Note: Descriptions are shown in the official language in which they were submitted.
lV79634
This invention relates to antigenic preparations
and processes for their preparation,
It is known that while a variety of inactivated
viruses, for example influenza virus, are good immunogens,
they are also pyrogenic. Attempts to remove the pyro-
genicity by purifying the immunogenic moiety of the ~irus
have been successful but the resulting viral subunits do not
provide avery active immunogen. There is therefore a need
for a suitable adjuvant for such subunits and other antigenic
proteins which would boost the immune response in the
vaccinated host.
There is an increasing interest in the use of lipo-
somes as carriers of drugs and enzymes and in their potential
as immunological adjuvants. Liposomes consist of aqueous
dispersions of concentric spheres consisting of phospholipid
bilayers separated by aqueous compartments. They are
essentially onion-like structures which have been fully
described in the literature. They can be produced when a
dried film of a phospholipid such as lecithin is shaken in
the presence of a buffer.
In previous publications on the use of liposomes,
the drugs, enzymes or antigens are described as entrapped
within the internal aqueous compartments of the multi-
lamellar lipid body,
It has now been found that antigenic proteins can
be bound to the exterior surface of the outer lipid layer
of liposomes and to the exterior surface of the single
lipid bilayer of similar unilamellar bodies, and that such
preparations have excellent antigenic properties compared
with the unbound protein. Where the antigenic protein is a
viral subunit, the orientation of the subunits seen under
. . .
,~, ' ' ' . . ., .,, ,~ ~ ; . !
1079634
the electron microscope suggests that they are arranged in
same manner as on the virus particle.
Accordingly, this invention provides an antigenic
preparation containing a plurality of microvesicles; each
microvesicle comprising at least one lipid bilayer upon the
exterior surface of which is bound an antigenic protein
derived from a pathogenic organism.
In particular the invention relates to an anti-
genic preparation containing a plurality of microvesicles,
each microvesicle being unilamellar in structure and com~
prising at least one lipid bilayer upon the exterior sur-
face of which is bound an antigenic protein derived from a
virus.
In another aspect of the invention there is pro-
vided a process for preparing the preparation comprising
(a) agitating a lipid in aqueous medium to form microvesicles,
followed by agitation of the microvesicles in the presence
of an antigenic protein such that the antigenic protein
binds to the outer surface of the lipid bilayer of the
microv~sicles; or (b) agitating a mixture of lipid, an
aqueous medium and an antigenic protein such that the
antigenic protein binds to the outer surface of the
lipid bilayer of the microvesicles,
~,, 1
` ~079634
The microvesicles may themselves be liposomes
or may be unilamellar bodies comprising a single lipid bi-
layer enclosing an aqueous compartment. The antigenic
protein derived from a single species type, different
types of the same species, or different species may be
attached to the same body, or a preparation may include
mixtures of these. Each microvesicle is preferably within
the size range of 40 ~o 100 nm, though larger and smaller
bodies are also within the scope of this invention, for
example, bodies of from 20 to 200 nm.
The antigenic protein may be derived from any
pathogen, includir.g protozoa, metazoa and bacteria, but
this invention is especially concerned with antigenic
protein derived from viruses. Protective surface antigens
derived from myxoviruses, such as influenza virus A, B
and C, Newcastle disease virus, and parainfluenza types
1, 2, 3 and 4 also other viruses such as measles virus,
mumps virus, cytomegalovirus and other herpes viruses,
corona viruses and capsomeres derived from picornaviruses
such as foot-and-mouth disease viruses, poliomyelitis
viruses, rhinoviruses, wart viruses, and enteroviruses,
are especially suitable.
D
..... . .. ..
. ;, .... .... . ......
~ ,.. , . . .. .. ,. ~.
` ` 10796~4 ~470
It is believed that the protein antigens are
bound to the microvesicles by hydrophobic bonding,
and where th~ antigenic protein does not itself possess
an exposed or exposable hydrophobic region, then a
hydrophobic group may be provided, or example to
picornaviruE ci~pso~eres, by re~ction with a reage~
whicl~ provides su~h a group, for 2xample a palmityi,
stearyl, lauryl, polyalanyl group or other long aliphatic
chain.
The microvesicles may ke made of any suitable
lipid material, conveniently a~e which is itself bio-
degradable and non-antigenic. Conventional materials
such as natural or synthetic leci~hLns or other phospho-
lipids are notably useful. Ot~ lipids may also be
included such as cholesterol as a strengthener, preferably
in an amount of less than 30 m~les Sw~w of the whole lipid
oomposition. An optional third material to provide a positive
or negative charge may also be in~lu~ed. Materials which provide
a negative charge include phosp~tidic acid, beef brain
ganglioside, dicetyl phosphate, ~sphatidyl serine,
and phosphatidyl inositol; materi~ls providing a positive
ch~rge are stearvlamine and other primary amines.
The microvesicles may optionally encapsulate a second
adjuvant such as one of the customary ad3u~ants kno~n in the art.
The antigenic protein may be adacd to the lipid materials
before or after the fo~mation of the n~cro~esicles, but
prefera~ly after to eliminate the possibility of the protein being
entrapped within the bodies rather thar. being bound to the exterior suraces.
.
, : :
. .: . .. . .
. . . ,: . ,: . :. . :
-: : : :.,:, : ~ . .
A470
. 10~79~;34
Mll7ti-lamellar microvesicles (liposomes) may be made by
any ~nown method, and conveniently by dissolv;ng the lipid start~ng
material(s) in a solvent and evaporating t~e solvent. The lipid
layer is then dispersed with aqueous saline or a buffer
(if it is intended to incorporate the prcte m after microvesicle
formation) or ~ith an aqueous suspension o~ the pr~tein (if t is ~
inten~ed to incorporate the protein prior to microvesicle formati~n), ~;
and the mixture then agitated. Protein may then be added~æte
it is not already incorporated, and the microvesicles agam -~
agitated.
If unilamellar microvesicles are required, then the
initial agitation should be prolonged to diminish the number ;
of ~iposomes present.
-- .
An alternative method is to add the lipid starting
materia~) to an aqueous phase, and slowly heat the mixture.
It is then agitated to form the liposomes. The aqueous phas~
may contain the antigenic prokein or it may be added oubsequ~nt~y.
A further method of preparing microvesicles (liposo~es~
2~ comprises the ~apid injection of an etha~olic solution of
- phospholipid into aqueous saline or a buffer which has be~n pr~viouslypurg~d ~rith nitrogen. The resulting liposome preparation is then
concentrated by ultra-filtration with-rapid stirring under nitrogen
at lo~r pressure to avoid the ~or~ation of larger non-hetcrogeneous
liposomes. The ethanol may be removed from the liposome fraction by
dialysis or washing on an `ultra-filter. The antigenic protein ~hich
is intended to be bound to the liposome may be present in the aqueous
solution, or alternatively the liposome fraction obtained after ultra
filtration may be lightly sonicated with the antig~nic prote m.
- 5 -
~. .. ...
,., , , . . . .. .. ~ :.. , ,, ,, . :
107963~
~70
The antig~nic preparations obtained in the manner described
above comp~ise aqueous dispersions of the microvesicles, and these
may be for~ula~ed into vaccines hy incorporating them ~1 a
sterilised :rol~n in sealed sing]e dose or multi dose containers.
Preservatives, stabilisers an~ o.4her con~entional vaccine excipients,
if desired, may be included.
Vaccines so produced may ~ ~dministered by the methods
customarily used for the admi,l~stration of the particular antigenjc
protein or proteins in the vaccine. This usually takes the form of
nasal application, intramuscular or subcutaneous injection into the
animal (including man). The dose is of course dependent upon the nature
of the antigen, the recipient an;~al, the vaccination schedule, and the
extent of adjuvenicity coDferred by the preparation, and is of course
at the discretion of the attendant physician or veterinary surgeon,
as appropriate.
In general a dose of the vaccine may be admanistered as a single
unit, or as a multiplicity of sub-dcx~s over a period of time.
Accordingly the invention pro~es the following:-
~a) an antigenic preparation c~mprising a plurality of microvesicles
upon the exterior surface of ~hich an antigenic protein is bound; ~-
(b) a process for preparing the antigenic preparation;
(c) pharmaceutical formulations containing the antigenic preparation,
~d) method of making such pharmaceutical formulations; and
(e) a method for the prophylaxis of an infection in a
mam~al, ind uding man which comprises the administration of a
non-toxic, prophylactic amount of the antigenic ^-
preparation.
The follownng Examples are provided by wa~ of an illustration of
the present invention and should not be construed as a limitation thereof.
-- 6 --
- - ................... ; , ... . ..... . .
,... . jj . -,... ::,:. :. , '.: ' :, ',
1079634 A470
Example 1
A. _eParatton of Influ~nza Virus Subunits
Highly purified (according to the method of J.J.Skehcle
and G.C. Schield, Virology, 1971, 44, 396-408) P~8 influenza ~ `
virus at 12 mg virus protein per ml was ~xed with non-ionic detergent
~onidet NP40) to a final detergent conce~tration of 5% v/v and
layered onto 11 ml gradients of ce~ium chloride from 24 - 45% w/v
cesium chloride (in 0.05M sodium phosphate ~uffer pH 7.0) with
0.3 ~1 sucrose overlays. The gradients were centrifuged ac lG~,"OOg
for three hours or more, fractionated and the fractions assa~ve~ ~or
haemagglutinating activity. High activity fractions were pooled,
vacuNn dialysed against phosphate buffered saline(PBS) and layered `-
onto a second gradient of 20 - 60~ w/v sucrose in PBS and cenlri~uged
at lOO,OOOg for 16 hours. The fractions were again assayed fr.r
haemagglutinating activity, high titres pooled and vacuum dia_ysed
against PBS. m e final solution had a protein concentration of
1.76n~/ml and a haemagglutinating activity of approximately 1~6 HAUhnl.
For production of liposomes the subunit mixture was adjusted t~ a ~;nal
protein concentration of 2CO~g/ml.
The phosphate buffered saline (PBS) had a oo~position ~,f
sodium chloride lOg/litre; potassium chloride 0.25g/litre; potas~ium
dihydr3gen phosphate 0.25g/litre: and disodium hydrogen phc3~hate
1.4375g/litre.
.
B. Preparation of Liposcmes
Dicetyl phosphate (2.~mg) and lecithin (22.;mg) were dissoiv~d
in chloroform (approximateLy 50 ml). The solution was evaporated to
dryness on a Buchi evaporator under a slight vacuum. Viral antigen (5ml)
preparation in PBS was added to the conter~ts of the flask and the flask
was agitated manually and mechanically until the lipid was susp~nded
- - 7 -
~ ;:
- .. .: . .:: .,
- . . ,:: .: .. .. .. : :
. . ~.. ~ : :.... ..
:: ... . ..
,. .,: : .: ,. ~ ,
1079634 A470
in the fluid. The mixture was trc~nsferred to a vial and subjected to
ultrasound for 1.5 n~nutes using a 1 cm probe and 8 ~ a~plitude.
C. Mollse Protection Test
m e liposome preparations were assayed for protection.
T~Y2nty mice were divided LntO four groups of five, and ~o
groups injected intra-~eri~oneally with the viral subunits produced
as ~escribed in Example 1~ c~bove, one group being dosed with ~ g/0.25rnl
ald ~.a other with Sj~/0.2~ ne second and third groups were
s~r.-ilarily dosed with the liposo~les produoed as described in
Ex~rl~le IB above, one group re oe iving 5~g and the other 50~g per 0.25ml.
Ten days later the mice were challenged with live virus of
the came strain and the lungs harvested t~ days later. m e lungs were
hon~g~nised and sonicated to release any virus present and the
suspensiQns assayed by allantois-on-shell cultures, as described by
Fazekas de St. Groth and White (1958) Journal of Hygeine 56 151,
(195~)-
~:
The results are shown in the Table. m e 5~ug dose ofli~oscma-bound subunits produced solid protection against homologous
vir~s c~.all~nge 10 days after immunization. Only a traoe of virus
growth was detected in the lungs of one of the five mice in this
grc~p. The 5~g dose of liposom~es produced a similar protection level
to challenge as did the SO~g dose of subunits alone indicating a ten-fold
increase in the protective effect due to liposome material.
-- 8 --
" ,::: , . .
,. ... .
. : . . .: :.. ,: " . . . . .
,: - : .. , . :. ..
'` ": , ':':: ' ;
M 70
~079634
T~le
A.~TIGEN DOS~ ~50USE VIRUS LUNG 10
No. TITRATI~ (Log )
. ,
:
1 .
PR8 Subunit6l5,~g. 3 - 4.21 .
-~ . 4 . .
.. _ ,
P~B Subunlts50i~p. 8 `- 3-39
. . 9 . .-
. 10
. ,::
. 11 ' . . ! ~
Liposome6 5~g 12 - 3-4
- . (PR8 Subunitsi p 13
Suspension made . . . .
by 1/10 dilution 14
of b~low 1~ -
___ . ~'
16
Liposomes 5~g 17 -1.76
i p- 10 ~no virus growth
(PR& Subunits) . detectable
0.5~ Lipid 19 except1/8 in one
. . 20 of the mouse
. lungs).
. _ _ : .
_ g _ .
, :
- . : . : - - . :. ,. . ... ,: :.:. ., :, , -
. . -: , ,, , ,.: . :; ,. .~.,, :. : - : :: : -
. . : ,. :, . ,, ::, :. .. . :.,. :
,, ,,. . : .:
107963~ A470
Exam~]e 2
A. Dicetyl phosphate (2.5 mg) and lecithin (22.5 m~)
were dissolv~d in chloroform (approximately 50 ml).
m e soluti~,~ was evaporated to dryness as in Example 1 - -
and p~.osphat~ buffered salm e (as described above) was added
to provide a lipid concentration of 16.6 ~/ml.
rr.is mixture was sonicated for 1.5 hour in a ultrasonic bath
at a frequency of 50 K~z.
. To the resulting p~eparation was added influenza
~rus s~units~prepared as described in Example lA, to a
concentration of 200 ug/ml. This mixture was then sonicated
for a further 15 minutes in a ultrasonic bath at a
frc~g~ncy of 50 KHz.
. Examination in the electron microsccpe by the
negative stalning technique of the microscopic bodies produ oe d
acco~ding to section A above, showed ~hat the vast majority were
3mal1 unilamell æ structures slightly contaminated by larger
~ult',Jamellar structures (i.e. ~iposomes). The majori~l of the
id ~iscs were in the size range 50 to 100 nm.
. Examination in the same nenner of the influenza subunit
p,re~aration used in Example 2B above revealed only the typical star
and cartwheel forms associated with hae~agluttinin and neuraminidase
subunits. Wo trace of viral membrane could be found.
E. Examination in the same-manner of ~he preparation resulting
f,rcm the ~rocedure described in section B above, shcwed that the
majority of subunits were now arranged on the surface of the
unilamellar bodies to give an appearance very like that of m fluenza virus.
.
- 10 -
,, ~ .
;
:. , . ;, :- . . . . ~ :
. ~ . . : ~ . ~ : ....... . . : . .
: : : . .
.. . . .
~L079634 A470
In the majority of mlcroscopic bcdies, the haemagluttinin subunits
were the prom ment feature, but in so~e areas the attac~nent of
neuranuLnidase subunits cou~d be seen. In further e~amination
of the~ microscopic bodies by immune electro microscopy, the
addition of hyperimmune infl-~enza A antiserum aggrc~gated the bodies
into large complexes.
Ex p~ ~le 3
Preparation of Ethanol Li~ s~nies
Egg lecithin~30 mg)in cr,-oroform was dried in a rotary ~-~
evaForator for lz hours in partial vacuum. Ethanol(2 ml)was added
to dissolve the dried lipid ~nd the resulting solution taken up
into a 2 ml syringe fitted wi~h a 27 gauge hypodermic needle.
Potassium chloride solution (30 ml, 0.16 ~ was gassed with
nitrogen for 1 hour and the ethanolic solution of lecithin
syringed rapidly through a;27 gauge needle into the potassium
~hloride solution.
The resulting liposome ~r~-aration was concentrated by ultra-
filtration to approximately 3 n~ using an Amicon model 52 ultra-
~iltration cell fitted with a ~ 30 membrane. {Lit. ref. B.B. Acta,
298 ~1973) 1015-1019}.
Example 4
Ethanol liposo~.es prepared by the method described m
Example 3, l~ere mixed with i~n equal volume of Influenza vi~us
Xal sub-units C250~g/ml) and lightl~ sonicated.
The resulting antigenic preparation ~Tas assayed for protection
in the mouse protection test, described in Example l~c).
. .: : ::. , ,. ,. i
1079G34
A470
The results are sho~n in the follo~ing table:-
- !
~iouse Virus Lung 10
Anti~en D~se No. Titration (Log
~,
Mean A~e
1 - I.75
42~g subunits 42~g 2 - 1.75 - 1.85
on alcohol. - 3 - - I.75+ 0.22
liposomes in 0.25ml 4 ~ - 1.75
X31 5 - 2.25
:
. 16 _ 3.31
42~g X31 ;1~ ~ 2.63` - 2.95
submits 42~g 18 - 3.88+ 0.78
in P~S in 0.25ml 19 - 3.13
~ - 1.81
:
21 - 4.06
22 ~ 4-50- 4.42
unimmunized ~ 23 - 4.50` l 0.21
24 - 4.56
-`4.50
.
- 12 -
.
:, .
. . . ,, , ~ . ,
, , ::: : , , ,. ~ . . . .
lQ79634 A470
Example 5
Parenteral Solution
: .
lecithin/dicetylp}losphate (9:1, ~/~3 lmg
influenza vi~us X31 protein subunits 50~g
sodium merthiola~e 4C~g
phosphate buffered saline ~pH 7.230.2ml
phosphate buffered saline contains:-
sodium chloride lOg/litre
potassium chloride 0.25g/litre
potassium dihydrogen phosphate0.25g/litre
disodium hydrogen phosphate1.4375g/litre
~'. '' ' " '.
~ 13 -
,, . ~ ,. , ~........................... ... .. ....
- ~ .; , ~,, .. . ;. ,, . ;
1079t;34
A470
Exa~'ple 6
Di~htheria_Toxoid Incorporated Lir)osomes
A. Preparation of Liposomes
Dicetyl phosphate (3 mg) anl Lecithin (27 mg~
ere dissolved in chloroform (4 ml) and stirre~.
Phosphate buffered saline (3 ml) was added and
the flask attached to a Buchi rotary evaporator.
The aqueous and non-aqueous phases were mixed unde
magnetic stirring and rotary motion for one hour
under low vacuum (lO0 mm/Hg) at 20C. The
chloroform was then removed by increasing the vacuum.
The resulting liposome preparation was then allowed
to equilibrate for 3 hours. Diphtheria Toxoid
~0.3 ml) (9 mg/ml) (Wellcome Laboratories) was adde~,
~and the mixture was then stirred again for half an
ho1~r to incorporate the Diphtheria Toxoid onto the
surface of the liposomes. Excess toxoid was removec
by centTifugation at 40,000 g. for 2 hours at 5C -
and the liposomes were resuspended in phosphate
buffered saline (3 ml). ~Bioch~. Biophys. Acta.,
1972, Z~, 320).
.. . .
- 14 -
::. . ... ~ .. , ,. . -;
.. ., , : ... . . . .
~079~3~ : .
~470 ;
B. Effect of Diphtheria Toxoid incorporated
-Liposomes on the Immune Response'of Mice
Groups of 5-12 CBA and Porton Albino male
mice were injected subcutaneously in the back with
varying doses of Diphtheria Toxoid or with similaT
doses of Diphtheria Toxoid incorporated liposomas, ~-~
at a lipid concentration of 1~ ~w!v~. In each
test, a group of uninjected mice was included as a
negative control. The animals were bled 21 days
later and the sera absorbed to remo,ve antibody to
sheep red blood cells and a2 macroglobulin. Sera
were then assayed by haemagglutination tests using
sheep red blood Gells sensitised with diphtheria
toxoid. (J. Immunol, 1968, 10Q, No. 2, 274).
The haemagglutination titre, was taken as that
serum dilution to show distinct agglutination.
All tests were run against dilutions of a standard
horse anti-diphtheria serum as positive control
and sensitised cells with no serum as negative ,
control. The uninjected control groups always gave
a negative response. Results are expressed as means
of the reciproGal of ~he haemagglutination titres
'
: '.
'
- 1 5 - ,
.
~079634
A470
of all animals in each group and are shown in
Tables l and 2.
C. Effect of Diphtheria Toxoid incorpora*d
Liposomes on the Immune -Response of Guinea ~s
Groups of 5 Porcellus ma~e guinea pigs were
injected subcutaneously on day 0 with Diphtheria
Toxoid (0.4 ~g) or with Diphtheria Toxoid incorpora~ed
liposomes, at a lipid concentration of 1% (w/v).
The animals were bled 21 days later. Both groups
were injected on day 28 wit~ Diphtheria Toxoid
~0.4 ~g) only and bled 7 days later. Sera were tested
in haemagglutination.assays as in section B and
results expressed as mean of reciprocal of the
haemagglutination titres of all animals in each
~group. Sera taken from the guinea pigs before the :-
first injection served as negative controls and
showed no response to Diphtheria Toxoid. Results
are sho~m in Table 3.
~' .
- 16 -
~, ~ - -, , -. .. : :., . ., , . , , . .. - ,
. - .
. , ~ ... . . .
., :: . . . :: , . . ., .. , :. :. ., .,~, , .... : ,
'. : ~ ' : ~ ` : ! :
,, , , ~ ,.... ... . . . ..
1079634
A470
TABI,E
,
Haema~luti~ tion Assay of Sera from Groups of 5 CBA Male Mice
`21 days__ft.er Subcutaneous Injection of Diphtheria Toxoid or
D iPhtheria Toxoid incorporated Liposomes
.. _ j . I :
¦ Haemagglutination Titre: Reciprocal .
. _ _ ~`
Concentration :.
Diphtheria.To- Negative Diphtheria Toxoid Diphtheria Toxoid
xoid in ~g Control : only : incorporated Liposomes .
_ _ _,, : '.. ''
. : ~2 : _ _ : :
2 5 8 0 - 12.~ '
10.0 . I _ ~ 20.8 64
. ' ''' :'
- : :
~ .
~ - 17 - -
-, .: ., : . .. ,, . ~ . , .
~079634
A470 . 3
ABLE 2
Haemagglu'~ ion Assay of Sera ~ o~. Groups of 12 Porton Albino
e, bled 21 days after Subc.~taneous Injection of
heri~ Toxoid or Diphtheria Toxoid incorporated Liposomes
. .
. _ .
Haemagglutination Titre: Reciprocal
.
Concentra~ion
Diphtheria To- Negative Diphtheria Toxoi.d Diphtheria Toxoid :
xoid i.n ~g Control onlyincorporated Liposomes .
,
,. ~
-
.
-- 1~ -- . . .
,' ~
.. :. - , . ., , , , . ,, . .. ... ; ; , . -
.: . . .. :.. : . , ~ . ~, : .
, . . . . ~ . ~. .
1079~
. A470
TA~LE 3 :
. ~'
~aemaggl~ ination Assay of Sera from Groups of S Guinea Pi~s
i) 2~. ~s after first Injection ,OL Piphtheria Toxoid or
~'ntheria Toxoid incorporated Liposomes
. .
ii) 7 dayc after Booster Injection of Di~htheria Toxoid only
, .
..
. Haemagglutination Titre: Reciprocal
Concentration _ :~
. Diphtheria To- Diphtheria Toxoid . '
. xoid in ~g . Diphtheria Toxoid incorporated Liposomes ~
. :~.
i~ Prinh~ry . . ',~
Response C.4 . <2 ~2
ii) Seco~dary . .
Response _ 16 150
.
. .
;j.
19
