STABLE IMMUNOGENIC COMPOSITIONS OF STAPHYLOCOCCUS
AUREUS ANTIGENS
Background of the Invention
[0001] Humans are the natural reservoirs for Staphylococcus aureus (S. aureus).
Healthy individuals can be colonized by S. aureus on the skin, in the nares and the
throat either persistently (10-35%), intermittently (20-75%) or be in a non-carriage
state (5-70%o) with no associated disease. See Vandenbergh et al, J. Clin. Micro.
37:3 133-3 140 (1999). Disease subsequently occurs when individuals become
immunocompromised due to breaches in immune barriers, such as during surgery,
placement of indwelling catheters or other devices, trauma, or wounds. The
resulting S. aureus infection can cause a wide range of diseases that range from
mild skin infections to endocarditis, osteomyelitis, bacteremia, sepsis, and other
forms of disease with accompanying high mortality rates. The large human
reservoir enhances opportunity for evolution and spread of adapted pathogenic
clonal types.
[0002] Invasive staphylococcal infections from the Gram positive cocci S. aureus
and S. epidermidis are of particular concern because they are an increasing public
health problem worldwide. Specifically, S. aureus is responsible for the majority
of hospital-acquired (nosocomial) infections, and its prevalence in communityonset
infections is increasing. For example, the incidence of invasive methicillinresistant
S. aureus (MRSA) was estimated at 31.8 per 100,000 persons, including
18,650 deaths in the United States in 2005. See Klevens R.M. et al, JAMA,
298:1763-71 (2007).
[0003] Staphylococcal diseases have seen a dramatic increase in the last 20
years; this increase parallels the use of intravascular devices and invasive
procedures. The rise in disease incidence is made more troubling because of the
parallel rise of antibiotic resistance; therefore, there is an urgent need for
immunogenic compositions for use in vaccines or to elicit polyclonal or
monoclonal antibodies to confer passive immunity as a means to prevent or treat
staphylococcal infection and associated diseases.
[0004] Clumping factor A (ClfA) is an S. aureus cell wall-associated adhesin
that mediates staphylococcal binding to fibrinogen and platelets. It is expressed on
the cell surface of the bacterium, where it is thought to promote pathogenesis by
binding to the fibrinogen and fibrin that is deposited at the site of tissue damage.
ClfA is well conserved, and even the most diverse form (-85% identity) exhibits
extensive cross-reactivity to both monoclonal and polyclonal antibodies. Thus,
ClfA is a reasonable candidate for a component of a vaccine against S. aureus.
However, given the structural instability of ClfA, a formulation of ClfA is
problematic since it can readily degrade over time in storage.
[0005] Full-length ClfA comprises several regions and domains: an N-terminal
secretory domain ("S" domain); followed by a ligand-binding A region, which
contains three domains (Nl, N2, which contains an EF-hand motif, and N3);
followed by an R region, which contains serine-aspartate dipeptide repeats;
followed by a cell wall-binding region ("W" region) containing an LPXTG motif;
a hydrophobic membrane-spanning domain ("M" region); and a charged Cterminus
("C" region) containing positively charged amino acids. The Nl region
contains a protease-sensitive site. Much of the instability of ClfA is attributed to
the clipping of ClfA at Nl, which results in fragments containing Nl and N2N3.
The structure and function of ClfA is disclosed in U.S. Patent Application
Publication No. 20070087014A1 (Pavliak et al, April 19, 2007), which is
incorporated herein by reference in its entirety.
[0006] Staphylococcal microorganisms capable of causing invasive disease
generally also are capable of producing a capsule polysaccharide (CP) that
encapsulates the bacterium and enhances its resistance to clearance by the host
innate immune system. The CP serves to cloak the bacterial cell in a protective
capsule that renders the bacteria resistant to phagocytosis and intracellular killing.
Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular
polysaccharides are frequently an important virulence factor for many bacterial
pathogens, including Haemophilus influenzae, Streptococcus pneumoniae and
Group B streptococci.
[0007] Most clinical isolates of S. aureus are encapsulated with either serotypes
5 or 8. Type 5 (CP5) and type 8 (CP8) capsular polysaccharides have similar trisaccharide
repeating units comprised of N-acetyl mannosaminuronic acid, N-acetyl
L-fucosamine, and N-acetyl D-fucosamine. See Fournier, J.M. et al., Infect.
Immun. 45:97-93 (1984) and Moreau, M., et al, Carbohydrate Res. 201 :285-297
(1990). The two CPs, which have the same sugars, but differ in the sugar linkages
and in sites of O-acetylation, each produce serologically distinct patterns of
immunoreactivity.
[0008] The S. aureus MntC protein (also known as Protein 305, P305, P305A,
and ORF305) is a component of a manganese ABC transporter. This protein is
expressed in vivo. S. aureus uses manganese as a cofactor for an enzyme that
enhances the survival of S. aureus in neutraphils. MntC is, therefore, important for
the in vivo survival of S. aureus during infection. Like ClfA, this protein is also
unstable in solution. However, unlike ClfA, which can aggregate, or clip via
hydrolysis, the primary mechanism of MntC degradation is deamidation when
subject to basic pH and/or temperature around room temperature (about 25°C) or
higher.
[0009] Accordingly, there is an urgent need not only for vaccines against S.
aureus infection, but in particular for vaccines with enhanced antigen stability.
Summary of the Invention
[0010] The present invention is directed towards a lyophilized or reconstituted
multi-antigen or multicomponent immunogenic composition comprising at least
one antigen isolated from a staphylococcal bacterium. The antigens, which are
polypeptides and polysaccharides, may be obtained, inter alia, directly from the
bacterium using isolation procedures known to those skilled in the art, or they may
be produced using synthetic protocols, or they may be recombinantly produced
using genetic engineering procedures also known to those skilled in the art, or
through a combination of any of the foregoing. In certain embodiments, a
lyophilized or reconstituted immunogenic composition of the invention comprises
an isolated S. aureus clumping factor A (ClfA). In certain embodiments, a
lyophilized or reconstituted immunogenic composition of the invention comprises
three or more antigens selected from an isolated S. aureus clumping factor A
(ClfA) polypeptide, an isolated S. aureus clumping factor B (ClfB) polypeptide, an
isolated S. aureus capsular polysaccharide type 5 (CP5) conjugated to a carrier
protein, an isolated S. aureus capsular polysaccharide type 8 (CP8) conjugated to a
carrier protein, and an isolated S. aureus MntC protein. In addition, the present
invention provides methods for inducing an immune response against a
staphylococcal bacterium; methods for preventing, reducing the severity, or
delaying onset of a disease caused by a staphylococcal bacterium; and methods for
preventing, reducing the severity, or delaying onset of at least one symptom of a
disease caused by infection with a staphylococcal bacterium.
[0011] Accordingly, in one embodiment, the invention provides a lyophilized or
reconstituted immunogenic composition comprising: an isolated S. aureus
clumping factor A (ClfA) polypeptide, an isolated S. aureus capsular
polysaccharide type 5 (CP5) conjugated to a carrier protein, and an isolated
S. aureus capsular polysaccharide type 8 (CP8) conjugated to a carrier protein.
[0012] In one embodiment, the invention provides a lyophilized or reconstituted
immunogenic composition comprising: an isolated S. aureus clumping factor A
(ClfA) polypeptide, an isolated S. aureus clumping factor B (ClfB) polypeptide, an
isolated S. aureus capsular polysaccharide type 5 (CP5) conjugated to a carrier
protein, and an isolated S. aureus capsular polysaccharide type 8 (CP8) conjugated
to a carrier protein.
[0013] In one embodiment, the invention provides a lyophilized or reconstituted
immunogenic composition comprising: an isolated S. aureus clumping factor A
(ClfA) polypeptide, an isolated S. aureus clumping factor B (ClfB) polypeptide, an
isolated S. aureus MntC protein, an isolated S. aureus capsular polysaccharide type
5 (CP5) conjugated to a carrier protein, and an isolated S. aureus capsular
polysaccharide type 8 (CP8) conjugated to a carrier protein.
[0014] In one embodiment, the invention provides a lyophilized or reconstituted
immunogenic composition comprising: an isolated S. aureus clumping factor A
(ClfA) polypeptide, an isolated S. aureus MntC protein, an isolated S. aureus
capsular polysaccharide type 5 (CP5) conjugated to a carrier protein, and an
isolated S. aureus capsular polysaccharide type 8 (CP8) conjugated to a carrier
protein.
[0015] In one embodiment, the invention provides a lyophilized or reconstituted
immunogenic composition comprising: an isolated S. aureus clumping factor B
(ClfB) polypeptide, an isolated S. aureus capsular polysaccharide type 5 (CP5)
conjugated to a carrier protein, and an isolated S. aureus capsular polysaccharide
type 8 (CP8) conjugated to a carrier protein.
[0016] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) at least three components selected from the group
consisting of an isolated Staphylococcus aureus clumping factor A ("ClfA")
polypeptide, an isolated Staphylococcus aureus clumping factor B ("ClfB")
polypeptide, a Capsular Polysaccharide Type 5 (CP5)-protein conjugate, a
Capsular Polysaccharide Type 8 (CP8)-protein conjugate, and an isolated
Staphylococcus aureus MntC polypeptide; (b) a buffer having a pKa of 6.0 ± 0.6,
and (c) a bulking agent. In certain embodiments, the ClfA polypeptide remains
substantially undegraded for at least 1 month at 37°C.
[0017] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) an isolated Staphylococcus aureus clumping factor A
("ClfA") polypeptide, (b) a Capsular Polysaccharide Type 5 (CP5)-protein
conjugate, (c) a Capsular Polysaccharide Type 8 (CP8)-protein conjugate, (d) a
buffer having a pKa of 6.0 ± 0.6, and (e) a bulking agent. In certain embodiments,
the ClfA polypeptide remains substantially undegraded for at least 1 month at
37°C.
[0018] In one embodiment, the lyophilized immunogenic composition comprises
water at less than 3 percent weight of the total weight of the immunogenic
composition (% w/w), wherein the ClfA polypeptide is between 0.09% ± 0 .027%
and 0.85% ± 0.26% w/w, the CP5-protein conjugate is between 0.04% ± 0.013%
and 0.42 ± 0.13% w/w, the CP8-protein conjugate is between 0.04% ± 0.013% and
0.42 ± 0.13% w/w, and the buffer is at 2.54% ± 0.76% w/w.
[0019] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) a ClfA polypeptide; (b) a CP5-protein conjugate; (c) a
CP8-protein conjugate; (d) a histidine buffer, pH 6.0 ± 0.5; (e) sucrose; (f
polysorbate 80; and (g) water. In certain embodiments, the ClfA polypeptide
remains substantially undegraded for at least 3 months at 37°C.
[0020] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) a ClfA polypeptide between 0.09%> ± 0.027% and
0.85% ± 0.26% w/w; (b) a CP5-protein conjugate between 0.04% ± 0.013% and
0.42 ± 0.13% w/w; (c) a CP8-protein conjugate between 0.04% ± 0.013% and 0.42
± 0.13% w/w; (d) a histidine buffer, pH 6.0 ± 0.5, at 2.54% ± 0.76% w/w; (e)
sucrose at 97% ± 2.0% w/w; (f polysorbate 80 at 0.21% ± 0.04% w/w; and (g)
water at 2% ± 1% w/w.
[0021] In one embodiment, the invention provides a liquid immunogenic triantigen
composition manufactured by reconstituting a lyophilized immunogenic
composition of the invention in an aqueous diluent, said reconstituted composition
having a final pH of 6.0 ± 0.5.
[0022] In one embodiment, the invention provides a liquid immunogenic
composition manufactured by reconstituting a lyophilized immunogenic
composition of the invention comprising: (a) the ClfA polypeptide at a
concentration of between 40 mg/ml ± 4mg/ml and 800mg/ml ± 80mg/ml; (b) the
CP5-proteinconjugate at a concentration of between 20mg/ml ± 2mg/ml and
400 mg/ml ± 40mg/ml; (c) the CP8-protein conjugate at a concentration of between
20mg/ml ± 2mg/ml and 400 mg/ml ± 40mg/ml; (d) the histidine buffer at a
concentration of lOmM ± 5mM; (e) polysorbate 80 at a concentration of 0.1% ±
0.05% weight to volume (w/v); and (f) sucrose at a concentration of 9% ± 4.5%
w/v. In another embodiment, the polysorbate 80 is at a concentration of 0.01% ±
0.005% weight to volume (w/v) and the sucrose is at a concentration of 4.5% ±
1.5% w/v.
[0023] In one embodiment, the invention provides a liquid immunogenic
composition comprising: (a) a reconstituted lyophilized ClfA polypeptide; (b) a
CP5-protein conjugate; (c) a CP8-protein conjugate; (d) a histidine buffer, pH 6.0
± 0.5; (e) polysorbate 80; (f sucrose; and (g) an aqueous diluent.
[0024] In one embodiment, the invention provides a liquid immunogenic
composition manufactured by reconstituting a lyophilized immunogenic
composition of the invention comprising: (a) the reconstituted lyophilized ClfA
polypeptide at a concentration of between 40 mg/ml ± 4mg/ml and 800mg/ml ±
80mg/ml; (b) the CP5-protein conjugate at a concentration of between 0mg/ml ±
mg/ml and 400mg/ml ± 40mg/ml; (c) the CP8-protein conjugate at a concentration
of between 20mg/ml ± 2mg/ml and 400mg/ml ± 40mg/ml; (d) the histidine buffer at
a concentration of lOmM ± 5mM; (e) polysorbate 80 at a concentration of 0.1% ±
0.05% weight to volume (w/v); (f sucrose at a concentration of 9% ± 4.5% w/v;
and (g) an aqueous diluent. In another embodiment, the polysorbate 80 is at a
concentration of 0.01% ± 0.005% weight to volume (w/v) and the sucrose is at a
concentration of 4.5% ± 1.5% w/v.
[0025] In one embodiment, the invention provides a process of making an
immunogenic composition comprising the steps of: (a) combining an aqueous
solution comprising: (i) a ClfA polypeptide, (ii) a CP5-protein conjugate, (iii) a
CP8-protein conjugate, (iv) a buffer having a pKa of 6.0 ± 0.6, and (v) a bulking
agent; and (b) lyophilizing the combination of step (a) to form a cake comprising
less than 3 percent water by weight. In certain embodiments, the process further
comprises combining (vi) a surfactant, at step (a). In certain embodiments, the
process further comprises a step of filter sterilizing the combination of step (a)
prior to lyophilization of step (b). In certain embodiments, the aqueous solution
comprises lOmM ± lmM histidine, pH 6.0 ± 0.5, sucrose at 9% ± 4.5% w/v and
polysorbate 80 at 0.1% ± 0.05% w/v. In another embodiment, the polysorbate 80
is at a concentration of 0.01% ± 0.005% w/v and the sucrose is at a concentration
of 4.5% ± 1.5% w/v.
[0026] In certain embodiments, the lyophilizing step (b) comprises the steps of:
(i) freezing the sterilized combination of step (a) at a rate of 0.3°C ± 0.03°C per
minute until reaching a temperature of -50°C ± 5°C at a pressure of 400 millibars ±
40 millibars; and then holding the combination at -50°C ± 5°C for 60 minutes ± 6
minutes; (ii) annealing the combination by increasing the temperature to -10°C ±
5°C at a rate of 0.3°C ± 0.03°C per minute; subsequently holding the temperature
at -10°C ± 5°C for 120 minutes ± 12 minutes, followed by decreasing the
temperature at a rate of 0.3°C ± 0.03°C per minute until reaching a temperature of -
50°C ± 5°C, and holding the temperature at -50°C ± 5°C for 180 minutes ± 18
minutes; (iii) drying the combination by decreasing the pressure to 50 milliTorrs
(mTorr) and holding for 30 minutes, followed by increasing the temperature to -
30°C ± 5°C at a rate of 0.2°C ± 0.02°C per minute, and subsequently holding the
temperature at -30°C ± 5°C for 1,920 minutes ± 192 minutes; (iv) further drying
the combination by increasing the temperature to 30°C ± 5°C at a rate of 0.2°C ±
0.02°C per minute and increasing the pressure to 200 mTorr, and subsequently
holding the temperature at 30°C ± 5°C for 720 minutes ± 72 minutes; and (v)
decreasing the temperature to 5°C ± 5°C at a rate of 0.5°C ± 0.05°C per minute. In
some embodiments, lyophilization takes place in a vial. In some embodiments, the
vial is stoppered after lyophilization. In certain embodiments, the vial is backfilled
with nitrogen gas prior to stoppering the vial. In certain embodiments, the process
further comprises the step of: (c) reconstituting the lyophilized combination of step
(b) in an aqueous medium. In certain embodiments, the osmolality of the
reconstituted combination of step (c) is between 250 mOsM ± 25mOsM and 300
mOsM ± 30 mOsM. In certain embodiments, an immunogenic composition of the
invention is manufactured according to any process of making an immunogenic
composition of the invention.
[0027] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) at least four components selected from the group
consisting of an isolated Staphylococcus aureus clumping factor A (ClfA)
polypeptide, an isolated Staphylococcus aureus clumping factor B (ClfB)
polypeptide, a CP5-protein conjugate, a CP8-protein conjugate, and an isolated
Staphylococcus aureus MntC polypeptide; (b) a buffer having a pKa of 6.0 ± 0.6,
and (c) a bulking agent. In certain embodiments, the ClfA polypeptide remains
substantially undegraded for at least 1 month at 37°C.
[0028] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) an isolated Staphylococcus aureus clumping factor A
(ClfA) polypeptide; (b) a Capsular Polysaccharide Type 5 conjugated to CRM 197
(CP5-CRM 197) ; (c) a Capsular Polysaccharide Type 8 conjugated to CRMi 7 (CP8-
CRM 19-7); (d) an isolated MntC polypeptide; (e) a buffer having a pKa of 6.0 ± 0.6,
and (f) a bulking agent. In certain embodiments, the ClfA polypeptide remains
substantially undegraded for at least 1 month at 37°C.
[0029] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: water at less than 3 percent weight of the total weight of
the immunogenic composition (% w/w), wherein the ClfA polypeptide is between
0.09% ± 0.027% and 0.84% ± 0.25% w/w, the CP5-CRMi 7 is between 0.04% ±
0.013% and 0.42 ± 0.13% w/w, the CP8-CRMi is between 0.04% ± 0.013% and
0.42 ± 0.13% w/w, the MntC is between 0.09% ± 0.027% and 0.84% ± 0.25%
w/w, and the buffer is at 3.3% ± 0.99% w/w.
[0030] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) a ClfA polypeptide; (b) a CP5-CRMi 97 conjugate; (c)
a CP8-CRM 19-7 conjugate; (d) an MntC polypeptide; (e) a histidine buffer; (f)
sucrose; (g) polysorbate 80; and (h) water. In certain embodiments, the ClfA
polypeptide remains substantially undegraded for at least 3 months at 37°C.
[0031] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) a ClfA polypeptide between 0.09%> ± 0.027% and
0.84% ± 0.25% w/w; (b) a CP5-CRMi conjugate between 0.04% ± 0.013% and
0.42 ± 0.13% w/w; (c) a CP8-CRMi 7 conjugate between 0.04% ± 0.0 13% and
0.42 ± 0.13% w/w; (d) an MntC polypeptide 0.09% ± 0.027% and 0.84% ± 0.25%
w/w; (e) a histidine buffer at 3.3% ± 0.99% w/w; (f) sucrose at 95% ± 2% w/w; (g)
polysorbate 80 at 0.21% ± 0.063% w/w; and (h) water at 2% ± 1% w/w. In certain
embodiments, the ClfA polypeptide remains substantially undegraded for at least 3
months at 37°C.
[0032] In one embodiment, the invention provides a liquid immunogenic tetraantigen
composition manufactured by reconstituting a lyophilized immunogenic
composition of the invention in an aqueous diluent, said reconstituted composition
having a final pH of 6.5 ± 0.5.
[0033] In one embodiment, the invention provides a liquid immunogenic
composition comprising: (a) the ClfA polypeptide at a concentration of between 40
mg/ml ± 4mg/ml and 800mg/ml ± 80mg/ml; (b) the CP5-CRMi 7 conjugate at a
concentration of between 20m /hi1± 2mg/ml and 400m /hi1± 40m /hi 1; (c) the
CP8-CRM197 conjugate at a concentration of between 20m /hi1± 2mg/ml and
400m /hi1± 40m /hi1; (d) the isolated MntC polypeptide at a concentration of
between 40 mg/ml ± 4m /hi1and 800m /hi1± 80m /hi 1; (e) the histidine buffer at a
concentration of lOmM ± 5mM; (f) polysorbate 80 at a concentration of 0.01% ±
0.005% weight to volume (w/v); and (g) sucrose at a concentration of 4.5% ± 1.5%
w/v. In another embodiment, the polysorbate 80 is at a concentration of 0.01% ±
0.005% w/v and the sucrose is at a concentration of 4.5% ± 1.5% w/v.
[0034] In one embodiment, the invention provides a liquid immunogenic
composition comprising: (a) a reconstituted lyophilized ClfA polypeptide; (b) a
CP5-CRMi97 conjugate; (c) a CP8-CRM197 conjugate; (d) a MntC polypeptide; (d)
a histidine buffer; (e) polysorbate 80; (f) sucrose; and (g) an aqueous diluent.
[0035] In one embodiment, the invention provides a liquid immunogenic
composition comprising: (a) the reconstituted lyophilized ClfA polypeptide at a
concentration of between 40 (b) the
CP5-CRMi97 conjugate at a concentration of between 20mg/ml ± 2mg/ml and
400mg/ml ± 40m /ih1; (c) the CP8-CRM197 conjugate at a concentration of between
20mg/ml ± 20mg/ml and 400mg/ml ± 40m /ih1; (d) the isolated MntC polypeptide at
a concentration of between 40 (e) the
histidine buffer at a concentration of lOmM ± 5mM; (f) polysorbate 80 at a
concentration of 0.1% ± 0.05% weight to volume (w/v); and (g) sucrose at a
concentration of 9% ± 4.5% w/v; and (h) the aqueous diluent. In another
embodiment, the polysorbate 80 is at a concentration of 0.01% ± 0.005% w/v and
the sucrose is at a concentration of 4.5% ± 1.5% w/v.
[0036] In one embodiment, the invention provides a process of making an
immunogenic composition comprising the steps of: (a) combining in an aqueous
solution: (i) a ClfA polypeptide, (ii) a CP5-CRMi 97 conjugate, (iii) a CP8-CRM197
conjugate, (iv) a MntC polypeptide, (v) a buffer having a pKa of 6.0 ± 0.6, and (vi)
a bulking agent; and (b) lyophilizing the combination of step (a) to form a cake
comprising less than 3 percent water by weight. In certain embodiments, the
process further comprises combining (vi) a surfactant, at step (a). In certain
embodiments, the process further comprises a step of filter sterilizing the
combination of step (a) prior to lyophilization of step (b). In certain embodiments,
the aqueous solution comprises lOmM ± 5mM histidine, pH 6.0 ± 0.5, sucrose at
9% ± 1% w/v and polysorbate 80 at 0.1% ± 0.02%> w/v. In other embodiments, the
polysorbate 80 is at a concentration of 0.01% ± 0.00 1% w/v and the sucrose is at a
concentration of 4.5% ± 0.45% w/v. In certain embodiments, the lyophilizing
step (b) comprises the steps of: (i) freezing the sterilized combination of step (a) at
a rate of 0.3°C ± 0.03°C per minute until reaching a temperature of -50°C ± 5°C at
a pressure of 400 millibars ± 40 millibars; and then holding the combination at -
50°C ± 5°C for 60 minutes ± 6 minutes; (ii) annealing the combination by
increasing the temperature to -10°C ± 5°C at a rate of 0.3°C ± 0.03°C per minute;
then holding the temperature at -10°C ± 5°C for 120 minutes ± 12 minutes; then
decreasing the temperature at a rate of 0.3°C ± 0.03°C per minute until reaching a
temperature of -50°C ± 5°C; and then holding the temperature at -50°C ± 5°C for
180 minutes ± 18 minutes; (iii) drying the combination by decreasing the pressure
to 50 milliTorrs (mTorr) and holding for 30 minutes; then increasing the
temperature to -30°C ± 5°C at a rate of 0.2°C ± 0.02°C per minute; and then
holding the temperature at -30°C ± 5°C for 1,920 minutes ± 192 minutes; (iv)
drying the combination by increasing the temperature to 30°C ± 5°C at a rate of
0.2°C ± 0.02°C per minute; and then increasing the pressure to 200 mTorr and
holding the temperature at 30°C ± 5°C for 720 minutes ± 72 minutes; (v)
decreasing the temperature to 5°C ± 5°C at a rate of 0.5°C ± 0.05°C per minute. In
some embodiments, lyophilization takes place in a vial. In some embodiments, the
vial is stoppered after lyophilization. In certain embodiments, the vial is backfilled
with nitrogen gas prior to stoppering the vial. In certain embodiments, the process
further comprises the step of: (c) reconstituting the lyophilized combination of step
(b) in an aqueous medium. In certain embodiments, the osmolality of the
reconstituted combination of step (c) is between 250 mOsM ± 25mOsM and 300
mOsM ± 30 mOsM. In certain embodiments, an immunogenic composition of the
invention is manufactured according to any process of making an immunogenic
composition of the invention.
[0037] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) an isolated Staphylococcus aureus clumping factor A
(ClfA) polypeptide comprising a fibrinogen domain, (b) a buffer having a pKa of
6.0 ± 0.6, and (c) a bulking agent. In certain embodiments, the ClfA polypeptide
remains substantially undegraded for at least 1 month at 37°C. In one
embodiment, the invention provides a lyophilized immunogenic composition
comprising water at less than 3 percent weight of the total weight of the
immunogenic composition (% w/w), wherein the ClfA polypeptide is at 0.52
percent ± 0.46 % w/w, and the buffer is at 0.28% ± 0.065% w/w. In certain
embodiments, the bulking agent is sucrose at 97% ± 2.0% w/w.
[0038] In one embodiment, the invention provides a lyophilized immunogenic
composition comprising: (a) a ClfA polypeptide at 0.52%> ± 0.46%> w/w, (b) a
succinate buffer at 0.28% ± 0.065% w/w, (c) sucrose at 97% ± 0.57% w/w, (d)
polysorbate 80 at 0.20% ± 0.042% w/w, and (e) water at 2.5% ± 0.5% w/w. In
certain embodiments, the ClfA polypeptide remains substantially undegraded for at
least 1 month at 37°C.
[0039] In one embodiment, the invention provides a liquid immunogenic ClfA
composition manufactured by reconstituting a lyophilized immunogenic
composition of the invention in an aqueous diluent, said reconstituted composition
having a final pH of 6.0 ± 0.3.
[0040] In one embodiment, the invention provides a liquid immunogenic ClfA
composition manufactured by reconstituting a lyophilized immunogenic
composition of the invention in an aqueous diluent, said reconstituted composition
having a final pH of 6.5 ± 0.3.
[0041] In one embodiment, the invention provides a liquid immunogenic
composition comprising: (a) the ClfA polypeptide at a concentration of between
20mg/ml ± 2mg/ml and 600 mg/ml ± m (b) the histidine buffer at 1OmM ±
5mM; (c) polysorbate 80 at 0.1% ± 0.05%> weight to volume (w/v); and (d) sucrose
at a concentration of 9% ± 4.5% w/v. In another embodiment, the polysorbate 80
is at a concentration of 0.01% ± 0.005% w/v and the sucrose is at a concentration
of 4.5% ± 1.5% w/v.
[0042] In one embodiment, the invention provides a process of making an
immunogenic composition comprising the steps of: (a) combining in an aqueous
solution, (i) a recombinant clumping factor A (rClfA) polypeptide, (ii) a buffer
having a pKa of 6.0 ± 0.6, and (iii) a bulking agent, and (b) lyophilizing the
combination of step (a) to form a cake comprising less than 3% water by weight.
In certain embodiments, the process further comprises combining (vi) a surfactant
at step (a). In certain embodiments, the process further comprises a step of filter
sterilizing the combination of step (a) prior to lyophilization of step (b). In certain
embodiments, the aqueous solution comprises lOmM ± ImM histidine, pH 6.0 ±
0.5, sucrose at 9% ± 1% w/v and 80 at 0.1% ± 0.02% w/v. In other embodiments,
the polysorbate 80 is at a concentration of 0.01% ± 0.00 1% w/v and the sucrose is
at a concentration of 4.5% ± 0.45% w/v. In certain embodiments, the lyophilizing
step (b) comprises the steps of: (i) freezing the sterilized combination of step (a) at
a rate of 0.3°C ± 0.03°C per minute until reaching a temperature of -50°C ± 5°C at
a pressure of 400 millibars ± 40 millibars; and then holding the combination at -
50°C ± 5°C for 60 minutes ± 6 minutes; (ii) annealing the combination by
increasing the temperature to -10°C ± 5°C at a rate of 0.3°C ± 0.03°C per minute;
then holding the temperature at -10°C ± 5°C for 120 minutes ± 12 minutes; then
decreasing the temperature at a rate of 0.3°C ± 0.03°C per minute until reaching a
temperature of -50°C ± 5°C; and then holding the temperature at -50°C ± 5°C for
180 minutes ± 18 minutes; (iii) drying the combination by decreasing the pressure
to 50 mTorrs (mTorr) and increasing the temperature to -25°C ± 5°C at a rate of
0.2°C ± 0.02°C per minute; and then holding the temperature at 25°C ± 5°C for
1,320 minutes ± 132 minutes; (iv) drying the combination by increasing the
temperature to 30°C ± 5°C at a rate of 0.2°C ± 0.02°C per minute; and then
increasing the pressure to 200 mTorr and holding the temperature at 30°C ± 5°C
for 720 minutes ± 72 minutes; (v) decreasing the temperature to 5°C ± 5°C at a rate
of 0.5°C ± 0.05°C per minute. In some embodiments, lyophilization takes place in
a vial. In some embodiments, the vial is stoppered after lyophilization. In certain
embodiments, the vial is backfilled with nitrogen gas prior to stoppering the vial.
In certain embodiments, the process further comprises the step of: (c)
reconstituting the lyophilized combination of step (b) in an aqueous medium. In
certain embodiments, the osmolality of the reconstituted combination of step (c)
reconstituting the dried combination of step (b) in an aqueous medium, wherein the
osmolality of the reconstituted combination is 300 mOsm ± 30 mOsm. In certain
embodiments, the aqueous medium comprises at least two components selected
from the group consisting of: (a) a Staphylococcus aureus clumping factor B
(ClfB) polypeptide, (b) a Capsular Polysaccharide Type 5 (CP5)-protein conjugate,
(c) a Capsular Polysaccharide Type 8 (CP8)-protein conjugate, and (d) a
Staphylococcus aureus MntC polypeptide. In certain embodiments, the aqueous
solution comprises a CP5-protein conjugate and a CP8-protein conjugate. In
certain embodiments, the aqueous solution comprises a CP-5 protein conjugate, a
CP8-protein conjugate, and an MntC polypeptide. In certain embodiments, an
immunogenic composition of the invention is manufactured according to any
process of making an immunogenic composition of the invention.
[0043] In certain embodiments, the ClfA polypeptide comprises an N domain. In
certain embodiments, the ClfA polypeptide comprises an Nl, N2 or N3 domain. In
certain embodiments, the ClfA polypeptide comprises an Nl, N2 and N3 domain.
In certain embodiments, the ClfA polypeptide comprises a fibrinogen binding
domain. In certain embodiments, the fibrinogen binding domain has been altered
so as to bind to fibrinogen at a reduced level compared to the binding observed to
fibrinogen with the native fibrinogen binding domain of ClfA. In certain
embodiments, the fibrinogen binding domain displays reduced binding to
fibrinogen through having an amino acid substitution at one or more of Tyr 338,
Tyr 256, Pro 336, Lys 389, Ala 254 and e 387. In certain embodiments, the
amino acid substitution at one or more of Tyr 338, Tyr 256, Pro 336, Lys 389, Ala
254 and e 387 is to Ala or Ser. In certain embodiments, the Tyr 338 is substituted
to Ala.
[0044] In certain embodiments, the CP5-protein conjugate is CP5-CRMi 7, CP5-
pneumolysin, or CP5-streptococcal C5a peptidase (SCP). In certain embodiments,
the CP8-protein conjugate is CP8-CRMi 7, CP8-pneumolysin, or CP8-
streptococcal C5a peptidase (SCP).
[0045] In certain embodiments, the buffer of the lyophilized immunogenic
composition comprises succinate at pH 6.0 ± 0.3. In certain embodiments, the
buffer comprises histidine at pH 6.5 ± 0.5. In certain embodiments, the buffer
comprises histidine at pH 6.0 ± 0.3. In certain embodiments, the bulking agent is
selected from the group consisting of sucrose, trehalose, mannitol, glycine and
sorbitol. In certain embodiments, the bulking agent is sucrose. In certain
embodiments, the bulking agent is sucrose at 96% ± 0.2% w/w. In certain
embodiments, the lyophilized immunogenic composition further comprises a
surfactant. In certain embodiments, the surfactant is selected from the group
consisting of a poloxamer, a polyoxyethylene alkyl ether and a polyoxyethylene
sorbitan fatty acid ester. In certain embodiments, the polyoxyethylene sorbitan
fatty acid ester is polysorbate 80. In certain embodiments, the polysorbate 80 is at
0.20% ± 0.041% w/w for ClfA and tri-antigen formulations. In certain
embodiments, the polysorbate 80 is at 0.1% ± 0.05%> w/w for tetra-antigen
formulations. In other embodiments, the polysorbate 80 is at a concentration of
0.01% ± 0.005% w/v.
[0046] In certain embodiments, the lyophilized immunogenic composition
further comprises an adjuvant. In certain embodiments, the adjuvant is
ISCOMATRIX™.
[0047] In certain embodiments, the aqueous diluent of the liquid composition is
water. In certain embodiments, the aqueous diluent is a low salt solution. In
certain embodiments, the low salt solution comprises 60mM ± lOmM sodium
chloride. In certain embodiments, the aqueous diluent comprises polysorbate 80.
In certain embodiments, the aqueous diluent comprises an adjuvant. In certain
embodiments, the adjuvant is ISCOMATRIX™.
Brief Description of the Drawings
[0048] Figure 1 depicts a schematic diagram of the various forms of recombinant
ClfA polypeptide and discloses SEQ ID NOs: 125 and 127-129.
[0049] Figure 2 depicts the change in concentration of various lots of lyophilized
ClfA protein compared to their liquid counterparts, expressed as ln[C], over time
under various conditions. Panel A: lot nos. L3605 1-37-1 and L3605 1-44. Panel B:
lot no. L36051-72. Panel C: lot no. L36051-81-1. Panel D: lot no. 36051-81-2.
[0050] Figure 3 depicts size exclusion chromatograms of lyophilized DS
L40 184-61 at t = 0 compared to cakes stored at 2-8°C, 25°C, and 37°C for three
months.
[0051] Figure 4 depicts size exclusion chromatograms comparing lyophilized
formulation to pre-lyophilized liquid ClfA lot L3605 1-81-1 .
[0052] Figure 5 depicts histograms of pH (panel A), percent moisture (panel B)
and optical density (panel C), which is a measure of aggregation, of lyophilized
ClfA at various times and temperature conditions.
[0053] Figure 6 depicts histograms of percent dimer (panel A), percent monomer
(panel B), and percent degradant (cleavage) (panel C) of ClfA, as determined by
SE-HPLC after 24 hours of agitation at room temperature. X-axis depicts
formulations containing different concentrations of polysorbate 80 (0.000, 0.0100,
0.005 and 0.100% w/w) and sucrose (3, 4.5 and 6% w/w). The formulation
containing no PS80 exhibited an increase in percent dimer formation after agitation
(panel D).
[0054] Figure 7 depicts histograms of percent degradant (cleavage) (panel A),
percent dimer (panel B), and percent monomer (panel C) of ClfA as determined by
SE-HPLC for various bulking agent formulations over time.
[0055] Figure 8 depicts the purity of ClfA (panel A), strength (antigenicity) of
CP5 (panel B), and strength (antigenicity) of CP8 at various times after
reconstitution of high dose lyophilized cakes.
[0056] Figure 9 depicts the purity of ClfA (panel A), strength (antigenicity) of
CP5 (panel B), and strength (antigenicity) of CP8 at various times after
reconstitution of low dose lyophilized cakes.
[0057] Figure 10 depicts the concentration (panel A) and percent purity (panel B)
of ClfA and the concentration of CP5 and CP8 (panel C) at 0, 4 and 24 hours at
room temperature post-reconstitution.
[0058] Figure 11 depicts the concentration (panel A) and percent purity (panel B)
of ClfA and the concentration of CP5 and CP8 (panel C) after 1, 2 and 3 freezethaw
cycles.
[0059] Figure 12 depicts circular dichroism (CD) scans of multiple lots of rClfA
at pH 6.0 (panel A) and 7.0 (panel B)
[0060] Figure 13 depicts CD melts of multiple lots of rClfA as a function of pH.
[0061] Figure 14 depicts intrinsic tryptophan fluorescence (panel A) and ANS
fluorescence (panel B) melts as a function of pH for rClfAm and rmClfA.
[0062] Figure 15 depicts differential scanning calorimetry (DSC) melts as a
function of pH for rClfAm and rmClfA.
[0063] Figure 16 depicts OD350 melts of rmClfA as a function of pH.
[0064] Figure 1 depicts size exclusion-HPLC as a function of pH for rmClfA
for six weeks at 25°C (panel A) and pH and OD350 stability for liquid rmClfA
formulations (panel B).
[0065] Figure 18 depicts intrinsic tryptophan fluorescence melts for MntC (panel
A) and tracking of Tml (panel B).
[0066] Figure 19 depicts tracking of Tml from DSC melts as a function of pH for
MntC (panel A) and thermograms of two main thermal events (panels B and C).
[0067] Figure 20 depicts OD350 melts of MntC as a function of pH.
[0068] Figure 2 1 depicts chromatograms of rmClfA (panel A) and MntC (panel
B) for monitoring stability.
[0069] Figure 22 depicts stability results of rmClfA with RP-HPLC at 5°C (panel
A) and 25°C (panel B) and of MntC with IEX-HPLC at 5°C (panel C) and 25°C
(panel D).
[0070] Figure 23 depicts the stability results of CP5-CRMi 7 at 5°C and 25°C
(panels A and C, respectively) and CP8-CRMi 7 at 5°C and 25°C (panels B and D,
respectively) at different pHs using nephelometry.
[0071] Figure 24 depicts osmolality (panel A) and OD350 (panel B)
measurements of post-rocked samples with varying bulking agents.
[0072] Figure 25 depicts stability testing as a function of bulking agent using RPHPLC
data for high dose (panel A) and low dose (panel B) lyophilized
formulations and IEX-HPLC data for MntC quality (panel C).
[0073] Figure 26 depicts post-agitation data of all four antigens: antigen
concentrations (rmClfA and MntC in panel A; CP5-CRMi 7 and CP8-CRMi 7 in
panel B), purity of rmClfA and MntC by RP-HPLC (panel B), purity of MntC by
IEX-HPLC (panel C), pH (panel D) and OD350 (panel E).
[0074] Figure 27 depicts pre-lyophilization and post- lyophilization data of all
four antigens: RP-HPLC data for rmClfA and MntC (panel A), nephelometry
results for CP5-CRMi 7 (panel B) and CP8-CRMi 7 (panel C), CEX-HPLC data
for MntC using 4.5% sucrose (panel D) and 4% mannitol/l%> sucrose (panel E),
DSC data (panel F) and percent potency of rmClfA (panel G) and conjugates
(panel H).
[0075] Figure 28 depicts the osmolality of high and low dose formulations.
[0076] Figure 29 depicts RP-HPLC purity data for MntC (panel A) and rmClfA
(panel B), IEX-HPLC purity data for MntC (panel C), and post-reconstitution
kinetics of MntC (panel D) and rmClfA degradation (panel E).
[0077] Figure 30 depicts post-reconstitution antigen concentration (panels A-D)
and pH stability (panel E).
[0078] Figure 31 depicts post-lyophilization process recoveries of antigen
concentration (panels A and B) and purity (panels C and D).
[0079] Figure 32 depicts post-reconstitution rmClfA (panel A) and MntC purity
(panel B), rmClfA (panel C) and MntC concentration (panel D), CP5-CRMi 7
(panel E) and CP8-CRM19-7 (panel F) concentration, and pH stability (panel G).
[0080] Figure 33 depicts post-freeze thaw rmClfA and MntC purity by RP-HPLC
(panel A), MntC purity by IEX-HPLC (panel B), concentrations of antigens
(panels C and D) and pH stability (panel E).
[0081] Figure 34 depicts post-agitation rmClfA and MntC purity by RP-HPLC
(panel A), MntC purity by IEX-HPLC (panel B), concentrations of antigens
(panels C and D), pH (panel E) and OD350 (panel F).
[0082] Figure 35 depicts the degradation of MntC reconstituted with
ISCOMATRIX™ at 2-8°C (panel A) and 25°C (panel B) analyzed by CEX-HPLC
after 24 hours, the purity of rmClfA reconstituted with ISCOMATRIX™ analyzed
by CEX-HPLC (panels C and D), the purity of MntC reconstituted with
ISCOMATRIX™ at 2-8°C (panel E) and 25°C (panel F) analyzed by RP-HPLC,
the CP5-CRMi97 and CP8-CRMi 7 conjugate concentrations with
ISCOMATRIX™ at 2-8°C (panel G) and 25°C (panel H), and rmClfA and MntC
concentrations with ISCOMATRIX™ at 2-8°C (panel I) and 25°C (panel J).
[0083] Figure 36 depicts ISCOMATRIX™ concentrations over 24 hours at 2-
8°C (panel A) and 25°C (panel B), particle size of ISCOMATRIX™ (panel C),
and pH stability (panel D) of reconstituted formulations.
[0084] Figure 37 depicts the purity (panel A) and deamidation of MntC
reconstituted with ISCOMATRIX™ analyzed by RP-HPLC after 4 hours and
antigen concentrations with ISCOMATRIX™ (panels C and D).
[0085] Figure 38 depicts the particle size of ISCOMATRIX™ (panel A) and pH
stability (panel B) of reconstituted formulations over 4 hours.
[0086] Figures 39A-J depict the alignment of ClfA between various strains of
S. aureus (SEQ ID NOs: 62, 64, 68, 84, 70, 104, 66, 78, 86, 88, 90, 72, 74, 76, 80,
94, 82, 92, 96, 98, 100, 102, 106, and 108, respectively, in order of appearance).
[0087] Figures 40A-I depict the alignment of ClfB between various strains of
S. aureus (SEQ ID NOs: 26, 28, 32, 18, 54, 34, 36, 30, 16, 20, 22, 24, 38, 40, 42,
44, 46, 48, 50, 52, 56, 58, and 60, respectively, in order of appearance).
[0088] Figure 41A-C depict the alignment of MntC between various strains of
S. aureus (SEQ ID NOs: 2, 8, 10, 4, 6, 14 and 12, respectively, in order of
appearance).
[0089] Figure 42 depicts a chart of ClfB sequences from different strains of
S. aureus.
[0090] Figure 43 depicts a chart of MntC sequences from different strains of
S. aureus.
Detailed Description of the Invention
[0091] Before the present methods and treatment methodology are described, it is
to be understood that this invention is not limited to particular methods, and
experimental conditions described, as such methods and conditions may vary. It is
also to be understood that the terminology used herein is for purposes of describing
particular embodiments only, and is not intended to be limiting.
[0092] Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the invention, the
preferred methods and materials are now described. All publications mentioned
herein are incorporated by reference in their entirety.
[0093] The terms used herein have the meanings recognized and known to those
of skill in the art, however, for convenience and completeness, particular terms and
their meanings are set forth below.
[0094] As used in this specification and the appended claims, the singular forms
"a", "an", and "the" include plural references unless the context clearly dictates
otherwise. Thus, for example, references to "the method" includes one or more
methods, and/or steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this disclosure and so
forth.
[0095] The term "about" or "approximately" means within a statistically
meaningful range of a value. Such a range can be within an order of magnitude,
typically within 20%, more typically still within 10%, and even more typically
within 5% of a given value or range. The allowable variation encompassed by the
term "about" or "approximately" depends on the particular system under study, and
can be readily appreciated by one of ordinary skill in the art. Whenever a range is
recited within this application, every whole number integer within the range is also
contemplated as an embodiment of the invention.
[0096] An "antibody" is an immunoglobulin molecule capable of specific
binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.,
through at least one antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, unless otherwise indicated by context,
the term is intended to encompass not only intact polyclonal or monoclonal
antibodies, but also engineered antibodies (e.g., chimeric, humanized and/or
derivatized to alter effector functions, stability and other biological activities) and
fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain
antibodies, including shark and camelid antibodies), and fusion proteins
comprising an antibody portion, multivalent antibodies, multispecific antibodies
(e.g., bispecific antibodies so long as they exhibit the desired biological activity)
and antibody fragments as described herein, and any other modified configuration
of the immunoglobulin molecule that comprises an antigen recognition site. An
antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class
thereof), and the antibody need not be of any particular class. Depending on the
antibody amino acid sequence of the constant domain of its heavy chains,
immunoglobulins can be assigned to different classes. There are five major classes
of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be
further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and
IgA2 in humans. The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and
mu, respectively. The subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0097] "Antibody fragments" comprise only a portion of an intact antibody,
wherein the portion preferably retains at least one, preferably most or all, of the
functions normally associated with that portion when present in an intact antibody.
[0098] The term "antigen" generally refers to a biological molecule, usually a
protein, peptide, polysaccharide, lipid or conjugate which contains at least one
epitope to which a cognate antibody can selectively bind; or in some instances to
an immunogenic substance that can stimulate the production of antibodies or T-cell
responses, or both, in an animal, including compositions that are injected or
absorbed into an animal. The immune response may be generated to the whole
molecule, or to one or more various portions of the molecule (e.g., an epitope or
hapten). The term may be used to refer to an individual molecule or to a
homogeneous or heterogeneous population of antigenic molecules. An antigen is
recognized by antibodies, T-cell receptors or other elements of specific humoral
and/or cellular immunity. The term "antigen" includes all related antigenic
epitopes. Epitopes of a given antigen can be identified using any number of
epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping
Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996)
Humana Press, Totowa, N. J . For example, linear epitopes may be determined by
e.g., concurrently synthesizing large numbers of peptides on solid supports, the
peptides corresponding to portions of the protein molecule, and reacting the
peptides with antibodies while the peptides are still attached to the supports. Such
techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871;
Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al.
(1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their
entireties. Similarly, conformational epitopes may be identified by determining
spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-
dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols,
supra. Furthermore, for purposes of the present invention, an "antigen" may also
be used to refer to a protein that includes modifications, such as deletions,
additions and substitutions (generally conservative in nature, but they may be nonconservative),
to the native sequence, so long as the protein maintains the ability to
elicit an immunological response. These modifications may be deliberate, as
through site-directed mutagenesis, or through particular synthetic procedures, or
through a genetic engineering approach, or may be accidental, such as through
mutations of hosts, which produce the antigens. Furthermore, the antigen can be
derived, obtained, or isolated from a microbe, e.g. a bacterium, or can be a whole
organism. Similarly, an oligonucleotide or polynucleotide, which expresses an
antigen, such as in nucleic acid immunization applications, is also included in the
definition. Synthetic antigens are also included, for example, polyepitopes,
flanking epitopes, and other recombinant or synthetically derived antigens
(Bergmann et al. (1993) Eur. J. Immunol. 23:2777 2781; Bergmann et al. (1996) J.
Immunol. 157:3242 3249; Suhrbier, A. (1997) Immunol and Cell Biol. 75:402 408;
Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28
- Jul. 3, 1998).
[0099] The term "adjuvant" refers to a compound or mixture that enhances the
immune response to an antigen as further described and exemplified herein.
[0100] "Bacteremia" is a transient presence of bacteria in the blood. A
bacteremia can progress to septicemia, or sepsis, which would be considered an
infection and is the persistent presence of bacteria in the blood with associated
clinical signs/symptoms. Not all bacteria are capable of surviving in the blood.
Those that do have special genetic traits that provide for that ability. Also, the host
factors play an important role as well.
[0101] "Capsular polysaccharide" or "capsule polysaccharide" refers to the
polysaccharide capsule that is external to the cell wall of most isolates of
Staphylococci. For example, S. aureus includes a cell wall component composed
of a peptidoglycan complex, which enables the organism to survive under
unfavorable osmotic conditions and also includes a unique teichoic acid linked to
the peptidoglycan. External to the cell wall a thin polysaccharide capsule coats
most isolates of S. aureus. This serologically distinct capsule can be used to
serotype various isolates of S. aureus. Many of the clinically significant isolates
have been shown to include two capsular types: serotype 5 (CP5) and serotype 8
(CP8).
[0102] As used herein, "conjugates" comprise a capsule polysaccharide usually
of a desired range of molecular weight and a carrier protein, wherein the capsule
polysaccharide is conjugated to the carrier protein. Conjugates may or may not
contain some amount of free capsule polysaccharide. As used herein, "free capsule
polysaccharide" refers to capsule polysaccharide that is non-covalently associated
with (i.e., non-covalently bound to, adsorbed to or entrapped in or with) the
conjugated capsular polysaccharide-carrier protein. The terms "free capsule
polysaccharide," "free polysaccharide" and "free saccharide" may be used
interchangeably and are intended to convey the same meaning. Regardless of the
nature of the carrier molecule, it can be conjugated to the capsular polysaccharide
either directly or through a linker. As used herein, "to conjugate", "conjugated"
and "conjugating" refers to a process whereby a bacterial capsular polysaccharide
is covalently attached to the carrier molecule. Conjugation enhances the
immunogenicity of the bacterial capsular polysaccharide. The conjugation can be
performed according to the methods described below or by processes known in the
art.
[0103] As described above, the present invention relates to conjugates
comprising S. aureus serotype 5 capsular polysaccharides (CP5) conjugated to
carrier proteins and conjugates comprising S. aureus serotype 8 capsular
polysaccharides (CP8) conjugated to carrier proteins. One embodiment of the
invention provides conjugates comprising a S. aureus serotype 5 capsular
polysaccharide conjugated to a carrier protein and a S. aureus serotype 8 capsular
polysaccharide conjugated to a carrier protein wherein: the type 5 capsular
polysaccharide has a molecular weight of between 50 kDa and 800 kDa; the type 8
capsular polysaccharide has a molecular weight of between 50 and 700 kDa; the
immunogenic conjugates have molecular weights of between about 1000 kDa and
about 5000 kDa; and the conjugates comprise less than about 30% free
polysaccharide relative to total polysaccharide. In one embodiment, the conjugates
comprise less than about 25%, about 20%>, about 15%, about 10%, or about 5%
free polysaccharide relative to total polysaccharide. In one embodiment, the
type 5 or 8 polysaccharide has a molecular weight between 20 about kDa and
about 1000 kDa; between about 50 kDa and about 500 kDa; between about 50 kDa
and about 200 kDa; and between about 75 kDa and about 150 kDa.
[0104] In one embodiment, the conjugate has a molecular weight of between
about 50 kDa and about 5000 kDa. In one embodiment, the conjugate has a
molecular weight of between about 200 kDa and about 5000 kDa. In one
embodiment, the immunogenic conjugate has a molecular weight of between about
400 kDa and about 2500 kDa. In one embodiment, the immunogenic conjugate
has a molecular weight of between about 500 kDa and about 2500 kDa. In one
embodiment, the immunogenic conjugate has a molecular weight of between about
600 kDa and about 2800 kDa. In one embodiment, the immunogenic conjugate
has a molecular weight of between about 700 kDa and about 2700 kDa. In one
embodiment, the immunogenic conjugate has a molecular weight of between about
1000 kDa and about 2000 kDa; between about 1800 kDa and about 2500 kDa;
between about 1100 kDa and about 2200 kDa; between about 1900 kDa and about
2700 kDa; between about 1200 kDa and about 2400 kDa; between about 1700 kDa
and about 2600 kDa; between about 1300 kDa and about 2600 kDa; between about
1600 kDa and about 3000 kDa.
[0105] Accordingly, in one embodiment, the carrier protein within the
immunogenic conjugate of the invention is CRM197, and the CRM197 is covalently
linked to the capsular polysaccharide via a carbamate linkage, an amide linkage, or
both. The number of lysine residues in the carrier protein that become conjugated
to a capsular polysaccharide can be characterized as a range of conjugated lysines.
For example, in a given immunogenic composition, the CRM197 may comprise 5 to
15 lysines out of 39 covalently linked to the capsular polysaccharide. Another way
to express this parameter is that 12% to 40% of CRM1 7 lysines are covalently
linked to the capsular polysaccharide. In some embodiments, the CRM197 portion
of the polysaccharide covalently bound to the CRM197 comprises 5 to 25 lysines
covalently linked to the polysaccharide. In some embodiments, the CRM197
portion of the polysaccharide covalently bound to the CRM197 comprises 5 to 20
lysines covalently linked to the polysaccharide. In some embodiments, the
CRM 1 7 portion of the polysaccharide covalently bound to carrier protein of
comprises 10 to 25 lysines covalently linked to the polysaccharide. In some
embodiments, the CRM 197 portion of the polysaccharide covalently bound to
carrier protein of comprises 8 to 15 lysines covalently linked to the polysaccharide.
For example, in a given immunogenic composition, the CRM 197 may comprise 18
to 22 lysines out of 39 covalently linked to the capsular polysaccharide. Another
way to express this parameter is that 40% to 60% of CRM1 7 lysines are covalently
linked to the capsular polysaccharide. In some embodiments, the CRM 197
comprises 5 to 15 lysines out of 39 covalently linked to CP8. Another way to
express this parameter is that 12% to 40% of CRM 197 lysines are covalently linked
to CP8. In some embodiments, the CRM 197 comprises 18 to 22 lysines out of 39
covalently linked to CP5. Another way to express this parameter is that 40% to
60% of CRM 197 lysines are covalently linked to CP5.
[0106] As discussed above, the number of lysine residues in the carrier protein
conjugated to the capsular polysaccharide can be characterized as a range of
conjugated lysines, which may be expressed as a molar ratio. For example, the
molar ratio of conjugated lysines to CRM 197 in the CP8 immunogenic conjugate
can be between about 18:1 to about 22:1. In one embodiment, the range of molar
ratios of conjugated lysines to CRM 197 in the CP8 immunogenic conjugate can be
between about 15:1 to about 25:1. In some embodiments, the range of molar ratios
of conjugated lysines to CRM 197 in the CP8 immunogenic conjugate can be
between about 14:1 to about 20:1; about 12:1 to about 18:1; about 10:1 to about
16:1; about 8:1 to about 14:1; about 6:1 to about 12:1; about 4:1 to about 10:1;
about 20:1 to about 26:1; about 22:1 to about 28:1; about 24:1 to about 30:1; about
26:1 to about 32:1; about 28:1 to about 34:1; about 30:1 to about 36:1; about 5:1 to
about 10:1; about 5:1 to about 20:1; about 10:1 to about 20:1; or about 10:1 to
about 30:1. Also, the molar ratio of conjugated lysines to CRM 197 in the CP5
immunogenic conjugate can be between about 3:1 and 25:1. In one embodiment,
the range of molar ratio of conjugated lysines to CRM 197 in the CP5 immunogenic
conjugate can be between about 5:1 to about 20: 1. In one embodiment, the range
of molar ratio of conjugated lysines to CRM 197 in the CP5 immunogenic conjugate
can be between about 4 :1 to about 20: 1; about 6 :1 to about 20: 1; about 7 :1 to about
20:1; about 8:1 to about 20:1; about 10:1 to about 20:1; about 11:1 to about 20:1;
about 12:1 to about 20:1; about 13:1 to about 20:1; about 14:1 to about 20:1; about
15:1 to about 20:1; about 16:1 to about 20:1; about 17:1 to about 20:1; about 18:1
to about 20:1; about 5:1 to about 18:1; about 7:1 to about 16:1; or about 9:1 to
about 14:1.
[0107] Another way to express the number of lysine residues in the carrier
protein conjugated to the capsular polysaccharide can be as a range of conjugated
lysines. For example, in a given CP8 immunogenic conjugate, the CRM 197 may
comprise 5 to 15 lysines out of 39 covalently linked to the capsular polysaccharide.
Alternatively, this parameter can be expressed as a percentage. For example, in a
given CP8 immunogenic conjugate, the percentage of conjugated lysines can be
between 10% and 50%. In some embodiments, 20%> to 50%> of lysines can be
covalently linked to CP8. Alternatively still, 30% to 50% of CRM 197 lysines can
be covalently linked to the CP8; 10% to 40% of CRM 197 lysines; 10% to 30% of
CRM 197 lysines; 20% to 40% of CRM 197 lysines; 25% to 40% of CRM 197 lysines;
30% to 40% of CRM 197 lysines; 10% to 30% of CRM 197 lysines; 15% to 30% of
CRM197 lysines; 20% to 30% of CRM197 lysines; 25% to 30% of CRM197 lysines;
10% to 15% of CRM197 lysines; or 10% to 12% of CRM197 lysines are covalently
linked to CP8. Also, in a given CP5 immunogenic conjugate, the CRM 197 may
comprise 18 to 22 lysines out of 39 covalently linked to the capsular
polysaccharide. Alternatively, this parameter can be expressed as a percentage.
For example, in a given CP5 immunogenic conjugate, the percentage of conjugated
lysines can be between 40%> and 60%>. In some embodiments, 40%> to 60%> of
lysines can be covalently linked to CP5. Alternatively still, 30% to 50% of
CRM197 lysines can be covalently linked to CP5; 20% to 40% of CRM197 lysines;
10% to 30% of CRM197 lysines; 50% to 70% of CRM197 lysines; 35% to 65% of
CRM197 lysines; 30% to 60% of CRM197 lysines; 25% to 55% of CRM197 lysines;
20% to 50% of CRM197 lysines; 15% to 45% of CRM197 lysines; 10% to 40% of
CRM 197 lysines; 40% to 70% of CRM 197 lysines; or 45% to 75% of CRM 197 lysines
are covalently linked to CP5 .
[0108] The frequency of attachment of the capsular polysaccharide chain to a
lysine on the carrier molecule is another parameter for characterizing conjugates of
capsule polysaccharides. For example, in one embodiment, at least one covalent
linkage between CRMi 7 and polysaccharide occurs for at least every 5 to 10
saccharide repeat units of the capsular polysaccharide. In another embodiment,
there is at least one covalent linkage between CRM 19-7 and capsular polysaccharide
for every 5 to 10 saccharide repeat units; every 2 to 7 saccharide repeat units, every
3 to 8 saccharide repeat units; every 4 to 9 saccharide repeat units; every 6 to 11
saccharide repeat units; every 7 to 12 saccharide repeat units; every 8 to 13
saccharide repeat units; every 9 to 14 saccharide repeat units; every 10 to 15
saccharide repeat units; every 2 to 6 saccharide repeat units, every 3 to 7
saccharide repeat units; every 4 to 8 saccharide repeat units; every 6 to 10
saccharide repeat units; every 7 to 11 saccharide repeat units; every 8 to 12
saccharide repeat units; every 9 to 13 saccharide repeat units; every 10 to 14
saccharide repeat units; every 10 to 20 saccharide repeat units; every 5 to 10
saccharide repeat units of the capsular polysaccharide. In another embodiment, at
least one linkage between CRMi 7 and capsular polysaccharide occurs for every 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 saccharide repeat
units of the capsular polysaccharide.
[0109] The chemical activation of the polysaccharides and subsequent
conjugation to the carrier protein may be achieved by conventional means. See,
for example, U.S. Pat. Nos. 4,673,574 and 4,902,506. Other activation and
conjugation methods may alternatively be used.
[0110] "Carrier protein" or "protein carrier" as used herein, refers to any protein
molecule that may be conjugated to an antigen (such as the capsular
polysaccharides) against which an immune response is desired. Conjugation of an
antigen such as a polysaccharide to a carrier protein can render the antigen
immunogenic. Carrier proteins are preferably proteins that are non-toxic and nonreactogenic
and obtainable in sufficient amount and purity. Examples of carrier
proteins are toxins, toxoids or any mutant cross-reactive material (CRM 19-7) of the
toxin from tetanus, diphtheria, pertussis, Pseudomonas species, E. coli,
Staphylococcus species, and Streptococcus species. Carrier proteins should be
amenable to standard conjugation procedures. In a particular embodiment of the
present invention, CRM 1 - is used as the carrier protein.
[0111] CRMi 7 (Wyeth/Pfizer, Sanford, NC) is a non-toxic variant (i.e., toxoid)
of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7
( b ΐ 97 ) grown in casamino acids and yeast extract-based medium. CRM 19-7 is
purified through ultra-filtration, ammonium sulfate precipitation, and ion-exchange
chromatography. A culture of Corynebacterium diphtheriae strain C7 (197), which
produces CRMi 7 protein, has been deposited with the American Type Culture
Collection, Rockville, Maryland and has been assigned accession number ATCC
53281. Other diphtheria toxoids are also suitable for use as carrier proteins.
[0112] Other suitable carrier proteins include inactivated bacterial toxins such as
tetanus toxoid, pertussis toxoid, cholera toxoid {e.g., as described in International
Patent Application WO2004/083251), E. coli LT, E. coli ST, and exotoxin A from
Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer
membrane protein complex c (OMPC), porins, transferrin binding proteins,
pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin
protein (PsaA), C. difficile enterotoxin (toxin A) and cytotoxin (toxin B) or
Haemophilus influenzae protein D, can also be used. Other proteins, such as
streptococcal C5a peptidase (SCP), ovalbumin, keyhole limpet hemocyanin
(KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin
(PPD) can also be used as carrier proteins.
[0113] 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 a variety of techniques. These
techniques include, e.g., concentration/diafiltration operations,
precipitation/elution, column chromatography, and depth filtration. See examples
below.
[0114] After the individual conjugates are purified, they may be combined to
formulate an immunogenic composition of the present invention, which may be
used, for example, in a vaccine. Formulation of the immunogenic composition of
the present invention can be accomplished using art-recognized methods.
[0115] It is noted that in this disclosure, terms such as "comprises", "comprised",
"comprising", "contains", "containing" and the like can have the meaning
attributed to them in U.S. patent law; e.g., they can mean "includes", "included",
"including" and the like. Such terms refer to the inclusion of a particular
ingredients or set of ingredients without excluding any other ingredients. Terms
such as "consisting essentially of and "consists essentially of have the meaning
attributed to them in U.S. patent law, e.g., they allow for the inclusion of additional
ingredients or steps that do not detract from the novel or basic characteristics of the
invention, i.e., they exclude additional unrecited ingredients or steps that detract
from novel or basic characteristics of the invention, and they exclude ingredients or
steps of the prior art, such as documents in the art that are cited herein or are
incorporated by reference herein, especially as it is a goal of this document to
define embodiments that are patentable, e.g., novel, non-obvious, inventive, over
the prior art, e.g., over documents cited herein or incorporated by reference herein.
And, the terms "consists of and "consisting of have the meaning ascribed to them
in U.S. patent law; namely, that these terms are close-ended. Accordingly, these
terms refer to the inclusion of a particular ingredient or set of ingredients and the
exclusion of all other ingredients.
[0116] A "conservative amino acid substitution" refers to the substitution of one
or more of the amino acid residues of a protein with other amino acid residues
having similar physical and/or chemical properties. Substitutes for an amino acid
within the sequence may be selected from other members of the class to which the
amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. Amino acids containing aromatic ring structures are phenylalanine,
tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged
(basic) amino acids include arginine, lysine and histidine. The negatively charged
(acidic) amino acids include aspartic acid and glutamic acid. Such alterations will
not be expected to affect apparent molecular weight as determined by
polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred
substitutions are: Lys for Arg and vice versa such that a positive charge may be
maintained; Glu for Asp and vice versa such that a negative charge may be
maintained; Ser for Thr such that a free —OH can be maintained; and Gin for Asn
such that a free NH2 can be maintained.
[0117] "Fragment" refers to proteins where only specific domains of a larger
protein are included. For example, ClfA and ClfB proteins contain as many as 8
domains each if the signal sequences are included. A polypeptide corresponding to
the N1N2N3, N2N3, N1N2, Nl, N2, or N3 domains are each considered to be
fragments of fA or ClfB. "Fragment" also refers to either a protein or
polypeptide comprising an amino acid sequence of at least 4 amino acid residues
(preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least
20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid
residues, at least 50 amino acid residues, at least 60 amino residues, at least 70
amino acid residues, at least 80 amino acid residues, at least 90 amino acid
residues, at least 100 amino acid residues, at least 125 amino acid residues, or at
least 150 amino acid residues) of the amino acid sequence of a parent protein or
polypeptide or a nucleic acid comprising a nucleotide sequence of at least 10 base
pairs (preferably at least 20 base pairs, at least 30 base pairs, at least 40 base pairs,
at least 50 base pairs, at least 50 base pairs, at least 100 base pairs, at least 200 base
pairs) of the nucleotide sequence of the parent nucleic acid.
[0118] "Functional activity" of an antibody or "functional antibody" as used
herein refers to an antibody that, at a minimum, can bind specifically to an antigen.
Additional functions are known in the art and may include additional components
of the immune system that effect clearance or killing of the pathogen such as
through opsonization, ADCC or complement-mediated cytotoxicity. After antigen
binding, any subsequent antibody functions can be mediated through the Fc region
of the antibody. The antibody opsonophagocytic assay (OPA) is an in vitro assay
designed to measure in vitro Ig complement-assisted killing of bacteria with
effector cells (white blood cells), thus mimicking a biological process. Antibody
binding may also directly inhibit the biological function of the antigen it binds,
e.g., antibodies that bind ClfA can neutralize its enzymatic function. In some
embodiments, a "functional antibody" refers to an antibody that is functional as
measured by the killing of bacteria in an animal efficacy model or an
opsonophagocytic killing assay that demonstrates that the antibodies kill the
bacteria.
[0119] The molecular weight of the S. aureus capsule polysaccharides is a
consideration for use in immunogenic compositions. For example, high molecular
weight capsule polysaccharides may be able to induce certain antibody immune
responses due to a higher valency of the epitopes present on the antigenic surface.
The isolation of "high molecular weight capsular polysaccharides" is contemplated
for use in the compositions and methods of the present invention. For example, in
one embodiment of the invention, the isolation of type 5 high molecular weight
polysaccharides ranging in size from about 50 to about 800 kDa in molecular
weight is contemplated. In one embodiment of the invention, the isolation of type
5 high molecular weight polysaccharides ranging in size from about 20 to about
1000 kDa in molecular weight is contemplated. In one embodiment of the
invention, the isolation and purification of type 5 high molecular weight capsular
polysaccharides ranging in size from about 50 to about 300 kDa in molecular
weight is contemplated. In one embodiment, the isolation and purification of type
5 high molecular weight capsular polysaccharide ranging from 70 kDa to 300 kDa
in molecular weight is contemplated. In one embodiment, the isolation and
purification of type 5 high molecular weight capsular polysaccharide ranging from
90 kDa to 250 kDa in molecular weight is contemplated. In one embodiment, the
isolation and purification of type 5 high molecular weight capsular polysaccharide
ranging from 90 kDa to 150 kDa in molecular weight is contemplated. In one
embodiment, the isolation and purification of type 5 high molecular weight
capsular polysaccharide ranging from 90 kDa to 140 kDa in molecular weight is
contemplated. In one embodiment, the isolation and purification of type 5 high
molecular weight capsular polysaccharide ranging from 80 kDa to 120 kDa in
molecular weight is contemplated. Other ranges of high molecular weight serotype
5 capsular polysaccharide that can be isolated and purified by the methods of this
invention include size ranges of about 70 kDa to about 100 kDa in molecular
weight; 70 kDa to 110 kDa in molecular weight; 70 kDa to 120 kDa in molecular
weight; 70 kDa to 130 kDa in molecular weight; 70 kDa to 140 kDa in molecular
weight; 70 kDa to 150 kDa in molecular weight; 70 kDa to 160 kDa in molecular
weight; 80 kDa to 110 kDa in molecular weight; 80 kDa to 120 kDa in molecular
weight; 80 kDa to 130 kDa in molecular weight; 80 kDa to 140 kDa in molecular
weight; 80 kDa to 150 kDa in molecular weight; 80 kDa to 160 kDa in molecular
weight; 90 kDa to 110 kDa in molecular weight; 90 kDa to 120 kDa in molecular
weight; 90 kDa to 130 kDa in molecular weight; 90 kDa to 140 kDa in molecular
weight; 90 kDa to 150 kDa in molecular weight; 90 kDa to 160 kDa in molecular
weight; 100 kDa to 120 kDa in molecular weight; 100 kDa to 130 kDa in
molecular weight; 100 kDa to 140 kDa in molecular weight; 100 kDa to 150 kDa
in molecular weight; 100 kDa to 160 kDa in molecular weight; and similar desired
molecular weight ranges.
[0120] As discussed above, the molecular weight of the S. aureus capsule
polysaccharides is a consideration for use in immunogenic compositions. For
example, high molecular weight capsule polysaccharides may be able to induce
certain antibody immune responses due to a higher valency of the epitopes present
on the antigenic surface. In one embodiment of the invention, the isolation and
purification of type 8 high molecular weight capsular polysaccharides ranging
from about 20 kDa to about 1000 kDa in molecular weight is contemplated. In one
embodiment of the invention, the isolation and purification of type 8 high
molecular weight capsular polysaccharides ranging from about 50 kDa to about
700 kDa in molecular weight is contemplated. In one embodiment of the
invention, the isolation and purification of type 8 high molecular weight capsular
polysaccharides ranging from 50 kDa to 300 kDa in molecular weight is
contemplated. In one embodiment, the isolation and purification of type 8 high
molecular weight capsular polysaccharide ranging from 70 kDa to 300 kDa in
molecular weight is contemplated. In one embodiment, the isolation and
purification of type 8 high molecular weight capsular polysaccharides ranging
from 90 kDa to 250 kDa in molecular weight is contemplated. In one embodiment,
the isolation and purification of type 8 high molecular weight capsular
polysaccharides ranging from 90 kDa to 150 kDa in molecular weight is
contemplated. In one embodiment, the isolation and purification of type 8 high
molecular weight capsular polysaccharides ranging from 90 kDa to 120 kDa in
molecular weight is contemplated. In one embodiment, the isolation and
purification of type 8 high molecular weight capsular polysaccharides ranging
from 80 kDa to 120 kDa in molecular weight is contemplated. Other ranges of
high molecular weight serotype 8 capsular polysaccharides that can be isolated and
purified by the methods of this invention include size ranges of about 70 kDa to
about 100 kDa in molecular weight; 70 kDa to 110 kDa in molecular weight;
70 kDa to 120 kDa in molecular weight; 70 kDa to 130 kDa in molecular weight;
70 kDa to 140 kDa in molecular weight; 70 kDa to 150 kDa in molecular weight;
70 kDa to 160 kDa in molecular weight; 80 kDa to 110 kDa in molecular weight;
80 kDa to 120 kDa in molecular weight; 80 kDa to 130 kDa in molecular weight;
80 kDa to 140 kDa in molecular weight; 80 kDa to 150 kDa in molecular weight;
80 kDa to 160 kDa in molecular weight; 90 kDa to 110 kDa in molecular weight;
90 kDa to 120 kDa in molecular weight; 90 kDa to 130 kDa in molecular weight;
90 kDa to 140 kDa in molecular weight; 90 kDa to 150 kDa in molecular weight;
90 kDa to 160 kDa in molecular weight; 100 kDa to 120 kDa in molecular weight;
100 kDa to 130 kDa in molecular weight; 100 kDa to 140 kDa in molecular
weight; 100 kDa to 150 kDa in molecular weight; 100 kDa to 160 kDa in
molecular weight; and similar desired molecular weight ranges.
[0121] An "immune response" to an immunogenic composition is the
development in a subject of a humoral and/or a cell-mediated immune response to
molecules present in the composition of interest (for example, an antigen, such as a
protein or polysaccharide). For purposes of the present invention, a "humoral
immune response" is an antibody-mediated immune response and involves the
generation of antibodies with affinity for the antigens present in the immunogenic
compositions of the invention, while a "cell-mediated immune response" is one
mediated by T-lymphocytes and/or other white blood cells. A "cell-mediated
immune response" is elicited by the presentation of antigenic epitopes in
association with Class I or Class II molecules of the major histocompatibility
complex (MHC). This activates antigen-specific CD4+ T helper cells or CD8+
cytotoxic T lymphocyte cells ("CTLs"). CTLs have specificity for peptide or lipid
antigens that are presented in association with proteins encoded by the major
histocompatibility complex (MHC) or CD1 and expressed on the surfaces of cells.
CTLs help induce and promote the intracellular destruction of intracellular
microbes, or the lysis of cells infected with such microbes. Another aspect of
cellular immunity involves an antigen-specific response by helper T-cells. Helper
T-cells act to help stimulate the function, and focus the activity of, nonspecific
effector cells against cells displaying peptide antigens in association with classical
or nonclassical MHC molecules on their surface. A "cell-mediated immune
response" also refers to the production of cytokines, chemokines and other such
molecules produced by activated T-cells and/or other white blood cells, including
those derived from CD4+ and CD8+ T-cells. The ability of a particular antigen or
composition to stimulate a cell-mediated immunological response may be
determined by a number of assays, such as by lymphoproliferation (lymphocyte
activation) assays, CTL cytotoxic cell assays, by assaying for T-lymphocytes
specific for the antigen in a sensitized subject, or by measurement of cytokine
production by T cells in response to restimulation with antigen. Such assays are
well known in the art. See, e.g., Erickson et al, J. Immunol. (1993) 151:4189-
4199; Doe et al, Eur. J. Immunol. (1994) 24:2369-2376.
[0122] The term "immunogenic" refers to the ability of an antigen or a vaccine to
elicit an immune response, either humoral or cell-mediated, or both.
[0123] An "immunogenic amount", or an "immunologically effective amount" or
"dose", each of which is used interchangeably herein, generally refers to the
amount of antigen or immunogenic composition sufficient to elicit an immune
response, either a cellular (T cell) or humoral (B cell or antibody) response, or
both, as measured by standard assays known to one skilled in the art.
[0124] The amount of a particular conjugate in a composition is generally
calculated based on total polysaccharide, conjugated and non-conjugated for that
conjugate. For example, a CP5 conjugate with 20% free polysaccharide will have
about 80 meg of conjugated CP5 polysaccharide and about 20 meg of nonconjugated
CP5 polysaccharide in a 100 meg CP5 polysaccharide dose. The
protein contribution to the conjugate is usually not considered when calculating the
dose of a conjugate. The amount of conjugate can vary depending upon the
staphylococcal serotype. Generally, each dose will comprise 0.01 to 100 meg of
polysaccharide, particularly 0.1 to 10 meg, and more particularly 1 to 10 meg. The
"immunogenic amount" of the different polysaccharide components in the
immunogenic composition, may diverge and each may comprise 0.01 meg,
0.1 meg, 0.25 meg, 0.5 meg, 1 meg, 2 meg, 3 meg, 4 meg, 5 meg, 6 meg, 7 meg, 8
meg, 9 meg, 10 meg, 15 meg, 20 meg, 30 meg, 40 mcg,50 meg, 60 meg, 70 meg,
80 meg, 90 meg, or about 100 meg of any particular polysaccharide antigen.
[0125] In another embodiment, the "immunogenic amount" of the protein
components in the immunogenic composition, may range from about 10 meg to
about 300 meg of each protein antigen. In a particular embodiment, the
"immunogenic amount" of the protein components in the immunogenic
composition, may range from about 20 meg to about 200 meg of each protein
antigen. The "immunogenic amount" of the different protein components in the
immunogenic composition may diverge, and each comprise 10 meg, 20 meg, 30
meg, 40 meg, 50 meg, 60 meg, 70 meg, 80 meg, 90 meg, 100 meg, 125 meg,
150 meg, 175 meg or about 200 meg of any particular protein antigen.
[0126] The effectiveness of an antigen as an immunogen can be measured by
measuring the levels of B cell activity by measuring the levels of circulating
antibodies specific for the antigen in serum using immunoassays,
immunoprecipitation assays, functional antibody assays, such as in vitro opsonic
assay and many other assays known in the art. Another measure of effectiveness
of an antigen as an T-cell immunogen can be measured by either by proliferation
assays, by cytolytic assays, such as chromium release assays to measure the ability
of a T cell to lyse its specific target cell. Furthermore, in the present invention, an
"immunogenic amount" may also be defined by measuring the serum levels of
antigen specific antibody induced following administration of the antigen, or, by
measuring the ability of the antibodies so induced to enhance the
opsonophagocytic ability of particular white blood cells, as described herein. The
level of protection of the immune response may be measured by challenging the
immunized host with the antigen that has been injected. For example, if the
antigen to which an immune response is desired is a bacterium, the level of
protection induced by the "immunogenic amount" of the antigen can be measured
by detecting the percent survival or the percent mortality after challenge of the
animals with the bacterial cells. In one embodiment, the amount of protection may
be measured by measuring at least one symptom associated with the bacterial
infection, for example, a fever associated with the infection. The amount of each
of the antigens in the multi-antigen or multi-component vaccine or immunogenic
compositions will vary with respect to each of the other components and can be
determined by methods known to the skilled artisan. Such methods would include,
for example, procedures for measuring immunogenicity and/or in vivo efficacy.
[0127] The term "immunogenic composition" relates to any pharmaceutical
composition containing an antigen, e.g. a microorganism, or a component thereof,
which composition can be used to elicit an immune response in a subject. The
immunogenic compositions of the present invention can be used to treat a human
susceptible to S. aureus infection, by means of administering the immunogenic
compositions via a systemic transdermal or mucosal route. These administrations
can include injection via the intramuscular (i.m.), intraperitoneal (i.p.), intradermal
(i.d.) or subcutaneous routes; application by a patch or other transdermal delivery
device; or via mucosal administration to the oral/alimentary, respiratory or
genitourinary tracts. In one embodiment, intranasal administration is used for the
treatment or prevention of nasopharyngeal carriage of S. aureus, thus attenuating
infection at its earliest stage. In one embodiment, the immunogenic composition
may be used in the manufacture of a vaccine or in the elicitation of a polyclonal or
monoclonal antibodies that could be used to passively protect or treat an animal.
[0128] Optimal amounts of components for a particular immunogenic
composition can be ascertained by standard studies involving observation of
appropriate immune responses in subjects. Following an initial vaccination,
subjects can receive one or several booster immunizations adequately spaced.
[0129] In one embodiment of the present invention, the S. aureus lyophilized
immunogenic composition comprises a recombinant S. aureus clumping factor A
(ClfA) fragment (N1N2N3, or combinations thereof). In a further embodiment of
the present invention, the S. aureus lyophilized immunogenic composition
comprises a recombinant S. aureus clumping factor A (ClfA) fragment, an isolated
capsular polysaccharides type 5 conjugated to CRM 197 and an isolated capsular
polysaccharides type 8 conjugated to CRM 197 .
[0130] The immunogenic compositions of the present invention can further
comprise one or more additional "immunomodulators", which are agents that
perturb or alter the immune system, such that either up-regulation or downregulation
of humoral and/or cell-mediated immunity is observed. In one particular
embodiment, up-regulation of the humoral and/or cell-mediated arms of the
immune system is preferred. Examples of certain immunomodulators include, for
example, an adjuvant or cytokine, or ISCOMATRIX™ (CSL Limited, Parkville,
Australia), described in U.S. Patent No. 5,254,339 among others. Non-limiting
examples of adjuvants that can be used in the vaccine of the present invention
include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels
such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions
such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer
(CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M
(Chiron, Emeryville Calif), AMPHIGEN® adjuvant, saponin, Quil A or other
saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant.
Non-limiting examples of oil-in-water emulsions useful in the vaccine of the
invention include modified SEAM62 and SEAM 1/2 formulations. Modified
SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1%
(v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) polysorbate 80 detergent
(ICI Surfactants), 2.5% (v/v) ethanol, 200 ΐ Quil A, 100 ΐ cholesterol,
and 0.5% (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion
comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v)
polysorbate 80 detergent, 2.5% (v/v) ethanol, 100 m hΐ Quil A, and 50 m hΐ
cholesterol. Other "immunomodulators" that can be included in the compositions
of the invention include, e.g., one or more interleukins, interferons, or other known
cytokines or chemokines. In one embodiment, the adjuvant may be a cyclodextrin
derivative or a polyanionic polymer, such as those described in U.S. patent
numbers 6,165,995 and 6,610,310, respectively. It is to be understood that the
immunomodulator and/or adjuvant to be used will depend on the subject to which
the vaccine or immunogenic composition will be administered, the route of
injection and the number of injections to be given.
[0131] In a still further embodiment of the present invention, the S. aureus
lyophilized immunogenic composition comprises a recombinant S. aureus
clumping factor A (ClfA) fragment, an isolated capsular polysaccharides type 5
conjugated to CRMi 7, an isolated capsular polysaccharides type 8 conjugated to
CRMi97, and a recombinant MntC protein (also known as rP305A). In some
embodiments, the MntC protein is lipidated. In other embodiments, the MntC
protein is not lipidated. In another embodiment, the S. aureus immunogenic
composition is a sterile formulation (liquid, lyophilized, DNA vaccine, intradermal
preparation) of recombinant S. aureus clumping factor (ClfA) fragment (N1N2N3,
or combinations thereof), recombinant S. aureus clumping factor B (ClfB)
fragment (N1N2N3, or combinations thereof), an isolated capsular polysaccharides
type 5 conjugated to CRM 197 and an isolated capsular polysaccharides type 8
conjugated to CRM 197 .
[0132] In one embodiment of the present invention, the S. aureus immunogenic
composition comprises a recombinant S. aureus clumping factor A (ClfA)
fragment (N1N2N3, or combinations thereof), S. aureus iron binding protein
MntC, an isolated capsular polysaccharides type 5 conjugated to CRMi 7 and an
isolated capsular polysaccharides type 8 conjugated to CRM 19-7. In one
embodiment, the S. aureus immunogenic composition is a sterile formulation
(liquid, lyophilized, DNA vaccine, intradermal preparation) of recombinant
S. aureus clumping factor (ClfA) fragment (N1N2N3, or combinations thereof),
recombinant S. aureus clumping factor B (ClfB) fragment (N1N2N3, or
combinations thereof), S. aureus iron binding protein MntC, an isolated capsular
polysaccharides type 5 conjugated to CRMi 7 and an isolated capsular
polysaccharides type 8 conjugated to CRMi 7.
[0133] In one embodiment of the present invention, the S. aureus immunogenic
composition comprises a recombinant S. aureus clumping factor B (ClfB) fragment
(N1N2N3, or combinations thereof), an isolated capsular polysaccharides type 5
conjugated to CRMi 7 and an isolated capsular polysaccharides type 8 conjugated
to CRM 19-7. In one embodiment of the present invention, the S. aureus
immunogenic composition comprises a recombinant S. aureus clumping factor B
(ClfB) fragment (N1N2N3, or combinations thereof), S. aureus iron binding
protein MntC, an isolated capsular polysaccharides type 5 conjugated to CRM 19-7
and an isolated capsular polysaccharides type 8 conjugated to CRMi 7 . In one
embodiment of the present invention, the S. aureus immunogenic composition
comprises a S. aureus iron binding protein MntC, an isolated capsular
polysaccharides type 5 conjugated to CRMi 7 and an isolated capsular
polysaccharides type 8 conjugated to CRMi 7.
[0134] S. aureus "invasive disease" is the isolation of bacteria from a normally
sterile site, where there is associated clinical signs/symptoms of disease. Normally
sterile body sites include blood, CSF, pleural fluid, pericardial fluid, peritoneal
fluid, joint/synovial fluid, bone, internal body site (lymph node, brain, heart, liver,
spleen, vitreous fluid, kidney, pancreas, ovary), or other normally sterile sites.
Clinical conditions characterizing invasive diseases include bacteremia,
pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock and more.
[0135] The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring or from its
host organism if it is a recombinant entity, or taken from one environment to a
different environment). For example, an "isolated" capsule polysaccharide, protein
or peptide is substantially free of cellular material or other contaminating proteins
from the cell or tissue source from which the protein is derived, or substantially
free of chemical precursors or other chemicals when chemically synthesized, or
otherwise present in a mixture as part of a chemical reaction. In the present
invention, the proteins or polysaccharides may be isolated from the bacterial cell or
from cellular debris, so that they are provided in a form useful in the manufacture
of an immunogenic composition. The term "isolated" or "isolating" may include
purifying, or purification, including for example, the methods of purification of the
proteins or capsular polysaccharides, as described herein. The language
"substantially free of cellular material" includes preparations of a
polypeptide/protein in which the polypeptide/protein is separated from cellular
components of the cells from which it is isolated or recombinantly produced. Thus,
a capsule polysaccharide, protein or peptide that is substantially free of cellular
material includes preparations of the capsule polysaccharide, protein or peptide
having less than about 30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of
contaminating protein or polysaccharide or other cellular material. When the
polypeptide/protein is recombinantly produced, it is also preferably substantially
free of culture medium, i.e., culture medium represents less than about 20%>, 10%>,
or 5% of the volume of the protein preparation. When polypeptide/protein or
polysaccharide is produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis of the protein or
polysaccharide. Accordingly, such preparations of the polypeptide/protein or
polysaccharide have less than about 30%, 20%, 10%>, 5% (by dry weight) of
chemical precursors or compounds other than polypeptide/protein or
polysaccharide fragment of interest.
[0136] A "non-conservative amino acid substitution" refers to the substitution of
one or more of the amino acid residues of a protein with other amino acid residues
having dissimilar physical and/or chemical properties, using the characteristics
defined above.
[0137] The term "pharmaceutically acceptable carrier" means a carrier approved
by a regulatory agency of a Federal, a state government, or other regulatory
agency, or listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, including humans as well as non-human
mammals. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle
with which the pharmaceutical composition is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including those of petroleum,
animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose
and glycerol solutions can be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene,
glycol, water, ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions, emulsion,
sustained release formulations and the like. Examples of suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
The formulation should suit the mode of administration.
[0138] The terms "protein", "polypeptide" and "peptide" refer to a polymer of
amino acid residues and are not limited to a minimum length of the product. Thus,
peptides, oligopeptides, dimers, multimers, and the like, are included within the
definition. Both full-length proteins and fragments thereof are encompassed by the
definition. The terms also include modifications, such as deletions, additions and
substitutions (generally conservative in nature, but which may be nonconservative),
to a native sequence, preferably such that the protein maintains the
ability to elicit an immunological response within an animal to which the protein is
administered. Also included are post-expression modifications, e.g. glycosylation,
acetylation, lipidation, phosphorylation and the like.
[0139] A "protective" immune response refers to the ability of an immunogenic
composition to elicit an immune response, either humoral or cell mediated, which
serves to protect the subject from an infection. The protection provided need not
be absolute, i.e., the infection need not be totally prevented or eradicated, if there is
a statistically significant improvement compared with a control population of
subjects, e.g. infected animals not administered the vaccine or immunogenic
composition. Protection may be limited to mitigating the severity or rapidity of
onset of symptoms of the infection. In general, a "protective immune response"
would include the induction of an increase in antibody levels specific for a
particular antigen in at least 50% of subjects, including some level of measurable
functional antibody responses to each antigen. In particular situations, a
"protective immune response" could include the induction of a two fold increase in
antibody levels or a four fold increase in antibody levels specific for a particular
antigen in at least 50% of subjects, including some level of measurable functional
antibody responses to each antigen. In certain embodiments, opsonising antibodies
correlate with a protective immune response. Thus, protective immune response
may be assayed by measuring the percent decrease in the bacterial count in an
opsonophagocytosis assay, for instance those described below. Preferably, there is
a decrease in bacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%,
90%, 95% or more.
[0140] The term "recombinant" as used herein simply refers to any protein,
polypeptide, or cell expressing a gene of interest that is produced by genetic
engineering methods. The term "recombinant" as used with respect to a protein or
polypeptide, means a polypeptide produced by expression of a recombinant
polynucleotide. The proteins used in the immunogenic compositions of the
invention may be isolated from a natural source or produced by genetic
engineering methods, such as, for example recombinant ClfA, recombinant ClfB or
recombinant MntC. "Recombinant," as used herein, further describes a nucleic
acid molecule, which, by virtue of its origin or manipulation, is not associated with
all or a portion of the polynucleotide with which it is associated in nature. The
term "recombinant" as used with respect to a host cell means a host cell into which
a recombinant polynucleotide has been introduced.
[0141] Recombinant ClfA (rClfA) and recombinant ClfB (rClfB) as used herein
refers to forms of ClfA or ClfB for use in the immunogenic compositions of the
invention. In certain embodiments, rClfA is a fragment of ClfA comprising one or
more of the N domains, for example, N1N2N3, N2N3, N2 or N3 and is referred to
herein as "recombinant ClfA" or "rClfA". In one embodiment, rClfB is a fragment
of ClfB comprising one or more of the N domains of ClfB, for example, N1N2N3,
N2N3, N2 or N3 and is referred to herein as "recombinant ClfB" or "rClfB".
[0142] The term "subject" refers to a mammal, bird, fish, reptile, or any other
animal. The term "subject" also includes humans. The term "subject" also
includes household pets. Non-limiting examples of household pets include: dogs,
cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes,
lizards, fish, turtles, and frogs. The term "subject" also includes livestock animals.
Non-limiting examples of livestock animals include: alpaca, bison, camel, cattle,
deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yak,
chickens, geese, and turkeys.
[0143] As used herein, "treatment" (including variations thereof, for example,
"treat" or "treated") refers to any one or more of the following: (i) the prevention of
infection or reinfection, as in a traditional vaccine, (ii) the reduction in the severity
of, or, in the elimination of symptoms, and (iii) the substantial or complete
elimination of the pathogen or disorder in question. Hence, treatment may be
effected prophylactically (prior to infection) or therapeutically (following
infection). In the present invention, prophylactic or therapeutic treatments can be
used. According to a particular embodiment of the present invention, compositions
and methods are provided which treat, including prophylactically and/or
therapeutically immunize, a host animal against a microbial infection (e.g. a
bacterium such as Staphylococcus species). The methods of the present invention
are useful for conferring prophylactic and/or therapeutic immunity to a subject.
The methods of the present invention can also be practiced on subjects for
biomedical research applications.
[0144] The terms "vaccine" or "vaccine composition", which are used
interchangeably, refer to pharmaceutical compositions comprising at least one
immunogenic composition that induces an immune response in an animal.
General Description
[0145] The present invention relates to lyophilized immunogenic compositions
comprising antigens from a staphylococcal organism, for example S. aureus. In
some embodiments, the lyophilized immunogenic composition comprises ClfA. In
some embodiments, the lyophilized immunogenic composition comprises at least
three antigens. In some embodiments, the lyophilized immunogenic composition
comprises at least four antigens. The antigens may be isolated from the organism
using biochemical isolation procedures, or they may be produced synthetically or
by recombinant means. The antigens may be polypeptides, or polysaccharides, or
a combination thereof. These immunogenic compositions may be used in the
manufacture of a vaccine to immunize subjects against infections caused by a
staphylococcal organism. The components suitable for use in these compositions
are described in greater detail below.
Staphylococcal Immunogenic Compositions
[0146] S. aureus is the causative agent of a wide variety of human diseases
ranging from superficial skin infections to life threatening conditions such as
pneumonia, sepsis and endocarditis. See Lowy N. Eng. J . Med. 339:580-
532(1998). In cases of invasive disease, S. aureus can be isolated from normally
sterile body sites including blood, cerebral spinal fluid CSF, pleural fluid,
pericardial fluid, peritoneal fluid, joint/synovial fluid, bone, internal body site
(lymph node, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovary), or
other normally sterile sites. This can lead to life threatening clinical conditions
such as bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, and septic
shock. Adults, elderly and pediatric patients are most at risk for S. aureus
infections.
[0147] Embodiments of the present invention describe a selected antigen or
antigens in lyophilized immunogenic compositions including an isolated S. aureus
clumping factor A (ClfA) polypeptide, an isolated S. aureus capsular
polysaccharide type 5 conjugated to a carrier protein, an isolated S. aureus capsular
polysaccharide type 8 conjugated to a carrier protein, an isolated S. aureus
clumping factor B (ClfB), and isolated S. aureus MntC protein. Some
formulations of the lyophilized immunogenic compositions were tested to
demonstrate increased stability of the ClfA protein. Some formulations of the
lyophilized immunogenic compositions were tested to demonstrate stability of the
MntC protein. Some formulations of the lyophilized compositions were also tested
to ensure that CP5-protein conjugates and CP8-protein conjugates were stable after
lyophilization.
[0148] Accordingly, one lyophilized immunogenic composition comprises: an
isolated S. aureus clumping factor A (ClfA) polypeptide. One lyophilized
immunogenic composition comprises: an isolated S. aureus clumping factor A
(ClfA) polypeptide, an isolated S. aureus capsular polysaccharide type 5
conjugated to a carrier protein, and an isolated S. aureus capsular polysaccharide
type 8 conjugated to a carrier protein. One lyophilized immunogenic composition
comprises: an isolated S. aureus clumping factor A (ClfA) polypeptide, an isolated
S. aureus clumping factor B (ClfB), isolated S. aureus capsular polysaccharide
type 5 conjugated to a carrier protein, and an isolated S. aureus capsular
polysaccharide type 8 conjugated to a carrier protein. One lyophilized
immunogenic composition comprises: an isolated S. aureus clumping factor A
(ClfA) polypeptide, an isolated S. aureus clumping factor B (ClfB) polypeptide, an
isolated S. aureus MntC protein, an isolated S. aureus capsular polysaccharide type
5 conjugated to a carrier protein, and an isolated S. aureus capsular polysaccharide
type 8 conjugated to a carrier protein. One lyophilized immunogenic composition
comprises: an isolated S. aureus clumping factor A (ClfA) polypeptide, an isolated
S. aureus MntC protein, an isolated S. aureus capsular polysaccharide type 5
conjugated to a carrier protein, and an isolated S. aureus capsular polysaccharide
type 8 conjugated to a carrier protein. One lyophilized immunogenic composition
comprises: an isolated S. aureus clumping factor B (ClfB) polypeptide, an isolated
S. aureus capsular polysaccharide type 5 conjugated to a carrier protein, and an
isolated S. aureus capsular polysaccharide type 8 conjugated to a carrier protein.
One lyophilized immunogenic composition comprises: an isolated S. aureus
clumping factor B (ClfB) polypeptide, an isolated S. aureus MntC protein, an
isolated S. aureus capsular polysaccharide type 5 conjugated to a carrier protein,
and an isolated S. aureus capsular polysaccharide type 8 conjugated to a carrier
protein. One lyophilized immunogenic composition comprises: an isolated
S. aureus MntC protein, an isolated S. aureus capsular polysaccharide type 5
conjugated to a carrier protein, and an isolated S. aureus capsular polysaccharide
type 8 conjugated to a carrier protein. One lyophilized immunogenic composition
comprises: an isolated S. aureus clumping factor A (ClfA) polypeptide, an isolated
S. aureus clumping factor B (ClfB) polypeptide, and an isolated S. aureus MntC
protein.
[0149] In some embodiments, the above combinations further comprise at least
one of the following antigens: EkeS, DsqA, KesK, KrkN, KrkN2, RkaS, RrkN,
KnkA, SdrC, SdrD, SdrE, Opp3a, DltD, HtsA, LtaS, IsdA, IsdB, IsdC, SdrF, SdrG,
SdrH, SrtA, SpA, Sbi, alpha-hemolysin (hla), beta-hemolysin, fibronectin-binding
protein A (fnbA), fibronectin-binding protein B (fnbB), coagulase, Fig, map,
Panton-Valentine leukocidin (pvl), alpha-toxin and its variants, gamma-toxin (hlg)
and variants, ica, immunodominant ABC transporter, Mg2+ transporter, Ni ABC
transporter, RAP, autolysin, laminin receptors, IsaA/PisA, IsaB/PisB , SPOIIIE,
SsaA, EbpS, SasA, SasF, SasH, EFB (FIB), SBI, Npase, EBP, bone sialo binding
protein II, aureolysin precursor (AUR)/Seppl, Cna, and fragments thereof such as
M55, TSST-1, mecA, poly-N-acetylglucosamine (PNAG/dPNAG)
exopolysaccharide, GehD, EbbA, EbhB, SSP-1, SSP-2, HBP, vitronectin binding
protein, HarA, EsxA, EsxB, Enterotoxin A, Enterotoxin B, Enterotoxin CI, and
novel autolysin.
Adjuvants
[0150] Lyophilized immunogenic compositions as described herein also
comprise, in certain embodiments, one or more adjuvants. In some embodiments,
the adjuvant is a component of the dried lyophilized composition. In other
embodiments, the adjuvant can be added to the reconstituted lyophilized
composition prior to administration of the composition to patient. An adjuvant is a
substance that enhances the immune response when administered together with an
immunogen or antigen. A number of cytokines or lymphokines have been shown
to have immune modulating activity, and thus are useful as adjuvants, including,
but not limited to, the interleukins 1-a, 1-b, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S.
Patent No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms); the
interferons-a, b and g ; granulocyte-macrophage colony stimulating factor (GMCSF)
(see, e.g., U.S. Patent No. 5,078,996 and ATCC Accession Number 39900);
macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating
factor (G-CSF); and the tumor necrosis factors a and b. Still other adjuvants that
are useful with the immunogenic compositions described herein include
chemokines, including without limitation, MCP-1, MIR - I , MIR - I b, and
RANTES; adhesion molecules, such as a selectin, e.g., L-selectin, P-selectin and
E-selectin; mucin-like molecules, e.g., CD34, GlyCAM-1 and MadCAM-1; a
member of the integrin family such as LFA-1, VLA-1, Mac-1 and pl50.95; a
member of the immunoglobulin superfamily such as PECAM, ICAMs, e.g.,
ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; co-stimulatory molecules such
as B7-1, B7-2,CD40 and CD40L; growth factors including vascular growth factor,
nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-
1, and vascular endothelial growth factor; receptor molecules including Fas, TNF
receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,
DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; and Caspase (ICE).
[0151] Suitable adjuvants used to enhance an immune response further include,
without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa,
Hamilton, MT), which is described in U.S. Patent No. 4,912,094. Also suitable for
use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine
phosphate compounds (AGP), or derivatives or analogs thereof, which are
available from Corixa (Hamilton, MT), and which are described in United States
Patent No. 6,1 13,918. One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino]
ethyl 2-Deoxy-4-0-phosphono-3-0-[(R)-3-tetradecanoyoxytetradecanoyl]-
2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is
also known as 529 (formerly known as RC529). This 529 adjuvant is formulated
as an aqueous form (AF) or as a stable emulsion (SE).
[0152] Still other adjuvants include muramyl peptides, such as N-acetylmuramyl-
L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-
2-( 1'-2' dipalmitoyl - /?-glycero-3 -hydroxyphosphoryloxy)-ethylamine (MTP-PE);
oil-in-water emulsions, such as MF59 (U.S. Patent No. 6,299,884) (containing 5%
Squalene, 0.5% polysorbate 80, and 0.5% SPAN™ 85 (optionally containing
various amounts of MTP-PE) formulated into submicron particles using a
microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA)),
and SAF (containing 10%> Squalene, 0.4%> polysorbate 80, 5% pluronic-b locked
polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or
vortexed to generate a larger particle size emulsion); incomplete Freund's adjuvant
(IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,
aluminum sulfate; Amphigen; Avridine; L121/squalene; D-lactidepolylactide/
glycoside; pluronic polyols; killed Bordetella; saponins, such as
STIMULON™ QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent
No. 5,057,540, ISCOMATRFX™ (CSL Limited, Parkville, Australia), described in
U.S. Patent No. 5,254,339, and immunostimulating complexes
(ISCOMATRFX™); Mycobacterium tuberculosis; bacterial lipopolysaccharides;
synthetic polynucleotides such as oligonucleotides containing a CpG motif {e.g.,
U.S. Patent No. 6,207,646); IC-31 (Intercell AG, Vienna, Austria), described in
European Patent Nos. 1,296,713 and 1,326,634; a pertussis toxin (PT) or mutant
thereof, a cholera toxin or mutant thereof {e.g., U.S. Patent Nos. 7,285,281,
7,332,174, 7,361,355 and 7,384,640); or an E. coli heat-labile toxin (LT) or mutant
thereof, particularly LT-K63, LT-R72 {e.g., U.S. Patent Nos. 6,149,919, 7,1 15,730
and 7,291,588).
[0153] In some embodiments, the adjuvant is ISCOMATRFX™ (see, e.g., Davis
et al, Proc. Nat'l. Acad. Sci., 2004, 101(29): 10697-10702).
Candidate Antigens :
ClfA: Domain organization
[0154] Clumping factor A (ClfA) is a S. aureus surface protein associated with
binding to host matrix proteins via a fibrinogen binding site. ClfA is a member of
a family of proteins containing the carboxyl terminal LPXTG (SEQ ID NO: 125)
motif that enables the protein to become covalently linked to the cell surface. ClfA
also belongs to another family of proteins (Microbial Surface Components
Recognizing Adhesive Matrix Molecule, or MSCRAMMs) that are associated with
binding host proteins such as fibrinogen (bound by ClfA), the fibronectin binding
proteins (FnbA and FnbB), the collagen binding protein (Cna) and others. These
proteins all share the amino terminal signal sequence that mediates transport to the
cell surface. The MSCRAMMs also include an A-domain that is the functional
region containing the active site for ligand binding {e.g., fibrinogen, fibronectin,
elastin, keratin). The A-domain is followed by a region composed of serine
aspartate repeats (SD repeat), which is thought to span the peptidoglycan layer.
The SD repeat is followed by a membrane-spanning region that includes the
LPXTG (SEQ ID NO: 125) motif for covalent linkage of the protein to
peptidoglycan. ClfA is described in U.S. Pat. No. 6,008,341.
[0155] The ligand binding region of ClfA comprising N1N2N3 of the A domain
(Figure 1) spans amino acids 40-559. The N domains of ClfA have been assigned
as follows: Nl encompasses residues 45-220; N2 encompasses residues 229-369;
and N3 encompasses residues 370-559. See Deivanayagam et al. EMBO J .
2 1:6660-6672 (2002). For ease of reference the N1N2N3 domains may be referred
to as N123, likewise N2N3 may be referred to as N23. In preparations of
recombinant N1N2N3, the Nl domain has been found to be protease sensitive and
is easily cleaved or hydrolyzed to leave the N2N3 as a stable ligand binding
recombinant fragment. See Deivanayagam et al. EMBO J . 2 1:6660-6672 (2002).
The crystal structure of the fibrinogen binding N2N3 fragment of ClfA A domain,
revealed that both N2 and N3 are dominated by anti-parallel beta strands. In
addition to the anti-parallel beta strands, the N2 domain contains a single turn
alpha helix and two 3io helices and the N3 domain contains three 3io helices. See
Deivanayagam et al. EMBO J . 2 1:6660-6672 (2002). Sequence alignment of N2
and N3 reveals only 13% sequence identity and 36%> sequence similarity over their
lengths. See Deivanayagam et al. EMBO J . 21:6660-6672 (2002). The topology
of the N2 and N3 domains are similar to the classic IgG fold and have been
proposed to be novel variants of the IgG fold. See Deivanayagam et al. EMBO J .
21:6660-6672 (2002).
[0156] Disclosed herein is a composition comprising a stable Staphylococcus
aureus clumping factor A ("ClfA") polypeptide, and methods for making same,
which is useful as an immunogenic composition or vaccine. Also disclosed herein
is a composition comprising a stable ClfA polypeptide, a CP5 conjugate, and a
CP8 conjugate ("tri-antigen"), and methods for making same, which is useful as an
immunogenic composition or vaccine. Further disclosed herein is a composition
comprising a stable ClfA polypeptide, a CP5 conjugate, a CP8 conjugate, and an
MntC polypeptide ("tetra-antigen"), and methods for making same, which is useful
as an immunogenic composition or vaccine. ClfA is known to be an unstable
protein, which readily undergoes clipping between the l and N2 domains. This
application is directed to a ClfA formulation which enables ClfA N1N2N3 to
remain substantially undegraded under accelerated stress conditions for at least
three months. In some embodiments, the formulation is a lyophilized composition
that comprises less than three percent water. The term "lyophilized composition"
refers to the formulation having less than 3% water and is not meant to limit the
composition to the method of which the composition was made, e.g., lyophilization
or any other method of drying a mixture, or compounding dry ingredients. By
accelerated stress conditions, what is meant are conditions of extreme temperature,
pH and/or ionic strength, generally used to test the stability of a formulation, such
as, e.g., 37°C for several weeks or more.

What is Claimed is:
1. A lyophilized immunogenic composition comprising:
(a) at least three components selected from the group consisting of an isolated
Staphylococcus aureus clumping factor A (ClfA) polypeptide, an isolated
Staphylococcus aureus clumping factor B (ClfB) polypeptide, a Capsular
Polysaccharide Type 5 (CP5)-protein conjugate, a Capsular Polysaccharide Type 8
(CP8)-protein conjugate, and an isolated Staphylococcus aureus MntC
polypeptide;
(b) a buffer having a pKa of 6.0 ± 0.6, and
(c) a bulking agent;
wherein said ClfA polypeptide remains substantially undegraded for at least 1
month at 37°C.
2. A lyophilized immunogenic composition comprising:
(a) an isolated Staphylococcus aureus clumping factor A (ClfA) polypeptide,
(b) a Capsular Polysaccharide Type 5 (CP5)-protein conjugate,
(c) a Capsular Polysaccharide Type 8 (CP8)-protein conjugate,
(d) a buffer having a pKa of 6.0 ± 0.6, and
(e) a bulking agent;
wherein said ClfA polypeptide remains substantially undegraded for at least 1
month at 37°C.
3. The lyophilized immunogenic composition of claim 2, wherein the
composition further comprises an isolated Staphylococcus aureus MntC
polypeptide.
4. The lyophilized immunogenic composition of claim 2 or claim 3, wherein the
composition further comprises an isolated Staphylococcus aureus clumping factor
B (ClfB).
5. The lyophilized immunogenic composition of any one of claims 1-4, wherein
the ClfA polypeptide comprises an N domain.
6. The lyophilized immunogenic composition of any one of claims 1-5, wherein
the ClfA polypeptide comprises a fibrinogen binding domain.
7. The lyophilized immunogenic composition of any one of claims 1-6, wherein
the fibrinogen binding domain has been altered so as to bind to fibrinogen at a
reduced level compared to the binding observed to fibrinogen with the native
fibrinogen binding domain of ClfA.
8. The lyophilized immunogenic composition of any one of claims 1-7, wherein
the fibrinogen binding domain displays reduced binding to fibrinogen through
having an amino acid substitution at one or more of Tyr 338, Tyr 256, Pro 336, Lys
389, Ala 254 and e 387.
9. The lyophilized immunogenic composition of claim 8, wherein the amino acid
substitution at one or more of Tyr 338, Tyr 256, Pro 336, Lys 389, Ala 254 and He
387 is to Ala or Ser.
10. The lyophilized immunogenic composition of claim 9, wherein the Tyr 338 is
substituted to Ala.
11. The lyophilized immunogenic composition of any one of claims 1-10, wherein
the CP5-protein conjugate is CP5-CRMi 97, CP5-pneumolysin, or CP5-
streptococcal C5a peptidase (SCP).
12. The lyophilized immunogenic composition of any one of claims 1-1 1, wherein
the CP8-protein conjugate is CP8-CRMi 7, CP8-pneumolysin, or CP8-
streptococcal C5a peptidase (SCP).
13. The lyophilized immunogenic composition of any one of claims 1-12, which
comprises water at less than 3 percent weight of the total weight of the
immunogenic composition (% w/w), wherein the ClfA polypeptide is between
0.09% ± 0.027% and 0.85% ± 0.26% w/w, the CP5-protein conjugate is between
0.04% ± 0.013% and 0.42 ± 0.13% w/w, the CP8-protein conjugate is between
0.04% ± 0.013% and 0.42 ± 0.13% w/w, and the buffer is at 2.54% ± 0.76% w/w.
14. The lyophilized immunogenic composition of any one of claims 1-13, wherein
the buffer comprises histidine at pH 6.0 ± 0.5.
15. The lyophilized immunogenic composition of any one of claims 1-14, wherein
the bulking agent is selected from the group consisting of sucrose, trehalose,
mannitol, glycine, and sorbitol.
16. The lyophilized immunogenic composition of claim 15, wherein the bulking
agent is sucrose.
17. The lyophilized immunogenic composition of claim 16, wherein the bulking
agent is sucrose at 96% ± 0.060% w/w.
18. The lyophilized immunogenic composition of any one of claims 1-17, further
comprising a surfactant.
19. The lyophilized immunogenic composition of claim 18, wherein the surfactant
is selected from the group consisting of a poloxamer, a polyoxyethylene alkyl
ether, and a polyoxyethylene sorbitan fatty acid ester.
20. The lyophilized immunogenic composition of claim 19, wherein the
polyoxyethylene sorbitan fatty acid ester is polysorbate 80.
21. The lyophilized immunogenic composition of claim 20, wherein the
polysorbate 80 is at 0.20% ± 0.041% w/w.
22. The lyophilized immunogenic composition of any one of claims 1-21, wherein
the composition further comprises an adjuvant.
23. The lyophilized immunogenic composition of claim 22, wherein the adjuvant is
ISCOMATRIX™.
24. A liquid immunogenic composition prepared by reconstituting the lyophilized
immunogenic composition of any one of claims 1-23 with an aqueous diluent,
wherein said liquid immunogenic composition has a final pH of 6.0 ± 0.5.
25. The liquid immunogenic composition of claim 24, wherein the aqueous diluent
is water.
26. The liquid immunogenic composition of claim 24 or claim 25, wherein the
liquid immunogenic composition comprises:
(a) the ClfA polypeptide at a concentration of between 40 mg/ml ± 4mg/ml and
80(^g/ml ± 8(^g/ml;
(b) the CP5-protein conjugate at a concentration of between 2(^g/ml ± 2mg/ml
and 40(^g/ml ± 4(^g/ml;
(c) the CP8-protein conjugate at a concentration of between 2(^g/ml ± 2mg/ml
and 40(^g/ml ± 4(^g/ml;
(d) the histidine buffer at a concentration of lOmM ± 5mM;
(e) the polysorbate 80 at a concentration of 0.1% ± 0.05% weight to volume
(w/v); and
(f the sucrose at a concentration of 9% ± 4.5% w/v.
27. The liquid immunogenic composition of any one of claims 24-26, wherein the
liquid immunogenic composition further comprises the MntC polypeptide at a
concentration of between 40 mg/ml ± 4mg/ml and 800mg/ml ± 80mg/ml.
28. The liquid immunogenic composition of any one of claims 24-27, wherein the
liquid immunogenic composition further comprises the ClfB polypeptide at a
concentration of between 40 mg/ml ± 4mg/ml and 800mg/ml ± 80mg/ml.