U.S. patent application number 09/951657 was filed with the patent office on 2002-05-09 for vaccine composition comprising a polysaccharide conjugate antigen adsorbed onto aluminium phosphate.
This patent application is currently assigned to SmithKline Beecham Biologicals, sa. Invention is credited to Hauser, Pierre, Peetermans, Julien.
Application Number | 20020054884 09/951657 |
Document ID | / |
Family ID | 27547241 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020054884 |
Kind Code |
A1 |
Peetermans, Julien ; et
al. |
May 9, 2002 |
Vaccine composition comprising a polysaccharide conjugate antigen
adsorbed onto aluminium phosphate
Abstract
The invention relates to a vaccine formulation for the
prevention of Haemophilus Influenzae Type B (Hib) infections and
where the antigen is adsorbed onto aluminium phosphate. The
invention also relates to a multivalent vaccine, that is a vaccine
for the amelioration or treatment of more than one disease states.
The present invention also relates to the production and use of
such vaccines in medicine.
Inventors: |
Peetermans, Julien;
(Rixensart, BE) ; Hauser, Pierre;
(Chaumont-Gistoux, BE) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham Biologicals,
sa
|
Family ID: |
27547241 |
Appl. No.: |
09/951657 |
Filed: |
September 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09951657 |
Sep 13, 2001 |
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09522234 |
Mar 9, 2000 |
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09522234 |
Mar 9, 2000 |
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08983271 |
Feb 11, 1998 |
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08983271 |
Feb 11, 1998 |
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PCT/EP96/02690 |
Jun 19, 1996 |
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Current U.S.
Class: |
424/196.11 ;
424/193.1; 424/197.11; 424/201.1; 424/204.1; 424/217.1; 424/226.1;
424/227.1; 424/234.1; 424/236.1; 424/239.1; 424/256.1; 514/54 |
Current CPC
Class: |
A61K 39/08 20130101;
A61K 39/102 20130101; A61K 2039/545 20130101; Y02A 50/464 20180101;
A61K 39/092 20130101; A61K 2039/6037 20130101; A61K 39/095
20130101; C12N 2730/10134 20130101; Y02A 50/30 20180101; A61K 39/29
20130101; A61K 39/05 20130101; A61K 39/13 20130101; Y02A 50/466
20180101; A61K 2039/70 20130101; A61K 39/0016 20130101; A61K
2039/55505 20130101; A61K 39/12 20130101 |
Class at
Publication: |
424/196.11 ;
514/54; 424/201.1; 424/193.1; 424/197.11; 424/204.1; 424/226.1;
424/227.1; 424/217.1; 424/234.1; 424/236.1; 424/239.1;
424/256.1 |
International
Class: |
A61K 039/385; A61K
031/715; A61K 039/295; A61K 039/02; A61K 039/102; A01N 043/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 23, 1995 |
GB |
9512827.8 |
Jul 1, 1996 |
GB |
9513443.3 |
Dec 15, 1995 |
GB |
9525657.4 |
Mar 22, 1996 |
GB |
9606032.2 |
Claims
1. A vaccine composition comprising a polysaccharide conjugate
antigen adsorbed onto aluminium phosphate.
2. A vaccine as claimed in claim 1, wherein the antigen is a
capsular polysaccharide from Haemophilius influenzae B conjugate
with a carrier protein.
3. A vaccine as claimed in claim 1 or 2, wherein the carrier is
selected from the group comprising Diphtheria toxoid, Diphtheria
CRM.sub.197 protein, meninogococcal outer membrane protein and
Tetanus toxoid.
4. A vaccine as claimed in claim 1, 2 or 3 wherein the carrier
protein is a tetanus toxoid.
5. A vaccine as claimed in any one of claims 1 to 4 wherein the
ratio of polysaccharide to protein is from 1:0.3 to 1:2
(weight:weight).
6. A vaccine as claimed in any of claims 1 to 5 contains at least
one other component to afford protection against a pathogen other
than Haemophilus influenzae B.
7. A vaccine as claimed in any of claims 1 to 6 wherein the other
component is selected from the group of antigens which afford
protection against one of Hepatitis A virus, diphtheria, tetanus,
pertussis, Hepatitis B and Polio.
8. A vaccine as claimed in any of claim 1 to 7 wherein the adsorbed
conjugate is freeze dried.
9. A vaccine as claimed in any of claim 1 to 7 wherein the adsorbed
conjugate is suspended in water for injection.
10. A kit comprising a container of a freeze dried vaccine
according to claim 8 and a second container with a vaccine against
a second pathogen.
11. A method of producing a vaccine according to claims 1 to 6
comprising conjugating a polysaccharide antigen to a protein
carrier and adsorbing said antigen onto aluminium phosphate.
12. A vaccine composition as defined in any of claims 1 to 9 for
use in medicine.
13. A method of treating a patient suffering from or susceptible to
Haemophilus influenzae b infection, comprising administering a safe
and efficacious amount of a vaccine composition according to any of
claims 1 to 9.
Description
[0001] The present invention relates to new vaccine formulations,
comprising a conjugated polysaccharide antigen linked to a carrier
protein. In particular the invention relates to a vaccine
formulation for the prevention of Haemophilus Influenzae Type B
(Hib) infections and where the antigen is adsorbed on to aluminium
phosphate. The invention also relates to a multivalent vaccine,
that is a vaccine for the amelioration or treatment of more than
one disease states. The present invention also relates to the
production and use of such vaccines in medicine.
[0002] Vaccines that utilise polysaccharides are known in the art.
For example a vaccine for the prevention of Haemophilus influenzae
b (Hib) infections are based on the capsular polysaccharide (PRP)
conjugated with a carrier protein. The polysaccharide is a polymer
of ribose, ribitol and phosphate. These vaccines are typically
presented as plain (ie without adjuvantation) formulations.
Although in one case, (Pedvax Hib produce by Merck) a diluent
containing aluminium hydroxide is utilised to reconstitute the
lyophilised conjugate. Typically the carrier protein is a
diphtheria or tetanus toxoid or an outer membrane protein of
N.menigitidis. Examples of such conjugate vaccine antigens are
disclosed in U.S. Pat. No. 4,365,170, U.S. Pat. No. 4,673,574, EP
208 375, EP 477508 and EP 161 188.
[0003] It is desirable to administer such conjugate vaccines with
other antigens or vaccines at the same time and this can involve
multiple injections. Problems associated with multiple injections
include a more complicated administration procedure and a large
total injection volume. This is a particularly acute problem when
the vaccine is intended for infants.
[0004] It has therefore been proposed to produce combination
vaccines. One well known combination vaccine provides protection
against Diphtheria, tetanus and B. pertussis infections. This
vaccine comprises a whole cell or an accellular pertussis component
which typically consists of two or three antigens--(detoxified PT,
FHA and often, but not exclusively 69kDa) although in certain
circumstances other B. pertussis antigens may also be present and
toxoided diphtheria and tetanus toxins. Such vaccines are often
referred to as DTPw or DTPa. Other antigens would desirable be
added to such a combination vaccine for the prevention of diseases
like hepatitis B. or Polio.
[0005] It would be desirable to add polysaccharide conjugate
vaccines to such a combination. However we have found that simple
mixing of the components results in a reduction of antibody titres
to the polysaccharide component.
[0006] The present inventors have discovered that this reduction
can be inhibited if the conjugate antigen is adsorbed on to
aluminium phosphate. In contrast, if the antigen is adsorbed on to
aluminium hydroxide, there is a complete reduction of antibody
titres to the polysaccharide component.
[0007] Accordingly the present invention provides a vaccine
composition comprising a polysaccharide conjugate antigen adsorbed
on to aluminium phosphate. Preferably the antigen is capsular
polysaccharide (PRP) from Hib conjugated with a carrier
protein.
[0008] Preferably the carrier protein is either diphtheria or
tetanus toxoid, Diphtheria Crm.sub.197 protein or an outer membrane
protein from a bacteria such as N.menigitidis.
[0009] The polysaccharide conjugate may be prepared by any known
coupling technique. For example the polysaccharide can be coupled
via a thioether linkage. This conjugation method relies on
activation of the polysaccharide with 1cyano-4-dimethylamino
pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The
activated polysaccharide may thus be coupled directly or via a
spacer group to an amino group on the carrier protein. Preferably,
the cyanate ester is coupled with hexane diamine and the
amino-derivatised polysaccharide is conjugated to the carrier
protein using heteroligation chemistry involving the formation of
the thioether linkage. Such conjugates are described in PCT
published application WO93/15760 Uniformed Services University.
[0010] The conjugates can also be prepared by direct reductive
amination methods as described in U.S. Pat. No. 4,365,170
(Jennings) and U.S. Pat. No.4,673,574 (Anderson). Other methods are
described in EP-0-161-188, EP-208375 and EP-0-40477508.
[0011] A further method involves the coupling of a cyanogen bromide
activated polysaccharide derivatised with adipic acid hydrazide
(ADH) to the protein carrier by carbodiimide condensation. Such
conjugation is described in Chu C. et al Infec Immunity, 1983 245
256.
[0012] In a preferred embodiment of the invention the ratio of PRP
polysaccharide to carrier protein is reduced from a typical 1:3 to
1:0.3 to 1:2. Such low ratio conjugates are advantageous, since
even in an unadjuvanted state, they do not suffer from interference
problems.
[0013] In a preferred embodiment of the invention the formulation
preferably contains at least one other component selected from
antigens which afford protection against one or more of the
following: Hepatitis A virus (HAV), diphtheria, tetanus, pertussis,
Hepatitis B and polio.
[0014] Particular combination vaccines within the scope of the
invention include a DTPa (diphtheria-tetanus-accellular
pertussis)--Hib combination vaccine formulation, an Hib-Hepatitis B
vaccine formulation, a DTPa-Hib-Hepatitis B vaccine formulation and
an IPV (inactivated polio vaccine)--DTPa-Hib-Hepatitis B vaccine
formulation.
[0015] The above combinations may optionally include a component
which is protective against Hepatitis A.
[0016] Suitable components for use in such vaccines are already
commercially available and details may be obtained from the World
Health Organisation. For example the IPV component may be the Salk
inactivated polio vaccine. The Diphtheria, Tetanus and Pertussis
vaccine may comprise an acellular product such as Infanrix DTPa
(SmithKline Beecham Biologicals). The component affording
protection against Hepatitis A is preferably the product known as
`Havrix` (SmithKline Beecham Biologicals) which is a killed
attenuated vaccine derived from the HM-175 strain of HAV [see
`Inactivated Candidate Vaccines for Hepatitis A` by F. E. Andre, A
Hepburn and E. D'Hondt, Prog Med. Virol. Vol 37, pages 72-95 (1990)
and the product monograph `Havrix` published by SmithKline Beecham
Biologicals (1991)]. The Hepatitis B component may comprise the `S`
antigen as in `Engerix-B`.
[0017] Advantageously the Haemophilus Influenzae B or combination
vaccine according to the invention is a paediatric vaccine.
[0018] Vaccine preparation is generally described in Vaccine
Design--The Subunit and adjuvant approach Ed Powell and Newman;
Pellum Press. Encapsulation within liposomes is described, for
example, by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of
proteins to macromolecules is disclosed, for example, by Likhite,
U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.
4,474,757.
[0019] 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 vaccinees.
Such amount will vary depending on which specific immunogens are
employed. Generally it is expected that each dose will comprise
1-1000 ug of total immunogen, preferably 2-100 ug, most preferably
4-40 ug. An optimal amount for a particular vaccine can be
ascertained by standard studies involving observation of antibody
titres and other responses in subjects. Following an initial
vaccination, subjects may receive one or two booster injections at
about 4 weeks intervals.
[0020] In a further aspect according to the invention, there is
provided a method of producing the vaccine comprising adsorbing the
conjugate antigen on to aluminium phosphate. The adsorbing is
preferably done at a pH of between 5 and 6, preferably at about
5.4. In an embodiment the vaccine is freeze dried after standing
for more than 24 hours. Alternatively, the vaccine of the invention
may be combined with other antigens in a liquid form.
[0021] The invention further provides the first medical use of such
a vaccine.
[0022] In a further embodiment the invention provides a method of
preventing or ameliorating Heamophilus Influenzae B infections, the
method comprising the administration of a non toxic, effective
amount of the vaccine of the invention.
[0023] The following examples illustrate the invention.
EXAMPLE 1
Vaccine Formulation Comprising HiB Polysaccharide Conjugated on
Tetanus Toxoid Adsorbed on to Aluminium Phosphate.
[0024] Synthesis of Haemophilus influenzae type B capsular
polysaccharide (PRP) Tetanus toxoid (TT) conjugate
[0025] 1.a Cyanogen Bromide Coupling
[0026] The covalent binding of PRP and TT is carried out by a
coupling chemistry developed at the NIH (Chu C. et al (1983),
further studies on the immunogenicity of Haemophilus influenzae
type b and pneumococcal type 6A polysaccharide protein conjugates.
Infec. Immunity, 245-256). The PRP is activated under controlled
conditions by cyanogen bromide and derivatised with an adipic
hydrazide spacer.
[0027] After derivatisation, the activated polysaccharide (PRP-AH)
is purified by diafiltration. The coupling of the two purified
components (PRP-AH and TT) is effected by carbodiimide
condensation. The conjugate is then purified by ultrafiltration and
gel filtration to remove the reagent and unconjugated PRP and
TT.
[0028] Synthesis of PRP-TT Conjugates
[0029] 1 .b CDAP coupling
[0030] 30mg of native Hib PRP were dissolved in 6 ml 2M NaCl. 225
mcl of CDAP (1 cyano4-dimethylamino-pyridinum tetrafluoroborate)
was added to the polysaccharide solution (from a 100 mg/ml stock
solution in acetonitrile). 90 seconds later, 450 mcl of 0.2 M
triethylamine was added. The activation was performed at pH 10.0
during 1 minute on ice and minute at room temperature.
[0031] 90 mg of tetanus toxoid (initial PS/protein ratio of 1/3)
were added to the activated polysaccharide and the coupling
reaction was performed at room temperature for 1 hour. Then, the
reaction was quenched with 3 ml of 1M glycine solution, pH 5.0 for
30 minutes at room temperature and overnight at 4.degree. C.
[0032] The conjugate was purified by gel filtration on a sephacryl
HR 500 column equilibrated in 0.2M NaCl. The carbohydrate and
protein content was determined in each fraction. The conjugate was
pooled and sterile filtered (membrane Minisart .sym. 0.222
.gamma.m).
[0033] Adsorption on to aluminium phosphate
[0034] 1.c To 0.15 mg of aluminium phosphate was added 12.5 mcg of
the polysaccharide conjugate of example 1(a). This was stirred for
two hours the pH is adjusted to 5.1. The mixture was left to stand
for one day at room temperature and the adsorbed conjugate then
left for a further 9 days at 2 to 8.degree. C. To prepare a freeze
dried product the adsorbed product is diluted in lactose (15.75 mg)
to give a final composition of 25 mcg polysaccharide/ml and 0.4 mg
Al/ml and the resulting composition was filled into 0.5 ml vials
and freezed dried.
[0035] To prepare a liquid product the adsorbed conjugate is
diluted in water for injection with 150 mM NaCl and 5 mg/ml phenoxy
ethanol to give a final composition of 20 mcg polysaccharide/ml and
0.32 mgAl/ml.
[0036] 1.d Formulation of a Diphtheria Tetanus and Pertussis
(acellular) vaccine with and without hepatitis B was done in
accordance to the methods of WO 93/24148 (SmithKline Beecham
Biologicals).
[0037] 1.e Preparation of a `low ratio` PRP-TT aluminium phosphate
pre-adsorbed conjugate.
[0038] The conjugate was prepared in an analogous manner to the
example of 1a, but with reduced amount of Tetanus used (30mcg, 60
mcg) to give a product with Polysaccharide:Protein ratio of 1:1 or
1:2. The conjugate is then adsorbed on to aluminium phosphate
according to the method of example 1c. The final freeze dried
preparation contains 12.5 .mu.g of conjugate, 0.15 mg ALPO.sub.4,
15.75 mg lactose. This is reconstituted in 0.5 ml water for
injection prior to use at a pH of 0.1+/-0.1.
EXAMPLE 2
Immunogenicity of PRP-TT Conjugate Preadsorbed on Aluminium
Phosphate and Combined with DTPa or DTPa-B
[0039] The Hib conjugate of example 1a), either plain or
pre-adsorbed on Al PO4 (both vaccines were lyophilized) was mixed
with DTPa or DTPa HB no more than 1 hour before injection and the
combination was injected in baby rats (1 week of age) by the
subcutaneous route at a dose corresponding to {fraction (1/20)}th a
human dose (0.5 .mu.g of PRP). The rats were boosted 2 weeks and 4
weeks later and the serum was collected was collected after each
immunization to measure anti-PRP antibodies. Controls included the
Hib vaccines (adsorbed or not on Al PO4) reconstituted in
saline.
[0040] Groups of 10 randomized baby rats (1 week of ago-OFA strain)
were immunized 3 times subcutaneously at 0-14-28 days with
{fraction (1/20)}th human dose of Hib vaccine, alone or combined
with DTPa or DTPa HB ({fraction (1/20)}th a human dose). The
reconstitution of the lyophilised Hib vaccine with saline or
combinations (DTPa or DTPa HB) was done less than 1 hour before
immunization.
[0041] The rats were bled under anesthesia at 14-28-42 and 56 days.
The anti-PRP antibodies were measured by ELISA in individual sera
and the titers were expressed in .gamma./ml using a calibrated
reference. The GMT was calculated for each group and for each time
point. The 95% confidence limits were calculated for the titers
obtained after that third immunization.
[0042] As shown in table 1, the adsorption of Hib conjugate on Al
PO4 does not modify its immunogenicity: some anti-PS were produced
after the second dose and a good booster effect is shown after the
third dose as seen in human babies. The mixing of Hib vaccine with
DTPa or DTPa HB reduces by 3 to 8 fold the anti-PRP response and,
in the case of DTPa-HB, this decrease is significant. In contrast,
the pre-adsorption of the Hib vaccine Al PO4 restores the anti-PRP
response to a level at least equivalent to that obtained with the
plain vaccine.
Conclusion
[0043] The Hib/aluminium phosphate formulation has thus the
potential to solve the compatibility problem encountered when
mixing Hib with other peadiatric combinations. Immunogenicity in a
baby rat model of PRP-TT conjugate pre adsorbed on AIP04 and
combined with DTPa or DTPa-HB
1 TABLE 1 Anti-PRP titre (.gamma./ml) at day Vaccine 14 (Post I) 28
(Post II) 42 (Post III) 56 (Post III 30) None (Nacl 0.9%) <0.05
<0.05 <0.05 <0.05 Hib-001 <0.05 0.06 12.9 (4-37) 10.9
(4-31) Hib/AlP04 (Dhib-024) <0.05 1.3 11.8 (5-29) 15.4 (7-35)
Hib-001 + DTPa (119) <0.05 0.16 3.4 (0-28) 1.4 (0.1-17)
Hib/AlP04 + DTPa (119) <0.05 1.9 20.9 (7-59) 19.7 (9.42) Hib-001
+ DTPa HB (16705) <0.05 0.14 2.8 (1-6) 3.9 (2-9) Hib/AlP04 +
DTPa HB (16705) <0.05 0.47 11.4 (5-27) 18.1 (9-38)
Hib/Al(OH.sub.3) <0.05 <0.05 <0.05 <0.14
* * * * *