GB2037165A

GB2037165A - Vaccines

Info

Publication number: GB2037165A
Application number: GB7942733A
Authority: GB
Original assignee: Sandoz AG
Current assignee: Sandoz AG
Priority date: 1978-12-15
Filing date: 1979-12-11
Publication date: 1980-07-09
Also published as: AU5379079A; FR2443839B1; CA1153309A; PH15412A; IL58947A0; IT7951051A0; IL58947A; SE7910089L; FR2443839A1; JPS5589231A; BE880620A; NL7908936A; ZA796820B; DE2949031A1

Abstract

Vaccines, for use against viral illnesses, e.g. Herpes-, Myxo or Paramyxo-viruses, comprise viral antigen/antibody immunocomplexes.

Description

SPECIFICATION Vaccines This invention relatestothe use of immunocomplexes as vaccines, or as components thereof, against viral illnesses, in particular illnesses caused by Herpes-, Myxo- or Paramyxo-viruses.

Immunocomplexes (or antibody-antigen complexes as they are often referred to) result from the 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 the formation of these immunocomplexes, an excess of antigen 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 of antibodies against the antigen in the immunocomplex. For this purpose, an antiserum is thereby employed which stems from the same species which is to be immunised with the complex. This 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 protection against the virus from which the immuno-complex 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 antibodylsoluble antigen derived from such a virus.

The immunocomplexes for use in the vaccines of the invention, may be obtained by combining material containing 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. Sandoglobulin s) may, for example, be employed. Alternatively, donors with high antibodytitres againstthevirus 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, specificallyforthis purpose, has been inoculated with the virus in question or immunised repeatedly with an inactivated virus or with an immunocomplex vaccine in accordance with the invention.

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 purified, 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 a) Inoculated cells release a series of vi rus proteins into the culture medium. After high-speed centrifugation of the culture medium to remove virus particles and cell fragments, the released virusspecific proteins can be concentrated and isolated from the supernatant by known biochemical methods; b), c) Crude virus suspensions may be obtained either from infected cells or from their culture medium. Virus may be released from infected cells after short soaking in hypotonic buffer and subsequent homogenisation of the cells in the Dounce homogeniser, and separated from particulate residual 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 particles may be solubilised by detergent treatment, e.g. with sodium dodecylsulphate or sodium desoxycholate or with a combination of such detergents.

To obtain material c), the surface antigens, in particularthe glycoproteins, may be solubilised by detergent treatment and separated from the residual viral particles by high-speed centrifugation; d) Membranes of infected cells may be obtained by known procedures. For example, infected cells may be washed with 10-3M calcium acetate buffer in physiological saline and then suspended in 0.02M Tris.HCI buffer (pH = 7.0) with 0.01 M 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 vesicles banded in this gradient after 20 hours centrifugation at 26.0009. The membrane vesicle-containing bands are raised, diluted 4 times in Tris buffer and the material is pelleted by high-speed centrifugation. The purified cell membrane can then be solubilised with suitable detergents or chaotropic ions (e.g. 5% Triton X-100 or 2.5M quanidine HCl); 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 37"C for 10-30 minutes and subsequent ultrasonification.The solubilised material can be separated from the insoluble residue by low- and, subsequently highspeed (60 minutes, 100,0009) centrifugation.

The solubilised antigen-containing material can be combined with antibodies in conventional manner, for example by incubation with, e.g. a y-globulin preparation, followed by centrifugation of the resulting immunocomplex. Alternatively, the immunocomplexes may be produced by crossed immunoelectrophoresis in agarose gel. The quantity of antibody source, e.g. anti-sera and antigencontaining material to be employed will depend on the desired properties of the immunocomplex to be produced. In general, insoluble immunocomplexes are preferred in the vaccines of the invention and in general the preferred immunocomplexes are of equivalence or antigen excess. More preferably, an antigen quantity is used that lies slightly above the 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 allows the separation of the desired viral antigens selectively from host cell materials and viral DNA and RNA.

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 practically 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 conventional immunological adjuvants, such as aluminium hydroxide. The dosage of vaccine, or more particularly, immunocomplex, to be administered will depend on many well-known 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 corresponding 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.

A particular vaccine in accordance with the invention is a vaccine against Herpes simplex virus and having the following composition: 1. An insoluble immunocomplex containing: 100,ug 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 HSV1, mainly 2 groups of glycoproteins in the respective M.W. ranges 120,000-130,000, and 55,000-65,000) and ca. 300 yg of human y-globulin.

2. TritonX-100: maximum 1 cos.

3. Physiological saline: 1 ml.

4. Al(OH)s 0.1% (optional).

The following Examples illustrate the invention.

EXAMPLE 1 In this test, the effectiveness of vaccination with Herpes-inoculated solubilised cells is compared with that with an immunocomplex vaccine obtained from such cells.

a) Oh ten tion of y-globulin from guinea pig antisera against Herpes Simplex virus White, outbred guinea pigs are inoculated with 104 plaque-building units (pfu) of Herpes simplex virus Type 2 (HSV2) in both hind paws. 6 weeks later, the animals receive 106 pfu in the neck. 10 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 centrifugation. The serum is inactivated by incubation at 56"C for 60 minutes and the y-globulin is precipitated from the serum by addition of satu rated ammonium sulphate [20 ml serum + 10 ml saturated (NH4)2SO4] .

After centrifugation, the precipitate is taken up in 10 ml of physiological saline (0.9 /O NaCI) and the residual (NH4)2SO4 is removed by dialysis for 48 hours against NaCI solution.

b) Vaccine preparation Embryonic guinea pig fibroblasts (GPF) are inoculated with HSV2 in a multiplicity of 0.04 pfu per cell.

The cultures are incubated for 48 hours at 36"C. The cells are then separated from the culture medium by low-speed centrifugation. Ca.7 x 107 cells are suspended in 35 ml of a 0.02M Tris-glycine buffer (pH = 8.4), which contains 5% Triton-X-100 and solubilised by incubation at 37"C for 10 minutes, followed by ultrasonification (3 x 15 seconds). The undissolved cell components (mainly the nuclei) are separated from dissolved supernatant by centrifugation at 9,0009. The resulting material is referred to as "LYSATE".

10 ml of this LYSATE is mixed with 20 ml of the y-globulin preparation, produced under a), from guinea pig anti-HSV serum. The mixture is incubated for 2 hours at370C, followed by 48 hours at4"C. The resulting immunocomplexes are centrifuged off at 50,0009 for 30 minutes and this precipitate is suspended in 10 ml of physiological saline. The resulting material is referred to as "1C".

c) Animal Test LYSATE and IC are each mixed with the same volume of incomplete Freund's adjuvant. 0.2 ml of each mixture is administered per dose. 20 guinea pigs per group are vaccinated twice s.c. at an interval of3 weeks. One week after the booster injection, a blood sample is taken from the animals for antibody determination. The animals are then inoculated intravaginallywith 104pfu of HSV2. 20 nonimmunised animals are also inoculated as control.

The clinical observation of the animals follows over 62 days p.i.

Both vaccines, LYSATE and IC, induce similar titres of neutralising antibodies and similar protection against the genital challenge infection, that is the primary illness is diminished and the recurring infections are also reduced.

EXAMPLE 2: In this test, the protective effect of immunocomplexes which contain 1 to 3 defined HSV2-antigens, is investigated.

A lysate from HSV2 inoculated GPF and y-globulin from guinea pig anti-HSV-serum, are prepared as in Example 1.

The immunocomplex is obtained by crossed immunoelectrophoresis in agarose gel, as follows:10 x 10 cm glass plates are coated with 15 ml of agarose solution at 48"C. This solution contains 1.5% Indubiose A37 (L'lndustrie Biologique Francaise, Clichy), electrophoresis buffer and 0.5 ml anti HSV-y-globulin. The electrophoresis buffer (pH = 8.4) contains per 100 ml: 0.224 g Diethyl barbituric acid 0.44gTris 0.01 g Ca-lactate 0.02 9 NaN3 1.0 ml Triton-X-100.

After solidification of the agarose, holes are stamped in the lower right-hand corner of the plates, 1 cm from the edge, and filled with 15 ml of the lysate. The antigens are separated and precipitated in a two dimensional electrophoretis (1.dimension 200 V, 2 hours; 2.dimension 100 V, 12 hours).

Four different precipitates are cut out of the plates and the identical precipitates from 16 plates are pooled. The gel is pulverised in frozen state and each probe is taken up in 2 ml of phosphate-buffered NaCI-solution.

In a parallel run, radioactively marked lysate is introduced as antigen and the resulting immunoprecipitates are dissolved and analysed by SDS Polyacrylamide gel electrophoresis (Method of Norrild and Vestergaard, J. Virol. 1977,22, 113).

From this analysis, the immunocomplexes cut out of the plates contain the following HSV-2 proteins: sample 1: glycoprotein of MW 80,000-85,000 sample 2: glycoprotein of MW 78,000-80,000 sample3: non-glycosilated protein of MW 131,000 sample 4: glycoproteins of MW 57,000, 115,000-126,000.

The immunocomplex gel suspensions are each mixed with the same quantity of incomplete Freund's adjuvant, and 0.2 ml of each mixture is administered twice at a 3 week interval s.c. to guinea pigs (10 animals per group). One week after the booster injection, the guinea pigs are inoculated with 104pfu of HSV2 s.c. in the sole of the left foot and the clinical symptoms are followed for 100 days p.i.

The animals vaccinated with samples 2 or4 showed protection against the appearance of recurring Herpetic lesions.

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.

y-globulin from guinea pig anti-HSV-serum is obtained as in Example 1.

Antigen Verocells are inoculated with HSV2 at a multiplicity of 0.01 pfu/cell and incubated at 34"C 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-y-globulin is mixed with 1 ml of Alu Gel S (Serva) and absorbed for 18 hours at4 C.

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 incu bated for a further 18 hours at 4"C and all non-bound material is then removed by washing 6times in PBS.

The gel is suspended in 10 ml of PBS. This resulting suspension is designated as "Ag-Ab-Alu vaccine".

An Alu Gel absorbed lysate, designated 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 4"C, unbound material is removed by washing 6 times in PBS and the gel 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 Gel 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 HSV2.

Both vaccines induced high titres of neutralising antibodies and good protection against the cutaneous Herpetic lesions. The Ag-Alu vaccine induced in addition a high titre of antibodies against the host cells, which are detectable as complementdependent cytotoxic antibodies as well by passive cutaneous anaphylaxis. The Ag-Ab-Alu-vaccine on the other hand, induced no antibodies against host cell material.

Claims

1. A vaccine against illnesses caused by a virus, comprising an immunocomplex of antibody/soluble antigen derived from such a virus.

2. A vaccine against illnesses caused by a virus in a particular species, comprising an immunocomplex of one or more soluble specific antigens of such a virus combined with antibodies to the virus from such a species.

3. Avaccine against illnesses caused by a virus in a particular species comprising all essential specific antigens of such a virus in the substantial absence of non-essential components thereof, the antigens being in the form of immunocomplex(es) of soluble specific antigen(s) with antibodies to the virus from such a species.

4. A vaccine according to any one of the preceding claims, against illnesses caused by a Herpes virus, and comprising an antibody/soluble antigen immunocomplex of such a virus.

5. A vaccine acccording to any one of the preced ing claims, against illnesses caused by a myxo- or paramyxo-virus, and comprising an antibody/soluble antigen immunocomplex of such a virus.

6. Avaccine against illnesses caused by Herpes simplex virus (HSV), comprising an HSV antibody/soluble antigen immunocomplex comprising soluble HSV-specific antigens complexed with human anti-HSV-y-globulin, the HSV-specific antigens containing, in the case of HSV2, at least three groups of glycoproteins having respective molecular weight ranges of 120,000-130,000; 80,000-90,000 and 55,000-60,000, and in the case of HSV1 at least two groups of glycoproteins having respective molecular weight ranges of 120,000-130,000 and 55,000-60,000.

7. A vaccine according to any one of the preceding claims, in which the immunocomplex is insoluble.

8. A vaccine according to any one of the preceding claims, in which the immunocomplex is at equivalence or antigen excess.

9. A vaccine according to any one of the preceding claims, which additionally comprises an immunological adjuvant.

10. Viral antibody/soluble antigen immunocomplexes as agents for protecting against viral illnesses.

11. A method of protecting against viral illnesses comprising administering to a subject in need of such protection an effective amount of a vaccine according to any one of Claims 1 to 10.