WO2001029194A1 - Porcine endogenous retrovirus (poerv) expressed in guinea pig and corresponding animal models - Google Patents

Abstract

The present invention relates to the infection of Cavia porcellus (guinea pig) with porcine endogenous retrovirus (PoERV) and uses of infected guinea pig as an animal model for amongst other things examining the kinetics and tissue profile of infection of PoERV, production of an attenuated PoERV vaccine, testing vaccines, as well as determining PoERV functioning and expression in response to immunosuppressive drugs.

Description

PORCINE ENDOGENOUS RETROVIRUS (POERV) EXPRESSED IN GUINEA PIG AND CORRESPONDING ANIMAL MODELS

The present invention relates to the infection of Cavia porcellus (guinea pig) with porcine endogenous retrovirus (PoERV) and uses of infected guinea pig as an animal model for amongst other things examining the kinetics and tissue profile of infection of PoERV, production of an attenuated PoERV vaccine, testing vaccines, as well as determining PoERV functioning and expression in response to immunosuppressive drugs.

Porcine endogenous retrovirus (PoERV) is an endogenous Gammaretrovirus present typically as a provirus found at several loci in the porcine genome where the proviral genome can be silent. Expression of virus was found associated with leukaemic pigs (Strandstrom et al . , 1974) and some continuous porcine cell lines have been shown to produce PoERV virions (Todaro et al . , 1974). The virus has been reported to infect cells from a variety of non-porcine origins including human cells and is, therefore, designated as a xenotropic, a photropic or polytropic virus (Lieber et al . , 1975; Strandstrom et al . , 1974; Todaro et al . , 1974; Galbraith et al . , 1997; Patience et al . , 1997). Three subgroups of PoERV have been described and are designated PoERV A, B and C dependent on the tropism of the virus and the related envelope gene structure (Onions et al . , 1998; Takeuchi et al . , 1998). Only subtypes A and B have been shown to be capable of consistently infecting human cells in vitro although one cell line has been reported to be susceptible to subgroup C. Since PoERV is expressed in pigs there is the potential for virus to be present in material prepared from pigs. Furthermore, as a consequence of xenotransplantation using porcine donor organs, there is the possibility that the endogenous virus will be expressed in vivo and be a potential risk of PoERV infection of the patient and the general population thereafter (Stoyle et al . , 1998). To date there is no evidence of this and in a preliminary study of 160 patients receiving porcine tissue, infection of the recipients with PoERV has not been demonstrated (Paradis et al . , 1999). Nevertheless, there remains a need for an in vivo model system of PoERV infection to allow informed risk assessment of the potential infection of porcine xenograft recipients and to allow development of prophylactic vaccines, therapeutic vaccines and anti-virals and other reagents leading to safe xenotransplantation using pigs.

It is among the objects of the present invention to provide an in vivo system that has the ability to model the situation of PoERV virion particles infecting an animal. This invention provides for the first time such an animal model for infection by an isolate of PoERV able to induce a productive infection of human cells and so provides for example a model for the safety assessment of xenotransplantation of porcine or other animal tissue harbouring an endogenous retrovirus, to humans.

According to a first aspect the present invention provides a guinea pig capable of expressing at least a portion of a porcine endogenous retrovirus (PoERV) genome. Cavia porcellus (guinea pig) is a convenient laboratory animal and characterisation of the guinea pig retrovirus indicates its genome is not closely related to those derived from a number of animal species (Michalides et al . , 1975). The present inventors therefore expected that problems of infection of guinea pigs by PoERV would be minimised and that the guinea pig may be used to raise antibodies to PoERV virions. It was somewhat surprising therefore that upon introduction of PoERV virion particles into the guinea pig that PoERV expression was observed.

"Capable of expressing at least a portion of a PoERV genome" is understood to relate to the generation of at least one peptide and/or protein from the PoERV genome. Typically the GAG, POL and/or ENV proteins of PoERV may be expressed and optionally infectious virions produced.

Without wishing to be bound by theory it is thought that the RNA PoERV genome is integrated into the genome of the guinea pig as a proviral DNA. The proviral DNA may then be transcribed and translated in order to generate PoERV peptides and/or proteins.

Thus, in a further aspect the present invention provides a genetically modified guinea pig wherein the genome of the guinea pig comprises PoERV proviral DNA capable of expression.

Preferably the guinea pig comprises the entire PoERV proviral DNA genome (ie. capable of expressing all necessary proteins for virion production) . Alternatively, the PoERV proviral DNA may only comprise a portion of the PoERV genome capable of expressing one or more PoERV peptides and/or proteins.

In a preferred embodiment, the guinea pig is infected with and is capable of expressing an isolate of PoERV shown to be capable of infecting human cells in vitro . It will be appreciated that such isolates of PoERV are of most interest because of concerns over the xenotropic nature of the virus.

In a further aspect the present invention provides a method for allowing observation of in vivo PoERV expression, comprising administrating an infectious isolate of PoERV to a guinea pig and observing any PoERV expression. This allows reagents such as immunosuppressive drug or vaccines to be added before or after PoERV administration and an effect if any on PoERV expression observed.

The PoERV guinea pig of the present invention finds use in a great many applications. For example, it may be possible to look at the kinetics of infection and the development of associated disease states, the distribution of the sites of infection and subsequent virus expression. The individual PoERV proviruses may produce virions with different properties in vivo because of differences in the long terminal repeat, primer binding site or other sequence. These sequences particularly those in the long terminal repeat could effect the leukaemogenic properties of these viruses (Neil and Onions, 1999) . Moreover the PoERV guinea pig model provides means for determining which proviruses may encode viruses of high pathogenecity. This finds utility in developing safe porcine xenotransplants or other porcine living tissue derived products where it may be advantageous to eliminate highly pathogenic proviruses from the porcine genome or to modulate their expression by means of transgenes. Other modifications of the model and/or method may be used such as immunosuppression of the animals before or during infection. For example it may be possible to determine how acquired or iatrogenic immunosuppression may affect replication, transmission, immune response to and/or pathogenicity of PoERVs in vivo .

In a further aspect the present invention provides a method for attenuation of an isolate of PoERV comprising the multiple passage of PoERV through the guinea pigs or other means to prepare an attenuated vaccine strain of PoERV.

A further additional aspect of the present invention is the use of the animal model as described herein for testing vaccines to PoERV. In one embodiment the animals can be first immunised with a potential vaccine based on attenuated or inactivated PoERV, single cycle PoERV vectors, Perv proteins, sub unit vaccines composed of recombinant PoERV proteins, or PoERV proteins expressed from viral, bacterial, naked DNA or other vector systems; and thereafter challenged with the isolated PoERV and examined for infection. Alternatively the animal model may be used to investigate the therapeutic potential of any of the abovementioned vaccines. In a further embodiment the animals being tested may be immunosuppressed during the vaccine regime in order to ascertain the effects of immunosuppression on PoERV expression.

The animal model of the present invention may be used for testing anti-viral drugs neutralising or polyclonal antibodies. For example an infection can be induced in the animal model with subsequent administration of the test drug/antibody and examination of the animals for PoERV expression. Other modifications of the regime described above may be used such that drugs/antibodies may be administered before or during viral infection or in combination with other therapeutic methods.

According to a still further aspect of the present invention is to allow testing of PoERV functions using full length and deleted or mutated recombinant PoERV nucleotide sequences embodied for example in Patent WO97/40167 (Galbraith et al . , 1997) and including polypeptides or peptides by either direct application of nucleic acids such as recombinant or other DNA or as packaged nucleic acid in a virus leading to subsequent expression of the test material in the animals. The use of such manipulated PoERV nucleic acids will allow development and testing of vaccines and clones suitable for generating PoERV knockout vectors or vectors leading to attenuation of infectivity of PoERV. An additional aspect of the present invention is for use as a diagnostic reagent in screening for the presence of infectious PoERV in a test sample where a test sample may comprise any biological tissue or body fluid (for example, cells, serum, plasma, semen, urine, saliva, sputum, cerebrospinal fluid) , and may be processed in any suitable manner in order to prepare the sample for testing. An induction step may be included prior to testing the sample in order to stimulate any retrovirus production. Other modifications of the screening may be used such that the animals may be immunosuppressed, vaccinated or subjected to a regime of chemical or other material prior to, or after inoculation of the test material. Such procedures may mimic drug regimes intended for use in the pre-operative and post-operative treatment of human, porcine-xenotransplant recipients.

The present invention will now be further described by way of non-limiting example and with reference to the following methods:

Preparation of PoERV virions

Human 293 cells (American Type Culture Collection [ATCC] # CRL1573) were infected with PoERV by exposure to polybrene (Sigma-Aldrich Co. Ltd.) and continued incubation with cell-free filtered supernatant from PK-15 (ATCC # CCL 33) cells previously shown to be infected with all three subgroups of PoERV. The 293 cells allow replication of the subgroup B of PoERV. The 293 cells were shown to be infected after passage by measurement of the reverse transcriptase activity of the cell supernatant and by a PoERV gragr-specific Polymerase Chain Reaction (PCR) (Shepherd and Smith 1999) . The resulting virus particles were isolated from the cell line supernatant as follows. Supernatant from exponentially growing cells was harvested and clarified by centrifugation at 10,000 g for 10 min followed by filtration through a 0.45 μm filter. The viral particles were pelleted by ultracentrifugation at 100,000 g for 60 min, followed by resuspension in DMEM (Life Technologies Ltd., UK).

Infection of animals

Approximately 0.5 ml containing 10⁸ virions of PoERV was injected subcutaneously into of five 42 day old Duncan Hartley strain of guinea pigs (Harlan) . After 28 the animals were re-inoculated and 14 days later the animals were killed and blood samples and spleen tissue taken for analyses.

Preparation of recombinant PoERV p30-gragr and env polypeptides

A general PoERV recombinant p30-gragr and env were designed and produced by for use as capture antigens and to produce anti-polypeptide sera. The required polypeptide portions of the gag and env genes were produced by PCR amplification, molecularly cloned into a prokaryotic expression vector and expressed as described below using standard techniques (Maniatis et al . , 1982).

PoERV p30-gagr

A fragment encompassing the p30 region of the gag ORF (PoERV gag is approximately 59.2 kDA) from nucleotide 1173- 1949 of the PoERV genome (Galbraith et al . , 1997; Gene Bank Accession # A66553) was amplified by PCR from cDNA generated from PK15 mRNA using ligation independent cloning oligonucleotide primers (pET-32 Ek/LIC cloning and expression vector; Novagen Inc. Catalogue # 69076-3) .

PoERV env

A fragment encompassing the region of the env ORF from nucleotide 5616-6304 (the PoERV env is 656 amino acids or 73.2 kDa) of the PoERV genome (Galbraith et al . , 1997; Gene Bank Accession # A66553) was amplified by PCR from cDNA generated from PK15 and PoERV-infected 293 cells mRNA using ligation independent cloning oligonucleotide primers (pET- 32 Ek/LIC cloning and expression vector; Novagen Inc. Catalogue # 69076-3) .

Recombinant PoERV protein was purified from cultures of Escherichia . coli AD494 (DE3) transformed with either of the two expression constructs. Preparations of purified gagr and env polypeptides were made according to the manufacturer's instructions (Novagen Inc. Catalogue # 69076-3) . Preparation of Western blot membranes

Recombinant p30-gagr polypeptide and env polypeptide were prepared, harvested and purified from an E. coli vector. The recombinant proteins were tested to determine an appropriate dilution of protein which yielded a positive result in the immunoassay. In addition, extracts from PoERV-infected 293 cells or purified PoERV virions were used as antigens. To obtain specific and reproducible Western blot assays, a number of parameters were required to be optimised for each assay, such as: Primary antibody dilution, incubation time, incubation temperature, secondary antibody dilution, incubation time, incubation temperature, washing buffers, blocking/dilution buffers, developing reagents. Recombinant polypeptides were added to 9 wells of a 10 lane 12% Tris/glycine acrylamide gel. Molecular weight markers were added to the first lane. The samples were electrophoresed and the gel electroblotted to a poly vinylidene fluoride (PVDF) membrane. (Gallagher, 1997; Gallagher et al . , 1997). The membrane was cut into strips each strip containing one lane of recombinant protein. These strips were used as the basis of the assay.

Preparation of Western blotting membranes and PoERV antibody detection

To block non-specific binding sites membrane strips each were placed in a 15 ml centrifuge tube and 2 ml blocking reagent (2.5 g skimmed dried milk in 50 ml PBS/ 0.05% v/v Tween 20) added. The strips were placed on a rotary shaker such that the strip moved slightly on each revolution and were incubated for 30 min at ambient temperature. The blocking reagent was removed and replaced with 5-10 μl of the diluted serum. The membrane was Incubated with shaking for 1 hour at ambient temperature. To stop incubation the strip was removed from diluted serum and placed into PBS/Tween 20 and washed with 3 changes of PBS/Tween 20 at ambient temperature with shaking.

The appropriate species-specific secondary antiserum conjugated to alkaline phosphatase was used as detector for guinea pig serum, an anti-guinea pig IgG alkaline phosphatase (AP) conjugate was used. The p30-gag positive control required anti-rabbit IgG AP conjugate for detection. The detection was done as follows; each strip was placed in an unused 15 ml centrifuge tube, 2 ml of 1:1000 dilution of secondary sera in blocking reagent was added and incubated with shaking at ambient temperature for 1 h. The strip was removed from the centrifuge tube, placed in PBS/Tween 20 and washed with 3 changes of PBS/Tween 20, at ambient temperature with shaking. The strips were then put into a 15 ml centrifuge tube and 2 ml of bromochlorindoyl phosphate/nitroblue tetrazolium (BCIP/NBT, Sigma-Aldrich Co. Ltd.) solution was added to each tube. The strips were shaken gently and allowed to develop for 5 min. The reaction was stopped by rinsing the membrane strip in purified water and the strips were removed from the water and allowed to air dry. Preparation dilutions of antisera

Samples were prepared in a Class 2 safety cabinet or other clean environments.

A typical negative control was prepared by making up to a 1:200 dilution of normal sera in blocking reagent.

A typical positive control was prepared by making a 1:500, 1:1000 or greater dilution of anti-PoERV p30-gag polypeptide serum.

A typical test serum was prepared by making up to a 1:200 dilution of sera.

PoERV RT-PCR PCR Preparation of RNA, DNA

RNA was purified from all samples using a commercial extraction kit, following the manufacturer's instructions. (RNeasy® Qiagen # 75161) , briefly, the samples were suspended in extraction buffer, the RNA was bound to a membrane, washed and eluted in purified water.

DNA was extracted from samples as described by Maniatis et al . (1982).

Precautions to prevent contamination of PCR reactions

To avoid contamination, all reagents and sterile plastic ware for RT-PCR were received, prepared and stored in a designated RT-PCR-product free area in a separate airspace from that used for RT-PCR amplifications and product analyses. The reagents treated as such include purified water, Tag polymerase, Tag polymerase reaction buffer, oligonucleotides, deoxyribonucleotides and DNA/RNA purification solutions. A one-way system was used to control the flow of materials and reagents from "clean areas" to "dirty" areas. Separate air spaces, UV treated where applicable, for reduction of potential contaminating RT-PCR amplifiable DNA (Ou et al . , 1991) were used for preparation of: reagents; negative control nucleic acids; test nucleic acids and positive control nucleic acids. Sentinel controls were used to detect airborne contamination (Saksena, 1991) . Three open reaction tubes containing all the assay components are carried through the addition of all test samples for environmental monitoring. The sentinel samples were processed with the test samples and examined for reaction product. Detection of the target sequence by RT-PCR in any of the sentinel or negative controls invalidates the test and is indicative of contamination.

Detection of PoERV proviral genome and PoERV expression

The specific oligonucleotide primers used to amplify the target region were derived from sequences on the PoERV genome which correspond to positions 1462 to 1442 (Gl) and to position 2104 to 2082 (G1A) , which are in a conserved region of the gag gene of PoERV (Galbraith et al . , 1997). RT-PCR or PCR using this primer pair amplifies a 662 bp fragment. The nested primers are derived from sequences which correspond to approximate positions 1808 to 1830 (G2) and to positions 2050 to 2069 (G2A) on the PoERV genome (Galbraith et al . , 1997). RT-PCR or PCR using this primer pair amplifies a 261 bp specific fragment.

The oligonucleotide primers used for this amplification were as follows:

Forward primer first round (Gl) S'-GCGACCCACGCAGTTGCATA-S' Forward primer second round (G2) S'-CAGTTCCTTGCCCAGTGTCCTT-S' Reverse primer first round (G1A) 5'-TGATCTAGTGAGAGAGGCAGAG-3' Reverse primer second round (G2A) 5'- CGCACACTGGTCCTTGTCG -3'.

The amplification cycle parameters were 30 cycles of 95°C 60 sec; 56°C 60 sec; 72°C 60 sec. An aliquot of the first round amplification was then added to a second round reaction mixture containing second primers. The reaction mix was amplified using the following conditions: 25 cycles consisting of denaturation at 95°C for 60 sec, annealing at 56°C for 60 sec and extension for 60 sec at 72°C, followed by a final extension at 72°C for 10 min.

Detection of spliced PoERV mRNA from guinea pig spleen

To distinguish between PoERV RNA in a virion and messenger RNA mRNA transcribed in the guinea pig an RT-PCR was used to detect the spliced env mRNA. The splice donor and acceptor sites were identified at positions 201 and 5385 respectively on the PoERV genome. The upstream oligonucleotide primer is from position 1-18 and the downstream primer is from position 5892-5909 on the PoERV genome. The primers would amplify a fragment of approximately 507 bp from a spliced mRNA and 5909 bp from PoERV genomic RNA or proviral DNA.

The oligonucleotide primers used for this amplification were as follows:

Upstream of splice donor 5 • -GTGGTGTACGACTGTGGC-3 '

Downstream of splice acceptor 5 ' -GCGGGGTTAATCAATCGG-3 •

The reverse transcription reaction was done as following the manufacturer's instructions (Superscript ™ Preamplification System; Life Technologies # 18089-011) . The test RNA was added to random hexamers in sterile water and heated to 70°C for 10 minutes. RT master mix was then added (dNTPs, reverse transcriptase buffer, MgCl₂ and DTT) and heated to 42°C for 5 minutes. After incubation the RT enzyme was added and the reaction mix was incubated at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min; RNase H was then added to the reaction mix and incubated at 37°C for 15 min. The cDNA was subjected to PCR.

The PCR cycle parameters were 95°C 1 min; 55°C 1 min; 72°C 1 min for 30 cycles.

Electrophoresis of PCR products

Aliquots of the finished reactions were electrophoresed through 5% (v/v) acrylamide gels, stained in ethidium bromide and examined and photographed under UV light (Maniatis et al . , 1982). EXAMPLE ONE Western Blotting for Antibodies to PoERV gag and PoERV env

Sera from guinea pigs inoculated with the PoERV detected the expected polypeptides of approximately 73 kd, 59 kd and 30 kd in extracts of PoERV-infected 293 or purified PoERV virions Recombinant p30-gag and recombinant env was also detected. The PoERV antibody could be detected at a dilution of 1/2000. No band of equivalent size to the gag or env polypeptide was detected in uninfected control cells. Serum from uninfected control animals did not detect PoERV viral-specific polypeptides. Therefore, these data show that PoERV can elicit an immune response in guinea pigs.

In the gammaretroviruses like PoERV the onset of anti- env antibodies is often associated with the transition from a productive infection with viraemia to a latent infection. Recrudence frokm latent infection can however be associated with subsequent immunosuppression. (Onions, 1994) .

EXAMPLE 2

Detection of PoERV Proviral DNA

The amplimers (Gl and G1A) amplified the expected fragment of approximately 662 bp in size from RNA from PoERV-infected cells and the DNA extracted from the spleens of the PoERV-inoculated guinea pigs. The nested primers (G2) and (G2A) amplified the expected fragment of 261 bp from DNA from the same samples. There was no evidence of amplification of a fragment from samples from uninoculated negative control animals, demonstrating that there were no sequences in the guinea pig genome that could be amplified by the PoERV-specific oligonucleotide primers.

Therefore, these data indicate the presence of PoERV proviral DNA in spleens of PoERV-inoculated animals and demonstrates that the endogenous guinea pig retrovirus does not interfere with infection of guinea pig cells by PoERV.

EXAMPLE 3

Detection of PoERV expression

The oligonucleotides allowed the amplification of the expected fragment of 507 bp in size from RNA from PoERV- infected cells and RNA from spleens of the animals inoculated with PoERV. There was no evidence of amplification of a fragment from negative control samples including samples without a prior reverse transcriptase treatment.

Therefore, these data indicate that the PoERV genome is expressed in the tissue of the guinea pigs.

REFERENCES

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Claims

1. A guinea pig capable of expressing at least a portion of a porcine endogenous retrovirus (PoERV) genome.

2. The guinea pig according to claim 1, wherein the guinea pig has been infected with PoERV virion particles.

3. A genetically modified guinea pig wherein the genome of the guinea pig comprises PoERV proviral DNA capable of expression.

4. The genetically modified guinea pig according to claim 3 wherein the genome of the guinea pig comprises the entire PoERV proviral DNA genome.

5. The genetically modified guinea pig according to claim 3 wherein the genome of the guinea pig comprises a portion of the PoERV genome capable of expression one or more PoERV peptides and/or proteins.

6. The guinea pig according to any preceding claim wherein the PoERV genome or portion thereof is obtained from an isolate of PoERV shown to be capable of infecting human cells in vitro .

7. A method for allowing observation of in vivo PoERV expression, comprising administering an infectious isolate of PoERV to a guinea pig and observing any PoERV expression.

8. The method according to claim 7 further comprising the step of adding a reagent, such as an immunosuppressive drug or vaccine, before or after PoERV administration in order that any effect on PoERV expression due to administration of the reagent is observed.

9. Use of the guinea pig according to any one of claims 1 to 6 in the study of the kinetics of infection, the development of associated disease states and/or the distribution of the sites of infection and subsequent virus expression.

10. Use of the guinea pig according to any one of claims 1 to 6 as a means for determining which proviruses may encode viruses of high pathogenicity.

11. Use according to either of claims 9 or 10 wherein the guinea pig is immunosuppressed before or during infection.

12. Use of the guinea pig according to any one of claims 1 to 6 for testing vaccines to PoERV.

13. Use according to claim 12 wherein the guinea pig is first immunised with a potential vaccine based on attenuated or inactivated PoERV, single cycle PoERV vectors, Perv proteins, sub unit vaccines composed of recombinant PoERV proteins, or PoERV proteins expressed from viral, bacterial, naked DNA or other vector systems; and thereafter challenged with the isolated PoERV and examined for infection.

14. Use according to claim 13 wherein the guinea pig is immunosuppressed during the vaccine regime.

15. Use of the guinea pig according to any one of claims 1 to 6 for testing anti-viral drugs, neutralising or polyclonal antibodies.

16. Use according to claim 15 wherein the anti-viral drugs, neutralising or polyclonal antibodies are administered before or during viral infection or in combination with other therapeutic methods.

17. Use of the guinea pig according to any one of claims 1 to 6 as a diagnostic reagent in screening for the presence of infectious PoERV in a test sample, wherein said test sample comprises a biological tissue or body fluid selected from the group consisting of cells, serum, plasma, semen, urine, saliva, sputum and cerebrospinal fluid.

18. Use according to claim 17 wherein an induction step is included prior to testing the sample in order to stimulate any retrovirus production.

19. Use according to claim 17 or 18 wherein the guinea pig is immunosuppressed, vaccinated or subjected to a regime of chemical or other material prior to, or after inoculation of the test material.

20. A method for attenuation of an isolate of PoERV comprising the multiple passage of PoERV through a guinea pig to prepare an attenuated vaccine strain of PoERV.