U.S. patent application number 13/183303 was filed with the patent office on 2012-01-12 for meningococcal and pneumococcal conjugate vaccine and method of using same.
Invention is credited to Che-Hung Robert Lee, Stanley Shih-Peng Tai.
Application Number | 20120009213 13/183303 |
Document ID | / |
Family ID | 42981141 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120009213 |
Kind Code |
A1 |
Tai; Stanley Shih-Peng ; et
al. |
January 12, 2012 |
Meningococcal And Pneumococcal Conjugate Vaccine And Method Of
Using Same
Abstract
This disclosure relates to vaccine formulations comprising an
immunogenic composition for inducing antibodies to both S.
pneumoniae and N. meningitides in a subject. In a preferred aspect,
the immunogenic composition comprises covalently conjugated
recombinant PsaA ("rPsaA") from S. pneumoniae and capsular
polysaccharide from N. meningitidis serogroup C. This disclosure
further relates to methods for producing the immunogenic
composition as well as methods for their use.
Inventors: |
Tai; Stanley Shih-Peng;
(Rockville, MD) ; Lee; Che-Hung Robert; (Silver
Spring, MD) |
Family ID: |
42981141 |
Appl. No.: |
13/183303 |
Filed: |
July 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12425232 |
Apr 16, 2009 |
8003112 |
|
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13183303 |
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Current U.S.
Class: |
424/197.11 |
Current CPC
Class: |
A61P 37/04 20180101;
Y10S 424/831 20130101; A61K 39/092 20130101; A61P 31/04 20180101;
A61K 2039/6068 20130101; A61K 2039/70 20130101; A61K 39/095
20130101 |
Class at
Publication: |
424/197.11 |
International
Class: |
A61K 39/385 20060101
A61K039/385; A61P 31/04 20060101 A61P031/04 |
Claims
1. A vaccine comprising capsular polysaccharide from N.
meningitidis conjugated to a pneumococcal protein.
2. The vaccine of claim 1 wherein the N. meningitidis capsular
polysaccharide is derived from at least one N. meningitidis
serogroup selected from the group consisting of serogroups A, B, C,
D, X, Y, Z, 29E, W-135, and combinations thereof.
3. The vaccine of claim 2 wherein the N. meningitidis capsular
polysaccharide is derived from N. meningitidis serogroup C.
4. The vaccine of claim 1 wherein the pneumococcal protein is
selected from the group consisting of PsaA, pneumolysin, PspA,
PspC, CbpA, pneumococcal histidine triad protein selected from the
group consisting of PhtA, BVH11-3, PhtB, PhpA, BVH-11, PhtE, BVH-3,
PhtD and BVH-11-2, PcpA, PppA, Dpr, NanA, NanB, PiuA, PiaA, LytA,
LytB, LytC, ClpP, PavA, and combinations thereof.
5. The vaccine of claim 4 wherein the pneumococcal protein is
PsaA.
6. A method for generating an immune response against N.
meningitidis and S. pneumoniae in an individual, the method
comprising administering to the individual an amount of vaccine
composition effective for generating an immune response to both N.
meningitidis and S. pneumoniae, wherein the vaccine composition
comprises N. meningitidis capsular polysaccharide conjugated to a
pneumococcal protein.
7. The method of claim 6 wherein the vaccine is administered by a
route selected from the group consisting of oral intravenous,
intramuscular, nasal, subcutaneous, intraperitoneal, and
combinations thereof.
8. The method of claim 6 wherein the N. meningitidis capsular
polysaccharide is derived from at least one N. meningitidis
serogroup selected from the group consisting of serogroups A, B, C,
D, X, Y, Z, 29E, W-135, and combinations thereof.
9. The method of claim 8 wherein the N. meningitidis polysaccharide
is derived from N. meningitidis serogroup C.
10. The method of claim 6 wherein the pneumococcal protein is
selected from the group consisting of PsaA, pneumolysin, PspA,
PspC, CbpA, pneumococcal histidine triad protein selected from the
group consisting of PhtA, BVH11-3, PhtB, PhpA, BVH-11, PhtE, BVH-3,
PhtD and BVH-11-2, PcpA, PppA, Dpr, NanA, NanB, PiuA, PiaA, LytA,
LytB, LytC, ClpP, PavA, and combinations thereof.
11. The method of claim 10 wherein the pneumococcal protein is
PsaA.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 12/425,232, filed Apr. 16, 2009, which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This disclosure relates to vaccine formulations that include
an immunogenic composition for inducing antibodies to the S.
pneumoniae PsaA protein and N. meningitidis capsular
polysaccharide. This disclosure further relates to methods for
producing the immunogenic composition as well as methods for their
use.
BACKGROUND OF THE INVENTION
[0003] Status of current pneumococcal vaccines. S. pneumoniae is a
gram-positive encapsulated diplococcus. Capsule, a layer of
polysaccharide (PS) surrounding the bacterial cell, is a major
virulence factor of S. pneumoniae. Based on the differences in
structure and immunological response to capsular polysaccharide, S.
pneumoniae can be divided into more than 90 different serotypes.
Capsular polysaccharides are the base for the currently used
vaccines. The FDA has approved two types of pneumococcal vaccines
for use in humans: a 23-valent PS vaccine and a 7-valent PS/protein
conjugate vaccine. The former is comprised of capsular
polysaccharide purified from 23 different serotypes of S.
pneumoniae, which account for almost 89 percent of disease cases.
PNEUMOVAX.RTM. (Merck) is an example of this group of vaccines.
However, PS elicits type-specific antibodies. Antibodies raised for
one serotype do not provide protection against infection of other
serotypes. The efficacy of the 23-valent vaccine is limited.
Furthermore, PS is a T-cell independent antigen which induces
short-term immunity without immune memory and is not effective in
children younger than two years of age (Greenwood B M et al., Trans
R Soc Trop Med Hyg, 1980, 74:756-760). It is only recommended for
high risk groups, such as the elderly and persons with underlying
disease. A recently approved pneumococcal vaccine is a mixture of
conjugates of 7 different individually prepared capsular
polysaccharides covalently linked with carrier protein CRM.sub.197,
which is a non-toxic and immunologically cross-reactive mutant of
diphtheria toxin (Uchida et al, J. Biol. Chem. 248:3838-3844, 1973)
and a component of the pediatric DPT (Diphtheria-Tetanus-Pertussis
toxin) vaccine. Upon conjugation to a carrier protein, the
otherwise T-cell independent PS becomes a T-cell dependent antigen
by obtaining the immunological property of the protein. (Schneerson
R et al., J Exp Med 1980, 152:361-376). The conjugate induces
long-lasting immunity with immune memory and is effective in young
infants. The 7 serotypes were selected for their prevalence in
pediatric diseases. A conjugate vaccine of 7 pneumococcal capsular
PS(PCV7) with CRM.sub.197 (Wyeth) is the only vaccine of this
family that is commercially available. It is only prescribed for
use in the prevention of pediatric invasive pneumococcal disease
because of its high cost and limited supply. The drawback of these
two families of vaccines is that they only provide protection
against infection by the specific serotypes of S. pneumoniae that
are included in the respective vaccine formulations.
[0004] Status of current meningococcal vaccine. N. meningitidis is
a gram-negative, encapsulated diplococcus. At least 13 different
serogroups have been identified based on the structure of capsular
PS, but serogroups A, B, C, Y, and W-135 account for almost all
cases of disease. Serogroup B organisms account for 46 percent of
all cases, serogroup C for 45 percent of all cases, and serogroups
W-135 and Y and strains that could not be serogrouped account for
most of the remaining cases. Like S. pneumoniae, the major
ingredient for meningococcal vaccines is capsular PS. Its vaccines
can be divided into two families: the capsular PS vaccine and
PS-protein conjugate vaccines. Three versions of PS vaccines are
commercially available.
[0005] Quadrivalent PS vaccine (GlaxoSmithKline and Sanofi-Pasteur)
is composed of capsular PS purified from serogroups A, C, Y, and
W-135. It is expensive and not affordable for developing countries.
Bivalent PS vaccine (GlaxoSmithKline and Sanofi-Pasteur) is
composed of capsular PS purified from serogroups A and C. Trivalent
PS vaccine (GlaxoSmithKline) is composed of capsular PS purified
from serogroups A, C, and W-135. This vaccine has been used in the
epidemics in the "Meningitis Belt" countries in Africa. Like
pneumococcal vaccine, PS vaccine is not efficacious in children
younger than two years of age. Such deficiency can be overcome by
PS-protein conjugates.
[0006] Two types of meningococcal vaccine conjugates are
commercially available or being developed. MENACTRA.RTM.
(Sanofi-Pasteur) is the first quadrivalent conjugate meningococcal
vaccine. It is a mixture of meningococcal polysaccharides (groups
A, C, Y, and W135) conjugated with diphtheria toxoid. A monovalent
meningococcal conjugate vaccine currently under development is a
conjugate of serogroup C polysaccharide-diphtheria toxoid (Chiron
and Wyeth), serogroup C PS-tetanus toxoid (Chiron, Baxter), and
serogroup A PS-tetanus toxoid (PATH-SII). Preliminary results of
clinical trials indicate these vaccines are efficacious.
[0007] With the burden of S. pneumoniae and N. meningitidis
infection on the public health system at a global scale, it is
desirable to have a single vaccine that is effective to prevent
disease resulting from the infection of both pathogens.
SUMMARY
[0008] This disclosure provides an immunogenic composition for
inducing an immune response to two different microorganisms, S.
pneumoniae and N. meningitidis. This disclosure further provides an
inoculum and/or vaccine comprising the immunogenic composition
dispersed and/or dissolved in a pharmaceutically acceptable
diluent. The vaccine includes at least one N. meningitidis capsular
polysaccharide conjugated to a pneumococcal protein. In a preferred
aspect, the immunogenic composition comprises recombinant PsaA
("rPsaA") from S. pneumoniae and capsular polysaccharide from N.
meningitidis serogroup C. Pneumococcal protein acts as an antigen
as well as a carrier protein for N. meningitidis capsular
polysaccharide in the vaccine. Thus, the vaccine is effective for
providing dual protection against infection by both S. pneumoniae
and N. meningitidis.
[0009] Several pneumococcal proteins are universally found in all
tested serotypes of S. pneumoniae, such as pneumococcal surface
antigen A (PsaA), pneumococcal surface protein A (PspA),
pneumococcal surface protein C (PspC), pneumolysin, and
histidine-triad proteins. Studies have shown that these proteins
are capable of eliciting protective antibodies in laboratory
animals. In particular, PsaA has been found by immunological and
PCR methods in all S. pneumoniae tested including 23 vaccine
serotypes as well as clinical isolates from various countries. PsaA
has a length of 309 amino acid residues. In an important aspect,
the rPsaA used in the immunogenic composition described herein
includes at least the amino acid residues at positions 21 to 319 of
SEQ ID NO:1.
[0010] The capsular polysaccharide (about 300,000 Da) of N.
meningitidis serogroup C comprises about 850 repeating units of
sialic acid with a(2.fwdarw.9) glycosidic linkage and about 80
percent O-acetylation at C7 or C8. The capsular polysaccharide of
N. meningitidis serogroup C and PsaA are provided in conjugated
form. In a preferred aspect, the capsular polysaccharide and PsaA
are conjugated by covalent linkage.
[0011] In another aspect, a method is provided for generating an
immune response in a subject against pneumococcal surface antigen A
(PsaA) and capsular polysaccharide from N. meningitidis serogroup
C. The method comprises administering to a subject an effective
amount for inducing production of antibodies specific to rPsaA and
capsular polysaccharide from N. meningitidis serogroup C.
Administering to a subject a combination of rPsaA and capsular
polysaccharide from N. meningitidis serogroup C in covalently
linked form is effective for generating an immune response in the
subject. In an important aspect, immunogenicity of the conjugated
pneumococcal surface antigen A (PsaA) and capsular polysaccharide
is significantly increased as compared to the immune response
observed when the antigens are administered individually. In this
aspect, more than a 40-fold increase in immunogenicity is seen for
conjugated PsaA as compared to non-conjugated PsaA, and more than a
170-fold increase in immunogenicity is seen for conjugated capsular
polysaccharide as compared to non-conjugated capsular
polysaccharide.
[0012] The immunogenic composition may be administered to a subject
by a number of different routes, including intramuscular
administration, intranasal administration, oral administration,
sub-cutaneous administration, transdermal administration, and
transmucosal administration.
[0013] Immunogenic compositions described herein are prepared by a
method comprising preparing recombinant PsaA ("rPsaA") and
conjugating rPsaA with capsular polysaccharide from N. meningitidis
serogroup C. rPsaA can be prepared using well-known recombinant
techniques. Capsular polysaccharide can be isolated from natural
sources or synthesized using a number of techniques which are well
known in the art.
[0014] The immunogenic compositions described herein advantageously
provide dual protection against S. pneumoniae and N. meningitidis
infection. The immunogenic composition described herein also
utilizes PsaA as a protein carrier for polysaccharide.
[0015] Advantageously, the conjugated immunogenic composition
provided herein can reduce the costs of preparing and administering
the vaccine. This is a particularly important benefit to developing
and underdeveloped countries because the vaccine will reduce the
economic and medical burden to the countries which have high rates
of pneumococcal and meningococcal disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 provides the nucleotide sequence (SEQ ID NO. 2) of a
cloned psaA fragment, including restriction endonuclease sites at
the 5' and 3' ends produced according to the Example.
[0017] FIG. 2 provides the deduced amino acid sequence (SEQ ID NO.
1) of recombinant PsaA protein produced according to the
Example.
[0018] FIG. 3 shows a photograph of a SDS-polyacrylamide gel
electrophoresis and Western blot analysis of rPsaA according to the
Example.
[0019] FIG. 4 is a chromatogram demonstrating that the protein
signal shifted from a low molecular weight position to a high
molecular weight for the conjugate produced according to the
Example.
[0020] FIG. 5A shows a photograph of an Immuno-dot blot according
to the Example. FIG. 5B shows a photograph of a Western blot
according to the Example.
DETAILED DESCRIPTION
[0021] This disclosure provides an immunogenic composition
comprising capsular polysaccharide from N. meningitidis and a
protein from S. pneumoniae (referred to as "Pn-Mn" vaccine). In a
preferred aspect, the S. pneumoniae protein is recombinant
pneumococcal surface antigen A ("rPsaA") and the N. meningitidis
capsular polysaccharide is serogroup C capsular polysaccharide.
PsaA is universally found in all tested serotypes of S. pneumoniae.
The immunogenic composition is useful for inducing production of
antibodies for diagnostic and therapeutic purposes. This disclosure
further provides an inoculum and vaccine comprising the immunogenic
composition dispersed or dissolved in a pharmaceutically acceptable
diluent. It is particularly preferred that the rPsaA from S.
pneumoniae is covalently conjugated to capsular polysaccharide from
N. meningitidis serogroup C.
[0022] The term "antibody" refers to a molecule that is a member of
a family of glycosylated proteins called immunoglobulins, which can
specifically bind to an antigen. The word "antigen" refers to an
entity that is bound by an antibody. "Immunogen" or "immunogenic
composition" refers to the entity that induces antibody production
or binds to the receptor.
[0023] The words "protein" and "polypeptide" are used
interchangeably throughout the specification and designate a series
of amino acid residues connected by peptide bonds.
[0024] Capsular Polysaccharide from N. meningitidis Serogroup C
[0025] Polysaccharide is a T cell-independent (T-I) antigen
inducing short-term immunity with little immune memory and is not
effective in infants younger than 2 years old. When covalently
linked to a carrier protein, the resulting PS component in a
conjugate vaccine becomes a T cell-dependent (T-D) antigen inducing
long-term immunity with immune memory even in infants and young
children.
[0026] The capsular polysaccharide of N. meningitidis serogroup C
comprises repeating units of sialic acid with a (2.fwdarw.9)
glycosidic linkage and about 80 percent O-acetylation at C7 or C8.
The size of the N. meningitidis group C polysaccharide is about 590
to about 1,030 sialic acid repeating units assuming the molecular
weight of a sialic acid repeating unit is 340 Daltons. The size of
the N. meningitidis serogroup C capsular polysaccharide
particularly useful in the invention is about 200 to about 350 kDa,
preferably about 250 to about 300 kDa, although other sizes may be
used, if desired, provided that the selected size of the
polysaccharide is effective to induce production of antibodies in a
subject after conjugation to a carrier protein.
[0027] The capsular polysaccharide can be isolated from natural
sources using a number of techniques which are well known in the
art. For example, N. meningitidis group C strain can be grown in a
defined medium for 18 hours and inactivated with 0.5 percent
formaldehyde. After centrifugation to precipitate the cells, the
polysaccharide in the removed supernatant can be precipitated by
0.1 percent cetavlon. The insoluble cetavlon complex is then
dissolved in 0.9 M calcium chloride and the crude polysaccharide is
precipitated with 5 volume ethanol. The precipitate is further
dissolved in phosphate buffer. After phenol extraction and
ribonuclease treatment, the sample is dialyzed against water and
concentrated (Bundle et al, J. Biol. Chem. 249:4797-4801, 1974,
which is incorporated herein by reference.)
[0028] In another aspect, the capsular polysaccharide derived from
N. meningitidis serogroup C may be substituted with capsular
polysaccharide derived from N. meningitidis serogroups A, B, D, X,
Y, Z, 29E, W-135, or a combination thereof, in the Pn-Mn conjugates
described herein. N. meningitidis serogroups A, B, C, D, X, Y, Z,
29E, and W-135 account for almost all cases of disease. Such
conjugates can be administered to a subject capable of inducing an
immune response to an antigen in order to provide protection
against infection of these serogroups. Meningococcal serogroup A
polysaccharide (about 300 kDa) is composed of N-acetyl mannosamine
6-phosphate repeating units with a (1.fwdarw.phosphate) glycosidic
linkage and about 70-90 percent O-acetylation at C3. Meningococcal
serogroup W135 polysaccharide (.about.300,000 Daltons) is composed
of (2.fwdarw.6) .alpha.-D-galactose (1.fwdarw.4) .alpha.-D-sialic
acid repeating units with about 70 percent O-acetylation at C7 or
C9 of the sialic acid residue. Meningococcal serogroup Y
polysaccharide (about 300 kDa) is composed of (2.fwdarw.6)
.alpha.-D-galactose (1-4) .alpha.-D-sialic acid repeating units
with about 70 percent O-acetylation at C7 or C9 of the sialic acid
residue. The size of the N. meningitidis capsular polysaccharide
particularly useful in the invention is about 200 to about 350 kDa,
preferably about 250 to about 300 kDa, although other sizes may be
used, if desired, provided that the selected size of the
polysaccharide is effective to induce production of antibodies in a
subject after conjugation to a carrier protein. The activation
conditions for these polysaccharides may be different from that for
group C polysaccharide due to differences in their structures.
[0029] Pneumococcal Protein
[0030] PsaA has a length of 309 amino acid residues. It is
preferred that the rPsaA used in the immunogenic composition
includes at least the residues at positions 21 to 319 of SEQ ID
NO:1.
[0031] Recombinant PsaA from S. pneumoniae can be prepared using
conventional recombinant techniques. Recombinant methodologies
required to produce a DNA encoding a desired protein are well known
and are routine to those of ordinary skill in the art. The nucleic
acid sequences used to practice this invention, whether cDNA,
genomic DNA, vectors, and the like, may be isolated from a variety
of sources, genetically engineered, amplified, and/or expressed
recombinantly. The nucleotide sequence for psaA is provided at
nucleotide positions 6 to 867 in SEQ ID NO:2. The coding sequence
of the desired protein can be cloned into a vector.
[0032] Any recombinant expression system can be used, including
bacterial, mammalian, yeast, insect, or plant cell expression
systems. Alternatively, these nucleic acids can be synthesized in
vitro by well-known chemical synthesis techniques. Double stranded
DNA fragments may then be obtained either by synthesizing the
complementary strand and annealing the strands together under
appropriate conditions, or by adding the complementary strand using
DNA polymerase with an appropriate primer sequence.
[0033] Nucleic acid amplification methods are well known in the
art. Oligonucleotide primers can be used to amplify nucleic acids
to generate psaA coding sequence used to prepare recombinant PsaA.
The coding sequence can be cloned into an expression cassette, such
as plasmids, recombinant viruses which can infect or transfect
cells in vitro, ex vivo, and/or in vivo, and other vectors which
can be used to express the PsaA polypeptide in vitro or in vivo.
Selection markers can be incorporated to confer a selectable
phenotype on transformed cells, such as antibiotic resistance. The
expressed rPsaA can be recovered and purified using conventional
techniques.
[0034] In another aspect and in addition to PsaA, other
pneumococcal proteins can be used as a component of the Pn-Mn
conjugate vaccine provided herein. Other S. pneumoniae proteins
that may be used include pneumolysin, pneumococcal surface protein
A (PspA), pneumococcal surface protein C (PspC or CbpA),
pneumococcal histidine triad proteins or similar proteins with
different nomenclatures such as PhtA or BVH11-3, PhtB or PhpA or
BVH-11, PhtE or BVH-3), PhtD or BVH-11-2, and, pneumococcal choline
binding protein A (PcpA), non-heme iron-containing ferritin or
pneumococcal protective proteins (PppA, Dpr), neuraminidase A
(NanA), neuraminidase B (NanB), iron transport proteins or
iron-compound-binding protein PiuA and PiaA,
N-acetylmuramoyl-L-alanine amidase or autolysin (LytA),
endo-.beta.-acetylglucosaminidase (LytB),
1,4-.beta.-N-acetylmuranminidase (LytC), caseinolytic protease or
serine proteases (ClpP), and adherence and virulence protein A
(PavA).
[0035] Conjugate Preparation
[0036] Polysaccharides contain hydroxyl groups, and occasionally
carboxyl and amino groups, and proteins contain amino and carboxyl
groups. Both polysaccharides and proteins are not active for
chemical reaction with each other in their natural form. Proper
pretreatment or activation of one or both of the polysaccharide and
protein is required to convert the otherwise non-reactive molecules
to a reactive form in order to produce the polysaccharide-protein
conjugate. Many methods are known in the art for conjugating a
protein to a polysaccharide. Polysaccharide can be activated by
cyanogen bromide to provide cyanate groups which react with
hydrazide-activated protein (Schneerson et al., J. Exp. Med. 1980;
152:361-3760). Polysaccharide can be activated by cyanogen bromide
to provide cyanate groups, which further reacts with di-hydrazide,
and then conjugates to protein in the presence of EDC (Chu et al.,
Infect. Immun 1983; 40:245-256). Polysaccharide can be partially
hydrolyzed and added with an amino group at the reducing terminus.
After a bifunctional linker is added to the amino group, the
activated polysaccharide is conjugated to the carrier protein
(Costantino et al., Vaccine 1992; 10:691-8). Polysaccharide can be
activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate
to provide cyanate groups which react with a carrier protein (Lees
A, Nelson B L, Mond J J. Vaccine 1996; 14:190-198).
[0037] In a preferred aspect, rPsaA is dialyzed before use, such as
against 30 mM NaCl at about 4.degree. C. for about 18 to about 24
hours. The dialyzed rPsaA is then treated to activate the protein,
such as with 0.1 M MES (pH 6.5), 0.5 M hydrazine (pH 7.0), and 20
mM 1-[3-dimethylamino propyl)-3-ethyl carbodiimide-HCl ("EDC" from
Sigma-Aldrich) in saline, and incubated for 4 hours. The treated
rPsaA is then neutralized, such as with 1 M NaOH, before dialyzing
the protein, such as dialyzing against buffer containing 3 mM
Na.sub.2CO.sub.3 and 30 mM NaCl at 4.degree. C. The dialyzed
activated rPsaA can be used immediately or stored at 4.degree.
C.
[0038] In a preferred aspect, the capsular polysaccharide is
treated with 6 mM sodium periodate and incubated for 4 hours at
room temperature to activate the capsular polysaccharide. The
activated capsular polysaccharide is then dialyzed against
deionized water, such as for about 18 to about 24 hours at
4.degree. C. The dialyzed activated capsular polysaccharide can be
used immediately or stored at 4.degree. C.
[0039] Activated rPsaA is lyophilized, redissolved in water.
Dialyzed activated capsular polysaccharide (is lyophilized,
redissolved in 0.2 M HEPES, pH 7.5, 30 mM EDTA. The protein
solution is added to the polysaccharide solution and incubated
overnight. NaBH.sub.4 is added to a final concentration of 50 mM
and incubated for about 4 to about 6 hours to reduce the C.dbd.N
double bonds in the polysaccharide-protein conjugate to C--N single
bonds, and to reduce the unreacted aldehyde to alcohol. The
conjugate is dialyzed against 150 mM NaCl, 10 mM HEPES (pH 7), 1 mM
EDTA at 4.degree. C. The dialyzed conjugate can then be evaluated,
such as by HPLC, for shift of protein signal (280 nm) from 19
minute position to 18 minute upon conjugation.
[0040] Method of Using Conjugate
[0041] The rPsaA/capsular polysaccharide conjugate provided herein
can be administered to a subject capable of inducing an immune
response to an antigen. The rPsaA/capsular polysaccharide conjugate
is administered to the subject in an effective amount for inducing
an antibody response. An "effective amount" is an amount of
rPsaA/capsular polysaccharide conjugate which assists a subject in
producing both anti-rPsaA and anti-capsular polysaccharide
antibodies. Such antibodies may prevent infection by S. pneumoniae
and N. meningitidis serotype C.
[0042] One of ordinary skill in the art can determine whether an
amount of the rPsaA/capsular polysaccharide conjugate is effective
to induce immunity in a subject using routine methods known in the
art. For example, the ability of an antigen to produce antibody in
a subject can be determined by screening for antibodies using
separate coating antigens rPsaA and capsular polysaccharide in the
respective ELISA assays.
[0043] In one aspect, a vaccine formulation is provided for N.
meningitidis serogroup C and S. pneumoniae. The vaccine formulation
is effective for generating an immune response in a subject to both
N. meningitidis serogroup C and S. pneumonia. The vaccine
formulation comprises rPsaA from S. pneumoniae and capsular
polysaccharide from N. meningitidis serogroup C. The conjugated
immunogenic composition can be provided with one or more additional
components, such as a pharmaceutically acceptable diluents,
carriers, adjuvants, and/or buffers. For example, the conjugate can
be dispersed or dissolved in a diluent.
[0044] The immunogenic composition may be prepared as a solution,
suspension, tablet, pill, capsule, sustained release formulation,
powder, or the like. The antigens and immunogenic composition may
be mixed with physiologically acceptable carriers which are
compatible therewith. These may include water, saline, dextrose,
glycerol, ethanol, combinations thereof, and the like. The vaccine
may further contain auxiliary substances, such as wetting or
emulsifying agents or pH buffering agents, to further enhance the
effectiveness. Administration of the conjugate in a vaccine
formulation can include delivery by various routes, such as, for
example, oral, intravenous, intramuscular, nasal, subcutaneous, and
intraperitoneal administration.
[0045] The immunogenic composition is administered in a manner
compatible with the dosage formulation, and in such amount as to be
therapeutically effective, protective, and immunogenic. The
quantity to be administered depends on the subject to the
immunized, including, for example, the capacity of the subject's
immune system to synthesize antibodies and, if needed, to produce a
cell-mediated immune response. Precise amounts of antigen and
immunogenic composition to be administered depend on the judgment
of the practitioner. However, suitable dosage ranges are readily
determinable by those skilled in the art and may be of the order of
micrograms to milligrams. Suitable regimes for initial
administration and booster doses are also variable but may include
an initial administration followed by subsequent administrations.
The dosage of the vaccine may also depend on the route of
administration and will vary according to the size of the
subject.
[0046] In an important aspect, the rPsaA/capsular polysaccharide
Pn-Mn conjugate provided herein may be used to prevent infection of
both S. pneumoniae and N. meningitidis serotype C, which are the
leading causes of otitis media and meningitis in young children.
Furthermore, the rPsaA/capsular polysaccharide conjugate provided
herein also could be used in the prevention of other pneumococcal
and meningococcal diseases, such as bacteremia, pneumoniae and
meningitis in the population of other age groups.
[0047] The examples that follow are intended to illustrate the
invention and not to limit it. All percentages used herein are by
weight unless otherwise indicated. All patents, patent
applications, and literature references cited herein are hereby
incorporated by reference in their entirety.
Example
[0048] A better understanding of the vaccine provided herein and
its many advantages is provided with the following example.
A. Preparation of Purified rPsaA
[0049] psaA gene cloning and expression. To prepare recombinant
pneumococcal PsaA (rPsaA) protein, the coding sequence of
pneumococcal psaA genes in E. coli was cloned in the expression
vector pET22b(+) (Novagen, Madison, Wis.). Sequence analysis
revealed that the coding sequence of psaA does not include BamHI
and HindIII restriction sites. For the purpose of cloning,
expression, and purification of rPsaA protein, a pair of primers
for PCR amplification were designed so that: 1) the PCR product
would have a BamHI and HindIII site at the 5' and 3' ends,
respectively; 2) the reading frame of cloned psaA would be in-frame
with that of the vector; and 3) the produced rPsaA protein would
have a His-tag at its C-terminal. The forward and reverse primers
(5'-GGGATCCTAGCGGAAAAAAAGATACA-3' (SEQ ID NO. 3),
5'-GCAAGCTTTGCCAATCCTTCAGCAATC-3' (SEQ ID NO. 4), respectively,
were intended to amplify a 868-bp fragment starting from nucleotide
no. 42 to no. 921 of the psaA coding sequence. The underlined
nucleotides indicate the positions of BamHI and HindIII sites in
these primers. The coded rPsaA protein would have 331 amino
residues and a predicted molecular mass of 36,940 daltons. The
nucleotide sequence of the cloned fragment is shown in FIG. 1 and
the predicted amino acid sequence for rPsaA in FIG. 2.
[0050] A typical PCR mixture contained 5 .mu.mole primers, 20 ng S.
pneumoniae serotype 4 chromosomal DNA and PCR Supermix (Life
Technologies, Rockville, Md.). The conditions for PCR were as
follows: DNA denaturation at 95.degree. C. for 40 seconds, primer
annealing at 42.degree. C. for 1 min, and DNA synthesis at
72.degree. C. for 1.5 min. After 30 cycles of synthesis, the
reaction was terminated with an extension at 72.degree. C. for 5
min. The PCR products were purified with the GeneClean kit
(Qbiogen, Carlsbad, Calif.), cloned into pGEM-T easy vector
(Promega, Madison, Wis.) and transformed into E. coli DH5.alpha..
The insert was isolated from the resultant plasmid after a double
digestion with restriction enzymes BamHI and HindIII, cloned into
the compatible site of pET22b(+) to generate plasmid pST648, and
transformed into E. coli BL21(DE3). To confirm that psaA gene on
pST648 was cloned as planned, the restriction map of the cloned PCR
product was determined. The results were consistent with those of
published psaA gene. The proper cloning of the BamHI-HindIII
restriction fragment into pET22b(+) was further confirmed by the
induction of recombinant protein and by the presence of His-tag at
the carboxyl end.
[0051] To induce the synthesis of recombinant protein,
isopropyl-13-D-thiogalactoside (IPTG, 0.1 mM) was added to the
log-phase culture (A600 nm.sup.=0.6) of E. coli BL21(DE3) harboring
pST648 and growth continued for another 2 hours. Cells were
harvested, washed, suspended in one-tenth volume of 50 mM Tris-HCl,
pH 7.9 containing 200 mM NaCl (TN buffer) at 4.degree. C., and
disrupted by sonication. After the removal of unbroken cells by
centrifugation, the supernatant was subject to SDS-PAGE analysis.
To confirm that recombinant protein had a His-tag, the proteins on
the SDS-gel were analyzed by western blotting against mouse
monoclonal anti-poly-histidine antibody (Sigma-Aldrich, St. Louis,
Mo.). The proteins on the gel were transferred onto nitrocellulose
paper and the paper was washed with blotto (20 mM Tris, 0.2M NaCl,
1.5 percent nonfat milk), incubated with monoclonal
anti-poly-histidine antibody in blotto (1:200 dilution) for 2
hours, washed with blotto three times, incubated with alkaline
phosphatase-conjugated goat anti-mouse antibody in blotto (1:5000
dilution), and washed with blotto and AP buffer (0.1 M NaCl, 0.1 M
Tris-C1, pH 9.5). The antibody-antigen interaction was visualized
by incubating with 0.1 percent naphthol and 1 percent fast blue
(Sigma-Aldrich). The results are shown in FIG. 3. The results
indicate that the overproduced protein was indeed rPsaA and the
crude cell lysate of E. coli BL2I(DE3)(pST648) could be used as the
source of rPsaA in protein purification.
[0052] Purification of rPsaA protein. To purify rPsaA protein,
crude cell lysate was loaded on a HIS-BIND.RTM. Column (Novagen,
Madison, Wis.). The resin was washed with binding buffer (TN buffer
containing 50 mM imidazole) and washing buffer (TN buffer
containing 200 mM imidazole) to remove excess and nonspecifically
bound proteins. The bound protein was eluted with elution buffer
(TN buffer containing 1 M imidazole) and analyzed by
SDS-polyacrylamide gel electrophoresis and Western blotting against
monoclonal anti-poly-histidine antibody as described above.
Fractions containing protein that reacts with anti-poly-histidine
monoclonal antibody were collected as purified rPsaA protein (FIG.
3).
B. Preparation of rPsaA-MCPS Conjugate
[0053] Activation of rPsaA. rPsaA was dialyzed against 30 mM NaCl
at 4.degree. C. overnight before use. The dialyzed rPsaA was mixed
with 1 M MES, pH 6.5, 5 M hydrazine, pH 7.0, 1 M EDC
(Sigma-Aldrich) in saline at the final concentration of 0.1 M, 0.5
M, and 20 mM, respectively. After incubation at room temperature
for 4 hours, 1 M NaOH (0.05 mL) was added to neutralize the
reaction before dialysis against buffer containing 3 mM
Na.sub.2CO.sub.3 and 30 mM NaCl at 4.degree. C. The protein
solution was stored at 4.degree. C.
[0054] MCPS activation. N. meningitidis type C capsular PS (MCPS,
10 mg/ML) was mixed with sodium periodate at a final concentration
of 6 mM. After incubation at room temperature for 4 hours, the
reaction mixture was dialyzed against deionized water overnight and
stored at 4.degree. C.
[0055] Conjugation of PsaA-MCPS. Aliquot activated rPsaA (0.25 mg)
was lyophilized and re-dissolved in 25 .mu.l water. Aliquot
activated MCPS (0.25 mg) was lyophilized and redissolved in 25
.mu.l of 0.2 M HEPES, pH 7.5 containing 30 mM EDTA. These two
solutions were combined. After incubation overnight at room
temperature, 5 .mu.l of 1 M NaBH.sub.4 was added and incubation
continued for another 6 hours. After dialysis against 150 mM NaCl,
10 mM HEPES, pH 7, 1 mM EDTA at 4.degree. C., the conjugate product
was stored at 4.degree. C. The conjugate of MCPS with rPsaA was
evaluated with HPLC analysis using a Waters Ultrahydrogel Linear
size-exclusion column and monitored at the wavelengths of 206 nm
and 280 nm. Upon conjugation, the protein signal shifted from low
molecular weight position to the high molecular weight in the
chromatogram, as shown in FIG. 4.
C. Characterization of rPsaA-MCPS Conjugate.
[0056] Immunogenicity. Mice (NIH-Swiss) were subcutaneously
immunized every two weeks with rPsaA, MCPS, or PsaA-MCPS conjugate,
respectively, at the dose of 1 .mu.g per mouse. Blood was collected
from optical vein two weeks after the third immunization and the
titers of antibodies were determined by enzyme-linked immunosorbent
assay (ELISA). Briefly, wells of microtiter plate (Dynatec, no. 1)
was coated with MCPS by adding 100 .mu.l of solution comprised of
antigen, 0.5 .mu.g/mL rPsaA or 5 .mu.g/mL native MCPS plus 5 mg/ml
methylated human serum albumin in PBS, pH 7.5 and incubated at room
temperature for at least 4 hours. Wells were washed three times
(150 .mu.l/well) with PBS containing 0.05 percent TWEEN.RTM. 20 and
0.02 percent NaN.sub.3. 100 .mu.L of diluent (5 percent calf serum
and 0.02 percent NaN.sub.3 in PBS) was added to each well and a
two-fold serial dilution of diluted (1:100) antiserum was prepared.
The reference serum, which was assigned with 3,200 units/mL IgG
against MCPS or rPsaA, was similarly treated in the same plate.
After incubating overnight at room temperature and washing three
times, 100 .mu.l of alkaline phosphate-conjugated goat anti-mouse
IgG Fc (1:3000 dilution) was added and incubated at room
temperature for 3 hours. Wells were washed three times and 100
.mu.l of substrate (p-nitrophenyl phosphate, 1 mg/mL in 1 M
Tris-HCl, pH 9.8 containing 0.3 mM MgCl.sub.2) was added. The plate
was incubated at room temperature for 20 minutes (it might vary
depending on the color development of sample and reference serum)
and the absorbances were measured at 405 nm. The respective
reference serum for MCPS and rPsaA was prepared in the laboratory
and were used as standards to determine the antibody level of the
sample serum. Results are shown in Table I below.
TABLE-US-00001 TABLE I Immunogenicity of rPsaA, MCPS, and
rPsaA-MCPS conjugate. IgG level* Antigen Dose anti-PsaA anti-MCPS
rPsaA 3 .times. 1 .mu.g 107 (9; 1678) -- MCPS 3 .times. 1 .mu.g --
533 (46; 6176) rPsaA- 3 .times. 1 .mu.g 4,418 (2006; 9734) 90,506
(50,421; 162,455) MCPS *The data that is not in parenthesis
represents the geometric mean of IgG antibody level in 10 antiserum
samples. The anti-rPsaA or anti-MCPS IgG antibody level of each
antiserum was measured by ELISA and compared with respective
reference serum, assigned with 3,200 unit/mL IgG antibody. The
numbers in parenthesis represents the confidence interval of one
standard deviation.
[0057] Both rPsaA and MCPS were immunogenic in mice in the absence
of adjuvant. Their immunogenicity increased significantly after
they were conjugated. When compared with each individual component,
the immunogenicity increased approximately 41-fold and 170-fold for
rPsaA and MCPS, respectively.
[0058] Reactivity of anti-rPsaA antibodies. It has been
demonstrated that active immunization of PsaA is effective to
protect laboratory animals from S. pneumoniae infection. To provide
protection, anti-PsaA should interact with all S. pneumoniae cells.
The cross-reactivity of the generated anti-rPsaA antibodies was
investigated by immuno-dot blotting and western blotting against
clinical isolates of S. pneumoniae, including serotypes 1, 2, 3, 4,
5, 6A, 6B, 7C, 8, 9A, 10A, 10B, 11A, 12A, 14, 15A, 15C, 16F, 18A,
18C, 19A, 19F, 20, 24, 22A, 23B, 23F, 23C and 35. Cells of S.
pneumoniae were cultured in 15 mL Todd-Hewitt broth overnight at
37.degree. C. in the presence of 5 percent CO.sub.2, harvested by
centrifugation, and suspended in 2 mL of TN buffer. Cells were
disrupted by sonication in ice bath at the energy level of 7, 50
percent cycle, for 5 minutes. The supernatant after centrifugation
at 10,000.times.g for 10 minutes was collected and used as the
source of S. pneumoniae proteins. For immuno-dot blotting, 5 .mu.l
cell lysate was spotted on the nitrocellulose paper. For Western
blot, randomly selected pneumococcal cell lysates were analyzed by
SDS-PAGE and transferred on nitrocellulose paper. The paper was
processed as described above, except anti-rPsaA antibody was used.
Results are shown in FIG. 5A and FIG. 5B. The anti-rPsaA antibody
cross-reacted with cells of all serotypes tested and reacted with a
single protein that has an apparent molecular weight comparable to
that of PsaA.
[0059] Bactericidal activity of anti-MCPS antibody. The biological
function of the induced MCPS-specific antibodies was determined by
bactericidal assay against N. meningitidis serogroup C (strain
C11). Briefly, bacteria were cultured overnight on brain heart
infusion (BHI) agar plates containing 5 percent normal horse serum
(NHS) and transferred to fresh plates and cultured for 5 hours the
second day. Bacteria from the 5 hour culture were suspended to
65-66 percent transmittance at 530 nm in DPBSG (1.times.PBS, pH
7.2, 0.5 mM MgCl.sub.2, 0.9 mM CaCl.sub.2, and 0.01 percent
gelatin) followed by 1:10,000 dilution with the same buffer to
contain approximately 4,000 cfu/mL. In the wells of a microtiter
plate, 50 .mu.l-fold dilutions of test and control sera were
prepared with DPBSG and mixed with 25 .mu.l bacterial suspension
and 25 .mu.l baby rabbit complement (Pel-Freez, Rogers, Ark.).
After incubation at 37.degree. C. for 60 min, 10 .mu.l of the
bacterial suspension was withdrawn from each well and spread on the
BHI/NHS plate. The colonies were enumerated after incubation
overnight at 37.degree. C. with 5 percent CO.sub.2. The
bactericidal titer was the reciprocal of the highest dilution of
the sample yielding a 50 percent reduction in CFU as compared to
the control well containing complement without antiserum. The
geometric means of the titer for each mouse group was calculated.
Results are shown in Table II below.
TABLE-US-00002 TABLE II Bactericidal activity of antisera against
MCPS, rPsaA-MCPS conjugate. Antigen Bactericidal activity titer*
MCPS 109 (63; 190) rPsaA-PCPS 5022 (1123; 22454) *The data that is
not in parenthesis represents the geometric mean of sera from 10
mice for each antigen. The numbers in parenthesis represents the
confidence interval of one standard deviation.
[0060] Sera for both MCPS and rPsaA-MCPS conjugates had
bactericidal activity, but the titer for the conjugates were
significantly higher (approximately 46-fold).
[0061] While the invention has been particularly described with
specific reference to particular process and product embodiments,
it will be appreciated that various alterations, modifications, and
adaptations may be based on the present disclosure, and are
intended to be within the spirit and scope of the invention as
defined by the following claims.
Sequence CWU 1
1
41331PRTStreptococcus pneumoniae 1Met Lys Tyr Leu Leu Pro Thr Ala
Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Met
Asp Ile Gly Ile Asn Ser Asp Pro Ser 20 25 30Gly Lys Lys Asp Thr Thr
Ser Gly Gln Lys Leu Lys Val Val Ala Thr 35 40 45Asn Ser Ile Ile Ala
Asp Ile Thr Lys Asn Ile Ala Gly Asp Lys Ile 50 55 60Asp Leu His Ser
Ile Val Pro Ile Gly Gln Asp Pro His Glu Tyr Glu65 70 75 80Pro Leu
Pro Glu Asp Val Lys Lys Thr Ser Glu Ala Asn Leu Ile Phe 85 90 95Tyr
Asn Gly Ile Asn Leu Glu Thr Gly Gly Asn Ala Trp Phe Thr Lys 100 105
110Leu Val Glu Asn Ala Lys Lys Thr Glu Asn Lys Asp Tyr Phe Ala Val
115 120 125Ser Asp Gly Val Asp Val Ile Tyr Leu Glu Gly Gln Asn Glu
Lys Gly 130 135 140Lys Glu Asp Pro His Ala Trp Leu Asn Leu Glu Asn
Gly Ile Ile Phe145 150 155 160Ala Lys Asn Ile Ala Lys Gln Leu Ser
Ala Lys Asp Pro Asn Asn Lys 165 170 175Glu Phe Tyr Glu Lys Asn Leu
Lys Glu Tyr Thr Asp Lys Leu Asp Lys 180 185 190Leu Asp Lys Glu Ser
Lys Asp Lys Phe Asn Lys Ile Pro Ala Glu Lys 195 200 205Lys Leu Ile
Val Thr Ser Glu Gly Ala Phe Lys Tyr Phe Ser Lys Ala 210 215 220Tyr
Gly Val Pro Ser Ala Tyr Ile Trp Glu Ile Asn Thr Glu Glu Glu225 230
235 240Gly Thr Pro Glu Gln Ile Lys Thr Leu Val Glu Lys Leu Arg Gln
Thr 245 250 255Lys Val Pro Ser Leu Phe Val Glu Ser Ser Val Asp Asp
Arg Pro Met 260 265 270Lys Thr Val Ser Gln Asp Thr Asn Ile Pro Ile
Tyr Ala Gln Ile Phe 275 280 285Thr Asp Ser Ile Ala Glu Gln Gly Lys
Glu Gly Asp Ser Tyr Tyr Ser 290 295 300Met Met Lys Tyr Asn Leu Asp
Lys Ile Ala Glu Gly Leu Ala Lys Leu305 310 315 320Ala Ala Ala Leu
Glu His His His His His His 325 3302868DNAStreptococcus pneumoniae
2gatcctagcg gaaaaaaaga tacaacttct ggtcaaaaac taaaagttgt tgctacaaac
60tcaatcatcg ctgatattac taaaaatatt gctggtgaca aaattgacct tcatagtatc
120gttccgattg ggcaagaccc acacgaatac gaaccacttc ctgaagacgt
taagaaaact 180tctgaggcta atttgatttt ctataacggt atcaaccttg
aaacaggtgg caatgcttgg 240tttacaaaat tggtagaaaa tgccaagaaa
actgaaaaca aagactactt cgcagtcagc 300gacggcgttg atgttatcta
ccttgaaggt caaaatgaaa aaggaaaaga agacccacac 360gcttggctta
accttgaaaa cggtattatt tttgctaaaa atatcgccaa acaattgagc
420gccaaagacc ctaacaataa agaattctat gaaaaaaatc tcaaagaata
tactgataag 480ttagacaaac ttgataaaga aagtaaggat aaatttaata
agatccctgc tgaaaagaaa 540ctcattgtaa ccagcgaagg agcattcaaa
tacttctcta aagcctatgg tgtcccaagt 600gcttacatct gggaaatcaa
tactgaagaa gaaggaactc ctgaacaaat caagaccttg 660gttgaaaaac
ttcgccaaac aaaagttcca tcactctttg tagaatcaag tgtggatgac
720cgtccaatga aaactgtttc tcaagacaca aacatcccaa tctacgctca
aatctttact 780gactctatcg cagaacaagg taaagaaggc gacagctact
acagcatgat gaaatacaac 840cttgacaaga ttgctgaagg attggcaa
868326DNAArtificial SequenceForward primer 3gggatcctag cggaaaaaaa
gataca 26427DNAArtificial SequenceReverse primer 4gcaagctttg
ccaatccttc agcaatc 27
* * * * *