Note: Descriptions are shown in the official language in which they were submitted.
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VACCINE
The present invention relates to new vaccine formulations comprising a
Respiratory Syncytial Virus (RSV) antigen and a 'CpG' containing
immunostimulating oligonucleotide, methods for preparing it and its use in
therapy.
In addition the present invention relates to new combination vaccines for
administration to children, to adults and to the elderly.
Human Respiratory Syncytial Virus (RSV) is a member of the
Paramyxoviridiae family of viruses and causes lower respiratory tract illness,
particularly in young children and babies. Recent report suggests that RSV is
an
important pathogen in adults, particularly the elderly.
RSV is an enveloped virus with a non-segmented, negative strand
ribonucleic acid (RNA) genome of 15,222 nucleotides that codes for 10
messenger
RNAs, each coding for a single polypeptide. Three of the ten proteins are
transmembrane surface proteins: the G (attachment), F (fusion) and SH
proteins.
Two proteins are virion matrix proteins (M and M2), three proteins are
components
of the nucleocapsid (N, P and L), and two proteins are nonstructural (NS1 and
NS2). Two antigenically distinct strains of RSV exist, designated strain A and
B.
Characterization of strains from these groups has determined that the major
differences reside on the G proteins, while the F proteins are conserved.
RSV occurs in seasonal outbreaks, peaking during the winter in temperate
climates and during the rainy season in warmer climates. Wherever the area,
RSV
tends to have a regular and predictable pattern and other respiratory viral
pathogens
that occur in outbreaks are rarely present concurrently.
RSV is a major cause of serious lower respiratory tract disease in children.
It is estimated that 40-50 % of children hospitalized with bronchiolitis and
25 % of
children hospitalized with pneumonia are hospitalized as a direct result of
RSV
infections. Primary RSV infection usually occurs in children younger than one
year
of age; 95 % of children have serologic evidence of past infection by two
years of
age and 100% of the population do so by adulthood.
In infants and young children, infection progresses from the upper to the
lower respiratory tract in approximately 40 % of cases and the clinical
presentation
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is that of bronchiolitis or pneumonia. Children two to six months of age are
at
greatest risk of developing serious manifestations of infection with RSV
(primarily
respiratory failure); however, children of any age with underlying cardiac or
pulmonary disease, premature infants, and infants who are immunocompromised,
are at risk for serious complications as well.
Symptomatic reinfection occurs throughout life and it has become
increasingly apparent that RSV is an important adult pathogen as well,
especiaMly~for
the elderly. RSV infection is almost certainly under-diagnosed in adults, in
part
because it is considered to be an infection of children. Consequently,
evidence of
the virus in adults is not sought in order to explain respiratory illness. In
addition,
RSV is difficult to identify in nasal secretions from individuals who have
some
degree of partial immunity to the virus, as do the large majority of adults.
Young
to middle-age adults typically develop a persistent cold-like syndrome when
infected
with RSV. Elderly individuals may develop a prolonged respiratory syndrome
which is virtually indistinguishable from influenza, with upper respiratory
symptoms which may be accompanied by lower respiratory tract involvement,
including pneumonia. Institutionalised elderly populations are of particular
concern, because they comprise large numbers of susceptible individuals
clustered
together. The spread of infection through such a population, many of whom have
multiple medical problems which may predispose them to a more severe course of
the disease, is difficult to control. Morbidity and mortality may be
considerable:
pneumonia has been reported in 33 to 67 % of cases, and mortality rates of 5
to 20
have been reported.
A number of studies have evaluated the contribution of RSV infection to
respiratory illness and mortality in nursing homes. Infection rates with RSV
in the
nursing homes has been reported as 9 % in a home in France of which 6 % died,
8
in South Carolina, 27 % in Rochester of which 5 % died and 21 % in Los
Angeles.
Finally, reports of recent studies evaluating the impact of RSV infection as a
cause
of hospitalisation in adults and in community dwelling healthy elderly further
point
to an important role of RSV infection in severe lower respiratory tract
disease in
these populations. Dowell identified RSV as one of the four most common
pathogens causing severe lower respiratory tract disease resulting in
hospitalisation
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of adults. Falsey demonstrated that serious RSV infections in elderly persons
are
not limited to nursing homes or outbreak situations. Rather, RSV infection is
a
predictable cause of serious illness among elderly patients residing in the
community. Similar to hospitalisations for influenza A, those related to RSV
infections were associated with substantial morbidity, as evidenced by
prolonged
hospital stays, high intensive care admission rates, and high ventilatory
support
rates.
Taken together, these studies point to the medical and economic need for an
effective vaccine which can prevent severe complications of RSV infection in
infants, adults and both community dwelling healthy and institutionalised
elderly.
RSV vaccination in the 1960s with whole formalin inactivated virus formulated
with
alum, led to exacerbation of disease in children subsequently exposed to
natural
RSV infection. There is therefore a need for a safe and effective vaccine to
provide
protection against this pathogen.
RSV has two envelope glycoproteins, the F protein having a molecular
weight of 68,000 to 70,000 Daltons and a larger G glycoprotein having a
molecular
weight of 84,000 to 90,000 Daltons (Collins et al J. of Virology Vol 49 pp 572-
578
(1984)).
FG fusion proteins, typically comprising the extracellular domain of both
proteins, are known (US 5,194,595 Upjohn).
Vaccine preparations comprising FG constructs and 3D-MPL are described
in WO 98/18819 (SmithKline Beecham Biologicals s.a.).
Immunomodulatory oligonucleotides contain unmethylated CpG
dinucleotides ("CpG") and are known (WO 96/02555, EP 468520). CpG is an
abbreviation for cytosine-guanosine dinucleotide motifs present in nucleic
acid.
Historically, it was observed that the DNA fraction of BCG could exert an anti-
tumour effect. In further studies, synthetic oligonucleotides derived from BCG
gene sequences were shown to be capable of inducing immunostimulatory effects
(both in vitro and in vivo). The authors of these studies concluded that
certain
palindromic sequences, including a central CG motif, carried this activity.
The
central role of the CG motif in immunostimulation was later elucidated in a
publication by Krieg, Nature 374, p546 1995. Detailed analysis has shown that
the
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CG motif has to be in a certain sequence context, and that such sequences are
common in bacterial DNA but are rare in vertebrate DNA.
It is currently believed that this evolutionary difference allows the
vertebrate
immune system to detect the presence of bacterial DNA (as occurring during an
infection) leading consequently to the stimulation of the immune system. The
immunostimulatory sequence as defined by Krieg is:
Purine Purine CG pyrimidine pyrimidine and where the CG motif is not
methylated. In certain combinations of the six nucleotides a palindromic
sequence
is present. Several of these motifs, either as repeats of one motif or a
combination
of different motifs, can be present in the same oligonucleotide. The presence
of one
or more of these immunostimulatory sequence containing oligonucleotides can
activate various immune subsets, including natural killer cells (which produce
interferon y and have cytolytic activity) and macrophages (Wooldrige et al Vol
89
(no. 8), 1977). Although other unmethylated CpG containing sequences not
having
this consensus sequence have now been shown to be immunomodulatory.
The present invention provides a vaccine preparation comprising an
immunostimulatory CpG oligonucleotide and a RSV antigen.
Preferably the RSV antigen is a RSV envelope glycoprotein or derivative
thereof derived from, preferably, strain A. More preferably the antigen is
selected
from F glycoprotein, G glycoprotein or a FG fusion protein or immunogenic
derivatives thereof. Alternatively the RSV antigen may be for example
inactivated
virus.
Typically immunogenic derivatives include wherein the protein is devoid of
the transmembrane domain ie F Otm, G Otm and F Otm GOtm. Preferably the
signal sequence is deleted from the G protein. It is preferred that at least
about
50 % or at least about 80 % (contiguous sequence) of the extracellular domain
of the
F or G protein is present. Particular examples include 1-526 or 1-489 amino
acid
of the F protein, amino acid 69-298 or alternative positions 97-279 of the G
protein,
and F~_SZ6G69-298 Vision protein. An alternative fusion comprises 1-489 amino
acid
from F followed by 97-279 of G protein.
The preferred oligonucleotides preferably contain two or more CpG motifs
separated by six or more nucleotides. The oligonucleotides of the present
invention
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are typically between 15-45 oligonucleotides in length and are typically
deoxynucleotides. In a preferred embodiment the internucleotide in the
oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate
bond,
although phosphodiester and other internucleotide bonds are within the scope
of the
invention including oligonucleotides with mixed internucleotide linkages.
Preferred oligonucleotides have the following sequences: The sequences
preferably contain all phosphorothioate modified internucleotide linkages.
WD000 1: TCC ATG ACG TTC CTG ACG TT
WD000 2: TCT CCC AGC GTG CGC CAT
WD000 3: ACC GAT AAC GTT GCC GGT GAC G
WD000 7: ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG
The CpG oligonucleotides utilised in the present invention may be
synthesized by any method known in the art (eg as described in EP 468520).
Conveniently, such oligonucleotides may be synthesized utilising an automated
synthesizer. Methods for producing phosphorothioate oligonucleotides or
phosphorodithioate are described in US5,666,153, US5,278,302 and W095/26204.
When CpG and aluminium salt, such as aluminium hydroxide or aluminium
phosphate (Alum) are both present this synergistically enhances anti RSV
antigen
specific antibody. In particular, such a combination adjuvant significantly
enhances
the levels of IgG2a antibody, a marker of a TH1 response. Moreover, the
adjuvant
combination of a CpG oligonucleotide and an aluminium salt allows a cell
mediated
response as determined by specific lymphoproliferation.
Accordingly, in one embodiment of the invention there is provided a vaccine
formulation comprising a RSV antigen and an immunostimulatory CpG
oligonucleotide in combination with an aluminium salt. Preferably the
aluminium
salt is aluminium hydroxide.
Other vaccine excipients may be added to the formulation for the invention.
Preferred additional immunostimulants include for example saponin adjuvants,
such
as QS21.
In the vaccine of the present invention, an aqueous solution of the proteins)
can be used directly for mixing with the adjuvant. Alternatively, the protein
can be
lyophilised.
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The antigens of the present invention may be expressed in any suitable host,
such as bacterial, mammalian, insect, yeast and fungal cells. The use of
insect cells
such as Sf9 cells is described by Du et al, BIOTECHNOLOGY 12,1994, 813-818.
Preferably the proteins of the invention are expressed in insect cells using a
recombinant baculovirus (Wathen et al J.Gen Virol 1989, 70 pp2625-2635). Most
preferably however the proteins of the invention are produced in eukaryotic
cells,
particularly in Chinese Hamster Ovary (CHO) cells and Vero cells and purified
by
the method as disclosed in W098/18819 (SmithKline Beecham Biologicals s.a.).
In a preferred embodiment of the invention the antigen is produced by
expression in mammalian cells from a DNA sequence having optimised mammalian
codon usage. Optimisation of the codon usage involves the replacement of at
least
one non-preferred or less preferred codon in a natural gene encoding a protein
by a
preferred codon encoding the same amino acid. Mammalian genes expressed at
high
levels typically have C or G at their degenerative position (third base in the
codon)
whereas the RSV or more generally paramyxoviridae codons have A or T. At least
one codon, and more preferably all the codons of the RSV protein can be
changed
to best fit mammalian cell usage, that is, the one (or ones) that is the most
prevalent
as shown below.
Ala: GCC Cys: TGC His: CAC Met: ATG Thr: ACC
Arg: CGC Gln: CAG Ile: ATC Phe: TTC Trp: TGG
AGG
CGG
Asn: AAC Glu: GAG Leu: CTG Pro: CCC Tyr: TAC
Asp: GAC Gly: GGC Lys: AAG Ser: AGC Val: GTG
TCC
Each amino acid encoded by one of these codons is then considered
optimised.
The antigen according to the invention may be expressed as a fusion protein.
For example, the antigen may be a heterochimeric fusion of an RSV envelope
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antigen or an immunogenic derivative thereof with an antigen from a different
pathogen. Particular examples include fusions with envelope antigens or
immunogenic derivatives from other Paramyxoviruses eg parainfluenza viruses
(PIV-1, 2 and 3) or mumps virus or measles virus.
The invention also provides DNA encoding such a protein or immunogenic
derivative thereof in which the codon usage of one or more nucleic acids has
been
substantially optimised and a process for expressing said DNA in a CHO or
insect
cell.
The antigens may also be made using a recombinant live microorganism, such
as a virus or bacterium as the expression system. The gene of interest can be
inserted
into the genome of a live recombinant virus or bacterium. Inoculation and in
vivo
infection with this live vector will lead to in vivo expression of the antigen
and
induction of immune responses. Viruses and bacteria used for this purpose are
for
instance: poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses
(Sindbis virus,
Semliki Forest Virus, Venezuelian Equine Encephalitis Virus), adenoviruses,
adeno-
associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses
(varicella zoster
virus, etc), Listeria, Salmonella , Shigella, BCG. These viruses and bacteria
can be
virulent, or attenuated in various ways in order to obtain live vaccines. Such
live
vaccines when formulated with a CpG oligo nucleotides also form part of the
invention.
The invention further relates to methods for constructing and expressing the
proteins of the invention and methods to optimise the codon usage of the
nucleic
acid sequences which encode such proteins.
The antigens of the present invention may also be presented as a nucleic acid
encoding said antigen and formulated with a CpG oligonucleotide.
The antigens of the present invention are not necessarily recombinant subunit
anitigens. The RSV antigen may for example be in the form of inactivated whole
virus. Such inactivated virus may be produced from RSV which has been
attenuated eg by passaging or by genetic manipulation. A recombinant RSV may
be
used which has been engineered to contain an envelope antigen from a different
strain of RSV eg an RSV strain A backbone with an RSV B envelope protein,
optionally attenuated.
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Vaccine preparation is generally described in New Trends and Developments
in Vaccines, edited by Voller et al, University Park Press, Baltimore,
Maryland,
USA 1978 and in Vaccine Design: The Subunit and Adjuvant Approach, edited by
Powell and Newman, Plenum Press, New York, 1995. Encapsulation within
liposomes is described, for example, by Fullerton, US Patent 4,235,877.
Conjugation of proteins to macromolecules is disclosed, for example by
Likhite, US
Patent 4,372,945 and by Armor et al, US Patent 4,474,757.
The amount of protein in each vaccine dose is selected as an amount which
induces an immunoprotective response without significant, adverse side effects
in
typical vaccinees. Such amount will vary depending upon which specific
immunogen is employed and how it is presented. Generally, it is expected that
each
dose will comprise 1-1000 pg of protein, preferably 2-100 p.g, most preferably
5-50
p.g. An optimal amount for a particular vaccine can be ascertained by standard
studies involving observation of appropriate immune responses in subjects.
Following an initial vaccination, subjects may receive one or several booster
immunisations adequately spaced.
Suitably the CpG will be present in the range 100p,g per dose to 3000~,g,
preferably 250-750p,g, such as SOO~g per dose.
Suitably the vaccine used in the present invention may comprise a carrier
such as an aluminium salt, eg aluminium hydroxide (A1(OH3), aluminium
phosphate or aluminium phosphate sulfate (alum), or a non-toxic oil in water
emulsion or a mixture thereof.
If an aluminium salt (preferably aluminium hydroxide) is used as a carrier it
is generally present in the range of 50 to 100pg (human: 500 to 1000p,g)
preferably
SOO~.g per dose.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, eg
squalene and an emulsifier such as polysorbitan monoleate (Tween 80), in an
aqueous carrier such as phosphate buffered saline.
If desired the vaccine used in the present invention may comprise an
additional adjuvant, preferably a saponin adjuvant such as QS21 as described
for
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example in WO 95/17210, optionally in the presence of a sterol, such as
cholesterol
as described for example in PCT/EP96/01464.
If desired, other antigens may be added, in any convenient order, to provide
multivalent vaccine compositions as described herebelow.
In a preferred aspect the vaccine formulation of the invention additionally
comprises a Streptococcus pneumoniae antigen.
Streptococcus pneumoniae is a gram positive bacteria responsible for
considerable morbidity and mortality, particularly in the young and aged.
Expansive colonisation of the respiratory tract, and middle ear, especially in
young
children, is the single most common cause for hospital visits in the US. The
bacteria may become invasive, infecting the lower lungs and causing pneumonia.
The rate of pneumococcal pneumonia in the US for persons over 60 years of age
is
estimated to be 3 to 8 per 100,000. In 20% of cases this leads to bacteremia,
and
other manifestations such as meningitis, with a mortality rate close to 30 %
even
with antibiotic treatment. There are 90 known serotypes of Streptococcus
pneumoniae which are determined by the structures of the capsular
polysaccharide
surrounding the bacteria, and this is its major virulence factor.
A 17 - valent pneumococcal vaccine (Moniarix) is known, based on the
purified polysaccharides of the pneumococcal serotypes most commonly involved
in
invasive disease. The method of purification of these polysaccharides was
disclosed
in European Patent 72513 B1. Vaccine efficacy trials with lower valent
vaccines
demonstrated a 70 to 90 % efficacy with respect to serotypes present in the
combination. Case controlled studies in the US in persons > 55 years using a
14
valent vaccine demonstrated 70% efficacy (Mills and Rhoads 1996). Inclusion of
additional polysaccharides (to make a 23-valent pneumococcal vaccine) were
accepted on the basis of an adequate serological response, even though there
was
clinical efficacy data lacking (Brown 1995).
Pneumococcal polysaccharides can be rendered more immunogenic by
chemically coupling them to protein carriers, and clinical efficacy trials are
being
performed to verify this concept for efficacy in preventing infant Otitis
media.
There are two conjugation methods generally used for producing
immunogenic polysaccharide constructs: (1) direct conjugation of carbohydrate
and
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protein; and (2) indirect conjugation of carbohydrates and protein via a
bifunctional
linker or spacer reagent. Generally, both direct and indirect conjugation
require
chemical activation of the carbohydrate moiety prior to derivatisation. See
for
example US 5,651,971 and Dick & Beurret, "Glycoconjugates of Bacterial
Carbohydrate Antigens," Conjugate Vaccines, J.M. Cruse & R.E. Lewes (eds),
Vol. 10, 48 - 114 (1989).
Typically the Streptococcus pneumoniae component in a vaccine of the
present invention will comprise polysaccharide antigens (preferably
conjugated),
wherein the polysaccharides are derived from at least four serotypes of
pneumococcus. Preferably the four serotypes include 6B, 14, 19F and 23F. More
preferably, at least 7 serotypes are included in the composition, for example
those
derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. More preferably
still, at
least 11 serotypes are included in the composition, for example the
composition in
one embodiment includes capsular polysaccharides derived from serotypes 1, 3,
4,
5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated). In another
preferred embodiment of the invention at least 13 polysaccharide antigens
(preferably conjugated) are included, although further polysaccharide
antigens, for
example 23 valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A,
11A,
12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated
by the invention. For elderly vaccination (for instance for the prevention of
pneumonia) it is advantageous to include serotypes 8 and 12F (and most
preferably
15 and 22 as well) with the 11 valent antigenic composition described above to
form
a 15 valent vaccine, whereas for infants or toddlers (where otitis media is of
more
concern) serotypes 6A and 19A are advantageously included to form a 13 valent
vaccine.
In another preferred aspect the vaccine composition of the invention
additionally comprises a Group B Streptococcus antigen.
Among the infants, Group B streptococci (GBS) are a main cause of life
threatening bacterial infections, eg pneumonia and meningitides. Additionally
GBS
has emerged as an important pathogen in adults, more especially the elderly
patients
or patients with chronic underlying diseases.
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All strains of GBS express a polysaccharide capsule. Among the nine
capsular serotypes identified, strains of the four classical serotypes (Ia,
Ib, II and
III) are responsible for most invasive neonatal infections. Approximately 90 %
of
these strains express either Rib or alpha, two members of the same family of
streptococcal cell surface proteins, and have been shown to confer protective
immunity against GBS in animal models.
Optionally the vaccine composition of the invention additionally comprises
one or more of a number of other antigens such as an antigen against influenza
virus
or PIV-3.
In order that this invention may be better understood, the following
examples are set forth. These examples are for purposes of illustration only,
and
are not to be construed as limiting the scope of the invention in any manner.
EXAMPLES
EXAMPLE 1 - Studies on RSV formulated with CpG containing
oli~onucleotides
1. Formulations:
FG antigen was expressed in CHO cells and purified according to W098/ 18819
1.1. CpG or MPL based formulations
When needed, Al(OH)3 was diluted in H20 before adsorption of the FG (2 ~cg)
antigen for 30 minutes. Subsequently MPL or CpG was added and incubated for
thirty minutes. The formulations were then buffered with 10 fold concentrated
P04-
NaCI pH 6.8. Thiomersal at 1 mg/ml or phenoxy at Smg/ml was added as
preservative.
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All incubations were carried out at room temperature with agitation. The
formulations were prepared simultaneously for the 2 injections with a 12-day
maturation of the finalized formulations before the first injection.
1.2. Composition of formulation constituents:
COMPONENT CONCENTRATION BUFFER
(~G/ML)
FG 484 P04/NaCI 10/ 150mM pH
6.8
MPL 1019 H O
CPG 5000 H20
A1P04 1000 NaCI 150mM pH6.1
Al(OH)3 10380 Hz0
2. Immunisation protocol:
9 groups of 10 mice were immunized by different routes (50 ~cl) at days 0 and
28
with various formulations (see Table 1). Groups 8 was immunized with live RSV
by
the intra-nasal route (60,1) . Sera were obtained at days 28 (28 d Post I) and
42 ( 14
d Post II). On day 42, spleen cells were taken from 5 mice of all groups for
CMI
analysis.
3. Humoral response:
3.1. Anti-FG antibodies:
All humoral results were performed for 10 mice/ group for the anti-FG titers
(individual responses except for groups 6, 7 and 9 and cellular results were
presented for 5 mice/group).
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Individual sera were obtained 28 days after the first immunization and 14 days
after
the second immunisation and were tested for the presence of FG specific Ig
antibodies and their isotype (IgG2a, IgGl) distribution.
The assay protocol was as follows: coating overnight at 4°C with 50 ~,1
of purified
FG 54/023 (l~,g/ml) per well, saturation lh at 37°C, incubation with
sera 1h30 at
37°C, incubation with anti-mouse Ig biotin 1/1500 (or IgGI, IgG2a
biotin 1/1000)
1h30 at 37°C, incubation with strepta-peroxydase 1/2500 30 min at
37°C,
incubation with OPDA Sigma 15 min at RT, stop with HZS04 2N.
OD were monitored at 490 nm and the titers determined by Softmaxpro (4
parameters
equation) referring to a standard curve and expressed in EU/ml.
Individual sera obtained 14d Post II were tested for the presence of
neutralising
antibodies using the following protocol: 50 ~1 of serial two-fold dilutions of
sera
(first dilution 1/250) were incubated for 1 hour at 37°C with 50 ~,l of
a mixture
containing 500 pfu of RSV-A/Long (Lot 14) and guinea pig complement (1/25
dilution) in a 96 well plate in duplicate. 100 ~,1 of a HEp-2 cell suspension
at 105
cells/ml were then added to each well and the plates were incubated for 4 days
at
37 ° C in the presence of 5 % C02.
The supernatants were then aspirated, and after addition of a 100 ~,1 of a WST-
1
preparation (dilution 1/12.5) the plates were further incubated for 24H at
37°C in
the presence of 5 % C02. The OD were monitored at 595 nm and the titers
determined by linear regression (y=a.logx + b): titer = serum dilution giving
50%
reduction of the maximal OD observed for the uninfected cells.
Controls in test included a pool of randomly chosen human sera (Human pool)
and
Sandoglobuline (lot 069, generic human IgG produced by Sandoz).
4. Cellular response
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Spleen cells were isolated 14d Post II from groups 1-8 and from naive mice
(Group
9) for use as a negative control for the FG-specific cellular response
analysis.
Samples were analyzed for both FG-specific lymphoproliferation and cytokine
(IFN-
y + IL-5) secretion.
Proliferation was evaluated after a 96h incubation of 4x105 cells/well of 96
well
plates with 200 ~l of media containing 10 to 0.03 ~,g/ml of FG (3-fold
dilutions).
Upon 3H-thymidine incorporation, the FG specific proliferation was measured
following our standard protocol.
Cytokine induction was evaluated after 96 h incubation of 2.5x106 cells per
well of
24 well with 1 ml of media containing 10~,g to O.Ol~cg of FG (10-fold
dilutions).
Supernatants were then harvested to determine the quantity of IFN-y and IL-5
induced by ELISA following our standard protocol.
RESULTS
1. Groups:
Nine groups received two immunisations of various formulations containing FG
formulated with alum 3D-MPL CpG or CpG-Alum. Group 9 constitutes the
negative control for the CMI studies. Groups 6 and 7 constitute controls for
the
immunogenicity that could be induced by immunization of the mice with the
adjuvants alone. The RSV live immunizations was a control for the immune
response induced upon natural RSV IN infection (Group 8).
2. Humoral response:
2.1. Ann-FG antibodies
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Analysis of the specific anti-FG Ig antibodies at 14 d Post II vaccination was
performed. The results show (table 2) that a clear synergistic effect is
observed by
combining CpG with Alum as FG formulated with CpG-Alum induces twice as
many anti-FG-specific Ig antibodies as FG formulated with each of the
adjuvants
alone. The results also show that alum CpG and alum 3D-MPL induce equivalent
levels of antibody.
The analysis of the isotype profiles shows that CpG-Alum induce a IgGl:Ig2a <
1
ratio while the three other formulations induce a > 1 ratio. CpG and CpG-Alum
induce similar IgGl titers which are two to three times (CpG < CpG-Alum < SB
ASli) lower than those induced by alum 3D-MPL and Alum. CpG-Alum induce
high IgG2a titers which are three to five times higher than those induced by
CpG
and alum 3D-MPL and thirty to fifty times higher than those induced by FG
Alum.
The addition of Alum to CpG thus not seem to affect the IgG 1 titers but
rather
increases the IgG2a titers by three fold as compared to FG CpG allowing the
IgGl/IgG2a ratio to go from > 1 to < 1.
3. Cellular response:
The induced FG-specific lymphoproliferation shows the induction of equivalent
stimulation indexes for CpG alum, 3D-MPL alum and alum except for FG CpG.
FG CpG similarly to the adjuvants alone groups and the naive mice control
group,
does not induce any detectable FG specific lymphoproliferation. As observed in
other experiments, RSV live IN does not induce a sufficiently high immune
response for the lymphoproliferation assay to be able to pick it up.
DISCUSSION AND CONCLUSIONS
The antibody analysis shows a clear linear synergistic effect is observed upon
formulating FG with CpG and Alum as compared to FG formulated with either of
the adjuvants.
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Based on the isotype analysis FG CpG-Alum, FG CpG and FG alum MPL all
induce a mixed Th profile as measured by the induction of both IgG2a and IgGl
antibodies. FG Alum however almost exclusively induces IgGl antibodies, marker
of a Th2 response. Based on the level of induced FG-specific IgG2a antibodies
the
results suggest FG CpG-Alum predominantly induce Thl antibody isotypes while
FG CpG and to a greater extend FG alum 3D-MPL predominantly induce Th2
antibody isotypes. The formulation of FG with both CpG and Alum leads to a
three-fold increase in IgG2a titer as compared to FG CpG alone.
Analysis of the induced FG specific cell mediated response suggests that FG
CpG
does not induce detectable lymphoproliferation. However, similarly to what is
observed for the FG-specific Ig antibody responses the formulation of FG with
both
CpG and Alum does allow the induction of a specific lymphoproliferation.
TABLE 1
Groups Antigen Adjuvant Route
'~
FG (2pg) CpG IM
FG (2pg) CpG Alum IM
4 FG (2pg) Alum MPL IM
FG (2~tg) Alum IM
6 ~ Control adjuvantIM
CpG (100 pg) IM
8 RSV Live (Lot 14: lOSPFL~
none none
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TABLE 2
VaccineGroup 14 days
Post
II analysis
of the
immunised
Balb/c
mice
serum
CandidatNo. (G~j
I
a
Anti-FG RSV-A ELIS IgGl lgG1 Ig lgG2b
/ / G2a
ELISA Neuua NeutraIgG2a
lg
ratio Ratio
FG CpG 2 66777 1.97 60139 30604 5121
FG CpG-3 122236 0.71 78557 11048815567
alum
FG SB 4 1608022 7.19 178569 24846 17771
alum
3DMPL
FG Alum5 91568 66.55 161254 2423 617
RSV 8 4623 0.82 1396 1705 823
live
EXAMPLE 2 - Studies on RSV formulated with CpG containing
oligonucleotides, together with additional antigens (polysaccharide from
Streptococcus pneumoniae)
1. Formulation process:
Formulations were prepared 4 days before the first injection.
1.1 AI(OH)3-based formulations
CpG (50,10 or 2 ~cg) was preadsorbed on A1(OH)3 as concentrated monobulk by
mixing the immunostimulants with the Aluminium salt one day before the final
formulation. Non-adsorbed 11-valent SP, conjugated to protein D, was prepared
by
mixing the 11 conjugated components by numeric order and adjusting the
concentration to 2~cg/val/ml with NaCI 150mM. The final formulations were
prepared by mixing H20 and Aluminium salt if needed and preadsorbed CpG. After
5 minutes of incubation non-adsorbed 11-valent SP (O.l~.g/val) and /or FG
(2tcg)
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(FG as for Example 1) were added and incubated for 30min. P04/NaCI buffer was
then added and incubated for Smin before addition of thiomersal (l~,g/ml)
which
was allowed to incubate for 30min.
1.2 Formulations without AI(OH)3
The non-adsorbed 11-valent polysaccharide (PS) was prepared by mixing the 11
valences by numeric order and adjusting the concentration at 2pg/val/ml with
NaCI
150mM. The final formulations were prepared by mixing H20 and PO4/NaCI bui~er
and Non-adsorbed 11-valent (0.1 ~g/val) and /or FG (2pg). After 5 minutes of
incubation CpG was added and incubated for 30min. Thiomersal (1 ~g/ml) was
then
added and allowed to incubate for 30min.
All incubations were carried out at room temperature with agitation. The
formulations
were prepared simultaneously for the 2 injections with a 7-day maturation of
the
finalized formulations before the first injection.
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2. Composition of formulation constituents:
TABLE 3
COMPONENT AG OR IMST AL+++ BUFFER
CONC CONC
(N.G/ML) (EcG/ML)
FG 484 P04/NaCI
10/150mM H 6.8
Clinical 11-valent
nonadsorbed
PS 1 144 NaCI 150mM H6.1
PS3 149 NaC1150mM H6.1
PS4 114 NaC1150mM H6.1
PSS 135 NaC1150mM H6.1
PS6b 200 NaC1150mM H6.1
PS7 168 NaC1150mM H6.1
PS9 175 NaC1150mM H6.1
PS 14 180 NaCI 150mM H6.1
PS 18 137 NaCI 150mM H6.1
PS 19 140 NaCI 150mM H6.1
PS23 158 NaC1150mM H6.1
QS21 2000 H O
' C G 1826 20000 H O
Al(OH) 10380 H O
2. Immunization protocol:
19 groups of 10 mice were immunized by the intramuscular route (100 pl) at
days 0
and 28 with various formulations (see Table ~. Sera were obtained at days 42
and 43
( 14/1 S d Post II). On day 43, spleen cells were taken from 5 mice of the
groups
immunized with FG containing formulations as well as from 5 naive Balb/c mice
(group 24, not immunized).
3. Humoral response.
3.1. Anti-FG antibodies:
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All humoral results were performed for 10 mice/group (individual response for
the
anti-FG Ig, IgGl and IgG2a and neutralization titers) and cellular results
were
presented for 5 mice/group on pool.
Individual sera were obtained 14/15 days after the second immunization and
were
tested for the presence of FG specific Ig antibodies and their isotype (IgG2a,
IgGl)
distribution.
Assays were performed as described in Example 1.
3.2. Anti-Pneumococcal Polysaccharide IgG:
Murine IgG to pneumococcal polysaccharide types 3, 6B, 7F, 14, 19F and 23F was
measured by ELISA in a method adapted from the CDC protocol. This protocol
includes the addition of soluble cell wall polysaccharide (CPS) to the sera to
inhibit the
measurement of CPS antibodies. CPS is a phosphoryl-choline containing teichoic
acid
common to all pneumococci. It is present under the capsule, and antibodies to
it are
only weakly protective. Since CPS is linked to the capsular polysaccharide,
there is
usually 0.5 to 1% CPS contaminating the purified capsular polysaccharide used
to coat
the ELISA plates. Thus, measurement of the CPS antibodies can confound the
interpretation ELISA results with respect to the capsular polysaccharide.
The ELISA was performed with polysaccharides coated at 20, 5, 5, 20 and 20
~g/ml in
PBS buffer for types 6B, 7F, 14, 19F and 23F respectively. Sera was pre-mixed
with
the equivalent of 500 pg/ml CPS in undiluted sera, and incubated for 30
minutes
before addition to the ELISA plate. Murine IgG was detected with Jackson
ImmunoLab goat anti-murine IgG (H+L) peroxidase at 1:5000 dilution. The
titration
curves were referenced to polysaccharide specific murine monoclonal antibodies
of
known concentration for each serotype using logistic log comparison by SoftMax
Pro.
The monoclonal antibodies used were HASP4, PS7/19, PS14/4, PS19/5 and PS23/22
for types 6B, 7F, 14, 19F and 23F respectively.
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For serotype 3, a similar ELISA was done except that immunoplates were first
coated
with methylated human serum albumin (1 pg/ml in PBS, 2 hours, 37°C) in
order to
improve the PS3 coating (2.5 pg/ml in PBS, overnight, 4°C). Monoclonal
antibody
PS3/6 was used as standard reference.
4. Cellular response
Spleen cells were isolated 14d Post II from groups immunized with FG
containing
formulations and from nave mice for use as a negative control for the FG-
specific
cellular response analysis. Samples were analyzed for both FG-specific IFN-y
and IL-5
cytokines secretion.
Assays were performed as described in Example 1.
RESULTS
1. GROUPS.
19 groups (A to S) received two immunizations of various formulations
containing
either the 11-valent conjugated vaccine or FG or a combination of both
antigens
formulated with CpG or CpG alum with a dose range of CpG: 50, 10 or 2 pg.
Groups
A to R represent these combinations. Group S, FG Alum constitutes a control
for anti-
RSV analysis. Finally group T constitutes the negative control for the CMI
analysis.
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TABLE 4
Grou s Anti en Ad'uvant
A SPN Undeca (0.1 pg) CpG SOp.g
B SPN Undeca (0.1 pg) CpG lOpg
C SPN Undeca (0.1 pg) CpG 2pg
D FG (2 pg) CpG SOpg
E FG (2pg) CpG lOpg
F FG (2pg) CpG 2pg
G SPN Undeca(0.1 pg)+FG(2ug)CpG SOpg
H SPN Undeca(0.1 pg)+FG(2pg)CpG l0ug
I SPN Undeca(0.1 p.g)+FG(2pg)CpG 2pg
J SPN Undeca (0.1 pg) CpG SOpg /Alum SOpg
K SPN Undeca (0.1 pg) CpG lOpg /Alum SOpg
L SPN Undeca (0.1 pg) CpG 2pg /Alum SOpg
M FG (2 ~tg) CpG SOpg /Alum SOpg
N FG (2pg) CpG l0ug /Alum SOUg
O FG (2pg) CpG 2pg /Alum SOpg
P SPN Undeca(0.1 pg)+FG(2pg)CpG SOpg /Alum SOpg
Q SPN Undeca(0.1 pg)+FG(Zp.g)CpG lOpg /Alum SOpg
R SPN Undeca(0.1 pg)+FG(2pg)CpG 2pg /Alum SOpg
S FG (2~tg) Alum
T none none
S 2. HUMORAL RESPONSE.
2.1. Ann-FG antibodies
None of the FG-specific responses were affected by the mixture with the 11-
valent
pneumococcal polysaccharide conjugate. For practical purposes the FG-specific
analysis below are thus only described for the FG formulations.
Analysis of the specific anti-FG Ig antibodies at 14d/15 Post II vaccination
was
performed. The results show the induction of FG-specific Ig Ab with all
formulations
(Figure 1). For the FG CpG formulations the FG-specific Ig titers decrease
with
decreasing doses of CpG (groups D-F). The FG-CpG Alum based formulations
induce
higher FG-specific Ig Ab responses than those observed for the FG CpG or FG
Alum
group. A synergistic effect can be observed upon formulating FG with CpG and
Alum
as compared to the FG formulated with either alone. The Ab levels induced by
FG-
CpG Alum formulations are not sensitive to the CpG doses tested.
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Analysis of the induced anti-RSV/A neutralizing antibodies leads to the same
conclusions as for the anti-FG Ig antibody response (Figure 2).
Analysis of individual anti-FG IgGI and IgG2a isotype titers showed similar
antibody
response profiles to the ones observed for the anti-FG Ig and neutralizing
antibody
titres for the FG CpG formulations (Figure 3). For the FG CpG Alum based
formulations the Ab profiles were also similar except that a slight decrease
in IgG2a
antibodies could be observed with decreasing doses of CpG. Here too a
synergistic
effect is observed upon formulating FG with CpG and Alum as compared to the FG
formulated with either CpG or alum alone. Further analysis showed that the
addition of
the 11-valent conjugated SP vaccine to FG CpG Alum or FG CpG does not affect
the
IgGl and IgG2a responses.
Analysis of the ratio of IgGl/IgG2a isotype responses indicates that the
greater the
CpG dose the greater the amount of both isotypes and in particular the IgG2a
antibodies in any of the tested formulations. In comparison to the CpG
formulations,
FG Alum induces much lower levels of IgG2a antibodies and much higher levels
of
IgGI antibodies.
2.2. Ann-Pneumococcal Polysaccharide IgG
For both adjuvant formulations, anti-PS IgG titers were at least similar in
animals given
the 11-valent PS conjugate together with FG, compared to the animals immunized
with
the 11-valent PS conjugate alone (Figure 4). This result demonstrated that
combining
pneumococcal PS conjugates and RSV FG antigen within a CpG or an Alum CpG
formulation did not reduce or alter the anti-pneumococcal immune response.
Antibody
titers to serotypes 7F and 14 were even significantly improved in the presence
of FG
using the CpG adjuvant formulation (with 2 gg and 50 ~g CpG doses).
Higher antibody responses were reached to serotypes 7F, 14 and 19F (and to a
lesser
extent to serotypes 3 and 6B), when administering the Alum CpG formulation
rather
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than CpG given alone at the same dose. Using the former formulation, 2 pg CpG
was
as good an adjuvant as 50 pg CpG, whereas a clear dose-response was observed
in
animals given the non-adsorbed CpG formulations.
3. CELLULAR RESPONSE.
None of the FG-specific responses were affected by the mixture with the 11-
valent
pneumococcal polysaccharide conjugate. For practical purposes the FG-specific
analysis below are thus only described for the FG formulations. Due to
experimental
problems no analysis was obtained from group G.
The analysis of the production of IL-5 and IFN-y (Figure 5) shows that FG CpG
formulations induce IFN-y and a weak amount of IL-5. FG CpG alum formulations
however do induce much higher amounts of IFN-y independently of the CpG dose
while IL-5 production seems to increase with decreasing CpG doses. This is
reflected
in the INF-y/11,-5 ratio by a dominant IFN-y production, marker of a Thl
response, for
both formulations. However, the FG CpG alum formulations induce much more of
the
Th 1 marker than FG CpG formulations alone.
DISCUSSION AND CONCLUSIONS
The analysis of the induced anti-FG Ig specific and anti-RSV/A neutralizing
antibody
titers shows that the combination of FG CpG alum and FG CpG with the S.
Pneumoniae 11-valent conjugated vaccine does not hamper the induction of the
humoral and cellular responses.
Thus the combination of FG CpG alum or FG CpG with Strep 11-valent conjugated
vaccine does not afFect the response observed with FG CpG alum or FG CpG and
therefore the characteristics of these formulations eg induction of high
secondary
neutralizing antibody responses and a Thl response as measured by the presence
of
IgG2a antibodies and the induction IFN-y are maintained.
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For practical purposes the conclusions are thus described below in relation to
FG
formulations.
The analysis of the induced anti-FG Ig specific and anti-RSV/A neutralizing
antibody
titers shows a dose dependent induction of these antibodies for FG CpG
formulations
which is not observed for the FG CpG alum formulations. In addition, a clear
synergistic effect is observed upon formulating FG with CpG alum as compared
to FG
formulated with either CPG or alum alone.
Based on the isotype analysis FG CpG-Alum, FG CpG and FG alum all induce a
mixed
Th profile as measured by the induction of both IgG2a and IgGl antibodies. FG
alum
however induces mostly IgGI antibodies, marker of a Th2 response while FG CpG
and
FG CpG-alum both induce more IgG2a, marker of a Thl response than IgGl. For
both
CpG formulations, the greater the CpG dose, the greater the predominance of
the Thl
marker.
Analysis of the induced specific cell mediated response suggests that,
similarly to what
was observed for the FG-specific Ig isotype responses, both FG CpG alum and FG
CpG formulations induce IFN-y, marker of a Thl response with FG CpG alum
inducing the highest amounts.
Results demonstrated that combining pneumococcal PS conjugates and RSV FG
antigen within a CpG or an Alum CpG formulation did not alter the anti-
pneumococcal
immune response. Antibody titers to serotypes 7F and 14 were even
significantly
improved in the presence of FG using the CpG adjuvant formulation. For several
serotypes, the Alum CpG formulations were clearly more potent than CpG given
alone
at the same dose. Using the former formulation, 2 ug CpG was as good an
adjuvant as
50 ~.g CpG, whereas a clear dose-response was observed in animals given the
non-
adsorbed CpG formulations.
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