WO1993002202A1

WO1993002202A1 - Compositions and methods for reproducing positive diagnostic indications

Info

Publication number: WO1993002202A1
Application number: PCT/US1992/006025
Authority: WO
Inventors: James E. Marich; Thomas W. Dubensky, Jr.
Original assignee: Syngene Inc
Current assignee: Syngene Inc
Priority date: 1991-07-19
Filing date: 1992-07-17
Publication date: 1993-02-04
Anticipated expiration: 1994-01-19

Abstract

The invention provides a functional vector for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms. The vector includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism. Host cells transformed with the functional vector and recombinant viruses produced by such transformed host cells are also provided. A method for detecting one or more target sequences indicative of one or more infectious or pathogenic organisms is provided. The method includes (a) providing a self-replicating functional vector. The vector includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism; (b) introducing the self-replicating functional vector into compatible host cells to produce infected cells capable of generating recombinant viruses having the self-replicating functional vector as their viral genome; (c) fixing the infected cells capable of generating recombinant viruses; and (d) detecting the one or more heterologous sequences characteristic of the infectious or pathogenic organism in the fixed infected cells.

Description

COMPOSITIONS AND METHODS FOR

REPRODUCING POSITIVE DIAGNOSTIC INDICATIONS

BACKGROUND OF THE INVENTION

Accurate evaluation of nucleic acid hybridization assays requires the use of controls which are tested simultaneously with samples during the assay. Controls which most closely represent actual samples provide the highest confidence that the assay is working properly and that the results are valid. For assays to detect the presence of pathogens, cultures of the pathogen are typically used as controls. However, these pathogens are often highly infectious or otherwise hazardous. Such hazardous organisms which are cultured for these reasons include, for example, human immunodeficiency virus (HIV), malaria (Plasmodium falciparum) and Mycobacteria tuberculosis. While it possible to culture these agents, the high costs of containment and risks to human health during the manufacture and subsequent use can be significant. Other hazardous organisms, such as human papillomavirus (HPV) and hepatitis virus (HBV, HCV), have not been successfully propagated in cell culture. Controls used for reproducing and detecting HPV samples, for example, have often used cancer-derived cell lines, such as CaSki and HeLa, which contain permanently integrated HPV sequences, Spence, R.P., et al., Cancer Res. 48:324-328, 1988. Few of these cell lines exist and the numbers of integrated HPV sequences are usually low and do not accurately represent the majority of clinically diagnosed HPV infections. Additionally, the HPV sequences are part of the host cell chromosomes and not present as independently replicating extrachromosomal elements, as is the case during an actual HPV infection, Awady, M.K., et al.. Virology 159:389-398, 1987, Schwarz, E., et al., Nature 314:111-114, 1985, Yiu, K.C. et al., Anticancer Res. 10:917-922, 1990. If viral infections cannot be performed and permanently transformed cell lines containing viral sequences are not available, cell lines can be transfected with viral DNA to either transiently express or permanently integrate viral sequences. The disadvantages of these procedures are that the techniques are inefficient, harsh and poorly reproducible, resulting in a relatively low percentage of transfected cells whose morphology is usually compromised for a period following chemical transfection. Mammalian cells transformed with cloned DNA can also be accomplished using retrovirus vectors. Miller, D.G. and Adam, M.A., Mol. Cell. Biol. 10:4239-4242, 1990. In this procedure, the retroviral vector containing a cloned DNA is transfected into a cell line which expresses all of the viral coat proteins necessary to package the construct. Such proteins enable the production of chimeric virus which is excreted from the transfected cells. The chimeric virus can then be used to infect other cell monolayers which contain the cloned DNA stably integrated into the host chromosome.

Although cells containing foreign nucleic acid sequences can be generated through direct infection or a variety of recombinant techniques, these cells do not ideally reproduce positive diagnostic indications for the presence of infectious or pathogenic organisms. Manufacturing of controls with human infectious agents requires thorough containment during culture and complete inactivation before shipment. Incomplete containment or inactivation of human infectious agents such as hepatitis virus represents a significant hazard to the health of personnel involved in the manufacture, packaging or shipment and, ultimately, to the consumer. Recombinant methods which generate transiently expressed sequences are inconsistent and levels of expression are not regulatable. Cells lines permanently transformed with foreign nucleic acid sequences do not contain levels of viral nucleic acid representative of typical pathogen infection.

There thus exists a need for compositions and methods to efficiently and reproducibly produce positive diagnostic indications of infectious or pathogenic organisms without risk to humans. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION The invention provides a functional vector for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms. The vector includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism. Host cells transformed with the functional vector and recombinant viruses produced by such transformed host cells are also provided. A method for detecting one or more target sequences indicative of one or more infectious or pathogenic organisms is provided. The method includes provision of a functional vector which includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism; introduction of the functional vector into compatible host cells to produce infected cells capable of generating recombinant viruses having the functional vector as their viral genome; fixation of the infected cells; and detection of the heterologous sequences characteristic of the infectious or pathogenic organism within the fixed infected cells. DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to vectors and to methods by which DNA sequences can be propagated in eukaryotic cells for subsequent use in nucleic acid hybridization assays. Sequences derived from any organism, particularly infectious or pathogenic organisms, can be inserted into the recombinant viral vector described herein and propagated at high titer as a virus. Virus produced from the recombinant vector containing the inserted heterologous sequence can be used to infect other cells. These infected cells are then used directly in methods which detect the specific inserted nucleic acid sequences by in situ hybridization. Alternatively, the nucleic acids can be purified from other cellular material and assayed for the presence of the foreign nucleic acid by, for example, solution or blot hybridization. Thus, the invention can be advantageously employed as a reference or control for the detection of any nucleic acid sequence. Such a method allows for the convenient detection of sequences derived from hazardous infectious or pathogenic organisms.

As used herein, the term "positive diagnostic indications" refers to the consistent reproduction of the necessary replication of a target sequence that is characteristic of an infectious or pathogenic organism. Consistent reproduction includes comparable levels and/or subcellular locations of the target sequence to that of the corresponding infectious or pathogenic organism.

As used herein, the term "functional vector," when used in reference to reproducing positive diagnostic indications, refers to a vector that is capable of, or can be made capable of, autonomously directing the production of infectious virions once introduced into a compatible host cell. The vector can be derived from a specific viral genome or can be composed of multiple, different viral genomes that together are capable of autonomously directing the production of infectious virions. Sequences other than those of viral origin can also be included within the vector so long as it remains functional. The ability of the functional vector to produce infectious virions is due to the presence of all viral encoded functions necessary for the replication of the vector as a viral genome and expression of the viral proteins. It is understood that a functional vector can be derived from a non-functional vector, such as a non-functional viral genome. A specific example of a non-functional viral genome which can be used to generate a functional vector is the abbreviated-leader mutant (ALM) virus described by Adami and Carmichael, J. Virol. 58:417-425 (1986), which is incorporated herein by reference. This virus is a polyoma variant lacking 48 essential bases of the late leader exon. Substitution of sequences into this essential region restores the functionality of the genome. Non-functional viral genomes other than ALM can also be used to generate functional vectors so long as the dysfunction is due to the viral genome and not to a host function necessary for virus production.

As used herein, the term "heterologous sequences" when used in reference to an insertion within a viral genome means a nucleotide sequence or sequences that are different from the authentic nucleotide sequence found at the same location within the wild type viral genome. Such heterologous sequences can, for example, correspond to sequences from a virus or organism different from the origin of the functional vector genome or to a different species than that of a compatible host cell. For example, if a mouse polyoma viral genome is used to derive the functional vector, heterologous sequences can be derived from viruses or organisms other than polyoma or from species other than mouse. Specific examples of such heterologous viruses and organisms include human herpes simplex, cytomegalo, hepatitis, polyoma JC, polyoma BK, and adeno viruses.

As used herein, the term "substantially" is used to modify the terms identical or complementary as applied to nucleotide sequences. "Identical" refers to the ability of the nucleotide sequence to detectably hybridize to a specific sequence under conditions known to one skilled in the art. For example, specific hybridization of short complementary sequences will occur rapidly under stringent conditions if there are no mismatches between the two sequences. If mismatches exist, specific hybridization can still occur if a lower stringency is used. Specificity of hybridization is also dependent on sequence length. For example, a longer sequence can have a greater number of mismatches with its complement than a shorter sequence without losing hybridization specificity. Such parameters are well known and one skilled in the art will know, or can determine, what sequences are substantially complementary to allow specific hybridization.

The invention provides a functional vector for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms. The vector includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of said infectious or pathogenic organism.

In one embodiment, a functional vector is described herein which is derived from a recombinant mouse polyomavirus (Py) called Py-ALM or ALM (Advanced Leader Mutant) which is not infectious to humans, Adami, G. R., Christopher, W. M., Barret, N.L., and Carmichael, G. G., J. Virol. 63:85-93 (1989) and Adami, G. R., and Carmichael, G. G., J. Virol. 58:417-425 (1986), both of which are incorporated herein by reference. The vector remains episomal in compatible host cells and replicates to high levels (up to about 1 x 10⁵ copies/cell). Py-ALM contains a deletion of forty-eight essential bases of the viral late leader sequence which is associated with expression of viral late gene products. The ALM recombinant vector cannot produce viable virions unless between about 24-100 bps are substituted for the deleted region. It has been shown that it is the length and not the sequence of the central leader region that is important for the ALM vector to be able to produce viable infectious virions. An advantage of this characteristic is that essentially any DNA sequence up to about 100 bp in length can be inserted into the ALM vector and propagated as virus.

The genome of such a virus could contain as the inserted sequence a heterologous sequence. Like wild type polyomavirus, this recombinant virus (ALM-fDNA) can be used to infect all cells which contain a Py receptor with both the inserted fDNA and Py sequences being expressed and replicated during growth of the virus. It is understood that expression and replication of heterologous sequences need not be limited to the use of Py-ALM for one skilled in the art to practice the invention. Other eukaryotic viral vectors which can be propagated in mammalian cell culture can also serve as a heterologous method for generating recombinant virions and propagating heterologous sequences. For example, various other cloned viral genomes can be altered to contain heterologous sequences and still be capable of producing infectious virions. Such functional vectors can also be used as vectors for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms. The invention will be described, however, with specific reference to the ALM viral vector.

Heterologous nucleic acid sequences characteristic of infectious or pathogenic organisms can be inserted into the ALM vector within the deleted late leader region. Such heterologous sequences can be, for example, recombinant sequences derived from cloned DNA or synthetic sequences chemically synthesized using methods known to one skilled in the art. The length of the heterologous sequence inserted into the deleted region of the ALM vector should be between about 24-100 nucleotides in order to restore viral replication and gene expression to the nonfunctional ALM vector. These sequences can be identical or substantially identical to sequences from one or more infectious or pathogenic organisms. Such sequences can be unique or repetitive so long as their detection by hybridization faithfully parallels that of an authentic infectious or pathogenic organism. Combinations of heterologous sequences corresponding to different infectious or pathogenic organisms can also be inserted into the deleted region for the simultaneous detection of multiple organisms. For example, positive diagnostic indications can be reproduced for two organisms by inserting heterologous sequences corresponding to both organisms into the same vector. Likewise, positive diagnostic indications can be reproduced for three or more organisms by inserting heterologous sequences for each of the desired organisms. Whether it is desired to reproduce conditions for a single or for multiple organisms, it is important that the total length of the heterologous sequences or sequences be no larger than about 100 nucleotides.

To generate the function vector, heterologous sequences can be inserted into the ALM vector, for example, by cloning into the unique Bel I site contained within the deleted late leader region. Heterologous sequences to be cloned in this fashion can be modified through a cloning strategy or by the addition of linkers to produce 5' and 3' ends complementary to the ALM Bel I site. Heterologous sequences can also be synthesized directly to contain such complementary ends. Additional modifications to the heterologous sequence or vector can also be performed to generate ligatable ends for cloning. Such modifications include, for example, the ligation of linkers or the creation of blunt ends by polymerase fill-in and are known to one skilled in the art.

The heterologous sequence-containing insert and vector are ligated under conditions favoring circular recombinants containing a single ALM and insert molecule. Ligation products can be subsequently transformed into transformation-competent cells, such as E. coli and recombinant clones screened by ampicillin resistance and hybridization to probes complementary to insert sequences. Putative positive clones can be further screened by restriction endonuclease digestion. A single positive clone is then selected and plasmid DNA purified to use for reproducing positive diagnostic conditions. Recombinant methods other than those described above can also be used to identify positive clones. Such methods are well known to one skilled in the art. The invention provides a host cell transformed with a functional vector for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms. The functional vector includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism. A recombinant virus useful for reproducing positive diagnostic indications of an infectious or pathogenic organism is also provided. The functional vector containing heterologous sequences can be used for the transfection of compatible host cells. Such transfected host cells are capable of producing recombinant virus encoded by the functional vector. The self-replicating viral genome is therefore indicative of an infectious or pathogenic condition. Recombinant virus produced from the transfected host cells can be isolated and used to infect additional host cells to reproduce positive diagnostic indications. Thus, it is only necessary to generate the functional vector and transfeet host cells once for reproducing a desired diagnostic condition. Recombinant virus produced from transfected cells can be subsequently used for reproducing further diagnostic conditions. The ALM vector described previously contains plasmid vector sequences (pUC19, BRL, Gaithersberg, MD) for propagation in bacterial organisms. For the vector to be functional in compatible host cells, these sequences should be removed after the heterologous sequences have been inserted and positive clones identified. To do this, the functional vector sequences are liberated from plasmid vector by digestion with Bam HI and separated according to molecular weight by agarose gel electrophoresis. The band migrating at the mobility predicted for the viral sequences (about 5300 bp) is excised from the gel and purified by electroelution. Purified viral DNA is then ligated, using T₄ ligase or other comparable ligase such as E. coli DNA ligase, under dilute conditions to promote circularization. The resultant ligated product is then transfected into mouse 3T6 cells using agents such as DEAE dextran, calcium phosphate or lipids. Such methods are well known within the art and can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY 2nd ed., 1989, which is incorporated herein by reference. At times ranging from 3-14 days post transfection, the cells can be used directly for reproducing positive diagnostic indications or a crude preparation of virus can be prepared to generate subsequent diagnostic cells or high titer virus stock. The crude preparation of virus can be prepared by, for example, first freeze-thawing the plates (including media) on which the cells are contained, followed by sonication and pelleting of the cellular debris. This preparation can then be used to infect fresh monolayers of 3T6 cells to generate high titers of virus. Virus titers are determined by conventional methods known to one skilled in the art on, for example, UC1B mouse fibroblasts or other cell lines permissive for mouse polyoma virus infection. Typically, about 10⁶-10⁸ plaque forming units/ml can be recovered.

Once the recombinant virus has been generated in high titer, it can be used immediately or stored and used as needed to infect cells. Cells infected with the recombinant virus produce infectious virions at levels comparable to wild type Py, up to 1 x 10⁵ virions/cell. The level of infection per cell can be controlled by, for example, variation in infection time.

The invention provides a method for detecting one or more target sequences indicative of one or more infectious or pathogenic organisms. The method includes:

(a) providing a functional vector, the functional vector includes a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism; (b) introducing the functional vector into compatible host cells to produce infected cells capable of generating recombinant viruses having the functional vector as their viral genome; (c) fixing the infected cells capable of generating recombinant viruses; and (d) detecting the one or more heterologous sequences characteristic of the infectious or pathogenic organism within the fixed infected cells. Host cells transfected with the functional vector or infected with recombinant virus as described above can be used for reproducing positive diagnostic conditions. Detecting the heterologous sequence contained within the functional vector is indicative of the infectious or pathogenic organism. Such a method is useful when there is a need to compare an unknown sample with a standard to determine whether the sample contains an infectious or pathogenic organism. For example, infected cells are harvested at appropriate times and used directly or stored until needed. Detection can be performed by, for example, in situ hybridization. This procedure is a method whereby the presence of specific nucleic acids are detected within intact permeabilized cells. Use of this methodology enables very small amounts of nucleic acid to be detected, as few as 1-2 copies/cell, Heiles, H.B.J., et al., Biotechniques 6:978-981, 1988, which is incorporated herein by reference. Additionally, localization of the target nucleic acid is possible within specific cells in a sample, Valentino, K.L., Eberwine, J.H., Barchas, J.D., Oxford University Press, New York, Oxford, 1987, which is incorporated herein by reference.

Infected cells are grown and/or fixed on slides for direct use without further modification. Detectable probes having attached reporter molecules are applied to the cells and allowed to hybridize. Reporter molecules can be, for example, isotopic, colorimetric, fluorescent or luminescent, Chesselet, M.F., CRC Press, Boca Raton, FL, 1990, which is incorporated herein by reference. Detection of the signal indicates a positive diagnostic condition for the heterologous sequences. The degree of signal obtained from in situ or other hybridization procedures (dependent on the number of molecules per infected cell) is regulated by varying the time, post infection, at which the cells are prepared for detection. Thus, cells can be generated which faithfully reproduce the appropriate level of target sequence that is indicative of an infectious or pathogenic organism. For DNA hybridization assays other than in situ hybridization, the nucleic acids are prepared and analyzed using methods known to one skilled in the art. The above- described method is also useful, for example, in testing and optimizing procedures for the detection of heterologous sequences in clinical samples. A specific example for using the method described herein as a control in a diagnostic in situ hybridization procedure is outlined below.

Py permissive cells are grown on microscope slides or in tissue cultureware and infected with recombinant virus containing heterologous sequences or wild type Py at a multiplicity of infection (MOI) ranging from about 10^-2 - 10³. Other cells are also grown but not infected with virus (mock infection). Cells are harvested at various times post infection (0 - 7 days) and hybridized with heterologous sequence-specific probes or a Py specific probe. Such probes are substantially complementary to the heterologous or Py sequences.

The heterologous sequence-specific probes hybridize in situ to cells infected with the recombinant virus but not to cells infected with wild type Py. The Py specific probe hybridizes in situ to cells infected with both viruses. No hybridization occurs in situ with any probes on cells mock infected. Positive signal is observed over the nucleus of the infected cells and is generally strongest, in combination with conserved cellular morphology, at 48 - 72 hours post infection.

The method described herein is applicable for the detection and reproduction of positive diagnostic conditions of a variety of viruses and intracellular pathogens. For example, sequences from herpes simplex virus (HSV), cytomegalovirus (CMV), hepatitis (HBV, HCV), malaria, Chlamydia, human immunodeficiency virus (HIV) and adenovirus can be inserted into the ALM vector described herein. Other sequences representing essentially any virus, bacteria, fungus, yeast, plant, protozoa or animal, including human, can also be used.

The invention also provides a system for detecting one or more target sequences indicative of one or more infectious or pathogenic organisms. The system includes: (a) a first component including one or more fixed cells containing a functional vector consisting essentially of a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of the infectious or pathogenic organism; and (b) a second component comprising one or more oligonucleotide probes substantially complementary to the one or more heterologous sequences of said first component. Cells previously infected with a recombinant virus having one or more heterologous sequences associated with its genome can be fixed and supplied with a probe to the sequence(s) in the form of a kit. Such a kit can be used as a control for the determination of an infectious or pathogenic organism within a clinical or laboratory sample. The previously infected cells should be fixed with an agent that preserves the integrity of the cell. Specific fixing agents can be, for example, methanol or glutaraldehyde. In addition to containing recombinant virus infected cells, the kit can contain, for example, wild type and mock infected cells. Such cells can be grown and fixed on separate slides or in different compartments on the same slide. Ancillary reagents such as buffers, dyes and probe labeling and detection reagents can also be included. The following examples are intended to illustrate but not limit the invention.

EXAMPLE I

Construction of functional vectors containing

heterologous sequences

This example describes the construction of five different functional vectors containing different heterologous sequences.

Construction of the non-functional Py-ALM viral genome: Py-ALM was constructed as described in Adami,

G.R. and Carmichael, G.G. J. Virol. 58:417-425, 1986 supra. The resulting construct had a 48 bp deletion relative to wild type polyoma. The ALM was cloned into pUC19 (BRL, Gaithersburg, MD) at a unique Bam HI site to facilitate propagation in E. coli. Heterologous sequences were inserted into the unique Bel I site in this late leader region deletion to restore viral gene expression as described below.

Construct 1 Two complementary synthetic oligonucleotides (SEQ

ID NOS: 1 & 2) were synthesized on an Applied Biosystems synthesizer using phosphoamidite chemistry. These oligonucleotides contained sequences complementary to nucleotides 835-854 and 730-750 of the E7 open reading frame (ORF) of HPV 16, Seedorf et al. Virology 145:181-185, 1985, which is incorporated herein by reference. Additionally, a Sph I linker was designed between the two regions of HPV 16 homology and each oligonucleotide terminated in Hind III and Pst I sequences so that when annealed, the two complementary oligonucleotides had overlapping complementary ends (SEQ ID NOS : 1 & 2). The complementary oligonucleotides were HPLC purified, combined and desalted on a G-25 column and eluted in TE. The mixture was heated to 100°C for 5 minutes and cooled at room temperature for 30 minutes. Py-ALM was linearized with Bel I for 1 hour at 50ºC (50 mM NaCl, 25mM Tris-HCl, pH 7.7, 10 mM MgCl₂, 10 mM β-mercaptoethanol, 100 μg/ml BSA). The ends of linearized Py-ALM and synthetic insert were made blunt ended using the fill-in reaction of klenow polymerase (100 mM dNTP's, 50 mM Tris-HCl, pH 7.6, 20 mM MgCl₂, 1 hour, 37ºC) and ligated to the annealed oligonucleotides with T₄ ligase (1 hour at room temperature, then overnight at 4°C, in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 20 mM DTT, ImM ATP, 50 μg/ml BSA) at a 100:1 molar ratio, insert:Py-ALM. Pst I Sph I Hind III

5'- GCCCATCTGTTCTCAGAAACCGCATGCTTTTGTTGCAAGTGTGACTCTA -3'

3' -ACCTCGGGTAGACAAGAGTCTTTGGCGTACGAAAACAACGTTCACATGAGATTCGA -5'

Construct 2 This construct contains a 23 bp, blunt ended, double stranded fragment of human immunodeficiency virus plasmid clone pBH10R3 (isolate BH10). The heterologous insert corresponds to nucleotides 1854 - 1877, and has the following sequence: 5'-AACATAATTGGAAGAAATCTGTT-3' (SEQ ID NO: 3). This sequence is generated by Hinc II restriction enzyme digestion in universal buffer (Stratagene) for 1 hour at 37°C, segregated by electrophoresis in a 2% agarose gel and purified by electroelution. Py-ALM is linearized with Bel I and the 5' and 3' ends filled in by incubation with Klenow as described above in construct 1. The isolated HIV heterologous sequence and Py-ALM axe ligated with T₄ ligase at a 1:1 (vector:insert) molar ratio for 2 hours at room temperature followed by overnight incubation at 4°C. After fill-in and ligation total insert size is 27 nucleotides.

Construct 3

The heterologous insert for construct 3 is a sequence complementary to a nucleic acid probe that is specific for repetitive DNA unique to Plasmodium falciparum. Two complementary oligonucleotides are synthesized with Bel I linkers, annealed and digested with Bel I as described in construct 1. The sequence of the top strand is as follows: 5'-TAGGTCTTAACTTGACTAACATGATCA-3' (SEQ ID NO: 4). Py-ALM is digested with restriction endonuclease Bel I and ligated to these oligonucleotides directly as described previously.

Construct 4

A 100 base pair DNA fragment, complementary to nucleotides 6470 - 6569 of HPV type 31, Goldborough et al., Virology 171:306-311, 1989, which is incorporated herein by reference, is generated with Bel I linkers using either oligonucleotide synthesis or the polymerase chain reaction (PCR) (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 200 μm dNTP's, 1.0 μM each primer, 2.5 units Taq Polymerase). About 10¹¹ copies of HPV 31 DNA are generated using 30 PCR cycles consisting of 45°C annealing, 72°C extension and 94°C denaturation. The specific primers (SEQ ID NOS: 5 & 6) are: forward, 5'-TGATCAATTTTTAATAAACCATATTGGATGC-3' reverse, 5'-TGATCATACGTGTGGTATCTACCACAGTAAC-3'

ALM and PCR product are digested with Bel I and ligated together as described previously. This construct contains sequences complimentary to HPV 6, 11, 18, 31, 33 and 35 and can be used to monitor the performance of probes to any of these virus types. Construct 5

The heterologous sequence insert of this construct are complementary oligonucleotides (SEQ ID NOS :

7 & 8 ) that are 75 base pairs in length and contain three contiguous sequences complementary to HIV (nucleotides

1854-1877), CMV (nucleotides 3712-3732) and EBV

(nucleotides 10007-10029). 5' and 3' ends of the oligonucleotides are synthesized with Bel I linkers. The

ALM vector is prepared and ligated to the synthetic insert as described previously in construct 3.

5'-GATCAAACATAATTGGAAGAAATCTGTTGCTGACCGACGCGTCAGGATGCCTAC GGCTCCGCCTGCGCAGGTT-3'

3'-TTTGTATTAACCTTCTTTAGACAACGACTGGCTGCGCAGTCCTACGGATGCCGA GGCGGACGCGTCCAACTAG-5'

Ligation products of these constructs were transformed into competent dam- E.coli GM2163 (New England Biolabs, Beverly, MA) that were treated with 0.1 M MgCl₂ and 0.1 M CaCl₂. Colonies were screened for positive clones by boiling lysis miniprep of a 1 ml overnight liquid culture followed by diagnostic restriction enzyme digestion. A single positive clone was cultured in 500 ml of LB broth and purified by alkaline lysis and CsCl density gradient centrifugation. pUC19 vector sequences were removed from the recombinant polyoma viral sequences by digestion with BamH I (1 hour, 37°C, 150 mM NaCl, 10 mM Tris-HCl, pH 7.9, 10 mM MgCl₂, 1 mM DTT) and separated by electrophoresis on a 1% agarose gel. The fragment corresponding to the functional vector was isolated from pUC19 sequences by electoelution. Purified 0.5 - 1 μg functional vector was circularized with T₄ ligase for 2 hours at room temperature followed by overnight

incubation at 4°C at a DNA concentration of 5nM. This circular product was used to perform transfection of NIH 3T6 cells .

EXAMPLE II

Transfection and viral culture

Mouse 3T6 cells were cultured in Dulbecco's modified eagles medium (DMEM) + 10% Fetal Calf serum

(FCS). One day prior to transfection, 100 mm plates were seeded with 1 x 10⁶ cells. After 24 hours growth, transfection inoculum was prepared as follows: lμg ligated DNA construct and 50 μl of 10 mg/ml DEAE dextran adjusted to 1 ml with TSM (30 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1.5 mM MgCl₂). Culture media was aspirated from 3T6 cells and plates were washed twice with 5 ml of TSM.

Final wash was aspirated and transfection inoculum was overlaid. Plates were rocked every 10 minutes for 40 minutes. TSM (5 ml) was added and aspirated followed by a 5 ml wash with DMEM + 10% FCS. DMEM + 10% FCS (10 ml) was added and cells were incubated overnight at 37°C. At 24 hours post transfection, media was exchanged and discarded. When cells reached confluency, plates and media together were frozen at -20°C and thawed, heated to 45°C for 15 minutes and then sonicated for 2 minutes.

Plates were scraped and cellular debris was separated from supernatant by centrifugation at 5000g for 15 minutes at room temperature. This viral supernatant was subsequently used to infect new plates seeded as

described above and virus was harvested as before once cellular cytopathic effect (CPE) was complete.

EXAMPLE III

Detection of heterologous sequences Infected cell controls for in situ

hybridization were prepared from the isolated recombinant virus stock of Example II. Microscope slides with attached cell culture chambers were inoculated with 3T6 cells (10⁴cells/chamber). After 24 hours of growth, one of two chambers was infected at an MOI of 5 with the virus produced from construct 1 (ALM-16). Infection was allowed to proceed for 96 hours. Chambers were removed from slides and cells were fixed in 100% MeOH for 10 minutes. Slides were either stored at -20°C - -70°C or used directly in a hybridization assay.

Experimental samples fixed on microscope slides as well as the ALM-16 control slides were assayed by in situ hybridization to HPV 16 probes. Detection is performed with cloned or synthetic complementary probes labelled with radionuclides (ie. ³H, ¹⁴C and ³²P), biotin, digoxygenin or enzymes (ie. alkaline phosphatase,

horseradish peroxidase and luciferase) using procedures known to one skilled in the art. For example, SNAP^® oligonucleotide probes (Syngene, San Diego, CA) were used to detect HPV 16 sequences according to the following protocol: Cells were fixed in 100% MeOH for 10 minutes at room temperature followed by denaturation for 10 minutes in 70% formamide, 1X SSC, 0.5% BSA. To remove denaturant, a wash was performed for 1 minute at room temperature in 1X SSC. Two complementary SNAP^®

oligonucleotide (5 nm of each) probes were hybridized at 50°C for 2 hours using a buffer consisting of 5X SSC, 0.5% BSA, 0.5% SDS. The cells were then washed six times by repeating each of the following procedures twice: (1) a 6 minute wash at 45°C in 1X SSC and 1% SDS; (2) a 6 minute wash at 45°C in 1X SSC, 0.5% Triton X-100, 0.075% Brij-35; and (3) a 6 minute wash at 45°C in 1X SSC,

0.075% Brij-35. The alkaline phosphatase reporter label was detected by incubating for 2 hours at 37°C in

developer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCl₂, 5% polyvinyl alcohol, 0.33 mg/ml NBT and 0.16 mg/ml BCIP), followed by 2 minutes at room temperature in water. The cells were then stained in 1% Eosin B plus 5% MeOH for 4 minutes at room temperature. Slides were examined by light microscopy for presence of purple stain indicating the presence of HPV 16 virus (samples) and ALM-16 (control slides).

EXAMPLE IV

In situ hybridization kit

An in situ hybridization kit for the detection of various HPV viral types is produced which contains the following components:

1) Glass slides containing fixed cells infected with ALM-HPV 6&11 virus, ALM-HPV 16, 18, 31, 33&35 virus and uninfected cells in separate locations or on separate slides prepared as described in Example III.

2) Two sets of oligonucleotide probes having

alkaline phosphatase reporter molecules and are complementary to HPV types 6&11 and to HPV types 16, 18, 31, 33 & 35.

Although the invention has been described with reference to the presently-preferred embodiment, it should be understood that various modifications can be made by those skilled in the art without departing from the invention. Accordingly, the invention is limited only by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: MARICH, JAMES E.

DUBENSKY, JR., THOMAS W.

(ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR REPRODUCING

POSITIVE DIAGNOSTIC INDICATIONS

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CAMPBELL AND FLORES

(B) STREET: 4370 LA JOLLA VILLAGE DRIVE, SUITE 700

(C) CITY: SAN DIEGO

(D) STATE: CALIFORNIA

(E) COUNTRY: UNITED STATES

(F) ZIP: 92122

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 17-JUL-1992

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: CAMPBELL, CATHRYN

(B) REGISTRATION NUMBER: 31,815

(C) REFERENCE/DOCKET NUMBER: FP-MB 9368

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 619-535-9001

(B) TELEFAX: 619-535-8949

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GCCCATCTGT TCTCAGAAAC CGCATGCTTT TGTTGCAAGT GTGACTCTA 49 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

AGCTTAGAGT CACACTTGCA ACAAAAGCAT GCGGTTTCTG AGAACAGATG GGCTGCA 57

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AACATAATTG GAAGAAATCT GTT 23

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TAGGTCTTAA CTTGACTAAC ATGATCA 27

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TGATCAATTT TTAATAAACC ATATTGGATG C 31

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

TGATCATACG TGTGGTATCT ACCACAGTAA C 31 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GATCAAACAT AATTGGAAGA AATCTGTTGC TGACCGACGC GTCAGGATGC CTACGGCTCC 60 GCCTGCGCAG GTT 73

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GATCAACCTG CGCAGGCGGA GCCGTAGGCA TCCTGACGCG TCGGTCAGCA ACAGATTTCT 60 TCCAATTATG TTT 73

Claims

We Claim:

1. A functional vector for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms comprising a viral genome having a deletion of a native sequence and an insertion of one or more heterologous nucleotide sequences characteristic of said infectious or pathogenic organism.

2. The vector of claim 1, wherein said viral genome having a deletion of a native sequence is a papovavirus.

3. The vector of claim 2, wherein said deletion of a native sequence is in the late leader region.

4. The vector of claim 1, wherein said

heterologous nucleotide sequence contains between about 24 to 100 base pairs.

5. The vector of claim 1, wherein said functional vector is self-replicating.

6. A host cell transformed with a functional vector for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms, said functional vector comprising a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of said infectious or pathogenic organism.

7. The host cell of claim 6, wherein said viral genome having a deletion of a native sequence is characteristic of the papovavirus family of viruses.

8. The host cell of claim 7, wherein said deletion of a native sequence is in the late leader region.

9. The host cell of claim 6, wherein said heterologous sequence is between about 24 to 100 base pairs.

10. The host cell of claim 6, wherein said functional vector is self-replicating.

11. The host cell of claim 6, wherein said host cell is fixed with an agent that preserves the integrity of a cell.

12. A recombinant virus useful for reproducing positive diagnostic indications of one or more infectious or pathogenic organisms comprising a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of said infectious or pathogenic organism.

13. The recombinant virus of claim 12, wherein said viral genome having a deletion of a native sequence is characteristic of the papovavirus family of viruses.

14. The recombinant virus of claim 13, wherein said deletion of a native sequence is in the late leader region.

15. The recombinant virus of claim 12, wherein said heterologous sequence is between about 24 to 100 base pairs.

16. The recombinant virus of claim 12, wherein said functional vector is self-replicating.

17. A system for detecting one or more target sequences indicative of one or more infectious or

pathogenic organisms comprising:

(a) a first component comprising one or more fixed cells containing a functional vector consisting essentially of a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of said

infectious or pathogenic organism; and

(b) a second component comprising one or more oligonucleotide probes substantially complementary to said one or more

heterologous sequences of said first component.

18. The system of claim 17, wherein said viral genome having a deletion of a native sequence of said first and second components are characteristic of the papovavirus family of viruses.

19. The system of claim 17, wherein said recombinant infectious virus of said first and second components are self-replicating.

20. The system of claim 17, wherein said oligonucleotide probes further comprise a detectable label.

21. A method for detecting one or more target sequences indicative of one or more infectious or pathogenic organisms comprising:

(a) providing a functional vector, said

functional vector comprising a viral genome having a deletion of a native sequence and an insertion of one or more heterologous sequences characteristic of said infectious or pathogenic organism; (b) introducing said functional vector into compatible host cells to produce infected cells capable of generating recombinant viruses having said functional vector as their viral genome;

(c) fixing said infected cells capable of

generating recombinant viruses; and

(d) detecting said one or more heterologous sequences characteristic of said

infectious or pathogenic organism in said fixed infected cells.

22. The method of claim 21 further comprising comparing the levels of said heterologous sequence within said fixed infected cells to that within a sample

suspected of containing an infectious or pathogenic organism.

23. The method of claim 21, wherein said viral genome having a deletion of a native sequence is

characteristic of the papovavirus family of viruses.

24. The method of claim 21, wherein step (d) further comprises the steps:

(d1) fixing said infected host cells with an agent that preserves the integrity of a cell; and

(d2) hybridizing an oligonucleotide probe

containing a detectable label to said one or more heterologous sequences

characteristic of said infectious or pathogenic organism.

25. The method of claim 21, wherein said functional vector is self-replicating.