase in GMT required by the European
Authorities. [0338] At day 21, the seroprotection rates in the
three groups ranged from 88.0% to 100% for the different virus
strains. This rate is largely superior to the seroprotection rate
of 60% required in the .gtoreq.60 years old population, by the
European Authorities. [0339] In this study, the seroconversion rate
was greater than 50% for the three groups. Overall response rates
for the three strains were essentially equal in the three
groups.
III.5.1.2 Neutralising Antibody Titers
[0340] In order to better characterise the immune response to
influenza vaccination in the elderly, the serum antibody responses
to the neutralising antigens was assessed. Results are shown in
Table 9 (Seroprotection rates and geometric mean titres (GMT) for
anti-neutralising antibody titres) and Table 10 (Seroconversion
rates for anti-neutralising at post vaccination day 21
(fold-increase=4)).
[0341] Titres of neutralising antibody against the three influenza
strains were measured in pre- and post-immunisation sera. The
following parameters were determined: [0342] Geometric mean titres
(GMTs) of serum neutralising antibodies with 95% confidence
intervals (95% CI) pre and post-vaccination [0343] Seroconversion
rates with 95% CI at day 21, defined as the percentage of vaccinees
with at least a 4-fold increase in HI titres on day 21 compared to
day 0, for each vaccine strain.
TABLE-US-00009 [0343] TABLE 9 >=18 1/DIL GMT 95% CI 95% CI
Antibody Group Timing N n % LL UL Value LL UL A/NEW_CALEDONIA 1 PRE
49 46 93.9 83.1 98.7 106.6 77.6 146.6 PI(D21) 49 49 100.0 92.7
100.0 870.2 608.5 1244.3 2 PRE 49 48 98.0 89.1 99.9 115.6 89.4
149.5 PI(D21) 49 49 100.0 92.7 100.0 955.8 649.5 1406.5 3 PRE 50 46
92.0 80.8 97.8 87.7 63.6 120.8 PI(D21) 50 50 100.0 92.9 100.0 375.4
271.2 519.6 A/PANAMA 1 PRE 49 49 100.0 92.7 100.0 724.7 558.0 941.1
PI(D21) 49 49 100.0 92.7 100.0 2012.8 1438.4 2816.5 2 PRE 49 49
100.0 92.7 100.0 727.8 556.1 952.6 PI(D21) 49 49 100.0 92.7 100.0
1597.7 1128.8 2261.5 3 PRE 50 50 100.0 92.9 100.0 512.0 409.3 640.6
PI(D21) 50 50 100.0 92.9 100.0 977.8 738.2 1295.0 B/SHANGDONG 1 PRE
49 29 59.2 44.2 73.0 25.6 18.8 35.0 PI(D21) 49 48 98.0 89.1 99.9
222.5 148.1 334.2 2 PRE 49 27 55.1 40.2 69.3 29.3 20.1 42.7 PI(D21)
49 49 100.0 92.7 100.0 190.4 127.6 284.3 B/SHANGDONG 3 PRE 50 31
62.0 47.2 75.3 33.4 23.1 48.4 PI(D21) 50 46 92.0 80.8 97.8 117.8
82.6 168.0 Group 1: Flu vaccine mix Adjuvant 2.times. conc Flu vac
Group 2: Flu vaccine Flu vaccine Group 3: Flu vaccine Flu WVV
vaccine 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 PRE = Pre-vaccination at Day 0 PI(D21) = Post-vaccination at
Day 21)
TABLE-US-00010 TABLE 10 Responders 95% CI Antibody Group N n % LL
UL A/New 1 49 29 59.2 44.2 73.0 Caledonia 2 49 30 61.2 46.2 74.8 3
50 21 42.0 28.2 56.8 A/Panama 1 49 12 24.5 13.3 38.9 2 49 9 18.4
8.8 32.0 3 50 9 18.0 8.6 31.4 B/Shangdong 1 49 29 59.2 44.2 73.0 2
49 26 53.1 38.3 67.5 3 50 19 38.0 24.7 52.8 Group 1: Flu vaccine
(DFLU58A16) mix Adjuvant (D621024A8) 2.times. conc Flu vac Group 2:
Flu vaccine (18854B9) Flu vaccine Group 3: Flu vaccine (DFLU59A2)
Flu WVV vaccine N = number of subjects with both pre and post
vaccination result available. n = number of responders % =
Proportion of responders (n/N .times. 100). 95% CI = exact 95%
confidence interval; LL = lower limit, UL = upper limit
[0344] The main findings are: [0345] For the three vaccines, at day
21, a seroprotection rate of 100% is obtained for both A strains.
For the B strain, the seroprotection rates in the three groups
ranged from 92% to 100%. [0346] Post vaccination, there was a
significant increase of GMT for all strains, in the three groups.
However, there was a trend for higher GMTs of neutralising antibody
for all three vaccine strains in the FluAS03 and FLUARIX.RTM.
groups than in the FIuWVV although there was some overlap of 95% CI
between the FLUARIX.RTM. group and the FIuWVV group. [0347] For the
seroconversion rates, overall response rates for the three strains
were essentially equal in the three groups.
[0348] In all groups, the results were consistent with those
obtained from the analysis performed for anti-hemagglutinin
antibodies.
III.5.1.3 Nauraminidase (NA) Antibody Titers
[0349] In order to better characterise the immune response to
influenza vaccination in the elderly population, the serum antibody
responses to the neuraminidase antigens was assessed. Similarly to
the HI antibody titre, the following endpoints were determined:
[0350] GMT (taking the anti-log of the mean of the log titre
transformations) [0351] Seroconversion rate defined as the
percentage of vaccinees with at least a 4-fold increase in HI
titres on day 21 compared to day 0, for each vaccine strain.
[0352] The GMTs and seroconversion rates for NI antibodies with 95%
CI are shown in Table 11 (anti-NA antibody GMT) and Table 12
(Seroconversion rates of NA at post-vaccination (day 21)
(4-fold-increase)).
TABLE-US-00011 TABLE 11 95% CI Antibody Group Timing N GMT LL UL
A/New FluAS03 PRE 49 77.8 61.8 97.9 Caledonia PI(D21) 48 270.0
212.9 342.3 FLUARIX .RTM. PRE 49 77.8 64.6 93.6 PI(D21) 49 249.1
190.0 326.5 FluWVV PRE 50 66.8 53.8 83.0 PI(D21) 50 159.2 122.8
206.4 A/Panama FluAS03 PRE 49 33.3 28.5 48.7 PI(D21) 48 156.8 124.8
196.9 FLUARIX .RTM. PRE 49 34.2 25.6 45.8 PI(D21) 49 133.7 100.9
177.3 FluWVV PRE 50 24.6 18.7 32.4 PI(D21) 49 78.9 59.4 104.7
B/Shangdong FluAS03 PRE 49 46.7 36.5 59.9 PI(D21) 49 204.2 156.4
266.7 FLUARIX .RTM. PRE 49 46.1 35.3 60.1 PI(D21) 49 133.7 100.9
177.3 FluWVV PRE 50 48.6 36.4 64.7 PI(D21) 49 128.2 101.7 161.6
FluAS03: Flu vaccine (DFLU58A16) mix with AS03 Adjuvant (D621024A8)
FLUARIX .RTM.: Flu vaccine (18854B9) FluWVV: Flu WVV vaccine
(DFLU59A2) PRE = Pre-vaccination, PI(D21) = Day 21 post vaccination
95% CI, LL, and UL = 95% confidence interval, lower and upper
limit
TABLE-US-00012 TABLE 12 Responders 95% CI Antibody Group N n % LL
UL A/New FluAS03 48 25 52.1 37.2 66.7 Caledonia FLUARIX .RTM. 49 24
49.0 34.4 63.7 FluWVV 49 18 36.7 23.4 51.7 A/Panama FluAS03 48 27
56.3 41.2 70.5 FLUARIX .RTM. 49 23 46.9 32.5 61.7 FluWVV 49 21 42.9
28.8 57.8 B/Shangdong FluAS03 48 26 54.2 39.2 68.6 FLUARIX .RTM. 49
23 46.9 32.5 61.7 FluWVV 49 16 32.7 19.9 47.5 FluAS03: Flu vaccine
(DFLU58A16) mix with AS03 Adjuvant (D621024A8), FLUARIX .RTM.: Flu
vaccine (18854B9), FluWVV: Flu WVV vaccine (DFLU59A2) N = number of
subjects with both pre and post vaccination result available, n =
number of responders. % = Proportion of responders (n/N .times.
100). 95% CI = exact 95% confidence interval; LL = lower limit, UL
= upper limit
[0353] The main findings are: [0354] Higher value of the GMT and
seroconversion rates were observed for hemagglutinin than for
neuraminidase. [0355] Pre-vaccination GMTs of antibodies for all
vaccine strains were within the same range in the three groups.
After vaccination, anti-neuraminidase antibody levels increased
significantly. As for the HI antibody titres, post vaccination,
there was a trend for higher GMTs of HI antibody for all three
vaccine strains in the FluAS03 and FLUARIX.RTM. groups although
there was some overlap of 95% CI between the Fluarix group and the
FIuWVV group. [0356] Regarding the seroconversion rates, overall
response rates for the three strains were essentially equal in the
three groups and for the three strains.
[0357] Our results show that healthy elderly vaccinated in this
study against influenza developed good antibody responses to
neuraminidase antigens whatever the influenza vaccine. However, the
response to the neuraminidase antigen is lower than the response to
the hemagglutinin antigen.
III.5.2. Cellular Immune Response
[0358] Peripheral blood antigen-specific CD4 and CD8 T cells can be
restimulated in vitro to produce IL-2, CD40L, TNF-alpha and
IFN.gamma. 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 as well as peptides derived from specific influenza
protein were used as antigen to restimulate Influenza-specific T
cells. Results are presented for the CD4 and CD8 T-cell response in
Tables 13 to 18.
TABLE-US-00013 TABLE 13 Antigen specific CD4 'T-cell responses
expressed into cells producing at least two different cytokines:
Descriptive Statistics on PRE and POST for
CD40L/IL2/TNF-.alpha./IFN-.gamma. (Total vaccinated cohort) Time
Secretion Antigen Gr point N Mean SD Min CD40L/IL2/ Peptide 1 Day 0
44 33.50 139.026 1.00 IFN.gamma./TNF.alpha. Influenza 1 Day 21 45
58.40 132.664 1.00 in CD4 2 Day 0 42 92.10 368.790 1.00 2 Day 21 44
88.36 272.528 1.00 3 Day 0 45 80.13 284.316 1.00 3 Day 21 47 91.40
382.967 1.00 Split 1 Day 0 47 1901.66 1596.203 102.00 Influenza 1
Day 21 48 6163.75 4265.900 773.00 2 Day 0 45 2151.04 2622.594
265.00 2 Day 21 49 4150.73 3712.469 328.00 3 Day 0 48 1678.44
916.329 142.00 3 Day 21 50 3374.60 1920.194 449.00 Whole 1 Day 0 48
3134.33 2568.369 507.00 Influenza 1 Day 21 47 9332.04 6875.403
1482.00 2 Day 0 47 3050.85 2654.936 486.00 2 Day 21 49 6760.31
6788.258 1852.00 3 Day 0 48 2955.33 2019.233 473.00 3 Day 21 50
5661.40 4530.321 635.00 Kruskall- Wallis Time test (P- Secretion
Antigen Gr point Q1 Median Q3 Max value) CD40L/IL2/ Peptide 1 Day 0
1.00 1.00 4.00 915.00 0.7631 IFN.gamma./TNF.alpha. Influenza 1 Day
21 1.00 1.00 56.00 733.00 in CD4 2 Day 0 1.00 1.00 54.00 2393.00 2
Day 21 1.00 1.00 69.50 1740.00 3 Day 0 1.00 1.00 65.00 1908.00 3
Day 21 1.00 1.00 63.00 2615.00 Split 1 Day 0 957.00 1560.00 2408.00
9514.00 0.0002 Influenza 1 Day 21 3468.00 4908.00 7624.00 21324.00
2 Day 0 930.00 1381.00 2274.00 16289.00 2 Day 21 2247.00 3036.00
4744.00 21924.00 3 Day 0 1086.00 1502.00 2189.00 3899.00 3 Day 21
2312.00 3040.00 4437.00 10431.00 Whole 1 Day 0 1730.00 2298.50
3876.00 15066.00 0.0040 Influenza 1 Day 21 4091.00 6523.00 14045.00
29251.00 2 Day 0 1190.00 2031.00 4161.00 11994.00 2 Day 21 3573.00
4621.00 7234.00 40173.00 3 Day 0 1421.50 2668.50 3411.50 10578.00 3
Day 21 2459.00 4315.00 7303.00 22053.00 Group 1: FluAS03: Flu
vaccine Fluarix .TM. mixed with AS03 Adjuvant Group 2: FLUARIX
.RTM.: Flu vaccine FLUARIX .RTM. Group 3: FluWVV: Flu WVV vaccine
SD = Standard Deviation; Min, Max = Minimum, Maximum Q1 = First
quartile; Q3 = Third quartile N = number of subjects with available
results P-value: Kruskall-Wallis Test (Non-parametric procedure) to
test location difference (Wilcoxon rank-sum test) between the 3
groups at Day 21.
TABLE-US-00014 TABLE 14 Antigen-specific CD4 T-cell responses
expressed into cells producing at least two different cytokines:
Descriptive Statistics on difference between PRE and POST ('Total
vaccinated cohort) Secretion Antigen Group N Mean SD Min
CD40L/IFN-.gamma./ Peptide 1 44 9.57 159.363 -860.00 TNF-.alpha. in
CD4 Influenza 2 42 -40.98 386.998 -2392.00 3 45 -50.73 256.596
-1664.00 Split 1 47 4307.02 4468.828 -8161.00 Influenza 2 45
1982.93 3802.332 -14318.0 3 48 1555.90 1596.216 -526.00 Whole 1 47
6197.98 7220.765 -11763.0 Influenza 2 47 3791.34 5820.894 -2128.00
3 48 2535.98 3966.345 -4766.00 CD40L/IFN-.gamma./ Peptide 1 42
-15.95 215.710 -451.00 TNF-.alpha. in CD8 Influenza 2 41 50.83
264.370 -614.00 3 44 -52.11 243.811 -684.00 Split 1 42 134.71
426.699 -603.00 Influenza 2 44 -65.05 822.036 -4938.00 3 45 2.49
330.700 -1094.00 Whole 1 39 189.38 1394.153 -2641.00 Influenza 2 44
-479.75 1790.094 -9455.00 3 44 -243.73 719.269 -1892.00 Secretion
Antigen Group Q1 Median Q3 Max P-value CD40L/IFN-.gamma./ Peptide 1
0.00 0.00 37.50 430.00 0.0765 TNF-.alpha. in CD4 Influenza 2 -15.00
0.00 26.00 514.00 3 -37.00 0.00 0.00 212.00 Split 1 1888.00 3396.00
6634.00 19555.00 <0.0001 Influenza 2 699.00 1490.00 2573.00
15169.00 3 466.00 1183.50 2186.50 7851.00 Whole 1 2170.00 4009.00
11681.00 25570.00 0.0003 Influenza 2 1246.00 2382.00 3992.00
33801.00 3 503.00 1382.50 3300.50 19337.00 CD40L/IFN-.gamma./
Peptide 1 -106.00 0.00 81.00 655.00 0.0932 TNF-.alpha. in CD8
Influenza 2 -58.00 13.00 202.00 703.00 3 -160.50 0.00 53.00 567.00
Split 1 -122.00 35.50 221.00 1387.00 0.2121 Influenza 2 -64.50 0.00
160.50 1252.00 3 -99.00 0.00 76.00 1060.00 Whole 1 -420.00 49.00
591.00 5045.00 0.0851 Influenza 2 -1016.00 -263.50 180.00 3743.00 3
-651.00 -86.50 180.00 1011.00
TABLE-US-00015 TABLE 15 Antigen-specific CD4 T-cell responses
expressed into cells producing at least CD40L and another cytokine:
Descriptive Statistics on difference between PRE and POST ('Total
vaccinated cohort) Secretion Antigen Group N Mean SD Min CD40L in
CD4 Peptide 1 44 10.09 153.007 -815.00 Influenza 2 42 -29.40
316.983 -1921.00 3 45 -43.73 251.146 -1629.00 Split 1 46 4266.20
4470.807 -8093.00 Influenza 2 45 2026.42 3511.508 -11482.0 3 47
1512.34 1576.133 -494.00 Whole 1 47 6071.96 7118.132 -11691.0
Influenza 2 47 3764.64 5740.762 -2114.00 3 48 2544.27 3959.879
-4390.00 CD40L in CD8 Peptide 1 44 -19.41 81.675 -370.00 Influenza
2 41 -3.98 100.998 -399.00 3 45 -5.56 64.666 -181.00 Split 1 43
39.53 190.122 -438.00 Influenza 2 44 27.61 91.173 -155.00 3 45
30.18 191.326 -291.00 Whole 1 41 -91.24 617.077 -1779.00 Influenza
2 44 -115.91 588.424 -2583.00 3 45 -150.89 367.300 -1239.00
Secretion Antigen Group Q1 Median Q3 Max P-value CD40L in CD4
Peptide 1 0.00 0.00 36.50 428.00 0.1233 Influenza 2 -8.00 0.00
27.00 494.00 3 -35.00 0.00 3.00 230.00 Split 1 1799.00 3156.50
6647.00 19480.00 <0.0001 Influenza 2 783.00 1485.00 2546.00
15021.00 3 469.00 1107.00 2035.00 7687.00 Whole 1 2109.00 4048.00
11472.00 25448.00 0.0004 Influenza 2 1212.00 2509.00 3957.00
33428.00 3 523.00 1392.00 3261.50 19478.00 CD40L in CD8 Peptide 1
-2.00 0.00 0.50 100.00 0.9721 Influenza 2 -28.00 0.00 24.00 231.00
3 -13.00 0.00 3.00 176.00 Split 1 -35.00 0.00 140.00 608.00 0.6175
Influenza 2 -18.50 0.00 77.50 326.00 3 -9.00 0.00 28.00 1188.00
Whole 1 -142.00 -8.00 175.00 2087.00 0.3178 Influenza 2 -195.50
-34.50 150.00 1258.00 3 -270.00 -103.00 88.00 588.00
TABLE-US-00016 TABLE 16 Antigen-specific CD4 T-cell responses
expressed into cells producing at least IFN.gamma. and another
cytokine: Descriptive Statistics on difference between PRE and POST
('Total vaccinated cohort) N Secretion Antigen Group N missing Mean
SD Min IFN.gamma. in CD4 Peptide 1 44 5 7.50 64.539 -171.00
Influenza 2 42 7 -30.67 277.984 -1766.00 3 45 5 -27.91 103.403
-639.00 Split 1 46 3 2712.87 2905.629 -4394.00 Influenza 2 45 4
1148.56 2526.536 -10586.0 3 47 3 871.00 1016.251 -764.00 Whole 1 47
2 4240.09 4811.891 -8272.00 Influenza 2 47 2 2445.38 4030.694
-3018.00 3 48 2 1535.48 2456.915 -3670.00 IFN.gamma. in CD8 Peptide
1 44 5 7.75 146.412 -226.00 Influenza 2 41 8 10.68 176.026 -420.00
3 44 6 -49.80 217.214 -699.00 Split 1 43 6 138.58 365.565 -470.00
Influenza 2 44 5 -112.82 793.746 -4919.00 3 44 6 29.91 238.157
-708.00 Whole 1 41 8 6.66 1642.577 -5610.00 Influenza 2 44 5
-471.55 1792.348 -9586.00 3 44 6 -189.05 685.291 -1879.00 Secretion
Antigen Group Q1 Median Q3 Max P-value IFN.gamma. in CD4 Peptide 1
-9.50 0.00 7.50 265.00 0.1541 Influenza 2 -5.00 0.00 24.00 222.00 3
-20.00 0.00 0.00 51.00 Split 1 1273.00 1644.00 4057.00 13296.00
<0.0001 Influenza 2 405.00 931.00 1757.00 9426.00 3 283.00
624.00 1114.00 5031.00 Whole 1 1610.00 2693.00 7437.00 17489.00
<0.0001 Influenza 2 723.00 1487.00 2983.00 21594.00 3 232.50
810.00 2218.50 11319.00 IFN.gamma. in CD8 Peptide 1 -52.50 0.00
40.00 615.00 0.3322 Influenza 2 -1.00 0.00 72.00 610.00 3 -172.00
0.00 90.50 424.00 Split 1 -46.00 42.00 294.00 1549.00 0.1257
Influenza 2 -62.00 0.00 74.00 1028.00 3 -59.50 26.50 123.00 643.00
Whole 1 -385.00 131.00 450.00 5068.00 0.1179 Influenza 2 -955.50
-221.00 177.00 3492.00 3 -476.50 -36.50 198.00 1299.00
TABLE-US-00017 TABLE 17 Antigen-specific CD4 T-cell responses
expressed into cells producing at least IL2 and another cytokine:
Descriptive Statistics on difference between PRE and POST ('Total
vaccinated cohort) Secretion Antigen Group N Mean SD Min IL2 in CD4
Peptide 1 44 2.82 118.164 -595.00 Influenza 2 42 0.90 84.255
-167.00 3 45 -28.62 191.709 -1222.00 Split 1 46 3456.15 3853.960
-7009.00 Influenza 2 45 1738.29 2406.045 -451.00 3 47 1210.02
1361.705 -634.00 Whole 1 47 4839.02 5978.277 -9178.00 Influenza 2
47 2891.00 4493.387 -1370.00 3 48 2042.50 3123.912 -3179.00 IL2 in
CD8 Peptide 1 42 -30.60 219.777 -630.00 Influenza 2 41 38.85
210.715 -674.00 3 45 -44.80 197.026 -526.00 Split 1 41 54.85
250.817 -336.00 Influenza 2 44 -2.36 423.957 -2272.00 3 45 -26.07
244.870 -1004.00 Whole 1 39 56.21 406.262 -704.00 Influenza 2 44
-151.02 822.384 -4304.00 3 45 -63.56 359.699 -1036.00 Secretion
Antigen Group Q1 Median Q3 Max P-value IL2 in CD4 Peptide 1 -1.50
0.00 31.50 324.00 0.0806 Influenza 2 -34.00 0.00 2.00 362.00 3
-19.00 0.00 0.00 253.00 Split 1 1309.00 2598.50 5926.00 16988.00
<0.0001 Influenza 2 453.00 1113.00 2049.00 12273.00 3 331.00
806.00 1596.00 6474.00 Whole 1 1516.00 3341.00 8955.00 21032.00
0.0006 Influenza 2 995.00 1942.00 3007.00 26358.00 3 371.50 1083.50
2624.50 14057.00 IL2 in CD8 Peptide 1 -111.00 0.00 103.00 412.00
0.1684 Influenza 2 -41.00 0.00 138.00 542.00 3 -150.00 -34.00 71.00
447.00 Split 1 -76.00 26.00 133.00 803.00 0.2311 Influenza 2 -78.50
0.00 121.50 1064.00 3 -93.00 -1.00 30.00 705.00 Whole 1 -167.00
63.00 261.00 1302.00 0.4586 Influenza 2 -444.50 -4.00 199.00
1398.00 3 -198.00 9.00 131.00 838.00
TABLE-US-00018 TABLE 18 Antigen-specific CD4 T-cell responses
expressed into cells producing at least TNF.alpha. and another
cytokine: Descriptive Statistics on difference between PRE and POST
('Total vaccinated cohort) Secretion Antigen Group N Mean SD Min
TNF-.alpha. in CD4 Peptide 1 44 9.48 92.992 -466.00 Influenza 2 42
-47.71 367.624 -2333.00 3 45 -37.38 179.147 -1169.00 Split 1 46
2343.11 2596.177 -4450.00 Influenza 2 45 703.87 2973.241 -14260.0 3
47 732.00 740.001 -611.00 Whole 1 47 3103.74 4248.997 -5146.00
Influenza 2 47 1658.38 3639.959 -1393.00 3 48 1010.15 1689.394
-1482.00 TNF-.alpha. in CD8 Peptide 1 42 11.71 201.031 -453.00
Influenza 2 41 37.46 245.241 -612.00 3 44 -42.95 210.185 -645.00
Split 1 41 138.54 362.601 -329.00 Influenza 2 44 -70.27 790.309
-4741.00 3 44 -39.75 348.803 -1044.00 Whole 1 39 279.59 1048.352
-1184.00 Influenza 2 44 -280.70 1562.095 -9070.00 3 44 -71.57
492.135 -1574.00 Secretion Antigen Group Q1 Median Q3 Max P-value
TNF-.alpha. in CD4 Peptide 1 -1.50 0.00 39.00 239.00 0.1836
Influenza 2 -4.00 0.00 12.00 277.00 3 -26.00 0.00 5.00 53.00 Split
1 862.00 1466.50 3931.00 9267.00 <0.0001 Influenza 2 251.00
698.00 1229.00 12275.00 3 191.00 540.00 1010.00 3288.00 Whole 1
868.00 1607.00 5266.00 17199.00 0.0008 Influenza 2 367.00 871.00
1584.00 23540.00 3 175.00 592.00 1385.50 8760.00 TNF-.alpha. in CD8
Peptide 1 -80.00 0.50 70.00 772.00 0.2759 Influenza 2 -81.00 0.00
155.00 791.00 3 -179.00 0.00 39.50 566.00 Split 1 -23.00 60.00
178.00 1468.00 0.0790 Influenza 2 -107.00 0.00 158.00 1286.00 3
-185.00 0.00 78.50 1021.00 Whole 1 -250.00 108.00 399.00 4601.00
0.1482 Influenza 2 -392.00 -56.50 205.00 3258.00 3 -233.50 -54.00
160.00 1543.00
[0359] Results were also expressed as a frequency of
cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell
sub-population and presented in FIG. 4 and FIG. 5.
[0360] In a similar analysis, the cross-reactive CD4 T-cells
response was evaluated using influenza antigen from drifted strains
(A/H1N1/Beijing/262/95 (H1N1d), A/H3N2/Sydney/5/97 (H3N2d),
B/Yamanashi/166/98 (Bd)) or shift strains (A/Singapore/1/57 (H2N2),
A/Hongkong/1073/99 (H9N2)). Results expressed as a frequency of
cytokine(s)-positive CD4 T cells are presented in FIG. 6.
[0361] The main findings are: [0362] Vaccination with FLUARIX.RTM.
or Whole virus slightly boosts the CD4 T-cell response. Vaccination
with Flu AS03 induces a strong CD4 T-cell response (FIG. 4), and
this is statistically significant. The same conclusion is made
after In Vitro stimulation with the split antigen or Whole virus,
and this with all cytokines investigated (IL-2, IFN.gamma.,
TNF.alpha., and CD40L). [0363] Most individuals have a CD8 T-cell
response against the whole flu, however the vaccination has no
measurable impact on the CD8 T-cell response (i.e. Pre=post),
whatever the group studied (FIG. 5).
[0364] Vaccination with FLUARIX.RTM. only induces low levels of
cross-reactive CD4 T-cell response (FIG. 6). Vaccination with
FluAS03 induces a strong CD4 T-cell response against drifted
influenza strains and this is statistically significant (FIG. 6). A
little response was detected against shift strains.
III.5.3. B-cells Elispot Memory
III.5.3.1 Objective
[0365] In order to better characterise the CMI response induced by
the AS03-adjuvanted influenza vaccine, the B-cells Elispot memory
response induced to differentiate into plasma cells in vitro using
influenza vaccine strains or anti-human immunoglobulin was evaluate
in order to enumerate anti-influenza or IgG secreting plasma. The
results are described in Table 19 and Table 20 and in FIG. 7.
[0366] A subset of 22 first subjects having received one dose of
FluAS03 vaccine and 21 first subjects having received one dose of
FLUARIX.RTM. vaccine was selected to evaluate the impact
[0367] of vaccination on influenza-specific memory B-cells using
the B-cell memory Elispot technology. The following endpoints were
determined [0368] At days 0 and 21: Influenza-specific memory
B-cells have been measured by B-cell Elispot in all subjects.
Results have been expressed as a frequency of Influenza
specific-antibody forming cells per million (10.sup.6) of antibody
forming cells. [0369] Difference between post (day 21) and pre (day
0) vaccination is also expressed as a frequency of Influenza
specific-antibody forming cells per million (10.sup.6) of antibody
forming cells.
III.5.3.2 Statistical Methods
[0370] Descriptive statistics for each vaccination group at days 0
and day 21 expressed as a frequency of Influenza specific-antibody
forming cells per million (10.sup.6) of antibody forming cells.
Descriptive statistics in individual difference between day 21 and
day 0 (Post-Pre) as a frequency of Influenza specific-antibody
forming cells per million (10.sup.6) of antibody forming cells.
[0371] A Wilcoxon test was used to compare the location of
difference between the two groups and the statistical p-value was
calculated for each of 3 strains (A/New Caledonia, A/Panama and
B/Shangdong).
III.5.3.3 Results
[0372] There is a tendency in favour of the influenza adjuvanted
AS03 vaccine compared to FLUARIX.RTM. group. For A/New Caledonia
strain, there is a statistical significant difference
(p-value=0.021) in favour of FluAS03 compared to FLUARIX.RTM.. No
statistical difference between the two groups was observed for
A/Panama and B/Shangdong strains.
TABLE-US-00019 TABLE 19 B-cells Memory: descriptive statistics on
pre (Day 0) and post (Day 21) and inferential statistics of post
(Day 21) frequency of antigen-plasma within a 10.sup.6 of
IgG-producing plasma cells (subset of subjects) Time- STRAIN Group
point N Mean SD Min A/NEW 1 Day 0 22 9751.58 6630.335 0.00
CALEDONIA 1 Day 21 22 22001.65 11308.261 3981.90 2 Day 0 21 9193.61
4339.421 1300.81 2 Day 21 21 12263.08 7285.698 789.47 A/PANAMA 1
Day 0 22 4329.17 2923.497 0.00 1 Day 21 22 18066.69 14604.842
714.29 2 Day 0 21 4860.41 3392.373 0.00 2 Day 21 21 13872.95
12052.163 0.00 B/SHANDONG 1 Day 0 22 3722.80 2347.315 0.00 1 Day 21
22 15949.60 12385.965 0.00 2 Day 0 21 3030.39 2206.589 640.57 2 Day
21 21 9714.03 5656.805 0.00 P-value Time- (Wilcoxon STRAIN Gr point
Q1 Median Q3 Max test) A/NEW 1 Day 0 4117.65 9606.46 13430.66
25570.78 0.0056 CALEDONIA 1 Day 21 11052.63 20450.55 30234.74
40526.32 2 Day 0 6363.64 9686.41 11698.11 19164.84 2 Day 21 7741.05
9545.45 17069.60 32000.00 A/PANAMA 1 Day 0 2275.45 4003.02 5764.55
10842.49 0.1814 1 Day 21 9347.37 13176.41 21471.39 54789.92 2 Day 0
2222.22 4545.45 7495.74 11698.11 2 Day 21 6231.88 10147.06 20540.54
52188.84 B/SHANDONG 1 Day 0 2058.82 2956.78 5972.22 7832.17 0.1483
1 Day 21 6860.47 12796.90 22947.37 48947.37 2 Day 0 1290.32 2113.82
4770.02 7783.25 2 Day 21 6590.91 9009.01 12774.87 21201.72 Group 1:
Flu vaccine FLUARIX .RTM. + AS03 oil-in-water emulsion adjuvant
Group 2: Flu vaccine FLUARIX .RTM. SD = Standard Deviation Min, Max
= Minimum, Maximum Q1 = First quartile Q3 = Third quartile N =
number of subjects with available results P-value: Wilcoxon Test
(Non-parametric procedure) to test location difference (Wilcoxon
rank-sum test) between the 2 groups at Day 21.
TABLE-US-00020 TABLE 20 B cells Memory: Descriptive and inferential
statistics on difference between POST (Day 21) and PRE (Day 0)
frequency of antigen-specific plasma within a 10 6 of IgG-producing
plasma cells (subset of subjects) STRAIN Group N Mean SD Min A/NEW
1 22 12250.07 12875.755 -4365.08 CALEDONIA 2 21 3069.46 7309.731
-10043.4 A/PANAMA 1 22 13737.52 13677.942 -188.29 2 21 9012.54
11489.012 -1551.05 B/SHANDONG 1 22 12226.81 12243.895 -2222.22 2 21
6683.64 6240.312 -2113.82 P-value (Wilcoxon STRAIN Gr Q1 Median Q3
Max test) A/NEW 1 2418.07 6776.65 26036.01 35059.98 0.0210
CALEDONIA 2 -1762.54 1694.51 6850.19 18579.97 A/PANAMA 1 4551.30
11039.04 16614.85 49881.94 0.1449 2 1522.85 6480.96 9214.67
47812.47 B/SHANDONG 1 1788.75 9322.70 18907.05 42134.18 0.1895 2
2117.44 5384.41 9897.27 19801.28 Group 1: Flu vaccine FLUARIX .RTM.
+ AS03 oil-in-water emulsion adjuvant Group 2: Flu vaccine FLUARIX
.RTM. SD = Standard Deviation Min, Max = Minimum, Maximum Q1 =
First quartile Q3 = Third quartile N = number of subjects with
available results P-value: Wilcoxon Test (Non-parametric procedure)
to test location difference (Wilcoxon rank-sum test) between the 2
groups at Day 21.
III.6. Overall Conclusions
III.6.1. Reactogenicity and Safety Results
[0373] While influenza immunisation significantly reduces the risk
of pneumonia and associated deaths, vaccination of elderly only
affords 23-72% protection against influenza disease. Formulation of
vaccine antigen with potent adjuvants is an attractive approach for
enhancing immune responses to subunit antigens. This study was
designed to evaluate (1) the safety and reactogenicity in healthy
elderly of an influenza vaccine adjuvanted with oil in water
emulsion, i.e. AS03, (2) the antibody and cell-mediated immune
responses. Reactogenicity data show that the influenza vaccine
adjuvanted with AS03 induced more local and general symptoms than
the two other vaccines. However regarding unsolicited adverse
events, no difference was observed between the three vaccines. From
these results, it can be concluded that the reactogenicity and
safety profile of the candidate vaccines is satisfactory and
clinically acceptable.
III.6.2. Immunogenicity Results
[0374] Regarding the immune response, the three vaccines exceeded
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
immuno-logical assessment of the annual strain
changes--CPMP/BWP/214/96). The three influenza vaccines tested in
this study were immunogenic in the healthy elderly, who developed a
good antibody response to influenza hemagglutinin and neutralising
antigens (Table 21).
TABLE-US-00021 TABLE 21 EU standard for Variable antibody response
Results Conversion factor >2.0 >6.1 Seroconversion rate
>30% >50% Protection rate >60% >88%
[0375] Regarding cell-mediated immunity (CMI) response, the
influenza vaccine adjuvanted with AS03 induced a significantly
stronger CD4 response (included drifted strains) than the two other
vaccines (FLUARIX.RTM. and whole influenza virus vaccine). However,
vaccination has no measurable impact on the CD8 response.
[0376] Regarding the B cell memory response, there is a tendency in
favour of the influenza adjuvanted vaccine compared to the
un-adjuvanted vaccine.
Example IV
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine containing a split influenza antigen preparation and AS03
adjuvant--Explo-Flu-002
[0377] A phase I/II, open, controlled study has been conducted in
order to evaluate the reactogenicity and the immunogenicity of the
GlaxoSmithKline Biologicals influenza candidate vaccine containing
the adjuvant AS03, in an elderly population aged over 65 years and
previously vaccinated in 2003 with the candidate vaccine in the
Explo-Flu-001 clinical trial. For immunogenicity and safety
evaluations, FLUARIX.RTM. vaccine (known as .alpha.-RIX.RTM. in
Belgium) has been used as reference.
IV.1. Objective
[0378] The humoral immune response (i.e. anti-hemagglutinin
antibody titres) and cell mediated immune response (CD4 and/or CD8
T cell responses) and B memory cell response were measured 21 days
after intramuscular administration of one dose of an AS03
adjuvanted vaccine. FLUARIX.RTM./.alpha.-RIX.RTM. was used as
reference.
[0379] The objectives were:
1) to determine if AS03 adjuvanted Flu (40 subjects) versus
FLUARIX.RTM. (18 subjects) confirm his strongest immunostimulating
activity on CD4- and/or CD8-mediated immunity of individuals
vaccinated with influenza antigens; 2) to investigate, using a
longitudinal analysis, the influence of AS03 adjuvanted on the
immune response in prevaccination 2004 (so response one year after
the first vaccination in 2003).
IV.2. Study Design, Vaccine Composition and End-Points
[0380] 40 subjects aged >65 years who have previously received
one dose of the AS03 adjuvanted influenza vaccine during the
Explo-Flu-001 clinical trial in 2003 (FluAS03) [0381] one control
group of about 20 subjects aged >65 years who have previously
received one dose of FLUARIX.RTM. during the Explo-Flu-001 clinical
trial in 2003 (FLUARIX.RTM.) IV.2.1. Vaccine composition
[0382] The vaccine composition is similar to that used for the
study Explo-Flu-001 except for the influenza strains included in
the vaccine (year 2004 vaccine). The strains are as follows: [0383]
A/New Caledonia/20/99 (IVR-116) (H1N1)=A/New Caledonia/(HINI)-like
strain [0384] A/Wyoming/3/2003 (X-147) (H3N2)=A/Fujian (H3N2)-like
strain [0385] B/Jiangsu/10/2003=B/Shanghai-like strain
IV.2.2. Immunogenicity (HI) End-Points
[0385] [0386] GMTs (taking the anti-log of the mean of the log
titre transformations) [0387] Conversion factors (the fold increase
in serum HI GMTs on day 21 compared to day 0) [0388] Seroconversion
rate (the percentage of vaccinees with at least a four-fold
increases in HI titers on day 21 compared to day 0, for each
vaccine strain) [0389] Protection rate (the percentage of vaccinees
with a serum HI.gtoreq.1: 40 at day 21)
IV.2.3. CMI-Endpoints
Observed Variable:
[0390] At days 0 and 21: frequency of cytokine-positive CD4/CD8
cells per 10.sup.6 into 4 different cytokines. Each test quantifies
the response of CD4/CD8 T cell to: [0391] Pool of the 3 following
antigens [0392] New Caledonia antigen [0393] Wyoming antigen [0394]
Jiangsu antigen.
Derived Variables:
[0395] Antigen-specific CD4 and CD8-T-cell response expressed into
the 5 different tests (cytokines):
1. cells producing at least two different cytokines (CD40L, IL-2,
IFN.gamma., TNF.alpha.) 2. cells producing at least CD40L and
another cytokine (IL-2, TNF.alpha., IFN.gamma.) 3. cells producing
at least IL-2 and another cytokine (CD40L, TNF.alpha., IFN.gamma.)
4. cells producing at least IFN.gamma. and another cytokine (IL-2,
TNF.alpha., CD40L) 5. cells producing at least TNF.alpha. and
another cytokine (IL-2, CD40L, IFN.gamma.)
IV.2.4. CMI Analysis
[0396] The first CMI analysis was based on the Total Vaccinated
cohort (N=40 subjects for FluAS03 group and N=18 subjects for
FLUARIX.RTM. group).
[0397] A longitudinal analysis was based on the Kinetic cohort of
the Explo-Flu-001 (split protein) and Explo-Flu-002 (pool flu
antigen) studies: [0398] Pre: N=36 subjects for FluAS03 group and
N=15 for FLUARIX.RTM. group. [0399] Post-Pre: N=34 subjects for
FluAS03 group and N=15 for FLUARIX.RTM. group. [0400] (a) The
frequency of CD4/CD8 T-lymphocytes secreting in response was
summarised by descriptive statistics for each antigen, for each
cytokine, for each vaccine group and at each timepoint (pre- and
post-vaccination). [0401] (b) Descriptive statistics in individual
difference between timepoints (Post-Pre) responses were tabulated
for each antigen, for each cytokine and for each vaccine group.
[0402] (c) For the timepoints post and (post-pre) vaccination,
non-parametric Wilcoxon's test was used to compare the location
differences between the two vaccine groups and to calculate the
statistical p-value regarding the 4 different cytokines on: [0403]
CD4 T-cell response to New Caledonia, Wyoming, Jiangsu and the pool
of the 3 strains. [0404] CD8 T-cell response to New Caledonia,
Wyoming, Jiangsu and the pool of the 3 strains. [0405] (d)
Non-parametric test (Wilcoxon-test) was also used: [0406] To
investigate the kinetic of the immune response at Pre (Day 0) in
term of frequency of specific CD4 between Explo-Flu-001 and
Explo-Flu-002 in each vaccine group [0407] To investigate the
kinetic of the immune response at Pre (Day 0) in term of frequency
of specific CD4 between the 2 vaccine groups in each of the study
Explo-Flu-001 and Explo-Flu-002 [0408] To investigate the kinetic
of the immune response in term of differences (Post-Pre) of
frequency of specific CD4 between Explo-Flu-001 and Explo-Flu-002
in each vaccine group. [0409] To investigate the kinetic of the
immune response in term of differences (Post-Pre) of frequency of
specific CD4 between the 2 vaccine groups in each of the study
Explo-Flu-001 and Explo-Flu-002
[0410] All significance tests were two-tailed. P-values less than
or equal to 0.05 were considered as statistically significant.
IV.3. Results
[0411] Results were expressed as a frequency of
cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell
sub-population.
IV.3.1. Antigen Specific CD4 T-Lymphocytes
[0412] The frequency of antigen-specific CD4 T-lymphocytes
secreting in response was summarised by descriptive statistics for
each antigen, for each cytokine, for each vaccine group and at each
timepoint (pre- and post-vaccination).
[0413] Descriptive statistics in individual difference between time
points (Post-Pre) in CD4 T-lymphocytes responses for each antigen
at each 5 different cytokines and for each vaccine group are shown
in Table 22.
TABLE-US-00022 TABLE 22 Descriptive Statistics on difference
between Post-vaccination (at Day 21) and Prevaccination (at Day 0)
for the antigen-specific CD4 T-lymphocyte responses (Total
vaccinated cohort) Vaccine Antigen Cytokine Group N Mean SD Min Q1
Median Q3 Max Pool Flu All FLUARIX .RTM. 18 1268.67 1051.744 197.00
724.00 863.00 1561.00 4676.00 double Flu AS03 36 1781.31 1484.860
-2379.00 929.50 1664.50 2821.00 4669.00 CD40L FLUARIX .RTM. 18
1260.11 1054.487 243.00 721.00 849.00 1602.00 4743.00 Flu AS03 36
1711.56 1433.113 -2359.00 838.00 1576.00 2759.50 4575.00 IFN.gamma.
FLUARIX .RTM. 18 762.94 813.884 -12.00 294.00 496.00 1061.00
3564.00 Flu AS03 36 1179.92 881.255 -817.00 692.50 1180.50 1865.50
2831.00 IL2 FLUARIX .RTM. 18 1019.06 917.905 -258.00 544.00 702.00
1174.00 3850.00 Flu AS03 36 1423.33 1359.471 -2702.00 651.00
1260.00 2200.50 4342.00 TNF.alpha. FLUARIX .RTM. 18 803.39 915.838
32.00 231.00 533.00 936.00 3892.00 Flu AS03 36 1078.28 1029.122
-1816.00 446.00 983.00 1836.00 3310.00 A/New All FLUARIX .RTM. 18
481.44 381.534 -241.00 282.00 448.50 598.00 1412.00 Caledonia
double Flu AS03 36 812.78 749.192 -828.00 215.50 911.50 1274.50
3206.00 CD40L FLUARIX .RTM. 18 450.78 360.378 -239.00 291.00 447.00
580.00 1248.00 Flu AS03 36 783.75 711.608 -760.00 242.00 808.00
1161.00 3050.00 IFN.gamma. FLUARIX .RTM. 18 316.28 279.662 -165.00
175.00 259.00 387.00 1111.00 Flu AS03 36 438.22 420.770 -685.00
125.00 393.00 733.50 1557.00 IL2 FLUARIX .RTM. 18 326.06 290.792
-294.00 193.00 330.00 488.00 834.00 Flu AS03 36 634.72 616.478
-557.00 179.50 678.50 952.00 2602.00 TNF.alpha. FLUARIX .RTM. 18
316.44 372.492 -140.00 50.00 278.00 542.00 1449.00 Flu AS03 36
449.17 591.796 -916.00 100.50 343.50 848.00 2452.00 A/Wyoming All
FLUARIX .RTM. 18 609.56 559.396 -176.00 257.00 510.50 957.00
1998.00 double Flu AS03 36 766.61 579.191 -568.00 316.00 864.50
1221.00 1662.00 CD40L FLUARIX .RTM. 18 616.33 550.853 -176.00
274.00 488.00 939.00 2017.00 Flu AS03 36 728.61 570.316 -670.00
260.00 789.50 1216.00 1675.00 IFN.gamma. FLUARIX .RTM. 18 407.06
424.758 -311.00 129.00 370.50 723.00 1372.00 Flu AS03 36 526.72
443.938 -770.00 219.00 556.50 776.00 1342.00 IL2 FLUARIX .RTM. 18
495.83 503.805 -187.00 88.00 540.50 801.00 1841.00 Flu AS03 36
572.89 533.728 -789.00 220.00 602.00 882.50 1512.00 TNF.alpha.
FLUARIX .RTM. 18 424.56 485.591 -260.00 110.00 359.50 461.00
1718.00 Flu AS03 36 550.58 538.461 -765.00 269.50 543.50 905.50
1678.00 B/Jiangsu All FLUARIX .RTM. 18 698.44 793.119 -306.00
233.00 433.00 961.00 2822.00 double Flu AS03 36 861.42 688.852
-223.00 339.00 745.00 1325.50 2284.00 CD40L FLUARIX .RTM. 18 678.39
777.259 -206.00 227.00 401.50 962.00 2878.00 Flu AS03 36 825.89
674.879 -223.00 305.00 722.00 1282.00 2337.00 IFN.gamma. FLUARIX
.RTM. 18 431.72 489.912 -95.00 191.00 272.50 382.00 1712.00 Flu
AS03 36 615.94 473.543 -286.00 288.50 501.50 897.50 1740.00 IL2
FLUARIX .RTM. 18 552.50 666.853 -234.00 155.00 278.50 833.00
2386.00 Flu AS03 36 696.19 622.931 -359.00 207.50 540.50 1146.50
2182.00 TNF.alpha. FLUARIX .RTM. 18 441.39 695.792 -338.00 97.00
269.50 564.00 2440.00 Flu AS03 36 500.03 448.636 -166.00 107.50
436.00 745.00 1626.00 SD = Standard Deviation Min, Max = Minimum,
Maximum Q1 = First quartile Q3 = Third quartile N = number of
subjects tested with available results
[0414] Vaccine-induced CD4 T-cells are shown to be able to persist
at least for one year since there is an observable difference in
prevaccination levels of CD4 T-cell responses between individuals
vaccinated with FLUARIX.RTM. has compared to those vaccinated with
FLUARIX.RTM./AS03 the year before. The results are also shown in
FIG. 8, showing the CD4 T-cell response to split Flu antigen before
and after revaccination. D0 corresponds to 12 months after first
year vaccination and thus indicates persistence.
[0415] Comparing the difference in the frequency of
antigen-specific CD4 T-lymphocytes between the 2 groups by Wilcoxon
test at post-vaccination, almost all p-values were less than 0.05
and were considered as statistically significant (see Table 23) in
favour of the FluAS03 group.
TABLE-US-00023 TABLE 23 Inferential statistics: p-values from
Wilcoxon rank-sum test between the two vaccine groups at Day 21 for
antigen-specific CD4 T-lymphocyte responses (Total vaccinated
cohort) P-value New Cytokine Pool Caledonia Wyoming Jiangsu All
0.0014 0.0023 0.0286 0.0133 double CD40L 0.0016 0.0014 0.0427
0.0155 INF.gamma. 0.0006 0.0366 0.0400 0.0041 IL2 0.0037 0.0024
0.0584 0.0162 TNF.alpha. 0.0031 0.0103 0.0918 0.0114 P-value:
Wilcoxon Test (Non-parametric procedure) to test location
difference (Wilcoxon rank-sum test) between the 2 groups at Day
21.
[0416] Comparing the difference of the individual difference
(Post-Pre) in the frequency of antigen-specific CD4-T-lymphocytes
responses between the 2 groups by Wilcoxon test, p-values less than
0.05 and considered as statistically significant occurred for the
following antigen-cytokine combinations: pool flu-all double, pool
flu-IFN.gamma. and Jiangsu-IFN.gamma. in favour of the FluAS03
group (Table 24).
TABLE-US-00024 TABLE 24 Inferential statistics: p-values calculated
by Wilcoxon rank-sum test between the different groups on the
difference between Post- vaccination (at Day 21) and Prevaccination
(at 0) for the antigen- specific CD4 T-lymphocyte responses (Total
vaccinated cohort) P-value New Cytokine Pool Caledonia Wyoming
Jiangsu All 0.0435 0.1124 0.2189 0.3085 double CD40L 0.0638 0.0781
0.2831 0.2872 INF.gamma. 0.0290 0.3589 0.2553 0.0435 IL2 0.1024
0.0563 0.3986 0.0435 TNF.alpha. 0.0693 0.4090 0.1232 0.3129
P-value: Wilcoxon Test (Non-parametric procedure) to test location
difference (Wilcoxon rank-sum test) between the 2 groups.
IV.3.2. Antigen specific CD8 T-Lymphocytes
[0417] The frequency of antigen-specific CD8 T-lymphocytes
secreting in response was summarised by descriptive statistics for
each antigen, for each cytokine, for each vaccine group and at each
timepoint (pre- and post-vaccination), similarly to the procedure
followed in respect of CD4 T cell response.
[0418] Comparing the difference in the frequency of
antigen-specific CD8 T-lymphocytes between the 2 groups by Wilcoxon
test at post-vaccination, all p-values were higher than 0.05 and
were not considered as statistically significant. Comparing the
difference of the individual difference (Post-Pre) in the frequency
of antigen-specific CD8-T-lymphocytes responses between the 2
groups by Wilcoxon test, all p-values were higher than 0.05 and
were not considered as statistically significant.
IV.3.3. Kinetic Analysis: Immune Response at Prevaccination (One
Year after the First Vaccination in 2003)
[0419] The frequency of antigen-specific CD4 T-lymphocytes
secreting in response at prevaccination was summarised by
descriptive statistics for each cytokine and for each vaccine group
and for each of the two studies in Table 25, for each of the two
studies study and for each vaccine group in Table 27. Inferential
statistics are given in Table 26 and Table 28.
TABLE-US-00025 TABLE 25 Descriptive Statistics on prevaccination
(Day 0) for the specific CD4 T- lymphocytes response vaccination
(Kinetic) Cytokine Group Study N Mean SD Min Q1 Median Q3 Max All
Flu EXPLO 001 36 2000.86 1783.474 102.00 911.50 1461.50 2791.00
9514.00 double AS03 EXPLO 002 36 2028.28 1427.000 55.00 1190.50
1647.50 2575.00 7214.00 FLUARIX .RTM. EXPLO 001 15 2152.87 2162.463
747.00 930.00 1354.00 2101.00 7868.00 EXPLO 002 15 1587.07 2123.841
192.00 468.00 735.00 1578.00 8536.00 CD40L Flu EXPLO 001 35 1946.66
1771.102 120.00 837.00 1340.00 2819.00 9462.00 AS03 EXPLO 002 35
1992.20 1440.721 77.00 1125.00 1590.00 2587.00 7286.00 FLUARIX
.RTM. EXPLO 001 15 2094.93 2076.632 745.00 902.00 1340.00 2077.00
7385.00 EXPLO 002 15 1561.73 2097.201 34.00 475.00 672.00 1579.00
8428.00 INF.gamma. Flu EXPLO 001 35 1068.63 1030.745 91.00 448.00
790.00 1503.00 5425.00 AS03 EXPLO 002 35 1259.23 890.590 312.00
725.00 984.00 1354.00 4146.00 FLUARIX .RTM. EXPLO 001 15 1248.07
1452.459 320.00 388.00 778.00 1227.00 5431.00 EXPLO 002 15 974.80
1394.044 52.00 252.00 337.00 1057.00 5576.00 IL2 Flu EXPLO 001 35
1690.20 1524.689 37.00 688.00 1211.00 2416.00 8235.00 AS03 EXPLO
002 35 1883.60 1361.337 14.00 1068.00 1413.00 2370.00 6891.00
FLUARIX .RTM. EXPLO 001 15 1888.40 2085.857 568.00 715.00 1136.00
1770.00 7403.00 EXPLO 002 15 1493.93 2037.139 58.00 444.00 755.00
1485.00 8193.00 TNF.alpha. Flu EXPLO 001 35 1174.74 1119.633 55.00
466.00 795.00 1720.00 5415.00 AS03 EXPLO 002 35 1545.40 1159.490
135.00 831.00 1203.00 1857.00 5354.00 FLUARIX .RTM. EXPLO 001 15
1444.20 1946.211 201.00 520.00 688.00 1254.00 7213.00 EXPLO 002 15
1304.73 1759.716 144.00 316.00 824.00 1171.00 7056.00 SD = Standard
Deviation Min, Max = Minimum, Maximum Q1 = First quartile Q3 =
Third quartile N = number of subjects tested with available
results
[0420] Comparing the difference in the frequency of
antigen-specific CD4 T-lymphocytes between the 2 studies by
Wilcoxon test for each vaccine group, p-values less than 0.05 and
considered as statistically significant (in favour of
Explo-Flu-002) occurred only for FluAS03 group and with TNF.alpha.
cytokine (see Table 26).
TABLE-US-00026 TABLE 26 Inferential statistics: p-values from
Wilcoxon rank- sum test between the different studies at Day 0 for
antigen-specific CD4 T-lymphocyte responses (Kinetic) Cytokine
Group p-value ALL FluAS03 0.5209 DOUBLE FLUARIX .RTM. 0.0712 CD40L
FluAS03 0.4957 FLUARIX .RTM. 0.0744 INF.gamma. FluAS03 0.0896
FLUARIX .RTM. 0.1103 IL2 FluAS03 0.1903 FLUARIX .RTM. 0.1647
TNF.alpha. FluAS03 0.0427 FLUARIX .RTM. 0.5476 P-value: Wilcoxon
Test (Non-parametric procedure) to test location difference
(Wilcoxon rank-sum test) between the 2 groups at Day 21.
TABLE-US-00027 TABLE 27 Descriptive Statistics on Prevaccination
(Day 0) for the specific CD4 T- lymphocytes response vaccination
(Kinetic) Cytokine Study Group N Mean SD Min Q1 Median Q3 Max All
EXPLO Flu 36 2000.86 1783.474 102.00 911.50 1461.50 2791.00 9514.00
double 001 AS03 FLUARIX .RTM. 15 2152.87 2162.463 747.00 930.00
1354.00 2101.00 7868.00 EXPLO Flu 36 2028.28 1427.000 55.00 1190.50
1647.50 2575.00 7214.00 002 AS03 FLUARIX .RTM. 15 1587.07 2123.841
192.00 468.00 735.00 1578.00 8536.00 CD40L EXPLO Flu 35 1946.66
1771.102 120.00 837.00 1340.00 2819.00 9462.00 001 AS03 FLUARIX
.RTM. 15 2094.93 2076.632 745.00 902.00 1340.00 2077.00 7385.00
EXPLO Flu 35 1992.20 1440.721 77.00 1125.00 1590.00 2587.00 7286.00
002 AS03 FLUARIX .RTM. 15 1561.73 2097.201 34.00 475.00 672.00
1579.00 8428.00 INF.gamma. EXPLO Flu 35 1068.63 1030.745 91.00
448.00 790.00 1503.00 5425.00 001 AS03 FLUARIX .RTM. 15 1248.07
1452.459 320.00 388.00 778.00 1227.00 5431.00 EXPLO Flu 35 1259.23
890.590 312.00 725.00 984.00 1354.00 4146.00 002 AS03 FLUARIX .RTM.
15 974.80 1394.044 52.00 252.00 337.00 1057.00 5576.00 IL2 EXPLO
Flu 35 1690.20 1524.689 37.00 688.00 1211.00 2416.00 8235.00 001
AS03 FLUARIX .RTM. 15 1888.40 2085.857 568.00 715.00 1136.00
1770.00 7403.00 EXPLO Flu 35 1883.60 1361.337 14.00 1068.00 1413.00
2370.00 6891.00 002 AS03 FLUARIX .RTM. 15 1493.93 2037.139 58.00
444.00 755.00 1485.00 8193.00 TNF.alpha. EXPLO Flu 35 1174.74
1119.633 55.00 466.00 795.00 1720.00 5415.00 001 AS03 FLUARIX .RTM.
15 1444.20 1946.211 201.00 520.00 688.00 1254.00 7213.00 EXPLO Flu
35 1545.40 1159.490 135.00 831.00 1203.00 1857.00 5354.00 002 AS03
FLUARIX .RTM. 15 1304.73 1759.716 144.00 316.00 824.00 1171.00
7056.00 SD = Standard Deviation Min, Max = Minimum, Maximum Q1 =
First quartile Q3 = Third quartile N = number of subjects tested
with available results
[0421] Comparing the difference in the frequency of
antigen-specific CD4 T-lymphocytes between the 2 vaccine groups by
Wilcoxon test for each study, all p-values for Explo-Flu-002 were
less than 0.05 and were considered as statistically significant (in
favour of FluAS03) (see Table 28).
TABLE-US-00028 TABLE 28 Inferential statistics: p-values from
Wilcoxon rank-sum test between the different groups at Day 21 for
antigen-specific CD4 T-lymphocyte responses (Kinetic) Cytokine
Study p-value ALL DOUBLE Explo Flu 001 0.9423 Explo Flu 002 0.0300
CD40L Explo Flu 001 0.8989 Explo Flu 002 0.0361 INF.gamma. Explo
Flu 001 0.8738 Explo Flu 002 0.0121 IL2 Explo Flu 001 0.9747 Explo
Flu 002 0.0216 TNF.alpha. Explo Flu 001 0.9916 Explo Flu 002 0.0514
P-value: Wilcoxon Test (Non-parametric procedure) to test location
difference (Wilcoxon rank-sum test) between the 2 groups at Day
21.
IV.3.4. Kinetic Analysis: Immune Response at Post Minus
Prevaccination
[0422] The frequency of antigen-specific CD4 T-lymphocytes
secreting in response at (post-pre) timepoint was summarised by
descriptive statistics for each cytokine and for each vaccine group
and for each study in Table 29, for each study and for each vaccine
group in Table 31. Inferential statistics are given in Table 30 and
Table 32.
TABLE-US-00029 TABLE 29 Descriptive Statistics on the difference
between Post-vaccination (Day 21) and Prevaccination (Day 0) for
the specific CD4 T-lymphocytes response vaccination (Kinetic)
Cytokine Group Study N Mean SD Min Q1 Median Q3 Max All Flu EXPLO
34 4837.56 4476.129 -609.00 1888.00 3483.50 8148.00 19555.00 double
AS03 001 EXPLO 34 1737.79 1450.177 -2379.00 936.00 1664.50 2743.00
4669.00 002 FLUARIX .RTM. EXPLO 15 3103.53 3726.645 436.00 800.00
2283.00 3226.00 15169.00 001 EXPLO 15 1369.00 1127.784 197.00
725.00 869.00 1808.00 4676.00 002 CD40L Flu EXPLO 33 4819.06
4489.788 -718.00 1799.00 3479.00 8288.00 19480.00 AS03 001 EXPLO 33
1694.73 1431.082 -2359.00 921.00 1659.00 2662.00 4575.00 002
FLUARIX .RTM. EXPLO 15 3090.00 3684.759 477.00 822.00 2189.00
3208.00 15021.00 001 EXPLO 15 1360.93 1131.051 243.00 725.00 860.00
1687.00 4743.00 002 IFN.gamma. Flu EXPLO 33 3127.09 2974.067
-453.00 1325.00 1721.00 5162.00 13296.00 AS03 001 EXPLO 33 1167.85
893.363 -817.00 633.00 1207.00 1803.00 2831.00 002 FLUARIX .RTM.
EXPLO 15 1660.13 1834.023 -84.00 480.00 1386.00 2284.00 7120.00 001
EXPLO 15 851.87 859.585 148.00 294.00 501.00 1222.00 3564.00 002
IL2 Flu EXPLO 33 3950.18 3878.538 -358.00 1309.00 2780.00 6635.00
16988.00 AS03 001 EXPLO 33 1404.67 1355.665 -2702.00 719.00 1341.00
2109.00 4342.00 002 FLUARIX .RTM. EXPLO 15 2413.87 3027.392 263.00
674.00 1672.00 2425.00 12273.00 001 EXPLO 15 1117.80 975.934
-258.00 575.00 714.00 1618.00 3850.00 002 TNF.alpha. Flu EXPLO 33
2627.36 2574.458 -825.00 862.00 1475.00 4764.00 9267.00 AS03 001
EXPLO 33 1072.36 1044.140 -1816.00 447.00 1000.00 1752.00 3310.00
002 FLUARIX .RTM. EXPLO 15 1460.53 3115.174 -1586.00 251.00 813.00
1314.00 12275.00 001 EXPLO 15 904.67 974.958 32.00 338.00 752.00
965.00 3892.00 002 SD = Standard Deviation Min, Max = Minimum,
Maximum Q1 = First quartile Q3 = Third quartile N = number of
subjects tested with available results
[0423] Comparing the difference in the frequency of
antigen-specific CD4 T-lymphocytes between the 2 studies by
Wilcoxon test for each vaccine group, all p-values for FluAS03
group were less than 0.05 and were considered as statistically
significant (in favour of Explo-Flu-001) (see Table 30).
TABLE-US-00030 TABLE 30 Inferential statistics on the difference
between Post-vaccination (Day 21) and Prevaccination (Day 0):
p-values from Wilcoxon rank-sum test between the different studies
at Day 21 for antigen-specific CD4 T-lymphocyte responses (Kinetic)
Cytokine Group p-value ALL FluAS03 0.0005 DOUBLE FLUARIX .RTM.
0.1300 CD40L FluAS03 0.0007 FLUARIX .RTM. 0.0890 INF.gamma. FluAS03
0.0012 FLUARIX .RTM. 0.1103 IL2 FluAS03 0.0025 FLUARIX .RTM. 0.1409
TNF.alpha. FluAS03 0.0327 FLUARIX .RTM. 0.6936 P-value: Wilcoxon
Test (Non-parametric procedure) to test location difference
(Wilcoxon rank-sum test) between the 2 groups at Day 21.
TABLE-US-00031 TABLE 31 Descriptive Statistics on the difference
between Post-vaccination (Day 21) and Prevaccination (Day 0) for
the specific CD4 T-lymphocytes response vaccination (Kinetic)
Cytokine Study Group N Mean SD Min Q1 Median Q3 Max All EXPLO Flu
34 4837.56 4476.129 -609.00 1888.00 3483.50 8148.00 19555.00 double
001 AS03 FLUARIX .RTM. 15 3103.53 3726.645 436.00 800.00 2283.00
3226.00 15169.00 EXPLO Flu 34 1737.79 1450.177 -2379.00 936.00
1664.50 2743.00 4669.00 002 AS03 FLUARIX .RTM. 15 1369.00 1127.784
197.00 725.00 869.00 1808.00 4676.00 CD40L EXPLO Flu 33 4819.06
4489.788 -718.00 1799.00 3479.00 8288.00 19480.00 001 AS03 FLUARIX
.RTM. 15 3090.00 3684.759 477.00 822.00 2189.00 3208.00 15021.00
EXPLO Flu 33 1694.73 1431.082 -2359.00 921.00 1659.00 2662.00
4575.00 002 AS03 FLUARIX .RTM. 15 1360.93 1131.051 243.00 725.00
860.00 1687.00 4743.00 IFN.gamma. EXPLO Flu 33 3127.09 2974.067
-453.00 1325.00 1721.00 5162.00 13296.00 001 AS03 FLUARIX .RTM. 15
1660.13 1834.023 -84.00 480.00 1386.00 2284.00 7120.00 EXPLO Flu 33
1167.85 893.363 -817.00 633.00 1207.00 1803.00 2831.00 002 AS03
FLUARIX .RTM. 15 851.87 859.585 148.00 294.00 501.00 1222.00
3564.00 IL2 EXPLO Flu 33 3950.18 3878.538 -358.00 1309.00 2780.00
6635.00 16988.00 001 AS03 FLUARIX .RTM. 15 2413.87 3027.392 263.00
674.00 1672.00 2425.00 12273.00 EXPLO Flu 33 1404.67 1355.665
-2702.00 719.00 1341.00 2109.00 4342.00 002 AS03 FLUARIX .RTM. 15
1117.80 975.934 -258.00 575.00 714.00 1618.00 3850.00 TFN.alpha.
EXPLO Flu 33 2627.36 2574.458 -825.00 862.00 1475.00 4764.00
9267.00 001 AS03 FLUARIX .RTM. 15 1460.53 3115.174 -1586.00 251.00
813.00 1314.00 12275.00 EXPLO Flu 33 1072.36 1044.140 -1816.00
447.00 1000.00 1752.00 3310.00 002 AS03 FLUARIX .RTM. 15 904.67
974.958 32.00 338.00 752.00 965.00 3892.00 SD = Standard Deviation
Min, Max = Minimum, Maximum Q1 = First quartile Q3 = Third quartile
N = number of subjects tested with available results
[0424] Comparing the difference in the frequency of
antigen-specific CD4 T-lymphocytes between the 2 vaccine groups by
Wilcoxon test for each study, p-value was less than 0.05 only for
Explo-Flu-001 and was considered as statistically significant (in
favour of FluAS03) (see Table 32).
TABLE-US-00032 TABLE 32 Inferential statistics: p-values from
Wilcoxon rank-sum test between the different groups at Day 21 for
antigen-specific CD4 T-lymphocyte responses (Kinetic) Cytokine
Study p-value ALL DOUBLE Explo Flu 001 0.0827 Explo Flu 002 0.0992
CD40L Explo Flu 001 0.0931 Explo Flu 002 0.1391 INF.gamma. Explo
Flu 001 0.0543 Explo Flu 002 0.1068 IL2 Explo Flu 001 0.0847 Explo
Flu 002 0.2254 TNF.alpha. Explo Flu 001 0.0375 Explo Flu 002 0.2009
P-value: Wilcoxon Test (Non-parametric procedure) to test location
difference (Wilcoxon rank-sum test) between the 2 groups at Day
21.
IV.4. HI Titers
[0425] Results are shown in FIG. 9 and in Tables 33 to 36.
TABLE-US-00033 TABLE 33 Geometric Mean Titers (GMT) and
seropositivity rates of anti-HI titers (GMTs calculated on
vaccinated subjects) 95% CI 95% CI Antibody Group Timing N S+ %
L.L. U.L. GMT L.L. U.L. New FLUARIX .RTM. PRE 18 17 94.4 72.6 99.9
63.5 38.1 105.9 Caledonia PI(D21) 18 18 100 81.5 100 131.9 77.1
225.6 FluAS03 PRE 40 39 97.5 86.8 99.9 70.3 50.5 97.7 PI(D21) 40 40
100 91.3 100 218.6 158.2 302.0 A/Fujian FLUARIX .RTM. PRE 18 18 100
81.5 100 95.0 51.0 176.9 PI(D21) 18 18 100 81.5 100 498.3 272.1
912.7 FluAS03 PRE 40 40 100 91.3 100 94.3 71.4 124.6 PI(D21) 40 40
100 91.3 100 735.1 564.4 957.5 B/Shanghai FLUARIX .RTM. PRE 18 16
88.9 65.3 98.6 23.3 15.2 35.8 PI(D21) 18 17 94.4 72.6 99.9 139.8
64.0 305.0 FluAS03 PRE 40 38 95.0 83.1 99.4 58.6 43.9 78.1 PI(D21)
40 40 100 91.3 100 364.4 269.7 492.4 PRE = Prevaccination, PI(D21)
= day 21 post vaccination 95% CI, LL, and UL = 95% confidence
interval, lower and upper limit S+ = number of seropositive
subjects
TABLE-US-00034 TABLE 34 Conversion factor of anti-HI titers (All
vaccinated subjects) A/N-Caledonia A/Fujian B/Shanghai GMR GMR GMR
Group N [95% CI] N [95% CI] N [95% CI] FLUARIX .RTM. 18 2.1 18 5.2
18 6.0 [1.4; 3.2] [3.0; 9.3] [3.5; 10.2] FluAS03 40 3.1 40 7.8 40
6.2 [2.4; 4.0] [5.6; 10.9] [4.7; 8.2] N = total number of subjects
GMR = Geometric Mean Ratio (antilog of the mean log day 21/day 0
titers ratios) 95% CI = 95% confidence interval
TABLE-US-00035 TABLE 35 Seroprotection rates of anti-HI titers (All
vaccinated subjects) >=40 Antibody Group Timing N n % 95% CI
A/New FLUARIX .RTM. PRE 18 14 77.8 52.4 93.6 Caledonia PI(D21) 18
16 88.9 65.3 98.6 FluAS03 PRE 40 32 80 64.4 90.9 PI(D21) 40 39 97.5
86.8 99.9 A/Fujian FLUARIX .RTM. PRE 18 14 77.8 52.4 93.6 PI(D21)
18 18 100 81.5 100 FluAS03 PRE 40 36 90 76.3 97.2 PI(D21) 40 40 100
91.2 100 B/Shanghai FLUARIX .RTM. PRE 18 6 33.3 13.3 59.0 PI(D21)
18 14 77.8 52.4 93.6 FluAS03 PRE 40 34 85 70.2 94.3 PI(D21) 40 40
100 91.2 100 PRE = Prevaccination, PI(D21) = day 21 post
vaccination N = number of subjects with available results. n =
number of subjects with titres within the specified range. % =
percentage of subjects with titres within the specified range
TABLE-US-00036 TABLE 36 Seroconversion rates at PI day
21(fold-increase = 4) (All vaccinated subjects) Responders 95% CI
Antibody Vaccine Group N n % LL UL A/New Caledonia FLUARIX .RTM. 18
3 16.7 3.6 41.5 FluAS03 40 19 47.5 31.5 63.9 A/Fujian FLUARIX .RTM.
18 13 72.2 46.5 90.3 FluAS03 40 34 85.0 70.2 94.3 B/Shanghai
FLUARIX .RTM. 18 12 66.7 41.0 86.7 FluAS03 40 31 77.5 61.5 89.2 N =
number of subjects with both pre and post vaccination result
available. n = number of responders. % = Proportion of responders
(n/N .times. 100). 95% CI = exact 95% confidence interval; LL =
lower limit, UL = upper limit
IV.5. Overall Conclusions
[0426] From this clinical study it is confirmed that the adjuvanted
vaccine Flu-AS03 is superior to the equivalent unadjuvated vaccine
FLUARIX.RTM. in terms of frequency of influenza specific CD4 T
cells, and also in terms of persistence of the immune response
elicited by the first Flu-AS03 vaccination (primo-vaccination in
Explo Flu 001) until D0 of the revaccination study (Explo Flu 002
i.e. +/-1 year later). Furthermore this response is capable to
recognise drifted influenza strains present in the new vaccine and
to recognise the strains of the 2004 influenza vaccine.
[0427] In contrast to first year vaccination, upon revaccination
individuals previously vaccinated with the adjuvanted FLUARIX.RTM.
showed increased HI titer responsiveness as compared to those
vaccinated with un-adjuvanted FLUARIX.RTM.. There is an observable
trend for 1.5- to 2-fold increase in HI titer directed against H1N1
and H3N2 strains and a demonstrated statistical increase in HI
titer directed against B strain.
Example V
Pre-Clinical Evaluation of Adjuvanted and Unadjuvanted Influenza
Vaccines in Ferrets
First Study--Efficacy of New Formulations AS03 and AS03+MPL
V.1. Rationale and Objectives
[0428] Influenza infection in the ferret model closely mimics human
influenza, with regards both to the sensitivity to infection and
the clinical response.
[0429] 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.
[0430] This study investigated the efficacy of various Trivalent
Split vaccines, adjuvanted or not, to reduce disease symptoms (body
temperature) and viral shedding in nasal secretions of ferrets
challenged with homologous strains.
[0431] The objective of this experiment was to demonstrate the
efficacy of an adjuvanted influenza vaccine compared to the plain
(un-adjuvanted) vaccine.
[0432] The end-points were:
1) primary end-point: Reduction of viral shedding in nasal washes
after homologous challenge: 2) secondary end-points: Analysis of
the humoral response by IHA and monitoring of the temperature
around the priming and the challenge.
V.2. Experimental Design
V.2.1. Treatment/Group (Table 37)
[0433] Female ferrets (Mustela putorius furo) (6 ferrets/group)
aged 14-20 weeks were obtained from MISAY Consultancy (Hampshire,
UK). Ferrets were primed on day 0 with heterosubtypic strain H1N1
A/Stockholm/24/90 (4 Log TCID.sub.50/ml). On day 21, ferrets were
injected intramuscularly with a full human dose (500 .mu.g vaccine
dose, 15 .mu.g HA/strain) of a combination of H1N1 A/New
Caledonia/20/99, H3N2 A/Panama/2007/99 and B/Shangdong/7/97.
Ferrets were then challenged on day 41 by intranasal route with an
homotypic strain H3N2 A/Panama/2007/99 (4.51 Log
TCID.sub.50/ml).
TABLE-US-00037 TABLE 37 Comments (schedule/ Antigen(s) +
Formulation + route/ Other Group dosage dosage challenge)
treatments 1 Trivalent Full HD: IM; Day 21 Priming H1N1 Plain 15
.mu.g (A/Stockolm/24/ HA/strain 90) Day 0 2 Trivalent Full HD: IM;
Day 21 Priming H1N1 AS03 15 .mu.g (A/Stockolm/24/ HA/strain 90) Day
0 3 Trivalent Full HD: IM; Day 21 Priming H1N1 AS03 + MPL 15 .mu.g
(A/Stockolm/24/ HA/strain 90) Day 0 4 PBS IM; Day 21 Priming H1N1
(A/Stockolm/24/ 90) Day 0
V.2.2. Preparation of the Vaccine Formulations
Formulation 1: Trivalent Plain (Un-Adjuvanted) Formulation (500
.mu.l):
[0434] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) are added to water for injection. The detergents
quantities reached are the following: 750 .mu.g TWEEN.RTM. 80, 110
.mu.g TRITON X-100.TM. and 100 .mu.g VES per 1 ml. After 5 min
stirring, 15 .mu.g of each strain H1N1, H3N2 and 17.5 .mu.g of B
strain are added in sequence with 10 min stirring between each
addition. The formulation is stirred for 15 minutes at room
temperature and stored at 4.degree. C. if not administered
directly.
Formulation 2: Trivalent Split Influenza Adjuvanted with AS03 (500
.mu.L):
[0435] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) is added to water for injection. The detergents quantities
reached are the following: 750 .mu.g TWEEN.RTM. 80, 110 .mu.g
TRITON X-100.TM. and 100 .mu.g VES per 1 ml. After 5 min stirring,
15 .mu.g of each strain H1N1, H3N2 and 17.5 .mu.g of B strain are
added with 10 min stirring between each addition. After 15 min
stirring, 250 .mu.l of SB62 emulsion (prepared as in taught in
Example II.1) is added. The formulation is stirred for 15 minutes
at room temperature and stored at 4.degree. C. if not administered
directly.
Formulation 3: Trivalent Split Influenza Adjuvanted with
AS03+MPL
[0436] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X-100.TM. and
VES (quantities taking into account the detergents present in the
strains) is added to water for injection. The detergents quantities
reached are the following: 750 .mu.g TWEEN.RTM. 80, 110 .mu.g
TRITON X-100.TM. and 100 .mu.g VES per 1 ml. After 5 min stirring,
15 .mu.g of each strain H1N1, H3N2 and 17.5 .mu.g of B strain are
added with 10 min stirring between each addition. After 15 min
stirring, 250 .mu.l of SB62 emulsion (prepared as in taught in
Example II.1) is added. The mixture is stirred again for 15 min
just prior addition of 25 .mu.g of MPL from a suspension prepared
as detailed in Example II.3.1. The formulation is stirred for 15
minutes at room temperature and stored at 4.degree. C. if not
administered directly.
[0437] 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.
V.2.3. Read-Outs (Table 38)
TABLE-US-00038 [0438] TABLE 38 Analysis Readout Timepoint
Sample-type I/P method Viral D - 1 to D + 7 Nasal washes In
Titration shedding Post priming D - 1 to D + 5 Post challenge
T.degree. D - 1 to D + 3 Implant in In Telemetry monitoring Post
priming peritoneal cavity D - 2 to D + 3 Post challenge IHA Pre,
Post priming, Post Serum In IHA imm, Post challenge In =
Individual/Po = Pool
V.3. Results
[0439] A schematic representation of the results is given in FIG.
10 and FIG. 11.
V.3.1. Temperature Monitoring
[0440] Individual temperature were monitored with the transmitters
and by the telemetry recording (according to the procedure detailed
under 1.2.2). All implants were checked and refurbished and a new
calibration was performed by DSI before placement in the
intraperitoneal cavity. All animals were individually housed in
single cage during these measurements. Temperature were monitored
from 3 days Pre-challenge until 5 days Post challenge every 15
minutes and an average has been calculated by mid-day. Results from
baseline to baseline body temperature are shown in FIGS. 10A
(results from -1 to +3 days are shown) and 10B (results from -2 to
+3 days are shown).
[0441] Post-challenge, a peak of body temperature only observed
after immunization with trivalent split plain or PBS. No peak
observed after immunization with trivalent split adjuvanted with
AS03 or AS03+MPL.
V.3.2. Viral shedding (FIG. 11)
[0442] Viral titration of nasal washes was performed on 6 animals
per group. The nasal washes were performed by administration of 5
ml of PBS in both nostrils in awake animals. The inoculation was
collected in a Petri dish and placed into sample containers at
-80.degree. C. (dry ice).
[0443] All nasal samples were first sterile filtered through Spin X
filters (Costar) to remove any bacterial contamination. 50 .mu.l of
serial ten-fold dilutions of nasal washes were 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. until cell confluence is reached for the control cells, e.g. for
5-7 days. After 6-7 days of incubation, the culture medium is
gently removed and 100 .mu.l of a 1/20 WST-1 containing medium is
added and incubated for another 18 hrs.
[0444] The intensity of the yellow formazan dye produced upon
reduction of WST-1 by viable cells is proportional to the number of
viable cells present in the well at the end of the viral titration
assay and is quantified by measuring the absorbance of each well at
the appropriate wavelength (450 nanometers). The cut-off is defined
as the OD average of uninfected control cells--0.3 OD (0.3 OD
correspond to +/-3 StDev of OD of uninfected control cells). A
positive score is defined when OD is <cut-off and in contrast a
negative score is defined when OD is >cut-off. Viral shedding
titers were determined by "Reed and Muench" and expressed as Log
TCID50/ml.
[0445] Lower viral shedding was observed Post-challenge with
Trivalent Split adjuvanted with AS03 or AS03+MPL compared to
Trivalent Split Plain or PBS. The protective effect was slightly
better with AS03 compared to AS03+MPL (see Day 2 Post-challenge).
Statistical significance could not be determined due to the low
number of animals per group.
V.3.3. Conclusion of the Experiment
[0446] Higher humoral responses (HI titers) were observed with
Trivalent Split adjuvanted with AS03 or AS03+MPL compared to the
Trivalent Split Plain for all 3 strains (at least 2-fold for 2 out
of 3 strains, i.e. H3N2 and B strains).
[0447] AS03 and AS03+MPL formulations showed added benefit in terms
of protective efficacy in ferrets (lower viral shedding and
temperature) (FIGS. 10 and 11).
[0448] Post-challenge, no boost of the humoral response was
observed after immunization with Trivalent Split adjuvanted with
AS03 or AS03+MPL.
Second Study--Heterotypic Challenge study in ferrets: demonstration
of efficacy of new formulation tested
V.4. Rationale and Objectives
[0449] This study investigated the efficacy of various Trivalent
Split vaccines, adjuvanted or not, by their ability to reduce
disease symptoms (body temperature) and their effects on viral
shedding in nasal secretions of immunized ferrets after a
heterologuous challenge.
V.5. Experimental Design
[0450] Female ferrets (Mustela putorius furo) (6 ferrets/group)
aged 14-20 weeks were obtained from MISAY Consultancy (Hampshire,
UK). Four groups were tested: [0451] FLUARIX.RTM. Trivalent Split
AS03 [0452] Trivalent Split AS03+MPL [0453] PBS
[0454] Ferrets were primed on day 0 with heterosubtypic strain H1N1
A/Stockholm/24/90 (4 Log TCID.sub.50/ml). On day 21, ferrets were
injected intramuscularly with a full human dose (500 .mu.g vaccine
dose, 15 .mu.g HA/strain) of a combination of H1N1 A/New
Caledonia/20/99, H3N2 A/Panama/2007/99 and B/Shangdong/7/97 (17.5
.mu.g HA). Ferrets were then challenged on day 43 by intranasal
route with an heterosubtypic strain H3N2 A/Wyoming/3/2003 (4.51 Log
TCID.sub.50/ml).
V.6. Results
[0455] A schematic representation of the results is given in FIG.
12 and in FIG. 13.
V.6.1. Temperature Monitoring
[0456] Individual temperature were monitored with the transmitters
and by the telemetry recording. All implants were checked and
refurbished and a new calibration was performed by DSI before
placement in the intraperitoneal cavity. All animals were
individually housed in single cage during these measurements.
[0457] The results (FIG. 12) show that: [0458] A high variability
from one group to another was observed around the priming. The
baseline seemed to be higher before priming than after priming.
[0459] Despite the high variability in the body temperature, a peak
was only observed Post-challenge in ferrets immunized with PBS (6/6
ferrets), Trivalent Split Plain (5/6 ferrets) and Trivalent Split
adjuvanted with AS03 (2/6 ferrets). No peak was observed after
immunization with trivalent split adjuvanted with AS03+MPL (0/6
ferrets). [0460] AS03 seemed to be less efficient than AS03+MPL
against heterologous strains in terms of fever prevention. We
cannot conclude the possibility that difference between adjuvant is
due to different level in pre-challenge antibody levels.
V.6.2. Viral Shedding (FIG. 13)
[0461] The nasal washes were performed by administration of 5 ml of
PBS in both nostrils in awake animals. The inoculation was
collected in a Petri dish and placed into sample containers at
-80.degree. C. (dry ice).
[0462] All nasal samples were first sterile filtered through Spin X
filters (Costar) to remove any bacterial contamination. 50 .mu.l of
serial ten-fold dilutions of nasal washes were 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. until cell confluence is reached for the control cells, e.g. for
5-7 days. After 6-7 days of incubation, the culture medium is
gently removed and 100 .mu.l of a 1/20 WST-1 containing medium is
added and incubated for another 18 hrs.
[0463] The intensity of the yellow formazan dye produced upon
reduction of WST-1 by viable cells is proportional to the number of
viable cells present in the well at the end of the viral titration
assay and is quantified by measuring the absorbance of each well at
the appropriate wavelength (450 nanometers). The cut-off is defined
as the OD average of uninfected control cells--0.3 OD (0.3 OD
corresponds to +/-3 St Dev of OD of uninfected control cells). A
positive score is defined when OD is <cut-off and in contrast a
negative score is defined when OD is >cut-off. Viral shedding
titers were determined by "Reed and Muench" and expressed as Log
TCID50/ml.
Viral Shedding after Priming
[0464] Viral shedding was measured for 12 ferrets from Day 1
Pre-priming--to Day 7 Post-priming. Results are expressed in
pool.
[0465] The viral clearance was observed on Day 7 Post-priming in
all ferrets.
Viral Shedding after Challenge
[0466] Viral shedding was measured for 6 ferrets/group from Day 1
Pre-challenge to Day 7 Post-challenge.
[0467] Two days Post-challenge, statistically significant lower
viral titers were observed in ferrets immunized with Trivalent
Split adjuvanted with AS03 and AS03+MPL compared to ferrets
immunized with Trivalent Split Plain and PBS (difference of
1.25/1.22 log and 1.67/1.64 log with adjuvanted groups
AS03/AS03+MPL compared to the Plain vaccine, respectively). On Day
50, no virus was detected in nasal washes.
V.6.3. Hemagglutination Inhibition Test (HI titers) (FIGS. 14A and
B)
[0468] Serum samples were collected 1 day before priming, 21 days
Post-priming, 22 days post-immunization and 14 days
post-challenge.
[0469] Anti-Hemagglutinin antibody titers to the H3N2 influenza
virus (vaccine and challenge strains) were 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). Sera were first treated with a
25% neuraminidase solution (RDE) and were heat-inactivated to
remove non-specific inhibitors. After pre-treatment, two-fold
dilutions of sera were incubated with 4 hemagglutination units of
each influenza strain. Chicken red blood cells were then added and
the inhibition of agglutination was scored. The titers were
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.
Results:
[0470] Results are shown in FIGS. 14A and 14B. After immunization
with H3N2 A/Panama, higher humoral responses (HI titers) were
observed in ferrets immunized with the trivalent split vaccine
adjuvanted with AS03 or AS03+MPL, as compared to the humoral
response observed after immunization of ferrets with the
un-adjuvanted (plain) trivalent split vaccine FLUARIX.RTM.).
[0471] Similar HI titers were observed in ferrets immunized with
H3N2 A/Panama adjuvanted with AS03 or AS03+MPL.
[0472] Cross-reactive HI titers to the heterologous strain
A/Wyoming H3N2 was only observed after immunization with A/Panama
H3N2 strain containing vaccine adjuvanted with AS03 or AS03+MPL
(not observed after immunization with Trivalent Split Plain). A
boost of A/Wyoming-specific HI titers was observed in ferrets
immunized with the heterologous strain A/Panama H3N2 and challenged
with A/Wyoming H3N2. As expected and contrary to the homologous
challenge, the heterologous challenge resulted in an increase of
A/Panama-specific HI titers in ferrets immunized with A/Panama H3N2
adjuvanted with AS03 and AS03+MPL.
V.6.4. Conclusion of this Experiment
[0473] As expected, a boost of anti-H3N2 HI titers was observed
after heterologous challenge compared to the situation after
homologous challenge (no boost).
[0474] However, similar protection (viral shedding) was observed
after heterologous and homologous challenge.
Example VI
Pre-Clinical Evaluation of Adjuvanted and Unadjuvanted Influenza
Vaccines in C57BI/6 Primed Mice
VI.1. Experimental Design and Objective
[0475] Significant higher CD4 T cell responses were observed, in
Explo-Flu-001 clinical study (see Example III), for Trivalent Flu
Split AS03 compared to FLUARIX.RTM. Plain (un-adjuvanted). No
difference was observed for both CD8 T cell and humoral responses
between these two groups.
[0476] The purpose was to select readouts to induce in mice similar
CMI responses than observed in humans. Particularly, the purpose
was to show higher CMI responses in mice by using Split AS03 or
split AS03+MPL compared to Split plain.
VI.1.1. Treatment/Group
[0477] Female C57BI/6 mice (15 mice/group) aged 6-8 weeks were
obtained from Harlan Horst, Netherland. The groups tested were:
[0478] Trivalent Split Plain [0479] Trivalent Split AS03 [0480]
Trivalent Split AS03+MPL [0481] PBS
[0482] Mice were primed on day 0 with heterosubtypic strains (5
.mu.g HA whole inactivated H1N1 A/Johnannesburg/82/96, H3N2
A/Sydney/5/97, B/Harbin/7/94). On day 28, mice were injected
intramuscularly with 1.5 .mu.g HA Trivalent split (A/New
Caledonia/20/99, A/Panama/2007/99, B/Shangdong/7/97) plain or
adjuvanted (see groups below).
VI.1.2. Preparation of the Vaccine Formulations
[0483] 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.
Split Trivalent Plain (Un-Adjuvanted):
[0484] Formulation 1 (for 500 .mu.l): PBS 10 fold concentrated (pH
7.4 when one fold concentrated) as well as a mixture containing
TWEEN.RTM. 80, TRITON X-100.TM. and VES (quantities taking into
account the detergents present in the strains) are added to water
for injection. The detergents quantities reached are the following:
750 .mu.g TWEEN.RTM. 80, 110 .mu.g TRITON X-100.TM. and 100 .mu.g
VES per 1 ml After 5 min stirring, 15 .mu.g of each strain H1N1,
H3N2 and B are added with 10 min stirring between each addition.
The formulation is stirred for 15 minutes at room temperature and
stored at 4.degree. C. if not administered directly.
Split Trivalent Adjuvanted with the Oil-in-Water Emulsion Adjuvant
AS03:
[0485] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X-100.TM. and
VES (quantities taking into account the detergents present in the
strains) is added to water for injection. The detergents quantities
reached are the following: 750 .mu.g TWEEN.RTM. 80, 110 .mu.g
TRITON X-100.TM. and 100 .mu.g VES per 1 ml. After 5 min stirring,
15 .mu.g of each strain H1N1, H3N2 and B are added with 10 min
stirring between each addition. After 15 min stirring, 250 .mu.l of
SB62 emulsion (prepared as taught in Example II.1) is added. The
formulation is stirred for 15 minutes at room temperature and
stored at 4.degree. C. if not administered directly.
Split Trivalent Adjuvanted with AS03+MPL:
[0486] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) is added to water for injection. The detergents quantities
reached are the following: 750 .mu.g TWEEN.RTM. 80, 110 .mu.g
TRITON X-100.TM. and 100 .mu.g VES per 1 ml After 5 min stirring,
15 .mu.g of each strain H1N1, H3N2 and B are added with 10 min
stirring between each addition. After 15 min stirring, 250 .mu.l of
SB62 emulsion (prepared as taught in Example II.1) is added. The
mixture is stirred again for 15 min just prior addition of 25 .mu.g
of MPL. The formulation is stirred for 15 minutes at room
temperature and stored at 4.degree. C. if not administered
directly.
VI.1.3. Read-Outs
CMI Analysis (ICS: CD4/CD8, IL-2/IFNg Staining)
[0487] PBMCs from primed mice were harvested 7 days
post-immunization. They were tested in pools/group.
VI.2. Results
[0488] Conditions that showed higher frequencies of CD4 and CD8+T
cells, as well as lower background, were determined by using
C57BI/6 primed mice and whole inactivated virus 1 .mu.g/ml as
re-stimulating antigen. Results are shown in FIG. 15 (CD4 T-cell
responses) and in FIG. 16 (CD8 T-cell response).
[0489] With these conditions, it was possible to induce: [0490]
Higher CD4 T cell responses for Split AS03 compared to Split Plain,
as observed in humans. [0491] Higher CD4 T cell responses for Split
AS03+MPL compared to Split Plain. [0492] Similar CD8 T cell
responses between Split Plain and Split AS03, as observed in
humans. [0493] Trend for higher CD8 T cell responses for AS03+MPL
compared to Split AS03 or Split Plain
Example VII
Pre-Clinical Evaluation of Adjuvanted and Unadjuvanted Split and
Sub-Unit Influenza Vaccines in C57BI/6 Mice Primed with
Heterologous Strains
VII.1. Experimental Design and Objective
[0494] Significant higher CD4 T cell responses were observed, in
Explo-Flu-001 clinical study (see Example III), for Trivalent Flu
Split AS03 compared to FLUARIX.RTM. Plain (un-adjuvanted). No
difference was observed for both CD8 T cell and humoral responses
between these two groups.
[0495] An animal model reproducing similar immune profiles than
observed in humans was developed by using C57BI/6 mice primed with
heterologous strains. For ICS (intracellular cytokine staining),
the re-stimulation is performed with an inactivated whole virus.
The purpose was to compare the CMI response induced by a
GlaxoSmithKline commercially available split vaccine (FLUARIX.RTM.)
versus a subunit vaccine (Chiron's vaccine FLUAD.RTM.) as well as
the CMI response obtained with these vaccines adjuvanted with AS03,
or AS03+MPL or another oil-in-water emulsion adjuvant (OW).
VII.1.1. Treatment/Group
[0496] Female C57BI/6 mice (24 mice/group) aged 6-8 weeks were
obtained from Harlan Horst, Netherland. Mice were primed
intranasally on day 0 with heterosubtypic strains (5 .mu.g HA whole
formaldehyde inactivated H1N1 A/Johnannesburg/82/96, H3N2
A/Sydney/5/97, B/Harbin/7/94). On day 29, mice were injected
intramuscularly with 1.5 .mu.g HA Trivalent split (A/New
Caledonia/20/99, A/Wyoming/3/2003, B/Jiangsu/10/2003) plain or
adjuvanted (see groups in Table 39 below).
TABLE-US-00039 TABLE 39 Gr Antigen / Formulation Other treatment 1
Trivalent split*/Plain Heterologous priming D0 (un-adjuvanted) =
FLUARIX .RTM. 2 Trivalent split*/OW Heterologous priming D0 3
Trivalent split*/AS03 Heterologous priming D0 4 Trivalent
split*/AS03 + MPL Heterologous priming D0 (2.5 .mu.g per dose) 5
Gripguard ( = FLUAD .RTM. = Heterologous priming D0 sub-unit in an
oil-in-water emulsion 6 AGGRIPAL .TM. (sub-unit)/AS03 Heterologous
priming D0 7 AGGRIPAL .TM. (sub-unit)/ Heterologous priming D0 AS03
+ MPL (2.5 .mu.g per dose) 8 AGGRIPAL .TM. (sub-unit)/OW**
Heterologous priming D0 9 AGGRIPAL .TM. (sub-unit) Heterologous
priming D0 10 PBS Heterologous priming D0 *FLUARIX .RTM. **OW
produced as explained in the section below
VII.1.2. Preparation of the vaccine formulations
Preparation of OW
[0497] An oil-in-water emulsion called OW is prepared following the
recipe published in the instruction booklet contained in Chiron
Behring FLUAD.RTM. vaccine.
[0498] Water for injection, 36.67 mg of Citric acid and 627.4 mg of
Na Citrate. 2H2O are mixed together and the volume is adjusted to
200 ml. 470 mg of TWEEN.RTM. 80 is mixed with 94.47 ml of this
buffer and this mixture is called "solution A". The oil mixture is
prepared by mixing 3.9 g of squalene and 470 mg of SPAN 85.TM.
(sorbitan trioleate) under magnetic stirring. Solution A is then
added to the oil mixture and the final volume obtained is 100 ml.
The mixture is then first passed trough a 18Gx 11/2 needle and is
then put in the M110S microfluidiser (from Microfluidics) in two
samples to reduce the size of the oil droplets. When a particle
size around 150 nm is obtained for each, the 2 samples are pooled
and filtrated on 0.2 .mu.m filter. A z average mean of 143 nm with
a polydispersity of 0.10 is obtained for the pooled sample at T0
and of 145 nm with a polydispersity of 0.06 after 4 months storage
at 4.degree. C. This size is obtained using the Zetasizer 3000HS
(from Malvern), under the following technical conditions: [0499]
laser wavelength: 532 nm (Zeta3000HS). [0500] laser power: 50 mW
(Zeta3000HS). [0501] scattered light detected at 90.degree.
(Zeta3000HS). [0502] temperature: 25.degree. C., [0503] duration:
automatic determination by the soft, [0504] number: 3 consecutive
measurements, [0505] z-average diameter: by cumulants analysis
Formulation for Group 1 (for 1 ml):
[0506] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X-100.TM. and
VES (quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml TWEEN.RTM.
80, 55 .mu.g/ml TRITON X100.TM. and 50 .mu.g/ml VES, are added to
water for injection. After 5 min stirring, 15 .mu.g of each strain
H1N1, H3N2 and B are added with 10 min stirring between each
addition. The formulation is stirred for 15 minutes and stored at
4.degree. C. if not administered directly.
Formulation for Group 2 (for 1 ml):
[0507] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml TWEEN.RTM.
80, 55 .mu.g/ml TRITON X-100.TM. and 50 .mu.g/ml VES, is added to
water for injection. After 5 min stirring, 15 .mu.g of each strain
H1N1, H3N2 and B are added with 10 min stirring between each
addition. After 15 min stirring, 250 .mu.l of OW emulsion is added.
The formulation is stirred for 15 minutes and stored at 4.degree.
C. if not administered directly.
Formulation for Group 3: for 1 ml:
[0508] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X-100.TM. and
VES (quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml TWEEN.RTM.
80, 55 .mu.g/ml TRITON X-100.TM. and 50 .mu.g/ml VES, is added to
water for injection. After 5 min stirring, 15 .mu.g of each strain
H1N1, H3N2 and B are added with 10 min stirring between each
addition. After 15 min stirring, 250 .mu.l of SB62 emulsion is
added. The formulation is stirred for 15 minutes and stored at
4.degree. C. if not administered directly.
Formulation for Group 4: for 1 ml:
[0509] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml TWEEN.RTM.
80, 55 .mu.g/ml TRITON X-100.TM. and 50 .mu.g/ml VES, is added to
water for injection. After 5 min stirring, 15 .mu.g of each strain
H1N1, H3N2 and B are added with 10 min stirring between each
addition. After 15 min stirring, 250 .mu.l of SB62 emulsion is
added. The mixture is stirred again for 15 min just prior addition
of 25 .mu.g of MPL. The formulation is stirred for 15 minutes and
stored at 4.degree. C. if not administered directly.
Formulation for Group 5: for 1 ml:
[0510] Equal volume of PBS and FLUAD.RTM./GRIPGUARD.TM. (commercial
vaccine) vaccine are mixed. The formulation is stirred for 15
minutes and stored at 4.degree. C. if not administered
directly.
Formulation for Group 6: for 1 ml: 250 .mu.l of PBS mod pH 7.4 are
added to a 500 .mu.l dose of AGGRIPAL.TM. (commercial vaccine).
After 15 min stirring, 250 .mu.l of SB62 is added (prepared
according to the methodology detailed for the scaled-up
production). The formulation is stirred for 15 minutes and stored
at 4.degree. C. if not administered directly.
Formulation for Group 7: for 1 ml:
[0511] PBS mod pH 7.4 (to reach a final volume of 1 ml) is added to
a 500 .mu.l dose of AGGRIPAL.TM. (commercial vaccine). After 15 min
stirring, 250 .mu.l of SB62 is added (prepared according to the
methodology detailed for the scaled-up production). 25 .mu.g of MPL
are then added. The formulation is stirred for 15 minutes and
stored at 4.degree. C. if not administered directly.
Formulation for Group 8: for 1 ml:
[0512] 250 .mu.l of PBS mod pH 7.4 are added to a 500 .mu.l dose of
AGGRIPAL.TM.. After 15 min stirring, 250 .mu.l of OW as prepared
for group 2 is added and the formulation is stirred 15 min and
stored at 4.degree. C. if not administered directly.
Formulation for Group 9: for 1 ml:
[0513] Equal volume of PBS mod pH 7.4 and AGGRIPAL.TM. are mixed.
The formulation is stirred for 15 minutes and stored at 4.degree.
C. if not administered directly.
VII.1.3. Read-Outs (Table 40)
CMI (ICS): 7 Days Post-Immunization.
IHA/Neutralization Assay: 21 Days Post-Immunization.
TABLE-US-00040 [0514] TABLE 40 Analysis Read-out Timepoint Sample
type UP method ICS D35 PBLs Po FACS analysis (CD4, CD8, IL-2,
IFN-.gamma.) Humoral D14, D44 Sera In IHA, neutra response In =
Individual/Po = Pool
CMI Analysis (ICS: CD4/CD8; IL-2/IFN-Gamma Staining)
[0515] PBMCs from 24 mice/group were harvested 7 days
post-immunization and tested in pools/group.
VII.2. Results
VII.2.1. Humoral Immunity
[0516] Haemagglutination inhibition activity against the 3 vaccine
strains was detected in sera from 24 animals per group at Day 14
after intranasal heterologous priming and at Day 16
Post-immunization.
[0517] For the 3 strains and for all groups, a boost of HI titers
was observed after immunization. [0518] For a same adjuvant and for
the 3 strains, similar HI titers were induced by the subunit
vaccine and the Split vaccine. [0519] Similar HI titers were
observed for FLUAD.RTM. compared to AGGRIPAL.TM. OW for the 3
strains [0520] No difference was observed between FLUARIX.RTM. and
AGGRIPAL.TM. for H1N1 and B strains. [0521] For the 3 strains,
statistically significant higher HI titers were observed when the
Flu vaccine (Split or subunit) was adjuvanted with AS03 with or
without MPL compared to the plain Flu vaccine. [0522] HI titers
were statistically significant higher for the Flu vaccine (Split or
subunit) adjuvanted with OW compared to the Flu vaccine plain only
for the A/Wyoming strain.
VII.2.2. Cell-Mediated Immune Response (ICS at Day 7 Post
Immunization)
CD4 T Cell Responses--FIG. 17 Upper Part
[0523] PBMCs from 24 mice per group were harvested at Day 7
Post-immunization and tested in one pool/group. Inactivated
trivalent whole viruses (1 .mu.g/ml) were used as re-stimulating
antigen. Results are shown in FIG. 17 upper part.
In Terms of Flu Whole Virus-Specific CD4+ T Cells Expressing IL-2,
IFN-.gamma. or Both Cytokines (FIG. 17 Upper Part):
[0524] 1. GSK adjuvants showed the same trend as previously
observed (Example VI): AS03+MPL was superior to AS03 which was in
turn superior to the result obtained with the plain vaccine. This
trend was observed both for the split or the subunit vaccine.
[0525] 2. Whatever the formulation (Plain, AS03 or AS03+MPL), the
split vaccine induced a higher CD4+ T cell responses than the
subunit vaccine. [0526] 3. FLUAD.RTM. (subunit+oil-in-water
emulsion OW--see preparation section) seemed to induce similar
frequencies than Fluarix Plain. [0527] 4. Formulations Trivalent
Split/AS03 or Trivalent Split/AS03+MPL induced higher CD4+T cell
responses than the formulation subunit/oil-in-water emulsion OW.
CD8 T cell Responses--FIG. 17 Lower Part
[0528] PBMCs from 24 mice per group were harvested at Day 7
Post-immunization and tested in one pool/group. Inactivated
trivalent whole viruses (1 .mu.g/ml) were used as re-stimulating
antigen.
[0529] In Terms of Flu Whole Virus-Specific CD8+T Cells Expressing
IL-2, IFN-.gamma. or Both Cytokines (FIG. 17 Lower Part): [0530]
The cut-off of this experiment was relatively high due to the high
background observed for the PBS negative control group. [0531]
However higher specific CD8 T cell responses were observed for mice
immunized with Trivalent Split/AS03+MPL compared to other vaccine
formulations.
VII.3. Summary of Results and Conclusions
[0532] The following results were obtained:
1) Flu-specific CD4+ T cells obtained by ICS at Day 7
post-immunization showed: [0533] 1. Similar responses were obtained
for FLUAD.RTM. compared to FLUARIX.RTM. [0534] 2. The adjuvanted
formulation induced higher immune response compared to the
un-adjuvanted vaccine, both for the split influenza vaccine (as
observed in humans) and for the subunit (AGGRIPAL.TM.) vaccine (not
assessed in humans). The oil-in-water emulsion adjuvant AS03
supplemented with MPL (groups 4 and 9) gave higher responses than
the oil-in-water emulsion adjuvant AS03 (groups 3 and 8). [0535] 3.
There is a trend of a higher CD4 responses with Split/AS03+MPL
compared to Split/AS03 (FIG. 17). [0536] 4. The responses induced
by the split vaccine were superior to the responses obtained with
the subunit vaccine (compare groups 1 to 4 and groups 5 to 9).
[0537] 5. The split vaccine, whether adjuvanted with AS03 with or
without MPL (groups 3 and 4) performed showed higher CD4+ T cell
responses than the sub-unit vaccine, either FLUAD.RTM. (group 5) or
AGGRIPAL.TM.+OW (group 7). 2) Flu-specific CD8+T cells obtained by
ICS at Day 7 post-immunization showed no differences are observed
between Split/AS3 and Split Plain (as observed in humans). There
was a trend for a higher CD8+T cell response by using
Split/AS03+MPL compared to Split/AS03 or Split Plain. 3) For a same
adjuvant and for the 3 strains, similar HI titers were induced by
the subunit vaccine and the split vaccine. For the 3 strains,
statistically significant higher titers were observed when the Flu
vaccine (subunit or split) was adjuvanted with AS03 or AS03+MPL
compared to the Flu vaccine plain (Flu vaccine OW>Flu vaccine
Plain only for the A/Wyoming strain).
Example VIII
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine Containing a Split Influenza Antigen Preparation and AS03
with or without MPL Adjuvant
VIII.1. Study Design
[0538] A phase I, open, randomised, controlled study in an elderly
population aged over 65 years 65 years-old) in order to evaluate
the reactogenicity and the immunogenicity of GlaxoSmithKline
Biologicals influenza candidate vaccines containing the adjuvant
AS03 or AS03+MPL, administered intramuscularly as compared to
FLUARIX.RTM. vaccine (known as .alpha.-RIX.RTM. in Belgium).
[0539] Three parallel groups were assessed: [0540] one group of 50
subjects receiving one dose of the reconstituted and AS03
adjuvanted SV influenza vaccine (Flu AS03) [0541] one group of 50
subjects receiving one dose of the reconstituted and Flu AS03+MPL
adjuvanted SV influenza vaccine (Flu AS03+MPL) [0542] one control
group of 50 subjects receiving one dose of FLUARIX.RTM.
VIII.2. Vaccine Composition and Administration
[0543] The strains used in the three vaccines were the ones that
had been recommended by the WHO for the 2004-2005 Northern
Hemisphere season, i.e. A/New Caledonia/20/99 (H1N1), A/New
California/3/2003 (H3N2) and B/Jiangsu/10/2003. Like
FLUARIX.RTM./.alpha.-RIX.RTM., the commercially available vaccine
used as a comparator, the adjuvanted vaccines (AS03, or AS03+MPL)
contain 15 .mu.g haemagglutinin (HA) of each influenza virus strain
per dose. The adjuvanted influenza candidate vaccines are 2
component vaccines consisting of a concentrated trivalent
inactivated split virion antigens presented in a type I glass vial
and of a pre-filled type I glass syringe containing the adjuvant
(AS03 or AS03+MPL). They have been prepared as detailed in Example
II. The three inactivated split virion antigens (monovalent bulks)
used in formulation of the adjuvanted influenza candidate vaccines,
are exactly the same as the active ingredients used in formulation
of the commercial FLUARIX.RTM./.alpha.-RIX.RTM..
AS03 Adjuvanted Vaccine:
[0544] The AS03-adjuvanted influenza candidate vaccine is a 2
components vaccine consisting of a concentrated trivalent
inactivated split virion antigens presented in a type I glass vial
(335 .mu.l) (antigen container) and of a pre-filled type I glass
syringe containing the SB62 emulsion (335 .mu.l) (adjuvant
container). Description and composition of the AS03 candidate
vaccine is explained in Example III.
AS03+MPL Adjuvanted Vaccine:
[0545] Briefly, the AS03+MPL-adjuvanted influenza candidate vaccine
is a 2 components vaccine consisting of a concentrated trivalent
inactivated split virion antigens presented in a type I glass vial
(335 .mu.l) (antigen container) and of a pre-filled type I glass
syringe containing the AS03+MPL adjuvant (360 .mu.l) (adjuvant
container). At the time of injection, the content of the antigen
container is removed from the vial by using the syringe containing
the AS03+MPL adjuvant, followed by gently 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 the AS03+MPL-adjuvanted influenza candidate vaccine
corresponds to 530 .mu.l. To obtain the 15 .mu.g HA for each
influenza strain at reconstitution of the AS03+MPL adjuvanted
vaccine, the inactivated split virion antigen are concentrated
two-fold in the antigen container (i.e. 60 .mu.g HA/ml) as compared
to FLUARIX.RTM. (i.e. 30 .mu.g HA/ml). The composition of one dose
of the reconstituted adjuvanted influenza vaccine is identical to
that reported in Table 45 (see Example XI) except for the influenza
strains. Both vaccines were given intramuscularly.
VIII.3. CMI Objective, End-Points and Results
[0546] The CMI objectives were to determine which immunogenic
composition between the formulation adjuvanted with AS03, or
AS03+MPL versus the composition without any adjuvant has the
strongest immunostimulating activity on CD4- and CD8-mediated
immunity of individuals vaccinated with influenza antigens.
VIII.3.1. CMI End Points and Results
Observed Variable
[0547] At days 0 and 21: frequency of cytokine-positive CD4/CD8
cells per 10.sup.6 into 5 different cytokines. Each test quantifies
the response of CD4/CD8 T cell to: [0548] Pool of the 3 following
antigens [0549] New Caledonia antigen [0550] Wyoming antigen [0551]
Jiangsu antigen.
Derived Variables:
[0552] Antigen-specific CD4 and CD8-T-cell response expressed into
the 5 different tests:
(a) cells producing at least two different cytokines (CD40L, IL-2,
IFN.gamma., TNF.alpha.) (b) cells producing at least CD40L and
another cytokine (IL-2, TNF.alpha., IFN.gamma.) (c) cells producing
at least IL-2 and another cytokine (CD40L, TNF.alpha., IFN.gamma.)
(d) cells producing at least IFN.gamma. and another cytokine (IL-2,
TNF.alpha., CD40L) (e) cells producing at least TNF.alpha. and
another cytokine (IL-2, CD40L, IFN.gamma.)
Analysis of the CMI Response:
[0553] The CMI analysis was based on the Total vaccinated cohort.
[0554] (a) For each treatment group, the frequency of CD4/CD8
T-lymphocytes secreting in response was determined for each
vaccination group, at each timepoint (Day 0, Day 21) and for each
antigen: New Caledonia, Wyoming and Jiangsu and the pooled of the 3
different strains. [0555] (b) Descriptive statistics in individual
difference between timepoint (POST-PRE) responses for each
vaccination group and each antigen at each 5 different cytokines.
[0556] (c) Comparison of the 3 groups regarding the 5 different
cytokines on: [0557] CD4 T-cell response to New Caledonia, Wyoming,
Jiangsu and the pool of the 3 strains [0558] CD8 T-cell response to
New Caledonia, Wyoming, Jiangsu and the pool of the 3 strains
[0559] (d) 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 cytokines. [0560] (e) A Wilcoxon test were use to test
pairwise comparison of 2 groups respectively between Flu AS03+MPL
versus FLUARIX.RTM., Flu AS03+MPL versus Flu AS03 and Flu AS03
versus FLUARIX.RTM. [0561] (f) All significance tests were
two-tailed. P-values less than or equal to 0.05 were considered as
statistically significant.
VIII.3.2. CMI Results
[0562] Results were expressed as a frequency of
cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell
sub-population.
Frequency of Antigen Specific CD4 T-Lymphocytes
[0563] (a) The frequency of antigen-specific CD4 T-lymphocytes
secreting in response was determined for each vaccination group, at
each time point (Day 0, Day 21) and for each antigen (Pool, New
Caledonia, Wyoming and Jiangsu), similarly to that performed in
Example III. [0564] (b) Comparing the difference in the frequency
of antigen-specific CD4 T-lymphocytes between the 3 groups by
Kruskall-Wallis test, all p-values were less than 0.05 and were
considered as statistically significant. [0565] (c) Comparing the
difference in the frequency of antigen-specific CD4 T-lymphocytes
between Flu AS03+MPL and FLUARIX.RTM. groups by the Wilcoxon test,
all p-values were less than 0.05 and were considered as
statistically significant. [0566] (d) Comparing the difference in
the frequency antigen-specific of CD4 T-lymphocytes between Flu
AS03 and FLUARIX.RTM. groups by the Wilcoxon test, all p-values
were less than 0.05 and were considered as statistically
significant. [0567] (e) Comparing the difference in the frequency
of antigen-specific CD4 T-lymphocytes between Flu AS03 and Flu
AS03+MPL groups by the Wilcoxon test, all p-values were more than
0.05 and were considered as no statistically significant.
Individual Difference Between Time Point (Post-Pre) in CD4
T-Lymphocytes
[0567] [0568] (a) Descriptive statistics in individual difference
between time point (POST-PRE) in CD4 T-lymphocytes responses was
calculated for each vaccination group and for each antigen at each
5 different cytokines, similarly to what has been done in Example
III. [0569] (b) Comparing the individual difference POST-PRE in the
antigen-specific CD4-T-lymphocytes responses between the 3 groups
by Kruskall-Wallis test, all p-values were less than to 0.001 and
were considered as highly statistically significant. [0570] (c)
Comparing the individual difference POST-PRE in the
antigen-specific CD4-T-lymphocytes responses between Flu AS03+MPL
and FLUARIX.RTM. using Wilcoxon test, all p-values were less than
to 0.05 and were considered as statistically significant. [0571]
(d) Comparing the individual difference POST-PRE in the
antigen-specific CD4-T-lymphocytes responses between Flu AS03 and
FLUARIX.RTM. using Wilcoxon test, all p-values were less than to
0.001 and were considered as highly statistically significant.
[0572] (e) Comparing the individual difference POST-PRE in the
antigen-specific CD4-T-lymphocytes responses between Flu AS03+MPL
and Flu AS03 using Wilcoxon test, all p-values were more than 0.05
and were considered as no statistically significant.
VIII.4. B Cell Memory Response Objective, End-Points and
Results
[0573] The objective of the study was to investigate whether the
frequency of memory B cell specific to Flu Antigen are
significantly induced upon one intramuscular vaccination with the
Flu candidate vaccine containing the Adjuvant AS03+MPL or AS03, as
compared to FLUARIX.RTM. in elderly population. The frequency of
memory B cell has been assessed by B cell Elispot assay.
VIII.4.1. B Cell Memory Response End-Points
[0574] The end points are: [0575] (a) At days 0, 21: cells
generated in vitro cultivated memory B-cells measured by B-cell
ELISPOST in all subjects in term of frequency of specific-antigen
plasma within a million (10.sup.6) of IgG producing plasma cells.
[0576] (b) Difference between post (day 21) and pre (day 0)
vaccination are also expressed as a frequency of Influenza
specific-antibody forming cells per million (10.sup.6) of antibody
forming cells.
VIII.4.2. B Cell Memory Response Results
[0577] The frequency of Influenza-specific antibody forming cells
per million (10.sup.6) of antibody forming cells were determined.
The results showed that the frequency of memory B cell specific to
Flu antigen between Flu AS03+MPL and FLUARIX.RTM. groups by the
Wilcoxon test was significantly (p<0.05) higher for B/Jiangsu
strain, whilst not for the other two strains (A strains New
Caledonia and Wyoming).
[0578] The individual difference between time point (post-pre) in
memory B cell specific to Flu antigen was also determined. The
results showed that individual difference between time point
(post-pre) in the frequency of memory B cell specific to Flu
antigen between Flu AS03+MPL and FLUARIX.RTM. groups by the by the
Kruskall-Wallis test was significantly (p<0.05) higher for
B/Jiangsu strain, whilst not for the other two strains (A strains
New Caledonia and Wyoming).
[0579] The results are shown in FIG. 18.
Example IX
Pre-Clinical Evaluation of Adjuvanted and Unadjuvanted Influenza
Vaccines in Ferrets (Study III)
IX.1. Rationale and Objectives
[0580] This study compared GSK commercial influenza trivalent split
vaccine, either un-adjuvanted (FLUARIX.RTM.) or adjuvanted with
AS03+MPL, with two other commercially available sub-unit vaccines:
[0581] FLUAD.RTM., Chiron's adjuvanted subunit vaccine (the
adjuvant is Chiron's MF59 adjuvant), [0582] AGRIPPAL.TM., Chiron
un-adjuvanted commercial sub-unit vaccine, which was in the present
study adjuvanted with AS03 adjuvant.
[0583] The objective of this experiment was to evaluate the ability
of these vaccines to reduce disease symptoms (body temperature and
viral shedding) in nasal secretions of ferrets challenged with
heterologous strains.
[0584] The end-points were:
1) Primary end-point: reduction of viral shedding in nasal washes
after heterologous challenge: 2) Secondary end-points: analysis of
the humoral response by IHA and monitoring of the temperature
around the priming and the heterologous challenge.
IX.2. Experimental Design
IX.2.1. Treatment/Group
[0585] Female ferrets (Mustela putorius furo) aged 14-20 weeks were
obtained from MISAY Consultancy (Hampshire, UK). Ferrets were
primed intranasally on day 0 with the heterosubtypic strain H1N1
A/Stockholm/24/90 (4 Log TCID.sub.50/ml). On day 21, ferrets were
injected intramuscularly with a full human dose (1 ml vaccine dose,
15 .mu.g HA/strain) of a combination of H1N1 A/New Caledonia/20/99,
H3N2 A/Wyoming/3/2003 and B/Jiangsu/10/2003. Ferrets were then
challenged on day 42 by intranasal route with a heterotypic strain
H3N2 A/Panama/2007/99 (4.51 Log TCID.sub.50/ml). The groups (6
ferrets/group) are illustrated in Table 41. The read-out that were
performed are detailed in Table 42.
TABLE-US-00041 TABLE 41 Comments (ex: schedule/ Antigen(s) +
Formulation + route/ Other Group dosage dosage challenge)
treatments 1 Trivalent Full HD: IM; Day 21 Priming H1N1 plain 15
.mu.g (A/Stockolm/24/90) FLUARIX .RTM.) HA/strain Day 0 2 Trivalent
Full HD: IM; Day 21 Priming H1N1 AS03 + MPL 15 .mu.g
(A/Stockolm/24/90) HA/strain Day 0 3 FLUAD .RTM. Full HD: IM; Day
21 Priming H1N1 15 .mu.g (A/Stockolm/24/90) HA/strain Day 0 4
AGRIPPAL .TM. Full HD: IM; Day 21 Priming H1N1 AS03 15 .mu.g
(A/Stockolm/24/90) HA/strain Day 0
IX.2.2. Preparation of the Vaccine Formulations
Split Trivalent Plain (Un-Adjuvanted): Formulation for 1 ml:
[0586] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) are added to water for injection. The detergents
quantities reached are the following: 375 .mu.g TWEEN.RTM. 80, 55
.mu.g TRITON X100.TM. and 50 .mu.g VES per 1 ml. After 5 min
stirring, 15 .mu.g of each strain H1N1, H3N2 and 17.5 .mu.g of B
strain are added with 10 min stirring between each addition. The
formulation is stirred for 15 minutes at room temperature and
stored at 4.degree. C. if not administered directly.
Split Trivalent Adjuvanted with AS03+MPL: Formulation for 1 ml:
[0587] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing TWEEN.RTM. 80, TRITON X100.TM. and
VES (quantities taking into account the detergents present in the
strains) is added to water for injection. The detergents quantities
reached are the following: 375 .mu.g TWEEN.RTM. 80, 55 .mu.g TRITON
X100.TM. and 50 .mu.g VES per 1 ml. After 5 min stirring, 15 .mu.g
of each strain H1N1, H3N2 and B are added with 10 min stirring
between each addition. After 15 min stirring, 250 .mu.l of SB62
emulsion (prepared as detailed in Example II.1) is added. The
mixture is stirred again for 15 minutes just prior addition of 25
.mu.g of MPL. The formulation is stirred for 15 minutes at room
temperature and stored at 4.degree. C. if not administered
directly.
FLUAD.RTM. Formulation: Formulation for 1 ml:
[0588] A 2 fold dilution of FLUAD.RTM. vaccine is made in PBS
buffer pH 7.4.
Agrippal.TM. AS03 Formulation: Formulation for 1 ml:
[0589] 250 .mu.l of PBS buffer pH 7.4 is added to one dose of
AGGRIPAL.TM.. After mixing, 250 .mu.l of SB62 emulsion (prepared as
detailed in Example II.1) is added. The mixture is stirred at room
temperature.
IX.2.2. Read-Outs
TABLE-US-00042 [0590] TABLE 42 Analysis Readout Timepoint
Sample-type I/Po method Viral D - 3 to D + 7 Nasal washes In
Titration Post priming shedding D + 1 to D + 5 Post challenge
T.degree. D - 3 to D + 4 Implant in In Telemetry monitoring Post
priming peritoneal D - 2 to D + 4 cavity Post challenge IHA Pre,
Post priming, Post Serum In IHA imm, Post challenge In =
Individual/Po = Pool
IX.3. Results (FIGS. 19 to 22)
IX.3.1. Temperature Monitoring
[0591] Individual temperatures were monitored with the transmitters
and by the telemetry recording. All implants were checked and
refurbished and a new calibration was performed by DSI before
placement in the intraperitoneal cavity. All animals were
individually housed in single cage during these measurements.
Temperature was monitored from 2 days Pre-challenge until 4 days
Post challenge every 15 minutes and an average temperature
calculated by mid-day. Results are shown in FIG. 19.
Results:
[0592] Post-challenge, a peak of body temperature was observed
after immunization of ferrets with the un-adjuvanted (plain)
trivalent split (FLUARIX.RTM.) or the sub-unit vaccine FLUAD.RTM.
(which contains MF59 oil-in-water emulsion). No peak was observed
after immunization of ferrets with the trivalent split vaccine
adjuvanted neither with AS03+MPL nor with sub-unit Agrippal.TM.
adjuvanted with AS03. In conclusion, an added value of the
AS03-containing vaccines in the prevention of body temperature rise
after challenge was shown for both the split and sub-unit tested
vaccines, by contrast to the inability of the MF59-containing
vaccines to prevent this temperature rise in ferrets after
challenge.
IX.3.2. Viral Shedding
[0593] Viral titration of nasal washes was performed on 6 animals
per group. The nasal washes were performed by the administration of
5 ml of PBS in both nostrils in awake animals. The inoculation was
collected in a Petri dish and placed into sample containers on dry
ice (-80.degree. C.).
[0594] All nasal samples were first sterile filtered through Spin X
filters (Costar) to remove any bacterial contamination. 50 .mu.l of
serial ten-fold dilutions of nasal washes were 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. After 5-7 days of incubation, the culture medium
is gently removed and 100 .mu.l of a 1/20 WST-1 containing medium
is added and incubated for another 18 hrs.
[0595] The intensity of the yellow formazan dye produced upon
reduction of WST-1 by viable cells is proportional to the number of
viable cells present in the well at the end of the viral titration
assay and is quantified by measuring the absorbance of each well at
the appropriate wavelength (450 nanometers). The cut-off is defined
as the OD average of uninfected control cells--0.3 OD (0.3 OD
corresponds to +/-3 St Dev of OD of uninfected control cells). A
positive score is defined when OD is <cut-off and in contrast a
negative score is defined when OD is >cut-off. Viral shedding
titers were determined by "Reed and Muench" and expressed as Log
TCID50/ml.
Results:
[0596] Results are shown in FIG. 20. Lower viral shedding was
observed post-challenge with the trivalent split vaccine adjuvanted
with AS03+MPL, or with the Agrippal.TM. sub-unit vaccine adjuvanted
with AS03, as compared to the very low viral shedding reduction
observed after immunization of ferrets with the un-adjuvanted
(plain) trivalent split vaccine (FLUARIX.RTM.) or with FLUAD.RTM.
sub-unit vaccine.
[0597] Similarly to what was discussed in respect of body
temperature rise, an added value of the AS03-containing vaccines
was observed compared to the MF59-containing vaccines.
IX.3.3. HI Titers
[0598] Anti-Hemagglutinin antibody titers to the H3N2 influenza
virus strains were 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). Sera were first treated with a 25% neuraminidase solution
(RDE) and were heat-inactivated to remove non-specific inhibitors.
After pre-treatment, two-fold dilutions of sera were incubated with
4 hemagglutination units of each influenza strain. Chicken red
blood cells were then added and the inhibition of agglutination was
scored. The titers were 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.
Results:
[0599] After immunization with H3N2 A/Wyoming, higher humoral
responses (HI titers) were observed in ferrets immunized with the
trivalent split vaccine adjuvanted with AS03+MPL or with the
Agrippal.TM. sub-unit vaccine adjuvanted with AS03, as compared to
the humoral response observed after immunization of ferrets with
the un-adjuvanted (plain) trivalent split vaccine (FLUARIX.RTM.) or
with FLUAD.RTM. sub-unit vaccine (FIG. 21). After immunization with
H3N2 A/Wyoming, higher humoral responses (HI titers) were also
observed against the drift strain H3N2 A/Panama, used as the
challenge strain, in ferrets immunized with Trivalent Split
adjuvanted with AS03+MPL or Agrippal.TM. adjuvanted with AS03
compared to ferrets immunized with Trivalent Split Plain or
FLUAD.RTM. (FIG. 22).
[0600] This cross-reaction observed with our adjuvant (AS03 or
AS03+MPL) against a heterologous strain correlated with the
protection observed in ferrets immunized with the trivalent split
vaccine adjuvanted with AS03+MPL or with the AGRIPPAL.TM. sub-unit
vaccine adjuvanted with AS03, and then challenged with this
heterologous strain. This cross-reactivity to heterologous strain
induced by AS03-containing vaccines was not induced by the MF59's
adjuvanted vaccines (FLUAD.RTM.).
Example X
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine Containing a Split Influenza Antigen Preparation and AS03
with or without MPL Adjuvant: Immunogenicity Persistence Data at
Day 90 and 180
X.1. Study Design
[0601] A phase I, open, randomised, controlled study in an elderly
population aged over 65 years 65 years-old) in order to evaluate
the reactogenicity and the immunogenicity of GlaxoSmithKline
Biologicals influenza candidate vaccines containing the adjuvant
AS03 or AS03+MPL, administered intramuscularly as compared to
FLUARIX.RTM. vaccine (known as .alpha.-Rix.TM. in Belgium). This
study follows that reported in Example VIII.
[0602] Three parallel groups were assessed: [0603] one group of 50
subjects receiving one dose of the reconstituted and AS03
adjuvanted SV influenza vaccine (Flu AS03) [0604] one group of 50
subjects receiving one dose of the reconstituted and Flu AS03+MPL
adjuvanted SV influenza vaccine (Flu AS03+MPL) one control group of
50 subjects receiving one dose of FLUARIX.RTM.
X.2. Immunogenicity Results
X.2.1. Humoral Immune Response Endpoints and Results
[0605] In order to evaluate the humoral immune response induced by
the AS03 and AS03+MPL adjuvanted vaccines and its persistence, the
following parameters were calculated for each treatment group.
[0606] At Days 0, 21, 90 and 180: serum
haemagglutination-inhibition (HI) antibody titres, tested
separately against each of the three influenza virus strains
represented in the vaccine (anti-H1N1, anti-H3N2 &
anti-B-antibodies). [0607] Serum HI antibody GMTs' with 95% CI at
Days 0, 21, 90 and 180 [0608] Seroconversion rates with 95% CI at
Days 21, 90 and 180 [0609] Conversion factors with 95% CI at Day 21
[0610] Seroprotection rates with 95% CI at Days 0, 21, 90 and
180
Results
[0611] The GMTs for HI antibodies with 95% CI are shown in FIG. 23.
Pre-vaccination GMTs of antibodies for all 3 vaccine-strains were
within the same range in the 3 groups. After vaccinations,
anti-haemagglutinin antibody levels increased significantly.
Post-vaccination GMTs of antibodies for the 3 vaccine strains
remained however within the same ranges for all vaccines. On Day
21, a slight tendency in favour of the 2 adjuvanted vaccines
compared to FLUARIX.RTM. was noted for the A/New Caledonia and the
B/Jiangsu strains and among the two adjuvanted vaccines, the higher
GMTs were observed with FLU AS03 for the A/Wyoming and B/Jiangsu
strains.
[0612] The same trends were observed at Day 90. On Day 180, GMTs of
antibodies for the 3 vaccine strains were within the same ranges
for the 3 vaccines.
[0613] All influenza vaccines fulfilled the requirements of the
European authorities for annual registration of influenza
inactivated vaccines ["Note for Guidance on Harmonisation of
Requirements for Influenza Vaccines for the immunological
assessment of the annual strain changes" (CPMP/BWP/214/96)] in
subjects aged over 60 years. Three months (90 days) and 6 months
(180 days) after vaccination, the seroprotection rates were still
higher than the minimum rate of 60% required by the European
Authorities whatever the study group considered. On Day 90, the
minimum seroconversion rate of 30% required by the European
Authorities was still achieved for all vaccines strains in the 3
vaccine groups except with FLUARIX.RTM. for the A/New Caledonia
strain. On Day 180, it was still achieved for the A/Wyoming and
B/Jiangsu strains with the 3 vaccines but not for the A/New
Caledonia strain (Table 43 and Table 44).
TABLE-US-00043 TABLE 43 Seroprotection rates as the percentage of
vaccinees with a serum haemagglutination inhibition titre superior
or equal to 1:40 (ATP cohort for immunogenicity) .gtoreq.1:40 95%
CI Antibody Group Timing N n % LL UL A/New Flu AS03 + MPL PRE 50 28
56.0 41.3 70.0 Caledonia PI(D21) 50 46 92.0 80.8 97.8 PI(D90) 50 43
86.0 73.3 94.2 PI(D180) 50 39 78.0 64.0 88.5 FLUARIX .RTM. PRE 50
26 52.0 37.4 66.3 PI(D21) 50 46 92.0 80.8 97.8 PI(D90) 50 38 76.0
61.8 86.9 PI(D180) 50 34 68.0 53.3 80.5 FluAS03 PRE 49 28 57.1 42.2
71.2 PI(D21) 49 48 98.0 89.1 99.9 PI(D90) 49 45 91.8 80.4 97.7
PI(D180) 49 38 77.6 63.4 88.2 A/ Flu AS03 + MPL PRE 50 33 66.0 51.2
78.8 Wyoming PI(D21) 50 47 94.0 83.5 98.7 PI(D90) 50 46 92.0 80.8
97.8 PI(D180) 50 45 90.0 78.2 96.7 FLUARIX .RTM. PRE 50 32 64.0
49.2 77.1 PI(D21) 50 50 100 92.9 100.0 PI(D90) 50 49 98.0 89.4 99.9
PI(D180) 50 50 100 92.9 100.0 FluAS03 PRE 49 34 69.4 54.6 81.7
PI(D21) 49 48 98.0 89.1 99.9 PI(D90) 49 46 93.9 83.1 98.7 PI(D180)
49 47 95.9 86.0 99.5 B/Jiangsu Flu AS03 + MPL PRE 50 19 38.0 24.7
52.8 PI(D21) 50 50 100 92.9 100.0 PI(D90) 50 47 94.0 83.5 98.7
PI(D180) 50 46 92.0 80.8 97.8 FLUARIX .RTM. PRE 50 17 34.0 21.2
48.8 PI(D21) 50 48 96.0 86.3 99.5 PI(D90) 50 47 94.0 83.5 98.7
PI(D180) 50 47 94.0 83.5 98.7 FluAS03 PRE 49 25 51.0 36.3 65.6
PI(D21) 49 49 100 92.7 100.0 PI(D90) 49 47 95.9 86.0 99.5 PI(D180)
49 46 93.9 83.1 98.7 N = number of subjects with available results
n/% = number/percentage of subjects with titre within the specified
range PRE = pre-vaccination titre PI(D21) = post-vaccination blood
sampling at Day 21 PI(D90) = post-vaccination blood sampling at Day
90 PI(D180) = post-vaccination blood sampling at Day 180
TABLE-US-00044 TABLE 44 Seroconversion rate for haemagglutination
inhibition (HI) antibody titres defined as the percentage of
vaccinees who have at least a 4-fold increase in serum HI titre at
each post-vaccination time point compared to Day 0 (ATP cohort for
immunogenicity) 4-fold 95% CI Vaccine strain Timing Group N n % LL
UL A/NEW CALEDONIA Day 21 Flu AS03 + MPL 50 30 60.0 45.2 73.6
FLUARIX .RTM. 50 25 50.0 35.5 64.5 Flu AS03 49 31 63.3 48.3 76.6
Day 90 Flu AS03 + MPL 50 19 38.0 24.7 52.8 FLUARIX .RTM. 50 14 28.0
16.2 42.5 Flu AS03 49 17 34.7 21.7 49.6 Day 180 Flu AS03 + MPL 50
12 24.0 13.1 38.2 FLUARIX .RTM. 50 11 22.0 11.5 36.0 Flu AS03 49 10
20.4 10.2 34.3 A/WYOMING Day 21 Flu AS03 + MPL 50 46 92.0 80.8 97.8
FLUARIX .RTM. 50 38 76.0 61.8 86.9 Flu AS03 49 40 81.6 68.0 91.2
Day 90 Flu AS03 + MPL 50 33 66.0 51.2 78.8 FLUARIX .RTM. 50 33 66.0
51.2 78.8 Flu AS03 49 31 63.3 48.3 76.6 Day 180 Flu AS03 + MPL 50
27 54.0 39.3 68.2 FLUARIX .RTM. 50 23 46.0 31.8 60.7 Flu AS03 49 26
53.1 38.3 67.5 B/JIANGSU Day 21 Flu AS03 + MPL 50 44 88.0 75.7 95.5
FLUARIX .RTM. 50 38 76.0 61.8 86.9 Flu AS03 49 43 87.8 75.2 95.4
Day 90 Flu AS03 + MPL 50 37 74.0 59.7 85.4 FLUARIX .RTM. 50 36 72.0
57.5 83.8 Flu AS03 49 37 75.5 61.1 86.7 Day 180 Flu AS03 + MPL 50
32 64.0 49.2 77.1 FLUARIX .RTM. 50 29 58.0 43.2 71.8 Flu AS03 49 31
63.3 48.3 76.6 N = number of subjects with both pre- and
post-vaccination results available n/% = number/percentage of
subjects with at least a 4-fold increase 95% CI = exact 95%
confidence interval; LL = lower limit, UL = upper limit
X.2.2. CMI Response Endpoints and Results
[0614] In order to evaluate the cellular immune response induced by
the adjuvanted vaccines and its persistence, the following
parameters were calculated for each treatment group: At each time
point (Days 0, 21, 90 and 180): frequency of cytokine-positive
CD4/CD8 cells per 10.sup.6 in different tests (New Caledonia,
Wyoming and Jiangsu antigens considered separately as well as
pooled at Days 0 and 21; New Caledonia, Wyoming, Jiangsu and New
York antigens considered separately as well as pooled at Days 90
and 180) [0615] All double: cells producing at least two different
cytokines (CD40L, IFN-.gamma., IL-2, TNF-.alpha.). [0616] CD40L:
cells producing at least CD40L and another cytokine (IFN-.gamma.,
IL-2, TNF-.alpha.). [0617] IFN-.gamma.: cells producing at least
IFN-.gamma. and another cytokine (CD40L, IL-2, TNF-.alpha.). [0618]
IL-2: cells producing at least IL-2 and another cytokine (CD40L,
IFN-.gamma., TNF-.alpha.). [0619] TNF-.alpha.: cells producing at
least TNF-.alpha. and another cytokine (CD40L, IFN-.gamma.,
IL-2).
Results
[0620] The main findings were (FIG. 24): [0621] (a) Twenty-one days
after the vaccination, the frequency of cytokine-positive CD4 T
cells (IL-2, CD40L, TNF-.alpha. and IFN-.gamma.) was significantly
higher in the two adjuvanted vaccine groups compared to the
FLUARIX.RTM. group. No significant difference was however detected
between the two adjuvants. [0622] (b) All statistical differences
between adjuvanted vaccines and FLUARIX.RTM. were maintained up to
Day 90 and Day 180 with the following exceptions at Day 180: [0623]
No statistically significant difference was found between
FluAS03/MPL and FLUARIX.RTM. for all double, CD40L, IFN-.gamma. and
IL2 (Wyoming strain only) and for all double, CD40L and TNF-.alpha.
(New York strain only) [0624] No statistically significant
difference was found between FluAS03 and FLUARIX.RTM. for IL2
(Jiangsu strain only) [0625] (c) The absence of statistically
significant difference between the two adjuvanted vaccines was
confirmed up to Day 90 and Day 180. [0626] (d) The difference
between pre and post-vaccination (Day 21) in CD4 T-lymphocytes
responses for all cytokines investigated (IL-2, CD40L, TNF-.alpha.
and IFN-.gamma.) was significantly higher with the two adjuvanted
vaccines compared to FLUARIX.RTM.. No significant difference was
however detected between both adjuvants. [0627] (e) The vaccination
had no measurable impact on the CD8 response whatever the treatment
group.
Example XI
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine Containing a Split Influenza Antigen Preparation and AS03
with MPL Adjuvant
XI.1. Study Design and Objectives
[0628] A phase I/II, open, controlled study was conducted in order
to evaluate the reactogenicity and the immunogenicity of
GlaxoSmithKline Biologicals influenza candidate vaccine containing
the AS03+MPL adjuvant in an elderly population aged over 65 years
(>65 years-old) previously vaccinated in 2004 with the same
candidate vaccine. For immunogenicity and safety evaluations,
FLUARIX.RTM. (known as .alpha.-RIX.RTM. in Belgium) vaccine was
used as reference.
[0629] Two parallel groups were assessed: [0630] One group of about
50 subjects who had previously received one dose of the
reconstituted adjuvanted influenza vaccine during the previous
clinical trial [0631] One control group (FLUARIX.RTM.) of about 50
subjects who had previously received one dose of FLUARIX.RTM.
during the previous clinical trial
[0632] One objective of this study was to evaluate the humoral
immune response (anti-haemagglutinin and anti-MPL titres) of the
revaccination with the adjuvanted influenza vaccine Flu AS03+MPL
administered about one year after administration of the first dose.
For comparison purposes, subjects who had already received
FLUARIX.RTM. in the previous trial received a dose of commercial
vaccine and formed the control group of this trial.
XI.2. Vaccine Composition and Administration
[0633] The strains used in the three vaccines were the ones that
had been recommended by the WHO for the 2005-2006 Northern
Hemisphere season, i.e. A/New Caledonia/20/99 (H1N1), A/New
California/7/2004 (H3N2) and B/Jiangsu/10/2003. Like
FLUARIX.RTM./.alpha.-RIX.RTM., the commercially available vaccine
used as a comparator, the (AS03+MPL-adjuvanted vaccine, hereinafter
in short "the adjuvanted vaccine") contains 15 .mu.g haemagglutinin
(HA) of each influenza virus strain per dose.
[0634] The adjuvanted influenza candidate vaccine is a 2 component
vaccine consisting of a concentrated trivalent inactivated split
virion antigens presented in a type I glass vial and of a
pre-filled type I glass syringe containing the AS03+MPL adjuvant.
It has been prepared according the method detailed in Example
II.
[0635] 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 adjuvanted influenza candidate vaccine
corresponds to 0.7 mL. The adjuvanted influenza candidate vaccine
is a preservative-free vaccine.
[0636] The composition of one dose of the reconstituted adjuvanted
influenza vaccine is given in Table 45. Both vaccines were given
intramuscularly.
TABLE-US-00045 TABLE 45 Composition of the reconstituted vaccine
adjuvanted (AS03 + MPL) influenza candidate vaccine Component
Quantity per dose Inactivated split virions A/New Caledonia/20/99
(H1N1) 15 .mu.g HA A/New California/7/2004 (H3N2) 15 .mu.g HA
B/Jiangsu/10/2003 15 .mu.g HA Adjuvant SB62 emulsion (squalene)
10.68 mg (DL-alpha-tocopherol) 11.86 mg (polysorbate 80 - TWEEN
.RTM. 4.85 mg 80) 25 .mu.g MPL
XI.3. Immunogenicity Results
XI.3.1. Anti-HA Humoral Immune Response Endpoints and Results
Observed Variables:
[0637] At days 0 and 21: serum haemagglutination-inhibition (HI)
antibody titres, tested separately against each of the three
influenza virus strains represented in the vaccine (anti-H1N1,
anti-H3N2 & anti-B-antibodies).
Derived variables (with 95% confidence intervals): [0638] (f)
Geometric mean titres (GMTs) of serum HI antibodies with 95%
confidence intervals (95% CI) pre and post-vaccination [0639] (g)
Seroconversion rates* with 95% CI at day 21 [0640] (h)
Seroconversion factors** with 95% CI at day 21 [0641] (i)
Seroprotection rates*** with 95% CI at day 21
[0642] *Seroconversion rate defined as the percentage of vaccinees
with either a pre-vaccination HI titre<1:10 and a
post-vaccination titre 1:40, or a pre-vaccination titre 1:10 and a
minimum 4-fold increase at post-vaccination titre, for each vaccine
strain.
[0643] **Seroconversion factor defined as the fold increase in
serum HI GMTs on day 21 compared to day 0, for each vaccine
strain.
[0644] ***Protection rate defined as the percentage of vaccinees
with a serum HI titre.gtoreq.40 after vaccination (for each vaccine
strain) that usually is accepted as indicating protection.
Results
[0645] As expected, the vast majority of subjects were already
seropositive for the three strains in both groups before
vaccination. Pre-vaccination GMTs for all 3 vaccine strains were
within the same range in the 2 groups. There was a trend for higher
GMTs at post-vaccination for all 3 vaccine strains in the Flu
AS03+MPL group compared to the FLUARIX.RTM. group, although 95% CI
were overlapping (FIG. 25). The two influenza vaccines fulfilled
the requirements of the European authorities for annual
registration of influenza inactivated vaccines ["Note for Guidance
on Harmonisation of Requirements for Influenza Vaccines for the
immunological assessment of the annual strain changes"
(CPMP/BWP/214/96)] in subjects aged over 60 years (Table 46).
TABLE-US-00046 TABLE 46 Seroprotection rates seroconversion rates
and conversion factors at day 21 (ATP cohort for immunogenicity)
Seroprotection rate Seroconversion rate Seroconversion (HI titre
.gtoreq. 40) (.gtoreq.4-fold increase) factor Strains Group N %
[95% CI] [95% CI] EU standard (>60 >60% >30% >2.0 A/New
Caledonia Flu + MPL-AS03 38 89.5 [75.20-97.06] 31.6 [17.5-48.7] 3.1
[2.2-4.4] FLUARIX .RTM. 45 82.2 [67.95-92.00] 31.1 [18.2-46.6] 2.5
[1.8-3.5] A/New York (H3N2) Flu + MPL-AS03 38 92.1 [78.62-98.34]
78.9 [62.7-90.4] 8.8 [6.1-12.5] FLUARIX .RTM. x 45 95.6
[84.85-99.46] 68.9 [53.4-818] 6.0 [4.4-8.3] B/Jiangsu (B) Flu +
MPL-AS03 38 100 [90.75-100] 57.9 [40.8-73.7] 5.1 [3.7-7.0] FLUARIX
.RTM. x 45 100 [92.13-100] 37.8 [23.8-53.5] 3.1 [2.4-4.0] N = total
number of subject; % = Percentage of subjects with titre at day 21
within the specified range; CI = confidence interval
Example XII
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine Containing a Split Influenza Antigen Preparation Adjuvanted
with AS03 and MPL at Two Different Concentrations
XII.1. Study Design and Objectives
[0646] An open, randomized phase I/II study to demonstrate the non
inferiority in term of cellular mediated immune response of
GlaxoSmithKline Biologicals influenza candidate vaccines containing
various adjuvants administered in elderly population (aged 65 years
and older) as compared to FLUARIX.RTM. (known as .alpha.-RIX.RTM.
in Belgium) administered in adults (18-40 years)
[0647] Four parallel groups were assigned: [0648] (a) 75 adults
(aged 18-40 years) in one control group receiving one dose of
FLUARIX.RTM. (FLUARIX.RTM. group) [0649] (b) 200 elderly subjects
(aged 65 years and older) randomized 3:3:2 into three groups:
[0650] one group with 75 subjects receiving influenza vaccine
adjuvanted with AS03+MPL (concentration 1-25 .mu.g) [0651] One
group with 75 subjects receiving influenza vaccine adjuvanted with
AS03+MPL (concentration 2-50 .mu.g) [0652] Reference Flu group with
50 subjects receiving one dose of FLUARIX.RTM..
Primary Objective
[0653] The primary objective is to demonstrate the non inferiority
21 days post-vaccination of the influenza adjuvanted vaccines
administered in elderly subjects (aged 65 years and older) as
compared to FLUARIX.RTM. administered in adults (aged 18-40 years)
in terms of frequency of influenza-specific CD4 T-lymphocytes
producing at least two different cytokines (CD40L, IL-2,
TNF-.alpha., IFN-.gamma.).
Secondary Objectives
[0654] The secondary objectives are [0655] (a) To evaluate the
safety and reactogenicity of vaccination with candidate influenza
vaccines adjuvanted during 21 days following the intramuscular
administration of the vaccine in elderly subjects (aged 65 years
and older). FLUARIX.RTM. is used as reference. [0656] (b) To
evaluate the humoral immune response (anti-haemagglutinin titre)
21, 90 and 180 days after vaccination with influenza candidate
vaccines adjuvanted. FLUARIX.RTM. is used as reference.
Tertiary Objective
[0657] The tertiary objective is to evaluate the cell mediated
immune response (production of IFN-.gamma., IL-2, CD40L, and
TNF-.alpha. and memory B-cell response) 21, 90 and 180 days after
vaccination with adjuvanted influenza-vaccines. FLUARIX.RTM. is
used as reference.
XII.2. Vaccine Composition and Administration
[0658] The influenza vaccine adjuvanted with AS03+MPL (25 .mu.g per
dose) system is also used in study illustrated in Example XI. The
influenza vaccine adjuvanted with AS03+MPL (50 .mu.g per dose)
system is of identical composition except that the concentration of
MPL is doubled. The process is the same as the one described in
Example VIII for the influenza vaccine adjuvanted with AS03+MPL,
with as only difference that the concentration of MPL is doubled.
Control: full dose of FLUARIX.RTM. by IM administration. [0659]
Four scheduled visits per subject: at days 0, 21, 90 and 180 with
blood sample collected at each visit to evaluate
immunogenicity.
[0660] Vaccination schedule: one injection of influenza vaccine at
day 0
XII.3. Immunogenicity Results
XII.3.1. CMI Endpoints and Results
Evaluation of the Primary Endpoint.
[0661] At day 21: CMI response in all subjects in terms of
frequency of influenza-specific CD4 T-lymphocyte per 10.sup.6 in
tests producing at least two different cytokines (IL-2,
IFN-.gamma., TNF-.alpha. and CD40L)
[0662] For evaluation of CMI response, frequency of
influenza-specific CD4 are analysed as follows: The GM ratio in
term of influenza-specific CD4 frequency between groups vaccinated
with adjuvanted vaccines and Flu YNG is obtained using an ANCOVA
model on the logarithm-transformed titres. The ANCOVA model
includes the vaccine group as fixed effect and the pre-vaccination
log-transformed titre as regressor. The GM ratio and their 98.75%
CI are derived as exponential-transformation of the corresponding
group contrast in the model. The 98.75% CI for the adjusted GM is
obtained by exponential-transformation of the 98.75% CI for the
group least square mean of the above ANCOVA model.
Results--Inferential Analysis (Table 47)
[0663] The adjusted GM and GM ratios (with their 98.75% CI) of
influenza-specific CD4 T-lymphocyte producing at least two
cytokines (IL-2, IFN-.gamma., TNF-.alpha. and CD40L) at day 21,
after in vitro restimulation with "pooled antigens II", are
presented in Table 47. For each adjuvanted influenza vaccine, the
upper limit of two-sided 98.75% CI of GM ratio is far below the
clinical limit of 2.0. This shows the non-inferiority of both
adjuvanted influenza vaccines administered to elderly subjects
compared to the FLUARIX.RTM. vaccine administered in adults aged
between 18 and 40 years in term of post-vaccination frequency of
influenza-specific CD4.
TABLE-US-00047 TABLE 47 Adjusted GM ratio of influenza-specific CD4
producing at least two cytokines, Day 21 (ATP cohort for
immunogenicity) Adjusted GM ratio (Flu YNG/AS03 + AS03 + MPL MPL
(conc. 1) Flu YNG (conc. 1) 98.8% CI N Adjusted GM N Adjusted GM
Value LL UL 70 1995.3 72 2430.0 0.82 0.65 1.04 Adjusted GM ratio
(Flu YNG/AS03 + AS03 + MPL MPL (conc. 2) Flu YNG (conc. 2) 98.8% CI
N Adjusted GM N Adjusted GM Value LL UL 70 1979.4 72 2603.8 0.76
0.59 0.98 Adjusted GM = geometric mean antibody adjusted for
baseline titre; N = Number of subjects with both pre- and
post-vaccination results available; 98.8% CI = 98.8% confidence
interval for the adjusted GM ratio (Ancova model: adjustment for
baseline); LL = lower limit, UL = upper limit
Results--Descriptive Analysis (FIG. 26)
[0664] The main findings were: [0665] Before vaccination the CMI
response if higher in young adults than in elderly [0666] After
vaccination, [0667] there was a booster effect of the influenza
vaccine on the CMI response in young adults (18-40 years) [0668]
CMI response in the elderly having received adjuvanted influenza
vaccine is comparable to the CMI response of young adults. [0669]
The difference between pre and post-vaccination in CD4
T-lymphocytes responses for all cytokines investigated (IL-2,
CD40L, TNF-.alpha. and IFN-.gamma.) was significantly higher with
the adjuvanted vaccines compared to FLUARIX.RTM. (18-40 years) for
all tests excepted for IFN.gamma. when we compare Fluarix (18-40
years) and Flu/AS03+MPL (conc. 1).
[0670] It should be noted that the in vitro stimulation was
performed with the Flu strains (i) B/Jiangsu, (ii) A/H3N2/New-York
and (iii) A/H3N2/Wyoming instead of A/H1N1/New-Caledonia included
in the vaccine. However, preliminary data including the A/H1N1/New
Caledonia vaccine strain from subsets of subjects indicate that the
results will be similar.
Results--Evaluation of the Tertiary End-Point (Table 48)
[0671] In order to evaluate the tertiary end point, the frequency
of influenza-specific CD4/CD8 T-lymphocytes and memory B-cells were
measured at days 0, 21, 90 and 180. The frequency of
influenza-specific cytokine-positive CD4/CD8 T-lymphocytes was
summarised (descriptive statistics) for each vaccination group at
days 0 and 21, for each antigen.
[0672] A Non-parametric test (Wilcoxon test) was used to compare
the location of difference between the two groups (influenza
adjuvanted vaccine versus FLUARIX.RTM.) and the statistical p-value
is calculated for each antigen at each different test.
[0673] Descriptive statistics in individual difference between day
21/day 0 (Post-/Pre-vaccination) responses is calculated for each
vaccination group and each antigen at each different test.
[0674] A Non-parametric test (Wilcoxon test) is used to compare the
individual difference Post-/Pre-vaccination) and the statistical
p-value will be calculated for each antigen at each different
test.
[0675] The p-values from Wilcoxon test used to compare the
difference in the frequency of influenza-specific CD4 T-lymphocytes
are presented in Table 48.
TABLE-US-00048 TABLE 48 Inferential statistics: p-values from
Kruskal-Wallis Tests for CD4 T cells at each time point (ATP Cohort
for immunogenicity) p-value Group1 and Group2 and Group1 and Group2
and Flu ELD Flu ELD Flu YNG Flu YNG day 0 day 21 day 0 day 21 day 0
day 21 day 0 day 21 ALL DOUBLES 0.4380 0.0003 0.4380 0.0003 0.0000
0.9014 0.0005 0.4889 CD4OL 0.3194 0.0002 0.3194 0.0002 0.0000
0.9841 0.0003 0.5412 IFN.gamma. 0.5450 0.0004 0.5450 0.0004 0.0000
0.5397 0.0001 0.7895 IL2 0.3701 0.0008 0.3701 0.0008 0.0003 0.8557
0.0022 0.4766 TFN.alpha. 0.3716 0.0004 0.3716 0.0004 0.0000 0.8730
0.0013 0.2114 Group 1: Influenza vaccine adjuvanted with AS03 + MPL
(conc. 1) Group 2: Influenza vaccine adjuvanted with AS03 + MPL
(conc. 2)
[0676] The main conclusions are: [0677] (a) Pre-vaccination GM
frequencies of influenza-specific CD4 were similar in all groups of
elderly subjects but superior in the adults aged between 18 and 40
years. [0678] (b) Post-vaccination (day 21) frequency of
influenza-specific CD4 T lymphocytes was similar in elderly
subjects vaccinated with adjuvanted vaccines and in adults aged
between 18 and 40 years vaccinated with FLUARIX.RTM. [0679] (c) In
elderly subjects, post-vaccination (day 21) frequency of
influenza-specific CD4 T lymphocytes was significantly higher after
vaccination with adjuvanted vaccines than with FLUARIX.RTM.. [0680]
(d) Pre-vaccination and post vaccination GM frequency of
influenza-specific CD8 T cell was essentially similar in all
groups.
Results--Evaluation of the Humoral Immune Response Endpoints
Observed Variables:
[0681] At days 0, 21, 90 and 180: serum
haemagglutination-inhibition (HI) antibody titres, tested
separately against each of the three influenza virus strains
represented in the vaccine (anti-H1N1, anti-H3N2 &
anti-B-antibodies). The cut-off value for HI antibody against all
vaccine antigens was defined by the laboratory before the analysis
(and equals 1:10). A seronegative subject is a subject whose
antibody titre is below the cut-off value. A seropositive subject
is a subject whose antibody titre is greater than or equal to the
cut-off value. Antibody titre below the cut-off of the assay is
given an arbitrary value of half the cut-off.
[0682] Based on the HI antibody titres, the following parameters
are calculated: [0683] (j) Geometric mean titres (GMTs) of HI
antibody at days 0 and 21, calculated by taking the anti-log of the
mean of the log titre transformations. [0684] (k) Seroconversion
factors (SF) at day 21 defined as the fold increase in serum HI
GMTs on day 21 compared to day 0. [0685] (l) Seroconversion rates
(SC) at day 21 defined as the percentage of vaccinees with either a
pre-vaccination HI titre<1:10 and a post-vaccination titre 1:40,
or a pre-vaccination titre 1:10 and a minimum 4-fold increase at
post-vaccination titre. [0686] (m) Seroprotection rates (SPR) at
day 21 defined as the percentage of vaccinees with a serum HI
titre:40.
[0687] The 95% CI for GM is obtained within each group separately.
The 95% CI for the mean of log-transformed titre is first obtained
assuming that log-transformed titres are normally distributed with
unknown variance. The 95% CI for the GM is then obtained by
exponential-transformation of the 95% CI for the mean of
log-transformed titre.
[0688] Missing serological result for a particular antibody
measurement is not replaced. Therefore a subject without
serological result at a given time point do not contribute to the
analysis of the assay for that time point.
Humoral Immune Response Results (FIG. 27 and Table 49)
[0689] Pre-vaccination GMTs of HI antibodies for all 3 vaccine
strains were within the same range in the 4 treatment groups. After
vaccination, there is clear impact of the 2 adjuvants which
increase the humoral response in elderly, compared to standard
FLUARIX.RTM. in the same population.
[0690] GMTs are [0691] significantly higher for H1N1 for AS03+MPL
(conc. 2), [0692] significantly higher for H3N2 and for B for both
adjuvants,
[0693] Twenty one days after vaccination, the subjects of
FLUARIX.RTM. (18-40 years) had a higher HI response for New
Caledonia and B/Jangsu strains.
[0694] As shown in Table 49, the adjuvanted influenza vaccines
exceeded the requirements of the European authorities for annual
registration of split virion influenza vaccines ["Note for Guidance
on Harmonization of Requirements for Influenza Vaccines for the
immunological assessment of the annual strain changes"
(CPMP/BWP/214/96)] in subjects aged over 60 years.
[0695] After vaccination, there was a statistically difference in
terms of seroprotection rates of HI antibodies between FLUARIX.RTM.
(65 years) group and [0696] Flu AS03+MPL (conc 2) for A/New
Caledonia strain
[0697] For each vaccine strain, the seroprotection rates for the 2
influenza adjuvanted vaccine groups are in the same range compared
to FLUARIX.RTM. (18-40 years) group.
[0698] There was a statistically difference in terms of
seroconversion rates of HI antibodies between FLUARIX.RTM. (65
years) group and [0699] Flu AS03+MPL (conc 2) for A/New Caledonia
strain [0700] Flu AS03+MPL (conc 1) for B/Jiangsu strain
[0701] For each vaccine strain, the seroconversion rates for the 2
influenza adjuvanted vaccine groups are in the same range compared
to FLUARIX.RTM. (18-40 years) group excepted for New Caledonia
strain.
TABLE-US-00049 TABLE 49 Seroprotection rates seroconversion rates
and conversion factors at day 21 (ATP cohort for immunogenicity)
Seroprotection rate Seroconversion Conversion (HI titre .gtoreq.
40) rate (.gtoreq.4-fold factor Strains Group N % increase) [95%
Cl] EU standard (>60 years) >60% >30% >2.0 EU standard
(<60 years) >70% >40% >2.5 A/New Flu Yng 75 100
[95.20-100] 77.3 [66.2-86.2] 35.1 [21.9-56.4] Flu Elderly 49 71.4
[56.74-83.42] 30.6 [18.3-45.4] 3.7 [2.4-5.7] Caledonia FluAS03 +
MPL (conc. 1) 75 90.5 [81.48-96.11] 55.4 [43.4-67.0] 6.4 [4.5-9.0]
(H1N1) FluAS03 + MPL (conc. 2) 75 98.7 [92.79-99.97] 74.7
[63.3-84.0] 9.2 [6.4-13.3] A/New York Flu Yng 75 93.3 [85.12-97.80]
76.0 [64.7-85.1] 9.2 [7.1-11.8] (H3N2) Flu Elderly 49 81.6
[67.98-91.24] 69.4 [54.6-81.7] 8.2 [5.7-11.8] FluAS03 + MPL (conc.
1) 75 94.6 [86.73-98.51] 90.5 [81.5-96.1] 19.2 [14.6-25.3] FluAS03
+ MPL (conc. 2) 75 93.3 [85.12-97.80] 82.7 [72.2-90.4] 15.0
[11.2-20.2] B/Jiangsu (B) Flu Yng 75 100 [95.20-100] 80.0
[69.2-88.4] 13.9 [10.1-19.1] Flu Elderly 49 93.9 [83.13-98.72] 81.3
[70.7-89.4] 4.3 [3.0-6.1] FluAS03 + MPL (conc. 1) 75 95.9
[88.61-99.16] 73.0 [61.4-82.6] 8.5 [6.5-11.2] FluAS03 + MPL (conc.
2) 75 98.7 [92.79-99.97] 66.7 [54.8-77.1] 7.6 [5.6-10.2] N = total
number of subject; % = Percentage of subjects with titre at day 21
within the specified range; CI = confidence interval
XII.3.2. Immunogenicity Conclusions
[0702] (a) Pre-vaccination frequency of influenza-specific CD4 was
significantly inferior in elderly adults compared to adults aged
between 18 and 40 years. After vaccination with FLUARIX.RTM.
post-vaccination frequency (day 21) remained inferior in elderly
adults compared to younger ones. On the contrary, the
non-inferiority in term of frequency of post-vaccination frequency
of influenza-specific CD4 after vaccination with adjuvanted
vaccines of elderly subjects was demonstrated compared to
vaccination with FLUARIX.RTM. in adults aged between 18 and 40
years. [0703] (b) Regarding the humoral immune response in term of
HI antibody response, all influenza vaccines fulfilled the
requirements of the European authorities for annual registration of
influenza inactivated vaccines ["Note for Guidance on Harmonisation
of Requirements for Influenza Vaccines for the immunological
assessment of the annual strain changes" (CPMP/BWP/214/96)]. In
elderly adults, adjuvanted vaccines mediated at least a trend for a
higher humoral immune response to influenza haemagglutinin
than--FLUARIX.RTM.. Significant difference between the humoral
immune response against each vaccine strain mediated in elderly
subjects by adjuvanted vaccines compared to--FLUARIX.RTM. are
summarised in Table 50. Compared to adults aged between 18 and 40
years vaccinated with FLUARIX.RTM., elderly subjects vaccinated
with the adjuvanted vaccines showed a trend for higher
post-vaccination GMTs and seroconversion factor at day 21 against
the A/New York strain.
TABLE-US-00050 [0703] TABLE 50 Significant difference in humoral
immune response between adjuvanted vaccines and FLUARIX .RTM. in
elderly subjects Post-vacc Seroconversion Seroprotection
Seroconversion GMT Factor rate Rate Flu AS03 + MPL A/New York A/New
York -- B/Jiangsu (conc. 1) B/Jiangsu Flu AS03 + MPL A/New York
A/New Caledonia A/New Caledonia A/New Caledonia (conc. 2) B/Jiangsu
A/New Caledonia
XII.4. Reactogenicity Results
XII.4.1. Recording of Adverse Events (AE)
[0704] Solicited symptoms (see Table 51) occurring during a 7-day
follow-up period (day of vaccination and 6 subsequent days) were
recorded. Unsolicited symptoms occurring during a 21-day follow-up
period (day of vaccination and 20+3 subsequent days) were also
recorded. Intensity of the following AEs was assessed as described
in Table 52.
TABLE-US-00051 TABLE 51 Solicited local/general adverse events
Solicited local AEs Solicited general AEs Pain at the injection
site Fatigue Redness at the injection site Fever Swelling at the
injection site Headache Haematoma Muscle ache Shivering Joint pain
in the arm of the injection Joint pain at other locations N.B.
Temperature was recorded in the evening. Should additional
temperature measurements performed at other times of day, the
highest temperature was recorded.
TABLE-US-00052 TABLE 52 Intensity scales for solicited symptoms in
adults Intensity Adverse Event grade Parameter Pain at injection
site 0 Absent 1 on touch 2 when limb is moved 3 prevents normal
activity Redness at injection site Record greatest surface diameter
in mm Swelling at injection site Record greatest surface diameter
in mm Haematoma at injection site Record greatest surface diameter
in mm Fever Record temperature in .degree. C./.degree. F. Headache
0 Absent 1 is easily tolerated 2 interferes with normal activity 3
prevents normal activity Fatigue 0 Absent 1 is easily tolerated 2
interferes with normal activity 3 prevents normal activity Joint
pain at the injection 0 Absent site and other locations 1 is easily
tolerated 2 interferes with normal activity 3 prevents normal
activity Muscle ache 0 Absent 1 is easily tolerated 2 interferes
with normal activity 3 prevents normal activity Shivering 0 Absent
1 is easily tolerated 2 interferes with normal activity 3 prevents
normal activity *Fever is defined as axillary temperature
.gtoreq.37.5.degree. C. (99.5.degree. F.)
[0705] The maximum intensity of local injection site
redness/swelling is scored as follows:
[0706] 0 is 0 mm; 1 is >0-.ltoreq.20 mm; 2 is >20-.ltoreq.50
mm; 3 is >50 mm.
[0707] The maximum intensity of fever is scored as follows:
[0708] 1 is >37.5-38.0.degree. C.; 2 is >38.0-39.0.degree.
C.; 3 is >39.0
[0709] The investigator makes an assessment of intensity for all
other AEs, i.e. unsolicited symptoms, including SAEs reported
during the study. The assessment is based on the investigator's
clinical judgement. The intensity of each AE recorded is assigned
to one of the following categories:
1 (mild)=An AE which is easily tolerated by the subject, causing
minimal discomfort and not interfering with everyday activities; 2
(moderate)=An AE which is sufficiently discomforting to interfere
with normal everyday activities; 3 (severe)=An AE which prevents
normal, everyday activities (In adults/adolescents, such an AE
would, for example, prevent attendance at work/school and would
necessitate the administration of corrective therapy).
XII.4.2. Recording of Adverse Events (AE)
[0710] The reactogenicity observed in elderly subjects with
adjuvanted vaccines, in terms of both local and general symptoms,
was found to be higher than with FLUARIX.RTM. in the same
population. However, it was shown to be similar to the level seen
in the adult population. The incidence and the intensity of
symptoms was increased after vaccination with adjuvanted vaccines
compared to the reactogenity seen in elderly subjects with
FLUARIX.RTM. (FIG. 28). In all cases, symptoms resolved
rapidly.
[0711] Grade 3 symptoms showed a trend to be higher in the group
who received the vaccine adjuvanted with the highest MPL
concentration compared to the group who received the adjuvanted
vaccine wherein the MPL is at a lower concentration. In all cases,
symptoms however resolved rapidly.
* * * * *