CA1153309A

CA1153309A - Vaccines

Info

Publication number: CA1153309A
Application number: CA000341913A
Authority: CA
Inventors: Bent F. Vestergaard; Ekke Liehl; Marianne Scriba
Original assignee: Sandoz AG
Current assignee: Sandoz AG
Priority date: 1978-12-15
Filing date: 1979-12-13
Publication date: 1983-09-06
Also published as: AU5379079A; BE880620A; IL58947A; SE7910089L; FR2443839B1; JPS5589231A; IL58947A0; IT7951051A0; NL7908936A; FR2443839A1; DE2949031A1; GB2037165A; ZA796820B; PH15412A

Abstract

Case 900-9234 VACCINES
Abstract of the Disclosure Viral antigen/antibody immunocomplexes for use as vaccines against viral illnesses.

Description

1153309 c~s~ 9~0-~234 - VACCINF.S
: ' ' 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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' ~lS3309 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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~153309 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-:

~1533~)9 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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1533~)9 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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` ~53309 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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- ~1533~)9 ` - 9 - 900-9234 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 p.i .

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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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~i3309 - 13 - ~00-9234 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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, ---- ~153309 sno-s234 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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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing a vaccine against illnesses caused by Herpes, Myxo- or Paramyxo-viruses, which comprises combining material containing dissolved viral antigens derived from Herpes, Myxo- or Paramyxo-virus, with the corresponding antibodies; to form an immuno-complex thereof and separating the immunocomplex thus obtained.

2. A process according to claim 1 wherein the antibodies are in the form of anti-serum or .gamma.-globulin.

3. An immunocomplex of a soluble antigen of a Herpes, Myxo- or Parmyxo-virus combined with antibodies to the virus, whenever produced by the process according to claim 1 or 2 or an obvious chemical equivalent.