GB2321103A

GB2321103A - Poxvirus antigen detection and recombinant expression vector

Info

Publication number: GB2321103A
Application number: GB9700512A
Authority: GB
Inventors: David Ulaeto
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 1997-01-11
Filing date: 1997-01-11
Publication date: 1998-07-15
Also published as: GB9700512D0

Abstract

A method of detecting poxviruses in a sample, which method comprises reacting said sample with a reagent which binds a surface antigen of a poxvirus and detecting the presence of bound reagent/antigen complex. The surface antigen is suitably encoded by an essential gene of the virus. For example the P37 antigen of vaccinia or antigen encoded by homologous genes in other poxvirus strains or species, for example the antigen encoded by the C17L gene of variola. The binding reagents are suitably high affinity antibodies, generated using a specific protocol. The protocol may involve the use of recombinant expression vectors which express the poxvirus surface antigen, such as adenovirus vectors.

Description

Poxvirus Detection System The present invention relates to the detection of poxviruses and in particular Orthopoxviruses as well as to antibodies and kits used in the detection methods, and the production of said antibodies.

There are several poxviruses which are present in the environment. These include Orthopoxviruses such as monkeypox and cowpox which are rodent viruses which can be transmitted to other species. It is known that many mammalian species including sheep, goats and rabbits are subject to infection with viruses of this type. Detection systems for such viruses would be useful in diagnosis.

Other poxviruses such as vaccinia and variola are presumed to have been eliminated from the environment. However, these viruses may be kept or used in laboratories for research purposes. Vaccinia in particular is commonly used in research laboratories, usually in a recombinant form, for example as a vector for transient gene expression in cells, and as a potential vaccine vector. Systems for detecting such strains would be useful in checking containment or decontamination procedures, as well as for detecting any release, whether accidental or malicious.

According to the present invention, there is provided a method of detecting poxviruses in a sample, which method comprises reacting said sample with a reagent which binds a surface antigen of a pox virus and detecting the presence of bound reagent/antigen complex.

In view of the fact that recombinant forms of these viruses are commonly used, it is preferable that the surface antigen is encoded by a gene which is essential for the viability, replication or infectivity of said virus, since this will ensure that accidental release of infectious (rather than nonviable) viruses is detected.

A particularly suitable surface antigen is the 37,000 Da protein (VP37) of vaccinia which is encoded by the Fl3L gene or homologues thereof (Paoletti-E, et al. The complete DNA sequence of vaccinia virus. Virology 1990 Nov; 179(1): 247-66). This surface antigen has been found to be essential for the formation of extracellular enveloped virions (EEV) (B.

Moss, J. Virol.(l991) 65(11) 5910-20), the form of the virus which is implicated both in the in vivo dissemination of the virus, and the in vitro dissemination of the virus in tissue culture (L.G. Payne, J. Gen. Virol. (1980) 50(1): 89-100, B.

Moss J. Virol.(1992), 66(7), 4170-9).

The term Shomologue as used herein means that the gene encodes a protein which is similar in structure and/or function to the VP37 protein but is derived from a different strain. In general such proteins will have a related amino acid sequence, for example at least 60% of the amino acids in the sequence will be similar, and more generally at least 80% of the amino acids will be similar. For example, the corresponding protein in the variola virus is encoded by the Cl7L gene (Esposito-JJ, et al. (1994). ": Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975." : Virology 201(2): 215-40).

Other suitable surface antigens include the gp42 or pl4K antigens of vaccinia, encoded by the B5R and A27L genes respectively, or the antigens encoded by the L1R or D8L genes of vaccinia, or homologues thereof.

Suitable reagents include antibodies including both monoclonal or polyclonal antibodies, preferably monoclonal antibodies, or binding fragments thereof, such as F(ab) and Fab' fragments.

They may bind similar antigens from one or more strain or species of poxvirus since in many of the circumstances envisaged, the nature of the poxvirus if one is present, would be known. However, it may be preferred that the reagents are specific for the antigen of one particular strain of poxvirus, so that identification as well as detection can be effected.

Preferably the reagent has a high binding affinity for the surface antigen, in order to generate an assay system with an extremely low limit of detection. High affinity antibodies can be generated by using a suitable immunisation schedule. In the present case, it has been found that such a schedule involves the following steps: i) inoculating a host animal (e.g. mouse) with protein comprising a purified pox virus surface antigen, preferably a purified P37 antigen of vaccinia; and ii) after a delay of from 3 weeks to 3 months, preferably after about 2 months, inoculating the animal with a non-infectious preparation of a complete or substantially complete poxvirus; and iii) isolating antibodies and antibody producing cells from said animal.

By employing this strategy, only those antibodies which bind the native antigen, as opposed to denatured or otherwise altered forms, are boosted in step (ii).

The specificity of the antibody obtained for antigens from strains other than that used in the immunisation schedule can be checked by applying the antibody to an antigen which corresponds to that produced by the target strain and detecting binding interaction therebetween. Such testing may be carried out using virus of the target strain as the antigen source, isolated antigen obtained from the strain, or antigen obtained by recombinant methods. This latter option may be preferable where the strain is highly pathogenic, for example variola virus, as it avoids the need to handle the pathogenic strain.

In the event that the antibody obtained is not specific for the target strain, the above-described inoculation schedule can be modified by adding a further step (iia), or (iv) between steps (ii) and (iii), or after step (iii) above as follows: iia),(iv) after a further delay of from 3 weeks to 3 months, preferably after about 2 months, further inoculating the animal with a poxvirus surface antigen or a variant thereof, in particular a variola virus antigen encoded by the Cl7L gene.

The term "variant" as used herein refers to any protein which produces an immune response in a host animal and said response includes the production of agents which bind the poxvirus surface antigen. If required, this antigen may be a strain specific antigen.

This steps achieves a further boost of those antibodies which bind the target antigen.

In step (iia) or (iv) above, the antigen may be applied per se, or alternatively, an expression vector which includes a nucleotide sequence which encodes the antigen may be administered. Expression of the nucleotide sequence encoding the antigen will be under the control of regulatory elements including but not confined to a promoter, such that the vector will express the desired antigen in vivo in the animal.

Suitable vectors include virus vectors such as adenoviruses.

Suitably recombinant adenoviruses are prepared by deleting genes such as ElA/ElB and inserting a nucleotide sequence which encodes the desired surface antigen into the adenovirus genome, for example the open reading frame of the Cl7L gene of variola, preferably at the site of gene deletion. The ElA/ElB genes are transcriptional regulators and are essential for virus replication, and so the recombinant virus is defective and will neither replicate nor induce disease in the inoculated animal.

In order to propagate the defective recombinant virus, a transfected cell line which constitutively expresses the ElA/ElB functions can be used.

Other modifications to the adenovirus genome may be effected in the recombinant which make it more suitable for its application as an immunogen. For instance, the E3 gene, which is responsible for downregulating the immune response of a host in vivo may be inactivated, for example by deletion.

This may heighten the animals immune response to the recombinant antigen expressed by the defective adenovirus and thus improve antibody production.

The nucleotide sequence which encodes the desired surface antigen should be under the control of appropriate transcriptional control elements such as promoters, enhancers etc. as understood in the art. A particularly preferred promoter for use in the adenovirus vector system is the cytomegalovirus (CMV) immediate early promoter (Wilkinson and Akrigg. 1992. Nucl. Acid. Res. 20: 2233-2239).

Recombinant expression vectors which express the above described surface antigen form a further aspect of the invention. In particular the invention provides a recombinant adenovirus which comprises a nucleotide sequence which encodes the amino acid sequence which is expressed by the C17L gene of variola under the control of a CMV immediate early promoter.

Expression vectors as described above, together with cell lines containing them form a further aspect of the invention.

Antibodies (both polyclonal and monoclonal) can be obtained using this protocol using conventional techniques.

Novel reagents which specifically bind a surface antigen of a pox virus, in particular antibodies produced using the abovedescribed immunisation schedule, form further aspects of the invention Detection of bound reagent can be carried out using methods which are conventional in the art. Such methods include immunoassays including both competitive and non-competitive immunoassay techniques. In general, an entity utilised in the assay will include a detectable label. Such labels may be radiolables, enzyme labels, fluorescent labels or particulate labels such as gold, latex as would be understood in the art.

Labelled reagents will generally comprise binding reagents which either bind with or compete with the binding of the target reagent. Such reagents include antibodies or binding fragments thereof.

Binding reagents used in the assays may be immobilised on supports, also as conventional in the art.

The invention will now be particularly described by way of example.

Example 1 Purification of P37 Antigen of Vaccinia Vaccinia virus EEV or IMV virions are purified by sequential centrifugation through discontinuous sucrose, continuous sucrose, and continuous Caesium Chloride gradients. Membranes of purified virions will be removed by incubation in 58 mM Mega 8 (Sigma) at 370C overnight, and virus cores removed by microcentrifugation. After boiling for 30 minutes, or ultraviolet (uv) irradiation for 15 minutes to destroy any remaining infectious virus, the solubilized membranes in the supernatant will be dialysed against water to remove the detergent. After dialysis the samples will be concentrated by vacuum evaporation under centrifugation.

Concentrated membrane samples of EEV and IMV are subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the P37 antigen identified by comparison of EEV, IMV and molecular weight marker lanes after coomassie blue staining. The gel is dialysed against water without dialysis membrane and the band corresponding to P37 is excised from the gel, desiccated under vacuum, and dialysed twice against chloroform without dialysis membrane. After dialysis against chloroform the band is desiccated under vacuum, rehydrated in approximately 100 p1 of water, and macerated with a pestle and mortar.

Example 2 Production of Antibodies The macerated material from Example 1 is emulsified with either Freund's complete or incomplete adjuvant and used for immunisation of animals.

After a suitable period (usually around two 2 to 3 months), animals are given a booster inoculation comprising the equivalent of approximately lx106 pfu of EEV virions, inactivated (rendered non-infectious) by uv irradiation.

After a further 2 to 3 months the animals are given a tertiary boost with the equivalent of approximately lox105 pfu of uv inactivated EEV virions, by the intravenous route. 5 days after tertiary boost, animals are culled and spleens harvested for hybridoma generation in the conventional manner.

Antibodies and particularly monoclonal antibodies are generated by these hybridomas, which may be used in the detection of poxviruses.

Claims

1. A method of detecting poxviruses in a sample, which method comprises reacting said sample with a reagent which binds a surface antigen of a poxvirus and detecting the presence of bound reagent/antigen complex.

2. A method according to claim 1 wherein the surface antigen is essential for the viability, replication or infectivity of said poxvirus.

3. A method according to claim 1 or claim 2 wherein the poxvirus is an Orthopoxvirus.

4. A method according to any one of the preceding claims wherein the said surface antigen is the 37,000 Da protein (VP37) of vaccinia which is encoded by the F13L gene or homologues thereof.

5. A method according to claim 4 wherein the surface antigen is encoded by the Cl7L gene of the variola virus.

6. A method according to any one of claims 1 to 3 wherein the surface antigen is selected from the gp42 or pl4K antigens of vaccinia, encoded by the B5R and A27L genes respectively, or the antigen encoded by the L1R or D8L genes of vaccinia, or homologues thereof.

7. A method according to any one of the preceding claims wherein the said reagent comprises an antibody or a binding fragment thereof.

8. A method according to claim 7 wherein the reagent comprises a monoclonal antibody.

9. A method according to claim 7 or claim 8 wherein the antibody has a high binding affinity for the surface antigen.

10. A method of generating an antibody, which method comprises the steps of i) inoculating a host animal with protein comprising a surface antigen of a pox virus; and ii) after a delay of from 3 weeks to 3 months, inoculating the animal with a non-infectious preparation of a complete or substantially complete form of said poxvirus; and iii) isolating antibodies from said animal.

11. A method according to claim 10 wherein the poxvirus surface antigen comprises a purified P37 antigen of vaccinia.

12. A method according to claim 10 or claim 11 wherein step (ii) is effected about 2 months after step (i).

13. A method according to any one of claims 10 to 12 wherein in step (ii), a non-infectious preparation of vaccinia virus or other poxvirus is used.

14. A method according to any one of the preceding claims which further comprises the step of testing the specificity of an antibody obtained in step (iii) for an antigen of a poxvirus strain other than that used in the method.

15. A method according to any one of claims 10 to 14 wherein a further step (iia) or (iv) is effected between steps (ii) and (iii), or after step (iii) above as follows: iia) after a further delay of from 3 weeks to 3 months, further inoculating the animal with a poxvirus surface antigen or a variant thereof.

16. A method according to claim 15 wherein the said further delay is about 2 months.

17. A method according to claim 15 or claim 16 wherein the said poxvirus surface antigen is a variola virus antigen encoded by the Cl7L gene.

18. A method according to any one of claims 15 to 17 wherein the animal is inoculated with an expression vector which includes a nucleotide sequence which encodes the said poxvirus surface antigen such that the desired antigen is expressed in vivo in the animal.

19. A method according to claim 18 wherein said expression vector comprises a recombinant adenovirus.

20. A method according to claim 19 wherein the vector comprises a recombinant adenovirus which includes the cytomegalovirus (CMV) immediate early promoter which is arranged to control expression of said nucleotide sequence.

21. A recombinant expression vector which is able to express a surface antigen of a poxvirus in vivo in a mammal.

22. A recombinant expression vector according to claim 21 which comprises a recombinant adenovirus which comprises a nucleotide sequence which encodes the amino acid sequence which is expressed by the Cl7L gene of variola under the control of a CMV immediate early promoter.

23. A cell line comprising a recombinant expression vector according to claim 21 or claim 22.

24. A reagent which specifically binds a surface antigen of a poxvirus.

25. A reagent according to claim 24 which comprises an antibody or a binding fragment thereof.

26. A reagent according to claim 25 wherein the antibody is obtainable by a method according to any one of claims 10 to 20.

27. A reagent according to claim 25 or claim 26 which comprises a monoclonal antibody.