Negative-stranded nonsegmented RNA viruses include: paramixoviruses such as Newcastle""s disease, mumps and parainfluenza; morbilliviruses such as measles, canine distemper and bovine rinderpest; pneumovirus or respiratory syncytial virus; vesiculovirus; and lyssaviruses such as rabies. The single-stranded negative sense genomic RNA of these viruses act as a direct template for both transcription and replication (Wunner et al. Rev Infect Dis 1988, 10, S771-S784). In rabies virus, for example, during the process of transcription a positive strand leader RNA and five monocistronic mRNA are synthesized. These are then translated into individual structural proteins. In the process of replication, the full-length positive strand RNA is synthesized and then becomes the template for the synthesis of progeny negative-stranded nonsegmented genomic RNA.
Vaccination provides a means for preventing infection by several of these more prevalent viruses, i.e., mumps and measles. However, vaccinations are not available for all of these viruses. Further, post-infection treatments are oftentimes unsatisfactory.
For example, it is estimated that more than 50,000 people die of rabies each year. The postexposure treatment recommended by World Health Organization includes administration of rabies vaccine together with antirabies immunoglobulin. The treatment regimen is very effective providing it is initiated within 24-48 hours after exposure. However, such rapid treatment is not always possible. Treatment with rabies vaccines after neuronal infection and dysfunction have occurred may lead to exaggerated immunopathologic disease (Murphy, F. A. Arch Virol 1977, 54, 279-297). Once clinical symptoms occur, the disease is usually fatal. There is no treatment or intervention currently available.
Oligodeoxynucleotides (ODN), which are complementary to certain message or viral sequences, have been reported to inhibit specific gene expression or virus infection (Zamecnik, P. C. and Stephenson, M. L. Proc Nat""l Acad Sci USA 1978, 75, 280-284). ODN hybridization to complementary RNA sequences inhibits the processing, nuclear transport, or translation of mRNA by blocking the access of functional machinery to requisite mRNA sequences, and the formation of DNA-RNA hybrids leads to RNA cleavage by means of RNase H activities (Agrawal et al. Proc Natl Acad Sci USA 1990, 87, 1401-1405). Thus, production of gene products is inhibited, reduced or shut off (Zamecnik, P. C. and Stephenson, M. L. Proc Nat""l Acad Sci USA 1978, 75, 280-284). ODN have been used widely to study gene expression (Wickstrom, E. Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS, Wiley-Liss, New York, 1991) and to inhibit tumor growth (Agrawal, S. Oligonucleotide Therapeutic Approach: Near Clinical Development, Humana Press, New York, 1996). Antisense ODN to oncogenes, such as c-myb, c-Ha-ras, bcr-abl, or NF-kB, have been reported to inhibit the growth of tumor cells in vitro and in vivo (Kitajima et al. J Biol Chem 1992, 267, 25881-25888; Gray et al. Cancer Res 1993, 53, 577-580; Skorski et al. Proc Natl Acad Sci 1994, 91, 4504-4508). One of the first successful demonstrations of using antisense to inhibit virus infection was performed in Rous sarcoma virus in cell culture (Zamecnik, P. C. and Stephenson, M. L. Proc Nat""l Acad Sci USA 1978, 75, 280-284). Subsequently, it has been shown that ODN specific to viruses, when added into culture medium, can protect cultured cells to varying extents from infection by a variety of viruses, such as influenza virus (Leiter et al. Proc Natl Acad Sci USA 1990, 87, 3430-3434), vesicular stomatitis virus (VSV) (Agris et al. Biochemistry 1986, 25, 6268-6275), duck hepatitis virus (Offensperger et al. EMBO J 1993, 12, 1257-1262), and human immunodeficiency virus type 1 (HIV-1) (Zaia et al. J Virol 1988, 62, 3914-3917). Most of the ODN used to inhibit virus infections are antisense DNA complementary to different viral transcripts. However, ODN to other targets have also been shown to inhibit virus infection, such as the TAR element of HIV-1 (Vickers et al. Nucleic Acid Res 1991, 19, 3359-3368). Further, in a brief abstract made available to the public in May of 1996, Fu et al. disclosed that ODNs complementary to rabies virus genomic RNA blocked almost completely rabies virus infection at concentrations as low as 2 xcexcM, while ODNs complementary to vial transcripts did poorly even at concentrations as high as 20 xcexcM (Fu et al., Abstract W15-9, page 110, presented at the 15th Annual Meeting of the American Society for Virology on Jul. 13-17, 1996). This abstract also teaches that the antigenomic ODNS inhibited cell-to-cell spread of the rabies virus and it is suggested that ODNS complementary to rabies virus genomic RNA may have potential to be used for therapy in clinical rabies.
It has now been found that antigenomic ODN targeted to negative strand genomic RNA of nonsegmented RNA viruses are effective inhibitors of viral transcription and may be useful in the treatment and prevention of infection by negative-stranded nonsegmented RNA viruses.
An object of the present invention is to provide oligodeoxynucleotides complementary to genomic RNA of negative-stranded nonsegmented RNA viruses.
Another object of the present invention is to provide a method of inhibiting infection of cells by a negative-stranded nonsegmented RNA virus which comprises exposing cells to an effective amount of an oligodeoxynucleotide complementary to genomic RNA of a negative-stranded nonsegmented RNA virus so that infection of the cells by the virus is inhibited.
Yet another object of the present invention is to provide a method of treating animals infected with a negative-stranded nonsegmented RNA virus which comprises administering to an animal infected with a negative-stranded nonsegmented RNA virus an effective amount of an oligodeoxynucleotide complementary to genomic RNA of the negative-stranded nonsegmented RNA virus.
Oligodeoxynucleotides (ODN) complementary to the genomic RNA of negative-stranded nonsegmented RNA viruses have now been found to block virus infection. Further, the oligodeoxynucleotides of the present invention have been found to be more effective inhibitors of viral transcription and replication than antisense oligodeoxynucleotides targeted to mRNA of the viruses.
The effects of antigenomic ODNs in blocking infection by negative-stranded nonsegmented RNA virus were studied with eight 16 mer ODNs targeted to rabies virus. The sequences and targets of these ODNs are listed in the following Table 1.
Inhibitory activities of these ODNs were tested in baby hamster kidney (BHK) and mouse neuroblastoma (NA, clone 1300) cells. BHK and NA cells grown in 24-well plates were infected with rabies virus ERA strain (derived from the same isolate as SAD B19, Sacramento et al. J Gen Virol 1992, 73, 1149-1158) at a multiplicity of infection (moi) of approximately 5 focus-forming units (ffu) per cell to ensure that 100% of the cells were infected. After a one hour incubation, the viral inoculum was removed and fresh medium was added with different ODNs to a final concentration of 20 xcexcM. ODNs were further added to culture medium at the same concentration at 3 and 5 hours post infection (p.i.). The cells were cultured for another 16 hours before harvesting. The cell pellets were used for RNA extraction and Northern blot hybridization, using rabies virus N cDNA or G3PDH cDNA as probes, and the supernatants were used for virus titration by the fluorescent focus forming assay.
Northern blot analysis revealed that rabies virus transcription was almost completely inhibited with ODN RH+1 (SEQ ID NO: 1), which is complementary to the 3xe2x80x2 end of rabies virus negative-strand genomic RNA. Antigenomic ODN to the positive-strand intermediate genomic RNA (RE-1; SEQ ID NO: 2) and antisense ODNs to different mRNAs showed little or no inhibitory activity at this concentrations. Virus titration revealed no infectious virus in the supernatants of cells (BHK and NA) infected with rabies virus and treated with antigenomic ODN RH+1 (SEQ ID NO: 1), while varying titers of virus were detected in cells treated with other ODNs. Thus, these studies indicate that antigenomic ODNs are most effective at inhibiting negative-stranded nonsegmented RNA viruses such as rabies virus in cell culture. See Table 2.
Additional experiments in cell culture indicate that the ability of the ODNs of the present invention to inhibit viral protein synthesis and production results from blocking of viral transcription. In these experiments, viral protein synthesis was measured in RH+1 treated NA cells. NA cells grown on 24 well plates were infected with ERA strain of rabies virus. Cells in one well were treated with ODN RH+1 while cells in a second well were left untreated. Cells were harvested 16 hours after addition of RH+1 and subjected to SDS-PAGE and Western blot analysis using anti-glycoprotein (G) antibody, Rabies virus G protein was detected in the untreated cells infected with rabies virus. However, the G protein was not detected in infected cells treated with RH+1.
Optimal concentrations of ODN resulting in inhibition of rabies virus were determined. RH+1 (SEQ ID NO: 1) was diluted and added to infected NA cells at final concentrations of 10, 5, 2, and 1 xcexcM, respectively. Cells were harvested 24 hours after virus infection for Northern Blot hybridization. Concentrations of RH+1 as low as 2 xcexcM showed strong inhibition of rabies virus transcription and at 1 xcexcM, RH+1 inhibited more than 70% of rabies virus transcription.
Phosphorothioate and methylphosphonate analogs have been reported to have stronger activity in gene inhibition compared to ordinary ODNs with phosphodiester linkage due to their relatively longer half-life and increased resistance to nuclease digestion (Akhtar et al. Life Sci 1991, 49, 1793-1801; Agris et al. Biochemistry 1986, 25, 6268-6275; Agrawal et al. Proc Nat""l Acad Sci USA 1990, 87, 1401-1405). Furthermore, while phosphodiester ODNs failed to inhibit replication of influenza viruses at concentrations up to 80 xcexcM, it was shown that phosphorothioate ODNs inhibited virus replication at concentrations as low as 1.25 xcexcM (Leiter et al. Proc Natl Acad Sci 1990, 87, 3430-3434). Accordingly, experiments were performed to determine whether phosphorothioate analogs of the antigenomic ODNs of the present invention are more efficacious. A phosphorothioate analog of antigenomic ODN RH+1 having the sequence 5xe2x80x2-AsTsCsAsAsAsGsAsAsAsAsAsAsCsAsG-3xe2x80x2 (SEQ ID NO: 1) referred to herein as RH+1S was synthesized. However, no inhibition of rabies virus at any of the concentrations of RH+1S including 10, 5, 2, and 1 xcexcM was observed.
The ability of the ODNs of the present invention to inhibit spread of these viruses from cell to cell in culture was also demonstrated. It was found that in NA cells treated with antigenomic ODN RH+1 at a concentration of 20 xcexcM, rabies virus remained in the originally infected cells and did not spread to neighboring cells even at 72 hours p.i. In contrast, rabies virus spread quickly in untreated NA cells, and all cells were infected with rabies virus by 72 hours p.i.
In vitro inhibition by ODN RH+1 has also been correlated to in vivo activity. Rats, intranasally infected with rabies virus CVS-24 strain, were used as this model with intranasal infections is used routinely for the study of rabies pathogenesis. All infected animals develop rabies 5 to 6 days following infection. See e.g., Fu et al. J Virol 1993, 67, 6674-6681. In these experiments, miniosmotic pumps filled with ODN RH+1, a random ODN, or saline were surgically implanted in the rats for continuous delivery for 7 days of micromolar quantities of the ODN directly to the brain, prior to infection with the rabies virus. All the infected animals were healthy until the 5th day following infection when one of the rats treated with random ODN was paralyzed. By the 6th post infection day all rats treated with random ODN and saline were found paralyzed. However, only three rats treated with RH+1 ODN were paralyzed while 2 of the RH+1 ODN treated rats remained healthy. Levels of viral transcript in brain tissue from these rats were 100 to 1000 times less as compared to the sick rats, thus indicating that rabies virus infection in the CNS of the two healthy rats was inhibited by the antigenomic ODN.
While these experiments were performed with the CVS-24 strain of rabies virus, sequence analysis of the 3xe2x80x2 end of rabies virus genomic RNA from multiple strains of rabies virus revealed conserved and variable regions thus indicating that any antigenomic ODNs complementary to at least a portion of the conserved regions would be effective in inhibiting multiple viral strains. The first 58 nucleotides of the 3xe2x80x2 end of rabies virus genomic RNA are transcribed into the leader RNA in infected cells (Tordo et al. Proc Natl Acad Sci USA 1986, 83, 3914-3918. Comparison of the 3xe2x80x2 end sequences of genomic RNA among rabies virus strains sequenced to date revealed only three nucleotide differences between PV and SAD B19 strains (at positions 36, 43 and 47, Tordo et al. 1986; Conzelmann et al. J Virol 1990, 175, 484-499), and six nucleotide differences between PV and AV01 strains (at positions 17, 20, 25, 29, 43, and 47; Tordo et al. 1986; Poch et al. Biochime 1988, 70, 1019-1029). The 3xe2x80x2 end of the genome of several virus variants was also sequenced and compared with the published sequences for SAD B19, PV and AV01. A comparison of the sequences is shown in Table 3. SAD B19 and ERA are derived from the same original isolate with different passage history and PV is the original Pasteur isolate (Sacramento et al. 1992). AV01 and F3 are derived from CVS-11 (Dietzschold et al. Proc Natl Acad Sci USA 1983, 80, 70-74; Poch et al. 1988). All these viruses are laboratory adapted and have gone through numerous passages in laboratory animals and cell cultures. Coyote street rabies virus (COSV) and Thailand dog rabies virus (TLDV) are wildtype viruses isolated from dogs (coyotes) from the United States and Thailand, respectively. Silver haired bat rabies strain (SHBV) is the silver haired bat isolate which has been associated with many of the recent human cases in the United States.
The first 11 nucleotides of the 3xe2x80x2 end of genomic RNA are assumed to be conserved. Sequences from nucleotides 12 to 36 showed some variation; sequences from 37 to 58 (the end of the leader transcript) were more conserved even among the wildtype isolates. Sequences from 59 to 73, which encodes the 5xe2x80x2 untranslated regions up to AUG codon of the N transcript are identical among all strains except for PV strains, which showed a one nucleotide differences. As will be obvious to those of skill in the art upon this disclosure, ODNs targeted to other conserved portions of the negative strand of the rabies virus genomic RNA can be designed in accordance with the teachings provided herein. Further, while the exemplified sequences used in the experiments described herein are 16 mer, it is believed that deoxynucleotides of ranging in length from about 10 to about 20 nucleotides would be useful. While absolute complementarity is not required, it is preferred that the deoxynucleotides have no more than one mismatch. Further, it is believed that ODNs targeted to the genomic RNA of other negative-stranded nonsegmented RNA viruses will also be effective as antiviral agents. Examples of other negative stranded nonsegmented RNA viruses include, but are not limited to, paramixoviruses such as Newcastle""s disease, mumps and parainfluenza; morbilliviruses such as measles, canine distemper and bovine rinderpest; pneumovirus or respiratory syncytial virus; and vesiculovirus. ODNs targeted to the genomic RNA of these viruses can be designed routinely by those of skill in the art in accordance with the disclosure provided herein.
The ODNs of the present invention are useful in inhibiting infection of cells by a negative-stranded nonsegmented RNA virus. In this method, cells are contacted with an effective amount of an oligodeoxynucleotide complementary to genomic RNA of a negative-stranded nonsegmented RNA virus so that infection of the cells by the virus is inhibited. By xe2x80x9ceffective amountxe2x80x9d it is meant a concentration of ODN which inhibits viral transcription and replication in cells. Such concentrations can be routinely determined in accordance with the methods described herein.
Further, as demonstrated by the in vivo data, ODNs of the present invention which are effective in inhibiting transcription and replication of a negative-strand nonsegmented RNA virus in cell culture are also expected to be useful in treating animals infected with this virus. Appropriate dosing regimes, route of administration and pharmaceutical vehicles can be routinely selected by one of skill in accordance with the virus being treated. For example, for treatment of rabies virus, it is preferred that the oligodeoxynucleotide be administered introcerebroventricularly in a sterile normal saline solution at a concentration of about 5 to about 75 mM.
The following nonlimiting examples are provided to further illustrate the present invention.