The present invention relates generally to the fields of biochemistry, molecular biology, and virology. More particularly, it relates to the identification of GB virus B (GBV-B)/HCV chimeras. The invention involves nucleic acid constructs and compositions encoding GBV-B/HCV chimera, including at least part of a 5' NTR derived from a HCV 5' NTR. This construct, and chimeric versions of it, may be employed to study GBV-B and related hepatitis family members, such as hepatitis C virus. The invention thus includes methods of preparing GBV-B/HCV chimeric sequences, constructs, and viruses, as well as methods of employing these compositions.

Description of the Invention

SUMMARY OF THE INVENTION

As discussed above, an infectious molecular clone of GBV-B would be very useful for the development of HCV preventative and therapeutic treatments. The construction of an infectious molecular clone of this virus will require the newly determined 3' sequence to be included in order for the clone to be viable. The inventors have elucidated the previously unrecognized 3' terminal sequence of GBV-B (SEQ ID NO:1). This sequence has been reproducibly recovered from tamarin serum containing GBV-B RNA, in RT-PCR protocols using several different primer sets, and as a fusion with previously reported 5' GBV-B sequences.

The newly identified 3' sequence is not included in published reports of the GBV-B sequence, nor described in patents relating to the original identification of the viral sequence (see U.S. Pat. No. 5,807,670 and references therein).

The invention has utility in that the inclusion of the sequence will be necessary for construction of an infectious molecular GBV-B clone. Such clones clearly have the potential to be constructed as chimeras including relevant hepatitis C virus sequences in lieu of the homologous GBV-B sequence, providing unique tools for drug discovery efforts. A full-length molecular clone of GBV-was constructed, as described in later sections of this specification.

GBV-B can be used as a model for HCV, and the GBV-B genome can be used as the acceptor molecule in the construction of chimeric viral RNAs containing sequences of both HCV and GBV-B. Such studies will allow one to investigate the mechanisms for the different biological properties of these viruses and to discover and investigate potential inhibitors of specific HCV activities (e.g., proteinase) required for HCV replication. However, all this work is dependent upon construction of an infectious clone of GBV-B, which is itself dependent on the incorporation of the correct 3' terminal nucleotide sequence within this clone. GBV-B has unique advantages over HCV in terms of its ability to replicate and cause liver disease in tamarins, which present fewer restrictions to research than chimpanzees, the only nonhuman primate species known to be permissive for HCV.

An infectious molecular clone of GBV-B is expected to have utility in liver-specific gene expression or in gene therapy. This application might be enhanced by the inclusion of HCV genomic sequence in the form of a GBV-B/HCV chimera. Further, an infectious GBV-B/HCV chimera expressing HCV envelope proteins can have utility as a vaccine immunogen for hepatitis C.

A full-length cDNA copy of the GBV-B genome was constructed to contain the newly identified 3' terminal sequences. RNA transcribed from this cDNA copy of the genome would be infectious when inoculated into the liver of a GBV-B permissive tamarin, giving rise to rescued GBV-B virus particles. A chimeric molecule would then be constructed from this infectious GBV-B clone in which the HCV NS3 proteinase or proteinase/helicase sequence (or other relevant HCV sequences of interest in drug discovery efforts) would be placed in frame in lieu of the homologous GBV-B sequence, and this chimeric cDNA would be used to generate infectious GBV-B/HCV chimeric viruses by intrahepatic inoculation of synthetic RNA in tamarins. Published studies indicate that the GBV-B and HCV proteinases have closely related substrate recognition and cleavage properties, making such chimeras highly likely to be viable. These newly generated chimeric GBV-B/HCV viruses could be used in preclinical testing of candidate HCV NS3 proteinase inhibitors.

Therefore, the present invention encompasses an isolated polynucleotide encoding a 3' sequence of the GBV-B genome. The polynucleotide may include the sequence identified as SEQ ID NO:1. It is contemplated that the polynucleotide may be a DNA molecule or it can be an RNA molecule. It is further contemplated that expression constructs may contain a polynucleotide that has a stretch of contiguous nucleotides from SEQ ID NO:1 and/or SEQ ID NO:2, for example, lengths of 50, 100, 150, 250, 500, 1000, 5000, as well as the entire length of SEQ ID NO:1 or 2, are considered appropriate. Such polynucleotides may also be contained in other constructs of the invention or be used in the methods of the invention. Polynucleotides employing sequences from SEQ ID NO:1 may alternatively contain sequences from SEQ ID NO:2 in the constructs and methods of the present invention.

The invention is also understood as covering a viral expression construct that includes a polynucleotide encoding a 3' sequence of the GBV-B genome. This expression construct is further understood to contain the sequence identified as SEQ ID NO:1. The present invention contemplates the expression construct as a plasmid or as a virus. Furthermore, the expression construct can express GBV-B sequences; alternatively it may express sequences from a chimeric GBV-B/HCV virus.

The identification and isolation of a 3' sequence of GBV-B additionally provides a method of producing a virus, particularly a full-length virus, by introducing into a host cell a viral expression construct containing a polynucleotide encoding a 3' sequence of GBV-B and by culturing the host cell under conditions permitting production of a virus from the construct. This method can be practiced using a prokaryotic cell as a host cell, or by using a eukaryotic cell as a host cell. Furthermore, the eukaryotic cell can be located within an animal.

A method of producing virus according to the claimed invention can also be employed using a polynucleotide that contains synthetic RNA and/or synthetic DNA. Moreover, a step can be added to the method by also isolating any virus produced from the host cell. The virus can then be purified to homogeneity.

In further embodiments, the present invention encompasses an oligonucleotide between about 10 and about 259 consecutive bases of SEQ ID NO:1. This oligonucleotide is contemplated to be about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 50 bases in length, about 100 bases in length, about 150 bases in length, about 200 bases in length, or about 259 bases in length.

Additional examples of the claimed invention include a method for identifying a compound active against a viral infection by providing a virus expressed from a viral construct containing a 3' sequence of a GBV-B virus, by contacting the virus with a candidate substance; and by comparing the infectious ability of the virus in the presence of the candidate substance with the infectious ability of the virus in a similar system in the absence of the candidate substance. It is contemplated that the invention can be practiced using GBV-B virus or a GBV-B/HCV chimera. Infectious ability of the virus is comparable to infectivity of the virus. Infectivity can be evaluated by evaluating or assessing the ability of a host cell to become infected by the virus or by the ability of the virus to replicate in the cell. However, it may also be evaluated by virus production, virus infectivity, viral load, or phenotype of the cell such as signs of CPE.

The present invention can also be understood to provide a compound active against a viral infection identified by providing a virus expressed from a viral construct containing a 3' sequence of a GBV-B virus; contacting the virus with a candidate substance; and comparing the infectious ability of the virus in the presence of the candidate substance with the infectious ability of the virus in a similar system in the absence of the candidate substance. In some embodiments an active compound is identified using a GBV-B virus, while in other embodiments an active compound is identified using a GBV-B/HCV chimera. Compounds considered active against viral infection would include, but are not limited to, those compounds that inhibit viral infection or that expedite clearance of the virus.

In various embodiments of the invention, a GBV-B polynucleotide may encode a GBV-B/HCV chimera that includes at least part of a 5' NTR sequence derived from a HCV 5' NTR. The 5' NTR may comprise at least one domain, i.e., domain I, II, III and/or IV derived from the 5' NTR of HCV. However, it is specifically contemplated that not all four domains are from HCV in some embodiments. In certain embodiments, the GVB-B/HCV chimera may include at least domain III of the 5' NTR derived from the 5'NTR of HCV. In yet other embodiments the infectious GBV-B clone may comprise domain III of the 5' NTR of HCV, which may or may not include one or more structural or non-structural genes of HCV also incorporated into the chimeric virus. The portions of the 5' NTR of the GVB-B/HCV chimeras will generally be replaced by analogous sequences from the 5' NTR of HCV. It will be understood that the portions or parts of the 5' NTR of GBV-B that may be replaced include all or part of domain I (including sub-region Ia and Ib of GBV-B), domain II, domain III, domain IV, or any combination thereof. Any combination of 5' NTR domains of GBV-B may be replaced with an analogous region of HCV. In certain embodiments, the replacement of a GBV-B region may be accompanied by the deletion of the 5' NTR GBV-B domain Ib region. In addition, any one, two, or three of the 5' NTR domains of GBV-B may be replaced in any combination with analogous sequences from HCV.

In further embodiments of the invention, a polynucleotide encoding a GBV-B/HCV chimera including a 5' NTR domain III sequence derived from a HCV 5' NTR may be propagated in vivo, in particular, in the liver of an appropriate host.

Various other embodiments may include isolated polynucleotides comprising a chimeric GBV-B genome, wherein at least part, but not all of a 5' NTR sequence is derived from a HCV 5' NTR. The polynucleotides may be synthetic RNA, RNA, DNA or the like.

Some embodiments include one or more virus, one or more hepatotropic virus, and/or one or more viral expression constructs comprising a chimeric GBV-B polynucleotides including at least a part of the 5' NTR sequence is derived from a HCV 5' NTR.

Methods of producing a chimeric GBV-B virus encoding at least part of a 5' NTR sequence derived from a HCV 5' NTR sequence comprising introducing into a host cell a viral expression construct comprising a chimeric GBV-B polynucleotide encoding at least part of a 5' NTR sequence derived from a HCV 5' NTR sequence and culturing said host cell under conditions permitting production of a virus from said construct are contemplated. The method may use a host cell that is a eukaryotic cell and the host cell may be in an animal. The method may further include the step of isolating virus from said host cell and in particular purify the virus to homogeneity.

In addition, methods for identifying a compound active against a viral infection comprising are contemplated. The methods may include providing a virus expressed from a viral construct comprising at least part of a 5' NR derived from a HCV 5' NTR, as described herein; contacting said virus with a candidate substance; and comparing the infectious ability of the virus in the presence of said candidate substance with the infectious ability of the virus in a similar system in the absence of said candidate substance. Each of the embodiments may use or include any of the 5' NTR chimeras described herein.

Other embodiments of the invention may include a compound active against a viral infection identified according to the method described above.

Furthermore, additional methods include providing or administering to a subject or patient in need of a compound active against viral infection the compound active against a viral infection, particularly a flavivirus such as HCV infection. The patient may have been diagnosed with HCV infection or be at risk for HCV infection, or another flavivirus infection. Alternatively, a subject or patient may be administered a vaccine that immunizes the subject or patient with respect to a flavivirus infection, such as HCV. The invention includes such methods.

It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. GVB-B Virus

The GBV-B genome structure is very similar to hepatitis C and these viruses share approximately 25% nucleotide identity (Simons et al., 1995; Muerhoff et al., 1995). As indicated above, this makes GBV-B more closely related to HCV than any other known virus. GBV-B genomic RNA is about 9.5 kb in length (Muerhoff et al., 1995) with a structured 5' noncoding region that contains an IRES that shares many structural features with the HCV IRES (Honda et al., 1996; Rijnbrand et al., 1999, each of which is incorporated herein by reference). As in HCV, this IRES drives the cap-independent translation of a long open reading frame. The polyprotein expressed from this reading frame appears to be organized identically to that of HCV, and processed to generate proteins with functions similar to those of HCV (Muerhoff et al., 1995). In fact, the major serine proteinases of these viruses (NS3) have been shown to have similar cleavage specificities (Scarselli et al., 1997). Finally, like HCV and distinct from the pestiviruses, the genomic RNA of GBV-B has a poly(U) tract located near its 3' terminus (Simons et al., 1995; Muerhoff et al., 1995). In addition, unreported sequences located at the extreme 3' end of the genome have been identified. This work indicates that the GBV-B RNA, like that of HCV (HCV (Tanaka et al., 1995; Kolykhalov et al., 1996), terminates in a lengthy run of heterogeneous bases (310 nts in GBV-B) possessing a readily apparent secondary structure

B. Nucleic Acids

The present invention provides a nucleic acid sequence encoding a 3' sequence of the GBV-B genome (SEQ ID NO:1).

It should be clear that the present invention is not limited to the specific nucleic acids disclosed herein. As discussed below, a "3' sequence of the GBV-B genome" may contain a variety of different bases and yet still be functionally indistinguishable from the sequences disclosed herein. Such functionally indistinguishable sequences are likely to maintain the basic structure depicted in FIGS. 1 and 2 (see Original Patent), which may be used to guide the prediction of viable nucleotide substitutions.

1. Polynucleotides Encoding the 3' Sequence of the GBV-B Genome

A 3' sequence of the GBV-1 genome disclosed in SEQ ID NO:1 is one aspect of the present invention. Nucleic acids according to the present invention may encode the 3' sequence of the GBV-B genome set forth in SEQ ID NO:1, the entire GBV-B genome, or any other fragment of a 3' sequence of the GBV-B genome set forth herein. The nucleic acid may be derived from genomic RNA as cDNA, i.e., cloned directly from the genome of GBV-B. cDNA may also be assembled from synthetic oligonucleotide segments.

It also is contemplated that a 3' sequence of the GBV-B genome may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, maintain the same general structure (see FIGS. 1 and 2) and perform the same function in RNA replication.

As used in this application, the term "a nucleic acid encoding a 3' sequence of the GBV-B genome" refers to a nucleic acid molecule that may be isolated free of total viral nucleic acid. In preferred embodiments, the invention concerns nucleic acid sequences essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. The term "as set forth in SEQ ID NO:1" means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1. It is contemplated that the techniques and methods described in this disclosure may apply to any of the sequences contained herein, including SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:1 will be sequences that are "as set forth in SEQ ID NO:1." Sequences that are essentially the same as those set forth in SEQ ID NO:1 may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:1 under standard conditions.

The nucleic acid segments and polynucleotides of the present invention include those encoding biologically functional equivalent 3' sequences of the GBV-B genome. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.

3' sequence of the GBV-B genome sequences also are provided. Each of the foregoing is included within all aspects of the following description. The present invention concerns cDNA segments reverse transcribed from GBV-B genomic RNA (referred to as "DNA"). As used herein, the term "polynucleotide" refers to an RNA or DNA molecule that may be isolated free of other RNA or DNA of a particular species.

"Isolated substantially away from other coding sequences" means that the 3' sequence of the GBV-B genome forms the significant part of the RNA or DNA segment and that the segment does not contain large portions of naturally-occurring coding RNA or DNA, such as large fragments or other functional genes or cDNA noncoding regions. Of course, this refers to the polynucleotide as originally isolated, and does not exclude genes or coding regions later added to the it by the hand of man.

In certain other embodiments, the invention concerns isolated DNA segments (cDNA segments reverse transcribed from GVB-B genomic RNA) and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1. The term "essentially as set forth in SEQ ID NO:1" is used in the same sense as described above.

It also will be understood that nucleic acid sequences may include additional residues, such as additional 5' or 3' sequences, and still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological activity. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include additional various non-coding sequences flanking either of the 5' or 3' portions of the coding region, which are known to occur within viral genomes.

Sequences that are essentially the same as those set forth in SEQ ID NO:1 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:1 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other RNA or DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO:1, such as about 15-24 or about 25-34 nucleotides and that are up to about 259 nucleotides being preferred in certain cases. Other stretches of contiguous sequence that may be identical or complementary to any of the sequences disclosed herein, including the SEQ ID NOS. include the following ranges of nucleotides: 50-9,399, 100-9,000, 150-8,000, 200-7,000, 250-6,000, 300-5,000, 350-4,000, 400-3,000, 450-2,000, 500-1000. RNA and DNA segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.

In a non-limiting example, one or more nucleic acid constructs may be prepared that include a contiguous stretch of nucleotides identical to or complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Such a stretch of nucleotides, or a nucleic acid construct, may be about, or at least about, 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39 about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 618, about 650, about 700, about 750, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 9,100, about 9,200, about 9,300, about 9,399, about 9,400, about 9,500, about 9,600, about 9,700, about 9,800, about 9,900, about 10,000, about 15,000, about 20,000, about 30,000, about 50,000, about 100,000, about 250,000, about 500,000, about 750,000, to about 1,000,000 nucleotides in length, as well as constructs of greater size, up to and including chromosomal sizes (including all intermediate lengths and intermediate ranges), given the advent of nucleic acids constructs such as a yeast artificial chromosome are known to those of ordinary skill in the art.

It will be readily understood that "intermediate lengths," in these contexts means any length between the quoted ranges, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; ranges, up to and including sequences of about 1,001, 1,250, 1,500, and the like.

In certain embodiments, GBV-B polynucleotides include chimeric GBV-B poynucleotides. Chimeric GBV-B polynucleotides may include GBV-B/HCV chimeras. GBV-B/HCV chimeras include, but are not limited to GBV-B polynucleotides in which portions of the GBV-B virus have been replaced with or mutated to resemble analogous sequences from the HCV virus. A GVB-B chimera may comprise all or part of the 5'NTR region of a HCV virus. The polynucleotide may comprise domain I, II. III and/or IV of the 5' NTR derived from a HCV 5'NTR. The polynucleotide may have the 5' NTR domain Ib of GBV-B is deleted. The polynucleotide may comprise domain II and domain III of the 5' NTR derived from a HCV 5'NTR. The polynucleotide may comprise domain II and domain IV of the 5' NTR derived from a HCV 5'NTR. The polynucleotide may compirse domain III and domain IV of the 5' NTR derived from a HCV 5'NTR. The polynucleotide may comprise domain II, domain m and domain IV of the 5' NTR derived from a HCV 5'NTR. Domain Ib of GBV-B may or may not be deleted form the polynucleotide. The polynucleotide may be DNA or RNA. The polynucleotide may further comprising at least part of a structural and/or nonstructural protein coding region of derived from HCV. It will be understood that the term "derived" indicates the origin of the sequence. Thus, the recited domains can be obtained from HCV.

In other embodiments a HCV/GBV-B chimera is contemplated. A HCV/GBV-B chimera will comprise a virus where one or more portions of the HCV virus have been replaced with or mutated to resemble the analogous GBV-B portions of the GBV-B virus.

The various probes and primers designed around the disclosed nucleotide sequences of the present invention may be of any length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all primers can be proposed: n to n+y where n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one, where n+y does not exceed the last number of the sequence. Thus, for a 20-mer, the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on. For a 30-mer, the probes correspond to bases 1 to 30, 2 to 31, 3 to 32 . . . and so on. For a 35-mer, the probes correspond to bases 1 to 35, 2 to 36, 3 to 37 . . . and so on.

2. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses RNA and DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementary rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO. 1 under relatively stringent conditions such as those described herein. Such sequences may encode the entire 3' sequence of the GBV-B genome or functional or non-functional fragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or 3431 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.

Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37.degree. C. to about 55.degree. C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20.degree. C. to about 55.degree. C. Thus, hybridization conditions can be readily manipulated and thus will generally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl.sub.2, 10 mM dithiothreitol, at temperatures between approximately 20.degree. C. to about 37.degree. C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures ranging from approximately 40.degree. C. to about 72.degree. C. Formamide and SDS also may be used to alter the hybridization conditions.

One method of using probes and primers of the present invention is in the search for other viral sequences related to GBV-B or, more particularly, homologs of the GBV-B sequence. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention is in site-directed, or site-specific, mutagenesis. The technique provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into complementary DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. There are newer and simpler site-directed mutagenesis techniques that can also be employed for this purpose. These include procedures marketed in kit form that are readily available to one of ordinary skill in the art.

The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

3. Antisense Constructs

In certain embodiments of the invention, the use of antisense constructs of the 3' sequence of the GBV-B genome is contemplated.

Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNAs, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense constructs could be used to block early steps in the replication of GBV-B and related viruses, by annealing to 3' terminal sequences and blocking their role in negative-strand initiation.

As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of 15 bases in length may be termed complementary when they have complementary nucleotides at 13 or 14 positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

4. Amplification and PCR.TM.

The present invention utilizes amplification techniques in a number of its embodiments. Nucleic acids used as a template for amplification are isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989). The nucleic acid may be genomic DNA or RNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA using reverse transcriptase (RT). In one embodiment, the RNA is genomic RNA and is used directly as the template for amplification. In others, genomic RNA is first converted to a complementary DNA sequence (cDNA) and this product is amplified according to protocols described below.

Pairs of primers that selectively hybridize to nucleic acids corresponding to GBV-B sequences are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term "primer," as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.

Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals.

A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR.TM.) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and each incorporated herein by reference in entirety.

Briefly, in PCR.TM., two or more primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.

A reverse transcriptase PCR.TM. amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed Dec. 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPA No. 320 308, incorporated herein by reference in its entirety. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, incorporated herein by reference, also may be used as still another amplification method in the present invention.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site also may be useful in the amplification of nucleic acids in the present invention.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences also can be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference.

Davey et al, EPA No. 329 822 (incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.

Miller et al., PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target ssDNA followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, 1990 incorporated by reference).

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, also may be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography that may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al., 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose or nylon, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

5. Expression Constructs

In some embodiments of the present invention, an expression construct that encodes a 3' sequence of GBV-B is utilized. The term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Expression includes both transcription of a gene and translation of mRNA into a gene product. Expression may also include only transcription of the nucleic acid encoding a gene of interest.

In some constructs, the nucleic acid encoding a gene product is under transcriptional control of promoter and/or enhancer. The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of nucleic acids, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of nucleic acids with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

6. Host Cells and Permissive Cells

As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of the present invention, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector or virus and/or expressing viral proteins. A host cell can, and has been, used as a recipient for vectors, including viral vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. A "permissive cell" refers to a cell that supports the replication of a given virus and consequently undergoes cell lysis. In the context of the present invention, such a virus would include HCV, GBV-B, or other hepatitis viruses. In a "nonpermissive cell," productive infection does not result, but the cell may become stably transformed. In some embodiments, methods employ permissive cells that are a cell line derived from liver cells (liver cell line).

Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5.alpha., JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE.RTM. Competent Cells and SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector or virus or virus particle may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector. It is contemplated that the present invention includes vectors composed of viral sequences, viruses, and viral particles in the methods of the present invention, and that they may be used interchangeably in these methods, depending on their utility.

Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

7. Pharmaceutical Compositions

The present invention encompasses the use of a 3' sequence of GBV-B in the production of or use as a vaccine to combat HCV infection. Compositions of the present invention comprise an effective amount of GBV-B clone as a therapeutic dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

The biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, or even intraperitoneal routes. The preparation of an aqueous composition that contains GVB-B nucleic acid sequences as an active component or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

A GBV-B clone of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. In terms of using peptide therapeutics as active ingredients, the technology of U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, each incorporated herein by reference, may be used.

The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like also can be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used, including cremes.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
 

Claim 1 of 14 Claims

1. An isolated polynucleotide comprising an infectious chimeric GBV-B polynucleotide encoding a virus comprising domain III of the 5' NTR from a HCV 5' NTR, and at least part of a structural protein coding region of HCV or at least part of a non-structural protein coding region of HCV.

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