EP0354213A1 - Transgenic animal cells resistant to viral infection - Google Patents

Abstract

Methods are used to produce animal cells resistant to viral infections. In particular, animal cells transformed with the gp50 glycoprotein encoding the genes of the pseudo-rabies virus are resistant to infections by herpes viruses, in particular the herpes simplex virus and the pseudo-rabies virus. .

Description

  
 



     TRAh'SCw IC    ANIMAL CELLS RESISTANT TO VIRAL INFECTION
FIELD OF   INV95-rION   
 This invention relates to transgenic animal cells. More particularly,   the    invention relates to transgenic animal cells resistant to   DiA    virus infection, and methods for producing such cells.



  BACKGROUND OF THE INVENTION
 Retroviruses characteristically induce a variety of neoplastic diseases and are very widely distributed among vertebrate species.



  They are recognized by their morphology, the structure of their RNA viral genomes, and their RNA-dependent DNA polymerase (reverse transcriptase) (D.R. Lowy, "Transformation and Oncogenesis: Retroviruses", in   i'irology,    B.N Fields, et al., eds., Raven Press,    NY    (1985)).



   The retrovirus life cycle involves integration of the viral genome into the host genome (proviruses) which often leads to expression of viral antigens. It has long been known that infection of a cell line with one retrovirus will interfere with subsequent infection by other viruses in the same serogroup (P.K Vogt and R.



  Ishizaki, Virology, 30, pp. 368-74, (1966)). This effect was shown to be due to interference with the early events in infection, such as adsorption of the retrovirus to cells and cell penetration (F.T.



  Steck and H. Rubin, Virology, 29, pp. 642-53, (1965)) and is commonly called "interference".



   H.L. Robinson et al., J. Virol., 40, pp. 745-51 (1981) demonstrated that interference by the endogenous retrovirus with infection by exogenous retroviruses of the same serogroup is due to expression of a viral glycoprotein. Robinson et al. showed that the synthesis of viral glycoprotein slowed penetration by exogenous retrovirus.



  Robinson et al. also demonstrated that interference reduced the replication of retroviruses in chickens in which the viral gene product was expressed.



   The retrovirus interference phenomenon is not unique to chickens. The resistance of DBA/2 mice to mink cell focus-inducing virus is due to expression of a mouse gene encoding a viral glycoprotein (S. Ruscetti et al., J. Exp. Med., 154, pp. 907-20 (1981));   R.H.    Bassin et al., Virology, 123, pp. 139-51 (1982)). The Fv-4 gene that makes   xice    resistant to murine leukemia retroviruses also  encodes a viral glycoprotein (K. Kai et al., virology, 150, pp. 50912 (1986)).



   A phenomenon similar in effect to interference has also been demonstrated in plants. Plants susceptible to a virus are made resistant by prior infection with a mild strain of the same virus.



  In plants the phenomenon is known as "cross protection*. The mechanism by which cross protection occurs is unknown but it probably does not involve insertion of viral cDNA into the host genome (P.



  Palukaitis and M. Zaitlin, "A Model to Explain the 'Cross-Protection'
Phenomenon Shown by Plant Viruses and   Viroidst,    pp. 420-29, in Plant   Hicrobe      Interactior)s,    T. Kosuge and E.W. Nester, eds., Macmillan,
Inc. (1984)).



  INFORMATION DISCLOSURE STATEMENT
 P.P. Abel et al., Science, 232, pp. 738-43 (1986) refer to plants made resistant to tobacco mosaic virus (TMV) by expression of the TMV coat protein gene cDNA cloned into the tobacco plant's genome. Seedlings that expressed the coat protein gene were delayed in symptom development upon subsequent infection with TMV and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments.



   Robinson et al., supra, has been cited in several general review articles on the potential for producing transgenic chickens. In these review articles the authors speculate that chickens expressing retrovirus-derived viral glycoprotein genes could become resistant to pathogenic retroviruses. L.B. Crittenden and D.W. Salter, "Gene
Insertion: Current Progress and Long-Term Goals", Avian Diseases, 30, pp. 43-46 (1985), and L.B. Crittenden and D.W. Salter, Genetic
Engineering to Improve Resistance to Viral Diseases of Poultry: A
Model for Application to Livestock Improvement", Can. J. Anim. Sci., 65, pp. 553-62 (1985), discuss, generally, the prospects for inserting genes into the chicken germ line to produce a more diseaseresistant phenotype.

  More specifically, they discuss the possibility of inserting the viral gene coding for the envelope antigen of subgroup A avian leukosis virus ("ALV", a retrovirus) which, they state, if expressed in the cell membrane, would interfere with infection by the most common subgroup of ALV in chicken flocks. They further suggest that the endogenous retroviral gene, ev 6, represents a naturally occurring model for such a host gene citing Robinson et  al., supra. P.M. Biggs, "Infectious Animal Disease and Its Control",
Phil. Trans. R. Soc. Lond. B, 310, pp. 259-74 (1985) refers to various means for controlling animal infections including, inter alia, a discussion of the use of the env gene of ALV to block receptors for virus infection in chickens as it does in nature. J.C.



  Sanford and S.A. Johnston, "The Concept of Parasite-Derived   Resis-    tance - Deriving Resistance Genes from the Parasite's Own Genome", J.



  Theor. Biol., 113, pp. 395-405 (1985), refers to the general concept of producing hosts resistant to a particular   parasiteby    introducing a gene of pathogenic origin into the host by cloning the appropriate parasite gene, possibly modifying its expression, and transforming it into the host genome. As a specific example of this approach, they discuss theoretically how the genes of the bacteriophage   Qss,    an RNA phage, could be used to make E. coli resistant to   Qss    infection.

  PCT application PCT/US86/00514, published 25 September 1986 (inventors are Johnston and Sanford, supra), also refers to a method for conferring resistance to a parasite to a host of a parasite, which comprises isolating a gene fragment from the parasite (including viruses) and inserting the gene fragment into the host where it is transcribed in an antisense direction, or wherein the gene fragment is expressed to produce a product capable of disrupting an essential activity of the parasite, or wherein the gene fragment acts as a binding site competing with a natural binding site of the parasite.

 

  The only concrete example set forth in this application relates to the   Qss    phage referred to above.



   None of these documents refers to inserting a gene encoding a glycoprotein of a DNA virus such as a herpesvirus into a host which, upon expression, confers resistance of cells and animals to a DNA virus. All of the above-cited references set forth actual examples relating only to RNA viruses or retroviruses which insert genes into the host genome, and thereby confer some resistance, during their normal life cycle.



  SUMMARY OF THE INVENTION
 The present invention relates to transgenic animal cells comprising a gene derived from a DNA virus which, upon expression, renders the animal cell resistant to infection by a related DNA virus.



   More specifically, the invention relates to transgenic mammalian  cells comprising a herpesvirus glycoprotein gene, which, upon expression, renders the transgenic mammalian cells resistant to infection by the herpesvirus.



     Hore    specifically, the invention relates to transgenic mammalian cells comprising the gp50 pseudorabies virus gene, which, upon expression, renders the transgenic mammalian cell resistant to infection by a herpesvirus, preferably a pseudorabies virus.



   The present invention also relates to methods for making an animal cell resistant to DNA virus infection, comprising transforming said animal cell with a glycoprotein gene derived from said DNA   virus   
DETAILED DESCRIPTION OF THE INVENTION
 As used herein "resistance" means a reduced ability of a parasite to infect a host Therefore, "resistance" includes complete and partial immunity to infection by the host to the virus.



   As used herein, "transgenic" refers to cells that have had transferred into them, and which subsequently maintain and express, genes from unrelated organisms (e.g., the expression of viral genes in animal cells), such genes being inherited by progeny cells on cell division.



   As used herein, "a related DNA virus" refers to a virus related to the DNA virus from which the gene used to produce the transgenic animal cell is derived. "Related" means having a relatively close phylogenetic relationship, for example, the herpesviruses.



   As used herein, "mammal" and mammalian includes all mammals except humans.



   Herpesvirus glycoproteins are well known to those skilled in the art and include, for example, herpes simplex virus-l glycoprotein D (HSV-1 gD), herpes simplex virus-2 glycoprotein D (HSV-2 gD), and pseudorabies virus glycoprotein 50 (PRV gp50). These three   glycopro-    teins are homologous. Other herpes simplex virus glycoproteins in both HSV-1 and HSV-2 include gB, gC, gE, gG, gH, and US7. Homologous
PRV proteins are known as   girl,    gIII, gI, and gX, respectively for the first four and gp63 for US7. Glycoproteins are also known for varicella, cytomegalovirus, infectious bovine rhinotracheitis virus, and Marek's disease virus. Other viruses include bovine mammilitis virus, malignant catarhal virus, infectious laryngotracheinis virus, equine herpesvirus 1 virus, and feline herpesvirus.  



   The instant invention relates to a method for making animal cells resistant to viral infection. We introduced DNA that directs expression of the pseudorabies virus (PRV) gp50 glycoprotein into cells in culture. Cells expressing gp50 were found to be resistant to infection by PRV and herpes simplex virus (HSV).



   E.A. Petrovskis et al., "DNA sequence of the Gene for Pseudorabies Virus gp50, a Glycoprotein without N-Linked Glycosylation", J.



  Virol., 59, pp. 216-23 (1986), and PCT patent application PCT/US86/01761, filed 28 August 1986, describe the gp50   nucleotide    sequence and its expression in Chinese hamster ovary cells. Both of these documents are incorporated herein by reference.



   Methods for producing transgenic animal cells are well known to those skilled in the art (C. Corman, "High Efficiency Gene Transfer into Mammalian Cells", in DNA Cloning Vol. 2, A Practical Approach,
D.M. Glover, ed., IRL Press, Oxford (1985) which is incorporated herein by reference). This is true not only at the cell line level, but also for whole animals (B. Hogan, et al., "Manipulating the Mouse
Embryo - A Laboratory Manual", Cold Spring Harbor Laboratory (1986);
T.E. Wagner, Can. J. Anim. Sci., 65, pp. 539-52 (1985) both of which are incorporated herein by reference).



   EXAMPLE 1
Cells and Viruses. HeLa cells were obtained from J. Ross, University of Wisconsin, Madison, WI. Porcine MVPK-1 cells [Swaney, Am. J. Vet.



  Res. 37:1319-1322] were obtained from M. Wathen, The Upjohn Co.,
Kalamazoo, MI. These cell lines were propagated in Dulbecco modified
Eagle Medium with   108    fetal calf serum. G-418 sulfate (Gibco) was added to 300   pg/ml    for growth of neomycin resistant derivatives. The propagation of Chinese hamster ovary (CHO) cells has been described previously (Petrovskis, et al., supra). Use of Vero cells to propagate PRV has been described previously Rea, et al., J. Virol.



  54:21-29 (1985), which is incorporated herein by reference. The Rice strain of PRV was obtained from D. P. Gustafson, Purdue University,
Lafayette, IN. Isolation of the HR strain of PRV has been described previously (Petrovskis, et al., supra.). Infection of HeLa and HeLaderivative cells was in medium 199 (Gibco) with 1% fetal calf serum, as described for infection of vero cells above. Virus was harvested by freezing and thawing infected flasks, adding an equal volume of sterile milk and sonicating briefly. The virus was titrated by  dilution in medium 199 and plating dilutions on monolayers of vero cells. Herpes simplex virus strain HSV-1 was propagated in vero cells the same as were PRV. The F+ strain of HSV-1 was provided by
B. Roizman at the University of Chicago. The F+ strain is a derivative of HSV-1 F (ATCC VR-733) that was adapted to grow at   370.   



   DNA. All recombinant DNA methods were done by standard techniques set forth, for example, in Maniatis, et al., Molecular   Cloning'-    A Laboratory Hanual, (1982), which is incorporated herein by reference. The isolation, characterization and expression of the
PRV gp50 gene has been previously described (PCT patent application
PCT/US 86/01761, supra, and Petrovskis, et al., supra). To construct plasmid pNIE5OPA, plasmid pSV2neo (ATCC No. 37149) was substituted for   pSV2dhfr.    Plasmid pNIE50PA is identical in every other respect to plasmid pDIESOPA (Petroviskis, et al, supra).



     Transfection    Plasmid pNIE50PA was introduced into HeLa and
MVPK cells by calcium phosphate coprecipitation with salmon sperm carrier DNA (Graham and Van Der Eb, Virology, 52, pp. 456-67). The transfected cells were selected in Dulbecco modified Eagle medium with 10% fetal calf serum and 300   Crg/ml    G-418 sulfate. The plasmid pNIE50TPA was introduced into HeLa and MVPK cells as previously described (Application PCT/US 86/01761 and Petrovskis, et al., supra).



   Antibodies and protein analysis. The isolation of monoclonal antibody 3A-4, which reacts with gp50, has been described previously (Petrovskis, et al., supra; patent application PCT/US86/02809, filed 19 December 1986). Polyclonal antiserum reacting with gp50 was isolated by immunizing CF-1 mice with a vaccinia virus recombinant expressing the gp50 gene (Application PCT/US 86/01761). Transfected
HeLa and CHO cell extracts were   immunoprecipitated    with 3A-4 as previously described (Petrovskis et al., supra). Transfected MVPK cell extracts were examined by Western blot (Towbin et al., Proc.

 

  Natl. Acad. Sci. USA, 76, pp. 4350-54 (1979)) using the polyclonal antiserum to gp50 to select for cells expressing gp50.



   Several HeLa cell lines expressing gp50 were produced as described above. One cell line so produced, gp50HeLa-9, was infected with several PRV strains and with HSV F+. As set forth in Table 1, resultant virus titers were 1-2 orders of magnitude lower in gp50
HeLa-9 infected with PRV and at least 4 orders of magnitude lower in  gp50HeLa-9 cells infected with HSV F+.



   TABLE 1
 Resistance of gp50HeLa Cell   Lines   
 to Infection with   Herpesvir¯ses   
 Virus MOI Cells Titera
 PRV Rice 0.1 gp50HeLa-9 5.0 x 105
 HeLa 4.5 x 106
 PRV HR 0.1 gp50HeLa-9 2.6 x 105
 - HeLa 1.3 x 107
 HSV F+ 0.1 gp50HeLa-9    <     104
 HeLa 2.4 x 108
 PRV Rice 0.01 gp50HeLa-9 1.2 x 104
 HeLa 3.0 x 105
 PRV HR 0.01 gp50HeLa-9  <  104
 HeLa 2.5 x 106
 HSV F+ 0.01 gp50HeLa-9  <  104
 HeLa 9.0 x 107 aPFU/ml
 Table 2 shows the resistance of other HeLa cell lines expressing gpS0 to infection by HSV and PRV over time as compared to nontransformed HeLa cells and HeLa cells expressing a non-viral polypeptide, bovine growth hormone   (bGH).     



   TABLE 2
 Resistance of gp50 Expressing HeLa Cell lines
 to Infection Over   TimQ   
 Days post-infection
Cells 2 3 4
 Experiment #1 - Infection with HSV F+   (MOI-0.001)   
HeLa 6.7 x 106 1.5 x 107 1.1 x 107 bGH HeLab 1.8 x 106 2.1 x 107 1.8 x 107   gp5OHeLa-9-2     <    103      S      103     <  103   gpSOHeLa-1-7  <  103 S 103 S  <  103   
 Experiment &num;

  ;2 - Infection with PRV HR   (MOI-0.001)   
HeLa 4.8 x 103 4.6 x 104 3.1 x 105 bGH HeLa 2.9 x 103 4.1 x 104 9.3 x 104 gp50HeLa-9-2  <  10  <  10  <  10   gp50HeLa-l-7     <  10  <  10  <  10 aPFU/ml bbGH Hela cells contain bovine growth hormone (bGH) genomic DNA expressed by the human cytomegalovirus immediate early promoter (United States patent application 758,517, filed 26 July 1985), cloned into plasmid pSV2neo.



   These results demonstrate that HeLa cell lines expressing gp50 are resistant as compared either to the parent HeLa line or a HeLa line transformed to produce a protein other than gp50 (e.g., bGH).



   To demonstrate that transgenic pig cells are similarly resistant to herpesvirus infection, pig MVPK-1 cells were transformed with the gene for gp50 as set forth above. Cell line MVPK-2 produces gp50.



  Cell line MVPK-4 is G418 resistant by transfection with pNIE5OPA, but does not produce gp50. MVPK-7 produces gp50, but at a lower level than does MVPK-2. MVPK-tPA is a transformed MVPK cell line that produces tissue plasminogen activator rather than gp50, and is also
G418 resistant. As seen in Table 3, MVPK-2 cells which produce the greatest amount of gp50 are the most resistant to subsequent infection by a herpesvirus.  



   TABLE 3
 Resistance of gp50 Expressing MVPK Cell Lines
 to Infection Over Timea
 Hours post-infection/M0I-0.01
Cells   18.5    27.5 45 60
MVPK-2 3.1x105 2.3x106 1.2x107   l.0x108   
MVPK-4 1.6x106   1.3xl07    2.5x108 3.6x108
MVPK-7 1.7x106   2.0x107    3.7x108 7.5x108   MVPK-tPA    1.6x106 2.3x107 2.0x108   - 7.4x108   
 Hours-post-infection with   PRV/MOI-0 .001   
 45
MVPK-2 6.7x106
MVPK-4 1.2x108
MVPK-7 2.2x108
MVPK- tPA 1.8x108
 Hours post-infection with   PRV/MOI=O.1   
 45
MVPK-2 2.6x108
MVPK-4 7.0x108
MVPK-7 6.3x108
MVPK-tPA   3.8xl08   
 Hours post-infection with   HSV/HOI=O.O1   
 72
MVPK-2   8.5xl02   
MVPK-4   l.0xl05   
MVPK-7 

   8.2x105
MVPK- tPA 1.3x105
 Hours post-infection with Vaccinia   virus/MOIsO.01   
 72
MVPK-2 2.6x106
MVPK-4 4.0xl05
MVPK-7 6.0x105
MVPK-tPA   1. 5xl06    aPFU/ml
 The introduction of the gp50 gene into a cell by transfection, microinjection, protoplast fusion, etc., will make that cell resistant to PRV infection if the gp50 gene is expressed. Resistance, as  previously defined, is demonstrated by infection of the gp50-producing cell line with PRV and then measuring virus replication by standard virus titration methods. Other genes encoding proteins such as gD-l and   gD-2,    can be used in the same manner.



   EXAMPLE 2
 In this example we set forth the production of a transgenic animal resistant to PRV infection upon expression of the gp50.



   Plasmid pMK contains the mouse metallothionein-I (HT-I) promoter (Cell, 27, pp. 223-31 (1981)), and is available from Dr. Richard
Palmiter, University of Washington, Seattle, Washington. pMK is cut with EcoRI, the ends made blunt with T4 DNA polymerase, BglII linkers are ligated onto the ends, and the construction is then digested with
BglII. The smaller piece produced by the BglII digestion, which is approximately 2 kb, contains the metallothionein promoter and is isolated by agarose gel electrophoresis.



   Plasmid pD50 (International Application No. PCT/US 86/01761 which is incorporated herein by reference) is cut with BamHI and the ends so produced are dephosphorylated using bacterial alkaline phosphatase to produce fragment 1. Fragment 1 is then ligated to the
MT-I promoter-containing- fragment from above to produce plasmid   pMT50.   



   Plasmid   pMT5O    is cut with EcoRI to produce fragment 2. Fragment 2 is dephosphorylated with bacterial alkaline phosphatase and mixed with fragment 12 produced as set forth in Application No. PCT/US 86/01761 to add the bovine growth hormone polyadenylation signal.



  The resulting plasmid is called pMT50PA.



   A preferred alternative method to produce   pMT5O    is as follows:
To destroy the BamHI site in plasmid pDIESOPA (Petrovskis, et al., supra), it is cut with BamHI, the ends are made blunt with DNA
Polymerase I (Klenow) and religated to produce plasmid   pDIE5OPAX.    A gp50 expression cassette is constructed in a three-piece ligation.



  Fragment 1, the vector, is generated by cutting pUCl9 (C. Yanisch
Perron, et al., Gene, 33, pp. 103-19 (1985)) with BamHI and EcoRI and by isolating the 2.7 kb fragment by agarose gel electrophoresis.



  Fragment 2, the upstream piece of the gpSO gene, is generated by cutting   pD50    with BamHI and SalI and by isolating the 0.5 kb fragment by agarose gel electrophoresis. Fragment 3, the downstream piece of the gpSO gene linked to the bovine growth hormone polyadenylation  signal, is generated by cutting pDIE50PAX with SalI and EcoRI and by isolating the 1.1 kb fragment. Fragments 1, 2, and 3 are ligated to produce plasmid pUC50PA.



   Plasmid pMK is cut with EcoRI, the ends made blunt with DNA
Polymerase I (Klenow), HindIII linkers are ligated onto the ends, and the construction is then digested with HindIII and BgIII. The approximately 2.0 kb fragment containing the metallothionein promoter is isolated by agarose gel electrophoresis.



   Plasmid pUC50PA is cut with BamHI and   HindIII,    and the 3.5 kb fragment is isolated by agarose gel electrophoresis. The MT-I promoter fragment from above is ligated to this fragment to generate plasmid pMT50PA.

 

   Plasmid pMT50PA is microinjected into mouse embryos as described in Hogan, et al., supra. Because the   MT-I-promoter    is expressed in many different tissues, and because the tissue specificity varies in different animals (reviewed by Palmiter and Brinster, Ann. Rev.



  Genet., 20, pp 465-99 (1986)), it is necessary to screen the transgenic offspring to find those where the tissue specificity produces
PRV resistance.



   Similar constructions can be made with other promoters. For example, the human metallothionein promoter can be used to obtain a different tissue distribution (Nature, 325, pp. 412-16 (1987)).



   Similar microinjection technology, as described by Hammer, et al, Nature, 315, pp. 680-83 (1985) can be used to generate transgenic livestock animals, including pigs. 

Claims

1. A transgenic animal cell comprising a gene derived from a DNA virus, which gene, upon expression, renders the animal cell resistant to infection by a related DNA virus.

2. A transgenic animal cell according to claim 1, wherein the animal cell is a transgenic mammalian cell comprising a herpesvirus glycoprotein gene, which, upon expression, renders the transgenic mammalian cells resistant to infection by a herpesvirus.

3. A transgenic animal cell according to claim 2, wherein the glycoprotein is selected from the group consisting of HSV-1 gD, HSV2 gD, and PRV gp50.

4. A transgenic mammalian cell according to claim 2, comprising the gp50 pseudorabies virus gene.

5. A transgenic mammalian cell according to claim 4 wherein the herpesvirus that the cell is resistant to is a pseudorabies virus.

6. A method for making animal cells resistant to DNA virus infection comprising transforming said cells with a gene derived from said DNA virus, which gene, upon expression by the animal cell, renders the animal cell resistant to infection a DNA virus.

7. A method according to claim 6, wherein said virus is a herpesvirus and the animal cell is transformed with a glycoprotein gene derived from said herpesvirus.

8. A method according to claim 7, wherein said herpesvirus is a pseudorabies virus and said glycoprotein is gp50.

9. A transgenic nonhuman animal comprising a gene derived from a DNA virus, which gene, upon expression, renders the animal resistant to infection by a related DNA virus.

10. A transgenic nonhuman animal according to claim 9 wherein the animal is a transgenic mammal comprising a herpesvirus glycoprotein gene, which, upon expression, renders the transgenic mammal resistant to infection by the herpesvirus.

11. A transgenic animal according to claim 10, wherein the glycoprotein is selected from the group consisting of HSV-1 gD, HSV-2 gD, PRV gp50.

12. A transgenic mammal according to claim 10, comprising the gp50 pseudorabies virus gene, which, upon expression, renders the transgenic mammal resistant tv infection by a herpesvirus.

13. A transgenic mammal according to claim 12, wherein the herpesvirus that the cell is resistant to is a pseudorabies virus.

14. A method for making animals resistant to DNA virus infection comprising transforming said animal with a gene derived from said DNA virus, which gene, upon expression by the animal, renders the animal resistant to infection.

15. A method according to claim 14, wherein said virus is a herpesvirus and the animal is transformed with a glycoprotein gene derived from said herpesvirus.

16. A method according to claim 15, wherein said herpesvirus is a pseudorabies virus and said glycoprotein is gp50.