# US 2014/0193423 A1 · Diagnosis and treatment of viral diseases

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- Number: US 2014/0193423 A1
- Document ID: US20140193423A1
- Status: Published application
- Kind: A1
- Application number: 13/920,964
- Filed: 2013-06-18
- Published: 2014-07-10
- Earliest claimed: 2013-01-08
- Assignees: Enzo Biochem, Inc.
- Applicants: Enzo Biochem, Inc.
- Inventors: GERARD NUOVO; VIRGINIA NIVAR
- Web page: https://patentgrep.com/patent/US20140193423A1

## CPC

- C12Q 1/705: {for herpetoviridae, e.g. herpes simplex, varicella zoster}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

This disclosure relates to methods of diagnosing a viral disease in a patient by identifying one or more virus-specific elements or a patient antibody to a virus-specific element, as well as to kits for diagnosing a viral disease in a patient. The disclosure further relates to methods of monitoring disease progression and/or the efficacy of therapy by measuring the levels of a virus-specific element in a sample from a patient. In addition, the disclosure relates to methods of identifying therapeutic agents that show efficacy in reducing levels of virus-specific agents in vitro. The disclosure further relates to methods of treating idiopathic pulmonary fibrosis, as well as to methods of preventing viral infection, including Herpesvirus saimiri infection.

## Claims

### Claim at position 1

1-19. (canceled)

### Claim 20

A method of detecting the presence of viral target sequences in a human clinical sample comprising the steps of:
a. providing
i. a human clinical sample suspected of having a viral infection,
ii. a labeled nucleic acid probe comprising one or more sequences derived from Herpesvirus saimiri,
b. contacting said clinical sample (i) with said labeled nucleic acid probe (ii),
c. allowing hybridization to take place between said labeled nucleic acid probe (ii) and said viral target sequences in said clinical sample (i) if present, and
d. detecting hybridization of said nucleic acid probe (ii) to said viral target sequences in said clinical sample (i).

### Claim 21 (depends on 20)

The method of claim 20, wherein said viral target sequences comprises mRNA.

### Claim 22 (depends on 20)

The method of claim 20, wherein said viral target sequences comprises DNA.

### Claim 23 (depends on 20)

The method of claim 20, wherein said nucleic acid probe is labeled with a radioactive label, a fluorescent label, a chemiluminescent label, a hapten label, a chromogenic label, or an energy transfer pair.

### Claim 24 (depends on 23)

The method of claim 23, wherein said labeled binding partner is biotin, avidin or streptavidin.

### Claim 25 (depends on 20)

The method of claim 20, wherein said human clinical sample is selected from blood, tissue, lavage, and combinations thereof.

### Claim 26 (depends on 25)

The method of claim 25, wherein said tissue sample is a lung biopsy.

### Claim 27 (depends on 25)

The method of claim 25, wherein said clinical sample comprises a paraffin embedded slide.

### Claim 28 (depends on 20)

The method of claim 20, wherein said method of detection comprises in situ hybridization or flow cytometry.

### Claim 29 (depends on 20)

The method of claim 20, wherein said providing step comprises isolation of nucleic acids from said clinical sample.

### Claim 30 (depends on 20)

The method of claim 20, further comprising a nucleic acid amplification step before or concurrently with step b.

### Claim 31 (depends on 30)

The method of claim 30, wherein said amplification step is carried out by an Eberwine amplification, a polymerase chain reaction (PCR) amplification, an AmpiProbe® amplification, a real time polymerase chain reaction (RT-PCR) amplification, a degenerate oligonucletide primer PCR (DOP-PCR) amplification, a multiple displacement amplification, a self-sustained sequence reaction (3SR) amplification, a nucleic acid based transcription assay (NASBA) amplification, a transcription mediated amplification (TMA), a strand displacement amplification (SDA), a helicase-dependent amplification (HDA), a loop-mediated isothermal amplification (LAMP), a stem-loop amplification, a signal mediated amplification of RNA technology (SMART), an isothermal multiple displacement amplification (IMDA), a single primer isothermal amplification (SPIA), or a circular helicase-dependent amplification (cHDA).

### Claim 32 (depends on 30, 31)

The method of claim 30 or claim 31, wherein said detection is carried out in a dot blot format, a slot blot format, a microarray format, a sandwich assay format, a primer extension format, or fluorescence resonance energy transfer (FRET).

### Claim 33 (depends on 20)

The method of claim 20, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 50% homologous with said labeled nucleic acid probe sequence.

### Claim 34 (depends on 33)

The method of claim 33, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 75% homologous with said labeled nucleic acid probe sequence.

### Claim 35 (depends on 34)

The method of claim 34, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 90% homologous with said labeled nucleic acid probe sequence.

### Claim 36 (depends on 20)

The method of claim 20, wherein said labeled nucleic acid probe comprises one or more sequences derived from Herpesvirus saimiri A, Herpesvirus saimiri B or Herpesvirus saimiri C.

### Claim 37

A method of detecting the presence of viral target sequences in a human clinical sample comprising the steps of:
a. providing
i. a human clinical sample suspected of containing nucleic acids comprising viral target sequences,
ii. a labeled nucleic acid probe comprising one or more sequences derived from a virus related to Herpesvirus saimiri, wherein said related virus has at least 50% nucleic acid sequence homology with Herpesvirus saimiri,
b. contacting said clinical sample (i) with said labeled nucleic acid probe (ii),
allowing hybridization to take place between said labeled nucleic acid probe (ii) and viral target
c. sequence nucleic acids in said clinical sample (i) if said viral target sequence nucleic acids are present, and
d. detecting hybridization of said nucleic acid probe (ii) to said viral target sequences in said clinical sample (i).

### Claim 38 (depends on 37)

The method of claim 37, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 75% homologous with said labeled nucleic acid probe sequence.

### Claim 39 (depends on 38)

The method of claim 38, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 90% homologous with said labeled nucleic acid probe sequence.

### Claim 40 (depends on 37)

The method of claim 37, wherein said viral target sequence comprises mRNA.

### Claim 41 (depends on 37)

The method of claim 37, wherein said viral target sequence comprises DNA.

### Claim 42 (depends on 36)

The method of claim 36, wherein said nucleic acid probe is labeled with a radioactive label, a fluorescent label, a chemiluminescent label, a hapten label, a chromogenic, label, or an energy transfer pair.

### Claim 43 (depends on 42)

The method of claim 42, wherein said labeled binding partner is biotin, avidin or streptavidin.

### Claim 44 (depends on 37)

The method of claim 37, wherein said nucleic acid probe comprises a nucleotide analogue.

### Claim 45 (depends on 37)

The method of claim 37, wherein said human clinical sample is selected from blood, tissue, lavage, and combinations thereof.

### Claim 46 (depends on 45)

The method of claim 45, wherein said tissue sample is a lung biopsy.

### Claim 47 (depends on 45)

The method of claim 45, wherein said clinical sample comprises a paraffin embedded slide.

### Claim 48 (depends on 37)

The method of claim 37, wherein said method of detection comprises in situ hybridization or flow cytometry.

### Claim 49 (depends on 37)

The method of claim 37, wherein said providing step comprises isolation of nucleic acids from said clinical sample.

### Claim 50 (depends on 49)

The method of claim 49, further comprising a nucleic acid amplification step.

### Claim 51 (depends on 50)

The method of claim 50, wherein said amplification step is carried out by an Eberwine amplification, a polymerase chain reaction (PCR) amplification, an AmpiProbe® amplification, a real time polymerase chain reaction (RT-PCR) amplification, a degenerate oligonucletide primer PCR (DOP-PCR) amplification, a multiple displacement amplification, a self-sustained sequence reaction (3SR) amplification, a nucleic acid based transcription assay (NASBA) amplification, a transcription mediated amplification (TMA), a strand displacement amplification (SDA), a helicase-dependent amplification (HDA), a loop-mediated isothermal amplification (LAMP), a stem-loop amplification, a signal mediated amplification of RNA technology (SMART), an isothermal multiple displacement amplification (IMDA), a single primer isothermal amplification (SPIA), or a circular helicase-dependent amplification (cHDA).

### Claim 52 (depends on 50, 51)

The method of claim 50 or claim 51, wherein said detection is carried out by gel electrophoresis, in a dot blot format, a slot blot format, a microarray format, a sandwich assay format, a primer extension format, fluorescence resonance energy transfer (FRET) or fluorescence derived from intercalation of a dye.

### Claim 53 (depends on 37)

The method of claim 37, wherein said labeled nucleic acid probe comprises one or more sequences from Herpesvirus saimiri A, Herpesvirus saimiri B or Herpesvirus saimiri C.

### Claim 54

A method of diagnosing idiopathic pulmonary fibrosis in a human patient comprising the steps of:
a. providing
i. a human clinical sample suspected of having idiopathic pulmonary fibrosis,
ii. a labeled nucleic acid probe comprising one or more sequences derived from Herpesvirus saimiri or a virus related to Herpesvirus saimiri, wherein said related virus has at least 50% nucleic acid sequence homology with Herpesvirus saimiri,
b. contacting said clinical sample (i) with said labeled nucleic acid probe (ii),
c. allowing hybridization to take place between said labeled nucleic acid probe (ii) and viral sequences in said clinical sample (i) if present, and
d. detecting hybridization of said nucleic acid probe (ii) to said viral sequences in said clinical sample (i), and
thereby diagnosing said patient as having idiopathic pulmonary fibrosis.

### Claim 55 (depends on 54)

The method of claim 54, wherein said viral sequences comprise mRNA.

### Claim 56 (depends on 54)

The method of claim 54, wherein said viral sequences comprise DNA.

### Claim 57 (depends on 54)

The method of claim 54, wherein said nucleic acid probe is labeled with a radioactive label, a fluorescent label, a chemiluminescent label, a hapten label, a chromogenic label, or an energy transfer pair.

### Claim 58 (depends on 57)

The method of claim 57, wherein said labeled binding partner is biotin, avidin or streptavidin.

### Claim 59 (depends on 57)

The method of claim 57, wherein said nucleic acid probe comprises a nucleic acid analogue.

### Claim 60 (depends on 54)

The method of claim 54, wherein said human clinical sample is selected from blood, tissue, lavage, and combinations thereof.

### Claim 61 (depends on 60)

The method of claim 60, wherein said tissue sample is a lung biopsy.

### Claim 62 (depends on 61)

The method of claim 61, wherein said clinical sample comprises a paraffin embedded slide.

### Claim 63 (depends on 54)

The method of claim 54, wherein said method of detection comprises in situ hybridization or flow cytometry.

### Claim 64 (depends on 54)

The method of claim 54, wherein said providing step comprises isolation of nucleic acids from said clinical sample.

### Claim 65 (depends on 64)

The method of claim 64, further comprising a nucleic acid amplification step.

### Claim 66 (depends on 64)

The method of claim 64, wherein said amplification step is carried out by an Eberwine amplification, a polymerase chain reaction (PCR) amplification, an AmpiProbe® amplification, a real time polymerase chain reaction (RT-PCR) amplification, a degenerate oligonucletide primer PCR (DOP-PCR) amplification, a multiple displacement amplification, a self-sustained sequence reaction (3SR) amplification, a nucleic acid based transcription assay (NASBA) amplification, a transcription mediated amplification (TMA), a strand displacement amplification (SDA), a helicase-dependent amplification (HDA), a loop-mediated isothermal amplification (LAMP), a stem-loop amplification, a signal mediated amplification of RNA technology (SMART), an isothermal multiple displacement amplification (IMDA), a single primer isothermal amplification (SPIA), or a circular helicase-dependent amplification (cHDA).

### Claim 67 (depends on 65, 66)

The method of claim 65 or claim 66, wherein said detection is carried out in a dot blot format, a slot blot format, a microarray format, a sandwich assay format, a primer extension format, or fluorescence resonance energy transfer (FRET).

### Claim 68 (depends on 54)

The method of claim 54, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 75% homologous with said labeled nucleic acid probe sequence.

### Claim 69 (depends on 68)

The method of claim 68, wherein said hybridization is carried out under conditions where said labeled probe hybridizes with a viral sequence that is at least 90% homologous with said labeled nucleic acid probe sequence.

### Claim 70 (depends on 54)

The method of claim 54, wherein said labeled nucleic acid probe comprises one or more sequences derived from Herpesvirus saimiri A, Herpesvirus saimiri B or Herpesvirus saimiri C.

### Claim 71

A method of diagnosing idiopathic pulmonary fibrosis in a human patient comprising the steps of:
a. providing
i. a human clinical sample suspected of having a viral infection,
ii. antibodies to at least two protein targets selected from DHFR, cyclin D, IL-17 and thymidylate synthase;
b. contacting said clinical sample (i) with said antibodies (ii),
c. allowing binding to take place between said antibodies (ii) and proteins in said clinical sample (i) if present, and
d. detecting binding of said antibodies (ii) to said proteins in said clinical sample (i), and thereby diagnosing said patient as having idiopathic pulmonary fibrosis.

### Claim 72 (depends on 71)

The method of claim 71, wherein said antibodies (ii) are labeled.

### Claim 73 (depends on 71)

The method of claim 71, wherein said antibodies (ii) are detected by binding labeled secondary antibodies to said antibodies (ii).

### Claim 74 (depends on 72, 73)

The method of claim 72 or claim 73, wherein said label is selected from a radioactive label, a fluorescent label, a chemiluminescent label, a hapten label, a chromogenic label, or an energy transfer pair.

### Claim 75 (depends on 71)

The method of claim 71, wherein said antibodies are monoclonal antibodies, polyclonal antibodies or combinations thereof.

### Claim 76 (depends on 75)

The method of claim 75, wherein said antibodies are polyclonal antibodies.

### Claim 77 (depends on 71)

The method of claim 71, wherein said antibodies are human antibodies, humanized antibodies or combinations thereof.

### Claim 78

A method of diagnosing idiopathic pulmonary fibrosis in a human subject comprising the steps of
a. providing
iii. a clinical sample from a subject who may have idiopathic pulmonary fibrosis,
iv. one or more antibodies to viral proteins expressed by Herpesvirus saimiri or a virus related to Herpesvirus saimiri, wherein said related virus has as at least 50% nucleic acid homology with Herpesvirus saimiri
b. contacting said clinical sample (i) with said one or more antibodies (ii),
c. allowing binding to take place between said one or more antibodies (ii) and said viral proteins in said clinical sample (i), and
d. detecting the binding of said one or more antibodies (ii) to said viral proteins in the clinical sample (i) and thereby diagnosing said subject as having idiopathic pulmonary fibrosis.

### Claim 79 (depends on 78)

The method of claim 78 wherein one of said viral proteins is IL-17.

### Claim 80 (depends on 78)

The method of claim 78 wherein one of said viral proteins is DHFR.

### Claim 81 (depends on 78)

The method of claim 78 wherein one of said viral proteins is cyclin D.

### Claim 82 (depends on 78)

The method of claim 78 wherein one of said viral proteins is thymidylate synthase.

### Claim 83 (depends on 78)

The method of claim 78 wherein one of said viral proteins is a viral capsid protein.

### Claim 84

A method of treating idiopathic pulmonary fibrosis in a subject comprising administering to said subject, one or more antibodies to one or more viral proteins expressed by Herpesvirus saimiri or a virus related to Herpesvirus saimiri wherein said related virus has at least 50% nucleic acid homology with Herpesvirus saimiri.

### Claim 85 (depends on 84)

The method of claim 84 wherein one of said viral proteins is IL17.

### Claim 86 (depends on 84)

The method of claim 84 wherein one of said viral proteins is DHFR.

### Claim 87 (depends on 84)

The method of claim 84 wherein one of said viral proteins is cyclin D.

### Claim 88 (depends on 84)

The method of claim 84 wherein one of said viral proteins is thymidylate synthase.

### Claim 89 (depends on 84)

The method of claim 84 wherein one of said viral proteins is a viral capsid protein.

### Claim 90

A method of preventing idiopathic pulmonary fibrosis in a subject comprising administering to said subject one or more antibodies to one or more proteins expressed by Herpesvirus saimiri or a virus related to Herpesvirus saimiri wherein said related virus has as at least 50% nucleic acid homology with Herpesvirus saimiri.

### Claim 91 (depends on 90)

The method of claim 90 wherein one of said viral proteins is IL-17.

### Claim 92 (depends on 90)

The method of claim 90 wherein one of said viral proteins is DHFR.

### Claim 93 (depends on 90)

The method of claim 90 wherein one of said viral proteins is cyclin D.

### Claim 94 (depends on 90)

The method of claim 90 wherein one of said viral proteins is thymidylate synthase.

### Claim 95 (depends on 90)

The method of claim 90 wherein one of said viral proteins is a viral capsid protein.

### Claim 96

A kit for detection of viral target sequences in a human clinical sample comprising:
a. a labeled nucleic acid probe selected from (i) a probe comprising one or more sequences derived from Herpesvirus saimiri, (ii) a probe derived from a virus related to Herpesvirus saimiri, wherein said related virus has at least 50% nucleic acid sequence homology with Herpesvirus saimiri, or a combination of (i) and (ii); and
b. reagents for carrying out hybridization of said probe to said nucleic acids in a clinical sample.

### Claim 97 (depends on 96)

The kit of claim 96, further comprising reagents for isolating said viral target sequences.

### Claim 98

A kit comprising
a. a labeled nucleic acid probe selected from (i) a probe comprising one or more sequences derived from Herpesvirus saimiri, (ii) a probe derived from a virus related to Herpesvirus saimiri, wherein said related virus has at least 50% nucleic acid sequence homology with Herpesvirus saimiri, or a combination of (i) and (ii); and
b. reagents for carrying out hybridization of said probe to a clinical sample
c. a primer comprising a sequence complementary to a sequence in one strand of the viral target sequence;
d. a primer comprising a sequence identical to a sequence in said strand of the viral target sequence; and
e. reagent for carrying out amplification of said viral target sequence.

### Claim 99 (depends on 96)

The kit of claim 96, further comprising a nucleic acid probe that is complementary to a viral target sequence.

### Claim 100 (depends on 96)

The kit of claim 96, further comprising an intercalator that increases fluorescence after binding to double-stranded DNA.

### Claim 101 (depends on 99, 100)

The kit of claim 99 or claim 100, wherein at least one primer or probe is labeled.

### Claim 102

A method of detecting the presence of viral target sequences in a human clinical sample comprising the steps of
a. providing a human clinical sample that may contain virally infected cells,
b. a reagent for isolating nucleic acids from said clinical sample,
c. a reagent for amplification of nucleic acids in said sample, wherein said reagent is capable of amplifying nucleic acids of Herpesvirus saimiri or a virus related to Herpesvirus saimiri wherein said related virus has at least 50% nucleic acid homology with Herpesvirus saimiri when said sample comprises nucleic acids of Herpesvirus saimiri or said related virus,
d. isolating nucleic acids from said clinical sample by said reagent (ii),
e. combining said isolated nucleic acids with said amplification reagent (iii),
f. amplifying said nucleic acids from Herpesvirus saimiri or said related virus, and
g. detecting the amplification of said nucleic acids of Herpesvirus saimiri or said related virus,
thereby detecting the presence of said viral target sequences.

### Claim 103 (depends on 102)

The method of claim 102, wherein amplification is carried out by an Eberwine amplification, a polymerase chain reaction (PCR) amplification, an AmpiProbe® amplification, a real time polymerase chain reaction (RT-PCR) amplification, a degenerate oligonucletide primer PCR (DOP-PCR) amplification, a multiple displacement amplification, a self-sustained sequence reaction (3SR) amplification, a nucleic acid based transcription assay (NASBA) amplification, a transcription mediated amplification (TMA), a strand displacement amplification (SDA), a helicase-dependent amplification (HDA), a loop-mediated isothermal amplification (LAMP), a stem-loop amplification, a signal mediated amplification of RNA technology (SMART), an isothermal multiple displacement amplification (IMDA), a single primer isothermal amplification (SPIA), or a circular helicase-dependent amplification (cHDA).

### Claim 104 (depends on 103)

The method of claim 103, wherein said detecting step (vii) is carried out using one or more labeled nucleotides, one or more labeled primers, one or more labeled probes, one or more intercalating dyes or a combination thereof.

### Claim 105

A kit for detecting at least two protein targets selected from DHFR, cyclin D, IL-17 and thymidylate synthase in a human clinical sample comprising:
a. an antibody to any two of DHFR, cyclin D, IL-17 and thymidilate synthase; and
b. reagents for the binding of said antibodies to proteins in said sample.

### Claim 106 (depends on 105)

The kit of claim 105, wherein said antibodies are monoclonal antibodies, polyclonal antibodies, or combinations thereof.

### Claim 107 (depends on 105)

The kit of claim 105, wherein said antibody is labeled.

### Claim 108 (depends on 105)

The kit of claim 105, further comprising a secondary antibody.

### Claim 109 (depends on 108)

The kit of claim 108, wherein the secondary antibody is labeled.

### Claim 110 (depends on 105)

The kit of claim 105, wherein the secondary antibody is conjugated to an enzyme.

### Claim 111 (depends on 110)

The kit of claim 110, further comprising reagents for signal amplification.

### Claim 112

A composition comprising a viral target sequence hybridized to (i) a non-radioactively labeled nucleic acid comprising one or more sequences derived from Herpesvirus saimiri, (ii) a non-radioactively labeled nucleic acid comprising one or more sequences derived from a virus related to Herpesvirus saimiri, wherein said related virus has at least 50% nucleic acid sequence homology with Herpesvirus saimiri, or a combination thereof, wherein said hybridization product is in a human cell of a clinical sample.

### Claim 113

A method of diagnosing idiopathic pulmonary fibrosis in a human patient comprising the steps of:
a. providing
i. a human clinical sample suspected of having a viral infection,
ii. an antibody to viral IL-17;
b. contacting said clinical sample (i) with said antibody (ii),
c. allowing binding to take place between said antibody (ii) and proteins in said clinical sample (i) if present, and
d. detecting binding of said antibody to said viral IL-17 in said clinical sample (i), and
thereby diagnosing said patient as having idiopathic pulmonary fibrosis.

### Claim 114 (depends on 112)

The composition of claim 112, wherein said labeled nucleic acid is partially hybridized to said one or more sequences derived from Herpesvirus saimiri.

### Claim 115 (depends on 112)

The composition of claim 112, wherein said labeled nucleic acid has more than 50% homology to said one or more sequences derived from Herpesvirus saimiri.

### Claim 116 (depends on 112)

The composition o claim 112, wherein said labeled nucleic acid comprises one or more nucleotide analogues.

### Claim 117 (depends on 116)

The composition of claim 116, wherein said one or more nucleotide analogues confers a properly to said labeled nucleic acid selected from differential melting, a detectable signal, and maintenance of stable hybridization with a nucleic acid that is not completely complementary to said labeled nucleic acid.