U.S. patent application number 10/878094 was filed with the patent office on 2005-05-26 for hepatitis a vaccines.
This patent application is currently assigned to SmithKline Beecham Biologicals S.A.. Invention is credited to D'Hondt, Erik.
Application Number | 20050112144 10/878094 |
Document ID | / |
Family ID | 10840772 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112144 |
Kind Code |
A1 |
D'Hondt, Erik |
May 26, 2005 |
Hepatitis A vaccines
Abstract
A process for the production of inactivated Hepatitis A virus
substantially free of host cell contamination is described, the
process comprising: a)culturing Hepatitis A virus and harvesting a
Hepatitis A preparation; b) treating said Hepatitis A preparation
with a protease; and thereafter c) separating intact virus from
protease-digested material; d) inactivating said virus. Also
described are vaccines comprising the inactivated Hepatitis A
virus, preferably in combination with strong adjuvants.
Inventors: |
D'Hondt, Erik; (Ottenburg,
BE) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham Biologicals
S.A.
|
Family ID: |
10840772 |
Appl. No.: |
10/878094 |
Filed: |
June 28, 2004 |
Current U.S.
Class: |
424/226.1 ;
435/239 |
Current CPC
Class: |
C12N 2770/32434
20130101; A61K 2039/5252 20130101; A61P 1/16 20180101; C12N 7/00
20130101; A61K 2039/55505 20130101; C12N 2770/32471 20130101; C12N
2770/32451 20130101; A61K 2039/55572 20130101; A61K 2039/70
20130101; A61P 31/12 20180101; C12N 2770/32463 20130101; C12N
2730/10134 20130101; A61K 39/12 20130101 |
Class at
Publication: |
424/226.1 ;
435/239 |
International
Class: |
A61K 039/29; C12N
007/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 16, 1998 |
GB |
9822714.3 |
Claims
1-16. (canceled)
17. An inactivated Hepatitis A virus substantially free of host
cell contaminants.
18. The inactivated Hepatitis A virus of claim 17 comprising less
than 10% contaminating host cell proteins detectable by scanning
SDS PAGE.
19. A Hepatitis A vaccine comprising an inactivated Hepatitis A
virus according to claim 17.
20. A vaccine according to claim 19 formulated with a Th1-type
inducing adjuvant.
21. A vaccine according to claim 20, wherein the adjuvant comprises
monophosphoryl lipid A or a derivative thereof.
22. A vaccine according to claim 21, further comprising QS21.
23. A vaccine according to claim 22, further comprising an oil in
water emulsion and tocopherol.
24. A vaccine according to claim 18, further comprising a Hepatitis
B antigen.
25. A vaccine according to claim 22, further comprising a Hepatitis
B antigen.
26. A vaccine according to claim 23, further comprising a Hepatitis
B antigen.
27. A vaccine according to claim 18, further comprising a
non-hepatitis antigen.
28. A vaccine according to claim 22, further comprising a
non-hepatitis antigen.
29. A vaccine according to claim 23, further comprising a
non-hepatitis antigen.
30. A vaccine according to claim 24, further comprising a
non-hepatitis antigen.
31. A vaccine according to claim 25, further comprising a
non-hepatitis antigen.
32. A vaccine according to claim 26, further comprising a
non-hepatitis antigen.
33. The virus of claim 17 wherein the virus is obtained by a
process comprising: a) culturing Hepatitis A virus and harvesting a
hepatitis A preparation; b) treating said hepatitis A preparation
with a protease, thereafter; c) separating intact virus from
protease-digested protein; and d) inactivating said virus.
34. A Hepatitis A vaccine comprising an inactivated Hepatitis A
virus according to claim 33.
Description
[0001] The present invention relates to new vaccine compositions,
processes for their manufacture and their use in medicine. In
particular, the present invention relates to improved Hepatitis A
vaccines adjuvanted with a potent immunostimulator, preferably such
as monophosphoryl lipid A or a derivative thereof The invention
also relates to combination vaccines in which the Hepatitis A
vaccine is a component.
[0002] Hepatitis A vaccines are known. For example the vaccine
Havrix (Trade Mark), from SmithKline Beecham Biologicals can be
used to prevent hepatitis A infections and is also formulated with
aluminium hydroxide as adjuvant. This vaccine is produced according
to the procedure of Andre et al. It comprises an attenuated strain
of the HM-175 Hepatitis A virus inactivated with formol
(formaldehyde); see Andre et al [Prog Med. Virol. 1990, vol 37;
-p72-95].
[0003] The vaccine Twinrix (Trade Mark) which is a combination of
the above hepatitis A and hepatitis B antigens may be used to
protect against Hepatitis A and Hepatitis B simultaneously. The
vaccine Hepatyrix (Trade Mark) which is a combination of the above
hepatitis A antigen and a Salmonella typhimurium purified Vi
polysaccharide may be used to protect against Hepatitis and typhoid
simultaneously.
[0004] International patent application WO93/19780 (SmithKline
Beecham Biologicals s.a.) discloses, inter alia, a Hepatitis A
vaccine adjuvanted with 3D-MPL.
[0005] European patent 0 339 667 (Chemo Sero) describes the general
concept of combining a hepatitis A antigen and a hepatitis B
antigen to make a combination vaccine. In that specification it is
stated that the adjuvant which is used is not critical: it must
only be capable of enhancing the immune activity to a desired
extent and not cause any side effects. It is stated that aluminium
gel may be used, in particular aluminium hydroxide gel and
aluminium phosphate gel.
[0006] It has now been found that traditional processes for
producing and purifying inactivated virus for hepatitis A vaccines
can leave a small residue of contaminants from the host cells in
which the hepatitis A virus was grown. Such host cell contaminants,
especially when they are from human origin, diploid in nature and
at a low level, provide no concern when the vaccine is adjuvanted
with aluminium salts. But when the vaccine is adjuvanted with
strong immunostimulants there is a theoretical possibility that a
vaccinee may raise an adverse immune response to the host
contaminants.
[0007] Accordingly there is a need for a method of manufacture
which removes substantially all traces of such host cell
proteins.
[0008] Accordingly in one aspect of the invention there is provided
a process for the production of inactivated Hepatitis A virus
substantially free of host cell contamination, the process
comprising:
[0009] a) culturing Hepatitis A virus and harvesting a hepatitis A
preparation;
[0010] b) treating said hepatitis A preparation with a protease;
and thereafter
[0011] c) separating intact virus from protease-digested
material;
[0012] d) inactivating said virus.
[0013] Surprisingly, the protease digestion treatment does not
adversely affect the Hepatitis A virus, but facilitates the
breakdown and separation of host cell contaminants from the
Hepatits A preparation.
[0014] Preferably the Hepatitis A virus is derived from HM-175
strain.
[0015] By substantially free of host cell contamination is meant
that less than 10%, preferably less than 8%, more preferably less
than 5% host cell protein can be detected by scanning of
silver-stained SDS PAGE. More importantly and as determined by slot
blot hybridisation one dose of HAV in the vaccine preferably
contains less than 10 ng of host cell proteins.
[0016] Preferably the protease used is trypsin. Other proteases
that may be utilised include pronase, papain, and pepsin.
[0017] The protease treatment is preferably carried out at above
room temperature, e.g. at about 37.degree. C. for about 2 hrs.
[0018] The separation of the intact virus from the protease and the
digested components can be achieved by a variety of suitable
methods, for example by permeation chromatography.
[0019] Alternatively the protease and digested components may be
separated by any separation method that separates on the basis of
size, for example ultra filtration. The product can then be further
purified by other steps to remove other contaminants. For example,
the product can be further purified by subjecting the product to
ion-exchange chromatography to remove any nucleic acid residue.
[0020] It is believed that the protease digestion step of the
method according to the invention can improve purification of the
hepatitis A preparation due to two effects. First, the protease
digests any contaminating host proteins such that they are easier
to separate in the chromatographic separation step that follows the
protease treatment. Second, the digestion of contaminating host
proteins allows better separation of other contaminating materials
which would otherwise be associated with undigested host proteins,
in particular nucleic acid, in the ion exchange step. It will be
appreciated that these observed effects do not necessarily limit
the invention in any way.
[0021] In another aspect of the present invention there is provided
an inactivated Hepatitis A virus substantially free of
contaminating host proteins, as defined above.
[0022] The inactivated hepatitis A virus may then be formulated
into a vaccine.
[0023] Thus the invention provides in a further aspect a Hepatitis
A vaccine comprising an inactivated hepatitis A virus substantially
free of host cell contaminants.
[0024] Such a vaccine may advantageously include a suitable
adjuvant. Suitable adjuvants include an aluminium salt such as
aluminium hydroxide gel 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.
[0025] Advantageously, the highly purified hepatitis A virus may be
formulated with strong adjuvant systems. Thus in the formulation of
the invention, it is preferred that the adjuvant composition
induces an immune response comprising Th1 aspects.
[0026] In general terms, a Th1-type response is characterised by
the production of IFN-.gamma. as opposed to a Th2-type response
which is characterised by the production of cytokines such as IL-4,
IL-5 and IL-10. The isotypic profile of the humoral response can
also be used as a marker for Th1 or Th2-type responses. In mice
Th1-type responses are often associated with the generation of
antibodies of the IgG2a subtype while IgG1 are markers of a
Th2-type response. The situation is not as clear in humans but data
suggest that IgG1 and IgG4 could respectively be markers of Th1-
and Th2-type responses.
[0027] Suitable adjuvant systems include for example a combination
of monophosphoryl lipid A, preferably 3-O-de-acylated
monophosphoryl lipid A (3D-MPL), and preferably formulated together
with an aluminium salt.
[0028] An enhanced system involves the combination of
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.
[0029] A particularly potent adjuvant formulation involving QS21,
3D-MPL and d,1-alpha-tocopherol in an oil in water emulsion is
described in WO 95/17210.
[0030] Other known adjuvants which may be included are CpG
containing oligonucleotides for example as disclosed in WO
96/02555.
[0031] Accordingly in a preferred embodiment of the present
invention there is provided a vaccine comprising a virus of the
present invention, adjuvanted with monophosphoryl lipid A or a
derivative thereof.
[0032] Preferably the vaccine additionally comprises a saponin,
more preferably QS21.
[0033] Preferably the formulation additionally comprises an oil in
water emulsion and d,1-alpha-tocopherol.
[0034] The present invention also provides a method for producing a
vaccine formulation comprising mixing a purified virus of the
present invention together with a pharmaceutically acceptable
excipient or carrier, such as 3D-MPL.
[0035] The purified virus of the invention may advantageously be
combined with other antigens so that it is effective in the
prophylaxis or treatment of other diseases in addition to hepatitis
A infections. A preferred combination involves a combination
containing a hepatitis B antigen.
[0036] The preparation of Hepatitis B surface antigen (HBsAg) is
well documented. See, for example, Harford et al in Develop. Biol.
Standard 54, page 125 (1983), Gregg et al in Biotechnology, 5, page
479 (1987), EP-A-0 226 846, EP-A-0 299 108 and references
therein.
[0037] As used herein the expression `Hepatitis B surface antigen`0
or `HBsAg` includes any HBsAg antigen or fragment thereof
displaying the antigenicity of HBV surface antigen. It will be
understood that in addition to the 226 amino acid sequence of the
HBsAg S antigen (see Tiollais et al, Nature, 317, 489 (1985) and
references therein) HBsAg as herein described may, if desired,
contain all or part of a pre-S sequence as described in the above
references and in EP-A-0 278 940. In particular the HBsAg may
comprise a polypeptide comprising an amino acid sequence comprising
residues 12-52 followed by residues 133-145 followed by residues
175-400 of the L-protein of HBsAg relative to the open reading
frame on a Hepatitis B virus of ad serotype (this polypeptide if
referred to as L*; see EP 0 414 374). HBsAg within the scope of the
invention may also include the preS1-preS2-S polypeptide described
in EP 0 198 474 (Endotronics) or analogues thereof such as those
described in EP 0 304 578 (McCormick and Jones). HBsAg as herein
described can also refer to mutants, for example the `escape
mutant` described in WO 91/14703 or EP 0 511 855 A1, especially
HBsAg wherein there is an amino acid substitution at position 145
to arginine from glycine.
[0038] Normally the HBsAg will be in particle form. The particles
may comprise for example S protein alone or may be composite
particles, for example (L*,S) where L* is as defined above and S
denotes the S-protein of HBsAg. The said particle is advantageously
in the form in which it is expressed in yeast.
[0039] The invention in a further aspect provides a vaccine
formulation as described herein for use in medical therapy,
particularly for use in the treatment or prophylaxis of hepatitis
viral infections. In a preferred aspect the vaccine accordingly to
the invention is a therapeutic vaccine useful for the treatment of
ongoing hepatitis infections, more especially hepatitis A and/or
hepatitis B infections in humans suffering therefrom.
[0040] In view of the surprisingly efficacious results obtained, in
a further preferred aspect the invention provides a vaccine
composition for the treatment or prophylaxis of Hepatitis A and/or
Hepatitis B infections.
[0041] Advantageously the hepatitis vaccine composition of the
invention contains other antigens so that it is effective in the
treatment or prophylaxis of one or more other bacterial, viral or
fungal infections.
[0042] Accordingly the hepatitis vaccine formulation according to
the invention preferably contains at least one other component
which may be selected from non-hepatitis antigens which are known
in the art to afford protection against one or more of the
following diseases:
[0043] diphtheria, tetanus, pertussis, Haemophilus influenzae b
(Hib), and polio.
[0044] Preferably the vaccine according to the invention includes
HBsAg as hereinabove defined.
[0045] Suitable components for use in such combination vaccines are
already commercially available and details may be obtained from the
World Health Organization. For example the polio component may be
the Salk inactivated polio vaccine (IPV). The pertussis vaccine
component may comprise a whole cell or acellular product.
[0046] Advantageously the hepatitis or combination vaccine
according to the invention is a paediatric or an adolescent
vaccine.
[0047] Preferred combination vaccines according to the invention
for adolescent use include one or more components selected from
antigens which are known in the art to provide protection against
one or more of the following diseases:
[0048] human papillomavirus (HPV), herpes simplex virus (HSV),
Epstein Barr virus (EBV), Varicella Zoster virus (VZV), human
cytomegalovirus (HCMV), Toxoplasma gondii.
[0049] The amount of each antigen in the vaccine dose is selected
as an amount that 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 0.01 to 1.0
.mu.g protein/dose for Hepatitis A, most preferably from between
0.06 to 0.220 .mu.g protein/dose. For antigens other than Hepatitis
A, for example HBsAg, HSV etc, the amount of protein per dose may
be higher, e.g. up to about 20 .mu.g per dose. An optimal amount of
each of one or more immunogens for a particular vaccine can be
ascertained by standard studies involving observation of antibody
titres and/or other responses in subjects. Following an initial
vaccination, subjects may receive a boost within about 4 weeks.
[0050] In a further aspect of the present invention there is
provided a method of manufacture of a vaccine effective in
preventing or treating hepatitis infection, wherein the method
comprises mixing the hepatitis antigen as defined herein with MPL
or a derivative thereof.
[0051] Using this method one or more additional components are
preferably admixed with the inactivated Hepatitis A vaccine to
provide a combination vaccine.
[0052] The following examples illustrate the invention and its
advantages.
EXAMPLES
Example 1
[0053] a) Purification Steps
[0054] The series of steps given in this Example may be performed
in different combinations in accordance with the method according
to the invention, but always involving a trypsin or other protease
digestion step.
[0055] Culture and Harvest
[0056] Hepatitis A virus HM175 is cultured on MRC5 cells (Andre et
al supra) and the virus is harvested after washing of the cell
layer to remove serum used in growth media. After freeze/thaw a
detergent (Tween 20) is added to extract the virus from the cell
debris. Cell debris is removed by filtration through a 0.22 .mu.m
membrane. Filtrate is further subjected to ultra-filtration. The
resulting concentrate can eventually be clarified by centrifugation
at 5-10,000.times.g for 1-2 hours.
[0057] Trypsination
[0058] The concentrate containing the HAV virus is treated with
purified trypsin extracted from pig pancreas. The trypsin used is
double crystallised and kept frozen before use. Before addition of
trypsin the concentrate is prewarmed at 37.degree. C. under
constant agitation. Trypsin is then added at a ratio of 440 IU per
ml of concentrate, and the mixture gently stirred for minimum 2hrs
at 37.degree. C. (maximum 2.5hrs).
[0059] Concentration
[0060] After trypsin treatment the product can be processed without
delay at ambient temperature on an ultrafiltration device in order
to reduce the volume. The membrane used is regenerated cellulose
with nominal cut-off of 30,000 Dalton, and up to a maximum of 8 ml
of trypsinated product per cm.sup.2 of membrane, is processed at a
transmembrane pressure between 0.2 and 0.6 bar to achieve a
concentration factor of between 8 and 12.
[0061] Permeation Chromatography
[0062] The aim of this step is to separate proteins from the intact
HAV virus. When a permeation chromatography step is conducted after
trypsin treatment, conditions have to be adapted to eliminate
residual trypsin as well. The separation gel used is Permeation
Sepharose 4BFF.
[0063] The virus is eluted at a smaller retention volume than the
smaller protein fragments which are eluted with larger retention
volume (closer to the total volume of the column).
[0064] Chromatography parameters are as follows:
[0065] Chromatographic medium: Sepharose 4B FF (from Pharmacia)
[0066] Injected volume: 1 to 5% of gel volume
[0067] Elution rate: 5-10 cm/h
[0068] Temperature: 10 to 16.degree. C.
[0069] Pool of fractions: target 100 ng prot/720 Elisa units
(.+-.10%)
[0070] Ion Exchange
[0071] The purpose of this purification step is to reduce the DNA
content (originating from MRC5 cells). This step is run according
to the batch principle.
[0072] The pool from the previous chromatographic step is adjusted
to 0.3M NaCl and then mixed with the ion exchange resin under mild
agitation for 1 hr (maximum 1.5 hr) at room temperature.
[0073] After DNA fixation the gel is eliminated by filtration. The
unfixed HAV virus suspension is then diluted to adjust the NaCl
concentration to 150 mM. Alternatively, the ion exchange
purification step can also be conducted by column
chromatography.
[0074] The final purified product is sterile filtered on 0.22 .mu.m
filter. Chromatographic parameters are as follows:
[0075] Load: 3% of gel compared to the volume of the pool
(vol/vol).
[0076] Temperature: ambient
[0077] Inactivation is carried out as described in Andre et al,
except that 250 .mu.g/ml of formol is used.
[0078] Formaldehyde Reduction
[0079] Within 48 hr after the end of the inactivation the product
is diafiltered and concentrated in order to reduce the formaldehyde
content and to be preadsorbed on an aluminium salt (preferably
aluminium hydroxide or aluminium phosphate).
[0080] Prior to use, the complete ultrafiltration device is
sanitised with 0.1N NaOH for at least 30 minutes. The device is
then thoroughly rinsed with diafiltration buffer and the membranes
are then coated with a buffer containing amino acids (Travasol).
Finally the device is rinsed with diafiltration buffer.
[0081] After diafiltration and concentration the final product is
sterile filtered on a 0.22 .mu.m filter.
[0082] b) Purification Schemes
[0083] The purification steps described above were combined in such
a way that a pure product was obtained in an economical way. Two
such purification schemes are presented in Scheme 1, both of which
yield a similar product. In one configuration of the steps as shown
in scheme 1, the steps were carried out in the order described in
Example 1a, and in the other configuration the trypinisation step
was carried out between the ultrafiltration and first permeation
chromatography steps. This meant that the second permeation
chromatography step could be eliminated.
Example 2
Characterisation
[0084] Samples of purified product were analysed by SDS PAGE 12.5%
acrylamide, 1% SDS in a stacking gel, migration for 15 h at 45-50
volts. The gel was stained with AgNO.sub.3 and the colour was
allowed to develop for 10 to 20 min and compared with traditional
HAV processes (Andre et al).
[0085] As can be seen from FIGS. 2 and 3, by subjecting the product
to protease treatment a majority of high molecular weight
contaminants are removed.
Example 3
HAV Vaccine Formulations
[0086] 3.1 HAV-alum 3D-MPL
[0087] The HAV particle of example 1 was first adsorbed on to
aluminium hydroxide (superfos) followed by the addition of free
3D-MPL. A 0.5 ml dose 720 ELU of Hepatitis A virus particle/0.025
mg A1.sup.3+ ion and 50 .mu.g of 3D-MPL.
[0088] 3.2 HAV+Hbs Ag Formulations
[0089] The following formulations were made:
[0090] 1. Hep B S Ag 20 .mu.g/AlP0.sub.4+HA 720/Al(OH).sub.3
[0091] 2. Hep B S Ag 20 .mu./AlPO.sub.4/3D-MPL 50 .mu.g+HA
1440/Al(OH).sub.3
[0092] 3. Hep B S Ag 20 .mu.g/AlPO.sub.4+3D-MPL 50
.mu.g/Al(OH).sub.3+HA 720/Al(OH).sub.3
[0093] 4. Hep B S Ag 20 .mu.g/AlPO.sub.4+3D-MPL
50.mu.g/AlPO.sub.4+HA 720 Al(OH).sub.3
[0094] 5. Hep B S Ag 20/.mu.g/AlPO.sub.4/3D-MPL 50 .mu.g+HA
720/Al(OH).sub.3
[0095] In group 1 the individual antigens were adsorbed on to the
aluminium salt 0.025 mg Al.sup.3+ ion (Al(OH).sub.3 Superfos) for
HA, 0.475 mg Al3+ion (AlPO.sub.4 Superfos type). In group 2 and 5,
50 .mu.g/dose of free 3D-MPL was added to adsorbed Hepatitis S
antigen to which the adsorbed hepatitis A component was added. In
group 3 and 4, 3D-MPL was separately adsorbed on to the aluminium
salt, and then the three adsorbed components were mixed
together.
Example 4
Immunogenicity Experiments
[0096] Balb/c Mice
[0097] Groups of 10 mice were immunised intramuscularly three times
at 2 weeks interval with HAV/HBs formualtions (1/10 HD). Antibody
response to Hbs were monitored by ELISA at 14 days post II and 14
days post III. The isotypic profile of the anti-HBs response was
analysed at 14 days post II. Antibody response to HAV was monitored
14 days post III.
[0098] NMRI Mice
[0099] Groups of 10 mice were immunised intraperitoneally once with
HAV/HBs formulations (1/2 HD). Antibody response to Hbs and HAV
were monitored by ELISA at 28 days post injection.
1 Formulations Group Vaccine lot Formulation 1 HAB112B6 HBs 20
.mu.g/AlPO4 + HAV 720/Al(OH)3 2 DHAB713 HBs 20 .mu.g/AlPO4/MPL 50 +
HAV 1440/Al(OH)3 3 DHAB717 HBs 20 .mu.g/AlPO4 + MPL 50/Al(OH)3 +
HAV 720/Al(OH)3 4 DHAB718 HBs 20 .mu.g/AlPO4 + MPL 50/AlPO4 + HAV
720/Al(OH)3 5 DHAB716 HBs 20 .mu.g/AlPO4/MPL 50 + HAV
720/Al(OH)3
[0100] HAV Mouse Serology
[0101] Quantitation of anti-Hepatitis A Virus antigen (HAV)
antibody was performed using Enzygnost kit from Behring (ref:
OQEC11). This assay is an ELISA based on the competitive test
principle, run in one step and initially developed for human
serology.
[0102] Two-fold dilution of mice sera (4 dilutions starting at
1/10) human anti-HAV reference (8 dilutions starting at 80 mlU/ml)
and controls were performed in anti-HAV negative human sera.
Mixtures of test/control samples (25 .mu.l), HAV antigen solution
(50 .mu.l) and anti-HAV mouse monoclonal conjugated with peroxidase
(50 .mu.l of 1/41 dilution performed in conjugate buffer) were
incubated on HAV pre-coated microplates for 2 hrs at 37.degree. C.
The plates were then washed and incubated for 30 min with a
solution of TMB (100 .mu.l). The reaction was stopped with
H.sub.2SO.sub.4 0.5N and read at 450/620 nm.
[0103] Anti-HAV antibody titers were calculated from the reference
by SoftmaxPro (using a four parameters equation) and expressed in
mlU/ml.
[0104] Results
[0105] The results are shown in FIG. 1.
[0106] In FIG. 1a results demonstrate that formulations containing
MPL induce significantly higher antibody responses to the hepatitis
A component than the aluminum salt group alone. Similarly the
results shown in FIG. 1b demonstrate that MPL containing
formulations induce higher antibody titres to HbsAg.
Example 5
Clinical Studies
[0107] HAV/HBs (HAB) formulations were administered to healthy
subjects.
[0108] Serum titres of anti-HAV antibodies were measured by ELISA
(Enzymun test from Boehringer Mannheim) and anti-HBs antibodies by
radioimmunoassay (RIA) using test kit AUSAB-Abbott. The assay
cut-off for anti-HAV antibodies was 33 mIU/ml and the assay cut-off
for anti-HBs antibodies was 1 mIU/ml.
[0109] Subjects with anti-HAV antibody titres of 33 mIU/ml were
considered to be seropositive for anti-HAV antibodies. Subjects
with anti-HBs antibody titres 1 mIU/ml were considered to be
seropositive for anti-HBs antibodies. Seroprotection rate for
anti-HBs was defined as the ratio of subjects with anti-HBs titres
10 mIU/ml.
2 Vaccine lots Commercial Combined HAB/MPL TwinrixTM (adult)
candidate Group 1 Group 3 Lot n.sup.o HAB 116C4/M1 DHAB 713A2
Inactivated hepA at least 720 EL.U at least 1440 (new process)
(strain HM175-RIT EL.U 4380) Recombinant HBsAg 20 .mu.g 20 .mu.g
MPL -- 50 .mu.g Al salt 0.45 mg 0.5 mg Volume/dose 1.0 ml 0.5
ml
[0110] Results:
[0111] In this phase I clinical trial where HAB/MPL was
administered to healthy subjects, there is a marked effect of MPL
acting as immunostimulant on the immune response.
[0112] MPL has a clear effect on anti-HAV kinetics. It induces a
faster and stronger immune response with a marked anamnestic
response observed at Mth 6.5 and Mth 7 (i.e. 14 and 30 days
respectively, after the last vaccine dose).
[0113] Within the limitations of the study, it can be concluded
that the candidate HAB/MPL vaccine exhibited a very good safety and
reactogenicity profile. It was very immunogenic after a full
vaccination course of 2 doses in the study cohort of healthy adults
aged 18-40 years. There was a strong priming and faster immune
response to both hep A & B antigens.
3 1. Anti-HAV Kinetics D7 D9 D11 D13 D15 Twinrix 720/20 SC % 5 10
30 65 74 0, 1, 6 M GMT 35 41 71 84 176 N = 20 1440 HAV (new
process) SC % 10 60 95 100 100 20 .mu.g HBsAg GMT 37 43 125 316 569
50 .mu.g MPL (mixed) 0.5 mg Alum - 0, 6M N = 20 2. Anti-HAV Titers
D15 M1 M7 Twinrix 720/20 GMT 176 349 7107 0, 1, 6 M N = 50 1440 HAV
(new process) GMT 569 888 13386 20 .mu.g HBsAg 50 .mu.g MPL (mixed)
0.5 mg Alum - 0, 6M N = 50
[0114]
4 Scheme 1 PURIFICATION PROCEDURES FOR HAV 175 strain HARVEST
THAWING ADDITION TWEEN 20 CLARIFICATION (0.22 .mu.m)
ULTRAFILTRATION ULTRAFILTRATION (cut-off 300.000) (cut-off 300.000)
TRYPSINISATION CHROMATOGRAPHY (440 UI/ML - 2 h - 37.degree. C.
(permeation Sepharose 4BFF) ION EXCHANGE POOL (DEAE Sepharose 6B)
TRYPSINATION CHROMATOGRAPHY (440 UI/ML - 2 h - 37.degree. C.) (gel
permeation Sepharose 4BFF) RECONCENTRATION FILTRATION (0.22 .mu.m)
(CUT-OFF 30.000) CHROMATOGRAPHY (permeation Sepharose 4BFF) POOL
ION EXCHANGE (DEAE Sepharose 6B) FILTRATION (0.22 .mu.m)
INACTIVATION FORMALDEHYDE REDUCTION and CONCENTRATION (cut-off
30.000) STERILE FILTRATION PREADSORPTION STORAGE FORMULATION
FILLING
* * * * *