U.S. patent application number 16/268287 was filed with the patent office on 2020-01-02 for immunogenic composition.
The applicant listed for this patent is GlaxoSmithKline Biologicals s.a.. Invention is credited to Ralph Leon Biemans, Dominique Boutriau, Carine Capiau, Philippe Denoel, Pierre Duvivier, Jan Poolman.
Application Number | 20200000911 16/268287 |
Document ID | / |
Family ID | 36716943 |
Filed Date | 2020-01-02 |
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United States Patent
Application |
20200000911 |
Kind Code |
A1 |
Biemans; Ralph Leon ; et
al. |
January 2, 2020 |
IMMUNOGENIC COMPOSITION
Abstract
The present application discloses an immunogenic composition
comprising at least 2 different N. meningitidis capsular
saccharides, wherein one or more is/are selected from a first group
consisting of MenA, MenC, MenY and MenW which is/are conjugated
through a linker to a carrier protein(s), and one or more different
saccharides is/are selected from a second group consisting of MenA,
MenC, MenY and MenW which is/are directly conjugated to a carrier
protein(s).
Inventors: |
Biemans; Ralph Leon;
(Rixensart, BE) ; Boutriau; Dominique; (Rixensart,
BE) ; Capiau; Carine; (Rixensart, BE) ;
Denoel; Philippe; (Rixensart, BE) ; Duvivier;
Pierre; (Rixensart, BE) ; Poolman; Jan;
(Rixensart, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlaxoSmithKline Biologicals s.a. |
Rixensart |
|
BE |
|
|
Family ID: |
36716943 |
Appl. No.: |
16/268287 |
Filed: |
February 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15265597 |
Sep 14, 2016 |
10245317 |
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16268287 |
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11917702 |
Jun 10, 2008 |
9486515 |
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PCT/EP2006/006269 |
Jun 23, 2006 |
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15265597 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/145 20130101;
A61K 39/05 20130101; A61K 2039/575 20130101; C12N 2760/16234
20130101; A61P 31/04 20180101; A61P 43/00 20180101; A61K 2039/545
20130101; A61P 11/04 20180101; A61K 2039/6037 20130101; A61K
2039/70 20130101; A61P 31/20 20180101; A61K 39/092 20130101; A61K
2039/62 20130101; A61K 2039/627 20130101; C12N 2730/10134 20130101;
A61K 2039/55 20130101; A61P 31/12 20180101; A61K 39/095 20130101;
A61K 39/12 20130101; C12N 7/00 20130101; A61K 39/0018 20130101;
A61P 37/04 20180101; A61K 39/099 20130101; A61K 39/102 20130101;
Y02A 50/30 20180101; A61P 11/14 20180101; A61K 39/08 20130101; A61K
39/292 20130101; Y02A 50/466 20180101; A61K 39/385 20130101; A61P
31/00 20180101; A61P 37/00 20180101; C12N 2760/16271 20130101; Y02A
50/484 20180101; C07H 3/00 20130101; C12N 2770/32634 20130101; A61K
39/0017 20130101; A61K 39/116 20130101 |
International
Class: |
A61K 39/385 20060101
A61K039/385; A61K 39/00 20060101 A61K039/00; A61K 39/095 20060101
A61K039/095; C07H 3/00 20060101 C07H003/00; A61K 39/102 20060101
A61K039/102; A61K 39/116 20060101 A61K039/116; A61K 39/05 20060101
A61K039/05; A61K 39/08 20060101 A61K039/08; A61K 39/09 20060101
A61K039/09; A61K 39/02 20060101 A61K039/02; A61K 39/29 20060101
A61K039/29; A61K 39/145 20060101 A61K039/145; C12N 7/00 20060101
C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 2005 |
GB |
0513069.5 |
Jun 27, 2005 |
GB |
0513071.1 |
Jul 28, 2005 |
GB |
0515556.9 |
Nov 28, 2005 |
GB |
0524204.5 |
Dec 21, 2005 |
GB |
0526040.1 |
Dec 21, 2005 |
GB |
0526041.9 |
Claims
1.-106. (canceled)
107. An immunogenic composition, comprising: (a) a Neisseria
meningitidis (N. meningitids) serogroup A capsular saccharide
conjugated to an adipic acid dihydrazide (ADH) linker, wherein the
linker is conjugated to tetanus toxoid carrier protein; (b) a N.
meningitidis serogroup C capsular saccharide conjugated to an
adipic acid dihydrazide (ADH) linker, wherein the linker is
conjugated to tetanus toxoid carrier protein; (c) a N. meningitidis
serogroup W capsular saccharide directly conjugated to tetanus
toxoid carrier protein in the absence of a linker; and (d) a N.
meningitidis serogroup Y capsular saccharide directly conjugated to
tetanus toxoid carrier protein in the absence of a linker; wherein
each N. meningitidis capsular saccharide is present at a dose of 5
.mu.g.
108. The immunogenic composition of claim 107, wherein the ratio of
Men W and/or Y saccharide to carrier protein is between 1:0.5 and
1:2 (w/w).
109. The immunogenic composition of claim 107, wherein the
composition does not include an adjuvant.
110. The immunogenic composition of claim 107, wherein the
composition does not include an aluminum salt adjuvant.
111. The immunogenic composition of claim 107, further comprising
sucrose.
112. The immunogenic composition of claim 107, further comprising a
N. meningitidis serogroup B outer membrane vesicle preparation or
capsular saccharide.
113. The immunogenic composition of claim 107, further comprising
Haemophilus influenza (H. influenzae) b capsular saccharide
conjugated to a carrier protein, the carrier protein selected from
the group consisting of tetanus toxoid, diphtheria toxoid, cross
reactive material 197, fragment C of tetanus toxoid and protein D,
wherein the H. influenzae b conjugate is present in a lower dose
than the dose of any other bacterial saccharide conjugate.
114. The immunogenic composition of claim 113, wherein the H.
influenzae b capsular saccharide (Hib) is conjugated to tetanus
toxoid.
115. A pharmaceutical composition, comprising the immunogenic
composition of claim 107 and a pharmaceutically acceptable
excipient.
116. A process of making a vaccine comprising the step of mixing
the immunogenic composition of claim 107 with a pharmaceutically
acceptable excipient.
117. A method of immunizing a human host against disease caused by
Neisseria meningitidis infection, comprising administering to the
host an immunoprotective dose of the immunogenic composition of
claim 107.
Description
[0001] The present invention relates to immunogenic compositions
comprising bacterial capsular saccharides conjugated to a carrier
protein, in particular those saccharides of N. meningitidis. It
additionally relates to vaccines and vaccine kits comprising such
saccharide conjugates, processes for making the immunogenic
compositions and vaccines and the use of the vaccines and
immunogenic compositions of the invention in therapy. It also
relates to methods of immunising against infection using the
saccharide conjugates and the use of the saccharide conjugates in
the manufacture of a medicament.
[0002] Neisseria meningitidis is a Gram-negative human pathogen
which causes bacterial meningitis. Based on the organism's capsular
polysaccharide, twelve serogroups of N. meningitidis have been
identified (A, B, C, H, I, K, L, 29E, W135, X, Y and Z). Serogroup
A (MenA) is the most common cause of epidemic disease in
sub-Saharan Africa. Serogroups B and C are responsible for the
majority of cases in developing countries, with the remaining cases
being caused by W135 and Y).
[0003] Immunogenic compositions comprising N. meningitidis
saccharides conjugated to carrier proteins are known in the art;
the carrier protein having the known effect of turning the
T-independent polysaccharide antigen into a T-dependent antigen
capable of triggering an immune memory response. For instance WO
02/58737 discloses a vaccine comprising purified capsular
polysaccharides from N. meningitidis serogroups A, C, W135 and Y
conjugated to a carrier protein. However, this application teaches
that all polysaccharides should essentially be conjugated in the
same way (through the same linker to the same protein carrier).
[0004] There remains a need to develop improved conjugate vaccines
against neisserial meningitis. The present invention concerns the
provision of a meningococcal polysaccharide conjugate vaccine where
conjugation of each polysaccharide is tailored (rather than being
uniform) to achieve an efficacious combination vaccine. In
particular it is advantageous to use linker molecules to conjugate
certain meningococcal saccharides to their protein carriers in
combination with others that are directly conjugated. In this way
polysaccharides that are less good immunogens may be presented to
the immune system via a linker, and those that are very good
immunogens may be directly conjugated so that they do not dominate
the immune response to the combination.
[0005] Accordingly, in one aspect of the present invention there is
provided an immunogenic composition comprising at least 2 different
N. meningitidis capsular saccharides, wherein one or more is/are
selected from a first group consisting of MenA, MenC, MenY and MenW
which is/are conjugated through a linker to a carrier protein(s),
and one or more different saccharides is/are selected from a second
group consisting of MenA, MenC, MenY and MenW which is/are directly
conjugated to a carrier protein(s).
[0006] In a MenAC vaccine, for example, MenA may be conjugated
through a linker and MenC directly. In a MenCY vaccine, MenC may be
conjugated through a linker and MenY directly. In a MenACWY vaccine
Men A may be conjugated through a linker and MenCWY directly, or
MenAC may be conjugated through a linker and MenWY directly.
[0007] A further consideration in a combination vaccine comprising
various saccharides conjugated to the same carrier is the issue of
carrier immune suppression: too much carrier may be used and the
immune response may be dampened. With a uniform approach to
conjugation the carrier will present a similar blend of B- and
T-cell epitopes to the immune system. However if conjugation takes
place at different chemical groups within the carrier protein for
one saccharide versus another, the protein carriers are likely to
be different to some extent in how they present themselves to the
immune system.
[0008] Accordingly, in a separate embodiment of the invention there
is provided an immunogenic composition comprising at least 2
different saccharides conjugated separately to the same type of
carrier protein (for instance tetanus toxoid), wherein one or more
saccharide(s) is/are conjugated to the carrier protein via a first
type of chemical group on the protein carrier, and one or more
saccharide(s) is/are conjugated to the carrier protein via a second
(different) type of chemical group on the protein carrier.
[0009] The first and second types of chemical group may be present
in the protein carrier on a mutually exclusive first and second set
of amino acids of the protein carrier (for instance certain
aspartic acid/glutamic acid residues in one set and certain lysine
residues in the second). One saccharide may be conjugated to a
carboxyl group on the carrier, and another on an amino group for
instance. Such conjugation may involve conjugation on separate B-
and/or T-cell epitopes for each different conjugate.
[0010] For instance in a MenAC vaccine, MenA may be linked to a
first type of chemical group (such as carboxyl) on the carrier
protein and MenC linked to a second (such as amino). In a MenCY
vaccine MenC may be linked to a first type of chemical group (such
as carboxyl) on the carrier protein and MenY linked to a second
(such as amino). In a MenACWY vaccine, MenAC may be linked to a
first type of chemical group (such as carboxyl) on the carrier
protein and MenWY linked to a second (such as amino), or MenA may
be linked to a first type of chemical group (such as carboxyl) on
the carrier protein and MenCWY linked to a second (such as
amino).
[0011] According to a further aspect of the invention there is
provided a method of immunising a human host against disease caused
by Neisseria meningitidis comprising administering to the host an
immunoprotective dose of the immunogenic composition or vaccine of
the invention.
[0012] According to a further aspect or the invention there is
provided an immunogenic composition of the invention for use in the
treatment or prevention of disease caused by Neisseria
meningitidis.
[0013] According to a further aspect or the invention there is
provided a use of the immunogenic composition or vaccine of the
invention in the manufacture of a medicament for the treatment or
prevention of diseases caused by Neisseria meningitidis.
DESCRIPTION OF FIGURES
[0014] FIG. 1--A--Bar chart showing GMC responses in an anti-MenY
ELISA. ENYTT012 is a MenY-TT conjugate prepared from native MenY
polysaccharide. ENYTT014 is a MenY-TT conjugate prepared from
microfluidised MenY polysaccharide which had undergone 40 cycles of
microfluidisation. ENYTT015bis is a MenY-TT conjugate prepared from
microfluidised MenY polysaccharide which had undergone 20 cycles of
microfluidisation. [0015] B--Bar chart showing GMT responses in an
anti-MenY SBA assay. ENYTT012 is a MenY-TT conjugate prepared from
native MenY polysaccharide. ENYTT014 is a MenY-TT conjugate
prepared from microfluidised MenY polysaccharide which had
undergone 40 cycles of microfluidisation. ENYTT015bis is a MenY-TT
conjugate prepared from microfluidised MenY polysaccharide which
had undergone 20 cycles of microfluidisation.
DETAILED DESCRIPTION
[0016] In one aspect of the present invention there is provided an
immunogenic composition comprising at least 2 different N.
meningitidis capsular saccharides, wherein one or more is/are
selected from a first group consisting of MenA, MenC, MenY and MenW
which is/are conjugated through a linker to a carrier protein(s),
and one or more different saccharides is/are selected from a second
group consisting of MenA, MenC, MenY and MenW which is/are directly
conjugated to a carrier protein(s).
[0017] More specifically, the first group may consist of MenA and
MenC, and the second group consist of MenC, MenY and MenW.
Particular embodiments of the invention are immunogenic
compositions comprising: MenA capsular saccharide conjugated
through a linker to a carrier protein and MenC capsular saccharide
directly conjugated to a carrier protein; MenC capsular saccharide
conjugated through a linker to a carrier protein and MenY capsular
saccharide directly conjugated to a carrier protein; MenA and MenC
capsular saccharides conjugated through a linker to a carrier
protein(s) and MenY and Men W capsular saccharides directly
conjugated to a carrier protein(s); MenA capsular saccharide
conjugated through a linker to a carrier protein and MenC, MenY and
Men W capsular saccharides directly conjugated to a carrier
protein(s). In any of these embodiments a Hib conjugate may also be
included, which is linked to a carrier protein (see list of
carriers above and below, for example TT) directly or through a
linker.
[0018] The term "saccharide" throughout this specification may
indicate polysaccharide or oligosaccharide and includes both.
Polysaccharides are isolated from bacteria or isolated from
bacteria and sized to some degree by known methods (see for example
EP497524 and EP497525) and optionally by microfluidisation.
[0019] 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.
[0020] Each N. meningitidis (and/or Hib) capsular saccharide may be
conjugated to a carrier protein independently selected from the
group consisting of TT, DT, CRM197, fragment C of TT and protein D.
A more complete list of protein carriers that may be used in the
conjugates of the invention is presented below. Although one or
more N. meningitidis (and/or Hib) capsular saccharide may be
conjugated to different carrier proteins from the others, in one
embodiment they are all conjugated to the same carrier protein. For
instance they may all be conjugated to the same carrier protein
selected from the group consisting of TT, DT, CRM197, fragment C of
TT and protein D. In this context CRM197 and DT may be considered
to be the same carrier protein as they differ by only one amino
acid. In an embodiment all the N. meningitidis (and/or Hib)
capsular saccharides present are conjugated to TT.
[0021] If the protein carrier is the same for 2 or more saccharides
in the composition, the saccharide 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; for example, a single carrier protein might be
conjugated to MenA and MenC; MenA and MenW; MenA and MenY; MenC and
MenW; MenC and MenY; Men W and MenY; MenA, MenC and MenW; MenA,
MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY; MenA,
MenC, MenW and MenY; Hib and MenA; Hib and MenC; Hib and MenW; or
Hib and MenY]. 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).
[0022] Immunogenic compositions of the first aspect of the
invention may also have any or all the additional characteristics
of the second aspect of the invention and vice versa.
[0023] In a second aspect of the invention there is presented an
immunogenic composition comprising at least 2 different saccharide
conjugates conjugated separately to the same type of carrier
protein, wherein one or more saccharide(s) is/are conjugated to the
carrier protein via a first type of chemical group on the protein
carrier, and one or more saccharide(s) is/are conjugated to the
carrier protein via a second (different) type of chemical group on
the protein carrier.
[0024] In one embodiment the 2 conjugates involve the same
saccharide linked to the same carrier, but by different conjugation
chemistries. In an alternative embodiment 2 different saccharides
are conjugated to different groups on the protein carrier.
[0025] By "conjugated separately to the same type of carrier
protein" it is meant that the saccharides are conjugated to the
same carrier individually (for example, MenA is conjugated to
tetanus toxoid through an amine group on the tetanus toxoid and
MenC is conjugated to tetanus toxoid through a carboxylic acid
group on a different molecule of tetanus toxoid.)
[0026] The capsular saccharide(s) may be conjugated to the same
carrier protein independently selected from the group consisting of
TT, DT, CRM197, fragment C of TT and protein D. A more complete
list of protein carriers that may be used in the conjugates of the
invention is presented below. In this context CRM197 and DT may be
considered to be the same carrier protein as they differ by only
one amino acid. In an embodiment all the capsular saccharides
present are conjugated to TT.
[0027] In one embodiment the first and second type of chemical
group on the protein carrier are present on separate B- and/or
T-cell epitopes on the carrier protein. That is, they are present
on a different set of B- and/or T-cell epitopes from each other. To
predict B-cell epitopes for a carrier known methods may be used
such as either or both of the following two methods: 2D-structure
prediction and/or antigenic index prediction. 2D-structure
prediction can be made using the PSIPRED program (from David Jones,
Brunel Bioinformatics Group, Dept. Biological Sciences, Brunel
University, Uxbridge UB8 3PH, UK). The antigenic index can be
calculated on the basis of the method described by Jameson and Wolf
(CABIOS 4:181-186 [1988]). The parameters used in this program are
the antigenic index and the minimal length for an antigenic
peptide. An antigenic index of 0.9 for a minimum of 5 consecutive
amino acids can be used as the thresholds in the program. T-helper
cell epitopes are peptides bound to HLA class II molecules and
recognized by T-helper cells. The prediction of useful T-helper
cell epitopes can be based on known techniques, such as the
TEPITOPE method describe by Sturniolo at al. (Nature Biotech. 17:
555-561 [1999]).
[0028] The saccharides may be selected from a group consisting of:
N. meningitidis serogroup A capsular saccharide (MenA), N.
meningitidis serogroup C capsular saccharide (MenC), N.
meningitidis serogroup Y capsular saccharide (MenY), N.
meningitidis serogroup W capsular saccharide (MenW), H. influenzae
type b capsular saccharide (Hib), Group B Streptococcus group I
capsular saccharide, Group B Streptococcus group II capsular
saccharide, Group B Streptococcus group III capsular saccharide,
Group B Streptococcus group IV capsular saccharide, Group B
Streptococcus group V capsular saccharide, Staphylococcus aureus
type 5 capsular saccharide, Staphylococcus aureus type 8 capsular
saccharide, Vi saccharide from Salmonella typhi, N. meningitidis
LPS (such as L3 and/or L2), M. catarrhalis LPS, H. influenzae LPS,
and from any of the capsular pneumococcal saccharides such as from
serotype: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,
15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F. In one embodiment the
immunogenic composition of the invention consists of or comprises
two or more different saccharides from the same genus of bacteria
(e.g. Neisseria, Streptococcus, Staphylococcus, or
Haemophilus).
[0029] The first and second chemical groups present on the protein
carrier are different from each other and are ideally natural
chemical groups that may be readily used for conjugation purposes.
They may be selected independently from the group consisting of:
carboxyl groups, amino groups, sulphydryl groups, Hydroxyl groups,
Imidazolyl groups, Guanidyl groups, and Indolyl groups. In one
embodiment the first chemical group is carboxyl and the second is
amino, or vice versa. These groups are explained in greater detail
below.
[0030] In a specific embodiment the immunogenic composition
comprises at least 2 different N. meningitidis capsular
saccharides, wherein one or more is/are selected from a first group
consisting of MenA and MenC which is/are conjugated to the carrier
protein via the first type of chemical group on the protein carrier
(for instance carboxyl), and one or more different saccharides
is/are selected from a second group consisting of MenC, MenY and
MenW which is/are conjugated to the carrier protein via the second
type of chemical group on the protein carrier (for instance
amino).
[0031] In a further embodiment the immunogenic composition of the
invention comprises MenA conjugated via the first type of chemical
group (for instance carboxyl), and MenC conjugated via the second
type of chemical group (for instance amino).
[0032] In another embodiment the immunogenic composition comprises
MenC conjugated via the first type of chemical group (for instance
carboxyl), and MenY conjugated via the second type of chemical
group (for instance amino).
[0033] In another embodiment the immunogenic composition comprises
MenA conjugated via the first type of chemical group (for instance
carboxyl), and MenC, MenY and MenW conjugated via the second type
of chemical group (for instance amino).
[0034] In another embodiment the immunogenic composition comprises
MenA and MenC conjugated via the first type of chemical group (for
instance carboxyl), and MenY and MenW conjugated via the second
type of chemical group (for instance amino).
[0035] In any of the above embodiments Hib may also be present also
conjugated to the same type of protein carrier. Hib may be
conjugated to the carrier by the first or second type of chemical
group. In one embodiment it is conjugated via a carboxyl group.
General Considerations in the Aspects of the Invention
[0036] The saccharides of the invention (in particular the N.
meningitidis saccharides and/or the Hib capsular saccharide)
included in pharmaceutical (immunogenic) compositions of the
invention are conjugated to a carrier protein such as tetanus
toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of
tetanus toxin [note all such variants of TT are considered to be
the same type of carrier protein for the purposes of this
invention], diphtheria toxoid (DT), CRM197, other non-toxic mutants
of diphtheria toxin [such as CRM176, CRM 197, 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 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 6,455,673; or
fragment disclosed in U.S. Pat. No. 5,843,711] (note all such
variants of DT are considered to be the same type of carrier
protein for the purposes of this invention), pneumococcal
pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), OMPC
(meningococcal outer membrane protein--usually extracted from N.
meningitidis serogroup B--EP0372501), 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.
difficile (WO 00/61761) or Protein D (EP594610 and WO
00/56360).
[0037] In an embodiment, the immunogenic composition of the
invention uses the same type of carrier protein (independently) in
at least two, three, four or each of the saccharides (e.g. N.
meningitidis capsular saccharides and/or Hib) contained therein. In
an embodiment where Hib and N. meningitidis capsular saccharides
are present, Hib may be conjugated to the same type of carrier
protein as the at least two, three, four or each of the N.
meningitidis saccharides. For example, 2, 3 or 4 of the N.
meningitidis saccharides (MenA,C,Y,W) are independently conjugated
to tetanus toxoid to make 2, 3 or 4 conjugates, and optionally Hib
is also conjugated to TT.
[0038] In an embodiment, the immunogenic composition of the
invention comprises a N. meningitidis saccharide conjugated to a
carrier protein selected from the group consisting of TT, DT,
CRM197, fragment C of TT and protein D. In an embodiment, the
immunogenic composition of the invention comprises a Hib saccharide
conjugated to a carrier protein selected from the group consisting
of TT, DT, CRM197, fragment C of TT and protein D.
[0039] The immunogenic composition of the invention optionally
comprises at least one meningococcal saccharide (for example MenA;
MenC; MenW; MenY; MenA and MenC; MenA and MenW; MenA and MenY; MenC
and Men W; Men C and MenY; Men W and MenY; MenA, MenC and MenW;
MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY or
MenA, MenC, MenW and MenY) conjugate having a ratio of Men
saccharide to carrier protein of between 1:5 and 5:1, between 1:2
and 5:1, between 1:0.5 and 1:2.5 or between 1:1.25 and 1:2.5
(w/w).
[0040] The immunogenic composition of the invention optionally
comprises a Hib saccharide conjugate having a ratio of Hib to
carrier protein of between 1:5 and 5:1; 1:2 and 2:1; 1:1 and 1:4;
1:2 and 1:3.5; or around or exactly 1:2.5 or 1:3 (w/w).
[0041] The ratio of saccharide to carrier protein (w/w) in a
conjugate may be determined using the sterilized conjugate. The
amount of protein is determined using a Lowry assay (for example
Lowry et al (1951) J. Biol. Chem. 193, 265-275 or Peterson et al
Analytical Biochemistry 100, 201-220 (1979)) and the amount of
saccharide is determined using ICP-OES (inductively coupled
plasma-optical emission spectroscopy) for MenA, DMAP assay for MenC
and Resorcinol assay for MenW and MenY (Monsigny et al (1988) Anal.
Biochem. 175, 525-530).
[0042] In an embodiment, the immunogenic composition of the
invention comprises N. meningitidis saccharide conjugate(s) and/or
the Hib saccharide conjugate wherein the N. meningitidis
saccharide(s) and/or the Hib 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 (Geyer
et al (1979) Med. Microbiol. Immunol. 165; 171-288), haloalkyl
halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat.
Nos. 4,673,574, 4,808,700), hexane diamine and 6-aminocaproic acid
(U.S. Pat. No. 4,459,286).
[0043] 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
holoacetylated carrier protein (for example using iodoacetimide or
N-succinimidyl bromoacetatebromoacetate). Optionally, 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 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.
[0044] Other suitable techniques use carbiinides, 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.
[0045] 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.
[0046] A further method involves the coupling of a cyanogen bromide
(or CDAP) activated saccharide derivatised with adipic acid
hydrazide (ADH) to the protein carrier by Carbodiimide condensation
(Chu C. et al Infect. Immunity, 1983 245 256), for example using
EDAC.
[0047] In an embodiment, a hydroxyl group (optionally an activated
hydroxyl group for example a hydroxyl group activated by a cyanate
ester) 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 optionally
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 Hib or N. meningitidis capsular saccharide(s)
(or saccharide in general) 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.
[0048] In general the following types of chemical groups on a
protein carrier can be used for coupling/conjugation:
[0049] 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.
[0050] 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.
[0051] 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.
[0052] D) Hydroxyl group (for instance via tyrosine). In one
embodiment this group is activated/modified with bis
diazobenzidine.
[0053] E) Imidazolyl group (for instance via histidine). In one
embodiment this group is activated/modified with bis
diazobenzidine.
[0054] F) Guanidyl group (for instance via arginine).
[0055] G) Indolyl group (for instance via tryptophan).
[0056] 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.
[0057] Direct Coupling Approaches:
[0058] Saccharide-OH+CNBr or CDAP - - - - - >cyanate
ester+NH2-Prot - - - - >conjugate
[0059] Saccharide-aldehyde+NH2-Prot - - - - >Schiff base+NaCNBH3
- - - - >conjugate
[0060] Saccharide-COOH+NH2-Prot+EDAC - - - - >conjugate
[0061] Saccharide-NH2+COOH-Prot+EDAC - - - - >conjugate
[0062] Indirect Coupling Via Spacer (Linker) Approaches:
[0063] Saccharide-OH+CNBr or CDAP - - - >cyanate ester+NH2 - - -
- NH2 - - - - >saccharide - - - - NH2+COOH-Prot+EDAC - - - - -
>conjugate
[0064] 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
[0065] Saccharide-OH+CNBr or CDAP - - - >cyanate ester+NH2 - - -
- SH - - - - - - >saccharide - - - - SH+maleimide-Prot
(modification of amino groups) - - - - >conjugate
[0066] Saccharide-COOH+EDAC+NH2 - - - - - NH2 - - - >saccharide
- - - - - - NH2+EDAC+COOH-Prot - - - - >conjugate
[0067] 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
[0068] Saccharide-COOH+EDAC+NH2 - - - - SH - - - - - >saccharide
- - - - SH+maleimide-Prot (modification of amino groups) - - - -
>conjugate
[0069] Saccharide-Aldehyde+NH2 - - - - - NH2 - - - - >saccharide
- - - NH2+EDAC+COOH-Prot - - - - >conjugate
[0070] Note: instead of EDAC above, any suitable carbodiimide may
be used.
[0071] 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).
[0072] In an embodiment, the Hib saccharide, where present, is
conjugated to the carrier protein using CNBr, or CDAP, or a
combination of CDAP and carbodiimide chemistry (such as EDAC), or a
combination of CNBr and carbodiimide chemistry (such as EDAC).
Optionally Hib is conjugated using CNBr and carbodiimide chemistry,
optionally EDAC. For example, CNBr is used to join the saccharide
and linker and then carbodiimide chemistry is used to join linker
to the protein carrier.
[0073] In an embodiment, at least one of the N. meningitidis
capsular saccharides (or saccharide in general) is directly
conjugated to a carrier protein; optionally Men W and/or MenY
and/or MenC saccharide(s) is directly conjugated to a carrier
protein. For example MenW; MenY; MenC; MenW and MenY; MenW and
MenC; MenY and MenC; or MenW, MenY and MenC are directly linked to
the carrier protein. Optionally, at least one of the N.
meningitidis capsular saccharides is directly conjugated by CDAP.
For example MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenY
and MenC; or MenW, MenY and MenC are directly linked to the carrier
protein by CDAP (see WO 95/08348 and WO 96/29094). In an
embodiment, all N. meningitidis capsular saccharides are conjugated
to tetanus toxoid.
[0074] In an embodiment, the ratio of Men W and/or Y saccharide to
carrier protein is between 1:0.5 and 1:2 (w/w) and/or the ratio of
MenC saccharide to carrier protein is between 1:0.5 and 1:4 or
1:0.5 and 1:1.5 (w/w), especially where these saccharides are
directly linked to the protein, optionally using CDAP.
[0075] In an embodiment, at least one of the N. meningitidis
capsular saccharide(s) (or saccharide in general) 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 amine group and a
reative carboxylic acid group, 2 reactive amine groups or 2
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.
[0076] In an embodiment, MenA; MenC; or MenA and MenC is conjugated
to a carrier protein (for example tetanus toxoid) via a linker.
[0077] In an embodiment, at least one N. meningitidis saccharide is
conjugated to a carrier protein via a linker using CDAP and EDAC.
For example, MenA; MenC; or MenA and MenC are 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.
For example, CDAP is used to conjugate the saccharide to a linker
and EDAC is used to conjugate the linker to a protein. Optionally
the conjugation via a linker results in a ratio of saccharide to
carrier protein of of between 1:0.5 and 1:6; 1:1 and 1:5 or 1:2 and
1:4, for MenA; MenC; or MenA and MenC.
[0078] In an embodiment, the MenA capsular saccharide, where
present is at least partially O-acetylated such that at least 50%,
60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated
at at least one position. O-acetylation is for example present at
least at the 0-3 position of at least 50%, 60%, 70%, 80%, 90%, 95%
or 98% of the repeat units.
[0079] In an embodiment, the MenC capsular saccharide, where
present is is at least partially O-acetylated such that at least
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of
(.alpha.2.fwdarw.9)-linked NeuNAc repeat units are O-acetylated at
at least one or two positions. O-acetylation is for example present
at the 0-7 and/or 0-8 position of at least 30%. 40%, 50%, 60%, 70%,
80%, 90%, 95% or 98% of the repeat units.
[0080] In an embodiment, the MenW capsular saccharide, where
present is is at least partially O-acetylated such that at least
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units
are O-acetylated at at least one or two positions. 0-acetylation is
for example present at the 0-7 and/or 0-9 position of at least 30%.
40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.
[0081] In an embodiment, the MenY capsular saccharide, where
present is at least partially O-acetylated such that at least 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units
are O-acetylated at at least one or two positions. 0-acetylation is
present at the 7 and/or 9 position of at least 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95% or 98% of the repeat units.
[0082] The percentage of O-acetylation refers to the percentage of
the repeat units containing O-acetylation. This may be measured in
the saccharide prior to conjugate and/or after conjugation.
[0083] In one embodiment of the invention the immunogenic
composition, saccharide present, or each N. meningitidis capsular
saccharide present, is conjugated to TT. In a further embodiment
each N. meningitidis capsular saccharide is separately conjugated
to a separate carrier protein. In a further embodiment each N.
meningitidis capsular saccharide conjugate has a saccharide:carrier
ratio of 1:5-5:1 or 1:1-1:4 (w/w). In a further embodiment at least
one, two or three N. meningitidis capsular saccharide conjugate(s)
is directly conjugated to a carrier protein. In a further
embodiment Men W and/or MenY, MenW and/or MenC, MenY and/or MenC,
or MenW and MenC and MenY are directly conjugated to a carrier
protein. In a further embodiment at least one, two or three N.
meningitidis saccharide conjugate(s) is directly conjugated by CDAP
chemistry. In a further embodiment the ratio of Men W and/or Y
saccharide to carrier protein is between 1:0.5 and 1:2 (w/w). In a
further embodiment the ratio of MenC saccharide to carrier protein
is between 1:0.5 and 1:2 (w/w). In a further embodiment at least
one, two or three N. meningitidis capsular saccharide(s) are
conjugated to the carrier protein via a linker (which may be
bifunctional such as having two reactive amino groups (such as ADH)
or two reactive carboxyl groups, or a reactive amino group at one
end and a reactive carboxyl group at the other). The linker can
have between 4 and 12 carbon atoms. In a further embodiment the or
each N. meningitidis capsular saccharide(s) conjugated via a linker
are conjugated to the linker with CDAP chemistry. In a further
embodiment the carrier protein is conjugated to the linker using
carbodiimide chemistry, for example using EDAC. In a further
embodiment the or each N. meningitidis capsular saccharide is
conjugated to the linker before the carrier protein is conjugated
to the linker. In a further embodiment MenA is conjugated to a
carrier protein via a linker (the ratio of MenA saccharide to
carrier protein may be between 1:2 and 1:5 (w/w)). In a further
embodiment MenC is conjugated to a carrier protein via a linker
(the ratio of MenC saccharide to carrier protein may be between 1:2
and 1:5 (w/w)).
[0084] 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 immungenicity which is
filterable through a 0.2 micron filter; 2) immune memory may be
enhanced (as in example three); 3) the alteration of the ratio of
polysaccharide to protein in the conjugate such that the ratio of
polysaccharide to protein (w/w) in the conjugate may be increased
(this can result in a reduction of the carrier suppression effect);
4) immunogenic conjugates prone to hydrolysis (such as MenA
conjugates) may be stabilised by the use of larger polysaccharides
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 meningococcal
(or saccharide) conjugate vaccines retaining a larger size of
saccharide can provide a good immune response against meningococcal
disease.
[0085] The immunogenic composition of the invention may thus
comprise one or more saccharide conjugates wherein the average size
of each saccharide before conjugation is above 50 kDa, 75 kDa, 100
kDa, 110 kDa, 120 kDa or 130 kDa. 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.
[0086] In particular, the immunogenic composition of the invention
comprises N. meningitidis capsular saccharides from at least one,
two, three or four of serogroups A, C, W and Y conjugated to a
carrier protein, wherein the average size (weight-average molecular
weight; Mw) of at least one, two, three or four or each N.
meningitidis saccharide is above 50 kDa, 60 kDa, 75 kDa, 100 kDa,
110 kDa, 120 kDa or 130 kDa.
[0087] The immunogenic composition may comprise N. meningitidis
capsular saccharides from at least one, two, three or four of
serogroups A, C, W and Y conjugated to a carrier protein, wherein
at least one, two, three or four or each N. meningitidis saccharide
is either a native saccharide or 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 relative to the weight average
molecular weight of the native polysaccharide.
[0088] For the purposes of the invention, "native polysaccharide"
refers to a saccharide that has not been subjected to a process,
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.
[0089] 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.
[0090] In an aspect of the invention, the immunogenic composition
comprises N. meningitidis capsular saccharides from at least one,
two, three or four of serogroups A, C, W and Y conjugated to a
carrier protein, wherein at least one, two, three or four or each
N. meningitidis saccharide is native polysaccharide.
[0091] In an aspect of the invention, the immunogenic composition
comprises N. meningitidis capsular saccharides from at least one,
two, three or four of serogroups A, C, W and Y conjugated to a
carrier protein, wherein at least one, two, three or four or each
N. meningitidis saccharide is sized by a factor up to .times.1.5,
.times.2, .times.3, .times.4, .times.5, .times.6, .times.7,
.times.8, .times.9 or .times.10.
[0092] The immunogenic compositions of the invention optionally
comprise conjugates of: N. meningitidis serogroup C capsular
saccharide (MenC), serogroup A capsular saccharide (MenA),
serogroup W135 capsular saccharide (MenW), serogroup Y capsular
saccharide (MenY), serogroup C and Y capsular saccharides (MenCY),
serogroup C and A capsular saccharides (MenAC), serogroup C and W
capsular saccharides (MenCW), serogroup A and Y capsular saccharide
(MenAY), serogroup A and W capsular saccharides (MenAW), serogroup
W and Y capsular saccharides (Men WY), serogroup A, C and W
capsular saccharide (MenACW), serogroup A, C and Y capsular
saccharides (MenACY); serogroup A, W135 and Y capsular saccharides
(MenAWY), serogroup C, W135 and Y capsular saccharides (MenCWY); or
serogroup A, C, W135 and Y capsular saccharides (MenACWY). This is
the definition of "one, two, three or four", or "at least one of"
of serogroups A, C, W and Y, or of each N. meningitidis saccharide
where mentioned herein.
[0093] In an embodiment, the average size of at least one, two,
three, four or each N. meningitidis saccharide is between 50 KDa
and 1500 kDa, 50 kDa and 500 kDa, 50 kDa and 300 KDa, 101 kDa and
1500 kDa, 101 kDa and 500 kDa, 101 kDa and 300 kDa as determined by
MALLS.
[0094] In an embodiment, the MenA saccharide, where present, has a
molecular weight of 50-500 kDa, 50-100 kDa, 100-500 kDa, 55-90 KDa,
60-70 kDa or 70-80 kDa or 60-80 kDa.
[0095] In an embodiment, the MenC saccharide, where present, has a
molecular weight of 100-200 kDa, 50-100 kDa, 100-150 kDa, 101-130
kDa, 150-210 kDa or 180-210 kDa.
[0096] In an embodiment the MenY saccharide, where present, has a
molecular weight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa,
100-150 kDa or 110-140 kDa, 50-100 kDa, 100-140 kDa, 140-170 kDa or
150-160 kDa.
[0097] In an embodiment the MenW saccharide, where present, has a
molecular weight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa,
100-150 kDa, 110-140 kDa, 50-100 kDa or 120-140 kDa.
[0098] The molecular weight or average molecular weight of a
saccharide herein refers to the weight-average molecular weight
(Mw) of the saccharide measured prior to conjugation and is
measured by MALLS.
[0099] The MALLS technique is well known in the art and is
typically carried out as described in example 2. For MALLS analysis
of meningococcal saccharides, two columns (TSKG6000 and
5000PW.times.I) 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).
[0100] In an embodiment the N. meningitidis saccharides are native
polysaccharides or native polysaccharides which have reduced in
size during a normal extraction process.
[0101] In an embodiment, the N. meningitidis 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 (fro example through
a 0.2 micron filter). Sizing is by a factor of no more than
.times.20, .times.10, .times.8, .times.6, .times.5, .times.4,
.times.3, .times.2 or .times.1.5.
[0102] In an embodiment, the immunogenic composition comprises N.
meningitidis conjugates that are made from a mixture of native
polysaccharides and saccharides that are sized by a factor of no
more than .times.20. For example, saccharides from MenC and/or MenA
are native. For example, saccharides from MenY and/or MenW are
sized by a factor of no more than .times.20, .times.10, .times.8,
.times.6, .times.5, .times.4, .times.3 or .times.2. For example, an
immunogenic composition contains a conjugate made from MenY and/or
MenW and/or MenC and/or MenA which is sized by a factor of no more
then .times.10 and/or is microfluidised. For example, an
immunogenic composition contains a conjugate made from native MenA
and/or MenC and/or MenW and/or MenY. For example, an immunogenic
composition comprises a conjugate made from native MenC. For
example, an immunogenic composition comprises a conjugate made from
native MenC and MenA which is sized by a factor of no more then
.times.10 and/or is microfluidised. For example, an immunogenic
composition comprises a conjugate made from native MenC and MenY
which is sized by a factor of no more then .times.10 and/or is
microfluidised.
[0103] In an embodiment, the polydispersity of the saccharide is
1-1.5, 1-1.3, 1-1.2, 1-1.1 or 1-1.05 and after conjugation to a
carrier protein, the polydispersity of the conjugate is 1.0-2.5,
1.0-2.0, 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2 or 1.5-2.0. All
polydispersity measurements are by MALLS.
[0104] Saccharides are optionally sized up to 1.5, 2, 4, 6, 8, 10,
12, 14, 16, 18 or 20 times from the size of the polysaccharide
isolated from bacteria.
[0105] In one embodiment each N. meningitidis saccharide is either
a native polysaccharide or is sized by a factor of no more than
.times.10. In a further embodiment each N. meningitidis capsular
saccharide is a native polysaccharide. In a further embodiment at
least one, two, three or four N. meningitidis capsular
saccharide(s) is sized by microfluidization. In a further
embodiment each N. meningitidis capsular saccharide is sized by a
factor of no more than .times.10. In a further embodiment the N.
meningitidis conjugates are made from a mixture of native
polysaccharides and saccharides that are sized by a factor of no
more than .times.10. In a further embodiment the capsular
saccharide from serogroup Y is sized by a factor of no more than
.times.10. In a further embodiment capsular saccharides from
serogroups A and C are native polysaccharides and saccharides from
serogroups W135 and Y are sized by a factor of no more than
.times.10. In a further embodiment the average size of each N.
meningitidis capular saccharide is between 50 kDa and 300 KDa or 50
kDa and 200 kDa. In a further embodiment the immunogenic
composition comprises a MenA capsular saccharide having an average
size of above 50 kDa, 75 kDa, 100 kDa or an average size of between
50-100 kDa or 55-90 KDa or 60-80 kDa. In a further embodiment the
immunogenic composition comprises a MenC capsular saccharide having
an average size of above 50 kDa, 75 kDa, 100 kDa or between 100-200
kDa, 100-150 kDa, 80-120 kDa, 90-110 kDa, 150-200 kDa, 120-240 kDa,
140-220 kDa, 160-200 kDa or 190-200 kDa. In a further embodiment
the immunogenic composition comprises a MenY capsular saccharide,
having an average size of above 50 kDa, 75 kDa, 100 kDa or between
60-190 kDa or 70-180 kDa or 80-170 kDa or 90-160 kDa or 100-150
kDa, 110-145 kDa or 120-140 kDa. In a further embodiment the
immunogenic composition comprises a MenW capsular saccharide having
an average size of above 50 kDa, 75 kDa, 100 kDa or between 60-190
kDa or 70-180 kDa or 80-170 kDa or 90-160 kDa or 100-150 kDa,
140-180 kDa, 150-170 kDa or 110-140 kDa.
[0106] The immunogenic composition of the invention may comprise a
H. influenzae b capsular saccharide (Hib) conjugated to a carrier
protein. This may be conjugated to a carrier protein selected from
the group consisting of TT, DT, CRM197, fragment C of TT and
protein D, for instance TT. The Hib saccharide may be conjugated to
the same carrier protein as for at least one, two, three or all of
the N. meningitidis capsular saccharide conjugates, for instance
TT. The ratio of Hib to carrier protein in the Hib capsular
saccharide conjugate may be between 1:5 and 5:1 (w/w), for instance
between 1:1 and 1:4, 1:2 and 1:3.5 or around 1:3 (w/w). The Hib
capsular saccharide may be conjugated to the carrier protein via a
linker (see above). The linker may bifunctional (with two reactive
amino groups, such as ADH, or two reactive carboxylic acid groups,
or a reactive amino group at one end and a reactive carboxylic acid
group at the other end). It may have between 4 and 12 carbon atoms.
Hib saccharide may be conjugated to the carrier protein or linker
using CNBr or CDAP. The carrier protein may be conjugated to the
Hib saccharide via the linker using a method comprising
carbodiimide chemistry, for example EDAC chemistry (thus using the
carboxyl chemical group on the carrier). The dose of the Hib
saccharide conjugate may be between 0.1 and 9 .mu.g, 1 and 5 .mu.g
or 2 and 3 .mu.g of saccharide.
[0107] In a further embodiment, the immunogenic composition of the
invention comprises a Hib saccharide conjugate and at least two N.
meningitidis saccharide conjugates wherein the Hib conjugate is
present in a lower saccharide dose than the mean saccharide dose of
the at least two N. meningitidis saccharide conjugates.
Alternatively, the Hib conjugate is present in a lower saccharide
dose than the saccharide dose of each of the at least two N.
meningitidis saccharide conjugates. For example, the dose of the
Hib conjugate may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or
80% lower than the mean or lowest saccharide dose of the at least
two further N. meningitidis saccharide conjugates.
[0108] The mean dose is determined by adding the doses of all the
further saccharides and dividing by the number of further
saccharides. Further saccharides are all the saccharides within the
immunogenic composition apart from Hib and can include N.
meningitidis capsular saccharides. The "dose" is in the amount of
immunogenic composition or vaccine that is administered to a
human.
[0109] A Hib saccharide is the polyribosyl phosphate (PRP) capsular
polysaccharide of Haemophilus influenzae type b or an
oligosaccharide derived therefrom.
[0110] At least two further bacterial saccharide conjugates is to
be taken to mean two further bacterial saccharide conjugates in
addition to a Hib conjugate. The two further bacterial conjugates
may include N. meningitidis capular saccharide conjugates.
[0111] The immunogenic compositions of the invention may comprise
further saccharide conjugates derived from one or more of Neisseria
meningitidis, Streptococcus pneumoniae, Group A Streptococci, Group
B Streptococci, S. typhi, Staphylococcus aureus or Staphylococcus
epidermidis. In an embodiment, the immunogenic composition
comprises capsular saccharides derived from one or more of
serogroups A, C, W135 and Y of Neisseria meningitidis. A further
embodiment comprises capsular saccharides derived from
Streptococcus pneumoniae. The pneumococcal capsular saccharide
antigens are optionally selected from serotypes 1, 2, 3, 4, 5, 6A,
6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,
22F, 23F and 33F (optionally from serotypes 1, 3, 4, 5, 6B, 7F, 9V,
14, 18C, 19F and 23F). A further embodiment comprises the Type 5,
Type 8 or 336 capsular saccharides of Staphylococcus aureus. A
further embodiment comprises the Type I, Type II or Type III
capsular saccharides of Staphylococcus epidermidis. A further
embodiment comprises the Vi saccharide from S. typhi. A further
embodiment comprises the Type Ia, Type Ic, Type II, Type III or
Type V capsular saccharides of Group B streptococcus. A further
embodiment comprises the capsular saccharides of Group A
streptococcus, optionally further comprising at least one M protein
and optionally multiple types of M protein.
[0112] The immunogenic compositions of the invention may also
comprise a DTPa or DTPw vaccine (for instance one containing DT,
TT, and either a whole cell pertussis (Pw) vaccine or an acellular
pertussis (Pa) vaccine (comprising for instance pertussis toxoid,
FHA, pertactin, and, optionally agglutinogins 2 and 3). Such
combinations may also comprise a vaccine against hepatitis B (for
instance it may comprise hepatitis B surface antigen [HepB],
optionally adsorbed onto aluminium phosphate). In one embodiment
the immunogenic composition of the invention comprises a
DTPwHepBHibMenAC vaccine where the HibMenAC component is as
described above.
[0113] Immunogenic compositions of the invention optionally
comprise additional viral antigens conferring protection against
disease caused by measles and/or mumps and/or rubella and/or
varicella. For example, immunogenic composition of the invention
contains antigens from measles, mumps and rubella (MMR) or measles,
mumps, rubella and varicella (MMRV). In an embodiment, these viral
antigens are optionally present in the same container as the
meningococcal and/or Hib saccharide conjugate(s). In an embodiment,
these viral antigens are lyophilised.
[0114] In an embodiment, the immunogenic composition of the
invention further comprises an antigen from N. meningitidis
serogroup B. The antigen is optionally a capsular polysaccharide
from N. meningitidis serogroup B (MenB) or a sized polysaccharide
or oligosaccharide derived therefrom, which may be conjugated to a
protein carrier. The antigen is optionally an outer membrane
vesicle preparation from N. meningitidis serogroup B as described
in EP301992, WO 01/09350, WO 04/14417, WO 04/14418 and WO
04/14419.
[0115] In general, the immunogenic composition of the invention may
comprise a dose of each saccharide conjugate between 0.1 and 20
.mu.g, 2 and 10 .mu.g, 2 and 6 .mu.g or 4 and 7 .mu.g of
saccharide.
[0116] In an embodiment, the immunogenic composition of the
invention contains each N. meningitidis capsular saccharide at a
dose of between 0.1-20 .mu.g; 1-10 .mu.g; 2-10 .mu.g, 2.5-5 .mu.g,
around or exactly 5 .mu.g; or around or exactly 2.5 .mu.g. In an
embodiment, the immunogenic composition of the invention comprises
MenA, MenC, MenW and MenY (optionally conjugated to tetanus toxoid)
in doses of 2.5, 2.5, 2.5 and 2.5 .mu.g respectively, 5, 5, 5 and 5
.mu.g respectively or 5, 5, 2.5 and 2.5 .mu.g respectively.
[0117] In an embodiment, the immunogenic composition of the
invention for example contains the Hib saccharide conjugate at a
saccharide dose between 0.1 and 9 .mu.g; 1 and 5 .mu.g or 2 and 3
.mu.g or around or exactly 2.5 .mu.g. In a further embodiment the
immunogenic composition of the invention for example contains the
Hib saccharide conjugate at a saccharide dose between 0.1 and 9
.mu.g; 1 and 5 .mu.g or 2 and 3 .mu.g or around or exactly 2.5
.mu.g and each of the N. meningitidis polysaccharide conjugates at
a saccharide dose of between 2 and 20 .mu.g, 3 and 10 .mu.g, or
between 4 and 7 .mu.g or around or exactly 5 .mu.g.
[0118] "Around" or "approximately" are defined as within 10% more
or less of the given figure for the purposes of the invention.
[0119] In an embodiment, the immunogenic composition of the
invention may contain a saccharide dose of the Hib saccharide
conjugate which is for example less than 90%, 80%, 75%, 70%, 60%,
50%, 40%, 30%, 20% or 10% of the mean saccharide dose of at least
two, three, four or each of the N. meningitidis saccharide
conjugates. The saccharide dose of the Hib saccharide is for
example between 20% and 60%, 30% and 60%, 40% and 60% or around or
exactly 50% of the mean saccharide dose of at least two, three,
four or each of the N. meningitidis saccharide conjugates.
[0120] In an embodiment, the immunogenic composition of the
invention contains a saccharide dose of the Hib saccharide
conjugate which is for example less than 90%, 80%, 75%, 70%, 60%,
50%, 40%, 30%, 20% or 10% of the lowest saccharide dose of the at
least two, three, four or each of the N. meningitidis saccharide
conjugates. The saccharide dose of the Hib saccharide is for
example between 20% and 60%, 30% and 60%, 40% and 60% or around or
exactly 50% of the lowest saccharide dose of the at least two,
three, four or each of the N. meningitidis saccharide
conjugates.
[0121] In an embodiment of the invention, the saccharide dose of
each of the at least two, three, four or each of the N.
meningitidis saccharide conjugates is optionally the same, or
approximately the same.
[0122] Examples of immunogenic compositions of the invention are
compositions consisting of or comprising:
[0123] Hib conjugate and MenA conjugate and MenC conjugate,
optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:6:3,
1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the saccharide dose
of MenA is greater than the saccharide dose of MenC.
[0124] Hib conjugate and MenC conjugate and MenY conjugate,
optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1,
1:8;4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w).
[0125] Optionally, the saccharide dose of MenC is greater than the
saccharide dose of MenY.
[0126] Hib conjugate and MenC conjugate and MenW conjugate,
optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1,
1:8;4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the
saccharide dose of MenC is greater than the saccharide dose of
MenW.
[0127] Hib conjugate and MenA conjugate and MenW conjugate,
optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1,
1:8;4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally, the
saccharide dose of MenA is greater than the saccharide dose of
MenW.
[0128] Hib conjugate and MenA conjugate and MenY conjugate,
optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:4:2, 1:4:1,
1:8:4, 1:6:3, 1:3:3, 1:4:4, 1:5:5, 1:6:6 (w/w). Optionally the
saccharide dose of MenA is greater than the saccharide dose of
MenY.
[0129] Hib conjugate and MenW conjugate and MenY conjugate,
optionally at saccharide dose ratios of 1:2:2, 1:2:1, 1:1:2, 1:4:2,
1:2:4, 1:4:1, 1:1:4, 1:3;6, 1:1:3, 1:6:3, 1:3:3, 1:4:4, 1:5:5,
1:6:6 (w/w). Optionally the saccharide dose of MenY is greater than
the saccharide dose of MenW.
[0130] MenA, MenC, MenW and MenY at saccharide dose ratios of
1:1:1:1 or 2:1:1:1 or 1:2:1:1 or 2:2:1:1 or 1:3:1:1 or 1:4:1:1
(w/w).
[0131] A further aspect of the invention is a vaccine comprising
the immunogenic composition of the invention and a pharmaceutically
acceptable excipient.
[0132] In an embodiment, the immunogenic composition of the
invention is adjusted to or buffered at, or adjusted to between pH
7.0 and 8.0, pH 7.2 and 7.6 or around or exactly pH 7.4.
[0133] The immunogenic composition or vaccines of the invention are
optionally lyophilised in the presence of a stabilising agent for
example a polyol such as sucrose or trehalose.
[0134] Optionally, the immunogenic composition or vaccine of the
invention contains an amount of an adjuvant sufficient to enhance
the immune response to the immunogen. Suitable adjuvants include,
but are not limited to, aluminium salts (aluminium phosphate or
aluminium hydroxide), squalene mixtures (SAF-1), muramyl peptide,
saponin derivatives, mycobacterium cell wall preparations,
monophosphoryl lipid A, mycolic acid derivatives, non-ionic block
copolymer surfactants, Quil A, cholera toxin B subunit,
polyphosphazene and derivatives, and immunostimulating complexes
(ISCOMs) such as those described by Takahashi et al. (1990) Nature
344:873-875.
[0135] For the N. meningitidis or HibMen combinations discussed
above, it may be advantageous not to use any aluminium salt
adjuvant or any adjuvant at all.
[0136] As with all immunogenic compositions or vaccines, the
immunologically effective amounts of the immunogens must be
determined empirically. Factors to be considered include the
immunogenicity, whether or not the immunogen will be complexed with
or covalently attached to an adjuvant or carrier protein or other
carrier, route of administrations and the number of immunising
dosages to be administered.
[0137] The active agent can be present in varying concentrations in
the pharmaceutical composition or vaccine of the invention.
Typically, the minimum concentration of the substance is an amount
necessary to achieve its intended use, while the maximum
concentration is the maximum amount that will remain in solution or
homogeneously suspended within the initial mixture. For instance,
the minimum amount of a therapeutic agent is optionally one which
will provide a single therapeutically effective dosage. For
bioactive substances, the minimum concentration is an amount
necessary for bioactivity upon reconstitution and the maximum
concentration is at the point at which a homogeneous suspension
cannot be maintained. In the case of single-dosed units, the amount
is that of a single therapeutic application. Generally, it is
expected that each dose will comprise 1-100 .mu.g of protein
antigen, optionally 5-50 .mu.g or 5-25 .mu.g. For example, doses of
bacterial saccharides are 10-20 .mu.g, 5-10 .mu.g, 2.5-5 .mu.g or
1-2.5 .mu.g of saccharide in the conjugate.
[0138] The vaccine preparations of the present invention may be
used to protect or treat a mammal (for example a human patient)
susceptible to infection, by means of administering said vaccine
via systemic or mucosal route. A human patient is optionally an
infant (under 12 months), a toddler (12-24, 12-16 or 12-14 months),
a child (2-10, 3-8 or 3-5 years) an adolescent (12-21, 14-20 or
15-19 years) or an adult. 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 if saccharides are present in a vaccine these could
be administered separately at the same time or 1-2 weeks after the
administration of a bacterial protein vaccine for optimal
coordination of the immune responses with respect to each other).
In addition to a single route of administration, 2 different routes
of administration may be used. For example, viral antigens may be
administered ID (intradermal), whilst bacterial proteins may be
administered IM (intramuscular) or IN (intranasal). If saccharides
are present, they 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.
[0139] 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.
[0140] A further aspect of the invention is a vaccine kit for
concomitant or sequential administration comprising two
multi-valent immunogenic compositions for conferring protection in
a host against disease caused by Bordetella pertussis, Clostridium
tetani, Corynebacterium diphtheriae and Neisseria meningitidis and
optionally Haemophilus influenzae. For example, the kit optionally
comprises a first container comprising one or more of:
[0141] tetanus toxoid (TT),
[0142] diphtheria toxoid (DT), and
[0143] whole cell or acellular pertussis components
[0144] and a second container comprising:
[0145] an immunogenic composition of the invention as described
above (for instance those comprising Men or HibMen saccharide
conjugate combinations).
[0146] A further aspect of the invention is a vaccine kit for
concomitant or sequential administration comprising two
multi-valent immunogenic compositions for conferring protection in
a host against diease caused by Streptococcus pneumoniae and
Neisseria meningitidis and optionally Haemophilus influenzae. For
example, the kit optionally comprises a first container
comprising:
[0147] one or more conjugates of a carrier protein and a capsular
saccharide from Streptococcus pneumoniae [where the capsular
saccharide is optionally from a pneumococcal serotype selected from
the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A,
11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F].
[0148] and a second container comprising:
[0149] an immunogenic composition of the invention as described
above (for instance those comprising Men or HibMen saccharide
conjugate combinations).
[0150] Examples of the Hib conjugate and the N. meningitidis
polysaccharide conjugates are as described above.
[0151] Typically the Streptococcus pneumoniae vaccine in the
vaccine kit of the present invention (or in any of the immunogenic
compositions of the invention described above) will comprise
saccharide antigens (optionally conjugated), wherein the
saccharides are derived from at least four serotypes of
pneumococcus chosen from the group consisting of 1, 2, 3, 4, 5, 6A,
6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,
22F, 23F and 33F. Optionally, the four serotypes include 6B, 14,
19F and 23F. Optionally, at least 7 serotypes are included in the
composition, for example those derived from serotypes 4, 6B, 9V,
14, 18C, 19F, and 23F. Optionally more than 7 serotypes are
included in the composition, for instance at least 10, 11, 12, 13
or 14 serotypes. For example the composition in one embodiment
includes 10 or 11 capsular saccharides derived from serotypes 1, 4,
5, 6B, 7F, 9V, 14, 18C, 19F and 23F, and optionally 3 (all
optionally conjugated). In an embodiment of the invention at least
13 saccharide antigens (optionally conjugated) are included,
although further saccharide antigens, for example 23 valent (such
as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,
15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also
contemplated by the invention.
[0152] The pneumococcal saccharides are independently conjugated to
any known carrier protein, for example CRM197, tetanus toxoid,
diphtheria toxoid, protein D or any other carrier proteins as
mentioned above.
[0153] Optionally, the vaccine kits of the invention comprise a
third component. For example, the kit optionally comprises a first
container comprising one or more of:
[0154] tetanus toxoid (TT),
[0155] diphtheria toxoid (DT), and
[0156] whole cell or acellular pertussis components
[0157] and a second container comprising:
[0158] one or more conjugates of a carrier protein and a capsular
saccharide from Streptococcus pneumoniae [where the capsular
saccharide is optionably from a pneumococcal serotype selected from
the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A,
11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F].
[0159] and a third container comprising:
[0160] an immunogenic composition of the invention as described
above (for instance those comprising Men or HibMen saccharide
conjugate combinations).
[0161] A further aspect of the invention is a process for making
the immunogenic composition or vaccine of the invention, comprising
the step of mixing the saccharides of the invention, for instance
mixing N. meningitidis capsular saccharides from at least one, two,
three or all four of serogroups A, C, W and Y conjugated to a
carrier protein with a pharmaceutically acceptable excipient.
[0162] A further aspect of the invention is a method of immunising
a human host against disease caused by bacteria, for example N.
meningitidis and optionally Haemophilus influenzae infection
comprising administering to the host an immunoprotective dose of
the immunogenic composition or vaccine or kit of the invention,
optionally using a single dose.
[0163] An independent aspect of the invention is a method of
immunising a human host with an immunogenic composition comprising
at least 2 different N. meningitidis capsular saccharide conjugates
selected from the group consisting of serogroup A, C, W and Y
(optionally MenA, C, W and Y) wherein a single dose administration
(optionally to teenagers, aldults or children) results in a blood
test taken one month after administration giving over 50%, 60%,
70%, 80%, 90% or 95% responders in an SBA assay measuring levels of
response against MenA, MenC, MenW and/or MenY. Optionally the SBA
assay is as described in Example 9 with responder assessed as
described in Example 9.
[0164] A further independent aspect of the invention is an
immunogenic composition comprising MenA MenC, MenW and/or MenY
conjugates which is capable of eliciting an immune response after a
single dose such that over 50%, 60%, 70%, 80%, 90% or 95% of human
subjects (children, teenagers or adults) inoculated are classified
as responders in an SBA assay on blood extracted a month after
inoculation (optionally using the criteria described in example
9).
[0165] Such an immunogenic composition optionally has the further
structural characteristics described herein.
[0166] A further aspect of the invention is an immunogenic
composition of the invention for use in the treatment or prevention
of disease caused by bacteria, for example N. meningitidis and
optionally Haemophilus influenzae infection.
[0167] 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 bacteria for example N. meningitidis and optionally Haemophilus
influenzae infection.
[0168] 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.
[0169] All references or patent applications cited within this
patent specification are incorporated by reference herein.
[0170] The invention is illustrated in the accompanying examples.
The examples below are carried out using standard techniques, which
are well known and routine to those of skill in the art, except
where otherwise described in detail. The examples are illustrative,
but do not limit the invention.
EXAMPLES
Example 1--Preparation of Polysaccharide Conjugates
[0171] The covalent binding of Haemophilus influenzae (Hib) PRP
polysaccharide to TT was carried out by a coupling chemistry
developed by Chu et al (Infection and Immunity 1983, 40 (1);
245-256). Hib PRP polysaccharide was activated by adding CNBr and
incubating at pH10.5 for 6 minutes. The pH was lowered to pH8.75
and adipic acid dihydrazide (ADH) was added and incubation
continued for a further 90 minutes. The activated PRP was coupled
to purifed tetanus toxoid via carbodiimide condensation using
1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC). EDAC was
added to the activated PRP to reach a final ratio of 0.6 mg EDAC/mg
activated PRP. The pH was adjusted to 5.0 and purified tetanus
toxoid was added to reach 2 mg TT/mg activated PRP. The resulting
solution was left for three days with mild stirring. After
filtration through a 0.45 .mu.m membrane, the conjugate was purifed
on a sephacryl S500HR (Pharmacia, Sweden) column equilibrated in
0.2M NaCl.
[0172] MenC-TT conjugates were produced using native
polysaccharides (of over 150 kDa as measured by MALLS) or were
slightly microfluidised. MenA-TT conjugates were produced using
either native polysaccharide or slightly microfluidised
polysaccharide of over 60 kDa as measured by the MALLS method of
example 2. MenW and
[0173] MenY-TT conjugates were produced using sized polysaccharides
of around 100-200 kDa as measured by MALLS (see example 2). Sizing
was by microfluidisation using a homogenizer Emulsiflex C-50
apparatus. The polysaccharides were then filtered through a 0.2
.mu.m filter.
[0174] Activation and coupling were performed as described in
WO96/29094 and WO 00/56360. Briefly, the polysaccharide at a
concentration of 10-20 mg/ml in 2M NaCl pH 5.5-6.0 was mixed with
CDAPsolution (100 mg/ml freshly prepared in acetonitrile/WFI,
50/50) to a final CDAP/polysaccharide ratio of 0.75/1 or 1.5/1.
After 1.5 minutes, the pH was raised with sodium hydroxide to
pH10.0. After three minutes tetanus toxoid was added to reach a
protein/polysaccharide ratio of 1.5/1 for MenW, 1.2/1 for MenY,
1.5/1 for MenA or 1.5/1 for MenC. The reaction continued for one to
two hours.
[0175] After the coupling step, glycine was added to a final ratio
of glycine/PS (w/w) of 7.5/1 and the pH was adjusted to pH9.0. The
mixture was left for 30 minutes. The conjugate was clarified using
a 10 .mu.m Kleenpak filter and was then loaded onto a Sephacryl
S400HR column using an elution buffer of 150 mM NaCl, 10 mM or 5 mM
Tris pH7.5. Clinical lots were filtered on an Opticap 4 sterilizing
membrane. The resultant conjugates had an average
polysaccharide:protein ratio of 1:1-1:5 (w/w).
Example 1a--Preparation of MenA and MenC Polysaccharide Conjugates
of the Invention
[0176] MenC-TT conjugates were produced using native
polysaccharides (of over 150 kDa as measured by MALLS) or were
slightly microfluidised. MenA-TT conjugates were produced using
either native polysaccharide or slightly microfluidised
polysaccharide of over 60 kDa as measured by the MALLS method of
example 2. Sizing was by microfluidisation using a homogenizer
Emulsiflex C-50 apparatus. The polysaccharides were then filtered
through a 0.2 .mu.m filter.
[0177] In order to conjugate MenA capsular polysaccharide to
tetanus toxoid via a spacer, the following method was used. The
covalent binding of the polysaccharide and the spacer (ADH) is
carried out by a coupling chemistry by which the polysaccharide is
activated under controlled conditions by a cyanylating agent,
1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). The
spacer reacts with the cyanylated PS through its hydrazino groups,
to form a stable isourea link between the spacer and the
polysaccharide.
[0178] A 10 mg/ml solution of MenA (pH 6.0) [3.5 g] was treated
with a freshly prepared 100 mg/ml solution of CDAP in
acetonitrile/water (50/50 (v/v)) to obtain a CDAP/MenA ratio of
0.75 (w/w). After 1.5 minutes, the pH was raised to pH 10.0. Three
minutes later, ADH was added to obtain an ADH/MenA ratio of 8.9.
The pH of the solution was decreased to 8.75 and the reaction
proceeded for 2 hours maintaining this pH (with temperature kept at
25.degree. C.).
[0179] The PSAAH solution was concentrated to a quarter of its
initial volume and then diafiltered with 30 volumes of 0.2M NaCl
using a Filtron Omega membrane with a cut-off of 10 kDa, and the
retentate was filtered.
[0180] Prior to the conjugation (carbodiimide condensation)
reaction, the purified TT solution and the PSAAH solution were
diluted to reach a concentration of 10 mg/ml for PSAAH and 10 mg/ml
for TT.
[0181] EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was
added to the PSAH solution (2 g saccharide) in order to reach a
final ratio of 0.9 mg EDAC/mg PSAAH. The pH was adjusted to 5.0.
The purified tetanus toxoid was added with a peristaltic pump (in
60 minutes) to reach 2 mg TT/mg PSAAH. The resulting solution was
left 60 min at +25.degree. C. under stirring to obtain a final
coupling time of 120 min. The solution was neutralised by addition
of 1M Tris-Hcl pH 7.5 ( 1/10 of the final volume) and left 30
minutes at +25.degree. C. then overnight at +2.degree. C. to
+8.degree. C. The conjugate was clarified using a 10 .mu.m filter
and was purified using a Sephacryl S400HR column (Pharmacia,
Sweden). The column was equilibrated in 10 mM Tris-HCl (pH 7.0),
0.075 M NaCl and the conjugate (approx. 660 mL) was loaded on the
column (+2.degree. C. to +8.degree. C.). The elution pool was
selected as a function of optical density at 280 nm. Collection
started when absorbance increased to 0.05. Harvest continued until
the Kd reached 0.30. The conjugate was filter sterilised at
+20.degree. C., then stored at +2.degree. C. to +8.degree. C. The
resultant conjugate had a polysaccharide:protein ratio of 1:2-1:4
(w/w).
[0182] In order to conjugate MenC capsular polysaccharide to
tetanus toxoid via a spacer, the following method was used. The
covalent binding of the polysaccharide and the spacer (ADH) is
carried out by a coupling chemistry by which the polysaccharide is
activated under controlled conditions by a cyanylating agent,
1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). The
spacer reacts with the cyanylated PS through its hydrazino groups,
to form a stable isourea link between the spacer and the
polysaccharide.
[0183] A 20 mg/ml solution of MenC (pH6.0) (3.5 g) was treated with
a freshly prepared 100 mg/ml solution of CDAP in acetonitrile/water
(50/50 (v/v)) to obtain a CDAP/MenC ratio of 1.5 (w/w). After 1.5
minutes, the pH was raised to pH 10.0. At activation pH 5M NaCl was
added to achieve a final concentration of 2M NaCl. Three minutes
later, ADH was added to obtain an ADH/MenC ratio of 8.9. The pH of
the solution was decreased to 8.75 and the reaction proceeded for 2
hours (retained at 25.degree. C.).
[0184] The PSCAH solution was concentrated to a minimum of 150 mL
and then diafiltered with 30 volumes of 0.2M NaCl using a Filtron
Omega membrane with a cut-off of 10 kDa, and the retentate was
filtered.
[0185] Prior to the conjugation reaction, the purified TT solution
and the PSCAH solution (2 g scale) were diluted in 0.2M NaCl to
reach a concentration of 15 mg/ml for PSCAH and 20 mg/ml for
TT.
[0186] The purified tetanus toxoid was added to the PSCAH solution
in order to reach 2 mg TT/mg PSCAH. The pH was adjusted to 5.0.
EDAC (16.7 mg/ml in Tris 0.1M pH 7.5) was added with a peristaltic
pump (in 10 minutes) to reach a final ratio of 0.5 mg EDAC/mg
PSCAH. The resulting solution was left 110 min at +25.degree. C.
under stirring and pH regulation to obtain a final coupling time of
120 min. The solution was then neutralized by addition of 1M
Tris-Hcl pH 9.0 ( 1/10 of final volume) and left 30 minutes at
+25.degree. C. then overnight at +2.degree. C. to +8.degree. C.
[0187] The conjugate was clarified using a 10 .mu.m filter and was
purified using a Sephacryl S400HR column (Pharmacia, Sweden). The
column was equilibrated in 10 mM Tris-HCl (pH 7.0), 0.075 M NaCl
and the conjugate (approx. 460 mL) was loaded on the column
(+2.degree. C. to +8.degree. C.). The elution pool was selected as
a function of optical density at 280 nm. Collection started when
absorbance increased to 0.05. Harvest continued until the Kd
reached 0.20. The conjugate was filter sterilised at +20.degree.
C., then stored at +2.degree. C. to +8.degree. C. The resultant
conjugate had a polysaccharide:protein ratio of 1:2-1:4 (w/w).
Example 2--Determination of Molecular Weight Using MALLS
[0188] Detectors were coupled to a HPLC size exclusion column from
which the samples were eluted. On one hand, the laser light
scattering detector measured the light intensities scattered at 16
angles by the macromolecular solution and on the other hand, an
interferometric refractometer placed on-line allowed the
determination of the quantity of sample eluted. From these
intensities, the size and shape of the macromolecules in solution
can be determined.
[0189] The mean molecular weight in weight (M.sub.w) is defined as
the sum of the weights of all the species multiplied by their
respective molecular weight and divided by the sum of weights of
all the species. [0190] a) Weight-average molecular weight:
-Mw-
[0190] M w = W i M i W i = m 2 m 1 ##EQU00001## [0191] b)
Number-average molecular weight: -Mn-
[0191] M n = N i M i N i = m 1 m 0 ##EQU00002## [0192] c) Root mean
square radius: -Rw- and R.sup.2w is the square radius defined
by:
[0192] R 2 w or ( r 2 ) w = m i r i 2 m i ##EQU00003## [0193]
(-m.sub.i- is the mass of a scattering centre i and -r.sub.i- is
the distance between the [0194] scattering centre i and the center
of gravity of the macromolecule). [0195] d) The polydispersity is
defined as the ratio -Mw/Mn-.
[0196] Meningococcal polysaccharides were analysed by MALLS by
loading onto two HPLC columns (TSKG6000 and 5000PW.times.I) used in
combination. 25 .mu.l of the polysaccharide were loaded onto the
column and was eluted with 0.75 ml of filtered water. The
polyaccharides are detected using a light scattering detector
(Wyatt Dawn DSP equipped with a 10 mW argon laser at 488 nm) and an
inferometric refractometer (Wyatt Otilab DSP equipped with a P100
cell and a red filter at 498 nm).
[0197] The molecular weight polydispersities and recoveries of all
samples were calculated by the Debye method using a polynomial fit
order of 1 in the Astra 4.72 software.
Example 3--Clinical Trial Comparing Immunisation with Meningitec or
a Larger Sized MenC-TT Conjugate
[0198] A phase II, open, controlled study was carried out to
compareGSK Biologicals meningococcal serogroup C conjugate vaccine
(MenC) with GSK Biological's Haemophilus influenzae b-meningococcal
serogroup C conjugate vaccine (Hib-MenC) or Meningitec.RTM.. Each
dose of Meningitec.RTM. contains 10 .mu.g of meningococcal
serogroup C oligosaccharide conjugated to 15 .mu.g of CRM197 and is
produced by Wyeth. The GSK MenC conjugates contained native
polysaccharides of about 200 kDa conjugated to tetanus toxoid
(TT).
[0199] The study consisted of five groups, each planned to contain
100 subjects, allocated to two parallel arms as follws:
[0200] In this present study, all subjects in both arms received
one-fifth (1/5) of a dose of Mencevax.TM. ACWY and a concomitant
dose of Infanrix.TM. hexa at 12-15 months of age (Study Month 0).
Two blood samples were collected from all subjects (Study Month 0
and Study Month 1). Arm 1 consisted of four groups from a primary
vaccination study who were primed at their age of 3, 4 and 5 months
with the following vaccines: [0201] Group K: MenC (10 .mu.g),
non-adsorbed (non-ads), tetanus toxoid (TT) conjugate and
Infanrix.TM. hexa (MenC10-TT+Infanrix.TM. hexa) [0202] Group L: Hib
(10 .mu.g)-MenC (10 .mu.g), non-ads TT conjugate and Infanrix.TM.
penta (Hib10-MenC10-TT+Infanrix.TM. penta) [0203] Group M: Hib (5
.mu.g)-MenC (5 .mu.g), non-ads, TT conjugate and Infanrix.TM. penta
(Hib5-MenC5-TT+Infanrix.TM. penta) [0204] Group N: Meningitec.TM.
and Infanrix.TM. hexa (Meningitec.TM.+Infanrix.TM. hexa)
[0205] The two Hib-MenC-TT vaccine groups (Groups L and M) were
kept blinded in the booster study as to the exact formulation of
the candidate vaccine.
[0206] Arm 2-(Group O) consisted of age-matched subjects not
previously vaccinated with a meningococcal serogroup C vaccine
(naive) but who had received routine pediatric vaccines according
to the German Permanent Commission on Immunization.
[0207] Criteria for Evaluation:
[0208] Immunogenicity:
[0209] Determination of bactericidal antibody titers against
meningococcal C (SBA-MenC) by a bactericidal test (cut-off: a
dilution of 1:8) and ELISA measurement of antibodies against
meningococcal serogroup C (assay cut-off: 0.3 .mu.g/ml), the Hib
polysaccharide PRP (assay cut-off: 0.15 .mu.g/ml) and tetanus
toxoid (assay cut-off: 0.1 IU/ml) in blood samples obtained prior
to vaccination and approximately one month after vaccination in all
subjects.
[0210] Statistical Methods:
[0211] Demographics:
[0212] Determination of mean age in months (with median, range and
standard deviation [SD]), and racial and gender composition of the
ATP and Total vaccinated cohorts.
[0213] Immunogenicity:
[0214] Two analyses of immunogenicity were performed based on the
ATP cohort for immunogenicity (for analyses of immune memory and
booster response) or the ATP cohort for safety (for analysis of
persistence). These included:
[0215] Evaluation of immune memory for MenC and booster response
for Hib and Tetanus (before and one month after administration of
1/5 dose of the plain polysaccharide vaccine): [0216] Determination
of geometric mean titers and concentrations (GMTs and GMCs) with
95% confidence intervals (95% CI) [0217] Determination of the
percentage of subjects with antibody titer/concentration above the
proposed cutoffs with exact 95% CI (seropositivity/seroprotection
rates) [0218] Investigation of antibody titers/concentration after
vaccination using reverse cumulative curves [0219] Computation of
standardized asymptotic 95% CI for the difference in
seropositivity/seroprotection rate [0220] between the primed group
(Groups K, L, M and N) and the unprimed group (Group O) [0221]
Determination of the geometric mean of individual ratio of SBA-MenC
titer over anti-PSC concentration, with 95% CI [0222] Determination
of the 95% CI for the post-vaccination GMT/C ratio between the
groups K, L, M and the control group N for anti-PRP and
anti-tetanus and between each primed group (Groups K, L, M and N)
and the unprimed group (Group O) for SBA-MenC and anti-PSC using an
ANOVA model
[0223] Results
TABLE-US-00001 TABLE 1 SBA-MenC titres and anti-PSC antibody
concentration after booster vaccination 95% CL Antibody Group N
GMT/C 95% CL LL UL SBA-MenC K-MenC- 71 3508.9 2580.1 4772.2 TT L-
79 2530.1 1831.7 3494.7 HibMenC M-HibMenC 81 5385.4 4425.0 6554.2
N- 85 1552.6 1044.4 2307.9 Meningitec O-Control 91 9.3 6.3 13.6
Anti-PSC K-MenC- 70 28.10 22.59 34.95 TT L- 71 30.01 24.09 37.38
HibMenC M-HibMenC 76 34.58 29.10 41.09 N- 78 16.59 12.98 21.21
Meningitec O-Control 94 3.05 2.36 3.93 Group K: subjects primed
with MenC10-TT + Infanrix. hexa; Group L: subjects primed with
Hib10-MenC10-TT + Infanrix. penta; Group M: subjects primed with
Hib5-MenC5-TT + Infanrix. penta; Group N: subjects primed with
Meningitec. + Infanrix. hexa; Group O: control subjects (i.e.
subjects not primed with MenC conjugate vaccine)
[0224] N: number of subjects with available results
[0225] Higher titres of antibodies against MenC and higher SBA
titres were achieved by priming with the larger sized MenC
polysaccharide conjugate vaccines (groups K, L and M) compared with
the Meningitec oligosaccharide conjugate vaccine.
TABLE-US-00002 TABLE 2 Geometric mean ratio for SBA MenC
titre/anti-PSC concentration Group Timing N GMR LL UL K Pre 70
49.470 34.939 70.044 Post 66 126.138 101.419 156.882 L Pre 76
36.528 25.849 51.621 Post 70 90.200 70.153 115.975 M Pre 77 51.298
36.478 72.139 Post 74 164.950 139.304 195.318 N Pre 84 22.571
16.521 30.837 Post 76 90.168 67.757 119.991 O Pre 3 91.634 0.651
12889.8 Post 87 2.708 1.767 4.149
[0226] In all four primed groups (Groups K, L, M and N), the GMR
increased significantly from pre to post booster vaccination
indicating the presence of antibody maturation and functionality.
GMR in the Group M (primed with Hib5-MenC5-TT) was higher than in
the Group N (primed with Meningitec.TM.).
TABLE-US-00003 TABLE 3 Persistence at 12-15 months of age just
prior to administration of the booster vaccines Endpoints Group N %
Group N % Difference Value % SBAMenC .gtoreq. K 79 88.6 N 91 80.2 N
- K -8.4 1:8 L 84 93.3 N 91 80.2 N - L -3.1 M 85 87.1 N 91 80.2 N -
M -6.8 SBAMenC .gtoreq. K 79 65.8 N 91 51.6 N - K -14.2 1:128 L 84
56.0 N 91 51.6 N - L -4.3 M 85 64.7 N 91 51.6 N - M -13.1 Anti-PSC
.gtoreq. K 79 100.0 N 91 100.0 N - K 0.0 0.3 .mu.g/ml L 84 100.0 N
91 100.0 N - L 0.0 M 88 98.9 N 91 100.0 N - M 1.1 Anti-PSC .gtoreq.
K 79 72.2 N 91 81.3 N - K 9.2 2 .mu.g/ml L 84 64.3 N 91 81.3 N - L
17.0 M 88 64.3 N 91 81.3 N - M 8.6 Anti-PRP .gtoreq. K 81 88.9 N 91
85.7 N - K -3.2 0.15 .mu.g/ml L 86 96.5 N 91 85.7 N - L -10.8 M 90
98.9 N 91 85.7 N - M -13.2 Anti-PRP .gtoreq. K 81 33.3 N 91 28.6 N
- K -4.8 1 .mu.g/ml L 86 55.8 N 91 28.6 N - L -27.2 M 90 74.4 N 91
28.6 N - M -45.9 Anti-tetanus .gtoreq. K 81 100.0 N 91 96.7 N - K
-3.3 0.1 IU/ml L 86 100.0 N 91 96.7 N - L -3.3 M 90 100.0 N 91 96.7
N - M -3.3 Group K: subjects primed with MenC10-TT + Infanrix .TM.
hexa; Group L: subjects primed with Hib10-MenC10-TT + Infanrix .TM.
penta; Group M: subjects primed with Hib5-MenC5-TT + Infanrix .TM.
penta; Group N: subjects primed with Meningitec .TM. + Infanrix
.TM. hexa; N: number of subjects with available results Higher SBA
titres against MenC were achieved by priming with the larger size
of MenC (groups K, L and M) compared to priming with the
MenC-oligosaccharide conjugate Meningitec .TM..
[0227] Immune Memory (ATP Cohort for Immunogenicity)
[0228] Administration of 1/5 dose of the plain polysaccharide ACWY
vaccine elicited very high SBA-MenC titer in all four primed groups
with 98.7-100% and 97.5-100% of subjects primed with a candidate
vaccine regimen exhibiting titers .gtoreq.8 and .gtoreq.128,
respectively. In the group primed with the Meningitec.TM. regimen,
there was a trend for a lower percentage of subjects with titers
.gtoreq.128 (91.8%). In comparison, 17.6% of unprimed subjects had
SBA MenC titers .gtoreq.1:8 and .gtoreq.128.
Example 4 Phase II Clinical Trial on HibMenAC-TT Conjugate Vaccine
Mixed with DTPw-HepB
[0229] Study Design:
[0230] Open, randomized (1:1:1:1:1), single centre study with five
groups. The five groups received the
[0231] following vaccination regimen respectively, at 6, 10 and 14
weeks of age. [0232] Tritanrix.TM.-HepB/Hib-MenAC 2.5/2.5/2.5:
henceforth referred to as 2.5/2.5/2.5 [0233]
Tritanrix.TM.-HepB/Hib-MenAC 2.5/5/5: henceforth referred to as
2.5/5/5 [0234] Tritanrix.TM.-HepB/Hib-MenAC 5/5/5: henceforth
referred to as 5/5/5 [0235] Tritanrix.TM.-HepB+Hiberix.TM.
henceforth referred to as Hiberix [0236]
Tritanrix.-HepB/Hiberix.TM. Meningitec.TM.: henceforth referred to
as Meningitec
[0237] Blood samples were taken at the time of the first vaccine
dose (Pre) and one month after the third vaccine dose (Post-dose
3).
[0238] Tritanrix is a DTPw vaccine marketted by GlaxoSmithKline
Biologicals S.A.
[0239] 105 subjects were used in each of the five groups giving a
total of 525 subjects in the study.
TABLE-US-00004 TABLE 4 Content of GSK vaccine formulations
Components per dose (0.5 ml) 2.5/2.5/2.5* 2.5/5/5 5/5/5 Hib
capsular polysaccharide PRP 2.5 .mu.g 2.5 .mu.g 5 .mu.g conjugated
to tetanus toxoid (TT) Neisseria meningitidis A capsular 2.5 .mu.g
5 .mu.g 5 .mu.g polysaccharide (PSA) conjugated to TT Neisseria
meningitidis C capsular 2.5 .mu.g 5 .mu.g 5 .mu.g polysaccharide
(PSC) conjugated to TT *The 2.5/2.5/2.5 vaccine was a dose dilution
of GSK Biologicals' Hib-MenAC 5/5/5 vaccine containing 2.5 .mu.g of
each of PRP-TT, MenA-TT and MenC-TT.
[0240] The Hib-MenAC vaccine formulations were mixed
extemporaneously with Tritanirix-HepB. GSK Biologicals' combined
diphtheria-tetanus-whole cell Bordetella pertussis--hepatitis B
(DTPw-HB) vaccine (Tritanrix-HepB) contains not less than 30
International Units (IU) of diphtheria toxoid, not less than 60 IU
of tetanus toxoid, not less than 4 IU of killed Bordetella
pertussis and 10 .mu.g of recombinant hepatitis B surface
antigen.
[0241] Reference Therapy, Dose, Mode of Administration, Lot
No.:
[0242] Vaccination Schedule/Site:
[0243] One group received Tritanrix.-HepB vaccine intramuscularly
in the left thigh and Hiberix. intramuscularly in the right thigh
at 6, 10 and 14 weeks of age. Another group received
Tritanrix.-HepB/Hiberix. vaccine intramuscularly in the left thigh
and Meningitec. vaccine intramuscularly in the right thigh at 6, 10
and 14 weeks of age.
[0244] Vaccine/Composition/Dose/Lot Number:
[0245] The Tritanrix.-HepB vaccine used was as described above.
[0246] One dose (0.5 ml) of GSK Biologicals' Haemophilus influenzae
type b conjugate vaccine: Hiberix.TM. contained 10 .mu.g of PRP
conjugated to tetanus toxoid. In the Hiberix.TM. Group, it was
mixed with sterile diluent and in the Meningitec.TM. Group it was
mixed with Tritanrix.TM.-HepB.
[0247] One dose (0.5 ml) of Wyeth Lederle's MENINGITEC.TM. vaccine
contained: 10 .mu.g of capsular oligosaccharide of meningococcal
group C conjugated to 15 .mu.g of Corynebacterium diphtheria CRM197
protein and aluminium as salts.
[0248] Results--Immune Responses Generated Against Hib, MenA and
MenC
TABLE-US-00005 TABLE 5a Anti-PRP (.mu.g/ml) Group 2.5/2.5/2.5
2.5/5/5 5/5/5 Hiberix .TM. Meningitec .TM. % 95% CL % 95% CL % 95%
CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL
UL GMC/T LL UL %.gtoreq.0.15 100 96.5 100 99.0 94.8 100 100 96.5
100 100 96.5 100 100 96.5 100 GMC 20.80 15.96 27.10 22.62 17.72
28.88 19.36 15.33 24.46 38.55 29.93 49.64 10.94 8.62 13.88
TABLE-US-00006 TABLE 5b SBA-MenC Group 2.5/2.5/2.5 2.5/5/5 5/5/5
Hiberix .TM. Meningitec .TM. % 95% CL % 95% CL % 95% CL % 95% CL %
95% CL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL
%.gtoreq.1:8 99 94.7 100 100 96.5 100 100 96.5 100 2.9 0.6 8.4 100
96.5 100 GMT 3132 2497 3930 4206 3409 5189 3697 3118 4384 4.7 3.9
5.6 4501 3904 5180
TABLE-US-00007 TABLE 5c SBA MenA Group 2.5/2.5/2.5 2.5/5/5 5/5/5
Hiberix .TM. Meningitec .TM. % 95% CL % 95% CL % 95% CL % 95% CL %
95% CL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL UL
%.gtoreq.1:8 99.7 91.9 99.7 100 95.8 100 100 96.2 100 6.8 2.5 14.3
9.1 4.0 17.1 GMT 316.7 251.4 398.9 418.5 358.6 488.5 363 310.5
424.4 5.6 4.3 7.4 5.6 4.4 7.2
TABLE-US-00008 TABLE 5d Anti-PSC (.mu.g/ml) Group 2.5/2.5/2.5
2.5/5/5 5/5/5 Hiberix .TM. Meningitec .TM. % 95% CL % 95% CL % 95%
CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL
UL GMC/T LL UL %.gtoreq.0.3 100 96.5 100 100 96.4 100 100 96.5 100
8.2 3.6 15.6 100 96.5 100 GMC 49.03 43.24 55.59 71.11 62.49 80.92
61.62 54.88 69.20 0.17 0.15 0.19 58.02 51.42 65.46
TABLE-US-00009 TABLE 5e Anti-PSA (.mu.g/ml) Group 2.5/2.5/2.5
2.5/5/5 5/5/5 Hiberix .TM. Meningitec .TM. % 95% CL % 95% CL % 95%
CL % 95% CL % 95% CL GMC/T LL UL GMC/T LL UL GMC/T LL UL GMC/T LL
UL GMC/T LL UL %.gtoreq.0.3 100 96.4 100 100 96.5 100 99.0 94.8 100
1.0 0.0 5.4 5.9 2.2 12.5 GMC 18.10 15.34 21.35 26.51 22.93 30.79
23.40 20.05 27.30 0.15 0.15 0.15 0.17 0.15 0.18
Conclusion
[0249] A comparison of the immunogenicity results achieved using
the oligosaccharide MenC-CRM197 conjugate vaccine and the three GSK
formulations which contain polysacharide MenA-TT and MenC-TT
conjugates showed that the polysaccharide Men conjugates were able
to elicit a good immunogenic response similar to that achieved
using the oligosaccharide conjugate vaccine Meningitec. All
formulations tested gave a response to MenC in 100% of
patients.
Example 5--Phase II Clinical Trial Administering Hib MenCY
Concomitantly with Infanrix Penta According to a 2, 3 and 4 Month
Schedule
[0250] Study Design:
[0251] A Phase II, open (partially double-blind*) randomized
controlled multi-center study with 5 groups receiving a three-dose
primary schedule with vaccines as follows:
[0252] Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5)+Infanrix.TM.
penta
[0253] Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10)+Infanrix.TM.
penta
[0254] Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5)+Infanrix.TM.
penta
[0255] Group Hib-MenC: Hib-MenC (5/5)+Infanrix.TM. penta
[0256] Group Menjugate: Menjugate.TM.**+Infanrix.TM. hexa
(control). *Hib-MenCY 2.5/5/5, Hib-MenCY 5/10/10 and Hib-MenC were
administered in a double-blind manner while the Hib-MenCY 5/5/5
group and the Menjugate group were open. The 2.5/5/5, 5/10/10 and
5/5/5 formulations of Hib-MenCY contain MenC native polysaccharides
and MenY polysaccharides which are microfluidized.**Menjugate.TM.
contains 10 .mu.g of MenC oligosaccharides conjugated to 12.5-25
.mu.g of CRM197 per dose and is produced by Chiron.
[0257] Vaccination at +/-2, 3, 4 months of age (Study Month 0,
Month 1 and Month 2), and blood samples (3.5 ml) from all subjects
prior to and one month post primary vaccination (Study Month 0 and
Month 3).
[0258] Study Vaccine, Dose, Mode of Administration, Lot Number:
[0259] Three doses injected intramuscularly at one month intervals,
at approximately 2, 3 and 4 months of age as follows:
TABLE-US-00010 TABLE 6 Vaccines administered (study and control),
group, schedule/site and dose Concomitant Vaccine dose vaccine
Schedule administered administered (months Site-Left upper Site
Right upper Group of age) thigh thigh Hib-MenCY 2, 3, and 4 Hib
(2.5 .mu.g)- DTPa-HBV-IPV 2.5/5/5 MenC-TT (5 .mu.g)- (Infanrix .TM.
penta) MenY-TT (5 .mu.g) Hib-MenCY 2, 3, and 4 Hib (5 .mu.g)-MenC-
DTPa-HBV-IPV 5/10/10 TT (10 .mu.g)-MenY- (Infanrix .TM. penta) TT
(10 .mu.g) Hib-MenCY 5/5/5 2, 3, and 4 Hib (5 .mu.g)-MenC-
DTPa-HBV-IPV TT (5 .mu.g)-MenY- (Infanrix .TM. penta) TT (5 .mu.g)
Hib-MenC 2, 3, and 4 Hib (5 .mu.g)-Men C DTPa-HBV-IPV (5 .mu.g)
(Infanrix .TM. penta) Menjugate .TM. 2, 3, and 4 Menjugate .TM.
DTPa-HBV- IPV/Hib (Infanrix .TM.hexa)
[0260] Immunogenicity:
[0261] Measurement of antibody titres/concentrations against each
vaccine antigen:
[0262] Prior to the first dose (Month 0) and approximately one
month after the third dose (Month 3) in all subjects for: SBA-MenC
and SBA-MenY, anti-PSC and anti-PSY, anti-PRP, anti-T, anti-FHA,
anti-PRN and anti-PT. Using serum bactericidal activity against N.
meningitidis serogroups C and Y (SBA-MenC and SBA-MenY cut-off: 1:8
and 1:128); ELISA assays with cut-offs: .gtoreq.0.3 .mu.g/ml and
.gtoreq.2 .mu.g/ml for anti-N. meningitidis serogroups C and Y
polysaccharides (anti-PSC IgG and anti-PSY IgG); .gtoreq.0.15
.mu.g/ml and .gtoreq.1.0 .mu.g/ml for Hib polysaccharide
polyribosil-ribitol-phosphate (anti-PRP IgG); 5ELU/ml for
anti-FHA,
[0263] anti-PRN, anti-PT; .gtoreq.0.1 IU/ml anti-tetanus toxoid
(anti-TT). Only at one month after the third dose (Month 3) in all
subjects for: anti-D, anti-HBs and anti-polio 1, 2 and 3.
[0264] Using ELISA assays with cut-offs: 0.1 IU/ml for
anti-diphtheria (anti-D); 0 mIU/ml for antihepatitis B (anti-HBs);
and microneutralization test cut-off: 1:8 for anti-polio type 1, 2
and 3 (anti-polio 1, 2 and 3).
[0265] Statistical Methods:
[0266] The seroprotection/seropositivity rates and geometric mean
concentrations/titres (GMCs/GMTs) with 95% confidence intervals
(95% CI) were computed per group, for SBA-MenC, anti-PSC, SBA-MenY,
anti-PSY, anti-PRP, anti-Tetanus, anti-PT, anti-FHA and anti-PRN
prior to and one month after vaccination; for anti-Diphtheria,
anti-HBs, anti-Polio 1, anti-Polio 2 and anti-Polio 3 one month
after vaccination. Vaccine response (appearance of antibodies in
subjects initially seronegative or at least maintenance of antibody
concentrations in subjects initially seropositive) with 95% CI for
anti-PT, anti-
[0267] PRN and anti-FHA were also computed one month after
vaccination. Reverse cumulative curves for each antibody at Month 3
are also presented. The differences between the Hib-MenCY and the
Hib-MenC groups, compared with the Menjugate.TM. control group were
evaluated in an exploratory manner for each antibody, except for
SBA-MenY and anti-PSY, in terms of (1) the difference between the
Menjugate.TM. group (minus) the Hib-MenCY and Hib-MenC groups for
the percentage of subjects above the specified cut-offs or with a
vaccine response with their standardized asymptotic 95% CI, (2) the
GMC or GMT ratios of the Menjugate.TM. group over the Hib-MenCY and
Hib-MenC groups with their 95% CI. The same comparisons were done
to evaluate the difference between each pair of Hib-MenCY
formulations for anti-PRP, SBA-MenC, anti-PSC, SBA-MenY, anti-PSY
and anti-TT antibodies.
[0268] The overall incidences of local and general solicited
symptoms were computed by group according to the type of symptom,
their intensity and relationship to vaccination (as percentages of
subjects reporting general, local, and any solicited symptoms
within the 8 days following vaccination and their exact 95% CI).
Incidences of unsolicited symptoms were computed per group. For
Grade 3 symptoms, onset .ltoreq.48 hours, medical attention,
duration, relationship to vaccination and outcomes were provided.
Serious Adverse Events were fully described.
[0269] Seroprotection/Seropositivity Rates & GMC/Ts (ATP Cohort
for Immunogenicity)
TABLE-US-00011 TABLE 7a Anti-PRP (.mu.g/ml) Group N % .gtoreq. 0.15
LL UL .gtoreq.1 LL UL GMC LL UL Hib MenCY 67 100.0 94.6 100.0 98.5
92.0 100.0 9.01 7.25 11.21 2.5/5/5 Hib MenCY 67 100.0 94.6 100.0
98.5 92.0 100.0 9.49 7.72 11.65 5/10/10 Hib MenCY 70 100.0 94.9
100.0 98.6 92.3 100.0 8.08 6.53 9.98 5/5/5 Hib MenC 74 100.0 95.1
100.0 98.6 92.7 100.0 10.44 8.49 12.83 Menjugate .TM. 71 100.0 94.9
100.0 80.3 69.1 88.8 2.60 1.97 3.43
TABLE-US-00012 TABLE 7b SBA-MenC (Titre) Group N % .gtoreq. 1:8 LL
UL .gtoreq.1:128 LL UL GMT LL UL Hib MenCY 2.5/5/5 70 100.0 94.9
100.0 95.7 88.0 99.1 1005.8 773.5 1308.0 Hib MenCY 67 100.0 94.6
100.0 94.0 85.4 98.3 1029.8 799.7 1326.0 5/10/10 Hib MenCY 5/5/5 71
100.0 94.9 100.0 94.4 86.2 98.4 906.9 691.3 1189.8 Hib MenC 74
100.0 95.1 100.0 95.9 88.6 99.2 871.0 677.3 1120.0 Menjugate .TM.
71 100.0 94.9 100.0 100.0 94.9 100.0 3557.6 2978.8 4248.8
TABLE-US-00013 TABLE 7c Anti-PSC (.mu.g/ml) Group N % .gtoreq. 0.3
LL UL .gtoreq.2 LL UL GMC LL UL Hib MenCY 2.5/5/5 69 100.0 94.8
100.0 100.0 94.8 100.0 21.70 18.36 25.65 Hib MenCY 66 100.0 94.6
100.0 100.0 94.6 100.0 27.26 23.26 31.95 5/10/10 Hib MenCY 5/5/5 70
100.0 94.9 100.0 100.0 94.9 100.0 19.02 16.49 21.93 Hib MenC 74
100.0 95.1 100.0 100.0 95.1 100.0 21.08 18.24 24.35 Menjugate .TM.
71 100.0 94.9 100.0 100.0 94.9 100.0 38.49 33.64 44.05
TABLE-US-00014 TABLE 7d SBA-MenY (Titre) Group N % .gtoreq. 1:8 LL
UL .gtoreq.1:128 LL UL GMT LL UL Hib MenCY 2.5/5/5 69 97.1 89.9
99.6 92.8 83.9 97.6 470.7 351.1 631.2 Hib MenCY 66 97.0 89.5 99.6
86.4 75.7 93.6 437.1 322.0 593.4.8 5/10/10 Hib MenCY 5/5/5 71 98.6
92.4 100.0 95.8 88.1 99.1 635.3 501.5 804.8 Hib MenC 74 21.6 12.9
32.7 13.5 6.7 23.5 9.3 6.3 13.7 Menjugate .TM. 71 19.7 11.2 30.9
9.9 4.1 19.3 7.5 5.4 10.4
TABLE-US-00015 TABLE 7e Anti-PSY (.mu.g/ml) Group N % .gtoreq. 0.3
LL UL .gtoreq.2 LL UL GMC LL UL Hib MenCY 2.5/5/5 69 100.0 94.8
100.0 100.0 94.8 100.0 26.86 22.86 31.56 Hib MenCY 66 100.0 94.6
100.0 100.0 94.6 100.0 37.02 31.84 43.04 5/10/10 Hib MenCY 5/5/5 70
100.0 94.9 100.0 100.0 94.9 100.0 23.57 19.94 27.86 Hib MenC 74 8.1
3.0 16.8 4.1 0.8 11.4 0.19 0.15 0.25 Menjugate .TM. 71 5.6 1.6 13.8
1.4 0.0 7.6 0.17 0.15 0.19
TABLE-US-00016 TABLE 7f Anti-tetanus (IU/ml) Group N % .gtoreq. 0.1
LL UL GMC LL UL Hib MenCY 2.5/5/5 68 100.0 94.7 100.0 3.06 2.63
3.55 Hib MenCY 67 100.0 94.6 100.0 3.25 2.88 3.68 5/10/10 Hib MenCY
5/5/5 70 100.0 94.9 100.0 2.97 2.59 3.41 Hib MenC 74 100.0 95.1
100.0 3.15 2.73 3.64 Menjugate .TM. 71 100.0 94.9 100.0 1.66 1.39
1.97 Group Hib-MenCY 2.5/5/5: Hib-MenCY (2.5/5/5) + Infanrix .TM.
penta Group Hib-MenCY 5/10/10: Hib-MenCY (5/10/10) + Infanrix .TM.
penta Group Hib-MenCY 5/5/5: Hib-MenCY (5/5/5) + Infanrix .TM.
penta Group Hib-MenC: Hib-Men (5/5) + Infanrix .TM. hexa Group
Menjugate: Menjugate .TM. + Infanrix .TM. penta N = number of
subjects with available results. % = percentage of subjects with
concentration/titre within the specified range GMC/T: geometric
mean concentration/titre 95% CI = 95% confidence interval; LL =
Lower Limit; UL = Upper Limit
Conclusion
[0270] The MenC and Y polysaccharide conjugates produced a good
immune response in all subjects with 100% of subjects producing
above 0.3 .mu.g/ml responses against MenC and MenY.
Example 6--Phase II Clinical Trial Comparing Three Formulations of
MenACWY-TT with Meningitec MenC-CRM197 Oligosaccharide-Conjugate
Vaccine
[0271] This example reports a phase II, open (partially-blind),
randomized, controlled dose-range study to evaluate the
Immunogenicity of three different formulations of GlaxoSmithKline
Biological's meningococcal serogroups A, C, W-135, Y tetanus toxoid
conjugate (MenACWY-TT) vaccine in comparison to a MenC
oligosaccharide-CRM197 conjugate vaccine (Meningitec.TM.) when
given as one dose to children aged 12-14 months.
[0272] The clinical trial was an open (partially double-blind*),
controlled, multicentric study in which eligible subjects of 12-14
months were randomized (1:1:1:1) to one of four parallel groups of
50 subjects to receive a single primary dose at Visit 1 as follows:
*The three different MenACWY-TT formulations were administered in a
double-blind manner.
[0273] Form 1T: MenACWY-TT at a dose of 2.5 .mu.g of MenA
polysaccharide conjugated to tetanus toxoid (TT), 2.5 .mu.g of MenC
polysaccharide conjugated to TT, 2.5 .mu.g of MenW polysaccharide
conjugated to TT and 2.5 .mu.g of MenY polysaccharide conjugated to
TT.
[0274] Form 2T: MenACWY-TT at a dose of 5 .mu.g of MenA
polysaccharide conjugated to TT, 5 .mu.g of MenC polysaccharide
conjugated to TT, 5 .mu.g of MenW polysaccharide conjugated to TT
and 5 .mu.g of MenY polysaccharide conjugated to TT.
[0275] Form 3T: MenACWY-TT at a dose of 2.5 .mu.g of MenA
polysaccharide conjugated to TT, 10 .mu.g of MenC polysaccharide
conjugated to TT, 2.5 .mu.g of MenW polysaccharide conjugated to TT
and 2.5 .mu.g of MenY polysaccharide conjugated to TT.
[0276] Ctrl T: 10 .mu.g MenC oligosaccharide conjugated to 12.5-25
.mu.g CRM197 (Meningitec).
[0277] Vaccination Schedule/Site:
[0278] A single vaccine dose was administered intramuscularly in
the left deltoid at Visit 1 (Study Month 0) according to randomized
assignment. All candidate vaccines were supplied as a lyophilized
pellet in a monodose vial (0.5 ml after reconstitution with the
supplied saline diluent).
[0279] Immunogenicity:
[0280] Measurement of titers/concentrations of antibodies against
meningococcal vaccine antigen components in blood samples obtained
prior to the study vaccine dose (Month 0) and approximately one
month after the study vaccine dose (Month 1) in all subjects.
Determination of bactericidal antibody titers against N.
meningitidis serogroups A, C, W-135 and Y (SBA-MenA, SBA-MenC,
SBA-MenW and SBA-MenY) by a bactericidal test (assay cut-offs: a
dilution of 1:8 and 1:128) and ELISA measurement of antibodies
against N. meningitidis serogroups A, C, W-135 and Y (anti-PSA,
anti-PSC, anti-PSW and anti-PSY, assay cut-offs .gtoreq.0.3.mu.g/ml
and .gtoreq.2 .mu.g/ml), and tetanus toxoid (anti-tetanus, assay
cut-off 0.1 IU/ml).
[0281] Results
[0282] Antibody response in terms of the percentage of SBA-MenA,
SBA-MenC, SBA-MenW and SBA-MenY responders one month after
vaccination (the primary endpoint) is shown in Table 8. A response
is defined as greater than or equal to a 4-fold increase for
seropositive subjects or seroconversion for seronegative subjects
before vaccination.
TABLE-US-00017 TABLE 8 Vaccine responses for SBA antibody one month
after vaccination Antibody Group N % LL UL SBA-MenA Form 1T 42 61.9
45.6 76.4 Form 2T 39 82.1 66.5 92.5 Form 3T 40 62.5 45.8 77.3
Meningitec .TM. 36 11.1 3.1 26.1 SBA-MenC Form 1T 46 97.8 88.5 99.9
Form 2T 43 100.0 91.8 100.0 Form 3T 44 95.5 84.5 99.4 Meningitec
.TM. 49 91.8 80.4 97.7 SBA-MenW Form 1T 45 100.0 92.1 100.0 Form 2T
43 97.7 87.7 99.9 Form 3T 45 100.0 92.1 100.0 Meningitec .TM. 46
15.2 6.3 28.9 SBA-MenY Form 1T 47 97.9 88.7 99.9 Form 2T 44 88.6
75.4 96.2 Form 3T 45 93.3 81.7 98.6 Meningitec .TM. 49 4.1 0.5
14.0
[0283] Table 9 shows the numbers of subjects achieving SBA titres
over cutoff points of 1:8 and 1:128 as well as GMTs.
TABLE-US-00018 TABLE 9 Seropositivity rates and GMTs for SBA
antibodies one month after vaccination .gtoreq.1:8 .gtoreq.1:128
Group N % LL UL % LL UL GMT SBA- Form 1T 46 100 92.3 100 100 92.3
100 1457.3 MenA Form2T 45 100 92.1 100 97.8 88.2 99.9 1776.9 Form3T
48 97.9 88.9 99.9 97.9 88.9 99.9 1339.5 Meningitec .TM. 41 51.2
35.1 67.1 43.9 28.5 60.3 42.8 SBA- Form 1T 47 97.9 88.7 99.9 78.7
64.3 89.3 281.3 MenC Form2T 45 100 92.1 100 84.4 70.5 93.5 428.6
Form3T 47 95.7 85.5 99.5 85.1 71.7 93.8 478.4 Meningitec .TM. 50
94.0 83.5 98.7 62.0 47.2 75.3 200.1 SBA- Form 1T 47 100 92.5 100
100 92.5 100 2529.1 MenW Form2T 45 100 92.1 100 100 92.1 100 2501.6
Form3T 48 100 92.6 100 97.9 88.9 99.9 2300.2 Meningitec .TM. 48
27.1 15.3 41.8 6.3 1.3 17.2 9.4 SBA- Form 1T 47 100 92.5 100 100
92.5 100 1987.4 MenY Form2T 45 100 92.1 100 100 92.1 100 2464.8
Form3T 48 100 92.6 100 97.9 88.9 99.9 2033.7 Meningitec .TM. 49
49.0 34.4 63.7 28.6 16.6 43.3 25.0
[0284] Vaccination with all three formulations of the ACWY-TT
polysaccharide conjugate led to good SBA responses against MenA,
MenC, MenW and MenY with 95-100% of subjects with titres greater
than 1:8. In particular, the 5/5/5/5 and 2.5/10/2.5/2.5
formulations of the polysaccharide conjugates produced a higher
response against MenC than the oligosaccharide Meningitic.TM.
vaccine as seen by a higher proportion of subjects having a titre
greater than 1:128 and the GMT readings.
TABLE-US-00019 TABLE 10 Seropositivity rates and GMCs for anti
polysaccharide antibodies one month after vaccination .gtoreq.0.3
.gtoreq.2 .mu.g/ml .mu.g/ml GMC Group N % LL UL % LL UL .mu.g/ml
Anti- Form 1T 47 93.6 82.5 98.7 68.1 52.9 80.9 2.35 MenA Form2T 45
100 92.1 100 64.4 48.8 78.1 3.11 Form3T 48 95.8 85.7 99.5 37.5 24.0
52.6 1.65 Meningitec .TM. 50 10.0 3.3 21.8 2.0 0.1 10.6 0.18 Anti-
Form 1T 47 100 92.5 100 100 92.5 100 9.57 MenC Form2T 45 100 92.1
100 100 92.1 100 12.53 Form3T 47 100 92.5 100 97.9 88.7 99.9 19.29
Meningitec .TM. 49 98.0 89.1 99.9 93.9 83.1 98.7 7.95 Anti- Form 1T
47 100 92.5 100 80.9 66.7 90.9 4.56 MenW Form2T 45 100 92.1 100
93.3 81.7 98.6 6.83 Form3T 48 93.8 82.8 98.7 72.9 58.2 84.7 2.88
Meningitec .TM. 50 0.0 0.0 7.1 0.0 0.0 7.1 0.15 Anti- Form 1T 47
100 92.5 100 97.9 88.7 99.9 8.90 MenY Form2T 45 100 92.1 100 100
92.1 100 12.78 Form3T 47 97.9 88.7 99.9 87.2 74.3 95.2 5.67
Meningitec .TM. 50 2.0 0.1 10.6 0.0 0.0 7.1 0.15
[0285] All three formulations of the ACWY-TT polysaccharide
conjugate vaccine produced good immune responses against MenA,
MenC, MenW and MenY with between 93% and 100% of subjects achieving
titres grater than 0.3 .mu.g/ml. Higher GMC readings were achieved
using the 5/5/5/5 and 2/5/10/2.5/2.5 formulations of the ACWY-TT
polysaccharide conjugate vaccine in comparison with
Meningitec.TM..
Example 7--Comparison of Immunogenicity of Native and Sized MenY
Polysaccharide Conjugates
[0286] Mice (female DBA/2 of 6-8 wk) received two injections, 2
weeks apart, of PSY-TT by the subcutaneous route. Blood samples
were taken 14 days after the second injection in order to perform
anti-PSY ELISA and SBA using S1975 menY strain. Per injection, mice
received 1 .mu.g of PSY-TT (lyo non-ads formulation).
[0287] The conjugates described in table 11 were used.
TABLE-US-00020 TABLE 11 Conjugates ENYTT012 ENYTT014 ENYTT015 bis
PSY NO Yes (40 cycles) Yes (20 cycles) microfluidisation TT/PS
ratio 1/1 1/1 1/1
[0288] Results
[0289] The results (FIG. 1) show a trend towards higher
immunogenicity for conjugates prepared using sized PSY. FIG. 1A
shows the GMC results obtained in an ELISA for antisera raised
against conjugates prepared from native MenY (ENYTT012),
microfluidised MenY-40 cycles (ENYTT014) and microfluidised MenY-20
cycles (ENYTT015 bis). Higher GMCs were obtained where the MenY-TT
was prepared from microfluidised MenY.
[0290] Similar results were obtained when the antisera were
assessed by SBA assay (FIG. 1B). Again the higher GMT values were
achieved using conjugates prepared from microfluidised MenY.
Example 8--Clinical Trial Assessing the Effect of a Linker in MenA
in a MenACWY Conjugate Vaccine
[0291] A single dose of different formulations of MenACWY vaccine
was administered to teenagers of 15-19 years in 5 groups of 25
subjects in a 1:1:1:1:1 randomised trial. The formulations tested
were:
[0292] F1--MenACWY conjugated to tetanus toxoid with the MenA
conjugate containing an AH spacer--5/5/5/5 .mu.g
[0293] F2--MenACWY conjugated to tetanus toxoid with the MenA
conjugate containing an AH spacer--2.5/5/2.5/2.5 .mu.g
[0294] F3--MenACWY conjugated to tetanus toxoid with the MenA
conjugate containing an AH spacer--5/5/2.5/2.5 .mu.g
[0295] F4--MenACWY conjugated to tetansus toxoid with no spacer in
any conjugate--5/5/5/5 .mu.g
[0296] Control group--Mencevax.TM. ACWY
[0297] On day 30 after inoculation, a blood sample was taken from
the patients.
[0298] The blood samples were used to asess the percentage of
SBA-MenA, SBA-MenC, SBA-MenW135 and SBA-MenY responders one month
after the vaccine dose. A vaccine response was defined as 1) for
initially seronegative subjects--a post-vaccination antibody titre
1/32 at 1 month or 2) for initially seropositive subjects--antibody
titre of 4 fold the pre-vaccination antibody titre.
[0299] Results
[0300] As shown in Table 13, the use of a spacer in the MenA
conjugate led to an increased immune response against MenA. The
percentage of responders rose from 66% to 90-95% when the AH spacer
was added. This was reflected in an increase in SBA GMT from 4335
to 10000 and an increase in GMC from 5 to 20-40. Surprisingly, the
use of a AH spacer also led to an increased immune response against
MenC as seen by an increase in the percentage of responders and an
increase in the SBA GMT. An increase could also be seen in the
SBA-GMT against MenY (6742-7122) and against MenW (4621-5418) when
a spacer was introduced.
TABLE-US-00021 TABLE 12 % SBA MenA SBA-MenA Anti-PSA GMC
Formulation responders GMT .mu.g/ml ELISA F 1 5AH/5/5/5 90.9 9805
20.38 F2 2.5AH/5/2.5/2.5 75 8517 29.5 F3 5AH/5/2.5/2.5 95.5 10290
47.83 F4 5/5/5/5 66.7 4335 5.46 Mencevax .TM. 85.7 8022 27.39 % SBA
MenC SBA-MenC Anti-PSC GMC Formulation responders GMT .mu.g/ml
ELISA F 1 5AH/5/5/5 69.6 3989 12.11 F2 2.5AH/5/2.5/2.5 81.8 3524
12.78 F3 5AH/5/2.5/2.5 81.8 3608 8.4 F4 5/5/5/5 73.9 2391 8.84
Mencevax .TM. 90.0 5447 38.71 % SBA MenW SBA-MenW Anti-PSW GMC
Formulation responders GMT .mu.g/ml ELISA F 1 5AH/5/5/5 95 5418
9.65 F2 2.5AH/5/2.5/2.5 85 4469 14.55 F3 5AH/5/2.5/2.5 95.5 4257
6.39 F4 5/5/5/5 95.5 4621 10.7 Mencevax .TM. 86.4 2714 13.57 % SBY
MenY SBA-MenY Anti-PSY GMC Formulation responders GMT .mu.g/ml
ELISA F 1 5AH/5/5/5 91.3 7122 16.3 F2 2.5AH/5/2.5/2.5 87.5 5755
12.52 F3 5AH/5/2.5/2.5 80 5928 8.88 F4 5/5/5/5 91.3 6742 13.88
Mencevax .TM. 91.7 4854 21.02
Example 9--Clinical Trial Assessing the Effect of a Linker in MenA
and MenC Conjugates in a MenACWY Conjugate Vaccine
[0301] A single dose of different formulations of MenACWY vaccine
was administered to teenagers of 15-19 years in 5 groups of 25
subjects in a 1:1:1:1:1 randomised trial. The formulations tested
were:
[0302] F1--MenACWY conjugated to tetanus toxoid with the MenA and
MenC conjugates containing an AH spacer--2.5/2.5/2.5/2.5 .mu.g
[0303] F2--MenACWY conjugated to tetanus toxoid with the MenA and
MenC conjugates containing an AH spacer--5/5/2.5/2.5 .mu.g
[0304] F3--MenACWY conjugated to tetanus toxoid with the MenA and
MenC conjugates containing an AH spacer--5/5/5/5 .mu.g
[0305] F4--MenACWY conjugated to tetansus toxoid with the MenA
conjugate containing an AH spacer--5/5/5/5 .mu.g
[0306] Control group--Mencevax.TM. ACWY
[0307] On day 30 after inoculation, a blood sample was taken from
the patients.
[0308] The blood samples were used to asess the percentage of
SBA-MenA, SBA-MenC, SBA-MenW135 and SBA-MenY responders one month
after the vaccine dose. A vaccine response was defined as 1) for
initially seronegative subjects--a post-vaccination antibody titre
.gtoreq. 1/32 at 1 month or 2) for initially seropositive
subjects--antibody titre of .gtoreq.4 fold the pre-vaccination
antibody titre.
[0309] Results
[0310] The introduction of an AH spacer into the MenC conjugate led
to an increase in the immune response against MenC as shown in
Table 14. This is demonstrated by an increase in SBA GMT from 1943
to 4329 and an increase in anti-PSC GMC from 7.65 to 13.13. Good
immune responses against MenA, MenW and MenY were maintained.
TABLE-US-00022 TABLE 13 % SBA MenA SBA-MenA Anti-PSA GMC
Formulation responders GMT .mu.g/ml ELISA F 12.5AH/2.5AH/ 75 8417
20.23 2.5/2.5 F2 5AH/5AH/2.5/2.5 72 6299 16.07 F3 5AH/5AH/5/5 87
9264 27.26 F4 5AH/5/5/5 77.3 9632 20.39 Mencevax .TM. 78.3 8284
12.93 % SBA MenC SBA-MenC Anti-PSC GMC Formulation responders GMT
.mu.g/ml ELISA F 12.5AH/2.5AH/ 88 3619 12.82 2.5/2.5 F2
5AH/5AH/2.5/2.5 88 2833 13.32 F3 5AH/5AH/5/5 95.8 4329 13.13 F4
5AH/5/5/5 95.8 1943 7.65 Mencevax .TM. 91.7 1567 16.55 % SBA MenW
SBA-MenW Anti-PSW GMC Formulation responders GMT .mu.g/ml ELISA F
12.5AH/2.5AH/ 100 5656 7 2.5/2.5 F2 5AH/5AH/2.5/2.5 96 4679 5.4 F3
5AH/5AH/5/5 91.3 4422 4.45 F45AH/5/5/5 88 4947 7.67 Mencevax .TM.
96 3486 11.93 % SBY MenY SBA-MenY Anti-PSY GMC Formulation
responders GMT .mu.g/ml ELISA F 1 75 3891 17.81 2.5AH/2.5AH/2.5/2.5
F2 5AH/5AH/2.5/2.5 92 3968 11.96 F3 5AH/5AH/5/5 79.2 2756 9.51 F4
5AH/5/5/5 80 3914 16.76 Mencevax .TM. 88 3056 21.41
* * * * *