AU776535B2 - Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening - Google Patents

Description

WO 97/27319 PCT/US97/01609 -1- COMPOSITIONS AND METHODS FOR DETERMINING ANTI-VIRAL DRUG SUSCEPTIBILITY AND RESISTANCE AND ANTI-VIRAL DRUG SCREENING Technical Field This invention relates to anti-viral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of viral infections. The invention further relates to novel vectors, host cells and compositions for carrying out these novel anti-viral drug susceptibility and resistance tests. This invention also relates to the screening of candidate drugs for their capacity to inhibit selected viral sequences and/or viral proteins. More particularly, the invention relates to the use of recombinant DNA technology to first construct a resistance test vector comprising a patient-derived segment and an indicator gene, then introducing the resistance test vector into a host cell, and determining the expression or inhibition of the indicator gene product in a target host cell in the presence of an anti-viral drug. This invention is also related to the means and methods of identifying anti-viral drugs which have distinct patterns of *ao0 resistance when compared with existing anti-viral drugs.

This invention also relates to methods and compositions for the identification and assessment of the biological effectiveness of potential therapeutic compounds. This invention is more particularly related to drug susceptibility and resistance tests useful in providing an optimal therapeutic regimen for the treatment of various viral diseases, including for example, HIV/AIDS and hepatitis.

SUBSTITUTE SHEET (RULE 26) Ai WO 97/27319 PCT/US97/01609 -2- Background of the Invention Viral Drug Resistance The use of anti-viral compounds for chemotherapy and chemoprophylaxis of viral diseases is a relatively new development in the field of infectious diseases, particularly when compared with the more than 50 years of experience with antibacterial antibiotics. The design of anti-viral compounds is not straightforward because viruses present a number of unique problems. Viruses must replicate intracellularly and often employ host cell enzymes, macromolecules, and organelles for the synthesis of virus particles. Therefore, safe and effective anti-viral compounds must be able to discriminate with a high degree of efficiency between cellular and virus-specific functions.

In addition, because of the nature of virus replication, evaluation of the in vitro sensitivity of virus isolates to anti-viral compounds must be carried out in a complex culture system consisting of living cells tissue culture). The results from such assay systems vary widely according to the type of tissue culture cells which are employed and the conditions of assay. Despite these complexities nine drugs have been approved for AIDS therapy, five reverse transcriptase inhibitors AZT, ddl, ddC, d4T, 25 3TC, one non-nucleoside reverse transcriptase inhibitor, nevirapine and three protease inhibitors saquinavir, ritonavir and indinovir and several additional anti-viral drug candidates have been recently developed such as nelfinavir, delaviridine, VX-478 and 1592.

Viral drug resistance is a substantial problem given the high rate of viral replication and mutation frequencies.

Drug resistant mutants were first recognized for poxviruses with thiosemicarbazone (Appleyard and Way (1966) Brit. J.

Exptl. Pathol. 47, 144-51), for poliovirus with guanidine WO 97/27319 PCT/US97/01609 -3- (Melnick et al. (1961) Science 134, 557), for influenza A virus with amantadine (Oxford et al. (1970) Nature 226, 82-83; Cochran et al. (1965) Ann. NY Acad Sci 130, 423-429) and for herpes simplex virus with iododeoxyuridine (Jawetz et al. (1970) Ann. NYAcad Sci 173, 282-291). Approximately HIV drug resistance mutations to various anti-viral agents have been identified to date (Mellors et al. (1995) Intnl. Antiviral News, supplement and Condra, J.H. et al.

(1996) J Virol. 70, 8270-8276).

The small and efficient genomes of viruses have lent themselves to the intensive investigation of the molecular genetics, structure and replicative cycles of most important human viral pathogens. As a consequence, the sites and mechanisms have been characterized for both the activity of and resistance to anti-viral drugs more precisely than have those for any other class of drugs. (Richman (1994) Trends Microbiol. 2, 401-407). The likelihood that resistant mutants will emerge is a function of at least four factors: 1) the viral mutation frequency; 2) the intrinsic mutability of the viral target site with respect to a specific anti-viral; 3) the selective pressure of the anti-viral drug; and, 4) the magnitude and rate of virus replication.

With regard to the first factor, for single stranded RNA 25 viruses, whose genome replication lacks a proofreading mechanism, the mutation frequencies are approximately 3x10 per base-pair per replicative cycle (Holland et al. (1992) Curr. Topics Microbiol Immunol. 176, 1-20; Mansky et al.

(1995) J Virol. 69, 5087-94; Coffin (1995) Science 267, 483-489). Thus, a single 10 kilobase genome, like that of human immunodeficiency virus (HIV), would be expected to contain on average one mutation for every three progeny viral genomes. As to the second factor, the intrinsic mutability of the viral target site in response to a specific anti-viral agent can significantly affect the WO 97/27319 PCT/US97/01609 likelihood of resistant mutants. For example, zidovudine (AZT) selects for mutations in the reverse transcriptase of HIV more readily in vitro and in vivo than does the other approved thymidine analog d4T (stavudine).

One, perhaps inevitable consequence of the action of an anti-viral drug is that it confers sufficient selective pressure on virus replication to select for drug-resistant mutants (Herrmann et al. (1977) Ann NY Acad Sci 284, 632-7). With respect to the third factor, with increasing drug exposure, the selective pressure on the replicating virus population increases to promote the more rapid emergence of drug resistant mutants. For example, higher doses of AZT tend to select for drug resistant virus more rapidly than do lower doses (Richman et al. (1990) J. AIDS.

3, 743-6). This selective pressure for resistant mutants increases the likelihood of such mutants arising as long as significant levels of virus replication are sustained.

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20 •"25 *o eoooo The fourth factor, the magnitude and rate of replication of the virus population, has major consequences on the likelihood of emergence of resistant mutants. Many virus infections are characterized by* high levels of virus replication with high rates of virus turnover. This is especially true of chronic infections with HIV as well as hepatitis B and C viruses. The likelihood of emergence of AZT resistance increases in HIV-infected patients with diminishing CD4 lymphocyte counts which are associated with increasing levels of HIV replication (Ibid).

Higher levels of virus increase the probability of preexisting mutants. It has been shown that the emergence of a resistant population results from the survival and selective proliferation of a previously existing subpopulation that randomly emerges in the absence of WO 97/27319 PCT/US97/01609 selective pressure. All viruses have a baseline mutation rate. With calculations of approximately 1010 new virions being generated daily during HIV infection (Ho et al. (1995) Nature 373, 123-126), a mutation rate of 10- 4 to 10 5 per nucleotide guarantees the preexistence of almost any mutation at any time point during HIV infection. Evidence is accumulating that drug resistant mutants do in fact exist in subpopulations of HIV infected individuals (Najera et al.

(1994) AIDS Res Hum Retroviruses 10, 1479-88; Najera et al.

(1995) J Virol. 69, 23-31). The preexistence of drug resistant picornavirus mutants at a rate of approximately 5 is also well documented (Ahmad et al. (1987) Antiviral Res. 8, 27-39).

Human Immunodeficiency Virus (HIV) Acquired immune deficiency syndrome (AIDS) is a fatal human disease, generally considered to be one of the more serious diseases to ever affect humankind. Globally, the numbers of human immunodeficiency virus (HIV) infected individuals and of AIDS cases increase relentlessly and efforts to curb the course of the pandemic, some believe, are of limited S. effectiveness. Two types of HIV are now recognized: HIV-1 and HIV-2. By December 31, 1994 a total of 1,025,073 AIDS cases had been reported to the World Health Organization.

This is only a portion of the total cases, and WHO estimates that as of late 1994, allowing for underdiagnosis, underreporting and delays in reporting, and based on the estimated number of HIV infections, there have been over million cumulative AIDS cases worldwide (Mertens et al.

30 (1995) AIDS 9 (Suppl S259-S272). Since HIV began its spread in North America, Europe and sub-Saharan Africa, over 19.5 million men, women and children are estimated to have been infected (Ibid). One of the distinguishing features of the AIDS pandemic has been its global spread within the last 20 years, with about 190 countries reporting AIDS cases WO 97/27319 PCT/US97/01609 -6today. The projections of HIV infection worldwide by the WHO are staggering. The projected cumulative total of adult AIDS cases by the year 2000 is nearly 10 million. By the year 2000, the cumulative number of HIV-related deaths in adults is predicted to rise to more than 8 million from the current total of around 3 million.

HIV-1 and HIV-2 are enveloped retroviruses with a diploid genome having two identical RNA molecules. The molecular organization of HIV is U3-R-U5-gag-pol-env-U3-R-U5 as shown in Fig. la. The U3, R, and U5 sequences form the long terminal repeats (LTR) which are the regulatory elements that promote the expression of the viral genes and sometimes nearby cellular genes in infected hosts. The internal regions of the viral RNA code for the structural proteins: gag (p55, p17, p24 and p7 core proteins), pol (pl0 protease, p66 and p51 reverse transcriptase and p32 integrase) and env (gpl20 and gp41 envelope glycoproteins).

Gag codes for a polyprotein precursor that is cleaved by a viral protease into three or four structural proteins; pol codes for reverse transcriptase (RT) and the viral protease and integrase; env codes for the transmembrane and outer glycoprotein of the virus. The gag and pol genes are expressed as a genomic RNA while the env gene is expressed 25 as a spliced subgenomic RNA. In addition to the env gene there are other HIV genes produced by spliced subgenomic RNAs that contribute to the replication and biologic activities of the virus. These genes include: tat which encodes a protein that activates the expression of viral and some cellular genes; rev which encodes a protein that promotes the expression of unspliced or single-spliced viral mRNAs; nef which encodes a myristylated protein that appears to modulate viral production under certain conditions; vif which encodes a protein that affects the ability of virus particles to infect target cells but does not appear to WO 97/27319 PCT/US97/01609 -7affect viral expression or transmission by cell-to-cell contact; vpr which encodes a virion-associated protein; and vpu which encodes a protein that appears to promote the extracellular release of viral particles.

No disease better exemplifies the problem of viral drug resistance than AIDS. Drug resistant HIV isolates have been identified for nucleoside and non-nucleoside reverse transcriptase inhibitors and for protease inhibitors. The emergence of HIV isolates resistant to AZT is not surprising since AZT and other reverse transcriptase inhibitors only reduce virus replication by about 90%. High rates of virus replication in the presence of the selective pressure of drug treatment provide ideal conditions for the emergence of drug-resistant mutants. Patients at later stages of infection who have higher levels of virus replication develop resistant virus with AZT treatment more quickly than those at early stages of infection (Richman et al. (1990) J AIDS 3, 743-6). The initial description of the emergence of resistance to AZT identified progressive and stepwise reductions in drug susceptibility (Larder et al. (1989) Science 243, 1731-1734) This was explained by the recognition of multiple mutations in the gene for reverse transcriptase that contributed to reduced susceptibility (Larder et al. (1989) Science 246, 1155-1158). These mutations had an additive or even synergistic contribution to the phenotype of reduced susceptibility (Kellam et al.

(1992) Proc. Natl. Acad. Sci. 89, 1934-1938). The cumulative acquisition of such mutations resulted in 30 progressive decreases in susceptibility. Similar effects have been seen with non-nucleoside reverse transcriptase inhibitors (Nunberg et al. (1991) J Virol 65, 4887-4892; Sardanna et al. (1992) J Biol Chem 267, 17526-17530).

Studies of protease inhibitors have found that the selection of HIV strains with reduced drug susceptibility occurs WO 97/27319 PCT/US97/01609 -8within weeks (Ho et al. (1994) J Virol 68, 2016-2020; Kaplan et al. (1994) Proc. Natl. Acad. Sci. 91, 5597-5601). While recent studies have shown protease inhibitors to be more powerful than reverse transcriptase inhibitors, nevertheless resistance has developed. (Condra et al., Id. and Report 3rd Conference on Retroviruses and Opportunistic Infections, March 1996). Subtherapeutic drug levels, whether caused by reduced dosing, drug interactions, malabsorption or reduced bioavailability due to other factors, or self-imposed drug holidays, all permit increased viral replication and increased opportunity for mutation and resistance. (Id.) The selective pressure of drug treatment permits the outgrowth of preexisting mutants. With continuing viral replication: in the absence of completely suppressive anti-viral drug activity, the cumulative acquisition of multiple mutations can occur over time, as has been described for AZT and protease inhibitors of HIV. Indeed viral mutants multiply resistant to different drugs have 20 been observed (Larder et al. (1989) Science 243, 1731-1734; Larder et al. (1989) Science 246, 1155-1158; Condra et al.

(1995) Nature 374, 569-71). With the inevitable emergence of resistance in many viral infections, as with HIV for example, strategies must be designed to optimize treatment '25 in the face of resistant virus populations. Ascertaining the contribution of drug resistance to drug failure is a difficult problem because patients who are more likely to develop drug resistance are more likely to have other confounding factors that will predispose them to a poor prognosis (Richman (1994) AIDS Res Hum Retroviruses 901-905). In addition patients contain mixtures of viruses with different susceptibilities.

WO 97/27319 PCT/US97/01609 -9- Hepatitis B (HBV) HBV is a causative agent for acute and chronic hepatitis, which strikes about 200 million patients worldwide.

Zuckerman A.J. Trans. R. Soc. Trop. Med. Hygiene (1982) 76, 711-718. HBV infection acquired in adult life is often clinically inapparent, and most acutely infected adults recover completely from the disease and clear the virus.

Rarely, however, the acute liver disease may be so severe that the patient dies of fulminant hepatitis. A small fraction, perhaps 5 to 10%, of acutely infected adults, becomes persistently infected by the virus and develops chronic liver disease of varying severity. Neonatally transmitted HBV infection, however, is rarely cleared, and more than 90% of such children become chronically infected.

Because HBV is commonly- spread from infected mother to newborn infant in highly populated areas of Africa and Asia, several hundred million people throughout the world are persistently infected by HBV for most of their lives and suffer varying degrees of chronic liver disease, which greatly increases their risk of developing cirrhosis and hepatocellular carcinoma (HCC). Indeed, the risk of HCC is increased 100-fold in patients with chronic hepatitis, and the lifetime risk of HCC in. males infected at birth approaches 40%. Beasley RP et al., Lancet (1981) 2, 25 1129-1133. Accordingly, a large fraction of the world's population suffers from and dies of these late complications of HBV infection. The development of anti-HBV drugs has been long awaited, but has been hampered by the extremely narrow host range of HBV: HBV replicates mainly in human and 30 chimpanzee livers and not in experimental animals or in cultured cells. Tiollais, P et al. Nature (London) (1985) 317, 489-495.

WO 97/27319 PCT/US97/01609 Hepatitis B virus is a DNA virus with a compact genomic structure; despite its small, circular, 3200 base pairs, HBV DNA codes for four sets of viral products and has a complex, multiparticle structure. HBV achieves its genomic economy by relying on an efficient strategy of encoding proteins from four overlapping genes: S, C, P, and X. HBV is one of a family of animal viruses, hepadnaviruses, and is classified as hepadnavirus type 1. Similar viruses infect certain species of woodchucks, ground and tree squirrels, and Pekin ducks. All hepadnaviruses, including HBV, share the following characteristics: 1) three distinctive morphological forms exist, 2) all members have proteins that are functional and structural counterparts to the envelope and nucleocapsid antigens of HBV, 3) they replicate within the liver but can also exist in extrahepatic sites, 4) they contain an endogenous DNA polymerase with both RNA- and DNAdependent DNA polymerase activities, 5) their genomes are partially double stranded circular DNA molecules, 6) they are associated with acute and chronic hepatitis and hepatocellular carcinoma and 7) replication of their genome goes through an RNA intermediate which is reverse transcribed into DNA using the virus's endogenous RNA-dependent DNA polymerase activity in a manner analogous to that seen in retroviruses. In the nucleus of infected 25 liver cells, the partially double stranded DNA is converted to a covalently closed circular double stranded DNA (cccDNA) by the DNA-dependent DNA polymerase. Transcription of the viral DNA is accomplished by a host RNA polymerase and gives rise to several RNA transcripts that differ in their 30 initiation sites but all terminate at a common 'polyadenylation signal. The longest of these RNAs acts as the pregenome for the virus as well as the message for the some of the viral proteins. Viral proteins are translated from the pregenomic RNAs, and the proteins and RNA pregenome are packaged into virions and secreted from the hepatocyte.

WO 97/27319 PCT/US97/01609 -11- Although HBV is difficult to cultivate in vitro, several cells have been successfully transfected with HBV DNA resulting in the in vitro production of HBV particles.

There are three particulate forms of HBV: non-infectious 22nm particles, which appear as either spherical or long filamentous forms, and 42nm double-shelled spherical particles which represent the intact infectious hepatitis B virion. The envelope protein, HBsAg, is the product of the S gene of HBV and is found on the outer surface of the virion and on the smaller spherical and tubular structures.

Upstream of the S gene open reading frame are the pre-S gene open reading frames, pre-Si and pre-S2, which code for the pre-S gene products, including receptors on the HBV surface for polymerized human serum albumin and the attachment sites for hepatocyte receptors. The intact 42nm virion can be disrupted by mild detergents and the 27nm nucleocapsid core particle isolated. The core is composed of two nucleocapsid proteins coded for by the C gene. The C gene has two initiation codons defining a core and a precore region. The major antigen expressed on the surface of the nucleocapsid core is coded for by the core region and is referred to as hepatitis B core antigen (HBcAg). Hepatitis B e antigen (HBeAg) is produced from the same C gene by initiation at 25 the precore ATG.

Also packaged within the nucleocapsid core is a DNA polymerase, which directs replication and repair of HBV DNA.

The DNA polymerase is coded for by the P gene, the third and largest of the HBV genes. The enzyme has both DNA-dependent DNA polymerase and RNA-dependent reverse transcriptase activities and is also required for efficient encapsidation of the pregenomic RNA. The fourth gene, X, codes for a small, non-particle-associated protein which has been shown to be capable of transactivating the transcription of both WO 97/27319 PCTIUS97/01609 -12viral and cellular genes.

Although HBV replication is fairly well understood, early steps in HBV infection have not been well defined. Cellular receptors or attachment sites on the virions cannot be studied without appropriate tissue culture assays. In an effort to address this problem, certain cell lines have been developed, human hepatoblastoma cells Huh (HB 611) (Ueda, K.

et al., Virology (1989) 169, 213-216) and HepG2 cells (Galle, P.R. and Theilmann, L. Arzneim-Forsch. Drug Res.

(1990) 40, 1380-1382) for evaluation of anti-HBV drugs.

Recently, attention has focused on molecular variants of HBV. Variation occurs throughout the HBV genome, and -clinical isolates of HBV that do not express viral-proteins have been attributed to mutation in individual or even multiple gene locations. For example, variants have been described which lack nucleocapsid proteins, envelope proteins, or both. Two mutants have attracted attention.

The first is found in certain patients with severe chronic HBV infection. These patients were found to be infected with an HBV mutant that contained an alteration in the precore region rendering the virus incapable of encoding HBeAg. The most commonly encountered mutation in such 25 patients is a single base substitution, from G to A, which occurs in the second to last codon of the pre-C gene at nucleotide 1896. This substitution results in the replacement of the TGG tryptophan codon by a stop codon (TAG),.which prevents the translation of HBeAg. Patients with such precore mutants that are unable to secrete HBeAg S"tend to have severe liver disease that progresses rapidly to cirrhosis and that does not respond readily to anti-viral therapy.

WO 97/27319 PCT/US97/01609 -13- The second category of HBV mutants consists of escape mutants, in which a single amino acid substitution, from glycine to arginine, occurs at position 145 of the immunodominant a determinant common to all subtypes of HBsAg. This change in HBsAg leads to a critical conformational change that results in a loss of neutralizing activity by anti-HBs antibody.

Presently Available Viral Resistance Assays The definition of viral drug susceptibility is generally understood to be the concentration of the anti-viral agent at which a given percentage of viral replication is inhibited the ICso for an anti-viral agent is the concentration at which 50% of virus replication is inhibited)-. Thus, a decrease in viral drug susceptibility is the hallmark that an anti-viral has selected for mutant virus that is resistant to that anti-viral drug. Viral drug resistance is generally defined as a decrease in viral drug susceptibility in a given patient over time. In the clinical context, viral drug resistance is evidenced by the anti-viral drug being less effective or no longer being clinically effective in a patient.

At present the tools available to the researcher and 25 clinician to assess anti-viral drug susceptibility and resistance are inadequate. The classical test for determining the resistance and sensitivity of HIV to an anti-viral agent is complex, time-consuming, expensive, and is hazardous in that it requires the culture of pathogenic virus from each and every patient (Barre-Sinoussi et al (1983) Science 220, 868-871; Popovic et al. (1984) Science 224, 497-500). In this procedure, the patient's peripheral blood mononuclear cells (PBMC) are first cultured to establish a viral stock of known multiplicity of infection (moi), and the viral stock thereby produced is used to WO 97/27319 PCT/US97/01609 -14infect a target indicator cell line.. The resulting burst of viral replication is then typically measured in the presence and absence of an anti-viral agent by determining the production of viral antigens in the cell culture. Such tests can be performed reliably only in the hands of expert investigators, and may take two to three months to carry out at a cost of thousands of dollars per patient for each agent tested. Furthermore, as viral stocks of sufficient moi cannot be established from the PBMC of some HIV patients, the classical test for HIV resistance cannot be performed on all HIV-infected individuals. More significantly, in the course of generating the viral stock by passage of the virus in culture, the characteristics of the viruses themselves can change and may therefore obscure the true nature of the pat-ient'.s virus. Thus, the application of the classical test has been limited to gathering information about trends in clinical trials and has not been available for a prospective analysis which could be used to custom tailor anti-viral therapy for a given patient. Notwithstanding 20 these limitations, the classical test has two important qualities: it is specific for the agent under evaluation, and it provides information on the phenotype of the patient's own virus, that is, the concentration of the drug which inhibits 50% of viral replication (IC 50 A number of attempts have been made to improve upon the classical test, but each of these has serious shortcomings.

The first type of these tests can be described as nonspecific in that they do not determine the characteristics of a patient's own virus at all, but rather •provide an independent measure of the course of the infection. Among these tests are those which measure the patient's CD4 T cell count, the hallmark of HIV disease progression (Goedert et al. (1987) JAMA 257, 331-334), those which measure viral antigen levels p24 core antigen WO 97/27319 PCTUS97/0609 (Allain et al. (1987) N. Engl. J. Med. 317, 1114-1121)), and those which measure viral RNA and DNA levels quantitative polymerase chain reaction and branched DNA assays (Piatak et al. (1993) Science 259, 1749-1754; Urdea (1993) Clin. Chem. 39, 725-726)). The primary disadvantage of such nonspecific tests is that they do not provide any information on viral drug resistance per se, but rather attempt to infer this information from the apparent course of the patient's disease. In addition, many factors other than viral drug resistance can affect the level of the parameter under consideration. In other words, CD4* T cell counts, p24 antigen levels and HIV viral RNA levels can vary for reasons other than drug resistance during the course of disease.

Another modified classical test amplifies the viral gene that is the target of the anti-viral agent. In this test the viral gene from a given patient is amplified and then recombined into a biologically active proviral clone of HIV.

This proviral clone is transfected into human cells to generate a viral stock of known moi which can then be used to infect a target indicator cell line. In the manner of the classical test, one then determines the production of viral antigens in the presence or absence of the anti-viral agent. One such assay described by Kellam and Larder (1994) Antimicrobial Agents and Chemo. 38, 23-30, involves PCR amplification of reverse transcriptase coding sequences from a patient, which is then introduced into a proviral DNA clone by homologous recombination to reconstitute the complete viral genome including the reverse transcriptase gene which was deleted. The resulting recombinant virus *Vt: produced from such clones is then cultured in T-cell lines, and the drug sensitivity is tested in the HeLa CD4' plaque reduction assay. However, this class of test still requires the culturing of virus to determine drug resistance, and is WO 97/27319 PCT/~S97/01609 -16thus difficult, lengthy and costly and requires the laboratory investigator to handle hazardous viral cultures.

Furthermore, given the attendant variation of the virus itself during the culture process, the results may be correspondingly inaccurate.

A second class of test attempts to provide specific information on the genotype of the patient's HIV, with the ultimate goal of correlating this genotypic information with the virus' drug resistant phenotype. Indeed, specific amino acid substitutions within viral genes such as reverse transcriptase and protease genes have been shown to correspond to specific levels of viral resistance to reverse transcriptase and protease inhibitors, respectively (Larder et al. (1994) J. Gen Virol.. .75, 951-957). A major shortcoming associated with such an analysis is that it is indirect and can be obfuscated by secondary mutations which have been shown to add to or counter the effects of the first mutation. It is the complex interplay of all amino acid residues within a given viral polypeptide which ultimately determines the gene product's activity in the presence or absence of an inhibitor. Thus, a database of vast and impractical proportions would be necessary to interpret the status of drug resistance or sensitivity of a 25 given genotype, given the number of potential amino acid changes in the HIV genome.

A third class of test, a recently developed bacterial-based assay *makes use of a molecularly cloned viral gene (specifically, the reverse transcriptase gene) which has been inserted into a bacterial expression vector. Upon Stransformation of special strains of E. coli which are deficient in the bacterial DNA polymerase I, the cloned reverse transcriptase gene can rescue the growth of the bacteria under selected growth conditions. In making E.

WO 97/27319 PCTIUS97/01609 -17coli dependent upon reverse transcriptase for their growth, one can ascertain the effects of certain reverse transcriptase inhibitors on the activity of the viral gene (PCT Application No. WO 95/22622). A major shortcoming with this approach, however, is that the inhibitor may be transported across the cell membrane and metabolized differently by the bacteria than it is by a human cell, and as a result the concentration of the true metabolic inhibitor of the reverse transcriptase may be grossly different in the bacterium than it would be in a relevant human cell target of infection, or the true inhibitor may be absent altogether. Indeed, nucleoside metabolism is known to differ markedly between human and bacterial cells.

Another significant shortcoming of this approach is that the assay measures- DNA-dependent DNA polymerase activity of reverse transcriptase but not the RNA-dependent DNA polymerase, strand transfer or RNAse H activities of the reverse transcriptase. Thus, an anti-viral compound which S. acts, at least in part, on these other activities would not 20 have its full inhibitory activity in this assay. Yet another difficulty with this approach is that it is a S* growth-based test; thus if an inhibitor a nucleoside analog) also blocks bacterial growth for reasons other than its effects on reverse transcriptase, it can not be adequately tested in this system.

Viral Vectors Viral vectors and particularly retroviral vectors have been used for modifying mammalian cells because of the high 30 efficiency with which retroviral vectors infect target cells and integrate into the target cell genome. Because of their ability to insert into the genome of mammalian cells much attention has focused on retroviral vectors for use in gene therapy. Details on retroviral vectors and their use can be found in patents and patent publications including European WO97127319 PCTIUS97/01609 -18- Patent Application EPA 0 178 220, U.S. Patent 4,405,712, PCT Application WO 92/07943, U.S. Patent 4,980,289, U.S. Patent 5,439,809 and PCT Application WO 89/11539. The teachings of these patents and publications are incorporated herein by reference.

One consequence of the emphasis on retroviral vector technology has been the development of packaging cell lines.

A major problem with the use of retroviruses is the possibility of the spread of replication-competent retrovirus. There is thus a need for producing helper vectors which could not be processed into virions. As a result packaging defective vectors and packaging cell lines were developed. Details on packaging-defective vectors and packaging cell lines can be found in patents and patent publications, including U.S. Patent 5,124,263, European Patent Application Pub. No. 0386 882, PCT Application No. WO 91/19798, PCT Application No. WO 88/08454, PCT Application No. WO 93/03143, U.S. Patent No. 4,650,764, U.S. Patent 20 4,861,719 and U.S. Patent 5,278,056, the disclosures of which are incorporated herein by reference.

It is an object of this invention to provide a drug susceptibility and resistance test capable of showing 25 whether a viral population in a patient is resistant to a given prescribed drug. Another object of this invention is to provide a test that will enable the physician to substitute one or more drugs in a therapeutic regimen for a patient that has become resistant to a given drug or drugs S 30 after a previous course of therapy. Yet another object of this invention is to provide a test that will enable selection of an effective drug regimen for the treatment of virus infections. Yet another object of this invention is to provide a safe, standardized, affordable, rapid, precise and reliable assay of drug susceptibility and resistance for WO 97/27319 PCT/US97/01609 -19clinical and research application. Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds which act on specific viral genes and/or viral proteins particularly with respect to viral drug resistance and cross resistance. It is also an object of this invention to provide the means and compositions for evaluating viral drug resistance and susceptibility. This and other objects of this invention will be apparent from the specification as a whole.

.e:e e eoeeo .°e.ei eeoeo oeoeo* WO 97127319 PCTIUS97/01609 Summary of the Invention Objects of the present invention are accomplished by a novel test for determining susceptibility for an anti-viral drug comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring expression of the indicator gene in a target host cell; and comparing the expression of the indicator gene from step with the expression of the indicator gene measured when steps are carried out in the absence of the anti-viral drug, wherein a test concentration of the anti-viral drug is present at steps at steps or at step (c) This invention also provides a method for determining anti-viral drug resistance in a patient comprising: (a) developing a standard curve of drug susceptibility for an anti-viral drug; determining anti-viral drug 20 susceptibility in the patient using the susceptibility test described above; and comparing the anti-viral drug susceptibility in step with the standard curve determined in step wherein a decrease in anti-viral susceptibility indicates development of anti-viral drug resistance in the patient.

This invention further provides a method for determining anti-viral drug resistance in a patient comprising: (a) determining anti-viral drug susceptibility in the patient at 30 a first time according to the above method, wherein the patient-derived segment is obtained from the patient at said time; determining anti-viral drug susceptibility of the same patient at a later time; and comparing the anti-viral drug susceptibilities determined in step and wherein a decrease in anti-viral drug susceptibility at WO 97/27319 PCTIUS97/01609 -21the later time compared to the first time indicates the development or progression of anti-viral drug resistance in the patient.

The assay of this invention enables a physician to assess whether a viral gene encoding a viral protein or a functional viral sequence, each of which maybe the target of an anti-viral agent, has mutated to render the drug less effective. More particularly, the novel assay of this invention enables one to determine whether a virus has become resistant to a particular anti-viral drug.

Furthermore, this invention enables a physician to assess drug susceptibility and resistance of combination therapy.

In addition the assay enables one to alter a therapeutic -regimen- prospectively by-. testing particular. drug(s) or combinations of drugs and determining whether these drugs, alone or in combination, inhibit one or more viral gene(s) and/or viral protein(s). This invention provides significant advantages over presently available assays by providing a safer, more affordable, more reliable, more rapid and more effective drug susceptibility and resistance assay to assess the therapeutic efficacy of particular anti-viral drug(s) or combinations of drugs enabling a physician to optimize treatment. The assay of this invention has the significant advantage of enabling the evaluation of resistance and susceptibility at all stages of drug development: 1) during preclinical evaluation of candidate compounds; 2) during clinical evaluation of new drugs;. 3) during patient therapy enabling design of an effective therapeutic regimen to overcome the problem of drug resistance; and 4) as part of epidemiologic surveillance, evaluating the prevalence of resistance during the use of approved and experimental drugs.

The present invention is directed to the methods and WO 97/27319 PCT/US97/01609 -22compositions of assessing drug susceptibility and resistance, including: a) the assay method of determining the susceptibility and resistance of a patient-derived segment to an anti-viral drug; b) compositions including resistance test vectors comprising a patient-derived segment and an indicator gene; and c) host cells containing the resistance test vectors. This invention is further directed to the compositions and methods of constructing the vectors and host cells which are used in the drug susceptibility and resistance assay of this invention.

In one aspect of the invention there is provided a method for determining susceptibility for an anti-HIV drug comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring expression of the indicator gene in a target host cell; and comparing the expression of the indicator gene from step with the expression of the indicator gene 20 measured when steps are carried out in the absence of the anti-HIV drug, wherein a test concentration of the anti-HIV drug is present at steps at steps or at step In one aspect of the invention there is provided a method for determining susceptibility for an anti-HIV drug comprising: introducing a resistance test vector 30 comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step (a) measuring an indicator in a target host cell wherein said indicator is a DNA or RNA structure; and comparing the measurement of the indicator from step with the measurement of the indicator when steps are carried WO 97/27319 PCT/US97/01609 -23out in the absence of the anti-HIV drug, wherein a test concentration of the anti-HIV drug is present at steps at steps or at step In one aspect of the invention there is provided a method for determining susceptibility for an anti-HBV drug comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring expression of the indicator gene in a target host cell; and comparing the expression of the indicator gene from step with the expression of the indicator gene measured when steps are carried out in the absence of the anti-HBV drug, wherein a test concentration of the anti-HBV drug is present at steps at steps or at step In one aspect of the invention there is provided a method for determining susceptibility for an anti-HBV drug comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring an indicator in a target host cell wherein said indicator is a DNA or RNA structure; and comparing 25 the measurement of the indicator from step with the measurement of the indicator when steps are carried out in the absence of the anti-HBV drug, wherein a test concentration of the anti-HBV drug is present at steps at steps or at step This invention also provides a method for determining anti-HIV drug resistance in a patient comprising:(a) developing a standard curve of drug susceptibility for an anti-HIV drug; determining anti-HIV drug susceptibility in the patient using the susceptibility test described WO 97/27319 PCT/US97/01609 -24above; and comparing the anti-HIV drug susceptibility in step with the standard curve determined in step wherein a decrease in anti-HIV susceptibility indicates development of anti-HIV drug resistance in the patient.

This invention also provides a method for determining anti-HBV drug resistance in a patient comprising: (a) developing a standard curve of drug susceptibility for an anti-HBV drug; determining anti-HBV drug susceptibility in the patient using the susceptibility test described above; and comparing the anti-HBV drug susceptibility in step with the standard curve determined in step wherein a decrease in anti-HBV susceptibility indicates development of anti-HBV drug resistance in the patient.

This invention also provides a method for evaluating the biological effectiveness of a candidate anti-viral drug compound comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring expression of the indicator gene in a target host cell; and comparing the expression of the indicator gene from step with the expression of the :indicator gene measured when steps are carried out 25 in the absence of the candidate anti-viral drug compound, wherein a test concentration of the candidate anti-viral drug compound is present at steps at steps or at step 30 This invention also provides a method for evaluating the biological effectiveness of a candidate anti-viral drug compound comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring an indicator in a target host cell WO 97/27319 PCT/US97/01609 wherein said indicator is a DNA or RNA structure; and (d) comparing the measurement of the indicator from step (c) with the measurement of the indicator when steps are carried out in the absence of the candidate anti-viral drug compound, wherein a test concentration of the candidate anti-viral drug compound is present at steps at steps or at step The resistance test vector comprising the patient-derived viral segment(s) HIV, HBV, etc.) and the indicator gene may additionally include one or more non-patient-derived segments. In one embodiment the resistance test vector is constructed from a genomic viral vector which may include a deletion in one or more genes.

-For example, in the case of HIV, env is deleted in a resistance test vector which otherwise preserves the mRNA expression and processing characteristics of the complete virus. Alternatively, the resistance test vector is constructed from a subgenomic viral vector which may include 20 only one or a few viral genes which are typically the target of the anti-viral drug. For example in the case of HBV, one or more of the HBV genes encoding certain structural and enzymatic functions necessary for HBV DNA replication and virus particle formation C, S and X genes) may be 25 deleted from a resistance test vector which otherwise preserves mRNA expression and processing characteristics of the complete virus. The resistance test vector further S. comprises either the native enhancer/promoter of the particular virus or a foreign enhancer/promoter for the 30 expression of the anti-viral target genes.

The expression of the indicator gene in the resistance test vector in the target cell is ultimately dependent upon the action of the patient-derived segment sequences. The indicator gene may be functional or non-functional. In the WO 97/27319 PCTIUS97/01609 -26case of a non-functional indicator gene, the indicator gene is not efficiently expressed in a host cell transfected by the resistance test vector until it is converted into a functional indicator gene through the action of one or more of the patient-derived segment products. In one embodiment, the indicator gene is rendered non-functional through use of a permuted promoter, i.e. a promoter that, although in the same transcriptional orientation as the indicator gene, follows rather than precedes the coding sequence of the indicator gene. In addition, the orientation of the non-functional indicator gene is opposite to that of the viral promoters or LTRs. Thus the coding sequence of the non-functional indicator gene can neither be transcribed by the permuted promoter nor by the viral promoters. In the case of HIV, the indicator gene is rearranged as a consequence of reverse transcription so that the permuted promoter now precedes the indicator gene sequence, which as a result can be functionally expressed. In the case of HBV, the indicator gene is rearranged as a consequence of circularization of the genome during HBV replication. In a second embodiment, the indicator gene is rendered non-functional through use of a permuted coding region, i.e.

an indicator gene coding region in which the 5' portion of the coding region follows rather than precedes the 3' portion of the coding region. In this configuration, no mRNA is expressed which can give rise to a functional indicator gene product. In the case of HIV, the indicator gene is rearranged as a consequence of reverse transcription so that the 5' coding region of the indicator gene now precedes the 3' coding region, and as a result the indicator gene can be functionally exrressed. In the case of HBV, the indicator gene is rearranged as a result of circularization of the genome during HBV replication. In a third embodiment, the indicator gene is rendered non-functional through use of an inverted intron, i.e. an intron inserted WO 97/27319 PCTIUS97/01609 -27into the coding sequence of the indicator gene with a transcriptional orientation opposite to that of the indicator gene. In addition, the indicator gene itself contains a functional promoter with the entire transcriptional unit oriented opposite to the viral promoters. Thus the non-functional indicator gene is in the wrong orientation to be transcribed by the viral LTRs in the case of HIV or the HBV enhancer-promoter and it cannot be functionally transcribed by its own promoter, as the inverted intron cannot be properly excised by splicing.

However, in the case of retroviruses and hepadnaviruses, and HIV and HBV specifically, transcription by the viral promoters result in the formation of mRNA in which removal of the inverted intron can occur by splicing. In retroviruses, as.a consequence of reverse transcription of the resulting spliced transcript and the integration of the resulting provirus into the host cell chromosome, the indicator gene can now be functionally transcribed by its own promoter. In HBV, as a consequence of reverse 20 transcription of the resulting spliced transcript and circularization of the genomic DNA in the host cell, the *indicator gene can now be functionally transcribed by its own promoter.

ooee0: e Resistance test vectors comprising a non-functional indicator gene can be used to carry out resistance tests in either, a particle-based or non-particle-based assay. The particle based assay. is based on resistance test vector viral particles, which' are replication defective, being produced by the resistance test vector host cells. The trans-acting factors necessary for production of the resistance test vector viral particles are provided by the packaging expression vectors which are transfected into the WO 97/27319 PCTIUS97/01609 -28packaging host cell. In contrast the non-particle based resistance test is performed by transfection of a single host cell with a resistance test vector in the absence of packaging expression vectors.

In the case of the functional indicator gene, the functional indicator gene is efficiently expressed in a first host cell transfected by the resistance test vector (referred to herein as a resistance test vector host cell). Thus, the function of the indicator gene in the resistance test vector host cell is not dependent on the patient-derived segment.

However, the capacity of the indicator gene to be expressed in a second host cell (referred to herein as a target host cell) is dependent on the production of functional resistance test vector viral particles in the resistance test vector host cell. Thus, the activity of the indicator gene in the target host cells is dependent on the activity of the patient-derived segments.

20 In another aspect this invention is directed to anti-viral drug susceptibility and resistance tests for HIV/AIDS or HBV. Particular resistance test vectors of the invention for use in the HIV/AIDS anti-viral drug susceptibility and resistance test are identified as well as resistance test 25 vector host cells. In yet another aspect this invention is directed to anti-viral drug susceptibility and resistance tests for hepatitis. Similarly in the case of HBV, particular resistance test vectors (also referred to herein as resistance test vector systems) of the invention for use :30 in the HBV anti-viral drug susceptibility and resistance test are identified as well as resistance test vector host 4 cells.

In yet another aspect this invention provides for the identification and assessment of the biological 29 effectiveness of potential therapeutic anti-viral compounds for the treatment of viral diseases. In still another aspect the invention is directed to a host cell transfected with one or more vectors to assess drug susceptibility. In another aspect, the invention is directed to a novel resistance test vector comprising a patient-derived viral gene(s) and an indicator gene.

In the specification the term "comprising" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term "comprising" such as "comprise" and "comprises".

*o.

oooo oooo •go• *o oo o*o •go oo o* *o *o• •o WO 97/27319 PCT/US97/01609 Brief Description of the Drawings .0 Fig. 1. A. Diagrammatic representation of the DNA genomic structure of HIV-1. Viral proteins are encoded in each of the three reading frames by the gag, pol, vif, vpr, tat, rev, vpu, env and nef genes.

The RNA is transcribed from viral DNA and processed by viral and cellular enzymes, giving rise to both genomic viral RNA and mRNA. The U3, R and U5 elements of the viral long terminal repeat (LTR) are indicated.

B. Generalized diagrammatic representation of the HIV genomic viral vector which contains the following elements in a 5' to 3' orientation: 1) an HIV-LTR U3 region, 2) an HIV-LTR R region, 3) an HIV-LTR U5 region 4) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, and 5) a 3' HIV-LTR.

C. Generalized diagrammatic representation of the HIV genomic viral vector which contains the following elements in a 5' to 3' orientation: 1) a CMV IE enhancer-promoter, 2) an HIV-LTR R region, 3) an HIV-LTR U5 region, 4) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, and 5) a 3'

HIV-LTR.

D. Generalized diagrammatic representation of the HIV subgenomic viral vector which contains the following elements in a 5' to 3' orientation: 1) an HIV-LTR U3 region, 2) an HIV-LTR R region, 3) an HIV-LTR U5 region, 4) the coding region of the HIV gag-pol gene, and 5) a 3' HIV- LTR.

WO 97/27319 PCTIUS97/01609 -31- E. Generalized diagrammatic representation of the HIV subgenomic viral vector which contains the following elements in a 5' to 3' orientation: 1) a CMV IE enhancer-promoter, 2) an HIV-LTR R region, 3) an HIV-LTR U5 region, 4) the coding region of the HIV gag-pol gene, and 5) a 3' HIV-

LTR.

Fig. 2. A. Diagrammatic representation of the DNA genomic structure of HIV-1.

B. Diagrammatic representation of the resistance test vector comprising a nonfunctional indicator gene comprising a permuted promoter having the following elements in a 5' to 3' orientation: 1) an HIV-LTR U3 region (pLG-lucPP-HS and pLG-lucPP-PB) or a CMV IE enhancer-promoter (pCG-lucPP-HS and pCG-lucPP-PB), 2) an HIV-LTR R region, 3) an HIV-LTR U5 region containing an inserted T7 phage RNA polymerase promoter (herein referred to as T7 promoter) with a transcriptional orientation .opposite to that of the LTRs, 4) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 5) a patient-derived segment(s) inserted into the patient sequence acceptor sites, 6) an indicator gene cassette inserted into the deleted env gene, and 7) a 3' HIV-LTR.

C. Diagrammatic representation showing the conversion of the non-functional indicator gene (permuted promoter) in the resistance test vector, described in 2 to a functional indicator gene by reverse transcriptase. The WO 97/27319 PCT/US97/01609 -32conversion to a functional indicator gene results from the repositioning of the T7 promoter relative to the indicator gene coding region.

Fig. 3. A. Diagrammatic representation of the DNA genomic structure of HIV-1.

B. Diagrammatic representation of the packaging expression vector pLTR-HIV3' which provides the vif, vpr, tat, rev, vpu and nef genes, each of which is expressed as a spliced subgenomic mRNA transcribed from the HIV LTR U3 region.

C. Diagrammatic representation of the packaging expression vector pCMV-HIV3' which provides the vif, vpr, tat, rev, vpu and nef genes, each of which is expressed as a spliced subgenomic mRNA transcribed from the CMV IE enhancer-promoter.

D. Diagrammatic representation of the packaging expression vector pVL-env4070A [pCXAS (4070A env)] which provides the amphotrophic MLV env gene product, by transcription from the CMV IE enhancer-promoter.

Fig. 4. A. Diagrammatic representation of the DNA genomic structure of HIV-1.

B. A generalized diagrammatic representation of the resistance test vectors comprising a .nonfunctional indicator gene comprising a permuted coding region containing the following elements in a 5' to 3' orientation: 1) an HIV-LTR U3 region (pLG-lucPC-HS and pLG-lucPC-PB) or a first CMV IE enhancer-promoter (pCG-lucPC-HS WO 97/27319 PCTIUS97/01609 -33and pCG-lucPC-PB), 2) the HIV-LTR R and regions, 3) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 4) a patient-derived segment(s) inserted into the patient sequence acceptor sites, 5) a first indicator gene cassette containing the 5' coding region of the luciferase gene, inserted into the deleted env gene, 6) a second indicator gene cassette containing the 3' coding region of the luciferase gene, inserted into a deleted 3' HIV-LTR U3 region, and 7) a 3' HIV-LTR R and U5 region.

C. Diagrammatic representation showing the conversion of the non-functional indicator gene (permuted coding region) in the resistance test vector, described in to a functional indicator gene by reverse transcriptase.

Following reverse transcription and strand transfer, the luciferase 3' coding region is copied from the 3' LTR to the 5' LTR, permitting the transcription of mRNA which can be spliced to generate a functional luciferase coding region.

Fig. 5. A. Diagrammatic representation of the DNA genomic structure of HIV-1.

SB. A generalized diagrammatic representation of the resistance test vectors comprising a nonfunctional indicator gene comprising an inverted intron containing the following elements in a 5' to 3' orientation: 1) an HIV-LTR U3 region (pLG-lucII-HS and pLG-lucII-PB) or a first CMV IE enhancer-promoter (pCG-lucII-HS and pCG-lucII-PB), 2) the HIV-LTR R and U5 regions, WO 97/27319 PCT/US97/01609 -34- 3) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 4) patient-derived segment(s) inserted into the patient sequence acceptor site, 5) an indicator gene cassette inserted into the deleted env gene, and 5) a 3' HIV-LTR.

C. Diagrammatic representation showing the conversion of the non-functional indicator gene (inverted intron) in the resistance test vector, described in to a functional indicator gene by reverse transcriptase. The overall transcriptional orientation of the indicator gene cassette is opposite to that of the first CMV enhancer-promoter and viral LTRs, while the orientation of the artificial intron is the same as the latter elements. Transcription of the indicator gene by the second CMV enhancer-promoter does not lead to the production 20 of functional transcripts as the inverted intron cannot be spliced in this orientation.

Transcription of the indicator gene by the viral LTR or the first CMV IE enhancer-promoter, however, leads to the removal of the inverted 25 intron by RNA splicing, although the indicator @000 gene is still not functionally expressed as the resulting transcript has an antisense orientation. Following the reverse transcription of this transcript and integration of the 30 resultant proviral DNA, the indicator gene can be functionally transcribed by the second CMV enhancer-promoter as the inverted intron has been previously removed.

WO 97/27319 PCT/US97/01609 Fig. 6. A. Diagrammatic representation of the DNA genomic structure of HIV-1 is shown above and (b) B. A generalized diagrammatic representation of the resistant test vectors comprising a functional indicator gene having the following elements in a 5' to 3' orientation: 1) an HIV-LTR U3 region (pLG-luc-HS-1 and pLG-luc-PB-1) or a first CMV IE enhancer-promoter (pCG-luc-HS-1 and pCG-luc-PB-1), 2) the HIV-LTR R and U5 regions, 3) the coding region of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 4) a functional indicator gene cassette inserted into the deleted env gene, with a transcriptional orientation opposite to the viral .LTRs and 5) a 3' HIV-LTR.

C. A generalized diagrammatic representation of the resistance test vectors comprising a functional indicator gene cassette (pLG-luc-HS-2, pLG-luc-PB-2, pCG-luc-HS-2 and pCG-luc-PB-2) in which the transcriptional orientation of the indicator gene cassette is the same as the viral LTRs.

20 25 Fig. 7. A. Demonstration of drug susceptibility using the resistance test vectors, pCG-CXCN(F-lucP) 2-AA and pCG-CXAT(F-lucP)2-AA. Data are presented as luciferase gene activity in target host cells as Relative Light Units (RLU) in the absence of AZT or in the presence of 5 mM AZT.

WO 97/27319 PCT/US97/01609 -36- B. Drug susceptibility and resistance test performed with the resistance test vector, pCG- CXCN(F-lucP)2-AA containing pre-AZT treatment and post-AZT treatment "test" patient-derived segments.

The resistance test vectors are derived from the genomic indicator gene viral vector, pCG-CXCN(FlucP)2-AA. Data are presented as percent inhibition of luciferase gene activity in target host cells vs. AZT concentration (log 10 pCG- CXCN(F-lucP)2-AA is plotted as solid boxes. pCG- CXCN(F-lucP)2-AA containing patient-derived segments prior to AZT treatment is plotted as solid circles and post-AZT treatment as solid triangles.

C. Drug susceptibility and resistance test performed with the resistance test vector, pCG- CXCN(F-lucP)2-AA containing the reverse transcriptase segment derived from the biologically active proviral clone, pNL4-3. Data are presented 20 as percent inhibition of luciferase gene activity in target host cells vs. nevirapine concentration (log 10 and is plotted as solid boxes.

D. Drug susceptibility and resistance test 25 performed with the resistance test vector, pCG- CXCN(F-lucP)2-AA containing the protease segment derived from the biologically active proviral clone, pNL4-3. Data are presented as percent inhibition of luciferase gene activity in target host cells vs. indinavir concentration (log 10 )and is plotted as solid boxes.

WO 97/27319 PCT/US97/01609 -37- 20 oeoo 25 ooooo Fig. 8 A. Diagrammatic representation of the RNA pregenomic structure of HBV. Pregenomic RNA is shown as a solid line. Direct repeat sequences (DR) are shown as closed rectangles. The positions of the encapsidation signal sequence is shown The C, P, S, and X genes are shown as open rectangles. The terminal protein (TP), spacer, DNA polymerase/reverse transcriptase (pol/RT), and RNase H regions of the P gene are indicated. Sites of C, P, S, and X translation initiation are indicated by shaded triangles.

B. A generalized diagrammatic representation of the subgenomic indicator gene viral vector, pCS-HBV(NF-IG)II-(PSAS-), a. component of the resistance test vector system, comprising an indicator gene cassette and an inverted intron containing the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome and the DR1 and 5' (the pre-C ORF translation initiation codon is eliminated), a non-functional indicator gene cassette in which the indicator gene ORF contains an inverted intron, the region of the HBV genome containing DR2, DR1*, the and the 3' HBV polyadenylation (pA) signal region.

C. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the subgenomic indicator gene viral vector, pCS-HBV(F-IG)II(PSAS-), containing a functional indicator gene cassette assembled as a result of HBV viral replication.

c WO 97/27319 PCT/US97/01609 -38- D. Diagrammatic representation of one example of a packaging vector, pPK-CPX, a component of the resistance test vector system comprising a patient-derived segment containing the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the region of the HBV genome spanning from the C ORF translation initiation codon to the 3' pA signal and including the C, P, S, and X genes. The C gene of the packaging vector, pPK-CPX, is modified such that it does not contain the pre-C ORF sequences and does not express the S proteins (as shown by the X at the translation initiation sites).

E. Diagrammatic representation of an additional packaging vector, pPK-S, providing the S gene proteins, that is cotransfected with the resistance test vector system comprising the 20 indicator gene viral vector, pCS-HBV(NF-IG) II-(PSAS-), and the packaging plasmid, pPK-CPX.

F. A generalized diagrammatic representation of 25 the resistance test vector, pCS-HBV(NF-IG)II-(PSAS+), comprising a non-functional indicator gene with an inverted intron containing the following elements in a to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome and the DR1 and 5' (the pre-C ORF translation initiation codon is eliminated), indicator gene cassette (containing an inverted intron) within the region of the HBV genome which contains a patient-derived P gene WO 97/27319 PCTIUS97/01609 -39segment, the region of the HBV genome containing DR2, DR1*, the 3'E, and the 3' HBV pA signal region.

G. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the resistance test vector, pCS-HBV(F-IG)II-(PSAS+), containing a functional indicator gene cassette and a patient-derived P gene segment assembled as a result of HBV viral replication.

H. Diagrammatic representation of a packaging vector, pPK-CSX, providing the C, S and X gene proteins, that is cotransfected with the resistance test vector, pCS-HBV(NF-IG)II- (PSAS+).

Fig. 9 A. Diagrammatic representation of the RNA pregenomic structure of HBV.

B. Diagrammatic representation of an HBV indicator gene viral vector containing a non-functional indicator gene cassette. Primer binding sites for the amplification of a target DNA sequence are shown. The location and :25 orientation of the forward primer (Pf) and reverse primer (Pr) binding sites do not constitute a functional amplification unit in the linear form of the vector that is used to transfect packaging host cells. The Pr binding site is designed to span the junction sequence that is generated by splicing of the pregenomic

RNA.

WO 97/27319 PCTIUS97/01609 o e C. Diagrammatic representation of the rc-DNA form of the indicator gene viral vector described in Primer binding sites for the amplification of a target DNA sequence are shown. The location and orientation of the Pf and Pr primer binding sites constitute a functional amplification unit in the plus strand DNA component of the rc-DNA form and the plus and minus strand DNA components of the cccDNA form of the vector that is generated by HBV DNA replication within the virus particles produced in packaging host cells. The Pr binding site is assembled by the splicing of the pregenomic RNA.

D. Diagrammatic representation of an HBV indicator gene viral vector containing a non-functional indicator gene cassette. Primer binding sites for the amplification of a target DNA sequence are shown. The location and orientation of the Pf and Pr binding sites constitute a functional amplification unit in the linear form of the vector that is used to transfect packaging host cells, but the Pf binding site is not adjacent to the binding site of the exonuclease detection probe (probe) in the unspliced linear form of the vector. This arrangement of Pf, Pr, and probe binding sites does not constitute an efficient exonuclease detection unit.

r r r ~r WO 97/27319 PCTUS97/01609 -41- E. Diagrammatic representation of the rc-DNA form of the indicator gene viral vector described in Fig. 9D. Primer binding sites for the amplification of a target DNA sequence are shown.

The location and orientation of the Pf and Pr primer binding sites constitute a functional amplification unit in the rc-DNA and cccDNA forms of the vector that are generated by HBV DNA replication in packaging host cells. The location of the Pf binding site is brought immediately adjacent to the binding site of the exonuclease detection probe (probe) in the rc-DNA and cccDNA forms of the vector. This arrangement of Pf, Pr, and probe binding sites constitute an efficient exonuclease detection unit.

F. Diagrammatic representation of an HBV indicator gene viral vector. Primer binding sites for the amplification of a target DNA sequence are shown. The location and orientation of the forward primer (Pf) and reverse primer (Pr) binding sites do not constitute a functional amplification unit in the linear form of the vector that is used to transfect packaging host S 25 cells.

G. Diagrammatic representation of the rc-DNA form of the indicator gene viral vector described in Primer binding sites for the amplification of a target DNA sequence are shown. The location and orientation of the Pf and Pr primer binding 'sites constitute a functional amplification unit in the plus strand DNA component of the rc-DNA form and the plus and minus strand DNA components WO 97/27319 PCT/US97/01609 -42of the cccDNA form of the vector that is generated by HBV DNA replication within the virus particles produced in packaging host cells.

Fig. 10 A. Diagrammatic representation of the RNA pregenomic structure of HBV.

B. A generalized diagrammatic representation of the subgenomic indicator gene viral vector, pCS-HBV(NF-IG)PP-(PSAS-), a component of the resistance test vector system comprising a non-functional indicator gene with a permuted promoter containing the following elements in a to 3' orientation: the CMV IE -enhancer promoter region, the region of the HBV genome and the DR1 and 5' (the pre-C ORF translation initiation codon is eliminated), (3) a non-functional indicator gene cassette assembled such that the promoter region is positioned i.e. downstream, of the indicator gene ORF, the 3' region of the HBV genome containing DR2, DR1*, the and the 3' HBV pA signal region (the pre-C ORF translation initiation codon is eliminated). The packaging :25 vector, pPK-CPX, a component of the resistance test vector system comprising a patient-derived P gene segment is shown in Fig. 8D and the S packaging vector, pPK-S, is shown in Fig. 8E.

C. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the subgenomic indicator gene viral vector, pCS-HBV(F-IG)PP(PSAS-), containing a functional indicator gene cassette assembled as a result of HBV viral replication.

WO 97/27319 PCTIUS97/01609 -43- D. A generalized diagrammatic representation of the resistance test vector, pCS-HBV(NF-IG)PP(PSAS+), comprising a non-functional indicator gene with a permuted promoter containing the following elements in a to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome and the DR1 and 5' copy of (the pre-C ORF translation initiation codon is eliminated) an enhancer-promoter region (permuted promoter), the P gene containing the patient-derived segment the indicator gene ORF an internal ribosome entry site (IRES), and the 3' region of the HBV.genome containing DR2, DR1*, the 3'E, and the 3' HBV pA signal region (the pre-C ORF translation initiation codon is eliminated). The packaging S. vector, pPK-CSX, providing the C, S and X genes .20 is cotransfected with the resistance test vector, pCS-HBV(NF-IG)PP(PSAS+), and is shown in Fig. 8H.

E. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the :25 resistance test vector, pCS-HBV(F-IG)PP(PSAS+), containing a functional indicator gene cassette and a patient-derived P gene segment assembled as a result of HBV viral replication.

Fig. 11 A. Diagrammatic representation of the RNA pregenomic structure of HBV.

WO 97/27319 PCT/US97/01609 -44- B. A generalized diagrammatic representation of the subgenomic indicator gene viral vector, pCS-HBV(NF-IG)PPTIS-(PSAS-), a component of the resistance test vector system comprising a non-functional indicator gene with a permuted promoter and translation initiation site containing the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5' (the pre-C ORF translation initiation codon is eliminated), (3) an indicator gene ORF lacking a translation initiation site, an enhancer-promoter region (permuted promoter) the 3' region of the HBV genome containing DR2, pre-C ORF translation initiation codon, DR1*, the and the 3' HBV pA signal region. The packaging vector, pPK-CPX, a component of the resistance test vector system comprising a patient-derived segment is shown in Fig. 8D and the S packaging vector, pPK-S, is shown in Fig. 8E.

S.C. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the 25 subgenomic indicator gene viral vector, pCS-HBV(F-IG)PPTIS(PSAS-), containing a functional indicator gene cassette assembled as a result of HBV viral replication.

D. A generalized diagrammatic representation of the resistance test vector, pCS-HBV(NF-IG)PPTIS(PSAS+), comprising a non-functional indicator gene with a permuted promoter containing the following elements in a 5' to 3' orientation: the CMV IE WO 97/27319 PCT/US97/01609 enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5' (the pre-C ORF translation initiation codon is eliminated), an indicator gene ORF lacking a translation initiation site, the P gene containing the patient-derived segment, an enhancer-promoter region (permuted promoter) (6) the 3' region of the HBV genome containing DR2, pre-C ORF translation initiation codon, DR1*, the 3'E, and the 3' HBV pA signal region. The packaging vector, pPK-CSX, providing the C, S and X genes is cotransfected with the resistance test vector, pCS-HBV(NF-IG)PPTIS(PSAS+), and is shown in Fig. 8H.

E. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the r e s i s t a n c e t e s t v e c t o r pCS-HBV(F-IG)PPTIS(PSAS+), containing a 20 functional indicator gene cassette and a patient-derived P gene segment assembled as a result of HBV viral replication.

Fig. 12 A. Diagrammatic representation of the RNA 25 pregenomic structure of HBV.

B. A generalized diagrammatic representation of the subgenomic indicator gene viral vector, pCS-HBV(NF-IG)PCR-(PSAS-), a component of the resistance test vector system comprising a non-functional indicator gene with a permuted coding region containing the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5' (the WO 97/27319 PCT/US97/01609 -46pre-C ORF translation initiation codon is eliminated), a non-functional indicator gene cassette assembled such that the promoter region and a 5' portion of the coding region are positioned i.e. downstream, of the remaining 3' portion of the coding region, the 3' region of the HBV genome containing DR2, DR1*, the and the 3' HBV pA signal region (the pre-C ORF translation initiation codon is eliminated). The packaging vector, pPK-CPX, a component of the resistance test vector system comprising a patient-derived P gene segment is shown in Fig. 8D and the S packaging vector, pPK-S, is shown in Fig. 8E.

C. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the subgenomic indicator gene viral vector, pCS-HBV(F-IG)PCR(PSAS-), containing a functional indicator gene cassette assembled as a result of HBV viral replication.

D. A generalized diagrammatic representation of the resistance test vector, pCS-HBV(NF-IG)PCR(PSAS+), comprising a non-functional indicator gene with a permuted coding region containing the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5' (the pre-C ORF translation initiation codon is eliminated), the 3' portion of the indicator gene ORF beginning with a splice acceptor sequence in the reverse orientation, the P gene containing the patient-derived segment, 20 25 WO 97/27319 PCT/US97/01609 -47an enhancer-promoter region, the 5' portion of the indicator gene ORF ending in a splice donor sequence in the reverse orientation, (7) the 3' region of the HBV genome containing DR2, DR1*, the and the 3' HBV pA signal region (the pre-C ORF translation initiation codon is eliminated). The packaging vector, pPK-CSX, providing the C, S and X genes is cotransfected with the resistance test vector, pCS-HBV(NF-IG) PCR(PSAS+) and is shown in Fig.

8H.

E. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form. of the resistance test vector, pCS-HBV(F-IG)PCR(PSAS+), containing a functional indicator gene cassette and a patient-derived P gene segment assembled as a result of HBV viral replication.

20 Fig. 13 A. Diagrammatic representation of the RNA pregenomic structure of HBV.

B. A generalized diagrammatic representation of the subgenomic indicator gene viral vector component, pCS-HBV(F-IG) (PSAS-), a component of the resistance test vector system comprising a functional indicator gene cassette containing the following elements in a 5' to 3' orientation: (1) the CMV IE enhancer-promoter region, the region of the HBV genome including the DR1 and ((the pre-C ORF translation initiation codon is Seliminated), a functional indicator gene cassette, the 3' region of the HBV genome containing DR2, DR1*, the 3'E, and the 3' HBV pA signal region (the pre-C ORF translation WO 97/27319 PCT/US97/01609 -48initiation codon is eliminated). The packaging vector, pPK-CPX, a component of the resistance test vector system comprising a patient-derived P gene segment is shown in Fig. 8D and the S packaging vector, pPK-S, is shown in Fig. 8E.

C. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the subgenomic indicator gene viral vector, pCS-HBV(F-IG)(PSAS-), containing a functional indicator gene.

D. A generalized diagrammatic representation of the resistance test vector, pCS-HBV(F-IG) (PSAS+), .comprising a functional indicator gene containing the following elements in a 5' to 3' orientation: the CMV IE enhancer promoter region, the region of the HBV genome including the DR1 and 5' (the pre-C ORF translation initiation codon 20 is eliminated), a functional indicator gene cassette, the P gene containing the patient-derived segment, the 3' region of the HBV genome containing DR2, DR1*, the 3'E, and the 3' HBV pA signal region (the pre-C ORF 25 translation initiation codon is eliminated). The packaging vector, pPK-CSX, providing the C, S and X genes, that is cotransfected with the resistance test vector, pCS-HBV(F-IG) (PSAS+), and is shown in Fig. 8H.

E. Diagrammatic representation of the covalently closed circular DNA (cccDNA) form of the resistance test vector, pCS-HBV(F-IG)(PSAS+), containing a functional indicator gene.

WO 97/27319 PCT/US97/01609 -49- Detailed Description of the Invention In order that the invention described herein may be more fully understood, the following description is set forth.

The present invention provides a novel drug susceptibility and resistance assay comprising the steps of: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step (c) measuring expression of the indicator gene in a target host cell; and comparing the expression of the indicator gene from step with the expression of the indicator gene measured when steps are carried out in the absence of the anti-viral drug, wherein a test concentration of the anti-viral drug is present at steps at steps or at step (c) In one aspect of the invention there is provided a method for determining susceptibility for an anti-viral drug 20 comprising: introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; culturing the host cell from step measuring an indicator in a target host cell wherein said indicator is a DNA or RNA structure; and comparing 25 the measurement of the indicator from step with the measurement of the indicator when steps are carried out in the absence of the anti-viral drug, wherein a test concentration of the anti-viral drug is present at steps at steps or at step (c) This invention also provides a method for determining anti-viral drug resistance in a patient comprising: (a) developing a standard curve of drug susceptibility for an anti-viral drug; determining anti-viral drug susceptibility in the patient using either of the WO 97/27319 PCT/US97/01609 susceptibility tests described above; and comparing the anti-viral drug susceptibility in step with the standard curve determined in step wherein a decrease in anti-viral susceptibility indicates development of anti-viral drug resistance in the patient.

This invention also provides a method for determining anti-viral drug resistance in a patient comprising: (a) determining anti-viral drug susceptibility in the patient at a first time according to either of the above methods, wherein the patient-derived segment is obtained from the patient at about said time; determining anti-viral drug susceptibility of the same patient at a later time; and (c) comparing the anti-viral drug susceptibilities determined in step -and wherein a decrease in anti-viral drug susceptibility at the later time compared to the first time indicates development or progression of anti-viral drug resistance in the patient.

S 20 The assay of this invention can be used for any viral disease where anti-viral drug susceptibility and resistance is a concern including, for example, HIV, herpes simplex virus, cytomegalovirus virus, varicella zoster virus, other human herpes (HIV) viruses,.influenza A virus, respiratory syncytial virus, hepatitis A, B and C viruses, rhinovirus, and human papilloma virus. The foregoing are representative of certain viruses for which there is presently available anti-viral chemotherapy, and represent the viral families retroviridae, herpesviridae, orthomyxoviridae, pneumovirus and hepadnaviridae. The assay of this invention would be used with other viral infections arising from infections due to other viruses within these families as well as viral infections arising from viruses in other viral families. In addition, the drug susceptibility and resistance test of this invention is useful for screening for compounds to WO 97/27319 PCT/US97/01609 -51treat viral diseases for which there is no currently available therapy.

The structure, life cycle and genetic elements of the viruses which could be tested in the drug susceptibility and resistance test of this invention would be known to one of ordinary skill in the art. It is useful to the practice of this invention, for example, to understand the life cycle of a retrovirus, as well as the viral genes required for retrovirus rescue and infectivity. Retrovirally infected cells shed a membrane virus containing a diploid RNA genome.

The virus, studded with an envelope glycoprotein (which serves to determine the host range of infectivity), attaches to a cellular receptor in the plasma membrane of the cell to be -infected. After receptor .binding, the virus is internalized and uncoated as it passes through the cytoplasm of the host cell. Either on its way to the nucleus or in the nucleus, the reverse transcriptase molecules resident in the viral core drive the synthesis of the double-stranded 20 DNA provirus, a synthesis that is primed by the binding of a tRNA molecule to the genomic viral RNA. The double-stranded DNA provirus is subsequently integrated in the genome of the host cell, where it can serve as a transcriptional template for both mRNAs encoding viralproteins and virion genomic RNA, which will be packaged into viral core particles. On their way out of the infected cell, core particles move through the cytoplasm, attach to the inside of the plasma membrane of the newly infected cell,, and bud, taking with them tracts of membrane containing the virally encoded envelope glycoprotein gene product. This cycle of infection reverse transcription, transcription, translation, virion assembly, and budding repeats itself over and over again as infection spreads.

The viral RNA and, as a result, the proviral DNA encode WO 97/27319 PCT/US97/01609 -52several cis-acting elements that are vital to the successful completion of the viral lifecycle. The virion RNA carries the viral promoter at its 3' end. Replicative acrobatics place the viral promoter at the 5' end of the proviral genome as the genome is reverse transcribed. Just 3' to the retroviral LTR lies the viral packaging site. The retroviral lifecycle requires the presence of virally encoded transacting factors. The viral-RNA-dependent DNA polymerase (pol)-reverse transcriptase is also contained within the viral core and is vital to the viral life cycle in that it is responsible for the conversion of the genomic RNA to the integrative intermediate proviral DNA. The viral envelope glycoprotein, env, is required for viral attachment to the uninfected cell and for viral spread.

There are also transcriptional trans-activating factors, so called transactivators, that can serve to modulate the level of transcription of the integrated parental provirus.

Typically, replication-competent (non-defective) viruses are .self-contained in that they encode all of these trans-acting factors. Their defective counterparts are not self-contained.

In the case of a DNA virus, such as a hepadnavirus, understanding the life cycle and viral genes required for infection is useful to the practice of this invention. The process of HBV entry has not been well defined. Replication of HBV uses an RNA intermediate template. In the infected cell the first step in replication is the conversion of the asymmetric relaxed circle DNA (rc-DNA) to covalently closed circle DNA (cccDNA). This process, which occurs within the nucleus of infected liver cells, involves completion of the DNA positive-strand synthesis and ligation of the DNA ends.

In the second step, the cccDNA is transcribed by the host RNA polymerase to generate a '3.5 kB RNA template (the pregenome). This pregenome is complexed with protein in the WO 97/27319 PCT/US97/01609 -53viral core. The third step involves the synthesis of the first negative-sense DNA strand by copying the pregenomic RNA using the virally encoded P protein reverse transcriptase. The P protein also serves as the minus strand DNA primer. Finally, the synthesis of the second positive-sense DNA strand occurs by copying the first DNA strand, using the P protein DNA polymerase activity and an oligomer of viral RNA as primer. The pregenome also transcribes mRNA for the major structural core proteins.

The following flow chart illustrates certain of the various vectors and host cells which may be used in this invention.

It is not intended to be all inclusive.

Vectors Indicator gene cassette Viral vector (functional/nonfunctional (genomic or subgenomic) indicator gene) Indicator Gene Viral Vector (functional/nonfunctional indicator gene) Patient sequence acceptor sites *6 S+ Patient-derived segments Resistance Test Vector (patient-derived segments indicator gene) 35 Host Cells Packaging Host Cell transfected with packaging expression vectors

S

40 Resistance Test Vector Host Cell a packaging host cell transfected with a resistance test vector Target Host Cell a host cell to be infected by a resistance test vector viral particle produced by the resistance test vector host cell WO 97/27319 PCT/US97/01609 -54- Resistance Test Vector "Resistance test vector" means one or more vectors which taken together contain DNA or RNA comprising a patient-derived segment and an indicator gene. In the case where the resistance test vector comprises more than one vector the patient-derived segment may be contained in one vector and the indicator gene in a different vector. Such a resistance test vector comprising more than one vector is referred to herein as a resistance test vector system for purposes of clarity but is nevertheless understood to be a resistance test vector. The DNA or RNA of a resistance test vector may thus be contained in one or more DNA or RNA molecules. In one embodiment, the resistance test vector is made by insertion of a patient-derived segment into an indicator gene viral vector. In another embodiment, the resistance test vector is made by insertion of a patient-derived segment into a packaging vector while the 20 indicator gene is contained in a second vector, for example an indicator gene viral vector. As used herein, "patient-derived segment" refers to one or more viral segments obtained directly from a patient using various means, for example, molecular cloning or polymerase chain 25 reaction (PCR) amplification of a population of patient-derived segments using viral DNA or complementary DNA (cDNA) prepared from viral RNA, present in the cells peripheral blood mononuclear cells, PBMC), serum or other bodily fluids of infected patients. When a viral 30 segment is "obtained directly" from a patient it is obtained without passage of the virus through culture, or if the virus is cultured, then by a minimum number of passages to essentially eliminate the selection of mutations in culture.

The term "viral segment" refers to any functional viral sequence or viral gene encoding a gene product a 0O 0 0000 5@0O 0 0

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5 0055 5 S S WO 97/27319 PCT/US97/01609 protein) that is the target cf an anti-viral drug. The term "functional viral sequence" as used herein refers to any nucleic acid sequence (DNA or RNA) with functional activity such as enhancers, promoters, polyadenylation sites, sites of action of trans-acting factors, such as tar and RRE, packaging sequences, integration sequences, or splicing sequences. If a drug were to target more than one functional viral sequence or viral gene product then patient-derived segments corresponding to each said viral gene would be inserted in the resistance test vector. In the case of combination therapy where two or more anti-virals targeting two different functional viral sequences or viral gene products are being evaluated, patient-derived segments corresponding to each functional viral sequence or viral gene product would be-.inserted in the resistance test vector. The patient-derived segments are inserted into unique restriction sites or specified locations, called patient se uence acceptor sites, in the indicator gene viral vector or for example, a packaging S 20 vector depending on the particular construction being used as described herein.

As used herein, "patient-derived segment" encompasses segments derived from human and various animal species.

25 Such species include, but arc not limited to chimpanzees, horses, cattles, cats and dogs.

Patient-derived segments car also be incorporated into resistance test vectors usinj any of several alternative 30 cloning techniques. For example, cloning via the introduction of class II res:riction sites into both the plasmid backbone and the palient-derived segments or by uracil DNA glycosylase primer cloning (refs).

The patient-derived segment may be obtained by any method of WO 97/27319 PCT/US97/01609 -56molecular cloning or gene almplification, or modifications thereof, by introducing patient sequence acceptor sites, as described below, at the ends of the patient-derived segment to be introduced into the resistance test vector. For example, in a gene amplification method such as PCR, restriction sites corresponding to the patient-sequence acceptor sites can be inccrporated at the ends of the primers used in the PCR react on. Similarly, in a molecular cloning method such as cDNA cloning, said restriction sites can be incorporated at the ends of the primers used for first or second strand cDNA ;ynthesis, or in a method such as primer-repair of DNA, whether cloned or uncloned DNA, said restriction sites can be incorporated into the primers used for the repair reaction. The patient sequence acceptor sites and primers are designed to improve the representation of patient-derived segments. Sets of resistance test vectors having designed patient sequence acceptor sites provide representation of Iatient-derived segments that would be underrepresented ii one resistance test vector 20 alone.

Resistance test vectors are prepared by modifying an indicator gene viral vector (c.escribed below) by introducing patient sequence acceptor ,:ites, amplifying or cloning 25 patient-derived segments anc: inserting the amplified or cloned sequences precisely inl.o indicator gene viral vectors at the patient sequence acceptor sites. The resistance test vectors are constructed from indicator gene viral vectors which are in turn derived from genomic viral vectors 30 or subgenomic viral vectors aid an indicator gene cassette, each of which is described below. Resistance test vectors are then introduced into a aost cell. Alternatively, a resistance test vector (alsc referred to as a resistance test vector system) is prepared by introducing patient sequence acceptor sites into .1 packaging vector, amplifying WO 97/27319 PCT/US97/01609 -57or cloning patient-derived segments and inserting the amplified or cloned sequences precisely into the packaging vector at the patient sequence acceptor sites and co-transfecting this packagirg vector with an indicator gene viral vector.

In one preferred embodiment, the resistance test vector may be introduced into packagiig host cells together with packaging expression vectors as defined below, to produce resistance test vector viral -articles that are used in drug resistance and susceptibility tests that are referred to herein as a "particle-based test." In an alternative preferred embodiment, the resistance test vector may be introduced into a host cell in the absence of packaging expression vectors to carry out a drug- resistance and susceptibility test that is referred to herein as a "non-particle-based test." As used herein a "packaging expression vector" provides the factors, such as packaging proteins structural pro-:eins such as core and envelope 20 polypeptides), transacting factors, or genes required by replication-defective retrov'rus or hepadnavirus. In such a situation, a replicatior.-competent viral genome is enfeebled in a manner such ti.at it cannot replicate on its own. This means that, although the packaging expression 25 vector can produce the trans-acting or missing genes required to rescue a defective viral genome present in a cell containing the enfeeblec genome, the enfeebled genome cannot rescue itself.

30 Indicator or Indicator Gene "Indicator or indicator gen'," refers to a nucleic acid encoding a protein, DNA or RNA structure that either directly or through a reactior gives rise to a measurable or noticeable aspect, e.g. a color or light of a measurable wavelength or in the case of ENA or RNA used as an indicator WO 97/27319 PCT/US97/01609 -58a change or generation of a specific DNA or RNA structure.

Preferred examples of an ind.cator gene is the E. coli lacZ gene which encodes beta-gali.ctosidase, the luc gene which encodes luciferase either from, for example, Photonis pyralis (the firefly) or Renilla reniformis (the sea pansy), the E. coli phoA gene which encodes alkaline phosphatase, green fluorescent protein ani the bacterial CAT gene which encodes chloramphenicol acatyltransferase. Additional preferred examples of an indicator gene are secreted proteins or cell surface proteins that are readily measured by assay, such as radioimmu.ioassay (RIA), or fluorescent activated cell sorting including, for example, growth factors, cytokines ard cell surface antigens (e.g.

growth hormone, 11-2 or CD< respectively). "Indicator gene" is understood to also .nclude a selection gene, also referred to as a selectable marker. Examples of suitable selectable markers for mammaLlian cells are dihydrofolate reductase (DHFR), thymidine kinase, hygromycin, neomycin, zeocin or E. coli gpt. I the case of the foregoing examples of indicator genes the indicator gene and the patient-derived segment are discrete, i.e. distinct and separate genes. In some cases a patient-derived segment may also be used as an indicator gene. In one such embodiment in which the patient-derivec segment corresponds to more than one viral gene which is the target of an anti-viral, one of said viral genes may also serve as the indicator gene. For example, a viral protease gene may serve as an indicator gene by virtue c.f its ability to cleave a chromogenic substrate or its Ebility to activate an inactive 30 zymogen which in turn cleaves a chromogenic substrate, giving rise in each case to a color reaction. In all of the above examples of indicator genes, the indicator gene may be either "functional" or "non-ft.nctional" but in each case the expression of the indicator gene in the target cell is ultimately dependent upon the action of the patient-derived WO 97/27319 PCT/US97/01609 segment.

Functional Indicator Gene In the case of a "functional indicator gene" the indicator gene may be capable of being expressed in a "packaging host cell/resistance test vector host cell" as defined below, independent of the patient-ierived segment, however the functional indicator gene could not be expressed in the target host cell, as defined below, without the production of functional resistance teEt vector particles and their effective infection of the target host cell. In one embodiment of a functional :.ndicator gene, the indicator gene cassette, comprising control elements and a gene encoding an indicator protein,, is inserted .into the indicator gene viral vector with the same or opposite transcriptional orientation as the native or foreign enhancer/promoter of the viral vector. One example of a functional indicator gene in :he case of HIV or HBV, places the indicator gene and its promoter (a CMV IE 20 enhancer/promoter) in the sane or opposite transcriptional orientation as the HIV-LTF or HBV enhancer-promoter, respectively, or the CMV IE enhancer/promoter associated with the viral vector.

25 Non-Functional Indicator Gene Alternatively the indicator gene, may be "non-functional" in that the indicator gene is not efficiently expressed in a packaging host cell transfec:ed with the resistance test vector, which is then referred to a resistance test vector 30 host cell, until it is converted into a functional indicator gene through the action of one or more of the patient-derived segment prodicts. An indicator gene is rendered non-functional through genetic manipulation according to this invention.

WO 97/27319 PCT/US97/01609 1. Permuted Promoter In one embodiment an indicator gene is rendered non-functional due :o the location of the promoter, in that, although the promoter is in the same transcriptional orientatior as the indicator gene, it follows rather than precedes the indicator gene coding sequence. This misplaced promoter is referred to as a "permuted promoter." In adc.ition to the permuted promoter the orientation of the non-functional indicator gene is opposite to that of the natire or foreign promoter/enhancer of the viral vector. Thus the coding sequence of the non-functional indicator gene can neither be transcribed by the permuted promoter nor by the viral promoters. The non-functional indicator gere and its permuted promoter is rendered functional by the action of one or more of the viral proteins. One example of a non-functional indicator gene with a permuted promoter in the case of HIV, places a T7 phage RNA polymerase pronoter (herein referred to as T7 promoter) promoter in the 5' LTR in the same transcriptional orientation as the indicator gene. The indicator gene 20 cannot be transcribed by the T7 promoter as the indicator gene cassette is positioned upstream of the T7 promoter.

The non-functional indicato:: gene in the resistance test vector is converted into a functional indicator gene by reverse transcriptase upon nfection of the target cells, 25 resulting from the repositioning of the T7 promoter, by copying from the 5' LTR to the 3' LTR, relative to the indicator gene coding region Following the integration of the repaired indicator gene :.nto the target cell chromosome by HIV integrase, a nuclear r7 RNA polymerase expressed by 30 the target cell transcribes the indicator gene. One example of a non-functional indicato:- gene with a permuted promoter in the case of HBV, places an enhancer-promoter region downstream or 3' of the indicator gene both having the same transcriptional orientation. The indicator gene cannot be transcribed by the enhancer-promoter as the indicator gene WO 97/27319 PCT/US97/01609 71cassette is positioned upstream. The non-functional indicator gene in the resistance test vector is converted into a functional indicator gene by reverse transcription and circularization of the H:AV indicator gene viral vector by the repositioning of tie enhancer-promoter upstream relative to the indicator gene coding region.

A permuted promoter may be any eukaryotic or prokaryotic promoter which can be transc::ibed in the target host cell.

Preferably the promoter wil:. be small in size to enable insertion in the viral gen me without disturbing viral replication. More preferably, a promoter that is small in size and is capable of transc:-iption by a single subunit RNA polymerase introduced into thLe target host cell, such as a bacteriophage promoter, will be used. Examples of such bacteriophage promoters and their cognate RNA polymerases include those of phages T3 and Sp6. A nuclear localization sequence (NLS) may be attached to the RNA polymerase to localize expre sion of the RNA polymerase to 20 the nucleus where they may be needed to transcribed the repaired indicator gene. Such an NLS may be obtained from any nuclear-transported protein such as the SV40 T antigen.

If a phage RNA polymerase is imployed, an internal ribosome entry site (IRES) such as the EMC virus 5' untranslated 25 region (UTR) may be added in front of the indicator gene, for translation of the transcripts which are generally uncapped. In the case of HIV the permuted promoter itself can be introduced at any posi:ion within the 5' LTR that is copied to the 3' LTR during reverse transcription so long as 30 LTR function is not disrupted. preferably within the U5 and R portions of the LTR, and most preferably outside of functionally important and h..ghly conserved regions of and R. In the case of HBV, the permuted promoter can be placed at any position that does not disrupt the cis acting elements that are necessarr for HBV DNA replication.

WO 97/27319 PCT/US97/01609 -62- Blocking sequences may be added at the ends of the resistance test vector should there be inappropriate expression of the non-func:ional indicator gene due to transfection artifacts (DNA concatenation). In the HIV example of the permuted T7 promoter given above, such a blocking sequence may consist of a T7 transcriptional terminator, positioned to bl.ock readthrough transcription resulting from DNA concatenation, but not transcription resulting from repositioning of the permuted T7 promoter from the 5' LTR to the 3' LTF during reverse transcription.

2. Permuted Coding Region In a second embodiment, an indicator gene is rendered non-functional due to the relative location of the 5' and 3' coding regions of the indicator gene, in that, tae 3' coding region precedes rather than follows the 5' coding region. This misplaced coding region is referred to as a "permuted coding region." The orientation of the non-finctional indicator gene may be the same or opposite to tiat of the native or foreign 20 promoter/enhancer of the viriLl vector, as mRNA coding for a functional indicator gene will be produced in the event of either orientation. The non-functional indicator gene and its permuted coding region Ls rendered functional by the action of one or more of the patient-derived segment 25 products. A second example of a non-functional indicator gene with a permuted codinc region in the case of HIV, places a 5' indicator gene ceding region with an associated promoter in the 3' LTR U3 region and a 3' indicator gene coding region in an upstrean location of the HIV genome, 30 with each coding region having the same transcriptional orientation as the viral LTRs. In both examples, the 5' and 3' coding regions may also have associated splice donor and acceptor sequences, respectively, which may be heterologous or artificial splicing signals. The indicator gene cannot be functionally transcribeI either by the associated o

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WO 97/27319 PCT/US97/01609 -63promoter or viral promoters, as the permuted coding region prevents the formation of functionally spliced transcripts.

The non-functional indicator gene in the resistance test vector is converted into a functional indicator gene by reverse transcriptase upon irfection of the target cells, resulting from the reposition:.ng of the 5' and 3' indicator gene coding regions relative :o one another, by copying of the 3' LTR to the 5' LTR. Following transcription by the promoter associated with the c coding region, RNA splicing can join the 5' and 3' coding regions to produce a functional indicator gene )roduct. One example of a non-functional indicator gene with a permuted coding region in the case of HBV, places a 3 indicator gene coding region upstream or 5' of the enhancer-promoter and the 5' coding region of the indicator cene. The transcriptional orientation of the indicator jene 5' and 3' coding regions are identical to one another, and the same as that of the indicator gene viral vector. However, as the indicator gene 5' and 3' coding regions are permuted in the resistance test 20 vectors the 5' coding region is downstream of the 3' coding region), no mRNA is transcribed which can be spliced to generate a functional inlicator gene coding region.

Following reverse transcription and circularization of the indicator gene viral vector, the indicator gene 3' coding 25 region is positioned doimstream or 3' to the enhancer-promoter and 5' coding regions thus permitting the transcription of mRNA which can be spliced to generate a functional indicator gene cod:.ng region.

3. Inverted Intron In a third embodiment, the indicator gene is rendered non-functional through use of an "inverted intron," i.e. an intron inserted into the coding sequence of the indicator gene with a transcriptional orientation opposite to that of the indicator gene. The overall transcriptional orientation of the indicator gene cassette WO 97/27319 PCT/US97/01609 S64including its own, linked promoter, is opposite to that of the viral control elements, while the orientation of the artificial intron is the same as the viral control elements.

Transcription of the indicator gene by its own linked promoter does not lead to the production of functional transcripts as the inverted :.ntron cannot be spliced in this orientation. Transcription of the indicator gene by the viral control elements does, however, lead to the removal of the inverted intron by RNA s:)licing, although the indicator gene is still not functionally expressed as the resulting transcript has an antisensc orientation. Following the reverse transcription of this transcript and integration of the resultant retroviral DNA, or the circularization of hepadnavirus DNA, the indicator gene can be functionally transcribed using its own lLnked promoter as the inverted intron has been previously removed. In this case, the indicator gene itself may contain its own functional promoter with the entire :ranscriptional unit oriented opposite to the viral control elements. Thus the 20 non-functional indicator gen.! is in the wrong orientation to be transcribed by the viral control elements and it cannot S. be functionally transcribed by its own promoter, as the inverted intron cannot be properly excised by splicing.

However, in the case of a r.trovirus and HIV specifically and hepadnaviruses, and HBV specifically, transcription by the viral promoters (HIV L.TR or HBV enhancer-promoter) results in the removal of the inverted intron by splicing.

As a consequence of reverse transcription of the resulting spliced transcript and the integration of the resulting 30 provirus into the host cell clromosome or circularization of the HBV vector, the indicatcr gene can now be functionally transcribed by its own prcnoter. The inverted intron, consisting of a splice donor and acceptor site to remove the intron, is preferably located in the coding region of the indicator gene in order to disrupt translation of the WO 97/27319 PCTUS97/01609 indicator gene. The splice donor and acceptor may be any splice donor and acceptDr. A preferred splice donor-receptor is the CMV I:I splice donor and the splice acceptor of the second exon 3f the human alpha globin gene ("intron Indicator Gene Viral Vector Construction As used herein, "indicator cene viral vector" refers to a vector(s) comprising an in.icator gene and its control elements and one or more viial genes. The indicator gene viral vector is assembled fiom an indicator gene cassette and a "viral vector," defined below. The indicator gene viral vector may additionally include an enhancer, splicing signals, polyadenylation sequences, transcriptional terminators, or other regulatory sequences. Additionally the indicator gene viral rector may be functional or nonfunctional. In the event that the viral segments which are the target of the anti-viral drug are not included in the indicator gene viral vE:ctor they are provided in a 20 second vector. An "indicatcr gene cassette" comprises an indicator gene and control elements. "Viral vector" refers to a vector comprising some or all of the following: viral genes encoding a gene prodLct, control sequences, viral packaging sequences, and in- the case of a retrovirus, 25 integration sequences. The viral vector may additionally include one or more viral secments one or more of which may be the target of an anti-viral drug. Two examples of a viral vector which contain viral genes are referred to herein as an "genomic viral vector" and a "subgenomic viral 30 vector." A "genomic viral rector" is a vector which may comprise a deletion of a one or more viral genes to render the virus replication inconpetent, but which otherwise preserves the mRNA expression and processing characteristics of the complete virus. In cne embodiment for an HIV drug susceptibility and resistance test, the genomic viral vector WO 97/27319 PCT/US97/01609 -66comprises the HIV gag-pol, vpr, tat, rev, vpu, and nef genes (some, most or all of env may be deleted) A "subgenomic viral vector" reEers to a vector comprising the coding region of one or more viral genes which may encode the proteins that are the ta:-get(s) of the anti-viral drug.

In the case of HIV, a prefezred embodiment is a subgenomic viral vector comprising the }:IV gag-pol gene. In the case of HBV a preferred embodiment is a subgenomic viral vector comprising the HBV P gene. In the case of HIV, two examples of proviral clones used for viral vector construction are: HXB2 (Fisher et al., (1986) Aature, 320, 367-371) and NL4-3, (Adachi et al., (1986) J. Vizol., 59, 284-291). In the case of HBV, a large number of fulL length genomic sequences have been characterized and could be used for construction of HBV viral vectors: GenBank Nos. M54923, M38636, J02203 and X59795. The viral coding genes may be under the control of a native enhancer/promoter c.r a foreign viral or cellular enhancer/promoter. A preferred embodiment for an HIV drug susceptibility and resistance test, is to place the genomic or subgenomic viral coding regions under the control of the native enhancer/promoter of the HIV-LTR U3 region or the CMV Simmediate-early (IE) enharcer/promoter. A preferred embodiment for an HBV drug susceptibility and resistance test, is to place the genomic or subgenomic viral coding S 25 regions under the control ol the CMV immediate-early (IE) enhancer/promoter. In the case of an indicator gene viral vector that contains one or nore viral genes which are the targets or encode proteins which are the targets of an anti-viral drug(s) then saidi vector contains the patient 30 sequence acceptor sites. The patient-derived segments are inserted in the patient sequence acceptor site in the indicator gene viral vector which is then referred to as the resistance test vector, as described above.

"Patient sequence acceptor s:tes" are sites in a vector for WO 97/27319 PCT/US97/01609 -67insertion of patient-derived segments and said sites may be: 1) unique restriction site:; introduced by site-directed mutagenesis into a vector; 2) naturally occurring unique restriction sites in the vector; or 3) selected sites into which a patient-derived segment may be inserted using alternative cloning methods UDG cloning). In one embodiment the patient sequence acceptor site is introduced into the indicator gene vira. vector. The patient sequence acceptor sites are preferably located within or near the coding region of the viral protein which is the target of the anti-viral drug. The viral sequences used for the introduction of patient sequence acceptor sites are preferably chosen so that no change, or a conservative change, is made in the amino acid coding sequence found at that position. Preferably the patient sequence acceptor sites are located within a relatively conserved region of the viral genome to fac:litate introduction of the patient-derived segments. Alternatively, the patient sequence acceptor sites are located between functionally important genes or regulator( sequences. Patient-sequence acceptor sites may be located at or near regions in the viral genome that are relatively conserved to permit priming by the primer used to introduce the corresponding restriction site into the patient-derived segment. -To improve the representation of patient-derived segments further, such primers may be designed as degenerate pools to accommodate viral sequence he:erogeneity, or may incorporate residues such as deoxyinosLne which have multiple base-pairing capabilities. sets of resistance test vectors having patient sequence acceltor sites that define the same or overlapping restriction site intervals may be used together in the drug resistarce and susceptibility tests to provide representation of patient-derived segments that contain internal restriction sites identical to a given patient sequence acceptor site, and would thus be WO 97/27319 PCTfUS97/01609 -58underrepresented in either resistance test vector alone.

Host Cells The resistance test vector is introduced into a host cell.

Suitable host cells are mamnalian cells. Preferred host cells are derived from human :issues and cells which are the principle targets of viral infection. In the case of HIV these include human cells such as human T cells, monocytes, macrophage, dendritic cells, 2Langerhans cells, hematopoeitic stem cells or precursor cells, and other cells. In the case of HBV, suitable host cells include hepatoma cell lines (HepG2, Huh primary huma: hepatocytes, mammalian cells which can be- infected by pseudotyped HBV, and other cells.

Human derived host cells wi l assure that the anti-viral drug will enter the cell efficiently and be converted by the cellular enzymatic machinery .nto the metabolically relevant form of the anti-viral inhib.tor. Host cells are referred to herein as a "packaging host cells, "resistance test vector host cells," or "target host cells." A "packaging 20 host cell" refers to a host cell that provides the trans-acting factors and vir.il packaging proteins required by the replication defective -riral vectors used herein, such as the resistance test vecto:rs, to produce resistance test vector viral -particles. T.ie packaging proteins may be 25 provided for by the expression of viral genes contained within the resistance test vector itself, a packaging expression vector(s), or bot:i. A packaging host cell is a host cell which is transfected with one or more packaging expression vectors and when transfected with a resistance 30 test vector is then referred :o herein as a "resistance test vector host cell" and is sometimes referred to as a packaging host cell/resistance test vector host cell.

Preferred host cells for use ;is packaging host cells for HIV include 293 human embryonic kidney cells (293, Graham, F.L.

et al., J. Gen Virol. 36: 5S, 1977), BOSC23 (Pear et al., WO 97/27319 PCTUS97/01609 -59- Proc. Natl. Acad. Sci. 90, 9392, 1993), tsa54 and tsa201 cell lines (Heinzel et al., J.Virol. 62, 3738,1988), for HBV HepG2 (Galle and Theilmann, L. Arzheim.-Forschy Drug Res.

(1990) 40, 1380-1382). (Huh, Ueda, K et al. Virology *1989) 169, 213-216). A "target host cell" refers to a cell to be infected by resistance test sector viral particles produced by the resistance test vectoi host cell in which expression or inhibition of the indicatcr gene takes place. Preferred host cells for use as target .lost cells include human T cell leukemia cell lines including Jurkat (ATCC T1B-152), H9 (ATCC HTB-176), CEM (ATCC CCL-119), HUT78 (ATCC T1B-161), and derivatives thereof.

Drug Susceptibility and Resi.;tance Tests The drug susceptibility ard resistance tests of this invention may be carried ou: in one or more host cells.

Viral drug susceptibility is determined as the concentration of the anti-viral agent at which a given percentage of indicator gene expression is inhibited the ICs 0 for an 20 anti-viral agent is the ccncentration at which 50% of indicator gene expression is inhibited). A standard curve for drug susceptibility of a given anti-viral drug can be developed for a viral segmeit. that is either a standard laboratory viral segment or from a drug-naive patient (i.e.

25 a patient who has not received any anti-viral drug) using the method of this invention. Correspondingly, viral drug resistance is a decrease in viral drug susceptibility for a given patient either by comparing the drug susceptibility to such a given standard or by making sequential measurement in 30 the same patient over time, as determined by increased inhibition of indicator gens expression decreased indicator gene expression).

In the first type of drug ;;usceptibility and resistance test, resistance test vector viral particles are produced by WO 97/27319 PCT/US97/01609 a first host cell (the res:.stance test vector host cell) that is prepared by transfec:ing a packaging host cell with the resistance test vectDr and packaging expression vector(s). The resistance test vector viral particles are then used to infect a secoid host cell (the target host cell) in which the expression of the indicator gene is measured. Such a two cell system comprising a packaging host cell which is transf cted with a resistance test vector, which is then referred to as a resistance test vector host cell, and a tarcet cell are used in the case of either a functional or non-functional indicator gene.

Functional indicator genes ire efficiently expressed upon transfection of the packaging host cell and would require infection of a target host co'll with resistance test vector host cell supernatant to carry out the test of this invention. Non-functional indicator genes with a permuted promoter, a permuted coding region, or an inverted intron are not efficiently expressed upon transfection of the packaging host cell and thus the infection of the target 20 host cell can be achieved eLther by co-cultivation by the L resistance test vector host cell and the target host cell or through infection of the target host cell using the resistance test vector host cell supernatant. In the second type of drug susceptibility and resistance test, a single 25 host cell (the resistance test vector host cell) also serves as a target host cell. The packaging host cells are transfected and produce resistance test vector viral particles and some of the packaging host cells also become the target of infection the resistance test vector 30 particles. Drug susceptibility and resistance tests 'employing a single host cel:, type are possible with viral resistance test vectors comprising a non-functional indicator gene with a permuted promoter, a permuted coding region, or an inverted intror Such indicator genes are not efficiently expressed upon transfection of a first cell, but WO 97/27319 PCT/US97/01609 1are only efficiently expressed upon infection of a second cell, and thus provide an opp)rtunity to measure the effect of the anti-viral agent under evaluation. In the case of a drug susceptibility and resiEtance test using a resistance test vector comprising a func:ional indicator gene, neither the co-cultivation procedu:-e nor the resistance and susceptibility test using a s:ngle cell type can be used for the infection of target cells. A resistance test vector comprising a functional indicator gene requires a two cell system using filtered superna:ants from the resistance test vector host cells to infect the target host cell.

In one embodiment of the invention in the case of HIV, a particle-based resistance tests are carried out with resistance test vectors derived from genomic viral vectors, pLG-lucPP-HS, pC3-lucPP-HS, pLG-lucPP-PB, pCG-lucPP-PB, pLG-lucPC-HS, pCG-lucPC-HS, pLG-lucPC-PB, pCG-lucPC-PB, pLG-lucII-HS, pCG-lucII-HS, pLG-lucII-PB, pCG-lucII-PB, and pCG-CXZN(F-lucP)2-AA which are 20 cotransfected with the packaging expression vector pVL-env4070A (also referred to as pCXAS-407OAenv) Alternatively, a particle- ased resistance test may be carried out with resistance test vectors derived from subgenomic viral vectors, pLS-lucPP-HS, pCS-lucPP-HS, pLS-lucPP-PB, pCS-luc-PP-PB, pLS-lucPC-HS, pCS-lucPC-HS, pLS-lucPC-PB, pCS-luc-PC-PB, pLS-lucII-HS, pCS-lucII-HS, pLS-lucII-PB, and pCS-luc-II -PB) which are cotransfected with the packaging expression rector pVL-env4070A and either pLTR-HIV3' or pCMV-HIV3'. n another embodiment of the 30 invention in the case of HIV, non-particle-based resistance tests are carried out using each of the above described resistance test vectors by transfection of selected host cells in the absence of packajing expression vectors.

In the case of the particle-based susceptibility and WO 97/27319 PCT/US97/01609 -72resistance test, resistance :est vector viral particles are produced by a first host ce.l (the resistance test vector host cell) that is prepared by transfecting a packaging host cell with the resistance test vector and packaging expression vector(s). The resistance test vector viral particles are then used to nfect a second host cell (the target host cell) in which the expression of the indicator gene is measured. In a second type of particle-based susceptibility and resistance test, a single host cell type (the resistance test vector host cell) serves both purposes: some of the packaging host cells in a given culture are transfected and produce resistance test vector viral particles and some of the host cells in the same culture are the target of infection by the resistance test vector particles thus produced. Resistance tests employing a single host cell type are aossible with resistance test vectors comprising a non-furctional indicator gene with a permuted promoter since such Lndicator genes are efficiently expressed upon infection of a permissive host cell, they are 20 not efficiently expressed upcn transfection of the same host cell type, and thus provide an opportunity to measure the effect of the anti-viral agent under evaluation. For similar reasons, resistance :ests employing two cell types may be carried out by co-cul:ivating the two cell types as an alternative to infecting the second cell type with viral particles obtained from the supernatants of the first cell type.

In the case of the non-particle-based susceptibility and 30 resistance test, resistance tests are performed by transfection of a single host cell with the resistance test vector in the absence of packaging expression vectors.

Non-particle based resistance tests are carried out using the resistance test vectors comprising non-functional indicator genes with either permuted promoters, permuted WO 97/27319 PCT/US97/01609 -73coding regions or inverted introns. These non-particle based resistance tests are rerformed by transfection of a single host cell type with each resistance test vector in the absence of packaging expression vectors. Although the non-functional indicator gnes contained within these resistance test vectors are :iot efficiently expressed upon transfection of the host cells, there is detectable indicator gene expression resulting from non-viral particle-based reverse transcription. Reverse transcription and strand transfer results in the conversion of the permuted, non-functional ind:cator gene to a non-permuted, functional indicator gene. As reverse transcription is completely dependent upon tle expression of the pol gene contained within each resistance test vector, anti-viral agents may be tested for their ability to inhibit the pol gene products encoded by :he patient-derived segments contained within the resista.ce test vectors. In the case of HIV, reverse transcription and strand transfer results in the conversion of the non-ftnctional indicator gene to a functional indicator gene. As reverse transcription is completely dependent upon the expression of the patient-derived segment contained within each resistance test vector, anti-viral age:lts may be tested for their S. ability to inhibit the ge:le products encoded by the patient-derived segments contained within the resistance test vectors.

The packaging host cells are transfected with the resistance test vector and the apprcpriate packaging expression vector(s) to produce resistance test vector host cells.

Individual anti-viral agen:s, including the reverse transcriptase inhibitors AZT, ddl, ddC, d4T and 3TC, and the protease inhibitors saquinavir, ritonavir and indinavir, as well as combinations thereof, are added to individual plates of packaging host cells at the time of their transfection, WO 97/27319 PCT/US97/01609 at an appropriate range of coi.centrations. Twenty-four to 48 hours after transfection, talget host cells are infected by co-cultivation with resistarce test vector host cells or with resistance test vector viral particles obtained from filtered supernatants of resistance test vector host cells.

Each anti-viral agent, or coibination thereof, is added to the target host cells prior 1:o or at the time of infection to achieve the same final conzentration of the given agent, or agents, present during the transfection.

Determination of the expression or inhibition of the indicator gene in the target host cells infected by co-cultivation or with filtered viral supernatants is made by assay of indicator gene expression, for example in the case where the indicator gene is the firefly luc gene, by measuring luciferase activity. The reduction in luciferase activity observed for target. host cells infected with a given preparation of resistanze test vector viral particles in the presence of a given antiviral agent, or agents, as compared to a control run in the absence of the antiviral agent, generally relates to tae log of the concentration of the antiviral agent as a sigrioidal curve. This inhibition curve is used to calculate the apparent inhibitory concentration (IC) of that agent, or combination of agents, for the viral target product encoded by the patient-derived segments present in the resistance test vector.

In the case of a one cell ;;usceptibility and resistance test, host cells are transfe:ted with the resistance test 30 vector and the appropriate packaging expression vector(s) to produce resistance test ve:tor host cells. Individual antiviral agents, or combinations thereof, are added to individual plates of transfecced cells at the time of their transfection, at an appropriate range of concentrations.

Twenty-four to 72 hours after transfection, cells are WO 97/27319 PCT/US97/01609 collected and assayed for fi:-efly luciferase activity. As transfected cells in the culture do not efficiently express the indicator gene, transfected cells in the culture, as well superinfected cells in the culture, can serve as target host cells for indicator gene expression. The reduction in luciferase activity observed for cells transfected in the presence of a given antiviral agent, or agents as compared to a control run in the abserce of the antiviral agent(s), generally relates to the loc of the concentration of the antiviral agent as a sigmoidaj curve. This inhibition curve is used to calculate the apparent inhibitory concentration (IC) of an agent, or combina:ion of agents, for the viral target product encoded by :he patient-derived segments present in the resistance test vector.

Antiviral Drugs/Drug Candidates The antiviral drugs being added to the test system are added at selected times depending uron the target of the antiviral drug. For example, in the ca.se of HIV protease inhibitors, including saquinavir, ritonav:.r, indinavir, and nelfinavir, they are added to individual plates of packaging host cells at the time of their transfection with a resistance test vector, at an appropriate rz.nge of concentrations.

HIV

protease inhibitors are also c.dded to the target host cells at the time of infection to achieve the same final concentration added during :ransfections. HIV reverse transcriptase inhibitors, inc.uding AZT, ddl, ddC, d4T, 3TC and nevaripine, are added to individual plates of target host cells at the time of infection by the resistance test 30 vector viral particles, it a test concentration.

Alternatively, the antiviral drugs may be present throughout :the assay. The test concentration is selected from a range of concentrations which is typ .cally between about 0. InM and about 100(M and more specifically for each of the following drugs: AZT, from about InM to iLbout 5(M; ddl, from about InM WO 97/27319 PCT/US97/01609 to about 25(M; 3TC, from abou- InM to about 50(M; d4T, from about InM to about 25(M; and, nevaripine, from about InM to about 100(M.

In another embodiment of 1.his invention, a candidate antiviral compound is tested the drug susceptibility and resistance test of this invenl.ion. The candidate antiviral compound is added to the t st system at an appropriate concentration and at select d times depending upon the protein target of the candidate anti-viral. Alternatively, more than one candidate antiv:.ral compound may be tested or a candidate antiviral compound may be tested in combination with an approved antiviral drug such as AZT, ddl, ddC, d4T, 3TC, saquinavir or a compound which is undergoing clinical trials such as ritonavir, or indinovir. The effectiveness of the candidate antiviral w:.ll be evaluated by measuring the expression or inhibition of the indicator gene. In another aspect of this embodinent, the drug susceptibility and resistance test may be usei to screen for viral mutants.

S 20 Following the identification of resistant mutants to either known anti-virals or candidate anti-virals the resistant mutants are isolated and the DNA is analyzed. A library of viral resistant mutants can tnus be assembled enabling the .screening of candidate anti-virals, alone or in combination.

This will enable one of ordina-y skill to identify effective anti-virals and design effective therapeutic regimens.

General Materials and Methods Most of the techniques useci to construct vectors, and transfect and infect cells, are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraphs may serve as a guiieline.

WO 97/27319 PCT/US97/01609 -77- "Plasmids" and "vectors" are designated by a lower case p followed by letters and/or nunbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, 3r can be constructed from available plasmids in accord ith published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

Construction of the vectorE of the invention employs standard ligation and restriction techniques which are well understood in the art (see Arsubel et al., (1987) Current Protocols in Molecular Biology, Wiley Interscience or Maniatis et al., (1992) in Mo.ecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, Isolated plasmids, DNA sequences, or syrthesized oligonucleotides are cleaved, tailored, and relega:ed in the form desired. The sequences of all DNA constructs incorporating synthetic DNA were confirmed by DNA sequei.ce analysis (Sanger et al.

20 (1977) Proc. Natl. Acad. Sci. 74, 5463-5467) "Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme :hat acts only at certain sequences, restriction sites, in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are known to the ordinarily skilled artisan.

For analytical purposes, typically 1(g of plasmid or DNA fragment is used with about 2 inits of enzyme in about 20(1 .30 of buffer solution. Alternatilely, an excess of restriction enzyme is used to insure corTplete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37 0 C are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroJorm, and may be followed by WO 97/27319 PCT/US97/01609 -78ether extraction, and the nucleic acid recovered from aqueous fractions by prec:.pitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using stardard techniques. A general description of size separations is found in Methods of Enzvmoloqv 65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 0 C in 50 mM Tris (ph7.6) 5C mM NaC1, 6 mM MgCl 2 6 mM DTT and 5-10 mM dNTPs. The Kl now fragment fills .in at sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only one of the dNTPs, or with selected dNTPs, within the limitations dictated by the nature of the sticky ends. After treatment 20 with Klenow, the mixture is e> tracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with S1 nuclease cr Bal-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 15-50 (1 volumes under the following standard conditicns and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 mM DTT, 33 mg/ml BSA, 10 mMmM NaC1, and either 40 mM 1.TP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticcy end" ligation) or ImM ATP, 30 0.3 0.6 (Weiss) units T4 INA ligase at 14 0 C (for "blunt end" ligation). Intermolecu.ar "sticky end" ligations are usually performed at 33-100 Ig/ml total DNA concentrations (5-100 mM total end concentration) Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 mM total ends concentration.

WO 97/27319 PCT/US97/01609 -*79- "Transient expression" refers to unamplified expression within about one day to twc weeks of transfection. The optimal time for transient expression of a particular desired heterologous protein may vary depending on several factors including, for example, any transacting factors which may be employed, translational control mechanisms and the host cell. Transient expression occurs when the particular plasmid that has been transfected functions, is transcribed and trai.slated. During this time the plasmid DNA which has entered the cell is transferred to the nucleus. The DNA is in a nor.integrated state, free within the nucleus. Transcription cf the plasmid taken up by the cell occurs during this perioi. Following transfection the plasmid DNA may become degraded or diluted by cell division.

Random integration.within the cell chromatin occurs.

In general, vectors containing promoters and control sequences which are derived fiom species compatible with the host cell are used with the particular host cell. Promoters suitable for use with prokiryotic hosts illustratively include the beta-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as tac promoter. However, other functional bacterial promoter.; are suitable. In addition to prokaryotes, eukaryotic micro.es such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly u.;ed eukaryotic microorganism, although a number of other strains are commonly available.

Promoters controlling trarscription from vectors in mammalian host cells may be oatained from various sources, for example, the genomes of viruses such as: polyoma, simian :virus 40 (SV40), adenovirus, r!troviruses, hepatitis B virus and preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. b-a:tin promoter. The early and late promoters of the SV 40 virus are conveniently obtained WO 97/27319 PCT/US97/01609 -83as an SV40 restriction fragmen: which also contains the viral origin of replication. The immediate early promoter of the human cytomegalovirus Ls conveniently obtained as a HindIII E restriction fragmen:. Of course, promoters from the host cell or related species also are useful herein.

The vectors used herein may contain a selection gene, also termed a selectable marker. A selection gene encodes a protein, necessary for the surrival or growth of a host cell transformed with the vectcr. Examples of suitable selectable markers for manmalian cells include the dihydrofolate reductase ge e (DHFR), the ornithine decarboxylase gene, the multi-drug resistance gene (mdr), the adenosine deaminase gene, and the glutamine synthase gene. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's 20 metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. The second category is referred to as dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection.

Examples of such dominant sel:ction use the drugs neomycin (Southern and Berg (1982) J. Molec. Appl. Genet. 1, 327), .30 mycophenolic acid (Mulligan and Berg (1980) Science 209, 1422), or hygromycin (Sugden et al. (1985) Mol. Cell. Biol.

5, 410-413). The three ecamples given above employ bacterial genes under euk.ryotic control to convey resistance to the appropriace drug neomycin (G418 or genticin), xgpt (mycopheno:.ic acid) or hygromycin, WO 97/27319 PCT/US97/01609 -61respectively.

"Transfection" means introduc:ing DNA into a host cell so that the DNA is expressed, whe:her functionally expressed or otherwise; the DNA may also replicate either as an extrachromosomal element or by chromosomal integration.

Unless otherwise provided, the method used herein for transformation of the host c(:lls is the calcium phosphate co-precipitation method of Graham and van der Eb (1973) Virology 52, 456-457. Alternative methods for transfection are electroporation, the DEAI;-dextran method, lipofection and biolistics (Kriegler (1990) Gene Transfer and Expression: A Laboratory Manu.l, Stockton Press) Host cells may be transfected irith the expression vectors of the present invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. Host cells are cultured in F12:DMEM (Gibco) 50:50 with added glutamine and without antibiotics. The culture conditions, such as temperature, pH and the like, Ere those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

*o The following examples merely illustrate the best mode now known for practicing the intention, but should not be construed to limit the invention. All literature references are expressly incorporated by reference.

WO 97/27319 PCT/US97/01609 -32- EXA IPLE 1 HIV Drug Susceptibil.ty And Resistance Test Using Resistance Test Vectors Comprising Patient-derived ;;egment(s) And A Non-Functional Indicator Gene With A Permuted Promoter.

Indicator Gene Viral Vector Construction Indicator gene viral vector;; containing a non-functional indicator gene with a permuted promoter were designed using both HIV genomic and subgen.mic viral vectors comprising viral genes which are the tirget(s) of anti-viral drugs.

The indicator gene viral vectors pLG-lucPP and pCG-lucPP are based on the genomic viral vectors pLG and pCG; each bears a deletion in the HIV env g!ne. Resistance test vectors derived from the genomic inidicator gene viral vectors, pLG-lucPP and pCG-lucPP, contiin a patient sequence acceptor site for insertion of the pazient-derived segment and are used in conjunction with a packaging expression vector encoding the amphotrophic ML\ 4070A env gene product. The indicator gene viral vectors pLS-lucPP and pCS-lucPP are based on the subgenomic virail vectors pLS and pCS; each encodes the HIV gag-pol gene only. Resistance test vectors 25 derived from the subgenomic .ndicator gene viral vectors, pLS-lucPP and pCS-lucPP, conl.ain a sequence acceptor site for insertion of patient-derLved segment and are used in conjunction with a first packaging expression vector encoding the HIV vif, vpr, tat, rev, vpu and nef genes and a second packaging vector e:lcoding the amphotrophic MLV 4070A env gene product.

a.

a a.

o* *o ooooo: 35 HIV Viral Vectors Genomic aid Subgenomic HIV viral vectors were designed using the sequences of the biologically active proviral clone, HXB2 (Fisher et al.

(1986) Nature 320, 367-371). Two types of viral vector were designed: genomic viral vectors with deletions in a single WO 97/27319 PCT/US97/01609 -33gene such as env, but whicl. otherwise preserve the mRNA expression and processing claracteristics of the complete virus, and subgenomic viral rectors which may include only one or a few genes that are :ypically the specific targets of susceptibility and resistance testing, such as gag-pol, or which may lack viral genrs altogether. Both types of vectors have a unique restriction site within the viral genome for the insertion of an indicator gene cassette as well as patient sequence acc:eptor sites, i.e. additional unique restriction sites near or within the anti-viral target gene pol) tD permit the insertion of patient-derived HIV sequence In addition, both types of vector were designed to ircorporate either the native enhancer-promoter of the HIV-LTR U3 region, or .a foreign enhancer-promoter from the CMI immediate-early (IE) region.

Standard methods are employed for the construction of plasmid DNAs (Ausubel et al. (1987) Current Protocols in Molecular Biology, Wiley-Interscience) The sequences of all DNA constructs incorporating synthetic DNA are confirmed by DNA sequence analysis (Sarger et al. (1977) Proc. Natl.

Acad. Sci. 74, 5463-5467).

SHIV sequences are obtained frcm plasmid pBS-HIV (Page et al.

(1990) J. Virol. 64, 5270-5::76) which contains the HXB2 proviral DNA sequence on an Hpal to XbaI restriction fragment inserted into the po:ylinker of the pBluescript KS plasmid cloning vector (St.ratagene, San Diego, CA). As this proviral clone contains uncharacterized, flanking human DNA from the site of proviriLl integration, two steps of 30 site-directed mutagenesis a:-e employed to remove such sequences using plasmid pBS-H3V as a template. In step one, human sequences adjacent to :he 5' LTR are removed using oligonucleotide 1 which contains the following sequences in a 5' to 3' direction: 1) a ,:equence complementary to the first 18 nucleotides of the integrated provirus, within the WO 97/27319 PCTfUS97/01609 -84left U3 region, 2) a six nucl!otide.SmaI site, and 3) an 18 nucleotide sequence complementary to the region in pBluescript KS just beyond the polylinker and phage T3 promoter. In step two, human sequences adjacent to the LTR are removed using oligonucleotide 2 which contains the following sequences to 1) an 18 nucleotide sequence complementary to the region in pBluescript KS just beyond the PvuI site, within the LacZ gene, 2) a six nucleotide XbaI site, and 3) a sequence complementary to the last 18 nucleotides of the in:egrated provirus, within the right U5 region. The resultirg plasmid is called pBS-HXB2.

Genomic and subgenomic viral rectors employing the HIV-LTR U3 region as an enhancer-prcmoter for the expression of anti-viral target genes (Fic 1) are each derived from plasmid pBS-HXB2 by a sii.gle step of site-directed mutagenesis. The genomic viral vector pLG, which is deleted for the env gene, is prepared using oligonucleotide 3 which contains the following sequences to 1) an 18 nucleotide sequence complementary to positions 7626 to 7643 of HXB2 within the env gene (aLl coordinates for HXB2 are by reference to GenBank, access:.on number K03455), 2) an 8 Snucleotide NotI site, and 3- an 18 nucleotide sequence complementary to positions- 63 34 to 6401 of HXB2 within the

S..

25 env gene. The subgenomic viral vector pLS, which is deleted for the env, tat, rev, vif, v:r and vpu genes, is prepared 0 using oligonucleotide 4 which contains the following Ssequences to 1) an 18 nucleotide sequence complementary to positions 76 6 to 7643 of HXB2 within the 30 env gene, 2) an 8 nucleotide NotI site, and 3) an 18

S

nucleotide sequence complement.ary to positions 5109 to 5126 of HXB2 within the vif gene.

S

Genomic and subgenomic viral vectors which employ the CMV IE region as an enhancer-promcter for the expression of WO 97/27319 PCT/US97/01609 anti-viral target genes are derived from plasmids pLG and pLS, and are called pCG and pC;, respectively (Fig. 1) The genomic viral vector pCG is p:epared in two steps. In the first step, an intermediate plismid is prepared from two DNA fragments: 1) a vector fragment 11.2 kB prepared from digesting plasmid pLG with Sma and treating the vector with alkaline phosphatase, and 2) a DNA fragment of 0.9 kB containing the CMV IE enhancer-promoter prepared by digesting plasmid pVL-1 (described below) with Smal.

Plasmids containing the CM IE region in the same transcriptional orientation as the viral LTRs are identified by restriction mapping. In tha second step, plasmid pCG is prepared from this intermedia.te plasmid by site-directed mutagenesis to join the CMV IE enhancer-promoter to the 5'-LTR R region at a position permitting transcription initiation to occur at the beginning of the R region, using oligonucleotide 5 which contains the following sequences to 1) an 18 nucleotice sequence complementary to positions 455 to 472 of HXB2 at the beginning of the R region, and 2) an 18 nucleotide sequence complementary to positions -18 to -1 of th CMV IE enhancer-promoter (coordinates referenced to Bo3hart et al. (1985) Cell 41, 521-530). The subgenomic virz.l vector pCS is derived from plasmid pCG and is prepared! Irom two DNA fragments: 1) a vector DNA of 9.1 kB prepared .y digesting plasmid pLS with iSmaI and Cla,. and 2) a DNA fragment of 1.3 kB prepared by digesting plasmid pCG with Smz.I and ClaI.

Genomic Indicator Gene Viral Vector- Permuted Promoter The indicator gene viral vectDrs pLG-lucPP and pCG-lucPP, and resistance test vectors derived therefrom, contain the following elements in a 5' to 3' orientation (Fig 2B): 1) an HIV-LTR U3 region (ILG-lucPP) or a CMV IE enhancer-promoter (pCG-lucPP), 2) an HIV-LTR R region, 3) an HIV-LTR U5 region containing ai inserted T7 promoter with a WO 97/27319 PCTIUS97/01609 -E6transcriptional orientation opposite to that of the LTRs, 4) the coding regions of the HIX gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 5) an indicator gene cassette inserted into the deleted env gene, and 6) a 3' HIV-LTR. The same indicator gene cassette is inserted in pLG-lucPP and pCG-lucPP and ccntains the following elements: 1) an EMC 5'-UTR region which permits internal ribosome entry, 2) the complete coding region of the luciferase (luc) gene, and 3) a T7 transcripticnal terminator. The indicator gene has a transcriptional orientation opposite to the HIV-LTR or CMV IE enhancer-promoter and therefore cannot be functionally transcribed by these elements. The indicator gene also cannot be transcribed by the T7 promoter as the indicator gene cassette is positioned upstream of the T7 promoter. Following rever:;e transcription and strand transfer, the T7 promoter is copied from the 5' LTR to the 3' LTR, permitting functional transcription of the indicator gene from the newly created T7 promoter by T7 RNA polymerase (Fig 2C).

Plasmid plucPP, which contain:; the indicator gene cassette, is prepared in three steps. In the first step, plasmid pVL-EMC/T7 which contains a cassette flanked by unique NotI sites comprising the EMC 5'-UTR element and T7 25 transcriptional terminator, is prepared from two DNA fragments: 1) a vector DNA of 3.0 kB prepared by digesting plasmid pVL (described below with NotI and treating the vector with alkaline phosphatase, and 2) a DNA fragment of 0.8 kB containing the EMC 5' TTR and T7 terminator prepared by PCR using plasmid pTM1 (Moss et al. (1990) Nature 348, 91-92) as a template and cligonucleotides 6 and 7 as primers, followed by digestionI with NotI. Oligonucleotides 6 and 7 each incorporate a NotI restriction site. In the second step, plasmid pVL-luc, which contains the coding region of the firefly lucif:rase gene inserted into the WO 97/27319 PCT/US97/01609 mammalian expression vector 3VL-1 (described below), is prepared from two DNA fragments: a vector DNA of 4.1 kB prepared by digesting plasmid IVL-1 with NruI and BglII, and 2) a DNA fragment of 1.7 cB containing the complete luciferase coding region, prepared by PCR using plasmid pGEM-luc (Promega, Madison, WI) as a template and oligonucleotides 8 and 9 as primers followed by digestion with NruI and BglII. Oligonucleotides 8 and 9 incorporate NruI and NcoI, and BglII aid XhoI restriction sites, respectively. In the third step, plasmid plucPP, which contains the coding region of the luciferase gene inserted between the EMC 5'-UTR element and T7 transcriptional terminator, is prepared from t'io DNA fragments: 1) a vector DNA of 3.8 kB prepared by digesting plasmid pVL-EMC/T7 with NcoI and SalI, and 2) a DNA f:agment of 1.7 kB containing the complete luciferase coding region, prepared by digesting plasmid pVL-luc with NcoI and KhoI.

Plasmid pLG-lucPP is prepared in two steps. In the first step, plasmid pLG-T7 is prepared by inserting a phage T7 promoter into the upstream HIV-LTR U5 region in plasmid pLG by site-directed mutagenesis u ing oligonucleotide 10 which contains the following sequences to 1) an 18 nucleotide'sequence complementary to positions 552 to 569 of HXB2 within the U5 region, 2) a 20 nucleotide sequence complementary to the T7 promoter, and 3) an 18 nucleotide sequence complementary to po itions 534 to 551 of HXB2 within the U5 region. In the se:cond step, plasmid pLG-lucPP is prepared from two DNA fragments: 1) a vector DNA of 11.2 kB prepared by digesting plzsmid pLG-T7 with NotI and Streating the resulting vector w..th alkaline phosphatase, and 2) a DNA fragment of 2.5 kE containing the luciferase indicator gene cassette preparei by digesting plasmid plucPP with NotI. Clones corresponding to pLG-lucPP, which contain the indicator gene cassette inserted into the viral vector WO 97/27319 PCT/US97/01609 38with a transcriptional orien:ation opposite to that of the viral LTRs, are identified by restriction mapping.

Plasmid pCG-lucPP is preparel in two steps. In the first step, plasmid pCG-T7 is prepared by inserting a phage T7 promoter into the upstream HIV-LTR U5 region in plasmid pCG by site-directed mutagenesis using oligonucleotide 10. In the second step, plasmid pCG-lucPP is prepared from two DNA fragments: 1) a vector DNA of 11.7 kB prepared by digesting plasmid pCG-T7 with NotI and treating the resulting vector with alkaline phosphatase, aid 2) a DNA fragment of 2.5 kB containing the luciferase incicator gene cassette prepared by digesting plasmid plucPP with NotI. Clones corresponding to pCG-lucPP, which contain the indicator gene cassette inserted into the viral vector with a transcriptional orientation opposite to that cf the CMV IE enhancer-promoter and viral LTRs, are identified by restriction mapping.

Subqenomic Indicator Gene Vizal Vector Permuted Promoter 20 The indicator gene viral vec:ors pLS-lucPP and pCS-lucPP, and resistance test vectors cerived therefrom, contain the following elements in a 5' tc 3' orientation (Fig 2B): 1) an HIV-LTR U3 region (pLS-lucPP) or a CMV IE enhancer-promoter (pCS-lucPP) 2) an HIV-LTR R region, 3) an 25 HIV-LTR U5 region containing inserted T7 promoter with a transcriptional orientation opposite to that of the LTRs, 4) the coding region of the HIV rag-pol gene, 5) the indicator gene cassette, 6) an RRE element from the HIV env gene o* containing a viral packaging sequence, and 7) a 3' HIV- LTR.

The indicator gene cassette of pLS-lucPP and pCS-lucPP is the same as in pLG-lucPP and pCG-lucPP. As for the latter vectors, the indicator genes of pLS-lucPP and pCS-lucPP cannot be functionally :ranscribed until reverse transcription and strand tran:;fer results in the copying of the T7 promoter from the 5' LFR to the 3' LTR (Fig 2C).

WO 97/27319 PCT/US97/01609 -89- Plasmid pLS-lucPP is prepared in two steps. In the first step, plasmid pLS-T7, which :ontains a phage T7 promoter inserted into the upstream HIV-LTR U5 region of plasmid pLS, is prepared from two DNA fragnents: 1) a vector DNA of 9.1 kB prepared by digesting plasmid pLS with SmaI and Clal, and 2) a DNA fragment of 0.8 conttining the HIV-LTR with an region containing an inserted T7 promoter, prepared by digesting plasmid pLG-T7 with SmaI and Clal. In the second step, plasmid pLS-lucPP is prepared from two DNA fragments: 1) a vector DNA of 9.9 kB prepared by digesting plasmid pLS-T7 with NotI and treating the resulting vector with alkaline phosphatase, and 2) a DNA fragment of 2.5 kB containing the luciferase ind:.cator gene cassette prepared by digesting plasmid plucPP wit.h NotI. Clones corresponding to pLS-lucPP, which contain the indicator gene cassette inserted into the viral vector with a transcriptional S* orientation opposite to that of the viral LTRs, are identified by restriction mapping.

:20 Plasmid pCS-lucPP is prepared from two DNA fragments: 1) a vector DNA of 11.6 kB pre-ared by digesting plasmid pLS-lucPP with SmaI and Clal, and 2) a DNA fragment of 1.3 containing the CMV IE promoter fused to the R5 region with an inserted T7 promoter, priepared by digesting plasmid 25 pCG-T7 with SmaI and Clal.

Resistance Test Vectors Construction Resistance test vectors are prepared by 1) modifying the indicator gene viral vectors pL3-lucPP, pCG-lucPP, pLS-lucPP and pCS-lucPP by introducinc unique restriction sites, called patient sequence acceptor sites, in or near the pol gene, 2) amplifying patient-derived segments corresponding to the HIV protease and reverse transcriptase coding regions by PCR using complementary DNA (cDNA) prepared from viral RNA or DNA present in the serum or cells of infected WO 97/27319 PCT/US97/01609 -Io0patients, and 3) inserting the: amplified sequences precisely into indicator gene viral vectors at patient sequence acceptor sites (Fig. 2B). Two sets of patient sequence acceptor sites are introduced by site-directed mutagenesis into each of the four indici.tor gene viral vectors. The first set of patient sequence acceptor sites consist of a HpaI site and a SalI site which define an interval comprising the entire proteas coding region and most of the reverse transcriptase coding region, resulting in plasmids pLG-lucPP-HS, pCG-lucPP-HS, pLS-lucPP-HS and pCS-lucPP-HS.

The second set of patient seqtence acceptor sites consist of a PvuI site and a BamHI site which define the same interval, resulting in plasmids pLG-lucPP-PB, pCG-lucPP-PB, pLS-lucPP-PB and pCS-lucPP-PB, respectively. Cognate pairs of resistance test vectors which define the same restriction site interval those derived from pLG-lucPP-HS and pLG-lucPP-PB) are used together in some resistance tests to improve the representation of :hose patient-derived segments that contain internal restriction sites identical to a given 20 patient sequence acceptor site, and would thus be underrepresented in either resistance test vector alone.

Plasmid pLG-lucPP-HS is prepared by three consecutive steps of site-directed mutagenesis using plasmid pLG-lucPP as a S 25 template. The first two s:eps are for the purpose of introducing two new restriction sites, one of which (HpaI) is unique to, and one of whic i (SalI) is already is present once in each indicator gene viral vector. The third step is for the purpose of deleting the pre-existing SalI site in each vector to make the introduced SalI site unique. In step 1, a HpaI site is introluced immediately upstream of the mature coding region of the HIV protease at position 2243 using oligonucleotide 11 which contains the following sequences to 1) an 18 nucleotide sequence complementary to positions 2:49 to 2266 of HXB2, 2) a six WO 97/27319 PCT/US97/01609 -9Lnucleotide HpaI site, which leaves the gag protein sequence at this position unaltered and introduces a conservative amino acid change (Phe to 'ral) into the pol precursor sequence, and 3) an 18 nucleotide sequence complementary to positions 2225 to 2242 of HXB2. In step 2, a SalI site is introduced at the carboxy-terninal coding region of the HIV reverse transcriptase at position 4190 using oligonucleotide 12 which contains the followiig sequences to 1) an 18 nucleotide sequence complementary to positions 4196 to 4213 of HXB2, 2) a six nucleot de SalI site which leaves the reverse transcriptase protei.i sequence at this position unaltered, and 3) an 18 nucleot.ide sequence complementary to positions 4172 to 4189 of HXB2. In step 3, the pre-existing SalI site within the vpr codi:ig region at position 5785 of HXB2 is deleted using oligonucleotide 13 which contains the following sequences to 1) an 18 nucleotide sequence complementary to positions 5791 to 5808 of HXB2, 2) the 6 S: nucleotide sequence GCCGAC wh..ch ablates the SalI site but leaves the vpr protein sequence at this position unaltered, 20 3) an 18 nucleotide sequence ccmplementary to positions 5767 to 5784 of HXB2.

Plasmids pCG-lucPP-HS, pLS-licPP-HS and pCS-lucPP-HS are derived from pLG-lucPP-HS as fDllows. Plasmid pCG-lucPP-HS 25 is prepared from two DNA fragrrents: 1) a vector DNA of 12.9 kB prepared by digesting plasiid pLG-lucPP-HS with SmaI and Clal, and 2) a DNA fragment of 1.3 kB prepared by digesting plasmid pCG-lucPP with SmaI and Clal. Plasmid pLS-lucPP-HS is prepared from two DNA fragrents: 1) a vector DNA of 11.1 kB prepared by digesting plasmid pLG-lucPP-HS.with NdeI and XhoI, and 2) a DNA fragment of 1.3 kB prepared by digesting plasmid pLS-lucPP with NdeI and XhoI. Plasmid pCS-lucPP-HS is prepared from two DNA fragments: 1) a vector DNA of 11.6 kB prepared by digesting plasmid pLS-lucPP-HS with SmaI and Clal, and 2) a DNA fragment of 1.3 kB prepared by digesting WO 97/27319 PCT/US97/01609 -92plasmid pCS-lucPP with SmaI aid Clal.

Plasmid pLG-lucPP-PB is prepared by four consecutive steps of site-directed mutagenesis using plasmid pLG-lucPP as a template. The first two steps are for the purpose of introducing two new restrictioa sites (PvuI and BamHI), each of which is already present once in each indicator gene viral vector. The third ard fourth steps are for the purpose of deleting the pre-existing PvuI and BamHI sites in each vector to make the newly introduced sites unique. In step 1, a PvuI site is introcuced immediately upstream of the mature coding region of :he HIV protease at position 2221 using oligonucleotide 14 which contains the following sequences to 1) an 18 nucleotide sequence complementary to positions 2227 to 2244 of HXB2, 2) a six nucleotide PvuI site, which leaves the gag and pol precursor protein sequences at this pos:.tion unaltered, and 3) an 18 nucleotide sequence complementary to positions 2203 to 2220 of HXB2. In step 2, a BamH site is introduced at the 20 carboxy-terminal coding rec-ion of the HIV reverse transcriptase at position 4212 using oligonucleotide which contains the following sequences to 1) an 18 nucleotide sequence complementary to positions 4218 to 4235 of HXB2, 2) a six nucleotide BamHI site which leaves the.

25 reverse transcriptase proteir sequence at this position unaltered, and 3) an 18 nucleotide sequence complementary to positions 4194 to 4211 of HXB2. In step 3, the pre-existing PvuI site within the b-lactamzse coding region at position 2413 (.coordinates references :o GenBank, accession number X52331) is deleted using oligcnucleotide 16 which contains the following sequences i.o 1) an 18 nucleotide sequence complementary to rositions 2395 to 2412 of pBluescript KS 2) the nucleotide sequence CAATCG which ablates the PvuI site but leaves the b-lactamase protein sequence at this position unaltered, and 3) an 18 WO 97/27319 PCT/US97/01609 -92nucleotide sequence complementary to.positions 2419 to 2436 of pBluescript KS In step 4, the pre-existing BamHI site within the HIV rev coding region at position 8474 of HXB2 is deleted using oligonuc.eotide 17 which contains the following sequences to 1) an 18 nucleotide sequence complementary to positions 8410 to 8497 of HXB2, 2) the 6 nucleotide sequence GGATTC whi:h ablates the BamHI site but leaves the HIV rev protein sequence at this position unaltered, and 3) an 18 nucleotLde sequence complementary to positions 8456 to 8473 of HXB2.

Plasmids pCG-lucPP-PB, pLS-lu:PP-PB and pCS-lucPP-PB are derived from pLG-lucPP-PB as fcllows. Plasmid pCG-lucPP-PB is prepared from two DNA fragments: 1) a vector DNA of 12.9 kB prepared by digesting plasmid pLG-lucPP-PB with SmaI and Clal, and 2) a DNA fragment of 1.3 kB prepared by digesting plasmid pCG-lucPP with SmaI anc. Clal. Plasmid pLS-lucPP-PB 'is prepared from three DNA fragments: 1) a vector DNA of 11.1 kB prepared by digesting p.asmid pLG-lucPP-PB with NdeI :20 and XhoI, 2) a DNA fragment of 0.5 kB prepared by digesting plasmid pLS-lucPP with NdeI and HindIII and 3) a DNA fragment of 0.8 kB prepared by digesting plasmid pLG-lucPP-PB with HindIII and XioI. Plasmid pCS-lucPP-PB is prepared from two DNA fragments: 1) a vector DNA of 11.6 kB prepared by digesting plasmid pLS-lucPP-PB with SmaI and Clal, and 2) a DNA fragment of 1.3 kB prepared by digesting plasmid pCS-lucPP with SmaI an.l Clal.

Patient-derived segment corresponding to the HIV protease and reverse transcriptase coding regions are amplified by the reverse transcription-polynerase chain reaction method (RT-PCR), using viral RNA i;olated from the serum of HIV-infected patients. Two R'7-PCR protocols are used as described. In the first method (Piatak et al. (1993) Science 259, 1749-1754), separate enzymes, Moloney murine WO 97/27319 PCT/US97/01609 -S4leukemia virus reverse transcriptase (BRL, Bethesda, MD) and Taq DNA polymerase (Roche Molecular Diagnostics, Ontario, Canada), are used for the preparation of cDNA and for the PCR reaction, respectively. in the second method (Mulder et al. (1994) J. Clin. MicrobLol. 32, 292-300), a single enzyme, Thermus thermophilus (Tth) DNA polymerase, is used to carry both cDNA synthesis and the PCR reaction. Two primer pairs, consisting of o.igonucleotides 18 and 19, and oligonucleotides 20 and are employed for the .0 amplification of patient-derived segments that can be inserted precisely into the indicator gene viral vectors containing the HpaI/SalI and PvuI/BamHI patient acceptor sites, respectively.

er 2 20 o A first set of four resistan:e test vectors incorporating the first primer pair is consi.ructed from the following two DNA preparations: 1) a vector DNA prepared from plasmid pLG-lucPP-HS, pCG-lucPP-HS, ]LS-lucPP-HS or pCS-lucPP-HS, digested with HpaI and SalI, and 2) an amplified DNA product of 2.0 kB prepared by RT-PCR using viral RNA isolated from the serum of an.HIV-infected individual as a template and oligonucleotides 18 and 19 as arimers, followed by digestion with HpaI and SalI. A second set of four resistance test vectors incorporating the seccnd primer pair are constructed from the following two DNA preparations: 1) a vector DNA prepared from plasmid LG-lucPP-PB, pCG-lucPP-PB, pLS-lucPP-PB or pCS-lucPP-PB, digested with PvuI and BamHI, and 2) an amplified DNA product of 2.0 kB prepared by RT-PCR using viral RNA isolated from the serum of an HIV-infected individual as a template and Dligonucleotides 18 and 19 as primers, followed by digescion with PvuI and BamHI.

Oligonucleotides 18, 19, 20 and 21 incorporate HpaI, SalI, PvuI and BamHI restriction si:es, respectively. To ensure that the plasmid DNA corresponding to each of the eight resulting resistance test vectors comprises a representative WO 97/27319 PCT/US97/01609 sample of the HIV viral quasi-species present in the serum of a given patient, at least ore hundred independent E. coli transformants obtained in the construction of a given resistance test vector are ised for the preparation of plasmid DNA.

To improve the representation of patient-derived segments, a third and fourth set of ::esistance test vectors are prepared using partially degenerate PCR primer pools, called oligonucleotides 22, 23, 24 ard 25, which are based on the sequences of oligonucleotides 18, 19, 20 and 21, respectively. Each primer pod is synthesized in a manner that incorporates more than on! nucleotide base A, T or C) at the each of the 18 nucleozide positions located at the 3' end of the parent primer tha: display sequence variations among the different patient i.olates cataloged in the Los Alamos HIV sequence database (Myers et al. (1993) Human Retroviruses and AIDS 1993, Lo:; Alamos National Laboratory, Los Alamos, NM). The third set of four resistance test 20 vectors is constructed using vectors prepared from plasmid pLG-lucPP-HS, pCG-lucPP-HS, p!jS-lucPP-HS or pCS-lucPP-HS, with amplified patient sequences prepared with oligonucleotides 22 and 23; the fourth set of four resistance test vectors is constructed using vectors prepared from plasmid p*G-lucPP-PB, pCG-lucPP-PB, pLS-lucPP-PB or pCS-lucPP-PI, with amplified patient sequences prepared with ol:gonucleotides 24 and Oligonucleotides 22, 23, 24 ani 25 incorporate HpaI, Sall, PvuI and BamHI restriction sites, respectively.

Host Cells Preparation Packaging Host Cells and REsistance Test Vector Host Cells Resistance test vectors are used to prepare resistance test vector host cells from packaging host cells expressing viral WO 97/27319 PCTfUS97/01609 -96packaging proteins. The packaging proteins may be provided for by the expression of v:ral genes contained within the resistance test vector itself, a packaging expression vector(s), or both. Either 1ransient or stable transfection of the packaging host cell may be employed to produce the packaging proteins. A packaging expression vector encoding an amphotrophic MLV env gen2 product enables production in a resistance test vector hos: cell of resistance test vector viral particles which can efficiently infect human target cells. Resistance test 'rectors derived from plasmids pLG-lucPP-HS, pLG-lucPP-PB, pCG-lucPP-HS and pCG-lucPP-PB encode all HIV genes with th2 exception of env, and are used to produce resistance te3t vector host cells. The pVL-env4070A packaging expression vector which encodes the amphotrophic MLV 4070A env gene product is used with the foregoing genomic-based resistance test vectors to enable production in the resistaice test vector host cell of resistance test vector viril particles. Resistance test vectors derived from plasmids pLS-lucPP-HS, pLS-lucPP-PB, S: 20 pCS-lucPP-HS and pCS-lucPP-PB encode the HIV gag-pol gene products only, and are us.'d to prepare resistance test vector host cells. The pVL-tnv4070A which provides env, and either the pLTR-HIV3' or the pCMV-HIV3' packaging expression vectors, each of which-provides the HIV vif, vpr, tat, rev, 25 vpu and nef genes are used with the foregoing subgenomic based resistance test vectors to enable production in the resistance test vector host cells of resistance test vector viral particles.

Plasmids pLTR-HIV3' and pCMV-HIV3' are each derived by removing most of the gag-pol coding region from the genomic viral vectors pLG and pCG, respectively. Plasmid pLTR-HIV3' (Fig. 3B) is prepared by site-directed mutagenesis using plasmid pLG as a template with oligonucleotide 26 which contains the following secuences to 1) an 18 WO 97/27319 PCT/US97/01609 -97nucleotide sequence complementary to positions 4712 to 4729 of HXB2 within the pol ge e, and 2) an 18 nucleotide sequence complementary to positions 925 to 942 of HXB2 within the gag gene. Plan;mid pCMV-HIV3' (Fig. 3C) is prepared from two DNA fragmencs: 1) a vector fragment of 6.8 kB prepared by digesting plcsmid pLTR-HIV3' with SmaI and ClaI, and 2) a DNA fragment cf 1.3 kB prepared by digesting plasmid pCG with SmaI and Clil.

Plasmid pVL-env4070A (Fig. is constructed from two DNA fragments: 1) a vector fragment of 4.3 kB prepared by digestion of the pVL-2 mammal.an expression vector with NruI and BglII, and 2) a DNA fracment of 2.0 kB containing the complete coding region of the MLV4070A env gene product (nucleotides 37 to 2001, cDordinates given in GenBank, accession number M33469, Ott et al. (1990) J. Virol. 64, 757-766) prepared by PCR using plasmid pCRIPamgag-2 (Danos and Mulligan (1988) Proc. Natl. Acad. Sci. 85, 6460) as a template with oligonucleotides 27 and 28 as primers, r20 followed by digestion with NruI and BglII. Oligonucleotide 27 incorporates a unique Nru:: site followed by a consensus sequence for mammalian trans..ation initiation Kozak (1991) J. Biol. Chem, 266, 19867-19870), while oligonucleotide 28 incorporates a unique BglII site.

The mammalian expression vector pVL-2 contains the following elements in a 5' to 2' direction: the CMV IE promoter/enhancer, the CMV II: first exon splice donor, the human (1 globin second exon splice acceptor, a cloning site polylinker, the polyadenylat:on site of the SV40 T antigen gene, and the SV40 origin of replication. Plasmid pVL-2 is constructed in four steps as follows. In the first step, plasmid pVL is prepared by replacing the cloning site polylinker and phage T7 aid T3 promoters of plasmid pBluescript II KS witl a cloning site polylinker WO 97/27319 PCT/US97/01609 -98containing BssHII, NotI, SmaI, HindIII, SphI, Smal, EcoRI, NruI, Apal, BglII, NheI, Not I, XhoI, and BssHII restriction sites. pVL is constructed from two DNA fragments: 1) a vector fragment of 3.0 kl; prepared by cutting plasmid pBluescript II KS w:.th BssHII, and treating the resulting vector with alkaline phosphatase, and 2) a DNA fragment prepared by annealing overlapping oligonucleotides 29 and 30, extending with Klenow DNA polymerase and digesting with BssHII. Pla3mids containing the HindIII to XhoI sites in a 5' to 3' orier relative to the pBluescript II KS plasmid map (GenBznk accession number X52327) are identified by restriction mapping analysis. In the second step, an intermediate plasmid is prepared from plasmid pVL by inserting the CMV IE erhancer-promoter and first exon splice donor, and the huma:1 (1 globin second exon splice acceptor. This intermediate plasmid is prepared from three DNA fragments: 1) a vector fragment of 3.0 kB prepared by digesting plasmid pVL with HindIII and EcoRI, 2) a DNA fragment of 0.9 kB containing a CMV IE promoter-enhancer and 20 first exon splice donor (nucleotides -674 to -19, coordinates referenced to ]oshart et al. (1985) Cell 41, 521-530), prepared by PCZ using the plasmid pCM5027 containing the PstI m-fragment from HCMV strain AD169 (Boshart et al., Ibid) as-template with oligonucleotides 31 25 and 32 as primers, followec by digestion with HindIII and SphI, and 3) a DNA fragment of 0.1 kB containing the human (1 globin second exon splic2 acceptor (nucleotides 6808 to 6916, coordinates by refererce to GenBank, accession number J00153) prepared by PCR usiig plasmid ppSVaHP (Treisman et al. (1983) Proc. Natl. Acid. Sci. 80, 7428-7432) as a template with oligonucleot.ides 33 and 34 as primers, followed by digestion with S:hI and EcoRI. Oligonucleotides 31, 32, 33 and 34 incorporate HindIII, SphI, SphI and EcoRI restriction sites at their respective ends. In the third step, plasmid pVL-1 is prepared by inserting the SV40 T WO 97/27319 PCT/US97/01609 -99antigen polyadenylation site into this intermediate plasmid.

Plasmid pVL-1 is prepared from two DNA fragments: 1) a vector fragment of 4.0 1.B prepared by cutting the intermediate plasmid with EglII and NheI, and 2) a DNA fragment of 0.2 kB containing the SV40 T antigen polyadenylation site (nucle)tides 2770 to 2533 of coordinates by reference to Raddy et al. (1978) Science 200, 494-502) prepared by PCR using plasmid pSV2 (Southern and Berg (1982) J. Mol. Appl. Ge r. 1, 327-341) as template with oligonucleotides 35 and 36 as primers, followed by digestion with BglII and NheI. Oligonucleotides 35 and 36 incorporate unique BglII and NheI restriction sites at their respective ends. In the fourth step, plasmid pVL-2 is prepared by inserting the SV40 origin of :eplication into plasmid pVL-1.

Plasmid pVL-2 is prepared from two DNA fragments: 1) a vector fragment of 4.2 kB prepared by digesting plasmid pVL-1 with NheI and XhoI, ard 2) a DNA fragment of 0.2 kB containing the SV40 origin of replication (nucleotides 5725 to 5578 of SV40, Ibid) prepared by PCR using plasmid pSV2 as 20 template with oligonucleotides 37 and 38 as primers, followed by digestion with NheI and SalI. Oligonucleotides 37 and 38 incorporate unique Hlhel and Sall restriction sites at their respective ends.

Target Host Cells Target host cells used for res:istance tests carried out with resistance test vectors derived from plasmids pLG-lucPP-HS, pLG-lucPP-PB, pCG-lucPP-HS, pCG-lucPP-PB, pLS-lucPP-HS, pLS-lucPP-PB, pCS-lucPP-HS or pCS-lucPP-PB are prepared from the human embryonic kidney :ell line 293 and the Jurkat leukemic T cell line (American Type Culture Collection, Rockville, MD). Each cell l:.ne is stably transfected with an expression vector encodLng a variant phage T7 RNA WO 97/27319 PCT/US97/01609 polymerase. This variant cortains an SV40 T antigen nuclear localization signal (NLS) fused in frame to the N-terminus of the T7 RNA polymerase, permitting its transport into, and function in, the cell nucleu.3 (Lieber et al. (1989) Nucleic Acids Res. 17, 8485-8493). The non-functional indicator gene in the resistance tes: vector is converted into a functional indicator gene by reverse transcriptase upon infection of the target cells, resulting from the repositioning of the T7 proroter relative to the indicator gene coding region. Following the integration of the repaired indicator gene intc the target cell chromosome by HIV integrase, the nuclear "7 RNA polymerase expressed by the target cell is capable ol functionally transcribing the indicator gene.

Plasmid pVL-T7RNAP-NLS is used to direct the expression of a variant T7 RNA polymerase linked to an NLS, in human and other mammalian cells and call lines. pVL-T7RNAP-NLS is prepared from three DNA fragnents: 1) a vector fragment of 20 4.3 kB prepared by digestion Df the plasmid pVL-2 with EcoRI and BglII, 2) a DNA fragmeit of 2.6 kB encoding T7 RNA polymerase amino acid residues 34 to 883 (nucleotides 267 to 2817, coordinates by reference to GenBank, accession number M38308,- Grachev and Pletnv (1984) Bioorg. Khim. 25 824-843) prepared by PCR using plasmid pT7-G1 (Deng et al.

(1994) Gene 143, 245-219) as a template with oligonucleotides 39 and 40 as primers, followed by digestion with NruI and BglII, and 3) a synthetic DNA fragment encoding the first three aimino acids of SV40 T antigen followed by amino acids 11E to 133 of the SV40 large T antigen containing the NLS (Lieber et al., Ibid), prepared by annealing the overlapping oligonucleotides 41 and 42, extending with Klenow DNA ]olymerase and digesting with EcoRI and NruI. Oligonucleotides 39, 40, 41, 42 incorporate NruI, BglII, EcoRI and NruI restrictions site, respectively.

WO 97/27319 PCT/US97/01609 01- Plasmid pVL-Neo is employed as a selectable marker for the establishment of stable traisfectants of human and other mammalian cells and cell lines by co-transfection. pVL-Neo directs the expression of nromycin phosphotransferase and confers resistance to the antibiotic G418. Plasmid pVL-Neo is prepared from two DNA fracments: 1) a vector fragment of 4.3 kB prepared by digestinc plasmid pVL-2 with EcoRI and BglII, and 2) a DNA fragment of 0.8 kB containing the complete Neo coding region (rucleotides 1551 to 2345 of the Tn5 transposon sequence, coordinates given in GenBank accession number U00004, Beck et al. (1982) Gene 19, 327-336) prepared by PCR usinj plasmid pSV2neo (Southern and Berg (1982) J. Mol. Appl. Gin. 1, 327-341) as a template with oligonucleotides 43 ani 44 as primers, followed by digestion with EcoRI and BglII. Oligonucleotide 43 incorporates a unique EcoRI site followed by a consensus sequence for mammalian translation initiation, while oligonucleotide 44 incorpora:es a unique BglII site.

20 pVL-T7RNAP is introduced by stable transfection into 293 cells by the calcium phosphate coprecipitation method (Wigler et al. (1979) Cell 16, 777) and into Jurkat cells by electroporation (Irving et itl. (1991) Cell 64, 891-901) 293 cells are maintained in DMEM medium (JRH Biosciences) 25 supplemented with 1 g/L glucose, 10% donor calf serum (Tissue Culture Biologics). Jurkat cells are maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Irvin Scientific), glutamine, penicillin and streptomycin.

-:00 Transfection cocktails for 2 )3 cells and Jurkat cells each contain a mixture of 10 micrograms of pVL-T7RNAP and the selectable marker pVL-Neo in a mass ratio of 10:1 to 20:1.

Twenty-four to 48 hours following transfection, cells are replated in the same media containing the antibiotic G418 (GIBCO, Grand Island, Independent 293 cell clones resistant to G418 are picked directly from selection plates WO 97/27319 PCT/US97/01609 L02after two weeks and are expanded for analysis. Independent Jurkat cell clones resistaLnt to G418 are obtained by limiting dilution after two t.o three weeks of drug selection and are expanded for analysis.

G418-resistant 293 and Jurket cell clones are screened for their level of expression of T7 RNA polymerase by determining the level of steady-state T7 RNA polymerase-specific mRNA syn:hesized by the cells using the Northern blot hybridization method (Ausubel et al. (1987) Current Protocols in Molecular Biology, Wiley-Interscience) 293 and Jurkat cell clones e::pressing optimal levels of the T7 RNA polymerase are then identified by determining their ability to support T7 RNA polymerase-specific transcription in transient transfections with plasmid pEMCLucbgAn (Deng et al. (1991) Gene 109, 193-201 in which the transcription of the luciferase gene is directed by a T7 promoter. 293 and Jurkat clones supporting tht! highest levels of luciferase gene expression are chosen for further use; these are 20 referred to as 293/T7RNAP-N-S cells and Jurkat/T7RNAP-NLS cells, respectively.

Drug Susceptibility and Resistance Tests Resistance- tests are carried -out with resistance- test vectors based on indicator gene viral vectors pLG-lucPP-HS, pLG-lucPP-PB, pCG-lucPP-HS, pCG-lucPP-PB, pLS-lucPP-HS, pLS-lucPP-PB, pCS-lucPP-HS or pCS-lucPP-PB, using either two types of host cell or one type of host cell. In the first type of resistance test, resistance test vector viral particles are produced by a Jiirst host cell (the resistance test vector host cell) that is prepared by transfecting a packaging host cell with the resistance test vector and packaging expression vector(;). The resistance test vector viral particles are then used to infect a second host cell (the target host cell) in which the expression of the WO 97/27319 PCTIUS97/0609 -L03indicator gene is measured. In the second type of resistance test, a single Lost cell type (the resistance test vector host cell) serves both purposes: some of the packaging host cells in a given culture are transfected and produce resistance test vector viral particles and some of the host cells in the sane culture are the target of infection by the resistance test vector particles thus produced. Resistance tests employing a single host cell type are possible with resis:ance test vectors comprising a non-functional indicator gene with a permuted promoter: while such indicator genes ire efficiently expressed upon infection of a permissive host cell, they are not efficiently expressed upon transfection of the same host cell type, and thus provide an opportunity to measure the effect of the anti-viral igent under evaluation. For similar reasons, resistance tests employing two cell types may be carried out by co-cultivating the two cell types as an alternative to infecting -:he second cell type with viral particles obtained from the supernatants of the first cell 20 type.

Susceptibility and Resistance Test Two Cell Resistance test vector hos:- cells are prepared by the cotransfection of a resistance test vector and the appropriate packaging expres;ion vector(s) using either the 293 cell line, the tsa54 or tsa201 cell lines (Heinzel et al. (1988) J. Virol. 62, 3738), or the BOSC 23 cell line (Pear et al. (1993) Proc. latl. Acad. Sci. 90, 8392) as packaging host cells. Resis:ance test vectors constructed by inserting patient-derived segment into pLG-lucPP-HS, pCG-lucPP-HS, pLG-lucPP-PA and pCG-lucPP-PB are cotransfected with the packaging expression vector pVL-env4070A, while resistaice test vectors prepared by inserting patient-derived segments into pLS-lucPP-HS, pCS-lucPP-HS, pLS-lucPP-PH and pCS-lucPP-PB are WO 97/27319 PCT/US97/01609 -104cotransfected with the Dackaging expression vectors pVL-env4070A and either pLTR-HIV3' or pCMV-HIV3'.

Jurkat/T7RNAP-NLS cells are employed as target host cells.

Packaging host cells are gro n in DMEM media, 1 g/L glucose, donor calf serum and pa:;saged at a 1:10 dilution every three days. Cells are plated 48 hours prior to transfection at 1x10 6 cells per 10 cm plate. Cells are transfected by the calcium phosphate copret:ipitation method using 5 to mg each of the resistance test vector and the appropriate packaging expression vector(s) to produce resistance test vector host cells. Individual anti-viral agents, including the reverse transcriptase irhibitors AZT, ddl, ddC, d4T and 3TC, and the protease inhib..tors saquinavir, ritonavir and indinavir, as well as comb..nations thereof, are added to individual plates of transfEcted cells at the time of their transfection, at an approp:riate range of concentrations.

Twenty-four to 48 hours after transfection, target host cells are infected by co-cultivation with resistance test 20 vector host cells or with viral particles obtained from filtered supernatants of resistance test vector host cells.

Each anti-viral agent, or combination thereof, is added tothe target host cells at the time of infection to achieve the same final concentratior of the given agent, or agents, 25 present during the transfeci.ion.

For infection by co-cultivation, media is removed from a cm plate of resistance test vector host cells prepared by transfection 24 to 48 hours earlier, and 0.5 to 1.0 x 106 Jurkat/T7RNAP-NLS target cells are added to the plate in Jurkat cell media containirg the anti-viral agent at the appropriate concentration. Target cells are co-cultivated with the resistance test vector host cells for 24 hours, then removed and added to f:reshly prepared resistance test vector host cells for a secoi.d co-cultivation in Jurkat cell WO 97/27319 PCT/US97/01609 media containing the anti-viral agent(s) at the appropriate concentration. Twenty-four hours later, the target host cells are harvested from the second co-cultivation, collected by centrifugaticn, washed three times with ice-cold phosphate-buffered saline (PBS), and assayed for luciferase activity. For infection with filtered supernatants, media is renoved from a 10 cm plate of resistance test vector host cells prepared by transfection 24 to 48 hours earlier. Tie media is filtered through a 0.45 mm filter at the time oE harvest, frozen at -70 0 C, and thawed before transduction. Jurkat/T7RNAP-NLS cells (0.5 to x 106) are added to 5 ml of an equal mixture of Jurkat cell media and the filtered ;;upernatant, made up to 8 mg/ml of polybrene (Sigma, St. Louis, MI) and the appropriate concentration of the anti-vi al agent(s). Twenty-four to 48 hours post-infection, the target host cells are collected by centrifugation, washed three times with ice-cold phosphate-buffered saline, z.nd assayed for indicator gene expression. Target host cells infected by co-cultivation or i. 20 with filtered viral supernatants are assayed for firefly luciferase activity as described (Ausubel et al. (1987) Current Protocols in Molecular Biology, Wiley-Interscience).

The reduction in luciferase activity observed for target host cells infected with a given preparation of resistance 25 test vector viral particles in the presence of a given anti-viral agent, or agents, as compared to a control run in the absence of the anti-vir;il agent, generally relates to the log of the concentratioz of the anti-viral agent as a sigmoidal curve. This inhibition curve is used to calculate the apparent inhibitory concentration (IC) of that agent, or combination of agents, for the viral target product encoded by the patient-derived segments present in the resistance test vector.

WO 97/27319 PCT/US97/01609 -106- Susceptibility and Resistance Test One Cell Resistance test vector host cells are prepared by the cotransfection of a resi3tance test vector and the appropriate packaging expression vector(s) using either 293/T7RNAP-NLS cells or Jurk.t/T7RNAP-NLS cells as packaging host cells. Resistance test vectors constructed by inserting patient-derived segments into pLG-lucPP-HS, pCG-lucPP-HS, pLG-lucPP-EB and pCG-lucPP-PB are cotransfected with the packaging expression vector pVL-env4070A, while resistance test vectors prepared by inserting patient-derived segments into pLS-lucPP-HS, pCS-lucPP-HS, pLS-lucPP-FB and pCS-lucPP-PB are cotransfected with the packaging expression vectors pVL-env4070A and either pLTI.-HIV3' or pCMV-HIV3'.

Cells are transfected usi g 5 to 10 mg each of the resistance test vector aid the appropriate packaging expression vector(s) to prodice resistance test vector host cells. 293/T7RNAP-NLS cells are transfected by the calcium 20 phosphate coprecipitation mel:hod and Jurkat/T7RNAP-NLS cells are transfected by electroparation. Individual anti-viral agents, or combinations thereof, are added to individual plates of transfected cells at the time of their transfection, at an appropriate range of concentrations.

25 Twenty-four to 72 hours after transfection, cells are collected by centrifugaticn, washed three times with ice-cold phosphate-buffered saline, and assayed for firefly luciferase activity as described. As transfected cells in the culture do not efficiently express the indicator gene, transfected cells in the culture, as well superinfected cells in the culture, can serve as target host cells for indicator gene expression. The reduction in luciferase activity observed for cells transfected in the presence of a given anti-viral agent, or agents as compared to a control run in the absence of the anti-viral agent(s), generally WO 97/27319 PCT/US97/01609 -107relates to the log of the concentration of the anti-viral agent as a sigmoidal curve. This inhibition curve is used to calculate the apparent inhibitory concentration (IC) of that agent, or combination cf agents, for the viral target product encoded by the patient-derived segments present in the resistance test vector.

EXA PLE 2 HIV Drug Susceptibil Lty And Resistance Tests Using Resistance Tost Vectors Comprising Patient-derived Segments And A Non-Functional Indicator Gene With A Permuted Coding Region Indicator Gene Viral Vector Construction The genomic indicator gene viral vectors with patient sequence acceptor sites, pLG-lucPC-HS, pLG-lucPC-PB, pCG-lucPC-HS and pCG-lucPC-B, and resistance test vectors derived therefrom, each contain the following elements in a to 3' orientation (Fig 1) an HIV-LTR U3 region (pLG-lucPC-HS and pLG-luc?C-PB) or a first CMV IE :enhancer-promoter (pCG-lucPC-HS and pCG-lucPC-PB), 2) the HIV-LTR R and U5 regions, 3) the coding regions of the HIV 25 gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef *genes, 4) a first indicator (!ene cassette containing the coding region of the lucif',rase gene, inserted into the deleted env gene, 5) a second indicator gene cassette containing the 3' coding rfgion of the luciferase gene, inserted into a deleted 3' ][IV-LTR U3 region, and 6) a 3' HIV-LTR R and U5 region. pLG-lucPC-HS and pCG-lucPC-HS o contain unique Hpal and Sall patient sequence acceptor sites at nucleotides 2243 and 4190 of HXB2, respectively; pLG-lucPC-PB and pCG-lucPC-P3 contain unique PvuI and BamHI patient sequence acceptor sites at nucleotides 2221 and 4212 of HXB2, respectively (see Example 1 for details). The first indicator gene cassette contains: 1) a second CMV WO 97/27319 PCT/US97/01609 -108enhancer-promoter, 2) the 5' coding region of the luciferase gene (amino acids 1 to 446) and 3) a CMV IE splice donor.

The second indicator gene cassette contains: 1) an _-globin gene second exon splice acce3tor, 2) the 3' coding region of the luciferase gene (amino a:ids 447 to 550), and 3) an polyadenylation site. The transcriptional orientation of the luciferase 5' and 3' cod:.ng regions are identical to one another, and opposite t: that of the first CMV enhancer-promoter and viral LTRs. However, as the luciferase 5' and 3' coding regions are permuted in the resistance test vectors the 5' coding region is downstream of the 3' coding region), no mRNA is transcribed which can be spliced to generate a functional luciferase coding region. Following reverse transcription and strand transfer, the luciferase 3' (:oding region is copied from the 3' LTR to the 5' LTR, permitting the transcription of mRNA which can be spliced to ge:ierate a functional luciferase coding region (Fig 4C).

20 The subgenomic indicator gene viral vectors with patient sequence acceptor sites, pLS-lucPC-HS, pLS-lucPC-PB, pCS-lucPC-HS and pCS-lucPC-IB, and resistance test vectors derived therefrom, each contain the following elements in a to 3' orientation (Fig IB): 1) an HIV-LTR U3 region 25 (pLS-lucPC-HS and pLS-luc?C-PB) or a first CMV IE enhancer-promoter (pCS-lucPC-HS and pCS-lucPC-PB), 2) the HIV-LTR R and U5 regions, 3 the coding region of the HIV gag-pol gene, 4) a first ind.cator gene cassette containing :the 5' coding region of tle luciferase gene, 5) an RRE element from the HIV env gere containing a viral packaging sequence, 6) a second indicator gene cassette containing the 3' coding region of the luciferase gene, inserted into a deleted 3' HIV-LTR U3 regior, and 7) a 3' HIV-LTR R and region. pLS-lucPC-HS and pCS-lucPC-HS contain unique HpaI and SalI patient sequence acceptor sites at nucleotides 2243 WO 97/27319 PCT/US97/01609 -L09and 4190 of HXB2, respectively; pLS-lucPC-PB and pCS-lucPC-PB contain unique ?vul and BamHI patient sequence acceptor sites at nucleot:.des 2221 and 4212 of HXB2, respectively. The first incicator gene cassette contains: 1) a second CMV enhancer-prcmoter, 2) the 5' coding region of the luciferase gene (amina acids 1 to 446), and 3) a CMV IE splice donor. The second indicator gene cassette contains: 1) an (-globin gere second exon splice acceptor, 2) the 3' coding region of the luciferase gene (amino acids 447 to 550), and 3) an SV40 polyadenylation site. As the luciferase 5' and 3' codinci regions are permuted in the resistance test vectors, reverse transcription and strand transfer must occur to generate non-permuted luciferase and 3' coding regions, permil.ting the transcription of .mRNA which can be spliced to gererate a functional luciferase coding region (Fig 4C).

Plasmid pVL-luc5', which contains the first indicator gene cassette, is prepared in three steps. In the first two 20 steps, the artificial intron contained in pVL-1 consisting of the CMV IE splice donor ani (-globin gene splice acceptor is subjected to site-dire:cted mutagenesis to create restriction sites which upon digestion yield a DNA fragment whose and 3' termini correspond precisely to the start 25 and end of the artificiil intron. In step one, site-directed mutagenesis is carried out with pVL-1 using oligonucleotide 45 which cortains the following sequences to 1) the 18 nucleot:.de sequence which precedes the CMV IE splice donor, 2) i TAC trinucleotide sequence corresponding to first half )f the SnaBI restriction site, and 3) the 18 nucleotide secuence at the beginning of the artificial intron. As the sequence of the first three nucleotides of the intron is GTA, the resulting plasmid pVL-SnaBI contains a SnaBI restriction site which upon digestion releases the 5' secuence of the intron as a blunt WO 97/27319 PCTfUS97/01609 DNA end. In step two, site-cirected mutagenesis is carried out with pVL-SnaBI using oligonucleotide 46 which contains the following sequences to 1) the 18 nucleotide sequence at the end of the artificial intron, 2) a CTG trinucleotide sequence corresponding to last half of the PvuII restriction site, and 3) the 18 nucleotide sequence following the _-globin splice acceptor. As the sequence of the last three nucleotides of the intron is CAG, the resulting plasmid pVL-SnaBI/PvuII contains a PvuII restriction site which upcn digestion releases the 3' sequence of the intron as a blunt DNA end. In the third step, plasmid pVL-luc5' is prepared from two DNA fragments: 1) a vector DNA of 5.3 kB prepared by digesting plasmid pVL-luc with EcoRV and Nhe: and treating the resulting vector with Klenow DNA polym rase and alkaline phosphatase, and 2) a DNA fragment of 0.1 cB containing the CMV IE splice donor, prepared by digesting plasmid pVL-SnaBI/PvuII with SnaBI and SmaI. Clones cor:.esponding to pVL-luc5', which contain the CMV IE splice ionor inserted in the correct orientation into the luciferase coding region, are identified by restriction mapping.

Plasmid pVL-luc3', which contains the second indicator gene cassette; is prepared in three steps. In step one, plasmid S 25 pBS-LTR, in which the 3'LTR of pBS-HXB2 is subcloned, is prepared from two DNA fragments: 1) a vector DNA of 3.0 kB prepared by digesting plasmLd pBluescript II KS with XhoI and XbaI, and 2) a DNA fragment of 0.8 kB containing the 3'LTR, prepared by digosting pBS-HXB2 with XhoI and XbaI. In step two, plasmid pBS-LTR-luc3', which contains the 3' coding region of lIciferase followed by an polyadenylation site, inserted into the deleted 3'LTR, is prepared from two fragments: 1) a vector DNA of 3.5 kB prepared by digesting pBS-LIR with EcoRV, and treating the resulting vector with alkaline phosphatase, and 2) a DNA WO 97/27319 PCT/US97/01609 -11Lfragment of 0.5 kB containirg the. 3' luciferase coding region and SV40 polyA site, prepared by digesting plasmid pVL-luc with EcoRV and NheI, ollowed by treatment of the resulting fragment with Klen w DNA polymerase. Clones having the 3' luciferase coding region inserted in the correct orientation cpposite to the direct of transcription in the 3'LTR) a:-e identified by restriction mapping. In step three, plasm:.d pVL-luc3' is prepared from two DNA fragments: 1) a vector DNA of 4.0 kB prepared by digesting plasmid pBS-LTR-luc3' with EcoRV,. followed by treatment of the resulting vector with alkaline phosphatase, and 2) a DNA fragment of 0.1 kB containing the (-globin gene second exon splice acceptor, prepared by digesting plasmid pVL-SnaBI/PvuII with PvuII and SmaI. Clones corresponding to pVL-luc3', which contain th(: (-globin second exon splice acceptor inserted in the correct orientation into the luciferase coding region, are identified by restriction mapping.

.20 Plasmids pLG-lucPC-HS, pLG-lucPC-PB, pLS-lucPC-HS and pLS-lucPC-PB are prepared by tie same three step procedure.

In step one, plasmids pLG-lucDP-HS, pLG-lucDP-PB, pLS-lucDP-HS and pLS-lucDP-PB are prepared from two DNA fragments: i) a vector DNA prepared by digesting plasmids pLG-lucPP-HS, pLG-lucPP-PB, :LS-lucPP-HS, pLS-lucPP-PB, respectively, with SmaI and CliI, and 2) a DNA fragment of 0.8 kB prepared by digesting pl. smid pLG with SmaI and Clal.

In step two, plasmids p LG-luc5'-HS, pLS-luc5'-HS and pLS-luc5'-PB are prepared from two DNA fragments: 1) a vector DNA prepared by digesting plasmids pLG-lucDP-HS, pLG-lucDP-PB, pL,;-lucDP-HS and pLS-lucDP-PB, respectively, and treating the resulting vectors with alkaline phosphatase, and 2) a DNA fragment of 2.5 kB containing the first indicator gene cassette prepared by digesting pVL-luc5' with NotI. Clones which contain the WO 97/27319 PCT/US97/01609 -1:2first indicator gene cassette inserted into the viral vector with a transcriptional orientation opposite to that of the viral LTRs are identified by restriction mapping. In step three, plasmids pLG-lucPC-HS, :LG-lucPC-PB, pLS-lucPC-HS and pLS-lucPC-PB are prepared from two DNA fragments: 1) a vector DNA prepared by digesting plasmids pLS-luc5'-HS ani pLS-luc5'-PB, respectively, with XhoI and XbaI, and 2) a DNA fragment of 1.1 kB containing the second indicator gene cassette, prepared by digesting plasmid pVL-luc3' with XhoI and XbaI.

Plasmids pCG-lucPC-HS, pCG-lucPC-PB, pCS-lucPC-HS and pCS-lucPC-PB are each preparedI from two DNA fragments: 1) a vector DNA prepared by digesting either .plasmids pLG-lucPC-HS, pLG-lucPC-PB, pLS-lucPC-HS and pLS-lucPC-PB, respectively, with SmaI and CLaI, and 2) a DNA fragment of 1.3 kB prepared by digesting plasmid pCG with SmaI and Clal.

Resistance Test Vector Construction 20 Resistance test vectors containing a non-functional indicator gene with a permuted coding region were designed using the HIV genomic and subgenomic viral vectors comprising anti-viral target !renes described in Example 1.

25 Resistance test vectors are prepared from plasmids pLG-lucPC-HS, pLG-lucPC-PB, pCG-lucPC-HS, pCG-lucPC-PB, pLS-lucPC-HS, pLSlucPC-PB, p S-lucPC-HS and pCS-lucPC-PB (Fig. 4B), by the procedure described in Example 1.

Resistance test vectors ara constructed with vectors prepared from plasmids DLG-lucPC-HS, pCG-lucPC-HS, pLS-lucPC-HS or pCS-lucPC-I:S using amplified patient sequences prepared with oligorucleotides 18 and 19, and with oligonucleotides 22 and 23. Resistance test vectors are constructed with vectors prepared from plasmids pLG-lucPC-PB, pCG-lucPC-PB, pLS-lucPC-PB or pCS-lucPC-PB WO 97/27319 PCT/US97/01609 -113using amplified patient sequences prepared with oligonucleotides 20 and 21, and with oligonucleotides 24 and Drug Susceptibility and Resisriance Test Resistance tests are carried out by the procedures described in Example 1 as follows. Res:.stance test vectors prepared from plasmids pLG-lucPC-HS, p]LG-lucPC-PB, pCG-lucPC-HS and pCG-lucPC-PB lack a functional HIV env gene, and are used in conjunction with the packaging expression vector pVL-env4070A. Resistance I.est vectors prepared from plasmids pLS-lucPC-HS, pLS-lucPC-PB, pCS-lucPC-HS and pCS-lucPC-PB encode the HIV gag-pol gene products only, and are used in conjunction with pVL-env4070A, and either the pLTR-HIV3' or pCMV-HIV3' packa.ging expression vectors. In resistance tests carried out using two host cell types, the 293 cell line, the tsa54 cell 3ine, the tsa201 cell line, or the BOSC 23 cell line are empl3yed as packaging host cells, and unmodified Jurkat cells are employed as target cells.

20 As the non-functional indicator genes with permuted coding regions contained within these resistance test vectors are not efficiently expressed upon transfection of the packaging host cells, infection of target host cells is carried out either by co-cultivation with packaging host cells, or by using virus from the packaginc host cell supernatant. For similar reasons, resistance tests carried out with these resistance test vectors may employ a single host cell type.

Resistance tests using a single host cell type are carried out using either 293, tsa54, tsa201, BOSC 23 or Jurkat cells.

WO 97/27319 PCT/US97/01609 EXAMPLE 3 HIV Drug Susceptibility And Resistance Test Using Resistance Tes Vectors Comprising Patient-derived Segment(s) And A Non-Functional Indic tor Gene With An Inverted Intron Resistance test vectors containing a non-functional indicator gene with an inverted intron were designed using the HIV genomic and subgenoric viral vectors comprising anti-viral target genes described in Example 1.

Indicator Gene Viral Vectors Inverted Intron The genomic indicator gene viral vectors with patient sequence acceptor sites, 3LG-lucII-HS, pLG-lucII-PB, pCG-lucII-HS and pCG-lucII-PB, and resistance test vectors derived therefrom, each contain the following elements in a 5' to 3' orientation (Fig 5B 1) an HIV-LTR U3 region (pLG-lucII-HS and pLG-lucII-PB) or a first CMV IE enhancer-promoter (pCG-lucII-][S and pCG-lucII-PB), 2) the HIV-LTR R and U5 regions, 3) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 4) an indicator gene cassette inserted into the deleted env gene, and 5) a 3 HIV-LTR. pLG-lucII-HS and 25 pCG-lucII-HS contain unique Hial and SalI patient sequence acceptor sites at nucleotidi s 2243 and 4190 of HXB2, respectively; pLG-lucII-PB anc pCG-lucII-PB contain unique PvuI and BamHI patient sequence acceptor sites at nucleotides 2221 and 4212 of HXB2, respectively (see Example 30 1 for details). The indicato2 gene cassette contains 1) a second CMV enhancer-promoter, 2) the coding region of the luciferase gene interrupted by an inverted artificial intron, and 3) an SV40 poladenylation sequence. The overall transcriptional orien:ation of the indicator gene cassette is opposite to that of the first CMV enhancer-promoter and viral LTRs, while the orientation of the artificial intron is the same as the latter elements.

WO 97/27319 PCT/US97/01609 -135- Transcription of the indica:or gene by the second CMV enhancer-promoter does not lead to the production of functional transcripts as the inverted intron cannot be spliced in this orientation. "ranscription of the indicator gene by the 5' viral LFR or the first CMV IE enhancer-promoter, however, .eads to the removal of the inverted intron by RNA splicing, although the indicator gene is still not functionally expressed as the resulting transcript has an antisense orientation. Following the reverse transcription of this transcript and integration of the resultant proviral DNA, the indicator gene can be functionally transcribed by th! second CMV enhancer-promoter as the inverted intron has been previously removed (Fig The subgenomic indicator gene viral vectors with patient sequence acceptor sites, pLS-lucII-HS, pLS-lucII-PB, pCS-lucII-HS and pCS-lucII-PB and resistance test vectors derived therefrom, each contain the following elements in a to 3' orientation (Fig 5B): 1) an HIV-LTR U3 region 20 (pLS-lucII-HS and pLS-lucII-PB) or a first CMV IE enhancer-promoter (pCS-lucII- S and pCS-lucII-PB), 2) the :HIV-LTR R and U5 regions, 3) the coding region of the HIV gag-pol gene, 4) the indicator gene cassette, 5) an RRE -element from the HIV env gene containing a viral packaging 25 sequence, and 6) a 3' H[V-LTR. pLS-lucII-HS and **pCS-lucII-HS contain unique HpaI and SalI patient sequence acceptor sites at nucleotides 2243 and 4190 of HXB2, respectively; pLS-lucII-PB and pCS-lucII-PB contain unique PvuI and BamHI patient s4equence acceptor sites at nucleotides 2221 and 4212 of HXB2, respectively. The indicator gene cassette c)ntains 1) a second CMV enhancer-promoter, 2) the coding region of the luciferase gene interrupted by an inverted artificial intron, and 3) an polyadenylation sequence As is the case for the pLG-lucII and pCG-lucII genomic: viral vectors, the indicator WO 97/27319 PCT/US97/01609 -115genes of pLS-lucII and pCS-licII cannot be functionally transcribed until after the irverted intron is removed and reverse transcription and proriral integration occur (Fig Plasmid pVL-lucII, which ccntains the indicator gene cassette, in which the artificial intron from pVL-SnaBI/PvuII is inserted into the luciferase coding region in an inverted orientat .on, is prepared from two DNA fragments: 1) a vector fragnent of 5.8 kB prepared by digesting pVL-luc with EcoRV and treating the resulting vector with alkaline phosphatase, and 2) a DNA fragment of 0.2 kB corresponding precisely to the artificial intron sequence prepared by digesting pVL-SnaBI/PvuII with Snal and PvuII. Clones corresponding to pVL-lucII which contain the S. artificial intron inserted intc the luciferase coding region in an inverted orientation are identified by restriction mapping.

20 Plasmids pLG-lucII-HS, pLG-:.ucII-PB, pLS-lucII-HS and pLS-lucII-PB are prepared from two DNA fragments: 1) a vector DNA prepared by digesting plasmids pLG-lucDP-HS, pLG-lucDP-PB, pLS-lucDP-HS and pLS-lucDP-PB, respectively, and treating the resulting vectors with alkaline phosphatase, and 2) a DNA fragnent of 3.2 kB containing the luciferase indicator gene cassette prepared by digesting pVL-lucII with NotI. Clones which contain the correct plucII indicator gene casseti.e inserted into the viral vector with a transcriptional Drientation opposite to that of the viral LTRs are identified by restriction mapping and Sare used for the preparation oE resistance test vectors.

Plasmids pCG-lucII-HS, pCG-JucII-PB, pCS-lucII-HS and pCS-lucII-PB are each prepared from two DNA fragments: 1) a vector DNA prepared by diges:ing plasmids pLG-lucII-HS, WO 97/27319 PCT/US97/01609 -117pLG-lucII-PB, pLS-lucII-HS anc pLS-lucII-PB, respectively, with SmaI and Clal, and 2) a DIA fragment of 1.3 kB prepared by digesting plasmid pCG with SmaI and Clal.

Resistance Test Vector Construction Resistance test vectors are prepared from plasmids pLG-lucII-HS, pLG-lucII-PB, pCG-lucII-HS, pCG-lucII-PB, pLS-lucII-HS, pLS-lucII-PB, pCS-lucII-HS and pCS-lucII-PB (Fig. 5B), by the procedure described in Example 1.

Resistance test vectors are constructed with vectors prepared from plasmids pLG-lucII-HS, pCG-lucII-HS, pLS-lucII-HS or pCS-lucII-HM using amplified patient sequences prepared with oligontcleotides 18 and 19, and with oligonucleotides 22 and 23. Resistance test vectors are constructed with vectors prepared from plasmids pLG-lucII-PB, pCG-lucII-PB, pLS-lucII-PB or pCS-lucII-PB using amplified patient sequences prepared with oligonucleotides 20 and 21, and with oligonucleotides 24 and Drug Susceptibility and Resist.mce Test Resistance tests are carried ou: by the procedures described in Example 1 as follows. Resistance test vectors prepared from plasmids pLG-lucII-HS, pL;-lucII-PB, pCG-lucII-HS and pCG-lucII-PB lack a functional IIV env gene, and are used in Sconjunction with the pac]:aging expression vector pVL-env4070A. Resistance teIst vectors prepared from plasmids pLS-lucII-HS, pLS-lacII-PB, pCS-lucII-HS and pCS-lucII-PB encode the HIV gag-pol gene products only, and are used in conjunction with pVL-env4070A, and either the pLTR-HIV3' or pCMV-HIV3' packacing expression vectors. In resistance tests carried out using two host cell types, the 293 cell line, the tsa54 cell line, the tsa201 cell line, or the BOSC 23 cell line are emplored as packaging host cells, and unmodified Jurkat cells ar! employed as target cells.

WO 97/27319 PCT/US97/01609 -118- As the non-functional indicatcr genes with inverted introns contained within these resistance test vectors are not efficiently expressed upon t:.ansfection of the packaging host cells, infection of target host cells is carried out either by co-cultivation with packaging host cells, or by using virus from the packagingj host cell supernatant. For similar reasons, resistance l.ests carried out with these resistance test vectors may enploy a single host cell type.

Resistance tests using a sing.e host cell type are carried out using either 293, tsa54. tsa201, BOSC 23 or Jurkat cells.

EXAMILE 4 Non-Particle Based HIV Drug Susceptibility And Resistance Test UIsing Resistance Test Vectors Comprising Patient-derived Segment(s) And A Non-functional Indicator Gene.

20 Drug Susceptibility and Resis :ance Test Non-particle based resistance tests are carried out using the resistance test vectors comprising non-functional indicator genes with either permuted promoters, permuted coding regions or inverted int:ons, described in Examples 1, 25 2 and 3. These non-particle based resistance tests are performed by transfection of a single host cell type with each resistance test vector in the absence of packaging expression vectors. Although the non-functional indicator genes contained within these resistance test vectors are not 30 efficiently expressed upon transfection of the host cells, there is detectable indicator ;.ene expression resulting from non-viral particle-based reverse transcription. Reverse transcription and strand tran:fer results in the conversion of the permiuted, non-functLonal indicator gene to a non-permuted, functional iniicator gene. As reverse transcription is completely dependent upon the expression of the pol gene contained within each resistance test vector, WO 97/27319 PCT/US97/01609 anti-viral agents may be testec for their ability to inhibit the pol gene products encoded by the patient-derived segments contained within the resistance test vectors.

Non-particle based resistance tests are carried out by the general procedures described ir Example 1 with the following modifications: 1) Resistance 1.est vectors are transfected into the appropriate host cel..s, 2) anti-viral agents, or combinations thereof, are added at appropriate concentrations to individual c:ultures of transfected host cells immediately after transfection, 3) host cells are harvested 24 to 72 hours folloing transfection and assayed for luciferase activity. Tie reduction in luciferase activity observed for host ce..ls transfected with a given resistance test vector in the presence of a given anti-viral agent, or agents, as compared to a control run in the absence of the anti-viral ageni:(s) is used to calculate the apparent inhibitory content (Ki) of that agent, or combination of agents, for the viral target gene product encoded by the patient-derived segments present in the .*20 resistance test vector.

se* Resistance Test Vector Construction 0 For non-particle based resistarce tests with resistance test vectors comprising a non-functional indicator gene with a permuted promoter, resistance test vectors are prepared as described in Example 1 us.ng plasmids pLG-lucPP-HS, pLG-lucPP-PB, pCG-lucPP-HS, pCG-lucPP-PB, pLS-lucPP-HS, pLS-lucPP-PB, pCS-lucPP-HS or pCS-lucPP-PB. Each resistance test vector is transfected inLto host cells expressing a cytoplasmic T7 RNA polymerasE 293/T7RNAP cells or Jurkat/T7RNAP cells). Such hcst cells are prepared by the stable transfection of 293 cells and Jurkat cells as described in Example 1, usinj plasmid pVL-T7RNAP, which directs the expression of a cytoplasmic phage T7 RNA polymerase in human and other mammalian cells and cell WO 97/27319 WO 9727319PCTIUS97/01609 -l1 0lines. pVL-T7RNAP is construc:ed from the following two DNA fragments: 1) a vector frapnment of 4.3 kB prepared by digestion of the mammalian :xpression vector pVL-2 with EcoRI and BglII, and 2) a DNA fragment of 2.6 kB containing the complete coding region of the T7 RNA polymerase (nucleotides 166 to 2815, c:oordinates given in GenBank accession number M38308, Graclev and Pletnev (1984) Bioorg.

Khiin. 10, 824-843) prepared b-i PCR using plasmid pT7-G1 as a template with oligonucleot ides 47 and 40 as primers, followed by digestion with EcoZI and BglII. Oligonucleotide 47 incorporates a unique EcoRl site followed by a consensus sequence for eukaryotic translation initiation Kozak (1991) J. Biol. Chemn, 266, 19867-19870), while oligonucleotide 40 incorporat~s a unique BglII site.

For non-particle based resista.-ce tests with resistance test see vect ors comprising a non-functional indicator gene with a permuted coding region or inverted intron, resistance test vectors are prepared as de 3cribed in Example 1 using 20 plasmids pLG-lucPC-{S, pLG-lucPC-PB, pCG-lucPC-HS, pC0lcC-B pLSuP-S L-uP-B C-uP-S pCG-lucPC-PB, pLS-lucPC-HS, pLS-lucPC-PB, pCS-lucPC-HS, pCS-lucPC-PB, pLG-lucII-HS, L-lucII-PB, pC-lucII-HS o pCS-lucII-p. Each resistanc'e test vector is transfected 25 into either 293, tsa54, tsa2OL, BOSC 23 or Jurkat cells.

EXAMILE 66666:HIV Drug Susceptibili-:y And Resistance Test Using Resistance Test Vectors Comprising 0:66:3 Patient-derived Segmeu~t(s) And A Functional Indicator Gene.

Indicator Gene Viral Vector Functional Indicator Gene The genomic indicator gene viral vectors with patient sequence acceptor sites, plasmids pLG-luc-HS-1, pLG-luc-HS-2, pLG-luc-PB-1, pLG-luc-PB-2, pCG-luc-1{S-1, WO 97/27319 PCTUS97/01609 -121pCG-luc-HS-2, pCG-luc-PB-1 and pCG-luc-PB-2, and resistance test vectors derived therefrom, each contain the following elements in a 5' to 3' orienta:ion (Fig 1) an HIV-LTR U3 region (pLG-luc-HS-1, pLG-luc-HS-2, pLG-luc-PB-1 and pLG-luc-PB-2) or a first CMV IE enhancer-promoter (pCG-luc-HS-l, pCG-luc-HS-2, pC3-luc-PB-landpCG-luc-PB-2), 2) the HIV-LTR R and U5 regioILs, 3) the coding regions of the HIV gag-pol, vif, vpr, tat, rev, vpu, deleted env, and nef genes, 4) an indicator gene cassette inserted into the deleted env gene, and 5) a 3' HIV-LTR. pLG-luc-HS-1, pLG-luc-HS-2, pCG-luc-HS-l and pCG-luc-HS-2 contain unique HpaI and SalI patient sequence tcceptor sites at nucleotides 2243 and 4190 of HXB2, ::espectively; pLG-luc-PB-1, pLG-luc-PB-2, pCG-luc-PB-1 and pCG-luc-PB-2 contain unique 15 PvuI and BamHI patient sequence acceptor sites at nucleotides 2221 and 4212 of HX:32, respectively (see Example S1 for details) The indica:or gene cassettes of each plasmid contain 1) a second CITV enhancer-promoter, 2) the coding region of the lucife::ase gene, and 3) an polyadenylation sequence. The indicator gene cassettes of pLG-luc-HS-1, pLG-luc-PB-1, pcG-luc-HS-1 and pCG-luc-PB-1 are inserted into the vector with a transcriptional orientation opposite to the .viral LTRs or first. CMV enhancer-promoter (Fig. GB), while the indicator gene cassettes of pLG-luc-HS-2, pL;-luc-PB-2, pCG-luc-HS-2 and pCG-luc-PB-2 are inserted into the vector with the same g orientation (Fig. 6C) The subgenomic indicator gene viral vectors with patient sequence acceptor sites, plasmids pLS-luc-HS-, pLS-luc-HS-2, pLS-luc-PB-, pLS-luc-PB-2, pCS-luc-HS-, pCS-luc-HS-2, pCS-luc-PB-l and pCS-luc-PB-2, and resistance test vectors derived therefron, each contain the following elements in a 5' to 3' orientation (Fig 1) an HIV-LTR U3 region (pLS-luc-HS-, pLS-luc-HS-2, pLS-luc-PB- and WO 97/27319 PCTIUS97/0 1609 2pLS-luc-PB-2) or a first CMV IE enhancer-promoter (pCS-luc-HS-1, pCS-luc-HS-2, p"S-luc-PB-1 and pCS-luc-PB-2), 2) the 1IV-LTR R and US region3, 3) the coding region of the HIV gag-pol gene, 4) the indicator gene cassette, 5) an RRE element from the HIV env gene containing a viral packaging sequence, and 6) a 3' HIV-LTR. pLS-luc-HS-l, pLS-luc-HS-2, pCS-luc-H-S-1 and pCS-luc-HS-2 contain unique HpaI and Sail patient sequence acceptor sites at nucleotides 2243 and 4190 of HXB2, respectively; ?LS-luc-PB-l, pLS-luc-PB-2,, pCS-luc-PB-1 and pCS-luc-PB-2 contain unique PvuI and Bam.HI patient sequence acceptor Sites at nucleotides 2221 and 4212 of HXB2, respectively. The inlicator gene cassettes of each plasmid contain 1) a second (74V enhancer -promoter, 2) the complete coding region of th lucif erase gene, and 3) an SV40 polyadenylation sequence. The indicator gene cassettes of pLS-luc-HS-1, pLS-luc-PB-1, pCS-luc-HS-1 and pCS-luc-PB-1 are inserted into the veczor with a transcriptional orientation opposite to thet viral LTRs or first CMV enhancer -promoter (Fig. 6B) while the indicator gene cassettes of pLS-luc-HS-2, pJS-luc-PB-2, pCS-luc-HS-2 and pCS-luc-PB-2 are inserted into the vector with the same orientation (Fig. 6C).

Plasmids pLG-luc-HS-1 and pI.G-luc-IIS-2, pLG-luc-PB-1 and pLG-luc-PB-2,' pCG-luc-IIS-1 aItd pCG-luc-HS-2, pCG-luc-PB-1 and pCG-luc-PB-2,. pLS-lu=-HS-1 and pLS-luc-HS-2, *pLS-luc-PB-1 and pLS-lue:-PB-2, pCS-luc-HS-1 and pCS-luc-HS-2. pCS-luc-PB-1 and pCS-luc-PB-2 are each prepared from two DNA fragments: 1) a vector DNA prepared by digesting either pLG-lucII-H!:, pLG-luII-PB, pCG-lucII-HS, pCG-lucII-PB, pLS-luc-HS, pLS-lucII-PB, pCS-lucII-HS and pCS-lucII-PB, respectively ,7ith NotI and treating the resulting vectors with alkal--ne phosphatase, and 2) a DNA fragment of 3.0 kB containing the lucif erase indicator gene cassette prepared by digestii.g pVL-luc with NotI. Clones WO 97/27319 PCTIUS97/01609 -12 3containing the indicator gene cassette inserted into a given viral vector in both transcriptional orientations relative to the viral LTRs are identified by restriction mapping pLG-luc-HS-1 and pLG-luc-HS-2).

Resistance Test Vector Construction Resistance test vectors contaLning a functional indicator gene were designed using the HIV genomic and subgenomic viral vectors comprising anti-viral target genes described in Example 1. Resistance test vectors are prepared from the above plasmids (Fig. 6) by the procedure described in Example 1. Resistance test 'ectors are constructed with vectors prepared from plasmids pLG-luc-HS-1, pLG-luc-HS-2, pCG-luc-HS-1, pCG-luc-HS-2, pLS-luc-HS-1, pLS-Luc-HS-2, 15 pCS-luc-HS-1 or pCS-luc-HS-:. using amplified patient sequences prepared with oligonicleotides 18 and 19, and with oligonucleotides 22 and 23. Resistance test vectors are constructed with vectors prepared from plasmids pLG-luc-PB-1, pLG-luc-PB-2, pCG-luc-PB-1, pCG-luc-PB-2, pLS-luc-PB-1, pLS-luc-PB-2, ICS-luc-PB-1 or pCS-luc-PB-1 using amplified patient sequences prepared with oligonucleotides 20 and 21, anc with oligonucleotides 24 and 25 Drug Susceptibility and Resistance Test Resistance tests are carried ott by the procedures described in Example 1 as follows. Resistance test vectors prepared from plasmids pLG-luc-HS-1, pLG-luc-HS-2, pLG-luc-PB-1, pLG-luc-PB-2, pCG-luc-HS-1, pCG-luc-HS-2, pCG-luc-PB-1 and pCG-luc-PB-2 lack a functional HIV env gene, and are used in conjunction with the packaging expression vector pVL-env4070A. Resistance test vectors prepared from plasmids pLS-luc-HS-1, p:S-luc-HS-2, pLS-luc-PB-1, pLS-luc-PB-2, pCS-luc-HS-1, pC(S-luc-HS-2, pCS-luc-PB-1 and pCS-luc-PB-2 encode the HIV gay-pol gene products only, and WO 97/27319 PCT/US97/01609 are used in conjunction with pVL-env4070A, and either the pLTR-HIV3' or pCMV-HIV3' packaging expression vectors.

Resistance tests are carried Dut with two host cell types, using the 293 cell line, the tsa54 cell line, the tsa201 cell line, or the BOSC 23 cell line as packaging host cells, and using unmodified Jurkat cells as target cells. The infection of target cells wi:h these or other resistance test vectors containing furctional indicator genes is carried out employing the procedure for infection with resistance test vector viral particles from the filtered supernatants obtained from res istance test vector indicator host cells, as described in Example 1. In contrast to resistance test vectors comprising non-functional indicator gene viral vectors with a pernuted promoter or an. inverted intron, those comprising a functional indicator genes are typically able to express taeir indicator genes in the transfected packaging host cells. Neither the co-cultivation procedure, noi the resistance test using a single cell type can therefore be readily adapted for the infection of target cells using resistance test vectors with functional indicator genes, as it would be difficult to S* distinguish between indicator gene expression in infected target host cells and the trar.sfected packaging host cells.

V All publications and patent applications cited in this specification are herein inco:rporated by reference in their entirety as if each indiv:.dual publication or patent application were specifically and individually indicated to be incorporated by reference.

As will be apparent to those Ekilled in the art to which the invention pertains, the prese.t invention may be embodied in forms other than those specifically disclosed above, for example to carry out the drug susceptibility and resistance test on other viruses, withou: departing from the spirit or WO 97/27319 PCT/US97/01609 -125essential characteristics of the invention. The particular embodiments of the invention described above, are, therefore to be considered as illustratLve and not restrictive. The scope of the present invent on is as set forth in the appended claims rather than leing limited to the examples contained in the foregoing description.

EXAMILE 6 HIV Drug Susceptibili:y And Resistance Test Using Resistance Test Vectors Comprising Patient-derived Segment(s) And A Functional Indi:ator Gene.

Drug Susceptibility and Resis:ance Tests Resistance tests were carried out with resistance test vectors based on the indicator gene viral vectors pCG-CXCN(F-lucP)2, and pCG-CXI.T(F-lucP)2, both of which are similar to the pCG-luc-2 described in Example 5, using two host cell types. In the c.se of pCG-CXCN(F-lucP)2 the 20 indicator gene viral vector was modified in that the indicator gene cassette lacked intron A (CMV/a-globin intron described above), not contain a polyadenylation signal and the downstream 3' U5 seqtence was ommitted in the construction. The downstream U5 was replaced with the poly A signal and origin replication regions. The indicator gene viral vector pC3-CXAT(F-lucP)2 differed from pCG-CXCN(F-lucP)2 in that the indicator gene cassette contained an artificial intron downstream of the CMV enhancer-promoter and a TK pclyadenylation signal region.

30 Resistance test vector viral particles were produced by a first host cell (the resistance test vector host cell) that was prepared by transfecting a packaging host cell with the resistance test vector and the packaging expression vector.

The resistance test vector viral particles were then used to infect a second host cell (tle target host cell) in which the expression of the indicat>r gene is measured.

WO 97/27319 PCT/US97/01609 -1 6- AZT Drug Susceptibility/Resis:ance Tests The resistance test vector pCG -luc-2 containing a functional luciferase gene cassette was constructed and host cells were transfected with the resistance test vector DNA. The resistant test vectors contained "test" patient-derived reverse transcriptase sequences that were either susceptible or resistant to the nucleoside reverse transcriptase inhibitor, AZT (Sigma). The resistance test vector viral particles produced by trans:ecting the resistance test vector DNA into host cells we:-e used to infect target host cells grown either in the absence of AZT or in the presence of increasing concentrations of the drug (ranging from approximately 0.0001lM to 1000uM). The amount of luciferase activity produced in infected target host cells in the presence of drug was compared to the amount of luciferase produced in infected target lost cells in the absence of drug. Drug resistance was measured as the amount of drug required to inhibit by 50% the luciferase activity detected in the absence of drug (inhibitory concentration 50%, IC 50 The IC 50 values were determined by plotting percent drug inhibition vs. logo drug concentration.

Host cells (293) were seeded in 10-cm-diameter dishes and were transfected several days ifter plating with resistance 25 test vector plasmid DNA and tle envelope expression vector pCXAS(4070A-env) Transfections were performed using a calcium-phosphate precipitatioa procedure. The cell culture media containing the DNA precipitate was replaced with fresh medium, from one to 24 hours, after transfection. Cell culture media containing res;istance test vector viral particles was harvested one to four days after transfection and was passed through a 0.45-nm filter before being stored at -800 C. HIV capsid protein [p24) levels in the harvested cell culture media were dete rmined by an EIA method as WO 97/27319 PCT/US97/01609 -127described by the manufacturer SIAC; Frederick, MD). Six to forty-eight hours before infe:tion, target cells (293 and 293/T) were plated in cell culture media containing no AZT or serial two fold dilutions c.f AZT beginning at 100pM and ending at 0.00005AM. The AZT concentrations were maintained throughout the infection. Tarcet host cells were inoculated with 90pl of transfected resi;tance test vector host cell supernatant. Control infecticns were performed using cell culture media from mock transfections (no DNA) or transfections containing the resistance test vector plasmid DNA without the envelope expression plasmid DNA (pCXAS(4070A-env)) One to :wenty-four hours after the inoculation, fresh medium was added to each well. Twelve to thirty-six hours later the media was completely replaced with fresh media. One to three or more days after infection the media was removed and cel.. lysis buffer (Promega) was added to each well. Cell lysates were diluted 100 fold in lysis buffer and each diluted cell lysate was assayed for luciferase activity (Fig. 7A) The inhibitory effect of AZT was determined using the following equation: luciferase inhibition 1 (RLUluc [AZT1 RLUluc) where RLUluc (AZT] is the Relative Light Unit of luciferase activity in infected cells in tie presence of AZT and RLUluc is the Relative Light Unit of luciferase activity in infected cells in the absence of AZT. ICs 5 values were obtained from the sigmoidal curves that were generated from the data by plotting the percent inhibition of luciferase activity vs. the logo dru3 concentration. The AZT inhibition curves are shown in (Fig. 7B).

Nevirapine Drug Susceptibility'Resistance Test The resistant test vector, basei on the indicator gene viral WO 97/27319 PCT/US97/01609 -123vector pCG-CXCN(F-lucP)2, contained the reverse transcriptase sequence derived from the biologically active proviral clone, pNL4-3, that is susceptible to the nonnucleoside reverse transcriptase inhibitor, nevirapine (BI- RG-587, Boehringer Ingleheit) Transfection of host packaging cells and infection of host target cells were performed as described for the AZT drug susceptibility/resistance tests as described above.

Nevirapine susceptibility/resistance was evaluated using nevirapine concentrations ranging from 0.0001M to 100AM.

The nevirapine inhibition curva was determined as described above for AZT and is shown in Figure 7C.

Indinavir Drug SusceptibilityResistance Tests .15 The resistant test vector, based on the indicator gene viral vector pCG-CXCN(F-lucP)2, contained the protease sequence derived from the biologically active proviral clone, pNL4-3, that is susceptible to the protease inhibitor, indinavir (MK-639, Merck). Transfectio. of host packaging cells and infection of host target cells: were performed as described for the AZT drug susceptibilit{/resistance test except that the protease inhibitor, ind:.navir, was present in the transfected packaging host ctll cultures as well as the infected target host cell cultures, as described above.

25 Indinavir susceptibility/resistance was evaluated using indinavir concentrations ranging from 1. 5pM to 3iM. The indinavir inhibition curve was determined as described above for AZT and is shown in Figure 7D.

WO 97/27319 PCT/US97/01609 -129- EXAMPLE 7 Hepatitis Drug Susceptibility And Resistance Test Using Resistance Test Vectors Comprising Patient-Derived Segment(s) And A Non- Functional Indic;.tor Gene Containing An Inverted Intron.

Indicator Gene Viral Vector Construction Indicator gene viral vectors containing a non-functional indicator gene with an inverted intron were designed using HBV subgenomic viral vectors comprising viral genes which are the target(s) of anti-viral drugs. The indicator gene viral vectors pCS-HBV(NF-IG)::I-(PSAS-), are based on the subgenomic viral vector pCS-H3V. The indicator gene viral vector contains a non-functional indicator gene cassette containing an inverted intron and all of the cis-acting regulatory elements that Ere necessary for HBV DNA replication (DR1, 5'E DZ2, DR1*, 3'pA) but lacks the HBV gene sequences C, P, S, X genes) that provide the trans-acting structural and mnzymatic functions that are :necessary for HBV DNA replication and virus particle formation (Fig. 8B). The C, P, S and X genes and patient sequence acceptor sites are contained within a packaging 25 vector pPK-CPX (described :>elow, Fig. 8D) and pPK-S (described below, Fig. 8E). In this embodiment the indicator gene viral vector pC£ -HBV(NF-IG) II- (PSAS-) and the packaging vector pPK-CPX const tute a resistance test vector system. The non-functional indicator gene viral vector 30 pCS-HBV(NF-IG)II-(PSAS-) conti.ins the following elements in a 5' to 3' orientation: :he CMV IE enhancer promoter region, the 5' region of the HBV genome and the DR1 and copy of the encapsidation signal region (the pre-C ORF translation initiation codon is eliminated), a non-functional indicator geie cassette in which the indicator gene ORF contains an inverted intron, the region of the HBV genome containing DR2, DR1*, the and WO 97/27319 PCTIUS97/01609 -1.10the 3' HBV polyadenylation (pA) signal region. The non-functional indicator g(:ne expression cassette is comprised of some or all of tle following elements arranged in the 5' to 3' orientation: a transcriptional enhancer-promoter region, an intron, an indicator gene containing an inverted :ntron, a transcriptional polyadenylation signal sequence SV40), (HSV-1 thymidine kinase gene). The indicator gene expression cassette has a transcriptionaL orientation opposite to the HBV sequence elements (Fig. 8B'. However, the intron within the indicator gene ORF ha.; the same transcriptional orientation as the HBV sequen:e elements.

In a second embodiment, the ron-functional indicator.gene viral vector, pCS-HBV(NF-IG)II(PSAS+), contains a non-functional indicator geie cassette containing an inverted intron, all of the c:.s-acting regulatory elements that are necessary for HBV DNA replication, and some or all of the HBV gene sequences C, P, S, X genes) that :20 provide the trans-acting struc:ural and enzymatic functions that are necessary for HBV DNA replication and virus particle formation (Fig. 8F Resistance test vectors derived from the indicator gene viral vector pCS-HBV(NF-IG)II(PSAS+) conta:.n patient sequence acceptor 25 sites (PSAS) and are used in conjunction with the packaging vector, pPK-CSX (Fig. 8H). In :his embodiment the indicator gene viral vector may also provide some or all of the packaging functions, such a; P. The structural and enzymatic activities that are not provided by the indicator gene viral vector, but that are necessary for HBV DNA replication and virus particle Eormation, are provided using additional packaging vector(s' pPK-CSX (described below, Fig. 8H). In this embodiment, the non-functional indicator gene viral vector, pCS-HBV(N -IG)II(PSAS+) contains the following elements in a 5' to 3' orientation: the CMV IE WO 97/27319 PCTIUS97/01609 -231enhancer-promoter region, the 5' region of the HBV genome and the DR1 and 5'v (the pre-C ORF translation initiation codon is elimina.ted), an indicator gene cassette, containing an inve:7ted intron, positioned within the region of the HBV genome which may contain some or all of the C, P, S, and X genes ;is well as a patient-derived P gene segment, the region of the HBV genome containing DR2, DR1*, the 3'E, and the 3'E HBV pA signal region.

Within the non-functional :.ndicator gene viral vector, pCS-HBV(NF-IG)II-(PSAS+) the indicator gene expression cassette has a transcriptionail orientation opposite to the HBV sequence elements (Fig. 8' 7 However, the intron within the indicator gene ORF h is the same transcriptional orientation as the HBV sequence elements.

In transfected cells the packaging vectors (Figs 8D, 8E and 8H) provide, in trans, the structural and enzymatic functions that are necessary for HBV DNA replication and virus particle formation, but. that are not provided by the resistance test vector or th: indicator gene viral vector.

In the embodiment in which an indicator gene viral vector such as pCS-HBV(NF-IG)PP-(PEAS-)is co-transfected with a packaging vector, such as pPK-CPX, the.combination of those vectors constitute a resistaice test vector system, it is 25 the packaging vector that contains patient sequence acceptor sites for insertion of the patient-derived P gene segment (described above). The packaging vector pPK-CPX contains the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the region of the HBV genome spanning from the C OR? translation initiation codon to the 3' pA signal and including the C, P, S, and X genes.

The C gene of the packaging vectors is modified such that it does not contain and/or express the pre-C ORF sequences and does not express the S proteins (described below).

WO 97/27319 PCT/US97/01609 -112- In HBV, RNA that encodes C and/or P proteins is preferentially packaged, in cis, and consequently could interfere with the efficient packaging of the resistance test vector containing a non- functional indicator gene or a non-functional indicator gene viral vector RNA that does not encode C and P proteins. Two steps are taken to prevent the encapsidation of RNA produced from packaging vectors and to improve the encapsidation eff Lciency of the resistance test vector or non-functional ind..cator gene viral vector RNA.

First, the RNA produced by packaging vectors does not contain the 5' encapsidation ;;ignal region (Figs 8D, 8E and 8H). Second, in cases wh,!re either the C gene and/or P gene packaging functions are provided by the resistance test vector or non-functional indicator gene viral vector, packaging vectors, such as pPK-S or pPK-CSK, that do not express the C and/or P gene p:-oducts are used (Figs. 8E and 8H).

e* Resistance Test Vectors Construction Resistance test vectors are prepared by 1) modifying the indicator gene viral vector pCS-HBV(NF-IG)II-(PSAS-) by Sintroducing unique restrict.ion sites, called patient sequence acceptor sites (PSAS) in the P gene region, 2) amplifying patient-derived se-ments corresponding to the HBV 25 drug target, e.g. reverse transcriptase or DNA polymerase, by the amplification of vira.. DNA present in the serum or cells of infected patients, and 3) inserting the amplified sequences precisely into the indicator gene viral vectors at patient sequence acceptor sites (Fig. 8F). Alternatively, resistance test vector system; are prepared by 1) modifying the packaging vectors, pPK-CPX, by introducing patient sequence acceptor sites in the P gene, 2) amplifying patient-derived segments corresponding to the HBV drug target, e.g. reverse transcriptase or DNA polymerase, by amplification using viral DNA present in the serum or cells WO 97/27319 PCT/US97/01609 -1:3of infected patients, and 3) inserting the amplified sequences precisely into the packaging vectors at patient sequence acceptor sites (Fig. 8D). In one embodiment, the PSAS is situated near the border of the spacer and RT domains of the P protein (imnediately downstream of the S protein translation initiation site) and the 3' PSAS is situated near the C-terminal 2nd of the RNase H domain of the P protein. Insertion cf a patient-derived P gene segment into the patient sequence acceptor sites results in the formation of a chimeric P 4ene sequence in which the TP and spacer domains are encoded by the vector P gene sequence while the reverse transcript ase/polymerase and RNase H domains are encoded by the patient-derived segments (Figs 8D and 8F).

In HBV the entire S gene ORF orerlaps the P gene ORF but is expressed using a different reading frame (Nassal, M. and Schaller, H. (1993) Trends in Microbiology 1, 221-228).

Thus, HBV P gene sequences (re-rerse transcriptase and RNase H domains) obtained from patients also contain the corresponding patient S gene sequences. Expression of the patient-derived S gene region from the overlapping S gene ORF is prevented by elimina:ing the three S gene ORF (pre-S1, pre-S2, and S) transltion initiation sites and/or introducing in-frame termination codons in the pCS-HBV(NF-IG)II-(PSAS+) or pPK-CPK vectors (Figs. 8F and 8D). S gene expression is provided, in trans, using a separate packaging vector that provides well-characterized S gene products (Figs 8E and 3H). The modifications that eliminate S gene expression are performed without introducing changes to the ove:-lapping amino acid sequences that are encoded by the terminal protein (TP) or spacer domains of the P gene.

WO 97/27319 PCT[US97/01609 -124- Drug Susceptibility and Resis:ance Tests Drug susceptibility and resi;;tance tests are carried out with a resistance test vector based on indicator gene viral vectors pCS-HBV(NF-IG)II-(PSA3+) or with a resistance test vector system based on an indicator gene viral vector, pCS-HBV(NF-IG)II-(PSAS-) and a packaging vector, pPK-CPX, using either one type of host cell or two types of host cell. Co-transfection of paccaging host cells either with a resistance test vector, suc as pCS-HBV(NF-IG)II-(PSAS+) and a packaging vector, pPK-C3X, or with an indicator gene viral vector, such as pCS-HBV(]F-IG)II-(PSAS-) and packaging vector-containing patient-der.ved segment, such as pPK-CPX, resistance test vectoi system) produces HBV viral particles containing an encapsidated indicator gene "pregenome" RNA, which as a result of splicing of the inverted intron, contains a functional indicator gene.

(Figs. 8B and 8C).

Replicate transfections are performed on a series of packaging host cell cultures maintained either in the absence of the anti-vira. drug or in increasing concentrations of the anti-HB'7 drug an HBV P protein reverse transcriptase or pclymerase inhibitor). After maintaining the packaging hos: cells for up to several days 25 in the presence or absence of the anti- HBV drug the level of drug susceptibility or res:.stance can be assessed either directly in the host packagin!g cell lysates, or in isolated HBV particles obtained by harvesting the host packaging cell culture media. Alternative approaches can be used to evaluate drug susceptibility and resistance in the cell lysates and the isolated HBV particles.

In one embodiment, referred to as the one cell assay, drug susceptibility or resistance is assessed by measuring indicator gene expression, e.j. luciferase activity, in the WO 97/27319 PCT/US97/01609 -135transfected packaging host cells in the presence or absence of anti-viral drug. A redtction in luciferase activity observed for cells transfectd in the presence of a given anti-viral agent, or combination of agents as compared to a control run in the absence of the anti-viral agent(s), generally relates to the loc of the concentration of the anti-viral agent as a sigmoical curve.

In a second embodiment, refeired to as the two cell assay, drug susceptibility or resistance is assessed by measuring indicator gene expression, e. j. luciferase activity, in the target host cells following infection or transfection with HBV particles or HBV particle DNA, respectively. HBV viral particles obtained from packaging host cells are used to infect the target host cells or DNA from those particles is used to transfect target host cells. At the time of infection or transfection the appropriate concentration of the anti-viral drug is added to the cell culture. Up to several days following the infection or transfection, the 20 target host cells are lysed ani indicator gene expression is measured. A reduction in ind:cator gene expression will be observed for cells transfected or infected in the presence of drugs which inhibit HBV replication, for example by inhibiting either the reverse transcriptase strand DNA) or the DNA polymerase strand DNA) activities of the HBV P protein as compared to a cDntrol run in the absence of drug.

In a .third embodiment, referred to as the DNA structure indicator assay, drug susceptibility or resistance is assessed by measuring the level of HBV DNA replication that has occurred within the transfected packaging host cells or within the virus particles pioduced by the packaging host cells. In transfected host calls the HBV subgenomic viral vector is transcribed and the RNA transcript is packaged as WO 97/27319 PCT/US97/0609 36- "pregenomic" RNA. During virus maturation, the pregenomic RNA is converted to the relaxed circular form of the genomic DNA (rc-DNA) consisting of a complete minus strand and partial plus strand DNA cop,. In a subsequent step, the rc-DNA is converted to a cova..ently closed circular DNA form (cccDNA). To measure HBV ENA replication, amplification primers are designed to amplify an HBV DNA structure that is formed by the splicing of pregenomic RNA and the conversion of this spliced RNA to the rc-DNA and cccDNA forms.

Formation of the correct arplification target structure within HBV particles requireE successful completion of HBV DNA replication resulting in the formation of rc-DNA and cccDNA. Anti-viral drugs that inhibit HBV DNA replication (reverse transcription and DIA polymerase activities) will limit the formation of the INA target sequence, which in turn, can be measured as a c.ecrease in the amplified DNA 6* product using a number of quantitative amplification assays.

In one example (Fig. 10B), the binding site of the reverse 20 primer (Pr) is separated into two components by an intron sequence that is inserted into an indicator gene ORF in the O* same transcriptional orientat on as the HBV sequences. The primer binding site of the forward primer (Pf) is located within the region of the viral vector that is flanked by the 25 DR2 and DR1* sequences. In th2 linear HBV vector the Pf and Pr primers direct DNA synthesis in opposite directions and are oriented outward with respect to each other. In this case, the Pr primer directs ENA synthesis in the upstream ~direction (toward the 5' copy of and the Pf primer directs DNA synthesis in the downstream direction (toward the 3' This arrangement of primers and template does not constitute a functional amplification unit in the linear indicator gene viral vector. Furthermore, the Pr binding site is not intact in the linear unspliced vector and thus Pr will not anneal to the tarc et DNA. In contrast, the Pf WO 97/27319 PCT/US97/01609 -137and Pr primers assume an inward orientation with respect to each other in the relaxed circle form of HBV DNA found in mature virions and splicinj of the RNA pregenome has assembled an intact Pr primer binding site. Both primers now direct DNA synthesis tcward the single copy of DR1 within the plus strand copy of rc-DNA and in either plus or minus strand or cccDNA. Th:.s arrangement of primers and template constitutes a functional amplification unit (Fig.

In an alternative embodiment of the DNA sequence indicator assay (Fig. 9D) the 5' exonuclease activity of the amplification enzyme Taq polymerase) is measured rather than the production of amplified DNA Heid et al., 1996. Genome Research 6:9E6-994). The 5' exonuclease activity is measured by monitoring the nucleolytic cleavage 0 g: of a fluorescently tagged oLigonucleotide probe that is o capable of binding to the anplified DNA template region flanked by the Pf and Pr bind:.ng sites. The performance of 20 this assay is dependent on the close proximity of the 3' end of the upstream primer (Pf) to the 5' end of the oligonucleotide probe. When the Pf primer is extended it displaces the 5' end of the oligonucleotide probe such that the 5' exonuclease activity of the polymerase cleaves the S 25 oligonucleotide probe. The purpose of the intron is to distance the Pf binding site sufficiently far away from the 5' end of the exonuclease probe sequence to essentially

S

eliminate detectable exonucliase digestion of the probe oligonucleotide in the unspliced target template. Removing the intron by splicing serves to position the 3' end of the Pf binding site immediately upstream of the probe 5' binding site. The latter rearrangement enables the quantitative detection of exonuclease activity of the amplified target template (Fig. 1E).

WO 97/27319 PCT/US97/01609 -138- Drug Screening Drug screening is carried out using an indicator gene viral vector containing a non-funct.ional indicator gene cassette with an inverted intron anc a packaging vector(s). In transfected packaging host cells, the indicator gene viral vector produces an encapsidation competent RNA transcript containing the irdicator gene. The packaging vector(s) provide, in trans, the structural and/or enzymatic viral functions that are not Irovided by the resistance test vector, but that are necessary for viral DNA replication and particle formation. Upon co-:ransfection of packaging host cells, the indicator gene viral vector and packaging vectors give rise to HBV viral partic .es containing an encapsidated indicator gene viral vector "pregenome" RNA, which as a result of splicing of the inverted intron, contains a functional indicator gene.

Drug screening is performed a£ follows: indicator gene viral vector and packaging vector DNA is used to transfect the packaging host cells. Replicate transfections are performed on a series of packaging h)st cell cultures maintained either in the absence or pre3ence of potential anti-viral compounds candidate HBV P protein reverse transcriptase or polymerase inhibitors). After maintaining the packaging host cells for up to several days in the presence or absence of the cindidate anti-viral drugs the level of inhibition of DNA replication is assessed either directly in the packaging hos: cell lysates, or in isolated HBV particles obtained by harvesting the host packaging cell 30 culture media. Either DNA detection or indicator gene activity methods, described above, can be used to evaluate potential anti-HBV drug candidates.

WO 97/27319 PCT/US97/01609 -139- EXAMPLE 8 Hepatitis Drug Suscept ibility And Resistance Test Using Resistance Test Vectors Comprising Patient-Derived Segment(s) And A Non-Functional Indicator Gene Containing A Permuted Promoter.

Indicator Gene Viral Vector Construction Indicator gene viral vectors containing a non-functional indicator gene with a permutec. promoter were designed using an HBV subgenomic viral vector comprising viral genes which are the target(s) of anti-viial drugs. The indicator gene viral vectors, pCS-HBV(NF-IG)PP-(PSAS-), are based on the subgenomic viral vector pCS-H3V. The indicator gene viral vector, pCS-HBV(NF-IG) PP- contains a non-functional indicator gene cassette with a permuted promoter and all of the cis-acting regulatory elements that are necessary for 20 HBV DNA replication DR1, 5'E DR2, DR1*, 3'pA) but lacks the HBV gene sequences C, P, S, X genes) that S: provide the trans-acting structural and enzymatic functions that are necessary for HBV DNA replication and virus particle formation (Fig. 10B) The C, P and X and patient sequence acceptor sites are contained within a packaging vector pPK-CPX (described in Example 7, see Fig. 8D). The S gene is contained within a packaging vector pPK-S (described in Example 7, see Fig. 8E). In this embodiment, the indicator gene viral vector, pCS-HBV(NF-IG)PP-(PSAS-) 30 and the packaging vector pPK CPX constitute a resistance test vector system. The non-functional indicator gene viral o* vector, pCS-HBV(NF-IG)PP-(PSA3-), contains the following elements in a 5' to 3' orienta:ion: the CMV IE enhancer promoter region, the 5' recion of the HBV genome and the DR1 and (the pre-C ORF translation initiation codon is eliminated), a non-functinal indicator gene cassette assembled such that the promo:er region is positioned 3', WO 97/27319 PCT/US97/01609 -14:0i.e. .downstream, of the indicator gene ORF, the 3' region of the HBV genome containing DR2, DR1*, the and the 3' HBV pA signal regior (the pre-C ORF translation initiation codon is eliminated). The non-functional indicator gene expression cassette is comprised of some or all of the following elements arranged in the 5' to 3' orientation: an internal ribosome entry site (IRES), an indicator gene, which m.iy contain an inverted intron, and a transcriptional po:yadenylation signal sequence HSV-1 thymidine kiz.ase gene, SV40) an enhancer-promoter region. Within the non-functional indicator gene viral vector, :he indicator gene expression cassette has a transcriptiona:. orientation either opposite to or the same as the HBV sequence elements. In cases where the indicator gene ORF contains an intron, the intron has the same transcriptional orientation as the HBV sequence elements.

In a second embodiment, the ron-functional indicator gene viral vector contains a no:i-functional indicator gene cassette containing a permutec. promoter region, all of the cis-acting regulatory element;; that are necessary for HBV DNA replication, and some or zll of the HBV gene sequences C, P, S, X genes) thiat provide the trans-acting structural and enzymatic functions that are necessary for HBV DNA replication and virus particle formation, pCS-HBV(NF-IG) PP- (PSAS+) (Fig. 10D) Resistance test vectors derived from the indica:or gene viral vector, :CS-HBV(NF-IG)PP-(PSAS+), contain patient sequence acceptor 30 sites and are used in conjunct..on with the packaging vector pPK-CSK (Fig. 8H). In this enbodiment the indicator gene viral vector may also provide some or all of the packaging functions. Furthermore, in this embodiment the structural and enzymatic activities tha: are not provided by the indicator gene viral vector, but that are necessary for HBV WO 97/27319 PCT/US97/01609 -14 1- DNA replication and virus par:icle formation, are provided using additional packaging vectors. In this embodiment, the non-functional indicator gene viral vector, pCS-HBV(NF-IG)PP- (PSAS+), contains the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome and the DR1 and (the pre-C ORF trans3ation initiation codon is eliminated), an enhancer-promoter region (permuted promoter), the P gene containing patient-derived segment the indicator gene ORF an internal ribosome entry site (IRES), and the 3 region of the HBV genome containing DR2, DR1*, the 3'E, and the 3' HBV pA signal region (the pre-C ORF tran:;lation initiation codon is eliminated) Within the non-functional indicator gene viral vector, the indicator gene expression cassette has a transcriptional orientation either in the reverse or forward direction with respect to thf: HBV sequence elements. In cases where the indicator gene contains an inverted intron, the intron has a transcriptioral orientation the same as to the HBV sequence elements.

Resistance Test Vector Cons:ruction Resistance test vectors c ntaining a non-functional indicator gene with a permutec promoter were designed using the HBV subgenomic viral vecto:- comprising anti-viral target genes as described in Example 8. The indicator gene viral vector or the packaging vector is modified to include patient sequence acceptor sites (PSAS) for the insertion of P gene containing patient-derived segments (PDS) (described 30 in Example 7, see Figs. 8D and 8F). The expression of the patient-derived S gene is elim.nated as described in Example 7 (see Fig. Uniform S gene expression is provided, in trans, using a separate packaging. vector that provides well-characterized S gene products (Fig. 8E).

WO 97/27319 PCT/US97/01609 -142- Drug Susceptibility and Resia;tance Test Drug susceptibility and res:.stance tests are carried out with a resistance test vector based on indicator gene viral vectors, pCS-HBV(NF-IG) PP-(PcAS+), or with a resistance test vector system comprising an indicator gene viral vector, pCS-HBV(NF-IG)PP-(PSAS-) and a packaging vector, pPK-CPX, using either one type of host cell or two types of host cell. Upon co-transfection cf packaging host cells, either with a resistance test vector and a packaging vector or with an indicator gene viral vector and packaging vectors (i.e.

resistance test vector system) HBV viral particles are produced containing an encapsidated indicator gene "pregenome" RNA containing a ion-functional indicator gene.

Within the transfected host cells, the non-functional indicator gene with the permuted promoter is converted to a functional indicator gene dtring the HBV DNA replication process (Figs. 10B and Drug susceptibility and resistance tests are performed as 20 described in Example 7 (.bove) The resistance or susceptibility of patient-darived reverse transcriptase and/or DNA polymerase activit:.es to various anti-viral drugs can be measured by measuring the levels of indicator gene expression in transfected or infected host cells.

Alternatively, resistance or susceptibility can be measured by quantitating the amount oJ HBV DNA replication that has taken place. The latter can be performed using quantitative DNA amplification assays. Ii one example of this type of assay., (Figs. 9F and 9G) thE primer binding site of the 30 reverse primer (Pr) is located in the region downstream of the 5'E. The primer binding Eite of the forward primer (Pf) is located within the regior flanked by the DR2 and DR1* sequences. In the linear HBV vector the Pf and Pr primers direct DNA synthesis in opposite directions. In this case, the Pr primer directs DNA synthesis in the upstream WO 97/27319 PCT/US97/01609 direction (toward the ai.d the Pf primer directs DNA synthesis in the downstream direction (toward the 3'C. This primer configuration does not constitute a functional amplification unit in the linear copy of the viral vector that is used for transfection. In contrast, the Pf and Pr primers assume an orientation toward each other in the rc-DNA form that is found in nature virions. Both primers now direct DNA synthesis toqard the single copy of DR1 within the plus strand copy oJ rc-DNA. This arrangement of primers and template constitutes a functional amplification unit.

Drug Screening Drug screening using an indi:ator gene viral vector that contains a non-functional indicator gene with a permuted promoter is performed essentially as described in Example 7 above.

EXAMPLE 20 Hepatitis Drug Susceptibility And Resistance Test Using Resistance Test Vectors Comprising Patient-derived Segments And A Non-Functional Indica:or Gene Containing A Permuted Promoter And 'ranslation Initiation 25 Sites.

Indicator Gene Viral Vector Indicator gene viral vectors containing a non-functional 30 indicator gene with a permuted promoter and translation initiation site, pCS-HBV(NF-IG)PPTIS were designed using HBV subgeromic viral vector comprising viral genes which are the target(s) of anti-viral drugs. The indicator gene viral vectors pCS-HBV(NF-IG)PPTIS(?SAS-), are based on the subgenomic viral vector pCS-HIV. The indicator gene viral vector contains a non-functional indicator gene cassette with permuted promoter and translation initiation regions, WO 97/27319 PCT/US97/01609 and all of the cis-acting iegulatory elements that are necessary for HBV DNA replication DR1, DR2, DR1*, 3'pA) but lacks the HBV gene sequences C, P, S, X genes) that provide the trans-acting structural and enzymatic functions that are necessary for HBV DNA replication and virus particle formation (Fig. 11B). The C, P and X genes and patient sequence acceptor sites are contained within a packaging vector pPK-CPX (Example 7, Fig.

8D). The S gene is contained in the packaging vector, pPK-S, (Example 7, Fig. 8E). In this embodiment the indicator gene viral vector pCS-HBV(NF-IG)PPTIS(PSAS-) and the packaging vector pPK-CPX :onstitute a resistance test vector system. The non-functional indicator gene viral vector, pCS-HBV(NF-IG)PPTIS(P£AS-), contains the following elements in a 5' to 3' orientation: the CMV IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5 C (the pre-C ORF translation initiation codon is eliminated), an indicator gene ORF lacking a translation initiation site, an *2 0 enhancer-promoter region (permuted promoter) the 3' region of the HBV genome containing DR2, a functional pre-C ORF translation initiation codn, DR1*, the and the 3' HBV pA signal region. The non-functional indicator gene cassette is comprised of sone or all of the following elements arranged in a 5' t3 3' orientation: an indicator gene ORF that doe3 not contain an in-frame translation initiation site, a transcriptional polyadenylation signal sequence HSV-1 thymidine kinase gene, .SV40), an enhancer-1romoter region. Within the non-functional indicator gene viral vector, pCS-HBV(NF-IG)PPTIS (PSAS-) the indicator gene expression cassette transcriptional orient ation is the same as the HBV sequence elements. In cases vhere the indicator gene ORF contains an intron, the intron has a transcriptional orientation the same as the HBT sequence elements.

WO 97/27319 PCT/US97/01609 -145- In a second embodiment, the non-functional indicator gene viral vector contains a non-functional indicator gene cassette containing a permuted promoter and translation initiation regions, all cf the cis-acting regulatory elements that are necessary for HBV DNA replication, and some or all of the HBV gene sequences C, P, S, X genes) that provide the trans-acting structural and enzymatic functions that ire necessary for HBV DNA replication and virus particle formation, pCS-HBV(NF-IG)PPTIS(PSAS+) (lig. 11D). The structural and enzymatic activities that are not provided by the indicator gene viral vector, but that are necessary for HBV DNA replication and virus particle formation, are provided using additional packaging vectors pPK-CSX (described in Example 7, see Fig. 8E). The C, S a:id X are contained within the packaging vector pPK-CSX (described in Example 7, see Fig.

8H). In this embodiment, the non-functional indicator gene viral vector, pCS-HBV(NF-IG)PPTIS(PSAS+), contains the following elements in a 5' to 3' orientation (Fig. 11D) (1) the CMV IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5'E (the pre-C ORF translation initiation codcn is eliminated), an indicator gene ORF lacking a translation initiation site, the P gene containing pat.ient-derived segment, an enhancer-promoter region (permuted promoter) the 3' region of the HBV genome containing DR2, pre-C ORF translation initiation codon, DR1*, the and the 3' HBV pA signal region. Within the ion-functional indicator gene viral.vector, the indicator g'ine expression cassette has a transcriptional orientation the same as the HBV sequence elements. In cases where th indicator gene contains an inverted intron, the intron has a transcriptional orientation the same as the HJ1V sequence elements.

WO 97/27319 PCT/US97/01609 -146- Resistance Test Vectors Construction Resistance test vectors cDntaining a non-functional indicator gene with a permutec promoter were designed using the HBV subgenomic viral vecto:- comprising anti-viral target genes as described in Example 7. The indicator gene viral vector or the packaging vector is modified to include patient sequence acceptor sites (PSAS) for the insertion of P gene containing patient-derived segments (PDS) (described in Example 7, see Figs. 8D and 8F). The expression of the patient-derived S gene is elim:.nated as described in Example 7 (see Fig. 8D). Uniform S geae expression is provided, in trans, using a separate packaging vector that provides well-characterized S gene products (Fig. 8E).

Drug Susceptibility and Resistance Test Drug susceptibility and resistance tests are carried out by the procedures described ii Examples 7 and 8. The .:non-functional indicator gene is converted to a functional indicator gene during HBV replication (Figs. 11B and 11C).

Drug Screening Drug screening using an indi(ator gene viral vector that contains a non-functional irdicator gene with permuted promoter and translation initiation regions is performed essentially as described in E>amples 7 and 8 (above).

EXAMPLE 1) Hepatitis Drug Suscept:.bility And Resistance Test Using Resistance Test Vectors Comprising Patient-de:.ived Segments And A Non-Functional Indicator Gene With Permuted Coding Regions.

Indicator Gene Viral Vector- Indicator gene viral vectors containing a non-functional indicator gene with permuted coding region were designed using HBV subgenomic viral WO 97/27319 PCT/US97/01609 -117vectors comprising viral geni s which are the target(s) of anti-viral drugs. The incicator gene viral vectors pCS-HBV(NF-IG)PCR(PSAS-), are based on the subgenomic viral vector pCS-HBV. The indicator gene viral vector contains a non-functional indicator gene cassette with a permuted coding region, and all of the :is-acting regulatory elements that are necessary for HBV DNA replication DR1, DR2, DR1*, 3'pA) but lacks the HBV gene sequences

C,

P, S, X genes) that provide tie trans-acting structural and enzymatic functions that Ere necessary for HBV DNA replication and virus particle formation (Fig. 12B) The C, P and X genes and patient sequence acceptor sites are contained within a packaging vector, pPK-CPX and the S gene is provided by the packaginc vector pPK-S (described in Example 7, see Figs. 8D and 3E). In this embodiment the indicator gene viral vector pC -HBV(NF-IG) PCR (PSAS-) and the packaging vector pPK-CPX const tute a resistance test vector system. The non-functional Lndicator gene viral vector, pCS-HBV(NF-IG)PCR(PSAS-), contains the following elements in a 5' to 3' orientation: :he CMV IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and 5'E (the pre-C ORF translation initiation codon is eliminated), a non-functiDnal indicator gene cassette assembled such that the promoter region and a 5' portion of the coding region are positioned i.e. downstream, of the remaining 3' portion of the coc.ing region, the 3' region of the HBV genome containing DZ2, DR1*, the 3'E, and the 3' HBV pA signal region (the pre-C ORF translation initiation codon is eliminated). The non-functional indicator gene 30 cassette is comprised of sorte or all of the following elements arranged in the 5' tc 3' orientation: the 3' region of an intron ending in a splice acceptor sequence (2) the 3' region of an indicator (reporter) ORF or selectable marker ORF, a transcript:.onal polyadenylation signal sequence HSV-1 thymidinE kinase gene, SV40), an WO 97/27319 PCT/US97/01609 -148enhancer-promoter region, the 5' region of an indicator gene ORF, the 5' region of an intron beginning in a splice donor sequence. Within the non-functional indicator gene viral vector, the indicator gene expression cassette has a transcriptional oriertation either the same or opposite the HBV sequence elements. In cases where the indicator gene ORF contains an intron, the intron is oriented in the same orientation with respect to the HBV sequence elements.

In a second embodiment, the ron-functional indicator gene viral vector contains a noa-functional indicator gene cassette containing a permuted coding region, all of the cis-acting regulatory elements that are necessary, for HBV DNA replication, and some or ll of the HBV gene sequences C, P, S, X genes) that provide the trans-acting structural and enzymatic func:ions that are necessary for HBV DNA replication and virus particle formation pCS-HBV(NF-IG)PCR(PSAS+) (Fiqf. 12D). Resistance test 20 vectors derived from the i dicator gene viral vector pCS-HBV(NF-IG)PCR(PSAS+) contaiin patient sequence acceptor sites (PSAS) and are used in cDnjunction with the packaging vector pPK-CSK (described in Example 7, see Fig. 8H). In this embodiment the indicato2 gene viral vector may also provide some or all of the packaging functions. In this embodiment the structural and enzymatic activities that are not provided by the indicator gene viral vector, but that are necessary for HBV DNA replication and virus particle formation, are provided using additional packaging vectors.

30 In this embodiment, the non-functional indicator gene viral vector contains the following elements in a 5' to 3' orientation: the CMV IE enhancer promoter region, (2) the region of the HBV genome :.mmediately downstream of the pre-C ORF translation initiation codon and the DR1 and the indicator gene casseti.e containing a region of the WO 97/27319 PCT/US97/01609 -149- HBV genome which may contain ;ome or all of the C, P, S, and X genes, the region of the HBV genome containing DR2, DR1*, the 3'C, and the 3' HBV pA signal region (the pre-C ORF initiation codon has bEen eliminated). Within the non-functional indicato2 gene viral vector, pCS-HBV(NF-IG)PCR(PSAS+) the indicator gene expression cassette has a transcriptionzl orientation either the same or opposite to HBV sequence ilements. In cases where the indicator gene contains an inverted intron, the intron is oriented in the same orientation with respect to the HBV sequence elements.

Resistance Test Vectors Construction Resistance test vectors containing a non-functional indicator gene with a permuted coding region were designed using the HBV subgenomic viral vector comprising anti-viral target genes as described in Ixample 7. The indicator gene viral vector or the packaging vector is modified to include patient sequence acceptor sites (PSAS) for the insertion of :20 P gene containing patient-derived segments (PDS) (described in Example 7, see Figs. 8D ani 8F). The expression of the patient-derived S gene is elim..nated as described in Example 7 (see Fig. 8D) Uniform S gene expression is provided, in oo trans, using a separate paccaging vector that provides 25 well-characterized S gene products (Fig. 8E).

Drug Susceptibility and Resistance Tests Drug susceptibility and resistance tests are carried out by the procedures described ii Examples 7 and 8. The *30 non-functional indicator gene is converted to a functional indicator gene during HBV replication (Figs. 12B and 12C).

Drug Screening Drug screening using an indicator gene viral vector that contains a non-functional irdicator gene with permuted WO 97/27319 PCT/US97/01609 -150promoter and translation in.tiation regions is performed essentially as described in E:xamples 7 and 8 (above).

EXAMPLE 11 Hepatitis Drug Susceptibility And Resistance Test Using Resistance Test Vector Comprising Patient-Derived Segment(s) And A Functional Indicator Gene.

Indicator Gene Viral Vector Construction Indicator gene viral vectors containing a functional indicator gene were designe using HBV subgenomic viral vectors comprising viral genes which are the target(s) of anti-viral drugs. The indicator gene viral vectors pCS-HBV(F-IG) (PSAS-), are bi.sed on the subgenomic viral vector pCS-HBV. The indicator gene viral vector, pCS-HBV(F-IG) (PSAS-), contaii.s a functional indicator gene cassette and all of the cis-a:ting regulatory elements that are necessary for HBV DNA replication DR1, 5'E DR2, 20 DR1*, 3'pA) but lacks the HBV gene sequences C, P, S, X genes) that provide the trans-acting structural and enzymatic functions that ire necessary for HBV DNA replication and virus particle, formation (Fig. 14B) The C, P and X genes and patient sequence acceptor sites are contained within a packaging vector pPK-CPX and the S gene is contained within the packaging vector, pPK-S, (described below see Figs. 8D and 8E.. In this embodiment the indicator gene viral vector pCS-HBV(F-IG) (PSAS-) and the packaging vector pPK-CPX constitute a resistance test vector system. The functional indicator gene viral vector pCS-HBV(F-IG) (PSAS-) contains the following elements in a to 3' orientation: the CM' IE enhancer-promoter region, the 5' region of the HBV genome including the DR1 and (the pre-C ORF tranulation initiation codon is eliminated), a functional indicator gene cassette, (4) the 3' region of the HBV genome containing DR2, DR1*, the and the 3' HBV pA signal region (the pre-C ORF w S *5

S

S

S

WO 97/27319 PCT/US97/01609 -l.;iltranslation initiation codon .s eliminated). The indicator gene expression cassette is comprised of some or all of the following elements arranged Ln the 5' to 3' orientation: a transcriptional enhancer-promoter region, an intron, an indicator gene ORF or selectable marker gene ORF, a transcriptional pclyadenylation signal sequence SV40) or unidirectional (HSV-1 thymidine kinase gene) The indicator gene expression :assette has a transcriptional orientation the same as or opposite to the HBV sequence elements.

In a second embodiment, the indicator gene viral vector contains a functional indicator gene cassette, all of the cis-acting regulatory elements that are necessary for HBV DNA replication, and some or ill of the HBV gene sequences C, P, S, X genes) that provide the trans-acting structural and enzymatic functions that are necessary for o o HBV DNA replication and virus particle formation **oo Se pCS-HBV(F-IG) (PSAS+) (Fig. 13D). Resistance test vectors S. SC :20 derived from the indicator gene viral vector pCS-HBV(F-IG) (PSAS+) contain patient sequence acceptor sites (PSAS) and are used in conjunct:ion with the packaging vector pPK-CSK. In this embodiment the indicator gene viral vector also provides some or all of :he packaging functions. The structural and enzymatic activities that are not provided by the indicator gene viral vector, but that are necessary for SHBV DNA replication and virus particle formation, are provided using additional packaging vectors pPK-CSX (described in Example 7, see Fig. 8H). In this embodiment, 30 the functional indicator gen? viral vector contains the following elements in a 5' to orientation: the CMV IE enhancer promoter region, the region of the HBV genome immediately downstream of the pre-C ORF translation initiation codon and the DR] and 5'E, a functional indicator gene cassette withir the region of the HBV genome WO 97/27319 PCT/US97/01609 -352that contains some or all of the C, P, S, and X genes, (4) the region of the HBV genome containing DR2, DR1*, the 3'E, and the 3' HBV pA signal reg.on. Within the indicator gene viral vector, the indicator !jene expression cassette has a transcriptional orientation either opposite to or the same as the HBV sequence elements 15 20 Resistance Test Vector Con:truction Resistance test vectors .:ontaining a non-functional indicator gene with a permuted promoter were designed using the HBV subgenomic viral vectcr comprising anti-viral target genes as described in Example 7. The indicator gene viral vector or the packaging ve:tor is modified to include patient sequence acceptor sitas (PSAS) for the insertion of P gene containing patient-derived segments (PDS) (described in Example 7, see Figs. 8D ard 8F). The expression of the patient-derived S gene is elininated as described in Example 7 (see Fig. 8D). Uniform S gene expression is provided, in trans, using a separate packaging vector that provides well-characterized S gene prcducts (Fig. 8E).

Drug Susceptibility and Resistance Test Drug susceptibility and resistance test are carried out with a resistance test vector based on a functional indicator gene viral vector, pCS-HBV(F-IG) (PSAS+) or with a resistance test vector system based on ai. indicator gene viral vector, pCS-HBV(F-IG)(PSAS-) and a packaging vector(s). In transfected packaging host cells, the indicator gene viral vector produces an encaps.dation competent

RNA

transcript containing a functional indicator gene cassette.

The packaging vector(s) provide, in trans, the structural and/or enzymatic viral functions that are not provided by the functional indicator gene viral vector, but that are necessary for viral DNA replication and particle formation.

Upon co-transfection of packaging host cells, the indicator WO 97/27319 PCT/US97/01609 -153gene viral vector and packaging vectors are capable of forming HBV particles containLng an encapsidated indicator gene viral vector "pregenome" RNA containing a functional indicator gene cassette.

In this example, the indicatoi gene viral vector contains a functional indicator gene cass tte and therefore can produce indicator gene activity in transfected cells in the absence of HBV DNA replication (Fic. 13). In the case of a functional indicator gene, the inhibition of HBV DNA replication by drugs can be evaluated by harvesting the virus particles produced in the packaging host cell and using the particles (or particle DNA) to infect (or transfect) a target host =ell. Alternatively, DNA *.15 replication can be measured directly in virus particles isolated from packaging host :ells by using the DNA as an indicator. A drug which inhilits HBV DNA replication will reduce the formation of virus particles containing the "mature" rc-DNA form of the functional indicator gene viral vector. Consequently, the fuictional indicator gene will not be efficiently transferred to the target host cells during infection/transfection and the amount of indicator gene viral vector cccDNA and indicator gene activity in these cells will be reduced. The detection of indicator gene expression in target host cells in a two cell assay is performed as described in Example 7. The detection of S. rc-DNA in virus particles is performed using DNA as an .indicator as described in Example 8 and illustrated in Figs.

9F and 9G.

Drug Screening Drug screening using an indicator gene viral vector that contains a non-functional iniicator gene with permuted promoter and translation initiation regions is performed essentially as described in Examples 7 and 8 (above).

Page(s)_fl 2 -VI-iae claims pages

A

they appear after the sequence listing WO 97127319 WO 9727319PCTfUS97/01609 -1:4 oligonucleotides 1) 5' -AGTGAATTAGCCCTTCCACCC SGGTCGAGCTTGGCGTAATCA- 3' (42-mer) (SEQ ID NO:l) 2) 5' -CTGTTGGGAAGGGCGATCTCT bGATGCTAGAGATTTTCCACA- 3' (42-mer) (SEQ ID NO:2) 3) 5' -CTCCTCCTCCAAGTCTGAGCG 3CCGCCTTTAGCATCTGATGCAC- 3' (44-rer) (SEQ ID NO:3) 4) CTCCTCCTCCAAGTCTGAGCG 3CCGCCATATGGTGTTTTACTAA~- 3' (44-mer) (SEQ ID NO:4) 5' -GGTCTAACCAGAGAGACCCGG rTCACTAAACGAGCT- 3' (36-mer) (SEQ ID 6) 5' -GAATTCGCGGCCGCAATTCCG 'CCCTCTCCCT- 3' (32-mer) (SEQ ID NO:6) 7) 5' -GTTAACGCGGCCGCGATATAG"TCCTCCTTTC-3' (32-mer) (SEQ ID NO:7) 5' -GAATTCTCGCGACCATGGAAG' CGCCAAAAAC- 3' (32-mer) (SEQ ID NO:8) 9) 5' -GTTAACAGATCTCTCGAGTTA :AATTTGGACTTTCC- 3' (36-mer) (SEQ ID NO:9) 5' -AGACGGGCACACACTACTAA'

ACGACTCACTATAGGG

TGAAGCACTCAAGGCAAG-3' (56-mer) (SEQ ID 11) 5' -AAGAGTGACCTGAGGGAAGTTIACGGATACAGTTCCTTGTCT- 3' (42-mer) (SEQ ID NO:11) 25 12) 5' -TCCAGCACTGACTAATTTGTCC ACTTGTTCATTTCCTCCAAT- 3' (42-mer) (SEQ ID NO:12) 13) 5' -TAACGCCTATTCTGCTATGCCC ACACCCAATTCTGAAAATGG- 3' (42-mer) (SEQ ID NO:13) 14) 5' -AAGGATACAGTTCCTTGTCGAJ'CGGCTCCTGCTCTGAGGGG- 31 (42-mer) (SEQ ID NO:14) 5 CTAAAAATAGTACTTTCCGGA9)CCCAGCACTGACTAATTTAT- 3' (42-trer) (SEQ ID 16) 5' -TTAGCTCCTTCGGTCCTCCAAI CGTTGTCAGAAGTAAGTTGG- 3' (42-rner) (SEQ ID NO:16) 17) 5' -GTCCCAGATAAGTGCCAAGGA1TCGTTCACTAATCGAATGGA- 3' WO 97/27319 WO 9727319PCT/US97/01609 -155- (42-mer) (SEQ. ID NO:17) 18) 5' -GAATTCGTTAACTTCCCTCA ;ATCACTCTTTGG- 3' (33-mer) (SEQ ID NO:18) 19) 5' -GTTAACGTCGACTTGTTCAr 'TCCTCCAAT- 3' (30-iner) (SEQ ID NO:19) 5' -GAATTCCGATCGACAAGGAA( :TGTATCCTTTAACTTCCC TCAGATCACTCTTTGG-3' (55-tier) (SEQ ID 21) 5' -GTTAACGGATCCCAGCACTGI .CTAATTTATCTACTTGTTC ATTTCCTCCAAT-3' (52-rner) (SEQ ID NO:21) 22) 5' -GAATTCGTTAACTTCCCTCA G/A) ATC CTCTTTGG-3' (33-mer pool) (SEQ ID NC':22) 23) 5' -GTTAACGTCGACTT(G/T) ('/C)TCATTTCCTCC(A/T)AT-3' pool) (SEQ ID NC :23) 24) 5' -GAATTCCGATCGACAAGGAAC TGTATCCTTTAACTTCCC TCA(G/A)ATC(A/C)CTCTTGC (55-mer pool) (SEQ ID 25)Y 5' -GTTAACGGATCCCAGCACTG7CTAATTTATCTACTT (G/T) TCATTTCCTCC AT- 3' (52-mer pool) (SEQ ID 26) 5' -ATCTCTTACCTGTCCTATCT; ACAGGCCAGGATTAA- 3' (36-mer) (SEQ ID NO:26) 27) 5' -GAATTCTCGCGACCACCATGC CGCGTTCAACGCTC -3' (SEQ ID NO:27) 28) 5' -GTTAACAGATCTTCATGGCTC GTACTCTAT-3' .25 (30-mer) (SEQ ID NO:28) 29) 5' -GAATTCGCGCGCAAGCGGCCC CAACCCGGGAAAAGCTT AAGCATGCAACCCGGGAAGAATTC PATCGCGAAA- 3' (72-ruer) (SEQ ID NO:29) 5' -GTTAACGCGCGCTTCTCGAGI TGCGGCCGCTTGCTAGCTT AGATCTT'GGGCCCTTCGCGAT7 SAATTCTT- 3' (72-mer) (SEQ ID 31) 5' -GAATTCAAGCTTGGCCATTGC ATACGTTGT- 3' (SEQ ID NO:31) 32) 5' -GTTAACGCATGCATAAGAAGC ZAA- 3' (24-mer) (SEQ ID NO:32) WO 97/27319 WO 9727319PCTIUS97/01609 -1!26 33) 5' -GAATTCGCATGCTCCCCTGCT:CGACCCGG-3' (SEQ ID NO: 33) 34) 5' -GTTAACGAATTCTCCTGCGGG 37AGAAGCAG- 3' (SEQ ID NO:34) 35) 5' -GAATTCAGATCTGCCATACCA':ATTTGTAG- 3' (SEQ ID 36) 5' -GTTAACGCTAGCTCCAGACAT YATAAGATA- 3' (SEQ ID NO:36) 37) 5' -GAATTCGCTAGCATCCCGCCC:'TAACTCCG-3' (30-mer) (SEQ ID NO:37) 38) 5' -GTTAACGTCGACGCAAAAGCcr[AGGCCTCC- 3' (SEQ ID NO:38) 39) 5' -GAATTCTCGCGAACAGTTGGC 'CT- 3' (24-rner) (SEQ ID NO:39) **S1 40) 5' -GTTAACAGATCTTTACGCGAA- GCGAAGTC- 3' (SEQ ID 5' -GTTAACGAATTCTTGCAAAAA' CTTTGCAAGATGGATA AAGTTTTAGAAACTCCAGTAGGA, TCC -3' "*(66-mer) (SEQ ID NO:41) 42) 5' -GAATTCTCGCGATCTAGACGT"

CTACCTTTCTCTTCTT

TTTTGGAGGAGTCCTACTGGAGTT' -3' (63-mer) (SEQ ID NO:42) 43) 5' V (35-rner) (SEQ ID NO:43) 25 44) 5' -GAATTCAGATCTTCAGAAGAACTrCGTCAAG-3' (SEQ ID NO:44) 5' -CCCCGTGCCAAGAGTGACTAC(;TAAGTACCGCCTATAGA- 3' (39-mer) (SEQ ID 46) 5' -CTCTGCTCTCCCCGCAGCTG( ;AGAATTCAATCGCGAAA- 3' (39-mer) (SEQ ID NO:46) 47) 5' -GTTAACGAATTCCCACCATGA itCACGATTAACATC (SEQ ID NO:47) WO 97/27319 PCT/US97/01609 -1 7- SEQUENCE LISTING GENERAL INFORMATION: APPLICANTS: Capon, Daniel J. and Christos J.

Petropoulous (ii) TITLE OF INVENTION: Compositions And Methods For Determining Anti-vi:-al Drug Susceptibility And Resistance (iii) NUMBER OF SEQUENCES: 47 25 30 30 e o (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Cooper Dunham LLP STREET: 1185 Ave.iue of the Americas CITY: New York STATE: New York COUNTRY: United States ZIP: 10036 COMPUTER READABLE FOIM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM PC-DOS/MS-DOS SOFTWARE: Patent:n Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii)ATTORNEY/AGENT INFOIMATION: NAME: White, Johi. P.

REGISTRATION NUMI.ER: 28,678 REFERENCE/DOCKET NUMBER: 50130-A-PCT/JPW/AKC (ix) TELECOMMUNICATION INIORMATION: TELEPHONE: 212-278-0400 TELEFAX: 212-391-0526 INFORMATION FOR SEQ ID NO 1: SEQUENCE CHARACTERISIICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 97/27319 PCT/US97/01609 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGTGAATTAG CCCTTCCACC CGGGTCGAGC TTGGCGTAAT CA 42 INFORMATION FOR SEQ ID NC:2: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTGTTGGGAA GGGCGATCTC TAGATGCFAG AGATTTTCCA CA S. 42 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISFICS: LENGTH: 44 base pairs 25 TYPE: nucleic a:id STRANDEDNESS: sLngle TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (jenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTCCTCCTCC AAGTCTGAGC GGCCGCCTT AGCATCTGAT GCAC 44 INFORMATION FOR SEQ ID NO 4: SEQUENCE CHARACTERIS"ICS: LENGTH: 44 base pairs TYPE: nucleic at:id STRANDEDNESS: s:.ngle TOPOLOGY: linea:- (ii) MOLECULE TYPE: DNA ((enomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO:4: CTCCTCCTCC AAGTCTGAGC GGCCGCCLTA TGGTGTTTTA CTAA 44 INFORMATION FOR SEQ ID WO 97/27319 PCT/US97/01609 -159- SEQUENCE CHARACTERIS"ICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: s:.ngle TOPOLOGY: lineal (ii) MOLECULE TYPE: DNA (cenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGTCTAACCA GAGAGACCCG GTTCACT)AA CGAGCT 36 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: lineal (ii) MOLECULE TYPE: DNA (cenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: .GAATTCGCGG CCGCAATTCC GCCCCTC CC CT 32 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERIS ICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (g-nomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTTAACGCGG CCGCGATATA GTTCCTCCT TC 32 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 32 base Dairs TYPE: nucleic acid STRANDEDNESS: si.gle TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (gnomic) WO 97/27319 PCT/US97/01609 -1 0- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAATTCTCGC GACCATGGAA GACGCCAkAA AC 32 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERIS"ICS: LENGTH: 36 base pairs TYPE: nucleic a:id STRANDEDNESS: s:.ngle TOPOLOGY: linea:- (ii) MOLECULE TYPE: DNA (qenomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO:9: GTTAACAGAT CTCTCGAGTT ACAATTT(;GA CTTTCC 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERIST'ICS: LENGTH: 56 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: lineal (ii) MOLECULE TYPE: DNA (cenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AGACGGGCAC ACACTACTTA ATACG CTCA CTATAGGGTG

AAGCACTCAA

GGCAAG 56 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISIICS: LENGTH: 42 base pairs 40 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AAGAGTGACC TGAGGGAAGT TAACGGATC AGTTCCTTGT CT 42 INFORMATION FOR SEQ ID NO:L2: WO 97/27319 WO 9727319PCTIUS97O 1609 -161- Wi SEQUENCE CHARACTERI:;TICs: LENGTH: 42 basi! pairs TYPE: nucleic itcid STRANDEDNESS: -;ingle TOPOLOGY: lineir- (ii) MOLECULE TYPE: DNA genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TCCAGCACTG ACTAATTTGT CGACTTC ;TTC ATTTCCTCCA AT 42 INFORMATION FOR SEQ ID NC':1.3: SEQUENCE CHARACTERI= ICS: LENGTH: 42 basE pairs TYPE: nucleic Ecid STRANDEDNESS: -ingle TOPOLOGY: linear (ii) MOLECULE TYPE: DNA igenomic) (xi) SEQUENCE DESCRIPTIOD: SEQ ID-NO:13: TAACGCCTAT TCTGCTATGC CGACACCCAA TTCTGAAAAT GG 42 INFORMATION FOR SEQ ID NC:14: SEQUENCE CHARACTERISTICS: CA) LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AAGGATACAG TTCCTTGTCG ATCGGCCT GCTTCTGAGG GG 42 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISrics: CA) LENGTH: 42 base pairs TYPE: nucleic a~id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (3enomic) WO 97/27319 PCT/US97/01609 -152- (xi) SEQUENCE DESCRIPTION: SEQ ID CTAAAAATAG TACTTTCCGG ATCCCACCAC TGACTAATTT AT 42 INFORMATION FOR SEQ ID NC:16: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TTAGCTCCTT CGGTCCTCCA ATCGTTGrCA GAAGTAAGTT GG 42 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERIS ICS LENGTH: 42 base pairs 25 TYPE: nucleic a:id STRANDEDNESS: sLngle TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (jenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTCCCAGATA AGTGCCAAGG ATTCGTT2AC TAATCGAATG GA 42 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs 40 TYPE: nucleic ac:id STRANDEDNESS: s:.ngle TOPOLOGY: linea:- (ii) MOLECULE TYPE: DNA ((fenomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO:18: GAATTCGTTA ACTTCCCTCA GATCACT(TT TGG 33 INFORMATION FOR SEQ ID NO 19: WO 97/27319 PCTfUS97/01609 -1 ;3- SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GTTAACGTCG ACTTGTTCAT TTCCTCCAAT .o INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 55 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GAATTCCGAT CGACAAGGAA CTGTATCC TT TAACTTCCCT CAGATCACTC TTTGG INFORMATION FOR SEQ ID NC:21: SEQUENCE CHARACTERISTICS: LENGTH: 52 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GTTAACGGAT CCCAGCACTG ACTAATI AT CTACTTGTTC ATTTCCTCCA AT 52

S

35 INFORMATION FOR SEQ ID NC:22: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 97/27319 PCT/US97/01609 -154- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GAATTCGTTA ACTTCCCTCA RATCMC CTT TGG 33 INFORMATION FOR SEQ ID NC:23: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GTTAACGTCG ACTTKYTCAT TTCCTCCJAT INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 55 base pairs TYPE: nucleic a:id STRANDEDNESS: s ngle TOPOLOGY: linea:- S(ii) MOLECULE TYPE: DNA (q!enomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO:24: GAATTCCGAT CGACAAGGAA CTGTATCCTT TAACTTCCCT CARATCMCTC TTTGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERIS-'ICS: LENGTH: 52 base pairs 40 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: lineal (ii) MOLECULE TYPE: DNA (cenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GTTAACGGAT CCCAGCACTG ACTAATTIAT CTACTTKYTC ATTTCCTCCW AT 52 INFORMATION FOR SEQ ID NO:26: WO 97/27319 PCT/US97/01609 5

S

S

S

SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATCTCTTACC TGTCCTATCT AACAGGC AG GATTAA 36 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISrICS: LENGTH: 35 base pairs TYPE: nucleic a=id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (3enomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GAATTCTCGC GACCACCATG GCGCGTT2AA CGCTC INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERIS ICS: LENGTH: 30 base pairs TYPE: nucleic a:id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Jenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GTTAACAGAT CTTCATGGCT CGTACTCrAT INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic a:id STRANDEDNESS: sLngle TOPOLOGY: linea- (ii) MOLECULE TYPE: DNA (jenomic)

S

S

WO 97/27319 PTU9/10 PCT/US97/01609 -166- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GAATTCGCGC .GCAAGCGGCC GC.AJCCCGGG AAAAGCTTAA

GCATGCAACC

CGGGAAGAAT TCAATCGCGA

AA

72 2S *too 40 C 0* 0 o: INFORMATION FOR SEQ ID b0O:30: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genotnic) (xi) SEQUENCE DESCRIPTIOIJ: SEQ ID GTTAACGCGC GCTTCTCGAG TTGC3GCCGC TTGCTAGCTT

AGATCTTTGG

GCCCTTTCGC GATTGAATTC

TT

72 INFORMATION FOR SEQ ID N'):31: SEQUENCE CHARACTERI,3TICS: LENGTH: 30 basi! pairs TYPE: nucleic acid STRANDEDNESS: :;ingle TOPOLOGY: lineiar (ii) MOLECULE TYPE: DNA genornic) (xi) SEQUENCE DESCRIPTIOVr: SEQ ID NO:31: GAATTCAAGC TTGGCCATTG CATACG',TGT INFORMATION FOR SEQ ID NC :32: SEQUENCE CHARACTERIE

TICS:

LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: WO 97/27319 PCT/US97/01609 -1)i7- GTTAACGCAT GCATAAGAAG CCAA 24 25 INFORMATION FOR SEQ ID NC:33: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Igenomic) (xi) SEQUENCE DESCRIPTIO SEQ ID NO:33: GAATTCGCAT GCTCCCCTGC TCCGACCCGG INFORMATION FOR SEQ ID NC:34: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTIOn: SEQ ID NO:34: GTTAACGAAT TCTCCTGCGG GGAGAACCAG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTIOn: SEQ ID GAATTCAGAT CTGCCATACC ACATTTCTAG INFORMATION FOR SEQ ID NC:36: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid 40 WO 97/27319 WO 9727319PCTIUS97/0 1609 -3 68- STRANDEDNESS: single TOPOLOGY: lineir (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTIO:I1: SEQ ID NO:36: GTTAACGCTA GCTCCAGACA TGATAA';ATA INFORMATION FOR SEQ ID Noi37: SEQUENCE CHAR-ACTERI:;TICs: LENGTH: 30 basfe pairs TYPE: nucleic itcid STRANDEDNESS: ,;ingle TOPOLOGY: lineir (ii) MOLECULE TYPE: DNA genomic) (xi) SEQUENCE DESCRIPTIO[: SEQ ID NO:37: GAATTCGCTA GCATCCCGCC CCTAAC7CCG INFORMATION FOR SEQ ID NC':38: SEQUENCE CHARACTERIc TICS: LENGTH: 30 basE pairs TYPE: nucleic zcid STRANDEDNESS: Eingle TOPOLOGY: linezr (ii) MOLECULE TYPE: DNA 'genomic) (xi) SEQUENCE DESCRIPTIOD: SEQ ID NO:38: GTTA.ACGTCG ACGCAAAAGC CTAGGCCTCC INFORMATION FOR SEQ ID NC:39: SEQUENCE CHARACTERISETICS: LENGTH: 24 base pairs TYPE: nucleic F-cid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTIOIS: SEQ ID NO:39: WO 97/27319 PCT/US97/01609 -11 9- GAATTCTCGC GAACAGTTGG CCCT 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISrICS: LENGTH: 30 base pairs TYPE: nucleic a:id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (3enomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GTTAACAGAT CTTTACGCGA ACGCGAA3TC INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic a id STRANDEDNESS: sLngle S: 25 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (jenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GTTAACGAAT TCTTGCAAAA AGCTT"GCAA GATGGATAAA GTTTTTAGAA ACTCCAGTAG

GACTCC

35 66 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERIS'ICS: 40 LENGTH: 63 base pairs TYPE: nucleic a,:id STRANDEDNESS: s..ngle TOPOLOGY: linea:" (ii) MOLECULE TYPE: DNA (!fenomic) (xi) SEQUENCE DESCRIPTION SEQ ID NO:42: GAATTCTCGC GATCTAGACG TTCTA :CTTT CTCTTCTTTT TTGGAGGAGT CCTACTGGAG TTT 63 WO 97/27319 WO 9727319PCT/US97O 1609 -170- INFORMATION FOR SEQ ID N0:43: Wi SEQUENCE CHARACTERI!;TICS: LENGTH: 35 baset pairs TYPE: nucleic iLcid STRANDEDNESS: ,;ingle TOPOLOGY: lineiur (ii) MOLECULE TYPE: DNA genomic) (xi) SEQUENCE DESCRIPTIOV: SEQ ID NO:43: GTTAACGAAT TCCCACCATG ATTGAA(!AAG ATGGA INFORMATION FOR SEQ ID NC1:44: SEQUENCE CHARACTERISTICS: LENGTH: 30 basE pairs TYPE: nucleic zcid STRANDEDNESS: ,ingle TOPOLOGY: 1ine~r (ii) MOLECULE TYPE: DNA 'genomic) (xi) SEQUENCE DESCRIPTIOk: SEQ ID NO:44: GAATTCAGAT CTTCAGAAGA ACTCGTCAAG INFORMATION FOR SEQ ID NC SEQUENCE CHARACTERISTICS:.

LENGTH: 39 basE pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTIOIS: SEQ ID CCCCGTGCCA AGAGTGACTA CGTAAGIACC GCCTATAGA INFORMATION FOR SEQ ID NC:46: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/27319 PCTIUS97/01609 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CTCTGCTTCT CCCCGCAGCT GGAGAA'IrCA ATCGCGAAA 39 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISrics: LENGTH{: 35 base pairs TYPE: nucleic a~id STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (3enomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GTTAACGAAT TCCCACCATG AACACGAVTA ACATC

Claims (63)

1. A method for determining susceptibility of a virus to an anti-viral drug said method comprising: culturing a host cell in the presence of said antiviral drug, wherein said host cell comprises a resistance test vector comprising a viral segment and an indicator gene, wherein expression of said indicator gene depends on the activity of said viral segment or a gene product encoded by said viral segment, and wherein said resistance test vector does not comprise all viral genes necessary to produce an infectious viral particle measuring the amount of expression of said indicator gene in said host cell; and comparing the amount of expression of said indicator gene as measured in step with a reference amount of expression of said S 15 indicator gene, wherein the difference between said amount of expression of said indicator gene measured in step relative said reference amount of expression correlates with the susceptibility of said virus to said antiviral drug, thereby determining the susceptibility of said virus to said antiviral drug.

2. The method of claim 1, wherein said reference amount of expression of said .•indicator gene is determined by performing the method of claim 1 in the absence of said antiviral drug.

3. The method of claim 1, wherein said reference amount of expression of said indicator gene is an amount of expression determined by performing the 25 method of claim 1 with a standard laboratory viral segment and in the absence :oooo of said antiviral drug.

4. The method of claim 1, wherein said resistance test vector comprises DNA of a genomic viral vector. The method of claim 1, wherein the resistance test vector comprises DNA of a subgenomic viral vector. C 9* 9 C 173

6. The method of claim 1, wherein the resistance test vector comprises DNA from a retrovirus. 5 7. The method of claim 1, wherein the resistance test vector comprises DNA from HIV.

8. The method of claim 1, wherein the resistance test vector comprises DNA encoding vif, vpr, tat, rev, vpu, and nef. 0

9. The method of claim 1, wherein said viral segment is a patient-derived segment. I (I 9.9.5 .9 #9 g9 9 91 9. 9~ 99 g e* 9 *.0 *C is. e g 9 sA 25 999 9 I9 9.. 30 The method of claim 1, wherein the said viral segment comprises a functional viral sequence.

11. The method of claim 1, wherein said viral segment encodes a protein that is a target of said anti-viral drug.

12. The method of claim 1, wherein said viral segment encodes two proteins that are target of said anti-viral drug.

13. The method of claim 1, wherein said viral segment comprises a retroviral gene.

14. The method of claim 1, wherein said viral segment comprises an HIV gene. The method of claim 1, wherein said viral segment comprises an HIV gag-pol gene.

16. The method of claim 1, wherein said indicator gene is a functional indicator gene and wherein said method additionally comprises the step of infecting said host cell with a resistance test vector viral particle prepared from filtered 9999 9 174 supernatants of a cell culture comprising a resistance test vector host cell, wherein said step of infecting is performed prior to step of the method of claim 1. 5 17. The method of claim 1, wherein said indicator gene is a non-functional indicator gene.

18. The method of claim 17, wherein said method additionally comprises the step of infecting said host cell with a resistance test vector viral particle prepared 10 from filtered supernatants of a cell culture comprising a resistance test vector S* host cells, wherein said step of infecting is performed prior to step of the method of claim 1. oo

19. The method of claim 17, wherein said host cell is cultured with a resistance test vector host cell.

20. The method of claim 1, wherein said indicator gene is a luciferase gene. U.

21. The method of claim 1, wherein said indicator gene is a lacZ gene.

22. The method of claim 18, wherein the said resistance test vector host cell is a human cell. 3

23. The method of claim 18, wherein said resistance test vector host cell is a 25 human embryonic kidney cell.

24. The method of claim 18, wherein said resistance test vector host cell is a 293 cell. 30 25. The method of claim 1, wherein said host cell is a human T cell.

26. The method of claim 1, wherein said host cell is derived from a human T cell leukemia cell line. S el o• 0•i' eel l 175

27. The method of claim 1, wherein said host cell is a Jurkat cell.

28. The method of claim 1 wherein said host cell is a H9 cell. 9 o 9**o

29. The method of claim 1, wherein said host cell is a CEM cell. o A resistance test vector comprising a viral segment and an indicator gene, wherein said resistance test vector does not comprise all viral genes 10 necessary to produce an infectious viral particle, and wherein expression of said indicator gene depends on the activity of a gene encoded by said viral **segment.

31. The resistance test vector of claim 30, wherein said viral segment comprises two genes.

32. The resistance test vector of claim 30, wherein said viral segment comprises a .retroviral gene. 20 33. The resistance test vector of claim 30, wherein said viral segment comprises an HIV gene. 9*

34. The resistance test vector of claim 30, wherein said viral segment comprises 0 in i an Hretrvial Vgene.n 20 35. The resistance test vector of claim 30, wherein said viral segment comprises 's.•an HIV gene. o 34. The resistance test vector of claim 30, wherein said indviral segment comprcator gene is aes :30 functional 25

35. The resistance test vector of claim 30, wherein said viral segment is a patient- functional indicator gene. .*9l i 30 functional indicator gene.

37. The resistance test vector of claim 30, wherein said indicator gene is a non- functional indicator gene. 999 o• 176

38. The resistance test vector of claim 30, wherein said indicator gene is a luciferase gene.

39. A packaging host cell that comprises the resistance test vector of claim The packaging host cell of claim 39, wherein said packaging host cell is a mammalian host cell.

41. The packaging host cell of claim 39, wherein said packaging host cell is a human host cell.

42. The packaging host cell of claim 39, wherein said packaging host cell is a human embryonic kidney cell.

43. The packaging host cell of claim 39, wherein said packaging host cell is a 293 cell.

44. The packaging host cell of claim 39, wherein said packaging host cell is derived from a human hepatoma cell line.

45. The packaging host cell of claim 39, wherein said packaging host cell is a ~HepG2 cell. 9 9i 25 46. The packaging host cell of claim 39, wherein said packaging host cell is a Huh7 cell.

47. A method for determining the susceptibility of a virus to an anti-viral drug said method comprising: culturing the host cell in the presence of said antiviral drug, wherein said host cell comprises a resistance test vector comprising a viral segment and a non-functional indicator gene, wherein expression of said non-functional indicator gene depends on the activity of said viral 177 segment or a gene product encoded by said viral segment and wherein said resistance test vector does not comprise all viral genes necessary to produce an infectious viral particle; measuring the amount of expression of said non-functional indicator gene in said host cell; and comparing the amount of expression of said non-functional indicator gene as measured in step with a reference amount of expression of said non-functional indicator gene, wherein the difference between said amount of expression of said non-functional indicator gene measured in step relative to that measured in step correlates with the susceptibility of said virus to said anti-viral drug.

48. The method of claim 47, wherein said reference amount of expression of said indicator gene is determined by performing steps and of the 15 method of claim 47 in the absence of said antiviral drug.

49. The method of claim 47, wherein said reference amount of expression of said indicator gene is a standard amount of expression determined from performing the method of claim 47 with a standard laboratory viral segment and in the absence of said antiviral drug. The method of claim 47, wherein said resistance test vector comprises DNA of a genomic viral vector. so goes 25 51. The method of claim 47 wherein said resistance test vector comprises DNA of a subgenomic viral vector.

52. The method of claim 47, wherein said resistance test vector comprises DNA derived from a retrovirus.

53. The method of claim 47, wherein said resistance test vector comprises DNA derived from HIV. a S 0a S S. a 5% r %55 S* **S eSS *ur G~\e. SE. CD 2 a a S. 0 O es 0 Dee 0G.. 25 egA 550 C eeetC@ 178

54. The method of claim 47, wherein the resistance test vector comprises DNA encoding vif, vpr, tat, rev, vpu, and nef, or a combination thereof.

55. The method of claim 47, wherein said viral segment encodes one protein.

56. The method of claim 47, wherein said viral segment encodes two proteins.

57. The method of claim 47, wherein said viral segment comprises a retroviral gene.

58. The method of claim 47 wherein said viral segment comprises an HIV gene.

59. The method of claim 47, wherein said viral segment comprises an HIV gag-pol gene.

60. The method of claim 47, wherein said viral segment is a patient-derived segment.

61. The method of claim 47, wherein said indicator gene is a luciferase gene.

62. The method of claim 47, wherein said non-functional indicator gene comprises a permuted promoter.

63. The method of claim 47, wherein said non-functional indicator gene comprises a permuted coding region.

64. The method of claim 47, wherein said non-functional indicator gene comprises an inverted intron. The method of claim 47, wherein said host cell is a human cell.

66. The method of claim 47, wherein said host cell is a human T cell. S 5 0 *S* a S 0 0 5 S *5 5 S S 0 0 *0 0 0 0 0 0 *000 0 179

67. The method of claim 47, wherein said host cell is cultured with a resistance test vector host cell.

68. The method of claim 47, wherein said method additionally comprises the step 5 of infecting said host cell with a resistance test vector viral particle prepared from filtered supernatants of a cell culture comprising a resistance test vector host cell.

69. The method of claim 68, wherein said resistance test vector host cell is a 10 human cell. The method of claim 68, wherein said resistance test vector host cell is a human embryonic kidney cell.

71. The method of claim 68, wherein said resistance test vector host cell is a 293 cell. o 0 001.10 *e000 0*000 000000 0** 0 .uee r .00 t.0 O .0 5 I 2 000 *000 ooo 2 *o 30 0**

72. The method of claim 68, wherein said resistance test vector host cell is derived from a human hepatoma cell line.

73. The method of claim 68, wherein said resistance test vector host cell is a HepG2 cell.

74. cell. The method of claim 68, wherein said resistance test vector host cell is a Huh7 The method of claim 68 wherein said host cell and said resistance test vector host cell are the same cell type.

76. A method for determining resistance of a virus infecting a patient to an antiviral drug, comprising: 00 0* 00 0 *0 0° 000 0 0 0 0 °0 go o o• 5 6:00 $0* 1 .06. 0 48-6-: .10* 5*w we 0* 15 S S0 S* e S 5*55* 0 *55 S* 5*5* 'w 25 *5 555*5S *550e 180 determining the susceptibility of said virus to said antiviral drug according to the method of claim 1, wherein said viral segment is a patient-derived segment; and comparing the susceptibility of said virus to said antiviral drug determined in step with a standard curve of susceptibility of said virus to said antiviral drug, wherein a decrease in the susceptibility of said virus to said antiviral drug relative to said standard curve indicates that said virus is resistant to said anti-viral drug and the amount of said decrease in susceptibility of said virus to said antiviral drug indicates the degree to which said virus is resistant to said antiviral drug.

77. A method for determining the resistance of a virus infecting a patient to an antiviral drug, comprising: determining the susceptibility of said virus to said antiviral drug according to the method of claim 47, wherein said viral segment is a patient-derived segment; and comparing the susceptibility of said virus to said antiviral drug determined in step with a standard curve of susceptibility of said virus to said antiviral drug, wherein a decrease in the susceptibility of said virus to said antiviral drug relative to said standard curve indicates that said virus is resistant to said antiviral drug and the amount of said decrease in susceptibility of said virus to said antiviral drug indicates the degree to which said virus is resistant to said anti-viral drug.

78. A method for determining the progression or development of resistance of a virus infecting a patient to an antiviral drug, comprising: determining the susceptibility of said virus at a first time according to the method of claim 1, wherein said viral segment is a patient-derived segment and said patient-derived segment is obtained from said patient at about said first time; assessing the effectiveness of said anti-viral drug as performed in step at a later second time; and S 0 S 4. a 5 6* 4 .00. %4 .0 0: 0.

966..: 10 20e *25 d4 d S30 4S* 4** 4 S 10 4* 4 4 5445~ S S @444 4, 44 *6.4 040.44 4S** 0* *r *4 *0+44. E. .4. 181 comparing the effectiveness of the anti-viral drug assessed in steps (a) and wherein a decrease in the susceptibility of said virus to said antiviral drug at said later second time as compared to said first time indicates development or progression of anti-viral drug resistance in said virus infecting said patient. 79. A method for determining the progression or development of resistance of a virus infecting a patient to an antiviral drug, comprising: determining the susceptibility of said virus to said drug at a first time according to the method of claim 47, wherein said viral segment is a patient-derived segment and said patient-derived segment is obtained from said patient at about said first time; assessing the effectiveness of said anti-viral drug as performed in step at a later second time; and comparing the effectiveness of the anti-viral drug assessed in steps (a) and wherein a decrease in the susceptibility of said virus to said antiviral drug at said later second time as compared to said first time indicates development or progression of anti-viral drug resistance in said virus infecting said patient. 80. The method of claim 1, wherein said resistance test vector comprises DNA of a hepadnavirus. 81. The method of claim 1, wherein said resistance test vector comprises DNA of HBV. 82. The method of claim 1, wherein said resistance test vector comprises DNA encoding C, P, or X, or a combination thereof. 83. The method of claim 1, wherein said viral segment comprises a P gene. 84. The method of claim 1, wherein said viral segment comprises an HBV gene. S *S 4* 0 *445 *44 S 5 **4 5 4** 0 4 54 182 The method of claim 1, wherein said viral segment comprises an HBV RT gene. 86. The method of claim 1, wherein said viral segment comprises an HBV DNA polymerase gene. 87. The resistance test vector of claim 30, wherein said viral segment comprises a hepadnaviral gene. 88. The resistance test vector of claim 30, wherein said viral segment comprises an HBV gene. 89. The resistance test vector of claim 30, wherein the patient-derived segment comprises an HBV P gene. 0: 90. The resistance test vector of claim 30, wherein said viral segment comprises an RT/DNA polymerase gene. 20 91. A method for determining the biological effectiveness of a candidate anti-viral compound comprising: :o culturing a host cell in the presence of said candidate antiviral compound, wherein said host cell comprises a resistance test vector comprising a viral segment and an indicator gene, wherein expression of said indicator gene depends on the activity of said viral segment or a gene product encoded by said viral segment and wherein said resistance test vector does not comprise all viral genes necessary to produce an infectious viral particle; measuring an amount of expression of said indicator gene in said host cell; and comparing said amount of expression of said indicator gene measured in step (b)with a reference amount of expression of said indicator gene, wherein the difference between the amount of expression of said indicator gene measured in step relative to that measured in step (b) 183 correlates with the effectiveness of said candidate antiretroviral compound, thereby determining the effectiveness of said candidate antiretroviral compound. 92 The method of Claim 91, wherein said reference amount of expression of said indicator gene is determined by performing steps and of the method of Claim 89 in the absence of said antiviral drug. 93. The method of claim 91, wherein said reference amount of expression of said indicator gene is a standard amount of expression determined from performing the method of claim 89 with a standard laboratory viral segment and in the absence of said antiviral drug. 15 94. The method of claim 91, wherein said resistance test vector comprises DNA *o from a retrovirus. ooo° 95. The method of claim 91, wherein said resistance test vector comprises DNA derived from HIV. 96. The method of claim 91, wherein said resistance test vector comprises DNA of a hepadnavirus. 97. The method of claim 91, wherein said resistance test vector comprises DNA of HBV. 98. The method of claim 91, wherein said resistance test vector comprises DNA encoding HIV gag-pol. 99. The method of claim 91, wherein said resistance test vector comprises DNA encoding HBV P protein. 100. The method of claim 91, wherein said viral segment encodes one protein. 184 101. The method of claim 91, wherein said viral segment encodes two proteins. 102. The method of claim 91, wherein said viral segment comprises a retroviral gene. 103. The method of claim 91, wherein said viral segment comprises an HIV gene. 104. The method of claim 91, wherein said viral segment comprises a hepadnaviral gene. 105. The method of claim 91, wherein said viral segment comprises an HBV gene. 106. A method for determining the susceptibility of a virus to an anti-viral drug, said 15 method comprising: culturing a host cell in the presence of said antiviral drug, wherein said S°host cell comprises a resistance test vector comprising a viral segment and an indicator, wherein expression of said indicator depends on the "00 activity of said viral segment or a gene product encoded by said viral segment and wherein that said resistance test vector does not comprise all viral genes necessary to produce an infectious viral particle; measuring the amount of expression of said indicator in said host cell; and comparing the amount of expression of said indicator as measured in 25 step with an amount of expression of said indicator measured in said host cell of step cultured in the absence of said antiviral drug, wherein the difference between said amount of expression of said indicator measured in step relative to that measured in step (c) correlates with the susceptibility of said virus to said antiviral drug, thereby determining the susceptibility of said virus to said anti-viral drug. 107. The method of claim 106, wherein said indicator comprises a DNA structure. 184a 108. The method of claim 106, wherein said indicator comprises a RNA structure. 109. A method for determining the biological effectiveness of a candidate anti-viral compound comprising: culturing a host cell in the presence of said candidate antiviral compound, wherein said host cell comprises a resistance test vector comprising a viral segment and an indicator, wherein expression of said indicator depends on the activity of said viral segment or a gene product encoded by said viral segment and wherein said resistance test vector does not comprise all viral genes necessary to produce an infectious viral particle; measuring an amount of expression of said indicator in said host cell; and 15 comparing said amount of expression of said indicator measured in step with an amount of expression of said indicator measured in the S. absence of said candidate antiviral compound, wherein the difference between the amount of expression of said indicator measured in step relative to that measured in step correlates with the effectiveness of said candidate antiretroviral compound, thereby determining the ."effectiveness of said candidate antiviral compound. loll 110. The method of claim 109, wherein said indicator comprises a DNA structure. 111. The method of claim 109, wherein said indicator comprises a RNA structure. DATED THIS NINTH DAY OF JULY 2004 VIROLOGIC INC. BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS