U.S. patent application number 15/730833 was filed with the patent office on 2018-09-06 for soluble needle arrays for delivery of influenza vaccines.
The applicant listed for this patent is Seqirus UK Limited. Invention is credited to Sung-Yun KWON, Derek O'HAGAN, Manmohan SINGH.
Application Number | 20180250381 15/730833 |
Document ID | / |
Family ID | 44764191 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180250381 |
Kind Code |
A1 |
O'HAGAN; Derek ; et
al. |
September 6, 2018 |
SOLUBLE NEEDLE ARRAYS FOR DELIVERY OF INFLUENZA VACCINES
Abstract
Influenza vaccines are administered using solid biodegradable
microneedles. The microneedles are fabricated from the influenza
vaccine in combination with solid excipient(s) and, after
penetrating the skin, they dissolve in situ and release the vaccine
to the immune system. The influenza vaccine is (i) a purified
influenza virus surface antigen vaccine, rather than a live vaccine
or a whole-virus or split inactivated vaccine (ii) an influenza
vaccine prepared from viruses grown in cell culture, not eggs,
(iii) a monovalent influenza vaccine e.g. for immunising against a
pandemic strain, (iv) a bivalent vaccine, (v) a tetravalent or
>4-valent vaccine, (vi) a mercury-free vaccine, or (vii) a
gelatin-free vaccine.
Inventors: |
O'HAGAN; Derek; (Cambridge,
MA) ; SINGH; Manmohan; (Cambridge, MA) ; KWON;
Sung-Yun; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seqirus UK Limited |
Berkshire |
|
GB |
|
|
Family ID: |
44764191 |
Appl. No.: |
15/730833 |
Filed: |
October 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15290349 |
Oct 11, 2016 |
9801935 |
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15730833 |
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13817814 |
Jun 27, 2013 |
9517205 |
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PCT/IB2011/002184 |
Aug 19, 2011 |
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15290349 |
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61401844 |
Aug 20, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/16111
20130101; A61K 39/12 20130101; C12N 2760/16134 20130101; C12N
2760/16234 20130101; A61K 2039/54 20130101; A61K 9/146 20130101;
A61M 37/0015 20130101; A61K 2039/5252 20130101; A61M 2037/0046
20130101; A61K 2039/70 20130101; A61M 2037/0023 20130101; A61B
17/205 20130101; A61B 2017/00004 20130101; A61K 39/145 20130101;
A61M 2202/30 20130101; G01N 2333/11 20130101; C12N 2760/16211
20130101; G01N 33/68 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61B 17/20 20060101 A61B017/20; A61M 37/00 20060101
A61M037/00; G01N 33/68 20060101 G01N033/68 |
Claims
1-2. (canceled)
3. An aqueous liquid or solid material comprising: (i) a biosoluble
and biodegradable matrix material and (ii) an influenza vaccine
selected from the group consisting of a purified influenza virus
surface antigen vaccine, an influenza vaccine prepared from viruses
grown in cell culture, a monovalent influenza vaccine, a bivalent
vaccine, a tetravalent or >4-valent vaccine, a mercury free
vaccine, and a gelatin-free vaccine.
4. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a purified influenza virus surface antigen
vaccine.
5-6. (canceled)
7. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is an influenza vaccine prepared from viruses
grown in cell culture.
8. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a monovalent influenza vaccine.
9. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a bivalent influenza vaccine.
10. aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a tetravalent influenza vaccine.
11. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a >4-valent influenza vaccine.
12. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a mercury-free influenza vaccine.
13. The aqueous liquid or solid material of claim 3, wherein the
influenza vaccine is a gelatin-free influenza vaccine.
14. The aqueous liquid or solid material of claim 3, wherein the
matrix material comprises one or more carbohydrates.
15. The aqueous liquid or solid material of claim 14, wherein the
matrix material comprises a cellulose and/or a dextrin and/or a
disaccharide.
16-19. (canceled)
20. The aqueous liquid or solid material of claim 3, comprising
between 0.5-50 .mu.g of detergent per pg of hemagglutinin.
21. aqueous liquid or solid material of claim 3, containing 1-15
.mu.g of hemagglutinin per influenza virus strain.
22-25. (canceled)
Description
[0001] This application claims the benefit of U.S. provisional
application 61/401,844 (filed. Aug. 20, 2010), the complete
contents of which are hereby incorporated herein by reference for
all purposes.
TECHNICAL FIELD
[0002] This invention is in the field of influenza vaccination.
BACKGROUND ART
[0003] Various forms of influenza virus vaccine are currently
available (e.g. see chapters 17 & 18 of reference 1) and
current vaccines are based either on inactivated or live attenuated
viruses. Inactivated vaccines are administered by intramuscular or
intradermal injection, whereas live vaccines are administered
intranasally.
[0004] It is an object of the invention to provide a different way
of administering inactivated influenza vaccines, and in particular
a more convenient way e.g. which does not require medical
personnel, and which may thus be sold in an over-the-counter
setting.
DISCLOSURE OF THE INVENTION
[0005] According to the invention, influenza vaccines are
administered using solid biodegradable microneedles. The
microneedles are fabricated from the influenza vaccine in
combination with solid excipient(s) and, after penetrating the
skin, they dissolve in situ and release the vaccine to the immune
system. In preferred embodiments the influenza vaccine is (i) a
purified influenza virus surface antigen vaccine, rather than a
live vaccine or a whole-virus or split inactivated vaccine (ii) an
influenza vaccine prepared from viruses grown in cell culture, not
eggs, (iii) a monovalent influenza vaccine e.g. for immunising
against a pandemic strain, (iv) a bivalent vaccine, (v) a
tetravalent or >4-valent vaccine, (vi) a mercury-free vaccine,
and/or (vii) a gelatin-free vaccine.
[0006] Thus the invention provides a skin patch comprising a
plurality of solid biodegradable microneedles, wherein the
microneedles comprise a mixture of (i) a biosoluble and
biodegradable matrix material and (ii) an influenza vaccine
selected from the group consisting of a purified influenza virus
surface antigen vaccine, an influenza vaccine prepared from viruses
grown in cell culture, a monovalent influenza vaccine, a bivalent
vaccine, a tetravalent or >4-valent vaccine, a mercury-free
vaccine, and a gelatin-free vaccine. The vaccine can have one or
more of these features. This patch can be used to deliver an
influenza vaccine to a subject via their skin, and so can be used
in a method for raising an immune response in a mammal.
[0007] The invention also provides a process for preparing a skin
patch comprising a plurality of solid biodegradable microneedles,
comprising steps of: (i) mixing a biosoluble and biodegradable
matrix material with an influenza vaccine selected from the group
consisting of a purified influenza virus surface antigen vaccine,
an influenza vaccine prepared from viruses grown in cell culture, a
monovalent influenza vaccine, a bivalent vaccine, a tetravalent or
>4-valent vaccine, a mercury-free vaccine, and a gelatin-free
vaccine; and (ii) adding the mixture from step (i) to a mold
containing cavities for forming microneedles.
[0008] The invention also provides an aqueous liquid or solid
material comprising (i) a biosoluble and biodegradable matrix
material and (ii) an influenza vaccine selected from the group
consisting of a purified influenza virus surface antigen vaccine,
an influenza vaccine prepared from viruses grown in cell culture, a
monovalent influenza vaccine, a bivalent vaccine, a tetravalent or
>4-valent vaccine, a mercury-free vaccine, and a gelatin-free
vaccine. This material is suitable for preparing a patch of the
invention.
[0009] The invention also provides a skin patch comprising a
plurality of solid biodegradable microneedles, wherein the
microneedles comprise a mixture of (i) a biosoluble and
biodegradable matrix material and (ii) an influenza virus
hemagglutinin, wherein the amount of influenza virus hemagglutinin
per patch is .ltoreq.16 .mu.g per strain. This patch can be used to
deliver an inactivated influenza vaccine to a subject via their
skin, and so can be used in a method for raising an immune response
in a mammal.
[0010] The invention also provides a process for preparing a skin
patch comprising a plurality of solid biodegradable microneedles,
comprising steps of: (i) mixing a biosoluble and biodegradable
matrix material with an influenza vaccine; and (ii) adding the
mixture from step (i) to a mold containing cavities for forming
microneedles, wherein the amount of mixture added in step (ii)
provides a patch having .ltoreq.16 .mu.g influenza virus
hemagglutinin per strain per patch.
[0011] The invention also provides an aqueous liquid or solid
material comprising (i) a biosoluble and biodegradable matrix
material and (ii) an influenza virus hemagglutinin at a
concentration of .ltoreq.16 .mu.g per strain. This material is
suitable for preparing a patch of the invention.
[0012] The invention also provides a process for determining the
amount of influenza hemagglutinin in a skin patch, wherein (a) the
patch comprises a biosoluble & biodegradable matrix material
and an influenza vaccine, and (b) the process comprises steps of:
(i) dissolving the patch in a solvent to provide a dissolved patch
solution; and (ii) assaying hemagglutinin in the dissolved patch
solution by enzyme-linked immunosorbent assay (ELISA).
[0013] The invention also provides a process for determining the
amount of influenza hemagglutinin in a skin patch, wherein (a) the
patch comprises a biosoluble & biodegradable matrix material
and an influenza vaccine, and (b) the process comprises steps of:
(i) dissolving the patch in a solvent to provide a dissolved patch
solution; (ii) precipitating proteins in the dissolved patch
solution; and (iii) assaying hemagglutinin after precipitation in
step (ii).
The Biodegradable Microneedles
[0014] Influenza vaccine is delivered via solid biodegradable
microneedles.
[0015] The microneedles are solid, such that they retain their
structural integrity during storage and can penetrate a subject's
skin when the patch is applied. The mechanical characteristics
which are required for skin penetration depend on the organism in
question, but they will usually have sufficient strength to
penetrate human skin. Materials for forming suitable solid needles
are readily available and these can be tested to determine
appropriate concentrations etc. for any particular need.
[0016] The microneedles are biosoluble and biodegradable. Thus the
solid material dissolves in the skin after the patch is applied, in
contrast to the coated metal microneedles used in references 2
& 3. Having dissolved, the material will then be metabolised to
give harmless end-products. The timescale for dissolving after
applying the patch can vary, but dissolving will typically commence
immediately after applying the patch (e.g. within 10 seconds) and
may continue for e.g. up to 1 minute, 5 minutes, 10 minutes, 20
minutes, 30 minutes, 1 hour, 5 hours, 10 hours, or 24 hours, until
the microneedle has fully dissolved. Materials with suitable in
vivo dissolving kinetics are readily available and these can be
varied and tested to determine appropriate concentrations etc. for
any desired dissolution profile.
[0017] Suitable matrix materials for forming the microneedles will
typically be biosoluble and biodegradable polymers, and these may
comprise one or more carbohydrates. For example, the material may
comprise a cellulose, a dextrin, a dextran, a disaccharide, a
chitosan, a chitin, etc., or mixtures thereof Other GRAS materials
may also be used.
[0018] Suitable celluloses include, but are not limited to,
cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose,
hydroxyethyl cellulose, and hydroxypropyl methylcellulose. Suitable
dextrins include, but are not limited to, maltodextrin,
cyclodextrin, amylodextrin, icodextrin, yellow dextrin, and white
dextrins. Suitable disaccharides include, but are not limited to,
sucrose, lactose, maltose, trehalose, turanose, and cellobiose.
[0019] Suitable mixtures for forming biosoluble and biodegradable
microneedles include, but are not limited to, mixtures of (i)
dextrin and trehalose, (ii) sucrose and sodium carboxymethyl
cellulose.
[0020] The microneedles can penetrate the skin. They should be long
enough to penetrate through the epidermis to deliver material into
the dermis (i.e. intradermal delivery), but are ideally not so long
that they can penetrate into or past the hypodermis. They will
typically be 100-2500 .mu.m long e.g. between 1250-1750 .mu.m long,
or about 1500 .mu.m. At the time of delivery the tip may penetrate
the dermis, but the base of the needle may remain in the
epidermis.
[0021] The microneedles can have various shapes and geometries.
They will typically be tapered with a skin-facing point e.g. shaped
as pyramids or cones. A tapered microneedle with a widest diameter
of <500 .mu.m is typical.
[0022] A single patch will typically include a plurality of
microneedles e.g. .gtoreq.10, .gtoreq.20, .gtoreq.30, .gtoreq.40,
.gtoreq.50, .gtoreq.60, .gtoreq.70, .gtoreq.80, .gtoreq.90,
.gtoreq.100, .gtoreq.200, .gtoreq.300, .gtoreq.400, .gtoreq.50,
.gtoreq.750, .gtoreq.1000 or more per patch. Where a patch includes
a plurality of microneedles, it may comprise a backing layer to
which all of the microneedles are attached. A unitary backing layer
with .gtoreq.20 projecting microneedles is typical. Where a patch
includes a plurality of microneedles, these can be arranged in a
regular repeating pattern or array, or nay be arranged irregularly.
A patch will typically have an area of 3 cm.sup.2 or less, for
example <2 cm.sup.2 or <1 cm.sup.2. A circular patch with a
diameter of between 0.5 cm and 1.5 cm is useful.
[0023] The density of microneedles on a patch can vary, but may be
.gtoreq.10 cm.sup.-2, .gtoreq.20 cm.sup.-2, .gtoreq.30 cm.sup.-2,
.gtoreq.40 cm.sup.-2, .gtoreq.50 cm.sup.-2, .gtoreq.60 cm.sup.-2,
.gtoreq.70 cm.sup.-2, .gtoreq.80 cm.sup.-2 or more.
[0024] A patch of the invention has a skin-facing inner face and an
environment-facing outer face. The inner face may include an
adhesive to facilitate adherence to a subject's skin. When present,
it is preferably not present on the microneedles themselves i.e.
the microneedles are adhesive-free. For example, a patch may have
an additional backing which provides an outer adhesive margin for
adhering the patch to skin e.g. as seen in sticking plasters or
nicotine patches.
[0025] Patches as described above can be made by following the
techniques and guidance in references 4-9. For instance, a mold
with 1.5 mm-long microneedle cavities can be prepared. A matrix
material of dextrin and trehalose can be combined with an influenza
vaccine and this aqueous material can be centrifugally cast in the
mold to foils an array of solid microneedles. A cellulose gel can
then be cast over the matrix/vaccine film to form a backing layer
on the patch. When this layer has dried, it can be removed to give
a patch from which the solid microneedles project. Thus a process
of the invention may include, after step (ii), further steps of:
(iii) letting the mixture set in the mold, to foils solid
microneedles; (iv) optionally, applying material to the set
microneedles to provide a backing layer; and (v) removing the
microneedles (and optional backing layer) from the mold.
[0026] Patches of the invention may be packaged into individual
pouches e.g. sealed under nitrogen, then heat sealed. They should
be stored carefully to avoid damage to the microneedles.
Surface Antigen Influenza Vaccines
[0027] Some embodiments of the invention use a surface antigen
influenza vaccine. Such vaccines contain fewer viral components
than a split or whole virion vaccine. They include the surface
antigens hemagglutinin and, typically, also neuraminidase.
Processes for preparing these proteins in purified form from
influenza viruses are well known in the art. The FLUVIRI.TM.,
AGRIPPAL.TM. and INFLUVAC.TM. products are examples of surface
antigen influenza vaccines.
[0028] The ability to administer surface antigen influenza vaccines
using solid biosoluble biodegradable microneedles is advantageous.
Other intradermal needle formats [10] have been found to be
incompatible with the high level of residual detergent that can be
present in surface antigen influenza vaccines, but the solid
biodegradable microneedle format is effective even in these
circumstances. Products of the invention may comprise detergent
(e.g. a non-ionic detergent) at between 0.0-50 .mu.g per .mu.g of
HA, e.g. as described in more detail below.
[0029] Where the invention uses a surface antigen influenza
vaccine, this virus may have been grown in eggs. The current
standard method for influenza virus growth for vaccines uses
embryonated SPF hen eggs, with virus being purified from the egg
contents (allantoic fluid). If egg-based viral growth is used then
one or more amino acids may be introduced into the allantoid fluid
of the egg together with the virus [16].
[0030] Virus is first grown in eggs. It is then harvested from the
infected eggs. Virions can be harvested from the allantoic fluid by
various methods. For example, a purification process may involve
zonal centrifugation using a linear sucrose gradient solution that
includes detergent to disrupt the virions. Antigens may then he
purified, after optional dilution, by diafiltration. Chemical means
for inactivating a virus include treatment with an effective amount
of one or more of the following agents: detergents, formaldehyde,
.beta.-propiolactone, methylene blue, psoralen, carboxyfullerene
(C60), binary ethylamine, acetyl ethyleneimine, or combinations
thereof. Non-chemical methods of viral inactivation are known in
the art, such as for example UV light or gamma irradiation.
Other Forms of Influenza Vaccines
[0031] Some embodiments of the invention (specifically those which
use cell-culture derived antigens, those which are not trivalent,
those which are mercury-free, and those which are gelatin-free) are
not restricted to using a surface antigen influenza vaccine. These
embodiments may thus use whole inactivated virus, split virus,
virosomes, live attenuated virus, or recombinant hemagglutinin.
These vaccines can easily be distinguished from surface antigen
vaccines by testing their antigens e.g. for the presence of extra
influenza virus proteins.
[0032] Whole inactivated virions can be obtained by harvesting
virions from virus-containing fluids (e.g. obtained from eggs or
from culture medium) and then treating them as described above.
[0033] Split virions are obtained by treating purified virions with
detergents (e.g. ethyl ether, polysorbate 80, deoxycholate,
tri-N-butyl phosphate, Triton X-100, Triton N101,
cetyltrimethylammonium bromide, Tergitol NP9, etc.) to produce
subvirion preparations, including the `Tween-ether` splitting
process. Methods of splitting influenza viruses, for example are
well known in the art e.g. see refs. 11-16, etc. Splitting of the
virus is typically carried out by disrupting or fragmenting whole
virus, whether infectious or non-infectious with a disrupting
concentration of a splitting agent. The disruption results in a
full or partial solubilisation of the virus proteins, altering the
integrity of the virus. Preferred splitting agents are non-ionic
and ionic (e.g. cationic) surfactants e.g. alkylglycosides,
alkylthioglycosides, acyl sugars, sulphobetaines, betains,
polyoxyethylene-alkylethers, N,N-di alkyl-Glucamides, Hecameg,
alkylphenoxy-polyethoxyethanols, NP9, quaternary ammonium
compounds, sarcosyl, CTABs (cetyl trimethyl ammonium bromides),
tri-N-butyl phosphate, myristyltrimethylammonium salts, lipofectin,
lipofectamine, and DOT-MA, the octyl- or nonylphenoxy
polyoxyethanols (e.g. the Triton surfactants, such as Triton X-100
or Triton N101), polyoxyethylene sorbitan esters (the Tween
surfactants), polyoxyethylene ethers, polyoxyethlene esters, etc.
One useful splitting procedure uses the consecutive effects of
sodium deoxycholate and formaldehyde, and splitting can take place
during initial virion purification (e.g. in a sucrose density
gradient solution). Thus a splitting process can involve
clarification of the virion-containing material (to remove
non-virion material), concentration of the harvested virions (e.g.
using an adsorption method, such as CaHPC.sub.4 adsorption),
separation of whole virions from non-virion material, splitting of
virions using a splitting agent in a density gradient
centrifugation step (e.g. using a sucrose gradient that contains a
splitting agent such as sodium deoxycholate), and then filtration
(e.g. ultrafiltration) to remove undesired materials. Split virions
can usefully be resuspended in sodium phosphate-buffered isotonic
sodium chloride solution. Examples of split vaccines are the
BEGRIVAC.TM., INTANZA.TM., FLUARIX.TM., FLUZONE.TM. and
FLUSHIELD.TM. products.
[0034] Virosomes are nucleic acid free viral-like liposomal
particles [17]. They can be prepared by solubilization of virus
with a detergent followed by removal of the nucleocapsid and
reconstitution of the a e brane containing the viral glycoproteins.
An alternative method for preparing virosomes involves adding viral
membrane glycoproteins to excess amounts of phospholipids, to give
liposomes with viral proteins in their membrane.
[0035] Live attenuated viruses are obtained from viruses (grown in
eggs or in cell culture), but the viruses are not inactivated.
Rather, the virus is attenuated ("art") e.g. so as not to produce
influenza-like illness in a ferret model of human influenza
infection. It may also be a cold-adapted ("ca") strain i.e. it can
replicate efficiently at 25.degree. C., a temperature that is
restrictive for replication of many wildtype influenza viruses. It
may also be temperature-sensitive ("ts") i.e. its replication is
restricted at temperatures at which many wild-type influenza
viruses grow efficiently (37-39.degree. C.). The cumulative effect
of the ca, ts, and att phenotype is that the virus in the
attenuated vaccine can replicate in the nasopharynx to induce
protective immunity in a typical human patient, but it does not
cause disease i.e it is safe for general administration to the
target human population. These viruses can be prepared by purifying
virions from virion-containing fluids e.g. after clarification of
the fluids by centrifugation, then stabilization with buffer (e.g.
containing sucrose, potassium phosphate, and monosodium glutamate).
Live vaccines include the FLUMIST.TM. product. Although live
vaccines can be used with the invention, it is preferred to use
non-live vaccines.
[0036] As an alternative to using antigens obtained from virions,
haemagglutinin can be expressed in a recombinant host (e.g. in an
insect cell line, such as Sf9, using a baculovirus vector) and used
in purified form [18-20] or in the form of virus-like particles
(VLPs; e.g. see references 21 & 22).
Influenza Vaccines From Cell Culture
[0037] Some embodiments of the invention use influenza vaccine
prepared from viruses which were grown in cell culture, rather than
in eggs.
[0038] When cell culture is used, the viral growth substrate will
typically be a cell line of mammalian origin. Suitable mammalian
cells of origin include, but are not limited to, hamster, cattle,
primate (including humans and monkeys) and dog cells. Various cell
types may be used, such as kidney cells, fibroblasts, retinal
cells, lung cells, etc. Examples of suitable hamster cells are the
cell lines having the names BHK21 or HKCC. Suitable monkey cells
are e.g. African green monkey cells, such as kidney cells as in the
Vero cell line. Suitable dog cells are e.g. kidney cells, as in the
MDCK cell line. Thus suitable cell lines include, but are not
limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; etc..
Preferred mammalian cell lines for growing influenza viruses
include: MDCK cells [23-26], derived from Madin Darby canine
kidney; Vero cells [27-29], derived from African green monkey
(Cercopithecus aethiops) kidney; or PER.C6 cells [30], derived from
human embryonic retinoblasts. These cell lines are widely available
e.g. from the American Type Cell Culture (ATCC) collection, from
the Coriell Cell Repositories, or from the European Collection of
Cell Cultures (ECACC). For example, the ATCC supplies various
different Vero cells under catalog numbers CCL-81 , CCL-81.2,
CRL-1586 and CRL-1587, and it supplies MDCK cells under catalog
number CCL-34. PER.C6 is available from the ECACC under deposit
number 96022940. As a less-preferred alternative to mammalian cell
lines, virus can be grown on avian cell lines [e.g. refs. 31-33],
including cell lines derived from ducks (e.g. duck retina) or hens.
Examples of avian cell lines include avian embryonic stem cells
[31,34] and duck retina cells [32]. Suitable avian embryonic stem
cells, include the EBx cell line derived from chicken embryonic
stem cells, EB45, EB14, and EB14-074 [35]. Chicken embryo
fibroblasts (CEF) may also be used.
[0039] The most preferred cell lines for growing influenza viruses
are MDCK cell lines. The original MDCK cell line is available from
the ATCC as CCL-34, but derivatives of this cell line may also be
used. For instance, reference 23 discloses a MDCK cell line that
was adapted for growth in suspension culture (MDCK 33016',
deposited as DSM ACC 2219). Similarly, reference 36 discloses a
MDCK-derived cell line that grows in suspension in serum-free
culture (B-702', deposited as FERM BP-7449). Reference 37 discloses
non-tumorigenic MDCK cells, including `MDCK-S` (ATCC PTA-6500),
`MDCK-SF101` (ATCC PTA-6501), `MDCK-SF 102` (ATCC PTA-6502) and
`MDCK-SF 103` (PTA-6503). Reference 38 discloses MDCK cell lines
with high susceptibility to infection, including `MDCK.5F1` cells
(ATCC CRL-12042). Any of these MDCK cell lines can be used.
[0040] Where virus has been grown on a mammalian cell line then
products of the invention will advantageously be free from egg
proteins (e.g. ovalbumin and ovomucoid) and from chicken DNA,
thereby reducing potential allergenicity.
[0041] Hemagglutinin in cell-derived products of the invention can
have a different glycosylation pattern from the patterns seen in
egg-derived viruses. Thus the HA (and other glycoproteins) may
include glycoforms that are not seen in chicken eggs. Useful HA
includes canine glycoforms.
[0042] The absence of egg-derived materials and of chicken
glycoforms provides a way in which vaccine prepared from viruses
grown in cell culture can be distinguished from egg-derived
products.
[0043] Where virus has been grown on a cell line then the culture
for growth, and also the viral inoculum used to start the culture,
will preferably be free from (i.e. will have been tested for and
given a negative result for contamination by) herpes simplex virus,
respiratory syncytial virus, parainfluenza virus 3, SARS
coronavirus, adenovirus, rhinovirus, reoviruses, polyomaviruses,
birnaviruses, circoviruses, and/or parvoviruses [39]. Absence of
herpes simplex viruses is particularly preferred. For growth on a
cell line, such as on MDCK cells, virus may be grown on cells in
suspension [23, 40, 41] or in adherent culture. One suitable MDCK
cell line for suspension culture is MDCK 33016 (deposited as DSM
ACC 2219). As an alternative, microcarrier culture can be used.
[0044] Cell lines supporting influenza virus replication are
preferably grown in serum-free culture media and/or protein free
media. A medium is referred to as a serum-free medium in the
context of the present invention in which there are no additives
from serum of human or animal origin. Protein-free is understood to
mean cultures in which multiplication of the cells occurs with
exclusion of proteins, growth factors, other protein additives and
non-serum proteins, but can optionally include proteins such as
trypsin or other proteases that may be necessary for viral growth.
The cells growing in such cultures naturally contain proteins
themselves.
[0045] Cell lines supporting influenza virus replication are
preferably grown below 37.degree. C. [42] during viral replication
e.g. 30-36.degree. C., at 31-35.degree. C., or at 33.+-.1.degree.
C.
[0046] The method for propagating virus in cultured cells generally
includes the steps of inoculating the cultured cells with the
strain to be cultured, cultivating the infected cells for a desired
time period for virus propagation, such as for example as
determined by virus titer or antigen expression (e.g. between 24
and 168 hours after inoculation) and collecting the propagated
virus. The cultured cells are inoculated with a virus (measured by
PFU or TCID.sub.50) to cell ratio of 1:500 to 1:1, preferably 1:100
to 1:5, more preferably 1:50 to 1:10. The virus is added to a
suspension of the cells or is applied to a monolayer of the cells,
and the virus is absorbed on the cells for at least 60 minutes but
usually less than 300 minutes, preferably between 90 and 240
minutes at 25.degree. C. to 40.degree. C., preferably 28.degree. C.
to 37.degree. C. The infected cell culture (e.g. monolayers) may be
removed either by freeze-thawing or by enzymatic action to increase
the viral content of the harvested culture supernatants. The
harvested fluids are then either inactivated or stored frozen.
Cultured cells may be infected at a multiplicity of infection
("m.o.i.") of about 0.0001 to 10, preferably 0.002 to 5, more
preferably to 0.001 to 2. Still more preferably, the cells are
infected at a m.o.i of about 0.01. Infected cells may be harvested
30 to 60 hours post infection. Preferably, the cells are harvested
34 to 48 hours post infection. Still more preferably, the cells are
harvested 38 to 40 hours post infection. Proteases (typically
trypsin) are generally added during cell culture to allow viral
release, and the proteases can be added at any suitable stage
during the culture.
[0047] A vaccine product including vaccine prepared from cell
culture preferably contains less than 10 ng (preferably less than 1
ng, and more preferably less than 100 pg) of residual host cell DNA
per dose, although trace amounts of host cell DNA may be
present.
[0048] It is preferred that the average length of any residual host
cell DNA is less than 5000 bp e.g. less than 400 bp, less than 300
bp, less than 200 bp, less than 100 bp, etc.
[0049] Contaminating DNA can be removed during vaccine preparation
using standard purification procedures e.g. chromatography, etc.
Removal of residual host cell DNA can be enhanced by nuclease
treatment e.g. by using a DNase. A convenient method for reducing
host cell DNA contamination is disclosed in references 43 & 44,
involving a two-step treatment, first using a DNase (e.g.
Benzonase). which may be used during viral growth, and then a
cationic detergent (e.g. CTAB), which may be used during virion
disruption. Treatment with an alkylating agent, such as
.beta.-propiolactone, can also be used to remove host cell DNA, and
advantageously may also be used to inactivate virions [45].
[0050] Influenza Vaccine Valency
[0051] Some embodiments of the invention use a monovalent influenza
vaccine (i.e. it includes hemagglutinin antigen from a single
influenza virus strain) but in some embodiments it may be a
multivalent vaccine, such as a trivalent vaccine, a tetravalent
vaccine, or a >4-valent vaccine (i.e. including hemagglutinin
from more than four different influenza virus strains). Monovalent
and multivalent vaccines are readily distinguished by testing for
multiple HA types, by amino acid sequencing, etc.
[0052] A monovalent vaccine is particularly useful for immunising
against a pandemic or potentially-pandemic strain, either during a
pandemic or in a pre-pandemic situation. Characteristics of these
strains are: (a) they contain a new hemagglutinin compared to the
hemagglutinins in currently-circulating human strains, i.e. one
that has not been evident in the human population for over a decade
(e.g. H2), or has not previously been seen at all in the human
population (e.g. H5, H6 or H9, that have generally been found only
in bird populations), such that the human population will be
immunologically naive to the strain's hemagglutinin; (b) they are
capable of being transmitted horizontally in the human population;
and (c) they are pathogenic to humans. These strains may have any
of influenza A HA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,
H11, H12, H13, H14, H15 or H16. A virus with H5 hemagglutinin type
is preferred for immunizing against pandemic influenza, or a H2, H7
or H9 subtype. The invention may protect against one or more of
influenza A virus NA subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9.
Thus possible strains include H5N1, H5N3, H9N2, H2N2, H7N1 and
H7N7, and any other emerging potentially pandemic strains. In some
embodiments, the invention does not use a monovalent vaccine based
on a H1N1 strain e.g. it does not use mouse-adapted A/PR/8/34 H1N1
strain.
[0053] A multivalent vaccine is more typical in a seasonal setting
e.g. a trivalent vaccine is typical, including hemagglutinins from
two influenza A virus strains and one influenza B virus strain,
such as from a H1N1 influenza A strain, a H3N2 influenza A virus
strain, and an influenza B virus strain. A tetravalent vaccine is
also useful [46] e.g. including antigens from two influenza A virus
strains and two influenza B virus strains, or three influenza A
virus strains and one influenza B virus strain. Thus a vaccine may
be bivalent, trivalent, tetravalent, etc. Except for monovalent
vaccines, it is usual to include hemagglutinin from both influenza
A and influenza B virus strains. In vaccines including only two
influenza A virus strains, these will usually he one H1 strain
(e.g. a H1N1 strain) and one H3 strain H3N2 strain). In some
embodiments, however, there may be one pandemic influenza A virus
strain and one H1 strain, or one pandemic influenza A virus strain
and one H3 strain.
[0054] Where a vaccine includes more than one strain of influenza,
the different strains are typically grown separately and are mixed
after the viruses have been harvested and antigens have been
prepared. Thus a process of the invention may include the step of
mixing antigens from more than one influenza strain.
[0055] As described in reference 46, exemplary tetravalent vaccines
can include hemagglutinin from two influenza A virus strains and
two influenza. B virus strains (`A-A-B-B`), or from three influenza
A virus strains and one influenza B virus strain (`A-A-A-B`).
[0056] Influenza B virus currently does not display different HA
subtypes, but influenza B virus strains do fall into two distinct
lineages. These lineages emerged in the late 1980s and have HAs
which can be antigenically and/or genetically distinguished from
each other [47]. Current influenza B virus strains are either
B/Victoria/2/87-like or B/Yamagata/16/88-like. Where a vaccine of
the invention includes two influenza B strains, this will usually
be one B Victoria/2/87-like strain and one B/Yamagata/16/88-like
strain. These strains are usually distinguished antigenically, but
differences in amino acid sequences have also been described for
distinguishing the two lineages e.g. B/Yamagata/16/88-like strains
often (but not always) have HA proteins with deletions at amino
acid residue 164, numbered relative to the `Lee40` HA sequence
[48].
[0057] Preferred A-A-B-B vaccines include hemagglutinins from: (i)
a H1N1 strain; (ii) a H3N2 strain; (iii) a B Victoria/2/87-like
strain; and (iv) B/Yamagata/16/88-like strain.
[0058] In vaccines including three influenza A virus strains, these
will usually be one H1 strain (e.g. a H1N1 strain) and two H3
strains (e.g. two H3N2 strains). The two H3 strains will have
antigenically distinct HA proteins e.g. one H3N2 strain that
cross-reacts with A/Moscow/10/99 and one H3N2 strain that
cross-reacts with A/Fujian/41 1/2002. The two H3 strains may be
from different clades (clades A, B and C of H3N2 strains are
disclosed in reference 49). In some embodiments, however, one of
these strains (i.e. H1, or one of the two H3 strains) may be
replaced by a pandemic strain.
[0059] Thus one preferred A-A-A-B vaccine includes hemagglutinins
from: (i) a H1N1 strain; (ii) a A/Moscow/10/99-like H3N2 strain;
(iii) a A/Fujian/41 1/2002-like H3N2 strain; and (iv) an influenza
B virus strain, which may be B/Victoria/2/87-like or
B/Yamagata/16/88-like. Another preferred A-A-A-B vaccine includes
hemagglutinins from: (i) a H1N1 strain, (ii) a H3N2 strain, (iii) a
H5 strain (e.g. a H5N1 strain) and (iv) an influenza B strain.
[0060] Another preferred A-A-A-B vaccine includes hemagglutinins
from: (i) two different H1 strains, (ii) a H3N2 strain, and (iii)
an influenza B strain.
[0061] Where antigens are present from two or more influenza B
virus strains, at least two of the influenza B virus strains may
have distinct hemagglutinins but related neuraminidases. For
instance, they may both have a B/Victoria/2/87-like neuraminidase
[50] or may both have a B/Yamagata/16/88-like neuraminidase. For
instance, two B/Victoria/2/87-like neuraminidases may both have one
or more of the following sequence characteristics: (1) not a serine
at residue 27, but preferably a leucine; (2) not a glutamate at
residue 44, but preferably a lysine; (3) not a threonine at residue
46, but preferably an isoleucine; (4) not a proline at residue 51,
but preferably a serine; (5) not an arginine at residue 65, but
preferably a histidine; (6) not a glycine at residue 70, but
preferably a glutamate; (7) not a leucine at residue 73, but
preferably a phenylalanine; and/or (8) not a proline at residue 88,
but preferably a glutamine. Similarly, in some embodiments the
neuraminidase may have a deletion at residue 43, or it may have a
threonine; a deletion at residue 43, arising from a trinucleotide
deletion in the NA gene, has been reported as a characteristic of
B/Victoria/2/87-like strains, although recent strains have
regained. Thr-43 [50]. Conversely, of course, the opposite
characteristics may be shared by two B/Yamagata/16/88-like
neuraminidases e.g. S27, E44, T46, P51 R65, G70, L73, and/or P88.
These amino acids are numbered relative to the `Lee40`
neuraminidase sequence [51]. Thus a A-A-B-B vaccine of the
invention may use two B strains that are antigenically distinct for
HA (one B/Yamagata/16/88-like, one B/Victoria/2/87-like), but are
related for NA (both B/Yamagata/16/88- like, or both B
Victoria/2/87-like).
[0062] In some embodiments, the invention does not encompass a
trivalent split vaccine containing hemagglutinin from each of A/New
Caledonia/20/99 (H1N1), A/Wyoming/03/2003 (H3N2) and
B/Jiangsu/10/2003 strains.
[0063] Strains whose antigens can usefully be included in the
compositions include strains which are resistant to antiviral
therapy (e.g. resistant to oseltamivir [52] and/or zanamivir),
including resistant pandemic strains [53].
Vaccines Free From Certain Additives
[0064] The preparation of vaccines without the use of certain
components and additives is disclosed in reference 54, thereby
ensuring that these materials are not present even in residual
amounts.
[0065] In some embodiments of the invention, a vaccine may include
a small amount of mercury-based preservative, such as thiomersal or
merthiolate. When present, such preservatives will typically
provide less than 5 .mu.g/ml mercury, and lower levels are possible
e.g. <1 .mu.g/ml, <0.5 .mu.g/ml. Preferred vaccines are free
from thiomersal, and are more preferably mercury-free [15,55]. Such
vaccines may include a non-mercurial preservative. Non-mercurial
alternatives to thiomersal include 2-phenoxyethanol or
.alpha.-tocopherol succinate [15]. Most preferably, a vaccine is
preservative-free.
[0066] In some embodiments, a vaccine may include a stabilising
amount of gelatin e.g. at less than 0.1%. In other embodiments,
however, a vaccine is gelatin-free. The absence of gelatin can
assure that the vaccine is safe in the small proportion of patients
who are gelatin-sensitive [56, 57].
[0067] In some embodiments, a vaccine may include one or more
antibiotics e.g. neomycin, kanamycin, polymyxin B. In preferred
embodiments, though, the vaccine is free from antibiotics. In some
embodiments, a vaccine may include formaldehyde. In preferred
embodiments, though, the vaccine is free from formaldehyde.
[0068] As mentioned above, in some embodiments a vaccine may
include egg components (e.g. ovalbumin and ovomucoid), but
preferred embodiments are free from egg components.
[0069] Where a vaccine is described herein as being free from any
particular component, the same limitation is also disclosed in
relation to patches, processes and materials of the invention.
Antigen Content
[0070] Hemagglutinin (HA) is the main immunogen in current
inactivated influenza vaccines, and.
[0071] vaccine doses are standardised by reference to HA levels,
typically measured by SRID. Existing vaccines typically contain
about 15 .mu.g of HA per strain, although lower doses can be used
e.g. for children, or in pandemic situations, or when using an
adjuvant. Fractional doses such as 1/2 (i.e. 7.5 .mu.g HA per
strain), and 1/4 and 1/8 have been used, as have higher doses (e.g.
3.times. or 9.times. doses [58,59]). These vaccines have a dosage
volume of 0.5 ml i.e. a typical HA concentration of 30
.mu.g/ml/strain. The trivalent INTANZA.TM. product contains 9 .mu.g
of HA per strain in a 0.1 ml volume i.e. a HA concentration of 90
.mu.g/ml/strain, giving a total HA concentration of 270
.mu.g/ml.
[0072] Products of the present invention can include between 0.1
and 50 .mu.g of HA per influenza strain per dose, preferably
between 0.1 and 50 .mu.g e.g. 1-20 .mu.g. Ideally a product has
.ltoreq.16 .mu.g hemagglutinin per strain e.g. 1-15 .mu.g, 1-10
.mu.g, 1-7 .mu.g, 1-5 .mu.g, etc. Particular HA doses per strain
include e.g. about 15, about 10, about 7.5, about 5, about 3.8,
about 1.9, about 1.5, etc.
[0073] For live vaccines, dosing is measured by median tissue
culture infectious dose (TCID.sub.50) rather than HA content e.g. a
TCID.sub.50 of between 10.sup.6 and 10.sup.8 (preferably between
10.sup.6.5-10.sup.7.5) per strain per dose.
[0074] Influenza strains used with the invention may have a natural
HA as found in a wild-type virus, or a modified HA. For instance,
it is known to modify HA to remove determinants (e.g. hyper-basic
regions around the HA1/HA2 cleavage site) that cause a virus to be
highly pathogenic in avian 5 species. The use of reverse genetics
facilitates such modifications.
Vaccine Products
[0075] Vaccine products of the invention can include components in
addition to the biosoluble and biodegradable matrix material and
influenza vaccine antigens.
[0076] As mentioned above, vaccine products may include a
detergent. The level of detergent can vary widely e.g. between
0.05-50 .mu.g detergent per .mu.g of HA (`.mu.g/.mu.g`). A low
level of detergent can be used e.g. between 0.1-1 .mu.g/.mu.g, or a
high level can be used e.g. between 5-30 .mu.g/.mu.g. The detergent
may be a single detergent (e.g. polysorbate 80, or CTAB) or a
mixture (e.g. both polysorbate 80 and CTAB). Preferred detergents
are non-ionic, such as polysorbate 80 (`Tween 80`) or octyl phenol
ethoxylate (`Triton XI 00`). Polysorbate 80 may be present at
between 0.05-50 .mu.g polysorbate 80 per .mu.g of HA e.g. between
0.1-1 .mu.g/.mu.g, 0.1-0.5 .mu.g/.mu.g, 5-40 .mu.g/.mu.g, or 8-25
.mu.g/.mu.g.
[0077] As mentioned above, some vaccine products may include
preservatives such as thiomersal or 2-phenoxyethanol, but preferred
vaccines are mercury- or preservative-free.
[0078] Vaccine products may include a physiological salt, such as a
sodium salt. Sodium chloride (NaCl) is preferred, which may be
present at between 1 and 20 mg/ml. Other salts that may be present
include potassium chloride, potassium dihydrogen phosphate,
disodium phosphate dehydrate, magnesium chloride, calcium chloride,
etc.
[0079] Vaccine products may include one or more buffers. Typical
buffers include: a phosphate buffer; a Tris buffer; a borate
buffer; a succinate buffer; a histidine buffer (particularly with
an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will
typically be included in the 5-20 mM range.
[0080] Vaccine products are preferably sterile. Vaccine products
are preferably non-pyrogenic e.g. containing <1 EU (endotoxin
unit, a standard measure) per dose, and preferably <0.1 EU per
dose. Vaccine products are preferably gluten-free.
[0081] Vaccine products can include immunostimulatory molecules.
These can be mixed with antigen before preparing a patch. Suitable
classes of immunostimulatory molecule include, but are not limited
to: TLR3 agonists; TLR4 agonists; TLR5 agonists; TLR7 agonists;
TLR8 agonists; TLR9 agonists; and CD1d agonists. Suitable
immunostimulatory molecules include, but are not limited to:
imidazoquinolines such as imiquimod ("R-837") [60,61] and
resiquimod ("R-848") [62], or salts thereof (e.g. the hydrochloride
salts); aminoalkyl glucosaminide phosphate derivatives, such as
RC-529 [63,64]; a-glycosylceramides, such as
.alpha.-galactosylceramide; `ER 804057` from reference 65; E5564
[66,67]; etc.
Methods of Treatment, and Administration of the Vaccine
[0082] Patches of the invention can be used to deliver an influenza
vaccine to a subject via their skin. Thus the invention provides a
method of raising an immune response in a subject, comprising the
step of applying a patch of the invention to the subject's skin,
such that the patch's microneedles penetrate the subject's
dermis.
[0083] The invention also provides a patch of the invention for use
in a method of intradermal vaccination of a subject. The invention
also provides the use of (i) a biosoluble and biodegradable matrix
material and (ii) an influenza vaccine selected from the group
consisting of a purified influenza virus surface antigen vaccine,
an influenza vaccine prepared from viruses grown in cell culture,
and a monovalent influenza vaccine, in the manufacture of a
medicament for raising an immune response in a subject.
[0084] Patches are suitable for administering vaccines to human or
non-human animal subjects
[0085] The immune response raised by these methods and uses will
generally include an antibody response, preferably a protective
antibody response. Methods for assessing antibody responses,
neutralising capability and protection after influenza virus
vaccination are well known in the art. Human studies have shown
that antibody titers against hemagglutinin of human influenza virus
are correlated with protection (a serum sample hemagglutination
inhibition titer of about 30-40 gives around 50% protection from
infection by a homologous virus) [68]. Antibody responses are
typically measured by hemagglutination inhibition, by
microneutralisation, by single radial immunodiffusion (SRID),
and/or by single radial hemolysis (SRH). These assay techniques are
well known in the art.
[0086] Patches may be applied to the skin by simple manual
application (e.g. as with a sticking plaster or with known skin
patches) or may be applied using a spring-driven injector.
[0087] Vaccines prepared according to the invention may be used to
treat both children and adults. Influenza vaccines are currently
recommended for use in pediatric and adult immunisation, from the
age of 6 months. Thus a human subject may be less than 1 year old,
1-5 years old, 5-15 years old, 15-55 years old, or at least 55
years old. Preferred subjects for receiving the vaccines are the
elderly (e.g. .gtoreq.50 years old, .gtoreq.60 years old, and
preferably .gtoreq.65 years), the young (e.g. .ltoreq.5 years old),
hospitalised subjects, healthcare workers, armed service and
military personnel, pregnant women, the chronically ill,
immunodeficient subjects, subjects who have taken an antiviral
compound (e.g. an oseltamivir or zanamivir compound; see below) in
the 7 days prior to receiving the vaccine, people with egg
allergies and people travelling abroad. The vaccines are not
suitable solely for these groups, however, and may be used more
generally in a population. For pandemic strains, administration to
all age groups is preferred.
[0088] Preferred compositions of the invention satisfy 1, 2 or 3 of
the CPMP criteria for efficacy. In adults (18-60 years), these
criteria are: (1) .gtoreq.70% seroprotection; (2) .gtoreq.40%
seroconversion; and/or (3) a GMT increase of .gtoreq.2.5-fold. In
elderly (>60 years), these criteria are: (1) .gtoreq.60%
seroprotection; (2) .gtoreq.30% seroconversion; and/or (3) a GMT
increase of .gtoreq.2-fold. These criteria are based on open label
studies with at least 50 patients.
[0089] Treatment can be by a single dose schedule or a multiple
dose schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. Administration
of more than one dose (typically two doses) is particularly useful
in immunologically naive patients e.g. for people who have never
received an influenza vaccine before, or for vaccinating against a
new HA subtype (as in a pandemic outbreak). Multiple doses will
typically be administered at least 1 week apart (e.g. about 2
weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks,
about 10 weeks, about 12 weeks, about 16 weeks, etc.).
Assays
[0090] The invention also provides assays for determining the
amount of influenza hemagglutinin in a skin patch which comprises a
biosoluble & biodegradable matrix material and an influenza
vaccine. As shown below, the matrix materials do not interfere with
an ELISA format and so this technique is suitable for analysing
patches of the invention, particularly for quantitative analysis of
HA content. A patch is first dissolved in a suitable solvent (e.g.
water or an aqueous buffer) to provide a dissolved patch solution.
The dissolved patch solution is then assayed by ELISA, for example
by a capture ELISA comprising immobilised anti-hemagglutinin
antibodies. If the patch contains a multivalent influenza vaccine
then the process may involve separate assays for each valence e.g.
by using strain- specific capture antibodies, one per strain.
[0091] After a patch is dissolved in a solvent the dissolved patch
solution can be treated to precipitate soluble proteins e.g. by
adding trichloroacetic acid (TCA), deoxycholate (DOC), acetone,
methanol, chloroform, or mixtures thereof. After precipitation the
proteins can be assayed; some analytical methods may first require
the proteins to be re-solubilised. As shown below, precipitation in
this manner can increase the recovery of protein for some
analytical purposes.
General
[0092] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0093] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0094] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.5%.
[0095] Unless specifically stated, a process comprising a step of
mixing two or more components does not require any specific order
of mixing. Thus components can be mixed in any order. Where there
are three components then two components can be combined with each
other, and then the combination may be combined with the third
component, etc.
[0096] Where animal (and particularly bovine) materials are used in
the culture of cells, they should be obtained from sources that are
free from transmissible spongiform encephalopathies (TSEs), and in
particular free from bovine spongiform encephalopathy (BSE).
Overall, it is preferred to culture cells in the total absence of
animal-derived materials.
[0097] Where a compound is administered to the body as part of a
composition then that compound may alternatively be replaced by a
suitable prodrug.
BRIEF DESCRIPTION OF THE DRAWINGS
[0098] FIGS. 1A-D shows scanning electron micrograph images of a
patch of the invention. FIGS. 1B, 1C & 1D show individual
needles from the patch shown in FIG. 1A.
[0099] FIG. 2 shows SDS-PAGE analysis of antigens, either in
solution or after formulation into a patch. Lanes are: (1) markers;
(2) 3-valent antigen at 30 .mu.g HA per strain; (3) 3-valent
antigen at 15 .mu.g HA/strain; (4) 3-valent antigen at 7.5 .mu.g
HA/strain; (5-7) monovalent HAs at 15 .mu.g; (8) empty patch after
TCA treatment; (9) patch after TCA treatment.
[0100] FIGS. 3A and 3B shows ELISA results for antigen from two
different strains. The circles show data for a trivalent vaccine.
The triangles show data with a dummy patch spiked with trivalent
vaccine. The squares show data for a patch with integral trivalent
vaccine. The crosses show a dummy patch.
[0101] FIG. 4 shows strain-specific IgG titers after immunisations.
Each of the seven triplets of bars shows titers for the three
strains in the trivalent vaccine. The triplets are, from left to
right: unadjuvanted injected vaccine at 0.1 .mu.g dose;
patch-administered vaccine at 0.1 .mu.g dose; adjuvanted injected
vaccine at 0.1 .mu.g dose; unadjuvanted injected vaccine at 0.01
.mu.g dose; patch-administered vaccine at 0.01 .mu.g dose;
adjuvanted injected vaccine at 0.01 .mu.g dose; naive mice.
[0102] FIG. 5 shows serum H1 titers. The bars are grouped as in
FIG. 4.
[0103] FIG. 6 shows strain-specific IgG titers. The five pairs of
bars show titers after 1 dose or 2 doses. The pairs are, from left
to right: unadjuvanted injected vaccine at 0.1 .mu.g dose;
patch-administered vaccine at 0.1 .mu.g dose; unadjuvanted injected
vaccine at 1 .mu.g dose; patch-administered vaccine at 1 .mu.g
dose; mice receiving PBS alone.
[0104] FIG. 7 shows serum HI titers against one vaccine strain. The
five groups are as in FIG. 6.
[0105] FIG. 8 shows % weight loss in mice after challenge. Diamonds
show data for unadjuvanted injected vaccine at 0.1 .mu.g (empty) or
lug (filled). Squares show data for patch-administered vaccine at
0.1 .mu.g (empty) or 1 .mu.g (filled). Crosses show data for mice
receiving PBS alone.
[0106] FIG. 9 shows microneutralization titers (IC80). The five
groups are as in FIG. 6.
MODES FOR CARRYING OUT THE INVENTION
Vaccine Patch Fabrication
[0107] An influenza virus vaccine was prepared using the MDCK cell
culture and antigen purification techniques used for manufacturing
the OPTAFLU.TM. product [69]. This provides a surface antigen
inactivated vaccine free from mercury, antibiotics, formaldehyde,
and egg-derived materials.
[0108] Bulk monovalent antigens from each of A/H1N1 A/H3N2 and B
strains included a high HA concentration (200-600 .mu.g/ml) with
about 0.5% w/v Tween 80. These three bulks were mixed to give a
trivalent bulk at high HA concentration. This bulk was mixed with
trehalose and sodium carboxymethylcellulose, and a microneedle
patch was prepared by filling a micromold with the mixture then
centrifuging at 4000 rpm for 5 minutes. The centrifuged material
was then dried to give the patch. Antigens were incorporated to
give a final concentration per patch of 0.0 .mu.g, 0.1 .mu.g, 1
.mu.g or 15 .mu.g of HA per strain.
[0109] FIGS. 1A-D shows scanning electron micrographs of a patch
after sputter coating with gold palladium alloy for two
minutes.
Assays for Antigen in Fabricated Patches
[0110] To confirm that vaccine antigens were properly incorporated
and stable, patches were characterized qualitatively by SDS-PAGE
and quantitatively by capture ELISA.
[0111] Patches containing trivalent antigen at 15.mu. per strain
were dissolved in 1 ml sterile water. Vials were vortexed for 10
minutes to ensure the entire patch was in solution. 100 .mu.l of
0.5% deoxycholate was added to the samples. Samples were allowed to
sit at room temperature for 10 minutes. After incubation 80 .mu.l
of 60% TCA was added to the sample. Samples were placed on
microcentrifuge for 20 minutes at room temperature at 12 k RPM. The
supernatant was removed and the pellet was dried. 60 .mu.l of
4.times. reducing loading buffer and 20 .mu.l of 1M Tris-HCl pH 8
was added to the pellet. The sample was vortexed and placed on a
heating block set at 90.degree. C. for 10 minutes. Samples were
allowed to cool to room temperature and were 9 .mu.l was added to
each well in a 4-20% SDS-PAGE gel. Gels were stained overnight,
de-stained in distilled water, and imaged. An antigen-free patch
was treated in the same way for comparison.
[0112] FIG. 2 shows results. Lanes 2-4 contain non-patch trivalent
antigen in lanes 2-4 at 2.times., 1.times. and 0.5.times. the
concentration in the patch. Lanes 5-7 show non-patch monovalent
antigens. Lane 8 shows an antigen-free patch, and lane 9 shows the
TCA-precipitated patch. The three individual antigens are clearly
visible in the patch.
[0113] Antigen content of the patches was analyzed by capture
ELISA. In this technique ELISA plates were coated to capture the
antigen. The dissolved patches were added to the plates and
incubated, followed by biotinylated IgG antibody for 30 min.
Subsequently, unbound IgG and antigen was washed off and a
streptavidin antibody conjugated to alkaline phosphatase was added.
Antigen content was then determined by enzymatic reaction with a
pNPP substrate. Absorbance was measured at 405 nm and antigen
concentration was extrapolated from antigen-specific standard
curves.
[0114] Results are shown in FIGS. 3A & 3B. The capture ELISA
was able to recover the full antigen content from patches,
confirming that the matrix excipients from the patch do not
interfere with the assay.
[0115] In contrast, mass spectrometry methods were able to recover
around 50% of the HA content. Recovery was calculated by comparing
the area of the peak in the patch sample with the area of the peak
in a standard mix sample, repeated with five different peptides for
each strain. This process was performed on patches which had been
treated with or without TCA to precipitate their proteins. Recovery
for one strain was 17% without TCA or 43% with TCA; for another
strain it was 24% without TCA or 49% with TCA. Spiking studies were
also used, and recovery was again poor (ranging from 41-55% across
three different strains). Thus mass spectrometry was not useful for
quantifying HA in the patches, presumably due to some interference
from the patch excipients.
Immunization and Challenge Studies
[0116] Patches for immunization studies had a much lower antigen
content (1, 0.1 or 0.01 .mu.g HA per strain) than the patches which
were used for antigen assays (15 .mu.g per strain).
[0117] In a first series of experiments patches were loaded at 0.1
or 0.01 .mu.g HA per strain per patch. Patches were applied to
shaved mice (female BalbC mice, 8-10 weeks old) with pressure for 3
minutes, and then removed 15 minutes later, by which time the tips
of the needles were completely dissolved. Two immunizations were
carried out 30 days apart and serum samples were collected before
the first immunization and two weeks after each immunization.
Individual serum samples were analyzed for IgG titers by ELISA
(FIG. 4) and hemagglutination inhibition (HI) titers (FIG. 5). The
results of the ELISA indicate comparable IgG titers upon
intramuscular injection of trivalent influenza vaccine or upon
patch administration at the 0.1 g dose.
[0118] In a second series of experiments patches were loaded at 0.1
or HA per strain per dose. Mice were immunised and assayed in the
same way as before. FIG. 6 shows strain-specific IgG titers, and
FIG. 7 shows HI results. In addition to these assays, two weeks
after the second immunization the animals were challenged with one
of the wild-type vaccine strains at 10 MLD.sub.50 (300,000
TCID.sub.50/mice). Animals were monitored every two days for weight
loss after challenge, and after 14 days neutralization titers were
determined to confirm protection.
[0119] FIG. 8 shows body weight. About 10-15% weight loss was
observed in the first three days after viral challenge, but mice in
the treated groups recovered within a week. In contrast, untreated
control group suffered a .about.20% weight loss and recovered only
to 97% of original weight after two weeks.
[0120] FIG. 9 shows neutralization titers, calculated as the sera
dilution at which 80% of the cells are protected against virus
infection. The titer is expressed as IC80 and calculated using a 4
parameter curve fitting. Administration of the vaccine via the
patch at 0.1 .mu.g dose resulted in neutralization titers slightly
lower than non-adjuvanted vaccine administered intramuscularly.
[0121] In conclusion, intradermal administration of influenza
vaccine by the patch induced HI titers for all three influenza
strains which were comparable to those achieved by intramuscular
administration of non-adjuvanted vaccine. This effect was seen with
HA doses as low as 0.1 .mu.g/strain. Additionally, ELISA results
indicated comparable IgG titers. In the challenge study, both
microneedle patches and non-adjuvanted influenza antigen at 0.1 and
1 .mu.g doses resulted in positive neutralization titers.
[0122] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
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