Utilizing intrinsic fluorescence to detect adenovirus

Details Utilizing intrinsic fluorescence to detect adenovirus Utilizing intrinsic fluorescence to detect adenovirus

2000-10-04

2002-09-10

Salimi, Ali R. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S008000, C435S320100, C424S233100

Reexamination Certificate

active

06447995

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention pertains to the detection and characterization of viral vector particles, particularly through the use of fluorescence.
BACKGROUND OF THE INVENTION
Viral vectors are of significant importance in several aspects of molecular biology and medicine. Numerous types of viral vectors have been developed for use as gene delivery vehicles. Examples of such viral vectors include vectors based on adenovirus (Ad), adeno-associated virus (AAV), baculovirus, herpes simplex virus (HSV), and murine leukemia virus (MLV). Compared to other methods of delivering genetic information (e.g., liposome-associated delivery techniques or naked DNA vectors), viral vectors offer several advantages, including higher rates of delivery and better targeting of specific tissues and/or cells. With the increased use of viral vectors for therapeutic, as well as diagnostic, applications there is an increasing need for better methods for quantification and characterization of viral vector particles.
Several techniques are known for the characterization and/or quantification of viral vector particles, including chromatographic methods and mass spectrometry (see, e.g., International Patent Application WO 99/54441, International Patent Application WO 00/40702, and U.S. Pat. No. 5,965,358). Presently, the quantification of viral vector particles is most commonly carried out by the use of ultraviolet (UV) radiation. For example, U.S. Pat. No. 5,837,520 discloses monitoring the absorbance of a chromatographic eluant of viral particles at a selected UV wavelength and comparing the absorbance value to a standard curve which relates absorbance to the number of viral vector particles. Ultraviolet absorbance is limited in its sensitivity and requires a large number of viral particles (typically about 5×10
9
particles/ml) for accurate detection (where the standard deviation in measurement is about 10% or less). Due to the large number of viral particles required for accurate quantification, ultraviolet absorbance is not useful in applications requiring small populations of viral particles, such as viral vector-based gene therapies where high quantities of viral vector particles can be undesirable.
Fluorescence-based detection and quantification of viral particles associated with fluorogenic dyes such as fluorescein isothiocynate (FITC) is known in the art. For example, Hara et al.,
Applied and Environmental Microbiology,
57(9), 2731-34 (1991), describes the use of epifluorescent microscopy on DAPI (4′,6′-diamidino-2-phenylindole)-treated water samples to determine numbers of bacteria, viruses, and DNA-associated particles. More recently, Hennes and Suttle,
Limnol Oceanogr.,
10(6), 1050-55 (1995), describes similar research using the cyanine-based dye, Yo-Pro-1.
Immunofluorescence, which combines antibody-antigen binding and fluorophore-associated fluorescence detection (see, e.g., Tanaka et al.,
J. Hepatology,
23, 742-45 (1995)), also has been used to detect and/or characterize viruses. For example, D'alessio et al.,
Applied Microbiology,
20(2), 233-39 (1970), discloses the use of fluorescein-based immunofluorescence techniques to detect influenza viruses, herpes simplex virus, and adenoviruses. More recently, Orito et al.,
Gut,
39, 876-880 (1996), described the use of a fluorescent enzyme immunoassay (FEIA) to quantify hepatitis C virus core protein levels in patients, and Wood et al.,
J. Medical Virol.,
51, 198-201 (1997), describes the use of FITC-based immunofluorescence to identify and type adenovirus isolates. Enzymatic techniques associated with fluorogenic dyes also are capable of detecting nucleic acids (see, e.g., U.S. Pat. No. 5,830,666).
The Green Fluorescent Protein (GFP), obtained from the jellyfish
Aequorea victoria
(see, e.g., Prasher et al.,
Gene,
111, 229-33 (1992)), which is intrinsically fluorogenic, has been used to characterize viruses by causing viruses to express GFP. For example, International Patent Application WO 00/08182 describes preparations of herpes virus expressing GFP fusion proteins to detect the progress of cell infection by the virus and to screen for neutralizing antibodies or inhibitors of infection. International Patent Application WO 99/54348 discloses the use of vectors transfected with short-lived GFP variants to assay activation or deactivation of promoters. International Patent Application WO 99/43843 teaches transfection with adenovirus vectors encoding GFP and tracking viral production by GFP-associated fluorescence.
Techniques for detecting or characterizing viral vector particles based on direct fluorescent dye-association with the viral particles, immunofluorescence, and GFP-associated viral fluorescence are limited in requiring either a fluorogenic dye or GFP to be associated with the viral vector particles. Because the use of fluorogenic dyes can be expensive, less sensitive than other techniques, and damaging to samples, direct dye-association techniques are often unsuitable. Immunofluorescence, while more sensitive than direct dye-based techniques, requires specific epitopes and antibodies. GFP-based techniques require either chemical or genetic modification to associate the viral vector particles with GFP.
Few fluorogenic methods have been used to study biological materials without the use of dyes or strong fluorogenic proteins such as GFP. U.S. Pat. No. 5,623,932 discloses the use of direct fluorogenic methods to differentiate between normal and abnormal cervical tissues. The '932 patent discloses using laser-induced fluorescence (LIF) to identify fluorogenic spectra associated with healthy tissue, relying on oxy-hemoglobin and NADH in the tissue as fluorophores, and further using such spectra to identify “abnormal” tissues by comparing spectra. The '932 patent suggests that such abnormal tissue could be inflamed or infected with human papilloma virus (HPV). However, the '932 patent fails to identify, characterize, or quantify HPV particles in such tissues.
Accordingly, there remains a need for techniques which allow for improved detection and characterization of viral vector particles. The present invention provides methods for such detection and characterization through fluorescence detection of viral vector particles and viral vector proteins. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of quantifying the number of viral vector particles in a medium. A medium containing a viral vector particle having an intrinsically fluorogenic portion is provided. The medium is contacted with an excitation energy, such that an electron associated with the intrinsically fluorogenic portion of the viral vector particle is raised to an excited energy state. The excited electron is permitted to emit radiation having one or more emission wavelengths and corresponding emission intensities. An emission wavelength, and the intensity of the a emitted radiation at the emission wavelength, are detected. The number of viral vector particles in the medium is quantified by evaluating the detected wavelength and intensity and comparing them to a provided standard signal.
The invention also provides a method of evaluating the viral vector particle content of a medium. A medium is provided and contacted with an excitation radiation having an excitation wavelength such that if a viral vector particle is in the medium an intrinsically fluorogenic portion of the viral vector particle will emit radiation having an emission wavelength at about 560-590 nm (e.g., about 575 nm). The viral vector particle content of the medium is evaluated by determining whether the medium emits radiation at about 560-590 nm.
The invention further provides a method of evaluating the adenoviral vector particle content of a medium. A medium is provided and contacted with an excitation radiation having one or more excitation wavelength

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