U.S. patent application number 11/216226 was filed with the patent office on 2006-01-05 for vaccine.
This patent application is currently assigned to SmithKlineBeecham Biologicals, S.A.. Invention is credited to Carine Capiau, Marguerite Deschamps, Pierre Michel Desmons, Craig AntonyJoseph Laferriere, Jan Poolman, Jean-Paul Prieels.
Application Number | 20060002961 11/216226 |
Document ID | / |
Family ID | 27451884 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060002961 |
Kind Code |
A1 |
Capiau; Carine ; et
al. |
January 5, 2006 |
Vaccine
Abstract
The present invention relates to the field of bacterial
polysaccharide antigen vaccines. In particular, the present
invention relates to vaccines comprising a pneumococcal
polysaccharide antigen, typically a pneumococcal polysaccharide
conjugate antigen, formulated with a protein antigen form
Streptococcus pneumoniae, and optionally a Th1-inducing
adjuvant.
Inventors: |
Capiau; Carine; (Rixensart,
BE) ; Deschamps; Marguerite; (Rixensart, BE) ;
Desmons; Pierre Michel; (Rixensart, BE) ; Laferriere;
Craig AntonyJoseph; (Rixensart, BE) ; Poolman;
Jan; (Rixensart, BE) ; Prieels; Jean-Paul;
(Rixensart, BE) |
Correspondence
Address: |
GLAXOSMITHKLINE;Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKlineBeecham Biologicals,
S.A.
|
Family ID: |
27451884 |
Appl. No.: |
11/216226 |
Filed: |
August 31, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09936985 |
Dec 19, 2001 |
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PCT/EP00/02467 |
Mar 17, 2000 |
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11216226 |
Aug 31, 2005 |
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Current U.S.
Class: |
424/244.1 |
Current CPC
Class: |
A61K 2039/55572
20130101; A61P 27/16 20180101; A61K 47/646 20170801; A61P 31/00
20180101; A61K 2039/55505 20130101; A61K 2039/70 20130101; A61K
39/095 20130101; A61K 2039/6075 20130101; A61K 39/155 20130101;
A61P 31/04 20180101; A61P 37/04 20180101; A61K 2039/6068 20130101;
A61P 11/00 20180101; A61P 27/06 20180101; Y02A 50/30 20180101; Y10S
424/831 20130101; A61K 39/102 20130101; A61K 39/12 20130101; A61K
2039/6037 20130101; A61K 39/092 20130101 |
Class at
Publication: |
424/244.1 |
International
Class: |
A61K 39/09 20060101
A61K039/09 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 15, 1999 |
GB |
9916677.9 |
Mar 19, 1999 |
GB |
9906437.0 |
Apr 20, 1999 |
GB |
9909077.1 |
Apr 23, 1999 |
GB |
9909466.6 |
Claims
1-15. (canceled)
16. An immunogenic composition comprising at least one
Streptococcus pneumoniae polysaccharide-protein conjugate, with at
least one unconjugated Streptococcus pneumoniae protein antigen,
and an adjuvant which is a preferential inducer of a TH1
response.
17. The immunogenic composition of claim 16, wherein the
unconjugated Streptococcus pneumoniae protein antigen is an outer
surface protein or a secreted protein of Streptococcus
pneumoniae.
18. The immunogenic composition of claim 16, wherein the
unconjugated Streptococcus pneumoniae protein antigen is a toxin,
adhesion or lipoprotein of Streptococcus pneumoniae.
19. The immunogenic composition of claim 16, wherein the
unconjugated Streptococcus pneumoniae protein antigen is selected
from the group consisting of: pneumolysin, PspA, PspC, PsaA,
glyceraldehyde-3-phosphate dehydrogenase, and CbpA.
20. The immunogenic composition of claim 16, wherein the protein
carrier in the polysaccharide-protein conjugate is selected from
the group consisting of: Diphtheria toxoid, Tetanus toxoid, CRM197,
Keyhole Limpet Haemocyanin (KLH), protein derivative of Tuberculin
(PPD), and protein D from H. influenzae.
21. An immunogenic composition of claim 16 which comprises at least
four pneumococcal polysaccharide antigens from different
serotypes.
22. An immunogenic composition as claimed in claim 16, wherein the
adjuvant comprises at least one of the following: 3D-MPL, a saponin
immunostimulant, or an immunostimulatory CpG oligonucleotide.
23. An immunogenic composition as claimed in claim 22, wherein the
adjuvant comprises a carrier selected from the group consisting of:
an oil in water emulsion, liposomes, and an aluminium salt.
24. A vaccine comprising the immunogenic composition of any one of
claims 16-23.
Description
FIELD OF INVENTION
[0001] The present invention relates to bacterial polysaccharide
antigen vaccines, their manufacture and the use of such
polysaccharides in medicines.
[0002] In particular the present invention relates to three
inter-related aspects: A--vaccines comprising a pneumococcal
polysaccharide antigen, typically a pneumococcal polysaccharide
conjugate antigen, formulated with a protein antigen from
Streptococcus pneumoniae and optionally a Th1 inducing adjuvant;
B--specific, advantageous pneumococcal polysaccharide conjugates
adjuvanted with a Th1 adjuvant; and C--bacterial polysaccharide
conjugates in general conjugated to protein D from H.
influenzae.
BACKGROUND OF INVENTION
[0003] Streptococcus pneumoniae is a Gram-positive bacteria
responsible for considerable morbidity and mortality (particularly
in the young and aged), causing invasive diseases such as
pneumonia, bacteremia and meningitis, and diseases associated with
colonisation, such as acute Otitis media. The rate of pneumococcal
pneumonia in the US for persons over 60 years of age is estimated
to be 3 to 8 per 100,000. In 20% of cases this leads to bacteremia,
and other manifestations such as meningitis, with a mortality rate
close to 30% even with antibiotic treatment.
[0004] Pneumococcus is encapsulated with a chemically linked
polysaccharide which confers serotype specificity. There are 90
known serotypes of pneumococci, and the capsule is the principle
virulence determinant for pneumococci, as the capsule not only
protects the inner surface of the bacteria from complement, but is
itself poorly immunogenic. Polysaccharides are T-independent
antigens, and can not be processed or presented on MHC molecules to
interact with T-cells. They can however, stimulate the immune
system through an alternate mechanism which involves cross-linking
of surface receptors on B cells.
[0005] It was shown in several experiments that protection against
invasive pneumococci disease is correlated most strongly with
antibody specific for the capsule, and the protection is serotype
specific.
[0006] Polysaccharide antigen based vaccines are well known in the
art. Four that have been licensed for human use include the Vi
polysaccharide of Salmonella typhi, the PRP polysaccharide from
Haemophilus influenzae, the tetravalent meningococcal vaccine
composed of serotypes A, C, W135 and Y, and the 23-Valent
pneumococcal vaccine composed of the polysaccharides corresponding
to serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,
15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33 (accounting for at
least 90% of pneumococcal blood isolates).
[0007] The latter three vaccines confer protection against bacteria
causing respiratory infections resulting in severe morbidity and
mortality in infants, yet these vaccines have not been licensed for
use in children less than two years of age because they are
inadequately immunogenic in this age group [Peltola et al. (1984),
N. Engl. J. Med. 310:1561-1566]. Streptococcus pneumoniae is the
most common cause of invasive bacterial disease and otitis media in
infants and young children. Likewise, the elderly mount poor
responses to pneumococcal vaccines [Roghmann et al., (1987), J.
Gerontol. 42:265-270], hence the increased incidence of bacterial
pneumonia in this population [Verghese and Berk, (1983) Medicine
(Baltimore) 62:271-285].
[0008] Strategies, which have been designed to overcome this lack
of immunogenicity in infants, include the linking of the
polysaccharide to large immunogenic proteins, which provide
bystander T-cell help and which induce immunological memory against
the polysaccharide antigen to which it is conjugated. Pneumococcal
glycoprotein conjugate vaccines are currently being evaluated for
safety, immunogenicity and efficacy in various age groups.
A) Pneumococcal Polysaccharide Vaccines
[0009] The 23-valent unconjugated pneumococcal vaccine has shown a
wide variation in clinical efficacy, from 0% to 81% (Fedson et al.
(1994) Arch Intern Med. 154: 2531-2535). The efficacy appears to be
related to the risk group that is being immunised, such as the
elderly, Hodgkin's disease, splenectomy, sickle cell disease and
agammaglobulinemics (Fine et al. (1994) Arch Intern Med.
154:2666-2677), and also to the disease manifestation. The
23-valent vaccine does not demonstrate protection against
pneumococcal pneumonia (in certain high risk groups such as the
elderly) and otitis media diseases.
[0010] There is therefore a need for improved pneumococcal vaccine
compositions, particularly ones which will be more effective in the
prevention or amelioration of pneumococcal disease (particularly
pneumonia) in the elderly and in young children.
[0011] The present invention provides such an improved vaccine.
B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL
Compositions
[0012] It is generally accepted that the protective efficacy of the
commercialised unconjugated pneumococcal vaccine is more or less
related to the concentration of antibody induced upon vaccination;
indeed, the 23 polysaccharides were accepted for licensure solely
upon the immunogenicity of each component polysaccharide (Ed.
Williams et al. New York Academy of Sciences 1995 pp. 241-249).
Therefore further enhancement of antibody responses to the
pneumococcal polysaccharides could increase the percentage of
infants and elderly responding with protective levels of antibody
to the first injection of polysaccharide or polysaccharide
conjugate and could reduce the dosage and the number of injections
required to induce protective immunity to infections caused by
Streptococcus pneumoniae.
[0013] Since the early 20.sup.th century, researchers have
experimented with a huge number of compounds which can be added to
antigens to improve their immunogenicity in vaccine compositions
[reviewed in M. F. Powell & M. J. Newman, Plenum Press, NY,
"Vaccine Design--the Subunit and Adjuvant Approach" (1995) Chapter
7 "A Compendium of Vaccine Adjuvants and Excipients"]. Many are
very efficient, but cause significant local and systemic adverse
reactions that preclude their use in human vaccine compositions.
Aluminium-based adjuvants (such as alum, aluminium hydroxide or
aluminium phosphate), first described in 1926, remain the only
immunologic adjuvants used in human vaccines licensed in the United
States.
[0014] Aluminium-based adjuvants are examples of the carrier class
of adjuvant which works through the "depot effect" it induces.
Antigen is adsorbed onto its surface and when the composition is
injected the adjuvant and antigen do not immediately dissipate in
the blood stream--instead the composition persists in the local
environment of the injection and a more pronounced immune response
results. Such carrier adjuvants have the additional known advantage
of being suitable for stabilising antigens that are prone to
breakdown, for instance some polysaccharide antigens.
[0015] 3D-MPL is an example of a non-carrier adjuvant. Its full
name is 3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylated
monophosphoryl lipid A or 3-O-desacyl-4' monophosphoryl lipid A)
and is referred to as 3D-MPL to indicate that position 3 of the
reducing end glucosamine is de-O-acylated. For its preparation, see
GB 2220211 A. Chemically it is a mixture of 3-deacylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains. It was
originally made in the early 1990's when the method to
3-O-deacylate the 4'-monophosphoryl derivative of lipid A (MPL) led
to a molecule with further attenuated toxicity with no change in
the immunostimulating activity.
[0016] 3D-MPL has been used as an adjuvant either on its own or,
preferentially, combined with a depot-type carrier adjuvant such as
aluminium hydroxide, aluminium phosphate or oil-in-water emulsions.
In such compositions antigen and 3D-MPL are contained in the same
particulate structures, allowing for more efficient delivery of
antigenic and immunostimulatory signals. Studies have shown that
3D-MPL is able to further enhance the immunogenicity of an
alum-adsorbed antigen [Thoelen et al. Vaccine (1998) 16:708-14; EP
689454-B1]. Such combinations are also preferred in the art for
antigens that are prone to adsorption (for instance, bacterial
polysaccharide conjugates), where adsorption onto alum tends to
stabilise the antigen. Precipitated aluminium-based adjuvants are
mostly used as they are the only adjuvants that are currently used
in licensed human vaccines. Accordingly, vaccines containing 3D-MPL
in combination with aluminium-based adjuvants are favoured in the
art due to their ease of development and speed of introduction onto
the market.
[0017] MPL (non 3-deacylated) has been evaluated as an adjuvant
with several monovalent polysaccharide-conjugate vaccine antigens.
Coinjection of MPL in saline enhanced the serum antibody response
for four monovalent polysaccharide conjugates: pneumococcal PS
6B-tetanus toxoid, pneumococcal PS 12-diphtheria toxoid, and S.
aureus type 5 and S. aureus type 8 conjugated to Pseudomonas
aeruginosa exotoxin A [Schneerson et al. J. Immunology (1991)
147:2136-2140]. The enhanced responses were taught as being
antigen-specific. MPL in an oil-in-water emulsion (a carrier type
adjuvant) consistently enhanced the effect of MPL in saline due to
the presence of MPL and antigen in the same particulate structure,
and was considered to be the adjuvant system of choice for optimal
delivery of other polysaccharide conjugate vaccines.
[0018] Devi et al. [Infect. Immun. (11991) 59:3700-7] evaluated the
adjuvant effect of MPL (non 3-deacylated) in saline on the murine
antibody response to a TT conjugate of Cryptococcus neoformans
capsular polysaccharide. When MPL was used concurrently with the
conjugate there was only a marginal increase in both the IgM- and
IgG-specific response to the PS; however MPL had a much larger
effect when administered 2 days after the conjugate. The
practicality of using an immunization scheme that requires a delay
in the administration of MPL relative to antigen, especially in
infants, is questionable. The adjuvant effect of MPL with
polysaccharides and polysaccharide-protein conjugates appears to be
composition-dependent. Again, the incorporation of MPL in a
suitable slow-release delivery systems (for instance using a
carrier adjuvant) provides a more durable adjuvant effect and
circumvents the problem of timing and delayed administration.
[0019] In summary, the state of the art has taught that, for
particular polysaccharide or polysaccharide-conjugate antigens,
where MPL or 3D-MPL is used as an adjuvant, it is advantageously
used in conjuction with a carrier adjuvant (for instance the
aluminium-based adjuvants) in order to maximise its
immunostimulatory effect.
[0020] Surprisingly, the present inventors have found that for
certain pneumococcal polysaccharide conjugates, the immunogenicity
of the vaccine composition is significantly greater when the
antigen is formulated with 3D-MPL alone rather than with 3D-MPL in
conjunction with a carrier adjuvant (such as an aluminium-based
adjuvant). Furthermore the observed improvement is independent of
the concentration of 3D-MPL used, and whether the particular
conjugates are in a monovalent composition or whether they are
combined to form a polyvalent composition.
C) Bacterial Polysaccharide-Protein D Conjugates
[0021] As mentioned above, polysaccharide antigen based vaccines
are well known in the art. The licensed polysaccharide vaccines
mentioned above have different demonstrated clinical efficacy. The
Vi polysaccharide vaccine has been estimated to have an efficacy
between 55% and 77% in preventing culture confirmed typhoid fever
(Plotkin and Cam, (1995) Arch Intern Med 155: 2293-99). The
meningococcal C polysaccharide vaccine was shown to have an
efficacy of 79% under epidemic conditions (De Wals P, et al. (1996)
Bull World Health Organ. 74: 407-411). The 23-valent pneumococcal
vaccine has shown a wide variation in clinical efficacy, from 0% to
81% (Fedson et al. (1994) Arch Intern Med. 154: 2531-2535). As
mentioned above, it is accepted that the protective efficacy of the
pneumococcal vaccine is more or less related to the concentration
of antibody induced upon vaccination.
[0022] Amongst the problems associated with the polysaccharide
approach to vaccination, is the fact that polysaccharides per se
are poor immunogens. Strategies which have been designed to
overcome this lack of immunogenicity include the linking of the
polysaccharide to large highly immunogenic protein carriers, which
provide bystander T-cell help.
[0023] Examples of these highly immunogenic carriers which are
currently commonly used for the production of polysaccharide
immunogens include the Diphtheria toxoid (DT or the CRM197 mutant),
Tetanus toxoid (TT), Keyhole Limpet Haemocyanin (KLH), and the
purified protein derivative of Tuberculin (PPD).
Problems Associated with Commonly-Used Carriers
[0024] A number of problems are associated with each of these
commonly used carriers, including in production of GMP conjugates
and also in immunological characteristics of the conjugates.
[0025] Despite the common use of these carriers and their success
in the induction of anti polysaccharide antibody responses they are
associated with several drawbacks. For example, it is known that
antigen specific immune responses may be suppressed (epitope
suppression) by the presence of preexisting antibodies directed
against the carrier, in this case Tetanus toxin (Di John et al;
(1989) Lancet, 2:1415-8). In the population at large, a very high
percentage of people will have pre-existing immunity to both DT and
TT as people are routinely vaccinated with these antigens. In the
UK for example 95% of children receive the DTP vaccine comprising
both DT and TT. Other authors have described the problem of epitope
suppression to peptide vaccines in animal models (Sad et al,
Immunology, 1991; 74:223-227; Schutze et al, J. Immunol. 135: 4,
1985; 2319-2322).
[0026] In addition, for vaccines which require regular boosting,
the use of highly immunogenic carriers such as TT and DT are likely
to suppress the polysaccharide antibody response after several
injections. These multiple vaccinations may also be accompanied by
undesirable reactions such as delayed type hyperresponsiveness
(DTH).
[0027] KLH is known as potent immunogen and has already been used
as a carrier for IgE peptides in human clinical trials. However,
some adverse reactions (DTH-like reactions or IgE sensitisation) as
well as antibody responses against antibody have been observed.
[0028] The selection of a carrier protein, therefore, for a
polysaccharide based vaccine will require a balance between the
necessity to use a carrier working in all patients (broad MHC
recognition), the induction of high levels of anti-polysaccharide
antibody responses and low antibody response against the
carrier.
[0029] The carriers used previously for polysaccharide based
vaccines, therefore have many disadvantages. This is particularly
so in combination vaccines, where epitope suppression is especially
problematic if the same carrier is used for various polysaccharide
antigens. In WO 98/51339, multiple carriers in combination vaccines
were used in order to try to get over this effect.
[0030] The present invention provides a new carrier for use in the
preparation of polysaccharide/polypeptide-based immunogenic
conjugates, that does not suffer from the aforementioned
disadvantages.
[0031] The present invention provides a protein D (EP 0 594 610 B1)
from Haemophilus influenzae, or fragments thereof, as a carrier for
polysaccharide based immunogenic compositions, including vaccines.
The use of this carrier is particularly advantageous in combination
vaccines.
SUMMARY OF THE INVENTION
A) Pneumococcal Polysaccharide Vaccines
[0032] Accordingly the present invention provides a vaccine
composition, comprising at least one Streptococcus pneumoniae
polysaccharide antigen (preferably conjugated) and a Streptococcus
pneumoniae protein antigen or immunologically functional equivalent
thereof, optionally with a Th1 adjuvant (an adjuvant inducing a Th1
immune response). Preferably both a pneumococcal protein and Th1
adjuvant are included. The compositions of the invention are
particularly suited in the treatment of elderly pneumonia.
[0033] Pneumococcal polysaccharide vaccines (conjugated or not) may
not be able to protect against pneumonia in the elderly population
for which the incidence of this disease is very high. The key
defense mechanism against the pneumococcus is opsonophagocytosis (a
humoral B-cell/neutrophil mediated event caused by the production
of antibodies against the pneumococcal polysaccharide, the
bacterium eventually becoming phagocytosed), however parts of the
involved opsonic mechanisms are impaired in the elderly, i.e.
superoxide production by PMN (polymorphonuclear cells), other
reactive oxygen species production, mobilization of PMN, apoptosis
of PMN, deformability of PMN. Antibody responses may also be
impaired in the elderly.
[0034] Contrary to the normally accepted dogma, normal levels of
anti-capsular polysaccharide antibodies may not be effective in
complete clearance of bacteria, as pneumococci may invade host
cells to evade this branch of the immune system.
[0035] Surprisingly, the present inventors have found that by
simultaneously stimulating the cell mediated branch of the immune
system (for instance T-cell meditated immunity) in addition to the
humoral brach of the immune system (B-cell mediated), a synergy (or
cooperation) results which is capable of enhancing the clearance of
pneumococci from the host. This is a discovery which will aid the
prevention (or treatment) of pneumococcal infection in general, but
will be particularly important for the prevention (or treatment) of
pneumonia in the elderly where polysaccharide based vaccines do not
show efficacy.
[0036] The present inventors have found that both arms of the
immune system may synergise in this way if a pneumococcal
polysaccharide (preferably conjugated) is administered with a
pneumococcal protein (preferably a protein expressed on the surface
of pneumococci, or secreted or released, which can be processed and
presented in the context of Class II and MHC class I on the surface
of infected mammalian cells). Although a pneumococcal protein can
trigger cell mediated immunity by itself, the inventors have also
found that the presence of a Th1 inducing adjuvant in the vaccine
formulation helps this arm of the immune system, and surprisingly
further enhances the synergy between both arms of the immune
system.
B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL
Compositions
[0037] Accordingly, the present invention also provides an
antigenic composition comprising one or more pneumococcal
polysaccharide conjugates adjuvanted with 3D-MPL and substantially
devoid of aluminium-based adjuvants, wherein at least one of the
pneumococcal polysaccharide conjugates is significantly more
immunogenic in compositions comprising 3D-MPL in comparison with
compositions comprising 3D-MPL in conjunction with an
aluminium-based adjuvant.
[0038] Preferred embodiments provided are antigenic compositions
comprising conjugates of one or more of the following pneumococcal
capsular polysaccharides: serotype 4, 6B, 18C, 19F, and 23F. In
such compositions, each of the polysaccharides are surprisingly
more immunogenic in compositions comprising 3D-MPL alone compared
with compositions comprising 3D-MPL and an aluminium-based
adjuvant.
[0039] Thus is one embodiment of the invention there is provided a
antigenic composition comprising the Streptococcus pneumoniae
capsular polysaccharide serotype 4, 6B, 18C, 19F or 23F conjugated
to an immunogenic protein and 3D-MPL adjuvant, wherein the
composition is substantially devoid of aluminium-based
adjuvants.
[0040] In a second embodiment, the present invention provides a
combination antigenic composition substantially devoid of
aluminium-based adjuvants and comprising 3D-MPL adjuvant and two or
more pneumococcal polysaccharide conjugates chosen from the group
consisting of: serotype 4; serotype 6B; serotype 18C; serotype 19F;
and serotype 23F.
C) Bacterial Polysaccharide-Protein D Conjugates
[0041] Accordingly, the present invention provides a polysaccharide
conjugate antigen comprising a polysaccharide antigen derived from
a pathogenic bacterium conjugated to protein D from Haemophilus
influenzae or a protein D fragment thereof. In addition, the
invention provides polyvalent vaccine compositions where one or
more of the polysaccharide antigens are conjugated to protein
D.
DESCRIPTION OF THE INVENTION
A) Pneumococcal Polysaccharide Vaccines
[0042] The present invention provides an improved vaccine
particularly for the prevention or amelioration of pnemococcal
infection of the elderly (and/or infants and toddlers).
[0043] In the context of the invention a patient is considered
elderly if they are 55 years or over in age, typically over 60
years and more generally over 65 years.
[0044] Thus in one embodiment of the invention there is provided a
vaccine composition, suitable for use in the elderly (and/or
Infants and toddlers) comprising at least one Streptococcus
pneumoniae polysaccharide antigen and at least one Streptococcus
pneumoniae protein antigen.
[0045] In a second, preferred, embodiment, the present invention
provides a vaccine (suitable for the prevention of pneumonia in the
elderly) comprising at least one Streptococcus pneumoniae
polysaccharide antigen and at least one Streptococcus pneumoniae
protein antigen and a Th1 adjuvant.
[0046] It is envisaged that such a vaccine will be also useful in
treating pneumococcal infection (for instance otitis media) in
other high risk groups of the population, such as for infants or
toddlers.
[0047] In a third embodiment there is provided a vaccine
composition comprising a pneumococcal polysaccharide antigen and a
Th1 adjuvant.
Streptococcus pneumoniae Polysaccharide Antigens of the
Invention
[0048] Typically the Streptococcus pneumoniae vaccine of the
present invention will comprise polysaccharide antigens (preferably
conjugated), wherein the polysaccharides are derived from at least
four serotypes of pneumococcus. Preferably the four serotypes
include 6B, 14, 19F and 23F. More preferably, at least 7 serotypes
are included in the composition, for example those derived from
serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. More preferably still,
at least 11 serotypes are included in the composition, for example
the composition in one embodiment includes capsular polysaccharides
derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F
(preferably conjugated). In a preferred embodiment of the invention
at least 13 polysaccharide antigens (preferably conjugated) are
included, although further polysaccharide antigens, for example 23
valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A,
11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are
also contemplated by the invention.
[0049] For elderly vaccination (for instance for the prevention of
pneumonia) it is advantageous to include serotypes 8 and 12F (and
most preferably 15 and 22 as well) to the 11 valent antigenic
composition described above to form a 15 valent vaccine, whereas
for infants or toddlers (where otitis media is of more concern)
serotypes 6A and 19A are advantageously included to form a 13
valent vaccine.
[0050] For the prevention/amelioration of pneumonia in the elderly
(+55 years) population and Otitis media in Infants (up to 18
months) and toddlers (typically 18 months to 5 years), it is a
preferred embodiment of the invention to combine a multivalent
Streptococcus pneumonia polysaccharide as herein described with a
Streptococcus pneumoniae protein or immunologically functional
equivalent thereof.
Pneumococcal Proteins of the Invention
[0051] For the purposes of this invention, "immunologically
functional equivalent" is defined as a peptide of protein
comprising at least one protective epitope from the proteins of the
invention. Such epitopes are characteristically surface-exposed,
highly conserved, and can elicit an bactericidal antibody response
in a host or prevent toxic effects. Preferably, the functional
equivalent has at least 15 and preferably 30 or more contiguous
amino acids from the protein of the invention. Most preferably,
fragments, deletions of the protein, such as transmembrane deletion
variants thereof (ie the use of the extracellular domain of the
proteins), fusions, chemically or genetically detoxified
derivatives and the like can be used with the proviso that they are
capable of raising substantially the same immune response as the
native protein.
[0052] Preferred proteins of the invention are those pneumococcal
proteins which are exposed on the outer surface of the pneumococcus
(capable of being recognised by a host's immune system during at
least part of the life cycle of the pneumococcus), or are proteins
which are secreted or released by the pneumococcus. Most
preferably, the protein is a toxin, adhesin, 2-component signal
tranducer, or lipoprotein of Streptococcus pneumoniae, or
immunologically functional equivalents thereof.
[0053] Particularly preferred proteins to be included in such a
combination vaccine, include but are not limited to: pneumolysin
(preferably detoxified by chemical treatment or mutation) [Mitchell
et al. Nucleic Acids Res. 1990 Jul. 11; 18(13): 4010 "Comparison of
pneumolysin genes and proteins from Streptococcus pneumoniae types
1 and 2.", Mitchell et al. Biochim Biophys Acta 1989 Jan. 23;
1007(1): 67-72 "Expression of the pneumolysin gene in Escherichia
coli: rapid purification and biological properties.", WO 96/05859
(A. Cyanamid), WO 90/06951 (Paton et al), WO 99/03884 (NAVA)]; PspA
and transmembrane deletion variants thereof (U.S. Pat. No.
5,804,193--Briles et al.); PspC and transmembrane deletion variants
thereof (WO 97/09994--Briles et al); PsaA and transmembrane
deletion variants thereof (Berry & Paton, Infect Immun 1996
December; 64(12):5255-62 "Sequence heterogeneity of PsaA, a
37-kilodalton putative adhesin essential for virulence of
Streptococcus pneumoniae"); pneumococcal choline binding proteins
and transmembrane deletion variants thereof; CbpA and transmembrane
deletion variants thereof (WO 97/41151; WO 99/51266);
Glyceraldehyde-3-phosphate-dehydrogenase (Infect. Immun. 1996
64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al. FEMS
Microbiol Lett 1998, 164:207-14); M like protein, SB patent
application No. EP 0837130; and adhesin 18627, SB Patent
application No. EP 0834568.
[0054] The proteins used in the present invention are preferably
selected from the group pneumolysin, PsaA, PspA, PspC, CbpA or a
combination of two or more such proteins. The present invention
also encompasses immunologically functional equivalents of such
proteins (as defined above).
[0055] Within the composition, the protein can help to induce a
T-cell mediated response against pneumococcal disease--particularly
required for protection against pneumonia--which cooperates with
the humoral branch of the immune system to inhibit invasion by
pneumococci, and to stimulate opsonophagocytosis.
[0056] Further advantages of including the protein antigen is
presentation of further antigens for the opsonophagocytosis
process, and the inhibition of bacterial adhesion (if an adhesin is
used) or the neutralisation of toxin (if a toxin is used).
[0057] Accordingly in an embodiment of the invention there is
provided a Streptococcus pneumoniae vaccine comprising a
pneumococcus polysaccharide conjugate vaccine comprising
polysaccharide antigens derived from at least four serotypes,
preferably at least seven serotypes, more preferably at least
eleven serotypes, and at least one, but preferably two,
Streptococcus pneumoniae proteins. Preferably one of the proteins
is Pneumolysin or PsaA or PspA or CbpA (most preferably detoxified
pneumolysin). A preferred combination contains at least pneumolysin
or a derivative thereof and PspA.
[0058] As mentioned above, a problem associated with the
polysaccharide approach to vaccination, is the fact that
polysaccharides per se are poor immunogens. To overcome this,
polysaccharides may be conjugated to protein carriers, which
provide bystander T-cell help. It is preferred, therefore, that the
polysaccharides utilised in the invention are linked to such a
protein carrier. Examples of such carriers which are currently
commonly used for the production of polysaccharide immunogens
include the Diphtheria and Tetanus toxoids (DT, DT CRM197 and TT
respectively), Keyhole Limpet Haemocyanin (KLH), OMPC from N.
meningitidis, and the purified protein derivative of Tuberculin
(PPD).
[0059] A number of problems are, however, associated with each of
these commonly used carriers (see section "Problems Associated with
Commonly-Used Carriers" above).
[0060] The present invention provides in a preferred embodiment a
new carrier for use in the preparation of polysaccharide-based
immunogen constructs, that does not suffer from these
disadvantages. The preferred carrier for the pneumococcal
polysaccharide based immunogenic compositions (or vaccines) is
protein D from Haemophilus influenzae (EP 594610-B), or fragments
thereof. Fragments suitable for use include fragments encompassing
T-helper epitopes. In particular a protein D fragment will
preferably contain the N-terminal 1/3 of the protein.
[0061] A further preferred carrier for the pneumococcal
polysaccharide is the pneumococcal protein itself (as defined above
in section "Pneumococcal Proteins of the invention").
[0062] The vaccines of the present invention are preferably
adjuvanted. Suitable adjuvants include an aluminium salt such as
aluminium hydroxide gel (alum) or aluminium phosphate, but may also
be a salt of calcium, iron or zinc, or may be an insoluble
suspension of acylated tyrosine, or acylated sugars, cationically
or anionically derivatised polysaccharides, or
polyphosphazenes.
[0063] It is preferred that the adjuvant be selected to be a
preferential inducer of a TH1 type of response to aid the cell
mediated branch of the immune response.
TH1 Adjuvants of the Invention
[0064] High levels of Th1-type cytokines tend to favour the
induction of cell mediated immune responses to a given antigen,
whilst high levels of Th2-type cytokines tend to favour the
induction of humoral immune responses to the antigen.
[0065] It is important to remember that the distinction of Th1 and
Th2-type immune response is not absolute. In reality an individual
will support an immune response which is described as being
predominantly Th1 or predominantly Th2. However, it is often
convenient to consider the families of cytokines in terms of that
described in murine CD4+ ve T cell clones by Mosmann and Coffman
(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells:
different patterns of lymphokine secretion lead to different
functional properties. Annual Review of Immunology, 7, p 145-173).
Traditionally, Th1-type responses are associated with the
production of the INF-.gamma. and IL-2 cytokines by T-lymphocytes.
Other cytokines often directly associated with the induction of
Th1-type immune responses are not produced by T-cells, such as
IL-12. In contrast, Th2-type responses are associated with the
secretion of Il-4, IL-5, IL-6, IL-10. Suitable adjuvant systems
which promote a predominantly Th1 response include, Monophosphoryl
lipid A or a derivative thereof, particularly 3-de-O-acylated
monophosphoryl lipid A, and a combination of monophosphoryl lipid
A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL)
together with an aluminium salt.
[0066] An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative, particularly the
combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO 96/33739.
[0067] A particularly potent adjuvant formulation involving QS21,
3D-MPL and tocopherol in an oil in water emulsion is described in
WO 95/17210, and is a preferred formulation.
[0068] Preferably the vaccine additionally comprises a saponin,
more preferably QS21. The formulation may also comprises an oil in
water emulsion and tocopherol (WO 95/17210).
[0069] The present invention also provides a method for producing a
vaccine formulation comprising mixing a protein of the present
invention together with a pharmaceutically acceptable excipient,
such as 3D-MPL.
[0070] Unmethylated CpG containing oligonucleotides (WO 96/02555)
are also preferential inducers of a TH1 response and are suitable
for use in the present invention.
[0071] Particularly preferred compositions of the invention
comprise one or more conjugated pneumococcal polysaccharides, one
or more pneumococcal proteins and a Th1 adjuvant. The induction of
a cell mediated response by way of a pneumococcal protein (as
described above) and the cooperation between both arms of the
immuen system may be aided using such a Th-1 adjuvant, resulting in
a particularly effective vaccine against pneumococcal disease in
general, and, importantly, against pneumococcal pneumonia in the
elderly.
[0072] In a further aspect of the present invention there is
provided an immunogen or vaccine as herein described for use in
medicine.
[0073] In a still further aspect of the invention, a composition is
provided comprising a pneumococcal polysaccharide conjugate and a
Th1 adjuvant (preferably 3D-MPL) which is capable of seroconverting
or inducing a humoral antibody response against the polysaccharide
antigen within a population of non-responders.
[0074] 10-30% of the population are known to be non-responders to
polysaccharide immunization (do not respond to more than 50% of
serotypes in a vaccine) (Konradsen et al., Scand. J. Immun 40:251
(1994); Rodriguez et al. JID, 173:1347 (1996)). This can also be
the case for conjugated vaccines (Musher et al. Clin. Inf. Dis.
27:1487 (1998)). This can be particularly serious for high risk
areas of the population (infants, toddlers and the elderly).
[0075] The present inventors have found that a combination of a
conjugated pneumococcal polysaccharide (which is prone to low
response in a particular population) with a Th1 adjuvant (see "Th1
adjuvants of the invention" above) can surprisingly overcome this
non-responsiveness. Preferably 3D-MPL should be used, and most
preferably 3D-MPL devoid of aluminium-based adjuvant (which
provides a better response still). The present invention thus
provides such compositions, and further provides a method of
treating non-responders to pneumococcal polysaccharides by
administering such compositions, and still further provides a use
of a Th1 adjuvant in the manufacture of a medicament comprising
conjugated pneumococcal polysaccharide antigens, in the treatment
against (or protection from) pneumococcal disease in individuals
which are non-responsive to the polysaccharide antigen.
[0076] In one embodiment there is a method of preventing or
ameliorating pneumonia in an elderly human comprising administering
a safe and effective amount of a vaccine, as described herein,
comprising a Streptoccocus pneumoniae polysaccharide antigen and
either a Th1 adjuvant, or a pneumococcal protein (and preferably
both), to said elderly patient.
[0077] In a further embodiment there is provided a method of
preventing or ameliorating otitis media in Infants or toddlers,
comprising administering a safe and effective amount of a vaccine
comprising a Streptococcus pneumoniae polysaccharide antigen and
either a Streptococcus pneumoniae protein antigen or a Th1 adjuvant
(and preferably both), to said Infant or toddler.
[0078] Preferably in the methods of the invention as descibed above
the polysaccharide antigen is present as a polysaccharide protein
conjugate.
Vaccine Preparations of the Invention
[0079] The vaccine preparations of the present invention may be
used to protect or treat a mammal susceptible to infection, by
means of administering said vaccine via systemic or mucosal route.
These administrations may include injection via the intramuscular,
intraperitoneal, intradermal or subcutaneous routes; or via mucosal
administration to the oral/alimentary, respiratory, genitourinary
tracts. Intranasal administration of vaccines for the treatment of
pneumonia or otitis media is preferred (as nasopharyngeal carriage
of pneumococci can be more effectively prevented, thus attenuating
infection at its earliest stage).
[0080] The amount of conjugate antigen in each vaccine dose is
selected as an amount which induces an immunoprotective response
without significant, adverse side effects in typical vaccines. Such
amount will vary depending upon which specific immunogen is
employed and how it is presented. Generally, it is expected that
each dose will comprise 0.1-100 .mu.g of polysaccharide, preferably
0.1-50 .mu.g, preferably 0.1-10 .mu.g, of which 1 to 5 .mu.g is the
most preferable range.
[0081] The content of protein antigens in the vaccine will
typically be in the range 1-100 .mu.g, preferably 5-50 .mu.g, most
typically in the range 5-25 .mu.g.
[0082] Optimal amounts of components for a particular vaccine can
be ascertained by standard studies involving observation of
appropriate immune responses in subjects. Following an initial
vaccination, subjects may receive one or several booster
immunisations adequately spaced.
[0083] Vaccine preparation is generally described in Vaccine Design
("The subunit and adjuvant approach" (eds Powell M. F. & Newman
M. J.) (1995) Plenum Press New York). Encapsulation within
liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
B) Selected Pneumococcal Polysaccharide Conjugate+3D-MPL
Compositions
[0084] For the purposes of this invention, the term "pneumococcal
polysaccharide conjugates of the invention" describes those
conjugates of Streptococcus pneumoniae capsular polysaccharides
which are more immunogenic in compositions comprising 3D-MPL in
comparison with compositions comprising 3D-MPL in conjunction with
an aluminium-based adjuvant (for example, conjugates of serotype 4;
serotype 6B; serotype 18C; serotype 19F; or serotype 23F).
[0085] For the purposes of this invention, the term "substantially
devoid of aluminium-based adjuvants" describes a composition which
does not contain sufficient aluminium-based adjuvant (for example
aluminium hydroxide, and, particularly, aluminium phosphate) to
cause any decrease in the immunogenicity of a pneumococcal
polysaccharide conjugate of the invention in comparison to an
equivalent composition comprising 3D-MPL with no added
aluminium-based adjuvant. Preferably the antigenic composition
should contain adjuvant that consists essentially of 3D-MPL.
Quantitities of aluminium-based adjuvant added per dose should
preferably be less than 50 .mu.g, more preferably less than 30
.mu.g, still more preferably less than 10 .mu.g, and most
preferably there is no aluminium-based adjuvant added to the
antigenic compositions of the invention.
[0086] For the purposes of this invention, the determination of
whether a pneumococcal polysaccharide conjugate is significantly
more immunogenic in compositions comprising 3D-MPL in comparison
with compositions comprising 3D-MPL in conjunction with an
aluminium-based adjuvant, this should be established as described
in Example 2. As an indication of whether a composition is
significantly more immunogenic when comprising 3D-MPL alone, the
ratio of GMC IgG concentration (as determined in Example 2) between
compositions comprising 3D-MPL alone versus an equivalent
composition comprising 3D-MPL in conjunction with aluminium
phosphate adjuvant should be more than 2, preferably more than 5,
more preferably more than 7, still more preferably more than 9, and
most preferably more than 14.
[0087] Amongst the problems associated with the polysaccharide
approach to vaccination, is the fact that polysaccharides per se
are poor immunogens. Strategies, which have been designed to
overcome this lack of immunogenicity, include the linking
(conjugating) of the polysaccharide to large protein carriers,
which provide bystander T-cell help. It is preferred that the
pneumococcal polysaccharides of the invention are linked to a
protein carrier which provides bystander T-cell help. Examples of
such carriers which may be used include the Diphtheria. Diphtheria
mutant, and Tetanus toxoids (DT, CRM197 and TT respectively),
Keyhole Limpet Haemocyanin (KLH), the purified protein derivative
of Tuberculin (PPD), and OMPC of Neisseria meningitidis.
[0088] Most preferably, protein D from Haemophilus influenzae (EP 0
594 610-B), or fragments thereof (see section C), is used as the
immunogenic protein carrier for the pneumococcal polysaccharides of
the invention.
[0089] In one embodiment the antigenic composition of the invention
comprises pneumococcal polysaccharide serotype (PS) 4 conjugated to
an immunogenic protein and formulated with 3D-MPL adjuvant, where
the composition is substantially devoid of aluminium-based
adjuvant. In further embodiments, the antigenic composition
comprises PS 6B, 18C, 19F, or 23F, respectively, conjugated to an
immunogenic protein and formulated with 3D-MPL adjuvant, where the
composition is substantially devoid of aluminium-based
adjuvant.
[0090] In a still further embodiment of the invention, a
combination antigenic composition is provided comprising two or
more pneumococcal polysaccharide conjugates from the group PS 4, PS
6B, PS 18C, PS19F, and PS 23F formulated with 3D-MPL adjuvant,
where the composition is substantially devoid of aluminium-based
adjuvant.
[0091] The immunogenicity of pneumococcal polysaccharide conjugates
of the invention is not significantly effected by combination with
other pneumococcal polysaccharide conjugates (Example 3).
Accordingly, a preferred aspect of the invention provides a
combination antigenic composition comprising one or more
pneumococcal polysaccharide conjugates of the invention in
combination with one or more further pneumococcal polysaccharide
conjugates, where the composition is formulated with 3D-MPL
adjuvant, but is substantially devoid of aluminium-based
adjuvant.
[0092] In further preferred embodiments of the invention,
combination antigenic compositions are provided which contain at
least one and preferably 2, 3, 4 or all 5 of the PS 4, 6B, 18C,
19F, or 23F pneumococcal polysaccharide conjugates, and in addition
any combination of other pneumococcal polysaccharide conjugates,
which are formulated with 3D-MPL adjuvant but substantially devoid
of aluminium-based adjuvant.
[0093] Typically the Streptococcus pneumoniae combination antigenic
composition of the present invention will comprise polysaccharide
conjugate antigens, wherein the polysaccharides are derived from at
least four, seven, eleven, thirteen, fifteen or twenty-three
serotypes (see "Streptococcus pneumoniae Polysaccharide Antigens of
the Invention" above for preferred combinations of serotypes
depending on the disease to be treated).
[0094] The antigenic compositions of the invention are preferably
used as vaccine compositions to prevent (or treat) pneumococcal
infections, particularly in the elderly and infants and
toddlers.
[0095] Further embodiments of the present invention include: the
provision of the above antigenic compositions for use in medicine;
a method of inducing an immune response to a Streptococcus
pneumoniae capsular polysaccharide conjugate, comprising the steps
of administering a safe and effective amount of one of the above
antigenic compositions to a patient; and the use of one of the
above antigenic compositions in the manufacture of a medicament for
the prevention (or treatment) of pneumococcal disease.
[0096] For the prevention/amelioration of pneumonia in the elderly
(+55 years) population and Otitis media in Infants (up to 18
months) and toddlers (typically 18 months to 5 years), it is a
further preferred embodiment of the invention to combine a
multivalent Streptococcus pneumonia polysaccharide conjugate
formulated as herein described with a Streptococcus pneumoniae
protein or immunologically functional equivalent thereof. See above
section "Pneumococcal Proteins of the invention" for preferred
proteins/protein combinations.
[0097] Preferably the antigenic compositions (and vaccines)
hereinbefore described are lyophilised up until they are about to
be used, at which point they are extemporaneously reconstituted
with diluent. More preferably they are lyophilsed in the presence
of 3D-MPL, and are extemporaneously reconstituted with saline
solution.
[0098] Lyophilising the compositions results in a more stable
composition (for instance it prevents the breakdown of the
polysaccharide antigens). The process is also surprisingly
responsible for a higher antibody titre still against the
pneumococcal polysaccharides. This has been shown to be
particularly significant for PS 6B conjugates. Another aspect of
the invention is thus a lyophilised antigenic composition
comprising a PS 6B conjugate adjuvanted with 3D-MPL and
substantially devoid of aluminium-based adjuvants.
[0099] For preparation of the vaccines, see above "Vaccine
Preparations of the Invention" section.
C) Bacterial Polysaccharide-Protein D Conjugates
[0100] The trend towards combination vaccines has the advantage of
reducing, discomfort to the recipient, facilitating scheduling, and
ensuring completion of regiment; but there is also the concomitant
risk of reducing the vaccine's efficacy (see above for discussion
on epitope suppression through overuse of carrier proteins). It
would be, therefore, advantageous to make vaccine combinations
which meet the needs of a population, and which, in addition, do
not exhibit immunogenic interference between their components.
These advantages may be realised by the immunogenic compositions
(or vaccines) of the invention, which are of particular benefit for
administration of combination vaccines to high risk groups such
infants, toddlers or the elderly.
[0101] The present invention provides a protein D from Haemophilus
influenzae, or fragments thereof, as a carrier for polysaccharide
based immunogenic composition, including vaccines. Fragments
suitable for use include fragments encompassing T-helper epitopes.
In particular protein D fragment will preferably contain the
N-terminal 1/3 of the protein.
[0102] Protein D is an IgD-binding protein from Haemophilus
influenzae (EP 0 594 610 B1) and is a potential immunogen.
[0103] Polysaccharides to be conjugated to Protein D contemplated
by the present invention include, but are not limited to the Vi
polysaccharide antigen against Salmonella typhi, meningococcal
polysaccharides (including type A, C, W135 and Y, and the
polysaccharide and modified polysaccharides of group B
meningococcus), polysaccharides from Staphylococcus aureus,
polysaccharides from Streptococcus agalactae, polysaccharides from
Streptococcus pneumoniae, polysaccharides from Mycobacterium e.g.
Mycobacterium tuberculosis (such as mannophosphoinisitides
trehaloses, mycolic acid, mannose capped arabinomannans, the
capsule therefrom and arabinogalactans), polysaccharide from
Cryptococcus neoformans, the lipopolysaccharides of non-typeable
Haemophilus influenzae, the capsular polysaccharide from
Haemophilus influenzae b, the lipopolysaccharides of Moraxella
catharralis, the lipopolysaccharides of Shigella sonnei, the
lipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi, the cancer
associated gangliosides GD3, GD2, the tumor associated mucins,
especially the T-F antigen, and the sialyl T-F antigen, and the HIV
associated polysaccharide that is structurally related to the T-F
antigen.
[0104] The polysaccharide may be linked to the carrier protein by
any known method (for example, by Likhite, U.S. Pat. No. 4,372,945
and by Armor et al., U.S. Pat. No. 4,474,757). Preferably, CDAP
conjugation is carried out (WO 95/08348).
[0105] In CDAP, the cyanylating reagent
1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP) is
preferably used for the synthesis of polysaccharide-protein
conjugates. The cyanilation reaction can be performed under
relatively mild conditions, which avoids hydrolysis of the alkaline
sensitive polysaccharides. This synthesis allows direct coupling to
a carrier protein.
[0106] The polysaccharide is solubilized in water or a saline
solution. CDAP is dissolved in acetonitrile and added immediately
to the polysaccharide solution. The CDAP reacts with the hydroxyl
groups of the polysaccharide to form a cyanate ester. After the
activation step, the carrier protein is added. Amino groups of
lysine react with the activated polysaccharide to form an isourea
covalent link.
[0107] After the coupling reaction, a large excess of glycine is
then added to quench residual activated functions. The product is
then passed through a gel permeation to remove unreacted carrier
protein and residual reagents. Accordingly the invention provides a
method of producing polysaccharide protein D conjugates comprising
the steps of activating the polysaccharide and linking the
polysaccharide to the protein D.
[0108] In a preferred embodiment of the invention there is provided
an immunogenic composition (or vaccine) formulation for the
prevention of Streptococcus pneumoniae infections.
[0109] The mechanisms by which pneumococci spread to the lung, the
cerebrospinal fluid and the blood is poorly understood. Growth of
bacteria reaching normal lung alveoli is inhibited by their
relative dryness and by the phagocytic activity of alveolar
macrophages. Any anatomic or physiological changes of these
co-ordinated defences tend to augment the susceptibility of the
lungs to infection. The cell-wall of Streptococcus pneumoniae has
an important role in generating an inflammatory response in the
alveoli of the lung (Gillespie et al. (1997), I&I 65:
3936).
[0110] Typically the Streptococcus pneumoniae vaccine of the
present invention will comprise protein D polysaccharide
conjugates, wherein the polysaccharide is derived from at least
four, seven, eleven, thirteen, fifteen or 23 serotypes. See above
"Streptococcus pneumoniae Polysaccharide Antigens of the Invention"
for preferred combinations of serotypes depending on the disease to
be treated.
[0111] In a further embodiment of the invention there is provided a
Neisseria meningitidis vaccine; in particular from serotypes A, B,
C W-135 and Y. Neisseria meningitides is one of the most important
causes of bacterial meningitis. The carbohydrate capsule of these
organisms can act as a virulence determinant and a target for
protective antibody. Carbohydrates are nevertheless well known to
be poor immunogens in young children. The present invention
provides a particularly suitable protein carrier for these
polysaccharides, protein D, which provides T-cell epitopes that can
activate a T-cell response to aid polysaccharide antigen specific
B-cell proliferation and maturation, as well as the induction of an
immunological memory.
[0112] In an alternative embodiment of the invention there is
provided a capsular polysaccharide of Haemophilus influenzae b
(PRP)-protein D conjugate.
[0113] The present invention also contemplates combination vaccines
which provide protection against a range of different pathogens. A
protein D carrier is surprisingly useful as a carrier in
combination vaccines where multiple polysaccharide antigens are
conjugated. As mentioned above, epitope suppression is likely to
occur if the same carrier is used for each polysaccharide. WO
98/51339 presented compositions to try to minimise this
interference by conjugating a proportion of the polysaccharides in
the composition onto DT and the rest onto TT.
[0114] Surprisingly, the present inventors have found protein D is
particularly suitable for minimising such epitopic suppression
effects in combination vaccines. One or more polysaccharides in a
combination may be advantageously conjugated onto protein D, and
preferably all antigens are conjugated onto protein D within such
combination vaccines.
[0115] A preferred combination includes a vaccine that affords
protection against Neisseria meningitidis C and Y (and preferably
A) infection wherein the polysaccharide antigen from one or more of
serotypes Y and C (and most preferably A) are linked to protein
D.
[0116] Haemophilus influenzae polysaccharide based vaccine (PRP
conjugated with preferably TT, DT or CRM197, or most preferably
with protein D) may be formulated with the above combination
vaccines.
[0117] Many Paediatric vaccines are now given as a combination
vaccine so as to reduce the number of injections a child has to
receive. Thus for Paediatric vaccines other antigens may be
formulated with the vaccines of the invention. For example the
vaccines of the invention can be formulated with, or administered
separately, but at the same time with the well known `trivalent`
combination vaccine comprising Diphtheria toxoid (DT), tetanus
toxoid (CT), and pertussis components [typically detoxified
Pertussis toxoid (PT) and filamentous haemagglutinin (FHA) with
optional pertactin (PRN) and/or agglutinin 1+2], for example the
marketed vaccine INFANRIX-DTPa.TM. (SmithKlineBeecham Biologicals)
which contains DT, TT, PT, FHA and PRN antigens, or with a whole
cell pertussis component for example as marketed by
SmithKlineBeecham Biologicals s.a., as Tritanrix.TM.. The combined
vaccine may also comprise other antigen, such as Hepatitis B
surface antigen (HBsAg), Polio virus antigens (for instance
inactivated trivalent polio virus--IPV), Moraxella catarrhalis
outer membrane proteins, non-typeable Haemophilus influenzae
proteins, N. meningitidis B outer membrane proteins.
[0118] Examples of preferred Moraxella catarrhalis protein antigens
which can be included in a combination vaccine (especially for the
prevention of otitis media) are: OMP106 [WO 97/41731 (Antex) &
WO 96/34960 (PMC)]; OMP21; LbpA & LbpB [WO 98/55606 (PMC)];
ThpA & TbpB [WO 97/13785 & WO 97/32980 (PMC)]; CopB
[Helminen M E, et al. (1993) Infect. Immun. 61:2003-2010]; UspA1/2
[WO 93/03761 (University of Texas)]; and OmpCD. Examples of
non-typeable Haemophilus influenzae antigens which can be included
in a combination vaccine (especially for the prevention of otitis
media) include: Fimbrin protein [(U.S. Pat. No. 5,766,608--Ohio
State Research Foundation)] and fusions comprising peptides
therefrom [eg LB 1(f) peptide fusions; U.S. Pat. No. 5,843,464
(OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673
(State University of New York)]; TbpA and TbpB; Hia; Hmw1,2; Hap;
and D15.
[0119] Preferred Peadiatric vaccines contemplated by the present
invention are: [0120] a) N. meningitidis C polysaccharide conjugate
and Haemophilus influenzae b polysaccharide conjugate, optionally
with N. meningitidis A and/or Y polysaccharide conjugate, provided
that at least one polysaccharide antigen, and preferably all are
conjugated to protein D. [0121] b) Vaccine a) with, DT, TT,
pertussis components (preferable PT, FHA and PRN), Hepatitis B
surface antigen and IPV (inactivated trivalent poliovirus vaccine).
[0122] c) Streptococcus pneumoniae polysaccharide antigens
conjugated to protein D. [0123] d) Vaccine c) with one or more
antigens from Moraxella catarrhalis and/or non-typeable Haemophilus
influenzae.
[0124] All the above combination vaccines, can benefit from the
inclusion of protein D as a carrier. Clearly, the more carriers
that are involved in a combination vaccine (for instance to
overcome epitope suppression), the more expensive and complex the
final vaccine. Having all, or the majority, of the polysaccharide
antigens of a combination vaccine conjugated to protein D thus
provides a considerable advantage
[0125] For the prevention of pneumonia in the elderly (+55 years)
population and Otitis media in Infants or toddlers, it is a
preferred embodiment of the invention to combine a multivalent
streptococcus pneumonia polysaccharide-protein D antigens as herein
described with a Streptococcus pneumoniae protein or
immunologically functional equivalent thereof. See above section
"Pneumococcal Proteins of the invention" for preferred
proteins/protein combinations that can be included in such a
combination.
[0126] Accordingly the present invention provides an immunogenic
composition comprising a Streptococcus pneumoniae
polysaccharide-protein D conjugate and a Streptococcus pneumoniae
protein antigen.
[0127] The polysaccharide-protein D conjugate antigens of the
present invention are preferably adjuvanted in the vaccine
formulation of the invention. Suitable adjuvants include an
aluminium salt such as aluminum hydroxide gel (alum) or aluminium
phosphate, but may also be a salt of calcium, iron or zinc, or may
be an insoluble suspension of acylated tyrosine, or acylated
sugars, cationically or anionically derivatised polysaccharides, or
polyphosphazenes.
[0128] For elderly vaccines it is preferred that the adjuvant be
selected to be a preferential inducer of a TH1 type of
response.
[0129] For particular Th1 adjuvants see "Th1 adjuvants of the
invention" above.
[0130] In a further aspect of the present invention there is
provided an immunogen or vaccine as herein described for use in
medicine.
[0131] For vaccine preparation/administration of the conjugate, see
"Vaccine Preparation of the Invention" above.
[0132] Protein D is also advantageously used in a vaccine against
otitis media, as it is in itself an immunogen capable of producing
B-cell mediated protection against non-typeable H. influenzae
(ntHi). ntHi may invade host cells, and evade the B-cell mediated
effects induced by the protein antigen. The present inventors have
surprisingly found a way of increasing the effectiveness of protein
D (either by itself or as a carrier for a polysaccharide) as an
antigen for an otitis media vaccine. This is done by adjuvanting
the protein D such that a strong Th1 response is induced in the
subject such that the cell mediated arm of the immune system is
optimised against protein D. This is surprisingly achieved using a
lyophilised composition comprising protein D and a Th1 adjuvant
(preferably 3D-MPL) which is reconstituted shortly before
administration. The invention thus also provides such compositions,
a process for making such compositions (by lyophilising a mixture
comprising protein D and a Th1 adjuvant), and a use of such a
composition in the treatment of otitis media.
[0133] In a broader sense, the inventors envisage that lyophilising
an immunogen in the presence of a Th1 adjuvant (see "Th1 adjuvants
of the invention"), preferably 3D-MPL, will generally augment the
Th1 immune response against the immunogen. The present invention is
therefore applicable to any immunogen to which a stronger Th1
immune response is required. Such immunogens comprise bacterial,
viral and tumour protein antigens, as well as self proteins and
peptides.
EXAMPLES
[0134] The examples illustrate, but do not limit the invention.
Example 1
S. pneumoniae Capsular Polysaccharide:
[0135] The 11-valent candidate vaccine includes the capsular
polysaccharides serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and
23F which were made essentially as described in EP 72513. Each
polysaccharide is activated and derivatised using CDAP chemistry
(WO 95/08348) and conjugated to the protein carrier. All the
polysaccharides are conjugated in their native form, except for the
serotype 3 (which was size-reduced to decrease its viscosity).
Protein Carrier:
[0136] The protein carrier selected is the recombinant protein D
(PD) from Non typeable Haemophilus influenzae, expressed in E
coli.
Expression of Protein D
Haemophilus influenzae Protein D
Genetic Construction for Protein D Expression
Starting Materials
The Protein D Encoding DNA
[0137] Protein D is highly conserved among H. influenzae of all
serotypes and non-typeable strains. The vector pHIC348 containing
the DNA sequence encoding the entire protein D gene has been
obtained from Dr. A. Forsgren, Department of Medical Microbiology,
University of Lund, Malmo General Hospital, Malmo, Sweden. The DNA
sequence of protein D has been published by Janson et al. (1991)
Infect. Immun. 59: 119-125.
The Expression Vector pMG1
[0138] The expression vector pMG1 is a derivative of pBR322 (Gross
et al., 1985) in which bacteriophage .lamda. derived control
elements for transcription and translation of foreign inserted
genes were introduced (Shatzman et al., 1983). In addition, the
Ampicillin resistance gene was exchanged with the Kanamycin
resistance gene.
The E. coli Strain AR58
[0139] The E. coli strain AR58 was generated by transduction of N99
with a P1 phage stock previously grown on an SA500 derivative
(galE::TN10, lambdaKil.sup.- cI857 .DELTA.H1). N99 and SA500 are E.
coli K12 strains derived from Dr. Martin Rosenberg's laboratory at
the National Institute of Health.
The Expression Vector pMG 1
[0140] For the production of protein D, the DNA encoding the
protein has been cloned into the expression vector pMG 1. This
plasmid utilises signals from lambdaphage DNA to drive the
transcription and translation of inserted foreign genes. The vector
contains the promoter PL, operator OL and two utilisation sites
(NutL and NutR) to relieve transcriptional polarity effects when N
protein is provided (Gross et al., 1985). Vectors containing the PL
promoter, are introduced into an E. coli lysogenic host to
stabilise the plasmid DNA. Lysogenic host strains contain
replication-defective lambdaphage DNA integrated into the genome
(Shatzman et al., 1983). The chromosomal lambdaphage DNA directs
the synthesis of the cI repressor protein which binds to the OL
repressor of the vector and prevents binding of RNA polymerase to
the PL promoter and thereby transcription of the inserted gene. The
cI gene of the expression strain AR58 contains a temperature
sensitive mutant so that PL directed transcription can be regulated
by temperature shift, i.e. an increase in culture temperature
inactivates the repressor and synthesis of the foreign protein is
initiated. This expression system allows controlled synthesis of
foreign proteins especially of those that may be toxic to the cell
(Shimataka & Rosenberg, 1981).
The E. coli Strain AR58
[0141] The AR58 lysogenic E. coli strain used for the production of
the protein D carrier is a derivative of the standard NIH E. coli
K12 strain N99 (F.sup.- su.sup.- galK2, lacZ.sup.- thr.sup.-). It
contains a defective lysogenic lambdaphage (galE::TN10,
lambdaKil.sup.- cI857 .DELTA.H 1). The Kil.sup.- phenotype prevents
the shut off of host macromolecular synthesis. The cI857 mutation
confers a temperature sensitive lesion to the cI repressor. The
.DELTA.H1 deletion removes the lambdaphage right operon and the
hosts bio, uvr3, and chlA loci. The AR58 strain was generated by
transduction of N99 with a P1 phage stock previously grown on an
SA500 derivative (galE::TN10, lambdaKil.sup.- cI857 .DELTA.H1). The
introduction of the defective lysogen into N99 was selected with
tetracycline by virtue of the presence of a TN10 transposon coding
for tetracyclin resistance in the adjacent galE gene.
Construction of Vector pMGMDPPrD
[0142] The pMG 1 vector which contains the gene encoding the
non-structural S1 protein of Influenzae virus (pMGNSI) was used to
construct pMGMDPPrD. The protein D gene was amplified by PCR from
the pHIC348 vector (Janson et al. 1991) with PCR primers containing
NcoI and XbaI restriction sites at the 5' and 3' ends,
respectively. The NcoI/XbaI fragment was then introduced into
pMGNS1 between NcoI and XbaI thus creating a fusion protein
containing the N-terminal 81 amino acids of the NS1 protein
followed by the PD protein. This vector was labeled pMGNS1 PrD.
[0143] Based on the construct described above the final construct
for protein D expression was generated. A BamHI/BamHI fragment was
removed from pMGNS1PrD. This DNA hydrolysis removes the NS1 coding
region, except for the first three N-terminal residues. Upon
religation of the vector a gene encoding a fusion protein with the
following N-terminal amino acid sequence has been generated:
TABLE-US-00001 -----MDP SSHSSNMANT----- NS1 Protein D
[0144] The protein D does not contain a leader peptide or the
N-terminal cysteine to which lipid chains are normally attached.
The protein is therefore neither excreted into the periplasm nor
lipidated and remains in the cytoplasm in a soluble form.
[0145] The final construct pMG-MDPPrD was introduced into the AR58
host strain by heat shock at 37.degree. C. Plasmid containing
bacteria were selected in the presence of Kanamycin. Presence of
the protein D encoding DNA insert was demonstrated by digestion of
isolated plasmid DNA with selected endonucleases. The recombinant
E. coli strain is referred to as ECD4.
[0146] Expression of protein D is under the control of the lambda
P.sub.L promoter/O.sub.L Operator. The host strain AR58 contains a
temperature-sensitive cI gene in the genome which blocks expression
from lambda P.sub.L at low temperature by binding to O.sub.L. Once
the temperature is elevated cI is released from O.sub.L and protein
D is expressed. At the end of the fermentation the cells are
concentrated and frozen.
[0147] The extraction from harvested cells and the purification of
protein D was performed as follows. The frozen cell culture pellet
is thawed and resuspended in a cell disruption solution (Citrate
buffer pH 6.0) to a final OD.sub.650=60. The suspension is passed
twice through a high pressure homogenizer at P=1000 bar. The cell
culture homogenate is clarified by centrifugation and cell debris
are removed by filtration. In the first purification step the
filtered lysate is applied to a cation exchange chromatography
column (SP Sepharose Fast Flow). PD binds to the gel matrix by
ionic interaction and is eluted by a step increase of the ionic
strength of the elution buffer.
[0148] In a second purification step impurities are retained on an
anionic exchange matrix (Q Sepharose Fast Flow). PD does not bind
onto the gel and can be collected in the flow through.
[0149] In both column chromatography steps fraction collection is
monitored by OD. The flow through of the anionic exchange column
chromatography containing the purified protein D is concentrated by
ultrafiltration.
[0150] The protein D containing ultrafiltration retentate is
finally passed through a 0.2 .mu.m membrane.
Chemistry:
Activation and Coupling Chemistry:
[0151] The activation and coupling conditions are specific for each
polysaccharide. These are given in Table 1. Native polysaccharide
(except for PS3) was dissolved in NaCl 2M or in water for
injection. The optimal polysaccharide concentration was evaluated
for all the serotypes.
[0152] From a 100 mg/ml stock solution in acetonitrile, CDAP
(CDAP/PS ratio 0.75 mg/mg PS) was added to the polysaccharide
solution. 1.5 minute later. 0.2M triethylamine was added to obtain
the specific activation pH. The activation of the polysaccharide
was performed at this pH during 2 minutes at 25.degree. C. Protein
D (the quantity depends on the initial PS/PD ratio) was added to
the activated polysaccharide and the coupling reaction was
performed at the specific pH for 1 hour. The reaction was then
quenched with glycine for 30 minutes at 25.degree. C. and overnight
at 4.degree. C.
[0153] The conjugates were purified by gel filtration using a
Sephacryl 500HR gel filtration column equilibrated with 0.2M
NaCl.
[0154] The carbohydrate and protein content of the eluted fractions
was determined. The conjugates were pooled and sterile filtered on
a 0.22 .mu.m sterilizing membrane. The PS/Protein ratios in the
conjugate preparations were determined.
Characterisation:
[0155] Each conjugate was characterised and met the specifications
described in Table 2. The polysaccharide content (.mu.g/ml) was
measured by the Resorcinol test and the protein content (.mu.g/ml)
by the Lowry test. The final PS/PD ratio (w/w) is determined by the
ratio of the concentrations.
Residual DMAP Content (ng/.mu.g PS):
[0156] The activation of the polysaccharide with CDAP introduces a
cyanate group in the polysaccharide and DMAP
(4-dimethylamino-pyridin) is liberated. The residual DMAP content
was determined by a specific assay developed at SB.
Free Polysaccharide Content (%):
[0157] The free polysaccharide content of conjugates kept at
4.degree. C. or stored 7 days at 37.degree. C. was determined on
the supernatant obtained after incubation with .alpha.-PD
antibodies and saturated ammonium sulfate, followed by a
centrifugation.
[0158] An .alpha.-PS/.alpha.-PS ELISA was used for the
quantification of free polysaccharide in the supernatant. The
absence of conjugate was also controlled by an
.alpha.-PD/.alpha.-PS ELISA. Reducing the quantity of free
polysaccharide results in an improved conjugate vaccine.
Antigenicity:
[0159] The antigenicity on the same conjugates was analyzed in a
sandwich-type ELISA wherein the capture and the detection of
antibodies were .alpha.-PS and .alpha.-PD respectively.
Free Protein Content (%):
[0160] The level of "free" residual protein D was determined by
using a method with SDS treatment of the sample. The conjugate was
heated 10 min at 100.degree. C. in presence of SDS 0.1% and
injected on a SEC-HPLC gel filtration column (TSK 3000-PWXL). As
protein D is dimer, there is a risk of overestimating the level of
"free" protein D by dissociation the structure with SDS.
Molecular Size (K.sub.av):
[0161] The molecular size was performed on a SEC-HPLC gel
filtration column (TSK 5000-PWXL).
Stability:
[0162] The stability was measured on a HPLC-SEC gel filtration (TSK
6000-PWXL) for conjugates kept at 4.degree. C. and stored for 7
days at 37.degree. C.
[0163] The 11-valent characterization is given in Table 2
[0164] The protein conjugates can be adsorbed onto aluminium
phosphate and pooled to form the final vaccine.
Conclusion:
[0165] Immunogenic conjugates have been produced, that have since
been shown to be components of a promising vaccine. The optimised
CDAP conditions for the best quality final conjugated pneumococcal
polysaccharide product was discovered for each of the 11 valencies.
Conjugates of these pneumococcal polysaccharides obtainable by the
above improved (optimised) CDAP process (regardless of the carrier
protein, but preferably protein D) is thus a further aspect of the
invention.
Example 2
Study of the Effect of Advanced Adjuvants on the Immunogenicity of
the 11-Valent Pneumococcal PS-PD Conjugate Vaccine in Infant
Rats
[0166] Infant rats were immunised with 11 valent pneumococcal PS-PD
conjugate vaccine at a dosage of 0.1 .mu.g each polysaccharide
(made according to the method of Example 1), and using the
following adjuvant formulations: none, AlPO.sub.4, 3D-MPL, 3D-MPL
on AlPO.sub.4.
[0167] The formulation with only 3D-MPL was statistically (and
surprisingly) more immunogenic (greatest GMC IgG) than for the
other formulations for 5 out of 11 antigens. This was true both at
high and low concentrations of 3D-MPL.
[0168] Opsonophagocytosis confirmed the GMC results.
Materials and Methods
Immunisation Protocol
[0169] Infant OFA rats were randomised to different mothers and
were 7 days old when they received the first immunisation. They
received 2 additional immunisations 14 and 28 days later. A bleed
was performed on day 56 (28 days post 111). All vaccines were
injected s.c., and there were 10 rats per vaccine group.
[0170] The rats were immunised with an 11 valent pneumococcal
conjugate vaccine comprising the following polysaccharide serotypes
conjugated onto protein D: 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F,
23F.
Formulation
[0171] To examine the effect of different advanced adjuvants, the
dosage of conjugate was held constant at 0.1 .mu.g of each
polysaccharide, and the adjuvants AlPO.sub.4 and 3D-MPL were
formulated in different dosages and combinations, including no
adjuvant at all. These are listed numerically in Table 3 for
reference.
Adsorption on AlPO.sub.4
[0172] The concentrated, adsorbed monovalents were prepared
according to the following procedure. 50 .mu.g AlPO.sub.4 (pH 5.1)
was mixed with 5 .mu.g conjugated polysaccharides for 2 hours. The
pH was adjusted to pH 5.1 and the mixture was left for a further 16
hours. 1500 mM NaCl was added to make up the salt concentration to
150 mM. After 5 minutes 5 mg/mL 2-phenoxyethanol was added. After a
further 30 minutes the pH was adjusted to 6.1, and left for more
than 3 days at 4.degree. C.
Preparation of Diluents
[0173] Three diluents were prepared in NaCl 150 mM/5 mg/mL
phenoxyethanol [0174] A: AlPO.sub.4 at 1 mg/ml. [0175] B: 3D-MPL on
AlPO.sub.4 at 250 and 1000 .mu.g/ml respectively Weight ratio
3D-MPL/AlPO.sub.4=5/20 [0176] C: 3D-MPL on AlPO.sub.4 at 561 and
1000 .mu.g/ml respectively Weight ratio 3D-MPL/AlPO.sub.4=50/89
Preparation of Adsorbed Undecavalent
[0177] The eleven concentrated, adsorbed PS-PD monovalents were
mixed at the correct ratio. The complement of AlPO.sub.4 was added
as the diluent A. When required, 3D-MPL was added either as an
aqueous solution (non adsorbed, Way 1 see below) or as the diluent
B or C (3D-MPL adsorbed on AlPO.sub.4 at 2 doses, Way 2, see
below).
Way 1
[0178] 3D-MPL was added to the combined adsorbed conjugates as an
aqueous suspension. It was mixed to the undecavalent for 10 minutes
at room temperature and stored at 4.degree. C. until
administration.
Way 2
[0179] 3D-MPL was preadsorbed onto AlPO.sub.4 before addition to
the combined adsorbed conjugates (diluent B and C). To prepare 1 ml
of diluent, an aqueous suspension of 3D-MPL (250 or 561 .mu.g) was
mixed with 1 mg of AlPO.sub.4 in NaCl 150 mM pH 6.3 for 5 min at
room temperature. This solution was diluted in NaCl pH 6.1/phenoxy
and incubated overnight at 4.degree. C.
Preparation of Non-Adsorbed Undecavalent
[0180] The eleven PS-PD conjugates were mixed and diluted at the
right ratio in NaCl 150 mM pH 6.1, phenoxy. When required, 3D-MPL
was added as a solution (non adsorbed).
[0181] The formulations for all injections were prepared 18 days
before the first administration.
ELISA
[0182] The ELISA was performed to measure rat IgG using the
protocol derived from the WHO Workshop on the ELISA procedure for
the quantitation of IgG antibody against Streptococcus pneumoniae
capsular polysaccharides in human serum. In essence, purified
capsular polysaccharide is coated directly on the microtitre plate.
Serum samples are pre-incubated with the cell-wall polysaccharide
common to all pneumococcus (substance C) and which is present in
ca. 0.5% in pneumococcal polysaccharides purified according to
disclosure (EP 72513 B1). Jackson ImmunoLaboratories Inc. reagents
were employed to detect bound murine IgG. The titration curves were
referenced to internal standards (monoclonal antibodies) modeled by
logistic log equation. The calculations were performed using
SoftMax Pro software. The maximum absolute error on these results
expected to be within a factor of 2. The relative error is less
than 30%.
Opsonophagocytosis
[0183] Opsonic titres were determined for serotypes 3, 6B, 7F, 14,
19F and 23F using the CDC protocol (Streptococcus pneumoniae
Opsonophagocytosis using Differentiated HL60 cells, version 1.1)
with purified human PMN and baby rabbit complement. Modification
included the use of in-house pneumococcal strains, and the
phagocytic HL60 cells were replaced by purified human neutrophils
PMN (there is a high degree of correlation between these phagocytic
cells). In addition, 3 mm glass beads were added to the microtitre
wells to increase mixing, and this allowed reduction of the
phagocyte:bacteria ratio which was recommended to be 400.
Results
IgG Concentrations
[0184] The geometric mean IgG concentrations determined for every
serotype, and PD are shown in Tables 4 to 10. For serotypes 6B, 14,
19F and 23F, previous results obtained using a tetravalent
formulation are included for comparison.
[0185] The highest IgG concentrations have been highlighted in
Tables 4 to 10. The statistical p value for 3D-MPL compositions vs.
3D-MPL/AlPO.sub.4 compositions is in Table 11. Adjuvant formulation
number 4 (non-adsorbed conjugates with high dose 3D-MPL) that gives
the highest GMC's for 9 out of 11 cases. In 5/11 cases, MPL at the
low dose is the second most immunogenic. In addition, adjuvantation
gives higher GMC's than by modifying the dose for all serotypes
(data not shown), and this is statistically significant for
serotypes 4, 6B, 7F, 18C and 23F (p<0.05 from 95% CI).
Opsonophagocytosis
[0186] Opsonophagocytosis results on pooled sera is shown for
serotypes 3, 6B, 7F, 14, 19F and 23F in Tables 4 to 8. For the most
part, these opsonic titres confirm the GMC IgG. Indeed, the
correlation with IgG concentration is greater than 85% for
serotypes 6B, 19F, 23F (data not shown). For serotype 3, it is
important to note that only the 3D-MPL group induced opsonic
activity above the threshold.
Conclusions
[0187] In this experiment, it was unexpected that the use of 3D-MPL
alone would induce the highest IgG concentrations.
[0188] The maximal GMC IgG obtained with modifying the adjuvant was
compared with the maximal GMC obtained by modifying the PS dosage,
and it was found that 3D-MPL could induce significantly higher
responses in 5/11 serotypes.
[0189] Table 11 shows that when 3D-MPL and 3D-MPL/AlPO.sub.4
compositions are compared (comparing the process of formulation,
and the dose of 3D-MPL), 5 of the polysaccharide conjugates are
significantly improved, in terms of immunogenicity, when formulated
with just 3D-MPL rather than 3D-MPL plus AlPO.sub.4: PS 4, PS 6B,
PS 18C, PS 19F, and PS 23.degree. F.
Example 3
Study of the Effect of Combination on the Immunogenicity of PS 4,
PS 6B, PS 18C, PS 19F, and PS 23F Conjugates in Adult Rats
[0190] Adult rats were immunised with pneumococcal
polysaccharide-protein D conjugate vaccines either individually, or
combined in a multivalent composition (either tetra-, penta-,
hepta-, or decavalent). Groups of 10 rats were immunised twice 28
days apart, and test bleeds were obtained on day 28 and day 42 (14
days after the 2.sup.nd dose).
[0191] The sera were tested by ELISA for IgG antibodies to the
pneumococcal polysaccharides. All conjugates induced specific IgG
antibodies as measured by ELISA. Table 12 shows the effect of
combination of monovalent PS 6B, PS 18C, PS 19F, and PS 23F protein
D conjugates on their immunogenicity in adult rats, as measured by
IgG concnetration at 14 days post 2.sup.nd dose.
[0192] Statistical analysis was performed on all samples to
determine if differences in antibody concentration upon combination
were significant. The combination of any of serotypes PS 6B, PS
18C, PS 19F, and PS 23F protein D conjugates in a multivalent
vaccine did not significantly change their immunogenicity.
TABLE-US-00002 TABLE 1 Specific activation/coupling/quenching
conditions of PS S. pneumoniae-Protein D conjugates 3 Serotype 1
(.mu.fluid.) 4 5 6B 7F PS 2.0 3.0 2.0 7.5 5.4 3.0 conc. (mg/ml) PS
dissolution NaCl NaCl 2M H.sub.2O H.sub.2O NaCl 2M NaCl 2M 2M PD
5.0 5.0 5.0 5.0 5.0 5.0 conc. (mg/ml) Initial PS/PD 1/1 1/1 1/1 1/1
1/1 1/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75 0.75 (mg/mg
PS) pH.sub.a = pH.sub.c = pH.sub.q 9.0/9.0/9.0 9.0/9.0/9.0
9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 Serotype 9V 14 18C
19F 23F PS 2.5 2.5 2.0 4.0 3.3 conc. (mg/ml) PS dissolution NaCl 2M
NaCl 2M H.sub.2O NaCl 2M NaCl 2M PD 5.0 5.0 5.0 5.0 5.0 conc.
(mg/ml) Initial PS/PD 1/0.75 1/0.75 1/1 1/0.5 1/1 Ratio (w/w) CDAP
conc. 0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pH.sub.a = pH.sub.c =
pH.sub.q 8.5/8.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 10/9.5/9.0
9.0/9.0/9.0
[0193] TABLE-US-00003 TABLE 2 Specifications of the 11 valent
pneumococcoal PS-PD vaccine (first numbers of the batch code
indicates serotype) Criteria D01PDJ227 D03PDJ236 D4PDJ228 D5PDJ235
D6PDJ209 Ratio PS/Prot (w/w) 1/0.66 1/1.09 1/0.86 1/0.86 1/0.69
Free polysac. content (%) 1 1 7 9 0 <10% Free protein content
(%) 8 <1 19 21 9 <15% DMAP content (ng/.mu.g PS) 0.2 0.6 0.4
1.2 0.3 <0.5 ng/.mu.g PS Molecular size (K.sub.av) 0.18 0.13
0.12 0.11 0.13 Stability no shift no shift no shift low shift no
shift D07PDJ225 D09PDJ222 D14PDJ202 D18PDJ221 D19PDJ206 D23PDJ212
Ratio PS/Prot (w/w) 1/0.58 1/0.80 1/0.68 1/0.62 1/0.45 1/0.74 Free
polysac. content (%) 1 <1 <1 4 4 0 <10% Free protein
content (%) 8 0.3 3 21 10 12 <15% DMAP content (ng/.mu.g PS) 0.1
0.6 0.3 0.2 0.1 0.9 <0.5 ng/.mu.g PS Molecular size (K.sub.av)
0.14 0.14 0.17 0.10 0.12 0.12 Stability no shift no shift no shift
no shift shift no shift
[0194] TABLE-US-00004 TABLE 3 Summary Table of Adjuvant
Formulations tested with 11-Valent Pneumococcal PS-PD in Infant
Rats Group AlPO4 MPL Method Description 1 None 2 100 AlPO4 3 5 MPL
low 4 50 MPL High 5 100 5 Way 1 Way 1 low 6 100 50 Way 1 Way 1 high
7 100 5 Way 2 Way 2 low 8 100 50 Way 2 Way 2 high
[0195] TABLE-US-00005 TABLE 4 Serotype 6B Geometric Mean IgG
Concentration, Seroconversion, and Mean Opsonic Titre on Day 28
Post III Immunisation of Infant Rats with 11-Valent PS-PD using
Different Adjuvants (And Comparison with Tetravalent Immunisation)
6B 6B GMC 6B GMC 6B IgG 6B Opso IgG 6B Opso AlPO4 MPL (.mu.g/ml)
Seroconversion Titre* (.mu.g/ml) Seroconversion Titre* Group .mu.g
.mu.g Method Tetravalent Undecavalent 1 0.047 2/10 12.5 0.004 1/10
<6.25 2 100 0.048 4/10 65 0.019 4/10 <6.25 3 5 1.345 10/10 43
4 50 4.927 10/10 192 5 100 5 1 0.042 7/10 <6.25 6 100 50 1 0.255
10/10 <6.25 7 100 5 2 0.033 3/10 <6.25 0.048 8/10 <6.25 8
100 50 2 0.057 8/10 <6.25
[0196] TABLE-US-00006 TABLE 5 Serotype 14 Geometric Mean IgG
Concentration, Seroconversion, and Mean Opsonic Titre on Day 28
Post III Immunisation of Infant Rats with 11-Valent PS-PD using
Different Adjuvants (And Comparison with Tetravalent Immunisation)
14 14 GMC 14 GMC 14 IgG 14 Opsonic IgG 14 Opsonic (.mu.g/ml)
Seroconversion Titre* (.mu.g/ml) Seroconversion Titre* Group AlPO4
MPL Method Tetravalent Undecavalent 1 0.046 3/10 64 0.022 3/10
<6.25 2 100 0.99 10/10 88 0.237 8/10 27 3 5 0.233 10/10 41 4 50
0.676 10/10 81 5 100 5 1 0.460 9/10 67 6 100 50 1 0.477 10/10 98 7
100 5 2 0.81 10/10 49 0.165 8/10 81 8 100 50 2 1.611 10/10 133
[0197] TABLE-US-00007 TABLE 6 Serotype 19F Geometric Mean IgG
Concentration, Seroconversion, and Mean Opsonic Titre on Day 28
Post III Immunisation of Infant Rats with 11-Valent PS-PD using
Different Adjuvants (And Comparison with Tetravalent Immunisation)
19F 19F GMC 19F GMC 19F IgG 19F Opsonic IgG 19F Opsonic AlPO4 MPL
(.mu.g/ml) Seroconversion Titre* (.mu.g/ml) Seroconversion Titre*
Group .mu.g .mu.g Method Tetravalent Undecavalent 1 0.04 2/10 64
0.021 2/10 <6.25 2 100 1.07 9/10 367 0.222 7/10 79 3 5 4.028
10/10 296 4 50 21.411 10/10 1276 5 100 5 1 1.649 10/10 172 6 100 50
1 2.818 10/10 208 7 100 5 2 1.09 10/10 193 0.766 10/10 323 8 100 50
2 3.539 10/10 241
[0198] TABLE-US-00008 TABLE 7 Serotype 23F Geometric Mean IgG
Concentration, Seroconversion, and Mean Opsonic Titre on Day 28
Post III Immunisation of Infant Rats with 11-Valent PS-PD using
Different Adjuvants (And Comparison with Tetravalent Immunisation)
23F 23F GMC 23F GMC 23F IgG 23F Opsonic IgG 23F Opsonic AlPO4 MPL
(.mu.g/ml) Seroconversion Titre* (.mu.g/ml) Seroconversion Titre*
Group .mu.g .mu.g Method Tetravalent Undecavalent 1 0.06 2/10
<6.25 0.152 3/10 <6.25 2 100 0.29 10/10 70 0.56 8/10 <6.25
3 5 2.296 9/10 389 4 50 4.969 10/10 >1600 5 100 5 1 0.462 5/10
17 6 100 50 1 0.635 8/10 54 7 100 5 2 0.38 10/10 <6.25 0.203
3/10 18 8 100 50 2 0.501 7/10 43
[0199] TABLE-US-00009 TABLE 8 Serotypes 3 and 7F Geometric Mean IgG
Concentration, Seroconversion, and Mean Opsonic Titre on Day 28
Post III Immunisation of Infant Rats with 11-Valent PS- PD using
Different Adjuvants 3 7F GMC 3 GMC 7F AlPO4 MPL IgG 3 Opsonic IgG
7F Opsonic Group .mu.g .mu.g Method (.mu.g/ml) Seroconversion
Titre* (.mu.g/ml) Seroconversion Titre* 1 0.003 1/10 <6.25 0.040
7/10 <6.25 2 100 0.008 6/10 <6.25 0.25 9/10 43 3 5 0.070
10/10 <6.25 2.435 10/10 477 4 50 0.108 10/10 18 2.569 10/10 332
5 100 5 1 0.015 10/10 <6.25 0.579 10/10 54 6 100 50 1 0.027
10/10 <6.25 0.611 9/10 59 7 100 5 2 0.006 10/10 <6.25 0.154
8/10 30 8 100 50 2 0.034 10/10 <6.25 0.638 9/10 140
[0200] TABLE-US-00010 TABLE 9 Serotypes 1, 4 and 5 Geometric Mean
IgG Concentration and Seroconversion on Day 28 Post III
Immunisation of Infant Rats with 11-Valent PS-PD using Different
Adjuvants 1 4 5 GMC GMC GMC AlPO4 MPL IgG 1 IgG 4 IgG 5 Group .mu.g
.mu.g Method (.mu.g/ml) Seroconversion (.mu.g/ml) Seroconversion
(.mu.g/ml) Seroconversion 1 0.026 4/10 0.005 0/10 0.040 3/10 2 100
0.282 8/10 0.052 5/10 0.774 9/10 3 5 1.614 10/10 3.452 10/10 7.927
10/10 4 50 2.261 10/10 7.102 10/10 13.974 10/10 5 100 5 1 0.568
10/10 0.676 10/10 3.015 10/10 6 100 50 1 1.430 10/10 0.419 9/10
5.755 10/10 7 100 5 2 0.478 10/10 0.267 9/10 2.062 10/10 8 100 50 2
1.458 10/10 0.423 10/10 5.009 10/10
[0201] TABLE-US-00011 TABLE 10 Serotypes 9V, 18C and PD Geometric
Mean IgG Concentration and Seroconversion on Day 28 Post III
Immunisation of Infant Rats with 11-Valent PS-PD using Different
Adjuvants 9V 18C PD GMC GMC GMC AlPO4 MPL IgG 9V IgG 18C IgG PD
Group .mu.g .mu.g Method (.mu.g/ml) Seroconversion (.mu.g/ml)
Seroconversion (.mu.g/ml) Seroconversion 1 0.018 0/10 0.013 1/10
0.003 0/10 2 100 0.489 6/10 0.092 5/10 0.993 10/10 3 5 0.482 7/10
6.560 10/10 3.349 10/10 4 50 11.421 10/10 14.023 10/10 5.446 10/10
5 100 5 1 2.133 9/10 0.690 10/10 11.407 10/10 6 100 50 1 2.558
10/10 1.771 10/10 1.258 10/10 7 100 5 2 1.536 10/10 0.528 10/10
1.665 8/10 8 100 50 2 2.448 9/10 0.980 10/10 5.665 10/10
[0202] TABLE-US-00012 TABLE 11 The statistical significance (p
value) of whether certain pneumococcal polysaccharide conjugates
had improved immunogenicity when formulated with 3D-MPL alone
versus with 3D-MPL/AlPO4. A p value under 0.01 is considered highly
significant. Way 1 and Way 2 indicate the method of formulation. 50
.mu.g 3D-MPL v 5 .mu.g 3D-MPL vs 3D-MPL/AlPO.sub.4
3D-MPL/AlPO.sub.4 serotype Way 1 Way 2 Way 1 Way 2 1 0.3 0.05 0.079
0.11 3 0.075 0.01 0.27 0.008 4 0.002 0.0003 0.02 0.003 5 0.04 0.002
0.1 0.12 6B 0.001 0.0001 0.001 0.0006 7F 0.13 0.15 0.01 0.005 9V
0.02 0.02 0.1 0.04 14 0.65 0.21 0.3 0.66 18C 0.0008 0.0002 0.006
0.004 19F 0.0009 0.006 0.21 0.04 23F 0.002 0.0004 0.01 0.0004
[0203] TABLE-US-00013 TABLE 12 Geometric Mean IgG concentration
(.mu.g/mL) on day 14 post 2.sup.nd dose after immunisation of adult
rats with 1.0 .mu.g polysaccharide-protein D conjugate alone or
combined in tetravalent, pentavalent, heptavalent or decavalent
vaccine. These data are combined from 5 separate experiments.
Serotypes Vaccines 4 6B 18C 19F 23F H T H T T Alone 9.3 0.11 15 5.2
2.5 Combined 4 0.23 3.7 3.7 2.8 T: combined in tetravalent (T) (PS
6B, 14, 19F, 23F), pentavalent (T plus PS 3), heptavalent (H) (T
plus PS 4, 9V and 18C), and decavalent (H plus PS 1, 5 and 7F)
combination vaccines. H: combined in heptavalent (H) (T plus PS 4,
9V and 18C), and decavalent (H plus PS 1.5 and 7F) combination
vaccines.
Example 4
Beneficial Impact of the Addition of Pneumolysin and 3D-MPL on the
Protective Effectiveness of PD-Conjugated 11-Valent Polysaccharide
Vaccine Against Pneumococcal Lung Colonization in Mice
Immunological Read-Outs
ELISA Dosage of Pneumolysin-Specific Serum IgG
[0204] Maxisorp Nunc immunoplates were coated for 2 hours at
37.degree. C. with 100 .mu.l/well of 2 .mu.g/ml recombinant native
pneumolysin (PLY) diluted in PBS. Plates were washed 3 times with
NaCl 0.9% Tween-20 0.05% buffer. Then, serial 2-fold dilutions (in
PBS/Tween-20 0.05%, 100 .mu.l per well) of an anti-PLY serum
reference added as a standard curve (starting at 670 ng/ml IgG) and
serum samples (starting at a 1/10 dilution) were incubated for 30
minutes at 20.degree. C. under agitation. After washing as
previously described, peroxydase-conjugated goat anti-mouse IgG
(Jackson) diluted 5000.times. in PBS/Tween-20 0.05% were incubated
(100 .mu.l/well) for 30 minutes at 20.degree. C. under agitation.
After washing, plates were incubated for 15 min at room temperature
with 100 .mu.l/well of revelation buffer (OPDA 0.4 mg/ml and
H.sub.2O.sub.2 0.05% in 100 mM pH 4.5 citrate buffer). Revelation
was stopped by adding 50 .mu.l/well HCl 1N. Optical densities were
read at 490 and 620 nm by using Emax immunoreader (Molecular
Devices). Antibody titre were calculated by the 4 parameter
mathematical method using SoftMaxPro software.
Hemolysis Inhibition
[0205] This assay was done for measuring the ability of serum
antibodies to inhibit the pneumolysin (PLY) hemolytic activity. In
order to eliminate the cholesterol (susceptible of interacting with
PLY), serum samples were treated 2.times. as follows: they were
mixed with 1 equal volume of chloroform and then incubated for 45
minutes under agitation. Supernatants were collected after
centrifugation for 10 minutes at 1000 rpm. Cholesterol-cleared sera
were diluted (serial 2-fold dilutions in 1 mM dithiothreitol, 0.01%
BSA, 15 mM TRIS, 150 mM NaCl, pH 7.5) in 96 well microplates
(Nunc). Fifty .mu.l of a solution containing 4 HU (Hemolysis Unit)
of PLY were added in each well and incubated for 15 minutes at
37.degree. C. Then, 100 .mu.l of sheep red blood cells (1%
solution) were added for 30 minutes at 37.degree. C. After
centrifugation for 10 minutes at 1000 rpm, supernatants (150 .mu.l)
were collected and put into another 96-well microplate for optical
density reading at 405 nm. Results were expressed as mid-point
dilution titers.
Pneumolysin Chemical Detoxification
[0206] Recombinant native pneumolysin (PLY) was dialyzed against
Phosphate 50 mM NaCl 500 mM pH 7.6 buffer. All following steps were
done at 39.5.degree. C. under episodic agitation. At day 1,
Tween-80 10% (1/250 v/v), N-acetyl tryptophan 57.4 mM pH 7.6 (3/100
v/v), glycin 2.2 M in Phosphate buffer (1/100 v/v) and formaldehyde
10% in Phosphate buffer (3/100 v/v) were added into PLY solution.
At days 2 and 3, formaldehyde 10% was added again, at 3/100 and
2/100 v/v ratio, respectively. Incubation at 39.5.degree. C. was
sustained until day 7 under episodic agitation. Finally, PLY was
dialyzed against Phosphate 50 mM NaCl 500 mM pH 7.6 buffer.
Complete inactivation of PLY was demonstrated in the hemolysis
assay.
Pneumococcal Intranasal Challenge in OF1 Mice
[0207] Seven week-old OF1 female mice were intranasally inoculated
under anesthesia with 5.10.sup.5 CFU of mouse-adapted S. pneumoniae
serotype 6B. Lungs were removed at 6 hours after challenge and
homogenized (Ultramax, 24000 rpm, 4.degree. C.) in Todd Hewith
Broth (THB, Gibco) medium. Serial 10-fold dilutions of lung
homogenates were plated overnight at 37.degree. C. onto Petri
dishes containing yeast extract-supplemented THB agar. Pneumococcal
lung infection was determined as the number of CFU/mouse, expressed
as logarithmic weighted-average. Detection limit was 2.14 log
CFU/mouse.
Example 4A
3D-MPL Adjuvant Effect on Anti-Pneumolysin Immune Response
[0208] In the present example, we evaluated the impact of 3D-MPL
adjuvantation on the immune response to native recombinant
pneumolysin (PLY, provided by J. Paton, Children's Hospital, North
Adelaide, Australia) and its chemically detoxified counterpart
(DPLY). Chemical detoxification was done as described above.
[0209] Groups of 10 female 6 week-old Balb/c mice were
intramuscularly immunized at days 0, 14 and 21 with 1 .mu.g PLY or
DPLY contained in either A: AlPO4 100 .mu.g; or B: AlPO4 100
.mu.g+5 .mu.g 3D-MPL (3 de-O-acylated monophosphoryl lipid A,
supplied by Ribi Immunochem). FIGS. 1A and 1B show ELISA IgG and
HemoLysis Inhibition titers (HL1) measured in post-III sera.
[0210] Whichever the antigen, best immune responses were induced in
animals vaccinated with 3D-MPL-supplemented formulations.
Interestingly, DPLY was as immunogenic as PLY when administered
with AlPO4+3D-MPL, while being a weaker immunogen in AlPO4
formulation. This showed the advantageous ability of 3D-MPL to
improve the antibody response to detoxified pneumolysin.
[0211] In compositions containing pneumolysin, it may be preferable
to use chemically detoxified pneumolysin rather than mutationally
detoxified pneumolysin. This is because detoxified mutants obtained
to date still have residual toxin activity--chemically detoxifed
pneumolysin does not. It is therefore considered another aspect of
the invention that, in general, compositions comprising pneumolysin
(or pneumolysin mutants) that has been chemically detoxified for
use in a vaccine, should be adjuvanted with a Th1 adjuvant,
preferably 3D-MPL. Such compositions are provided by the invention.
A method of increasing the immune response of chemically-detoxifed
pneumolysin within an immunogenic composition comprising the steps
of adding a Th1 adjuvant (preferably 3D-MPL) to the composition, is
also envisaged.
Example 4B
Beneficial Impact of the Addition of an Attenuated Mutant of
Pneumolysin and 3D-MPL Adjuvant on the Protective Effectiveness of
PD-Conjugated 11-Valent Polysaccharide Vaccine Against Pneumococcal
Lung Colonization in OF1 Mice Intranasally Challenged with Serotype
6B
[0212] In the present example, we evaluated the prophylactic
efficacy of a vaccine containing the 11-valent
polysaccharide-protein D conjugate, attenuated mutant pneumolysin
antigen (PdB, WO 90/06951) and AlPO4+3D-MPL adjuvants, compared to
the classical AlPO4-adsorbed 11-valent polysaccharide-protein D
conjugate formulation.
[0213] Groups of 12 female 4 week-old OF1 mice were immunized
subcutaneously at days 0 and 14 with formulations containing A: 50
.mu.g AlPO4; B: 0.1 .mu.g PS/serotype of PD-conjugated 11-valent
polysaccharide vaccine+50 .mu.g AlPO4; or C, 0.1 .mu.g PS/serotype
of PD-conjugated 11-valent polysaccharide vaccine+10 .mu.g PdB
(provided by J. Paton, Children's Hospital, North Adelaide,
Australia)+50 .mu.g AlPO4+5 .mu.g 3D-MPL (supplied by Ribi
Immunochem). Challenge was done at day 0.21 as described above.
[0214] As shown in FIG. 1C, a very significant protection
(p<0.007) was conferred by the 11-valent polysaccharide
conjugate vaccine supplemented with PdB and adjuvanted with
AlPO4+MPL (black bars represent the arithmetic mean). On the
contrary, no significant protection was observed in animals
immunized with the 11-valent polysaccharide conjugate/AlPO4
formulation. This result proved that the addition of pneumolysin
antigen (even attenuated) and 3D-MPL adjuvant enhanced the
effectiveness of the 11-valent polysaccharide conjugate vaccine
against pneumonia.
Example 4C
Immune Correlates of the Protection Showed in Example 4B
[0215] In order to establish the immune correlates of protection
conferred in example 4B, by the 11-valent polysaccharide conjugate
vaccine supplemented with attenuated mutant pneumolysin (PdB) and
3D-MPL, pre-challenge serological antibody responses to
polysaccharide 6B and PdB were measured as described above.
[0216] Antibody titers were then compared to bacteria colony
numbers measured in lungs of the corresponding animals collected at
6 hours post-challenge. R.sup.2 were calculated on Log/Log linear
regressions.
[0217] Calculated R.sup.2 were equal to 0.18 and 0.02 for anti-PdD
and anti-6B antibody responses, respectively. This showed the
absence of correlation between humoral immune responses and
protection for both antigens. Anti-6B antibody titers were not
significantly different in the groups immunized with the 11-valent
conjugate vaccine (GMT=0.318 ng/ml) or with the same vaccine
supplemented with PdD and 3D-MPL (GMT=0.458 ng/ml). Therefore, the
protection improvement seen with formulation C was not solely due
to a higher antibody response to polysaccharide 6B.
[0218] Taken together, the results suggest that protection was not
mediated by humoral immune responses alone, but rather also by a
cell-mediated immunity induced by the PdB antigen in the presence
of 3D-MPL. This gave additional support to the addition of protein
antigen(s) and potent adjuvant(s) in the pneumococcal
polysaccharide conjugate vaccine, so as to coordinate both arms of
the immune system for optimal protection.
Example 5
The Cooperation of Both Arms of the Immune System in Mice Actively
Immunised with Pneumolysin and Passively Immunised with Antibodies
Against Pneumococcal PS
Example 5A
Find the Concentration of Passively Administered
Anti-6B-Polysaccharide (Anti-PS) Antibody Protecting Against
Pneumonia
Method
[0219] Vaccine Groups: Four groups of 16 mice were passively
immunised (i.p.) on day -1 with 100 .mu.l of undiluted rat
anti-polysaccharide antisera according to the groups detailed
below. (total 64 mice) TABLE-US-00014 IgG Concentration in Group
Specificity Antisera G1 .alpha.-PS -6B 5 .mu.g/ml. G2 .alpha.-PS
-6B 2 .mu.g/ml. G3 .alpha.-PS -6B 0.75 .mu.g/ml. G4 Control 0
.mu.g/ml.
[0220] Animals: 64 male CD-1 mice from Charles River. Canada,
weighing approx 35 g (approx 10 weeks old).
[0221] Anesthesia: Mice were anesthetized with isoflurane (3%) plus
O2(1 L/min).
[0222] Organism: S. pneumoniae N1387 (serotype 6) was harvested
from trypticase soy agar plates (TSA) supplemented with 5% horse
blood and suspended in 6 ml of PBS. Immediately prior to infection,
1 ml bacterial suspension was diluted into 9 ml of cooled molten
nutrient agar (BBL) and kept at 41.degree. C. Mice received approx
6.0 log 10 cfu/mouse in a volume 50 ul.
[0223] Infection: On day 0 mice were anesthetized as described
above and infected with S. pneumoniae N1387 (50 .mu.l cooled
bacterial suspension) by intra-bronchial instillation via
non-surgical intra-tracheal intubation. This method was described
by Woodnut and Berry (Antimicrob. Ag. Chemotherap. 43: 29
(1999)).
[0224] Samples: On day 3 post infection, 8 mice/group were
sacrificed by CO.sub.2 overdose and lungs were excised and
homogenized in 1 ml PBS. Tenfold serial dilutions were prepared in
PBS to enumerate viable bacterial numbers. Samples were inoculated
(20 .mu.l) in triplicate onto TSA plates supplemented with 5% horse
blood and incubated overnight at 37.degree. C. prior to evaluation.
Further sets of mice were sacrificed on day 7 and sampled as
above.
[0225] Results: TABLE-US-00015 Bacterial numbers IgG conc (log 10
cfu/lungs) (ug/ml) at days post infection in rat sera 3 8 5 6.7
.+-. 0.7 (1/7) 7.2 .+-. 0.7 (5/8) 2 6.5 .+-. 0.7 (1/7) 6.9 .+-. 1.8
(4/7) 0.75 7.7 .+-. 0.5 (5/8) 4.8 .+-. 1.4 (2/8) 0 6.7 .+-. 1.5
(3/6) 6.3 .+-. 1.5 (3/9) Figures in parenthesis are numbers of
animals that died prior to sample time.
[0226] Conclusion: In general, there was no significant difference
in bacterial numbers isolated from any of the treatment groups.
This indicates that no measurable protection was afforded by the
anti-polysaccharide at concentrations up to and including 5
.mu.g/ml.
[0227] This is similar to what is observed in some human clinical
trials, that is, anti-polysaccharide body is insufficient to
protect against pneumococcal pneumonia in some populations.
Example 5B
Determine the Protection from Pneumonia Afforded by Active
Administration of Ply (Pneumolysin) with or without Adjuvant, and
Synergy with sub-Optimal Anti-PS Antibody
Method
[0228] Animals: 128 male CD-1 mice (6 weeks old at old at
immunisation, 10 weeks old at infection) from Charles River, St.
Constant, Quebec, Canada. Animals weighed approx 20 gm at 6 weeks
and 38 g at 10 weeks.
[0229] Immunisations: Six groups of 16 mice were immunised by
subcutaneous injection on days -22 and -14 with 100 ul of vaccine
as detailed below. (Total 128 mice). PdB (WO 90/06951) was obtained
courtesy of Dr. James Paton, Australia. 3D-MPL was obtained from
Ribi/Corixa.
[0230] On day -1, specific groups (see Table below) were immunised
(i.p. 100 .mu.l) passively with a concentration of 4.26 .mu.g/ml (4
ml of 5 .mu.g/ml+1.3 ml of 2 .mu.g/ml) mouse anti-polysaccharide
antibody. TABLE-US-00016 Injection Vaccine given Injection Passive
Volume days -22, -14 Volume IgG Group Active (Dosage .mu.g) Passive
(day -1) 1-1 100 .mu.l s.c. PdB/AlPO4 (10/50) None 1-2 100 .mu.l
s.c. PdB/MPL/AlPO4 (10/5/50) None 1-3 100 .mu.l s.c. PdB/AlPO4
(10/50) 100 .mu.l i.p. .alpha.-PS 1-4 100 .mu.l s.c. PdB/MPL/AlPO4
(10/5/50) 100 .mu.l i.p. .alpha.-PS 1-5 100 .mu.l s.c. MPL/AlPO4
(5/50) 100 .mu.l i.p. .alpha.-PS 1-6 100 .mu.l s.c. MPL/AlPO4
(5/50) None
[0231] Infection: On day 0, mice were anesthetized (3% isoflurane
plus 1 L/min O2). Bacterial inocula were prepared by harvesting
growth of S. pneumoniae N1387 (serotype 6) from trypticase soy agar
plates (TSA) supplemented with 5% horse blood and suspending in 6
ml of PBS. A ten-fold dilution (1 ml plus 9 ml) was prepared in
cooled molten nutrient agar (kept at 41.degree. C.) immediately
prior to infection. Mice were infected by intra-bronchial
instillation via intra-tracheal intubation and received
approximately 6.0 log 10 cfu/mouse in a volume of 50 .mu.l. This
method was described by Woodnut and Berry (Antimicrob. Ag.
Chemotherap. 43: 29 (1999)).
[0232] Samples: At 72 post infection, 8 mice/group were sacrificed
by CO2 overdose and the lungs were excised and homogenized in 1 ml
PBS. Tenfold serial dilutions were prepared in PBS to enumerate
viable bacterial numbers. Samples were inoculated (20 .mu.l) in
triplicate onto TSA plates supplemented with 5% horse blood and
incubated overnight 37.degree. C. prior to evaluation. Further sets
of mice were sacrificed on day 8 post-infection and samples as
above.
Analysis of Data
[0233] The outcome measure for comparison of treatment was the
number of bacteria in the lungs at 3 and 7 day post infection.
Results are presented as group means with standard deviations.
Statistical analysis was performed using the Students t-test where
a P value of <0.05 was considered significant.
Results:
72 h Post Infection
[0234] Bacterial counts from group 1-4 were significantly lower
(p<0.05) than those from group 1-3.
[0235] Bacterial counts from group 1-4 were significantly lower
(p<0.05) than those from group 1-5.
168 h Post Infection
[0236] Bacterial numbers in all groups were approx 2 logs lower at
8 days than at 3 days, indicating that the infection was
resolving.
[0237] Bacterial counts from group 1-2 were significantly lower
(p<0.05) than those from group 1-5. TABLE-US-00017 Day 3 Day 8
Log Standard Log Standard Group CFU/lung Deviation CFU/lung
Deviation 1-1 6.93 0.61 5.23 1.28 1-2 6.59 1.25 4.08 1.34 1-3 7.09
0.8 5.32 1.26 1-4 6.09 1.43 4.46 2.32 1-5 7.19 0.89 5.42 1.05 1-6
6.68 1.14 5.01 1.48
[0238] As demonstrated above, anti-polysaccharide antibody alone
(group 1-5) does not afford protection against growth of
pneumococci in the lung. PdB adjuvanted with AlPO4 does not confer
protection either, but at day 8 there is a trend to protection when
PdB is combined with 3D-MPL (group 1-2).
[0239] At Day 3, the group most significantly protected, group 1-4,
had all three elements, PdB, 3D-MPL and passively administered
anti-polysaccharide antibody. This conclusion is supported by the
mortality rate. Group 14 had only 2/8 deaths compared to 5/10 for
groups 1-5 and 1-3.
Conclusion:
[0240] As the experiment was done with passively immunised animals,
the synergistic effect of also actively immunising with pneumolysin
and MPL cannot be due to an increase in the level of antibodies
against the polysaccharide antigen.
[0241] As the animals were only passively immunised against
pneumococcal polysaccharide, by day 8 levels of such antibody would
have largely dissipated from the host.
[0242] Even so, significant protection against pneumococcal
pneumonia could be seen in groups immunised with pneumolysin plus
3D-MPL and especially in groups immunised with pneumolysin plus
3D-MPL plus passively administered anti-polysaccharide antibody,
indicating the synergy of this combination.
[0243] If the anti-polysaccharide immunisation had been carried out
actively (preferably with conjugated polysaccharide), the effect
would have been even more marked, as the effect of B-cell memory,
and constant levels of anti-PS antibody would have contributed to
the immune response cooperation (see for example FIG. 1C where many
of the animals actively immunised with polysaccharide and protein
was shown to have no bacteria in the lungs after challenge).
Example 6
Immunogenicity in 1-Year-Old Balb/C Mice of 11-Valent
Pneumococcal-Polysaccharide Protein D Conjugate Vaccine Adjuvanted
with 3D-MPL
Introduction & Objective(s):
[0244] Protection against pneumococcal infection is mediated by
serotype specific antibody through opsonophagocytosis. It may be
surmised that increases in the antibody concentration will result
in greater protection, and therefore much effort has been expended
to find ways to increase the humoral response. One strategy that
has been applied successfully to conjugate vaccines in pre-clinical
studies is the use of immunostimulating adjuvants (reviewed in
Poolman et al. 1998, Carbohydrate-Based Bacterial Vaccines. In:
Handbook of Experimental Pharmacology eds. P. Perlmann and H.
Wigsell. Springer-Verlag, Heidelberg, D).
[0245] The data presented in this section show the results of the
latest experiment using clinical lots in a protocol designed to
mimic a clinical trial.
Protocol:
[0246] One-year-old balb/c mice were immunised with 1/10th of the
human dose of pneumococcal-polysaccharide protein D conjugate
vaccine, or 23-valent plain polysaccharide vaccine. The vaccines
used were clinical lots DSP009, DSP013 or DSP014 corresponding to
the 1 mcg dosage of serotypes 6B and 23F and 5 mcg of the remaining
serotypes of the 11-valent conjugated vaccine, the 0.1 mcg dosage
of the 11valent conjugated vaccine, or the 0.1 mcg dosage of the
11-valent conjugated vaccine adjuvanted with 5 mcg 3D-MPL,
respectively. All 11-valent conjugated vaccines were also
adjuvanted with 50 .mu.g AlPO.sub.4.
[0247] Groups of 20 mice were immunised intramuscularly. Injections
of the groups listed in the following table were performed on days
0 and 21. Test bleeds were obtained on day 35, (14 days after the
second dose). TABLE-US-00018 TABLE Immunisation Schedule for
1-year-old Balb/c mice immunised with clinical lots of
pneumococcal-polysaccharide Protein D conjugate vaccine. Day 0 Day
21 Vaccine Vaccine Number Group Dose 1 Dose 2 of mice 1
Pneumovax-23 Buffer 20 2.5 mcg 2a 11-valent Pn-PD Buffer 20 0.1 mcg
2b 11-valent Pn-PD 11-valent Pn-PD 20 0.1 mcg 0.1 mcg 3a 11-valent
Pn-PD + MPL Buffer 20 0.1 mcg + 5 mcg 3b 11-valent Pn-PD + MPL
11-valent Pn-PD + MPL 20 0.1 mcg + 5 mcg 0.1 mcg + 5 mcg 4a
11-valent Pn-PD Buffer 20 1/0.5 mcg 4b 11-valent Pn-PD 11-valent
Pn-PD 20 1/0.5 mcg 1/0.5 mcg Control Buffer Buffer 20
[0248] The sera were tested by ELISA for IgG antibodies to the
pneumococcal polysaccharides following the CDC/WHO consensus
protocol, that is, after neutralisation of the sera with cell-wall
polysaccharide. The ELISA was calibrated to give antibody
concentrations in mcg/ml using serotype specific IgG1 monoclonal
antibodies.
[0249] Statistical analyses of comparisons were calculated using
UNISTAT version 5.0 beta. ANOVA by the Tukey-HSD method was
performed on log transformed IgG concentrations. Pairwise
comparison of seroconversion rates was performed using Fisher's
exact test.
Results:
[0250] The GMC IgG and 95% confidence interval against the 11
serotypes and protein D induced 14 days after the second
immunisation (dose 2) are shown in the following table.
Seroconversion rates are shown where a 95% confidence interval
could not be calculated.
[0251] Group 1 shows the effect of immunisation with plain
polysaccharides, which normally induce only IgM in animals. Most
IgG levels are below the threshold of detection; nevertheless,
balb/c mice were able to make IgG to a few pneumococcal
polysaccharides, notably serotypes 3, 19F and 14.
[0252] Immunisation with conjugate vaccines induced IgG antibody
with high seroconversion rates against all serotypes except
23F.
[0253] A dosage-dependent response (group 4 vs group 2) was
observed only for serotypes 7F and 19F, but these observations were
not statistically significant. A greater response was observed
after two doses (b groups vs a groups) for serotypes 3, 6B, 7F and
19F, and PD, and these observations were statistically significant
in many cases with all 3 formulations.
[0254] Most interesting is the effect of 3D-MPL. Two doses of the
3D-MPL formulated vaccine (group 3b) induced the highest GMC of
specific IgG, and this was statistically significant for all
serotypes except 23F, in which case it had a significantly higher
seroconversion rate (p=0.02 group 3b vs 2b, Fisher's exact).
TABLE-US-00019 TABLE Geometric Mean [IgG] and 95% Confidence
Intervals to Selected Pneumococcal Serotypes and Protein D in
1-Year-Old Balb/c 14 days Post II Immunisation with 11- valent
PS-PD Conjugate Vaccine Group 1 2a 2b 3a 3b 4a 4b GM [IgG] GM [IgG]
GM [IgG] GM [IgG] GM [IgG] GM [IgG] GM [IgG] .mu.g/ml .mu.g/ml
.mu.g/ml .mu.g/ml .mu.g/ml .mu.g/ml .mu.g/ml Serotype (95% CI) (95%
CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) 3 0.24 0.18 0.84
0.72 4.84 0.22 0.95 (0.16-0.6) (0.11-0.27) (0.47-1.5) (0.51-1.0)
(3.0-7.9) (0.14-0.35) (0.19-1.8) 6B 0.02 0.04 0.19 0.14 0.74 0.09
0.11 0/20.sup.# 8/19 (0.09-0.41) (0.07-0.27) (0.29-1.9) (0.05-0.16)
(0.05-0.23) 7F 0.04 0.07 0.19 0.15 0.97 0.09 0.45 0/20.sup.#
(0.04-0.12) (0.10-0.39) (0.10-0.22) (0.49-2.0) (0.06-0.14)
(0.20-1.02) 14 0.15 4.5 6.2 12.9 13.6 4.0 6.9 3/20.sup.# (2.5-8.1)
(3.6-10.5) (7.8-21.2) (9.4-19.7) (2.0-8.0) (4.6-10.6) 19F 1.2 6.7
12.1 10.1 58.5 5.9 22.0 (0.56-2.6) (3.6-12.5) (7.6-19.3) (5.5-18.5)
(42-81) (3.5-9.9) (16.0-30.2) 23F 0.07 0.08 0.08 0.07 0.17 0.06
0.10 1/20.sup.# .sup. 3/20.sup.# 2/19.sup.# 2/10.sup.# 9/20.sup.#
1/18.sup.# 4/20.sup.# PD* 0.25 5.2 11.9 13.5 98.0 10.9 38.7
1/20.sup.# (3.3-8.3) (6.9-20.7) (9.5-19.0) (49.1-195.) (6.4-18.4)
(21.3-70.3) *In EU/ml; # Seroconversion rate, defined as 2 standard
deviations above the average of the negative control. Please refer
to previous table for group definitions.
Conclusion:
[0255] The data presented here demonstrates that the addition of
3D-MPL to the 11-valent pneumococcal-polysaccharide Protein D
conjugate vaccine increased the immune response in elderly balb/c
mice to all serotypes tested.
[0256] In most cases, two doses of vaccine induced higher geometric
mean IgG concentrations that one dose. Since this is not observed
using plain polysaccharide vaccine, even in humans, it is
considered an indication of a T-cell dependent immune response and
the induction of immune memory.
[0257] These data support a vaccine administation scheme using
conjugated pneumococcal polysaccharides adjuvanted with Th1
adjuvants (preferably 3D-MPL), whereby at least two doses of the
adjuvanted vaccine are administered, preferably 1-12 weeks apart,
and most preferably 3 weeks apart. Such an administration scheme is
considered a further aspect of the invention.
[0258] The mice used in the experiment were non-responsive to PS 23
(plain or conjugated). Interestingly, although antibody levels
against the polysaccharide remained low regardless of the vaccine
composition used, many more mice responded to PS 23 when 3D-MPL was
used as the adjuvant (the seroconversion being significantly
higher). A use of Th1 adjuvants, particularly 3D-MPL, in vaccine
compositions comprising conjugated pneumococcal polysaccharides in
order to relieve non-responsiveness to a pneumococcal
polysaccharide in a vaccinee is a still further aspect of the
invention. A method of relieving non-responsiveness with the
aforementioned composition using the two dose administration scheme
described above is yet another aspect.
Example 7
Neisseria Meningitidis C Polysaccharide-Protein D Conjugate
(PSC-PD)
A: Expression of Protein D
[0259] As for Example 1.
B: Manufacture of Polysaccharide C
[0260] The source of group C polysaccharide is the strain C11 of N.
meningitidis. This is fermented using classical fermentation
techniques (EP 72513). The dry powder polysaccharides used in the
conjugation process are identical to Mencevax (SB Biologicals
s.a.).
[0261] An aliquot of C11 strain is thawed and 0.1 ml of suspension
is streaked on one Mueller Hinton medium petri dish supplemented
with yeast extract dialysate (10%, v/v) and incubated for 23 to 25
hrs at 36.degree. C. in a water saturated air incubator.
[0262] The surface growth is then re-suspended in sterilized
fermentation medium and inoculated with this suspension on one Roux
bottle containing Mueller Hinton medium supplemented with yeast
extract dialysate (10%, v/v) and sterile glass beads. After
incubation of the Roux bottle during 23 to 25 hrs at 36.degree. C.
in a water saturated air incubator, the surface growth is
re-suspended in 10 ml sterile fermentation medium and 0.2 to 0.3 ml
of this suspension are inoculated onto 12 other Mueller Hinton
medium Roux bottles.
[0263] After incubation during 23 to 25 hrs at 36.degree. C. in a
water saturated air incubator, surface growth is re-suspended in 10
ml sterile fermentation medium. The bacterial suspension is pooled
in a conical flask.
[0264] This suspension is then aseptically transferred into the
fermenter using sterile syringes.
[0265] The fermentation of meningococcus is performed in fermenters
contained in a clean room under negative pressure. The fermentation
is generally completed after 10-12 hrs corresponding to
approximately 10.sup.10 bacteria/ml (i.e. the early stationary
phase) and detected by pH increase.
[0266] At this stage, the entire broth is heat inactivated (12 min
at 56.degree. C.) before centrifugation. Before and after
inactivation, a sample of the broth is taken and streaked onto
Mueller Hinton medium petri dishes.
C: PS Purification
[0267] The purification process is a multi-step procedure performed
on the entire fermentation broth. In the first stage of
purification, the inactivated culture is clarified by
centrifugation and the supernatant is recovered.
[0268] Polysaccharide purification is based on precipitation with a
quaternary ammonium salt (Cetyltrimethylammonium
Bromide/CTAB,CETAVLON R). CTAB forms insoluble complexes with
polyanions such as polysaccharides, nucleic acid and proteins
depending on their pI. Following ionic controlled conditions, this
method can be used to precipitate impurities (low conductivity) or
polysaccharides (high conductivity).
[0269] The polysaccharides included in clarified supernatant are
precipitated using a diatomaceous earth (CELITE.RTM. 545) as matrix
to avoid formation of insoluble inert mass during the different
precipitations/purifications.
Purification Scheme for N. meningitidis Polysaccharide C:
[0270] Step 1: PSC-CTAB complex fixation on CELITE.RTM. 545 and
removal of cells debris, nucleic acids and proteins by washing with
CTAB 0.05%.
[0271] Step 2: Elution of PS with EtOH 50%. The first fractions
which are turbid and contain impurities and LPS are discarded. The
presence of PS in the following fractions is verified by
floculation test.
[0272] Step 3: PS-CTAB complex re-fixation on CELITE.RTM. 545 and
removal of smaller nucleic acids and proteins by CTAB 0.05%
washing.
[0273] Step 4: Elution of PS with EtOH 50%. The first turbid
fractions are discarded. The presence of PS in the following
fractions is verified by floculation test.
[0274] The eluate is filtered and the filtrate containing crude
polysaccharide collected.
[0275] The polysaccharide is precipitated from the filtrate by
adding ethanol to a final concentration of 80%. The polysaccharide
is then recovered as a white powder, vaccum dried and stored at
-20.degree. C.
D: CDAP Conjugation
Conjugation of PSC and PD
[0276] For conjugation of PSC and PD, the CDAP conjugation
technology was preferred to the classical CNBr activation and
coupling via a spacer to the carrier protein. The polysaccharide is
first activated by cyanylation with
1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). CDAP
is a water soluble cyanylating reagent in which the
electrophilicity of the cyano group is increased over that of CNBr,
permitting the cyanylation reaction to be performed under
relatively mild conditions. After activation, the polysaccharide
can be directly coupled to the carrier protein through its amino
groups without introducing any spacer molecule. The unreacted
estercyanate groups are quenched by means of extensive reaction
with glycine. The total number of steps involved in the preparation
of conjugate vaccines is reduced and most importantly potentially
immunogenic spacer molecules are not present in the final
product.
[0277] Activation of polysaccharides with CDAP introduces a cyanate
group in the polysaccharides and dimethylaminopyridine (DMAP) is
liberated. The cyanate group reacts with NH2-groups in the protein
during the subsequent coupling procedure and is converted to a
carbamate.
PSC Activation and PSC-PD Coupling
[0278] Activation and coupling are performed at +25.degree. C.
[0279] 120 mg of PS is dissolved for at least 4 h in WFI.
[0280] CDAP solution (100 mg/ml freshly prepared in acetonitrile)
is added to reach a CDAP/PS (w/w) ratio of 0.75.
[0281] After 1 min 30, the pH is raised up to activation pH (pH 10)
by addition of triethylamine and is stabilised up to PD
addition.
[0282] At time 3 min 30, NaCl is added to a final concentration of
2M.
[0283] At time 4 min, purified PD is added to reach a PD/PS ratio
of 1.5/1; pH is immediately adjusted to coupling pH (pH 10). The
solution is left for 1 h under pH regulation.
Quenching
[0284] 6 ml of a 2M glycine solution is added to the PS/PD/CDAP
mixture. The pH is adjusted to the quenching pH (pH 8.8). The
solution is stirred for 30 min at the working temperature, then
overnight at +2-8.degree. C. with continuous slow stirring.
PS-PD Purification
[0285] After filtration (5 .mu.m), the PS-PD conjugate is purified
in a cold room by gel permeation chromatography on a S400HR
Sephacryl gel to remove small molecules (including DMAP) and
unconjugated PD: Elution--NaCl 150 mM-pH 6.5; Monitoring--UV 280
nm, pH and conductivity.
[0286] Based on the different molecular size of the reaction
components, PS-PD conjugates are eluted first followed by free PD
and finally DMAP. Fractions containing conjugate as detected by
DMAB (PS) and .mu.BCA (protein) are pooled. The pooled fractions
are sterile filtered (0.2 .mu.m)
E: Formulation of PSC-PD Adsorbed Conjugate Vaccine
Washing of AlPO.sub.4
[0287] In order to optimize the adsorption of PSC-PD conjugate on
AlPO.sub.4, the AlPO.sub.4 is washed to reduce the PO.sub.4.sup.3-
concentration: [0288] AlPO4 is washed with NaCl 150 mM and
centrifuged (4.times.); [0289] the pellet is then resuspended in
NaCl 150 mM then filtrated (100 .mu.m); and [0290] the filtrate is
heat sterilized. This washed AlPO.sub.4 is referred to as WAP
(washed autoclaved phosphate). Formulation Process
[0291] The PSC-PD conjugate bulk is adsorbed on AlPO4 WAP before
the final formulation of the finished product. AlPO.sub.4 WAP was
stirred with PSC-PD for 5 minutes at room temperature. The pH was
adjusted to 5.1, and the mixture was stirred for a further 18 hours
at room temperature. NaCl solution was added to 150 mM, and the
mixture was stirred for 5 minutes at room temperature.
2-phenoxyethanol was added to 5 mg/mL and the mixture was stirred
for 15 minutes at room temperature, then adjusted to pH 6.1.
[0292] Final Composition/Dose TABLE-US-00020 PSC-PD: 10 .mu.g PS
AlPO4 WAP: 0.25 mg Al.sup.3+ NaCl: 150 mM 2-phenoxy-ethanol: 2.5 mg
Water for Injection: to 0.5 ml pH: 6.1
F: Preclinical Information Immunogenicity of Polysaccharide
Conjugate in Mice
[0293] The immunogenicity of the PSC-PD conjugate has been assessed
in 6- to 8-weeks-old Balb/C mice. The plain (unadsorbed) conjugate
or the conjugate adsorbed onto AlPO4 was injected as a monovalent
vaccine. Anti-PSC antibodies induced were measured by ELISA whilst
functional antibodies were analysed using the bactericidal test,
both methods being based on the CDC (Centers for Disease Control
and Prevention, Atlanta, USA) protocols. Results from two different
experiments performed to assess the response versus the dose and
adjuvant (AlPO.sub.4) effect are presented.
Dose-Range Experiment
[0294] In this experiment, the PSC-PD was injected twice (two weeks
apart) in Balb/C mice. Four different doses of conjugate formulated
on AlPO4 were used: 0.1; 0.5; 2.5; and 9.6 .mu.g/animal. The mice
(10/group) were bled on days 14 (14 Post I), 28 (14 Post II) and 42
(28 Post II). Geometric mean concentrations (GMCs) of
polysaccharide C specific antibodies measured by ELISA were
expressed in .mu.g IgG/ml using purified IgG as reference.
Bactericidal antibodies were measured on pooled sera and titres
expressed as the reciprocal of the dilution able to kill 50% of
bacteria, using the N. meningitidis C11 strain in presence of baby
rabbit complement.
[0295] The dose-response obtained shows a plateau from the 2.5
.mu.g dose. Results indicate that there is a good booster response
between 14 Post I and 14 Post II. Antibody levels at 28 Post II are
at least equivalent to those at 14 Post II. Bactericidal antibody
titres are concordant with ELISA concentrations and confirm the
immunogenicity of the PSC-PD conjugate.
Effect of Adjuvant
[0296] In this experiment, one lot of PSC-PD conjugate formulated
on AlPO4 was assessed, the plain (non-adjuvanted) conjugate was
injected for comparison. 10 mice/group were injected twice, two
weeks apart, by the subcutaneous route, with 2 .mu.g of conjugate.
Mice were bled on days 14 (14 Post I), 28 (14 Post II) and 42 (28
Post II), and ELISA and functional antibody titres measured (only
on 14 Post II and 28 Post II for the bactericidal test). The AlPO4
formulation induces up to 10 times higher antibody titres as
compared to the non-adjuvanted formulations.
Conclusions
[0297] The following general conclusions can be made from the
results of the experiments described above: [0298] PSC-PD conjugate
induces an anamnestic response demonstrating that PSC, when
conjugated, becomes a T cell dependent antigen. [0299] Anti-PSC
antibody concentrations measured by ELISA correlate well with
bactericidal antibody titres showing that antibodies induced by the
PSC-PD conjugate are functional against N. meningitidis serogroup
C. [0300] Approximately 2.5 .mu.g of conjugate adsorbed onto AlPO4
appears to elicit an optimum antibody response in mice. [0301] The
CDAP chemistry appears to be a suitable method for making
immunogenic PSC-PD conjugates.
Example 8
Preparation of a Polysaccharide from N. meningitidis Serogroup A-PD
Conjugate
[0302] A dry powder of polysaccharide A (PSA) is dissolved for one
hour in NaCl 0.2 M solution to a final concentration of 8 mg/ml. pH
is then fixed to a value of 6 with either HCl or NaOH and the
solution is thermoregulated at 25.degree. C. 0.75 mg CDAP/mg PSA (a
preparation to 100 mg/ml acetonitrile) is added to the PSA
solution. After 1.5 minutes without pH regulation, NaOH 0.2 M is
added to obtain a pH of 10. 2.5 minutes later, protein D
(concentrated to 5 mg/ml) is added according to a PD/PSA ratio of
approximately 1. A pH of 10 is maintained during the coupling
reaction period of 1 hour. Then, 10 mg glycine (2 M pH 9.0)/mg PSA
is added and pH regulated at a value of 9.0 for 30 minutes at
25.degree. C. The mixture is then conserved overnight at 4.degree.
C. before purification by exclusion column chromatography
(Sephacryl S400HR from Pharmacia). The conjugate elutes first
followed by unreacted PD and by-product (DMAP, glycine, salts). The
conjugate is collected and sterilized by a 0.2 .mu.m filtration on
a Sartopore membrane from Sartorius.
Example 9
In Vitro Characterisations of the Products of Examples 7 and 8
[0303] The major characteristics are summarized in the table here
below: TABLE-US-00021 PS/protein Conjugate Protein and PS ratio
Free Protein Free PS N.sup.o description content (.mu.g/ml) (w/w)
(%) (%) 1 PS C - PD PD: 210 1/0.68 <2 8-9 NaOH for pH PS: 308
regulation 2 PS C - PD PD: 230 1/0.65 <2 5-6 TEA for pH PS: 351
regulation 3 PS A - PD PD: 159 1/1.07 5 5-9 NaOH for pH PS: 149
regulation
In Vivo Results
[0304] Balb/C mice were used as animal model to test the
immunogenicity of the conjugates. The conjugates were adsorbed
either onto AlPO.sub.4 or Al(OH).sub.3 (10 .mu.g of PS onto 500
.mu.g of Al.sup.3+) or not adsorbed. The mice were injected as
followed: 2 injections at two week intervals (2 .mu.g
PS/injection).
[0305] From these results, we can conclude first that free PS
influences greatly the immune response. Better results have been
obtained with conjugates having less than 10% free PS. The above
improvements to the CDAP process is thus a further aspect of the
invention.
[0306] The formulation is also important. AlPO.sub.4 appears to be
the most appropriate adjuvant in this model. The conjugates induce
a boost effect which is not observed when polysaccharides are
injected alone.
Conclusions
[0307] Conjugates of N. meningitidis A and C were obtained with a
final PS/protein ratio of 1 and 0.6-0.7 (w/w) respectively. Free PS
and free carrier protein were below 10% and 15% respectively.
Polysaccharide recovery is higher than 70%. Conjugates of PSA and
PSC obtainable by the above improved (optimised) CDAP process
(regardless of the carrier protein, but preferably protein D) is
thus a further aspect of the invention.
Example 10
Preparation of a Polysaccharide from H. influenzae b-PD
Conjugtate
[0308] H. influenzae b is one of the major causes of meningitis in
children under 2 years old. The capsular polysaccharide of H.
influenzae (PRP) as a conjugate onto tetanus toxoid is well known
(conjugated by chemistry developed by J. Robbins). CDAP is an
improved chemistry. The following is account of optimal CDAP
conditions found for conjugating PRP, preferably to PD.
[0309] The parameters influencing the reaction of conjugation are
the following: [0310] The initial concentration of polysaccharide
(which can have a double impact on the final levels of free
polysaccharide and on the sterile filtration step). [0311] The
initial concentration of the carrier protein. [0312] The initial
ratio of polysaccharide to protein (which can also have the double
impact on the final levels of free polysaccharide and on the
sterile filtration step). [0313] The quantity of CDAP used (usually
in large excess). [0314] The temperature of the reaction (which can
influence the breakdown of the polysaccharide, the kinetics of the
reaction, and the breakdown of the reactive groups). [0315] The pH
of activation and coupling. [0316] The pH of quenching (influencing
the level of residual DMAP). [0317] The time of activation,
coupling and quenching.
[0318] The present inventors have found that the 3 most critical
parameters to optimise the quality of the end product are: the
initial ratio of polysaccharide/protein; the initial concentration
of polysaccharide: and the coupling pH.
[0319] A reaction cube was thus designed with the above 3
conditions as the three axes. The central points (and experimented
value range) for these axes were: PS/protein ratio-1/1 (.+-.0.3/1);
[PS]=5 mg/ml (.+-.2 mg/ml); and coupling pH=8.0 (.+-.1.0 pH
unit).
[0320] The less essential parameters were fixed at the following:
30 mg of polysaccharide were used; temperature 25.degree. C.;
[CDAP]=0.75 mg/mg PS; pH titrated with 0.2M NaOH; activation
pH=9.5; temperature for activation=1.5 minutes; coupling
temperature-1 hour; [protein]=10 mg/ml; quench pH=9.0; temperature
of quenching=1 hour; temperature of dissolving PS in solvent=1 hour
in 2M NaCl; purification on Sephacryl S-400HR eluted with NaCl 150
mM at 12 cm/hour; and filter sterilising with a SARTOLAB P20 at 5
ml/min.
[0321] The data looked at to establish optimised conditions when
making products within the aforementioned reaction cube were:
process data--maximum yield after filtration, maximum level of
protein incorporated; and quality of product data--final ratio
PS/protein, level of free PS, level of free protein, minimum levels
of residual DMAP (a breakdown product of CDAP).
Output from Filtration
[0322] The factor which affects the output after filtration is the
interaction between the initial [PS] and the coupling pH and
initial PS/protein ratio. At low [PS] there is little interaction
with the latter 2 factors, and good filterability always results
(approx. 95% for all products). However, at high concentrations
filterability diminishes if the pH and the initial ratio increase
(high [PS], lowest ratio, lowest pH=99% filtration; but high [PS],
highest ratio and pH=19% filtration).
Level of Incorporation of the Protein
[0323] The ratio of the final ratio PS/protein with respect to the
initial ratio is a measure of the efficiency of coupling. At high
[PS], pH does not effect the ratio of ratios. However the initial
ratio does (1.75 at low initial ratio, 1.26 at high initial
ratios). At low [PS], the ratio of ratios is for the most part
lower, however pH now has more of an affect (low pH, low
ratio=0.96; low pH, high ratio=0.8; high pH, low ratio=1.4; and
high pH, high ratio=0.92).
Final PS/Protein Ratio
[0324] The final ratio depends on the initial ratio and the [PS].
The most sizeable final ratios are obtained with a combination high
initial ratios and high [PS]. The effect of pH on the final ratio
is not as significant as a weak [PS].
Level of Free Protein D
[0325] The least amounts of free protein D is observed at high pH
and high [PS] (levels approaching 0.0). The effect of high [PS]
becomes especially marked when pH is low. The raising of the
initial ratio contributes a little bit to the increase in free
protein D.
Residual DMAP
[0326] The initial ratio does not have a significant effect. In
contrast, the level of DMAP increases with the [PS], and decreases
when the pH is raised.
Conclusions
[0327] The most preferable conjugation conditions are thus the
following: coupling pH=9.0; [PS]=3 mg/ml; and initial ratio=1/1.
With such conditions the characteristics of the final product are
as follows: TABLE-US-00022 PS Output Free Final ratio from protien
D DMAP levels PS/protein filtration (%) Ratio of ratios (%) (ng/10
.mu.g PS) value range value Range value Range value range value
range 1.10 0.91-1.30 92.6 50-138 1.16 1.03-1.29 0.71 0-10.40 4.95
2.60-7.80
[0328] Conjugates of PRP obtainable by the above improved
(optimised) CDAP process (regardless of the carrier protein, but
preferably protein D) is thus a further aspect of the
invention.
Example 11
Protein D as an Antigen--how its Protective Efficacy Against
Non-Typeable H. influenzae can be Improved by Formulating it with
3D-MPL
[0329] Female Balb/c Mice (10 per group) were immunized
(intramuscularly) with the eleven valent pneumococcal
polysaccharide-protein D conjugate vaccine for a first time at the
age of 20 weeks (D0) and received a second immunization two weeks
later (D14). Blood was collected 7 days after the second
immunization. Antibody titres against protein D were measured in
terms of the quantity of IgG1, IgG2a and IgG2b type antibodies.
[0330] Freeze-dried undecavalent vaccines (without AlPO.sub.4) were
prepared by combining the conjugates with 15.75% lactose, stirring
for 15 minutes at room temperature, adjusting the pH to 6.1.+-.0.1,
and lyophilising (the cycle usually starting at 69.degree. C.,
gradually adjusting to -24.degree. C. over 3 hours, then retaining
this temperature for 18 hours, then gradually adjusting to
-16.degree. C. over 1 hour, then retaining this temperature for 6
hours, then gradually adjusting to +34.degree. C. over 3 hours, and
finally retaining this temperature over 9 hours).
[0331] Composition of formulations and reconstituants for
lyophilisates are presented in Table 13.
[0332] The most characteristic measurement as to whether a Th1-type
cell mediated immune response has occurred is known to be
correlated with the level of IgG2a antibody. As can be seen from
the data, a surprisingly large increase in IgG2a results if the
protein D has been lyophilised with a Th1 adjuvant (in this case
3D-MPL). TABLE-US-00023 TABLE 13 Composition of formulations (per
human dose), and antibody titres against protein D in mice (with
1/10 dose) Physical PS AlPO.sub.4 Caking IgG1 IgG2a IgG2b IgG1
IgG2a IgG2b state (/500 .mu.l) (/500 .mu.l) Immunostimulant agent
Preservative Reconstituant .mu.g/ml % liquid 1 .mu.g 500 .mu.g no
no 2-PE.sup.3 no 76 0.425 0.24 99.1 0.554 0.313 liquid 5 .mu.g 500
.mu.g no no 2-PE no 66 0.284 0.176 99.3 0.427 0.265 liquid 1 .mu.g
0 .mu.g no no 2-PE no 6.6 0.207 0.036 96.4 3.02 0.526 liquid 5
.mu.g 0 .mu.g no no 2-PE no 5.2 0.169 0.043 96.1 3.12 0.795
freeze-dried 1 .mu.g 0 .mu.g no lactose no NaCl 150 5.2 0.147 0.046
96.4 2.73 0.853 3.15% mM.sup.1 freeze-dried 5 .mu.g 0 .mu.g no
lactose no NaCl 150 11.1 0.11 0.168 97.6 0.967 1.477 3.15% mM.sup.1
freeze-dried 1 .mu.g 0 .mu.g no lactose no AlPO.sub.4 45 1.86 0.075
95.9 3.96 0.160 3.15% 500 .mu.g.sup.2 freeze-dried 5 .mu.g 0 .mu.g
no lactose no AlPO.sub.4 19 0.077 0.119 99.0 0.401 0.620 3.15% 500
.mu.g.sup.2 freeze-dried 1 .mu.g 0 .mu.g no lactose no MPL 50
.mu.g.sup.1 45 2.6 3.5 88.1 5.09 6.849 3.15% freeze-dried 5 .mu.g 0
.mu.g no lactose no MPL 50 .mu.g.sup.1 135 25 5.1 81.8 15.1 3.089
3.15% freeze-dried 1 .mu.g 0 .mu.g MPL (50 .mu.g) lactose no
buffer.sup.1 43 22 5.7 60.8 31.1 8.062 3.15% liquid 1 .mu.g 500
.mu.g MPL (50 .mu.g) no 2-PE no 441 7.1 9.1 96.5 1.55 1.990 liquid
5 .mu.g 500 .mu.g MPL (50 .mu.g) no 2-PE no 299 1.4 0.899 99.2
0.465 0.298 .sup.1before injection; .sup.2+/- 2 hours before
injection; .sup.32-phenoxyethanol
* * * * *