U.S. patent application number 13/577810 was filed with the patent office on 2012-11-29 for 15-valent pneumococcal polysaccharide-protein conjugate vaccine composition.
Invention is credited to Patrick J. Ahl, Jeffrey T. Blue, Jayme L. Cannon, Michael J. Caulfield.
Application Number | 20120301502 13/577810 |
Document ID | / |
Family ID | 44353903 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301502 |
Kind Code |
A1 |
Caulfield; Michael J. ; et
al. |
November 29, 2012 |
15-VALENT PNEUMOCOCCAL POLYSACCHARIDE-PROTEIN CONJUGATE VACCINE
COMPOSITION
Abstract
The present invention provides a multivalent immunogenic
composition having 15 distinct polysaccharide-protein conjugates.
Each conjugate consists of a capsular polysaccharide prepared from
a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A,
6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F) conjugated to a
carrier protein, preferably CRM197. The immunogenic composition,
preferably formulated as a vaccine on an aluminum-based adjuvant,
provides broad coverage against pneumococcal disease, particularly
in infants and young children.
Inventors: |
Caulfield; Michael J.; (Ft.
Washington, PA) ; Ahl; Patrick J.; (Princeton,
NJ) ; Blue; Jeffrey T.; (Telford, PA) ;
Cannon; Jayme L.; (Lexington Park, PA) |
Family ID: |
44353903 |
Appl. No.: |
13/577810 |
Filed: |
February 3, 2011 |
PCT Filed: |
February 3, 2011 |
PCT NO: |
PCT/US11/23526 |
371 Date: |
August 8, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61302726 |
Feb 9, 2010 |
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Current U.S.
Class: |
424/197.11 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 2039/55505 20130101; A61P 37/04 20180101; A61P 31/04 20180101;
A61K 2039/6037 20130101; A61K 39/092 20130101 |
Class at
Publication: |
424/197.11 |
International
Class: |
A61K 39/385 20060101
A61K039/385; A61P 31/04 20060101 A61P031/04 |
Claims
1-9. (canceled)
10. An immunogenic composition comprising: (1) a multivalent
polysaccharide-protein conjugate mixture consisting of capsular
polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,
19A, 19F, 22F, 23F, and 33F of Streptococcus pneumoniae conjugated
to about 32 .mu.g CRM.sub.197 carrier protein; and (2) a
pharmaceutically acceptable carrier.
11. The immunogenic composition of claim 10 wherein CRM.sub.197 is
present at 32 .mu.g.+-.5 .mu.g.
12. The immunogenic composition of claim 10 wherein the immunogenic
composition is formulated as a single 0.5 mL dose containing 2
.mu.g of each saccharide, except for 6B at 4 .mu.g.
13. The immunogenic composition of claim 10 further comprising
0.125 mg of elemental aluminum adjuvant, about 150 mM sodium
chloride and about 20 mM L-histidine buffer.
14. The immunogenic composition of claim 13 wherein the sodium
chloride is present at 150 mM.+-.25 mM and the L-histidine buffer
is present at 20 mM.+-.5 mM.
15. A method of inducing an immune response to a Streptococcus
pneumoniae capsular polysaccharide, comprising administering to a
human an immunologically effective amount of the immunogenic
composition of claim 10.
16. A method of inducing an immune response to a Streptococcus
pneumoniae capsular polysaccharide, comprising administering to a
human an immunologically effective amount of the immunogenic
composition of claim 12.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] None
FIELD OF INVENTION
[0002] The present invention provides a multivalent immunogenic
composition having 15 distinct polysaccharide-protein conjugates.
Each conjugate consists of a capsular polysaccharide prepared from
a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A,
6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F) conjugated to a
carrier protein, preferably CRM.sub.197. The immunogenic
composition, preferably formulated as a vaccine on an
aluminum-based adjuvant, provides broad coverage against
pneumococcal disease, particularly in infants and young
children.
BACKGROUND OF THE INVENTION
[0003] Streptococcus pneumoniae is a significant cause of serious
disease world-wide. In 1997, the Centers for Disease Control and
Prevention (CDC) estimated there were 3,000 cases of pneumococcal
meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000
cases of pneumococcal otitis media and 500,000 cases of
pneumococcal pneumonia annually in the United States. See Centers
for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997,
46(RR-8):1-13. Furthermore, the complications of these diseases can
be significant with some studies reporting up to 8% mortality and
25% neurologic sequelae with pneumococcal meningitis. See Arditi et
al., 1998, Pediatrics 102:1087-97.
[0004] The multivalent pneumococcal polysaccharide vaccines that
have been licensed for many years have proved valuable in
preventing pneumococcal disease in adults, particularly, the
elderly and those at high-risk. However, infants and young children
respond poorly to unconjugated pneumococcal polysaccharides. The
pneumococcal conjugate vaccine, Prevnar.RTM., containing the 7 most
frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F)
causing invasive pneumococcal disease in young children and infants
at the time, was first licensed in the United States in February
2000. Following universal use of Prevnar.RTM. in the United States,
there has been a significant reduction in invasive pneumococcal
disease in children due to the serotypes present in Prevnar.RTM..
See Centers for Disease Control and Prevention, MMWR Morb Mortal
Wkly Rep 2005, 54(36):893-7. However, there are limitations in
serotype coverage with Prevnar.RTM. in certain regions of the world
and some evidence of certain emerging serotypes in the United
States (for example, 19A and others). See O'Brien et al., 2004, Am
J Epidemiol 159:634-44; Whitney et al., 2003, N Engl J Med
348:1737-46; Kyaw et al., 2006, N Engl J Med 354:1455-63; Hicks et
al., 2007, J Infect Dis 196:1346-54; Traore et al., 2009, Clin
Infect Dis 48:S181-S189.
[0005] U.S. Patent Application Publication No. US 2006/0228380 A1
describes a 13-valent pneumococcal polysaccharide-protein conjugate
vaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,
19A, 19F and 23F. Chinese Patent Application Publication No. CN
101590224 A describes a 14-valent pneumococcal
polysaccharide-protein conjugate vaccine including serotypes 1, 2,
4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F.
[0006] Other PCVs have covered 7, 10, 11, or 13 of the serotypes
contained in PCV-15, but immune interference has been observed for
some serotypes (e.g. lower protection for serotype 3 in GSK's
PCV-11) and lower response rates to serotype 6B in Pfizer's PCV-13.
See Prymula et al., 2006, Lancet 367:740-48 and Kieninger et al.,
Safety and Immunologic Non-inferiority of 13-valent Pneumococcal
Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate
Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers,
presented at the 48.sup.th Annual ICAAC/ISDA 46.sup.th Annual
Meeting, Washington D.C., Oct. 25-28, 2008.
SUMMARY OF THE INVENTION
[0007] The present invention provides an immunogenic composition
comprising (1) a multivalent polysaccharide-protein conjugate
mixture consisting of capsular polysaccharides from 15 different
serotypes of S. pneumoniae conjugated to a carrier protein, and (2)
a pharmaceutically acceptable carrier. More specifically, the
present invention provides a 15-valent pneumococcal conjugate
vaccine (PCV-15) composition comprising a multivalent
polysaccharide-protein conjugate mixture consisting of capsular
polysasccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,
18C, 19A, 19F, 22F, 23F, and 33F of S. pneumoniae conjugated to a
carrier protein; and a pharmaceutically acceptable carrier. In one
specific embodiment, the immunogenic composition contains capsular
polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,
19A, 19F, 22F, 23F and 33F and the carrier protein is
CRM.sub.197.
[0008] In certain embodiments, the composition further comprises an
adjuvant. In certain embodiments, the adjuvant is an aluminum-based
adjuvant, such as aluminum phosphate, aluminum sulfate or aluminum
hydroxide. In a particular embodiment of the invention, the
adjuvant is aluminum phosphate.
[0009] The present invention also provides a method of inducing an
immune response to a S. pneumoniae capsular polysaccharide,
comprising administering to a human an immunologically effective
amount of the above immunogenic composition.
[0010] The present invention further provides an immunogenic
composition administered as a single 0.5 mL dose formulated to
contain: 2 .mu.g of each polysaccharide, except for 6B at 4 .mu.g;
about 32 .mu.g CRM.sub.197 carrier protein; 0.125 mg of elemental
aluminum (0.5 mg aluminum phosphate) adjuvant; 150 mM sodium
chloride and 20 .mu.M L-histidine buffer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1: Comparison of GMCs for PCV-15 relative to
Prevnar.RTM. in Infant Rhesus Monkeys (Prevnar serotypes, PD-2 and
PD-3). Error bars denote 2 standard errors.
[0012] FIG. 2: Serotype-specific GMCs to non-Prevnar.RTM. serotypes
in infant rhesus monkeys immunized with PCV-15. Error bars denote 2
standard errors.
[0013] FIG. 3: NZWR-1: Comparison of geometric mean titers in
rabbits immunized with PCV-15 without (0xA) or with APA (B1)
(Post-dose 2). Error bars denote 95% CI on the geometric mean
fold-difference (PCV-15 without APA/PCV-15 with APA).
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides a multivalent immunogenic
composition comprising, consisting essentially of, or
alternatively, consisting of 15 distinct polysaccharide-protein
conjugates, wherein each of the conjugates contains a different
capsular polysaccharide conjugated to a carrier protein, and
wherein the capsular polysaccharides are prepared from serotypes 1,
3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of S.
pneumoniae, together with a pharmaceutically acceptable carrier. In
certain embodiments, the carrier protein is CRM.sub.197. The
immunogenic composition may further comprise an adjuvant, such as
an aluminum-based adjuvant, such as aluminum phosphate, aluminum
sulfate and aluminum hydroxide. The present invention also provides
a method of inducing an immune response to a S. pneumoniae capsular
polysaccharide conjugate, comprising administering to a human an
immunologically effective amount of the above multivalent
immunogenic composition.
[0015] As illustrated in the Examples, infra., preclinical studies
in infant rhesus monkeys demonstrated robust antibody responses to
all 15 serotypes in PCV-15 which are comparable to the responses
for the 7 common serotypes in Prevna.RTM.. Applicants' finding that
a 15 valent pneumococcal conjugate vaccine including the addition
of new polysaccharide-protein conjugates containing serotypes 22F
and 33F provides robust antibody responses demonstrates the
feasibility of expanding coverage of pneumococcal serotypes not
covered by existing pneumococcal vaccines.
[0016] The term "comprises" when used with the immunogenic
composition of the invention refers to the inclusion of any other
components (subject to limitations of "consisting of" language for
the antigen mixture), such as adjuvants and excipients. The term
"consisting of" when used with the multivalent
polysaccharide-protein conjugate mixture refers to a mixture having
those 15 particular S. pneumoniae polysaccharide protein conjugates
and no other S. pneumoniae polysaccharide protein conjugates from a
different serotype.
Streptococcus Pneumoniae Capsular Polysaccharide--Protein
Conjugates
[0017] Capsular polysaccharides from Steptococcus pneumoniae can be
prepared by standard techniques known to those skilled in the art.
For example, polysaccharides can be isolated from bacteria and may
be sized to some degree by known methods (see, e.g., European
Patent Nos. EP497524 and EP497525) and preferably by
microfluidisation. Polysaccharides can be sized in order to reduce
viscosity in polysaccharide samples and/or to improve filterability
for conjugated products. In the present invention, capsular
polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F,
9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae.
[0018] In one embodiment, each pneumococcal polysaccharide serotype
is grown in a soy-based medium. The individual polysaccharides are
then purified through standard steps including centrifugation,
precipitation, and ultra-filtration. See, e.g., U.S. Patent
Application Publication No. 2008/0286838 and U.S. Pat. No.
5,847,112.
[0019] Carrier proteins are preferably proteins that are non-toxic
and non-reactogenic and obtainable in sufficient amount and purity.
A carrier protein can be conjugated or joined with a S. pneumoniae
polysaccharide to enhance immunogenicity of the polysaccharide.
Carrier proteins should be amenable to standard conjugation
procedures. In a particular embodiment of the present invention,
CRM.sub.197 is used as the carrier protein. In one embodiment, each
capsular polysaccharide is conjugated to the same carrier protein
(each capsular polysaccharide molecule being conjugated to a single
carrier protein). In another embodiment, the capsular
polysaccharides are conjugated to two or more carrier proteins
(each capsular polysaccharide molecule being conjugated to a single
carrier protein). In such an embodiment, each capsular
polysaccharide of the same serotype is typically conjugated to the
same carrier protein.
[0020] CRM.sub.197 is a non-toxic variant (i.e., toxoid) of
diphtheria toxin. In one embodiment, it is isolated from cultures
of Corynebacterium diphtheria strain C7 (.beta.197) grown in
casamino acids and yeast extract-based medium. In another
embodiment, CRM.sub.197 is prepared recombinantly in accordance
with the methods described in U.S. Pat. No. 5,614,382. Typically,
CRM.sub.197 is purified through a combination of ultra-filtration,
ammonium sulfate precipitation, and ion-exchange chromatography. In
some embodiments, CRM.sub.197 is prepared in Pseudomonas
fluorescens using Pfenex Expression Technology.TM. (Pfenex Inc.,
San Diego, Calif.).
[0021] Other suitable carrier proteins include additional
inactivated bacterial toxins such as DT (Diphtheria toxoid), TT
(tetanus toxid) or fragment C of TT, pertussis toxoid, cholera
toxoid (e.g., as described in International Patent Application
Publication No. WO 2004/083251), E. coli LT, E. coli ST, and
exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane
proteins such as outer membrane complex c (OMPC), porins,
transferrin binding proteins, pneumococcal surface protein A (PspA;
See International Application Patent Publication No. WO 02/091998),
pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or
Group B streptococcus, or Haemophilus influenzae protein D,
pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63;
2706-13) including ply detoxified in some fashion for example
dPLY-GMBS (See International Patent Application Publication No. WO
04/081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE
and fusions of Pht proteins for example PhtDE fusions, PhtBE
fusions (See International Patent Application Publication Nos. WO
01/98334 and WO 03/54007), can also be used. Other proteins, such
as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin
(BSA) or purified protein derivative of tuberculin (PPD), PorB
(from N. meningitidis), PD (Haemophilus influenzae protein D; see,
e.g., European Patent No. EP 0 594 610 B), or immunologically
functional equivalents thereof, synthetic peptides (See European
Patent Nos. EP0378881 and EP0427347), heat shock proteins (See
International Patent Application Publication Nos. WO 93/17712 and
WO 94/03208), pertussis proteins (See International Patent
Application Publication No. WO 98/58668 and European Patent No.
EP0471177), cytokines, lymphokines, growth factors or hormones (See
International Patent Application Publication No. WO 91/01146),
artificial proteins comprising multiple human CD4+ T cell epitopes
from various pathogen derived antigens (See Falugi et al., 2001,
Eur J Immunol 31:3816-3824) such as N19 protein (See Baraldoi et
al., 2004, Infect Immun 72:4884-7), iron uptake proteins (See
International Patent Application Publication No. WO 01/72337),
toxin A or B of C. difficile (See International Patent Publication
No. WO 00/61761), and flagellin (See Ben-Yedidia et al., 1998,
Immunol Left 64:9) can also be used as carrier proteins.
[0022] Other DT mutants can be used, such as CRM176, CRM228, CRM 45
(Uchida et al., 1973, J Biot Chem 218:3838-3844); CRM 9, CRM 45,
CRM102, CRM 103 and CRM107 and other mutations described by
Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel,
Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp,
Gin or Ser and/or Ala 158 to Gly and other mutations disclosed in
U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950,740; mutation of at
least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534
and other mutations disclosed in U.S. Pat. No. 5,917,017 or U.S.
Pat. No. 6,455,673; or fragment disclosed in U.S. Pat. No.
5,843,711.
[0023] The purified polysaccharides are chemically activated to
make the saccharides capable of reacting with the carrier protein.
Once activated, each capsular polysaccharide is separately
conjugated to a carrier protein to form a glycoconjugate. The
polysaccharide conjugates may be prepared by known coupling
techniques.
[0024] In one embodiment, the chemical activation of the
polysaccharides and subsequent conjugation to the carrier protein
are achieved by means described in U.S. Pat. Nos. 4,365,170,
4,673,574 and 4,902,506. Briefly, that chemistry entails the
activation of pneumococcal polysaccharide by reaction with any
oxidizing agent which oxidizes a terminal hydroxyl group to an
aldehyde, such as periodate (including sodium periodate, potassium
periodate, or periodic acid). The reaction leads to a random
oxidative cleavage of vicinal hydroxyl groups of the carbohydrates
with the formation of reactive aldehyde groups.
[0025] Coupling to the protein carrier (e.g., CRM.sub.197) can be
by reductive amination via direct amination to the lysyl groups of
the protein. For example, conjugation is carried out by reacting a
mixture of the activated polysaccharide and carrier protein with a
reducing agent such as sodium cyanoborohydride. Unreacted aldehydes
are then capped with the addition of a strong reducing agent, such
as sodium borohydride.
[0026] In another embodiment, the conjugation method may rely on
activation of the saccharide with 1-cyano-4-dimethylamino
pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The
activated saccharide may thus be coupled directly or via a spacer
(linker) group to an amino group on the carrier protein. For
example, the spacer could be cystamine or cysteamine to give a
thiolated polysaccharide which could be coupled to the carrier via
a thioether linkage obtained after reaction with a
maleimide-activated carrier protein (for example using GMBS) or a
haloacetylated carrier protein (for example using iodoacetimide
[e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or
STAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally
made by CDAP chemistry) is coupled with hexane diamine or adipic
acid dihydrazide (ADH) and the amino-derivatised saccharide is
conjugated to the carrier protein using carbodiimide (e.g. EDAC or
EDC) chemistry via a carboxyl group on the protein carrier. Such
conjugates are described in International Patent Application
Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu
et al., 1983, Infect. Immunity 40:245-256.
[0027] Other suitable techniques use carbodiimides, hydrazides,
active esters, norborane, p-nitrobenzoic acid,
N-hydroxysuccinimide, S--NHS, EDC, TSTU. Many are described in
International Patent Application Publication No. WO 98/42721.
Conjugation may involve a carbonyl linker which may be formed by
reaction of a free hydroxyl group of the saccharide with CDI (See
Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al.,
1981, J. Chromatogr. 218:509-18) followed by reaction of with a
protein to form a carbamate linkage. This may involve reduction of
the anomeric terminus to a primary hydroxyl group, optional
protection/deprotection of the primary hydroxyl group, reaction of
the primary hydroxyl group with CDI to form a CDI carbamate
intermediate and coupling the CDI carbamate intermediate with an
amino group on a protein.
[0028] In one embodiment, prior to formulation, each pneumococcal
capsular polysaccharide antigen is individually purified from S.
pneumoniae, activated to form reactive aldehydes, and then
covalently conjugated using reductive amination to the carrier
protein CRM.sub.197.
[0029] After conjugation of the capsular polysaccharide to the
carrier protein, the polysaccharide-protein conjugates are purified
(enriched with respect to the amount of polysaccharide-protein
conjugate) by one or more of a variety of techniques. Examples of
these techniques are well known to the skilled artisan and include
concentration/diafiltration operations, ultrafiltration,
precipitation/elution, column chromatography, and depth filtration.
See, e.g., U.S. Pat. No. 6,146,902.
Pharmaceutical/Vaccine Compositions
[0030] The present invention further provides compositions,
including pharmaceutical, immunogenic and vaccine compositions,
comprising, consisting essentially of, or alternatively, consisting
of 15 distinct polysaccharide-protein conjugates, wherein each of
the conjugates contains a different capsular polysaccharide
conjugated to a carrier protein, and wherein the capsular
polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F,
9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae, together
with a pharmaceutically acceptable carrier and an adjuvant. These
pneumococcal conjugates are prepared by separate processes and bulk
formulated into a single dosage formulation.
[0031] As defined herein, an "adjuvant" is a substance that serves
to enhance the immunogenicity of an immunogenic composition of the
invention. An immune adjuvant may enhance an immune response to an
antigen that is weakly immunogenic when administered alone, e.g.,
inducing no or weak antibody titers or cell-mediated immune
response, increase antibody titers to the antigen, and/or lowers
the dose of the antigen effective to achieve an immune response in
the individual. Thus, adjuvants are often given to boost the immune
response and are well known to the skilled artisan. Suitable
adjuvants to enhance effectiveness of the composition include, but
are not limited to:
[0032] (1) aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc.;
[0033] (2) oil-in-water emulsion formulations (with or without
other specific immunostimulating agents such as muramyl peptides
(defined below) or bacterial cell wall components), such as, for
example, (a) MF59 (International Patent Application Publication No.
WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span
85 (optionally containing various amounts of MTP-PE) formulated
into submicron particles using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing
10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and
thr-MDP either mierofluidized into a submicron emulsion or vortexed
to generate a larger particle size emulsion, (c) Ribi.TM. adjuvant
system (RAS), (Corixa, Hamilton, Mont.) containing 2% Squalene,
0.2% Tween 80, and one or more bacterial cell wall components from
the group consisting of 3-O-deaylated monophosphorylipid A
(MPL.TM.) described in U.S. Pat. No. 4,912,094, trehalose
dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS
(Detox.TM.); and (d) a Montanide ISA;
[0034] (3) saponin adjuvants, such as Quil A or STIMULON.TM. QS-21
(Antigenics, Framingham, Mass.) (see, e.g., U.S. Pat. No.
5,057,540) may be used or particles generated therefrom such as
ISCOM (immunostimulating complexes formed by the combination of
cholesterol, saponin, phospholipid, and amphipathic proteins) and
Iscomatrix (having essentially the same structure as an ISCOM but
without the protein);
[0035] (4) bacterial lipopolysaccharides, synthetic lipid A analogs
such as aminoalkyl glucosamine phosphate compounds (AGP), or
derivatives or analogs thereof, which are available from Corixa,
and which are described in U.S. Pat. No. 6,113,918; one such AGP is
2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl
2-Deoxy-4-O-phosphono-3-O--[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-
-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside, which is
also known as 529 (formerly known as RC529), which is formulated as
an aqueous form or as a stable emulsion
[0036] (5) synthetic polynucleotides such as oligonucleotides
containing CpG motif(s) (U.S. Pat. No. 6,207,646); and
[0037] (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g.,
gamma interferon), granulocyte macrophage colony stimulating factor
(GM-CSF), macrophage colony stimulating factor (M-CSF), tumor
necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc;
and
[0038] (7) complement, such as a trimer of complement component
C3d.
[0039] In another embodiment, the adjuvant is a mixture of 2, 3, or
more of the above adjuvants, e.g., SBAS2 (an oil-in-water emulsion
also containing 3-deacylated monophosphoryl lipid A and QS21).
[0040] Muramyl peptides include, but are not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanine-2-(1'-2'
dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
etc.
[0041] In certain embodiments, the adjuvant is an aluminum salt.
The aluminum salt adjuvant may be an alum-precipitated vaccine or
an alum-adsorbed vaccine. Aluminum-salt adjuvants are well known in
the art and are described, for example, in Harlow, E. and D. Lane
(1988; Antibodies: A Laboratory Manual Cold Spring Harbor
Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in
Immunology 143:489-493). The aluminum salt includes, but is not
limited to, hydrated alumina, alumina hydrate, alumina trihydrate
(ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos,
Amphogel, aluminum (III) hydroxide, aluminum hydroxyphosphate
sulfate (Aluminum Phosphate Adjuvant (APA)), amorphous alumina,
trihydrated alumina, or trihydroxyaluminum.
[0042] APA is an aqueous suspension of aluminum hydroxyphosphate.
APA is manufactured by blending aluminum chloride and sodium
phosphate in a 1:1 volumetric ratio to precipitate aluminum
hydroxyphosphate. After the blending process, the material is
size-reduced with a high-shear mixer to achieve a target aggregate
particle size in the range of 2-8 .mu.m. The product is then
diafiltered against physiological saline and steam sterilized.
[0043] In certain embodiments, a commercially available
Al(OH).sub.3 (e.g. Alhydrogel or Superfos of Denmark/Accurate
Chemical and Scientific Co., Westbury, N.Y.) is used to adsorb
proteins in a ratio of 50-200 g protein/mg aluminum hydroxide.
Adsorption of protein is dependent, in another embodiment, on the
pI (Isoelectric pH) of the protein and the pH of the medium. A
protein with a lower pI adsorbs to the positively charged aluminum
ion more strongly than a protein with a higher pI. Aluminum salts
may establish a depot of Ag that is released slowly over a period
of 2-3 weeks, be involved in nonspecific activation of macrophages
and complement activation, and/or stimulate innate immune mechanism
(possibly through stimulation of uric acid). See, e.g., Lambrecht
et al., 2009, Curr Opin Immunol 21:23.
[0044] Monovalent bulk aqueous conjugates are typically blended
together and diluted to target 8 .mu.g/mL for all serotypes except
6.beta., which will be diluted to target 16 .mu.g/mL. Once diluted,
the batch will be filter sterilized, and an equal volume of
aluminum phosphate adjuvant added aseptically to target a final
aluminum concentration of 250 .mu.g/mL. The adjuvanted, formulated
batch will be filled into single-use, 0.5 mL/dose vials.
[0045] In certain embodiments, the adjuvant is a CpG-containing
nucleotide sequence, for example, a CpG-containing oligonucleotide,
in particular, a CpG-containing oligodeoxynucleotide (CpG ODN). In
another embodiment, the adjuvant is ODN 1826, which may be acquired
from Coley Pharmaceutical Group.
[0046] "CpG-containing nucleotide," "CpG-containing
oligonucleotide," "CpG oligonucleotide," and similar terms refer to
a nucleotide molecule of 6-50 nucleotides in length that contains
an unmethylated CpG moiety. See, e.g., Wang et al., 2003, Vaccine
21:4297. In another embodiment, any other art-accepted definition
of the terms is intended. CpG-containing oligonucleotides include
modified oligonucleotides using any synthetic internucleoside
linkages, modified base and/or modified sugar.
[0047] Methods for use of CpG oligonucleotides are well known in
the art and are described, for example, in Sur et al., 1999, J.
Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol. Med.
127:139-58; and Yasuda et al., 2006, Crit. Rev Ther Drug Carrier
Syst. 23:89-110.
Administration/Dosage
[0048] The compositions and formulations of the present invention
can be used to protect or treat a human susceptible to pneumococcal
infection, by means of administering the vaccine via a systemic or
mucosal route. In one embodiment, the present invention provides a
method of inducing an immune response to a S. pneumoniae capsular
polysaccharide conjugate, comprising administering to a human an
immunologically effective amount of an immunogenic composition of
the present invention. In another embodiment, the present invention
provides a method of vaccinating a human against a pneumococcal
infection, comprising the step of administering to the human an
immunogically effective amount of a immunogenic composition of the
present invention.
[0049] "Effective amount" of a composition of the invention refers
to a dose required to elicit antibodies that significantly reduce
the likelihood or severity of infectivitiy of S. pneumoniae during
a subsequent challenge.
[0050] The methods of the invention can be used for the prevention
and/or reduction of primary clinical syndromes caused by S.
pneumoniae including both invasive infections (meningitis,
pneumonia, and bacteremia), and noninvasive infections (acute
otitis media, and sinusitis).
[0051] Administration of the compositions of the invention can
include one or more of: injection via the intramuscular,
intraperitoneal, intradermal or subcutaneous routes; or via mucosal
administration to the oral/alimentary, respiratory or genitourinary
tracts. In one embodiment, intranasal administration is used for
the treatment of pneumonia or otitis media (as nasopharyngeal
carriage of pneumococci can be more effectively prevented, thus
attenuating infection at its earliest stage).
[0052] The amount of conjugate in each vaccine dose is selected as
an amount that induces an immunoprotective response without
significant, adverse effects. Such amount can vary depending upon
the pneumococcal serotype. Generally, each dose will comprise 0.1
to 100 .mu.g of each polysaccharide, particularly 0.1 to 10 .mu.g,
and more particularly 1 to 5 .mu.g. For example, each dose can
comprise 100, 150, 200, 250, 300, 400, 500, or 750 ng or 1, 1.5, 2,
3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22,
25, 30, 40, 50, 60, 70, 80, 90, or 100 .mu.g.
[0053] Optimal amounts of components for a particular vaccine can
be ascertained by standard studies involving observation of
appropriate immune responses in subjects. For example, in another
embodiment, the dosage for human vaccination is determined by
extrapolation from animal studies to human data. In another
embodiment, the dosage is determined empirically.
[0054] In one embodiment, the dose of the aluminum salt is 10, 15,
20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 .mu.g, or
1, 1.2, 1.5, 2, 3, 5 mg or more. In yet another embodiment, the
dose of alum salt described above is per .mu.g of recombinant
protein.
[0055] In a particular embodiment of the present invention, the
PCV-15 vaccine is a sterile liquid formulation of pneumococcal
capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,
14, 18C, 19A, 19F, 22F, 23F and 33F individually conjugated to
CRM.sub.197. Each 0.5 mL dose is formulated to contain: 2 .mu.g of
each saccharide, except for 6B at 4 .mu.g; about 32 .mu.g
CRM.sub.197 carrier protein (e.g., 32 .mu.g.+-.5 .mu.g, .+-.3
.mu.g, .+-.2 .mu.g, or .+-.1 .mu.g); 0.125 mg of elemental aluminum
(0.5 mg aluminum phosphate) adjuvant; and sodium chloride and
L-histidine buffer. The sodium chloride concentration is about 150
mM (e.g., 150 mM.+-.25 mM, 120 mM, .+-.15 mM, .+-.10 mM, or .+-.5
mM) and about 20 mM (e.g, 20 mM.+-.5 mM, .+-.2.5 mM, .+-.2 mM,
.+-.1 mM, or .+-.0.5 mM) L-histidine buffer.
[0056] According to any of the methods of the present invention and
in one embodiment, the subject is human. In certain embodiments,
the human patient is an infant (less than 1 year of age), toddler
(approximately 12 to 24 months), or young child (approximately 2 to
5 years). In other embodiments, the human patient is an elderly
patient (>65 years). The compositions of this invention are also
suitable for use with older children, adolescents and adults (e.g.,
aged 18 to 45 years or 18 to 65 years).
[0057] In one embodiment of the methods of the present invention, a
composition of the present invention is administered as a single
inoculation. In another embodiment, the vaccine is administered
twice, three times or four times or more, adequately spaced apart.
For example, the composition may be administered at 1, 2, 3, 4, 5,
or 6 month intervals or any combination thereof. The immunization
schedule can follow that designated for pneumococcal vaccines. For
example, the routine schedule for infants and toddlers against
invasive disease caused by S. pneumoniae is 2, 4, 6 and 12-15
months of age. Thus, in a preferred embodiment, the composition is
administered as a 4-dose series at 2, 4, 6, and 12-15 months of
age.
[0058] The compositions of this invention may also include one or
more proteins from S. pneumoniae. Examples of S. pneumoniae
proteins suitable for inclusion include those identified in
International Patent Application Publication Nos. WO 02/083855 and
WO 02/053761.
Formulations
[0059] The compositions of the invention can be administered to a
subject by one or more method known to a person skilled in the art,
such as parenterally, transmucosally, transdermally,
intramuscularly, intravenously, intra-dermally, intra-nasally,
subcutaneously, intra-peritonealy, and formulated accordingly.
[0060] In one embodiment, compositions of the present invention are
administered via epidermal injection, intramuscular injection,
intravenous, intra-arterial, subcutaneous injection, or
intra-respiratory mucosal injection of a liquid preparation. Liquid
formulations for injection include, solutions and the like.
[0061] The composition of the invention can be formulated as single
dose vials, multi-dose vials or as pre-filled syringes.
[0062] In another embodiment, compositions of the present invention
are administered orally, and are thus formulated in a form suitable
for oral administration, i.e., as a solid or a liquid preparation.
Solid oral formulations include tablets, capsules, pills, granules,
pellets and the like. Liquid oral formulations include solutions,
suspensions, dispersions, emulsions, oils and the like.
[0063] Pharmaceutically acceptable carriers for liquid formulations
are aqueous or non-aqueous solutions, suspensions, emulsions or
oils. Examples of nonaqueous solvents are propylene glycol,
polyethylene glycol, and injectable organic esters such as ethyl
oleate.
[0064] Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Examples of oils are those of animal, vegetable, or synthetic
origin, for example, peanut oil, soybean oil, olive oil, sunflower
oil, fish-liver oil, another marine oil, or a lipid from milk or
eggs.
[0065] The pharmaceutical composition may be isotonic, hypotonic or
hypertonic. However it is often preferred that a pharmaceutical
composition for infusion or injection is essentially isotonic, when
it is administrated. Hence, for storage the pharmaceutical
composition may preferably be isotonic or hypertonic. If the
pharmaceutical composition is hypertonic for storage, it may be
diluted to become an isotonic solution prior to administration. The
isotonic agent may be an ionic isotonic agent such as a salt or a
non-ionic isotonic agent such as a carbohydrate. Examples of ionic
isotonic agents include but are not limited to NaCl, CaCl.sub.2,
KCl and MgCl.sub.2. Examples of non-ionic isotonic agents include
but are not limited to mannitol, sorbitol and glycerol.
[0066] It is also preferred that at least one pharmaceutically
acceptable additive is a buffer. For some purposes, for example,
when the pharmaceutical composition is meant for infusion or
injection, it is often desirable that the composition comprises a
buffer, which is capable of buffering a solution to a pH in the
range of 4 to 10, such as 5 to 9, for example 6 to 8.
[0067] The buffer may for example be selected from the group
consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate,
phosphate, citrate, carbonate, glycinate, histidine, glycine,
succinate and triethanolamine buffer.
[0068] The buffer may furthermore for example be selected from USP
compatible buffers for parenteral use, in particular, when the
pharmaceutical formulation is for parenteral use. For example the
buffer may be selected from the group consisting of monobasic acids
such as acetic, benzoic, gluconic, glyceric and lactic; dibasic
acids such as aconitic, adipic, ascorbic, carbonic, glutamic,
malic, succinic and tartaric, polybasic acids such as citric and
phosphoric; and bases such as ammonia, diethanolamine, glycine,
triethanolamine, and TRIS.
[0069] Parenteral vehicles (for subcutaneous, intravenous,
intraarterial, or intramuscular injection) include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Examples are sterile liquids
such as water and oils, with or without the addition of a
surfactant and other pharmaceutically acceptable adjuvants. In
general, water, saline, aqueous dextrose and related sugar
solutions, glycols such as propylene glycols or polyethylene
glycol, and Polysorbate-80 are preferred liquid carriers,
particularly for injectable solutions. Examples of oils are those
of animal, vegetable, or synthetic origin, for example, peanut oil,
soybean oil, olive oil, sunflower oil, fish-liver oil, another
marine oil, or a lipid from milk or eggs.
[0070] The formulations of the invention may also contain a
surfactant. Preferred surfactants include, but are not limited to:
the polyoxyethylene sorbitan esters surfactants (commonly referred
to as the Tweens), especially polysorbate 20 and polysorbate 80;
copolymers of ethylene oxide (EO), propylene oxide (PO), and/or
butylene oxide (BO), sold under the DOWFAX.TM. tradename, such as
linear BO/PO block copolymers; octoxynols, which can vary in the
number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with
octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol)
being of particular interest; (oetylphenoxy)polyethoxyethanol
(IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine
(lecithin); nonylphenol ethoxylates, such as the Tergitol.TM. NP
series; polyoxyethylene fatty ethers derived from lauryl, cetyl,
stearyl and oleyl alcohols (known as Brij surfactants), such as
triethyleneglycol monolautyl ether (Brij 30); and sorbitan esters
(commonly known as the SPANs), such as sorbitan trioleate (Span 85)
and sorbitan monolaurate. A preferred surfactant for including in
the emulsion is Tween 80 (polyoxyethylene sorbitan monooleate).
[0071] Mixtures of surfactants can be used, e.g. Tween 80/Span 85
mixtures. A combination of a polyoxyethylene sorbitan ester such as
polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol
such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also
suitable. Another useful combination comprises laureth 9 plus a
polyoxyethylene sorbitan ester and/or an octoxynol.
[0072] Preferred amounts of surfactants (% by weight) are:
polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in
particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such
as Triton X-100, or other detergents in the Triton series) 0.001 to
0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as
laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1
to 1% or about 0.5%.
[0073] In another embodiment, the pharmaceutical composition is
delivered in a controlled release system. For example, the agent
can be administered using intravenous infusion, a transdermal
patch, liposomes, or other modes of administration. In another
embodiment, polymeric materials are used; e.g. in microspheres in
or an implant.
[0074] Also comprehended by the invention are compounds modified by
the covalent attachment of water-soluble polymers such as
polyethylene glycol, copolymers of polyethylene glycol and
polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl
alcohol, polyvinylpyrrolidone or polyproline. Such modifications
may increase the compound's solubility in aqueous solution,
eliminate aggregation, enhance the physical and chemical stability
of the compound, and greatly reduce the reactogenicity of the
compound. In another embodiment, the desired in vivo biological
activity is achieved by the administration of such polymer-compound
abducts less frequently or in lower doses than with the unmodified
compound.
[0075] In a preferred embodiment, the vaccine composition is
formulated in L-histidine buffer with sodium chloride.
[0076] Having described various embodiments of the invention with
reference to the accompanying description and drawings, it is to be
understood that the invention is not limited to those precise
embodiments, and that various changes and modifications may be
effected therein by one skilled in the art without departing from
the scope or spirit of the invention as defined in the appended
claims.
[0077] The following examples illustrate, but do not limit the
invention.
EXAMPLES
Example 1
Preparation of S. Pneumoniae Capsular Polysaccharides
[0078] Methods of culturing pneumococci are well known in the art.
See, e.g., Chase, 1967, Methods of Immunology and Immunochemistry
1:52. Methods of preparing pneumococcal capsular polysaccharides
are also well known in the art. See, e.g., European Patent No.
EP0497524. Isolates of pneumococcal subtypes are available from the
ATCC.
[0079] The bacteria are identified as encapsulated, non-motile,
Gram-positive, lancet-shaped diplococci that are alpha-hemolytic on
blood-agar. Subtypes are differentiated on the basis of Quelling
reaction using specific antisera. See, e.g., U.S. Pat. No.
5,847,112.
Cell Banks
[0080] Cell banks representing each of the S. pneumococcus
serotypes present in PCV-15 were obtained from the Merck Culture
Collection (Rahway, N.J.) in a frozen vial.
Inoculation
[0081] A thawed seed culture was transferred to the seed fermentor
containing an appropriate pre-sterilized growth media.
Seed Fermentation
[0082] The culture was grown in the seed fermentor with temperature
and pH control. The entire volume of the seed fermentor was
transferred to the production fermentor containing pre-sterilized
growth media.
Production Fermentation
[0083] The production fermentation was the final cell growth stage
of the process.
[0084] Temperature, pH and the agitation rate was controlled.
Inactivation
[0085] The fermentation process was terminated via the addition of
an inactivating agent. After inactivation, the batch was
transferred to the inactivation tank where it was held at
controlled temperature and agitation.
Purification
[0086] Cell debris was removed using a combination of
centrifugation and filtration. The batch was ultrafiltered and
diafiltered. The batch was then subjected to solvent-based
fractionations that remove impurities and recover
polysaccharide.
Example 2
Preparation of Pneumococcal Polysaccharide-CRM.sub.197
Conjugates
Activation Process
[0087] The different serotype saccharides are individually
conjugated to the purified CRM.sub.197 carrier protein using a
common process flow. In this process the saccharide is dissolved,
sized to a target molecular mass, chemically activated and
buffer-exchanged by ultrafiltration. The purified CRM.sub.197 is
then conjugated with the activated saccharide and the resulting
conjugate is purified by ultrafiltration prior to a final 0.2 .mu.m
membrane filtration. Several process parameters within each step,
such as pH, temperature, concentration, and time are
serotype-specific as described in this example.
[0088] Step 1: Dissolution
[0089] Purified polysaccharide was dissolved in water to a
concentration of 2-3 mg/mL. The dissolved polysaccharide was passed
through a mechanical homogenizer with pressure preset from 0-1000
bar. Following size reduction, the saccharide was concentrated and
diafiltered with sterile water on a 10 kDa MWCO ultrafilter. The
permeate was discarded and the retentate was adjusted to a pH of
4.1 with a sodium acetate buffer, 50 .mu.M final concentration. For
serotypes 4 and 5, 100 mM sodium acetate at pH 5.0 was used. For
serotype 4, the solution was incubated at 50.degree..+-.2.degree.
C. Hydrolysis was stopped by cooling to 20-24.degree. C.
[0090] Step 2: Periodate Reaction
[0091] The required sodium periodate molar equivalents for
pneumococcal saccharide activation was determined using total
saccharide content. With thorough mixing, the oxidation was allowed
to proceed between 3-20 hours at 20-24.degree. C. for all serotypes
except 5, 7F, and 19F for which the temperature was 2-6.degree.
C.
[0092] Step 3: Ultrafiltration
[0093] The oxidized saccharide was concentrated and diafiltered
with 10 mM potassium phosphate, pH 6.4 (10 mM sodium acetate, pH
4.3 for serotype 5) on a 10 kDa MWCO ultrafilter. The permeate was
discarded and the retentate was adjusted to a pH of 6.3-8.4 by
addition of 3 M potassium phosphate buffer.
Conjugation Process
[0094] Step 1: Conjugation Reaction
[0095] The concentrated saccharide was mixed with CRM.sub.197
carrier protein in a 0.2-2 to 1 charge ratio. The blended
saccharide-CRM.sub.197 mixture was filtered through a 0.2 .mu.m
filter.
[0096] The conjugation reaction was initiated by adding a sodium
cyanoborohydride solution to achieve 1.8-2.0 moles of sodium
cyanoborohydride per mole of saccharide. The reaction mixture was
incubated for 48-120 hours at 20-24.degree. C. (8-12.degree. C. for
serotypes 3, 5, 6A, 7F, 19A, and 19F).
[0097] Step 2: Borohydride Reaction
[0098] At the end of the conjugation incubation the reaction
mixture was adjusted to 4-8.degree. C., and a pH of 8-10 with
either 1.2 M sodium bicarbonate buffer or 3 M potassium phosphate
buffer (except serotype 5). The conjugation reaction was stopped by
adding the sodium borohydride solution to achieve 0.6-1.0 moles of
sodium borohydride per mole of saccharide (0 moles of borohydride
added for serotype 5). The reaction mixture was incubated for 45-60
minutes.
[0099] Step 3: Ultrafiltration Steps
[0100] The reaction mixture was diafiltered on a 100 kDa MWCO
ultrafilter with a minimum of 20 volumes of 100 mM potassium
phosphate, pH 8.4 buffer. The retentate from the 100 kDa
ultrafilter was diafiltered on a 300 kDa MWCO ultrafilter with a
minimum of 20 diavolumes of 150 mM sodium chloride at 20-24.degree.
C. The permeate was discarded.
[0101] Step 4: Sterile Filtration
[0102] The retentate from the 300 kDa MWCO diafiltration was
filtered through a 0.2 .mu.m filter and filled into borosilicate
glass containers at appropriate volumes for release testing,
in-process controls, and formulation (except serotype 19F). The
serotype 19F conjugate was passed through a 0.2 .mu.m filter into a
holding tank and incubated at 20-24.degree. C. Following
incubation, the conjugate was diafiltered on a 300 kDa MWCO
ultrafilter with a minimum of 20 diavolumes of 150 mM sodium
chloride at 20-24.degree. C. The permeate was discarded, and the
retentate was filtered through a 0.2 .mu.m filter and filled into
borosilicate glass containers at appropriate volumes for release
testing, in-process controls, and formulation. The final bulk
concentrates were stored at 2-8.degree. C.
Example 3
Formulation of a 15-Valent Pneumococcal Conjugate Vaccine
[0103] The required volumes of bulk concentrates were calculated
based on the batch volume and the bulk saccharide concentrations.
The combined 15 conjugates were further diluted to a target
adsorption concentration by the addition of a sodium chloride and
L-histidine, pH 5.8, containing buffer. After sufficient mixing,
the blend was sterile filtered through a 0.2 .mu.m membrane. The
sterile formulated bulk was mixed gently during and following its
blending with bulk aluminum phosphate. The formulated vaccine was
stored at 2-8.degree. C.
[0104] In an alternate process, the combined 15 conjugates were
further diluted to a target concentration by the addition of a
sodium chloride and L-histidine, pH 5.8, containing buffer.
Polysorbate 80 was added to a final concentration of 0.005%, to the
diluted buffered conjugate mixture prior to sterile filtration.
Following sterile filtration, the formulated vaccine was stored at
2-8.degree. C.
[0105] Table 1 shows the final composition of the adjuvanted and
non-adjuvanted form of PCV-15.
TABLE-US-00001 TABLE 1 Composition of Adjuvanted and Non-Adjuvanted
15-valent Pneumococcal Conjugate Vaccine Formulations Clinical
Formulations, unit/0.5 mL, dose Non-adjuvanted Description of
Ingredients Adjuvanted PCV-15 PCV-15 Active Pneumococcal 32 .mu.g
of total 32 .mu.g of total Ingredients polysaccharide antigens
polysaccharide polysaccharide (2 .mu.g of each of the (2 .mu.g of
each of the following following polysaccharide polysaccharide
serotypes 1, 3, 4, 5, 6A, serotypes 1, 3, 4, 5, 7F, 9V, 14, 18C,
19A, 6A, 7F, 9V, 14, 18C, 19F, 22F, 23F, 33F; 19A, 19F, 22F, 23F, 4
.mu.g of serotype 6B 33F; polysaccharide) 4 .mu.g of serotype 6B
polysaccharide)) Carrier protein CRM.sub.197 ~32 .mu.g ~32 .mu.g
Other Aluminum (.mu.g).sup.a 125 0 Ingredients Polysorbate-80
(.mu.g) 0 2.5 L-histidine (mM) 20 20 Sodium Chloride (mM) 150 150
Water for Injection Q.S..sup.b Q.S..sup.b .sup.aQuantity of
elemental aluminum in APA. .sup.bQuantify sufficient to 0.5 mL.
Example 4
Immunogenicity Studies
[0106] Experiments were designed to evaluate the immunogenicity of
multiple formulations of pneumococcal conjugate vaccines in the
infant rhesus monkeys (IRM) and New Zealand White Rabbits (NZWR)
animal models. Experiments in infant rhesus monkeys were designed
to closely match the recommended schedule for pneumococcal
conjugate vaccine in United States, with the infant series given at
2, 4, and 6 months of age. Thus, infant rhesus monkeys were
immunized starting at 2-3 months of age and administered vaccine at
2-month intervals. The 4th dose, which is also part of the
recommended schedule for U.S. children was not administered. Adult
rabbits (NZWR) were used to evaluate multiple vaccine formulations.
NZWR studies were performed using two vaccine doses given in an
accelerated (2-week interval) immunization regimen. For the
preclinical evaluation of immune responses, a full human dose was
delivered to rabbits whereas infant monkeys received a half-human
dose. The rationale for selecting a half-human dose for infant
monkeys was due to limitations in the volume that can be
administered to infant rhesus monkeys in a single intramuscular
site.
Assessment of Serotype-specific IgG Responses
[0107] A multiplexed electrochemiluminescence (ECL) assay was
developed for use with rabbit and rhesus monkey serum based on a
human assay using Meso Scale Discovery (MSD) technology which
utilizes a SULFO-TAG.TM. label that emits light upon
electrochemical stimulation. See Marchese et al., 2009, Clin
Vaccine Immunol 16:387-96. Using a dedicated ECL plate reader, an
electrical current is placed across the plate-associated electrodes
resulting in a series of electrically induced reactions leading to
luminescent signal. The multi-spot configuration used in
development and validation was 10 spots/well in a 96-well
plate-format, and each well was coated with 5 ng pneumococcal (Pn)
polysaccharide (Ps) per spot. Two plate formats were used to ensure
that crossreacting polysaccharides (i.e., 6A and 6B, and 19A and
19F) were tested in separate plates. Plate format 1 contained
serotypes 3, 4, 6B, 9V, 14, 18C, 19F, and 23F whereas plate format
2 contained serotypes 1, 5, 6A, 7F, 19A, 22F and 33F. Each well
also contained two bovine serum albumin (BSA) spots which were used
to assess the background reactivity of the assay (i.e., the
response associated with serum and labeled secondary antibody in
the absence of PnPs). Assay standard (89SF-2), controls, and test
sera were diluted to appropriate levels in phosphate buffered
saline (PBS) containing 0.05% Tween 20, 1% BSA, 5 .mu.g/ml
C-polysaccharide (CPs), 10 .mu.g/ml serotype 25 polysaccharide
(PnPs25) and 10 .mu.g/ml serotype 72 polysaccharide (PnPs72) and
incubated overnight at 4.degree. C. (2 to 8.degree. C.) or at
ambient temperature for 45 minutes. Human antibody reagents and
standards were used when testing the infant monkey samples whereas
SULFO-TAG.TM.-labeled anti-rabbit IgG was used as the secondary
antibody when testing rabbit serum samples. Each antigen coated
plate was incubated at ambient temperature for 1 hour on a shaker
platform with blocking agent. Plates were washed with 0.05% PBS-T
and 25 .mu.L per well of the pre-adsorbed and diluted test sera was
added and incubated for 45 min at ambient temperature on a shaker
platform. Plates were washed with 0.05% PBS-T and then MSD
SULFO-TAG.TM. labeled-goat anti-human IgG secondary antibody (for
rhesus monkey serum) and labeled goat anti-rabbit IgG secondary
antibody (for rabbit serum) was added to each well and incubated 1
hour at ambient temperature on a shaker platform. Plates were
washed with 0.05% PBS-T and 150 .mu.L of MSD Read Buffer-T 4.times.
(with surfactant) diluted 1:4 in water added to each well. The
plates were read using a MSD Sector Imager Model No. 2400 or 6000.
For rabbit studies, the results are presented as geometric mean
titers (GMTs) or ratios of GMTs. For infant rhesus monkey studies,
the results were expressed as geometric mean concentrations read
from a standard curve using the serotype-specific IgG
concentrations assigned to the human reference standard (89
SF-2).
Assessment of Functional (Opsonophagocytic) Responses
[0108] Samples from infant rhesus monkey study 2 were tested in a
4-plexed MOPA assay (MOPA-4). See Burton et al., 2006, Clin Vaccine
Immunol 13:1004-9. The assay uses bacterial strains selected to be
resistant to one of 4 antibiotics so that the first part of the
assay (opsonization and uptake into differentiated HL-60 cells) can
be performed with up to 4 serotypes at a time. The read-out for
bacterial killing is done in parallel in the presence of each of
the 4 antibiotics to which the corresponding strains are resistant
in order to determine killing titers for each specific serotype.
Results are expressed as the reciprocal dilution at which 50%
killing is observed (after interpolation).
Statistical Methodology for Preclinical Studies
[0109] Both animal models have limitations related to sample size.
In general, 8 infant monkeys or 8 rabbits were used per study aim.
With 8 animals per arm a critical fold difference in geometric mean
titer between treatment arms of 2.5 fold was regarded as a
meaningful response threshold. The 2.5-fold difference was
determined based on the assumption that for each serotype, the
standard deviation of the natural log transformed titers within a
treatment arm is ln(2). Letting Y.sub.i denote the mean of the ln
transformed titers in the i.sup.th treatment arm, n.sub.i the
number of animals within the i.sup.th treatment arm,
.sigma..sub.i.sup.2 the known variance of the ln transformed titers
among animals within the i.sup.th treatment arm, and setting
n.sub.i=8 and .sigma..sub.i.sup.2=(ln(2)).sup.2 for all i, then the
value of 2.5 is obtained by solving for e.sup. Y.sup.j.sup.-
Y.sup.k where
Y _ j - Y _ k .sigma. j 2 n j + .sigma. k 2 n k = Z 0.995 ,
##EQU00001##
and Z.sub.0.995 denotes the inverse of the standard normal
cumulative distribution, with a probability of 0.995 (i.e.,
Z.sub.0.995=2.576). Note that the calculated value of 2.44 is
rounded to 2.5 as 2.5 also provides for a convenient reciprocal in
0.4.
Serotype-specific IgG Response of Infant Rhesus Monkeys (IRMs) to
PCV-15
[0110] A pilot immunogenicity study (IRM-1) was conducted to
determine whether infant rhesus monkeys (IRMs) would be a good
model in which to evaluate Pn polysaccharide CRM.sub.197 conjugate
vaccines. The primary goal of the experiment was to determine
whether IRMs (like human infants) would be unresponsive to free Pn
polysaccharides but respond well to conjugate vaccines. Groups of 5
IRMs were injected starting at 2-3 months of age with either Pn
polysaccharide, Prevnar.RTM. or PCV-15. Three doses of vaccine were
administered intramuscularly (IM) at 2 month intervals, and
serotype-specific IgG responses were measured prior to the first
dose and at 1 month postdose 2 and at 1 month postdose 3 using a
multiarray electrochemiluminescence (ECL) assay (data not
shown).
[0111] The results indicated that IRMs responded poorly, if at all,
to free Pn polysaccharide but very well to the conjugate vaccines.
The results indicated that induction of an IgG response to Pn
polysaccharides in infant rhesus monkeys was dependent upon
conjugation of the polysaccharides to a carrier protein and
therefore was a classic T-cell dependent response. Thus, the IRM
model was determined to be suitable for evaluating PCV-15
formulations.
[0112] A second study (IRM-2) was conducted to evaluate a
formulation of PCV-15 using a bulk conjugation process that
minimized free (unconjugated) polysaccharide and unconjugated
CRM.sub.197. FIG. 1 shows the postdose 2 (PD-2) and postdose 3
(PD-3) IgG responses to PCV-15 versus Prevnar.RTM. for the 7
serotypes contained in Prevnar.RTM. (4, 6B, 9V, 14, 18C, 19F, 23F).
PD-2 responses to PCV-15 were equivalent or slightly lower than the
corresponding responses to Prevnar.RTM. whereas PD-3 responses to
PCV-15 were somewhat higher than those elicited by Prevnar.RTM. for
nearly all serotypes.
[0113] IRM responses to the non-Prevnar serotypes in PCV-15 are
shown in FIG. 2. PD-2 responses to the non-Prevnar serotypes in
PCV-15 were all at least 10-fold higher than baseline
(pre-vaccination) IgG concentrations, and titers continued to rise
at PD-3.
[0114] The results indicate that antibody responses to PCV-15 and
Prevnar.RTM. were comparable for the 7 common serotypes and that
post-vaccination responses to PCV-15 were >10-fold higher than
baseline for the 8 added serotypes.
Functional (Opsonophagocytic) Immune Response of IRMs to PCV-15
[0115] In order to determine whether PCV-15 induced functional
antibody responses in infant monkeys, an opsonophagocytic killing
(OPA) assay was performed on sera from IRM-2. Pre-vaccination,
PD-2, and PD-3 responses to PCV-15 and Prevnar.RTM. are shown in
Table 2. The results shown are the GMTs from serum samples from 7-8
monkeys per time point assayed in duplicate. Also shown are the
percent responders (i.e., those with OPA titers .gtoreq.8) at the
PD-3 time point. PCV-15 induced a high PD-2 GMT for all serotypes
except types 1 and 33F. After 3 vaccine doses, PCV-15 induced high
OPA GMTs to each serotype and a 100% OPA response rate for all 15
serotypes contained in the vaccine. Of note, PCV-15 also induced a
good crossreactive OPA response to serotype 6C, which is not in the
vaccine. Prevnar.RTM. induced high OPA titers and a 100% response
rate for all serotypes contained in that vaccine, but it induced
only a weak crossreactive response to serotypes 6A and 6C in a
fraction of monkeys.
TABLE-US-00002 TABLE 2 Serotype-Specific OPA GMTs in Infant Rhesus
Monkeys after Vaccination with PCV-15 or Prevnar .RTM.
(Pre-vaccination, PD-2, and PD-3 geometric mean titers and PD-3
percent responders with a titer .gtoreq.8) Prevnar PCV-15 PD-3 PD-3
Responders Responders Serotype Pre PD-2 PD-3 (titers .gtoreq.8) Pre
PD-2 PD-3 (titers .gtoreq.8) 1 n.d. n.d. n.d. n.d. 4 65 340 100% 3
n.d. n.d. n.d. n.d. 5 1442 1548 100% 4 4 11459 5004 100% 4 5280
3453 100% 5 4 4 4 0% 4 1879 1719 100% 6A 4 21 113 57% 4 1188 7807
100% 6B 8 8294 6043 100% 4 2477 9601 100% 6C 4 11 16 29% 4 1038
5134 100% 7F 4 4 71 43% 4 7541 10092 100% 9V 4 1779 748 100% 4 625
1297 100% 14 8 12395 7782 100% 4 11366 9891 100% 18C 4 5571 1718
100% 4 1934 1701 100% 19A 4 15 4 0% 4 2210 1895 100% 19F 4 1365 432
100% 4 2555 4021 100% 22F n.d. n.d. n.d. n.d. 4 2489 7298 100% 23F
4 1789 2126 100% 4 3093 2465 100% 33F n.d. n.d. n.d. n.d. 4 14
11548 100% Note: Serotypes contained in Prevnar are bolded. Results
for serotype 6C are shown in italics since that serotype is not
contained in PCV-15. n.d. not determined
Evaluation of PCV-15 Formulations in New Zealand White Rabbits
[0116] PCV-15 formulations were evaluated in 4 studies in adult New
Zealand White Rabbits (NZWRs) using a compressed immunization
regimen in which rabbits received a full human dose of vaccine at
day 0 and day 14, and serum was collected at day 0, 14 and 28 for
analysis. All studies were benchmarked with Prevnar.RTM., and as
summarized in Table 3 (NZWR Experiments 1-4).
[0117] Results are shown in Table 3 for Post-dose 2 responses of
New Zealand white rabbits expressed as a ratio of the geometric
mean IgG responses to Merck PCV-15 over Prevnar.RTM. for serotypes
in common between the vaccines.
TABLE-US-00003 TABLE 3 Post-dose 2 IgG Response Ratios
(PCV-15:Prevnar .TM.) of Lead PCV-15 Formulations Tested in NZWR
Serotype NZWR-1 NZWR-2 NZWR-3 NZWR-4 4 0.70 0.59 0.63 1.06 6B 1.35
0.49 1.53 0.45 9V 2.07 1.79 1.70 1.31 14 2.37 0.58 2.32 2.55 18C
0.87 0.6 0.52 0.27 19F 0.66 0.76 2.70 1.25 23F 0.36 0.30 1.22
0.41
[0118] Serotype-specific IgG responses were generally within
2.5-fold of the corresponding responses to Prevnar.RTM.. An
exception was serotype (23F), which was >2.5-fold lower than
that to Prevnar.RTM. in 2 of 4 experiments. The fold-rise in
antibody levels to the non-Prevnar.RTM. serotypes from Day 0 to Day
28 (Post-dose 2, PD-2) are summarized in Table 4.
TABLE-US-00004 TABLE 4 Fold-rise (Post-dose 2:Pre-dose 1) in IgG
Responses to Non-Prevnar .TM. Serotypes of PCV-15 Lead Formulations
Tested in NZWR Serotype NZWR-1 NZWR-2 NZWR-3 NZWR-4 1 14.9 30.5
55.1 59.9 3 33.6 16.2 61.5 28.5 5 12.8 70.2 112.0 134.0 6A 21.3
77.8 143.0 123.0 7F 42.0 83.8 194.0 108.0 19A 40.5 79.1 450.0 314.0
22F 45.7 87.8 243.0 135.0 33F 21.7 47.9 98.8 69.4
Effect of Polysaccharide Conjugate Vaccine Dose on Immunogenicity
in NZWRs
[0119] The immunogenicity of an increased dose (double dose,
2.times.) of polysaccharide conjugates was also evaluated for all
serotypes contained in PCV-15 compared with the planned human dose
(1.times.) of the vaccine. For the 2.times. polysaccharide
conjugate formulation, the APA concentration was increased to
1.5.times. in order to assure that most of the conjugate would be
bound to the aluminum adjuvant. As shown in Table 5, there did not
appear to be a significant benefit in increasing the amount of
polysaccharide-conjugate in the vaccine. Differences across all
serotypes were within 2-fold, and the geometric mean fold-ratio
(1.times.PCV-15/2.times.PCV-15/2+1.5x APA) was 1.1.
TABLE-US-00005 TABLE 5 Post-dose 2 Geometric Mean IgG titers (95%
confidence intervals) with Prevnar .RTM., 1x Human dose of PCV-15*
or 2x Human Dose of PCV-15.sup..dagger. in NZWR Fold- Difference
Ratio of Treatment Arm 2x PCV- Prevnar .TM. 1x PCV-15 2x PCV-15
15/1x PCV- Serotype (n = 8) (n = 8) (n = 8) 15 4 736,400 436,000
472,100 1.1 (483200, 1122400) (199700, 951700) (246800, 902900) 6B
363,600 176,500 196,900 1.1 (205000, 644800) (72800, 427800)
(85900, 451400) 9V 298,200 534,700 580,600 1.1 (173800, 511700)
(362800, 788100) (366300, 920200) 14 345,200 198,900 273,600 1.4
(200200, 595000) (94500, 418700) (229500, 326100) 18C 954,500
573,000 455,900 0.8 (815700, 1116800) (396900, 827400) (245100,
848000) 19F 720,100 548,000 544,300 1.0 (475700, 1090200) (367700,
816800) (269000, 1101400) 23F 816,300 246,200 188,500 0.8 (565100,
1179100) (117200, 517100) (78100, 454800) 1 5,300 91,500 72,200 0.8
(3100, 9200) (62600, 133600) (46700, 111600) 3 12,000 32,300 23,600
0.7 (8600, 16800) (19600, 53000) (14100, 39400) 5 5,700 245,600
224,600 0.9 (4100, 7900) (114200, 528200) (136700, 369100) 6A
525,900 186,700 251,800 1.3 (275000, 1005600) (71300, 488600)
(102300, 620100) 7F 4,600 326,900 212,200 0.6 (4000, 5200) (238000,
449000) (134200, 335500) 19A 432,800 260,900 276,100 1.1 (237800,
787800) (145500, 468000) (153200, 497600) 22F 6,000 359,800 345,300
1.0 (4400, 8100) (239000, 541700) (221200, 539000) 33F 6,600
177,400 138,500 0.8 (4700, 9300) (118300, 266200) (68300, 280900)
*Formulated with 1x aluminum adjuvant (APA) .sup..dagger.Formulated
with 1.5x APA
Effect of Aluminum Adjuvant on Immunogenicity of PCV-15 in
NZWRs
[0120] The impact of aluminum adjuvant (APA) on antibody responses
was evaluated in one rabbit study. PCV-15 formulated with the
planned human dose of APA (PCV-15 1.times.APA), with double the
planned human dose of APA (PCV-15 2.times.APA), and without any
aluminum adjuvant (PCV-15 0.times.APA), were tested. A Prevnar.RTM.
group was also included in the study.
[0121] The PD-2 results indicated that doubling the concentration
of APA had little impact on the serotype-specific IgG response to
PCV-15. The fold-difference in titer (1x APA/2.times.APA) ranged
from 0.6 (serotype 6B) to 2.3 (serotype 22F) and geometric mean
fold-ratio across the 15 serotypes was 1.1. In the absence of
aluminum adjuvant antibody titers appeared lower for many of the
serotypes relative to PCV-15 with 1.times.APA. The fold-difference
in titer (1x/0x) ranged from 0.5 (serotype 5) to 2.9 (serotype 23F)
and the geometric mean fold-ratio across the 15 serotypes was 1.4.
Overall, there does not appear to be a genuine advantage to
doubling the level of aluminum adjuvant and there appears to be a
disadvantage to eliminating the adjuvant (Table 6) in this animal
model.
[0122] The PD-2 results indicated that there was a decrease in
antibody titers for many of the serotypes in the arm that did not
contain Aluminum Phosphate Adjuvant (APA) when compared to PCV-15
containing APA (FIG. 3) indicating a requirement for the inclusion
of an aluminum adjuvant for optimal PCV-15 immunogenicity in
rabbits. In addition, no benefit was found when double the amount
of APA was included in the vaccine (data not shown).
TABLE-US-00006 TABLE 6 Post-dose 2 Geometric Mean IgG Titers (95%
confidence intervals) of PCV-15 Formulated with 1x, 2x or 0x
Aluminum Adjuvant (APA) in NZWR Fold Difference Relative to
Treatment Arm PCV-15 PCV-15 PCV-15 PCV-15 (1x APA) Prevnar .TM. 1x
APA 2x APA 0x APA 1x/2x 1x/0x Sero (n = 8) (n = 8) (n = 8) (n = 6)
APA APA 4 380,700 268,400 196,200 404,600 1.4 0.7 (226100, 641000)
(205100, 351200) (84000, 458800) (105500, 1550700) 6B 105,900
143,300 236,200 93,800 0.6 1.5 (59800, 187400) (90700, 226300)
(102000, 547100) (46300, 189700) 9V 271,600 562,000 595,600 569,200
0.9 1.0 (177400, 415700) (382700, 825400) (390200, 908900) (239900,
1350500) 14 142,500 337,600 453,000 167,600 0.7 2.0 (75200, 269700)
(174000, 654900) (237400, 864400) (65100, 431700) 18C 323,000
282,200 248,700 175,200 1.1 1.6 (250400, 416500) (210400, 378500)
(193900, 319000) (91800, 334000) 19F 302,500 199,800 243,400
127,700 0.8 1.6 (232700, 393200) (132200, 302100) (87600, 676600)
(35100, 463700) 23F 328,200 117,200 77,600 40,200 1.5 2.9 (178200,
604300) (60300, 227700) (31300, 192300) (10100, 160900) 1 5,600
91,700 67,800 53,300 1.4 1.7 (4400, 7200) (46800, 179800) (38900,
118100) (14800, 191600) 3 6,300 156,100 155,700 104,000 1.0 1.5
(4800, 8100) (104800, 232500) (75300, 322200) (45000, 240000) 5
4,800 81,700 60,700 176,100 1.3 0.5 (4000, 5700) (50800, 131300)
(29100, 126400) (72100, 430300) 6A 84,500 163,600 226,800 99,300
0.7 1.6 (50300, 141900) (114000, 234900) (92500, 556200) (48800,
202000) 7F 5,200 216,800 282,100 212,200 0.8 1.0 (3300, 8400)
(141500, 332000) (158200, 503200) (64200, 701900) 19A 95,100
238,300 207,400 125,700 1.1 1.9 (54300, 166700) (161000, 352700)
(69800, 615900) (34000, 465100) 22F 6,500 348,900 149,400 336,800
2.3 1.0 (4900, 8500) (267200, 455500) (90100, 247600) (184200,
615700) 33F 7,100 235,600 163,600 222,000 1.4 1.1 (5000, 10100)
(106500, 521300) (92700, 288800) (125100, 393800)
Discussion and Conclusions
[0123] The preclinical data demonstrate that a formulation of
PCV-15 (formulated on APA) is highly immunogenic in two species
(infant rhesus monkeys and rabbits). Serotype-specific responses to
PCV-15 were comparable to those elicited by Prevnar.RTM. for the 7
serotypes in common between the vaccines. For the 8 new serotypes
in PCV-15, there was a robust response elicited in both infant
rhesus monkeys and in rabbits, with .gtoreq.10-fold rise in IgG
responses for all serotypes after 2 vaccine doses in both species.
Limited dose-ranging experiments indicated that a 2-fold increase
in the amount of polysaccharide conjugates did not result in an
increased antibody response. Similarly, a 2-fold increase in
aluminum adjuvant concentration did not appear to significantly
improve the immunogenicity profile of PCV-15. Elimination of the
adjuvant did, however, result in lower responses to some serotypes
suggesting the potential need for an adjuvant in humans. Functional
(OPA) antibody responses were elicited by PCV-15 to all 15
serotypes in the vaccine as well as to Serotype 6C, which is not a
component of PCV-15.
* * * * *