U.S. patent application number 11/909414 was filed with the patent office on 2009-03-26 for composition.
This patent application is currently assigned to GlaxoSmithKline Biologicals s.a.. Invention is credited to Emmanuel Jules Hanon, Jean Stephenne.
Application Number | 20090081253 11/909414 |
Document ID | / |
Family ID | 36441219 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081253 |
Kind Code |
A1 |
Hanon; Emmanuel Jules ; et
al. |
March 26, 2009 |
COMPOSITION
Abstract
The present invention relates to influenza vaccine formulations
and vaccination regimes for immunising against various diseases. In
particular the invention relates to vaccine formulations comprising
an oil-in-water emulsion adjuvant and 3D-MPL, their use in
medicine, in particular their use in augmenting immune responses to
various antigens, and to methods of preparation, wherein the
oil-in-water emulsion comprises a sterol, a metabolisable oil and
an emulsifying agent.
Inventors: |
Hanon; Emmanuel Jules;
(Rixensart, BE) ; Stephenne; Jean; (Rixensart,
BE) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION;CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Assignee: |
GlaxoSmithKline Biologicals
s.a.
|
Family ID: |
36441219 |
Appl. No.: |
11/909414 |
Filed: |
March 21, 2006 |
PCT Filed: |
March 21, 2006 |
PCT NO: |
PCT/EP2006/002838 |
371 Date: |
September 21, 2007 |
Current U.S.
Class: |
424/206.1 ;
424/184.1 |
Current CPC
Class: |
A61K 2039/70 20130101;
A61K 39/12 20130101; A61P 43/00 20180101; C12N 2760/16234 20130101;
A61P 31/00 20180101; A61P 31/14 20180101; C12N 7/00 20130101; A61K
39/39 20130101; A61P 37/04 20180101; A61K 2039/55566 20130101; A61K
2039/55572 20130101; A61P 31/12 20180101; A61K 39/145 20130101;
C12N 2760/16034 20130101; A61K 2039/57 20130101; A61K 2039/55
20130101; C12N 2760/16134 20130101; A61P 31/16 20180101; A61K
2039/55511 20130101 |
Class at
Publication: |
424/206.1 ;
424/184.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 31/00 20060101 A61P031/00 |
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. An immunogenic composition comprising: (a) an antigen or
antigenic composition; (b) an oil-in-water emulsion adjuvant; and
(c) 25 vg (w/v) 3D MPL per composition dose, wherein said
oil-in-water emulsion comprises a metabolisable oil, a sterol
and/or Alpha-tocopherol, and an emulsifying agent, wherein said
emulisifying agent is Polyoxyethylene sorbitan monooleate (Tween
80.TM.).
2. The immunogenic composition according to claim 1, wherein said
metabolisable oil is present in an amount of from about 0.5% to
About 20% of the total volume of said immunogenic composition.
3. The immunogenic composition according to claim 1, wherein said
metabolisable oil is present in an amount of from about 1.0% to
about 10% of the total volume of said immunogenic composition.
4. (canceled)
5. The immunogenic composition as claimed in claims 1, wherein said
sterol or alpha-tocopherol is present in an amount of from about
1.0% to about 20% of the total volume of said immunogenic
composition.
6. the immunogenic composition as claimed in claim 5, wherein said
alpha-tocopherol is present in an amount of from about 1.0% to
about 50% of the total volume of said immunogenic composition.
7. The immunogenic composition as claimed in claim 6, wherein the
alpha-tocopherol is present in an amount of about 2.5% of the total
volume of said immunogenic composition.
8. The immunogenic composition as claimed in claim 1, wherein said
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. (canceled)
10. The immunogenic composition as claimed in claim 1, wherein said
oil-in-water emulsifying comprises: abpit 2% to 10% squalene, about
2% to 10% alpha-tocopherol and about 0.3% to 3% polyoxyethylene
sorbitan monooleate (Tween 80.TM.).
11. The immunogenic composition as claimed in claim 1, wherein said
oil-in-water emulsion consists: squalene, alpha-tocopherol,
polyoxyethylene sorbitan monooleate (tween 80.TM.), and PBS.
12. A method of preventing influenza infection in a human and/or a
disease associated with exposure of a human to influenza or a
pathogen comprising influenza or a variant thereof, said method
comprising the step of administering to the human an immunogenic
composition comprising: (a) an antigen or antigenic composition (b)
an oil-in-water emulsion adjuvant; and (c) 25 .mu.g (w/v) 3D MPL
per composition dose, wherein said oil-in-water emulsion adjuvant
comprises a metabolisable oil, a sterol and/or alph-tocopherol, and
an emulsifying agent, and wherein said emulsifying agent is
polyoxethylene sorbitan monooleate (Tween 80.TM.).
13. A method of vaccination comprising delivery of an antigen or
antigenic composition, 25 .mu.g 3D MPL per composition does and an
oil-in-water emulsion adjuvant as defined in claim 1 to an
individual or population in need thereof.
14. The method according to claim 12, wherein said immunogenic
composition induces at least one response in said human chosen from
the group of: i) an improved CD4 T-cell response; ii) an improved B
cell memory response; and iii) an improved antibody response
against said antigen.
15. (canceled)
16. The method according to claim 12, wherein said immunogenic
composition protects said human against infection or disease caused
by a pathogen that is a variant of the pathogen from which the
antigen in said immunogenic composition is derived.
17. The method according to claim 12, wherein said immunogenic
composition protects said human against infection or disease caused
by a pathogen that comprises an antigen that is a variant of that
antigen in said immunogenic composition.
18. (canceled)
19. The method according to claim 1, wherein said immunogenic
composition comprises an antigen chosen from the group: an antigen
with at least one CD4 T cell epitope and an antigen with at least
one B cell epitope.
20. (canceled)
21. A method of revaccinating a human previously vaccinated with an
antigen or antigenic composition, said method comprising the step
of administering to said human an immunogenic composition
comprising an antigen or antigenic composition thereof, or a
fragment or variant thereof,wherein said antigen or antigenic
composition for said previous vaccination comprises an antigen or
antigenic composition, or a fragment or variant thereof, 25 .mu.g
(w/v) 3D MPL per compsition dose, and an oil-in-water emulsion
adjuvant.
22. The method according to claim 21, wherein the Antigen for
revaccination shares common CD4 T-cell epitopes with an antigen or
Antigenic compsoition used in said human for a previous
vaccination.
23. The method according to claim 21 Wherein said antigen or
antigenic composition for revaccination is adjuvanted.
24. The method according to claim 23, wherein the Adjuvant is
chosen from the group of: an oil-in-water emulsion; 3D-MPL; a
Combination of an oil-in-water emulsion adjuvant and 3D-MPL; and
wherein said oil-in-water Emulsion adjuvant comprises a
metabolisabie oil, a sterol and/or alpha-tocopherol, And an
emulsifying agent, wherein said emulsifying agent is
Polyoxyethylene sorbitan monooleate (Tween 80.TM.).
25. (canceled)
26. A method for preparing an immunogenic composition comprising,
said method comprising the step of: combining an oil-in-water
emulsion adjuvant, wherein said adjuvant comprises a metabolisable
oil, a sterol and/or alpha-tocopherol and an emulsifying agent, and
wherein said emulsifying agent is polyoxyethylene sorbitan
monooleate (Tween 80.TM.) with an antigen or antigenic composition
and 25 .mu.g (w/v) 3D-MPL per composition dose.
27. The immunogenic composition according to claim 1, wherein said
composition is combined with a pharmaceutically acceptable
carrier.
28. A method for protecting a human against a pathogen, said method
comprising the step of administering to said human an immunogenic
composition that comprises: (a) an antigen derived from said
pathogen; (b) an oil-in-water emuslsion adjuvant, wherein said
adjuvant comprises a metabolisable oil, a sterol and/or
alpha-tocopherol, and an emulisifying agent, and wherein said
emulsifying agent is polyoxyethylene sorbitan monooleate (Tween
80.TM.); and (c) 25 .mu.g (w/v) 3D MPL per composition dose.
29. The method according to claim 1, wherein said antigen or
antigenic preparation is chosen from the group of: influenza virus,
and HPV.
30. The method according to claim 29, wherein said influenza
antigen is chosen from the group Of: a split influenza virus, a
whole influenza virus, a sub-unit influenza virus, an Influenza
virosome, and an antigenic preparation thereof.
31-34. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to influenza vaccine
formulations and vaccination regimes for immunising against various
diseases. In particular the invention relates to vaccine
formulations comprising an oil-in-water emulsion adjuvant and
3D-MPL, their use in medicine, in particular their use in
augmenting immune responses to various antigens, and to methods of
preparation, wherein the oil in water emulsion comprises a sterol,
a metabolisable oil and an emulsifying agent.
TECHNICAL BACKGROUND
[0002] New compositions or vaccines with an improved immunogenicity
are always needed. As one strategy, adjuvants have been used to try
and improve the immune response raised to any given antigen.
[0003] By way of example, influenza vaccines and vaccines against
human papilloma virus (HPV) have been developed with adjuvants.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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).
[0009] 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.
[0010] 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.
[0011] 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.).
[0012] 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).
[0013] 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).
[0014] 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.
[0015] 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).
[0016] Papillomaviruses are small DNA tumour viruses, which are
highly species specific. So far, over 100 individual human
papillomavirus (HPV) genotypes have been described. HPVs are
generally specific either for the skin (e.g. HPV-1 and -2) or
mucosal surfaces (e.g. HPV-6 and -11) and usually cause benign
tumours (warts) that persist for several months or years. Such
benign tumours may be distressing for the individuals concerned but
tend not to be life threatening, with a few exceptions.
[0017] Some HPVs are also associated with cancers. The strongest
positive association between an HPV and human cancer is that which
exists between HPV-16 and HPV-18 and cervical carcinoma. Cervical
cancer is the most common malignancy in developing countries, with
about 500,000 new cases occurring in the world each year. It is now
technically feasible to actively combat primary HPV-16 infections,
and even established HPV-16-containing cancers, using vaccines. For
a review on the prospects for prophylactic and therapeutic
vaccination against HPV-16 see Cason J., Clin. Immunother. 1994;
1(4) 293-306 and Hagenesee M. E., Infections in Medicine 1997 14(7)
555-556, 559-564.
[0018] Although minor variations do occur, all HPVs genomes
described have at least eight early genes, E1 to E8 and two late
genes L1 and L2. In addition, an upstream regulatory region harbors
the regulatory sequences which appear to control most
transcriptional events of the HPV genome.
[0019] HPV L1 based vaccines are disclosed in WO94/00152,
WO94/20137, WO93/02184 and WO94/05792. Such a vaccine can comprise
the L1 antigen as a monomer, a capsomer or a virus like particle.
Methods for the preparation of VLPs are well known in the art, and
include VLP disassembly-reassembly approaches to provide enhanced
homogeneity, for example as described in WO9913056 and U.S. Pat.
No. 6,245,568. Such particles may additionally comprise L2
proteins. L2 based vaccines are described, for example, in
WO93/00436. Other HPV vaccine approaches are based on the early
proteins, such as E7 or fusion proteins such as L2-E7.
[0020] There is still a need for improved vaccines, such as
influenza or HPV vaccines.
STATEMENT OF THE INVENTION
[0021] In first aspect of the present invention, there is provided
an immunogenic composition comprising: [0022] (a) an antigen,
[0023] (b) an oil-in-water emulsion adjuvant; and [0024] (c) 3D
MPL. wherein said oil-in-water emulsion comprises a metabolisable
oil, a sterol and an emulsifying agent.
[0025] The invention also relates to use of a composition
comprising: [0026] (a) an antigen, and [0027] (b) an oil-in-water
emulsion adjuvant; and [0028] (c) 3D MPL wherein said oil-in-water
emulsion comprises a metabolisable oil, a sterol and an emulsifying
agent, in the manufacture of an immunogenic composition for the
prevention of infection and/or disease.
[0029] The invention also relates to a method of vaccination
comprising delivery of an antigen, an oil in water emulsion
adjuvant as defined herein and 3D-MPL.
[0030] The invention also relates to a method for the preparation
of an immunogenic composition comprising combining an oil in water
emulsion as defined herein with an antigen and 3D-MPL.
[0031] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
LEGEND TO FIGURES
[0032] 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.
[0033] FIG. 2: Schematic illustration of the preparation of MPL
bulk.
[0034] FIG. 3: Schematic illustration of the preparation of
AS03+MPL adjuvant.
[0035] FIG. 4: Explo Flu-001 clinical trial. CD4 T cell response to
split influenza antigen (Q1=first quartile, Q3=third quartile).
[0036] FIG. 5: Explo Flu-001 clinical trial. CD8 T cell response to
split influenza antigen (Q1=first quartile, Q3=third quartile).
[0037] FIG. 6: Explo Flu-001 clinical trial. Cross-reactive CD4
T-cell response to split influenza virus antigen after vaccination
with Fluarix+AS03.
[0038] FIG. 7: Explo Flu-001 clinical trial. B cell memory response
post vaccination.
[0039] FIG. 8: Explo Flu-002 clinical trial. CD4 T cell response
against split influenza antigen following revaccination.
[0040] FIG. 9: Explo Flu-002 clinical trial. Anti-HI titers
following revaccination.
[0041] FIG. 10: Ferret study 1. Temperature monitoring (priming and
challenge). FIG. 10A is priming, FIG. 10B is challenge.
[0042] FIG. 11: Ferret study 1. Viral shedding.
[0043] FIG. 12: Ferret study II. Temperature monitoring (priming
and challenge). FIG. 12A is priming, FIG. 12B is challenge.
[0044] FIG. 13: Ferret study II. Viral shedding.
[0045] FIG. 14: Ferret study II. HI titers to H3N2 A/Panama
(vaccine strain) (FIG. 14A) and to H3N2 A/Wyoming (challenge
strain) (FIG. 14B).
[0046] 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).
[0047] 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).
[0048] 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).
[0049] FIG. 18: Human clinical trial. B cell memory response
post-vaccination of elderly with Fluarix, Fluarix+AS03,
Fluarix+AS03+MPL (difference between pre- and post-).
[0050] FIG. 19: Ferret study III. Temperature monitoring before and
after challenge.
[0051] FIG. 20: Ferret study III. Viral shedding before and after
challenge.
[0052] FIG. 21: Ferret study II. HI titers to H3N2 A/Woming
(vaccine strain).
[0053] FIG. 22: Ferret study III. HI titers to H3N2 A/Panama
(challenge strain).
[0054] FIG. 23: Human clinical trial. HI titers (GMTs) at days 21,
90 and 180 post vaccination (persistence).
[0055] FIG. 24: Human clinical trial. CD4 response--all double
test--Pool antigen at days 21, 90 and 180 post vaccination
(persistence).
[0056] FIG. 25: Human clinical trial. HI titers in a revaccination
clinical trial with AS03+MPL compared to Fluarix.
[0057] FIG. 26: Human clinical trial. CMI for CD4 response--all
double test--Pool antigen at days 0 and 21.
[0058] FIG. 27: Human clinical trial with AS03+MPL at two
concentrations. HI titers at days 0 and 21.
[0059] FIG. 28: Human clinical trial with AS03+MPL at two
concentrations. Reactogenicity.
DETAILED DESCRIPTION
[0060] The principles of the present invention are demonstrated in
respect of a sub-unit or split influenza antigen or with various
cancer-associated HPV antigens in the form of VLP, combined with an
oil in water emulsion and 3D-MPL.
[0061] In one aspect of the invention, the present inventors have
discovered that an influenza formulation comprising a sub-unit or
split influenza virus or antigenic preparation thereof together
with an oil-in-water emulsion adjuvant and 3D-MPL is capable of
improving the CD4 T-cell immune response and/or the B cell memory
response, against said antigen or antigenic composition in a human
compared to that obtained with the un-adjuvanted sub-unit or split
virus or split virus antigenic preparation thereof.
[0062] Said compositions thus provide improved influenza
vaccines.
[0063] The claimed formulations may be used to induce
anti-influenza CD4-T cell responses 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).
[0064] The present inventors have discovered that an influenza
formulation comprising a split influenza virus or split virus
antigenic preparation thereof together with an oil-in-water
emulsion adjuvant as defined herein and 3D MPL is capable of
inducing at least a trend for a higher B cell memory response
following the first vaccination of a human subject, compared to the
un-adjuvanted composition.
[0065] The adjuvanted influenza compositions according to the
invention have several advantages: [0066] 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); [0067] 2) An improved cross-protection profile:
increased cross-protection against variant (drifted) influenza
strains; [0068] 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).
[0069] In another aspect of the invention, the inventors have also
discovered that an oil-in-water emulsion adjuvant as defined herein
+3D MPL demonstrates immunogenicity results for both antibody
production and B cell memory which are equivalent to, or sometimes
greater than, those generated with an adjuvant devoid of the
oil-in-water emulsion component.
[0070] These findings can be applied to other forms of the same
antigens, and to other antigens.
Antigens
[0071] Antigens that may be used in the present invention
include:
[0072] Streptococcal antigens such as from Group A Streptococcus,
or Group B Streptococcus, but is most preferably from Streptococcus
pneumoniae. A protein and/or saccharide antigen is most preferably
used. The Streptococcus pneumoniae saccharide antigen and/or at
least one Streptococcus pneumoniae protein antigen(s) is most
preferably selected from the group consisting of: pneumolysin, PspA
or transmembrane deletion variants thereof, PspC or transmembrane
deletion variants thereof, PsaA or transmembrane deletion variants
thereof, glyceraldehyde-3-phosphate dehydrogenase, CbpA or
transmembrane deletion variants thereof, PhtA, PhtD, PhtB, PhtE,
SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, or
immunologically functional equivalent thereof.
[0073] Also preferred at least one (2, 3, 4, 5, 6, 7, 8, 9 or 10)
Streptococcus pneumoniae saccharide antigen(s) and/or Streptococcus
pneumoniae protein antigen preferably selected from the group of
protein antigens listed above.
[0074] Certain compositions are described in WO 00/56359 and WO
02/22167 and WO 02/22168 (incorporated by reference herein).
[0075] The antigen may comprise capsular saccharide antigens
(preferably conjugated to a carrier protein), wherein the
saccharides (most preferably polysaccharides) are derived from at
least four serotypes of pneumococcus. Preferably the four serotypes
include 6B, 14, 19F and 23F. More preferably, at least 7 serotypes
are included in the composition, for example those derived from
serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. More preferably still,
at least 11 serotypes are included in the composition, for example
the composition in one embodiment includes capsular saccharides
derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F
(preferably conjugated to a carrier protein). In a preferred
embodiment of the invention at least 13 saccharide antigens
(preferably conjugated to a carrier protein) are included, although
further saccharide antigens, for example 23 valent (such as
serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,
17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by
the invention. For elderly vaccination (for instance for the
prevention of pneumonia) it is advantageous to include serotypes 8
and 12F (and most preferably 15 and 22 as well) to the 11 valent
antigenic composition described above to form a 15 valent
composition, whereas for infants or toddlers (where otitis media is
of more concern) serotypes 6A and 19A are advantageously included
to form a 13 valent composition.
[0076] Although the above saccharides are advantageously in their
full-length, native polysaccharide form, it should be understood
that size-reduced polysaccharides may also be used which are still
immunogenic (see for example EP 497524 and 497525) if necessary
when coupled to a protein carrier.
[0077] For the prevention/amelioration of pneumonia in the elderly
(+55 years) population and Otitis media in Infants (up to 18
months) and toddlers (typically 18 months to 5 years), it is a
preferred embodiment of the invention to combine a multivalent
Streptococcus pneumonia saccharide as herein described with a
Streptococcus pneumoniae protein preferably selected from the group
of proteins listed above. A combination of pneumococcal proteins
may also be advantageously utilised as described below.
Pneumococcal Proteins
[0078] Streptococcus pneumoniae antigens are preferably selected
from the group consisting of: a protein from the polyhistidine
triad family (Pht), a protein from the Lyt family, a choline
binding protein, proteins having an LPXTG motif (where X is any
amino acid), proteins having a Type II Signal sequence motif of
LXXC (where X is any amino acid), and proteins having a Type I
Signal sequence motif. Preferred examples within these categories
(or motifs) are the following proteins (or truncate or
immunologically functional equivalent thereof):
[0079] The Pht (Poly Histidine Triad) family comprises proteins
PhtA, PhtB, PhtD, and PhtE. The family is characterised by a
lipidation sequence, two domains separated by a proline-rich region
and several histidine triads, possibly involved in metal or
nucleoside binding or enzymatic activity, (3-5) coiled-coil
regions, a conserved N-terminus and a heterogeneous C terminus. It
is present in all strains of pneumococci tested. Homologous
proteins have also been found in other Streptococci and Neisseria.
Preferred members of the family comprise PhtA, PhtB and PhtD. More
preferably, it comprises PhtA or PhtD. It is understood, however,
that the terms Pht A, B, D, and E refer to proteins having
sequences disclosed in the citations below as well as
naturally-occurring (and man-made) variants thereof that have a
sequence homology that is at least 90% identical to the referenced
proteins. Preferably it is at least 95% identical and most
preferably it is 97% identical.
[0080] With regards to the Pht proteins, PhtA is disclosed in WO
98/18930, and is also referred to Sp36. As noted above, it is a
protein from the polyhistidine triad family and has the type II
signal motif of LXXC.
[0081] PhtD is disclosed in WO 00/37105, and is also referred to
Sp036D. As noted above, it also is a protein from the polyhistidine
triad family and has the type II LXXC signal motif. PhtB is
disclosed in WO 00/37105, and is also referred to Sp036B. Another
member of the PhtB family is the C3-Degrading Polypeptide, as
disclosed in WO 00/17370. This protein also is from the
polyhistidine triad family and has the type II LXXC signal motif. A
preferred immunologically functional equivalent is the protein Sp42
disclosed in WO 98/18930. A PhtB truncate (approximately 79 kD) is
disclosed in WO99/15675 which is also considered a member of the
PhtX family.
[0082] PhtE is disclosed in WO00/30299 and is referred to as
BVH-3.
[0083] SpsA is a Choline binding protein (Cbp) disclosed in WO
98/39450.
[0084] The Lyt family is membrane associated proteins associated
with cell lysis. The N-terminal domain comprises choline binding
domain(s), however the Lyt family does not have all the features
found in the choline binding protein family (Cbp) family noted
below and thus for the present invention, the Lyt family is
considered distinct from the Cbp family. In contrast with the Cbp
family, the C-terminal domain contains the catalytic domain of the
Lyt protein family. The family comprises LytA, B and C. With
regards to the Lyt family, LytA is disclosed in Ronda et al., Eur J
Biochem, 164:621-624 (1987). LytB is disclosed in WO 98/18930, and
is also referred to as Sp46. LytC is also disclosed in WO 98/18930,
and is also referred to as Sp91. A preferred member of that family
is LytC.
[0085] Another preferred embodiment are Lyt family (particularly
LytA) truncates wherein "Lyt" is defined above and "truncates"
refers to proteins lacking 50% or more of the Choline binding
region. Preferably such proteins lack the entire choline binding
region.
[0086] Sp125 is an example of a pneumococcal surface protein with
the Cell Wall Anchored motif of LPXTG (where X is any amino acid).
Any protein within this class of pneumococcal surface protein with
this motif has been found to be useful within the context of this
invention, and is therefore considered a further protein of the
invention. Sp125 itself is disclosed in WO 98/18930, and is also
known as ZmpB--a zinc metalloproteinase.
[0087] Sp101 is disclosed in WO 98/06734 (where it has the
reference # y85993. It is characterised by a Type I signal
sequence.
[0088] Sp133 is disclosed in WO 98/06734 (where it has the
reference # y85992. It is also characterised by a Type I signal
sequence.
[0089] Sp128 and Sp130 are disclosed in WO 00/76540.
[0090] The proteins used in the present invention are preferably
selected from the group PhtD, PhtA and PhtE, or a combination of 2
or all 3 of these proteins (i.e. PhtA+D, A+E, D+E or A+D+E).
[0091] Further pneumococcal protein antigens that may be included
are one or more from the group consisting of: pneumolysin (also
referred to as Ply; preferably detoxified by chemical treatment or
mutation) [WO 96/05859, WO 90/06951, WO 99/03884], PsaA and
transmembrane deletion variants thereof (Berry & Paton, Infect
Immun 1996 December; 64(12):5255-62), PspA and transmembrane
deletion variants thereof (U.S. Pat. No. 5,804,193, WO 92/14488, WO
99/53940), PspC and transmembrane deletion variants thereof (WO
97/09994, WO 99/53940), a member of the Choline binding protein
(Cbp) family [e.g. CbpA and transmembrane deletion variants thereof
(WO 97/41151; WO 99/51266)],
Glyceraldehyde-3-phosphate-dehydrogenase (Infect. Immun. 1996
64:3544), HSP70 (WO 96/40928), PcpA (Sanchez-Beato et al. FEMS
Microbiol Lett 1998, 164:207-14), M like protein (SB patent
application No. EP 0837130), and adhesin 18627 (SB Patent
application No. EP 0834568). The present invention also encompasses
immunologically functional equivalents or truncates of such
proteins (as defined above).
[0092] Concerning the Choline Binding Protein family, members of
that family were originally identified as pneumococcal proteins
that could be purified by choline-affininty chromatography. All of
the choline-binding proteins are non-covalently bound to
phosphorylcholine moieties of cell wall teichoic acid and
membrane-associated lipoteichoic acid. Structurally, they have
several regions in common over the entire family, although the
exact nature of the proteins (amino acid sequence, length, etc.)
can vary. In general, choline binding proteins comprise an N
terminal region (N), conserved repeat regions (R1 and/or R2), a
proline rich region (P) and a conserved choline binding region (C),
made up of multiple repeats, that comprises approximately one half
of the protein. As used in this application, the term "Choline
Binding Protein family (Cbp)" is selected from the group consisting
of Choline Binding Proteins as identified in WO 97/41151, PbcA,
SpsA, PspC, CbpA, CbpD, and CbpG. CbpA is disclosed in WO 97/41151.
CbpD and CbpG are disclosed in WO 00/29434. PspC is disclosed in WO
97/09994. PbcA is disclosed in WO 98/21337. Preferably the Choline
Binding Proteins are selected from the group consisting of CbpA,
PbcA, SpsA and PspC.
[0093] If a Cbp is the further protein utilised it may be a Cbp
truncate wherein "Cbp" is defined above and "truncate" refers to
proteins lacking 50% or more of the Choline binding region (C).
Preferably such proteins lack the entire choline binding region.
More preferably, the such protein truncates lack (i) the choline
binding region and (ii) a portion of the N-terminal half of the
protein as well, yet retain at least one repeat region (R1 or R2).
More preferably still, the truncate has 2 repeat regions (R1 and
R2). Examples of such preferred embodiments are NR1.times.R2,
R1.times.R2, NR1.times.R2P and R1.times.R2P as illustrated in
WO99/51266 or WO99/51188, however, other choline binding proteins
lacking a similar choline binding region are also contemplated
within the scope of this invention. Cbp truncate-Lyt truncate
chimeric proteins (or fusions) may also be used in the composition
of the invention. Preferably this comprises NR1.times.R2 (or
R1.times.R2 or NR1.times.R2P or R1.times.R2P) of Cbp and the
C-terminal portion (Cterm, i.e., lacking the choline binding
domains) of Lyt (e.g., LytCCterm or Sp91Cterm). More preferably Cbp
is selected from the group consisting of CbpA, PbcA, SpsA and PspC.
More preferably still, it is CbpA. Preferably, Lyt is LytC (also
referred to as Sp91).
[0094] A PspA or PsaA truncate lacking the choline binding domain
(C) and expressed as a fusion protein with Lyt may also be used.
Preferably, Lyt is LytC.
[0095] In a pneumococcal composition it is possible to combine
different pneumococcal proteins of the invention.
[0096] Preferably the combination of proteins of the invention are
selected from 2 or more (3 or 4) different categories such as
proteins having a Type II Signal sequence motif of LXXC (where X is
any amino acid, e.g., the polyhistidine triad family (Pht)),
choline binding proteins (Cbp), proteins having a Type I Signal
sequence motif (e.g., Sp101), proteins having a LPXTG motif (where
X is any amino acid, e.g., Sp128, Sp130), toxins (e.g., Ply), etc.
Preferred examples within these categories (or motifs) are the
proteins mentioned above, or immunologically functional equivalents
thereof. Toxin+Pht, toxin+Cbp, Pht+Cbp, and toxin+Pht+Cbp are
preferred category combinations.
[0097] Preferred beneficial combinations include, but are not
limited to, PhtD+NR1.times.R2, PhtD+NR1.times.R2-Sp91Cterm chimeric
or fusion proteins, PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA,
PhtA+NR1.times.R2, PhtA+NR1.times.R2-Sp91Cterm chimeric or fusion
proteins, PhtA+Ply, PhtA+Sp128, PhtA+PsaA, PhtA+PspA,
NR1.times.R2+LytC, NR1.times.R2+PspA, NR1.times.R2+PsaA,
NR1.times.R2+Sp128, R1.times.R2+LytC, R1.times.R2+PspA,
R1.times.R2+PsaA, R1.times.R2+Sp128, R1.times.R2+PhtD,
R1.times.R2+PhtA. Preferably, NR1.times.R2 (or R1.times.R2) is from
CbpA or PspC. More preferably it is from CbpA.
[0098] A particularly preferred combination of pneumococcal
proteins comprises Ply (or a truncate or immunologically functional
equivalent thereof)+PhtD (or a truncate or immunologically
functional equivalent thereof) optionally with NR1.times.R2 (or
R1.times.R2 or NR1.times.R2P or R1.times.R2P). Preferably,
NR1.times.R2 (or R1.times.R2 or NR1.times.R2P or R1.times.R2P) is
from CbpA or PspC. More preferably it is from CbpA.
[0099] The antigen may be a pneumococcus saccharide conjugate
comprising polysaccharide antigens derived from at least four
serotypes, preferably at least seven serotypes, more preferably at
least eleven serotypes, and at least one, but preferably 2, 3, or
4, Streptococcus pneumoniae proteins preferably selected from the
group of proteins described above. Preferably one of the proteins
is PhtD (or an immunologically functional equivalent thereof)
and/or Ply (or an immunologically functional equivalent thereof). A
problem associated with the polysaccharide approach to vaccination,
is the fact that polysaccharides per se are poor immunogens. To
overcome this, saccharides may be conjugated to protein carriers,
which provide bystander T-cell help. It is preferred, therefore,
that the saccharides utilised in the invention are linked to such a
protein carrier. Examples of such carriers which are currently
commonly used for the production of saccharide immunogens include
the Diphtheria and Tetanus toxoids (DT, DT CRM197 and TT
respectively), Keyhole Limpet Haemocyanin (KLH), OMPC from N.
meningitidis, and the purified protein derivative of Tuberculin
(PPD).
[0100] A preferred carrier for the pneumococcal saccharide based
immunogenic compositions (or vaccines) is protein D from
Haemophilus influenzae (EP 594610-B), or fragments thereof.
Fragments suitable for use include fragments encompassing T-helper
epitopes. In particular a protein D fragment will preferably
contain the N-terminal 1/3 of the protein. A protein D carrier is
useful as a carrier in compositions where multiple pneumococcal
saccharide antigens are conjugated. One or more pneumococcal
saccharides in a combination may be advantageously conjugated onto
protein D.
[0101] A further preferred carrier for the pneumococcal saccharide
is the pneumococcal protein itself (as defined above in section
"Pneumococcal Proteins of the invention").
[0102] The saccharide may be linked to the carrier protein by any
known method (for example, by Likhite, U.S. Pat. No. 4,372,945 and
by Armor et al., U.S. Pat. No. 4,474,757). Preferably, CDAP
conjugation is carried out (WO 95/08348).
[0103] Preferably the protein:saccharide (weight:weight) ratio of
the conjugates is 0.3:1 to 1:1, more preferably 0.6:1 to 0.8:1, and
most preferably about 0.7:1.
[0104] Particularly preferred compositions of the invention
comprise one or more conjugated pneumococcal saccharides, and one
or more pneumococcal proteins of the invention In addition,
pneumococcal saccharides and proteins can be stably stored as bulk
components adsorbed onto aluminium phosphate in a liquid form.
[0105] Other antigens are suitably derived from HIV-1, (such as gag
or fragments thereof such as p24, tat, nef, gp120 or gp160 or
fragments of any of these), human herpes viruses, such as gD or
derivatives thereof or Immediate Early protein such as ICP27 from
HSV1 or HSV2, cytomegalovirus ((esp Human)(such as gB or
derivatives thereof), Rotavirus (including live-attenuated
viruses), Epstein Barr virus (such as gp350 or derivatives
thereof), Varicella Zoster Virus (such as gpI, II and IE63), or
from a hepatitis virus such as hepatitis B virus (for example
Hepatitis B Surface antigen or a derivative thereof), hepatitis A
virus, hepatitis C virus and hepatitis E virus, or from other viral
pathogens, such as paramyxoviruses: Respiratory Syncytial virus
(such as F, N, M and G proteins or derivatives thereof),
parainfluenza virus, measles virus, mumps virus, human papilloma
viruses (for example HPV6, 11, 16, 18,), flaviviruses (e.g. Yellow
Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese
Encephalitis Virus) or Influenza virus (whole live or inactivated
virus, split influenza virus, grown in eggs or MDCK cells, or whole
flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,
915-920) or purified or recombinant proteins thereof, such as HA,
NP, NA, or M proteins, or combinations thereof), or derived from
bacterial pathogens such as Neisseria spp, including N. gonorrhea
and N. meningitidis (for example capsular saccharides and
conjugates thereof, transferrin-binding proteins, lactoferrin
binding proteins, PiIC, adhesins); S. pyogenes (for example M
proteins or fragments thereof, C5A protease, lipoteichoic acids),
S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, including M
catarrhalis, also known as Branhamella catarrhalis (for example
high and low molecular weight adhesins and invasins); Bordetella
spp, including B. pertussis (for example pertactin, pertussis toxin
or derivatives thereof, filamenteous hemagglutinin, adenylate
cyclase, fimbriae), B. parapertussis and B. bronchiseptica;
Mycobacterium spp., including M. tuberculosis (for example ESAT6,
Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.
paratuberculosis, M. smegmatis; Legionella spp, including L.
pneumophila; Escherichia spp, including enterotoxic E. coli (for
example colonization factors, heat-labile toxin or derivatives
thereof, heat-stable toxin or derivatives thereof),
enterohemorragic E. coli, enteropathogenic E. coli (for example
shiga toxin-like toxin or derivatives thereof); Vibrio spp,
including V. cholera (for example cholera toxin or derivatives
thereof); Shigella spp, including S. sonnei, S. dysenteriae, S.
flexnerii; Yersinia spp, including Y. enterocolitica (for example a
Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp,
including C. jejuni (for example toxins, adhesins and invasins) and
C. coli; Salmonella spp, including S. typhi, S. paratyphi, S.
choleraesuis, S. enteritidis; Listeria spp., including L.
monocytogenes; Helicobacter spp, including H. pylori (for example
urease, catalase, vacuolating toxin); Pseudomonas spp, including P.
aeruginosa; Staphylococcus spp., including S. aureus, S.
epidermidis; Enterococcus spp., including E. faecalis, E. faecium;
Clostridium spp., including C. tetani (for example tetanus toxin
and derivative thereof), C. botulinum (for example botulinum toxin
and derivative thereof), C. difficile (for example clostridium
toxins A or B and derivatives thereof); Bacillus spp., including B.
anthracis (for example botulinum toxin and derivatives thereof);
Corynebacterium spp., including C. diphtheriae (for example
diphtheria toxin and derivatives thereof); Borrelia spp., including
B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii
(for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA,
OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA,
DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agent
of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including
R. rickettsii; Chlamydia spp., including C. trachomatis (for
example MOMP, heparin-binding proteins), C. pneumoniae (for example
MOMP, heparin-binding proteins), C. psittaci; Leptospira spp.,
including L. interrogans; Treponema spp., including T. pallidum
(for example the rare outer membrane proteins), T. denticola, T.
hyodysenteriae; or derived from parasites such as Plasmodium spp.,
including P. falciparum; Toxoplasma spp., including T. gondii (for
example SAG2, SAG3, Tg34); Entamoeba spp., including E.
histolytica; Babesia spp., including B. microti; Trypanosoma spp.,
including T. cruzi; Giardia spp., including G. lamblia; Leshmania
spp., including L. major; Pneumocystis spp., including P. carinii;
Trichomonas spp., including T. vaginalis; Schisostoma spp.,
including S. mansoni, or derived from yeast such as Candida spp.,
including C. albicans; Cryptococcus spp., including C.
neoformans.
[0106] Other preferred specific antigens for M. tuberculosis are
for example Tb Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL,
mTTC2 and hTCC1 (WO 99/51748). Proteins for M. tuberculosis also
include fusion proteins and variants thereof where at least two,
preferably three polypeptides of M. tuberculosis are fused into a
larger protein. Preferred fusions include Ra12-TbH9-Ra35,
Erd14-DPV-MTI, DPV-MTI-MSL, Erd14-DPV-MTI-MSL-mTCC2,
Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO
99/51748).
[0107] Most preferred antigens for Chlamydia include for example
the High Molecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP
366 412), and putative membrane proteins (Pmps). Other Chlamydia
antigens of the composition can be selected from the group
described in WO 99/28475.
[0108] Preferred bacterial compositions comprise antigens derived
from Haemophilus spp., including H. influenzae type B (for example
PRP and conjugates thereof), non typeable H. influenzae, for
example OMP26, high molecular weight adhesins, P5, P6, protein D
and lipoprotein D, and fimbrin and fimbrin derived peptides (U.S.
Pat. No. 5,843,464) or multiple copy varients or fusion proteins
thereof.
[0109] Derivatives of Hepatitis B Surface antigen are well known in
the art and include, inter alia, those PreS1, PreS2 S antigens set
forth described in European Patent applications EP-A-414 374;
EP-A-0304 578, and EP 198-474. In one preferred aspect the vaccine
formulation of the invention comprises the HIV-1 antigen, gp120,
especially when expressed in CHO cells. In a further embodiment,
the composition of the invention comprises gD2t as hereinabove
defined.
[0110] In a preferred embodiment of the present invention
compositions contain an antigen derived from the Human Papilloma
Virus (HPV) considered to be responsible for genital warts (HPV 6
or HPV 11 and others), and the HPV viruses responsible for cervical
cancer (HPV16, HPV18 and others).
[0111] Particularly preferred forms of genital wart prophylactic,
or therapeutic, compositions comprise L1 particles or capsomers,
and fusion proteins comprising one or more antigens selected from
the HPV proteins E1, E2, E5 E6, E7, L1, and L2.
[0112] The most preferred forms of fusion protein are: L2E7 as
disclosed in WO 96/26277, and proteinD(1/3)-E7 disclosed in GB
9717953.5 (PCT/EP98/05285).
[0113] A preferred HPV cervical infection or cancer, prophylaxis or
therapeutic compositions may comprise HPV 16 or 18 antigens. For
example, L1 or L2 antigen monomers, or L1 or L2 antigens presented
together as a virus like particle (VLP) or the L1 alone protein
presented alone in a VLP or caposmer structure. Such antigens,
virus like particles and capsomer are per se known. See for example
WO94/00152, WO94/20137, WO94/05792, and WO93/02184.
[0114] Additional early proteins may be included alone or as fusion
proteins such as E7, E2 or preferably E5 for example; particularly
preferred embodiments of this includes a VLP comprising L1E7 fusion
proteins (WO 96/11272).
[0115] Particularly preferred HPV 16 antigens comprise the early
proteins E6 or E7 in fusion with a protein D carrier to form
Protein D-E6 or E7 fusions from HPV 16, or combinations thereof; or
combinations of E6 or E7 with L2 (WO 96/26277).
[0116] Alternatively the HPV 16 or 18 early proteins E6 and E7, may
be presented in a single molecule, preferably a Protein D-E6/E7
fusion. Such a composition may optionally contain either or both E6
and E7 proteins from HPV 18, preferably in the form of a Protein
D-E6 or Protein D-E7 fusion protein or Protein D E6/E7 fusion
protein.
[0117] The composition of the present invention may additionally
comprise antigens from other HPV strains, preferably from strains
HPV 31 or 33.
[0118] Compositions of the present invention further comprise
antigens derived from parasites that cause Malaria. For example,
preferred antigens from Plasmodia falciparum include
circumsporozoite protein (CS protein), RTS,S MSP1, MSP3, LSA1,
LSA3, AMA1 and TRAP. RTS is a hybrid protein comprising
substantially all the C-terminal portion of the circumsporozoite
(CS) protein of P. falciparum linked via four amino acids of the
preS2 portion of Hepatitis B surface antigen to the surface (S)
antigen of hepatitis B virus. Its full structure is disclosed in
the International Patent Application No. PCT/EP92/02591, published
under Number WO 93/10152 claiming priority from UK patent
application No. 9124390.7. When expressed in yeast RTS is produced
as a lipoprotein particle, and when it is co-expressed with the S
antigen from HBV it produces a mixed particle known as RTS,S. TRAP
antigens are described in the International Patent Application No.
PCT/GB89/00895, published under WO 90/01496. A preferred embodiment
of the present invention is a Malaria vaccine wherein the antigenic
preparation comprises a combination of the RTS,S and TRAP antigens.
Other plasmodia antigens that are likely candidates to be
components of a multistage Malaria vaccine are P. faciparum MSP1,
AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332,
LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27/25, Pfs16,
Pfs48/45, Pfs230 and their analogues in Plasmodium spp. One
embodiment of the present invention is a composition comprising
RTS, S or CS protein or a fragment thereof such as the CS portion
of RTS, S in combination with one or more further malarial antigens
which may be selected for example from the group consisting of
MPS1, MSP3, AMA1, LSA1 or LSA3.
[0119] The compositions may also contain an anti-tumour antigen and
be useful for the immunotherapeutic treatment of cancers. For
example, the antigen may be a tumour rejection antigens such as
those for prostrate, breast, colorectal, lung, pancreatic, renal or
melanoma cancers. Exemplary antigens include MAGE 1, 3 and MAGE 4
or other MAGE antigens such as disclosed in WO99/40188, PRAME,
BAGE, Lage (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or
GAGE (Robbins and Kawakami, 1996, Current Opinions in Immunology 8,
pps 628-636; Van den Eynde et al., International Journal of
Clinical & Laboratory Research (submitted 1997); Correale et
al. (1997), Journal of the National Cancer Institute 89, p293.
Indeed these antigens are expressed in a wide range of tumour types
such as melanoma, lung carcinoma, sarcoma and bladder
carcinoma.
[0120] MAGE antigens for use in the present invention may be
expressed as a fusion protein with an expression enhancer or an
Immunological fusion partner. In particular, the Mage protein may
be fused to Protein D from Heamophilus infuenzae B or a lipidated
derivative thereof. In particular, the fusion partner may comprise
the first 1/3 of Protein D. Such constructs are disclosed in
Wo99/40188.
[0121] Other tumour-specific antigens include, but are not
restricted to KSA (GA733) tumour-specific gangliosides such as GM
2, and GM3 or conjugates thereof to carrier proteins; or said
antigen may be a self peptide hormone such as whole length
Gonadotrophin hormone releasing hormone (GnRH, WO 95/20600), a
short 10 amino acid long peptide, useful in the treatment of many
cancers, or in immunocastration.
[0122] In a preferred embodiment prostate antigens are utilised,
such as Prostate specific antigen (PSA), PAP, PSCA (PNAS 95(4)
1735-1740 1998), PSMA or antigen known as Prostase.
[0123] Prostase is a prostate-specific serine protease
(trypsin-like), 254 amino acid-long, with a conserved serine
protease catalytic triad H-D-S and a amino-terminal pre-propeptide
sequence, indicating a potential secretory function (P. Nelson, Lu
Gan; C. Ferguson, P. Moss, R. Gelinas, L. Hood & K. Wand,
"Molecular cloning and characterisation of prostase, an
androgen-regulated serine protease with prostate restricted
expression, In Proc. Natl. Acad. Sci. USA (1999) 96, 3114-3119). A
putative glycosylation site has been described. The predicted
structure is very similar to other known serine proteases, showing
that the mature polypeptide folds into a single domain. The mature
protein is 224 amino acids-long, with one A2 epitope shown to be
naturally processed.
[0124] Prostase nucleotide sequence and deduced polypeptide
sequence and homologs are disclosed in Ferguson, et al. (Proc.
Natl. Acad. Sci. USA 1999, 96, 3114-3119) and in International
Patent Applications No. WO 98/12302 (and also the corresponding
granted patent U.S. Pat. No. 5,955,306), WO 98/20117 (and also the
corresponding granted patents U.S. Pat. No. 5,840,871 and U.S. Pat.
No. 5,786,148) (prostate-specific kallikrein) and WO 00/04149
(P703P). The present invention provides compositions comprising
prostase protein fusions based on prostase protein and fragments
and homologues thereof ("derivatives"). Such derivatives are
suitable for use in therapeutic vaccine formulations which are
suitable for the treatment of a prostate tumours. Typically the
fragment will contain at least 20, preferably 50, more preferably
100 contiguous amino acids as disclosed in the above referenced
patent and patent applications.
[0125] A further preferred prostate antigen is known as P501S,
sequence ID no 113 of Wo98/37814. Immunogenic fragments and
portions thereof comprising at least 20, preferably 50, more
preferably 100 contiguous amino acids as disclosed in the above
referenced patent application. See for example PS108 (WO 98/50567).
Other prostate specific antigens are known from Wo98/37418, and
WO/004149. Another is STEAP PNAS 96 14523 14528 7-12 1999.
[0126] Other tumour associated antigens useful in the context of
the present invention include: Plu-1 J. Biol. Chem. 274 (22)
15633-15645, 1999, HASH-1, HasH-2, Cripto (Salomon et al Bioessays
199, 21 61-70,U.S. Pat. No. 5,654,140) Criptin U.S. Pat. No.
5,981,215, Additionally, antigens particularly relevant for therapy
of cancer also comprise tyrosinase and survivin.
[0127] Mucin dervied peptides such as Muc1 see for example U.S.
Pat. No. 5,744,144 U.S. Pat. No. 5,827,666 WO 8805054, U.S. Pat.
No. 4,963,484. Specifically contemplated are Muc 1 derived peptides
that comprise at least one repeat unit of the Muc 1 peptide,
preferably at least two such repeats and which is recognised by the
SM3 antibody (U.S. Pat. No. 6,054,438). Other mucin derived
peptides include peptide from Muc 5.
[0128] The antigen of the invention may be a breast cancer antigens
such as her 2/Neu, mammaglobin (U.S. Pat. No. 5,668,267) or those
disclosed in WO/00 52165, WO99/33869, WO99/19479, WO 98/45328. Her
2 neu antigens are disclosed inter alia, in U.S. Pat. No.
5,801,005. Preferably the Her 2 neu comprises the entire
extracellular domain (comprising approximately amino acid 1-645) or
fragments thereof and at least an immunogenic portion of or the
entire intracellular domain approximately the C terminal 580 amino
acids. In particular, the intracellular portion should comprise the
phosphorylation domain or fragments thereof. Such constructs are
disclosed in WO00/44899. A particularly preferred construct is
known as ECD PD a second is known as ECD PD See Wo/00/44899. The
her 2 neu as used herein can be derived from rat, mouse or
human.
[0129] The compositions may contain antigens associated with
tumour-support mechanisms (e.g. angiogenesis, tumour invasion) for
example tie 2, VEGF. It is foreseen that compositions of the
present invention may use antigens derived from Borrelia sp. For
example, antigens may include nucleic acid, pathogen derived
antigen or antigenic preparations, recombinantly produced protein
or peptides, and chimeric fusion proteins. In particular the
antigen is OspA. The OspA may be a full mature protein in a
lipidated form virtue of the host cell (E. Coli) termed (Lipo-OspA)
or a non-lipidated derivative. Such non-lipidated derivatives
include the non-lipidated NS1-OspA fusion protein which has the
first 81 N-terminal amino acids of the non-structural protein (NS1)
of the influenza virus, and the complete OspA protein, and another,
MDP-OspA is a non-lipidated form of OspA carrying 3 additional
N-terminal amino acids.
[0130] Compositions of the present invention may be used for the
prophylaxis or therapy of allergy. Such vaccines would comprise
allergen specific (for example Der p1) and allergen non-specific
antigens (for example peptides derived from human IgE, including
but not restricted to the stanworth decapeptide (EP 0 477 231
B1)).
[0131] Compositions of the present invention may also be used for
the prophylaxis or therapy of chronic disorders others than
allergy, cancer or infectious diseases. Such chronic disorders are
diseases such as atherosclerosis, and Alzheimer.
[0132] The compositions of the present invention are particularly
suited for the immunotherapeutic treatment of diseases, such as
chronic conditions and cancers, but also for the therapy of
persistent infections. Accordingly the compositions of the present
invention are particularly suitable for the immunotherapy of
infectious diseases, such as Tuberculosis (TB), AIDS and Hepatitis
B (HepB) virus infections.
[0133] Also, in the context of AIDS, there is provided a method of
treatment of an individual susceptible to or suffering from AIDS.
The method comprising the administration of a vaccine of the
present invention to the individual, thereby reducing the amount of
CD4+ T-cell decline caused by subsequent HIV infection, or slowing
or halting the CD4+ T-cell decline in an individual already
infected with HIV.
[0134] Other antigens include bacterial (preferably capsular)
saccharides other than (or in addition to) those pneumococcal
antigens described above. Polysaccharide antigens are conveniently
stored in liquid bulk adsorbed onto aluminium phosphate--it is
therefore straightforward to generate vaccine compositions of the
invention by admixing said liquid bulk with the oil emulsions of
the invention extemporaneously. Preferably the other bacterial
saccharides are selected from a group consisting of: N.
meningitidis serogroup A capsular saccharide (MenA), N.
meningitidis serogroup C capsular saccharide (MenC), N.
meningitidis serogroup Y capsular saccharide (MenY), N.
meningitidis serogroup W-135 capsular saccharide (MenW), Group B
Streptococcus group I capsular saccharide, Group B Streptococcus
group II capsular saccharide, Group B Streptococcus group III
capsular saccharide, Group B Streptococcus group IV capsular
saccharide, Group B Streptococcus group V capsular saccharide,
Staphylococcus aureus type 5 capsular saccharide, Staphylococcus
aureus type 8 capsular saccharide, Vi saccharide from Salmonella
typhi, N. meningitidis LPS, M. catarrhalis LPS, and H. influenzae
LPS. By LPS it is meant either native lipo-polysaccharide (or
lipo-oligosaccharide), or lipo-polysaccharide where the lipid A
portion has been detoxified by any of a number of known methods
(see for example WO 97/18837 or WO 98/33923), or any molecule
comprising the O-polysaccharide derived from said LPS. By N.
meningitidis LPS it is meant one or more of the 12 known
immunotypes (L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11 or
L12).
[0135] Particularly preferred combinations are compositions
comprising: 1) conjugated Hib, conjugated MenA and conjugated MenC;
2) conjugated Hib, conjugated MenY and conjugated MenC; 3)
conjugated Hib and conjugated MenC; and 4) conjugated MenA,
conjugated MenC, conjugated MenY and conjugated MenW-135. The
amount of PS in each of the above conjugates may be 5 or 10
.quadrature.g each per 0.5 mL human dose. Preferably Hib, MenA,
MenC, MenW-135 and MenY are TT conjugates.
[0136] A problem associated with the polysaccharide approach to
vaccination, is the fact that polysaccharides per se are poor
immunogens. To overcome this, saccharides of the invention may be
conjugated to protein carriers, which provide bystander T-cell
help. It is preferred, therefore, that the saccharides utilised in
the invention are linked to such a protein carrier. Examples of
such carriers which are currently commonly used for the production
of saccharide immunogens include the Diphtheria and Tetanus toxoids
(DT, DT CRM197 and TT respectively), Keyhole Limpet Haemocyanin
(KLH), protein D from Haemophilus influenzae (EP 594610-B), OMPC
from N. meningitidis, and the purified protein derivative of
Tuberculin (PPD).
[0137] The saccharide may be linked to the carrier protein by any
known method (for example, by Likhite, U.S. Pat. No. 4,372,945 and
by Armor et al., U.S. Pat. No. 4,474,757). Preferably, CDAP
conjugation is carried out (WO 95/08348).
[0138] Preferably the protein:saccharide (weight:weight) ratio of
the conjugates is 0.3:1 to 1:1, more preferably 0.6:1 to 0.8:1, and
most preferably about 0.7:1.
[0139] Combinations of antigens which provide protection against
pneumococcus and a different pathogen are included in the present
invention. Many Paediatric vaccines are now given as a combination
vaccine so as to reduce the number of injections a child has to
receive. Thus for Paediatric vaccines other antigens from other
pathogens may be formulated with the pneumococcal vaccines of the
invention. For example the vaccines of the invention can be
formulated with (or administered separately but at the same time)
the well known `trivalent` combination vaccine comprising
Diphtheria toxoid (DT), tetanus toxoid (TT), and pertussis
components [typically detoxified Pertussis toxoid (PT) and
filamentous haemagglutinin (FHA) with optional pertactin (PRN)
and/or agglutinin 1+2], for example the marketed vaccine
INFANRIX-DTPa.TM. (SmithKlineBeecham Biologicals) which contains
DT, TT, PT, FHA and PRN antigens, or with a whole cell pertussis
component for example as marketed by SmithKlineBeecham Biologicals
s.a., as Tritanrix.TM.. The combined vaccine may also comprise
other antigen, such as Hepatitis B surface antigen (HBsAg), Polio
virus antigens (for instance inactivated trivalent polio
virus--IPV), Moraxella catarrhalis outer membrane proteins,
non-typeable Haemophilus influenzae proteins, N. meningitidis B
outer membrane proteins.
[0140] Examples of preferred Moraxella catarrhalis protein antigens
which can be included in a combination vaccine (especially for the
prevention of otitis media) are: OMP106 [WO 97/41731 (Antex) &
WO 96/34960 (PMC)]; OMP21; LbpA &/or LbpB [WO 98/55606 (PMC)];
TbpA &/or TbpB [WO 97/13785 & WO 97/32980 (PMC)]; CopB
[Helminen M E, et al. (1993) Infect. Immun. 61:2003-2010]; UspA1
and/or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR
(PCT/EP99/03824); PilQ (PCT/EP99/03823); OMP85 (PCT/EP00/01468);
lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11 (GB
9918302.2); lipo18 (GB 9918038.2); P6 (PCT/EP99/03038); D15
(PCT/EP99/03822); OmplA1 (PCT/EP99/06781); Hly3 (PCT/EP99/03257);
and OmpE. Examples of non-typeable Haemophilus influenzae antigens
which can be included in a combination vaccine (especially for the
prevention of otitis media) include: Fimbrin protein [(U.S. Pat.
No. 5,766,608--Ohio State Research Foundation)] and fusions
comprising peptides therefrom [eg LB1 (f) peptide fusions; U.S.
Pat. No. 5,843,464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638
(Cortecs)]; P6 [EP 281673 (State University of New York)]; TbpA
and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15
(WO 94/12641); protein D (EP 594610); P2; and P5 (WO 94/26304).
[0141] Other combinations contemplated are the pneumococcal
saccharide & protein of the invention in combination with viral
antigens, for example, from influenza (attenuated, split, or
subunit [e.g., surface glycoproteins neuraminidase (NA) and
haemagglutinin (HA). See, e.g., Chaloupka I. et al, Eur. Journal
Clin. Microbiol. Infect. Dis. 1996, 15:121-127], RSV (e.g., F and G
antigens or F/G fusions, see, eg, Schmidt A. C. et al, J Virol, May
2001, p4594-4603), PIV3 (e.g., HN and F proteins, see Schmidt et
al. supra), Varicella (e.g., attenuated, glycoproteins I-V, etc.),
and any (or all) component(s) of MMR (measles, mumps, rubella).
[0142] A preferred Paediatric combination vaccine contemplated by
the present invention for global treatment or prevention of otitis
media comprises: one or more Streptococcus pneumoniae saccharide
antigen(s) (preferably conjugated to protein D), one or more
pneumococcal proteins (preferably those described above), and one
or more surface-exposed antigen from Moraxella catarrhalis and/or
non-typeable Haemophilus influenzae. Protein D can advantageously
be used as a protein carrier for the pneumococcal saccharides (as
mentioned above), and because it is in itself an immunogen capable
of producing B-cell mediated protection against non-typeable H.
influenzae (ntHi). The Moraxella catarrhalis or non-typeable
Haemophilus influenzae antigens can be included in the vaccine in a
sub-unit form, or may be added as antigens present on the surface
of outer membrane vesicles (blebs) made from the bacteria.
Preferred Antigens
[0143] As mentioned above, in one aspect the invention relates to
use of a composition comprising: [0144] (a) an antigen, and [0145]
(d) an oil-in-water emulsion adjuvant; and [0146] (e) 3D MPL
wherein said oil-in-water emulsion comprises a metabolisable oil, a
sterol and an emulsifying agent, in the manufacture of an
immunogenic composition for the prevention of infection and/or
disease.
[0147] The composition of the invention is thus used for infections
and/or diseases which are capable of being prevented or ameliorated
by that composition, and suitably in which the antigen is derived
from or associated with a pathogen (such as a bacteria or virus)
which is associated with the disease.
[0148] Antigens from influenza virus A and B, HPV antigens, RSV A
and B, SARS, streptococcus, VZV, rhinovirus, parainfluenza virus
are preferred for use in the present invention, such as split
influenza, VZV gE, VZV IE63, and PhtD from Streptococcus pneumonia.
However, any suitable antigen may be used.
[0149] In one embodiment the composition used in the invention does
not comprise an influenza subunit antigen with the MF59.TM.
adjuvant.
[0150] For all aspects of the invention it is preferred that
antigens comprise a CD4 T cell epitope, or a B cell epitope or
suitably both.
Influenza Viral Strains and Antigens
[0151] 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.
[0152] 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.
[0153] In another embodiment, the influenza virus preparation is in
the form of a purified sub-unit influenza. 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.
[0154] 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.
[0155] Said influenza virus or antigenic preparation thereof may be
egg-derived or tissue-culture derived.
[0156] 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.
[0157] The influenza virus antigen or antigenic preparation thereof
may be produced by any of a number of commercially applicable
processes, for example the split flu process described in patent
no. DD 300 833 and DD 211 444, incorporated herein by reference.
Traditionally split flu was produced using a solvent/detergent
treatment, such as tri-n-butyl phosphate, or diethylether in
combination with Tween.TM. (known as "Tween-ether" splitting) and
this process is still used in some production facilities. Other
splitting agents now employed include detergents or proteolytic
enzymes or bile salts, for example sodium deoxycholate as described
in patent no. DD 155 875, incorporated herein by reference.
Detergents that can be used as splitting agents include cationic
detergents e.g. cetyl trimethyl ammonium bromide (CTAB), other
ionic detergents e.g. laurylsulfate, taurodeoxycholate, or
non-ionic detergents such as the ones described above including
Triton X-100 (for example in a process described in Lina et al,
2000, Biologicals 28, 95-103) and Triton N-101, or combinations of
any two or more detergents.
[0158] 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.
[0159] Preferred split flu vaccine antigen preparations according
to the invention comprise a residual amount of Tween 80 and/or
Triton X-100 remaining from the production process, although these
may be added or their concentrations adjusted after preparation of
the split antigen. Preferably both Tween 80 and Triton X-100 are
present. The preferred ranges for the final concentrations of these
non-ionic surfactants in the vaccine dose are:
Tween 80:0.01 to 1%, more preferably about 0.1% (v/v) Triton
X-100:0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02%
(w/v).
[0160] In a specific embodiment, the final concentration for Tween
80 ranges from 0.045%-0.09% w/v. In another specific embodiment,
the antigen is provided as a 2 fold concentrated mixture, which has
a Tween 80 concentration ranging from 0.045%-0.2% (w/v) and has to
be diluted two times upon final formulation with the adjuvanted (or
the buffer in the control formulation).
[0161] In another specific embodiment, the final concentration for
Triton X-100 ranges from 0.005%-0.017% w/v. In another specific
embodiment, the antigen is provided as a 2 fold concentrated
mixture, which has a Triton X-100 concentration ranging from
0.005%-0.034% (w/v) and has to be diluted two times upon final
formulation with the adjuvanted (or the buffer in the control
formulation).
[0162] 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.
[0163] A preferred composition contains three inactivated split
virion antigens prepared from the WHO recommended strains of the
appropriate influenza season.
[0164] 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 (335111) (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.
[0165] Preferred compositions of the invention comprise antigens
having CD4 T cell epitopes and optionally B cell epitopes.
HPV Antigens
[0166] In another aspect of the present invention compositions
contain an antigen derived from the Human Papilloma Virus (HPV),
for example from a virus considered to be responsible for genital
warts (HPV 6 or HPV 11 and others), or the HPV viruses responsible
for cervical cancer (HPV16, HPV18 and others). In one aspect
prophylactic or therapeutic compositions comprise HPV 16 or 18
antigens. Infection by HPV 16 and HPV 18 is related to development
of cancer.
[0167] Combinations of antigens from different HPV genotypes may be
employed in the invention, such as a combination of HPV 16 and HPV
18 antigens, suitably in the form of VLP. Antigens from additional
VLP types that may be included with 16 and/or 18 include antigens
from other known cancer-causing types such as HPV 31, 45, 33, 58
and 52.
[0168] In one aspect the HPV antigens are L1 or L2 antigen
monomers. In one aspect the invention relates to a combination of
HPV L1 and L2 antigens from the same genotype presented together as
a capsomer or virus like particle (VLP). In one aspect the HPV
antigen is an L1 protein (absent an L2 antigen) in the form of a
VLP or capsomer structure. Such antigens, virus like particles and
capsomer are per se known. See, for example, WO94/00152,
WO94/20137, WO94/05792, and WO93/02184.
[0169] In one aspect a truncated L1 protein may be used in the
invention, for example as disclosed in WO 96/11272. Preferably a
C-terminal truncation of L1 is used, for example a 34 amino acid
C-terminal truncation of HPV 16, or an equivalent truncation from
other HPV type.
[0170] In one aspect of the invention a composition comprises a
combination of HPV virus like particles or capsomers from HPV 16
and HPV 18, together with HPV 31 and/or HPV 45. In one aspect of
the invention a composition comprises a combination of HPV virus
like particles or capsomers from HPV 16 and HPV 18, together with
HPV 33 and/or HPV 58. In one aspect of the invention a composition
comprises a combination of HPV virus like particles or capsomers
from HPV 16 and HPV 18, together with VLPs or capsomers from one or
more cancer causing HPV types, such as one, two, three, four or all
of HPV 31, 33, 45, 52, and 58.
[0171] The invention thus relates in one aspect to a composition
comprising virus like particles from HPV 16, 18, 31 and 45 in
combination with an adjuvant comprising 3D MPL and an oil in water
emulsion as described herein.
[0172] In one aspect of the invention a composition comprises a
mixture of HPV 16, 18, 31, 33, 45, 52, and 58 L1-only virus like
particles or capsomers. L1 or L2 proteins may be provided in the
form of fusion proteins.
[0173] Particularly suitable forms of genital wart prophylactic, or
therapeutic, compositions comprise L1 particles or capsomers, and
fusion proteins comprising one or more antigens selected from the
HPV 6 and HPV 11 proteins, for example E6, E7, L1, and L2.
[0174] HPV antigens from cancer types may be combined with antigens
from genital warts types, such as HPV 16 and/or 18 with HPV 6
and/or 11. For example, a composition comprising HPV 16, 18, 6 and
11 is contemplated. In one aspect of the invention a combination of
HPV 16, 18, 31, 33, 45, 52, and 58 L1-only virus like particle or
capsomers may be used in combination with virus like particles or
capsomers from HPV 6 and/or HPV 11. In one aspect early proteins
such as E7, E2 or E5 for example may be included alone, in
combinations, or may be fusion proteins; an embodiment of this
includes a VLP comprising L1E7 fusion proteins (WO 96/11272).
[0175] In one aspect the fusion protein is L2E7 as disclosed in WO
96/26277, or proteinD (1/3)-E7 disclosed in GB 9717953.5
(PCT/EP98/05285).
[0176] In one aspect HPV 16 antigens comprise the early proteins E6
or E7 in fusion with a protein D carrier to form Protein D-E6 or E7
fusions from HPV 16, or combinations thereof; or combinations of E6
or E7 with L2 (WO 96/26277).
[0177] Alternatively the HPV 16 or 18 early proteins E6 and E7, may
be presented in a single molecule, such as a Protein D-E6/E7
fusion. Such a composition may optionally contain either or both E6
and E7 proteins from HPV 18, such as in the form of a Protein D-E6
or Protein D-E7 fusion protein or Protein D E6/E7 fusion
protein.
Oil-in-Water Emulsion Adjuvant Component
[0178] 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.
[0179] 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).
[0180] 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).
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] The oil in water emulsion 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.
[0186] Suitably the sterol is alpha-tocopherol or a derivative
thereof such as alpha-tocopherol succinate (also known as vitamin E
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.
[0187] The oil in water emulsion 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.
[0188] The emulsifying agent may suitably be polyoxyethylene
sorbitan monooleate (Tween 80). In a specific embodiment, a 0.5 ml
vaccine dose volume contains 1% (w/w) Tween 80, and a 0.7 ml
vaccine dose volume contains 0.7% (w/w) Tween 80. In another
specific embodiment the concentration of Tween 80 is 0.2%
(w/w).
[0189] 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 80. The oil in water emulsion may also contain span 85
and/or Lecithin. Typically the oil in water will comprise from 2 to
10% squalene of the total volume of the immunogenic composition,
from 2 to 10% alpha tocopherol and from 0.3 to 3% Tween 80, and may
be produced according to the procedure described in WO 95/17210.
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%.
3D-MPL
[0190] The composition comprise an additional adjuvant, 3
de-O-acylated monophosphoryl lipid A (3D-MPL). 3D MPL is a TRL-4
ligand adjuvant, a non-toxic derivative of lipid A.
[0191] 3D-MPL is sold under the trademark MPL.RTM. by Corixa
corporation and is referred throughout the document as MPL. It
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 WO 94/21292 and in Example
II.
[0192] 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.
[0193] 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.
[0194] 3D MPL is a TRL-4 ligand, a non-toxic derivative of lipid A.
The present invention contemplates use of other suitable TLR-4
ligands in place of 3D-MPL, including lipopolysaccharide (LPS) and
derivatives, MDP (muramyl dipeptide) and F protein of RSV.
Non-toxic derivatives of lipid A, particularly monophosphoryl lipid
A, are also contemplated.
[0195] Synthetic derivatives of lipid A are known, some being
described as TLR-4 agonists, which might be suitable for use in the
present invention and include, but are not limited to: [0196] OM174
(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phos-
phono-.beta.-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.alpha.-
-D-glucopyranosyldihydrogenphosphate), (WO 95/14026) [0197] OM 294
DP (3S, 9
R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydr-
oxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)
(WO99/64301 and WO 00/0462) [0198] OM 197 MP-Ac DP (3S-,
9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxyt-
etradecanoylamino]decan-1,10-diol, 1-dihydrogenophosphate
10-(6-aminohexanoate) (WO 01/46127)
Immunogenic Properties of the Immunogenic Composition Used for the
First Vaccination of the Present Invention
[0199] The composition of the invention suitably induces 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 is un-adjuvanted, i.e. does not contain any exogeneous
adjuvant (herein also referred to as `plain composition`) or at
least lacks one of both components (either 3D-MPL or the
oil-in-water emulsion adjuvant) of the adjuvant composition.
[0200] 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 or lacking one of the two components of the adjuvant
composition. For example, a higher CD4 T-cell response is obtained
in a human patient upon administration of an immunogenic
composition comprising a split influenza virus or split virus
antigenic preparation thereof together with an oil-in-water
emulsion adjuvant as herein defined, compared to the response
induced after administration of an immunogenic composition
comprising a split influenza virus or split virus antigenic
preparation thereof. 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.
[0201] For influenza, preferably said immunological response
induced by an adjuvanted split influenza composition of 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.
[0202] In particular, but not exclusively, said `improved CD4
T-cell immune response` is suitably obtained in an immunologically
"unprimed" patient, i.e. a patient who is seronegative to the
antigen. This seronegativity may be the result of said patient
having never faced such an antigen (for example, not having been
infected with a virus or bacteria containing said antigen--a
so-called `naive` patient) or, alternatively, having failed to
respond to said antigen once encountered.
[0203] An improved CD4 T-cell immune response may be assessed by
measuring the number of cells producing any of the following
cytokines: [0204] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0205] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0206] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0207] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0208] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
[0209] 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.
[0210] An improved CD4 T-cell immune response conferred by an
adjuvanted 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 a disease eg
influenza, 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.
[0211] Thus, the present invention also relates to use of a
composition comprising: [0212] (a) an antigen, and [0213] (f) an
oil-in-water emulsion adjuvant; and [0214] (g) 3D MPL wherein said
oil-in-water emulsion comprises a metabolisable oil, a sterol and
an emulsifying agent, in the manufacture of an immunogenic
composition for inducing an improved CD4 T-cell response against
said antigen.
[0215] The invention also relates to a method of inducing an
improved CD4 T-cell response against an antigen comprising delivery
of a composition comprising: [0216] (a) an antigen, and [0217] (b)
an oil-in-water emulsion adjuvant as herein defined; and [0218] (c)
3D-MPL.
[0219] 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 or with a composition lacking one of the two components
of the adjuvant 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).
[0220] In a still further specific embodiment, the vaccination with
the composition for the first vaccination, adjuvanted, has no
measurable impact on the CD8 response.
[0221] Preferably said improved CD4 T-cell immune response is
obtained in an immunocompromised subject such as an elderly
individual, typically at least 50, 55, 60 or 65 years of age or
above, or an adult younger than 55 years of age with a high risk
medical condition (`high risk` adult), or a child under the age of
two.
[0222] The Applicants have surprisingly found that an oil-in-water
emulsion adjuvant comprising a metabolisable oil, a sterol and an
emulsifying agent, and 3D MPL is effective in promoting T cell
responses in an immuno-compromised human population. As the
Applicants have demonstrated for influenza, 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 re-vaccination against influenza in a
human elderly population, than does the vaccination with an
un-adjuvanted split 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 (eg 50, 55, 60, 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 to induce 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.
[0223] 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).
[0224] 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.
[0225] These findings with influenza are applicable to other
diseases and antigens as it has also been demonstrated for HPV
antigens (see Example XIII).
[0226] Thus, the present invention also relates to the use of the
composition of the invention in an immunocompromised human
individual or population such as high risk adults or elderly, and
in the manufacture of an immunogenic composition for vaccination of
a human immuno-compromised individual or population, such as a high
risk adult or a elderly population.
[0227] We have determined that use of an antigen with an oil in
water emulsion as herein defined can generate a cross reactive
response against a variant antigen.
[0228] 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.
[0229] For influenza, for example, available influenza vaccines are
usually effective only against infecting strains of influenza virus
that have haemaglutinins 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.
[0230] 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.
[0231] In another aspect of the present invention, there is
provided the use of the composition of the invention for protection
against infections or disease caused by a pathogen which is a
variant of the pathogen from which the antigen in the first
composition is derived. Also provided issue of the composition of
the invention for protection against infection or disease caused by
a pathogen which comprises an antigen which is a variant of that in
the composition of the invention.
[0232] Variant pathogens and/or antigens suitably have antigens
with common CD4 T cell epitopes and/or B cell epitopes with the
first pathogen or antigen, but which are not identical.
Detection of Cross-Reactive CD4 T-Cells (eg Following Vaccination
with Influenza Vaccine)
[0233] 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 Calcdonia/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).
[0234] 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.
[0235] CD4 T-cells that are able to recognize both homologous and
drifted Influenza strains have been named in the present document
"cross-reactive".
[0236] 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).
[0237] 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
[0238] The composition may comprise additional adjuvants, suitably
such as TRL-4 ligand adjuvants or a non-toxic derivative of lipid
A. Suitable TLR-4 ligands are lipopolysaccharide (LPS) and
derivatives, MDP (muramyl dipeptide) and F protein of RSV.
[0239] Synthetic derivatives of lipid A are known, some being
described as TLR-4 agonists, and include, but are not limited to:
[0240] OM174
(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-O--
phosphono-.beta.-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.a-
lpha.-D-glucopyranosyldihydrogenphosphate), (WO 95/14026) [0241] OM
294 DP (3S, 9
R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-
-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)
(WO99/64301 and WO 00/0462) [0242] OM 197 MP-Ac DP (3S--,
9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxyt-
etradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate
10-(6-aminohexanoate) (WO 01/46127)
[0243] Other suitable TLR-4 ligands are, for example,
lipopolysaccharide and its derivatives, muramyl dipeptide (MDP) or
F protein of respiratory syncitial virus.
[0244] 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, p243-254) 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.
[0245] 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
[0246] An aspect of the present invention provides the use of an
antigen in the manufacture of an immunogenic composition for
revaccination of humans previously vaccinated with the antigen or
fragment or variant thereof with 3D MPL and an oil-in-water
emulsion adjuvant as herein defined.
[0247] 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.
[0248] Preferably, there is provided the use of:
(1) an antigen and (2) an oil-in-water emulsion adjuvant in the
manufacture of an immunogenic composition for revaccination of
humans previously vaccinated with the antigen, or fragment or
variant thereof, 3D MPL and an oil-in-water emulsion adjuvant as
herein defined.
[0249] Preferably, there is provided the use of:
(1) an antigen and (2) an oil-in-water emulsion adjuvant, and
(3) 3D MPL
[0250] in the manufacture of an immunogenic composition for
revaccination of humans previously vaccinated with the antigen, or
fragment or variant thereof, 3D MPL and an oil-in-water emulsion
adjuvant as herein defined.
[0251] In a preferred embodiment, the invention provides for the
use of: [0252] (a) an antigen, and [0253] (b) an oil-in-water
emulsion adjuvant, and [0254] (c) 3D MPL
[0255] in the manufacture of an immunogenic composition for
revaccination of humans previously vaccinated with said antigen or
fragment or variant thereof.
[0256] In a specific embodiment, the composition for revaccination
suitably shares common CD4 T-cell epitopes with the composition
used for the first vaccination. In this respect it is a considered
a variant of the antigen used in first vaccination.
[0257] The immunogenic composition for re-vaccination (the boosting
composition) may contain the same type of antigen preparation--eg
subunit/split/whole inactivated virus--as the immonogenic
composition used for the first vaccination. Alternatively the
boosting composition may contain another type of antigen
preparation, For example, for influenza the first vaccination may
be with a split preparation and the booster vaccination with
another inactivated influenza antigen, such as inactivated whole
virus or purified HA and NA (subunit vaccine). The booster
composition may be adjuvanted or un-adjuvanted. For influenza the
un-adjuvanted booster composition may be
Fluarix.TM./.alpha.-Rix.RTM. given intramuscularly. The formulation
contains three inactivated split virion antigens prepared from the
WHO recommended strains of the appropriate influenza season.
[0258] A variant may be an antigen which shares common CD4 T-cell
epitopes (generally considered as antigenic determinants recognized
and bound by the T-cell receptor) with the antigenic composition
used for the first vaccination, but which is not identical to that
antigenic composition.
[0259] A variant may be an antigen which shares common B-cell
epitopes (generally considered as antigenic determinants recognized
and bound by the B-cell receptor) with the antigenic composition
used for the first vaccination, but which is not identical to that
antigenic composition.
[0260] T cell and B cell epitopes may be predicted using techniques
well known in the art or inferred from immune responses using
techniques as described herein.
[0261] 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.
[0262] In a specific embodiment for influenza, the immunogenic
composition for revaccination (also called herein below the
`boosting composition`) contains a split influenza virus or split
virus antigenic preparation thereof which shares a common CD4
T-cell epitope with the split influenza virus or split virus
antigenic preparation thereof used for the first vaccination.
[0263] Generally, for all antigens, 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).
[0264] In the context of influenza, a preferred embodiment, the
influenza strain may be associated with a pandemic outbreak or have
the potential to be associated with a pandemic outbreak. In
particular, when the vaccine is a multivalent vaccine such as a
bivalent or a trivalent 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.
[0265] Typically a booster composition, where used, is given at the
next season, e.g. approximately one year after the first
immunogenic composition. The booster composition may also be given
every subsequent year (third, fourth, fifth vaccination and so
forth). The boosting composition may be the same as the first
composition. Suitably, the boosting composition contains an strain
or antigenic preparation therefrom which is a variant of the strain
or antigen used for the first vaccination.
[0266] 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 for example.
[0267] For all antigens of the invention re-vaccination suitably
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
split influenza virus or split virus antigenic preparation thereof.
Preferably the immunological responses induced after re-vaccination
with the adjuvanted split influenza virus or split virus antigenic
preparation thereof as herein defined, are higher than the
corresponding response induced after the re-vaccination with the
un-adjuvanted composition. Preferably the immunological responses
induced after re-vaccination with an un-adjuvanted, preferably
split, influenza virus are higher in the population first
vaccinated with the adjuvanted split influenza composition than the
corresponding response in the population first vaccinated with the
un-adjuvanted split influenza composition.
[0268] In another aspect of the present invention, there is
provided the use of: [0269] (a) an antigen from a first viral or
bacterial strain and [0270] (b) an oil-in-water emulsion adjuvant
[0271] (c) 3D MPL in the manufacture of an immunogenic composition
for protection against infections or disease caused by a viral or
bacterial strain which is a variant of said first strain.
[0272] For example, for influenza, the adjuvanted composition of
the invention is capable of inducing a better protection against
drifted strain (the influenza strain from the next influenza
season) compared to the protection conferred by the control
vaccine.
Influenza Viral Strains and Antigens Thereof
[0273] For compositions comprising a split influenza virus or split
virus antigenic preparation thereof the composition is suitably
monovalent or multivalent such as bivalent or trivalent or
quadrivalent. Preferably the split influenza virus or split virus
antigenic preparation thereof is trivalent or quadrivalent, having
an antigen from three different influenza strains.
[0274] Optionally at least one strain is associated with a pandemic
outbreak or has the potential to be associated with a pandemic
outbreak.
[0275] 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.
[0276] 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
(Hong-Kong Flu pandemic) by the emergence of the H3N2 influenza
subtype, after which the N2 NA continued to drift along with the H3
HA (Nakajima et al., 1991, Epidemiol. Infect. 106, 383-395).
[0277] 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.
[0278] 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] In a further embodiment the invention relates to a
vaccination regime in which the first vaccination is made with a
split influenza composition containing at least one influenza
strain that could potentially cause a pandemic outbreak and the
re-vaccination is made with a circulating strain, either a pandemic
strain or a classical strain.
CD4 Epitope in HA
[0283] 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.
[0284] 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).
[0285] In a specific embodiment, the re-vaccination is made by
using a booster composition which contain an influenza virus or
antigenic preparation thereof which shares common CD4 T-cell
epitopes with the split influenza virus antigen or split virus
antigenic preparation thereof used for the first vaccination.
[0286] The invention thus relates to the use of the immunogenic
composition comprising a split influenza virus or split virus
antigenic preparation thereof, an oil-in-water emulsion adjuvant as
herein defined and 3D MPL 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 split influenza virus antigen or split virus
antigenic preparation thereof of the dose given at the first
vaccination.
Vaccination
[0287] 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.
[0288] The intramuscular delivery route is preferred for the
adjuvanted influenza composition.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] Preferred devices for intranasal administration of the
vaccines according to the invention are spray devices. Suitable
commercially available nasal spray devices include Accuspray.TM.
(Becton Dickinson). Nebulisers produce a very fine spray which can
be easily inhaled into the lungs and therefore does not efficiently
reach the nasal mucosa. Nebulisers are therefore not preferred.
[0293] 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.
[0294] 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.
[0295] 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.
[0296] Alternatively, the epidermal or transdermal vaccination
route is also contemplated in the present invention.
[0297] In a specific aspect of the present invention, the
adjuvanted immunogenic composition for the first administration may
be given intramuscularly, and the booster 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 booster 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
[0298] The target population is preferably a human population or
individual.
[0299] 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.
[0300] 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
infection or vaccination. Preferably the target population is
elderly persons suitably aged 55 years and over, younger high-risk
adults (i.e. between 18 and 54 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
[0301] The amount of saccharide (or conjugate thereof) antigen in
each vaccine dose is selected as an amount which induces an
immunoprotective response without significant, adverse side effects
in typical vaccines. Such amount will vary depending upon which
specific immunogen is employed and how it is presented. Generally,
it is expected that each dose will comprise 0.1-100 .mu.g of
saccharide, preferably 0.1-50 .mu.g, preferably 0.1-10 .mu.g, of
which 1 to 5 .mu.g is the most preferable range.
[0302] The content of protein antigens in the vaccine will
typically be in the range 1-100 .mu.g, preferably 5-50 .mu.g, most
typically in the range 5-25 .mu.g.
[0303] Optimal amounts of components for a particular vaccine can
be ascertained by standard studies involving observation of
appropriate immune responses in subjects. Following an initial
vaccination, subjects may receive one or several booster
immunisations adequately spaced.
[0304] Vaccine preparation is generally described in Vaccine Design
("The subunit and adjuvant approach" (eds Powell M. F. & Newman
M. J.) (1995) Plenum Press New York). Encapsulation within
liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
Influenza Antigen Dose
[0305] For influenza, 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] The influenza medicament of the invention preferably meets
certain international criteria for vaccines.
[0310] 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.
[0311] 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.
[0312] 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 [0313] 1) selecting an
antigen containing CD4+ epitopes, and [0314] 2) combining said
antigen with an oil-in-water emulsion adjuvant as defined herein
above with 3D MPL, wherein said vaccine upon administration in said
mammal is capable of inducing an enhanced CD4 T cell response in
said mammal.
[0315] The teaching of all references in the present application,
including patent applications and granted patents, are herein fully
incorporated by reference.
[0316] 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.
[0317] The invention will be further described by reference to the
following, non-limiting, examples:
[0318] Example I describes immunological read-out methods used in
mice, ferret and human studies.
[0319] Example II describes the preparation and characterization of
the oil in water emulsion and adjuvant formulations used in the
studies exemplified.
[0320] 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
[0321] 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.
[0322] 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.
[0323] Example VI shows a pre-clinical evaluation of adjuvanted and
un-adjuvanted influenza vaccines in C57BI/6 naive and primed
mice.
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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.
[0330] Example XIII shows a pre-clinical evaluation of two
adjuvanted HPV vaccines in mice. Antibody and B cell memory
responses were measured.
EXAMPLE I
Immunological Read-out Methods
[0331] I.1. Mice methods
I.1.1. Hemagglutination Inhibition Test
Test Procedure
[0332] 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
[0333] Statistical analysis were performed on post vaccination HI
titers using UNISTAT. The protocol applied for analysis of variance
can be briefly described as follow: [0334] Log transformation of
data [0335] Shapiro-Wilk test on each population (group) in order
to verify the normality of groups distribution [0336] Cochran test
in order to verify the homogenicity of variance between the
different populations (groups) [0337] Two-way Analysis of variance
performed on groups [0338] Tukey HSD test for multiple
comparisons
I.1.2. Intracellular Cytokine Staining
[0339] 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.
[0340] 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.
[0341] 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 Fl (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).
[0342] 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.
[0343] 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.
I.2. Ferrets Methods
I.2.1. Hemagglutination Inhibition Test (HI)
Test Procedure.
[0344] 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.
[0345] 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: [0346] Log
transformation of data. [0347] Shapiro-wilk test on each population
(group) in order to verify the normality of groups distribution.
[0348] Cochran test in order to verify the homogeneity of variance
between the different populations (groups). [0349] Test for
interaction of one-way ANOVA. [0350] Tukey-HSD Test for multiple
comparisons.
I.2.2. Body Temperature Monitoring
[0351] 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.
[0352] Temperatures were recorded every 15 minutes 4 days before
challenge until 7 days Post-challenge.
I.2.3. Nasal Washes
[0353] 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
[0354] 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.
[0355] 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.
[0356] 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
[0357] 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).
[0358] 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.
[0359] 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. 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
[0360] 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
[0361] 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)
[0362] 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
I.3.5.1. Primary Endpoints
[0363] 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. [0364] 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.
[0365] Occurrence of serious adverse events during the entire
study.
I.3.5.2. Secondary Endpoints
For the Humoral Immune Response:
Observed Variables:
[0365] [0366] 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). [0367] 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): [0368] Geometric
mean titres (GMTs) of serum HI antibodies with 95% confidence
intervals (95% CI) pre and post-vaccination [0369] Seroconversion
rates* with 95% CI at day 21 [0370] Conversion factors** with 95%
CI at day 21 [0371] Seroprotection rates*** with 95% CI at day 21
[0372] Serum NI antibody GMTs' (with 95% confidence intervals) at
all timepoints. *Seroconversion rate defined as the percentage of
vaccinees who have at least a 4-fold increase in serum HI titres on
day 21 compared to day 0, for each vaccine strain. **Conversion
factor defined as the fold increase in serum HI GMTs on day 21
compared to day 0, for each vaccine strain. ***Protection rate
defined as the percentage of vaccinees with a serum HI titre=40
after vaccination (for each vaccine strain) that usually is
accepted as indicating protection.
For the Cell Mediated Immune (CMI) Response
Observed Variable
[0373] 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: [0374] Peptide Influenza (pf)
antigen (the precise nature and origin of these antigens needs to
be given/explained [0375] Split Influenza (sf) antigen [0376] Whole
Influenza (wf) antigen.
Derived Variables:
[0376] [0377] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0378] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0379] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0380] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0381] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
I.3.5.3. Analysis of Immunogenicity
[0382] The immunogenicity analysis was based on the total
vaccinated cohort. For each treatment group, the following
parameters (with 95% confidence intervals) were calculated: [0383]
Geometric mean titres (GMTs) of HI and NI antibody titres at days 0
and 21 [0384] Geometric mean titres (GMTs) of neutralising antibody
titres at days 0 and 21. [0385] Conversion factors at day 21.
[0386] 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. [0387] Protection
rates at day 21 defined as the percentage of vaccinees with a serum
HI titre=1:40. [0388] 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)). [0389] 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. [0390] 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
[0391] Unless otherwise stated, the oil/water emulsion used in the
subsequent examples is composed an organic phase made of 2 oils
(alpha-tocopherol and squalene), and an aqueous phase of PBS
containing Tween 80 as emulsifying agent. Unless otherwise stated,
the oil in water emulsion adjuvant formulations used in the
subsequent examples were made comprising the following oil in water
emulsion component (final concentrations given): 2.5% squalene
(v/v), 2.5% alpha-tocopherol (v/v), 0.9% polyoxyethylene sorbitan
monooleate (v/v) (Tween 80), see WO 95/17210. This emulsion, termed
AS03 in the subsequent examples, was prepared as followed as a
two-fold concentrate.
II.1. Preparation of Emulsion SB62
II.1.1. Lab-Scale Preparation
[0392] Tween 80 is dissolved in phosphate buffered saline (PBS) to
give a 2% solution in the PBS. To provide 100 ml two-fold
concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of
squalene are vortexed to mix thoroughly. 90 ml of PBS/Tween
solution is added and mixed thoroughly. The resulting emulsion is
then passed through a syringe and finally microfluidised by using
an M110S microfluidics machine. The resulting oil droplets have a
size of approximately 120-180 nm (expressed as Z average measured
by PCS). The other adjuvants/antigen components are added to the
emulsion in simple admixture.
II.1.2. Scaled-Up Preparation
[0393] The preparation of the SB62 emulsion is made by mixing under
strong agitation of an oil phase composed of hydrophobic components
(.alpha.-tocopherol and squalene) and an aqueous phase containing
the water soluble components (Tween 80 and PBS mod (modified), pH
6.8). While stirring, the oil phase (1/10 total volume) is
transferred to the aqueous phase (9/10 total volume), and the
mixture is stirred for 15 minutes at room temperature. The
resulting mixture then subjected to shear, impact and cavitation
forces in the interaction chamber of a microfluidizer (15000 PSI-8
cycles) to produce submicron droplets (distribution between 100 and
200 nm). The resulting pH is between 6.8.+-.0.1. The SB62 emulsion
is then sterilised by filtration through a 0.22 .mu.m membrane and
the sterile bulk emulsion is stored refrigerated in Cupac
containers at 2 to 8.degree. C. Sterile inert gas (nitrogen or
argon) is flushed into the dead volume of the SB62 emulsion final
bulk container for at least 15 seconds.
[0394] The final composition of the SB62 emulsion is as
follows:
[0395] Tween 80:1.8% (v/v) 19.4 mg/ml; Squalene: 5% (v/v) 42.8
mg/ml; .alpha.-tocopherol: 5% (v/v) 47.5 mg/ml; 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
[0396] 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
[0397] 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 11.2.4). Samples were diluted 4000.times.-8000.times. in
PBS 7.4.
[0398] As a control, PL-Nanocal Particle size standards 100 nm (cat
n.degree. 6011-1015) was diluted in 10 mM NaCl.
II.2.3. Malvern Zetasizer 3000HS Size Measurements
[0399] All size measurements were performed with both Malvern
Zetasizer 3000HS.
[0400] 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: [0401] either real particle refractive
index of 0 and imaginary one of 0. [0402] 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).
[0403] The technical conditions were: [0404] laser wavelength: 532
nm (Zeta3000HS). [0405] laser power: 50 mW (Zeta3000HS). [0406]
scattered light detected at 900 (Zeta3000HS). [0407] temperature:
25.degree. C., [0408] duration: automatic determination by the
soft, [0409] number: 3 consecutive measurements, [0410] z-average
diameter: by cumulants analysis [0411] size distribution: by the
Contin or the Automatic method.
[0412] The Automatic Malvern algorithm uses a combination of
cumulants, Contin and non negative least squares (NNLS)
algorithms.
[0413] 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 [0414] TABLE 2 Sample Dilution Record Count rate ZAD
Polydispersity SB62 5000 1 7987 153 0.06 2 7520 153 0.06 3 6586 152
0.07 average 7364 153 0.06 SB62 8000 1 8640 151 0.03 (Example IV) 2
8656 151 0.00 3 8634 150 0.00 average 8643 151 0.01 SB62 + 8000 1
8720 154 0.03 MPL 25 .mu.g (*) 2 8659 151 0.03 3 8710 152 0.02
average 8697 152 0.02 (*) Prepared as follows: Water for injection,
PBS 10x concentrated, 250 .mu.l of SB62 emulsion and 25 .mu.g of
MPL are mixed together to reach a final volume of 280 .mu.l.
[0415] 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.
[0416] The count rate (CR) is a measure of scattered light: it
corresponds to thousands of photons per second.
[0417] The polydispersity (Poly) index is the width of the
distribution. This is a dimensionless measure of the distribution
broadness.
Contin and Automatic Analysis:
[0418] 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.
[0419] 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.
[0420] 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
[0421] 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.
[0422] 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
[0423] 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.
[0424] 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: [0425] suspend
the MPL powder in water for injection [0426] desaggregate any big
aggregates by heating (thermal treatment) [0427] reduce the
particle size between 100 nm and 200 nm by microfluidization [0428]
prefilter the preparation on a Sartoclean Pre-filter unit, 0.8/0.65
.mu.m [0429] sterile filter the preparation at room temperature
(Sartobran P unit, 0.22 .mu.m)
[0430] 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:
n.times.quantity of MPL to treat (ml)/flow rate (ml/min)
[0431] 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.
[0432] 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.
[0433] 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
[0434] To the AS03 adjuvant formulation, MPL is added at a final
concentration of between 10 and 50 .mu.g per vaccine dose.
[0435] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a SB62 mixture containing Tween, Triton X-100 and VES
(vitamin E succinate, i.e. alpha-tocopherol 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 80, 110 .mu.g/ml Triton X-100
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.
[0436] 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 Per human dose Name Component
Concentration Quantity Other SB62 781 .mu.l/ml 250 .mu.l Squalene
(solution 43 mg/ml) 10.68 mg Tocopherol (solution 48 mg/ml) 11.86
mg Tween 80 (solution 20 mg/ml) 4.85 mg MPL** (solution 1 mg/ml) 78
.mu.g/ml or 25 .mu.g or 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 for Ad 320 .mu.l injection pH 6.8 +/- 0.1 *PBS mod
10x 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
[0437] 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.g+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
[0438] 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 80, Triton
X-100 and .alpha.-tocopheryl hydrogen succinate are diluted in
water for injection. The three concentrated monobulks (A/New
Calcdonia, A/New York, B/Jiangsu) are then successively diluted in
the resulting phosphate buffered saline/Tween 80-Triton
X-100-.alpha.-tocopheryl hydrogen succinate solution (pH 7.4, 137
mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 990 .mu.g/ml
Tween 80, 150 .mu.g/ml Triton X-100 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.
[0439] 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.
[0440] 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).
[0441] 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
[0442] To the SB62 emulsion of 11.1 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)
[0443] 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
[0444] Three groups of subjects in parallel received the following
vaccine intramuscularly: [0445] one group of 50 subjects receiving
one dose of the reconstituted and adjuvanted SV influenza vaccine
(FluAS03) [0446] one group of 50 subjects receiving one dose of
whole virus influenza vaccine (FluWVV) [0447] one group of 50
subjects receiving one dose of Fluarix.TM. (Fluarix)=control
[0448] 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.
[0449] The standard trivalent split influenza vaccine--Fluarix.TM.
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
[0450] 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.
[0451] 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.TM./.alpha.-Rix.RTM. vaccine and contains 10.68 mg
squalene, 11.86 mg DL-alpha tocopherol, and 4.85 mg polysorbate 80
(Tween 80).
Preparation
[0452] 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.
[0453] 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 80, Triton
X-100 and .alpha.-tocopheryl hydrogen succinate are diluted in
water for injection. The three concentrated monobulks (strain A/New
Calcdonia-, strain A/Panama- and strain B/Shangdong-) are then
successively diluted in the resulting phosphate buffered
saline/Tween 80-Triton X-100-.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 KH.sub.2PO.sub.4, 1500 .mu.g/ml Tween
80, 220 .mu.g/ml Triton X-100 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.
[0454] 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.
[0455] Aqueous phase: while stirring, 902 ml of Tween 80 is mixed
with 44105 ml of PBS-mod buffer (pH=6.8 after adjustment with HCl).
[0456] Oil phase: while stirring, 2550 ml of squalene is added to
2550 ml of .alpha.-tocopherol. [0457] 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.
[0458] 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. [0459] 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.
[0460] 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.
[0461] The final composition of the SB62 emulsion is as
follows:
TABLE-US-00005 TABLE 5 Tween 80: 1.8% (v/v) 19.4 mg/ml Squalene: 5%
(v/v) 42.8 mg/ml alpha-tocopherol: 5% (v/v) 47.5 mg/ml 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
[0462] 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.
[0463] 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 [0464] TABLE 6 Vaccine Formulation Group Fluarix
.TM. HA from 3 influenza strains (total HA = 45 .mu.g) Fluarix
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 + 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)
[0465] 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
[0466] A total of 148 subjects were enrolled in this study: 49
subjects in the FluAS03 group, 49 subjects in the Fluarix group and
50 subjects in the FluWVV 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
[0467] 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
[0468] Analysis of immunogenicity was performed on the total
vaccinated cohort.
III.5.1. Humoral Immune Response
[0469] 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:
[0470] Geometric mean titres (GMTs) of HI and NI antibody titres at
days 0 and 21 [0471] Geometric mean titres (GMTs) of neutralising
antibody titres at days 0 and 21. [0472] 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.
[0473] 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.
[0474] 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)
[0475] 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 groups although there was some overlap of 95% CI between
the Fluarix group and the FluWVV 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 Fuarix 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 Fuarix 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 Fuarix 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(D 21) =
Post-vaccination at Day 21)
b) Conversion Factors of Anti-HI Antibody Titres, Seroprotection
Rates and Seroconversion Rates (Correlates for Protection in
Human)
[0476] Results are presented in Table 8.
[0477] 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. The seroprotection rates represent the proportion of
subjects with a serum HI titre .gtoreq.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
240 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 .gtoreq.60 years old population, by the
European Authorities.
[0478] 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 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 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/ Flu AS03 49
100.0 [92.7-100.0] 73.5 [58.9-85.1] 11.6 [7.2-18.6] shangdong
Fluarix 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:
[0479] Post vaccination, there was a trend for higher GMTs of HI
antibody for all three vaccine strains in the FluAS03 and Fluarix
groups although there was some overlap of 95% CI between the
Fluarix group and the FluWVV group. [0480] 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. [0481] 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.
[0482] 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
[0483] 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)).
[0484] Titres of neutralising antibody against the three influenza
strains were measured in pre- and post-immunisation sera. The
following parameters were determined: [0485] Geometric mean titres
(GMTs) of serum neutralising antibodies with 95% confidence
intervals (95% CI) pre and post-vaccination [0486] 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 [0486] 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(D 21) 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(D 21) 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(D 21) 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(D 21) 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(D 21) 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(D 21) 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(D 21) 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(D 21) 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(D 21) 50
46 92.0 80.8 97.8 117.8 82.6 168.0 Group 1: Flu vaccine mix
Adjuvant 2x 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(D 21) =
Post-vaccination at Day 21)
TABLE-US-00010 TABLE 10 Responders 95% CI Antibody Group N n % LL
UL A/New Caledonia 1 49 29 59.2 44.2 73.0 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) 2x 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
[0487] The main findings are: [0488] 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%. [0489] 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 groups
than in the FluWVV although there was some overlap of 95% CI
between the Fluarix group and the FluWVV group. [0490] For the
seroconversion rates, overall response rates for the three strains
were essentially equal in the three groups.
[0491] 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
[0492] 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:
[0493] GMT (taking the anti-log of the mean of the log titre
transformations) [0494] 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.
[0495] 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 Caledonia FluAS03 PRE 49 77.8 61.8 97.9 PI(D 21) 48 270.0
212.9 342.3 Fluarix PRE 49 77.8 64.6 93.6 PI(D 21) 49 249.1 190.0
326.5 FluWVV PRE 50 66.8 53.8 83.0 PI(D 21) 50 159.2 122.8 206.4
A/Panama FluAS03 PRE 49 33.3 28.5 48.7 PI(D 21) 48 156.8 124.8
196.9 Fluarix PRE 49 34.2 25.6 45.8 PI(D 21) 49 133.7 100.9 177.3
FluWVV PRE 50 24.6 18.7 32.4 PI(D 21) 49 78.9 59.4 104.7
B/Shangdong FluAS03 PRE 49 46.7 36.5 59.9 PI(D 21) 49 204.2 156.4
266.7 Fluarix PRE 49 46.1 35.3 60.1 PI(D 21) 49 133.7 100.9 177.3
FluWVV PRE 50 48.6 36.4 64.7 PI(D 21) 49 128.2 101.7 161.6 FluAS03:
Flu vaccine (DFLU58A16) mix with AS03 Adjuvant (D621024A8) Fluarix:
Flu vaccine (18854B9) FluWVV: Flu WVV vaccine (DFLU59A2) PRE =
Pre-vaccination, PI(D 21) = 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 Caledonia FluAS03 48 25 52.1 37.2 66.7 Fluarix 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 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 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: 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
[0496] The main findings are: [0497] Higher value of the GMT and
seroconversion rates were observed for hemagglutinin than for
neuraminidase. [0498] 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 groups although there
was some overlap of 95% CI between the Fluarix group and the FluWVV
group. [0499] Regarding the seroconversion rates, overall response
rates for the three strains were essentially equal in the three
groups and for the three strains.
[0500] 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
[0501] 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/IFN.gamma./
Peptide 1 Day 0 44 33.50 139.026 1.00 TNF.alpha. in CD4 Influenza 1
Day 21 45 58.40 132.664 1.00 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 Influenza 1 Day 0 47 1901.66 1596.203
102.00 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
Influenza 1 Day 0 48 3134.33 2568.369 507.00 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/ Peptide 1
Day 0 1.00 1.00 4.00 915.00 0.7631 IL2/ Influenza 1 Day 21 1.00
1.00 56.00 733.00 IFN.gamma./TNF.alpha. 2 Day 0 1.00 1.00 54.00
2393.00 in CD4 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: Flu vaccine Fluarix .TM. 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./TNF- Peptide 1 44 9.57 159.363 -860.00 .alpha. in
CD4 Influenza 2 42 -40.98 386.998 -2392.00 3 45 -50.73 256.596
-1664.00 Split Influenza 1 47 4307.02 4468.828 -8161.00 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./TNF- Peptide 1 42
-15.95 215.710 -451.00 .alpha. in CD8 Influenza 2 41 50.83 264.370
-614.00 3 44 -52.11 243.811 -684.00 Split Influenza 1 42 134.71
426.699 -603.00 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- Peptide 1 0.00 0.00 37.50
430.00 0.0765 .gamma./TNF-.alpha. in Influenza 2 -15.00 0.00 26.00
514.00 CD4 3 -37.00 0.00 0.00 212.00 Split Influenza 1 1888.00
3396.00 6634.00 19555.00 <0.0001 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- Peptide 1
-106.00 0.00 81.00 655.00 0.0932 .gamma./TNF-.alpha. in Influenza 2
-58.00 13.00 202.00 703.00 CD8 3 -160.50 0.00 53.00 567.00 Split
Influenza 1 -122.00 35.50 221.00 1387.00 0.2121 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 Influenza 1 46
4266.20 4470.807 -8093.00 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
Influenza 1 43 39.53 190.122 -438.00 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 Influenza 1 1799.00
3156.50 6647.00 19480.00 <0.0001 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 Peptide 1 -2.00
0.00 0.50 100.00 0.9721 CD8 Influenza 2 -28.00 0.00 24.00 231.00 3
-13.00 0.00 3.00 176.00 Split Influenza 1 -35.00 0.00 140.00 608.00
0.6175 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 Influenza 1 46 3 2712.87 2905.629 -4394.00 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 Influenza 1 43 6 138.58 365.565
-470.00 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
-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 Influenza 1 1273.00 1644.00 4057.00
13296.00 <0.0001 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 Influenza 1 -46.00 42.00 294.00 1549.00 0.1257 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 Influenza 1 46 3456.15
3853.960 -7009.00 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 Influenza 1 41
54.85 250.817 -336.00 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 Influenza 1 1309.00 2598.50 5926.00
16988.00 <0.0001 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 Influenza 1 -76.00 26.00 133.00 803.00 0.2311 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
Influenza 1 46 2343.11 2596.177 -4450.00 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 Influenza 1 41 138.54 362.601 -329.00 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 Peptide 1 -1.50 0.00 39.00 239.00 0.1836 CD4
Influenza 2 -4.00 0.00 12.00 277.00 3 -26.00 0.00 5.00 53.00 Split
Influenza 1 862.00 1466.50 3931.00 9267.00 <0.0001 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
Peptide 1 -80.00 0.50 70.00 772.00 0.2759 CD8 Influenza 2 -81.00
0.00 155.00 791.00 3 -179.00 0.00 39.50 566.00 Split Influenza 1
-23.00 60.00 178.00 1468.00 0.0790 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
[0502] 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.
[0503] 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.
[0504] The main findings are: [0505] Vaccination with Fluarix or
Whole virus slightly boosts the CD4 T-cell response. Vaccination
with FluAS03 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). [0506] 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).
[0507] Vaccination with Fluarix 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
[0508] 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.
[0509] A subset of 22 first subjects having received one dose of
FluAS03 vaccine and 21 first subjects having received one dose of
Fluarix vaccine was selected to evaluate the impact of vaccination
on influenza-specific memory B-cells using the B-cell memory
Elispot technology. The following endpoints were determined [0510]
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 (106) of antibody forming cells. [0511]
Difference between post (day 21) and pre (day 0) vaccination is
also expressed as a frequency of Influenza specific-antibody
forming cells per million (106) of antibody forming cells.
III.5.3.2 Statistical Methods
[0512] 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.
[0513] 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 Calcdonia, A/Panama and
B/Shangdong).
III.5.3.3 Results
[0514] There is a tendency in favour of the influenza adjuvanted
AS03 vaccine compared to Fluarix group. For A/New Calcdonia strain,
there is a statistical significant difference (p-value=0.021) in
favour of FluAS03 compared to Fluarix. 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 .TM. + AS03 oil-in-water emulsion adjuvant
Group 2: Flu vaccine Fluarix .TM. 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 STRAIN Gr Q1 Median Q3 Max
(Wilcoxon 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 .TM.
+ AS03 oil-in-water emulsion adjuvant Group 2: Flu vaccine Fluarix
.TM. 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
[0515] 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
[0516] 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 Variable EU standard for antibody response
Results Conversion factor >2.0 >6.1 Seroconversion rate
>30% >50% Protection rate >60% >88%
[0517] 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 and whole influenza virus vaccine). However,
vaccination has no measurable impact on the CD8 response.
[0518] 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
[0519] 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.TM. vaccine (known as .alpha.-rix.TM. in
Belgium) has been used as reference.
IV.1. Objective
[0520] 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.TM. was used as reference.
[0521] The objectives were:
1) to determine if AS03 adjuvanted Flu (40 subjects) versus Fluarix
(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
[0522] 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) [0523] one control
group of about 20 subjects aged >65 years who have previously
received one dose of Fluarix.TM. during the Explo-Flu-001 clinical
trial in 2003 (Fluarix)
IV.2.1. Vaccine Composition
[0524] 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: [0525]
A/New Calcdonia/20/99 (IVR-116) (H1N1)=A/New Calcdonia/(HINI)-like
strain [0526] A/Wyoming/3/2003 (X-147) (H3N2)=A/Fujian (H3N2)-like
strain [0527] B/Jiangsu/10/2003=B/Shanghai-like strain
IV.2.2. Immunogenicity (HI) End-Points
[0527] [0528] GMTs (taking the anti-log of the mean of the log
titre transformations) [0529] Conversion factors (the fold increase
in serum HI GMTs on day 21 compared to day 0) [0530] 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) [0531] Protection rate (the percentage of vaccinees
with a serum HI.gtoreq.1:40 at day 21)
IV.2.3. CMI-Endpoints
Observed Variable:
[0532] At days 0 and 21: frequency of cytokine-positive CD4/CD8
cells per 106 into 4 different cytokines. Each test quantifies the
response of CD4/CD8 T cell to: [0533] Pool of the 3 following
antigens [0534] New Calcdonia antigen [0535] Wyoming antigen [0536]
Jiangsu antigen.
Derived Variables:
[0537] 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
[0538] The first CMI analysis was based on the Total Vaccinated
cohort (N=40 subjects for FluAS03 group and N=18 subjects for
Fluarix group).
[0539] A longitudinal analysis was based on the Kinetic cohort of
the Explo-Flu-001 (split protein) and Explo-Flu-002 (pool flu
antigen) studies: [0540] Pre: N=36 subjects for FluAS03 group and
N=15 for Fluarix group. [0541] Post-Pre: N=34 subjects for FluAS03
group and N=15 for Fluarix group. [0542] (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). [0543] (b) Descriptive statistics in individual
difference between timepoints (Post-Pre) responses were tabulated
for each antigen, for each cytokine and for each vaccine group.
[0544] (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: [0545]
CD4 T-cell response to New Calcdonia, Wyoming, Jiangsu and the pool
of the 3 strains. [0546] CD8 T-cell response to New Calcdonia,
Wyoming, Jiangsu and the pool of the 3 strains. [0547] (d)
Non-parametric test (Wilcoxon-test) was also used: [0548] 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 [0549] 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 [0550] 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.
[0551] 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
All significance tests were two-tailed. P-values less than or equal
to 0.05 were considered as statistically significant.
IV.3. Results
[0552] 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
[0553] 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).
[0554] 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 18 1268.67 1051.744 197.00
724.00 863.00 1561.00 4676.00 double Flu 36 1781.31 1484.860
-2379.00 929.50 1664.50 2821.00 4669.00 AS03 CD40L Fluarix 18
1260.11 1054.487 243.00 721.00 849.00 1602.00 4743.00 Flu 36
1711.56 1433.113 -2359.00 838.00 1576.00 2759.50 4575.00 AS03
IFN.gamma. Fluarix 18 762.94 813.884 -12.00 294.00 496.00 1061.00
3564.00 Flu 36 1179.92 881.255 -817.00 692.50 1180.50 1865.50
2831.00 AS03 IL2 Fluarix 18 1019.06 917.905 -258.00 544.00 702.00
1174.00 3850.00 Flu 36 1423.33 1359.471 -2702.00 651.00 1260.00
2200.50 4342.00 AS03 TNF.alpha. Fluarix 18 803.39 915.838 32.00
231.00 533.00 936.00 3892.00 Flu 36 1078.28 1029.122 -1816.00
446.00 983.00 1836.00 3310.00 AS03 A/New All Fluarix 18 481.44
381.534 -241.00 282.00 448.50 598.00 1412.00 Caledonia double Flu
36 812.78 749.192 -828.00 215.50 911.50 1274.50 3206.00 AS03 CD40L
Fluarix 18 450.78 360.378 -239.00 291.00 447.00 580.00 1248.00 Flu
36 783.75 711.608 -760.00 242.00 808.00 1161.00 3050.00 AS03
IFN.gamma. Fluarix 18 316.28 279.662 -165.00 175.00 259.00 387.00
1111.00 Flu 36 438.22 420.770 -685.00 125.00 393.00 733.50 1557.00
AS03 IL2 Fluarix 18 326.06 290.792 -294.00 193.00 330.00 488.00
834.00 Flu 36 634.72 616.478 -557.00 179.50 678.50 952.00 2602.00
AS03 TNF.alpha. Fluarix 18 316.44 372.492 -140.00 50.00 278.00
542.00 1449.00 Flu 36 449.17 591.796 -916.00 100.50 343.50 848.00
2452.00 AS03 A/Wyoming All Fluarix 18 609.56 559.396 -176.00 257.00
510.50 957.00 1998.00 double Flu 36 766.61 579.191 -568.00 316.00
864.50 1221.00 1662.00 AS03 CD40L Fluarix 18 616.33 550.853 -176.00
274.00 488.00 939.00 2017.00 Flu 36 728.61 570.316 -670.00 260.00
789.50 1216.00 1675.00 AS03 IFN.gamma. Fluarix 18 407.06 424.758
-311.00 129.00 370.50 723.00 1372.00 Flu 36 526.72 443.938 -770.00
219.00 556.50 776.00 1342.00 AS03 IL2 Fluarix 18 495.83 503.805
-187.00 88.00 540.50 801.00 1841.00 Flu 36 572.89 533.728 -789.00
220.00 602.00 882.50 1512.00 AS03 TNF.alpha. Fluarix 18 424.56
485.591 -260.00 110.00 359.50 461.00 1718.00 Flu 36 550.58 538.461
-765.00 269.50 543.50 905.50 1678.00 AS03 B/Jiangsu All Fluarix 18
698.44 793.119 -306.00 233.00 433.00 961.00 2822.00 double Flu 36
861.42 688.852 -223.00 339.00 745.00 1325.50 2284.00 AS03 CD40L
Fluarix 18 678.39 777.259 -206.00 227.00 401.50 962.00 2878.00 Flu
36 825.89 674.879 -223.00 305.00 722.00 1282.00 2337.00 AS03
IFN.gamma. Fluarix 18 431.72 489.912 -95.00 191.00 272.50 382.00
1712.00 Flu 36 615.94 473.543 -286.00 288.50 501.50 897.50 1740.00
AS03 IL2 Fluarix 18 552.50 666.853 -234.00 155.00 278.50 833.00
2386.00 Flu 36 696.19 622.931 -359.00 207.50 540.50 1146.50 2182.00
AS03 TNF.alpha. Fluarix 18 441.39 695.792 -338.00 97.00 269.50
564.00 2440.00 Flu 36 500.03 448.636 -166.00 107.50 436.00 745.00
1626.00 AS03 SD = Standard Deviation Min, Max = Minimum, Maximum Q1
= First quartile Q3 = Third quartile N = number of subjects tested
with available results
[0555] 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 has compared to those vaccinated with
Fluarix/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.
[0556] 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.
[0557] 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
[0558] 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.
[0559] 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)
[0560] 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 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
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 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 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 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
[0561] 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 0.0712 CD40L
FluAS03 0.4957 Fluarix 0.0744 INF.gamma. FluAS03 0.0896 Fluarix
0.1103 IL2 FluAS03 0.1903 Fluarix 0.1647 TNF.alpha. FluAS03 0.0427
Fluarix 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 001 Flu 36 2000.86 1783.474 102.00 911.50 1461.50 2791.00
9514.00 double AS03 Fluarix 15 2152.87 2162.463 747.00 930.00
1354.00 2101.00 7868.00 EXPLO 002 Flu 36 2028.28 1427.000 55.00
1190.50 1647.50 2575.00 7214.00 AS03 Fluarix 15 1587.07 2123.841
192.00 468.00 735.00 1578.00 8536.00 CD40L EXPLO 001 Flu 35 1946.66
1771.102 120.00 837.00 1340.00 2819.00 9462.00 AS03 Fluarix 15
2094.93 2076.632 745.00 902.00 1340.00 2077.00 7385.00 EXPLO 002
Flu 35 1992.20 1440.721 77.00 1125.00 1590.00 2587.00 7286.00 AS03
Fluarix 15 1561.73 2097.201 34.00 475.00 672.00 1579.00 8428.00
INF.gamma. EXPLO 001 Flu 35 1068.63 1030.745 91.00 448.00 790.00
1503.00 5425.00 AS03 Fluarix 15 1248.07 1452.459 320.00 388.00
778.00 1227.00 5431.00 EXPLO 002 Flu 35 1259.23 890.590 312.00
725.00 984.00 1354.00 4146.00 AS03 Fluarix 15 974.80 1394.044 52.00
252.00 337.00 1057.00 5576.00 IL2 EXPLO 001 Flu 35 1690.20 1524.689
37.00 688.00 1211.00 2416.00 8235.00 AS03 Fluarix 15 1888.40
2085.857 568.00 715.00 1136.00 1770.00 7403.00 EXPLO 002 Flu 35
1883.60 1361.337 14.00 1068.00 1413.00 2370.00 6891.00 AS03 Fluarix
15 1493.93 2037.139 58.00 444.00 755.00 1485.00 8193.00 TNF.alpha.
EXPLO 001 Flu 35 1174.74 1119.633 55.00 466.00 795.00 1720.00
5415.00 AS03 Fluarix 15 1444.20 1946.211 201.00 520.00 688.00
1254.00 7213.00 EXPLO 002 Flu 35 1545.40 1159.490 135.00 831.00
1203.00 1857.00 5354.00 AS03 Fluarix 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
[0562] 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
[0563] 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
001 34 4837.56 4476.129 -609.00 1888.00 3483.50 8148.00 19555.00
double AS03 EXPLO 002 34 1737.79 1450.177 -2379.00 936.00 1664.50
2743.00 4669.00 Fluarix EXPLO 001 15 3103.53 3726.645 436.00 800.00
2283.00 3226.00 15169.00 EXPLO 002 15 1369.00 1127.784 197.00
725.00 869.00 1808.00 4676.00 CD40L Flu EXPLO 001 33 4819.06
4489.788 -718.00 1799.00 3479.00 8288.00 19480.00 AS03 EXPLO 002 33
1694.73 1431.082 -2359.00 921.00 1659.00 2662.00 4575.00 Fluarix
EXPLO 001 15 3090.00 3684.759 477.00 822.00 2189.00 3208.00
15021.00 EXPLO 002 15 1360.93 1131.051 243.00 725.00 860.00 1687.00
4743.00 IFN.gamma. Flu EXPLO 001 33 3127.09 2974.067 -453.00
1325.00 1721.00 5162.00 13296.00 AS03 EXPLO 002 33 1167.85 893.363
-817.00 633.00 1207.00 1803.00 2831.00 Fluarix EXPLO 001 15 1660.13
1834.023 -84.00 480.00 1386.00 2284.00 7120.00 EXPLO 002 15 851.87
859.585 148.00 294.00 501.00 1222.00 3564.00 IL2 Flu EXPLO 001 33
3950.18 3878.538 -358.00 1309.00 2780.00 6635.00 16988.00 AS03
EXPLO 002 33 1404.67 1355.665 -2702.00 719.00 1341.00 2109.00
4342.00 Fluarix EXPLO 001 15 2413.87 3027.392 263.00 674.00 1672.00
2425.00 12273.00 EXPLO 002 15 1117.80 975.934 -258.00 575.00 714.00
1618.00 3850.00 TNF.alpha. Flu EXPLO 001 33 2627.36 2574.458
-825.00 862.00 1475.00 4764.00 9267.00 AS03 EXPLO 002 33 1072.36
1044.140 -1816.00 447.00 1000.00 1752.00 3310.00 Fluarix EXPLO 001
15 1460.53 3115.174 -1586.00 251.00 813.00 1314.00 12275.00 EXPLO
002 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
[0564] 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 0.1300
CD40L FluAS03 0.0007 Fluarix 0.0890 INF.gamma. FluAS03 0.0012
Fluarix 0.1103 IL2 FluAS03 0.0025 Fluarix 0.1409 TNF.alpha. FluAS03
0.0327 Fluarix 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 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 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 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 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 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 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 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 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 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
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
[0565] 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
[0566] 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 PRE 18 17 94.4 72.6 99.9 63.5
38.1 105.9 Caledonia PI (D 21) 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 (D 21) 40 40 100
91.3 100 218.6 158.2 302.0 A/Fujian Fluarix PRE 18 18 100 81.5 100
95.0 51.0 176.9 PI (D 21) 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 (D 21) 40 40 100
91.3 100 735.1 564.4 957.5 B/Shanghai Fluarix PRE 18 16 88.9 65.3
98.6 23.3 15.2 35.8 PI (D 21) 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 (D 21) 40 40 100
91.3 100 364.4 269.7 492.4 PRE = Prevaccination, PI (D 21) = 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 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 PRE 18 14 77.8 52.4 93.6 Caledonia PI (D 21) 18 16
88.9 65.3 98.6 FluAS03 PRE 40 32 80 64.4 90.9 PI (D 21) 40 39 97.5
86.8 99.9 A/Fujian Fluarix PRE 18 14 77.8 52.4 93.6 PI (D 21) 18 18
100 81.5 100 FluAS03 PRE 40 36 90 76.3 97.2 PI (D 21) 40 40 100
91.2 100 B/Shanghai Fluarix PRE 18 6 33.3 13.3 59.0 PI (D 21) 18 14
77.8 52.4 93.6 FluAS03 PRE 40 34 85 70.2 94.3 PI (D 21) 40 40 100
91.2 100 PRE = Prevaccination, PI (D 21) = 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 18 3
16.7 3.6 41.5 FluAS03 40 19 47.5 31.5 63.9 A/Fujian Fluarix 18 13
72.2 46.5 90.3 FluAS03 40 34 85.0 70.2 94.3 B/Shanghai Fluarix 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
[0567] From this clinical study it is confirmed that the adjuvanted
vaccine Flu-AS03 is superior to the equivalent unadjuvated vaccine
Fluarix 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.
[0568] In contrast to first year vaccination, upon revaccination
individuals previously vaccinated with the adjuvanted Fluarix.TM.
showed increased HI titer responsiveness as compared to those
vaccinated with un-adjuvanted Fluarix.TM.. 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
[0569] First Study--Efficacy of new formulations AS03 and
AS03+MPL
V.1. Rationale and Objectives
[0570] Influenza infection in the ferret model closely mimics human
influenza, with regards both to the sensitivity to infection and
the clinical response.
[0571] 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.
[0572] 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.
[0573] The objective of this experiment was to demonstrate the
efficacy of an adjuvanted influenza vaccine compared to the plain
(un-adjuvanted) vaccine.
[0574] 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 reponse by IHA and monitoring of the temperature around
the priming and the challenge.
V.2. Experimental Design
V.2.1. Treatment/Group (Table 37)
[0575] 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
Calcdonia/20/99, H3N2A/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 Formulation + (schedule/route/
Group Antigen(s) + dosage dosage challenge) Other treatments 1
Trivalent Full HD: 15 .mu.g IM; Day 21 Priming H1N1 Plain HA/strain
(A/Stockolm/24/ 90) Day 0 2 Trivalent Full HD: 15 .mu.g IM; Day 21
Priming H1N1 AS03 HA/strain (A/Stockolm/24/90) Day 0 3 Trivalent
Full HD: 15 .mu.g IM; Day 21 Priming H1N1 AS03 + MPL HA/strain
(A/Stockolm/24/ 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):
[0576] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 110 .mu.g
Triton X-100 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):
[0577] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 110 .mu.g Triton
X-100 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 11.1) 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
[0578] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 110 .mu.g Triton
X-100 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.
[0579] 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 [0580] TABLE 38 Sample- Analysis Readout Timepoint
type I/P method Viral D - 1 to D + 7 Post priming Nasal In
Titration shedding D - 1 to D + 5 Post challenge washes T .degree.
D - 1 to D + 3 Post priming Implant in In Telemetry monitoring D -
2 to D + 3 Post challenge peritoneal cavity IHA Pre, Post priming,
Post Serum In IHA imm, Post challenge In = Individual/Po = Pool
V.3. Results
[0581] A schematic representation of the results is given in FIG.
10 and FIG. 11.
V.3.1. Temperature Monitoring
[0582] Individual temperature were monitored with the transmitters
and by the telemetry recording (according to the procedure detailed
under I.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.
[0583] 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).
[0584] 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)
[0585] Viral titration of nasal washes was performed on 6 animals
per group.
[0586] 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).
[0587] 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.
[0588] 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.
[0589] 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
[0590] 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).
[0591] AS03 and AS03+MPL formulations showed added benefit in terms
of protective efficacy in ferrets (lower viral shedding and
temperature) (FIGS. 10 and 11).
[0592] 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
[0593] 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
heterologous challenge.
V.5. Experimental Design
[0594] Female ferrets (Mustela putorius furo) (6 ferrets/group)
aged 14-20 weeks were obtained from MISAY Consultancy (Hampshire,
UK). Four groups were tested: [0595] Fluarix [0596] Trivalent Split
AS03 [0597] Trivalent Split AS03+MPL [0598] PBS 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 Calcdonia/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
[0599] A schematic representation of the results is given in FIG.
12 and in FIG. 13.
V.6.1. Temperature Monitoring
[0600] 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.
[0601] The results (FIG. 12) show that: [0602] A high variability
from one group to another was observed around the priming. The
baseline seemed to be higher before priming than after priming.
[0603] 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). [0604] 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)
[0605] 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).
[0606] 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.
[0607] 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
[0608] Viral shedding was measured for 12 ferrets from Day 1
Pre-priming- to Day 7 Post-priming. Results are expressed in
pool.
[0609] The viral clearance was observed on Day 7 Post-priming in
all ferrets.
Viral Shedding after Challenge
[0610] Viral shedding was measured for 6 ferrets/group from Day 1
Pre-challenge to Day 7 Post-challenge.
[0611] 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).
[0612] On Day 50, no virus was detected in nasal washes.
V.6.3. Hemagglutination Inhibition Test (HI Titers) (FIGS. 14A and
B)
[0613] Serum samples were collected 1 day before priming, 21 days
Post-priming, 22 days post-immunization and 14 days
post-challenge.
[0614] 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:
[0615] 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.TM.).
[0616] Similar HI titers were observed in ferrets immunized with
H3N2 A/Panama adjuvanted with AS03 or AS03+MPL.
[0617] 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).
[0618] 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
[0619] As expected, a boost of anti-H3N2 HI titers was observed
after heterologous challenge compared to the situation after
homologous challenge (no boost).
[0620] 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
[0621] 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 Plain (un-adjuvanted). No difference
was observed for both CD8 T cell and humoral responses between
these two groups.
[0622] 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
[0623] Female C57BI/6 mice (15 mice/group) aged 6-8 weeks were
obtained from Harlan Horst, Netherland. The groups tested were:
[0624] Trivalent Split Plain [0625] Trivalent Split AS03 [0626]
Trivalent Split AS03+MPL [0627] PBS
[0628] 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
Calcdonia/20/99, A/Panama/2007/99, B/Shangdong/7/97) plain or
adjuvanted (see groups below).
VI.1.2. Preparation of the Vaccine Formulations
[0629] In each formulation, PBS 10 fold concentrated is added to
reach isotonicity and is 1 fold concentrated in the final volume.
H.sub.2O volume is calculated to reach the targeted volume.
Split Trivalent Plain (Un-Adjuvanted):
[0630] Formulation 1 (for 500 .mu.l): PBS 10 fold concentrated (pH
7.4 when one fold concentrated) as well as a mixture containing
Tween 80, Triton X-100 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 80, 110 .mu.g Triton X-100 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:
[0631] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 110 .mu.g Triton
X-100 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:
[0632] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 110 .mu.g Triton
X-100 and 100 .mu.g VES per 1 ml After 5 min stirring, 15 .mu.g of
each strain H1N1, H.sub.3N.sub.2 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
[0633] CMI analysis (ICS: CD4/CD8, IL-2/IFNg staining)
[0634] PBMCs from primed mice were harvested 7 days
post-immunization. They were tested in pools/group.
VI.2. Results
[0635] 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).
With these conditions, it was possible to induce: [0636] Higher CD4
T cell responses for Split AS03 compared to Split Plain, as
observed in humans. [0637] Higher CD4 T cell responses for Split
AS03+MPL compared to Split Plain. [0638] Similar CD8 T cell
responses between Split Plain and Split AS03, as observed in
humans. [0639] 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
[0640] VII.1. Experimental design and objective
[0641] 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 Plain (un-adjuvanted). No difference
was observed for both CD8 T cell and humoral responses between
these two groups.
[0642] 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.
[0643] The purpose was to compare the CMI response induced by a
GlaxoSmithKline commercially available split vaccine (Fluarix.TM.)
versus a subunit vaccine (Chiron's vaccine Fluad.TM.) 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
[0644] 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
Calcdonia/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 (un-adjuvanted) = Fluarix .TM. Heterologous
priming D0 2 Trivalent split*/OW Heterologous priming D0 3
Trivalent split*/AS03 Heterologous priming D0 4 Trivalent
split*/AS03 + MPL (2.5 .mu.g per dose) Heterologous priming D0 5
Gripguard (=Fluad .TM.) = sub-unit in an oil-in-water Heterologous
emulsion priming D0 6 Aggripal .TM. (sub-unit)/AS03 Heterologous
priming D0 7 Aggripal .TM. (sub-unit)/AS03 + MPL (2.5 .mu.g per
Heterologous dose) priming D0 8 Aggripal .TM. (sub-unit)/OW**
Heterologous priming D0 9 Aggripal .TM. (sub-unit) Heterologous
priming D0 10 PBS Heterologous priming D0 *Fluarix .TM. **OW
produced as explained in the section below
VII.1.2. Preparation of the Vaccine Formulations
Preparation of OW
[0645] An oil-in-water emulsion called OW is prepared following the
recipe published in the instruction booklet contained in Chiron
Behring FluAd vaccine.
[0646] 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 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 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: [0647] laser wavelength: 532 nm
(Zeta3000HS). [0648] laser power: 50 mW (Zeta3000HS). [0649]
scattered light detected at 900 (Zeta3000HS). [0650] temperature:
25.degree. C., [0651] duration: automatic determination by the
soft, [0652] number: 3 consecutive measurements, [0653] z-average
diameter: by cumulants analysis
Formulation for Group 1 (for 1 Ml):
[0654] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 and VES
(quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml Tween 80,
55 .mu.g/ml Triton X-100 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):
[0655] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 and VES
(quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml Tween 80,
55 .mu.g/ml Triton X-100 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:
[0656] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 and VES
(quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml Tween 80,
55 .mu.g/ml Triton X-100 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:
[0657] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 and VES
(quantities taking into account the detergents present in the
strains) to reach a final concentration of 375 .mu.g/ml Tween 80,
55 .mu.g/ml Triton X-100 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:
[0658] Equal volume of PBS and FluAd.TM./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:
[0659] 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 methodoly 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:
[0660] 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
methodoly 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:
[0661] 250 .mu.l of PBS mod pH 7.4 are added to a 500 .mu.l dose of
Aggripal. 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:
[0662] Equal volume of PBS mod pH 7.4 and Aggripal 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.
[0663] IHA/neutralization assay: 21 Days Post-immunization.
TABLE-US-00040 TABLE 40 Analysis Read-out Timepoint Sample type I/P
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)
[0664] PBMCs from 24 mice/group were harvested 7 days
post-immunization and tested in pools/group.
VII.2. Results
VII.2.1. Humoral Immunity
[0665] 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.
[0666] For the 3 strains and for all groups, a boost of HI titers
was observed after immunization. [0667] For a same adjuvant and for
the 3 strains, similar HI titers were induced by the subunit
vaccine and the Split vaccine. [0668] Similar HI titers were
observed for Fluad compared to Aggripal OW for the 3 strains [0669]
No difference was observed between Fluarix and Aggripal for H1N1
and B strains. [0670] 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. [0671] 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
[0672] 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.
[0673] In terms of Flu whole virus-specific CD4+ T cells expressing
IL-2, IFN-.gamma. or both cytokines (FIG. 17 upper part): [0674] 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. [0675] 2. Whatever the
formulation (Plain, AS03 or AS03+MPL), the split vaccine induced a
higher CD4+ T cell responses than the subunit vaccine. [0676] 3.
Fluad (subunit +oil-in-water emulsion OW--see preparation section)
seemed to induce similar frequencies than Fluarix Plain. [0677] 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
[0678] 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.
[0679] In terms of Flu whole virus-specific CD8+ T cells expressing
IL-2, IFN-.gamma. or both cytokines (FIG. 17 lower part): [0680]
The cut-off of this experiment was relatively high due to the high
background observed for the PBS negative control group. [0681]
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
[0682] The following results were obtained:
1) Flu-specific CD4+ T cells obtained by ICS at Day 7
post--immunization showed: [0683] 1. Similar responses were
obtained for Fluad compared to Fluarix. [0684] 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) 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). [0685] 3.
There is a trend of a higher CD4 responses with Split/AS03+MPL
compared to Split/AS03 (FIG. 17). [0686] 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).
[0687] 5. The split vaccine, whether adjuvanted with AS03 with or
without MPL (groups 3 and 4) showed higher CD4+ T cell responses
than the sub-unit vaccine, either Fluad (group 5) or Aggripal +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
[0688] A phase I, open, randomised, controlled study in an elderly
population aged over 65 years (.gtoreq.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.TM. vaccine (known as .alpha.-Rix.TM. in
Belgium).
[0689] Three parallel groups were assessed: [0690] one group of 50
subjects receiving one dose of the reconstituted and AS03
adjuvanted SV influenza vaccine (Flu AS03) [0691] one group of 50
subjects receiving one dose of the reconstituted and Flu AS03+MPL
adjuvanted SV influenza vaccine (Flu AS03+MPL) [0692] one control
group of 50 subjects receiving one dose of Fluarix.TM.
(Fluarix)
VIII.2. Vaccine Composition and Administration
[0693] 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 Calcdonia/20/99 (H1N1), A/New
California/3/2003 (H.sub.3N.sub.2) and B/Jiangsu/10/2003. Like
Fluarix.TM./.alpha.-Rix.TM., 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.
[0694] 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.TM./.alpha.-Rix.
AS03 Adjuvanted Vaccine:
[0695] 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:
[0696] 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.TM. (i.e. 30 .mu.g HA/ml).
[0697] 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
[0698] 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
[0699] 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: [0700] Pool of the 3 following
antigens [0701] New Calcdonia antigen [0702] Wyoming antigen [0703]
Jiangsu antigen.
Derived Variables:
[0704] 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:
[0705] The CMI analysis was based on the Total vaccinated cohort.
[0706] (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 Calcdonia, Wyoming and Jiangsu and the pooled of the 3
different strains. [0707] (b) Descriptive statistics in individual
difference between timepoint (POST-PRE) responses for each
vaccination group and each antigen at each 5 different cytokines.
[0708] (c) Comparison of the 3 groups regarding the 5 different
cytokines on: [0709] CD4 T-cell response to New Calcdonia, Wyoming,
Jiangsu and the pool of the 3 strains [0710] CD8 T-cell response to
New Calcdonia, Wyoming, Jiangsu and the pool of the 3 strains
[0711] (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. [0712] (e) A Wilcoxon test were use to test
pairwise comparison of 2 groups respectively between Flu AS03+MPL
versus Fluarix, Flu AS03+MPL versus Flu AS03 and Flu AS03 versus
Fluarix [0713] (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
[0714] 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
[0715] (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
Calcdonia, Wyoming and Jiangsu), similarly to that performed in
Example Ill. [0716] (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. [0717] (c) Comparing the
difference in the frequency of antigen-specific CD4 T-lymphocytes
between Flu AS03+MPL and Fluarix groups by the Wilcoxon test, all
p-values were less than 0.05 and were considered as statistically
significant. [0718] (d) Comparing the difference in the frequency
antigen-specific of CD4 T-lymphocytes between Flu AS03 and Fluarix
groups by the Wilcoxon test, all p-values were less than 0.05 and
were considered as statistically significant. [0719] (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
[0719] [0720] (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
Ill. [0721] (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. [0722] (c)
Comparing the individual difference POST-PRE in the
antigen-specific CD4-T-lymphocytes responses between Flu AS03+MPL
and Fluarix using Wilcoxon test, all p-values were less than to
0.05 and were considered as statistically significant. [0723] (d)
Comparing the individual difference POST-PRE in the
antigen-specific CD4-T-lymphocytes responses between Flu AS03 and
Fluarix using Wilcoxon test, all p-values were less than to 0.001
and were considered as highly statistically significant. [0724] (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
[0725] 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 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
[0726] The end points are: [0727] (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.
[0728] (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
[0729] 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 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 Calcdonia and
Wyoming).
[0730] 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 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 Calcdonia and Wyoming).
[0731] 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
[0732] This study compared GSK commercial influenza trivalent split
vaccine, either unadjuvanted (Fluarix.TM.) or adjuvanted with
AS03+MPL, with two other commercially available sub-unit vaccines:
[0733] Fluad.TM., Chiron's adjuvanted subunit vaccine (the adjuvant
is Chiron's MF59 adjuvant),
[0734] Agrippal.TM., Chiron un-adjuvanted commercial sub-unit
vaccine, which was in the present study adjuvanted with AS03
adjuvant.
[0735] 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.
[0736] 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
[0737] 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 Calcdonia/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 Formulation + (ex: schedule/ Group
Antigen(s) + dosage dosage route/challenge) Other treatments 1
Trivalent plain Full HD: 15 .mu.g IM; Day 21 Priming H1N1 (Fluarix
.TM.) HA/strain (A/Stockolm/24/ 90) Day 0 2 Trivalent Full HD: 15
.mu.g IM; Day 21 Priming H1N1 AS03 + MPL HA/strain
(A/Stockolm/24/90) Day 0 3 Fluad .TM. Full HD: 15 .mu.g IM; Day 21
Priming H1N1 HA/strain (A/Stockolm/24/ 90) Day 0 4 Agrippal .TM.
Full HD: 15 .mu.g IM; Day 21 Priming H1N1 AS03 HA/strain
(A/Stockolm/24/ 90) Day 0
IX.2.2. Preparation of the Vaccine Formulations
Split Trivalent Plain (Un-Adjuvanted): Formulation for 1 ml:
[0738] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 55 .mu.g
Triton X-100 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:
[0739] PBS 10 fold concentrated (pH 7.4 when one fold concentrated)
as well as a mixture containing Tween 80, Triton X-100 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 80, 55 .mu.g Triton
X-100 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.TM. Formulation: Formulation for 1 ml:
[0740] A 2 fold dilution of FluAd.TM. vaccine is made in PBS buffer
pH 7.4.
Agrippal.TM. AS03 Formulation: Formulation for 1 ml:
[0741] 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 [0742] TABLE 42 Sample- I/ Analysis Readout
Timepoint type Po method Viral D - 3 to D + 7 Post priming Nasal In
Titration shedding D + 1 to D + 5 Post challenge washes T .degree.
D - 3 to D + 4 Post priming Implant in In Telemetry monitoring D -
2 to D + 4 Post challenge peritoneal cavity 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
[0743] 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:
[0744] Post-challenge, a peak of body temperature was observed
after immunization of ferrets with the un-adjuvanted (plain)
trivalent split (Fluarix.TM.) or the sub-unit vaccine Fluad.TM.
(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
[0745] 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.).
[0746] 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.
[0747] 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:
[0748] 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.TM.) or with Fluad.TM.
sub-unit vaccine.
[0749] 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
[0750] 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:
[0751] After immunization with H.sub.3N.sub.2 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.TM.) or
with Fluad.TM. sub-unit vaccine (FIG. 21).
[0752] 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 (FIG. 22).
[0753] 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.TM.).
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
[0754] A phase I, open, randomised, controlled study in an elderly
population aged over 65 years (.gtoreq.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.TM. vaccine (known as .alpha.-Rix.TM. in
Belgium). This study follows that reported in Example VIII.
[0755] Three parallel groups were assessed: [0756] one group of 50
subjects receiving one dose of the reconstituted and AS03
adjuvanted SV influenza vaccine (Flu AS03) [0757] one group of 50
subjects receiving one dose of the reconstituted and Flu AS03+MPL
adjuvanted SV influenza vaccine (Flu AS03+MPL) [0758] one control
group of 50 subjects receiving one dose of Fluarix.TM.
(Fluarix)
X.2. Immunogenicity Results
X.2.1. Humoral Immune Response Endpoints and Results
[0759] 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.
[0760] 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). [0761] Serum HI antibody GMTs' with 95% CI at
Days 0, 21, 90 and 180 [0762] Seroconversion rates with 95% CI at
Days 21, 90 and 180 [0763] Conversion factors with 95% CI at Day 21
[0764] Seroprotection rates with 95% CI at Days 0, 21, 90 and
180
Results
[0765] 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 was noted for the A/New Calcdonia 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.
[0766] 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.
[0767] 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.
[0768] 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 for the A/New Calcdonia 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 Calcdonia 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 Caledonia Flu PRE 50 28
56.0 41.3 70.0 AS03 + PI (D 21) 50 46 92.0 80.8 97.8 MPL PI (D 90)
50 43 86.0 73.3 94.2 PI (D 180) 50 39 78.0 64.0 88.5 Fluarix PRE 50
26 52.0 37.4 66.3 PI (D 21) 50 46 92.0 80.8 97.8 PI (D 90) 50 38
76.0 61.8 86.9 PI (D 180) 50 34 68.0 53.3 80.5 FluAS03 PRE 49 28
57.1 42.2 71.2 PI (D 21) 49 48 98.0 89.1 99.9 PI (D 90) 49 45 91.8
80.4 97.7 PI (D 180) 49 38 77.6 63.4 88.2 A/Wyoming Flu PRE 50 33
66.0 51.2 78.8 AS03 + PI (D 21) 50 47 94.0 83.5 98.7 MPL PI (D 90)
50 46 92.0 80.8 97.8 PI (D 180) 50 45 90.0 78.2 96.7 Fluarix PRE 50
32 64.0 49.2 77.1 PI (D 21) 50 50 100 92.9 100.0 PI (D 90) 50 49
98.0 89.4 99.9 PI (D 180) 50 50 100 92.9 100.0 FluAS03 PRE 49 34
69.4 54.6 81.7 PI (D 21) 49 48 98.0 89.1 99.9 PI (D 90) 49 46 93.9
83.1 98.7 PI (D 180) 49 47 95.9 86.0 99.5 B/Jiangsu Flu PRE 50 19
38.0 24.7 52.8 AS03 + PI (D 21) 50 50 100 92.9 100.0 MPL PI (D 90)
50 47 94.0 83.5 98.7 PI (D 180) 50 46 92.0 80.8 97.8 Fluarix PRE 50
17 34.0 21.2 48.8 PI (D 21) 50 48 96.0 86.3 99.5 PI (D 90) 50 47
94.0 83.5 98.7 PI (D 180) 50 47 94.0 83.5 98.7 FluAS03 PRE 49 25
51.0 36.3 65.6 PI (D 21) 49 49 100 92.7 100.0 PI (D 90) 49 47 95.9
86.0 99.5 PI (D 180) 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 (D
21) = post-vaccination blood sampling at Day 21 PI (D 90) =
post-vaccination blood sampling at Day 90 PI (D 180) =
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 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 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 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
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 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 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 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 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
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
[0769] 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 106 in different tests (New Calcdonia, Wyoming
and Jiangsu antigens considered separately as well as pooled at
Days 0 and 21; New Calcdonia, Wyoming, Jiangsu and New York
antigens considered separately as well as pooled at Days 90 and
180) [0770] All double: cells producing at least two different
cytokines (CD40L, IFN-.gamma., IL-2, TNF-.alpha.). [0771] CD40L:
cells producing at least CD40L and another cytokine (IFN-.gamma.,
IL-2, TNF-.alpha.). [0772] IFN-.gamma.: cells producing at least
IFN-.gamma. and another cytokine (CD40L, IL-2, TNF-.alpha.).
[0773] IL-2: cells producing at least IL-2 and another cytokine
(CD40L, IFN-.gamma., TNF-.alpha.).
[0774] TNF-.alpha.: cells producing at least TNF-.alpha. and
another cytokine (CD40L, IFN-.gamma., IL-2).
Results
[0775] The main findings were (FIG. 24): [0776] (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
group. No significant difference was however detected between the
two adjuvants. [0777] (b) All statistical differences between
adjuvanted vaccines and Fluarix were maintained up to Day 90 and
Day 180 with the following exceptions at Day 180: [0778] No
statistically significant difference was found between FluAS03/MPL
and Fluarix for all double, CD40L, IFN-.gamma. and IL2 (Wyoming
strain only) and for all double, CD40L and TNF-.alpha. (New York
strain only) [0779] No statistically significant difference was
found between FluAS03 and Fluarix for IL2 (Jiangsu strain only)
[0780] (c) The absence of statistically significant difference
between the two adjuvanted vaccines was confirmed up to Day 90 and
Day 180. [0781] (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.TM.. No significant difference was however detected between
both adjuvants. [0782] (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
[0783] 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 (known as .alpha.-Rix.TM. in Belgium) vaccine was used as
reference.
[0784] Two parallel groups were assessed: [0785] One group of about
50 subjects who had previously received one dose of the
reconstituted adjuvanted influenza vaccine during the previous
clinical trial [0786] One control group (Fluarix) of about 50
subjects who had previously received one dose of Fluarix.TM. during
the previous clinical trial
[0787] 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.TM. in the previous trial received a dose of commercial
vaccine and formed the control group of this trial.
XI.2. Vaccine Composition and Administration
[0788] 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 Calcdonia/20/99 (H1N1), A/New
California/7/2004 (H3N2) and B/Jiangsu/10/2003. Like
Fluarix.TM./.alpha.-Rix.TM., 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.
[0789] 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.
[0790] 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.
[0791] 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 80)
4.85 mg MPL 25 .mu.g
XI.3. Immunogenicity Results
XI.3.1. Anti-HA Humoral Immune Response Endpoints and Results
Observed Variables:
[0792] 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): [0793] (f)
Geometric mean titres (GMTs) of serum HI antibodies with 95%
confidence intervals (95% CI) pre and post-vaccination [0794] (g)
Seroconversion rates* with 95% CI at day 21 [0795] (h)
Seroconversion factors** with 95% CI at day 21 [0796] (i)
Seroprotection rates*** with 95% CI at day 21 * Seroconversion rate
defined as the percentage of vaccinees with either a
pre-vaccination HI titre <1:10 and a post-vaccination titre
.gtoreq.1:40, or a pre-vaccination titre .gtoreq.1:10 and a minimum
4-fold increase at post-vaccination titre, for each vaccine strain.
** Seroconversion factor defined as the fold increase in serum HI
GMTs on day 21 compared to day 0, for each vaccine strain. ***
Protection rate defined as the percentage of vaccinees with a serum
HI titre .gtoreq.40 after vaccination (for each vaccine strain)
that usually is accepted as indicating protection.
Results
[0797] 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 group, although 95% CI were
overlapping (FIG. 25).
[0798] 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)
Seroconversion rate Seroconversion Seroprotection rate
(.gtoreq.4-fold increase) factor Strains Group N (HI titre .gtoreq.
40) % [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 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 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
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
[0799] 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.TM. (known as .alpha.-Rix.TM. in
Belgium) administered in adults (18-40 years)
[0800] Four parallel groups were assigned: [0801] (a) 75 adults
(aged 18-40 years) in one control group receiving one dose of
Fluarix.TM.(Fluarix group) [0802] (b) 200 elderly subjects (aged 65
years and older) randomized 3:3:2 into three groups: [0803] one
group with 75 subjects receiving influenza vaccine adjuvanted with
AS03+MPL (concentration 1-25 .mu.g) [0804] One group with 75
subjects receiving influenza vaccine adjuvanted with AS03+MPL
(concentration 2-50 .mu.g) [0805] Reference Flu group with 50
subjects receiving one dose of Fluarix.TM.
Primary Objective
[0806] 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.TM. 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
[0807] The secondary objectives are [0808] (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.TM. is used as reference. [0809] (b) To
evaluate the humoral immune response (anti-haemagglutinin titre)
21, 90 and 180 days after vaccination with influenza candidate
vaccines adjuvanted. Fluarix.TM. is used as reference.
Tertiary Objective
[0810] 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.TM. is used
as reference.
XII.2. Vaccine Composition and Administration
[0811] 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.
[0812] Control: full dose of Fluarix.TM. by IM administration.
[0813] Four scheduled visits per subject: at days 0, 21, 90 and 180
with blood sample collected at each visit to evaluate
immunogenicity. [0814] 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.
[0815] 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)
[0816] For evaluation of CMI response, frequency of
influenza-specific CD4 are analysed as follows:
[0817] 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)
[0818] 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.TM. 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 + MPL (conc. 1) Flu
YNG AS03 + MPL (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 + MPL (conc. 2) Flu YNG AS03 + MPL (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)
[0819] The main findings were: [0820] Before vaccination the CMI
response if higher in young adults than in elderly [0821] After
vaccination, [0822] there was a booster effect of the influenza
vaccine on the CMI response in young adults (18-40 years) [0823]
CMI response in the elderly having received adjuvanted influenza
vaccine is comparable to the CMI response of young adults. [0824]
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.TM. (18-40 years) for
all tests excepted for IFN.gamma. when we compare Fluarix (18-40
years) and Flu/AS03+MPL (conc. 1).
[0825] 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-Calcdonia included
in the vaccine. However, preliminary data including the A/H1N1/New
Calcdonia vaccine strain from subsets of subjects indicate that the
results will be similar.
Results--Evaluation of the Tertiary End-Point (Table 48)
[0826] 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.
[0827] 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.
[0828] A Non-parametric test (Wilcoxon test) was used to compare
the location of difference between the two groups (influenza
adjuvanted vaccine versus Fluarix.TM.) and the statistical p-value
is calculated for each antigen at each different test.
[0829] 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.
[0830] 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.
[0831] 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 Group 1 and Group 2 and Group 1 and
Group 2 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)
[0832] The main conclusions are: [0833] (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. [0834] (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.TM.. [0835] (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.TM.. [0836]
(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
[0837] Observed variables:
[0838] 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.
[0839] Based on the HI antibody titres, the following parameters
are calculated: [0840] (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. [0841] (k) Seroconversion
factors (SF) at day 21 defined as the fold increase in serum HI
GMTs on day 21 compared to day 0. [0842] (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. [0843] (m) Seroprotection rates
(SPR) at day 21 defined as the percentage of vaccinees with a serum
HI titre >1:40.
[0844] 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.
[0845] 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)
[0846] 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 in the same population.
GMTs are
[0847] significantly higher for H1N1 for AS03+MPL (conc. 2), [0848]
significantly higher for H.sub.3N.sub.2 and for B for both
adjuvants,
[0849] Twenty one days after vaccination, the subjects of Fluarix
(18-40 years) had a higher Hi response for New Calcdonia and
B/Jangsu strains.
[0850] 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.
[0851] After vaccination, there was a statistically difference in
terms of seroprotection rates of HI antibodies between Fluarix
(.gtoreq.65 years) group and [0852] Flu AS03+MPL (conc 2) for A/New
Calcdonia strain
[0853] For each vaccine strain, the seroprotection rates for the 2
influenza adjuvanted vaccine groups are in the same range compared
to Fluarix (18-40 years) group.
[0854] There was a statistically difference in terms of
seroconversion rates of HI antibodies between Fluarix (>65
years) group and [0855] Flu AS03+MPL (conc 2) for A/New Calcdonia
strain [0856] Flu AS03+MPL (conc 1) for B/Jiangsu strain
[0857] For each vaccine strain, the seroconversion rates for the 2
influenza adjuvanted vaccine groups are in the same range compared
to Fluarix (18-40 years) group excepted for New Calcdonia
strain.
TABLE-US-00049 TABLE 49 Seroprotection rates seroconversion rates
and conversion factors at day 21 (ATP cohort for immunogenicity)
Seroconversion rate (.gtoreq.4-fold Conversion Seroprotection rate
increase) factor Strains Group N (HI titre .gtoreq. 40) % [95% CI]
[95% CI] % 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 {circumflex over (
)}21.9-56.4] Caledonia Flu Elderly 49 71.4 [56.74-83.42] 30.6
[18.3-45.4] 3.7 [2.4-5.7] (H1N1) FluAS03 + MPL (conc. 1) 75 90.5
[81.48-96.11] 55.4 [43.4-67.0] 6.4 [4.5-9.0] 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
[0858] (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.TM.,
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.TM. in adults aged between 18 and 40
years. [0859] (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.TM.. Significant difference between the humoral immune
response against each vaccine strain mediated in elderly subjects
by adjuvanted vaccines compared to Fluarix.TM. are summarised in
Table 50. Compared to adults aged between 18 and 40 years
vaccinated with Fluarix.TM., 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 [0859] TABLE 50 Significant difference in humoral
immune response between adjuvanted vaccines and Fluarix in elderly
subjects Seroconversion Seroprotection Seroconversion Post-vacc 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)
[0860] 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 0 Absent injection site and 1 is easily tolerated other
locations 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.)
[0861] The maximum intensity of local injection site
redness/swelling is scored as follows:
0 is 0 mm; 1 is >0-.ltoreq.20 mm; 2 is >20-.ltoreq.50 mm; 3
is >50 mm.
[0862] The maximum intensity of fever is scored as follows:
1 is >37.5-.ltoreq.38.0.degree. C.; 2 is
>38.0-.ltoreq.39.0.degree. C.; 3 is >39.0
[0863] 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)
[0864] 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.TM. 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.TM. (FIG. 28). In all cases, symptoms resolved rapidly.
[0865] 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.
EXAMPLE XII
Preclinical Studies in Mice Using HPV Vaccine Adjuvanted with
AS03+MPL
XIII.1. Introduction
[0866] BALB/C mice were injected with an adjuvanted mixture
containing 2.5 .mu.g of each of 4 different antigens, HPV 16 L1,
HPV 18 L1, HPV 31 L1, and HPV 45 L1, in the form of virus like
particles. Each L1 protein was a C terminal truncate removing, in
the case of HPV 16, 34 amino acids (or the equivalent region in the
other sequences). HPV proteins were expressed and purified in
baculovirus expression systems, for example as described in WO
03/077942.
[0867] The tetravalent VLP combination was combined with one of 2
different adjuvants.
[0868] The adjuvants tested were: [0869] 1 A mixture of aluminium
hydroxide and 3-D MPL (termed AS04). [0870] 2 A mixture of
AS03+3D-MPL, prepared essentially as Example 2.
[0871] The adjuvant AS04 was used in a vaccine comprising HPV 16
and HPV 18 L1-only virus like particles, tested in phase II
clinical trials as described in The Lancet, vol 364, issue 947, 13
Nov. 2004, 1757-1765. It thus provides a good basis for comparison
with AS03+3D MPL. This adjuvant can be made as described in, for
example, WO 01/17751 and WO 00/23105.
[0872] An analysis of antibody titres was carried out for each
component of the vaccine. In addition, an analysis of B memory
cells specific for HPV 16 and HPV 18 was made within the total
population of IgG molecules.
XIII.2. Material and Methods
XIII.2.1. Animal Model
[0873] Two groups of BALB/c mice (n=10) were immunised
intramuscularly in one leg (days 0 and 28) with 2.5 .mu.g of
HPV-16/18/31/45 L1, formulated with AS04 (Al(OH).sub.3 50 .mu.g+MPL
5 .mu.g) or a mixture of AS03+3D-MPL, prepared essentially as
Example 2, containing 50 .mu.l emulsion+MPL 5 .mu.g.
XIII.2.2. Anti-HPV-16/18/31/45 L1 Serology: Ig
[0874] Quantitation of anti-HPV-16/18/31/45 L1 antibody was
performed by ELISA using HPV-16 L1 (lot E16L1P093), HPV-18 L1 (lot
E18L1P079), HPV-31 L1 (lot EA31 L1P329) and HPV-45 L1 (lot
EA45L1P328) as coating antigen. HPV-16/18/31/45 L1 and antibody
solutions were used at 50 .mu.l per well; only the saturation
solution was used at 100 .mu.l per well. HPV-16/18/31/45 L1 were
diluted at a final concentration of 0.5 .mu.g/ml in PBS and were
adsorbed overnight at 4.degree. C. to the wells of 96 wells
microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). After
removal of coating solution, the plates were then incubated for 1
hr at 37.degree. C. with PBS containing 1% bovine serum albumin
(saturation buffer). Two-fold dilutions of mice sera in the
dilution buffer (saturation buffer+0.1% Tween20) were added to the
coated plates after removal of saturation solution and incubated
for 1 hr 30 min at 37.degree. C. The plates were washed four times
with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse Ig 1/1000
(Dako) diluted in saturation buffer, was added to each well and
incubated for 1 hr 30 min at 37.degree. C. After a washing step,
avidin-horseradish peroxydase complex (Dako, UK) diluted 1/3000 in
saturation buffer was added for an additional 30 min at 37.degree.
C. Plates were washed four times as above and incubated for 20 min
at room temperature with a solution of o-phenylenediamine (Sigma
MO, USA) 0.04% H.sub.2O.sub.2 0.03% in 0.1% Tween 20 0.05M citrate
buffer pH4.5. The reaction was stopped with the addition of
H.sub.2SO.sub.4 2N and the plates were read at 490/630 nm.
ELISA Titer Calculation
[0875] The optical densities (OD's) were measured using a
microplate reader connected to a computer. Data were captured with
the SoftMaxPro software. In order to titrate each sample, a
standard is included on each plate. A four parameters logistic log
function is used to calculate the standard curve. Antibody
concentrations were calculated at each dilution of the test sample
by interpolation of the standard curves.
[0876] The antibody titers were obtained by averaging the values
from all dilutions that fall within the working range (20-80% OD)
of the standard curve. ELISA titers are expressed in EU/ml.
XIII.2.3. B Memory Cell Elispot
[0877] Thirty-three or seventy-five days after the second
immunization, mice were sacrificed; spleen cells were separated by
a lymphoprep gradient. PBMCs were then resuspended in RPMI 1640
medium (Gibco) containing additives (sodium pyruvate 1 mM, MEM
non-essential amino acids, Pen/Strep, Glutamine and .beta.-2
mercaptoethanol), 5% fetal calf serum, 50 U/ml rhlL-2 (eBioscience)
and 3 .mu.g/ml CpG (phosphothioated CpG ODN-7909-5'-TCG TCG TTT TGT
CGT TTT GTC GTT-3'-SEQ ID NO.1). Other CpG sequences are also
suitable for use in this B memory evaluation method. Cells were
cultured five days at a final concentration of 10.sup.6 cells/ml,
in 5 ml per flat-bottomed 6 wells. After an activation step with
ethanol, nitrocellulose plates (Multiscreen-IP; Millipore) were
coated with 10 .mu.g/ml of HPV-16/18 L1 or goat anti-mouse Ig (GAM;
Sigma) diluted 1/200 in PBS. After a saturation step with complete
medium, 100 .mu.l of 2.10.sup.6 cells/ml were added to HPV-16/18 L1
coated plates and 100 .mu.l of 10.sup.6 and 5.10.sup.5 cells/ml
were added to GAM plates. After an incubation time of 2 hrs at
37.degree. C., plates were stored overnight at 4.degree. C. Plates
were washed four times with PBS 0.1% Tween 20 and anti-mouse Ig
Biot diluted 1/200 in PBS 1% BSA 5% FCS (dilution buffer) was
distributed to plates and incubated for 2 hrs at 37.degree. C.
After a washing step, Extravidin HRP (Sigma) diluted 1/550 in
dilution buffer was added for an additional 1 hr at 37.degree. C.
Plates were washed as above and incubated for 10 min at room
temperature with a solution of AEC (Sigma). Reaction was stopped by
rinsing plates gently under tap water. After drying, plates were
read with an automated ELISPOT image analysis system (Zeiss
KS400).
[0878] The percentage of B memory cells specific for HPV-16/18 L1
corresponds to the ratio of HPV-16/18 L1 positive spots compared to
the total IgG spots.
XIII.3. Serological results (Tables 53-55)
TABLE-US-00053 TABLE 53 Anti-VLPs titers (EU/ml) at day 14 post II
HPV16 HPV18 HPV31* HPV45 VLPs 2.5 .mu.g/AS04 528441 512290 28671
42224 VLPs 2.5 .mu.g/AS03 + 3D MPL 824773 460800 77232 65716
TABLE-US-00054 TABLE 54 Anti-VLPs titers (EU/ml) at day 75 post II
HPV16 HPV18 HPV31 HPV45* VLPs 2.5 .mu.g/AS04 230425 241983 15846
9812 VLPs 2.5 .mu.g/AS03 + 3D MPL 415657 292199 29054 32230
*statistical difference between groups
TABLE-US-00055 TABLE 55 B cell memory HPV16 HPV18 Frequency of HPV
specific memory B cells in total IgG memory B cells at day 33 post
II VLPs 2.5 .mu.g/AS04 0.4% 0.2% VLPs 2.5 .mu.g/AS03 + 3D MPL 0.7%
0.4% Frequency of HPV specific memory B cells in total lgG memory B
cells at day 75 post II VLPs 2.5 .mu.g/AS04 2.0% 0.6% VLPs 2.5
.mu.g/AS03 + 3D MPL 3.8% 0.5%
XIII.4. Conclusions
[0879] In the animal model system tested the AS03+3D-MPL adjuvant
demonstrates immunogenicity results for both antibody production
and B cell memory which are equivalent to, or sometimes greater
than, those generated with AS04, depending upon the HPV type being
assessed.
Sequence CWU 1
1
1124DNAArtificial SequenceSynthetic sequence, unmethylated CG
containing sequence 1tcgtcgtttt gtcgttttgt cgtt 24
* * * * *