U.S. patent application number 15/313393 was filed with the patent office on 2017-07-13 for novel semi-synthetic meningococcal conjugate vaccine.
The applicant listed for this patent is BIOLOGICAL E LIMITED. Invention is credited to Mahima Datla, Akshay Goel, Eswara Kowlakuntla, Narendra Dev Mantena, Santosh Nukuntla.
Application Number | 20170198004 15/313393 |
Document ID | / |
Family ID | 53776914 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170198004 |
Kind Code |
A1 |
Goel; Akshay ; et
al. |
July 13, 2017 |
NOVEL SEMI-SYNTHETIC MENINGOCOCCAL CONJUGATE VACCINE
Abstract
The present invention relates to novel semi-synthetic
meningococcal conjugate vaccine comprising novel synthetic
oligosaccharide conjugated to a carrier protein. The present
invention also relates to novel synthetic meningococcal
oligosaccharide and a process for its preparation.
Inventors: |
Goel; Akshay; (Hyderabad,
IN) ; Nukuntla; Santosh; (Hyderabad, IN) ;
Kowlakuntla; Eswara; (Hyderabad, IN) ; Datla;
Mahima; (Hyderabad, IN) ; Mantena; Narendra Dev;
(Hyderabad, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOLOGICAL E LIMITED |
Hyderabad, Telangana |
|
IN |
|
|
Family ID: |
53776914 |
Appl. No.: |
15/313393 |
Filed: |
May 22, 2015 |
PCT Filed: |
May 22, 2015 |
PCT NO: |
PCT/IN2015/000218 |
371 Date: |
November 22, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/6037 20130101;
A61K 39/095 20130101; C07H 1/00 20130101; C07H 15/04 20130101; A61P
31/04 20180101 |
International
Class: |
C07H 15/04 20060101
C07H015/04; A61K 39/095 20060101 A61K039/095; C07H 1/00 20060101
C07H001/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 24, 2014 |
IN |
1570/CHE/2014 |
Claims
1. A novel synthetic meningococcal C oligosaccharide of formula (I)
##STR00027## Wherein m is an integer from 2-10 and n is an integer
from 6-10; R1 and R2 are same or different and independently
represent H, C1-6alkyl, acetyl; R3 represents azide, NR5R6, wherein
R5 and R6 are same or different and independently represent H, C1-6
alkyl, aryl; R4 represents H, C1-6 alkyl, alkali metal cation
selected from Li, Na, K and Cs.
2. A novel semisynthetic meningococcal C vaccine of the formula
(II) wherein m is an integer from 2-10 and n is an integer from
6-10; R1 and R2 are same or different and independently represent
H, C1-6alkyl, acetyl; R5 represent H, C1-6 alkyl, aryl; R4
represents H or C1-6 alkyl; CP represents a carrier protein; L1 is
a bond, --O--, --S--, --NR8-, --C(.dbd.O)--, --NR8C(.dbd.O)--,
--NR8C(.dbd.O)O--, --C(.dbd.O)NR8-, --OC(.dbd.O)NR8-,
--SC(.dbd.O)--, --C(.dbd.O)S--, --OC(.dbd.O)--, --C(.dbd.O)O--,
--NR8C(.dbd.S)--, --C(.dbd.S)NR8-, trans-CR9=CR9, cis-CR9=CR9,
--C.ident.C--, --OC(R9)2-, --C(R9)20-, --NR8C(R9)2-, --C(R9)2NR8-
--SC(R9)2-, --C(R9)2S--, --S(.dbd.O)2O--, --OS(.dbd.O)2-,
--S(.dbd.O)2NR8-, --NR8S(.dbd.O)2-, or an optionally substituted
C1-20 hydrocarbon chain, optionally wherein one or more carbon
units of the hydrocarbon chain is replaced with --O--, --S--,
--NR8-, --C(.dbd.O)--, NR8C(.dbd.O)--, --NR8C(.dbd.O)O--,
--C(.dbd.O)NR8-, --OC(.dbd.O)NR8- --SC(.dbd.O)--, --C(.dbd.O)S--,
--OC(.dbd.O)--, --C(.dbd.O)O--, --NR8C(.dbd.S)--, --C(.dbd.S)NR8-,
trans-CR9=CR9-, cis-CR9=CR9-, --C.dbd.C--S(.dbd.O)2O--,
--OS(.dbd.O)2-, --S(.dbd.O)2NR8-, or --NR8S(.dbd.O)2, wherein R8 is
hydrogen, optionally substituted C1-6 alkyl, or a nitrogen
protecting group, or R8 is joined with the adjacent carbon atom to
form an optionally substituted heterocyclic ring, and wherein each
occurrence of R9 is independently selected from the group
consisting of hydrogen, halogen, optionally substituted C1-10
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, and optionally
substituted heteroaryl, or R9 is joined with the adjacent carbon or
nitrogen or oxygen atom to form an optionally substituted
carbocyclic or heterocyclic ring, or two R9 groups are joined to
form an optionally substituted carbocyclic or optionally
substituted heterocyclic ring; L2 is a moiety derived from a
crosslinking reagent capable of crosslinking the carrier and L1 and
is selected from ##STR00028## R7 is independently hydrogen,
optionally substituted C1-6 alkyl, optionally substituted acyl, or
a nitrogen protecting group.
3. A process for the preparation of novel semisynthetic
meningococcal conjugate vaccine as claimed in claim 2, which
comprises the steps of: a) synthesizing the meningococcal
oligosaccharide of formula (I) of claim 1, b) activating the
oligosaccharide, c) derivatizing a carrier protein, d) conjugating
the meningococcal oligosaccharide obtained in step (b) with the
carrier protein to form a conjugate vaccine and e) purifying the
conjugate vaccine obtained in step (d).
4. The process as claimed in claim 3, wherein the step of
activating oligosaccharide is carried out using bromoacetic
N-hydroxysuccinimide (NHS) ester, 6-Maleimidohexanoic acid NHS
ester, 6-(iodoacetamido)caproic acid NHS ester, maleimidopropionoic
acid NHS ester, maleimidoacetic acid NHS ester, maleimidobenzoic
acid NHS ester.
5. The process as claimed in claim 3, wherein the step of
derivatizing the carrier protein is carried out using 6-3-(acetyl
thio) propionic acid N-hydroxysuccinimide ester,
acetylthio-hexadecanoic acid NHS ester.
6. The semisynthetic meningococcal C conjugate vaccine of claim 2,
wherein the carrier protein is selected from Tetanus toxoid,
diphtheria toxoid or CRM197.
7. The synthetic meningococcal oligosaccharides of claim 1,
selected from: ##STR00029##
8. An immunogenic composition comprising novel semisynthetic
meningococcal C conjugate of claim 6 is selected from: ##STR00030##
wherein CRM197 is cross reacting moiety 197, DT is diphtheria
toxoid and TT is tetanus toxoid.
9. A pharmaceutical composition comprising (a) a novel
semisynthetic meningococcal C conjugate vaccine as claimed in claim
2 and (b) a pharmaceutically acceptable carrier.
10. The composition of claim 9, further comprising a saccharide
antigen from one or more of serogroups A, W135 and Y of N.
meningitidis, the saccharide being an oligosaccharide and being
conjugated to the carrier protein.
11. The composition of claim 10, further comprising a vaccine
adjuvant.
12. The composition of claim 11, which is a vaccine against a
disease caused by Neisseria meningitidis.
13. (canceled)
14. The composition of claim 9 further comprising 1 .mu.g to 10
.mu.g of each polysaccharide selected from Meningococcal serogroups
A, Y and W-135, conjugated individually to 5 to 20 .mu.g of carrier
protein.
Description
PRIORITY CLAIM
[0001] This application is a 371 U.S. National Stage Application of
International Patent Application No. PCT/IN2015/000218 filed May
22, 2015, which claims priority to Indian Patent Application Serial
No. 1570/CHE/2014 filed on May 24, 2014, the entire contents of
which are incorporated herein by reference and relied upon.
FIELD OF THE INVENTION
[0002] The present invention relates to novel semi-synthetic
meningococcal conjugate vaccine comprising novel synthetic
oligosaccharide conjugated to a carrier protein. The present
invention also relates to novel synthetic meningococcal
oligosaccharide and a process for its preparation.
BACKGROUND OF THE INVENTION
[0003] Neisseria meningitidis is a leading cause of bacterial
meningitis and sepsis throughout the world. Meningococcal bacteria
incorporate polysaccharides into their surface structure. Thus a
large majority of bacteria are covered with capsule or glycocalyx
polysaccharide which induces an immunological response in humans.
The outer membrane of gram-negative Neisseria meningitidis (NM)
bacterium consists, inter alia, of lipopolysaccharide (LPS). Such
polysaccharides (PS) are formed on the basis of repeating units in
which, the constituents and the bonds are defined and which are
each characteristics of the NM serogroups. Hence, these repeating
units contain the epitopes or the antigenicity determining
structures.
[0004] Conjugated immunogenic composition comprises of a target
antigen (polysaccharide) component chemically conjugated to a
carrier protein. These vaccines are known to be highly immunogenic
in all age groups including infants. A conjugate comprising an
oligosaccharide covalently bound to a carrier protein is also
referred to as glyco-conjugate.
[0005] The immunogenicity of the capsular polysaccharides can be
improved by covalently coupling them to a carrier protein. When
covalently linked to a carrier protein, the resulting PS component
in a conjugate vaccine becomes a T cell-dependent (TD) antigen,
inducing long-term immunity with immune memory even in infants and
young children.
[0006] WO 02/058737 disclosed immunological compositions for
treatment of meningococcal polysaccharide-protein conjugates caused
by pathogenic Neisseria meningitidis, which comprises two or more
protein-polysaccharide conjugates, wherein each of the conjugates
comprises a capsular polysaccharide from N. meningitidis conjugated
to a carrier protein.
[0007] WO 03/007985 disclosed a process for purifying a bacterial
capsular polysaccharide, comprising the steps of (a) precipitation
of the polysaccharide, followed by (b) solubilisation of the
precipitated polysaccharide using an alcohol.
[0008] Lin, Chang-Ching; et al. (Tetrahedron (2009), 65(24),
4714-4725) disclosed the synthesis of .alpha.-(2.fwdarw.9)
tetrasialic acid using phosphite donor and iterative sialylations
to elongate the sugar chain from the non-reducing end to the
reducing end.
##STR00001##
[0009] Chang-Ching Lin, et al. (J. Org. Chem., 2010, 75 (15),
4921-4928) disclosed an efficient .alpha.-selective method for
synthesis of .alpha.-(2.fwdarw.9) tetrasialic acid using
5-N,4-O-carbonyl-protected thioglycoside sialvl donors.
##STR00002##
[0010] Chu, Kuo-Ching; et al. (Angewandte Chemie, International
Edition (2011), 50(40), 9391-9395) disclosed the synthesis of
.alpha. (2.fwdarw.9)oligosialic acids: from monomers to dodecamers,
which are considered to be the current vaccines against
meningococcal C diseases. The synthetic route of tetrasialoside and
hexasialoside can be used to synthesize the proposed Men C pentamer
using 5-N,4-O-carbonyl-protected glycosyl phosphate sialyl
donors.
##STR00003##
[0011] Several meningococcal C conjugate vaccines available in the
market as monovalent (e.g. Neisvac-C.RTM., Menjugate.RTM.) as well
as multivalent products (e.g. Menactra.RTM., Mencevax, Nimenrix,
Menveo.RTM.). The polysaccharide component in all these vaccines is
derived from the fermentation and purification of the actual
pathogenic bacteria. The saccharide moiety in glycoconjugate
vaccines is usually a functionalised bacterial capsular
polysaccharide (CPS). In such processes the various challenges that
have to be met include, the need for fermentation using the
pathogenic host, stringent control of additives and physiological
parameters; cumbersome purification steps to remove impurities such
as protein, endotoxins, nucleic acids, need for reduction in size
of purified oligosaccharide, need for attachment of a linker to the
purified oligosaccharide and low conjugation yields. Hence, a major
disadvantage of these conventional biological processes is
requirement of repeated purifications, which results in low
yields.
[0012] WO 2014/097099 A2 discussed, that prior to activation step
the polysaccharide of the invention may be sized to achieve an
appropriate molecular weight. This is done either mechanically or
simple hydrolyzation.
[0013] In order to overcome the disadvantages associated with the
preparation of conventional meningococcal oligosaccharides, the
inventors of the present invention have found that the conjugation
of carrier protein with synthetically made immunogenic
oligosaccharide will result in development of a cost effective
meningococcal vaccine with non-inferior immunogenicity compared to
existing vaccines.
[0014] Synthetic polysaccharides have a number of potential
advantages over native polysaccharides. There is an ease of
production of these polysaccharides without the need for
fermentation. Naturally derived carbohydrates are heterogeneous
mixtures and may include small amounts of natural impurities and
contaminants. In contrast, synthetic carbohydrates can be produced
as homogeneous single compounds in a controlled manner, with little
or no batch-to-batch variability. Another advantage is that they
can be made to include functional groups for derivatization or
modification of the carbohydrate moiety that are difficult or
impossible to perform with native polysaccharide.
OBJECTIVE OF THE INVENTION
[0015] The main objective of the present invention is to provide
novel semi-synthetic Men C conjugate vaccine, with non-inferior
immunogenicity.
[0016] Yet another objective is to provide semi-synthetic Men C
conjugate vaccine without using the cumbersome fermentation process
and thereby repeated multistep purifications.
[0017] Yet another objective of the present invention is to provide
impurity free and economically viable Men C conjugate vaccine
production.
SUMMARY OF THE INVENTION
[0018] Accordingly, the present invention provides a novel
synthetic meningococcal C oligosaccharide of formula (I)
##STR00004##
wherein m and n are integers ranging from 1 to 10; R.sub.1 and
R.sub.2 are same or different and independently represent H,
C.sub.1-6alkyl, acetyl; R.sub.3 represents azide, NR.sub.5R.sub.6,
wherein R.sub.5 and R.sub.6 are same or different and independently
represent H, C.sub.1-6 alkyl, aryl; R.sup.4 represents H, C.sub.1-6
alkyl, alkali metal cation selected from Li, Na, K and Cs; with a
proviso that when m=1 to 2, then n is not 1 to 5.
[0019] In another embodiment, the present invention provides a
novel semi-synthetic meningococcal C conjugate vaccine of the
formula (II)
##STR00005##
wherein m and n are integers ranging from 1 to 10; R.sub.1 and
R.sub.2 are same or different and independently represent H,
C.sub.1-6alkyl, acetyl; R.sup.4 represents H, C.sub.1-6 alkyl,
alkali metal cation selected from Li, Na, K and Cs; R.sub.5
represent H, C.sub.1-6 alkyl, aryl; CP represents a carrier
protein; L.sub.1 is a bond, --O--, --S--, --NR.sub.8,
--C(.dbd.O)--, --NR.sub.8C(.dbd.O)--, --NR.sub.8C(.dbd.O)O--,
--C(.dbd.O)NR.sub.8--, --OC(.dbd.O)NR.sub.8--, --SC(.dbd.O)--,
--C(.dbd.O)S--, --OC(.dbd.O)--, --C(.dbd.O)O--,
--NR.sub.8C(.dbd.S)--, --C(.dbd.S)NR.sub.8--,
trans-CR.sub.9.dbd.CR.sub.9, cis-CR.sub.9.dbd.CR.sub.9,
--C.ident.C--, --OC(R.sub.9)2-, --C(R.sub.9).sub.2O--,
--NR.sub.8C(R.sub.9).sub.2--, --C(R.sub.9).sub.2NR.sub.8--
--SC(R.sub.9).sub.2--, --C(R.sub.9).sub.2S--, --S(.dbd.O).sub.2O--,
--OS(O).sub.2--, --S(.dbd.O).sub.2NR.sub.8--,
--NR.sub.8S(.dbd.O).sub.2--, or an optionally substituted
C.sub.1-20hydrocarbon chain, optionally wherein one or more carbon
units of the hydrocarbon chain is replaced with --O--, --S--,
--NR.sub.8--, --C(.dbd.O)--, NR.sub.8C(.dbd.O)--,
--NR.sub.8C(.dbd.O)O--, --C(.dbd.O)NR.sub.8--,
--OC(.dbd.O)NR.sub.8-- --SC(.dbd.O)--, --C(.dbd.O)S--
--OC(.dbd.O)--, --C(.dbd.O)O--, --NR.sub.8C(.dbd.S)--,
--C(.dbd.S)NR.sub.8--, trans-CR.sub.9.dbd.CR.sub.9--,
cis-CR.sub.9.dbd.CR.sub.9--, --C.dbd.C-- --S(.dbd.O).sub.2O--,
--OS(.dbd.O).sub.2--, --S(.dbd.O).sub.2NR.sub.8--, or
--NR.sub.8S(.dbd.O).sub.2, wherein R.sub.8 is hydrogen, optionally
substituted C.sub.1-6 alkyl, or a nitrogen protecting group, or
R.sub.8 is joined with the adjacent carbon atom to form an
optionally substituted heterocyclic ring, and wherein each
occurrence of R.sub.9 is independently selected from the group
consisting of hydrogen, halogen, optionally substituted C.sub.1-10
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, and optionally
substituted heteroaryl, or R.sub.9 is joined with the adjacent
carbon or nitrogen or oxygen atom to form an optionally substituted
carbocyclic or heterocyclic ring, or two R.sub.9 groups are joined
to form an optionally substituted carbocyclic or optionally
substituted heterocyclic ring; L.sub.2 is a moiety derived from a
crosslinking reagent capable of crosslinking the carrier and
L.sub.1; R.sub.7 is independently hydrogen, optionally substituted
C.sub.1-6 alkyl, optionally substituted acyl, or a nitrogen
protecting group. with a proviso that when m is 1 to 2, then n is
not 1 to 5.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1: .sup.1H NMR of novel synthetic oligosaccharide.
[0021] FIG. 2: Molecular weight of synthetic oligosaccharide
(HRMS).
[0022] FIG. 3: Analytical confirmation (TOCSY) of oligosaccharide
in semi-synthetic bulk conjugate.
[0023] FIG. 4: Quantification of Sialic acid in semi-synthetic
Meningococcal-C Conjugate using sialic acid as reference standard
by HPAEC-PAD Method.
[0024] FIG. 5: The individual anti-meningococcal polysaccharide IgG
antibody levels measured by serotype specific ELISA.
[0025] FIG. 6: Geometric mean anti-MenC IgG concentrations for
group of mice following immunisation with test and reference
vaccine formulations.
[0026] FIG. 7: Relative rSBA titre for sera obtained following
immunisation with sMenC where the geometric mean is displayed as
bar.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The capsular polysaccharide of Neisseria meningitidis
serogroup C is a homopolymer of N-acetyl neuraminic acid bound to
-2.fwdarw.9,.fwdarw.9)-D-NeupNAc(7/8OAc)-.alpha.-(2.fwdarw., with
O-acetyl group in position C7 and C8 in variable percentage.
[0028] The present invention provides a novel synthetic
meningococcal C oligosaccharide of formula (I)
##STR00006##
wherein m and n are integers ranging from 1 to 10; R.sub.1 and
R.sub.2 are same or different and independently represent H,
C.sub.1-6alkyl, acetyl; R.sub.3 represents azide, NR.sup.5R.sup.6,
wherein R.sup.5 and R.sup.6 are same or different and independently
represent H, C.sub.1-6 alkyl, aryl; R.sup.4 represents H, C.sub.1-6
alkyl,alkali metal cation selected from Li, Na, K and Cs; with a
proviso that when m=1 to 2 then n is not 1 to 5.
[0029] The present invention provides novel semi-synthetic
meningococcal C conjugate vaccine of the formula (II)
##STR00007##
wherein m and n are integers ranging from 1 to 10; R.sub.1 and
R.sub.2 are same or different and independently represent H,
C.sub.1-6alkyl, acetyl; R.sup.4 represents H, C.sub.1-6 alkyl,
alkali metal cation selected from Li, Na, K and Cs; R.sub.5
represent H, C.sub.1-6 alkyl, aryl; CP represents a carrier
protein; L.sub.1 is a bond, --O--, --S--, --NR.sub.8--,
--C(.dbd.O)--, --NR.sub.8C(.dbd.O)--, --NR.sub.8C(.dbd.O)O--,
--C(.dbd.O)NR.sub.8--, --OC(.dbd.O)NR.sub.8--, --SC(.dbd.O)--,
--C(.dbd.O)S--, --OC(.dbd.O)--, --C(.dbd.O)O--,
--NR.sub.8C(.dbd.S)--, --C(.dbd.S)NR.sub.8--,
trans-CR.sub.9.dbd.CR.sub.9, cis-CR.sub.9.dbd.CR.sub.9,
--C.ident.C--, --OC(R.sub.9)2-, --C(R.sub.9).sub.2O--,
--NR.sub.8C(R.sub.9).sub.2--, --C(R.sub.9).sub.2NR.sub.8--
--SC(R.sub.9).sub.2--, --C(R.sub.9).sub.2S--, --S(.dbd.O).sub.2O--,
--OS(O).sub.2--, --S(.dbd.O).sub.2NR.sub.8--,
--NR.sub.8S(.dbd.O).sub.2--, or an optionally substituted
C.sub.1-20hydrocarbon chain, optionally wherein one or more carbon
units of the hydrocarbon chain is replaced with --O--, --S--,
--NR.sub.8--, --C(.dbd.O)--, NR.sub.8C(.dbd.O)--,
--NR.sub.8C(.dbd.O)O--, --C(.dbd.O)NR.sub.8--,
--OC(.dbd.O)NR.sub.8-- --SC(.dbd.O)--, --C(.dbd.O)S--
--OC(.dbd.O)--, --C(.dbd.O)O--, --NR.sub.8C(.dbd.S)--,
--C(.dbd.S)NR.sub.8--, trans-CR.sub.9.dbd.CR.sub.9--,
cis-CR.sub.9.dbd.CR.sub.9--, --C.dbd.C-- --S(.dbd.O).sub.2O--,
--OS(.dbd.O).sub.2--, --S(.dbd.O).sub.2NR.sub.8--, or
--NR.sub.8S(.dbd.O).sub.2, wherein R.sub.8 is hydrogen, optionally
substituted C.sub.1-6 alkyl, or a nitrogen protecting group, or
R.sub.8 is joined with the adjacent carbon atom to form an
optionally substituted heterocyclic ring, and wherein each
occurrence of R.sub.9 is independently selected from the group
consisting of hydrogen, halogen, optionally substituted C.sub.1-10
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, and optionally
substituted heteroaryl, or R.sub.9 is joined with the adjacent
carbon or nitrogen or oxygen atom to form an optionally substituted
carbocyclic or heterocyclic ring, or two R.sub.9 groups are joined
to form an optionally substituted carbocyclic or optionally
substituted heterocyclic ring; L.sub.2 is a moiety derived from a
crosslinking reagent capable of crosslinking the carrier and
L.sub.1; R.sub.7 is independently hydrogen, optionally substituted
C.sub.1-6 alkyl, optionally substituted acyl, or a nitrogen
protecting group. with a proviso that when m is 1 to 2, then n is
not 1 to 5.
[0030] In preferred embodiment, L.sub.1 is of formula.
##STR00008##
[0031] In preferred embodiment, L.sub.2 is of formula.
##STR00009##
[0032] In a preferred embodiment, the present invention relates to
a synthetic meningococcal C oligosaccharide of formula (IA).
##STR00010##
wherein m and n are integers ranging from 1 to 10; R.sub.1 and
R.sub.2 are same or different and independently represent H,
C.sub.1-6alkyl, acetyl; R.sup.4 represents H, C.sub.1-6 alkyl,
alkali metal cation selected from Li, Na, K and Cs; with a proviso
that when m is lto 2 n is not 1 to 5.
[0033] In an embodiment, the present invention also provides a
process for the preparation of novel semi-synthetic meningococcal
conjugate vaccine comprising the steps of: [0034] a) synthesising
meningococcal oligosaccharide of formula (I), [0035] b) activation
of oligosaccharide, [0036] c) derivatization of carrier protein,
[0037] d) conjugation of meningococcal oligosaccharide obtained in
step (b) with carrier protein and [0038] e) purification of
conjugate vaccine obtained in step (d).
[0039] In a preferred embodiment, the synthetic oligomer (sMenC) is
activated using 6-maleimidohexonoic acid N-hydroxysuccinimide
ester. The carrier protein is derivatized using 3-(acetyl thio)
propionic acid N-hydroxysuccinimide ester. Crosslinking reagents
suited to the invention are widely known in the art (see, for
example, 1994 Pierce Technical Handbook: cross-linking available at
http://www.piercenet.com/resources/browse.cfm?fldID=184).
[0040] In preferred embodiment the crosslinking reagent used to
derivatize the carrier protein is
##STR00011##
[0041] In certain embodiments, the invention provides a method of
preparing a conjugate vaccine of formula (II) described herein,
comprising coupling a compound of formula
##STR00012##
with a compound of the formula
##STR00013##
wherein L.sub.2 is a crosslinking reagent capable of crosslinking
an amino group and --SH.
[0042] In another embodiment, the preferred semi-synthetic
conjugate vaccine is of the formula as given below:
##STR00014##
[0043] In another embodiment, the carrier is a protein, a lipid, a
lipolized protein, a virus, a peptide comprising a T cell epitope,
or a dendrimer of glycopeptides. In certain embodiments, the
carrier is a toxin protein selected from the group consisting of
diphtheria toxin cross-reacting material 197 (DT-CRM197),
diphtheria toxoid (DT), tetanus toxoid (TT), and outer-membrane
protein (OMP). Preferably, the carrier protein is Tetanus toxoid,
diphtheria toxoid or CRM197.
[0044] The activation of oligosaccharide is carried out using
brotnoacetic N-hydroxysuccinimide (NHS) ester, 6-Maleimidohexanoic
acid NHS ester, 6-(iodoacetamido)caproic acid NHS ester,
maleimidopropionoic acid NHS ester, maleimidoacetic acid NHS ester,
maleimidobenzoic acid NHS ester and the like.
[0045] The derivatization of carrier protein is carried out using
3-(acetyl thio) propionic acid N-hydroxysuccinimide ester,
acetylthio-hexadecanoic acid NHS ester and the like.
[0046] The polysaccharide component manufactured using chemical
process is referred to as synthetic oligosaccharide or oligomer.
The conjugate construct using the synthetic oligosaccharide and a
carrier protein is referred to as semi-synthetic conjugate.
[0047] As used herein the term "Men C" is referred to Neisseria
meningitidis serogroup C component of the vaccine. The Men C
Capsular polysaccharide component manufactured using chemical
process is hereby referred to as sMenC Oligomer (or sMenC). sMenC
is further conjugated to a carrier protein to give a semi-synthetic
immunogenic composition.
[0048] As used herein the term "Oligomer" or "capsular saccharide"
is referred to Oligosaccharides. The terms can be used
interchangeably.
[0049] Examples of protecting groups and details of their usage are
available in, for example, Greene, T. W., and Wuts, R G. M.,
Protective Groups in Organic Synthesis, 2.sup.nd ed. (1991).
[0050] Following compounds are the preferred novel synthetic
meningococcal oligomers of the present invention:
##STR00015##
[0051] Given below are the preferred novel semi-synthetic
meningococcal C conjugate vaccines of the present invention:
##STR00016##
[0052] In yet another embodiment, the present invention provides a
process for the preparation of sMenC Oligomer of formula (I) as
shown in the scheme I given as follows:
##STR00017##
where A represents leaving group such as halogen (such as F, Cl,
Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy,
alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g.,
acetoxy), arylcarbonyloxy, aryloxy, methoxy,
N,O-dimethylhydroxylamino, pixyl, and haloformates. In some cases,
the leaving group is a sulfonic acid ester, such as
toluenesulfonate (tosylate, -OTs), methanesulfonate (mesylate,
-OMs), .rho.-bromobenzenesulfonyloxy (brosylate, -OBs), or
trifluoromethanesulfonate (triflate, -OTf); R.sub.1 is
chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate.
[0053] Step (i): The coupling of compound (1) and (2), followed by
acetylation to yield compound of formula (3). The coupling is
carried out in the presence of solvents such as dichloromethane,
acetonitrile, propionitrile, ether, acetone, chloroacetonitrile,
N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide and the
like or a mixture thereof using N-Iodosuccinimide (NIS),
N-Bromosuccinimide (NBS) and the like. The reaction may be carried
out in the presence of catalysts such as TMSOTf, TESOTf, TfOH,
Tf.sub.2O, AgOTf, BF.sub.3.Et.sub.2O, lanthanide triflates and the
like, at a temperature in the range of -78.degree. C. to 25.degree.
C. for a period in the range of 1-12 hours.
[0054] Acetylation of the hydroxyl groups of the coupled product is
carried out using acetic anhydride, acetyl chloride in the presence
of a base such as DIPEA, TEA, imidazole, lutidine, pyridine and the
like as well as TfOH, TMSOTf, BF.sub.3.Et.sub.2O, lanthanide
triflates and the like.
[0055] Step (ii): Dechloroacetylation is carried out in the
presence of solvents such as methanol, ethanol, isopropanol,
acetone, acetonitrile and the like using bases such as DIPEA, TEA,
imidazole, pyridine and the like.
[0056] Step (iii): Repetition of the sialylation, acetylation, and
dechloroacetylation reactions to produce high order sialosides.
This three-step sequence provides the tri- and tetrasialic
acids.
[0057] Step (iv): Deprotection and deesterification: The reaction
is carried out using solvents such as ethanol, methanol, propanol,
i-propanol, water and the like; and bases such as lithium
hydroxide, sodium hydroxide, potassium hydroxide and the like. The
reaction is carried out at a temperature in the range of 25.degree.
C. to 80.degree. C. The resulting amines were N-acetylated using
acetylating agents such as acetic anhydride, acetylchloride and the
like in the presence of bases such as sodium hydroxide, potassium
hydroxide, NaHCO.sub.3, DIPEA, TEA, imidazole, pyridine and the
like.
[0058] In yet another embodiment, the compound of formula (I)
wherein R.sub.3 represents azide is converted to amino. The
reaction is carried out using reducing agents such as Pd/C, Pt/C,
LiAlH.sub.4, sodium borohydride and the like in a conventional
method.
[0059] In an embodiment, the present invention also provides a
process for the preparation of novel semi-synthetic conjugate
vaccine of formula (II), which comprises the steps of:
[0060] Step 1: Derivatization of carrier protein.
[0061] To carrier protein 3-(Acetyl thio) propionic acid
N-succinimidyl ester is added and incubated. This mixture is
desalted and buffer exchanged with Phosphate-buffered saline (PBS).
Further, the solution is treated with hydroxylamine hydrochloride
and incubated. This mixture is desalted and concentrated.
[0062] Step 2: Activation of Oligosaccharide.
[0063] The oligosaccharide with linker is activated with
6-maleimidohexonoic acid N-hydroxysuccinimide ester and then
incubated for the reaction to occur. This mixture is further
desalted and concentrated.
[0064] Step 3: Conjugation:
[0065] Derivatized carrier protein and activated synthetic
oligosaccharide are mixed and incubated to form the conjugate
vaccine. The conjugate is purified by using diafiltration to
generate bulk conjugate.
[0066] Physico-chemical aspects as well as the immunological
response of the novel semi-synthetic construct is characterized by
analytical methods.
[0067] The novel semi-synthetic conjugate vaccine of the present
invention is physically and chemically characterized to confirm and
quantify the presence of synthetic oligosaccharide. The construct
is also characterized using 2D Total Correlated Spectroscopy
(TOCSY) to confirm the presence of sialic acid in the
semi-synthetic Meningococcal C conjugate bulk.
[0068] Physico-chemical aspects of the semi-synthetic conjugate
vaccine is measured by the following techniques: [0069] a. NMR
spectra of the synthetic oligosaccharide (FIG. 1); [0070] b. High
Resolution Mass Spectroscopy (HRMS) of the synthetic Meningococcal
C oligosaccharide. The FIRMS confirms the molecular weight of the
glycan construct. (FIG. 2); [0071] c. TOCSY (120 ins) of
semi-synthetic Meningococcal C bulk conjugate. (FIG. 3); [0072] d.
Quantification of Sialic acid in semi-synthetic Meningococcal-C
Conjugate using sialic acid as reference standard by HPAEC-PAD
Method (FIG. 4).
[0073] The immunogenic activity of oligosaccharides described
herein is often enhanced by conjugation to a protein. Thus the
invention includes conjugating the oligosaccharide to a protein
through a linker moiety, which may be L.sub.1 and L.sub.2 in
formula (II).
[0074] The immunological response was measured in mice after each
dose in a 3-dose regime of semi-synthetic Meningococcal C
conjugate, as compared to references such as non conjugated Quadri
Meningo and conjugated Menectra.RTM..
[0075] Mouse immunogenicity by ELISA was also done. The antibody
response was measured by indirect ELISA using commercial
polysaccharide coating antigen in the NUNC covalink plates.
Readings were measured by using 96 well plate reader.
[0076] Identification of antigenicity by Immunodiffusion assay was
also done. The antigenicity of conjugate was evaluated by use of a
Meningococcal group C antisera by immunodiffusion as compared with
negative purified polysaccharide of N. meningitidis C, which served
as Positive Reference. A clear zone of antigen Vs antibody
precipitate was observed.
[0077] The immunogenic compositions of the invention are suitable
for use in adult humans as well as in children. Optionally, such a
composition may be administered in combination with other
pharmaceutically active substances, and frequently it will be
administered in combination with other vaccines as part of a
childhood vaccination program. Compositions for administration may
beneficially include other types of immunogenic compounds such as
glycoconjugates that elicit an immune response against other
meningitis pathogens.
[0078] In yet another embodiment, the present invention also
provides pharmaceutical compositions comprising novel
semi-synthetic meningococcal C conjugate vaccine of the invention
admixed with at least one pharmaceutically acceptable
excipient.
[0079] The novel semi-synthetic conjugate of the present invention
is demonstrated to be immunogenic in an established animal model
(mice). Sera isolated after primary vaccination is demonstrated to
contain antibodies specific to Meningococcal C polysaccharide
(ELISA). In addition, immunodiffusion assays shows a clear
precipitation band corresponding to neutralizing antibodies against
meningococcal C polysaccharide.
[0080] The advantages of semi-synthetic Meningococcal conjugate
vaccine, of the present invention includes: [0081] 1. The
polysaccharide component is synthetically manufactured instead of
being derived from a biological process. Linker with multiple
carbon in the synthetic oligosaccharide construct is attached to
the oligomer without the involvement of a separate process step;
[0082] 2. the process of conjugation using thiol chemistry with
synthetic oligosaccharide construct resulting in high yields.
[0083] 3. the construction of a T-cell independent antigen
(oligomer or glycan) which is transformed into a t-cell dependent
antigen upon conjugation; [0084] 4. the oligosaccharides are small
and has potential to conjugate with different carrier proteins.
[0085] The invention also provides vaccines and immunogenic
compositions comprising synthetic capsular saccharide from N.
meningitidis serogroup C and capsular saccharides from at least two
of serogroups A, W135 and Y of N. meningitidis, wherein said
capsular saccharides are conjugated to carrier protein(s) methods
of which are known in the art. For example the following reference
provide the conjugation of polysaccharides to carrier protein.
[0086] 1. Joanna Kubler-Kielb and Vince Pozsgay, National Institute
of Child Health and Human Development, National Institutes of
Health, 31 Center Dr. MSC 2423 Bethesda, Md.; [0087] 2. Joanna
Kubler-Kielb et al., Oligosaccharide conjugates of Bordetella
pertussis and bronchiseptica induce bactericidal antibodies, an
addition to pertussis vaccine, PNAS 2011, 108:4087-92; [0088] 3.
Joanna Kubler-Kielb and Vince Pozsgay, A New Method for Conjugation
of Carbohydrates to Proteins Using an Aminooxy-Thiol
Heterobifunctional Linker, J. Org. Chem. 2005, 70, 6987-6990;
[0089] 4. J. Kubler-Kielb, E. Vinogradov, G. Ben-Menachem, V.
Pozsgay, J. B. Robbins, R. Schneerson, Saccharide/protein conjugate
vaccines for Bordetella species: preparation of saccharide,
development of new conjugation procedures, and physico-chemical and
immunological characterization of the conjugates. Vaccine 2008, 26:
3587-93.
[0090] The present invention provides a kit comprising: (a)
synthetic capsular saccharide from N. meningitidis serogroup C;
conjugated to a carrier protein (b) capsular saccharides from N.
meningitidis serogroups A, W135 and Y, conjugated to a carrier
protein.
[0091] In yet another embodiment, the conjugated synthetic capsular
saccharide from N. meningitidis serogroup C has a
saccharide:protein ratio (w/w) of between 0.1:1 and 2:1.
[0092] In yet another embodiment, the present invention provides an
immunogenic composition comprising novel semi-synthetic conjugate
vaccine containing the 1 .mu.g to 10 .mu.s of synthetic men C
oligosaccharide conjugated to 5 to 20 .mu.g of carrier protein. The
present invention further comprises, 1 .mu.g to 10 .mu.g of each
polysaccharide selected from Meningococcal serogroups A, Y and
W-135, each conjugated individually to 5 to 20 .mu.g of carrier
protein.
[0093] The vaccines and pharmaceutical compositions of the
invention are intended for parenteral, topical, oral or local
administration. Preferably, they are administered parenterally,
e.g., intravenously, subcutaneously, intradermally, or
intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the
immunogenic moiety dissolved or suspended in an acceptable
excipient, preferably a primarily aqueous carrier. A variety of
aqueous carriers may be used, e.g., water, buffered water, 0.8%
saline, 0.3% glycine, hyaluronic acid and the like. These
compositions may be sterilized by conventional sterilization
techniques, or may be sterile filtered. The resulting aqueous
solutions may be packaged for use as is, or lyophilized, the
lyophilized preparation being combined with a sterile solution
prior to administration.
[0094] The invention provides an immunogenic composition comprising
semi-synthetic meningococcal C oligosaccharide conjugate, and
further comprising (i) an aluminium phosphate or an aluminium
hydroxide adjuvant (ii) a buffer preferably a phosphate buffer and
optinally one or more excipeints selected from sucrose, polysorbate
and trometamol and the like
[0095] The invention also provides the use of a synthetic capsular
saccharide from N. meningitidis serogroup C, or of a conjugate
thereof, in the manufacture of a medicament for preventing or
treating a disease caused by capsulate bacteria. Diseases caused by
Neisseria include meningitis, septicemia and gonorrhea. Diseases
caused by H. influenzae include otitis media, bronchitis,
pneumonia, cellulitis, pericarditis, and meningitis. Diseases
caused by Pneumococcus include meningitis, sepsis and
pneumonia.
[0096] The following examples are provided to illustrate the
invention and are merely for illustrative purpose only and should
not be construed to limit the scope of the invention.
Example 1
Preparation of Compound 2
##STR00018##
[0098] To a stirred solution of 6-bromo-1-hexanol (25 g, 0.138 mol)
in DMF, sodium azide (18 g, 0.277 mol) was added at 0.degree. C.
and stirred RT for 24 h. The reaction mixture was quenched with
ice-cold water and extracted with diethyl ether. Organic layer was
washed with brine, separated, dried over Na.sub.2SO.sub.4 and
concentrated. 6-azido-1-hexanol was obtained as colorless liquid
(17 g, 75%).
##STR00019##
[0099] To a stirred solution of Compound-1 (40 g, 62.30 mmoles) and
6-azido-1-hexanol (10.6 g) in DCM (265 ml) and acetonitrile (135
ml) was added MS 4 A.degree. (40 g), cooled to -78.degree. C. To
this mixture NIS (20.04 g, 89.09 mmoles), TfOH (2.65 ml, 18.6
mmloes) were added. Reaction temperature was raised to -50.degree.
C. and stirred for 30 mins. The reaction mixture was quenched with
saturated hypo solution (150 ml), filtered through celite, organic
layer was separated, washed with water, dried Na.sub.2SO.sub.4 and
concentrated. Crude compound was purified by FCC by eluting with
10-20% ethyl acetate and hexanes, compound-1a was obtained as an
off-white sticky solid (28 g, 65%).
[0100] To a stirred solution of compound-1a (34 g, 51.5 mmol) in
methanol (1.3 lit), triethyl amine (7.1 ml, 51.5 mmol) was added to
the reaction mixture at 0.degree. C. and stirred for 30 minutes at
RT. After completion, the reaction mixture was neutralized (pH 7)
with 10% HCl solution and reaction mixture was concentrated under
reduced pressure. Crude compound was purified by FCC, by eluting
with (1:1:0.1) ethyl acetate, hexanes and methanol. Compound 2 was
obtained as sticky solid (14 g, 60%).
Preparation of Compound 3
##STR00020##
[0102] (i) To a stirred mixture of Compound 1 (40 g, 62.30 mmoles,
prepared as described in J. Org. Chem., 2010, 75, 4921-4928) and
Compound 2 (10.65 g) in DCM (140 ml) and acetonitrile (70 ml) was
added MS 3 A.degree. (20 g), cooled to -78.degree. C. To this
mixture NIS (6.937 g, 44.102 mmoles), Triflic acid (1.28 ml, 14.494
mmoles) were added. Reaction temperature was raised to -50.degree.
C. and stirred for 30 mins. The reaction mixture was quenched with
saturated Na.sub.2S.sub.2O.sub.3 solution (150 ml), filtered
through celite, organic layer was separated, washed with water,
dried Na.sub.2SO.sub.4 and concentrated. Crude compound (31 g) was
put into next step.
[0103] (ii) To the solution of the crude material in
CH.sub.2Cl.sub.2 (400 ml), cooled to 0.degree. C., Ac.sub.2O (11.65
ml, 123.364 mmoles) and TfOH (0.272 ml, 3.084 mmoles) were added.
Reaction was stirred at 0.degree. C. for 15 min and then quenched
with saturated NaHCO.sub.3 solution. The organic layer was
separated, washed with brine solution, dried over Na.sub.2SO.sub.4
and concentrated. The dimer 3 (17 g, 50% for 2 steps) was isolated
by FCC eluting with 50-60% ethyl acetate; hexanes as an off-white
solid.
Preparation of Compound 4
##STR00021##
[0105] To a stirred solution of dimer 3 (16 g, 15.46 mmol) in MeOH
(600 ml) and EtOAc (100 ml), Et.sub.3N (2.15 ml, 15.46 mmoles) was
added to the reaction mixture at 0.degree. C. and stirred for 30
minutes at room temperature.
[0106] Then the reaction mixture was neutralized to pH 7 with 10%
HCl solution and concentrated. The product 4 was isolated by FCC
eluting with (1:1:0.1) ethyl acetate, hexanes and methanol as white
solid (6 g, 50%).
Preparation of Compound 5
##STR00022##
[0108] Compound 1 (5 g, 7.453 mmoles) and compound 4 (5 g, 6.211
mmoles) were coupled using similar method described for dimer 3
step i (DCM 40 ml, CH.sub.3CN 20 ml, MS 3 A.degree. 3.1 g, MS 2.5
g, TfOH 0.275 ml). The Crude compound (10 g) was put into next step
without purification.
[0109] The crude product was treated in a similar method as
described for dimer 3 step ii (DCM 100 ml, Ac.sub.2O 2.95 ml, TfOH
0.137 ml). The trimer 5 (4.3 g, 40% for 2 steps) was isolated by
FCC eluting with 50-60% ethyl acetate; hexanes as an off-white
solid.
Preparation of Compound 6
##STR00023##
[0111] Trimer 5 (4.8 g, 3.409 mmol) was treated in a similar method
as described for compound 4 (MeOH 190 ml, EtOAc 37 ml, Et.sub.3N
0.473 ml, 3.409 mmoles). The product 6 (2 g, 45%) was isolated by
FCC eluting with (1:1:0.2) ethyl acetate, hexanes and methanol as
white solid.
Preparation of Compound 7
##STR00024##
[0113] (i) Compound 1 (1.308 g, 2.307 mmoles) and Compound 6 (2 g,
1.697 mmoles) were coupled using similar method described for dimer
3 step i (DCM 16 ml, CH.sub.3CN 8 ml, MS 3 A.degree. 0.85 g, NIS
0.687 g, TfOH 0.072 ml). Crude compound (3 g) was put to next step
without purification.
[0114] (ii) The crude compound was treated in a similar method as
described for compound 4 (MeOH 100 ml, EtOAc 15 ml, Et.sub.3N 0.25
ml). Crude compound (3 g) was put to next step without
purification.
[0115] (iii) To a stirred solution of above crude product in
pyridine (4 ml), acetic anhydride (2 ml) was added and stirred at
RT for 24 h. Tetrasialoside 7 (1 g, 35%) was isolated by FCC
eluting with (1:1:0.2) ethyl acetate, hexanes and methanol as white
solid.
Preparation of Compound 8
##STR00025##
[0117] (i) To a stirred solution of tetrasialoside 7 (1 g, 0.5963
mmoles) in ethanol (60 ml) and water (60 ml) was added lithium
hydroxide monohydrate (0.75 g, 17.889 mmoles) and reaction was
maintained at 80.degree. C. for 24 h. Reaction mixture was
neutralized with 10% HCl solution and then was concentrated.
[0118] (ii) The residue (1.8 g) was dissolved in water (20 ml), and
then NaHCO.sub.3 (1 g, 11.926 mmoles) followed by acetic anhydride
(0.608 g, 5.963 mmoles) were added at RT. After being stirred for
additional 16 h, the solvent was evaporated under reduced pressure
and the crude was put into next step
[0119] (iii) The residue was dissolved in methanol (20 ml) and then
NaOMe (0.65 g, 11.926 mmoles) was added at RT. After being stirred
for 16 h, the reaction mixture was neutralized with Dowex W.times.B
50*8 resin and the neutralized solution fitered and was
concentrated. The residue was purified by P2 Biogel, eluting with
water. Obtained product was lyophilized to give tetrasialic acid 8
as white solid (0.5 g, 60% for 3 steps).
Preparation of Tetramer of Compound 9
##STR00026##
[0121] To a stirred solution of tetrasialic acid 8 (0.5 g, 0.3825
mmoles) in ethanol (20 ml) and water (10 ml), were added acetic
acid (10 ml) and 10% Pd/C (0.2 g). After purging with N.sub.2, the
reaction mixture was filtered through celite and concentrated under
reduced pressure at RT. Product was purified by Sephadex-G-10,
eluting with water. Obtained product 9 was lyophilized to get the
required product as white solid (0.3 g, 60%).
Example 2
Derivatization of Carrier Protein for Conjugation
[0122] Tetanus toxoid was concentrated and buffer exchanged with 10
mM PBS to 5 to 20 mg/ml. To this 25 molar equivalent of 3-(acetyl
thio) propionic acid N-hydroxysuccinimidyl ester was added against
the amine molar concentration. After which it was finally incubated
at room temperature for 2 hours.
[0123] After the incubation period, the reaction mixture was
desalted and buffer exchanged with 10 mM PBS using a 10 kD cut-off
membrane filter. It was deacetylated using hydroxylamine
hydrochloride and then incubated at room temperature for 2 hours.
Finally it was desalted and concentrated with 10 mM PBS.
Example 3
Activation of Oligosaccharide
[0124] Firstly the amine concentration of oligomer was estimated by
TNBS assay. Then 1.5 molar equivalent of 6-maleimidohexonoic acid
N-hydroxysuccinimide ester was added respective to oligomer amine
molar concentration. This reaction mixture was incubated at room
temperature for 5 hours. After this, the derivatized oligomer was
dried using vacuum for one hour. It was then desalted and buffer
exchanged with 10 mM PBS using chromatography and concentrated to
about 5 to 10 mg/ml.
Example 4
Conjugation of Synthetic Oligosaccharide with Carrier Protein
[0125] The synthetically prepared oligomer was conjugated with
carrier protein (TT) using thiol chemistry, where both the
activated oligomer and derivatized TT prepared as above were mixed
and incubated at 2 to 8.degree. C. for 96 hours.
[0126] After the conjugate was formed, it was purified and followed
by buffer exchange with 1.times.PBS for which 10 kD cut-off
membrane filter was used. Finally it was filtered through 0.2.mu.
filter.
Example 5
Test for Immunogenicity of Semi-Synthetic Oligosaccharide
Vaccine
[0127] Mouse immunogenicity study of the semi-synthetic
meningococcal serogroup C conjugate formulation was done. It was
administered through subcutaneous route. A group of 10 mice were
immunised with 3 doses of vaccine and each time the terminal bleed
was taken after 7 days. Control group receiving Menactra received 2
doses. Following this, anti-meningococcal C (Men-c) polysaccharide
IgG antibody levels were measured by a serotype specific ELISA.
Similarly sera from terminal bleeds of 10 mice immunized with
Menactra were pooled and used. Antibody concentration for standard
was assigned with arbitrary value of 100 units/ml. Antibody
concentration for test vaccine formulation were calculated in
relation to the standard.
[0128] Geometric mean antibody concentration following First dose
of semi-synthetic Men C was found to be 1.4, a significant increase
was observed following the Second dose to give a mean antibody
concentration of 176.23 units/ml. This was analogous with a memory
response and all mice showed increased antibody content. Mean
antibody concentration increased following the Third dose to 412.5
units/ml. (FIG. 5 and FIG. 6). This indicates that semi-synthetic
formulation elicited an antibody response with a similar profile to
a classical conjugate vaccine.
[0129] To determine functional antibodies in response to
vaccination Serum Bactericidal Assay (SBA) was performed. The sera
was acquired following immunization with semi-synthetic Men C
conjugate composition, Control Sera was obtained from mice
immunised with Quadri Meningo and Menactra.RTM.. Analysis of
variance between all the groups showed a significant difference
(p=0.00). Interrogation of the difference between the groups,
performed using Dunnerr test with multiple comparisons,
demonstrated significantly higher titres for the CONTROL groups
i.e. Quadri Meningo Vaccine sera (p-0.00) and Menactra.RTM. vaccine
sera (p=0.003) (FIG. 7).
[0130] Hence, high concentration of functional IgG antibodies
capable of killing Neisseria meningitidis in the SBA was found for
sMen-C preparation given at a 2.5 .mu.g immunisation dosage.
* * * * *
References