U.S. patent application number 15/394085 was filed with the patent office on 2017-04-20 for nucleic acid amplification controls.
The applicant listed for this patent is ZeptoMetrix Corporation. Invention is credited to Gregory R. Chiklis, James C.D. Hengst.
Application Number | 20170107585 15/394085 |
Document ID | / |
Family ID | 22908986 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170107585 |
Kind Code |
A1 |
Hengst; James C.D. ; et
al. |
April 20, 2017 |
NUCLEIC ACID AMPLIFICATION CONTROLS
Abstract
The present invention discloses positive control material for
nucleic acid amplification based detection of microorganisms in
biological samples. The control material comprises purified
microorganism that is rendered non-infectious but is amenable to
nucleic acid amplification. Also disclosed is a process for making
and using the control material.
Inventors: |
Hengst; James C.D.;
(Getzville, NY) ; Chiklis; Gregory R.; (Franklin,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZeptoMetrix Corporation |
Buffalo |
NY |
US |
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|
Family ID: |
22908986 |
Appl. No.: |
15/394085 |
Filed: |
December 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14841053 |
Aug 31, 2015 |
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15394085 |
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11929123 |
Oct 30, 2007 |
9127048 |
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14841053 |
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09981506 |
Oct 17, 2001 |
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11929123 |
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11929089 |
Oct 30, 2007 |
9120849 |
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14841053 |
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09981506 |
Oct 17, 2001 |
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11929089 |
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60241038 |
Oct 17, 2000 |
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60241038 |
Oct 17, 2000 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/689 20130101;
C12N 2740/16222 20130101; C12N 2770/24363 20130101; C12N 7/00
20130101; C12Q 1/70 20130101; C12N 2740/16063 20130101; C12N
2730/10122 20130101; C12Q 2600/166 20130101; Y02A 50/54 20180101;
C12N 2730/10163 20130101; C12Q 1/6888 20130101; Y02A 50/30
20180101; C12N 2770/24322 20130101; C12N 2740/15022 20130101; C07K
14/005 20130101; Y02A 50/59 20180101; C12N 2740/16022 20130101 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12N 7/00 20060101 C12N007/00 |
Claims
1. A method for making a full process positive control material for
detection of virus in biological samples comprising: a) purifying
an intact virus from a source; b) exposing the purified intact
virus to an aldehyde at a temperature and for a time such that one
or more surface proteins are irreversibly modified while leaving
the nuclear components substantially intact thereby rendering the
purified intact virus non-pathogenic and wherein nucleic acids of
the purified intact virus are amenable to amplification; and c)
identifying that the purified intact virus can be used as a full
process positive control by confirming absence of active virus and
an ability of viral nucleic acids in the purified intact virus to
be amplified.
2. The method of claim 1, wherein the aldehyde is selected from the
group consisting of paraformaldehyde, formaldehyde, acetaldehyde,
propionaldehyde, n-butyraldehyde, benzaldehyde,
p-nitrobenzaldehyde, p-tolualdehyde, salicylaldehyde,
phenylacetaldehyde, 2-methylpentanal, 3-methylpentanal, and
4-methylpentanal.
3. The method of claim 1, wherein the time is controlled by
quenching the aldehyde to provide the exposing for the time such
that one or more surface proteins are irreversibly modified while
leaving the nuclear components substantially intact.
4. The method of claim 3, wherein the quenching is carried out by
glycine.
5. The method of claim 1, further comprising storing the purified
intact virus at a refrigeration temperature.
6. The method of claim 1, further comprising suspending the
purified intact virus in a liquid matrix comprising a buffer, a
biological fluid, or a synthetic biological fluid.
7. A non-pathogenic intact virus prepared by the method of claim 1,
wherein the nucleic acids in the non-pathogenic intact virus are
amenable to amplification.
8. A non-pathogenic intact virus prepared by the method of claim 1,
wherein the nucleic acids in the non-pathogenic intact virus are
amenable to amplification after storage at a refrigeration
temperature.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. non-provisional
application Ser. No. 14/841,053, filed Aug. 31, 2015, which is a
continuation of U.S. non-provisional application Ser. No.
11/929,123, now U.S. Pat. No. 9,127,048, filed Oct. 30, 2007, which
is a divisional application of U.S. non-provisional application
Ser. No. 09/981,506, filed on Oct. 17, 2001, which in turn claims
priority to U.S. provisional application No. 60/241,038, filed on
Oct. 17, 2000, the disclosures of which are incorporated herein by
reference.
[0002] U.S. non-provisional application Ser. No. 14/841,053 also is
a continuation of U.S. non-provisional application Ser. No.
11/929,089, now U.S. Pat. No. 9,120,849, filed Oct. 30, 2007, which
is a divisional application of U.S. non-provisional application
Ser. No. 09/981,506, filed on Oct. 17, 2001, which in turn claims
priority to U.S. provisional application No. 60/241,038, filed on
Oct. 17, 2000, the disclosures of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the area of virus
detection in biological samples. More particularly, the present
invention relates to a composition of matter that can serve as a
reliable control in the detection of viruses by nucleic acid
amplification methods.
DESCRIPTION OF RELATED ART
[0004] The presence of virus, such as the human immunodeficiency
virus (HIV), in biological samples is typically identified by
indirect methods, i.e., by detecting antibodies directed against a
particular virus or a component of the virus. This method of
detection is limited in its ability to detect minute amounts of
virus because antibodies typically do not develop in detectable
levels until after the virus has grown or reproduced considerably
inside the body. Thus, for example, in methods of screening blood
supplies for transfusion, existing indirect methods may not be
adequate to screen infected blood. In an effort to diagnose viral
infections at an earlier stage, nucleic acid amplification
techniques are being developed for detecting and quantifying
viruses in biological samples. Such techniques include, polymerase
chain reaction (PCR), transcription mediated amplification (TMA),
nucleic acid signal based amplification (NASBA) and ligase chain
reaction (LCR). These technologies are useful in the diagnosis of
viral infection and to monitor viral load in infected individuals
during treatment. Further, these technologies are useful in
screening of blood prior to transfusion.
[0005] Beginning in the spring of 1999, the American Red Cross and
16 member laboratories of America's Blood Centers began testing
donor blood for the human immunodeficiency virus (HIV) type-1 and
the hepatitis C virus with a new genetic test designed to detect
viral infections in their very early stages. These tests, called
Nucleic Acid Testing (NAT), are able to detect small amounts of a
virus before the blood donor's body mounts an immune response. The
power of NAT is its ability to detect the presence of infection by
directly testing for viral genomic nucleic acids rather than by
indirectly testing for the presence of antibodies. Furthermore, NAT
is much more sensitive than other direct detection methods such as
HIV p24 antigen detection assay in that NAT can detect as low as 50
viral particles in clinical specimens. NAT could potentially detect
HIV in blood approximately 10 days post-infection. The U.S. Food
and Drug Administration (FDA) is strongly encouraging blood banks
to begin NAT testing and hospitals to use NAT-screened blood
(Kornman et al., 1999, Cancer Control, Volume 6, Number 5).
[0006] Several commercial NAT kits and services are available for
the testing of HIV, hepatitis C virus (HCV) and hepatitis B virus
(HBV), such as those marketed by Roche Diagnostics (Indianapolis,
Ind.), National Genetics Institute, Inc. (Los Angeles, Calif.),
Bayer Corporation (Tarrytown, N.Y.) and Gen-Probe, Inc. (San Diego,
Calif.). These kits and testing services employ multistep assays
wherein the initial step is extraction or partial purification of
the target nucleic acid, followed by amplification and detection of
the nucleic acid. The positive controls developed for NAT based
detection thus far are: 1) plasma or serum from infected
individuals and 2) synthetic and cloned nucleic acids. These
controls have several drawbacks. For example, although plasma or
serum from infected individuals serves as a full process control
for all steps in a diagnostic procedure, it contains infectious
substances and is not stable at refrigerator temperatures
(2-8.degree. C.). Synthetic or cloned nucleic acids do not serve as
controls for the extraction step and are therefore, not full
process controls. Moreover, the synthetic and cloned nucleic acids
are extremely sensitive to nucleases and therefore require special
care in handling. A nuclease resistant "armored RNA" has been
developed. However, this armored material is not contained within
an intact virus particle and thus does not extract similar to
virally infected plasma. Thus, the armored material does not serve
as a control for the extraction process and also does not amplify
with TMA or LCR.
[0007] The Center for Biologics Evaluation and Research (CBER)
branch of the FDA has released guidelines for HIV NAT control
materials. The guidelines (Guidance for Industry, In the
Manufacture and Clinical Evaluation of in vitro Tests to Detect
Nucleic Acid Sequences of Human Immunodeficiency Viruses Types 1
and 2, BBS, FDA, CBER, December 1999) provide the FDA's
recommendations for the format and performance of controls and
calibrators for NAT based kits. According to these guidelines, the
control material should act as a full process control, i.e., it
should act as a control for all the steps of the sample handling
and detection process. Further, under the guidelines, the material
should be noninfectious and based on a well-validated
microorganism. Accordingly, the ideal control should provide an
indication of the steps of ultracentrifugation, extraction,
amplification, hybridization, quantitation, and of possible
contamination.
[0008] Thus, in view of the FDA guidelines and the drawbacks
identified with the existing control materials, there is a need in
the area of viral detection techniques for the development of
positive controls that can serve as full process controls, are
stable at refrigerator temperatures, and are safe to handle.
[0009] Accordingly, an object of the present invention is to
provide a noninfectious positive full process control for detection
of microorganisms by nucleic acid amplification techniques.
[0010] Another object of the present invention is to provide a
method for producing the internal controls of the invention.
[0011] Another objective of the invention is to provide a method of
screening biological samples for the presence of microorganisms by
nucleic acid amplification techniques.
[0012] These and additional objectives are satisfied by the present
invention which comprises nondisrupted inactivated microorganism
particles used as positive control material in nucleic acid
amplification detection techniques. The control material is
comprised of nondisrupted inactivated microorganism and can be
formulated in a stabilized plasma matrix. The control material is
noninfectious, stable under nonfrozen storage (such as 2-8.degree.
C.) and yields reproducible results in nucleic acid amplification
assays. The control material of the present invention can be stored
at refrigerator temperatures. Further, since the control material
comprises whole microorganism particles, it is run as a full
process control and thus is handled and processed exactly the same
way as the biological sample being tested. As a full process
control, the material can be used in the sample preparation step
and carried through the entire detection procedure. The control
materials of the present invention, thus, qualify for meeting the
FDA guidelines.
[0013] The references cited throughout the specification are hereby
incorporated herein in their entirety to more fully describe the
state of the art to which the invention applies.
SUMMARY OF THE INVENTION
[0014] The present invention provides a positive control material,
which can serve as a reliable control for nucleic acid
amplification techniques. The positive control material of the
invention generally comprises a virus or parasite that has been
rendered noninfectious, but retains the nuclear components
substantially intact so as to be amenable to nucleic acid
amplification and detection processes.
[0015] Thus, this invention provides a purified microorganism
comprising surface proteins and substantially intact nuclear
components, wherein one or more surface proteins have been
irreversibly modified such that the microorganism is thereby
rendered non-pathogenic.
[0016] This invention also provides a purified microorganism
comprising surface proteins and substantially intact nuclear
components, wherein one or more surface proteins have been
irreversibly modified by covalent attachment of a compound
comprising one or more reactive functional groups to one or more
reactive sites on said surface proteins, such that said
microorganism is thereby rendered non-pathogenic.
[0017] This invention further provides a composition of matter
comprising a purified microorganism comprising surface proteins and
intact nuclear components, wherein one or more surface proteins
have been irreversibly modified such that the microorganism is
thereby rendered non-pathogenic, and a liquid matrix that simulates
a biological fluid.
[0018] This invention also provides a method for producing a
non-pathogenic purified microorganism comprising surface proteins
and intact nuclear components and irreversibly modifying one or
more surface proteins while leaving the nuclear components
substantially unmodified, such that the microorganism is thereby
rendered non-pathogenic.
[0019] This invention also provides a method for detection of a
microorganism comprising surface proteins in a biological sample by
amplification of nuclear components of said microorganism, which
method comprises amplification of the nuclear components of a
purified control sample of said microorganism, wherein one or more
surface proteins of said control microorganism have been
irreversibly modified such that said control microorganism is
thereby rendered non-pathogenic.
[0020] In addition, this invention provides a kit for analyzing a
biological sample for the presence of a microorganism having
surface proteins, wherein the kit comprises a positive control
composition comprising a purified microorganism comprising surface
proteins and intact nuclear components, wherein one or more surface
proteins have been irreversibly modified such that the
microorganism is thereby rendered non-pathogenic.
[0021] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0022] This invention provides a purified microorganism comprising
surface proteins and substantially intact nuclear components,
wherein one or more surface proteins have been irreversibly
modified such that the microorganism is thereby rendered
non-pathogenic.
[0023] The microorganisms contemplated in the practice of the
invention are those that are pathogenic, and the detection of which
would aid in the detection or treatment of an ailment. As used
herein the term "microorganism" includes any infectious microscopic
organism that contains surface proteins which aid or assist in the
organism's ability to infect a host. For example, gp120 is present
on the surface of the HIV virus and acts as a receptor protein that
allows the virus to attach to monocytes and lymphocytes through
binding to their CD4 receptors. (Maddon et al., 1986, Cell, 47:333;
MacDougal et al., 1986, Science, 231:382; Moore et al., 1993, In J.
Bentz (ed.), Viral Fusion Mechanisms, CRC Press, Boca Raton, Fla.
In separately preferred embodiments, the microorganism can be a
virus or an intracellular parasite. As used herein the term "virus"
is meant to include either enveloped or nonenveloped viruses and
those containing either RNA or DNA as the nuclear material.
Examples include, but are not limited to, the human
immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus,
hepatitis C virus, cytomegalovirus, human lymphotrophic virus,
Epstein-Barr virus, parvovirus, herpes simplex virus, human herpes
virus 8. Examples of intracellular parasites include, but are not
limited to, Chlamydia trachomatis, Chlamydia psittaci, Rickettsia
prowazeki, Rickettsia typhi, Rickettsia rickettsi, Rickettsia
sibtricus, Rickettsia conori, Rickettsia australis, Rickettsia
akari, Rickettsia tsutsugamushi, Coxiella bumeti and Rochalimaea
quintana.
[0024] For the preparation of the control material of the
invention, the desired microorganism can be grown by standard
methods. For example, for the HIV virus, methods for growth are
disclosed in U.S. Pat. No. 5,135,864, which describes the
production and purification of HIV. As an alternative source,
microorganism can be isolated from infected biological fluids, such
as from the blood from an infected animal. The techniques are
similar to those used when purifying virus from cell culture. See
Davis et al., Microbiology, 2d Ed. (Harper & Row, 1980). Thus,
for example, hepatitis B and C viruses can be purified from the
blood of infected individuals by this method. In addition, bulk
production of the desired microorganism can be derived from a
chronically infected cell lines which are available from, for
example, the AIDS Research and Reference Reagent Program of the
National Institutes of Health (NIH) and the American Type Culture
Collection (ATCC).
[0025] Once sufficient culture material is available, the
microorganism can be purified using techniques known to those of
ordinary skill in the art. A typical method of purification is as
follows. The first step in purification of microorganisms involves
the removal of cells and cell debris. This can be achieved by
separation techniques based on size or mass, such as filtration or
low-speed centrifugation. Following this, the microorganism can be
concentrated by filtration using a suitable pore size or high speed
centrifugation to form a partially purified microorganism
preparation. The partially purified preparation can then be
subjected to ultracentrifugation and density gradient purification
techniques to obtain a purified microorganism preparation. The bulk
purified material obtained following purification is generally
stored at -70.degree. C. and may be tested for viral or parasitic
activity by culture techniques, and for nucleic acid integrity by
amplification techniques according to methods known in the art or
as described herein.
[0026] After purification, the microorganism is rendered
non-pathogenic according to the methods of this invention by
selective modification of its surface proteins whereby the
microorganism is rendered non-infectious while the nuclear
components are left substantially intact. Thus, the invention
provides a method for producing a nonpathogenic microorganism which
comprises providing a purified microorganism comprising surface
proteins and intact nuclear components and irreversibly modifying
one or more surface proteins while leaving the nuclear components
substantially unmodified, such that the microorganism is thereby
rendered non-pathogenic.
[0027] As used herein, the term "substantially intact" allows for
minimal contact of the microorganism's intracellular components
with the modifying agents so as to preserve enough of the nuclear
contents, in particular the nucleic acid content of the cell, from
degradation by the modifying reagents such that the nucleic acid is
amenable to the nucleic acid amplification techniques discussed
herein. As used herein, the term "non-pathogenic" means that as a
result of the modification of the surface proteins according to the
methods of the invention, the microorganism is not able to infect
cells, replicate or cause disease despite having its nuclear
contents substantially intact.
[0028] In a preferred embodiment of the invention, the purified
microorganism is modified by covalent attachment of a compound
comprising one or more reactive functional groups to one or more
reactive sites on the surface proteins, such that the microorganism
is thereby rendered non-pathogenic. As used herein, the "compounds"
used are those capable of covalently conjugating to a surface
protein or cross-link two or more surface proteins on the
microorganism. Surface proteins typically contain several reactive
sites at which covalent attachment of compounds and crosslinking
are feasible. For example, amine groups can be modified by
acylation; sulfhydryl groups can be modified by addition reactions
and alkylations; carbonyl and carboxyl groups can be modified by
acylation; and aldehyde and hydroxyl groups can be modified by
amination and reductive amination. One or more of these
modification reactions can be used in the preparation of the
non-pathogenic microorganisms of the invention.
[0029] According to this embodiment of the invention, surface
protein modification generally involves covalent binding to the
protein via reactions with exposed amino acid residues. Although
any type of covalent modification or crosslinking known to those
skilled in the art may be used, it is preferable to use covalent
modification or crosslinking of amino, sulfhydryl, and carboxylic
acid residues. For example, crosslinking agents that modify the
amine groups include gluteraldehyde and paraformaldehyde. Each of
these are readily attacked at pH>7 by the nucleophilic free
amines from either the protein terminus or by internal lysine side
residues. The resultant product is a Schiff's base with a free
amine of the protein. Gluteraldehyde is also attacked specifically
by these reactive amine residues and leads to a high degree of
cross linking.
[0030] Suitable reagents for modification of proteins through their
sulfhydryl residues are the irreversible sulfhydryl blocking agent
N-ethylmaleimide (NEM) or an n-substituted bismaleimide
crosslinker. NEM is an alkylating reagent that reacts with
sulfhydryl bonds forming a stable thioether bond. This reaction may
be carried out at pH 6.0 to prevent reaction with free amines.
Other sulfhydryl reactive reagents such as bismaleimidohexane (BMH)
may also be used. BMH is a homobifunctional crosslinker with a six
carbon spacer between active sites. This cross linker also forms a
stable thioether bond at pH 7.0.
[0031] In separately preferred embodiments, the chemical modifying
agents that can used include monofunctional crosslinkers, which
contain a single reactive functional group, such as an aldehyde
group. Examples of agents containing aldehyde functional groups
include, but are not limited to, formalin (formaldehyde),
acetaldehyde, propionaldehyde, n-butyraldehyde, benzaldehyde,
p-nitrobenzaldehyde, p-tolualdehyde, salicylaldehyde,
phenylacetaldehyde, 2-methylpentanal, 3-methylpentanal and
4-methylpentanal. Alternatively, the chemical modifying agents may
include functional groups such as NHS imidate, imidoester,
maleimide, chloroacetyl, fluoroacetal, iodoacetyl, bromoacetyl,
amine, hydrazide, carbodiimide, and derivatives of these groups. In
a preferred embodiment the chemical modifying agent will comprise
two or more of such reactive functional groups so as to facilitate
multiple covalent attachment with reactive sites on a single
surface proteins or crosslinking of separate surface proteins.
Compounds containing two same functional groups are referred to
herein as having homobifunctional structure and those containing
two different functional groups are referred to as having
heterobifunctional structure. Particularly preferred
homobifunctional reagents include dialdehydes such as
paraformaldehyde, glyoxal, malondialdehyde, succinialdehyde,
adipaldehyde, gluteraldehyde and phthaldehyde.
[0032] Several preferred cross linking and bifunctional conjugating
agents useful in the practice of this invention are commercially
available from Pierce Chemical Company and can be used according to
the methods described in the available product literature, the
contents of which are incorporated herein in their entirety to
describe the state of the art. Examples of such compounds and
general methods are as follows.
[0033] N-(.beta.-Maleimidopropionic acid) hydrazide (BMPH) (Pierce
Chemical Co., Product No. 22297). BMPH is a sulfhydryl-reactive and
carbonyl-reactive heterobifunctional reagent. The hydrazide group
can be covalently coupled to carbohydrate residues in glycoproteins
and other glycoconjugates after oxidation to produce aldehydes.
Sugar groups can be oxidized either by the use of specific oxidases
(such as galactose oxidase) or by the use of sodium periodate.
Treatment of glycoproteins with 1 mM sodium periodate at 0.degree.
C. oxidizes sialic acid groups to possess carbonyls. Reaction with
10 mM sodium periodate at room temperature (RT) will create
aldehydes on all sugars containing diols. The reaction of BMPH with
these aldehydes creates hydrazone linkages. The maleimide end of
the crosslinker can be reacted with sulfhydryl groups on surface
proteins. If sulfhydryls are not present, they may be created
through disulfide reduction or through thiolation with
2-iminothiolane or SATA. Maleimides react with sulfhydryl groups at
a pH of 6.5-7.5, forming stable thioether linkages. Maleimide
reaction is typically complete in 2 hours at RT or in about 4 hours
at 4.degree. C.
[0034] 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide Hydrochloride
(CAS #25952-53-8) (EDC) (Pierce Chemical Co. Product Nos. 22980,
22981), EDC reacts with a carboxyl group first and forms an
amine-reactive intermediate, an O-acylisourea. In two-step
conjugation procedures using aqueous solutions, stabilization of
the intermediate is achieved using N-hydroxysuccinimide. Reaction
with an amine will result in hydrolysis of the intermediate,
regeneration of the carboxyl, and release of an N-substituted urea.
A side reaction is the formation of an N-acylurea, which is usually
restricted to carboxyls located in hydrophobic regions of
proteins.
[0035] Imidoester Crosslinkers (Pierce Chemical Co. Product Nos.
20660, 20663, 21667, 21666, 20700, 20665). Homobifuctional
imidoesters possess two identical groups which can react with
primary amine groups to form stable covalent bonds. These include
dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl
suberimidate (DMS) and dimethyl 3,3'-dithiobisproprionimidate
(DTBP). Unlike other coupling chemistries, imidoesters have minimal
cross-reactivity toward other nucleophilic groups in proteins. In
mildly alkaline pH's (7-10), imidoesters react only with primary
amines to form imidoamides. Imidoester conjugation is usually
performed between pH 8.5-9.0. Imidoesters have an advantage over
other crosslinking reagents since they do not affect the overall
charge of the protein. They carry a positive charge at
physiological pH, as do the primary amines they replace. Imidoester
reactions are carried out at 0.degree. C. or room temperature
because elevated temperatures can contribute to poor yields with
these reactions. Homobifunctional imidoesters are available with
varying distances between the groups for different crosslinking
needs (e.g., to measure inter-residue distances of proteins and
macromolecular complexes and near neighbor relationships between
proteins). DTBP, a thiol-cleavable, homobifunctional crosslinker,
is used in conjunction with the non-cleavable forms of these
crosslinkers to study near neighbor relationships. DMP has been
used to crosslink antibodies to Protein A immobilized on an agarose
support.
[0036] N-hydroxysuccinimide Esters (NHS-esters) (Pierce Chemical
Co. Product Nos. 21555, 21580, 21655, 21658, 22311, 22312, 22416,
22317, 22309, 22324, 22307, 22308). Disuccinimidyl suberate (DSS)
is a water-insoluble, homobifunctional NHS-ester;
Bis(sulfosuccinimidyl) suberate (BS.sup.3) is its water-soluble
analog. These crosslinkers are non-cleavable and widely used for
conjugating radiolabeled ligands to cell surface receptors.
Additional examples include maleimide species such as
m-Maleimidobenzoyl-N-hydoxysuccinimide ester (MBS),
m-Maleimidobenzoyl-N-hydoxysuccinimide ester (sulfo-MBS),
succinimidyl 4-[p-maleimidophenyl]butyrate (SMBP),
sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (sulfo-SMBP),
N-[ymaleimidobutyryloxy]succinimide ester (GMBS),
N-[.gamma.maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS),
N-[.epsilon.-maleimidocaproyloxy]succinimide ester (EMCS), and
N-[.epsilon.maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS)
have maleimide groups.
[0037] Primary amines are principal targets for NHS-esters.
Accessible .alpha.-amine groups present on the N-termini of
peptides and proteins react with NHS-esters. However,
.alpha.-amines are seldom available on a protein, so the reaction
with side chains of amino acids becomes important. While five amino
acids have nitrogen in their side chains, only the .epsilon.-amine
of lysine reacts significantly with NHS-esters. A covalent amide
bond is formed when the NHS-ester conjugation reagent reacts with
primary amines. The reaction results in the release of
N-hydroxysuccinimide.
[0038] NHS-ester crosslinking reactions are most commonly performed
in phosphate, carbonate/bicarbonate, HEPES and borate buffers.
Other buffers can also be used provided they do not contain primary
amines. Primary amines are found in the structure of Tris, making
it an unacceptable buffer for NHS-ester reactions. A large excess
of Tris at neutral-to-basic pH can be added at the end of the
reaction to quench it. Glycine is a primary amine that can be used
in a similar manner.
[0039] NHS-esters can be broadly grouped into two separate classes
with essentially identical reactivity toward primary amines,
water-soluble and water-insoluble forms. Water-soluble NHS-ester
solubility is due to the sulfonate (--SO.sub.3--) group on the
N-hydroxysuccinimide ring. Sulfonated NHS-ester crosslinking
reagents are supplied as sodium salts and are soluble in water to a
concentration of 10 mM.
[0040] The non-sulfonated forms of N-hydroxysuccinimide ester
conjugation reagents are not water-soluble. These compounds are
first dissolved in an organic solvent then added to the aqueous
reaction mixture. Water-insoluble NHS-ester crosslinkers do not
possess a charged group. They are lipophilic and therefore
membrane-permeable. They are useful for intracellular and
intramembrane conjugation.
[0041] Water-insoluble NHS-esters can be dissolved in an organic
solvent such as DMSO or DMF. The cross linker solution is then
added to the aqueous reaction mixture so that the final volume
contains up to 10% organic solvent. Crosslinkers begin to fall out
of solution at high concentrations as noted by the appearance of a
milky, turbid solution. While crosslinking still may occur under
such conditions, the protocol may be modified to ensure complete
dissolution of the NHS-ester. For example, the aqueous phase can be
supplemented with additional organic solvents.
[0042] The maleimide group is most selective for sulfhydryl groups
when the pH of the reaction mixture is kept between 6.5 and 7.5. At
pH 7, the rate of reaction of maleimides with sulfhydryls is
1000-fold faster than with amines. Above this pH range, the
reaction rate with primary amines becomes more significant.
Maleimides do not react with tyrosines, histidines or methionines
as do iodoacetamides. A stable thioether linkage between the
maleimide group and the reacted sulfhydryl is formed, which cannot
be cleaved under physiological conditions. Hydrolysis of maleimides
to a nonreactive maleamic acid can compete with thiol modification,
especially above pH 8.0. Hydrolysis can occur prior or subsequent
to thiol conjugation.
[0043] .beta.-mercaptoethanol, dithiothreitol, mercaptoethylamine,
cysteine, and other thiol compounds must be removed prior to
coupling. Excess maleimides can be quenched at the end of the
incubation period by the addition of free thiols such as cysteine
or .beta.-mercapto-ethanol. EDTA can be included in coupling buffer
to prevent the reoxidation of disulfides.
[0044] 1,4-bis-Maleimidobutane (BMB) (Pierce Chemical Co. Product
No. 22331). BMB is an intermediate length sulfhydryl-reactive
homobifunctional crosslinker. The maleimide ends of the crosslinker
can be reacted with sulfhydryl groups on surface proteins. If
sulfhydryls are not present, they may be created through disulfide
reduction or through thiolation with 2-iminothiolane or SATA.
Maleimides react with --SH groups at a pH of 6.5-7.5, forming
stable thioether linkages. Maleimide reaction is complete in 2
hours at room temperature or about 4 hours at 4.degree. C.
[0045] Following reaction with the crosslinking or conjugating
agent, the reaction is quenched by using a suitable agent such as
one having the same active group as that contained in the reactive
group that is being covalently modified. For example, glycine may
be used for quenching crosslinking with paraformaldehyde.
[0046] The purified virus can also be inactivated by conjugation of
amino acids to carboxylic acid residues on the protein. An example
of a conjugating agent is sodium periodate which oxidizes
carbohydrate hydroxyl and terminal carboxylic acids on the protein
to form active aldehyde intermediates. This activated group is then
exposed to a nucleophile at elevated pH in the form of glycine or
lysine, which results in an irreversible conjugation of the amino
acid to the protein. Variation of coupling time, temperature, and
rocking speed to optimize the coupling protocols is well within the
purview of those skilled in the art.
[0047] In a separately preferred embodiment, the microorganism can
be rendered non-pathogenic by enzyme digestion of the surface
proteins by commercially available methods. Enzymes that can be
used for surface protein modification include, but are not limited
to, bromelin, chymotrypsin, clostripain, collagenase, elastase,
ficin, kallikrein, metalloendopeptidase, proteinase,
aminopeptidase, carboxypeptidase, factor Xa, papain, chymopapain,
pepsin, staphylococcus aureaus protease (V-8 strain), trypsin,
either alone or in combination. In this aspect of the invention,
the microorganisms are reacted with enzyme preparations in a
reaction mixture under conditions and for a period of time
sufficient to at least partially digest the surface proteins of the
microorganism to render it non-pathogenic, yet retain the nucleic
acid content of the microorganism substantially intact. Methods of
preparing enzyme solutions for such reactions and methods of
carrying out the reactions are known to those of ordinary skill in
the art and guidance can be found in such texts as The Worthington
Enzyme Manual (Worthington Biochemical Corporation).
[0048] Choice of any particular enzyme may depend on such factors
as availability, ease of use and substrate specificity, each of
which can be assessed by those of ordinary skill in the art.
Reactions can be controlled by length of time the microorganism is
exposed to the enzyme or by quenching reactions with enzyme
inhibitors. For example, Papain, and similarly chymopapain,
hydrozyzes a number of peptide and ester bonds and is activated for
example by cysteine, sulfide, and sulfite and is inhibited by
addition of reagents such as sulfhydryl reagents including heavy
metals, carbonyl reagents, and ascorbic acid. Chymotrypsin, an
endopeptidase, readily acts on amides and esters of susceptible
amino acids, has specificity for bonds involving aromatic amino
acids, and catalyzes hydrolysis of bonds of leucyl, methionyl,
asparaginyl, and glutamyl residues. The enzyme is inhibited for
example by heavy metals, and organophosphorus compounds. Trypsin, a
proteolytic enzyme, catalyzes the hydrolysis of peptide bonds
between carboxy group of arginine or lysine and the amino group of
another amino acid, and is inhibited for example by
organophosphorus compounds, benzyl 4-guanidiobenzoate, and
4'-nitrobenzyl 4-guanidinobenzoate. Pepsin is an endopeptidase that
catalyzes the hydrolysis of a variety of peptide bonds and is
inhibited by phenylacyl bromides, aliphatic alcohols, and
diphenyldiazomethane.
[0049] The virus material that has been inactivated by the above
procedure is separated from the other materials present in the
final reaction mixture. This may be achieved by standard techniques
of purification including, but not limited to, dialysis, gel
filtration and tangential filtration. The final purified,
inactivated preparations may be tested for the presence of active
viruses by methods known to those of ordinary skill in the art. A
convenient method of testing for active viruses is to test for the
ability of the virus to replicate. For HIV, for example, this may
be done by monitoring the production of HIV p24 antigen in culture
media. The purified, inactivated microorganisms of the present
invention can be stored at nonfrozen temperatures, such as from
2-8.degree. C.
[0050] The invention further provides a composition of matter
comprising purified microorganism comprising surface proteins and
intact nuclear components, wherein one or more surface proteins
have been irreversibly modified such that the microorganism is
thereby rendered non-pathogenic, and a liquid matrix. Thus, when
prepared as a positive control material, the purified, inactivated
microorganism is preferably suspended in a liquid matrix comprising
stabilized biological fluids which correspond to fluids from which
biological samples will be analyzed. Such fluids include, but are
not limited to, serum, plasma, defibrinated plasma, stabilized
plasma pool, cerebral spinal fluid (CSF), urine, saliva, semen, and
sputum. Alternatively the liquid matrix can comprise synthetic
matrices formulated to simulate such biological fluids. Methods of
preparing synthetic biological fluids are well known in the art.
Further, the liquid matrix can contain additives such as
antioxidants, buffer salts, preservatives, antibiotics, and matrix
stabilizing fillers such as sugars (monosaccharides and
polysaccharides), proteins (including albumin, ovalbumin, gamma
globulin, red blood cell lysates, casein, dry powdered milk, and/or
other serum proteins), and synthetic stabilizers such as
poly-vinylpyrrolidine, poly-1-lysine, and methylated Bovine Serum
Albumin (BSA). The liquid matrix can also be modified for
lyophilization for long term storage and stability by addition of,
for example, sucrose and mannose.
[0051] In addition, this invention provides a kit for analyzing a
biological sample for the presence of a microorganism having
surface proteins, wherein the kit comprises a positive control
composition comprising a purified sample of said microorganism
comprising surface proteins and substantially intact nuclear
components, wherein one or more surface proteins have been
irreversibly modified such that the microorganism is thereby
rendered non-pathogenic. In a preferred embodiment, the kit
comprises the non-pathogenic microorganism control in a liquid
matrix as described above. In the practice of the invention, the
kit can comprise additional materials necessary for conducting
nucleic acid amplification techniques known to those of ordinary
skill in the art. Further, the invention is intended to encompass
the addition of the non-pathogenic microorganism of the invention
to existing kits and services used in nucleic acid amplification
techniques. Such kits and services include, but are not limited to,
those marketed by Roche Diagnostics (Indianapolis, Ind.) under the
COBAS AMPLICOR tradename and the NAT screening services marketed by
National Genetics Institute, Inc. (Los Angeles, Calif.) Bayer
Corporation (Tarrytown, N.Y.) and Gen-Probe, Inc. (San Diego,
Calif.).
[0052] The non-infectious, inactivated control material of the
present invention serve as a positive control for the entire
process of nucleic acid amplification techniques. For example, the
control material of the present invention comprising inactivated
HIV virus can be processed through the steps of (1) sample
preparation (chaotrophic salt/solvent extraction), (2) reverse
transcription of RNA to produce cDNA if necessary, (3)
amplification of the nucleic acids and (4) detection of amplified
nucleic acids.
[0053] Thus the invention also provides a method for detection of a
microorganism comprising surface proteins in a biological sample by
amplification of nuclear components of said microorganism, which
method comprises amplification of the nuclear components of a
purified control material of this invention. In a preferred
embodiment, the method comprises the steps (a) preparing a sample
by addition of the non-pathogenic microorganism control of the
invention to a biological sample to be tested for the presence of a
corresponding microorganism, (b) extracting target nucleic acid to
be amplified, (c) amplifying target nucleic acid, (d) hybridizing
the amplified target nucleic acid with detectably labeled nucleic
acid probes, and (e) detecting the hybridized amplified target
nucleic acid. Such individual method steps are well known to those
of ordinary skill in the art. For example, amplification of target
nucleic acid can be accomplished by polymerase chain reaction (PCR)
on DNA or on RNA after reverse transcription of the RNA to cDNA.
Hybridization and detection can be accomplished by use of, for
example, alkaline phosphatase-labeled nucleic acid probes, or by
detection of amplicons with energy transfer methodology. In a
particularly preferred embodiment, the method can further comprises
quantification of the target nucleic acid contained in the
biological sample.
[0054] In the practice of the invention, the method can be used as
a screening method, such as to screen biological fluids prior to
transfusion or transplantation, a diagnostic tool, such as where
biological fluids from individuals suspected of harboring
pathogenic microorganisms are analyzed for the presence of such
microorganisms, or as a therapy monitoring tool, such as where
biological samples from a patient undergoing treatment for
infection is analyzed for presence and amount of microorganism.
[0055] The various embodiments and advantages of the present
invention will be better understood from the examples presented
below, which are intended to be illustrative and not
restrictive.
Example 1
[0056] This example describes the large scale manufacture of
purified HIV particles. A chronically infected cell line,
designated BP-1, was used to provide the virus. This cell line is a
high producing variant of the original H-9 cell line and was
provided by Dr. Bernard Poiesz. The cell line was maintained in
RPMI-1640 medium supplemented with FBS and antibiotics (penicillin
and streptomycin). Master Cell Banks (MCB) and Working Cell Banks
(WCB) of each cell line were established and maintained according
to accepted USFDA guidelines for manufacture of biologics.
Typically, a MCB has 30 to 50 vials of frozen cells. A vial of the
MCB is thawed, grown in culture and used to prepare 30 to 50 vials
of frozen cells that constitute the WCB. MCB and WCB are stored in
secure liquid nitrogen containers and logs are maintained to ensure
traceability.
[0057] For HIV manufacturing, a WCB vial of a cell line was thawed
and expanded in culture containing RPMI-1640, fetal bovine serum
(PBS) and antibiotics. Initially, the cells were grown in static
cell culture flasks until a volume of 1 liter was reached. At this
point the cells were transferred to 2 liter roller baffles,
containing 1 liter of culture fluid per bottle, and were expanded
further to reach a production scale of 60 to 70 roller bottles.
Roller bottle cultures were maintained according to written
Standard Operating Procedures and routine testing was done to check
for mycoplasma and other adventitious agents.
[0058] Once the cultures reached production scale, the roller
bottles were harvested once per week. Typically, 90% of each
culture was harvested, however, this may vary slightly according to
cell density at the time of harvest. Harvested cell cultures were
processed in a dual flow path tangential flow system consisting of
two filtration loops. The first loop used a 10 square foot 0.45 um
membrane to remove cells and cell debris and the second flow path
uses a 10 square foot 300 Kd cut off membrane to concentrate the
virus containing culture supernatants. The system was washed twice
with tris buffered saline and virus containing supernatants were
concentrated to approximately 2 liters.
[0059] Virus was isolated from concentrated supernatants by
ultracentrifugation (30,000.times.g). Pelleted virions were
resuspended in 50 ml of tris buffered saline. This material was
then loaded onto a 1.2 liter 22-66% linear sucrose gradient and
virus was purified by density gradient ultracentrifugation. Virion
containing gradient fractions were identified by refractive index
and pooled fractions were diluted in tris buffered saline and
virions were again pelleted. Virions were then resuspended in
phosphate buffered saline.
[0060] All manufacturing activities involving growth and
purification of viable viruses were conducted in Biological Level 3
(BL-3) laboratories. Liquid wastes generated by these procedures
were treated with hypoclorite or activated iodine before discharge
into municipal sewage. Solid wastes were treated by autoclaving
before being removed from the BL-3 laboratories.
Example 2
[0061] This example describes one method of inactivation of the
purified virus according to the invention by crosslinking of
surface proteins. As an illustration, the crosslinking of virion
envelope proteins with paraformaldehyde, a commonly used fixative
for tissue and cell preparation, was tested. Titrations of the
fixative were preformed above and below the concentration
recommended by the CDC for inactivation of virus in biological
fluids (May 8, 1992/41 (RR-8); 001 CDC Guidelines for the
Performance of CD4+ T-Cell Determinations in Persons with Human
Immunodeficiency Virus Infection). Initially, purified virus was
slowly thawed at 2-8.degree. C. for 6-8 hours to minimize lysis of
the virus. Next, while on ice, virus was diluted with cold PBS to a
final concentration of approximately 1.times.10.sup.10 copies/mL
(cp/mL). Diluted virus was then divided into four different 5 ml
aliquots and freshly prepared paraformaldehyde was added to each at
a final concentration of either 0.625, 1.25, 2.5, or 5%. The
reaction mixtures were then incubated at 2-8.degree. C. for 60
minutes while gently rocking. To quench the reaction, 1 mL of 0.5 M
glycine (pH 7.4) was added and allowed to react with any residual
paraformaldehyde. After the addition of glycine the reactions were
gently rocked for 2 hr. at 2-8.degree. C. To remove glycine and any
excess unquenched fixative each reaction mixture was then dialyzed
(10-14K m.w. cutoff) against 1 L PBS (pH 7.4, 2-8.degree. C.) for 4
changes. Dialyzed virus was then transferred into a 15 mL tube and
centrifuged for 10 min. at 3,000.times.g to pellet any precipitated
virus. A sample of each supernatant was then tested in cell culture
for the presence of infectious virus.
[0062] The remaining inactivated virus was titrated into a
stabilized plasma matrix that consisted of the following: human
source plasma collected in 4% Sodium Citrate, 1 mL EDTA, 1 U/mL
Ribonuclease Inhibitor (Human Placenta), 0.09% NaN.sub.3, 1 mM
Dithiothreitol, 0.05% Gentamicin, in Phosphate Buffered Saline pH
7.4. In order to optimize the matrix for other viruses, one or more
of the additives can be deleted.
Example 3
[0063] The modified virus produced as described in Example 2 was
tested to ensure the process inactivated the virus. As an
illustration, the CEM cell line, obtained from the American Type
Culture Collection was used as a host cell for viral infectivity
studies. Advantages of using this line are that establishment of in
vitro HIV infection requires relatively few virions and that the
infection is chronic rather than lytic, facilitating analysis. For
these studies, infection is detected and followed using the
ZeptoMetrix HIV p24 Antigen EIA (ZeptoMetrix, Buffalo N.Y.).
[0064] CEM cells are cultured in RPMI-1640 containing 10% FBS in 24
well plates Cells are plated at 10.sup.5 cells per well in a 1 ml
volume and 100 ul of various dilutions of virus, either treated or
untreated, are added to individual wells. Cultures are fed twice
weekly by 50% media replacement and culture supernatants are
assayed for HIV p24. All cultures initially contain HIV p24.
However, in cultures where virus is noninfectious HIV p24 levels
decrease over time eventually reaching background levels. Cultures
containing infectious virions exhibit increasing levels of HIV p24
over time allowing easy discrimination of cultures containing
infectious versus noninfectious virus. Thus, by comparing the
results obtained using different dilutions of virus, one can
estimate how many logs of infectious HIV have been inactivated by a
given treatment procedure.
[0065] Table 1 summarizes data from experiments where purified HIV
was treated with different concentrations of paraformaldehyde for
60 minutes. Here, HIV was aliquoted and treated with 0.625%, 1.25%,
2.5% or 5.0% paraformaldehyde. Different dilutions (1:8,000,
1:80,000, and 1:800,000) of treated virus were then placed into
cultures of CEM cells and p24 assays were performed on culture
supernatants on days 3, 7, 10, and 14 post-inoculation. Data are
expressed as pg/ml of HIV p24 protein in the culture supernatants.
The standard curve for the p24 assay is linear from 7.8 pg/ml to
125 pg/ml. HIV p24 concentrations below 7.8 pg/ml are expressed as
"<7.8 pg/ml" to indicate sensitivity of the assay, and values
above 125 pg/ml are expressed as ">125 pg/ml" since the assay is
nonlinear above 125 pg/ml.
TABLE-US-00001 TABLE 1A Virus Inactivation Using Different
Concentrations of Paraformaldehyde 3 Days Post-Inoculation Final
Dilution of Treated HIV in Culture % Paraformaldehyde 1:8,000
1:80,000 1:800,000 0 332 pg/ml 209 pg/ml 212 pg/ml 0.625 71.8 pg/ml
<7.8 pg/ml <7.8 pg/ml 1.25 41.9 pg/ml <7.8 pg/ml <7.8
pg/ml 2.5 <7.8 pg/ml <7.8 pg/ml <7.8 pg/ml 5.0 <7.8
pg/ml <7.8 pg/ml <7.8 pg/ml
TABLE-US-00002 TABLE 1B Virus Inactivation Using Different
Concentrations of Paraformaldehyde 7 Days Post-Inoculation % Final
Dilution of Treated HIV in Culture Paraformaldehyde 1:8,000
1:80,000 1:800,000 0 287 pg/ml 266 pg/ml 128 pg/ml 0.625 109 pg/ml
<7.8 pg/ml <7.8 pg/ml 1.25 34.2 pg/ml <7.8 pg/ml <7.8
pg/ml 2.5 <7.8 pg/ml <7.8 pg/ml <7.8 pg/ml 5.0 <7.8
pg/ml <7.8 pg/ml <7.8 pg/ml
TABLE-US-00003 TABLE 1C Virus Inactivation Using Different
Concentrations of Paraformaldehyde 10 Days Post-Inoculation % Final
Dilution of Treated HIV in Culture Paraformaldehyde 1:8,000
1:80,000 1:800,000 0 368 pg/ml 278 pg/ml .sup. 32 pg/ml 0.625 22.9
pg/ml <7.8 pg/ml <7.8 pg/ml 1.25 <7.8 pg/ml <7.8 pg/ml
<7.8 pg/ml 2.5 <7.8 pg/ml <7.8 pg/ml <7.8 pg/ml 5.0
<7.8 pg/ml <7.8 pg/ml <7.8 pg/ml
TABLE-US-00004 TABLE 1D Virus Inactivation Using Different
Concentrations of Paraformaldehyde 14 Days Post-Inoculation % Final
Dilution of Treated HIV in Culture Paraformaldehyde 1:8,000
1:80,000 1:800,000 0 276 pg/ml 275 pg/ml .sup. 52 pg/ml 0.625
<7.8 pg/ml <7.8 pg/ml <7.8 pg/ml 1.25 <7.8 pg/ml
<7.8 pg/ml <7.8 pg/ml 2.5 <7.8 pg/ml <7.8 pg/ml <7.8
pg/ml 5.0 <7.8 pg/ml <7.8 pg/ml <7.8 pg/ml
Untreated virus grew in all cultures at all dilutions tested as
evidenced by the presence of HIV p24 in all cultures during the 14
day period. On the other hand, treatment of virus with
paraformaldehyde resulted in non-detectable amounts of p24 in the
culture supernatants by day 14. Levels of the HIV p24 Ag that could
be detected on days 3, 7 and 10 were due to addition of inactivated
virus to the cultures as evidenced by a progressive decline in
these levels over the 14 day period. Table 2 shows the effects of
inactivation methods of this invention according to the exposure
time in minutes that the virus is incubated with the
crosslinker.
TABLE-US-00005 TABLE 2A Virus Inactivation at Different Exposure
Times 3 Days Post-Inoculation Exposure Final Dilution of Treated
HIV in Culture Time 1:8,000 1:80,000 1:800,000 0 >125 pg/ml
>125 pg/ml >125 pg/ml 15 minutes >125 pg/ml 27.9 pg/ml
<7.8 pg/ml 30 minutes >125 pg/ml 22.6 pg/ml <7.8 pg/ml 60
minutes >125 pg/ml 59.8 pg/ml <7.8 pg/ml Overnight >125
pg/ml 16.3 pg/ml <7.8 pg/ml
TABLE-US-00006 TABLE 2B Virus Inactivation at Different Exposure
Times 7 Days Post-Inoculation Exposure Final Dilution of Treated
HIV in Culture Time 1:8,000 1:80,000 1:800,000 0 >125 pg/ml
>125 pg/ml >125 pg/ml 15 minutes >125 pg/ml 18.0 pg/ml
<7.8 pg/ml 30 minutes >125 pg/ml 10.3 pg/ml <7.8 pg/ml 60
minutes >125 pg/ml 36.0 pg/ml <7.8 pg/ml Overnight >125
pg/ml 17.5 pg/ml <7.8 pg/ml
TABLE-US-00007 TABLE 2C Virus Inactivation at Different Exposure
Times 10 Days Post-Inoculation Exposure Final Dilution of Treated
HIV in Culture Time 1:8,000 1:80,000 1:800,000 0 >125 pg/ml
>125 pg/ml 32 pg/ml 15 minutes 43.9 pg/ml <7.8 pg/ml <7.8
pg/ml 30 minutes 46.2 pg/ml <7.8 pg/ml <7.8 pg/ml 60 minutes
87.4 pg/ml <7.8 pg/ml <7.8 pg/ml Overnight 27.6 pg/ml <7.8
pg/ml <7.8 pg/ml
TABLE-US-00008 TABLE 2D Virus Inactivation at Different Exposure
Times 14 Days Post-Inoculation Exposure Final Dilution of Treated
HIV in Culture Time 1:8,000 1:80,000 1:800,000 0 >125 pg/ml
>125 pg/ml 52 pg/ml 15 minutes <7.8 pg/ml <7.8 pg/ml
<7.8 pg/ml 30 minutes <7.8 pg/ml <7.8 pg/ml <7.8 pg/ml
60 minutes <7.8 pg/ml <7.8 pg/ml <7.8 pg/ml Overnight
<7.8 pg/ml <7.8 pg/ml <7.8 pg/ml
Example 4
[0066] This example provides a method of preparing the control
material of the invention by enzyme digestion of surface proteins.
Purified virus (for example, produced as in Example 1) is incubated
for approximately 2 hours at 37.degree. C. in a mixture comprising
0.25% bovine trypsin and 0.1% EDTA in Hank's Buffered Saline
Solution. At the end of the incubation, the reaction is stopped by
adding an equal volume of fetal bovine serum. The material is then
purified according to known techniques and non-pathogenicity is
confirmed by methods as set out in Example 3.
Example 5
[0067] This example demonstrates that the viral nucleic acids are
intact so as to be amenable to amplification following the
inactivation procedures described in Example 3. As an illustration,
experiments were conducted to examine whether chemically
inactivated HIV could still be detected by PCR. The COBAS
AmpliScreen HIV-1 Monitor assay (Roche), a PCR based clinical kit
that quantitates HIV RNA copy number, was used. These experiments
were performed by titrating material from the inactivation
reactions described in Example 3 into a stabilized plasma
formulation and then dispensing it into DNAse, RNAse, and pyrogen
free PCR tubes (Chasma Scientific, Cambridge, Mass.). Quantitation
of HIV RNA copy number was determined on samples of the deep frozen
virus using the Roche COBAS assay. Samples were diluted 1:1000 and
data is expressed as the copy number at this dilution. This data is
summarized in Table 3 below.
TABLE-US-00009 TABLE 3 Paraformaldehyde conc. HIV RNA copy no.
0.63% 325,000 1.25% 48,000 2.50% 12,678 5.00% 7,192
Example 6
[0068] Several crosslinking reagents were tested for their efficacy
in inactivating HIV at various concentrations. Table 4 lists the
materials used in the experiments, their concentration, volume,
molecular weight and mass.
TABLE-US-00010 TABLE 4 Volume Molecular Materials Concentration
(mL) Weight Mass DMA-PBS 20 mM 10 245.15 49 mg BMB-DMSO 20 mM 10
248.23 49.6 mg BMPH-PBS 40 mM 2 297.19 23.7 mg EDC-MES 10 10 g
Sodium Periodate 100 mM 10 213.9 213 mg Desalting columns PD-10 MES
Buffer As per packet PBS Buffer as per packet Glycine 0.5M 10 75.07
375 mg DMSO Neat DMF Neat
[0069] To prepare the HIV buffer pre dilutions, the live HIV is
thawed and set on ice. In one dilution, 0.5 mL of HIV is added to
20 mL of PBS buffer, then gently mixed and stored on ice. In
another dilution, 0.5 mL of HIV is added to 20 mL of MES buffer,
then gently mixed and stored on ice.
[0070] For the crosslinking reaction, HIV was combined with one of
the crosslinking reagents and/or diluent (PBS or MES) as per Tables
5 (for DMA samples) and 6 (for BMB samples) and mixed gently at
room temperature (x,y,z rotator) for 2 hours. The reaction was
quenched by addition of 0.5 mL (0.5 M) of glycine, where indicated
in Tables 5 and 6, for 1 hour at RT. Then 2.4 mL reaction mixture
was added to PD-10 Desalting column (preequilibrated with saline).
The virus was eluted with 3.5 mL of saline.
TABLE-US-00011 TABLE 5 DMA Diluted Glycine DMA Conc. Volume HIV
Virus Quench Sample PBS mL mL mL 1 0.02 mM 1 1 0.5 2 0.2 mM 1 1 0.5
3 2 mM 1 1 0.5 4 20 mM 1 1 0.5
TABLE-US-00012 TABLE 6 DMA Conc. BMB Glycine in DMSO Volume Diluent
PBS Diluted HIV Quench Sample (mM) (uL) (uL) (mL) mL 5 1 200 800 1
0.5 6 2.5 200 800 1 0.5 7 5 200 800 1 0.5 8 20 200 800 1 0.5
[0071] For cross linking with BMPH, the HIV was first pre-treated
with Na Periodate to oxidize carbohydrates. Na Periodate and HIV
are combined and incubated in the dark for 30 minutes. To crosslink
with BMPH, BMPH is added to the pre-treated HIV and mixed gently at
RT for 2 hours. The reaction was quenched by addition of 0.5 rnL
(0.5 M) of glycine. Incubate for 1 hour at RT. Add 2.5 mL of
reaction mixture to Desalting column, eluted with 3.5 rnL of
saline, then aliquoted into 0.5 rnL fractions. HIV was diluted 1:10
with defibrinated plasma pool. Table 7 lists the materials and
quantities tested.
TABLE-US-00013 TABLE 7 Na Periodate Diluted HIV BMPH Glycine (100
mM) Virus BMPH PBS Volume Quench Sample (uL) (mL) (mM) (mL) mL 9
100 1 2 0.9 0.5 10 100 1 10 0.9 0.5 11 100 1 20 0.9 0.5 12 100 1 40
0.9 0.5
[0072] For crosslinking with EDC, HIV is combined with glycine and
mixed for 2 minutes. EDC is added to water, titrated, then 900 uL
was added to the reaction mixture, and incubated for 2 hours at RT.
The reaction mixture was purified over desalting columns, eluted
with 3.5 mL of saline, then aliquoted into 0.5 mL fractions. HIV
was diluted 1:10 with defibrinated plasma pool. Table 8 lists the
materials and quantities tested.
TABLE-US-00014 TABLE 8 Diluted HIV EDC H20 EDC Volume Sample
Glycine 0.5M (mL) (mg) (mL) 13 100 uL 1 0.1 0.9 14 100 uL 1 1 0.9
15 100 uL 1 10 0.9 16 100 uL 1 100 0.9
[0073] The control materials thus produced were tested in the Roche
Cobas HIV-1 Monitor Assay described in Example 5. Results are
described in Table 9.
TABLE-US-00015 TABLE 9 Roche Coupling Monitor Functional Chemical
HIV RNA Sample Coupling Group Reactive Protein Group Concentration
cp/mL ID# Reagent Structure Group Reactivity mM (1:10 in *NHP) 1
DMA Ho I --NH.sub.2 (Amine) 0.02 >7.5 .times. 105 2 DMA Ho I
--NH.sub.2 (Amine) 0.2 >7.5 .times. 105 3 DMA Ho I --NH.sub.2
(Amine) 2 >7.5 .times. 105 4 DMA Ho I --NH.sub.2 (Amine) 20
>7.5 .times. 105 5 BMB Ho M SH 1 Negative 6 BMB Ho M SH 2.5
Negative 7 BMB Ho M SH 5 Negative 8 BMB Ho M SH 20 1.6 .times. 105
9 BMPH He M + H SH + CO 2 >7.5 .times. 105 10 BMPH He M + H SH +
CO 10 1.29 .times. 104 11 BMPH He M + H SH + CO 20 3.61 .times. 103
12 BMPH He M + H SH + CO 40 5.58 .times. 102 13 EDC Ho C -COOH +
NH2 groups 0.1 (mg/mL) >7.5 .times. 105 14 EDC Ho C -COOH + NH2
groups 1 (mg/mL) >7.5 .times. 105 15 EDC Ho C -COOH + NH2 groups
10 (mg/mL) >7.5 .times. 105 16 EDC Ho C -COOH + NH2 groups 100
(mg/mL) >7.5 .times. 105 *Roche Cobas HIV-1 Monitor Assay (Range
400-750,000 cp/mL) **NHP = defibrinated normal human plasma
He-Heterobifunctional Ho-homobifunctional I-imidoester M-maleimide
H-hydrizide C-carbodiimide SH-sulfhydryl CO-carbonyl (aldehyde)
Example 7
Inactivation of Hepatitis B Virus
[0074] In this example the method of the invention is applied to
inactivation of the hepatitis B virus using paraformaldehyde.
Purified virus, prepared as described herein, was inactivated using
the method described in Example 2. Duck hepatitis B virus (DHBV)
was employed as surrogate to human hepatitis B virus (HHBV) in HBV
inactivation studies as ethical concerns, availability and cost
limit the use of chimpanzee in HBV inactivation studies. This model
uses primary duck hepatocytes (PDHs) as host (Pugh et al., 1999).
We isolated and maintained PDHs from less than one week-old
seronegative White Pekin ducklings (Anas dornesticus) with
modifications from previous procedures (Berry and Friend, 1969,
Seglen. 1976, Pugh and Summers, 1989, Pugh et al., 2000, Tuttleman
et al., 1986). Briefly, ducks were euthanized by exposing them to
C0.sub.2 and livers were perfused twice via the atrium of the heart
with approximately 200 mL each of two solutions. The first solution
consisted of SWIM's S-77 medium (Sigma) supplemented with 0.5
mmol/L EGTA, 2 mmol/L Hepes (pH 7.45) and 50 ug/mL penicillin and
streptomycin (Biofluids, Rockville, Md.). The second solution
contained SWIM's S-77 medium supplemented with 2.5 mmol/L
CaCl.sub.2, 0.5 mg of collagenase/mL (type I, Sigma, St. Louis) and
50 ug/mL penicillin and streptomycin. These perfusates were
administered at approximately 15 mL/minute using a peristaltic pump
and the temperature of the perftisate was adjusted to maintain the
liver at 37.degree. C. The liver was removed, minced with scissors
and repeatedly mixed with a 25 mL pipette for hepatocyte dispersion
into complete L-15 medium (Biofluids, Rockville, Md.) supplemented
with 0.05-percent (w/v) sodium bicarbonate (Biofluids), 1 mmol/L
glutamine (Biofluids), 20 mmol/L HEPES (Biofluids), 50 ug/mL
penicillin and streptomycin (Biofluids), I (g/mL insulin (Sigma,
St. Louis), and 10 (moi/L hydrocortisone-hemisuccinate (Sigma).
Cells were filtered through sterile gauze, centrifuged at
50.times.g for 4 minutes, and the resulting cell pellet was washed
three times with L-15 medium and resuspended in L-15 medium.
12-well plates were seeded with approximately two-mL of the cell
suspension (ca. 105 cells). Media on the plated cells were
aspirated every two days and replaced with fresh L-15 media.
[0075] Serum of four week-old congenitally infected duckling served
as the source of virus used in these experiments. Treated and
control DHBV spiked red cell samples were serially diluted 10-fold
in L-15 medium and one-mL volumes were subsequently inoculated into
PDH monolayers in quadruplicate. The infected cultures were
incubated at 37.degree. C.+20.degree. C. with 5+1% C0.sub.2
overnight for virus attachment and entry. The inoculum was then
removed, cell monolayers washed once with complete L-15 medium to
remove excess red blood cells, and then each well was overlaid with
approximately 2-mL of fresh L-15 medium. Infected monolayers were
incubated an additional 10-14 days at 37(+20.degree. C. with 5+1%
C0.sub.2, with media changes every 2 days.
[0076] We detected the presence of DHB V-infected duck hepatocytes
by an IFA. Briefly, the medium from the PDH monolayer was removed
by aspiration, and the monolayers washed with agitation for 5-10
minutes with 1-2 mL of phosphate buffered saline (PBS). The wash
solution was then removed by aspiration and replaced with 1-2 mL of
-20+2.degree. C. ethanol. The ethanol overlaid samples were fixed
for 2-48 hours at 4+1.degree. C. prior to removal of the ethanol,
and subsequent to a 1-2 mL PBS wash, agitated monolayers were
incubated at room temperature for at least 2 hours with 0.25 mL of
a 1:2 dilution of anti-DHBV monoclonal antibody directed against
pre-S domain of DHVB envelope (1 H. 1) (Pugh et al., 1995) in PBS
containing 0.5% fetal bovine serum to each well. The antibody
solution was removed by aspiration, and the primary antibody
stained monolayer washed with PBS. Following removal of the wash,
0.25 mL of a 1:200 dilution of goat anti-Mouse 1 gG-FITC
(fluorescein isothiocyanate conjugate) (Jackson ImmnuroResearch
Laboratories, Inc., West Grove. Pa.), in PBS containing 0.5% fetal
bovine serum was added to each well and incubated with agitation
for 2 hours at room temperature. The secondary antibodies were
removed by aspiration, and the flourescently-stained monolayer
washed with PBS. Following removal of the PBS, inverted wells were
examined by UV light microscopy using a Nikon Diaphot microscope,
arid monolayers that contained one or more DHBV surface antigen
positive hepatocytes were scored positive. Virus titers were
determined by scouring flourescence focus-forming unit dose 50
using Reed and Muench method (1938).
[0077] As shown in Table 10, no virus infected cells were detected
in any of the samples tested.
TABLE-US-00016 TABLE 10 Effect of paraformaldehyde on inactivation
of duck hepatitis B virus (DHBV) in suspension for 60 minutes at
ambient room temperature (20.degree. C.). Paraformaldehyde/FFFUs
observed in dilutions assayed Dilution Input virus 1 2 3 4
10.sup.-1 ++++ ---- ---- ---- ---- 10.sup.-2 ++++ ---- ---- ----
---- 10.sup.-3 ++++ ---- ---- ---- ---- 10.sup.-4 ++++ ---- ----
---- ---- 10.sup.-5 ++++ ---- ---- ---- ---- 10.sup.-6 ++-- ----
---- ---- ---- 10.sup.-7 ---- ---- ---- ---- ---- *FFFUD.sub.50/mL
10.sup.6.00 0 0 0 0 (log.sub.10) *FFFUD.sub.50/ML = Fluorescence
focus forming unit dose fifty as determined by Reed and Muench
(1938). (+) presence or (-) absence of FFFU. +, DHBV infected cells
detected; -, no DHBV infected cells detected.
Example 8
Inactivation of Bovine Viral Diarrhea Virus (BVDV)
[0078] In this example bovine viral diarrhea virus (BVDV), biotype
1 (CPE), genotype 1 Host: Madin-Darby bovine kidney (MDBK) cells
were inactivated according to the methods discussed in Example
2.
[0079] Bovine viral diarrhea virus (BVDV), biotype 1 (CPE),
genotype 1 was used in these studies which infect Madin-Darby
bovine kidney (MDBK) cells. The procedure for assaying infectious
BVDV was the same as described for DHBV (see above) with the
following differences: post-treatment of both test and control BVDV
samples with 2.5% paraformaldehyde, the reaction was quenched with
0.5 M glycine. These samples (0.5 mL) were loaded into pre-spun
sephacryl columns. The columns were spun for 4 minutes at 1000 rpm.
The samples were aseptically removed from the columns and ten-fold
serial dilutions were then prepared in minimal essential medium
(MEM) and adsorbed on the monolayer of host cells seeded in tissue
culture plates for one hour at 37.+-.2.degree. C. and 5.+-.1%
C0.sub.2. Post-adsorption, the monolayers were washed with EBSS and
replaced with fresh MEM and incubated for 5-7 days at
37.+-.2.degree. C. and 5.+-.1% C0.sub.2. The presence of infectious
viruses was determined as described below: post-incubation, the
plates were washed with 3.times. with PBS and fixed with TC grade
alcohol and stained with direct porcine polyclonal anti-BVDV
conjugated antibody. The stained plate were scored using UV
microscopy using a Nikon Diaphot microscope, and monolayers that
contained one or more BVDV surface antigen positive hepatocytes
were scored positive. Four wells per dilution were inoculated and
results were recorded as the FFFUD.sub.50 calculated by Reed and
Munch (1938).
[0080] As shown in Table 11, no virus infected cells were detected
in any of the samples tested.
TABLE-US-00017 TABLE 11 Effect of paraformaldehyde on inactivation
of bovine viral diarrhea virus (BVDV) in suspension for 60 minutes
at ambient room temperature (20.degree. C.). Paraformaldehyde/FFFUs
observed in dilutions assayed Dilution Input virus 1 2 3 4
10.sup.-1 ++++ ---- ---- ---- ---- 10.sup.-2 ++++ ---- ---- ----
---- 10.sup.-3 ++++ ---- ---- ---- ---- 10.sup.-4 ++++ ---- ----
---- ---- 10.sup.-5 ++++ ---- ---- ---- ---- 10.sup.-6 ++-- ----
---- ---- ---- 10.sup.-7 ---- ---- ---- ---- ---- *FFFUD.sub.50/mL
10.sup.6.77 0 0 0 0 (log.sub.10) *FFFUD.sub.50/ML = Fluorescence
focus forming unit dose fifty as determined by Reed and Muench
(1938). (+) presence or (-) absence of FFFU. Table Footnote: +,
BVDV infected cells detected; -, no BVDV infected cells
detected.
REFERENCES
[0081] Pugh J C, Summers J W. Infection and uptake of duck
hepatitis B virus by duck hepatocytes maintained in the presence of
dimethyl sulfoxide. Virology 1989; 172:564.572. [0082] Pugh, J. C.,
Ijaz, M. K., Suchmann D. B. Use of surrogate models for testing
efficacy of disinfectants against Hepatitis B virus. Am J Infect
Cont 1999; 27:373-6. Pugh, J. C., Suchmann, [0083] D. B., Ijaz, M.
K. Hepatitis B virus efficacy testing: Qualification of an avian
Hepadavirus in vitro system that uses primary duck hepatocyte
cultures. 10th International Symposium on Viral Hepatitis and Liver
Diseases. Atlanta. USA, 2000, Abstract B 139. [0084] Berry, M. N.,
Friend, D. S. High-yield preparation of isolated rat liver
parenchymal cells. J. Cell Biology 1969; 43:506-520. [0085] Seglen,
P. Preparation of isolated rat liver cells. Methods Cell Biol 1971;
3:29-83. [0086] Pugh, J. C., Di, Q. U., Mason W. S., Simmons H.
Susuceptibility to duck hepatitis B virus infection is associated
with the presence of cell surface receptor sites that efficiently
bind virus particles. J Virol 1995; 69:4814-22. [0087] Reed, L.,
Muench, H. A. A simple method of estimating fifty percent end
points. Am J of Hyg 1938; 27:493-497.
[0088] It should be understood that while the invention has been
described in detail herein, the examples provided are for
illustrative purposes only. Other modifications of the embodiments
of the present invention that are obvious to those of ordinary
skill in the art are intended to be within the scope of the
invention, which is more fully defined in the claims which follow
hereafter.
* * * * *