EP0859846A1

EP0859846A1 - Rabies recombinant adenovirus

Info

Publication number: EP0859846A1
Application number: EP96931711A
Authority: EP
Inventors: Oksana K. Yarosh; Ludvik Prevec; Frank L. Graham
Original assignee: Microbix Biosystems Inc
Current assignee: Microbix Biosystems Inc
Priority date: 1995-10-05
Filing date: 1996-10-04
Publication date: 1998-08-26
Also published as: WO1997012981A1; AU7081896A; CA2233983A1

Abstract

Described herein is a recombinant adenovirus adapted to raise in a mammal an immune response to rabies, the adenovirus having a functionally disrupted e1 region and comprising an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host.

Description

RABIES RECOMBINANT ADENOVIRUS

TECHNICAL FIELD

The present invention relates to vaccines and more particularly to vaccines against rabies virus.

BACKGROUND ART

The rhabdovirus rabies is known to be spreading among selected wild animal species in Eastern Central North America. According to figures released in an August 1993 issue of the New York Times, 1098 people were treated for rabies in New York State in 1992 compared to only 81 in 1989.

Vaccination continues to be the only effective way to prevent or to treat rabies and other rhabdovirus infections. The objective of vaccinating wildlife species against rabies is to reduce the number of non-protected animals to the point where the disease cannot sustain itself in nature. Rabies vaccines, consisting of killed or highly attenuated rabies virus, have been available since the time of Rasteur. Attenuated rabies strains currently used to vaccinate North American wildlife fail to effectively immunize several vector species and are pathogenic in rodents. Attempts have been made to overcome these problems with limited success.

It is an object of the present invention to provide an improved rabies vaccine.

DISCLOSURE OF THE INVENTION

Briefly stated, the invention involves a recombinant adenovirus adapted to raise in a mammal an immune response to rabies, comprising a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host.

In another aspect of the invention, there is provided a vaccine for rabies virus to be administered to mammals, comprising recombinant adenovirus having a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host in an amount effective to generate an immune response in the recipient mammal, and a vehicle suitable for delivering the adenovirus to the mammal.

In still another aspect of the present invention, there is provided a method useful to immunize a mammal against rabies, comprising the step of delivering a dosage of an adenovirus sufficient to generate an immune response to rabies in the mammal, the adenovirus comprising a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host.

In still another aspect of the present invention, there is provided a method of producing a defective adenovirus vaccine comprising the steps of: culturing in an E1 producing eukaryotic host, an adenovirus having a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in the eukaryotic host,

and harvesting the replicated adenovirus. BRIEF DESCRIPTION OF THE DRAWINGS

Several preferred embodiments of the present invention will now be described, by way of example only, with reference to the appended drawings in which:

Figure 1 is a schematic representation of recombinant adenovirus construct;

Figure 2 is a schematic representation of certain plasmids involved in the preparation of the construct illustrated in Figure 1; and

Figure 3 is an autoradiogram of immuno-precipitated protein expressed by the construct of Figure 1. BEST MODE FOR CARRYING OUT THE INVENTION

The invention concerns a recombinant adenovirus of the type having a linear double stranded genome ranging in size from 30 to 40 kbp. The virion is a non-enveloped icosahedron, approximately 70 nm in diameter composed of proteins and DNA. The double stranded genome has what is known as a right strand and a left strand. In its wild type, the adenovirus is known to include early regions E1 and E3 in the right strand. A particular feature of the present recombinant adenovirus is the presence of an a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host.

'Eukaryotic hosts' include adenovirus hosts including mammalian cells including cell lines such as 293, MRC-5, MDCK and HeLa and most preferably E1 producing hosts such as 293 cells.

'Expression controls' are DNA sequences that serve to drive the transcription of the rabies glycoprotein-encoding DNA and include such functional elements as a promoter and a terminator.

Preferably, the promoter is from cytomegalovirus (CMV) and more preferably from a human cytomegalovirus (hCMV) and most desirably an hCMV immediate early (IE) promoter. While hCMV is preferable, other promoters may be used including, for example, murine cytomegalovirus immediate early promoter or adenovirus type 2 major late promoter. Suitable terminators are well known and include SV40 polyadenylation sequence and termination region.

An 'immune response' is considered 'raised' when neutralizing antibodies can be detected in serum taken within about three to four months post immunization from a recipient mammal using such detection techniques as an ELISA assay, or other suitable methods of detection such as a rabies fluorescence inhibition microtest (FIMT) described by Zalan et al., (1979 Biological Standards, vol. 7, pp 213-220).

By 'functionally disrupted' it is meant that E1 is no longer able to function in its capacity to replicate the genome. In structural terms, this may mean that, while the E1 DNA remains in the genome, it incorporates additional DNA, the presence of which prevents production of functional E1. Alternatively, this may mean that substantially all or part of the E1 DNA has been deleted.

Preferably, the expression cassette is incorporated within a cloning site available in the functionally disrupted E1 region of the adenovirus and more desirably, the early region E1 is deleted and the expression cassette is substituted therefor. However, there may be other suitable sites within the adenovirus genome in which the rabies glycoprotein-encoding DNA or the expression cassette may be inserted, so long as the insertion does not interfere with the ability of the recombinant virus to replicate in suitable cells.

Preferably, the rabies glycoprotein-encoding DNA encodes for rabies glycoprotein G, and most desirably for rabies glycoprotein G derived from ERA strain of rabies, though rabies glycoprotein-encoding DNA from other strains may also be used including those derived from arctic fox rabies variants.

The expression cassette may be formed by a number of plasmid vector routes. In a first route, the rabies glycoprotein-encoding DNA is obtained from plasmid pSV2RGla and the recombinant adenovirus is formed by ligating the rabies glycoprotein-encoding DNA into a suitable cloning site of plasmid pHMCVspl3 and co-transfecting the plasmid pHMCVspl3 with plasmid pJM17 into 293 cells. More particularly, the glycoprotein-encoding DNA and accompanying SV40 polyadenylation and termination sequences may be isolated by ligating the approximately 1.8 kb fragment obtained from plasmid pSV2RGla after complete digestion with XbaI, to form a rabies glycoprotein-encoding DNA fragment which may be gel purified and ligated into the XbaI site of pHCMVsp13 in an orientation parallel to its HCMV IE promoter. The resultant plasmid pDCHCMV13RG may then be co-transfected with pJM17 into 293 cells according to the protocol described by Graham and Prevec (Mol. Biotechnology vol. 3, 207-220 (1995)) and Hitt et al ('Methods in Molecular Genetics' Academic Press Vol. 7B, pp 13-30 (1995)), and then rescued as Ad5HMCV13RG. In a second route, the rabies glycoprotein-encoding DNA sequences may be inserted into any one of pCA3, pCA4, pCA13 or pCA14 (available from MICROBIX BIOSYSTEMS INC.) using one or more of the available cloning sites between the HCMV promoter in each and polyadenylation sequences in each of these plasmids to form a shuttle plasmid. The shuttle plasmid can then be combined with one of pJM17, pBHG10 pBHG11 , or pBHGE3 by co-transfection of 293 cells using the calcium phosphate technique (Graham and van der Eb (1973) Virology 52:456-467) to produce a recombinant Ad5HMCV13RG vector.

Though the resulting vectors from the second route have a deleted E1 region and therefore are defective, some have a disrupted E3 region, including a deleted or substituted E3 region structure. For example, when formed from pJM17, the resulting vector has an E3 region essentially identical to that produced by the first route. When formed with pBHG10 or pBHG11, the resulting vector has a deleted E3 region while with pBHGE3, the resulting vector has an intact E3 region.

In another aspect of the invention, there is provided a vaccine for rabies virus to be administered to mammals, comprising an amount of recombinant adenovirus having a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host, and a vehicle suitable for delivering said adenovirus in the mammal.

In still another aspect of the present invention, there is provided a method useful to immunize a mammal against rabies, comprising the step of delivering a dosage of an adenovirus sufficient to generate an immune response to rabies in the mammal, said adenovirus comprising a functionally disrupted E1 region and an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host.

To form the vaccine, the resulting defective recombinant adenovirus is cultured by infecting E1 -producing eukaryotic host cells such as 293 cells available from MICROBIX BIOSYSTEMS INC (Graham et al, (1977) J. General Virology, vol 36, pp 59-72). Virus is harvested from the infected cells and purified if necessary by procedures described in Graham and Prevec (Mol. Biotechnology vol. 3, 207-220 (1995)).

The aim of the vaccine is to deliver to the mammal, an amount of the recombinant adenovirus effective to generate an immune response in the mammal to rabies virus. The vaccine may be effective at a strength of 10⁶ or more plaque forming units (pfu). For instance, a strength of about 10⁸ pfu is suitable for use in vaccinating skunks. The dosage will of course otherwise vary with the animal treated and will also vary according to how the vaccine is administered and may also be effective at lower dosages.

For example, the vaccine may be injected intramuscularly, intraperitoneally, or intravenously with a suitable vehicle such as phosphate buffered saline (PBS) solution. As an alternative to injection that is acceptable for treating wild animals, the recombinant adenovirus may be administered orally by direct oral installation or by way of an edible bait material with sufficient virus in a single edible bait material, to generate a immune response in wild animals such as skunks or raccoons.

Though not intended to limit the scope of the invention, the following examples were undertaken to prepare a defective recombinant adenovirus and to test the immunogenic properties of the defective recombinant adenovirus as well as a rabies vaccine comprising the defective recombinant adenovirus.

Example 1: HAd5 E1 RABIES GLYCOPROTEIN GENE RECOMBINANT As will be described with reference to Figures 1 and 2, the vector

Ad5HCMB13RG contains the rabies glycoprotein gene driven by the human cytomegalovirus (HCMV) immediate early (IE) promoter inserted into an E1 region deletion. The expression cassette is constructed with the rabies glycoprotein-encoding DNA flanked upstream by the HCMV immediate early promoter and flanked downstream by the SV40 polyadenylation and terminator sequences. The expression cassette containing the rabies glycoprotein- encoding DNA replaces E1 region sequences from 0.9 to 9.8 map units (mu) in the adenovirus genome. As will be described, the vector Ad5HCMB13RG is derived from the following: pJM17

pJM17 (available from MICROBIX BIOSYSTEMS INC.) is a derivative of pFG140 (available from MICROBIX BIOSYSTEMS INC.), which is itself derived from the Ad5 mutant dll309 (Jones and Shenk, (1978) Cell 13:181-188; (1979) Cell 17:683-689). The detailed differences between d1309 and wild type Ad5 in the E3 region have been described by Bett et al. in Virus Research (in press, 1995) and in general terms involve s small deletion of the Ad5 E3 region and insertion of non-Ad5 DNA sequences which removes the EcoRI site at 83.6 mu and the XbaI site at 84.4 mu and a base substitution in the XbaI site at 78.3 mu. pSV2X3

pSV2X3 contains the SV40 early promoter and polyadenylation sequences separated by a multi-cloning site (HindIII BamHI EcoRI SmaI XhoI) (Prevec et al., (1990) J. Infect. Disease 161:27-30) pSV2RG1

pSV2RGl (Prevec et al., (1990) J. Infect. Disease 161:27-30) is derived from pSV2X3 and contains an approximately 1.7 kb EcoRI to BamHI fragment of rabies (ERA strain) glycoprotein G cDNA cloned into the unique SmaI site and oriented parallel to the SV40 promoter. pSV2RG1a

To form pSV2RG1a, pSV2RG1 was cut with NcoI and a self-annealed XbaI linker AB1185 (5'CATGTCTAGA 3' ) was ligated into this site. E. coli cells were transformed with the ligation mix and plasmid cDNA was screened for an additional XbaI site replacing a 120 bp NcoI fragment to form pSV2RG1a, the modifications to which were confirmed by sequencing with primer AB 1180. pHCMVsp13

pHCMVsp13 is a derivative of the generic pBR322 plasmid and contains left-end sequences of Ad5 (nt-1-5788) with an AflII/SlpI E1 region deletion (nt 342-3533). The deletion was replaced with the SlpI binding site for protein IX, the human cytomegalovirus (HCMV) immediate early (IE) promoter, and a poly-linker region. The hCMV IE promoter and poly-linker sequences were oriented so that transcription from the hCMV promoter of genes inserted into the poly-linker site is toward the left end of the resultant adenovirus vector.

Ad5HCMV13RG

Rabies glycoprotein and SV40 poly A sequences were first isolated by cutting pSV2RG1a with XbaI to form the 1.8 kb XbaI fragment, which was later gel-purified and ligated into the XbaI site of pHCMVsp13 in an orientation parallel to the HCMV IE promoter. The resultant plasmid pDCHCMVBRG was co-transfected with pJM17 into 293 cells according to the protocol developed by Graham and van der Eb ((1973) Virology 52:456-467) as described by Graham and Prevec (Mol. Biotechnology vol. 3, 207-220 (1995)). Large 150mm dishes of 293 cells were washed with IX citrate-saline, and set up into 60mm dishes for 70-80% confluence at the time of use, usually the following day. Five dishes were prepared per DNA concentration per plasmid. For the co-transfections, sufficient IX HEPES buffered saline (20 mM HEPES (nN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄ 2H₂O, 6mM glucose, final pH 7.1, autoclaved and stored at 4 degrees Celsius) was prepared with 10 μg/ml or 10 μg/0.5 ml of each plasmid used for the co-transfection, and mixed well. 2.5 M CaCl₂ was added to a final concentration of 125 mM (50 μl/ml), the tubes were inverted several times and incubated at room temperature for 15 to 30 minutes until a fine precipitate was observed. The mixture was added drop-wise thoroughly over the cells: 0.5 ml of mix per 60mm dish containing 5 ml of medium. Cells were incubated in a 5% CO₂ in air (90% humidity) incubator for 4.5 hours at 37 degrees Celsius, medium was then replaced with 10 ml of overlay of 0.5 % agarose and F11 supplemented with 5 % horse serum, penicillin-streptomycin (100 Units/ml and 100 μg/ml final concentration, respectively, 25 Units/ml nystatin suspension and 0.2% yeast extract (prepared as 5% stock and autoclaved.) Incubation was continued at 37 degrees Celsius until plaques were visible after 5-7 days. The recombinant virus was rescued as Ad5HMCV13RG. The resultant Ad5HCMV13RG carries the rabies glycoprotein gene flanked by the HCMV IE promoter and the SV40 polyA addition sequences within a deletion of the E1 region of an Ad5 virus derived from pJM17 and with modified E3 region as described above under pJM17.

All procedures for virus growth, purification or DNA isolation are carried out as described by Graham and Prevec (Mol. Biotechnology vol. 3, 207-220 (1995)) and Hitt et al ('Methods in Molecular Genetics' Academic Press Vol. 7B, pp 13-30 (1995).

Example 2: EXPRESSION OF RABIES GLYCOPROTEIN IN CELL

CULTURE

Levels of rabies glycoprotein provided by the recombinant adenovirus vector Ad5HMCV13RG was examined by immuno-precipitation of rabies glycoprotein from radio-labelled infected cell monolayers. This now well-established process is reiterated briefly in Graham and Prevec (Mol. Biotechnology vol. 3, 207-220 (1995)).

Rabies glycoprotein expression by Ad5HMCV13RG was examined in cell lines that were either able or unable to provide E1 helper function in trans. E1 region coding sequences are required in trans for Ad5HMCV13RG replication as the virus contains a deletion within E1. Cell lines 293, MRC-5, MDCK and HeLa were infected with either Wild Type hAd5 or with recombinant Ad5HMCV13RG at a multiplicity of infection (MOI) of 100 pfu/cell. The Wild Type hAd5 was strain d1309, which contains an E3 deletion which removes both the EcoRI site (83.6 mu) and XbaI site (84.8 mu) and a base substitution at the 78.3 mu XbaI site (Jones and Shenk, (1979) Cell 17:683-689).

Infected cells were metabolically labelled with [³⁵S] methionine prior to harvesting at either 9 or 25 hours post immunization. Rabies glycoprotein was immuno-precipitated with 3 μl of ascites-derived monoclonal antibodies. Although an E1 deleted vector of this type can infect all these cell types, it is unable to replicate in all cell lines except for 293. Any detectable rabies glycoprotein expression occurs as a result of the hCMV promoter and, in the absence of replication, the levels of glycoprotein expression depend on the MOL

As shown in Figure 3, rabies glycoprotein expression was detected in human 293 cells or HeLa cells or MRC-5 cells and in canine MDCK cells. As expected the highest levels of expression were obtained with 293 cells and these levels were approximately 10 to 100 fold higher than levels in cells non-permissive for virus replication. Cells, other than 293, infected with Ad5HCMV13RG, failed to exhibit a cytopathic effect for an extended period of time as compared to wild type virus, and would therefore be expected to continue to produce the glycoprotein for extended periods.

Example 3: FORMULATION OF VACCINE

Vaccine was prepared in the following manner and according to the technique described by Hitt et al. (1994 Cell Biology: A laboratory handbook, Academic Press pp 479-490). 150mm dishes of 293 cells were grown to be 80 to 90% confluent at the time of infection. The medium was removed from the

293 cells and the cells were infected with Ad5HCMV13RG at a multiplicity of infection of 1-10 PFU per cell (1 ml virus suspension per 150mm dish).

Absorption was allowed to occur for 30 to 60 minutes with a mixture of complete MEM F11 and 5% HS added thereafter. The cells were incubated at

37 degrees Celsius and cytopathic effect was initially observed after 1-2 days by the rounding of cells and cell detachment from the substrate. Thereafter the cells were examined for signs that the cytopathic effect was nearly complete which was found to be complete after 3-4 days. At this point, the cells were harvested by scraping the cells off the dish and combining the cells with spent medium and centrifuging at 800 g for 15 minutes. The medium was then aspirated the cell pellet resuspended in 2 ml PBS+ + (the + + signifying the presence of magnesium and calcium ions in solution), or alternatively PBS+ + and 10% glycerol per 150mm dish. The crude virus stock was frozen at -70 degrees Celsius and thawed at 37 degrees Celsius three times prior to titration.

Adenovirus titres were determined by assaying for viral plaques on 293 cells. Cells were set up in 60mm dishes one day prior to use so as to have the cells at about 90% confluence at the time of infection. Generally, eight 60mm dishes of 293 cells were seeded from each 150mm dish. Virus dilutions (1:10 series) were prepared in PBS+ + . Medium was removed, the cells were washed once with PBS+ + , and 0.2 ml of virus dilution was added per dish. The dishes were tilted to spread the virus evenly, and adsorption was carried out for 30- to 60 minutes in a 37 degree Celsius incubator. Overlay, 10 ml per dish, consisted of a 1:1 ratio of 1 % (w/v) agarose in sterile water and 2X MEM F11 (supplemented with 8 ml of penicillin/streptomycin solution and 25 Unit/ml nystatin suspension). On 293 cells plaques were usually visible after 5-7 days and were visually counted.

The appropriate virus concentration was then diluted in PBS for oral installation into animals.

Example 4: EVALUATION OF RABIES GLYCOPROTEIN VECTORS IN

SKULLS

Recombinant vector AdHCMV13RG was evaluated for its ability to induce an immune response in striped skunks (Mephitis mephitis). Skunks were given a 2 ml dosage of virus DIOC by direct instillation into the mouth. At biweekly or monthly intervals, post immunization serum was tested for the production of rabies virus neutralizing antibodies (VNA). VNA determinations were carried out according to an ELISA developed by Barton and Campbell ((1988) J. Wildlife Dis. 24:246-258) or by a rabies fluorescence inhibition microtest (FIMT). Purified rabies glycoprotein was used as the antigen in an ELISA Assay. Absorbance units from the ELISA assay were converted to FIMT equivalent units (E.U.) from a standard curve (Barton and Campbell, (1988) J. Wildlife Dis. 24:246-258). One FIMT E.U. was defined as the inverse of the highest twofold serum dilution resulting in a reduction of fluorescent foci of ERA strain rabies virus grown under standard conditions by at least 50 %. FIMT E.U. were divided by 46 to obtain international units (I.U.)

Within two months of the administration, all vectors stimulated the production of VNA to high levels. Titers observed in individual animals are presented in table 1. As seen in table 1 , a response was usually detected by 14 days post-immunization and maximum VNA titers measured at either 30 or 60 days. Several VNA positive animals failed to develop an immune response until day 90.

In another case, as shown in tables 2, 3 and 4, immunizations and serum analysis were carried out three groups of eight skunks. Each group was given respectively given 2 ml 10⁵, 10⁷ and 10⁸ dosages, respectively, of the recombinant vector AdHCMV13RG directly into the oral cavity (DIOC) . At biweekly or monthly intervals, post immunization serum was tested for the production of VNA.

All eight skunks given the 10⁸ dosage responded after one month post infection. Three of the eight skunks given the 10⁷ dosage responded after one month post infection, while two of the eight skunks given the 10⁵ dosage responded after three months post infection.

Claims

What is claimed is:

1. A recombinant adenovirus adapted to raise in a mammal an immune response to rabies, the adenovirus having a functionally disrupted E1 region and comprising an expression cassette in which DNA coding for rabies glycoprotein is linked operably with expression controls functional in a eukaryotic host.

2. An adenovirus as defined in claim 1 wherein the expression cassette is incorporated within said functionally disrupted E1 region.

3. An adenovirus as defined in claim 2 wherein said expression controls include a CMV promoter.

4. An adenovirus as defined in claim 3 wherein said CMV promoter is an hCMV promoter.

5. An adenovirus as defined in claim 4 wherein said hCMV promoter is an hCMV immediate early (IE) promoter.

6. An adenovirus as defined in claim 5 wherein said early region E1 is deleted and said expression cassette is substituted therefor.

7. An adenovirus as defined in claim 6 wherein said DNA codes for rabies glycoprotein G derived from ERA strain of rabies.

8. A vaccine for rabies virus comprising an amount of the adenovirus of claim 1 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

9. A vaccine for rabies virus comprising an amount of the adenovirus of claim 2 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

10. A vaccine for rabies virus comprising an amount of the adenovirus of claim 3 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

11. A vaccine for rabies virus comprising an amount of the adenovirus of claim 4 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

12. A vaccine for rabies virus comprising an amount of the adenovirus of claim 5 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

13. A vaccine for rabies virus comprising an amount of the adenovirus of claim 6 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

14. A vaccine for rabies virus comprising an amount of the adenovirus of claim 7 effective to generate an immune response in said mammal, and a vehicle suitable for delivering the adenovirus in said mammal.

15. A vaccine as defined in claim 14 wherein said vehicle is an edible bait material.

16. A mediod useful to immunize a mammal against rabies, comprising the step of delivering a dosage of the adenovirus of claim 1 sufficient to generate an immune response in said mammal to rabies.

17. A method of producing a defective adenovirus vaccine comprising the steps of culturing the adenovirus of claim 1 in an E1 producing eukaryotic host in order to promote the replication of said adenovirus, and harvesting the replicated adenovirus.