U.S. patent application number 12/097303 was filed with the patent office on 2009-01-08 for pneumococcal polysaccharide conjugate vaccine.
Invention is credited to Ralph Leon Biemans, Nathalie Marie-Josephe Garcon, Philippe Vincent Hermand, Jan Poolman, Marcelle Paulette Van Mechelen.
Application Number | 20090010959 12/097303 |
Document ID | / |
Family ID | 39133667 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090010959 |
Kind Code |
A1 |
Biemans; Ralph Leon ; et
al. |
January 8, 2009 |
Pneumococcal Polysaccharide Conjugate Vaccine
Abstract
The present invention is in the field of pneumococcal capsular
saccharide conjugate vaccines. Specifically, a multivalent
Streptococcus pneumoniae immunogenic composition is provided with
various conjugated capsular saccharides from different S.
pneumoniae serotypes conjugated to 2 or more different carrier
proteins, where the composition comprises serotype 19F capsular
saccharide conjugated to diphtheria toxoid (DT) or CRM197,
optionally wherein 19F is the only saccharide in the composition
conjugated to diphtheria toxoid (DT) or CRM197.
Inventors: |
Biemans; Ralph Leon;
(Rixensart, BE) ; Garcon; Nathalie Marie-Josephe;
(Rixensart, BE) ; Hermand; Philippe Vincent;
(Rixensart, BE) ; Poolman; Jan; (Rixensart,
BE) ; Van Mechelen; Marcelle Paulette; (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: |
39133667 |
Appl. No.: |
12/097303 |
Filed: |
December 20, 2006 |
PCT Filed: |
December 20, 2006 |
PCT NO: |
PCT/EP06/69974 |
371 Date: |
June 13, 2008 |
Current U.S.
Class: |
424/197.11 |
Current CPC
Class: |
A61K 39/092 20130101;
A61K 39/385 20130101; A61P 25/00 20180101; A61P 27/14 20180101;
A61K 2039/545 20130101; A61K 47/61 20170801; A61K 2039/627
20130101; A61K 2039/6037 20130101; Y02A 50/30 20180101; A61P 27/16
20180101; A61K 2039/55566 20130101; A61P 11/00 20180101; A61P 43/00
20180101; A61K 2039/555 20130101; A61P 37/04 20180101; A61K 2039/70
20130101; A61K 47/6415 20170801; A61K 2039/55 20130101; A61K 47/646
20170801; A61K 2039/575 20130101; A61K 2039/6087 20130101; A61P
27/02 20180101; A61K 2039/6068 20130101; A61K 2039/6031 20130101;
A61P 37/00 20180101; A61P 31/00 20180101; A61P 31/04 20180101; A61P
29/00 20180101; A61P 7/00 20180101; A61K 2039/62 20130101 |
Class at
Publication: |
424/197.11 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 43/00 20060101 A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2005 |
GB |
0526232.4 |
Apr 7, 2006 |
GB |
0607087.4 |
Apr 7, 2006 |
GB |
0607088.2 |
May 18, 2006 |
GB |
0609902.2. |
Oct 12, 2006 |
GB |
0620336.8 |
Oct 12, 2006 |
GB |
0620337.6 |
Oct 19, 2006 |
GB |
062816.9 |
Oct 19, 2006 |
GB |
0620815.1 |
Claims
1. A Streptococcus pneumoniae immunogenic composition comprising 9
or more capsular saccharides from different S. pneumoniae serotypes
conjugated to 2 or more different carrier proteins, wherein the
composition comprises serotype 19F capsular saccharide conjugated
to diphtheria toxoid (DT) or CRM197.
2. An immunogenic composition according to claim 1 wherein serotype
19F is conjugated to Diphtheria toxoid.
3. (canceled)
4. An immunogenic composition according to claim 1 wherein 19F
capsular saccharide is directly conjugated to the carrier
protein.
5. An immunogenic composition according to claim 1 wherein 19F
capsular saccharide is conjugated to the carrier protein via a
linker.
6-10. (canceled)
11. The immunogenic composition of claim 1 wherein the 19F
saccharide is conjugated to the carrier protein or to the linker
using CDAP chemistry.
12. The immunogenic composition of claim 1 wherein the ratio of
carrier protein to 19F saccharide is between 5:1 and 1:5 (w/w).
13. The immunogenic composition of claim 1 wherein the average size
of the 19F saccharide is above 100 kDa.
14-16. (canceled)
17. The immunogenic composition of claim 1 wherein the dose of the
19F saccharide conjugate is between 1 and 10 .mu.g of
saccharide.
18-19. (canceled)
20. An immunogenic composition according to claim 1 comprising 2
different carrier proteins.
21. (canceled)
22. An immunogenic composition according to claim 1 wherein one or
more or all carrier proteins is selected from the group consisting
of DT, CRM 197, TT, Fragment C, dPly, PhtA, PhyB, PhtD, PhtE,
PhtDE, OmpC, PorB and Haemophilus influenzae Protein D.
23-26. (canceled)
27. An immunogenic composition according to claim 1 wherein the
composition comprises capsular saccharide 18C conjugated to TT.
28-116. (canceled)
117. A method of immunising a human host against disease caused by
Streptococcus pneumoniae infection comprising administering to the
host an immunoprotective dose of the immunogenic composition of
claim 1.
118-130. (canceled)
131. A method of immunising a human host against disease caused by
Streptococcus pneumoniae serotype 19A infection comprising the
steps of administering to the host an immunoprotective dose of the
immunogenic composition of claim 1 which comprises a capsular
saccharide conjugate of serotype 19F but does not comprise capsular
saccharide from serotype 19A.
132-150. (canceled)
151. The immunogenic composition of claim 1 comprising serotype 19F
capsular saccharide but not comprising capsular sacccharide from
serotype 19A.
152. A Streptococcus pneumoniae immunogenic composition comprising
9 or more capsular saccharides from different S. pneumoniae
serotypes conjugated to 2 or more different carrier proteins,
wherein the composition comprises serotype 19F capsular saccharide
conjugated to diphtheria toxoid (DT) or CRM197, wherein 19F is the
only saccharide in the composition conjugated to diphtheria toxoid
(DT) or CRM197.
153. An immunogenic composition according to claim 20 wherein one
or more or all carrier proteins is selected from the group
consisting of DT, CRM 197, TT, Fragment C, dPly, PhtA, PhyB, PhtD,
PhtE, PhtDE, OmpC, PorB and Haemophilus influenzae Protein D.
154. An immunogenic composition according to claim 1 wherein the
composition comprises capsular saccharide 18C conjugated to TT,
wherein 18C is the only saccharide in the composition conjugated to
tetanus toxoid (TT).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an improved Streptococcus
pneumoniae vaccine.
BACKGROUND OF THE INVENTION
[0002] Children less than 2 years of age do not mount an immune
response to most polysaccharide vaccines, so it has been necessary
to render the polysaccharides immunogenic by chemical conjugation
to a protein carrier. Coupling the polysaccharide, a T-independent
antigen, to a protein, a T-dependent antigen, confers upon the
polysaccharide the properties of T dependency including isotype
switching, affinity maturation, and memory induction.
[0003] However, there can be issues with repeat administration of
polysaccharide-protein conjugates, or the combination of
polysaccharide-protein conjugates to form multivalent vaccines. For
example, it has been reported that a Haemophilus influenzae type b
polysaccharide (PRP) vaccine using tetanus toxoid (TT) as the
protein carrier was tested in a dosage-range with simultaneous
immunization with (free) TT and a pneumococcal polysaccharide-TT
conjugate vaccine following a standard infant schedule. As the
dosage of the pneumococcal vaccine was increased, the immune
response to the PRP polysaccharide portion of the Hib conjugate
vaccine was decreased, indicating immune interference of the
polysaccharide, possibly via the use of the same carrier protein
(Dagan et al., Infect Immun. (1998); 66: 2093-2098)
[0004] The effect of the carrier-protein dosage on the humoral
response to the protein itself has also proven to be multifaceted.
In human infants it was reported that increasing the dosage of a
tetravalent tetanus toxoid conjugate resulted in a decreased
response to the tetanus carrier (Dagan et al supra). Classical
analysis of these effects of combination vaccines have been
described as carrier induced epitopic suppression, which is not
fully understood, but believed to result from an excess amount of
carrier protein (Fattom, Vaccine 17: 126 (1999)). This appears to
result in competition for Th-cells, by the B-cells to the carrier
protein, and B-cells to the polysaccharide. If the B-cells to the
carrier protein predominate, there are not enough Th-cells
available to provide the necessary help for the B-cells specific to
the polysaccharide. However, the observed immunological effects
have been inconsistent, with the total amount of carrier protein in
some instances increasing the immune response, and in other cases
diminishing the immune response.
[0005] Hence there remain technical difficulties in combining
multiple polysaccharide conjugates into a single, efficacious,
vaccine formulation.
[0006] Streptococcus pneumoniae is a Gram-positive bacterium
responsible for considerable morbidity and mortality (particularly
in the young and aged), causing invasive diseases such as
pneumonia, bacteraemia and meningitis, and diseases associated with
colonisation, such as acute Otitis media. 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
bacteraemia, and other manifestations such as meningitis, with a
mortality rate close to 30% even with antibiotic treatment.
[0007] Pneumococcus is encapsulated with a chemically linked
polysaccharide which confers serotype specificity. There are 90
known serotypes of pneumococci, and the capsule is the principle
virulence determinant for pneumococci, as the capsule not only
protects the inner surface of the bacteria from complement, but is
itself poorly immunogenic. Polysaccharides are T-independent
antigens, and can not be processed or presented on MHC molecules to
interact with T-cells. They can however, stimulate the immune
system through an alternate mechanism which involves cross-linking
of surface receptors on B cells.
[0008] It was shown in several experiments that protection against
invasive pneumococci disease is correlated most strongly with
antibody specific for the capsule, and the protection is serotype
specific.
[0009] Streptococcus pneumoniae is the most common cause of
invasive bacterial disease and Otitis media in infants and young
children. Likewise, the elderly mount poor responses to
pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol.
42:265-270], hence the increased incidence of bacterial pneumonia
in this population [Verghese and Berk, (1983) Medicine (Baltimore)
62:271-285].
[0010] The major clinical syndromes caused by S. pneumoniae are
widely recognized and discussed in all standard medical textbooks
(Fedson D S, Muscher D M. In: Plotkin S A, Orenstein W A, editors.
Vaccines. 4rth edition. Philadelphia WB Saunders Co, 2004a:
529-588). For instance, Invasive pneumococcal disease (IPD) is
defined as any infection in which S. pneumoniae is isolated from
the blood or another normally sterile site (Musher D M.
Streptococcus pneumoniae. In Mandell G L, Bennett J E, Dolin R
(eds). Principles and Practice of Infectious diseases (5th ed). New
York, Churchill Livingstone, 2001, p2128-2147). Chronic obstructive
pulmonary disease (COPD) is recognised as encompassing several
conditions (airflow obstruction, chronic bronchitis, bronchiolitis
or small airways disease and emphysema) that often coexist.
Patients suffer exacerbations of their condition that are usually
associated with increased breathlessness, and often have increased
cough that may be productive of mucus or purulent sputum (Wilson,
Eur Respir J 2001 17:995-1007). COPD is defined physiologically by
the presence of irreversible or partially reversible airway
obstruction in patients with chronic bronchitis and/or emphysema
(Standards for the diagnosis and care of patients with chronic
obstructive pulmonary disease. American Thoracic Society. Am J
Respir Crit. Care Med. 1995 November; 152(5 Pt 2):S77-121).
Exacerbations of COPD are often caused by bacterial (e.g.
pneumococcal) infection (Sethi S, Murphy T F. Bacterial infection
in chronic obstructive pulmonary disease in 2000: a
state-of-the-art review. Clin Microbiol Rev. 2001 April;
14(2):336-63).
[0011] It is thus an object of the present invention to develop an
improved formulation of a multiple serotype Streptococcus
pneumoniae polysaccharide conjugate vaccine.
BRIEF DESCRIPTION OF FIGURES
[0012] FIG. 1 Bar chart showing 11 valent conjugate immunogenicity
in elderly Rhesus monkeys. The lighter bars represent the GMC after
two inoculations with 11 valent conjugate in aluminium phosphate
adjuvant. The darker bars represent the GMC after two inoculations
with 11 valent conjugate in adjuvant C.
[0013] FIG. 2 Bar chart showing memory B cells for PS3 after
inoculation with the 11 valent conjugate in adjuvant C or aluminium
phosphate adjuvant.
[0014] FIG. 3 Bar chart showing anti polysaccharide 19F
immunogenicity in Balb/C mice for the 4-valent plain
polysaccharides and the 4-valent dPly conjugates.
[0015] FIG. 4 Bar chart showing anti polysaccharide 22F
immunogenicity in Balb/C mice for the 4-valent plain
polysaccharides and the 4-valent PhtD conjugates.
[0016] FIG. 5 Bar chart showing anti-22F IgG response in Balb/c
mice FIG. 6 Bar chart showing anti-22F opsono-phagocytosis titres
in Balb/c mice.
[0017] FIG. 7 Bar chart comparing IgG responses induced in young
C57B1 mice after immunisation with 13 Valent conjugate vaccine
formulated in different adjuvants.
[0018] FIG. 8 Bar chart showing the protective efficacy of
different vaccine combinations in a monkey pneumonia model.
[0019] FIG. 9 Bar chart showing anti PhtD IgG response in Balb/c
mice after immunisation with 22F-PhtD or 22F-AH-PhtD
conjugates.
[0020] FIG. 10 Protection against type 4 pneumococcal challenge in
mice after immunisation with 22F-PhtD or 22F-AH-PhtD.
DESCRIPTION OF THE INVENTION
[0021] The present invention provides an improved Streptococcus
pneumoniae vaccine comprising 10 or more (e.g. 11, 12, 13, 14, or
15 or more) capsular saccharides from different S. pneumoniae
serotypes conjugated to 2 or more carrier proteins, wherein the
vaccine comprises serotype 19F capsular saccharide conjugated to
diphtheria toxoid or CRM197, and wherein the vaccine optionally
further comprises protein D from Haemophilus influenzae as free
protein or as a further carrier protein or both.
[0022] For the purposes of this invention, "immunizing a human host
against exacerbations of COPD" or "treatment or prevention of
exacerbations of COPD" or "reduction in severity of COPD
exacerbations" refers to a reduction in incidence or rate of COPD
exacerbations (for instance a reduction in rate of 0.1, 0.5, 1, 2,
5, 10, 20% or more) or a reduction in severity of COPD
exacerbations as defined above, for instance within a patient group
immunized with the compositions or vaccines of the invention.
[0023] Typically the Streptococcus pneumoniae vaccine of the
present invention will comprise capsular saccharide antigens
(preferably conjugated), wherein the saccharides are derived from
at least ten serotypes of S. pneumoniae. The number of S.
pneumoniae capsular saccharides can range from 10 different
serotypes (or "V", valences) to 23 different serotypes (23V). In
one embodiment there are 10, 11, 12, 13, 14 or 15 different
serotypes. In another embodiment of the invention, the vaccine may
comprise conjugated S. pneumoniae saccharides and unconjugated S.
pneumoniae saccharides. Preferably, the total number of saccharide
serotypes is less than or equal to 23. For example, the invention
may comprise 10 conjugated serotypes and 13 unconjugated
saccharides. In a similar manner, the vaccine may comprise 11, 12,
13, 14, 15 or 16 conjugated saccharides and 12, 11, 10, 9, 8 or 7,
respectively, unconjugated saccharides.
[0024] In one embodiment the multivalent pneumococcal vaccine of
the invention will be selected from the following 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, although it is appreciated that one or
two other serotypes could be substituted depending on the age of
the recipient receiving the vaccine and the geographical location
where the vaccine will be administered, e.g. serotype 6A may be
included on the list. For example, an 10-valent vaccine may
comprise polysaccharides from serotypes 1, 4, 5, 6B, 7F, 9V, 14,
18C, 19F and 23F. An 11-valent vaccine may also include saccharides
from serotype 3. A 12 or 13-valent paediatric (infant) vaccine may
also include the 10 or 11 valent formulation supplemented with
serotypes 6A and 19A, or 6A and 22F, or 19A and 22F, or 6A and 15B,
or 19A and 15B, or 22F and 15B, whereas a 13-valent elderly vaccine
may include the 11 valent formulation supplemented with serotypes
19A and 22F, 8 and 12F, or 8 and 15B, or 8 and 19A, or 8 and 22F,
or 12F and 15B, or 12F and 19A, or 12F and 22F, or 15B and 19A, or
15B and 22F. A 14 valent pediatric vaccine may include the 10
valent formulation described above supplemented with serotypes 3,
6A, 19A and 22F; serotypes 6A, 8, 19A and 22F; serotypes 6A, 12F,
19A and 22F; serotypes 6A, 15B, 19A and 22F; serotypes 3, 8, 19A
and 22F; serotypes 3, 12F, 19A and 22F; serotypes 3, 15B, 19A and
22F; serotypes 3, 6A, 8 and 22F; serotypes 3, 6A, 12F and 22F; or
serotypes 3, 6A, 15B and 22F.
[0025] The composition in one embodiment includes capsular
saccharides derived from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C,
19F and 23F (preferably conjugated). In a further embodiment of the
invention at least 11 saccharide antigens (preferably conjugated)
are included, for example capsular saccharides derived from
serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. In a
further embodiment of the invention, at least 12 or 13 saccharide
antigens are included, for example a vaccine may comprise capsular
saccharides derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,
18C, 19A, 19F and 23F or capsular saccharides derived from
serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F,
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.
[0026] The vaccine of the present invention may comprise protein D
(PD) from Haemophilus influenzae (see e.g. EP 0594610). Haemophilus
influenzae is a key causative organism of otitis media, and the
present inventors have shown that including this protein in a
Streptococcus pneumoniae vaccine will provide a level of protection
against Haemophilus influenzae related otitis media (reference POET
publication). In one embodiment, the vaccine composition comprises
protein D. In one aspect, PD is present as a carrier protein for
one or more of the saccharides. In another aspect, protein D could
be present in the vaccine composition as a free protein. In a
further aspect, protein D is present both as a carrier protein and
as free protein. Protein D may be used as a full length protein or
as a fragment (WO0056360). In a further aspect, protein D is
present as a carrier protein for the majority of the saccharides,
for example 6, 7, 8, 9 or more of the saccharides may be conjugated
to protein D. In this aspect, protein D may also be present as free
protein.
[0027] The vaccine of the present invention comprises two or more
different types of carrier protein. Each type of carrier protein
may act as carrier for more than one saccharide, which saccharides
may be the same or different. For example, serotypes 3 and 4 may be
conjugated to the same carrier protein, either to the same molecule
of carrier protein or to different molecules of the same carrier
protein. In one embodiment, two or more different saccharides may
be conjugated to the same carrier protein, either to the same
molecule of carrier protein or to different molecules of the same
carrier protein.
[0028] Each Streptococcus pneumoniae capsular saccharide may be
conjugated to a carrier protein independently selected from the
group consisting of TT, DT, CRM197, fragment C of TT, PhtD, PhtDE
fusions (particularly those described in WO 01/98334 and WO
03/54007), detoxified pneumolysin and protein D, other than
saccharide from serotype 19F which is always conjugated to DT or
CRM 197, preferably DT. A more complete list of protein carriers
that may be used in the conjugates of the invention is presented
below
[0029] If the protein carrier is the same for 2 or more saccharides
in the composition, the saccharides could be conjugated to the same
molecule of the protein carrier (carrier molecules having 2 more
different saccharides conjugated to it) [see for instance WO
04/083251]. Alternatively the saccharides may each be separately
conjugated to different molecules of the protein carrier (each
molecule of protein carrier only having one type of saccharide
conjugated to it).
[0030] The carrier protein conjugated to one or more of the S.
pneumoniae capsular saccharides in the conjugates present in the
immunogenic compositions of the invention is optionally a member of
the polyhistidine triad family (Pht) proteins, fragments or fusion
proteins thereof. The PhtA, PhtB, PhtD or PhtE proteins may have an
amino acid sequence sharing 80%, 85%, 90%, 95%, 98%, 99% or 100%
identity with a sequence disclosed in WO 00/37105 or WO 00/39299
(e.g. with amino acid sequence 1-838 or 21-838 of SEQ ID NO: 4 of
WO 00/37105 for PhtD). For example, fusion proteins are composed of
full length or fragments of 2, 3 or 4 of PhtA, PhtB, PhtD, PhtE.
Examples of fusion proteins are PhtA/B, PhtA/D, PhtA/E, PhtB/A,
PhtB/D, PhtB/E. PhtD/A. PhtD/B, PhtD/E, PhtE/A, PhtE/B and PhtE/D,
wherein the proteins are linked with the first mentioned at the
N-terminus (see for example WO01/98334).
[0031] Where fragments of Pht proteins are used (separately or as
part of a fusion protein), each fragment optionally contains one or
more histidine triad motif(s) and/or coiled coil regions of such
polypeptides. A histidine triad motif is the portion of polypeptide
that has the sequence HxxHxH where H is histidine and x is an amino
acid other than histidine. A coiled coil region is a region
predicted by "Coils" algorithm Lupus, A et al (1991) Science 252;
1162-1164. In an embodiment the or each fragment includes one or
more histidine triad motif as well as at least one coiled coil
region. In an embodiment, the or each fragment contains exactly or
at least 2, 3, 4 or 5 histidine triad motifs (optionally, with
native Pht sequence between the 2 or more triads, or intra-triad
sequence that is more than 50, 60, 70, 80, 90 or 100% identical to
a native pneumococcal intra-triad Pht sequence--e.g. the
intra-triad sequence shown in SEQ ID NO: 4 of WO 00/37105 for
PhtD). In an embodiment, the or each fragment contains exactly or
at least 2, 3 or 4 coiled coil regions. In an embodiment a Pht
protein disclosed herein includes the full length protein with the
signal sequence attached, the mature full length protein with the
signal peptide (for example 20 amino acids at N-terminus) removed,
naturally occurring variants of Pht protein and immunogenic
fragments of Pht protein (e.g. fragments as described above or
polypeptides comprising at least 15 or 20 contiguous amino acids
from an amino acid sequence in WO00/37105 or WO00/39299 wherein
said polypeptide is capable of eliciting an immune response
specific for said amino acid sequence in WO00/37105 or
WO00/39299).
[0032] In particular, the term "PhtD" as used herein includes the
full length protein with the signal sequence attached, the mature
full length protein with the signal peptide (for example 20 amino
acids at N-terminus) removed, naturally occurring variants of PhtD
and immunogenic fragments of PhtD (e.g. fragments as described
above or polypeptides comprising at least 15 or 20 contiguous amino
acids from a PhtD amino acid sequence in WO00/37105 or WO00/39299
wherein said polypeptide is capable of eliciting an immune response
specific for said PhtD amino acid sequence in WO00/37105 or
WO00/39299 (e.g. SEQ ID NO: 4 of WO 00/37105 for PhtD).
[0033] If the protein carrier is the same for 2 or more saccharides
in the composition, the saccharides could be conjugated to the same
molecule of the protein carrier (carrier molecules having 2 more
different saccharides conjugated to it) [see for instance WO
04/083251]. Alternatively the saccharides may each be separately
conjugated to different molecules of the protein carrier (each
molecule of protein carrier only having one type of saccharide
conjugated to it).
[0034] Examples of carrier proteins which may be used in the
present invention are DT (Diphtheria toxoid), TT (tetanus toxid) or
fragment C of TT, DT CRM197 (a DT mutant) other DT point mutants,
such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218;
3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and
other mutations described by Nicholls and Youle in Genetically
Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion
or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and
other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.
No. 4,950,740; mutation of at least one or more residues Lys 516,
Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in
U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment
disclosed in U.S. Pat. No. 5,843,711, pneumococcal pneumolysin (Kuo
et al (1995) Infect Immun 63; 2706-13) including ply detoxified in
some fashion for example dPLY-GMBS (WO 04081515, PCT/EP2005/010258)
or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and fusions
of Pht proteins for example PhtDE fusions, PhtBE fusions (WO
01/98334 and WO 03/54007), (Pht A-E are described in more detail
below) OMPC (meningococcal outer membrane protein--usually
extracted from N. meningitidis serogroup B--EP0372501), PorB (from
N. meningitidis), PD (Haemophilus influenzae protein D--see, e.g.,
EP 0 594 610 B), or immunologically functional equivalents thereof,
synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO
93/17712, WO 94/03208), pertussis proteins (WO 98/58668,
EP0471177), cytokines, lymphokines, growth factors or hormones (WO
91/01146), artificial proteins comprising multiple human CD4+ T
cell epitopes from various pathogen derived antigens (Falugi et al
(2001) Eur J Immunol 31; 3816-3824) such as N19 protein (Baraldoi
et al (2004) Infect Immun 72; 4884-7) pneumococcal surface protein
PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or
B of C. difflcile (WO 00/61761).
[0035] Nurkka et al Pediatric Infectious Disease Journal.
23(11):1008-14, 2004 Nov. described an 11 valent pneumococcal
vaccine with all serotypes conjugated to PD. However, the present
inventors have shown that opsonophagocytic activity was improved
for antibodies induced with conjugates having 19F conjugated to DT
compared with 19F conjugated to PD. In addition, the present
inventors have shown that a greater cross reactivity to 19A is seen
with 19F conjugated to DT. It is therefore a feature of the
composition of the present invention that serotype 19F is
conjugated to DT or CRM 197. In one aspect, serotype 19F is
conjugated to DT. The remaining saccharide serotypes of the
immunogenic composition may all be conjugated to one or more
carrier proteins that are not DT (i.e. only 19F is conjugated to
DT), or may be split between one or more carrier proteins that are
not DT and DT itself. In one embodiment, 19F is conjugated to DT or
CRM 197 and all of the remaining serotypes are conjugated to PD. In
a further embodiment, 19F is conjugated to DT or CRM 197, and the
remaining serotypes are split between PD, and TT or DT or CRM 197.
In a further embodiment, 19F is conjugated to DT or CRM 197 and no
more than one saccharide is conjugated to TT. In one aspect of this
embodiment, said one saccharide is 18C or 12F. In a further
embodiment, 19F is conjugated to DT or CRM 197 and no more than two
saccharides are conjugated to TT. In a further embodiment, 19F is
conjugated to DT or CRM 197, and the remaining serotypes are split
between PD, TT and DT or CRM 197. In a further embodiment, 19F is
conjugated to DT or CRM 197, and the remaining serotypes are split
between PD, TT and pneumolysin. In a further embodiment, 19F is
conjugated to DT or CRM 197, and the remaining serotypes are split
between PD, TT and CRM 197. In a further embodiment, 19F is
conjugated to DT or CRM197 and the remaining serotypes are split
between PD, TT, pneumolysin and optionally PhtD or PhtD/E fusion
protein. In a further embodiment, 19F is conjugated to DT or
CRM197, 19A is conjugated to pneumolysin or TT, one (two or three)
further saccharide(s) is conjugated to TT, one further saccharide
is conjugated to PhtD or PhtD/E and all further saccharides are
conjugated to PD. In a further embodiment 19F is conjugated to DT
or CRM197, 19A is conjugated to pneumolysin, one (two or three)
further saccharide(s) is conjugated to TT, one further saccharide
is conjugated to pneumolysin, 2 further saccharides are conjugated
to PhtD or PhtD/E and all further saccharides are conjugated to
PD.
[0036] In one embodiment, the immunogenic composition of the
invention comprises protein D from Haemophilus influenzae. Within
this embodiment, If PD is not one of the carrier proteins used to
conjugate any saccharides other than 19F, for example 19F is
conjugated to DT whilst the other serotypes are conjugated to one
or more different carrier proteins which are not PD, then PD will
be present in the vaccine composition as free protein. If PD is one
of the carrier proteins used to conjugate saccharides other than
19F, then PD may optionally be present in the vaccine composition
as free protein.
[0037] The term "saccharide" throughout this specification may
indicate polysaccharide or oligosaccharide and includes both.
Polysaccharides are isolated from bacteria and may be sized to some
degree by known methods (see for example EP497524 and EP497525) and
preferably by microfluidisation. Polysaccharides can be sized in
order to reduce viscosity in polysaccharide samples and/or to
improve filterability for conjugated products. Oligosaccharides
have a low number of repeat units (typically 5-30 repeat units) and
are typically hydrolysed polysaccharides
[0038] Capsular polysaccharides of Streptococcus pneumoniae
comprise repeating oligosaccharide units which may contain up to 8
sugar residues. For a review of the oligosaccharide units for the
key Streptococcus pneumoniae serotypes see JONES, Christopher.
Vaccines based on the cell surface carbohydrates of pathogenic
bacteria. An. Acad. Bras. Ci nc., June 2005, vol. 77, no. 2, p.
293-324. ISSN 0001-3765.
[0039] In one embodiment, a capsular saccharide antigen may be a
full length polysaccharide, however in others it may be one
oligosaccharide unit, or a shorter than native length saccharide
chain of repeating oligosaccharide units. In one embodiment, all of
the saccharides present in the vaccine are polysaccharides. Full
length polysaccharides may be "sized" i.e. their size may be
reduced by various methods such as acid hydrolysis treatment,
hydrogen peroxide treatment, sizing by Emulsiflex.RTM. followed by
a hydrogen peroxide treatment to generate oligosaccharide fragments
or microfluidization. The inventors have also noted that the focus
of the art has been to use oligosaccharides for ease of conjugate
production. The inventors have found that by using native or
slightly sized polysaccharide conjugates, one or more of the
following advantages may be realised: 1) a conjugate having high
immunogenicity which is filterable, 2) the ratio of polysaccharide
to protein in the conjugate can be altered such that the ratio of
polysaccharide to protein (w/w) in the conjugate may be increased
(which can have an effect on the carrier suppression effect), 3)
immunogenic conjugates prone to hydrolysis may be stabilised by the
use of larger saccharides for conjugation. The use of larger
polysaccharides can result in more cross-linking with the conjugate
carrier and may lessen the liberation of free saccharide from the
conjugate. The conjugate vaccines described in the prior art tend
to depolymerise the polysaccharides prior to conjugation in order
to improve conjugation. The present inventors have found that
saccharide conjugate vaccines retaining a larger size of saccharide
can provide a good immune response against pneumococcal
disease.
[0040] The immunogenic composition of the invention may thus
comprise one or more saccharide conjugates wherein the average size
(e.g. weight-average molecular weight; M.sub.w) of each saccharide
before conjugation is above 80 kDa, 100kDa, 200 kDa, 300 kDa, 400
kDa, 500 kDa or 1000 kDa. In one embodiment one or more saccharide
conjugates of the invention should have an average size of
saccharide pre-conjugation of 50-1600, 80-1400, 100-1000, 150-500,
or 200-400 kDa (note that where average size is M.sub.w, `kDa`
units should be replaced herein with `.times.10.sup.3`). In one
embodiment the conjugate post conjugation should be readily
filterable through a 0.2 micron filter such that a yield of more
than 50, 60, 70, 80, 90 or 95% is obtained post filtration compared
with the pre filtration sample.
[0041] For the purposes of the invention, "native polysaccharide"
refers to a saccharide that has not been subjected to a process
(e.g. post-purification), the purpose of which is to reduce the
size of the saccharide. A polysaccharide can become slightly
reduced in size during normal purification procedures. Such a
saccharide is still native. Only if the polysaccharide has been
subjected to sizing techniques would the polysaccharide not be
considered native.
[0042] For the purposes of the invention, "sized by a factor up to
.times.2" means that the saccharide is subject to a process
intended to reduce the size of the saccharide but to retain a size
more than half the size of the native polysaccharide. .times.3,
.times.4 etc. are to be interpreted in the same way i.e. the
saccharide is subject to a process intended to reduce the size of
the polysaccharide but to retain a size more than a third, a
quarter etc. the size of the native polysaccharide.
[0043] In an aspect of the invention, the immunogenic composition
comprises Streptococcus pneumoniae saccharides from at least 10
serotypes conjugated to a carrier protein, wherein at least 1, 2,
3, 4, 5, 6, 7, 8, 9 or each S. pneumoniae saccharide is native
polysaccharide.
[0044] In an aspect of the invention, the immunogenic composition
comprises Streptococcus pneumoniae saccharides from at least 10
serotypes conjugated to a carrier protein, wherein at least 1, 2,
3, 4, 5, 6, 7, 8, 9 or each S. pneumoniae saccharide is sized by a
factor up to .times.2, .times.3, .times.4, .times.5, .times.6,
.times.7, .times.8, .times.9 or .times.10. In one embodiment of
this aspect, the majority of the saccharides, for example 6, 7, 8
or more of the saccharides are sized by a factor up to .times.2,
.times.3, .times.4, .times.5, .times.6, .times.7, .times.8,
.times.9 or .times.10.
[0045] The molecular weight or average molecular weight (or size)
of a saccharide herein refers to the weight-average molecular
weight (M.sub.w) of the saccharide measured prior to conjugation
and is measured by MALLS.
[0046] The MALLS technique is well known in the art and is
typically carried out as described in example 2. For MALLS analysis
of pneumococcal saccharides, two columns (TSKG6000 and 5000PWxl)
may be used in combination and the saccharides are eluted in water.
Saccharides are detected using a light scattering detector (for
instance Wyatt Dawn DSP equipped with a 10 mW argon laser at 488
nm) and an inferometric refractometer (for instance Wyatt Otilab
DSP equipped with a P100 cell and a red filter at 498 nm).
[0047] In an embodiment the S. pneumoniae saccharides are native
polysaccharides or native polysaccharides which have been reduced
in size during a normal extraction process.
[0048] In an embodiment, the S. pneumoniae saccharides are sized by
mechanical cleavage, for instance by microfluidisation or
sonication. Microfluidisation and sonication have the advantage of
decreasing the size of the larger native polysaccharides
sufficiently to provide a filterable conjugate. Sizing is by a
factor of no more than .times.20, .times.10, .times.8, .times.6,
.times.5, .times.4, .times.3 or .times.2.
[0049] In an embodiment, the immunogenic composition comprises S.
pneumoniae conjugates that are made from a mixture of native
polysaccharides and saccharides that are sized by a factor of no
more than .times.20. In one aspect of this embodiment, the majority
of the saccharides, for example 6, 7, 8 or more of the saccharides
are sized by a factor of up to .times.2, .times.3, .times.4,
.times.5 or .times.6.
[0050] In an embodiment, the Streptococcus pneumoniae saccharide is
conjugated to the carrier protein via a linker, for instance a
bifunctional linker. The linker is optionally heterobifunctional or
homobifunctional, having for example a reactive amino group and a
reactive carboxylic acid group, 2 reactive amino groups or two
reactive carboxylic acid groups. The linker has for example between
4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible linker is
ADH. Other linkers include B-propionamido (WO 00/10599),
nitrophenyl-ethylamine (Gever et al (1979) Med. Microbiol. Immunol.
165; 171-288), haloalkyl halides (U.S. Pat. No. 4,057,685),
glycosidic linkages (U.S. Pat. No. 4,673,574, U.S. Pat. No.
4,808,700), hexane diamine and 6-aminocaproic acid (U.S. Pat. No.
4,459,286). In an embodiment, ADH is used as a linker for
conjugating saccharide from serotype 18C. In an embodiment, ADH is
used as a linker for conjugating saccharide from serotype 22F.
[0051] The saccharide conjugates present in the immunogenic
compositions of the invention may be prepared by any known coupling
technique. The conjugation method may rely on activation of the
saccharide with 1-cyano-4-dimethylamino pyridinium
tetrafluoroborate (CDAP) to form a cyanate ester. The activated
saccharide may thus be coupled directly or via a spacer (linker)
group to an amino group on the carrier protein. For example, the
spacer could be cystamine or cysteamine to give a thiolated
polysaccharide which could be coupled to the carrier via a
thioether linkage obtained after reaction with a
maleimide-activated carrier protein (for example using GMBS) or a
haloacetylated carrier protein (for example using iodoacetimide
[e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or
SIAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally
made by CDAP chemistry) is coupled with hexane diamine or ADH and
the amino-derivatised saccharide is conjugated to the carrier
protein using carbodiimide (e.g. EDAC or EDC) chemistry via a
carboxyl group on the protein carrier. Such conjugates are
described in PCT published application WO 93/15760 Uniformed
Services University and WO 95/08348 and WO 96/29094
[0052] Other suitable techniques use carbodiimides, hydrazides,
active esters, norborane, p-nitrobenzoic acid,
N-hydroxysuccinimide, S--NHS, EDC, TSTU. Many are described in WO
98/42721. Conjugation may involve a carbonyl linker which may be
formed by reaction of a free hydroxyl group of the saccharide with
CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al J.
Chromatogr. 1981. 218; 509-18) followed by reaction of with a
protein to form a carbamate linkage. This may involve reduction of
the anomeric terminus to a primary hydroxyl group, optional
protection/deprotection of the primary hydroxyl group' reaction of
the primary hydroxyl group with CDI to form a CDI carbamate
intermediate and coupling the CDI carbamate intermediate with an
amino group on a protein.
[0053] The conjugates can also be prepared by direct reductive
amination methods as described in U.S. Pat. No. 4,365,170
(Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods
are described in EP-0-161-188, EP-208375 and EP-0-477508.
[0054] A further method involves the coupling of a cyanogen bromide
(or CDAP) activated saccharide derivatised with adipic acid
dihydrazide (ADH) to the protein carrier by Carbodiimide
condensation (Chu C. et al Infect. Immunity, 1983 245 256), for
example using EDAC.
[0055] In an embodiment, a hydroxyl group (preferably an activated
hydroxyl group for example a hydroxyl group activated to make a
cyanate ester [e.g. with CDAP]) on a saccharide is linked to an
amino or carboxylic group on a protein either directly or
indirectly (through a linker). Where a linker is present, a
hydroxyl group on a saccharide is preferably linked to an amino
group on a linker, for example by using CDAP conjugation. A further
amino group in the linker for example ADH) may be conjugated to a
carboxylic acid group on a protein, for example by using
carbodiimide chemistry, for example by using EDAC. In an
embodiment, the pneumococcal capsular saccharide(s) is conjugated
to the linker first before the linker is conjugated to the carrier
protein. Alternatively the linker may be conjugated to the carrier
before conjugation to the saccharide.
[0056] A combination of techniques may also be used, with some
saccharide-protein conjugates being prepared by CDAP, and some by
reductive amination.
[0057] In general the following types of chemical groups on a
protein carrier can be used for coupling/conjugation:
A) Carboxyl (for instance via aspartic acid or glutamic acid). In
one embodiment this group is linked to amino groups on saccharides
directly or to an amino group on a linker with carbodiimide
chemistry e.g. with EDAC. B) Amino group (for instance via lysine).
In one embodiment this group is linked to carboxyl groups on
saccharides directly or to a carboxyl group on a linker with
carbodiimide chemistry e.g. with EDAC. In another embodiment this
group is linked to hydroxyl groups activated with CDAP or CNBr on
saccharides directly or to such groups on a linker; to saccharides
or linkers having an aldehyde group; to saccharides or linkers
having a succinimide ester group. C) Sulphydryl (for instance via
cysteine). In one embodiment this group is linked to a bromo or
chloro acetylated saccharide or linker with maleimide chemistry. In
one embodiment this group is activated/modified with bis
diazobenzidine. D) Hydroxyl group (for instance via tyrosine). In
one embodiment this group is activated/modified with bis
diazobenzidine. E) Imidazolyl group (for instance via histidine).
In one embodiment this group is activated/modified with bis
diazobenzidine. F) Guanidyl group (for instance via arginine). G)
Indolyl group (for instance via tryptophan).
[0058] On a saccharide, in general the following groups can be used
for a coupling: OH, COOH or NH2. Aldehyde groups can be generated
after different treatments known in the art such as: periodate,
acid hydrolysis, hydrogen peroxide, etc.
Direct Coupling Approaches:
[0059] Saccharide-OH+CNBr or CDAP---------->cyanate
ester+NH2-Prot-------->conjugate
Saccharide-aldehyde+NH2-Prot-------->Schiff
base+NaCNBH3-------->conjugate
Saccharide-COOH+NH2-Prot+EDAC-------->conjugate
Saccharide-NH2+COOH-Prot+EDAC-------->conjugate
Indirect Coupling Via Spacer (Linker) Approaches:
[0060] Saccharide-OH+CNBr or CDAP------>cyanate
ester+NH2--------NH2-------->saccharide--------NH2+COOH-Prot+EDAC------
----->conjugate
Saccharide-OH+CNBr or CDAP-------->cyanate
ester+NH2----------SH---------->saccharide--------SH+SH-Prot
(native Protein with an exposed cysteine or obtained after
modification of amino groups of the protein by SPDP for
instance)---------->saccharide-S--S-Prot
Saccharide-OH+CNBr or CDAP------>cyanate
ester+NH2--------SH-------------->saccharide--------SH+maleimide-Prot
(modification of amino groups)-------->conjugate
Saccharide-OH+CNBr or CDAP------>cyanate
ester+NH2----------SH------>Saccharide-SH+haloacetylated-Prot--------&-
gt;Conjugate
Saccharide-COOH+EDAC+NH2----------NH2------>saccharide------------NH2-
+EDAC+COOH-Prot-------->conjugate
Saccharide-COOH+EDAC+NH2--------SH---------->saccharide--------SH+SH--
Prot (native Protein with an exposed cysteine or obtained after
modification of amino groups of the protein by SPDP for
instance)---------->saccharide-S--S-Prot
Saccharide-COOH+EDAC+NH2--------SH---------->saccharide------------SH-
+maleimide-Prot (modification of amino groups)-------->conjugate
Saccharide-COOH+EDAC+NH2--------SH------>Saccharide-SH+haloacetylated--
Prot-------->Conjugate
Saccharide-Aldehyde+NH2----------NH2-------->saccharide------NH2+EDAC-
+COOH-Prot-------->conjugate
[0061] Note: instead of EDAC above, any suitable carbodiimide may
be used.
[0062] In summary, the types of protein carrier chemical group that
may be generally used for coupling with a saccharide are amino
groups (for instance on lysine residues), COOH groups (for instance
on aspartic and glutamic acid residues) and SH groups (if
accessible) (for instance on cysteine residues.
[0063] Preferably the ratio of carrier protein to S. pneumoniae
saccharide is between 1:5 and 5:1; e.g. between 1:0.5-4:1,
1:1-3.5:1, 1.2:1-3:1, 1.5:1-2.5:1; e.g. between 1:2 and 2.5:1; 1:1
and 2:1 (w/w). In an embodiment, the majority of the conjugates,
for example 6, 7, 8, 9 or more of the conjugates have a ratio of
carrier protein to saccharide that is greater than 1:1, for example
1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1 or 1.6:1.
[0064] In an embodiment, at least one S. pneumoniae saccharide is
conjugated to a carrier protein via a linker using CDAP and EDAC.
For example, 18C or 22F may be conjugated to a protein via a linker
(for example those with two hydrazino groups at its ends such as
ADH) using CDAP and EDAC as described above. When a linker is used,
CDAP may be used to conjugate the saccharide to a linker and EDAC
may then be used to conjugate the linker to a protein or,
alternatively EDAC may be used first to conjugate the linker to the
protein, after which CDAP may be used to conjugate the linker to
the saccharide.
[0065] In general, the immunogenic composition of the invention may
comprise a dose of each saccharide conjugate between 0.1 and 20
.mu.g, 1 and 10 .mu.g or 1 and 3 .mu.g of saccharide.
[0066] In an embodiment, the immunogenic composition of the
invention contains each S. pneumoniae capsular saccharide at a dose
of between 0.1-20 .mu.g; 0.5-10 .mu.g; 0.5-5 .mu.g or 1-3 .mu.g of
saccharide. In an embodiment, capsular saccharides may be present
at different dosages, for example some capsular saccharides may be
present at a dose of exactly 1 .mu.g or some capsular saccharides
may be present at a dose of exactly 3 .mu.g. In an embodiment,
saccharides from serotypes 3, 18C and 19F (or 4, 18C and 19F) are
present at a higher dose than other saccharides. In one aspect of
this embodiment, serotypes 3, 18C and 19F (or 4, 18C and 19F) are
present at a dose of around or exactly 3 .mu.g whilst other
saccharides in the immunogenic composition are present at a dose of
around or exactly 1 .mu.g.
[0067] "Around" or "approximately" are defined as within 10% more
or less of the given figure for the purposes of the invention.
[0068] In an embodiment, at least one of the S. pneumoniae capsular
saccharides is directly conjugated to a carrier protein (e.g. using
one of the chemistries described above). Preferably the at least
one of the S. pneumoniae capsular saccharides is directly
conjugated by CDAP. In an embodiment, the majority of the capsular
saccharides for example 5, 6, 7, 8, 9 or more are directly linked
to the carrier protein by CDAP (see WO 95/08348 and WO
96/29094)
[0069] The immunogenic composition may comprise Streptococcus
pneumoniae proteins, herein termed Streptococcus pneumoniae
proteins of the invention. Such proteins may be used as carrier
proteins, or may be present as free proteins, or may be present
both as carrier proteins and as free proteins. The Streptococcus
pneumoniae proteins of the invention are either surface exposed, at
least during part of the life cycle of the pneumococcus, or are
proteins which are secreted or released by the pneumococcus.
Preferably the proteins of the invention are selected from the
following 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 (PhtX)), choline binding proteins
(CbpX), 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), and toxins (e.g., Ply). Preferred examples
within these categories (or motifs) are the following proteins, or
immunologically functional equivalents thereof.
[0070] In one embodiment, the immunogenic composition of the
invention comprises at least 1 protein selected from the group
consisting of the Poly Histidine Triad family (PhtX), Choline
Binding Protein family (CbpX), CbpX truncates, Lytx family, LytX
truncates, CbpX truncate-LytX truncate chimeric proteins (or
fusions), pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125
and Sp133. In a further embodiment, the immunogenic composition
comprises 2 or more proteins selected from the group consisting of
the Poly Histidine Triad family (PhtX), Choline Binding Protein
family (CbpX), CbpX truncates, Lytx family, LytX truncates, CbpX
truncate-LytX truncate chimeric proteins (or fusions), pneumolysin
(Ply), PspA, PsaA, and Sp128. In one more embodiment, the
immunogenic composition comprises 2 or more proteins selected from
the group consisting of the Poly Histidine Triad family (PhtX),
Choline Binding Protein family (CbpX), CbpX truncates, LytX family,
LytX truncates, CbpX truncate-LytX truncate chimeric proteins (or
fusions), pneumolysin (Ply), and Sp128.
[0071] The Pht (Poly Histidine Triad) family comprises proteins
PhtA, PhtB, PhtD, and PhtE. The family is characterized 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.
In one embodiment of the invention, the Pht protein of the
invention is 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.
[0072] With regards to the PhtX 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. 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. PhtE is disclosed in WO00/30299 and is referred to as
BVH-3. Where any Pht protein is referred to herein, it is meant
that immunogenic fragments or fusions thereof of the Pht protein
can be used. For example, a reference to PhtX includes immunogenic
fragments or fusions thereof from any Pht protein. A reference to
PhtD or PhtB is also a reference to PhtDE or PhtBE fusions as
found, for example, in WO0198334.
[0073] Pneumolysin is a multifunctional toxin with a distinct
cytolytic (hemolytic) and complement activation activities (Rubins
et al., Am. Respi. Cit Care Med, 153:1339-1346 (1996)). The toxin
is not secreted by pneumococci, but it is released upon lysis of
pneumococci under the influence of autolysin. Its effects include
e.g., the stimulation of the production of inflammatory cytokines
by human monocytes, the inhibition of the beating of cilia on human
respiratory epithelial, and the decrease of bactericidal activity
and migration of neutrophils. The most obvious effect of
pneumolysin is in the lysis of red blood cells, which involves
binding to cholesterol. Because it is a toxin, it needs to be
detoxified (i.e., non-toxic to a human when provided at a dosage
suitable for protection) before it can be administered in vivo.
Expression and cloning of wild-type or native pneumolysin is known
in the art. See, for example, Walker et al. (Infect Immun,
55:1184-1189 (1987)), Mitchell et al. (Biochim Biophys Acta,
1007:67-72 (1989) and Mitchell et al (NAR, 18:4010 (1990)).
Detoxification of ply can be conducted by chemical means, e.g.,
subject to formalin or glutaraldehyde treatment or a combination of
both (WO 04081515, PCT/EP2005/010258). Such methods are well known
in the art for various toxins. Alternatively, ply can be
genetically detoxified. Thus, the invention encompasses derivatives
of pneumococcal proteins which may be, for example, mutated
proteins. The term "mutated" is used herein to mean a molecule
which has undergone deletion, addition or substitution of one or
more amino acids using well known techniques for site directed
mutagenesis or any other conventional method. For example, as
described above, a mutant ply protein may be altered so that it is
biologically inactive whilst still maintaining its immunogenic
epitopes, see, for example, WO90/06951, Berry et al. (Infect Immun,
67:981-985 (1999)) and WO99/03884.
[0074] As used herein, it is understood that the term "Ply" refers
to mutated or detoxified pneumolysin suitable for medical use
(i.e., non toxic).
[0075] Concerning the Choline Binding Protein family (CbpX),
members of that family were originally identified as pneumococcal
proteins that could be purified by choline-affininty
chromatography. All of the choline-binding proteins are
non-covalently bound to phosphorylcholine moieties of cell wall
teichoic acid and membrane-associated lipoteichoic acid.
Structurally, they have several regions in common over the entire
family, although the exact nature of the proteins (amino acid
sequence, length, etc.) can vary. In general, choline binding
proteins comprise an N terminal region (N), conserved repeat
regions (R1 and/or R2), a proline rich region (P) and a conserved
choline binding region (C), made up of multiple repeats, that
comprises approximately one half of the protein. As used in this
application, the term "Choline Binding Protein family (CbpX)" is
selected from the group consisting of Choline Binding Proteins as
identified in WO97/41151, PbcA, SpsA, PspC, CbpA, CbpD, and CbpG.
CbpA is disclosed in WO97/41151. CbpD and CbpG are disclosed in
WO00/29434. PspC is disclosed in WO97/09994. PbcA is disclosed in
WO98/21337.5 psA is a Choline binding protein disclosed in WO
98/39450. Preferably the Choline Binding Proteins are selected from
the group consisting of CbpA, PbcA, SpsA and PspC.
[0076] Another preferred embodiment is CbpX truncates wherein
"CbpX" is defined above and "truncates" refers to CbpX 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 and R1xR2 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.
[0077] The LytX family is membrane associated proteins associated
with cell lysis. The N-terminal domain comprises choline binding
domain(s), however the LytX family does not have all the features
found in the CbpA family noted above and thus for the present
invention, the LytX family is considered distinct from the CbpX
family. In contrast with the CbpX family, the C-terminal domain
contains the catalytic domain of the LytX protein family. The
family comprises LytA, B and C. With regards to the LytX 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.
[0078] Another preferred embodiment are LytX truncates wherein
"LytX" is defined above and "truncates" refers to LytX proteins
lacking 50% or more of the Choline binding region. Preferably such
proteins lack the entire choline binding region. Yet another
preferred embodiment of this invention are CbpX truncate-LytX
truncate chimeric proteins (or fusions). Preferably this comprises
NR1xR2 (or R1xR2) of CbpX and the C-terminal portion (Cterm, i.e.,
lacking the choline binding domains) of LytX (e.g., LytCCterm or
Sp91Cterm). More preferably CbpX is selected from the group
consisting of CbpA, PbcA, SpsA and PspC. More preferably still, it
is CbpA. Preferably, LytX is LytC (also referred to as Sp91).
Another embodiment of the present invention is a PspA or PsaA
truncates lacking the choline binding domain (C) and expressed as a
fusion protein with LytX. Preferably, LytX is LytC.
[0079] With regards to PsaA and PspA, both are know in the art. For
example, PsaA and transmembrane deletion variants thereof have been
described by Berry & Paton, Infect Immun 1996 December;
64(12):5255-62. PspA and transmembrane deletion variants thereof
have been disclosed in, for example, U.S. Pat. No. 5,804,193, WO
92/14488, and WO 99/53940.
[0080] Sp128 and Sp130 are disclosed in WO00/76540. 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 characterized by a
Type I signal sequence. Sp133 is disclosed in WO 98/06734 (where it
has the reference # y85992). It is also characterized by a Type I
signal sequence.
[0081] 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 or fragments thereof (WO 00/8910); 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); OmpIA1
(PCT/EP99/06781); Hly3 (PCT/EP99/03257); and OmpE. Examples of
non-typeable Haemophilus influenzae antigens or fragments thereof
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); P2; and P5 (WO 94/26304).
[0082] The proteins of the invention may also be beneficially
combined. By combined is meant that the immunogenic composition
comprises all of the proteins from within the following
combinations, either as carrier proteins or as free proteins or a
mixture of the two. For example, in a combination of two proteins
as set out hereinafter, both proteins may be used as carrier
proteins, or both proteins may be present as free proteins, or both
may be present as carrier and as free protein, or one may be
present as a carrier protein and a free protein whilst the other is
present only as a carrier protein or only as a free protein, or one
may be present as a carrier protein and the other as a free
protein. Where a combination of three proteins is given, similar
possibilities exist. Preferred 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, NRIxR2+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. Other
combinations include 3 protein combinations such as
PhtD+NR1xR2+Ply, and PhtA+NR1xR2+PhtD. In one embodiment, the
vaccine composition comprises detoxified pneumolysin and PhtD or
PhtDE as carrier proteins. In a further embodiment, the vaccine
composition comprises detoxified pneumolysin and PhtD or PhtDE as
free proteins.
[0083] The present invention further provides a vaccine containing
the immunogenic compositions of the invention and a
pharmaceutically acceptable excipient.
[0084] The vaccines of the present invention may be adjuvanted,
particularly when intended for use in an elderly population but
also for use in infant populations. Suitable adjuvants include an
aluminum salt such as aluminum hydroxide gel or aluminum phosphate
or alum, but may also be a salt of calcium, magnesium, iron or
zinc, or may be an insoluble suspension of acylated tyrosine, or
acylated sugars, cationically or anionically derivatized
saccharides, or polyphosphazenes.
[0085] It is preferred that the adjuvant be selected to be a
preferential inducer of a TH1 type of response. Such high levels of
Th1-type cytokines tend to favour the induction of cell mediated
immune responses to a given antigen, whilst high levels of Th2-type
cytokines tend to favour the induction of humoral immune responses
to the antigen.
[0086] The distinction of Th1 and Th2-type immune response is not
absolute. In reality an individual will support an immune response
which is described as being predominantly Th1 or predominantly Th2.
However, it is often convenient to consider the families of
cytokines in terms of that described in murine CD4+ ve T cell
clones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R. L.
(1989) TH1 and TH2 cells: different patterns of lymphokine
secretion lead to different functional properties. (Annual Review
of Immunology, 7, p145-173). Traditionally, Th1-type responses are
associated with the production of the INF-.gamma. and IL-2
cytokines by T-lymphocytes. Other cytokines often directly
associated with the induction of Th1-type immune responses are not
produced by T-cells, such as IL-12. In contrast, Th2-type responses
are associated with the secretion of 11-4, IL-5, IL-6, IL-10.
Suitable adjuvant systems which promote a predominantly Th1
response include: Monophosphoryl lipid A or a derivative thereof,
particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (for
its preparation see GB 2220211 A); and a combination of
monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl
lipid A, together with either an aluminum salt (for instance
aluminum phosphate or aluminum hydroxide) or an oil-in-water
emulsion. In such combinations, antigen and 3D-MPL are contained in
the same particulate structures, allowing for more efficient
delivery of antigenic and immunostimulatory signals. Studies have
shown that 3D-MPL is able to further enhance the immunogenicity of
an alum-adsorbed antigen [rhoelen et al. Vaccine (1998) 16:708-14;
EP 689454-B1].
[0087] An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative, particularly the
combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO 96/33739. A particularly potent
adjuvant formulation involving QS21, 3D-MPL and tocopherol in an
oil in water emulsion is described in WO 95/17210. In one
embodiment the immunogenic composition additionally comprises a
saponin, which may be QS21. The formulation may also comprise an
oil in water emulsion and tocopherol (WO 95/17210). Unmethylated
CpG containing oligonucleotides (WO 96/02555) and other
immunomodulatory oligonucleotides (WO0226757 and WO03507822) are
also preferential inducers of a TH1 response and are suitable for
use in the present invention.
[0088] Particular adjuvants are those selected from the group of
metal Salts, oil in water emulsions, Toll like receptors agonist,
(in particular Toll like receptor 2 agonist, Toll like receptor 3
agonist, Toll like receptor 4 agonist, Toll like receptor 7
agonist, Toll like receptor 8 agonist and Toll like receptor 9
agonist), saponins or combinations thereof.
[0089] An adjuvant that can be used with the vaccine compositions
of the invention are bleb or outer membrane vesicle preparations
from Gram negative bacterial strains such as those taught by
WO02/09746--particularly N. meningitidis blebs. Adjuvant properties
of blebs can be improved by retaining LOS (lipooligosacccharide) on
its surface (e.g. through extraction with low concentrations of
detergent [for instanct 0-0.1% deoxycholate]). LOS can be
detoxified through the msbB(-) or htrB(-) mutations discussed in
WO02/09746. Adjuvant properties can also be improved by retaining
PorB (and optionally removing PorA) from meningococcal blebs.
Adjuvant properties can also be improved by truncating the outer
core saccharide structure of LOS on meningococcal blebs--for
instance via the IgtB(-) mutation discussed in WO2004/014417.
Alternatively, the aforementioned LOS (e.g. isolated from a msbB(-)
and/or IgtB(-) strain) can be purified and used as an adjuvant in
the compositions of the invention.
[0090] A further adjuvant which may be used with the compositions
of the invention may be selected from the group: a saponin, lipid A
or a derivative thereof, an immunostimulatory oligonucleotide, an
alkyl glucosaminide phosphate, an oil in water emulsion or
combinations thereof. A further preferred adjuvant is a metal salt
in combination with another adjuvant. It is preferred that the
adjuvant is a Toll like receptor agonist in particular an agonist
of a Toll like receptor 2, 3, 4, 7, 8 or 9, or a saponin, in
particular Qs21. It is further preferred that the adjuvant system
comprises two or more adjuvants from the above list. In particular
the combinations preferably contain a saponin (in particular Qs21)
adjuvant and/or a Toll like receptor 9 agonist such as a CpG
containing immunostimulatory oligonucleotide. Other preferred
combinations comprise a saponin (in particular QS21) and a Toll
like receptor 4 agonist such as monophosphoryl lipid A or its 3
deacylated derivative, 3 D-MPL, or a saponin (in particular QS21)
and a Toll like receptor 4 ligand such as an alkyl glucosaminide
phosphate.
[0091] Particularly preferred adjuvants are combinations of 3D-MPL
and QS21 (EP 0 671 948 B1), oil in water emulsions comprising
3D-MPL and QS21 (WO 95/17210, WO 98/56414), or 3D-MPL formulated
with other carriers (EP 0 689 454 B1). Other preferred adjuvant
systems comprise a combination of 3 D MPL, QS21 and a CpG
oligonucleotide as described in U.S. Pat. No. 6,558,670, U.S. Pat.
No. 6,544,518.
[0092] In an embodiment the adjuvant is (or comprises) a Toll like
receptor (TLR) 4 ligand, preferably an agonist such as a lipid A
derivative particularly monophosphoryl lipid A or more particularly
3 Deacylated monophoshoryl lipid A (3 D-MPL).
[0093] 3D-MPL is available from GlaxoSmithKiine Biologicals North
America and primarily promotes CD4+ T cell responses with an IFN-g
(Th1) phenotype. It can be produced according to the methods
disclosed in GB 2 220 211 A. Chemically it is a mixture of
3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated
chains. Preferably in the compositions of the present invention
small particle 3 D-MPL is used. Small particle 3 D-MPL has a
particle size such that it may be sterile-filtered through a 0.22
.mu.m filter. Such preparations are described in International
Patent Application No. WO 94/21292. Synthetic derivatives of lipid
A are known and thought to be TLR 4 agonists including, but not
limited to: [0094] OM174
(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-O--
phosphono-.beta.-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-.a-
lpha.-D-glucopyranosyldihydrogenphosphate), (WO 95/14026) [0095] OM
294 DP
(3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-h-
ydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)
(WO99/64301 and WO 00/0462) [0096] OM 197 MP-Ac DP (3S--,
9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxyt-
etradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate
10-(6-aminohexanoate) (WO 01/46127)
[0097] Other TLR4 ligands which may be used are alkyl Glucosaminide
phosphates (AGPs) such as those disclosed in WO9850399 or U.S. Pat.
No. 6,303,347 (processes for preparation of AGPs are also
disclosed), or pharmaceutically acceptable salts of AGPs as
disclosed in U.S. Pat. No. 6,764,840. Some AGPs are TLR4 agonists,
and some are TLR4 antagonists. Both are thought to be useful as
adjuvants.
[0098] Another preferred immunostimulant for use in the present
invention is Quil A and its derivatives. Quil A is a saponin
preparation isolated from the South American tree Quilaja Saponaria
Molina and was first described as having adjuvant activity by
Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv. fur die
gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin,
p243-254). 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.
[0099] Particular formulations of QS21 have been described which
are particularly preferred, these formulations further comprise a
sterol (WO96/33739). The saponins forming part of the present
invention may be separate in the form of micelles, mixed micelles
(preferentially, but not exclusively with bile salts) or may be in
the form of ISCOM matrices (EP 0 109 942 B1), liposomes or related
colloidal structures such as worm-like or ring-like multimeric
complexes or lipidic/layered structures and lamellae when
formulated with cholesterol and lipid, or in the form of an oil in
water emulsion (for example as in WO 95/17210). The saponins may
preferably be associated with a metallic salt, such as aluminium
hydroxide or aluminium phosphate (WO 98/15287).
[0100] Preferably, the saponin is presented in the form of a
liposome, ISCOM or an oil in water emulsion.
[0101] An enhanced system involves the combination of a
monophosphoryl lipid A (or detoxified lipid A) and a saponin
derivative, particularly the combination of QS21 and 3D-MPL as
disclosed in WO 94/00153, or a less reactogenic composition where
the QS21 is quenched with cholesterol as disclosed in WO 96/33739.
A particularly potent adjuvant formulation involving tocopherol
with or without QS21 and/or 3D-MPL in an oil in water emulsion is
described in WO 95/17210. In one embodiment the immunogenic
composition additionally comprises a saponin, which may be
QS21.
[0102] Immunostimulatory oligonucleotides or any other Toll-like
receptor (TLR) 9 agonist may also be used. The preferred
oligonucleotides for use in adjuvants or vaccines of the present
invention are CpG containing oligonucleotides, preferably
containing two or more dinucleotide CpG motifs separated by at
least three, more preferably at least six or more nucleotides. A
CpG motif is a Cytosine nucleotide followed by a Guanine
nucleotide. The CpG oligonucleotides of the present invention 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. Also included within the scope of the invention are
oligonucleotides with mixed internucleotide linkages. Methods for
producing phosphorothioate oligonucleotides or phosphorodithioate
are described in U.S. Pat. No. 5,666,153, U.S. Pat. No. 5,278,302
and WO95/26204.
[0103] Examples of preferred oligonucleotides have the following
sequences. The sequences preferably contain phosphorothioate
modified internucleotide linkages.
TABLE-US-00001 OLIGO 1 (SEQ ID NO:1): TCC ATG ACG TTC CTG ACG TT
(CpG 1826) OLIGO 2 (SEQ ID NO:2): TCT CCC AGC GTG CGC CAT (CpG
1758) OLIGO 3 (SEQ ID NO:3): ACC GAT GAC GTC GCC GGT GAC GGC ACC
ACG OLIGO 4 (SEQ ID NO:4): TCG TCG TTT TGT CGT TTT GTC GTT (CpG
2006) OLIGO 5 (SEQ ID NO:5): TCC ATG ACG TTC CTG ATG CT (CpG 1668)
OLIGO 6 (SEQ ID NO:6): TCG ACG TTT TCG GCG CGC GCC G (CpG 5456)
[0104] Alternative CpG oligonucleotides may comprise the preferred
sequences above in that they have inconsequential deletions or
additions thereto.
[0105] The CpG oligonucleotides utilised in the present invention
may be synthesized by any method known in the art (for example see
EP 468520). Conveniently, such oligonucleotides may be synthesized
utilising an automated synthesizer.
[0106] The adjuvant may be an oil in water emulsion or may comprise
an oil in water emulsion in combination with other adjuvants. The
oil phase of the emulsion system preferably comprises a
metabolisable oil. The meaning of the term metabolisable oil is
well known in the art. Metabolisable can be defined as "being
capable of being transformed by metabolism" (Dorland's Illustrated
Medical Dictionary, W. B. Sanders Company, 25.sup.th edition
(1974)). The oil may be any vegetable oil, fish, oil, animal or
synthetic oil, which is not toxic to the recipient and is capable
of being transformed by metabolism. Nuts, seeds, and grains are
common sources of vegetable oils. Synthetic oils are also part of
this invention and can include commercially available oils such as
NEOBEE.RTM. and others. Squalene
(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an
unsaturated oil which is found in large quantities in shark-liver
oil, and in lower quantities in olive oil, wheat germ oil, rice
bran oil, and yeast, and is a particularly preferred oil for use in
this invention. Squalene is a metabolisable oil by virtue of the
fact that it is an intermediate in the biosynthesis of cholesterol
(Merck index, 10.sup.th Edition, entry no. 8619). Tocols (e.g.
vitamin E) are also often used in oil emulsions adjuvants (EP 0 382
271 B1; U.S. Pat. No. 5,667,784; WO 95/17210). Tocols used in the
oil emulsions (preferably oil in water emulsions) of the invention
may be formulated as described in EP 0 382 271 B1, in that the
tocols may be dispersions of tocol droplets, optionally comprising
an emulsifier, of preferably less than 1 micron in diameter.
Alternatively, the tocols may be used in combination with another
oil, to form the oil phase of an oil emulsion. Examples of oil
emulsions which may be used in combination with the tocol are
described herein, such as the metabolisable oils described
above.
[0107] Oil in water emulsion adjuvants per se have been suggested
to be useful as adjuvant compositions (EP 0 399 843B), also
combinations of oil in water emulsions and other active agents have
been described as adjuvants for vaccines (WO 95/17210; WO 98/56414;
WO 99/12565; WO 99/11241). Other oil emulsion adjuvants have been
described, such as water in oil emulsions (U.S. Pat. No. 5,422,109;
EP 0 480 982 B2) and water in oil in water emulsions (U.S. Pat. No.
5,424,067; EP 0 480 981 B). All of which form preferred oil
emulsion systems (in particular when incorporating tocols) to form
adjuvants and compositions of the present invention.
[0108] Most preferably the oil emulsion (for instance oil in water
emulsions) further comprises an emulsifier such as TWEEN 80 and/or
a sterol such as cholesterol. A preferred oil emulsion (preferably
oil-in-water emulsion) comprises a metabolisible, non-toxic oil,
such as squalane, squalene or a tocopherol such as alpha tocopherol
(and preferably both squalene and alpha tocopherol) and optionally
an emulsifier (or surfactant) such as Tween 80. A sterol
(preferably cholesterol) may also be included. The method of
producing oil in water emulsions is well known to the man skilled
in the art. Commonly, the method comprises mixing the
tocol-containing oil phase with a surfactant such as a
PBS/TWEEN80.TM. solution, followed by homogenisation using a
homogenizer, it would be clear to a man skilled in the art that a
method comprising passing the mixture twice through a syringe
needle would be suitable for homogenising small volumes of liquid.
Equally, the emulsification process in microfluidiser (M110S
Microfluidics machine, maximum of 50 passes, for a period of 2
minutes at maximum pressure input of 6 bar (output pressure of
about 850 bar)) could be adapted by the man skilled in the art to
produce smaller or larger volumes of emulsion. The adaptation could
be achieved by routine experimentation comprising the measurement
of the resultant emulsion until a preparation was achieved with oil
droplets of the required diameter. In an oil in water emulsion, the
oil and emulsifier should be in an aqueous carrier. The aqueous
carrier may be, for example, phosphate buffered saline.
[0109] The size of the oil droplets found within the stable oil in
water emulsion are preferably less than 1 micron, may be in the
range of substantially 30-600 nm, preferably substantially around
30-500 nm in diameter, and most preferably substantially 150-500 nm
in diameter, and in particular about 150 nm in diameter as measured
by photon correlation spectroscopy. In this regard, 80% of the oil
droplets by number should be within the preferred ranges, more
preferably more than 90% and most preferably more than 95% of the
oil droplets by number are within the defined size ranges. The
amounts of the components present in the oil emulsions of the
present invention are conventionally in the range of from 0.5-20%
or 2 to 10% oil (of the total dose volume), such as squalene; and
when present, from 2 to 10% alpha tocopherol; and from 0.3 to 3%
surfactant, such as polyoxyethylene sorbitan monooleate. Preferably
the ratio of oil (preferably squalene):tocol (preferably
.alpha.-tocopherol) is equal or less than 1 as this provides a more
stable emulsion. An emulsifier, such as Tween80 or Span 85 may also
be present at a level of about 1%. In some cases it may be
advantageous that the vaccines of the present invention will
further contain a stabiliser.
[0110] Examples of preferred emulsion systems are described in WO
95/17210, WO 99/11241 and WO 99/12565 which disclose emulsion
adjuvants based on squalene, .alpha.-tocopherol, and TWEEN 80,
optionally formulated with the immunostimulants QS21 and/or 3D-MPL.
Thus in a particularly, preferred embodiment of the present
invention, the adjuvant of the invention may additionally comprise
further immunostimulants, such as LPS or derivatives thereof,
and/or saponins. Examples of further immunostimulants are described
herein and in "Vaccine Design--The Subunit and Adjuvant Approach"
1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F.,
and Newman, M. J., Plenum Press, New York and London, ISBN
0-306-44867-X.
[0111] In a preferred aspect the adjuvant and immunogenic
compositions according to the invention comprise a saponin
(preferably QS21) and/or an LPS derivative (preferably 3D-MPL) in
an oil emulsion described above, optionally with a sterol
(preferably cholesterol). Additionally the oil emulsion (preferably
oil in water emulsion) may contain span 85 and/or lecithin and/or
tricaprylin. Adjuvants comprising an oil-in-water emulsion, a
sterol and a saponin are described in WO 99/12565.
[0112] Typically for human administration the saponin (preferably
QS21) and/or LPS derivative (preferably 3D-MPL) will be present in
a human dose of immunogenic composition in the range of 1 .mu.g-200
.mu.g, such as 10-100 .mu.g, preferably 10 .mu.g-50 .mu.g per dose.
Typically the oil emulsion (preferably oil in water emulsion) will
comprise from 2 to 10% metabolisible oil. Preferably it will
comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and
from 0.3 to 3% (preferably 0.4-2%) emulsifier (preferably tween 80
[polyoxyethylene sorbitan monooleate]). Where both squalene and
alpha tocopherol are present, preferably the ratio of
squalene:alpha tocopherol is equal to or less than 1 as this
provides a more stable emulsion. Span 85 (Sorbitan trioleate) may
also be present at a level of 0.5 to 1% in the emulsions used in
the invention. In some cases it may be advantageous that the
immunogenic compositions and vaccines of the present invention will
further contain a stabiliser, for example other
emulsifiers/surfactants, including caprylic acid (merck index
10.sup.th Edition, entry no. 1739), of which Tricaprylin is
particularly preferred.
[0113] Where squalene and a saponin (preferably QS21) are included,
it is of benefit to also include a sterol (preferably cholesterol)
to the formulation as this allows a reduction in the total level of
oil in the emulsion. This leads to a reduced cost of manufacture,
improvement of the overall comfort of the vaccination, and also
qualitative and quantitative improvements of the resultant immune
responses, such as improved IFN-.gamma. production. Accordingly,
the adjuvant system of the present invention typically comprises a
ratio of metabolisable oil:saponin (w/w) in the range of 200:1 to
300:1, also the present invention can be used in a "low oil" form
the preferred range of which is 1:1 to 200:1, preferably 20:1 to
100:1, and most preferably substantially 48:1, this vaccine retains
the beneficial adjuvant properties of all of the components, with a
much reduced reactogenicity profile. Accordingly, the particularly
preferred embodiments have a ratio of squalene:QS21 (w/w) in the
range of 1:1 to 250:1, also a preferred range is 20:1 to 200:1,
preferably 20:1 to 100:1, and most preferably substantially 48:1.
Preferably a sterol (most preferably cholesterol) is also included
present at a ratio of saponin:sterol as described herein.
[0114] The emulsion systems of the present invention preferably
have a small oil droplet size in the sub-micron range. Most
preferably the oil droplet sizes will be in the range 120 to 750
nm, and most preferably from 120-600 nm in diameter.
[0115] A particularly potent adjuvant formulation (for ultimate
combination with AIPO4 in the immunogenic compositions of the
invention) involves a saponin (preferably QS21), an LPS derivative
(preferably 3D-MPL) and an oil emulsion (preferably squalene and
alpha tocopherol in an oil in water emulsion) as described in WO
95/17210 or in WO 99/12565 (in particular adjuvant formulation 11
in Example 2, Table 1).
[0116] Examples of a TLR 2 agonist include peptidoglycan or
lipoprotein. Imidazoquinolines, such as Imiquimod and Resiquimod
are known TLR7 agonists. Single stranded RNA is also a known TLR
agonist (TLR8 in humans and TLR7 in mice), whereas double stranded
RNA and poly IC (polyinosinic-polycytidylic acid--a commercial
synthetic mimetic of viral RNA). are exemplary of TLR 3 agonists.
3D-MPL is an example of a TLR4 agonist whilst CPG is an example of
a TLR9 agonist.
[0117] The immunogenic composition may comprise an antigen and an
immunostimulant adsorbed onto a metal salt. Aluminium based vaccine
formulations wherein the antigen and the immunostimulant
3-de-O-acylated monophosphoryl lipid A (3D-MPL), are adsorbed onto
the same particle are described in EP 0 576 478 B1, EP 0 689 454
B1, and EP 0 633 784 B1. In these cases then antigen is first
adsorbed onto the aluminium salt followed by the adsorption of the
immunostimulant 3D-MPL onto the same aluminium salt particles. Such
processes first involve the suspension of 3D-MPL by sonication in a
water bath until the particles reach a size of between 80 and 500
nm. The antigen is typically adsorbed onto aluminium salt for one
hour at room temperature under agitation. The 3D-MPL suspension is
then added to the adsorbed antigen and the formulation is incubated
at room temperature for 1 hour, and then kept at 4.degree. C. until
use.
[0118] In another process, the immunostimulant and the antigen are
on separate metal particles, as described in EP 1126876. The
improved process comprises the adsorption of immunostimulant, onto
a metallic salt particle, followed by the adsorption of the antigen
onto another metallic salt particle, followed by the mixing of the
discrete metallic particles to form a vaccine. The adjuvant for use
in the present invention may be an adjuvant composition comprising
an immunostimulant, adsorbed onto a metallic salt particle,
characterised in that the metallic salt particle is substantially
free of other antigen. Furthermore, vaccines are provided by the
present invention and are characterised in that the immunostimulant
is adsorbed onto particles of metallic salt which are substantially
free from other antigen, and in that the particles of metallic salt
which are adsorbed to the antigen are substantially free of other
immunostimulant.
[0119] Accordingly, the present invention provides an adjuvant
formulation comprising immunostimulant which has been adsorbed onto
a particle of a metallic salt, characterised in the composition is
substantially free of other antigen. Moreover, this adjuvant
formulation can be an intermediate which, if such an adjuvant is
used, is required for the manufacture of a vaccine. Accordingly
there is provided a process for the manufacture of a vaccine
comprising admixing an adjuvant composition which is one or more
immunostimulants adsorbed onto a metal particle with an antigen.
Preferably, the antigen has been pre-adsorbed onto a metallic salt.
Said metallic salt may be identical or similar to the metallic salt
which is adsorbed onto the immunostimulant. Preferably the metal
salt is an aluminium salt, for example Aluminium phosphate or
Aluminium hydroxide. The present invention further provides for a
vaccine composition comprising immunostimulant adsorbed onto a
first particle of a metallic salt, and antigen adsorbed onto a
metallic salt, characterised in that first and second particles of
metallic salt are separate particles.
[0120] LPS or LOS derivatives or mutations or lipid A derivatives
described herein are designed to be less toxic (e.g. 3D-MPL) than
native lipopolysaccharides and are interchangeable equivalents with
respect to any uses of these moieties described herein. They may be
TLR4 ligands as described above. Other such derivatives are
described in WO020786737, WO9850399, WO01 34617, WO0212258,
WO03065806.
[0121] In one embodiment the adjuvant used for the compositions of
the invention comprises a liposome carrier (made by known
techniques from a phospholipids (such as dioleoyl phosphatidyl
choline [DOPC]) and optionally a sterol [such as cholesterol]).
Such liposome carriers may carry lipid A derivatives [such as
3D-MPL--see above] and/or saponins (such as QS21--see above). In
one embodiment the adjuvant comprises (per 0.5 mL dose) 0.1-10 mg,
0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (e.g. 0.4-0.6, 0.9-1.1, 0.5 or 1
mg) phospholipid (for instance DOPC), 0.025-2.5, 0.05-1.5,
0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25
or 0.125 mg) sterol (for instance cholesterol), 5-60, 10-50, or
20-30 .mu.g (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 .mu.g) lipid A
derivative (for instance 3D-MPL), and 5-60, 10-50, or 20-30 .mu.g
(e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 .mu.g) saponin (for
instance QS21).
[0122] This adjuvant is particularly suitable for elderly vaccine
formulations. In one embodiment the vaccine composition comprising
this adjuvant comprises saccharide conjugates derived from at least
all the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F
(and may also comprise one or more from serotypes 3, 6A, 19A, and
22F), wherein the GMC antibody titre induced against one or more
(or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is
not significantly inferior to that induced by the Prevnar.RTM.
vaccine in human vaccinees.
[0123] In one embodiment the adjuvant used for the compositions of
the invention comprises an oil in water emulsion made from a
metabolisable oil (such as squalene), an emulsifier (such as Tween
80) and optionally a tocol (such as alpha tocopherol). In one
embodiment the adjuvant comprises (per 0.5 mL dose) 0.5-15, 1-13,
2-11, 4-8, or 5-6 mg (e.g. 2-3, 5-6, or 10-11 mg) metabolisable oil
(such as squalene), 0.1-10, 0.3-8, 0.6-6, 0.9-5, 1-4, or 2-3 mg
(e.g. 0.9-1.1, 2-3 or 4-5 mg) emulsifier (such as Tween 80) and
optionally 0.5-20, 1-15, 2-12, 4-10, 5-7 mg (e.g. 11-13, 5-6, or
2-3 mg) tocol (such as alpha tocopherol).
[0124] This adjuvant may optionally further comprise 5-60, 10-50,
or 20-30 .mu.g (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 .mu.g) lipid
A derivative (for instance 3D-MPL).
[0125] These adjuvants are particularly suitable for infant or
elderly vaccine formulations. In one embodiment the vaccine
composition comprising this adjuvant comprises saccharide
conjugates derived from at least all the following serotypes: 4,
6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or
more from serotypes 3, 6A, 19A, and 22F),
[0126] wherein the GMC antibody titre induced against one or more
(or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is
not significantly inferior to that induced by the Prevnar.RTM.
vaccine in human vaccinees.
[0127] This adjuvant may optionally contain 0.025-2.5, 0.05-1.5,
0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25
or 0.125 mg) sterol (for instance cholesterol), 5-60, 10-50, or
20-30 .mu.g (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 .mu.g) lipid A
derivative (for instance 3D-MPL), and 5-60, 10-50, or 20-30 .mu.g
(e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 .mu.g) saponin (for
instance QS21).
[0128] This adjuvant is particularly suitable for elderly vaccine
formulations. In one embodiment the vaccine composition comprising
this adjuvant comprises saccharide conjugates derived from at least
all the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F
(and may also comprise one or more from serotypes 3, 6A, 19A, and
22F), wherein the GMC antibody titre induced against one or more
(or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is
not significantly inferior to that induced by the Prevnar.RTM.
vaccine in human vaccinees.
[0129] In one embodiment the adjuvant used for the compositions of
the invention comprises aluminium phosphate and a lipid A
derivative (such as 3D-MPL). This adjuvant may comprise (per 0.5 mL
dose) 100-750, 200-500, or 300-400 .mu.g Al as aluminium phosphate,
and 5-60, 10-50, or 20-30 .mu.g (e.g. 5-15, 40-50, 10, 20, 30, 40
or 50 .mu.g) lipid A derivative (for instance 3D-MPL).
[0130] This adjuvant is particularly suitable for elderly or infant
vaccine formulations. In one embodiment the vaccine composition
comprising this adjuvant comprises saccharide conjugates derived
from at least all the following serotypes: 4, 6B, 9V, 14, 18C, 19F,
23F, 1, 5, 7F (and may also comprise one or more from serotypes 3,
6A, 19A, and 22F), wherein the GMC antibody titre induced against
one or more (or all) the vaccine components 4, 6B, 9V, 14, 18C, 19F
and 23F is not significantly inferior to that induced by the
Prevnar.RTM. vaccine in human vaccinees.
[0131] The vaccine preparations containing immunogenic compositions
of the present invention may be used to protect or treat a mammal
susceptible to infection, by means of administering said vaccine
via systemic or mucosal route. These administrations may include
injection via the intramuscular, intraperitoneal, intradermal or
subcutaneous routes; or via mucosal administration to the
oral/alimentary, respiratory, genitourinary tracts. Intranasal
administration of vaccines for the treatment of pneumonia or otitis
media is preferred (as nasopharyngeal carriage of pneumococci can
be more effectively prevented, thus attenuating infection at its
earliest stage). Although the vaccine of the invention may be
administered as a single dose, components thereof may also be
co-administered together at the same time or at different times
(for instance pneumococcal saccharide conjugates could be
administered separately, at the same time or 1-2 weeks after the
administration of the any bacterial protein component of the
vaccine for optimal coordination of the immune responses with
respect to each other). For co-administration, the optional Th1
adjuvant may be present in any or all of the different
administrations. In addition to a single route of administration, 2
different routes of administration may be used. For example,
saccharides or saccharide conjugates may be administered IM (or ID)
and bacterial proteins may be administered IN (or ID). In addition,
the vaccines of the invention may be administered IM for priming
doses and IN for booster doses.
[0132] The content of protein antigens in the vaccine will
typically be in the range 1-100 .mu.g, preferably 5-50 .mu.g, most
typically in the range 5-25 .mu.g. Following an initial
vaccination, subjects may receive one or several booster
immunizations adequately spaced.
[0133] Vaccine preparation is generally described in Vaccine Design
("The subunit and adjuvant approach" (eds Powell M. F. & Newman
M. J.) (1995) Plenum Press New York). Encapsulation within
liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
[0134] The vaccines of the present invention may be stored in
solution or lyophilized. Preferably the solution is lyophilized in
the presence of a sugar such as sucrose or lactose. It is still
further preferable that they are lyophilized and extemporaneously
reconstituted prior to use. Lyophilizing may result in a more
stable composition (vaccine) and may possibly lead to higher
antibody titers in the presence of 3D-MPL and in the absence of an
aluminum based adjuvant.
[0135] In one aspect of the invention is provided a vaccine kit,
comprising a vial containing an immunogenic composition of the
invention, optionally in lyophilised form, and further comprising a
vial containing an adjuvant as described herein. It is envisioned
that in this aspect of the invention, the adjuvant will be used to
reconstitute the lyophilised immunogenic composition.
[0136] Although the vaccines of the present invention may be
administered by any route, administration of the described vaccines
into the skin (ID) forms one embodiment of the present invention.
Human skin comprises an outer "horny" cuticle, called the stratum
corneum, which overlays the epidermis. Underneath this epidermis is
a layer called the dermis, which in turn overlays the subcutaneous
tissue. Researchers have shown that injection of a vaccine into the
skin, and in particular the dermis, stimulates an immune response,
which may also be associated with a number of additional
advantages. Intradermal vaccination with the vaccines described
herein forms a preferred feature of the present invention.
[0137] The conventional technique of intradermal injection, the
"mantoux procedure", comprises steps of cleaning the skin, and then
stretching with one hand, and with the bevel of a narrow gauge
needle (26-31 gauge) facing upwards the needle is inserted at an
angle of between 10-15.degree.. Once the bevel of the needle is
inserted, the barrel of the needle is lowered and further advanced
whilst providing a slight pressure to elevate it under the skin.
The liquid is then injected very slowly thereby forming a bleb or
bump on the skin surface, followed by slow withdrawal of the
needle.
[0138] More recently, devices that are specifically designed to
administer liquid agents into or across the skin have been
described, for example the devices described in WO 99/34850 and EP
1092444, also the jet injection devices described for example in WO
01/13977; 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. Alternative methods of intradermal administration of the
vaccine preparations may include conventional syringes and needles,
or devices designed for ballistic delivery of solid vaccines (WO
99/27961), or transdermal patches (WO 97/48440; WO 98/28037); or
applied to the surface of the skin (transdermal or transcutaneous
delivery WO 98/20734; WO 98/28037).
[0139] When the vaccines of the present invention are to be
administered to the skin, or more specifically into the dermis, the
vaccine is in a low liquid volume, particularly a volume of between
about 0.05 ml and 0.2 ml.
[0140] The content of antigens in the skin or intradermal vaccines
of the present invention may be similar to conventional doses as
found in intramuscular vaccines (see above). However, it is a
feature of skin or intradermal vaccines that the formulations may
be "low dose". Accordingly the protein antigens in "low dose"
vaccines are preferably present in as little as 0.1 to 10 .mu.g,
preferably 0.1 to 5 .mu.g per dose; and the saccharide (preferably
conjugated) antigens may be present in the range of 0.01-1 .mu.g,
and preferably between 0.01 to 0.5 .mu.g of saccharide per
dose.
[0141] As used herein, the term "intradermal delivery" means
delivery of the vaccine to the region of the dermis in the skin.
However, the vaccine will not necessarily be located exclusively in
the dermis. The dermis is the layer in the skin located between
about 1.0 and about 2.0 mm from the surface in human skin, but
there is a certain amount of variation between individuals and in
different parts of the body. In general, it can be expected to
reach the dermis by going 1.5 mm below the surface of the skin. The
dermis is located between the stratum corneum and the epidermis at
the surface and the subcutaneous layer below. Depending on the mode
of delivery, the vaccine may ultimately be located solely or
primarily within the dermis, or it may ultimately be distributed
within the epidermis and the dermis.
[0142] The present invention further provides an improved vaccine
for the prevention or amelioration of Otitis media caused by
Haemophilus influenzae by the addition of Haemophilus influenzae
proteins, for example protein D in free or conjugated form. In
addition, the present invention further provides an improved
vaccine for the prevention or amelioration of pneumococcal
infection in infants (e.g., Otitis media), by relying on the
addition of one or two pneumococcal proteins as free or conjugated
protein to the S. pneumoniae conjugate compositions of the
invention. Said pneumococcal free proteins may be the same or
different to any S. pneumoniae proteins used as carrier proteins.
One or more Moraxella catarrhalis protein antigens can also be
included in the combination vaccine in a free or conjugated form.
Thus, the present invention is an improved method to elicit a
(protective) immune response against Otitis media in infants.
[0143] In another embodiment, the present invention is an improved
method to elicit a (protective) immune response in infants (defined
as 0-2 years old in the context of the present invention) by
administering a safe and effective amount of the vaccine of the
invention [a paediatric vaccine]. Further embodiments of the
present invention include the provision of the antigenic S.
pneumoniae conjugate compositions of the invention for use in
medicine and the use of the S. pneumoniae conjugates of the
invention in the manufacture of a medicament for the prevention (or
treatment) of pneumococcal disease.
[0144] In yet another embodiment, the present invention is an
improved method to elicit a (protective) immune response in the
elderly population (in the context of the present invention a
patient is considered elderly if they are 50 years or over in age,
typically over 55 years and more generally over 60 years) by
administering a safe and effective amount of the vaccine of the
invention, preferably in conjunction with one or two S. pneumoniae
proteins present as free or conjugated protein, which free S.
pneumoniae proteins may be the same or different as any S.
pneumoniae proteins used as carrier proteins.
[0145] A further aspect of the invention is a method of immunising
a human host against disease caused by S. pneumoniae and optionally
Haemophilus influenzae infection comprising administering to the
host an immunoprotective dose of the immunogenic composition or
vaccine or kit of the invention.
[0146] A further aspect of the invention is an immunogenic
composition of the invention for use in the treatment or prevention
of disease caused by S. pneumoniae and optionally Heemophilus
influenzae infection.
[0147] A further aspect of the invention is use of the immunogenic
composition or vaccine or kit of the invention in the manufacture
of a medicament for the treatment or prevention of diseases caused
by S. pneumoniae and optionally Haemophilus influenzae
infection.
[0148] The terms "comprising", "comprise" and "comprises" herein
are intended by the inventors to be optionally substitutable with
the terms "consisting of", "consist of" and "consists of",
respectively, in every instance.
[0149] Embodiments herein relating to "vaccine compositions" of the
invention are also applicable to embodiments relating to
"immunogenic compositions" of the invention, and vice versa.
[0150] All references or patent applications cited within this
patent specification are incorporated by reference herein.
[0151] 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
Expression of Protein D
Haemophilus Influenzae Protein D
Genetic Construction for Protein D Expression
[0152] Starting Materials
The Protein D encoding DNA
[0153] Protein D is highly conserved among H. influenzae of all
serotypes and non-typeable strains. The vector pHIC348 containing
the DNA sequence encoding the entire protein D gene has been
obtained from Dr. A. Forsgren, Department of Medical Microbiology,
University of Lund, Malmo General Hospital, Malmo, Sweden. The DNA
sequence of protein D has been published by Janson et al. (1991)
Infect. Immun. 59: 119-125.
The Expression Vector pMG1
[0154] The expression vector pMG1 is a derivative of pBR322 (Gross
et al., 1985) in which bacteriophage .lamda. derived control
elements for transcription and translation of foreign inserted
genes were introduced (Shatzman et al., 1983). In addition, the
Ampicillin resistance gene was exchanged with the Kanamycin
resistance gene.
The E. Coli Strain AR58
[0155] The E. coli strain AR58 was generated by transduction of N99
with a P1 phage stock previously grown on an SA500 derivative
(galE::TN10, lambdaKil.sup.- cl857 .DELTA.H1). N99 and SA500 are E.
coli K12 strains derived from Dr. Martin Rosenberg's laboratory at
the National Institute of Health.
The Expression Vector pMG 1
[0156] For the production of protein D, the DNA encoding the
protein has been cloned into the expression vector pMG 1. This
plasmid utilises signals from lambdaphage DNA to drive the
transcription and translation of inserted foreign genes. The vector
contains the promoter PL, operator OL and two utilisation sites
(NutL and NutR) to relieve transcriptional polarity effects when N
protein is provided (Gross et al., 1985). Vectors containing the PL
promoter, are introduced into an E. coli lysogenic host to
stabilise the plasmid DNA. Lysogenic host strains contain
replication-defective lambdaphage DNA integrated into the genome
(Shatzman et al., 1983). The chromosomal lambdaphage DNA directs
the synthesis of the cl repressor protein which binds to the OL
repressor of the vector and prevents binding of RNA polymerase to
the PL promoter and thereby transcription of the inserted gene. The
cl gene of the expression strain AR58 contains a temperature
sensitive mutant so that PL directed transcription can be regulated
by temperature shift, i.e. an increase in culture temperature
inactivates the repressor and synthesis of the foreign protein is
initiated. This expression system allows controlled synthesis of
foreign proteins especially of those that may be toxic to the cell
(Shimataka & Rosenberg, 1981).
The E. Coli Strain AR58
[0157] The AR58 lysogenic E. coli strain used for the production of
the protein D carrier is a derivative of the standard NIH E. coli
K12 strain N99 (F.sup.- su.sup.- galK2, lacZ.sup.- thr.sup.-). It
contains a defective lysogenic lambdaphage (galE::TN10, lambdaKil
c1857 .DELTA.H1). The Kil.sup.- phenotype prevents the shut off of
host macromolecular synthesis. The c1857 mutation confers a
temperature sensitive lesion to the cl repressor. The .DELTA.H1
deletion removes the lambdaphage right operon and the hosts bio,
uvr3, and chIA loci. The AR58 strain was generated by transduction
of N99 with a P1 phage stock previously grown on an SA500
derivative (gaIE::TN10, lambdaKil.sup.- cl857 .DELTA.H1). The
introduction of the defective lysogen into N99 was selected with
tetracycline by virtue of the presence of a TN10 transposon coding
for tetracyclin resistance in the adjacent galE gene.
Construction of Vector pMGMDPPrD
[0158] The pMG 1 vector which contains the gene encoding the
non-structural S1 protein of Influenzae virus (pMGNSI) was used to
construct pMGMDPPrD. The protein D gene was amplified by PCR from
the pHIC348 vector (Janson et al. 1991 Infect. Immun. 59:119-125)
with PCR primers containing NcoI and XbaI restriction sites at the
5' and 3' ends, respectively. The NcoI/XbaI fragment was then
introduced into pMGNS1 between NcoI and XbaI thus creating a fusion
protein containing the N-terminal 81 amino acids of the NS1 protein
followed by the PD protein. This vector was labelled pMGNS1
PrD.
[0159] Based on the construct described above the final construct
for protein D expression was generated. A BamHI/BamHI fragment was
removed from pMGNS1PrD. This DNA hydrolysis removes the NS1 coding
region, except for the first three N-terminal residues. Upon
religation of the vector a gene encoding a fusion protein with the
following N-terminal amino acid sequence has been generated:--
--MDP SSHSSNMANT--
NS1 Protein D
[0160] The protein D does not contain a leader peptide or the
N-terminal cysteine to which lipid chains are normally attached.
The protein is therefore neither excreted into the periplasm nor
lipidated and remains in the cytoplasm in a soluble form.
[0161] The final construct pMG-MDPPrD was introduced into the AR58
host strain by heat shock at 37.degree. C. Plasmid containing
bacteria were selected in the presence of Kanamycin. Presence of
the protein D encoding DNA insert was demonstrated by digestion of
isolated plasmid DNA with selected endonucleases. The recombinant
E. coli strain is referred to as ECD4.
[0162] Expression of protein D is under the control of the lambda
P.sub.L promoter/O.sub.L Operator. The host strain AR58 contains a
temperature-sensitive cl gene in the genome which blocks expression
from lambda P.sub.L at low temperature by binding to OL Once the
temperature is elevated cl is released from O.sub.L and protein D
is expressed.
Small-Scale Preparation
[0163] At the end of the fermentation the cells are concentrated
and frozen.
[0164] The extraction from harvested cells and the purification of
protein D was performed as follows. The frozen cell culture pellet
is thawed and resuspended in a cell disruption solution (Citrate
buffer pH 6.0) to a final OD.sub.600=60. The suspension is passed
twice through a high pressure homogenizer at P=1000 bar. The cell
culture homogenate is clarified by centrifugation and cell debris
is removed by filtration. In the first purification step the
filtered lysate is applied to a cation exchange chromatography
column (SP Sepharose Fast Flow). PD binds to the gel matrix by
ionic interaction and is eluted by a step increase of the ionic
strength of the elution buffer.
[0165] In a second purification step impurities are retained on an
anionic exchange matrix (Q Sepharose Fast Flow). PD does not bind
onto the gel and can be collected in the flow through.
[0166] In both column chromatography steps fraction collection is
monitored by OD. The flow through of the anionic exchange column
chromatography containing the purified protein D is concentrated by
ultrafiltration.
[0167] The protein D containing ultrafiltration retentate is
finally passed through a 0.2 .mu.m membrane.
Large Scale Preparation
[0168] The extraction from harvested cells and the purification of
protein D was performed as follows. The harvested broth is cooled
and directly passed twice through a high pressure homogenizer at a
Pressure of around 800 bars.
[0169] In the first purification step the cell culture homogenate
is diluted and applied to a cation exchange chromatography column
(SP Sepharose Big beads). PD binds to the gel matrix by ionic
interaction and is eluted by a step increase of the ionic strength
of the elution buffer and filtrated.
[0170] In a second purification step impurities are retained on an
anionic exchange matrix (Q Sepharose Fast Flow). PD does not bind
onto the gel and can be collected in the flow through.
[0171] In both column chromatography steps fraction collection is
monitored by OD. The flow through of the anionic exchange column
chromatography containing the purified protein D is concentrated
and diafiltrated by ultrafiltration.
[0172] The protein D containing ultrafiltration retentate is
finally passed through a 0.2 .mu.m membrane.
Example 1b
Expression of PhtD
[0173] The PhtD protein is a member of the pneumococcal
histidine-triad (Pht) protein family characterized by the presence
of histidine-triads (HXXHXH motif). PhtD is a 838 aa-molecule and
carries 5 histidine triads (see MedImmune WO00/37105 SEQ ID NO: 4
for amino acid sequence and SEQ ID NO: 5 for DNA sequence). PhtD
also contains a proline-rich region in the middle (amino acid
position 348-380). PhtD has a 20 aa-N-terminal signal sequence with
a LXXC motif.
Genetic Construct
[0174] The gene sequence of the mature Medimmune PhtD protein (from
aa 21 to aa 838) was transferred recombinantly to E. coli using the
in-house pTCMP14 vector carrying the p.lamda. promoter. The E. coli
host strain is AR58, which carries the cI857 thermosensitive
repressor, allowing heat-induction of the promotor.
[0175] Polymerase chain reaction was realized to amplify the phtD
gene from a MedImmune plasmid (carrying the phtD gene from
Streptococcus pneumoniae strain Norway 4 (serotype 4)-SEQ ID NO: 5
as described in WO 00/37105). Primers, specific for the phtD gene
only, were used to amplify the phtD gene in two fragments. Primers
carry either the NdeI and KpnI or the KpnI and XbaI restriction
sites. These primers do not hybridize with any nucleotide from the
vector but only with phtD specific gene sequences. An artificial
ATG start codon was inserted using the first primer carrying the
NdeI restriction site. The generated PCR products were then
inserted into the pGEM-T cloning vector (Promega), and the DNA
sequence was confirmed, Subcloning of the fragments in the TCMP14
expression vector was ten realized using standard techniques and
the vector was transformed into AR58 E. coli.
PhtD Purification
Phtd Purification is Achieved as Follows:
[0176] Growth of E. coli cells in the presence of Kanamycin: growth
30 hours at 30.degree. C. then induction for 18 hours at
39.5.degree. C. [0177] Breakage of the E. coli cells from whole
culture at OD.+-.115 in presence of EDTA 5 mM and PMSF 2 mM as
protease inhibitors: Rannie, 2 passages, 1000 bars. [0178] Antigen
capture and cells debris removal on expanded bed mode Streamline Q
XL chromatography at room temperature (20.degree. C.); the column
is washed with NaCl 150 mM+Empigen 0.25% pH 6.5 and eluted with
NaCl 400 mM+Empigen 0.25% in 25 mM potassium phosphate buffer pH
7.4. [0179] Filtration on Sartobran 150 cartridge (0.45+0.2 .mu.m)
[0180] Antigen binding on Zn.sup.++ Chelating Sepharose FF IMAC
chromatography at pH 7.4 in presence of 5 mM imidazole at 4.degree.
C.; the column is washed with Imidazole 5 mM and Empigen 1% and
eluted with 50 mM imidazole, both in 25 mM potassium phosphate
buffer pH 8.0. [0181] Weak anion exchange chromatography in
positive mode on Fractogel EMD DEAE at pH 8.0 (25 mM potassium
phosphate) at 4.degree. C.; the column is washed with 140 mM NaCl
and eluted at 200 mM NaCl while contaminants (proteins and DNA)
remain adsorbed on the exchanger. [0182] Concentration and
ultrafiltration with 2 mM Na/K phosphate pH 7.15 on 50 kDa
membrane. [0183] Sterilising filtration of the purified bulk on a
Millipak-20 0.2 .mu.m filter cartridge.
Example 1c
Expression of Pneumolysin
[0184] Pneumococcal pneumolysin was prepared and detoxified as
described in WO2004/08 1515 and WO2006/032499.
Example 2
Preparation of Conjugates
[0185] It is well known in the art how to make purified
pneumococcal polysaccharides. For the purposes of these examples
the polysaccharides were made essentially as described in EP072513
or by closesly-related methods. Before conjugation the
polysaccharides may be sized by microfluidisation as described
below.
[0186] The activation and coupling conditions are specific for each
polysaccharide. These are given in Table 1. Sized polysaccharide
(except for PS5, 6B and 23F) was dissolved in NaCl 2M, NaCl 0.2M 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. Two alternative serotype 22F conjugates were
made; one conjugated directly, one through an ADH linker.
[0187] From a 100 mg/ml stock solution in acetonitrile or
acetonitrile/water 50%/50% solution, CDAP(CDAP/PS ratio 0.5-1.5
mg/mg PS) was added to the polysaccharide solution. 1.5 minute
later, 0.2 M-0.3 M 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,
PhtD, pneumolysin 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.
[0188] Preparation of 18C:
[0189] 18C was linked to the carrier protein via a linker-Adipic
acid dihydrazide (ADH)
[0190] Polysaccharide serotype 18C was microfluidized before
conjugation.
[0191] Derivatization of tetanus toxoid with EDAC
[0192] 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.
[0193] 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
[0194] Details of the conjugation parameters can be found in Table
1.
[0195] 2 grams of microfluidized PS were diluted at the defined
concentration in water and adjusted to 2M NaCl by NaCl powder
addition.
[0196] CDAP solution (100 mg/ml freshly prepared in 50/50 v/v
acetonitrile/NFI) was added to reach the appropriate CDAP/PS
ratio.
[0197] 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.
[0198] 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.
[0199] For quenching, a 2M glycine solution, was added to the
mixture PS/TT.sub.AH/CDAP.
[0200] The pH was adjusted to the quenching pH (pH 9.0).
[0201] The solution was stirred for 30 min at 25.degree. C., and
then left overnight at 2-8.degree. C. with continuous slow
stirring.
PS22F.sub.AH-PhtD Conjugate
[0202] In a second conjugation method for this saccharide (the
first being the direct PS22-PhtD conjugation method shown in Table
1), 22F was linked to the carrier protein via a linker--Adipic acid
dihydrazide (ADH). Polysaccharide serotype 22F was microfluidized
before conjugation.
PS 22F Derivatization
[0203] Activation and coupling are performed at 25.degree. C. under
continuous stirring in a temperature-controlled waterbath.
[0204] Microfluidized PS22F was diluted to obtain a final PS
concentration of 6 mg/ml in 0.2M NaCl and the solution was adjusted
at pH 6.05.+-.0.2 with 0.1N HCl.
[0205] CDAP solution (100 mg/ml freshly prepared in
acetonitrile/WFI, 50/50) was added to reach the appropriate CDAP/PS
ratio (1.5/1 ww).
[0206] The pH was raised up to the activation pH 9.00.+-.0.05 by
the addition of 0.5M NaOH and was stabilised at this pH until
addition of ADH.
[0207] After 3 minutes, ADH was added to reach the appropriate
ADH/PS ratio (8.9/1 w/w); the pH was regulated to coupling pH 9.0.
The solution was left for 1 hour under pH regulation.
[0208] The PS.sub.AH derivative was concentrated and
diafiltrated.
Coupling
[0209] PhtD at 10 mg/ml in 0.2M NaCl was added to the PS22F.sub.AH
derivative in order to reach a PhtD/PS22F.sub.AH ratio of 4/1
(w/w). The pH was adjusted to 5.0.+-.0.05 with HCl. The EDAC
solution (20 mg/ml in 0.1 M Tris-HCl pH 7.5) was added manually in
10 min (250 .mu.l/min) to reach 1 mg EDAC/mg PS22F.sub.AH. The
resulting solution was incubated for 150 min (though 60 mins was
also used) at 25.degree. C. under stirring and pH regulation. The
solution was neutralized by addition of 1 M Tris-HCl pH 7.5 ( 1/10
of the final volume) and let 30 min at 25.degree. C.
[0210] Prior to the elution on Sephacryl S400HR, the conjugate was
clarified using a 5 .mu.m Minisart filter.
[0211] The resulting conjugate has a final PhtD/PS ratio of 4.1
(w/w), a free PS content below 1% and an antigenicity
(.alpha.-PS/.alpha.-PS) of 36.3% and anti-PhtD antigenicity of
7.4%.
Purification of the Conjugates:
[0212] The conjugates were purified by gel filtration using a
Sephacryl S400HR 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, PS-PhtD, PS-pneumolysin
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).
[0213] 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.
Specific Activation/Coupling/Quenching Conditions of PS S.
Pneumoniae-Protein DITT/DT/PhtD/Plyconjugates
[0214] Where ".mu.fluid" appears in a row header, it indicates that
the saccharide was sized by microfluidisation before conjugation.
Sizes of saccharides following microfluidisation are given in table
2.
TABLE-US-00002 TABLE 1 Specific activation/coupling/quenching
conditions of PS S. pneumoniae-Protein D/TT/DT/PhtD/Plyconjugates
Serotype 1 4 7F .mu.fluid .mu.fluid 5 6A 6B .mu.fluid PS 2.5 2.5
7.1 5.0 5.0 5.0 conc.(mg/ml) PS WFI WFI WFI NaCl 2M NaCl 2M NaCl 2M
dissolution PD 10.0 10.0 5.0 5.0 5.0 10.0 conc.(mg/ml) Initial
PD/PS 1.5/1 1.5/1 1/1 1/1 1.1/1 1.2/1 Ratio (w/w) CDAP conc. 0.50
0.50 0.79 0.83 0.83 0.75 (mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q
9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.0
9.5/9.5/9.0 Serotype 9V 14 18C 19A 19F 22F .mu.fluid .mu.fluid
.mu.fluid .mu.fluid .mu.fluid .mu.fluid 23F PS 5.0 5.0 4.5 15.0 9.0
6.0 2.38 conc.(mg/ml) PS NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2M
NaCl 0.2M NaCl 2M dissolution Carrier 10.0 10.0 20.0 (TT) 10.0 20.0
10.0 5.0 protein (Ply) (DT) (PhtD) conc.(mg/ml) Initial carrier
1.2/1 1.2/1 2/1 2.5/1 1.5/1 3/1 1/1 protein/PS Ratio (w/w) CDAP
conc. 0.50 0.75 0.75 1.5 1.5 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
9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 Note: pHa, c, q corresponds to
the pH for activation, coupling and quenching, respectively
Characterisation:
[0215] Each conjugate was characterised and met the specifications
described in Table 2. The polysaccharide content (.mu.g/ml) was
measured by the Resorcinol test and the protein content (.mu.g/ml)
by the Lowry test. The final PS/PD ratio (w/w) is determined by the
ratio of the concentrations.
Free Polysaccharide Content (%):
[0216] The free polysaccharide content of conjugates kept at
4.degree. C. or stored 7 days at 37.degree. C. was determined on
the supernatant obtained after incubation with .alpha.-carrier
protein antibodies and saturated ammonium sulfate, followed by a
centrifugation.
[0217] An .alpha.-PS/.alpha.-PS ELISA was used for the
quantification of free polysaccharide in the supernatant. The
absence of conjugate was also controlled by an .alpha.-carrier
protein/.alpha.-PS ELISA.
Antigenicity:
[0218] The antigenicity on the same conjugates was analyzed in a
sandwich-type ELISA wherein the capture and the detection of
antibodies were .alpha.-PS and .alpha.-Protein respectively.
Free Protein Content (%)
[0219] Unconjugated carrier protein can be separated from the
conjugate during the purification step. The content of free
residual protein was determined using size exclusion chromatography
(TSK 5000-PWXL) followed by UV detection (214 nm). The elution
conditions allowed separating the free carrier protein and the
conjugate. Free protein content in conjugate bulks was then
determined versus a calibration curve (from 0 to 50 .mu.g/ml of
carrier protein). Free carrier protein in % was obtained as
follows: % free carrier =(free carrier (1 g/ml)/(Total
concentration of corresponding carrier protein measured by Lowry
(.mu.g/ml)*100%).
Stability:
[0220] Molecular weight distribution (K.sub.av) and stability was
measured on a HPLC-SEC gel filtration (TSK 5000-PWXL) for
conjugates kept at 4.degree. C. and stored for 7 days at 370C.
[0221] The 10/11/13/14-valent characterization is given in Table 2
(see comment thereunder).
[0222] The protein conjugates can be adsorbed onto aluminium
phosphate and pooled to form the final vaccine.
Conclusion:
[0223] Immunogenic conjugates have been produced, that have since
been shown to be components of a promising vaccine.
TABLE-US-00003 TABLE 2 characteristics of the conjugates PS PS size
Carrier/PS Free PS Antigenicity Conj. Size Conjugates (Dax10.sup.3)
Ratio (Elisa) Free Carrier (Elisa) (kDa) PS1-PD 349-382* 1.5-1.6
1.0%-1.2%.sup. 3.9%-4.8% 87%-95% 1499-1715 PS4-PD 93-100* 1.5-1.6
4.7-6.5% 3.2%-4.0% 90%-96% 1303-1606 PS5-PD*** 367-443 0.80
8.7-11.2% 2.2%-3.8% 93%-108% 1998-2352 PS6A-PD 1100-1540 0.61 4.5%
Not done 45.9% Not done PS6B-PD*** 1069-1391 0.7-0.8 1.3-1.6%
<2.0% 68%-75% 4778-5235 PS7F-PD 255-264* 1.1-1.2 <1% <1.4%
58% 3907-4452 PS9V-PD 258-280* 1.3-1.5 <1% <1.3% 67%-69%
9073-9572 PS14-PD 232-241* 1.4 <1% <1.5% 70% 3430-3779
PS18C-TT 89-97* 2.2-2.4 1.5-2.2% <4% 46%-56% 5464-6133
PS19A-Ply* 151 3.2 <1% 29% PS19F-DT 133-143* 1.4-1.5
4.1%-5.9%.sup. <1.2%-<1.3% 82%-88% 2059-2335 PS22F-PhtD*
159-167 2.17 5.8 Not done 37% Not done PS22F-AHPhtD* 159-167
3.66-4.34 <1% Not done .sup. 28-31% Not done PS23F-PD*** 914-980
0.5 1.4-1.9% 3.7%-4.9% 137%-154% 2933-3152 *PS size following
microfluidization of the native PS
[0224] A 10 valent vaccine was made by mixing serotype 1, 4, 5, 6B,
7F, 9V, 14, 18C, 19F and 23F conjugates (e.g. at a dose of 1, 3, 1,
1, 1, 1, 1, 3, 3, 1 .mu.g of saccharide, respectively per human
dose). An 11 valent vaccine was made by further adding the serotype
3 conjugate from Table 5 (e.g. at 1 .mu.g of saccharide per human
dose). A 13 valent vaccine was made by further adding the serotypes
19A and 22F conjugates above (with 22F either directly linked to
PhtD, or alternatively through an ADH linker) [e.g. at a dose of 3
.mu.g each of saccharide per human dose]. A 14 valent vaccine may
be made by further adding the serotype 6A conjugate above [e.g. at
a dose of 1 .mu.g of saccharide per human dose.
Example 3
Evidence that Inclusion of Haemphilus influenzae Protein D in an
Immunogenic Composition of the Invention can Provide Improved
Protection Against Acute Otitis Media (AOM)
Study Design.
[0225] The study used an 11Pn-PD vaccine--comprising serotypes 1,
3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F each conjugated to
protein D from H. influenzae (refer to Table 5 in Example 4).
Subjects were randomized into two groups to receive four doses of
either the 11 Pn-PD vaccine or Havrix at approximately 3, 4, 5 and
12-15 months of age. All subjects received GSK Biologicals'
Infanrix-hexa (DTPa-HBV-IPV/Hib) vaccine concomitantly at 3, 4 and
5 months of age. Infanrix-hexa is a combination of Pediarix and Hib
mixed before administration. Efficacy follow-up for the
"According-to-Protocol" analysis started 2 weeks after
administration of the third vaccine dose and continued until 24-27
months of age. Nasopharyngeal carriage of S. pneumoniae and H.
influenzee was evaluated in a selected subset of subjects.
[0226] Parents were advised to consult the investigator if their
child was sick, had ear pain, spontaneous perforation of the
tympanic membrane or spontaneous ear discharge. If the investigator
suspected an episode of AOM, the child was immediately referred to
an Ear, Nose and Throat (ENT) specialist for confirmation of the
diagnosis. A clinical diagnosis of AOM was based on either the
visual appearance of the tympanic membrane (i.e. redness, bulging,
loss of light reflex) or the presence of middle ear fluid effusion
(as demonstrated by simple or pneumatic otoscopy or by microscopy).
In addition, at least two of the following signs or symptoms had to
be present: ear pain, ear discharge, hearing loss, fever, lethargy,
irritability, anorexia, vomiting, or diarrhea. If the ENT
specialist confirmed the clinical diagnosis, a specimen of middle
ear fluid was collected by tympanocentesis for bacteriological
testing.
[0227] For subjects with repeated sick visits, a new AOM episode
was considered to have started if more than 30 days had elapsed
since the beginning of the previous episode. In addition, an AOM
episode was considered to be a new bacterial episode if the
isolated bacterium/serotype differed from the previous isolate
whatever the interval between the two consecutive episodes.
Trial Results
[0228] A total of 4968 infants were enrolled, 2489 in the 11 Pn-PD
group and 2479 in the control group. There were no major
differences in the demographic characteristics or risk factors
between the two groups.
Clinical Episodes and AOM Case Definition
[0229] During the per protocol follow-up period, a total of 333
episodes of clinical AOM were recorded in the 11 Pn-PD group and
499 in the control group. Table 3 presents the protective efficacy
of the 11 Pn-PD vaccine and both 7-valent vaccines previously
tested in Finland (Eskola et al N Engi J Med 2001; 344: 403-409 and
Kilpi et al Clin Infect Dis 2003 37:1155-64) against any episode of
AOM and AOM caused by different pneumococcal serotypes, H.
influenzae, NTHi and M. catarrhalis. Statistically significant and
clinically relevant reduction by 33.6% of the overall AOM disease
burden was achieved with 11 Pn-PD, irrespective of the etiology
(table 3). The overall efficacy against AOM episodes due to any of
the 11 pneumococcal serotypes contained in the 11 PN-PD vaccine was
57.6% (table 3).
[0230] Another important finding in the current study is the 35.6%
protection provided by the 11 Pn-PD vaccine against AOM caused by
H. influenzae (and specifically 35.3% protection provided by NTHi).
This finding is of major clinical significance, given the increased
importance of H. influenzae as a major cause of AOM in the
pneumococcal conjugate vaccine era. In line with the protection
provided against AOM, the 11 Pn-PD vaccine also reduced
nasopharyngeal carriage of H. influenzae following the booster dose
in the second year of life. These findings are in contrast with
previous observations in Finland where, for both 7-valent
pneumococcal conjugate vaccines, an increase in AOM episodes due to
H. influenzee was observed, (Eskola et al and Kilpi et al) as
evidence of etiological replacement.
[0231] A clear correlation between protection against AOM episodes
due to Hi and antibody levels against the carrier Protein D could
not be established, as post-primary anti-PD IgG antibody
concentrations in 11 Pn-PD vaccinees, that remained Hi AOM
episode-free, were essentially the same as post-primary anti-PD IgG
antibody levels measured in 11 Pn-PD vaccinees that developed at
least one Hi AOM episode during the efficacy follow-up period.
However, although no correlation could be established between the
biological impact of the vaccine and the post-primary IgG anti-PD
immunogenicity, it is reasonable to assume that the PD carrier
protein, which is highly conserved among H. influenzae strains, has
contributed to a large extent in the induction of the protection
against Hi.
[0232] The effect on AOM disease was accompanied by an effect on
nasopharyngeal carriage that was of similar magnitude for vaccine
serotype pneumococci and H. influenzae (FIG. 1), This reduction of
the nasopharyngeal carriage of H. influenzae in the PD-conjugate
vaccinees supports the hypothesis of a direct protective effect of
the PD-conjugate vaccine against H. influenzae, even if the
protective efficacy could not be correlated to the anti-PD IgG
immune responses as measured by ELISA.
[0233] In a following experiment a chinchilla otitis media model
was used with serum pools from infants immunised with the 11 valent
formulation of this example or with the 10 valent vaccine of
Example 2 (see also Table 1 and 2 and comments thereunder). Both
pools induce a significant reduction of the percentage of animals
with otitis media versus the pre-immune serum pool. There is no
significant difference beteen the 10 and 11 valent immune pools.
This demonstrates that both vaccines have a similar potential to
induce protection against otitis media caused by non typeable H.
influenzae in this model.
TABLE-US-00004 TABLE 3 11Pn-PD Prevnar in FinOM.sup.(Eskola et al)
7v-OMP in FinOM.sup.(Kilip et. al.) n VE n VE n VE Type of AOM
11Pn- 95% Cl 7v- 95% Cl 7v- 95% Cl episode PD Control % LL UL CRM
Control % LL UL OMP Control % LL UL N 2455 2452 786 794 805 794 Any
AOM 333 499 33.6 20.8 44.3 1251 1345 6 -4 16 1364 1345 -1 -12 10
Any AOM with MEF 322 474 32.4 19.0 43.6 1177 1267 7 -5 17 1279 1267
0 -12 10 Culture confirmed 92 189 51.5 36.8 62.9 271 414 34 21 45
314 414 25 11 37 pneumococcus Vaccine 60 141 57.6 41.4 69.3 107 250
57 44 67 110 250 56 44 66 pneumococcal serotypes* Other bacterial
pathogens H. influenzae 44 68 35.6 3.8 57.0 315 287 -11 -34 8 315
287 -9 -32 10 Non-typeable 41 63 35.3 1.8 57.4 NP NP NP NP NP NP NP
NC NP NP H. influenzae (NTHi) M. catarrhalls 31 34 9.4 -52.5 46.1
379 381 -1 -19 15 444 381 -16 -36 2 NP = Not published; N = number
of subjects in ATP efficacy cohort; n = number of episodes *Vaccine
pneumococcal serotypes for 11Pn-PD = 11 serotypes, for Prevnar and
7v-OMP = 7 serotypes MEF = Middle ear fluid
Example 4
Selection of Carrier Protein for Serotype 19F
ELISA Assay Used
[0234] The 22F inhibition ELISA method was essentially based on an
assay proposed in 2001 by Concepcion and Frasch and was reported by
Henckaerts et al., 2006, Clinical and Vaccine Immunology
13:356-360. Briefly, purified pneumococcal polysaccharides were
mixed with methylated human serum albumin and adsorbed onto Nunc
Maxisorp.TM. (Roskilde, D K) high binding microtitre plates
overnight at 4.degree. C. The plates were blocked with 10% fetal
bovine serum (FBS) in PBS for 1 hour at room temperature with
agitation. Serum samples were diluted in PBS containing 10% FBS, 10
.mu.g/mL cell-wall polysaccharide (SSI) and 2 .mu.g/mL of
pneumococcal polysaccharide of serotype 22F (ATCC), and further
diluted on the microtitre plates with the same buffer. An internal
reference calibrated against the standard serum 89-SF using the
serotype-specific IgG concentrations in 89-SF was treated in the
same way and included on every plate. After washing, the bound
antibodies were detected using peroxidase-conjugated anti-human IgG
monoclonal antibody (Stratech Scientific Ltd., Soham, UK) diluted
in 10% FBS (in PBS), and incubated for 1 hour at room temperature
with agitation. The color was developed using ready-to-use single
component tetramethylbenzidine peroxidase enzyme immunoassay
substrate kit (BioRad, Hercules, Calif., US) in the dark at room
temperature. The reaction was stopped with H2SO4 0.18 M, and the
optical density was read at 450 nm. Serotype-specific IgG
concentrations (in .mu.g/mL) in the samples were calculated by
referencing optical density points within defined limits to the
internal reference serum curve, which was modelized by a
4-parameter logistic log equation calculated with SoftMax Pro.TM.
(Molecular Devices, Sunnyvale, Calif.) software. The cut-off for
the ELISA was 0.05 .mu.g/mL IgG for all serotypes taking into
account the limit of detection and the limit of quantification.
Opsonophagocytosis Assay
[0235] At the WHO consultation meeting in June 2003, it was
recommended to use an OPA assay as set out in Romero-Steiner et al
Clin Diagn Lab Immunol 2003 10 (6): pp 1019-1024. This protocol was
used to test the OPA activity of the serotypes in the following
tests.
Preparation of Conjugates
[0236] In studies 11Pn-PD&Di-007 and 11Pn-PD&Di-007, three
11-valent vaccine formulations (Table 4) were included in which 3
.mu.g of the 19F polysaccharide was conjugated to diphtheria toxoid
(19F-DT) instead of lpg polysaccharide conjugated to protein D
(19F-PD). Conjugation parameters for the studies 11Pn-PD, 11
Pn-PD&Di-001 and 11 Pn-PD&Di-007 are disclosed in Tables 5,
6 and 7 respectively.
[0237] Anti-pneumococcal antibody responses and OPA activity
against serotype 19F one month following primary vaccination with
these 19F-DT formulations are shown in Table 8 and 9
respectively.
[0238] Table 10 shows 22F-ELISA antibody concentrations and
percentages of subjects reaching the 0.2 .mu.g/mL threshold before
and after 23-valent plain polysaccharide booster vaccination.
[0239] The opsonophagocytic activity was shown to be clearly
improved for antibodies induced with these 19F-DT formulations as
demonstrated by higher seropositivity rates (opsonophagocytic
titers .gtoreq.1:8) and OPA GMTs one month following primary
vaccination (Table 9). One month after 23-valent plain
polysaccharide booster vaccination, opsonophagocytic activity of
19F antibodies remained significantly better for children primed
with 19F-DT formulations (Table 11).
[0240] Table 12 presents immunogenicity data following a 11Pn-PD
booster dose in toddlers previously primed with 19F-DT or 19F-PD
conjugates compared to a 4.sup.th consecutive dose of Prevnar.RTM..
Given the breakthrough cases reported after the introduction of
Prevnar.RTM. in the US, the improved opsonophagocytic activity
against serotype 19F when conjugated to the DT carrier protein may
be an advantage for the candidate vaccine.
[0241] Table 13 provides ELISA and OPA data for the 19F-DT
conjugate with respect to the cross-reactive serotype 19A. It was
found that 19F-DT induces low but significant OPA activity against
19A.
TABLE-US-00005 TABLE 4 Pneumococcal conjugate vaccine formulations
used in clinical studies. Pneumococcal serotype .mu.g/carrier
protein Al.sup.3+ Formulation 1 3 4 5 6B 7F 9V 14 18C 19F 23F mg
11Pn-PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD
<0.8 19F-DT Form 1 3/PD 3/PD 3/PD 3/PD 10/DT 3/PD 3/PD 3/PD 3/PD
3/DT 5/DT .ltoreq.0.35 19F-DT Form 2 3/PD 2/PD 2/PD 3/PD 5/DT 3/PD
2/PD 2/PD 2/PD 3/DT 5/DT .ltoreq.0.35 19F-DT Form 3 3/PD 3/PD 3/PD
3/PD 3/PD 3/PD 3/PD 3/PD 3/PD 3/DT 3/PD =0.5
TABLE-US-00006 TABLE 5 Specific activation/coupling/quenching
conditions of PS S. pneumoniae-Protein D/TT/DT conjugates Serotype
1 3 4 5 6B 7F Native .mu.fluid Native Native Native Native PS 1.5 2
2.0 7.5 5.5 3.0 conc.(mg/ml) PS NaCl NaCl 2M WFI WFI NaCl 2M NaCl
2M dissolution 150 mM PD 5.0 5.0 5.0 5.0 5.0 5.0 conc.(mg/ml)
Initial PS/PD 1/0.7 1/1 1/1 1/1 1/1 1/1 Ratio (w/w) CDAP conc. 0.75
0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q
9.0/9.0/9.0 9.5/9.5/9.0 8.8/8.8/9.0 9.0/9.0/9.0 9.5/9.5/9.0
9.0/9.0/9.0 Coupling time 60 mins 60 mins 45 mins 40 mins 60 mins
60 mins Serotype 9V 14 18C 19F 23F Native Native Native Native
Native PS 1.75 2.5 1.75 4.0 2.5 conc.(mg/ml) PS dissolution NaCl 2M
NaCl 2M WFI NaCl 2M NaCl 2M PD 5.0 5.0 5.0 5.0 5.0 conc.(mg/ml)
Initial PS/PD 1/0.75 1/0.75 1/1.2 1/1 1/1 Ratio (w/w) CDAP conc.
0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q
8.5/8.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.0
Coupling time 60 mins 60 mins 45 mins 30 mins 60 mins
TABLE-US-00007 TABLE 6 Specific activation/coupling/quenching
conditions of PS S. pneumoniae-Protein D/DT conjugates for the 11
Pn-PD&Di-007 study Serotype 1 3 4 5 6B 7F .mu.fluid .mu.fluid
.mu.fluid .mu.fluid .mu.fluid Native PS 4 2.0 2.5 7.5 10 3.0
conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2M
NaCl 2M PD 10.0 5.0 5.0 5.0 20 (DT) 5.0 conc.(mg/ml) NaCl 2M NaCl
2M Initial PD/PS 1.2/1 1/1 1/1 1/1 1.5/1 1/1 Ratio (w/w) CDAP conc.
1.50 0.75 1.5 2 1.5 0.75 (mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q
9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9/9/9
Coupling time 60 mins 60 mins 60 mins 60 mins 60 mins 60 mins
Serotype 9V 14 18C 19F 23F Native Native .mu.fluid .mu.fluid
.mu.fluid PS 1.75 2.5 5.0 9.0 10 conc.(mg/ml) PS dissolution NaCl
2M NaCl 2M NaCl 2M NaCl 2M NaCl 2M Carrier protein 5.0 5.0 5.0 20
(DT) 10 (DT) conc.(mg/ml) Initial carrier 0.75/1 0.75/1 1.2/1 1.5/1
1.5/1 protein/PS Ratio (w/w) CDAP conc. 0.75 0.75 1.5 1.5 0.75
(mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q 8.5/8.5/9.0 9.0/9.0/9.0
9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 Coupling time 60 mins 60 mins
30 mins 60 mins 60 mins
TABLE-US-00008 TABLE 7 Specific activation/coupling/quenching
conditions of PS S. pneumoniae-Protein D/DT conjugates for the 11
Pn-PD&Di-007 study Serotype 1 3 4 5 6B 7F Native .mu.fluid
Native Native Native .mu.fluid PS 1.5 2.0 2 7.5 5.5 5.0
conc.(mg/ml) PS dissolution NaCl NaCl 2M WFI WFI NaCl 2M NaCl 2M
150 mM PD 5.0 5.0 5.0 5.0 5 10 conc.(mg/ml) Initial PD/PS 0.7/1 1/1
1 1/1 1/1 1.2/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75
0.75 (mg/mg PS) pH.sub.a = pH.sub.c = pH.sub.q 9.0/9.0/9.0
9.5/9.5/9.0 8.8/8.8/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5./9.5/9 Coupling
time 60 mins 60 mins 45 mins 40 mins 60 mins 60 mins Serotype 9V 14
18C 19F 19F 23F .mu.fluid .mu.fluid Native .mu.fluid .mu.fluid
.mu.fluid PS 5.0 5.0 1.75 9.0 10.0 9.5 conc.(mg/ml) PS dissolution
NaCl 2M NaCl 2M WFI NaCl 2M NaCl 2M NaCl 2M Carrier protein 10 10.0
5.0 20 (DT) 5.0 (PD) 10 conc.(mg/ml) Initial carrier 1.2/1 1.2/1
1.2/1 1.5/1 1.2/1 1/1 protein/PS Ratio (w/w) CDAP conc. 0.5 0.75
0.75 1.5 0.75 0.75 (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 9.0/9.0/9.0
9.5/9.5/9.0 Coupling time 60 mins 60 mins 45 mins 120 mins 120 mins
60 mins
TABLE-US-00009 TABLE 8 Percentage of subjects with 19F antibody
concentration .gtoreq. 0.20 .mu.g/mL and 19F antibody Geometric
mean antibody concentrations (GMCs with 95% Cl; .mu.g/mL) one month
following 1 .mu.g 19F-PD, 3 .mu.g 19F-DT or Prevnar (2 .mu.g
19F-CRM) primary vaccination (Total cohort) 11Pn-PD&Di-001
(22F-ELISA) 11Pn-PD&Di-007 (22F-ELISA) % .gtoreq. 0.20 .mu.g/mL
GMC (.mu.g/mL) % .gtoreq. 0.20 .mu.g/mL GMC (.mu.g/mL) Group N (95%
Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 152 98.7 (95.3-99.8) 1.93
(1.67-2.22) 50 100 (92.9-100) 2.78 (231-3.36) 19F-DT Form
1.sup..GAMMA. 146 99.3 (96.2-100) 2.88 (2.45-3.38) -- -- -- 19F-DT
Form 2.sup..GAMMA. 150 96.0 (91.5-98.5) 2.43 (2.01-2.94) -- -- --
19F-DT Form 3.sup..GAMMA. -- -- -- 50 96.0 (86.3-99.5) 3.70
(2.58-5.30) Prevnar 148 98.6 (95.2-99.8) 2.98 (2.60-3.41) 41 97.6
(87.1-99.9) 2.91 (2.15-3.94) .sup..GAMMA.The composition of the
different formulations is provided in table 4.
TABLE-US-00010 TABLE 9 Percentage of subjects with 19F OPA titer
.gtoreq. 1:8 and 19F OPA GMTs one month following primary
vaccination with 1 .mu.g 19F-PD, 3 .mu.g 19F-DT or Prevnar (2 .mu.g
19F-CRM) (Total cohort) 11Pn-PD&Di-001 11Pn-PD&Di-007
.gtoreq.1:8 GMT .gtoreq.1:8 GMT Group N (95% Cl) (95% Cl) N (95%
Cl) (95% Cl) 11Pn-PD 136 84.6 (77.4-90.2) 77.8 (58.1-104.4) 46 95.7
(85.2-99.5) 167.8 (118.1-238.6) 19F-DT Form 1.sup..GAMMA. 137 95.6
(90.7-98.4) 263.2 (209.4-330.7) -- -- -- 19F-DT Form 2.sup..GAMMA.
139 92.1 (86.3-96.0) 218.9 (166.5-287.9) -- -- -- 19F-DT Form
3.sup..GAMMA. -- -- -- 49 91.8 (80.4-97.7) 403.1 (225.7-719.9)
Prevnar 131 86.3 (79.2-91.6) 82.6 (61.1-111.6) 38 81.6 (65.7-92.3)
65.0 (37.7-112.2) .sup..GAMMA.The composition of the different
formulations is provided in Table 4.
TABLE-US-00011 TABLE 10 Percentage of subjects with 19F antibody
concentration .gtoreq. 0.20 .mu.g/mL and 19F antibody GMCs
(.mu.g/mL) prior to and one month following 23-valent plain
polysaccharide booster in children primed with 1 .mu.g 19F-PD, 3
.mu.g 19F-DT or Prevnar (2 .mu.g 19F-CRM) (Total cohort)
11Pn-PD&Di-002 (22F ELISA) Prior to booster vaccination One
month post 23-valent PS booster % .gtoreq. 0.20 .mu.g/mL GMC
(mg/ml) % .gtoreq. 0.20 .mu.g/mL GMC (mg/ml) Primary group N (95%
Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 70 77.1 (65.6-86.3) 0.67
(0.45-0.98) 67 94.0 (85.4-98.3) 11.50 (7.76-17.03) 19F-DT Form
1.sup..GAMMA. 68 91.2 (81.8-96.7) 0.71 (0.54-0.94) 69 98.6
(92.2-100) 14.50 (10.47-20.07) 19F-DT Form 2.sup..GAMMA. 74 81.1
(70.3-89.3) 0.59 (0.43-0.80) 72 95.8 (88.3-99.1) 9.90 (6.74-14.54)
Prevnar 65 64.6 (51.8-76.1) 0.40 (0.27-0.60) 67 100 (94.6-100) 9.40
(6.95-12.71) .sup..GAMMA.The composition of the different
formulations is provided in Table 4
TABLE-US-00012 TABLE 11 Percentage of subjects with 19F OPA titer
.gtoreq. 1:8 and 19F OPA GMTs prior to and one month following
23-valent plain polysaccharide booster in children primed with 1
.mu.g 19F-PD, 3 .mu.g 19F-DT or Prevnar (2 .mu.g 19F-CRM) (Total
cohort) 11Pn-PD&Di-002 Prior to booster vaccination One month
post 23-valent PS booster % .gtoreq. 1:8 GMT % .gtoreq. 1:8 GMT
Primary group N (95% Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 29
27.6 (12.7-47.2) 10.9 (5.0-23.7) 28 82.1 (63.1-93.9) 408.0
(157.3-1058.3) 19F-DT Form 1.sup..GAMMA. 19 47.4 (24.4-71.1) 18.1
(7.2-45.7) 18 94.4 (72.7-99.9) 1063.8 (386.6-2927.5) 19F-DT Form
2.sup..GAMMA. 27 33.3 (16.5-54.0) 8.5 (4.7-15.3) 28 100 (87.7-100)
957.6 (552.8-1659.0) Prevnar 24 12.5 (2.7-32.4) 8.1 (3.4-19.6) 23
82.6 (61.2-95.0) 380.9 (133.2-1089.5) .sup..GAMMA.The composition
of the different formulations is provided in Table 4.
TABLE-US-00013 TABLE 12 Percentage of subjects with antibody
concentrations .gtoreq. 0.2 .mu.g/mL, OPA .gtoreq. 1:8 and
GMCs/GMTs against 19F pneumococci one month following 11Pn-PD or
Prevnar booster in children primed with 1 .mu.g 19F-PD, 3 .mu.g
19F-DT or Prevnar (2 .mu.g 19F-CRM) (Total cohort)
11Pn-PD&Di-002 22F-ELISA assay OPA assay % .gtoreq. 0.20
.mu.g/mL GMC (.mu.g/ml) % .gtoreq. 1:8 GMT Primary group N (95% Cl)
(95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 70 100 (94.9-100) 4.52
(3.7-5.5) 21 100 (83.9-100) 255.6 (135.5-481.9) 19F-DT Form
1.sup..GAMMA. 66 98.5 (91.8-100) 3.45 (2.8-4.3) 23 95.7 (78.1-99.9)
374.0 (192.6-726.2) 19F-DT Form 2.sup..GAMMA. 70 98.6 (92.3-100)
3.80 (2.9-4.9) 29 96.6 (82.2-99.9) 249.1 (144.7-428.7) Prevnar 69
97.1 (89.9-99.6) 2.56 (2.0-3.3) 31 96.8 (83.3-99.9) 528.7
(319.4-875.2) .sup..GAMMA.The composition of the different
formulations is provided in Table 4.
TABLE-US-00014 TABLE 13 Percentage of subjects with antibody
concentrations .gtoreq. 0.2 .mu.g/mL, OPA .gtoreq. 1:8 and
GMCs/GMTs against 19A pneumococci one month following primary
vaccination with 1 .mu.g 19F-PD, 3 .mu.g 19F-DT or Prevnar (2 .mu.g
19F-CRM) (Total cohort) 11Pn-PD&Di-001 22F-ELISA assay OPA
assay % .gtoreq. 0.20 .mu.g/mL GMC (.mu.g/mL) % .gtoreq. 1:8 GMT
Group N (95% Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 45 28.9
(16.4-44.3) 0.09 (0.07-0.11) 52 7.7 (2.1-18.5) 5.2 (4.0-6.8) 19F-DT
Form 2.sup..GAMMA. 51 29.4 (17.5-43.8) 0.11 (0.08-0.16) 59 27.1
(16.4-40.3) 12.4 (7.6-20.3) Prevnar 55 18.2 (9.1-30.9) 0.10
(0.08-0.12) 61 3.3 (0.4-11.3) 4.6 (3.8-5.6) .sup..GAMMA.The
composition of the different formulations is provided in Table
4
Example 5
Adjuvant Experiments in Preclinical Models: Impact on the
Immunogenicity of Pneumococcal 11-Valent Polysaccharide Conjugates
in Elderly Rhesus Monkeys
[0242] To optimize the response elicited to conjugate pneumococcal
vaccines in the elderly population, GSK formulated an 11-valent
polysaccharide (PS) conjugate vaccine with a novel adjuvant
Adjuvant C--see below.
[0243] Groups of 5 elderly Rhesus monkeys (14 to 28 years-old) were
immunized intramuscularly (IM) at days 0 and 28 with 500 .mu.l of
either 11-valent PS conjugates adsorbed onto 315 .mu.g of AIPO4 or
11-valent PS conjugates admixed with Adjuvant C.
[0244] In both vaccine formulations, the 11-valent PS conjugates
were each composed of the following conjugates PS1-PD, PS3-PD,
PS4-PD, PS5-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-PD, PS19F-PD,
PS23F-DT and PS6B-DT. The vaccine used was 1/5 dose of the human
dose of the vaccine (5 .mu.g of each saccharide per human dose
except for 6B [10 .mu.g]) conjugated according to Table 6
conditions (Example 4), except 19F was made according to the
following CDAP process conditions: sized saccharide at 9 mg/ml, PD
at 5 mg/ml, an initial PD/PS ratio of 1.2/1, a CDAP concentration
of 0.75 mg/mg PS, pHa=pHc=pHq 9.0/9.0/9.0 and a coupling time of 60
min.
[0245] Anti-PS ELISA IgG levels and opsono-phagocytosis titres were
dosed in sera collected at day 42. Anti-PS3 memory B cell
frequencies were measured by Elispot from peripheral blood cells
collected at day 42.
[0246] According to the results shown here below, Adjuvant C
significantly improved the immunogenicity of 11-valent PS
conjugates versus conjugates with AIPO4 in elderly monkeys. The
novel adjuvant enhanced the IgG responses to PS (FIG. 1) and the
opsono-phagocytosis antibody titres (Table 14). There was also
supportive evidence that the frequency of PS3-specific memory B
cells is increased by the use of Adjuvant C (FIG. 2).
TABLE-US-00015 TABLE 14 Conjugate immunogenicity in elderly Rhesus
monkeys (post-II opsonophagocytosis titres) PS1 PS3 PS4 PS5 PS6B
PS7F PS9V PS14 PS18C PS19F PS23F 11-valent Pre-immune <8 5 <8
5 <8 16 <8 <8 <8 <8 <8 AlPO4 day 14 post II 8 181
64 49 64 4096 42 37 169 64 <64 11 valent Pre-immune 5 9 <8 5
8 37 <8 <8 <8 <8 <8 Adj-C day 14 post II 776 1351
891 676 6208 16384 111 161 7132 2048 <64
[0247] B Cell Elispot
[0248] The principle of the assay relies on the fact that memory B
cells mature into plasma cells in vitro following cultivation with
CpG for 5 days. In vitro generated antigen-specific plasma cells
can be easily detected and therefore be enumerated using the B-cell
elispot assay. The number of specific plasma cells mirrors the
frequency of memory B cells at the onset of the culture.
[0249] Briefly, in vitro generated plasma cells are incubated in
culture plates coated with antigen. Antigen-specific plasma cells
form antibody/antigen spots, which are detected by conventional
immuno-enzymatic procedure and enumerated as memory B cells. In the
present study, Polysaccharides have been used to coat culture
plates in order to enumerate respective memory B cells. Results are
expressed as a frequency of PS specific memory B cells within a
million of memory B cells.
[0250] The study shows that Adjuvant C may be able to alleviate the
known problem of PS3 boostability (see 5th International Symposium
on Pneumococci and Pneumococcal Diseases, Apr. 2-6 2006, Alice
Springs, Central Australia.
[0251] Specificities of immune responses against a serotype 3
pneumococcal conjugate. Schuerman L, Prymula R, Poolman J. Abstract
book p 245, PO10.06).
Example 6
Effectiveness of Detoxified Pneumolysin (DPly) as a Protein Carrier
to Enhance the Immunogenicity of PS 19F in Young Balb/c Mice
[0252] Groups of 40 female Balb/c mice (4-weeks old) were immunized
IM at days 0, 14 and 28 with 50 .mu.l of either 4-valent plain PS
or 4-valent dPly-conjugated PS, both admixed with Adjuvant C.
[0253] Both vaccine formulations were composed of 0.1 .mu.g
(quantity of saccharide) of each of the following PS: PS8, PS12F,
PS19F and PS22F.
[0254] Anti-PS ELISA IgG levels were dosed in sera collected at day
42.
[0255] The anti-PS19F response, shown as an example in FIG. 3, was
strongly enhanced in mice given 4-valent dPly conjugates compared
to mice immunized with the plain PS. The same improvement was
observed for the anti-PS8, 12F and 22F IgG responses (data not
shown).
Example 7
Effectiveness of Pneumococcal Histidine Triad Protein D (PhtD) as a
Protein Carrier to Enhance the Immunogenicity of Ps 22F in Young
Balb/C Mice
[0256] Groups of 40 female Balb/c mice (4-weeks old) were immunized
IM at days 0, 14 and 28 with 50 .mu.l of either 4-valent plain PS
or 4-valent PhtD-conjugated PS, both admixed with Adjuvant C.
[0257] Both vaccine formulations were composed of 0.1 .mu.g
(quantity of saccharide) of each of the following PS: PS8, PS12F,
PS19F and PS22F.
[0258] Anti-PS ELISA IgG levels were dosed in sera collected at day
42.
[0259] The anti-PS22F response, shown as an example in FIG. 4, was
strongly enhanced in mice given 4-valent PhtD conjugates compared
to mice immunized with the plain PS. The same improvement was
observed for the anti-PS8, 12F and 19F IgG responses (data not
shown).
Example 8
Immunogenicity in Elderly C57BI Mice of 13-Valent PS Conjugates
Containing 19A-dPly and 22F-PhtD
[0260] Groups of 30 old C57Bl mice (>69-weeks old) were
immunized IM at days 0, 14 and 28 with 50 .mu.l of either 11-valent
PS conjugates or 13-valent PS conjugates, both admixed with
Adjuvant C (see below).
[0261] The 11-valent vaccine formulation was composed of 0.1 .mu.g
saccharide of each of the following conjugates: PS1-PD, PS3-PD,
PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-TT,
PS19F-DT and PS23F-PD (see Table 1 and comment on 11 valent vaccine
discussed under Table 2). The 13-valent vaccine formulation
contained in addition 0.1 .mu.g of PS19A-dPly and PS22F-PhtD
conjugates (see Table 1 and comment on 13 valent vaccine discussed
under Table 2 [using directly-conjugated 22F]). In group 2 and 4
the pneumolysin carrier was detoxified with GMBS treatment, in
group 3 and 5 it was done with formaldehyde. In groups 2 and 3 PhtD
was used to conjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion
(the construct VP147 from WO 03/054007) was used. In group 6 19A
was conjugated to diphtheria toxoid and 22F to protein D.
[0262] Anti-PS19A and 22F ELISA IgG levels were dosed in individual
sera collected at day 42. The ELISA IgG response generated to the
other PS was measured in pooled sera. 19A-dPly and 22F-PhtD
administered within the 13-valent conjugate vaccine formulation
were shown immunogenic in old C57BI mice (Table 15). The immune
response induced against the other PS was not negatively impacted
in mice given the 13-valent formulation compared to those immunized
with the 11-valent formulation.
TABLE-US-00016 TABLE 15 PS immunogenicity in old C57Bl mice
(post-III IgG levels) Old C57 Black mice GROUP 2 GROUP 3 GROUP 4
GROUP 5 11V 11V 11V 11V GROUP 6 19A-dPly 19A-dPly 19A-dPly 19A-dPly
11V GROUP 1 gmbs formol gmbs formol 19A-DT 11V 22F-PhtD 22F-PhtD
22F-PhtD-E 22F-PhtD-E 22F-PD 0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l
0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l 0.1
.mu.g/50 .mu.l ELISA Adj C Adj C Adj C Adj C Adj C Adj C 1 average
19.30 20.20 24.40 12.80 12.10 13.60 Pool 3 average 6.32 4.84 5.21
6.74 2.38 2.54 Pool 4 average 60.9 67.1 51.4 47.4 45.5 41.1 Pool 5
average 1.34 3.81 3.06 2.75 1.26 1.23 Pool 6B average 4.41 4.12
5.88 1.58 2.31 5.64 Pool 7F average 0.83 0.81 1.65 1.98 0.89 0.99
Pool 9V average 13.8 23.7 20.0 13.1 15.5 9.6 Pool 14 average 25.73
42.96 34.12 32.53 23.97 15.60 Pool 18C average 13.4 20.1 11.9 9.1
8.3 8.4 Pool 19F average 57.5 90.0 63.8 36.5 47.0 69.1 Pool 23F
average NR NR NR NR NR NR Pool 19A GMC 0.06 0.09 0.25 0.08 0.23
0.19 IC 0.04-0.1 0.05-0.14 0.15-0.41 0.06-0.12 0.14-0.38 0.09-0.3 %
sero 33% 47% 83% 53% 80% 73% 22F GMC NR 5.81 3.76 0.54 0.85 2.02 IC
3.2-106 1.8-7.9 0.3-1.1 0.4-1.7 1.2-3.4 % sero 0% 97% 90% 77% 87%
97%
Example 9
Immunogenicity in Young Balb/c Mice of 13-Valent PS Conjugates
Containing 19A-dPly and 22F-PhtD
[0263] Groups of 30 young Balb/c mice (4-weeks old) were immunized
IM at days 0, 14 and 28 with 50 .mu.l of either 11-valent PS
conjugates or 13-valent PS conjugates, both admixed with Adjuvant C
(see below).
[0264] The 11-valent vaccine formulation was composed of 0.1 .mu.g
saccharide of each of the following conjugates: PS1-PD, PS3-PD,
PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-Tf,
PS19F-DT and PS23F-PD (see Table 1 and comment on 11 valent vaccine
discussed under Table 2). The 13-valent vaccine formulation
contained in addition 0.1 .mu.g of PS19A-dPly and PS22F-PhtD
conjugates (see Table 1 and comment on 13 valent vaccine discussed
under Table 2 [using directly-conjugated 22F]). In group 2 and 4
the pneumolysin carrier was detoxified with GMBS treatment, in
group 3 and 5 it was done with formaldehyde. In groups 2 and 3 PhtD
was used to conjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion
(the construct VP147 from WO 03/054007) was used. In group 6 19A
was conjugated to diphtheria toxoid and 22F to protein D.
[0265] Anti-PS19A and 22F ELISA IgG levels were dosed in individual
sera collected at day 42. The ELISA IgG response generated to the
other PS was measured in pooled sera. 19A-dPly and 22F-PhtD
administered within the 13-valent conjugate vaccine formulation
were shown immunogenic in young Balb/c mice (Table 16). The immune
response induced against the other PS was not negatively impacted
in mice given the 13-valent formulation compared to those immunized
with the 11-valent formulation.
TABLE-US-00017 TABLE 16 PS immunogenicity in young Balb/c mice
(post-Ill IgG levels) BalbC mice GROUP 2 GROUP 3 GROUP 4 GROUP 5
11V 11V 11V 11V GROUP 6 19A-dPly 19A-dPly 19A-dPly 19A-dPly 11V
GROUP 1 gmbs formol gmbs formol 19A-DT 11V 22F-PhtD 22F-PhtD
22F-PhtD-E 22F-PhtD-E 22F-PD 0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l
0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l 0.1
.mu.g/50 .mu.l ELISA Adj C Adj C Adj C Adj C Adj C Adj C 1 average
131.70 101.20 83.00 82.40 67.90 85.50 Pool 3 average 21.85 10.38
12.53 8.83 8.73 14.98 Pool 4 average 147.4 127.0 104.4 95.0 113.6
114.2 Pool 5 average 21.38 20.29 18.26 18.95 18.02 23.04 Pool 6B
average 1.97 4.76 3.72 2.35 1.43 1.05 Pool 7F average 7.69 4.58
4.77 4.24 3.92 3.94 Pool 9V average 30.1 30.7 26.5 21.4 23.4 28.3
Pool 14 average 28.78 27.67 26.23 21.54 24.34 13.73 Pool 18C
average 53.4 52.37 46.5 57.8 47.8 75.8 Pool 19F average 186.6 157.7
169.3 178.9 181.9 223.2 Pool 23F average 4.98 3.9 5.11 0.57 3.13
4.57 Pool 19A GMC 0.4 32.8 25.1 21.6 18.9 23.5 IC 0.2-0.6 26.4-40.7
20.6-30.6 17.5-26.7 15.1-23.5 19.5-28.5 % sero 93% 100% 100% 100%
100% 100% 22F GMC NR 3.99 3.76 6.27 8.70 18.76 IC 0% 1.9-8.42 1.8-8
3.8-10.4 5.4-13.9 15.2-23.1 % sero 93% 100% 100% 100% 100%
Example 10
Immunogenicity in Guinea Pigs of 13-Valent PS Conjugates Containing
19A-dPly and 22F-PhtD
[0266] Groups of 20 young Guinea Pigs (Hartley Strain; 5 weeks old)
were immunized IM at days 0, 14 and 28 with 125 .mu.l of either
11-valent PS conjugates or 13-valent PS conjugates, both admixed
with Adjuvant C (see below).
[0267] The 11-valent vaccine formulation was composed of 0.25 .mu.g
saccharide of each of the following conjugates: PS1-PD, PS3-PD,
PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-TT,
PS19F-DT and PS23F-PD (see Table 1 and comment on 11 valent vaccine
discussed under Table 2). The 13-valent vaccine formulation
contained in addition 0.1 .mu.g of PS19A-dPly and PS22F-PhtD
conjugates (see Table 1 and comment on 13 valent vaccine discussed
under Table 2 [using directly-conjugated 22F]). In group 2 and 4
the pneumolysin carrier was detoxified with GMBS treatment, in
group 3 and 5 it was done with formaldehyde. In groups 2 and 3 PhtD
was used to conjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion
(the construct VP147 from WO 03/054007) was used. In group 6 19A
was conjugated to diphtheria toxoid and 22F to protein D.
[0268] Anti-PS19A and 22F ELISA IgG levels were dosed in individual
sera collected at day 42. The ELISA IgG response generated to the
other PS was measured in pooled sera.
TABLE-US-00018 TABLE 17 PS immunogenicity in young Balb/c mice
(post-III IgG levels) Guinea pigs GROUP 2 GROUP 3 GROUP 4 GROUP 5
11V 11V 11V 11V GROUP 6 19A-dPly 19A-dPly 19A-dPly 19A-dPly 11V
GROUP 1 gmbs formol gmbs formol 19A-DT 11V 22F-PhtD 22F-PhtD
22F-PhtD-E 22F-PhtD-E 22F-PD 0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l
0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l 0.1 .mu.g/50 .mu.l 0.1
.mu.g/50 .mu.l ELISA Adj C Adj C Adj C Adj C Adj C Adj C 1 average
78.00 77.21 76.15 68.77 68.59 81.04 Pool 3 average 7.75 9.31 12.73
7.94 4.75 9.59 Pool 4 average 130.7 94.4 132.6 166.8 85.0 101.3
Pool 5 average 109.10 117.10 110.70 158.40 74.10 100.40 Pool 6B
average 3.14 4.26 14.4 7.63 6.3 7.52 Pool 7F average 154.2 216.0
240.0 181.0 142.0 179.1 Pool 9V average 90.69 105.45 98.20 93.45
54.12 73.05 Pool 14 average 71.19 77.18 46.53 59.67 38.47 53.69
Pool 18C average 109.4 122.3 137.1 79.9 73.7 83.1 Pool 19F average
73.9 102.5 112.2 75.5 62.3 72.1 Pool 23F average 19.19 30.74 29.44
31.52 19.13 24.94 Pool 19A GMC 0.4 25.58 41.49 14.25 27.49 6.74 IC
0.24-0.68 12-54.5 24.4-70.5 5.9-34.6 16.6-45.4 4-11.3 % sero 75%
100% 100% 100% 100% 100% 22F GMC 0.12 2.51 3.67 45.74 30.68 96.38
IC 0.09-0.16 0.94-6.73 1.59-8.42 29.3-71.4 17-53.3 73.5-126.4 %
sero 10% 95% 95% 100% 100% 100%
Example 11
Formulations being Made and Tested
[0269] a) The following formulations are made (using the 13 valent
vaccine from table 1 and serotype 3 from table 5--see comment on 14
valent vaccine discussed under Table 2 [using directly-conjugated
22F or through an ADH linker]). The saccharides are formulated with
aluminium phosphate and 3D-MPL as shown below.
TABLE-US-00019 14V 25 .mu.g MPL 14V 10 .mu.g MPL Sum of BAC
Aluminium content -> FF Sum of BAC Aluminium content -> FF
Per Dose: Per Dose: ratio PS/Al ratio PS/Al PS carrier .mu.g PS
.mu.g MPL l/x .mu.g Al PS carrier .mu.g PS .mu.g MPL l/x .mu.g Al 1
PD 1 10 10 1 PD 1 10 10 3 PD 1 10 10 3 PD 1 10 10 4 PD 3 10 30 4 PD
3 10 30 5 PD 1 10 10 5 PD 1 10 10 6A PD 1 10 10 6A PD 1 10 10 6B PD
1 10 10 6B PD 1 10 10 7F PD 1 10 10 7F PD 1 10 10 9V PD 1 10 10 9V
PD 1 10 10 14 PD 1 10 10 14 PD 1 10 10 18C TT.sub.AH 3 15 45 18C
TT.sub.AH 3 15 45 19A dPly 3 10 30 19A dPly 3 10 30 19F DT 3 10 30
19F DT 3 10 30 22F PhtD 3 10 30 22F PhtD 3 10 30 23F PD 1 10 10 23F
PD 1 10 10 BAC MPL 50/200 25 4 100 BAC MPL 50/200 10 4 40 FF
Aluminium content Sum = 355 FF Aluminium content Sum - 295
b) The same saccharide formulation is adjuvanted with each of the
following adjuvants:
[0270] In the table herebelow the concentration of the emulsion
components per 500 .mu.l dose is shown.
TABLE-US-00020 Adjuvant A1 Adjuvant A2 Adjuvant A3 250 .mu.l o/w
125 .mu.l o/w 50 .mu.l o/w Ingredients emulsion emulsion emulsion
alpha 11.88 mg 5.94 mg 2.38 mg Tocopherol Squalene 10.7 mg 5.35 mg
2.14 mg Tween 80 4.85 mg 2.43 mg 0.97 mg Adjuvant A4 Adjuvant A5
Adjuvant A6 Adjuvant A7 250 .mu.l o/w 250 .mu.l o/w 125 .mu.l o/w
50 .mu.l o/w Ingredients emulsion emulsion emulsion emulsion alpha
11.88 mg 11.88 mg 5.94 mg 2.38 mg Tocopherol Squalene 10.7 mg 10.7
mg 5.35 mg 2.14 mg Tween 80 4.85 mg 4.85 mg 2.43 mg 0.97 mg 3D-MPL
50 .mu.g 25 .mu.g 25 .mu.g 10 .mu.g
c) The saccharides are also formulated with two liposome based
adjuvants:
Composition of Adjuvant Bi
[0271] Qualitative Quantitative (per 0.5 mL dose)
Liposomes:
[0272] DOPC 1 mg
[0273] cholesterol 0.25 mg
3DMPL 50 .mu.g
QS21 50 .mu.g
KH.sub.2PO.sub.4 13.124 mg Buffer
Na.sub.2HPO.sub.4 1 0.290 mg Buffer
NaCl 2.922 mg
[0274] (100 mM)
WFI q.s. ad 0.5 ml Solvent
[0275] pH 6.1 1. Total PO.sub.4 concentration=50 mM
Composition of Adjuvant B2
[0276] Qualitative Quantitative (per 0.5 mL dose)
Liposomes:
[0277] DOPC 0.5 mg
[0278] cholesterol 0.125 mg
3DMPL 25 .mu.g
QS21 25 .mu.g
KH.sub.2PO.sub.4 1 3.124 mg Buffer
Na.sub.2HPO.sub.4 1 0.290 mg Buffer
NaCl 2.922 mg
[0279] (100 mM)
WFI q.s. ad 0.5 ml Solvent
[0280] pH 6.1
[0281] d) The saccharides are also formulated with Adjuvant C (see
above for other compositions where this adjuvant has been
used):
Qualitative Quantitative (per 0.5 mL dose) Oil in water emulsion:
50 .mu.l
[0282] squalene 2.136 mg
[0283] .alpha.-tocopherol 2.372 mg
[0284] Tween 80 0.97 mg
[0285] cholesterol 0.1 mg
3DMPL 50 .mu.g
QS21 50 .mu.g
KH.sub.2PO.sub.4 1 0.470 mg Buffer
Na.sub.2HPO.sub.4 1 0.219 mg Buffer
NaCl 4.003 mg
[0286] (137 mM)
KCl 0.101 mg
[0287] (2.7 mM)
WFI q.s. ad 0.5 ml Solvent
[0288] pH 6.8
Example 12
Impact of Conjugation Chemistry on 22F-PhtD Conjugate
Immunogenicity in Balb/c Mice
[0289] Groups of 30 female Balb/c mice were immunised by the
intramuscular (IM) route at days 0, 14 and 28 with 13-valent PS
formulations containing PS 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A,
19F, 22F and 23F (dose: 0.3 .mu.g saccharide/PS for P .delta. 4,
18C, 19A, 19F and 22F and 0.1 .mu.g saccharide/PS for the other
PS).
[0290] PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria
Toxoid, 19A to formol-detoxified Ply, 22F to PhtD and the other PS
to PD.
[0291] Two formulations, constituted of either 22F-PhtD prepared by
direct CDAP chemistry or 22F-AH-PhtD (ADH-derivitized PS), were
compared. See Example 2, Table 1 and comment under Table 2 for
characteristics of 13 valent vaccine made either with 22F directly
conjugated or via an ADH spacer. The vaccine formulations were
supplemented with adjuvant C.
[0292] Anti-PS22F ELISA IgG levels and opsono-phagocytosis titres
were measured in sera collected at day 42.
[0293] 22F-AH-PhtD was shown much more immunogenic than 22F-PhtD in
terms of both IgG levels (FIG. 5) and opsono-phagocytic titres
(FIG. 6).
Example 13
Impact of New Adjuvants on Immunogenicity of Streptoccoccus
Pneumoniae Capsule PS Conjuqates
[0294] Groups of 40 female Balb/c mice were immunised by the IM
route at days 0, 14 and 28 with 13-valent PS formulations
containing PS 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and
23F (dose: 0.3 .mu.g/PS for P .delta. 4, 18C, 19A, 19F and 22F and
0.1 .mu.g/PS for the other PS).
[0295] PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria
Toxoid, 19A to formol-detoxified Ply, 22F to PhtD and the other PS
to PD. See Example 2, Table 1 and comment under Table 2 for
characteristics of 13 valent vaccine made with 22F directly
conjugated.
[0296] Four formulations, supplemented with either AlPO.sub.4,
adjuvant Al, adjuvant A4 or adjuvant A5, were compared.
[0297] Anti-PS, Ply, PhtD and PD ELISA IgG levels were measured in
sera collected at day 42 and pooled per group. The following ratio
was calculated for each antigen:IgG level induced with the new
adjuvant tested/IgG level induced with AIPO.sub.4.
[0298] All the new adjuvants tested improved at least 2-fold the
immune responses to 13-valent conjugates compared to the classical
AIPO.sub.4 formulation (FIG. 7).
Example 14
Protective Efficacy of a PhtD/Detoxified Ply Combo in a
Pneumococcal Monkey Pneumonia Model
[0299] Groups of 6 Rhesus monkeys (3 to 8 years-old), selected as
those having the lowest pre-existing anti-19F antibody levels, were
immunized intramuscularly at days 0 and 28 with either 11-valent PS
conjugates (i.e. 1 .mu.g of PS 1, 3, 5, 6B, 7F, 9V, 14 and 23F, and
3 .mu.g of PS 4, 18C and 19F) or PhtD (10 .mu.g)+ formol-detoxified
Ply (10 .mu.g) or the adjuvant alone.
[0300] PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria
Toxoid and the other PS to PD. See Example 2, Table 1 and comment
under Table 2 for characteristics of 11 valent vaccine. All
formulations were supplemented with adjuvant C.
[0301] Type 19F pneumococci (5.10.sup.8 cfu) were inoculated in the
right lung at day 42. Colonies were counted in broncho-alveolar
lavages collected at days 1, 3 and 7 post-challenge. The results
were expressed as the number of animals per group either dead, lung
colonized or cleared at day 7 after challenge.
[0302] As shown in FIG. 8, a good protection close to statistical
significance (despite the low number of animals used) was obtained
with 11-valent conjugates and the PhtD+dPly combo (p<0.12,
Fisher Exact test) compared to the adjuvant alone group.
Example 15
Impact of Conjugation Chemistry on the Anti-PhtD Antibody Response
and the Protective Efficacy Against a Type 4 Challenge Induced by
22F-PhtD Conjugates
[0303] Groups of 20 female OF1 mice were immunised by the
intramuscular route at days 0 and 14 with 3 .mu.g of either
22F-PhtD (prepared by direct CDAP chemistry) or 22F-AH-PhtD
(ADH-derivitized PS), or the adjuvant alone. Both monovalent 22F
conjugates were made by the processes of Example 2 (see also Table
1 and Table 2). Each formulation was supplemented with adjuvant
C.
[0304] Anti-PhtD ELISA IgG levels were measured in sera collected
at day 27.
[0305] Mice were challenged intranasally with 5.10.sup.6 cfu of
type 4 pneumococci at day 28 (i.e. a pneumococcal serotype not
potentially covered by the PS present in the vaccine formulation
tested). The mortality induced was monitored until day 8
post-challenge.
[0306] 22F-AH-PhtD induced a significantly higher anti-PhtD IgG
response and better protection against type 4 challenge than
22F-PhtD.
* * * * *