U.S. patent application number 12/096838 was filed with the patent office on 2008-11-13 for vaccine compositions comprising a saponin adjuvant.
Invention is credited to Pierre Vandepapeliere.
Application Number | 20080279926 12/096838 |
Document ID | / |
Family ID | 37876836 |
Filed Date | 2008-11-13 |
United States Patent
Application |
20080279926 |
Kind Code |
A1 |
Vandepapeliere; Pierre |
November 13, 2008 |
Vaccine Compositions Comprising a Saponin Adjuvant
Abstract
An immunogenic composition in a dose volume suitable for human
use comprising an antigen or antigenic preparation, in combination
with an adjuvant which adjuvant comprises an immunologically active
saponin fraction derived from the bark of Quillaja Saponaria Molina
presented in the form of a liposome and a lipopolysaccharide
wherein said saponin fraction and said lipopolysaccharide are both
present in said human dose at a level of below 30 .mu.g.
Inventors: |
Vandepapeliere; Pierre;
(Rixensart, BE) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B482
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
37876836 |
Appl. No.: |
12/096838 |
Filed: |
December 12, 2006 |
PCT Filed: |
December 12, 2006 |
PCT NO: |
PCT/GB2006/004634 |
371 Date: |
June 10, 2008 |
Current U.S.
Class: |
424/450 ;
424/184.1; 424/204.1; 424/209.1; 424/230.1; 424/244.1;
424/272.1 |
Current CPC
Class: |
C12N 2710/16134
20130101; A61P 35/00 20180101; A61K 39/12 20130101; A61P 31/20
20180101; C12N 2710/16771 20130101; A61K 39/102 20130101; A61K
2039/6068 20130101; Y02A 50/412 20180101; A61K 39/092 20130101;
A61K 39/39 20130101; C12N 2710/16734 20130101; C12N 2760/16171
20130101; A61P 33/06 20180101; C12N 2710/20071 20130101; C12N
2760/16234 20130101; A61K 39/1045 20130101; A61P 31/12 20180101;
A61K 2039/70 20130101; A61P 43/00 20180101; C12N 2730/10134
20130101; A61K 2039/55505 20130101; Y02A 50/30 20180101; A61K
39/015 20130101; A61P 31/04 20180101; A61P 31/22 20180101; C12N
2760/16134 20130101; A61K 2039/5258 20130101; A61K 2039/57
20130101; A61K 2039/6037 20130101; A61P 31/16 20180101; A61P 37/00
20180101; C12N 2710/10134 20130101; A61P 15/00 20180101; A61P 31/18
20180101; C12N 2710/16171 20130101; A61K 2039/545 20130101; A61K
39/145 20130101; A61K 39/00 20130101; C12N 2760/16271 20130101;
C12N 2760/20034 20130101; A61K 2039/55555 20130101; A61P 37/04
20180101; A61K 2039/55572 20130101; A61K 2039/55577 20130101; A61K
2039/55 20130101 |
Class at
Publication: |
424/450 ;
424/184.1; 424/230.1; 424/244.1; 424/272.1; 424/209.1;
424/204.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/00 20060101 A61K039/00; A61K 39/25 20060101
A61K039/25; A61K 39/09 20060101 A61K039/09; A61K 39/015 20060101
A61K039/015; A61K 39/145 20060101 A61K039/145; A61K 39/12 20060101
A61K039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2005 |
GB |
0525321.6 |
May 18, 2006 |
GB |
0609902.2 |
Oct 12, 2006 |
GB |
0620336.8 |
Oct 12, 2006 |
GB |
0620337.6 |
Claims
1.-91. (canceled)
92. An immunogenic composition in a dose volume suitable for human
use comprising an antigen or antigenic preparation, in combination
with an adjuvant which adjuvant comprises an immunologically active
saponin fraction derived from the bark of Quillaja Saponaria Molina
presented in the form of a liposome and a lipopolysaccharide
wherein said saponin fraction and said lipopolysaccharide are both
present in said human dose at a level of below 30 .mu.g.
93. An immunogenic composition as claimed in claim 92, wherein said
dose volume suitable for human use is between 0.5 and 1.5 ml.
94. An immunogenic composition as claimed in claim 93 wherein said
dose volume is 0.5 ml.
95. An immunogenic composition as claimed in claim 93 wherein said
dose volume is 0.7 ml.
96. An immunogenic composition as claimed in claim 93 wherein said
dose volume is 1.0 ml.
97. An adjuvant composition in a volume suitable for use in a human
dose of an immunogenic composition comprising between 1 and 30
.mu.g of a lipopolysaccharide and between 1 and 30 .mu.g of an
immunologically active saponin fraction presented in the form of a
liposome.
98. An adjuvant composition according to claim 97 wherein said
human dose suitable volume is 250 .mu.l.
99. An immunogenic composition according to claim 92 wherein said
adjuvant further comprises a sterol, wherein the ratio of
saponin:sterol is from 1:1 to 1:100 w/w.
100. An immunogenic composition according to claim 99 wherein the
ratio of saponin:sterol is from 1:1 to 1:10 w/w.
101. An immunogenic composition according to claim 99 wherein the
ratio of saponin:sterol is from 1:1 to 1:5 w/w.
102. An immunogenic composition according to claim 92 wherein said
immunologically active saponin fraction is QS21.
103. An immunogenic composition according to claim 99 wherein said
sterol is cholesterol.
104. An immunogenic composition according to claim 92 wherein said
lipopolysaccharide is a lipid A derivative.
105. An immunogenic composition according to claim 104 wherein said
lipid A derivative is 3D-MPL.
106. An immunogenic composition according to claim 92 wherein said
lipopolysaccharide and said immunologically active saponin fraction
are present in the adjuvant composition at the same amount.
107. An immunogenic composition according to claim 106 wherein the
ratio of QS21:3D-MPL is 1:1.
108. An immunogenic composition according to claim 92 wherein said
lipopolysaccharide is present at an amount of 1-30 .mu.g.
109. An immunogenic composition according to claim 108 wherein said
lipopolysaccharide is present at an amount of 25 .mu.g.
110. An immunogenic composition according to claim 108 wherein said
lipopolysaccharide is present at an amount of 1-15 .mu.g.
111. An immunogenic composition according to claim 110 wherein said
lipopolysaccharide is present at an amount of 10 .mu.g.
112. An immunogenic composition according to claim 110 wherein said
lipopolysaccharide is present at an amount of 5 .mu.g.
113. An immunogenic composition according to claim 92 wherein said
saponin is present at an amount of 1-25 .mu.g.
114. An immunogenic composition according to claim 113 wherein said
saponin is present at an amount of 25 .mu.g.
115. An immunogenic composition according to claim 113 wherein said
saponin is present at an amount of 1-10 .mu.g.
116. An immunogenic composition according to claim 115 wherein said
saponin is present at an amount of 10 .mu.g.
117. An immunogenic composition according to claim 115 wherein said
saponin is present at an amount of 5 .mu.g.
118. An immunogenic composition according to claim 92 wherein said
antigen or antigenic preparation is derived from varicella zoster
virus (VZV).
119. An immunogenic composition according to claim 92 wherein said
antigen or antigenic preparation is derived from Streptococcus
pneumoniae.
120. An immunogenic composition according to claim 92 wherein said
antigen or antigenic preparation is derived from Cytomegalovirus
(CMV).
121. An immunogenic composition according to claim 92 wherein said
antigen or antigenic preparation is derived from Plasmodium
falciparum.
122. An immunogenic composition according to claim 92 wherein said
antigen or antigenic preparation is derived from influenza
virus.
123. An immunogenic composition according to claim 92 wherein said
antigen or antigenic preparation is derived from human
papillomavirus (HPV).
Description
TECHNICAL FIELD
[0001] The present invention relates to improved vaccine
compositions, methods for making them, and their use in medicine.
In particular the invention relates to adjuvanted vaccine
compositions wherein the adjuvant is a liposomal formulation,
comprising a saponin and a lipopolysaccharide. The present
invention further relates to influenza vaccine formulations and
vaccination regimes for immunizing against influenza disease.
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] Lipopolysaccharides (LPS) are the major surface molecule of,
and occur exclusively in, the external leaflet of the outer
membrane of gram-negative bacteria. LPS impede destruction of
bacteria by serum complements and phagocytic cells, and are
involved in adherence for colonisation. LPS are a group of
structurally related complex molecules of approximately 10,000
Daltons in size and consist of three covalently linked regions:
[0004] (i) an O-specific polysaccharide chain (O-antigen) at the
outer region [0005] (ii) a core oligosaccharide central region
[0006] (iii) lipid A--the innermost region which serves as the
hydrophobic anchor, it comprises glucosamine disaccharide units
which carry long chain fatty acids.
[0007] The biological activities of LPS, such as lethal toxicity,
pyrogenicity and adjuvanticity, have been shown to be related to
the lipid A moiety. In contrast, immunogenicity is associated with
the O-specific polysaccharide component (O-antigen). Both LPS and
lipid A have long been known for their strong adjuvant effects, but
the high toxicity of these molecules has precluded their use in
vaccine formulations. Significant effort has therefore been made
towards reducing the toxicity of LPS or lipid A while maintaining
their adjuvanticity.
[0008] The Salmonella minnesota mutant R595 was isolated in 1966
from a culture of the parent (smooth) strain (Luderitz et al. 1966
Ann. N.Y. Acad. Sci. 133:349-374). The colonies selected were
screened for their susceptibility to lysis by a panel of phages,
and only those colonies that displayed a narrow range of
sensitivity (susceptible to one or two phages only) were selected
for further study. This effort led to the isolation of a deep rough
mutant strain which is defective in LPS biosynthesis and referred
to as S. minnesota R595.
[0009] In comparison to other LPS, those produced by the mutant S.
minnesota R595 have a relatively simple structure. [0010] (i) they
contain no O-specific region--a characteristic which is responsible
for the shift from the wild type smooth phenotype to the mutant
rough phenotype and results in a loss of virulence [0011] (ii) the
core region is very short--this characteristic increases the strain
susceptibility to a variety of chemicals [0012] (iii) the lipid A
moiety is highly acylated with up to 7 fatty acids.
[0013] 4'-monophosphoryl lipid A (MPL), which may be obtained by
the acid hydrolysis of LPS extracted from a deep rough mutant
strain of gram-negative bacteria, retains the adjuvant properties
of LPS while demonstrating a toxicity which is reduced by a factor
of more than 1000 (as measured by lethal dose in chick embryo eggs)
(Johnson et al. 1987 Rev. Infect Dis. 9 Suppl:S512-S516). LPS is
typically refluxed in mineral acid solutions of moderate strength
(e.g. 0.1 M HCl) for a period of approximately 30 minutes. This
process results in dephosphorylation at the 1 position, and
decarbohydration at the 6' position, yielding MPL.
[0014] 3-O-deacylated monophosphoryl lipid A (3D-MPL), which may be
obtained by mild alkaline hydrolysis of MPL, has a further reduced
toxicity while again maintaining adjuvanticity, see U.S. Pat. No.
4,912,094 (Ribi Immunochemicals). Alkaline hydrolysis is typically
performed in organic solvent, such as a mixture of
chloroform/methanol, by saturation with an aqueous solution of weak
base, such as 0.5 M sodium carbonate at pH 10.5.
[0015] Further information on the preparation of 3D-MPL is
available in, for example, U.S. Pat. No. 4,912,094 and WO02/078637
(Corixa Corporation).
[0016] Quillaja saponins are a mixture of triterpene glycosides
extracted from the bark of the tree Quillaja saponaria. Crude
saponins have been extensively employed as veterinary adjuvants.
Quil-A is a partially purified aqueous extract of the Quillaja
saponin material. QS21 is a Hpic purified non toxic fraction of
Quil A and its method of its production is disclosed (as QA21) in
U.S. Pat. No. 5,057,540.
[0017] By way of example, influenza vaccines and vaccines against
human papilloma virus (HPV) have been developed with adjuvants.
[0018] 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.
[0019] 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.
[0020] 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 that enable the virus to escape
the immune system causing the well known, almost annual,
epidemics.
[0021] 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.
[0022] 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).
[0023] 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.
[0024] 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.
[0025] 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.).
[0026] 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). 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).
[0027] 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.
[0028] 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).
[0029] 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.
[0030] 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).
[0031] 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.
[0032] 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.
[0033] 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.
[0034] Although minor variations do occur, all HPV 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.
[0035] 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.
[0036] There is still a need for improved vaccines, especially in
the case of influenza and in particular influenza pandemics and for
the elderly population, or in the case of HPV vaccines.
[0037] Adjuvants containing combinations of lipopolysaccharide and
Quillaja saponins have been disclosed previously, for example in
EP0671948. This patent demonstrated a strong synergy when a
lipopolysaccharide (3D-MPL) was combined with a Quillaja saponin
(QS21). It has now been found that good adjuvant properties may be
achieved with combinations of lipopolysaccharide and quillaja
saponin as immunostimulants in an adjuvant composition even when
the immunostimulants are present at low amounts in a human
dose.
STATEMENT OF THE INVENTION
[0038] In first aspect of the present invention, there is provided
an immunogenic composition comprising an antigen or antigenic
preparation thereof, in combination with an adjuvant composition
comprising an immunologically active saponin fraction derived from
the bark of Quillaja Saponaria Molina presented in the form of a
liposome and a lipopolysaccharide.
[0039] In a second aspect of the present invention, there is
provided an immunogenic composition comprising an influenza virus
or antigenic preparation thereof, in combination with a saponin
adjuvant presented in the form of a liposome. In a specific
embodiment of this aspect, the immunogenic composition further
comprises a Lipid A derivative, such as 3D-MPL.
[0040] Suitably the saponin adjuvant in the form of a liposome
according to the invention comprises an active fraction of the
saponin derived from the bark of Quillaja Saponaria Molina, such as
QS21, and a sterol, such as cholesterol, in a ratio saponin:sterol
from 1:1 to 1:100w/w.
[0041] In particular, said immunogenic composition comprises an
antigen with a CD4 T cell epitope. Alternatively, said immunogenic
composition comprises an antigen with a B cell epitope.
[0042] The invention also relates to the use of an influenza virus
or antigenic preparation thereof, and an adjuvant comprising an
immunologically active saponin fraction derived from the bark of
Quillaja Saponaria Molina presented in the form of a liposome and a
lipopolysaccharide in the manufacture of an immunogenic composition
for the prevention of influenza virus infection and/or disease.
[0043] The invention also relates to the use of a human papilloma
virus antigen or antigens or antigenic preparation thereof, and an
adjuvant comprising an immunologically active saponin fraction
derived from the bark of Quillaja Saponaria Molina presented in the
form of a liposome and a lipopolysaccharide in the manufacture of
an immunogenic composition for the prevention of human papilloma
virus infection and/or disease.
[0044] The invention also relates to the use of a Cytomegalovirus
antigen or antigens or antigenic preparation thereof, and an
adjuvant comprising an immunologically active saponin fraction
derived from the bark of Quillaja Saponaria Molina presented in the
form of a liposome and a lipopolysaccharide in the manufacture of
an immunogenic composition for the prevention of Cytomegalovirus
infection and/or disease.
[0045] The invention also relates to the use of a Streptococcus
pneumonaie antigen or antigens or antigenic preparation thereof,
and an adjuvant comprising an immunologically active saponin
fraction derived from the bark of Quillaja Saponaria Molina
presented in the form of a liposome and a lipopolysaccharide
defined in the manufacture of an immunogenic composition for the
prevention of Streptococcus pneumonaie infection and/or
disease.
[0046] The invention also relates to the use of a Plasmodium
falciparum antigen or antigens or antigenic preparation thereof,
and an adjuvant comprising an immunologically active saponin
fraction derived from the bark of Quillaja Saponaria Molina
presented in the form of a liposome and a lipopolysaccharide in the
manufacture of an immunogenic composition for the prevention of
Plasmodium falciparum infection and/or malarial disease.
[0047] The invention also relates to the use of a Varicella Zoster
virus antigen or antigens or antigenic preparation thereof, and an
adjuvant comprising an immunologically active saponin fraction
derived from the bark of Quillaja Saponaria Molina presented in the
form of a liposome and a lipopolysaccharide in the manufacture of
an immunogenic composition for the prevention of Varicella Zoster
virus infection and/or disease.
[0048] In another aspect there is provided the use of (a) an
antigen or antigenic preparation thereof, and (b) an adjuvant as
hereinabove defined in the manufacture of an immunogenic
composition for inducing, in a human, at least one, or at least
two, or all of the following: (i) an improved CD4 T-cell immune
response against said antigen or antigenic preparation thereof,
(ii) an improved humoral immune response against said antigen or
antigenic preparation thereof, (iii) an improved B-memory cell
response against said antigen or antigenic preparation thereof.
[0049] In particular said antigen is an influenza virus, HPV,
Cytomegalovirus (CMV), Varicella zoster virus (VZV), Streptococcus
pneumoniae or malaria antigen or antigenic preparation thereof, and
said human is an immuno-compromised individual or population, such
as a high risk adult, an elderly adult or an infant. In a specific
embodiment, there is provided the use of an antigen or antigenic
preparation thereof and an adjuvant as herein defined in the
preparation of an immunogenic composition for vaccination of human,
in particular a human elderly adult, against the pathogen from
which the antigen in the immunogenic composition is derived.
Specifically said antigen is an influenza virus, human papilloma
virus, Cytomegalovirus, Varicella Zoster virus, Streptococcus
pneumoniae, Plasmodium parasite, antigen or antigens or antigenic
preparation thereof.
[0050] There is also provided a method of vaccination comprising
delivery of an antigen or antigenic composition, in particular an
influenza virus or HPV, Cytomegalovirus, Varicella Zoster virus,
Streptococcus pneumoniae, Plasmodium parasite, or antigenic
preparation thereof and an adjuvant as hereinabove defined to an
individual or population in need thereof.
[0051] In a specific embodiment, the immunogenic composition is
capable of inducing an improved CD4 T-cell immune response against
said antigen or antigenic preparation thereof, and in particular is
further capable of inducing either a humoral immune response or an
improved B-memory cell response or both, compared to that obtained
with the un-adjuvanted antigen or antigenic composition.
Specifically said CD4 T-cell immune response involves the induction
of a cross-reactive CD4 T helper response. Specifically said
humoral immune response involves the induction of a cross-reactive
humoral immune response.
[0052] In a further embodiment, there is provided a method or use
as hereinabove defined, for protection against infection or disease
caused by a pathogen which is a variant of the pathogen from which
the antigen in the immunogenic composition is derived. In another
embodiment, there is provided a method or use as hereinabove
defined for protection against infections or disease caused by a
pathogen which comprises an antigen which is a variant of that
antigen in the immunogenic composition. In a specific embodiment,
there is provided the use of an antigen, in particular an influenza
or HPV, or antigenic preparation thereof in the manufacture of an
immunogenic composition for revaccination of humans previously
vaccinated with an immunogenic composition comprising an antigen,
in particular an influenza or HPV or antigenic preparation thereof,
in combination with an adjuvant as herein described.
[0053] In a specific embodiment, the composition used for the
revaccination may additionally contain an adjuvant. In another
specific embodiment, the immunogenic composition for revaccination
contains an antigen which shares common CD4 T-cell epitopes with an
antigen or antigenic composition used for a previous vaccination.
Specifically, the immunogenic composition for revaccination
contains an influenza virus or antigenic preparation thereof which
shares common CD4 T-cell epitopes with the influenza virus or virus
antigenic preparation thereof used for the first vaccination.
[0054] In one aspect, the revaccination is made in subjects who
have been vaccinated the previous season against influenza.
Typically revaccination is made at least 6 months after the first
vaccination, preferably 8 to 14 months after, more preferably at
around 10 to 12 months after. In another aspect the revaccination
is made in subjects who have been vaccinated with a composition
comprising an influenza virus or antigenic preparation thereof
wherein at least one strain is associated with a pandemic outbreak
or has the potential to be associated with a pandemic outbreak.
[0055] In a further aspect of the present invention, there is
provided the use of an influenza virus or antigenic preparation
thereof from a first influenza strain in the manufacture of an
immunogenic composition as herein defined for protection against
influenza infections caused by a variant influenza strain.
[0056] The invention also relates to a method of vaccination
comprising delivery of an influenza virus or antigenic preparation
thereof and an adjuvant as herein defined.
[0057] In another aspect, there is provided a method of vaccination
of an immuno-compromised human individual or population such as
high risk adults or elderly, comprising administering an influenza
immunogenic composition comprising an influenza virus or antigenic
preparation thereof in combination with an adjuvant as herein
defined.
[0058] In still another embodiment, the invention provides a method
for revaccinating humans previously vaccinated with an influenza
immunogenic composition comprising an influenza antigen or
antigenic preparation thereof from at least one influenza virus
strains in combination with an adjuvant as herein defined, said
method comprising administering to said human an immunogenic
composition comprising an influenza virus or antigenic preparation
thereof, either adjuvanted or un-adjuvanted.
[0059] The invention also relates to a method for the preparation
of an immunogenic composition comprising combining a saponin
adjuvant in the form of a liposome with an influenza virus or
antigenic preparation thereof, and optionally with 3D-MPL.
[0060] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
DESCRIPTION OF FIGURES
[0061] FIG. 1--diagrammatic representation of MPL preparation.
[0062] FIG. 2--Humoral response against various strains of
influenza following immunization of ferrets with experimental
formulations: Hemagglutination Inhibition Test (GMT+/-IC95) before
and after heterologous priming (H1N1 A/Stockholm/24/90), after
immunization (H1N1 A/New Caledonia/20/99, H3N2 A/Panama/2007/99 and
B/Shangdong/7/97) and after heterologous challenge (H3N2
A/Wyoming/3/2003)
[0063] FIG. 3--Ferret study: Viral titration in nasal washes after
challenge (Day 42)
[0064] FIG. 4--Mice study: Humoral response against the three
vaccine strains of influenza following immunization of mice with
experimental formulations: Hemagglutination Inhibition Test
(GMT+/-IC95) 21 days after immunization (H1N1 A/New
Caledonia/20/99, H3N2 A/Wyoming/3/2003 and B/Jiangsu/10/2003).
[0065] FIG. 5--Mice study: Cell mediated immune response:
Flu-specific CD4+ T cell responses on Day 7 Post-immunization.
[0066] FIG. 6--Mice study: CMI for CD4--Pooled strain (all
double)--Day 0 and Day 21
[0067] FIG. 7--GMTs at days 0 and 21 for HI antibodies
[0068] FIG. 8: Incidence of local and general symptoms in humans
(Total and grade 3 related) reported during the 7-day follow up
period following immunisation with adjuvanted influenza virus
formulations, comparing adjuvants having two different
concentrations of immunostimulants.
[0069] FIG. 9: Humoral responses to HPV 16 and 18 L1 in mice
following immunisation with adjuvanted HPV formulations, comparing
adjuvants having two different concentrations of
immunostimulants
[0070] FIG. 10: Cell mediated immune response in mice:
Intracellular Cytokine Staining--VLP16 and 18 CD4+ T cells
following immunisation with adjuvanted HPV formulations, comparing
adjuvants having two different concentrations of
immunostimulants
[0071] FIG. 11: Production of Specific B Memory cells following
immunisation with adjuvanted HPV formulations, comparing adjuvants
having two different concentrations of immunostimulants
[0072] FIG. 12: Preclinical comparison of adjuvanted S. pneumonaie
vaccines in mice, comparing adjuvants having two different
concentrations of immunostimulants.
[0073] FIG. 13: Guinea pig Anti-gB ELISA titers following
immunisation with adjuvanted Gb vaccine, comparing adjuvants having
two different concentrations of immunostimulants.
[0074] FIG. 14: Guinea Pig Anti CMV neutralizing titers following
immunisation with adjuvanted Gb vaccine, comparing adjuvants having
two different concentrations of immunostimulants.
[0075] FIG. 15: Mice Anti-gB ELISA titers following immunisation
with adjuvant gB vaccine.
[0076] FIG. 16: Mice anti CMV neutralising titers following
immunisation with adjuvanted gB vaccine.
[0077] FIG. 17: Mice study: Cell Mediated immunity--CMV specific
CD4+ and CD8+ cells following re-stimulation with a pool of gB
peptides (7 days post second immunisation)
[0078] FIG. 18: Mice study. Cell Mediated immunity--CMV specific
CD4+ cells following re-stimulation with two different dosages of a
pool of gB peptides (21 days post second immunisation).
[0079] FIG. 19: Mice study. Cell Mediated immunity--CMV specific
CD8+ cells following re-stimulation with two different dosages of a
pool of gB peptides (21 days post second immunisation).
[0080] FIG. 20: Geometric mean antibody titers (GMT) against
Circumsporozoite protein CSP following immunization with adjuvanted
RTS,S vaccine in mice; comparing adjuvants having immunostimulants
at two different concentrations.
[0081] FIG. 21: Geometric mean antibody titers (GMT) against
Hepatitis B surface antigen (HBs) following immunization with
adjuvanted RTS,S vaccine in mice; comparing adjuvants with
immunostimulants at two different concentrations.
[0082] FIG. 22: Ex vivo expression of IL-2 and/or IFN gamma by
CSP-specific CD4 and CD8 T cells following immunization with an
adjuvanted RTS,S immunogenic composition, comparing adjuvants with
immunostimulants at two different concentrations.
[0083] FIG. 23: Ex vivo expression of IL-2 and/or IFN gamma by
HBs-specific CD4 and CD8 T cells following immunization with an
adjuvanted RTS,S immunogenic composition, comparing adjuvants with
immunostimulants at two different concentrations.
[0084] FIG. 24: Humoral responses in mice following immunisation
with adjuvanted trivalent split influenza vaccine (A/New Caledonia,
A/Wyoming, B/Jiangsu), immunostimulants at two different
concentrations.
[0085] FIG. 25: Cell mediated immune response in mice following
immunisation with adjuvanted trivalent influenza vaccine (A/New
Caledonia, A/Wyoming, B/Jiangsu), immunostimulants at two different
concentrations.
[0086] FIG. 26: Preclinical results in mice comparing VZV gE
vaccines adjuvant with AS01B or AS01E.
[0087] FIG. 27: viral nasal wash titres following priming and
challenge with influenza virus antigens--immunisation with A/New
Caledonia, A/Wyoming, B/Jiangsu either plain or adjuvanted with
adjuvant compositions comprising immunostimulants at two different
concentrations, in ferrets
[0088] FIG. 28: Body temperature monitoring in ferrets following
priming and challenge with influenza antigens. Immunisation with
A/New Caledonia, A/Wyoming, B/Jiangsu either plain or adjuvanted
with adjuvant compositions comprising immunostimulants at two
different concentrations,
[0089] FIG. 29: Anti HI titers for the A strains in the trivalent
vaccine formulation following immunisation and challenge with
influenza antigen preparations. Immunisation with A/New Caledonia,
A/Wyoming, B/Jiangsu either plain or adjuvanted with adjuvant
compositions comprising immunostimulants at two different
concentrations,
[0090] FIG. 30: Anti HI titres for B/Jiangsu and the drift strain
used for challenge following immunisation and challenge with
influenza antigen preparations. Immunisation with A/New Caledonia,
A/Wyoming, B/Jiangsu either plain or adjuvanted with adjuvant
compositions comprising immunostimulants at two different
concentrations,
DETAILED DESCRIPTION
[0091] The present inventors have discovered that an adjuvant
composition which comprises a saponin presented in the form of a
liposome, and a lipopolysaccharide, where each immunostimulant is
present at a level at or below 30 .mu.g per human dose can improve
immune responses to an antigenic preparation, whilst at the same
time having lower reactogenicity than some of the prior art
formulations where the immunostimulants were present at higher
levels per human dose.
[0092] The present inventors have further found that an influenza
formulation comprising an influenza virus or antigenic preparation
thereof together with an adjuvant comprising a saponin presented in
the form of a liposome, and optionally additionally with a lipid A
derivative such as 3D-MPL, was capable of improving the CD4 T-cell
immune response against said antigen or antigenic composition
compared to that obtained with the un-adjuvanted virus or antigenic
preparation thereof. The formulations adjuvanted with saponin
presented in the form of a liposome are advantageously 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 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).
[0093] It is a specific embodiment of the present invention that
the compositions for use in the present invention may be able to
provide, in humans, better sero-protection against influenza
following revaccination, as assessed by the number of human
subjects meeting the influenza correlates of protection.
Furthermore, it is another specific embodiment that the composition
for use in the present invention will also be able to induce a
higher B cell memory response following the first vaccination of a
human subject, and a higher humoral response following
revaccination, compared to the un-adjuvanted composition.
[0094] The adjuvanted influenza compositions according to the
invention have several advantages: [0095] 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); [0096] 2) An improved cross-protection profile:
increased cross-protection against variant (drifted) influenza
strains; [0097] 3) They will also allow a reduced antigen dosage to
be used for a similar response, thus ensuring an increased capacity
in case of emergency (pandemics for example).
[0098] In another aspect of the invention, the inventors have
discovered that the adjuvant composition as defined herein
demonstrates immunogenicity results for both antibody production
and post-vaccination frequency of influenza-specific CD4 which are
equivalent to, or sometimes greater than, those generated with
non-adjuvanted vaccine. This effect is in particular of value in
the elderly population and can be achieved with an adjuvant as
herein defined containing a lower dose of immunostimulants. In
addition, reactogenicity symptoms showed a trend to be higher in
the group who received the vaccine adjuvanted with the highest
immunostimulants concentration compared to the group who received
the adjuvanted vaccine wherein the immunostimulants is at a lower
concentration.
[0099] These findings can be applied to other forms of the same
antigens, and to other antigens.
Saponin Adjuvant
[0100] The adjuvant composition of the invention comprises a
saponin adjuvant presented in the form of a liposome.
[0101] A particularly suitable saponin for use in the present
invention is Quil A and its derivatives. Quil A is a saponin
preparation isolated from the South American tree Quillaja
Saponaria Molina and was first described by Dalsgaard et al. in
1974 ("Saponin adjuvants", Archiv. fur die gesamte Virusforschung,
Vol. 44, Springer Verlag, Berlin, p 243-254) to have adjuvant
activity. Purified fragments of Quil A have been isolated by HPLC
which retain adjuvant activity without the toxicity associated with
Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7
and QA21). QS-21 is a natural saponin derived from the bark of
Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells
(CTLs), Th1 cells and a predominant IgG2a antibody response and is
a preferred saponin in the context of the present invention.
[0102] In a suitable form of the present invention, the saponin
adjuvant within the immunogenic composition is a derivative of
saponaria molina quil A, preferably an immunologically active
fraction of Quil A, such as QS-17 or QS-21, suitably QS-21. In one
embodiment the compositions of the invention contain the
immunologically active saponin fraction in substantially pure form.
Preferably the compositions of the invention contain QS21 in
substantially pure form, that is to say, the QS21 is at least 90%
pure, for example at least 95% pure, or at least 98% pure.
[0103] In a specific embodiment, QS21 is provided in its less
reactogenic composition where it is quenched with an exogenous
sterol, such as cholesterol for example. Several particular forms
of less reactogenic compositions wherein QS21 is quenched with an
exogenous cholesterol exist. In a specific embodiment, the
saponin/sterol is in the form of a liposome structure (WO 96/33739,
Example 1). In this embodiment the liposomes suitably contain a
neutral lipid, for example phosphatidylcholine, which is suitably
non-crystalline at room temperature, for example eggyolk
phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or
dilauryl phosphatidylcholine. The liposomes may also contain a
charged lipid which increases the stability of the lipsome-QS21
structure for liposomes composed of saturated lipids. In these
cases the amount of charged lipid is suitably 1-20% w/w, preferably
5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol),
suitably 20-25%.
[0104] Suitable sterols include .beta.-sitosterol, stigmasterol,
ergosterol, ergocalciferol and cholesterol. In one particular
embodiment, the adjuvant composition comprises cholesterol as
sterol. These sterols are well known in the art, for example
cholesterol is disclosed in the Merck Index, 11th Edn., page 341,
as a naturally occurring sterol found in animal fat.
[0105] Adjuvant compositions of the invention comprising QS21 and a
sterol, cholesterol in particular, show a decreased reactogenicity
when compared to compositions in which the sterol is absent, while
the adjuvant effect is maintained. Reactogenicity studies may be
assessed according to the methods disclosed in WO 96/33739. The
sterol according to the invention is taken to mean an exogenous
sterol, i.e. a sterol which is not endogenous to the organism from
which the antigenic preparation is taken but is added to the
antigen preparation or subsequently at the moment of formulation.
Typically, the sterol may be added during subsequent formulation of
the antigen preparation with the saponin adjuvant, by using, for
example, the saponin in its form quenched with the sterol. Suitably
the exogenous sterol is associated to the saponin adjuvant as
described in WO 96/33739.
[0106] Where the active saponin fraction is QS21, the ratio of
QS21:sterol will typically be in the order of 1:100 to 1:1 (w/w),
suitably between 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1
(w/w). Suitably excess sterol is present, the ratio of QS21:sterol
being at least 1:2 (w/w). In one embodiment, the ratio of
QS21:sterol is 1:5 (w/w). The sterol is suitably cholesterol.
[0107] Other useful saponins are derived from the plants Aesculus
hippocastanum or Gyophilla struthium. Other saponins which have
been described in the literature include Escin, which has been
described in the Merck index (12.sup.th ed: entry 3737) as a
mixture of saponins occurring in the seed of the horse chestnut
tree, Lat: Aesculus hippocastanum. Its isolation is described by
chromatography and purification (Fiedler, Arzneimittel-Forsch. 4,
213 (1953)), and by ion-exchange resins (Erbring et al., U.S. Pat.
No. 3,238,190). Fractions of escin have been purified and shown to
be biologically active (Yoshikawa M, et al. (Chem Pharm Bull
(Tokyo) 1996 August; 44(8):1454-1464)). Sapoalbin from Gypsophilla
struthium (R. Vochten et al., 1968, J. Pharm. Belg., 42, 213-226)
has also been described in relation to ISCOM production for
example.
[0108] A key aspect of the present invention is the fact that the
immunologically active saponin, which is preferably QS21, can be
used at lower amounts than had previously been thought useful,
suitably at below 30 .mu.g, for example between 1 and 30 .mu.g, per
human dose of the immunogenic composition.
[0109] The invention therefore provides a human dose of an
immunogenic composition comprising immunologically active saponin,
preferably QS21, at a level of 30 .mu.g or less, for example
between 1 and 30 .mu.g.
[0110] In one embodiment, an immunogenic composition in a volume
which is suitable for a human dose which human dose of the
immunogenic composition comprises QS21 at a level of around 25
.mu.g, for example between 20-30 .mu.g, suitably between 21-29
.mu.g or between 22 and 28 .mu.g or between 23 and 27 .mu.g or
between 24 and 26 .mu.g, or 25 .mu.g. In another embodiment, the
human dose of the immunogenic composition comprises QS21 at a level
of around 10 .mu.g per, for example between 5 and 15 .mu.g,
suitably between 6 and 14 .mu.g, for example between 7 and 13 .mu.g
or between 8 and 12 .mu.g or between 9 and 11 .mu.g, or 10
.mu.g.
[0111] In a further embodiment, the human dose of the immunogenic
composition comprises QS21 at a level of around 5 .mu.g, for
example between 1 and 9 .mu.g, or between 2 and 8 .mu.g or suitably
between 3 and 7 .mu.g or 4 and 6 .mu.g, or 5 .mu.g.
[0112] A suitable amount of QS21 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, .mu.g (w/v) per human dose of the
immunogenic composition.
[0113] By the term "human dose" is meant a dose which is in a
volume suitable for human use. Generally this is between 0.3 and
1.5 ml. In one embodiment, a human dose is 0.5 ml. In a further
embodiment, a human dose is higher than 0.5 ml, for example 0.6,
0.7, 0.8, 0.9 or 1 ml. In a further embodiment, a human dose is
between 1 ml and 1.5 ml. The invention is characterised in that
each human dose contains 30 .mu.g or less, for example between 1
and 30 .mu.g, of QS21.
[0114] The invention further provides an adjuvant composition
comprising 30 .mu.g or less, for example between 1 and 30 .mu.g, of
QS21. Typically such an adjuvant composition will be in a human
dose suitable volume. Where the adjuvant is in a liquid form to be
combined with a liquid form of an antigenic composition, the
adjuvant composition will be in a human dose suitable volume which
is approximately half of the intended final volume of the human
dose, for example a 360 .mu.l volume for an intended human dose of
0.7 ml, or a 250 .mu.l volume for an intended human dose of 0.5 ml.
The adjuvant composition is diluted when combined with the antigen
composition to provide the final human dose of vaccine. The final
volume of such dose will of course vary dependent on the initial
volume of the adjuvant composition and the volume of antigen
composition added to the adjuvant composition. In an alternative
embodiment, liquid adjuvant is used to reconstitute a lyophilised
antigen composition. In this embodiment, the human dose suitable
volume of the adjuvant composition is approximately equal to the
final volume of the human dose. The liquid adjuvant composition is
added to the vial containing the lyophilised antigen composition.
The final human dose can vary between 0.5 and 1.5 ml. In a
particular embodiment the human dose is 0.5 ml, in this embodiment
the vaccine composition of the invention will comprise a level of
QS21 at or below 30 .mu.g, for example between 1 and 30 .mu.g, per
0.5 ml human dose, furthermore in this embodiment an adjuvant
composition of the invention will comprise a level of QS21 at or
below 30 .mu.g, for example between 1 and 30 .mu.g, per 250 .mu.l
of adjuvant composition, or per 500 .mu.l of adjuvant composition
dependent on whether the adjuvant composition is intended to be
combined with a liquid or lyophilised antigen composition
respectively.
[0115] Specifically when combined with an influenza antigen, an
amount of QS21 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 QS21 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, QS21
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 QS21 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 QS21 is contained per ml of vaccine composition.
Specifically, a 0.5 ml vaccine dose volume contains 25 .mu.g or 50
.mu.g of QS21 per dose.
[0116] The dose of QS21 is suitably able to enhance an immune
response to an antigen in a human. In particular a suitable QS21
amount is that which improves the immunological potential of the
composition compared to the unadjuvanted composition, or compared
to the composition adjuvanted with another QS21 amount, whilst
being acceptable from a reactogenicity profile.
3D-MPL Adjuvant
[0117] The composition further comprises an additional adjuvant
which is a lipopolysaccharide, suitably a non-toxic derivative of
lipid A, particularly monophosphoryl lipid A or more particularly
3-Deacylated monophosphoryl lipid A (3D-MPL).
[0118] 3D-MPL is sold under the name MPL by GlaxoSmithKline
Biologicals N.A. and is referred throughout the document as MPL or
3D-MPL. see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;
4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4+ T cell
responses with an IFN-g (Th1) phenotype. 3D-MPL 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 3D-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.
[0119] A key aspect of the present invention is the fact that the
lipopolysaccharide, which is preferably 3D-MPL, can be used at
lower amounts than had previously been thought useful, suitably at
a level of 30 .mu.g or less, for example between 1 and 30 .mu.g,
per human dose of the immunogenic composition.
[0120] The invention therefore provides a human dose of an
immunogenic composition comprising lipopolysaccharide, preferably
3D-MPL, at a level of 30 .mu.g or less, for example between 1 and
30 .mu.g.
[0121] In one embodiment, the human dose of the immunogenic
composition comprises 3D-MPL at a level of around 25 .mu.g, for
example between 20-30 .mu.g, suitably between 21-29 .mu.g or
between 22 and 28 .mu.g or between 23 and 27 .mu.g or between 24
and 26 .mu.g, or 25 .mu.g.
[0122] In another embodiment, the human dose of the immunogenic
composition comprises 3D-MPL at a level of around 10 .mu.g, for
example between 5 and 15 .mu.g, suitably between 6 and 14 .mu.g,
for example between 7 and 13 .mu.g or between 8 and 12 .mu.g or
between 9 and 11 .mu.g, or 10 .mu.g.
[0123] In a further embodiment, the human dose of the immunogenic
composition comprises 3D-MPL at a level of around 5 .mu.g, for
example between 1 and 9 .mu.g, or between 2 and 8 .mu.g or suitably
between 3 and 7 .mu.g or 4 and 6 .mu.g, or 5 .mu.g.
[0124] 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, .mu.g (w/v) per human dose of
the immunogenic composition.
[0125] In one embodiment, the volume of the human dose is 0.5 ml.
In a further embodiment, the immunogenic composition is in a volume
suitable for a human dose which volume is higher than 0.5 ml, for
example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, the
human dose is between 1 ml and 1.5 ml. The invention is
characterised in that each human dose contains 30 .mu.g or less,
for example between 1 and 30 .mu.g of 3D-MPL.
[0126] The invention further provides an adjuvant composition
comprising 30 .mu.g or less, for example between 1 and 30 .mu.g, of
3D-MPL. Typically such an adjuvant composition will be in a human
dose suitable volume. Where the adjuvant is in a liquid form to be
combined with a liquid form of an antigenic composition, the
adjuvant composition will be in a human dose suitable volume which
is approximately half of the intended final volume of the human
dose, for example a 360 .mu.l volume for an intended human dose of
0.7 ml, or a 250 .mu.l volume for an intended human dose of 0.5 ml.
The adjuvant composition is diluted when combined with the antigen
composition to provide the final human dose of immunogenic
composition. The final volume of such dose will of course vary
dependent on the initial volume of the adjuvant composition and the
volume of antigen composition added to the adjuvant composition. In
an alternative embodiment, liquid adjuvant composition is used to
reconstitute a lyophilised antigen composition. In this embodiment,
the human dose suitable volume of the adjuvant composition is
approximately equal to the final volume of the human dose. The
liquid adjuvant composition is added to the vial containing the
lyophilised antigen composition. The final human dose can vary
between 0.5 and 1.5 ml. In a particular embodiment the human dose
is 0.5 ml, in this embodiment the vaccine composition of the
invention will comprise a level of 3D-MPL at or below 30 .mu.g, for
example between 1 and 30 .mu.g, per 0.5 ml human dose, furthermore
in this embodiment an adjuvant composition of the invention will
comprise a level of 3D-MPL at or below 30 .mu.g, for example
between 1 and 30 .mu.g, per 250 .mu.l of adjuvant composition, or
per 500 .mu.l of adjuvant composition dependent on whether the
adjuvant composition is intended to be combined with a liquid or
lyophilised antigen composition respectively.
[0127] When the immunogenic composition contains an influenza virus
or antigenic preparation thereof, the adjuvant composition which
comprises a saponin in the form of a liposome optionally
additionally contains a lipid A derivative, particularly
monophosphoryl lipid A or more particularly 3D-MPL. In this
embodiment, 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 one 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 another embodiment, a final
concentration of 35.7 .mu.g or 71.4 .mu.g of 3D-MPL is contained
per ml of vaccine composition. Specifically, a 0.5 ml vaccine dose
volume contains 25 .mu.g or 50 .mu.g of 3D-MPL per dose.
[0128] The dose of 3D-MPL is suitably able to enhance an immune
response to an antigen in a human. In particular a suitable 3D-MPL
amount is that which improves the immunological potential of the
composition compared to the unadjuvanted composition, or compared
to the composition adjuvanted with another MPL amount, whilst being
acceptable from a reactogenicity profile.
[0129] Suitable compositions of the invention are those wherein
liposomes are initially prepared without MPL (as described in WO
96/33739), and MPL is then added, suitably as small particles of
below 100 nm particles or particles that are susceptible to sterile
filtration through a 0.22 .mu.m membrane. The MPL is therefore not
contained within the vesicle membrane (known as MPL out).
Compositions where the MPL is contained within the vesicle membrane
(known as MPL in) also form an aspect of the invention. The antigen
can be contained within the vesicle membrane or contained outside
the vesicle membrane. Suitably soluble antigens are outside and
hydrophobic or lipidated antigens are either contained inside or
outside the membrane.
[0130] In one embodiment the adjuvant composition of the invention
comprises both lipopolysaccharide and immunologically active
saponin. In a specific embodiment of the invention, the
lipopolysaccharide is 3D-MPL and the immunologically active saponin
is QS21. In a further embodiment of the invention, the adjuvant
composition consists essentially of a lipopolysaccharide and
immunologically active saponin in a liposomal formulation. Suitably
in one form of this embodiment, the adjuvant composition consists
essentially of 3D-MPL and QS21, with optionally sterol which is
preferably cholesterol.
[0131] In a further embodiment of the invention, the adjuvant
composition comprises in a liposomal formulation lipopolysaccharide
and immunologically active saponin in combination with one or more
further immunostimulants or adjuvants. Suitably in one form of this
embodiment the lipopolysaccharide is 3D-MPL and the immunologically
active saponin is QS21.
[0132] In a specific embodiment, QS21 and 3D-MPL are present in the
same final concentration per human dose of the immunogenic
composition. In one aspect of this embodiment, a human dose of
immunogenic composition comprises a final level of 25 .mu.g of
3D-MPL and 25 .mu.g of QS21. In a further embodiment, a human dose
of immunogenic composition comprises a final level of 10 .mu.g each
of MPL and QS21. In a further specific embodiment is provided an
adjuvant composition having a volume of 250 .mu.l and comprising a
level of 25 .mu.g of 3D-MPL and 25 .mu.g of QS21, or 10 .mu.g each
of MPL and QS21.
[0133] Antigens that may be used with the adjuvant compositions of
the present invention include viral, parasitic, bacterial or tumour
associated antigens, for example:
[0134] An influenza virus or antigenic preparation thereof for use
according to the present invention, which 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.
[0135] 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.
[0136] Alternatively, the influenza virus may be in the form of a
whole virus vaccine. This may prove to be an advantage over a split
virus vaccine for a pandemic situation as it avoids the uncertainty
over whether a split virus vaccine can be successfully produced for
a new strain of influenza virus. For some strains the conventional
detergents used for producing the split virus can damage the virus
and render it unusable. Although there is always the possibility to
use different detergents and/or to develop a different process for
producing a split vaccine, this would take time, which may not be
available in a pandemic situation. In addition to the greater
degree of certainty with a whole virus approach, there is also a
greater vaccine production capacity than for split virus since
considerable amounts of antigen are lost during additional
purification steps necessary for preparing a suitable split
vaccine.
[0137] In another embodiment, the influenza virus preparation is in
the form of a purified sub-unit influenza vaccine. Sub-unit
influenza vaccines generally contain the two major envelope
proteins, HA and NA, and may have an additional advantage over
whole virion vaccines as they are generally less reactogenic,
particularly in young vaccines. Sub-unit vaccines can be produced
either recombinantly or purified from disrupted viral
particles.
[0138] 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.
[0139] Said influenza virus or antigenic preparation thereof may be
egg-derived or cell-culture derived.
[0140] 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 cell or
cell 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.
[0141] 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.
[0142] 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.
[0143] 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).
[0144] In a specific embodiment, the final concentration for Tween
80 ranges from 0.025%-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 0.025%-0.2% (w/v) and has to
be diluted two times upon final formulation with the adjuvanted (or
the buffer in the control formulation).
[0145] In another specific embodiment, the final concentration for
Triton X-100 ranges from 0.004%-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).
[0146] 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.
[0147] A preferred composition contains three inactivated split
virion antigens prepared from the WHO recommended strains of the
appropriate influenza season.
[0148] Preferably the influenza virus or antigenic preparation
thereof and the adjuvant according to the invention 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 adjuvant according to the invention are contained in separate
containers or vials and admixed shortly before or upon
administration into the subject. It is referred to as `two vials
approach`. By way of example, when the vaccine is a 2 components
vaccine for a total dose volume of 0.7 ml, the concentrated
antigens (for example the concentrated trivalent inactivated split
virion antigens) are presented in one vial (335 .mu.l) (antigen
container) and a pre-filled syringe contains the adjuvant (360
.mu.l) (adjuvant container). At the time of injection, the content
of the vial containing the concentrated trivalent inactivated split
virion antigens is removed from the vial by using the syringe
containing the adjuvant followed by gentle mixing of the syringe.
Prior to injection, the used needle is replaced by an intramuscular
needle and the volume is corrected to 530 .mu.l. In this example,
one dose of the reconstituted adjuvanted influenza vaccine
candidate corresponds to 530 .mu.l.
[0149] In one aspect of the invention, where there is a multivalent
composition, then at least one influenza strain in said multivalent
immunogenic composition as herein defined is associated with a
pandemic outbreak or have the potential to be associated with a
pandemic outbreak. Such strain may also be referred to as `pandemic
strains` in the text below. In particular, when the vaccine is a
multivalent vaccine such as a bivalent, or a trivalent or a
quadrivalent vaccine, at least one strain is associated with a
pandemic outbreak or has the potential to be associated with a
pandemic outbreak. Suitable strains are, but not limited to: H5N1,
H9N2, H7N7, and H2N2.
[0150] Said influenza virus or antigenic preparation thereof is
suitably multivalent such as bivalent or trivalent or quadrivalent.
Preferably the influenza virus or antigenic preparation thereof is
trivalent or quadrivalent, having an antigen from three different
influenza strains, at least one strain being associated with a
pandemic outbreak or having the potential to be associated with a
pandemic outbreak.
[0151] The features of an influenza virus strain that give it the
potential to cause a pandemic outbreak are: it contains a new
haemagglutinin compared to the haemagglutinin in the currently
circulating strains; it is capable of being transmitted
horizontally in the human population; and it is pathogenic for
humans. A new haemagglutinin may be one which has not been evident
in the human population for an extended period of time, probably a
number of decades, such as H2. Or it may be a haemagglutinin that
has not been circulating in the human population before, for
example H5, H9, H7 or H6 which are found in birds. In either case
the majority, or at least a large proportion of, or even the entire
population has not previously encountered the antigen and is
immunologically naive to it.
[0152] 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.
[0153] 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.
[0154] 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, H9N.sub.2, H7N7, and H2N2.
[0155] Optionally the composition may contain more than three
valencies, for example three non pandemic strains plus a pandemic
strain. Alternatively the composition may contain three pandemic
strains. Preferably the composition contains three pandemic
strains.
[0156] Also examples of antigens for the immunogenic composition of
the invention are Streptococcal antigens such as from Group A
Streptococcus, or Group B Streptococcus, but most preferably from
Streptococcus pneumoniae. At least one protein and/or at least one
saccharide antigen is most preferably used. The 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 (for instance fusions
of domains of the above proteins, for instance the PhtDE fusion
proteins described in WO01/98334 and WO 03/54007).
[0157] Certain compositions are described in WO 00/56359 and WO
02/22167 and WO 02/22168 (incorporated by reference herein).
[0158] 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. In one embodiment the four
serotypes include 6B, 14, 19F and 23F. In a further embodiment, at
least 7 serotypes are included in the composition, for example
those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. In a
further embodiment, at least 10 serotypes are included in the
composition, for example the composition in one embodiment includes
capsular saccharides derived from serotypes 1, 4, 5, 6B, 7F, 9V,
14, 18C, 19F and 23F (preferably conjugated to a carrier protein).
In another embodiment, the immunogenic composition comprises
capsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V,
14, 18C, 19F and 23F 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.
[0159] 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.
[0160] 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
[0161] 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):
[0162] 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.
[0163] 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.
[0164] 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.
[0165] PhtE is disclosed in WO00/30299 and is referred to as
BVH-3.
[0166] SpsA is a Choline binding protein (Cbp) disclosed in WO
98/39450.
[0167] 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.
[0168] 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.
[0169] 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. Sp101 is disclosed in WO
98/06734 (where it has the reference # y85993. It is characterised
by a Type I signal sequence.
[0170] Sp133 is disclosed in WO 98/06734 (where it has the
reference # y85992. It is also characterised by a Type I signal
sequence.
[0171] Sp128 and Sp130 are disclosed in WO 00/76540. 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).
[0172] 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). Concerning the Choline Binding Protein
family, members of that family were originally identified as
pneumococcal proteins that could be purified by choline-affinity
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.
[0173] 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 NR1xR2, R1xR2,
NR1xR2P and R1xR2P 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 NR1xR2 (or R1xR2 or NR1xR2P or R1xR2P) 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).
[0174] 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.
[0175] In a pneumococcal composition it is possible to combine
different pneumococcal proteins of the invention. 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. Preferred beneficial combinations include,
but are not limited to, PhtD+NR1xR2, PhtD+NR1xR2-Sp91Cterm chimeric
or fusion proteins, PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA,
PhtA+NR1xR2, PhtA+NR1xR2-Sp91Cterm chimeric or fusion proteins,
PhtA+Ply, PhtA+Sp128, PhtA+PsaA, PhtA+PspA, NR1xR2+LytC,
NR1xR2+PspA, NR1xR2+PsaA, NR1xR2+Sp128, R1xR2+LytC, R1xR2+PspA,
R1xR2+PsaA, R1xR2+Sp128, R1xR2+PhtD, R1xR2+PhtA. Preferably, NR1xR2
(or R1xR2) is from CbpA or PspC. More preferably it is from
CbpA.
[0176] 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 NR1xR2 (or R1xR2 or
NR1xR2P or R1xR2P). Preferably, NR1xR2 (or R1xR2 or NR1xR2P or
R1xR2P) is from CbpA or PspC. More preferably it is from CbpA.
[0177] 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 ten 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).
[0178] 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).
[0179] 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.
[0180] A further preferred carrier for the pneumococcal saccharide
is the pneumococcal protein itself (as defined above in section
"Pneumococcal Proteins of the invention").
[0181] 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).
[0182] 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.
[0183] 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.
[0184] In another aspect of the invention, the vaccine composition
may comprise a human cytomegalovirus (HCMV) antigen. HCMV is a
human DNA virus belonging to the family of herpes viruses, and is a
major cause of congenital defects in newborns and also causes
serious medical conditions in immunocompromised patients. Clinical
disease causes a variety of symptoms including fever, hepatitis,
pneumonititis and infectious mononucleosis.
[0185] In one embodiment, the HCMV antigen is a chimeric fusion
protein or an immunogenic derivative thereof comprising a portion
of an HCMV glycoprotein fused to a portion of an HSV glycoprotein.
The HCMV glycoprotein is typically gB, and the HSV glycoprotein is
typically gD, in particular HSV type 2 gD (gD2). The fusion is
typically between an amino acid in the N-terminal part of a portion
of the HCMV gB protein and an amino acid at the C terminus of a
portion of the HSV gD protein. Both the HCMV gB protein and the HSV
gD protein components of the fusion protein may lack a membrane
anchor domain.
[0186] The portion of the HCMV gB protein may comprise a
non-cleavable form of HCMV gB. Suitably this is achieved by
changing one or more amino acids at a cleavage site of the protein,
for example by exchanging Arg458 and Arg459 for Glu and Thr,
respectively. The portion of the HSV protein may comprise the
signal sequence of gD2 (amino acids 1 to 25) and optionally amino
acids 26 to 52 of gD2 and/or the sequence from gD2 which is
PEDSALLEDPED or functionally equivalents thereof, which may be
shorter or longer. Further sequences from HSV gD may be added to
the fusion protein, for example at the C terminus of the HCMV gB
protein.
[0187] In one embodiment, the fusion protein comprises amino acids
1 to 52 of the HSV gD2 protein fused to residues 28 to 685 of the
HCMV gB protein. Such a fusion protein is designated HCMV gB685*.
In a further embodiment, the amino acid sequence PEDSALLEDPED,
which is derived from an internal gD2 sequence, may be included at
the C terminal end of the protein HCMV gB685* to produce the
protein designated HCMV gB685**. These specific fusion proteins are
described in more detail in WO 95/31555.
[0188] Another immunogen suitable for use as an HCMV vaccine is
pp65, an HCMV matrix protein as described in WO 94/00150 (City of
Hope).
[0189] In a further embodiment of the present invention,
immunogenic compositions contain an antigen or antigenic
preparation derived from the Human Papilloma Virus (HPV) considered
to be responsible for genital warts (HPV 6 or HPV 11 and others),
and/or the HPV viruses responsible for cervical cancer (HPV16,
HPV18 and others).
[0190] In one embodiment the 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.
[0191] In one embodiment the 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).
[0192] A preferred HPV cervical infection or cancer, prophylactic
or therapeutic composition 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 protein alone
presented in a VLP or caposmer structure. Such antigens, virus like
particles and capsomers are per se known. See for example
WO94/00152, WO94/20137, WO94/05792, and WO93/02184.
[0193] 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).
[0194] In one embodiment the 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).
[0195] 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.
[0196] The composition of the present invention may additionally
comprise antigens from other HPV types, preferably from HPV 31 or
33.
[0197] Oncogenic HPV types include HPV 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 66 and 68. Thus the composition of the present
invention may comprise antigens from one or more of these HPV
types, in addition to HPV 16 and/or HPV 18.
[0198] HPV L1 VLPs or capsomers useful in the invention may
comprise or consist of full length L1 or an immunogenic fragment of
L1. Where the VLP or capsomer comprises or consists of an
immunogenic fragment of L1, then suitable immunogenic fragments of
HPV L1 include truncations, deletions, substitution, or insertion
mutants of L1. Such immunogenic fragments are suitably capable of
raising an immune response, said immune response being capable of
recognising an L1 protein such as a virus like particle, from the
HPV type from which the L1 protein was derived.
[0199] Suitable immunogenic L1 fragments include truncated L1
proteins. In one aspect the truncation removes a nuclear
localisation signal. In another aspect the truncation is a C
terminal truncation. In a further aspect the C terminal truncation
removes fewer than 50 amino acids, such as fewer than 40 amino
acids. Where the L1 is from HPV 16 then in another aspect the C
terminal truncation removes 34 amino acids from HPV 16 L1. Where
the L1 is from HPV 18 then in a further aspect the C terminal
truncation removes 35 amino acids from HPV 18 L1.
[0200] Suitable truncated HPV 16 and 18 L1 sequences are given in
WO 06/114312. The HPV 16 sequence may also be that disclosed in
WO9405792 or U.S. Pat. No. 6,649,167, for example, suitably
truncated. Suitable truncates are truncated at a position
equivalent to that discussed above, as assessed by sequence
comparison.
[0201] An alternative HPV 18 sequence is disclosed in WO9629413,
which may be suitably truncated. Suitable truncates are truncated
at a position equivalent to that described above, as assessed by
sequence comparison.
[0202] Other HPV 16 and HPV 18 sequences are well known in the art
and may be suitable for use in the present invention.
[0203] Where there is an L1 protein from another HPV type then C
terminal truncations corresponding to those made for HPV 16 and HPV
18 may be used, based upon DNA or protein sequence alignments.
Suitable truncations of HPV 31 and 45 L1 proteins are given in WO
06/114312.
[0204] Suitable truncations of, for example, HPV 31, 33, 35, 39,
45, 51, 52, 56, 58, 59, 66 and 68 may also be made, in one aspect
removing equivalent C terminal portions of the L1 protein to those
described above, as assessed by sequence alignment.
[0205] The L1 protein or immunogenic fragment of the invention may
optionally be in the form of a fusion protein, such as the fusion
of the L1 protein with L2 or an early protein.
[0206] The HPV L1 protein is suitably in the form of a capsomer or
virus like particle (VLP). In one aspect HPV VLPs may be used in
the present invention. HPV VLPs and methods for the production of
VLPs are well known in the art. VLPs typically are constructed from
the L1 and optionally L2 structural proteins of the virus, see for
example WO9420137, U.S. Pat. No. 5,985,610, WO9611272, U.S. Pat.
No. 6,599,508B1, U.S. Pat. No. 6,361,778B1, EP 595935. Any suitable
HPV VLP may be used in the present invention which provides cross
protection, such as an L1 or L1+L2 VLP.
[0207] Suitably the VLP is an L1-only VLP.
[0208] The composition of the invention may contain a combination
of HPV 16 L1 VLPs and HPV 18 L1 VLPs, or a combination of HPV L1
VLPs from HPV 16, 18, 31 and 45, or larger combinations, and
includes HPV 16 and 18 or HPV 16, 18, 31 and 45 L1 VLPs, or large
combinations, wherein the L1 is optionally truncated as described
herein.
[0209] In a particular embodiment of the invention, one or more
additional antigens from cancer-causing HPV types are used with HPV
16 and/or 18 antigens, the antigens being selected from the
following HPV types: HPV 31, 45, 33, 58 and 52. As described
herein, the antigen may in each case be L1 for example in the form
of L1 VLPs or capsomers. Thus HPV antigens for use in the
compositions, methods and uses described herein may comprise or
consist of L1 VLPs or capsomers from HPV 16, 18, 31, 45, 33, 58 and
52. The L1 VLPs may be L1-only VLPs or in combination with another
antigen such as L2 in L1+L2 VLPs. The L1 protein may suitably be
truncated as described herein.
[0210] VLP formation can be assessed by standard techniques such
as, for example, electron microscopy and dynamic laser light
scattering.
[0211] The VLP may comprise full length L1 protein. In one aspect
the L1 protein used to form the VLP is a truncated L1 protein, as
described above.
[0212] VLPs may be made in any suitable cell substrate such as
yeast cells or insect cells e.g. in a baculovirus expression
system, and techniques for preparation of VLPs are well known in
the art, such as WO9913056, U.S. Pat. No. 6,416,945B1, U.S. Pat.
No. 6,261,765B1 and U.S. Pat. No. 6,245,568, and references
therein, the entire contents of which are hereby incorporated by
reference. VLPS may be made by disassembly and reassembly
techniques, which can provide for more stable and/or homogeneous
papillomavirus VLPs. For example, McCarthy et al, 1998
"Quantitative Disassembly and Reassembly of Human Papillomavirus
Type 11 Virus like Particles in Vitro" J. Virology 72(1):33-41,
describes the disassembly and reassembly of recombinant L1 HPV 11
VLPs purified from insect cells in order to obtain a homogeneous
preparation of VLP's. WO9913056 and U.S. Pat. No. 6,245,568 also
describe disassembly/reassembly processes for making HPV VLPs.
[0213] In one aspect HPV VLPS are made as described WO9913056 or
U.S. Pat. No. 6,245,568
[0214] Compositions of the present invention may comprise antigens
or antigenic preparations derived from parasites that cause Malaria
such as Plasmodium falciparum or Plasmodium vivax. For example,
possible antigens derived from Plasmodium 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
MSP1, MSP3, AMA1, LSA1 or LSA3. Possible antigens from P vivax
include circumsporozoite protein (CS protein) and Duffy antigen
binding protein and fragments thereof, such as PvRII (see eg
WO02112292).
[0215] Other possible antigens that may be used in the immunogenic
compositions of the present invention include:
[0216] Streptococcal antigens such as from Group A Streptococcus,
or Group B Streptococcus, antigens are suitably derived from HIV-1,
(such as F4 antigen or fragments thereof or 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 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, PilC,
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.
[0217] 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), Mtb72F and M72. 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, Erd
14-DPV-MTI-MSL-mTCC2, Erd 14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2,
TbH9-DPV-MTI (WO 99/51748). A particular Ra12-Tbh9-R35 sequence
that may be mentioned is defined by SEQ ID No 6 of WO2006/117240
together with variants in which Ser 704 of that sequence is mutated
to other than serine, eg to Ala, and derivatives thereof
incorporating an N-terminal His tag of an appropriate length (eg
SEQ ID No 2 or 4 of WO2006/117240)".
[0218] Exemplary antigens for Chlamydia sp eg C trachomatis are
selected from CT858, CT 089, CT875, MOMP, CT622, PmpD, PmpG and
fragments thereof, SWIB and immunogenic fragments of any one
thereof (such as PmpDpd and PmpGpd) and combinations thereof.
Preferred combinations of antigens include CT858, CT089 and CT875.
Specific sequences and combinations that may be employed are
described in WO2006/104890. 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
variants or fusion proteins thereof.
[0219] 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.
[0220] 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, p 293.
Indeed these antigens are expressed in a wide range of tumour types
such as melanoma, lung carcinoma, sarcoma and bladder
carcinoma.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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 U.S. Pat. No. 5,955,306), WO 98/20117 (and also the
corresponding granted U.S. Pat. No. 5,840,871 and U.S. Pat. No.
5,786,148) (prostate-specific kallikrein) and WO 00/04149
(P703P).
[0226] 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.
[0227] 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).
[0228] Other prostate specific antigens are known from Wo98/37418,
and WO/004149. Another is STEAP PNAS 96 14523 14528 7-12 1999.
[0229] 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.
[0230] Mucin derived 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.
[0231] 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.
[0232] The compositions may contain antigens associated with
tumour-support mechanisms (e.g. angiogenesis, tumour invasion) for
example tie 2, VEGF.
[0233] 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.
[0234] 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)).
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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 adjuvant 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).
[0239] 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.
[0240] 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).
[0241] 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).
[0242] 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.
[0243] 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.
[0244] 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).
[0245] 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, p 4594-4603), 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).
[0246] 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.
Immunogenic Properties of the Immunogenic Composition Used for the
Vaccination of the Present Invention
[0247] In the present invention the immunogenic composition is
preferably capable of inducing an improved CD4 T-cell immune
response against at least one of the component antigen(s) or
antigenic composition compared to the CD4 T-cell immune response
obtained with the corresponding composition which in un-adjuvanted,
i.e. does not contain any exogeneous adjuvant (herein also referred
to as `plain composition`). In a specific embodiment, where the
immunogenic composition is an influenza composition and where the
influenza vaccine preparation is from several influenza strains,
one of which being a pandemic starin, said improved CD4 T-cell
immune response is against the pandemic influenza strain.
[0248] By "improved CD4 T-cell immune response" is meant that a
higher CD4 response is obtained in a mammal after administration of
the adjuvanted immunogenic composition than that obtained after
administration of the same composition without adjuvant. For
example, a higher CD4 T-cell response is obtained in a human
patient upon administration of an immunogenic composition
comprising an influenza virus or antigenic preparation thereof
together with adjuvant according to the invention, compared to the
response induced after administration of an immunogenic composition
comprising an influenza virus or antigenic preparation thereof
which is un-adjuvanted. Such formulation will advantageously be
used to induce anti-influenza CD4-T cell response capable of
detection of influenza epitopes presented by MHC class II
molecules.
[0249] In particular but not exclusively, said `improved CD4 T-cell
immune response` is obtained in an immunologically unprimed
patient, i.e. a patient who is seronegative to said influenza virus
or antigen. This seronegativity may be the result of said patient
having never faced such virus or antigen (so-called `naive`
patient) or, alternatively, having failed to respond to said
antigen once encountered. In a specific aspect said CD4 T-cell
immune response is obtained in an immunocompromised subject such as
an elderly, typically 65 years of age or above, or an adult younger
than 65 years of age with a high risk medical condition (`high
risk` adult), or a child under the age of two.
[0250] The improved CD4 T-cell immune response may be assessed by
measuring the number of cells producing any of the following
cytokines: [0251] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0252] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0253] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0254] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0255] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
[0256] 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.
[0257] The improved CD4 T-cell immune response conferred by an
adjuvanted influenza composition of the present invention may be
ideally obtained after one single administration. The single dose
approach will be extremely relevant for example in a rapidly
evolving outbreak situation. In certain circumstances, especially
for the elderly population, or in the case of young children (below
9 years of age) who are vaccinated for the first time against
influenza, or in the case of a pandemics, it may be beneficial to
administer two doses of the same composition for that season. The
second dose of said same composition (still considered as
`composition for first vaccination`) may be administered during the
on-going primary immune response and is adequately spaced.
Typically the second dose of the composition is given a few weeks,
or about one month, e.g. 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6
weeks after the first dose, to help prime the immune system in
unresponsive or poorly responsive individuals.
[0258] In another embodiment, the administration of said
immunogenic composition induces an improved B-memory cell response
in patients administered with the adjuvanted immunogenic
composition compared to the B-memory cell response induced in
individuals immunized with the un-adjuvanted composition. An
improved B-memory cell response is intended to mean an increased
frequency of peripheral blood B lymphocytes capable of
differentiation into antibody-secreting plasma cells upon antigen
encounter as measured by stimulation of in-vitro
differentiation.
[0259] In another embodiment, the administration of said
immunogenic composition induces an improved humoral response in
patients administered with the adjuvanted immunogenic composition
compared to the humoral response induced in individuals immunized
with the un-adjuvanted composition. Said humoral immune response
may be measured according to any of the procedure detailed in
Example I, and especially in sections I.1 (I.1.1), I.2 (I.2.1) and
I.3 (I.3.5.2). When the immunogenic composition is an influenza
composition specifically said humoral response is obtained against
homologous and heterologous strains. In particular, said
heterologous humoral immune response means a humoral response
between influenza strains, and is termed `cross-reactive` humoral
immune response. Said `cross-reactive` humoral immune response
involves the induction of response against an influenza strain
which is a variant (a drift) of the influenza strain used for
vaccination. An example of such a response is illustrated in
Example III.3.1 and in FIG. 2.
[0260] In a specific embodiment, the administration of said
adjuvanted immunogenic composition induces at least two of the
following responses: (i) an improved CD4 T-cell immune response,
(ii) an improved B-memory cell response, (iii) an improved humoral
response, against at least one of the component antigen(s) or
antigenic composition compared to either immune response obtained
with the corresponding composition which in un-adjuvanted, i.e.
does not contain any exogeneous adjuvant (herein also referred to
as `plain composition`).
[0261] In a still further specific embodiment, the vaccination with
the composition for the first vaccination, adjuvanted, has no
measurable impact on the CD8 response.
[0262] It is a specific embodiment of the invention that the
composition comprising an influenza virus or antigenic preparation
thereof formulated with saponin adjuvant presented in the form of a
liposome, in particular QS21 saponin in its quenched form with
cholesterol, is effective in promoting T cell responses in an
immuno-compromised human population. In one embodiment, said
adjuvant further comprises 3D-MPL. In particular, the
administration of a single dose of the immunogenic composition for
first vaccination as described in the invention, is capable of
providing better sero-protection, as assessed by the correlates of
protection for influenza vaccines, following revaccination against
influenza in a human elderly population, than does the vaccination
with an un-adjuvanted influenza vaccine. The claimed adjuvanted
formulation has also been able to induce an improved CD4 T-cell
immune response against influenza virus compared to that obtained
with the un-adjuvanted formulation. This finding can be associated
with an increased responsiveness upon vaccination or infection
vis-a-vis influenza antigenic exposure. Furthermore, this may also
be associated with a cross-responsiveness, i.e. a higher ability to
respond against variant influenza strains. This improved response
may be especially beneficial in an immuno-compromised human
population such as the elderly population (65 years of age and
above) and in particular the high risk elderly population. This may
result in reducing the overall morbidity and mortality rate and
preventing emergency admissions to hospital for pneumonia and other
influenza-like illness. This may also be of benefit to the infant
population (below 5 years, preferably below 2 years of age).
Furthermore it may allow inducing a CD4 T cell response which is
more persistent in time, e.g. still present one year after the
first vaccination, compared to the response induced with the
un-adjuvanted formulation.
[0263] In a specific aspect, 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.
[0264] Usually, available influenza vaccines are effective only
against infecting strains of influenza virus that have
haemagglutinin of similar antigenic characteristics. When the
infecting (circulating) influenza virus has undergone minor changes
(such as a point mutation or an accumulation of point mutations
resulting in amino acid changes in the for example) in the surface
glycoproteins in particular haemagglutinin (antigenic drift variant
virus strain) the vaccine may still provide some protection,
although it may only provide limited protection as the newly
created variants may escape immunity induced by prior influenza
infection or vaccination. Antigenic drift is responsible for annual
epidemics that occur during interpandemic periods (Wiley &
Skehel, 1987, Ann. Rev. Biochem. 56, 365-394). The induction of
cross-reactive CD4 T cells provides an additional advantage to the
composition of the invention, in that it may provide also
cross-protection, in other words protection against heterologous
infections, i.e. infections caused by a circulating influenza
strain which is a variant (e.g. a drift) of the influenza strain
contained in the immunogenic composition. This may be advantageous
when the circulating strain is difficult to propagate in eggs or to
produce in cell 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.
[0265] The adjuvanted influenza immunogenic composition as herein
defined has therefore a higher ability to induce sero-protection
and cross-reactive CD4 T cells in vaccinated elderly subjects. This
characteristic may be associated with a higher ability to respond
against a variant strain of the strain present in the immunogenic
composition. This may prove to be an important advantage in a
pandemic situation. For example a multivalent influenza immunogenic
composition comprising any or several of H5, a H2, a H9, H7 or H6
strain(s) may provide a higher ability to respond against a
pandemic variant, i.e. a drift strain of said pandemic strain(s),
either upon subsequent vaccination with or upon infection by said
drift strain.
Detection of Cross-Reactive CD4 T-Cells Following Vaccination with
Influenza Vaccine
[0266] CD4 T-cells that are able to recognize both homologous and
drifted Influenza strains have been named in the present document
"cross-reactive". The adjuvanted influenza compositions as
described herein have been capable to show heterosubtypic
cross-reactivity since there is observable cross-reactivity against
drifted Influenza strains. As said above, the ability of a pandemic
vaccine formulation to be effective against drift pandemic strains
may prove to be an important characteristic in the case of
pandemics.
[0267] Consistently with the above observations, CD4 T-cell
epitopes shared by different Influenza strains have been identified
in human (Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder C
M et al. 1996 J. Virol. 70(7):4787-90; Gelder C M et al. 1995 J.
Virol. 1995 69(12):7497-506).
[0268] 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.
Revaccination and Composition Used for Revaccination (Boosting
Composition)
[0269] In one embodiment, the invention provides for the use of an
influenza virus or antigenic preparation thereof in the manufacture
of an immunogenic composition for revaccination of humans
previously vaccinated with an immunogenic composition as claimed
herein.
[0270] In one aspect of the present invention, there is provided
the use of an influenza virus or antigenic preparation thereof,
from a first pandemic influenza strain, in the manufacture of an
adjuvanted immunogenic composition as herein defined for protection
against influenza infections caused by a influenza strain which is
a variant of said first influenza strain.
[0271] In another aspect, the invention provides for the use of an
influenza virus or antigenic preparation thereof in the manufacture
of an influenza immunogenic composition for revaccination of humans
previously vaccinated with an adjuvanted influenza composition as
claimed herein or with an adjuvanted influenza composition
comprising a variant influenza strain, the adjuvant being as
defined herein.
[0272] In another aspect the present invention provides for a
method for vaccinating a human population or individual against one
influenza virus strain followed by revaccination of said human or
population against a variant influenza virus strain, said method
comprising administering to said human (i) a first composition
comprising an influenza virus or antigenic preparation thereof from
a first influenza virus strain and an adjuvant as herein defined,
and (ii) a second immunogenic composition comprising a influenza
virus strain variant of said first influenza virus strain. In a
specific embodiment said first strain is associated with a pandemic
outbreak or has the potential to be associated with a pandemic
outbreak. In another specific embodiment said variant strain is
associated with a pandemic outbreak or has the potential to be
associated with a pandemic outbreak. In particular, the
re-vaccination is made with an influenza composition comprising at
least one strain which is a circulating pandemic strain. Both the
priming composition and the boosting composition can be
multivalent, i.e. can contain at least two influenza virus strains.
When the composition(s) is (are) multivalent, at least one strain
is associated with a pandemic outbreak or has the potential to be
associated with a pandemic outbreak. 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.
[0273] The immunogenic composition for revaccination (the boosting
composition) may contain any type of antigen preparation, either
inactivated or live attenuated. It may contain the same type of
antigen preparation e.g. a split influenza virus or antigenic
preparation thereof, a whole virion, or a purified HA and NA
(sub-unit) vaccine, as the immunogenic composition used for the
first vaccination. Alternatively the boosting composition may
contain another type of influenza antigen than that used for the
first vaccination. Preferably a split virus is used. The boosting
composition may be adjuvanted or un-adjuvanted. The un-adjuvanted
boosting composition may be
Fluarix.TM./.alpha.-Rix.RTM./Influsplit.RTM. given intramuscularly.
The formulation contains three inactivated split virion antigens
prepared from the WHO recommended strains of the appropriate
influenza season.
[0274] The boosting composition may be adjuvanted or un-adjuvanted.
In a specific embodiment, the boosting composition comprises a
saponin adjuvant which is as defined herein.
[0275] In a specific embodiment, the immunogenic composition for
revaccination (also called herein below the `boosting composition`)
contains an influenza virus or antigenic preparation thereof which
shares common CD4 T-cell epitopes with the influenza virus or
antigenic preparation thereof used for the first vaccination. A
common CD4 T cell epitope is intended to mean
peptides/sequences/epitopes from different antigens which can be
recognised by the same CD4 cell (see examples of described epitopes
in: Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder C M et
al. 1996 J. Virol. 70(7):4787-90; Gelder C M et al. 1995 J. Virol.
1995 69(12):7497-506).
[0276] In an embodiment according to the invention, the boosting
composition is a monovalent influenza composition comprising an
influenza strain which is associated with a pandemic outbreak or
has the potential to be associated with a pandemic outbreak. In
particular said strain in the boosting composition is a circulating
pandemic strain. Suitable strains are, but not limited to: H5N1,
H9N2, H7N7, and H2N2. Said strain may be the same as that, or one
of those, present in the composition used for the first
vaccination. In an alternative embodiment said strain may be a
variant strain, i.e. a drift strain, of the strain present in the
composition used for the first vaccination.
[0277] In another specific embodiment, the boosting composition is
a multivalent influenza vaccine. In particular, when the boosting
composition is a multivalent vaccine such as a bivalent, trivalent
or quadrivalent vaccine, at least one strain is associated with a
pandemic outbreak or has the potential to be associated with a
pandemic outbreak. In a specific embodiment, two or more strains in
the boosting composition are pandemic strains. In another specific
embodiment, the at least one pandemic strain in the boosting
composition is of the same type as that, or one of those, present
in the composition used for the first vaccination. In an
alternative embodiment the at least one strain may be a variant
strain, i.e. a drift strain, of the at least one pandemic strain
present in the composition used for the first vaccination. In
particular the at least one strain in the boosting composition is a
circulating pandemic strain. The boosting composition may be
adjuvanted or not.
[0278] Typically a boosting composition, where used, is given at
the next influenza season, e.g. approximately one year after the
first immunogenic composition. The boosting composition may also be
given every subsequent year (third, fourth, fifth vaccination and
so forth). The boosting composition may be the same as the
composition used for the first vaccination. Suitably, the boosting
composition contains an influenza virus or antigenic preparation
thereof which is a variant strain of the influenza virus used for
the first vaccination. In particular, the influenza viral strains
or antigenic preparation thereof are selected according to the
reference material distributed by the World Health Organisation
such that they are adapted to the influenza strain which is
circulating on the year of the revaccination.
[0279] The influenza antigen or antigenic composition used in
revaccination preferably comprises an adjuvant, suitably as
described above. The adjuvant may be a saponin presented in the
form of a liposome, as herein above described, which is preferred,
optionally containing an additional adjuvant such as 3D-MPL.
[0280] In one aspect revaccination induces any, preferably two or
all, of the following: (i) an improved CD4 response against the
influenza virus or antigenic preparation thereof, or (ii) an
improved B cell memory response or (iii) an improved humoral
response, compared to the equivalent response induced after a first
vaccination with the un-adjuvanted influenza virus or antigenic
preparation thereof. Preferably the immunological response(s)
induced after revaccination with the adjuvanted influenza virus or
antigenic preparation thereof as herein defined, is (are) higher
than the corresponding response induced after the revaccination
with the un-adjuvanted composition. Preferably the immunological
responses induced after revaccination with an un-adjuvanted,
preferably split, influenza virus are higher in the population
first vaccinated with the adjuvanted, preferably split, influenza
composition than the corresponding response in the population first
vaccinated with the un-adjuvanted, preferably split, influenza
composition.
[0281] In a specific embodiment, the revaccination of the subjects
with a boosting composition comprising an influenza virus and a
saponin adjuvant in the form of a liposome, as defined herein
above, shows higher antibody titers than the corresponding values
in the group of people first vaccinated with the un-adjuvanted
composition and boosted with the un-adjuvanted composition. The
effect of the adjuvant in enhancing the antibody response to
revaccination is especially of importance in the elderly population
which is known to have a low response to vaccination or infection
by influenza virus. The adjuvanted composition-associated benefit
was also marked in terms of improving the CD4 T-cell response
following revaccination.
[0282] Specifically, the adjuvanted composition of the invention is
capable of inducing a better cross-responsiveness against drifted
strain (the influenza strain from the next influenza season)
compared to the protection conferred by the control vaccine. Said
cross-responsiveness has shown a higher persistence compared to
that obtained with the un-adjuvanted formulation. The effect of the
adjuvant in enhancing the cross-responsiveness against drifted
strain is of important in a pandemic situation.
[0283] In a further embodiment the invention relates to a
vaccination regime in which the first vaccination is made with an
influenza composition, preferably a split influenza composition,
containing at least one influenza strain that could potentially
cause a pandemic outbreak and the revaccination is made with a
circulating strain, either a pandemic strain or a classical
strain.
CD4 Epitope in HA
[0284] 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.
[0285] 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).
[0286] In a specific embodiment, the revaccination is made by using
a boosting composition which contains an influenza virus or
antigenic preparation thereof which shares common CD4 T-cell
epitopes with the influenza virus antigen or antigenic preparation
thereof used for the first vaccination. The invention thus relates
to the use of the immunogenic composition comprising a pandemic
influenza virus or antigenic preparation thereof and a saponin in
the form of a liposome, in particular QS21 in its detoxified form
with cholesterol optionally with 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 pandemic influenza virus antigen or virus
antigenic preparation thereof of the dose given at the first
vaccination.
Vaccination Means
[0287] The immunogenic compositions 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 immunogenic 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 boosting composition, either
adjuvanted or not, may be administered through a different route,
for example intradermal, subcutaneous or intranasal. In another
specific embodiment, an immunogenic composition comprising
specifically an influenza virus antigen or antigenic preparation
thereof for the first administration may contain a standard HA
content of 15 .mu.g per influenza strain, and the boosting
influenza 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 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.
[0299] Preferably the target population is a population which is
unprimed against influenza, either being naive (such as vis a vis a
pandemic strain), or having failed to respond previously to
influenza infection or vaccination. Preferably the target
population is elderly persons suitably aged 65 years and over,
younger high-risk adults (i.e. between 18 and 64 years of age) such
as people working in health institutions, or those young adults
with a risk factor such as cardiovascular and pulmonary disease, or
diabetes. Another target population is all children 6 months of age
and over, especially children 6-23 months of age who experience a
relatively high influenza-related hospitalization rate. Preferably
the target population is elderly above 65 years of age.
Vaccination Regimes, Dosing and Additional Efficacy Criteria
[0300] Suitably the immunogenic compositions according to the
present invention are a standard 0.5 ml injectable dose in most
cases, and, when an influenza composition, 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. For influenza vaccines, slight adaptation of
the dose volume will be made routinely depending on the HA
concentration in the original bulk sample.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] The influenza medicament of the invention preferably meets
certain international criteria for vaccines.
[0305] 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.
[0306] 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.
[0307] 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 [0308] 1) selecting an
antigen containing CD4+ epitopes, and [0309] 2) combining said
antigen with saponin adjuvant in the form of a liposome as defined
herein above, wherein said vaccine upon administration in said
mammal is capable of inducing an enhanced CD4 T cell response in
said mammal.
[0310] The teaching of all references in the present application,
including patent applications and granted patents, are herein fully
incorporated by reference.
[0311] 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.
[0312] The invention will be further described by reference to the
following, non-limiting, examples:
[0313] Example I describes immunological read-out methods used in
mice, ferret and human studies.
[0314] Example II describes preparation of the MPL/QS21 liposomal
adjuvant
[0315] Example III describes a pre-clinical evaluation of
adjuvanted and unadjuvanted influenza vaccines in ferrets.
[0316] Example IV shows a pre-clinical evaluation of adjuvanted and
un-adjuvanted influenza vaccines in C57BI/6 naive and primed
mice.
[0317] Example V describes a comparison of adjuvanted influenza
vaccine with 3D-MPL at two different concentrations in mice.
[0318] Example VI describes a comparison of adjuvanted influenza
vaccine with 3D-MPL at two different concentrations in elderly
humans.
[0319] Example VII describes the pre-clinical evaluation of
adjuvanted HPV vaccines in mice.
[0320] Example VIII describes a pre-clinical evaluation of
adjuvanted and non-adjuvanted cytomegalovirus immunogenic
compositions.
[0321] Example IX describes the pre-clinical evaluation of an
adjuvanted RTS,S vaccine composition with 3D-MPL at two different
concentrations.
[0322] Example X describes the clinical evaluation of an adjuvanted
RTS,S vaccine with 3D-MPL at two different concentrations.
EXAMPLE I
Immunological Read-Out Methods
I.1. Mice Methods
I.1.1. Hemagglutination Inhibition Test
Test Procedure
[0323] 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
[0324] Statistical analysis were performed on post vaccination HI
titers using UNISTAT. The protocol applied for analysis of variance
can be briefly described as follow: [0325] Log transformation of
data [0326] Shapiro-Wilk test on each population (group) in order
to verify the normality of groups distribution [0327] Cochran test
in order to verify the homogeneity of variance between the
different populations (groups) [0328] Two-way Analysis of variance
performed on groups [0329] Tukey HSD test for multiple
comparisons
I.1.2. Intracellular Cytokine Staining
[0330] 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.
[0331] 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.
[0332] In vitro antigen stimulation of PBMCs was carried out at a
final concentration of 1.times.10.sup.6 cells/ml (tube FACS) with
Flu trivalent split on .mu.beads (5 .mu.g HA/strain) or Whole FI (1
.mu.gHA/strain) and then incubated 2 hrs at 37.degree. C. with the
addition of anti-CD28 and anti-CD49d (1 .mu.g/ml for both).
[0333] The addition of both antibodies, increased proliferation and
cytokine production by activated T and NK cells and can provide a
costimulatory signal for CTL induction.
[0334] In addition, PBMCs were also stimulated overnight with Flu
trivalent split (30 .mu.g HA/strain)- or Whole FI (5
.mu.gHA/strain)-pulsed BMDCs (1.times.10.sup.5 cells/ml), which
were prepared by pulsing BMDCs with Flu split (60 .mu.g/HA strain)
or Whole Flu trivalent FI (10 .mu.gHA/strain) for 6 hrs at
37.degree. C. Following the antigen restimulation step, PBMC are
incubated O.N. at 37.degree. C. in presence of Brefeldin (1
.mu.g/ml) at 37.degree. C. to inhibit cytokine secretion.
[0335] 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.50,000
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.
[0336] 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 using tears for reading. 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.
[0337] 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: [0338] Log
transformation of data. [0339] Shapiro-wilk test on each population
(group) in order to verify the normality of groups distribution.
[0340] Cochran test in order to verify the homogenicity of variance
between the different populations (groups). [0341] Test for
interaction of one-way ANOVA. [0342] Tuckey-HSD Test for multiple
comparisons.
I.2.2. Nasal Washes
[0343] The nasal washes were performed by administration of 5 ml of
PBS in both nostrils in awake animals. The inoculum was collected
in a Petri dish and placed into sample containers on dry ice.
Viral Titration in Nasal Washes
[0344] 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 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.
[0345] 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
[0346] 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).
[0347] 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.
[0348] 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
[0349] 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
[0350] 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)
[0351] 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
[0352] 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. [0353] 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.
[0354] Occurrence of serious adverse events during the entire
study.
I.3.5.2. Secondary Endpoints
For the Humoral Immune Response:
Observed Variables:
[0354] [0355] 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). [0356] 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): [0357]
Geometric mean titres (GMTs) of serum HI antibodies with 95%
confidence intervals (95% CI) pre and post-vaccination [0358]
Seroconversion rates* with 95% CI at day 21 [0359] Conversion
factors** with 95% CI at day 21 [0360] Seroprotection rates*** with
95% CI at day 21 [0361] Serum NI antibody GMTs' (with 95%
confidence intervals) at all timepoints. [0362] * 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. [0363] **Conversion factor defined as the fold
increase in serum HI GMTs on day 21 compared to day 0, for each
vaccine strain. [0364] ***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
[0365] 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: [0366] Peptide Influenza (pf)
antigen (the precise nature and origin of these antigens needs to
be given/explained [0367] Split Influenza (sf) antigen [0368] Whole
Influenza (wf) antigen.
Derived Variables:
[0368] [0369] cells producing at least two different cytokines
(CD40L, IL-2, IFN.gamma., TNF.alpha.) [0370] cells producing at
least CD40L and another cytokine (IL-2, TNF.alpha., IFN.gamma.)
[0371] cells producing at least IL-2 and another cytokine (CD40L,
TNF.alpha., IFN.gamma.) [0372] cells producing at least IFN.gamma.
and another cytokine (IL-2, TNF.alpha., CD40L) [0373] cells
producing at least TNF.alpha. and another cytokine (IL-2, CD40L,
IFN.gamma.)
I.3.5.3. Analysis of Immunogenicity
[0374] The immunogenicity analysis was based on the total
vaccinated cohort. For each treatment group, the following
parameters (with 95% confidence intervals) were calculated: [0375]
Geometric mean titres (GMTs) of HI and NI antibody titres at days 0
and 21 [0376] Geometric mean titres (GMTs) of neutralising antibody
titres at days 0 and 21. [0377] Conversion factors at day 21.
[0378] 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. [0379] Protection
rates at day 21 defined as the percentage of vaccinees with a serum
HI titre=1:40. [0380] 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)). [0381] 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. [0382] 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 of the MPL/QS21 Liposomal Adjuvant
II.3 Preparation of MPL Liquid Suspension
[0383] 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 3D-MPL 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. 1.
[0384] 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: [0385] suspend
the MPL powder in water for injection [0386] desaggregate any big
aggregates by heating (thermal treatment) [0387] reduce the
particle size between 100 nm and 200 nm by microfluidization [0388]
prefilter the preparation on a Sartoclean Pre-filter unit, 0.8/0.65
.mu.m [0389] sterile filter the preparation at room temperature
(Sartobran P unit, 0.22 .mu.m)
[0390] MPL powder is lyophilized by microfluidisation resulting in
a stable colloidal aqueous dispersion (MPL particles of a size
susceptible to sterile filtration). 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)
[0391] 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.
[0392] 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.
[0393] 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.2 Preparation of MPL/QS21 Liposomal Adjuvant
[0394] This adjuvant, named AS01, comprises 3D-MPL and QS21 in a
quenched form with cholesterol, and was made as described in WO
96/33739, incorporated herein by reference. In particular the AS01
adjuvant was prepared essentially as Example 1.1 of WO 96/33739.
The AS01B adjuvant comprises: liposomes, which in turn comprise
dioleoyl phosphatidylcholine (DOPC), cholesterol and 3D MPL [in an
amount of 1000 .mu.g DOPC, 250 .mu.g cholesterol and 50 .mu.g
3D-MPL, each value given approximately per vaccine dose], QS21 [50
.mu.g/dose], phosphate NaCl buffer and water to a volume of 0.5 ml.
The AS01E adjuvant comprises the same ingredients than AS01B but at
a lower concentration in an amount of 500 .mu.g DOPC, 125 .mu.g
cholesterol, 25 .mu.g 3D-MPL and 25 .mu.g QS21, phosphate NaCl
buffer and water to a volume of 0.5 ml.
[0395] In the process of production of liposomes containing MPL the
DOPC (Dioleyl phosphatidylcholine), cholesterol and MPL are
dissolved in ethanol. A lipid film is formed by solvent evaporation
under vacuum. Phosphate Buffer Saline (9 mM Na.sub.2HPO.sub.4, 41
mM KH.sub.2PO.sub.4, 100 mM NaCl) at pH 6.1 is added and the
mixture is submitted to prehomogenization followed by high pressure
homogenisation at 15,000 psi (around 15 to 20 cycles). This leads
to the production of liposomes which are sterile filtered through a
0.22 .mu.m membrane in an aseptic (class 100) area. The sterile
product is then distributed in sterile glass containers and stored
in a cold room (+2 to +8.degree. C.).
[0396] In this way the liposomes produced contain MPL in the
membrane (the "MPL in" embodiment of WO 96/33739).
[0397] QS21 is added in aqueous solution to the desired
concentration.
EXAMPLE III
Pre-Clinical Evaluation of Adjuvanted and Unadjuvanted Influenza
Vaccines in Ferrets
III.1. Rationale and Objectives
[0398] Influenza infection in the ferret model closely mimics human
influenza, with regards both to the sensitivity to infection and
the clinical response.
[0399] 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.
[0400] 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.
[0401] The objective of this experiment was to demonstrate the
efficacy of an adjuvanted influenza vaccine compared to the plain
(un-adjuvanted) vaccine.
[0402] The end-points were:
1) Primary end-point: reduction of viral shedding in nasal washes
after homologous challenge: 2) Secondary end-points: Analysis of
the humoral response by HI titers.
III.2. Experimental Design
III.2.1. Treatment/Group (Table 1)
[0403] Female ferrets (Mustela putorius furo) aged 14-20 weeks were
obtained from MISAY Consultancy (Hampshire, UK). Ferrets were
primed on day 0 with heterosubtypic strain H1N1A/Stockholm/24/90 (4
Log TCID.sub.50/ml). On day 21, ferrets were injected
intramuscularly with a full human dose (500 .mu.g vaccine dose, 15
.mu.g HA/strain) of a combination of H1N1 A/New Caledonia/20/99,
H3N2 A/Panama/2007/99 and B/Shangdong/7/97. Ferrets were then
challenged on day 42 by intranasal route with an heterosubtypic
strain H3N2 A/Wyoming/3/2003 (4.5 Log TCID.sub.50/ml).
TABLE-US-00002 TABLE 1 Comments Antigen(s) + Formulation +
(schedule/route/ Group dosage dosage challenge) In/Po Other
treatments 1 Trivalent Full HD: 15 .mu.g IM; Day 21 In Priming H1N1
Plain HA/strain (A/Stockolm/24/ 90) Day 0 2 Trivalent/ Full HD: 15
.mu.g IM; Day 21 In Priming H1N1 MPL-QS21 in HA/strain
(A/Stockolm/24/90) liposomes Day 0 6 ferrets/group. In/Po =
Individual/pool
III.2.2. Preparation of the Vaccine Formulations (Table 2)
Formulation 1: Trivalent Split Plain (Un-Adjuvanted)
Formulation:
[0404] 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. After 5 min stirring, 1
.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 minimum 15 minutes and stored at 4.degree. C. if not
administered directly.
Formulation 2: Trivalent Split Influenza Adjuvanted with MPL/QS21
in Liposomes:
[0405] 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. 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. A premix of so called "DQS21-MPLin is
added to the formulation which is then stirred for minimum 15
minutes. The DQS21-MPLin premix is a mixture of liposomes (made of
DOPC 40 mg/ml, cholesterol 10 mg/ml, MPL 2 mg/ml) and the
immunostimulant QS21. This premix is incubated for a minimum of 15
minutes prior to addition to the trivalent split mixture. The
concentration of MPL and QS21 in the final formulation is 50 .mu.g
per 500 .mu.l. The formulation is stored at 4.degree. C. if not
administered directly.
[0406] 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.
TABLE-US-00003 TABLE 2 Final composition of formulations 1 and 2
(Formulations prepared with split strains (for 500 .mu.l)) Tween
Triton X- Formulation Antigen 80 100 VES DOPC Cholesterol MPL QS21
1 H1N1: 15 .mu.g 375 .mu.g 55 .mu.g 50 .mu.g -- -- -- -- H3N2: 15
.mu.g B: 17.5 .mu.g 2 H1N1: 15 .mu.g 375 .mu.g 55 .mu.g 50 .mu.g 1
mg 250 .mu.g 50 .mu.g 50 .mu.g H3N2: 15 .mu.g B: 17.5 .mu.g
III.2.3. Read-Outs (Table 3)
TABLE-US-00004 [0407] TABLE 3 Readout Timepoint Sample-type
Analysis method Viral shedding D + 1 to D + 7 Post Nasal washes
Titration challenge Anti-HI Pre, Post priming, Sera
Hemagglutination antibodies (HI Post immunization, inhibition test
titers) Post challenge
III.3. Results
[0408] A schematic representation of the results is given in FIGS.
1 and 2.
III.3.1. Humoral Immunity (FIG. 1).
[0409] Haemagglutination inhibition activity against the H3N2
vaccine strains (vaccine strain A/Panama/2007/99 and challenge
strain A/Wyoming/3/2003) was detected in sera from 6 animals per
group at Day 17 after intranasal heterologous priming and at Day 21
Post-immunization and Day 13 Post-challenge.
[0410] Anti-Hemagglutinin antibody titers to the three influenza
virus strains were determined using the hemagglutination inhibition
test (HI) as detailed under Example I.2.1. The conclusions are as
follows: [0411] For the two A/H3N2 strains and for all groups, a
boost of HI titers was observed in all vaccinated groups after
immunization. [0412] Post-immunization with A/Panama/2007/99,
statistically significant higher anti-A/Panama/2007/99 HI titers
were observed when the Trivalent Split vaccine was adjuvanted with
MPL/QS21 in liposomes compared to the Trivalent Split Plain
vaccine. [0413] After immunization with A/Panama/2007/99, only the
Trivalent Split adjuvanted with MPL/QS21 in liposomes was able to
significantly increase HI titers to the heterologous strain
A/Wyoming/3/2003 (cross-reactivity before challenge with this drift
strain). [0414] After challenge with A/Wyoming3/2003, an
significant increase of anti-A/Wyoming/3/2003 HI titers was
observed for both Trivalent Split Plain and Trivalent Split
adjuvanted with MPL/QS21 in liposomes. [0415] For A/New
Caledonia/20/99 and B/Shangdong/7/97 strains, statistically
significant higher HI titers were observed when the Trivalent Split
was adjuvanted with MPL/QS21 in liposomes compared to the Trivalent
Split Plain vaccine.
III.3.2. Viral Shedding (FIG. 3).
[0416] Viral titration of nasal washes was performed on 6 animals
per group as detailed under Example I.2.3. 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. [0417] Two days
after challenge, statistically significant lower viral shedding was
observed with Trivalent Split adjuvanted with MPL/QS21 in liposomes
compared to Trivalent Split Plain. [0418] On Day 49 (7 days
Post-challenge), no virus was detected in nasal washes.
III.3.3. Conclusion of the Experiment
[0419] Higher humoral responses (HI titers) were observed with
Trivalent Split adjuvanted with MPL/QS21 in liposomes compared to
the Trivalent Split Plain for all 4 strains.
[0420] After immunization with A/Panama/2007/99, only the Trivalent
Split adjuvanted with MPL/QS21 in liposomes was able to
significantly increase HI titers to the heterologous strain
A/Wyoming/3/2003 (cross-reactivity before challenge with this
strain).
[0421] MPL/QS21 in liposomes formulations showed added benefit in
terms of protective efficacy in ferrets (lower viral shedding after
heterologous challenge). The cross-reaction observed after
immunization with Trivalent Split MPL/QS21 in liposomes against the
drift strain used for the challenge seemed to correlate with the
protection effect observed in these ferrets.
EXAMPLE IV
Pre-Clinical Evaluation of Adjuvanted and Unadjuvanted Influenza
Vaccines in C57BI/6 Primed Mice
IV.1. Experimental Design and Objective
[0422] C57BI/6 mice primed with heterologous strains were used for
this experiment.
[0423] The purpose was to compare the humoral (HI titers) and CMI
(ICS, intracellular cytokine staining) immune responses induced by
a GlaxoSmithKline commercially available Trivalent split vaccine
(Fluarix.TM.) versus a Trivalent subunit vaccine (Chiron's vaccine
Agrippal.TM.) as well as the CMI response obtained with these
vaccines adjuvanted with Liposomes containing 3D-MPL alone, DQS21
(QS21 in liposomes, i.e. detoxified QS21) alone or MPL/QS21 in
liposomes. In the example hereinbelow, formulations were prepared
starting from the split monobulks to reach the same composition
than in the Fluarix vaccine and not from commercially available
Fluarix doses. The formulations obtained were called "Fluarix
like".
IV.1.1. Treatment/Group
[0424] Female C57BI/6 mice aged 6-8 weeks were obtained from Harlan
Horst, Netherland. Mice were primed on day 0 with heterosubtypic
strains (5 .mu.g HA whole inactivated H1N1 A/Beijing/262/95, H3N2
A/Panama/2007/99, B/Shangdong/7/97). On day 28, mice were injected
intramuscularly with 1.5 .mu.g HA Trivalent split (A/New
Caledonia/20/99, A/Wyoming/3/2003, B/Jiangsu/10/2003) plain or
adjuvanted (see groups in Tables 4 to 6 below).
TABLE-US-00005 TABLE 4 Gr Antigen/Formulation Other treatment 1
Trivalent split*/Plain (un-adjuvanted) = Heterologous priming D0
Fluarix like 2 Trivalent split*/Liposomes containing Heterologous
priming D0 3D-MPL 3 Trivalent split*/DQS21 Heterologous priming D0
4 Trivalent split*/MPL/QS21 in liposomes Heterologous priming D0 5
Aggripal .TM. (sub-unit) Heterologous priming D0 6 Aggripal .TM.
(sub-unit)/Liposomes Heterologous priming D0 containing 3D-MPL 7
Aggripal .TM. (sub-unit)/DQS21 Heterologous priming D0 8 Aggripal
.TM. (sub-unit)/MPL/QS21 in Heterologous priming D0 liposomes 9 PBS
Heterologous priming D0 *Fluarix like. 16 mice/group
IV.1.2. Preparation of the Vaccine Formulations
Formulation for Group 1:
[0425] 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. After 5 min stirring, 15
.mu.g of each strain H1N1, H3N2 and 15 .mu.g of B strain are added
with 10 min stirring between each addition. The formulation is
stirred for minimum 15 minutes and stored at 4.degree. C. if not
administered directly.
Formulation for Group 2:
[0426] 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. After 5 min stirring, 15
.mu.g of each strain H1N1, H3N2 and 15 .mu.g of B strain are added
with 10 min stirring between each addition. The formulation is
stirred for 15 minutes. Concentrated liposomes containing 3D-MPL
(made of DOPC 40 mg/ml, Cholesterol 10 mg/ml, 3D-MPL 2 mg/ml) are
added to reach a final MPL concentration of 50 .mu.g per dose. The
formulation is then stirred minimum 15 minutes and stored at
4.degree. C. if not administered directly.
Formulation for Group 3:
[0427] 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. After 5 min stirring, 15
.mu.g of each strain H1N1, H3N2 and 15 .mu.g of B strain are added
with 10 min stirring between each addition. The formulation is
stirred for 15 minutes. A premix made of liposomes (made of DOPC 40
mg/ml, Cholesterol 10 mg/ml) and QS21 called "DQS21" is then added
to reach a QS21 concentration of 50 .mu.g per dose. This premix is
incubated at least for 15 minutes prior to addition to the
trivalent split mixture. The formulation is stirred for minimum 15
minutes and stored at 4.degree. C. if not administered
directly.
Formulation for Group 4:
[0428] 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. After 5 min stirring, 15
.mu.g of each strain H1N1, H3N2 and 15 .mu.g of B strain are added
with 10 min stirring between each addition. The formulation is
stirred for 15 minutes. A mixture made of liposomes containing
3D-MPL (made of DOPC 40 mg/ml, Cholesterol 10 mg/ml, 3D-MPL 2
mg/ml) and QS21 is then added to reach QS21 and MPL concentrations
of 50 .mu.g per dose. This mixture is incubated at least for 15
minutes prior to addition to the trivalent split mixture. The so
called "trivalent split MPL/QS21 in liposomes" formulation is
stirred for minimum 15 minutes and stored at 4.degree. C. if not
administered directly.
[0429] Remark: In groups 1 to 4, 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.
Formulation for Group 5:
[0430] One Aggripal.TM. dose is mixed with equal volume of PBS mod
pH 7.4. The formulation is stirred for minimum 15 minutes and
stored at 4.degree. C. if not administered directly.
Formulation for Group 6:
[0431] PBS pH 7.4 and one Aggripal.TM. dose are mixed. Liposomes
containing 3D-MPL (made of DOPC 40 mg/ml, Cholesterol 10 mg/ml,
3D-MPL 2 mg/ml) are then added under stirring to reach the
equivalent of 50 .mu.g of MPL per dose. The formulation is stirred
for minimum 15 minutes and stored at 4.degree. C. if not
administered directly.
[0432] Remark: PBS is added to reach isotonicity in the final
volume. Aggripal is half the formulation volume.
Formulation for Group 7:
[0433] PBS pH 7.4 and one Aggripal.TM. dose are mixed. A premix of
liposomes (made of DOPC 40 mg/ml, Cholesterol 10 mg/ml) and QS21 so
called "DQS21" is then added under stirring to reach the equivalent
of 50 .mu.g of QS21. This premix is incubated for at least 15
minutes prior to addition. The formulation is stirred minimum 15
minutes and stored at 4.degree. C. if not administered
directly.
[0434] Remark: PBS is added to reach isotonicity in the final
volume. Aggripal.TM. is half the formulation volume.
Formulation for Group 8:
[0435] PBS pH 7.4 and one Aggripal.TM. dose are mixed. A premix of
so called "DQS21-MPLin" is added under stirring to the formulation.
The DQS21-MPLin premix is a mixture of liposomes (made of DOPC 40
mg/ml, cholesterol 10 mg/ml, MPL 2 mg/ml) and the immunostimulant
QS21. This premix is incubated for at least 15 minutes prior to
addition to the Aggripal/PBS mixture. The quantity of MPL and QS21
in the formulation is 50 .mu.g each. The formulation is stirred
minimum 15 minutes and stored at 4.degree. C. if not administered
directly.
[0436] Remark: PBS is added to reach isotonicity in the final
volume. Aggripal is half the formulation volume.
TABLE-US-00006 TABLE 5 Final composition of the formulations 1 to 4
prepared with split strains (for 1 ml) Triton Group Antigen Tween
80 X-100 VES DOPC Cholesterol MPL QS21 1 H1N1: 15 .mu.g 750 .mu.g
110 .mu.g 100 .mu.g -- -- -- -- H3N2: 15 .mu.g B: 17.5 .mu.g 2
Identical to 1 Identical to 1 110 .mu.g 100 .mu.g 1 mg 250 .mu.g 50
.mu.g -- 3 Identical to 1 Identical to 1 110 .mu.g 100 .mu.g 1 mg
250 .mu.g -- 50 .mu.g 4 Identical to 1 Identical to 1 110 .mu.g 100
.mu.g 1 mg 250 .mu.g 50 .mu.g 50 .mu.g
TABLE-US-00007 TABLE 6 Final composition of the formulations 5 to 8
prepared with Aggripal .TM. vaccine (1 ml) Group Antigen DOPC
Cholesterol MPL QS21 5 1dose of Aggripal -- -- -- -- vaccine 6
Identical to 5 1 mg 250 .mu.g 50 .mu.g -- 7 Identical to 5 1 mg 250
.mu.g -- 50 .mu.g 8 Identical to 5 1 mg 250 .mu.g 50 .mu.g 50
.mu.g
IV.1.3. Read-Outs (Table 7)
TABLE-US-00008 [0437] TABLE 7 Sample Read-out Timepoint type In/Po
Analysis method Anti-HI antibodies Day 21 Post- Sera In
Hemagglutination (HI titers) Immunization Inhibition test (Day 49)
CD4, CD8, IL-2, Day 7 Post- PBLs Po Intracellular IFN-.gamma.
(FACS) Immunization cytokine staining (Day 35) (ICS) In =
Individual/Po = Pool
IV. 2. Results
IV.2.1. Humoral Response (HI Titers 21 Days Post Immunization).
Humoral Responses by HI Titers--FIG. 4.
[0438] Haemagglutination inhibition activity against the three
vaccine strains (A/New Caledonia/20/99, A/Wyoming/3/2003,
B/Jiangsu/10/2003) was detected in sera from 8 animals per group at
Day 21 Post-immunization. [0439] Compared to mice immunized with
PBS, an increase in HI titers was observed after immunization with
all Flu vaccine candidates tested for all three strains (Trivalent
split or Trivalent subunit vaccine). [0440] For all three strains,
statistically significant higher HI titers were observed in mice
immunized with Trivalent split adjuvanted with DQS21 alone or
MPL/QS21 in liposomes compared to mice immunized with Trivalent
split Flu plain or adjuvanted with Liposomes containing 3D-MPL
alone. The ranking for the humoral response was as follow:
(MPL/QS21 in liposomes=DQS21 alone)>(Liposomes containing 3D-MPL
alone=Plain)>PBS [0441] For all three strains, statistically
significant higher HI titers were observed in mice immunized with
Trivalent subunit adjuvanted with DQS21 alone, Liposomes containing
3D-MPL alone or MPL/QS21 in liposomes compared to mice immunized
with Trivalent split plain. The ranking for the humoral response
was as follow: (MPL/QS21 in liposomes=DQS21 alone=Liposomes
containing 3D-MPL alone)>Plain >PBS. [0442] Trivalent split
and Trivalent subunit induced similar HI titers when formulations
were not adjuvanted or adjuvanted with DQS21 alone or MPL/QS21 in
liposomes.
IV.2.2. Cell-Mediated Immune Response (ICS at day 7 Post
Immunization).
CD4 T Cell Responses--FIG. 5
[0443] PBMCs from 8 mice per group were harvested at Day 7
Post-immunization and tested in 4 pools of 2 mice/group.
[0444] In terms of Flu whole virus-specific total CD4+ T cells
(expressing IL-2, IFN-.gamma. and both cytokines): [0445] Whatever
the formulation, identical CD4+ T cell responses were observed
between the Trivalent split and Trivalent subunit vaccines. [0446]
Higher CD4+ T cell responses were observed for Trivalent
formulations (split or subunit) adjuvanted with MPL/QS21 in
liposomes when compared to Trivalent formulations (split or
subunit) plain or adjuvanted with Liposomes containing 3D-MPL alone
or DQS21 alone. [0447] For the cellular response induced by a
Trivalent formulation (split or subunit), there is a synergic
effect of Liposomes containing 3D-MPL+DQS21 compared to DQS21 alone
or Liposomes containing 3D-MPL alone. [0448] The ranking for the
cellular response was as follow: MPL/QS21 in liposomes
>(Liposomes containing 3D-MPL alone=DQS21 alone=Plain
.dbd.PBS).
IV.3. Summary of Results and Conclusions
[0448] [0449] For all three strains, statistically significant
higher HI titers were observed in mice immunized with Trivalent
formulations (split or subunit) adjuvanted with DQS21 alone or
MPL/QS21 in liposomes compared to mice immunized with Trivalent
formulations (split or subunit) plain. Liposomes containing 3D-MPL
alone seemed to induced higher humoral response when formulated
with Trivalent subunit than Trivalent split. [0450] Whatever the
formulation, similar CD4+ T cell responses were obtained for
Trivalent split (Fluarix) and Trivalent subunit (Agrippal). [0451]
Trivalent formulations (split or subunit) adjuvanted with MPL/QS21
in liposomes induced higher CD4+ T cell responses compared to
Trivalent formulations (split or subunit) plain or adjuvanted with
Liposomes containing 3D-MPL alone or QS21 in liposomes (DQS21)
alone.
EXAMPLE V
Preclinical Comparison of a Vaccine Containing a Split Influenza
Antigen Preparation Adjuvanted with 3D-MPL/QS21 in a Liposomal
Formulation (3D-MPL at Two Different Concentrations)
V.1--Mice.
V.1.1--Experimental Design and Objective.
[0452] C57B1/6 mice primed with heterologous strains were used for
this experiment. The purpose was to analyse the humoral (HI titers)
and CMI (ICS, intracellular cytokine staining) immune responses
induced by a GlaxoSmithKline commercially available Trivalent split
vaccine (Fluarix.TM.) in un-adjuvanted form, and when adjuvanted
with liposomes containing two different concentrations of 3D-MPL
and QS21.
V.1.2 Treatment/Group
[0453] Female C57B1/6 mice aged 8 weeks were obtained from Harlan
Horst, Netherlands. Mice were primed intranasally on day 0 with
heterosubtypic strains (whole inactivated A/Beijing/262/95, H3N2
A/Panama/2007/99, B/Shandong/7/97). On day 28, mice were injected
intramuscularly with Trivalent Split (A/New Caledonia, A/Wyoming,
B/Jiangsu) plain or adjuvanted with two different concentrations of
immunostimulants in liposomal formulations (see groups in table 8
below).
TABLE-US-00009 TABLE 8 Formulation + Group Antigen(s) + dosage
dosage Other treatments 1 Trivalent Split Flu - 1.5 .mu.g/ Plain
Heterologous priming D0 strain/50 .mu.l whole inactivated 5
.mu.g/20 .mu.l intranasally 2 Trivalent Split Flu - 1.5 .mu.g/
Liposomes Heterologous priming D0 strain/50 .mu.l containing 3D-MPL
whole inactivated 50 .mu.g per 0.5 ml dose 5 .mu.g/20 .mu.l
intranasally 3 Trivalent Split Flu - 1.5 .mu.g/ DQS21 Heterologous
priming D0 strain/50 .mu.l 50 .mu.g per 0.5 ml dose whole
inactivated 5 .mu.g/20 .mu.l intranasally 4 Trivalent Split Flu -
1.5 .mu.g/ MPL and QS21 Heterologous priming D0 strain/50 .mu.l 25
.mu.g per 0.5 ml dose whole inactivated 5 .mu.g/20 .mu.l
intranasally 5 Trivalent Split Flu - 1.5 .mu.g/ MPL and QS21
Heterologous priming D0 strain/50 .mu.l 50 .mu.g per 0.5 ml dose
whole inactivated 5 .mu.g/20 .mu.l intranasally 6 PBS None
Heterologous priming D0 whole inactivated 5 .mu.g/20 .mu.l
intranasally
[0454] Formulations were prepared as in example IV.
V. 1.3--Results.
Humoral Responses by HI Titers --FIG. 24.
[0455] Hemagglutination inhibition activity against the 3 vaccine
strains was detected in sera from 9 animals/group on day 21 post
immunisation. [0456] Compared to mice immunized with PBS, an
increase in HI titres was observed after immunization with all Flu
vaccine candidates tested for all three strains. [0457] For all
three strains, statistically significant higher HI titers were
observed in mice immunized with Trivalent Split adjuvant with MPL
and QS21 at either concentration compared to mice immunized with
the Trivalent Flu Split Plain (p value max=0.03) [0458] No
statistically significant difference was observed between the two
liposomal adjuvant groups adjuvant groups
Cell-Mediated Immune Response (ICS at Day 7
Post-Immunisation)--FIG. 25.
[0459] PBMC's from 9 mice/group were harvested 7 days
post-immunisation and tested in three pools of 3 mice/group. In
terms of whole Flu virus-specific CD4+ T cells expressing IL-2,
IFN-.gamma. or both cytokines:
[0460] As can be seen from FIG. 25 the highest IFN-.gamma. CD4+ T
cell-specific responses were obtained after immunization with
trivalent split adjuvanted with the highest concentration of
immunostimulants. However, IL2 and IL2+IFN-.gamma. T cell responses
were similar between the two concentrations of
immunostimulants.
EXAMPLE VI
Clinical Trial in an Elderly Population Aged Over 65 Years with a
Vaccine Containing a Split Influenza Antigen Preparation Adjuvanted
with MPL/QS21 in a Liposomal Formulation (3D-MPL at Two Different
Concentrations)
VI.1. Study Design and Objectives
[0461] 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)
[0462] Four parallel groups were assigned: [0463] 75 adults (aged
18-40 years) in one control group receiving one dose of
Fluarix.TM.(Fluarix Group) [0464] 200 elderly subjects (aged 65
years and older) randomized 3:3:2 into three groups: [0465] one
group with 75 subjects receiving influenza vaccine adjuvanted with
AS01B [0466] One group with 75 subjects receiving influenza vaccine
adjuvanted with AS01E [0467] Reference Flu group with 50 subjects
receiving one dose of Fluarix.TM.
Primary Objective
[0468] 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
[0469] The secondary objectives are:
1) 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. 2) 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
[0470] 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.
VI.2. Vaccine Composition and Administration
[0471] Two different adjuvants have been used: [0472] 1. AS01B a
liposome-based adjuvant containing 50 .mu.g MPL and QS21 [0473] 2.
AS01E a two-fold diluted formulation of AS01B Control: full dose of
Fluarix.TM. by IM administration.
[0474] All vaccines are intended for intramuscular administration.
The strains used in the five vaccines are the ones that have been
recommended by the WHO for the 2005-2006 Northern Hemisphere
season, i.e. A/New Caledonia/20/99 (H1N1), A/New York/7/2004 (H3N2)
and B/Jiangsu/10/2003.
[0475] The three inactivated split virion antigens (monovalent
bulks) used in formulation of the adjuvanted influenza candidate
vaccine, are exactly the same as the active ingredients used in
formulation of the commercial Fluarix.TM./.alpha.-Rix.TM.-GSK Bio's
split virion inactivated influenza vaccine. They are derived from
egg-grown viruses. The influenza strains are the recommended ones
for the 2005/2006 season, as used in the formulation of the
commercial Fluarix.TM./.alpha.-Rix.TM. 2005/2006.
[0476] The strains used in the three vaccines are the ones that
have been recommended by the WHO for the 2005-2006 Northern
Hemisphere season i.e. [0477] A/New Caledonia/20/99
(H.sub.1N.sub.1) IVR-116 [0478] A/New York/55/2004 (H3N2) NYMC
X-157 [0479] B/Jiangsu/10/2003 Like Fluarix.TM./.alpha.-Rix.TM. the
adjuvanted vaccine contains 15 .mu.g haemagglutinin (HA) of each
influenza virus strain per dose.
VI.2.1. Description of the AS01B Adjuvanted Vaccine Lots
[0480] The AS01B-adjuvanted influenza candidate vaccine is a 2
components vaccine consisting of a concentrated trivalent
inactivated split virion antigens presented in a glass vial and of
a glass vial containing the AS01B adjuvant. At the time of
injection, the content of the adjuvant vial is withdrawn and
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. The used needle is replaced by an
intramuscular needle and the volume is corrected to 1 ml. One dose
of the reconstituted AS01B-adjuvanted influenza candidate vaccine
corresponds to 1 mL.
[0481] The AS01B-adjuvanted influenza candidate vaccine is a
preservative-free vaccine.
VI.2.2. Composition of the AS01B Adjuvanted Clinical Lot
[0482] One dose of the reconstituted AS01B-adjuvanted influenza
vaccine corresponds to 1 mL. Its composition is given in Table 8.
It contains 15 .mu.g HA of each influenza virus strain as in the
registered Fluarix.TM./.alpha.-Rix.TM. vaccine.
TABLE-US-00010 TABLE 8 Composition (influenza and adjuvant
components) of the reconstituted AS01B adjuvanted influenza
candidate vaccine Analytical Component Quantity per dose Reference
ACTIVE INGREDIENTS Inactivated split virions A/New Caledonia/20/99
(H1N1) 15 .mu.g HA Ph. Eur. 158 IVR-116 A/New York/55/2004 (H3N2)
15 .mu.g HA Ph. Eur. 158 NYMC X-157 B/Jiangsu/10/2003 15 .mu.g HA
Ph. Eur. 158 AS01B ADJUVANT Liposomes dioleyl phosphatidylcholine
1000 .mu.g GSK Bio 3217 (DOPC) Cholesterol 250 .mu.g Ph. Eur. 0993
MPL 50 .mu.g GSK Bio 2972 QS21 50 .mu.g GSK Bio 3034
VI.2.3. Production Method of the AS01B Adjuvanted Vaccine Lot
[0483] The manufacturing of the AS01B-adjuvanted influenza vaccine
consists of three main steps: [0484] Formulation of the trivalent
final bulk (2.times. concentrated) without adjuvant and filling in
the antigen container [0485] Preparation of the AS01B adjuvant
[0486] Extemporaneous reconstitution of the AS01B adjuvanted split
virus vaccine. Formulation of the Trivalent Final Bulk without
Adjuvant and Filling in the Antigen Container
[0487] 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 1320 ml. Concentrated
phosphate buffered saline PO4 Na/K.sub.2 (80 .mu.l/dose) 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 Caledonia/20/99 IVR-116, A/New
York/55/2004 NYMC X-157, B/Jiangsu/10/2003) are then successively
diluted in the resulting phosphate buffered saline/Tween 80-Triton
X-100-.alpha.-tocopheryl hydrogen succinate solution (pH 7.8, 81 mM
NaCl, 1.56 mM KCl, 4.79 mM Na.sub.2HPO.sub.4, 0.87 mM
KH.sub.2PO.sub.4, 7.2 mM NaH2PO4, 72.8 mM K.sub.2HPO.sub.4, 750
.mu.g/ml Tween 80, 110 .mu.g/ml Triton X-100 and 100 .mu.g/ml
.alpha.-tocopheryl hydrogen succinate) in order to have a final
concentration of 30 .mu.g HA of A (H1N1 and H3N2) strains per ml of
trivalent final bulk (15 .mu.g HA/A strain/500 .mu.l trivalent
final bulk) and 35 .mu.g HA of B strain (17.5 .mu.g HA/B strain/500
.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 7.65.+-.0.25 with HCl
or NaOH.
[0488] 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 600 .mu.l (500 .mu.l+100.mu. overfill).
Preparation of AS01B Adjuvant Bulk and Filling in the Adjuvant
Container
[0489] The adjuvant AS01B is prepared by mixing of two components:
QS21 and liposomes containing MPL. The preparation of each of these
components is summarized below. QS21 is a triterpene glycoside,
obtained from the tree bark of Quillaja saponaria, and is produced
by Aquila Worchester, Mass., USA (now Antigenics).
[0490] QS21 is provided to GSK Biologicals as a lyophillised
powder. The preparation of QS21 at GSK Bio consists of suspension
of QS21 powder in water for injection at a concentration of
approximately 5 mg/mL, pH adjustment to pH 6.0.+-.0.2 and sterile
filtration. The liquid bulk QS21 is stored at -20.degree. C. in
polyethylene containers.
[0491] MPL is the 3-O-deacyl-4'-monophosphoryl lipid A obtained by
sequential acid and base hydrolyses of the lipopolysaccharide from
the Re595 strain of Salmonella minnesota. It is produced by GSK
Biologicals, Hamilton, Mont. Bulk MPL is supplied as the
lyophilized salt of triethylamine (TEA).
[0492] In the process of production of MPL-containing liposomes,
DOPC (Dioleyl phosphatidylcholine), cholesterol and MPL are
dissolved in ethanol. A lipid film is formed by solvent evaporation
under vacuum. Phosphate Buffer Saline made of 9 mM
Na.sub.2HPO.sub.4, 41 mM KH.sub.2PO.sub.4, 100 mM NaCl at pH 6.1 is
added and the mixture is submitted to prehomogenization followed by
high pressure homogenization at 15,000 psi (+/-20 cycles). This
leads to the production of liposomes, which are sterile filtered
through a 0.22 .mu.m membrane in an aseptic (class 100) area. The
sterile product is then distributed in sterile glass containers and
stored in the cold room (+2 to +8.degree. C.).
[0493] Sterile bulk preparation of liposomes is mixed with sterile
QS21 bulk solution. After 30 min stirring, this mixture is added to
a mixture of water for injection and phosphate 500 mM, NaCl 1M pH
6.1 when diluted 10 times. Quantity of the phosphate 500 mM, NaCl
1M pH 6.1 when diluted 10 times, is calculated to reach isotonicity
in the final volume. The pH is checked. The adjuvant is then
sterile filtered (0.22 .mu.m) and aseptically distributed into
vials. The vials are stored at +2 to +8.degree. C.
[0494] The AS01B diluent is an opalescent colorless liquid, free
from foreign particles, contained in a sterile, type 1 glass vial.
The target fill for each vial is 0.7 ml in order to meet the
specification (.gtoreq.0.5 ml).
Extemporaneous Reconstitution of the AS01B Adjuvanted Split Virus
Vaccine
[0495] At the time of injection, the content of the vial containing
the adjuvant is withdrawn and 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, and the
volume is corrected to 1 ml. One dose of the reconstituted
AS01B-adjuvanted influenza candidate vaccine corresponds to 1
mL.
VI.2.4. Description of the AS01E Adjuvanted Vaccine Lots
[0496] The AS01E adjuvanted influenza candidate vaccine is a 3
components vaccine consisting of a concentrated trivalent
inactivated split virion antigens presented in a glass vial, a
glass vial containing the AS01B adjuvant and a glass vial
containing the diluent (sodium chloride solution for injection) for
the two-fold dilution of AS01B.
[0497] To prepare the AS01E adjuvant the content of the diluent
vial is withdrawn with a syringe and injected into the vial
containing the AS01B adjuvant, followed by mixing. At the time of
injection, 600 .mu.l AS01E adjuvant is withdrawn with a syringe
from the AS01E vial and 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
AS01B-adjuvanted influenza candidate vaccine corresponds to 1
mL.
[0498] The AS01E-adjuvanted influenza candidate vaccine is a
preservative-free vaccine.
VI.2.5. Composition of the AS01E Adjuvanted Clinical Lot
[0499] One dose of the reconstituted AS01E-adjuvanted influenza
vaccine corresponds to 1 mL. Its composition is given in Table 9.
It contains 15 .mu.g HA of each influenza virus strain as in the
registered Fluarix.TM./.alpha.-Rix.RTM. vaccine.
TABLE-US-00011 TABLE 9 Composition (influenza and adjuvant
components) of the reconstituted AS01E adjuvanted influenza
candidate vaccine Analytical Component Quantity per dose Reference
ACTIVE INGREDIENTS Inactivated split virions A/New Caledonia/20/99
(H1N1) 15 .mu.g HA Ph. Eur. 158 IVR-116 A/New York/55/2004 (H3N2)
15 .mu.g HA Ph. Eur. 158 NYMC X-157 B/Jiangsu/10/2003 15 .mu.g HA
Ph. Eur. 158 AS01B ADJUVANT Liposomes dioleyl phosphatidylcholine
500 .mu.g GSK Bio 3217 (DOPC) Cholesterol 125 .mu.g Ph. Eur. 0993
MPL 25 .mu.g GSK Bio 2972 QS21 25 .mu.g GSK Bio 3034
VI.2.6. Production Method of the AS01E Adjuvanted Vaccine Lot
[0500] The manufacturing of the AS01B-adjuvanted influenza vaccine
consists of three main steps: [0501] Formulation of the trivalent
final bulk (2.times. concentrated) without adjuvant and filling in
the antigen container [0502] Preparation of the AS01B adjuvant
[0503] Preparation of the AS01E adjuvant followed by extemporaneous
reconstitution of the AS01E adjuvanted split virus vaccine.
Formulation of the Trivalent Final Bulk without Adjuvant and
Filling in the Antigen Container
[0504] Reference is made to section V.2.3 for the AS01B adjuvanted
influenza vaccine.
Preparation of AS01B Adjuvant Bulk and Filling in the Adjuvant
Container
[0505] Reference is made to section V.2.3 for the AS01B adjuvanted
influenza vaccine.
Extemporaneous Reconstitution of the AS10E Adjuvanted Split Virus
Vaccine
[0506] To prepare the AS01E adjuvant the content of the diluent
vial is withdrawn with a syringe and injected into the vial
containing the AS01B adjuvant, followed by mixing. At the time of
injection, 600 .mu.l AS01E adjuvant is withdrawn with a syringe
from the AS01E vial and 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
AS01E-adjuvanted influenza candidate vaccine corresponds to 1
mL.
[0507] Four scheduled visits per subject: at days 0, 21, 90 and 180
with blood sample collected at each visit to evaluate
immunogenicity.
[0508] Vaccination schedule: one injection of influenza vaccine at
day 0
VI.2.7--Immunological Assays
Haemagglutination--Inhibition Assay
[0509] The immune response is determined by measuring
Haemagglutination inhibition (HI) antibodies using the method
described by the WHO collaborating Centre for influenza, Centres
for Diseases Control, Atlanta, USA (1991). 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. 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
shows complete inhibition (100%) of hemagglutination. All assays
were performed in duplicate.
[0510] Cytokine Flow Cytometry) CFC) used to evaluate the
frequencey of cytokine(s)--positive CD4 or CD8 T lymphocytes.
[0511] Peripheral blood antigen-specific CD4 and CD8 T cells can be
restimulated in vitro to produce CD40L, IL-2, 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 antigens will be used as antigens to restimulate
influenza-specific T cells. Results will be expressed as a
frequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4
or CD8 T cell sub-population.
ELISPOT Used to Evaluate Frequency of Memory B-Cell
[0512] The B cell Elispot technology allows the quantification of
memory B cells specific to a given antigen. Memory B cells can be
induce to differentiate into plasma cells in-vitro following
cultivation with CpG for 5 days. In-vitro generated
antigen-specific plasma cells can therefore be enumerated using the
B-cell elispot assay. Briefly, in-vitro generated plasma cells are
incubated in culture plates coated with antigen. Antigen-specific
plasma cells will form antibody/antigen spots, which can be
detected by conventional immuno-enzymatic procedure. In the present
study, influenza vaccine strains or anti-human immunoglobulin are
used to coat culture plates in order to enumerate anti-influenza or
IgG secreting plasma cells, respectively. Results are expressed as
a frequency of antigen-specific plasma within a million of
IgG-producing plasma cells.
Exploratory Characterisation of PBMCs
[0513] The expression of selected surfact/activation markers (i.e.
CD4., CD8, CD45RO, CD45 RA, CD28, CD27 or some KIR) can be
performed. The function of vaccine-induced T lymphocytes can be
addressed by the analysis of homing markers (i.e. CCR7, CXCR5), of
cytokines (T helper 1 or T helper 2 cytokines), or by analysing the
expression of factors associated with regulatory functions such as
Foxp3, CTLA-4, or TGF.beta.. In particular, the CD8+CD28-
population or other regulatory T cell populations can be analysed
in relation the humoral, B and T cell responses to the vaccine
antigen.
VI.3. Immunogenicity Results
VI.3.1. CMI Endpoints and Results
[0514] In order to characterize the CMI response after vaccination
with the adjuvanted influenza vaccines, CD4 and CD8 T-lymphocytes
were restimulated in vitro using antigens from the three vaccine
strains (used individually or pooled). Influenza-specific CD4/CD8
T-lymphocytes were enumerated by flow cytometry following
conventional immunofluorescence labelling of intracellular
cytokines production (IL-2, IFN-.gamma., TNF-.alpha. and
CD40L).
Evaluation of the Primary Endpoint.
[0515] At day 21: CMI response in all subjects in terms of
frequency of influenza-specific CD4 T-lymphocyte per 106 in tests
producing at least two different cytokines (IL-2, IFN-.gamma.,
TNF-.alpha. and CD40L).
[0516] For the evaluation of CMI response, frequency of
influenza-specific CD4 are analysed as follows:
[0517] Using the non-inferiority approach, the non inferiority of
at least one influenza adjuvanted candidate vaccine (administered
to elderly aged .gtoreq.65 years--the group termed Flu elderly or
Flu ELD) compared to Fluarix.TM. (administered to adults aged 18-40
years--the group termed Flu Young or Flu YNG) was reached when the
upper limit of two-sided 98.75% confidence interval on Geometric
Mean (GM) ratio (between the Fluarix.TM. (18-40 years) group and
the influenza adjuvanted candidate vaccine (.gtoreq.65 years group)
in terms of frequency of influenza-specific CD4 T-cells producing
at least two cytokines at day 21) was below 2.0
UL 98.75 % CI ( GM Fluarix adults GM influenzaAdjuvanted ) < 2
##EQU00001##
[0518] The 98.75% CI of GM ratios, 21 days after vaccination, was
computed using an analysis of covariance (ANCPVA) model on the
logarithm 10 transformation of the frequencies. The ANCOVA model
included the vaccine group as fixed effect (Fluarix.TM. (18-40
years) versus the influenza adjuvanted candidate vaccine
(.gtoreq.65 years)) and the pre-vaccination frequency as a
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 10)
[0519] 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 10. 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-00012 TABLE 10 Adjusted GM ratio of influenza-specific CD4
T cellsproducing at least two cytokines after restimulation with
pooled vaccine antigens, Day 21 (ATP cohort for immunogenicity) GM
ratio (Flu YNG/AS01B) Flu YNG AS01B 98.8% CI N GM N GM Value LL UL
74 2844.8 71 2725.6 1.04 0.79 1.38 GM ratio (Flu YNG/AS01E) Flu YNG
AS01E 98.8% CI N GM N GM Value LL UL 74 2879.6 74 2697.0 1.07 0.79
1.44 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; Data source = Appendix table
IIIA
Results--Descriptive Analysis (FIG. 6)
[0520] The main findings were:
1) Before vaccination the CMI response is higher in young adults
than in elderly 2) After vaccination: [0521] there was a booster
effect of the influenza vaccine on the CMI response in young adults
(18-40 years) [0522] the CMI response in the elderly having
received the adjuvanted influenza vaccine is comparable to the CMI
response of young adults.
[0523] 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. for all tests.
Analysis of the Tertiary Objective:
[0524] 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. [0525] 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. [0526] 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. [0527] 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. [0528] 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.
[0529] The p-values from Wilcoxon test used to compare the
difference in the frequency of influenza-specific CD4 T-lymphocytes
are presented in Table 11.
Results--Evaluation of the Tertiary End-Point (Table 11)
[0530] The main conclusions are: [0531] Pre-vaccination GM
frequencies of influenza-specific CD4 T cells were similar in all
groups of elderly subjects but superior in the adults aged between
18 and 40 years. [0532] 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.. [0533] Post-vaccination frequency of
influenza-specific CD4 T lymphocytes remained lower in elderly
subjects vaccinated with AS01B or AS01E adjuvanted vaccines than in
adults aged between 18 and 40 years vaccinated with Fluarix.TM..
[0534] Pre-vaccination and post vaccination GM frequency of
influenza-specific CD8 T cell was essentially similar in all
groups.
TABLE-US-00013 [0534] TABLE 11 Inferential statistics: p-values
from Kruskal-Wallis Tests for CD4 T cells at each time point (ATP
Cohort for immunogenicity) P-value AS01B vs. AS01E vs. Flu YNG Flu
YNG Test description day 0 day 21 day 0 day 21 ALL DOUBLES
<0.0001 0.0070 <0.0001 0.0025 CD4OL <0.0001 0.0056
<0.0001 0.0015 IFN.gamma. <0.0001 0.0009 <0.0001 0.0006
IL2 <0.0001 0.0029 <0.0001 0.0021 TFN.alpha. <0.0001
0.0295 <0.0001 0.0378 AS01B vs. AS01E vs. Flu ELD Flu ELD day 0
day 21 day 0 day 21 ALL DOUBLES 0.6165 0.0004 0.8744 0.0018 CD4OL
0.7560 0.0005 0.9504 0.0021 IFN.gamma. 0.9936 0.0008 0.9835 0.0029
IL2 0.6702 0.0011 0.7855 0.0023 TFN.alpha. 0.5450 0.0022 0.6688
0.0040
Results--Evaluation of the Humoral Immune Response Endpoints
Observed Variables:
[0535] At days 0, 21, 90 and 180: serum hemagglutination-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).
[0536] 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.
[0537] Based on the HI antibody titres, the following parameters
are calculated: [0538] 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. [0539] Seroconversion factors (SF)
at day 21 defined as the fold increase in serum HI GMTs on day 21
compared to day 0. [0540] 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
.gtoreq.1:40, or a pre-vaccination titre .gtoreq.1:10 and a minimum
4-fold increase at post-vaccination titre. [0541] Seroprotection
rates (SPR) at day 21 defined as the percentage of vaccinees with a
serum HI titre .gtoreq.1:40.
[0542] 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.
[0543] 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. 7 and Table 12)
[0544] 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 (FIG. 7, shown on a linear scale,
but same impact obviously seen if shown on a logarithmic
scale).
[0545] GMTs are [0546] significantly higher for H1N1 for AS01E
[0547] significantly higher for H3N2 for both adjuvants. [0548] No
significant difference was observed in terms of post-vaccination
GMTs between the two groups of subjects having received the
adjuvanted vaccines.
[0549] Twenty one days after vaccination, the subjects of Fluarix
(18-40 years) had a higher HI response for New Caledonia and
B/Jangsu strains.
[0550] As shown in Table 12 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.
[0551] After vaccination, there was a statistically difference in
terms of seroprotection rates of HI antibodies between Fluarix
(.gtoreq.65 years) group and [0552] Flu/AS01B and Flu/AS01E for
A/H1N1/New Caledonia strain
[0553] 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.
[0554] 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 Caledonia
strain.
TABLE-US-00014 TABLE 12 Seroprotection rates seroconversion rates
and conversion factors at day 21 (ATP cohort for immunogenicity)
Seroconversion rate Seroprotection rate (.gtoreq.4-fold increase)
Conversion 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) Flu AS01B 75 97.3 [90.70-99.68]
48.0 [36.5-59.8] 4.5 [3.3-6.1] Flu AS01E 75 93.3 [85.12-97.80] 52.0
[40.2-63.7] 5.0 [3.6-6.9] 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] Flu AS01B 75 96.0
[88.75-99.17] 85.3 [75.3-92.4] 13.1 [10.0-17.1] Flu AS01E 75 93.3
[85.12-97.80] 80.0 [69.2-88.4] 14.5 [10.4-20.2] B/Jiangsu (B) Flu
Yng 75 100 [95.20-100] 81.3 [70.7-89.5] 13.9 [10.1-19.1] Flu
Elderly 49 93.9 [83.13-98.72] 44.9 [30.7-59.8] 4.3 [3.0-6.1] Flu
AS01B 75 100 [95.20-100] 65.3 [53.5-76.0] 5.2 [4.2-6.5] Flu AS01E
75 97.3 [90.70-99.68] 70.7 [59.0-80.6] 6.7 [5.1-8.9] N = total
number of subject; % = Percentage of subjects with titre at day 21
within the specified range; CI = confidence interval
VI.3.2. Immunogenicity Conclusions
[0555] 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. [0556] 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 13. 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-00015 [0556] TABLE 13 Influenza strains for which
significantly higher humoral immun response (based on
non-overlapping of 95% Cl) was observed in elderly subjects
vaccinated with the different adjuvanted vaccines compared to
Fluarix in the same population. Post-vacc Seroconversion
Seroprotection Seroconversion GMT Factor rate Rate FluAS01B A/New
A/New York Caledonia FluAS01E A/New A/New Caledonia Caledonia A/New
York Post-vacc GMT = Geometric Mean Titre at post-vaccination
VI.4 Reactogenicity Conclusions
VI.4.1. Recording of Adverse Events (AE)
[0557] Solicited symptoms (see Table 14) 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 15.
TABLE-US-00016 TABLE 14 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-00017 TABLE 15 Intensity scales for solicited symptoms in
adults Intensity Adverse Event grade Parameter Pain at injection
site 0 Absent 1 on touch 2 when limb is moved 3 prevents normal
activity Redness at injection site Record greatest surface diameter
in mm Swelling at injection site Record greatest surface diameter
in mm Haematoma at injection site Record greatest surface diameter
in mm Fever* Record temperature in .degree. C./.degree. F. Headache
0 Absent 1 is easily tolerated 2 interferes with normal activity 3
prevents normal activity Fatigue 0 Absent 1 is easily tolerated 2
interferes with normal activity 3 prevents normal activity Joint
pain at the injection 0 Absent site and other locations 1 is easily
tolerated 2 interferes with normal activity 3 prevents normal
activity Muscle ache 0 Absent 1 is easily tolerated 2 interferes
with normal activity 3 prevents normal activity Shivering 0 Absent
1 is easily tolerated 2 interferes with normal activity 3 prevents
normal activity *Fever is defined as axillary temperature
.gtoreq.37.5.degree. C. (99.5.degree. F.)
[0558] 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.
[0559] 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
[0560] 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).
VI.4.2. Recording of Adverse Events (AE)
[0561] In elderly subjects, the reactogenicity observed with
adjuvanted vaccines, in terms of both local and general symptoms
was higher than with Fluarix.TM.. Not only the incidence but also
the intensity of symptoms was increased after vaccination with
adjuvanted vaccines (FIG. 8). Grade 3 symptoms showed a trend to be
higher in the group who received the vaccine adjuvanted with the
highest immunostimulants (MPL, QS21) concentration compared to the
group who received the adjuvanted vaccine wherein the
immunostimulants is at a lower concentration. In all cases,
symptoms however resolved rapidly.
EXAMPLE VII
Pre-Clinical Evaluation of Adjuvanted HPV Vaccines in Mice
[0562] This study used a bivalent antigenic composition from human
papillomavirus (HPV), combining virus like particles (VLPs) formed
from L1 of HPV 16 and L1 from HPV 18 as the antigen. The objective
of the study was to compare the efficacy of this antigenic
preparation when formulated with AS01B and a 1/5 dilution of AS01B,
benchmarked against the current adjuvant found in GSK's cervical
cancer vaccine, AS04 (MPL on alum).
VII.1--Vaccination
[0563] Mice (n=12 per group) were injected at 0 and 28 days with
vaccine formulations composed of HPV16/18 L1 (2 .mu.g or 0.5 .mu.g
each) derived from Hi-5 80/80L process and formulated with AS04 (50
.mu.g MPL formulated with alum or AS01B (50 .mu.g QS21-50 .mu.g MPL
in 0.5 ml) 1/10 and 1/50 Human dose. As the studies were carried
out in mice, 1/10 human dose can be taken to be equivalent to the
AS01B human formulation, i.e. 50 .mu.g QS21 and 50 .mu.g MPL in 0.5
ml and 1/50 can be taken to be a 1/5 dilution of the AS01B human
formulation i.e. 10 .mu.g QS21 and 10 .mu.g MPL in 0.5 ml. Blood
samples were taken at 14 and 45 days post dose II to assay for
total anti-L1 type specific antibodies in individual sera.
Intracellular cytokines staining were measured at days 7 and 14
post II on PBMC and at day 45 post II using spleen cells. Frequency
of VLPs specific memory B cells were measured at day 45 post II
using spleen cells.
VII.2--Anti-HPV 16/18 L1 ELISA
[0564] Quantification of anti-HPV-16 and HPV-18 L1 antibodies was
performed by ELISA using HPV-16 and HPV-18 L1 as coating. Antigens
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 (Maxisorp Immuno-plate, Nunc, Denmark). The
plates were then incubated for 1 hr at 37.degree. C. with PBS
containing 1% Bovine Serum Albumine (saturation buffer). Sera
diluted in buffer containing PBS+0.1% Tween 20+1% BSA were added to
the HPV L1-coated plates 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 (Dako, UK) diluted at 1/1000
in saturation buffer was added to each well and incubated for 1 hr
30 at 37.degree. C. After a washing step, streptavidin-horseradish
peroxydase (Dako, UK), diluted 1/3000 in saturation buffer was
added for an additional 30 min at 37.degree. C. Plates were washed
as indicated above and incubated for 20 min at room temperature
with a solution of 0.04% o-phenylenediamine (Sigma) 0.03%
H.sub.2O.sub.2 in 0.1% Tween 20, 0.05M citrate buffer pH 4.5. The
reaction was stopped with 2N H2SO4 and read at 492/620 nm. ELISA
titers were calculated from a reference by SoftMaxPro (using a four
parameters equation) and expressed in EU/ml.
VII.3--Intracellular Cytokines Staining (ICS)
[0565] Intracellular staining of cytokines of T cells was performed
on PBL at days 7 and 14 post II and on spleen cells at day 45 after
the second immunisation. PBMCs (1 pool/group) or spleen cells (4
pools of 3 organs per group) were collected from mice. In vitro
antigen stimulation of spleen cells were carried out at a final
concentration of 5 10.sup.6 cells/ml (microplate 96 wells) with VLP
16 or 18, (5 .mu.g/ml)+CD49d CD28 antibodies (1 .mu.g/ml) and then
incubated 3H at 37.degree. C. Following the antigen restimulation
step, cells were incubated overnight in presence of Brefeldin (1
.mu.g/ml) at 37.degree. C. to inhibit cytokine secretion. Cell
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-APC (1/50) and anti-CD8 perCp (1/50)
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 anti-IF.gamma. APC (1/50)+anti-IL-2 FITC (1/50) diluted
in PermWash. After 2 H incubation 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). The percentages of IF.gamma.+ or IL2+were
calculated on CD4+ and CD8+ gated populations.
VII.4--B Cell Memory
[0566] Forty-five days after the second immunisation, mice were
sacrificed, spleens cells were separated by a lymphoprep gradient
(Cedarlane). B cells 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% foetal calf serum, 50 U/ml rhIL-2
(eBioscience) and 3 .mu.g/ml CpG. 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 VLPs or with 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 VLPs coated plates and
100 .mu.l of 106 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 is stopped by rinsing plates gently under tap
water. When plates are dried, read with KS400.
VII.5--Statistical Analysis
[0567] The formulation means were compared using a one-way analysis
of variance (ANOVA 1). The analysis was conducted on log 10
transformed data for normalization purpose. When a significant
difference between process means was detected (p-value
.ltoreq.0.05), pair wise comparisons among means were performed at
a 0.05 significant level (Student-Newman-Keuls multiple comparison
test).
VII.6--Results
[0568] Mice were immunized as in VII.1 above. The following groups
were used:
TABLE-US-00018 Group Antigen Adjuvant Adjuvant dilution 1 HPV 16-18
L1 2 .mu.g AS04 1/10 human dose (equivalent to 50 .mu.g MPL per 0.5
ml HD) 2 HPV 16-18 L1 AS04 1/50 human dose (equivalent 0.5 .mu.g to
10 .mu.g MPL per 0.5 ml HD) 3 HPV 16-18 L1 2 .mu.g AS04 1/10 human
dose (equivalent to 50 .mu.g MPL per 0.5 ml HD) 4 HPV 16-18 L1 AS04
1/50 human dose (equivalent 0.5 .mu.g to 10 .mu.g MPL per 0.5 ml
HD) 5 HPV 16-18 L1 2 .mu.g AS01B 1/10 human dose (equivalent to 50
.mu.g MPL per 0.5 ml HD) 6 HPV 16-18 L1 AS01B 1/50 human dose
(equivalent 0.5 .mu.g to 10 .mu.g MPL per 0.5 ml HD) 7 HPV 16-18 L1
2 .mu.g AS01B 1/10 human dose (equivalent to 50 .mu.g MPL per 0.5
ml HD) 8 HPV 16-18 L1 AS01B 1/50 human dose (equivalent 0.5 .mu.g
to 10 .mu.g MPL per 0.5 ml HD)
VII.6.1--Humoral Responses
[0569] No significant dose range was observed for the two tested
doses of antigens with either dilution of adjuvant for either anti
HPV 16-L1 antibody titers or anti HPV 18-L1 antibody titers (FIG.
9)
[0570] No significant dose range was observed for the two tested
doses of each adjuvant whatever the dose of antigen for anti HPV
16-L1 antibody titers.
[0571] When looking at anti HPV 18-L1 antibody titers, a slight
increase in titer was seen for AS01B (1/10 HD) compared to AS01B
(1/50 HD) as measured at day 14 post II (2.5 fold dose range, p
value=0.0035), however this range was observed only for 2 .mu.g
antigen and not for 0.5 .mu.g antigen (p value=0.0867), By day 45
post II, no significant dose range was seen for the two tested
doses of each adjuvant whatever the dose of antigen.
VII.6.2--Cellular Responses
Intracellular Cytokine Staining
[0572] No dose range effect of antigen was observed for the two
tested doses of antigens whatever the dose of adjuvant for HPV
18-L1. Similar frequencies of VLP16 specific CD4+ T cells were
obtained with the two tested doses of antigens with different doses
of adjuvants. (FIG. 10).
[0573] A slight dosage effect (2.6 fold, p value=0.0009 for HPV
18-L1, 2 fold, p value=0.0187 for HPV 16-L1) was seen for AS01B
(1/10 HD) compared to AS01B (1/50 HD), however this range was
observed only for 2 .mu.g antigen and not for 0.5 .mu.g
antigen.
Specific B Memory Cells
[0574] No dose range effect of antigen was observed for the two
tested doses of antigens whatever the dose of adjuvant for HPV 16
or 18 L1 (FIG. 11)
[0575] No dose range effect of adjuvant was observed for the two
tested doses of adjuvants whatever the dose of antigen for HPV 17
or 18 L1.
[0576] As can be seen from the above results, a 1/5 dilution of
AS01B produces a formulation which has equivalent efficacy in
immunogenic compositions to AS01B itself.
EXAMPLE VIII
Preclinical Evaluation of Adjuvanted S. pneumoniae Vaccines in
Mice
[0577] The pneumococcal vaccine used in this study was an 11-valent
adjuvanted pneumococcal conjugate vaccines (11 PCV/AS) consisting
of a mixture of 11 pneumococcal polysaccharide conjugates
adjuvanted either with AS01B or AS01E. The conjugates consist of
the S. pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F
and 23F purified polysaccharides, each individually conjugated to a
carrier protein, either diphtheria toxoid (DT), tetanus toxoid (TT)
or protein D from H. influenzae (PD). The vaccines are presented as
a freeze-dried powder to be reconstituted with one of the liquid
adjuvants.
11PCV/AS is Produced as Follows:
[0578] The activation and coupling conditions are specific for each
polysaccharide. These are given in the table below. Sized
polysaccharide (except for PS5, 6B and 23F) was dissolved in NaCl
2M or in water for injection (WFI). The optimal polysaccharide
concentration was evaluated for all the serotypes. All serotypes
except serotype 18C were conjugated directly to the carrier protein
as detailed below.
[0579] From a 100 mg/ml stock solution in acetonitrile or
acetonitrile/water 50%/50% solution, CDAP (CDAP/PS ratio 0.75 mg/mg
PS) was added to the polysaccharide solution. 1.5 minute later,
0.2M-0.3M NaOH was added to obtain the specific activation pH. The
activation of the polysaccharide was performed at this pH during 3
minutes at 25.degree. C. Purified protein (protein D or DT) (the
quantity depends on the initial PS/carrier protein ratio) was added
to the activated polysaccharide and the coupling reaction was
performed at the specific pH for up to 2 hour (depending upon
serotype) under pH regulation. In order to quench un-reacted
cyanate ester groups, a 2M glycine solution was then added to the
mixture. The pH was adjusted to the quenching pH (pH 9.0). The
solution was stirred for 30 minutes at 25.degree. C. and then
overnight at 2-8.degree. C. with continuous slow stirring.
[0580] Preparation of 18C:
[0581] 18C was linked to the carrier protein via a linker--Adipic
acid dihydrazide (ADH) Polysaccharide serotype 18C was
microfluidized before conjugation.
Derivatization of Tetanus Toxoid with EDAC
[0582] For derivatization of the tetanus toxoid, purified TT was
diluted at 25 mg/ml in 0.2M NaCl and the ADH spacer was added in
order to reach a final concentration of 0.2M. When the dissolution
of the spacer was complete, the pH was adjusted to 6.2. EDAC
(1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was then added to
reach a final concentration of 0.02M and the mixture was stirred
for 1 hour under pH regulation. The reaction of condensation was
stopped by increasing pH up to 9.0 for at least 30 minutes at
25.degree. C. Derivatized TT was then diafiltrated (10 kDa CO
membrane) in order to remove residual ADH and EDAC reagent.
[0583] TT.sub.AH bulk was finally sterile filtered until coupling
step and stored at -70.degree. C.
Chemical Coupling of TT.sub.AH to PS18C
[0584] Details of the conjugation parameters can be found in Table
1.
[0585] 2 grams of microfluidized PS were diluted at the defined
concentration in water and adjusted to 2M NaCl by NaCl powder
addition.
[0586] CDAP solution (100 mg/ml freshly prepared in 50/50 v/v
acetonitrile/WFI) was added to reach the appropriate CDAP/PS
ratio.
[0587] The pH was raised up to the activation pH 9.0 by the
addition of 0.3M NaOH and was stabilised at this pH until addition
of TT.sub.AH.
[0588] After 3 minutes, derivatized TT.sub.AH (20 mg/ml in 0.2 M
NaCl) was added to reach a ratio TT.sub.AH/PS of 2; the pH was
regulated to the coupling pH 9.0. The solution was left one hour
under pH regulation.
[0589] For quenching, a 2M glycine solution, was added to the
mixture PS/TT.sub.AH/CDAP.
[0590] The pH was adjusted to the quenching pH (pH 9.0).
[0591] The solution was stirred for 30 min at 25.degree. C., and
then left overnight at 2-8.degree. C. with continuous slow
stirring.
Purification of the Conjugates:
[0592] The conjugates were purified by gel filtration using a
Sephacryl 500HR gel filtration column equilibrated with 0.15M NaCl
(S500HR for 18C) to remove small molecules (including DMAP) and
unconjugated PS and protein. Based on the different molecular sizes
of the reaction components, PS-PD, PS-TT or PS-DT conjugates are
eluted first, followed by free PS, then by free PD or free DT and
finally DMAP and other salts (NaCl, glycine).
[0593] Fractions containing conjugates are detected by UV.sub.280
nm. Fractions are pooled according to their Kd, sterile filtered
(0.22 .mu.m) and stored at +2-8.degree. C. The PS/Protein ratios in
the conjugate preparations were determined.
TABLE-US-00019 Specific activation/coupling/quenching conditions of
PS S. pneumoniae-Protein D/TT/DTconjugates 1 3 4 7F Serotype
.mu.fluid (.mu.fluid.) .mu.fluid 5 6B .mu.fluid PS 2.5 3.0 2.5 7.1
5.0 5.0 conc. (mg/ml) PS WFI NaCl 2M WFI WFI NaCl 2M NaCl 2M
dissolution PD 10.0 5.0 10.0 5.0 5.0 10.0 conc. (mg/ml) Initial
PS/PD 1.5/1 1/1 1.5/1 1/1 1.1/1 1.2/1 Ratio (w/w) CDAP conc. 0.50
0.75 0.50 0.79 0.83 0.75 (mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q
9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0
9.5/9.5/9.5 9V 14 18C 19F Serotype .mu.fluid .mu.fluid .mu.fluid
.mu.fluid 23F PS 5.0 5.0 4.5 9.0 2.38 conc. (mg/ml) PS NaCl 2M NaCl
2M NaCl 2M NaCl 2M NaCl 2M dissolution Carrier 10.0 10.0 20.0 (TT)
20.0 (DT) 5.0 protein conc. (mg/ml) Initial carrier 1.2/1 1.2/1 2/1
1.5/1 1/1 protein/PS Ratio (w/w) CDAP conc. 0.50 0.75 0.75 1.5 0.79
(mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q 9.5/9.5/9.0 9.5/9.5/9.0
9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0
[0594] The 11 conjugates were then mixed together, and the final
antigenic preparation mixed with the appropriate adjuvant before
immunisation.
[0595] Groups of 40 female 4-weeks old Balb/c mice were immunized
IM at days 0, 14 and 28 with 0.1 .mu.g of 11-valent PS conjugates
formulated with either AS01B or AS01E. Anti-PS IgG antibodies were
dosed by ELISA in sera collected at day 42.
[0596] As can be seen from FIG. 12, comparable responses were seen
between the diluted AS01E formulation compared to the AS01B
formulation except for PS 14 where a higher response was seen with
AS01B, and PS 19F where a higher response was seen with AS01E.
EXAMPLE IX
Preclinical Evaluation of Adjuvanted and Non-Adjuvanted
Cytomegalovirus Immunogenic Compositions
IX.1: Guinea Pigs
IX.1.1 Elisa Anti-gB
[0597] Quantification of anti-gB antibodies was performed by ELISA
using gB as a coating antigen. Antigen was diluted at a final
concentration of 4 .mu.g/ml in PBS and 100 .mu.l was incubated
overnight at 4.degree. C. in 96 well microtiter plates. Plastes
were then saturated for 1 hour at 37.degree. C. with 200 .mu.l of
PBS containing 1% bovine serum albumin. Two-fold serial dilutions
of sera were added (100 .mu.l/well) and incubated for 1 hour 30
minutes at 37.degree. C. The plates were washed 4 times with PBS
0.1% Tween 20 and 100 .mu.l of horseradish peroxidase anti-guinea
pig IgG (Dako, UK) was added to each well and incubated for 1 hour
30 minutes at 37.degree. C. Plates were washed 4 times with PBS
0.1% Tween 20 and 1 time with water. Then they were incubated for
20 min at 22.degree. C. with 100 .mu.l of a solution of
o-phenylenediamine (Sigma) in 0.1M citrate buffer pH 4.2. This
reaction was stopped with 100 .mu.l of H.sub.2SO.sub.4 2N and read
at 490/620 nm. Elisa titers were determined by interpolation of OD
values from a sample reference by SoftMaxPro. Titers were expressed
in EU/ml.
[0598] Statistical analyses were performed on days 14 post 2 Elisa
data using UNISTAT. The protocol applied for analysis of variance
can be briefly described as follows: [0599] 1) Log transformation
of the data [0600] 2) Shapiro-Wilk test on each population (group)
in order to verify the normality [0601] 3) Cochran test in order to
verify the homogeneity of variance between different populations
(groups) [0602] 4) Analysis of variance on selected data (one way)
[0603] 5) Tuckey-HSD test for multiple comparison.
IX.1.2--Neutralization Assay
[0604] Prior to the assay, MRC5 cells (10000 cells/200 .mu.l MEM
medium) were distributed in 96 well microplates and incubated for 3
days at 37.degree. C. with CO.sub.2. Two-fold dilutions of
inactivated sera (30 min at 56.degree. C.) were realized and
incubated with 100 .mu.l of viral solution (800/ml) for 1 hour at
37.degree. C. After incubation, 100 .mu.l of serium/virus mixture
was inoculated in 96 wells microplates containing MRC5 monolayer.
The plates were centrifuged at 2000 RPM for 1 hour at 35.degree. C.
After an overnight incubation at 37.degree. C., the plates were
fixed with an acetone 80% solution (20 minutes at -20.degree. C.).
The acetone solution was discarded and CMV positive cells were
detected using a specific monoclonal anti-immediate early antigen
for 1 hour at 37.degree. C. The plates were washed 3 times with PBS
and biotin-conjugated anti-mouse Ig was added to each well and
incubated for 1 hour at 37.degree. C. After a washing step,
streptavidin-horseradish peroxidase was added for an addition 30
minutes at 37.degree. C. Plates were washed 4 times and incubated
for 10 minutes with a solution of True-blue. Specific coloured
signals were recorded by examination under microscope. Neutralizing
titers were expressed as the reverse of the highest dilution of
serium giving 50% reduction of CMV positive cells as compared to a
virus control (CMV plus cells without serum).
IX.1.3--Immunization Protocols
[0605] 4 groups were immunised. Each group contained 8 female
Hartley Crl:(ha) Guinea pigs 5-8 weeks old, except for a control
group (group 4) containing only 4 subjects. Subjects were immunised
IM at 0 and 28 days. Serum samples were collected 28 days after the
first immunization and 14 days after the second immunization.
Elisas were performed as described above on serum taken at 28 post
I and 14 post II. Neutralisation assays were performed as described
above at 14 post II. Groups were as below:
TABLE-US-00020 Group Antigen Adjuvant 1 gB NaCl 2 gB AS01B 3 gB
AS01E 4 NaCl NaCl
[0606] The antigen was prepared as follows: The vaccine antigen is
expressed in Chinese Hamster Ovary (CHO) cells as gB**, a truncated
chimera containing peptide sequences from glycoprotein gD of Herpes
Simplex virus 2 (HSV2) at its N and C-terminus. The gB** is
truncated at its C-terminal domain that contains the membrane
anchoring sequence and is therefore secreted into the culture
supernatant.
[0607] For the first three groups, 15 .mu.g gB** made up in 500
.mu.l of either PBS, AS01B or AS01E (prepared as in example II.2
above) was injected intramuscularly. In group 4, PBS alone was
administered intramuscularly.
IX.1.4--Results
[0608] As can be seen in FIG. 13, Significantly higher anti-gB
ELISA titres were observed for the two adjuvanted groups as
compared to the gB plain (8 and 5.5-fold higher for gB and AS01B
and gb/AS01E respectively). Post dose II antibody titers were very
slightly higher (1.5 fold) in the gB/AS01B group compared to the
gB/AS01E group.
TABLE-US-00021 Multiple comparison: Tuckey - HSD Group Cases Mean
Plain AS01E AS01B Plain 8 4.7917 ** ** AS01E 8 5.5293 ** AS01B 8
5.6942 ** Plain < AS01E = AS01B
[0609] In respect of neutralising titres (FIG. 14): [0610] No
specific neutralising antibodies were observed in the gB plain
group [0611] Specific neutralising antibodies were detected in both
adjuvanted groups [0612] Similar levels of neutralising antibodies
were observed in both adjuvanted groups.
IX.2--Mice
IX.2.1--ELISA Anti gB
[0613] Quantification of anti-gB antibodies was performed by ELISA
using gB as a coating antigen. Antigen was diluted at a final
concentration of 1 .mu.g/ml in PBS and 100 .mu.l was incubated
overnight at 4.degree. C. in 96 well microtiter plates. Plastes
were then saturated for 1 hour at 37.degree. C. with 200 .mu.l of
PBS containing 1% bovine serum albumin. Two-fold serial dilutions
of sera were added (100 .mu.l/well) and incubated for 1 hour 30
minutes at 37.degree. C. The plates were washed 4 times with PBS
0.1% Tween 20 and 100 .mu.l of streptavidin-horseradish peroxidase
was added to each well for an additional 30 minutes at 37.degree.
C. Plates were washed 4 times with PBS 0.1% Tween 20 and 1 time
with water. Then they were incubated for 10 min at 22.degree. C.
with 100 .mu.l of tetra-methyl-benzidine 75% in 0.1M citrate buffer
pH 5.8. This reaction was stopped with 100 .mu.l of H.sub.2SO.sub.4
0.4N and read at 450/620 nm. Elisa titers were determined by
interpolation of OD values from a sample reference by SoftMaxPro.
Titers were expressed in EU/ml.
[0614] Statistical analyses were performed on days 14 post 2 Elisa
data using UNISTAT. The protocol applied for analysis of variance
can be briefly described as follows: [0615] 1) Log transformation
of the data [0616] 2) Shapiro-Wilk test on each population (group)
in order to verify the normality [0617] 3) Cochran test in order to
verify the homogeneity of variance between different populations
(groups) [0618] 4) Analysis of variance on selected data (one way)
[0619] 5) Tuckey-HSD test for multiple comparison.
IX.2.2--Neutralization Assay
[0620] Prior to the assay, MRC5 cells (10000 cells/200 .mu.l, MEM
medium) were distributed in 96 well microplates and incubated for 3
days at 37.degree. C. with 5% CO.sub.2. Two-fold dilutions (60
.mu.l) of inactivated sera (30 min at 56.degree. C.) were incubated
with 60 .mu.l of viral solution (800 IPU/ml) for 1 hour at
37.degree. C. After incubation, 100 .mu.l of sera-virus mixture was
inoculated in 96 well microplates containing MRC5 cells. The plates
were centrifuged at 2000 RPM for 1 hour at 35.degree. C. After an
overnight incubation at 37.degree. C., the plates were fixed with
an acetone 80% solution (20 minutes at -20.degree. C.). The acetone
solution was discarded and CMV positive cells were detected using a
specific monoclonal anti-immediate early I (IE-I) antigen for 1
hour at 37.degree. C. The plates were washed 3 times with PBS and
biotin-conjugated anti-mouse Ig was added to each well and
incubated for 1 hour at 37.degree. C. After a washing step,
streptavidin-horseradish peroxidase was added for an addition 30
minutes at 37.degree. C. Plates were washed 4 times and incubated
for 10 minutes with a solution of True-blue. Specific coloured
signals were recorded by examination under microscope. Neutralizing
titers were expressed as the reverse of the highest dilution of
serium giving 50% reduction of CMV positive cells as compared to a
virus control (CMV plus cells without serum).
IX.2.3--Intracellular Cytokine Staining
[0621] Intracellular detection of T cells cytokines were performed
on PBLs on days 7 and 21 after the second immunization. PBLs were
collected from mice and pooled (1 pool per group). In vitro antigen
stimulation of lymphocytes (final concentration of 10*7 cells/ml)
were done either with a pool of peptide covering the CMV sequence
or with the gB protein. PBLs/antigen mix was incubated 2H at
37.degree. C. Cells were then incubated overnight in the presence
of Brefelding (1 .mu.g/ml) at 37.degree. C. to inhibit cytokine
secretion.
[0622] Cell staining was performed as follows: Cell suspensions
were washed, resuspended in 50 .mu.l of PBS 1& FCS containing
2% Fc blocking reagent. After 10 min incubation at 4.degree. C., 50
.mu.l of a mixture of anti-CD4 PE and anti-CD8 perCp was added and
incubated 30 min at 4.degree. C. After a washing step in PBS 1%
FCS, cell membranes were permeabilised by resuspension in 200 .mu.l
of Cytofix=Cytoperm (kit Beckton Dickinson) and incubated 20 min at
4.degree. C. Cells were then washed with Perm Wash (kit BD) and
resuspended with 50 .mu.l of an anti-IFN-gamma APC+anti-IL-2 FITC
diluted in PermWash. After 2 hours incubation at 4.degree. C.,
cells were resuspended in PBS 1% FCS+1% paraformaldehyde.
[0623] Sample analysis was performed by FACS. Live cells were gated
and acquisition was performed on +/-20000 events. The percentages
of IFNg+ or IL2+were calculated on CD4+ and CD8+ gated
populations.
IX.2.4--Immunisation Protocols
[0624] 4 groups were immunised. Each group contained 12 female
C57BI/6 mice of 4-10 weeks old.
TABLE-US-00022 Group Antigen Adjuvant 1 gB PBS 2 gB AS01B 3 gB
AS01E 4 NaCl NaCl
[0625] The antigen was prepared as follows: The vaccine antigen is
expressed in Chinese Hamster Ovary (CHO) cells as gB**, a truncated
chimera containing peptide sequences from glycoprotein gD of Herpes
Simplex virus 2 (HSV2) at its N and C-terminus. The gB** is
truncated at its C-terminal domain that contains the membrane
anchoring sequence and is therefore secreted into the culture
supernatant.
[0626] For each group gB** at a concentration of 1.5 .mu.g/dose was
made up in 625 .mu.l of PBS or adjuvant AS01B or AS01E (prepared as
in example II.2 above having a concentration of 100 .mu.l of
immunostimulants per ml or 50 .mu.l of immunostimulants per ml
respectively). 50 .mu.l (i.e. 1/10 of a human dose of 0.5 ml) was
injected intramuscularly. One control group of mice was injected
with saline. Injections were performed at days 0 and 28. Serum
samples were collected 14 days after the second injections for
ELISA and Neutralisation assays. PBLs were collected 7 days and 21
days post second injections for ICS.
IX.2.5--Results
Anti-gB ELISA Titers (FIG. 15).
[0627] A very weak to undetectable level of anti-gB antibodies was
observed in the unadjuvanted gB group. However, a high antibody
response (65 and 66 fold higher) was observed in both adjuvant
groups, AS01B and AS01E respectively. There was no statistical
significance between the AS01B and AS01E group.
Multiple Comparison: Tuckey--HSD
TABLE-US-00023 [0628] Group Cases Mean Plain AS01E AS01B Plain 12
2.1132 ** ** AS01E 12 3.9317 ** AS01B 12 3.9375 ** Plain < AS01E
= AS01B
Anti-CMV Neutralizing Titers (FIG. 16)
[0629] Significantly higher anti-gB neutralising titers were
observed for the two adjuvanted groups as compared to the gB plain
group. No significant difference in neutralising antibody titres
was observed between the AS01B and AS01E formulations.
Cell Mediated Immunity.
[0630] Due to the very low level of response observed after
restimulation of 7 post II samples, no discrimination can be done
between groups and no conclusive response for CD4 and CD8
stimulation can be seen (FIG. 17). These low to undetectable
responses were probably due to a technical issue during sample
preparations. However, responses could be seen 21 days post second
injection. The CD4 data (FIG. 18) shows no difference after
restimulation by gB (5 .mu.g/ml) or peptides (2 .mu.g/ml or 4
.mu.g/ml). A similar cytokine profile is seen for AS01E and AS01B.
No conclusive response can be seen for CD8 stimulation (FIG.
19)
[0631] These experiments show that for another antigenic
composition and in two different organisms, an adjuvant having
lower levels of immunostimulants is as immunologically effective as
that having higher levels.
X: Preclinical Evaluation of Adjuvanted RTS,S Vaccine
X.1--Formulation
[0632] The antigenic composition, RTS, S is produced in
Saccharomyces cervisiae and consists of two proteins, RTS and S,
that intracellularly and spontaneously assemble into mixed
polymeric particulate structures that are each estimated to
contain, on average, 100 polypeptides. RTS is a 51 kDa hybrid
polypeptide chain of 424 amino acids consisting of 189aa derived
from a sporozoite surfact antigen of the malaria parasite P.
falciparum strain NF53 (the CSP antigen, a.a. 207 to 395), fused to
the amino terminal end of the hepatitis B virus S protein. S is a
24 kDa polypeptide (226 amino acids long) corresponding to the
surfact antigen of hepatitis B virus. The lyophilised antigen
pellet contains approximately 50 .mu.g (when designed to be
formulated in 0.5 ml with AS01B) or 25 .mu.g (when designed to be
formulated in 0.5 ml with AS01E) of antigen.
[0633] AS01B and AS01E were prepared by mixing the various
components (PBS, liposomes, MPL and QS21) in a tank, and stirring
under aseptic conditions. The product was then sterile filtered
before filling into vials or syringes. The liquid adjuvant was
stored at +2.degree. C. to +8.degree. C. before being used to
reconstitute the lyophilised antigen pellet.
X.2--Mice Experiments
[0634] Two experiments in mice were performed aiming at comparing
the immune responses specific to RTS,S induced by RTS,S/AS01B as
compared to RTS,S formulated with AS01E. In each experiment,
C57BI/6 mice (10 mice/group) were immunised intramuscularly three
times two weeks apart with 10, 5 or 2.5 .mu.g of RTS,S formulated
with AS01B or AS01E adjuvants. AS controls, two groups were
immunised with either AS01B or AS01E alone. The antibody responses
specific to HBs and CS were assessed for each mouse by ELISA 15
days after the third immunisation. The geometric mean antibody
titres and their 95% confidence intervals were calculated for all
the mice receiving the same treatment in both experiments.
Statistical analyses to evaluate adjuvant effect and antigen dose
effects were performed on pooled data from both experiments. The
CD4 and CD8 specific T cell responses were measured by flow
cytometry 7 days after the second and third immunizations on pools
of blood cells from 5 mice per group. Thus two values were
generated for each group in each experiment.
Humoral Immune Response
[0635] As shown in FIGS. 20 and 21, both AS01B and AS01E adjuvants
induce strong comparable antibody responses against CSP and
HBs.
[0636] A three-way ANOVA on anti-CSP GMTs showed that there was no
significant differences between AS01B and AS01E for the 5 or 2.5
.mu.g doses of RTS,S. For the 10 .mu.g dose, AS01B adjuvant was
found to induce higher anti-CS titers than AS01E and the GMT ration
"AS01B group/AS01E group" was 1.93 (95% CI: 1.33-2.79; p=0.001)
Cell Mediated Specific Immune Response
[0637] FIGS. 22 and 23 show the levels of CD4 and CD8 T cells
specific for CSP and HBs that express IL-2 and/or IFN gamma.
[0638] The CD4 response specific for CSP tends to be higher with
AS01B as compared to AS01E after three immunizations whereas the
CD8 T cell response with AS01E are equivalent to or better than
with AS01B.
[0639] The CD4 response specific for HBs tends to be higher with
AS01B as compared to AS01E after three immunizations except for the
lower dose of RTS,S where the levels of CD4 T cells are comparable
between the two adjuvants. The HBs specific CD8 T cell responses
induced by RTS,S formulated with AS01E are equivalent to or better
than the responses induced by RTS,S formulated with AS01B.
[0640] These differences are thought to be within the expected
variability of cellular immunology assays.
[0641] Pre-clinical evaluation of the RTS,S/AS01E vaccine in mice
revealed an acceptable safety profile, similar to that of
RTS,S/AS01B.
XI: Clinical Evaluation of RTS,S/AS01E.
[0642] Formulations are prepared as in example X above. Sucrose is
used as an excipient in the lyophillised antigen pellet. As in
example X, the liquid adjuvant is used to reconstitute the
lyophillised antigen. AS01E was prepared as described in example
II.2, and stored at +2 to +8.degree. C. until needed for
reconstitution.
[0643] A phase II randomized double-blind study of the safety and
immunogenicity of RTS,S adjuvanted with AS01E is currently underway
in children aged 18 months to 4 years living in Gabon. The
vaccination schedule is a 0, 1, 2-month vaccination schedule.
Objectives are as follows for RTS,S/AS01E when administered as 3
doses intramuscularly on a 0, 1, 2-month schedule to children aged
18 months to 4 years living in a malaria-endemic area:
Coprimary
[0644] to assess safety until one month post Dose 3. [0645] To
demonstrate non-inferiority to an oil in water emulsion adjuvanted
RTS,S vaccine in terms of anti-CS antibody response one month post
Dose 3.
Secondary
[0645] [0646] to assess reactogenicity until one month post Dose 3
[0647] to demonstrate non-inferiority to an oil in water emulsion
adjuvanted RTS,S vaccine in terms of anti HBs antibody response one
month post Dose 3 [0648] to describe seroprotection against
hepatitis B up to one month post Dose 3 [0649] to describe the
anti-CS response up to one month post Dose 3
Tertiary
[0649] [0650] Safety between 1 month post Dose 3 until 12 months
post Dose 3 [0651] Humoral immune response to CS antigen at 12
months post Dose 3 [0652] Humoral immune response to HBs antigen at
12 months post Dose 3
Exploratory
[0652] [0653] to evaluate T-cell mediated immune response to CS
antigen up to 12 months post dose 3 [0654] to evaluate B-cell
memory immune response to CS antigen up to 12 months post dose 3
[0655] to describe the anti-CS response up to one month post Dose 3
according to documented HBV immunization status at screening.
[0656] 180 subjects were enrolled, 90 were given a vaccine
adjuvanted with a previously validated proprietary oil in water
emulsion adjuvant (termed "control" in the tables below) and 90
were given a vaccine adjuvanted with AS01E. Healthy male and female
children aged 18 months to 4 years of age were screened. Vaccines
were administered by the IM route to the left deltoid.
TABLE-US-00024 Incidence and nature of symptoms (solicited and
unsolicited) reported during the 7- day (Days 0-6) post-vaccination
period following each dose and overall (Total vaccinated cohort)
Any symptom General symptoms Local symptoms 95% CI 95% CI 95% CI
Group N n % LL UL N n % LL UL N n % LL UL Dose 1 Gr 1 90 40 44.4
34.0 55.3 90 23 25.6 16.9 35.8 90 20 22.2 14.1 32.2 Gr 2 90 47 52.2
41.4 62.9 90 26 28.9 19.8 39.4 90 32 35.6 25.7 46.3 Dose 2 Gr 1 88
50 56.8 45.8 67.3 88 36 40.9 30.5 51.9 88 35 39.8 29.5 50.8 Gr 2 87
53 60.9 49.9 71.2 87 39 44.8 34.1 55.9 87 34 39.1 28.8 50.1 Dose 3
Gr 1 83 78 94.0 86.5 98.0 83 34 41.0 30.3 52.3 83 76 91.6 83.4 96.5
Gr 2 85 82 96.5 90.0 99.3 85 50 58.8 47.6 69.4 85 79 92.9 85.3 97.4
Overall/dose Gr 1 261 168 64.4 58.2 70.2 261 93 35.6 29.8 41.8 261
131 50.2 44.0 56.4 Gr 2 262 182 69.5 63.5 75.0 262 115 43.9 37.8
50.1 262 145 55.3 49.1 61.5 Overall/subject Gr 1 90 87 96.7 90.6
99.3 90 60 66.7 55.9 76.3 90 83 92.2 84.6 96.8 Gr 2 90 85 94.4 87.5
98.2 90 70 77.8 67.8 85.9 90 84 93.3 86.1 97.5 Gr 1 = RTS, S.AS01E
Gr 2 = control LL = lower limit UL = upper limit For each dose and
overall/subject: N = number of subjects with at least one
administered dose n/% = number/percentage of subjects presenting at
least one type of symptom whatever the study vaccine administered
For overall/dose: N = number of administered doses n/% =
number/percentage of doses followed by at least one type of symptom
whatever the study vaccine administered 95% CI = exact 95%
confidence interval, LL = Lower Limit, UL = Upper Limit
[0657] These data demonstrate that an AS01E adjuvanted RTS,S
vaccine gave acceptable reactogenicity results in a paediatric
population when compared with a control formulation.
[0658] Serological responses were measured by evaluating antibody
responses to HBs and to CSP repeats (anti R32LR). Serum for
antibody determination was collected at screening, at day 60 and at
day 90 (at second vaccination and at third vaccination). Antibody
levels against CS were measured by standard ELISA methodology using
plate adsorbed R32LR antigen with a standard reference antibody as
a control according to SOPs from the laboratory. Results are
reported in EU/mL.
[0659] Antibody to hepatitis B surface antigen was measured using a
commercially available ELISA immunoassay (AUSAB EIA test kit from
Abbott) or equivalent according to the assay instructions. Results
are reported in mIU/mL.
TABLE-US-00025 Seropositivity rates and GMCs for anti-CS antibodies
(Total vaccinated cohort) >=0.5 ELU/ML GMC 95% CI 95% CI
Antibody Group Timing N n % LL UL value LL UL Min Max Anti-CS Gr 1
SCREENING 89 0 0.0 0.0 4.1 0.3 0.3 0.3 <0.5 <0.5 PII(D60) 78
78 100 95.4 100 81.9 64.9 103.2 4.6 568.6 PIII(D90) 75 75 100 95.2
100 215.6 178.8 259.9 14.3 1922.3 Gr 2 SCREENING 90 1 1.1 0.0 6.0
0.3 0.2 0.3 <0.5 0.5 PII(D60) 78 78 100 95.4 100 56.9 45.7 70.9
3.6 2380.9 PIII(D90) 80 80 100 95.5 100 164.8 134.1 202.6 6.3
2093.6 Gr 1 = RTS, S.AS01E Gr 2 = Control GMC = geometric mean
antibody concentration calculated on all subjects N = number of
subjects with available results n/% = number/percentage of subjects
with concentration within the specified range 95% CI = 95%
confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX =
Minimum/Maximum
TABLE-US-00026 Seropositivity rates and GMCs for anti-HBs
antibodies (Total vaccinated cohort) >=10 MIU/ML GMC 95% CI 95%
CI Antibody Group Timing N n % LL UL value LL UL Min Max Anti-HBs
Gr 1 SCREENING 89 43 48.3 37.6 59.2 40.8 23.3 71.4 <10.0 46421.6
PII(D60) 78 77 98.7 93.1 100 8936.4 4684.2 17048.7 <10.0 1615367
PIII(D90) 75 75 100 95.2 100 24527.7 15316.5 39278.5 21.1 1694306
Gr 2 SCREENING 90 37 41.1 30.8 52.0 20.0 12.8 31.0 <10.0 30796.4
PII(D60) 78 77 98.7 93.1 100 3640.0 1963.1 6749.3 <10.0 1508114
PIII(D90) 80 80 100 95.5 100 19485.0 13511.3 28099.9 178.6 1103974
Gr 1 = RTS, S.AS01E Gr 2 = Control GMC = geometric mean antibody
concentration calculated on all subjects N = number of subjects
with available results n/% = number/percentage of subjects with
concentration within the specified range 95% CI = 95% confidence
interval; LL = Lower Limit, UL = Upper Limit MIN/MAX =
Minimum/Maximum
[0660] These data demonstrate that an AS01E adjuvanted RTS,S
vaccine formulation gave acceptable humoral immune responses in a
paediatric population when compared to a validated control.
EXAMPLE XII
Preclinical Evaluation of Varicella Zoster Virus with AS01B
Compared to AS01E
[0661] The candidate vaccine is composed of a truncated VZV
envelope protein, gE, produced in CHO cells.
[0662] For this study C57BL/6 mice (n=48) were primed with one
human dose (HD) of Varilrix (.about.4 log pfu/dose) administered
sub-cutaneously. Five weeks after priming with Varilrix, mice were
divided into to 5 groups of 12 mice and injected intramuscularly
(tibias) on days 0 and 28 with 5 .mu.g of gE alone, 5 .mu.g
gE+AS01E* (1/10 HD) or 5 .mu.g gE+AS01B (1/10 HD). The control
group of mice (primed only) was injected with saline (0.9% NaCl).
Immune responses were evaluated at 14 and/or 30 days following the
second vaccination. Levels of gE specific total antibodies and the
frequency of cytokine producing (IL2/IFN.quadrature.) CD4 and CD8 T
cells were evaluated.
gE Specific Antibody Responses:
[0663] An ELISA was developed to detect and quantify gE-specific
antibodies in mice sera, using gE protein as the coating antigen.
The ELISA titers were defined as the reciprocal of the serum
dilution, which produced an absorbance (optical density) measure
equal to 50% of the maximal absorbance value. ELISA titers were
calculated by regression analysis.
[0664] The data demonstrate that gE AS01E and gE AS01B induced
similar levels of gE specific antibodies (pvalues >0.05). Both
formulations induced significantly higher responses compared to the
gE antigen alone (10-13 fold, pvalues <0.05) at both 14 and 30
days post II (FIG. 26)
TABLE-US-00027 14 days post II 95% CI 30 days post II 95% CI GMT
GMT Group (EU/ml) LL UL (EU/ml) LL UL gE 12067 5960 24433 3832 911
16115 gE/AS01E 125934 95504 166059 50439 38071 66825 gE/AS01B
131728 88112 196934 47589 36158 62635 Varilrix 34 11 105 33 10
102
gE Specific CD4 and CD8 Responses
[0665] Cytokine production was evaluated in CD4 and CD8 T cells
using an intra-cellular cytokine staining technique. Spleen cells
were isolated from each group of 12 mice at 30 days post II and
pooled into 4 groups of 3 spleens. Spleen cells (1.times.10.sup.6)
were incubated for 2 hours in the presence of gE peptides (63
peptides) spanning the complete gE protein (20 aa peptides/10 aa
overlap) and then incubated overnight in the presence of brefeldin.
Subsequently cells were stained with fluorescent mAb specific for
cell surface CD4/CD8 and following permeabilization intracellular
cytokines IL-2 and IFN.gamma..
[0666] As shown in FIG. 26 although similar cytokine profiles
(IL2/IFN.gamma.) were induced with both gE AS01B and gE AS01E
formulations, the AS01B formulation induced a higher magnitude of
both CD4 and CD8 cytokine producing cells (2 fold, p>0.05 for
CD4, 3.6 fold, p>0.05 for CD8). Due to unexpectedly high
variability of the T cell responses the power to detect a
significant difference between adjuvant doses was very limited
(<50%). Importantly both gE formulated with AS01B or AS01E
induced cytokine producing CD4 T cells of a significantly higher
magnitude (13.3 fold, p<0.05) compared to gE alone. Higher
levels of CD8 cells were also induced by gE formulated with AS01B
or AS01E (3.8 fold, p>0.05) compared to gE antigen alone.
EXAMPLE XIII
Preclinical Evaluation of AS01B v AS01E in an Influenza Ferret
Model
Materials and Methods
[0667] Female ferrets (Mustela putorius furo) aged 4-6 months were
obtained from MISAY Consultancy (Hampshire, UK). Ferrets were
primed on day 0 with heterosubtypic strain H1N1 A/Stockholm/24/90,
(4LogTCID50/ml), 250 .mu.l administered intranasally. On day 21,
ferrets were injected intramuscularly with a full human dose (1000
.mu.l vaccine dose, 15 .mu.g HA per A strain, 17.5 .mu.g B strain)
of a combination of H1N1A/New Caledonia C/20/99 (15 .mu.g/ml), H3N2
A/Wyoming/3/2003 (15 .mu.g/ml) and B/Jiangsu/10/2003 (17.5
.mu.g/ml). Ferrets were then challenged on day 42 by intranasal
route with 250 .mu.l of a heterosubtypic strain Wh.A/NY/55/04 (4.51
Log TCID50/ml).
[0668] Vaccinations on day 21 were either with the plain trivalent
formulation ("plain" in the tables below) or with the trivalent
formulation adjuvanted with AS01B ("AS01B" in the tables below) or
AS01E ("AS01E" in the tables below). Formulations were prepared as
set out in example 3 above.
Body Temperature Monitoring:
[0669] Individual temperatures were monitored during the challenge
period and were assessed using telemetry implants which recorded
each individual animal temperature every 15 minutes before and
after the challenge. 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. Temperatures were recorded
every 15 minutes 6 days before priming until 4 days post-priming,
as well as 3 days before challenge until 7 days post-challenge.
Hemagglutination Inhibition Test (HI).
Test Procedure
[0670] 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 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 using tears for reading. 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
[0671] Statistical analysis was performed on HI titers using
UNISTAT. The protocol applied for analysis of variance can be
briefly described as followed: [0672] Log transformation of data.
[0673] Shapiro-wilk test on each population (group) in order to
verify the normality of groups distribution. [0674] Cochran test in
order to verify the homogenicity of variance between the different
populations (groups). [0675] One-way analysis of variance performed
on groups. [0676] Tuckey-HSD Test for multiple comparisons.
Viral Titration in Nasal Washes
[0677] 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 6-7 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 hours. 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 TCID.sub.50/ml.
Lymphoproliferation Assay.
[0678] PBMC were collected by density gradient centrifugation (20
min at 2500 rpm and 4.degree. C.) on Ficoll Cedarlane lympholyte
mammal solution. PBMC were resuspended in 5 ml culture medium
(RPMI/Add at 4.degree. C.) and 10% of normal ferret serum.
Additives were composed by 100 mM sodium pyruvate, non essential
amino acids MEM, Penicillin/streptamycine, glutamine and
1000.times. concentrated .beta.2-mercaptoethanol. Freshly isolated
PBMC were immediately used for in vitro proliferation assays. The
cells were placed in 96-well flat bottom tissue culture plates at
2.times.10.sup.5 cells/well and cultured with different
concentrations of antigen (0.1 to 1 .mu.g HA of whole inactivated
virus) for 44 to 96 h and then were pulse labeled with 0.5 .mu.Ci
of [.sup.3H]thymidine. Incorporation of radiolabel was estimated 4
to 16 h later by .beta.-emission spectroscopy.
Results
[0679] Viral Load in Nasal Washes after Challenge.
[0680] Nasal washes were collected 2 days before priming
(priming=day 0) 1, 2 and 7 days post priming, as well as 4 days
before challenge (challenge=day 42) and for a period of 7 days post
challenge.
TABLE-US-00028 Group -2 0 +1 +2 +7 39 42 43 44 45 47 49 Plain 0.82
1.84 5.35 1.85 0.8 1.82 5.77 4.44 1.97 0.9 AS01E 0.82 2.11 5.83
1.65 0.8 1.62 4.93 4.15 2.4 0.85 AS01B 0.81 2.26 5.38 1.91 0.82
1.74 2.25 1.89 1.350 0.9
[0681] See results in FIG. 27.
Viral Shedding after Priming
[0682] A peak of viral shedding was observed in all ferrets 2 days
after the priming. 7 days post priming, only residual viral load
was observed in all groups.
Viral Shedding after Challenge
[0683] The peak of viral shedding was observed 24 hours after
challenge. Viral titration 3 days post-challenge showed high viral
titers (no protection) in ferrets immunized with trivalent split
plain. Lower reduction of viral shedding was observed in ferrets
immunised with trivalent split AS01E than was seen with trivalent
split adjuvanted with AS01B.
Temperature Monitoring:
[0684] Body temperature was monitored from 6 days pre-priming
(priming=day 0) until 4 days post-priming as well as from 3 days
pre-challenge until 7 days post challenge (challenge=day 42).
Measurements were taken every 15 minutes and an average calculated
by mid-day for each group. Results can be seen in FIG. 28.
Post Priming
[0685] Body temperature monitored before, during and after priming
did show an increase in temperature in all groups.
Post-Challenge
[0686] Interpretation of body temperature monitoring is difficult.
A slight increase of body temperature was observed post-challenge
in ferrets immunized with trivalent split plain and trivalent split
AS01E, but not with trivalent split AS01B. The score below was
obtained by the number of ferrets with an increase of body
temperature >0.4.degree. C.
Increase in Temperature Post Challenge
[0687] Trivalent plain: 5/8 (+0.4, +0.4, +0.5, +0.7, +0.8)
Trivalent AS01B 0/8
Trivalent AS01E 6/8 (+0.4, +0.4, +0.5, +0.5, +0.9, +1.6)
[0688] This read out is less robust than other read outs used in
ferrets.
Haemagglutination Inhibition Test (HI)
[0689] Serum samples were collected 4 days before priming, 17 days
post-priming, 21 days post-immunization and 13 days post challenge.
Results can be seen in FIGS. 29 and 30. For all three vaccine
strains, statistically significantly higher HI titers were observed
in ferrets immunised with trivalent split adjuvanted with AS01B or
AS01E compared to trivalent split plain. No difference was observed
between the two adjuvanted groups. Compared to other groups
statistically significantly higher cross-reactive HI titers to
A/New York H3N2 (challenge strain) were observed after immunisation
of ferrets with trivalent split vaccines adjuvanted with AS01B.
* * * * *