U.S. patent application number 11/874647 was filed with the patent office on 2008-07-31 for novel vaccine composition.
This patent application is currently assigned to Saechsisches Serumwerk Dresden. Invention is credited to Uwe Eichhorn.
Application Number | 20080181914 11/874647 |
Document ID | / |
Family ID | 30445235 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080181914 |
Kind Code |
A1 |
Eichhorn; Uwe |
July 31, 2008 |
NOVEL VACCINE COMPOSITION
Abstract
An inactivated influenza virus preparation is described which
comprises a haemagglutinin antigen stabilised in the absence of
thiomersal, or at low levels of thiomersal, wherein the
haemagglutinin is detectable by a SRD assay. The influenza virus
preparation may comprise a micelle modifying excipient, for example
.alpha.-tocopherol or a derivative thereof in a sufficient amount
to stabilise the haemagglutinin.
Inventors: |
Eichhorn; Uwe; (Dresden,
DE) |
Correspondence
Address: |
GLAXOSMITHKLINE;Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
Saechsisches Serumwerk
Dresden
|
Family ID: |
30445235 |
Appl. No.: |
11/874647 |
Filed: |
October 18, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10480952 |
Jun 22, 2004 |
7316813 |
|
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PCT/EP02/05883 |
May 29, 2002 |
|
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11874647 |
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Current U.S.
Class: |
424/209.1 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 31/12 20180101; A61K 2039/70 20130101; C12N 2760/16234
20130101; A61K 2039/55 20130101; A61P 31/16 20180101; C12N 7/00
20130101; C12N 2760/16161 20130101; C12N 2760/16134 20130101; A61K
2039/5252 20130101; A61K 39/12 20130101; A61K 2039/55577 20130101;
A61K 2039/55572 20130101; A61K 39/145 20130101 |
Class at
Publication: |
424/209.1 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61P 31/16 20060101 A61P031/16 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2001 |
GB |
0113083.0 |
Feb 21, 2002 |
GB |
0204116.8 |
Claims
1-35. (canceled)
36. An influenza vaccine comprising an inactivated influenza virus
preparation and an adjuvant, wherein said inactivated influenza
virus preparation comprises hemagglutinin antigen (HA), 0 .mu.g/ml
to 5 .mu.g/ml of thiomersal and at least one stabilising excipient,
and wherein said adjuvant is selected from the group of: an oil in
water emulsion, a non-toxic derivative of lipid A, a saponin or
derivative thereof, and a combination of two or more of said
adjuvants.
37. The influenza vaccine according to claim 36, wherein said
non-toxic derivative of lipid A is 3D-MPL.
38. The influenza vaccine according to claim 36, wherein 3D-MPL is
in the form of an emulsion having a small particle size less than
0.2 .mu.m in diameter.
39. The influenza vaccine according to claim 36, wherein said said
excipient comprises .alpha.-tocopherol or a derivative thereof.
40. The influenza vaccine according to claim 36, wherein said
excipient comprises .alpha.-tocopherol succinate.
41. The influenza vaccine according to claim 36, wherein said
excipient comprises a positively or negatively or zwitterionic
charged amphiphilic molecule.
42. The influenza vaccine according to claim 36, wherein said
excipient comprises a non-ionic amphiphilic molecule from the group
of: octyl- or nonylphenoxy polyoxyethanols, polyoxyethylene
sorbitan esters and polyoxyethylene ethers or esters of general
formula (I): HO(CH2CH2O).sub.n-A-R (I) wherein n is 1-50, A is a
bond or --C(O)--, R is C.sub.1-50 alkyl or phenyl C.sub.1-50 alkyl;
and combinations of two or more of these.
43. The influenza vaccine according to claim 42, wherein said
non-ionic amphiphilic molecule is selected from the group of:
Triton X-45, t-octylphenoxy polyethoxyethanol (Triton X-100.TM.),
Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton
X-305, Triton N-57, Triton N-101, Triton N-128, Breij 35,
polyoxyethylene-9-lauryl ether (laureth 9),
polyoxyethylene-9-stearyl ether (steareth 9), polyoxyethylene
sorbitan ester, and polyoxyethylene sorbitan monooleate (Tween
80.TM.).
44. The influenza vaccine according to claim 42, wherein said
non-ionic amphiphilic molecule is selected from the group: Triton
X-100.TM., Tween 80.TM., and a combination of both.
45. The influenza vaccine according to claim 39, wherein the
a-tocopherol or derivative thereof is present at a concentration
between 1 .mu.g/ml and 10 mg/ml.
46. The influenza vaccine according to claim 36, wherein the
.alpha.-tocopherol is present at a concentration between 1 .mu.g/ml
and 10 mg/ml.
47. The influenza vaccine according to claim 36, wherein said
influenza virus antigen preparation is selected from the group
consisting of: split virus antigen preparations, subunit antigens,
chemically or otherwise inactivated whole virus and recombinantly
produced haemagglutinin antigen.
48. The influenza vaccine according to claim 36, wherein the
concentration of haemagglutinin antigen for each strain of
influenza is 1-1000 .mu.g per ml, as measured by a Single radial
Immunodiffusion (SRD) assay.
49. The influenza vaccine according to claim 36, wherein said
influenza virus antigen preparation is derived from the embryonated
egg method or derived from cell culture.
50. The influenza vaccine according to claim 36, wherein said
influenza virus antigen preparation comprises a haemagglutinin
antigen that is produced recombinantly.
51. An inactivated influenza virus preparation, comprising
hemagglutinin antigen (HA), 0 .mu.g/ml to 5 pg/ml of thiomersal,
and at least one of .alpha.-tocopherol or a derivative thereof in
an amount sufficient to stabilize said HA.
52. The inactivated influenza virus preparation of claim 50,
wherein said influenza virus is selected from the group of: split
virus antigen preparations, subunit antigens, chemically or
otherwise inactivated whole virus, and recombinantly produced
haemagglutinin antigen.
53. The inactivated influenza virus preparation of claim 50,
wherein said preparation comprises .alpha.-tocopherol or
.alpha.-tocopherol succinate.
54. The inactivated influenza virus preparation of claim 50,
wherein the at least one of .alpha.-tocopherol or a derivative
thereof is present in an amount such that the HA of said
preparation remains stable for at least 6 months after said
preparation is produced as determined by the presence of an amount
of HA detectable by Single radial Immunodiffusion (SRD) assay.
55. The inactivated influenza virus preparation of claim 50,
wherein the at least one of a-tocopherol or a derivative thereof
further comprises at least one of a non-ionic amphiphilic molecule
from the group of: octyl- or nonylphenoxy polyoxyethanols,
polyoxyethylene sorbitan esters and polyoxyethylene ethers or
esters of general formula (I): HO(CH2CH2O).sub.n-A-R (I) wherein n
is 1-50, A is a bond or --C(O)--, R is C.sub.1-50 alkyl or phenyl
C.sub.1-50 alkyl; and combinations of two or more of these.
56. The inactivated influenza virus preparation of claim 55,
wherein said non-ionic amphiphilic molecule is selected from the
group of: Triton X-45, t-octylphenoxy polyethoxyethanol (Triton
X-100.TM.), Triton X-102, Triton X-114, Triton X-165, Triton X-205,
Triton X-305, Triton N-57, Triton N-101, Triton N-128, Breij 35,
polyoxyethylene-9-lauryl ether (laureth 9),
polyoxyethylene-9-stearyl ether (steareth 9), polyoxyethylene
sorbitan ester, and polyoxyethylene sorbitan monooleate (Tween
80.TM.).
57. The inactivated influenza virus preparation of claim 55,
wherein said non-ionic amphiphilic molecule is selected from the
group: Triton X-100.TM., Tween 80.TM., and a combination of
both.
58. The inactivated influenza virus preparation according to claim
50, wherein the .alpha.-tocopherol or derivative thereof is present
at a concentration between 1 pg/ml and 10 mg/ml.
59. A vaccine comprising the inactivated influenza virus
preparation of claim 50.
60. An influenza vaccine according to claim 58, wherein the vaccine
additionally comprises an adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of co-pending
application Ser. No. 10/480,952, filed 22 Jun. 2004, which is a
National Stage Entry of International Application No.
PCT/EP02/05883, filed 29 May 2002, the contents of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to novel influenza virus antigen
preparations, methods for preparing them and their use in
prophylaxis or therapy. In particular the invention relates to
inactivated influenza vaccines which are disrupted rather than
whole virus vaccines and which are stable in the absence of
organomercurial preservatives. Moreover, the vaccines contain
haemagglutinin which is stable according to standard tests. The
vaccines can be administered by any route suitable for such
vaccines, such as intramuscularly, subcutaneously, intradermally or
mucosally e.g. intranasally.
BACKGROUND OF THE INVENTION
[0003] Influenza virus is one of the most ubiquitous viruses
present in the world, affecting both humans and livestock. The
economic impact of influenza is significant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 shows the results of the HI (haemagglutinin
inhibition) assay done in Example 10.
[0005] FIG. 2 shows the results of the HI (haemagglutinin
inhibition) assay done in Example 12.
SUMMARY OF THE INVENTION
[0006] The influenza virus is an RNA enveloped virus with a
particle size of about 125 nm in diameter. It consists basically of
an internal nucleocapsid or core of ribonucleic acid (RNA)
associated with nucleoprotein, surrounded by a viral envelope with
a lipid bilayer structure and external glycoproteins. The inner
layer of the viral envelope is composed predominantly of matrix
proteins and the outer layer mostly of host-derived lipid material.
The surface glycoproteins neuraminidase (NA) and haemagglutinin
(HA) appear as spikes, 10 to 12 nm long, at the surface of the
particles. It is these surface proteins, particularly the
haemagglutinin, that determine the antigenic specificity of the
influenza subtypes.
DETAILED DESCRIPTION OF THE INVENTION
[0007] Currently available influenza vaccines are either
inactivated or live attenuated influenza vaccine. Inactivated flu
vaccines are composed of three possible forms of antigen
preparation: inactivated whole virus, sub-virions where purified
virus particles are disrupted with detergents or other reagents to
solubilise the lipid envelope (so-called "split" vaccine) or
purified HA and NA (subunit vaccine). These inactivated vaccines
are given intramuscularly (i.m.) or intranasally (i.n.). There is
no commercially available live attenuated vaccine.
[0008] Influenza vaccines, of all kinds, are usually trivalent
vaccines. They generally contain antigens derived from two
influenza A virus strains and one influenza B strain. A standard
0.5 ml injectable dose in most cases contains 15 .mu.g of
haemagglutinin antigen component from each strain, as measured by
single radial immunodiffusion (SRD) (J. M. Wood et al.: An improved
single radial immunodiffusion technique for the assay of influenza
haemagglutinin antigen: adaptation for potency determination of
inactivated whole virus and subunit vaccines. J. Biol. Stand. 5
(1977) 237-247; J. M. Wood et al., International collaborative
study of single radial diffusion and immunoelectrophoresis
techniques for the assay of haemagglutinin antigen of influenza
virus. J. Biol. Stand. 9 (1981) 317-330).
[0009] The influenza virus strains to be incorporated into
influenza vaccine each season are determined by the World Health
Organisation in collaboration with national health authorities and
vaccine manufacturers.
[0010] Typical influenza epidemics cause increases in incidence of
pneumonia and lower respiratory disease as witnessed by increased
rates of hospitalisation or mortality. The elderly or those with
underlying chronic diseases are most likely to experience such
complications, but young infants also may suffer severe disease.
These groups in particular therefore need to be protected.
[0011] Current efforts to control the morbidity and mortality
associated with yearly epidemics of influenza are based on the use
of intramuscularly administered inactivated influenza vaccines. The
efficacy of such vaccines in preventing respiratory disease and
influenza complications ranges from 75% in healthy adults to less
than 50% in the elderly.
[0012] Standards are applied internationally to measure the
efficacy of influenza vaccines. The European Union official
criteria for an effective vaccine against influenza are set out in
the table below. Theoretically, to meet the European Union
requirements, an influenza vaccine has to meet only one of the
criteria in the table, for all strains of influenza included in the
vaccine. However in practice, at least two or all three of the
criteria will need to be met for all strains, particularly for a
new vaccine such as a new vaccine for delivery via a different
route. Under some circumstances two criteria may be sufficient. For
example, it may be acceptable for two of the three criteria to be
met by all strains while the third criterion is met by some but not
all strains (e.g. two out of three strains). The requirements are
different for adult populations (18-60 years) and elderly
populations (>60 years).
TABLE-US-00001 18-60 years >60 years Seroconversion rate*
>40% >30% Conversion factor** >2.5 >2.0 Protection
rate*** >70% >60% *Seroconversion rate is defined as the
percentage of vaccinees who have at least a 4-fold increase in
serum haemagglutinin inhibition (HI) titres after vaccination, for
each vaccine strain. **Conversion factor is defined as the fold
increase in serum HI geometric mean titres (GMTs) after
vaccination, for each vaccine strain. ***Protection rate is defined
as the percentage of vaccinees with a serum HI titre equal to or
greater than 1:40 after vaccination (for each vaccine strain) and
is normally accepted as indicating protection.
[0013] For a novel flu vaccine to be commercially useful it will
not only need to meet those standards, but also in practice it will
need to be at least as efficacious as the currently available
injectable vaccines. It will also need to be commercially viable in
terms of the amount of antigen and the number of administrations
required.
[0014] The current commercially available influenza vaccines are
either split or subunit injectable vaccines. These vaccines are
prepared by disrupting the virus particle, generally with an
organic solvent or a detergent, and separating or purifying the
viral proteins to varying extents. Split vaccines are prepared by
fragmentation of whole influenza virus, either infectious or
inactivated, with solubilizing concentrations of organic solvents
or detergents and subsequent removal of the solubilizing agent and
some or most of the viral lipid material. Split vaccines generally
contain contaminating matrix protein and nucleoprotein and
sometimes lipid, as well as the membrane envelope proteins. Split
vaccines will usually contain most or all of the virus structural
proteins although not necessarily in the same proportions as they
occur in the whole virus. Subunit vaccines on the other hand
consist essentially of highly purified viral surface proteins,
haemagglutinin and neuraminidase, which are the surface proteins
responsible for eliciting the desired virus neutralising antibodies
upon vaccination.
[0015] Many vaccines which are currently available require a
preservative to prevent deterioration. A frequently used
preservative is thimerosal which is a mercury-containing compound.
Some public concerns have been expressed about the effects of
mercury containing compounds. There is no surveillance system in
place to detect the effects of low to moderate doses of
organomercurials on the developing nervous system, and special
studies of children who have received high doses of
organomercurials will take several years to complete. Certain
commentators have stressed that the potential hazards of
thimerosal-containing vaccines should not be overstated (Offit; P.
A. JAMA Vol. 283; No:16). Nevertheless, it would be advantageous to
find alternative methods for the preparation of vaccines to replace
the use of thiomerosal in the manufacturing process. There is thus
a need to develop vaccines which are thimerosal-free, in particular
vaccines like influenza vaccines which are recommended, at least
for certain population groups, on an annual basis.
[0016] It has been standard practice to date to employ a
preservative for commercial inactivated influenza vaccines, during
the production/purification process and/or in the final vaccine.
The preservative is required to prevent microorganisms from growing
through the various stages of purification. For egg-derived
influenza vaccines, thiomersal is typically added to the raw
allantoic fluid and may also be added a second time during the
processing of the virus. Thus there will be residual thiomersal
present at the end of the process, and this may additionally be
adjusted to a desirable preservative concentration in the final
vaccine, for example to a concentration of around 100 .mu.g/ml.
[0017] A side-effect of the use of thiomersal as a preservative in
flu vaccines is a stabilisation effect. The thiomersal in
commercial flu vaccines acts to stabilise the HA component of the
vaccine, in particular but not exclusively HA of B strain
influenza. Certain A strain haemagglutinins e.g. H3 may also
require stabilisation. Therefore, although it may be desirable to
consider removing thiomersal from influenza vaccines, or at least
reducing the concentration of the thiomersal in the final vaccine,
there is a problem to overcome in that, without thiomersal, the HA
will not be sufficiently stable.
[0018] It has been discovered in the present invention that it is
possible to stabilise HA in inactivated influenza preparations
using alternative reagents that do not contain organomercurials.
The HA remains stabilised such that it is detectable over time by
quantitative standard methods, in particular SRD, to a greater
extent than a non-stabilised antigen preparation produced by the
same method but without stabilising excipient. The SRD method is
performed as described hereinabove. Importantly, the HA remains
stabilised for up to 12 months which is the standard required of a
final flu vaccine. In a first aspect the present invention provides
an inactivated influenza virus preparation comprising a
haemagglutinin antigen stabilised in the absence of thiomersal, or
at low levels of thiomersal, wherein the haemagglutinin is
detectable by a SRD assay.
[0019] Low levels of thiomersal are those levels at which the
stability of HA derived from influenza B is reduced, such that a
stabilising excipient is required for stabilised HA. Low levels of
thiomersal are generally 5 .mu.g/ml or less.
[0020] Generally, stabilised HA refers to HA which is detectable
over time by quantitative standard methods, in particular SRD, to a
greater extent than a non-stabilised antigen preparation produced
by the same method but without any stabilising excipient.
Stabilisation of HA preferably maintains the activity of HA
substantially constant over a one year period. Preferably,
stabilisation allows the vaccine comprising HA to provide
acceptable protection after a 6 month storage period, more
preferably a one year period.
[0021] Suitably, stabilisation is carried out by a stabilising
excipient, preferably a micelle modifying excipient. A micelle
modifying excipient is generally an excipient that may be
incorporated into a micelle formed by detergents used in, or
suitable for, solubilising the membrane protein HA, such as the
detergents Tween 80, Triton X100 and deoxycholate, individually or
in combination.
[0022] Without wishing to be constrained by theory, it is believed
that the excipients work to stabilise HA by interaction with the
lipids, detergents and/or proteins in the final preparation. Mixed
micelles of excipient with protein and lipid may be formed, such as
micelles of Tween and deoxycholate with residual lipids and/or
Triton X-100. It is thought that surface proteins are kept
solubilised by those complex micelles. Preferably, protein
aggregation is limited by charge repulsion of micelles containing
suitable excipients, such as micelles containing negatively charged
detergents.
[0023] Suitable micelle modifying excipients include: positively,
negatively or zwitterionic charged amphiphilic molecules such as
alkyl sulfates, or alkyl-aryl-sulfates; non-ionic amphiphilic
molecules such as alkyl polyglycosides or derivatives thereof, such
as Plantacare.RTM. (available from Henkel KGaA), or alkyl alcohol
poly alkylene ethers or derivatives thereof such as Laureth-9.
[0024] Preferred excipients are .alpha.-tocopherol, or derivatives
of .alpha.-tocopherol such as .alpha.-tocopherol succinate. Other
preferred tocopherol derivatives for use in the invention include
D-.alpha. tocopherol, D-.delta. tocopherol, D-.gamma. tocopherol
and DL-.alpha.-tocopherol. Preferred derivatives of tocopherols
that may be used include acetates, succinates, phosphoric acid
esters, formiates, propionates, butyrates, sulfates and gluconates.
Alpha-tocopherol succinate is particularly preferred. The
.alpha.-tocopherol or derivative is present in an amount sufficient
to stabilise the haemagglutinin.
[0025] Other suitable excipients may be identified by methods
standard in the art, and tested for example using the SRD method
for stability analysis as described herein.
[0026] In a preferred aspect the invention provides an influenza
virus antigen preparation comprising at least one stable influenza
B strain haemagglutinin antigen.
[0027] The invention provides in a further aspect a method for
preparing a stable haemagglutinin antigen which method comprises
purifying the antigen in the presence of a stabilising micelle
modifying excipient, preferably .alpha.-tocopherol or a derivative
thereof such as .alpha.-tocopherol succinate.
[0028] Further provided by the invention are vaccines comprising
the antigen preparations described herein and their use in a method
for prophylaxis of influenza infection or disease in a subject
which method comprises administering to the subject a vaccine
according to the invention.
[0029] The vaccine may be administered by any suitable delivery
route, such as intradermal, mucosal e.g. intranasal, oral,
intramuscular or subcutaneous. Other delivery routes are well known
in the art.
[0030] Intradermal delivery is preferred. Any suitable device may
be used for intradermal delivery, for example short needle devices
such as those described in U.S. Pat. No. 4,886,499, U.S. Pat. No.
5,190,521, U.S. Pat. No. 5,328,483, U.S. Pat. No. 5,527,288, U.S.
Pat. No. 4,270,537, U.S. Pat. No. 5,015,235, U.S. Pat. No.
5,141,496, U.S. Pat. No. 5,417,662. Intradermal vaccines may also
be administered by devices which limit the effective penetration
length of a needle into the skin, such as those described in
WO99/34850 and EP1092444, incorporated herein by reference, and
functional equivalents thereof. Also suitable are jet injection
devices which deliver liquid vaccines to the dermis via a liquid
jet injector or via a needle which pierces the stratum corneum and
produces a jet which reaches the dermis. Jet injection devices are
described for example in U.S. Pat. No. 5,480,381, U.S. Pat. No.
5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No. 5,993,412, U.S.
Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S. Pat. No.
5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397, U.S.
Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No.
5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S.
Pat. No. 5,520, 639, U.S. Pat. No. 4,596,556, U.S. Pat. No.
4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO
97/37705 and WO 97/13537. Also suitable are ballistic
powder/particle delivery devices which use compressed gas to
accelerate vaccine in powder form through the outer layers of the
skin to the dermis. Additionally, conventional syringes may be used
in the classical mantoux method of intradermal administration.
However, the use of conventional syringes requires highly skilled
operators and thus devices which are capable of accurate delivery
without a highly skilled user are preferred.
[0031] The invention thus provides a method for the prophylaxis of
influenza infection or disease in a subject which method comprises
administering to the subject intradermally an influenza vaccine
according to the invention.
[0032] The invention also extends to intradermal devices in
combination with a vaccine according to the present invention, in
particular with devices disclosed in WO99/34850 or EP1092444, for
example.
[0033] Also provided is the use of a micelle modifying excipient,
preferably .alpha.-tocopherol or a derivative thereof as a
haemagglutinin stablilser in the manufacture of an influenza
vaccine.
[0034] The invention applies particularly but not exclusively to
the stabilisation of B strain influenza haemagglutinin.
[0035] Preferably the stabilised HA of the present invention is
stable for 6 months, more preferably 12 months.
[0036] Preferably the .alpha.-tocopherol is in the form of an
ester, more preferably the succinate or acetate and most preferably
the succinate.
[0037] Preferred concentrations for the .alpha.-tocopherol or
derivative are between 1 .mu.g/ml-10 mg/ml, more preferably between
10 .mu.g/ml-500 .mu.g/ml.
[0038] The vaccine according to the invention generally contains
both A and B strain virus antigens, typically in a trivalent
composition of two A strains and one B strain. However, divalent
and monovalent vaccines are not excluded. Monovalent vaccines may
be advantageous in a pandemic situation, for example, where it is
important to get as much vaccine produced and administered as
quickly as possible.
[0039] The non-live flu antigen preparation for use in the
invention may be selected from the group consisting of split virus
antigen preparations, subunit antigens (either recombinantly
expressed or prepared from whole virus), inactivated whole virus
which may be chemically inactivated with e.g. formaldehyde,
.beta.-propiolactone or otherwise inactivated e.g. U.V. or heat
inactivated. Preferably the antigen preparation is either a split
virus preparation, or a subunit antigen prepared from whole virus,
particularly by a splitting process followed by purification of the
surface antigen. Most preferred are split virus preparations.
[0040] Preferably the concentration of haemagglutinin antigen for
each strain of the influenza virus preparation is 1-1000 .mu.g per
ml, more preferably 3-300 .mu.g per ml and most preferably about 30
.mu.g per ml, as measured by a SRD assay.
[0041] The vaccine according to the invention may further comprise
an adjuvant or immunostimulant such as but not limited to
detoxified lipid A from any source and non-toxic derivatives of
lipid A, saponins and other reagents capable of stimulating a TH1
type response.
[0042] It has long been known that enterobacterial
lipopolysaccharide (LPS) is a potent stimulator of the immune
system, although its use in adjuvants has been curtailed by its
toxic effects. A non-toxic derivative of LPS, monophosphoryl lipid
A (MPL), produced by removal of the core carbohydrate group and the
phosphate from the reducing-end glucosamine, has been described by
Ribi et al (1986, Immunology and Immunopharmacology of bacterial
endotoxins, Plenum Publ. Corp., NY, p 407-419) and has the
following structure:
##STR00001##
[0043] A further detoxified version of MPL results from the removal
of the acyl chain from the 3-position of the disaccharide backbone,
and is called 3-O-Deacylated monophosphoryl lipid A (3D-MPL). It
can be purified and prepared by the methods taught in GB 2122204B,
which reference also discloses the preparation of diphosphoryl
lipid A, and 3-O-deacylated variants thereof.
[0044] A preferred form of 3D-MPL is in the form of an emulsion
having a small particle size less than 0.2 .mu.m in diameter, and
its method of manufacture is disclosed in WO 94/21292. Aqueous
formulations comprising monophosphoryl lipid A and a surfactant
have been described in WO9843670A2.
[0045] The bacterial lipopolysaccharide derived adjuvants to be
formulated in the compositions of the present invention may be
purified and processed from bacterial sources, or alternatively
they may be synthetic. For example, purified monophosphoryl lipid A
is described in Ribi et al 1986 (supra), and 3-O-Deacylated
monophosphoryl or diphosphoryl lipid A derived from Salmonella sp.
is described in GB 2220211 and U.S. Pat. No. 4,912,094. Other
purified and synthetic lipopolysaccharides have been described
(Hilgers et al., 1986, Int. Arch. Allergy. Immunol., 79(4):392-6;
Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549 074
B1). A particularly preferred bacterial lipopolysaccharide adjuvant
is 3D-MPL.
[0046] Accordingly, the LPS derivatives that may be used in the
present invention are those immunostimulants that are similar in
structure to that of LPS or MPL or 3D-MPL. In another aspect of the
present invention the LPS derivatives may be an acylated
monosaccharide, which is a sub-portion to the above structure of
MPL.
[0047] Saponins are taught in: Lacaille-Dubois, M and Wagner H.
(1996. A review of the biological and pharmacological activities of
saponins. Phytomedicine vol 2 pp 363-386). Saponins are steroid or
triterpene glycosides widely distributed in the plant and marine
animal kingdoms. Saponins are noted for forming colloidal solutions
in water which foam on shaking, and for precipitating cholesterol.
When saponins are near cell membranes they create pore-like
structures in the membrane which cause the membrane to burst.
Haemolysis of erythrocytes is an example of this phenomenon, which
is a property of certain, but not all, saponins.
[0048] Saponins are known as adjuvants in vaccines for systemic
administration. The adjuvant and haemolytic activity of individual
saponins has been extensively studied in the art (Lacaille-Dubois
and Wagner, supra). For example, Quil A (derived from the bark of
the South American tree Quillaja Saponaria Molina), and fractions
thereof, are described in U.S. Pat. No. 5,057,540 and "Saponins as
vaccine adjuvants", Kensil, C. R., Crit Rev Ther Drug Carrier Syst,
1996, 12 (1-2):1-55; and EP 0 362 279 B1. Particulate structures,
termed Immune Stimulating Complexes (ISCOMS), comprising fractions
of Quil A are haemolytic and have been used in the manufacture of
vaccines (Morein, B., EP 0 109 942 B1; WO 96/11711; WO 96/33739).
The haemolytic saponins QS21 and QS17 (HPLC purified fractions of
Quil A) have been described as potent systemic adjuvants, and the
method of their production is disclosed in U.S. Pat. No. 5,057,540
and EP 0 362 279 B1. Other saponins which have been used in
systemic vaccination studies include those derived from other plant
species such as Gypsophila and Saponaria (Bomford et al., Vaccine,
10(9):572-577, 1992).
[0049] An enhanced system involves the combination of a non-toxic
lipid A derivative 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.
[0050] A particularly potent adjuvant formulation involving QS21
and 3D-MPL in an oil in water emulsion is described in WO 95/17210
and is a preferred formulation.
[0051] Accordingly in one embodiment of the present invention there
is provided a vaccine comprising an influenza antigen preparation
of the present invention adjuvanted with detoxified lipid A or a
non-toxic derivative of lipid A, more preferably adjuvanted with a
monophosphoryl lipid A or derivative thereof.
[0052] Preferably the vaccine additionally comprises a saponin,
more preferably QS21.
[0053] Preferably the formulation additionally comprises an oil in
water emulsion. The present invention also provides a method for
producing a vaccine formulation comprising mixing an antigen
preparation of the present invention together with a
pharmaceutically acceptable excipient, such as 3D-MPL.
[0054] The vaccines according to the invention may further comprise
at least one surfactant which may be in particular a non-ionic
surfactant. Suitable non-ionic surfactants are selected from the
group consisting of the octyl- or nonylphenoxy polyoxyethanols (for
example the commercially available Triton.TM. series),
polyoxyethylene sorbitan esters (Tween.TM. series) and
polyoxyethylene ethers or esters of general formula (I):
HO(CH.sub.2CH.sub.2O).sub.n-A-R (I)
wherein n is 1-50, A is a bond or --C(O)--, R is C.sub.1-50 alkyl
or phenyl C.sub.1-50 alkyl; and combinations of two or more of
these.
[0055] Preferred surfactants falling within formula (I) are
molecules in which n is 4-24, more preferably 6-12, and most
preferably 9; the R component is C.sub.1-50, preferably
C.sub.4-C.sub.20 alkyl and most preferably C.sub.12 alkyl.
[0056] Octylphenoxy polyoxyethanols and polyoxyethylene sorbitan
esters are described in "Surfactant systems" Eds: Attwood and
Florence (1983, Chapman and Hall). Octylphenoxy polyoxyethanols
(the octoxynols), including t-octylphenoxypolyethoxyethanol (Triton
X-100.TM.) are also described in Merck Index Entry 6858 (Page 1162,
12.sup.th Edition, Merck & Co. Inc., Whitehouse Station, N.J.,
USA; ISBN 0911910-12-3). The polyoxyethylene sorbitan esters,
including polyoxyethylene sorbitan monooleate (Tween 80.TM.) are
described in Merck Index Entry 7742 (Page 1308, 12.sup.th Edition,
Merck & Co. Inc., Whitehouse Station, N.J., USA; ISBN
0911910-12-3). Both may be manufactured using methods described
therein, or purchased from commercial sources such as Sigma
Inc.
[0057] Particularly preferred non-ionic surfactants include Triton
X-45, t-octylphenoxy polyethoxyethanol (Triton X-100), Triton
X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305,
Triton N-57, Triton N-101, Triton N-128, Breij 35,
polyoxyethylene-9-lauryl ether (laureth 9) and
polyoxyethylene-9-stearyl ether (steareth 9). Triton X-100 and
laureth 9 are particularly preferred. Also particularly preferred
is the polyoxyethylene sorbitan ester, polyoxyethylene sorbitan
monooleate (Tween 80.TM.).
[0058] Further suitable polyoxyethylene ethers of general formula
(I) are selected from the following group:
polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether,
polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl
ether.
[0059] Alternative terms or names for polyoxyethylene lauryl ether
are disclosed in the CAS registry. The CAS registry number of
polyoxyethylene-9 lauryl ether is: 9002-92-0. Polyoxyethylene
ethers such as polyoxyethylene lauryl ether are described in the
Merck index (12.sup.th ed: entry 7717, Merck & Co. Inc.,
Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Laureth 9 is
formed by reacting ethylene oxide with dodecyl alcohol, and has an
average of nine ethylene oxide units.
[0060] Two or more non-ionic surfactants from the different groups
of surfactants described may be present in the vaccine formulation
described herein. In particular, a combination of a polyoxyethylene
sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween
80.TM.) and an octoxynol such as t-octylphenoxypolyethoxyethanol
(Triton) X-100.TM. is preferred. Another particularly preferred
combination of non-ionic surfactants comprises laureth 9 plus a
polyoxyethylene sorbitan ester or an octoxynol or both.
[0061] Non-ionic surfactants such as those discussed above have
preferred concentrations in the final vaccine composition as
follows: polyoxyethylene sorbitan esters such as Tween 80.TM.: 0.01
to 1%, most preferably about 0.1% (w/v); octyl- or nonylphenoxy
polyoxyethanols such as Triton X-100.TM. or other detergents in the
Triton series: 0.001 to 0.1%, most preferably 0.005 to 0.02% (w/v);
polyoxyethylene ethers of general formula (I) such as laureth 9:
0.1 to 20%, preferably 0.1 to 10% and most preferably 0.1 to 1% or
about 0.5% (w/v).
[0062] For certain vaccine formulations, other vaccine components
may be included in the formulation. As such the formulations of the
present invention may also comprise a bile acid or a derivative
thereof, in particular in the form of a salt. These include
derivatives of cholic acid and salts thereof, in particular sodium
salts of cholic acid or cholic acid derivatives. Examples of bile
acids and derivatives thereof include cholic acid, deoxycholic
acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic
acid, hyodeoxycholic acid and derivatives such as glyco-, tauro-,
amidopropyl-1-propanesulfonic-,
amidopropyl-2-hydroxy-1-propanesulfonic derivatives of the
aforementioned bile acids, or N,N-bis (3Dgluconoamidopropyl)
deoxycholamide. A particularly preferred example is sodium
deoxycholate (NaDOC) which may be present in the final vaccine
dose.
[0063] Also provided by the invention are pharmaceutical kits
comprising a vaccine administration device filled with a vaccine
according to the invention. Such administration devices include but
are not limited to needle devices, liquid jet devices, powder
devices, and spray devices (for intranasal use).
[0064] The influenza virus antigen preparations according to the
invention may be derived from the conventional embryonated egg
method, or they may be derived from any of the new generation
methods using tissue culture to grow the virus or express
recombinant influenza virus surface antigens. Suitable cell
substrates for growing the virus include for example dog kidney
cells such as MDCK or cells from a clone of MDCK, MDCK-like cells,
monkey kidney cells such as AGMK cells including Vero cells,
suitable pig cell lines, or any other mammalian cell type suitable
for the production of influenza virus for vaccine purposes.
Suitable cell substrates also include human cells e.g. MRC-5 cells.
Suitable cell substrates are not limited to cell lines; for example
primary cells such as chicken embryo fibroblasts are also
included.
[0065] The influenza virus antigen preparation may be produced by
any of a number of commercially applicable processes, for example
the split flu process described in patent no. DD 300 833 and DD 211
444, incorporated herein by reference. Traditionally split flu was
produced using a solvent/detergent treatment, such as tri-n-butyl
phosphate, or diethylether in combination with Tween.TM. (known as
"Tween-ether" splitting) and this process is still used in some
production facilities. Other splitting agents now employed include
detergents or proteolytic enzymes or bile salts, for example sodium
deoxycholate as described in patent no. DD 155 875, incorporated
herein by reference. Detergents that can be used as splitting
agents include cationic detergents e.g. cetyl trimethyl ammonium
bromide (CTAB), other ionic detergents e.g. laurylsulfate,
taurodeoxycholate, or non-ionic detergents such as the ones
described above including Triton X-100 (for example in a process
described in Lina et al, 2000, Biologicals 28, 95-103) and Triton
N-101, or combinations of any two or more detergents.
[0066] The preparation process for a split vaccine will include a
number of different filtration and/or other separation steps such
as ultracentrifugation, ultrafiltration, zonal centrifugation and
chromatography (e.g. ion exchange) steps in a variety of
combinations, and optionally an inactivation step, e.g., with heat,
formaldehyde or .beta.-propiolactone or U.V. which may be carried
out before or after splitting. The splitting process may be carried
out as a batch, continuous or semi-continuous process.
[0067] Preferred split flu vaccine antigen preparations according
to the invention comprise a residual amount of Tween 80 and/or
Triton X-100 remaining from the production process, although these
may be added or their concentrations adjusted after preparation of
the split antigen. Preferably both Tween 80 and Triton X-100 are
present. The preferred ranges for the final concentrations of these
non-ionic surfactants in the vaccine dose are: [0068] Tween 80:
0.01 to 1%, more preferably about 0.1% (v/v) [0069] Triton X-100:
0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02% (w/v).
[0070] Alternatively the influenza virus antigen preparations
according to the invention may be derived from a source other than
the live influenza virus, for example the haemagglutinin antigen
may be produced recombinantly.
[0071] The invention will now be further described in the
following, non-limiting examples.
EXAMPLES
Example 1
Preparation of Influenza Virus Antigen Preparation Using
.alpha.-Tocopherol Succinate as a Stabiliser for a
Preservative-Free Vaccine (Thiomersal-Reduced Vaccine)
[0072] Monovalent split vaccine was prepared according to the
following procedure.
Preparation of Virus Inoculum
[0073] On the day of inoculation of embryonated eggs a fresh
inoculum is prepared by mixing the working seed lot with a
phosphate buffered saline containing gentamycin sulphate at 0.5
mg/ml and hydrocortisone at 25 .mu.g/ml. (virus strain-dependent).
The virus inoculum is kept at 2-8.degree. C.
Inoculation of Embryonated Eggs
[0074] Nine to eleven day old embryonated eggs are used for virus
replication. Shells are decontaminated. The eggs are inoculated
with 0.2 ml of the virus inoculum. The inoculated eggs are
incubated at the appropriate temperature (virus strain-dependent)
for 48 to 96 hours. At the end of the incubation period, the
embryos are killed by cooling and the eggs are stored for 12-60
hours at 2-8.degree. C.
Harvest
[0075] The allantoic fluid from the chilled embryonated eggs is
harvested. Usually, 8 to 10 ml of crude allantoic fluid is
collected per egg.
Concentration and Purification of Whole Virus From Allantoic
Fluid
[0076] 1. Clarification
[0077] The harvested allantoic fluid is clarified by moderate speed
centrifugation (range: 4000-14000 g).
[0078] 2. Adsorption Step
[0079] To obtain a CaHPO.sub.4 gel in the clarified virus pool, 0.5
mol/L Na.sub.2HPO.sub.4 and 0.5 mol/L CaCl.sub.2 solutions are
added to reach a final concentration of CaHPO.sub.4 of 1.5 g to 3.5
g CaHPO.sub.4/litre depending on the virus strain.
[0080] After sedimentation for at last 8 hours, the supernatant is
removed and the sediment containing the influenza virus is
resolubilised by addition of a 0.26 mol/L EDTA-Na.sub.2 solution,
dependent on the amount of CaHPO.sub.4 used.
[0081] 3. Filtration
[0082] The resuspended sediment is filtered on a 6 .mu.m filter
membrane.
[0083] 4. Sucrose Gradient Centrifugation
[0084] The influenza virus is concentrated by isopycnic
centrifugation in a linear sucrose gradient (0.55% (w/v))
containing 100 .mu.g/ml Thiomersal. The flow rate is 8-15
litres/hour.
[0085] At the end of the centrifugation, the content of the rotor
is recovered by four different fractions (the sucrose is measured
in a refractometer):
TABLE-US-00002 fraction 1 55-52% sucrose fraction 2 approximately
52-38% sucrose fraction 3 38-20% sucrose* fraction 4 20-0% sucrose
*virus strain-dependent: fraction 3 can be reduced to 15%
sucrose.
[0086] For further vaccine preparation, only fractions 2 and 3 are
used.
[0087] Fraction 3 is washed by diafiltration with phosphate buffer
in order to reduce the sucrose content to approximately below 6%.
The influenza virus present in this diluted fraction is pelleted to
remove soluble contaminants.
[0088] The pellet is resuspended and thoroughly mixed to obtain a
homogeneous suspension. Fraction 2 and the resuspended pellet of
fraction 3 are pooled and phosphate buffer is added to obtain a
volume of approximately 40 litres. This product is the monovalent
whole virus concentrate.
[0089] 5. Sucrose Gradient Centrifugation With Sodium
Deoxycholate
[0090] The monovalent whole influenza virus concentrate is applied
to a ENI-Mark II ultracentrifuge. The K3 rotor contains a linear
sucrose gradient (0.55% (w/v)) where a sodium deoxycholate gradient
is additionally overlayed. Tween 80 is present during splitting up
to 0.1% (w/v) and Tocopherol succinate is added for
B-strain-viruses up to 0.5 mM. The maximal sodium deoxycholate
concentration is 0.7-1.5% (w/v) and is strain dependent. The flow
rate is 8-15 litres/hour.
[0091] At the end of the centrifugation, the content of the rotor
is recovered by three different fractions (the sucrose is measured
in a refractometer) Fraction 2 is used for further processing.
Sucrose content for fraction limits (47-18%) varies according to
strains and is fixed after evaluation:
[0092] 6. Sterile Filtration
[0093] The split virus fraction is filtered on filter membranes
ending with a 0.2 .mu.m membrane. Phosphate buffer containing
0.025% (w/v) Tween 80 and (for B strain viruses) 0.5 mM Tocopherol
succinate is used for dilution. The final volume of the filtered
fraction 2 is 5 times the original fraction volume.
[0094] 7. Inactivation
[0095] The filtered monovalent material is incubated at
22.+-.2.degree. C. for at most 84 hours (dependent on the virus
strains, this incubation can be shortened). Phosphate buffer
containing 0.025% (w/v). Tween 80 is then added in order to reduce
the total protein content down to max. 250 .mu.g/ml. For B strain
viruses, a phosphate buffered saline containing 0.025% (w/v) Tween
80 and 0.25 mM Tocopherol succinate is applied for dilution to
reduce the total protein content down to 250 .mu.g/ml. Formaldehyde
is added to a final concentration of 50 .mu.g/ml and the
inactivation takes place at 20.degree. C..+-.2.degree. C. for at
least 72 hours.
[0096] 8. Ultrafiltration
[0097] The inactivated split virus material is concentrated at
least 2 fold in a ultrafiltration unit, equipped with cellulose
acetate membranes with 20 kDa MWCO. The Material is subsequently
washed with phosphate buffer containing 0.025% (w/v) Tween 80 and
following with phosphate buffered saline containing 0.01% (w/v)
Tween. For B strain virus a phosphate buffered saline containing
0.01% (w/v) Tween 80 and 0.1 mM Tocopherol succinate is used for
washing.
[0098] 9. Final Sterile Filtration
[0099] The material after ultrafiltration is filtered on filter
membranes ending with a 0.2 .mu.m membrane. Filter membranes are
rinsed and the material is diluted if necessary such that the
protein concentration does not exceed 500 .mu.g/ml with phosphate
buffered saline containing 0.01% (w/v) Tween 80 and (for B strain
viruses) 0.1 mM Tocopherol succinate.
[0100] 10. Storage
[0101] The monovalent final bulk is stored at 2-8.degree. C. for a
maximum of 18 months.
Stability
TABLE-US-00003 [0102] TABLE 1 Comparison of time dependent HA
content (.mu.g/ml) measured by SRD in monovalent final bulks. After
4 weeks 6 month 12 month at Strain Stabiliser production at
30.degree. C. at 2-8.degree. C. 2-8.degree. C. B/Yamanashi/166/98
Tocopherylsuccinate 169 139 172 ND (residual mercury 3 (82%)
(>100%) .mu.g/ml) B/Yamanashi/166/98 Thiomersal 192 160 186 178
(108 .mu.g/ml) (83%) (97%) (93%) B/Yamanashi/166/98 None 191 122
175 154 (residual mercury 3 (60%) (92%) (81%) .mu.g/ml)
B/Johannesburg/5/99 Tocopherylsuccinate 166 183 158 179 (residual
mercury 4 (>100%) (95%) (>100%) .mu.g/ml) B/Johannesburg/5/99
Tocopherylsuccinate 167 179 158 178 (residual mercury 4 (>100%)
(95%) (>100%) .mu.g/ml) B/Johannesburg/5/99 Tocopherylsuccinate
144 151 130 145 (residual mercury 3 (>100%) (90%) (>100%)
.mu.g/ml) B/Johannesburg/5/99* Thiomersal 159 ND 172 154 (>100%)
(97%) B/Johannesburg/5/99** None 169 107 153 ON (63%) (90%)
*produced according to licensed FLUARIX .TM., **produced according
to example 1 without Tocopherylsuccinate, ON: Ongoing, ND not
determined
Example 2
Preparation of Influenza Vaccine Using .alpha.-Tocopherol Succinate
as a Stabiliser for a Thiomersal-Reduced Vaccine
[0103] Monovalent final bulks of three strains, A/New
Caldonia/20/99 (H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17
and B/Yamanashi/166/98 were produced according to the method
described in Example 1.
Pooling
[0104] The appropriate amount of monovalent final bulks was pooled
to a final HA-concentration of 30 .mu.g/ml for A/New Caldonia/20/99
(H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17, respectively and
of 39 .mu.g/ml for B/Yamanashi/166/98. Tween 80 and Triton X-100
were adjusted to 580 .mu.g/ml and 90 .mu.g/ml, respectively. The
final volume was adjusted to 3 l with phosphate buffered saline.
The trivalent pool was filtered ending with 0.8 .mu.m cellulose
acetate membrane to obtain the trivalent final bulk. Trivalent
final bulk was filled into syringes at least 0.5 mL in each.
TABLE-US-00004 TABLE 2 Comparison of time dependent HA content
measured by SRD in trivalent final bulks which was recovered from
syringes. Vaccine 0 2 4 6 formul. Strain months months months
months Influenza A/NCal/20/99 33 32 36 31 vaccine (32-34) (31-33)
(34-38) (30-32) without stabilizer A/Pan/2007/99 29 31 34 32
(27-31) (28-34) (32-36) (31-33) B/Yam/166/98 36 33 32 31 (34-38)
(32-34) (30-34) (29-33) Influenza A/NCal/20/99 31 32 36 32 vaccine
(30-32) (31-33) (34-38) (31-33) containing alpha- A/Pan/2007/99 33
33 36 33 tocopherol (30-36) (30-36) (35-37) (31-35) succinate
B/Yam/166/98 37 36 38 36 (35-39) (34-38) (35-41) (33-39)
Example 3
SRD Method Used to Measure Haemagglutinin Content
[0105] Glass plates (12.4-10.0 cm) are coated with an agarose gel
containing a concentration of anti-influenza HA serum that is
recommended by NIBSC. After the gel has set, 72 sample wells (3 mm
O) are punched into the agarose. 10 microliters of appropriate
dilutions of the reference and the sample are loaded in the wells.
The plates are incubated for 24 hours at room temperature (20 to
25.degree. C.) in a moist chamber. After that, the plates are
soaked overnight with NaCl-solution and washed briefly in distilled
water. The gel is then pressed and dried. When completely dry, the
plates are stained on Coomassie Brillant Blue solution for 10 min
and destained twice in a mixture of methanol and acetic acid until
clearly defined stained zones become visible. After drying the
plates, the diameter of the stained zones surrounding antigen wells
is measured in two directions at right angles. Alternatively
equipment to measure the surface can be used. Dose-response curves
of antigen dilutions against the surface are constructed and the
results are calculated according to standard slope-ratio assay
methods (Finney, D. J. (1952). Statistical Methods in Biological
Assay. London: Griffin, Quoted in: Wood, J M, et al (1977). J.
Biol. Standard. 5, 237-247).
Example 4
Clinical Testing of .alpha.-Tocopherol Stabilised Influenza Vaccine
(Reduced Thiomersal)
[0106] Syringes obtained as described in Example 2 are used for
clinical testing
[0107] H3N2: A/Panama/2007/99 RESVIR-17
[0108] H1N1: A/New Caledonia/20/99 (H1N1) IVR-116
[0109] B: B/Yamanashi/166/98
TABLE-US-00005 TABLE 3 thio- thio- Adults 18-60 reduced plus years
H3N2 H1N1 B H3N2 H1N1 B pre- GMT 47 41 111 55 37 102 vacc. Titer
<10 [%] 10.3% 13.8% 1.7% 5.3% 12.3% 8.8% Titer .gtoreq.40, SPR
60.3% 55.2% 75.9% 70.2% 52.6% 75.4% [%] post- Seroconv. rate 10.3%
13.8% 1.7% 5.3% 12.3% 8.8% vacc. [%] Significant 58.6% 74.1% 58.6%
63.2% 73.7% 52.6% Increase in antibody titer [%] Seroconversions
58.6% 74.1% 58.6% 63.2% 73.7% 52.6% [%] GMT 328 525 766 324 359 588
Fold GMT 7.3 13.0 6.9 5.9 9.8 5.9 Titer .gtoreq.40, SPR 100.0%
100.0% 100.0% 100.0% 100.0% 100.0% [%] n.d. = C.I. for proportion p
= n/N is not defined, because p*(1 - p)*N < 9 n/N = responders
(n) as part of number of subjects of the (sub)population (N), i.e.
seroconversions or significant increase, see also: CPMAP/BWP/214/96
12 Mar. 1997, p. 17ff GMT = geometric mean titer, reciprocal 95%
C.I. = 95% confidence interval, SPR = Seroprotection rate:
proportion of subjects with a protective titer pre- or
postvaccination .gtoreq.40 titer = HI-antibody titer Seroconversion
rate = proportion of subjects with antibody increase from <10
prevaccination to .gtoreq.40 postvaccination fold GMT = fold
increase of GMT Significant increase = proportion of subjects with
an antibody titer <10 prevaccination and 4-fold antibody
increase postvaccination (two steps of titer) req. = EU requirement
Seroconversions = neg to pos or g.e. 4-fold (neg: titer <10,
pos: titer .gtoreq.40) = proportion of subjects with either
seroconversion (<10 to .gtoreq.40) or significant increase.
[0110] Results show that the vaccine is able to offer equivalent
protection to vaccines containing thiomersal as a preservative.
Example 5a
Preparation of Influenza Virus Antigen Preparation Using
.alpha.-Tocopherol Succinate as a Stabiliser for a Thiomersal-Free
Vaccine
[0111] Monovalent split vaccine was prepared according to the
following procedure.
Preparation of Virus Inoculum
[0112] On the day of inoculation of embryonated eggs a fresh
inoculum is prepared by mixing the working seed lot with a
phosphate buffered saline containing gentamycin sulphate at 0.5
mg/ml and hydrocortisone at 25 .mu.g/ml. (virus strain-dependent).
The virus inoculum is kept at 2-8.degree. C.
Inoculation of Embryonated Eggs
[0113] Nine to eleven day old embryonated eggs are used for virus
replication. Shells are decontaminated. The eggs are inoculated
with 0.2 ml of the virus inoculum. 60,000 inoculated eggs are
incubated at the appropriate temperature (virus strain-dependent)
for 48 to 96 hours. At the end of the incubation period, the
embryos are killed by cooling and the eggs are stored for 12-60
hours at 2-8.degree. C.
Harvest
[0114] The allantoic fluid from the chilled embryonated eggs is
harvested. Usually, 8 to 10 ml of crude allantoic fluid is
collected per egg.
Concentration and Purification of Whole Virus From Allantoic
Fluid
Clarification
[0115] The harvested allantoic fluid is clarified by moderate speed
centrifugation (range: 4000-14000 g).
Precipitation Step
[0116] Saturated ammonium sulfate solution is added to the
clarified virus pool to reach a final ammonium sulfate
concentration of 0.5 mol/L. After sedimentation for at least 1
hour, the precipitate is removed by filtration on depth filters
(typically 0.5 .mu.m)
Filtration
[0117] The clarified crude whole virus bulk is filtered on filter
membranes ending with a validated sterile membrane (typically 0.2
.mu.m).
Ultrafiltration
[0118] The sterile filtered crude monovalent whole virus bulk is
concentrated on a cassettes equipped with 1000 kDa MWCO BIOMAX.TM.
membrane at least 6 fold. The concentrated retentate is washed with
phosphate buffered saline at least 1.8 times.
Sucrose Gradient Centrifugation
[0119] The influenza virus is concentrated by isopycnic
centrifugation in a linear sucrose gradient (0.55% (w/v)). The flow
rate is 8-15 litres/hour.
[0120] At the end of the centrifugation, the content of the rotor
is recovered by four different fractions (the sucrose is measured
in a refractometer):
TABLE-US-00006 fraction 1 55-52% sucrose fraction 2 approximately
52-38% sucrose fraction 3 38-20% sucrose* fraction 4 20-0% sucrose
*virus strain-dependent: fraction 3 can be reduced to 15%
sucrose.
[0121] For further vaccine preparation, either only fraction 2 is
used or fraction 2 together with a further purified fraction 3 are
used.
[0122] Fraction 3 is washed by diafiltration with phosphate buffer
in order to reduce the sucrose content to approximately below 6%.
Optionally this step may be omitted. The influenza virus present in
this diluted fraction is pelleted to remove soluble
contaminants.
[0123] The pellet is resuspended and thoroughly mixed to obtain a
homogeneous suspension. Fraction 2 and the resuspended pellet of
fraction 3 are pooled and phosphate buffer is added to obtain a
volume of approximately 40 litres. This product is the monovalent
whole virus concentrate.
Sucrose Gradient Centrifugation With Sodium Deoxycholate
[0124] The monovalent whole influenza virus concentrate is applied
to a ENI-Mark II ultracentrifuge. The K3 rotor contains a linear
sucrose gradient (0.55% (w/v)) where a sodium deoxycholate gradient
is additionally overlayed. Tween 80 is present during splitting up
to 0.1% (w/v) and Tocopherylsuccinate is added for B-strain viruses
up to 0.5 mM. The maximal sodium deoxycholate concentration is
0.7-1.5% (w/v) and is strain dependent. The flow rate is 8-15
litres/hour.
[0125] At the end of the centrifugation, the content of the rotor
is recovered by three different fractions (the sucrose is measured
in a refractometer) Fraction 2 is used for further processing.
Sucrose content for fraction limits (47-18%) varies according to
strains and is fixed after evaluation:
Sterile Filtration
[0126] The split virus fraction is filtered on filter membranes
ending with a 0.2 .mu.m membrane. Phosphate buffer containing
0.025% (w/v) Tween 80 and (for B strains) 0.5 mM
Tocopherylsuccinate is used for dilution. The final volume of the
filtered fraction 2 is 5 times the original fraction volume.
Inactivation
[0127] The filtered monovalent material is incubated at
22.+-.2.degree. C. for at most 84 hours (dependent on the virus
strains, this incubation can be shortened). Phosphate buffer
containing 0.025% (w/v) Tween 80 is then added in order to reduce
the total protein content down to max. 450 .mu.g/ml. For B-strains
a phosphate buffered saline containing 0.025% (w/v) Tween 80 and
0.25 mM Tocopherylsuccinate is applied for dilution to reduce the
total protein content down to 450 .mu.g/ml. Formaldehyde is added
to a final concentration of 100 .mu.g/ml and the inactivation takes
place at 20.degree. C..+-.2.degree. C. for at least 72 hours.
Ultrafiltration
[0128] The inactivated split virus material is concentrated at
least 2 fold in a ultrafiltration unit, equipped with cellulose
acetate membranes with 20 kDa MWCO. The Material is subsequently
washed with phosphate buffer containing 0.025% (w/v) Tween 80 and
following with phosphate buffered saline containing 0.01% (w/v)
Tween. For B-strain viruses a phosphate buffered saline containing
0.01% (w/v) Tween 80 and 0.1 mM Tocopherylsuccinate is used for
washing.
Final Sterile Filtration
[0129] The material after ultrafiltration is filtered on filter
membranes ending with a 0.2 .mu.m membrane. Filter membranes are
rinsed and the material is diluted if necessary that the protein
concentration does not exceed 500 .mu.g/ml with phosphate buffered
saline containing 0.01% (w/v) Tween 80 and, specific for B strains,
0.1 mM Tocopherylsuccinate.
Storage
[0130] The monovalent final bulk is stored at 2-8.degree. C. for a
maximum of 18 months.
Stability
TABLE-US-00007 [0131] TABLE 4 Comparison of time dependent HA
content (.mu.g/ml) measured by SRD in monovalent final bulks. After
4 weeks 6 month Strain Stabiliser production at 30.degree. C. at
2-8.degree. C. B/Johannesburg/ Tocopherol 214 196 206 5/99
succinate (92%) (96%) B/Johannesburg/ None 169 107 153 5/99** (63%)
(90%) **produced according to example 1 without
Tocopherylsuccinate.
Example 5b
Preparation of Influenza Virus Antigen Preparation Using
.alpha.-Tocopherol Succinate as a Stabiliser for a Thiomersal-Free
Vaccine
[0132] A preferred variation of the method described in Example 5a
is as follows:
[0133] Harvesting of the whole virus is followed by the
precipitation step (ammonium sulfate precipitation). This is
followed by the clarification step where the fluid is clarified by
moderate speed centrifugation (range 4000-14000 g). Thus the order
of the precipitation and clarification steps is reversed compared
to Example 5a.
[0134] Sterile filtration, ultrafiltration and ultracentrifugation
(sucrose gradient centrifugation) steps follow as for Example 5a.
However, there is no need for reprocessing step of the fractions
resulting from the ultracentrifugation step.
[0135] The remaining steps in the process are as described in
Example 5a.
[0136] Thus, the summarised process in this example is as follows:
[0137] Harvest [0138] Precipitation (ammonium sulfate) [0139]
Clarification [0140] Sterile filtration [0141] Ultrafiltration
[0142] Ultracentrifugation [0143] Splitting (preferably sodium
deoxycholate) [0144] Sterile filtration [0145] Inactivation [0146]
Ultrafiltration [0147] Final sterile filtration
[0148] Another preferred variation of Example 5a involves a
prefiltration step before the first sterile filtration. This uses a
membrane which does not sterile filter but which enables the
removal of contaminants e.g. albumin prior to sterile filtration.
This can result in a better yield. A suitable membrane for
prefiltration is about 0.8 .mu.m to about 1.8 .mu.m, for example
1.2 .mu.m. The prefiltration step can be used in the scheme of
Example 5a or Example 5b.
Example 6
Preparation of Influenza Vaccine Using .alpha.-Tocopherol Succinate
as a WStabiliser for a Thiomersal-Free Vaccine
[0149] Monovalent final bulks of three strains, A/New
Caldonia/20/99 (H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17
and B/Yamanashi/166/98 were produced according to the method
described in Example 5.
Pooling
[0150] The appropriate amount of monovalent final bulks was pooled
to a final HA-concentration of 30 .mu.g/ml for A/New Caldonia/20/99
(H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17, respectively and
of 36 .mu.g/ml for B/Johannesburg/5/97. Tween 80 and Triton X-100
were adjusted to 580 .mu.g/ml and 90 .mu.g/ml, respectively. The
final volume was adjusted to 3 l with phosphate buffered saline.
The trivalent pool was filtered ending with 0.8 .mu.m cellulose
acetate membrane to obtain the trivalent final bulk. Trivalent
final bulk was filled into syringes at least 0.5 mL in each.
TABLE-US-00008 TABLE 5 Comparison of time dependent HA content
(.mu.g/ml) measured by SRD in trivaient final bulks. 0 4 weeks 6
months Vaccine formul. Strain months at 30.degree. C. at
2-8.degree. C. Influenza vaccine A/NCal/20/99 31 32 30 without
stabilizer A/Pan/2007/99 31 34 33 B/Joh/5/99* 35 25 31 Influenza
vaccine A/NCal/20/99 34 35 34 containing alpha-tocopherol
A/Pan/2007/99 33 33 34 succinate B/Joh/5/99** 29 25 28 *Formulation
was based on target concentration of 39 .mu.g/ml. **Formulation was
based on target concentration of 34 .mu.g/ml.
Example 7
Preparation of Influenza Virus Antigen Preparation Using Sodium
Lauryl Sulfate as a Stabiliser for a Preservative-Free Vaccine
(Thiomersal-Reduced Vaccine)
Monovalent Whole Virus Concentrate of B/Johannesburg/5/99 was
Obtained as Described in Example 1.
Sucrose Gradient Centrifugation With Sodium Deoxycholate
[0151] The monovalent whole influenza virus concentrate is applied
to a ENI-Mark II ultracentrifuge. The K3 rotor contains a linear
sucrose gradient (0.55% (w/v)) where a sodium deoxycholate gradient
is additionally overlayed. Tween 80 is present during splitting up
to 0.1% (w/v). The maximal sodium deoxycholate concentration is
0.7-1.5% (w/v) and is strain dependent. The flow rate is 8-15
litres/hour. At the end of the centrifugation, the content of the
rotor is recovered by three different fractions (the sucrose is
measured in a refractometer) Fraction 2 is used for further
processing. Sucrose content for fraction limits (47-18%) varies
according to strains and is fixed after evaluation:
Sterile Filtration
[0152] A sample of fraction 2 of 10 ml was taken for further
processing. The split virus fraction is filtered on filter
membranes ending with a 0.2 .mu.m membrane. Phosphate buffer
containing 0.025% (w/v) Tween 80 and 0.5 mM sodium lauryl sulfate
is used for dilution. The final volume of the filtered fraction 2
is 5 times the original fraction volume.
Inactivation
[0153] The filtered monovalent material is incubated at
22.+-.2.degree. C. for at most 84 hours (dependent on the virus
strains, this incubation can be shortened). Phosphate buffered
saline containing 0.025% (w/v) Tween 80 and 0.5 mM sodium
laurylsulfate is then added in order to reduce the total protein
content down to max. 250 .mu.g/ml. Formaldehyde is added to a final
concentration of 50 .mu.g/ml and the inactivation takes place at
20.degree. C..+-.2.degree. C. for at least 72 hours.
Ultrafiltration
[0154] The inactivated split virus material is concentrated at
least 2 fold in a ultrafiltration unit, equipped with cellulose
acetate membranes with 20 kDa MWCO. The Material is subsequently
washed with 4 volumes phosphate buffered saline containing 0.01%
(w/v) Tween and 0.5 mM sodium lauryl sulfate.
Final Sterile Filtration
[0155] The material after ultrafiltration is filtered on filter
membranes ending with a 0.2 .mu.m membrane. Filter membranes are
rinsed and the material is diluted if necessary that the protein
concentration does not exceed 500 .mu.g/ml with phosphate buffered
saline containing 0.01% (w/v) Tween 80 and 0.5 mM sodium lauryl
sulfate.
Storage
[0156] The monovalent final bulk is stored at 2-8.degree. C.
TABLE-US-00009 TABLE 6 Comparison of time dependent HA content
measured by SRD in monovalent final bulks. 4 weeks stabiliser After
production at 30.degree. C. B/Johannesburg/5/99 None* 182 139 (77%)
B/Johannesburq/5/99 Sodium lauryl 288 264 (92%) sulfate *produced
according to Example 7 without addition of sodium lauryl
sulfate
Example 8
Preparation of Influenza Virus Antigen Preparation Using Plantacare
or Laureth-9 as a Stabiliser for a Preservative-Free Vaccine
(Thiomersal-Reduced Vaccine)
Monovalent Whole Virus Concentrate of B/Yamanashi/166/98 was
Obtained as Described in Example 1.
Fragmentation
[0157] The monovalent whole influenza virus concentrate is diluted
to a protein concentration of 1,000 .mu.g/ml with phosphate
buffered saline pH 7.4. Either Plantacare.RTM. 2000 UP or Laureth-9
is added to a final concentration of 1% (w/v). The material is
slightly mixed for 30 min. Then the material is overlayed on a
sucrose cushion 15% (w/w) in a bucket. Ultracentrifugation in a
Beckman swing out rotor SW 28 is performed for 2 h at 25,000 rpm
and 20.degree. C.
Sterile Filtration
[0158] A supernatant was taken for further processing. The split
virus fraction is filtered on filter membranes ending with a 0.2
.mu.m membrane.
Inactivation
[0159] Phosphate buffered saline is added if necessary in order to
reduce the total protein content down to max. 500 .mu.g/ml.
Formaldehyde is added to a final concentration of 100 .mu.g/ml and
the inactivation takes place at 20.degree. C..+-.2.degree. C. for
at least 6 days.
Ultrafiltration
[0160] Tween 80 and Triton X 100 is adjusted in the inactivated
material to 0.15% and 0.02% respectively. The inactivated split
virus material is concentrated at least 2 fold in a ultrafiltration
unit, equipped with cellulose acetate membranes with 30 kDa MWCO.
The Material is subsequently washed with 4 volumes phosphate
buffered saline.
Final Sterile Filtration
[0161] The material after ultrafiltration is filtered on filter
membranes ending with a 0.2 .mu.m membrane. Filter membranes are
rinsed and the material is diluted that the protein concentration
does not exceed 500 .mu.g/ml with phosphate buffered saline
Storage
[0162] The monovalent final bulk is stored at 2-8.degree. C.
TABLE-US-00010 TABLE 7 Comparison of time dependent HA content
measured by SRD in monovalent final bulks. 4 weeks stabiliser After
production at 30.degree. C. B/Yamanashi/166/98 None 143 98 (68%)
B/Yamanashi/166/98 Plantacare .RTM. 476 477 (100%) 2000 UP
B/Yamanashi/166/98 Laureth-9 468 494 (>100%)
Example 9
Clinical Testing of .alpha.-Tocopherol Stabilised Influenza Vaccine
(Reduced Thiomersal) in the Elderly via ID and IM
Administration
A Preparation of Influenza Virus Antigen Preparation
[0163] Monovalent split vaccine was prepared according to the
following procedure.
Preparation of Virus Inoculum
[0164] On the day of inoculation of embryonated eggs a fresh
inoculum is prepared by mixing the working seed lot with a
phosphate buffered saline containing gentamycin sulphate at 0.5
mg/ml and hydrocortisone at 25 .mu.g/ml. (virus strain-dependent).
The virus inoculum is kept at 2-8.degree. C.
Inoculation of Embryonated Eggs
[0165] Nine to eleven day old embryonated eggs are used for virus
replication. Shells are decontaminated. The eggs are inoculated
with 0.2 ml of the virus inoculum. The inoculated eggs are
incubated at the appropriate temperature (virus strain-dependent)
for 48 to 96 hours. At the end of the incubation period, the
embryos are killed by cooling and the eggs are stored for 12-60
hours at 2-8.degree. C.
Harvest
[0166] The allantoic fluid from the chilled embryonated eggs is
harvested. Usually, 8 to 10 ml of crude allantoic fluid is
collected per egg.
Concentration and Purification of Whole Virus From Allantoic
Fluid
[0167] 1. Clarification
[0168] The harvested allantoic fluid is clarified by moderate speed
centrifugation (range: 4000-14000 g).
[0169] 2. Adsorption Step
[0170] To obtain a CaHPO.sub.4 gel in the clarified virus pool, 0.5
mol/L Na.sub.2HPO.sub.4 and 0.5 mol/L CaCl.sub.2 solutions are
added to reach a final concentration of CaHPO.sub.4 of 1.5 g to 3.5
g CaHPO.sub.4/litre depending on the virus strain.
[0171] After sedimentation for at last 8 hours, the supernatant is
removed and the sediment containing the influenza virus is
resolubilised by addition of a 0.26 mol/L EDTA-Na.sub.2 solution,
dependent on the amount of CaHPO.sub.4 used.
[0172] 3. Filtration
[0173] The resuspended sediment is filtered on a 6 .mu.m filter
membrane.
[0174] 4. Sucrose Gradient Centrifugation
[0175] The influenza virus is concentrated by isopycnic
centrifugation in a linear sucrose gradient (0.55% (w/v))
containing 100 .mu.g/ml Thiomersal. The flow rate is 8-15
litres/hour.
[0176] At the end of the centrifugation, the content of the rotor
is recovered by four different fractions (the sucrose is measured
in a refractometer):
TABLE-US-00011 fraction 1 55-52% sucrose fraction 2 approximately
52-38% sucrose fraction 3 38-20% sucrose* fraction 4 20-0% sucrose
*virus strain-dependent: fraction 3 can be reduced to 15%
sucrose.
[0177] For further vaccine preparation, only fractions 2 and 3 are
used.
[0178] Fraction 3 is washed by diafiltration with phosphate buffer
in order to reduce the sucrose content to approximately below 6%.
The influenza virus present in this diluted fraction is pelleted to
remove soluble contaminants.
[0179] The pellet is resuspended and thoroughly mixed to obtain a
homogeneous suspension. Fraction 2 and the resuspended pellet of
fraction 3 are pooled and phosphate buffer is added to obtain a
volume of approximately 40 litres, a volume appropriate for 120,000
eggs/batch. This product is the monovalent whole virus
concentrate.
[0180] 5. Sucrose Gradient Centrifugation With Sodium
Deoxycholate
[0181] The monovalent whole influenza virus concentrate is applied
to a ENI-Mark II ultracentrifuge. The K3 rotor contains a linear
sucrose gradient (0.55% (w/v)) where a sodium deoxycholate gradient
is additionally overlayed. Tween 80 is present during splitting up
to 0.1% (w/v) and Tocopherol succinate is added for
B-strain-viruses up to 0.5 mM. The maximal sodium deoxycholate
concentration is 0.7-1.5% (w/v) and is strain dependent. The flow
rate is 8-15 litres/hour.
[0182] At the end of the centrifugation, the content of the rotor
is recovered by three different fractions (the sucrose is measured
in a refractometer) Fraction 2 is used for further processing.
Sucrose content for fraction limits (47-18%) varies according to
strains and is fixed after evaluation:
[0183] 6. Sterile Filtration
[0184] The split virus fraction is filtered on filter membranes
ending with a 0.2 .mu.m membrane. Phosphate buffer containing
0.025% (w/v) Tween 80 and (for B strain viruses) 0.5 mM Tocopherol
succinate is used for dilution. The final volume of the filtered
fraction 2 is 5 times the original fraction volume.
[0185] 7. Inactivation
[0186] The filtered monovalent material is incubated at
22.+-.2.degree. C. for at most 84 hours (dependent on the virus
strains, this incubation can be shortened). Phosphate buffer
containing 0.025% (w/v). Tween 80 is then added in order to reduce
the total protein content down to max. 250 .mu.g/ml. For B strain
viruses, a phosphate buffered saline containing 0.025% (w/v) Tween
80 and 0.25 mM Tocopherol succinate is applied for dilution to
reduce the total protein content down to 250 .mu.g/ml. Formaldehyde
is added to a final concentration of 50 .mu.g/ml and the
inactivation takes place at 20.degree. C..+-.2.degree. C. for at
least 72 hours.
[0187] 8. Ultrafiltration
[0188] The inactivated split virus material is concentrated at
least 2 fold in a ultrafiltration unit, equipped with cellulose
acetate membranes with 20 kDa MWCO. The Material is subsequently
washed with phosphate buffer containing 0.025% (w/v) Tween 80 and
following with phosphate buffered saline containing 0.01% (w/v)
Tween. For B strain virus a phosphate buffered saline containing
0.01% (w/v) Tween 80 and 0.1 mM Tocopherol succinate is used for
washing.
[0189] 9. Final Sterile Filtration
[0190] The material after ultrafiltration is filtered on filter
membranes ending with a 0.2 .mu.m membrane. Filter membranes are
rinsed and the material is diluted if necessary such that the
protein concentration does not exceed 1,000 .mu.g/ml but
haemagglutinin concentration exceeds 180 .mu.g/ml with phosphate
buffered saline containing 0.01% (w/v) Tween 80 and (for B strain
viruses) 0.1 mM Tocopherol succinate.
[0191] 10. Storage
[0192] The monovalent final bulk is stored at 2-8.degree. C. for a
maximum of 18 months.
B Preparation of Influenza Vaccine
[0193] Monovalent final bulks of three strains, A/New
Caldonia/20/99 (H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17
and B/Johannesburg/5/99 were produced according to the method
described in part A above.
Pooling
[0194] The appropriate amount of monovalent final bulks was pooled
to a final HA-concentration of 60 .mu.g/ml for A/New Caldonia/20/99
(H1N1) IVR-116, A/Panama/2007/99 (H3N2) Resvir-17, respectively and
of 68 .mu.g/ml for B/Johannesburg/5/99. Tween 80, Triton X-100 and
Tocopherol succinate were adjusted to 1,000 .mu.g/ml, 110 .mu.g/ml
and 160 .mu.g/ml, respectively. The final volume was adjusted to 3
with phosphate buffered saline. The trivalent pool was filtered
ending with 0.8 .mu.m cellulose acetate membrane to obtain the
trivalent final bulk. Trivalent final bulk was filled into syringes
at least 0.165 mL in each.
Vaccine Administration
[0195] The vaccine was supplied in pre-filled syringes and was
administered intradermally in the deltoid region. The intradermal
(ID) needle was as described in EP1092444, having a skin
penetration limiter to ensure proper intradermal injection. Since
formation of a wheal (papule) at the injection site demonstrates
the good quality of ID administration, the investigator with the
subject measured the exact size of the wheal 30 minutes after
vaccination.
[0196] One dose (100 .mu.l) contained the following components:
TABLE-US-00012 HEMAGGLUTININ FROM THREE INFLUENZA STRAINS A/NEW
CALEDONIA/20/99 6.0 .mu.g A/PANAMA/2007/99 6.0 .mu.g B/JOHANNESBURG
5/99 6.0 .mu.g THIOMERSAL PRESERVATIVE 0.4 .mu.g-0.8 .mu.g
B The Above Vaccine Was Compared a Standard Trivalent Split
Influenza Vaccine:
[0197] Fluarix.TM.. The Fluarix vaccine was supplied in pre-filled
syringes and was administered intramuscularly in the deltoid
muscle. A needle of at least 2.5 cm/1 inch in length (23 gauge) was
used to ensure proper intramuscular injection.
[0198] One dose (0.5 ml) contained the following components:
TABLE-US-00013 HEMAGGLUTININ FROM THREE INFLUENZA STRAINS A/NEW
CALEDONIA/20/99 15.0 .mu.g A/PANAMA/2007/99 15.0 .mu.g
B/JOHANNESBURG 5/99 15.0 .mu.g THIOMERSAL PRESERVATIVE 50.0
.mu.g
Results
[0199] The mean age of the total cohort at the time of vaccine
administration was 70.4.+-.6.2 years Standard Deviation (S.D.), the
female/male ratio was 1.7:1.
TABLE-US-00014 Immunogenicity results: Analysis of derived
immunogenicity variables was as follows: Variable Flu-red ID (N =
65) Fluarix .TM. IM (N = 65) GMT GMT LL UL GMT LL UL A/NEW
CALEDONIA PRE 99.5 76.9 128.7 90.0 70.1 115.7 POST 165.1 129.2
211.0 174.3 133.3 227.9 A/PANAMA PRE 75.5 54.7 104.2 69.2 51.9 92.4
POST 128.6 99.1 166.8 164.3 126.0 214.1 B/JOHANNESBURG PRE 236.0
187.7 296.8 222.6 176.9 280.2 POST 341.2 276.0 421.7 402.4 312.1
518.9 Seroconversion rate % LL UL % LL UL A/NEW CALEDONIA 15.4 7.6
26.5 18.5 9.9 30.0 A/PANAMA 20.0 11.1 31.8 29.2 18.6 41.8
B/JOHANNESBURG 9.2 3.5 19.0 16.9 8.8 28.3 Conversion factor GMR LL
UL GMR LL UL A/NEW CALEDONIA 1.7 1.4 2.0 1.9 1.6 2.3 A/PANAMA 1.7
1.4 2.1 2.4 1.9 3.0 B/JOHANNESBURG 1.4 1.2 1.7 1.8 1.5 2.1
Seroprotection rate % LL UL % LL UL A/NEW CALEDONIA PRE 87.7 77.2
94.5 90.8 81.0 96.5 POST 92.3 83.0 97.5 96.9 89.3 99.6 A/PANAMA PRE
75.4 63.1 85.2 81.5 70.0 90.1 POST 90.8 81.0 96.5 93.8 85.0 98.3
B/JOHANNESBURG PRE 98.5 91.7 100.0 96.9 89.3 99.6 POST 100.0 94.5
100.0 98.5 91.7 100.0 N: number of subjects with available results;
%: percentage of subjects within the given parameter; LL/UL: lower
and upper limit of 95% Cl; Pre: at the time of vaccine
administration; Post: 21 days after the vaccine dose
[0200] Injection site pain, reported by 10/65 (15.4%) vaccinees,
was the most common symptom following IM administration of
Fluarix.TM.. In the ID group, pain was reported by 3/65 (4.6%)
vaccinees. This difference was statistically significant (p=0.038;
Fisher exact test). Accordingly the ID delivery of a thiomersal
reduced product is preferred.
Conclusions
[0201] Both ID and IM administration of a thio-reduced flu vaccine
in an elderly population can provide 100% seroprotection.
[0202] A comparable response to vaccination in terms of geometric
mean titers, seroprotection rates, seroconversion rates and
conversion factors was found in IM and ID vaccinated individuals
where the ID group received 2.5-fold less antigen. There was no
discernible difference in the overall incidence of vaccine-related
solicited/unsolicited systemic symptoms in the two treatment
groups.
Example 10
Intradermal Delivery of a Thiomersal-Reduced Influenza Vaccine
[0203] Immunogenicity of the thiomersal reduced split influenza
vaccine prepared as described in Example 9 (except that the pooling
was done independently and the vaccine was not filled into
syringes) was assessed by ID delivery in guinea pigs using a
standard needle.
[0204] Groups of 5 animals each were primed intranasally with whole
inactivated trivalent influenza virus containing 5 .mu.g of each HA
in a total volume of 200 .mu.l. Twenty-eight days after priming the
animals were vaccinated by either the intradermal or intramuscular
routes. Intradermal doses containing 0.1, 0.3, or 1.0 .mu.g
trivalent thiomersal-reduced split Flu in 0.1 ml were administered
in the back of the guinea pig using a standard needle An
intramuscular dose of 1.0 .mu.g trivalent thiomersal-reduced split
Flu was administered in the hind leg of the guinea pig in a volume
of 0.1 ml. The groups were as follows: [0205] Group 1--0.1 .mu.g
trivalent thiomersal-reduced split Flu ID; [0206] Group 2--0.3
.mu.g trivalent thiomersal-reduced split Flu ID; [0207] Group
3--1.0 .mu.g trivalent thiomersal-reduced split Flu ID [0208] Group
4--1.0 .mu.g trivalent thiomersal-reduced split Flu IM
[0209] Fourteen days after vaccination the animals were bled and
the antibody titers induced by the vaccination were assessed using
a standard hemagglutination inhibition assay (HI). The results are
shown in FIG. 1. Strong HI responses to all three strains were
induced by vaccination. No clear dose response was noted suggesting
that very low doses of thiomersal-reduced antigen can still induce
very potent HI antibody responses when administered by the ID
route. There was no significant difference between the HI titers
induced by ID or IM vaccination. Thus, the results obtained in
guinea pigs confirmed that the thimerosal-reduced trivalent split
influenza antigens induce similar levels of HI antibodies in
animals when delivered by the ID route compared to the IM
route.
Example 11
Intradermal Delivery of a Thiomersal-Reduced, Adjuvanted Influenza
Vaccine
Protocol
[0210] Guinea pigs were primed on Day 0 with 5 .mu.g trivalent
whole inactivated Flu virus in 200 .mu.l, intranasally.
[0211] Vaccination--Day 28--Vaccine containing 0.1 .mu.g HA per
strain trivalent split Flu prepared as described in Example 9
(except that the pooling step resulted in a final concentration for
each antigen of 1.0 .mu.g/ml to give a dose of 0.1 .mu.g in 100
.mu.l compared to 60 .mu.g/ml in Example 9). The final trivalent
formulation was administered intradermally using tuberculin
syringes, either adjuvanted or unadjuvanted, in 100 .mu.l.
[0212] Bleeding--Day 42.
[0213] The effect of adjuvantation was assessed by measuring
antibody responses by HI assay (day 0, 28, 42).
[0214] All ID experiments were carried out using a standard
needle.
Results
[0215] G1-G5 refer to 5 groups of guinea pigs, 5 per group.
[0216] G1 Split trivalent thiomersal reduced 0.1 .mu.g
[0217] G2 Split trivalent thio red 0.1 .mu.g+3D-MPL 50 .mu.g
[0218] G3 Split trivalent thio red 0.1 .mu.g+3D-MPL 10 .mu.g
[0219] G4 Split trivalent thio red 0.1 .mu.g+3D-MPLin 50 .mu.g+QS21
50 .mu.g
[0220] G5 Split trivalent thio red 0.1 .mu.g+3D-MPLin 10 .mu.g+QS21
10 .mu.g
[0221] Note 3D-MPLin+QS21 refers to an adjuvant formulation which
comprises a unilamellar vesicle comprising cholesterol, having a
lipid bilayer comprising dioleoyl phosphatidyl choline, wherein the
QS21 and the 3D-MPL are associated with, or embedded within, the
lipid bilayer. Such adjuvant formulations are described in EP 0 822
831 B, the disclosure of which is incorporated herein by
reference.
[0222] HI Titres anti-A/New Caledonia/20/99
TABLE-US-00015 NC Pre-immun Pre-boost Post-boost G1 5 10 92 G2 5 10
70 G3 5 11 121 G4 7 9 368 G5 5 10 243
[0223] HI Titres anti-A/Panama/2007/99
TABLE-US-00016 P Pre-immun Pre-boost Post-boost G1 5 485 7760 G2 5
279 7760 G3 5 485 8914 G4 7 485 47051 G5 5 320 17829
[0224] HI Titres anti-B/Johannesburg/5/99
TABLE-US-00017 J Pre-immun Pre-boost Post-boost G1 5 23 184 G2 5 11
121 G3 5 11 70 G4 6 15 557 G5 5 13 320
[0225] Thus, whether adjuvanted or unadjuvanted the
thiomersal-reduced trivalent split Flu antigen is a potent
immunogen and capable of inducing strong HI responses when
administered by the ID or IM route. These responses appear to be at
least as potent as the responses induced by the standard Fluarix
preparation.
Example 12
Comparison of Thiomersal-Containing and Thiomersal-Free Vaccine
Delivered Intradermally in Pigs
[0226] In order to assess the immunogenicity of the split Flu
vaccine (plus and minus thiomersal) administered by the ID route
the primed pig model was used. As the vast majority of the
population has experienced at least one infection with influenza an
influenza vaccine must be able to boost a pre-existing immune
response. Therefore animals are primed in an effort to best
simulate the human situation.
[0227] In this experiment 4 week old pigs were primed by the
intranasal route. Six groups of five animals each were primed as
follows:
[0228] Group 1--two primings of trivalent whole inactivated virus
(50 .mu.g each HA) at day 0 and 14; Group 2--two primings of
trivalent whole inactivated virus (50 .mu.g each HA) at day 0 and
14; Group 3--single priming with trivalent whole inactivated virus
(50 .mu.g each HA) at day 0; Group 4--two primings of trivalent
whole inactivated virus (25 .mu.g each HA) at day 0 and 14; Group
5--single priming of trivalent whole inactivated virus (25 .mu.g
each HA) at day 0; Group 6--two primings of trivalent whole
inactivated virus (12.5 .mu.g each HA) at day 0 and 14.
[0229] On day 28 post final priming, the animals were vaccinated
with 3 .mu.g each HA trivalent split antigen (strains A/New
Caledonia H1N1, A/Panama H3N2, and B/Johannesburg) in 100 .mu.l by
the ID route. Group 1 received standard Fluarix.TM. containing
thiomersal preservative as vaccine antigen. All other groups
received the preservative-free antigen.
[0230] The HI results obtained in this experiment are shown in FIG.
2 (Anti-Influenza Hemagglutination Inhibition Titers Induced in
Pigs Primed with a Variety of Antigen Doses and Vaccinated with 3
Micrograms Trivalent Influenza Antigen Plus or Minus Thiomersal by
the Intradermal Route).
[0231] Relatively low HI titers are induced to the B strain in this
experiment and the background against the A/H3N2 strain is high. A
beneficial effect in terms of response to vaccination is observed
when the priming dose is reduced. In almost all cases, reduction in
the antigen concentration or number of priming doses (from the two
primings with 50 .mu.g) resulted in a heightened response to
vaccination. While the response of the animals in Groups 1 and 2,
which were primed twice with 50 .mu.g, to vaccination is not so
evident, it appears that the preservative-free antigen (Group 2)
functions at least as well as Fluarix.TM. (Group 1) under these
conditions. A strong response to vaccination with preservative-free
trivalent influenza antigen administered by the ID route in the
alternatively primed animals (Groups 3-6) is clear and this
response is seen even in the B strain, although the HI titers
remain low.
* * * * *