Note: Descriptions are shown in the official language in which they were submitted.
1153309 c~s~ 9~0-~234
- VACCINF.S
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This inven~ion relates to ~he u~se of immuno-
complexes as vaccines, or as components thereof, against
viral illnesses, in particular illnesses caused by Herpes-,
~ Myxo- or Paramyxo-viruses.
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Immunocomplexes (or antibody-antigen complexes as
they are often referred to) result from tbe combination
of an antibody with its corresponding antigen. Depending
on the quantity of the components employed, soluble or
high-molecular insoluble immunocomplexes may result. For
10~ the formation of these immunocomplexes, an excess of anti-
gen is preferably employed, in particular an amount
slightly above the equivalence region of the precipitation
curve.
It is known that immunisation with immunocomplexes
leads to the formation o~ antibodies against the antigen
in the immunocomplex. For this purpose,
an antiserum is thereby employed which st.ems from the same
species which is to be .immunised with the complex. This
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method may also be employed with viral antigens for the
obtention of monospecific antibodies.
The present invention is based on the finding that
vaccination with immunocomplexes can provide effective
peotection against the virus from which the immunocomplex
is derived, and therefore against illnesses caused by this
virus.
The invention accordingly provides a vaccine against
illnesses caused by a virus comprising an immunocomplex of
an antibody/soluble antigen derived from such a virus.
The immunocomplexes for use in the vaccines of the
invention, may be obtained by combining material contain-
ing the dissolved viral antigens, with the corresponding
antibodies. The necessary antibodies are obtained from
sera of the species in which the vaccine is to be used,
obtained after natural infection by the virus, or after
immunisation. In the case of human vaccines, commercially
available human Y-globulin (e.g. Sandoglubulin ~ ) may,
for example, be employed. Alternatively, donors with high
antibody titres against the virus in question may be sought
and their sera pooled. In the case of animal vaccines (for
example against pseudorabies in pigs), the necessary sera
may be obtained from the animal species in question which,
specifically for this purpose, has been inoculated with the
virus in question or immunised repeatedly with an inactiv-
ated virus or with an immunocomplex vaccine in accordance
with the invention.
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More specifically, the invention provides a vaccine
against illness caused by a virus in a species comprising
an immunocomplex consisting of one or more soluble antigens
of such a virus combined with antibodies to the virus in
such a species.
The immunocomplexes for use in the vaccines of the
invention are, as indicated obtained in known manner by
combination of antibodies, e.g. in the form of anti-serum
or Y-globulin, with the viral soluble antigen-containing
material. The latter can involve, for example, antigens
which are soluble as such or which are solubilised, e.g.,
- a) soluble viral antigens which are secreted in the
culture medium;
b) solubilised purified, or partially puriEied, virus;
c) solubilised surface antigens of purified or partially
purified virus;
d) solubilised membrane of infected cells; or
e) solubilised infected cells.
Processes for the production of all of these materials
are well-known, for example as follows, in the case of
Herpes simplex virus for each type a) to e):-
a) In~ected cells release a series of virus proteins into
the culture medium. After high-speed centrifugation of
the culture medium to remove virus particles and all
fragments, the released virus-specific proteins can be
concentrated and isolated from the supernatant by
known biochemical methods;
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b), c) Crude virus suspensions may be obtained either from
injected cells or from their culture medium. Virus
may be released from injected cells after short soaking
in hypotonic buffer and subsequent homogenisation of
the cells in the Dounce homogeniser, and separated from
particulate cesidual components of the cells by low-
speed centrifugation. Alternatively, the virus may be
pelleted by high-speed centrifugation of the culture
medium. The virus suspension thus obtained may be
purified by centrifugation in a sucrose- or dextran-
density gradient. To obtain material b), the purified
virus particules may be solubilised by detergent
treatment, e.g. with sodium dodecylsulphate or sodium
desoxycholate. or with a combination of such deter- `
lS gents. To obtain material c), the surface antigens,
in particular the glycoproteins, may be solubilised by
detergent treatment and separated from the residual
viral particles by high-speed centrifugation;
d) Membranes of injected cells may be obtained by known
procedures, for example, injected cells may be washed
with 10 3M calcium acetate buffer in physiological
saline and then suspended in 0.02M Tris.HCl buffer (pH
= 7.0) with O.OlM EDTA. The cells are broken up in a
Dounce homogeniser and the nuclei removed by low-speed
centrifugation. Sucrose is mixed with the supernatant
to an end concentration of 45% (w/w). This solution
is introduced as the lowest layer into a discontinuous
sucrose gradient and the cell-membrane
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vesicles banded in this gradient a~ter 20 hours
centrifugiation at 26.000g. The membrane vesicle-
containing bands are raised, diluted 4 times in Tris
buffer and the material pelleted by high-speed
centrifugation. The purified cell membrane can
then be solubilised ~lith suitable detergents or
chaotropic ions (e.g. 5~ Triton X-l`00 or 2.5~5
guanldine KCl);
e) Inoculated cells are taken up in approximately the
same volume of a Tris-glycine buffer (pH = 8.4),
containing 5~ Triton or 2.5M guanidine hydrochloride,
and solubilised by incubation at 37C for 10-30
minutes and subsequent u1trasonification. The sol-
ubilised material can be separated from the insol-
uble residue by low- and, subsequently hi~h-speed
~ (60 minutes, lOO,OOOg) centrifugation.
; The solubilised antigen-containing material can be
combined witll antibodies in conventional manner, for exam-
ple by incubation with, e.g. a ~-globulin preparation,
20 followed by centrifugation of the resulting immunocomDlex.
~lternatively, the immunocomplexes may he produced by cros-
sed i~nunoelectrophoresis in agarose gel. The quantity
of antibody source, e.g. anti~sera and antigen-containing
mate~ial to be employed wi]l depend on the desired proper-
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ties of the immunocomplex to be produced. In general,
insoluble immunocomplexes are preferred in the vaccines
of the invention and in general the preferred immuno-
complexes are of equivalence or antigen excess. ~lore
preferably, an antigen quantity is used that lies slightly
above tlle equivalence range of the precipitation curve.
-~ The resulting immunocomplexes may be separated in
conventional manner from undesired residual material, e.g.
by centrifugation.
The formation of immunocomplexes in this manner
allo~s the separation of the desired viral antigens sel-
ectively from host cell materials and viral D~ and R~A.
The use of immunocomplexes is thus a new way of
producing vaccines which contain only those antigens which
are essential for protection. Such vaccines are practic-
ally free of host cell material, which can lead to
side-reactions and undesired sensibilisation. They are
also practically free of viral nucleic acids and are
therefore also indicated for potential oncogenic virus
types.
- The vaccines of the invention may be formulated in
conventional manner and may, for example, contain conven-
tional immuno]ogical adjuvants, such as aluminium hydrox-
ide. The dosage of vaccine, or more particularly, immuno-
~5 comple~,to be administered wjll depend on many ~ell~ no"n
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factors, such as the virus to be protected against, the
species to be protected, and the level of immune response
desired. In general, however, the appropriate dosage for
any particular vaccine may be determined in conventional
studies. The standard doses of conventional vaccines for
the same virus as far as these are known may be regarded
as a basis for determination of the appropriate corres-
ponding dose of the vaccines of the invention. However,
on the one hand, the vaccines of the invention may provide
a higher immunity than some conventional vaccines, so that
effectively lower dosages may be employed to obtain the
same effect.
The vaccines of the invention are suitably administered
s .c .
As indicated, vaccines of the invention are
particularly indicated for use in protecting against
illnesses caused by Herpes, Myxo- or Paramyxo-viruses,
more particularly Herpes viruses, and the corresponding
preferred vaccines therefore contain immunocomplexes
derived from these viruses.
The Herpes vaccines are particularly preferred since
purification of Herpes virus is extremely laborious and
difficult. The use of Herpes immunocomplexes permits
relatively simple purification and isolation of the
essential antigens from undesirable components.
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A particular vaccine in accordance with the invention
is a vaccine against Herpes simplex and having the
following composition:~
1. An insoluble immunocomplex containing:-
100 ~g HSV-specific proteins (comprising in the case
of HSV2 mainly 3 groups of glycoproteins in the
respective M.W. ranges 120,000-130,000,
80,000-90,000, and 55,000-65,000 and in the case of
HSVl mainly 2 groups of glycoproteins in the
respective M.W. ranges 120,000-130,000 and
55,000-65,000Jand ca. 300 ~9 of human Y-globulin.
B 2. Triton X-100; maximum 1 ~9.
3. Physiological saline: 1 ml.
4. Al~OH)3 0.1~ (optional).
The following Examples illustrate the invention.
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EXA~IPI~ l
In this test, the effectiveness of vaccination with
~ erPeSinoculated solubili~ed cells is compared with that
with an immunocomplex vaccine obtained from such cells.
a) Ob~ention of ~-globulin from guinea pi~ ant1-sera
against Herves Simple~ virus
hite ou' bred guinea pigs are inoculated with l0
plaque-building units (pfu) of I~erpes simplex virus Type 2
(HSV2) in both hind paws. 6 weeks later, the animals
receive 106 pfu in the neck. l0 days after the 2nd
immunisation, the animals' blood is removed by heart
puncture and the serum is separated from solid blood
components by low-speed centrifug2tion. The serum is
inactivated by incubation at 56C for 60 minutes and the
~-globulin is precipitated from the serum by addition of
saturated ammonium sulphate [20 ml serum ~ l0 ml satur-
ated (NH4)2SO4]. After centrifugation, the precipitate
is taken up in l0 ml of physiological saline (0.9g~ NaCl)
and the residual (NH4)2SO4 is removed by dialysis for 48
hours against NaCl solution.
b) Vaccine pre~aration
Embryonic guinea pig fibroblasts (GPF) are inocul-
ated ~ith ~ISV2 in a multiplicity of 0.04 pfu per cell.
The cultures are incubated for 48 hours at 36C. The
cells are then separated from the culture meaiu~ by lo-~-
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speed centrifugation. Ca. 7X107 cells are suspended in
35 ml of a 0.02~l Tris-glycine buffer (pll = 8.4), which
contains 5~ Triton-X-100 and solubilised by incubation
at 37C for 10 minutes, followed by ultrasonification
- 5 (3 x 15 seconds). The undissolved cell components (mainly
the nuclei) are separated from dissolved supernatant by
centrifugation at 9,000g. The resulting material is
referred to as "LYSATE".
10 ml of this LYSATE is mixed with 20 ml of the
~-globulin preparation, produced under a),from guinea pig
anti-HSV serum. The mixture is incubated for 2 hours at
37C, follot~ed by 48 hours at 4C. The resulting immuno-
complexes are centrifuged off at 50,000g for 30 minutes
and this precipitate is suspended in 10 ml of physiological
saline. The resulting material is referred to as "lC".
c) 'Animal Test
LYSATE and IC are each mixed wi,th the same volume of
incomplete Freund's adjuvant. 0.2 ml of each mixture is
administered per dose. 20 guinea pigs per group are vac-
cinated twice s.c. at an interval of 3 weeks. One weekafter the booster injection, a blood sample is taken from
the animals for antibody determination. The animals are
then inoculated intravaginally with 104pfu of HSV2~ 20
non-immunised animals are also inoculated as control. The
clinical observatioll of the animals follows over 62 days
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Both vaccines, LYS~TE and IC, induce similar titres
of neutralisinc3 antibodies and simi.lar protection against
the genital challenge infection, that is the primary
illness is diminished and the recurring infections are
also reduced.
EXA~IPLE 2:
In this test, the protective effect of immunocom-
plexes which contain 1 to 3 defined HS~72-antigens, is
investigated. ~.
A lysate from ~SV2 inoculated GPF and ~-globulin
from yuinea pig anti-HSV-serum, are prepared as in
Example 1.
The immunocomplex is obtained by crossed immuno-
electrophoresis in agarose gel, as follo~s:-
10 x 10 cm glass plates are coated with 15 ml of agarosesolution at 48C. This solution contains 1.5~ Indubiose
A37 (L'Industrie Biologi~ue Francaise, Clichy), electro-
phoresis buffer and 0.5 ml anti-HSV-~- globulin. The
electrophoresis buffer (pll = 8.4) contains per 100 ml:-
0.224 g Diethyl barbituric acid
0.44 g Tris
0.01 g Ca-lactate
0.02 g NaN3
1.0 ml Triton-X-100.
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After solidification of the agarose, holes are
stamped in the lower right-hand corner of the plates,
1 cm from the edge, and each filled with 15 ml of
the lysate~ The antigens are separated and
5precipitated in a two dimensional electrophoresis
(l.dimension 200 V, 2 hours; 2.dimension lQ0 V, 12 hours).
Four different precipita~es are cut out of the plates
and the iden~ical precipitates from 16 plates are pooled.
The gel is pulverised in frozen state and each probe is
10 taken up in 2 ml of phosphate-buffered NaCl-solution.
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In a parallel run, radioactively marked lysate is
- introduced as antigen and the resulting immuno-precipitates
v are dissolved and analysed by SDS-Polyacrylamide gel
electrophoresis (Method of Norrild and Vestergaard,
15 J. Virol. 1977, 22, 113).
From this analysis, the immunocomplexes cut out of
the plates contain the following ITr>V-2 proteins:-
,~ sample 1: glycoprotein of MW 80,000-85,000
sample 2: glycoprotein of M~ 78,000-80,000
sample 3: non-glycosilated protein of MW 131,000
sample 4: glycoproteins of MW 57,000, 115,000-126,000.
Tlle irnmunocomplex gel suspensions are each mixed with
the sarne quantity of incomplete ~reund~sacljuvant~ and 0.2n~
of each mi~:ture is administered twice at a 3 ~Jeek interval
25 s.c. to guinea picTs (10 animals per c3roup). One week after
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the booster inj~ction, the guinea pigs are inoculated ~lith
104pfu of l~SV2 s.c. in the sole of the left foot and the
clinical symptoms are followed for 100 days y.i.
The animals vaccinated with samples 2 or 4 showed protect-
ion against thc appearance ~f recurring Herpetic lesions.
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EXAMPLE 3:
In this test, the antibodies are first absorbed on
Al(OH)3 and then the viral antigens are combined with the
absorbed antibodies.
~-globulin from guinea pig anti-HSV-serum is obtained
as in Example 1.
Anti~en
Verocells are inoculated with HSV2 at a multiplicity
of 0.01 pfu/cell and incubated at 34C for 96 hours. The
cells are separated from the culture medium by low-speed
centrifugation and a lysate is prepared as in Example 1.
9 ml of anti-HSV-~-globulin is mixed with 1 ml of
Alu Gel S (Serva)and absorbed for 18 hours at 4C. Non-
absorbed protein is removed by washing 6 times with PBS.
The gel is suspended in 10 ml of PBS and mixed with 10 ml
of lysate. The mixture is incubated for a further 18
hours at 4C and all non-bound mclterial is thell removed by
washing 6 times in L'BS. The gel is susl~ended in 10 ml of
PBS. This resulting suspellsion is designated as "Ag-Ab-Alu
vaccine".
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An Alu Gel absorbed lysate, desigrlated as "Ag-Alu-
vaccine" serves as control. To obtain this, 1 ml of Alu
Gel S is diluted with 9 ml of PBS and mixed with 10 ml of
lysate. After incubation for 18 hours at 4C, u~bound
material is removed by washing 6 times in PBS and the yel
is suspended in 10 ml of PBS.
Groups of 5 guinea pigs are inoculated twice, at a
3 week interval with 0.5 ml of the respective vaccine.
5 guinea pigs act as control and receive 0.2% Alu ~el in
PBS. One week after the second immunisation, blood samples
are taken for antibody determination. Finally, the animals
are inoculated intradermally in the flank with HS~72.
Both vaccines induced high titres of neutralising
antibodies and good protection against the cutaneous
15 Herpet~c lesions. The Ag-Alu-vaccine induced in addition a
high titre of antibodies against the host cells, which
are detectable as complement-dependent cytotoxic anti-
bodies as well by passive cutaneous anaphylaxis. The
Ag-Ab-Alu-vaccine on the other hand, induced no antibodies
against host cell material.
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